Encyclopedia of Respiratory Medicine, Four-Volume Set, Volume 1-4

INTRODUCTION R espiratory diseases represent one of the largest health problems word wide. Diseases such as asthma an...

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INTRODUCTION

R

espiratory diseases represent one of the largest health problems word wide. Diseases such as asthma and the smoking related diseases are already common and increasing so we urgently need better approaches to treat or cure these diseases. At the same time, new respiratory diseases such as those associated with viruses threaten pandemics that challenge our national health systems. With these continued challenges for new treatment with better patient care, clinical and respiratory researchers have sought better approaches to all aspects of patient care from improved diagnoses to superior therapies. This has lead to an explosion of new research with an increasingly better understanding of how to diagnose diseases and then develop new therapies. Thus, for example ever improving technologies for imaging lung disease have lead to increasingly better diagnoses, although challenges remain as we seek to further improve resolution. At the same time, the revolution in molecular biology, culminating with the publication of the complete human genome, has lead to hopes for finding more precise clues to disease susceptibility pathogenesis in genetic analysis. This is leading to new concepts in pharmacogenomics as we start to use new drugs, including those used for lung cancers, being directed at mutations associated with disease. This is the first Encyclopaedia of Respiratory Medicine. It is our hope that it is comprehensive and captures the key aspects of current patient care, as well as the exciting developments in respiratory science that we all believe will eventually lead to better patient care in the twenty first century. This encyclopaedia is comprehensive in scope and provides clinician and researcher with a snap shot of the current state of knowledge in respiratory medicine. All entries have adhered to a structured layout, starting with an abstract crystallizing the key facts and finishing with reading lists for those who want to delve further into the subject. In addition, most entries have a colour diagram designed to help understanding and provide a valuable aid for undergraduate and post-graduate teaching. These are exciting times for respiratory medicine. We hope this encyclopedia will become a valuble tool for clinicians and researches at all stages of their careers from those beginning their carreers to those established but wanting to update themselves on the new developments. Finally, we would like to thank our Advisory Editorial Board who helped so much in shaping the contents of this works, as well as the authors who wrote the articles and faced the challenge of condensing areas of respiratory medicine, often the subject of entire textbooks, into a short article of 4000 words or less.

GEOFFREY J. LAURENT STEVEN D. SHAPIRO

FOREWORD

Animals live by two principal things, food and breath. Of these, by far the most important is the respiration, for if it is stopped, the man will not endure long, but immediately dies. – Aretaeus the Cappocian (150–200 AD)

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f course, not all medical specialists would agree with this statement, and those who disagree would be quick to posit that it is the failure of ‘‘their’’ particular organ that tends to cause immediate death. However, that is not the issue. The point of this quotation is to illustrate that the proper functioning of the lung has been a subject of great interest for centuries. The Greek physician Aretaeus devoted many of his observations to diabetes, but his manuscript ‘‘On the Causes and Indications of Acute and Chronic Diseases’’ also discussed lung diseases, such as pneumonia. Since his time, great numbers of physicians from all continents and cultures have contributed to our knowledge of respiratory diseases. While acknowledging our rich history of discoveries about pulmonary and respiratory medicine— discoveries that were made by men and women whose names symbolize the great journey of this specialty—one must concede that the field experienced an extraordinary growth spurt beginning in the 1940s. Knowledge of respiratory physiology, which developed very fast during World War II, created a tidal wave of interest that continued for years afterward. The ability to measure and understand respiratory physiology and its alterations became a diagnostic tool, and it opened the door to therapeutic or respiratory support procedures. But, then, in the 1950s and 1960s cell biology and subcellular research entered the scene. The potential of molecular biology and genetics was quickly recognized, and respiratory medicine appreciated that a better understanding of normal and disordered biological respiratory processes hinged on use of these new approaches. Lung and respiratory researchers, impelled in part by the ever-increasing public health burdens of respiratory diseases, seized the opportunity. The stage was set for progress to occur. The architects of this ‘‘revolution’’ in respiratory medicine are well known; it is our good fortune that many have contributed to these four volumes. Four volumes! y Encyclopedia! y Indeed, these four volumes truly constitute an encyclopedia of pulmonary biology and respiratory medicine! Respiratory medicine is still growing. Because it is such a dynamic and exciting field, new investigators will almost surely want to be part of it. However, to do so they will need to know about the established state of knowledge that will be the basis of their work. New investigators in the science of respiratory medicine, whether interested in fundamental research or clinical research or application, will find ideas and inspiration in these volumes. All of the tools of the trade are assembled therein. As noted, respiratory medicine has been a progressive and expanding field but, as is the case with many fields of medicine, the transfer of what we know to the general practice of medicine has been slow and limited. Translation, as it is called, is an emerging discipline in need of assistance; fortunately, the breadth of the knowledge presented in these volumes provides tools to facilitate this translation process. This four-volume encyclopedia is, at once, both a tribute to the centuries of pioneering investigations in the field of respiratory medicine and a foundation for even greater accomplishments in the future. The presentation of all this knowledge in these excellent and comprehensive volumes can only serve to stimulate further work of equal or surpassing significance. The editors and the authors are to be commended for their contributions to this singular effort. Because of their work, respiratory science and medicine will advance faster and patients worldwide will be the beneficiaries. Claude Lenfant, MD Gaithersburg, Maryland

Notes on the Subject Index To save space in the index, the following abbreviations have been used: ALI

acute lung injury

ARDS

acute respiratory distress syndrome

BAL

bronchoalveolar lavage

BPD

bronchopulmonary dysplasia

CAP

community-acquired pneumonia

CFTR

cystic fibrosis transporter regulation

COP

cryptogenic organizing pneumonia

COPD

chronic obstructive pulmonary disease

CWP

coal workers’ pneumoconiosis

G-CSF

granulocyte colony-stimulating factor

GERD

gastroesophageal reflux disease

GM-CSF

granulocyte-macrophage colony-stimulating factor

HUVS

hypocomplementemic urticarial vasculitis syndrome

IL

interleukin

IPF

idiopathic pulmonary fibrosis

IPH

idiopathic pulmonary hemosiderosis

MCP

monocyte chemoattractant protein

M-CSF

macrophage colony-stimulating factor

MIP

macrophage inflammatory protein

MMP

matrix metalloproteinase

NSCLC

non-small cell lung carcinoma

PPAR

peroxisome proliferator-activated receptor

SCLC

small-cell lung carcinoma

SP

surfactant protein

TGF

transforming growth factor

TIMP

tissue inhibitor of metalloproteinases

TNF

tumor necrosis factor

VEGF

vascular endothelial growth factor

Editorial Advisory Board Kenneth B. Adler, North Carolina State University, Raleigh, NC, USA Peter J. Barnes, Imperial College London, UK Paul Borm, Zuyd University, Heerlen, The Netherlands Arnold R. Brody, Tulane Medical School, New Orleans, LA, USA Rachel C. Chambers, University College London, UK Augustine M. K. Choi, University of Pittsburgh, PA, USA Jack A. Elias, Yale University School of Medicine, New Haven, CT, USA Patricia W. Finn, University of California San Diego, La Jolla, CA, USA Stephen T. Holgate, University of Southampton, Southampton, UK Steven Idell, The University of Texas Health Center at Tyler, TX, USA Sebastian L. Johnston, National Heart and Lung Institute, Imperial college London, UK Talmadge E. King, Jr, University of California, San Francisco, CA, USA Stella Kourembanas, Children’s Hospital Boston, Harvard Medical School, Boston, MA, USA Y. C. Gary Lee, University College London, UK Richard Marshall, University College London, UK Sadis Matalon, University of Alabama, Birmingham, AL, USA Joel Moss, National Institutes of Health, Bethesda, MD, USA William C. Parks, University of Washington, Seattle, WA, USA Charles G. Plopper, University of California, Davis, CA, USA Bruce W. S. Robinson, The University of Western Australia, Nedlands, Australia Neil Schluger, Columbia University College of Physicians and Surgeons, New York, NY, USA Edwin K. Silverman, Brigham and Women’s Hospital Boston, MA, USA Eric S. Silverman, Brigham and Women’s Hospital, Boston, MA, USA Peter Sly, Institute for Child Health Research, West Perth, Australia Kingman Strohl, Case Western Reserve University, Cleveland, OH, USA Teresa D. Tetley, Imperial College London, UK John B. West, University of California, San Diego, CA, USA

Editors Geoffrey J Laurent, Royal Free and University College Medical School, London, UK Steven D Shapiro, Brigham and Woman’s Hospital, Boston, USA

Dedication To my family, Lal, Guy, David and Gabrielle (GJL). My contribution to this work would not have been possible without the love and support from my wife Nicole and my daughters Calli, Tess, Skylar, and Ellery. I also thank my mentors and trainees for my continual education and the Division of Pulmonary and Critical Care Medicine at Brigham and Women’s Hospital who took care of our patients allowing me the time to undertake this project (SDS)

Permission Acknowledgments The following material is reproduced with kind permission of Lippincott Williams and Wilkins Figure 4 and 8 of ARTERIAL BLOOD GASES Table 1 of ARTERIES AND VEINS Figure 2 and 3 of BREATHING | Breathing in the Newborn Figure 2a, 2b, 3, 4 and 5 of DRUG-INDUCED PULMONARY DISEASE Table 1, 2 and 3 of DRUG-INDUCED PULMONARY DISEASE Figure 2 of ENVIRONMENTAL POLLUTANTS | Diesel exhaust particles Figure 4 of EXERCISE PHYSIOLOGY Figure 2 of FLUID BALANCE IN THE LUNG Figure 2 of GASTROESOPHAGEAL REFLUX Figure 2 of GENE REGULATION Figure 2 of HIGH ALTITUDE, PHYSIOLOGY AND DISEASES Figure 2 and 3 of IDIOPATHIC PULMONARY HEMOSIDEROSIS Table 1 of IDIOPATHIC PULMONARY HEMOSIDEROSIS Figure 1, 2 and 3 of OXYGEN-HEMOGLOBIN DISSOCIATION CURVE Figure 10a, 10b and 11a of SYSTEMIC DISEASE | Eosinophilic Lung Diseases http://www.lww.com

The following material is reproduced with kind permission of Nature Publishing Group Figure 2 of COAGULATION CASCADE | iuPA, tPA, uPAR Figure 1 of COAGULATION CASCADE | Tissue Factor Figure 1a of MATRIX METALLOPROTEINASES Figure 1 of MYOFIBROBLASTS

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Figure 1 of VESICULAR TRAFFICKING http://www.nature.com/nature and http://www.nature.com/reviews

The following material is reproduced with kind permission of Taylor & Francis Ltd Figure 2 of AUTOANTIBODIES Table 1 of BASAL CELLS Figure 1 of NEUROPHYSIOLOGY | Neuroendocrine Cells Table 1 of NEUROPHYSIOLOGY | Neuroendocrine Cells Figure 1 and 2 of SURFACANT | Overview Tables 1, 2, 3 and 4 of SURFACANT | Overview http://www.tandf.co.uk/journals

A ACETYLCHOLINE J Zaagsma and H Meurs, University of Groningen, Groningen, The Netherlands & 2006 Elsevier Ltd. All rights reserved.

Abstract In the airways, acetylcholine is a neurotransmitter in parasympathetic ganglia and in postganglionic parasympathetic nerves, as well as a nonneural paracrine mediator in various cells in the airway wall. Ganglionic transmission by acetylcholine is mediated by nicotinic receptors, which are ligand-gated in channels, whereas postganglionic transmission is through G-protein-coupled muscarinic receptors. Of the five mammalian muscarinic receptor subtypes, mainly M1, M2, and M3 receptors are involved in airway functions. Gq-coupled M1 receptors facilitate ganglionic transmission mediated by nicotinic receptors and modulate surfactant production and fluid resorption in the alveoli. Prejunctional Gi/o-coupled M2 receptors in parasympathetic nerve terminals attenuate acetylcholine release upon nerve stimulation. M2 receptors are also abundantly present in airway smooth muscle; however, the major function of these postjunctional M2 receptors is unknown. Postjunctional Gq-coupled M3 receptors mediate airway smooth muscle contraction and mucus secretion. Dysfunction of the prejunctional M2 autoreceptor induced by allergic airway inflammation has been implied in exaggerated vagal reflex activity and airway hyperresponsiveness in asthma. Inflammation-induced increased M3 receptor stimulation may be involved in airway remodeling in chronic asthma. Possible mechanisms include potentiation of growth factor-induced proliferation of airway smooth muscle cells and induction of a contractile phenotype of these cells. Exaggerated M3 receptor stimulation may also cause reduced responsiveness to b2-adrenoceptor agonists by transductional cross-talk between phosphoinositide metabolism and adenylyl cyclase, which involves protein kinase C-induced uncoupling of the b2adrenoceptor from the effector system. Muscarinic receptor antagonists have been shown to be effective in airway diseases like asthma and, especially, chronic obstructive pulmonary disease. Of these, tiotropium bromide is particularly useful, due to its long duration of action as well as its kinetic selectivity for the M3 receptor.

Introduction Acetylcholine is a neurotransmitter in the central and peripheral nervous system where it plays a major role in the afferent neurons of both the autonomic and somatic (voluntary) branches. As a chemical transmitter, it has been identified as ‘Vagusstoff’ in 1921 by Otto Loewi showing its release from an isolated frog heart following stimulation of the

vagosympathetic trunk; when applied to a second, unstimulated heart, the perfusate slowed its rate, resembling the effect of vagus stimulation. In 1926 Loewi provided evidence for identification of Vagusstoff as acetylcholine. Acetylcholine is the neurotransmitter of all sympathetic and parasympathetic autonomic ganglia and of the postganglionic parasympathetic nerves. In the airways, the parasympathetic ganglia are located near or within the airway wall. Ganglionic transmission mediated by acetylcholine is through nicotinic receptors which belong to the family of ligand-gated ion channels. Postganglionic transmission by acetylcholine, released from parasympathetic nerve terminals, is through muscarinic receptors of which five different subtypes have been identified, all being G-protein-coupled receptors. During periods of airway inflammation vagal release of acetylcholine may be increased by various mechanisms. Hence, both in asthma and (particularly) in chronic obstructive pulmonary disease (COPD) blockade of postjunctional muscarinic receptors is the key to reversing airway obstructions.

Synthesis, Storage, and Release Acetylcholine is synthesized from choline and acetylcoenzyme A (acetyl-CoA) in the cytoplasm of the nerve terminal through the enzyme choline acetyltransferase (ChAT). Choline is taken up by the nerve terminal from the extracellular fluid through a sodium-dependent carrier; this transport is the ratelimiting process in acetylcholine synthesis. Acetyl-CoA is synthesized in mitochondria which are abundantly present in the nerve endings. Most of the synthesized acetylcholine is actively transported from the cytosol into synaptic vesicles by a specific transporter; this vesicular (‘quantal’) package of acetylcholine reaches up to 50 000 molecules per vesicle. Release of acetylcholine is initiated by influx of Ca2 þ ions through voltage-operated N- or P-type calcium channels. The increased intracellular Ca2 þ ions bind to a vesicle-associated protein (synaptotagmin) which favors association of a second vesicle protein (synaptobrevin) with one or more proteins in the plasma membrane of the nerve terminal. Following

2 ACETYLCHOLINE

this vesicle-docking process, fusion between vesicle membrane and plasma membrane occurs, followed by exocytosis. After the expulsion of acetylcholine the empty vesicle is recaptured by endocytosis and can be reused. In the synaptic cleft, the released acetylcholine will associate with post- and prejunctional receptors and is also subject to rapid hydrolysis by the enzyme acetylcholinesterase into choline and acetate. Over 50% of the choline formed will be taken up again by the nerve terminal and reused for neurotransmitter synthesis. Acetylcholine is also present in nonneuronal cells. In recent years it has become clear that in the airways the majority of cells express ChAT and contain acetylcholine, including epithelial cells, smooth muscle cells, mast cells, and migrated immune cells such as alveolar macrophages, granulocytes, and lymphocytes. However, the regulatory role of this nonneuronal acetylcholine in inflammatory airways diseases has yet to be established.

Regulation of Synaptic Transmission and Activity Ganglionic

Preganglionic nerves innervating the parasympathetic ganglia in the airways evoke action potentials during normal breathing with relatively high frequencies, in the range of 1–20 Hz. As a result, basal airway smooth muscle tone in vivo is mediated to a significant extent by cholinergic nerve activity. The pattern of ganglionic action potential bursts coincides with respiration, suggesting that the respiratory centers in the brainstem govern preganglionic nerve activity. However, in addition to this central drive, reflex stimulation through mechanically sensitive afferent nerve terminals in the lungs during respiration is importantly involved as well. The fidelity by which preganglionic impulses are translated into action potentials in the postganglionic neurons is relatively low in parasympathetic airway ganglia, implying a filtering function of these ganglia. This filtering function can be diminished by various inflammatory mediators. Thus, histamine, prostaglandin D2 (PGD2), and bradykinin are able to enhance ganglionic cholinergic transmission and the same is true for tachykinins (substance P, neurokinin A) released by nonmyelinated sensory C-fibers in the airways. Postganglionic

The release of acetylcholine from parasympathetic nerve terminals is regulated by a variety of prejunctional receptors, which may inhibit or facilitate transmitter outflow. In the airways, autoinhibitory

muscarinic M2 receptors, activated by acetylcholine itself, represent an important negative feedback, limiting further release, at higher firing rates in particular. In animal models of allergic airway inflammation and asthma as well as in human asthma, dysfunction of these M2 autoreceptors has been found to contribute to exaggerated acetylcholine release from vagal nerve endings, to increased cholinergic reflex activity in response to inhaled stimuli, and to contribute to airway hyperresponsiveness. Most of this receptor dysfunction is thought to be caused by activated eosinophils that migrate to cholinergic nerves and release major basic protein (MBP) which acts as an allosteric antagonist of muscarinic M2 receptors. Since eosinophilic inflammation is far less prominent in COPD and since M2 autoreceptors are more prominent in larger airways, it is no surprise that these receptors are still functional in patients with stable COPD; however, this does not exclude a dysfunction during acute exacerbations. In addition to M2 autoreceptors, a variety of heteroreceptors modulating acetylcholine release have been identified on cholinergic nerve endings. Catecholamines may inhibit or facilitate acetylcholine overflow through prejunctional a2- and b2-adrenoceptors, respectively. Neurokinins like substance P may enhance cholinergic transmission through facilitatory neurokinin 1 (NK1) and/or 2 (NK2) receptors. Interestingly, substance P may also induce MBP release from eosinophils, causing M2 receptor dysfunction, which could act synergistically to direct facilitation. Allergic inflammation-derived prostanoids, including PGD2, PGF2a, and thromboxane A2, as well as histamine, can also augment acetylcholine release through prejunctional receptors. Taken together, the above observations indicate that parasympathetic acetylcholine release is governed by various regulatory systems, the set-point of which is subject to environmental modulations. During periods of airway inflammation these modulations often result in enhanced cholinergic transmission.

Receptors and Biological Function In the ganglia, acetylcholine interacts with nicotinic receptors. These receptors consist of five polypeptide subunits, together forming a cylindric structure of about 8 nm diameter, which acts as an ion channel. Each subunit passes the membrane four times, so the central pore is surrounded by 20 membranespanning helices. The subunits have been subdivided into five classes, designated a, b, g, d, and e. Of the a and b subunits, 10 and 4 different subtypes have been identified, respectively. Peripheral ganglionic

ACETYLCHOLINE 3

receptors consist of only a and b subunits, the main subtype being (a3)2(b4)3. Each a subunit possesses a binding site for acetylcholine; they need to be occupied both to induce channel opening, which will enhance Na þ and K þ permeability. This results in an inward flux of mainly Na þ ions, causing depolarization and action potential generation in the postganglionic cell (provided the acetylcholine concentration is high enough). Acetylcholine released by postganglionic parasympathetic nerves may choose between five muscarinic receptor subtypes, designated M1 to M5, to interact with. Most organs and tissues express more than one subtype and this is true for many individual cells as well. The five subtypes can be subdivided into two main classes, the odd-numbered receptors (M1, M3, M5), which couple preferentially to heterotrimeric Gqproteins, and the even-numbered (M2, M4) receptors which show selectivity for Gi/o type of G-proteins. The principal signaling route of Gq-coupled receptors is the activation of phospholipase C, mediating hydrolysis of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) (Figure 1, left). IP3 mobilizes Ca2 þ ions from intracellular stores, which generates a rapid and transient rise of the free Ca2 þ concentration in the cytosol. DAG triggers the translocation and activation of protein kinase C (PKC) which is able to phosphorylate a variety of different protein substrates. The main signal transduction of Gi/o-coupled receptors is to inhibit adenylyl cyclase activity, which reduces the intracellular cyclic AMP concentration.

In airway smooth muscle, activation of Gi/o by M2 receptors may also diminish opening of calcium-activated potassium channels (KCa or maxi-K channels) induced by (Gs-coupled) b-adrenoceptors. Thus, K þ efflux, membrane hyperpolarization, and subsequent smooth muscle relaxation, initiated by b-agonist administration, is under restraint of acetylcholine through this mechanism. In the airways of most mammalian species, including human, M1, M2, and M3 receptors are the most important ones. So far, M4 receptors have only been detected convincingly in bronchiolar smooth muscle and alveolar walls of the rabbit, whereas muscarinic M5 receptors, now known to mediate dilatation of cerebral arteries and arterioles, are absent in the lungs. M1 receptors have been found in alveolar walls, parasympathetic ganglia, and submucosal glands. Rat and guinea pig lung studies have indicated their presence in type II alveolar cells, mediating surfactant production and fluid reabsorption, respectively. In parasympathetic airway ganglia of several species, including human, M1 receptor stimulation is able to facilitate ganglionic transmission mediated by nicotinic receptors. Thus, vagal bronchoconstriction, induced by inhalation of SO2, has been found especially sensitive to inhaled pirenzepine, a M1-selective antagonist. In submucosal glands, M1 receptors are not involved in mucus secretion, which appears to be mediated solely by M3 receptors. M2 and M3 receptors represent the major receptor populations, both in intra- and extrapulmonary Epinephrine, 2-agonists

Acetylcholine

PIP2

M3 Gq

PLC

DAG

2 PKC −

IP3

Ca2+

Contraction

AC Gs

+

P

P

−

P

− ATP

P

ARK

cAMP

Relaxation

Figure 1 Cross-talk between M3 muscarinic receptors and b2-adrenoceptors in airway smooth muscle. Generation of 1,2-diacylglycerol (DAG) by M3 receptor-induced phosphoinositide (PIP2) metabolism causes activation of protein kinase C (PKC). PKC may phosphorylate the b2-adrenoceptor as well as Gs, causing uncoupling of the receptor from the effector system. Moreover, PKC may phosphorylate b-adrenoceptor kinase(s) (bARK), which amplifies b-agonist-induced desensitization mediated by bARK-induced phosphorylation of the receptor. AC, adenylyl cyclase; cAMP, cyclic adenosine 30 , 50 -monophosphate; IP3, inositol 1,4,5-trisphosphate; PLC, phospholipase C.

4 ACETYLCHOLINE

airways. As already discussed, inhibitory M2 autoreceptors located at parasympathetic nerve terminals have an important regulatory role in limiting acetylcholine release. Postjunctional muscarinic receptor populations in airway smooth muscle are a mixture of M2 and M3 receptors, the M2 subtype being predominant, particularly in the large airways. Contraction, however, is primarily mediated by M3 receptors (even in those smooth muscle preparations where the ratio of M2 : M3 receptors is 90 : 10), the M2 receptor population having at most a minor supporting role. This is confirmed in airway preparations from M2 receptor knockout mice, in which carbachol, a muscarinic agonist, was hardly less potent than in preparations from wildtype mice. Cross-talk between Gi-coupled M2 receptors and Gs-coupled b-adrenoceptors (having opposing effects on cyclic AMP accumulation or maxi-K channel opening) has no major effects in modulating muscarinic agonist induced contraction or b-agonist induced relaxation, at least under physiological circumstances. However, in inflammatory conditions such as asthma, in which Gi-proteins may be upregulated, the situation may change. In contrast to M2 receptors, Gq-coupled M3 receptors, generating IP3 and DAG by stimulating phosphoinositide metabolism, may have a major influence on b2-adrenoceptor function, even in noninflamed airways. This is due to DAG-induced activation of PKC which may (1) phosphorylate the b2-adrenoceptor as well as Gs, causing receptor uncoupling and desensitization, and (2) phosphorylate and activate b-adrenoceptor kinase(s) (bARKs, which are members of the G-protein receptor kinase (GRK) family), amplifying homologous, b-agonist induced desensitization (Figure 1). These processes may explain the well-known attenuation of b-agonist efficacy during episodes of severe bronchoconstriction, for example, during exacerbations. Airway Remodeling

In addition to phosphoinositide metabolism, inhibition of adenylyl cyclase and maxi K-channel activation, stimulation of M3 and/or M2 receptors in airway smooth muscle cells has been shown to activate different promitogenic signaling pathways, including the p42/p44 mitogen-activated protein kinase, Rho/Rho kinase, and PI3 kinase pathways. In vitro studies have revealed that muscarinic agonists do not induce airway smooth muscle cell proliferation by themselves, but enhance the proliferative response induced by peptide growth factors. This effect, which is solely mediated by the M3 receptor,

indicates that acetylcholine may contribute to airway remodeling as observed in asthma and COPD. Indeed, in an animal model of chronic asthma it was recently demonstrated that the long-acting muscarinic antagonist tiotropium bromide inhibited increased airway smooth muscle mass, enhanced airway smooth muscle contractility, and increased expression of contractile proteins in the lung upon repeated allergen challenge. This implies that, in addition to their bronchodilating properties, muscarinic receptor antagonists could be beneficial in the treatment of asthma by preventing chronic airway hyperresponsiveness and decline of lung function. Although not fully established, a more extensive role of acetylcholine in airway remodeling, including airway smooth muscle proliferation, contractile protein expression, promitogenic signaling, and regulation of secretory functions as well as cell migration, has recently been proposed (Figure 2).

Acetylcholine in Respiratory Diseases As indicated above, the parasympathetic nervous system represents a major constrictory pathway of the airways; even basal bronchomotor tone is partly governed by acetylcholine. Both its release and its postjunctional effects, including smooth muscle contraction and mucus secretion, are regulated and mediated, respectively, by muscarinic receptors. So far, no evidence for upregulation of postjunctional M3 and M2 receptors has been found in hyperresponsive airways of patients with asthma and COPD. In contrast, dysfunctional autoreceptors, leading to exaggerated vagal reflexes in the airways, are well established in allergic asthma. M1 receptors, facilitating nicotinic neurotransmission in parasympathetic airway ganglia, do not appear to contribute significantly to bronchomotor tone in humans, either with or without obstructive airway diseases. Hence, the principle therapeutic muscarinic receptor target in asthma and COPD is the M3 receptor. In COPD, muscarinic receptor antagonists like ipratropium, a quarternary nonselective antagonist, are very effective in causing bronchodilatation. A recently introduced antagonist is tiotropium, which acutely occupies both M2 and M3 receptors; however, while it dissociates rapidly from M2 receptors during washout, blockade of M3 receptors persists for hours. This kinetically based M3 receptor subtype selectivity could be of therapeutic advantage since blockade of prejunctional M2 receptors enhances acetylcholine outflow. Both in COPD and, particularly, in allergic asthma, in which M2 autoreceptors are already dysfunctional, this is an unwanted side effect.

ACID–BASE BALANCE 5

ACh Extracellular matrix proteins

Migration

Hypercontractility

Chemotaxis activation

Contraction

Hyperplasia

Figure 2 Proposed mechanisms by which acetylcholine (ACh) could affect airway smooth muscle remodeling. Acetylcholine has been shown to affect airway smooth muscle contractility, contractile protein expression, promitogenic signaling, and proliferation. In addition, like several other G-protein-coupled receptor agonists, acetylcholine could also be involved in airway smooth muscle cell migration, extracellular matrix protein production, and secretion of cytokines and chemokines. Altogether, these effects could contribute to airway remodeling in asthma and COPD. Reproduced from Gosens R, Zaagsma J, Grootte Bromhaar M, Nelemans SA, and Meurs H (2004) Acetylcholine: a novel regulator of airway smooth muscle remodelling. European Journal of Pharmacology 500: 193–201, with permission from Elsevier.

See also: Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Neurophysiology: Neural Control of Airway Smooth Muscle; Neuroanatomy; Neurons and Neuromuscular Transmission.

Further Reading Berge RE ten, Santing RE, Hamstra JJ, Roffel AF, and Zaagsma J (1995) Dysfunction of muscarinic M2 receptors after the early allergic reaction: possible contribution to bronchial hyperresponsiveness in allergic guinea-pigs. British Journal of Pharmacology 114: 881–887. Billington CK and Penn RB (2002) M3 muscarinic acetylcholine receptor regulation in the airway. American Journal of Respiratory Cell and Molecular Biology 26: 269–272. Coulson FR and Fryer AD (2003) Muscarinic acetylcholine receptors and airway diseases. Pharmacology and Therapeutics 98: 59–69. Gosens R, Bos ST, Zaagsma J, and Meurs H (2005) Protective effect of tiotropium bromide in the progression of airway

smooth muscle remodeling. American Journal of Respiratory and Critical Care Medicine 171: 1096–1102. Gosens R, Zaagsma J, Grootte Bromhaar M, Nelemans SA, and Meurs H (2004) Acetylcholine: a novel regulator of airway smooth muscle remodelling. European Journal of Pharmacology 500: 193–201. Racke´ K and Matthiesen S (2004) The airway cholinergic system: physiology and pharmacology. Pulmonary Pharmacology and Therapeutics 17: 181–198. Wess J (2004) Muscarinic acetylcholine receptor knockout mice: novel phenotypes and clinical implications. Annual Review of Pharmacology and Toxicology 44: 423–450. Wessler I, Kilbinger H, Bittinger F, Unger R, and Kirkpatrick CJ (2003) The nonneural cholinergic system in humans: expression, function and pathophysiology. Life Sciences 72: 2055–2061. Zaagsma J, Meurs H, and Roffel AF (eds.) (2001) Muscarinic Receptors in Airways Diseases. Basel: Birkhauser Verlag. Zaagsma J, Roffel AF, and Meurs H (1997) Muscarinic control of airway function. Life Sciences 60: 1061–1068.

ACID–BASE BALANCE O Siggaard-Andersen, University of Copenhagen, Copenhagen, Denmark & 2006 Elsevier Ltd. All rights reserved.

Abstract The acid–base balance or neutrality regulation maintains a pH around 7.4 in the extracellular fluid by excreting carbon dioxide (carbonic acid anhydride) in the lungs and noncarbonic acid or base in the kidneys. The result is a normal acid–base status in blood and extracellular fluid, i.e., a normal pH, a normal carbon dioxide tension (pCO2 ), and a normal concentration of

titratable hydrogen ion (ctH þ ). A pH, log pCO2 chart illustrates the acid–base status of the arterial blood. The chart shows normal values as well as values to be expected in typical acid– base disturbances, i.e., acute and chronic respiratory acidosis and alkalosis, and acute and chronic nonrespiratory (metabolic) acidosis and alkalosis. The chart allows estimation of the concentration of titratable H þ of the extended extracellular fluid (including erythrocytes), ctH þ Ecf. This quantity is also called standard base deficit but the term base does not directly indicate that the quantity refers to the excess or deficit of hydrogen ions. ctH þ Ecf is the preferred indicator of a nonrespiratory acid–base disturbance being independent of acute changes in pCO2 in vivo. While pH and pCO2 are directly measured, ctH þ Ecf

6 ACID–BASE BALANCE is calculated from pH and pCO2 using the Henderson–Hasselbalch equation and the Van Slyke equation.

Description The acid–base balance or neutrality regulation maintains a pH around 7.4 in the extracellular fluid by excreting carbon dioxide (carbonic acid anhydride) in the lungs and noncarbonic acid or base in the kidneys. The result is a normal acid–base status in blood and extracellular fluid, i.e., a normal pH, a normal carbon dioxide tension (pCO2 ), and a normal concentration of titratable hydrogen ion (ctH þ ). Figure 1 illustrates the acid–base status of the blood, especially the relationships among the three key variables. pH and the Hydrogen Ion Concentration (cH þ )

pH and cH þ of the plasma are both indicated on the abscissa of Figure 1. pH is the negative dacadic logarithm of molal hydrogen ion activity. Concentration of free hydrogen ion (cH þ ) is calculated as 109 pH nmol l 1. pH and pOH are closely related: pH þ pOH ¼ pKw ¼ 13.622 at 371C, where Kw is the ionization constant of water. If H þ is considered a key component of an aqueous solution, then OH is a derived component. Accounting for H þ and H2O indirectly accounts for OH as well. It is the author’s conviction that the relevant component is the hydrogen ion, not hydrogen ion binding groups (base) nor hydroxyl ions. The Carbon Dioxide Tension of the Blood (pCO2 )

pCO2 , i.e., the partial pressure of carbon dioxide in a gas phase in equilibrium with the blood, is shown on the ordinate on a logarithmic scale in Figure 1. When pCO2 increases, the concentration of dissolved carbon dioxide and carbonic acid increases, and hence the hydrogen ion concentration increases: CO2 þ H2 O-H2 CO3 -Hþ þHCO 3 The Concentration of Titratable Hydrogen Ion (ctH þ )

ctH þ is indicated on the scale in the upper left corner of Figure 1. ctH þ is a measure of added noncarbonic acid or base. The amount of hydrogen ion added or removed in relation to a reference pH of 7.40 may be determined by titration to pH ¼ 7.40 at pCO2 ¼ 5:33 kPa ( ¼ 40 mmHg) and T ¼ 371C using strong acid or base, depending upon the initial pH. Titratable hydrogen ion is also called base deficit, or with the opposite sign base excess. Unfortunately, the term ‘base’ is ambiguous (it has previously been associated

with cations) and does not directly indicate that the relevant chemical component is the hydrogen ion. The term hydrogen ion excess or acronym HX may also be used. Note: by definition, ctH þ of blood refers to the actual hemoglobin oxygen saturation, not the fully oxygenated blood. Acid and base are defined by the equilibrium: Acidz # Hþ þ Basez1 where Acidz and Basez 1 is a conjugate acid–base pair. The charge number z may be positive, zero, or negative. A strong acid, e.g., HCl, dissociates completely: HCl-H þ þ Cl . The anion that follows the hydrogen ion is called an aprote, nonbuffering, or strong anion. At physiological pH, even lactic acid is a strong acid and lactate an aprote anion. A base is a molecule containing a hydrogen ion-binding group. A strong base, e.g. OH , associates completely with hydrogen ion: OH þ H þ -H2O. The cation that follows the hydroxyl ion is called an aprote, nonbuffering, or strong cation, e.g., Na þ or K þ . A weak acid (buffer acid) is in equilibrium with its conjugate weak base (buffer base), e.g.: H2 CO3 # Hþ þ HCO 3 hemoglobinz # Hþ þ hemoglobinz1 The concentration of titratable hydrogen ion may be determined for plasma (P), whole blood (B), or a model of the extended extracellular fluid, i.e., blood plus interstitial fluid (Ecf). The model consists of blood diluted threefold with its own plasma to get a hemoglobin concentration similar to the one obtained if the red cells were evenly distributed in the whole extracellular volume. An acute increase in pCO2 in vivo causes a rise in ctH þ B and a fall in ctH þ P while ctH þ Ecf remains constant. For example, an acute rise in pCO2 from 5.33 to 10.66 kPa (40–80 mmHg) causes a rise in whole blood ctH þ of about 5 mmol l 1, a fall in plasma ctH þ of about 3 mmol l 1, while the extracellular ctH þ remains independent of acute changes in pCO2 . The cause is a redistribution of hydrogen ions within the extended extracellular volume. The hydrogen ion concentration increases more in the poorly buffered interstitial fluid than in the blood plasma, where it increases more than inside the erythrocytes, where hemoglobin binds the hydrogen ions. Hydrogen ions diffuse from the poorly buffered interstitial fluid into the blood plasma and further into the erythrocytes. Very little transfer of hydrogen ions occurs between the intracellular space and the extracellular space, so ctH þ Ecf remains virtually constant during acute


No

rm

–5

0 +5 ex ce ss

pCO2 in arterial blood Concentration of titratable mmHg kPa hydrogen ion in extracellular fluid 20.0 150 mmol l–1 19.0 Siggaard-Andersen 140 0 5 0 5 0 18.0 –1 –1 –2 –2 –3 acid–base chart 130 17.0

H+ deficit

al

120

0 +1

ca ia

ia

pn

pn

60

11.0

9.0 8.0 7.0 6.0

50

40

s ces

5.0

Normal

40

Area

30

20

ex

15

ion

12.0

50

Normal

10 en rog

13.0

10.0 70

it ic defic n ro n Ch n io e g o dr hy

Concentration of in plasma +25 bicarbonate mmol l–1

16.0 15.0 14.0 Hypercapnia

80

er

ca er

0 +2

90

p hy

p hy

ic on

e ut

5 +1

100

r Ch

Ac

H+

110

35

e

ut 3.0

nia

20

onic

2.5

Chr

15

Hypocapnia

ap

oc

p hy

ion

25

Ac

hyd roge n

4.0 3.5

capnia

exc ess

30

Chronic hypo

yd te h

Acu

2.0

+30

6.8

pH in arterial plasma 6.9 7.0

140 120 100 90 Concentration of free hydrogen ion in plasma nmol l–1

7.1

80

7.2

70

7.3

60 50 Acidemia

7.4

40 35 Normal

7.5

7.6

30 25 Alkalemia

7.7

1.5

20

Figure 1 Acid–base chart for arterial blood with normal and pathophysiological reference areas. The acid–base status is shown as a point with three coordinates: pH (abscissa), pCO2 (ordinate), and c tH þ (oblique coordinate). The bands radiating from the normal area (the central ellipse) show reference areas for typical acute and chronic, respiratory and nonrespiratory, acid–base disturbances. Hyper- and hypocapnia are also called respiratory acidosis and alkalosis, respectively. Hydrogen ion excess and deficit, i.e., increased and decreased c tH þ , are also called nonrespiratory (or metabolic) acidosis and alkalosis, respectively. Reproduced from Siggaard-Andersen O (1971) An acid-base chart for arterial blood with normal and pathophysiological reference areas. Scandinavian Journal of Clinical and Laboratory Investigation 27: 239–245. Copyright & 1970, 1974 by Radiometer Copenhagen A/S, A˚kandevej 21, Brønshøj, Denmark.

changes in pCO2 in vivo. ctH þ Ecf is also called standard base deficit (SBD), or with the opposite sign standard base excess (SBE), but the term base is deprecated by the author. It is important to use ctH þ Ecf

rather than ctH þ B (whole blood titratable hydrogen ion) as a measure of a nonrespiratory acid–base disturbance, especially in neonatology where high hemoglobin concentrations and high pCO2 values may be

8 ACID–BASE BALANCE

encountered. The ctH þ B may then be considerably higher than ctH þ Ecf (as much as 4 mmol l 1) causing an erroneous diagnosis of metabolic acidosis, when the situation is merely a redistribution of hydrogen ions within the extended extracellular volume. Projections to the ctH þ scale in the upper left corner of Figure 1 should be made along the slanting so-called vivo-CO2 titration curves, which are virtually straight lines (slightly convex upwards). The slope of the lines depends on the concentration of nonbicarbonate buffers, i.e., mainly hemoglobin. In the chart, the slope corresponds to a hemoglobin concentration of 3 mmol l 1 corresponding to the hemoglobin concentration of the extended extracellular fluid. Variations in the slope due to variations in blood hemoglobin concentration are small and generally without clinical significance. Variations in the concentration of other buffers, e.g., albumin, are even less significant. In summary, the hydrogen ion status of the blood is described by a point in the acid–base chart: the x,y coordinates indicate cH þ and pCO2 , the oblique coordinate is ctH þ Ecf. The Henderson–Hasselbalch Equation

Often a description of acid–base balance is based on the Henderson–Hasselbalch equation, derived from the law of mass action: pH ¼ pK þ log10 ðcHCO 3 =ðaCO2 pCO2 ÞÞ where pK ¼ 6.10 and aCO2 ¼ 0.23 mmol l 1 kPa 1 ¼ 0.0306 mmol l 1 mmHg 1 (solubility coefficient of carbon dioxide in plasma at 371C). aCO2 pCO2 gives the concentration of H2CO3 plus CO2. pH is determined by two variables, pCO2 and cHCO3 ,

representing respiratory and metabolic disturbances. cHCO3 is shown in Figure 1 on a horizontal logarithmic scale along the pCO2 ¼ 5:33 kPa line. Projections to the scale should be made at an angle of 451. However, cHCO3 is not independent of pCO2 . For this reason, standard bicarbonate was introduced, i.e., the bicarbonate concentration in plasma of whole blood equilibrated with a gas mixture with a normal pCO2 (5.33 kPa ¼ 40 mmHg) at 371C. However, even the standard bicarbonate is not completely independent of acute changes in pCO2 in vivo, decreasing slightly in acute hypercapnia. To be independent, the equilibration should be performed with a model of the extended extracellular fluid. Projecting from a given point in the chart to the bicarbonate scale along the slanting vivo-CO2 equilibration lines gives the standard bicarbonate concentration of the extended extracellular fluid. The Van Slyke Equation

Blood gas analyzers measure pH with a glass electrode and pCO2 with a membrane-covered glass electrode (Stow-Severinghaus electrode). ctH þ Ecf is calculated from pH, pCO2 , and cHb (concentration of hemoglobin) using a model of the titration curve called the Van Slyke equation (Table 1). The equation calculates the change in buffer base concentration (bicarbonate plus protein anion plus phosphate) from the value at the reference point: pHPy ¼ 7:40; PyCO2 ¼ 5:33 kPa; and T y ¼ 37 C Buffer base (BB) is the difference between the concentrations of buffer anions and buffer cations (the latter being virtually zero at physiological pH). Strong ion difference (SID) is the difference between

Table 1 Van Slyke equation for calculation of the concentration of titratable hydrogen ion in the extended extracellular fluid, ctH þ Ecf þ c tH þ Ecf ¼ ð1 cHbEcf=cHby Þ ðDcHCO 3 P þ bH Ecf DpHPÞ c HbEcf ¼ c HbB V B/V Ecf concentration of hemoglobin in the extended extracellular fluid V B/V Ecf ¼ 1/3 (default value) ratio between the volume of blood and volume of extended extracellular fluid c Hby ¼ 43 mmol l 1 empirical parameter accounting for an unequal distribution of hydrogen ions between plasma and erythrocytes Dc HCO3 P ¼ c HCO3 P c HCO3 Py y c HCO3 Py ¼ 24.5 mmol l 1 concentration of bicarbonate in plasma at pHPy ¼ 7.40, PCO ¼ 5:33 kPa, T y ¼ 37:0 C 2 DpHP ¼ pHP pHPy bHþ Ecf ¼ bm Hby cHbEcf þ bP bm Hby ¼ 2.3 apparent molar buffer capacity of hemoglobin monomer in whole blood bP ¼ 7.7 mmol l 1 (default value) buffer value of nonbicarbonate buffers in plasma for a normal plasma protein (albumin) concentration c HbB ¼ rHbB/MmHb (substance) concentration of hemoglobin in blood (unit: mmol l 1) as a function of the mass concentration, rHbB (unit: g l 1) MmHb ¼ 16 114 g mol 1 molar mass of hemoglobin monomer

Ecf refers to the extended extracellular fluid, B to whole blood, P to plasma. Replacing cHbEcf by cHbB gives ctH þ B; replacing cHbEcf by zero gives ctH þ P. Note: if cHbB ¼ 9.0 mmol l 1 3 rHbB ¼ 14.5 g dl 1, then the Van Slyke equation simplifies to c tH þ Ecf ¼ 0.93 (Dc HCO3 P þ DpHP 14.6 mmol l 1).


the concentrations of nonbuffer cations and nonbuffer anions (see Figure 2). According to the law of electroneutrality, the value of BB and SID must be identical. Buffer base is not a suitable indicator of a nonrespiratory acid–base disturbance; although independent of pCO2 , it varies with the albumin and hemoglobin concentrations, which are unrelated to acid–base disturbances.

Normal Acid–Base Balance

Concentration of ions in arterial plasma (mmol l −1)

Acid–base balance refers to the balance between input (intake and production) and output (elimination) of hydrogen ion. The body is an open system in equilibrium with the alveolar air where the partial pressure of carbon dioxide pCO2 is identical to the carbon dioxide tension in the blood. pCO2 is directly proportional to the CO2 production rate (at constant

150

Mg2+ Ca2+ K+

HCO3−

SID+

BB− Pr

−

100

Na+

Cl −

Cations

Anions

HPO42− +H2PO4− SO42− Organic anions

50

Figure 2 Electrolyte balance of arterial plasma showing columns of cations and anions of equal height (law of electroneutrality). The equality of the strong ion difference (SID) and buffer base (BB) is illustrated. The change in concentration of buffer base from normal (at pH ¼ 7.40, pCO2 ¼ 5:3 kPa, and T ¼ 371C) with opposite sign equals the concentration of titratable hydrogen ion.

alveolar ventilation and CO2 free inspired air) and inversely proportional to the alveolar ventilation (at constant CO2 production rate and CO2 free inspired air). CO2 is constantly produced in the oxidative metabolism at a rate of about 10 mmol min 1 ( ¼ 224 ml min 1) and eliminated in the lungs at the same rate so that the pCO2 remains at about 5.33 kPa ( ¼ 40 mmHg). Hydrogen ions associated with any anion other than bicarbonate or exchanging with a cation are eliminated by the kidneys. In the oxidative metabolism of sulfur-containing amino acids, hydrogen ions are produced together with sulfate ions at a rate of about 70 mmol day 1 depending upon the protein intake. Amino acids are oxidized to carbon dioxide and water, and the amino nitrogen, liberated as NH3, combines with carbon dioxide in the liver via the Krebs urea cycle to form neutral urea. Therefore, there is no production of base (ammonia) except in the kidneys, where ammonia formed from glutamine diffuses into the urine where it binds a hydrogen ion (NH3 þ H þ -NH4þ ) thereby preventing an excessively low urine pH. Normal values for the acid–base status of arterial blood are given in Table 2. The lower pCO2 in women than men is probably a progesterone effect on the respiratory center. The values are independent of age except at birth, where babies tend to have higher pCO2 , lower pH, and slightly increased ctH þ Ecf, approaching normal values for adults in the course of a few hours. In the last trimester of pregnancy, the pCO2 is lower (about 1 kPa ¼ 7.5 mmHg), compensated by a slightly increased ctH þ Ecf. A protein-rich diet causes a higher ctH þ Ecf (1–2 mmol l 1) and a slightly lower pH due to production of sulfuric acid from sulfur-containing amino acids. A diet rich in vegetables and fruit causes a lower (negative) ctH þ Ecf and a slightly higher pH due to organic anions binding H þ in the metabolism to carbon dioxide and water. High-altitude hypoxia stimulates ventilation; at 5 km above sea level, pCO2 is decreased to about 3.3 kPa ¼ 25 mmHg. The hypocapnia is compensated by increased ctH þ Ecf, so pH is only slightly elevated. The values fall in the area of chronic hypocapnia in the acid–base chart (Figure 1).

Table 2 Reference values for arterial blood Women þ

cH P c tH þ Ecf pCO2 cHCO3 P

Men 1

36.3–41.7 nmol l (pH: 7.38–7.44) 2.3 to þ 2.7 mmol l 1 4.59–5.76 kPa (33.8–42.4 mmHg) 21.2–27.0 mmol l 1

37.2–42.7 nmol l 1 (pH: 7.37–7.43) 3.2 to þ 1.8 mmol l 1 4.91–6.16 kPa (36.8–46.2 mmHg) 22.2–28.3 mmol l 1

cH þ P: conc. of (free) hydrogen ion in plasma; c tH þ Ecf: conc. of titratable hydrogen ion in extracellular fluid (also called standard base deficit, SBD); pCO2 : tension of carbon dioxide; cHCO3 P: conc. of bicarbonate in plasma.

10

ACID–BASE BALANCE

Acid–Base Disturbances Respiratory Acid–Base Disturbances

Acute respiratory acid–base disturbances are characterized by an acute change in pCO2 associated with an acute change in pH but with unchanged ctH þ Ecf. The relationship between pCO2 and pH is illustrated by the oblique in vivo CO2 equilibration lines in the acid–base chart (Figure 1). Primary increase and decrease in pCO2 are compensated by secondary renal decrease and increase in ctH þ Ecf, respectively. The acid–base chart shows the expected values in chronic hypercapnia and chronic hypocapnia. The effect of the compensation is a return of pH about two-thirds towards normal, slightly more in acute hypocapnia. Nonrespiratory Acid–Base Disturbances

Primary increase and decrease in ctH þ Ecf are compensated by secondary decrease and increase in pCO2 . A very acute rise in ctH þ Ecf, for example, due to anaerobic exercise with lactic acid formation, is only partly compensated because only peripheral chemoreceptors react promptly to a fall in blood pH. It takes about an hour before H þ equilibrium between blood and brain extracellular fluid is achieved and the central chemoreceptors are maximally stimulated. The acid–base values in acute nonrespiratory acidemia are illustrated in Figure 1 by the area labeled ‘acute hydrogen ion excess’. The outline of the area is dotted because it is less well-defined than the other areas of the chart. The compensations in more slowly developing nonrespiratory acidemia or alkalemia are illustrated by the areas labeled ‘chronic hydrogen ion excess’ and ‘deficit’, respectively. The effect of the respiratory compensation is a return of pH one-third to halfway towards normal. Once an increase in ctH þ Ecf has been detected, the question is: what caused the metabolic acidosis? It may be a production of lactic acid due to anaerobic metabolism or acetoacetic acid (ketoacidosis) due to diabetes mellitus or starvation. In both cases the diagnosis may be verified by direct measurement of blood lactate or acetoacetate. When these analyses are unavailable, calculation of the concentration of undetermined anions may be useful, i.e., the sum of the concentrations of measured cations (Na þ and K þ ) minus the sum of the concentrations of measured and calculated anions (Cl and HCO3 ). This equals the sum of the concentrations of unmeasured anions (mainly Protein , SO24 , HPO24 , fatty carboxylate, lactate, acetoacetate)minus the sum of the concentrations of unmeasured cations (Ca2 þ and Mg2 þ ). A metabolic acidosis with a major increase in undetermined anions usually indicates

organic acidosis. A hyperchloremic acidosis may be a renal acidosis with retention of H þ and Cl or an intestinal loss of Na þ þ HCO3 with subsequent intake of saline (Na þ þ Cl ). A hypochloremic alkalosis may be due to loss of H þ and Cl by vomiting. Hypokalemic alkalosis is due to inability of the kidneys to retain hydrogen ions in the presence of potassium depletion. See also: Arterial Blood Gases. Carbon Dioxide. Peripheral Gas Exchange. Ventilation: Overview.

Further Reading Astup P and Severinghaus JW (1986) The History of Blood Gases Acids and Bases. Copenhagen: Munksgaard International Publishers. Davenport HW (1969) The ABC of Acid–Base Chemistry, 5th edn. Chicago: The University of Chicago Press. Grogono AW (1986) Acid–base balance. International Anesthesiology Clinics, Problems and Advances in Respiratory Therapy 24(1). Halperin ML and Goldstein MB (1988) Fluid, Electrolyte, and Acid–Base Emergencies. Philadelphia: Saunders. Hills AG (1973) Acid–Base Balance. Chemistry, Physiology, and Pathophysiology. Baltimore: Williams & Wilkins. International Federation of Clinical Chemistry and International Union of Pure and Applied Chemistry (1987) Approved Recommendation (1984) on Physico-Chemical Quantities and Units in Clinical Chemistry. Journal of Clinical Chemistry and Clinical Biochemistry 25: 369–391. Masoro EJ and Siegel PD (1971) Acid–Base Regulation. Its Physiology and Pathophysiology. Philadelphia: Saunders. Nahas G and Schaefer KE (eds.) (1974) Carbon Dioxide and Metabolic Regulations. New York: Springer. Rooth G (1975) Acid–Base and Electrolyte Balance. Lund: Studentlitteratur. Severinghaus JW and Astrup P (1987) History of Blood Gas Analysis. Boston: Little, Brown and Company. Shapiro BA, Peruzzi WT, and Templin R (1994) Clinical Application of Blood Gases, 5th edn. St Louis: Mosby – Year Book. Siggaard-Andersen O (1971) An acid-base chart for arterial blood with normal and pathophysiological reference areas. Scandinavian Journal of Clinical and Laboratory Investigation 27: 239–245. Siggaard-Andersen O (1974) The Acid–Base Status of the Blood, 4th edn. Copenhagen: Munksgaard and Baltimore: Williams & Wilkins Company. Siggaard-Andersen O (1979) Hydrogen ions and blood gases. In: Brown SS, Mitchell FL, and Young DS (eds.) Chemical Diagnosis of Disease, pp. 181–245. London: Elsevier, NorthHolland Biomedical Press. Siggaard-Andersen O and Fogh-Andersen N (1995) Base excess or buffer base (strong ion difference) as measure of a non-respiratory acid–base disturbance. Acta Anaesthesiologica Scandinavica 39(supplement 107): 123–128. Thomson WST, Adams JF, and Cowan RA (1997) Clinical Acid– Base Balance. New York: Oxford University Press. West JB (1974) Respiratory Physiology, the Essentials. Oxford: Blackwell. West JB (2001) Pulmonary Physiology and Pathophysiology. An Integrated, Case-Based Approach. Philadelphia: Lippincott Williams & Wilkins.

ACUTE RESPIRATORY DISTRESS SYNDROME 11

Acute Exacerbations

see Asthma: Acute Exacerbations. Chronic Obstructive Pulmonary Disease:

Acute Exacerbations.

Acute Lung Injury

see Acute Respiratory Distress Syndrome.

ACUTE RESPIRATORY DISTRESS SYNDROME G Bellingan, University College London, London, UK S J Finney, Imperial College London, London, UK

Table 1 Clinical triggers of ARDS Etiology

Percentage

Sepsis (including pulmonary sepsis) Aspiration of gastric contents Pulmonary contusion Bacteremia Head injury Multiple bony fractures requiring ICU admission Blood transfusion exceeding 10 units in 24 h Cardiopulmonary bypass Burns (including smoke inhalation) Acute pancreatitis Lung reperfusion injury (e.g., posttransplant) Near-drowning

43–52 22–36 8–26 4–12 6–11 5–12 5–8 2 2 1

& 2006 Elsevier Ltd. All rights reserved.

Abstract The acute respiratory distress syndrome (ARDS) is the devastating manifestation of the diffuse pulmonary inflammation that may occur following a wide range of life-threatening systemic illnesses. The rapid onset of inflammation and bilateral nonhydrostatic alveolar edema results in severe hypoxemia and reduced pulmonary compliance often mandating mechanical ventilation. The clinical features, radiology, and pathogenesis are reviewed in this article. The management of patients comprises primarily of ventilatory support while the lung injury resolves. The techniques of ventilatory support can propagate the lung injury and adversely affect outcome; the techniques are discussed in detail here. By contrast, pharmacotherapy has a less clear role in ARDS. Corticosteroids may be beneficial after the acute phases, whilst other anti-inflammatory agents have not proved beneficial. Mortality is determined primarily by the underlying trigger for ARDS, but is approximately 30–40%. Follow-up of survivors has demonstrated that lung function often improves considerably, whereas nonpulmonary morbidities persist even 12 months after discharge from the intensive care unit.

Introduction The acute respiratory distress syndrome (ARDS) and its less extreme manifestation, acute lung injury (ALI), are devastating conditions that result in sudden bilateral nonhydrostatic alveolar edema and severe hypoxemia. Pulmonary failure is usually so severe that patients require mechanical ventilation of their lungs. ARDS and ALI are the consequence of the diffuse pulmonary inflammation that can be triggered by an insult either to the lung itself or more commonly at a distant site. As such, they form part of the spectrum of systemic inflammation that can occur following many life-threatening insults (see Table 1). The constellation of severe respiratory distress, refractory hypoxemia, decreased pulmonary compliance,

The figures in this were based on studies at the university of Washington and Colorado.

and diffuse radiological changes was first appreciated by Ashbaugh and co-workers in 1967. They referred to the clinical scenario as the adult respiratory distress syndrome. Since an identical condition can also occur in children, it is now referred to as the acute respiratory distress syndrome. Subsequently, the clinical syndrome has been increasingly recognized and estimates of the annual incidence of ARDS range from 8 to 70 cases per 100 000 population in developed countries. Overall, mortality in patients with ARDS is approximately 30–40%, although death is usually attributable to the underlying etiology rather than pulmonary failure per se.

Etiology The many possible triggers for ARDS are outlined in Table 1. Sepsis accounts for the majority of cases in general intensive care units (ICUs). Patients with multiple risk factors are more likely to develop ARDS. Intriguingly, ARDS only occurs in a subset of patients who suffer apparently similar insults. It is not clear what explains a particular individual going on

12

ACUTE RESPIRATORY DISTRESS SYNDROME

to develop ARDS. Although environmental factors such as age, sex, smoking history, and intercurrent pulmonary disease may be important, it is widely considered that genetic factors are critical. Candidate genes for which associations have been described include those that encode tumor necrosis factor alpha (TNF-a), angiotensin-converting enzyme, interleukin-6 (IL-6), surfactant protein B, and Toll-like receptors. Nevertheless, it has not been possible to draw definitive conclusions since these associations are complicated by tight linkage disequilibrium to other genes, lack of clear functional effects, and small patient numbers. Moreover, the heterogeneity of ARDS suggests that many genes with varying phenotypes and penetrance may underpin an individual’s susceptibility. Functional genomic approaches may help elucidate these complex genotypes.

After the initial injury, myofibroblasts are observed in the interstitium and then the airspace, and start to produce new matrix substance. Indeed, the lung collagen content can double by 2 weeks. With time, type II alveolar epithelial cells increase in number and may represent a stem cell population of the lung; they differentiate into type I alveolar epithelial cells and repopulate the denuded alveolar basement membrane during healing. Pulmonary function and computed tomography (CT) suggest that many patients subsequently return to have structurally normal lungs although this can take months. Some patients however develop severe fibrosis involving both the alveolar space and interstitium. At its most extreme, the ‘honeycomb’ of advanced fibrotic lung disease may form. Many of these patients succumb to intercurrent infection associated with a prolonged ICU stay. A few can recover with incomplete resolution of pulmonary damage.

Pathology Traditionally, the histological development of ARDS has been described as having three sequential phases which affect the lungs in a diffuse manner: exudative, proliferative, and fibrotic. It is now evident that these stages overlap considerably and fibrotic changes are initiated very early. Within the first 24 h, the lungs appear macroscopically edematous and congested. At this point, light microscopy reveals edema within the airspaces, alveolar walls, and septae. Type I alveolar epithelial cells are swollen, necrotic, and often detached from the underlying basement membrane. Pulmonary endothelial cells may also be swollen, with fibrin thrombi occluding alveolar capillaries. Neutrophils are also initially observed within alveolar capillaries, but inflammatory cells then accumulate rapidly in the edematous alveoli. Over the next few days, the lungs become more uniformly red as alveolar wall and airspace edema increase and red cells leak into the airspaces. Histologically, hyaline membranes form from fibrin-rich edema fluid and line the alveoli. The number of neutrophils increases rapidly as they move via the interstitium into the airspace and there is increased disruption of vascular structures with further neutrophil and fibrin plugs occluding capillaries.

Clinical Features Definition

The diagnosis of ARDS is based on the criteria developed at the 1992 American–European consensus conference and is illustrated in Table 2. Since the definition does not consider current management strategies, the relevance of scenarios in which suboptimal mechanical ventilation and management can influence whether criteria are met or not is not clear. The cutoffs for severity of hypoxemia are arbitrary, the definition of ‘acute’ onset lacks clarity and the use of the chest X-ray opens the way for individual interpretation. Further limitations of the definition include the inclusion of other conditions that probably have different pathological processes (such as severe pneumocystis carinii infection, diffuse alveolar hemorrhage), and exclusion of unilateral disease that may occur following pulmonary lobectomy. Nevertheless, the definition is simple to use and is supported by extensive literature. Post-mortem studies have demonstrated that the definition is 84% sensitive and 94% specific for diffuse alveolar damage; its performance in patients who survive is not clear.

Table 2 American–European consensus criteria for diagnosing ALI and ARDS ALI Chest radiography Clinical scenario Left atrial pressure Oxygenation

Bilateral airspace shadowing Acute onset and associated with a condition known to cause ALI/ARDS No direct or clinical evidence of left atrial hypertension (PAOP o18 mmHg) PaO2/FiO2 o39.9 kPa (300 mmHg)

PAOP, pulmonary artery occlusion pressure.

ARDS

PaO2/FiO2 o26.6 kPa (200 mmHg)

ACUTE RESPIRATORY DISTRESS SYNDROME 13 Natural History

The clinical picture of ARDS is dominated initially by severe hypoxemia due to mismatching of ventilation and perfusion. Indeed, intrapulmonary shunting may result in oxygen saturations that are relatively refractory to increases in the inspired oxygen content. Decreased pulmonary compliance increases the work of breathing and most patients require endotracheal intubation and mechanical ventilation. From a pulmonary perspective, the high oxygen requirements persist for some time. Further increases in the alveolar–arterial oxygen gradient occur with ongoing pulmonary inflammation, particularly in the setting of a positive fluid balance, but may also reflect a superimposed ventilatory pneumonia or pneumothorax. Pneumothoraces tend to occur after the first week and may tension rapidly. Since the inflamed lung may tether to the chest wall, pneumothoraces can be loculated and anterior, and thus easily missed on plain chest radiography. Thoracic CT may be required to locate pneumothoraces accurately. Surprisingly, despite devastating pulmonary failure, oxygenation often slowly improves allowing withdrawal of mechanical ventilation; for many, this may take weeks or months and often necessitate temporary tracheostomy. Although disease can remain compartmentalized with isolated lung failure, ARDS is usually part of the spectrum of systemic inflammation; hence, patients often demonstrate peripheral vasodilation, increased cardiac outputs, and systemic hypotension which may require the administration of vasopressors such as norepinephrine. Secondary pulmonary hypertension can occur and result in acute right ventricular failure. Renal dysfunction is also common as is the need for acute renal support. Other organs can also fail as part of this process and outcome is related to the number of failing organs. The course of ARDS is not a smooth wave of deterioration and recovery. Rather, it is interspersed by episodes of deterioration (commonly linked with intercurrent infection such as ventilator-associated pneumonia or line-related sepsis) and many patients need repeated episodes of inotrope and other organ support prior to final recovery (or demise).

Radiology

The appearance of the plain chest radiograph, although forming part of the consensus definition for ARDS, can be relatively non-specific. Clues distinguishing ARDS from cardiogenic pulmonary edema include normal cardiac dimensions, a normal vascular pedicle width, a peripheral distribution of airspace shadowing, and the absence of septal lines.

Chest CT demonstrates that the lungs are affected in a heterogenous manner. Typically, there is a gradient of opacification from apparently normally aerated lung, through ground-glass appearances, to densely consolidated lung. In the supine patient, this gradient occurs both in ventrodorsal and cephalocaudal directions. These gradients typically reverse within a few minutes if the patient is moved to the prone position. Since alveolar edema would not redistribute so quickly, some of these appearance are not due to increased edema in dependent zones but due to collapse of these areas due to the weight of the overlying lung. Thus ground-glass appearances most likely represent airspace edema, with densely opacified areas representing collapsed edematous lung. Regions of dense opacification in nondependent areas may signify collapse/consolidation due to infection or retained secretions. At later stages, groundglass appearances may be accompanied by bronchial dilatation which persists into recovery, suggesting established fine intralobular fibrosis (Figure 1). Other Pulmonary Investigations

Bronchoalveolar lavage samples are dominated by the granulocytic cell population initially. As the disease evolves, the proportion of granulocytes declines and a greater proportion of macrophages is seen. Persistent neutrophilia often portends a poor prognosis. Since most patients are mechanically ventilated, there are few data about classical pulmonary function tests in patients with ARDS. Re-breathing techniques have been used during positive pressure ventilation and have demonstrated marked reductions in the functional residual capacity, carbon monoxide diffusing capacity (DLCO), and diffusing coefficient (KCO). Lower values of DLCO and KCO tend to be associated with nonsurvival. Disease Severity

There are several scoring systems that evaluate the severity of ARDS. Although these systems have only limited clinical utility in individuals, they describe well the degree of physiological disturbance and act as useful descriptors of disease severity in clinical studies. The scoring systems include the acute physiology and chronic health evaluation II and the lung injury score. The former is used to evaluate all critically ill patients whereas the latter is specifically designed for patients with ARDS.

Pathogenesis Since neutrophils appear early in histological specimens and dominate in bronchoalveolar fluid samples,

14


also key initiators of pulmonary inflammation in ARDS. Studies of patient groups at risk of ARDS who do or do not progress to develop refractory hypoxemia suggest compartmentalized intrapulmonary inflammatory changes (e.g., increased IL-8) may precede a systemic inflammatory response. There is widespread activation and/or dysfunction of many cell types within the lung which results in the clinical manifestations of ARDS (Figure 2). Thus endothelial dysfunction and loss of epithelial integrity reduce the barrier function of the alveolar wall and result in alveolar edema. Alveolar edema is further exacerbated by the loss of epithelial cells which normally promote fluid transport out of the alveolus through apical sodium pumps. Surfactant is lost early during ARDS due to reduced production by damaged epithelial cells along with neutralization of preexisting surfactant by the protein-rich edema fluid. Surfactant loss contributes to alveolar collapse, intrapulmonary shunt, and hypoxemia. Endothelial and smooth muscle cell dysfunction result in impaired hypoxic pulmonary vasoconstriction and, along with microthrombi, contribute to the development of secondary pulmonary hypertension and also impacts on outcome.

Animal Models

Figure 1 Typical appearances of ARDS with (a) plain radiology and (b) computed tomography.

it has been considered that they are important in the pathogenesis of ARDS. It is possible that activated and thus rigid 7.5 mm neutrophils may get stuck in pulmonary capillaries where they release a plethora of inflammatory mediators that include chemokines, cytokines, and proteases. Activation may occur at remote sites and/or by circulating cytokines. However, since ARDS can occur in neutropenic patients, neutrophils cannot be absolutely required for the development of ARDS. Circulating inflammatory mediators (e.g., TNF-a, IL-1b, IL-6, IL-8, leukotrienes) along with the changes that occur within the coagulation system during systemic inflammation are

Animal models allow the study of the pathophysiology of ARDS and the effects of interventions whilst tightly controlling both insults and genetic background. However, the multiple models that exist for ARDS/ALI illustrate that none are ideal. Models in which the lung is injured directly include hyperoxia, high tidal-volume mechanical ventilation, and instillation of oleic acid, bleomycin, or thiourea. Alternatively, models mimic systemic sepsis in which the lung is also injured; these include systemic injections of lipopolysaccharide and caecal ligation and puncture. Some authors suggest that two-hit models are more clinically appropriate and combine models of sepsis with hypovolemia, burns, or high tidal-volume ventilation.

Management and Current Therapy The primary management aims for a patient with ARDS are to maintain adequate oxygenation and to support any other failing organs whilst the lung injury resolves. In general, respiratory failure is sufficiently severe to mandate mechanical ventilation, the techniques of which significantly influence mortality. By contrast, few, if any, therapies directed at modifying the pathogenesis and evolution of ARDS, have been demonstrated to effect outcome. A detailed


Inflammatory trigger

Mechanical ventilation

E.g., bacterial sepsis, massive transfusion Stretch-induced cell signaling

Activated neutrophils

Stretch-induced cell damage

Soluble mediators E.g., cytokines, chemokines, coagulation factors, eicosanoids, ROS

Smooth muscle cells

Endothelial cells

Vascular dysfunction

Reduced HPV Pulmonary hypertension

Epithelial cells

Barrier dysfunction

Production

Surfactant

Alveolar edema Neutralization

Alveolar collapse Figure 2 Pathogenesis of ARDS and ALI. HPV, hypoxic pulmonary vasoconstriction; ROS, reactive oxygen species.

description of optimal mechanical ventilation along with the pharmacotherapeutic approaches that have been explored are outlined below. Conventional Mechanical Ventilation

The manner in which the injured lung is ventilated influences mortality and may perpetuate lung injury. The origins of such ventilator-induced lung injury (VILI) are multifactorial but include the shear stresses exerted on alveoli during overdistension and cyclical collapse/re-inflation, and oxygen toxicity. VILI is illustrated by chest CT of those patients who have survived ARDS: relatively normal lung architecture is often present in previously densely consolidated areas, whereas a reticular pattern of fibrosis is seen in those regions (often nondependent) that were exposed to mechanical ventilation. VILI results in the ongoing release of inflammatory mediators that may spill over into the circulation and propagate systemic inflammation, thereby influencing mortality. Indeed, increased plasma levels of cytokines have been demonstrated in animal models and in patients receiving injuriously large tidal-volume ventilation. This theory has stimulated the development of socalled ‘protective’ strategies for mechanical ventilation which minimize VILI.

Tidal volume Delivery of a normal tidal volume to extensively consolidated lungs inevitably results in overdistension of the remaining lung units. Experimental work demonstrated that this may be a significant trigger for VILI, and resulted in the National Institutes of Health (NIH)-sponsored study of low tidal-volume ventilation in 861 patients. This landmark study showed that mortality could be reduced by the use of lower tidal volumes (4–6 ml kg 1, ideal body mass) or at least the avoidance of more ‘traditional’ tidal volumes (10–12 ml kg 1). The corresponding plateau inspiratory pressures were 25 and 35 cmH2O, respectively. Plasma and bronchoalveolar lavage cytokine and chemokines levels were greater in those patients receiving higher tidal volumes. The study protocol has been adopted by some as the definitive ventilatory strategy. More correctly, it provided excellent evidence for VILI and should form the basis of lung-protective strategies. Indeed, it has been suggested that targeting lower-end expiratory alveolar pressures may be more sensible, since the specific pulmonary compliance (compliance corrected for accessible lung volume) has been reported as normal in patients with ALI/ARDS. Smaller tidal volumes may reduce VILI by virtue of the reduced cyclical volume per se, or through a reduction in the end-inspiratory volume. It is not clear

16


which is the important factor although in vitro experiments on mechanically deformed epithelial layers have demonstrated that cyclical changes are more damaging than constant stretch to a high volume. How these results translate to the in vivo scenario is not clear. In the absence of increased respiratory rates, reductions in tidal volume will reduce alveolar ventilation and result in a hypercapnic acidosis. Permissive hypercapnia is generally well tolerated except in the setting of a marked metabolic acidosis and increased intracranial pressure. Hypercapnia may also reduce myocardial contractility, and possibly increase the need for sedation and/or paralysis. The effects on the immune response are still unclear. The degree of hypercapnic acidosis allowable is unclear although many clinicians accept arterial pH values not less than 7.20. When titrating respiratory rate to pCO2 , it must be remembered that increases in respiratory rate will have less influence on alveolar ventilation in ARDS due to the increased dead space, and that reductions in respiratory rate can paradoxically increase CO2 clearance when inspiratory times are particularly prolonged. Tracheal insufflation of oxygen and administration of weak bases are sometimes used to combat the acidosis. Fractional inspired oxygen High fractions of inspired oxygen (FiO2) cause absorption atelectasis and may be cytotoxic to the lung per se. The standard practice is to titrate the FiO2 to arterial saturations of 88–92%, rather than a specific pO2 which may be less relevant to oxygen delivery. Lower targets may be appropriate in those patients whose cardiac output is high or where an improvement of SaO2 would require an unacceptably injurious pattern of ventilation (e.g., through a higher FiO2, positive end expiratory pressure (PEEP), or tidal volume). The benefits of further reductions in FiO2 afforded by the use of inhaled nitric oxide (iNO), prone positioning, and related maneuvers are unknown and still subject to review (vide infra). Intrapulmonary shunting may result in arterial oxygen saturations being relatively independent of FiO2 between 0.8 and 1.0. Positive end expiratory pressure The application of PEEP during mechanical ventilation increases FRC and thus prevents the collapse of alveoli at end expiration. This will reduce intrapulmonary shunt and improve oxygenation. Furthermore, the shear forces required to repeatedly open collapsed alveoli during inspiration are considerable and most likely contribute to VILI. Thus, by keeping these alveoli open throughout the respiratory cycle, PEEP can limit

VILI. Since the lung is heterogeneously affected in ARDS, excessively high levels of PEEP can lead to overexpansion of those regions whose compliance is higher, an effect that may exacerbate VILI. Other detrimental effects of PEEP include reduced cardiac preload and cerebral perfusion. The method to determine the optimal level of PEEP in ARDS is still not established. A randomized multicenter trial in 549 patients, titrating PEEP according to the required FiO2, found no outcome differences between a lower and higher PEEP algorithm. Many clinicians set PEEP by inferring the region of maximal slope on the pressure–volume curve by assessing maximal tidal volume as PEEP is adjusted with a constant pressure control. Mathematical modeling suggests that this is best determined using a decremental rather than an incremental PEEP trial. Ventilatory modes The NIH trial of lower tidal-volume ventilation used a mandatory constant flow, volume control mode of mandatory ventilation. There is considerable interest in the role of descending flow/ pressure control modes since these may promote more even distribution of gas, as flow is slower at the end of expiration and thus more likely to be laminar. Pressure-control modes do not influence peak alveolar pressure, although peak airway pressures may be reduced. The most likely advantage afforded by pressure control ventilation and inverse ratio ventilation is that these may be associated with a greater proportion of the respiratory cycle spent at plateau inspiratory pressure. Greater plateau times promote more homogenous ventilation by allowing slow-filling regions to inter-fill from areas with faster time constants. The recruitment of previously collapsed or partially collapsed slow-filling alveoli reduces shunt and improves oxygenation. Prolongation of inspiration can also improve CO2 clearance by forcing expiration to be delayed to a point where alveolar pCO2 has risen to a maximum. Faster respiratory rates reduce the efficacy of inverse ratio ventilation by shortening the inspiratory time. Excessive prolongation of inspiration may reduce expiratory time to a point at which expiration is not completed. This may result in dynamic hyperinflation and the generation of autoPEEP. Most clinicians consider auto-PEEP undesirable since levels may be unpredictable thus leading to cardiovascular compromise. There are theoretical advantages to modes that allow spontaneous ventilation such as biphasic positive airway pressure and airway pressure release ventilation. These modes allow continued diaphragmatic and intercostal function and the ventilation of dependent, better-perfused regions. Although pressure support modes also allow spontaneous ventilation,


some argue against them as they preclude any plateau time. No study to date, excluding those examining weaning, has demonstrated any outcome differences according to ventilatory mode. Drainage of pneumothoraces Multiple pneumothoraces may complicate mechanical ventilation of patients with ARDS. Pneumothoraces can be complex and localized as the inflamed lung may tether to the chest wall. Indeed, localization can be difficult by conventional radiography and it may be necessary to insert drains under the guidance of CT. Intercostal drains are generally left in situ until the patient is well established on a spontaneous ventilatory weaning program. Supplemental Techniques for Mechanical Ventilation

Prone positioning Ventilation in the prone position improves oxygenation in approximately 70% of patients. It is not possible to predict which patients will respond, and nonresponders may improve on subsequent turns. Experienced teams can easily turn patients, even with multiple intercostal drains and intravascular catheters, with few complications. Improvements in oxygenation continue in 50% of patients after they are returned to the supine position and are probably the consequence of expansion of previously collapsed lung. More homogenous ventilation may prevent regional overdistension and limit VILI. Improved secretion drainage and altered diaphragmatic mechanics may also contribute to clinical improvement. Despite improvements in oxygenation, prone positioning has not been demonstrated to improve outcome in ARDS. Recruitment maneuvers Sustained and high airway pressures of up to 60 cmH2O may be required to open (or recruit) some regions of collapsed lung. There has been considerable enthusiasm for the use of such recruitment maneuvers which, in combination with the application of PEEP, may keep these newly recruited lung units open. Techniques vary from sophisticated determination of recruitment and derecruitment by stepwise alteration of ventilatory pressures and repeated blood gas sampling through to simply temporarily increasing PEEP to 30 cmH2O for 30 s. Improvements in oxygenation have often been demonstrated, although these may not be sustained. Such high intrathoracic pressures are often accompanied by brief episodes of reduced cardiac output and hypotension, but rarely barotrauma. The role of recruitment maneuvers still has to be clarified in ARDS.

Partial liquid ventilation Partial liquid ventilation has been proposed to improve oxygenation and limit VILI. Perfluorocarbons are dense liquids that have little biological activity but very low surface tension and exceptionally high-solubility coefficients for oxygen and carbon dioxide. The lung is filled (partially or fully) with the perfluorocarbon and then normal mechanical ventilation applied simultaneously. Since the perfluorocarbon is volatile, it evaporates over time through the ventilator circuit. Animal studies suggest that perfluorocarbons reduce atelectasis, improve perfusion matching, and improve secretion clearance. Some studies suggest they may also be anti-inflammatory, reducing permeability rises and neutrophil activation. Human studies have revealed an increase in the incidence of pneumothoraces, mucus plugging, and a disruption of the normal surfactant system. They are ingested by macrophages and their effect on immune function is unclear. A phase II/III study of partial-liquid ventilation is underway in France currently. Extracorporeal gas exchange Traditionally, extracorporeal gas exchange (ECGE) has only been used as a last resort in patients in whom oxygenation cannot be maintained by any other means. Devices vary from those which oxygenate and support the whole cardiac output, to those that provide partial oxygenation, and carbon dioxide removal to supplement conventional ventilation at lower pressures (extracorporeal lung assist, ECLA). Hemorrhage and coagulopathies have been the most significant complications, occurring in about 67% of adult patients. Although no mortality advantage has ever been shown for ECGE, new technology such as heparinbonded circuits, nonocclusive roller pumps, pumpless circuits, and better anticoagulation control, that may reduce complications is leading to a re-evaluation of this modality. Surfactant administration Alveolar surfactant is lost early in ARDS. The administration of exogenous surfactant either via a bronchoscope or nebulizer may reduce surface tension within alveoli and prevent cyclical collapse, improving oxygenation, and ameliorating VILI. Multiple administrations appear necessary due to the neutralization of surfactant by edema fluid. Despite improvements in oxygenation and phase I/II survival benefits, no large studies of surfactant have demonstrated any mortality advantage and thus it is not administered to patients with ARDS. However, there are many outstanding questions regarding the optimal formulation, patient selection, and dosing regimen.

18


Inhaled vasodilators iNO and prostacyclin cause selective vasodilatation of the regions of the lung to which they are delivered by ventilation, thus improving ventilation/perfusion matching and oxygenation. Their short half-lives limit systemic vasodilatation and hypotension. Both agents typically improve arterial pO2 by about 20% in responders, and this may be enhanced by the administration of intravenous almitrine bismesylate, an agent that is reported to enhance hypoxic pulmonary vasoconstriction. iNO is usually maximally effective at inspired concentrations of less than 10 parts per million, although the dose–response curve may vary considerably after 24 h. iNO and prostacyclin may also favorably influence inflammation, platelet activity, and vascular permeability. Although frequently used for their beneficial effects on oxygenation, no survival benefit has been shown for either agent in controlled studies. High-frequency ventilation The corollary of the fact that low tidal-volume ventilation is beneficial is that high-frequency ventilation may further protect against VILI. Tidal volumes delivered by jet ventilators and oscillators are less than the anatomical dead space, and respiratory rates are greater than 1 Hz. Gas transport occurs via diffusion rather than bulk flow. To date, no mortality advantage has been demonstrated for these ventilatory techniques in adults. Noninvasive ventilation Noninvasive ventilation (NIV) avoids the need for endotracheal intubation and the associated risks of ventilator-associated pneumonias, sinusitis, and sedation. The use of NIV in hypoxemic respiratory failure is controversial and a consensus conference into the role of NIV in acute respirator failure found little data to support its use in ARDS. Indeed, the natural history of ARDS is often longer than the time for which many patients are able to tolerate tight-fitting masks continuously. It may thus only delay endotracheal intubation. Manipulation of the Alveolar Fluid Balance

High pulmonary capillary pressures increase extravascular lung water and result in worsening oxygenation. Indeed, a persistently positive fluid balance is associated with a worse outcome in ARDS. It is difficult to ascertain whether this association is due to detrimental effects of administered fluids per se or simply a reflection of the severity of the underlying illness requiring fluids for cardiovascular support. Nevertheless, whilst it is critical to maintain organ perfusion with an adequate cardiac output, the administration of intravenous fluids in the absence of

evidence of organ hypoperfusion would seem inappropriate. The careful use of vasopressors may allow the limitation of fluid administration. Apical sodium and probably chloride channels in alveolar epithelial cells play an important role in normal alveoli in maintaining a flux of water into the circulation. A subgroup of the sodium channels can be stimulated by b-agonists such as salbutamol and terbutaline which may be administered either as inhaled preparations or as continuous intravenous infusion. Recent data suggest that salbutamol infusions can reduce extravascular lung water. How this influences outcome in ARDS is not known and thus these agents remain experimental. Manipulation of the Inflammatory Process

Corticosteroids Corticosteroids can reduce inflammation by repressing transcription and destabilizing pro-collagen mRNA. Initially evaluated in large doses in patients considered at risk of ARDS or early in ARDS, they have no beneficial effects on mortality. By contrast, their administration later when repair and remodeling have commenced, may be helpful. Possible negative effects include nosocomial infection, hyperglycemia and an exacerbation of critical illness poly(myo)neuropathy. In a study of only 24 patients, corticosteroids caused a significant improvement in oxygenation and reduced mortality in patients with unresolving ARDS. Methylprednisolone (2 mg kg 1 day 1) was administered from day 8 for the shorter of either the duration of mechanical ventilation or 14 days. Although the study was methodologically limited, many clinicians administer a similar regimen to patients with persistent ARDS after 7 days and in whom there is little evidence for systemic sepsis. The results of the North American Late Steroid Rescue Study (LaSRS) will shortly provide better evidence regarding the role of corticosteroids in ARDS. Other agents Other pharmacological agents that have been assessed in ARDS/ALI are outlined in Table 3. None have been demonstrated to improve mortality despite good rationale for their use and promising preliminary studies.

Outcome Over the last ten years, mortality has fallen from around 60% to around 30% in the current major trials. Although multifactorial in origin, this is in part due to improvement in the whole critical-care process but a greater appreciation that inappropriate mechanical ventilation can further injure the inflamed lung. Most patients succumb to multi-organ failure

ADAMs AND ADAMTSs 19 Table 3 Other Pharmacological agents assessed in ARDS/ALI Agent

Rationale

Effects

Prostaglandin E1

A direct pulmonary vasodilator, inhibitor of platelet aggregation, and inhibitor of neutrophil adhesion

Mortality unaltered More rapid improvement in oxygenation

Dazoxiben

Inhibitor of thomboxane synthase and thus acts as a pulmonary vasodilator and inhibitor of platelet aggregation

Mortality unaltered

Ketoconazole

Inhibitor of thomboxane synthase and 5-lipoxygenase, thus reducing production of leukotrienes, neutrophil chemokines

Mortality unaltered

N-acetyl cysteine

Antioxidant that reduces damage by reactive oxygen species

Mortality unaltered Improved oxygenation and compliance

Lisofylline

Inhibitor of phosphatidic acid which increases cytokine production and activates neutrophils

Mortality unaltered

or sepsis rather than specific pulmonary failure and an inability to maintain adequate oxygenation. Follow-up of patients who survive ARDS has demonstrated progressive improvement in their lung function that continues to improve even one year after discharge from the ICU. Lung volumes seem to improve more rapidly than diffusing capacity and the distance walked in six min. Radiologically, previously densely consolidated areas frequently appear normal, with fibrosis of nondependent regions. By contrast, survivors often have considerable long-term nonrespiratory morbidities, particularly related to physical strength and to psychosocial, functional, and stress indices. Indeed, less than 50% return to work within 12 months. See also: Corticosteroids: Therapy. Extracorporeal Membrane-Gas Exchange. Fluid Balance in the Lung. Genetics: Gene Association Studies. Hypoxia and Hypoxemia. Leukocytes: Neutrophils. Lung Imaging. Nitric Oxide and Nitrogen Oxides. Oxygen Therapy. Oxygen Toxicity. Surfactant: Overview. Ventilation, Mechanical: Positive Pressure Ventilation.

Further Reading Brower RG, Lanken PN, MacIntyre N, et al. (2004) Higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome. New England Journal of Medicine 351: 327–336. Evans TW, Griffiths MJD, and Keogh BF (eds.) (2002) European Respiratory Monograph: ARDS, vol. 70(20). Sheffield: ERS Journals. Herridge MS, Cheung AM, Tansey CM, et al. (2003) One-year outcomes in survivors of the acute respiratory distress syndrome. New England Journal of Medicine 348: 683–693. Meduri GU, Headley AS, Golden E, et al. (1998) Effect of prolonged methylprednisolone therapy in unresolving acute respiratory distress syndrome: a randomized controlled trial. Journal of the American Medical Association 280: 182–183. Pinhu L, Whitehead T, Evans T, and Griffiths M (2003) Ventilatorassociated lung injury. Lancet 361: 332–340. The Acute Respiratory Distress Syndrome Network (2000) Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. New England Journal of Medicine 342: 1301–1308. Ware LB and Matthay MA (2000) The acute respiratory distress syndrome. New England Journal of Medicine 342: 1334–1349.

ADAMs AND ADAMTSs C P Blobel, Cornell University, New York, NY, USA S S Apte, Cleveland Clinic Foundation, Cleveland, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Proteolysis has emerged as a key posttranslational regulator of the function of molecules on the cell surface and in the

extracellular milieu. In principle, proteolysis can activate or inactivate a substrate, or can change its functional properties. ADAM (a disintegrin and metalloprotease) and ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin type 1 repeats) proteases are related members of a superfamily of metalloendopeptidases that also includes matrix metalloproteinases (MMPs) and astacins. ADAMs are integral membrane proteins that typically cleave other membrane anchored proteins, whereas ADAMTS proteases lack a membrane anchor, and process both secreted and cell surface molecules.

20

ADAMs AND ADAMTSs

ADAMs are implicated in fertilization, neurogenesis, regulation of the function of ligands for the epidermal growth factor receptor, and the release of proteins such as the proinflammatory cytokine tumor necrosis factor alpha (TNF-a) from the plasma membrane. ADAMTS proteases have key roles in the molecular maturation of von Willebrand factor and procollagen and are implicated in the pathogenesis of osteoarthritis. Here, we provide a general overview of the biochemical properties and physiological functions of ADAMs and ADAMTS proteases and describe their relevance to lung and airway disorders.

ADAMs Introduction

ADAMs (a disintegrin and metalloprotease) are a family of membrane anchored glycoproteins that have been implicated in cleaving and releasing proteins from the cell surface. This process, which is referred to as protein ectodomain shedding, affects the function of molecules with key roles in development and disease, including growth factors such as transforming growth factor alpha (TGF-a), heparinbinding epidermal growth factor (HB-EGF), cytokines such as tumor necrosis factor alpha (TNF-a), and receptors such as the tumor necrosis factor receptor 1(TNFR1). The first recognized ADAMs were the two subunits of a sperm protein termed fertilin, which has a critical role in fertilization. Other ADAMs were subsequently identified based on their

sequence homology to fertilin, or based on functional assays, such as their ability to shed the proinflammatory cytokine TNF-a or process and activate the cell surface receptor Notch. Structure

The typical domain organization of an ADAM is shown in Figure 1. Of the over 30 ADAMs that have been identified to date, only about half contain a catalytic site consensus sequence and are therefore predicted to be catalytically active. The remaining ADAMs that are not catalytically active are thought to function mainly in cell–cell or cell–matrix interactions. Regulation of Production and Activity

All catalytically active ADAMs are synthesized with a pro-domain that helps the metalloprotease domain fold in the endoplasmic reticulum (ER), and keeps it inactive until the pro-domain is cleaved, usually just before reaching the trans-Golgi network. Once the pro-domain is removed, additional mechanisms, including phorbol esters, phosphatase inhibitors, calcium ionophores, and activation of G-protein-coupled receptors, regulate the catalytic activity of ADAMs. The disintegrin domain and cysteine-rich region of ADAMs are thought to have a role in cell–cell interaction and potentially also in

Cell

ADAMs Pro

Catalytic

Disintegrin

CRD

EGF-like

TSR

TSR

TM

Cytosolic

(0−14)

ADAMTSs Pro

Catalytic Protease domain

Disintegrin-like

CRD

Spacer

Ancillary domain

Figure 1 All ADAMs are membrane-anchored glycoproteins that are characterized by a conserved domain structure: an N-terminal signal sequence followed by pro (Pro), metalloprotease, and disintegrin domains, a cysteine-rich region (CRD), usually containing an EGF repeat, and finally a transmembrane (TM) domain and cytoplasmic tail. Only about half of the known ADAMs have a catalytic site consensus sequence, and are therefore predicted to be catalytically active. The disintegrin domain was first identified in snake venom toxins that bind to platelet integrins and function as anticoagulants, but is now known to be a characteristic feature of all ADAM and ADAMTS proteins. ADAMTS proteins are similar to ADAMs in the prometalloprotease domain, but unlike ADAMs, all ADAMTSs are catalytically active. ADAMTSs have a functionally critical ancillary domain containing specific modules not found in ADAMs, e.g., thrombospondin type 1 repeats (TSRs) (see text for details). A critical difference between these protease families is the absence of an integral membrane segment in ADAMTS proteases.

ADAMs AND ADAMTSs 21

substrate recognition. Finally, the cytoplasmic domain of catalytically active ADAMs usually contains signaling motifs such as potential phosphorylation sites and proline-rich Src-homology 3 (SH3) ligand domains, and several molecules that interact with the cytoplasmic domains of ADAMs and might regulate their maturation and function have been identified. The catalytic activity and substrate selectivity of ADAMs has been explored using both biochemical and cell biological approaches. One important conclusion from biochemical studies was that individual ADAMs do not have a clear consensus cleavage site in vitro. Since ADAMs and their substrates are both membrane anchored, cell-based assays are critical tools for understanding the substrate selectivity and regulation of ADAMs in the context of the plasma membrane. Studies using cells from ADAM knockout mice, or cells treated with small inhibitory RNA (siRNA) against different ADAMs, have revealed that these enzymes display substrate selectivity in cells, although the mechanism underlying this remains to be established. A common feature, however, is that ADAMs frequently cleave their substrates close to the plasma membrane, resulting in release or ‘ectodomain shedding’ of the substrate’s soluble ectodomain. Biological Function

As noted above, ADAM-dependent ectodomain shedding can profoundly affect the function of the released substrate protein. Ectodomain shedding can enable the released molecule to act at a distance from the cell that it was shed from, which is referred to as paracrine signaling. For example, processing of the EGF receptor (EGFR) ligands TGF-a and HB-EGF by ADAM17 is critical for activation of the EGFR during development, and therefore mice lacking

ADAM17 resemble mice lacking TGF-a or HB-EGF or EGFR. Ectodomain shedding is also a key mediator of the role of TNF-a in autoimmune diseases such as rheumatoid arthritis. Interestingly, receptors can be either inactivated by ectodomain shedding, such as the TNFR1, or activated, such as Notch. Mutations in the cleavage site of the TNFR1 that decrease its shedding cause accumulation of the receptor, leading to increased susceptibility to TNF in patients with TNF-receptor associated periodic febrile syndrome (TRAPS). Function of ADAMs in Lung Development and in Respiratory Diseases

Currently, ADAM17 is the only ADAM with a clearly established role in lung development (Figure 2). Mice lacking ADAM17 are born with respiratory distress, presumably caused by abnormal alveoli with septation defects and thickened mesenchyme, as well as impaired branching morphogenesis and delayed vasculogenesis, and thus reduced surface for gas exchange. Since similar defects are observed in mice lacking HB-EGF or the EGFR, the abnormal lung development in Adam17 / mice is most likely explained by a lack of HB-EGF shedding. With respect to respiratory diseases, smoking has been implicated in the activation of ADAMs and the release of EGFR ligands such as amphiregulin. The resulting activation of the EGFR can presumably contribute to the pathogenesis of lung cancer by stimulating cell proliferation and DNA replication at the same time that mutagens are delivered in smoke. Moreover, Grampositive bacteria stimulate the G-protein-coupled platelet activating receptor (PAR) in patients with cystic fibrosis, which in turn activates ADAMdependent release of HB-EGF, and thus mucin production. Therefore, inhibitors of ADAMs, such as hydroxamic acid type metalloprotease inhibitors,

HB-EGF

HB-EGF ADAM17

Extracellular space

Cell membrane

Cytoplasm Figure 2 Proteolytic processing of heparin-binding EGF-like growth factor (HB-EGF) by ADAM17 releases this growth factor from its membrane tether. The ADAM17-dependent ectodomain shedding of HB-EGF is thought to be critical for the function of this growth factor during lung and heart development.

22

ADAMs AND ADAMTSs

might be useful in the treatment of cystic fibrosis. Finally, mutations in the ADAM33 gene have been linked to asthma susceptibility, although the mechanism underlying the role of ADAM33 in asthma remains to be determined. In light of the key roles of ADAMs in regulating signaling via the EGF receptor and other cell surface signaling pathways, and the critical roles for ADAMs in lung development and in asthma and cystic fibrosis, it appears likely that further studies of this protein family in the context of respiratory disease will uncover novel functions, thus hopefully also providing new targets for drug design.

ADAMTSs Introduction

ADAMTS (a disintegrin-like and metalloprotease domain (reprolysin type) with thrombospondin type 1 repeats) comprises a family of 19 secreted metalloproteases that is distinct from the membrane-anchored ADAMs. The founding member of this family, ADAMTS1, was described very recently, in 1997. ADAMTS1 was so named because it resembled the ADAMs in the metalloprotease domain and disintegrin-like module and was thought to be a variant ADAM. Its main point of distinction from ADAMs, apart from the absence of a transmembrane segment, was the presence of three modules resembling thrombospondin type 1 repeats (TSRs). Soon afterwards, it became clear that all 19 ADAMTS proteases shared these major features. Structure

A typical ADAMTS consists of prometalloprotease and ancillary domains. The prometalloprotease domains resemble ADAMs, since the active site sequence is of the reprolysin (snake venom) type. Basic amino acid rich sequences providing sites for removal of the pro-domain by subtilisin-like proprotein convertases (SPCs) are present in the prometalloprotease domain and at its junction with the catalytic domain. The ancillary domain (from N-terminus to C-terminus) consists of a disintegrinlike module, a central TSR, a cysteine-rich module, a cysteine-free spacer, and a variable number of additional TSRs, ranging from 0 (ADAMTS4) to 14 (ADAMTS9 and 20) (Figure 1). An interesting feature of ADAMTS proteases is their clear grouping into distinct subfamilies. Proteases within a subfamily have an identical modular organization, gene structure, and active site sequence, suggesting evolution by gene duplication from a common precursor. Each subfamily has a distinct modular organization,

for example, ADAMTS7 and ADAMTS12, constituting one such subfamily, are the only metalloproteases known to have mucin modules and glycosaminoglycan attachment sites. Regulation of Production and Activity

Transcriptional regulation appears to be very important, since many ADAMTS mRNAs are highly regulated during embryogenesis, for example, ADAMTS2, 3, and 14, or induced in specific circumstances such as inflammation, for example, ADAMTS1. ADAMTS proteases are synthesized as zymogens and undergo removal of the prometalloprotease domain by SPCs either within the secretory pathway or at the cell surface. Subsequent to SPC processing, proteolysis within the ancillary domain appears to be an important posttranslational regulator of activity, resulting in further activation or inactivation of the enzymes. Several ADAMTS proteases bind to the cell surface through unknown mechanisms that suggest activity as operational cell surface proteases, and indicate locations at which posttranslational processing may occur. Unlike ADAMs, the ADAMTSs typically attack specific cleavage sites in their substrates. For instance, ADAMTS4 is a glutamyl endopeptidase that processes Glu–Xaa bonds in many proteoglycans and ADAMTS13 specifically cleaves von Willebrand factor (vWF) at the Tyr1605–Met1606 peptide bond. An important concept in the ADAMTS family is that their catalytic domains alone do not retain activity towards natural substrates. The ancillary domain is critical for the recognition and binding of substrates. In many instances, this requires specific posttranslational modifications in the substrates (such as glycosylation of proteoglycans, triplehelicity in the case of procollagens, and physical unfolding of vWF by fluid shear force). In addition, the minimal substrate binding sites are quite extended in length and difficult to reproduce in synthetic peptides. This makes the development of assays for ADAMTS activity and inhibition quite challenging. The only known ADAMTS inhibitors are tissue inhibitor of metalloprotease 3 (TIMP-3) and a2-macroglobulin. Biological Function

Unexpectedly diverse functions for ADAMTS proteases have been revealed through human genetic disorders and transgenic animals. All ADAMTS genetic disorders are recessive, as is typical of enzyme deficiencies. Inherited thrombocytopenic purpura results from ADAMTS13 mutations, with retention of unusually large polymers of vWF and failure to

ADAMs AND ADAMTSs 23

process these into forms that are optimal for coagulative homeostasis. Acquired thrombocytopenic purpura may result from circulating anti-ADAMTS13 autoantibodies. Ehlers–Danlos syndrome (type VIIC or the dermatosparactic type) is a consequence of ADAMTS2 mutations. In this disorder, tissue (especially skin) fragility results from lack of complete procollagen processing and a decrease in structurally competent collagen fibrils. ADAMTS1 is needed for mouse urinary tract development and fertility, and inhibits angiogenesis in vitro and in vivo by binding to vascular endothelial growth factor (VEGF). ADAMTS4 and ADAMTS5 null mice are developmentally normal, but ADAMTS5 mice are resistant to both mechanically induced and immune arthritis, suggesting that ADAMTS5 is a major cartilage-degrading enzyme. ADAMTS10 mutations cause Weill–Marchesani syndrome (WMS), comprising short stature, brachydactyly, cardiovascular defects, and ectopia lentis. Intriguingly, several aspects of WMS are the opposite of those seen in Marfan syndrome, a fairly common inherited connective tissue disorder. ADAMTS20 mediates melanoblast migration from the neural crest and is deficient in a natural mouse mutant named Belted because of the belt of white fur across the torso. Receptors

Although some ADAMTS proteases can bind to the cell surface, a specific receptor, syndecan-1, has been identified only for ADAMTS4. Nevertheless, because of the affinity of many ADAMTS for heparin and chondroitin sulfate, cell surface proteoglycans may constitute a broad category of receptors for this family. ADAMTS Proteases in Respiratory Diseases

Since thrombocytopenic purpura is a systemic coagulation disorder, patients can develop microthrombi in their lungs and have sometimes manifested with acute respiratory distress syndrome. Pneumothorax has occasionally been reported in Ehlers–Danlos syndrome type VIIC patients and Adamts2 null mice have widening of their distal air spaces. These mice show deficiency of procollagen I as well as procollagen III processing. Both collagens are structurally major components of the lung. Although ADAMTS10 is highly expressed in the lung, WMS cases are not reported to have lung problems.

See also: Asthma: Overview. Epidermal Growth Factors. Matrix Metalloproteinases. Tumor Necrosis Factor Alpha (TNF-a ).

Further Reading Apte SS (2004) A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motifs: the ADAMTS family. International Journal of Biochemistry and Cell Biology 36: 981–985. Becherer JD and Blobel CP (2003) Biochemical properties and functions of membrane-anchored metalloprotease-disintegrin proteins (ADAMs). Current Topics in Developmental Biology 54: 101–123. Blobel CP (2005) ADAMs: key players in EGFR-signaling, development and disease. Nature Reviews: Molecular Cell Biology 6: 32–43. Dagoneau N, Benoist-Lasselin C, Huber C, et al. (2004) ADAMTS10 mutations in autosomal recessive Weill–Marchesani syndrome. American Journal of Human Genetics 75: 801–806. Gao G, Plaas AH, Thompson VP, et al. (2004) ADAMTS4 (aggrecanase-1) activation on the cell surface involves C-terminal cleavage by GPI-anchored MT4-MMP and binding of the activated proteinase to chondroitin sulfate and heparan sulfate on syndecan-1. Journal of Biological Chemistry 279: 10042–10051. Kheradmand F and Werb Z (2002) Shedding light on sheddases: role in growth and development. BioEssays 24: 8–12. Kuno K, Kanada N, Nakashima E, et al. (1997) Molecular cloning of a gene encoding a new type of metalloproteinase-disintegrin family protein with thrombospondin motifs as an inflammation associated gene. Journal of Biological Chemistry 272: 556–562. Lapiere CM and Nusgens BV (1993) Ehlers–Danlos type VII-C, or human dermatosparaxis: the offspring of a union between basic and clinical research. Archives of Dermatological Research 129: 1316–1319. Lemjabbar H and Basbaum C (2002) Platelet-activating factor receptor and ADAM10 mediate responses to Staphylococcus aureus in epithelial cells. Nature Medicine 8: 41–46. Lemjabbar H, Li D, Gallup M, et al. (2003) Tobacco smoke-induced lung cell proliferation mediated by tumor necrosis factor alpha-converting enzyme and amphiregulin. Journal of Biological Chemistry 278: 26202–26207. Levy GG, Nichols WC, Lian EC, et al. (2001) Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature 413: 488–494. Porter S, Clark IM, Kevorkian L, and Edwards DR (2005) The ADAMTS metalloproteinases. Biochemical Journal 386: 15–27. Shapiro SD and Owen CA (2002) ADAM-33 surfaces as an asthma gene. New England Journal of Medicine 347: 936–938. Stanton H, Rogerson FM, East CJ, et al. (2005) ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro. Nature 434: 648–652. White JM (2003) ADAMs: modulators of cell–cell and cell–matrix interactions. Current Opinion in Cell Biology 15: 598–606. Zhao J, Chen H, Peschon JJ, et al. (2001) Pulmonary hypoplasia in mice lacking tumor necrosis factor-alpha converting enzyme indicates an indispensable role for cell surface protein shedding during embryonic lung branching morphogenesis. Developmental Biology 232: 204–218.

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ADENOSINE AND ADENINE NUCLEOTIDES

ADENOSINE AND ADENINE NUCLEOTIDES R Polosa, University of Catania, Catania, Italy D Zeng, CV Therapeutics, Palo Alto, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Adenosine is a purine nucleoside. A growing body of evidence has emerged in support of a proinflammatory and immunomodulatory role for adenosine in the pathogenic mechanisms of chronic inflammatory disorders of the airways such as asthma and chronic obstructive pulmonary disease (COPD). The fact that adenosine enhances mast cell allergen-dependent activation, that elevated levels of adenosine are present in chronically inflamed airways, and that adenosine given by inhalation causes dose-dependent bronchoconstriction in subjects with asthma and COPD emphasizes the importance of adenosine in the initiation, persistence, and progression of these common inflammatory disorders of the airways. These distinctive features of adenosine have been recently exploited in the clinical and research setting to identify innovative diagnostic applications for asthma and COPD. In addition, because adenosine exerts its multiple biological activities by interacting with four adenosine receptor subtypes, selective activation or blockade of these receptors may lead to the development of novel therapies for asthma and COPD. In this article, the evidence for the roles of adenosine receptors in the pathophysiology of chronic inflammatory airway diseases such as asthma and COPD are reviewed.

Introduction The cardiovascular actions of adenosine were first described in a classic publication by Drury and SzentGyorgyi in 1929. It was shown that adenosine causes coronary vasodilation, hypotension, and bradycardia. In 1963, Berne proposed the hypothesis that adenosine mediates the metabolic regulation of coronary blood flow. Since then, a large body of literature has supported the critical role of adenosine in modulating numerous cardiovascular functions. Two well-characterized effects of adenosine have successfully found clinical applications. First, adenosine causes bradycardia, slows A-V nodal conduction, and reduces atrial contractility, and is used as a rapid intravenous bolus for the acute termination of re-entrant supraventricular arrhythmias. Second, adenosine causes coronary vasodilation and increased blood flow, and it is used with radionuclide imaging in the heart to detect underperfused areas of myocardium. The action of adenosine in pulmonary diseases was first discovered in the late 1970s. Holgate and colleagues observed that adenosine and related synthetic analogs were potent agents in augmenting

IgE-dependent mediator release from isolated rodent mast cells. A few years later, adenosine (but not its metabolite inosine or the unrelated nucleoside guanosine) administered by inhalation was shown to be a powerful bronchoconstrictor of asthmatic but, importantly, not of normal airways. Asthma and chronic obstructive pulmonary disease (COPD) are complex syndromes sharing clinical heterogeneity and a number of pathogenic traits, which include variable degrees of airflow obstruction, bronchial hyperresponsiveness (BHR), and chronic airway inflammation. In addition to its known effect as a bronchoconstrictor, a growing body of evidence has emphasized the importance of adenosine in the initiation, progression, and control of chronic inflammation and remodeling of the airways. The key evidence can be summarized as follows: 1. Adenosine is generated in high concentrations at sites of tissue injury (e.g., hypoxia and inflammatory cell activation). 2. Elevated levels of adenosine are present in chronically inflamed airways; they have been observed both in the bronchoalveolar lavage (BAL) fluid and the exhaled breath condensate (EBC) of patients with asthma, and adenosine concentrations are also increased after specific allergen challenge in the plasma of atopic individuals and in the BAL fluid obtained from sensitized rabbits. 3. The progressive accumulation of adenosine in the lungs of mice lacking the purine catabolic enzyme adenosine deaminase (ADA) is strongly associated with development of lung inflammation, tissue eosinophilia, airway hyperresponsiveness mucus metaplasia, airway remodeling, and emphysemalike injury of the lung parenchyma. 4. Adenosine administration by inhalation induces concentration-dependent bronchoconstriction in subjects with asthma and COPD whereas the nucleoside had no discernible effect on airway caliber in normal individuals. 5. Adenosine augments allergen-induced mediator release from human mast cells in vitro, and potentiates the immediate response to allergen in asthmatics. 6. Airway hyperresponsiveness to adenosine better reflects the inflammatory status of the lung in asthma compared with directly acting bronchospasmogens including methacholine; the exquisite sensitivity of adenosine challenge to detect inflammatory changes in human airways has been

ADENOSINE AND ADENINE NUCLEOTIDES 25

beneficially exploited to evaluate modifications in the level of airway inflammation in a prospective and noninvasive manner. 7. Dipyridamole, a blocker of facilitated adenosine uptake, may precipitate asthma. 8. Theophylline, an established asthma therapy, is an adenosine receptor antagonist. Low dose of theophylline blocks AMP-induced bronchoconstriction in asthmatics; this effect is unlikely due to phosphodiesterase inhibition.

In light of these observations, adenosine has been proposed to be an important mediator of asthma. The evidence for the role of adenosine receptor signaling in the pathogenesis of chronic inflammatory disorders of the airways such as asthma and COPD is reviewed below.

Adenosine Metabolic Pathways Adenosine is an endogenous nucleoside consisting of the purine base, adenine, in glycosidic linkage with the sugar ribose (Figure 1). Adenosine is present at low concentrations in the extracellular space and its levels are greatly increased under metabolically stressful conditions as a result of enzymatic cleavage of the nucleotide adenosine 50 -monophosphate (AMP) by the 50 -nucleotidase. This increase in adenosine formation may also occur during inflammation when a large number of infiltrating inflammatory cells are competing for a limited oxygen supply. Intracellular levels of adenosine are kept low principally by its conversion to AMP by the enzyme adenosine kinase. Adenosine may also be degraded to inosine by ADA (Figure 2). It has been proposed that adenosine may function as a ‘retaliatory metabolite’. During injury, the imbalance of O2 supply and demand triggers the increased formation of adenosine. Adenosine may exert its protective effects by decreasing the energy demand of the tissue via a direct inhibitory effect on parenchymal

NH2 N

N

HO

N

N O

OH Figure 1 Structure of adenosine.

OH

cell function and indirectly by providing a more favorable environment for parenchymal cells, the best example of which is adenosine-mediated augmentation of nutrient availability via vasodilatation. Moreover, adenosine helps to maintain tissue integrity by modulating the function of the immune system.

Adenosine Receptors Adenosine elicits its biological activities by interacting with four adenosine receptor subtypes designated as A1, A2A, A2B, and A3 adenosine receptors. The genes for these receptors have been cloned from humans and several animal species. Tissue distributions of these receptors have been determined at the mRNA levels using Northern blot or in situ hybridization techniques or at the protein levels using subtype-selective radioligands or antibodies. In general, these receptors are widely expressed in many tissues. For example, high levels of A1 receptors are found in brain, adrenal gland, and adipose tissue whereas high levels of A2A receptors are found in spleen, thymus, striatum, and blood vessels. In addition, these receptors are often found to co-express in the same tissues or even on the same cells. The relative expression levels of these receptors have been found to be modulated by physiological and/or pathophysiological tissue environments. Although adenosine is the natural agonist for the four adenosine receptors, the ability of adenosine to activate these receptor subtypes varies. In many tissues, A1 and A2A receptors have relatively higher receptor reserves for adenosine, and can be activated by the physiological levels of adenosine and thus mediate the tonic action of adenosine. On the other hand, A2B and A3 receptors appear to have relatively lower affinities and/or receptor reserves for adenosine and require higher concentrations of adenosine for activation. However, the tissue adenosine levels in many pathophysiological conditions can reach significantly high levels to activate the A2B and A3 receptors. Numerous subtype-selective agonists and antagonists of adenosine receptors have been synthesized and are used as pharmacological tools. Although these ligands were classified as selective ligands based on their differential binding affinities for the four adenosine receptors, these compounds are often poorly characterized and not as selective as suggested. There are pharmacological reasons for the lack of functional selectivity. For example, potencies of an agonist for one given receptor could vary from one tissue to another depending on the receptor expression levels and the coupling efficiencies of the cells. As indicated before, adenosine receptors are widely distributed and their expression levels may be modified during disease processes. This

26

ADENOSINE AND ADENINE NUCLEOTIDES Ecto-adenosine deaminase

Ecto-5′-nucleotidase

AMP

Adenosine

Extracellular space

Nucleoside transporter

Adenosine deaminase

5′-nucleotidase

AMP

Inosine

Adenosine

Inosine

Adenosine kinase SAH hydrolase

SAH

Figure 2 Adenosine production and removal occur both intracellularly and extracellularly. Intracellularly, adenosine is formed by the action of 50 -nucleotidase, which dephosphorylates AMP, or by the action of S-adenosyl-homocysteine (SAH) hydrolase. When adenosine concentrations are high, it is phosphorylated to AMP by adenosine kinase (AK) or degraded to inosine by adenosine deaminase (ADA). Both 50 -nucleotidase and ADA are found in the extracellular space where they mediate the formation and degradation of adenosine. The intracellular and extracellular pools of adenosine are kept in equilibrium by the actions of bi-directional nucleoside transporters (NT). Inhibition of AK, ADA, or NT by drugs or genetic deletions raises the tissue adenosine level.

certainly adds complexity in predicting functional selectivity of agonists. In the case of antagonists, the functional blocking effects of competitive antagonists are dependent on the tissue levels of adenosine. If the levels of adenosine are too low to activate a given receptor, an antagonist for this receptor will not have any functional effects regardless of its binding affinity. In addition to these pharmacological issues, there are pharmacokinetic issues. In many cases, the half-life and tissue distributions of adenosine receptor ligands are poorly understood, making it difficult to draw conclusions on the role of receptor subtypes based on the absence of effects of ‘selective ligands’ in animal models. In spite of these limitations, selective agonists and antagonists of adenosine receptors are commonly utilized to establish the functions mediated by adenosine receptor subtypes. The four adenosine receptors also differ in their ability to couple to G-proteins and activate various intracellular signaling pathways. In most cells, A1 and A3 receptors couple to Gi/o and inhibit adenylate cyclase activity whereas A2A and A2B receptors couple to Gs proteins and increase adenylate cyclase activity and intracellular cAMP levels. While the adenylate cyclase–cAMP–protein kinase A axis is the most well-studied second messenger system involved in adenosine receptor function, it is clear that adenosine receptors utilize other signaling pathways as well. These include members of the mitogen-activated protein-kinase family, such as p38, p42/p44 (ERK 1/2), and c-jun terminal kinase, as well as various phospholipases, protein phosphatases, and ion channels.

Adenosine Biological Function Many cell types that play important roles in the pathogenesis of chronic inflammatory airway diseases are known to express adenosine receptors and to exhibit relevant effects through adenosine receptor signaling. These cell types include various inflammatory cells, such as mast cells, eosinophils, lymphocytes, neutrophils, and macrophages, and the structural cells in the lung, such as bronchial epithelial cells, smooth muscle cells, lung fibroblasts, and endothelial cells. The role of the adenosine receptors in these inflammatory and structural cells has been studied extensively using isolated cultured cells in vitro. In addition, numerous animal models have been developed and are extremely useful in determining the potential role of adenosine receptor subtypes in pulmonary functions. A1 Adenosine Receptor

The A1 receptor has been implicated in both pro- and anti-inflammatory aspects of disease processes. It has been shown that activation of the A1 receptor can promote activation of human neutrophils and monocytes and thus, their activation leads to proinflammatory responses. On the other hand, A1 receptors have been reported to be involved in anti-inflammatory and protective pathways in experimental models of injury in the heart, nerves, and kidney. The early evidence suggesting that the A1 receptor plays a role in asthma came from experimental work in rabbits rendered allergic by immunization

ADENOSINE AND ADENINE NUCLEOTIDES 27

protocols with allergen. These animals exhibited a bronchoconstrictor response to adenosine that was attenuated by pretreatment with A1 receptor blockers. In the same model, an antisense that targets the initiation codon of A1 receptor mRNA reduced the bronchoconstrictor response to adenosine and, more importantly, to the early response to allergen. However, the relevance of these observations to human asthma have been questioned due to the fundamental mechanistic difference between adenosine-induced bronchoconstriction in the allergic rabbit, which is due to activation of A1 receptors on the bronchial smooth muscle, and that in man, which appears to be dependent on activation of mast cells that do not express the A1 receptor. Furthermore, it has been shown recently that genetic removal of the A1 receptor gene from ADA-deficient mice resulted in enhanced pulmonary inflammation along with increased mucus metaplasia and alveolar destruction, thus indicating that in this experimental model, the activation of A1 receptors serves an anti-inflammatory and/or protective role in the regulation of pulmonary disorders triggered by elevated adenosine levels. A2A Adenosine Receptor

It is now well established that activation of A2A receptors on lymphoid cells leads to inhibition of an inflammatory response; this is largely due to its ability to induce accumulation of intracellular cyclic AMP in activated immune cells. Perhaps the strongest evidence for the critical role of A2A receptors in the regulation of inflammation in vivo originated from the elegant study of Ohta et al. using mice deficient in A2A receptors. In this model, the absence of the A2A receptor resulted in enhanced tissue inflammation and damage, thus suggesting a negative regulatory role for the A2A adenosine receptor subtype. In the airways, A2A receptors are present on most of the immunoinflammatory cells that have been implicated in asthma. A2A receptors are expressed on mast cells, and their activation results in increases in the intracellular cAMP concentrations, which are known to inhibit the biochemical pathways implicated in the release of histamine and tryptase from human mast cells. Stimulation of A2A receptors on neutrophils inhibits neutrophil adherence to the endothelium, prevents upregulation of integrin expression stimulated with formyl-Met-Leu-Phe and inhibits degranulation of activated neutrophils and monocytes. Activation of T lymphocytes, which plays a key role in the recruitment of leukocytes to the lung in clinical asthma, is also suppressed by A2A receptor activation. Thus, there are a multitude of mechanisms by which activation at A2A receptors

could result in suppression of airway inflammation in asthma and COPD. These findings support the hypothesis that A2A agonists could be potentially useful in controlling the inflammatory processes. A2B Adenosine Receptor

The initial evidence for the role of A2B receptors in asthma and COPD came from selectivity studies of enprofylline, a methylxanthine structurally closely related to theophylline. It was shown that enprofylline is a selective antagonist for the A2B receptors whereas theophylline has similar binding affinities for A1, A2A, and A2B receptors. The therapeutic concentrations of theophylline and enprofylline match their affinities for A2B receptors. Thus, it has been proposed that A2B receptors are possible targets for the long-term clinical benefit achieved with relatively low doses of theophylline and enprofylline. On the other hand, because the anti-inflammatory effects of endogenous adenosine might be mediated by A1 and A2A receptors, blockade of A1 and A2A receptors could be disadvantageous. It has been proposed that a more selective antagonist of the A2B receptors may be more effective and safer than theophylline. Recently, A2B receptors have been shown to have proinflammatory properties in many pulmonary cells. For example, functional human adenosine A2B receptors have been identified in mast cells, endothelial cells, bronchial smooth muscle cells, lung fibroblasts, and bronchial epithelium. Adenosine, via activation of A2B receptors, increases the release of various inflammatory cytokines from human mast cells (HMC-1), from human bronchial smooth muscle cells, human lung fibroblasts, and human airway epithelial cells. These cytokines, in turn, induce IgE synthesis from human B lymphocytes, and promote differentiation of lung fibroblasts into myofibroblasts. These findings provide strong support for the hypothesis that adenosine, via activation of A2B receptors, could enhance airway hyperresponsiveness and the inflammatory responses associated with asthma. Thus, an A2B antagonist could potentially be beneficial in the treatment of asthma and other pulmonary inflammatory diseases. A3 Adenosine Receptor

The functional role of the A3 receptor in the pathogenesis of chronic airway inflammatory diseases remains controversial due in large part to differences in the pharmacology of A3 receptors from different species. For instance, in rodents, mast cell degranulation and/or enhancement of mast degranulation in response to allergen appears to be dependent on A3 receptor activation. In humans, a relatively high density of functionally active A3 receptors is expressed in

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ADENOSINE AND ADENINE NUCLEOTIDES

eosinophils. Transcript levels for the A3 receptor are elevated in lung biopsies of patients with asthma or COPD and appear to be involved in the inhibition of eosinophil chemotaxis when stimulated. Furthermore, inhibition of important proinflammatory functions of human eosinophils by the selective A3 receptor agonist, IBMECA, has been reported. Because asthmatic inflammation is characterized by extensive infiltration of the airways by activated eosinophils, it is possible that the elevated adenosine concentrations associated with asthma could contribute to inhibition of eosinophil activation through stimulation of A3 receptors. In contrast, in their effort of dissecting out specific signaling pathways involved in adenosine-mediated pulmonary inflammation and airway remodeling in ADA-deficient mice, Young and colleagues have recently demonstrated that mice treated with the selective A3 receptor antagonists MRS 1523 resulted in a marked attenuation of pulmonary inflammation, reduced eosinophil infiltration into the airways, and decreased airway mucus production.

Adenosine in Respiratory Diseases Adenosine may play a critical role in the pathogenesis of chronic inflammatory disorders of the airways such as asthma and chronic obstructive pulmonary disease (COPD). Elevated levels of adenosine are present in chronically inflamed airways; they have been observed both in the BAL fluid and the EBC of patients with asthma. Adenosine levels are also raised after allergen exposure in the plasma and during experimental exacerbation of asthmatic symptoms due to exercise in atopic individuals. The observed adenosine elevations indicate that adenosine signaling may regulate aspects of acute and chronic airway disease. Consistent with the hypothesis of adenosine playing a critical role in the pathogenesis of chronic inflammatory disorders, mice deficient in ADA develop features of severe pulmonary inflammation and airway remodeling in association with elevations of adenosine concentrations in the lung. Features of the pulmonary phenotype noted include the accumulation of eosinophils and activated macrophages in the airways, mast cell degranulation, mucus metaplasia in the bronchial airways, and emphysema-like injury of the lung parenchyma. Although the histology seen in ADA-deficient mice fails to accurately resemble that of human asthma, since no epithelial shedding, subepithelial fibrosis, or muscle/submucosal gland hypertrophy were observed in this model, the ADA-deficient mouse is a useful tool to study the pathogenetic role of adenosine in chronic airway inflammation. The role of adenosine in the pathogenesis of asthma is not just limited to its biological effects in

Adenosine

A2B receptor

Mast cell

Cytokines

Prostaglandins and leukotrienes Histamine

Bronchoconstriction Figure 3 Mechanism of adenosine-induced bronchoconstriction. Adenosine, possibly via activation of A2B adenosine receptors, enhances the activation of airway mast cells, leading to increases in the release of potent contractile mediators such as histamine, leukotrienes, prostaglandins, and cytokines. These mediators in turn cause bronchoconstriction in human airways.

airway inflammation and remodeling. Adenosine administration by inhalation is known to elicit concentration-related bronchoconstriction in subjects with asthma and COPD whereas the nucleoside had no discernible effect on airway caliber in normal individuals. Since these initial observations were made, a considerable effort has been directed at revealing the fine mechanisms of adenosine-induced bronchoconstriction, which appear to involve a selective interaction with activated airway mast cells with subsequent release of preformed and newly formed mediators (Figure 3). An important development from adenosine research is the use of an adenosine (or AMP) inhalation challenge as an innovative diagnostic test for asthma and COPD. Compared with other noninvasive surrogate markers of airway inflammation, monitoring of the responsiveness of airways to inhaled adenosine appears to have the selective ability to probe changes in airway inflammation and it has been shown to be very useful when evaluating the effectiveness of different treatment regimens with inhaled corticosteroids. Because the airway response to inhaled adenosine is very sensitive to the effect of inhaled corticosteroids and is a good marker of disease activity, bronchoprovocation with adenosine has been proposed as a convenient and accurate biomarker to monitor corticosteroid requirements in asthma and to establish the appropriate dose needed to control airway inflammation.

ADHESION, CELL–CELL / Vascular 29

Conclusions and Future Direction Over the course of 20 years, the initial observation of the bronchoconstrictive effect of inhaled adenosine has evolved to provide the basis for a new asthma therapy as well as a diagnostic test. Recognition of the potential role of adenosine receptor signaling in the pathogenesis of chronic airway inflammatory diseases advocates the principle that modulating adenosine receptor signaling is likely to constitute a considerable advance in the management of asthma and COPD. The clinical evaluation of selective agonists or antagonists for the adenosine receptors should help elucidate the multiple roles of adenosine and its receptors in the pathophysiology of asthma and COPD. See also: Asthma: Overview. Bronchoalveolar Lavage. Chronic Obstructive Pulmonary Disease: Overview.

Further Reading Blackburn MR, Lee CG, Young HWJ, et al. (2003) Adenosine mediates IL-13-induced inflammation and remodeling in the lung and interacts in an IL-13-adenosine amplification pathway. Journal of Clinical Investigation 112(3): 332–344. Blackburn MR, Volmer JB, Thrasher JL, et al. (2000) Metabolic consequences of adenosine deaminase deficiency in mice are associated with defects in alveogenesis, pulmonary inflammation and airway obstruction. Journal of Experimental Medicine 192(2): 159–170. Cronstein BN (1994) Adenosine, an endogenous anti-inflammatory agent. Journal of Applied Physiology 76: 5–13. Cushley MJ, Tattersfield AE, and Holgate ST (1983) Inhaled adenosine and guanosine on airway resistance in normal and asthmatic subjects. British Journal of Clinical Pharmacology 15(2): 161–165.

Feoktistov I and Biaggioni I (1995) Adenosine A2b receptors evoke interleukin-8 secretion in human mast cells. An enprofyllinesensitive mechanism with implications for asthma. Journal of Clinical Investigation 96(4): 1979–1986. Feoktistov I, Polosa R, Holgate ST, and Biaggioni I (1998) Adenosine A2B receptors: a novel therapeutic target in asthma? Trends in Pharmacological Sciences 19: 148–153. Fozard JR (2003) The case for a role for adenosine in asthma: almost convincing? Current Opinion in Pharmacology 3(3): 264–269. Fredholm BB, Ijzerman AP, Jacobson KA, Klotz KN, and Linden J (2001) International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors. Pharmacological Reviews 53: 527–552. Ohta A and Sitkovsky M (2001) Role of G protein-coupled adenosine receptors in down-regulation of inflammation and protection from tissue damage. Nature 414: 916–920. Polosa R, Ng WH, Crimi N, et al. (1995) Release of mast cellderived mediators after endobronchial adenosine challenge in asthma. American Journal of Respiratory and Critical Care Medicine 151: 624–629. Rorke S, Jennison S, Jeffs JA, et al. (2002) Role of cysteinyl leukotrienes in adenosine 50 -monophosphate induced bronchoconstriction in asthma. Thorax 57(4): 323–327. Ryzhov S, Goldstein AE, Matafonov A, et al. (2004) Adenosineactivated mast cells induce IgE synthesis by B lymphocytes: an A2B-mediated process involving Th2 cytokines IL-4 and IL-13 with implications for asthma. Journal of Immunology 172(12): 7726–7733. Shryock JC and Belardinelli L (1997) Adenosine and adenosine receptors in the cardiovascular system: biochemistry, physiology and pharmacology. American Journal of Cardiology 79(12A): 2–10. Spicuzza L, Bonfiglio C, and Polosa R (2003) Research applications and implications of adenosine in diseased airways. Trends in Pharmacological Sciences 24(8): 409–413. Zhong H, Belardinelli L, Maa T, and Zeng D (2005) Synergy between A2B adenosine receptors and hypoxia in activating human lung fibroblasts. American Journal of Respiratory Cell and Molecular Biology 32(1): 2–8.

ADHESION, CELL–CELL Contents

Vascular Epithelial

Vascular H M DeLisser, University of Pennsylvania School of Medicine, Philadelphia, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Four distinct families of adhesion molecules (cadherins, immunoglobulin superfamily members, selectins, and integrins) mediate

vascular cell–cell adhesion. These molecules are required for the formation of the junctional complexes that enable the assembly of endothelial cells into functional vascular networks; they mediate the leukocyte–endothelial adhesive interactions involved in the trafficking of leukocytes out of the circulation; and they contribute to contacts between pericytes and the endothelium that are important to the regulation of endothelial cell proliferation. In the lung these vascular cell–cell interactions are the bases of the host defense to lung infection or injury, and the regulation of lung vascular permeability, but are disturbed or dysregulated in diseases such as asthma or the acute respiratory distress syndrome.

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Proper cadherin expression and function are essential for normal cell segregation during embryonic development and are required for the initiation and maintenance of normal tissue architecture. The extracellular domain has a variable number of homologous repeats of B110 amino acids with putative Ca2 þ binding sequences (cadherin-repeats), while the cytoplasmic tail forms complexes with cytoplasmic proteins of the catenin family (a-catenin, b-catenin, g-catenin/plakoglobin, and p120) that mediate association with the actin cytoskeleton. Endothelial cells principally express two cadherins: vascular endothelial-cadherin (VE-cadherin), a molecule expressed almost exclusively by endothelial cells, as well as the more widely distributed N-cadherin. They appear to be functionally different, with VE-cadherin mediating endothelial cell–cell adhesion, while N-cadherin may promote endothelial cell adhesion to pericytes and smooth cells. Other nonclassical cadherins, such as T-cadherin and VE-cadherin-2 (protocadherin 12), have been described on

Description Families of Adhesion Molecules

A diverse group of protein molecules mediate cell– cell as well as cell–matrix adhesion. These cell adhesion molecules are grouped into four major families: cadherins, immunoglobulin (Ig) superfamily members, selectins, and integrins (Figure 1).

Cadherins Cadherins were initially identified as a family of single-pass, transmembrane proteins responsible for mediating calcium-dependent, homophilic, cell–cell adhesion. These ‘classical cadherins’ are now recognized to be part of a larger cadherin superfamily that also includes the structural and functionally related desmosomal cadherins as well as other subfamilies with cytoplasmic domains that are unrelated to those of the classical cadherins. (Unless otherwise specified, the term cadherin will refer to the classical protein.)

Cadherins

Ig superfamily

Selectins

Integrins

P-selectin

VE-cadherin

E-selectin

PECAM-1 MAdCAM-1

L-selectin JAM-A C C

C C C C C C

C C C C C C C C C

Extracellular repeating elements

lg-like domain

Lectin-like domain

Integrin subunit

Ca2+ binding sites

Mucin-like domain

EGF-like domain

Integrin subunit

Transmembrane domain Anchoring domains

Transmembrane and cytoplasmic domains

Cytoplasmic domain

PDZ binding motif

C

Short consensus repeat



Figure 1 Families of cell adhesion molecules. Ilustrated are structural features that distinguish the members of each of the four major families of cell adhesion molecules. The extracellular domains of cadherins are composed of a variable number of extracellular repeats with putative Ca2 þ binding sites, and conserved sequences in the cytoplasmic domain. Immunoglobulin superfamily members share a common structure in which a variable number of Ig-like homology domains are present in the extracellular domains of the molecule. The presence of other structural motifs such as mucin-like domains contribute to the structural diversity of these receptors. The selectins are composed of an NH2-terminal lectin-like binding domain, followed by an EGF-like motif, a variable number of short consensus repeats, a single-pass transmembrane domain, and a short cytoplasmic tail. Integrins are heterodimeric proteins composed of two noncovalently linked a and b subunits, each with large extracellular domains and smaller but functionally important cytoplasmic tails.


the endothelium, but their functions are currently not known. Immunoglobulin superfamily The Ig superfamily represents a very diverse group of receptors that function in a variety of cell types and in a number of biological processes. Members of this family are defined by the presence of a variable number of Iglike homology domains in the extracellular domains of these molecules. The Ig-like motif represents a sandwich-like structure of two b-sheets each consisting of antiparallel b strands containing 5–10 amino acids. Appropriately positioned cysteine residues mediate the formation of intrachain disulfide bonds and stabilization of each Ig-like domain. The presence of additional structural domains such as mucinlike regions (e.g., mucosal adressin cell adhesion molecule-1 (MAdCAM-1)) contribute to the structural diversity of this family. Several Ig superfamily members, including intercellular cell adhesion molecule-1 (ICAM-1), platelet endothelial cell adhesion molecule-1 (PECAM-1), MAdCAM-1, vascular cell adhesion molecule-1 (VCAM-1), and the junctional adhesion molecules (JAMs), are expressed on the endothelium and play important roles in leukocyte– endothelial adhesion. Ligand binding for these endothelial Ig superfamily molecules may be homophilic (PECAM-1 and the JAMs) or involve interactions with b2 (ICAM-1, JAM-A, and JAM-C) or a4 integrins (MAdCAM-1, JAM-B, and VCAM-1). Selectins The selectins are a small family of receptors found only on vascular-related cells that mediate the initial adhesive interactions of circulating leukocytes with the endothelium at sites of inflammation or in lymphoid tissues. Selectin structure includes an N2-terminal lectin-like binding domain, followed by an epidermal growth factor (EGF)-like motif, a variable number of short consensus repeats, a single-pass transmembrane domain, and a short cytoplasmic tail. Three selectins (P-selectin, E-selectin, and L-selectin) have been identified, each differing in their patterns of expression and regulation. P-selectin is stored in cytoplasmic granules in platelets and endothelial cells, but is rapidly mobilized to the cell surface, where it is transiently expressed (minutes), in response to agonists such as histamine or thrombin. E-selectin is not present under resting conditions, but is synthesized and expressed briefly (hours) on vascular endothelial cells following cytokine stimulation. In contrast, L-selectin is constitutitively expressed on most leukocytes but is proteolytically cleaved from the surface following cellular activation. Selectins recognize sialylated and fucosylated oligosaccharides (e.g., sialyl Lewis x (sLex) and related

tetrasaccharides), as well as sulfated molecules that lack sialic acid or fucose, such as sulfatides and heparin glycosaminoglycans. Thus, in vitro, selectins are able to bind to a variety of glycoproteins on leukocytes and endothelial cells bearing these structural motifs. Several of these are mucin-like glycoproteins with many serine or threonine residues that are potential sites for attachment of O-linked glycans. In vivo, however, P-selectin glycoprotein ligand-1 (PSGL-1) is the dominant ligand for P- and L-selectin in inflammatory settings and may also be physiologically relevant for E-selectin. Recent reports also suggest that for neutrophils, CD44 may be also be a physiological E-selectin ligand. Additional L-selectin ligands have been identified on high endothelial venules (HEV) of lymphatic tissues and on some activated endothelial cells. These include CD34, glycosylated cell adhesion molecule (GlyCAM-1), and podocalyxin. Integrins The integrins are a family of heterodimeric membrane glycoproteins composed of noncovalently associated a and b subunits. To date 18 a and 8 b subunits have been identified and more than 24 a/b pairs have been described in vertebrate tissue. The combination of a and b subunits determines the ligand specificity. Although most integrins bind ligands that are components of the extracellular matrix, certain integrins (b2 and a4 integrins) bind receptors of the immunoglobulin superfamily (e.g., ICAM-1, MAdCAM-1, VCAM-1, and the JAMs). Significant redundancy, however, exists in that most integrins are capable of binding several different matrix or cell surface proteins and many of these ligands bind to multiple integrins. Through their cytoplasmic domains integrins are able to bind to a complex of cytoplasmic proteins, including a-actinin, vinculin, talin, and paxillin, linked to actin filaments. These integrin–cytoskeletal assemblies in turn form the foundation for intracellular signaling cascades. Endothelial Cell–Cell Adhesion

Endothelial cells adhere to one another through two morphologically distinct junctional structures, adherens junctions (AJs) and tight junctions (TJs), each composed of characteristic molecules (Figure 2). Although very similar structures are present in epithelial cells, their distribution within intercellular junction differs in the two cell types. In epithelial junctions TJs are located toward the apical region of the intercellular contact with the AJs positioned below the TJs, while AJs and TJs are intermingled with each other along the endothelial intercellular junction. Gap junctions represent a third type of junctional complex formed by endothelial cells.

32

ADHESION, CELL–CELL / Vascular Apical/luminal surface Junctional complexes

Associated transmembrane proteins

Tight junctions

Occludin, claudins, JAMs

Adherens junctions

Cadherins

Gap junctions

Connexins

Nonjunctional intercellular proteins

PECAM-1, endoglin

Basal lamina Figure 2 Adhesion structures found in epithelial and endothelial cells. Shown are intercellular junctional complexes (TJs and AJs) and their associated transmembrane proteins. Gap junctions mediate intercellular communication and other proteins such as PECAM-1 and endoglin mediate cell adhesion but are not present in specific junctional complexes.

However, unlike AJs and TJs, these structures mediate cell–cell communication rather than cell–cell adhesion. Epithelial cells also express another type of adhesive complex, the desmosone, but these structures are absent from endothelial cells. Adherens junctions These junctions are formed by clusters of dimerized cadherin molecules that bind to cadherin dimers on adjacent cells. Cadherins localize at AJs only when cells contact one another. The short cytoplasmic domain of cadherins interacts through a C-terminal domain with b-catenin and plakoglobin (g-catenin). b-catenin and plakoglobin associate with a-catenin which in turn binds to actin microfilaments. This association with the catenins is required for the adhesive function of cadherins and thus is essential to the assembly of AJs. An additional binding partner is p120, an src substrate that is homologous to b-catenin and plakoglobin. In contrast to b-catenin and plakoglobin, p120 binds loosely to a membrane proximal region of the cadherin cytoplasmic tail, associating with a-catenin or actin cytoskeleton. It appears that p120 is involved in the regulation of the cadherin turnover and stability. The formation of AJs provides the cell–cell attachments that are required for the assembly of the endothelium into patent tubular networks. AJs also contribute to maintenance of endothelial barrier function, in addition to the regulation provided by TJs (see below). Anti-VE-cadherin antibodies, both in vitro and in vivo are found to increase vascular permeability by disrupting VE-cadherin clustering at the junctions while leaving other junctional structures

intact. Further, AJs are required for the organization and/or maintenance of TJs and gap junctions and thus are critical to the integrity of the entire intercellular junctional complex. In addition, at least two other nonadhesive functions have been ascribed to AJs. First, b-catenin, plakoglobin, and p120 are all able to translocate to the nucleus, where in conjunction with other transcription factors they are able to modulate gene expression. Consequently, the association of these catenins with VE-cadherin in AJs may maintain them at the membrane and thus prevent their nuclear translocation. Second, VE-cadherin engagement promotes endothelial cell quiescence by modulating vascular endothelial growth factor (VEGF) receptor-mediated signaling – inhibiting the receptor-dependent proliferative responses, while activating receptor-mediated survival signals. Tight junctions These junctions were originally identified in epithelial cells by electron microscopy as dense regions in which membrane leaflets between the adjacent cells are closely opposed such that the membrane bilayers at the junctions are indistinguishable. In freeze-fracture preparations, TJs have the appearance of fibrillar strands within the membrane. They form a continuous seal around the apical region of the lateral membranes of adjoining cells, subdividing it into apical and basolateral domains. This is somewhat less so for endothelial cells, where the position of TJs relative to other intercellular junctional complexes can vary. Further, the level and extent of TJs depend on the vessel type and location. Endothelial cell TJs are well developed in arteries


and arterioles, but significantly less so in veins and postcapillary venules, the principal site for the extravasation of fluid and inflammatory leukocytes. TJs are also well developed in the vessels of the brain where they contribute to the blood–brain barrier, but are much less organized in the vasculature of other organs. Like other junctional structures, TJs are composed of both transmembrane and intracellular molecules. Three types of TJ-associated integral membrane proteins have been identified. These are occludin, the claudins, and several Ig superfamily members, including the JAMs, endothelial cell-selective adhesion molecule (ESAM), and coxsackie- and adenovirus receptor (CAR). Occludin and the claudins (of which more than 20 have been identified) define the presence of this junction and constitute the molecular basis of the TJ strands, while JAM-A may serve accessory functions such as the recruitment of TJ-associated proteins. The intracellular domains of occludin, the claudins, and the JAMs interact with zonula occludens-1 (ZO-1), a member of a family of membrane-associated, PDZ, and guanylate kinase domain-containing proteins. ZO-1 can bind to actin filaments and thus anchor TJs to the cell’s cytoskeleton. In addition to ZO-1, other PDZ-containing and/or actin binding proteins are recruited that contribute to the formation and function of TJs. Tight junctions restrict both the diffusion of solutes through intercellular spaces (‘barrier function’) and the movement of molecules between the apical and basolateral domains of the plasma membrane (‘fence function’). As such, TJs are the major regulator of cell permeability via the paracellular pathway (i.e., the extracellular space between the lateral membranes of neighboring cells) and are important to the maintenance of cell polarity. TJs are also likely involved in leukocyte transendothelial migration, possibly through interactions mediated by the JAMs. Gap junctions Gap junctions constitute another junctional complex that bridges the intercellular space between adjacent endothelial cells. However, unlike the other junctional structures described above, which mediate cell–cell adhesion, gap junctions promote intercellular communication. Metabolites, ions, and second messengers, including Ca2 þ , cAMP, and inositol triphosphate, are able to pass through gap junction channels from one cell to another, enabling the coordination of multicellular responses. Gap junction channels are dodecameric structures made up of the connexin family of proteins. Six individual connexin proteins oligomerize in the plasma membrane of one cell to form a hemichannel or connexon, and the docking of two connexons, one from each

opposing cell, results in a complete gap junction channel. Other intercellular junction proteins Endothelial cells express several other adhesive proteins that are concentrated in intercellular junctions but are not specifically located in either AJs or TJs. These include (1) PECAM-1, the loss of which compromises vascular barrier function and angiogenesis; (2) S-dndo 1, an Ig superfamily member and a mediator of homophilic adhesion; and (3) endoglin, a regulator of transforming growth factor beta (TGF-b) signaling, the absence of which leads to a phenotype reminiscent of VE-cadherin-null mice. Endothelial Cell–Leukocyte Adhesion

The recruitment of leukocytes to sites of infection or injury begins with a well-defined set of sequential adhesive interactions between the circulating leukocyte and the activated endothelium (Figure 3). A similar cascade of events is believed to also mediate the trafficking of lymphocytes across high endothelial venules in lymphoid tissues. Endothelial activation Endothelial activation is characterized by the enhanced expression of endothelial selections (P- and E-selectin) and members of the Ig superfamiy (ICAM-1, MAdCAM-1, and VCAM-1). This typically involves de novo synthesis, with peak expression occurring over several hours: 4–6 h for E-selectin and 12–24 h for VCAM-1 and ICAM-1. For P-selectin, however, endothelial surface expression occurs within minutes following stimulation with noncytokine mediators such as thrombin and histamine as preformed P-selectin is mobilized from Weibel–Palade bodies. The expression of other molecules such as PECAM-1 and the JAMs that have been implicated in leukocyte trafficking do not appear to be affected by inflammatory mediators. Leukocyte rolling The initial leukocyte–endothelial adhesive event is one in which the circulating leukocyte rolls along the surface of the inflamed endothelium (and on already adherent leukocytes). This process involves an initial capture and fast rolling of the leukocyte, followed by a period of slow rolling and deceleration prior to leukocyte arrest. This step has long been recognized as principally mediated by selectins, with each L- and P-selectin mediating the capture and fast rolling, while E-selectin is preferentially involved in the slow rolling. Interestingly, L-selectin-mediated capture and rolling requires a critical threshold of shear stress to occur, while rolling adhesion mediated P- and E-selectin

34


Leukocyte rolling

Capture

Fast rolling

Firm adhesion

Diapedesis

Slow rolling

L-selectin P-selectin E-selectin 2 integrins/ICAM-1/2 41/IVCAM-1 or 47/MAdCAM-1 PECAM-1 JAMs CD99 Figure 3 Leukocyte–endothelial interactions. The sequence of adhesive interactions involved in the emigration of leukocytes from the circulation across the endothelium to extravascular sites is illustrated. The steps of these interactions, in order, are leukocyte rolling, firm adhesion, and diapedesis (transendothelial migration). Each step is mediated, depending on the cell type and context, by the interactions of specific cell adhesion molecules and their ligands.

demonstrates weak or no dependence on a shear threshold. The basis for this appears to reside in the fact that L-selectin engages its ligands through exceptionally labile adhesive bonds that are only significantly stabilized above a critical shear threshold. Although originally thought to mediate only firm adhesion and transendothelial migration (see below), b2 integrins are now recognized to cooperate with Eselectin in the deceleration of the rolling leukocyte. Depending on the cell type, there is also evidence that a4b1, a4b7, and CD44 are able to respectively promote rolling on VCAM-1, MAdCAM-1, and hyaluronate-covered surfaces. Activation–firm adhesion As the leukocyte rolls, interactions between selectins and their ligands and between chemokines and chemoattractants associated with the endothelium and G-protein-coupled receptors on the leukocyte surface, activate b2 (and probably a4) integrins on the leukocyte surface. This activation, which involves upregulation of integrin expression and adhesive activity and the shedding of L-selectin, enables the arrest and firm adhesion of the leukocyte to the endothelium. This phase of leukocyte recruitment is mediated by the binding of endothelial Ig superfamily members to (activated)

leukocyte integrins (ICAM-1/aMb2, ICAM-1, -2/a1, b2, VCAM-1/a4b1, and MAdCAM-1/a4b7). Transendothelial migration (diapedesis) The adherent leukocyte then ‘crawls’ over the luminal surface, a process that involves the cyclic modulation of integrin receptor avidity. Once a junction has been located, the emigrating leukocyte squeezes between the closely opposed endothelial cells, crossing the basement membrane to enter the extravascular tissue. Significantly, this occurs without disrupting the integrity of the endothelium. This process of leukocyte transendothelial migration or diapedesis is mediated by at least five cell–cell adhesion molecules: Ig superfamily members PECAM-1 and JAM-A, -B and -C, as well as CD99, a molecule with a unique structure. With the exception of JAM-B, all of these molecules are expressed on both leukocytes and endothelial cells and thus are capable of homophilic adhesion. The JAMs also bind leukocyte integrins, heterophilic interactions that have also been implicated in their activity in leukocyte trafficking. PECAM-1 appears to be required for the initial entrance or penetration of the junction, while CD99 appears to be involved more distally in the passage through the intercellular junction. While traversing the junction, homophilic


interactions of leukocyte and endothelial PECAM-1 also upregulate a6b1 on transmigrating leukocytes, an integrin that is required for the passage of the leukocyte through the matrix of the basement membrane. Although animal studies have implicated the JAMs in leukocyte diapedesis, their specific roles remain to be determined. Pericyte–Endothelial Cell Adhesion

Pericytes are perivascular cells imbedded within the basement membrane of the endothelium of capillaries and postcapillary venules. Contact between the endothelial cell and the pericyte is made by cytoplasmic processes of the pericyte indenting the endothelial cell, and vice versa. This results in the so-called ‘peg and socket’ contact. The normally intervening basal lamina is absent at these pericyte–endothelial cell interdigitations and growth factors (e.g., epidermal growth) factor may concentrate at these contacts. In some tissues, TJs between pericytes and endothelial cells have been reported and ‘adhesion plaques’ have been described in the pericyte membrane. An inverse correlation exists between endothelial cell proliferation and the extent of pericyte coverage, with tissues demonstrating the slowest rate of endothelial cell turnover having the greatest pericyte coverage. This suggests that pericytes are a major negative modulator of capillary growth and hence a promoter of vessel quiescence. This may be mediated by the pericyte-derived TGF-b, an inhibitor of endothelial cell growth and migration, the activation of which is dependent on pericyte–endothelial cell contact. Pericytes are also contractile, and thus points of cell–cell contact may permit contractions of the pericyte to be transmitted to the endothelial cell to reduce the caliber of the vessel or alter vascular permeability.

Vascular Cell–Cell Adhesion in Normal Lung Function Lung Defenses

The recruitment of leukocytes is an essential component of the lung’s defense against infection or inflammatory insults. Although data certainly indicate that the leukocyte–endothelial interactions described above may operate in the lung to regulate the emigration of circulating leukocytes, there is also evidence that leukocyte recruitment in the pulmonary circulation may differ, depending on the stimulus, from that which occurs in postcapillary venules at extrapulmonary sites. Although the b2 integrins have been shown to be required for neutrophil emigration in various models of acute inflammation involving

the systemic vascular bed, including neutrophil recruitment mediated Streptococcus pneumoniae, HCl, and C5a, these stimuli mediate neutrophil extravasation from the pulmonary circulation that is independent of the b2 integrins. An even more complex picture is suggested by studies of mice deficient in both P-selectin and E-selectin or P-selectin and ICAM-1. In these mutant animals, neutrophil emigration into the peritoneal cavity was completely inhibited during S. pneumoniae-induced peritonitis. In contrast, neutrophil extravasation into the alveolar space during S. pneumoniae-induced pneumonia was intact. Together these data suggest that leukocyte recruitment from the pulmonary circulation can occur through pathways that do not require selectins, b2 integrins, or ICAM-1. These observations may be due in part to the fact that primary site for leukocyte extravasation is in the pulmonary capillaries and not in postcapillary venules as occurs in the systemic vasculature. This is significant because the average diameter of the leukocyte (particularly the neutrophil) approaches or exceeds that of the capillaries. As a result, intravascular leukocytes must deform in order to transit through the pulmonary microvasculature and thus are in close apposition with the pulmonary endothelium. Consequently, processes that change the physical properties of the leukocyte or compromise their ability to deform may be sufficient to slow and arrest the leukocyte in the microcirculation of the lung. Thus, it should not be surprising that in the lung there may be less stringent requirements for molecular-meditated processes to capture and attach the intravascular leukocyte to inflamed pulmonary endothelium. Lung Permeability

In the lung, as in other vascular beds, endothelial cell permeability depends largely on the regulation of paracellular fluid and solute transport through intercellular junctions. The degree of ‘openness’ of this pathway is governed by the balance of centrally directed contractile forces and opposing forces generated by cell–cell and cell–matrix adhesive complexes that tether endothelial cells to each other and to the basement membrane. Of the various junctional complexes, TJs are recognized as the primary determinants of the endothelial barrier function. However, disruption of VE-cadherin function results in interstitial edema in the lung and there is evidence that in the setting of hyperosmolarity, E-cadherin expression may be upregulated on the endothelium of the lung microvasculature where it acts to promote barrier function. These data suggest that cadherin-dependent

36


activity mediated through AJs may also contribute to the regulation of vascular permeability in the lung. Barrier integrity varies with vessel type, as the endothelium of the pulmonary microcirculation is much more restrictive than that of the pulmonary arteries or veins. This suggests that there may well be site-specific differences in junctional architecture and/or cytoskeletal machinery in the pulmonary vasculature.

Vascular Cell–Cell Adhesion in Respiratory Diseases Asthma

Asthma is an inflammatory disease characterized by increased infiltration of various inflammatory cells in the bronchial mucosa and airways. In allergic asthma, antigen exposure results in the activation of mast cells and TH2 lymphocytes and their subsequent release of a number of cytokines and lymphocyte or eosinophil chemoattractants. These inflammatory mediators alter the expression and activity of cell adhesion molecules on these asthma-associated leukocytes and on the bronchial microvasculature in specific patterns that target their recruitment out of the circulation into the bronchial tissues. Analyses of bronchial tissue from allergic and other asthmatics, as well as studies of murine models of asthma indicate that interactions between E-selectin and its ligands, 1CAM-1 and aLb2 and VCAM-1 and a4b1, are involved in the recruitment of eosinophils in the allergic airway response. It should be noted however, that there are reports in which acute antibody inhibition of a4b1, b2 integrin, or ICAM-1 may reduce airway hyperresponsiveness without reducing eosinophil or lymphocyte accumulation. This suggests that these molecules may mediate processes other than leukocyte–endothelial interactions, such as leukocyte activation or antigen presentation, that contribute to the development of the asthmatic phenotype. Acute Lung Injury

A large and diverse group of microbial and inflammatory insults may acutely injure the lung culminating in the acute respiratory distress syndrome (ARDS) and respiratory failure. A key feature of this syndrome is endothelial cell injury and subsequent dysfunction manifested by impaired barrier function and increased vascular permeability. This dysfunction must necessarily involve destabilization of intercellular junctions, but the exact molecular basis is still being defined. A variety of inflammatory agents including histamine, lipopolysaccharide, leukotriene B4, platelet activating factor, nitric oxide thrombin,

as well as cytokines (TNF-a, IL-1, and g-IFN) increase transendothelial permeability, at least in part, by inducing endothelial cell contraction and the resultant formation of interendothelial gaps. These alterations in endothelial morphology require the recruitment and activation of calcium-dependent cytoskeletal proteins (e.g., actin and myosin) that alter endothelial cell contour and reorganize intercellular junctional complexes. The alterations in endothelial permeability induced by inflammatory mediators may involve changes in the concentration of VE-cadherin and PECAM-1 in endothelial intercellular junctions as cytokines have been shown to redistribute these molecules out of endothelial cell–cell contacts with a concomitant increase in permeability. An important contributor to the pathogenesis of endothelial dysfunction in ARDS appears to be activated neutrophils sequestered in and adherent to the pulmonary capillaries. These trapped leukocytes release reactive oxygen species and granular constituents that undermine the normal barrier function of the endothelium. These products may also contribute the inflammatory cascade by activating monocytes and macrophages leading to release of additional proinflammatory mediators. Inhibition of b2 integrins, ICAM-1, and selectins blocks neutrophil recruitment and permeability injury in animal models of acute lung injury or infection although there do appear to be alternative pathways as noted above. This has made these molecules attractive therapeutic targets for the treatment of ARDS. However, the potential for inhibition of host defenses and reparative responses continues currently to be an obstacle to the widespread application of this approach.

Acknowledgments This work was supported by the Department of Defense (PR043482), National Institutes of Health (HL079090), and the Philadelphia Veterans Medical Center. See also: Acute Respiratory Distress Syndrome. Adhesion, Cell–Cell: Epithelial. Adhesion, Cell– Matrix: Integrins. Asthma: Overview. Endothelial Cells and Endothelium. Leukocytes: Neutrophils; Monocytes; T Cells.

Further Reading Allt G and Lawrenson JG (2001) Pericytes: cell biology and pathology. Cells Tissues Organs 169: 1–11. Angst BD, Marcozzi C, and Magee AI (2001) The cadherin superfamily. Journal of Cell Science 114: 629–641.

ADHESION, CELL–CELL / Epithelial 37 Bazzoni G and Dejana E (2004) Endothelial cell-to-cell junctions: molecular organization and role in vascular homeostasis. Physiological Reviews 84: 869–901. Boitano S, Safdar Z, Welsh DG, Bhattacharya J, and Koval M (2004) Cell–cell interactions in regulating lung function. American Journal Physiology (Lung Cell Molecular Physiology) 287: L455–L459. Dudek SM and Garcia JG (2001) Cytoskeletal regulation of pulmonary vascular permeability. Journal of Applied Physiology 91: 1487–1500. Hogg JC and Doerschuk CM (1995) Leukocyte traffic in the lung. Annual Review of Physiology 57: 97–114. Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110: 673–687. Juliano RL (2002) Signal transduction by cell adhesion receptors and the cytoskeleton: functions of integrins, cadherins, selectins, and immunoglobulin-superfamily members. Annual Review of Pharmacology and Toxicology 42: 283–323. Ley K (2003) The role of selectins in inflammation and disease. Trends in Molecular Medicine 9: 263–268. Mandell KJ and Parkos CA (2005) The JAM family of proteins. Advanced Drug Delivery Reviews 57(6): 857–867. Muller WA (2003) Leukocyte–endothelial-cell interactions in leukocyte transmigration and the inflammatory response. Trends in Immunology 24: 327–334. Sohl G and Willecke K (2004) Gap junctions and the connexin protein family. Cardiovascular Research 62: 228–232. Vincent PA, Xiao K, Buckley KM, and Kowalczyk AP (2004) VEcadherin: adhesion at arm’s length. American Journal Physiology Cell Physiology 286: C987–C997. Vorbrodt AW and Dobrogowska DH (2003) Molecular anatomy of intercellular junctions in brain endothelial and epithelial barriers: electron microscopist’s view. Brain Research Reviews 42: 221–242.

Epithelial J K McGuire, Children’s Hospital and Regional Medical Center, Seattle, WA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract A continuous epithelial sheet lines the respiratory tract to provide a barrier to the outside world and protect against invasion by microorganisms. Central to these functions are the complex junctions that regulate adhesion between adjacent epithelial cells. These junctions create epithelial polarity, regulate paracellular transfer of molecules across the epithelial barrier, and maintain the integrity of the epithelial layer. Far more than just ‘cellular glue’, the major junctional complexes that regulate cell–cell adhesion, i.e., tight junctions, adherens junctions, and desmosomes, are critical in tissue patterning and differentiation during development, epithelial cell sensing of the pericellular environment, and modulation of intercellular signaling. Dozens of different proteins have been associated with cell–cell adhesion functions in mammalian epithelia and only recently have the structure and function of specific proteins expressed at cell–cell junctions in the lung and airways been identified. For example, the claudins are major transmembrane proteins regulating extracellular interaction at tight junctions, and the E-cadherin/b-catenin complexes that form adherens junctions

are critical in cell motility and differentiation and their loss may be associated with epithelial carcinogenesis. However, little is known about how specific changes in cell–cell adhesion contribute to respiratory disease, and this remains an area of intense study.

Description From the upper airway to distal alveoli, an essentially continuous epithelial sheet comprising of several specialized cell types lines the respiratory tract to create a semipermeable barrier to the outside world and provide protection from invading microorganisms. Essential to these functions are the complex structures that produce cell–cell adhesion between individual epithelial cells. Far more than just ‘cellular glue’ holding epithelial cells together, cell–cell adhesion complexes organize development and differentiation of the lung and respiratory tract, promote and maintain structural and functional integrity of the epithelial surface, and regulate cellular responses to injury and disease. Integral to the consideration of cell–cell adhesion in respiratory epithelia are the specific structural and functional characteristics of these cells. Respiratory epithelial cells are polarized, with distinct apical and basolateral domains, with the apical domain facing the external environment or lumen and the basolateral domain forming contacts with the substratum or neighboring cells. In addition to regulating cell shape, polarization contributes to the asymmetric distribution of organelles and molecules within epithelial cells and to the arrangement of cytoskeletal networks. Cell–cell adhesion complexes are key determinants of epithelial cell polarity and are essential in major respiratory epithelial functions such as maintenance of the barrier to the external environment and other critical epithelial functions including secretion and repair. A high degree of similarity exists across different species in the structure of cell–cell junctions and as in most vertebrate epithelia, three major types of junctions mediate intercellular adhesion in respiratory epithelial cells: tight junctions, adherens junctions, and desmosomes. Additionally, all epithelia studied thus far have an adhesive belt called the zonula adherens that encircles the cell just below the apical surface and that is characterized by an electron-dense cytoplasmic actin plaque (Figure 1). Along with the tight junction, which is just apical to the zonula adherens, this adhesive belt delineates the apical and basolateral epithelial compartments and is an important contributor to regulation of epithelial permeability and barrier function. Although the various epithelial cell–cell junctions are composed of unique proteins and serve distinct functions, in general, they

38

ADHESION, CELL–CELL / Epithelial

Tight junction

Adherens junction

Desmosome

Figure 1 Cell–cell junctions in respiratory epithelia. Schematic diagram of the three major types of junctions mediating cell adhesion in epithelial cells. Tight junction transmembrane components occludin (in red above), claudin, and JAM (blue) interact in the extracellular space and cytosolic plaque proteins (green) interact with the actin cytoskeleton (pink). Adherens junctions are formed by transmembrane cadherins (orange) that bind the actin cytoskeleton (pink) via the catenins (yellow and blue). Desmosomes are formed by the desmosomal cadherins (blue and pink) and cytosolic plaque proteins (aqua) that interact with intermediate filaments (green). Electron micrograph of mouse tracheal epithelium shows ultrastructure of zonula adherens near the apical/luminal epithelial surface.

Table 1 Major cell–cell junction proteins Junction

Transmembrane proteins

Cytosolic proteins

Cytoskeletal linker proteins

Tight junction

Occludin Claudins Junctional adhesion molecule (JAM) Cadherins Desmosomal cadherins

Plaque proteins

PDZ domain proteins (zonula occludens, PAR)

b-Catenin Plakoglobin, plakophilins, p0071

a-Catenin Plakins

Adherens junction Desmosome

share common structural features; principal among these are transmembrane receptors, usually glycoproteins that bind proteins on the extracellular surface and determine the specificity of intercellular interactions. The cytosolic domains of these receptors associate with a variety of cytoplasmic proteins that link them structurally to the cytoskeleton and regulate interaction with intracellular signaling pathways (Table 1).

Epithelial Cell–Cell Adhesion in Normal Lung Function Tight Junctions

Early electron microscopic assessment of the contacts between epithelial and endothelial cells identified a

series of apparent fusions between the outer plasma membranes of adjacent cells where the intercellular space disappeared. These initial ultrastructural studies suggested and subsequent studies have confirmed that tight junctions are a central component of the barrier to the paracellular passage of solutes, molecules, and inflammatory cells. Thus, an understanding of tight junction structure is important in considering the regulation of lung barrier function. Over 40 different proteins have been localized to the tight junction in epithelial, endothelial, and myelinated cells that can be further classified as tight junction integral transmembrane proteins and cytoplasmic plaque proteins. The transmembrane proteins include the tetraspan protein occludin and members of the claudin family, which contain four transmembrane domains, two extracellular domains,

ADHESION, CELL–CELL / Epithelial 39

and are oriented with both the N- and C-terminal ends towards the cytoplasm, and the single transmembrane Ig-domain-containing, junctional adhesion molecule (JAM). All of these interact directly with cytoplasmic PDZ domain-containing plaque proteins such as the zonula occludens (ZO) and PAR proteins, which function as adapters to recruit cytoskeletal or signaling molecules. The tetraspan extracellular domains interact in the paracellular space with extracellular domains of tight junction proteins on neighboring cells and occludin, the first integral membrane protein to be found in tight junctions of many cell types, also directly binds F-actin via the last 150 amino acids of its carboxyl tail. The ‘tightness’ of tight junctions appears to be regulated by changes in the combination and expression ratios of different claudin family members, which can engage in homotypic or heterotypic interactions between several of the 24 distinct claudin gene products thus far identified in humans. One defined role of claudins in regulating permeability is the formation of ionselective pores, and different types of claudins appear to determine the specificity of the pores for individual ions such as Na þ , Cl , or Ca2 þ . Whereas tight junctions can form in the absence of occludin, claudins appear to be essential for tight junction formation. It is likely that JAM has an important role in tight junction assembly and resealing after disassembly in epithelia by engaging in homophilic interactions in the paracellular space. Tight junction plaque proteins have important roles in coordinating intracellular signaling by recruiting cytosolic proteins to the tight junction during assembly, and recent reports of ZO proteins localizing to the nucleus, in addition to their tetraspanbinding at tight junction plaques, suggest that they transduce signals from the cell membrane to the nucleus where they may regulate gene expression. Recruitment of plaque proteins to the tight junction may also have a role in regulating cell motility and proliferation. Consequently, although tight junctions perform key functions in regulating epithelial permeability, it is becoming increasingly clear that tight junctions are multifunctional complexes involved in many vital epithelial cell functions. Only recently have specific patterns of tight junction protein expression in the respiratory tract been characterized, and much work remains to further define how tight junctions are regulated in normal and abnormal lung function. Adherens Junctions

Adherens junctions are ancient structures present across eukaryotes and even in related structures in

single-celled organisms such as yeast, and as such, adherens junctions have critical functions in coordinating cell polarity, cytoskeletal dynamics, cell sorting, and differentiation. In addition to regulating organization within epithelia, adherens junctions are also important in transmitting information from the environment to the interior of cells. At the core of adherens junctions are the calcium-dependent transmembrane glycoprotein cadherins, and the major epithelial cadherin is E-cadherin, the best characterized member of the cadherin family and the primary cadherin expressed in respiratory epithelium. The extracellular domain of E-cadherin, like other classical cadherins, is comprised of five calcium-binding cadherin repeats that participate in homotypic interactions with neighboring cells. There is a single-pass transmembrane domain and the cadherin cytosolic domain is linked to cytoskeletal structures via the catenins. Cadherin adhesive activity is regulated in several ways including the level of cadherin gene expression, which correlates with the strength of adhesion, and the type of cadherin expressed, which affects the specificity of cell interaction and plays a role in embryo patterning and determination of cell fate. Posttranscriptional mechanisms regulating cadherin adhesion include modulation of cadherin clustering at the cell surface and changes in cadherin interaction with the catenins. Proteolysis of cadherin domains is now recognized as an important means of posttranslational cadherin modification with extracellular domains being cleaved by metalloproteinases, the transmembrane domains targeted by g-secretases, and intracellular domains degraded by caspases. Cadherins are key mediators of morphogenesis, epithelial sheet integrity, growth control, and maintenance of the terminally differentiated phenotype. Differential expression of different cadherin classes is likely to be important in cell sorting and embryo patterning in development and specific cadherins may be associated with the determination of cell fate. For example, E-cadherin is the primary cadherin expressed in lung epithelia, whereas N-cadherin is expressed at cell– cell junctions in pleural mesothelial cells. The cytoplasmic E-cadherin domain binds the armadillo family member protein b-catenin, and this binding induces a tertiary structure that in turn binds a-catenin, which nucleates the assembly of a multimeric complex that links E-cadherin/b-catenin complexes to the actin cytoskeleton. This process both stabilizes the intercellular junctions and allows for coordination of cytoskeletal dynamics with changes in cell–cell adhesion. These protein interactions are regulated by phosphorylation events of both the E-cadherin cytoplasmic tail (increase interaction) and

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b-catenin (decrease interaction) and by the recruitment of various other potential adherens junctionassociated proteins. Therefore, this complexity allows for many levels of regulation of adherens junctionbased cell adhesion. In addition to its structural role in adherens junctions, b-catenin also functions as a noncadherin-dependent signaling factor. In concert with activation of the Wnt signaling pathway, free cytoplasmic b-catenin translocates to the nucleus and binds to transcription factors of the lymphocyte enhancer-binding factor-1/T-cell factor (LEF-TCF) pathway to regulate expression of downstream target genes associated with cell migration and proliferation including matrilysin (MMP-7), c-myc, WISP, and cyclin D1. A comprehensive consideration of b-catenin in cell signaling is beyond the focus of this discussion of cell adhesion, but recent studies have suggested a role for b-catenin in terminal differentiation of alveolar epithelium. Adherens junctions at the cell surface associate with several other types of intercellular junctions and membrane receptors. Ultrastructural studies of epithelial sheets reveal closely apposed membranes at sites of cell–cell contact where adherens junctions alternate with desmosomes, and formation of adherens junctions has been shown to be necessary for desmosome and tight junction formation. Adherens junction-specific cadherins also associate with other cell surface receptors that participate in signaling such as connexins, Notch, receptor tyrosine kinases, and phosphatases, suggesting that adherens junctions can be regulated by extracellular cues. Desmosomes

Desmosomes are specialized junctions that anchor stress-bearing intermediate filaments at sites of strong intercellular adhesion. This provides a scaffold that gives integrity to tissues such as skin, heart, and respiratory epithelium that is subject to mechanical stress. At the core of desmosomes are proteins of the cadherin (the desmogleins and desmocollins) and armadillo (plakoglobin, the plakophilins, and p0071) families, which form membrane-associated complexes that are tethered to intermediate filaments via the plakins. Analogous to adherens junction cadherins, the single-pass transmembrane desmosomal cadherin extracellular domains interact in the extracellular space; however, in contrast to adherens junction cadherins, desmosomal cadherins interact in a heterotypic manner as each desmosome must contain at least one desmoglein and one desmocollin. Desmocollins and desmogleins are expressed in a tissueand differentiation-specific manner, and thus it has been proposed that desmosomal cadherins may

function in directing epithelial differentiation. The cytoplasmic cadherin tails directly bind an armadillo protein, usually plakoglobin (g-catenin), and this complex in turn associates with a cytoskeletal linking protein, e.g., desmoplakin, that mediates attachment to the intermediate filaments. The result is a series of connections between adjacent cells that maintains tissue tensile strength. Similarities exist between the regulation of adherens junction and desmosomal adhesion; as for adherens junctions, desmosomal adhesion during development is regulated by the timing of and type of desmosomal cadherin or armadillo protein expressed, and as discussed above, desmosome assembly appears to be downstream of and dependent on adherens junction assembly. Posttranslational mechanisms regulating desmosomal adhesion include proteolytic processing of intracellular cadherin domains by caspases and cleavage of extracellular domains by metalloproteinases. Although desmosomes are present throughout the airway and alveolar epithelium, little is known about the specific regulation of desmosome-based cell adhesion in the respiratory tract.

Cell–Cell Adhesion in Respiratory Diseases Although the net permeability of the lung and airways is jointly regulated by the epithelium and endothelium, disruption of paracellular permeability and leakage of fluid and proteins across the airway or alveolar epithelium are hallmarks of a variety of respiratory diseases including asthma, allergic rhinitis, acute lung injury, and pneumonia among others. Furthermore, pathogens may directly target junctional proteins to facilitate entry into the host. However, the specific mechanisms by which changes in tight and adherens junctions and the proteins that regulate these junctions in diseases of the airways and lung remain largely undefined. It has been known for a few years that some types of lung cancer, like other carcinomas, show altered staining patterns for several adherens junction proteins, and an increasing amount of evidence suggests that inactivation of E-cadherincatenin complexes plays a significant role in carcinogenesis. Additionally, altered b-catenin signaling has been implicated in idiopathic pulmonary fibrosis. Thus, these pathways may be attractive targets for therapy in these historically difficult to treat diseases. The available knowledge on the structure, assembly, and function of intercellular junctions in normal and abnormal processes is rapidly increasing. These complexes clearly have more than a structural role and perform critical functions in tissue development,

ADHESION, CELL–MATRIX / Focal Contacts and Signaling 41

homeostasis, and response to injury. However, our understanding of the regulation of epithelial cell–cell adhesion in respiratory tract development and maintenance of normal lung function, and in injury and disease remains limited. Thus, this remains an area of intense investigation. See also: Adhesion, Cell–Cell: Vascular. Asthma: Overview. Basal Cells. Bronchiectasis. Bronchiolitis. Connexins, Tissue Expression. Contractile Proteins. Defense Systems. Epithelial Cells: Type I Cells; Type II Cells. Gene Regulation. Lung Development: Overview. Matrix Metalloproteinases. Panbronchiolitis. Signal Transduction. Transcription Factors: Overview. Tumors, Malignant: Overview; Metastases from Lung Cancer.

Further Reading Braga VMM (2002) Cell–cell adhesion and signalling. Current Opinion in Cell Biology 14: 546–556.

Bremnes RM, Veve R, Hirsch FR, and Franklin WA (2002) The E-cadherin cell–cell adhesion complex and lung cancer invasion, metastasis, and prognosis. Lung Cancer 36: 115–124. Godfrey RWA (1997) Human airway epithelial tight junctions. Microscopy Research and Technique 38: 488–499. Gonzalez-Mariscal L, Betanzos A, Nava P, and Jaramillo BE (2003) Tight junction proteins. Progress in Biophysics and Molecular Biology 81: 1–44. Perez-Moreno M, Jamora C, and Fuchs E (2003) Sticky business: orchestrating cellular signals at adherens junctions. Cell 112: 535–548. Schneeberger EE and Lynch RD (2004) The tight junction: a multifunctional complex. American Journal of Physiology. Cell Physiology 286: C1213–C1228. Wheelock MJ and Johnson KR (2003) Cadherins as modulators of cellular phenotype. Annual Review of Cell and Developmental Biology 19: 207–235. Yin T and Green KJ (2004) Regulation of desmosome assembly and adhesion. Seminars in Cell and Developmental Biology 15: 665–677. Zhurinsky J, Shtutman M, and Ben-Ze’ev A (2000) Plakoglobin and b-catenin: protein interactions, regulation and biological roles. Journal of Cell Science 113: 3127–3139.

ADHESION, CELL–MATRIX Contents

Focal Contacts and Signaling Integrins

Focal Contacts and Signaling G D Rosen and D S Dube, Stanford University Medical Center, Stanford, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Focal adhesions (FAs) are contact points for the cell with the extracellular matrix. These complex structures regulate communication of the cell with the surrounding extracellular environment and signaling through these FAs regulates diverse cellular processes, including proliferation, migration, apoptosis, spreading, and differentiation. The principal components of the FAs are integrins, which are ab heterodimers that regulate cell– matrix and cell–cell interactions. Focal adhesion kinase (FAK) is a kinase that localizes to FAs and regulates signaling in FAs. FAK communicates signals between integrins and intracellular proteins, which regulate diverse cellular processes such as cell polarity, migration, and invasion. FAs play a central role in regulating normal developmental processes such as blood vessel morphogenesis but increased activity of FA can incite aberrant events such as tumor cell invasion and pulmonary fibrosis. A further exploration of the role of FA in health and disease will provide greater insight into the regulation of normal developmental processes and into the pathogenesis of lung diseases such

as lung cancer, acute respiratory distress syndrome, and pulmonary fibrosis.

Introduction This article explores the physiology of focal contacts, and cell signaling in normal and pathological states. Focal adhesions (FAs) are specialized multimolecular structures that exist at the points of contact between the cell membrane and the extracellular matrix. Our current understanding of the physiological roles of FAs suggests that in addition to mediating cell adhesion, they also function as a physiological signaling link between the cytoplasm and the extracellular milieu by promoting the bidirectional transmission of biochemical signals. Integrins are the core components of FAs whose orderly function underlies the diverse roles associated with FAs. Integrins are a family of a/b heterodimeric glycoprotein receptors that span the cell membrane. Morphologically, these receptors exhibit an intracellular domain, a transmembrane domain, and an ectodomain that is in association with the cell matrix or other cells. The

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intracytoplasmic domains of integrins can associate with actin, adaptor proteins, and enzymatic components of signaling cascades and their substrates. This complex association of the cell matrix, integrins, and cytosolic proteins forms the molecular conglomerates that are termed FAs and mediates the bidirectional flow of signals between the cell matrix and the cell. The binding of ligands to integrins in either the extracellular or intracellular domains induces conformational changes within the integrins that affect the binding of ligands on the reciprocal end of the integrin molecule. The activation of FAs by paracrine and endocrine stimuli culminates in the activation of specific intracytoplasmic signaling cascades, including tyrosine phosphorylation, focal adhesion kinase (FAK)-mediated signaling, mitogen-activated protein-kinase (MAPK), and the generation of phosphatidylinositol-4, 5-bisphosphate. These intracellular signals result in a multitude of cytoplasmic responses that regulate, for example, cell proliferation, motility, and differentiation. Based on the multiplicity of functions attributed to FAs, the strict regulation of their function forms the basis of orderly organ structure and tissue function. A better understanding of the mechanisms that are involved in the regulation of FAs has led to the development of potent antithrombosis and anti-inflammatory therapies. Moreover, the multiple roles of FAs further promise to aid the elucidation of diverse pathological processes associated with FAs. Some of these processes include tumor growth, pathological organ remodeling, cell barrier defects, and embryogenesis as well as many other pathological entities that are regulated by cell–matrix and cell–cell contact.

Focal Contacts: Structure and Function Interference reflection microscopy first identified FAs as dark areas at the interface between the ventral cell surface and the extracellular matrix. Diverse types of focal adhesions have since been described. Subsequently, advances in gene cloning have led to the determination of the primary structures of many of the constituents of FAs. Early analysis of FA structure identified integrins as core glycoproteins intimately associated with the structure of FAs. The elucidation of the primary structure of integrins revealed three domains: an extracellular domain, a transmembrane domain, and an intracellular domain as well as sites for tyrosine phosphorylation. In parallel with the elucidation of the primary structure, the solution of the crystal structures of the components of FAs further advanced testable hypotheses on structure– function relationships of the individual protein components of FAs. Studies of the conformational

rearrangements within integrin molecules using diverse tools such as negative stain microscopy, nuclear magnetic resonance, and spectroscopy have further aided the understanding of the function of focal adhesion molecules. These studies have allowed the evaluation of conformational changes within integrins using ligand analogs that bind to ligand-induced binding sites (LIBSs). Site-directed mutagenesis has also aided the characterization of the roles of submolecular components of protein components of FAs, while in vivo studies of these mutagenesis studies have led to the discovery of unique phenotypes, thus further allowing the determination of the functional roles of individual components of FAs. Most recently, the use of fluorescent green protein (FGP) tagged proteins has allowed the determination of stoichiometric relationships of the various components of FAs.

Components of Focal Adhesions: Integrins and Extracellular Matrix Integrins are a large family of heterodimeric transmembrane glycoprotein receptors uniquely found among metazoans whose quaternary structure is characterized by a noncovalent union between a- and b-subunits. There are 18 a-subunits (MW 120– 180 kDa) and 8 b-subunits (MW 90–110 kDa) that assemble in various permutations to generate 24 distinct integrin heterodimers. These various subunit combinations expand the repertoire of intracellular and extracellular ligands for integrins. In turn, the expanded receptor capability created by the many combinations of a- and b-subunits accounts for the diverse functional roles of integrins. Integrins are central to the structure of FAs. The significance of integrins in metazoan cell structure and tissue organization is emphasized by the observation that the number of integrins parallels the evolutionary complexity of an organism. It has been proposed that the evolution of metazoans as multicellular organisms hinged upon the parallel evolution of integrins as anchor apparatus to the cell matrix and intercellular molecular adhesives. Figure 1 shows the subclassification of the integrin receptor family. Integrins have a large extracellular domain, a transmembrane domain, and an intracytoplasmic domain. The ectodomains of integrins exist in three major conformational states. The first of these states is a bent conformation that is similar to the crystal structure of avb3. This conformation is considered to be a low-affinity state. The second conformation has been referred to as a ‘closed head piece’ conformation and is considered to represent an intermediate affinity conformational state. Finally, there is the


ΙΙb

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Figure 1 The integrin receptor family. Integrins are ab heterodimers that span the cell membrane. This figure shows the mammalian subunits and their ab associations. Asterisks denote alternatively spliced cytoplasmic domains. Reproduced from Hynes RD (2002) Integrins: bi-directional, allosteric signaling machines. Cell 110: 673–687, with permission from Elsevier.

high-affinity ‘open head piece’ conformation, which is induced by ligand binding. The high-affinity conformation is thought to be acquired by the extension of the angle between the b- and hybrid domains. A detailed discussion of the crystal structures of the various integrins and their interacting partners is outside the scope of this article. The evidence so far supports a model whereby the binding of a ligand to the ectodomain mediates the extension and simultaneous separation of the a- and b-subunits of the integrin heterodimers within the transmembrane and intracytoplasmic domains. This model also suggests that the binding of a ligand in the intracellular domain of integrins by adaptor proteins such as talin also mediates counter conformational alterations that cause the exposure of LIBS in the ectodomain. It has further been proposed that these conformational changes are dynamic and dependent on regulatory factors such as growth factors.

Regulation of Integrins Intracytoplasmic Association between the a- and b-Chains

A considerable body of experimental data reveals that the interaction between the a- and b-subunits in the cytoplasm regulates the activation states of the integrin receptors. Studies utilizing the platelet integrin aIIbb3, for example, have revealed that the aIIb short intracytoplasmic domain negatively regulates the activation of the integrin receptors. The experimental deletion of the intracytoplasmic aIIb domain or the conserved GFFKR sequence leads to

constitutively activated integrin receptors. Similarly, deletions of conserved intracytoplasmic b3 sequences also culminate in constitutively activated integrin receptors. These observations have led to the conclusion that the interaction between these two cytoplasmic domains negatively controls the activation of integrins. This interaction seems to occur through a salt bridge between the R995 of the aIIb and the D723 in the b3. In support of this idea, the experimental mutation of either amino acid to the charge neutral alanine leads to a constitutively active integrin. Nuclear magnetic resonance (NMR) studies have demonstrated that the intracytoplasmic portions of the a- and b-domains interact with each other. Furthermore, it has been shown that point mutations in F992A or R995D impair the interaction of the a- and b-domains. The Role of Talin in Integrin Activation

Studies support the conclusion that talin, a cytoskeletal actin-binding protein that co-localizes with active forms of integrins, is a crucial element in the activation of integrins. Talin is a homodimer consisting of two antiparallel subunits, each approximately 270 kDa. Each of the talin subunits is comprised of a 220 kDa C-terminal and a 70 kDa globular head, which is thought to contain the binding sites for integrins. Talin has been demonstrated to bind the b1D, b2, b3, b5, and b7 intracytoplasmic integrins at their N-terminus. Structural analysis of the PTB-like 96 amino acid subdomain of the talin FERM (4.1, ezrin, radixin, moesin) domain shows a major integrin-binding site. Besides the ubiquitous interaction

44


of talin with diverse integrins, the overexpression of a talin fragment containing the head domain leads to the activation of integrin molecules. The role of talin in integrin activation is further supported by the observation that talin knockdown abrogates aIIb3 activation by endogenous agonists. This observation suggests that talin is a critical common downstream target in the activation of at least some integrins. Molecular mechanisms of the role of talin in the regulation of integrins are complex and still being explored. Vonogradova and colleagues have demonstrated that the talin head domain alters the NMR spectrum for the interaction between the b3 and aIIb domains. Other investigators have shown that the talin head domain attenuates the fluorescence resonance energy transfer (FRET) between fluorophoretagged a- and b-integrins within viable cells. These observations support a model whereby the binding of the talin phosphotyrosine-binding (PTB) domain to integrin disrupts the inhibitory interaction between a- and b-membrane proximal domains.

Regulation of Talin–Integrin Interactions The preceding section considers that the interaction of talin with intracytoplasmic domains of integrin is pivotal to the regulation of integrin function and hence FAs. A variety of mechanisms have been advanced to account for how these interactions are regulated. The talin head has a sixfold higher affinity in comparison to the intact talin molecule. This observation has led to the hypothesis that in the whole talin molecule, integrin binding sites are obscured. Therefore, a potential mechanism whereby talin regulates integrin activation is through the modulation of its conformations by physiological factors, which then reveal or mask integrin binding sites. Evidence supporting this possibility accrues from observations made from the function of the ERM (ezrin, radixin, moesin) family of proteins. The major integrin-binding site in talin lies within the talin FERM domain, and binding occurs via a variant of the classical PTB domain–NPxY interaction. In the ERM family, the intramolecular interaction of the N-terminal FERM domain with the C-terminal obscures ligandbinding sites. Intriguingly, ERM phosphorylation, PIP2 (phosphatidyl inositol 4,5-bis-phosphate), and rho-dependent pathways abrogate the native inhibition in the unstimulated ERM molecules presumably by unmasking the interaction of the FERM domain. Although the role of phosphorylation in the activation of integrins is still debated, it is notable that the binding of PIP2 to talin has been shown to mediate a conformational change within the talin molecule that culminates in the exposure of integrin-binding

sites in the FERM domain. The role of PIP in the regulation of talin is complicated by the observation that talin directly stimulates a splice variant of PIP2producing enzyme PIPKIg-90 (phosphatidylinositol phosphate kinase type Ig-90) thereby increasing PIP2 production. Interestingly, PIPKIg-90 and integrin b tails have overlapping binding sites on the talin F3 subdomain. This suggests a novel control strategy whereby PIPKIg-90 and talin may displace each other from integrin-binding sites following activation. Another regulatory mechanism for talin–integrin interaction is thought to involve calpain-mediated proteolysis of integrin b tails at sites straddling the NPXY/NXXY motifs that are necessary for the anchorage of integrins to the cytoskeleton and FAs. Calpains are calcium-dependent thiol proteases that have a diverse substrate spectrum that includes constituents of the cytoskeletal apparatus and cell-signaling proteins. Structurally, they are composed of an 80 kDa catalytic subunit and a 30 kDa regulatory subunit. Although calpain was historically considered to be involved in the detachment of migrating cells, accumulating evidence suggests that calpain also functions by regulating actin remodeling during cell migration. Supportive data have been obtained through experimental overexpression of calpain in aortic endothelial cells. These experiments reveal that overexpression of calpain leads to formation of focal adhesions. In contrast, when calpain is inhibited in fibroblasts and endothelial cells, adherence is maintained while cell dispersion is attenuated. Additional evidence points to the fact that the regulation of actin filament formation in endothelial cells is dependent on calpain-mediated proteolysis. The capacity of calpain to cleave integrins with diverse molecular structures suggests that calpains recognize higherorder protein structure rather than primary protein sequence. Calpains also cleave tyrosine kinases (PTK) such as focal adhesion kinase (FAK) and src and phosphatases (PTP) PTP-1B, Shp-1, and PTP-MEG. Other investigators have reported that calpain cleaves the N-terminal 47 kDa of talin, which is known to be hyperphosphorylated by protein kinase A.

Focal Adhesion Structure and Signaling FAs exhibit dynamic spatial–temporal variations in function. A large group of proteins are core to the structural behavior of focal adhesions. This is in keeping with the diversity of roles that are associated with FAs. The structural integrity of FA molecules relies on adaptor molecules that perform core scaffolding functions. Multiple other molecules also interact with the FAs and perform both structural and enzymatic chores of FAs. The overall function


and net effect of these scaffolds is to juxtapose kinases with their substrates and thereby facilitate FA-mediated signaling. The full repertoire of proteins that have been observed to interact with FAs is large and continuously expanding. In general, the relative expression or activation of different scaffolding proteins depends on the tissue of origin and cellular stimulus. Therefore, scaffolding proteins not only confer on FA structural integrity but are also responsible for the functional diversity associated with FAs. A core component of the FA complex is the p130Cas protein. The structure of p130Cas includes an SH3 domain, a proline-rich motif, and a very complex substrate-binding region that has the Src-SH2 binding region. The p130Cas SH3 domain interacts with the FAK possibly through interactions with the YDYV motif. This same YDYV motif also binds c-Src. It is thought that c-Src mediates the phosphorylation of the substrate domain of p130Cas. It is intriguing to also note that FAK has the capability to phosphorylate an Src binding site within p130Cas. The phosphorylation of the substrate-binding site within p130Cas achieves two major roles. First it allows the localization of p130Cas to the FA and second it allows p130Cas to interact with other proteins containing the SH2 domain such as protein tyrosine phosphatase (PTP)-PEST, NcK, and CrK. Crk/CrkL is an adaptor protein that interacts with phosphorylated p130Cas via an SH2 domain. The interaction of Crk with P130Cas leads to the formation of a complex that activates Dock 180, which is a novel exchange factor. Ultimately, the binding of Dock 180 to Crk and ELMO culminates in the activation of Rac, which augments the activation of p130Cas phosphorylation. In general, the association of p130Cas with Crk activates GTPases that are involved in the maintenance and reorganization of the actin cytoskeleton, which are important for cell motility and are crucial to establishing cellular structure. Paxillin is another adaptor molecule that interacts with a diverse range of proteins such as FAK, PKL, PTP-PEST, and b1 and a4 integrin intracytoplasmic tails. Similar to P130Cas, paxillin also complexes with a wide range of distinct exchange factors in a phosphorylation-dependent and tissue-specific manner. In summary, scaffold proteins bring into close proximity protein moieties important for the function of the FAs. The functional diversity conferred by the combinations afforded through these proteins results in tissue-specific function. Kinases

Tyrosine phosphorylation of focal adhesion components is considered to be the core event in the

generation of focal adhesion-mediated cell signaling. Tyrosine phosphorylation creates sites for binding of Src-homology domain (SH2-containing proteins). Moreover, tyrosine phosphorylation directly regulates a variety of kinases and phosphatases. The principal kinases that regulate focal adhesions are FAK and Src. Multiple other kinases (ser/thr kinases, Abl, PYK2,Csk) also probably play a role but their precise role in regulation of focal adhesions has not been fully elucidated.

Focal adhesion kinase FAK is a 125 kDa protein kinase that was initially shown to localize to FAs. Moreover, it was observed to be phosphorylated in relation to Src-mediated transformation. FAK is an FERM domain protein and is known to bind to many different signaling proteins. In general, it is considered that phosphorylation of FAK is the sentinel event in FA-mediated cell signaling. In response to integrin association with a ligand, FAK is activated by autophosphorylation at tyrosine Y397. One model proposes that subsequent to this autophosphorylation, Y397 acts as a docking site for Src that in turn mediates the phosphorylation of FAK at Y576 and Y577 and potentially at other sites. The phosphorylation of these tyrosines creates molecular contact points for proteins containing SH2 domains. As a result of this, the Ras-regulated MAPK pathway is activated (Figure 2). In support of this model, it has been observed that phosphorylation of Y926 in FAK creates an interaction site (pYNQV) for the SH2 domain of Grb2, an adapter protein that regulates growth factor signaling through Ras/SOS through binding of its SH2 and SH3 domains. This event is considered to be critical for the activation of the MAPK pathway. Grb2 can be phosphorylated at the Y926 position only when FAK is dissociated from the focal adhesion complex. This observation suggests that the phosphorylation of FAK causes it to dissociate from the FA complex and then phosphorylates its target proteins and in turn initiates signaling cascades. The interaction of FAK with FAs occurs through the FA-targeting domain (FAT) of FAK. Indeed, when the FAT domain in FAK is mutated, the FAK molecules are incapable of autophosphorylation and cannot phosphorylate known endogenous substrates. Although the role of FAK as a regulatory kinase is widely accepted, it is intriguing that FAK molecules lacking the kinase domain are still functional in some cell-signaling pathways. This has led to the proposal that FAK may predominantly perform a structural role by acting as a scaffold for diverse constituents of FAs. However, these same data have been interpreted

46


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Figure 2 Signaling through focal adhesion kinase (FAK). Association of FAK with integrins activates signaling pathways that regulate cell proliferation, apoptosis, spreading, and migration. Reproduced from Laboratory of Molecular Biophysics http://biop.ox.ac.uk, with permission.

to imply that other kinases have the ability to duplicate the kinase functions of FAK. Src tyrosine kinase The c-Src tyrosine kinase performs a pivotal role in the regulation of the function of FAs and the cytoskeleton. In transformed cells, vSrc is localized at three distinct locations: perinuclear, the actin cytoskeleton, and focal adhesions. Studies utilizing vSrc mutants in LA 29 rat fibroblasts, which express a temperature-sensitive (ts) v-src mutant, with D1025 rat fibroblasts, transfected with a ts mutant of v-fps, have demonstrated that at restrictive temperatures vSrc is inactive and localizes to the perinuclear regions where it is intimately associated with the microtubular cytoskeleton. In contrast, at more permissive temperatures v-Src localizes to focal adhesions at the cell periphery where it interacts with RhoA-induced actin fibers. An interesting observation is that targeting of v-Src is independent of its own tyrosine kinase function; rather it depends on its SH3 domain. However, this localization is dependent on the binding of the p85 regulatory subunit of phosphatidylinositol kinase (P13 K) to the v-Src SH3 domain as well as on P13 K kinase function. Although not completely analogous to v-Src, overexpressed c-Src in fibroblasts localizes to a perinuclear location and associates with microtubules and organelles such as endosomes and the trans-Golgi network. The localization of c-Src is dependent on

GTPse activity, which directs c-Src to diverse cellular locations. The extrapolation of the function of c-Src from the observations of v-Src is limited by the observation that c-Src does not associate with p85 of p13 K. Thus, the role of c-Src still needs to be fully defined.

Focal Adhesion Molecules in Respiratory Disease A characterization of the role of focal adhesion kinases in normal cellular function and in disease is an area of active investigation. Focal adhesion molecules such as integrins and FAK play key roles in the regulation of cell–cell communications, structure, and remodeling during development of the lung and other tissues. Perturbations of the tightly controlled functions of focal adhesions have been shown to affect tumorigenesis and metastasis but the role of focal adhesions in diseases of the lung and other organs is less well defined. However, studies have begun to reveal a role for focal adhesions in cellular function and disease states. For example, focal adhesions have been shown to be important in lymphocyte homing to sites of inflammation as b7 integrins have been shown to be crucial in the localization of lymphocytes to sites of inflammation in the gastrointestinal tract. Focal adhesions are also important

ADHESION, CELL–MATRIX / Integrins 47

in hemostasis as GII3b integrins are crucial in platelet aggregation and their blockade has given rise to a potent class of antagonists that are used to prevent platelet aggregation following interventional procedures in cardiology. Also, FAK is required for blood vessel morphogenesis and migration of vascular smooth muscle cells. FAK has been shown to be required for microtubule organization, nuclear movement, and neuronal migration during brain development and to be a negative regulator of axonal branching and synapse formation. In the acute respiratory distress syndrome, perturbation of the cell surface barrier is thought to be mediated through impaired focal adhesion function thus leading to increased permeability. Additionally, TGF-b-induced activation of FAK has been shown to mediate the TGF-b-mediated suppression of apoptosis in lung fibroblasts, which likely plays a role in the pathogenesis of idiopathic pulmonary fibrosis. We among others have shown that FAK is cleaved during apoptosis and a recent study revealed that FAK interacts with p53 and inhibits p53-mediated apoptosis. Further studies will elucidate the precise role for focal adhesions in normal cellular function and in disease. See also: Adhesion, Cell–Cell: Vascular ; Epithelial. Adhesion, Cell–Matrix: Integrins. Apoptosis. Cell Cycle and Cell-Cycle Checkpoints. Cytoskeletal Proteins. Endothelial Cells and Endothelium. Epithelial Cells: Type I Cells; Type II Cells. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils. Platelets.

Further Reading Calderwood DA (2004) Integrin activation. Journal of Cell Science 117(5): 657–666. Calderwood DA (2004) Talin controls integrin activation. Biochemical Society Transactions 32(3): 434–437. (Review: PMID: 15157154: PubMed – indexed for MEDLINE.) Calvete JJ (2004) Structures of integrin domains and concerted conformational changes in the bidirectional signaling mechanism of alphaIIbbeta3. Experimental Biology and Medicine (Maywood) 229(8): 732–744. Diagne I, Hall SM, Kogaki S, Kielty CM, and Haworth SG (2003) Paxillin-associated focal adhesion involvement in perinatal pulmonary arterial remodelling. Matrix Biology 22(2): 193–205. Frame MC, Fincham VJ, Carragher NO, and Wyke JA (2002) vSrc’s hold over actin and cell adhesions. Nature Reviews: Molecular Cell Biology 3(4): 233–245. Hynes RD (2002) Integrins: bi-directional, allosteric signaling machines. Cell 110: 673–687. Kanda S, Miyata Y, and Kanetake H (2004) Role of focal adhesion formation in migration and morphogenesis of endothelial cells. Cellular Signalling 16(11): 1273–1281.

Mould AP and Humphries MJ (2004) Cell biology: adhesion articulated. Nature 432(7013): 27–28. Petit V and Thiery JP (2000) Focal adhesions: structure and dynamics. Biology of the Cell 92(7): 477–494. Schlaepfer DD, Mitra SK, and Ilic D (2004) Control of motile and invasive cell phenotypes by focal adhesion kinase. Biochimica et Biophysica Acta 1692(2–3): 77–102. Schoenwaelder SM and Burridge K (1999) Bidirectional signaling between the cytoskeleton and integrins. Current Opinion in Cell Biology 11(2): 274–286. Takagi J, Petre BM, Walz T, and Springer TA (2002) Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell 110(5): 599–611. Vinogradova O, Velyvis A, Velyviene A, et al. (2002) A structural mechanism of integrin alpha(IIb)beta(3) ‘‘inside-out’’ activation as regulated by its cytoplasmic face. Cell 110(5): 587–597. Wen LP, Fahrni JA, Troie S, et al. (1997) Cleavage of focal adhesion kinase by caspases during apoptosis. Journal of Biological Chemistry 272(41): 26056–26061. Wozniak MA, Modzelewska K, Kwong L, and Keely PJ (2004) Focal adhesion regulation of cell behavior. Biochimica et Biophysica Acta 1692(2–3): 103–119.

Integrins L M Schnapp, University of Washington, Seattle, WA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Integrins are a large family of cell adhesion receptors that mediate cell–cell and cell–extracellular matrix adhesion. Integrin members are expressed on virtually every cell and provide essential links between the extracellular environment and intracellular signaling pathways. In doing so, integrins play a role in most essential cell behaviors, including cell survival, apoptosis, differentiation, and transcriptional regulation. Phenotypes of integrin knockout mice illustrate important roles of integrins in lung development, pulmonary fibrosis, and emphysema. Integrins contribute to immune response in the lung by mediating trafficking of leukocytes to areas of inflammation. In addition, they function as receptors for a variety of pulmonary pathogens. Because of their central role in different disease processes, therapeutic agents are being developed to target integrin–ligand interactions.

Integrins are a major family of cell adhesion receptors. The term integrin was first used in the 1980s to describe cell surface receptors that ‘integrated’ the cytoskeleton of one cell with that of another cell or extracellular matrix protein. Since the first description, it is apparent that integrins are essential for many important biological processes including, but not limited to, development, cell proliferation, cell survival, migration, immune function, and wound healing. Integrins are found in all metazoa, vertebrates, and invertebrates. No homologs have been

48

ADHESION, CELL–MATRIX / Integrins

Platelet integrin

1

IIb

Leukocyte integrins E

L

3

3

M X

2

2

4 5 6

7

D

4

1 V

7

8

5 6

8 9 10 11

Figure 1 Integrin family. a/b associations of mammalian subunits are illustrated. Subunits in red recognize the RGD motif, subunits in blue are collagen receptors, and subunits in purple are laminin receptors. Some integrins have restricted cell type expression (i.e., leukocyte integrins and platelet integrin).

Table 1 Nomenclature of b2 leukocyte integrins Leukocyte integrin

Names

aLb2

Lymphocyte function-associated antigen (LFA)-1, CD11a/CD18 Macrophage adhesion molecule (Mac)-1, CD11b/CD18, CR3 p150,95, CD11c/CD18 CD11d/CD18

aMb2 aXb2 aDb2

identified to date in prokaryotes, plants, or fungi. The number of integrin members roughly correlates with the complexity of the organisms. Currently, there are 18 known a subunits and 8 known b subunits, which combine to form 24 different heterodimers in mammals (Figure 1). Different a/b combinations alter the ligand specificity of the receptor. Additional complexity arises due to alternatively spliced variants of several integrin subunits. Integrins can be roughly divided into subfamilies according to their shared b subunit, although a subunits can sometimes bind to more than one b subunit, thus blurring the original subfamilies. The receptors are generally named according to the subunit composition (i.e., a8b1). However, older nomenclature still exists (Table 1). For example, the b1 subfamily of integrins was originally termed VLA (very late activation) antigens because they were originally detected on T cells ‘very late after’ mitogen stimulation (i.e., a4b1 is also referred to as VLA-4). All mammalian cells express some member of the integrin family. In addition, some integrins have cell-restricted expression, most notably the

b2-containing integrins, which are found exclusively on leukocytes, and the integrin aIIbb3 (GPIIb/IIIa), found exclusively on megokaryocytes and platelets.

Structure Integrins are heterodimeric glycoproteins that are composed of two noncovalently associated subunits, a and b (Figure 2). Each a and b subunit contains a large extracellular domain, a single pass transmembrane domain, and a short cytoplasmic domain (20–60 amino acids, except for the b4 subunit cytoplasmic domain, which contains 1000 amino acids). The cytoplasmic domain interacts with the cytoskeleton and/or cytoplasmic-signaling molecules. A subset of a subunits contains an inserted (I) domain that is homologous to the A domain of von Willebrand factor and is important in ligand binding. The extracellular domains of both subunits form the ligandbinding site. Divalent cations such as Ca2 þ or Mg2 þ are required for functional activity. Divalent cations can promote or suppress ligand binding, change ligand specificity, or stabilize integrin structure. In general, Mg2 þ promotes cell adhesion, Ca2 þ decreases it, and Mn2 þ increases ligand affinity. Insight into the structure of the extracellular domain was obtained from X-ray crystal structure analysis. The extracellular domain resembles a globular head atop two legs, one from each subunit (Figure 2). The globular heads form the main contact between the subunits and the ligand. The head of the a subunit is composed of a seven-bladed b-propeller


A domain

-propeller Hybrid domain PSI domain Thigh domain

EGF repeats

Calf domain

Calf domain Transmembrane domains

domain, followed by an immunoglobulin (Ig)-like ‘thigh’ domain and two ‘calf’ domains. The b subunit head has a bA domain (similar to the I domain of a subunits) followed by an Ig-like hybrid domain, PSI domain (plexin, semaphorin, and integrin), and four EGF-like domains. Recently, the crystal structure of the extracellular domain of avb3 bound to a protypical Arg–Gly–Asp (RGD) ligand was determined. The RGD peptide is inserted in a crevice between the heads of the a subunit and the b subunit. Amino acid residues previously predicted to be important in ligand binding by mutational analysis were confirmed to participate in ligand binding. Furthermore, occupation by ligand resulted in a conformational change of the extracellular domain that is likely to be transmitted to the cytoplasmic domain and contribute to outside-in signaling.

Regulation of Activity

Cytoplasmic domains Figure 2 Schematic representation of integrin based on structure of avb3. Ligand-binding pocket is formed by the b-propeller and bA domain of a and b subunits, respectively, and is indicated by the arrow.

Integrins can exist in different conformational states, and many integrins exist at rest in a low-affinity state (Figure 3). However, in response to agonists, signaling pathways are activated that cause a conformational change in the integrins to allow high-affinity binding (‘affinity modulation’). This inside-out signaling refers to the rapid reversible change in integrin affinity in response to external agonists. The classic

ECM

Extracellular

Intracellular

Actin cytoskeleton

Inactive low affinity

Conformational change/ high affinity

Clustering/ increased avidity

Figure 3 Schematic representation of integrin affinity and avidity modulation in response to inside-out signaling. In the resting (inactive) state, interaction of cytoplasmic tails via salt bridge (black bar) inhibits interaction with cytoskeletal and signaling partners. After agonist-induced signaling, the salt bridge is broken by cytoplasmic proteins such as talin. This results in a conformational change in the extracellular domain, allowing high-affinity binding to ligand. Diffusion of integrin within the plasma membrane leads to clustering and increased avidity.

50


example of inside-out signaling occurs in platelets. On a resting platelet, the integrin aIIbb3 exists in a low-affinity state and is unable to bind soluble fibrinogen. After platelet activation (by agonists such as thrombin, collagen, ADP, or epinephrine), aIIbb3 undergoes a conformational change to a high-affinity state and binds soluble fibrinogen, causing platelet aggregation. Regulation of integrin activation is also critical for leukocyte trafficking. Leukocytes circulate in a nonadhesive state and require activation (i.e., by cytokines or chemokines) to allow targeted integrinmediated adhesion to the vascular endothelial cells and subsequent transmigration. Activation of b2 integrins is also critical for phagocytosis, antigen presentation, and T-cell killer function. Integrin cytoplasmic domains regulate the activation state of the integrin. Truncation of the a or b integrin cytoplasmic domain results in a constitutively active receptor. In the resting (inactive) state, the cytoplasmic tails of a and b subunits are thought to be in close proximity to each other, connected by a salt bridge formed between several highly conserved residues within the cytoplasmic domain. When the salt bridge is broken, the integrin becomes activated. Several cytoplasmic proteins can bind to cytoplasmic tails and activate integrins, including talin, calcium and integrin binding protein (CIB), and b3 endonexin. Ligand binding can also be strengthened by increased lateral mobility of the integrin within the plasma membrane. This results in clustering of the integrin and increased ligand binding, referred to as avidity modulation.

as mechanotransducers. Integrins also coordinate signaling with many receptor tyrosine kinases and therefore determine cellular responses to soluble growth factors and cytokines. In addition, integrins can associate with other transmembrane or membrane-associated proteins (e.g., PDGF-bR, uPAR, and TM4SF) to influence cell signaling.

Integrin Ligands: More Than Just Glue Originally described as receptors that mediated adhesion to extracellular matrix proteins such as fibronectin, collagen, and laminin, it is increasingly evident the integrins play a broader role in homeostasis, and their ligand repertoire is much larger than originally appreciated. Many integrins recognize the tripeptide sequence RGD found in many matrix proteins, including fibronectin, vitronectin, and fibrinogen, and other extracellular proteins, including the latency-associated peptide (LAP)-TGF-b1. Integrins bind to a variety of counterreceptors (i.e., VCAM, ICAM, and E-cadherin), illustrating the importance of integrins in cell–cell adhesion. An increasing number of different substrates have been identified as biologically relevant ligands for integrins, including VEGF, matrix metalloproteinase (MMP)-2, sperm fertilin, and the endogenous angiogenesis inhibitors angiostatin, endostatin, and tumstatin. Identification of novel ligands has expanded the spectrum of biological processes that integrins regulate.

Integrins in Respiratory Diseases Biological Function Outside-in signaling occurs after ligand binding to integrins. The cytoplasmic tails do not contain any intrinsic catalytic activity but act as organizing centers and scaffolding for other signaling components and cytoskeletal proteins. Most integrins activate focal adhesion kinase, which subsequently activates a number of classic signaling pathways, such as mitogen-activated protein kinase, PI3-kinase, protein kinase C, and c-JUN kinase. These pathways regulate diverse cell behaviors, including survival, proliferation, migration, and transcriptional activity. Integrins are important links to the actin cytoskeleton through binding of the cytoplasmic tails to actinbinding proteins such as talin, filamin, integrin-linked kinase, and a-actinin. Integrin-mediated adhesion can activate the Rho family of small GTPases that are involved in actin cytoskeleton rearrangement. Integrin activation of RhoA, Rac-1, or CDC42 results in the formation of stress fibers, lamellopodia, and filopodia, respectively. Thus, integrins are situated to act

The nonredundant, important function of the different integrins is illustrated by the distinct phenotypes of the knockouts. In addition, several human genetic diseases illustrate the clinical importance of the integrin family. Leukocyte adhesion deficiency (LAD1) is a rare autosomal dominant disorder in which patients suffer from recurrent life-threatening bacterial infections, despite normal numbers of circulating leukocytes. LAD-1 is caused by lack of functional b2 integrins on leukocytes, which results in the inability of leukocytes to migrate to tissue sites of inflammation and infection. Glanzmann’s thrombasthenia is an autosomal recessive disorder in which patients suffer from severe mucocutaneous bleeding due to a lack of functional aIIbb3 on platelets. Mutations in a4b6, normally expressed on the basal surface of keratinocytes, cause some cases of the skin blistering disease epidermolysis bullosa. Mutation of the a7 integrin subunit, expressed primarily on skeletal and cardiac muscle, is responsible for some cases of congenital myopathy.


Because of their diverse roles in many processes, integrins are attractive targets in many diseases. Drugs aimed at directly blocking receptor–ligand interactions have been developed and include monoclonal antibodies and small molecule ligand mimetics (based on RGD). The platelet integrin aIIbb3 antibody, abciximab, was the first anti-integrin drug to be approved for clinical use. Several agents targeting integrins are being tested for use in asthma, cancer, and inflammatory diseases. Integrins in Lung Development

Early studies using in vitro models of lung branching showed that administration of RGD peptides decreased branching morphogenesis. These studies suggested a critical role of cell–matrix interactions, mediated through integrins in lung development. Further studies on knockout mice have confirmed the importance of several integrins in lung development. Mice lacking the a3 integrin subunit exhibit decreased branching morphogenesis and immature bronchiolar epithelium, in addition to abnormal kidney development. The underlying epithelial basement membrane was severely disrupted, demonstrating a critical role of a3 in basement membrane assembly. Mice lacking the a9 subunit die soon after birth due to bilateral chylothorax, suggesting abnormalities in lymphatic development. Recently, VEGF-C and VEGF-D, key effector proteins in lymphatic development, were identified as novel ligands for a9b1. a9b1–VEGF interactions mediated endothelial cell adhesion and migration, suggesting a mechanism for phenotype found in a9-deficient mice. The absence of lung phenotype in other integrin knockouts does not necessarily imply the absence of a role in lung development. In mice, lung development begins at approximately E12.5 and continues until approximately 4 weeks after birth. Knockouts of several potentially important integrins for lung development were embryonic lethal before lung development was completed (i.e., b1, a5); thus, determining their contribution to lung development is difficult. In addition, upregulation of other integrins or related proteins may compensate for the loss of an integrin subunit during development. Integrins and Fibrosis

The integrin avb6 is normally expressed at low levels on airway epithelium. Mice that lack the integrin subunit b6 develop exaggerated inflammation in the lung at baseline. Interestingly, following bleomycin administration, which typically results in lung injury and fibrosis, the b6-null mice were protected from development of pulmonary edema and fibrosis. This

was due to a defect in the activation of TGF-b1, an important mediator of pulmonary injury and fibrosis. TGF-b1 is secreted in a latent form, complexed with LAP, and is unable to signal through its receptor. LAP contains an RGD site that binds to avb6 and results in activation of TGF-b1. The activation of TGF-b1 by avb6 requires contact between avb6-expressing cells (i.e., epithelial cells) and TGF-b receptor-expressing cells (i.e., macrophages); it also requires an intact integrin cytoplasmic tail. Like avb6, avb8 also binds to the RGD sequence in LAP–TGF-b1 and activates TGF-b1. However, in contrast to avb6, avb8 activation requires metalloproteinase activity and results in free active TGF-b. Thus, integrin-mediated regulation of TGF-b activity can occur by several distinct mechanisms. Another interesting finding in the b6-null mice is the development of emphysema over time. Backcrossing the b6-null mice onto a MMP-12-null background eliminates the development of emphysema, suggesting that the development of emphysema is due to increased expression of MMP-12, a macrophage elastase. The findings suggest a model wherein active TGF-b1 normally suppresses MMP-12 expression by macrophages. In the absence of avb6-mediated TGF-b activation, MMP-12 expression is no longer suppressed and ultimately results in a matrixdegrading phenotype in the lung. Integrins and Asthma

In asthma, integrins, including aLb2 and a4b1, contribute to trafficking of eosinophils and other inflammatory cells to the lung during an asthma exacerbation. Therefore, integrins are attractive targets for novel anti-inflammatory therapies in asthma. Efalizumab, a humanized anti-aL monoclonal antibody, inhibits leukocyte migration to areas of inflammation and is approved for use in moderate to severe psoriasis. Efalizumab was tested in patients with mild asthma. Despite a significant decrease in activated eosinophils in the lung, there was no significant difference in pulmonary function after allergen challenge. There has been much interest in targeting the a4 integrin subunit in asthma and other chronic inflammatory diseases, such as Crohn’s disease, rheumatoid arthritis, and multiple sclerosis. In multiple sclerosis, treatment with the monoclonal a4 antibody natalizumab resulted in a significant reduction in relapses, leading to Food and Drug Administration approval of natalizumab. However, since approval, several unexpected cases of progressive multifocal leukoencelphalopathy (PML) have been reported in patients receiving natalizumab. PML is a rare, fatal demyelinating disease caused

52


by reactivation of human polyoma virus JC. The contribution of natalizumab to the development of PML is not entirely clear, but it likely relates to preventing migration of immune cells that normally repress latent virus into the central nervous system. These findings have raised serious concerns regarding the safety of a4 antagonists and put into question any future work targeting the a4 subunit. Integrins and Acute Lung Injury

The general paradigm for leukocyte trafficking occurs in several steps: 1. selectin-mediated rolling of leukocytes on endothelium, 2. activation of leukocyte b2 integrins by chemoattractants, 3. integrin-mediated firm adhesion of leukocytes to endothelium, and 4. integrin-mediated transmigration of leukocytes into the interstitium. In the lung, leukocyte trafficking differs from the general model in several aspects: the classical selectinmediated rolling does not occur; transmigration of leukocytes occurs at pulmonary capillaries, not postcapillary venules; and transmigration of leukocytes into interstitium and alveoli may follow a b2-dependent or b2-independent pathway. In the lung, involvement of b2 integrins for leukocyte migration depends on the stimulus. For example, leukocyte trafficking in response to Escherichia coli, Pseudomonas aeruginosa, lipopolysaccharide (LPS), and IL-1 is dependent on b2 integrins, whereas leukocyte trafficking in response to Streptococcus pneumoniae, group B streptococcus, Staphylococcus aureus, and hydrochloric acid is independent of b2 integrins. Integrins and Pathogens

Many pathogens utilize host integrins to facilitate cell attachment and infection (Table 2). Many viruses have RGD motifs in capsid proteins, which facilitates attachment and internalization of organisms. Hantavirus Table 2 Virus–integrin interactions Virus

Integrin receptor

Hantavirus Echovirus Rotavirus Papillomavirus Coxsackievirus Foot and mouth disease virus Adenovirus HHV-8 (KSHV)

avb3, aIIbb3 a2b1 a2b1, axb2, a4b1 a6b1 avb3, avb6 avb3, avb6 avb3, avb5, avb1 a3b1

causes severe pulmonary disease and thrombocytopenia and infects pulmonary epithelial cells and platelets. Pathogenic strains of hantaviruses attach to cells via b3-containing integrins. Another pathogen, foot and mouth disease virus (FMDV), causes a devastating disease of cattle. The primary route of infection by FMDV is through the epithelial cells and associated lymphoid tissues in the upper respiratory tract, followed by dissemination to other epithelial tissues. The outer capsid of FMDV contains a highly conserved RGD tripeptide motif on an exposed loop that interacts with av-containing integrins and facilitates viral attachment and infection of cells. Adenovirus is important both for the diseases it causes in humans, including respiratory infections, and because replicationdefective variants of adenovirus are being used as vectors for gene therapy. Adenovirus contains a conserved RGD sequence within a highly variable region in the penton base protein. In contrast to the viruses just mentioned, adenoviruses do not use integrins for initial attachment. Instead, adenovirus attaches to cells through a common coxsackievirus adenovirus receptor and then the RGD site interacts with av-containing integrins to facilitate internalization. Likewise, the causative virus of Kaposi’s sarcoma, human herpes virus 8 (HHV-8), interacts with a3b1 on target cells via RGD sequence on an outer envelope to facilitate viral entry and uptake. Thus, viruses and other pathogens have exploited integrins for their advantage. Integrins and Malignancy

Most adherent cells are anchorage dependent; that is, they require integrin-mediated signaling to stimulate cell-cycle progression, promote cell survival, and respond to soluble growth factors such as EGF and PDGF. Loss of integrin-mediated signaling causes a subtype of apoptosis, termed ‘anoikoisis’ from the Greek word meaning ‘homelessness’. Following malignant transformation, the requirement of integrin ligation is often bypassed by mutations in oncogenes or tumor suppressor genes, thus allowing anchorage-independent growth. However, integrin signaling still contributes to tumorigenesis and tumor progression. Depending on the integrin, cell type, and signaling pathway, activated integrins may facilitate tumor growth, metastasis, and attachment at distant sites or suppress tumor activity. In general, tumor cells, either through direct signaling mechanisms or through selection process, increase expression of integrins that promote cell proliferation, survival, and migration and decrease expression of integrins that promote a quiescent, differentiated state. Although it is difficult to make generalizations, a2b1 and a3b1 expression is associated with a tumor

ADRENERGIC RECEPTORS 53

suppressor phenotype, whereas avb3, avb6, and a6b4 expression is associated with tumor progression. The development of blood supply is essential for tumor survival and growth, and it is a potential target of antitumor agents. The endothelial integrin avb3 is upregulated in tumor neovasculature. In vitro and animal studies showed that avb3 antagonists (antibodies or peptides) caused tumor regression, inhibited neovascularization, and induced apoptosis of endothelial cells. Several anti-avb3 agents show promise as antiangiogenic therapy for cancer treatment. See also: Adhesion, Cell–Cell: Vascular; Epithelial. Adhesion, Cell–Matrix: Focal Contacts and Signaling. Asthma: Overview. CD11/18. Epithelial Cells: Type I Cells; Type II Cells. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Fibroblasts. Matrix Metalloproteinases.

Further Reading Calderwood DA (2004) Integrin activation. Journal of Cell Science 117: 657. Doerschuk CM (2000) Leukocyte trafficking in alveoli and airway passages. Respiration Research 1: 136. Guo W and Giancotti FG (2004) Integrin signalling during tumour progression. Nature Reviews: Molecular Cell Biology 5: 816. Humphries MJ, McEwan PA, Barton SJ, et al. (2003) Integrin structure: heady advances in ligand binding, but activation still makes the knees wobble. Trends in Biochemical Sciences 28: 313. Hynes R (2002) Integrins. Bidirectional, allosteric signaling machines. Cell 110: 673. Plow EF, Haas TA, Zhang L, Loftus J, and Smith JW (2000) Ligand binding to integrins. Journal of Biological Chemistry 275: 21785. Schwartz MA (2001) Integrin signaling revisited. Trends in Cell Biology 11: 466. Sheppard D (2000) In vivo functions of integrins: lessons from null mutations in mice. Matrix Biology 19: 203. Sheppard D (2003) Functions of pulmonary epithelial integrins: from development to disease. Physiological Reviews 83: 673. Sheppard D (2004) Roles of alphav integrins in vascular biology and pulmonary pathology. Current Opinion in Cell Biology 16: 552.

ADRENERGIC RECEPTORS A E Tattersfield, Nottingham University, Nottingham, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Adrenergic receptors, which includes a, b and dopamine receptors, belong to the large family of G-protein-coupled, seven transmembrane domain receptors. b-Adrenoceptors are the best characterized and predominant adrenoceptors in the lung, with both b1 and b2 receptors being widely distributed. b2Adrenoceptors are an important therapeutic target and their polymorphisms may influence the response to b2 agonist treatment. Their numbers and functions are regulated by b-agonist stimulation and by drugs, such as corticosteroids, and cytokines. a-Adrenoceptors are found on vascular smooth muscle, presynaptic nerve endings, airways, and submucus glands, and they may help to condition inspired air. There is evidence for D1 dopamine expression on alveolar cells, where they help to clear lung edema, and for D2 receptors on sensory nerves in the lung, where they may modulate neurogenic inflammation and reflexmediated symptoms.

Introduction Adrenergic receptors include a, b, and dopamine receptors; they are part of the large family of G-proteincoupled, seven transmembrane domain receptors. b-Adrenoceptors are the best characterized and predominant adrenoceptors in the lung; the role of a-adrenoceptors and dopamine receptors is less well established.

b-Adrenoceptors b1 and b2 receptors were initially identified on the basis of tissue selectivity to a range of agonists. Both b-adrenoceptors were subsequently characterized and cloned, as was a third (b3) receptor and there are putative claims for a b4 receptor in the heart. There is 54% homology between b1 and b2 receptors. Structure

The b2-adrenoceptor contains 413 amino acids with seven transmembrane-spanning domains, three intraand three extracellular loops, an intracellular C-terminus, and an extracellular N-terminus (Figure 1). The third intracellular loop is important for linking to G-proteins. Site-directed mutagenesis studies show that b-agonists bind to residues on the hydrophobic region within the cell membrane between the third and sixth transmembrane domains as shown in Figure 1. The salmeterol molecule anchors to an exosite on the fourth transmembrane domain, allowing the molecule to stimulate the receptor repetitively. The intracellular third loop and tail of the b-adrenoceptor contains phosphorylation sites that are involved in receptor downregulation. Polymorphisms

Nine single-base mutations or polymorphisms in the coding region of the b2-adrenoceptor gene on

54

ADRENERGIC RECEPTORS

Y

Y

2-Agonist

NH2

Cell membrane I

II

III

IV

V

VI VII P

s

P

cAMP PKA PKC PTK

P = Phosphorylation sites P P

H COO

P

+

arrestin

P ARK (GRK)

Inhibits receptor function

Figure 1 Schematic representation of the human beta 2-adrenoceptor showing the 7 trans membrane domains and the site on the third, fifth, and sixth domains that are required for beta-agonist binding. P marks the phosphorylation sites concerned with receptor ancapling. The blue circles indicate the amino acids that are essential for b-agonist binding. Adapted from ‘‘b-adrenergic receptors and their regulation’’ by PJ Barnes.

chromosome 5Q have been identified, though only four of these cause amino acid substitutions – at codons 16, 27, 34, and 167. The two most common polymorphisms are at codon 16, where glycine is substituted for arginine, and codon 27 where glutamic acid is substituted for glutamine. Both polymorphisms are common, with 37% and 23% of subjects being homozygous for the gly 16 and glu 27 polymorphisms, respectively, in a large general population study. There is linkage disequilibrium between polymorphisms at codons 16 and 27, that is, they are more likely to occur together, and this can cause difficulties when trying to determine the functional effects of a specific polymorphism. b-Adrenoceptor Activation

When a b-agonist binds to the b-adrenoceptor/Gs complex guanosine diphosphate (GDP) is released, allowing guanosine triphosphate (GTP) to bind and activate the a-subunit of Gs, which then dissociates from the b-receptor and is free to stimulate adenylate cyclase. Adenylate cyclase catalyzes the conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (AMP), which in turn activates protein kinase A. Cyclic AMP has a range of actions that promote smooth muscle relaxation in airways and other activities such as inhibition of mediator release in inflammatory cells (see Figure 2 and Table 1). Most of the effects of b-agonists are mediated via cyclic AMP and activation of protein kinase A, but some, such as the opening of maxi K þ channels, may, in part at least, be due to direct activation by the Gs a-subunit.

Protein kinase A is also able to phosphorylate and activate a transcription factor, CREB or cyclic AMP response element binding protein. By binding to the cyclic AMP response element (CRE) on the promoter region of target genes, CREB is able to cause transcription of various genes including the b-receptor gene. b2-Receptor expression may therefore be increased transiently by b-agonist stimulation. CREB also interacts with other transcription factors within the nucleus including some proinflammatory cytokines such as activating protein (AP-1) and nuclear factor kappa B (NF-kB). b-Adrenoceptor Regulation and Activity

Until recently, it was thought that b-adrenoceptors existed in high- or low-affinity states, the high-affinity state being increased in the presence of b-agonists and associated with an increased binding affinity for b-agonists. The affinity for b-adrenoceptor antagonists did not differ between high- and low-affinity states. b-Adrenoceptor activation with this model only occurred when an agonist was present. However, recent work suggests that receptors may have constitutive activity, that is, activity when no agonist is present. Some b-adrenoceptor antagonists are now thought to act by stabilizing this constitutive activity rather than as a competitive antagonist and hence they can be described as inverse agonists. The number of b-adrenoceptors in the airways and other tissues depends on the rates at which they are synthesized and degraded; their function also depends on the extent to which they are coupled to the catalytic unit. Both receptor numbers and functions

ADRENERGIC RECEPTORS 55

K+ -Agonist

Gs

-Adrenoceptor

Gs

ATP

Phosphorylation of myosin light chain kinase

AC

Inhibition of PI hydrolysis

cAMP

Cell membrane

Inactive PKA

Active PKA

Ca2+ extrusion and sequestration Stimulation of Na+/K+ ATPase Membrane hyperpolarization

Smooth muscle relaxation

Inhibition of mediator release

Activation of Ca2+ gated potassium channels Figure 2 Schematic representation of the signaling pathway following stimulation of the b-receptor; this causes coupling of the receptor to the Gs protein and stimulation of adenylate cyclase (AC). This in turn causes increased accumulation of cyclic-AMP (cAMP) and protein kinase A (PKA) and a fall in intracellular calcium. The most important cellular effects for asthma are smooth muscle relaxation and inhibition of mediator release from inflammatory cells.

Table 1 Main actions of b-adrenergic agonists at different receptor subtypes

underlie this development:

Tissue

Receptor

Response

Airways

b2 mainly

Heart Blood vessels

b1/b2 b2

Uterus Metabolic

b2 b2/b3

Muscle

b2

Bronchodilatation, reduction in mediator release from mast cells, increased mucus production, increased ciliary activity Tachycardia, inotropic action Dilatation, fall in blood pressure, compensatory reflex increase in heart rate Relaxation Increase in glucose, insulin, lactate, pyruvate, nonesterified fatty acids, glycerol, and ketone bodies; decrease in potassium, phosphate, calcium, and magnesium Tremor

1. Receptor uncoupling. Exposure of a b-receptor to a b-agonist causes phosphorylation of sites on the 3rd intracellular loop and tail of the b-adrenoceptor, causing the receptor to uncouple from its Gs protein and catalytic unit. Receptor uncoupling occurs within minutes and is rapidly reversible. At least two kinases are involved: the cyclic AMP-dependent protein kinase A and the G-protein-dependent b-adrenergic receptor kinase (bARK); protein kinase G may also contribute. Protein kinase A is activated by fairly low concentrations of b-agonist, as seen with therapeutic doses, whereas activation of bARK requires higher agonist concentrations and the cofactor b-arrestin. bARK causes homologous b-receptor desensitization, that is, to b-agonists only, whereas protein kinase A causes heterologous desensitization, so that the response to other agonists that use the same catalytic unit, for example, forskolin, is reduced. 2. Sequestration of receptors. Following exposure to a b-agonist, b-adrenoceptors may be internalized within the cell, and sequestrated into vesicles. This can also occur within minutes and is again reversible, the receptors being recycled to the cell surface. 3. Downregulation. With longer term exposure to bagonists, however, b-receptors may be internalized and degraded and hence are not available for recycling. Polymorphisms at codon 16 and 27 have been shown to affect b-adrenoceptor downregulation

are modified by b-agonists and by other factors, of which cytokines and corticosteroids are particularly relevant to asthma.

Regulation by b-agonists With repeated or continuous exposure to a b-agonist there is a reduction in the response, a phenomenon known as desensitization or tolerance. Several mechanisms have been shown to

56

ADRENERGIC RECEPTORS

in vitro. Receptors with the gly 16 polymorphism have shown increased downregulation whilst those with the glu 27 polymorphism were protected against downregulation. 4. Reduced synthesis. b-Adrenoreceptor numbers may also be lower as a result of a reduction in receptor mRNA, following continuous exposure. Regulation by other factors 1. Corticosteroids. Corticosteroids have a number of effects in vitro that may increase or decrease the response to a b-agonist. They are able to increase b-receptor numbers by inhibiting downregulation of b-receptors, and by increasing b-receptor gene transcription. However, the activity of CREB can be inhibited by glucocorticoid transcription factors, thus providing a possible mechanism whereby glucocorticoids might reduce the efficacy of a b-agonist. 2. Cytokines. The proinflammatory cytokine interleukin 1b has been shown to attenuate b-receptor responsiveness by uncoupling the receptor from its catalytic unit; this appears to be due to induction of the inducible form of cyclooxygenase (COX 2) and release of inhibitory prostanoids. Distribution of b-Adrenoceptors

The distribution and type of b-receptor within the lung has been determined by receptor-binding studies; airway epithelium and alveoli have the highest receptor density. Some 70% of the b-adrenoceptors in the lung are of the b2-receptor subtype, the remainder being b1-receptors. Only b2-receptors are found on airway and vascular smooth muscle whereas both b1- and b2-receptors are found on submucosal glands and alveoli. b-Receptors are distributed widely throughout the body and some of the more important sites are shown in Table 1.

patients with asthma who are homozygous for arginine at codon 16.

a-Adrenoceptors Classifying a-adrenoceptors has been less straightforward than classifying b-adrenoceptors. The two main classes are the a1-adrenoceptors, classic postsynaptic excitatory receptors, and a2-adrenoceptors, which are usually, though not invariably, presynaptic, where they inhibit neuronal mediator release. There are three fully defined a1-adrenoceptor subtypes (a1A, a1B, a1D, and a putative a1L) and four subtypes of the a2-adrenoceptor (a2A, a2B, a2C, and a2D). Most of the subtypes have now been cloned. a-Adrenoceptor Activation

The postsynaptic a1-adrenoceptor is coupled to a Gq protein and produces physiological effects via activation of phospholipase C, which mobilizes intracellular calcium by increasing inositol 1, 4, 5-trisphosphate concentrations, and diacylglycerol, which activates protein kinase C. a1-Adrenoceptors also activate other pathways such as the mitogen-activated protein kinase pathway. A single base polymorphism has been described in a1A but no effect on receptor function has been identified as yet. Activation of a2 receptors usually causes inhibition of adenylate cyclase through coupling with membrane-linked pertussis toxin-sensitive Gi/o protein. Distribution

Alpha-adrenoceptors are widely distributed on neuronal and non-neuronal tissues. Within the lung, aadrenoceptors have been located predominantly in smaller airways, on serous cells in submucus glands, and on pulmonary blood vessels. a1-Adrenoceptors are thought to be present on bronchial blood vessels and a2-adrenoceptors on airway ganglia and presynaptic cholinergic nerve endings.

b-Receptor Numbers and Function in Disease

Positron emission tomography studies indicate that there are normal numbers of b-receptors in the lungs of people with mild asthma, and they may even increase in patients dying from asthma. The function of b-receptors in the lungs of patients with asthma also appears to be normal, although it may be affected by previous b-agonist exposure and receptor polymorphisms. There is no evidence to suggest that the presence of asthma is associated with b2-receptor polymorphisms or haplotypes. However, there is some evidence that the response to b2-agonists is reduced in

Function

The role of a-adrenoceptors in the lung is not entirely clear and work is hampered by lack of specificity of many of the ligands used to investigate a-adrenoceptor subtypes. a1-Adrenoceptors classically cause smooth muscle contraction and could therefore help control bronchial blood flow and conditioning of inspired air. Other possible roles include inhibition of histamine release from mast cells and acetylcholine release from cholinergic nerve endings, and modulating the content of airway secretions through serous cell stimulation.

ADRENERGIC RECEPTORS 57 Function in Disease

The possibility that a-adrenergic mechanisms might contribute to asthma was explored several years ago, particularly when the density of a-receptors was found to be higher in patients with asthma. a-Adrenoceptor agonists caused bronchoconstriction in some but not all studies although whether this is a specific or non-specific effect is not clear. Similarly nonselective a-adrenoceptor antagonists such as indoramin caused some degree of bronchodilatation or protected against induced bronchoconstriction in some studies, but all the drugs studied had additional pharmacological effects that could have been responsible. None of the a-adrenoceptor antagonists available caused worthwhile bronchodilatation in asthma when given regularly or by inhalation. These data and the fact that norepinephrine when infused causes bronchodilatation rather than bronchoconstriction suggest that a-adrenergic mechanisms are not making an important contribution to the bronchoconstriction seen in asthma.

Dopamine Receptors Of the five subtypes of dopamine receptors identified (D1–D5), D1 and D5 receptors have similar homology and both stimulate adenylate cyclase. D2, D3, and D4 receptors are also homologous but they inhibit adenylate cyclase. Most work relates to the D1 and D2 subtypes. Distribution and Function

Dopamine receptors are found in the central and peripheral nervous system. They have been studied in the brain predominantly and in the context of the lung there is interest in the role that central dopaminergic pathways might play in the development of nicotine addiction. Within the lung there is evidence for D1 receptors on alveoli in the rat, which, when stimulated, increase the clearance of lung edema. D2 receptor mRNA and protein are expressed in sensory ganglia in the airways and dopamine receptor activation has been shown to inhibit depolarization of the vagus in animals and man, and neuropeptide release from nerve endings. D2 dopamine receptors may therefore have a role in modulating neurogenic inflammation and reflex-mediated symptoms such as cough. D2 receptor agonists have reduced cough, mucus production, and tachypnea in animal models and there is interest in whether they might reduce symptoms such as cough and

breathlessness in patients with chronic obstructive pulmonary disease. Dopamine in low doses stimulates dopamine receptors but as the dose is increased it stimulates b1adrenoceptors followed by a1- and a1-adrenoceptors. See also: Asthma: Overview. Bronchodilators: Beta Agonists. G-Protein-Coupled Receptors.

Further Reading Barnard ML, Ridge KM, Saldias F, et al. (1999) Stimulation of the dopamine 1 receptor increases lung edema clearance. American Journal of Respiratory and Critical Care Medicine 160: 982– 986. Barnes PJ (1986) Neural control of human airways in health and disease. American Review of Respiratory Diseases 134: 1289– 1314. Barnes PJ (1995) Beta-adrenergic receptors and their regulation. American Journal of Respiratory and Critical Care Medicine 152: 838–860. Birrell MA, Crispino N, Hele DJ, et al. (2002) Effect of dopamine receptor agonists on sensory nerve activity: possible therapeutic targets for the treatment of asthma and COPD. British Journal of Pharmacology 136: 620–628. Carswell H and Nahorski SR (1983) b-Adrenoceptor heterogeneity in guinea pig airways: comparison of functional and receptor labelling studies. British Journal of Pharmacology 79: 965–971. Goidie RG, Papidimitriou SM, Paterson SW, et al. (1986) Autoradiographic localisation of b-adrenoceptors in pig lung using [125I]-iodocyanopindolol. British Journal of Pharmacology 88: 621–628. Green SA, Spasoff AP, Coleman RA, Johnson M, and Liggett SB (1996) Sustained activation of a G protein coupled receptor via ‘anchored’ agonist binding. Molecular localization of the salmeterol exosite within the b-adrenergic receptor. Journal of Biological Chemistry 39: 24029–24035. Green SA, Turki J, Innis M, and Liggett SB (1994) Amino-terminal polymorphisms of the human b-adrenergic receptor impart distinct agonist-promoted regulatory properties. Biochemistry 33: 9414–9419. Hall IP and Tattersfield AE (1998) b-Adrenoceptor agonists. In: Barnes PJ, Rodger IW, and Thomson NC (eds.) Asthma. Basic Mechanisms and Clinical Management, 3rd edn, pp. 651–676. London: Academic Press. Koshimizu T, Tanoue A, Hirasawa A, Yamauchi J, and Tsujimoto G (2003) Recent advances in a1-adrenoceptor pharmacology. Pharmacology and Therapeutics 98: 235–244. Lands AM, Arnold A, McAuliff JP, et al. (1967) Differentiation of receptor systems activated by sympathomimetic amines. Nature 214: 597–598. Reihsaus E, Innis M, MacIntyre N, and Liggett SB (1993) Mutations in the gene encoding for the b2-adrenergic receptor in normal and asthmatic subjects. American Journal of Respiratory Cell and Molecular Biology 8: 334–339. Schwartz J, Carlsson A, Caron M, et al. (1998) The IUPHAR compendium of receptor characterisation and classification: dopamine receptors. IUPHAR Media (London) 142–151. Strader CD, Sigal IS, and Dixon AF (1989) Mapping the functional domains of the b-adrenergic receptor. American Journal of Respiratory Cell and Molecular Biology 1: 81–86.

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AEROSOLS S P Newman, Nottingham, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract The inhaled route is used to deliver drugs as aerosols for the maintenance therapy of asthma, chronic obstructive pulmonary disease, and other conditions. The deposition of aerosol particles in the respiratory tract is an important prerequisite to obtaining a good clinical effect. Generally, inhaler devices should deliver particles smaller than approximately 5 mm in diameter in order to enter the lungs. A variety of inhaler devices are available for inhalation therapy. Pressurized metered dose inhalers (pMDIs) have been widely used for 50 years, but many patients have problems using them correctly. They are currently being reformulated with ozone-friendly propellants. Breath-actuated inhalers and spacer attachments may be useful supplements to pMDIs for some patients. Dry powder inhalers (DPIs) are easier to use correctly than pMDIs, and they do not require propellants. Many pharmaceutical companies seem to be prioritizing DPIs above pMDI reformulation, and they are also preferred by many patients. Nebulizers continue to be used widely, but the limitations of jet and ultrasonic nebulizers have led to the development of novel systems, sometimes involving vibrating meshes. Finally, a new class of inhalers (soft mist inhalers) is emerging, composed of multidose devices containing liquid formulations, some of which could challenge pMDIs and DPIs in the portable inhaler market.

Inhaled Drug Delivery The pulmonary route may be used to deliver drugs for the maintenance therapy of some lung diseases, most notably asthma and chronic obstructive pulmonary disease (COPD). Drugs are also given by inhalation to treat other chest problems, including respiratory tract infections in cystic fibrosis. In addition, it is hoped that inhaled drugs intended to have a systemic action in the body (e.g., insulin) will soon be marketed. The potential benefits of the inhaled route have long been recognized, but the importance of good quality inhaler devices that deliver drugs reliably to the lungs has only been appreciated during the past 25 years.

Aerosol Properties An understanding of aerosol properties and aerosol deposition is an important prerequisite for optimizing inhalation therapy. Drugs are given by inhalation as aerosols of solid particles or liquid droplets, but for simplicity the term ‘particle’ may be used to describe both solid and liquid dispersions. The most important property of an aerosol particle is its

size, and this is best expressed as the aerodynamic diameter, which also takes into account particle density and shape. For spherical particles, aerodynamic diameter (Da) and physical diameter (Dp) are related by the formula Da ¼ DpOr, where r is the specific gravity of the material from which the particles are made. In practice, aerosol particles are seldom spherical; for instance, micronized drug particles are often highly irregular in shape. Aerosol systems found in medicine are usually heterodisperse, indicating that the particles in a particular spray or cloud have a wide range of sizes. Monodisperse aerosols, in which all the particles have approximately the same size, are not normally found in pharmaceutical products, although they can be made using specialized equipment. It is preferable to describe the mass or volume distribution of an aerosol rather than the distribution of particles by number since many small particles may contain much less drug than a few large particles. In practice, particle size spectra from inhaler devices often approximate to log-normal distributions. The mass median aerodynamic diameter (MMAD) may be used to express the average aerosol size. This diameter is such that half the aerosol mass is contained in larger particles and half in smaller particles. The spread of particle sizes may be expressed as a geometric standard deviation (GSD), a dimensionless quantity. A perfectly monodisperse aerosol has a GSD of 1. A typical pharmaceutical aerosol may contain particles ranging in size from o0.5 to 410 mm, with an MMAD of 3–4 mm and a GSD of 2.0–2.5. As explained later, deposition of aerosols depends critically on particle size. The fraction of the aerosol mass contained in particles o5 mm in diameter is usually termed the respirable fraction or fine particle fraction (FPF). These are the particles with the greatest likelihood of reaching the lungs in adults, although even smaller particles may be needed for drug therapies in small children. In adults, particles o3 mm in diameter are needed in order to deliver drugs to the alveolated regions – for instance, to deliver inhaled a1 antitrypsin to the alveoli of patients with emphysema. Particle size distributions of aerosols intended for pulmonary delivery may be quantified by several methods. The approach favored within the pharmaceutical industry is the cascade impactor, through which the aerosol is drawn by a vacuum pump, and particles of different sizes are collected on a series of stages. Each stage can be washed out with a solvent

AEROSOLS 59

so that the amount of drug associated with different size bands may be quantified by an analytical technique. Supplementary particle size data may be provided by optical methods, the best known of which is laser diffraction. This involves passing the aerosol cloud through a laser beam, and the angle of diffraction of the laser light is inversely proportional to particle size. It is important to remember that these in vitro measurements are undertaken primarily for purposes of quality control and product release, and they may not predict accurately drug delivery to the lungs in vivo.

Deposition of Pharmaceutical Aerosols Several mechanisms cause aerosol particles to deposit in the respiratory tract, but the two most important ones relating to pharmaceutical aerosols are inertial impaction and gravitational sedimentation. Inertial impaction takes place mainly in the oropharynx and at the bifurcations between major airways, when the aerosol particle has too much inertia to follow the air stream as it changes direction. The probability of inertial impaction occurring is proportional to D2a Q, where Q is the inhaled flow rate. Deposition in central lung regions may be enhanced by the effects of air turbulence, especially at fast inhaled flow rates. Gravitational sedimentation takes place mainly in smaller conducting airways and in the alveoli, when particles settle onto the airway surface under gravity either during slow steady breathing or during breath-holding. The probability of gravitational sedimentation occurring is proportional to D2a T, where T is the residence time of the particle in the airways. A third deposition mechanism (Brownian diffusion) is also important for aerosol particles o1 mm in diameter, which may be pushed in a random direction toward airway walls by collisions with gas molecules. Some particles (especially those o1 mm in diameter) are not deposited, and after inhalation they are simply exhaled. In addition to particle size, the patient’s inhalation also plays a major part in determining the site of aerosol deposition. The inhaled flow rate is particularly important, with slow inhalation usually being recommended in order to reduce impaction losses in the oropharynx. Deep inhalation and a period of breathholding help to increase gravitational sedimentation in the peripheral parts of the lungs. For most pharmaceutical aerosols, lung deposition is enhanced by a combination of aerosol particles o5 mm in diameter and a slow inhaled flow rate (20–30 l min 1). As will be explained later, there is an exception to this rule for dry powder inhalers, where faster inhalation

may preferable. Particles are filtered efficiently from the inhaled air by the nasal passages, so wherever practicable it is better to deliver an inhaled aerosol via a mouthpiece (with mouth breathing) than via a face mask (with nose breathing). The airways of the patient who inhales the aerosol particles also determine the site and extent of deposition in two major ways. First, random variations in airway geometry between different individuals will lead to random variations in the deposition pattern. Hence, for aerosols delivered from any inhaler device, considerable intersubject variability of deposition is to be expected. Second, in patients with asthma, COPD, and other obstructive conditions, the airways may be narrowed by bronchospasm, inflammation, and mucus hypersecretion so that aerosol particles may deposit preferentially in the larger airways of the lungs, with less deposition in the peripheral airways. Both electrostatic charge and humidity affect aerosol deposition in a variety of ways. The most striking effect of humidity is that dry particles composed of water-soluble materials are likely to absorb water when they enter the respiratory tract and, hence, to increase in size. The deposition of pharmaceutical aerosols may be quantified by radionuclide imaging (gamma scintigraphy, single photon emission computed tomography (SPECT), and positron emission tomography (PET)). SPECT and PET are three-dimensional imaging methods and provide information about the distribution pattern within the lungs. However, PET is relatively complex and is probably not practical for use on a regular basis. Certain pharmacokinetic methods are also useful for assessing delivery of some drugs to the lungs. For instance, the plasma or urinary concentrations of albuterol in the first 30 min after inhalation are considered to result solely from pulmonary absorption.

Pressurized Metered Dose Inhalers The pressurized metered dose inhaler (pMDI) has been the backbone of inhalation therapy for asthma for approximately 50 years, since its introduction by 3 M Riker Laboratories in 1956. Patients and physicians recognized the convenience of the pMDI, which contains 100–200 doses in a small portable device that is immediately ready for use (Figure 1). The pMDI consists of an aluminum can mounted in a plastic actuator. Individual doses (25–100 ml) are delivered as a spray via a sophisticated metering valve. The drug is usually a micronized suspension of drug particles but may be a solution dissolved in propellants, ethanol, or another excipient as a co-solvent.

60

AEROSOLS Patient presses canister while breathing in Canister Actuator

Drug formulation in propellants

Spray plume

Metering valve Actuator nozzle

Figure 1 Design and operation of a typical pressurized metered dose inhaler.

The best known pMDI therapies include the b-agonists albuterol, terbutaline, and salmeterol and the glucocorticosteroids beclomethasone dipropionate, budesonide, and fluticasone propionate. Successful pMDI therapy is highly dependent on the patient’s inhalation technique, and patient education about their use is essential. In most pMDI products, it is necessary for the patient to press the pMDI at the same time as inhaling. Failure to do this is sometimes described as poor coordination or hand–lung dyscoordination, and it is probably the most important problem patients have with pMDIs. A second major problem using pMDIs is the so-called cold Freon effect, where the patient stops inhaling when the cold propellant spray is felt on the back of the throat. Freon is one of the trade names of chlorofluorocarbon (CFC) propellants. In order to optimize lung deposition from pMDIs, patients also need to inhale slowly and deeply and to hold the breath for several seconds. Even with perfect inhalation technique, no more than 10–20% of the dose from a CFC pMDI is deposited in the lungs, with the majority of the dose being deposited in the oropharynx. However, the lung dose will vary from product to product according to the nature of the formulation and the diameter of the actuator orifice. Until recently, all pMDIs were formulated in CFC propellants, giving the pMDI an internal pressure of approximately 300 kPa (3 atm) and a spray velocity at the nozzle exceeding 30 m s 1. However, it is possible to reduce the spray velocity by modifications to the actuator design, for instance, in the Spacehaler device (formerly known as Gentlehaler). During the past few years, the pharmaceutical industry has been forced to start reformulating pMDIs in non-CFC propellants, consisting of one of two hydrofluoroalkanes (HFA-134a or HFA-227). This challenge arose

following the discovery that the degradation of CFCs damages stratospheric ozone and has proved to be a major stimulus to the development of novel inhaler technologies. The switch to HFA-powered pMDIs is in progress and will take several more years to complete. In the meantime, CFCs have been granted an essential-use exemption in pMDIs under the Montreal Protocol of 1987, reflecting their importance to the well-being of society. HFAs are greenhouse gases, and despite the fact that their contribution to global warming is small, this issue could restrict their future use. The development of novel HFA pMDI formulations has not been a simple manner, owing to a range of technical factors and the need to demonstrate clinical efficacy and safety for the reformulated products. Individual companies have adopted one of two strategies. One strategy involves making a product that is bioequivalent with the CFC pMDI that is to be replaced so that the HFA pMDI can be used in exactly the same doses as the CFC pMDI. The alternative strategy is to make a product that deposits drug in the lungs more efficiently than a CFC pMDI. This usually involves formulating a corticosteroid product as a solution, enabling a very small particle size to be achieved as the propellant evaporates. With such a product, it is also possible to reduce the spray velocity and to deposit up to half the dose in the patient’s lung, with greatly reduced oropharyngeal deposition, so that asthma control may be achieved using only a fraction of the CFC pMDI dose. A formulation of beclomethasone dipropionate (Qvar) was the first of these products to reach the market, and several similar products are either already marketed or in development. Breath-actuated pMDIs may be helpful in patients with poor coordination, who cannot actuate the pMDI at the same time as inhaling. These devices contain triggering mechanisms that are operated by the patient’s inhalation via the mouthpiece. However, it is unlikely that breath-actuated pMDIs confer any additional benefit on patients who can use a conventional pMDI successfully.

pMDIs with Spacer Devices Spacer devices are widely used with pMDIs. These vary greatly in size and shape, with volumes of commercially available models ranging from 50 to 750 ml. The concept of a spacer is to place some distance between the point at which the aerosol is generated and the patient’s mouth, allowing the propellant to evaporate and the rapidly moving aerosol cloud to slow down before it is inhaled (Figure 2). The most successful spacers have a one-way valve in

AEROSOLS 61

Formulation: ordered mixture of drug and carrier

DPI device

Powder de-aggregated by patient’s inhalation

Figure 3 Principle of operation of a dry powder inhaler (DPI). The formulation most frequently consists of an ordered mixture of micronized drug and carrier lactose, which is de-aggregated by the patient’s inhalation through the device.

Figure 2 pMDI connected to a large volume spacer device.

the mouthpiece, which allows the pMDI to be actuated into the spacer, with a brief pause before the patient inhales so that it is not necessary to actuate and inhale simultaneously. Some spacers function effectively if the patient takes a series of relaxed tidal breaths from the device immediately after actuating a dose. Spacers reduce oropharyngeal deposition of drug and may increase lung deposition, but the majority of the dose is often deposited on the walls of the spacer. This may allow the reduction of the total body burden of inhaled corticosteroids compared with a standard pMDI. Large volume spacers, such as the Volumatic and Nebuhaler, have a well-accepted role in hospital emergency rooms for treating acute asthmatic attacks. Specially designed spacers with a volume of 200–300 ml are available for treating young children. Most spacer devices are made of plastic, which may acquire a static charge during handling. This results in a suspended aerosol cloud being attracted to the spacer walls, with a marked reduction in the dose available for inhalation. Specific handling and washing techniques are usually recommended, and at least one lightweight metal spacer is available that is not susceptible to the effects of static charge. With correct use, including control over electrostatic charge effects, large volume (4500 ml) spacer devices may deposit more than 30% of the dose from a CFC pMDI in the patient’s lungs.

Dry Powder Inhalers Dry powder inhalers (DPIs) have been available commercially since approximately 1970, although the earliest prototypes were described several decades earlier. DPIs contain a powder formulation, which most frequently consists of an ordered mixture of micronized drug (o5 mm in diameter) and larger

carrier lactose particles that are required to improve powder flow properties. The patient’s inhalation through the device is used to disperse the powder and to ensure that some of the dose is carried into the lungs (Figure 3). An alternative type of formulation used in some DPIs consists either of micronized drug particles alone loosely aggregated into small spherules or of cospheronized drug and lactose. DPIs are basically of three types: (1) unit-dose devices, in which an individual dose in a gelatin capsule or blister is loaded by the patient immediately before use; (2) multiple unit-dose devices, which contain a series of blisters or capsules; and (3) reservoir devices, in which powder is metered from a storage unit by the patient before inhalation. Unit-dose devices, including Spinhaler and Rotahaler, were the only DPIs available until the mid-1980s. Patients generally find multiple unit-dose devices, such as the Diskus (Accuhaler), and reservoir DPIs, such as the Turbuhaler, to be more convenient than unit-dose DPIs since they provide several weeks’ treatment. DPIs tend to deposit a greater fraction of the dose in the lungs compared with CFC pMDIs, but in practice lung deposition varies widely between devices (Figure 4). Powder formulations are susceptible to the effects of moisture, and protecting the formulation against these effects is an important part of DPI design. By the end of 2004, at least 16 DPIs had been marketed in different areas of the world for asthma and COPD therapy, involving a range of unit-dose, multiple unit-dose, and reservoir systems. A further 20–30 DPIs were also known to be in development. The anticipated expansion of the generics market for inhaled asthma and COPD drugs is likely to result in a number of these novel DPIs reaching the market. It is interesting to note that the major pharmaceutical companies with an interest in inhaled asthma and COPD drugs appear to be prioritizing the DPI over reformulated HFA pMDIs products. In particular,

62

AEROSOLS 100 90

Percentage of dose

80 70 60 50 40 30 20 10 D is kh U ale ltr ah r al Pu er lv Sp inal in ha Ae ler ro liz e Ai r rm M AG ax Tu hal rb er uh Ea aler sy ha C lic ler kh al e N ov r ol iz e Ta r ifu Fl n ow ca p Ec s lip se

0

Figure 4 Mean percentage of the dose deposited in the lungs from 14 dry powder inhalers (DPIs), obtained in scintigraphic studies. The high lung deposition from the Flowcaps and Eclipse DPIs probably reflects the properties of the formulation as much as the DPI.

combination products in DPIs containing a longacting b-agonist and a corticosteroid (e.g., Advair Diskus and Symbicort Turbuhaler) have been very successful. However, DPIs tend to be more expensive than pMDIs, and this may limit their use, especially in developing countries. DPIs have two major advantages over pMDIs. First, they do not contain propellants. Second, all currently marketed models are breath-actuated, and patients find them easier to use correctly than pMDIs. However, this second advantage is closely linked to a disadvantage. In order to disperse the powder as efficiently as possible, and hence to maximize lung deposition, it may be necessary for patients to inhale as forcefully as possible via the DPI, and some patients may be either unable or unwilling to do this. All DPIs exhibit some degree of inhaled flow rate dependence, with forceful (fast) inhalation tending to give higher lung deposition than more gentle (slow) inhalation. For instance, in the Turbuhaler DPI, a reduction in peak inhaled flow rate from 60 to 30 l min 1 was shown to result in a reduction in lung deposition from 27% to 14% of the dose. In this respect, DPIs present a paradox since fast inhalation per se is generally associated with enhanced deposition in the oropharynx, as described previously. Low inspiratory effort through a DPI may result in a reduced emitted dose and poor particle deaggregation. The actual magnitude of the peak inhaled flow rate associated with forceful inhalation will vary between devices from o30 to 4100 l min 1, according to the resistance to airflow of each device. Not only the peak inhaled flow rate achieved through the DPI but

also the time taken to reach the peak flow will determine how efficiently particles are deaggregated. In practice, it seems that almost all patients with stable asthma or COPD can inhale sufficiently well via DPIs to benefit from them. Several so-called active DPIs have been developed, in which the powder is dispersed by some mechanism other than the patient’s inhalation – for instance, by an internal source of compressed air or by a fan driven by an electric motor. These active DPIs are generally more complex than breath-actuated DPIs and may come to be used primarily for therapies that require very efficient and reproducible targeting of drugs to specific lung regions, such as inhaled peptides for systemic therapy. Sophisticated formulations for use in DPIs are also in development. These include drug/lactose blends, in which the surface of the lactose particles has been smoothed in order to aid dispersion, or particles made by processes other than micronization. For instance, a spray-dried formulation of the antibiotic tobramycin is under development for the treatment and prevention of respiratory tract infections in patients with cystic fibrosis, consisting of low-density spherical particles that disperse efficiently with minimal inspiratory effort. An advantage of these sophisticated formulations is that often they can be delivered efficiently to the lungs using very simple and inexpensive DPI devices.

Nebulizers Drugs may often be formulated as solutions in water or ethanol, and they may be delivered by nebulizers

AEROSOLS 63 Signal from piezoelectric crystal

Drug formulation in reservoir

Mouthpiece Baffle Venturi

Mesh

Drug formulation in nebulizer cup

Compressed air

To mouthpiece

Figure 5 Design and operation of a typical jet nebulizer.

Figure 6 Principle of operation of a mesh-based nebulizer system. A mesh or grid is vibrated by a piezoelectric crystal, and a dispersion of micron-sized liquid droplets is formed.

that convert the solution into a spray. A variety of devices may be used to form the spray, and the three most common are jet nebulizers, ultrasonic nebulizers, and vibrating mesh nebulizers. An important advantage of nebulizers is that they can be used with relaxed tidal breathing. This makes them attractive for delivering inhaled drugs to children, the elderly, and those undergoing acute asthmatic attacks, who may not be able to use pMDIs or DPIs successfully. Currently, nebulizers represent the most practical way to deliver very large drug doses (4100 mg) that are occasionally needed for some inhaled antibiotics. Jet nebulizers are operated by compressed air passing through a narrow constriction (a venturi). A single dose contained in a volume of typically 2–4 ml in a cup within the nebulizer is drawn up a feed tube and is fragmented into droplets (Figure 5). Only the smallest droplets are delivered directly to the patient; larger droplets impact on baffle structures situated close to the nozzle and are returned to the cup to be nebulized again. Several minutes are required to nebulize the entire dose, and even at completion of treatment the majority of the dose remains within the device as large droplets on internal walls. There are major differences in performance between different commercially available nebulizers, with lung deposition ranging from o2% to 20% of the dose. Jet nebulizers can also be used to aerosolize micronized suspensions of corticosteroids. Recent developments in technology have included breath-enhanced nebulizers, in which passage of inhaled air through the device is used to increase aerosol output, and adaptive aerosol delivery systems, in which aerosol generation is synchronized to coincide with the first part of the patient’s inhalation. Adaptive aerosol delivery systems seem to be

able to reduce the intersubject variability of aerosol delivery. Ultrasonic nebulizers have many properties similar to jet nebulizers, but the aerosol is formed in a different way. A piezoelectric crystal is located beneath the cup, and a fountain of droplets is generated. Ultrasonic nebulizers are less popular now than a few years ago, possibly for several reasons. They may not handle either suspensions or viscous solutions well, and there is evidence that they damage some drug molecules, probably by heat generated during the nebulization process. Jet and ultrasonic nebulizers cannot compete with pMDIs and DPIs in the portable inhaler market, partly because they are singledose devices and partly because they generally need either a compressor or a power source in order to function. Several novel nebulizers are available in which the spray is formed by the passage of drug solution through a vibrating mesh or grid of micron-sized holes (Figure 6). The mesh is usually vibrated by a piezoelectric crystal, but unlike ultrasonic nebulizers, there is no evidence that this process damages drug molecules. Mesh-based systems deliver a higher proportion of the dose, and achieve higher lung deposition, compared to jet or ultrasonic nebulizers. A smaller percentage of the dose is retained in the device at the end of treatment, and this can result in less wastage for expensive drug substances. Nebulization time is short compared to that of jet and ultrasonic nebulizers, which should improve patient compliance. Some vibrating mesh nebulizers are small, compact, and battery operated, giving them practical advantages over jet and ultrasonic nebulizers. Careful cleaning of all nebulizers is essential in order to avoid bacterial contamination and to ensure that the working parts (particularly narrow nozzles) function correctly.

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Soft Mist Inhalers A recent development in inhaler technology has been the development of low-velocity sprays known as soft mist inhalers. These devices represent a new class of multidose inhaler devices and contain liquid formulations similar to those in nebulizers. A variety of principles are utilized, including forcing liquid under pressure through a nozzle array, ultrasonics, vibrating meshes, and several novel approaches, such as condensation of vapors to form particle dispersions. Many of these devices are able to achieve extremely high lung deposition (450% of the dose), and they are capable of delivering drugs to the deepest parts of the lungs. This may allow them to play a major future role in inhalation therapy, particularly in situations in which precise aerosol targeting is needed. In 2004, one soft mist inhaler (Respimat) was launched in Europe for asthma and COPD therapy as a direct replacement for the same drugs given either in a CFC pMDI or in a DPI. The spray is formed by passing a metered dose (typically 15 ml) via a sophisticated nozzle system under pressure. The velocity of the spray is only a fraction of that found in a CFC pMDI. This device deposits a greater percentage of the drug in the lungs compared to a CFC pMDI (Figure 7), and it is clinically effective

Exhaled

100%

Device

80%

Oropharynx 60%

Lungs

40% 20% 0% CFC pMDI

Respimat

Figure 7 Fractionation of the dose from a novel Respimat soft mist inhaler compared to that from a pMDI formulated with chlorofluorocarbon (CFC) propellants. Data from Newman SP et al. (1998) Lung deposition of fenoterol and flunisolide delivered using a novel device for inhaled medicines. Chest 113: 957–963.

using smaller doses. It is probable that other soft mist inhalers will be marketed in the relatively near future, and some could mount a significant challenge to pMDIs and DPIs in the portable inhaler market. See also: Asthma: Overview. Bronchodilators: Anticholinergic Agents; Beta Agonists. Chronic Obstructive Pulmonary Disease: Overview: Emphysema, Alpha-1Antitrypsin Deficiency. Corticosteroids: Therapy. Cystic Fibrosis: Overview. Particle Deposition in the Lung.

Further Reading Adjei AL and Gupta PK (1997) Inhalation Delivery of Therapeutic Peptides and Proteins. New York: Dekker. Bisgaard H, O’Callaghan C, and Smaldone GC (eds.) (2003) Drug Delivery to the Lung. New York: Dekker. Dalby RN, Byron PR, Peart J, and Farr SJ (eds.) (2002) Respiratory Drug Delivery VIII. Raleigh, NC: Davis Horwood. Dalby RN, Byron PR, Peart J, Suman JD, and Farr SJ (eds.) (2004) Respiratory Drug Delivery IX. River Grove, IL: Davis Healthcare. Dolovich M, MacIntyre NR, Dhand R, et al. (2000) Consensus conference on aerosols and delivery devices. Respiratory Care 45: 588–776. Hickey AJ (ed.) 2003. Aerosol delivery and asthma therapy (theme issue). Advanced Drug Delivery Reviews 55, 777–928. Mitchell JP and Nagel MW (2003) Cascade impactors for size characterization of aerosols from medical inhalers: their uses and limitations. Journal of Aerosol Medicine 16: 341–377. Moreń F, Dolovich MB, Newhouse MT, and Newman SP (eds.) (1993) Aerosols in Medicine: Principles, Diagnosis and Therapy. Amsterdam: Elsevier. Newman SP, et al. (1998) Lung deposition of fenoterol and flunisolide delivered using a novel device for inhaled medicines. Chest 113: 957–963. Newman SP and Newhouse MT (1996) Effect of add-on devices for aerosol drug delivery: deposition studies and clinical aspects. Journal of Aerosol Medicine 9: 55–70. O’Callaghan C and Barry PW (1997) The science of nebulised drug delivery. Thorax 52(supplement 2): S31–S44. Pauwels R, Newman SP, and Borgstro¨m L (1997) Airway deposition and airway effects of antiasthma drugs delivered from metered dose inhalers. European Respiratory Journal 10: 2127– 2138. Smith IJ and Parry-Billings M (2003) The inhalers of the future? A review of dry powder devices on the market today. Pulmonary Pharmacology and Therapeutics 16: 79–95.

Allergic Bronchopulmonary Aspergillosis see Asthma: Allergic Bronchopulmonary Aspergillosis.

ALLERGY / Overview 65

ALLERGY Contents

Overview Allergic Reactions Allergic Rhinitis

Overview J A Grant and C C Horner, University of Texas Medical Branch, Galveston, TX, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Allergy is the term used for a collection of diseases mediated by immunologic mechanisms. Allergic disorders include allergic rhinitis, conjunctivitis, asthma, urticaria, angioedema, food allergy, drug allergy, and anaphylaxis. The prevalence of allergic diseases has been increasing in recent times. They are a major cause of morbidity and decreased quality of life. The development of allergic diseases is influenced by heritable and environmental factors. Diagnosis often involves documenting responses to allergen such as in skin testing, radioallergosorbant allergen testing, or bronchial provocation testing. Allergic disorders share the common pathology of inflammation of affected tissues. Allergy requires sensitization to an allergen and response on reexposure to that same allergen. Pathogenesis involves production and release of cytokines, chemokines, and lipid mediators which cause tissue damage and recruit inflammatory cells. Current therapies include allergen avoidance, antihistamines, leukotriene modifiers, corticosteroids, phosphodiesterase inhibitors, humanized monoclonal anti-IgE, and immunotherapy.

Introduction Allergy encompasses a wide variety of disorders that share immunologic mechanisms. For centuries, patients had allergic symptoms but the causative agent for sensitivity to allergens was unknown. Allergic symptoms were first described by Leonardo Bottallo in sixteenth century Europe. Wyman identified ragweed as the trigger of the ‘autumnal catarrh’ and Blackely identified hay fever as being initiated by grass pollen in the 1870s. Twenty years later Behring described the adverse skin reactions to tubercle bacillus as ‘hypersensitivity’. Portier and Richet attempted to confer immunity to sea anemone toxin by injecting dogs with subsequent doses of toxin. They employed the term ‘anaphylaxis’ (denoting antiprotection) in 1902 to describe a lethal reaction after the dogs received the second dose of the toxin. Von Pirquet used the term ‘allergy’ in 1906 to describe the skin reaction to cowpox vaccine at 24 h post-vaccination. His definition of allergy described an organism’s alteration

by contact with an organic agent. Four years later, Noon observed reactions to pollen extracts on abraded skin and began immunotherapy with these extracts. Dale and Laidlaw produced respiratory distress and anaphylaxis in animals by histamine infusion in 1919. Another major discovery was the identification of cytokines. This class includes chemokines that are important in the recruitment of inflammatory cells. In 1921, Prausnitz and Kustner demonstrated that a factor could be transferred to a nonsensitized person’s skin and confer sensitivity. This factor was called ‘reagin’ by Coca and Cooke. In 1966, Ishizaka’s discovery of IgE established a scientific basis for the specific reactions. Concurrently, S G O Johannson independently identified a protein in multiple myeloma that was also elevated in allergic patients. Subsequently, the WHO determined that both proteins were IgE. In 1968, Gell and Coomb described four classes of immunologic reactions. These classes are IgE-mediated immediate hypersensitivity (e.g., anaphylactic shock), IgG- or IgM-mediated cytotoxic reactions (e.g., immune hemolytic anemia), immune complexmediated reactions (e.g., serum sickness), and delayed hypersensitivity (e.g., contact dermatitis). Allergic diseases can be mediated by any of these mechanisms. Allergic disorders include allergic rhinitis, conjunctivitis, asthma, urticaria, angioedema, food allergy, drug allergy, and anaphylaxis.

Etiology Atopy refers to the tendency of patients to be sensitive to allergens. Atopic diseases include eczema, allergic rhinitis, and asthma. Over the past century, genetics has been thought to affect allergic disease occurrence. The incidence of allergic disease in patients with an allergic family history is higher than in those without one. Atopy is thought to be autosomally transmitted and multigenic. It is probably a complex trait influenced by both heritable and environmental factors. Environmental exposures likely have effects on gene expression and the development of allergic disease. The hygiene hypothesis proposes

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that in Western countries the developing immune system is deprived of environmental microbial antigens that stimulate Th1 cells. This lack of stimulation increases the presence of atopic disease. Other factors such as diet and exposure to high pollen counts at birth may also increase allergic disease. Linkage studies for allergy and asthma have produced 420 distinct chromosomal regions with linkage to asthma or related traits. The lack of distinct phenotypes, the inexact definitions of allergic diseases, and the presumed influence of numerous genes have made positional cloning with linkage studies problematic. The linkages reproduced most frequently are on 6p, 5q, 12q, and 13q. Linkage on 14q and 7p was identified in founder populations in Iceland and Finland. Chromosome 2q14–2q32, which includes the IL-1 gene, has been linked to asthma. Candidate gene analysis is a promising technique. Candidate genes encode biochemical markers that affect allergic diseases. Many of these genes control IgE and cytokine production. Candidate genes include one loci on 5q near the gene cluster for IL-4, IL-5, IL-9, and IL-13. Polymorphisms of the IL-9 gene are associated with human asthma. 11q13, which encodes the b chain of the high-affinity IgE receptor has been linked to asthma. Polymorphisms of the IL-4 receptor a chain are associated with atopic asthma. PHF11, present in the locus for total IgE on 13q14, is expressed in many immune-related tissues. It is associated with total IgE and has been linked with asthma in multiple genome screens. The ADAM33 gene on 20p13 encodes a disintegrin and metalloprotease. This gene was mapped in 2002 as an asthma and airway hyperresponsiveness gene by Van Eerdewegh and co-workers. A cluster of single nucleotide polymorphisms (SNPs) was identified in the ADAM33 gene and demonstrated significant associations with asthma. Howard and co-workers reproduced the association seen by Van Eerdewegh’s group and lent support to the potential role of ADAM33 in asthma susceptibility (see Genetics: Gene Association Studies).

Pathology Allergic inflammation is present in all allergic diseases. Most tissues exhibit vasodilation and increased vascular permeability. Eosinophils, neutrophils, CD4 þ T cells, and basophils eventually infiltrate the site of allergy. Asthma is a disorder which also involves an increase in mucous glands, mucus hypersecretion, smooth muscle hypertrophy, and airway remodeling. Recruitment of eosinophils is a prominent feature of allergic diseases (see Leukocytes: Eosinophils). Eosinophils migrate across blood vessels into tissues

by binding endothelial cell adhesion molecules. Major basic protein, lipid mediators, and cytokines enable the eosinophil proinflammatory effects. Eosinophils may also repair damage to mucosal surfaces with fibrogenic growth factor and matrix metalloprotease. This repair mechanism may result in remodeling of airway tissue as seen in asthma. Mast cells are present in tissues throughout the body. Features of an allergic reaction vary with the anatomic site. The site of allergen contact determines the tissue or organ involved. The concentration of mast cells at the site determines the severity. The wheal and flare is the typical cutaneous allergic response. When allergen binds to specific IgE on mast cells, mast cell mediators are released and cause local blood vessel dilation. These vessels leak fluid and macromolecules and produce redness and swelling (known as the wheal). Dilation of vessels on the edge of the swelling causes redness (known as the flare).

Clinical Features Patients with allergic rhinitis typically experience rhinorrhea, congestion, sneezing, and itchy nose. Patients also may report postnasal drip and associated eye, ear, and throat symptoms. Patients commonly exhibit reactions to dust mite, animal dander, molds, and pollens. Small molecular weight chemicals can also react with self-proteins and become allergens. Reactivity to allergens can be determined by immediate hypersensitivity skin testing. Drops of allergen are placed into the dermis by various methods. Most skin testing employs a prick device composed of metal or plastic to introduce allergen into the skin. Allergen binds IgE and the mast cells in the skin release histamine to produce the wheal and flare response. The skin response can be compared to controls of saline (negative control) and histamine (positive control). Radioallergosorbant allergen testing (RAST) quantitates allergenspecific IgE in a patient’s serum. RAST is usually reserved for patients who have a contraindication to skin testing or are taking medications that either interfere with testing (antihistamines) or interfere with treatment should a reaction occur (beta blockers or ace inhibitors). Nasal cytology can reveal the presence or absence of inflammatory cells, especially eosinophils. Adjunct tests such as rhinoscopy and rhinomanometry can provide further characterization of the nasal airway (see Allergy: Allergic Rhinitis). Patients with asthma report symptoms of wheezing, shortness of breath, cough, and chest tightness. These patients’ airways show hyperresponsiveness


with reversibility. Patients may experience asthmatic symptoms in response to allergens, infections, exercise, nonsteroidal anti-inflammatory drugs, gastroesophageal reflux disease, stress, and irritants. Lung function can be assessed by spirometry before and after treatment with bronchodilators. Bronchial hyperresponsiveness can be investigated with bronchial provocation testing such as with methacholine or exercise challenge testing (see Asthma: Overview). Urticaria is localized edema in skin or mucous membranes. Patients have pale to pink wheals that are extremely pruritic. These lesions are transient and usually resolve within 24 h. Angioedema involves local edema in deeper areas of skin or mucous membranes. These lesions are often painful. Urticaria/angioedema can be triggered by temperature, sun, direct pressure, medication, infections, foods, or systemic diseases. Atopic eczema is a skin disorder with pruritis, erythematous macules or papules, and xerosis. Lesions may become excoriated with crust and exudates. Skin tends to be dry and more permeable to allergens and bacteria. Chronic irritation may cause lichenification of the skin and scaling patches or papules. Young children typically have lesions on the face, scalp, and extensor surfaces. Older children and adults have flexor surface involvement. Food allergy is most frequent in young children. Symptoms may include urticaria, angioedema, rash, flushing, rhinitis, wheezing, or anaphylaxis after ingestion of the allergenic food. Some patients experience the oral allergy syndrome which is usually confined to the oropharynx. Symptoms may consist of pruritis and angioedema of the tongue, lips, palate, and throat. Patients with birch pollen-induced rhinitis may develop oral allergy symptoms after eating raw potato, carrot, apple, celery, or hazelnut. Patients with ragweed pollen-induced rhinitis may develop symptoms after eating melons or bananas. Major food allergens in children are milk, egg, peanuts, soybeans, wheat, fish, and tree nuts. Major food allergens in adults are peanuts, tree nuts, fish, and shellfish. Conformational and sequential food epitopes are responsible for food allergy. Patients with IgE to sequential epitopes react to all forms of a food and tend to have persistent allergy, whereas those with IgE to conformational epitopes tolerate small amounts of food after heating or partial hydrolysis because these conformational epitopes are destroyed. These patients tend to have clinical tolerance. Prick skin testing for foods can be performed as described previously. Negative prick skin tests have a high negative predictive value. Positive skin tests are not conclusive. RAST may also be performed to aid in diagnosis.

Drug allergy reactions most commonly consist of urticaria, morbilliform rash, or anaphylaxis. Skin testing for drug allergy is not standardized and the predictive value of this technique is unclear. Anaphylaxis is an immediate generalized systemic allergic reaction in response to an allergen. Symptoms may include flushing, pruritis, urticaria, angioedema, wheezing, shortness of breath, chest tightness, abdominal pain, nausea, vomiting, diarrhea, laryngeal edema, arrhythmia, myocardial infarction, and hypotension. Serum tryptase can be measured within 6 h of a reaction to determine if mast cell degranulation has occurred during the reaction. Serum histamine and urinary LTE4 may be quantitated to provide evidence of anaphylaxis.

Pathogenesis Immunology plays a large role in allergic diseases. T lymphocytes are the major effector cells of these immune responses (see Leukocytes: T cells). CD4 þ lymphocytes are present in two predominant types, Th1 and Th2 cells. Th1 are helper cells that produce IL-2 and interferon gamma (IFN-g) and promote cellmediated reactions. Th2 are helper cells that produce IL-4, IL-5, IL-6, IL-10, and IL-13 and are involved in humoral immunity and allergic inflammation. Th2 cytokines augment antibody production (especially IgE), enhance eosinophil production, and are associated with allergic and antibody-driven responses (Figure 1). Th1 and Th2 cells suppress each other. Patients with an allergic phenotype generate responses to allergens that favor Th2 responses. Another type of T cell is the regulatory T cell (T reg). Thymus-derived CD4 þ CD25 þ regulatory cells are termed naturally occurring T regs. Adaptive T regs are T-cell populations induced by in vitro or in vivo manipulation. Active T-cell suppression by T regs promotes immunologic tolerance, but the mechanism of this suppression is controversial. There is a potential role for T regs in the control or prevention of Th2 responses. CD4 þ CD25 þ T cells and IL-10producing T regs have been shown in humans to prevent T-cell activation by allergens and are possibly deficient in atopic patients. T regs may also secrete the immunosuppressive cytokines IL-10 and TGF-b. IL-10 inhibits macrophage activation and antagonizes IFN-g while TGF-b inhibits T- and B-cell proliferation. IgE-mediated allergic responses occur through numerous steps. First, a patient is exposed to an allergen. Then antigen-presenting cells internalize the allergen which is then processed and presented to Th2 cells by class II MHC molecules. T-cell receptors bind the presented allergen thus activating the Th2

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ALLERGY / Overview APC

Th2 cell

Allergen IL-4

Specific IgE

Re-exposure to allergen B cell Arachidonic acid

Prostaglandins, leukotrienes

Mast cell Preformed mediators (histamine, TNF-)

Vascular leak, bronchoconstriction, inflammation, tissue damage, late response

Early response

Figure 1 Allergen is processed by antigen presenting cells (APCs) and presented to Th2 cells. Stimulated by IL-4, B cells produce allergen-specific IgE. Upon re-exposure to allergen, allergen cross-links specific IgE on mast cells and activates allergic responses.

cells. The activated cells produce IL-4. Atopic patients have more allergen-specific IL-4 secretory T cells in circulation than nonatopics and produce more IL-4 per cell. B cells, in turn, are stimulated by IL-4 to class-switch to produce IgE, specific to the allergen. Approximately 20% of an exposed population will generate specific IgE to an allergen. This IgE binds to receptors on mast cells. When the patient is subsequently exposed to the allergen, the allergen binds the IgE already present on the mast cell surfaces and cross-links the antibodies. Antibody cross-linking of mast cells activates a variety of responses. The early phase of immediate hypersensitivity occurs within minutes and involves the release of preformed mediators from granules. These mediators include biogenic amines (histamine), enzymes (tryptase, chymase), carboxypeptidase, cathepsin G, acid hydrolases, and tumor necrosis factor alpha (TNF-a) (see Leukocytes: Mast Cells and Basophils). Histamine causes endothelial cells to contract and allow plasma to leak into tissue. Endothelial cells also produce prostacyclin and nitric oxide which promote vascular smooth muscle relaxation and lead to vasodilation. Bronchial smooth muscle constriction is also caused by histamine. Antibody cross-linking also triggers the initiation of several pathways which lead to cytokine, prostaglandin, and leukotriene production. Arachadonic acid is converted to prostaglandin D2 and the cysteinyl leukotrienes (LTC4, LTD4, LTE4) (see Lipid Mediators: Leukotrienes). These mediators are responsible for vascular leak, bronchoconstriction, inflammation, and tissue damage. These substances lead to inflammatory changes hours after exposure, referred to as the late-phase response. The late phase involves recruitment and infiltration of the mucosa

with neutrophils, eosinophils, basophils, and Th2 cells. TNF-a activates endothelial expression of adhesion molecules E-selectin and ICAM-1 and promotes cell infiltration. IL-3 promotes mast cell proliferation. IL-5 stimulates eosinophil production and activation (see Interleukins: IL-5). The chemokines eotaxin and monocyte chemotactic protein-5 from epithelial cells recruit eosinophils. IL-4 and IL13 induce Th2 differentiation. While mast cells are responsible for the majority of leukotriene production in the early response, basophils and eosinophils produce most of the leukotrienes in the late-phase response.

Animal Models Mouse models have been used in the study of allergy because mice are readily available, have a well-characterized immune system, and strains are genetically characterized. Knockout mice can be used to evaluate the role of a cell type or mediator. Many allergists propose that allergic rhinitis and asthma may represent a continuum of inflammation and should be considered as a united allergy airway disorder. Mouse models have been used to investigate this concept. Mice were systemically sensitized with allergen (most often ovalbumin) and then challenged with airway allergen. The inflammatory response in the mouse nose resembles human allergic rhinitis. Nasal mucosal thickening can be seen on imaging. Experimental asthma in mice also mimics human asthma. Bronchial hyperresponsiveness can be documented by plethysmography. Most of the inflammation in mice is seen in the lower airways where the minority of allergen is deposited. This may indicate increased sensitivity in the lower airways. Inhaled


allergen causes an increase in allergen-specific IgE and eosinophils in the blood and increases bone marrow eosinopoiesis. Currently, it is unclear why sensitization causes symptoms in the nose, the lower airway, or both. There may be a genetic cause so studying different strains of mice may be useful. Mouse models have also been used in the study of atopic eczema. Mice have been sensitized epicutaneously with ovalbumin on shaved skin. Elevated serum total and specific IgE and IgG1 and increased dermal IL-4, IL-5, and IFN-g were observed. Deficiencies of these cytokines decreased the eczematous phenotype. For example, IL-4-deficient mice showed Th1biased skin inflammation with decreased eosinophils and eotaxin mRNA in the dermal infiltrate. IL-5deficient mice similarly had less pronounced epidermal and dermal thickening and the dermal infiltrate lacked eosinophils. IFN-g-deficient mice showed only slight dermal thickening. The necessity of certain lymphocytes in eczema was demonstrated. ab T cells are essential since T-cell receptor a-chain-deficient mice did not develop a dermal infiltrate or induction of IL-4 or IgE. Mice that lack gd T cells showed no change in infiltrate. Likewise, mice that lack B cells still develop an infiltrate and elevated IL-4. Numerous animal models have been utilized in food allergy. Rats and mice have been used to assess foodinduced anaphylaxis. Animals ingested ovalbumin by gavage or in drinking water and were subsequently challenged intraperitoneally. The route, dose, and age of the animal were shown to influence sensitization. Rats and swine sensitized to allergens and subsequently challenged orally, demonstrated alterations in small intestinal pathology. Tolerance is dose-dependent for specific antigens. Mice fed ovalbumin or peanuts required a 50-fold higher dose of peanuts to develop tolerance; low doses of peanuts were more likely to induce sensitization. The importance of an intact mucosal barrier was shown when mice, fed a novel dietary protein while their gastrointestinal tracts were inflamed, developed sensitization and high serum IgE. Allergic conjunctivitis has been studied in guinea pigs, rats, and mice. Mice exposed to ragweed by topical contact with conjunctival and nasal mucosa developed signs of allergic conjunctivitis and ragweedspecific IgE. Regulators of vascular permeability are important in allergic conjunctivitis. Substance P has been shown to be a mediator of allergic conjunctivitis and acts through NK1 receptors on blood vessels to produce conjunctival hyperpermeability. Nitric oxide has been shown to play a major role in regulating vascular permeability and stimulating prostaglandin E2 production. T-cell adhesion molecules are integral in allergic conjunctivitis. Guinea pig models with ovalbumin have shown that the integrin very

late activation antigen-4 (VLA-4) plays a critical role in eosinophil infiltration. Other mice studies showed that antibodies against the integrin intercellular adhesion molecule-1 (ICAM-1) and its ligand leukocyte function-associated antigen-1 (LFA-1) inhibited clinical and histological signs of conjunctivitis. The significance of IL-1 for inflammatory changes in conjunctivitis was demonstrated using an IL-1 receptor antagonist in mice exposed to cat dander antigens. Studies in rats using ovalbumin showed that IFN-g suppresses the development of allergic conjunctivitis during the induction phase.

Management and Current Therapy One of the cornerstones of allergy treatment is avoidance. Avoiding or reducing allergen exposure prevents or minimizes the body’s response to allergen. Environmental control measures help one decrease exposure. For example, patients with house dust-mite allergy can encase their mattresses and pillows in special covers to minimize exposure to dust mites while asleep. One study showed that in inner-city children with atopic asthma, a comprehensive environmental intervention decreased exposure to indoor allergens and reduced asthma-associated morbidity. Avoiding allergic foods or drugs can prevent reactions (Figure 2). Depending on one’s sensitivities, allergen avoidance may range from simple to extremely difficult. Other therapies available to treat allergic disorders include antihistamines, leukotriene modifiers, corticosteroids, phosphodiesterase inhibitors, humanized monoclonal anti-IgE, and immunotherapy. H1-antihistamines have been used for decades for the relief or prevention of allergic symptoms. Antihistamines have recently received the designation of inverse agonists because they stabilize the inactive form of the H1 histamine receptor. First-generation antihistamines have marked sedation; second-generation antihistamines that are relatively nonsedating Therapy algorithm Allergy diagnosis

Asthma diagnosis

Avoidance of allergen

Avoidance of triggers, allergens

Pharmacotherapy Pharmacotherapy Immunotherapy Immunotherapy Figure 2 Therapy for both allergic rhinitis and asthma starts with avoidance of allergens. Pharmacotherapy can be added. Some patients with allergic rhinitis and asthma benefit considerably from immunotherapy.

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ALLERGY / Overview

have also been identified. Antiallergic activities include inhibiting the release of mast cell mediators probably through direct inhibition of Caþ channels. Anti-inflammatory effects include inhibiting cell adhesion molecule expression and inhibiting inflammatory cell chemotaxis (e.g., eosinophil chemotaxis). These inhibitions probably involve the downregulation of NF-kB, a transcription factor that regulates adhesion proteins and proinflammatory cytokines. Antihistamines have established roles in the treatment of allergic rhinitis and urticaria. A potential role exists for the treatment of anaphylaxis or asthma. The cysteinyl leukotrienes and LTB4 are products of arachadonic acid metabolism by 5-lipoxygenase. Leukotrienes cause airway inflammation and obstruction by affecting mucus production, smooth muscle contraction, and vascular permeability. They may also affect airway remodeling. The effects of leukotrienes are most likely mediated through the CysLT1 receptor. The CysLT1 receptor antagonists montelukast, pranlukast, and zafirlukast block the actions of LTC4, LTD4, and LTE4. A nonselective antagonist of the CysLT1 and CysLT2 receptors is not yet clinically available. Zileuton is a 5-lipoxygenase inhibitor and decreases the production of leukotrienes. Leukotriene modifiers are useful in allergic rhinitis and asthma. Leukotriene modifiers inhibit an important part of the inflammatory cascade and decrease eosinophil survival, goblet cell hyperplasia, mucus release, collagen deposition, and airway smooth muscle proliferation. Corticosteroids block the production of inflammatory cytokines (see Corticosteroids: Therapy). They may be given topically at the site of inflammation (nose, lungs, or skin) or delivered systemically. Glucocorticoids are the most potent therapy for treating all allergic disorders. They are liposoluble

hormones that enter the cell and bind a cytoplasmic glucocorticoid receptor (see Corticosteroids: Glucocorticoid Receptors). This receptor translocates to the nucleus and binds a glucocorticoid-response element in the promoter region of target genes. The glucocorticoid receptor can also bind transcription factors like NF-kB and AP-1 and prevent these factors from binding their DNA-response elements. Glucocorticoids control airway inflammation by inhibiting transcriptional activity of genes encoding proinflammatory molecules such as cytokines, chemokines, adhesion molecules, and mediator-synthesizing enzymes. They may suppress histone acetylation and stimulate histone deacetylation. They may also interfere with signal transduction pathways, such as MAP kinase enzymatic cascades involved in the regulation of transcription factors. Theophylline is a nonselective phosphodiesterase inhibitor with a narrow therapeutic ratio and significant drug interactions and has been used exclusively in asthma (see Bronchodilators: Theophylline). Inhibitors of phosphodiesterase type 4 have recently been developed. These inhibitors increase the intracellular concentration of CAMP and exhibit a broad range of anti-inflammatory effects on effector cells. Blocking the PDE4B receptor subtype appears responsible for anti-inflammatory properties of these agents. Cilomast and roflumilast are PDE4 inhibitors that are in late phase III clinical trials. Roflumilast has demonstrated more selectivity and a superior therapeutic ratio (Figure 3). The humanized monoclonal anti-IgE omalizumab is a new therapy which decreases the amount of IgE available for reactions and downregulates the number of IgE receptors on mast cells. This therapy is currently available for the treatment of severe asthma. Its use in food allergy is being investigated.

Clinical features • Allergic rhinitis • Asthma • Urticaria • Angioedema • Atopic eczema • Food allergy • Oral allergy syndrome • Drug allergy • Anaphylaxis

Rhinorrhea, congestion, sneezing, itchy nose Wheezing, shortness of breath, cough, chest tightness Pruritic wheals Deep local swelling of skin or mucus membranes Pruritis, erythematous maculesor papules, xerosis, lichenification Urticaria/angioedema, rash, flushing, rhinitis, wheezing, anaphylaxis Pruritis/angioedema of tongue, lips, palate, throat Uticaria, morbilliform rash, anaphylaxis Flushing, pruritis, urticaria/angioedema, wheezing, shortness of breath, abdominal pain, nausea, vomiting, diarrhea, laryngeal edema, arrhythmia, myocardial infarction, hypotension

Figure 3 Clinical features associated with various allergic disorders.

ALLERGY / Overview 71 Diagnostic workup Allergic rhinitis

Asthma

History and physical examination

History and physical examination

Immediate hypersensitivity skin testing or RAST

Spirometry

Medication trial Nasal cytology

Rhinoscopy or rhinomanometry

Bronchial provocation testing

Figure 4 Diagnostic workups start with the history and physical exam. Initial testing for allergic rhinitis consists of skin testing or RAST. Adjunct tests include nasal cytology, rhinoscopy, and rhinomanometry. Lung function in asthma is assessed by spirometry and a medication trial is begun. Bronchial provocation testing can assess bronchial hyperresponsiveness if needed.

Patients with allergic rhinitis and/or asthma who are (Figure 4) poorly controlled on medications may find immunotherapy (allergy shots) a feasible alternative. Immunotherapy involves administering injections of allergens to which a patient is sensitive. Increasing doses of allergen are given weekly until the patient is at a maintenance dose. This maintenance dose is continued monthly for 3 to 5 years. Patients receive relief from nasal allergy symptoms and their asthma may improve. The mechanisms of immunotherapy are not well defined. Immunotherapy may induce specific T-cell tolerance or a shift from a Th2 to a Th1 phenotype. Possibly, the Th2 response is inhibited, Th1 response is upregulated, or both. Immunotherapy also increases the production of IL-10 and TGF-b by T cells including T regs. IgG is also produced in response to antigen instead of the typical IgE. Allergen-specific IgG or blocking antibodies may compete with IgE for allergen and inhibit IgE activation of mast cells. IgG can also bind epitopes on allergen that are not recognized by IgE. This IgG binding may prevent cross-linking of IgE. Allergen-IgG complexes on antigen-presenting cells might impair antigen processing or the co-stimulation of T cells and render patients anergic. Immunotherapy is a type of desensitization, where small amounts of allergen are given until the patient tolerates the allergen. The same theory is used in treatment of drug allergies by giving increasing doses of drug until a therapeutic dose is tolerated. Unmethylated CG dinucleotides, or CpG motifs, are responsible for the immunostimulatory effect of

bacterial DNA and induce a Th1 type response in humans. Synthetic oligodeoxynucleotides mimic the bacterial DNA immunostimulatory sequences. These synthetic nucleotides can be conjugated to allergen to produce an allergen vaccine that is more immunogenic but less allergenic than allergen alone. These vaccines are currently under clinical trials. Recently, there has been an interest in pharmacogenetics. This field recognizes that medications may work more efficaciously in certain patients because of their genetic makeup. Single nucleotide polymorphisms (SNPs) may signal a change in proteins or amino acids in an individual. These changes may alter a drug’s target, uptake, metabolism, or excretion. A polymorphism in Gly 16 promotes bronchodilator resistance while the Arg 16 polymorphism potentiates a greater response to bronchodilators. An alternatively spliced form of glucocorticoid receptor b is present with higher frequency in corticosteroid-resistant patients. A polymorphism in the 5-lipoxygenase promoter decreases the response to the 5-lipoxygenase inhibitor zileuton. Pharmacogenetics is an exciting area that may shape the future of treatment for allergic diseases. See also: Allergy: Allergic Reactions; Allergic Rhinitis. Asthma: Overview. Bronchodilators: Theophylline. Chemokines. Corticosteroids: Glucocorticoid Receptors; Therapy. Genetics: Gene Association Studies. Immunoglobulins. Interleukins: IL-5. Leukocytes: Mast Cells and Basophils; Eosinophils; T cells. Lipid Mediators: Leukotrienes; Prostanoids. Matrix Metalloproteinases.

Further Reading Abbas AK and Lichtman AH (2003) Cellular and Molecular Immunology, 5th edn. Philadelphia: Saunders. Adelman DC, Casale TB, and Corren J (2002) Manual of Allergy and Immunology, 4th edn. Philadelphia: Lippincott Williams & Wilkins. Broide DH (2001) Molecular and cellular mechanisms of allergic disease. Journal of Allergy and Clinical Immunology 108: S65–S71. Cakebread JA, et al. (2004) The role of ADAM33 in the pathogenesis of asthma. Springer Seminars in Immunopathology 25: 361–375. Creticos PS, Chen Y, and Schroeder JT (2004) New approaches in immunotherapy: allergen vaccination with immunostimulatory DNA. Immunology and Allergy Clinics North America 24: 569–581. Groneberg DA, et al. (2003) Animal models of allergic and inflammatory conjunctivitis. Allergy 58: 1101–1113. Gutermuth J, et al. (2004) Mouse models of atopic eczema critically evaluated. International Archives of Allergy and Immunology 135: 262–276. Hallstrand TS and Henderson WR (2002) Leukotriene modifiers. Medical Clinics of North America 86: 1009–1033. Hellings PW and Ceuppens JL (2004) Mouse models of global airway allergy: what have we learned and what should we do next? Allergy 59: 914–919. Helm RM and Burks AW (2002) Animal models of food allergy. Current Opinion in Allergy and Clinical Immunology 2: 541–546.

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Holgate ST (2004) Pharmacogenetics: the new science of personalizing treatment. Current Opinion in Allergy and Clinical Immunology 4: 37–38. Joad J and Casale TB (1988) Histamine and airway caliber. Annals of Allergy 61: 1–7. Kay AB (2001) Allergy and allergic diseases: first of two parts. New England Journal of Medicine 344(1): 30–37. Kim DS and Drake-Lee AB (2003) Allergen immunotherapy in ENT: historical perspective. Journal of Laryngology & Otology 117: 940–945. Lipworth BJ (2005) Phosphodiesterase-4 inhibitors for asthma and chronic obstructive pulmonary disease. Lancet 365: 167–175. Morgan WJ, et al. (2004) Results of a home-based environmental intervention among urban children with asthma. New England Journal of Medicine 351: 1068–1080. Pelaia G, et al. (2002) Molecular mechanisms of corticosteroid action in chronic inflammatory airway diseases. Life Sciences 72: 1549–1561. Robinson DS, Larche M, and Durham SR (2004) Tregs and allergic disease. Journal of Clinical Investigation 114: 1389–1397. Sampson HA (2004) Update on food allergy. Journal of Allergy and Clinical Immunology 113(5): 805–819. Shearer WT and Li JT (2003) Primer on allergic and immunologic diseases. Journal of Allergy and Clinical Immunology 111(2): S441–S778. Simons FER (2004) Advances in H1-antihistamines. New England Journal of Medicine 351: 2203–2217. Till SJ, et al. (2004) Mechanisms of immunotherapy. Journal of Allergy and Clinical Immunology 113(6): 1025–1034. Weiss ST and Raby BA (2004) Asthma genetics 2003. Human Molecular Genetics 13: R83–R89.

Allergic Reactions S H Arshad, University Hospital of North Staffordshire, Stoke-on-Trent, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Allergy is a harmful response to an otherwise innocuous substance. Allergic reactions are immunologically mediated reactions to external substances, usually proteins. These are often, but not always, mediated by immunoglobulin E (IgE) antibody. Initial exposure to an allergen results in sensitization with the production of allergen-specific IgE antibodies. These antibodies circulate in the blood but are largely bound to high-affinity receptors on the surface of basophils and mast cells and lowaffinity receptors on eosinophils, macrophages, and platelets. On further exposure, the allergen reacts with the IgE bound to mast cells and basophils, causing degranulation and release of preformed mediators such as histamine and tryptase. These mediators cause the immediate phase of the type I reaction, which occurs within a few minutes (immediate hypersensitivity). Other mediators and cytokines are released and eosinophils are attracted to the site of activity, precipitating the late phase of the type I reaction, which starts 4–6 h after exposure. Immediate hypersensitivity reaction occurs in asthma, rhinitis, and anaphylaxis. In a sensitized asthmatic, early and late phase asthmatic reaction can be observed following bronchial allergen challenge. Repeated or continued exposure to allergen results in chronic airway inflammation, which is characteristic of asthma.

Systemic allergic reactions vary in severity from mild (such as generalized urticaria) to severe and life-threatening reactions with cardiovascular collapse and death. Anaphylaxis is the clinical syndrome that represents the life-threatening systemic allergic reaction. It results from the immunologically induced release of mast cell and basophil mediators after exposure to a specific antigen in previously sensitized individuals. Clinically indistinguishable reactions, caused by non-IgE-mediated immune mechanisms, are termed anaphylactoid reactions. Common causes of anaphylaxis are foods, drugs, insect stings, latex, and allergen extracts used for immunotherapy. The symptoms of anaphylaxis occur within a few minutes of exposure and are generally related to the skin, gastrointestinal tract, respiratory tract, and cardiovascular systems. Common manifestations include generalized urticaria/angioedema, nausea, vomiting, stridor, wheezing, hypotension, and syncope. Anaphylaxis requires immediate treatment with epinephrine, given intramuscularly. The dosage for adults is 0.3–0.5 ml, and for children is 0.01 ml kg 1, of a 1:1000 solution. The dose can be repeated at 5–15 min intervals. Supportive treatment includes cardiopulmonary resuscitation (if required), intravenous fluids, oxygen, antihistamine, and corticosteroids. Following the first episode, an assessment by an allergist is essential to establish the cause and for appropriate advice on preventive measures including avoidance of the offending agent and self-injectable epinephrine.

Immune Responses Atopy is defined as the genetic predisposition to form immunoglobulin (IgE) antibodies on exposure to allergens. The production of IgE is central to the induction of allergic diseases. Allergens are proteins with the capability to react to the immune system through their antigenic determinants. Initial exposure to the antigen results in sensitization. Antigens enter the body through the respiratory and gastrointestinal mucosa and the skin. Allergenic proteins are engulfed by antigen-presenting cells (APCs) such as monocytes, macrophages, and dendritic cells inducing primary immune response. The antigen is broken down to reveal the specific part of the molecule called antigenic determinant or epitope. Once processed in this way, the antigen is bound to the MHC class II molecules on the surface of these cells and the complex is presented to the T lymphocyte cell receptor. Bacterial antigens favor the production of Th1 cells with the secretion of its profile of cytokines, particularly interferon gamma (IFN-g). In atopic individuals, and in the presence of co-stimulatory signals, naı¨ve T cells are converted to activated CD4 þ T-helper-2 (Th2) cells. T lymphocytes play a central role in orchestrating the allergic reaction. Th2 cells produce cytokines, such as interleukin-4 (IL-4) and IL-13. These cytokines cause proliferation and switching of B cells to IgE producing B and plasma cells, specific to the antigen (Figure 1). Some of these cells have a long life and are called memory cells. IL-4 is the most

ALLERGY / Allergic Reactions 73 Antigen

IgE

Antigen-presenting cell

Naive T cell

Th2 cell

B cell

Antigen

Release of mediators such as histamine and tryptase Mast cell

important pro-allergy cytokine. Apart from switching of B cells to IgE production, it also stimulates T cells and macrophages, and enhances the expression of low-affinity IgE receptor (FceR2) on B cells and adhesion molecules on endothelial cells. This latter action promotes the movement of cells out of the blood vessels. It also inhibits other types of immune reaction, such as antibody-dependent cell-mediated cytotoxicity. IL-13 has similar but weaker biological activity though it lasts longer than IL-4. IFN-g inhibits allergic responses and its effects are opposite to IL-4 and IL-13. Therefore, it is crucial in the regulation of IgE production. Other cytokines, which inhibit allergic responses, are IL10, IL-12, TGF-b, and IL-8. The direction of immune response on exposure to allergen depends on the balance of Th1 and Th2 reactivity and ensuing cytokine milieu. In atopic individuals the balance is tilted towards the production of Th2 type cytokines (IL-4 and IL-13) as opposed to Th1 type, such as IFN-g. The Th2 differentiation and production of IgE is also suppressed by regulatory T cells (CD4 þ CD25 þ ). Thus, an inappropriately weak T regulatory mechanism would facilitate Th2 dominance and production of IgE and allergic disease. It is likely that allergen exposure in early childhood results in a lifelong T cell memory pool. In atopic individuals, the immunological memory is dominated by Th2 type cells leading to allergic reactivity, whereas in nonatopic subjects, Th1 cells dominate the memory pool. In addition to genetic predisposition, environmental factors (infections, diet, etc.) may also influence the outcome of these initial responses by altering the cellular and cytokine milieu within the lymph nodes. In atopic subjects with their Th2 skew, IgE is formed specific to the antigenic protein following initial exposure (sensitization). The IgE circulates in the blood in small quantities but is mostly present in the tissues bound to high-affinity receptors (FceR1) on

FEV1

Figure 1 A schematic representation of immediate (IgE-mediated) hypersensitivity reaction.

4.5 4 3.5 3 2.5 2 1.5 1 0

1

2

3

4

5

6

7

8

9

10 11 12

Hours (postallergen exposure) Figure 2 Early and late-phase asthmatic reaction following allergen inhalation challenge. FEV1, forced expiratory volume.

the surface of mast cells and basophils and FceR2 on eosinophils, macrophages, and platelets. This IgE can be detected in the serum by immune assays or in the skin by allergy skin tests. On further exposure, multivalent antigens bind and cross-link IgE bound to FceR1 on cell surface leading to the signaling cascade that causes rapid release of preformed mediators such as histamine, tryptase, and heparin (Figure 1). These mediators cause the immediate phase of the type I hypersensitivity reaction, which occurs within a few minutes (hence immediate hypersensitivity). There is also induction of rapid synthesis of arachidonic acid metabolites such as prostaglandins and leukotrienes and expression of cytokines (IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, and tumor necrosis factor alpha (TNF-a)) and chemokines. Eosinophils are attracted to the site of activity, precipitating the late phase of the type I reaction, which starts 4 6 h after exposure (Figure 2). Immediate hypersensitivity is central to all IgEmediated allergic reactions and occurs in asthma, rhinitis, and anaphylaxis. During acute allergic reactions, the process is acute with the release of huge quantities of histamine causing typical symptoms and signs, such as acute bronchospasm or anaphylaxis. However, the process may be subacute or chronic and localized to one site such as lung or nose.

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Repeated exposure to allergens leads to the induction of a more chronic inflammatory process with the influx of inflammatory cells including T lymphocytes and eosinophils. Cytokines, produced by a variety of inflammatory cells, including T cells, regulate the inflammatory process. Proliferation of Th2 subsets producing predominantly IL-4 and IL-5 results in differentiation and isotype switching of the naı¨ve B cells to IgE-producing plasma cells as well as activation and influx of inflammatory effector cells such as eosinophils. Eosinophils are potentially tissue damaging, particularly after priming with IL-5. Various cytokines upregulate adhesion molecules on endothelial and epithelial cells, thereby enhancing migration of eosinophils into the mucosa.

Allergic Reaction in the Lung Allergic reaction in the lung results in airway inflammation. Exposure to allergen is recognized as important in initiating and maintaining allergic airway inflammation in atopic asthmatics. In the appropriate setting of repeated allergen exposure and Th2 type immune responses, a cytokine milieu is created with upregulation of adhesion molecules and continuous recruitment and activation of inflammatory cells from the bloodstream towards the bronchial mucosa. The release of cytokines and inflammatory mediators by activated cells causes amplification and persistence of the inflammatory process. However, structural cells such as epithelial cells and smooth muscle cells are not merely passive recipients of immune-related tissue damage but are active participants of the complex inflammatory cascade, which may well have initiated at the epithelial/ mesenchymal level within the airways. Nonatopics show similar inflammation in their airways and thus IgE-mediated allergy in not a prerequisite for airway inflammation in asthma or rhinitis. Early Asthmatic Reaction

The effect of allergen exposure can be observed and studied in a controlled fashion during bronchial allergen challenge. In an atopic asthmatic, inhalation of allergen to which the patient is sensitized, results in an immediate hypersensitivity reaction with the release of mast cell mediators in the bronchial mucosa. These mediators enhance vascular dilatation, increase permeability of the venule, and increase mucus secretion, resulting in edema and congestion, typical of an acute phase reaction. Histamine and leukotrienes are potent bronchoconstrictors. Histamine stimulates local type c neurones leading to the release of several neuropeptides, including substance

P, which further increase vascular permeability and cause stimulation of parasympathetic reflexes augmenting mucous secretion and bronchoconstriction. These changes manifest clinically in cough, wheeze, and dyspnea. Late Asthmatic Response

Clinically, the effect of early asthmatic reaction diminishes after 30 min (Figure 2). This is followed by a relatively asymptomatic period during which a plethora of cytokines and mediators draw leukocytes to the tissues. Events initiated during the early response result in vascular dilatation and increased permeability, edema formation, and the accumulation of cells. IL-5, secreted from mast cells, lymphocytes, and eosinophils is the most important cytokine for eosinophils. Besides attracting them to the site of inflammation, it also causes their proliferation, activation, and increased survival. Other eosinophilic cytokines are IL3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and chemokines. Upon activation, eosinophils release mediators such as eosinophilic cationic proteins, major basic proteins, leukotrienes, and prostaglandins. These and other mediators enhance inflammation and cause epithelial damage. This results in bronchoconstriction clinically 4–12 h later and prolonged bronchial hyperresponsiveness, mucus secretion, and edema formation. Further secretion of a host of cytokines including IL-3, IL-4 and IL-5, contribute to an ongoing inflammation. Chronic Inflammation

With continued or repeated exposure to allergen, a state of chronic inflammation develops with increased numbers of activated Th2 cells, expressing mRNA for the secretion of IL-3, IL-4, IL-5, and GMCSF. These cytokines are important in the continuation of inflammation and the attraction of mast cells and eosinophils. These cells cause further increase in histamine, prostaglandins, and eosinophilic toxic products, causing epithelial damage. There is upregulation of intercellular adhesion molecules in the blood vessels promoting stickiness of the endothelium to leukocytes and facilitating their passage across, into the tissues. Increased permeability and cellular infiltration causes mucosal edema. Even in patients with mild, intermittent asthma, a state of low-grade inflammation persists, in the absence of symptoms. It is hypothesized that almost continuous exposure to very small amounts of allergens, such as house dust-mite, or pollen during summer, contributes to this ongoing allergic reaction without causing symptoms. Under the influence of IL-4 from mast cells, more B cells are switched to the

ALLERGY / Allergic Reactions 75

production of IgE antibodies, thus maintaining allergic reaction. Bronchoscopy studies reveal increased numbers of activated inflammatory cells and cytokines in the respiratory mucosa and secretions. Clinical Effects

The clinical features of asthma are due to the airway narrowing causing obstruction to airflow. This airway obstruction has three elements: 1. Excessive bronchial smooth muscle contraction. Inflammatory mediators such as histamine, bradykinin, prostaglandins, and leukotrienes act directly on their specific receptors to cause bronchoconstriction. In asthma, the smooth muscles contract easily and excessively following exposure to inflammatory mediators perhaps due to heightened sensitivity of their receptors. On the other hand, b2-receptors may have a diminished response. This feature is called bronchial hyperresponsiveness. 2. Thickening of bronchial wall. Bronchial wall thickening is due to inflammatory and fibrotic changes. Increased capillary permeability allows plasma exudation into the mucosa causing edema and cellular infiltration. Proliferation of fibroblast and myofibroblast leads to thickening of the basement membrane with deposition of collagen and hypertrophy of bronchial smooth muscles (airway remodeling). This leads to irreversible airway obstruction in chronic asthma. 3. Excessive luminal secretions and cellular debris. There is excess mucus secretion due to glandular hyperplasia. The epithelium is fragile and damaged epithelial cells are found in the sputum. Impaired ciliary function encourages retention of thick mucus in the lumen. During severe exacerbation, the lumen of the airway is blocked by thick mucus, plasma proteins, and cell debris. Allergic reactions in the nose follow a similar process with inflammation of the nasal mucosa, resulting in rhinorrhea, sneezing, and nasal blockage.

Systemic Allergic Reactions and Anaphylaxis Allergic reactions vary widely in severity from mild pruritis and urticaria to circulatory collapse and death. An acute systemic allergic reaction with one or more life-threatening features, such as stridor or hypotension, is termed anaphylaxis. Allergic reactions with troublesome but not life-threatening reactions, such as generalized urticaria/angioedema and

bronchospasm of mild to moderate severity, may be called severe allergic reactions. Traditionally, the term anaphylaxis is used for IgE-mediated reactions. Systemic reactions that clinically resemble anaphylaxis but are caused by non-IgE-mediated mediator release from mast cells and basophils are referred to as anaphylactoid reactions. Anaphylaxis occurs in 30/100 000 population/year with a mortality of 1–2%. Offending agents include foods, drugs, insect stings, and exercise but in 20% of cases no cause can be found (idiopathic). Pathogenesis

Systemic allergic reaction occurs as a result of degranulation of mast cells and basophils. Mast cells in respiratory and gastrointestinal tract, skin, and perivascular tissues are involved in both IgE and non-IgEmediated allergic reaction. IgE-mediated release is caused by antigen-specific cross-linking of IgE molecules on the surface of tissue mast cells and peripheral blood basophils. Non-IgE-mediated release may be due to direct stimulation of mast cells. Mast cells produce both histamine and tryptase while basophils secrete histamine but not tryptase. Histamine is a primary mediator of anaphylaxis and signs and symptoms of anaphylaxis can be reproduced by histamine infusion. Clinical Features

Anaphylaxis is a rapidly developing generalized reaction that involves respiratory, cardiovascular, cutaneous, and gastrointestinal systems. Clinical manifestations vary depending on the cause of anaphylaxis, route of entry, host factors (such as degree of sensitization, associated factors such as exercise, comorbidity, etc.) and the amount of allergen exposure. The initial symptoms, such as nasal congestion or pruritis, can quickly progress to collapse or death. Laryngeal edema, cardiovascular collapse, and severe bronchospasm are life-threatening features. In one large series of fatal anaphylactic reactions, 70% of the deaths were from respiratory causes, and 24% were from cardiovascular causes. In 10–20% of cases, skin may not be involved. Anaphylaxis can be confused with septic or other forms of shock, asthma, airway foreign body, and panic attacks (Table 1). Symptoms commonly occur within a few seconds or minutes of exposure and death may occur within minutes. Speed of onset of symptoms is indicative of the severity. Occasionally, the onset of symptoms may be delayed for 2 h or more. In general, the later the symptoms begin after exposure to a causative agent, the less severe the reaction. Food allergens may have slower onset or slow progression and gastrointestinal

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Table 1 Differential diagnosis of anaphylaxis Acute cardiac event Vasovagal syncope Acute angioedema Acute severe asthma Pulmonary embolism Foreign body inhalation Carcinoid syndromes Pheochromocytoma Seizure Systemic mastocytosis Panic attack Vocal cord dysfunction

symptoms are more common. Onset of anaphylaxis to insect stings or allergen injections is usually rapid: 70% begin in o20 min and 90% in o40 min.

Table 2 Clinical features of anaphylaxis Symptoms

Physical examination

Cardiovascular

Faintness, syncope, palpitations

Throat

Throat tightness, hoarseness, inspiratory stridor, difficulty in swallowing Nasal congestion, rhinorrhea, sneezing Pruritis, lacrimation Generalized warmth, tingling, pruritis, rash Chest tightness, wheezing, cough, shortness of breath Nausea, vomiting, abdominal pain, bloating, cramps, diarrhea Dizziness, a sense of impending doom, lightheadedness

Hypotension, tachycardia, arrhythmias Laryngeal edema

Upper respiratory tract Ocular Skin

Chest

Management Gastrointestinal

A quick initial assessment should determine the nature and progression of the clinical event (Table 2). Continuous monitoring is essential as progression from a mild to a severe episode may occur rapidly. Epinephrine injected intramuscularly into the thigh provides the most efficient absorption (Figure 3). If there is no response to several doses of intramuscular epinephrine, intravenous administration may be needed, by using a formulation of 1:10 000 (0.1 mg ml 1) at 1 mg min 1, which can be increased to 2–10 mg min 1. If the response is still inadequate, transfer the patient to an intensive care unit for close monitoring and endotracheal intubation, if required. Specific treatment for coexisting medical conditions (e.g., coronary artery disease) may be necessary. There may be complete resolution of the reaction. However, if there are concerns, continued monitoring for remaining or recurring symptoms is essential. A short course of corticosteroids may reduce the risk of recurring or protracted symptoms of a biphasic reaction but this is not proven. Patients receiving badrenergic blocking agents may not respond adequately to epinephrine. They require continued fluid replacement and may respond to glucagon. Patients receiving angiotensin-converting enzyme inhibitors may also be at increased risk of anaphylaxis and be more refractory to treatment with epinephrine. If there is any doubt regarding the diagnosis, blood should be taken for plasma histamine or serum tryptase levels within the first 4 h after the onset of symptoms. Elevated serum levels of b-tryptase indicate mast cell activation and degranulation in both IgE-mediated (anaphylaxis) and non-IgE-mediated (anaphylactoid) reactions. b-tryptase is useful in differentiating anaphylaxis from other events having

Neurologic

Conjunctival injection Flushing, urticaria, swelling of the lips, tongue or uvula Wheezing, tachypnea, cyanosis, respiratory arrest

Loss of consciousness, seizures

similar clinical manifestations, particularly if hypotension is present. Blood for plasma histamine needs to be processed immediately to avoid detecting artificially high levels due to spontaneous basophil histamine release. If this is not possible, urinary histamine (or metabolites) levels can be checked for up to 24 h. After initial treatment of acute anaphylaxis, the patient should be followed-up closely for the possibility of recurrent episodes. For mild to moderate episodes and good response to treatment, further monitoring can be done at home. However, following a severe episode, in-patient monitoring may be required for late-phase reactions. Subsequent Assessment

All individuals who have had a known or suspected anaphylactic episode require a careful allergy evaluation. The aims are to review the diagnosis of anaphylaxis and prevent or minimize the risk of future anaphylactic episodes by identifying the cause, educate the patient and/or family members regarding avoidance of the offending agent, education and training to deal with future inadvertent exposures, and consideration of desensitization, if appropriate. The level of confidence in the diagnosis of the original episode should be reviewed with details of the


Evaluate: breathing status, pulse, blood pressure, and level of consciousness Recognize life-threatening features such as stridor, shock, arrhythmia, seizure, and loss of consciousness

• Institute CPR if there is loss of circulation or respiration • Maintain airway with an airway device or tracheotomy • Oxygen, if there is circulatory or respiratory compromise

• Epinephrine (1:1000) 0.3−0.5 ml i.m. (children: 0.15 ml), repeat every 10−15 min, as needed • Chlorpheniramine 10 mg i.v. (then 6 hourly) • Hydrocortisone 200 mg i.v.

Improve

Problems persist

Late-phase symptoms

Monitor

Discharge home

• Hypotension: intravenous fluids (colloids) and, if needed, vasopressor agents (e.g., dopamine) • Bronchospasm: nebulized 2-agonist, consider use of i.v. aminophylline. Mechanical ventilation may be required • Urticaria/angioedema: oral/i.v. antihistamines and steroids Figure 3 Emergency management of anaphylactic and anaphylactoid reactions.

events before and during the episode. Results of any laboratory tests (e.g., serum tryptase or urine histamine) may be helpful in supporting the diagnosis of anaphylaxis and differentiating it from other entities. However, a careful and comprehensive history is the most useful part of the assessment. Information on any previous similar episodes, known food or drug allergy, and medication record should be sought. The history might suggest a specific cause such as insect sting or peanut consumption just before the episode. However, the cause may not be obvious from history and sometimes no cause can be found despite thorough searching (idiopathic anaphylaxis). Diagnostic Tests

Skin prick tests (SPTs) or determination of specific IgE in serum (in vitro test) is helpful in identifying a

specific cause of anaphylaxis in cases of food, insect, and some cases of drug (penicillin, insulin) allergy. An SPT is more sensitive than in vitro testing and is the diagnostic procedure of choice. When possible, standardized extracts should be used. If skin tests or in vitro tests do not provide a cause, then challenge to the suspected agent/agents should be considered. Challenge procedures are helpful in IgE-mediated allergic reaction where standardized test material is not available and in non-IgE anaphylactic reactions (such as to aspirin). Prevention

Once the offending agent has been identified (e.g., food, medication, or insect sting), patients should be educated regarding the specific exposures and be counseled on avoidance measures (Table 3). All those

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Table 3 Specific measures to reduce allergic reactions to common provoking agents Provoking agents

Specific preventive methods

Foods

Patients should be taught to read and interpret food labels They should be encouraged to ask about ingredients in restaurant meals They should be provided with a list of alternative foods School personnel should be fully informed of the pupil’s allergy history Patients should carry a medical identification bracelet or card Use alternatives, when possible, but avoid crossreactive antibiotics If penicillin is essential, desensitization could be performed Remove any hives or nests in the garden Wear long shoes and full trousers when walking in the fields Be alert to the presence of insects when outdoors Discontinue exercise at the earliest symptom Avoid any exacerbating factors such as aspirin or NSAIDs Avoid exercise for 4–6 h after eating if there is a history of anaphylaxis after food ingestion Patients should carry a medical identification bracelet or card All procedures should be conducted in a latex-free environment Food with known crossreactivity to latex should be avoided Consider an alternative procedure that does not require RCM Use low osmolar RCM Consider pretreatment with steroids, antihistamine, and ephedrine Allergen immunotherapy should only be administered under the supervision of a trained allergist Consider alternative therapy in those who are at higher risk (Table 4) Care should be taken to avoid dosing errors Observe patients for at least 20 min after the injection Consider reducing the dose, if interval between injections had been longer than planned or on opening of new vials Adjust dose if large local reaction occurs Reduce dose in highly sensitive patients or if there is concomitant high allergen exposure

Penicillin

Insect sting

Exercise

Latex

Radiocontrast material (RCM) Allergen extracts

with a risk of future anaphylaxis outside the medical settings should carry and be educated in the use of self-injectable epinephrine and antihistamines. Self-injectable epinephrine is available in two different strengths (for adults, 0.3 ml of 1:1000 solution and for children, 0.3 ml of 1:2000 solution) in readyto-use syringes. Antihistamine (such as chlorpheniramine 4–8 mg orally) may be sufficient for a mild episode but epinephrine should not be held back if symptoms are severe from the outset or response to antihistamine is inadequate. Humanized, monoclonal anti-IgE antibody has shown protection against peanut-induced anaphylaxis.

Allergic Reactions Foods

Food allergic reactions are common in children and presents clinically with systemic involvement (as described above), although oral (itching, numbness and tingling of lips and mouth) and gastrointestinal symptoms may be more prominent. Although any food can cause a reaction, commonly implicated foods are milk, egg, peanuts, tree nuts, fish, and shellfish. Symptoms often occur within minutes of ingestion and certainly within 2 h. Assessment of specific IgE to

suspected food, either by skin prick test or in vitro test, in the presence of a suggestive history, is sufficient to make a definitive diagnosis. However, food challenges (single or double blind) may be required. Strict avoidance of the offending food is essential. Patients should also carry, and be trained, in the use of epinephrine in an emergency following inadvertent exposure. Penicillin

Allergic reaction to penicillin is the most common cause of anaphylaxis. Severe reactions are usually attributable to parenteral administration. Atopy and family history of penicillin allergy does not increase the risk of a reaction. A history of penicillin allergy is not reliable as nearly 80% of these individuals tolerate penicillin without ill effects. However, these subjects should have a skin test to major and minor determinants before penicillin is administered. The risk of a reaction following a negative test is less than 2%. Skin tests do not resensitize a patient to penicillin. A positive skin test in the presence of a history of reaction to penicillin indicates a more than 50% risk of an allergic reaction and penicillin should be avoided, or if penicillin is mandatory, desensitization should be considered. Cross-reactivity exists between


cephalosporins and penicillins due to the common b-lactam ring structure. Aspirin and Nonsteroidal Anti-Inflammatory Drugs

Aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) can induce life-threatening systemic, nonIgE-mediated reaction causing rhinoconjunctivitis, bronchospasm, urticaria, and laryngeal edema. In vitro or skin tests are not available and oral challenges are required for confirmation. If the diagnosis is confirmed, aspirin and NSAIDs should be strictly avoided. Desensitization may be performed if these drugs are considered essential. Insect Sting

Stinging insects belong to the order Hymenoptera. Common stinging insects include honeybees, wasps, yellow jackets, hornets, and fire ants. The self-reported prevalence of insect sting allergy is approximately 1%. Insect venoms contain several well-characterized allergens that can trigger anaphylactic reactions. Localized reaction may occur at the site of the sting and a large local reaction may involve, for example, the whole limb. However, these do not predispose to systemic allergic reaction. Urticaria and angioedema are key features of systemic allergic reactions caused by insect sting. Skin test is the preferred method of confirming allergy and identifying the responsible insect species. However, careful interpretation is needed, as falsepositive reactions are common. Following a systemic allergic reaction, only about half of the patients will react to a future sting. Patients should carry self-injectable epinephrine for early treatment of a sting, and allergen immunotherapy should be considered, where risk of future sting is substantial. The immunotherapy is 490% effective but optimal duration is not known. Intraoperative Drugs

Common agents responsible for intraoperative anaphylaxis are neuromuscular blocking agents, latex, antibiotics, anesthesia induction agents, radiocontrast material, and opioids. Neuromuscular blocking agents and thiopental are responsible for most anaphylactic reactions during general anesthesia. Both IgE-mediated (muscle relaxants, latex) and direct stimulation of mast cell (opioids, radiocontrast material) occurs. Clinical manifestations of intraoperative reactions differ from anaphylactic reactions due to other causes as cardiovascular collapse, airway obstruction, and flushing is prominent. It may be difficult to differentiate allergic reaction from the pharmacologic effects of a variety of medications

administered during general anesthesia. Plasma tryptase is useful in differentiating allergic reaction (due to mast cell release of mediators) from pharmacologic or other causes. Skin testing is used for diagnosis where IgE-mediated mechanism is suspected. Otherwise, graded challenge may be required. Latex

IgE-mediated allergic reactions to natural rubber latex became common during the 1990s due to a sudden increase in the use of rubber gloves. Risk factors for latex allergy include atopy and previous repeated exposure to latex (e.g., multiple surgical procedures, healthcare workers). Skin tests are indicated for investigation of latex allergy in those who have a history of possible latex allergic reaction and for screening those who are at high risk. However, in the last few years, the use of latex-free gloves and other products have been effective in reducing the occurrence of latex allergic reactions. Blood Transfusions

Allergic reactions may complicate 1–3% of blood transfusions. Most reactions are mild, are associated with cutaneous manifestations such as pruritis, maculopapular, or urticarial rash and flushing, and require no specific treatment except discontinuation of transfusion and perhaps antihistamine. However, severe or life-threatening reactions with hypotension and bronchoconstriction may occur occasionally. Most of these reactions are due to IgE or IgM antibodies to serum proteins (e.g., albumin, complement components, IgG, and IgA). Other mechanisms include transfusion of allergen, IgE antibodies, bacterial components or inflammatory substances such as cytokines, histamine, or bradykinin. Blood components containing large amounts of plasma, such as fresh frozen plasma, may be associated with more severe allergic reactions. Serum tryptase, measured soon after the reaction, may differentiate allergic from transfusion-related reaction. Allergen Extract

Allergen extracts are injected during skin test and allergen-specific immunotherapy. The risk of allergic reaction is extremely low with skin prick test but not insignificant when these extracts are injected for treatment. In a survey between 1985 and 1989 in the US, there were 17 deaths from allergen immunotherapy but none from skin testing. Mild, localized reactions are relatively common and respond to antihistamine. Factors that increase the risk of an allergic reaction should be kept in mind by those involved

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ALLERGY / Allergic Rhinitis

Table 4 Risk factors for systemic reactions during allergen immunotherapy Severe asthma Highly sensitive patient Too rapid increase in the dose of allergenic extract Starting a new vial of extract A history of previous systemic reactions to allergen immunotherapy Asthmatic symptoms present immediately before receiving an injection Administration of pollen extracts during high environmental pollen exposure Concomitant treatment with b-adrenergic blocking agents Fever or an upper respiratory tract infection at the time of administration of allergenic extracts

Lieberman P (2002) Anaphylactic reactions during surgical and medical procedures. Journal of Allergy and Clinical Immunology 110(supplement 2): S64–S69. Moffitt JE (2003) Allergic reactions to insect stings and bites. Southern Medical Journal 96(11): 1073–1079. Moneret-Vautrin DA and Kanny G (2002) Anaphylaxis to muscle relaxants: rational for skin tests. Allergic Immunology (Paris) 34(7): 233–240. Noone MC and Osguthorpe JD (2003) Anaphylaxis. Otolaryngologic Clinics of North America 36(5): 1009–1020. Sampson HA (2003) Anaphylaxis and emergency treatment. Pediatrics 111(6): 1601–1608. Sicherer SH and Leung D (2004) Advances in allergic skin disease, anaphylaxis, and hypersensitivity reactions to foods, drugs, and insect stings. Journal of Allergy and Clinical Immunology 114(1): 118–124. Tang AW (2003) A practical guide to anaphylaxis. American Family Physician 68(7): 1325–1332.

in administration of allergen-specific immunotherapy (Table 4). Exercise

Allergic Rhinitis

Exercise-induced anaphylaxis is a physical form of allergy that may occur in isolation or in combination with ingestion of food or drug, such as aspirin or NSAIDs. The episode resembles typical anaphylaxis and emergency treatment is the same as for anaphylaxis due to other causes. Patients should carry self-injectable epinephrine. Exercise may also cause urticaria or bronchospasm without anaphylaxis.

P van Cauwenberge, J-B Watelet, T Van Zele, and H Van Hoecke, Ghent University Hospital, Ghent, Belgium

See also: Allergy: Overview. Asthma: Overview. Chemokines, CXC: IL-8. Immunoglobulins. Interferons. Interleukins: IL-4; IL-10; IL-12; IL-13. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumor Necrosis Factor Alpha (TNF-a ).

Further Reading Cockcroft DW (1998) Airway responses to inhaled allergens. Canadian Respiratory Journal 5(supplement A): 14A–17A. El Biaze M, Boniface S, Koscher V, et al. (2003) T cell activation, from atopy to asthma: more a paradox than a paradigm. Allergy 58(9): 844–853. Gould HJ, Sutton BJ, Beavil AJ, et al. (2003) The biology of IgE and the basis of allergic disease. Annual Review of Immunology 21: 579–633. Holgate ST, Davies DE, Puddicombe S, et al. (2003) Mechanisms of airway epithelial damage: epithelial–mesenchymal interactions in the pathogenesis of asthma. European Respiratory Journal 44(supplement): 24s–29s. Joint Task Force on Practice Parameters, American Academy of Allergy, Asthma and Immunology, American College of Allergy, Asthma and Immunology, and the Joint Council of Allergy, Asthma and Immunology (1998) The diagnosis and management of anaphylaxis. Journal of Allergy and Clinical Immunology 101(6 Pt 2): S465–S528. Kemp SF and Lockey RF (2002) Anaphylaxis: a review of causes and mechanisms. Journal of Allergy and Clinical Immunology 110(3): 341–348.


Abstract Over the last decades, the prevalence of allergic rhinitis has risen to epidemic proportions. Nasal symptoms involve sneezing, nasal itch, rhinorrhea, and nasal congestion. These symptoms result from an immunologically mediated (usually IgE-mediated) inflammation of the nasal mucosa, following allergen exposure in sensitized patients. Although allergic rhinitis is often trivialized, it has become clear that the disease can cause serious morbidity beyond the nasal manifestations, that it has a significant impact on quality of life and substantial socioeconomic consequences, and that it is associated with multiple comorbidities, including asthma, conjunctivitis, sinusitis, and otitis media. Recently, the Allergic Rhinitis and its Impact on Asthma (ARIA) Working Group proposed a new classification for allergic rhinitis, based on the duration of symptoms, rather than on the type of exposure. The severity of the disease is categorized based on the impact of symptoms on quality of life parameters. Early and correct diagnosis is the basis for the management of allergic rhinitis and starts with a thorough clinical history and physical examination. To confirm the allergic origin of rhinitis symptoms, allergy tests are performed. The test of first choice is the skin prick test, which has a good sensitivity and specificity. Environmental control measures (allergen avoidance), pharmacological treatment, immunotherapy, and education are the cornerstones of therapeutic management of allergic rhinitis. Nowadays, many effective pharmacological agents are available and new potential targets for pharmacotherapy, new routes of administration, and alterations in treatment dosages and schedules are continuously being investigated. To facilitate and standardize the management of allergic rhinitis and to improve the patient care, satisfaction, and compliance, several clinical practice guidelines have been developed. Among those, the ARIA guidelines provide stepwise treatment recommendations, based on the best available evidence from research.

ALLERGY / Allergic Rhinitis 81

Introduction Allergic rhinitis (AR) is defined as a nasal disease with the presence of immunologically mediated hypersensitivity symptoms of the nose, for example, itching, sneezing, increased secretion, and blockage. The great majority of cases are immunoglobulin E (IgE) antibody-mediated. In the recent Allergic Rhinitis and its Impact on Asthma (ARIA) report, AR is considered as a major chronic respiratory disease because of its high prevalence in all countries, its significant impact on quality of life or work performance, its considerable economic burden, and its association with multiple comorbidities, asthma in particular. Several important epidemiological surveys (e.g., European Community Respiratory Health Survey, International Study of Asthma and Allergies in Childhood (ISAAC), and Swiss Study on Air Pollution and Lung Diseases in Adults) have recently improved our knowledge about the prevalence of rhinitis. The majority of monocentric studies have reported a prevalence of seasonal AR ranging from 1% to 40% and a prevalence of perennial rhinitis ranging from 1% to 18%. Furthermore, the ISAAC study phase 1 confirmed this large variation in the prevalence of rhinitis symptoms throughout the world. Over the last 40 years the prevalence of AR, like other allergic disorders, has risen to truly epidemic proportions. Despite the increasing insight into the pathophysiology of allergy and the availability of more effective treatment options, this upward trend in the incidence of AR continues, being most prominent in countries with a Western lifestyle, especially among children and adolescents. As a consequence, great attention is being paid to the identification of the factors responsible for this disease. It is well established that allergic diseases tend to occur within families and have a genetic basis. However, numerous generations are needed before changes in the gene pool occur. Therefore, the recent increase in prevalence of AR and allergy in general cannot be explained by genetic factors and is largely attributed to alterations in the environment. Diverse environmental and lifestyle factors (prenatal maternal influences, allergen exposure, active and passive smoking, viral and other respiratory infections, early life microbial exposure, indoor air quality/house dampness, outdoor air pollution, urban vs farming living environment, socioeconomic status, dietary factors, etc.) have been identified as possible risk factors or as protective factors in the pathogenesis of allergy, although the evidence supporting their involvement varies widely. In order to allow the introduction of individualized primary and secondary

prevention strategies and to meet the challenge of the growing impact of allergy, further assessment of the complex interactions within and between genetic and environmental determinants is required.

Etiology Allergens are antigens that induce and react through an IgE-mediated inflammation. The number of identified allergens has expanded enormously. They originate from animals, insects, plants, or fungi. In AR, airborne allergens are the most common provoking agents and the increase in time spent indoors in recent years is thought to be responsible for the rise in incidence of allergic diseases. The most common indoor allergens are derived from mites, domestic animals, insects, or plants. Mites, such as Dermatophagoides pteronyssinus or D. farinae, usually induce asthma or perennial AR. They feed on human skin and multiply under hot and humid conditions. Other types of mite, such as Tyrophagus putrecentiae and Acarus siro, are present in flour. The dander and secretions of many animals contain powerful allergens capable of inducing severe reactions. They can remain airborne for prolonged periods of time. Specific allergens have been identified in cat, dog, horse, cattle, rabbit, and other rodents. Outdoor allergens include pollen and molds. Pollen can be categorized into two groups based on their mode of transport: anemophilous pollen are carried by the wind and can be transported over long distances, while entemophilous pollen are carried by insects. The nature and number of pollen vary with geography, temperature, and climate. Fungi, molds, and yeasts can liberate large quantities of allergenic spores into the atmosphere. Their development and growth are faster in hot and humid conditions. The main atmospheric molds are Cladosporium and Alternaria. Finally, it has been noted that inhalation of insect waste or some bacteria seems to be able to induce an IgE-mediated reaction. AR can also be triggered by food and occupational allergens. Many allergens have enzymatic activity. Simultaneous exposure to both allergenic and proteolytic activity probably allows greater access to cells of the immune system and enhances sensitization and allergic inflammatory reactions.

Pathology Classification of Allergic Rhinitis

Before the publication of the ARIA report, AR was subdivided into seasonal AR, perennial AR, and by extension occupational AR, based on the time of

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exposure to the offending allergen(s). Seasonal AR is related to a wide variety of outdoor allergens such as pollens and molds. Perennial AR is most frequently caused by indoor allergens such as house dust mite, molds, cockroaches, and animal dander. Occupational AR occurs in response to airborne allegens in the workplace. Common causes are laboratory animals, wood dust (particularly hard woods), chemicals, and solvents. The distinction between seasonal and perennial AR, however, is not applicable in all patients and in all countries because *

*

*

*

symptoms of perennial rhinitis may not be present all year round; pollen and molds are perennial allergens in some parts of the world; many patients are sensitized to multiple allergens and present with symptoms during a number of periods in the year or even throughout the year; and symptoms of seasonal AR do not always occur strictly within the defined allergen season, due to a ‘priming effect’ and the concept of ‘minimal persistent inflammation’.

Therefore, this classification was considered to be inaccurate and the ARIA Working Group proposed a major change in the classification of AR. The ARIA classification for AR uses the terms ‘intermittent’ and ‘persistent’ to describe the duration of symptoms. Based on the impact of symptoms on quality-of-life parameters, the severity of the disease is classified as ‘mild’ or ‘moderate–severe’ (Table 1).

Histopathology of Allergic Rhinitis

Pollen-induced rhinitis is the most characteristic IgE-mediated allergic disease and is triggered by the interaction of mediators released by cells that are implicated in both allergic inflammation and nonspecific hyperreactivity. AR is characterized by a huge inflammatory reaction. The resulting inflammatory cell infiltrate is made up of different cells. The cellular response begins with chemotaxis, selective recruitment, and transendothelial migration of cells. These cells can be localized within the different compartments of the nasal mucosa and represent an activation state. Their survival is prolonged and they release a large amount of inflammatory mediators. They also participate in the regulation of IgE synthesis and communicate with the immune system. Biopsies from allergic nasal tissue demonstrate a thicker basement membrane and a greater number of intraepithelial monocytes, subepithelial eosinophils, and neutrophils (Figure 1).

Clinical Features Symptoms of rhinitis include rhinorrhea, nasal obstruction, nasal itch, and sneezing; often, patients with rhinitis are subdivided into ‘sneezers and runners’ and ‘blockers’, based on the main symptom(s).

Table 1 ARIA classification for allergic rhinitis ‘Intermittent’ rhinitis Symptoms are present: p4 days a week or p4 consecutive weeks

‘Persistent’ rhinitis Symptoms are present: 44 days a week and 44 consecutive weeks

‘Mild rhinitis’

‘Moderate/severe’ rhinitis X1 of following items are present:

None of following items are present: sleep disturbance impairment of daily activities, leisure, and/or sport impairment of work or school work troublesome symptoms In untreated patients

Adapted from Bousquet J, Van Cauwenberge P, Khaltaev N, Aria Workshop Group, World Health Organization (2001) Allergic rhinitis and its impact on asthma. Journal of Allergy and Clinical Immunology 108(5 supplement): S147–S334.

Figure 1 Hematoxylin-eosin staining (magnification 40) of normal nasal mucosa demonstrating epithelial cells, seromucous glands, and subepithelial lymphoid layer.


These rhinitis symptoms, however, do not necessarily have an allergic origin. In the differential diagnosis, AR must be differentiated from several types of nonallergic rhinitis and other nasal inflammatory conditions (Tables 2 and 3). The clinical history remains the most essential step for establishment of the diagnosis. Apart from the classical rhinitis symptoms, the patient must be questioned about the presence of other symptoms commonly associated with rhinitis, such as loss of smell, snoring, sleep disturbance, postnasal drip, cough, sedation, conjunctivitis, and lower respiratory symptoms. The history should also contain an evaluation of the severity and duration of the problem, the impact on daily life, and the response to treatment, and should document potential allergic and nonallergic triggers; it must also include a family and occupational history.

Clinical examination starts with a general inspection of the nose, ears, and throat. A complete and systematic nasal examination is required, especially in patients with persistent rhinitis. However, anterior rhinoscopy gives only limited information. Nasal endoscopy is therefore an essential complementary investigation, not to confirm AR, but to exclude other conditions, such as polyps, foreign bodies, tumors, and septal deformations. During allergen exposure, the sinonasal mucosa of patients with AR can demonstrate a bilateral, but not always symmetrical, swelling. Often, mucosal changes in color are seen, from a purplish to a more common pale coloration. An increase in vascularity is also commonly noticed. In the absence of allergen exposure, the nasal mucosa may appear completely normal, but in patients who have suffered from rhinitis for several years, irreversible mucosal hyperplasia and/or viscous secretions may also occur (Figures 2–6).

Table 2 Classification of rhinitis Allergic rhinitis Infectious rhinitis: viruses, bacteria, fungi Occupational rhinitis: allergic and nonallergic Drug-induced rhinitis, e.g., aspirin Hormonal rhinitis: puberty, pregnancy, menstruation, endocrine disorders Emotional rhinitis Atrophic rhinitis Irritant-induced rhinitis Food-induced rhinitis, e.g., red pepper NARES: nonallergic rhinitis with eosinophilic syndrome Rhinitis associated with gastroesophagel reflux Idiopathic rhinitis Adapted from International Rhinitis Management Working Group (1994) International Consensus Report on the Diagnosis and Management of Rhinitis. Allergy 49 (supplement 9): 5–34.

Diagnostic Evaluation

To confirm the allergic origin of rhinitis symptoms, the ARIA Working Group states that allergy tests should be performed. In vivo and in vitro tests for the diagnosis of allergic diseases are directed towards the detection of free or cell-bound IgE. Immediate hypersensitivity skin tests are a major diagnostic tool to demonstrate IgE-mediated allergic reactions. If properly performed, skin tests are the best available method for detecting the presence of allergen-specific IgE. The first choice of test is the skin prick test, which has a good sensitivity and specificity (Figures 7 and 8). It is very important that skin tests are performed carefully and interpreted correctly. Therefore,

Table 3 Differential diagnosis of rhinitis Mechanical factors Deviated septum Adenoidal hypertrophy Hypertrophic turbinates Foreign bodies Choanal atresia Tumors Benign Malignant Granulomas Wegener’s granulomatosis Sarcoid Infectious (tuberculosis, leprosy) Malignant – midline destructive granuloma Ciliary defects Cerebrospinal rhinorrhea Adapted from International Rhinitis Management Working Group (1994) International Consensus Report on the Diagnosis and Management of Rhinitis. Allergy 49(supplement 9): 5–34.

Figure 2 Profuse watery anterior rhinorrhea in a child with allergic rhinitis.

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Figure 3 The ‘nasal salute’ refers to habitual rubbing of the nose due to constant irritation. It usually signifies an underlying allergic phenomenon of producing a profuse rhinorrhea requiring frequent wiping. It is most marked in children.

Figure 4 Habitual rubbing of the nose usually produces a horizontal ‘nasal crease’ below the bridge of the nose.

Before allergen challenge

After allergen challenge

Figure 5 Schematic presentation of sinonasal mucosa swelling before and after allergen challenge.

it is recommended that trained healthcare professionals carry them out. The skin reaction can be affected by the quality of the allergen extract, the patient’s age, the use of some pharmacological agents (e.g., oral antihistamines and topical skin corticosteroids), and can also demonstrate seasonal variations. In addition, the possibility of false-positive and false-negative results must be considered (Table 4). Measurement of total serum IgE lacks specificity and is of little predictive value in allergy screening in rhinitis. On the contrary, serum-specific IgE is as valuable as skin testing. Skin tests, however, are less expensive, have a greater sensitivity, allow a wide allergen selection, and give results in less than half an hour. Serum-specific IgE is indicated in young children, in patients with dermographism or widespread dermatitis, in patients who are noncompliant for skin testing, or in those who did not continue with antihistamine treatment (short-acting antihistamines for 36–48 h, long-acting antihistamines for 4–6 weeks), as this can result in false-negative skin test results. Serum-specific IgE measurement is also a safer option in patients who are very allergic and where an anaphylactic reaction to skin testing is a possible risk. It is important to remember that positive in vivo or in vitro tests for allergen-specific IgE must always be interpreted in relation to the entire clinical presentation. The presence of allergen-specific IgE antibodies is not sufficient for the diagnosis of allergic disease, as patients can be sensitized in the absence of (or prior to) the development of any symptoms. Nasal challenge tests are used in particular for research purposes and are important in the diagnosis of occupational rhinitis. The International Committee on Objective Assessment of Nasal Airways has set up guidelines concerning the indications, techniques, and evaluation of nasal challenge tests. In addition to allergen provocations, nasal challenge tests with aspirin, non-specific agents (histamine, metacholine), and occupational agents can be performed. Imaging (sinus plain radiographs, computed tomography, and magnetic resonance imaging) is not indicated for the diagnosis of AR, but may be necessary to exclude other conditions or complications. Other diagnostic tests that are employed to assess the nasal airways include nasal peak flow, rhinomanometry, and acoustic rhinometry, but these are rarely used in the diagnosis of AR. Objective testing of a patient’s ability to smell can be performed by olfactory testing. Mucociliary function can be measured by nasomucociliary clearance, ciliary beat frequency, or electron microscopy, but these tests are of little relevance in the diagnosis of AR.


Figure 6 Nasoendoscopic visualization (right nasal cavity: Hopkins 301) of nasal mucosa swelling in an allergic patient before (a) and (b) after allergen challenge. Table 4 Causes of false-positive and false-negative skin tests Causes of false-positive skin tests: * Dermographism * Irritant reactions * Non-specific enhancement of a nearby strong reaction * Improper technique/material Causes of false-negative skin tests: * Poor initial potency or loss of potency of extracts * Use of drugs modulating allergic reaction * Diseases attenuating skin response * Decreased activity of the skin (infants and elderly patients) * Improper technique/material

Figure 7 Kit with standardized allergen extracts and positive and negative control solutions used for allergy skin tests.

Adapted from Bousquet J, Van Cauwenberge P, Khaltaev N, Aria Workshop Group, World Health Organization (2001) Allergic rhinitis and its impact on asthma. Journal of Allergy and Clinical Immunology 108(5 supplement): S147–S334.

Comorbidities

Figure 8 The technique used for skin prick testing involves introducing a drop of diluted allergen followed by puncturing the skin with a calibrated lancet (1 mm) at an angle of 451. The drops should be placed 2 cm apart. All patients undergoing skin prick testing should also have a positive (histamine) and negative diluent (saline) control test included. Skin tests should be read at the peak of their reaction by measuring (in mm) the wheal and the flare around 15 min after pricking. The relevance of skin prick testing should always be interpreted in the context of the patient’s history. Positive results can occur in people without symptoms and, similarly, false-negative results may also occur.

It must be emphasized that AR should be evaluated as a global systemic disease. Allergic inflammation does not necessarily limit itself to the nasal airway, but the treating physician must also be aware of the possible comorbidities of AR, including asthma, conjunctivitis, sinusitis, and otitis media. The link between asthma and rhinitis in particular has gained much interest. Epidemiological studies have shown that up to 80% of patients with asthma demonstrate symptoms of rhinitis, while approximately 20–40% of patients with AR have clinical asthma. There is growing evidence that rhinitis is a risk factor for the development of asthma, independent of atopy. Additionally, the airway mucosa of nose and bronchi have many similarities and the clinical and pathophysiological changes in asthma and AR are often very comparable. Although there are still some differences that should be highlighted, the strong relationship between rhinitis and asthma has introduced the concept of ‘the united airway disease’.

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Based on these findings the ARIA guidelines recommend that patients with persistent AR should be evaluated for asthma by history, chest examination, and, if possible and where necessary, by the assessment of airflow obstruction before and after using a bronchodilator, whereas patients with asthma should be evaluated for rhinitis by history and physical examination. Another frequent comorbidity of AR is conjunctivitis. It is estimated that 42% of patients with AR experience symptoms of allergic conjunctivitis and that 33–56% of the cases of allergic conjunctivitis occur in association with AR. This coexistence, referred to as ‘rhinoconjunctivitis’, seems to be a typical feature in patients with seasonal pollen allergy. As eye symptoms substantially contribute to the burden of allergic rhinoconjunctivitis, adequate assessment and treatment of conjunctivitis should be part of the overall management. AR is also considered as a contributing factor in acute and chronic rhinosinusitis. Up to 54% of adults with chronic rhinosinusitis have symptoms of AR. Similarly, a high concordance (between 25% and 75%) of these disorders is found in children. Conversely, there is a high prevalence of sinus disease in patients with AR: abnormal sinus radiographs occur in over 50% of adults and children with perennial AR and acute rhinosinusitis occurs often during the allergy season. There is still some controversy regarding the etiological role of AR in otitis media with effusion. Most epidemiological data suggest an association between these two diseases. However, the available evidence is compromised by a possible referral bias and by the lack of prospective, controlled studies. It is still not clear whether AR predisposes to the development of otitis or whether nasal dysfunction worsens otitis. It can be concluded that although the exact pathophysiological links between AR and its several comorbidities still need to be elucidated, AR is not an isolated disorder but is part of a systemic disease process. Hence, the ARIA Working Group recommends a coordinated diagnostic and therapeutic approach instead of a fragmented, organ-based management.

Pathogenesis Symptoms of AR develop upon inhalation of allergens in individuals previously exposed to such allergens and against which they have made IgE antibodies. The pathophysiological process of AR can be subdivided in two phases: the initial sensitization phase during which allergen presentation results in primary allergen-specific IgE antibody formation

by B lymphocyte, and the clinical disease phase during which symptoms in response to subsequent antigen exposure become manifest. The clinical disease phase, in turn, can be subdivided into two distinct phases: an early phase largely mediated through mast cells; and a late phase, which involves cellular infiltration and mediator release. Sensitization Phase

The development of sensitivity to an allergen requires IgE antibody production directed at the epitope. After allergen exposure, antigen-presenting cells present the allergens to CD4 þ cells. A subset of these CD4 þ cells, the T-helper type 2 (Th2) lymphocytes, generate Th2 cytokines, including IL-4 and IL-13, which stimulate IgE synthesis in combination with B-cell–T-cell ligand–receptor interactions that are pivotal in the B-cell isotype switching toward IgE synthesis. These B-cell–T-cell interactions include a major histocompatibility complex class II and T cell receptor/CD3 interaction and a binding of the CD40L on T lymphocytes and CD40 expressed on B cells. As a result, allergen-specific IgE antibodies are produced and these sensitize mast cells and other IgE receptor-bearing cells. There is now increasing evidence that IgE is produced locally in the nasal mucosa since nasal B cells, in the presence of IL-4 and CD40L positive mast cells, are able to produce IgE locally. Clinical Disease Phase

The clinical disease phase consists of two phases: an early phase and a late phase. The early phase is largely mediated through mast cells. In sensitized patients, allergen re-exposure mediates cross-linkage, of adjacent IgE molecules bound to mast cell surfaces. If a mast cell is activated by IgE cross-linking, it releases granule products containing histamine, tryptase, chymase, and cytokines such as interleukin-4 (IL-4), IL-5, IL-8, IL-13, and tumor necrosis factor alpha (TNF-a) into the extracellular environment. Second upon activation mast cells generate arachidonic acid products including cysteinyl-leukotrienes (LTC4, LTD4, LTE4) and prostaglandin D2 (PGD2) from the phospholipid cell membrane. Nasal challenge with allergens shows the local release within 10– 15 min of histamine, tryptase, PGD2, LTB4, and LTC4. These mediators cause the characteristic watery rhinorrhea by stimulating gland and globet cell secretion, vasodilation, and blood vessel leakage, which is characteristic for the early phase. Histamine is the most important mediator in AR and induces symptoms of nasal itching, sneezing, discharge, and transient nasal blockage whereas leukotrienes appear to be relatively more important than histamine in


inducing nasal blockage. In addition, the mast cell is thought to contribute to other features in rhinitis, namely the eosinophilic mucosal inflammation. Eosinophils are present in nasal mucosal biopsies within the submucosa and epithelium in active rhinitis. They generate vasoactive mediators and have the capacity to produce cytotoxic proteins, including major basic protein, eosinophil peroxidase, eosinophil-derived neurotoxin, and eosinophil cationic protein. Although the eosinophils feature heavily in the late-phase allergic response, their role in the early-phase allergic response is not clear. The late phase starts 4–8 h after allergen exposure. Clinically, it can be similar to the early phase but, in general, nasal congestion is more prominent. The late response involves a process of cellular accumulation. The expression of adhesion molecules and the presence of cytokines IL-3, IL-4, IL-5, IL-8, granulocytemacrophage colony-stimulating factor, and TNF-a enhances cell activation, accumulation of neutrophils, eosinophils, and T lymphocytes, and prolongs the survival of eosinophils within the nasal mucosal tissue. There is evidence showing that, in persistent rhinitis, there is an upregulation of the adhesion mechanisms with increased expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1. During this late phase, activated basophils are responsible for histamine release. All these events amplify the allergic inflammatory response, leading to a real cascade of reactions (Figures 9 and 10).

Although the inflammatory reaction in AR is triggered by allergen exposure, it has been demonstrated that even in cases of subliminal exposure to the allergen(s), in the absence of symptoms, a certain degree of inflammatory infiltration at the mucosal level persists. This is called the ‘minimal persistent inflammation’. The Priming Effect

The ‘priming effect’ refers to the phenomenon where the amount of allergens necessary to evoke an immediate response decreases with repeated allergen challenges or exposures. Nasal challenge induces an immediate clinical response in allergic subjects and a concomitant appearance of an inflammatory infiltrate. The mucosal inflammation may persist 48–72 h after allergen exposure. If the subjects are rechallenged within this period the response is more pronounced: the so-called priming effect. This effect is hypothesized to be a result of the influx and subsiding activity of inflammatory cells during the latephase allergic response. Clinically, this explains the observation that decreasing allergen quantities are required to elicit symptoms as the pollen season progresses. In patients allergic to tree and grass pollen, the tree pollen season has a priming effect on the subsequent grass pollen season and these patients can often develop symptoms early in the grass pollen season when pollen counts are still very low.

Histamine Concentration

Leukotrienes Tachykinins

Early phase

Late phase

Time

Blockage Rhinorrhea Sneezing Itching Figure 9 Nasal allergen provocation results in a significant increase in mediators (histamine, leukotrienes, and tachykinins) and immediate nasal symptoms (blockage, rhinorrhea, sneezing, and itching) in allergic rhinitis patients. Between 2 and 24 h after allergen provocation, late-phase nasal symptoms, especially blockage, are observed in allergic rhinitis patients.

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IL-4 IL-13

Allergen

IgE Mast cell

IL-5 IL-4

Th2

IL-6 Th0

Eosinophil

IL-4

APC IL-12

Th1

IFN- IL-2

IL-18 Figure 10 Allergic inflammation is characterized by a preponderance of T-helper type 2 (Th2) lymphocytes over T-helper type 1 (Th1) cells. APC, antigen-presenting cell; IL, interleukin; IFN-g, interferon gamma; IgE, immunoglobulin e; Th0, precursor T cell.

Systemic Component in the Allergic Rhinitis Response

In addition to the local events in the nose, there is a systemic element to the inflammatory process of AR. A variety of mechanisms have been proposed to explain the pathophysiological link between the upper and lower airways, including the loss of nasal protective function, altered breathing pattern, postnasal drip causing pulmonary aspiration of nasal contents, the presence of a nasal–bronchial reflex, and the progression of systemic inflammation. Recent data suggests that bidirectional systemic inflammation involving the bone marrow is likely to be important. Local allergen provocation (in the nose or bronchi) leads to upregulation and release from the bone marrow of hemopoietic eosinophil/basophil progenitor cells, which migrate to both nose and lungs where they can undergo differentiation and activation in situ.

Animal Models Besides clinical and in vitro studies, animal models of allergic airway disease may provide useful information concerning upper airway inflammation and serve as a model for relatively invasive experimental procedures. Although AR in animals is rare, chronic rhinitis associated with Aspergillus infection has been reported in rodents, poultry, dogs, and horses. In the absence of a common naturally occurring model of AR, sensitivity to allergens must be induced in healthy animals. A number of research groups are currently applying different experimental protocols to

induce experimental airway allergy. In guinea pigs, ovalbumin (OVA) is the most common sensitizing agent. To induce sensitization OVA is repeatedly injected into the peritoneum over the course of several weeks; this is followed by exposure to aerosolized OVA, which leads to allergen-specific IgE production. In addition to systemic sensitization, intranasal applications of OVA may induce allergen-specific IgE production but this exclusive local sensitization has been described in only a small number of studies. The upper airways of mice are less attractive for research compared to the lower airways, and this is for several reasons. First, mice are obligatory nose breathers, resulting in a significant baseline inflammation. Second, eosinophilic inflammation present in the nose of mice with experimental airway inflammation is rather limited compared to the inflammatory response in the lower airways. Several animal models of AR have shed light on the cellular and cytokine profiles associated with airway inflammation. They demonstrate that the relative importance of mast cells, eosinophils, IgE, and the cytokines IL-4 and IL-5 in the development of allergic inflammation varies with the sensitization protocol used and with the animal strain.

Management and Current Therapy Environmental control measures (allergen avoidance), pharmacological treatment, immunotherapy, and education are the cornerstones of therapeutic management of AR. In select cases, nasal surgery may be recommended as an adjunctive intervention.

ALLERGY / Allergic Rhinitis 89 Prevention

The treatment of patients with AR starts with the identification of possible allergens and subsequent prevention. If possible, allergen avoidance is the first step in the management of AR. The most common indoor allergens are dust mites and dog and cat dander. The use of simple measures to avoid the allergens can relieve the symptoms but there is still a paucity of data relating to the effectiveness of avoidance. Clinical improvement can be expected within weeks. However, some allergens require a longer period before they are effectively removed. Pharmacological Agents

Intranasal steroids Intranasal steroids are a potent and highly effective treatment for patients with AR. Their effect is based on localized repression of the inflammatory response. They reduce the number of eosinophils, the amount of eosinophilic cationic protein present, and the number of mast cell progenitors. Their multiple sites of action may account for their extreme potency. Clinically their efficacy exceeds that of antihistamines, decongestants, and cromoglycin. Compared to antihistamines they are more effective in reducing nasal blockage. However, they have a limited effect on associated eye symptoms and a relatively slow onset of action. There is some evidence that intranasal steroids have a beneficial effect on asthma symptoms. At the recommended doses, intranasal steroids cause few side effects. Systemic side effects have not been observed, and the risk of systemic absorption and possible effect on growth in children has been extensively studied but has shown no effect at the recommended doses. Despite these findings, nasal steroids should be used at the lowest possible dose in children. To date, there is no convincing evidence that doses greater than the recommended maximum increase efficacy. Antihistamines Antihistamines typically reduce itching, sneezing, and rhinorrhea but have no or little effect on nasal congestion associated with the late-phase reaction. The currently used antihistamines are clearly less effective than topical nasal steroids. They are subdivided into first- and second-generation histamines. First- and second-generation antihistamines differ in their side effects. First-generation antihistamines produce sedation and other central nervous system symptoms in X20% of patients and may cause drying of the mouth and urinary hesitancy. Secondgeneration antihistamines also have varying degrees of anticholinergic, antimuscarinic, and antiadrenergic effects, but to a lesser extent than first-generation antihistamines.

Cromoglycate and nedocromil Sodium cromoglycate and nedocromil sodium are both drugs that have been demonstrated to influence mast cell degranulation in in vitro studies. However, no inhibitory effect on histamine release has been demonstrated in mast cells recovered from nasal tissue. These drugs also inhibit the intermediate conductance pathways of mast cells, eosinophils, epithelial cells, fibroblasts, and sensory neurons. Cromoglycate blocks symptoms of the immediate and late phase and is effective even when used shortly before allergen inhalation. Cromoglycate is more effective than placebo; however, most studies show it to be less effective than nasal steroids. Both drugs do not induce any serious side effects. Ipratropium Ipratropium bromide blocks the activity of atropine and therefore reduces rhinorrhea when used intranasally. It does not block sneezing, pruritus, or nasal obstruction and is thus of greatest use in nonallergic rhinitis with predominant rhinorrhea. Vasoconstrictors Local vasoconstrictors reduce nasal blockage and are therefore indicated to relieve the symptoms of AR. However, they create a tachyphylaxis with rebound rhinitis as a symptom that develops after 3–7 days. Excessive consumption of nasal decongestants can cause rhinitis medicamentosa. Therefore, nasal decongestants are not recommended in the treatment of AR and should preferably be used for a short period when symptomatic vascular decongestion is indicated for the alleviation of nasal obstruction. Oral pseudoephedrine as a sustained release preparation has been shown to decrease both the nasal congestion and nasal airway resistance in patients with hay fever but only at doses where there is a high risk of systemic side effects. Leukotriene modifiers While sneezing and nasal itching correlate best with histamine levels in experimental AR, nasal congestion correlates with LTC4 levels. Leukotriene receptor antagonists provide some benefits in patients with AR. However, as demonstrated by some recent studies, leukotriene modifiers may be less effective than intranasal steroids. In children, leukotriene receptor antagonists have been shown to reduce exercise-induced asthma especially in combination with inhaled steroids. Allergen-specific immunotherapy Allergen-specific immunotherapy is the practise of administering gradually increasing doses of therapeutic vaccines of standardized allergen extracts until reaching an arbitrary dose that is maintained for several years. Traditionally, allergen-specific immunotherapy

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is administered subcutaneously. There is now good evidence that immunotherapy with seasonal and perennial allergens is clinically effective for the treatment of AR, but it should only be considered in patients with severe symptoms of AR, when allergen avoidance and pharmacotherapy have failed to reduce symptoms or when pharmacotherapy has been associated with unacceptable side effects. In addition, specific immunotherapy for AR, when administered early in the disease process, has been demonstrated to modify the long-term progress of the allergic inflammation and disease, by preventing the development of new sensitizations and by preventing the development of asthma. The exact mechanisms at the basis of the beneficial effects of allergen-specific immunotherapy are complex and are still not completely understood. Recent evidence suggests both a shift away from a Th2-type response as well as the generation of regulatory T cells. As immunotherapy is not free of risk and may provoke systemic reactions (severe asthma attacks and anaphylaxis in particular), it can only be carried out by or under the supervision of trained specialists, with direct access to the necessary rescue medication. Because of these risks, patients must be closely observed for 20–30 min after injection. More recently, other administration routes for allergen-specific immunotherapy have been investigated, including nasal, sublingual-swallow, and oral immunotherapy. Sublingual administration has shown to be effective. Novel treatments Increasing insights into the pathophysiology of AR, the roles of diverse cells and their cytokine products, and receptors and mediators involved in allergic inflammation has provided new (potential) targets for pharmacotherapy. Modulation of allergic response through antimediators and antireceptors has gained much interest (e.g., anti-IgE, anti-IL-5, anti-CCR3). Role of Surgery in the Management of Allergic Rhinitis

diagnostic tests have been developed, many effective pharmacological agents are currently available, and new potential targets for pharmacotherapy, new routes of administration, and alterations in treatment dosages and schedules are continuously being investigated. To provide and disseminate this knowledge from research into practice, to facilitate and standardize the management of AR, and to improve the patient care and, consequently, the patient satisfaction and compliance several clinical guidelines have been developed. Before 1998, European and American guidelines for the management of AR were developed, based on expert opinion. The European guidelines are in many respects very similar to the American guidelines, but a Table 5 Classification schemes of statements of evidence Category of evidence Ia: Evidence from meta-analysis of randomized controlled trials Ib: Evidence from at least one randomized controlled trial IIa: Evidence from at least one controlled study without randomization IIb: Evidence from at least one other type of quasi-experimental study III: Evidence from nonexperimental descriptive studies, such as comparative studies, correlation studies, and case–control studies IV: Evidence from expert committee reports or opinions or clinical experience of respected authorities or both Strength of evidence of recommendations A: Directly based on category I evidence B: Directly based on category II evidence or extrapolated recommendation from category I evidence C: Directly based on category III evidence or extrapolated recommendation from category I or II evidence D: Directly based on category IV evidence or extrapolated recommendation from category I, II, or III evidence Adapted form Shekelle PG, Woolf SH, Eccles M, and Grimshaw J (1999) Developing guidelines. British Medical Journal 318: 593– 596.

Table 6 Strength of evidence for the treatment of rhinitis

Surgery does not relieve allergic inflammation and should only be used in case of turbinate hypertrophy or cartilaginous or bony obstruction, contributing to or aggravating rhinitis symptoms, especially nasal obstruction. In these cases, a conchotomy and/or septo(rhino)plasty is recommended. In cases of secondary and independent sinus disease, functional endoscopic sinus surgery can be performed.

Intervention

Clinical Guidelines for the Management of Allergic Rhinitis

SIT, allergen-specific immunotherapy. Adapted from Bousquet J, Van Cauwenberge P, Khaltaev N, Aria Workshop Group, World Health Organization (2001) Allergic rhinitis and its impact on asthma. Journal of Allergy and Clinical Immunology 108(5 supplement): S147–S334.

Our insight into the pathophysiology of AR has increased in recent years. Highly sensitive and specific

Oral anti-H1 Intranasal anti-H1 Cromones Antileukotrienes Subcutaneous SIT Sublingual/nasal SIT Allergen avoidance

Seasonal AR

Perennial AR

Adults

Children

Adults

Children

A A A A A A D

A A A A A A D

A A A

A A A

A A D

A A


prominent feature of the former is the stepwise approach recommended for the treatment of rhinitis, which is similar to the Global Initiative for Asthma guidelines for the treatment of asthma. In 1999, the ARIA Working Group was founded, under the initiative of the World Health Organization. Unlike the European and American guidelines,

the ARIA guidelines are formulated on evidencebased medicine, categorized by Shekelle. Based on these categories of evidence, the strength of evidence for a certain recommendation is graded from A to D (Table 5). The evidence for the recommended pharmacotherapy and immunotherapy for AR treatment is particularly strong (category A evidence strength).

Diagnosis of allergic rhinitis Allergen avoidance

Intermittent symptoms

Mild

Moderate/ severe

Persistent symptoms

Moderate/severe

Mild

Intranasal corticosteroids Not in preferred order: • oral H1-blocker • intranasal H1-blocker • and/or decongestant

Review patient after 2− 4 weeks Not in preferred order: • oral H1-blocker • intranasal H1-blocker • and/or decongestant • intranasal corticosteroid • (cromones)

In persistent rhinitis: review the patient after 2−4 weeks

If failure: step-up If improved: continue for 1 month

failure

improved

Step-down and continue treatment for 1 month

Review diagnosis, compliance, query infections or other infections, or other causes

↑ Intranasal CS dose

Itch/sneeze: +H1 blocker

Rhinorrhea: + ipratropium Blockage: + decongestant or oral CS (short term)

Failure Surgical referral If conjunctivitis add : Oral H1-blocker or intraocular H1-blocker or intraocular cromones (or saline) Consider specific immunotherapy Figure 11 Stepwise treatment algorithm for allergic rhinitis in adolescents and adults, as recommended by ARIA. CS, corticosteroids. Adapted from Bousquet J, Van Cauwenberge P, Khaltaev N, Aria Workshop Group, World Health Organization (2001) Allergic rhinitis and its impact on asthma. Journal of Allergy and Clinical Immunology 108 (5 supplement): S147–S334.

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The evidence for allergen avoidance, on the other hand, is limited (category D evidence strength) (Table 6). Similar to the European guidelines, a stepwise approach for the treatment of AR is recommended by ARIA with the following first-line treatment approaches (Figure 11): oral or intranasal H1-antihistamines, with limited use of decongestants, for mild intermittent rhinitis; oral or intranasal H1-antihistamines or intranasal corticosteroids, with limited use of decongestants and cromones, for moderate to severe intermittent and mild persistent rhinitis; and intranasal corticosteroids, with step-down and step-up options, in conjunction with H1-antihistamines, decongestants, ipratropium, and eventually oral corticosteroids, for moderate to severe persistent rhinitis. Additionally, specific immunotherapy should be considered in persistent disease and when the symptoms are moderate to severe and do not respond to conventional treatment. When conjunctivitis is present, oral or intraocular H1-antihistamines should be used. Whereas the European guidelines did not take into account the costs and availability of the treatment strategies in different countries, the ARIA guidelines are developed for the whole world and recognize that the outcome of disease management largely depends on compliance with the suggested treatment, which in turn is influenced by the availability and affordability of the specific interventions. Therefore, a flexible stepwise approach is recommended, based on the four cornerstones of patient education, allergen avoidance, pharmacotherapy, and immunotherapy, but modifiable in low-income countries. See also: Allergy: Overview; Allergic Reactions. Asthma: Overview. Chemokines. Chymase and Tryptase. Histamine. Immunoglobulins. Interleukins: IL-4; IL-5; IL-10; IL-13. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils; T cells. Lipid Mediators: Leukotrienes; Prostanoids.

Further Reading Aalberse RC (2000) Molecular mechanisms in allergy and clinical immunology. Structural biology of allergens. Journal of Allergy and Clinical Immunology 106: 228–238. Barnes PJ (2003) Pathophysiology of allergic inflammation. In: Adkinson F Jr, Yunginger JW, Busse WW, et al. (eds.) Middleton’s Allergy Principles and Practice, pp. 483–499. Pennsylvania: Mosby. Bousquet J, Van Cauwenberge P, Khaltaev N, Aria Workshop Group, World Health Organization (2001) Allergic rhinitis and its impact on asthma. Journal of Allergy and Clinical Immunology 108 (5 supplement): S147–S334. Howarth PH (2003) Allergic and nonallergic rhinitis. In: Adkinson F Jr, Yunginger JW, Busse WW, et al. (eds.) Middleton’s Allergy Principles and Practice, pp. 1391–1410. Pennsylvania: Mosby. International Rhinitis Management Working Group (1994) International Consensus Report on the Diagnosis and Management of Rhinitis. Allergy 49(supplement 9): 5–34. Johansson SG, Bieber T, Dahl R, et al. (2004) A revised nomenclature for allergy for global use: Report of the Nomenclature Review Committee of the World Allergy Organization. Journal of Allergy and Clinical Immunology 113: 823–826. Malm L, Gerth van Wijk R, and Bachert C (1999) Guidelines for nasal provocations with aspects on nasal patency, airflow and airflow resistance. Rhinology 37: 133–135. Salib RJ and Howarth PH (2003) Remodelling of the upper airways in allergic rhinitis: is it a feature of the disease? Clinical and Experimental Allergy 33: 1629–1633. Shekelle PG, Woolf SH, Eccles M, and Grimshaw J (1999) Developing guidelines. British Medical Journal 318: 593–596. Van Cauwenberge P, Bachert C, Passalacqua G, et al. (2000) Consensus statement on the treatment of allergic rhinitis. Allergy 55: 116–134. Wheatley LM and Platts-Mills TAE (1999) Allergens. In: Naclerio RM, Durham SR, and Mygind N (eds.) Rhinitis, pp. 45–58. New York: Marcel Dekker, Inc. Worldwide variation in prevalence of symptoms of asthma, allergic rhinoconjunctivitis, and atopic eczema (1998) ISAAC. The International Study of Asthma and Allergies in Childhood (ISAAC) Steering Committee. Lancet 351: 1225–1232.

Relevant Website http://www.ginasthma.com – Global Initiative for Asthma. Global strategy for asthma management and prevention (2003).

ALVEOLAR HEMORRHAGE O C Ioachimescu, Cleveland Clinic Foundation, Cleveland, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Bleeding into the alveoli characterizes the syndrome of diffuse alveolar hemorrhage (DAH) and represents a potential

life-threatening condition. There are many causes of DAH, including vasculitides, such as Wegener’s granulomatosis, microscopic polyangiitis, Good pasture’s syndrome, connective tissue disorders, and other conditions. Pathologically, the syndrome is due to pulmonary vasculitis, to a ‘bland’ alveolar hemorrhage, or it represents the nondominant pathology, as seen in diffuse alveolar damage from acute respiratory distress syndrome (ARDS). Most patients present with dyspnea, cough, hemoptysis (the latter in only 66% of the cases), anemia, and

ALVEOLAR HEMORRHAGE 93 new pulmonary infiltrates. Urgent bronchoscopy and bronchoalveolar lavage is generally required to confirm the diagnosis, with a superior yield when it is performed in the first 48 h. In patients with evidence of DAH and renal involvement (pulmonary–renal syndrome), kidney biopsy may be considered to identify the etiology and direct the therapy. This chapter will describe the mean features of the DAH syndrome and review in short the main causes of alveolar bleeding.

Introduction Hemoptysis is most commonly due to disruption of the bronchial circulation of various (endo)bronchial conditions such as bronchitis, bronchiectasis, neoplastic disorders, or (rarely) disorders of the pulmonary circulation. Hemorrhage from a bronchial source can be rapidly fatal, since a brisk bleeding can lead to a large amount of blood to occupy anatomic and functional dead space of the respiratory tract; in this setting the alveoli can also be flooded quickly, mimicking the true alveolar hemorrhage. Anatomical injury at the alveolar–capillary basement membrane level of the pulmonary microcirculation may cause hemoptysis from arteriolar, venular, or capillary source. Bleeding into the alveoli characterizes the syndrome of diffuse alveolar hemorrhage (DAH). Although alveolar hemorrhage may be focal, generally there are multiple areas affected, hence the term DAH. Diffuse alveolar hemorrhage should always be considered a potentially life-threatening condition; it requires early recognition, care in stabilizing the patient (airway protection, sometimes selective intubation, mechanical ventilation, etc.) and specific treatment once the etiology is established. Synonyms with DAH that can be found in the literature are: (intra)pulmonary hemorrhage, pulmonary alveolar hemorrhage, pulmonary capillary hemorrhage, alveolar bleeding, and microvascular pulmonary hemorrhage. The DAH syndrome is relatively rare, although no studies addressed its specific epidemiology. Currently, our understanding of the etiopathogenesis and the appropriate management relies mainly on case reports or case series of specific disorders leading to DAH. Unfortunately, the therapeutic approach is rather non-specific, with a few notable exceptions (Wegener’s granulomatosis (WG), Goodpasture’s syndrome, systemic lupus erythematosus (SLE), etc.)

Etiology Various diseases can lead to DAH syndrome (Table 1). To date, no prospective studies of DAH have estimated the relative frequency of various etiologies. A wellknown retrospective review of 34 cases of DAH suggested that the most common cause was WG, which

Table 1 Causes of diffuse alveolar hemorrhage (DAH) DAH associated with vasculitis Wegener’s granulomatosis Microscopic polyangiitis Goodpasture’s syndrome Isolated pauci-immune pulmonary capillaritis Connective tissue disorders Antiphospholipid antibody syndrome Mixed cryoglobulinemia Henoch–Scho¨nlein purpura, IgA nephropathy Pauci-immune or immune complex-associated glomerulonephritis Behçet’s syndrome Acute lung graft rejection Thrombotic thrombocytopenic purpura and idiopathic thrombocytopenic purpura ‘Bland’ DAH Mitral stenosis and mitral regurgitation Anticoagulants, antiplatelet agents, or thrombolytics; disseminated intravascular coagulation Pulmonary venoocclusive disease Infections: human immunodeficiency virus (HIV) and infective endocarditis Toxins: trimellitic anhydride, isocyanates, crack cocaine Drugs: propylthiouracil, diphenylhydantoin, amiodarone, mitomycin, penicillamine, sirolimus, methotrexate, nitrofurantoin, gold, all-trans retinoic acid (ATRA), bleomycin (especially with high-flow O2), montelukast, zafirlukast, infliximab Idiopathic pulmonary hemosiderosis DAH as nondominant pathology Diffuse alveolar damage (DAD) Malignant conditions (e.g., pulmonary angiosarcoma) Lymphangioleiomyomatosis/tuberous sclerosis Pulmonary capillary hemangiomatosis Lymphangiography

accounted for one-third of cases, followed by Goodpasture’s syndrome (13%), idiopathic pulmonary hemosiderosis (IPH) (13%), collagen vascular disease (13%), and microscopic polyangiitis (9%). A review of 29 cases of DAH associated with capillaritis found that the most common cause was isolated pulmonary capillaritis. Other conditions associated with DAH are briefly discussed below. Overall, three different histologic patterns may be seen. DAH Associated with Vasculitis

This pathologic pattern is characterized by neutrophilic infiltration of the interalveolar and peribronchiolar septal vessels (pulmonary interstitium). This sequentially leads to anatomic disruption of the capillaries, and red blood cell extravasation into the alveoli and interstitium. This leads to neutrophil fragmentation and apoptosis, with subsequent release of the intracellular proteolytic enzymes and reactive oxygen species, which begets more inflammation,

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intra-alveolar neutrophilic nuclear dust, fibrin and inflammatory exudate, and fibrinoid necrosis of the interstitium. DAH Associated with ‘Bland’ Pulmonary Hemorrhage

This pattern is characterized by intra-alveolar red cell extravasation without any evidence of inflammation or destruction of the alveolar capillaries, venules, and arterioles; the epithelial lesions are usually discrete. DAH Associated with Other Conditions (Nondominant Pathology)

Here, the DAH is secondary to diffuse alveolar damage (DAD), lymphangioleiomyomatosis (LAM), drug-induced lung injury, metastatic neoplasia to the lungs, mitral stenosis, etc. DAD is the main underlying lesion of the acute respiratory distress syndrome (ARDS), and is characterized by intra-alveolar hyaline membranes, interstitial edema with minimal inflammation, and at times by ‘secondary’ DAH.

Clinical Presentation The syndrome of DAH may present with a constellation of symptoms, signs, and laboratory results that may suggest the underlying etiology (e.g., WG, Goodpasture’s syndrome, drug-related vasculitis, etc.) or only establish the diagnosis of the syndrome without a specific etiology. Symptoms

The onset of DAH is most often acute or subacute (less than 1 week). Dyspnea, cough, and fever are the common initial symptoms. Some patients, however, present with ARDS requiring mechanical ventilation. Hemoptysis may be absent at the time of presentation in up to one-third of patients with DAH syndrome. When present, it ranges from fulminant and life-threatening to mild and intermittent in nature. Physical Examination

The lung examination is usually non-specific, unless there are physical signs of an underlying systemic vasculitis or collagen vascular disorder (rashes, purpura, eye lesions, hepatosplenomegaly, clubbing, etc.).

The presence of Kerley B lines points toward mitral valve disease or pulmonary venoocclusive disease. Computed tomographic imaging studies may show areas of consolidation alternating with areas of ground-glass attenuation and preserved, normal areas. Gallium scan, rarely ordered today for this purpose, used to be performed in the past to reveal areas of active vasculitis or inflammatory activity (rather non-specific and with unclear sensitivity, too). Other nuclear studies, geared to reveal breakdown of the microcirculatory integrity and extravasation of red blood cell out of the vessels, have not been confirmed by the ultimate test of time. Pulmonary Function Tests

The DAH leads to various degrees of oxygen transfer impairment and hypoxemia, sometimes severe enough to require ventilatory support. A sensitive marker for DAH is a sequential increase in diffusing lung capacity for carbon monoxide (DLCO). In this setting, due to an increased availability of intraalveolar hemoglobin, capable of binding with highaffinity carbon monoxide, the DLCO is generally normal or elevated. Unfortunately, the severe condition of these patients and the associated hemoptysis generally preclude any DLCO measurements. An obstructive lung disease associated with recurrent DAH may be due to lymphangioleiomatosis, histiocytosis X, or pulmonary capillaritis in the setting of microscopic polyangiitis or WG. Recurrent episodes of DAH generally lead to interstitial fibrosis and ventilatory restrictive defects, as seen in idiopathic pulmonary hemosiderosis. Laboratory Abnormalities

The blood work generally reveals acute and/or chronic anemia, ‘stress’ leukocytosis, elevated erythrocyte sedimentation rate, and C-reactive protein (particularly in the cases of DAH caused by systemic diseases or associated with a pattern of vasculitis). Since several causes of DAH may present as pulmonary–renal syndromes (i.e., association of pulmonary hemorrhage with different types of glomerulonephritis), blood urea nitrogen (BUN) and creatinine concentration elevations, and abnormal urine sediments (red blood cells/casts, white blood cells/casts, proteinuria of glomerular origin) can be seen.

Imaging Studies

The radiographic findings are generally non-specific and show new and/or old, patchy or diffuse alveolar opacities. Recurrent episodes of DAH lead to reticular interstitial opacities due to pulmonary fibrosis, usually with minimal honeycombing (if any).

Diagnosis DAH represents a medical emergency, since it represents a potentially fatal condition. Despite recent advances and refinements of the diagnostic and therapeutic tools in DAH, it remains a highly morbid

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condition, with substantial fatality. One must have a low threshold to entertain the diagnosis, to confirm it and to thoroughly look for the underlying etiology. Methodically, two separate steps are to be followed. Establish the Diagnosis of DAH Syndrome

The triad of elevated DLCO, hypoxemia, and new pulmonary infiltrates in the setting of hemoptysis and dyspnea suggests the diagnosis of DAH. Patients who present with hemoptysis need to be screened for focal sources of pulmonary hemorrhage (i.e., bronchitis, bronchiectasis, infection, neoplastic processes, etc.); upper airway and gastrointestinal sources must also be excluded carefully. It is important to remember that most disorders manifested with severe hemoptysis can cause alveolar infiltrates. In patients without hemoptysis, the clinical evaluation needs also to screen for evidence of congestive heart failure, pneumonia, inhalational or drug-related lung injury, and other causes of bleeding. Most patients suspected of having DAH need a bronchoscopic examination, which serves two purposes: documentation of alveolar hemorrhage by visual inspection and bronchoalveolar lavage (BAL; especially if there is no hemoptysis) and exclusion of an associated infection. This procedure has a higher yield if it is performed early (within 48 h) rather than later. An increasing hematocrit or progressively bloodier aspect of three sequential BAL aliquots from an affected area is diagnostic of DAH. In subacute or recurrent episodes of DAH, counting the hemosiderin-laden macrophages (siderophages) as demonstrated by Prussian Blue staining on a pooled BAL specimen centrifugate may be useful for diagnosis (generally more than 30% siderophages). BAL specimens should be sent for routine bacterial, mycobacterial, fungal, viral, and Pneumocystis carinii microscopic studies and cultures. The use of transbronchial biopsy in patients with suspected DAH is of unclear value due to the small size of the specimens and possible sampling bias. Unless another clinical condition is suspected, transbronchial biopsy is generally not useful clinically. Identify the Specific Etiology

Identifying the specific etiology is generally equivalent to the process of differential diagnosis. Serologic studies need to be performed early during the course of the disease, although the results are generally not available in a timely manner for immediate management of the disease. Complement fractions C3 and C4 and anti-dsDNA, anti-glomerular basement membrane (GBM), antineutrophil cytoplasmic antibodies (ANCA), and antiphospholipid antibodies

represent the basic panel to be checked in DAH patients (see Autoantibodies). If the diagnosis of DAH is still not clear or the underlying etiology is still not known after a thorough clinical evaluation, imaging studies, serologies, and bronchoscopy, surgical biopsy should be entertained. Besides, the results of a surgical biopsy may become available faster than the serologic tests. The ‘ideal’ site to biopsy is dependent on the pretest probability of the underlying disorder: for WG a nasal biopsy may suffice, while a kidney biopsy (with immunofluorescent studies) may be less invasive than others in diagnosing Goodpasture’s syndrome, microscopic polyangiitis, or systemic lupus erythematosus. In isolated pulmonary disease or pauci-immune pulmonary capillaritis a lung biopsy is a mandatory test. Immunofluorescence reveals linear deposition of immunoglobulins and immune complexes along the basement membrane in Goodpasture’s syndrome and granular deposits in SLE, whereas the systemic vasculitides appears pauci-immune. In cases of pulmonary–renal syndrome, the kidney biopsy shows focal segmental necrotizing glomerulonephritis. Additionally, skin biopsy of a lesion can demonstrate leukocytoclastic vasculitis or Henoch–Scho¨nlein purpura (in the latter case, immunoglobulin A (IgA) deposits are suggestive of the diagnostic).

Current Therapy Corticosteroids are currently the backbone of DAH syndrome therapy, especially if associated with systemic or pulmonary vasculitis, Goodpasture’s syndrome, and connective tissue disorders. Most authors recommend intravenous methylpredisolone (up to 500 mg every 6 h, although lower doses seem to have similar efficacy) for approximately 4–5 days, followed by a gradual taper to maintain doses of oral steroids. In the setting of pulmonary–renal syndrome, the therapy needs to be initiated as soon as possible, since the potential of reversibility is lost exponentially in the first week of disease activity. Other immunosuppressive drugs can be used in DAH (e.g., cyclophosphamide, azathioprine, mycophenolate mofetil, etanercept, etc.) depending on the disease severity, failures to respond to corticosteroids and the underlying disease (e.g., WG, Goodpasture’s syndrome). Intravenously administered cyclophosphamide (2 mg kg 1 day 1, adjustable to renal function) is generally the preferred adjunctive immunosuppressive drug in the initiation phase of the treatment, and continued several weeks until blood marrow suppression, infections, or other limiting effects require discontinuation and thereafter switch to consolidative

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and/or maintenance therapy with methotrexate or other agents. Plasmapheresis is indicated in the treatment of DAH associated with Goodpasture’s syndrome or other vasculitis processes when the titers of pathogenetic immunoglobulins and immune complexes are very high (e.g., plasmapheresis in the setting of ANCA-associated vasculitis with overwhelming endothelial injury and/or hypercoagulable state). However, its utility in DAH syndromes other than Goodpasture’s syndrome has not been evaluated in prospective studies. If intravenous immunoglobulin (IVIG) therapy adds anything to the treatment of DAH due to vasculitis or other connective tissue disease is yet unclear. Besides the treatment of vasculitis and underlying disorder, stabilization of the patients with moderate and severe hemoptysis entails supplemental oxygen, bronchodilators, reversal of any coagulopathy, red blood cell transfusion, intubation with bronchial tamponade/protective strategies for the less involved lung, mechanical ventilation, etc. Several case reports showed success in treating alveolar hemorrhage due to allogeneic hemaopoietic stem cell transplantation, ANCA-associated vasculitis, systemic lupus erythematosus, and antiphospholipid syndrome with recombinant-activated human factor VIIa, which may become a new tool in the (otherwise poor) armamentarium available for this condition.

Prognosis Recurrent episodes of DAH may lead to various degrees of interstitial fibrosis, especially in patients with underlying WG, mitral stenosis, long-standing and severe mitral regurgitation, and idiopathic pulmonary hemosiderosis. A post-DAH syndrome has also been described, particularly in microscopic polyangiitis, and idiopathic pulmonary hemosiderosis and consists of progressive obstructive ventilatory defects and anatomic emphysema. Mortality

The disease fatality varies with the underlying cause of DAH; patients with systemic lupus erythematosus, anti-GBM antibody disease and several forms of ANCA-associated vasculitis can have a high mortality (up to 50%) due to the disease activity, infections, and cardiovascular morbidity (e.g., Churg–Strauss syndrome).

Experimental Models Experimental models of different conditions (e.g., microscopic polyangiitis, anti-GBM antibody disease,

etc.) have allowed the indirect study of DAH syndrome and its close interrelation with iron metabolism. Iron is of crucial importance in the inflammatory and anti-infectious defense of the respiratory tract and lung parenchymal homeostasis. Local macrophages have a major contribution to iron recycling from red blood cells, through the phagocytosis of the intra-alveolar erythrocytes and eventual return of the electrolyte to the bone marrow erythron, where iron is incorporated in the structure of heme in maturing red blood cells. The understanding of alveolar pathology in DAH has been enriched lately by a literature explosion in the field of ferroproteins and genetics of the regulatory factors involved in the iron metabolism. It is beyond the scope of this section to review exhaustively the respiratory iron metabolism, hence mention will only be made of the main facts. The free iron’s presence in the extracellular milieu together with local hypoxia induces reactive oxygen species and other free radical production, with subsequent peroxidation of the cellular membranes, cell apoptosis, and preinflammatory cytokine release. The phagocytosis capacity of the alveolar macrophages seems to be easily exhaustible, becoming hemosiderin-laden macrophages (siderophages), which in turn leads to more free iron and heme available in alveolar spaces and pulmonary interstitium. Recently, J774 macrophages have been used among other experimental models to investigate the presence of different ferroproteins and their role. A recently discovered peptide, hepcidin, seems to have a major ferrostat role in local and general iron overload syndromes; its role (if any) is yet unclear in the DAH syndromes. Another interesting protein, ferroportin, seems to be involved in macrophage iron recycling from engulfed erythrocytes. It is possible that macrophage’s iron retentive mechanism is abnormal, similar to what is seen in human hereditary hemochromatosis type 4, or ferroportin disease.

Specific Causes of DAH Wegener’s Granulomatosis

WG is a systemic disease characterized by necrotizing granulomatous inflammation of the small vessels, involving predominantly the respiratory tract and kidneys. The organ involvements can be recalled using the mnemotechnic JERKS: (1) Joint disease % (arthralgias and arthritis); (2) ENT (rhinosinusitis) % (both upper and and eye disease; (3) Respiratory % about 70–95% of cases); lower respiratory tracts, (4) Kidney involvement (lesions of focal segmental % necrotizing glomerulonephritis); (5) Skin and other % of the lung organs (systemic). The gross appearance


ANCA-associated small vessel vasculitis who were treated with plasma exchange in conjunction with induction immunosuppressive regimens had a remission of the DAH. Other therapies, such as trimethoprim– sulfamethoxazole, tumor necrosis factor inhibitors, and anti-CD20 monoclonal antibodies have been tried with unproven efficacy or are under scrutiny. Mortality is considerable and most often caused by infections, respiratory failure, and renal failure. Microscopic Polyangiitis

Figure 1 Diffuse alveolar hemorrhage associated with, necrotizing pulmonary vasculitis (shown) in a patient with Wegener’s granulomatosis.

in WG with DAH is dark red, sometimes associated with multiple nodules and cavitary lesions (exceptionally solitary pulmonary nodules), and much more frequently with areas of consolidations and focal or geographic necrosis (Figure 1). The chest radiograms show fluffy alveolar and/or interstitial infiltrates reflecting the diffuse microvasculature disorder. The diagnosis of WG is established based on clinical presentation and serologic confirmation, that is, positive ANCA antibodies (c- or p-ANCA). In organlimited WG there is ANCA positivity in 60% of the cases, while in generalized disease in 90–95% of cases. c-ANCA (directed against proteinase-3) is present in 85–90% of generalized forms of the disease. If diagnosis is still in doubt, surgical biopsy of affected organs (kidneys, lungs, nasal mucosa) may be useful for diagnosis. In WG, the DAH due to pulmonary capillaritis may mark the disease onset or occur later during the course, in a subclinical and/or recurrent fashion. The progressively bloodier BAL aliquots with large numbers of red blood cells and hemosiderin-laden macrophages in the absence of an infectious etiology confirm the diagnosis of DAH. The standard inductive therapy of DAH is high-dose corticosteroids (e.g., 1 g methylprednisolone daily for 3 days) and daily intravenous cyclophosphamide. Azathioprine generally substitutes cyclophosphamide in about 6 months or after remission in an inductivemaintenance approach and/or after a cumulative high dose of cyclophosphamide is reached. IVIG or therapeutic plasma exchange may be used for persistent disease. In one study, 20 out of 20 patients with

Microscopic polyangiitis (MP), a small-vessel variant of polyarteritis nodosa, is sometimes difficult to differentiate from WG due to similar clinical presentations, serologic and pathologic findings. The typical pathologic feature in microscopic pauci-immune neutrophilic polyangiitis (found in virtually 100% of cases) is a focal and segmental necrotizing glomerulonephritis, renal lesion also seen in WG, Goodpasture’s syndrome, and other connective tissue disorders. The lack of granulomatous inflammation differentiates pathologically between WG and MP. A positive serum perinuclear ANCA (p-ANCA), directed against myeloperoxidase’s epitopes, strongly supports the diagnosis, but it can be also seen in 20–30% of WG cases. Microscopic polyangiitis causes relatively frequently a syndrome of severe DAH. Treatment with corticosteroids and cyclophosphamide followed by azathioprine is similar to WG therapy. Plasmapheresis and IVIG may be also useful in difficult-to-treat cases. As in other hemorrhagic conditions, factor VIIa has also been used with various success. The short-term and long-term (5-year) mortality rates from DAH in MP are approximately 25% and 40%, respectively. Goodpasture’s Syndrome

Goodpasture’s syndrome is a form of anti-basement membrane antibody disease and is characterized by a combination of DAH and glomerulonephritis. Anti-GBM antibody, the pathognomonic immunoglobulin, is directed against alpha-3 (IV) collagen from GBM and is found in the serum of more than 90% of patients and part of the linear immunofluorescent deposits in the glomerular membrane noted in the disease. The syndrome typically involves DAH in a smoker (usually a young male, although older patients, women, and nonsmokers can also have it). Isolated, renal-sparing DAH, a rare occurrence, may have anti-GBM both in the serum and in the glomerular membrane in a typical linear antibody deposition. The treatment of Goodpasture’s syndrome includes urgent plasmapheresis and corticosteroids,

98

ALVEOLAR HEMORRHAGE

cyclophosphamide, and/or azathioprine. The isolated DAH seems to respond well to corticosteroids alone. Alveolar bleeding secondary to Goodpasture’s syndrome has a 2-year survival rate close to 50%, while patients presenting with renal insufficiency have a worse outcome. Churg–Strauss Syndrome

Churg–Strauss syndrome (allergic and granulomatous angiitis) is a systemic disorder characterized by asthma, peripheral blood eosinophilia, and systemic vasculitis of small-caliber vessels with extravascular necrotizing granulomas. It involves mainly the upper respiratory tract, the lungs, and the peripheral nerves. Tissue eosinophilia may involve the lungs or the gastrointestinal tract. Pulmonary hemorrhage is rare in this condition, while radiographic abnormal findings are described in 50–90% of the cases. p-ANCA positivity occurs in approximately 35–40%. Histologically, it is distinguished by small- and medium-vessel involvement and eosinophil-rich infiltrates (sometimes presenting with an aspect of chronic eosinophilic pneumonia). Of note, a Churg–Strauss-like syndrome can occur due to chronic ingestion of carbamazepine, quinine, or macrolides, while potential risk of aggravation of the disorder can occur on cysteinil leukotriene receptor blockers. Isolated Pauci-Immune Pulmonary Capillaritis

An interesting case of a DAH syndrome is represented by pauci-immune pulmonary alveolar capillaritis in the absence of any other systemic involvement or abnormalities. Several cases have been shown to have elevated serum titers of p-ANCA, but this finding may reveal in fact a fruste form of MP. Isolated, pauci-immune pulmonary capillaritis has been also seen in association with the drug called all-trans retinoic acid (ATRA). Available literature on pauciimmune pulmonary alveolar capillaritis shows that respiratory failure necessitating mechanical ventilation is frequent in this condition and the response to corticosteroids and immunosuppressive agents is favorable. Connective Tissue Disorders

Diffuse alveolar bleeding has been described in collagen vascular diseases such as SLE, scleroderma, rheumatoid arthritis, polymyositis/dermatomyositis, Henoch–Scho¨nlein syndrome, Behçet disease, and mixed connective tissue disorder. In SLE, pulmonary complications occur in more than 50% of cases, while DAH can occur in up to 5% of the patients

(although it is the most frequent disorder in this category presenting with alveolar bleeding); DAH has generally a poor prognostic connotation (50% fatality rate) and is due to a process of pulmonary capillaritis. When DAH occurs in SLE, contrary to WG, glomerulonephritis is generally absent. Immunofluorescent studies show granular deposits of IgG and complement (C3) in the pulmonary interstitium, alveolar blood vessels, and the GBM. Of note, the syndrome of DAH is clinically and pathologically distinct from acute lupus pneumonitis, which presents similarly and may be the inaugural presentation of SLE. Therapy consists of corticosteroids and cyclophosphamide and/or azathioprine. Plasmapheresis has no proven benefit. In rheumatoid arthritis, the syndrome of DAH secondary to pulmonary vasculitis generally occurs late, in ‘burnout’ disease. Hematologic Conditions

DAD seems to be the dominant lesion in the lungs of the patients who undergo chemotherapy for leukemia or stem cell transplantation; this lesion can be accompanied by DAH, sometimes fulminant and fatal, even in the absence of hemoptoic sputa. The offending factors seem to be the chemotherapeutic agents associated with actinic lesions, thrombocytopenia, and superimposed infections. The therapy includes platelet transfusions, reversal of coagulation abnormalities, and high-dose corticosteroids. DAH of the Immunocompromised Patient

Alveolar bleeding can occur in immunocompromised hosts due to a myriad of factors (infectious, chemotherapeutic, and immunosuppressive drugs, radiation therapy, thrombocytopenia, pulmonary edema, lung malignancy, other lung comorbidities, etc.) that have a common denominator: the endothelial injury. The exact frequency of the syndrome in the immunocompromised patients is currently unknown, partly because the hemorrhage can be subclinical, and because empirical therapy is instituted early, since the risk of invasive evaluation outweighs the benefits. While subclinical DAH in this setting may have minimal impact on survival, the alveolar hemorrhage associated with ‘serious’ causes like Kaposi’s sarcoma, invasive fungal infections (Aspergillus, Pseudoallescheria boydii, etc.), Mycobacterium, Legionella, or other invasive bacterial species may have catastrophic consequences. In human immunodeficiency virus (HIV) infection or acquired immunodeficiency syndrome (AIDS) patients, the cytomegalovirus (CMV) and Kaposi’s sarcoma represent the main risk factors for developing DAH. In a study of


HIV-infected patients with radiographic infiltrates, in up to 44% of them more than 20% of the BAL cells were hemosiderin-laden macrophages. Drug-Induced DAH

Many drugs have been associated with DAH, including anticoagulants (warfarin, heparin, etc.), thrombolytic agents, and platelet antiaggregant agents; as a rule, in order to produce DAH, a ‘second hit’ (infectious, inflammatory, inhalatory, etc.) is required. Several drugs can produce lung injury and DAD, with secondary DAH (e.g., sirolimus, methotrexate, nitrofurantoin, etc.). Other drugs causing DAH trigger a process of pulmonary capillaritis (e.g., propylthyouracil, phenytoin, mitomycin, and ATRA). Penicillamine can cause pulmonary capillaritis associated with glomerulonephritis with granular immunofluorescent deposits (pulmonary– renal syndrome). Interestingly, these agents are also responsible for p-ANCA generation. The standard therapy is discontinuation of the presumed offending drug and (rarely) plasma exchange. Other Causes of DAH

Other conditions associated with alveolar bleeding are: different coagulopathies, toxic exposures (isocyantes, trimellitic anhydride, crack cocaine, etc.) primary antiphospholipid antibody syndrome, mixed cryoglobulinemia, Behçet’s syndrome, Henoch– Scho¨nlein purpura, lung transplant acute rejection, mitral stenosis and regurgitation, pulmonary venoocclusive disease, pulmonary capillary hemangiomatosis, LAM, and tuberous sclerosis Bourneville. In patients with primary antiphospholipid syndrome (APS), the thromboembolic complications occur in up to 15% of cases, pulmonary hypertension in about 2% of the cases, interstitial lung disease in up to 1% of patients, with DAH in less than 1% of cases. Idiopathic Pulmonary Hemosiderosis

Idiopathic pulmonary hemosiderosis (IPH) is a diagnosis of exclusion (see Idiopathic Pulmonary Hemosiderosis); it is a rare disease characterized by recurrent episodes of DAH and ‘bland’ alveolar hemorrhage (Figure 2). IPH occurs most frequently in children (80% of cases), but adult cases with onset up to the eighth decade of life have been reported (20% of cases). The pathogenesis of IPH is largely unknown. Some cases have been linked to fungi (Stachybotrys atra), others to environmental insecticides; it seems that several cases have initially been called IPH, when in fact coagulopathies such as van Willebrand’s disease have been found upon

Figure 2 Diffuse alveolar hemorrhage without any evidence of pulmonary vasculitis (‘bland’ alveolar hemorrhage) in a patient with idiopathic pulmonary hemosiderosis.

further scrutiny. Corticosteroids, hydroxychloquine, azathyoprine, and other immunosuppressive agents have been used with favorable effects. Lung transplantation has been reported as rather unsuccessful in a couple of patients with progressive disease and significant pulmonary fibrosis, due to recurrent pulmonary bleeding. Survival with IPH varies widely, although recent data has suggested better outcomes, most likely due to more aggressive immunosuppressive therapies.

Summary of Therapeutic Options DAH syndromes represent a serious condition with possible catastrophic consequences, caused by a myriad of conditions, associated or not with pulmonary capillaritis. Dyspnea, cough, hemoptysis and new alveolar, fluffy infiltrates in conjunction with bronchoscopic findings of bloody BAL, with numerous erythrocytes and siderophages make the syndrome diagnosis evident. Rarely, a surgical biopsy from the lung or another organ involved by the underlying condition may be necessary. The advent of ANCA has revolutionarized the diagnosis of WG, microscopic polyangiitis, Churg–Strauss syndrome, and other ANCA-associated conditions. The therapy in DAH targets both the autoimmune destruction of the alveolar capillary membrane and the underlying condition; corticosteroids and immunosuppressive agents are still the ‘gold standard’ of therapy in the majority of cases. Factor VIIa seems to be a promising new therapy for DAH, although further evaluation is needed.

100 ALVEOLAR HEMORRHAGE See also: Acute Respiratory Distress Syndrome. Autoantibodies. Bronchoalveolar Lavage. Granulomatosis: Wegener’s Disease. Idiopathic Pulmonary Hemosiderosis. Systemic Disease: Diffuse Alveolar Hemorrhage and Goodpasture’s Syndrome.

Further Reading Afessa B, Cowart RG, and Koenig SM (1997) Alveolar hemorrhage in IgA nephropathy treated with plasmapheresis. Southern Medical Journal 90: 237–239. Afessa B, Tefferi A, Litzow MR, and Peters SG (2002) Outcome of diffuse alveolar hemorrhage in hematopoietic stem cell transplant recipients. American Journal of Respiratory and Critical Care Medicine 166: 1364–1368. Agusti C, Ramirez J, Picado C, et al. (1995) Diffuse alveolar hemorrhage in allogeneic bone marrow transplantation: a postmortem study. American Journal of Respiratory and Critical Care Medicine 151: 1006–1010. Bar J, Ehrenfeld M, Rozenman J, et al. (2001) Pulmonary–renal syndrome in systemic sclerosis. Seminars in Arthritis and Rheumatism 30: 403–410. Collard HR and Schwarz MI (2004) Diffuse alveolar hemorrhage. Clinics in Chest Medicine 25: 583–592. vii. Dhillon SS, Singh D, Doe N, et al. (1999) Diffuse alveolar hemorrhage and pulmonary capillaritis due to propylthiouracil. Chest 116: 1485–1488. Dweik RA, Arroliga AC, and Cash JM (1997) Alveolar hemorrhage in patients with rheumatic disease. Rheumatic Diseases Clinics of North America 23: 395–410. Dweik RA and Stoller JK (1999) Role of bronchoscopy in massive hemoptysis. Clinics in Chest Medicine 20: 89–105. Franks TJ and Koss MN (2000) Pulmonary capillaritis. Current Opinion Pulmonary Medicine 6: 430–435. Gertner E (1999) Diffuse alveolar hemorrhage in the antiphospholipid syndrome: spectrum of disease and treatment. Journal of Rheumatology 26: 805–807. Green RJ, Ruoss SJ, Kraft SA, et al. (1996) Pulmonary capillaritis and alveolar hemorrhage: update on diagnosis and management. Chest 110: 1305–1316. Henke D, Falk RJ, and Gabriel DA (2004) Successful treatment of diffuse alveolar hemorrhage with activated factor VII. Annals of Internal Medicine 140: 493–494. Ioachimescu O (2003) Idiopathic pulmonary hemosiderosis in adults. Pneumologia 52: 38–43. Ioachimescu OC, Kotch A, and Stoller JK (2005) Idiopathic pulmonary hemosiderosis in adults. Clinical Pulmonary Medicine 12: 16–25. Ioachimescu OC, Sieber S, and Kotch A (2004) Idiopathic pulmonary hemosiderosis revisited. European Respiratory Journal 24: 162–170. Jennings CA, King TE Jr, Tuder R, Cherniack RM, and Schwarz MI (1997) Diffuse alveolar hemorrhage with underlying

Alveolar Proteinosis

isolated, pauciimmune pulmonary capillaritis. American Journal of Respiratory and Critical Care Medicine 155: 1101– 1109. Klemmer PJ, Chalermskulrat W, Reif MS, et al. (2003) Plasmapheresis therapy for diffuse alveolar hemorrhage in patients with small-vessel vasculitis. American Journal of Kidney Diseases 42: 1149–1153. Lai RS, Lin SL, Lai NS, and Lee PC (1998) Churg–Strauss syndrome presenting with pulmonary capillaritis and diffuse alveolar hemorrhage. Scandinavian Journal of Rheumatology 27: 230–232. Lauque D, Cadranel J, Lazor R, et al. (2000) Microscopic polyangiitis with alveolar hemorrhage: a study of 29 cases and review of the literature-Groupe d’Etudes et de Recherche sur les Maladies ‘‘Orphelines’’ Pulmonaires (GERM ‘‘O’’ P). Medicine (Baltimore) 79: 222–233. Leatherman JW (1988) The lung in systemic vasculitis. Seminars in Respiratory Infections 3: 274–288. Murray RJ, Albin RJ, Mergner W, and Criner GJ (1988) Diffuse alveolar hemorrhage temporally related to cocaine smoking. Chest 93: 427–429. Nadrous HF, Yu AC, Specks U, and Ryu JH (2004) Pulmonary involvement in Henoch–Scho¨nlein purpura. Mayo Clinic Proceedings 79: 1151–1157. Pastores SM, Papadopoulos E, Voigt L, and Halpern NA (2003) Diffuse alveolar hemorrhage after allogeneic hematopoietic stem-cell transplantation: treatment with recombinant factor VIIa. Chest 124: 2400–2403. Schwarz MI and Brown KK (2000) Small vessel vasculitis of the lung. Thorax 55: 502–510. Schwarz MI and Fontenot AP (2004) Drug-induced diffuse alveolar hemorrhage syndromes and vasculitis. Clinics in Chest Medicine 25: 133–140. Schwarz MI, Mortenson RL, Colby TV, et al. (1993) Pulmonary capillaritis: the association with progressive irreversible airflow limitation and hyperinflation. American Review of Respiratory Diseases 148: 507–511. Schwarz MI, Zamora MR, Hodges TN, et al. (1998) Isolated pulmonary capillaritis and diffuse alveolar hemorrhage in rheumatoid arthritis and mixed connective tissue disease. Chest 113: 1609–1615. Segal SL, Lenchner GS, Cichelli AV, et al. (1988) Angiosarcoma presenting as diffuse alveolar hemorrhage. Chest 94: 214–216. Specks U (2001) Diffuse alveolar hemorrhage syndromes. Current Opinion in Rheumatology 13: 12–17. Travis WD, Colby TV, Lombard C, and Carpenter HA (1990) A clinicopathologic study of 34 cases of diffuse pulmonary hemorrhage with lung biopsy confirmation. American Journal of Surgical Pathology 14: 1112–1125. Zamora MR, Warner ML, Tuder R, and Schwarz MI (1997) Diffuse alveolar hemorrhage and systemic lupus erythematosus: clinical presentation, histology, survival, and outcome. Medicine (Baltimore) 76: 192–202.

see Interstitial Lung Disease: Alveolar Proteinosis.

ALVEOLAR SURFACE MECHANICS 101

ALVEOLAR SURFACE MECHANICS S B Hall and S Rugonyi, Oregon Health & Science University, Portland, OR, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The major component of the recoil forces that tend to deflate the lungs is the surface tension of a thin liquid layer that lines the alveoli. This surface tension is well below the value for a clean air/water interface, indicating the presence of a surfactant. The surface tensions in situ specify that the surfactant films must have certain characteristics. Following large expansions of the air/water interface during deep inhalations, the surfactant must adsorb rapidly to form the interfacial film. When compressed by the shrinking surface area during exhalation, the films must be sufficiently rigid to resist the tendency to collapse from the interface. Materials washed from the lungs show that pulmonary surfactant is a mixture containing mostly lipids with some proteins that is synthesized and secreted by the type II pneumocyte. The disorder in which an abnormality of pulmonary surfactant most clearly plays a role is the respiratory distress syndrome of premature babies, although altered surfactant function may also contribute to the acute respiratory distress syndrome that occurs in patients of all ages.

Description The importance of alveolar surface mechanics is readily evident from the pressure–volume (P–V) characteristics of the lungs. Pressures required to maintain any given volume are substantially higher for lungs inflated with air than with saline. The fundamental difference between the two procedures is that saline eliminates an air/water interface, the surface tension of which contributes to the inward recoil forces for lungs inflated with air. The presence of

surface tension implies that the curved surfaces of the pulmonary airspaces are lined by a layer of liquid. Electron microscopy has demonstrated that such a layer coats the alveoli, and that it is thin, with an average thickness of 0.2 mm, and continuous. The surface tension resulting from the air/water interface of this alveolar lining represents the major component of contractile forces in the lungs. Surface tension results from an imbalance of forces on molecules close to an interface between two separated phases. For molecules deep within a substance, attractive forces towards neighboring constituents are equal in all directions (Figure 1). Within a few molecular diameters of the interface, however, components experience a reduced attraction towards constituents in the adjoining phase, resulting in a net inward pull and a force per unit length, or surface tension, that tends to contract the interfacial area. The force along a curved surface, such as in the alveoli, translates directly into a difference in pressures across the interface. For a spherical interface with radius R, mechanical equilibrium between surface tension, s, and the difference in pressures, p p0, occurs (Figure 2) when pR2 ðp p0 Þ ¼ 2pRs which leads directly to the law of Young and Laplace for a sphere: Dp ¼ 2s=R Inflation of lungs with air therefore requires higher pressure to overcome interfacial forces that are absent during inflation with saline.

Air Water

Figure 1 Origin of surface tension: molecules in the bulk water experience an attractive force towards neighboring molecules that is equal in all directions, resulting in no net force. Within a few molecular diameters of the interface, however, the absence of neighbors towards the surface results in a net inward pull that tends to shrink the interfacial area. A film of surfactant at the interface tends to spread, resulting in an opposing force that lowers surface tension.

102 ALVEOLAR SURFACE MECHANICS p0 p

R

Figure 2 Relationship of surface tension to hydrostatic pressure across a spherical surface. Mechanical equilibrium occurs when the inward recoil force of surface tension (s) and the opposing force of hydrostatic pressure (p p0) are equal. At the midsection of a spherical bubble with radius R, surface tension will produce a force (length s) ¼ 2pRs, and the force from hydrostatic pressure will be [area (p p0)] ¼ pR2(p p0). The equality leads directly to the law of Young and Laplace, Dp ¼ 2s/R.

Several methods have been used to determine surface tension in the lungs. The difference in P–V curves between air- and saline-filled lungs can provide surface tensions, either with simple assumptions concerning alveolar geometries and tissue forces, or with an energetic analysis that makes those assumptions unnecessary. Rinsing the lungs with a series of detergents or liquids provides an air/liquid interface with constant known surface tensions, and the intersection of P–V curves from these and normal lungs indicates points of common surface tension. Fluorocarbon droplets deposited on the interfacial film in peripheral alveoli spread according to the relative surface tensions of the fluorocarbon and film, and the shape of droplets containing different materials has provided the most direct estimates of surface tensions in situ. These different approaches have produced remarkably consistent results. During excursions through large volumes, surface tension shows a major hysteresis between inflation and deflation that explains the hysteresis of the P–V mechanics. Deflation from total lung capacity to functional residual capacity lowers surface tension from B30 mN m 1 to o5 mN m 1. Over tidal volumes, surface tension varies between 10 mN m 1 and values as low as 1 mN m 1. These surface tensions are well below the values for a clean air/water interface, which would be 70 mN m 1, and indicate the presence of a surfactant. Surfactants are the general class of compounds that have higher concentrations at the surface than in the aqueous medium. They are amphipathic, with distinct hydrophilic and hydrophobic regions of the molecule, and best satisfy the energetics of both portions by orientation across an interface. Once trapped within the two-dimensional surface, surfactants tend to spread, resulting in a force that expands the interface and opposes surface tension. Surfactant films with higher densities produce larger reductions in surface

tension. The very low surface tensions in the lungs indicate films with particularly high densities. Surfactants can lower surface tension by adsorbing to an interface only to a limited extent. Adsorbed surfactants reach a maximum density, above which further constituents form a three-dimensional bulk phase at the interface rather than adding to the twodimensional film. Surface tensions in the lungs reach values well below this minimum equilibrium value, and they also vary with volume. These observations indicate that the low surface tensions and high densities of the films result not from insertion of more constituents, but from a decrease in surface area during deflation. The low surface tensions reached during deflation are impressively stable in static lungs for prolonged periods. The low magnitude and particularly the stability of the surface tensions indicate that the compressed films have specific characteristics. When compressed above the maximum equilibrium density, films that can flow from the surface will collapse to form the three-dimensional bulk phase, thereby reestablishing equilibrium surface tensions. Only films that are solid, defined by their inability to flow, can resist the tendency to collapse and demonstrate the behavior observed in the lungs. When compressed sufficiently, even solid films collapse. An interface without surface tension has no basis for existence, and at sufficiently high densities, solid films must also rupture. The hysteresis of surface tension, and of hydrostatic pressures, between deflation and inflation over large volumes reflects at least partially material lost from the interface during collapse. Lavaging the lungs produces the increase in recoil pressures expected from removal of a surfactant and a resulting increase in surface tension. Material recovered from the lungs can form films with characteristics indicated by the in situ measurements. Material obtained from alveolar foam, when suspended in saline, can form small bubbles that persist for prolonged periods, indicating that the pressuredifference across their surface, which would tend to dissolve the gas in the surrounding liquid, must be low, and that according to the law of Young and Laplace, surface tension must also be small. When compressed in vitro by changing their surface area, films formed from lavaged material can reach and sustain surface tension below the minimum equilibrium value. Material purified from lavaged material can also restore the P–V mechanics of the original lungs. These observations provide the basis for the identification of the surfactant in the lungs. Material recovered by lavage contains two sets of phospholipid vesicles that differ in size. The larger


Air

Liquid layer

Type I cell

Molecular view

Lamellar body Tubular myelin

Small vesicles

Surfactant film

Type II cell

Figure 3 Schematic of pulmonary surfactant in the alveolus. Constituents of pulmonary surfactant are synthesized in type II pneumocytes and assembled into lamellar bodies, which are secreted into a thin liquid layer that lines the alveolus. The vesicles unravel and first form tubular myelin, which appears to represent the immediate precursor of a film at the air/water interface. Lavage of the lungs recovers both the large multilamellar vesicles, which contain mostly phospholipids with small amounts of cholesterol and proteins, and small unilamellar vesicles, which contain only the lipids.

form has the same morphological appearance as a subcellular organelle, the lamellar body, of the type II pneumocytes (Figure 3). Organic extracts of these larger particles can restore the P–V mechanics of lavaged lungs, which perhaps best defines these vesicles as ‘pulmonary surfactant’. The smaller particles may represent material excluded from the interface during compression to very low surface tensions. Vesicles of both sizes contain the same set of phospholipids with small amounts of cholesterol. The larger particles are much more capable of lowering surface tension in vitro than the smaller vesicles. Four proteins copurify with the larger particles but are absent from the smaller forms. The organic extracts of the large particles, which function well in lavaged lungs, lack surfactant proteins SP-A and SP-D, and based on a variety of assays, their primary function appears related to processes other than the lowering of surface tension. SP-B and SP-C, which are sufficiently hydrophobic to extract with the lipids into organic solvents, determine the difference in surface activity between the large and small particles.

Normal Physiological Processes To achieve the surface tensions observed in the lungs, pulmonary surfactant must satisfy conflicting constraints. Vesicles must first adsorb rapidly to form the interfacial film. Pulmonary mechanics become normal during the first few breaths following the initial air-inflation of fluid-filled lungs, suggesting that adsorption to the newly created air/water interface forms a film having the equilibrium density within seconds. The low surface tensions reached during deflation indicate that the compressed film avoids the reverse process of desorption to re-establish the equilibrium density. Replicating in vitro the full

performance of films in the lungs has been difficult, and the mechanisms by which pulmonary surfactant functions in the alveoli remain the subject of active investigation. Adsorption

The adsorption of pulmonary surfactant is fundamentally different from the process for other more common surfactants, which insert into the interface as individual molecules. Surfactants share the general characteristic that they exist in solution as individual monomers only up to a certain concentration, above which they aggregate into structures such as micelles and bilayers (Figure 4), the nature of which depends on the effective shape of the particular molecule. The ‘critical micelle concentration’ at which phospholipids aggregate is less than 10 9 M. Constituents of pulmonary surfactant therefore exist in the alveolar lining exclusively as vesicles, which insert as collective units into the interface. Because the vesicles themselves are stable in aqueous medium, an energy barrier limits adsorption, which occurs quite slowly for lipid vesicles without the hydrophobic proteins. In the alveolar lining, lamellar bodies secreted by the type II pneumocytes unravel to form a distinct intermediate structure known as tubular myelin that apparently represents the direct precursor of the interfacial film (Figure 3). SP-A is required in vitro for the reconstruction of tubular myelin, which is absent from extracted surfactants and transgenic animals that lack SP-A. Extracted surfactants, however, function well in surfactant-deficient lungs, and mice without SP-A have normal pulmonary mechanics. The functional significance of tubular myelin for adsorption is therefore unclear. The absence of SP-B, whether in transgenic animals or in patients with

104 ALVEOLAR SURFACE MECHANICS

Figure 4 Aggregation of surfactants. Above a certain concentration, the hydrophobic portion of surfactants makes them insoluble as individual molecules, and causes their aggregation into structures that expose their hydrophilic components to the aqueous medium while sequestering the hydrophobic segments. The particular form of the aggregate depends on the effective shape of the specific compounds. Single chain surfactants tend to form micelles. Biological phospholipids form bilayers that may stack into the concentric layers of a multilamellar vesicle.

genetic abnormalities, does produce abnormal pulmonary function, consistent with the crucial role suggested by in vitro studies of this protein for adsorption. Stability of Compressed Film

Under equilibrium conditions, attempts to increase the density of surfactant monolayers above a maximum density, either by adding more constituents or by decreasing interfacial area, instead forms a threedimensional bulk phase that coexists with the twodimensional film. The bulk phase of phospholipids is a liquid-crystal, in which layers of material stack in the regularly repeating manner characteristic of a crystal, but with each layer having the disordered structure that is characteristic of liquids. Slowly compressed monolayers of pulmonary surfactant in vitro flow into these stacked structures (Figure 5), and their ability to flow indicates that the films are fluid. In the lungs, the prolonged low surface tensions, well below the minimum equilibrium values, indicate films that resist flow, and that therefore have the defining characteristic of a solid. The structure of two-dimensional solid films could be either highly ordered, analogous to a three-dimensional crystal, or amorphous, like a glass. At physiological temperatures, a single constituent of pulmonary surfactant, dipalmitoyl phosphatidylcholine (DPPC), can form highly ordered films that approach the structure of a two-dimensional crystal. DPPC has the unusual characteristic relative to other biological phospholipids that both acyl chains are fully saturated and that it constitutes an unusually large amount (30–50%) of pulmonary

surfactant. A widely held view contends that the functional film in the lung consists of essentially pure DPPC. The difference in composition between the secreted vesicles and the hypothetical functional film of DPPC could result either from selective adsorption of DPPC or from selective collapse of other constituents. Both processes are difficult to reconcile with current understandings of how adsorption and collapse occur. One prediction of the model is that P–V curves should change abruptly over a narrow range of temperatures. Films of DPPC, like three-dimensional crystals, melt from solid to fluid structures at specific temperatures. At surface tensions below the minimum equilibrium value, rates of collapse increase when solid films melt, resulting in increased surface tensions that would produce higher recoil pressures. Measurements of the temperature dependence for P–V curves have yielded conflicting results, and the presence of a highly ordered film remains unconfirmed. Although films containing only DPPC would explain the stability of low surface tensions in the lungs, experimental evidence to support that possibility is limited. The solid films that sustain low surface tensions in situ could also have a structure that resembles a two-dimensional glass. Three-dimensional liquids, if cooled fast enough and far enough below their freezing temperatures, retain their disordered structure but become frozen in place, forming amorphous solids, or glasses. Two-dimensional fluid films, defined by their ability to flow into collapsed structures, similarly become jammed into a form that resists collapse if supercompressed to sufficiently high


Figure 5 Liquid-crystalline collapse. Under equilibrium conditions, surfactant films reach a minimum surface tension at which they undergo a phase transition to form a three-dimensional bulk phase. Phospholipids form bulk smectic liquid crystals, and compression of fluid phospholipid films at the minimum equilibrium surface tension can cause the film to flow into stacked structures. Solid films, which can resist flow and remain at the interface below the minimum equilibrium surface tension, eventually also collapse from the interface at very low surface tensions, although probably by a process more like fracture.

densities and low surface tensions. These supercompressed films retain their solid behavior and slow rates of collapse when expanded, even when returned to the surface tensions at which they originally collapsed. If they reach low surface tensions in the lungs during a single exhalation, the films would be transformed, and their ability to avoid collapse could persist through multiple cycles of tidal breathing. To achieve low surface tensions, however, the initially fluid films must be compressed faster than they can collapse. The required rates may occur during normal breathing, but they are faster than rates in quasi-static experiments with excised lungs. The supercompressed films, which would require no compositional change, could explain surface tensions observed in the lungs, but like the films of pure DPPC, the process by which they would form remains unclear. Original views concerning surface tension in the lungs considered an interfacial film with the thickness of one molecule. Although perhaps difficult to explain how they might form, monolayers that have the characteristics of films in the lungs are well described, and more complicated structures were unnecessary to explain the observed behavior. Electron microscopy, however, has demonstrated that in situ, at least parts of the interface are occupied by films that are multilayered. Whether these structures are formed during adsorption or collapse, and the extent to which the additional material might affect the mechanical characteristics of the film, are both unknown.

Physiological Processes in Respiratory Diseases The disorder in which an abnormality of pulmonary surfactant most clearly represents a major pathogenic factor is the respiratory distress syndrome (RDS) that occurs in premature babies. Ventilation of immature lungs that lack adequate amounts of surfactant injures the alveolocapillary barrier, resulting in pulmonary edema and respiratory failure. Two

mechanisms, both involving shear stresses, could explain how a deficiency of pulmonary surfactant would produce the injury. First, elevated surface tension would produce an increased tendency for small alveoli to collapse, and the shear stresses involved in reopening the closed airspaces could produce the injury. Second, the meniscus of any fluid column in the small airways would have an increased surface tension, and the greater pressure-difference across the interface could rupture the epithelial cells over which it passes. Elevated surface tensions would also lower interstitial and pericapillary pressures, resulting in a greater transmural pressure-difference that would increase the flow of fluid across the alveolocapillary membrane. The most direct evidence that deficient surfactant causes RDS comes from manipulation of surfactant levels. Subsequent to removing surfactant by lavage, ventilation of animals produces an injury that replicates RDS. Conversely, giving exogenous surfactant to premature babies at risk for RDS prevents or reverses the disorder. The acute respiratory distress syndrome (ARDS) was originally called adult RDS to point out the clinical similarities between adults with injured lungs and the infants with RDS, and to suggest that the common presentation resulting from a variety of insults might reflect an abnormality of surfactant acting as the final common pathway. Although the primary defect in ARDS is an inflammatory process, abnormal surfactant might perpetuate the initial injury by the same processes that result from an elevated surface tension in RDS. Surfactant function could be altered in ARDS by either deficiency or inhibition. Injured lungs have reduced levels of the large active surfactant vesicles, suggesting a deficiency. Pulmonary surfactant could also be inhibited by the large number of extraneous compounds, such as plasma proteins and membrane lipids, that reach the alveolus in injured lungs and that can act as surfactants. Direct evidence for elevated surface tensions in ARDS, however, is limited. The presence of edema complicates the interpretation

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of P–V mechanics, and the distinction of small lungs, caused by fluid-filled airspaces that occur with any pulmonary edema, from stiff lungs, caused by increased surface tension, has been difficult. Evidence that exogenous surfactants can mitigate ARDS has also been lacking. The larger doses required to treat adults with ARDS relative to premature babies with RDS has limited the therapeutic agents that can be used. Initial trials with surfactants that lack SP-B have produced no improvement, just as early attempts to treat babies with RDS using aerosolized DPPC had no benefit. The role of therapeutic surfactants in ARDS, and of abnormal surfactant in its pathogenesis, therefore remains unresolved. See also: Acute Respiratory Distress Syndrome. Alveolar Hemorrhage. Alveolar Wall Micromechanics. Breathing: Breathing in the Newborn; Fetal Lung Liquid; First Breath. Bronchoalveolar Lavage. Drug-Induced Pulmonary Disease. Epithelial Cells: Type I Cells; Type II Cells. Fluid Balance in the Lung. Infant Respiratory Distress Syndrome. Lung Anatomy (Including the Aging Lung). Lung Imaging. Surfactant: Overview; Surfactant Protein A (SP-A); Surfactant Proteins B and C (SP-B and SP-C); Surfactant Protein D (SP-D).

Further Reading Bastacky J, Lee CY, Goerke J, et al. (1995) Alveolar lining layer is thin and continuous: low-temperature scanning electron

microscopy of rat lung. Journal of Applied Physiology 79: 1615–1628. Goerke J and Clements JA (1985) Alveolar surface tension and lung surfactant. In: Macklem PT and Mead J (eds.) Handbook of Physiology The Respiratory System, vol. III, part 1, pp. 247– 261. Washington, DC: American Physiological Society. Hoppin J, Frederic G, Joseph C, et al. (1986) Lung recoil: elastic and rheological properties. In: Fishman AE (ed.) Handbook of Physiology: A Critical, Comprehensive Presentation of Physiological Knowledge and Concepts, pp. 195–215. Bethesda, MD: American Physiological Society. Keough KMW (1992) Physical chemistry of pulmonary surfactant in the terminal air spaces. In: Robertson B, van Golde LMG, and Batenburg JJ (eds.) Pulmonary Surfactant: from Molecular Biology to Clinical Practice, pp. 109–164. Amsterdam, New York: Elsevier. Lewis JF and Jobe AH (1993) Surfactant and the adult respiratory distress syndrome. American Review of Respiratory Diseases 147: 218–233. Piknova B, Schram V, and Hall SB (2002) Pulmonary surfactant: phase behavior and function. Current Opinion in Structural Biology 12: 487–494. Robertson B (1984) Pathology and pathophysiology of neonatal surfactant deficiency (‘‘respiratory distress syndrome,’’ ‘‘hyaline membrane disease’’). In: Robertson B, Van Golde LMG, and Batenburg JJ (eds.) Pulmonary Surfactant, pp. 383–418. Amsterdam: Elsevier. Schu¨rch S, Green FH, and Bachofen H (1998) Formation and structure of surface films: captive bubble surfactometry. Biochimica et Biophysica Acta 1408: 180–202. Stamenovic D (1990) Micromechanical foundations of pulmonary elasticity. Physiological Reviews 70: 1117–1134. Wilson TA (1981) Mechanics of the pressure-volume curve of the lung. Annals of Biomedical Engineering 9: 439–449.

ALVEOLAR WALL MICROMECHANICS F G Hoppin Jr, Brown University, Providence, RI, USA

Introduction


The alveolar wall is the site of gas exchange by passive diffusion between inspired gas and the pulmonary capillary blood. Its tiny scale supports passive diffusion by providing an immense net-diffusing area and a short path length. Because it is elastically extensible, the gas-exchanging lung units can readily expand and contract during breathing. How does this diaphanous structure meet these functional needs and at the same time avoid structural weakness, configurational instability, and fluid accumulation?

Abstract The site of gas exchange in the lung is the alveolar wall. Its structure supports passive diffusion of the respiratory gases by spreading the pulmonary capillary bed over an immense surface area and by having an extremely thin air/blood barrier. It enables the alveolar gas spaces to inflate and deflate by its great extensibility. Yet it remains stiff enough to withstand the severe collapsing forces of surface tension acting in tiny confines and to transmit elastic tensions throughout the network of alveolar walls, maintaining by those tensions the fine and gross configuration of the lung and a reasonable distribution of ventilation within the lung. It can modulate local ventilation by activation of its smooth muscle. The mechanical properties that meet these exacting requirements can be understood in terms of the behavior of elastic networks and the elastic properties of the wall’s solid components and of pulmonary surfactant. Dysfunction at the level of the alveolar wall lies at the core of most disorders of the lung by impairing diffusion, ventilation, and perfusion.

Normal Mechanics The lung, like a parachute or a spinnaker, is a tensed structure (Figure 1). Patency of the airspaces and a relatively even distribution of expansion and contraction during breathing depend on satisfactory

ALVEOLAR WALL MICROMECHANICS 107

50 µm E J B

Figure 1 Photomicrograph of inflated lung, showing the network of alveolar walls, partitioning the lung into alveolar gas spaces. The predominant termination of the profile of the alveolar wall is a triple junction with two other walls (J). Where alveolar gas spaces open into an alveolar duct (right top), the profiles either appear bent (B) or simply end (E), and these edges of the wall are invariably reinforced with connective tissue cables, often accompanied by smooth muscle. Reproduced from Butler JP, Oldmixon EH, and Hoppin FG Jr (1996) Dihedral angles of septal ‘bend’ structures in lung parenchyma. Journal of Applied Physiology 81: 1800–1806, used with permission of The American Physiological Society.

transmission of tensions (lung elastic recoil) throughout. The evidence for the network of alveolar walls being the major tension-bearing structure in the lung include the following: *

*

*

*

It contains the majority of the lung’s tension-bearing material. Its tension-bearing structures are continuous throughout the lung. Distortions of the inflated lung whether by gravity, local indentation of the surface, or local internal expansions or contractions behave as expected for a diffuse, isotropic elastic network. Estimates of lung elastic recoil, based on stereological data and elastic properties of the tensionbearing components of the alveolar wall are close to observed lung-distending pressure differences over a range of inflation volumes.

The other tension-bearing structures, acting mechanically in parallel, include the lung’s outer rind (pleura), which has been thought to account for only B20% of the work of inflation, and the fibrous connective tissue systems of the airway and bronchovascular trees, which have been estimated to bear B10%. The network of alveolar walls is a cable/membrane structure. The membrane portion of the alveolar wall is tensed at its edges either by other walls or by an embedded cable of relatively heavy connective tissue. It is thin, polygonal, and pseudoplanar. It has three

main tension-bearing components: the fine, isotropic fibrous network; the basement membranes of the epithelium and capillary endothelium; and the air-liquid interfaces on either side (Figure 2). Along most of its perimeter (B66%), the wall joins two others (J in Figure 1). At this junction, there is no cable; the fine fibrous network, basement membranes, and air-liquid interfaces are directly continuous from one wall to the adjacent walls. The tensions of the walls at such junctions are resolved by the balance of the three walls pulling in three directions. Another mechanism is needed where the alveolar spaces open into the alveolar duct. At these locations, the wall either meets one other wall (B24% of its perimeter, B in Figure 1) or simply ends (B9% of the perimeter, E in Figure 1) and it requires some ancillary support. This is provided by relatively heavy connective tissue cables that are curved so as to oppose the wall’s tensions. The fine fibrous network of the walls connects directly with these cables. The cables of adjoining walls form a lattice that frames the alveolar duct. Centrally, their connective tissues merge with the still heavier connective tissues of the terminal bronchioles. The difference between the E and B configurations, incidentally, probably does not reflect a functional difference beyond the geometric complexity of space packing of alveoli and of the branching airway system. The cables and membranes, being continuous structures throughout the network, are capable of transmitting tensions from membrane to membrane or cable to cable across the lung in any direction. However, there is also a distinct serial connection between the cables and membranes at the level of the alveolar duct and its surrounding alveoli, where the membranes pull radially against the cables. This is dramatically apparent as dilation of the ducts of lungs that have been washed with detergent to increase the wall’s interfacial tension. Normally, however, the elastic properties of these very different systems are sufficiently matched so that expansion and contraction of the duct and its surrounding alveoli are relatively symmetrical during breathing. Lying alongside most of the cables are bundles of smooth muscle. Its function relates to its characteristic stiffening when it is activated and passively length-cycled at typical breathing frequencies. As a result of the series relationship of the alveolar duct and its surrounding alveoli (above), stiffening of the cables reduces the compliance of the respiratory unit. Indeed, the lung is substantially stiffened in response to low levels of carbon dioxide (hypocapnia). This suggests a homeostatic mechanism. Relatively under’ lung units are characteristically ’ Q) perfused (high V= hypocapnic. A local response that stiffens the lung

108 ALVEOLAR WALL MICROMECHANICS

Alveolar lining liquid Epithelial cell Basement membrane Interstitium Basement membrane Endothelial cell

Epithelial cell nucleus Fine fiber network Endothelial cell nucleus Red blood cell Plasma

Endothelial cell Fused basement membrane Epithelial cell Alveolar lining liquid Figure 2 Schematic depiction of the structure of an alveolar wall. It is pseudoplanar and has dimensions on the order of 100 100 10 mm. In the middle is the interstitium, containing the supporting fine fiber network, the marsh-like pulmonary capillary bed, and small amounts of interstitial fluid. Outside the interstitium on both sides of the wall is a basement membrane supporting the single layer of thin alveolar epithelial cells and, outside that, a fluid alveolar lining. The capillary itself is a single thin endothelial cell and its supporting basement membrane, fused on one side of the capillary with the basement membrane of the epithelium.

locally reduces the ventilation of such units. This hypocapnic pneumoconstriction improves the overall efficiency of gas exchange, much as hypoxic pulmonary vasoconstriction reduces the perfusion of un’ units. ’ Q) derventilated (low V= Tensions in adjoining walls are quite uniform. This conclusion is drawn from a vector analysis of the tensions at J and B junctions, the assumption that the walls are free to rotate relative to each other, and data for the configuration of fixed inflated specimens. At J junctions, the distribution of dihedral angles between walls, extremely narrow around 1201, is consistent with B2% variation of wall tensions. At B junctions, the inferred distribution is somewhat wider, but has a notably sharp cutoff below 1201. Open soap films supported on complex wire frames show these several features at their J and B junctions, where they are well understood to reflect (1) locally uniform film tensions and (2) a mechanism for adopting the least energy configuration, that is, minimal surface area. This finding suggests, intriguingly, that lung structure during growth and development (perhaps even during remodeling of the adult lung?) responded to these same physical principles. Local uniformity of wall

tensions, of course, does not imply global uniformity, which varies systematically over large distances in the lung, for example, vertically in the gravity field. The wall is highly extensible, undergoing up to about twofold change in linear dimensions for the deepest breaths, that is, about eightfold change of volume. (Birds followed a different evolutionary route by having unidirectional air flow through a relatively stiff gas exchange apparatus.) The risks inherent in such extensibility include instability, collapse, and gross unevenness of ventilation among the lung’s different air compartments. These risks are limited by the wall’s positive compliance and by the effects of network geometry. A given distortion of the elastic network stretches the walls on the side away from the displacement and realigns them closer to the direction of the displacement. As the walls are positively compliant, the effect is to increase tension on that side, and the effect of that increase is enhanced by the realignment of the walls to better oppose the displacement. The converse occurs on the side towards the displacement. This mechanism acts to stabilize configuration at the fine (alveolus) and gross (regional) levels.

ALVEOLAR WALL MICROMECHANICS 109

The wall’s positive compliance is conferred independently by both the air–liquid interface and the solid components (fine fibrous network and basement membranes). Interfacial tension modulated by pulmonary surfactant varies monotonically from near 0 to B30 dynes cm 1 over a fourfold change of surface area. The fine fibrous connective tissues are collagen fibers (stiff as steel in isolated fibers) and elastin (the prototypical rubber). They are substantially entangled and cross-linked, and the compressibility of a proteoglycan matrix modifies the extent to which collagen fibers fold and stretch. The resulting solid components are capable of more than the about twofold linear extension, that is, of maintaining tension over the full operating range of lung volume. The tensions in the interface interact with those of the solid elements of the wall through curvature. There is no reason to postulate shear forces between them. The interface tenses the cables at E and B junctions by curvature that is convex to the airspace, that is by draping over the cable region like washing on a clothes line. At the inner corners of the alveolus (J junctions and the inner side of the B junctions), by contrast, the curvature of the interface is sharply concave to the airspace, and therefore interfacial tension reduces the pressure in (i.e., sucks out on) the junctional tissues, transmitting tension to the cable and/or other septae at that junction. In lungs prepared in protocols that permit collapse, the freedom to shear permits the solid elements of the wall to pleat or fold beneath a smoothly curved interface in the corners of alveoli. This is probably not the configuration in vivo. Transiently low distending pressures (o2–3 cmH2O) permit folding. High distending pressures (416– 20 cmH2O) are required to resolve the folds. With the appropriate protocols, the wall’s fine fiber network remains quite planar right into the corners of the alveoli even down to 2–3 cmH2O. This is consistent with the capability of the fine fiber network to bear tension over the full feasible range of lung inflation, and with its roles of supporting the capillary network and of maintaining the configuration of the alveolus, particularly at low inflation volumes. The pulmonary capillaries are notably affected by the mechanical state of the wall. The majority of the capillary bed lies within the flat portions of the wall. There, the capillary distending pressure (the difference between intracapillary and alveolar gas space pressures) is in equilibrium with the combined opposing forces of intervening structures, namely the basement membranes of the capillary and alveolar epithelium, the fine fiber network, and the air–liquid interface. The effects are evident in the prominent bulging of the pulmonary capillaries into the alveolar space when hydrostatic capillary pressure increases

or interfacial tension is reduced (e.g., in the salinefilled lung). Conversely, the planar forces of the wall’s solid structures and interfacial tension flatten the capillaries when the wall is stretched at high lung volumes. By contrast, the vessels in the corners, for example, within the J junctions, where the curvatures of the solid structures and interface are concave to the alveolar space, are distended at high lung volumes, and may remain open even when the vessels within the flat portions are compressed. Under those conditions, blood–flow in such regions is not completely eliminated and there is gas exchange evidence compatible with very low perfusion units. Fluid balance is also impacted by mechanical factors, again with different mechanisms in the flat portions of the wall and the corners. Throughout the body, the volume of fluid in the interstitial spaces is controlled by the balance between hydrostatic and osmotic factors across the capillary walls (Starling equilibrium). The lung, however, is a special case because of the powerful forces that develop when interfacial tension acts on very tight curvatures. In particular, the tissue pressure in the alveolar corners (J junctions) is lowered. This may be beneficial by helping to draw interstitial fluid from the flat portions of the alveolar wall into the corners, whence it may be cleared by the lymphatics. But it is also counterproductive insofar as it opposes the fluid-absorptive forces across the microvascular walls and inhibits lymphatic drainage. Of greatest concern, it can lead to fluid accumulation within the alveolus. Ordinarily, a simple mechanical negative-feedback loop limits the accumulation of fluid any increase of alveolar fluid initially decreases the curvature of the interface, reducing the pressure-lowering effect of interfacial tension and thereby favoring the return of fluid to the capillaries and lymph. If, however, the fluid gathers to the point that the corners of the alveolar space are filled, the feedback loop reverses; any further gathering of fluid increases the curvature, leading to further alveolar filling or collapse. This problem is substantially mitigated by the interfacial tension-lowering effect of pulmonary surfactant, which has enabled evolution of alveolar walls at a much smaller scale than would otherwise have been possible, providing an immense surface area for gas exchange within a reasonably sized chest.

The Mechanical Role of the Alveolar Wall in Respiratory Diseases Mechanical dysfunction of the alveolar wall is at the core of most major acute and chronic lung diseases, as readily predictable from the above discussion and seen in the following examples.

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The essence of emphysema is loss of alveolar walls. This impairs gas exchange by reducing surface area of the pulmonary capillary bed and increasing the path lengths for passive diffusion. It also relaxes the network of alveolar walls. This has the effect of reducing the dilating effects of the network on the embedded and extrapulmonary airways and blood vessels. Narrowing of the airways increases their airflow resistance and forces the subject to breathe at disadvantageously high chest wall volumes in order to mount sufficient elastic tensions to hold the airways open. Inhomogeneity of damage to the network of alveolar walls and its effects on airway and blood vessel calibers cause substantial maldistribution of ventilation and perfusion, itself a major cause of inefficiency of gas exchange. A range of mechanical dysfunctions at the level of the alveolar wall permits blood to shunt through the lung without exchanging gas. Fluid can gather (pulmonary edema) secondary to the high hydrostatic pressures of heart failure, or even in normal individuals at very high altitudes. Toxic, infectious, or immunological entities can allow excessive fluid or protein leakage from damaged capillaries. Airspaces can collapse (atelectasis) from local underinflation and gas absorption, from dysfunctional surfactant, from trauma, or from chest wall dysfunction. High distending forces (e.g., deep breaths) are required for reopening. Repeated opening and closing of airspaces in a patient breathing with ventilator support can have its own direct physical and inflammatory consequences. Pulmonary fibrosis due to a wide range of causes can thicken and stiffen the alveolar wall, impairing

Amyloidosis

gas exchange and placing a mechanical load on breathing. See also: Alveolar Surface Mechanics. Atelectasis. Chronic Obstructive Pulmonary Disease: Overview; Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General. Diffusion of Gases. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens; Matrix Proteoglycans. Fluid Balance in the Lung. Interstitial Lung Disease: Overview; Idiopathic Pulmonary Fibrosis. Lung Development: Overview. Myofibroblasts. Occupational Diseases: Overview. Pulmonary Fibrosis. Smooth Muscle Cells: Airway. Stress Distribution in the Lung. Surfactant: Overview. Ventilation: Uneven.

Further Reading Butler JP, Oldmixon EH, and Hoppin FG Jr (1996) Dihedral angles of septal ‘bend’ structures in lung parenchyma. Journal of Applied Physiology 81: 1800–1806. Greaves IA, Hildebrandt J, Hoppin FG Jr (1986) Micromechanics of the lung. In: Fishman AP, Macklem PT, Mead J, and Geiger S (eds.) Handbook of Physiology. The Respiratory System, vol. III(1), pp. 217–231. Bethesda: American Physiological Society. Hoppin FG Jr, Stothert JC Jr, Greaves IA, Lai Y-L, Hildebrandt J (1986) Lung recoil: elastic and rheological properties. In: Fishman AP, Macklem PT, Mead J, and Geiger S (eds.) Handbook of Physiology. The Respiratory System, vol. III(1), pp. 195–215. Bethesda: American Physiological Society. Mead J (1961) Mechanical properties of lungs. Physiological Reviews 41: 281–330. Stamenovic´ D (1990) Micromechanical foundations of pulmonary elasticity. Physiological Reviews 70: 1117–1134. West JB (2003) Thoughts on the pulmonary blood–gas barrier. American Journal of Physiology. Lung Cellular and Molecular Physiology 285: L501–L513.

see Interstitial Lung Disease: Amyloidosis.

ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS P A D’Amore, Harvard Medical School, Boston, MA, USA M K Sakurai, Boston Children’s Hospital, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Angiogenesis is crucial in pulmonary development to establish the groundwork structure for the lungs’ main function of

gas exchange across a thin blood gas barrier. This network of blood and air conduits begins to form in early gestation and continues through early childhood, requiring intricate epithelial–mesenchymal interactions that are regulated by various angiogenic and growth factors. Disruptions in this complex system lead to inadequate gas exchange and diseases of respiratory distress. At this time, the precise roles of these growth factors and interactions between the epithelial and mesenchymal cells are poorly understood. A better understanding of these processes may lead to improved therapies for various respiratory diseases.

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Introduction The basic function of the lung is gas exchange, and efficient gas exchange requires a thin air–blood interface. Hence, epithelial–mesenchymal interactions are crucial to the formation of a blood gas. Lung development, which begins in early gestation and continues into early childhood, requires complex interactions that are regulated by a number of angiogenic and growth factors. Although it is not whether mesenchymal development drives epithelial growth or vice versa, several factors have been identified as important for the development of a functional blood gas barrier. Any disruption to this intricate and highly orchestrated process can cause ineffective gas exchange with resulting respiratory distress. Further knowledge of the factors influencing pulmonary development may lead to therapeutic interventions for various respiratory diseases.

General Embryology Lung development is divided into five stages, starting in early gestation and ending postnatally in early childhood. Basic organogenesis occurs during the initial embryonic phase. The next three phases – pseudoglandular, canalicular, and saccular – describe the morphogenesis of primitive lung parenchyma during those stages. The final alveolar stage extends into childhood. Concurrent with mesenchymal development, the lung vasculature develops from mesodermal cells by a combination of vasculogenesis and angiogenesis. Vasculogenesis is the process of de novo blood vessel development from mesenchymal precursor cells, whereas angiogenesis involves new vessels sprouting from existing vasculature. Initially, the blood lakes, formed by vasculogenesis, create a capillary plexus and connect to drain into the pulmonary veins. Eventually, the pulmonary arteries bud off the aortic arches by angiogenesis and grow into the mesenchyme where they anastamose with this capillary plexus. This vascular plexus surrounds the developing airways. The time frame of these phases overlaps, given the independent development of each lobe. During the embryonic stage, the initial lung buds originate from ventral diverticuli of the primitive foregut and proliferate into the surrounding mesenchyme. These buds undergo branching morphogenesis to create a complex network of airways that are lined with columnar epithelium. Simultaneously, vascular lakes of hematopoietic cells that have formed by vasculogenesis appear in the mesenchyme. Neither the pulmonary artery nor veins have formed at this time. At first the capillaries only drain into

systemic veins but eventually connect to the pulmonary veins that arise from the primitive cardiac atrium during the pseudoglandular phase. Bronchial tree formation and acinar structure development at approximately 5–17 weeks into gestation mark the pseudoglandular stage. Epithelial– mesenchymal interactions determine and regulate the branching patterns. Transplantation experiments have demonstrated that the mesenchyme directs the growth and branching of epithelial tubules forming the conductive airways. In vitro experiments have shown that removing the mesenchyme from the tip of a budding epithelial tube prevents further branching and that transplanted mesenchyme can induce new budding. During the pseudoglandular stage, differentiation of the cells comprising the airway walls occurs proximal to distal. These cells become ciliated, nonciliated, goblet, and basal epithelial cells. At the distal ends, cuboidal epithelial cells line the forming acini, the mesenchyme begins to thin, and a network of capillaries develops. During this stage, the venous system connects to the heart. Later in the pseudoglandular phase, the pulmonary artery buds from the aorta and primitive arterial branches begin to develop alongside the airways. At this point, the basic airway pattern has been established. The canalicular stage at gestational weeks 16–26 encompasses acini and pulmonary vasculature development, as well as the initiation of surfactant synthesis. At the crucial gas-exchange region, acini development involves widening of the peripheral tubules, further thinning of the mesenchyme and increased vascularization. The cuboidal epithelium thins to form the blood gas barrier and differentiates into type I and type II pneumocytes. The type II pneumocytes initiate surfactant production. During the saccular stage, which occurs during 24–38 weeks of gestation, continued expansion of the air spaces occurs and is accompanied by increased vasculature and decreased interstitial tissue as the lung prepares for its main function of gas exchange. At this phase, the airways end in thin-walled terminal sacs, hence the name saccular stage. The vessels continue to grow and elongate. With the lessening of interstitial tissue, the airspaces begin to approach one another and a capillary bilayer is formed in the intersaccular septa. The surfactant system continues to mature in preparation for the function of gas exchange. The alveolar stage starts at the end of gestation and continues for 1–2 years postnatally. Less than 10% of alveoli are present at birth and a majority of alveolar formation occurs via the process of septation after birth. The walls of the airspaces are called primary septa and contain a distinct double capillary

112 ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS

network. Secondary septa form to partition the saccules and form alveoli. This process increases the alveolar surface area and thus the gas-exchange area. These thick septa transform into a thin epithelial– endothelial cell lining via a process that is not well understood. The exact mechanisms that underlie the five stages of lung development have yet to be elucidated; however, various angiogenic growth factors and developmental factors have been demonstrated to play important roles in this process.

Angiogenic Growth Factors, Developmental Factors, and Their Roles Epithelial–mesenchymal interactions are crucial in the formation of a thin blood gas interface during lung development. Multiple angiogenic and growth factors interact throughout the five stages of pulmonary development. A variety of in vivo and in vitro techniques have been used to elucidate the role of particular growth factors in this process. These studies point to the importance of maintaining a delicate balance of growth factors during the process of pulmonary development. Vascular Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is a wellestablished angiogenic factor. Differential splicing of a single gene generates three distinct VEGF isoforms in the mouse and at least five in humans. VEGF acts to stimulate the proliferation and migration of vascular endothelium as well as to increase vascular permeability. In the lung, VEGF is expressed by type II pulmonary epithelial cells and localizes to the basement membrane. An analysis of the various VEGF isoforms in the mouse revealed that the lung produces primarily VEGF188. This isoform includes two domains, encoded by exons 6 and 7, which are highly charged and bind with high affinity to the heparan sulfate, thus accounting for the basement membrane localization. This specific localization is postulated to be important for inducing capillary development. VEGF interacts with tyrosine kinase receptors to exert its cellular functions. These receptors (VEGFR1 or Flt-1; VEGFR2 or Flt-2 and VEGFR3) are localized on endothelial cells. Recent studies have revealed that they are also expressed by a variety of nonendothelial cell types. VEGF expressed by the pulmonary epithelium induces vascular development via the VEGFR2 on the endothelial precursor cells. Alterations in VEGF expression lead not only to abnormal vasculature but also to abnormal lung morphology. Additional evidence indicates that VEGF is important for maintenance of newly formed blood vessels as well.

The tight regulation of VEGF presumably contributes to the formation and maintenance of a normal blood gas barrier. Experiments involving transgenic mice have revealed the importance of precise regulation of VEGF expression. Targeted disruption of VEGF expression results in embryonic mortality, and inactivation of just a single VEGF allele causes abnormal blood vessel formation, leading to early embryonic lethality. Furthermore, overexpression of VEGF by the respiratory epithelium produces abnormal vascular formation, which also leads to disruption of lung morphology and embryonic lethality. Hypoxia increases VEGF production by lung epithelial cells, whereas hyperoxia depresses the expression of VEGF as well as its receptors. Platelet-Derived Growth Factors

Platelet derived growth factor (PDGF) is composed of dimers of A and/or B chains and acts via two tyrosine kinase receptors PDGFR-a and -b. Early in gestation, PDGF protein localizes to airway epithelial cells as well as to mesenchymal cells. PDGF is produced by the epithelium and interacts with PDGF receptors (PDGFRs) on interstitial mesenchymal cells. Expression of PDGF appears to change throughout gestation, increasing during the pseudoglandular phase with minimal production by the saccular stage. The localization of PDGF as well as the gestation-dependent expression pattern indicates an important role during lung development. PDGF is also involved in the formation of the vessel wall. PDGF B produced by immature endothelial cells acts as a mitogen and chemoattractant for undifferentiated mesenchymal, recruiting them to the nascent vessel. Upon contact with the endothelium, latent transforming growth factor beta (TGF-b) is activated. The activated TGF-b induces the differentiation of the mesenchymal to become smooth muscle cells/pericytes. It also acts upon the endothelial cells to inhibit their proliferation and migration, and it induces the production of basement membrane. Taken together, these factors lead to the formation of a stable, mature vasculature. PDGF appears to regulate the generation of myofibroblasts. The development and analysis of PDGF A and PDGF B knockout mice has revealed a role for PDGF in lung development. Homozygous PDGF A null mutants do not develop alveolar myofibroblasts and therefore have reduced elastin deposition, resulting in abnormal alveolarization. Failure of alveolar septation leads to emphysema in the surviving mutant mice. PDGF B-deficient mice are embryonic lethal, due to generalized hemorrhage and edema, most likely secondary to defective blood wall formation.

ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS 113

Precise regulation of PDGF levels during each phase of development is also important. Overexpression of PDGF A by the respiratory epithelium causes overgrowth of the mesenchyme and results in death from abnormal lung architecture. Transforming Growth Factor Beta

The TGF-b family of cytokines regulates cell proliferation, differentiation, recognition, and death via serine/threonine kinase activity receptors. The TGF-b1, TGF-b2, and TGF-b3 have been identified in the developing lung where they have been shown to stimulate extracellular matrix deposition by lung fibroblasts and inhibit epithelial cell proliferation. Spatial and temporal patterns of expression during lung development suggest a role for TGF-b in branch morphogenesis. Both mesenchymal and epithelial cells of the primitive lung express TGF-b1. TGF-b2 expression localizes to the tips of developing bronchioles, while TGF-b3 has been demonstrated in the proximal respiratory tract epithelium as well as the terminal growing buds. Signaling via the TGF-b type I receptor (TGFbRI) directs the formation of branch points while activation of the TGF-b type II receptor (TGFbRII) exhibits an inhibitory effect. The inhibitory effect of TGF-b on lung branching is mediated by the stimulation of the production and deposition of extracellular matrix proteins and inhibition of production of collagenase and proteolytic enzymes. Control over these molecules allows TGF-b to modulate lung branching. In culture, TGF-b1 and TGF-b2 decrease lung explant size and inhibit branching. TGF-b3 null mice, which die at birth, have delayed lung development and exhibit cleft palate as well as pulmonary epithelial, mesenchymal, and vascular dysplasia. In contrast to TGF-b3-deficient mice, TGF-b1 knockout mice have normal lung development but die by 1 month of age from fulminant pulmonary inflammatory infiltrates. The normal lung development has been attributed either to functional redundancy provided by the other TGF-b isoforms or to rescue via transplancental transfer of maternal TGF-b. Overexpression of TGF-b1 results in neonatal lethality and hypoplastic lungs, with decreased saccular formation and epithelial differentiation. Conversely, inhibition of TGFbRIIs stimulates lung development. Insulin-Like Growth Factors

Insulin-like growth factor-I ( IGF-I) and Insulin-like growth factor-II (IGF-II) are peptides similar in structure to proinsulin. These growth factors modulate cell proliferation and differentiation via two receptors,

IGF-IR and IGF-IIR. IGF-IR is a tyrosine kinase receptor, whereas IGF-IIR is a cation-independent mannose 6-phosphate receptor. In addition, IGF binding proteins (IGFBPs), which modulate IGF function, have been identified. IGF-I, IGF-II, and IGF-IR have been localized during early gestation to endothelial cells lining the primary vascular plexus, suggesting a role for these factors in pulmonary vascular development. Studies suggest that IGFs may function as survival factors during pulmonary vascular development. In vitro experiments involving the treatment of fetal lung explants with IGF-IR inhibitors exhibit not only a reduction in endothelial cell number but also an increase in mesenchymal cell apoptosis. In transgenic mice, disruption of the IGF-IR gene is lethal postnatally from respiratory failure. Lungs in these mutants appear hypoplastic although no gross defect in lung architecture has been observed. Other studies have suggested that VEGF may act downstream of IGF-1 in vascular development. IGF is important in pulmonary development as a survival factor perhaps via signaling through other growth factors. Epidermal Growth Factor and Transforming Growth Factor Alpha

Epidermal growth factor (EGF) and TGF-a are members of the EGF family that act through the EGF receptor (EGFR). EGF and TGF-a are produced by the mesenchyme and appear to act on the EGFR located on pulmonary epithelial cells as well as in neighboring mesenchyme. The source of these factors and the localization of EGFR indicate an important role for EGF and TGF-a in epithelial– mesenchymal interactions. EGF is also expressed by alveolar epithelial cells and regulates type 2 cell proliferation in an autocrine manner. Both EGF and TGF-a affect growth and branching of the pulmonary tree. In vitro experiments demonstrate that administration of exogenous EGF increases cellular proliferation as well as branching, whereas inhibition of EGF leads to decreased branching in explants. Targeted disruption of TGF-a demonstrates no adverse effects, most likely due to the functional redundancy provided by EGF. However, disruption of EGFR results in respiratory distress, leading to neonatal mortality. The lungs of these mutants exhibit septal thickening, decreased branching, deficient alveolarization, and reduced epithelial differentiation. Overexpression in the lung of TGF-a disrupts alveolar development and causes fibrotic lesions. Clearly the IGFs have an important role in epithelial–mesenchymal interactions during lung development.

114 ANGIOGENESIS, ANGIOGENIC GROWTH FACTORS AND DEVELOPMENT FACTORS Fibroblast Growth Factors

The fibroblast growth factors (FGFs) are a family of proteins that mediate a variety of processes, including cell proliferation, differentiation, cell angiogenesis, and development. These growth factors interact with specific tyrosine kinase receptors to mediate their effects. Binding to heparin sulfate proteoglycans on cell surfaces and within the extracellular matrix potentiates the effects of some FGFs. Specific FGFs and FGF receptors (FGFRs) regulated temporally and spatially during lung development implicate them in epithelial–mesenchymal interactions. Abnormal FGF expression and signaling during pulmonary development lead to anomalous epithelial branching and differentiation. FGF-10 and keratinocyte growth factor (KGF or FGF-7) induce pulmonary epithelial proliferation and branching. FGF-10 regulates patterning by chemotaxis, whereas KGF influences patterning by promoting epithelial growth. FGF-10 null mice display perinatal mortality from aberrant lung development. Disruption of FGFR2 expression results in pulmonary atresia distal to the main stem bronchi. Interruption of FGFR3 and FGFR4 signaling leads to excess elastin deposition, aberrant alveolization, and postnatal lethality. Transgenic overexpression of KGF results in pulmonary malformations that resemble cystic adenomatoid malformations. The function of KGF appears to overlap with other factors since KGF knockout mice have no gross pulmonary abnormalities. FGF signaling is critical not only for lung development but for postnatal repair following lung injury as well.

Diseases and Therapeutic Considerations Pulmonary development is a complicated system involving multiple growth factors and extensive epithelial–mesenchymal signaling. Any disruptions in this system can lead to inadequate gas exchange and diseases of respiratory distress. Both the vascular and airway development are affected in diseases with abnormal lung formation such as pulmonary hypoplasia. A better understanding of these developmental processes may elucidate the mechanisms underlying various respiratory diseases and thus lead to improved therapies. Congenital Diaphragmatic Hernia

Congenital diaphragmatic hernia (CDH) is a condition that affects approximately 1 in every 3000–5000 infants and is one of the most common causes of fetal hypoplastic lungs. In CDH, improper diaphragmatic development leads to abdominal content herniation into the thoracic cavity. This results in impaired lung

growth due to the loss of opposition to the lung’s inherent tendency to collapse. Intrusion of the abdominal viscera into the thoracic cavity is a secondary event that may further hinder lung growth and development. In addition to being small in size, the lungs in CDH are also immature. Despite timely repair of the diaphragmatic defect, many infants suffer postnatally from respiratory compromise due to underdeveloped lungs. Survival and long-term outcome is dependent on the severity of pulmonary hypoplasia, the extent of bronchopulmonary dysplasia from ventilatory injury, and other associated anomalies. Lungs from patients with pulmonary hypoplasia associated with CDH exhibit significantly decreased number of airways, decreased vessel density, and more muscularized arteries. A murine model phenotypically similar to CDH has been developed using nitrofen exposure. Lungs from nitrofen-treated mice have decreased levels of VEGF, which may contribute to their immature state. Nitric oxide, important in the regulation of smooth muscle cell proliferation, and nitric oxide synthase are also reduced. The decreased production and diffusion of nitric oxide may contribute to the muscularized arteries, which in turn lead to pulmonary hypertension. The possibility of growth-factor-induced pulmonary growth is currently under investigation in hopes of a therapeutic intervention for CDH. Bronchopulmonary Dysplasia

Bronchopulmonary dysplasia (BPD), also known as neonatal chronic lung disease, is an important cause of respiratory illness, especially in preterm infants. In BPD, acute injury of the lung may be caused by multiple factors, including pulmonary oxygen toxicity, barotrauma from mechanical ventilation and cellular immaturity. BPD is more common among premature infants because of higher susceptibility of the immature lungs to injury. The acute injury causes an acute inflammatory reaction, which leads to interstitial fibrosis and emphysema with associated histologic features including airway mucosal metaplasia, smooth muscle hyperplasia, and atelectasis. Long-term histologic changes include decreased alveolar numbers and surface area. Airway lavage samples from patients with BPD contain increased levels of TGF-b, which are associated with an adverse prognosis. As described above, in vivo experimental models have shown that excessive expression of TGF-b inhibits lung morphogenesis and induces pulmonary fibrosis; EGF and PDGF are also thought to be involved in the development of pulmonary fibrosis in BPD. Decreased levels of VEGF in the presence of alveolar damage are also

ANTICOAGULANTS 115

likely to contribute to the abnormal pulmonary vasculature associated with lung injury. Manipulation of TGF-b may lead to therapeutic interventions to prevent the development of chronic pulmonary fibrosis associated with BPD.

Acknowledgments The authors were supported by CA45548 (PD’A) and EY05318 (PD’A). See also: Bronchopulmonary Dysplasia. Epidermal Growth Factors. Fibroblast Growth Factors. Insulin-Like Growth Factors. Keratinocyte Growth Factor. Platelet-Derived Growth Factor. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Vascular Endothelial Growth Factor.

Further Reading Alescio T and Cassini A (1962) Induction in vitro of tracheal buds by pulmonary mesenchyme grafted on tracheal epithelium. Journal of Experimental Zoology 150: b83–b94. Burri PH (1984) Fetal and postnatal development of the lung. Annual Review of Physiology 46: 617–628.

Carmeliet P, Ferreira V, et al. (1996) Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380: 435–439. Chinoy MR (2003) Lung growth and development. Frontiers in Bioscience 8: 392–415. deMello DE, Sawyer D, et al. (1997) Early fetal development of lung vasculature. American Journal of Respiratory Cell and Molecular Biology 16: 568–581. Gaultier C, Bourbon J, and Post M (eds.) (1999) Lung Development. New York: Oxford University Press. Han RN, Post M, et al. (2003) Insulin-like growth factor-I receptor-mediated vasculogenesis/angiogenesis in human lung development. American Journal of Respiratory Cell and Molecular Biology 28: 159–169. Jankov RP and Keith A (2004) Tanswell. Growth factors, postnatal lung growth and bronchopulmonary dysplasia. Paediatric Respiratory Reviews 5(supplement A): S265–S275. Kumar VH and Ryan RM (2004) Growth factors in the fetal and neonatal lung. Frontiers in Bioscience 9: 464–480. Shannon JM (1997) Epithelial–mesenchymal interactions in lung development. In: McDonald JA (ed.) Lung Growth and Development, pp. 81–118. New York: Dekker. Warburton D and Bellusci S (2004) The molecular genetics of lung morphogenesis and injury repair. Paediatric Respiratory Reviews 5(supplement A): S283–S287. Warburton D, Schwarz M, et al. (2000) The molecular basis of lung morphogenesis. Mechanisms of Development 92: 55–81.

ANTICOAGULANTS A Gu¨nther and C Ruppert, University of Giessen Lung center (UGLC), Giessen, Germany & 2006 Elsevier Ltd. All rights reserved.

Abstract Anticoagulants are widely used for the prevention and treatment of venous and/or arterial thrombosis. Anticoagulants comprise a chemically heterogeneous group of drugs acting at different steps within the coagulation cascade. Heparin and heparin-based anticoagulants are indirect anticoagulants that bind to antithrombin and enhance the inhibitory capacity of this natural anticoagulant. Coumarin derivatives (e.g., warfarin) interfere with the hepatic synthesis of coagulation factors (vitamin K antagonists). A third class comprises direct inhibitors of enzymes of the clotting cascade, primarily thrombin. Classic anticoagulants such as unfractionated heparin and coumarins have some clinical drawbacks. Heparin requires parenteral application, has serious adverse effects, and is difficult to dose and monitor due to variable and unpredictable pharmacokinetics. The orally active coumarin derivatives have a narrow therapeutic window and multiple interactions with food and drugs, necessitating individualized dosing and monitoring. During the past 10 years, the search for safer and more effective antithrombotic agents resulted in the development or discovery of new molecules, including fondaparinux and idraparinux (both selective inhibitors of factor Xa), thrombin inhibitors for parenteral use such as recombinant hirudin and hirulogs, and the orally active ximelagatran.

The first effective agent for therapy of thromboembolism was unfractionated (UF) heparin. It has become the anticoagulant of choice for the treatment and prevention of arterial and venous thrombotic diseases, including pulmonary embolism. Due to the problems accompanying heparin therapy (unpredictable pharmacokinetics, low bioavailability at low doses, unpredictable dose response, need for careful laboratory monitoring, bleeding, and thrombocytopenia), the search for improved anticoagulants resulted in the introduction of low-molecular-weight (LMW) heparins (Table 1). These heterogeneous compounds have been shown to produce a more predictable anticoagulant response and fewer adverse effects than UF heparin, reflecting the improved pharmacokinetic properties (greater bioavailability after subcutaneous injection (B30%), longer half-life, and dose-independent clearance). The expectation, however, that these new compounds might eliminate the risk of bleeding was not confirmed. Another newer anticoagulant is danaparoid sodium, a heparinoid that is often used for treatment of heparin-induced thrombocytopenia.

116 ANTICOAGULANTS Table 1 Heparins and heparinoids INN name

Brand name

Manufacturer

Unfractionated heparin Heparin-sodium Generic (e.g., Liquemin) Low-molecular-weight heparins Ardeparin Normiflo

Wyeth–Ayerst

Certoparin

Mono-Embolex

Novartis

Dalteparin

Fragmin

Pharmacia

Enoxaparin

Clexane

Aventis-Pharma

Nadroparin

Fraxiparine

Reviparin

Clivarine

Sanofi– Synthelabo Abbott

Tinzaparin

Innohep

LeoLabs

Heparinoids Danaparoid

Orgaran

Celltech Pharma

Pentosanpolysulfate

Elmiron

Ortho-McNeal

Hemoclar Fibrezym

Sanofi Aventis Bene Arzneimittel

Oral anticoagulants (4-hydroxycoumarin derivatives), the second major class of traditional anticoagulants, were developed in parallel to heparin, based on a serendipitous discovery. Today, several coumarins are used as agents of choice for longterm anticoagulant therapy (Table 2). For historical and marketing reasons, some countries use more warfarin (e.g., United States, Canada, and United Kingdom), others phenprocoumon (e.g., Germany), and yet others acenocoumarol. Another class of drugs, called indaniones, are similar to coumarins in terms of pharmacodynamics and are mostly used in France. The traditional anticoagulants UF heparin and coumarin derivatives are highly effective agents and relatively safe when administered at appropriate dosages and carefully monitored. LMW heparins improved and simplified anticoagulant therapy by obviating the need for routine coagulation monitoring. Newer agents, including fondaparinux and

Method of preparation

Mean molecular weight

Anti-Fxa: anti-FIIa ratio

Extraction from porcine mucosa

14 000 (range: 5 000–40 000)

1.0

Peroxidative depolymerization of UF heparin Isoamylnitrite depolymerization of UF heparin Nitrous acid depolymerization of UF heparin Benzylation and alkaline depolymerization of UF heparin Nitrous acid depolymerization of UF heparin Nitrous acid depolymerization of UF heparin and size exclusion chromatography Haparinase digestion of UF heparin

5 300

2.0

5 200

2.2

6 000

1.9–3.2

4 500

3.3–5.3

4 300

2.5–4.0

3 900

3.6–6.1

6 500

1.5–2.5

6 500

Anti-FXa 4 anti-FIIa n.a.

Extraction from porine mucosa Sulfation of pentosan derived from beech tree bark

6 000

idraparinux, represent the first agents of a new class of FXa inhibitors. Both are fully synthetic analogs of the unique antithrombin-binding pentasaccharide sequence found in UF and LMW heparin. Advances in molecular biology and biotechnology have led to the recombinant production of hirudin, the most potent naturally occurring direct thrombin inhibitor. Hirudin also serves as a standard for the development of LMW inhibitors of thrombin. Argatroban was the first synthetic thrombin inhibitor approved for prophylaxis and treatment of various thrombotic disorders mainly in patients with heparin-induced thrombocytopenia. The major drawback of argatroban and hirudins is their requirement for parenteral administration. Ximelagatran and its active metabolite, melagatran, are members of a class of newly developed oral direct thrombin inhibitors. These agents have been approved for anticoagulant treatment very recently and expand the treatment options for thrombotic disorders. Whether these new agents

ANTICOAGULANTS 117 Table 2 Coumarin and indandione derivatives INN

Brand name

Manufacturer

Pharmacokinetics t 12 (h)

Duration of action (days)

Acenocoumarol (Nicoumalone) Ethylbiscoumacetate Dicumarol Phenprocoumon Tioclomarol Warfarin

Sintrom Tromexane Generic Marcumar Apegmone Coumadin Warfilone

Novartis Ciba-Geigy Generic Roche Merck Lipha Bristol-Myers Squibb Merck Frosst

9 2.5 n.a. 150 24 35–45

2–4 1–2 n.a. 7–10 2–3 4–5

Anisindione Fluindione Phenindione

Miradon Previscan Dindevan Pindione

Schering Procter & Gamble Goldshield Merck Lipha

9 6 6

3–4 2–3 2–3

will be more effective and safer than traditional anticoagulants remains to be determined.

Chemical Structure Heparin and Heparinoids

Heparin is a naturally occurring, highly sulfated glycosaminoglycan that has been found in mast cells in a large number of mammalian and nonmammalian vertebrates. Material for clinical use is derived from bovine lungs or pig intestinal mucosa and is prepared either as UF heparin or depolymerized LMW heparin. Heparin is a heterogeneous mixture of unbranched polysaccharide chains composed of 15–100 alternating 1-4-linked mucosaccharide units of D-glucosamine and L-iduronic acid or D-glucuronic acid (Figure 1(a)). Heparin is the most negatively charged molecule in the human body. The molecular weight of UF heparin ranges from 5 to 40 kDa with an average of B14 kDa. LMW heparins are much smaller (mean MW, B5 kDa) and are produced from UF heparin by chemical or enzymatic depolymerization. The chemical composition is similar but not identical. The anticoagulant effect is mediated by a unique pentasaccharide unit (Figure 1(b)) that binds antithrombin with high affinity and activates the inhibitor by approximately 1000-fold. This pentasaccharide sequence is found in B30% of the chains of UF heparin (known as high-affinity heparin) but in only 15–25% of the chains of LMW heparin. Danaparoid sodium is an LMW heparinoid isolated from porcine mucosa with a mean MW of 6 kDa (range, 4–12 kDa). It consists of a mixture of 84% heparan sulfate (of which 4% has high antithrombin activity), 12% dermatan sulfate, and 4% chondroitin sulfate. The characteristic disaccharide

repeat units of these glycosaminoglycans are depicted in Figure 1(c). Pentosanpolysulfate (PPS) is a highly sulfated, semisynthetic polysaccharide derived from beech tree bark with a mean molecular weight of 6 kDa (range, 2–9 kDa). The degree of sulfation is higher than in heparin, resulting in a higher negative charge density. Pentosan consists of b-1-4-linked D-xylose units branched with 4-O-methyl-D-glucuronic acid in a ratio of 1 uronic to 9 xylose units (Figure 1(d)). In contrast to heparin and other glycosaminoglycans, PPS is orally bioavailable. FXa Inhibitors

Fondaparinux is a fully synthetic analog of the unique pentasaccharide sequence found in heparins. Idraparinux represents the second generation of this class. The O-methylation and O-sulfation result in a higher affinity for antithrombin and a higher negative charge, which in turn accounts for the tight binding of antithrombin and the prolonged half-life of idraparinux (80 h vs. 17 h for fondaparinux) (Figure 2). Vitamin K Antagonists

The clinically used vitamin K antagonists are derivatives of either 4-hydroxycoumarin or indan-1,3dione (Figure 3). The substituent at the 3 position of the coumarin backbone or side-chain variations influence pharmacokinetic and pharmacodynamic properties (Table 2). Pharmaceutical preparations of warfarin, acenocoumarol, and phenprocoumon contain a racemic mixture of R- and S-enantiomers. The stereoisomers exhibit different pharmacodynamics and pharmacokinetics since they undergo stereoselective biotransformation in the liver.

118 ANTICOAGULANTS k

CH2OSO3 O

O

COOk 4

1

4

O

OH

1 OH

O

k

NHSO3k

OSO3

(a)

n

Heparin CH2OSO3k O

CH2OSO3k O

COOk O

O OH

O

OH

O

k

OSO3

COOk

O

OH

O

OH OSO3k

NHSO3k

OH

NHCOCH3

CH2OSO3k O

O

O NHSO3k

Heparin pentasaccharide

(b)

COOk O

CH2OSO3k O

O

O 4

1

4

OH

O

COO

1

4

OH

1

n

O

NHCOCH3

n

Dermatan sulfate

R1O 4

1

1 3

OH

Heparan sulfate COOk O

O

O3SO 4

OH OH

4

CH2OH O

k

k

OH

CH2OR2 O

Chondroitin sulfate O

A: R1 = SO3k R2 = H C: R1 = H R2 = SO3k

1 3

O NHCOCH3

OH

(c)

O O

OSOk 3 OSO3k

O

O O

n

O

OSOk 3

O O

OSOk 3

OSO3k COOk O

OSOk 3 OSO3k

O O

OSOk 3

O

OSO3k

O

H3CO OSO3 OSO3k

(d)

Pentosanpolysulfate

Figure 1 Chemical structures of heparin and other members of the glycosaminoglycan family. (a) The major repeat unit of heparin is depicted, which is found in up to 90% in heparin from beef lung and up to 70% in heparin from pig mucosa. The disaccharide consists of 1-4 linked sulfated L-iduronic acid and sulfated D-glucosamine. (b) The specific pentasaccharide sequence that is responsible for highaffinity binding to antithrombin is depicted. (c) The major repeat units of heparan sulfate, dermatan sulfate, and chondroitin sulfate A are depicted. (d) A typical sequence of pentosanpolysulfate is shown.

Hirudin and Hirulogs

Hirudin is a naturally occurring inhibitor of thrombin that is a single-chain polypeptide of 65 amino acid residues with an apparent molecular weight of 7 kDa. The structure is stabilized by three intramolecular disulfide bridges. In 1986, recombinant hirudin (r-hirudin) became available. Desirudin

([Val1, Val2]-63-desulfohirudin) and lepirudin ([Leu12]-63-desulfohirudin) are therapeutically used recombinant hirudins produced in yeast (Table 3). They are not sulfated and differ only in the first two amino acids of the N terminus. Both natural and recombinant hirudins show almost identical pharmacodynamic and pharmacokinetic properties.

ANTICOAGULANTS 119 COOk O

CH2OSO3k O

CH2OSO3k O

CH2OSO3k O

O COOk

HO OH

O

OH

O NHSO3k

O

OSO3k

O

NHSO3k

OH

OH

OH

OCH3

OSO3k

NHSO3k

Fondaparinux

COOk

CH2OSO3k O

CH2OSO3k O

O

CH2OSO3k O

O COOk

O H3CO OCH3

OSO3k

OCH3

O OCH3

OSO3k

OCH3

O O

OCH3

OSO3 OCH3k

OCH3 OSO3k

Idraparinux

Figure 2 Chemical structure of FXa inhibitors. The structures of fondaparinux and idraparinux are given.

OH 4 12 O

O 1 2 3

3

O

O

4-Hydroxycoumarin

Indan-1, 3-dione

O O

OH

O

O

O OH

Warfarin

OH

Phenindione

O O O

O

OH

O

O

F

Dicumarol NO2

O

OH

O

OH

Fluindione Acenocoumarol O

O O

O

OH

O Ethylbiscoumacetate

O

O

OCH3 O Anisindione

Phenprocoumon

Figure 3 Chemical structures of coumarin and indandione derivatives. The lead structures and derivatives are given. Clinically used coumarin derivatives are substituted at the 3-position, and indandiones are substituted at the 2-position. Asymmetric carbons are indicated by asterisks.

120 ANTICOAGULANTS Table 3 Direct thrombin inhibitors INN name

Brand name

Manufacturer

Route of administration

Half-life

Desirudin Lepirudin Bivalirudin Argatroban Melagatran Ximelagatran

Revasc Refludan Angiomax Novastan Melagatran Exanta

Aventis/Novartis Schering, Berlex The Medicine Company Texas Biotechnology AstraZeneca AstraZeneca

Parenteral (i.v., s.c.) Parenteral (i.v.) Parenteral (i.v.) Parenteral Parenteral Oral

2–3 h 1.3 h 25 min 45 min 3–5 h 3–5 h

1 H2N

Val Val Tyr Thr Asp Cys Thr Glu Ser

20

Gly 10 Val Asn Ser Gly Glu Cys Leu Cys Leu Asn Gln Cys

40 Thr Val Cys Gly

Gly Gln Gly Asn Lys Cys IIe Leu 30

Gln Asn Lys Glu Gly Asp Ser Gly

Glu Gly

65 60 50 Thr Pro Lys Pro Gln Ser His Asn Asp Gly Asp Phe Glu Glu IIe Pro Glu Glu Tyr Leu Gln

COOH

SO3−

44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Gly Thr Pro Lys Pro Gln Ser His Asn Asp Gly Asp Phe Glu Glu IIe Pro Glu Glu Tyr Leu Gln Hirudin

D−Phe Pro Arg Pro Gly Gly Gly Gly Asn Gly Asp Phe Glu Glu IIe Pro Glu Glu Tyr Leu Bivalirudin Figure 4 Structure of hirudin and hirulogs. The amino acid sequences of natural hirudin and the hirulog bivalirudin are given. The C-terminal sequences blocking the anion-binding exosite (amino acids 54–65 of hirudin) are depicted in blue, and the sequences binding to the active site of thrombin (amino acids 45–48 of hirudin) are depicted in light blue.

The discovery of the bifunctional inhibition mechanism of hirudin resulted in the development of a new type of bivalent oligopeptide inhibitors, termed hirulogs. Bivalirudin, also termed hirulog-1, is a semisynthetic 20-amino acid peptide consisting of the N-terminal and the C-terminal active peptide domains from hirudin joined by a glycine linker (Figure 4).

mimicking the cleavage site in fibrinogen. The arginine-derived structures are shown in Figure 5. The pharmaceutical preparation consists of a mixture of R and S stereoisomers at a ratio of approximately 65:35. Ximelagatran has a molecular weight of 473.6 Da and is a pro-drug without intrinsic anticoagulant activity. Following resorption, it is rapidly converted into its active metabolite, melagatran (MW, 429.5 Da).

Synthetic Active Site Inhibitors of Thrombin

Argatroban and the orally active ximelagatran are LMW active site inhibitors of thrombin. They are the result of structure-based drug design and belong to the peptidomimetic (arginominetic) group of inhibitors

Mode of Action The classical and new anticoagulants interact with different clotting factors at different levels of blood

ANTICOAGULANTS 121 O H2N

OH

NH H2N

NH

Arginine

H3C

NH O

OH

O S O

NH

N O O

NH

HN

N

O CH3

O HO

O

N

NH2

NH H2N

CH3

NH

Ximelagatran Argatroban

Hydrolysis

Reduction

NH N O O

HN O OH

HN

NH2

Melagatran Figure 5 Chemical structures of synthetic thrombin inhibitors. The chemical structures of the arginomimetics argatroban, ximelagatran, and melagatran are shown. Bioconversion of ximelagatran into its active metabolite, melagatran, involves hydrolysis of the carboxyl ester and reduction of the hydroxyamidino group.

coagulation. A summary of their targets within the coagulation cascade is shown in Figure 6. Heparin and Heparinoids

Heparin acts as an accelerator of antithrombin (formerly called antithrombin III). The pentasaccharide sequence of heparin binds to the lysine site in antithrombin inducing a conformational change of

the antithrombin molecule, which facilitates binding to specific clotting factors and accelerates the rate at which antithrombin inhibits these factors by approximately 1000 times. UF heparin inhibits factors Xa, IIa (thrombin), IXa, XIa, and XIIa, with FXa and thrombin representing the most responsive and most critical factors within the clotting cascade. Heparin, antithrombin, and thrombin form a ternary complex in which thrombin initially binds to the

122 ANTICOAGULANTS Extrinsic pathway Vascular injury

Coumarins, indandiones

Tissue factor (TF) FVII

Intrinsic pathway

HMW kininogen Prekallikrein FXIIa FXI

FVII TF FVIIa

FIX

TFPI, FVIIai Coumarins, indandiones

Ca2+

FX

FXIa

UF heparin, LMW heparin Danaparoid, fondaparinux

Surface

FIXa

FVIII

Ca2+, PL

PL, Ca2+ Tenase complex

FIXa, thrombin

Antithrombin Hirudin, hirulogs Argatroban, melagatran

FVIIIa FXa 2+

APC

Prothrombin FV

Coumarins, indandiones

PL, Ca Prothrombinase complex

2+ Ca , PL, FXa

Thrombin FXIII

FVa

FXIIIa Heparin cofactor II

APC

Fibrinogen Danaparoid Dermatan sulfate

Fibrin

Ca2+

Crosslinked fibrin

Figure 6 Overview of the coagulation cascade and targets of anticoagulants. The targets of anticoagulants within the clotting cascade are given.

heparin–antithrombin complex in a non-specific manner to any site of the heparin molecule and then slides along the surface until it binds to the inhibitor. The affinity of heparin for the antithrombin–thrombin complex is much lower than that for unreacted antithrombin. Thus, heparin can dissociate from the complex and bind to additional unreacted antithrombin molecules, resulting in a continuing anticoagulant effect. The complex, however, is not effective in inhibiting fibrin-bound thrombin. UF heparin also blocks the thrombin-induced feedback activation of factors V and VIII. The sliding mechanism requires heparin chain lengths consisting of at least 18 saccharide units. The lower content of glycosaminoglycan chains 418 saccharide units in LMW heparin accounts for the greater relative activity against factor Xa. This difference is described in an activity ratio (anti-FXa:anti-FIIa ratio), which is 1:1 for UF heparin and 1.5:1 to 6:1 for LMW heparin (see Table 1). The anticoagulant activity of UF heparin is expressed in relation to the fourth international standard: Pharmaceutical preparations have specific activities of 150–190 U mg 1. The

anticoagulant activity of LMW heparin is expressed relative to the first international standard for LMW heparin; the specific activity ranges between 80 and 120 anti-FXa U mg 1 and between 35 and 45 antithrombin U mg 1. In addition to the interaction with antithrombin, both UF and LMW heparin enhance the release of tissue factor pathway inhibitor (TFPI), which forms a complex and inactivates FXa and subsequently FVIIa. UF and LMW heparins also bind to platelet factor 4, which is the predicate for heparin-induced thrombocytopenia (HIT), a severe complication of heparin therapy that is associated with paradoxic clotting, including deep vein thrombosis, pulmonary embolism, or occasional arterial thromboses. In HIT, antibodies form against the heparin–platelet factor 4 complex, leading to platelet activation and aggregation. LMW heparins bind less extensively to platelet factor 4 as well as to plasma proteins, endothelial cells, and macrophages. However, LMW heparins should not be used in patients with established HIT since HIT antibodies have an almost 100% cross-reactivity with LMW heparin.

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The heparinoid danaparoid sodium exerts its pharmacological effects in a similar manner via binding and activation of the serpin inhibitor heparin cofactor II. Danaparoid has little effect on platelet function and exhibits low cross-reactivity with heparin-induced antibodies, making it an option for the treatment of HIT. Similar to LMW heparins, danaparoid also exerts a higher inhibitory activity against FXa. The overall antithrombotic activity is much lower compared to that of UF heparin (anti-FXa activity B10% and antithrombin activity B1% of that of UF heparin). PPS may act via an antithrombin or heparin cofactor II-independent pathway, but the exact mechanism of action is not known. It has been used since the 1960s as an anticoagulant but does not seem to play a major role in today’s clinical medicine with regard to anticoagulation. In the United States, PPS is approved for pain relief in the management of interstitial cystitis. Fondaparinux and Idraparinux

Fonaparinux and idraparinux are selective indirect inhibitors of FXa. Their activity also requires the presence of antithrombin, but inhibition is achieved solely through binding of the pentasaccharide sequence to antithrombin and induction of a conformational change. Vitamin K Antagonists

Coumarin and indandione derivatives act as vitamin K antagonists and exert their anticoagulatory effect by interfering with the hepatic synthesis of vitamin K-dependent clotting factors. Synthesis of factors II (prothrombin), VII, IX, and X involves carboxylation of glutamate residues to g-carboxyglutamates by a specific carboxylase, which is required for their biological activity. These g-carboxyglutamate residues promote binding of the clotting factors to phospholipids, thereby accelerating coagulation. Coumarin and indandione derivatives interfere with the vitamin K cycle by inhibiting vitamin K epoxide reductase. By this mechanism, coumarins decrease the synthesis of coagulation factors by 30–50%. The anticoagulant response to coumarins is delayed by 2 or 3 days since previously formed coagulation factors circulate with a long half-life (4–6 h for FVII, 20–50 h for factors IX and X, and 50–70 h for thrombin). A major disadvantage of vitamin K antagonists is the narrow therapeutic index and the need for intensive monitoring. The anticoagulatory effect is assessed by the international normalized ratio (INR), which is defined as the ratio of the patient prothrombin time (PT) compared to the mean PT of normal donors normalized to the international sensitivity index, a correction factor for

the response of different thromboplastin reagents. The therapeutic INR range is 2.0–3.0. An INR o2.0 may be ineffective in preventing thrombotic events, and an INR 43.0 is associated with increased bleeding. The manifold interaction of coumarins with food and drugs has an important impact on the anticoagulatory response by either increasing or decreasing anticoagulant activity. Direct Thrombin Inhibitors (Hirudin, Hirulogs, and Synthetic Inhibitors)

Hirudin is a highly potent (Ki ¼ 22 fM) inhibitor specific for the serine protease thrombin. It forms a stable noncovalent 1:1 stoichiometric complex with the B-chain of a-thrombin, thereby abolishing its ability to cleave fibrinogen (Figure 7). Hirudin inhibits both free and fibrin-bound thrombin. During inhibition, hirudin displaces fibrin from thrombin. Bivalirudin is a bivalent inhibitor (Ki ¼ 1:9 nM) blocking both the active site and the substrate recognition site (anion-binding exosite). Argatroban (Ki ¼ 39 nM) and melagatran (Ki ¼ 2 nM) are peptidomimetics of the sequence of fibrinopeptide A that interact with the active site of thrombin. They are reversible, noncovalent, competitive inhibitors blocking the enzyme’s interaction with its substrate. The pro-drug ximelagatran has no intrinsic anticoagulant activity but is rapidly converted into melagatran following gastrointestinal resorption. The direct thrombin inhibitors are recommended agents for prevention and treatment of HIT. Lepirudin and argatroban are approved for the treatment of HIT in the United States. Bivalirudin, which is approved for anticoagulation during percutaneous coronary intervention, appears to be promising in patients with HIT. Although displaying pharmacologic advantages, its use for this indication is only weakly recommended due to the limited data available. Novel Anticoagulants

In addition to the previously described anticoagulants, novel agents that directly target specific steps within the coagulation cascade have been developed and tested in clinical phase II or phase III studies or are currently undergoing preclinical and clinical testing. They include LMW inhibitors of FXa and recombinant forms of naturally occurring anticoagulants. Recombinant tissue factor pathway inhibitor (tifacogin) Tifacogin is a recombinant form of the endogenous extrinsic pathway inhibitor TFPI, also referred to as anticonvertin or lipoprotein-associated coagulation inhibitor. TFPI is a 276-amino acid residue, high-affinity Kunitz-type serine protease inhibitor.

+ + +

+ + +

Fibrin

+ + +

Hirudin

Heparin recognition site = anion-binding exosite II

+ + +

Fibrin

124 ANTICOAGULANTS

Bivalirudin

Thrombin + + +

+ + +

Substrate recognition site = anion-binding exosite I

Binding of fibrin(ogen), PAR-1, heparin cofactor III thrombomodulin

Binding of heparin, prothrombin 2 fragment (F2) platelet gp Ib

+ + +

Active site

+ + +

Fibrin

Specificity pocket

Synthetic inhibitors (Argatroban, melagatran) Figure 7 Mechanism of action of thrombin inhibitors. The interaction of hirudin, hirologs, and synthetic thrombin inhibitors with thrombin is shown.

Inhibition of the tissue factor-mediated extrinsic coagulation pathway occurs in a two-step manner: TFPI directly binds to FXa at or near its active site via the second Kunitz domain and produces a FXadependent feedback inhibition of the TF–FVIIa catalytic complex by formation of a quaternary complex (FXa–TFPI–TF/FVIIa), in which the second Kunitz domain binds to FXa and the first Kunitz domain binds FVIIa (Figure 8). Despite showing promising results in animal thrombosis models, a phase III trial failed to demonstrate significant benefit in outcome in sepsis patients. Recombinant nematode anticoagulant protein c2 Recombinant nematode anticoagulant protein c2 (rNAPc2) is a potent 85-amino acid residue anticoagulant protein originally identified in the hookworm Ancylostoma caninum. Like TFPI, rNAPc2 inhibits initiation of the extrinsic coagulation pathway by binding to a noncatalytic exosite on FX or

FXa prior to the formation of the quaternary inhibitory complex with tissue factor–FVIIa. In a phase II study on patients who underwent knee replacement surgery, a low dose of rNAPc2 decreased the rate of deep vein thrombosis and major bleeding.

Active site inhibited factor VII Active site inhibited factor VII (FVIIai; ASIS) is a recombinant variant of activated factor VII in which the catalytic function is irreversibly blocked. The resulting molecule retains its TF binding capacity but is enzymatically inactive. FVIIai exerts its effects by competing with plasma FVII(a) for tissue factor binding on cell surfaces. The formation of an inactive binary FVIIai–TF complex attenuates the initiation of coagulation. However, although recombinant FVIIai was shown to inhibit TF-mediated injury in animal models, it failed to elicit a beneficial response in coronary patients in a phase II trial.

ANTICOAGULANTS 125 3 TFPI 1

2 FXa

Ca2+

TFPI

FXa

Ca2+

TFPI

FVIIa

FVIIa Ca2+

Ca2+

TF

FXa

TF Ca2+

Figure 8 Mechanism of action of tissue factor pathway inhibitor (TFPI). TFPI binds to FXa in solution forming a binary complex. Subsequent binding of FXa–TFPI to the TF–FVIIa complex results in the final quaternary complex inhibiting initiation of coagulation.

Direct FXa inhibitors DX-9065a (Daiichi) is a synthetic, LMW, nonpeptidic, direct reversible and parenteral inhibitor of factor Xa. BAY 59-7939 (Bayer), razaxaban (Bristol-Myers Squibb), LY 517717 (Eli Lilly), and YM-60828 (Yamanouchi Pharmaceutical) are orally active synthetic FXa inhibitors. Activated protein C (drotrecogin alpha (activated)) The protein C system also regulates coagulation by modulation of the activity of the two clotting factors, FVa and FVIIIa. Protein C, the key component of this system, is a vitamin K-dependent zymogen of an anticoagulant protease, which is activated on the surface of intact endothelial cells by thrombin that has bound to the integral membrane protein thrombomodulin. Activated protein C (APC) can cleave the phospholipid membrane-bound (activated) clotting factors FVa and FVIIIa, which blocks the function of the prothrombinase and tenase

complexes and thus inhibits the propagation of coagulation (Figure 9). Activation of protein C is augmented by the endothelial cell protein C receptor, and the anticoagulant activity of APC is supported by protein S, a vitamin K-dependent cofactor. The protein C system also has anti-inflammatory and antiapoptotic properties. Drotrecogin alpha (activated) is a recombinant version of APC. Drotrecogin alpha was approved for treatment of severe sepsis in adults based on a phase III trial in which 28-day mortality was reduced. Thrombomodulin Thrombomodulin is a complex multifunctional endothelial cell surface glycoprotein receptor. High-affinity binding to thrombin involving EGF domains 4–6 converts thrombin from a procoagulant into an anticoagulant state that can activate protein C. Thrombomodulin not only regulates hemostasis but also plays an important role in the

126 ANTICOAGULANTS Protein C APC TM

FV

Protein S

FVIIIi FVIIIa A P C

EPCR

Protein S

Th

FVi FVa A P C

Figure 9 Protein C pathway: mechanism of action of protein C and thrombomodulin. (Top) Thrombin (Th) generated in the vicinity of intact endothelial cells binds to thrombomodulin (TM) and activates protein C. This process is enhanced by the endothelial cell protein C receptor (EPCR). (Bottom) Activated protein C (APC) and protein S form a complex on the membrane of endothelial cells that cleaves and inactivates FVIIIa (-FVIIIi) and FVa (-FVi) and thus inhibits coagulation. In the case of FVIIIa, this process is further enhanced by FV, which in this context functions as an anticoagulant cofactor protein.

modulation of inflammation. The EGF 3–6 domains are involved in activation of thrombin-activatable fibrinolysis inhibitor, a natural anti-inflammatory molecule that inhibits vasoactive peptides such as the complement anaphalotoxin C5a. Soluble thrombomodulin is a recombinant variant that is undergoing clinical testing.

Role of Anticoagulants in Respiratory Medicine Thromboembolic events, including deep vein thrombosis and pulmonary embolism, are commonly encountered in clinical practice. In addition to the occlusion of a vessel, ‘periembolic’ processes, such as activation of circulating platelets and inflammatory cells at the surface of thrombi and the liberation of vasoactive mediators (thromboxan, serotonin, and leukotriene B4) and fibrin(ogen)-derived split products, markedly contribute to cardiopulmonary dysfunction, with increased pulmonary vascular resistance, elevated right heart pressure, and gas exchange disturbances. Anticoagulants are used for prevention and treatment of a variety of indications, including prevention of deep vein thrombosis and pulmonary embolism postoperatively after general or orthopedic surgery (e.g., hip replacement/fracture), in patients with acute spinal cord injuries, multiple trauma, ischemic stroke, after coronary angioplasty, heart valve replacement, and in many other medical conditions as well as for treatment of established deep vein thrombosis, unstable angina, and ischemic stroke. In a meta-analysis,

subcutaneous LMW heparin was shown to be as effective and safe as intravenous UF heparin for the initial treatment of nonmassive pulmonary embolism. Intravascular clot formation due to predominance of a procoagulant and suppression of fibrinolytic factors is a key event in a variety of inflammatory and infectious diseases, such as septic organ failure. Microthrombosis/microembolism is commonly found in patients with acute respiratory distress syndrome (ARDS), of which sepsis is the major underlying cause, and in patients with chronic pulmonary hypertension. The lung microvascular compartment represents the largest surface of the pulmonary vascular bed and may be of crucial importance in the dissolution of microthrombi. Under physiological conditions, patency of capillaries is provided by a high concentration of antithrombin that is ‘activated’ by glycosaminoglycans (e.g., heparan sulfate) and thrombomodulin on the surface of endothelial cells. In addition, clearance of thrombi is achieved through the production and secretion of tPA and uPA by microvascular endothelial cells. Under inflammatory or infectious conditions (e.g., sepsis and ARDS), this balance between anticoagulation and fibrinolysis regulation is shifted toward coagulation with a decrement in fibrinolysis, mainly due to activation of the extrinsic tissue factor/FVII-dependent coagulation pathway and secretion of plasminogen activator inhibitor-1 (PAI-1). In experimental models of acute lung injury (ALI), APC prevented intravascular coagulation, edema formation, and inflammatory cell recruitment in the lung. Although administration of APC has been shown to decrease mortality in patients

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with severe sepsis, clinical studies demonstrating its efficacy in ARDS are lacking. In ALI/ARDS, fibrin deposition occurs in both intravascular and extravascular locations. In patients with ALI/ARDS, the alveolar hemostatic balance is shifted toward predominance of procoagulant activity, which is mainly attributable to the extrinsic pathway enzymes tissue factor and FVII. In contrast, the fibrinolytic capacity of the alveolar space is markedly reduced. In lavage fluid from patients with ARDS, concentrations of urokinase, representing the predominant plasminogen activator in this compartment, were markedly decreased, whereas elevated activities of PAI-1 and a2-antiplasmin were consistently encountered. Under these conditions, fibrinogen leaking into the alveolar space due to an impaired barrier function is rapidly converted into fibrin. The function of fibrin formation in the alveolar space is largely unsettled. It may well exert beneficial effects by preventing pulmonary hemorrhage and serve as a primary matrix of wound repair. On the other hand, alveolar fibrin is a potent inhibitor of surfactant function, thus promoting atelectasis formation and contributing to the severe impairment of gas exchange properties. In vitro studies have demonstrated an incorporation of hydrophobic surfactant compounds into nascent fibrin strands, resulting in an almost complete loss of the surface tension-lowering properties. Alveolar/interstitial fibrin deposition and aberrant local fibrin turnover have likewise been shown in patients with chronic interstitial lung disease, including idiopathic pulmonary fibrosis, sarcoidosis, and hypersensitivity pneumonitis. Alveolar fibrin appears to be a key factor in triggering the fibroproliferative process in the lung. Delayed clearance of fibrin from the lung may promote fibroblast activation, proliferation, and tissue remodeling (i.e., replacement of the primary fibrin matrix by a secondary collagenous matrix associated with scarring and honeycombing). Thrombin also acts as a chemoattractant for inflammatory cells and fibroblasts and stimulates the release of proinflammatory cytokines. Moreover, it induces the release of growth factors and fibrogenic mediators, thereby stimulating fibroblast proliferation and connective tissue synthesis. Although the use of anticoagulants is not part of the standard clinical practice for these conditions, blockade of the extrinsic coagulation pathway and prevention of extravascular fibrin formation may be a promising approach for acute inflammatory and chronic interstitial lung disease. Experimental studies addressing anticoagulant intervention (e.g., by administration of APC, heparin, and direct thrombin inhibitor) in animal models of ALI and pulmonary fibrosis have been shown to

protect the lung and to reduce the fibroproliferative response. In addition, different anticoagulants are being tested in ongoing experimental and clinical studies. A phase II study of recombinant TFPI in patients with severe sepsis showed improvement in lung function and a trend toward improved survival in the subset of patients with ARDS. A follow-up phase III study, however, failed to demonstrate improved survival. Similarly, the beneficial effects of recombinant antithrombin observed in animal models could not be confirmed in humans. Heparin is being evaluated in humans with idiopathic pulmonary fibrosis. In a similar vein, heparin is considered an alternate agent in the treatment of allergic asthma. Glycosaminoglycans such as heparan sulfate are expressed as part of a proteoglycan on cell surfaces and are thought to play a role in the regulation of the inflammatory response (e.g., by binding chemokines). Heparin, which is synthesized by and stored exclusively in mast cells, has been found to exert anti-inflammatory effects in animal models and in human disease. Heparin binds and inhibits a variety of cytotoxic and inflammatory mediators, including eosinophilic cation protein and peroxidase. Furthermore, heparin has been associated with the inhibition of lymphocyte activation, neutrophil chemotaxis, smooth muscle growth, and vascular tone. The first clinical trial with inhaled heparin was performed in 1969, but further human studies using inhaled heparin were not published until the early 1990s. Three smaller studies of patients with exercise-induced asthma showed that inhaled heparin preserved specific airway conductance better than inhaled cromolyn or placebo following exercise. Studies investigating the effects of inhaled heparin on bronchial reactivity following inhaled allergen challenge have yielded mixed results.

Side Effects and Contraindications The most devastating complication of anticoagulant therapy is bleeding. The occurrence of bleeding, primarily in the gastrointestinal tract and the central nervous system, is a major cause of morbidity and mortality. Consequently, anticoagulants are contraindicated in any case in which the risk of hemorrhage may be greater than the potential clinical benefit of anticoagulation. Risk factors for bleeding include advanced age, serious illness (cerebral, cardiac, kidney, or liver disease), cerebrovascular or peripheral vascular disease, and an unstable anticoagulant effect. Whereas the action of unfractionated heparin may be antagonized by protamine sulfate, no specific antidote is available for LMW heparins, danaparoid, fondaparinux, or the direct thrombin inhibitors

128 ANTICOAGULANTS

hirudin and ximelagatran. The anticoagulant effect of coumarin derivatives may be antagonized by low doses of vitamin K and substitution with fresh frozen plasma. Other adverse effects of heparins are heparin-induced thrombocytopenia and osteoporosis, which can occur after long-term treatment and increase the risk for fractures. An absolute contraindication for coumarins is pregnancy because they cause fetal bone abnormalities by interfering with g-carboxylation of proteins synthesized in the bone. Heparins do not cross the placental barrier and do not cause these problems. Long-term use of ximelagatran carries the risk of severe liver injury. Administration in patients with hepatic disease is not recommended. As outlined previously, HIT is an antibody-mediated adverse effect of heparin associated with an increased thrombotic risk. According to the seventh American College of Clinical Pharmacology consensus conference guidelines, alternative, nonheparin anticoagulants should be used in patients with strongly suspected or confirmed HIT. Recommendations include direct thrombin inhibitors as well as danaparoid. Although withdrawn from the US market, danaparoid remains approved and available for prevention and treatment of HIT in Canada, Europe, Australia, and Japan. Because fondaparinux does not cross-react with HIT antibodies, it may also be useful for this indication. A general recommendation, however, cannot be made due to the minimal data available. Antihirudin antibodies are commonly generated during treatment with lepirudin. Although they are usually not clinically significant, the European Agency for the Evaluation of Medical Products recommended the use of nonhirudin anticoagulants in patients who have previously been exposed to lepirudin. Coumarins should not be used in patients with confirmed or strongly suspected HIT because they can contribute to skin necrosis and venous limb gangrene. For patients receiving coumarins at the time of diagnosis of HIT, the use of vitamin K for reversal of coumarin-induced anticoagulation is recommended. See also: Acute Respiratory Distress Syndrome. Coagulation Cascade: Overview; Antithrombin III;

Antioxidants

Factor X; Protein C and Protein S; Thrombin. Interstitial Lung Disease: Overview; Idiopathic Pulmonary Fibrosis. Pulmonary Thromboembolism: Deep Venous Thrombosis; Pulmonary Emboli and Pulmonary Infarcts. Thrombolytic Therapy.

Further Reading Ansell J, Hirsh J, Poller L, et al. (2004) The pharmacology and management of the vitamin K antagonists: the seventh ACCP conference on antithrombotic and thrombolytic therapy. Chest 126: 204S–233S. Bussey H, Francis JL, and the Heparin Consensus Group (2004) Heparin overview and issues. Pharmacotherapy 24: 103S–107S. Desai UR (2004) New antithrombin-based anticoagulants. Medical Research Reviews 24: 151–181. Ginsberg JS, Greer I, and Hirsh J (2001) Use of antithrombotic agents during pregnancy. Chest 119: 122S–131S. Happel KI, Nelson S, and Summer W (2004) The lung in sepsis: Fueling the fire. American Journal of the Medical Sciences 328: 230–237. Hirsh J and Raschke R (2004) Heparin and low-molecular-weight heparin: The seventh ACCP conference on antithrombotic and thrombolytic therapy. Chest 126: 188S–203S. Hirsh J, Fuster V, Ansell J, and Halperin JL (2003) American Heart Association/American College of Cardiology Foundation guide to warfarin therapy. Circulation 107: 1692–1711. Idell S (2001) Anticoagulants for acute respiratory distress syndrome. Can they work? American Journal of Respiratory and Critical Care Medicine 164: 517–520. Idell S (2002) Adult respiratory distress syndrome: Do selective anticoagulants help? American Journal of Respiratory Medicine 1: 383–391. Laterre PF, Wittebole X, and Dhainaut JF (2003) Anticoagulant therapy in acute lung injury. Critical Care Medicine 31(supplement): S329–S336. Nowak G (2002) Pharmacology of recombinant hirudin. Seminars in Thrombosis and Hemostasis 28: 415–423. Srivastava S, Goswami LN, and Dikshit DK (2005) Progress in the design of low molecular weight thrombin inhibitors. Medical Research Reviews 25: 66–92. Warkentin TE and Greinacher A (2004) Heparin-induced thrombocytopenia: Recognition, treatment, and prevention. The seventh ACCP conference on antithrombotic and thrombolytic therapy. Chest 126: 311–337. Weitz JI (1997) Low-molecular weight heparins. New England Journal of Medicine 337: 688–698. Weitz JI, Hirsh J, and Samama MM (2004) New anticoagulant drugs. The seventh ACCP conference on antithrombotic and thrombolytic therapy. Chest 126: 265S–286S. Wittkowsky AK (2003) Warfarin and other coumarin derivatives: Pharmacokinetics, pharmacodynamics, and drug interactions. Seminars in Vascular Medicine 3: 221–230.

see Oxidants and Antioxidants: Antioxidants, Enzymatic; Antioxidants, Nonenzymatic.

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ANTIVIRAL AGENTS M A Parniak, E N Vergis, and M E Abram, University of Pittsburgh, Pittsburgh, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Most respiratory tract infections (RTIs) are viral in origin; numerous different viruses cause RTIs, many of which can be quite serious. However, only five drugs are approved for their treatment, and these drugs are directed at only two viruses. Four drugs – amantadine, rimantadine, zanamivir, and oseltamivir – are used for the prophylaxis and treatment of influenza virus infection. These drugs target two different stages of influenza replication. Orally administered amantadine and rimantadine act only against influenza A and block the ion channel formed by the viral M2 protein, thereby preventing viral ribonucleoprotein release. Zanamivir and oseltamivir are active against influenza A and B, and they are competitive inhibitors of the viral neuraminidase. These drugs block nascent viral release from the infected cell and thus viral spread by preventing neuraminidase-catalyzed removal of sialic acid residues from membrane glycoproteins. Oseltamivir is administered orally, whereas zanamivir must be delivered topically by inhalation of the dry powder. The fifth approved drug, the ribonucleoside analog ribavirin, is administered by nebulization and is approved for the treatment of pediatric respiratory syncytial virus infection. Its mechanism of action is uncertain, but it may involve alteration of cellular nucleotide pools and inhibition of viral RNA synthesis.

Introduction Viruses are the cause of most respiratory tract infections (RTIs); numerous different viruses can infect the respiratory tract. Many of these infections can be quite serious, especially in the young, the elderly, chronically ill, and immunocompromised individuals. However, there are only five drugs approved for the treatment of viral RTIs, and these drugs are directed at only two viruses. Four of the approved drugs are used for the treatment or prophylaxis of influenza, and one is used for the treatment of pediatric respiratory syncytial virus infection.

Drugs for the Treatment of Influenza Influenza virus mainly infects the upper respiratory tract, and most individuals recover within approximately 1 week without medical treatment. Nevertheless, RTI due to influenza is a major cause of morbidity and mortality worldwide. The World Health Organization estimates that influenza can account for up to 15% of worldwide upper RTIs

annually, with up to 500 000 deaths. Influenza viruses that cause human disease are classified into groups A–C. Influenza A is the most serious pathogen since it leads to large recurrent epidemics with significant morbidity and mortality. Four drugs (Figure 1) – the adamantane derivatives amantadine and rimantadine and the neuraminidase inhibitors zanamivir and oseltamivir – are used for the treatment or prophylaxis of influenza virus infection. These drugs target different stages of influenza virus replication (Figure 2). Amantadine and rimantadine inhibit an early stage involved in the uncoating of the viral ribonucleoprotein, whereas zanamivir and oseltamivir inhibit a viral enzyme needed to allow nascent virus release from the infected cell. Amantadine and Rimantadine

The inhibitory activity of amantadine (adamantan-1ylamine hydrochloride) against influenza A was first identified in 1964, and this drug was first marketed in 1966. The close analog, rimantadine (1-adamantan-1-yl-ethylamine hydrochloride), was developed subsequently due to the adverse neurological side effects associated with the use of amantadine. These structurally similar compounds are used primarily for prophylaxis of influenza A infection, particularly for individuals with high risk of exposure such as healthcare workers, and prevent influenza-like illness in up to 80% of treated individuals. Both drugs can also reduce the duration of symptoms of established influenza A infection if therapy is initiated within 48 h of discernible symptoms. The adamantanes are effective only against influenza A. However, since influenza A is the source of most serious cases of influenza-related RTI, the adamantanes are valuable drugs for the treatment and especially prophylaxis of influenza infections. Amantadine and rimantadine are administered orally, once or twice daily with adult dosages of 100–200 mg per day. For prophylaxis, the drugs are administered for up to 7 days during high-risk periods of an epidemic. The two drugs have very different pharmacokinetic profiles despite their similarity in structure. Amantadine is rapidly absorbed after oral delivery, has a half-life of approximately 15 h, is not metabolized, and is excreted almost entirely in the urine. In contrast, rimantadine is more slowly absorbed, has a half-life twice that of amantadine, and undergoes extensive hepatic metabolism. Although peak plasma levels of rimantadine are less than those

130 ANTIVIRAL AGENTS H3C

NH2 HCl

NH2 HCl

Rimantadine

Amantadine

O

OH

O

OH

NH

O H

H

HO N H

NH2

NH2

O HN

HN OH OH

O

Zanamivir

O

Oseltamivir

Figure 1 Drugs used for prophylaxis or treatment of influenza virus infection. The drug oseltamivir is shown as the active caboxylic acid form, although it is administered as the ethyl ester pro-drug.

of amantadine, the former is more highly concentrated in respiratory secretions than amantadine, thereby providing better access to the viral target. Mechanism of action Amantadine and rimantadine target the influenza A virus M2 membrane protein. The influenza virus enters the cell via endocytosis, and fusion of the virus membrane envelope with the endosome membrane provides a mechanism for the release of the viral ribonucleoprotein (RNP) into the cytoplasm and thus to the nucleus, where replication of the viral genomic RNA can take place. The RNP interacts with the viral M1 protein that underlies the viral envelope. Furthermore, M1 masks a nuclear localization signal on the RNP. Unless the M1–RNP interactions are disrupted, the RNP can neither be released into the cytoplasm nor imported into the nucleus. Disassembly of the complex may also be important for activation of the viral RNA polymerase. The influenza M2 membrane protein is critical in enabling disruption of the M1–RNP complex. The M2 protein is a homotetramer that is considered to form an ion channel in the viral membrane envelope (Figure 3). During acidification of the viruscontaining endosome, the M2 protein enables protons to enter the virus particle. Acidification of

the interior of the virus particle results in dissociation of the RNP from the M1 protein. This allows release of the RNP into the cytoplasm and subsequent import into the nucleus. Amantadine and rimantadine bind to the viral M2 membrane protein, blocking the channel and preventing proton transport into the viral particle. The M2 protein is not found in influenza B virus, which accounts for the lack of antiviral activity of amantadine and rimantadine against influenza B. Contraindications Central nervous system side effects are noted in significant numbers (up to 10%) of amantadine-treated individuals. Symptoms include dizziness, anxiety, difficulty in concentrating, and insomnia, all of which are problematic for healthcare workers, a major target group for prophylactic treatment during influenza epidemics. Central nervous system side effects reverse once treatment is stopped. Severe toxic reactions or death can occur in patients with renal insufficiency and include serious neurotoxicity (mental status changes, hallucinations, tremor, myoclonus, seizures, and coma) or cardiac arrhythmias. Dosages of the drugs, especially amantadine, must be lowered and carefully monitored in such patients. Rimantadine is significantly better tolerated than amantadine.


Zanamivir, oseltamivir

Amantadine, rimantadine

Figure 2 Influenza virus replication cycle showing stages inhibited by approved therapeutic drugs. Following attachment, endocytosis, and acidification of the virion-containing endosome, the virion core is also acidified in a process mediated by the viral M2 membrane proton channel protein. This enables a pH-dependent release of viral ribonucleoprotein (RNP). Adamantane drugs target the virus M2 protein, preventing virion acidification and release of RNP. Nascent virus release from an infected cell requires cleavage of terminal sialic acid residues from cell surface and virion envelope proteins. Neuraminidase inhibitors target this stage of replication, preventing spread of virus to uninfected cells.

Neuraminidase Inhibitors

The viral neuraminidase inhibitors zanamivir (5-acetylamino-4-gunadino-6-(1,2,3-trihydroxypropyl)-5,6dihydro-4H-pyran-2-carboxylic acid) and oseltamivir (4-acetylamino-5-amino-3-(1-ethylpropoxy)-cyclohex1-ene carboxylic acid) were approved for therapeutic use in 1999. Both drugs are reversible competitive inhibitors of influenza virus neuraminidase. The drugs are effective against both influenza A and influenza B and also against strains of influenza A virus resistant to

the adamantane drugs. Zanamivir and oseltamivir have virtually no activity against human cell neuraminidases at levels that provide potent antiviral activity. Viral resistance to zanamivir or oseltamivir is uncommon. Both drugs are approved for treatment of influenza in adults and children (1 year or older for oseltamivir and 7 years or older for zanamivir) within 48 h of onset of discernible symptoms. Oseltamivir can also be used for prophylaxis in adults. Zanamivir has low oral bioavailability and is administered topically by dry powder inhalation, whereas oseltamivir can be

132 ANTIVIRAL AGENTS H+

NH2HCl V

V H

A S H

Viral membrane envelope

H

G

A S G

M2 protein subunits Figure 3 Schematic of the mechanism of adamantane drug inhibition. The drugs interact with specific residues of the influenza A virus M2 transmembrane protein. This protein forms a channel for the influx of protons into the virion. Only two of the four M2 subunits that form the channel are shown. Binding of amantadine or rimantadine blocks the channel, preventing intravirion acidification.

administered orally (bioavailability 460%, with a plasma half-life of approximately 8 h). Oseltamivir is not metabolized and is excreted almost entirely in the urine. Oseltamivir is administered as the ethyl ester pro-drug that lacks antiviral activity. This is hydrolyzed to the active oseltamivir carboxylate by esterases in the intestinal mucosal epithelia and in the liver. Mechanism of action Zanamivir and oseltamivir target the viral enzyme neuraminidase. Neuraminidase, also called sialidase, is one of the two major influenza viral proteins (the other is hemagglutinin, which is important for binding and fusion of the virus envelope with the host cell). Neuraminidase is essential for release of newly formed virus from infected cells and for virus spread throughout the respiratory tract of the infected host. Nascent virions bud off from the infected cell encased in an envelope composed of a lipid bilayer derived from the cell plasma membrane with associated viral envelope proteins. Many cell surface glycoproteins of mammalian cells possess sialic acid as the terminal sugar, a residue added during glycosylation events in subcellular Golgi organelles. Influenza virus envelope proteins are formed in the same subcellular compartment and are therefore also sialylated. However, the cell surface receptor for influenza virus attachment is sialic acid. Removal of the cell surface and viral envelope protein sialic acid residues is essential to prevent reattachment of the nascent virions

to the same cell and to prevent aggregation of the virus particles. Neuraminidase cleaves terminal sialic acid residues from cellular and viral membrane glycoproteins, thus destroying the cellular receptors recognized by the viral hemagglutinin. Influenza neuraminidase is a tetramer of identical subunits (Figure 4). The active site in each subunit is a deep groove on the protein surface that comprises residues that are apparently identical in all strains of influenza A and B. The inhibitors zanamivir and oseltamivir result from rational drug design based on the neuraminidase sialic acid-binding site determined by crystallography. Both drugs bind with high affinity to the active site of viral neuraminidase (Figure 4), thus preventing binding of the normal sialic acid glycoprotein substrate. However, the inhibition mechanisms of the two drugs differ. Zanamivir is an analog of sialic acid and acts as a classical competitive inhibitor to prevent substrate binding to the unliganded enzyme. Oseltamivir is a transition-state analog of sialic acid cleavage and thus interferes with neuraminidase conformational changes needed to allow substrate binding. Nonetheless, the result is the same, and both drugs have similar antiviral potency. Contraindications Few serious clinical toxicities have been reported with zanamivir or oseltamivir. Inhaled zanamivir is generally well tolerated, although exacerbations of asthma may occur. Orally administered oseltamivir may cause nausea and


Figure 4 Structure of influenza B virus neuraminidase bound with the inhibitor zanamivir (in red). The structure was based on pdb1A4G. Adapted from Taylor NR, Cleasby A, Singh O et al. (1998) Dihydropyrancarboxamides related to zanamivir: a new series of inhibitors of influenza virus sialidases. 2. Crystallographic and molecular modeling study of complexes of 4-amino-4H-pyran-6-carboxamides and sialidase from influenza virus types A and B. Journal of Medicinal Chemistry 41: 798–807.

gastrointestinal discomfort, although these are transient and less likely if the drug is administered with food.

NH2 O N

Drug for the Treatment of Respiratory Syncytial Virus Infection

N N O

Ribavirin HO

The synthetic nucleoside ribavirin (1-(3,4-dihydroxy5-hydroxymethyl-tetrahydrofuran-2-yl)-1H-[1,2,4]triazole-3-carboxylic acid amide) is a guanosine analog that is approved for the treatment of pediatric respiratory syncytial virus (RSV) infection (Figure 5). RSV is the most common cause of severe lower respiratory tract disease in infants, causing up to 90% of bronchiolitis and 40% of bronchopneumonia cases. Disease symptoms are much milder in older children and adults. Although orally administered ribavirin is rapidly absorbed with good bioavailability (approximately 45%), treatment of pediatric RSV infection requires the drug to be administered by continuous aerosol (nebulizer) for 12– 18 h daily for 3–6 days. This therapy is expensive and requires specialized equipment and monitoring. Its use is therefore generally reserved for treatment of RSV infection in high-risk infants (e.g., premature infants, immunocompromised children, or infants with congenital heart disease).

antiviral activity of ribavirin is unclear and may differ for different types of viruses. However, it is generally thought that ribavirin therapy alters cellular nucleotide pools, thereby affecting viral mRNA synthesis. It is likely that ribavirin must be phosphorylated by cellular kinases to provide antiviral activity. Ribavirin-50 -monophosphate inhibits the cellular enzyme inosine-50 -phosphate dehydrogenase, blocking the conversion of inosine-50 -monophosphate to xanthosine-50 -monophosphate. This decreases levels of guanosine triphosphate (GTP), which impacts on both RNA and DNA metabolism. Ribavirin-50 triphosphate may also be a competitive inhibitor of GTP-dependent 50 -capping of viral mRNA.

Mechanism of action Ribavirin is in fact a broadspectrum antiviral agent that inhibits a wide range of RNA and DNA viruses. The mechanism for the

Contraindications Aerosolized ribavirin may cause bronchospasm or conjunctival irritation. It requires close supervision, especially with mechanical

HO

OH

Figure 5 Structure of ribavirin, which is used for the treatment of high-risk pediatric respiratory syncytial virus infection.

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ventilation, because precipitation of the drug may occur. Healthcare workers involved in the administration and monitoring of aerosol ribavirin treatment may occasionally experience irritation of the eyes and respiratory tract. Ribavirin is mutagenic, teratogenic, and embryotoxic; thus, aerosolized ribavirin is a risk to pregnant healthcare workers. See also: Aerosols. Bronchiolitis. Pneumonia: Viral. Upper Respiratory Tract Infection. Vaccinations: Viral. Viruses of the Lung.

Further Reading Abdel-Magid AF, Maryanoff CA, and Mehrman SJ (2001) Synthesis of influenza neuraminidase inhibitors. Current Opinion in Drug Discovery and Development 4: 776–791. Broughton S and Greenough A (2004) Drugs for the management of respiratory virus infection. Current Opinion in Investigative Drugs 5: 862–865. De Clercq E (2004) Antiviral drugs in current clinical use. Journal of Clinical Virology 30: 115–133. Dreitlein WB, Maratos J, and Brocavich J (2001) Zanamivir and oseltamivir: two new options for the treatment and prevention of influenza. Clinical Therapeutics 23: 327–355.

Garman E and Laver G (2004) Controlling influenza by inhibiting the virus’s neuraminidase. Current Drug Targets 5: 119–136. Johnston SL (2002) Anti-influenza therapies. Virus Research 82: 147–152. Kandel R and Hartshorn KL (2001) Prophylaxis and treatment of influenza virus infection. BioDrugs 15: 303–323. Lew W, Chen X, and Kim CU (2000) Discovery and development of GS4104 (oseltamivir): an orally active influenza neuraminidase inhibitor. Current Medical Chemistry 7: 663–672. Oxford JS, Bossuyt S, Balasingam S, et al. (2003) Treatment of epidemic and pandemic influenza with neuraminidase and M2 proton channel inhibitors. Clinical Microbiology and Infection 9: 1–14. Snell NJC (2001) New treatments for viral respiratory tract infections – opportunities and problems. Journal of Antimicrobial Chemotherapy 47: 251–259. Tam RC, Lau JY, and Hong Z (2001) Mechanisms of action of ribavirin in antiviral therapies. Antiviral Chemistry and Chemotherapy 12: 261–272. Taylor NR, Cleasby A, Singh O, et al. (1998) Dihydropyrancarboxamides related to zanamivir: a new series of inhibitors of influenza virus sialidases. 2. Crystallographic and molecular modeling study of complexes of 4-amino-4H-pyran6-carboxamides and sialidase from influenza virus types A and B. Journal of Medicinal Chemistry 41: 798–807. Torrence PF and Powell LD (2002) The quest for an efficacious antiviral for respiratory syncytial virus. Antiviral Chemistry and Chemotherapy 13: 325–344.

APOPTOSIS A M K Choi and X Wang, University of Pittsburgh, Pittsburgh, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The phenomenon of programmed cell-death, which is more commonly known as apoptosis, was first described by C Vogt in 1842. Apoptosis, the term was proposed by Kerr and colleagues in 1972, is an active process of cellular self-destruction with distinctive morphological and biochemical features . It is indispensable in the development and maintenance of homeostasis within all multicellular organisms. The molecular machinery of apoptosis is also important for the regulation of homeostasis during adulthood, which is important for the control of neoplasia and autoimmunity. Understanding how apoptosis occurs in different situations will help to understand the pathogenesis of a number of human diseases and therefore provide clues to the treatment.

Apoptotic Pathways Caspases are involved in various programmed celldeath pathways reported in the literature. They are a

family of cysteine proteases, and many of them are implicated as important initiators or effectors of the apoptosis process. To date, at least 14 members of this family have been identified, but only a subset of them are partially or fully characterized. Like many enzymes, caspases are synthesized in the cell as inactive precursors and can be cleaved to active form comprised of two associated heterodimers. They are activated or inactivated through a series of intracellular steps, or pathways, in response to death or survival signals, which are subject to multiple regulations. Clearly, the discovery of diverse apoptosis pathways involving signals primarily via the death receptors (extrinsic pathway) or the mitochondria (intrinsic pathway) using caspases as effector molecules has dominated the field. Lately, a more sophisticated view of signaling pathways has arisen which leads to the observation that cell death can occur even in the absence of caspases. The death receptor pathway. The extrinsic pathway is initiated at the cell surface through the

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and caspase-8 are the key components of the Fas DISC. Once caspase-8 associates with FADD, the high local concentration of caspase-8 is believed to lead to its autoproteolytic cleavage to p43/41 and p20, and activation. Studies of knockout mice and mutant cell lines established that the DISC is essential for Fas apoptosis signaling. DISC assembly differs between cell types in ways that influence the efficiency of Fas signaling. In ‘type I’ cell lines and re-stimulated primary T cells, the DISC forms efficiently, and apoptosis can be induced with bivalent anti-Fas stimuli without additional cross-linking. The activated caspase-8 directly cleaves caspase-3 in its downstream. However, in ‘type II’ cell lines and recently activated primary T cells, the DISC is formed inefficiently, hypercrosslinking of Fas is necessary to induce apoptosis, and caspase-8 does not activate caspase-3 and cleaves Bid directly instead. The truncated Bid (tBid) translocates to mitochondria (Figure 1). TNF receptor type I (TNFR1) is another important member in the death receptor family. TNF is a highly pleiotropic cytokine that elicits diverse cellular responses ranging from proliferation and differentiation to activation of apoptosis. The different biological activities are mediated by two distinct cell surface receptors: TNFR1 and TNFR2. TNFR1 appears to be the key mediator of TNF signaling. Upon binding of its ligand, TNFR1 recruits the adaptor protein TRADD directly to its cytoplasmic DD. In turn, TRADD serves as an assembly platform to diverge TNFR1 signaling from the DD: interaction of

Fas/TNF-R1 family protein. Ligation of Fas either by its ligand, FasL, or by its agonistic antibodies triggers the homotrimeric association of the receptors. The clustering of the death domains (DDs) in the intracellular portion of the receptors recruits the adapter molecule, Fas-associated DD containing protein (FADD), which then recruits procaspase-8. Activation of procaspase-8 through self-cleavage leads to a series of downstream events, including activation of procaspase 3, cleavage of multiple caspase substrates, and induction of mitochondrial damages. Fas (CD95/APO-1) is the best-characterized member of the tumor necrosis factor (TNF) superfamily of receptors. Its main and best-known function in signaling is the induction of apoptosis. Fas receptors are expressed on the surface of cells as preassociated homotrimers. Fas is a prototype death receptor characterized by the presence of an 80 amino acid DD in its cytoplasm tail. This domain is essential for the recruitment of a number of signaling components upon activation by either agonistic anti-Fas antibodies or the cognate Fas ligand that initiate apoptosis. The complex of proteins that form upon triggering of Fas is called the death-inducing signaling complex (DISC). The DISC consists of an adaptor protein and initiator caspases and is essential for induction of apoptosis. A number of proteins have been reported to regulate formation or activity of the DISC. In the DISC, the adaptor molecule FADD is bound to Fas. FADD has been shown to interact with several proteins through its death effector domain (DED), including caspase-8. These two molecules, FADD

FasL Fas Membrane

}

FADD

Caspase-8 Activated caspase-8

DISC

Bid

Bax

tBid Activated-Bax Mitochondria

Cytochrome c Caspase-9 Caspase-3 Cell death Figure 1 A schematic diagram outlining the cell death pathways.

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TRADD with RIP-1 and TRAF-2 leads to nuclear factor kappa B (NF-kB) activation. Alternatively, TRADD can recruit FADD and procaspase-8, which is subsequently activated to initiate apoptosis. Under native conditions, a controversy has been reported about the formation of a TNFR1-associated DISC at the plasma membrane. It was reported that TNFR1-induced apoptosis involves two sequential signaling complexes. The initial plasma membrane bound complex (complex I) consists of TNFR1, the adaptor TRADD, the kinase RIP1, and TRAF2 and rapidly signals activation of NF-kB. In a second step, TRADD and RIP1 associate with FADD and caspase-8, forming a cytoplasmic complex (complex II) in human fibrosarcoma cells, HT1080. Recently, it has been reported that the DISC forms at the plasma membrane in several cell lines. We agree with this report in that the difference between the results of Micheau and co-workers and Harper and co-workers may be based on the different compositions of the lysis buffers used and the fact that different labeling and precipitation protocols are employed; we also discovered that the DISC formed at the plasma membrane in primary cultured hepatocytes (our unpublished results), which is consistent with the results of Schneider-Brachert and co-workers. The mitochondrial pathway. Many death stimuli do not seem to depend on the death receptor pathway. Instead, the death signals are transmitted to mitochondria through unique intracellular signaling pathways, where release of cytochrome c is induced. Cytochrome c activates Apaf-1, in the presence of dATP, which in turn activates procaspase-9. Activated caspase-9 can then cleave downstream effector caspases. Mitochondria apoptosis pathway is involved in many types of cell death induced by stress signals, such as irradiation, DNA-damaging drugs, hormone, or growth factor withdrawal. It has to be pointed out that cytochrome c release and caspase activation may not be the only effects caused by the various insults on mitochondria. Others include mitochondrial depolarization and free-radical generation. Death stimuli transmitted through the Fas/TNFR1 death receptor family are mainly mediated directly by caspase cascades in cytosol. However, in certain types of cells, such as hepatocytes, the effector caspases may not be efficiently activated by caspase-8 and thus the mitochondria pathway mediated by Bid becomes critical. As mentioned above, Bid is cleaved by caspase-8 and translocated to mitochondria to induce cytochrome c release. The mitochondria pathway is subject to regulation by Bcl-2 family proteins. This family of proteins consists of both death antagonists (Bcl-2, Bcl-XL, Bcl-W, Bfl-1, and Mcf-1) and death agonists (Bax, Bak, Bid,

Bim, Bnip3, Bnip3L (Nix), Bad, Bik, and Bok). They share structural homology in Bcl-2 homology (BH)1, 2, 3, and 4 domains, although not all members have all domains. The BH1 and BH2 domains of the antagonists are required to heterodimerize with the death agonists and repress cell death. Conversely, the BH3 domain of the death agonists is required for them to heterodimerize with the death antagonists and to promote cell death. Bcl-2 family proteins can regulate caspase activation through the regulation of cytochrome c release from mitochondria, which is inhibited by the death antagonists (Bcl-2 or Bcl-XL), and promoted by the death agonists (Bax or Bid). However, the exact mechanisms by which Bcl-2 proteins modulate apoptosis are still subject to much debate and controversy. One hypothesis is that both proapoptotic and antiapoptotic Bcl-2 proteins bind directly to components of the mitochondrial pore, leading to either its opening or closure, respectively. A second hypothesis is that upon activation, proapoptotic members such as Bax and Bak insert into the outer mitochondrial membrane where they oligomerize to form a protein-permeant pore of their own. Regulation of Bcl-2 proteins can occur at multiple levels. For example, Bid is cleaved by caspase-8 to form tBid, which then translocates to the mitochondrion and induces permeability transition, suggesting a linkage between the extrinsic and intrinsic pathways. In the context of signal transduction, phosphorylation also plays a critical role in regulating Bcl-2 proteins. In general, serine phosphorylation of Bad by multiple kinases causes its sequestration and hence inactivation by 14-3-3 proteins, and phosphorylation of Bim can target it for proteosomal degradation (13). The other apoptosis is the endoplasmic reticulum (ER)-involved pathway. As mentioned above, two major apoptotic cascades are triggered by specific initiator caspases: the death receptor pathway and the mitochondrial pathway. These are activated by caspase-8 and caspase-9, respectively. The key caspase in the ER pathway is caspase-12. Proper folding of polypeptide into a three-dimensional structure is essential for cellular function, and protein malfolding can threaten cell survival. Various conditions can perturb the protein folding in the ER, which is collectively called ER stress. In order to adapt ER stress conditions, the cells respond in three distinct ways such as transcriptional induction of ER chaperones, translational attenuation, and ER-associated degradation. After ER functions are severely impaired, the cell is eliminated by apoptosis via transcriptional induction of CHOP/GADD153, the activation of c-Jun NH2-terminal kinase, and/or the activation of caspase-12. This type of cellular stress is receiving increased attention because it is considered a cause of

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pathologically relevant apoptosis and is especially implicated in neurodegenerative disorders. ER stress activates caspase-12 on the surface of the ER, and caspase-12-deficient cells are resistant to ER stress inducers, indicating that caspase-12 is significant in ER stress-induced apoptosis. However, the mechanism responsible for caspase-12 activation is largely unknown, unlike in the case of both caspase-8 and caspase-9 whose activation mechanisms have been revealed at the molecular level.

Organelle Dysfunction Mitochondria. As the main energy source for all cellular processes, the proper function of the mitochondrion is important to the survival of the cell. The dysfunction of this organelle can usually be attributed to depolarization of the membrane, resulting in the loss of its property of selective mitochondrial membrane permeability (MMP), usually resulting in cell death. Many proapoptotic signals and antiapoptotic defenses converge in the mitochondrion. The congregation of proteins like Bax on the outer membrane cause alteration in the membrane permeability and compromise the integrity of the mitochondrial membrane, where MMP ensue. On the other hand, the mitochondrion can mount a defense through the antideath members of the Bcl-2 family, namely Bcl-2 and Bcl-XL. For example, these proteins usually reside at the mitochondrial membrane and can inhibit the function of Bax and consequently prevent the onset of MMP. It has been reported that caspase-8 resides in mitochondria. In the human breast carcinoma cell line MCF7, caspase-8 predominantly colocalizes with and binds to mitochondria. After induction of apoptosis through Fas or TNFRI, active caspase-8 translocate to plectin, a major cross-linking protein of the three main cytoplasmic filament systems, whereas the caspase-8 prodomain remain bound to mitochondria. Golgi apparatus. To date, there is no direct evidence suggesting that the Golgi apparatus is directly involved in the initiation of any apoptotic pathway. However, it has been reported that many apoptosissignaling proteins are enriched at the Golgi membrane. Among them are caspase-2, TNFR, Fas, and TNF-related apoptosis-inducing ligand receptor 1. Recently, we have found that the DISC (Fas/FADD/ caspase-8) forms first in Golgi complex, and then translocates to plasma membrane. The blockage of the DISC transport dramatically decreases the DISC level in plasma membrane. Plasma membrane. The plasma membrane is the first line of defense for the cell against extracellular

stimuli. With the right type of stimulus, the corresponding receptor will instigate a signal cascade that results in cell death. To date, three different types of death receptors have been identified: the TNFR, the Fas, and the APO-3. There was a report that Fas in type I lymphocytes associates with glycosphingolipid-enriched detergent-resistant membrane microdomains termed lipid rafts. Although there is a controversy if the DISC formation is involved in lipid raft, we have found that the DISC formation localizes in the lipid raft of lung endothelial cells exposed to hypoxia/reoxygenation. The lipid-based machinery may be involved in the formation of carriers trafficking from the Golgi complex to the cell surface.

Bcl-XL It is well known that Bcl-XL protects cells from apoptosis mediated by a variety of stimuli, but the mechanisms are not fully understood. Overexpression of Bcl-XL reportedly confers protection upon mitochondria, making it more difficult for numerous stimuli to induce permeability transition pore opening; or Bcl-XL tends to form small channels that assume a mostly closed conformation, preferring cations; Bcl-XL sequesters BH3 domain-only molecules in stable mitochondria complexes, preventing the activation of Bax. Bcl-XL was reported to block Bax translocation. Studies by others reported that Bcl-XL could not block activation of Fas by FasLinduced apoptosis, especially caspase-8 activation. A role for caspase-8 mediating Bid cleavage accompanied by cytochrome c release was observed in focal cerebral ischemia endothelial cells and focal cerebral ischemia in rats. It has been established that Bcl-XL blocks Bid cleavage and functions downstream of caspase-8 to inhibit Fas-induced apoptosis of MCF7 breast carcinoma cells. Recently, we have found that Bcl-XL inactivates caspase-8 by disrupting DISC formation in the plasma membrane, Golgi complex, and nucleus. Bcl-XL retains the DISC in mitochondria where caspase-8 is inactivated. Bcl-XL breaks the physical association of Fas and caspase-8 with GRASP65, a Golgi-apparatus-related protein. This indicates that Bcl-XL downregulates the transfer of DISC to the plasma membrane by the Golgi component, at the same time diverting the DISC formation to the mitochondria. FLIP (FLICE (Fas-associated death-domain-like IL-1beta-converting enzyme)-inhibitory protein)) was identified as an inhibitor of Fas signals. At least four splice variants have been identified (31–34). The largest variant FLIP long (FLIPL), a protein of 440 amino acids, is highly homologous to caspase-8. In fact, FLIPL contains a

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caspase-like domain at the carboxy-terminus but, as indicated by the name, was originally characterized as a molecule with inhibitory activity on caspase-8, in which FLIPL inhibits the final cleavage between the prodomain and the p20 subunit of the p43/41 intermediate. In addition to inhibition of receptor-mediated apoptosis, more recently, it has been reported that FLIP also promotes activation of NF-kB and Erk signaling pathway. Therefore, FLIP is not simply an inhibitor of death-receptor-induced apoptosis but also mediates the activation of NF-kB. The death effector domain (DED) of FLIP binds to Fas/FADD complexes and inhibits the recruitment and activation of procaspase-8 and therefore acts as antiapoptotic molecule. We have reported that, in addition to inhibiting the recruitment of caspase-8 into the DISC by decreasing DISC formation in the plasma membrane, FLIP blocks the transfer of the DISC formed in the Golgi to the plasma membrane. FLIP expression also inhibits Bax activation and Bax-induced apoptotic cell death by promoting the association of the inactive form of PKC to Bax, which inactivates Bax.

The Effect of Tyrosine Kinases on Apoptosis Tyrosine kinases are important mediators of the signaling cascade, determining key roles in diverse biological processes such as growth, differentiation, metabolism, and apoptosis in response to external and internal stimuli. Tyrosine kinases are a family of enzymes, which catalyze phosphorylation of select tyrosine residues in target proteins, using ATP. Tyrosine kinases are primarily classified as receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR) and c-Met, and nonreceptor tyrosine kinases, such as SRC, ABL, FAK, and Janus kinase. The receptor tyrosine kinases are not only cell surface transmembrane receptors, but also enzymes having kinase activity. The structural organization of the receptor tyrosine kinase exhibits a multidomain extracellular ligand for conveying ligand specificity, a single pass transmembrane hydrophobic helix, and a cytoplasmic portion containing a tyrosine kinase domain. The kinase domain has regulatory sequence both on the N- and C-terminal end. Nonreceptor tyrosine kinases are cytoplasmic proteins, exhibiting considerable structural variability. The nonreceptor tyrosine kinase has a kinase domain and often possesses several additional signaling or protein–protein interacting domains such as Src homology (SH)2, SH3, and the phosphotyrosine homology (PH)

domain. Generally, receptor tyrosine kinases activate many signaling pathways to inhibit apoptosis by regulating Bcl-2 family member protein expression and their phosphorylation. For example, receptor tyrosine kinases activate the serine/threonine kinase Akt, which inhibits apoptosis through the Bad phosphorylation. Here, we focus on recent novel findings that tyrosine kinases are involved in apoptotic regulation. The initiation of Fas-mediated apoptotic pathway is to stimulate Fas activities through Fas phosphorylation. The phosphorylation at the Fas tyrosine is thought to be prerequisite for Fas membrane trafficking and DISC formation in hepatocytes exposed to hyperosmolarity and Fas ligand in endothelial cells of the lung exposed to hypoxia/reoxygenation (our unpublished results). EGFR is reported to associate with Fas and induce Fas phosphorylation at the tyrosine site through EGFR tyrosine kinase activity. Hepatocyte growth factor (HGF) receptor, c-Met, can inhibit Fas-mediated apoptosis by physically binding to Fas, which may block the Fas conformation change required for trafficking and DISC formation in hepatocytes. HGF can upregulate the c-Met/Fas association through c-Met-signaling pathway in lung epithelial and endothelial cells (our unpublished results). HGF also inhibits Bax and Bid activation by activating p38 MAPK that induces Bax and Bid phosphorylation, which decreases their conformation change and activation. The Src kinase family comprises three ubiquitously expressed members (Src, Fyn, Yes), which share functional domains such as an amino-terminal myristoylation sequence for membrane targeting, SH2 and SH3 domains, a family member-specific unique region, and a kinase domain and a carboxy-terminal noncatalytic domain. Recent studies on vascular smooth muscle cells and endothelial cells indicated an involvement of c-Src, but not of Fyn, in the c-Jun NH2-terminal kinase (JNK) activation in response to oxidative stress. Also, oxidative stress-induced activation of Erk-5 in fibroblasts was shown to be Src-dependent, but not Fyn-dependent, whereas Fyn, but not Src, was required for activation of p90 ribosomal S6 kinase by reactive oxygen species. It has been reported that hydrophobic bile acids rapidly activate Yes but not Fyn, Src, or Lck. Activated Yes associates with the EGFR in a protein kinase A-sensitive way and acts as an EGFR-activating kinase, thereby triggering a decisive event in bile acid-induced apoptosis. Phosphorylation of the p53 tumor suppressor protein is a critical event in the upregulation and activation of p53 during cellular stress. It was demonstrated that the signaling pathway linking oxidative stress to p53 through PDGFb receptor. Hydrogen peroxide-induced phosphorylation of the

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PDGFb receptor and p53 activation was inhibited by kinase-inactive forms of the PDGFb receptor.

Concluding Remarks Apoptosis plays an important role in cell growth, differentiation, and homeostasis. It is obvious that our current knowledge in terms of the mechanism of apoptosis and its regulation is far from complete. For example, we do not have conclusive answers to questions such as: how does the DISC formation transport from Golgi complex to plasma membrane? What is the importance/significance of the DISC formed first in Golgi complex? How does FLIP affect the Bax/mitochondria apoptotic pathway? What is the mechanism of the dual function (antiapoptosis and proapoptosis) of tyrosine kinases? Much more effort is required to dissect and document these pathways. Studies of the apoptotic regulation will help understand the mechanisms of cell death or cancer development. We must better characterize the novel pathways of cell death and further our understanding of the pathologies underlying a variety of human health problems. See also: CD14. Cysteine Proteases, Cathepsins. DNA: Repair. Extracellular Matrix: Collagens. Hepatocyte Growth (Scatter) Factor. Ion Transport: Overview. Myofibroblasts. NADPH and NADPH Oxidase. Oncogenes and Proto-Oncogenes: jun Oncogenes. Transcription Factors: AP-1; NF-kB and Ikb. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading Anto RJ, Mukhopadhyay A, Denning K, and Aggarwal BB (2002) Curcumin (diferuloylmethane) induces apoptosis through activation of caspase-8, BID cleavage and cytochrome c release: its suppression by ectopic expression of Bcl-2 and Bcl-xl. Carcinogenesis 23: 143–150. Bin L, Li X, Xu LG, and Shu HB (2002) The short splice form of casperc-FLIP is a major cellular inhibitor of TRAIL-induced apoptosis. FEBS Letters 510: 37–40. Cao XX, Mohuiddin I, Chada S, et al. (2002) Adenoviral transfer of mda-7 leads to BAX up-regulation and apoptosis in mesothelioma cells, and is abrogated by over-expression of BCL-XL. Molecular Medicine 8: 869–876. Chen K, Albano A, Ho A, and Keaney JF Jr (2003) Activation of p53 by oxidative stress involves platelet-derived growth factorbeta receptor-mediated ataxia telangiectasia mutated (ATM) kinase activation. Journal of Biological Chemistry 278: 39527– 39533. Cheng EH, Wei MC, Weiler S, et al. (2001) BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAXand BAK-mediated mitochondrial apoptosis. Molecular Cell 8: 705–711. Creagh EM, Conroy H, and Martin SJ (2003) Caspase-activation pathways in apoptosis and immunity. Immunological Reviews 193: 10–21.

Eramo A, Sargiacomo M, Ricci-Vitiani L, et al. (2004) CD95 death-inducing signaling complex formation and internalization occur in lipid rafts of type I and type II cells. European Journal of Immunology 34: 1930–1940. Gniadecki R (2004) Depletion of membrane cholesterol causes ligand-independent activation of Fas and apoptosis. Biochemical and Biophysical Research Communications 320: 165–169. Harper N, Hughes M, MacFarlane M, and Cohen GM (2003) Fasassociated death domain protein and caspase-8 are not recruited to the tumor necrosis factor receptor 1 signaling complex during tumor necrosis factor-induced apoptosis. Journal of Biological Chemistry 278: 25534–25541. Hsu H, Shu HB, Pan MG, and Goeddel DV (1996) TRADDTRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84: 299–308. Huang DC, Hahne M, Schroeter M, et al. (1999) Activation of Fas by FasL induces apoptosis by a mechanism that cannot be blocked by Bcl-2 or Bcl-x (L). Proceedings of the National Academy of Sciences, USA 96: 14871–14876. Kataoka T, Budd RC, Holler N, et al. (2000) The caspase-8 inhibitor FLIP promotes activation of NF-kappaB and Erk signaling pathways. Current Biology 10: 640–648. Kerr JF, Wyllie AH, and Currie AR (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. British Journal of Cancer 26: 239–257. Kroemer G and Reed JC (2000) Mitochondrial control of cell death. Nature Medicine 6: 513–519. Krueger A, Schmitz I, Baumann S, Krammer PH, and Kirchhoff S (2001) Cellular FLICE-inhibitory protein splice variants inhibit different steps of caspase-8 activation at the CD95 death-inducing signaling complex. Journal of Biological Chemistry 276: 20633–20640. Liou AK, Clark RS, Henshall DC, Yin XM, and Chen J (2003) To die or not to die for neurons in ischemia, traumatic brain injury and epilepsy: a review on the stress-activated signaling pathways and apoptotic pathways. Progress in Neurobiology 69: 103–142. Los M, Wesselborg S, and Schulze-Osthoff K (1999) The role of caspases in development, immunity, and apoptotic signal transduction: lessons from knockout mice. Immunity 10: 629–639. Micheau O and Tschopp J (2003) Induction of TNF receptor Imediated apoptosis via two sequential signaling complexes. Cell 114: 181–190. Momoi T (2004) Caspases involved in ER stress-mediated cell death. Journal of Chemical Neuroanatomy 28: 101–105. Muppidi JR and Siegel RM (2004) Ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T cell death. Nature Immunology 5: 182–189. Paul MK and Mukhopadhyay AK (2004) Tyrosine kinase – role and significance in Cancer. International Journal of Medical Science 1: 101–115. Peter ME and Krammer PH (2003) The CD95(APO-1/Fas) DISC and beyond. Cell Death and Differentiation 10: 26–35. Plesnila N, Zinkel S, Amin-Hanjani S, Qiu J, Korsmeyer SJ, and Moskowitz MA (2002) Function of BID – a molecule of the bcl2 family – in ischemic cell death in the brain. European Surgical Research 34: 37–41. Reed JC (1998) The Bcl-XL family proteins. Oncogene 17: 3225– 3226. Reinehr R, Schliess F, and Haussinger D (2003) Hyperosmolarity and CD95L trigger CD95/EGF receptor association and tyrosine phosphorylation of CD95 as prerequisites for CD95 membrane trafficking and DISC formation. FASEB Journal 17: 731–733. Scaffidi C, Fulda S, Srinivasan A, et al. (1998) Two CD95 (APO-1/ Fas) signaling pathways. The EMBO Journal 17: 1675–1687.

140 AQUAPORINS Scaffidi C, Schmitz I, Krammer PH, and Peter ME (1999) The role of c-FLIP in modulation of CD95-induced apoptosis. Journal of Biological Chemistry 274: 1541–1548. Schneider-Brachert W, Tchikov V, Neumeyer J, et al. (2004) Compartmentalization of TNF receptor 1 signaling: internalized TNF receptosomes as death signaling vesicles. Immunity 21: 415–428. Srinivasan A, Li F, Wong A, et al. (1998) Bcl-xL functions downstream of caspase-8 to inhibit Fas- and tumor necrosis factor receptor 1-induced apoptosis of MCF7 breast carcinoma cells. Journal of Biological Chemistry 273: 4523–4529. Stegh AH, Barnhart BC, Volkland J, et al. (2002) Inactivation of caspase-8 on mitochondria of Bcl-xL-expressing MCF7-Fas cells: role for the bifunctional apoptosis regulator protein. Journal of Biological Chemistry 277: 4351–4360. Stegh AH, Herrmann H, Lampel S, et al. (2000) Identification of the cytolinker plectin as a major early in vivo substrate for caspase 8 during CD95- and tumor necrosis factor receptor-mediated apoptosis. Molecular and Cellular Biology 20: 5665–5679. Szegezdi E, Fitzgerald U, and Samali A (2003) Caspase-12 and ERstress-mediated apoptosis: the story so far. Annals of the New York Academy of Sciences 1010: 186–194. Tschopp J, Irmler M, and Thome M (1998) Inhibition of fas death signals by FLIPs. Current Opinion in Immunology 10: 552–558. Wajant H, Pfizenmaier K, and Scheurich P (2003) Tumor necrosis factor signaling. Cell Death and Differentiation 10: 45–65.

Wang X, DeFrances MC, Dai Y, et al. (2002) A mechanism of cell survival: sequestration of Fas by the HGF receptor Met. Molecular Cell 9: 411–421. Wang X, Wang Y, Zhang J, et al. (2005) FLIP protects against hypoxia/reoxygenation-induced endothelial cell apoptosis by inhibiting Bax activation. Molecular and Cellular Biology 25: 4742–4751. Wang X, Zhang J, Kim HP, et al. (2004) Bcl-XL disrupts deathinducing signal complex formation in plasma membrane induced by hypoxia/reoxygenation. FASEB Journal 18: 1826–1833. Wang X, Zhou Y, Kim HP, et al. (2004) Hepatocyte growth factor protects against hypoxia/reoxygenation-induced apoptosis in endothelial cells. Journal of Biological Chemistry 279: 5237– 5243. Xiao C, Yang BF, Asadi N, Beguinot F, and Hao C (2002) Tumor necrosis factor-related apoptosis-inducing ligand-induced deathinducing signaling complex and its modulation by c-FLIP and PEDPEA-15 in glioma cells. Journal of Biological Chemistry 277: 25020–25025. Yin XM (2000) Signal transduction mediated by Bid, a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways. Cell Research 10: 161–167. Yin XM and Ding WX (2003) Death receptor activation-induced hepatocyte apoptosis and liver injury. Current Molecular Medicine 3: 491–508.

AQUAPORINS A S Verkman and Y Song, University of California, San Francisco, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Aquaporins (AQP) are a family of water transporting proteins expressed in many epithelial, endothelial, and other tissues. AQP1 is expressed in microvascular endothelia throughout the lung/airways, AQP3 in basal cells in large airways, AQP4 at the basolateral membrane of epithelia throughout the airways, and AQP5 at the apical membrane of type I alveolar epithelial cells and submucosal gland acinar cells. The expression of some of these aquaporins increases near the time of birth and appears to be regulated by growth factors, inflammation, and osmotic stress, suggesting a role of aquaporins in lung physiology. However, studies in transgenic mouse models of AQP deficiency have provided evidence against an important physiological role for aquaporins in many lung functions. Although AQP1 and AQP5 provide the principal route for osmotically driven alveolar water transport, alveolar fluid clearance in the neonatal and adult lung is not affected by AQP deletion, nor is lung CO2 transport or fluid accumulation in experimental models of lung injury. In the airways, AQP3 and AQP4 facilitate water transport; however, airway hydration, airway surface liquid layer volume and composition, and airway fluid absorption are not impaired by AQP3/AQP4 deletion. In airway submucosal glands, AQP5 deletion significantly reduced the rate and increased the protein content of fluid secretions. Thus, although AQPs have many important extrapulmonary physiological functions, in the lung/ airways AQPs appear to be important mainly in airway submucosal gland function. The substantially slower rates of

fluid transport in airways, pleura, and lung compared to renal and some secretory epithelia may account for the apparent lack of functional significance of AQPs at these sites. However, the possibility remains that AQPs may play a role in lung physiology under conditions of stress/injury not yet tested or in functions unrelated to transepithelial fluid transport.

Description Fluid movement between the distal airspace and interstitial and vascular compartments of the lung/airways is important in the maintenance of airspace hydration, the absorption of airspace fluid near the time of birth and in pulmonary edema, and the secretion of fluid onto the airway surface by submucosal glands. Aquaporins (AQPs), a family of small (B30 kDa monomer) integral membrane water channel proteins, are expressed in many cell types involved in fluid transport. There are more than 10 aquaporins in mammals, at least four of which are expressed in the respiratory system (Figure 1). AQP1 is expressed in microvascular endothelia near airways and alveoli, as well as in microvessels and mesothelial cells of visceral and parietal pleura. AQP3 is expressed in the basolateral membrane of basal epithelial cells lining the trachea and large airways, and at the basal membrane in human (but not rodent) alveoli. AQP4 is expressed in the basolateral membrane

AQUAPORINS 141 AQP3 AQP4 AQP5 Nasopharnyx (upper airways) Submucosal gland

Airway surface liquid

Microvessels

AQP1 Trachea

AQP4 AQP3

Pleura Lung Distal airway

AQP1 Alveolus

Type I

Microvessels

Type II

AQP5

AQP1

Figure 1 Aquaporin expression in lung and airways. AQP1 is expressed in microvascular endothelial and pleural membranes; AQP3 in large airway epithelia; AQP4 at the basolateral membrane in epithelia throughout the airways; and AQP5 at the apical membrane of alveolar type I cells and of serous acinar cells in submucosal glands.

High osmolality

Low osmolality

Water

Figure 2 Tetrameric structure of AQPs. Each AQP tetramer consists of four B30 kDa monomers, each of which contains a narrow water-transporting pore. Water moves across AQP pores in response to osmotic gradients.

of ciliated columnar cells in bronchial, tracheal, and nasopharyngeal epithelia. AQP5 is expressed in the apical membrane of type I alveolar epithelial cells and of acinar cells in submucosal glands. AQP5 has also been reported in human lung to be expressed in bronchial and nasopharyngeal acinar and ciliated duct columnar cells. This expression pattern in fluid

transporting cells provides indirect evidence for a role of aquaporins in lung/airway physiology. AQPs 1, 4, and 5 are water selective, and consist of tetramers of four monomeric B30 kDa subunits, each of which contains an independent narrow pore pathway for water transport (Figure 2), whereas AQP3 (an ‘aquaglyceroporin’) transports both water and glycerol.

142 AQUAPORINS

Normal Physiological Processes Developmental Regulation of Aquaporin Expression

Rodent studies have shown developmental regulation of lung aquaporin expression with distinct patterns for each aquaporin. AQP1 is detectable just before birth in rodents, increasing several-fold perinatally and into adulthood. Functional measurements in rabbit showed significantly increased lung water permeability in the perinatal period in parallel to increasing AQP1 expression. AQP1 expression is also upregulated by treatment with corticosteroids. In contrast, little AQP5 is expressed at birth and gradually increases until adulthood, whereas AQP4 expression strongly increases just after birth and is upregulated by -agonists and glucocorticoids. Regulation of Aquaporin Expression in Adult Lung

Regulated aquaporin expression is also seen in the adult lung. As in prenatal lung, AQP1 expression can be upregulated by corticosteroids. AQP1 and AQP5 expression are reduced in rodent lung following adenoviral infection, and AQP5 expression is increased after bleomycin exposure. Reduced AQP5 expression was found after exposure of a mouse lung epithelial cell line (MLE-12) to tumor necrosis factor alpha (TNF-a), suggesting a possible mechanism for its downregulation in viral infection in vivo. AQP5 expression was increased in MLE-15 and alveolar epithelial cells exposed to hypertonicity. Although potentially interesting, the physiological relevance of many of these observations is unclear, as the airway/ lung is probably not exposed to significant hypertonicity, and regulated AQP expression is a general phenomenon not specific to the lung/airways. Role of Aquaporins from Functional Studies in Knockout Mice

Transgenic mice lacking each of the lung aquaporins (AQP1, AQP3, AQP4, and AQP5) were generated in our laboratory both individually and in combinations. Comparative studies were done in wild-type and AQPdeficient mice to investigate the physiological role of aquaporins in the lung/airways. These mice have been quite informative in elucidating important roles of various aquaporins outside the lung. For example, mice lacking AQPs 1–4 are defective in their ability to concentrate urine, mice lacking AQP1 have impaired angiogenesis and cell migration, mice lacking AQP3 have reduced epidermal hydration, mice lacking AQP4 have altered brain water balance and impaired neural signal transduction, mice lacking AQP5 have impaired saliva secretion, and mice lacking AQP7 manifest progressive fat accumulation and adipocyte hypertrophy.

Fluid Transport in Distal Lung

The proposed aquaporin functions in distal lung include alveolar fluid absorption at the time of birth and in the adult lung, gas (CO2, O2) exchange, and formation/resolution of lung edema in response to acute and subacute lung injury. AQP5 is expressed in type I alveolar epithelial cells and AQP1 in microvascular endothelial cells. Osmotic water permeability between alveolus and capillary was B10-fold reduced in lungs of AQP1 and AQP5 null mice compared to wild-type mice, and 30-fold reduced by AQP1/AQP5 codeletion (in double knockout mice). Interestingly, AQP1 deletion mildly reduced hydrostatic lung edema in an isolated perfused lung preparation, and computerized tomographic analysis of two humans lacking AQP1 showed a blunted increase in airway wall thickness following saline infusion compared to control subjects. Reduced hydrostatic lung edema in AQP1 deficiency may be related to an abnormality in microvasculature deficiency, since on theoretical grounds hydrostatic driving forces should be unable to produce significant net fluid movement across a water-only pathway. Alveolar fluid clearance is an important function of the alveolar epithelium. Fluid absorption is the result of sodium absorption through the epithelial sodium channel (ENaC) in response to the electrochemical driving force created by the basolateral membrane Na-K-ATPase of type II alveolar epithelial cells. The consequent osmotic imbalance drives water absorption primarily through the type I cells. Alveolar fluid clearance is measured in fluid-filled lung models from the kinetics of increasing concentration of an airspace volume marker such as radiolabeled albumin. Remarkably, even with maximal stimulation of alveolar fluid absorption with betaagonists and pretreatment with keratinocyte growth factor (to increase the number of type II cells), AQP1 or AQP5 deletion did not reduce alveolar fluid clearance. Further, the rapid absorption of fluid from the airspace just after birth was not impaired by aquaporin deletion, nor was lung edema following acidinduced epithelial cell injury, thiourea-induced endothelial cell injury, or hyperoxic subacute lung injury. The much slower rate of maximal alveolar fluid absorption (0.016 ml min 1cm 2) compared to fluid absorption in kidney proximal tubule or saliva secretion in salivary gland (410 ml min 1 cm 2) may account for the lack of effect of AQP1 and AQP5 deletion on alveolar fluid clearance. Because rates of fluid transport are relatively low in lung, the low intrinsic (aquaporin independent) water permeability of the alveolar epithelial and capillary membranes appears to be adequate to allow fluid

AQUAPORINS 143

transport to occur without impairment under normal physiological conditions and in response to clinically relevant stresses. Fluid Transport in the Pleura

Fluid is continuously secreted into and reabsorbed from the pleural space. Little fluid is present in the pleural space (0.2 ml kg 1) despite its large surface area (4000 cm2 in man, 10 cm2 in mouse). Fluid entry into the pleural space involves filtration across microvascular endothelia near the pleural surface, and movement across a mesothelial barrier lining the pleural space, whereas fluid clearance is thought to occur primarily by lymphatic drainage. Pleural fluid can accumulate in pathological conditions such as congestive heart failure, lung infection, lung tumor, and the acute respiratory distress syndrome. AQP1 is expressed in microvascular endothelia near the visceral and parietal pleura and in mesothelial cells in visceral pleura. Osmotic water permeability across the pleural barrier, measured from the kinetics of pleural fluid osmolality changes after instillation of hypertonic or hypotonic fluid into the pleural space, was rapid in wild-type mice (50% osmotic equilibration in 2 min), and slowed by fourfold in AQP1 knockout mice. However, the clearance of saline instilled in the pleural space was not affected by AQP1 deletion, nor was the accumulation of pleural fluid in a fluid overload model produced by intraperitoneal saline administration or in a thiourea model of acute endothelial injury. Thus, although rapid osmotic equilibration across the pleural surface is facilitated by AQP1, as found in distal lung, AQP1 does not appear to play a major role in physiologically important mechanisms of pleural fluid accumulation or clearance. Fluid Transport in the Airways

Potential functions of aquaporins in the airways include humidification of inspired air, regulation of airway surface liquid (ASL) volume and composition, and absorption of fluid from the airways. Evaporative water loss in the airways is thought to drive water influx from capillaries and interstitium into the ASL by the generation of an osmotic gradient. The depth and ionic composition of the ASL should depend theoretically on the ion transporting properties of the airway epithelium and the rate of evaporative water loss, as well as the water permeability of the airway-capillary barrier. Osmotic water permeability in upper airways, measured by dilution of an airway volume marker in response to an osmotic gradient, was reduced in mice lacking AQP3 and/or AQP4. However, there was little effect of AQP3/AQP4 deletion on humidification of lower airways, as measured from the moisture content of expired air during mechanical ventilation with dry

air through a tracheotomy, or of upper airways, as measured from the moisture content of dry air passed through the upper airways in mice breathing through a tracheotomy. Also, the depth and salt concentration of the ASL in the trachea, as measured in vivo using fluorescent probes and confocal microscopy, was not altered by AQP3/AQP4 deficiency. Finally, active isosmolar fluid absorption, measured in nasopharyngeal airways (using a volume marker as done for alveolar fluid clearance) was not impaired by aquaporin deletion. Thus, although AQP3/AQP4 facilitate osmotic water transport in the airways, they play at most a minor role in airway humidification, ASL hydration, and isosmolar fluid absorption. Interestingly, one study showed increased airway reactivity in response to bronchoconstricting agents in AQP5 null mice. The mechanism of this phenotype was not established, but may be related to indirect effects of AQP5 deletion on agonist-induced fluid secretion from submucosal glands as described below. Fluid Secretion by Airway Submucosal Glands

Submucosal glands in mammalian airways secrete a mixture of water, ions, and macromolecules onto the airway surface. Glandular secretions are important in establishing ASL fluid composition and volume, and in antimicrobial defense mechanisms. Abnormally viscous gland secretions in cystic fibrosis have been proposed to promote bacterial adhesion and inhibit bacterial clearance. Submucosal glands contain serous tubules, where active salt secretion into the gland lumen creates a small osmotic gradient driving water transport across a water-permeable epithelium, as well as mucous cells and tubules, where viscous glycoproteins are secreted. AQP5 is expressed at the luminal membrane of the serous epithelial cells. Pilocarpinestimulated fluid secretion was found to be reduced by twofold in AQP5 null mice, as determined by nasopharyngeal fluid collections and video imaging of fluid droplets (covered with mineral oil) secreted by individual submucosal glands. Analysis of secreted fluid showed a twofold increase of total protein concentration in AQP5 null mice, suggesting intact protein and salt secretion across a relatively water-impermeable epithelial barrier. There was no significant difference of submucosal gland morphology or density in wildtype versus AQP5 knockout mice. AQP5 thus facilitates fluid secretion in submucosal glands, indicating that the luminal membrane of serous epithelial cells is the rate-limiting barrier to water movement. CO2 Transport by Aquaporins in Lung

AQP1-dependent CO2 transport has been proposed based on measurements in AQP1-overexpressing

144 ARTERIAL BLOOD GASES

Xenopus oocytes; however, subsequent studies showed unimpaired CO2 permeability in AQP1-deficient erythrocytes, where rapid CO2 transport occurs by a passive membrane solubility-diffusion mechanism. Measurements of CO2 movement in the perfused and in vivo mouse lung showed no effect of AQP1 deletion, providing direct evidence against the role of AQP1 in lung CO2 transport.

Aquaporins and Respiratory Disease A small number of AQP1-deficient humans have been identified. Although initially reported to have no phenotype, subsequent studies showed that they manifest a urine concentrating defect that is qualitatively similar to that found in AQP1-deficient mice. As mentioned above, AQP1-deficient subjects have also been found to have a small reduction in the increase in bronchiolar wall thickness following intravenous volume overload compared to normal controls, though other lung phenotypes have not been reported. The significance of this observation is unclear. Together with a considerable body of data in transgenic mice, the functional studies suggest that aquaporins play at most a minor role in normal lung physiology and clinically relevant states of lung injury, with the possible exception of AQP5 in submucosal fluid secretion. It remains unresolved why aquaporins are expressed at multiple sites of fluid movement in the lung/airways, and why the expression of lung aquaporins appears to be altered in states of stress. When available, nontoxic aquaporin-selective inhibitors will be useful to examine effects of acute aquaporin inhibition, which may reveal lung/ airway phenotypes that might not be manifest in mice or humans with chronic aquaporin deficiency. If significant aquaporin-dependent phenotypes are found, then pharmacological modulation of aquaporin function may have clinical applications, such as AQP5 inhibition in reducing glandular fluid secretions in allergic and infectious rhinitis, or increasing AQP5

expression/function to reduce secretion/ASL viscosity in cystic fibrosis. See also: Basal Cells. Fluid Balance in the Lung.

Further Reading Agre P, King LS, Yasui M, et al. (2002) Aquaporin water channels from atomic structure to clinical medicine. Journal of Physiology 542: 3–16. Bai C, Fukuda N, Song Y, et al. (1999) Lung fluid transport in aquaporin-1 and aquaporin-4 knockout mice. Journal of Clinical Investigation 103: 555–561. Borok Z and Verkman AS (2002) Lung edema clearance: 20 years of progress. Invited review: role of aquaporin water channels in fluid transport in lung and airways. Journal of Applied Physiology 93: 2199–2206. Dobbs L, Gonzalez R, Matthay MA, et al. (1998) Highly waterpermeable type I alveolar epithelial cells confer high water permeability between the airspace and vasculature in rat lung. Proceedings of the National Academy of Sciences, USA 95: 2991–2996. Folkesson H, Matthay MA, Frigeri A, and Verkman AS (1996) High transepithelial water permeability in microperfused distal airways: evidence for channel-mediated water transport. Journal of Clinical Investigation 97: 664–671. King LS, Nielsen S, and Agre P (1996) Aquaporin-1 water channel protein in lung-ontogeny, steroid-induced expression, and distribution in rat. Journal of Clinical Investigation 97: 2183– 2191. King LS, Nielsen S, Agre P, and Brown RH (2002) Decreased pulmonary vascular permeability in aquaporin-1-null humans. Proceedings of the National Academy of Sciences USA 99: 1059–1063. Krane CM, Fortner CN, Hand AR, et al. (2001) Aquaporin-5 deficient mouse lungs are hyperresponsive to cholinergic stimulation. Proceedings of the National Academy of Sciences, USA 98: 14114–14119. Ma T, Fukuda N, Song Y, Matthay MA, and Verkman AS (2000) Lung fluid transport in aquaporin-5 knockout mice. Journal of Clinical Investigation 105: 93–100. Saadoun S, Papadopoulos M, Hara-Chikuma M, and Verkman AS (2005) Targeted AQP1 gene deletion impairs angiogenesis and cell migration. Nature 434: 786–792. Song Y and Verkman AS (2001) Aquaporin-5 dependent fluid secretion in airway submucosal glands. Journal of Biological Chemistry 276: 41288–41292. Verkman AS (2002) Physiological importance of aquaporin water channels. Annals of Medicine 34: 192–200.

ARTERIAL BLOOD GASES J W Severinghaus, UCSF, San Francisco, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Blood gas analyzers consist of three electrodes measuring pH, PCO2 , and PO2 at 371C. They were introduced in about 1960

following inventions by R Stow (CO2) and L Clark (PO2) both dating from 1954. From these outputs, internal computers calculate O2 saturation, base excess, bicarbonate, and other derived variables such as the compensation by the body for acid–base abnormalities. Arterial PO2 and PCO2 can be approximated using heated skin surface ‘transcutaneous’ electrodes, which are commonly used in premature infants and nurseries. Hemoglobin oxygen saturation, SO2%, is also directly measured by

ARTERIAL BLOOD GASES 145 multiwavelength blood oximeters. Arterial SO2 is approximated by pulse oximeters, which detect the arterial pulsatile variations in red and infrared light penetrating a finger, ear, or other tissue, a method invented by T Aoyagi in Tokyo in 1973 that became commercially available in 1983. Interpretation of blood gases and acid–base balance is briefly discussed. Figures include schema of the three electrodes, a pulse oximeter probe, an acid–base compensation diagram, and photographs of the first three-function blood gas analyzer, a combined PO2 PCO2 transcutaneous electrode in use on a child, and a pulse oximeter probe on a finger.

Henderson–Hasselbalch (HH) equation: pH ¼ pK0 þ log½HCO 3 =SPCO2 In plasma, pK0 ¼ 6.1, the effective dissociation constant of H2CO3 (carbonic acid), calculated as S PCO2 . S is CO2 solubility, 0.031 mM l 1 Torr 1. HCO3 is plasma bicarbonate content calculated as plasma [CO2 content – SPCO2 ].

Introduction

Electrodes

Arterial blood gas analyzers directly measure pH, PCO2 and PO2 , (mmHg or Torr) and calculate standard base excess (SBE), bicarbonate (HCO3 ), oxygen saturation (SO2), and other variables useful in diagnosis and clinical management of patients in emergencies, anesthesia, surgery, recovery, and intensive care. These tests were rarely done until the 1950s when electrodes were invented and developed. Understanding of acid–base and blood gas theory depended on discoveries in physical chemistry.

pH Electrode

Ionic Theory

Electrochemistry and physical chemistry of solutions of acids, alkalis, metals, and salts were transformed from empiricism to theory by the 1884 thesis of Svante Arrhenius in Uppsala, Sweden. Wilhelm Ostwald then used a platinum electrode to measure hydrogen ion strength electrically. In 1893, Ostwald’s student Walther Nernst applied the longestablished laws of gases to ions in solution to calculate the electrical potential of batteries or cells. Buffers

Shortly after 1900, Lawrence J Henderson at Harvard adapted the mass action law to relate [H þ ] to PCO2 and HCO3 :

In 1905, Max Cremer noted that hydrogen ions permeated some kinds of very thin glass, developing electrical potential gradients across the glass. Fritz Haber and Z Klemensiewicz made the first glass pH electrode in 1909. Its potential was a linear function of pH, not H þ ion concentration. In 1925, the first glass cup-shaped blood pH electrode was produced by Phyllis T Courage. By 1933, capillary blood pH electrodes were being made commercially. Blood pH was corrected to 371C with the Rosenthal factor ( 0.0147 1C 1) until thermostatted blood pH electrodes became available in the 1950s. A pH and reference electrode is schematically shown in Figure 1. Details Special glass compositions permit hydrogen ions to diffuse through imperfectly annealed submicroscopic cracks, probably exchanging loci with loosely bound alkali cations, especially lithium. Some pH electrodes are sensitive to very high Na þ concentrations. At 371C, the electromotive force (EMF) across the glass measured with reference electrodes (usually silver–silver chloride) is 61.5 mV per pH unit change (a 10-fold change in H þ ion concentration). Sample

K ¼ ½Hþ ½HCO 3 =H2 CO3 Glass body

He demonstrated how respiration could buffer metabolic acids by reducing PCO2 . The kidneys can also change blood and extracellular fluid buffer base to partially normalize pH in respiratory acidosis or alkalosis.

AgCl internal reference H+ permeable glass Liquid junction Saturated KCl

Origin of pH

In order to define H þ ion concentrations such as 0.000 000 04 moles liter 1 (e.g., in blood) more elegantly, in 1907, S P L Sørensen suggested defining pH as the negative log of hydrogen ion concentration (or activity). In 1915, K A Hasselbalch converted Henderson’s equation to log form, later dubbed the

AgCl reference

Sample Figure 1 Schema of a blood pH electrode with a liquid junction to a reference electrode in a thermostatted cuvette.

146 ARTERIAL BLOOD GASES

For use in blood, the reference electrode usually contacts blood via a liquid junction containing saturated KCl. Rapid diffusion of K þ into red cells causes an often-ignored error of 0.01 pH at the liquid junction when calibration is done with aqueous buffers. PCO2 Measurement

Until the worldwide polio epidemics of 1950–52, PCO2 was measured by the HH equation. This required measuring plasma CO2 content by acidification and extraction in a manometric Van Slyke apparatus and measuring pH and correcting it to 371C. In Copenhagen’s communicable disease hospital in 1950–51, sometimes up to 100 patients at a time were manually ventilated by volunteers using a bag and mask with O2. The laboratory director Poul Astrup, needing a faster analytic method than the HH equation, devised a new simple method. He measured pH before and after equilibrating the sample with known PCO2 . He then computed patient PCO2 by extrapolation. His method became the standard for the rest of the decade. PCO2 Electrode

At Ohio State University in 1954, while also trying to resolve the polio problem, Richard Stow invented a PCO2 electrode. He covered a glass bulb-shaped pH electrode with a rubber glove (through which CO2 can diffuse but H þ cannot), over a film of distilled water. However, Stow’s electrode drifted because various cations in pH glass altered the distilled water pH. This electrode was stabilized by John Severinghaus at the National Institute of Health (NIH) by adding bicarbonate and salt, and was manufactured by many firms from 1959 onwards. A blood PCO2 electrode is illustrated in Figure 2.

Details An internal, nearly flat, glass pH electrode is separated from the sample by a membrane permeable to CO2 but not ions (e.g., Teflon or silastic). Under the membrane, a spacer (e.g., lens-cleaning tissue paper) holds a film of electrolyte containing about 10 mM NaHCO3 and usually 0.1 M salt or KCl. Thermostatted to 37oC, the output signal is a log function of PCO2 , about 30 mV per decade in distilled water (Stow’s design), doubling to 61 mV per decade with bicarbonate electrolyte. Oxygen Electrode

In 1950, Leland Clark at Antioch College, Ohio used perfused isolated liver to study steroid metabolism. He needed oxygenated blood so he built a bubble oxygenator and discovered how to defoam the blood using silicone oil on glass wool. The journal Science rejected his paper on the basis that he hadn’t measured the PO2 in the oxygenated blood; to this end, Clark invented a polarographic oxygen electrode. He covered a platinum disc cathode sealed in glass with cellophane to keep blood protein from poisoning the cathode. It served his purpose but was not accurate, requiring very high flow past the sensor due to the depletion of oxygen at the membrane surface due to its consumption by the cathode. In October 1954, a sudden inspiration led Clark to substitute a less O2-permeable and electrically insulating polyethylene membrane for cellophane, by mounting a reference electrode and cathode in electrolyte in a sealed probe (Figure 3). His invention was presented at a meeting of the FASEB in April 1956. Details In a polarographic oxygen electrode, a negatively biased platinum cathode donates electrons to

Stow–Severinghaus PCO2 electrode Sample

AgCl external reference electrode AgCl internal reference H+ permeable glass 0.1M KCl + 0.01M KHCO3

Glass body

Teflon or silastic

membrane

Electrolyte in paper spacer Sample Figure 2 Schema of a blood PCO2 electrode with Teflon CO2 permeable membrane and spacer to contain electrolyte for pH measurement.

ARTERIAL BLOOD GASES 147 Clark-type oxygen electrode 25 µm polypropylene membrane

Sample

0.1 m KCl electrolyte 10 µm Platinum wire

Solid glass AgCl reference Sample Figure 3 Schema of Clark’s polarographic PO2 electrode, after cathode size was greatly reduced to avoid ‘stirring’ effect.

dissolved oxygen: O2 þ 4e þ 2H2 O ) 4OH The only major functional change since Clark’s original invention has been to reduce the cathode diameter from 2 mm to about 10 mm, requiring far more sensitive current analysis, which was not available 50 years ago. This almost eliminated the need for the sample to be rapidly stirred. The electrolyte usually contains KCl, and may have added agents for viscosity. No separator is needed between cathode and membrane. The cathode is biased to about 0.65 V at which all oxygen molecules reaching the cathode are reduced. Cathode current is a linear function of the membrane surface PO2 .

Figure 4 The first blood gas analyzer containing three electrodes in a water bath at 371C with tonometer for preparing blood for calibration of PO2 electrode. Reproduced from Severinghaus JW (2002) The invention and development of blood gas apparatus. Anaesthesiology 97: 253–256, with permission from Lippincott Williams & Wilkins.

Blood Gas Analyzers

In 1958, Severinghaus and Bradley created the first three-function blood gas analyzer by mounting a Clark PO2 electrode with a tiny stirring paddle, a Stow–Severinghaus PCO2 electrode, and a commercial pH electrode in a water bath at 371C (Figure 4). A small tonometer was included in which blood could be equilibrated with air or a known gas to calibrate the Clark electrode. Modern blood gas analyzers compute many variables from the three measured values. Transcutaneous PO2

PaO2 can be estimated transcutaneously using a flat Clark type PO2 electrode, typically internally heated to about 431C. Heating causes sufficient dermal vasodilation to raise skin capillary PO2 to nearly equal arterial PO2 . Heating also raises blood PO2 by about 7% per degree, while skin oxygen consumption reduces surface PO2 , these two factors approximately canceling each other out. No correction factors for

Figure 5 Transcutaneous combined PO2 and PCO2 electrode monitoring a patient recovering from anesthesia.

temperature or skin metabolism are thus needed. Introduced in the mid-1970s, these devices are widely used on premature and term infants to help control oxygen therapy and prevent blindness following retinal vascular growth interference (Figure 5). Transcutaneous PCO2

Arterial PCO2 can also be estimated transcutaneously using flat CO2 electrodes heated (e.g., to 431C) to increase skin capillary blood flow. The signal must be corrected 4.7% per degree to 371C, and reduced about 4 Torr to compensate for skin metabolism and electrode surface cooling.

148 ARTERIAL BLOOD GASES Hemoglobin Oxygen Saturation

Prior to about 1970, this was measured by vacuum extraction of oxygen from blood, before and after equilibrating a sample with air. Multiwavelength Oximetry

Compared with oxygenated blood, desaturated blood strongly absorbs red light (at about 660 nM wavelength). At the isobestic point, 805 nM (near infrared), absorption is unaffected by oxygenation. Multiwavelength oximeters use the ratio of optical density of a thin film of hemolyzed blood at red and infrared wavelengths to calculate saturation and hemoglobin concentration, with small corrections for other pigments such as bilirubin, detected at other wavelengths. A typical laboratory ‘bench’ oximeter using at least five wavelengths can be precise to at least 0.1% saturation. Some oximeters use filters to select wavelengths while others use more stable and precisely defined diffusion-grating monochromators, avoiding the need for user calibration. Confirmatory testing with dyes is recommended. Some blood gas analyzers include a multiwavelength oximeter (often termed CO-oximeter because it also measures the fraction of hemoglobin bound to carbon monoxide). Pulse Oximetry

Light passing through a finger or ear is partly absorbed by the blood in its path. Arteries expand with each pulse, absorbing a bit more light. Pulse oximeters measure the amplitude of the pulsatile variation of light as a fraction of total transmitted red (e.g., 660 nM) and infrared (e.g., 900–950 nM) light (Figures 6 and 7). The ratio of these two ratios was shown by T Aoyagi in 1973 (Nihon Kohden Co, Tokyo) to be a unique function of arterial oxygen saturation, theoretically independent of venous or capillary saturation, or skin color, tissue thickness, or other pigments. In the late 1940s, Earl Wood (Mayo Clinic) modified G Millikan’s 1942 original ear oximeter by adding a pneumatic pressure capsule. Wood showed that when blood was readmitted to a pressure-blanched ear, the ratios of the decreases in red and infrared light passing through the ear were unique functions of oxygen saturation.

Indications Blood gas analysis has become so commonplace that its use is nearly universal in diagnosis during admission, in emergencies, trauma, intensive care, anesthesia, and surgery. It is considered by physicians to

Figure 6 Pulse oximeter probe taped on fingertip with red and infrared LEDs on nail side and a photo diode on the dorsal side.

Photodiode

Finger

Red and infrared LEDs

Figure 7 Schema of pulse oximeter probe on a finger.

be the most useful and important diagnostic procedure available. Its indications are global and will not be listed here.

Common Patterns of Results and Interpretation Diagnostic Terminology

A pH of less than 7.35 is called acidemia, while that over 7.45 is termed alkalemia. A PCO2 over 45 Torr indicates respiratory acidosis or hypercapnia, while values under 35 (males) or 30 (females) indicate respiratory alkalosis or hypocapnia. A standard base excess (SBE) more negative than 5 mM is metabolic acidosis and over þ 5 mM is metabolic alkalosis. Presence of compensatory responses to chronic acid– base respiratory or metabolic imbalances can be predicted and used in diagnosis (Figure 8). Normal PO2 at sea level in young adults is 90– 100 mmHg. It falls with age to 60–70 mmHg at age 80. There is no consensus on what PO2 level is defined as ‘hypoxia’. Arterial oxyhemoglobin ‘functional’ saturation (100 x HbO2/[HbO2 þ HHb]) is normally 97–98% (i.e., 2–3% deoxyhemoglobin,

ARTERIAL BLOOD GASES 149 30

7.7

7.6

7.5

7.4

Metabolic SBE (mM)

M

20 CR

10 0 −10 −20

AR

AR

7.3 Metabolic alkalosis 7.2

Metabolic acidosis 7.1

CR

7.0 pH

M

−30 10

20

Respiratory alkalosis

Respiratory acidosis

30 40 50 60 70 Respiratory PaCO2 (Torr)

80

90

Figure 8 Acid–base compensation diagram predicting in vivo compensation for respiratory and metabolic acid–base imbalance. AR and CR, acute and chronic respiratory, respectively; M, metabolic. Reproduced from Schlichtig R, Grogono AW, and Severinghaus JW (1998) Human PaCO2 and standard base excess compensation of blood gas apparatus. Critical Care Medicine 26: 1173–1179, with permission from Lippincott Williams & Wilkins.

HHb). Carboxyhemoglobin or methemoglobin or other abnormal forms, if present, reduces ‘fractional’ saturation (or % oxyhemoglobin) computed as 100 x HbO2/total Hb but is not counted in ‘functional’ saturation. The terms ‘hypoxia’ or ‘hypoxemia’ generally imply that SaO2 is at least 5% lower than expected (at that age and altitude). Temperature Correction

Clinicians often ask whether they should instruct the laboratory to correct blood gas values to patient temperature. In general, this is not necessary. The appropriate pH and PCO2 for optimal physiologic function is the same function of temperature as are the in vitro temperature correction factors. At 301C in a hypothermic patient, the appropriate pH is 7.4 measured at 371C, or 7.55 corrected to 301C. Animals with a normal body temperature of 301C have a pH of 7.55. Fish in Antarctic waters at 01C have a pH of 8.0, as does normal human blood cooled in vitro to 01C. Blood at 90% SaO2 with PO2 ¼ 60 Torr will have PO2 ¼ 30 Torr at 251C, but delivers oxygen at least as effectively to tissues in hypothermia (as shown by decreased tissue lactate). However, physiologists who wish to study pulmonary gas transport and compare alveolar and arterial blood gas tension gradients must correct blood values from the laboratory (at 371C) to the body temperature under study. SID or SBE?

The interpretation of acid–base balance divides clinicians into two ‘schools of thought’. Strong ion

difference (SID) can identify the causes of many metabolic abnormalities in addition to obtaining an approximation of the degree of plasma metabolic acid– base abnormality, provided that all anions and cations are measured. This unfortunately is not a measure of the whole body extracellular fluid acid–base condition. For that one needs the SBE, which is computed from directly measured arterial pH, PCO2 , and PO2 without separate ion measurements. SBE is thus a quantitative analysis of imbalance while SID is an approximation of imbalance with additional causative suggestions. Hydrogen Ion Concentration or pH?

pH is used widely both for convenience and because chemical activities and potentials are log functions of concentrations. All animals no matter what their normal body temperature maintain their pH 0.6 unit above neutrality, i.e., a ratio of [OH ]/[H þ ] of about 16 whereas [H þ ] varies by a factor of more than 4 between 01C (fish) and 401C (hummingbirds). Buffering is a linear function of pH, an exponential function of [H þ ] making straight pH lines on acid– base compensation plots. Some clinicians prefer to interpret acid–base abnormalities using an approximation of Hendersen’s equation: Hþ ¼ 24PCO2 =HCO 3 using nM l 1 for H þ , mmHg for PCO2 and mM l 1 for HCO3 . This requires a constant 24 combining the dissociation constant K, CO2 solubility, equating carbonic acid with dissolved CO2, and a factor for the three different units.

Oxygen Dissociation Curve Computations and Corrections Blood gas analytic apparatus commonly provides a computed value of SO2 (oxygen saturation). The observed PO2 is first corrected from observed pH to pH ¼ 7.4 by obs obs ÞÞ logP7:4 O2 ¼ logPO2 ð0:48ð7:4 pH

At pH ¼ 7.40, 371C, the relationship of SO2 to PO2 is most simply and accurately expressed by SO2 % ¼ 100ð23; 400ðP3O2 þ 150PO2 Þ1 þ 1Þ1 No temperature correction (e.g., to patient temperature) is needed, all measurements being at 371C.

150 ARTERIES AND VEINS

SO2 in a blood sample does not vary with sample temperature.

State of the Art Electrodes versus Optical Sensors

Optodes can measure pH, PCO2 and PO2 using dyes, fluorescence, quenching, and other optically responsive material, permitting the use of extremely small sensors on optical fiber tips in blood at catheter tips or inside tissue cells. They rival electrodes in accuracy and cost, in particular providing portable and disposable sensors (e.g., for cardiac bypass oxygenator control, bedside and field blood gas analysis). See also: Acid–Base Balance. Diffusion of Gases. Diving. Erythrocytes. High Altitude, Physiology and Diseases. Oxygen–Hemoglobin Dissociation Curve. Permeability of the Blood–Gas Barrier. Ventilation: Control.

Further Reading Geha DG (1990) Blood gas monitoring. In: Blitt CDK (ed.) Monitoring in Anesthesia and Critical Care Medicine. New York: Churchill-Livingston. Nunn JF (1993) Nunn’s Applied Respiratory Physiology, 4th edn. Oxford: Butterworth-Heinemann. Schlichtig R, Grogono AW, and Severinghaus JW (1998) Human PaCO2 and standard base excess compensation for acid-base imbalance. Critical Care Medicine 26: 1173–1179. Severinghaus JW (1979) Simple, accurate equations for human blood O2 dissociation computations. Journal of Applied Physiology 46: 599–602. Severinghaus JW (2002) The invention and development of blood gas apparatus. Anaesthesiology 97: 253–256. Severinghaus JW and Astrup PB (1987) History of Blood Gas Analysis. International Anesthesiology Clinics, vol. 25(4). Boston: Little Brown. Severinghaus JW, Astrup P, and Murray J (1998) Blood gas analysis and critical care medicine. American Journal of Respiratory Critical Care Medicine 157: S114–S122. Severinghaus JW and Bradley AF Jr (1958) Electrodes for blood PO2 and PCO2 determination. Journal of Applied Physiology 13: 515–520.

ARTERIES AND VEINS D E deMello, Saint Louis University Health Sciences Center, St Louis, MO, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The lung’s vasculature is different from that of most other organs because it has a double arterial supply and a double venous drainage system. The pulmonary artery which carries deoxygenated blood to the lungs branches alongside the airways into conventional and supernumerary arteries before it empties into the vast capillary network in the alveolar walls where gas exchange occurs across air–blood barriers. Conventional and supernumerary pulmonary veins drain oxygenated blood to the left atrium of the heart. The smaller (intra-acinar) vessels develop at the same time as do the acini of the lung and a major component of acinar lung development occurs postnatally. Alveoli increase from about 20 million at birth to about 300 million by the time lung growth is complete in adolescence. Consequently, a large component of intra-acinar vessels develop postnatally and during this critical growth phase are susceptible to local influences such as increased blood flow from intracardiac left to right shunts which can curtail vessel growth. Arterial wall structure is composed of endothelial cells that line the lumen, smooth muscle cells that make up the media, and fibroblasts that contribute the adventitial fibrous sheath. The wall structure changes from proximal to distal vessels in the lung, and alterations in the normal structure may occur during intrauterine life or postnatally in response to a variety of stimuli. Altered wall structure results in functional changes reflected in pulmonary artery pressure and resistance.

Blood vessel assembly begins in primitive mesenchymal cells that undergo a complex series of steps before the mature vessel structure and function is attained. These stages in vessel development are under the control of a large number of transcriptional and growth factors. The timing and dose of some of these ‘angiogenic’ factors is critical to normal embryonic and fetal development; absence or even reduced expression of some factors is lethal for the developing embryo.

Anatomy, Histology, and Structure Unlike most other organs, the lung, because of its gas exchange function, has a double arterial supply and double venous drainage (Figure 1). One of the arterial systems, the pulmonary arterial tree, serves as a conduit for deoxygenated blood from the body to the alveoli where it drains into a vast capillary network within which gas exchange and oxygenation occur. From the alveoli, oxygenated blood is transported to the left atrium of the heart via the pulmonary veins. The other arterial system is the bronchial arterial tree which serves a nutrient function to the airways and perihilar structures. The arterial supply to the pleura, except at the hilar region, is from the pulmonary artery. For the intrapulmonary structures there are no bronchial veins, so all intrapulmonary structures drain to the pulmonary vein, resulting in a small amount of venous admixture in the left atrium. The hilar structures, however, drain to true bronchial

ARTERIES AND VEINS 151

Pulmonary artery

Alveoli Respiratory bronchiolus

Pulmonary vein

Bronchioli Bronchial artery Bronchi

Precapillary shunts

To left atrium

Capillary bed

To right atrium

Azygos vein

Figure 1 The lung’s vasculature consists of a double arterial and a double venous system. The pulmonary artery supplies the alveoli whereas the bronchial artery supplies the airways, the pulmonary artery, and the gas exchange region. Both systems drain via the pulmonary veins to the left atrium, except for the ‘true’ bronchial veins at the hilum which drain via the azygos veins to the right atrium. Reproduced from Reid L, Fried R, Geggel R, and Langleben D (1986) Anatomy of pulmonary hypertensive states. In: Bergofsky EH (ed.) Abnormal Pulmonary Circulation, vol. 4, pp. 221–263. New York: Churchill Livingstone, with permission from Elsevier.

veins and then to the azygos system and the right atrium.

Segmental hilum

Lateral pathway

Conventional and Supernumerary Arteries

The main pulmonary artery that forms the outflow path of the right ventricle divides into the right and left pulmonary arteries which enter the lung at the hilum. Within the lung the pulmonary arteries travel alongside the airway branches within a connective tissue sheath. The arterial branching pattern is similar to that of the airway it accompanies, but dissection has revealed many more branches from the pulmonary artery than from its accompanying airway. Two types of arterial branches exist. Those that branch dichotomously next to the airways are conventional branches, the longest of which are the axial pathways running the longest possible course from hilum to distal pleura. The lateral branches of the conventional pulmonary arteries supply the alveoli near the axial pathways (Figure 2). The other type of branch is the supernumerary artery which is short, arises at right angles to the axis of the pulmonary artery, and supplies the alveoli in the immediate vicinity of the artery (Figure 3). Supernumerary arteries are more numerous toward the periphery of any arterial pathway. Over the preacinar length of an axial artery, the ratio of the number of supernumerary to conventional

Axial pathways

Pleura

Figure 2 The path of axial and lateral airways in the human lung. The pulmonary arteries are alongside the airways within the bronchoarterial sheath and follow the same path. Reproduced from Davies P, deMello DE, and Reid LM (1990) Structural methods in the study of development of the lung. In Gil J (ed.) Models of Lung Disease: Microscopy and Structural Methods, vol. 47 of Lung Biology in Health and Disease, pp. 409–472. New York: Dekker, with permission.

branches is of the order of 3 : 1 and the lumen crosssection area of the supernumerary side branches is about one-third of the total cross-sectional area of all side branches (Figure 4). Supernumerary arteries have

152 ARTERIES AND VEINS 19-week fetus Hilum

Conventional artery Supernumerary Airway

1

3

1 2 2

3

4 5 4 6 5 6

7 8 7 8

9 10 10

Airways Conventional arteries Supernumerary arteries Figure 3 The anatomy of the pulmonary arterial tree. Conventional arteries divide alongside the airways at acute angles to the main axis and supply the respiratory region at the end of the axial pathway. Supernumerary arteries are short, arise at right angles to the main axis, and supply the airspaces adjacent to the axial vessel. Modified from deMello DE and Reid L (2002) Vascular development of lung. In: Tomanek R (ed.) Assembly of the Vasculature and its Regulation, vol. 2, pp. 211–237. Boston: Birkhauser, with permission.

a special sphincter at their point of origin which probably serves a functional purpose in recruitment. This valve is responsive to vasoconstrictive agents suggesting that it may be responsible for regulation of blood flow into the supernumerary artery. Because the diameter of the supernumerary vessel is smaller than that of the conventional artery from which it arises, it has a higher critical opening pressure, providing a built-in mechanism for recruitment as pulmonary artery pressure rises. It is noteworthy that the two types of pulmonary artery have different pharmacological profiles; for example, supernumerary arteries are 30 times more sensitive in their response to a vasoconstrictor such as 5-hydroxytryptamine (5-HT) compared to conventional arteries, and endogenous nitric oxide selectively attenuates the vasoconstrictor response to 5-HT in the supernumerary but not in the conventional artery. In pathological conditions, these short vessels provide an alternate route for blood in the conventional artery obstructed by thromboemboli. The capillary bed between an artery proximal to the block can open to conduct blood to an arterial bed distal to the block. Blood flow can produce remodeling of a vessel whether artery or vein, and injection techniques have demonstrated that such compensatory or collateral flow produces arcades between axial pulmonary arteries in a number of pathological states (Figure 5).

11 11

12 12

13 14 14

18 19 21.23 21 23 25

9

15 15 16 17 17 20 20 22 24 Terminal bronchiolus

Figure 4 Posterior basal lung segment in a 19-week fetus. Airway and accompanying conventional artery branches are numbered. The more numerous supernumerary arteries (58) are also shown. Reproduced from Hislop A and Reid L (1972) Intrapulmonary arterial development in fetal life: branching pattern and structure. Journal of Anatomy 113: 35–48, with permission from Blackwell Publishing Ltd.

Conventional and Supernumerary Veins

The venous drainage resembles the arterial pattern in that there are more venous tributaries than airway branches. The ‘conventional’ veins arise from the points of division of an airway and pass to the periphery of the unit, combining as tributaries to form progressively larger venous conduits. The supernumerary veins are short and drain small regions around the conventional veins. Additional tributaries to the pulmonary veins arise from pleura and connective tissue septa within the lung. Lung development is unique in that there is a set timetable for the growth and maturation of its major tissue components, airways, alveoli, and blood vessels, and that a large portion of its development continues postnatally until about 12 years of age. Laws of Lung Development

The template for the growth pattern of the lung is reflected in the three ‘laws of lung development’.

ARTERIES AND VEINS 153 Airway

Artery

Hilum

Vein

m Preacinar Resistance artery TB Intraacinar

RB AD A

pm Precapillary unit

Postcapillary unit

nm Capillary

Figure 5 Lung arteriogram from a premature infant who died at 9 months of age with pulmonary hypertension and vascular hypo plasia. Note dilated, tortuous vessels and interpulmonary artery arcades (arrows). Reproduced from Rendas A et al. (1980) American Review of Respiratory Disease 121: 873–880, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission.

Figure 6 Human lung arteriograms. Top, newborn; bottom left, 18 months; right, adult. Increase in radiodensity with age reflects progressive postnatal increase in number of intra-acinar arteries. Reproduced from Reid L (1978) The pulmonary circulation: remodeling in growth and disease. The 1978 J Burns Amberson Lecture. American Review of Respiratory Disease 119: 531–546, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission.

These generalizations are also predictive of the effect of perturbation at different times during development and they offer a framework against which to assess vascular growth patterns and to interpret anomalous development. Law I – Airways. The airways (i.e., bronchi and bronchioli) are present by the 16th week of intrauterine life.

Figure 7 The arteriovenous loop between the right and left side of the heart. The acinus is a unit of lung and includes that portion of lung that is subtended by the terminal bronchiole, i.e., respiratory bronchioles, alveolar ducts, and alveoli. The pulmonary artery that accompanies the airways is shown, including the resistance segment that is immediately precapillary in the newborn child when the acinus is 1 mm in diameter. In the adult the acinus is 1 cm in diameter and the resistance vessels are further downstream at the level of the alveolar ducts and alveoli. m, muscular; pm, partially muscular; nm, nonmuscular; TB, terminal bronchiole; RB, respiratory bronchiole; AD, alveolar duct; A, alveolus. Reproduced from deMello DE and Reid LM (1991) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientific Foundations, pp. 767–777. New York: Raven Press, with permission from Lippincott Williams & Wilkins.

Law II – Alveoli. At birth the ‘alveolar’ spaces are more primitive than in the adult. They have been described as ‘primitive saccules’. Of these saccules about 20 106 are present at birth. After birth the alveoli multiply, so that by the age of 8 the number is about 300 106 and within the adult range. Law III – Vascular. The ‘vascular law’ reflects the first two. The preacinar branches of the pulmonary artery (i.e., those that accompany bronchi or bronchioli), as well as the preacinar venous tributaries, appear at virtually the same time as do the accompanying airways. The intra-acinar vessels appear as alveoli grow (Figure 6). Preacinar Arteries: Structure and Size

The cellular structure of any vessel is composed of three cell types that make up the three coats of a vessel: the intima is constituted by endothelial cells, the media is composed of smooth muscle cells or their precursors, intermediate cells or pericytes, and the adventitia is made up of fibroblasts. Intercellular products also contribute to the composition of each coat. Regional and organ specificity dictate functional differences. A number of mediators, cytokines, growth factors, or hormones determine and modulate these differences.

154 ARTERIES AND VEINS Conventional branches Diameter of axial artery and side branches (µm)

Supernumerary branches 1000

External diameter of axial pathway 500

Acinus Lobule Figure 8 The relative sizes and numbers of conventional and supernumerary pulmonary arteries. Reproduced from deMello DE and Reid LM (1991) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientific Foundations, pp. 767–777. New York: Raven Press, with permission from Lippincott Williams & Wilkins.

Partially muscular

Muscular

Non muscular

Capillary (a)

M

I P E Artery

(b)

Lumen

M

(c) Figure 9 (a) Diagram depicting the light microscopic appearance of the structure of a pulmonary artery. In its path from the hilum to the periphery, the muscle coat gradually disappears so that the most peripheral segment still precapillary, is nonmuscular. (b) Diagrammatic illustration of the ultrastructural features of the pulmonary artery. From the hilum toward the periphery, muscle cells (M) are replaced by intermediate cells (I), and in the immediate precapillary segment, pericytes (P) are present. (c) Same as (a), in cross-section. Reproduced from deMello DE and Reid LM (1991) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientific Foundations, pp. 767–777. New York: Raven Press, with permission from Lippincott Williams & Wilkins.

ARTERIES AND VEINS 155 Fetus 100 60

Percentage arterial population

20

Child 100 60 NM

M

PM

20

Adult 100 60 20 100

200

300

400

Arterial size (ED, µm) Figure 10 The structure of vessels of different sizes at different ages in the human. The distribution of muscular (M), partially muscular (PM), and nonmuscular (NM) arteries is similar in the fetus and adult; however, in the child, larger arteries are nonmuscular and partially muscular. ED, external diameter. Reproduced with permission from the BMJ Publishing Group Davies G and Reid L (1970) Thorax 25: 669–681.

In the lung, the vessels proximal to the capillary bed are arteries and those distal to the capillary bed are veins. The immediate pre- and postcapillary vascular segments are functionally specialized and serve to protect the capillary bed (Figure 7). They are the key reactive site for remodeling in disease. Preacinar artery structure is established early in fetal life as elastic, transitional, or muscular and the program for structure is a regional or topographic one. In the adult along an axial pathway, the first seven generations which are vessels down to a diameter of 3000 mm (distended) are elastic. Vessels from 3000 to 2000 mm in diameter representing generations seven to nine have a transitional structure and smaller vessels down to a diameter of 30 mm have a muscular structure. The largest partially muscular and nonmuscular arteries are found in diameters up to 150 and 130 mm respectively (Figure 8). Along any arterial pathway, a complete muscular coat gives way to an incomplete coat which at first consists of a spiral before disappearing from vessels, still larger than capillaries, to produce a nonmuscular artery (Figure 9). On the arterial side of the capillary bed, nonmuscular, partially muscular, and muscular small arteries are identified. On the venous side a similar arrangement occurs. By

electron microscopy intermediate cells and pericytes, each a precursor of the smooth muscle cell, are identified: the intermediate cell in the nonmuscular part of the partially muscular artery and the pericyte in the nonmuscular wall. Whereas the pericyte lies within the basement membrane of the endothelial cell, the intermediate cell, like a smooth muscle cell, has its own basement membrane, but, unlike the latter does not have dense bodies. Characteristics of an artery include size, structure, and the thickness of the media as well as its position within the branching pattern. Because vessel remodeling occurs with growth and in disease, it is best to characterize a pulmonary artery by its position in the branching pattern defined by the structure of the accompanying airway, a landmark that remains relatively constant from fetal life. In arteries, the relative circumferences of the internal and external elastic laminae appear to influence the size and morphology of the endothelial cell and its microenvironment. For example, in vessels larger than 200 mm, the external lamina is shorter than the internal suggesting that it is the former that restricts distension and that the internal lamina may never be smoothed out. In the smaller arteries the situation is


reversed suggesting that the internal lamina is the limiting structure. The balance between these is changed in disease. The functional significance of this, in pulsatile flow and propagation of the pulse wave, has not been established. Postnatally, the length and diameter of the entire pulmonary vascular tree changes, but structural changes occur mainly at the sites of alveolarization in the acinar region, that is, in vessels distal to the level of the terminal bronchiole. In the newborn lung the resistance arteries, that is, vessels with a smooth muscle wall, are upstream from the alveolar wall. Normally before birth pulmonary arterial muscularization stops at the beginning of the acinus, so that the peripheral vessels are muscularized later (Figures 10 and 11). In conditions such as idiopathic pulmonary hypertension of the newborn, congenital heart lesions associated with increased pulmonary artery flow or pressure before birth, and pulmonary hypoplasia, a well-developed muscle coat is found in more peripheral and smaller arteries than is normal. The functional correlate of this structural change is an elevation in pulmonary artery pressure and resistance. AD

Species differences in structure and rate of lung growth occur and need to be considered when interpreting experimental results. In general, animals like the sheep that can walk or run within hours of birth have a prenatal burst in lung development, preparing the animal as it were for considerable, immediate postnatal activity. In mammals like the rat, mouse, and pig a similar burst in lung growth occurs postnatally, and in the human, postnatal lung growth continues for several years until adolescence. Postacinar Veins: Structure and Size

The veins are present at the periphery of an acinus within their own connective tissue sheath. As additional tributaries are added in their course toward the hilum, they increase progressively in size. Conventional axial veins enter the larger veins at an acute angle and are the main pathways from periphery to hilum. Their numbers are equivalent to the airway generations and to conventional arteries. The supernumerary tributaries are shorter, drain the lung immediately around the axial vein, and connect with RB

TB

Pleura

Alv

Species Variation

Normal Fetus 3 days 10 months 3 years 10 years 19 years Cases PPHN Mec Asp (fatal) HLHS IDTAPVR Figure 11 Top: diagram of an acinus of the lung which includes the terminal bronchiole (TB), respiratory bronchioles (RB), alveolar ducts (AD), and alveoli (alv). Below: the open bars indicate the level beyond which at different ages the arteries are nonmuscular. The blue bars indicate the extent of muscularization (‘precocious muscularization’) in different disease processes: PPHN, persistent pulmonary hypertension of the newborn; Mec Asp (fatal), fatal cases of PPHN with meconium aspiration; HLHS, hypoplastic left heart syndrome; IDTAPVR, infradiaphragmatic total anomalous venous return. Reproduced from Reid L, Fried R, Geggel R, and Langleben D (1986) Anatomy of pulmonary hypertensive stages. In: Bergofsky EH (ed.) Abnormal Pulmonary Circulation, vol. 4, pp. 221–263. New York: Churchill Livingstone, with permission from Elsevier.

ARTERIES AND VEINS 157 Pathway 1

1200

∗

800

External diameter of axial pathway

Diameter of axial vein and tributaries (µm)

400

Hilum Pathway 2 1200

800 + + +

400

Hilum Pathway 3 Conventional veins 400 Supernumerary veins

Figure 12 Diagrammatic reconstruction of conventional and supernumerary venous tributaries in a 20 week fetus. Pathway 2 is 9.31 mm in length. *Point where pathway 2 joins pathway 1. þ Point where pathway 1 joins pathway 2. z Point where pathway 3 joins pathway 2. There is a higher density of venous tributaries at the periphery. Reproduced from Hislop A (1971) The fetal and childhood development of the pulmonary circulation and its disturbance in certain types of congenital heart disease. PhD Thesis, London University, with permission from A A Hislop.

the conventional veins at right angles (Figure 12). The preacinar tributaries are developed by 20 weeks gestation, and subsequent tributaries arise within the acinus. Supernumerary tributaries exceed conventional, their ratio being 3.5 : 1, and more vessels drain from the capillary bed than enter. Postcapillary veins have an endothelial lining, but no muscle coat. Larger veins contain an internal elastic lamina with only occasional medial smooth muscle fibers. The medial thickness increases in yet larger veins, but even the largest veins do not have a well-defined external elastic lamina. Elastin and

collagen are present between the smooth muscle cells. The largest nonmuscular veins are approximately 150 mm in diameter and as with the arteries, overlap in size groups occurring between muscular, partially muscular, and nonmuscular veins. At 20 weeks gestation, the vein structure consists of endothelial cells resting on collagen mixed with an occasional elastic fiber. Even the largest veins have no muscle in their wall. Scattered muscle fibers appear by 28 weeks, but a continuous layer is only developed at term, and even then, no elastic lamina is present. Postnatally, the thickness of the muscle


increases but not as much as in arteries. At birth the smallest muscular vein measures about 105 mm in diameter, at 3 years the smallest is 130 mm, and at 10 years 70 mm. No significant structural change occurs after this time. Intra-Acinar Arteries and Veins: Structure and Size

By 5 years of age, axial pathways have finished development and future growth occurs in the more peripheral and smaller arteries that appear as alveoli are formed, but in these vessels, muscularization is slower, so that during childhood the transition from muscular to partially to nonmuscular occurs in vessels of a larger size than in the fetus or adult (Figure 10). Therefore, in the child, the structure of intra-acinar arteries cannot be predicted by their size. Bronchial Arteries

Early in gestation, primitive bronchial arteries arise from the dorsal aorta in the neck region near the celiac axis and are distributed with the early branches of the airways (Table 1). When lobar or segmental airway branches are present at about the 6th week of embryonic life, the central bronchial artery branches disappear. Between the 9th and 12th weeks, definitive bronchial arteries arise from the aorta, pass along the superior surface of the airways, and communicate with the pre-existing capillary bed in the distal airways. Sometimes the primitive bronchial arteries persist and migrate with their point of origin, to a site below the diaphragm, still near the Table 1 The development of the bronchial arteries Week of gestation

Airway

Blood vessels

4th

Main

5th

Lobar

6th 9th to 12th

Segmental

Primitive ventral aorta Pulmonary vein links to heart 6th arch supplies lung Paired systemic arteries from dorsal aorta Only blood from right ventricle to pulmonary artery Systemic arteries disappear Bronchial arteries enter peribronchial plexus

The primitive paired bronchial arteries arise from the dorsal aorta near the celiac axis in the neck. These have usually disappeared before the 5th week; if they persist, they then migrate with the celiac axis to below the diaphragm as seen in certain abnormal conditions (e.g., sequestered segment). Reproduced from deMello DE and Reid LM (1997) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientific Foundations, 2nd edn., pp. 1117–1127. Philadelphia: Lippincott-Raven Press, with permission from Lippincott Williams & Wilkins.

celiac axis. A portion of the lung bud may remain attached and manifests later as a lung sequestration.

Arteries and Veins in Normal Lung Function Blood Vessel Assembly

Two processes are involved in the development of blood vessels: (1) angiogenesis, the branching of new vessels from pre-existing ones, and (2) vasculogenesis, the formation of blood lakes that undergo transformation into vessels. In the lung, blood vessel development must be coordinated temporally and spatially with airway and alveolar development. Angioblasts are the precursor cells from which blood vessels are derived and they are of mesodermal origin. In the development of the pulmonary vasculature, angioblasts must interact with epithelial and mesenchymal components of the lung. Central events determine for a given vessel, the direction of blood flow, its distribution or drainage and its central connections, and local or peripheral events serve as modifiers. These determine the fine structure of the vessels as they supply the ultimate functioning unit of the lung. Vessel branching occurs at the end of a pathway and lengthening or widening results from cell multiplication. Chimeric experiments have shown that angioblasts migrate widely and to considerable distances from their sites of origin. Biologic differences in vessel cells, for example, endothelial cells from different sites in the vascular tree, exist, reflecting the influence of the local milieu. The transcription factor TAL1/SCL is a marker of angioblasts. In the presence of extracellular matrix, endothelial cell precursors form protrusions that result in the formation of vascular cords and then vessels. This process requires integrin-mediated adhesions. Vascular endothelial growth factor (VEGF) plays a role as a mitogen and as a vascular morphogen in vasculogenesis. Whereas vascular smooth muscle cells arise through progenitors within the mesoderm, they are also derived from endothelial cells. In the fetal mouse, transmission electron microscopy shows that between 9 and 10 days, primitive angioblast precursors within the mesenchyme surrounding the lung bud, form vascular lakes that have hematopoietic cells in their lumen (Figures 13(a)– 13(c)). Mercox pulmonary vascular injections combined with scanning electron microscopy of the vascular casts indicate that conventional and supernumerary branches from the main pulmonary artery are derived by angiogenesis and that the more distal vessels, those of the future alveolar region, arise by vasculogenesis, that is, from a lumen that appears


Figure 13 Transmission electron micrographs of fetal mouse thoraces. (a) 9 day fetus: a space between densely packed mesenchymal cells contains membrane-bound vesicles. Magnification 8750. (b) 10 day fetus: mesenchymal cells around intercellular spaces appear thin and endothelial-like. Magnification 5250. (c) 10 day fetus: some intercellular spaces contain hematopoietic precursor cells. Magnification 5250. Reproduced from deMello DE, Sawyer D, Galvin N, and Reid LM (1997) Early fetal development of lung vasculature. American Journal of Respiratory Cell and Molecular Biology 16: 568–581, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission.

locally within the mesenchyme: at 12 days, there are four generations of central arterial branches but no luminal connection is seen between these vessels and the lakes in the peripheral lung mesenchyme, where already a dense collection of lakes containing hematopoietic cells is present. By 14 days, five to seven generations of central artery branches, supernumerary and conventional, are present. There is now a connection between the central vessels and the peripheral system so that casts of the peripheral vessels are obtained by central injection (Figures 14(a)– 14(c)). Between 15 days and term, there is increasing complexity of the peripheral vascular casts reflecting an increase in the connections between the central and peripheral systems (Figure 14(d)). Whereas the processes of angiogenesis and vasculogenesis occur separately but concurrently, a third process, fusion, between these two systems is necessary for the circulation to be established.

In the human, examination of serial sections of embryos and fetuses of different ages in the Carnegie Collection of Human Embryos housed in the Carnegie Institute of Washington, DC (now located in the Museum of Human Development in the Armed Forces Institute of Pathology in Washington, DC) revealed that the blood lakes are the first to form and are present in the primitive mesenchyme around the lung bud in the neck between stages 14 and 18 (32– 44 days of gestation). As gestation progresses, abundant lakes appear in the subpleural mesenchyme. At stage 23 (56.5 days), pulmonary artery branches accompany the airways but lag behind the airway by two to three generations. The thick-walled arteries end blindly in a solid cord of cells. Between 12 and 16 weeks, an extensive capillary network is present in the subpleural mesenchyme surrounding the most distal airway buds but separated from them by mesenchyme. By 22 to 23 weeks, the capillary network


S

S

R

L

R

PA

PA L

PA

PA

Aorta Aorta

13 days

12 days

(a)

(b)

PA

S

L

R

PA L

PV R

PV

CL

15 days (c)

16 days (d)

Figure 14 Photomicrographs of pulmonary arterial mercox casts of mouse fetuses. (a) 12 day fetus: up to four generations of arterial branches are present. (b) 13 day fetus: isolated patches of peripheral vessels are filled with mercox. (c) 15 day fetus: mercox filling of more peripheral vessels results in visualization of several generations of arterial branches with an increase in diameter. (d) 16 day fetus: mercox filling of the expanded peripheral vascular network results in a markedly dense cast that obscures the proximal vessels. S, systemic; R, right; L, left; PA, pulmonary artery; PV, pulmonary vein; CL, cardiac lobe. Reproduced from deMello DE, Sawyer D, Galvin N, and Reid LM (1997) Early fetal development of lung vasculature. American Journal of Respiratory Cell and Molecular Biology 16: 568–581, Official Journal of the American Thoracic Society, & American Thoracic Society, with permission.

approaches the airway epithelium and bulges into the air space indicating that blood barriers for future gas exchange have formed. At this time, the pulmonary artery accompanies even the most distal airway branch just beneath the pleura. So from this study, it appears that in the human also, the three processes of vascular development, that is, angiogenesis, vasculogenesis, and fusion, contribute to the establishment of the pulmonary circulation. Other studies using markers for endothelial cell precursors in

mouse embryos and three-dimensional reconstruction of serial sections of human embryos suggest that the intrapulmonary vascular tree is predominantly developed by the process of vasculogenesis. Controlling Mechanisms: Genes and Factors Involved in Vessel Assembly

The path from angioblast precursor within primitive mesenchyme to mature blood vessel is complex and


Primitive mesenchyme

Hemangioblasts

Endothelial cell commitment ('blood island')

Migration and tube formation

Smooth muscle recruitment and differentiation

Mature blood vessel

Proteases

SCL/tal-1

Ets-1 Fra1 Vezf1

ARNT ELF-1 EPAS Fli-1 GATA2 GATA3 HIF-1 HOXD3 NERF-2

Ets-1

AML-1 COUP-TFII HESR1 HOXB3 PPAR-

dHAND MEF2C SMAD5 SmLIM

LKLF

Transcription factors Growth factors and receptors bFGF Flk-1 Flt-1 integrin V3 P/GH TGF- TIE2 VEGF

Angiopoietin-1 TIE2

Figure 15 The multiple steps involved in vessel assembly from primitive mesenchyme requires the sequential action of numerous factors. Reproduced from Pediatric and Developmental Pathology vol. 7, 2004, pp. 422–424, A matter of life and breath: context article, deMello DE, figure 1, with kind permission of Springer Science and Business Media.

involves a number of steps including commitment to differentiate into an endothelial cell, the action of proteases, migration and tube formation, recruitment and differentiation of smooth muscle cells, and acquisition of mature vessel structure and function. This complex process is under the coordinated control and influence of a large number of genes and growth factors (Figure 15), which have been identified by experiments involving overexpression or knockout of growth factors or genes. A fine-tuned and delicate balance in the temporal and spatial expression of a variety of genes and factors is essential for both normal intrauterine vessel development and postnatal vessel maintenance. For example, knockout of the VEGF gene or even its reduced expression in heterozygosity results in lethal defects in vessel formation in the mouse embryo, whereas overexpression of VEGF in the lung results in the formation of oversized vessels and disordered airway morphogenesis. Even the relative ratios of VEGF isoforms is critical for normal vessel and airway development. This was demonstrated in the VEGF 120 mouse in which the other VEGF isoforms, VEGF 164 and VEGF 188, are not expressed. Homozygous VEGF 120 mice have a severe reduction in the number of intra-acinar arteries and air–blood barriers and a

delay in lung development resulting in hypoplastic lungs (Figures 16 and 17). Knockout of the Flt-1 (VEGF receptor) gene produces a lethal defect in angiogenesis and knockout of the Flk-1 (VEGF receptor) gene results in a lethal failure of vasculogenesis. Transforming growth factor beta (TGF-b) is important for the eventual wall structure of the developing vasculature and influences growth, migration, and differentiation of endothelial, smooth muscle, and mesenchymal cells and pericytes. The receptor tyrosine kinase gene (tie-1) and receptor (Tie -2) are involved in the regulation of vasculogenesis and angiogenesis. Transitional Circulation and Perinatal Adaptation in Humans

At birth, as the lung expands with air, structural changes occur within the resistance segment of the pulmonary arteries producing an increase in compliance, and drop in resistance and pulmonary artery pressure as a result of an increase in external diameter and decrease in wall thickness. By 4 months the resistance arteries lose their fetal wall thickness, but in vessels less than 200 mm in diameter, wall thickness gradually increases.


1 mm

E13.5

1 mm

E15.5

1 mm

1 mm

1 mm

E18.5

E18.5

E18.5

Figure 16 Photographs of fetal mouse lungs from three gestational ages: top, E13.5; middle, E15.5; bottom, E18.5. Lungs of wild-type (VEGF þ / þ , left) and heterozygous (VEGF 120/ þ , middle) fetuses do not differ in size. At all gestational ages, the lungs of homozygous fetuses (VEGF 120/120, right) are smaller than the lungs of wild-type and heterozygous littermates. Reproduced from Galambos C, Ng Y-S, Ali A, et al. (2002) Defective pulmonary development in the absence of heparin-binding vascular endothelial growth factor isoforms. American Journal of Respiratory Cell and Molecular Biology 27: 194–203, with permission.

Structure

The distribution of preacinar elastic and transitional arteries is unchanged during childhood, although the size range varies with age. Intra-acinar arteries which develop as new alveoli are formed postnatally, are nonmuscular initially, and acquire muscle coats gradually with time. By 10 years of age, even alveolar wall level arteries have muscle coats.

Arteries and Veins in Respiratory Diseases Abnormalities of pulmonary arteries or veins can result from disorders of vessel assembly or from postnatal events that interfere with postnatal vascular growth or cause structural remodeling of the existing vasculature.

Veins

Disorders of Vessel Assembly

During childhood, the veins increase in size and peripheral veins increase in number. Within an acinus, the number of veins exceeds that of the arteries presumably to facilitate blood flow through the capillary bed.

These disorders are the consequence of aberrations in the processes involved in vessel assembly, that is, angiogenesis or vasculogenesis and result in failure of growth, overgrowth, or structural/functional abnormalities.


1 mm (a)

1 mm (b)

1 mm (a)

1 mm (c)

1 mm (b)

1 mm (c)

Figure 17 Lung mercox vascular casts of VEGF 120/120 fetal mouse littermates. Genotypes: (a), wild-type; (b), heterozygous; (c), homozygous. Gestational ages: top, E17; bottom, E18. The homozygous fetal casts are smaller, and fewer peripheral vessels make the casts less dense than those of the wild type or heterozygous littermates. Reproduced from Galambos C, Ng Y-S, Ali A, et al. (2002) Defective pulmonary development in the absence of heparin-binding vascular endothelial growth factor isoforms. American Journal of Respiratory Cell and Molecular Biology 27: 194–203, with permission.

Aberrant angiogenesis Absence of the main pulmonary artery or its branches The main pulmonary artery or one of its branches fails to grow and the lung is supplied instead by collateral vessels from the systemic circulation. Usually the pattern of intrapulmonary artery branching is unaffected so it can be presumed that central sprouting or angiogenesis fails, but that peripheral vasculogenesis is normal and the systemic collaterals assume the role of the intrapulmonary arterial tree. Misalignment of blood vessels This disorder often accompanies another serious condition, alveolar capillary dysplasia (Figure 18). The central pulmonary arteries and veins are ‘misaligned’ so that the veins are displaced from their normal location at the periphery of a lung lobule and instead share the bronchoarterial sheath. When present in all lobes of

the lung and in association with alveolar capillary dysplasia, the condition is fatal. Hypoplastic vascular tree: dwarfism and congenital diaphragmatic hernia The small thoracic cage in most forms of dwarfism or skeletal dysplasia is associated with small lungs. The main structural components of the lung, airways, alveoli, and vessels are hypoplastic but additional abnormalities suggest a direct metabolic effect as well. In congenital diaphragmatic hernia, the restricted space available for fetal lung growth produces hypoplastic lungs with a reduced number of airways and alveoli. Because the pulmonary arteries travel and branch with the airways, their number is also reduced and the size is small but appropriate for the smaller lung volume. Arterial structure is also altered so that the walls are thicker and muscle extends into smaller, more peripheral vessels.


(a)

(b)

(c) Figure 18 (a) Post-mortem lung barium angiograms. (left) Preterm neonatal lung reveals filling of preacinar and intra-acinar arteries. (right) Lung of a term infant with alveolar capillary dysplasia; the angiogram has the look of a ‘pruned tree’ because of a reduced number of intra-acinar arteries. (b) In the normal lung (left), the pulmonary artery (long arrow) is present within the bronchovascular sheath and the vein (short arrow) lies within the pulmonary septum at the periphery of the lung lobule. In misalignment of the pulmonary vessels (right), the pulmonary arteries (long arrows) and veins (short arrows) lie within the same bronchovascular sheath. The pulmonary arteries are filled with barium (post-mortem injection) and the veins are empty. Movat pentachrome stain. Magnification 100. (c) The normal term lung (left) has numerous air–blood barriers (arrows) within alveolar walls. In alveolar capillary dysplasia (right) air–blood barriers are absent and vessels larger than normal capillaries are present in the middle of thickened alveolar septa (arrows). Movat pentachrome stain. Magnification 200. Reproduced from deMello DE (2004) Pulmonary pathology. Seminars in Neonatology 9: 311– 329, with permission.

Disordered vasculogenesis Alveolar capillary dysplasia In this rare and fatal disorder, air–blood barriers which are critical for gas exchange fail to form within alveolar walls. When the entire lung is affected, the condition is incompatible with independent air-breathing existence (Figure 18). Sometimes, however, a single lobe is

affected suggesting failure of a local inductive mechanism for triggering capillary growth. Fusion Intrapulmonary arteriovenous malformations point to aberrant connections between the central and peripheral pulmonary vasculature during fetal development. This is usually a circumscribed


lesion suggesting that while overall angiogenesis and vasculogenesis has occurred normally, the mechanism that regulates fusion (? via chemotaxis) is faulty and results in communications between arteries and veins. Rarely, the process may involve an entire lobe. Postnatal Disorders Idiopathic (persistent) pulmonary hypertension of the newborn (PPHN), with or without meconium aspiration (also known as meconium aspiration syndrome) In both instances, the overall pattern of arterial growth is normal, but significant functional abnormalities at birth result from structural alterations in preacinar and intra-acinar arteries. The vessels are often small and have thick muscle walls and adventitial coats. There is precocious muscularization of intra-acinar arteries so muscle coats are present within smaller, normally nonmuscularized arteries. Congenital heart disease with left-to-right shunts In many types of congenital heart disease, the pulmonary circulation develops normally in utero, but adaptational changes occur after birth. An increase in pulmonary blood flow from left to right shunts interferes with postnatal intra-acinar artery growth and if left uncorrected will result in a reduction in intra-acinar artery number, increased pulmonary vascular resistance, and pulmonary hypertension. Systemic arteriovenous anastomoses in which high pulmonary blood flow occurs before birth will result in an abnormal vascular structure at birth. See also: Acute Respiratory Distress Syndrome. Alveolar Hemorrhage. Angiogenesis, Angiogenic Growth Factors and Development Factors. Arterial Blood Gases. Bronchial Circulation. Bronchopulmonary Dysplasia. Chronic Obstructive Pulmonary Disease: Acute Exacerbations. Coagulation Cascade: Factor VII. Diffusion of Gases. Epithelial Cells: Type I Cells; Type II Cells. Hypoxia and Hypoxemia. Infant Respiratory Distress Syndrome. Lung Anatomy (Including the Aging Lung). Lung Development: Overview; Congenital Parenchymal Disorders; Congenital Vascular Disorders. Lymphatic System. Neonatal Circulation. Nitric Oxide and Nitrogen Oxides. Oxygen Therapy. Oxygen Toxicity. Pediatric Pulmonary Diseases. Permeability of the Blood–Gas Barrier. Pulmonary Circulation. Pulmonary Edema. Pulmonary Vascular Remodeling. Smooth Muscle Cells: Vascular. Surgery: Transplantation. Systemic Disease: Diffuse Alveolar Hemorrhage and Goodpasture’s Syndrome. Vascular Disease. Vascular Endothelial Growth Factor. Vasculitis: Overview; Microscopic Polyangiitis.

Further Reading Davies G and Reid L (1970) Thorax 25: 669–681. Davies P, deMello DE, and Reid LM (1990) Methods in experimental pathology of pulmonary vasculature. In: Gil J (ed.) Models of Lung Disease: Microscopy and Structural Methods, vol. 47 of Lung Biology in Health and Disease, pp. 843–904. New York: Dekker. deMello DE (1999) Structural elements of human fetal and neonatal lung vascular development. In: Control Mechanisms in the Fetal and Neonatal Pulmonary Circulation, Proceedings of the Ninth Pulmonary Circulation Conference, Sedalia, CO, Oct 1999, pp. 37–64. deMello DE (2004) A matter of life and breath: context article. Pediatric and Developmental Pathology 7: 422–424. deMello DE (2004) Pulmonary pathology. Seminars in Neonatology 9: 311–329. deMello DE and Reid L (2000) Embryonic and early fetal development of human lung vasculature and its functional implications. Pediatric and Developmental Pathology 3: 439–449. deMello DE and Reid L (2004) Pre- and post natal development of the pulmonary circulation. In: Haddad GG, Abman SH, and Chernick V (eds.) Basic Mechanisms of Pediatric Respiratory Disease, pp. 77–101. Ontario: BC Decker. deMello DE and Reid LM (1991) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientific Foundations, pp. 767–777. New York: Raven Press. deMello DE and Reid LM (1991) Pre and post-natal development of the pulmonary circulation. In: Chernick V and Mellins RB (eds.) Basic Mechanisms of Pediatric Respiratory Disease: Cellular and Integrative, pp. 36–54. Philadelphia: BC Decker. deMello DE and Reid LM (1997) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, and Weibel ER (eds.) The Lung: Scientific Foundations, 2nd edn., pp. 1117–1127. Philadelphia: Lippincott-Raven. deMello DE and Reid LM (2002) Vascular development of lung. In: Tomanek R (ed.) Assembly of the Vasculature, vol. 2, pp. 211–237. Boston: Birkhauser. deMello DE, Sawyer D, Galvin N, and Reid LM (1997) Early fetal development of lung vasculature. American Journal of Respiratory Cell and Molecular Biology 16: 568–581. Galambos G, Ng YS, Ali A, et al. (2002) Defective pulmonary development in the absence of heparin-binding vascular endothelial growth factor isoforms. American Journal of Respiratory Cell and Molecular Biology 27: 194–203. Hall SM, Hislop AA, Pierce CM, and Haworth SG (2000) Prenatal origins of human intrapulmonary arteries. American Journal of Respiratory Cell and Molecular Biology 23: 194–203. Hislop A (1971) The fetal and childhood development of the pulmonary circulation and its disturbance in certain types of congenital heart disease. PhD Thesis, London University. Hislop A and Reid L (1972) Intra-pulmonary arterial development during fetal life: branching pattern and structure. Journal of Anatomy 113: 35–48. Hislop A and Reid L (1973) Fetal and childhood development of the intrapulmonary veins in man: branching pattern and structure. Thorax 28: 313–319. Hislop A and Reid L (1973) Pulmonary arterial development during childhood: branching pattern and structure. Thorax 28: 129–135. Oettgen P (2001) Transcriptional regulation of vascular development. Circulation Research 89: 380–388.

166 ASTHMA / Overview Reid L (1978) The pulmonary circulation: remodeling in growth and disease. The 1978 J Burns Amberson Lecture. American Review of Respiratory Disease 119: 531–546. Reid L, Fried R, Geggle R, and Langleben D (1986) Anatomy of pulmonary hypertensive states. In: Bergofsky EH (ed.) Abnormal Pulmonary Circulation, Vol. 4, pp. 221–263. New York: Churchill Livingstone.

Asbestos

Rendas A, et al. (1980) American Review of Respiratory Disease 121: 873–880. Schachtner SK, Wang YQ, and Baldwin SH (2000) Quantitative analysis of embryonic pulmonary vessel formation. American Journal of Respiratory Cell and Molecular Biology 22: 157–165.

see Occupational Diseases: Asbestos-Related Lung Disease.

ASTHMA Contents

Overview Allergic Bronchopulmonary Aspergillosis Aspirin-Intolerant Occupational Asthma (Including Byssinosis) Acute Exacerbations Exercise-Induced Extrinsic/Intrinsic

Overview P Chanez, Hoˆpital Arnaud de Villeneuve, Montpellier, France & 2006 Elsevier Ltd. All rights reserved.

Asthma is one of the most frequent chronic diseases. It is responsible for absenteeism from school and work, thereby handicapping daily life. Its management is based on drug therapy, control of the environment, therapeutic education, and management of triggering factors.

Abstract Asthma is one of the most prevalent chronic diseases in most of the countries in the world. Its continuous increase is clearly described in most places, confirming the potential importance of the environment. These environmental factors interact in a susceptible individual with some complex polygenic background, to reveal the asthma phenotype. Several triggers including inhaled allergens, viruses and some indoor and outdoor pollutants have been pointed as potential culprits to induce, perpetuate, or exacerbate asthma. Inflammation and structural changes are hallmarks of asthmatic airways occurring from the nose to the distal part of the lung. The relationships between those structural changes and clinical and functional abnormalities clearly deserve further investigations. Those findings led to the reinforcement of the use of inhaled corticosteroids as the pivotal treatment for asthma. The adjunction of long-acting b2 agonists has been shown to be the next logical step of pharmacology, in case of poor control when using inhaled steroids alone. The long-term management should include some tailor-made environment control and educational measures leading to a better partnership with the patients. In some occasions, the addition of leukotriene receptor antagonists, specific immunotherapy, and more recently, subcutaneous anti-IgE may offer better control for a subset of patients. A better understanding of the mechanisms, especially in the most severe forms of the disease, is paramount to develop better preventive strategies and innovative therapies.

Physiopathology Asthma is an inflammatory disorder accompanied by remodeling of the airways. This inflammation is secondary to polymorphic inflammatory infiltration, rich in mast cells, and eosinophils. In genetically predisposed subjects, this inflammation may cause symptoms which, in general, are related to diffuse variable bronchial obstruction that is spontaneously reversible or subsides under the influence of treatment. This inflammation is also associated with bronchial hyperresponsiveness to a wide variety of stimuli. This definition of asthma is supported by the physiopathological and clinical findings on the disease. The variability and reversibility of the airflow impairment, under the influence of bronchodilators and glucocorticoids, distinguish asthma from other bronchial disorders. Chronic rhinosinusitis is very often associated with asthma (approximately 80% of cases) and must be investigated, not only by questioning, but also by careful nasal examination.

ASTHMA / Overview 167

Several arguments support a link between persistent rhinosinusitis and asthma: the common characteristics of inflammation and tissue reorganization, similar epidemiology and chronicity, higher risk of asthma in cases of rhinitis, higher risk of bronchial hyperreactivity in patients suffering from rhinitis, occurrence of bronchial inflammation after nasal challenge with an allergen, and the same aggravating and triggering factors. The efficacy of local corticoid therapy constitutes another link. On the other hand, there is no solid evidence in favor of the bronchial efficacy of nasal treatment. Systemic treatments also require additional evaluation of their joint efficacy in rhinosinusitis and asthma.

Diagnosis Positive Diagnosis

This must be based on the definition of asthma. The two characteristic elements are clinical (chronicity, variability, and reversibility of symptoms), and functional. Tests of respiratory function clearly confirm the diagnosis provided that: 1. spirometry shows a reversible obstructive ventilatory disorder from 12% to 15% in comparison with theoretical values, or at least 180 ml in absolute value, with short-acting b2-mimetics; 20% or 250 ml during a 10-day test with glucocorticoids; 2. peak expiratory flow (PEF) has a variability X20%; and 3. bronchial hyperreactivity is another feature of the diagnosis. There have been few studies on the sensitivity and specificity of the functional signs of asthma and their predictive values seem insufficient. Those maintained in the international recommendations are cough, dyspnea, sibilant rhonchi (wheezing), chest tightness, and expectoration. There may be one or several symptoms. Symptoms may be absent at the time of the examination. Definition of reversibility or its significance is only obtained from expert opinions or by consensus. The same is true for the usefulness of the corticosteroid test to identify asthmatic patients; a short course of oral corticosteroid is used generally. The dose and duration of steroid treatment given in the literature are highly variable. However, these criteria are generally considered to be sufficient to propose the diagnosis of asthma and to potentially qualify nonresponding asthmatic patients as steroidresistant.

Table 1 Differential diagnosis of asthma in the child and adult In the child

In adults

Obliterating bronchiolitis Cystic fibrosis Foreign body Tracheobronchomalacia Bronchial inhalations Vocal cord dysfunction Upper airway abnormalities Immunodeficiency (IgA, IgG2, IgG4) Ciliary dyskinesia Abnormal aortic arch

Bronchiectasis Cystic fibrosis Foreign body Tracheobronchomalacia Bronchial inhalations Vocal cord dysfunction COPD Heart failure Bronchial amyloidosis Abnormal aortic arch Tracheobronchial cancer Bronchiolitis

Alternative Diagnosis

The main differential diagnoses are summarized in Table 1.

Clinical Definitions From the point of view of the terminology, three essential terms must be used and seem to be operational in clinical medicine and for the follow-up of asthmatics – the seriousness of the asthma which refers to the current state of the patient (serious acute asthma), the control of the asthma which refers to recent events (symptoms of brief duration and exacerbations), and the severity of the asthma which is mainly evaluated over the past year. Exacerbations

The definition of an exacerbation is variable and it is based on the unanticipated use of drugs, the persistent symptoms (repetition of short-lasting symptoms generally on two consecutive days), the increased bronchial obstruction, and the requirement for substantial change in treatment with oral corticoids being the most frequent. Exacerbations form part of poor control of the asthma, but differ from it. The permanence of shortlasting symptoms with return to the baseline state between these symptoms defines simple poor control, whereas an exacerbation is characterized by a persistent worsening without a return to the baseline state. Control

Several asthma evaluation control questionnaires have been developed and concern the events occurring during the 1–4 weeks before the visit. Some even cover a period of 3 months, or even the time since the previous visit. There are no data in the literature pointing to one or the other of these periods.

168 ASTHMA / Overview

Juniper showed that her questionnaire (ACQ) was more discriminating than a daily diary. The authors clearly specified that this comparison concerned clinical trials and no conclusion could be deduced for daily clinical practice. These indexes are often correlated with generic and specific quality of life questionnaires: AQLQ, SF 36, Saint Georges questionnaire. The definition of optimal (or excellent) and suboptimal (or acceptable) control is based on experts’ agreement and their clinical experience.

Table 2 Factors aggravating controlled asthma

Severity

Table 3 Examples of candidate genes implicated in the development of asthma

The notion of severity was initially based on the same parameters as control, though these were assessed retrospectively over a longer period, often including the number of successful anti-inflammatory treatments and the best level of respiratory function. At present, the assessment of asthma severity must be made more objectively, according to the control and the quantity of drugs required to obtain and maintain it. Difficult Asthma

Difficult asthma may only be defined after answering questions about its diagnostic reality, patient adherence to treatment, and the existence of major comorbidity which may interfere with management, making it impossible to obtain an acceptable control. Positive answers to these questions aid in diagnosing severe asthma. Severe asthma is characterized by frequent and serious exacerbations (stays in intensive care units), the persistence of airway obstruction, resorting to high doses of inhaled corticosteroids or even oral steroid dependence, and in some cases, by lack of steroid sensitivity. It should be pointed out that patients, nursing staff (nurses, educators in asthma schools etc.) , general practitioners, and even respiratory physicians are not always familiar with these notions of seriousness, control, exacerbations, and severity. They, therefore, often differ in their assessment of the same clinical situation. Efforts to provide information, education, and coherence are therefore required more than ever. Precipitating Factors

The risk factors of loss of control of asthma are summarized in Table 2. Serious Acute Asthma

From the patient’s point of view, the seriousness of an attack may be defined by the following characters: * *

it is unusual and a doctor must be called; it leads to the discontinuation of current activity (work, school, or play); and

Interruption of anti-inflammatory treatment Viral infections Administration of aspirin or a betablocker Hormonal factors with premenstrual recurrence in woman Contact with allergen Atmospheric pollution Domestic pollution Meteorological factors Stress

Chromosome

Candidate gene

5q 6q

Th2 related cytokines TNF-a

11q

Clara cell protein CC10 FceRIb: IgE highaffinity receptor Interferon g

12q 14q 16q

T-lymphocytes receptor IL-4 receptor a

20q

ADAM 33

*

Potential relation with asthma Airway inflammation Airway inflammation, treatment Airway inflammation, treatment

Airway inflammation Airway inflammation, treatment Airway remodeling

Certain authors note that an attack leading to a change in primary therapy must be considered to be potentially serious.

For the doctor, it is always a potentially fatal medical emergency. Table 3 presents the clinical signs of serious acute asthma (SAA). Various items have been recognized as essential to management of SAA: 1. Value of evaluation protocols. 2. Requirement to measure respiratory function (PEF) and blood gases. 3. Efficacy of treatment with inhaled short-acting b2mimetics, systemic glucocorticoids, inhaled anticholinergics (48 h mainly in the child), and oxygen therapy. 4. Value of second-line therapy with intravenous b2mimetics, nebulization using helium oxygen as vector (heliox), intravenous magnesium. 5. Requirement for regular clinical re-evaluation. 6. The criteria for hospitalization are not validated, though they are widely used and consist of PEF o60% of the predicted value or o100 l min 1, no clinical improvement, severe underlying asthma,

ASTHMA / Overview 169 Table 4 Clinical signs of serious acute asthma

Table 6 Why new therapies are needed in asthma?

Threatening syndrome Worsening in a few days Increase in the frequency of attacks Increase of severity of attacks Resistance to treatment Increase in drug consumption Disease-free intervals less and less frequent Progressive decrease in PEF

Therapy

Intermittent mild persistent

Moderate persistent

Severe persistent

Side effects

None

Few potential

Efficiency Compliance

High Low

Variable Variable

Highest frequency Low Usually better

Signs of immediate seriousness Unusual and/or progressive dyspnea Difficulty in speaking or coughing Agitation Sweats and/or cyanosis SCM muscle permanently tensed RR 430 min 1 HR 4120 min 1 Paradoxical pulse 420 mmHg PEF o150 l min 1 Gain in PEF under treatment o60 l min PaCO2 440 mmHg

Table 7 Goals for asthma treatment Minimize symptoms Achieve best lung function Prevent asthma exacerbations Obtain treatment with the best therapeutic balance Educate patients for best partnership Avoid decline in lung function 1

Signs of distress Consciousness disorders Collapsus Pause in breathing Respiratory silence PEF, peak expiratory flow; HR, heart rate; RR, respiratory flow; SCM, sternocleidomastoid.

Table 8 ICS side effects usually not clinically significant if low doses are used Frequent and mild Oral candidosis Hoarseness Potential and mild Cough Skin bruising

Table 5 Risk factors of mortality in serious acute asthma Severe asthma (bronchial obstruction during intercritical period or frequent exacerbations) Patients presenting major rapid variations in bronchial obstruction (variation in PEF 430%) Major reversibility under bronchodilators Psychosocial instability Use of three drugs (or more) for asthma Frequent resort to ER for SAA, with hospitalization and/or intensive care Poor perception of symptoms Elderly patients Patients with hypereosinophilia Poor compliance and denial of disease

and psychosocial problems. The same is true for criteria concerning the return home (PEF 4 60% or 300 l min 1). After an episode of SAA, oral glucocorticoid therapy must be prescribed for from 7 to 14 days, combined with inhaled glucocorticoid therapy and follow-up with therapeutic education. The risk factors for SAA mortality are given in Tables 4 and 5. Suissa showed that the death rate due to asthma decreased by 21% per additional vial of inhaled corticoids received during the previous year. The death rate due to SAA therefore decreased if low doses of

Rare and potentially severe Surrenal insufficiency Biologically proven but long-term clinical significance unknown Osteoporosis Impaired growth velocity Cataract

inhaled corticoids were given as primary therapy. These data validate a fundamental aspect of the longterm therapeutic management of asthma (Tables 6, 7, and 8). Asthma Associations

Bronchopulmonary allergic aspergillosis This entity is defined by the following major symptoms: 1. recurrent lung infiltrates with eosinophils, sensitive to corticoids, 2. highly characteristic proximal bronchiectasis, 3. sometimes high blood eosinophilia (41000), 4. increased total IgE and specific IgE (measured by RAST) and IgG (precipitins) immune reaction against Aspergillus fumigatus, and 5. Aspergillus can sometimes be present in sputum.


These point to severe asthma, usually justifying long-term oral corticoid therapy and, in certain circumstances, antifungal treatment. Churg–Strauss syndrome This is a granulomatous and necrosing vasculitis. It is a rare form of asthma characterized by the severity of the respiratory symptoms, the levels of blood eosinophilia (generally above 1500 mm3), and the existence of extrarespiratory signs (neurological and cutaneous). It is often oral steroid-dependent and sometimes requires immunosuppressive agents.

Triggering and Aggravating Factors Asthma is a multifactorial syndrome. It is more appropriate to speak of triggering factors rather than etiological factors. The diagnostic procedure should take place in two stages – first, by investigating for the presence of such a factor in a patient and second, by evaluating its role in the global evaluation of the asthma (seriousness, control, and severity). Genetic Component

The genetic component is indisputable. It is well known that asthma runs in families. Conventionally, this hereditary constituent seems to be associated with atopy. However, recent work has demonstrated that even asthmatics with no demonstrable personal or family allergic factor (intrinsic asthma) had asthmatics in their family. Asthma is almost certainly polygenic in origin. Association with numerous polymorphisms have been reported at different loci in relation with or not with the known physiopathological data for the disease. The finding that different patients have different responses to antiasthmatic treatments has made it possible to develop potentially promising strategies for pharmacogenetic studies. For instance, data concerning the polymorphism of b2-mimetic receptor and the potential severity of the disease represent an interesting approach. Some examples of candidate genes implicated in the development of asthma are reported in Table 3. Environmental Component

Inhaled allergens These are allergens present in the ambient air. When inhaled in small quantities, they are capable of sensitizing subjects and triggering symptoms when they reach the level of the respiratory mucosa. Dermatophagoides pteronissimus. These form one of the major allergens of household dust. Animal proteins. Proteins produced by pets, laboratory experiments, or leisure activities.

Arthropods. They are insects such as locusts or cockroaches, which can cause asthma. Atmospheric molds and yeasts. They are an important source of allergens. Allergens present in insalubrious habitats are associated with more severe asthma. Food allergens Food and drinks may cause respiratory reactions after allergic sensitization, but nonallergic reactions or toxic reactions may also occur due to non-specific histamine release. Drugs Very often, drugs behave as haptens. Atopic subjects and asthmatics do not have any predisposition to hypersensitivity to drug products but when these occur, they are more violent. Aspirin intolerance This sometimes involves severe asthma, often with a late-onset with pan-rhino sinusitis with polyposis and systemic reactions, often with anaphylactic shock after ingestion of aspirin or nonsteroidal anti-inflammatory drugs. Occupational sensitizers They may be allergenic, but they may also act by toxic, irritative, or pharmacological mechanisms. The most important directly allergic allergen is wheat flour, responsible for bakers’ asthma. Another classical example of occupational asthma is due to work in contact with isocyanates. Demonstration that the asthma is caused by an occupational agent requires the use of a coherent, clinical, and functional diagnostic procedure, sometimes including bronchial challenge tests in the laboratory. Other factors Importance of viruses This is variable as a function of age in the triggering of asthma exacerbations. In the infant, wheezing bronchitis is usually caused by viruses though the possibility of asthma must not be over- or underestimated. Certain studies show that more than 60% of asthma exacerbations are related to a viral respiratory infection. Viral infections can be involved at various levels: * * * *

occurrence of exacerbations, prevention of the occurrence of asthma, induction of asthma, and persistence and development of chronic asthma due to chronic infections.

Bacterial infection This plays a secondary role in the physiopathology of asthma and as a triggering factor in exacerbations. The presence of endotoxins in the environment may be involved in the triggering

ASTHMA / Overview 171

of exacerbations in asthmatic patients, though it may also potentially protect an individual from the development of asthma symptoms (hygiene theory). Pollution SO2, NO2, and ozone act in synergy to trigger an attack. These different pollutants are responsible for exacerbations, sometimes serious or even very serious, especially in a child. The main sources of pollution are boiler plants (using fuel-oil and coal), household and industrial refuse incinerators, and motor vehicles (diesel engine). Particulate pollution has been shown to be associated in industrialized areas with an increase in asthma exacerbations. Diesel exhaust particles worsen allergic symptoms by amplifying the release of inflammatory mediators. Indoor pollution should not be forgotten and in particular that produced by home boilers. There are several evidences indicating that indoor pollution indicating a potential role for endotoxins, cockroaches, and parents’ smoking are major contributors for asthma exacerbations and lack of control in children with asthma. There is no definitive argument proving the involvement of atmospheric pollution in the acquisition of the asthma phenotype of a given subject. Gastroesophageal reflux Gastroesophageal reflux (GER) is more frequent in asthmatics than in a normal population. The acidity of the lower esophagus induces reflex bronchoconstriction. Contamination of the bronchi may also occur following incompetence of the cardia. Controlled studies have not shown the value of systematic treatment of GER to improve the asthma, even in the most severely affected patients. Psychological and/or psychiatric factors These are certainly involved, both in the expression of symptoms and beliefs on treatment, and therefore therapeutic compliance. They are, however, always difficult to evaluate. Influence of endocrine factors This is probable and the role of sex hormones is always mentioned first (effect of puberty, episodes of genital life in woman). Premenstrual exacerbations are rare events which can contribute to the severity of the disease. It has been shown recently that severe asthma is more frequent in women than in men. Menopause is an important period for asthma revival, poor control, or late-onset, but the exact sustaining mechanisms are yet to be understood. Sexual female hormonal replacement has been shown in one epidemiological study to be associated with an increase in emergency room attendance for asthma.

Exercise Induced Asthma

This is characterized by the occurrence of bronchial obstruction at the end of exercise. In certain cases, the asthma occurs during exercise though it is then possible to ‘run through the asthma’ if the exercise is continued. This is practically always present in children and adolescents and it may constitute a marker of bronchial hyperreactivity and inflammation. It is essential to control this form of asthma to allow normal physical activity and a harmonious physical and psychological development. Intercritical bronchial obstruction may be responsible for exertional dyspnea though this is not exertional asthma in the strict sense of the term. Smoking

Tobacco smoke (active and passive) by itself induces inflammation of the airways with hypersecretion, paralysis, and ciliary destruction, and infiltration of the distal aerial spaces by neutrophils. Active smoking is associated with a more severe asthma which responds less well to corticosteroids. It is not always easy to distinguish between asthma in the smoker and chronic obstructive pulmonary disease (COPD) with a certain degree of variability of the respiratory function. Maternal smoking may induce more asthma or atopy-associated diseases in children. Passive smoking by children is clearly responsible for uncontrollable asthma and this factor must be always looked for in the child. Many asthmatic adolescents are smokers and they have a high risk of loss of control and SAA. Adapted educational strategies must always be proposed.

Treatment Asthma treatment is based on three main axes: drug therapy, control of aggravating and triggering factors, and education of patients and health professionals. The objectives must be discussed with the patient, though the main aim is to obtain an acceptable control of the disease. Pharmacology

Corticocorticosteroids The efficacy of treatment with inhaled corticocorticosteroids is well established. It decreases functional signs, exacerbations, deaths, and costs, and improves the level of respiratory function and bronchial reactivity. All these effects have been demonstrated in comparison with placebo. Inhaled corticoids considerably improve the risk–benefit ratio of long-term treatment (Figure 1). They are indicated in persistent, mild, moderate, or


severe asthma. The local side effects of inhaled corticoids (candidiasis, hoarse voice) are effectively prevented by rinsing the mouth after inhalation or by the use of a spacer. At recommended dosages, in persistent mild to moderate asthma (and up to 1500 mg day 1 of beclomethasone equivalents), the risks of systematic adverse effects (adrenal insufficiency, osteoporosis) are low. The main adverse effects are susceptibility to bruises and skin atrophy. In the child, the mean dosage of 400 mg day 1 of budesonide does not modify growth. However, questions still remain about inhaled corticosteroids their efficacy in modifying the severity of asthma, their interaction with the natural history of the disease, or the long-term systemic side effects are still a topic of debate.

100

75

Side effects

Efficacy

50

25

0 Mild

Moderate

Severe

Figure 1 Risk–benefit ratio potential for drugs in the treatment of persistent asthma.

Bronchodilators b2-mimetics are very potent bronchodilators. They stimulate bronchial smooth muscle b2-receptors. Short-acting b2-agonists (from 4 to 6 h) may be distinguished from long-acting b2-agonists (12 h and more). This class of drug has no or very few anti-inflammatory effects, and causes little tachyphylaxis. Side effects are minor with mainly tremors, which disappear during treatment, and usually nonserious tachycardia-type palpitations. In persistent asthma, on-demand treatment with short-acting b2-agonists must be combined with continuous anti-inflammatory treatment. In case of suboptimal or unacceptable control in patients requiring daily doses of these short-acting b2-agonists and/or if symptoms occur at night, a long-acting b2-agonist may be prescribed. The use of shortacting b2-agonists is then reserved for the treatment of episodes of dyspnea occurring in spite of wellconducted treatment. A high consumption is then associated with loss of control. It is better to give them on demand, rather than systematically, and they are not associated with a risk of an increased death rate. The value of long-acting b2-agonists is demonstrated by a high level of evidence. These agents improve the control of asthma when inhaled corticoid therapy is insufficient alone. Their combination with inhaled corticoid therapy is better than a double dose of the latter drug. This strategy improves the control of the asthma, decreases the incidence and seriousness of exacerbations, and improves patient quality of life. At present, LABAs should never be considered as the sole treatment for asthma (Figures 2 and 3).

BHR

FEV1

Hours

Days

Weeks

Symptoms

∆% DEP

Months

Exacerbations

Figure 2 Improvement of control components after first treatment initiation in asthma.

…Years

ASTHMA / Overview 173 + High-dose ICS ± OCS ± Theophylline ± AntiIGE ± Steroid sparing ± Ipratropium ± Leukotriene antagonists

+ LA 2 agonists + Moderate-dose ICS ± Leukotriene antagonists

+ Low-dose ICS SA 2 agonists as required

Mild controlled

Severe uncontrolled

Education Environment control ± specific immunotherapy Comorbidities treatment Figure 3 Treatment of asthma according to control.

Figure 4 Pathology of asthma.

Inhaled corticoid–long-acting b2-agonist combination Combination in a same inhaler of a corticoid and a long-acting b2-agonist follows recommendations for the treatment of persistent, moderate to severe asthma, with facilitated administration, and the objective of improving compliance, notably of corticoid therapy. Two combinations have been developed: fluticasone–salmeterol and formoterol– budesonide. The benefit of these fixed combinations in comparison with separate combination of longacting b2-agonists and inhaled corticocorticosteroids

is not completely established and nor is the value of a fixed combination administered systematically or modulated on demand (Figure 4). Cysteinyl–leukotriene receptor antagonists The cysteinyl–leukotriene receptor antagonists used at present are montelukast and zafirlukast. They are used orally and potentially can treat asthma and rhinosinusitis. These agents have been shown to be useful in exercise induced-asthma, as well as a singleagent therapy of mild or moderate asthma. They lead


to bronchodilation and have been able to improve asthma control in long-term placebo-controlled studies. Their equivalence with low-dose inhaled corticoid therapy (400 mg day 1 of beclomethasone) is suspected but not fully demonstrated. The equivalence of a combination of an antileukotriene in comparison with a long-acting b2-agonist in noncontrolled patients treated with inhaled corticoid therapy alone is not proven. Their role in severe asthma is uncertain; it should be interesting, though, to follow their benefit in aspirin-induced asthma, which was recently demonstrated in some clinical studies. Theophylline This is a less potent bronchodilator than the b2-mimetics. It also has numerous other actions and in particular an anti-inflammatory activity. Its effects are strictly dependent on its serum concentration, which must be maintained in a relatively narrow range between 10 and 20 mg l 1. Side effects are also dose-dependent. At present it is not used much for primary treatment of asthma, except in the most severely affected patients. Anticholinergics Ipratropium and oxitropium are antagonists of muscarinic receptors. They are bronchodilators but are less potent and slower to act than b2-agonists. They are indicated in the treatment of SAA and exacerbations of chronic asthma as a complement to short-acting b2-agonists. Their efficacy beyond the first 48 h of the treatment is unknown. Proof of their efficacy exists especially for treatment in children. There are few adverse effects. Their efficacy in the management of chronic asthma has not been evaluated. Tioptropium (Spiriva) is a long acting anticholinergic drug, which has been developed for the treatment of COPD. Its potential interest in asthma and specifically severe asthma deserves further investigations. Control of the Environment

Although environmental control seems useful, there is no complete evidence of its efficacy. It comprises the reduction of exposure to allergens and non-specific irritants and the prevention of active and passive smoking. Allergy is one of the major factors associated with asthma and the most important for children of school age. Prevention of exposure to allergens is logically the first measure to be taken. This obviously concerns pets though this is not necessarily well accepted by patients or close relatives and friends. It is possible to try to eradicate house-dust mites though this is rarely complete. Diagnosis of occupational

asthma with a maximum of evidence may force a patient to change jobs. It is more difficult to adopt outdoor pollution-control measures which mainly concern the authorities. Immunotherapy

Specific immunotherapy is of some help in allergic asthmatic patients if simple rules are applied. Patients with persistent severe rhinitis and persistent mild asthma benefit most from this treatment. It is contraindicated in uncontrolled and severe asthma. On the other hand, the development of anti-IgE strategies may give hope to better control in severely allergic patients. Education

Education of patients is indicated in all asthmatics, though it should be adapted to the severity, importance of triggering factors, and specific personality of each patient. It must be based on a precise educational diagnosis. Objectives must be defined in partnership with the patient. The educational methods used depend on these objectives. This therapeutic education may be individual or may be provided in institutions such as asthma schools.

Conclusion Asthma is a bronchial inflammatory disease that alters the structure of the airways including the upper airways, which may sometimes have systemic repercussions. It is chronic, variable, and reversible and also a multifactorial, polygenic, and environmental disease. Management must be based on a long-term strategy. The major objective is to obtain satisfactory control to allow an optimal quality of life. Treatment involves use of a potent and well-tolerated pharmacopoeia, but may only be implemented in partnership with the patient. See also: Asthma: Allergic Bronchopulmonary Aspergillosis; Aspirin-Intolerant; Occupational Asthma (Including Byssinosis); Acute Exacerbations; Exercise-Induced; Extrinsic/Intrinsic. Bronchodilators: Anticholinergic Agents. Capsaicin. Carbon Monoxide. CD14. Chemokines. Chymase and Tryptase. Cilia and Mucociliary Clearance. Cyclic Nucleotide Phosphodiesterases. DNA: Repair. Dust Mite. Environmental Pollutants: Oxidant Gases. Eotaxins. Epidermal Growth Factors. Extracellular Matrix: Surface Proteoglycans. Fibroblasts. Gastroesophageal Reflux. G-Protein-Coupled Receptors. Histamine. Immunoglobulins. Interferons. Kinins and Neuropeptides: Vasoactive Intestinal Peptide; Other Important Neuropeptides. Leukocytes: Eosinophils. Lipid Mediators: Overview; Leukotrienes; Prostanoids; Platelet-Activating Factors. Oxidants and Antioxidants: Oxidants. Pediatric Pulmonary

ASTHMA / Overview 175 Diseases. Proteinase-Activated Receptors. Signs of Respiratory Disease: Lung Sounds. Symptoms of Respiratory Disease: Dyspnea. Tumor Necrosis Factor Alpha (TNF-a ).

Further Reading Abramson MJ, Puy RM, and Weiner JM (2003) Allergen immunotherapy for asthma. Cochrane Database of Systematic Reviews 4: CD001186. Agertoft L and Pedersen S (2000) Effect of long-term treatment with inhaled budesonide on adult height in children with asthma. New England Journal of Medicine 343: 1064–1069. American Thoracic Society (1995) Standardization of spirometry, 1994 update. American Journal of Respiratory and Critical Care Medicine 152: 1107–1136. ANAES (2004) Recommandations pour le suivi me´dical des patients asthmatiques adultes et adolescents. Paris 169pp (with an english translation on site www.anaes.fr). Anderson SD and Holzer K (2000) Exercise-induced asthma: is it the right diagnosis in elite athletes? Journal of Allergy and Clinical Immunology 106: 419–428. Barnes PJ (1995) Inhaled glucocorticoids for asthma. New England Journal of Medicine 332: 868–875. Barnes PJ (2004) Corticoresistance in airway disease. Proceedings of the American Thoracic Society 1: 264–269. Bateman ED, Boushey HA, Bousquet J, et al. (2004) GOAL Investigators Group. Can guideline-defined asthma control be achieved? The gaining optimal asthma control study. American Journal of Respiratory and Critical Care Medicine 170: 836–844. British Thoracic Society (2003) British guideline on the management of asthma. Thorax 58(supplement 1): S1–S83. Chung KF, Godard P, Adelroth E, et al. (1999) Difficult/therapyresistant asthma: the need for an integrated approach to define clinical phenotypes, evaluate risk factors, understand pathophysiology and find novel therapies. European Respiratory Journal 13: 1198–1208. Cookson W (1999) The alliance of genes and environment in asthma and allergy. Nature 402(supplement 6760): B5–B11. Cottin V and Cordier JF (1999) Churg–Strauss syndrome. Allergy 54: 535–551. D’Amato G, Liccardi G, D’Amato M, and Cazzola M (2002) Outdoor air pollution, climatic changes and allergic bronchial asthma. European Respiratory Monograph 21: 30–51. De Blay F, Casel S, Pauli G, and Bessot JC (2000) Respiratory allergies and household allergenic environment. Revue des Maladies Respiratories 17: 167–176. Ducharme FM (2003) Inhaled glucocorticoids versus leukotriene receptor antagonists as single agent asthma treatment: systematic review of current evidence. British Medical Journal 326: 621. Eggleston PA, Bush RK, and American Academy of Asthma, Allergy and Immunology (2001) Environmental allergen avoidance: an overview. Journal of Allergy and Clinical Immunology 107(supplement 3): S403–S405. Everard ML, Bara A, Kurian M, Elliott TM, and Ducharme F (2002) Anticholinergic drugs for wheeze in children under the age of two years. Cochrane Database of Systematic Reviews 1: CD001279. Gern JE and Busse WW (2000) The role of viral infections in the natural history of asthma. Journal of Allergy and Clinical Immunology 106: 201–212. Gibson PG, Coughlan J, and Wilson AJ (1998) The effects of self management and asthma education and regular practioner

review in adults with asthma. Cochrane Database of Systematic Reviews 3: CD001279. Gibson PG, Henry RL, and Coughlan JL (2003) Gastro-oesophageal reflux treatment for asthma in adults and children. Cochrane Database of System Review 2: CD001496. Holgate ST (1999) Genetic and environmental interaction in allergy and asthma. Journal of Allergy and Clinical Immunology 104: 1139–1146. Juniper EF, O’Byrne PM, Ferrie PJ, King DR, and Roberts JN (2000) Measuring asthma control. Clinic questionnaire or daily diary? American Journal of Respiratory and Critical Care Medicine 162: 1330–1334. Juniper EF, O’Byrne PM, Guyatt GH, Ferrie PJ, and King DR (1999) Development and validation of a questionnaire to measure asthma control. European Respiratory Journal 14: 902–907. Kips JC, O’Connor BJ, Inman MD, et al. (2000) A long-term study of the anti-inflammatory effect of low-dose budesonide plus formoterol versus high-dose budesonide in asthma. American Journal of Respiratory and Critical Care Medicine 161: 996– 1001. Kips JC, Peleman RA, and Pauwels RA (1999) The role of theophylline in asthma management. Current Opinion in Pulmonary Medicine 5: 88–92. Lemiere C, Bai T, Balter M, et al. (2004) Adult asthma consensus guidelines update 2003. Canadian Respiratory Journal 11(supplement A): A9–A18. Meslier N, Racineux JL, Six P, and Lockhart A (1989) Diagnostic value of reversibility of chronic airway obstruction to separate asthma from chronic bronchitis: a statistical approach. European Respiratory Journal 2: 497–505. O’Hollaren MT, Yunginger JW, Offord KP, et al. (1991) Exposure to an aeroallergen as a possible precipitating factor in respiratory arrest in young patients with asthma. New England Journal of Medicine 324: 359–363. Parker JM and Guerrero ML (2004) Airway function in women: bronchial hyperesponsiveness, cough and vocal cord dysfunction. Clinics in Chest Medicine 25: 321–330. Pauwels RA, Lofdahl CG, Postma DS, et al. (1997) Effect of inhaled formoterol and budesonide on exacerbations of asthma. New England Journal of Medicine 337: 1405–1411. Reddel H, Ware S, Marks G, et al. (1999) Differences between asthma exacerbations and poor asthma control. Lancet 353: 364–369. Rosenstreich DL, Eggleston P, Kattan M, et al. (1997) The role of cockroach allergy and exposure to cockroach allergen in causing morbidity among inner-city children with asthma. New England Journal of Medicine 336: 1356–1363. Salmeron S, Liard R, Elkharrat D, et al. (2001) Asthma severity and adequacy of management in accident and emergency departments in France: a prospective study. Lancet 358: 629–635. Shrewsbury S, Pyke S, and Britton M (2000) Meta-analysis of increased dose of inhaled steroid or addition of salmeterol in symptomatic asthma (MIASMA). British Medical Journal 320: 1368–1373. Sistek D, Tschopp JM, Schindler C, et al. (2001) Clinical diagnosis of current asthma: predictive value of respiratory symptoms in the SAPALDIA study. Swiss study on air pollution and lung diseases in adults. European Respiratory Journal 17: 214–219. Suissa S, Ernst P, Benayoun S, Baltzan M, and Cai B (2000) Lowdose inhaled corticocorticosteroids and the prevention of death from asthma. New England Journal of Medicine 343: 332–336. ten Brinke A, Ouwerkerk ME, Zwinderman AH, Spinhoven P, and Bel EH (2001) Psychopathology in patients with severe asthma is associated with increased health care utilization. American Journal of Respiratory and Critical Care Medicine 163: 1093–1096.

176 ASTHMA / Allergic Bronchopulmonary Aspergillosis Thomas NS, Wilkinson J, Lio P, et al. (2000) Genetic factors involved in asthma and atopy. Studies in British families. Revue des Maladies Respiratories 17: 177–182. Thomson NC, Chaudhuri R, and Livingston E (2004) Asthma and cigarette smoking. European Respiratory Journal 24: 734–739. Venables KM and Chan-Yeung M (1997) Occupational asthma. Lancet 349: 1465–1469. Vignola AM, Chanez P, Godard P, and Bousquet J (1998) Relationships between rhinitis and asthma. Allergy 53: 833–839. Vollmer WM, Markson LE, O’Connor E, et al. (1999) Association of asthma control with health care utilization and quality of life. American Journal of Respiratory and Critical Care Medicine 160: 1647–1652. Wark PA and Gibson PG (2001) Allergic bronchopulmonary aspergillosis: new concepts of pathogenesis and treatment. Respirology 6: 1–7. WHO/NHLBI Workshop Report (1995) Global Strategy for Asthma Management and Prevention. National Institutes of Health, National Heart, Lung and Blood Institute, Publication Number 95-3659 (revised 2002). Wilson AJ, Gibson PG, and Coughlan J (2003) Long acting beta-agonists versus theophylline for maintenance treatment of asthma. Cochrane Database of Systematic Reviews 3: CD001281. Woolcock AJ and Peat JK (1997) Evidence for the increase in asthma worldwide. Ciba Foundation Symposium 206: 122–134.

Allergic Bronchopulmonary Aspergillosis P Wark, Southampton University, Southampton, UK & 2006 Elsevier Ltd. All rights reserved.

Introduction Allergic bronchopulmonary aspergillosis (ABPA) is a complex condition that results from hypersensitivity to the fungus Aspergillus fumigatus (Af). ABPA was first described in the UK in 1952, and has been estimated to occur in 1–2% of chronic asthmatics and up to 15% of patients with cystic fibrosis. It is unclear whether the prevalence of ABPA has declined with the widespread use of inhaled corticosteroids to treat asthma. There is an excessively high prevalence of ABPA in cystic fibrosis where evidence suggests that ABPA may cause a faster decline in lung function. In chronic asthma, ABPA follows a more variable course with recurrent exacerbations and, at least in some patients, it leads to proximal bronchiectasis and irreversible fibrotic lung disease. Exposure to Af can cause a wide range of pulmonary diseases, which are summarized in Figure 1, including: severe life-threatening pneumonia in the immunosuppressed; subacute infections such as the

formation of aspergilloma in those with pre-existing pulmonary cavities; a form of extrinsic allergic alveolitis; and hypersensitivity diseases. Subjects with asthma or cystic fibrosis (CF) may become sensitized to Af following inhalation of spores; once sensitized this results in a type I, IgE-mediated reaction and a spectrum of clinical responses from acute exacerbations of asthma to a more sustained and intense inflammatory response that shows features of both type I and type III hypersensitivity and leads to ABPA. ABPA is unique in these disorders as there is evidence of persistence of the organism within the airways resulting in an intense local and systemic immune response that is associated with mucus impaction and may lead to bronchiectasis and pulmonary fibrosis.

Etiology Fungi belonging to the genus Aspergillus are ubiquitous spore-forming filamentous fungi and while any member of the group can cause allergic sensitization most disease is attributable to Af. The organism is present in outdoor environments where it readily grows in decomposing organic matter, such as decaying vegetation in soil, mulches, wood chips, freshly mown grass, and sewage treatment debris. Indoors, Af can be found in damp areas especially on walls or ceilings where water damage has occurred; in addition, spores can be found in very large quantities in bird excreta. Acute inhalation of spores is known to trigger acute asthma in sensitized asthmatics, while in subjects with ABPA inhalation of Af antigens leads to a dramatic worsening of the acute and late-phase response with falls in FEV1 of 50–79%. Exacerbations of clinical disease have been reported to occur coincident with high outdoor spore counts but a direct temporal relationship to inhalation and an acute exacerbation of ABPA remains unclear. A genetic predisposition to ABPA is also possible, with reports of familial occurrence. Two small studies have shown a higher carriage rate of at least one mutation of the cystic fibrosis transmembrane regulator (CFTR) gene in subjects with ABPA, compared to subjects who are skin-test positive to Af and those with asthma alone. The alleles HLA-DR2 and -DR5 have also been linked to severity of disease in ABPA. Most recently, a polymorphism of surfactant proteins has been shown to be associated with increased serum IgE and blood eosinophilia in patients with ABPA. These findings suggest there may be a link between ABPA and an inability to effectively clear the organism in a group whose immune response is likely to be one associated with atopy and a T-helper (TH)-2 response.

ASTHMA / Allergic Bronchopulmonary Aspergillosis 177

No previous lung disease

Asthma

Cystic fibrosis

Cavitatary lung disease

Immunocompromised

Inhale Af spores

Acute asthma (those sensitized up to 30%)

No immune response

Colonization with Af

Mucoid impaction extrinsic allergic alveolitis (all extremely rare)

ABPA (1− 2%)

ABPA (10 −15%)

Aspergilloma

Invasive pulmonary aspergillosis

Figure 1 A summary of the wide range of pulmonary diseases caused by exposure to Aspergillus fumigatus. Aspergillus fumigatus is a ubiquitous environmental fungus and spores are regularly inhaled. For most individuals with no previous lung disease, this appears to cause no immune response and no symptoms. High-dose exposure or exposure of susceptible individuals may trigger extrinsic allergic alveolitis or, more rarely, colonization leading to an intense immune response as in ABPA with areas of mucoid impaction and inflammation. Up to 30% of subjects with asthma are sensitized to Af. In those individuals exposure to Af may initiate an acute asthma response. A proportion of asthmatics seem to be particularly prone to the effects of Af and are unable to clear the organism. It persists in the lower airways and initiates the intense immune response resulting in ABPA, which occurs in 1–2% of asthmatics. In CF, particularly in those with atopy or a history of asthma-like symptoms, sensitization to Af occurs. In at least 10–15% of CF individuals the organism is not cleared and colonization ensues. This initiates an immune response like that seen in asthma but is associated also with the endobronchial inflammation and chronic infection already present in CF. An aspergilloma (fungal ball) consists of masses of fungal mycelia, inflammatory cells, fibrin, mucus, and tissue debris, these usually developing in a preformed lung cavity. In a study of 549 subjects with pulmonary cavities due to old tuberculosis, aspergillomas were present in 11%. Subjects with immunosuppression subsequent to chemotherapy or the use of immunosuppressives or with disorders such as chronic granulomatous disease are unable to clear Af. In these cases the immune response is so ineffective that direct fungal invasion occurs, leading to a severe acute pneumonia, sepsis, and often death.

Pathology In the early stages of ABPA, pulmonary lesions consist of bronchial mucoid impaction, and areas of eosinophilic pneumonia. In mucoid impaction, bronchi are dilated and filled with mucus that contains necrotic eosinophils, Curschman’s spirals, Charcot-Leyden crystals, and occasionally fungal hyphae. Eosinophilic pneumonia is synonymous with radiographic infiltrates, composed of focal areas of alveoli filled with eosinophils and macrophages. As the disease progresses, endobronchial inflammation increases, which begins as a mixed eosinophilic/neutrophilic obliterative bronchiolitis. This then progresses to the complete replacement of bronchial structures by inflammatory cells (histiocytes, lymphocytes, and plasma cells),

known as bronchocentric granulomatosis. At the center of these areas are necrotic inflammatory cells and Af hyphae. Destruction of the bronchial wall matrix and subsequent scarring and repair are thought to account for the development of bronchiectasis and fibrosis. The intensity of the inflammatory infiltrate present in the airways of subjects with ABPA certainly correlates with the severity of bronchiectasis present. Pathological features of ABPA are demonstrated in Figure 2.

Clinical Features Diagnosis and Disease Staging

The criteria for diagnosis were standardized in 1977 by Patterson and co-workers and are summarized in

178 ASTHMA / Allergic Bronchopulmonary Aspergillosis Table 1 Diagnostic criteria for ABPA Essential criteria Asthma Immediate skin-prick test (SPT) positive to Af Total serum IgE 4 1000 ng ml 1 (400 IU ml 1) Serum specific IgG and IgE antibodies to Af (or positive precipitins) Presence of bronchiectasis Yes, define as ABPA-CB No, define as ABPA-S

(a)

(b)

(c) Figure 2 (a) Hyphae of Aspergillus fumigatus grown in vitro; (b) mucoid impaction. The dilated bronchus is filled with mucus that contains eosinophils, cell debris, and the products of eosinophil degranulation (Charcot-Leyden crystals). The bronchial epithelium is heavily infiltrated by inflammatory cells. (c) Bronchocentric granulomatosis: the epithelium of the bronchus becomes completely replaced by a granulomatous infiltrate of histiocytes, lymphocytes, plasma cells, and eosinophils. In addition, there are necrotic inflammatory cells and mucus within the dilated airway and marked generalized bronchial wall thickening. Slides Courtesy of Dr B Addis, Southampton General Hospital.

Table 1. They require the patient to have a pre-existing diagnosis of asthma (or cystic fibrosis), immediate-type skin reactivity to Af, and, at least during exacerbations or in the absence of treatment,

Nonessential criteria Transient pulmonary infiltrates on chest radiograph Blood eosinophilia Precipitating antibodies to Af Expectoration of mucus plugs

peripheral blood eosinophilia, precipitating antibodies to Af antigen, elevated serum IgE, and IgG antibodies against Af. Radiological evidence of proximal bronchiectasis is frequently found with ABPA, but is no longer felt to be a prerequisite for diagnosis. However, the presence of bronchiectasis along with skin-test reactivity and eosinophilia is quite specific for ABPA. ABPA has been subdivided into five stages. Stage I is the initial acute presentation, with eosinophilia, immediate-type skin reactivity to Af, total serum IgE greater than 2500 ng ml 1, and pulmonary infiltrates on a chest radiograph. Stage II is the disease in remission, where there is persistent immediate-type skin reactivity and precipitating antibodies to Af antigens. In stage III there is an exacerbation of symptoms with all the characteristics of stage I but with a twofold rise in serum IgE and new pulmonary infiltrates. Stage IV patients have asthma where control of symptoms is dependent on chronic use of highdose corticosteroids. In stage V, chronic disease has progressed to predominately fixed airflow obstruction with extensive bronchiectasis and fibrosis. Skin-prick testing is a useful screening test to identify potential patients with ABPA. It is highly sensitive but not specific. Consequently, a negative skin-prick test for Af can rule out ABPA whereas a positive test warrants further investigation, particularly in the case of an asthmatic with frequent exacerbations or parenteral corticosteroid dependence. Patients with a positive skin test should be evaluated with serology including measurement of total serum IgE, specific IgG, and IgE antibodies to Af and precipitins. Patients with an IgE 4 1000 ng ml 1 (400 IU ml 1) and positive IgE Af and IgG Af are likely to have ABPA and warrant further investigation with a high-resolution computed tomography (CT) scan of the chest to determine the presence of bronchiectasis. Patients with central bronchiectasis are classed as

ASTHMA / Allergic Bronchopulmonary Aspergillosis 179

ABPA-CB (ABPA with central bronchiectasis) and those without are classed as ABPA-S (ABPA serology). In milder disease, a high index of suspicion is required, with the criteria fulfilled only during an exacerbation or when off parenteral corticosteroids. Radiology

During acute disease flares chest radiographs may demonstrate transient pulmonary infiltrates that occur as a result of eosinophilic pneumonitis and areas of mucoid impaction that appear as areas of atelectasis with toothpaste shadows and the finger-in-glove appearance of bronchi filled with inspissated mucus. Permanent radiographic changes are a feature of more advanced disease with the development of central bronchiectasis and in some areas of parenchymal fibrosis. While bronchial wall thickening and minimal bronchiectasis (limited to 1–2 segments) has been described in asthma, the presence of widespread central bronchiectasis, which is best seen on high-resolution CT scans, is characteristic of ABPA. In addition, areas of bronchial wall thickening, mucoid impaction, circular opacities, and areas of atelectasis are common. ABPA and Cystic Fibrosis

Diagnosis of ABPA in CF is more difficult though the criteria have recently been reviewed and standardized by a consensus conference; these criteria are summarized in Table 2. The diagnosis in CF is complicated by the pre-existing presence of bronchiectasis and chronic endobronchial infection. A multiple regression analysis of over 14 000 individuals with CF determined that wheezing, a diagnosis of bronchial asthma, and colonization with Pseudomonas aeruginosa were independent risk factors for ABPA. The development of ABPA in someone with cystic fibrosis heralds a more complicated clinical course and is associated with more bacterial colonization, lower lung function, and an increase in pulmonary complications.

Pathogenesis Af is an effective pathogen in humans as intrinsic qualities of the organism enhance its ability to infect Table 2 Diagnostic criteria for ABPA in cystic fibrosis An acute or subacute deterioration in clinical symptoms (cough, wheeze, exercise capacity, change in pulmonary function, or increase in sputum) not attributable to another cause Immediate skin test reactivity to Af A total serum IgE 4 400 IU ml 1 Along with one of the following: precipitins to Af or specific IgG to Af new or recent infiltrates, mucus plugging, or proximal bronchiectasis on either chest radiograph or CT scan

the lungs and cause disease. The spores of Af are 2– 5 mm in size with a hydrophobic coat that allows them to be inspired into the lungs. Once inspired, the conidia of Af are able to bind to surfactant molecules in the distal airway lumen, as well as complement (C3) and fibrinogen. The conidia germinate within the airways and form focal areas of mycelia. The mature organisms are then capable of releasing allergens, virulence factors, and proteases. These factors contribute to: (1) impaired mucociliary clearance; (2) impaired action of fungicidal proteins and complement in the airway lining fluid; and (3) inhibition of phagocytosis and the killing capacity of phagocytic cells (macrophages, neutrophils). As the organism is ubiquitous in the environment, sensitized individuals are likely to regularly inhale spores and this will always represent a fresh source of antigenic stimulus. However, Af is unique as an aeroallergen and inhalation alone is insufficient to lead to ABPA. Persistence of viable Af within the airways appears to be an important factor in determining the development of ABPA. Viable Af has been found growing on and between bronchial epithelial cells, despite an intense inflammatory cell infiltrate while Af proteases lead to the release of proinflammatory mediators from epithelial cells. These proteases also have the ability to detach epithelial cells from their basement membrane and are particularly potent. This loss of epithelial integrity may lead to exposure of the underlying matrix, to which Af can also adhere allowing direct damage of the airways and the development of bronchiectasis; in addition, the disruption of mucosal integrity enhances antigen uptake and activation of T cells. In addition to its direct effect on the airway epithelium, Af appears to be able to elicit a powerful TH2 immune response. The presence of Af infection in mice induces a TH2 lymphocyte response, while the extracts from Af when co-cultured with B cells elicit the release of IgE and subjects with ABPA develop specific TH2 CD4 þ cells in response to exposure. Epithelial cell activation and a TH2 immune response favors recruitment and activation of eosinophils. Eosinophil activation and release of toxic granular proteins are likely to contribute to damage to both the epithelium and airway matrix. In asthma TH2 cells also release IL-4 and IL-13, which induces the release of TGF-b from epithelial cells, leading to a repair response with myofibroblast activation, fibrosis, and airway wall remodeling. This intense recruitment of inflammatory cells to the airway lumen and the development of progressive airway wall damage is in keeping with airway inflammation found in patients with ABPA and the development of radiographic bronchiectasis.

180 ASTHMA / Allergic Bronchopulmonary Aspergillosis

1

Mucoid impaction

Af conidia

Tenacious mucus

Alveolar macrophage 3

2

Proteases

Epithelial detachment

Mycelia

Fibroblast 8 Allergens

Inflammatory cytokines/chemokines

4

6 5

IgE IL-5

B cell

TH2 lymphocyte

IL-4 & IL-13

Eosinophil

7

Figure 3 A model for the pathogenesis of ABPA. 1: The conidia of Af are just 2–5 mm in diameter and are easily respired into the distal airways. 2: The conidia are trapped within the tenacious mucus bilayer, binding to surfactants, complement, and fibrinogen. The conidia attach to bronchial epithelial cells. Here they germinate and mature, forming mycelia or small fungal balls. 3: Mature Af produces proteases and allergens. Aspergillus proteases are extremely potent. They activate epithelial cells, releasing proinflammatory cytokines and chemokines, leading to the hypersecretion of mucus, thus damaging epithelial cells, which results in widespread epithelial detachment and the loss of mucosal integrity. This allows the proteases to damage the airway matrix also. In addition, proteases can block the ability of alveolar macrophages to ingest Af, the net effect being impaired mucociliary clearance and facilitated colonization. 4: Activated epithelial cells release proinflammatory mediators, such as interleukin-8 (IL-8), IL-6, RANTES, and MCP-1, activating and attracting lymphocytes, eosinophils, and neutrophils to the airways. 5: Aspergillus allergens are potent activators of TH2 type immune response from TH cells. These cells release IL-5, which recruits and activates eosinophils. 6: Eosinophils migrate to the airways and infiltrate. Once activated, they release toxic granular proteins that further damage the epithelium and airway matrix. 7: Activated TH2 lymphocytes produce IL-4 and IL-13, which induce isotype switching of bells to produce IgE antibodies. In addition, IL-4 and IL-13 have been shown to induce epithelial cells to secrete the anti-inflammatory but pro-fibrotic mediator TGF-b. 8: Epithelial and airway matrix damage elicits a wound-repair response in the airways. Release of TGF-b has been shown to induce fibroblasts to release growth factors and collagen. This is thought, at least in part, to induce airway wall remodeling in asthma.

A model for the pathogenesis of ABPA is proposed in Figure 3. ABPA appears to develop in susceptible individuals who are sensitized to Af; they are unable to clear the organism, which then adheres to the bronchial epithelium releasing allergens and proteases. This activates an intense TH2 immune response and leads to progressive airway destruction and remodeling.

Animal Models A number of investigators have employed animal models to determine the role of Af antigens in triggering the immune response and in determining the

constitutive components responsible for this response. Most investigators have used crude Af extract to sensitize animals, often followed by ongoing exposure to viable conidia or Af spores. Uniquely, sensitization with Af extract does not require an adjunct; it is thought that Af proteases effectively disrupt the mucosal barrier enhancing antigen presentation. Recently, recombinant antigen components of Af extract have been used; identifying Asp f 1, 3, and 4 as independent inducers of airway inflammation and bronchial hyperresponsiveness (BHR). The most widely employed animal model has been murine. As in humans, sensitization to Af leads to a

ASTHMA / Aspirin-Intolerant Table 3 Components of the immune response to Aspergillus elucidated by murine models of ABPA Immune mediator

Response observed in the model

Block IL-13

Reduced pulmonary eosinophilia and goblet cell hyperplasia No change to subepithelial fibrosis Reduced IgE and pulmonary eosinophila More pronounced TH1 response and consequent pulmonary inflammation Inhibit all aspects of the TH2 inflammatory response to Af Reduced pulmonary eosinophils Airway inflammation occurs with monocyte infiltrate and BHR still increased (a) Treated at time of Af sensitization, increased airway inflammation, and BHR (b) Treated at 14–30 postsensitization, reduced airway inflammation, and BHR Increased TH2-mediated inflammation Increased IgE production Reduced TH1 response with lower levels of interferon-g and reduced pulmonary neutrophils Reduced clearance of Af

Block IL-4

Addition of IL-10 Block IL-5

Inhibit MCP-1 or inactivate receptor CCR2 CCR2 knockout mice

potent response with production of IgE, IgG1, pulmonary eosinophilia, and TH2 activation and their recruitment to the airways. In addition, such models have identified Af as potent inducers of BHR, airway remodeling, goblet cell hyperplasia, and mucus hypersecretion. The ability to use knockout animals and block specific cytokines, chemokines, and their receptors in murine models has allowed a greater understanding of the specific components of the immune response to Af; the most important of these are summarized in Table 3. The profound TH2 immune response to Af has been well demonstrated in these models along with the suggestion that such a response is relatively ineffective in clearing the organism. See also: Chemokines, CXC: IL-8. Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Interleukins: IL-4; IL-13. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading Bateman ED (1994) A new look at the natural history of aspergillus hypersensitivity in asthmatics. Respiratory Medicine 88: 325. Bosken CH, Myers JL, et al. (1988) Pathological features of allergic bronchopulmonary aspergillosis. American Journal of Surgical Pathology 12(3): 216–222. Chetty A (2003) Pathology of allergic bronchopulmonary aspergillosis. Frontiers in Bioscience 8: e110–e114. Greenberger P, Miller T, et al. (1993) Allergic bronchopulmonary aspergillosis in patients with and without evidence of bronchiectasis. Annals of Allergy 70: 333–338.

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Grunig G and Kurup VP (2003) Animal models of allergic bronchopulmonary aspergillosis. Frontiers in Bioscience 8: e157–e171. Kauffman HF (2003) Immunopathogenesis of allergic bronchopulmonary aspergillosis and airway remodelling. Frontiers in Bioscience 8: e190–e196. Kauffman HF and Tomee JF (1999) Inflammatory cells and airway defense against Aspergillus fumigatus. Immunology and Allergy Clinics of North America 18: 619–640. Kurup VP and Banerjee B (1996) Allergic aspergillosis: antigens and immunodiagnosis. Advances in Medical Mycology 2: 133–154. Patterson R, Greenberger P, et al. (1982) Allergic bronchopulmonary aspergillosis: staging as an aid to management. Annals of Internal Medicine 96: 286–291. Soubani AO and Chandrasekar PH (2002) The clinical spectrum of pulmonary aspergillosis. Chest 121: 1988–1999. Stevens D, Moss RB, et al. (2003) Allergic bronchopulmonary aspergillosis in cystic fibrosis state of the art: Cystic Fibrosis Foundation Consensus Conference. Clinical Infectious Diseases 37(supplement 3): S225–S264. Varkey B (1998) Allergic bronchopulmonary aspergillosis: clinical perspectives. Immunology and Allergy Clinics of North America 18(3): 479–501. Wark PAB and Gibson PG (2001) Allergic bronchopulmonary aspergillosis: new concepts of pathogenesis and treatment. Respirology 6: 1–7. Wark PAB, Gibson PG, et al. (2003) Azoles for allergic bronchopulmonary aspergillosis associated with asthma. Cochrane Database System Review 4(CD001108).

Aspirin-Intolerant A P Sampson, University of Southampton School of Medicine, Southampton, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Aspirin-intolerant asthma (AIA) is a phenotype experienced by 10–20% of persistent asthmatics, in whom acute bronchoconstriction is induced by ingestion of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs). These drugs share the ability to inhibit synthesis of prostanoids by blockade of cyclooxygenase (COX). Acute reactions to NSAIDs can be life threatening and may be associated with rhinoconjunctival and dermal symptoms. Drugs that selectively inhibit COX-2 appear to be better tolerated than nonselective inhibitors of COX-1 and COX-2. Patients with AIA usually have persistent underlying asthma, often associated with nasal polyposis. Pathologically, the bronchial and nasal airways of AIA subjects show chronic eosinophilia, with evidence of activation of eosinophils and mast cells during acute reactions. The etiology of AIA is unclear, but the proposed mechanism focuses on the inhibition by NSAIDs of the synthesis of a prostanoid, putatively prostaglandin E2, that would normally suppress local inflammatory reactions. The consequent synthesis of cysteinyl-leukotrienes and other leukocyte-derived mediators contributes to bronchoconstriction and other acute features. Treatment of AIA involves avoidance of NSAIDs combined with conventional management of underlying asthma, with 75% of AIA patients requiring corticosteroids. Controlled desensitization with regular doses of an NSAID can provide protection against acute reactions.

182 ASTHMA / Aspirin-Intolerant

Introduction The classical aspirin-intolerant asthma (AIA) syndrome was described by Samter and Beers in 1968 as a triad of rhinosinusitis (often with nasal polyps), asthma, and aspirin sensitivity. Patients with fullblown AIA are about twice as likely to be female as male, but no more likely to be atopic than the general population. Patients typically present in early middle age with nasal congestion, anosmia, and rhinorrhea, and many develop nasal polyps, often with secondary infection of the paranasal sinuses. Bronchoconstriction and airway inflammation usually emerge later, and this becomes severe, perennial asthma, with about 75% of patients needing oral or inhaled corticosteroids to maintain control of their symptoms. Acute respiratory reactions to nonsteroidal anti-inflammatory drugs (NSAIDs) may be accompanied by rhinoconjunctival symptoms and by dermal symptoms such as facial flushing and exacerbation of preexisting urticaria. Aspirin challenges reveal that the tendency to bronchoconstrict in response to therapeutic doses of NSAIDs is more prevalent than the full-blown ‘aspirin triad’ recognized clinically. Based on patient history alone, the prevalence of NSAID intolerance in adult asthmatics is 3–5%, but it rises to 19% when consecutive asthmatic patients are challenged with oral aspirin. Prevalence in asthmatic children is less than 2% based on history alone, but 13–16% of postpubertal asthmatic children respond adversely when aspirin challenged. NSAID intolerance is overrepresented in the severe asthmatic population. Among patients who have experienced a near-fatal asthma exacerbation requiring treatment in the intensive care unit, 24% are aspirin-sensitive based on history alone. Most life-threatening acute exacerbations in AIA patients are directly due to inadvertent use of NSAIDs, and in a few cases the precipitating NSAID was prescribed by the patient’s own physician. However, over 40% of life-threatening asthma exacerbations in AIA patients cannot be attributed to NSAID ingestion, illustrating the severity of the underlying chronic asthma in these patients.

Etiology Aspirin (acetylsalicylic acid) and other members of the family of NSAIDs inhibit formation of the prostanoid family of lipid mediators by blocking isozymes of cyclooxygenase (COX). They are used therapeutically for their mild analgesic, antipyretic, and anti-inflammatory actions. NSAIDs are variably associated with adverse reactions including prolonged bleeding, nephritis, and gastric ulceration.

Table 1 Chemical classes of some nonsteroidal anti-inflammatory drugs (NSAIDs) Acetic acids Indomethacin Sulindac Diclofenac

Fenamates Mefenamic acid

Oxicams Piroxicam

Propionic acids Ibuprofen

Pyrazoles Phenylbutazone

Benoxaprofen

Oxyphenbutazone

Salicylates Aspirin (acetylsalicylic acid) Sodium salicylate

In 1919, Cooke recognized that in some patients with asthma, aspirin may also precipitate life-threatening acute exacerbations. Such reactions have now been recognized in response to a wide variety of NSAIDs of diverse chemical classes (Table 1). A family history of AIA is reported by only 6% of AIA patients, suggesting that any genetic influences are subtle and expressed phenotypically only in the presence of relevant environmental factors. Leukotriene C4 synthase is the terminal enzyme for the synthesis of the bronchoconstrictor cysteinylleukotrienes postulated to contribute to airway narrowing in AIA. A biallelic polymorphism in the promoter region of the LTC4 synthase gene has been reported, involving an A to C transversion at a position 444 bp upstream of the transcription start site. The variant 444C allele may lead to enhanced transcription of the LTC4 synthase gene in relevant cells. The prevalence of the variant allele has been reported to be significantly elevated in AIA patients from a Polish population, but this has not been replicated in US Caucasian or Japanese populations.

Pathology A classification system for allergic and pseudoallergic reactions to NSAIDs was proposed by Stevenson, Sanchez-Borges, and Szczeklik in 2001. AIA is classified as a type 1 pseudoallergic reaction in which asthmatic patients with a high frequency of sinusitis and nasal polyps experience lower respiratory tract reactions and/or rhinoconjunctival symptoms after exposure to therapeutic doses of aspirin and other NSAIDs. Reactions are dose-dependent and may be life threatening. The type 2 category describes urticarial reactions or angioedema induced by NSAIDs in patients with pre-existing chronic urticaria, and types 3 and 4 describe urticarial or sporadic reactions in patients who are otherwise normal. In contrast to types 1–4 above, in which patients show extensive cross-reactivity to many NSAIDs, patients classified in types 5–8 react adversely only to a single

ASTHMA / Aspirin-Intolerant

drug. These reactions include urticaria/angioedema, anaphylaxis, aseptic meningitis, and hypersensitivity pneumonitis. A consistent, although not diagnostic, pathological finding in AIA patients is chronic eosinophilia in the blood, nasal polyps, and bronchoalveolar lavage (BAL) fluid. Immunohistochemical studies in bronchial biopsies have confirmed a marked bronchial mucosal eosinophilia in AIA patients, with eosinophil counts three- to fourfold higher than in aspirintolerant asthmatics and 10- to 15-fold higher than in normal subjects. After NSAID challenge, further increases in eosinophils, their activation marker ECP (eosinophil cationic protein), and histamine have been described in the nasal airways, BAL fluid, and plasma of AIA patients, suggesting activation of eosinophils and mast cells. In the nasal airway, aspirin challenge of AIA patients is associated with increments in nasal tryptase and histamine, strongly suggesting mast cell activation. Tryptase and histamine levels also rise in the serum of patients experiencing systemic reactions to oral aspirin, but not in those with localized respiratory reactions. Release of tryptase is a recognized marker of mast cell activation in the pulmonary airway, and occurs within 5 min of allergen challenge in allergic asthmatics. However, tryptase did not rise in the BAL fluid of AIA patients challenged with inhaled or endobronchial lysine-aspirin. In contrast, a rise in urinary levels of the PGD2 metabolite 9a, 11b-PGF2 following endobronchial lysine-aspirin challenge has been interpreted as evidence for mast cell activation in AIA. Together, the evidence suggests that inflammatory mediators released both from mast cells and from eosinophils contribute to the pulmonary and rhinoconjunctival symptoms following NSAID exposure in AIA patients.

Clinical Features As there are no acceptable in vitro tests for AIA, confirmation of aspirin intolerance can only be obtained by NSAID challenge under controlled conditions. Lung function is monitored while patients ingest incremental oral doses of aspirin or inhaled doses of a lysine-aspirin conjugate or sulpyrine. Concomitant use of b2-adrenergic agonists, cromones, and inhaled corticosteroids may mask responses to NSAID challenge, leading to a high rate of false-negative results. In a 3-day oral challenge protocol, incremental doses of aspirin are given at 3-hourly intervals up to a maximum of 650 mg. The challenge is terminated when forced expiratory volume in 1 s (FEV1) falls by at least 20%. Reactions begin around

183

50 min after oral aspirin ingestion, ranging from 20 to 120 min. A shorter protocol involves the inhalation of incremental aerosolized doses of lysine-aspirin, a soluble and nonirritant form (this is not available in the US). Respiratory reactions to inhaled lysineaspirin often occur within 1 min, so shorter dosing intervals of 30–60 min allow the entire challenge to be completed within 1 day. Inhaled lysine-aspirin challenges are safer than oral challenges as reactions are localized to the airways and are easily reversible with inhaled b2-agonists. The sensitivity of inhaled lysine-aspirin challenge is similar to oral aspirin challenge.

Pathogenesis The Cyclooxygenase Theory

The most successful model to explain acute respiratory reactions to NSAIDs is the cyclooxygenase theory promulgated by Szczeklik in 1975. This postulates that reactions are related directly to the pharmacological activity of NSAIDs in inhibiting isozymes of COX, the key enzymes in the synthesis of the prostanoid family of lipid-inflammatory mediators. Intolerance to an individual NSAID in vivo was shown to be predictable by its potency in inhibiting COX in vitro, with strong inhibitors (including aspirin, indomethacin, mefenamic acid, ibuprofen, and piroxicam) being common precipitants of adverse reactions, while weak inhibitors (such as sodium salicylate) precipitated reactions rarely or only at high doses. The concept that inhibition of prostanoid synthesis is the trigger for NSAID-induced reactions is recognized as a key advance. The Role of Cysteinyl-Leukotrienes: The Shunting Hypothesis and the PGE2 Brake Hypothesis

The second key advance was the elucidation in 1979 of the structure of slow-reacting substance of anaphylaxis (SRS-A) as a mixture of potent bronchoconstrictor products of a related family of lipid mediators, the cysteinyl-leukotrienes (cysteinyl-LTs). The cysteinyl-LTs – LTC4, LTD4, and LTE4 – are now recognized to have important bronchoconstrictor and proinflammatory roles in many phenotypes of asthma. Their particular relevance to AIA emerged from the recognition that their biosynthetic pathway, the 5lipoxygenase (5-LO) pathway, shares arachidonic acid as a common precursor with the prostanoid pathway (Figure 1). The leukotriene pathway is not inhibited by NSAIDs. Specific blockade of the prostanoid pathway by NSAIDs was therefore proposed to shunt arachidonate away from conversion into


NSAIDs

Membrane phospholipids Phospholipase A2

− Cyclooxygenase-1

Arachidonic acid −

Zileuton

5-lipoxygenase FLAP LTA4

PGG2 PGH2

Cyclooxygenase-2

Prostanoid synthases & isomerases

LTC4 synthase LTC4 LTD4 LTE4 (Cysteinyl-leukotrienes)

− Montelukast Pranlukast Zafirlukast

CysLT1 receptors

PGE2

TXA2

PGD2 PGF2

Prostanoid receptors (EP1−4, TP, DP, CRTH2, FP)

Bronchoconstriction Vascular permeability Edema Mucus secretion Eosinophil migration

Figure 1 The 5-lipoxygenase and cyclooxygenase pathways of eicosanoid metabolism.

prostanoids, which have relatively little bronchoconstrictor activity, towards the formation of cysteinylLTs, which are highly potent bronchoconstrictors. This ‘shunting’ hypothesis is superficially attractive but measurements of lipid mediators in isolated leukocytes treated with NSAIDs argues against such a simple mechanism. It has therefore been superseded by an alternative notion, the ‘PGE2 brake’ hypothesis. This proposes that NSAIDs block the formation of an anti-inflammatory prostanoid, PGE2, which is otherwise known to suppress leukotriene synthesis by leukocytes. Exposure to NSAIDs may thus liberate the 5-LO pathway from suppression by endogenous PGE2 in vivo, at least in susceptible individuals (Figure 2). There are three main lines of experimental evidence supporting this model: 1. The triggering effect of NSAIDs on LT synthesis can be mimicked in vitro in a number of inflammatory leukocyte subtypes. Endogenous PGE2 suppresses, and NSAIDs consequently enhance, leukotriene synthesis in eosinophils, neutrophils, basophils, and macrophages, but apparently not in human lung mast cells. Eosinophils themselves generate sufficient PGE2 to suppress their own LTC4 synthesis by about 90%. Treatment of eosinophils with indomethacin inhibits endogenous PGE2 synthesis and stimulates LTC4 release. Replacement experiments showed that

exogenous PGE2 restores the braking effect, returning LTC4 synthesis to the levels seen before indomethacin treatment. PGE2 may act in an autocrine or paracrine manner at EP2 receptors on the cell surface, followed by an increase in intracellular cAMP and activation of protein kinase A, but the subsequent steps by which 5-LO activity is inhibited are unknown. 2. Eicosanoids can be measured in biological fluids, including bronchoalveolar lavage (BAL) fluid, and urine. Endoscopic challenge with lysine-aspirin reduces PGE2 and thromboxane A2 levels in the BAL fluid of AIA patients and aspirin-tolerant asthmatics, but a dramatic rise in BAL fluid cysteinyl-LTs is seen only in the AIA group. Following challenge with oral aspirin or inhaled lysine-aspirin, urinary LTE4 levels, used as a marker of whole-body cysteinyl-LT production, rise three to sevenfold in AIA patients, but not in aspirin-tolerant asthmatics; this response is not seen after methacholine-induced bronchoconstriction or placebo challenge. At the same time, there is a fall in urinary markers of prostanoid synthesis, such as 11-dehydro-thromboxane A2. In AIA patients, preinhalation of PGE2 before challenge with inhaled lysine-aspirin completely ablates the rise in urinary LTE4 and prevents the consequent bronchoconstriction, providing strong evidence that cysteinyl-LT synthesis has a functional role in the NSAID-induced airway narrowing. The

ASTHMA / Aspirin-Intolerant

185

Mechanism of acute NSAID reactions Mast cell / eosinophil

NSAID COX-1

FLAP 5-LO

LTC4 synthase

LTC4

cAMP

ECP

Histamine Tryptase IL-5

EP2

PGE2

Bronchoconstriction Eosinophillia Vasodilation / edema Remodeling / BHR

Figure 2 In this model, PGE2 derived from constitutive cyclooxygenase-1 (COX-1) normally acts via cell surface EP2 receptors to suppress the activity of mast cells and eosinophils, including the activity of the 5-lipoxygenase pathway. However, when an NSAID ingested by a susceptible individual inhibits COX-1, the reduced synthesis of PGE2 is insufficient to suppress the leukocyte effectively. The result is a surge in the synthesis and release of LTC4 and other mediators, leading to bronchoconstriction and inflammations, AIA patients may be susceptible to this response because they have exaggerated LTC4 synthase expression, and/or a defect in the PGE2 brake, based perhaps on insufficient COX-1 activity or more likely on impaired signaling from EP2 receptors.

protective effect of inhaled PGE2 does not correlate with its relatively weak bronchodilator activity, confirming that PGE2 preinhalation protects by restoring the suppression of cysteinyl-LT synthesis, not by dilating airways directly. 3. The effector role of cysteinyl-LTs in adverse respiratory and rhinitic reactions to NSAIDs in most AIA patients has been confirmed with placebocontrolled clinical trials of specific leukotriene modifier drugs. The 5-LO inhibitors zileuton and ZD-2138 markedly blocked the rise in urinary LTE4 and the fall in FEV1 following oral aspirin challenge of AIA. Rhinoconjunctival and dermal reactions to oral aspirin are also blocked by zileuton. Antagonists of CysLT1 receptors block oral NSAID-induced respiratory reactions in AIA patients. The PGE2 Brake is Derived from COX-1

The cyclooxygenase theory and the PGE2 brake hypothesis are thus crucial in understanding how NSAIDs trigger LT synthesis and bronchoconstriction, but they do not explain why only some asthmatics are susceptible to these responses. AIA patients tolerate selective COX-2 inhibitors including nimesulide, meloxicam, and rofecoxib, and respond adversely most often to NSAIDs with a greater selectivity for COX-1, such as aspirin and indomethacin. This suggests that cytoprotective/anti-inflammatory PGE2 in the lung is produced by constitutive COX-1. COX-1 is expressed in a large number of cell types,

including mast cells, eosinophils, macrophages, vascular endothelial cells, bronchial epithelium, and bronchial smooth muscle. The exact cellular sources of the putative PGE2 brake remain unclear. AIA patients may overproduce cysteinyl-LTs chronically and acutely after NSAID exposure because of a defect in endogenous PGE2 synthesis. However, inhaled lysineaspirin equieffectively inhibits airway PGE2 synthesis both in AIA patients and in aspirin-tolerant asthmatics. Baseline levels of PGE2 and other prostanoids are not consistently different in the BAL fluid and urine of AIA patients and control groups. Immunohistochemical studies of AIA bronchial biopsies have found little evidence for a meaningful anomaly in the cellular expression of COX isozymes. One possibility is that a defective PGE2 brake may derive not from a failure of PGE2 synthesis, but of leukocytes to be suppressed by PGE2. An anomaly in EP2 receptor structure, expression, or signaling might be one explanation, and this would also be consistent with the lack of clinical benefit shown in AIA patients treated with the stable PGE1 analog misoprostol. Suggestive evidence of an anomaly within the cysteinyl-LT biosynthetic pathway itself is that baseline cysteinyl-LT synthesis appears to be two- to sevenfold higher in AIA patients than in control groups, even in the absence of exposure to NSAIDs, as demonstrated by measurements of cysteinyl-LTs in BAL fluid, induced sputum, and urine (Figure 3). The increases seen after NSAID exposure are superimposed upon this chronically elevated baseline. Immunohistochemical analysis of bronchial biopsies from

Cys-LT production


NSAID AIA

ATA N 0

1

2

3

Time (h) Figure 3 Schematic diagram showing that cysteinyl-leukotriene production is chronically elevated in patients with aspirin-intolerant asthma (AIA) compared to those with aspirin-tolerant asthma (ATA) and normal subjects (N). A further rise in cysteinyl-LT production after exposure to a nonsteroidal anti-inflammatory drug (NSAID) occurs only in the AIA group.

AIA and aspirin-tolerant asthmatic subjects shows that counts of cells immunostaining for LTC4 synthase, the terminal enzyme for cysteinyl-LT synthesis, were fivefold higher in AIA biopsies than in aspirintolerant asthma biopsies and 18-fold higher than in normal biopsies. The numbers of LTC4 synthasepositive cells in the bronchial mucosa correlated with elevated levels of cysteinyl-LTs in the BAL fluid and with bronchial responsiveness to inhaled lysine-aspirin. Persistent overproduction of cysteinyl-LTs in steady-state AIA may therefore be related to overexpression of LTC4 synthase in the bronchial wall, much of it within eosinophils and mast cells. This may also contribute to the further surge in cysteinylLT synthesis when PGE2 suppression is removed by NSAIDs.

Animal Models Although the anti-inflammatory actions of NSAIDs and their effects of prolonged bleeding, nephritis, and gastric ulceration can be replicated in animal models, there has been relatively little interest in developing animal models of AIA. This may reflect, at least in part, the difficulty of replicating the diverse immunopathological features of asthma, especially chronic remodeling of structural airway tissues, and also of replicating the typically high degree of disease severity and glucocorticoid dependency observed in AIA patients.

Management and Current Therapy National and international management guidelines for asthma (e.g., GINA guidelines) are based on disease severity and make no therapeutic distinctions between AIA and other asthma phenotypes. Asthmatics should

avoid using NSAIDs either prescribed inadvertently or purchased over the counter. NSAID-induced acute reactions in susceptible asthmatics can be treated with nebulized b-2 adrenergic agonists, repeated frequently over several hours where necessary. Decongestants and antihistamines (topical or oral) can be used for associated rhinoconjunctival symptoms. In the most severe cases, intubation and mechanical ventilation in the intensive treatment unit may be required. Treatment of chronic AIA focuses on anti-inflammatory therapy of the upper and lower airways using topical and inhaled/insufflated corticosteroids. Antibiotics may be required when purulent nasal secretions indicate infection, and many AIA patients require repeated polypectomies. Some AIA patients may require NSAIDs for concomitant inflammatory disease such as arthritis, and in such cases, aspirin desensitization may be an option. Acute respiratory reactions to NSAIDs in AIA patients are always followed by a refractory period lasting 2–5 days during which further reactions to NSAIDs cannot be induced. Sensitivity to NSAIDs re-emerges within a week, but regular low-dose NSAIDs can maintain the refractory state indefinitely. Daily or alternate-day dosing of NSAIDs is therefore used clinically to desensitize AIA patients to inadvertent ingestion of NSAIDs and to allow NSAID therapy of concomitant diseases such as arthritis. Cross-desensitization occurs such that repeated dosing with one NSAID provides protection against adverse reactions to other NSAIDs. Chronic desensitization reduced the numbers of acute exacerbations and hospital admissions in AIA patients compared to a control group of AIA patients who avoided NSAIDs, associated with reductions in corticosteroid use. The mechanism of desensitization is unknown, but is tentatively linked to reductions both in the quantity of cysteinyl-LTs synthesized following NSAID exposure, and with reduced bronchial responsiveness to cysteinyl-LTs. In the nasal airway, desensitization has been linked to a reduction in expression of the CysLT1 receptor on infiltrating leukocytes. In small studies in AIA patients, the antiallergic cromone sodium cromoglycate, the antiviral agent acyclovir, the antibiotic roxithromycin, and the longacting b2-agonist salmeterol have all been reported to block not only the bronchial responses to inhaled NSAIDs, but also the associated rise in urinary LTE4 excretion. The mechanism of these drugs is difficult to understand in this context, but may involve prevention of mast cell degranulation. On the basis of the pathophysiological evidence of a central role of cysteinyl-LTs in AIA, clinical trials of 5-LO inhibitors and CysLT1 receptor antagonists have been reported. Zileuton improved lung function and rescue beta-2

ASTHMA / Occupational Asthma (Including Byssinosis) 187

agonist use and restored the sense of smell in a 6week study of 40 AIA patients. In an 8-week crossover trial of montelukast in 80 AIA patients, there were significant improvements in lung function, use of rescue therapy, symptom scores, night-time awakenings, and asthma quality-of-life (QOL) scores compared with placebo. In the clinic, treatment response to leukotriene modifiers is variable, but a treatment trial is advised in patients with AIA uncontrolled by topical corticosteroids. See also: Allergy: Overview. Asthma: Overview. Bronchoalveolar Lavage. Bronchoscopy, General and Interventional. Chymase and Tryptase. Genetics: Overview. Histamine. Immunoglobulins. Leukocytes: Mast Cells and Basophils; Eosinophils; Neutrophils. Pulmonary Function Testing in Infants.

Further Reading Drazen JM, Israel E, and O’Byrne PM (1999) Treatment of asthma with drugs modifying the leukotriene pathway. New England Journal of Medicine 340: 197–206. Pavord ID and Tattersfield AE (1994) Bronchoprotective role for endogenous prostaglandin E2. Lancet 345: 436–438. Samter M and Beers RF (1968) Intolerance to aspirin: clinical studies and consideration of its pathogenesis. Annals of Internal Medicine 68: 975–983. Sanak M and Szczeklik A (2000) Genetics of aspirin-induced asthma. Thorax 55(supplement 2): S45–S47. Stevenson D, Sanchez-Borges M, and Szczeklik A (2001) A classification of allergic and pseudoallergic reactions to drugs that inhibit cyclooxygenase enzymes. Annals of Allergy, Asthma and Immunology 87: 1–4. Stevenson DD, Simon RA, and Zuraw BL (2003) Sensitivity to aspirin and nonsteroidal antiinflammatory drugs. In: Adkinson NF, Bochner BS, Yunginger JW, et al. (eds.) Middleton’s Allergy: Principle and Practice, 6th edn., pp. 1695–1710. St Louis, MO: Mosby. Szczeklik A, Gryglewski RJ, and Czerniawska-Mysik G (1975) Relationship of inhibition of prostaglandin biosynthesis of analogues to asthma attacks in aspirin sensitive patients. British Medical Journal 1: 66–69. Szczeklik A and Stevenson DD (2003) Aspirin-induced asthma: advances in pathogenesis, diagnosis, and management. Journal of Allergy and Clinical Immunology 111: 913–921.

Occupational Asthma (Including Byssinosis) D J Hendrick, Royal Victoria Infirmary, Newcastle upon Tyne, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Respirable agents in the workplace are responsible for about 10% of asthma arising in adult life, and for an annual incidence

of occupational asthma (OA) of 25–100 per million employed in industrially developed countries. For 5–10% of cases, toxic mechanisms are responsible, and asthma is the result of accidents that release agents such as chlorine, sulfur dioxide, and acetic acid into occupational environments. For the remaining 90–95%, asthma appears to arise through hypersensitivity mechanisms. Many of the several hundred causal ‘asthmagens’ are reactive chemicals of low molecular weight, though some are naturally occurring allergens of high molecular weight. Some agents (e.g., di-isocyanates, epoxy resins, flour) have such sensitizing potency that at current exposure levels in some occupations (e.g., spray painting and baking) the risk of OA exceeds that of asthma arising spontaneously. OA is thus important in an epidemiologic sense, and it serves as a useful model of asthma in general. Asthma of occupational origin is like asthma resulting from any other cause. However, when due to hypersensitivity mechanisms, it has one unusual characteristic. If the diagnosis is recognized within 6–24 months and exposure ceases, there is a meaningful possibility that active disease will resolve. Therefore, the onus is on physicians to consider the possibility of an occupational cause in every adult presenting with asthma.

Introduction It was not until the twentieth century that investigatory techniques acquired the sophistication to distinguish airway disorders from those of the lung parenchyma/interstitium. The definition of asthma, the elucidation of its clinical characteristics and pathogenic mechanisms, and the evolution of strategies for its recognition, management, and prevention have consequently been comparatively recent events. Nevertheless, it is likely that asthma was the explanation for many of the respiratory disorders of antiquity and the middle ages that were recognized to afflict (and sometimes devastate) workers in certain trades and industries. The realization that asthma may arise as a direct consequence of inhaled occupational agents has been the focus of particular attention over the last three to four decades, and our understanding of occupational asthma (OA) has largely arisen during this period. OA has no recognized differences from asthma in general with respect to its pathology and genetic etiology, and so this article will focus on its features of special interest. Definitions and Classification

Asthma is a disease of the intrathoracic airways that is characterized, and often defined, by its means of clinical expression (diffuse airway obstruction that varies in degree over time) and its underlying pathogenic basis (a state of enhanced airway responsiveness). OA is caused by exposure to agents, almost exclusively airborne, that are encountered primarily in the workplace. Such exposure in susceptible individuals causes the level of airway responsiveness to

188 ASTHMA / Occupational Asthma (Including Byssinosis)

increase into the asthmatic range. Two distinct mechanisms are recognized. First, hypersensitivity mechanisms appear responsible since there is a latency period of presumed sensitization between exposure onset and symptom onset; this accounts for 90–95% of cases. Second, acute toxicity is the initiating event; this is generally a consequence of inhalation of toxic agents during an industrial accident. Asthma following the latter is sometimes identified as the reactive airways dysfunction syndrome (RADS) or, more simply, irritant asthma. Neither term is fully satisfactory. The first may be interpreted, incorrectly, to indicate a disorder other than asthma, while the second may be mistaken for pre-existing asthma that is exacerbated non-specifically at work (e.g., because of exertion in cold air); this is ‘work-aggravated’ not ‘occupational’ asthma, and is not associated with an increase in airway responsiveness. It is currently unclear whether repeated exposures to toxic agents at dose levels insufficient to provoke clinical reactions might nevertheless cause minor increases in airway responsiveness. If so, the cumulative effect might elevate airway responsiveness to a level at which asthma eventually becomes inevitable (‘low-level RADS’). This would simulate the latency period association with presumed hypersensitivity mechanisms, and ongoing exposure might then provoke acute symptoms in a non-specific fashion. This would appear to simulate hypersensitivity mechanisms also, but such exposure should provoke reactions in any asthmatic individual with a sufficient level of airway responsiveness. Key Clinical Features

When hypersensitivity mechanisms are responsible, further exposures to the particular inducing asthmagen may provoke specific asthmatic reactions. Symptoms are dependent on the degree of hypersensitivity, the magnitude of the provoking stimulus, the level of airway responsiveness, and the ease with which the affected individual perceives changing respiratory sensations. Airway responsiveness varies in level from subject to subject over a considerable range, and can be quantified throughout the population at large. Its distribution (like that of most biologic parameters) is unimodal, and it follows the common biologic pattern of a ‘bell-shaped’ curve. The tail in which responsiveness is most marked gives rise to asthma, but the adjacent segment implies vulnerability, and the opposite tail implies comparative impunity. It is misleading to think of asthmatic and nonasthmatic subjects being fundamentally different because of the presence or absence of airway hyperresponsiveness;

the issue, rather, is whether an individual’s level of airway responsiveness is sufficiently high to make meaningful bronchoconstriction likely when there are appropriate provoking stimuli. Whether the resulting degree of bronchoconstriction is perceived to be distressing (or is perceived at all), is very dependent on psychological factors. This adds an important further level of complexity and variability, since at all degrees of asthmatic severity as defined physiologically, there will be a wide spectrum of perceived disability. This is particularly so in OA because of resentment, even anger, over the possible liability of a third party (the employer), and the possibility of compensation. Longstanding OA, like asthma generally, is commonly associated with airway obstruction that has a reduced capability for reversal. It may come to simulate chronic obstructive pulmonary disease (COPD). Epidemiology

Over recent decades, OA has proved consistently to be the commonest type of newly diagnosed occupational lung disease in industrially developed countries, though the various disorders attributable to asbestos have an equal cumulative incidence. Between them, asthma and asbestos account for 65–70% of all incident respiratory disorders of occupational origin. In most outbreaks of OA no more than a few per cent of exposed workers become affected, but there are examples with both pathogenic pathways of prevalences approaching 50%. The reported incidence of OA varies widely at a global level, depending on local employment patterns and diagnostic criteria. Estimates from statutory notification schemes, compensation registers, voluntary surveillance schemes, and general population surveys, suggest that 5–15% (median 9–10%) of asthma beginning in adult life is occupational. When asthma arises for the first time in a working adult, the background odds favoring an occupational cause over a nonoccupational cause are consequently of the order 1 in 10 only. This is consistent with the estimate from SWORD (Surveillance of Work-Related and Occupational Respiratory Disease) data that about 40 cases of OA per million employed workers arise in the UK each year. SWORD has usefully considered the incidence of new cases within specific working groups for which there are particular occupational exposures. For example, among spray painters, who may use asthmagenic di-isocyanate, epoxy resin, and acrylic paints, the average annual incidence of OA from 1992 to 1997 was 1464 per million – more than threefold the UK national average for all cases of incident asthma.

ASTHMA / Occupational Asthma (Including Byssinosis) 189

Thus, in the case of spray painters developing asthma in adult life, the background odds favor an occupational cause over a coincidental cause. Other settings associated similarly with greater than even odds include baking, metal treatment, chemical processing, and plastics manufacture. Most national estimates range from 25 to 100 million per year.

Table 1 Commonly reported causes of occupational asthma Animal sources Arthopods/ insects Birds Mammals Marine species

Bees, locusts, mites, silkworms, weevils Feather bloom, excreta Rodent urinary protein, pancreatic enzyme supplements Corals, crabs, fishmeal, oysters, prawns, sponges

Etiology Many occupational agents (some 400) have been reported to be definite or probable sensitizers capable of inducing asthma. Some of the most prominent are classified in Table 1, and the most common reports to SWORD over a 9-year period are listed in Table 2. Notable agents reported to cause RADS over recent years have been acetic acid, chlorine/chlorine dioxide/hydrochloric acid, di-isocyanates, dinitrogen tetroxide, endotoxin, fire smoke, freons, hydrobromic acid, Iraq/Iran war gases, pentamidine, phosphoric acid, silo and swine confinement gases, sulfur dioxide/sulfuric acid, tear gas, and welding fume.

Clinical Features Once a diagnosis of asthma is suspected from the clinical history or physical examination and confirmed objectively (by the demonstration of reversible airway obstruction or the measurement of airway responsiveness), the diagnostic issue turns to whether it has arisen occupationally. If the toxicity pathway has provided the mechanism, the diagnosis is clear and needs no further investigation. Asthma becomes evident during the recovery phase from what is usually a combination of conjunctivitis, rhinitis, pharyngolaryngitis, tracheobronchitis, and (possibly) pneumonitis induced by a major exposure to a toxic chemical or organic dust. In survivors, full recovery is the rule. If asthma arises, it is generally the only persisting respiratory problem, though occasionally bronchiectasis or an obliterative bronchiolitis is also detectable. If hypersensitivity has provided the mechanism, the diagnosis is more challenging and may be extremely difficult. This is partly because the latency period during which sensitization occurs (usually 3–24 months) may be very short (days or weeks) or very prolonged (several years), and partly because in most working environments associated with OA, most cases of incident asthma are coincidental and quite unrelated to occupation. What follows addresses this diagnostically more challenging variety of OA. Clinical History

The history provides an obvious starting point, but may be importantly distorted. Affected workers

Vegetable sources Beans Castor, coffee, soy Colophony Pine resin solder Enzymes Bromelain (pineapple), papain (papaw) Grain/flour Gums Acacia, arabic, karaya Mushrooms Oil mists Tea Tobacco Wood Iroko, latex, redwood, western red cedar Microbial sources Endotoxin Gram-negative organisms Enzymes Bacillus subtilis (alcalase, maxatase, subtilisin) Spores Chemical sources Inorganic Salts/oxides of aluminum, chromium, cobalt, nickel, platinum, vanadium Stainless-steel welding fume Organic Acid anhydrides/phthalates, acrylates, amines, azodicarbonamide, benzalkonium, chloramine, di-isocyanates, fluxes, formaldehyde, furfuryl alcohol, glutaraldehyde, isothiazolinones, organophosphate pesticides, plexiglass, polyvinyl pyrolysis fume, reactive dyes, styrene, sulphone chloramides, tannic acid Pharmaceutical sources Intermediates 6-amino penicillanic acid Drugs Cimetidine, cephalosporins, ispaghula (psyllium), penicillins, piperazine, sulphonamides, tetracycline

Table 2 Causes of occupational asthma reported to SWORD over 9 years Agent Di-isocyanates Laboratory animals/insects Flour/grain Fluxes/glues/resins/solders Aldehydes Wood dust Welding fume Enzymes Latex Others Total

1990–98 (%) 16 9 8 7 5 5 2

47 100

The figures are rounded to the nearest whole number.

1998 (%) 13 12 7 9 5

14 6 34 100


anxious to remain employed may deny or minimize relevant symptoms; they may also exaggerate or falsify critical aspects. An overwhelming belief that occupational exposure (and/or employer negligence) is responsible may, curiously, make a diagnosis of OA the ‘independent variable’ on which the symptoms depend: ‘‘if improvement during holidays/vacations is a cardinal diagnostic feature of OA then, yes, this must be so in my case since I know I have OA.’’ For a classical case, there is exposure to a known asthmagen and other exposed workers are affected. For the affected individual, there is recognition that symptoms arise or worsen after a minimum of 1–2 hours and a maximum of 8 h from the onset of each sufficiently strong exposure, and persist for hours or days. Such ‘late’ asthmatic reactions are characteristically associated with increases in airway responsiveness and so are much more definitive of OA than ‘immediate’ reactions, which may simply result non-specifically through ‘irritant’ mechanisms. Mild late reactions may resolve within hours so that there is full recovery by the following day, but more commonly the rise in airway responsiveness is sufficient to worsen asthmatic severity for several days. A weekend away from work may therefore be insufficient to allow full recovery, and the association between occupational exposure and symptoms may not be recognized until there is a 2-week period of vacation (or sick leave). Even then gradual recovery, particularly cessation of disturbed sleep, may not become obvious until the second week, only to be reversed within a matter of days of returning to work. Serologic Investigations

Laboratory investigation for diagnostic IgE antibodies to relevant asthmagenic agents has proved disappointing for two reasons. First, many occupational asthmagens are reactive chemicals of low molecular weight. They are not thought to act as sensitizers until coupled with appropriate haptogenic body proteins, and it has proved difficult to produce suitable complexes for antibody detection. Second, exposed individuals may develop IgE responses without any apparent ill effect. Antibodies appear to correlate more closely with exposure than disease. Nevertheless, when radioallergosorbant allergen testing to relevant asthmagens is available, positive tests increase the probability of OA, even if sensitivity and specificity are limited. Peak Expiratory Flow

More popular and more readily available is peak expiratory flow (PEF) monitoring. Test subjects take measurements on several occasions each day

for periods of several weeks, so that any differences in pattern between work days and rest days can be detected. Statistical software can aid interpretation. The unsupervised recordings may lack reliability and precision, and work-related changes may reflect nonspecific ‘irritant’ reactions rather than specific hypersensitivity responses. There is consequently some difference of opinion over the value of PEF monitoring. In practice, however, it provides the most widely used diagnostic tool for OA. Figure 1 illustrates a typical positive outcome. Inhalation Challenge Tests

The greatest diagnostic confidence comes from laboratory inhalation challenge tests that are monitored by both supervised serial measurements of spirometry and repeated measurements of airway responsiveness. When there is deteriorating ventilatory function and increasing airway responsiveness, and the changes can be evaluated statistically, a diagnosis of OA can be considered ‘confirmed’ with considerable confidence, especially if the outcome is shown to be repeatable and the tests are carried out in a double-blind fashion. Thus, neither test subject nor the immediately supervising physician knows whether the challenge exposure involved the suspected asthmagen or a dummy ‘placebo’. These principles, with two methods of statistical evaluation, are illustrated in Figures 2 and 3. In practice, such tests require sophisticated equipment, are very time-consuming, pose potential risks, and are inevitably restricted to a few centers. They involve a fraction of 1% of all cases, but are particularly valuable (arguably indispensable) when hitherto unrecognized occupational asthmagens are first investigated. Figure 3 illustrates one such case involving a novel asthmagen. Return-to-Work Studies

A useful, and practical, compromise is the ‘return-towork’ challenge test. For this, the test subject is kept from work (or at least from exposure to the suspected asthmagen) for a period of 2–3 weeks, during which time any asthmatic medication is reduced to a minimum (ideally discontinued). In true OA, some improvement is likely (or there is no deterioration with treatment reduction), and can be demonstrated by serial measurements of spirometry and airway responsiveness. Hourly spirometric monitoring over the 3 days prior to the return-to-work generates confidence limits, and so allows the detection of any statistically significant deterioration subsequently (usually within a few days only if the asthma is occupational). If airway responsiveness too increases significantly, OA in the individual is reasonably ‘confirmed’. The method

Diurnal variation (%)

ASTHMA / Occupational Asthma (Including Byssinosis) 191 50 20 460 440 420

PEF (l min–1)

400 380 360 340 320 300 280

M T W T F S S M T W T F S S M T W T F S S M T W T F S S M T W T F S S M T W T F S S 18 19 20 21 22 23 24 25 26 27 28 29 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 11 12 Readings 9 9 9 8 9 8 7 9 8 7 9 8 8 7 5 9 9 8 8 8 8 9 8 3 6 8 6 7 8 9 9 7 7 9 8 6 9 7 8 8 7 6 W W W W W Comments

Figure 1 Serial PEF measurements in a laboratory technician sensitized to a floor cleaning material. Serial 2-hourly PEF measurements, waking to sleeping, for 6 weeks. Top panel: diurnal variation expressed as % predicted. Middle panel: daily maximum, mean, and minimum PEF. Bottom panel: date and number of PEF readings per day. W, days without waking measurement. Dotted line: PEF ¼ 359 represents the predicted PEF. The shaded columns represent work days. Reproduced from Hendrick DJ, Burge PS, Beckett WS, and Churg A (eds.) (2002) Occupational Disorders of the Lung: Recognition, Management, and Prevention, p. 54. London: Saunders, with permission from Elsevier.

does not confirm the identity of the asthmagenic agent, and is only suitable if the test subject is still employed and the employer cooperates fully. In many cases the possibility of an occupational cause does not arise until after the affected individual has ceased employment or the work environment has changed. Figure 4 illustrates this methodology in a newly appointed factory safety officer who found himself working with di-isocyanates.

FEV1 (l)

4

3

2

0 −40 0

Animal Models 80 2 Min

4

6

8 10 12

24 H

Figure 2 Inhalation challenge test with nebulized ceftazidime (3.2 mg) in a production worker. Upper plot: mean FEV1 at each hourly measurement point from 3 control days. Middle plot: lower 95% confidence band for the hourly control FEV1 derived from the pooled variance. Lower plot: FEV1 following nebulized ceftazidime administered in a double-blind fashion. The lower confidence band is breached for well over an hour, indicating a significant decrement consistent with a late asthmatic reaction. Reproduced from Hendrick DJ, Burge PS, Beckett WS, and Churg A (eds.) (2002) Occupational Disorders of the Lung: Recognition, Management, and Prevention, p. 64. London: Saunders, with permission from Elsevier.

Several animal species have provided invaluable insight as to how asthma arises following exposure to occupational agents. ‘Sensitization’ has been achieved by inhaled, dermal, and/or peritoneal routes for many occupational asthmagens (notably acid anhydrides, colophony, di-isocyanates, latex, plicatic acid), and both immediate and late asthmatic reactions have been provoked by subsequent inhalation challenge. The airways are then characterized by inflammation, eosinophil infiltration, mucus hypersecretion, and hyperresponsiveness. Hypersensitivity mechanisms have been confirmed by transferring lymphocytes or serum from affected to unaffected animals, which then respond to inhaled challenge in a similar fashion

192 ASTHMA / Occupational Asthma (Including Byssinosis) 5.0 4.0 FEV1 (l)

3.0 2.0 1.0 0 −40

(a)

0

60

Min

2

4

6

8

10 12

24

H

FEV1 area decrement 2−12 h (l × h)

9 8

Confidence limits

7 6 5

99%

4 3

95%

2 1 0 1

(b)

2 Control days

3 SINOS (32 µg)

Figure 3 Inhalation challenge test with nebulized SINOS (32 mg) in a research and development technician. (a) The FEV1-time plot following challenge with a newly developed low-temperature bleach-activating agent, SINOS. The shaded zone defines the 2–12 h area decrement (2–12 hAD). It provides a summary measure to quantify a late asthmatic reaction. The line demarcating the upper boundary represents the mean of the measurements during the 40 min before challenge. (b) Comparison of the 2–12 hAD following the SINOS challenge with those following three double-blind control challenges with nebulized solvent alone. It exceeds their 95% and 99% confidence limits, confirming that a significant late asthmatic reaction has occurred. The outcome was reproduced when the procedure was repeated. Reproduced from Hendrick DJ, Burge PS, Beckett WS, and Churg A (eds.) (2002) Occupational Disorders of the Lung: Recognition, Management, and Prevention, p. 64. London: Saunders, with permission from Elsevier.

to the donor animals. Specific IgE antibodies to the inducing agents (or hapten conjugates) have been evident commonly, but not invariably. Occasionally specific IgG antibodies are reported. There is involvement of both CD4 þ and CD8 þ T cells, with a dominant T-helper-2 cells (Th2) response. The mechanisms include deposition of excess extracellular matrix (and increased activity of matrix metalloprotease, MMP) and activation of the vascular endothelium associated with the release of vascular endothelial growth factor (VEGF). Experiments with inhibitors of MMP and VEGF have shown substantial reductions of the markers of asthmatic activity, possibly pointing a way to novel strategies for future management. Animal models have additionally confirmed that airway inflammation induced by exposure to toxic levels of certain reactive chemicals may also induce airway hyperresponsiveness. This simulates the RADS pathway. Ozone and chlorine exert their effects

through oxidative stress, which is associated with increased expression of inducible nitric oxide synthase and an increase in airway responsiveness. Ozone has also been shown to suppress Th1 responses (possibly thereby enhancing Th2 responses), and acute exposure to ozone or nitrogen dioxide amplifies asthmatic responses to allergenic agents in already-sensitized animals. Animal models have also been used to investigate the exposure threshold levels at which airway inflammation and hyperresponsiveness first develop. The importance of this for occupationally induced asthma in humans is readily evident, though extrapolation from animals is necessarily fraught with uncertainty. The threshold levels of exposure that trigger meaningful responses once sensitization has occurred may differ critically from those that are responsible for initial sensitization, and are likely to differ appreciably from individual to individual.

ASTHMA / Occupational Asthma (Including Byssinosis) 193 3.00

FEV1 (l s–1)

2.00

Test day 3

1.00

Mean of three control days Lower boundary for control days Work

0.00 07:30 08:30 09:30 10:30 11:30 12:30 13:30 14:30 15:30 16:30 17:30 18:30 19:30 20:30 21:30 22:30 23:30 Time (h) Figure 4 Return-to-work study of a safety officer employed by a factory using di-isocyanates. He developed asthma within months of employment onset. He was kept from work for several weeks during which airway responsiveness improved steadily (PD20.methacholine; 3.2-25.0 mg). An increase in PD20 of twofold or more indicates a significant change. The figure illustrates the third successive day of the return-to-work study. The lowered initial FEV1 measurement reflects an equivocal late reaction from the preceding day. On Day 4, following the 3 days at work, the PD20 was significantly decreased to 9.0 mg (i.e., to less than half the pretest value of 25.0 mg).

Management and Current Therapy Drug and ancillary therapies for OA are those of asthma of any cause, but OA arising by the hypersensitivity route offers an additional and critical means of management. If the relevance of an occupational sensitizer is recognized within 6–24 months of symptom onset, and if exposure then ceases, there is a meaningful probability of the asthmatic state resolving entirely (i.e., the level of airway responsiveness falls into the nonasthmatic range). This is almost unknown for adults with nonoccupational asthma, unless it is drug induced (beta-blockers, nonsteroidal anti-inflammatory drugs (NSAID). There is inevitably much variability from individual to individual, but if exposure ceases within as short a period as 6 months, the probability of complete resolution may exceed 50%. If the period exceeds 24 months, it is more likely that active asthma will continue, even in the absence of ongoing exposure. Most individuals developing OA have unskilled or semiskilled jobs. Their susceptibility to develop asthma with exposure to sensitizing agents will inevitably put them at a disadvantage in the labor market,

and this will be enhanced if active asthma persists. It is unfortunate that most lose their current jobs and never become gainfully employed again. This poses several management challenges. A correct diagnosis is the cornerstone, since mild asthma rarely leads to job loss of itself. A false-positive diagnosis may have devastating but unnecessary economic consequences for both the affected individual and the employer. A dilemma arises when asthma is occupational, but is mild, has been active for several years, and does not improve much after an experimental period away from the workplace. If the affected individual cannot find (or does not wish for) alternative employment, the risk of continuing exposure needs to be assessed. It will certainly diminish the long-term probability of regaining a nonasthmatic level of airway responsiveness, but this may be minimal anyway. More importantly it may cause a further and permanent increase in airway responsiveness, and hence worsening asthma with the possibility of remodeling and fixed airway obstruction. A period of close surveillance, with objective serial measurements of spirometry and airway responsiveness, may help the affected individual and his medical advisors to judge whether the


obvious benefits justify the uncertain risks. There is anecdotal evidence that tolerance develops in some affected individuals and that they have little to lose by continued exposure. Complete cessation of exposure offers the best outcome, especially if this can be achieved by using an alternative, nonsensitizing agent for the particular industrial process. If this is not possible or practical, it may be that exposure levels can be reduced by modifications to job plans, task sharing/exchanging, improved industrial hygiene (ventilation/extraction), or the use of respiratory protection equipment. Prevention

Prevention offers the best means of control. When a risk of OA is identified, surveillance programs may identify emergent cases at a point when exposure cessation is likely to produce cure. This assumes such programs are efficient. Some employers rely primarily on environment measurements, and reassure themselves (inappropriately) that if they have complied with regulatory limits, any emergent cases of asthma must be nonoccupational. When a clear risk of OA is recognized, the onus should lie with disproving the diagnosis when new cases of asthma arise, rather than the converse. It is the very nature of ‘allergy’ that some affected individuals become sensitized at exposure levels that are harmless to the majority, and that lie within regulatory limits. Compensation

Compensation is inevitably an issue for affected workers, particularly those who become disabled and unemployed. Equally inevitably, compensation systems differ from country to country. Regretfully, few include provision for retraining and re-placement. The physician managing OA needs to be familiar with local procedures in order to offer proper advice. At a time of increasing litigation, the physician too may find him- or herself the focus of a legal (negligence) suit if he or she has failed to provide correct advice.

Byssinosis The respiratory effects of dust from cotton and other biologic fibers became prominent in the nineteenth and early/mid-twentieth centuries. They appeared initially to be specific to the cloth manufacturing industry and the appellation, byssinosis, arose to identify the disorder characterized by them. This has had the misleading consequence of segregating cotton dust asthma from the many other types of OA that

were identified later, and of disguising the other respiratory disorders that may result from cotton dust. A very characteristic feature of reported byssinosis has been a prominence of symptoms on the first work day following periods without exposure. These tend to diminish or even cease on subsequent days, at least in some individuals, despite similar levels of ongoing exposure. While chest tightness and breathlessness may be a consequence of asthma, some affected individuals describe an influenza-like reaction with fever and malaise (‘Monday fever’). The latter may occur on the very first employment day, that is, before any possibility of sensitization. Such symptoms have also been generated in experimental conditions when naı¨ve volunteers are exposed. The same febrile reaction may occur in subjects heavily exposed to metal fume or the fume arising from certain polymers. It is closely related to neutrophil inflammation and the release into plasma of pyrogenic interleukin6 (IL-6). These non-specific systemic symptoms are features of ‘inhalation fevers’ generally. They occur widely with exposure to a variety of organic dusts, and in these contexts are now known more commonly by the term ‘organic dust toxic syndrome (ODTS)’. Products from microbial overgrowth appear to be responsible, and high airborne concentrations of respirable spores and endotoxins are characteristically seen. This is consistent with the recognition that byssinosis is most prevalent among those who harvest, transport, store, sort, process, and clean cotton before its ‘decontaminated’ fibers are woven into cloth. The matter is complex since heavy exposure of naı¨ve subjects to organic dust may also provoke cough, chest tightness, and bronchoconstriction. These symptoms are rarely severe, and rarely last for more than a day or two. Nevertheless, this response simulates RADS, and no clear sensitizer has been incriminated in the pathogenesis of byssinosis. Some individuals affected by other forms of OA may also have more prominent symptoms on Mondays that peter out as the working week progresses. A further ‘characteristic’ of byssinosis has also proved to be non-specific, namely the development of COPD. Early studies of byssinosis were necessarily cross-sectional, and it was assumed that subjects with COPD had initially experienced the typical acute and reversible features (i.e., asthma and/or inhalation fever), which had progressed to produce ‘chronic byssinosis’. It seems more likely now that COPD and asthma (and inhalation fever/ODTS) are independent disorders arising from cotton dust exposure, which result from different levels of susceptibility within exposed workers and different patterns of cellular response. Even so, it is to be expected that COPD might develop

ASTHMA / Acute Exacerbations 195

because of asthma as well as independently of it, and there is convincing evidence to support this from recent longitudinal studies.

Acute Exacerbations S L Johnston, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

See also: Chronic Obstructive Pulmonary Disease: Overview. Interleukins: IL-6. Occupational Diseases: Asbestos-Related Lung Disease.

Further Reading Beach JR, Young CL, Avery AJ, et al. (1993) Measurement of airway responsiveness to methacholine: relative importance of the precision of drug delivery and the method of assessing response. Thorax 48: 239–243. Brooks SM, Weiss MA, and Bernstein IL (1985) Reactive airways dysfunction syndrome (RADS). Persistent asthma syndrome after high level irritant exposures. Chest 88: 376–384. Burge PS, Pantin CFA, Newton DT, et al. (1999) Development of an expert system for the interpretation of serial peak expiratory flow measurements in the diagnosis of occupational asthma. Occupational and Environmental Medicine 56: 758–764. Cote J, Kennedy S, and Chan-Yeung M (1990) Outcome of patients with cedar asthma with continuous exposure. American Review of Respiratory Diseases 141: 373–376. Glindmeyer HW, Lefante JJ, Jones RN, et al. (1994) Cotton dust and across-shift change in FEV1 as predictors of annual change in FEV1. American Journal of Respiratory and Critical Care Medicine 149: 584–590. Hendrick DJ and Burge PS (2002) Occupational asthma. In: Hendrick DJ, Burge PS, Beckett WS, and Churg A (eds.) Occupational Disorders of the Lung – Recognition, Management, and Prevention, pp. 33–76. London: Saunders. Herrick CA, Xu L, Wisnewski AV, et al. (2002) A novel mouse model of diisocyanate-induced asthma showing allergic-type inflammation in the lung after inhaled antigen challenge. Journal of Allergy and Clinical Immunology 109: 873–878. Lee KS, Jin SM, Kim SS, and Lee YC (2004) Doxycycline reduces airway inflammation and hyperresponsiveness in a murine model of toluene diisocyanate-induced asthma. Journal of Allergy and Clinical Immunology 113: 902–909. Lee YC, Kwak YG, and Song CH (2002) Contribution of vascular endothelial growth factor to airway hyperresponsiveness and inflammation in a murine model of toluene diisocyanate-induced asthma. Journal of Immunology 168: 595–600. Meyer JD, Holt DL, Cherry NM, and McDonald JC (1999) SWORD ’98: surveillance of work-related and occupational respiratory disease in the UK. Occupational Medicine 47: 485–489. Newman Taylor A (2000) Asthma. In: McDonald JC (ed.) Epidemiology of Work Related Diseases, 2nd edn., ch. 8, pp. 149– 174. London: BMJ Books. Schilling RSF, Hughes JPW, Dingwall-Fordyce I, et al. (1995) An epidemiological study of byssinosis among Lancashire cotton workers. British Journal of Industrial Medicine 12: 217–227. Stenton SC, Avery AJ, Walters EH, and Hendrick DJ (1994) Technical note: statistical approaches to the identification of late asthmatic reactions. European Respiratory Journal 7: 806–812. Stenton SC, Dennis JH, Walters EH, and Hendrick DJ (1990) The asthmagenic properties of a newly developed detergent ingredient – sodium iso-nonanoyl oxybenzene sulphonate. British Journal of Industrial Medicine 47: 405–410. Venables KM, Topping MD, Howe W, et al. (1985) Interaction of smoking and atopy in producing specific IgE antibody against a hapten protein conjugate. British Medical Journal 290: 201–204.

Abstract Acute exacerbations of asthma are acute worsenings of asthma symptoms accompanied by reductions in lung function, normally provoked by some external event or combination of events. Exacerbations may be relatively mild or severe. The most severe may lead to asthma death. Symptoms include increased breathlessness, wheeze, cough, or chest tightness. Severity is graded on a combination of symptoms, clinical signs, and lung function. The majority of asthma exacerbations, particularly in children, are precipitated by acute respiratory tract viral infections. These may interact with a number of other cofactors such as allergen exposure, air pollution, and exercise. Exacerbations are more likely to occur on a background of poorly controlled rather than well-controlled underlying disease. Pathology involves increased airway inflammation with most inflammatory cell types implicated in pathogenesis. Most prominent, however, are neutrophils, lymphocytes, and eosinophils. A wide variety of acute inflammatory mediators are increased during exacerbations. Severity of virus infection is the major determinant of severity of exacerbation. Asthmatics have increased susceptibility to viral and bacterial infection. Treatment includes optimal control of underlying disease, inhaled/ oral steroids, and bronchodilators. The role of anti-infective therapy is under investigation.

Introduction Asthma is itself a heterogeneous disease. Asthma exacerbations also, therefore, are by definition heterogeneous as exacerbations are defined as a worsening of a pre-existing state. Given that the etiology (see below) of exacerbations is also heterogeneous, heterogeneity of both the underlying cause and of the etiology of the exacerbation makes the exacerbation itself very varied in its presentation. One of the difficulties in clinical practice and clinical research is defining exacerbations accurately and differentiating them from poor control of the underlying disease. There is no agreed definition of an exacerbation, but most clinicians and clinical researchers would agree on something like ‘episodes of relatively sudden onset and rapidly progressive increase in symptoms of shortness of breath, cough, wheeze, or chest tightness accompanied by reduction in lung function and normally provoked by some external event or combination of events’. Exacerbations of asthma are very important as they are the major cause of morbidity and mortality in asthma and are also responsible for the greater part of healthcare costs associated with asthma treatment. Currently available treatments for asthma exacerbations include supportive care, oxygen if required, bronchodilators, and oral/inhaled corticosteroids.

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This treatment is at best only partially effective. Understanding the causes and mechanisms of asthma exacerbations is therefore of great importance as there is an urgent need for more effective preventive and interventional therapy.

Etiology In infants and young children the vast majority (approaching 100%) of exacerbations are precipitated by acute respiratory tract virus infections. Respiratory syncytial virus (RSV) is the major cause of acute wheezing illness severe enough to require hospitalization in infants and 1–2-year-old children. There is, however, increasing evidence that rhinoviruses also play an important role and most recent data suggest RSV is implicated in 70–80% of hospitalized wheezing lower respiratory tract infections and rhinoviruses in around 40%. The incidence of dual infection with these two viruses and with a variety of other respiratory viruses is likely to be as high as 20–30%. There is much less data available on community lower respiratory wheezing illness but birth cohort studies have now reported that rhinoviruses are the dominant cause of acute attacks of wheezing in this age group, perhaps causing around three times as many episodes of wheezing illness as RSV and other respiratory viruses. In school-age children and teenagers, viruses precipitate at least 80% of acute asthma exacerbations. Rhinoviruses again are by far the most common virus implicated accounting for around two-thirds of viruses detected. In adults, viruses likely cause around two-thirds to three-quarters of asthma exacerbations. In adults in particular where virus loads tend to be much lower than in children, the evidence is less strong than it is in children and further work is required to clarify the role of viruses. In all these age groups, viruses can interact with a number of other factors in provoking asthma exacerbations. There are good data in both adults and children that virus infection and exposure to an allergen to which the patient is sensitized interact in a synergistic manner in increasing the risk of exacerbation. There is also evidence that air pollution interacts with virus infection in increasing the risk of lower respiratory illness when infected and it is possible that a number of other cofactors also play a role. Poor control of the underlying disease is a major risk factor for asthma exacerbation and treatment with prophylactic therapy, principally with inhaled corticosteroids, but also with leukotriene receptor antagonists and long-acting beta-agonists is a major protective factor against exacerbation.

Asthma exacerbations show marked seasonality in all age groups, peaking 1–2 weeks after school return, most especially in the autumn and most especially in school-age children. This seasonal pattern is important as it emphasizes the times at which prophylactic therapy is most needed. Asthmatics have recently been shown to have increased susceptibility to virus infection through impaired innate immunity and there is a biological rationale that they are also likely to have impaired acquired antiviral immunity. Clinical studies confirm that asthmatics are more susceptible to virus infection and bacterial infection, having a greater risk of invasive pneumococcal disease. To date, very little is known about susceptibility to infection in asthma but this is an important area for future research. Genetic susceptibility is also an underresearched area and very little is known in this regard. Finally, there is increasing evidence that chronic infection with the atypical bacteria Chlamydophila pneumoniae and Mycoplasma pneumoniae may play a role in exacerbations of asthma and a recent study confirms therapeutic benefit of treating asthma exacerbations with an antibiotic active against these organisms. Further studies are required to confirm these findings and to shed further light on the role of chlamydia and mycoplasma.

Pathology Relatively little is known about the pathology of asthma exacerbations for an obvious reason: sampling the lower respiratory tract during an exacerbation is extremely difficult. In one study asthmatic patients were bronchoscoped during naturally occurring colds, and eosinophilic, neutrophilic, and CD8 þ T-cell inflammatory responses were found. Studies of post-mortem samples from patients dying of asthma exacerbation also reveal CD8 þ T cells, activated CD8 þ T cells, increased perforin expression, and impaired ratios of interferon gamma (IFN-g) to interleukin-4 (IL-4), possibly implicating impaired antiviral immunity in asthma death. Noninvasive methods of sampling the lower airway include measurement of exhaled breath condensate or exhaled nitric oxide, and sputum sampling. Studies with sputum again show both eosinophilic and neutrophilic inflammation with increased levels of IL-8 and fibrinogen. Markers of both eosinophil and neutrophil activation are also increased and sputum lactate dehydrogenase (LDH) as a marker of virus-induced cytotoxicity is also markedly elevated. Levels of sputum LDH and eosinophil cationic protein were associated with longer hospital stay, indicating that virus-induced cell damage was the major predictor of

ASTHMA / Acute Exacerbations 197

the severity of asthma exacerbation and that eosinophilia as a consequence of either viral and/or allergen exposure was also an important contributor.

Clinical Features Asthma exacerbations present with a sudden worsening of wheeze, cough, shortness of breath, and chest tightness with a reduction in lung function. Exacerbations can range from mild to severe, the most severe leading to asthma death. Diagnosis is normally made on the clinical history with the assistance of peak expiratory flow measurement and/or spirometry. Clinical examination will normally reveal a distressed, anxious patient with tachycardia, tachypnea, and audible wheeze on auscultation (though in the most severe exacerbations the chest can be almost silent). Patients will have prolonged expiration, and signs of severe exacerbation include inability to talk in complete sentences, tachycardia above 110 beats min 1, a respiratory rate above 25 breaths min 1, and a peak expiratory flow below 50% of predicted or best. Presence of pulsus paradoxus also indicates a severe attack and life-threatening attacks are suggested by a silent chest, presence of cyanosis, bradycardia, or hypotension, and a peak expiratory flow below 30% of predicted or best. Measurement of blood gases should be carried out. Hypoxia is usual. In milder exacerbations, hypocapnia is common due to hyperventilation while in more severe and life-threatening exacerbations, PCO2 starts to rise. A raised PCO2 or rising PCO2 is an indication for intensive care. Chest X-rays should be performed to exclude pneumonia and pneumothorax. The response to therapy and the progress of the exacerbation are monitored with serial peak expiratory flow or spirometry testing. Routine hematology and biochemistry are indicated but other blood testing is not normally required.

Pathogenesis Asthma exacerbations are always mixed in their pathogenesis. They involve a mixture of acute and chronic inflammation provoked by virus infection and other stimuli including allergen exposure, air pollution, tobacco smoke, etc. Much of the information we have gained regarding the pathogenesis of asthma exacerbations has come from experimental studies of rhinovirus infection in asthmatic and normal subjects. These studies have implicated neutrophils, CD4 þ and CD8 þ lymphocytes, and eosinophils, but in addition mast cells, macrophages, and mediators of acute inflammation are also shown to play important roles, including leukotrienes. Most cytokines/chemokines

that have been investigated have been found to be elevated in exacerbations. Perhaps the most prominent are interleukin-8, interleukin-1b, tumor necrosis factor alpha (TNF-a), the regulated upon activation normally T-cell expressed and secreted factor (RANTES), and IFN-g. The mechanisms of induction of lower airway inflammation in the context of respiratory virus infection are of great interest as these represent potential targets for interventional therapy. Nuclear factor kappa B (NF-kB) is very strongly implicated in virus-induced inflammation, as is activating protein 1 (AP-1). However, a number of other transcription factors are also implicated. Mechanisms of induction of mucus secretion are also of great interest – complex pathways are involved, but NF-kB, Sp1 transcription factors, and the epidermal growth factor receptor signaling pathway are all implicated. Airway obstruction is a result of acute smooth muscle contraction, chronic smooth muscle hypertrophy, mucus secretion, acute tissue edema, and chronic tissue inflammation with airway fibrosis/remodeling. An important aspect of pathogenesis is host resistance to infection as virus infection is the major precipitant to exacerbation. Recent studies have confirmed that asthmatic bronchial epithelial cells mount defective apoptotic and IFN-b innate immune responses to rhinovirus infection. In consequence, rhinovirus infection is robust while in normal epithelial cells infection is largely abortive. The clinical studies indicating increased susceptibility to virus infection in asthma are consistent with this biological evidence, and more recent evidence that asthmatics are also susceptible to invasive pneumococcal disease possibly indicates a more generalized immune deficit. There is an urgent need to increase our understanding of host immunity to both viral and bacterial infection in asthma.

Experimental Models There are no small-animal models of rhinovirus infection as rhinoviruses only infect humans and nonhuman primates. There are animal models of other virus infections including influenza, RSV, and parainfluenza, and these models have been combined in some instances with allergen exposure to try to increase our understanding of the pathogenesis of virus-induced asthma exacerbations. These studies indicate that impaired T helper (Th)-1 immune responses increase susceptibility to virus infection and that airway inflammation, airway obstruction, and bronchial hyperreactivity are increased when virus infection occurs in the presence of ongoing allergic inflammation. Other studies have confirmed an increased risk of developing allergen sensitization if

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allergen exposure occurs in the context of an acute respiratory virus infection. Studies with RSV indicate that the major protective immune responses are production of neutralizing antibody, natural killer (NK) cell, and CD8 þ T-cell IFN-g responses. A great deal of further work is needed to increase our understanding of the interaction between virus infection and allergen exposure in the context of asthma exacerbations. In the absence of an animal model, investigators have carried out human rhinovirus experimental infections in asthmatic and normal subjects, and have demonstrated bronchial hyperactivity and airway obstruction in asthmatic volunteers. These studies have also implicated Th1 responses in increased susceptibility to virus infection and further work in these models is ongoing to try to increase our understanding of the pathogenesis of virus-induced asthma. In vitro models of host immunity to virus infection and of virus-induced lower airway inflammation include infection of airway epithelial cells and macrophages with a variety of respiratory viruses including rhinoviruses, RSV, and influenza. These studies demonstrate that virus infection induces many proinflammatory cytokines and chemokines, dependent upon transcription factor activation such as NF-kB, AP-1, and NF-IL-6. In turn, much of the activation of transcription factors is dependent upon activation of oxidant pathways. Production of nitric oxide appears to exert some degree of protection against virus infection.

Management and Current Therapy Perhaps the most important aspect to management of asthma exacerbations is prevention of asthma exacerbations. Optimal control of underlying disease with optimal therapy including inhaled corticosteroids, leukotriene modifiers, and long-acting betaagonists has been shown to reduce exacerbation frequency by approximately 50%. Once exacerbations occur, the initial response is to give short-acting bronchodilators, if mild via inhalation through a spacer, and if more severe by nebulization. Short-acting beta-agonists are initial therapy. The addition of anticholinergic bronchodilators has been shown to further improve lung function and to reduce the need for hospitalization. In mild exacerbations, increasing the dose of inhaled corticosteroids can reduce the severity of the exacerbation and speed recovery. However, evidence indicates that doubling the dose is usually not adequate and that quadrupling the dose or giving high-dose therapy may give better responses. In moderate to

severe exacerbations, oral/systemic corticosteroid therapy is indicated. Intravenous magnesium has been shown to be of some benefit in more severe exacerbations failing to respond to initial therapy. Oxygen and supportive care should be given to all exacerbations where hypoxia is a feature. Leukotriene modifiers have been shown to be more beneficial in exacerbations in infants and young children. Their intravenous use in the acute setting has also been shown to produce some benefit, though there is not much evidence available as yet. Use of standard antibiotics in two placebo-controlled studies has produced no evidence of benefit. However, a recent study with an antibiotic active against atypical bacteria and with anti-inflammatory properties has shown clinically significant benefit over placebo. The most severe exacerbations require high dependency unit or intensive care monitoring and some require invasive ventilation. Hospitalization with acute exacerbation is a major risk factor for asthma mortality, and preventive therapy including inhaled corticosterioids and self-management plans are strongly indicated in such patients. See also: Chemokines. Leukocytes: Eosinophils; Neutrophils. Viruses of the Lung.

Further Reading British Thoracic Society, Scottish Intercollegiate Guidelines Network (2003) British guidelines on the management of asthma. Thorax 58(Supplement 1): 11–94. Corne JM, Marshall C, Smith S, et al. (2002) Frequency, severity, and duration of rhinovirus infections in asthmatic and nonasthmatic individuals: a longitudinal cohort study. Lancet 359(9309): 831–834. Gern JE (2002) Rhinovirus respiratory infections and asthma. American Journal of Medicine 112(Supplement 6A): 19S–27S. Gern JE and Busse WW (2002) Relationship of viral infections to wheezing illnesses and asthma. Nature Reviews: Immunology 2(2): 132–138. Gern JE and Lemanske RF Jr. (2003) Infectious triggers of pediatric asthma. Pediatric Clinics of North America 50(3): 555–575. Green RM, Custovic A, Sanderson G, et al. (2002) Synergism between allergens and viruses and risk of hospital admission with asthma: case-control study. British Medical Journal 324(7340): 763. (Erratum British Medical Journal 324 (7346): 1131.) Johnston SL and Martin RJ (2005) Chlamydophila pneumoniae and Mycoplasma pneumoniae: a role in asthma pathogenesis? American Journal of Respiratory and Critical Care Medicine, doi: 10.1164/rccm.200412–1743pp. Message SD and Johnston SL (2002) Viruses in asthma. British Medical Bulletin 61: 29–43. Message SD and Johnston SL (2004) Host defense function of the airway epithelium in health and disease: clinical background. Journal of Leukocyte Biology 75(1): 5–17. Talbot TR, Hartert TV, Mitchel E, et al. (2005) Asthma as a risk factor for invasive pneumococcal disease. New England Journal of Medicine 352(20): 2082–2090.

ASTHMA / Exercise-Induced 199 Wark PA, Johnston SL, Bucchieri F, et al. (2005) Asthmatic bronchial epithelial cells have a deficient innate immune response to infection with rhinovirus. Journal of Experimental Medicine 201(6): 937–947. Wark PA, Johnston SL, Moric I, et al. (2002) Neutrophil degranulation and cell lysis is associated with clinical severity in virusinduced asthma. European Respiratory Journal 19(1): 68–75.

Exercise-Induced N C Thomson and M Shepherd, University of Glasgow, Glasgow, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Exercise-induced asthma (EIA) is described in asthmatics, elite athletes, and the general population alike. Difficulties reconciling the variety of presentations complicate our understanding of the disease process and prevalence. Exercise-induced bronchospasm (EIB) can be measured during and after exercise in susceptible individuals, but whether this signifies EIA is unclear. Exercise can cause an inflammatory response in the airway that appears to be dose dependent and exaggerated by breathing cold dry air. Classical understanding of the pathogenesis of EIA suggests that drying of the airway mucosa leading to an osmotic gradient across the epithelium and basement membrane combined with cooling of the bronchial wall triggers bronchospasm in EIA. Modern investigation suggests that airway surface stress can be communicated to the bronchial wall by chemical signals arising from the epithelium. Managing a patient with EIA requires careful investigation to ensure the correct diagnosis and consideration of alternative disease processes should be made. Pharmacological intervention can both treat and limit the extent of EIB, but behavioral modifications can also be introduced. Finally, clinicians managing EIA should be aware of the conflicts that arise when regular asthma medications are used by patients engaging in competitive sport.

Introduction Increased airway resistance triggered by vigorous exercise is variously called exercise-induced asthma (EIA) or exercise-induced bronchospasm (EIB). Although a consensus exists on the criteria for making a diagnosis of EIB, the range of circumstances in which it has been recognized mean a precise clinical definition of EIA is difficult to achieve. EIB has been described in asthmatics and those with no history of asthma and subjects ranging from school children to elite athletes. It is not clear whether the disease is the same in all cases. While it may be more accurate to reserve the term EIA for exercise-induced, symptomatic bronchial narrowing occurring in previously diagnosed asthma we will use the term broadly to describe asthmatic symptoms or physiological changes consistent with asthma developing during or immediately after exercise.

Epidemiology EIA is common in known asthmatic subjects and exercise has been recognized as a trigger for ‘asthma’ since the seventeenth century. At least 90% of asthma sufferers will experience a fall in forced expiratory volume (FEV1) or peak flow during or shortly after an appropriate exercise challenge. Some authorities believe that all asthmatics will have demonstrable bronchospasm following sufficient exercise challenge. In atopic patients who suffer only from allergic rhinitis prevalence drops to 40%. Studies of other groups are complicated by the definition of EIA used. For example, comparing the prevalence of EIB, hypersensitivity to methacholine, and self-reported symptoms of EIA consistently identify different groups within a given population. Most of the prevalence data provided are therefore relatively generalized (Table 1). EIB is a useful model for clinical asthma and it has been used as a screening tool. These cohorts are frequently drawn from populations engaged in regular exercise so may not represent a truly unselected population. Based on these studies the prevalence of EIA in the general adult population is estimated at between 6% and 13% and this varies geographically with higher levels in the UK and Australia and lower levels in developing countries. In cohorts of normal school children challenged with exercise then tested by spirometry up to 12% are shown to have EIA. The identification of exercise-induced bronchial narrowing is often a surprise to both child and adult subjects, who may not have been aware of any such problem. A poor perception of airflow restriction is generally recognized in asthma but also casts some doubt on the definition of EIA based on spirometry alone. It is not known if otherwise normal subjects who experience a fall in FEV1 with exercise will benefit from asthma therapy or will go on to develop clinical asthma later in life. Failure to recognize potentially reversible EIA may not be a trivial matter. Children who suffer distressing

Table 1 Estimated prevalence (percent study population) of exercise induced asthma based on a number of studies using different methodology Study population

Children (o18 years) (%)

Adults (%)

General population Asthma Atopic nonasthmatic Athletes Elilte Recreational

9 45–80 15

4–20 70–90 40

19

4.2–20 11–50

EIA prevalence has been reported using physiological based parameters and self-reporting questionnaires.

200 ASTHMA / Exercise-Induced

symptoms will tend to avoid exercise leading to possible psychosocial and neurological development problems. Similarly, the importance of correct identification of EIA is highlighted by studies demonstrating lower self-esteem scores in children who perceive themselves to be socially disadvantaged by asthma. A surprisingly high prevalence of EIA is reported in both recreational and elite athletes (Table 2). Anecdotally, this association has been explained by the focus of some asthmatic children on ventilatory control and on athletic pursuit to achieve this. Whatever the cause, around 23% of recreational athletes report asthma induced by exercise while within the elite ranks prevalence rates of between 16% in summer outdoor events and 50% in winter events such as cross-country skiing or ice skating are reported. While asthmatics frequently state that swimming tends to cause less bronchospasm than other sports, a particularly high prevalence of EIA has been observed in elite swimmers in whom up to 79% have been reported to have demonstrable bronchial hyperresponsiveness to methacholine and 33% have asthma. The prevalence of EIA reported in elite athletes has raised concern among sporting governing bodies regarding the potential for inappropriate use of performance enhancing medications. As a result a considerable body of research has focused on EIA developing in elite athletes. However, elite athletes are subject to a variety of additional possible triggers of EIB including the stress of competition. Thus, whether this research is equally applicable to recreational athletes or nonathletic sufferers from EIA is not known. Therefore, it appears that EIA, defined by a fall in FEV1, occurs in a relatively high proportion of the general population. This prevalence is increased by underlying atopy, asthma, and athletic pursuit,

Table 2 The prevalence of EIA based on sporting pursuit Athletes

Season

Olympians

Summer Winter

Elite/events

Summer Winter

All seasons a

Activity

Asthmaa (%)

EIB (%)

23 22 Track/field Indoor Nordic skiing Skating Nonendurance Swimming

19 12 60 6.4 1 44–50

12 50 35

Definition of asthma varies among reports and includes selfreporting of ever diagnosed asthma and current or ever used asthma medications.

particularly outdoor winter endurance events. Accurate assessments of prevalence are hampered by difficulty in definition stemming from the range of possible criteria for diagnosis and the variety of presenting complaints, which will be outlined later. Further difficulties stem from poor perception and variable self-reporting. This difficulty in precise definition has also limited understanding of the pathology underlying EIA.

Pathogenesis Pathophysiology

The normal respiratory responses to exercise result in an increase in ventilation of up to 200 l min1 . In order to facilitate the increased airflow, mild bronchodilation occurs early during an exercise event and this can be measured using spirometry. Classically, EIA was described as taking place after the exercise challenge was completed, but it is clear that airway narrowing can occur during exercise lasting more than 12 min and in the postexercise period. The ‘stop–start’ nature of some sporting events means that symptoms developing during exercise are entirely consistent with a diagnosis of EIA. A variable period of protection from further exercise-induced bronchial narrowing known as the refractory period is well described. A refractory period may last from 30 min to 2 h and may be due in part to the bronchodilator properties of prostaglandin E2 (PGE2). The cause of EIA and the refractory period are not fully understood; however, considerable evidence points to a cellular response to increased ventilation triggering an inflammatory reaction in the airway. EIA may therefore be considered as a subset of chronic asthma in which exercise is the trigger to inflammation. While this hypothesis is attractive, a variety of studies have produced conflicting data regarding the inflammatory character of EIA. The three-stage model proposed by R Gotshall serves as a useful framework to consider the pathogenesis of EIA. In this model an exercise challenge serves as a trigger sensed in the airway that ultimately signals to the cells of the airway controlling caliber. Hyperventilation alone can cause bronchoconstriction in human and canine subjects and has been identified as the key element of exercise that triggers EIA. How this trigger leads to bronchoconstriction is a matter of controversy. Airway Cooling and Hyperosmolarity

In 1864, H H Slater noted that cold air triggered asthma and offered pulmonary vascular congestion as a possible explanation. This theory has been

ASTHMA / Exercise-Induced 201

developed by McFadden and others. Airway cooling in response to inspired cold air may trigger vasoconstriction of the bronchial vasculature. Subsequent reflex vasodilation and hyperemia with extravasated fluid leading to mucosal edema could lead to airway narrowing. Although attractive, this theory fails to acknowledge the capacity of the upper airways to warm inspired air such that it is unlikely that cold air is delivered to medium-sized airways, the main site of narrowing in asthma. However, cooling of the airways could take place if evaporation of mucosal water exceeded the replacement capacity of the airway. Exercise is associated with mouth breathing, which bypasses the humidifying effect of the nasal mucosa, and dry air has been shown to increase the capacity of an exercise challenge to trigger EIB. Evaporation of water from the upper airways might take place as a result of a large increase in ventilation of relatively dry air. This would cause an increase in the osmolarity of the airway mucosa. This concept of airway hyperosmolarity has been developed by Anderson and colleagues and has become popular in current literature. Inhaling hyperosmolar mannitol or saline solutions can trigger bronchospasm in the absence of an exercise stimulus. It has been difficult to separate the specific elements of these two processes and it may be that both are partly responsible for the effect of hyperventilation on the airway in EIA. Since cold air holds less moisture than warm air, this may add to the drying of the airway. Alternative mechanisms for transducing the proasthmatic stimulus to a bronchoconstrictive response can be postulated. These include stretching of the airway wall cells and altered pressure dynamics of the airway lumen. Ultimately, a signal to alter the diameter of the airway lumen is generated that results in a fall in FEV1. The nature of this signal is also controversial but biochemical mediators associated with inflammation offer a possible explanation. Inflammation in EIA

It has become clear that an inflammatory process in the airway leads to bronchial hyperresponsiveness and chronic asthma. Exacerbations of asthma can be

caused by a variety of inflammatory triggers such as allergen exposure or viral infection. While less intuitive it seems possible that EIA may also have an inflammatory basis. Table 3 details the variety of inflammation markers that have been studied with reference to EIA. Biological markers of inflammation A variety of studies have demonstrated elevated inflammatory cells in the airway lumen and bronchial wall from athletes engaged in a wide spectrum of athletic pursuits. These cells include eosinophils, T lymphocytes, macrophages, and mast cells all of which are recognized as key cellular elements of the asthmatic inflammatory response. Changes in airway inflammatory cell number correlate with changes in bronchial reactivity and some groups have demonstrated that the degree of bronchospasm in response to exercise challenge more closely reflects the level of airway eosinophilia than the prechallenge methacholine sensitivity. Bronchial biopsies taken from resting cross-country skiers demonstrated increased inflammatory cells in the bronchial wall compared to nonathlete controls. Regular exercise may therefore lead to a persistent airway inflammation beyond the acute effects of exercise challenge. Cell-based studies appear to support the role of exercise as a trigger for airway inflammation. The study of winter athletes described above demonstrated that airway wall inflammation occurred even in the absence of symptomatic EIB. This suggests that airway inflammation is not sufficient to cause EIA and that exercise per se can induce inflammation in normal subjects. Unfortunately, none of these studies offer an explanation for why some athletes are susceptible to the inflammatory effects of exercise while others are not. Inflammatory cells in the airway are presumed to cause bronchial hyperresponsiveness by the production of chemical mediators of inflammation. A variety of such mediators have been studied in asthma and EIA (Table 3). Early studies investigating the role of the mast cell stabilizers such as nedcromil and sodium cromoglycate identified histamine as a likely

Table 3 Inflammatory mediators in EIA Mediator

Role in asthma

Induced by exercise

Efficacy of inhibitor

Histamine Leukotrienes Prostaglandin D2

Bronchoconstrictor Bronchoconstriction, smooth muscle proliferation, chemoattractant

þ þ

? þþ Variable

Various inflammatory mediators have been investigated for their capacity to induce or maintain airway inflammation in EIA. Evidence for induction during exercise comes from studies looking at plasma, urine, or sputum levels of the mediator or product. The role of histamine has recently been challenged due to the overriding effect of leukotriene antagonists with no additional effect of antihistamine and a consistent difficulty in demonstrating exercise induction.

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trigger for EIA although this has been challenged recently. The role of other chemical mediators of inflammation including cysteinyl leukotrienes (LTD4 and LTC4) and prostaglandins (PGD2 and PGF2) have been supported by the discovery of increased levels in postexercise plasma and urine and by the effects of specific antagonists. The role of inflammatory cells and their products in the development of EIA is supported by studies demonstrating the capacity of exercise to increase their availability to airway cells. However, not all investigators have found increased levels of proasthmatic mediators in response to exercise, and the presence of a particular agent does not imply a causal link. Further evidence is available from interventions aimed at reducing airway inflammation. Do Anti-Inflammatory Therapies Prevent EIA?

Inhaled corticosteroids (ICs) are the most widely used airway-delivered anti-inflammatory agents. Among well-controlled asthmatic subjects who use regular ICs to abolish normal symptoms 50% will still experience EIB on testing. This suggests that EIA is relatively resistant to standard doses of IC therapy. This hypothesis is supported by early studies demonstrating only a 50% reduction in exercise-induced fall of FEV1 when ICs were introduced. When a short-acting b-adrenergic receptor agonist was added to the IC therapy, this further reduced EIB by 30%, hinting that increased airway resistance in EIA is a complex phenomenon. The introduction of modern anti-inflammatories has reinforced the theory that EIA is an inflammation-mediated phenomenon. Leukotrienes and in particular LTD4 are regarded as among the most potent proasthmatic inflammatory mediators. Leukotriene receptor antagonists (LTRAs) inhibit the effect of these mediators at their specific receptors. The introduction of LTRAs to common practice suggested that they might have efficacy in treating EIA, a suggestion supported by subsequent examination. LTRA therapy provides a dose-dependent protection from EIB and shortens the time to recovery of FEV1. In contrast antihistamine therapy does not appear to add any further protection over LTRA medication. Several lines of evidence support the role of inflammatory processes in EIA. Exercise can trigger and maintain airway inflammation that is associated with bronchial hyperresponsiveness. Intervention studies show that combating inflammation can improve EIA. What is unclear, however, is what predisposes an individual to develop exercise-induced airway inflammation and why otherwise potent antiinflammatory agents are only partially effective in

preventing EIA despite improved control in underlying asthma. Finally, various chemical triggers of asthma have been investigated for their ability to trigger or enhance bronchoconstriction on exercise challenge. Of these adenosine, the product of ATP breakdown, seems worthy of further investigation. Exercise increases circulating adenosine levels suggesting that any proasthmatic activity of this chemical could then be enhanced by exercise. A correlation between the fall in FEV1 following exercise challenge in asthmatic subjects and plasma adenosine levels has been reported. Adenosine is believed to trigger inflammatory cell degranulation, and in the context of asthma this may enhance a mild asthmatic response making it considerably more severe. Such studies may ultimately explain why exercise can be the only trigger for symptomatic EIA in otherwise nonasthmatic subjects. Integrating the Two Pathogenic Theories

It can be concluded that exercise-induced hyperventilation can trigger physical changes in the airway that are subsequently transduced to an inflammatory signal in the bronchial wall, which can be assumed to lead to bronchoconstriction and possibly chronic inflammation in susceptible people. Can physical changes at the surface of the airway communicate to the bronchial wall to cause inflammation and bronchial hypersensitivity? Evidence for pathways communicating signals between different airway compartments has been accumulating over the last decade, offering possible mechanisms to complete the model. As yet these pathways are untested in the context of EIA but do offer intriguing possibilities. It is becoming clear that airway epithelial stress can cause the release of inflammatory mediators that promote airway inflammation. Recent research supports the development of an integrated epithelium– mesenchyme complex (the epithelial mesenchymal trophic unit) reminiscent of the embryonic lung. Epithelial monolayers in vitro can trigger inflammatory signals in fibroblasts and smooth muscle cells of the airway supporting such a communication. Inflammatory cells found superficially in the airway, such as eosinophils, can be activated by osmolar stress and release mediators capable of eliciting an asthmatic response offering a more direct line of communication. Thus, drying and cooling of surface epithelium could in theory at least trigger generalized airway inflammation as seen in chronic asthma. The hyperemia following airway rewarming would support an inflammatory process by supplying exudates and inflammatory cells (Figure 1).


Hyperventilation/exercise

Cooling of the airway & bronchial vasoconstriction

Reactive vasodilitation & mucosal hyperemia

Hyperosmolarity of airway mucous layer

Mucosal congestion & exudate formation

Accumulation of inflammatory cells in the airway lumen

Osmotic stress communicated to superficial cells of the airway

Degranulation Degranulation of inflammatory cells – release of inflammatory mediators LTD4 and PGE2

Release of proinflammatory cytokines from epithelial cells

Bronchial wall inflammation, airway smooth muscle contraction, mucosaledema, possible epithelial cell shedding Figure 1 Summary diagram of the proposed pathogenesis of EIA. A series of pathological events following increased ventilation in exercise are proposed that trigger an asthmatic response in susceptible individuals.

Clinical Presentation Symptoms associated with exercise can arise from a variety of sources including the lungs, heart, and gastrointestinal tract. A careful history can help to identify the most likely source, but EIA may present with classical symptoms such as dyspnea, wheeze, and cough or with more obscure symptoms making the diagnosis difficult. Complaints such as headache, abdominal pain, chest pain, cramps, and severe fatigue may all respond to treatment for EIA. Symptoms may develop early during an exercise event or following its completion. The association with exercise should

alert the clinician to the possibility of EIA although the complaint may seem unrelated to the lungs. Frequently patients believe they are simply ‘out of condition’ and even well-controlled asthmatics may not associate their exercise-induced symptoms with asthma. EIA can be the only manifestation of hyperresponsive airways so the absence of a formal diagnosis of asthma does not exclude the possibility of EIA.

Differential Diagnosis Since the diagnosis of EIA is not always straightforward to make a high index of clinical suspicion, it

204 ASTHMA / Exercise-Induced Table 4 Differential diagnosis of the patient presenting with exercise-induced breathlessness System

Disease

Respiratory

EIA COPD Pulmonary fibrosis Pulmonary vascular hypertension Angina Ventricular dysfunction Dysrhythmia Poor fitness

Cardiovascular

General

COPD, chronic obstructive pulmonary disease; EIA, exerciseinduced asthma.

should be combined with an open mind regarding the variety of other potential sources of symptoms. In particular, true vocal cord dysfunction can be difficult to distinguish from EIA when symptoms predominantly present with exercise. A list of alternative diagnoses for exercise-induced dyspnea is presented (Table 4). It is not yet clear if patients who have demonstrable exercise-induced bronchoconstriction but no symptoms will benefit from pharmacological treatment. Examination of the lungs of a subject with exerciseinduced dyspnea will usually be normal in the context of asthma or lone EIB. The presence of wheeze or hyperinflation might point to chronic airflow obstruction that has gone unrecognized. A full examination might point to an alternative diagnosis as listed. Again quiescent asthma or lone EIB will usually be associated with normal pulmonary function tests while abnormalities may point to alternative pulmonary diagnoses or chronic airflow obstruction.

Investigation The need for further investigation will be directed by the clinical findings but may fall into two groups. In the first a pragmatic trial of pharmacological intervention prior to a predictable exercise trigger of symptoms can be the most useful and easiest test. In some cases, however, detailed pulmonary function in response to exercise is desirable either to monitor treatment, to make a difficult diagnosis, or if the presence of EIA would limit the performance of essential life-saving work (American Thoracic Society (ATS) Guideline). Other uses for detailed exercise testing include the diagnosis of asthma in elite athletes for drug monitoring or performance testing. In a pragmatic trial a patient should be prescribed either a preventative or a reliever inhaled therapy and advised to use this prior to or during exercise that would normally trigger the reported symptoms. Antiinflammatory drugs such as inhaled corticosteroids

or cromoglycate have been shown to have some preventative efficacy in this setting. b2-adrenergic agonists have been shown to relieve exercise-induced bronchoconstriction but their efficacy is reduced in EIB. Patients should be able to record the effect of treatment on symptoms or on peak flow with a small degree of training. Where it is desirable to direct investigations towards a specific diagnosis of EIA, a variety of alternative procedures, ranging from exercise provocation to bronchial challenge, are available. Various procedures have been described that are capable of triggering airway narrowing in asthma consistent with a diagnosis of EIA. The principal element of all these tests is to raise minute ventilation as occurs with exercise. It is possible to trigger bronchial narrowing by hyperventilation and this has been recommended as a surrogate for more formal exercise challenge. More exercise specific tests have been developed for both field and laboratory. While testing in the field has the advantage of replicating the conditions that normally trigger asthma in the subject, the equipment required to make useful measurements has to be portable. This has led to the development of protocols designed to measure pulmonary responses to exercise under laboratory conditions that replicate the essential features of outdoor activity. Guidelines recommending the best practice for performing these tests are available, the essential features of which are summarized below. Patient Instructions

Subjects should avoid bronchoprotective or bronchoreliever medication for 48 h and have eaten only a light meal. Antihistamines and caffeine should also be avoided. Exercise should be kept to a minimum prior to the test, as around 50% of EIA sufferers will experience a refractory period of up to 4 h after vigorous exercise. Any medical or orthopedic contraindications to exercise should be considered and the ability of the patient to fulfill the physiological requirements should be ensured. Exercise

Exercise can be performed on an electronic treadmill or stationary bicycle as both methods have been validated. The desired level of exercise is based on 80–90% of maximal heart rate (based on an HR 220 – age) or 50–60% of maximal voluntary ventilation. The degree of exercise required to achieve this level of exercise response will vary considerably among subjects and it is advised that a period of rapid progressive increase in workload is used to achieve these targets and then maintained for at least 4 min. The


total exercise time for adults should be around 6– 8 min. During the exercise, heart rate and where possible minute ventilation should be measured to ensure that an adequate stimulus to the airway is being achieved. Climatic Considerations

Outdoor exercises are generally better than indoor exercises at triggering bronchospasm. This is believed to be due to lower humidity and temperature and cool dry air is known to trigger asthma in exercise sensitive subjects better than indoor room air. Maintaining the ambient temperature at 20–251C and relative humidity at 50% is satisfactory. Exercise should be performed with a nose clip to prevent nasal humidification of inspired air. Severe responses to exercise have been described in susceptible people and it is advisable to have medical supervision available to monitor patients’ responses and administer bronchodilator therapies as required. Measurements

FEV1 (% fall)

FEV1 is the most useful and convenient measurement to make in the laboratory. Where an alternative diagnosis is suspected full flow-volume loops can offer additional information, while peak expiratory flow might be used if field testing is performed. FEV1 should be measured before exercise and following the exercise challenge. Intervals of 5, 10, 15, 20, and 30 min are recommended by the ATS; however, additional measurements can be taken and should be in the event of severe symptoms of bronchial obstruction. Changes in FEV1 can then be observed by plotting percent resting FEV1 against time (Figure 2).

20 18 16 14 12 10 8 6 4 2 0

What constitutes a ‘positive’ exercise test is controversial due to the variety of techniques described for measurement. Most authorities consider a fall of more than 10% of resting FEV1 as abnormal especially as the ‘normal’ response to exercise is bronchodilation. Some groups require a greater than 15% fall in FEV1 to diagnose EIA and this appears to be the most frequently quoted value. Plotting the FEV1 against time allows an area under the graph calculation to be made that improves the reliability of the test. Alternative Testing Procedures

Other techniques to mimic the airway response to exercise have been described. These are generally thought to be more convenient than full exercise challenge, but are necessarily less specific. Osmotic drying of the airway has been achieved by inhalation of mannitol and this method has received some attention from testers from the field of elite sport. Eucapnic hyperventilation achieves a ventilation rate that mimics that achieved by exercise and has also been used as an outpatient measure of the airway responses. Management

The efficacy of asthma medications for exerciseinduced symptoms will be dealt with elsewhere. Patients should be encouraged to manage their symptoms rather than stop the exercise activity that triggers it. Occasionally, altering the exercise environment is helpful, for example, changing to indoor from outdoor activity. Some elite athletes have learned to manage their asthma by introducing a targeted warm up to trigger a mild episode of EIA and the resultant refractory period elite may allow the completion of the competitive element of the activity. Sporting authorities restrict the use of most inhaled therapeutic agents for asthma. It is sensible for athletes and their coaches to be aware of these restrictions, which can be accessed easily. A range of websites for agencies involved in regulating drugs in sport is given at the end of this article.

Conclusion 0

10 20 30 40 50 60 70 Time from start of exercise (min)

Figure 2 A plot of change in FEV1 as percent baseline with time exercised. Exercise begins at time 0 and ceases at the point marked by the arrow. FEV1 is measured and the percent change is calculated. , change in FEV1; , the effect of an agent , the effect of an agent that changes maximum fall in FEV1; that reduces the duration of EIB. As can be seen it is frequently more accurate to represent changes to EIB by describing the area under the curve than any single element of the plot.

EIA may be a specific disease entity in some circumstances or may be the only manifestation of latent asthma. It is common in asthma sufferers and in athletes of all capabilities. The basis for the development and persistence of exercise triggered airway pathology is not fully understood but probably reflects the ability of hyperventilation to trigger airway inflammation. Diagnosis can be complicated by the variety of presentations and detailed investigations

206 ASTHMA / Extrinsic/Intrinsic

are sometimes required. Asthma management is not always successful at controlling symptoms and behavioral changes may have to be made to achieve control of the disease. See also: Exercise Physiology. Lipid Mediators: Leukotrienes.

Further Reading Anderson SD and Brannan JD (2002) Exercise-induced asthma: is there still a case for histamine? Journal of Allergy and Clinical Immunology 109(5): 771–773. Anderson SD and Daviskas E (2000) The mechanism of exerciseinduced asthma is. Journal of Allergy and Clinical Immunology 106(3): 453–459. Gotshall RW (2002) Exercise-induced bronchoconstriction. Drugs 62(12): 1725–1739. Milgrom H (2004) Exercise-induced asthma: ways to wise exercise. Current Opinion in Allergy and Clinical Immunology 4: 147–153. Seale JP (2003) Science and physicianly practice: are they compatible? Clinical and Experimental Pharmacology and Physiology 30(11): 833–835. Storms WW (2003) Review of exercise-induced asthma. Medicine and Science in Sports and Exercise 35(9): 1464–1470. Tan RA and Spector SL (2002) Exercise-induced asthma: diagnosis and management. Annals of Allergy, Asthma, and Immunology 89(3): 226–235.

which many cells and cellular elements play a role, in particular, eosinophils, mast cells, T lymphocytes, neutrophils, and epithelial cells. Some patients develop structural changes of the airway, a process known as remodeling, possibly due to ongoing inflammation and abnormal repair processes. Susceptible individuals experience recurrent episodes of wheezing, breathlessness, chest tightness, and cough, particularly at night and in the early morning. These episodes are usually associated with widespread but variable airflow obstruction, which is often reversible, and bronchial hyperresponsiveness to a variety of stimuli. Acute asthma is a common medical emergency and requires prompt assessment and treatment. Advances in the understanding of the genetic and environmental factors that account for asthma and its pathogenesis should lead to improved management strategies.

Introduction Historical Perspective

The symptoms of asthma were described by Aretaeus over 2000 years ago. However, despite significant progress in our understanding of its pathogenesis and considerable improvements in pharmacological treatment, we have been unable to halt the relentless increase in prevalence that has taken place over the last 30 years.

Definition Relevant Websites http://www.olympic.org – Home page of the International Olympic Movement, offering insight into the role of antidoping authorities in elite sport. It includes useful links to national Olympic committees detailing specific national guidelines for athletes. http://www.wada-ama.org Homepage of the world antidoping authority. Including details of the use of prescribed proscribed therapeutics in sport. http://www.asthma.org.uk Asthma UK link to exercise induced asthma with tips for patients and some general information regarding school and preschool sporting activities for asthma sufferers.

Extrinsic/Intrinsic N C Thomson and G Vallance, University of Glasgow, Glasgow, UK & 2006 Elsevier Ltd. All rights reserved.

Several different definitions have been devised that describe the asthma phenotype. In 1997 the National Asthma Education and Prevention Program Expert Panel Report defined asthma as: ‘‘A chronic inflammatory disorder of the airways in which many cells and cellular elements play a role, in particular, mast cells, eosinophils, T lymphocytes, neutrophils, and epithelial cells. In susceptible individuals, this inflammation causes recurrent episodes of wheezing, breathlessness, chest tightness, and cough, particularly at night and in the early morning. These episodes are usually associated with widespread but variable airflow obstruction that is often reversible either spontaneously or with treatment. The inflammation also causes an associated increase in the existing bronchial hyperresponsiveness to a variety of stimuli.’’

Epidemiology Prevalence of Asthma and Atopy

Abstract Asthma is one of the most common chronic diseases, affecting 300 million people worldwide. There has been a significant increase in prevalence over the last 30 years, particularly in the West. Complex relationships between genetic and environmental factors, such as viral infections, allergens, and occupational agents, influence the origin and progression of the disease. Asthma is a chronic inflammatory disorder of the airway in

Asthma is one of the most common chronic conditions affecting 300 million people worldwide. The Global Initiative for Asthma (GINA) estimates that one in 20 people in the world now have asthma, with a significant increase in the prevalence of disease over the last 30 years. This is in parallel with an increase in other atopic diseases, such as allergic rhinitis and

Country

ASTHMA / Extrinsic/Intrinsic

207

Scotland Jersey Guernsey Wales Isle of Man England New Zealand Australia Republic of Ireland Canada Peru Trinidad & Tobago Costa Rica Brazil United States of America Fiji Paraguay Uruguay Israel Barbados Panama Kuwait Ukraine Ecuador South Africa Finland Malta Czech Republic Ivory Coast Colombia Turkey Lebanon Kenya Germany France Japan Norway Thailand Sweden Hong Kong United Arab Emirates Philippines Belgium Austria Saudi Arabia Argentina Iran Estonia Nigeria Spain Chile Singapore Malaysia Portugal Uzbekistan FYR Macedonia Italy Oman Pakistan Tunisia Latvia Cape Verde Poland Algeria South Korea Bangladesh Morocco Occupied Territory of Palestine Mexico Ethiopia Denmark India Taiwan Cyprus Switzerland Russia China Greece Georgia Romania Nepal Albania Indonesia Macau

0

5

10

15

20

25

30

35

40

Prevalence of asthma symptoms (%) Figure 1 Ranking of the prevalence of current asthma symptoms in childhood by country: written questionnaire. Reproduced with permission from GINA (2004) Self-reported wheezing in the previous 12 month period in 13- to 14-year-old children. Global Burden of Asthma, p. 6.

atopic dermatitis. These increases have been most noticeable in affluent countries with a mild climate such as the UK, New Zealand, Australia, and North America (Figure 1) and correlate with urbanization and the adoption of a westernized life style. It is more common in children than in adults. The clearest risk factor for the development of asthma is atopy. Atopy is the genetic predisposition for the development of an IgE-mediated response to common aeroallergens. Complex relationships between atopy and

environmental factors such as viral infections, allergens, and occupational agents influence the origin and progression of the disease. Morbidity

The morbidity from asthma is considerable. Surveys of patients with asthma indicate that many have poorly controlled symptoms, impaired indices of quality of life, and are often receiving inadequate


treatment. Hospital admission rates for asthma, particularly in children, increased from the 1970s until the mid 1980s and have since then remained stable. In the US during 2002 there were 13.9 million outpatient visits, 1.9 million emergency room visits, and 484 000 hospitalizations. Many children and adults with asthma lose time from school and work, respectively. The financial impact of asthma is considerable; in the US the estimated total cost exceeds $6 billion per annum, with hospitalization and emergency room visits making up 50% of that figure.

Classification Asthma can be classified on the basis of severity and etiology. Severity

The severity of asthma can be graded by assessing the frequency and severity of symptoms and measurements of lung function before treatment is started or by the level of treatment required to achieve asthma control (Table 1). Etiology

Mortality

International mortality figures for asthma are often unreliable due to misclassification of the cause of death. In Western countries, where studies were restricted to the 5–34 years age group, the mortality from asthma increased steadily from the mid-1970s to the late 1980s. More recent studies suggest a plateau or decline in deaths from asthma. Risk factors for increased morbidity and potential mortality include socioeconomic deprivation, ethnicity, urban dwelling, and comorbid issues such as drug abuse. The vast majority of deaths occur among those with chronic severe asthma; few deaths occur among those with previously mild disease. Deaths are associated with inadequate treatment with inhaled or oral steroid and with poor follow-up and monitoring. Natural History

The findings of the Tucson Children’s Respiratory Study suggested three clinical phenotypes of childhood asthma. Transient infant wheezing occurs during infancy, but not after the age of three years. These children have no family history of atopy and have a good prognosis. The second phenotype is the nonatopic wheeze of the toddler and early school years, after an early lower respiratory tract infection. The third phenotype is persistent atopic wheeze, which describes children who continue to wheeze at age 10 and have associated atopy and airway hyperresponsiveness. Many children have a favorable outcome with spontaneous remission in their adolescence. Risk factors for progression into adulthood include early onset with severe symptoms, poor lung function, and airway hyperresponsiveness. It occurs more commonly in girls with associated atopy. Most adults with mild-to-moderate asthma appear to continue to have symptoms of a similar severity. Remission of adult asthma is rare. Irreversible airflow obstruction can develop in nonsmokers with asthma particularly in those individuals with severe symptoms and mucus hypersecretion. Smokers with asthma have an accelerated decline in lung function.

In 1947, Rackeman was the first to subdivide asthma into intrinsic and extrinsic asthma. He noted that extrinsic asthma, now described as allergic asthma, started before the age of 30 years and was associated with atopy. Intrinsic or nonallergic asthma was noted to begin in middle age and was not associated with allergy, but with nasal polyps. It is more common in women.

Etiology Genetic

Twin and family studies have demonstrated that atopic diseases cluster in families and have a genetic basis. Genome-wide scans have shown that many genes determine the risk of asthma. The region of chromosome 5q31-33 controls the production of interleukin (IL)-4 and IL-13 and has been linked to atopy. Linkage studies have implicated other candidate genes on chromosomes 2, 3, 4, 6, 7, 11, 12, 13, 17, and 19. Classical positional cloning approaches have led to the identification of new genes of potential significance such as ADAM33, IL4RA, and CD14. This may contribute to our understanding of the mechanism of disease, such as the role of ADAM33 in airway hyperresponsiveness. They may also cast light on different individual responses to therapy, as there are polymorphisms of a number of common drug targets. The delineation of the precise relationships between these genetic factors and environmental agents is now required. Environment

Hygiene hypothesis In 1989, Strachan noted an inverse relationship between family size and hay fever, with children with more siblings having a lower risk of atopy. It has been suggested that childhood exposure to infection may protect against risk of atopy. Low levels of infection in infancy, associated with improvements in public health, may deprive the immune system of the Th1 stimulus that normally balances the Th2 predominance of the neonate. This


209

Table 1 Classification of asthma severity by daily medication regimen and response to treatment Patient symptoms and lung function on current therapy

Current treatment step Step 1: intermittent

Step 2: mild persistent

Step 3: moderate persistent

Level of severity Step 1: intermittent Symptoms less than once a week Brief exacerbations Nocturnal symptoms not more than twice a month Normal lung function between episodes

Intermittent

Mild persistent

Moderate persistent

Step 2: mild persistent Symptoms more than once a week but less than once a day Nocturnal symptoms more than twice a month but less than once a week Normal lung function between episodes

Mild persistent

Moderate persistent

Severe persistent

Step 3: moderate persistent Symptoms daily Exacerbations may affect activity and sleep Nocturnal symptoms at least once a week 60%oFEV1 o80% predicted OR 60%oPEFo80% of personal best

Moderate persistent

Severe persistent

Severe persistent

Step 4: severe persistent Symptoms daily Frequent exacerbations Frequent nocturnal asthma symptoms FEV1 p60% predicted OR PEFp60% of personal best

Severe persistent

Severe persistent

Severe persistent

Reproduced with permission from GINA (2004) Global Strategy for Asthma Management and Prevention, Chapter 7, Part 4A, Figures 5–7, p. 7.

unrestrained Th2 response is postulated to predispose to allergic disease. European studies showing lower levels of atopic disease amongst children raised in rural communities have suggested that exposure to bacterial endotoxin may play a role in the development of tolerance to common allergens. However, the high levels of asthma associated with cockroach allergy in inner-city areas of the US, and the simultaneous increase in Th1 diseases, such as type 1 diabetes, illustrate that our understanding of the relationship between environmental exposure and disease is not yet complete. Allergen exposure Sensitization to the house dust mite Dermataphagoides pteronnysinus is the most common risk factor for the development of asthma in adults and children. Early exposure to the dust mite antigen Der p1 has been shown to increase the risk of asthma fivefold. Peat elegantly demonstrated the doseresponse relationship between dust mite level and severity of asthma symptoms across six different regions

of Australia with increasing humidity levels. Dust mites thrive in a humid environment, and improved westernized building construction techniques, favoring colonization by house dust mites, may have contributed to the increase in asthma. Studies at altitudes where the levels of house dust mite are extremely low have suggested that a low allergen environment does improve symptoms of asthma. However, simple measures such as mattress covers and carpet cleaning have shown little effect on reducing the domestic burden of dust mite and asthma symptoms. Exposure to other indoor allergens from animal dander and cockroach are also important risk factors for asthma. Removal of the pet from the family is advised to reduce exposure to the allergen. However, early exposure to cat allergen has recently been demonstrated to protect against development of asthma and the full intricacies of the relationship remain to be elucidated. Cockroach infestation has become rampant in inner-city areas of the US and sensitization to the


German cockroach Blatella germanica has become an important risk factor for asthma. Outdoor air environment Sensitization to the fungus Alternaria is a risk factor for asthma. It has been suggested that outbreaks of asthma after thunderstorms are related to release of fungal spores of Cladosporium. The relationship between air pollution and asthma is complex as there has been considerable reduction in air pollution over the time period when asthma prevalence has increased. Extremely high levels of asthma have been noted in the rural Highlands of Scotland. It has been suggested that particulate pollution may protect against incidence but exacerbate symptoms in those sensitized. Diet Changes in the modern processed diet have been linked with an increased risk of asthma. High levels of omega-3 oils associated with eating fresh fish have been shown to reduce the risk of asthma. Breast-feeding to 3 months has also been associated with lower levels of asthma. Cigarette smoking There is much evidence to suggest that exposure to tobacco smoke in utero and in early childhood increases the risk of allergy and wheeze. Nevertheless, as the total numbers of smokers in the UK have fallen during the last 30 years, it is not the whole answer to the rise in asthma prevalence.

Pathology Fatal Asthma

In cases of fatal asthma the lungs are hyperinflated due to air trapping caused by plugging of the medium

to small airways with mucus and inflammatory cells, particularly eosinophils. Histologically, the airways show characteristic changes: an intense infiltration by inflammatory cells, particularly eosinophils and T lymphocytes, sloughing of the surface epithelium, thickening of the reticular basement, increase in the airway smooth muscle mass, increased numbers of epithelial goblet cells, vasodilatation, and edema (Figure 2). Neutrophils are found also in those who die suddenly from acute asthma. Chronic Asthma

Information on the pathology of asthma has been obtained from bronchial biopsies obtained from patients with mainly mild asthma. The histological changes are similar although less pronounced than those obtained from cases of fatal asthma. The similarity of the histological changes in allergic or extrinsic and nonallergic or intrinsic asthma suggests a final common pathogenic mechanism in both types of asthma. Neutrophils are found more commonly in patients with severe asthma.

Clinical Features Acute Asthma

Acute asthma is a common medical emergency. It is characterized by a progressive increase in dyspnea, cough, or wheeze. The decrease in expiratory airflow can be quantified by a fall in peak expiratory flow (PEF) or forced expiratory volume in 1 s (FEV1). Deterioration usually progresses over hours to days, although in some cases it may be more sudden and require rapid treatment within minutes. The severity of exacerbation is highly variable. Respiratory tract

Mucus Epithelial cells and goblet-cell hyperplasia Thickening of sub-basement membrane Cellular infiltrate

Hypertrophy of smooth muscle

Vascular congestion Figure 2 Autopsy specimen of airway from a subject who died from acute asthma showing characteristic histological changes. Hematoxylin and Eosin, 40. Photograph courtesy of Dr F Roberts, Department of Pathology, Western Infirmary Glasgow.


infections are thought frequently to precipitate attacks of asthma, particularly in children. Infections are mainly viral, especially human rhinoviruses but also respiratory syncytial virus, adenoviruses, parainfluenza, and influenza viruses. The role of infection in provoking asthma attacks in adults is less certain. Clinical assessment Clinical features A short history of the features of an exacerbation should be elicited. The aims are to ascertain duration and severity of symptoms, with the perspective of current medication and prior admissions. Assessment must be rapid and accurate to permit prompt treatment. The history is usually one of increasing breathlessness and wheeze. Patients often have difficulty speaking and sleep is disturbed by the severity of these symptoms. There is increasing need for bronchodilator treatment, which becomes less effective. The patient may show signs of exhaustion and reduced conscious level. There is invariably an associated tachycardia, increase in respiratory rate, and auscultation of the chest may reveal severe wheeze or absent breath sounds that indicates very severe airflow obstruction. The chest becomes hyperinflated and patients may use accessory respiratory muscles. Investigations Measurement of pulse oximetry is necessary in acute asthma to evaluate oxygen saturation. The aim is to maintain SpO2492%. Arterial blood gas analysis is necessary if SpO2o92%. If oxygenation remains inadequate despite supplemental oxygen, additional complications should be considered, particularly pneumonia. The earliest abnormality is respiratory alkalosis and hypocarbia, but normal oxygen tension. As airflow obstruction increases there is uneven distribution of inspired air and changes in the normal ventilation-to-perfusion ratio. As severity increases, hypoxemia develops. The presence of normal levels of arterial carbon dioxide tension is ominous as it indicates the patient is becoming exhausted. Monitoring response to treatment should be on the basis of PEF and clinical examination. A chest radiograph may show evidence of hyperinflation, mucus plugging, and atelectasis in an acute exacerbation, but these findings may add little to management. A chest radiograph should be performed if pneumothorax is suspected. Levels of severity Asthma guidelines have been developed to ensure prompt, systematic history and examination, and ensure accurate assessment of severity. The British Guideline on the Management of Asthma defines a moderate exacerbation as one presenting with increasing symptoms of wheeze,

211

dyspnea, or breathlessness and a fall in PEF to 50–75% of the best or predicted. However, if the PEF falls to 33–55% best or predicted and is accompanied by an inability to speak in complete sentences, tachypnea of 425 breaths per min, or tachycardia of 110 beats per min, the exacerbation is classified as severe. Life-threatening features are a PEF less than one-third of best or predicted or hypoxia demonstrated by arterial oxygen saturations of less than 92% on air or arterial partial pressure of less than 8 kPA. Normal levels of CO2, a silent chest on examination, or feeble respiratory effort, cyanosis, bradycardia, dysrhythmia, hypotension, exhaustion, confusion, or coma are all signs of a near fatal episode. Chronic Asthma

The diagnosis based on a history of episodic respiratory symptoms especially after exercise or during the night is usually not difficult. Demonstration of reversible airflow obstruction gives a simple, reliable, and objective diagnosis of asthma. Evidence of reversibility can be found through the history of symptoms of episodic cough, wheeze, chest tightness, or dyspnea, measurement of PEF, or spirometry, and trials of therapy. Conditions to be considered in the differential diagnosis are listed in Table 2. Clinical assessment Clinical features Asthma may present with wheeze, shortness of breath, cough, or chest tightness. The hallmark of asthma is that these symptoms tend to be variable and intermittent. They are often worse at night and early morning and provoked by triggers such as allergens or exercise (Table 3). Less common factors are rhinitis, bacterial sinusitis, menstruation, gastroesophageal reflux, and pregnancy. When cough is the predominant symptom without wheeze, this is Table 2 Differential diagnosis of asthma Disease

Children

Adults

Cystic fibrosis Gastroesophageal reflux Bronchiectasis Ciliary dyskinesia Developmental disorder of the airway Inhaled foreign body Chronic obstructive pulmonary disease Left ventricular function Pulmonary thromboembolism Vocal cord dysfunction Upper airway obstruction Pulmonary eosinophilia Bronchial carcinoid

O O O O O O

O O O

O, Diagnosis should be considered.

O O O O

O O O O O O O O

212 ASTHMA / Extrinsic/Intrinsic Table 3 Triggers of asthma Infections, particularly viral Allergens, e.g., house dust mite, pollens, animals Occupational agents, e.g., isocyanate-containing paints, flour Environmental pollutants, e.g., cigarette smoke, sulfur dioxide Drugs, e.g., beta-blockers Exercise Cold air Hyperventilation Foods Psychological factors

referred to as cough-variant asthma. The physical sign of wheezing (usually expiratory, bilateral, polyphonic, and diffuse) is associated with asthma but has low sensitivity and specificity, and in many patients examination will be normal. Investigation A simple measure of pulmonary function by PEF is helpful, not only for initial assessment, but also to monitor symptoms, alert to deterioration in airflow obstruction, and evaluate response to treatment. Twice-daily recording of PEF for two weeks is a simple and cheap method of demonstrating variation in airflow obstruction, with a diurnal variation of greater than 15% confirming the diagnosis. However, in patients with mild asthma, the PEF may show normal variability. Spirometry demonstrates an obstructive pattern with reduction of the ratio of FEV1 to forced vital capacity (FVC). The key feature is that this airway obstruction may be reversible. Administration of a bronchodilator typically causes an increase in FEV1 of 12–15%. However, failure to demonstrate reversibility does not exclude asthma, or prove irreversible disease. Airway hyperresponsiveness is a characteristic feature of asthma and can be demonstrated by bronchial provocation techniques. The most common methods are provocation by inhalation of methacholine or histamine and exercise challenge. Fall in FEV1 is measured by serial spirometry after inhalation of increasing concentrations of methacholine. Results are expressed as the concentration of the agent that elicits a fall of 20% in FEV1. This concentration defines the degree of bronchial responsiveness and severity of disease. Skin prick testing, measurement of total and specific IgE levels, and blood eosinophilia are difficult to interpret in asthma because they have variable sensitivity and specificity. Routine chest radiographs in asthma may yield no new information and may be normal in chronic asthma. Assessment of control Asthma control can be determined by assessing symptoms, inhaled b2 adrenoceptor agonists use, lung function as well as rate

of exacerbations, number of emergency consultations for asthma, and hospital admissions. Good control is described as the presence of minimal symptoms during day and night, minimal need for reliever medication, no exacerbations, no limitation of physical activity, and normal lung function. It may not be possible to achieve good asthma control in patients with moderate or severe persistent asthma (Table 1). Specific clinical problems Asthma during pregnancy The course of asthma during pregnancy varies, with a similar proportion of women improving, remaining stable, or worsening. The risk of an exacerbation of asthma is high immediately postpartum, but the severity of asthma usually returns to preconception level after delivery. Changes in b2-adrenoceptor responsiveness and changes in airway inflammation induced by high levels of circulating progesterone have been proposed as possible explanations for the effects of pregnancy on asthma. Gastroesophageal reflux Gastroesophageal reflux can trigger attacks of asthma although the incidence is unclear. The mechanism is unknown; possibilities include aspiration or an esophagio-bronchial reflux triggered by acid irritation of the esophageal mucosa.

Pathogenesis The pathogenesis of asthma involves acute and chronic inflammation as well as remodeling (Figure 3). The underlying process is one of inflammation involving eosinophils and T lymphocytes, with the release of various mediators and cytokines, although recent evidence indicates a role for other cells including mast cells, neutrophils, macrophages, and epithelial cells. Some patients develop structural changes of the airway, a process known as remodeling. The mechanisms involved in remodeling remain to be clarified but probably consist of ongoing inflammation and abnormal repair processes. Remodeling occurs in many asthmatic patients, although the extent varies. It is thought that remodeling may play an important role in causing symptoms and loss of lung function in severe asthma, although this hypothesis remains to be established. Airway Inflammation

There are two distinct responses to inhalation of an allergen. The immediate hypersensitivity reaction, in which wheeze occurs within minutes, is comparable to the wheal-and-flare response of skin. Further wheeze is caused by the late phase response, mounted between 6 and 9 h after allergen provocation.


Inducers of asthma

213

Inflammatory mediators and asthma

Allergen Antigen presenting cell Dendritic cell Lymph node Acute airway inflammation

Chronic airway inflammation

Th2 lymphocyte

Airway remodeling

Cytokines (e.g., IL-4, IL-5, IL-13)

Symptoms Exacerbations Disability Figure 3 In susceptible individuals allergens, occupational agents, and other known and unknown inducers of asthma cause airway inflammation. The inflammation may be acute and resolved, but in most individuals the inflammation is chronic and may be associated with airway remodeling. Airway inflammation and remodeling cause asthma symptoms, exacerbations, and disability.

The immediate reaction involves the activation of mast cells by allergen cross-linking two IgE antibodies (Figure 4). IgE antibodies are produced by B cells in response to processed antigen, which is presented by airway dendritic cells in the draining lymph node. The IgE antibodies bind mast cells by their high-affinity receptors (FceRI). Cross-linking by allergen of IgE antibodies initiates signal transduction by the FceRI effecting degranulation of the mast cell. This promotes release of mediators such as histamine, tryptase, eicosanoids, and reactive oxygen species. These spasmogens cause secretion of mucus, smooth muscle constriction, and vasodilation. Leakage of plasma protein causes edema of the airway wall, impedes clearance of mucus, and causes formation of plugs; all result in reduced airway conductance. The late phase reaction includes the accumulation of activated eosinophils, lymphocytes, macrophages, neutrophils, and basophils. The ability of cytokines to induce the expression of adhesion molecules provides a mechanism for cell migration from the circulation to the airway. Eosinophils are thought to play a central role in the pathogenesis of chronic asthma. IL-5 controls the production of eosinophils by the bone marrow and their subsequent release into the circulation. They migrate from circulation to the airway under the influence of chemokines and release toxic granule proteins, including major basic proteins, eosinophil peroxidase and eosinophil cationic protein, Th2

Mast cell (e.g., leukotrienes, histamine, tryptase)

Epithelium

Eosinophil

(e.g., prostaglandins, IL-6, IL-8)

(e.g., leukotrienes, major basic proteins)

Inflammatory mediators

BronchoMucosal constriction edema

Mucus hypersecretion

Bronchial reactivity

Airway remodeling

Symptoms of asthma Figure 4 Possible pathways in the development of airway inflammation in asthma following exposure to allergen.

cytokines, and leukotrienes. Major basic proteins causes direct airway damage with epithelial shedding. Leukotrienes increase vascular permeability and constrict smooth muscles. Challenge of the airway with allergen increases the local levels of IL-5, which correlates directly with the degree of airway eosinophilia. Recently, doubts have arisen about the role of eosinophils in causing airway hyperresponsiveness in asthma. Treatment of patients with allergic asthma using anti-interleukin-5 monoclonal antibody does not prevent allergen-induced bronchoconstriction or airway hyperresponsiveness, despite markedly suppressing eosinophil numbers within the airways. T cells are found in abundance in the inflamed airways of asthma patients and it is widely held that the Th2 subset is a driving force in allergic inflammation. The T helper subsets were characterized by Mosman on the basis of their signature cytokines. Th2 cell cytokines include IL-4, IL-5, and IL-13. IL-5 is involved in eosinophil maturation and activation, whereas IL-4 and IL-13 control synthesis of IgE. However, the Th2 paradigm for allergic asthma is now thought to be too simplistic and an additional role for Th1 cells has been postulated.


The mast cell degranulation is crucial to the acute response to allergen, with release of histamine and synthesis of mediators effecting early recruitment, adhesion, and proliferation of cells. It may also contribute to remodeling as it contains proteoglycans with various functions, including support structures for remodeling. The role of other inflammatory cells in the pathogenesis of asthma including neutrophils and macrophages is less clearly characterized. There are also complex interactions between neural control of airways and inflammation. Airway Remodeling

There has been considerable evidence that inflammation alone may not explain the full pathophysiology of asthma. Two large longitudinal studies of inhaled corticosteroids have shown little effect on the natural history of asthma. Acute airway inflammation should be resolved to permit resumption of normal function. In chronic asthma, a state of increased injury and repair is thought to lead to remodeling. It is coordinated by inflammatory cells, such as eosinophils, mast cells and T cells, and structural cells such as fibroblasts. Growth factors are secreted in response to epithelial damage and cause thickening of the smooth muscle and basement membrane, resulting in airway narrowing. It is thought that a primary abnormality of the epithelium predisposes to such damage by toxins. The interaction between the thickened basement membrane and altered submucosa, known as the epithelial-mesenchymal trophic unit, is postulated to be important early in the origins of disease. This hypothesis is supported by the recent identification of ADAM33 as an asthma susceptibility gene expressed abundantly by the smooth muscle and fibroblasts, but not by inflammatory cells.

Animal Models Animal models have been used to study both the pathogenesis and treatment of asthma. Asthma does not occur spontaneously in animals although both horses and the Berenji greyhound develop a respiratory condition that has some features of asthma. Most animal models involve sensitization to an allergen such as ovalbumin or house dust mite allergen challenge. The species commonly used include mice, guinea pigs, sheep, rats, monkeys, and dogs. The mouse is often used in these models, mainly because this species allows for the application in vivo of gene deletion technology as well as the low cost and availability of inbred species with known characteristics. Animal models of allergen-induced airway

inflammation and hyperresponsiveness have provided important information on the acute inflammatory response to allergen exposure but have been less relevant to the study of mechanisms of chronic asthma and airway remodeling. The interpretation of data from experiments in animal models is influenced by the protocol used to sensitize and challenge the animal and the strain of animal used. See also: ADAMs and ADAMTSs. Allergy: Overview. Angiogenesis, Angiogenic Growth Factors and Development Factors. Arterial Blood Gases. Asthma: Overview; Allergic Bronchopulmonary Aspergillosis; Aspirin-Intolerant; Occupational Asthma (Including Byssinosis); Acute Exacerbations; Exercise-Induced. Bronchoalveolar Lavage. Bronchodilators: Anticholinergic Agents; Beta Agonists. Carbon Dioxide. Chymase and Tryptase. Corticosteroids: Therapy. Dendritic Cells. Dust Mite. Endothelial Cells and Endothelium. Environmental Pollutants: Overview. Epidermal Growth Factors. Genetics: Overview; Gene Association Studies. Histamine. Immunoglobulins. Leukocytes: Eosinophils; Neutrophils; Monocytes; T cells; Pulmonary Macrophages. Lipid Mediators: Overview. Matrix Metalloproteinases. Neurophysiology: Neural Control of Airway Smooth Muscle. Oxygen Therapy. Pneumothorax. Respiratory Muscles, Chest Wall, Diaphragm, and Other. Signs of Respiratory Disease: Breathing Patterns; General Examination; Lung Sounds. Smooth Muscle Cells: Airway. Symptoms of Respiratory Disease: Cough and Other Symptoms. Tumor Necrosis Factor Alpha (TNF-a ). Upper Airway Obstruction. Upper Respiratory Tract Infection.

Further Reading Barnes P, Drazen J, Rennard S, and Thomson NC (eds.) (2002) Asthma and COPD – Basic Mechanisms and Clinical Management. London: Academic Press. Bel E (2004) Clinical phenotypes of asthma. Current Opinion in Pulmonary Medicine 10(1): 44–50. Bousquet JP, Jeffery Busse W, Johnson M, and Vignola A (2000) Asthma – from bronchoconstriction to airways inflammation and remodeling. American Journal of Respiratory and Critical Care Medicine 161: 1720–1745. British Thoracic Society (2003) British guideline on the management of asthma. Thorax 58: i1–i94. Busse W (2001) Asthma. New England Journal of Medicine 5: 350–362. GINA (2004) Self-reported wheezing in the previous 12 month period in 13- to 14-year-old children. Global Burden of Asthma, p. 6. GINA (2004) Global Strategy for Asthma Management and Prevention, Chapter 7, Part 4A, Figure 5–7, p.7. Kay AB (2001) Allergy and allergic diseases. New England Journal of Medicine 344: 30–37. Kips JC, Anderson GP, Fredberg JJ, et al. (2003) Murine models of asthma. European Respiratory Journal 22(2): 374–382. Larche M, Robinson R, and Kay AB (2003) The role of T lymphocytes in asthma. Journal of Allergy and Clinical Immunology 111: 450–459.

ATELECTASIS 215 McFadden E (2003) Acute severe asthma. American Journal of Respiratory and Critical Care Medicine 168: 740–759. National Asthma Education and Prevention Program Expert Panel Report: guidelines for the diagnosis and management of asthma, Update on Selected Topics – 2002. Journal of Allergy and clinical Immunology 110(5 pt 2): S141–S219. NHLBI/WHO (2004) Global initiative for asthma: global strategy for asthma management and prevention. NHLBI/WHO Workshop Report NIH 02-3659. Bethesda: NIH.

O’Byrne PM and Thomson NC (eds.) (2001) Manual of Asthma Management, 2nd edn. London: W B Saunders. Rodrigo G, Rodrigo M, and Jesse B (2004) Acute asthma in adults: a review. Chest 125(3): 1081–1102. Tattersfield A, Knox A, Britton J, and Hall I (2002) Asthma. Lancet 360: 1313–1322. Thomson NC, Chaudhuri R, and Livingston E (2004) Asthma and cigarette smoking. European Respiratory Journal 24: 822–833.

ATELECTASIS P A Kritek, Brigham and Women’s Hospital at Harvard Medical School, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Atelectasis is the loss of volume resulting from decreased gas in a given portion of lung. The mechanisms that cause atelectasis can be divided into three categories: passive, adhesive, and resorptive. Passive atelectasis results from space-occupying lesions in either the pleural space or the parenchyma compressing adjacent normal lung tissue. Adhesive atelectasis is caused by a decrease in the level or activity of surfactant leading to an increase in surface tension in the alveolus and subsequent collapse. Resorptive atelectasis ensues when there is partial or complete occlusion of flow of gas between alveoli and the trachea. Oxygen, carbon dioxide, and nitrogen will diffuse from the alveolus into the capillary until all gas is removed from the alveolar space. Small areas of atelectasis can be found in normal lungs due to the effects of gravity. Pneumothoraces or large bullae can result in passive atelectasis. Adhesive atelectasis is a feature of respiratory distress syndrome of the neonate as well as acute respiratory distress syndrome in adults. Resorptive atelectasis is found distal to an obstructive lesion, such as a tumor or mucus plug. Atelectasis associated with anesthesia is complex and caused by a combination of these mechanisms.

thereby creating a smaller space in which to maintain the inflated lung (Figure 1). As a result, adjacent lung tissue will lose gas and subsequently collapse. This form of atelectasis is referred to as passive because the collapsed lung is not inherently abnormal but is being affected by an adjacent pathologic process. If the inciting cause of the atelectasis is resolved (e.g., a pneumothorax is evacuated), the underlying atelectatic lung should reexpand and return to normal function. It should be noted, however, that after a segment of lung has collapsed, there are local changes in permeability, inflammatory markers, alveolar macrophage function, and surfactant associated with all types of atelectasis that may predispose to abnormal function upon reinflation. Adhesive Atelectasis

Atelectasis is the loss of volume resulting from decreased gas in a given portion of lung. These changes can be small, affecting only subsegmental regions, or more dramatic, leading to collapse of an entire lung. The mechanisms that result in atelectasis can be divided into three categories: passive, adhesive, and resorptive. Each of these is discussed individually. Note that although discussions of radiographic descriptions of atelectasis are included, the discussion is grounded in these pathophysiologic aspects of atelectasis.

Adhesive atelectasis results from the absence, loss, or decreased activity of surfactant within the alveoli (Figure 2). Surfactant, produced by type II alveolar cells, decreases surface tension as alveolar surface area decreases and balances the retraction forces of the lung in order to avoid end-expiratory alveolar collapse. When surfactant is decreased or inactivated, the balance is upset and atelectasis ensues. In this situation, there is loss of gas volume based on mechanical forces within the alveolus as opposed to external compression. This form of atelectasis can occur in the neonatal infant in the setting of immature type II alveolar cells and decreased surfactant production. Alternatively, certain adult disease states, including ventilator-associated pneumonia and acute respiratory distress syndrome (ARDS), are associated with decreases in absolute surfactant levels or its activity.

Passive Atelectasis

Resorptive Atelectasis

Passive atelectasis results from a space-occupying lesion within the pleural space or the parenchyma

Resorptive atelectasis results when there is partial or complete occlusion of flow of gas between alveoli

Description

216 ATELECTASIS

Pneumothorax

Pleural space

Normal alveolus

Passive atelectasis

Figure 1 Normal alveolus and passive atelectasis.

Edema and decreased surfactant

Normal alveolus

Adhesive atelectasis

Figure 2 Normal alveolus and adhesive atelectasis.

and the trachea (Figure 3). As the section of obstructed lung continues to be perfused, the partial pressure of oxygen in the alveolus equilibrates with that in the alveolar capillary. The loss of oxygen in the alveolus leads to increased concentrations of nitrogen and carbon dioxide in the alveolus, and subsequent gradients result in movement of both gases from the alveolus into the capillary. This process will continue until all gas has been removed from the airspace, a phenomenon that takes 18–24 h in normal volunteers with a completely occluded lobe. Resorptive atelectasis can result from any cause of obstruction to airflow. Common etiologies include

tumor, mucus plug, and foreign body. The process of resorptive atelectasis is thought to occur more quickly in the setting of oxygen-rich gas because the first step of oxygen absorption is more rapid and there is a larger portion of gas that is absorbed initially.

Atelectasis in Normal Lung Function By definition, atelectasis is caused by loss of gas from a normally gas-filled section of lung. However, there is a form of passive atelectasis that is commonly seen in ‘normal’ lungs that is referred to as dependent atelectasis. This is the loss of volume in alveoli and

ATELECTASIS 217

Tumor

Normal alveolus

Resorptive atelectasis

Figure 3 Normal alveolus and resorptive atelectasis.

small airways in the lower lung zones based on gravity-related decreases in transpulmonary pressures. Although these areas of atelectasis may have physiologic consequences in patients with underlying lung disease (e.g., ARDS), the small loss of volume most likely has no physiologic consequence in normal subjects. The increased use of computed tomography (CT) has led to a greater awareness of these changes. Dependent atelectasis will resolve with position change, as demonstrated by repeated tomography with the patient in the prone position.

Atelectasis in Respiratory Diseases

reserved for collapse of lung associated with pleural thickening, most commonly found in association with asbestos exposure. The etiology of rounded atelectasis is not well understood but is thought to result from pleural fibrosis contracting and causing adjacent lung to curl upon itself and collapse. The radiographic findings are best characterized on chest tomography. The lesions are classically peripheral, subpleural, uniform in density, and mass-like in appearance. They often have a curvilinear opacity of bronchi and vessels (termed a comet tail) extending toward the hilum. Care needs to be taken in diagnosing a lung nodule as rounded atelectasis purely on CT findings because there are many reports of malignancy masquerading as rounded atelectasis.

Passive Atelectasis

Passive atelectasis can occur in a generalized or localized manner. In the setting of a large pneumothorax, the majority of the parenchyma of the ipsilateral lung will undergo passive atelectasis. In contrast, lung tissue adjacent to a bleb or a parenchymal cyst can collapse in a more localized form of passive atelectasis. Both examples illustrate a space-occupying phenomenon that causes subsequent loss of gas and collapse. With relief of a large space-occupying lesion, such as a pneumothorax, the underlying lung may develop reexpansion pulmonary edema. This process is thought to result from changes in pulmonary capillary permeability but is not well understood. The pulmonary edema is usually transient and resolves with supportive care. There is a unique form of passive atelectasis termed rounded atelectasis. This description is

Adhesive Atelectasis

The classic example of absorptive atelectasis is that of respiratory distress syndrome (RDS) of the neonate. As discussed previously, type II alveolar cells are often immature and not fully functional in the preterm infant, predisposing the infant to RDS. There have been significant decreases in morbidity and mortality in RDS with the initiation of surfactant (natural or synthetic) replacement in the immediate neonatal period. This improvement is in part due to the marked decrease in atelectasis and resulting improved gas exchange with surfactant therapy. Many studies demonstrate decreased surfactant levels in adults with ARDS. There is also experimental evidence for decreased surfactant activity in the setting of leakage of plasma proteins into the alveolar space. At the same time, studies using CT have

218 ATELECTASIS

demonstrated areas of atelectasis in ARDS. The etiology of this atelectasis is likely multifactorial. There are increased changes in dependent lung zones suggesting a component of passive atelectasis accompanying the adhesive atelectasis, the latter presumably from decreased quantity or activity of surfactant. Some believe that the recurrent opening and closing of small, atelectatic lung units contributes to ventilator-induced lung injury (termed ‘atelectrauma’). In response to this, there has been increasing research on how to avoid or overcome the atelectasis associated with ARDS. Efforts to replace surfactant through a variety of modalities have not demonstrated a mortality benefit, although some studies have shown improved gas exchange. Lung ventilation strategies aimed at maintaining an ‘open lung’, such as recruitment maneuvers, high-frequency jet ventilation, and conventional ventilation with high positive end expiratory pressure (PEEP), have shown transient rises in oxygenation but no improvement in mortality. There is no conclusive evidence supporting any specific treatment of atelectasis associated with ARDS. Resorptive Atelectasis

Endobronchial tumors are a common cause of resorptive atelectasis, often resulting in segmental or lobar collapse. One retrospective review reported an incidence of atelectasis in one of five patients with small cell lung cancer. Because these are gradual processes occurring over weeks to months, the atelectatic lung often does not completely reexpand or function normally if the obstruction is relieved. However, because there is a risk for postobstructive pneumonitis and there is evidence for increased bacterial growth in atelectatic lung, clinicians will often attempt to minimize the obstruction. In contrast to the more gradual development of atelectasis with tumor growth, resorptive atelectasis can occur rapidly with acute occlusions of large airways. Mucus plugs, in both patients with asthma and those with altered secretion clearance, have been described as causes of resorptive atelectasis. Resorptive atelectasis has also been reported with malpositioned endotracheal tubes that selectively intubate the right lung. In a short period of time, the entire left lung can undergo atelectasis that will resolve with repositioning of the endotracheal tube.

by CT) and is often clinically significant. Lobar atelectasis can result in parenchymal shunt and be manifest as marked hypoxemia. There is evidence of a positive correlation between the amount of atelectatic lung and the degree of hypoxemia. The atelectasis associated with anesthesia is caused by multiple mechanisms. The first contributor is dependent atelectasis (a form of passive atelectasis) from prolonged recumbent positioning with exaggerated effects of gravity. There is also evidence of changes in diaphragm position and shape with supine positioning for anesthesia contributing to dependent atelectasis. These effects are more pronounced when neuromuscular blockade is used in conjunction with anesthesia. All these changes are especially pronounced in morbidly obese patients. Some anesthesiologists advocate for intermittent use of large tidal volume breaths to overcome atelectasis intraoperatively, whereas others advocate for elevated levels of PEEP with ventilation. Administration of supplemental oxygen as part of mechanical ventilation contributes to resorptive atelectasis. Results are mixed with regard to whether higher inspired fractions (80–100%) during induction and maintenance of anesthesia increase the likelihood of atelectasis compared to lower oxygen/ nitrogen mixtures. There is also evidence of early airway closure during anesthesia contributing to the resorptive mechanism of atelectasis. It has been suggested that repeated episodes of atelectasis, from the previously mentioned means, can also lead to impaired surfactant function resulting in a component of adhesive atelectasis as well. The issues of atelectasis often persist into the postoperative period, resulting in persistent hypoxemia. The resorptive atelectasis associated with higher inspired fractions of oxygen persists after extubation despite attempts at recruitment at the end of the operation. Dependent atelectasis, which in the normal subject will resolve with changes in position and deep breathing, often persists due to decreased mobility, pain, and sedation in the postoperative period. These conditions also commonly lead to impaired clearance of secretions predisposing to obstruction of airways and resorptive atelectasis. Deep breathing, incentive spirometry, vibration beds, chest physiotherapy, noninvasive ventilation, and therapeutic bronchoscopy all seem to have similar results in terms of resolution of postoperative atelectasis.

Atelectasis Associated with General Anesthesia

Atelectasis associated with general anesthesia deserves special mention because it occurs at rates as high as 90% of anesthetized patients (as detected

See also: Alveolar Surface Mechanics. Breathing: Breathing in the Newborn. Diffusion of Gases. Lung Imaging. Mucus. Oxygen Therapy. Peripheral Gas Exchange. Physiotherapy. Signs of Respiratory

AUTOANTIBODIES 219 Disease: Lung Sounds. Surfactant: Overview. Ventilation, Mechanical: Positive Pressure Ventilation.

Further Reading Brower RG, et al. (2003) Effects of recruitment maneuvers in patients with acute lung injury and acute respiratory distress syndrome ventilated with high positive end-expiratory pressure. Critical Care Medicine 31(11): 2592–2597. Fraser RS and Paraê PD (1999) Fraser and Paraê’s Diagnosis of Diseases of the Chest, 4th edn. Philadelphia: Saunders. Gunther A, et al. (2001) Surfactant alteration and replacement in acute respiratory distress syndrome. Respiration Research 2(6): 353–364. Hallman M, Glumoff V, and Ramet M (2001) Surfactant in respiratory distress syndrome and lung injury. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 129(1): 287–294. Hedenstierna G and Rothen HU (2000) Atelectasis formation during anesthesia: causes and measures to prevent it. Journal of Clinical Monitoring and Computing 16(5–6): 329–335.

Kreider ME and Lipson DA (2003) Bronchoscopy for atelectasis in the ICU: a case report and review of the literature. Chest 124(1): 344–350. Magnusson L and Spahn DR (2003) New concepts of atelectasis during general anaesthesia. British Journal of Anaesthesia 91(1): 61–72. Peroni DG and Boner AL (2000) Atelectasis: mechanisms, diagnosis and management. Paediatric Respiratory Reviews 1(3): 274–278. Rouby JJ, et al. (2003) Acute respiratory distress syndrome: lessons from computed tomography of the whole lung. Critical Care Medicine 31(4 supplement): S285–S295. Spragg RG, et al. (2004) Effect of recombinant surfactant protein C-based surfactant on the acute respiratory distress syndrome. New England Journal of Medicine 351(9): 884– 892. Vaaler AK, et al. (1997) Obstructive atelectasis in patients with small cell lung cancer. Incidence and response to treatment. Chest 111(1): 115–120. Woodring JH and Reed JC (1996) Types and mechanisms of pulmonary atelectasis. Journal of Thoracic Imaging 11(2): 92–108.

AUTOANTIBODIES O C Ioachimescu, Cleveland Clinic Foundation, Cleveland, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Autoimmunity represents an immune response directed against self-antigens, which may be (artificially) separated into T-cell and B-cell responses. The importance of the B cells in autoimmunity is correlated to the production of several autoantibodies, which have a role in diagnosis, pathogenesis, guiding therapy, and predicting outcome of the patients with these conditions. Among these autoantibodies, antineutrophil cytoplasm antibodies (ANCAs) have been distinguished as very important in several conditions, such as Wegener’s granulomatosis, microscopic polyangiitis, Goodpasture’s syndrome, drug-related vasculitides, etc. We review in this article the importance of different classes of autoantibodies and their relationship to underlying disorders.

Introduction: Autoimmunity and Lung Disorders The phenomenon of autoimmunity has been the object of exploration for over a century. Recent technical and methodological advances in the study of cellular and biochemical processes of autoimmunity have led to a publication ‘big bang’. Nevertheless, the etiopathogenesis of most autoimmune disorders remains, to date, largely unknown. In this article, we discuss the basic mechanisms leading to autoimmunity, as we understand them today, and then the

relevance of several classes of autoantibodies in various lung conditions. An autoimmune disorder is a pathologic condition caused by an autoimmune response, which is critically dependent upon antigen quality, concentration, and persistence, and the magnitude of the interaction between self and foreign components. The autoimmune response leads to end-organ organ-damage through cellular or humoral mechanisms. The humoral response is represented by antibody-mediated injury, which could be differentiated in direct, cytotoxic antibody damage or immune complex-related damage. Despite their diverse etiology, there are several pathogenetic mechanisms which are common to all autoimmune conditions. With few exceptions, they require the presence of self-reactive CD4 positive lymphocytes. The immune system is naturally endowed with myriad mechanisms responsible for recognition of and defense against foreign assaults. Direct and rapid responses can be mediated by a set of germline-encoded receptors, called Toll-like receptors (TLRs), which recognize specifically the molecular determinants of various pathogens. Activation of the innate immune defense mechanisms leads to a response from different cell types (local dentritic cells, natural killer lymphocytes, neutrophils, monocytes, macrophages, basophils, eosinophils, and mastocytes), ranging from production of chemokines, cytokines, adhesion molecules, antimicrobial, pro- and antiapoptoic factors. This response is reproducible (acts

220 AUTOANTIBODIES

by the same intensity at each antigenic exposure), is not antigen specific, and creates a pool of memory cells for long-term immunity. The adaptive immunity requires contributions from cells whose receptors are generated by VDJ recombination, the T and B lymphocytes. Furthermore, inducible expression of TLRs in B cells may provide a link between the innate and adaptive branches of the immune system. Genetic susceptibility to autoimmune diseases may occur at several levels: the major histocompatibility complex (MHC) haplotype and polymorphisms of genes involved in establishing self-tolerance and immune regulation, for example, the autoimmune regulator (AIRE), the T-cell immunoglobulin and mucin-domain-containing (TIM) family, and cytolytic T-lymphocyte-associated antigen 4 (CTLA-4). There are several major pathways that can initiate or modulate autoimmunity: 1. molecular mimicry of self-proteins by viral agents can activate specific T cells that attack both the specific virus and the host; 2. bacterial infections can cause intense inflammation, with secondary polyclonal activation of ‘bystander’ T cells, which will act in concert in intensifying the local injury; 3. epitope exposure and maintenance in the local milieu as a result of self local damage may perpetuate the immune response; and 4. drug metabolism or infection can cause the formation of proteins which are seen as ‘foreign’ and thus result in a neo-antigen-specific response to the modified self. There is also a range of possible posttranslational modifications (PTMs) of proteins that can allow immune recognition of neo-self epitopes. The most direct way the posttranslational changes can influence T-cell reactivity is to generate a new peptide–MHC (pMHC) complex that can stimulate stronger binding to T lymphocytes through T-cell receptors. In general, the immune recognition can be modified by posttranslational changes of the antigens in a number of ways: 1. Different additions (glycosylation, methylation, phosphorilation, etc.), as in collagen-induced arthritis. 2. Enzymatic or spontaneous conversions (deamidation or citrullination) as in celiac disease or rheumatoid arthritis (RA). In the latter, multiple autoantibodies have been described: antiperinuclear factor, antifilaggrin, and antikeratin antibodies. The target antigens are the result of PTM, namely deimination of the natural aminoacid

arginine to the amino acid citrulline by the activity of peptidylarginine deiminase (PAD). This discovery led to the development of a new serologic test for RA, that is, antibody against cyclic citrullinated peptide (CCP), which has a high specificity (497%) for the disease. Anti-CCP binding also seems predictive of progression to RA in recent-onset arthritis, or retrospectively in blood samples positive for rheumatoid factor (RF), donated years before onset of symptoms of the disease. There is also evidence that citrullination may play a role in T-cell autoreactivity in RA by enhancing the strength of the bonds to MHC II molecules. Therefore, expression of PAD4 and citrullinated proteins, presence of anticitrulline antibody-secreting plasma cells in the inflamed synovium, the strong MHC–peptide bonds, and the predictive strength of anti-CCP testing, all prove that this autoimmune cascade is involved in the progression of RA. 3. Extracellular modifications by proteolysis or antibody binding. The current understanding is that differential PTMs might therefore provide the means of provoking a local autoaggressive immune response as a consequence of an infection. This would be an alternative explanation of the autoimmunity by self-mimicking microbial antigens, for which definitive proof remains elusive in humans as yet.

ANCAs and Associated Lung Disorders In 1982, antineutrophil cytoplasm antibodies (ANCAs) were first described in patients with pauci-immune glomerulonephritis. By 1985, ANCA had already been linked to Wegener’s granulomatosis (WG); within a few more years, a link with microscopic polyangiitis (MP), and ‘renal-limited’ vasculitis has been made. As of today, ANCAs have become very important factors in the diagnosis, pathogenesis, and classification of vasculitides. ANCAs can be determined in two different ways: indirect immunofluorescence assay (IIA) and enzyme-linked immunosorbent assay (ELISA). While IIA is more sensitive than ELISA, the latter is clearly more specific; hence, in clinical testing for ANCA it is recommended to screen with IIA, and to confirm all positive results with ELISA directed against the specific antigens, if possible in a reference laboratory. Furthermore, and unfortunately, there is a significant subjective component to the interpretation of the immunofluorescence tests, so confirmation by other tests is needed before releasing the definitive ‘positive test’. The main target antigens for ANCAs are

AUTOANTIBODIES 221

proteinase 3 or PR3 (PR3–ANCA) and myeloperoxidase or MPO (MPO–ANCA). PR3 and MPO are located in the azurophilic granules of neutrophils, and the peroxidase-positive lysosomes of monocytes, respectively. Other azurophilic granule proteins can cause p-ANCA autoantibodies: lactoferrin, elastase, cathepsin G, bactericidal permeability inhibitor, catalase, lysozyme, azurocidin, beta-glucuronidase, etc.

*

specimens, because formalin-fixed neutrophils do not cluster charged cellular components around the nucleus. Mixed or atypical pattern, seen mostly in patients with other immune-mediated conditions than systemic vasculitis (e.g., connective tissue disorders, inflammatory bowel disease, and autoimmune hepatitis). Such patterns may be confused with p-ANCA patterns.

Immunofluorescence Assay

When the sera of patients with ANCA-associated vasculitis (AAV) or other conditions are incubated with ethanol-fixed human neutrophils, three major IIA patterns are observed (Figure 1): *

*

c-ANCA pattern (cytoplasmic), with diffuse staining of the cytoplasm. In most cases, the responsible antibodies are PR3–ANCA in the setting of WG; rarely, MPO–ANCA can present in the cytolasmic, uniform immunofluorescence. p-ANCA pattern (perinuclear), which results from staining around the nucleus, which in fact is fixation artifact when ethanol is used. With ethanol fixation, positively charged granule constituents cluster around the negatively charged nuclear membrane, leading to perinuclear fluorescence. The usual antibody responsible for this pattern is MPO– ANCA (rarely PR3–ANCA), generally in the setting of MP. A positive p-ANCA immunofluorescence staining pattern may also be detected in a wide variety of inflammatory illnesses; it has a low specificity for vasculitis. The p-ANCA pattern on IIA can be similar to that caused by antinuclear antibodies (ANAs), the reason why individuals with ANA frequently have ‘false-positive’ results on ANCA testing by immunofluorescence. Rigorous IIA testing procedures for ANCA (especially when a diagnosis of vasculitis is entertained) entails the use of both formalin- and ethanol-fixed neutrophil

Enzyme-Linked Immunosorbent Assay

Specific ELISA kits for antibodies directed against PR3, MPO, and other azurophilic granule components are commercially available. PR3–ANCA and MPO–ANCA should be part of any standardized approach to the testing for ANCA; they are associated with substantially higher specificities and positive predictive values than their corresponding IIA patterns (c- and p-ANCA, respectively). Antineutrophil cytoplasm antibodies are mainly associated with Wegener’s granulomatosis, microscopic polyangiitis, Churg–Strauss syndrome (CSS), ‘renal-limited’ vasculitis (pauci-immune glomerulonephritis without evidence of extrarenal disease), and drug-induced vasculitides. In these conditions, there is either PR3–ANCA or MPO–ANCA, but almost never both. ANCA with different antigen specificities may be detected in various other rheumatologic and gastrointestinal disorders. Wegener’s Granulomatosis

The histopathologic hallmark of WG is necrotizing granulomatous inflammation that involves the respiratory tract, kidneys, skin, and/or the joints. Nearly 90% of patients with generalized, active WG are ANCA-positive, while in limited disease (restricted to upper or lower respiratory tract disease and no renal involvement) only 40–60% of patients may be ANCA-positive. Thus, the absence of ANCA does

Figure 1 Indirect immunofluorescence assay patterns on ethanol-fixed human neutrophils. (a) Diffuse, cytoplasmic cANCA; (b) perinuclear pANCA; (c) antinuclear (ANA) illustrated as control.

222 AUTOANTIBODIES

not exclude the diagnosis; among WG patients with ANCA positivity, 80–95% have PR3–ANCA by ELISA, the rest nearly always have MPO–ANCA. The diagnostic performance of PR3–ANCA for WG (positive and negative predictive values) is related mainly to disease prevalence in the population searched, and the disease activity. Persistently, high or rising titers of ANCAs are often associated with disease relapses. However, this association may not occur in 10–30% or more of those with such ANCA profiles during one or more years of follow-up. Microscopic Polyangiitis

Approximately 70% of patients with MP are ANCA positive. Most ANCA-positive MP patients have MPO–ANCA, with a minority having PR3–ANCA. ANCA serologies are useful in distinguishing MP from classic polyarteritis nodosa (PAN), a vasculitis of medium-sized muscular arteries. In general, PAN is associated with neither PR3–ANCA, nor MPO– ANCA. Since PR3–ANCA or MPO–ANCA may occur in both WG and MP, it is important to know that these diseases cannot be distinguished solely on ANCA tests. However, distinction between MP and WG is not clinically that important, since their treatment and prognosis are similar. Churg–Strauss Syndrome

Both PR3–ANCA and MPO–ANCA have been detected in patients with CSS; overall, 50% of CSS patients are ANCA positive, whereas in those with active, untreated disease the percentage is even higher. So far, no identification has been made of any consistent clinical differences with therapeutic or prognostic implications between ANCA-positive CSS and ANCA-negative CSS. Renal-Limited Vasculitis

Pauci-immune vasculitis limited to the kidney is characterized by focal and segmental glomerular inflammation and necrosis with little or no deposition of immunoreactants (IgG, IgA, IgM, and complement fractions). Almost all patients are ANCA positive, and up to 80% of them have MPO–ANCA. Some consider this disorder as part of the WG/MP spectrum because the renal histologic findings are indistinguishable, and because some patients with renal-limited vasculitis eventually develop extrarenal manifestations of either WG or MP.

positive. One study, for example, found that 38 of 100 sera with anti-GBM antibodies also had ANCA; of these, 25 had MPO–ANCA, 12 had PR3–ANCA and 1 both types of ANCAs. Almost all such patients have both ANCAs and anti-GBM antibodies at the first serum examination. The clinical significance of combined ANCA and anti-GBM serologies is unclear. In some, the titers of ANCAs are low and there are no clinical manifestations of vasculitis. In others, however, there are disease features of anti-GBM antibody disease, but quite typical of systemic vasculitis, including purpura, arthralgias, and granulomatous inflammation, suggesting the concurrence of two disease processes. The incriminated self-antigen for the anti-GBM antibodies is the same as in patients with anti-GBM antibody disease alone, suggesting that the inciting epitopes are the same. Alternatively, the production of ANCA could precede that of anti-GBM antibodies, with pulmonary or renal damage caused by ANCA leading to secondary anti-GBM antibody formation. Other Rheumatologic Disorders

ANCAs have been reported in virtually all rheumatic diseases, including RA, systemic lupus erythematosus (SLE), Sjogren’s syndrome (SS), scleroderma, inflammatory myopathies, dermatomyositis, relapsing polychondritis, and the antiphospholipid syndrome. In most cases, the IIA pattern is p-ANCA. Many reports of ANCAs in these diseases preceded the era of reliable ELISA assays for PR3– and MPO–ANCA, and used only IIA. Various target antigens have been described in these disorders, such as lactoferrin, elastase, lysozyme, cathepsin G, and others. In other cases, the specific target antigens have not been identified yet. Cystic Fibrosis

Non-MPO p-ANCAs are common in patients with cystic fibrosis (CF), particularly among those with bacterial airway infections. The ANCA is generally directed against BPI (bactericidal/permeability-increasing) protein. In one series of 66 patients with CF, BPI-IgG and BPI-IgA ANCAs were found in 91% and 83%, respectively. Anti-BPI titers were directly related to the severity of airway destruction. It is unclear whether this relationship represents an epiphenomenon or a response to overwhelming infection, with major release of endotoxins.

Anti-GBM Antibody Disease (Goodpasture’s Syndrome)

Others

Up to 40% of patients with antiglomerular basement membrane (anti-GBM), antibody disease are ANCA

ANCA has also been observed in isolated patients with autoimmune hepatitis, Bu¨rger’s disease,

AUTOANTIBODIES 223

(pre)eclampsia, subacute bacterial endocarditis, leprosy, malaria, and chronic graft-versus-host disease. ANCA positivity is seen in 60–80% of patients with ulcerative colitis and in primary sclerosing cholangitis. It can be observed in only 10–27% of patients with Crohn’s disease, in whom only low titers are present. The p-ANCA is the predominant appearance, and is directed against a myeloid cell-specific 50 kDa nuclear envelope protein. Other reported antigens include BPI, lactoferrin, cathepsin G, elastase, lysozyme, and PR3. The pathogenetic significance of these antibodies is unclear. The titers of ANCAs do not vary with the activity or severity of the disease and, in ulcerative colitis, do not fall even after colectomy. Drug-Induced ANCA-Associated Vasculitis

Several medications can induce various forms of AAV. Most patients diagnosed with drug-induced AAV have high titers of MPO–ANCA. In addition, most have also antibodies to elastase or lactoferrin. Relatively few have PR3–ANCA positivity. Many cases of drug-induced AAV are associated with constitutional symptoms, arthralgias/arthritis, and cutaneous vasculitis. However, the full range of clinical features associated with ANCAs, including crescentic glomerulonephritis and alveolar hemorrhage, can also occur. Discontinuation of the offending agent may be the only intervention necessary for mild cases of AAV induced by medications; such cases have in general only constitutional symptoms, arthralgias/arthritis, and/or cutaneous vasculitis. Some patients, however, require high doses of corticosteroids and even cyclophosphamide because of more severe manifestations. The most renowned medications capable of causing AAV are propylthiouracil (PTU), carbimazole, thiamazole, hydralazine, procainamide, minocycline, penicillamine, allopurinol, phenytoin, and clozapine. Drug-induced AAV is quite rare; consequently, the above medications should not be incriminated until other etiologies have been thoroughly excluded. Propylthiouracil

PTU may be the most common offending agent causing drug-induced AAV. Generally, the medication is taken for long periods of time before the complication occurs (months to years). Vasculitis is a rare complication, whereas a much higher percentage develops serological evidence of ANCA. In a cross-sectional study, 27% of patients receiving long-term treatment with PTU developed MPO–ANCA. The postulated mechanism by which PTU leads to AAV stems from the observation that PTU accumulates in

neutrophils and then it binds to MPO altering its structure, which could lead to ANCA production in susceptible individuals. Discontinuation of the offending drug may be the only intervention necessary in mild cases. Some patients, however, require high doses of corticosteroids and even cyclophosphamide, while others require maintenance therapy. ANCA titers usually persist at low levels, even after active vasculitis goes into resolution, which points out that previous ingestion of PTU is an important piece of information in the history of patients with other pathologies and prior PTU-induced, ‘by-stander’ ANCA. Hydralazine

Hydralazine may cause drug-induced lupus and drug-induced AAV. Unlike hydralazine-induced lupus syndrome, hydralazine-induced AAV is frequently associated with a pauci-immune glomerulonephritis, antibodies to double stranded DNA (dsDNA), high titers of MPO–ANCA, and is a more ‘serious’ condition. Minocycline

Minocycline had produced fever, livedo reticularis, arthritis, and ANCA in a seven-patient case series. The p-ANCA IIA pattern associated with this disorder is usually directed against ‘minor’ antigens such as cathepsin G, elastase, and bactericidal/permeability increasing (BPI) protein, rather than against ‘major’ antigens, such as MPO. Symptoms typically resolve after minocycline discontinuation, and recur with drug rechallenge. Some patients require treatment with corticosteroids for short periods of time. More serious manifestations of minocyclineinduced AAV are crescentic glomerulonephritis, lupus-like syndrome, and cutaneous ‘classic’ PAN. Pathogenesis of ANCA-Associated Disorders

Substantial evidence in animal models and human observations supports a significant pathogenetic role of ANCA in producing widespread tissue damage. The hypothesis is that antibodies produce a necrotizing vasculitis by inciting a respiratory burst with diffuse endothelial damage, chemotaxis, and degranulation of neutrophils and monocytes. If autoantibody (ANCA) generation is secondary to a cryptic epitope exposure or a primary event, it is still unclear. After a cryptic antigen exposure, the epitope spread may occur, leading to a more generalized, systemic reaction (Figure 2). The number of activated B lymphocytes seems to correlate with the activity score of the disease (e.g., Birminham vasculitis score), which supports the hypothesis that B lymphocytes

224 AUTOANTIBODIES AAT ANCA

Insult

PR3 or MPO Macrophage

MMP-12 Neutrophil

TNF- IL-8 Monocyte

Endothelial cell Figure 2 A simplified model of ANCA pathogenesis and neutrophil/monocyte priming. Of note, alpha-1 antitrypsin (AAT) is a natural tissue inhibitor of proteinase 3 (PR3), myeloperoxidase (MPO), or other lysosomal enzymes. In addition, binding ANCA to the cell surface is mediated by specific FcgR receptors for immunoglobulins. IL-8, interleukin-8; MMP-12, matrix metalloproteinase 12; TNF-a, tumor necrosis factor alpha. Copyright (2005) from Journal of COPD by Ioachimescu O and Stoller J. Reproduced by permission of Taylor & Francis Group, LLC., http://www.taylorandfrancis.com.

are involved in the disease pathogenesis. Furthermore, while in normal conditions MPO and PR3 mRNA transcripts are found almost exclusively in early promyelocytes, it was noted that in AAV disorders (and not in SLE or other conditions), both MPO and PR3 mRNA are found in high concentrations in the peripheral neutrophils, and this correlates well with neutrophil total number and disease activity, but not with ANCA titer, ‘left shift’, or cytokine levels, including tumor necrosis factor alpha (TNF-a). Cell membrane PR3 and MPO expression has been found in the affected glomeruli of the ANCA-associated diseases (e.g., WG, MP), but not in normal glomeruli, suggesting that local factors also play an important role in gauging the organ involvement. In CSS, it is still unclear what the eosinophil’s contribution to the disease pathogenesis is.

Autoantibodies in Connective Tissue Disorders Laboratory screening is commonly used for evaluation of connective tissue disorders (CTDs), although there are rare tests which are sensitive or specific enough to establish the diagnosis. For example, Westergren sedimentation rate and C-reactive protein are commonly elevated in infections, malignant, or inflammatory conditions; therefore, a high value does not add too much towards the diagnosis, while a low titer may make an active CTD seem unlikely. Table 1 illustrates a synopsis of autoantibodies found in different immune conditions.

Table 1 The main pathogenic autoantibodies in different lung conditions Antibody

Lung disease

ANCA ANA Anti-Sm, Anti-dsDNA Anti-U1-RNP RF CCP Anticentromere Anti-Scl Anti-Jo1 Cryoglobulins Anti-GM-CSF

WG, MP, CSS, drug-related AAV SLE, SS, scleroderma, RA, MCTD, UCTD SLE MCTD RA, SLE, MCTD, UCTD RA CREST syndrome Scleroderma DM/PM – ILD Essential and secondary cryoglobulinemia PAP

ANAs are found positive in most of CTD patients, with different frequencies (from 30% in RA, to 95% in SLE and scleroderma). On the other hand, other lung conditions such as idiopathic pulmonary fibrosis and coal worker’s pneumoconiosis may have positive ANAs in titers above 1:40 in up to 33% of the cases, as opposed to 2–3% of the general population. An extractable nuclear antigen (ENA) panel is available in most of the reference laboratories, which will guide the testing against common antigens if ANA is positive. Anti-dsDNA and anti-Smith (Sm) antibodies are relatively specific for SLE: 95–97%, and 50–99%, respectively. While anti-dsDNA antibodies seem to correlate with the disease activity, anti-Sm antibodies tend to persist after normalization of anti-dsDNA titers. An interstitial lung disease (ILD)

AUTOANTIBODIES 225

identical to usual interstitial pneumonia (UIP) or non-specific interstitial pneumonia (NSIP) can be seen in SLE, RA, scleroderma, or dermatomyositis, while bronchiolitis obliterans with organizing pneumonia (BOOP) can be frequently seen in RA. In SLE, the most frequent pulmonary complications are pleurisy and pleuritis, lupus pneumonitis, shrinking lung syndrome, bacterial pneumonia, diffuse alveolar hemorrhage (DAH), thromboembolic disease, and pulmonary arterial hypertension. Traditionally, RF is found with higher frequency in patients with RA and long-standing disease, with multiple extra-articular manifestations. In other lung conditions, such as hypersensitivity pneumonitis (bird fancier’s lung) or idiopathic pulmonary fibrosis (IPF), RF can be positive in up to half of the cases. Since part of the workup for interstitial lung disorders is exclusion of associated rheumatic conditions, reliance on more specific testing is generally required to diagnose a systemic disease like RA. This can be achieved by anticyclic citrullinated peptide (CCP) antibodies, which have a much higher specificity, of 91–98%. In RA, the lung involvement can take the form of ILD (UIP, NSIP, BOOP), DAH, pleuritis and pleural effusion, rheumatoid nodules (follicular, constrictive, or obliterative) bronchiolitis, cricoarythenoid arthritis with upper airway obstruction or drug-induced pneumonitis (methotrexate, gold, cyclophosphamide, rituximab etc.). In the workup of disorders associated with myopathy or myositis, the determination of muscle creatinphosphokinase (or aldolase) is important. Polymyositis (PM) and amyotrophic dermatomyositis (ADM) can both present with lung disease in more than 65% of cases, as one study found at the time of diagnosis; it is important to know that, contrary to rheumatoid lung disease, in PM or ADM, the pulmonary involvement may be inaugural. In DM/PM, ANA is positive in up to 30% of the patients; one in three patients will also have antibodies against an ENA called histidyl tRNA synthetase, or Jo-1 antibodies. Other antiaminoacyl tRNA synthetases have been identified to date: anti-PL7 or anti-treonyl tRNA synthetase, anti-PL12 or anti-alanyl tRNA synthetase, anti-OJ or anti-isoleucyl tRNA synthetase, anti-EJ or anti-glycyl tRNA synthetase, anti-KS or anti-asparaginyl tRNA synthetase, and anti-Wa directed against a 48 kDa protein yet uncharacterized, but known to be bound to acetylated tRNA. It has been noted that patients with anti-synthetase syndrome (arthritis, Raynaud’s syndrome, mechanic’s hands, and anti-Jo-1 antibodies) have a much higher incidence of pulmonary disease. Pathologic pictures of NSIP, UIP, or BOOP can be seen in these settings.

The interstitial lung disease in scleroderma can occur with both limited and diffuse disease. ANA is generally positive in both forms of the disease, anticentromere or anti-TO/TH antibodies found in limited disease, while anti-Scl antibodies are more often found in the diffuse variants of scleroderma. The cellular and fibrotic form of NSIP can together account for 70% of patients with scleroderma, suggesting a high rate of pulmonary involvement. Sjogren’s syndrome is an autoimmune exocrinopathy and disorder of the epithelia characterized by lymphocytic infiltration of the glandular and nonglandular subepithelial tissue. It presents with xerostomia (dry mouth), xerophtalmia (dry eyes) and keratoconjunctivitis, xerotrachea (dry trachea, presenting as dry cough and frequent infections), and arthritis. The pulmonary involvement in SS manifests as lymphocytic bronchitis, lymphocytic follicular bronchiolitis, bronchial-associated lymphoid tissue lymphoma, lymphocytic interstitial pneumonia (LIP) or cystic lung disease. The serologic markers of this condition (either primary or secondary SS) are represented by SS-A (anti-Ro) and SS-B (anti-La) antibodies. Mixed connective tissue disorder (MCTD) is an overlap syndrome with features of RA, SLE, scleroderma, and DM/PM, which do not meet the criteria for the individual disorders, in the presence of antiribonucleoprotein (anti-RNP) antibodies on ENA testing, directed against U1 RNP, which is rich in uridine. Pulmonary involvement is common in patients with MCTD, and has feature of UIP or NSIP, with significant septal thickening, less ground-glass attenuation and honeycombing on CT scans. ‘Incomplete’ rheumatic conditions can occur and are usually called undifferentiated connective tissue disorder (UCTD) and may involve the respiratory tract in a fashion similar to MCTD, although without any evidence of anti-RNP antibodies. In cryoglobulinemia, a condition characterized by cryoglobulins in the serum, there is a systemic inflammatory response with involvement of small and medium-size vessels, and generated by cryoglobulincontaining immune responses. By definition, cryoprecipitation is a phenomenon of protein precipitation at temperatures lower than 371C, and it can be present when serum proteins or plasma proteins precipitate (cryofibrinogens and cryoglobulins respectively). Cryoglobulins are a mixture of immunoglobulins and complement components, which generally precipitate upon refrigeration of the serum. Three types of cryoglobulinemic conditions have been described (Brouet’s classification): type I, characterized by isolated monoclonal immunoglobulins (Ig) and seen mostly in hematologic conditions; type II with polyclonal Ig seen in hepatitis C, HIV

226 AUTOANTIBODIES

and other viral conditions; and type III, with mixed cryoglobulins, encountered in CTDs. Pulmonary involvement may be commonly seen in type III cryoglobulinemia and, rarely, in type I, and is mostly subclinical. In up to 50% of cases, there may be cough, pleuritic chest pain, and/or dyspnea. Pulmonary function tests usually show evidence of reactive small airway disease, and occasionally a decreased diffusing lung capacity for CO. Pulmonary vasculitis, DAH, or BOOP can also be found (though rarely). Another rare condition is hypocomplementemic urticarial vasculitis syndrome, which presents with urticarial vasculitis, arthritis, glomerulonephritis, and obstructive lung disease; it is thought to be caused by serum IgG against C1q fraction of the complement, which will decrease significantly (similar to SLE, but more commonly, with angioedema and eye inflammation).

Autoantibodies in Other Conditions Lung Cancer

The antibodies responsible for the paraneoplastic syndromes are discussed in articles Tumors, Malignant: Overview; Bronchogenic Carcinoma. Sarcoidosis

A possible relationship between sarcoidosis and autoimmunity was described more than a century ago, although is still not accepted to be an autoimmune condition. Good preliminary results of the CD20 targeting in several autoimmune conditions (sarcoidosis, SLE, RA, type II cryoglobulinemia, neuropathies, WG, Goodpasture’s syndrome, etc.) have opened important avenues for research, conceivably capable of improving our understanding of the pathogenesis of these conditions and the effectiveness of pathogenesis-directed therapy. Pulmonary Alveolar Proteinosis

Animal and human studies have confirmed a pivotal role played by granulocyte-macrophage colony-stimulating factor (GM-CSF) in pulmonary alveolar proteinosis (PAP) pathogenesis. A decreased GM-CSF pathway activity seems to be the common pathogenic pathway. It was shown that a neutralizing (or blocking) anti-GM-CSF IgG antibody can be found in bronchoalveolar lavage fluid and sera of patients with idiopathic PAP. The sensitivity of the serum anti-GM-CSF assay is close to 100% and the specificity too is close to 100% when using a cutoff titer of 1:400. Furthermore, anti-GM-CSF antibodies are increasingly used as a diagnostic tool in

PAP. To what degree a high end-titer of anti-GM-CSF represents an indication to treat more aggressively, or with a larger dose of GM-CSF, remains to be proven.

Conclusions During the past century, much has been accomplished in defining, understanding, and treatment of autoimmune conditions, yet more is to be learned. Autoantibodies play an important role in these immune processes, and of particular importance are ANCAs, involved in the pathogenesis of the so-called ANCA-associated vasculitides. The importance of the autoantibodies stems not only from their contribution to the diagnosis of different conditions, but also from their role in pathogenesis and the importance in monitoring the disease progression. See also: Cryoglobulinemia. Cystic Fibrosis: Overview. Granulomatosis: Wegener’s Disease. Interstitial Lung Disease: Overview. Systemic Disease: Diffuse Alveolar Hemorrhage and Goodpasture’s Syndrome. Toll-Like Receptors. Tumors, Malignant: Overview. Vasculitis: Overview.

Further Reading Bonfield TL, Russell D, Burgess S, et al. (2002) Autoantibodies against granulocyte macrophage colony-stimulating factor are diagnostic for pulmonary alveolar proteinosis. American Journal of Respiratory Cell and Molecular Biology 27: 481–486. Csernok E (2003) Anti-neutrophil cytoplasmic antibodies and pathogenesis of small vessel vasculitides. Autoimmunity Reviews 2: 158–164. Imbert-Masseau A, Hamidou M, Agard C, Grolleau JY, and Cherin P (2003) Antisynthetase syndrome. Joint, Bone, Spine 70: 161–168. Ioachimescu O and Stoller J (2005) A review of alpha-1 antitrypsin deficiency. Journal of COPD 2(2): 263–275. Mahadeva R, Dunn AC, Westerbeek RC, et al. (1999) Anti-neutrophil cytoplasmic antibodies (ANCA) against bactericidal/ permeability-increasing protein (BPI) and cystic fibrosis lung disease. Clinical and Experimental Immunology 117: 561–567. Miescher PA, Zavota L, Ossandon A, and Lagana B (2003) Autoimmune disorders: a concept of treatment based on mechanisms of disease. Springer Seminars in Immunopathology 25(supplement 1): S5–S60. Paran D, Fireman E, and Elkayam O (2004) Pulmonary disease in systemic lupus erythematosus and the antiphospholpid syndrome. Autoimmunity Reviews 3: 70–75. Quintana FJ and Cohen IR (2004) The natural autoantibody repertoire and autoimmune disease. Biomedicine & Pharmacotherapy 58: 276–281. Schmitt WH (2004) Newer insights into the aetiology and pathogenesis of myeloperoxidase associated autoimmunity. Japanese Journal of Infectious Diseases 57: S7–S8. Seo P and Stone JH (2004) The antineutrophil cytoplasmic antibody-associated vasculitides. American Journal of Medicine 117: 39–50. Sharma OP (2002) Sarcoidosis and other autoimmune disorders. Current Opinion in Pulmonary Medicine 8: 452–456.

AUTOANTIBODIES 227 Strange C and Highland KB (2004) Interstitial lung disease in the patient who has connective tissue disease. Clinics in Chest Medicine 25: 549–559. Uchida K, Nakata K, Trapnell BC, et al. (2004) High-affinity autoantibodies specifically eliminate granulocyte-macrophage colony-stimulating factor activity in the lungs of patients with idiopathic pulmonary alveolar proteinosis. Blood 103: 1089–1098.

Wiik A (2003) Autoantibodies in vasculitis. Arthritis Research & Therapy 5: 147–152. Wisnieski JJ, Baer AN, Christensen J, et al. (1995) Hypocomplementemic urticarial vasculitis syndrome. Clinical and serologic findings in 18 patients. Medicine (Baltimore) 74: 24–41.

B BASAL CELLS M J Evans, University of California, Davis, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Despite differences in basal cell distribution, the unifying feature in all animal species is that the number of basal cells present is related to the height of the columnar epithelium. This relationship is associated

Abstract Basal cells are an integral part of pulmonary airway epithelium. They exist as a separate layer of cells covering most of the basement membrane zone. In this central position, they can interact with columnar epithelium, neurons, the basement membrane zone, and underlying mesenchymal cells. In addition, they interact with inflammatory cells, lymphocytes, and dendritic cells. The interactions with trafficking leukocytes and neurons take place in the lateral intercellular space between basal cells. In this central position basal cells become a very important part of the epithelial–mesenchymal trophic unit of larger airways. Structurally, basal cells function in attachment of columnar epithelium with the basement membrane zone. Failure of attachment between columnar and basal cells is thought to be responsible for sloughing of the columnar epithelium in asthmatics. They also function in regulation of fibroblast growth factor-2 signaling from the basement membrane zone, neurogenic inflammation, the inflammatory response, transepithelial water movement, and oxidant defense of the tissue and formation of the lateral intercellular space for airway epithelium. A subpopulation of basal cells (parabasal cells) has the potential to function as progenitor cells. Clinically, basal cells are probably also involved with the formation of squamous cell carcinoma and, possibly, the progression to organized carcinoma.

LIS LIS

*

*

LIS BC

BC

PCB

*

BC BMZ (a)

Introduction Basal cells are derived from undifferentiated columnar epithelium in the developing airway. They are characterized by their basal position in the columnar epithelium, the presence of hemidesmosomes (characterized by alpha 6 beta 4 integrins), cytokeratins 5 and 14, and the nuclear protein p63 (Figure 1(a)). The distribution of basal cells varies by airway level and animal species. Airways that are larger in diameter have more basal cells than airways with smaller diameters. For example, the largest numbers of basal cells are found in the trachea. As the airway decreases in diameter, the number of basal cells also decreases, and none are present in the terminal bronchioles. As basal cells increase in number, they displace columnar cells on the basement membrane zone (BMZ). In human airways, 90–95% of the BMZ is covered by basal cells from the trachea to bronchioles 1–3 mm in diameter (Figure 1(b)).

(b) Figure 1 (a) Electron micrograph of tracheal epithelium from a young rhesus monkey demonstrating basal cells (BC), parabasal cells (PCB), the lateral intercellular space (LIS), and the basement membrane zone (BMZ). The lateral intercellular space is the open area between cells (asterisks). The lateral intercellular space is reduced or absent between ciliated cells and ciliated cells next to secretory cells (arrowheads). Magnification 1500. Copyright 2001 from Cellular and molecular characteristics of basal cells in airway epithelium. Experimental Lung Research 27: 401–415 by Evans MJ, Van Winkle LS, Fanucchi MV, et al. Reproduced by permission of Taylor & Francis, Inc. (b) Scanning electron micrograph of a sheep tracheal whole mount treated with EDTA. The columnar epithelium has been released leaving a layer of basal cells attached to the BMZ. The basal cells cover between 90% and 95% of the BMZ. Magnification 1100.

230 BASAL CELLS

with the role of basal cells in attachment of columnar epithelium to the BMZ. Functionally, large airway epithelium is stratified, with a layer of basal cells attached to the BMZ and a layer of columnar epithelium attached to the basal cells. Thus, basal cells act as a separate layer of cells in a central position, that can interact with columnar epithelium, neurons, BMZ, and the underlying mesenchymal cells. In addition, they can interact with inflammatory cells, lymphocytes, and dendritic cells in the epithelium. These interactions take place in the lateral intercellular space. The lateral intercellular space is a distinct space between basal cells, basal and adjacent secretory cells, and secretory cells (Figure 1(a)). The lateral intercellular space is hydrated with the aid of the proteoglycan hyaluronan, which is bound to CD44 adhesion molecules on the surface of basal cells. In this central position, basal cells become a very important and integral part of the epithelial–mesenchymal trophic unit of large airways. The large number of receptors found on basal cells that bind growth-regulating proteins and trafficking leukocytes (Table 1) supports this concept.

Basal Cell Functions in the Normal Lung Junctional Adhesion

The structural role of basal cells in the airways is for attachment of columnar epithelium to the BMZ. Epithelial cells are attached to the BMZ by hemidesmosomes and cell adhesion molecules. Cytokeratins 5 and 14 link anchoring junctions of basal cells with the cytoskeletons of adjacent cells through desmosomes and to the BMZ with hemidesmosomes. This arrangement of junctional adhesion provides mechanical stability to a group of cells or tissue. In airway epithelium, basal cells are the only cells that form hemidesmosome junctions with the BMZ. Columnar cells are attached to the BMZ via desmosome attachment with basal cells. The significance of basal cells in junctional adhesion can be demonstrated by treating the tissue with ethylenediaminetetraacetic acid (EDTA). Desmosome junctions are dependent on calcium. When the tissue is treated with EDTA, the calcium is removed from the desmosome, and the columnar epithelium is released leaving the basal cells attached to the BMZ (Figure 1(b)). The number of basal cells present at a particular airway level and their morphology is related to their role in junctional adhesion. When the columnar epithelium increases in height, there is an increase in the size and shape of basal cells along with a corresponding increase in desmosome attachment with the columnar epithelium and hemidesmosome attachment with the BMZ. These changes maintain a

Table 1 Cellular and molecular characteristics of basal cells Cell surface characteristics Alkaline phosphatase Aquaporin 3 transmembrane water channels b-adrenergic receptors CD44 transmembrane glycoproteins Epidermal growth factor receptor Fibroblast growth factor receptor-1 Fas receptors and ligand ICAM-1 IgE receptor Integrins (a6b4) Lectins LEEP-CAM MRP transmembrane transporters MUC 1,4,8 Neurokinin-1 receptor Neutral endopeptidase Syndecan-4 4-1 BB receptor Intracellular characteristics Adrenomedullin receptor (mRNA) Autotaxin (mRNA) Annexin II Bcl-2 protein Extracellular SOD (mRNA) Leukemic inhibitory factor P63 nuclear protein Reproduced from Evans MJ, Van Winkle LS, Fanucchi MV, et al. (2001) Cellular and molecular characteristics of basal cells in airway epithelium. Experimental Lung Research 27: 401–415.

constant amount of junctional adhesion between the columnar epithelium and the BMZ. Thus, the relationship between basal cell junctional adhesions and height of the epithelium is constant and not related to airway level or animal species. Fibroblast Growth Factor-2 Signaling

Fibroblast growth factor-2 is stored in the BMZ of the airways where it binds with perlecan, a heparan sulfate proteoglycan that is an intrinsic constituent of the BMZ. Fibroblast growth factor-2 is released from perlecan in response to various conditions and becomes an important cytokine within the local microenvironment of the epithelial–mesenchymal trophic unit. In airway epithelium, basal cells are the only cell type involved with fibroblast growth factor-2 signaling. Fibroblast growth factor-2 signals by forming a ternary complex with fibroblast growth factor receptor-1 (FGFR-1) and syndecan-4. When the fibroblast growth factor-2 ternary complex is formed, it initiates tyrosine kinase signaling associated with cell proliferation, migration, and differentiation. Basal cells express the cell surface receptors FGFR-1 and syndecan-4 whereas columnar cells do not (Figure 2). Presumably, fibroblast growth factor-2 is stored in airway BMZ as an intact growth factor to aid in rapid cellular responses to changes in local environmental

BASAL CELLS 231 Syndecan-4 FGFR-1

Basal cell FGF-BP FGF-2

Perlecan

Collagen I, III, & V

Perlecan

Basement membrane zone Figure 2 Ternary signaling complex in airway epithelium. In this illustration, BMZ-bound FGF-2 is released, and formation of the FGF-2 ternary complex with basal cells occurs via diffusion or binding with FGF-binding protein (FGF-BP). This is an example of how basal cells may function in growth factor signaling, neurogenic inflammation, and the inflammatory response through interactions with substances in the lateral intercellular space. Reproduced from Evans MJ, Fanucchi MV, Baker GL, et al. (2003) Atypical development of the tracheal basement membrane zone of infant rhesus monkeys exposed to ozone and allergen. American Journal of Physiology: Lung, Cellular and Molecular Physiology 285: L931–L939, with permission from The American Physiological Society.

conditions such as sloughing of damaged columnar epithelium or damage to the BMZ by leukocyte trafficking. Neurogenic Inflammation

Basal cells are involved with regulation of neurogenic inflammation. In response to various inhaled foreign materials, axons in the airway epithelium release neuropeptides into the lateral intercellular space, initiating the process of neurogenic inflammation (increased vascular permeability, neutrophil adhesion, vasodilatation, gland secretion, ion transport, smooth muscle contraction, increased cholinergic transmission, and cough). Basal cells contain the protein leukemic inhibitory factor. Leukemic inhibitory factor is thought to function in neurogenic inflammation by stimulating the release of neuropeptides (tachykinins) from axons and the formation of neurokinin receptors. Neutral endopeptidase is a cell surface enzyme also associated with the process of neurogenic inflammation. The enzyme neutral endopeptidase is expressed mainly on the surface of basal cells. The enzyme neutral endopeptidase cleaves neuropeptides in the lateral intercellular space. Cleavage of neuropeptides by neutral endopeptidase modulates the neurogenic inflammatory responses in the airways. Inflammation

Basal cells participate in the inflammatory response by upregulating expression of receptors for migratory

inflammatory cells and lymphocytes. Human basal cells upregulate intercellular adhesion molecule-1. Human basal cells can also upregulate expression of IgE receptors indicating that they may be involved with allergic responses of the airway. An unusual cell adhesion molecule, lymphocyte endothelial–epithelial cell adhesion molecule, is expressed in the basal cell layer of human bronchial epithelium. Lymphocyte adhesion to epithelia and endothelia is mediated by lymphocyte endothelial–epithelial cell adhesion molecule. Human basal cells also express the receptor 4-1BB. The receptor 4-1BB is a member of the tumor necrosis factor receptor super-family and is associated with T cell activation. Human basal cells express the Fas receptor and its ligand FasL. Ligation of the Fas receptor by migratory inflammatory cells can lead to their apoptosis. Expression of these molecules by basal cells may play an important role in regulation of the inflammatory response. They interact with inflammatory cells when the latter are moving through the lateral intercellular space of airway epithelium. Transepithelial Water Movement

Basal cells have the aquaporin water channel AQP3, which is not found in the columnar epithelial cells. Instead, columnar epithelial cells have the aquaporin water channel AQP4. Water transfer between cells and the matrix occurs through these water channels. AQP3 is found in a basolateral position in the airways and in other tissues, implying movement of water between the extracellular matrix and epithelium. The presence of AQP3 water channels in the membranes of basal cells of normal airway epithelium demonstrates a unique role for the basal cell in fluid modulation of airway surface liquids and also in the lateral intercellular space. The cellular distribution of AQPs 3 and 4 in airway epithelium implies the presence of cell-specific pathways for transcellular water movement between the extracellular matrix and epithelium. The cystic fibrosis transmembrane conductance regulator protein is a regulator of AQP3 water channels in basal cells suggesting a role in this disease. Downregulation of AQP 3 is thought to play a role in the pathogenesis of bronchiectasis. Oxidant Defense of the Tissue

Basal cells in normal human airway epithelium express extracellular superoxide dismutase mRNA. Extracellular superoxide dismutase is a secreted protein found in the extracellular matrix responsible for metabolizing superoxide free radicals. The physiologic functions are not fully defined but it is thought to be critical for the protection of extracellular matrix elements against oxidative damage. The multidrug resistance-associated protein transmembrane transporter is

232 BASAL CELLS

found in bronchial epithelium and plays a major role in cell detoxification and defense against oxidant stress via efflux of glutathione conjugates into the lateral intercellular space. The pattern of multidrug resistance-associated protein transmembrane transporter expression differs markedly according to cell type. In basal cells it is distributed over the entire circumference of the cell whereas in ciliated cells it is restricted to the basolateral surface. Expression of extracellular superoxide dismutase and multidrug resistance-associated protein transmembrane transporter by basal cells in normal subjects indicates that basal cells participate in defense of the tissue against oxidative stress. Progenitor Cells

The basal cell has the capacity to be the progenitor of columnar airway epithelium. This has been demonstrated in studies where denuded airways were repopulated with enriched populations of basal cells, in biphasic organotypic cultures, and in vivo following loss of the columnar epithelium. Under these conditions, basal cells dedifferentiate into a highly proliferative cell phenotype from which a mucociliary epithelium redifferentiates. In vivo there are two populations of proliferating basal cells (basal and parabasal cells). Basal cells have their nuclei next to the basement membrane. Parabasal cells are taller, and their nuclei are above the layer of basal cell nuclei. Basal cells make up 31% of the cell population in large airways and the taller parabasal cells make up 7%. However, parabasal cells have a proliferative fraction 4 to 5 times greater than basal cells. The higher proliferative fraction in parabasal cells suggests they may be more active in reparative proliferation following injury when compared with basal cells. Parabasal cells probably are the intermediate cells seen with electron microscopy. Intermediate cells are known to be the primary proliferating cells following injury to the columnar epithelium. During growth of the airway, the basal cell has a high rate of proliferation. The purpose of such proliferation in the growing airway is for the formation of new basal cells. The increase in basal cells per millimeter is related to their role in attaching columnar epithelium to the BMZ. Increased proliferation of parabasal cells during development is probably associated with formation of the columnar epithelium. In the normal adult airway epithelium, the rate of basal cell proliferation is low. Such proliferation may be for replacement of dying basal cells. When basal cells are lost due to injury or apoptosis, proliferation of surviving basal cells occurs, and they are replaced. Proliferation of parabasal cells is most likely associated with normal turnover of the columnar epithelium.

Basal Cells in Respiratory Disease Asthma

Defining the mechanism of columnar cell attachment to the BMZ was critical to the understanding of asthma and other disease conditions associated with sloughing of epithelium. In conditions where columnar cells are sloughed from the epithelium of larger airways, basal cells remain attached to the basal lamina. Desmosomal attachment between the columnar epithelium and basal cells represents a plane of cleavage between the two cell populations. Failure of desmosomal attachment between columnar and basal cells is thought to be responsible for sloughing of the columnar epithelium in asthmatics. The mechanism of desmosomal failure between airway cells is not known. However, it may not be failure per se but rather a specific protective function of basal cells. Shortly after the columnar epithelium has been sloughed, basal cells flatten out and form a protective barrier. This is considered to be an important protective function of the basal cell along with the ability to quickly release desmosomal attachments to damaged columnar epithelial cells. It has also been speculated that stimulation of the epithelial neural system causes edema of the lateral intercellular space with a subsequent local sloughing of the epithelium. In a similar manner, an influx of inflammatory cells could overwhelm the lateral intercellular space and cause local sloughing of the epithelium. However, these scenarios are speculations and the reasons for or mechanisms of epithelial sloughing are not known at this time. Bronchiogenic Cancer

The p63 nuclear protein is specific for basal cells. Its functions are not clear, however, it has been used as a marker for tumors of basal cell origin in several tissues. In the lung, p63 was shown to be expressed in basal cells of normal tissue, in areas of squamous metaplasia, and in squamous cell carcinomas of the bronchi. In poorly organized carcinomas, p63 is expressed in most of the cells. However, it is only found in the basalar external cells of organized carcinomas. In adenocarcinomas, p63 is present occasionally but is not present in small cell carcinomas. These findings indicate that basal cells are probably involved with the formation of squamous cell carcinoma and, possibly, the progression to organized carcinoma. Basal cells have also been implicated in abnormal epithelium found in idiopathic pulmonary fibrosis when p63 was used as a marker. See also: Adhesion, Cell–Cell: Vascular; Epithelial. Adhesion, Cell–Matrix: Focal Contacts and Signaling; Integrins. Aquaporins. Cell Cycle and Cell-Cycle

BREATHING / Breathing in the Newborn 233 Checkpoints. Epidermal Growth Factors. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. Fibroblast Growth Factors. Stem Cells.

Further Reading Boers JE, Ambergen AW, and Thunnissen FB (1998) Number and proliferation of basal and parabasal cells in normal human airway epithelium. American Journal of Respiratory Critical Care and Medicine 157(6 Pt 1): 2000–2006. Evans MJ, Fanucchi MV, Baker GL, et al. (2003) Atypical development of the tracheal basement membrane zone of infant rhesus monkeys exposed to ozone and allergen. American Journal of Physiology: Lung, Cellular and Molecular Physiology 285: L931–L939. Evans MJ and Moller PC (1991) Biology of airway basal cells. Experimental Lung Research 17: 513–531. Evans MJ and Shami SG (1989) Lung cell kinetics. In: Lenfant C and Massaro M (eds.) Lung Cell Biology, vol. I. of the series Lung Biology in Health and Disease, pp. 1–36. New York: Marcel Dekker, Inc.

Basement Membranes

Evans MJ, Van Winkle LS, Fanucchi MV, and Plopper CG (1999) The attenuated fibroblast sheath of the respiratory tract epithelial-mesenchymal trophic unit. American Journal of Respiratory Cell and Molecular Biology 21: 655–657. Evans MJ, Van Winkle LS, Fanucchi MV, et al. (2001) Cellular and molecular characteristics of basal cells in airway epithelium. Experimental Lung Research 27: 401–415. Ford JR and Terzaghi-Howe M (1992) Basal cells are the progenitors of primary tracheal cultures. Experimental Cell Research 198: 69–77. Inayama Y, Hook GE, Brody AR, et al. (1988) The differentiation potential of basal cells. Laboratory Investigation 58: 706–717. Johnson NF and Hubbs AF (1990) Epithelial progenitor cells in rat trachea. American Journal of Respiratory Cell and Molecular Biology 3: 579–585. Mercer RR, Russell ML, Roggli VL, and Crapo JD (1994) Cell number and distribution in human and rat airways. American Journal of Respiratory Cell and Molecular Biology 10: 613–624. Persson CGA and Erjefalt JS (1997) Airway epithelial restitution after shedding and denudation. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung, pp. 2611–2627. Philadelphia: Lippincott-Raven.

see Extracellular Matrix: Basement Membranes.

Berylliosis

see Occupational Diseases: Hard Metal Diseases – Berylliosis and Others.

Bradykinin

see Kinins and Neuropeptides: Bradykinin.

BREATHING Contents

Breathing in the Newborn Fetal Breathing Fetal Lung Liquid First Breath

Breathing in the Newborn J A Adams, Mount Sinai Medical Center, Miami Beach, FL, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The complex physiology of breathing in the newborn has important developmental and maturational aspects. Early in fetal

life the respiratory system and controllers respond to intrauterine stimuli (PaCO2 , PaO2 , and lung inflation). The complex network of control of breathing can be summarized into three basic components: (1) controllers, (2) effectors, and (3) sensors. At birth a multitude of inputs are responsible for initiation of rhythmic ventilation, and adaptation of the pulmonary circulation to extrauterine life. The normal physiology of breathing in the newborn is affected by posture, sleep state, and gestational age. Pathophysiological aspects of breathing in the newborn can be divided into: (1) fetal pathologies that occur during fetal life and ultimately result in abnormal lung development,

234 BREATHING / Breathing in the Newborn (2) immediate postnatal events that occur primarily as a result of poor or abnormal transition to extrauterine life, and (3) neonatal events occurring in the neonatal period that lead to intrinsic lung disease or deranged control of breathing such as apnea. This article emphasizes the general importance of understanding the influence of age, posture, sleep state, and maturation on breathing in the newborn, and provides a general overview of these. Paramount to the interpretation of normative and study data is the understanding of the influence of these factors.

Description The undertaking of a complex vital physiological function such as breathing in the newborn is a marvelous feat. Initiation of breathing in the newborn requires a multitude of appropriately synchronized events and signaling pathways. In preparation for extrauterine life and in order to assume the responsibility for extrauterine gas exchange, an anatomical, maturational, and functional process must occur. The alveoli are developed by the 25th week of gestation and by the 35th week of gestation, adequate quantities of surfactant (surface active material that keeps alveoli open and maintains alveoli stability) are present. Pulmonary circulation parallels alveolar development, and thus by the 25th week of gestation alveolar gas exchange can take place, but requires assisted ventilation. During fetal life, respiration is present in human fetuses from 10 weeks onward. These fetal breathing movements are not as well synchronized as those occurring in the newborn period. In fetal life, breathing is discontinuous and becomes continuous after birth. The fetus spends nearly 30% of its time engaged in discoordinate form of breathing associated with rapid irregular electrocortical activity, as seen in active sleep state. In addition, since the entire airways are filled with amniotic fluid, at term almost 600 ml of amniotic fluid is ‘inhaled’ per day. Maintenance of normal amniotic fluid volume, and respiratory activity, are crucial in the development of the airway, and maturation of lung structure and function. The fetus is also capable of modulating breathing movements in response to: (1) PaCO2 (hypercarbia increases fetal breathing), (2) PaO2 (hypoxia abolishes fetal breathing in sleep, and (3) pulmonary reflexes (inflation reflex of Herring–Bauer lung distension with saline infusion decreases frequency of breathing). The overall framework for the control of breathing can be schematically viewed as: (1) controller (central nervous system and brainstem); (2) effectors (respiratory muscles and airway); and (3) feedback (chemoreceptor, mechanoreceptors, i.e., stretch receptors) (Figure 1). Functional maturity of the controller, effectors, and feedback mechanism allows for extremely premature newborns to survive, albeit requiring ventilatory assistance. The maturational process continues during gestation until term at 38–40

weeks of gestation, but even after birth the continual adaptation to air-breathing is an ongoing process. The First Breaths

During vaginal birth but less so during Cesarean section delivery, the thoracic cage is compressed to as much as 160 cmH2O pressure; this produces an ejection of tracheal fluid via the airways. The recoil of the chest wall causes a passive inspiration and establishes an air–liquid interface. The first active breath is made slightly easier by the fact that some fetal lung fluid is retained in the alveoli and smaller airway, thus requiring less distending pressure than a totally collapsed lung. In addition, in near-term and term newborns, surfactant produced by type II pneumocytes decreases alveolar surface tension, which prevents the lungs from total collapse at the lower transpulmonary pressures that occur in the subsequent breaths (Figure 2). The stimuli for the first active inspiration is debatable but is likely to be a multifactorial set of events with which the newborn is confronted including change in temperature, light, noise, gravity, hypercapnea, sudden change in PaO2 , etc. A complete discussion on the control of breathing during the first breaths is beyond the scope of this article, but suffice it to say that environmental nonrespiratory stimuli will enhance or facilitate the general tone of the respiratory neurons. In addition to the mechanical effects of the first breath, the pulmonary circulation that parallels lung development and is maintained at high pulmonary vascular resistance during fetal life must also transition from a fetal to a newborn circulation. The latter is characterized by a gradual lowering of the pulmonary vascular resistance and eventual closure and redirection of blood flow via the foramen ovale and ductus arteriosus, and a change from a parallel circulation to one in series (Figure 3). The resultant effect is ultimately to match ventilation to perfusion for the most efficient oxygen extraction and delivery and carbon dioxide removal. Sleep state modulates control of breathing. The sleep and waking cycles begin to develop during fetal life. In the premature newborn at 24 weeks’ gestation, rapid eye movement (REM) periods account for nearly 80% of the total sleep time. At term, REM sleep is 60–65% of sleep time, while in adulthood it accounts for only 20–25%. There are major differences in the control of breathing between awake and sleep, which are independent of age. During sleep the brain is controlled more by stimulation related to gas exchange than any other stimuli. When comparing the breathing patterns and pulmonary mechanics

BREATHING / Breathing in the Newborn 235

Controllers Effectors

Cerebrum Cerebellum Midbrain

Sensors

Brainstem

Chemoreceptors

Lung receptors

Upper airway

Spinal cord Proprioreceptors

Vagal resp. motor efferents

Upper airway receptors

Resp. muscles

Lung

Figure 1 Schematic representation of the control of breathing. Adapted from Givan DC (2003) Physiology of breathing and related pathological processes in infants. Seminars in Pediatric Neurology 10: 271–280, with permission from Elsevier.

among newborns, measurements should always be made in the same sleep state and in the same posture. Noninvasive methods to quantify these breathing patterns over various sleep states can serve as a basis for comparison. Breathing is affected by gestational age. Maturation of ventilatory response to chemical and mechanical stimuli is also influenced by gestational age. The ventilatory response to hypoxia in newborns is biphasic. It is characterized by an initial phase of hyperventilation followed by hypoventilation below baseline levels. The etiology of such biphasic response is unclear, and may be related to metabolic demands or neurotransmitters (Figure 4). This response is clearly different to that which occurs in older infancy and adults. The ventilatory response to carbon dioxide in premature infants is also different from that in term newborns. In premature infants the ventilatory response to hypercapnea is quantitatively and qualitatively different from that in older term neonates and adults. Term infants and adults increase ventilation through an increase in tidal volume

and frequency; premature infants do not increase frequency in response to hypercarbia, and have a prolonged expiration. This response appears to be centrally mediated at the level of the brainstem and probably involves the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Furthermore, the ventilatory response to hypercapnea is influenced by sleep state in term newborns. There is a greater response in minute ventilation during quiet state compared to REM (Figure 5). Breathing is affected by posture in newborns. The characteristics of a very compliant chest wall in newborns have a significant mechanical effect on breathing as efficiency of ventilation is reduced due to increased chest wall compliance. In full term infants, a change from supine to prone posture increases minute ventilation and respiratory drive, with a concomitant decrease in thoracoabdominal asynchrony. In premature infants (o35 weeks’ gestation) supine posture is associated with higher respiratory rate, lower arterial oxygen saturation, lower ventilatory response to hypercapnea, and increased thoracoabdominal

236 BREATHING / Breathing in the Newborn Saline

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Figure 2 (a) Pressure–volume curves after saline and air expansion of the lung. Lower pressures are required to expand a saline-filled lung compared to air. The deflation curve in air is not superimposable on the inflation curve (hysteresis) due to mobilization and orientation of surfactant during deflation decreasing alveolar surface tension as the alveolar surface contracts. Adapted from Nelson NM (1999) The onset of respiration. In: Avery GB, Fletcher MA, and McDonald MG (eds.) Neonatology Pathophysiology & Management of the Newborn, pp. 257–278. Philadelphia: Lippincott Williams & Wilkins, with permission from Lippincott Williams & Wilkins. (b) Pressure– volume loops of a normal lung (red) and surfactant-deficient lung. Note that the slope of the loop is decreased in the surfactant-deficient lung. A greater amount of pressure is required for a lower change in lung volume. (Compliance ¼ D volume/D pressure.)

synchrony. Thus, posture appears to play a role in pulmonary mechanics, primarily in the efficiency of ventilation. Whether these changes occur as a result of conferring greater chest wall stability or diaphragmatic mechanical advantage in the prone posture in both full term and preterm infants remains to be clearly elucidated. In spite of this, conferring a mechanical advantage in the prone posture for full term infants does not justify its use in the care of the full term newborns upon hospital discharge. Sudden infant death rates have clearly decreased in the US as a result of the ‘Back to Sleep Campaign’.

Pathophysiology In order to understand the effects of various pathological states on breathing in the newborn it is useful to describe three distinct time periods for occurrence of pulmonary pathology: (1) fetal, (2) immediate postnatal, and (3) neonatal.

Fetal

To a large extent, lung growth is dependent on amniotic fluid production and volume, and the mechanical


Pulmonary vascular resistance

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Left atrial pressure

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Peripheral vascular resistance Figure 3 Schematic of the transition to neonatal circulation once ventilation is established. L–R, left to right; ret, return. Multiple factors play a role in the conversion of this circulation, including prostaglandins, endothelin, and endothelial-derived nitric oxide. Adapted from Nelson NM (1999) The onset of respiration. In: Avery GB, Fletcher MA, and McDonald MG (eds.) Neonatology Pathophysiology & Management of the Newborn, pp. 257–278. Philadelphia: Lippincott Williams & Wilkins, with permission from Lippincott Williams & Wilkins.

% Change in baseline

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−30 Time (s) Figure 4 The biphasic ventilatory response to hypoxia. Note the increase in ventilation, followed by a marked decrease in ventilation below baseline values. Adapted from Martin RJ, Di Fiore JM, Jana L, et al. (1998) Persistence of the biphase ventilatory response to hypoxia in preterm infants. Journal of Pediatrics 132: 960–964, with permission from Elsevier.

shear it exerts on the primitive airways and alveolar ducts. Thus, a spectrum of pulmonary hypoplasia due to multiple etiologies is the major pathological problem in the fetal period.

Immediate Postnatal

Failure to transition from a fluid-filled to a gas-filled pulmonary tree and to reabsorb the fetal lung fluid results in a transient condition called transient

238 BREATHING / Breathing in the Newborn

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pulmonary hypertension with increased pulmonary vascular resistance and right to left shunting via the patent ductus arteriosus and foramen ovale. This condition has several underlying etiologies including intrauterine hypoxia, pneumonia, and other pulmonary vascular pathologies.

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Figure 5 Ventilatory response to hypercapnea. Note the difference in slope of the line between active state (REM) and quiet state in full term infants. End tidal CO2 ¼ PET CO2. Reproduced from Cohen G, Xu C, and Henderson-Smart D (1991) Ventilatory response of the sleeping newborn to CO2 during normoxic rebreathing. Journal of Applied Physiology 71(1): 168–174, with permission from The American Physiological Society.

tachypnea of the newborn (TTN or respiratory distress syndrome (RDS) type II). This condition is characterized by the early appearance of tachypnea and a variable degree of respiratory distress in an otherwise ‘healthy appearing’ newborn. The greater occurrence of TTN in newborns delivered by Cesarean section make this condition rather common. Onset of respiratory distress after the initial newborn transitional period (about 4–6 h) usually signifies intraparenchymal lung pathology due to pneumonia (viral or bacterial). Medications administered during labor and delivery can also have an effect on breathing; these include narcotics or magnesium sulfate, both of which can depress respiratory drive. In the premature infant, failure to transition from fetal to extrauterine life can be due to pulmonary insufficiency secondary to RDS type I (surfactant deficiency). Surfactant administration has markedly improved mortality and morbidity of this disease process and has radically changed the outcomes for premature newborns. In both premature and term newborns, failure of the pulmonary circulation to transition from intrauterine fetal circulation to that of the newborn results in the well known persistent pulmonary hypertension of the newborn (PPHN) or persistent fetal circulation (PFC) characterized by

In premature infants, particularly those born at less than 34 weeks’ gestation who have immature lungs or an intrinsic pulmonary pathology, a frequent respiratory problem is apnea. Apnea is typically defined as cessation of breathing for more than 15 s; however, the literature is replete with various definitions for the duration of cessation of breathing. Apnea is most commonly seen in premature infants, and the younger the gestational age the more common its occurrence (Figure 6). Apnea in the premature infant can be caused by intracranial pathology, metabolic derangements, and infection, among others. The diagnosis of apnea of prematurity is one of exclusion and thus an exhaustive undertaking of diagnostic tests should be done prior to categorizing apnea as such (Figure 7). In addition to cessation of breathing or ineffective ventilation, clinically relevant apneas decrease arterial oxygen saturation and cause bradycardia. With the advent of computerized monitoring and noninvasive technologies to measure breathing and arterial oxygen saturation, long-term studies in newborns have become possible in the home environment. It has been found that apneas can occur at any gestational age and that their severity can only be detected if various physiological parameters are simultaneously monitored. Apnea has been typically classified into three types: (1) central (no respiratory efforts and thus no airflow), (2) obstructive (respiratory efforts against a partially or totally occluded airway), and (3) mixed (combination of central and obstructive within the same event). With the advent of more refined monitoring techniques, it is evident that purely obstructive apneas in the newborn are rare, and that most apneas in premature infants have a mixed/obstructive characteristic. Cessation of airflow is present followed by obstructive breaths or obstructive breaths are followed by cessation of respiratory effort. Perhaps the most clinically relevant and important aspect of apnea relates to the changes in arterial oxygen saturation that it can cause and bradycardia (Figures 8(a)–8(c)). Using respiratory inductive plethysmography that measures both ribcage and abdominal excursions and can be calibrated in the newborn to provide a relative estimate of tidal volume, we have been able to study apneas in premature newborns. In


Relative incidence of apnea

Apnea of prematurity

Prematurity

Term 1 mo 2 mo Term 1 mo 2 mo 6 mo Age Figure 6 Relative incidence of apnea as a function of maturation. Note the dramatic decrease in apnea after term gestation. Reproduced with permission from NeoReviews, Vol. 3, pages e66–e70, Copyright 2002.

Immaturity

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Figure 7 A schematic representation of the etiology of apnea in premature newborns. Examples of cause of apneas are highlighted in yellow. IVH, intraventicular hemorrhage. Adapted with permission from NeoReviews, Vol. 3, page(s) e66–e70 and NeoReviews, Vol. 3, page(s) e59–e65, Copyright 2002.

addition to apneas, newborns also experience hypopnea (decreased tidal volumes below 25% of the unimpeded baseline value). Hypopnea is a commonly recognized problem in sleep disorder breathing in adults but not commonly appreciated in newborns due to the lack of ability to measure changes in tidal volume over prolonged periods. The end result of both apneas and hypopneas is to cause decreased functional residual capacity (FRC). Since newborns typically have relatively low FRC and oxygen stores, the concomitant decrease in arterial oxygen saturation during apneas or hypopneas is not surprising. Additionally, we have found using noninvasive methods that during periods of apnea in the premature newborn the cardiac output can decrease by as much as 50%. Cerebral blood flow fluctuations have also been shown to occur to a greater degree during periodic breathing, apnea, and REM sleep. Whether cardiac output and episodic arterial oxygen desaturation and changes in cerebral blood flow will have untoward long-term neurological effects needs to be determined.

Conclusion Disordered breathing in the premature and term infant can be ascribed to a multitude of causes, which

240 BREATHING / Breathing in the Newborn 30.5 mm s–1

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(b) Figure 8 Examples of respiratory inductive plethysmographic recordings: (a) normal tidal breathing in the newborn; (b) central apnea of 14 s duration; and (c) mixed/obstructive apnea of 21 s duration (note a decrease in HR to 90 bpm, followed by a decrease in arterial oxygen saturation to a nadir of 80%). Vt, the tidal volume from calibrated respiratory inductive plethysmography derived as a percent of the calibration value. RC, the volume contribution of the ribcage to tidal volume. AB, the abdominal volume contribution to tidal volume. EPANG, the phase angle between the RC and AB, obtained from a plot of RC vs. AB; values of 180o denote complete paradoxical motion of the RC and AB in opposite directions. ECG, electrocardiogram. HR, heart rate derived from the electrocardiogram. OxiP, the arterial pulse obtained from the pulse oximeter. SAT, percent arterial oxygen saturation.

BREATHING / Breathing in the Newborn 241 5.6 mm s–1

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have their basis in pulmonary developmental problems, intrinsic pulmonary disease, or deranged control of breathing. The influence of sleep state, posture, and gestational age are paramount in the interpretation of both normative and study data. See also: Breathing: Fetal Breathing; Fetal Lung Liquid; First Breath. Infant Respiratory Distress Syndrome. Sudden Infant Death Syndrome.

Further Reading Adams JA, Zabaleta IA, and Sackner MA (1994) Comparison of supine and prone noninvasive measurements of breathing patterns in fullterm newborns. Pediatric Pulmonology 18: 8–12. Adams JA, Zabaleta IA, and Sackner MA (1997) Hypoxemic events in spontaneously breathing premature infants: etiologic basis. Pediatric Research 42: 463–471. Adams JA, Zabaleta IA, Stroh D, Johnson P, and Sackner MA (1993) Tidal volume measurements in newborns using respiratory inductive plethysmography. American Review of Respiratory Diseases 148: 585–588. Avery GB, Fletcher MA, and McDonald MG (1999) Neonatology Pathophysiology & Management of the Newborn. Philadelphia: Lippincott Williams & Wilkins. Baird TM, Martin RJ, and Abu-Shaweesh JM (2002) Clinical associations, treatment, and outcome of apnea of prematurity. NeoReviews 3(4): e66–e70. Campbell AJ, Bolton DP, Taylor BJ, and Sayers RM (1998) Responses to an increasing asphyxia in infants: effects of age and sleep state. Respiration Physiology 112: 51–58.

Chernick V (1981) The fetus and the newborn. In: Hornbein T (ed.) Regulation of Breathing, Part II, pp. 1141–1179. New York: Dekker. Cohen G, Xu C, and Henderson-Smart D (1991) Ventilatory response of the sleeping newborn to CO2 during normoxic rebreathing. Journal of Applied Physiology 71(1): 168–174. Curzi-Dascalova L (1992) Physiological correlates of sleep development in premature and full-term neonates. Neurophysiology Clinincs 22: 151–166. Fanaroff AA and Martin RJ (2002) Neonatal–Perinatal Medicine Disease of the Fetus and Infant. St Louis: Mosby. Givan DC (2003) Physiology of breathing and related pathological processes in infants. Seminars in Pediatric Neurology 10: 271–280. Guilleminault C and Robinson A (1996) Developmental aspects of sleep and breathing. Current Opinion in Pulmonary Medicine 2: 492–499. Martin RJ, Abu-Shaweesh JM, and Baird T (2002) Pathophysiologic mechanisms underlying apnea of prematurity. NeoReviews 3: e59–e65. Martin RJ, Di Fiore JM, Jana L, et al. (1998) Persistence of the biphasic ventilatory response to hypoxia in preterm infants. Journal of Pediatrics 132: 960–964. Mortola JP and Saiki C (1996) Ventilatory response to hypoxia in rats: gender differences. Respiration Physiology 106: 21–34. Nelson NM (1999) The onset of respiration. In: Avery GB, Fletcher MA, and McDonald MG (eds.) Neonatology Pathophysiology & Management of the Newborn, pp. 257–278. Philadelphia: Lippincott Williams & Wilkins. Polin RA and Fox WW (1992) Fetal and Neonatal Physiology. Philadelphia: Saunders. Rigatto H (1992) Maturation of breathing. Clinical Perinatology 19: 739–756. Stocks J, Sly PD, Teppers RS, and Morgan WJ (1996) Infant Respiratory Function Testing. New York: Wiley-Liss.

242 BREATHING / Fetal Breathing

Fetal Breathing R Harding and S B Hooper, Monash University, Melbourne, VIC, Australia C A Albuquerque, Santa Clara Valley Medical Center, San Jose, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Fetal breathing movements (FBMs) are breathing-like movements that occur episodically in healthy mammalian fetuses. As with postnatal breathing, FBMs are centrally organized rhythmic contractions of the diaphragm, but may also involve other skeletal muscles such as those of the chest wall and upper respiratory tract. Owing to the airways being filled with liquid, FBMs cause only minor changes in lung volume but typically lower intrathoracic pressure by up to 5 mmHg and alter the shape of the fetal chest. By altering intrathoracic pressure they affect blood flow within the fetus and fluid movement within the fluid-filled fetal airways. FBMs are characteristically highly variable in frequency and amplitude, but become more organized with increasing gestational age and relate to fetal behavioral states. They are inhibited by fetal hypoxia and hence can be used in the diagnosis of fetal compromise. FBMs are critical for normal in utero lung growth and development as they maintain the fetal lung in an expanded state by opposing lung recoil: in the absence of FBMs the fetal lungs tend to ‘deflate’ leading to lung hypoplasia. At birth, breathing becomes continuous, possibly by removal of inhibitory substances produced by the placenta or fetal brain and by increased carbon dioxide production.

Introduction Fetal breathing movements (FBMs) are breathinglike movements that occur episodically in healthy fetuses during much of gestation. FBMs have been observed in many mammalian species, including man, as well as in birds and reptiles. As with postnatal breathing, FBMs are centrally organized rhythmic contractions of the diaphragm, but may also involve other skeletal muscles such as those of the chest wall and upper respiratory tract. They play no role in fetal gas exchange, as the fetal ‘airways’ are filled with liquid; in sheep, they typically lower intrathoracic pressure by up to 5 mmHg. Most of the available information on FBMs has been obtained from two species, humans and sheep. In humans, FBMs can be detected by ultrasonography from about 10 weeks of gestation. They are observed as rhythmic descending movements of the diaphragm and are usually coincident with inward movement of the chest wall and outward movement of the abdominal wall. In chronically catheterized fetal sheep, FBMs can be detected as electrical activity of the diaphragm muscle, rhythmic reductions in intratracheal or intrathoracic pressure, or fluid movement within the trachea.

Two other types of inspiratory efforts have been recognized in the fetus; isolated deep inspiratory efforts and asphyxial gasping. Isolated deep inspiratory efforts, often termed hiccups, are commonly observed in healthy fetal humans, pigs, and sheep; typically, these occur in low-frequency bouts. Asphyxial gasps are strong inspiratory efforts initiated by fetal asphyxia and involve intense activation of many respiratory muscles.

Central and Peripheral Control of FBMs FBMs are an expression of rhythmic activation of neurons in the fetal brainstem. These brainstem neurons generate rhythmic bursts of activity in phrenic motoneurons and in vagal preganglionic fibers destined for dilator muscles of the upper respiratory tract. With development, episodes of FBMs become temporally associated with fetal behavioral states involving body movements, rapid eye movement (REM) sleep, or arousal. During the last third of gestation, FBMs are usually infrequent or absent in association with the fetal state resembling quiet, or non-REM, sleep (low activity state). The periods of FBM/REM sleep/activity and the intervening periods of apnea/non-REM sleep/inactivity are of similar duration, both occupying B50% of the time. Compared to postnatal life, respiratory drive in the fetus is relatively low and is regulated mainly by fetal CO2 levels, as the incidence and amplitude of FBMs are increased by elevated fetal PaCO2 (partial pressure of carbon dioxide in arterial blood) levels and decreased by low fetal PaCO2 levels. It is assumed that this CO2 drive is mediated by acidification of the fluid surrounding the central chemoreceptors, as altering the pH of fetal blood or cerebrospinal fluid (CSF) can have effects similar to those of alterations in PaCO2 levels. FBMs are inhibited by moderate to severe hypoxia (Figure 1), by what is thought to be a central inhibitory mechanism that also inhibits other forms of fetal skeletal muscle activity and alters fetal behavioral state favoring reduced activity. This inhibitory mechanism may override the stimulatory effects of mild hypoxia. Although peripheral chemoreceptors are active in the fetus and do respond to hypoxia, they do not apparently play a role in the hypoxic inhibition of FBMs. Recent studies suggest that central adenosine receptors play a major role in the central inhibition of FBMs and alteration in fetal behavior by hypoxia.

Effects of FBMs on Airway Fluid Movement and Lung Volume As FBMs alter fetal intrathoracic pressure, it is to be expected that they affect fluid movement within the

BREATHING / Fetal Breathing 243 50

Incidence of FBMs (min h–1)

40

30

20

∗ ∗

10 ∗

∗

∗

∗ ∗ ∗ ∗

∗ 24 h normoxia

∗ ∗

24 h hypoxia

0 −4

0

4

8

12

16

20

24

28

Period of hypoxia (h) Figure 1 Effects of 24 h of fetal hypoxia on the incidence of FBMs in a late gestational fetal sheep. Hypoxia causes a profound but transient inhibition of FBMs, with normal values returning after 14–18 h of hypoxia. Asterisks show values that differ significantly from control values. Reproduced from Hooper SB and Harding R (1990) Changes in lung liquid dynamics induced by prolonged fetal hypoxemia. Journal of Applied Physiology 69: 127–135, used with permission from the American Physiological Society.

respiratory tree. Oscillatory fluid flows synchronous with diaphragm movements have been detected in the fetal trachea and at the nose in humans. Although there is some controversy as to the volume of fluid moved with each fetal ‘breath’, it is clear that ‘tidal volume’ in the fetus is much smaller than after birth, which can be attributed to the much greater viscosity of liquid relative to air. In fetal sheep, for example, ‘tidal volumes’ measured by flow meters in the trachea are much less than 1 ml, whereas after birth, in the same species, tidal volumes are typically 30– 50 ml. Thus, FBMs are essentially isovolumic, causing only very small changes in thoracic volume with each breath. However, in human fetuses, ‘tidal volumes’ of about 2 ml have been estimated by ultrasound, in the trachea and at the nose. The reason for the larger ‘tidal volume’ in humans is unknown but may be due to the slower breathing rates of human fetuses compared to fetal sheep. Although FBMs individually may have little effect on volume flow within the airways, it is clear that episodes of FBM can result in the movement of much greater volumes, relative to total lung fluid volume. This effect on fluid movement can be largely attributed to FBM-related changes in transpulmonary pressure and upper airway resistance. Studies in sheep have shown that net fluid movement to and from the fetal lungs is affected by FBM episodes such that most of the efflux occurs during these episodes; during episodes of FBM the laryngeal dilator muscles

are rhythmically active (in phase with the diaphragm) and laryngeal adductor muscles are largely quiescent. Hence, the resistance to tracheal fluid movement offered by the upper airway is lowered during FBMs, allowing fluid that has accumulated within the airways to flow into the pharynx, from where it is either swallowed or flows into the amniotic sac. During periods of fetal apnea, the laryngeal dilator muscles become quiescent and adductor tone is usually present, resulting in raised resistance of the upper airway. Together with a lack of inspiratory muscle activity, this results in low rates of fluid efflux from the trachea, although increased efflux may occur with fetal postural adjustments or other activities, including those resembling straining movements or Valsalva maneuvers that are common in the fetus. The fetal upper airway plays a critical role in regulating the flow of fluid to and from the lungs, thereby preserving lung volume and the composition of liquid within the airways. Removal of the influence of the upper airway by creating a tracheo-amniotic bypass results in a major loss of lung liquid during periods of apnea and a large ingress of amniotic fluid during FBM episodes. This loss of upper airway function not only allows near total lung deflation during periods of fetal apnea, it also allows large influxes of amniotic fluid during FBM episodes. This brings the alveolar epithelium into contact with undiluted amniotic fluid with potentially harmful effects, especially if the amniotic fluid contains meconium.

Although the net flow of liquid within the fetal trachea is away from the lungs, primarily due to continuous liquid secretion, periods of influx may occur, especially in association with periods of vigorous FBMs. This may explain the entry of substances into the lungs following their deposition in the amniotic fluid.

Effects of FBMs on Fetal Blood Flows As FBMs alter intrathoracic pressure, it is to be expected that they will affect blood flows within the fetus. FBM-related changes in blood flows have been observed in the umbilical vein, fetal vena cava, foramen ovale and, more recently, in the pulmonary artery. In the pulmonary artery, episodes of vigorous FBMs are associated with increased blood flow, likely to be a consequence of reduced resistance in the pulmonary capillaries as a result of an altered transmural pressure.

Functional Importance of FBMs A major function of FBMs is to maintain lung liquid volume, and hence lung expansion, which is known to be essential for normal growth and structural maturation of the fetal lung. This role of FBMs has been demonstrated in experimental models that have eliminated FBMs while preserving the integrity of the diaphragm. The long-term abolition or suppression of normal FBM results in lung hypoplasia and structural immaturity of the lungs, which is probably due to a chronic reduction in lung expansion rather than abolition of the small phasic movements of the chest wall. A chronic reduction in fetal lung expansion leads to a reduction in lung tissue growth and alterations in the structure of the alveolar wall and epithelium; similar changes in lung development are caused by the abolition of FBMs. Abolishing the diaphragmatic movements that cause FBM is thought to reduce the force that normally opposes the inherent elastic recoil of the lungs, thereby allowing the fetal lungs to ‘deflate’; a further reduction occurs if the resistance offered by the upper airway is removed (Figure 2).

Use of FBMs in Clinical Assessment In the healthy human fetus, FBMs occur at an average frequency of 60 per min and are accompanied by increased body movements and heart rate variability. FBMs are first detectable at about 10–12 weeks of gestation when they are usually irregular and sporadic. From about 28 weeks of gestation onwards, FBMs become more regular and organized

Lung volume (% of intact fetus)

244 BREATHING / Fetal Breathing

100 80 60 40 20 0

Intact fetus

No No FBMs Collapsed FRC FBMs & lung newborn no UA

Figure 2 Lung luminal volumes in fetal and neonatal sheep, expressed in relation to values in the intact, late gestation ovine fetus in utero. Lung liquid volume, and hence lung expansion, is reduced by the prolonged absence of FBMs (no FBMs) induced by phrenic nerve blockade or a high section of cervical spinal cord. A further reduction occurs if the fetal upper airway is bypassed, allowing direct continuity between the fetal lungs and the amniotic sac (no FBMs & no UA). Lung luminal volume is reduced further when the lungs are removed from the fetus (collapsed lung) due to unopposed recoil of the fluid-filled lung. Functional residual capacity (FRC) in the air-breathing newborn is shown for comparison. Data from Harding R and Hooper SB (1996) Regulation of lung expansion and lung growth before birth. Journal of Applied Physiology 81: 209–224.

into discrete episodes. Using color Doppler and spectral ultrasonography analyses, the breath-to-breath interval and the inspiratory phase of the respiratory cycle have been shown to increase from 22 to 35 weeks gestation, but then decrease towards term. FBMs are used as one component of the ‘fetal biophysical profile’ which is widely used to assess fetal health; the occurrence of FBMs is observed, together with assessments of fetal heart rate, amniotic fluid volume, fetal body movements, and fetal body dimensions. If FBMs are not detected, the fetus may be hypoxic, may have a neural disorder affecting the brainstem and phrenic motor nerves, or it may have an abnormality of skeletal muscle function. However, it must be recognized that the inhibition of FBMs by chronic fetal hypoxia may be only transient, as has been shown in sheep (Figure 1). FBMs have also proven to be useful for the diagnosis of intrauterine infection in patients with preterm rupture of membranes as they have a high negative predictive value for intra-amniotic infection; a depression in FBM incidence is non-specific but is suggestive of infection. Studies of patients with premature rupture of membranes have shown a decrease in FBM incidence in those patients positive for amniotic fluid infection, clinical chorioamnionitis, or neonatal sepsis. This may be related to inflammatory cytokines affecting fetal behavioral states.

BREATHING / Fetal Breathing 245

Fetal Breathing, Amniotic Fluid Volume, and Lung Growth There is conflicting evidence on the role of FBMs in the development of pulmonary hypoplasia in human fetuses with preterm rupture of membranes and reduced amniotic fluid volume (oligohydramnios). Studies examining the role of FBM in the lung hypoplasia associated with premature rupture of membranes have shown either no change or a reduction in the incidence of FBM. However it is likely that both the oligohydramnios following membrane rupture and a reduced incidence of FBMs may contribute to the associated fetal lung hypoplasia. In humans, it has been shown that a prolonged reduction in FBMs at critical periods of development (i.e., o24 weeks of gestation) can lead to pulmonary hypoplasia; similarly, pulmonary hypoplasia can develop in human fetuses after prolonged (more than 6 days) membrane rupture. It is likely that oligohydramnios leads to lung hypoplasia due to increased flexion of the fetal trunk, which compresses the fetal lungs causing their deflation and reduced growth rates.

Effects of Labor on FBMs At term, the incidence of FBMs decreases. In sheep, the incidence of FBMs decreases 2–3 days before the onset of labor and remains reduced until delivery. This may be related to increased circulating concentrations of prostaglandin E2, which is known to inhibit FBMs. Similarly, in humans, fetal apnea of 20–60 min duration is considered to be a reliable indicator of premature labor with delivery within 48–72 h. During spontaneous or induced labor, the incidence of FBMs is decreased to less than 10% of the time during the latent phase and is further decreased during the active phase of labor. In human pregnancy, the rupture of fetal membranes at term does not appear to affect FBMs. However, a study before term showed a significant reduction in FBMs for the first 2 weeks of membrane rupture, compared to controls, with a return to near normal by the third week. With increased uterine contractility there is a decrease in the incidence of FBMs.

Maternal Smoking and Fetal Breathing As with most drugs taken by the mother, tobacco smoking has been shown to affect fetal behavior. Nicotine readily crosses the placenta, and is thought to reduce utero-placental and/or umbilico-placental blood flow, contributing to fetal hypoxemia; furthermore, the formation of carboxyhemoglobin as a result

of maternal smoking reduces the oxygen carrying capacity of fetal blood. FBMs are inhibited by maternal tobacco smoking, and the inhibition may continue for up to an hour following a single cigarette. It is thought that this effect is due mostly to fetal hypoxemia, or more specifically to reduced cerebral oxygen delivery. Maternal smoking has recently been shown to increase the resistance in the fetal cerebral artery, and this may also contribute to reduced cerebral oxygen delivery. The inhibition of FBMs by maternal smoking may contribute to the known adverse effects of smoking on fetal lung development. The same may apply to other drugs taken by the mother, such as narcotics, sedatives, or analgesics.

Respiratory Transition at Birth The physiological mechanisms underlying the transition from discontinuous fetal breathing to continuous postnatal breathing are complex and not fully understood. During parturition and when the umbilical cord is cut at birth, the neonate may become profoundly hypoxemic, hypercapnic, and acidemic. It is also exposed to lower environmental temperatures, leading to increased heat loss, and to a greatly increased degree of external sensory stimuli, thereby changing the behavioral state to one of arousal. It is also possible that the removal of circulating inhibitory or suppressive substances that originate in the placenta (e.g., prostaglandin E2, adenosine, neuroactive progesterone metabolites) may contribute to the onset of continuous breathing. For example, their removal may lead to an increase in metabolic activity (e.g., by stimulating thermogenesis), and hence an increase in the rate of CO2 production, or to an increase in the sensitivity of central chemoreceptors to CO2. It has also been suggested that endocrine changes at birth, such as a large increase in circulating catecholamines, lead to respiratory stimulation, possibly via an increase in fetal metabolism and hence CO2 production. Studies of fetal sheep maintained ex utero by extracorporeal oxygenation also support the notion that CO2 plays a crucial role in the maintenance of continuous breathing after birth. The integrity of the vagus nerves has been shown to be essential for the onset of adequate breathing at birth. Although the critical pathways have not yet been identified, it is likely that volume receptive feedback from the lungs is involved. Studies in neonatal lambs have shown that a reduction in end-expiratory lung volume (functional residual capacity, FRC), which reduces vagal sensory traffic from pulmonary stretch receptors, results in profound hypoventilation, periodic breathing, and active glottic adduction during periods of apnea. This indicates

246 BREATHING / Fetal Lung Liquid

that receptive vagal feedback of lung volume at endexpiration, which is normally maintained by an adequate FRC, is essential for continuous breathing in the newborn, and explains, at least in part, the benefits of positive end-expiratory pressure (PEEP) in the treatment of infantile apnea. See also: Breathing: Fetal Lung Liquid. Lung Development: Overview.

stretch stimulus that is essential for normal fetal lung growth. If the distending influence of fetal lung liquid is absent, the lungs fail to grow, which is the primary mechanism for fetal lung hypoplasia in humans. At birth, the airways are cleared of liquid to allow the entry of air and the onset of air breathing, due to a reversal of the transepithelial ionic gradient that promotes reabsorption into the lung parenchyma; this is stimulated by the release of stressrelated hormones during labor. Increases in transpulmonary pressure, due to changes in fetal posture, are likely to also contribute to the clearance of fetal lung liquid at birth.

Further Reading

Description

Bissonnette JM (2000) Mechanisms regulating hypoxic respiratory depression during fetal and postnatal life. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 278: R1391–R1400. Bocking AD (2003) Assessment of fetal heart rate and fetal movements in detecting oxygen deprivation in-utero. European Journal of Obstetrics and Gynecology 110: S108–S112. Champagnat J and Fortin G (1997) Primordial respiratory-like rhythm generation in the vertebrate embryo. Trends in Neuroscience 20: 119–124. Cosmi EV, Anceschi MM, Cosmi E, et al. (2003) Ultrasonographic patterns of fetal breathing movements in normal pregnancy. International Journal of Gynaecology and Obstetrics 80: 285–290. Harding R (1997) Fetal breathing movements. In: Crystal RG, West JB, Weibel ER, et al. (eds.) The Lung, Scientific Foundations, pp. 2093–2103. Philadelphia: Lippincott-Raven. Harding R and Bocking AD (2001) Fetal Growth and Development. Cambridge: Cambridge University Press. Harding R and Hooper SB (1996) Regulation of lung expansion and lung growth before birth. Journal of Applied Physiology 81: 209–224. Hooper SB and Harding R (1990) Changes in lung liquid dynamics induced by prolonged fetal hypoxemia. Journal of Applied Physiology 69: 127–135. Laudy JA and Wladimiroff JW (2000) The fetal lung. 1: Developmental aspects. Ultrasound in Obstetrics and Gynecology 16: 284–290. Rigatto H (1996) Regulation of fetal breathing. Reproduction, Fertility and Development 8: 23–33.

Throughout embryonic and fetal development, the future airways of the lung are filled with liquid and the lungs take no part in gas exchange. This liquid (fetal lung liquid) is a unique secretory product of the lung and is quite unlike fetal plasma or amniotic fluid in composition; it has a low pH, a high Cl concentration, and low protein content. Fetal lung liquid is secreted across the pulmonary epithelium and leaves the lungs by flowing out of the trachea.

Fetal Lung Liquid S B Hooper and R Harding, Monash University, Melbourne, VIC, Australia & 2006 Elsevier Ltd. All rights reserved.

Abstract Fetal lung liquid is secreted across the pulmonary epithelium and enters the future airspaces due to an osmotic gradient created by the transepithelial flux of ions into the lung lumen. This liquid leaves the lungs by flowing out of the trachea, whereby it is either swallowed or contributed to amniotic fluid volume. The high resistance to liquid movement through the fetal upper airway promotes the retention of lung liquid within the future airways, which provides a small (1 or 2 mmHg) internal distending pressure on the lungs. This acts as an internal hydrostatic splint that maintains the fetal lungs in a distended state and provides a

Control of Fetal Lung Liquid Secretion

Fetal lung liquid is formed by the net movement of Cl and Na þ across the epithelium into the lumen, which provides an osmotic gradient for the movement of water in the same direction. This mechanism is thought to be driven by Na þ /K þ ATPase, which creates an electrochemical gradient for Na þ to enter epithelial cells via the Na þ /K þ /2Cl cotransporter. The resultant Na þ -linked Cl entry across the basolateral membrane increases intracellular Cl concentrations that passively exit the cell, down its electrochemical gradient, via selective channels located in the apical surface. The net movement of Cl into the lung lumen generates a small transepithelial potential difference (lumen negative) that also promotes the movement of Na þ . As a result, water moves down an osmotic gradient generated by the net movement of Na þ and Cl (Figure 1). However, water movement across the epithelium must also depend on the intraluminal hydrostatic pressure existing, which will oppose its movement into the lung lumen. At rest, a small hydrostatic distending pressure (1 or 2 mmHg above ambient pressure) is present within the lung lumen and, therefore, the secretion of lung liquid must normally occur against a small hydrostatic pressure. When intraluminal pressures are increased above 5 or 6 mmHg, lung liquid secretion ceases, indicating that the osmotic pressure driving lung liquid can be counterbalanced by a hydrostatic pressure of 5 or 6 mmHg. On the other hand, when the fetal lungs are partially deflated and the intraluminal hydrostatic pressure is reduced, fetal lung liquid secretion rates increase.

BREATHING / Fetal Lung Liquid 247

Interstitial space

Epithelial cell

Lung lumen

H2O

Na+

K+

2Cl− Cl−

2K+ 3Na+ Na+

Figure 1 The proposed mechanism for fetal lung secretion across the pulmonary epithelium. Na þ /K þ ATPase, located on the basolateral surface of pulmonary epithelial cells, provides the free energy for Na þ to enter the cell via the Na þ /K þ /2Cl cotransporter, which promotes the entry of Cl against its electrochemical gradient. Cl exits the cell across the apical membrane down its electrochemical gradient, which causes a transepithelial potential difference (lumen negative) that promotes the movement of Na þ into the lung lumen. The combined net flux of Na þ and Cl into the lung lumen provides an osmotic gradient for water to flow in the same direction.

Although the mechanisms for fetal lung liquid secretion are relatively clear, the precise cellular origin is less clear. Early in gestation most epithelial cells are likely to contribute, but later in gestation the most likely candidates are the epithelial cells residing in the smaller, terminal airways because they constitute the vast majority (o90%) of the internal surface area of the lung. This is further supported by the finding that fetal bronchial epithelial cells predominantly absorb Na þ , whereas cultured fetal type II epithelial cells exhibit similar ionic fluxes as that described in the intact fetal lung. However, the relative contribution of type I and type II cells to fetal lung liquid secretion is unknown. Control of Fetal Lung Liquid Volume

Fetal lung liquid exits the lung by flowing out of the trachea, whereby it is either swallowed or enters the amniotic sac. The factors regulating liquid movement within the fetal airways, particularly during late gestation, are complex but primarily involve the

transpulmonary pressure gradient, the degree of lung recoil, and the resistance to liquid efflux offered by the fetal glottis and upper airway. During apnea, when fetal breathing movements (FBMs) are absent, active adduction of the glottis increases the resistance to lung liquid efflux and promotes its retention within the future airways. This promotes lung expansion, and the resultant increase in lung recoil generates the small intraluminal hydrostatic pressure (1 or 2 mmHg above ambient pressure) that characterizes fetal apneic periods. During FBMs, phasic abduction of the glottis greatly reduces the resistance to lung liquid efflux via the trachea and, therefore, the liquid leaves the lungs at a greater rate; usually two or three times the rate observed during apnea (Figure 2). Individual FBMs are essentially isovolumetric because the chest wall partially collapses when the diaphragm contracts, giving rise to small changes in thoracic volume with each movement. This is mainly due to the high viscosity of lung liquid, compared with air, which greatly increases the resistance to fluid movement through the fetal airways, particularly at high flow rates. Because the fetal chest wall is very compliant, it is unable to sustain the transpulmonary pressure gradients required to move liquid at flow rates equivalent to the postnatal flow of air. As a result, the tidal volume in the fetus is very small (o0.3 ml kg 1) compared to that of the newborn (7–10 ml kg 1). The transpulmonary pressure gradient is a major determinant of fetal lung liquid volume and is influenced by a number of factors, particularly fetal posture. For example, when intrauterine space is limiting, which can occur in the absence of amniotic fluid (oligohydramnios), the fetus is forced into a flexed position that greatly increases the curvature of the thoracoabdominal spine. This chronically increases abdominal pressure, elevates the diaphragm, and increases the transpulmonary pressure gradient leading to a decrease in lung liquid volume. The resultant decrease in lung expansion is considered to be the primary mechanism for the lung hypoplasia induced by oligohydramnios. Other changes in fetal posture (e.g., stretching), resulting in a decrease in spinal curvature, explain the influxes of lung liquid that are occasionally observed during periods of FBMs. Because fetal body movements most commonly occur during FBMs, reductions in transpulmonary pressure associated with these movements would promote the influx of lung liquid when upper airway resistance is low. Fetal Lung Liquid Clearance at Birth

Fetal lung liquid must be removed rapidly at birth to allow the onset of air breathing, and failure to do so

248 BREATHING / Fetal Lung Liquid Glottis adducted High resistance to liquid efflux

Dilated glottis Low resistance to liquid efflux

0 mmHg

Intraluminal distending pressure 1–2 mmHg

Lung liquid

Intraluminal pressure ∼ 0 mmHg

Lung liquid

Figure 2 Control of fetal lung liquid volumes during periods of apnea and FBMs. During apnea, the glottis is actively adducted, which restricts the efflux of lung liquid and promotes the accumulation within the future airways, thereby maintaining an intraluminal distending pressure of 1 or 2 mmHg above ambient pressure (amniotic sac pressure). During periods of FBMs, the glottis phasically dilates, which greatly reduces the resistance to lung liquid efflux. As a result, the liquid leaves the lungs at a higher rate, causing a reduction in lung liquid volume and the distending pressure (at end-expiration) reduces to ambient pressure.

is a major cause of respiratory morbidity in newborn infants. In particular, it is relatively common in preterm infants and near-term infants delivered by cesarean section in the absence of labor. Thus, the processes responsible for lung liquid clearance at birth mature relatively late in gestation, and labor plays a major role in activating and/or facilitating these processes. One mechanism for lung liquid clearance at birth results from a reversal of the osmotic gradient driving liquid secretion. This process is initiated by a cAMP-mediated activation of amiloride-inhibitable Na þ channels, located on the apical surface of lung epithelial cells, resulting in an increase in Na þ entry into the cell. This increases Na/K ATPase activity, located within the basolateral membrane, resulting in increased Na þ and Na þ -linked Cl flux across the epithelium, from lung lumen to interstitium. This reverses the osmotic gradient and promotes the uptake of lung liquid into the interstitial tissue, from where it is gradually cleared via the circulation and lymphatics. The increase in intracellular cAMP levels is thought to be due to an epinephrine-mediated increase in b-adrenoceptor activity because epinephrine is a potent stimulator of lung liquid reabsorption in late gestation. The rigors of labor, particularly during delivery of the head, induce a major stress

response within the fetus, leading to a large increase in a number of stress-related hormones, including epinephrine and vasopressin. Because vasopressin can also stimulate lung liquid reabsorption, it is likely that a number of stress-related hormones act through the same pathway. The ability of epinephrine to induce lung liquid reabsorption matures late in gestation and is mediated by the prepartum increase in circulating glucocorticoids (perhaps in synergy with thyroid hormones), which also mature many other aspects of the lung. This explains why preterm infants commonly suffer from liquid retention within the pulmonary airways after birth and explains one of the many beneficial effects that antenatal glucocorticoids, administered to women at risk of preterm labor, have on neonatal respiratory outcome in preterm infants. The maturational effect of glucocorticoids on lung liquid reabsorption involves an increase in the response of epithelial cells to epinephrine as well as an increase in the intracellular machinery responsible for lung liquid secretion/ reabsorption; this includes Na/K ATPase activity as well as Na channel expression. Although liquid uptake across the epithelium is a major mechanism for clearing liquid from the terminal airways, it is unlikely to be the only mechanism.

BREATHING / Fetal Lung Liquid 249

The maximum reabsorption rates (8–10 ml h 1 kg 1) demonstrated experimentally cannot account for the large volume of liquid that must be cleared, particularly because this process does not begin until delivery of the head. Similarly, the measured increase in lung tissue water content at birth cannot account for the total volume of liquid that is cleared from the airways. Although it was once assumed that lung liquid was forced from the airways due to compression of the chest as it passed through the birth canal, this theory has been discounted because the head and shoulders offer the widest obstacle for human delivery. However, because transpulmonary pressure is a major factor regulating the volume of fetal lung liquid, the effects of labor on fetal posture are likely to significantly contribute to liquid loss from the airways. Reduced amniotic fluid volumes, particularly following rupture of the membranes, as well as uterine contractions during labor, are known to cause posture-related increases in transpulmonary pressure and lung liquid loss. This may explain the large loss of liquid that occurs after labor has begun but before (by many hours) second-stage labor commences and delivery of the head begins (i.e., when stress-related hormones are released).

The Role of Fetal Lung Liquid in Development and Disease Fetal lung liquid plays a major role in the growth and development of the fetal lung by acting as an internal splint that maintains the lungs in an expanded state. The luminal volume of the fetal lung is higher than the end-expiratory lung volume (functional residual capacity) of the postnatal air-filled lung. The decrease in resting lung volume at birth (by 5–15 ml kg 1) is due to the removal of the distending influence of lung liquid in addition to the entry of air into the airways. The latter causes an air/liquid interface to form, which creates surface tension and, despite the presence of surfactant, increases lung recoil. This causes partial collapse of the lung and the formation of a subatmospheric intrapleural pressure after birth, which does not exist before birth. During fetal life, the distending influence of the lung liquid provides a stretch stimulus that is essential for lung growth and determines the three-dimensional tissue structure of the lung as well as the differentiated state of alveolar epithelial cells. If the distending influence of lung liquid is removed, growth and structural development of the lung ceases, particularly that of the terminal air sacs, and alveolar epithelial cells differentiate into the surfactant secreting type II cell phenotype. On the

other hand, overdistension of the fetal lungs induces a rapid increase in lung growth that can result in a near doubling in lung cell number within 7 days. Similarly, structural development accelerates and alveolar epithelial cells differentiate into type I cells following prolonged overdistension, which can reduce the proportion of type II alveolar epithelial cells to o2% of all alveolar epithelial cells. Because lung growth and development are very sensitive to the degree of fetal lung expansion, it is not surprising that most instances of fetal lung hypoplasia in humans are caused by conditions that reduce fetal lung expansion. These conditions include oligohydraminos, whether it is induced by premature rupture of the membranes or urinary tract abnormalities (which reduce or prevent the flow of fetal urine into the amniotic sac), congenital diaphragmatic hernia, or other space-occupying thoracic lesions (e.g., tumors and cysts). As indicated previously, oligohydramnios causes an increase in fetal trunk flexion, which increases the transpulmonary pressure gradient and reduces the degree of lung expansion. Herniation of the diaphragm allows abdominal contents to migrate into the fetal thorax, which acts as a space-occupying lesion that prevents lung expansion. In both conditions, the reduction in lung growth can be so severe that, after birth, the resulting respiratory insufficiency is fatal. The presence of lung liquid within the fetal airways may also affect the pulmonary circulation. Studies indicate that the distending influence of lung liquid, and the small hydrostatic pressure it generates, may contribute to the high pulmonary vascular resistance (PVR) in the fetus. As a result of the high PVR, B88% of right ventricular output bypasses the fetal lungs and enters the systemic circulation via the ductus arteriosus, which connects the main pulmonary artery with the descending aorta. With the clearance of lung liquid and the onset of ventilation at birth, PVR markedly decreases, allowing a rapid increase in pulmonary blood flow, to accept the entire output of the right ventricle, and the ductus arteriosus to close. The mechanisms responsible for the high PVR near term and the sudden decrease at birth are multifactorial. However, evidence indicates that the small hydrostatic distending pressure, caused by the retention of liquid within the future airways, may be a major contributing factor by causing compression and closure of perialveolar capillaries. Loss of this distending pressure at birth and the increase in lung recoil may contribute to the rapid decline in PVR when air first enters the lungs. See also: Breathing: Breathing in the Newborn; Fetal Breathing; First Breath. Lung Development: Overview.

250 BREATHING / First Breath

Further Reading Barker PM and Olver RE (2002) Invited review: clearance of lung liquid during the perinatal period. Journal of Applied Physiology 93: 1542–1548. Bland RD and Nielson DW (1992) Developmental changes in lung epithelial ion transport and liquid movement. Annual Review of Physiology 54: 373–394. Harding R and Hooper SB (1996) Regulation of lung expansion and lung growth before birth. Journal of Applied Physiology 81: 209–224. Harding R and Hooper SB (2004) Physiologic mechanisms of normal and altered lung growth. In: Polin RA, Fox WW, and Abman SH (eds.) Fetal and Neonatal Physiology, pp. 802–811. Philadelphia: Saunders. Hooper SB and Harding R (1995) Fetal lung liquid: a major determinant of the growth and functional development of the fetal lung. Clinical and Experimental Pharmacology and Physiology 22: 235–247. Olver RE, Walters DV, and Wilson M (2004) Developmental regulation of lung liquid transport. Annual Review of Physiology 66: 77–101.

attempts to separate the first breath from the breathing-like activities preceding birth. In fact, it is wellknown that the fetus presents intermittent breathing from at least the second trimester of gestation (see Breathing: Fetal Breathing). Breathing-like movements in utero are not for gas exchange, which is provided by the placenta, but are important in controlling the pulmonary fluid and, with it, fetal lung growth. In egg-laying animals such as birds, the definition of first breath is blurry because, even before hatching, breathing contributes to gas exchange, in conjunction with that provided by the chorioallantoic membrane. Small marsupials are another special case. In fact, these newborn mammals do not need to ventilate the lungs at birth because gas exchange occurs by diffusion through their skin. Hence, in these neonates, breathing acts occur sparsely, seemingly randomly, for several days after birth.

Physiological Processes

First Breath

Mechanics of the Onset of Breathing

J P Mortola, McGill University, Montreal, QC, Canada

The fetal lung is filled by fluid of its own production. At birth, some of the fluid is squeezed out of the upper airways during the passage through the pelvic canal. However, this mechanism is not crucial because differences in the mode of delivery (by breech or head, by cesarean section or vaginal) have no major impact on the cardiopulmonary adjustments at birth. Indeed, in many species with large litters, the mode of delivery alternates among pups, and in marine mammals delivery by tail first is the norm. What is important, though, is the occurrence of labor because the hormonal surge accompanying this phase of delivery is responsible for the switch from the prenatal production of the fetal pulmonary fluid to its postnatal absorption. Quantitative analyses of the mechanical events accompanying the first breath are based on observations and measurements performed in infants and, to a lesser extent, in lambs; very little information is available on other species. The first inspiration does not accomplish an immediate and homogeneous aeration of the lungs. In fact, although some alveolar areas pop open, many others will be ventilated only several minutes or even hours later. The work that the inspiratory muscles need to produce to expand the lungs with air is quite high, probably 8–10 times more than the respiratory work required during quiet breathing at older ages or in adults. The distortion of the thorax during inspiration, favored by the high compliance of the chest wall, is an additional factor that increases the total work of the inspiratory muscles.


Abstract In mammals and other vertebrates, intermittent breathing activities originate early during development; however, the term ‘first breath’ is used to indicate the first breathing act of a new independent organism. Usually, this coincides with the establishment of continuous pulmonary ventilation, although this is not always the case. What triggers the onset of regular breathing at birth remains an unsolved question. The general state of stress, created by all the new stimuli and events surrounding the time of birth, and the sudden increase in oxygenation, raises metabolic rate. The gaseous component of metabolism could be the common mechanism for the establishment of a sustained ventilation after birth. The respiratory work of the first inspiration is high because of the friction of the column of pulmonary fluid as it is displaced toward the peripheral airways. The formation of the air–liquid interface generates surface pressure, which is the primary determinant of the recoil pressure of the lungs. Lung expansion with air and the rise in oxygenation are the main factors responsible for the changes in pulmonary circulation at birth. The early breathing pattern is irregular, and the expiratory flows are briefly interrupted by closure of the vocal folds. This mechanism raises intra-airway pressure and contributes to the clearing of the pulmonary fluid.

Description First breath indicates the first breathing act of a new independent organism. When applied to mammals, it refers to the first active muscle effort aiming to pump air into the lungs at birth. The specifications ‘independent organism’ and ‘at birth’ are semantic

BREATHING / First Breath 251

Lung volume (ml) A few days

P ∝ /r

100

· P ∝ V ·R

50 First breath

−40

−30

−20

−10

0

10

20

30

40

Pleural pressure (cmH2O) Figure 1 Schematic representation of the changes in pleural pressure and lung volume during the first breath and during some of the following breaths (dashed lines) until a few days. The arrows indicate the direction of the loop, and the areas of the loop are an approximate indication of the external work of the respiratory muscles. (Left) The silhouette of the lungs indicates that during the first breath (bottom) of the total pressure (P ) required to generate flow (V ), the largest fraction is to overcome airflow resistance (R ). Later (top), the largest component of the inspiratory pressure is due to surface tension (g ) and the radius of curvature (r ) of the air–liquid interface.

The total inspiratory pressure is mainly contributed by the airflow resistance and by the surface pressure (Figure 1). This latter is determined by the product of the surface tension at the air–liquid interface and the radius of curvature of the interface. In the liquid-filled lung before the first breath, because there is no interface, the surface pressure is nil. The interface forms with the entrance of air during the first inspiration, and as the air progresses toward the small peripheral airways, the surface pressure continues to rise because the curvature of the interface continues to decrease. Eventually, after the first few breaths, the pressure determined by surface forces will be the primary reason for the tendency of the lungs to deflate. Surfactants produced by specialized cells of the alveolar epithelium play a fundamental role in lowering the surface tension and the propensity of the lungs to deflate. This reduction in lung recoil pressure is important for the progressive establishment of the end expiratory reserve of air, or functional residual capacity (see Surfactant: Overview). Indeed, the first breath contributes 10–20% to what will be the functional residual capacity in an infant a few days old. The airflow resistance of the first inspiration is high because of the viscosity of the column of pulmonary fluid. The incoming air displaces this fluid toward the lung peripheral airways. Eventually, the fluid will pass in the pulmonary interstitium, where it will be cleared by the pulmonary circulation and, to

a lesser extent, the lymphatics. The process of clearing the airways from the fluid takes a few hours in human infants and probably a longer period of time in larger species. During this time, air and fluid mix with the formation of foam, a process favored by the presence of surfactant. The air trapped in the pulmonary liquid in the form of foam contributes to gas exchange, and this explains why blood oxygenation rises rapidly in the minutes after birth, despite the much longer time required for the clearance of the fluid. Once the airways are liquid-free, the pressure to overcome airflow resistance is small. This is the main reason for the large decrease in the work of breathing of the following breaths compared to the first breath. Theoretically, at birth several mechanisms may help the newborn’s respiratory muscles in generating the large pressures required for the first breathing acts. For example, the contraction of the upper airway muscles, such as during sucking, or the outward recoil of the chest during recovery after the squeeze through the pelvic passage can produce subatmospheric pressures. Also, the erection of the pulmonary capillaries with the rise in pulmonary blood flow may contribute some negative (and therefore inspiratory) pressure within the airways. In reality, when subjected to experimental scrutiny, none of these mechanisms have demonstrated an appreciable contribution to the pressure required for the first inspirations.

252 BREATHING / First Breath Changes in Pulmonary Circulation

Birth is accompanied by dramatic vascular changes. The lung shifts from receiving less than 10% of the cardiac output during fetal life to receiving practically all of it after birth. To a great extent, this change is due to the closure of the ductus arteriosus, which in fetal life permits a large fraction of the blood of the pulmonary trunk to bypass the pulmonary circulation, and to the drop in resistance of the pulmonary vessels (Figure 2). Because the ductus arteriosus enters the aortic arch, its postnatal closure reduces blood-flow to the descending aorta, in favor of the upper body and brain. The first breath and the onset of continuous ventilation play a crucial role in the cardiovascular changes accompanying birth, mostly because of two mechanisms – lung expansion and oxygenation. The increase in lung volume, by itself and independent from the rise in oxygen pressure, increases pulmonary blood-flow approximately fivefold, according to animal experiments. This may seem astonishing given that lung volume does not differ much before and after the first breath; in fact, the volume of the fetal liquid-filled lung is not lower, and most probably larger, than that of the air-filled postnatal lung. However, after birth, the surface tension created at the air–liquid interface reduces the pressure in the

lung interstitial tissue, which promotes vascular expansion and a decrease in pulmonary vascular resistance. Lung ventilation also promotes the secretion of pulmonary prostaglandins, with vasodilating effects. The importance of the rise in oxygen levels in lowering pulmonary vascular resistance has been appreciated for a long time. Sustained hypoxia delays this process, leading to right ventricular hypertrophy. It also seems that the respiratory alkalosis accompanying the onset and establishment of pulmonary ventilation contributes to the vascular adjustments at birth. The Early Breathing Pattern

Despite the mechanical constraints, in infants the first inspiration is approximately 40 ml deep – one of the deepest of the first few days (Figure 1). It is not clear why this is the case. Among the various possibilities is a change in the threshold of the mechanisms normally controlling tidal volume, notably the airway slowly adapting stretch receptors and the central integration of their inputs. Although the activity of these receptors is related to changes in lung volume, their stimulus is transpulmonary pressure. In utero, the lungs are liquid-filled; hence, the transpulmonary pressure is low because of the absence of surface forces. At birth, with the entrance of air,

Fetus

Newborn

Aorta

Aorta

Ductus arteriosus × 0.05 Pulmonary trunk

Right ventricle

Pulmonary ×7

Left ventricle

Right ventricle

Lungs

Lungs

Left atrium Right atrium Foramen ovale

Left ventricle

Left atrium

Descending aorta × 0.5

Figure 2 Schematic representations of the main features of cardiopulmonary circulation before birth (left) and after birth (right). Numbers indicate the approximate changes in blood flow occurring at birth as the result of lung expansion and oxygenation.

surface forces immediately increase transpulmonary pressure. Therefore, intrapulmonary airway receptors may have to reset to the new mechanical condition, and in the process, the control of tidal volume may not be as accurate as it will be in the following breaths. Peripheral chemoreceptors are also known to undergo resetting with the abrupt change in stimulus. In the case of peripheral chemoreceptors, the abrupt change at birth is caused by the rise in arterial oxygenation with the onset of ventilation. Another set of airway receptors, the rapidly adapting ones, may be involved in the deep inspiration of the first breath. These receptors are normally activated by a sudden lung stretch, and their reflex effect (called Head’s reflex) is that of promoting the activity of the inspiratory muscles. Hence, the increase in transpulmonary pressure with the first air expansion may provoke this reflex, resulting in further activation of the inspiratory muscles. During the minutes following birth, the breathing pattern is very irregular, and short apneas are interspersed with bursts of high-frequency and shallow breathing. Tachypnea, with average rates between 70 and 90 breaths per minute, has been observed after both vaginal and cesarean section deliveries, and it usually subsides within a few hours. In infants who have difficulty clearing pulmonary fluid, transient tachypnea can last much longer. It seems likely that the high-frequency pattern is the reflex response resulting from the stimulation of a group of receptors located in the pulmonary interstitium. These receptors are known to cause rapid and shallow breathing when activated by an increase in pulmonary interstitial pressure, such as during lung congestion. After the first breath, and for much of the following hours, during expiration the flow is decreased, and often briefly interrupted, by the narrowing of the vocal folds. Some widening and narrowing of the glottis with inspiration and expiration, respectively, are normal events during breathing. However, in the neonatal period narrowing is exaggerated, and in the first minutes after birth interruptions of expiration at various times are common (Figure 3). This expiratory pattern, or grunting, is controlled by neural information from the lungs and serves important purposes. By delaying expiration, it maintains the average air volume within the lungs higher than it would be with unobstructed expiration, and it raises the end expiratory level. Because it increases the intra-airway pressure, grunting contributes to drainage of pulmonary fluid from the peripheral airways into the lung interstitium, where it is picked up by the lymphatic circulation. This mechanism can be effective because the rise in intra-airway pressure is

Tidal volume inspiration

BREATHING / First Breath 253

Obstructed expirations

Unobstructed expiration

End-expiratory volume

Figure 3 Schematic representation of a breath with unobstructed expiration (blue) and of several types of obstructed expirations (red). In the unobstructed condition, the lung volume decreases during expiration along a quasi-exponential trajectory. In case of obstruction, the expiratory trajectory is distorted, lung volume takes longer to decrease, and the end expiratory volume rises.

small. If airway pressure reached higher values, as can occur during crying or other forced expiratory maneuvers or during assisted positive airway pressure ventilation, it would hinder the pulmonary circulation and aggravate gas exchange. What Triggers the Onset of Regular Breathing at Birth?

This question is still unanswered. Once outside the shielded intrauterine environment, the newborn is bombarded by many stimuli – visual, acoustic, thermal, tactile, and pressure – either totally new or of unusual intensity. Also, neural and chemical information present before birth assumes a new dimension after birth. For example, as air enters the lungs, the increase in transpulmonary pressure brings the airway slowly adapting receptors to a new level of activity. The arterial chemoreceptors, sensing a major increase in the partial pressure of oxygen, decrease or cease their prenatal activity; it will take a few days before they reset to the new oxygen level. The prenatal surge of many antioxidant enzymes testifies to the uniqueness of birth as a hyperoxic event. The relative contribution of so many stimuli and new conditions to the onset of ventilation and the establishment of a steady pattern is difficult to quantify. Various experimental approaches have emphasized or dismissed one or the other mechanism. It is worth noting that at birth, as a result of the multiple stimuli and of the general state of stress, the newborn’s metabolic rate rises much higher than the fetal value. Also, the level of the metabolically produced carbon dioxide increases, which may play a fundamental role in the maintenance of pulmonary ventilation. In animal fetuses exteriorized and kept alive with extracorporeal circulation to provide for their gas exchange, continuous breathing did not

254 BREATHING / First Breath

Birth

2nd

1st breath

3rd 4th 5th 6th

Metabolic level

Thermal, mechanical and sensory stimuli, neural and chemical inputs

Figure 4 Many new stimuli are produced during the events surrounding birth. Either individually or by creating a general state of stress, they increase the metabolic level, which may represent the common mechanism for the maintenance of a continuous breathing pattern. Sensory stimuli include tactile, pressure, and pain stimuli. Neural inputs refer to the facilitation originating from the airway rapidly adapting receptors, as well as to neurochemical substances that may be increased within the brainstem as a result of air-breathing. Chemical inputs refer to the putative role of the chemoreceptors and to the state of relative hyperoxia. The spirometric recording at the top is from a newborn infant delivered by cesarean section. Time bars of 1 s are plotted along the zero volume line. Starting from the first breath, the amount of air exhaled is less than the amount inhaled – a surfactant-dependent process that forms the functional residual capacity.

start even after umbilical cord occlusion as long as carbon dioxide was not allowed to rise. Hence, it is possible that the many stimuli thought to be involved in the onset of breathing do so, ultimately, by favoring the increase in metabolic rate (Figure 4). Also, the relative hyperoxic condition accompanying birth may cause increased metabolism since hyperoxia has a hypermetabolic effect in young animals. Metabolic rate plays a key role in controlling the level of pulmonary ventilation in many circumstances, ranging from muscle exercise to responses to cold or hypoxia. Therefore, its crucial involvement in the maintenance of continuous respiration at birth would not be a special case. Contrary to the rather abrupt onset of continuous breathing observed at birth in the majority of mammals, in birds and other egg-laying animals breathing commences gradually. At first, breathing is intermittent, and it becomes more regular and continuous during the many hours of the hatching process, in parallel with the progressive decline of the gas exchange function of the chorioallantoic membrane. This process is similar to that observed in some small marsupials, in which pulmonary ventilation becomes continuous over a period of several days, whereas gas exchange through the skin gradually regresses. These examples support the view that the establishment of continuous ventilation at birth is not necessarily an event timed with a particular stage of development. Rather, it may be so closely linked to gaseous

metabolism that as long as extrapulmonary organs take care of gas exchange, pulmonary ventilation does not need to become a continuous process. See also: Breathing: Breathing in the Newborn; Fetal Breathing. Lung Development: Overview. Oxidants and Antioxidants: Antioxidants, Enzymatic; Antioxidants, Nonenzymatic; Oxidants. Pulmonary Circulation. Signs of Respiratory Disease: Breathing Patterns. Surfactant: Overview.

Further Reading Bland RD (1997) Fetal lung liquid and its removal near birth. In: Crystals RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientific Foundation, 2nd edn., pp. 2115–2127. Philadelphia: Lippincott-Raven. Hodson WA (1997) The first breath. In: Crystals RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientific Foundation, 2nd edn., pp. 2105–2114. Philadelphia: LippincottRaven. Lagercrantz H and Slotkin TA (1986) The ‘stress’ of being born. Scientific American 254: 100–107. Mortola JP (2001) Respiratory Physiology of Newborn Mammals. A Comparative Perspective. Baltimore: Johns Hopkins University Press. Strang LB (1991) Fetal lung liquid: secretion and reabsorption. Physiology Review 71: 991–1016. Tod ML and Cassin S (1997) Fetal and neonatal pulmonary circulation. In: Crystals RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientific Foundation, 2nd edn., pp. 2129– 2139. Philadelphia: Lippincott-Raven.

BRONCHIAL CIRCULATION 255

BRONCHIAL CIRCULATION E M Wagner, Johns Hopkins Asthma and Allergy Center, Baltimore, MD, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The bronchial circulation is the systemic vascular supply to the lung, and it supplies blood to conducting airways down to the level of the terminal bronchioles as well as nerves, lymph nodes, visceral pleura, and the walls of large pulmonary vessels. Within the airway wall, the circulation is composed of parallel vascular plexuses adjacent to airway smooth muscle. The density of this vascular network predicts its role in the clearance of aerosols delivered to the airway mucosa. As in other systemic vascular beds, inflammatory cells are recruited to the airway wall through postcapillary venules. Inflammatory proteins are largely vasodilatory in their effect on bronchial vascular smooth muscle tone. Most vasodilatory action is mediated at least partially through endothelial cell-derived nitric oxide. Inflammatory proteins play a major role in the loss of bronchial endothelial barrier function causing airway wall edema, as demonstrated in conditions of asthma and allergy. The most prominent pathologic feature of the bronchial circulation is its proliferative capacity. Unlike the pulmonary vasculature, the bronchial circulation is pro-angiogenic in asthma, chronic pulmonary thromboembolism, interstitial pulmonary fibrosis, cystic fibrosis, and other inflammatory conditions. Although the recruitment of new bronchial vessels to ischemic lung parenchyma may prove beneficial, hemoptysis resulting from rupture of abnormal, bronchial vessels can be life-threatening.

Anatomy, Histology, and Structure The variability in origin and number of systemic arteries perfusing the airways both among and within species has contributed to the difficulty in study and the relative paucity of information concerning the physiological function of the bronchial vasculature. Leonardo da Vinci is frequently credited with providing the first anatomical drawings of the bronchial circulation. Although he understood the need for this circulation as described in his notebooks that ‘‘nature gave a vein and an artery to the trachea which would be sufficient for its life and nourishment,’’ reproduction of his efforts demonstrated that his drawings were those of pulmonary veins. The first complete anatomical drawings of the bronchial circulation in 1721 should be credited to the Dutch anatomist Frederich Ruysch. Since that time, the anatomical arrangement of the bronchial circulation has been described for humans, sheep, pigs, dogs, opossum, rabbits, and several small rodents. The bronchial vasculature originates from the aorta, subclavian, or

intercostal arteries (Figure 1). The bronchial artery courses to the dorsal aspect of the carina, where it bifurcates and sends branches down the mainstem bronchi. This vascular bed perfuses the airways from the level of the carina to the terminal bronchioles. The bronchial arteries send arterioles throughout the airway adventitia that perfuse capillaries that are prominent in both the adventitia and the mucosa of the airway wall. Thus, the bronchial vasculature forms parallel vascular plexuses, situated on either side of the airway smooth muscle. In extraparenchymal airways, bronchial venules drain into bronchial veins that subsequently drain into the right heart. In the intraparenchymal airways, postcapillary venules collect bronchial venous drainage into pulmonary venules and/or alveolar capillaries that drain into pulmonary veins and the left atrium. Bronchial anastomoses with pulmonary precapillary vessels have also been reported. Additionally, the bronchial artery sends branches to large pulmonary vessels as vasa vasorum, nerves, lymph nodes, and the visceral pleura. Values reported for absolute bronchial blood flow vary according to the method used to measure flow and the species evaluated. However, all reports demonstrate that flow through the bronchial vasculature represents less than 3% of cardiac output. The vasculature of the airway wall, which constitutes the major perfusion pathway, is tortuous and forms a dense network of vessels. Morphometry of postmortem or resected lung specimens from normal human subjects showed that blood vessels of the mucosa comprise o1% of the airway wall area and within the advential area approximately 8% of the wall area. Subjects with inflammatory airways disease show a significant increase in the overall number of airway vessels. However, several studies have suggested that the increase is proportionate to the increase in wall area. Few studies of the embryonic development of the bronchial vasculature exist. In humans, between weeks 9 and 12 of gestation, the bronchial artery arises as an outgrowth from the aorta. This process occurs later than the development of the pulmonary vasculature. One or two vessels extend from the dorsal aorta and form along the cartilaginous plates of the large airways. The vessels develop longitudinally along the airways to the lung periphery as far as the terminal bronchioles. However, the process by which the parallel vascular plexuses interdigitate and proliferate on either side of airway smooth

256 BRONCHIAL CIRCULATION

Aortic arch Bronchial artery

Carina

Descending aorta

Figure 1 The bronchial artery originates from the aorta and courses to the dorsal aspect of the carina, where it bifurcates and sends branches down the mainstem bronchi. This vascular bed perfuses the airways from the level of the carina to the terminal bronchioles. In extraparenchymal airways, bronchial venules drain into bronchial veins that subsequently drain into the right heart. In the intraparenchymal airways, postcapillary venules collect bronchial venous drainage into pulmonary venules and/or alveolar capillaries that drain into pulmonary veins and the left atrium. Additionally, the bronchial artery sends branches to the pulmonary artery as vasa vasorum, nerves, lymph nodes, and the visceral pleura.

muscle has not been studied. Whether the fine capillary networks within the airway wall, the vasa vasorum within large pulmonary vessel walls, or vessels anastomosing with pulmonary vessels form as a continual outgrowth of arterial sprouting or as de novo formation from mesoderm is unknown.

Table 1 Methods to measure bronchial blood flow Method

Species

Flow probe Microspheres Artery cannulated/perfused Isolated vascular pouch Anastomotic blood collection Inert gas

Sheep Dog, sheep Sheep Sheep Dog, sheep Human

Bronchial Circulation in Normal Lung Function Given the separate structures within the lung that are perfused by branches of the bronchial vasculature, it is likely that the bronchial circulation regulates and supports diverse physiologic functions. However, due to the difficulty in accessing this vascular bed in most species, its complex anatomy within the lung, and its small proportion of blood flow relative to pulmonary flow, the role of the bronchial circulation in supporting airway and lung function is incompletely described. Most published studies have focused on characterizing regulators of bronchial blood flow, with few directed at understanding function

(Tables 1 and 2). However, several specific physiological functions have been defined and supported by experimental evidence, including that of airway cell nutrition, clearance of inhaled substances, recruitment of inflammatory cells, and conditioning inspired air. The evidence for each of these functions is presented in the following sections. Airway Cell Nutrition

Although all vascular beds exist to support the metabolic needs of the tissue perfused, assessment of

BRONCHIAL CIRCULATION 257 Table 2 Bronchial blood flow responses Vasoconstrictors

Vasodilators

a-Adrenergic agonists Endothelin Glucocorticoids Increased airway pressure Increased left atrial pressure Vasopressin

b-Adrenergic agonists Adenosine Antigen Bradykinin Histamine Hypercarbia Hypoxia Nitric oxide Prostanoids

cellular function during acute changes in perfusion can provide suggestive evidence for the importance of maintaining a critical level of blood flow. Within the airway wall, it is questionable whether oxygen delivery by the blood is necessary given the proximity to airway luminal oxygen. In animal models, bronchial vascular smooth muscle demonstrated significant reduction in tone when exposed to hypoxemia and hypercarbia. Additionally, when the sheep bronchial artery was perfused with an artificial perfusate without calcium, a prompt reduction in airway smooth muscle tone was noted. In an effort to determine the importance of bronchial perfusion on mucociliary function in sheep, clearance of an insoluble tracer (99mTc-sulfur colloid) was determined. When bronchial blood flow was substantially reduced from a normal level, mucociliary activity was significantly slowed. Whether this response was due to changes in mucus gland production, ciliary function, or neural control mechanisms, each could be related to alterations in substrate delivery to specific cells of the airway wall supplied by the bronchial artery. These studies demonstrate the importance of normal bronchial perfusion to maintain diverse cell function within the airway. Clearance

The normal function of any vascular bed is to support the removal of excess metabolites from a tissue. In addition, the bronchial circulation is juxtaposed between the airway epithelial barrier to the external environment of inhaled substances and the internal environment of the bloodstream. This circulation could be important in the systemic uptake of soluble, inhaled substances. Studies have shown that both large increases or decreases in bronchial blood flow decreased the blood uptake of a soluble tracer (99mTc-DTPA) deposited on airway epithelial surface. Subsequent work demonstrated that increased flow accompanied by increased pressures caused a hydrostatic edema that limited soluble tracer uptake.

These studies demonstrate the complexity of the interaction between aerosol uptake and the underlying airway vasculature. Vasomotor and endothelial barrier changes of the airway vasculature can impact aerosol uptake of both therapeutic and toxicological substances. Several studies have shown that the level of blood flow through the bronchial circulation can affect both the magnitude and the time course of agonistinduced airway smooth muscle constriction by contributing to the passive washout of delivered agonist. Furthermore, it is interesting to note that most airway smooth muscle agonists cause bronchial vasodilation, thus suggesting a homeostatic mechanism for preserving normal airway smooth muscle tone. Another aspect of clearance by this vasculature is confirmed by the study of the metabolic capacity of the bronchial endothelium. Angiotensin-converting enzyme (ACE) inhibitor significantly depressed metabolism of a synthetic peptide substrate for ACE in an in situ perfused bronchial preparation. This study concluded that the bronchial circulation is pharmacokinetically and metabolically active with respect to bradykinin and that the enzymes responsible for this metabolic activity line the vascular lumen. Recent work confirmed the importance of ACE activity in vivo by demonstrating inhibition of bradykinin vasodilation after treatment with an ACE inhibitor. This result further suggests an important regulatory role for bronchial endothelial ACE in the metabolism of kinin peptides known to contribute to airway pathology. Each aspect of clearance, from inhaled substances removed from an active site causing physiologic changes to endothelial metabolism, suggests an important, albeit incompletely characterized, function. Recruitment of Inflammatory Cells

Recruitment of leukocytes to the airway provides an important defense function due to their antimicrobial, secretory, and phagocytic functions. Leukocyte recruitment in systemic organs involves an orchestrated series of molecular events between rolling leukocytes and postcapillary venular endothelium. Since the tracheal/bronchial circulations are systemic circulatory beds, the same endothelial cell adhesion molecules likely are responsible for the welldocumented leukocyte recruitment of inflammatory airways diseases. Although this is generally assumed, only recently have studies using intravital microscopy to document specific molecules and mechanisms of recruitment in vivo been performed in airways. Research suggests that ventilatory stresses imposed on airway endothelium may exert

258 BRONCHIAL CIRCULATION

additional cell stimulation that is not predicted by static endothelial cell culture conditions, thus confirming the molecules and mechanisms responsible for specific leukocyte recruitment in models that are relevant for airways disease appear to be essential. Conditioning Inspired Air

Typically ascribed to the airway circulation is the function of heating and humidifying inspired air. Both the tracheal and the bronchial circulations have been shown to dilate when healthy human subjects or animals are challenged with dry air hyperventilation. However, whether the airway circulation contributes significantly to heating and humidification has been debated because the pulmonary circulation provides a huge heat sink. In experimental systems in which bronchial perfusion could be limited, little effect on exhaled temperatures or on airway obstruction was observed. However, measurements of water exchange demonstrated that the bronchial vasculature served an important role in hydrating the airway mucosal surface and interstitial compartments of peribronchial tissues and limited the degree of airway mucosal injury after hyperpnea. Thus, it appears that the airway circulation is important in preserving normal humidification of airway cells.

Bronchial Circulation in Respiratory Diseases Proliferation

The unique proliferative capacity of the bronchial circulation compared to the pulmonary vasculature in a variety of lung diseases has long been recognized. Chronic inflammatory conditions and chronic pulmonary thromboembolism are the two most cited conditions leading to bronchial vascular proliferation. Inflammation can contribute significantly to bronchial vascular remodeling and life-threatening hemoptysis. Hemoptysis in the vast majority of patients originates from the systemic rather than the pulmonary vasculature, and the bronchial vessels are almost universally involved. Techniques for identifying and embolizing bronchial vessels have been well described. However, the etiology of hemoptysis requiring therapeutic embolization is variable, and the pathology of bronchial vessels leading to this acute condition is poorly understood. Several studies have focused on the chronic inflammation of asthma and airway vascular remodeling. Li and Wilson demonstrated increased vascular density in biopsy specimens of subjects with mild asthma compared to normal control volunteers. However, others have shown that only biopsy specimens from

asthmatic subjects with concurrent Mycoplasma pneumoniae infections demonstrated significantly increased vessel numbers. In studies of alterations in airway vascularity in models of inflammation, rodent models have shown increased vessel numbers, size, and permeability characteristics. Airway remodeling following Mycoplasma pulmonis infection showed sustained alterations in the tracheal vasculature that could be reversed with corticosteroid treatment. Additionally, tracheal vessels of rats with chronic M. pulmonis infection demonstrated significantly increased permeability when challenged with substance P. These results suggest abnormal endothelial cell barrier function after inflammationinduced proliferation. Perhaps the most extensively studied form of systemic vascular proliferation within the lung is that which occurs after pulmonary artery embolization. In 1847, Virchow recognized that the bronchial circulation could proliferate and sustain lung tissue distal to a pulmonary embolism. Bronchial arteriograms in patients with chronic thromboembolic disease demonstrate the unique capacity of systemic vessels to proliferate and to invade the ischemic lung parenchyma. Both a dilated bronchial artery and a fine meshwork of vessels distal to the pulmonary occlusion can be seen. Neovascularization of the systemic circulation into the lung after pulmonary artery obstruction has been confirmed and studied in humans, sheep, dog, pig, guinea pig, rat, and mouse. Systemic blood flow to the lung has been shown to increase as much as 30% compared to the original pulmonary blood flow after pulmonary artery occlusion. Although precise mechanisms of vascular proliferation are still under investigation, in a model of chronic pulmonary thromboembolism in the mouse, the pro-angiogenic CXC chemokines appear to play a major role in vascular proliferation. This observation further supports a growing body of evidence, reported by Strieter and colleagues, from studies of non-small cell lung cancer and idiopathic pulmonary fibrosis that the ELR þ CXC chemokines are important pro-angiogenic factors within the lung. Loss of Endothelial Barrier Function

Numerous studies on a variety of models, as well as autopsy specimens of human lungs, demonstrate the propensity for the systemic airway circulation to contribute to fluid accumulation within and around airways. In particular, inflammatory cytokines have been shown to cause the postcapillary venular endothelium to form gaps and allow transudation of plasma and protein into the interstitium. Changes in airway wall geometry due to fluid and inflammatory

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cell accumulation and vasodilation of the bronchial vasculature have contributed to a geometric theory of asthma. Whether mechanisms are related to physical encroachment on small airway lumen or the loss of parenchymal tethering, inflammationinduced changes involving bronchial vascular fluid exudates likely contribute to the pathology of asthma. Another means by which the bronchial circulation can alter barrier function within the lung is through a portal-like response. Several studies have demonstrated that inhalation of injurious chemicals can result in loss of pulmonary endothelial barrier function. Pulmonary vascular leak due to smoke inhalation has been shown to be attenuated when the bronchial vasculature is occluded. Authors of separate studies concluded that inflammatory proteins released within the large airways after smoke inhalation are carried by the bronchial vasculature to pulmonary tissue, where they impact pulmonary endothelial barrier function. However, these results are in contrast to those of Pearse and colleagues, who showed that bronchial perfusion attenuated the loss of pulmonary endothelial barrier function in ischemia–reperfusion. These authors suggested that bronchial endothelial-derived nitric oxide helps to preserve pulmonary barrier function. However, in each of these models, the bronchial vasculature serves as a conduit to transport substances to the pulmonary circulation where function is altered.

Bronchial Infections

See also: Angiogenesis, Angiogenic Growth Factors and Development Factors. Asthma: Exercise-Induced. Bronchodilators: Beta Agonists. Chemokines, CXC: IL-8. Cilia and Mucociliary Clearance. Endothelial Cells and Endothelium. Fluid Balance in the Lung. Leukocytes: Neutrophils. Lung Development: Overview. Neonatal Circulation. Pulmonary Thromboembolism: Deep Venous Thrombosis; Pulmonary Emboli and Pulmonary Infarcts.

Further Reading Butler J (ed.) (1992) The bronchial circulation. In: Lung Biology in Health and Disease. New York: Dekker. Cudkowicz L (1968) The Human Bronchial Circulation in Health and Disease. Baltimore: Williams & Wilkins. Deffebach ME, Charan NB, Lakshminarayan S, and Butler J (1987) The bronchial circulation – small, but a vital attribute of the lung. American Review of Respiratory Disease 135: 463–481. Wagner EM (1995) The role of the tracheobronchial circulation in aerosol clearance. Journal of Aerosol Medicine 8(1): 1–5. Wagner EM (1997) Bronchial circulation. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientific Foundations, pp. 1093–1105. New York: Lippincott–Raven. Wagner EM (1997) Bronchial vessels. In: Barnes PJ, Grunstein MM, Leff AR, and Woolcock AJ (eds.) Asthma. New York: Lippincott–Raven. Wanner A (1989) Circulation of the airway mucosa. Journal of Applied Physiology 67: 917–925. Widdicombe J (ed.) (2003) Archives of Physiology and Biochemistry. Eighth International Meeting of the DaVinci Society, Nimes, France, pp. 287–368.

see Bronchiectasis. Bronchiolitis. Panbronchiolitis.

BRONCHIECTASIS R Wilson, Royal Brompton and Harefield NHS Trust, London, UK R Boyton, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Bronchiectasis is abnormal chronic dilatation of one or more bronchi due to loss of structural elements in the bronchial wall. There are a number of known causes and several associated conditions, but the underlying cause is not known in many cases. The prevalence is unknown, but thin-section computed tomography has increased its recognition. Symptoms are of chronic productive cough, breathlessness, recurrent exacerbations usually provoked by infection, and tiredness. There may be crackles over affected areas but not in all cases, and there may be wheezes due to airflow obstruction that is largely irreversible. Subsegmental

airways are permanently dilated, tortuous, and contain excess secretions. Characteristically, the elastin layer of the wall is deficient, and muscle and cartilage show signs of destruction. Increased mucus production and impaired clearance predispose to bacterial infection, which stimulates chronic inflammation in which lymphocytes predominate within the wall and neutrophils in the lumen. The inflammation causes more damage, which in turn further impairs the lung defenses, leading to more infection and a vicious cycle of events. Treatment involves daily physiotherapy to drain affected areas and appropriate antibiotics to treat infection and thus reduce inflammation.

Introduction Bronchiectasis is defined as abnormal chronic dilatation of one or more bronchi. This structural abnormality predisposes the bronchi to bacterial

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infection. The most common symptoms are chronic cough and sputum production. Recurrent bronchial infections occur that may become chronic, causing daily purulent sputum production. Cases of cystic or saccular bronchiectasis, in which severe loss of bronchial wall structure leads to large balloon-like dilatations, are now infrequently seen in developed countries. This type of bronchiectasis often followed severe lung infections, usually in childhood, and was characterized by production of very large volumes of sputum and finger clubbing. Improved socioeconomic conditions, vaccination programs, and ready availability of antibiotics are responsible for the reduced incidence of cystic or saccular bronchiectasis. Much more common today is a cylindrical form in which the damage to the bronchial wall is less severe. This type of bronchiectasis, which is usually bilateral and may be diffuse, although the lower lobes are usually worst affected, has been called ‘modern’ bronchiectasis. In varicose bronchiectasis, there are localized constrictions caused by scarring superimposed on cylindrical changes. Traction bronchiectasis occurs in fibrotic lung conditions such as fibrosing alveolitis, in which the airway walls are pulled apart by the fibrotic process. Interestingly, in these cases the bronchiectasis is usually asymptomatic, probably because the mucosa is unaffected. The current prevalence of bronchiectasis is unknown. Published data from older studies were based on chest radiographs, which have been shown to be very insensitive for detecting bronchiectasis. The availability of thin-section, high-resolution computed tomography (CT), which is a much easier investigation to perform than the old ‘gold standard’ of bronchography, has led to increased recognition in patients who might otherwise have been undiagnosed or classified as having chronic bronchitis or asthma. CT has enabled a diagnosis of bronchiectasis to be made in much milder cases, and this must be kept in mind when comparing older studies to those published recently. Indeed, this can cause confusion in terminology because some patients with asthma or smoking-related chronic bronchitis have bronchiectatic airways by CT criteria. A high prevalence of bronchiectasis has been reported in specific ethnic groups (e.g., Native Americans in North America, New Zealand Maoris, and Western Samoans). However, it is unclear whether genetic predisposition, environmental factors, or social practices are responsible. Most patients with bronchiectasis are nonsmokers. This may be because a history of childhood respiratory problems is common, and consequently starting smoking is less likely, but it could also be influenced by referral practice because a smoker presenting with a chronic

productive cough is more likely to be advised on smoking cessation rather than be referred for investigation.

Etiology There are a number of known causes of bronchiectasis (Table 1) and several other conditions that have been associated with bronchiectasis (Table 2) without a clear understanding of the pathological link. Congenital

Several types of congenital bronchiectasis occur because of the absence or deficiency of elements of the bronchial wall that are crucial to the retention of its normal architecture. In Williams–Campbell syndrome, there is no or much reduced cartilage in the bronchi, generally extending beyond the first division. Congenital tracheobronchomegaly (Munier– Kuhn syndrome) is another example of this type, and bronchiectasis can occur in Marfan’s and Ehlers– Danlos syndromes. Patients with intralobar pulmonary sequestration have blind-ending bronchi that Table 1 Causes of bronchiectasis Congenital (e.g., deficiency of structural elements of the bronchial wall) Infective (e.g., tuberculosis, whooping cough, or nontuberculous mycobacteria) Mechanical obstruction within lumen (e.g., inhaled foreign body) or external compression (e.g., tuberculous lymph node) Deficient immune response (e.g., common variable immune deficiency or human immunodeficiency virus) Inflammatory pneumonitis (e.g., aspiration of gastric contents or inhalation of toxic gases) Excessive immune response (e.g., allergic bronchopulmonary aspergillosis or lung transplant rejection) Abnormal mucus clearance (e.g., primary ciliary dyskinesia, cystic fibrosis, or young’s syndrome) Fibrosis (e.g., fibrosing alveolitis, sarcoidosis, or radiation pneumonitis)

Table 2 Conditions associated with bronchiectasis Infertility (e.g., primary ciliary dyskinesia, cystic fibrosis, or Young’s syndrome) Inflammatory bowel disease (e.g., ulcerative colitis, Crohn’s disease, or coeliac disease) Connective tissue disorders (e.g., rheumatoid arthritis, systemic lupus erythematosus, or Sjogren’s disease) Malignancy (e.g., acute or chronic lymphatic leukemia) Diffuse panbronchiolitis, predominantly reported in Japan Yellow nail syndrome I dystrophic and colored (usually yellow) nails, lymphedema, and pleural effusions a1-Antiproteinase deficiency, usually causes emphysema Mercury poisoning may cause Young’s syndrome (obstructive azoospermia, sinusitis, and bronchiectasis)

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trap mucus and are prone to repeated infections that lead to bronchiectasis. Postinfective

A severe pneumonia of any sort can damage the bronchial wall sufficiently to leave residual bronchiectasis. Mycobacterium tuberculosis, Bordetella pertussis, and measles virus are particularly likely to cause damage, but the diagnosis can be difficult to make because the patient’s history of the illness is vague. Chronic cough and sputum production should follow the severe lung infection, and bronchiectasis should be confined to the affected area. Some patients give a history of childhood pneumonia when they present in adult life with bronchiectasis, but they have diffuse disease on CT and have had an asymptomatic period in between. The importance of the childhood history in such cases is uncertain, but it may be that the earlier event provides a starting point for later problems if a bronchial infection occurs and is not cleared from the damaged area. Bronchiectasis is a common but not universal finding in Sawyer–James (Macleod’s) syndrome. This condition is a form of obliterative bronchiolitis that most commonly follows an infectious insult to the developing lung during the first 8 years of life. There is unilateral hyperlucent, hypovascular lung with marked air trapping. Nontuberculous mycobacteria, particularly Mycobacterium avium complex, can cause bronchiectasis. This type of infection seems to occur particularly in middle-aged females who present with a long history of dry cough. Mechanical Obstruction

Localized bronchiectasis can occur as a result of bronchial obstruction from any cause, and the obstruction can either be in the lumen of the airway (e.g., inhaled foreign body) or due to compression from outside (e.g., tuberculous lymph node at the origin of the right middle lobe bronchus). The obstruction leads sequentially to impaired mucus clearance, distal infection, chronic inflammation, damage to the bronchial wall, and bronchiectasis. Middle lobe syndrome, which involves recurrent acute infections at this site, has been described in cases without evidence of obstruction. It may be that the anatomy of the right middle lobe, which is long and acutely angulated with a collar of lymph nodes at its origin, is the reason. Immune Deficiency

Children with severe immune deficiencies do not usually survive, although a proportion develop

bronchiectasis before they die. Bronchiectasis is a frequent complication of the rare X-linked hypogammaglobulinemia, in which there is complete absence of the B-lymphocyte system. Common variable hypogammaglobulinemia, in which there is a reduction in the amount of antibody present in one or more of the major classes, is much more commonly found when investigating an adult population with bronchiectasis. These patients will often give a history of acute severe respiratory infections (pneumonia) in addition to their chronic symptoms. Solitary IgA deficiency occurs in approximately 0.2% of the population and can be present without any clinical problems, although some patients suffer frequent viral-like illnesses. IgG subclass deficiency may be transient and can also occur in healthy subjects. More important is functional antibody deficiency, which involves failure to mount an antibody response to polysaccharide antigens (e.g., Streptococcus pneumoniae capsular antigen). This is associated with low IgG-2 levels, low pre-immunization pneumococcal antibody levels, and failure to mount a sustained antibody response to pneumococcal vaccination. An acquired immunodeficient state can occur secondary to multiple myeloma, chronic lymphatic leukemia, protein-losing enteropathy, nephrotic syndrome, or lymphoma. Bronchiectasis can also complicate HIV infection, and this may be seen more frequently now that patients are surviving much longer with antiretroviral therapy. Inflammatory Pneumonitis

Inhalation of toxic gases, such as ammonia or smoke by victims caught in fires, and aspiration of gastric contents can cause sufficient damage to lead to bronchiectasis. Acid reflux in patients with hiatus hernia can cause cough and wheeze, and may aggravate bronchiectasis symptoms, but is unlikely to be the sole cause of the disease. Excessive Immune Response

Bronchiectasis in patients with allergic bronchopulmonary aspergillosis (ABPA) typically affects the proximal bronchial tree and the upper lobes, although more generalized disease is not uncommon. It is caused by an immune reaction involving eosinophils and fungus colonizing the airways. Atelectasis occurs because of obstruction by plugs of inspissated secretions containing fungal hyphae. Acute episodes of fever, wheeze, expectoration of viscid sputum plugs, and pleuritic pain, sometimes associated with consolidation in different areas on the chest radiograph, merge insidiously over time as bronchiectasis develops into chronic purulent sputum production,

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where exacerbations of ABPA are difficult to distinguish from infective exacerbations of bronchiectasis. Some disorders causing constrictive obliterative bronchiolitis also damage the large airways. This pathology is seen in lung transplant rejection, chronic graft versus host disease, rheumatoid arthritis, and after inhalation of toxic gases. Abnormal Mucociliary Clearance

Three different forms of impaired mucociliary clearance result in bronchiectasis: primary ciliary dyskinesia (Kartagener’s syndrome), cystic fibrosis (CF), and Young’s syndrome. Forms of infertility are associated with all three. Primary ciliary dyskinesia is rare and thought to be an autosomal recessive condition with incomplete penetrance involving numerous genes. There are usually, but not always, ultrastructural abnormalities in the cilia that make them move in a slow, disordered manner or the cilia may be completely static. Most commonly, there is the absence of one or both dynein arms (Figure 1), but abnormalities of the microtubules or radial spokes have been described. In cases with normal ultrastructure, the abnormality may be beyond the resolution of the electron microscope or may involve the orientation of the cilia on the cell surface. Cilia

line the nose, paranasal sinuses, and bronchi as far as the respiratory bronchioles and also form the tails of spermatozoa. Impaired ciliary function occurs at all these sites, and diffuse bronchiectasis is usually associated with chronic sinusitis, middle-ear disease, and often, but not invariably, male infertility. Females may have some reduction in fertility, and an increased risk of ectopic pregnancy, due to abnormal ciliary movement in the fallopian tubes. The condition may present in the neonatal period with pneumonia or segmental collapse due to mucus impaction or in childhood with recurrent infections. Patients cough continuously because this is their only form of mucus clearance. Approximately 50% of cases have dextracardia and a smaller proportion full situs inversus, which is thought to be due to abnormal cellular microtubules that are involved in rotation of organs in the embryo. Kartagener’s syndrome is bronchiectasis, chronic sinusitis, and dextracardia named after the German pediatrician who first described it. Young’s syndrome is bronchiectasis, chronic sinusitis, and azoospermia due to a functional blockage of the sperm in the caput epididymis, which is usually enlarged and palpable in the scrotum. The cause is unknown, and it may occur later in life after successful parenthood. The respiratory secretions are viscid, making ciliary clearance inefficient. The Outer arm Inner arm

Normal cilium

}

Dynein

Nexin link

Radial spoke

Cell membrane

Figure 1 (Top) Diagram of normal ultrastructure of a cilium and (bottom) electron micrograph of an abnormal cilium from a patient with primary ciliary dyskinesia that has no dynein arms.

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syndrome has been linked to mercury poisoning in childhood (pink disease), and its incidence has decreased since calomel (mercurous chloride) was removed from teething powders and worm medication. A Young’s syndrome equivalent is recognized in women who had mercury poisoning in childhood. CF is caused by mutation of a gene on the long arm of chromosome 7 that codes for the CF transmembrane conductance regulator (CFTR), a cyclic-AMPdependent chloride channel that has wide ranging effects, including hydration of the airways. Most cases present in childhood and are associated with severe disease. However, late presentation has been described with atypical CF alleles, some of which are associated with milder bronchiectasis, normal pancreatic function, normal sweat electrolytes, and male fertility. Upper lobe bronchiectasis and culture of Staphylococcus aureus are clues in this type of patient, and CF genotyping should be performed in suspected cases. Fibrosis

Traction bronchiectasis is common in the CT scans of many patients with all forms of pulmonary fibrosis, but this is rarely symptomatic. When symptomatic bronchiectasis does occur, it is most common in association with rheumatoid arthritis. In these cases, there may be an immunological explanation, or bacterial infection may establish itself within damaged lung, particularly if patients are immunosuppressed or if bronchial architecture is distorted. Infection causes neutrophilic airway inflammation and mucus hypersecretion that can become self-perpetuating if the infection is not eradicated. Associated Conditions

Pulmonary involvement is a frequent extraarticular manifestation of rheumatoid arthritis, and bronchiectasis may occur in the absence of interstitial lung disease. Airflow obstruction and constrictive obliterative bronchiolitis may be associated with the bronchiectasis. Other connective tissue diseases, including Sjogren’s syndrome and systemic lupus erythematosus, have varied pulmonary manifestations, including bronchiectasis. Bronchiectasis is associated with ulcerative colitis, Crohn’s disease, and celiac disease. The association of bowel and lung disease may be related to an underlying immune system dysregulation, with cells migrating between both sites and exposure of both epithelia to common environmental antigens and shared epithelial antigens. The association with ulcerative colitis is best established and two presentations are recognized. First, patients with a history

of severe colitis eventually undergo colectomy and then develop abrupt onset of cough and sputum production soon afterwards. Second, patients with one condition develop the other several years later. In the latter patients, flare ups of their bowel disease may or may not be associated with exacerbations in their lungs. Bronchiectasis associated with ulcerative colitis is varied in severity, but in some cases it is associated with florid diffuse neutrophilic airway inflammation and very large volumes of purulent sputum, which is often sterile. Another feature is that bronchiectasis regresses with anti-inflammatory medication, which contradicts the concept that bronchiectasis is always an irreversible condition. Onset of chronic cough and sputum production has also been linked to bowel resection in Crohn’s disease. Diffuse panbronchiolitis is a chronic airway condition first described in Japan, where it seems to be much more common. Patients present in middle age with a productive cough and breathlessness. The condition has subsequently been found in Korea and China, and occasional Caucasian patients with a similar syndrome have been described. There is inflammatory hypertrophy of the walls of the respiratory bronchioles, which involves infiltration with plasma cells, lymphocytes, and foamy cells. If the condition is untreated, it progresses to bronchiectasis. There is a remarkable response to treatment with long-term low-dose erythromycin, which has led to the investigation of macrolide antibiotic treatment for other forms of bronchiectasis. Alpha-1-antiproteinase deficiency predisposes to emphysema, particularly in cigarette smokers. Sometimes, bronchiectasis is also present, and occasionally it is the predominant pathology. There is a proteinase–antiproteinase imbalance in the airways of all patients with bronchiectasis, and possibly these patients were predisposed to airway as well as alveolar problems following an infection that caused initial airway damage. Yellow nail syndrome is a rare condition involving yellow discoloration of dystrophic nails, primary lymphedema, and pleural effusions. Chronic rhinosinusitis is common, and a significant proportion of patients have bronchiectasis. Idiopathic Bronchiectasis

Even at centers that perform a full set of investigations into possible causes of bronchiectasis, approximately half of patients remain idiopathic. There is a predominance of females in this group and also in many cases a history of wheezy bronchitis in childhood. This is followed by a period of good health before the onset of chronic cough and sputum

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production in adult life, often in their 20s or 30s. Patients may describe the onset of their problems as a severe ‘viral-like’ illness that went into their chest and did not resolve. Patients usually have chronic rhinosinusitis, which suggests that whatever abnormality is predisposing to the illness is present throughout the respiratory tract. These patients suffer from profound tiredness and difficulty concentrating, much more so than patients with bronchiectasis due to a postinfective cause or primary ciliary dyskinesia. This condition may involve a dysregulated innate and/or adaptive immune response.

Pathology In parts of the lung with bronchiectasis, subsegmental airways are permanently dilated, tortuous, and partially or totally obstructed by copious amounts of secretions. Side branches of the airways are frequently obliterated. Structural proteins are lost from the bronchial wall, and there is a variable amount of fibrosis. Characteristically, the elastin layer of the wall is deficient or absent, and the muscle and cartilage layers show signs of destruction. These changes weaken the wall and lead to the distortion of the normal architecture. The process often involves bronchioles, and long-standing obstruction may result in complete fibrosis of small airways. The airway epithelium is damaged and ciliated cells are lost. There is goblet cell hyperplasia and mucus gland hypertrophy. There may be peribronchial pneumonic changes with evidence of parenchymal damage. The pulmonary arteries may thrombose and can recanalize. With long-standing disease there is hypertrophy of the bronchial arteries with anastamosis and sometimes considerable shunting of blood to the pulmonary arteries. There is usually chronic inflammation, in which lymphocytes predominate in the bronchial wall and neutrophils in the lumen. The walls of bronchi and bronchioles contain lymphoid follicles and nodes containing B lymphocytes, CD4 þ and CD8 þ T lymphocytes, and mature macrophages.

Bacteriology The bacterial species isolated in bronchiectasis are listed in Table 3. Mixed infections are common. Bronchiectatic airways are often chronically colonized, and for long periods a balance may be struck in which the bacteria colonize the mucosal surface without stimulating a systemic inflammatory response. Patients carry the same strain for long periods, and acquisition of a new strain is not necessarily associated with an exacerbation. The species

Table 3 Bacteria isolated from sputum of patients with bronchiectasis Common Hemophilus influenzae Hemophilus parainfluenzae Pseudomonas aeruginosa Less common Streptococcus pneumoniae Moraxella catarrhalis Stenotrophomonas maltophilia Staphylococcus aureus Mycobacterium species Other Gram-negative bacilli

of bacteria and their concentration in the lumen are both important determinants of the level of inflammation. Host factors and the presence of additional viral infection will also influence when an exacerbation occurs, and it may be a combination of events that upsets the balance and provokes an exacerbation. This may lead to invasion of the mucosa and peribronchial pneumonia. The etiology and severity of the bronchiectasis influence the type of bacterial infection. For example, S. aureus is associated with CF and ABPA, and Pseudomonas aeruginosa is associated with extensive lung disease and severe airflow obstruction. The initial infection with P. aeruginosa is usually a nonmucoid strain, which becomes a mucoid phenotype when chronic infection occurs. More than any other species, P. aeruginosa is likely to establish chronic infection, and once this has occurred it usually persists for the rest of the patient’s life. However, compared to CF, there is less evidence that this is associated with more rapid disease progression. Patients with chronic P. aeruginosa infection do have worse quality of life, and this may be due to several factors: their underlying disease is worse; treatment is more often required in hospital for intravenous antibiotics because ciprofloxacin is the only active oral antibiotic; because of antibiotic resistance, treatment is less effective and therefore exacerbations occur more frequently; and long-term antibiotic prophylaxis is used more frequently. Tuberculosis is a rare complication of bronchiectasis, but when it does occur the classic clinical and radiological features may not be present. A high index of suspicion should be maintained in patients presenting with persistent low-grade fever, weight loss, or hemoptysis. Environmental nontuberculous mycobacterial species are sometimes isolated from bronchiectatic sputum. This may be a ‘one-off’ isolate indicating environmental exposure just before the sputum sample was obtained. The mycobacterium may establish itself in the airway without apparently affecting the patients’ condition. However,

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long-term follow-up is required to ensure this is true colonization and not low-grade infection. Some species are more pathogenic and therefore more likely to require treatment (e.g., M. avium complex and M. kansasii). These species can cause infection in patients without pre-existing lung disease or demonstrable immune deficiency. The infection may cause bronchiectasis, and in such cases it can be difficult to determine if the infection is primary or secondary. When there is a combination in a CT scan of mild to moderate cylindrical bronchiectasis and peripheral nodules, some of which may be cavitating, sputum samples should be sent for mycobacterial microscopy and culture.

Clinical Features Symptoms

The most common symptoms are chronic cough and sputum production. Exacerbations are caused by bronchial infections, which may be viral or bacterial or both, or there may be chronic bacterial infection causing daily purulent sputum production. Usually, an exacerbation is associated with increased sputum volume and purulence, although the volume can decrease because the sputum becomes more viscous and therefore difficult to clear. Low-grade temperature is common, whereas a high temperature is suspicious of pneumonia. Chronic rhinosinusitis is very common, and most patients have expiratory airflow obstruction, the severity of which correlates with the severity of bronchiectasis. This is usually largely irreversible, although some patients do have an asthmatic component. Chest pains are common and are usually described as an ache, but they may be pleuritic during exacerbations. Hemoptysis is usually minor and complicates an exacerbation; serious hemoptysis requiring selective embolization or surgery is rare, probably because of the ready availability of antibiotics and because cystic bronchiectasis is less common. Undue tiredness and difficulty concentrating occur in poorly controlled disease. Physical Signs

There may be coarse inspiratory crackles heard over the site of bronchiectasis, but sometimes there are no signs to suggest the diagnosis. Airflow obstruction can cause wheezes, and there may be late inspiratory squeaks indicating small airway disease. Clubbing is unusual in ‘modern’ bronchiectasis because it is associated with more severe disease. A 24 h sputum collection can be very informative because patients tend to be inaccurate in their description of the color and volume of what they produce.

Investigations

These are listed in Table 4. Suspicion of bronchiectasis should lead to a high-resolution thin-section (e.g., fast scan time of 1 s or less, 1 mm sections, and 10-mm spacing) CT scan. Investigation may discover a treatable cause of bronchiectasis. Younger patients, those with associated conditions, and those in whom respiratory function is deteriorating and/or infective exacerbations are becoming more frequent or prolonged should be seen by a respiratory physician with a special interest in bronchiectasis who has access to all the investigations listed in Table 4. Chest radiographs are an insensitive test for bronchiectasis; in one study, they detected less than half of patients who subsequently had positive bronchography. The CT findings are related to the presence of dilated air-filled bronchi, dilated secretion-filled bronchi, and loss of volume resulting from parenchymal loss, which leads to crowding of the bronchi. The appearance depends on the orientation of the bronchi to the scanning plane. When they lie in the same plane, they appear as tram lines that do not decrease (taper) in diameter in the usual way as they progress to the outside of the lung (Figure 2(a)). The easiest way to determine whether a bronchus is dilated is to compare it to the adjacent pulmonary artery. Dilated bronchi that are perpendicular to the Table 4 Investigation of bronchiectasis All patients Chest radiograph PA and lateral Sinus radiograph Lung function tests High-resolution thin-section CT scan Sputum microscopy to determine cell type (neutrophils or eosinophils) Sputum culture and sensitivities Sputum smear and culture for acid fast bacilli Skin tests (atopy, aspergillus) Sweat test, nasal potentials, CF genotyping Cilia studies (if nasal mucociliary clearance is prolonged or nasal nitric oxide is low, proceed to light microscopy of ciliary beat frequency and then electron microscopy) Blood investigationsa Selected patients Fiberoptic bronchoscopy Videofluoroscopy of swallowing to detect aspiration Ph study for acid reflux Semen analysis Tests for associated conditions Blood tests for rarer immune deficiencies a Full blood count with differential white cell count; C-reactive protein; erythrocyte sedimentation rate; total immunoglobulin (Ig) levels of IgG, IgM, IgA, and IgE and specific antibodies to pneumococcus and tetanus antigens; aspergillus radioallergosorbent test (IgE) and precipitins (IgG); rheumatoid factor; protein electrophoretic strip; and a1-antiproteinase.

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Figure 2 High-resolution thin-section computed tomography of bronchiectasis demonstrating (a) a nontapering bronchus, (b) the signet ring sign, (c) tree-in-bud pattern, and (d) mosaic perfusion.

scanning plane have a circular appearance, and then the smaller pulmonary artery gives a ‘signet ring’ appearance (Figure 2(b)). Mucus-filled bronchi appear as branching tubes or nodules, and many filled small airways (exudative bronchiolitis) give a ‘treein-bud’ appearance (Figure 2(c)). End-expiratory scans exaggerate increased transradiency in areas where gas trapping has occurred because of obstruction of small airways. Since gas trapping is usually patchy, this gives a pattern of mosaic perfusion (Figure 2(d)). Peripheral blood inflammatory markers (neutrophil count and C-reactive protein) and sputum appearance can be used to monitor the response of an exacerbation to treatment. Most patients with bronchiectasis require long-term follow-up because disease progression can occur insidiously or in a stepwise rather than gradual manner. A history of more frequent and prolonged exacerbations, not responding well to treatment, is usually associated with an increase in sputum volume and purulence, increased breathlessness, as well as increased tiredness. CT scan features associated with subsequent disease progression are bronchial wall thickening, mucus plugging, and tree-in-bud pattern. Lung function may show a decrease in FEV1 and gas transfer. These changes are reversible, but mosaic perfusion is not. Health status questionnaires, such as the St George’s Respiratory Questionnaire, and measures of exercise capacity, such as the shuttle walking test, can also be used to monitor the patient’s progress. A

deterioration should lead to a review of sputum bacteriology (including mycobacteria) and aspergillus serology, as well as physiotherapy practice, before considering a change to the management regimen. P. aeruginosa is often isolated for the first time in these circumstances, but it may reflect the patient’s poor condition rather than be the cause of it.

Pathogenesis Excess mucus is produced in the dilated, tortuous bronchiectatic airways. The mucus is poorly cleared partly because of the abnormal anatomy but also because cilia are lost when the epithelium is damaged, and the mucus is less elastic and more viscous largely due to its inflammatory cell DNA content. Bacteria adhere avidly to the stationary mucus and multiply so that they are often present in very high concentrations. Large numbers of neutrophils are attracted into the bronchial lumen from the circulation by chemotactic products of the bacteria and also mediators released by host cells (e.g., interleukin (IL)-8, C5a, and leukotriene B4). Serum levels of adhesion molecules are elevated, suggesting that endothelial activation also occurs in the lung. Activated neutrophils spill proteolytic enzymes and reactive oxygen species while trafficking through the lung and during phagocytosis. These may overwhelm the body’s ability to neutralize them and cause tissue damage in the affected area. The exuberant inflammation contains infection within the

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Impaired host defences

Tissue damage

Microbial infection

agar beads to establish chronic infection after they are instilled into the airway, but a considerable amount of pneumonia also occurs. Another model achieved the same result by partially ligating a bronchus and instilling bacteria distally, but this was technically difficult. Future work may utilize candidate genes to generate inducible transgenic animal models that mimic the abnormal inflammatory responses seen in the lung in bronchiectasis.

Management and Therapy Surgical Treatment Inflammation

Figure 3 A vicious cycle of bacterial infection-driven, host inflammation-mediated lung tissue damage in bronchiectasis.

lung, so bacteremia and spread of infection outside the lung are very rare. However, the inflammation may fail to eradicate the infection due to impaired local host defenses, so the infection becomes chronic, and it may slowly spread to involve adjacent normal bystander airways. Immune complexes form between bacterial antigens and antibodies that are produced locally and arrive by transudation. These stimulate other inflammatory processes. Epithelial cells, lymphocytes, and macrophages release cytokines and other factors that orchestrate and perpetuate the inflammation. Bronchiectatic secretions contain large amounts of IL-8, IL-1a, IL-1b, tumor necrosis factor-a, IL-6, and granulocyte colony-stimulating factor. The lung defenses are impaired by the damage caused directly by bacterial products and by inflammation, which in turn promotes continued infection. This has been termed a ‘vicious cycle’ of events (Figure 3). Bronchiectasis may occur because of an acute insult to the bronchial wall (e.g., whooping cough in a young child or inhalation of a toxic gas), and the damaged area may subsequently become infected. In other cases, there may be a recognized deficiency of the host defenses that renders the patient prone to infection (e.g., primary ciliary dyskinesia or common variable immune deficiency). However, in many cases the starting point of the vicious cycle may be obscure. It seems more likely that as yet unknown deficiencies in the host’s immune defense system, or an abnormality in the body’s ability to control the inflammatory response, will explain these cases, and cases in which bronchiectasis progresses rapidly, rather than the direct effect of a virulent infectious process. There are no satisfactory animal models of bronchiectasis. A rat model uses bacteria embedded in

The only curative treatment of bronchiectasis is surgical resection. Surgery is only appropriate when the bronchiectasis is localized and there is no underlying condition that predisposes to generalized bronchiectasis, such as primary ciliary dyskinesia. Good results are obtained from resection of severe localized bronchiectasis when the bronchiectatic area contributes little to lung function, which is otherwise reasonably preserved. Modern cylindrical bronchiectasis is usually bilateral, and surgery is rarely considered for this reason. Palliative surgical resection may be considered if a localized area of severe bronchiectasis defies medical management and acts as a sump for infection of other areas, even if less severe bronchiectasis is present elsewhere. Lung transplantation (usually two lungs or heart–lung) is used successfully to treat respiratory failure due to bronchiectasis. Medical Treatment

Patients with daily sputum production must perform postural drainage at least once daily and do this two or three times daily during exacerbations. They are taught to adopt the correct position to drain affected areas and to clear mucus by controlled breathing techniques sometimes aided by chest percussion. Patients need to be reminded about and encouraged to perform physiotherapy, and their technique should be regularly reviewed. Physical exercise should also be encouraged because it aids mucus clearance. A number of conditions that are recognized as causes or associations of bronchiectasis may require specific treatment (e.g., hypogammaglobulinemia and allergic bronchopulmonary aspergillosis). In patients with an asthmatic component, inhaled bronchodilators and corticosteroids are used in the usual way, and beta-agonists are often used to improve mucociliary clearance. However, there is little evidence to support the widespread use of inhaled corticosteroids in the absence of asthma. If this approach is used, then careful measure of objective benefit should be sought (e.g., lung function). Short

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courses of systemic corticosteroids may be used during severe exacerbations. Patients with chronic respiratory failure require long-term oxygen treatment. Carbon dioxide retention is sometimes a problem. Nasal intermittent positive-pressure ventilation is effective in these cases and surprisingly well tolerated despite a history of sinusitis. This approach is also effective in assisting mucus clearance. Antibiotics are given during infective exacerbations associated with purulent sputum, breathlessness, and malaise. The choice of antibiotic is influenced by the likely bacterial species (Table 3), (previous) sputum culture results, and the presence or absence of P. aeruginosa. As a general rule, because of the damaged airways and the level of infection, the antibiotic dosage has to be higher and the course longer than usual. The amount of bacterial resistance is also high due to previous antibiotic prescription (e.g., b-lactamase production in Hemophilus influenzae). Intravenous antibiotics are used for severe exacerbations, resistant species, and when oral antibiotics have failed. Continuous antibiotic treatment can be given as prophylaxis by the oral or nebulized route or as regular planned courses of intravenous treatment. This improves patient well-being, probably by reducing the frequency of exacerbations and the number of bacteria in the lung (and thus the level of inflammation). However, there is no evidence that it alters disease progression, and there is a definite risk that it will drive the lung flora toward more resistant strains and species, such as P. aeruginosa and Stenotrophomonas maltophilia. This approach should be reserved for patients who, despite optimum medical management, continue to have very frequent (more than six per year) exacerbations, some of which involve admission to the hospital. There has been great interest in the past few years in macrolide antibiotic prophylaxis. The benefit is thought not to be due to their action of killing bacteria but via effects on inflammatory cells and possibly mucus production. Several studies have

provided encouraging results, and this is a promising area of future research. See also: Bronchiolitis. Bronchomalacia and Tracheomalacia. Cilia and Mucociliary Clearance. Cystic Fibrosis: Overview; Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene. Defense Systems. Immunoglobulins. Panbronchiolitis. Pneumonia: Mycobacterial. Primary Ciliary Dyskinesia.

Further Reading Amsden GW (2005) Anti-inflammatory effects of macrolides – an underappreciated benefit in the treatment of community-acquired respiratory tract infections and chronic inflammatory pulmonary conditions? Journal of Antimicrobial Chemotherapy 55: 10–21. Barker AF (2002) Bronchiectasis. New England Journal of Medicine 346: 1383–1393. Bush A, Cole PJ, Hariri M, et al. (1998) Primary ciliary dyskinesia – diagnosis and standards of care. European Respiratory Journal 12: 982–988. Evans D, Bara AI, and Greenstone M (2005) Prolonged Antibiotics for Purulent Bronchiectasis, the Cochrane Collaboration. New York: Wiley. Hansell DM (1998) Imaging of obstructive pulmonary disease. Bronchiectasis Radiology Clinics of North America 36: 107–128. Hermaszewski RA and Webster ADB (1993) Primary hypogammaglobulinaemia: a survey of clinical manifestations and complications. Quarterly Journal of Medicine 86: 31–42. Homma H, Yamanake A, Tanimoto S, et al. (1983) Diffuse panbronchiolitis. A disease of the transitional zone of the lung. Chest 83: 63–69. Mukhopadhyay S, Singh M, Cater JI, et al. (1996) Nebulised antipseudomonal antibiotic therapy in cystic fibrosis: a metaanalysis of benefits and risks. Thorax 51: 364–368. Pasteur MC, Helliwell SM, Houghton SJ, et al. (2000) An investigation into causative factors in patients with bronchiectasis. American Journal of Respiratory and Critical Care Medicine 162: 1277–1284. Wilson R (2003) Bronchiectasis. In: Gibson GJ, Geddes DM, Costabel U, Sterk PJ, and Corrin B (eds.) Respiratory Medicine, pp. 1445–1464. London: Saunders/Elsevier. Wilson R and Abdallah S (1997) Pulmonary disease caused by non-tuberculous mycobacteria in immunocompetent patients. European Respiratory Monograph 12: 247–272.

BRONCHIOLITIS R L Smyth and S P Brearey, Alder Hey Children’s Hospital, Liverpool, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Bronchiolitis is a common respiratory tract infection usually affecting infants and young children during annual epidemics. It

is characterized by wheeze, respiratory distress, and poor feeding. Respiratory syncytial virus (RSV) is the most common cause for bronchiolitis and is amongst the most important pathogens causing respiratory infection in infants worldwide. The healthcare burden of bronchiolitis is large, due to large numbers of hospitalized infants and the high risk of nosocomial spread during epidemics. Most children will suffer only mild, short-lived symptoms. A small proportion will need admission to hospital, where treatment is generally supportive until the

BRONCHIOLITIS 269 illness resolves. Some will require ventilatory support for which mortality can be up to 10%. Infants at high risk of severe disease include those born prematurely, those with chronic lung disease, and immunocompromised infants. RSV infects ciliated epithelial cells, causing sloughing of the epithelium, cytokine and inflammatory mediator release, increases in mucus production, and interstitial edema. Clinical manifestations of bronchiolitis are a combined result of viral toxicity and the immune response to infection. Innate immune responses are important to the pathogenesis of bronchiolitis, as severe infection tends to occur after maternal antibody protection has waned and before the infant’s adaptive immune responses have matured. Immunoprophylaxis, in the form of intramuscular anti-RSV IgG1, is effective in reducing rates of hospitalization for high-risk infants.

Introduction Bronchiolitis, meaning inflammation of the bronchioles, is a clinical complex usually affecting children less than 2 years old. It is characterized by wheezing, dyspnea, tachypnea, and poor feeding. The clinical characteristics, originally termed ‘congestive catarrhal fever’, have been recognized for over 150 years. It was not until the late 1950s that the epidemiology and viral etiology of the illness were described. Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and is amongst the most important pathogens causing respiratory infection in infants worldwide. Epidemics occur during the winter months in temperate climates and during the rainy season in tropical climates (Figure 1). In the US, more than 120 000 infants are hospitalized annually with RSV infection, with more than 200 deaths

attributed to RSV lower respiratory tract disease. Total hospital charges for RSV-coded primary diagnoses over the 4-year period from 1997 to 2000 have been estimated at US$2.6 billion. Within hospitals, there is a high risk of nosocomial spread of RSV during epidemics, which can put vulnerable infants at risk of severe disease. Infection in infancy also predisposes children to the development of recurrent wheeze, thus increasing the long-term healthcare burden of this disease. Prophylaxis is available as a recombinant monoclonal antibody, but is expensive and currently limited to infants at highest risk of severe disease. Development of an effective vaccine would have a dramatic effect on morbidity and healthcare costs, but this is unlikely to occur in the near future. Treatment is generally supportive until the infection runs its natural course.

Etiology RSV is the primary cause of bronchiolitis. Seroepidemiological studies show that over 90% of children are infected with RSV by 2 years of age. Infection is spread by droplet exposure or direct contact with secretions. Fomites are infectious outside the body for up to 12 h. RSV was first isolated from chimpanzees in 1955 and was originally called chimpanzee coryza agent (CCA). Subsequently, RSV has been shown to cause 75–85% of cases of bronchiolitis. Other pathogens known to cause bronchiolitis are shown in Table 1. Historically, in over 10% of cases of bronchiolitis, no pathogen is detected. More

5000 4500 4000

Number of reports

3500 3000 2500 2000 1500 1000 500 0 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 Year Figure 1 Laboratory reports to Communicable Disease Surveillance Centre, Health Protection Agency, UK of infections due to respiratory syncytial virus in England and Wales, 1990–2004 (4 weekly). Reproduced with permission from HPA.

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recently, polymerase chain reaction (PCR) techniques have identified other pathogens, such as human metapneumovirus (hMPV) and coronavirus, which may have gone undetected previously. Co-infection with Table 1 Bronchiolitis pathogens Respiratory syncytial virus Rhinovirus Parainfluenza viruses (1,2,3) Influenza viruses (A and B) Human metapneumovirus Coronavirus (NL-63) Adenovirus Bordatella pertussis Mycoplasma pneumoniae

hMPV and RSV may be associated with a higher risk of severe disease. RSV is a negative-sense single-stranded RNA pneumovirus from the Paramyxoviridae family (Figures 2 and 3). The viral genome contains only 10 genes that encode for 11 viral proteins. Nine of these are structural proteins and glycoproteins that form the viral coat and bring about attachment to host cells, whilst the other two direct viral replication within the host cell. Table 2 describes the functions of each protein. Based on immunological techniques, two different strains of the virus have been identified: A and B. Subsequently, it has been demonstrated that the two strains are also genetically distinct. Most variability between strains is due to differences in the amino acid sequence of the G protein on the viral coat. Strain A is seen more commonly in the UK and North America. There is some evidence that strain A results in more severe infections. The F protein, responsible for fusion of infected cells with adjacent uninfected cells, facilitates cell-to-cell transmission of the virus and results in epithelial cell syncytia (appearance of apparently large multinucleate cells), which give the virus its name.

Pathology

Figure 2 Negative stain electron micrograph of RSV. Scale ¼ 100 nm. Reproduced with permission from Professor C A Hart.

Envelope proteins

G SH F

Nucleocapsid complex (N, P, and L proteins)

Matrix M1 proteins M2 Negative-sense RNA genome Lipid envelope

Figure 3 A schematic diagram of the RSV virion.

RSV initially causes inflammation of the bronchioles and destruction of ciliated epithelial cells. The submucosa becomes edematous, whilst cellular debris and mucus form plugs within the bronchioles. Neutrophils and alveolar macrophages are the

BRONCHIOLITIS 271 Table 2 RSV proteins and their functions Protein Surface glycoproteins F

Function

G SH

Virus penetration and fusion of infected cells with uninfected cells Virus attachment Unknown

Nucleocapsid-associated proteins N P L M2-1 M2-2

Encapsidates genome Phosphoprotein RNA polymerase Transcription elongation factor Regulation of transcription

Matrix protein M1

Non structural proteins NS1 NS2

May mediate association of nucleocapsid with envelope

Antagonize interferon-induced antiviral response Unknown

predominant inflammatory cells in the small airways. There is a peribronchiolar infiltration with lymphocytes that is associated with edema fluid accumulating within the alveoli. Severe disease involves destruction of the respiratory epithelium, parenchymal necrosis, and formation of hyaline membranes. The bronchiolar epithelium regenerates after 3–4 days, but cilia do not reappear until up to 15 days after the illness has resolved.

Clinical Features The spectrum of severity for infected children is wide. After an incubation period of 1–2 days, most infants exhibit predominantly upper respiratory tract symptoms. These include rhinorrhea, cough, and low-grade fever that often persists for several weeks, before resolving, without any other symptoms. Thirty to fifty percent of infants will progress to develop lower respiratory tract signs and symptoms within 2–3 days of infection. Infants may develop a rapid respiratory rate, wheeze, and other signs of respiratory distress (subcostal and intercostal recession, tracheal tug, and nasal flaring). Apnea at presentation is common, especially in infants less than 6 weeks old, those with a history of apnea of prematurity, and those with congenital heart disease. Approximately 2–3% of infected children require admission to hospital. Reasons for admission include hypoxia, inadequate fluid intake, apnea, and signs of imminent respiratory failure. A more severe cough,

pharyngitis, conjunctivitis, and otitis media may also be present. Irritability, signs of cardiovascular compromise, lethargy, and exhaustion are late signs and may be followed by respiratory arrest if no clinical interventions are made. One to two percent of infants hospitalized with bronchiolitis require ventilatory support and mortality in this group may be up to 10%. Premature birth, chronic lung disease of prematurity, congenital heart disease, cystic fibrosis, and immunodeficiency all predispose infants to a high risk of severe disease. In addition to this, boys are more likely to be hospitalized than girls. Infants exposed to high levels of particulate air pollution or cigarette smoke are also more likely to suffer from severe bronchiolitis. A chest radiograph will typically show hyperinflation, a flattened diaphragm, and patchy peribronchial infiltration. The chest radiograph can be normal even in severe cases. A reduction in oxygen saturations is associated with increased respiratory rate and is an accurate indicator for reduced gas exchange that is seen in severe disease. Arterial or capillary blood gas analysis is helpful in assessing changes in the clinical status, particularly if ventilatory support is being considered. Most infants, given adequate supportive care, improve clinically in 3–4 days. Within 2 weeks of the height of their illness most infants will have a normal respiratory rate and any radiological abnormalities will have cleared. However, up to 20% of infants may suffer from persistent wheezing and airway obstruction for several months, especially those that required hospital admission. In the longer term, airway reactivity is increased in children after infection and persists for at least 5–8 years. It is still uncertain whether RSV causes asthma in later life. RSV infection does not confer lasting immunity to reinfection. However, subsequent infections are usually less severe and are more likely to be limited to the upper respiratory tract.

Pathogenesis RSV first infects the ciliated epithelial cells of the respiratory tract. Subsequent disease manifestations are caused by a combination of viral cytotoxicity and the immune response to infection. These effects are summarized in Figure 4. On contact with epithelial cells, the G protein attaches the virus to epithelial cells and allows viral RNA and enzymes to enter the cell and initiate production of new viral RNA and proteins. The F protein mediates the formation of syncytia that enable the virus to spread rapidly. Multiple new viruses are assembled within cells, which are ultimately

272 BRONCHIOLITIS Soluble mediators: surfactant protein, immunoglobulin, sCD14

RSV

Opsonization Virus aggregation Increased phagocytosis

Epithelial cell sloughing

Airway inflammation Bronchoconstriction Mucus accumulation Surfactant inhibition

Macrophage

Epithelial cell

Inflammatory cytokines and chemokines

Neutrophil Eosinophil

Macrophage

NK cell

Dendritic cell

+ B lymphocyte CD4 T-helper cell

CD8+ cytotoxic T cell

Th2 response Th1 response

Figure 4 Pathogenesis of RSV bronchiolitis. RSV first infects ciliated epithelial cells of the respiratory tract. Inflammatory cytokines and chemokines, secreted by infected epithelial cells and macrophages, attract inflammatory cells and cause inflammation and bronchoconstriction in the airway. The predominant inflammatory cells in the airway are neutrophils and alveolar macrophages. Dendritic cells are the main antigen-presenting cells that stimulate T-lymphocyte function. Neutrophil survival is prolonged and IL-9 secretion promotes mucus production. CD4 þ T-helper lymphocytes may be skewed to produce proinflammatory (Th2) cytokines. The combination of epithelial cell sloughing, mucus plugging, and airway inflammation leads to the clinical presentation of acute bronchiolitis.

destroyed, leading to sloughing of the epithelium. As the cells are destroyed they release proinflammatory substances and cytokines that increase capillary permeability and attract inflammatory cells such as neutrophils (interleukin-8 (IL-8) secretion), macrophages (IL-1b, MIP-1a), eosinophils (RANTES, MIP1a), and natural killer cells (interferon (IFN)-a/b). Some secreted mediators, such as IL-9 stimulate mucus production, whilst others, such as leukotrienes, cause bronchoconstriction. Pulmonary surfactant function is also inhibited. Increased production and impaired clearance of cellular debris and mucus results in plugging of the small airways, air trapping, and atelectasis. For infants with preexisting poor lung function, such as those with chronic lung disease, this airway obstruction is likely to have more clinical significance and cause severe disease. The immune response to RSV includes innate and adaptive components. Innate immune responses are now recognized as more important than previously thought and are capable of influencing the subsequent adaptive immune response.

Innate immunity is important in defense against RSV and other viruses that cause bronchiolitis as the adaptive immune responses of infants are relatively immature. The innate immune response is rapid and recruits effector molecules and phagocytic cells to the site of infection through the release of cytokines. Pulmonary surfactant provides early defense from viral infection. Surfactant proteins A and D are members of the collectin family that can bind to the surface of a number of pathogens including RSV. The virus is thus opsonized and capable of binding to complement and receptors on phagocytic cells (neutrophils and macrophages), eosinophils, and natural killer cells. Toll-like receptors (TLRs) are the most important of these receptors. They are present on epithelial cells and phagocytic cells and recognize distinctive nonhuman pathogen-associated molecular patterns (PAMPs). Although their precise role is still being elucidated, TLRs are important receptors to the innate and adaptive immune responses to RSV. TLR2 and TLR4 are receptors for surfactant proteins A and D, both of which bind to RSV. TLR4 and CD14 have been shown to be co-receptors for the

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RSV F protein. Genetic polymorphisms in the TLR4 gene are associated with a risk of severe disease. TLR3 recognizes double-stranded RNA in endosomes of the cell and initiates an immune response to RSV. Pulmonary dendritic cells also express TLRs and are the predominant antigen-presenting cells in RSV infection. Circulating dendritic cells are recruited to sites of viral replication in the lungs. They produce large amounts of type I interferon (IFN-a and -b) and stimulate subsequent T-lymphocyte responses to RSV infection. Regulatory T lymphocytes (Treg) are known to dampen inflammatory responses and could be stimulated by RSV either via TLRs expressed on their cell surface or via pulmonary dendritic cells. Therefore, they provide a way by which the T-lymphocyte response to RSV can be influenced by both innate and adaptive immunity. Alveolar macrophages have phagocytic functions, but act mainly as antigen-presenting cells, interacting with helper and cytotoxic T lymphocytes. Along with respiratory epithelial cells they respond, via TLR and other signaling pathways, by secreting cytokines that increase tissue permeability as well as attracting and activating additional inflammatory cells. Neutrophils are the predominant airway leukocytes in RSV bronchiolitis, representing about 90% of cells in the upper airway and 80% of cells in the lower airway. Neutrophil chemotaxis is dependent on the production of the potent chemotractant, IL-8, by airway epithelial cells and macrophages. IL-8 is secreted very soon after infection, leading to an almost immediate inflammatory response before the infection is established. A later peak in IL-8 secretion is dependent on viral replication. Genetic polymorphisms in the IL-8 gene are associated with a risk of severe disease. Neutrophil recruitment and adherence to epithelial cells is increased by persistence of RSV infection. Neutrophils, whose survival is prolonged, secrete products such as myeloperoxidase and neutrophil elastase, which amplify viral cytotoxicity. Neutrophils have also been shown to secrete IL-9 in large quantities in the lungs of severely infected infants. IL-9 is a potent proinflammatory cytokine that is known to cause eosinophilic inflammation, bronchial hyperresponsiveness, and increased mucus production. Neutrophil function therefore plays an important role in the pathological changes that occur in bronchiolitis. Eosinophils might be expected to have an important role in the pathogenesis of bronchiolitis. They have antiviral activity and eosinophil chemoattractants are secreted by infected respiratory epithelial cells. Following the clinical trials of the formalininactivated vaccine, postmortem examinations revealed massive eosinophilic infiltrates. However,

subsequent clinical studies have not reported significant numbers of eosinophils in the airways of infants with RSV bronchiolitis. Adaptive immunity to RSV is generally composed of protective humoral immunity (B cells and antibodies) and viral clearance (T cells). The humoral response to RSV infection results in the production of IgG, IgM, and IgA antibodies in both blood and airway secretions. Protective benefits of these antibodies are demonstrated by the reduced likelihood of infection in the first month of life in term babies, due to the carriage of maternal IgG antibodies. Prophylaxis with monoclonal anti-RSV IgG (palivizumab), which has been shown to reduce the incidence and frequency of infections in high-risk infants, also demonstrates the role of the humoral response. Evidence from animal models confirms that antibody protects against RSV disease, but once infection is established, the T-cell response promotes viral clearance. The role of cell-mediated immunity is demonstrated in children with deficient cellular immunity who shed the virus for many months after initial infection, compared to 2 weeks for immunocompetent infants. The cell-mediated response is composed of the CD8 þ cytotoxic T lymphocytes and CD4 þ T-helper lymphocytes. Cytotoxic lymphocytes are important in both recovery from and the pathogenesis of RSV bronchiolitis. They promote clearance of RSV from the lungs but also cause pulmonary injury. Functional studies in T-lymphocyte-depleted mice demonstrated prolonged RSV replication, yet no overt evidence of illness. CD4 þ T-helper cells have traditionally been subdivided further according to the cytokine profiles they secrete: T-helper-1 (Th1) cells produce interferon gamma (IFN-g) and other antiviral cytokines. Th2 cells produce cytokines that induce eosinophil proliferation, and the release of leukotrienes and IgE antibodies leading to an enhanced inflammatory response. The idea that RSV bronchiolitis might be a Th2-type disease, and that this may explain airway obstruction and postbronchiolitic wheeze, has somewhat limited evidence to support it. Multiple factors are more likely to influence the T-lymphocyte response to RSV infection.

Animal Models Several experimental animal models have been used to improve our understanding of the pathophysiology of RSV bronchiolitis. The variety of animal models used reflects the diversity of clinical manifestations of RSV disease, dependent on an individual’s age, genotype, phenotype, immune status, and concurrent disease. RSV has been shown to replicate in

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animal models: primates, cotton rats, mice, calves, guinea pigs, ferrets, and hamsters have been used. The need to investigate immunological responses to RSV, using good animal models, was highlighted after the unsuccessful development of the first formalin-inactivated (FI) RSV vaccine in the 1960s. When natural RSV infection occurred in children given the vaccine, 80% of these children were hospitalized and two children died. Interpretation of the trial results was hampered by the lack of a small animal model, in which vaccine-enhanced disease could be studied. It is now apparent that other vaccine formulations are also capable of stimulating inappropriate immune responses to RSV. Therefore, comprehensive studies using animal models are essential before any further human vaccine trials are undertaken and are key to our understanding of the pathogenesis of bronchiolitis. Cotton rats have proved to be useful animal models, as they are uniformly susceptible to pulmonary infection through adulthood and are more immunologically responsive than mice to RSV. Studies using the cotton rat model were largely responsible for the development of RSV-neutralizing IgG in preventing pulmonary infection and treatment with ribavarin. Despite this, cotton rats have no congenic, transgenic, or knockout strains and there is only limited availability of reagents used to characterize immunological responses to RSV. Mice have been the most widely used model, due to a wide array of inbred, congenic, transgenic, and knockout strains. They also have the most reagents available, specific to RSV immunology, and have relatively low maintenance costs. Many insights have been gained into the immune response to RSV, which could not have been achieved using other models. The BALB/c mouse is particularly important, as it develops similar airway inflammatory responses to humans after intranasal RSV infection. Mice studies have been useful in investigating the immunology of RSV infection, especially the cytokine responses to the virus and for experimental vaccines.

Management and Current Therapy After admission to hospital, infants should be monitored and assessed regularly. Pulse oximetry, fluid balance, and respiratory rate are useful in assessing changes in the infant’s condition. Barrier nursing and stringent hand-washing policies have been shown to reduce nosocomial spread of RSV. Rapid RSV antigen testing, by either immunofluorescence or enzyme immunoassay, of nasopharyngeal secretions is useful in determining RSV status in hospital.

Despite considerable efforts to develop effective treatments for RSV bronchiolitis, no effective measures exist other than supportive care. The cornerstones of supportive care are supplemental oxygen to correct hypoxia and adequate fluid administration. Infants with inadequate fluid intake may need nasogastric feeding or, if this is unsuccessful, intravenous fluids. Intravenous fluids should be given with care due to the possibility of worsening lung fluid accumulation and left ventricular overload. A large number of trials and meta-analyses have investigated the efficacy of b2 agonists and ipratropium for patients with bronchiolitis. There is no compelling evidence that bronchodilators are useful in the treatment of bronchiolitis. There may be a subgroup of patients for which bronchodilators are safe and efficacious. However, no criteria exist to identify this subgroup and a significant number of studies found that patients deteriorated after receiving bronchodilators. The inconsistency of these results can be explained by our knowledge of the causes of wheeze in bronchiolitis. Bronchodilators will have no effect on increased mucus production, sloughed epithelial cells in the airway, or interstitial edema. Ribavarin is an antiviral preparation administered by aerosol and designed to inhibit the synthesis of viral structural proteins. Although early trials of ribavarin supported its use, later trials found no significant positive effect. In addition, it is potentially teratogenic and labor intensive to administer. Other treatments that have failed to demonstrate consistent benefits in the treatment of bronchiolitis include epinephrine, inhaled or systemic corticosteroids, recombinant human DNase, interferon-a, vitamin A, and antibiotics.

Prevention Two immunoprophylactic agents are currently available that are recommended for infants at risk of severe infection. RSV immune globulin intravenous (RSV-IGIV) is a polyclonal hyperimmune globulin, prepared from donors selected for having high serum titers of RSV neutralizing antibody. Palivizumab is a humanized murine monoclonal IgG1 antibody against the RSV F-glycoprotein and is given as a monthly intramuscular injection during the RSV season. RSV-IGIV was the first immunoprophylactic agent to be licensed for prevention of severe RSV bronchiolitis and clinical trials demonstrated a 41–63% reduction in hospital admissions attributable to RSV lower respiratory tract infections. However, RSV-IGIV has a number of disadvantages that have resulted in a decline in its use in favor of palivizumab. Early trials reported an increase in postoperative mortality in patients with cyanotic

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congenital heart disease who received RSV-IGIV. Administration required intravenous access and a 4-h infusion every month. There are concerns regarding volume overload, interference with live vaccines, and theoretical risks of infection from human donors. Trials of palivizumab have also demonstrated benefits to high-risk infants, with early trials demonstrating a 45–55% reduction in hospital admissions attributable to RSV lower respiratory tract infections. It is free from potential risk of infection from human donors, does not interfere with immune response to vaccines, and can be administered easily, without the need for hospital admission. Clinical trials also demonstrated its safety for infants with hemodynamically significant congenital heart disease. However, no trial has demonstrated a significant reduction in mortality due to RSV infection. Although palivizumab was shown to reduce rates of hospitalization and intensive care admissions in large double-blind randomized control trials, several studies have questioned its cost-effectiveness. The American Academy of Pediatrics recommends its use for certain high-risk groups: children under 2 years with chronic lung disease requiring medical therapy; infants born before 28 weeks’ gestation in the first year of life; and infants born at 29–32 weeks’ gestation up to 6 months of age. Infants born at 32–35 weeks’ gestation have a lower risk of hospitalization and palivizumab is only recommended for these infants if they have two other risk factors such as child care attendance, school age siblings, parental smoking, or congenital abnormalities of the airways. Infants with hemodynamically significant congenital heart disease are also likely to benefit and palivizumab is recommended in this group, particularly those with cyanotic heart disease, congestive heart failure, or pulmonary hypertension. There is little knowledge regarding its benefits for immunocompromised infants, children with cystic fibrosis, or prophylaxis in the second year of life.

Bronchiolitis Obliterans

See also: Antiviral Agents. Bronchodilators: Beta Agonists. Chemokines. Cilia and Mucociliary Clearance. Epithelial Cells: Type I Cells; Type II Cells. Genetics: Gene Association Studies. Immunoglobulins. Infant Respiratory Distress Syndrome. Leukocytes: Eosinophils; Pulmonary Macrophages. Mucus. Pediatric Pulmonary Diseases. Surfactant: Overview. Toll-Like Receptors. Vaccinations: Viral. Viruses of the Lung.

Further Reading American Academy of Pediatrics Committee on Infectious Diseases and Committee of Fetus and Newborn (2003) Revised indications for the use of palivizumab and respiratory syncytial virus immune globulin intravenous for the prevention of respiratory syncytial virus infections. Pediatrics 112(6): 1442–1446. Arden KE, Nissen MD, Sloots TP, and Mackay IM (2005) New human coronavirus, HCoV-NL63, associated with severe lower respiratory tract disease in Australia. Journal of Medical Virology 75: 455–462. Black CP (2003) Systematic review of the biology and medical management of respiratory syncytial virus infection. Respiratory Care 48(3): 209–233. Byrd LG and Prince GA (1997) Animal models of respiratory syncytial virus infection. Clinical Infectious Diseases 25: 1363–1368. McNamara PS and Smyth RL (2002) The pathogenesis of respiratory syncytial virus disease in childhood. British Medical Bulletin 61: 13–28. Semple MG, Cowell A, Dove W, et al. (2005) Dual infection of infants by human metapneumovirus and human respiratory syncytial virus is strongly associated with severe bronchiolitis. Journal of Infectious Diseases 191: 382–386. Welliver RC (2003) The burden of respiratory syncytial virus (RSV) and the value of prevention. Journal of Pediatrics 143(5): S111–S162. Welliver RC (2004) Bronchiolitis and infectious asthma. In: Feigin RD, Demmier GJ, Cherry JD, and Kaplan SL (eds.) Textbook of Pediatric Infectious Diseases, 5th edn., pp. 273–285. Philadelphia: Elsevier. Wohl MB (1998) Bronchiolitis. In: Chernick V, Boat TF, and Kendig EL (eds.) Kendig’s Disorders of the Respiratory Tract in Children, 6th edn, pp. 473–485. Philadelphia: Harcourt. Wright JR (2005) Immunoregulatory functions of surfactant proteins. Nature Reviews: Immunology 5: 58–68.

see Interstitial Lung Disease: Cryptogenic Organizing Pneumonia.

BRONCHOALVEOLAR LAVAGE M Drent and J A Jacobs, University Hospital Maastricht, Maastricht, The Netherlands & 2006 Elsevier Ltd. All rights reserved.

Abstract Provided its limitations are kept in mind, there is a place for bronchoalveolar lavage (BAL) in the evaluation of diffuse lung

diseases. The analysis of cells obtained by BAL may be helpful in narrowing the differential diagnosis. The cellular analysis of the BAL fluid (BALF) relies on an accurate identification and differential counting of the cells obtained. Universal standards are not available; consequently, each institution should ensure that the various readers of BALF samples have been properly trained in cellular analysis. Using a standard technique for performing the BAL and handling the sample will also reduce variability. To explore the possible role for BALF analysis in the

BRONCHODILATORS / Anticholinergic Agents 285

Bronchocentric Granulomatosis

see Systemic Disease: Sarcoidosis.

BRONCHODILATORS Contents

Anticholinergic Agents Beta Agonists Theophylline

Anticholinergic Agents N J Gross, Hines VA Hospital, Chicago, IL, USA

the routine treatment of airways obstruction particularly for chronic obstructive pulmonary disease (COPD) but also for asthma in certain circumstances.

Published by Elsevier Inc.

Chemistry Abstract Anticholinergic agents have bronchodilator effects on the human airways and have a role in the treatment of obstructive airways diseases, particularly chronic obstructive pulmonary disease (COPD). Those in approved clinical use are synthetic quaternary ammonium congeners of atropine, and include ipratropium bromide and tiotropium bromide, each of which is very poorly absorbed when given by inhalation. Ipratropium has a relatively short duration of action and has been widely used for many years, either alone or in combination with the short-acting b-adrenergic agent albuterol for the maintenance treatment of stable COPD. Tiotropium, which was introduced in the early 2000s, has a duration of action of at least 1–2 days making it suitable for once-daily maintenance treatment of COPD. Both agents have a wide therapeutic margin and are very well tolerated by patients.

Introduction Naturally occurring anticholinergic agents such as atropine and scopolamine which are present in many plants, for example, belladonna spp. have been used in traditional medical cultures to relieve breathlessness for millennia. These agents, which are all tertiary ammonium alkaloids, and which were smoked or chewed, are well absorbed into the systemic circulation and thus have multiple systemic side effects that limited their clinical usefulness as bronchodilators. In the last century it was discovered that synthetic quaternary ammonium analogs of atropine were very poorly absorbed but retained topical anticholinergic activity. This enabled the development of quaternary ammonium analogs of atropine such as ipratropium, oxytropium, and tiotropium bromides for clinical use as inhaled bronchodilators. In the last two decades, these agents have become important components in

The structure of some natural and synthetic anticholinergic agents is shown in Figure 1. The parent compound, atropine, is a tertiary ammonium alkaloid due to a 3-valent nitrogen atom on the tropine ring. Although widely used for respiratory purposes in previous eras, its rapid absorption and widespread systemic distribution resulted in unacceptable systemic effects. Atropine and other natural tertiary agents are no longer used for respiratory diseases and will not be further considered. Synthetic analogs such as ipratropium bromide and tiotropium bromide are quaternary ammonium compounds and carry a charge on the 5-valent nitrogen atom which renders them less able to cross tissue barriers. However, they are able to penetrate mucosal surfaces sufficiently to exert pharmacologic actions on local structures such as smooth muscle.

Mode of Action Anticholinergic agents compete with acetylcholine for muscarinic receptors, inhibiting the numerous

N

Br −•H2O + C H CH3 N 3 7

CH3

O CH2OH O C CH

Atropine

O CH2OH O C CH

Ipratropium bromide

Figure 1 The molecular structure of tertiary and quaternary ammonium compounds.

286 BRONCHODILATORS / Anticholinergic Agents

‘housekeeping’ actions of the parasympathetic branch of the autonomic nervous system. When taken by the inhalational route, the poorly absorbed synthetic quaternary ammonium agents have actions that are limited to the sites of their deposition, namely the mouth where they inhibit salivary gland secretions, and the airways where they relax bronchial smooth muscle. For reasons that are not well understood, they tend not to inhibit respiratory gland secretions nor mucociliary clearance. Unlike other bronchodilators such as b-adrenergic agents or methyl xanthines, inhaled anticholinergic agents have no demonstrated effects other than as airway smooth muscle relaxants. They do not have clinically significant anti-inflammatory effects. They do not have significant cardiac, or pulmonary vascular actions, nor systemic effects. They have not been shown to have long-term effects on disease progression (although trials with tiotropium that explore such an effect are underway at present). Cholinergic Activity in the Lungs

In humans, airway caliber is largely controlled by the cholinergic parasympathetic system via branches of the vagus nerve, which stimulates smooth muscle contraction. At rest, parasympathetic activity provides bronchomotor tone, a low level of bronchial smooth muscle activity. There is evidence that resting bronchomotor tone is increased in both asthma and COPD. In addition to supplying bronchomotor tone, cholinergic activity can also be reflexly increased by numerous stimuli such as inhalation of cold dry air, mechanical factors, irritant particles, aerosols and gases, and specific mediators such as histamine and bronchoconstricting eicosanoids. Clinically, the use of an anticholinergic agent aims both to abolish cholinergic tone of airway smooth muscle and to inhibit the phasic increases in reflex bronchoconstriction.

Three muscarinic receptor subtypes, called Ml, M2, and M3, are expressed in human lungs (Figure 2). M1 receptors are found in peribronchial ganglia, and M3

Preganglionic nerve

Ganglion

M2

Postganglionic nerve

Available Agents

In the US, ipratropium bromide is available as a metered-dose inhaler (MDI) and as a nubilizable solution. Combinations with albuterol are also available in both forms. Its duration of action, both as monotherapy and as a combination, is 4–6 h. Tiotropium bromide is available as a dry powder inhaler; its duration of action is greater than 24 h, probably at least 48 h. Oxitropium bromide is available in several countries outside the US, and has a duration of action of 6–8 h. All of these agents are functionally selective for M1 and M3 muscarinic receptor subtypes, and tend to spare M2 receptors by dissociating rapidly from the latter. The half-life of the tiotropium– M3 receptor complex is 35 h compared with 0.3 h for ipratropium. Apart from tiotropium’s somewhat slower onset of action, much longer duration of action, and possibly slightly greater bronchodilator action at peak effect, the actions and side effects of each of these agents are very similar.

Clinical Role and Uses

Muscarinic Receptors in Lungs

M1

receptors are present on bronchial smooth muscle cells. Both are believed to mediate bronchomotor tone and reflex bronchoconstriction. M2 receptors, in contrast, are located on postganglionic autonomic fibers and are believed to be autoreceptors whose stimulation provides feedback inhibition of further acetylcholine release from terminals of these nerves, tending to limit vagally mediated bronchoconstriction. An implication of this scheme is that M1 and M3 receptors would be appropriate targets for anticholinergic bronchodilators, while M2 receptors should be spared. Indeed, there is evidence that M2 receptors are selectively damaged by certain viruses as well as eosinophil products, perhaps accounting for the bronchospasm associated with viral infections and asthma.

M3

Smooth muscle

Figure 2 The presumed location of muscarinic receptors in the parasympathetic pathway of human lungs.

Stable COPD

The major clinical use of inhaled anticholinergic agents is for the routine treatment of stable COPD. Both ipratropium, given three to four times daily, and tiotropium given once daily qam, are approved and recommended by current guidelines as options for first-line therapy for stable COPD of all degrees of severity. In practice, tiotropium is generally preferred because its prolonged duration of action provides more consistent bronchodilation around the clock, and its once-daily use is more convenient for patients. The onset of bronchodilation is slower than with inhaled short-acting b-adrenergic agents, peak effects being reached at 30–60 min with ipratropium

BRONCHODILATORS / Anticholinergic Agents 287

and at about 3–4 h with tiotropium. The magnitude of bronchodilation, measured as the peak of FEV1 improvement, is very approximately 0.15–0.25 l following a single dose of 40 mg ipratropium by MDI, 0.30 l following 400 mg of ipratropium by nebulization, and 0.30 l following 18 mg of tiotropium dry powder inhaler (DPI). Uniquely, regular use of tiotropium results in a rise in the prebronchodilator (trough) FEV1 of 0.1–0.15 l after about 1 week of once-daily use, and a peak FEV1 level (3 h after the morning dose) that is about 0.05 l higher than that after the first day of use, indicating a further benefit with regular use. These improvements with tiotropium compare favorably with those obtained with long-acting b-adrenergic bronchodilators. In parallel with improvements in FEV1, a decrease in functional residual capacity (a marker of hyperinflation) occurs by 4 weeks of regular treatment with tiotropium. No tachyphylaxis has been observed with long-term use of anticholinergic agents. In addition to these effects on lung function, measurable symptomatic improvements have been consistently reported with regular use of tiotropium such as clinically significant improvements in dyspnea, exercise capacity, health status (quality of life), and most importantly, an approximately 20–25% reduction in the frequency of acute exacerbations of COPD. The mechanism of the latter, which is shared by some other treatments for COPD, is not well understood. Acute Exacerbations of COPD

Studies comparing bronchodilator treatments for acute exacerbations of COPD do not show consistent benefit for treatments that include anticholinergic agents. Nevertheless, most current guidelines recommend the addition of ipratropium to a short-acting b-agonist in the initial treatment regimen for these serious events. Tiotropium is not appropriate as monotherapy for these events; however, it should be continued through the exacerbation when patients have been receiving it already.

agent. It has not been possible to predict reliably which asthmatics will benefit from ipratropium other than by an individual trial. Ipratropium may also be a useful alternative bronchodilator for those rare asthmatic patients who cannot tolerate the palpitations or tachycardia that a b-adrenergic agent may produce. The role of tiotropium has not been studied in asthma. Acute Severe Asthma

Although short-acting b-adrenergic agents are the bronchodilators of choice in the initial management of acute severe asthma, several studies and a metaanalysis suggest that the inclusion of ipratropium in the initial treatment of these serious events provides more rapid improvement in lung function and may avoid prolonged emergency room treatments and hospitalization. Current guidelines recommend that ipratropium (usually by nebulization) be added when the response to initial treatments with a short-acting b-agonist is ‘less than complete’ or ‘poor’. In practice, it is very common for the combination of albuterol and ipratropium to be given routinely as initial treatment for episodes of acute severe asthma, at least for the first few treatments. Pediatric Airways Disease

For pediatric asthma, both stable and acute severe forms, the role of ipratropium is similar to that in adults. Although studies with adequate statistical power or long duration in children are lacking, adult doses have been used without adverse effects in children down to 4 years of age. As in adults, anticholinergic agents have a very limited role in asthma. The role of tiotropium has not been studied in pediatric asthma. There are occasional reports of the use of ipratropium in other pediatric diseases such as cystic fibrosis, viral bronchiolitis, exercise-induced asthma, and bronchopulmonary dysplasia. Although these sometimes suggest a benefit from the use of ipratropium, the body of evidence is insufficient to make any recommendation in these conditions.

Stable Asthma

Numerous studies have compared ipratropium with a variety of short-acting b-adrenergic agents for the regular treatment of stable asthma. Almost uniformly, these reports show that ipratropium is a less potent bronchodilator than a short-acting b-adrenergic agent in asthmatic patients. No anticholinergic agent has been approved for use in asthma in the US. However, a few patients with otherwise typical asthma respond well to it, and it may occasionally add to the bronchodilation obtained with an adrenergic

Side Effects and Adverse Events Being very poorly absorbed, inhaled anticholinergic agents have a very wide therapeutic margin and are very well tolerated, even in rare instances when massive doses have accidentally been given. In normal clinical use, dryness of the mouth is common to all agents in this class but is rarely sufficiently severe for the patient to discontinue use. Bad taste is an occasional complaint, as is a brief coughing spell shortly

288 BRONCHODILATORS / Beta Agonists

after inhalation. One serious but rare idiosyncratic effect is paradoxical bronchospasm whose mechanism is unknown. It has been reported to occur in perhaps 0.3% of patients following use of any of the above anticholinergic bronchodilators. The drug should be discontinued in patients who experience wheezing, chest tightness, and dyspnea within an hour of the inhalation. Ipratropium was extensively studied for possible adverse effects on mucociliary clearance from the lungs, urinary outflow, and increased intraocular pressure (well-known side effects of atropine). These were found not to be problems with inhaled ipratropium, nor have they been found with oxitropium or tiotropium. However, each of these agents can cause dilatation of the pupil and, possibly, precipitate acute glaucoma, if they are placed directly onto the eye. Care should be taken that neither the mist from a nebulized anticholinergic treatment nor even minute amounts of tiotropium dry powder accidentally find their way into the eye. See also: Acetylcholine. Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview.

Further Reading Cattapan SE and Gross NJ (2002) Anticholinergic agents. In: Barnes PJ, Drazen JM, Rennard S, and Thomson NC (eds.) Asthma and COPD: Basic Mechanisms and Clinical Management, pp. 527–534. New York: Academic Press. Celli BR and MacNee W (2004) Standards for the diagnosis and treatment of patients with COPD: a summary of the ATS/ERS position paper. European Respiratory Journal 23: 932–946. Coulson FR and Fryer AD (2003) Muscarinic acetylcholine receptors and airway diseases. Pharmacology & Therapeutics 98: 59–69. Global Initiative for Chronic Obstructive Lung Disease (2003) Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: Update. Bethesda: National Institutes of Health, National Heart, Lung, and Blood Institute. Gross NJ (2004) Tiotropium bromide. Chest 126: 1946–1953.

airway epithelial cells. Drugs targeted at b2-adrenoceptors fall into two main classes: short-acting b-agonists (SABAs) such as salbutamol and terbutaline and long-acting b2-agonists (LABAs) such as salmeterol and formoterol. These agents are in general composed of a benzene ring with a chain of two carbon atoms and either an amine head group or a substituted amine head group. Beta2-agonists interact selectively at the b2-adrenoceptor to stimulate elevation in cell cyclic AMP content which is responsible for the majority of downstream signaling events following from receptor stimulation. The major clinical effect of b2-agonists is airway smooth muscle relaxation which produces the acute bronchodilator effects of these drugs. In addition, b2adrenoceptor agonists are bronchoprotective agents, that is, they inhibit bronchoconstriction induced by irritant challenge, for example, with histamine or methacholine. The b2-adrenoceptor is highly polymorphic and potential functional effects which may be of clinical relevance have been described for some of these polymorphisms. SABAs are first-line bronchodilators agents used in the management of asthma: LABAs should be used in the management of moderately severe asthma in combination with inhaled steroids. Side effects of these agents include tremor, tachycardia, and hypokalemia: there have also been concerns that regular use of some b2-adrenoceptor agonists (e.g., fenoterol) may be associated with asthma exacerbation and very rarely death.

Introduction Beta-adrenoceptors were initially separated into b1- and b2-subtypes based upon differences in physiological response profiles in the heart, blood vessels, and airways. Subsequently a b3-subtype was identified and shown to be present in a number of tissues, most notably adipocytes. The main consequences of activation of these three subtypes are shown in Table 1. Given the profile of receptor mediated effects shown in Table 1, the most effective b-agonists in the management of airway disease can be seen to be selective b2-adrenoceptor agonists. These fall into two main groups: the short-acting b2-agonists (SABAs) and long-acting b2-agonists (LABAs). Frequently used SABAs include salbutamol (albuterol) and terbutaline, whilst the most frequently used LABAs

Table 1 Effects of the three subtypes of b-adrenoceptor

Beta Agonists I P Hall, Nottingham University, Nottingham, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Beta2-adrenoceptor agonists are the mainstay bronchodilator agents used in the treatment of asthma and chronic obstructive pulmonary disease. Three b-adrenoceptors have been characterized using molecular pharmacology and molecular physiological approaches: b2-adrenoceptors are the major receptor subclass mediating both bronchodilatation and effects on

b1-Adrenoceptor

b2-Adrenoceptor

b3-Adrenoceptor

Tachycardia Positive inotropy

Bronchodilatation Inhibition of mediator release from mast cells Mucus secretion Increased ciliary beat frequency Relaxation of uterine smooth muscle Venous and arterial dilatation

Lipolysis

BRONCHODILATORS / Beta Agonists 289

are formoterol and salmeterol. In the 1960s–70s other drugs were used, including isoprenaline and fenoterol. However, because of concerns over an increase in asthma deaths associated with use of the nonselective b-agonist isoprenaline, this is no longer in general use in clinical practice: similar concerns regarding asthma deaths associated with fenoterol led to its withdrawal from the market in New Zealand and some other countries in the 1990s. Beta2-adrenoceptor agonists can also be classified according to whether they are partial agonists or full agonists. In most assay systems salbutamol and salmeterol act as partial agonists whereas terbutaline and formoterol are generally full or near full agonists. A number of novel b-adrenoceptor agonists are currently in development. In general these are full agonists with prolonged duration of action.

kinase A activation. In airway smooth muscle, these effects include phosphorylation of key targets, including myosin light chain kinase and calcium activated potassium channels, increased calcium sequestration and/or removal and inhibition of contractile signaling pathways (Figure 2). However, the correlation between whole cell cyclic AMP content and physiological effects (e.g., smooth muscle relaxation) is not precise which has led to the suggestion that some effects of receptor stimulation are cyclic AMP independent. These effects could potentially be mediated directly by the a-subunit of Gs or by non-Gs coupled pathways. This may be relevant to some of the longer-term effects of b2-adrenoceptor stimulation on gene transcription.

Chemical Structure

Beta2-adrenoceptor agonists have two main clinically relevant effects. First, they provide the mainstay bronchodilator therapy used in the management of asthma and other airway diseases where reversible airflow obstruction may be present, for example, chronic obstructive pulmonary disease (COPD). As required SABAs are first-line agents for the management of mild asthma: most guidelines (e.g., those produced by the British Thoracic Society) suggest ‘as required’ usage is appropriate as monotherapy as long as patients are not requiring more than an average of one dose per day. For those patients requiring frequent doses (i.e., more than one daily of SABAs), an inhaled steroid should be added to the treatment. In general, the LABAs should be used for those patients who are inadequately controlled on moderate doses of inhaled steroid (typically 400– 800 mg of inhaled beclomethasone or equivalent) where control is inadequate. These drugs should not be used as monotherapy. Salbutamol, terbutaline, salmeterol, and formoterol are all available in both aerosol form administered via metered dose inhalers (MDIs) and also as dry powder formulations in most countries. Beta2-agonists are also used in the management of acute asthma where they are generally given by nebulizer (salbutamol or terbutaline) or in severe acute asthma by intravenous infusion. In the past, oral preparations of both salbutamol and terbutaline were used in the management of asthma but these have been superseded by long-acting inhaled formulations. A very small number of patients have been treated with subcutaneous b2-agonists (usually terbutaline): whilst there are anecdotal reports of benefit there are no high-quality randomized control trials utilizing this

The chemical structure of the four most frequently used b-agonists is shown in Figure 1. Most are based on the naturally occurring catecholamine epinephrine. The structure activity relationship of these drugs depends on the presence or substitution of hydroxyl (OH) groups on the benzene ring and on the substituted head group. Substitution of the hydroxyl groups at position 3 and 4 on the benzene ring generally results in production of a less potent catecholamine: however, these drugs are more resistant to breakdown by catechol-O-methyl transferase (COMT). Substitutions on the a carbon atom prevent oxidation by monoamine oxidase (MAO). The action of b-agonists within tissue is terminated as a consequence of uptake into sympathetic nerve endings (uptake 1) or uptake into other innervated tissues such as smooth muscle (uptake 2) and subsequent degradation by COMT and MAO. Beta-adrenoceptor agonists can also be conjugated to sulfates or glucuronides in the liver, gut wall, and lung.

Mode of Action Beta2-adrenoceptor agonists mediate clinical effects by stimulation of the b2-adrenoceptor. This receptor is one of the superfamilies of G-protein-coupled receptors (GPCRs). The b2-adrenoceptor is coupled via the stimulatory G protein Gs to adenylate cyclase. Increases in adenylate cyclase activity result in elevation of intracellular cyclic AMP content and subsequently activation of protein kinase A. The majority of the downstream events stimulated by b2-agonists are therefore a consequence of protein

Role of b2-Adrenoceptor Agonists in Respiratory Medicine

290 BRONCHODILATORS / Beta Agonists HO OH HO

CHCH2NHCH3

Epinephrine H H CHOH−CH2−NH–C–CH2

HO

OCH3

CH3 OHCHN Formoterol HOH2C OH HO

CH3

CHCH2NH–C–CH3 CH3

Salbutamol CH(OH)CH2NH(CH2)6O(CH2)4Ph

CH2OH OH Salmeterol HO OH

CH3

CHCH2NH–C–CH3 CH3 HO Terbutaline Figure 1 Structure of a range of b-agonists.

approach which should only be initiated by specialist asthma clinics dealing with severe patients. The use of SABAs and LABAs is also recommended in most COPD guidelines: again SABAs should be used for rapid relief of symptoms and

LABAs reserved for patients with more severe disease. In general, the therapeutic effect in terms of both bronchodilatation and symptom relief is much smaller than in true asthma. Some patients with severe COPD have been treated with regular (four

BRONCHODILATORS / Beta Agonists 291 2AR

Gs

AC

ATP

PKA activation

cAMP

Downstream effects (e.g., relaxation of airway smooth muscle)

5′AMP Phosphodiesterases Figure 2 Activity of b2-agonists. AC, adenylate cyclase; AMP, adenosine monophosphate; ATP, adenosine triphosphate; b2AR, b2-adrenoceptor; Gs, stimulatory G protein; PKA, protein kinase A.

times daily) nebulized b2-agonists (sometimes in combination with an anticholinergic); this approach should be reserved for patients with severe disease and where there is objective evidence of benefit. In addition to the bronchodilator effects of b2-agonists, this class of drugs also protects against the actions of bronchoconstrictor stimuli; this may be part of the basis for the beneficial effects of LABAs seen when used regularly. These bronchoprotective effects can be readily demonstrated in the lung function laboratory by the administration of b2-agonists before bronchial provocation tests. The degree of protection seen is maximal within the first 12–24 h when regular dosing is instituted with both SABAs and LABAs; subsequently, the degree of bronchoprotection afforded falls somewhat although still remains above placebo levels. Contraindications

In general, b2-agonists are safe and effective drugs in the management of reversible airflow obstruction. However, there have been a number of concerns over the last 50 years regarding their usage. Arrhythmias have been reported following both intravenous and inhaled administration of b2-agonists. These are probably due to a direct effect of b2-adrenoceptor agonist stimulation in the heart and in addition hypokalemia which occurs as a consequence of stimulation of sodium potassium ATPase. These effects are likely to be related to relative selectivity for b2- over b1-receptors and dose; it is possible that arrhythmias were related to the excess of asthma deaths seen following the introduction of isoprenaline and fenoterol in the 1960s–70s. Troublesome side effects with b2-agonists are relatively rare. The main limiting factor is tremor which again is dose related. There have been persistent concerns regarding regular usage of SABAs and LABAs in terms of over all

asthma control. These concerns arose from a number of studies where regular (generally four times a day) SABAs were used: in these studies overall asthma control and/or lung function deteriorated in groups taking regular as opposed to as required treatment. These studies were in part responsible for the advice to use SABAs on an as-required rather than regular basis in the management of asthma. Very recently these concerns have extended to LABAs resulting in a black box warning from the FDA. One possible explanation underlying this effect is the presence of polymorphic variation within the b2-adrenoceptor. The receptor is highly polymorphic, with nine known single nucleotide polymorphisms (SNPs) in its coding region. Four of these code for amino acid substitutions of which three may have functional relevance (Arg/Gly16, Gln/ Glu27, Thr/Ile164). The Thr164 polymorphism alters agonist-binding properties of the receptor but is rare (allelic frequency 3% in Caucasian populations). The Arg16 polymorphism has been associated in several studies with worse overall asthma control and in a recent prospective trial individuals given regular salbutamol who were Arg16 homozygous showed markedly reduced responses compared with individuals homozygous for the Gly16 form of the receptor. Arg16 homozygous individuals account for roughly 15% of the Caucasian population. See also: Adrenergic Receptors. Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. G-Protein-Coupled Receptors. Leukocytes: Mast Cells and Basophils.

Further Reading British Thoracic Society, Scottish Intercollegiate Guidelines Network (2003) British guideline on the management of asthma. Thorax 58(supplement I): 1–94. Drazen JM, Israel E, Boushey HA, et al. (1996) Comparison of regularly scheduled with as-needed use of albuterol in mild asthma. New England Journal of Medicine 335: 841–847.

292 BRONCHODILATORS / Theophylline Fenech A and Hall IP (2002) Pharmacogenetics of Asthma. British Journal of Clinical Pharmacology 53(1): 3–15. Hall IP (2004) The beta-agonist controversy revisited. Lancet 363(9404): 183–184. Hall IP and Tattersfield AE (1992) b-agonists. In: Clark TJ, Godfrey S, and Lee T (eds.) Asthma, 3rd edn., pp. 341–366. London: Chapman and Hall. Israel E, Chinchilli VM, Ford JG, et al. (2004) Use of regularly scheduled albuterol treatment in asthma: genotype-stratified, randomised, placebo-controlled cross-over trial. Lancet 364: 1505–1512. Kobilka BK, Dixon RAF, Frielle T, et al. (1987) cDNA for the human b2 adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor. Proceedings of the National Academy of Sciences of the United States of America 84: 46–50. National Collaborating Centre for Chronic Conditions (2004) Chronic obstructive pulmonary disease: national clinical guideline on management of chronic obstructive pulmonary disease in adults in primary and secondary care. Thorax 59(supplement I): 1–232. Reihsaus E, Innis M, MacIntyre N, and Liggett SB (1993) Mutations in the gene encoding for the beta 2-adrenergic receptor in normal and asthmatic subjects. American Journal of Respiratory Cell and Molecular Biology 8: 334–339. Sears MR, Taylor DR, Print CG, et al. (1990) Regular inhaled bagonist treatment in bronchial asthma. Lancet 336: 1391–1396. Tattersfield AE, Knox AJ, Britton JR, and Hall IP (2002) Asthma. Lancet 360: 1313–1322. van Schayck CP, Dompeling E, van Herwaarden CLA, et al. (1991) Bronchodilator treatment in moderate asthma or chronic bronchitis: continuous or on demand? A randomized controlled study. British Medical Journal 303: 1426–1431. Vathenen AS, Higgins BG, Knox AJ, et al. (1988) Rebound increase in bronchial responsiveness after treatment with inhaled terbutaline. Lancet 1: 554–558.

Theophylline P J Barnes, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

not controlled by bronchodilator therapy. Side effects are related to plasma concentrations and include nausea, vomiting, and headaches due to phosphodiesterase inhibition and, at higher concentrations, to cardiac arrhythmias and seizures due to adenosine A1 receptor antagonism.

Theophylline has been used in the treatment of airway disease since 1930. Indeed, theophylline is still the most widely used antiasthma therapy worldwide because it is inexpensive. Theophylline became more useful with the availability of rapid plasma assays and the introduction of reliable slow-release preparations. However, the frequency of side effects and the relatively low efficacy of theophylline have recently led to reduced usage since inhaled b2 agonists are far more effective as bronchodilators and inhaled steroids have a greater anti-inflammatory effect. However, in patients with severe asthma and chronic obstructive pulmonary disease (COPD), it remains a very useful drug. There is increasing evidence that theophylline has an anti-inflammatory or immunomodulatory effect.

Chemical Structure Theophylline is a methylxanthine similar in structure to the common dietary xanthines, caffeine and theobromine (Figure 1). Several substituted derivatives have been synthesized but none has any advantage over theophylline, apart from the 3-propyl derivative enprofylline, which is more potent as a bronchodilator and may have fewer toxic effects. Many salts of theophylline have also been marketed, with the most common being aminophylline, which is the ethylenediamine salt used to increase solubility at neutral pH. Salts such as choline theophyllinate do not have any advantage and others, such as acepifylline, are virtually inactive so that theophylline remains the only methylxanthine in clinical use.

Mode of Action Abstract Theophylline is a methylxanthine that has been used to treat airway diseases for more than 70 years. It was originally used as a bronchodilator, but the relatively high doses required were associated with frequent side effects, so its use declined as inhaled b2 agonists became more widely used. Recently, it has been shown to have anti-inflammatory effects in asthma and chronic obstructive pulmonary disease (COPD) at lower concentrations. The molecular mechanism of bronchodilatation is non-specific phosphodiesterase inhibition, but the anti-inflammatory effect may be due to histone deacetylase activation, resulting in switching off of activated inflammatory genes. Theophylline is given systemically (orally as slow-release preparations for chronic treatment and intravenously for acute exacerbations of asthma), and blood concentrations are related to hepatic metabolism. This may be increased or decreased in several diseases and by concomitant drug therapy. Theophylline is usually used as an add-on therapy in asthma patients not well controlled on inhaled corticosteroids and in COPD patients with severe disease

Although theophylline has been in clinical use for more than 70 years, its mechanism of action is still uncertain. In addition to its bronchodilator action, theophylline has many other actions that may be relevant to its antiasthma effect (Figure 2). Many of these effects are seen only at high concentrations that far exceed the therapeutic range. There is increasing evidence that theophylline has anti-inflammatory effects, with a reduction in eosinophils and T lymphocytes in asthma and of neutrophils in COPD. Conversely, withdrawal of theophylline results in clinical deterioration and increased inflammation in patients with severe asthma and COPD. Several molecular mechanisms of action have been proposed for theophylline (Table 1). Phosphodiesterases, which break down cyclic nucleotides (cyclic

BRONCHODILATORS / Theophylline 293 O H3C

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Figure 1 Structures of naturally occurring methylxanthines.

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Figure 2 The inhibitory effect of theophylline on phosphodiesterases (PDE) may result in bronchodilatation and inhibition of inflammatory cells. ATP, adenosine triphosphate; AMP, adenosine monophosphate; PKA, protein kinase A; GTP, guanosine triphosphate; GMP, guanosine monophosphate; PKG, protein kinase G.

Table 1 Mechanisms of action of theophylline Phosphodiesterase inhibition (nonselective) Adenosine receptor antagonism (A1, A2A, and A2B receptors) Increased interleukin-10 release Stimulation of catecholamine (epinephrine) release Mediator inhibition (prostaglandins, tumor necrosis factor-a) Inhibition of intracellular calcium release Inhibition of NF-kB (decreased nuclear translocation) Increased apoptosis Increased histone deacetylase activity (increased efficacy of corticosteroids)

AMP and cyclic GMP) in the cell, are weakly inhibited by theophylline, and this accounts for the bronchodilator effect of theophylline (predominantly phosphodiesterase-3 (PDE3) and PDE5) (Figure 2). However, it is unlikely to account for the nonbronchodilator effects of theophylline that are seen at subbronchodilator doses. Theophylline inhibits adenosine receptors at therapeutic concentrations and an inhibitory action on A2B receptors may account for its inhibitory effect on mast cells. Adenosine antagonism is unlikely to account for the anti-inflammatory effects

of theophylline but may be responsible for serious side effects, including cardiac arrhythmias and seizures. Theophylline enhances the release of interleukin-10, which has anti-inflammatory effects from inflammatory cells, but this is secondary to PDE inhibition and requires high concentrations. It inhibits the proinflammatory transcription factor nuclear factor kappa B (NF-kB) but this also requires concentrations above the therapeutic range. Theophylline promotes apoptosis in eosinophils, neutrophils, and T lymphocytes associated with a reduction in the antiapoptotic protein Bcl-2. Recruitment of histone deacetylase-2 (HDAC2) by glucocorticoid receptors switches off inflammatory genes. Theophylline is an activator of HDAC at therapeutic concentrations, thus enhancing the anti-inflammatory effects of corticosteroids (Figure 3). This mechanism is independent of PDE inhibition or adenosine antagonism, and the antiinflammatory effects of theophylline are inhibited by an HDAC inhibitor trichostatin A. Low doses of theophylline increase HDAC activity in bronchial biopsies of asthmatic patients and correlate with the reduction in eosinophil numbers in the biopsy.

294 BRONCHODILATORS / Theophylline Inflammatory stimuli (e.g., IL-1, TNF-)

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+

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Figure 3 Theophylline directly activates histone deacetylases (HDACs), which deacetylate core histones that have been acetylated by the histone acetyltransferase (HAT) activity of coactivators such as CREB-binding protein (CBP). This results in suppression of inflammatory genes and proteins, such as granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-8 (IL-8), that have been switched on by proinflammatory transcription factors such as nuclear factor kappa B (NF-kB). Corticosteroids also activate HDACs, but they do so through a different mechanism resulting in the recruitment of HDACs to the activated transcriptional complex via activation of the glucocorticoid receptors (GR), which function as a molecular bridge. This predicts that theophylline and corticosteroids may have a synergistic effect in repressing inflammatory gene expression.

Pharmacokinetics

Table 2 Factors affecting clearance of theophylline

There is a close relationship between improvement in airway function and serum theophylline concentration. Below 10 mg l 1 bronchodilatation is minimal and above 25 mg l 1 additional benefits are outweighed by side effects, so the therapeutic range for bronchodilatation was usually taken as 10– 20 mg l 1. It is now clear that theophylline has antiasthma effects other than bronchodilatation and that these may be seen below 10 mg l 1. A more useful therapeutic range is 5–15 mg l 1. The dose of theophylline required to give these therapeutic concentrations varies between subjects, largely because of differences in clearance. In addition, there may be differences in bronchodilator response to theophylline and, with acute bronchoconstriction, higher concentrations may be required to produce bronchodilatation. Theophylline is rapidly and completely absorbed, but there are large interindividual variations in clearance due to differences in hepatic metabolism (Table 2). Theophylline is metabolized in the liver by the cytochrome P450 microsomal enzyme system (mainly CYP1A2), and a large number of factors may influence hepatic metabolism. Increased clearance is seen in children (1–16 years old) and in cigarette and marijuana smokers. Concurrent administration of phenytoin and phenobarbitone increases activity of P450, resulting in increased metabolic breakdown so that higher doses may be required.

Increased clearance Enzyme induction (rifampicin, phenobarbitone, and ethanol) Smoking (tobacco and marijuana) High-protein, low-carbohydrate diet Barbecued meat Childhood Decreased clearance Enzyme inhibition (cimetidine, erythromycin, ciprofloxacin, allopurinol, zileuton, and zafirlukast) Congestive heart failure Liver disease Pneumonia Viral infection and vaccination High-carbohydrate diet Old age

Reduced clearance is found in liver disease, pneumonia, and heart failure, and doses need to be reduced to half and plasma levels monitored carefully. Increased clearance is also seen with certain drugs, including erythromycin, certain quinolone antibiotics (ciprofloxacin but not ofloxacin), allopurinol, cimetidine (but not ranitidine), fluoxamine, and zafirlukast, which interfere with cytochrome P450 function. Thus, if a patient on maintenance theophylline requires a course of erythromycin, the dose of theophylline should be halved. Viral infections and vaccination may also reduce clearance, and this may be particularly important in children. Because of these variations in clearance, individualization of

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theophylline dosage is required and plasma concentrations should be measured 4 h after the last dose with slow-release preparations when steady state has usually been achieved. There is no significant circadian variation in theophylline metabolism, although there may be delayed absorption at night, which may relate to the supine posture.

Role of Theophylline in Respiratory Medicine Acute Severe Asthma

In patients with acute severe asthma, intravenous aminophylline is less effective than nebulized b2 agonists and should therefore be reserved for those patients who fail to respond to b agonists. Theophylline should not be added routinely to nebulized b2 agonists since it does not increase the bronchodilator response and may only increase their side effects. It is of little proven benefit in exacerbations of COPD.

Theophylline is a less preferred option than inhaled corticosteroids and is recommended as a second-line choice of controller in the management of asthmatic patients. Although LABAs are more effective as an add-on therapy, theophylline is considerably less expensive and may be the only affordable add-on treatment when the costs of medication are limiting. Chronic Obstructive Pulmonary Disease

Theophylline is still used as a bronchodilator in COPD, but inhaled anticholinergics and b2 agonists are preferred. Theophylline tends to be added to these inhaled bronchodilators in more severe patients and has been shown to give additional clinical improvement when added to a LABA. A theoretical advantage of theophylline is that its systemic administration may have effects on small airways, resulting in a reduction of hyperinflation and thus a reduction in dyspnea. Sleep Apnea

Asthma

Theophylline has little or no effect on bronchomotor tone in normal airways but reverses bronchoconstriction in asthmatic patients, although it is less effective than inhaled b2 agonists and is more likely to have unwanted effects. Theophylline and b2 agonists have additive effects, even if true synergy is not seen, and there is evidence that theophylline may provide an additional bronchodilator effect even when maximally effective doses of b2 agonist have been given. This means that if adequate bronchodilatation is not achieved by a b agonist alone, theophylline may be added to the maintenance therapy with benefit. Addition of low-dose theophylline to either high- or low-dose inhaled corticosteroids in patients who are not adequately controlled provides better symptom control and lung function than doubling the dose of inhaled steroid, although it is less effective as an add-on therapy than a long-acting b2 agonist (LABA). Theophylline may be useful in patients with nocturnal asthma since slow-release preparations are able to provide therapeutic concentrations overnight, although it is less effective than a LABA. Studies have also documented steroid-sparing effects of theophylline. Although theophylline is less effective than a b2 agonist and corticosteroids, a minority of asthmatic patients appear to derive unexpected benefit, and even patients on oral steroids may show a deterioration in lung function when theophylline is withdrawn. Theophylline has been used as a controller in the management of mild persistent asthma, although it is usually found to be less effective than low doses of inhaled corticosteroids.

Theophylline has been used to treat sleep apnea in adults and apnea in neonates because it increases ventilation rate. However, there is no convincing evidence that it is effective for these indications, and it cannot be routinely recommended.

Adverse Effects and Contraindications Unwanted effects of theophylline are usually related to plasma concentration and tend to occur when plasma levels exceed 20 mg l 1. However, some patients develop side effects even at low plasma concentrations. To some extent, side effects may be reduced by gradually increasing the dose until therapeutic concentrations are achieved. The most common side effects are headache, nausea, and vomiting (due to inhibition of PDE4), abdominal discomfort, and restlessness (Table 3). There may also be increased acid secretion and diuresis (due to inhibition of adenosine A1 receptors). There was concern that theophylline, even at therapeutic concentrations, may lead to behavioral disturbance and learning difficulties in schoolchildren, although

Table 3 Side effects of theophylline Nausea and vomiting Headaches Gastric discomfort Diuresis Behavioral disturbance (?) Cardiac arrhythmias Epileptic seizures

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it is difficult to design adequate controls for such studies. At high concentrations, cardiac arrhythmias may occur as a consequence of PDE3 inhibition and adenosine A1 receptor antagonism, and at very high concentrations seizures may occur (due to central A1 receptor antagonism). Use of low doses of theophylline that give plasma concentrations of 5–10 mg l 1 largely avoids side effects and drug interactions and makes it unnecessary to monitor plasma concentrations (unless checking for compliance). Care must be taken when using theophylline in children, elderly patients, those taking drugs that inhibit cytochrome P450, and those with heart and liver disease because of the pharmacokinetic considerations discussed previously.

Future Directions The use of theophylline has been declining, partly because of the problems with side effects but mainly because more effective therapy with inhaled corticosteroids has been introduced. Oral theophylline is still a useful treatment in some patients with difficult asthma and appears to have effects beyond those provided by steroids. Rapid-release theophylline preparations are cheap and are the only affordable antiasthma medication in some developing countries. Theophylline has anti-inflammatory effects at doses that are lower than those needed for bronchodilatation, and plasma levels of 5–10 mg l 1 are now recommended instead of the previously recommended 10–20 mg l 1. Because the molecular mechanisms for the anti-inflammatory effects of theophylline are better understood (HDAC activation), there is a strong scientific rationale for combining low-dose theophylline with inhaled corticosteroids, particularly in patients with more severe asthma. The synergistic effect of low-dose theophylline and corticosteroids on inflammatory gene expression may

account for the add-on benefits of theophylline in asthma, whereas in COPD theophylline appears to overcome corticosteroid resistance in vitro and therefore may be a useful anti-inflammatory therapy in the future. New drugs based on this molecular action of theophylline may also be developed in the future. See also: Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Cyclic Nucleotide Phosphodiesterases. Gene Regulation.

Further Reading Barnes PJ (1996) The role of theophylline in severe asthma. European Respiratory Review 6: 154S–159S. Barnes PJ (2003) Theophylline: new perspectives on an old drug. American Journal of Respiratory and Critical Care Medicine 167: 813–818. Barr RG, Rowe BH, and Camargo CA Jr (2003) Methylxanthines for exacerbations of chronic obstructive pulmonary disease: meta-analysis of randomised trials. British Medical Journal 327: 643. Cosio BG, Tsaprouni L, Ito K, et al. (2004) Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages. Journal of Experimental Medicine 200: 689–695. Hansel TT, Tennant RC, Tan AJ, et al. (2004) Theophylline: mechanism of action and use in asthma and chronic obstructive pulmonary disease. Drugs of Today (Barcelona) 40: 55–69. Kankaanranta H, Lahdensuo A, Moilanen E, and Barnes PJ (2004) Add-on therapy options in asthma not adequately controlled by inhaled corticosteroids: a comprehensive review. Respiratory Research 5: 17. Rabe K, Magnussen H, and Dent G (1996) Theophylline and selective PDE inhibitors as bronchodilators and smooth muscle relaxants. European Respiratory Journal 8: 637–642. Ram FS, Jones PW, Castro AA, et al. (2002) Oral theophylline for chronic obstructive pulmonary disease. Cochrane Database of Systematic Reviews CD003902. Shah L, Wilson AJ, Gibson PG, and Coughlan J (2003) Long acting beta-agonists versus theophylline for maintenance treatment of asthma. Cochrane Database of Systematic Reviews CD001281. Weinberger M and Hendeles L (1996) Theophylline in asthma. New England Journal of Medicine 334: 1380–1388.

BRONCHOMALACIA AND TRACHEOMALACIA P N Mathur and J R Ladwig, Indiana University Hospital, Indianapolis, IN, USA & 2006 Elsevier Ltd. All rights reserved.

tracheobronchomalacia. Diagnosis is best performed by bronchoscopy but improvements in computed tomography has made radiographic diagnosis easier. Treatment varies from various means of stenting the airway to surgical correction.

Abstract Tracheomalacia and bronchomalacia are a loss of cartilaginous support of the trachea and large airways. This can occur as a congenital defect or as a result of acquired disease processes. The loss of cartilaginous support leads to airway obstruction upon expiration. Symptoms of dyspnea and cough result from

Introduction Tracheomalacia is a condition of weakened cartilaginous support of the trachea. The term ‘bronchomalacia’ refers to loss of supportive structure for

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the large airways. With the loss of structural support, the trachea does not maintain its diameter and the membranous portion of the trachea collapses anteriorly, causing obstruction of airflow during expiration.

Etiology Tracheomalacia and bronchomalacia can be the result of multiple etiologies. In infants, the cause is often a congenital defect in cartilaginous development which usually resolves spontaneously as the child reaches 6 months of age and older. It may also be the result of congenital vascular rings causing compression of the airways resulting in abnormal development or erosion of cartilaginous rings. In adults, tracheobronchomalacia may also be the result of previously unrecognized congenital abnormalities, or acquired anatomic or pathologic processes. Substernal goiter may present with cough in tracheomalacia. The enlargement of thyroid tissue can lead to compressive erosion of tracheal rings. Neoplasm may also exert pressure on the tracheal rings or cause destruction by local invasion resulting in tracheobronchomalacia. Inflammatory processes may also be a cause of tracheomalacia. Relapsing polychondritis, an autoimmune disease directed against cartilage, may affect the larynx and cartilaginous rings of the large airways. It is most often seen in the fourth decade; airway symptoms are more common amongst women with the disease. Patients with severe chronic obstructive pulmonary disease (COPD) may develop atrophy of cartilage in the airway, leading to easy collapsibility of airways. The diagnosis of tracheomalacia in a patient with COPD may go unrecognized until progression of disease prompts bronchoscopy or other imaging. Patients who have had prolonged intubation or require tracheostomy may also develop tracheomalacia. The local pressure effect of the inflated balloon at the distal end of the tube can cause local inflammation, overdistention, or local tissue ischemia resulting in erosion of the cartilaginous rings.

Clinical Features Diagnosis of tracheobronchomalacia includes a thorough history and physical examination to exclude other conditions or elicit clinical features typical of tracheobronchomalacia. Clinical findings are nonspecific and often manifest in dyspnea on exertion and cough. The cough of tracheobronchomalacia has been characterized as seal-like. Diagnosis of tracheobronchomalacia is aided by pulmonary function testing, imaging, and direct visualization via bronchoscopy. Pulmonary Function Testing

Patients with tracheomalacia show reduced expiratory flow measurements. Flow–volume curves often demonstrate expiratory flow limitation (Figure 1). Inspiratory flow is preserved while the expiratory loop is similar to that of COPD. Diagnostic Imaging of the Trachea

As a general rule, plain chest radiographs are not useful for diagnosis of tracheomalacia. In extreme cases of airway narrowing, a large air column above the obstruction may be seen. A simple computed tomography (CT) scan of the chest may not be useful for tracheobronchomalacia. However, it is very useful for identification of

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Pathology Congenital tracheobronchomalacia represents a failure or incomplete development of the cartilaginous rings of the airway. In adults, tracheobronchomalacia is acquired more commonly as described above. The acquired insult often leads to compression and local destruction of the tracheal rings.

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extrinsic causes of tracheal compression which may result in tracheobronchomalacia such as substernal goiter, vascular abnormalities, and neoplasm. One method of making CT more beneficial in diagnosing tracheobronchomalacia is to compare inspiratory and expiratory images. This aids in demonstrating the dynamic narrowing of the trachea and it may then be more obvious on the CT scan (Figures 2(a) and 2(b)). Newer advances in CT imaging show significant sensitivity and specificity for identifying airway stenosis. A ‘virtual bronchoscopy’ can be performed with the images acquired from a routine CT scan that is then reconstructed through computer software (Figures 2(c) and 2(d)). The imaging is enhanced when a multidetector CT image is used instead of a single detector one. There are different methods to render the data received by the CT scan. Data may be surface or volume rendered. Surface rendering is faster and requires less computer power. However, volume rendering provides more

detail of the mucosal-airway interface allowing more detail of the mucosa. Virtual bronchoscopy has been found to have high sensitivity (90%) and specificity (96.6%) for central airway stenosis. The virtual bronchoscopy provides the benefit of detailed imaging in patients that might not be candidates for routine bronchoscopy. It also provides the bronchoscopist a noninvasive method to acquire information allowing for planning prior to an interventional procedure or foregoing a procedure for lesions not amenable to repair. The gold standard diagnostic tool for tracheobronchomalacia is bronchoscopy. If a patient is able to tolerate bronchoscopy with minimal sedation, various respiratory maneuvers can be performed (forced expiration, cough) to exaggerated or elicit airway obstruction. Once the diagnosis of tracheobronchomalacia is made, consideration of therapeutic options is undertaken. Correction of underlying extrinsic processes (substernal goiter, vascular rings, and neoplasm),

Figure 2 (a) Inspiration, (b) expiration, (c) virtual bronchoscopy inspiration, (d) virtual bronchoscopy expiration. Courtesy of C Mayer, University of Cincinnati.

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systemic illness (relapsing polychondritis), and airway disease (COPD) should be treated appropriately. If the causative process cannot be eliminated or when the tracheomalacia already results in airway compromise, several options exist to palliate the obstruction.

airway pressure (CPAP) to maintain a patent airway. In one case of relapsing polychondritis, it provided a temporary measure, delaying the time to invasive procedures. If further investigation identifies the type of patient most likely to benefit from CPAP, it may prove a useful bridge prior to a more invasive therapy.

Management and Current Therapy Balloon Bronchoplasty

Surgery

Airways may be dilated by rigid or inflatable balloon dilators. The modality is best used for extrinsic or intrinsic compression of the airways. Balloon bronchoplasty is easily performed through a flexible bronchoscope. Alone, the modality does not provide sustained benefit. However, it has been described as a useful tool in optimizing the airway caliber prior to stent placement.

Multiple surgical approaches to tracheomalacia have been described. One surgical procedure used for treatment of tracheobroncomalacia is a membranous-wall tracheoplasty. This is done through a right thoracotomy. The procedure involves sewing a polypropylene mesh to the membranous wall of the trachea which then provides external support of the tracheal wall. Another external wall stent procedure has been investigated in an animal model using reabsorbable stents. Although still experimental, this may provide another alternative in the right patient population.

Airway Stents

Tracheobronchomalacia is one of the indications for airway stenting, as described by the ERS/ATS statement on interventional pulmonology. There are a number of different types of airway stents, each with pros and cons. Generally stents can be categorized as silicone, metal, or a hybrid of the two materials. Silicone stents have the benefit of being cheaper and more easily removed. However, they require knowledge of rigid bronchoscopy for placement and have higher rates of migration. Currently, the most widely used stent is the Dummon stent, which is a silicone stent that has studs on the outer surface to retard migration. Metal stents have the advantage of being placed via flexible bronchoscopy and are less prone to migration. Metal stents may be uncovered or covered by a silastic or polyurethane coating. However, uncovered stents should not be used in benign disease such as tracheobronchomalacia, as they allow ingrowth of malignant or granulation tissue. Metal stents are very difficult to remove, may fracture, and pose a risk if future interventional bronchoscopy is needed (laser, APC) because of the potential for conduction of current/heat or airway fire. In choosing a stent, the airway should be evaluated by bronchoscopy and/or CT scan to determine the extent of the obstruction. The optimal stent is one that exceeds the margins of the stenosis and whose diameter is larger than that of the airway. Placement of a stent is expected to bridge the extent of the abnormal airway. Inadequate stenting may only result in moving the ‘choke point’, the area of airflow obstruction, distal to the stent. One method of noninterventional stenting still under investigation is the use of continuous positive

See also: Bronchiectasis. Bronchoscopy, General and Interventional. Relapsing Polychondritis. Trauma, Chest: Postpneumonectomy Syndrome. Upper Airway Obstruction.

Further Reading Adliff M, et al. (1997) Treatment of diffuse tracheomalacia secondary to relapsing polychondritis with continuous positive airway pressure. Chest 112: 1701–1703. Aquino SL, Shepard J-AO, Ginns LC, et al. (2001) Acquired tracheomalacia: detection by expiratory ct scan. Journal of Computer Assisted Tomography 25(3): 394–399. Bolliger CT and Mathur PN (2002) ERS/ATS statement on interventional pulmonology. European Respiratory Journal 19: 356–373. Davis S, et al. (1998) Effect of continuous positive airway pressure on forced expiratory flows in infants with tracheomalacia. American Journal of Respiratory and Critical Care Medicine 158: 148–152. Ernst A, et al. (2004) Central airway obstruction. American Journal of Respiratory and Critical Care Medicine 169: 1278–1297. Hoppe H, et al. (2004) Grading airway stenosis down to the segmental level using virtual bronchoscopy. Chest 125: 704–711. Hopper KD, et al. (2000) Mucosal detail at CT virtual reality: surface versus volume rendering. Radiology 214: 517–522. Miyazawa T, et al. (2004) Stenting at the flow-limiting segment in tracheobronchial stenosis due to lung cancer. American Journal of Respiratory and Critical Care Medicine 169: 1096–1102. Murray JF and Nadel JA (eds.) Textbook of Respiratory Medicine, 3rd edn., pp. 1368–1373. Sewall A, Gregory K, et al. (2003) Comparison of resorbable poly-L-lactic acid – polyglycolic acid and internal palmaz stents for the surgical correction of severe tracheomalacia. The Annals of Otology, Rhinology, and Laryngology 112: 515–521. Wright CD (2003) Tracheomalacia. Chest Surgery Clinics of North America 13: 349–357.

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BRONCHOPULMONARY DYSPLASIA E Bancalari, University of Miami School of Medicine, Miami, FL, USA

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Bronchopulmonary dysplasia (BPD) is one of the most common sequelae in premature infants who survive after prolonged mechanical ventilation. Its pathogenesis is multifactorial and includes prematurity, perinatal infections, pulmonary volutrauma, oxygen toxicity, and increased pulmonary blood flow due to patent ductus arteriosus. It is characterized by chronic respiratory failure with abnormalities in lung function that can persist into adulthood. This is due to severe alterations in lung development characterized by decreased alveolar and capillary formation plus emphysema, fibrosis, and airway obstruction in more severe cases. The prevention of BPD is based on the avoidance of all the factors that are implicated in its pathogenesis.

Bronchopulmonary dysplasia (BPD) was first described by Northway and co-workers in 1967 and has become a major complication in premature infants who require prolonged mechanical ventilation. The natural course of severe respiratory distress syndrome (RDS) in the premature infant was altered in the 1960s by the introduction of mechanical ventilation. As a result, smaller and sicker infants survive, but many of these survivors are left with chronic lung damage. These infants frequently remain oxygen- and ventilator-dependent for prolonged periods of time, and the mortality rate can be as high as 30–40%. In surviving infants, pulmonary function may remain abnormal for years, and they have increased risk of neurodevelopmental sequelae and impaired growth curves. The incidence of BPD is closely and inversely related to both, birth weight and gestational age (Figure 1). For this reason, with increasing survival of extremely premature infants, the number of patients at risk for developing BPD has increased.

Pathology Most of the pathologic descriptions of BPD represent the most severe form of chronic lung damage since those infants with milder forms of BPD rarely die. Macroscopically, the lungs are firm, heavy, and dark in color, with a grossly abnormal appearance showing emphysematous areas alternating with areas of collapse. Histologically, the lungs show areas of emphysema, with abnormal alveoli that have coalesced into larger cystic areas, surrounded by areas of atelectasis (Figure 2). Widespread bronchial and

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Birth weight (g) Figure 1 Incidence of BPD (% of total births) in the NICHD Neonatal Research Network, Jan. 1995 to Dec. 1996. PMA, post menstrual age. Reproduced from Lemons J, Bauer CR, Oh W, et al. (2001) Very low birth weight outcomes of the National Institute of Child Health and Human Development Research Network January 1995 through December 1996. Pediatrics 107: 1–8.

Figure 2 Low-magnification view from a lung with BPD showing areas of emphysema alternating with areas of partial collapse. Reproduced from Kluwer Academic Publishers Advances in Perinatal Medicine, 1982, p. 181, Barotrauma to the lung, Bancalari E and Goldman SL, figure 29, with kind permission of Springer Science and Business Media.

bronchiolar mucosal hyperplasia and metaplasia reduce the lumen in many small airways. In addition, there is interstitial edema and an increase in fibrous tissue with focal thickening of the interstitial spaces. Lymphatics may be dilated and torturous. There may be vascular evidence of pulmonary hypertension, such as medial muscle hypertrophy, elastic degeneration, and reduction in the branching of the pulmonary vascular bed. The heart frequently shows evidence of biventricular hypertrophy. Infants dying with severe BPD show a marked reduction in the number of alveoli and capillaries, with a reduction in the gas exchange surface area. Infants who have mild

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BPD at the time of death have only mild or moderate alveolar septal fibrosis, lack of severe airway epithelial lesions, and normal-appearing pulmonary vessels. Nonetheless, they show evidence of inhibition of acinar and capillary development.

Clinical Features The diagnosis of BPD is based on clinical and radiographic manifestations, which are not specific. With rare exceptions, BPD occurs in preterm infants and is preceded by the use of prolonged mechanical ventilation. Radiographic findings include hyperinflation and nonhomogeneity of pulmonary tissues, with fine or coarser densities extending to the periphery in the more severe forms (Figure 3). In milder forms, the radiographic changes are also milder, revealing mainly diffuse haziness. Once lung damage has occurred, these infants continue to require mechanical ventilation and increased oxygen concentration for months or sometimes years. Because of the increased use of antenatal steroids and exogenous surfactant, many small infants have mild initial respiratory disease and require ventilation with low pressures and oxygen concentration. After a few days or weeks of mechanical ventilation, these infants frequently show progressive deterioration in lung function, an increase in their ventilatory and oxygen requirements accompanied by signs of respiratory failure, and ultimately develop BPD.

This deterioration is frequently triggered by bacterial or viral infections or respiratory failure secondary to a patent ductus arteriosus (PDA). Infants with BPD who survive show a slow but steady improvement in lung function and roentgenographic changes, with gradual weaning from ventilator and oxygen therapy. Signs of respiratory distress, such as chest retractions and tachypnea, frequently persist long after extubation. More severe BPD may evolve into progressive respiratory failure, and even death, as a result of severe lung damage and pulmonary hypertension or intercurrent infections. Some of these infants may also develop anastomoses between systemic and pulmonary circulations, which may aggravate their pulmonary hypertension. Acute pulmonary infection, either bacterial or viral, frequently complicates the course of BPD and, in many cases, is the precipitating cause of death. Because of chronic hypoxia and higher energy expenditure required by their increased work of breathing, weight gain in infants with BPD is usually below normal, even when receiving appropriate caloric intake for their age. In addition, poor feeding tolerance and a tendency toward fluid retention and pulmonary edema often requires fluid restriction as well as diuretic therapy, further limiting the calories that can be provided.

Pathogenesis Because BPD occurs almost exclusively in premature infants who have received mechanical ventilation and increased oxygen concentrations, prematurity, baro/volutrauma, and oxygen toxicity have been considered crucial factors in BPD pathogenesis. Other factors that may play an important role in BPD pathogenesis include perinatal infections, pulmonary edema resulting from a PDA or excessive fluid administration, and nutritional deficiencies. Prematurity

BPD occurs almost exclusively in the extremely premature infant. This suggests that the vulnerability of the very preterm infant may be related to the immature state of lung development. It is likely that premature birth followed by therapeutic interventions disrupts lung development during this crucial period, producing an interruption and/or disruption in the development of alveoli, capillaries, and lung matrix. Barotrauma/Volutrauma Figure 3 Chest radiograph from an infant with bronchopulmonary dysplasia, showing areas of mild hyperinflation alternating with coarse densities.

Almost all infants who develop BPD have received prolonged mechanical ventilation; therefore, it is


likely that mechanical trauma also plays an important role in the pathogenesis of BPD. The presence of an endotracheal tube may also contribute to BPD pathogenesis by increasing the risk of pulmonary infections. Studies on preterm experimental animals have demonstrated that only a few breaths with excessive tidal volumes given prior to surfactant replacement produce lung damage with a decrease in lung compliance. Oxygen Toxicity

Pulmonary oxygen toxicity is also considered an important factor in the pathogenesis of BPD. High inspired oxygen concentrations can increase the production of cytotoxic oxygen free radicals, which overwhelm the antioxidant defenses in the capillary endothelial cells and the alveolar epithelial cells of the premature lung because of incomplete development of the pulmonary antioxidant enzyme system. The precise concentration of oxygen that is toxic to the premature lung is unknown, but it is possible that any concentration in excess of room air may increase the risk of lung damage when administered over prolonged periods of time. Infection and Inflammation

Evidence has been mounting to support a role for infection and inflammation in the pathogenesis of BPD, particularly in very small infants who develop BPD after receiving prolonged mechanical ventilation for poor respiratory effort rather than because of severe underlying lung disease. The evidence of an association between ureaplasma urealyticum tracheal colonization and the development of BPD has been inconsistent. However, maternal infections, specifically chorioamnionitis, are associated with an increased risk of BPD. Several inflammatory cytokines are present in higher concentrations in fetal cord blood and in the amniotic fluid of mothers who deliver infants who subsequently develop BPD. An inflammatory response in the lung can also be triggered by other factors, including oxygen free radicals, ventilation with excessive tidal volumes, and increased pulmonary blood flow due to a PDA. Among the markers of inflammation found in high concentrations in tracheobronchial secretions in infants who develop BPD are neutrophils, macrophages, leukotrienes, platelet-activating factor (PAF), interleukin (IL)-6, IL-8, and tumor necrosis factor. The increased concentration of inflammatory mediators may be responsible for the bronchoconstriction, vasoconstriction, and increased vascular permeability characteristic of these infants. The potential role of inflammation in the pathogenesis of

BPD is supported by the documented beneficial effects of steroids in these infants. Patent Ductus Arteriosus and Pulmonary Edema

Infants with RDS who receive greater fluid intake or do not have a diuretic phase in early life have a higher incidence of BPD. High fluid intake increases the incidence of PDA, which then produces increased pulmonary blood flow, an increase in interstitial lung fluid, and causes a decrease in pulmonary compliance and increased resistance. This deterioration in lung mechanics may prolong the requirement for mechanical ventilation and high inspired oxygen concentrations, thereby increasing the risk of BPD. In addition, the increased pulmonary blood flow can induce endothelial damage with neutrophil margination and activation in the lung and contribute to the progression of the inflammatory cascade. These interrelated factors may explain the strong association between the duration of the PDA and the increased risk of BPD. Infants with established BPD have a predisposition for fluid accumulation in their lungs. Capillary permeability may be increased due to the oxygen toxicity, volutrauma, or infection. Infants with BPD can also have increased plasma levels of vasopressin with a reduced urine output and decrease in free water clearance. With excessive accumulation of lung fluid, lung function is further compromised, perpetuating a cycle in which more aggressive respiratory support is required, thereby resulting in additional lung injury. Figure 4 shows the relative influence of different factors in the pathogenesis of BPD. Increased Airway Resistance

Infants with severe BPD frequently have a marked increase in airway resistance. This can impair distribution of the inspired gas and thereby favor uneven lung expansion. The airway obstruction may be secondary to bronchiolar epithelial hyperplasia, metaplasia, and mucosal edema, and it may also relate to pulmonary edema secondary to PDA or fluid overload. These infants may also have bronchoconstriction resulting from smooth muscle hypertrophy. Other possible factors producing increased airway resistance in infants with BPD include inflammatory mediators such as leukotrienes and PAF, which have been found in high concentrations in the airways of infants with BPD. Finally, tracheobronchomalacia, which may be present in some infants with severe BPD, can produce marked airway obstruction, particularly during periods of agitation and increased intrathoracic pressure.

BRONCHOPULMONARY DYSPLASIA 303 Other Factors

OR for BPD

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Figure 4 Perinatal and postnatal risk factors for the development of BPD defined as X28 days’ duration of oxygen-dependency during hospitalization. Obtained by logistic regression analysis from all extremely premature infants born at UM/JMH during the period 1995–2000 (N ¼ 505 alive at 28 days). Birth weight (Bw), 500–1000 g; gestational age (GA), 23–32 weeks; PDA, patent ductus arteriousus; RDS, respiratory distress syndrome; OR, odds ratio. Reproduced from Bancalari E, Claure N, and Sosenko IRS (2003) Bronchopulmonary dysplasia: changes in pathogenesis, epidemiology and definition. Seminars in Neonatology 8: 63, with permission from Elsevier.

Other factors have been proposed as having a pathogenetic role in BPD. One of these factors is a genetic predisposition to abnormal airway reactivity since there have been reports of a stronger family history of asthma in infants with BPD. Vitamin A deficiency may also play a role because infants who develop BPD have lower vitamin A levels than those who recover without BPD. This possible association is supported by the similarities between some of the airway epithelial changes observed both in BPD and in vitamin A deficiency and by clinical evidence that vitamin A administration in the first weeks of life reduces the incidence of BPD. Another factor suggested to play a role in the development of BPD is early adrenal insufficiency because infants with BPD have lower cortisol levels in the first week of life. Figure 5 summarizes the most important factors that contribute to the pathogenesis of BPD.

Prematurity-respiratory failure mechanical ventilation

Excessive tidal volume Decreased lung compliance Volutrauma

Increased inspired oxygen Deficient antioxidant systems Nutritional deficiencies Oxygen toxicity

Pre/postnatal infections PMN’s activation Inflammatory mediators elastase/proteinase inhibitors imbalance

Patent ductus arteriosus Excessive fluid intake

Increased pulmonary blood flow-lung edema

Acute lung injury inflammatory response

Airway damage Metaplasia Smooth muscle hypertrophy ↑ Mucus secretion

Airway obstruction Emphysema-atelectasis

Vascular injury Increased permeability Smooth muscle hypertrophy

Pulmonary edema Pulmonary hypertension

Bronchopulmonary dysplasia Figure 5 Factors contributing to the pathogenesis of bronchopulmonary dysplasia.

Interstitial damage ↑ Fibronectin ↑ Elastase ↓ Alveolar septation ↓ Vascular development

Matrix damage ↑ Fibrosis Decreased number of alveoli and capillaries


Animal Models Because BPD is a chronic process with multiple factors involved in its pathogenesis, it has been extremely difficult to develop suitable animal models. However, there are two models that closely resemble human BPD. The first model was developed by Drs Robert de Lemos and Jacqueline Coalson in preterm baboons. When these animals are delivered at approximately 75% gestation and are subsequently ventilated over several weeks with positive pressure and high oxygen concentrations, they develop physiologic and morphologic changes that closely resemble those of human BPD. This is a very labor-intensive and expensive model that requires a full intensive care setting for these animals to survive. A similar model has been developed in preterm lambs that, after several weeks of mechanical ventilation, also develop histopathological changes very similar to human BPD. These models are used extensively by investigators who are exploring various aspects of lung development, injury, and repair as well as possible strategies to prevent BPD.

Prevention and Management Strategies to prevent BPD focus on eliminating or reducing the multiplicity of factors known to contribute to lung injury (Figure 5). Since lung immaturity is the main predisposing factor in the pathogenesis of BPD, prevention of BPD should start prenatally by attempting to avoid premature birth. When preterm birth is imminent, administration of antenatal steroids reduces the incidence and severity of RDS and the subsequent development of severe BPD. Minimizing volutrauma and exposure to high inspired oxygen concentrations is also critical to reduce BPD. Surfactant replacement therapy in infants with RDS improves their respiratory course, enabling mechanical ventilation with lower inspiratory pressures and oxygen concentrations. The use of positive end expiratory pressure (PEEP) is critical because insufficient PEEP is associated with increased ventilatorassociated lung damage. Ventilation with high frequency has not been conclusively shown to reduce BPD, although data from experimental animals suggest that this ventilation modality may reduce lung injury. Data demonstrating an increased risk of BPD in infants exposed to prenatal and postnatal infections suggest that prevention and aggressive management of these infections should also play a preventive role in BPD. Avoidance of excessive fluid intake is also important in the prevention of BPD. Diuretics, specifically loop diuretics, may be indicated to improve pulmonary fluid balance and reduce interstitial lung

water. Prompt closure of a PDA using prostaglandin inhibitors or by surgical ligation is important in reducing the risk and/or severity of BPD. Bronchodilators, most of them b-agonists, administered by inhalation have been shown to reduce airway resistance in infants with BPD. Because their effect is short-lived and often associated with cardiovascular side effects (e.g., tachycardia, hypertension, and possible arrhythmias), their use tends to be limited to acute exacerbations of airway obstruction. Adequate nutrition is important in BPD prevention. General undernutrition, particularly an insufficiency in dietary protein, may increase the vulnerability of the preterm infant to oxidant lung injury and the development of BPD. Additional nutrients, including those that may increase intracellular glutathione (e.g., sulfur-containing amino acids), inositol to serve as substrate for surfactant, and selenium and other trace minerals to function as essential cofactors for the pulmonary antioxidant enzymes, may provide added protection to the premature infant against the development of BPD. Clinical trials in preterm infants with severe RDS have shown that supplemental vitamin A administration reduces the incidence and severity of BPD. Although the use of corticosteroids clearly has short-term benefits in lung function in infants with BPD, the optimal age of treatment, dose schedule, and duration of therapy have not been established. Most alarming are recent follow-up data demonstrating that infants who received short or prolonged steroid therapy had worse neurological outcome compared to controls. Table 1 summarizes some of the strategies to prevent BPD. Future directions in BPD-prevention may include exogenous administration of specific antioxidants, genetic manipulation, and strategies for maturing the preterm lung that have greater selectivity and fewer adverse effects than the use of glucocorticoids. Table 1 Potential strategies to prevent BPD Avoid premature birth Antenatal steroids Surfactant replacement Minimize volutrauma and duration of mechanical ventilation Minimize oxygen exposure Prevention/aggressive management of pre- and postnatal infections Avoid excessive fluid administration Prompt patent ductus asteriosus closure Maintain normal serum vitamin A levels Investigational Corticosteroids and other anti-inflammatory drugs Exogenous administration or induction of antioxidant enzymes Gene therapy/genetic manipulation

BRONCHOSCOPY, GENERAL AND INTERVENTIONAL 305 See also: Epithelial Cells: Type II Cells. Fluid Balance in the Lung. Infant Respiratory Distress Syndrome. Lung Development: Overview. Pediatric Pulmonary Diseases. Peripheral Gas Exchange.

Further Reading Bancalari E (2004) Pathophysiology of chronic lung disease. In: Polin R, Fox W, and Abman S (eds.) Fetal and Neonatal Physiology, 3rd edn., vol. 1, pp. 954–961. Philadelphia: Saunders. Bancalari E (2005) Neonatal chronic lung disease. In: Fanaroff AA and Martin RJ (eds.) Neonatal–Perinatal Medicine Diseases of the Fetus and Infant, 8th edn. St. Louis: Mosby. Bancalari E, Claure N, and Sosenko IRS (2003) Bronchopulmonary dysplasia: changes in pathogenesis, epidemiology and definition. Seminars in Neonatology 8: 63–71. Bancalari E and Goldman SL (1982) Barotrauma to the lung. Advances in Perinatal Medicine 181. Bancalari E, Wilson-Costello D, and Iben SC (2005) Management of bronchopulmonary dysplasia. In: Maalouf E (ed.) Best Practice Guidelines, Early Human Development. Oxford: Elsevier. Bland RD and Coalson JJ (2000) Chronic Lung Disease in Early Infancy. New York: Dekker. Coalson JJ, Winter VT, Gerstmann DR, et al. (1992) Pathophysiologic, morphologic, and biochemical studies of the premature baboon with bronchopulmonary dysplasia. American Review of Respiratory Disease 145: 872–881. D’Angio CT and Maniscalco WM (2004) Bronchopulmonary dysplasia in preterm infants: pathophysiology and management strategies. Paediatric Drugs 6: 303–330. Gonzalez A, Sosenko IRS, Chandar J, et al. (1996) Influence of infection on patent ductus arteriosus and chronic lung disease in premature infants weighting 1000 grams or less. Journal of Pediatrics 128: 470–478. Grier DG and Halliday HL (2003) Corticosteroids in the prevention and management of BPD. Seminars in Neonatology 8: 83–91.

Husain AN, Siddiqui NH, and Stocker JT (1998) Pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia. Human Pathology 29: 710–717. Jobe AH and Bancalari E (2001) Bronchopulmonary dysplasia. American Journal of Respiratory and Critical Care Medicine 163: 1723–1729. Lemons J, Bauer CR, Oh W, et al. (2001) Very low birth weight outcomes of the National Institute of Child Health and Human Development Research Network January 1995 through December 1996. Pediatrics 107: 1–8. Northway WH, Moss RB, Carlisle KB, et al. (1990) Late pulmonary sequelae of bronchopulmonary dysplasia. New England Journal of Medicine 323: 1793–1799. Northway WH Jr, Rosen RC, and Porter DY (1967) Pulmonary disease following respirator therapy of hyaline membrane disease: bronchopulmonary dysplasia. New England Journal of Medicine 276: 357–368. Pierce MR and Bancalari E (1995) The role of inflammation in the pathogenesis of bronchopulmonary dysplasia. Pediatric Pulmonology 19: 371–378. Rojas MA, Gonzalez A, Bancalari E, et al. (1995) Changing trends in the epidemiology and pathogenesis of neonatal chronic lung disease. Journal of Pediatrics 126: 605–610. Sosenko I and Bancalari E (2003) Bronchopulmonary dysplasia (BPD). In: Greenough A and Milner A (eds.) Neonatal Respiratory Disorders, 2nd edn., pp. 399–422. London: Arnold. Watterberg KL, Demers LM, Scott SM, et al. (1996) Chorioamniotis and early lung inflammation in infants in whom bronchopulmonary dysplasia develops. Pediatrics 97: 210–215. Watterberg KL, Scott SM, Backstrom C, et al. (2000) Links between early adrenal function and respiratory outcome in preterm infants: airway inflammation and patent ductus arteriosus. Pediatrics 150: 320–324. Yoon BH, Romero R, Jun JK, et al. (1997) Amniotic fluid cytokines (interleukin-6, tumor necrosis factor-a, interleukin-1b, and interleukin-8) and the risk for the development of bronchopulmonary dysplasia. American Journal of Obstetrics and Gynecology 177: 825–830.

BRONCHOSCOPY, GENERAL AND INTERVENTIONAL D J Feller-Kopman and R Bechara, Harvard Medical School, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Bronchoscopy refers to examination of the tracheobronchial tree via a rigid or flexible bronchoscope. This chapter will review both diagnostic and therapeutic bronchoscopy, with a focus on the available technology and procedural techniques used to diagnose and treat a variety of lung diseases. As ‘Bronchoscopy: Regular and Interventional’ is a topic for which textbooks are available, the reader is encouraged to explore the relevant literature, with some pertinent references listed in the further reading section.

Introduction Gustav Killian has been described as the ‘Father of Bronchoscopy’. With the vision of using a metallic

tube, electric light, and topical cocaine to prevent glottic closure, Killian removed a pork bone from a farmer’s airway in 1897. Over the subsequent years, Killian went on to develop bronchoscopes, laryngoscopes, and endoscopes, as well as describe techniques such as using fluoroscopy and X-ray to define endobronchial anatomy. Over the next 150 years, bronchoscopic techniques and instruments continued to be refined. In 1966, Shigeto Ikeda, presented the first prototype flexible fiberoptic bronchoscope at the 9th International Congress on Diseases of the Chest in Copenhagen. In 1968 Machita and Olympus introduced the first commercially available fiberoptic bronchoscopes. In 1980, Dumon presented his use of the neodymium:yttrium aluminum garnet (Nd:YAG) laser via the fiberoptic bronchoscope, and since that time the flexible bronchoscope has been widely utilized as both a diagnostic and therapeutic tool for both diseases of the parenchyma and central airways.

306 BRONCHOSCOPY, GENERAL AND INTERVENTIONAL

With the miniaturization of electronic devices, Ikeda was able to incorporate the video chip into the bronchoscope, and Pentax introduced the first video bronchoscope in 1987. Suddenly, endoscopic pictures could be printed out and shared, and the physicians no longer needed to look through an eyepiece, but instead the endoscopic image could be projected onto monitors, allowing everyone in the room to visualize what was happening in the airway.

Modern Video Bronchoscopes Little has changed in the appearance of bronchoscopes since 1968. The external diameter of the flexible bronchoscope varies from 2.7 to 6.3 mm diameter. The diameter of the working channel ranges from 1.2 to 3.2 mm. A working channel X2:8 mm is recommended for more therapeutic flexible bronchoscopy, as well as endobronchial ultrasound (EBUS), as it allows for better suction and the passage of larger instruments. Most flexible bronchoscopes can flex 1801 up and 1301 down. It is important to note the relative anatomy at the tip of the bronchoscope. By convention, as viewed from the operator’s perspective, the camera is at 9:00, suction at 6:00 and the working channel at 3:00. These landmarks play a role when navigating the airways as the bronchoscope may need to be rotated in order to visualize the intended target or guide a tool to its intended target.

Airway Anatomy It is crucial that the bronchoscopist become an expert in airway anatomy. Anatomical knowledge should include the naso and oropharynx, as well as the larynx as these structures are visualized, yet often overlooked, by the pulmonologist, who is typically more concerned about lower airway/parenchymal disease. Additionally, the segmental anatomy of the lungs, from both an external and internal perspective, is required, and we recommend knowledge of both the name and number system. One must also be familiar with the anatomy external to the airway, primarily the intrathoracic vessels and lymph nodes, as these will serve as reference points for transbronchial needle aspiration and EBUS, and will help avoid injury should the bronchoscopist use therapy such as the Nd:YAG laser or brachytherapy. The use of endoscopic simulators has been associated with a more rapid acquisition of bronchoscopic expertise and we recommend the novice bronchoscopist use a simulator as much as possible.

Preparation Prior to the procedure, it is crucial that the bronchoscopist review the patient’s relevant history,

physical examination, and imaging studies. A distinct plan should be in place regarding the sequence of sampling techniques, and everyone participating in the procedure should be familiar with both this plan and also their particular roles during the procedure. Topical anesthesia is crucial, and we typically use 1% lidocaine, keeping the total dosage o400 mg. Stronger concentrations do not provide additional sensory anesthesia and will only limit the volume of lidocaine one can apply to the airways. Most procedures are performed under conscious sedation with a narcotic and a benzodiazepine. All practitioners involved in the administration of conscious sedation are required to undergo formal training by their institution and should have a thorough understanding of the effects, side effects, and typical dosages, as well as antagonist drugs. The physician should also be comfortable in airway management including the use of bag-valve mask, oral airways, and endotracheal intubation. If one plans to perform bronchoscopy through an endotracheal tube (or tracheostomy), the internal diameter should ideally be 47.5 mm in order to accommodate the bronchoscope and maintain patient ventilation. Depending on the length of the procedure, it may be necessary to remove the bronchoscope intermittently to ensure adequate ventilation.

Diagnostic Bronchoscopy There are many indications for diagnostic bronchoscopy. The most common indication is for the diagnosis and staging of suspected lung cancer. Other indications include evaluation of diffuse lung disease, infiltrates in the immunocompromised host, hemoptysis, and cough. Additionally, bronchoscopy with bronchoalveolar lavage (BAL) and/or brushing is useful for the diagnosis of community and healthcare associated pneumonia. Cancer Diagnosis and Staging

Lung cancer is the leading cause of cancer deaths in the US, and the incidence and number of lung cancer deaths continues to increase amongst women in the US. Bronchoscopy provides a minimally invasive approach for the diagnosis of tumors in the central airways. Though the yield is somewhat lower for solitary parenchymal lesions, advances in navigation with techniques such as computed tomography (CT) fluoroscopy and electromagnetic guidance (to be discussed later) have significantly improved the yield for peripheral tumors. The bronchoscopic evaluation of patients with suspected malignancy is guided by clinical symptoms as well as radiographic findings. Depending upon the

BRONCHOSCOPY, GENERAL AND INTERVENTIONAL 307

history and chest CT findings, bronchoscopy may be the diagnostic modality of choice as it can both make the diagnosis and stage the patient at the same time. If appropriate staging is to be performed, biopsy of the lesion that will place the patient in the highest clinical stage should occur prior to other biopsies. For example, if a patient presents with a left lower lobe mass and a right paratracheal lymph node (4R), the appropriate procedure would be a bronchoscopy with transbronchial needle aspiration (TBNA) of the 4R node, with attention then turned to the left lower lobe lesion, as a positive TBNA would stage the patient as IIIb and therefore change the treatment plan. Initial biopsy of the mass could contaminate the working channel and make the TBNA a false positive, precluding the patient from curative surgery. There are several available tools by which to obtain specimens during bronchoscopy including forceps biopsy, brushing, bronchial wash/lavage, and TBNA. Electrocautery snare forceps removal of a pedunculated airway lesion can also provide excellent tissue for the pathologist. The choice of the above modalities is primarily determined by the location of the pathology; however, data support the use of the combination of techniques to improve diagnostic yield, as opposed to using them in isolation. Endobronchial needle aspiration should be used with all visible lesions, as its use in combination with conventional techniques has been associated with an improvement in the diagnostic yield. If the lesion is not visible endoscopically, BAL can be performed. Briefly, the bronchoscope is wedged in the target segmental or subsegmental bronchus leading to the lesion. Two or three aliquots of 40–60 ml of normal saline solution are instilled, and then aspirated. Ideally, the return should be between 40% and 60% of the instilled volume, however this is dependent upon the segment lavaged, with better return coming from less dependant locations. BAL is thought to sample approximately 1 million alveoli and the cellular and noncellular contents of the lavage fluid have been shown to closely correlate with the inflammatory nature of the entire lower respiratory tract. Transbronchial biopsy, brushing, and TBNA can also be performed for peripheral lesions. The diagnostic yield of bronchoalveolar lavage for peripheral cancer ranges from 4% to 68%. Without advanced guidance (discussed below), the yield for transbronchial biopsy ranges from 49% to 77%. The yield from brushing alone is 26–57%. The combination of all the three techniques results in a combined yield of upto 68%. TBNA of peripheral nodules has been shown to have a higher diagnostic yield than other sampling techniques, and should be used to biopsy

peripheral lesions, as well as mediastinal and hilar lymph nodes. Despite TBNA being introduced more than 25 years ago, it remains an underutilized technique, with only 12% of pulmonologists reporting its routine use for the diagnosis and staging of lung cancer. The technique, however, is incredibly useful, and may preclude further invasive surgery in 29% of patients. The yield with TBNA has been associated with tumor cell type (small cell 4 non-small cell 4 lymphoma), lymph node size, and lymph node location. The use of CT fluoroscopy to guide TBNA and transbronchial biopsy has several advantages over standard fluoroscopy. As opposed to standard fluoroscopy, which is typically used only in two dimensions, CT provides the ability to visualize the target in three dimensions. With standard fluoro, either the patient or the C-arm needs to be rotated to confirm the biopsy tool is not anterior or posterior to the target. CT fluoro provides real-time three dimensional confirmation of the appropriate (Figure 1) and inappropriate (Figure 2) biopsy sites. The use of CT fluoro to guide TBNA has been associated with an accuracy of 88%, including patients who have previously undergone a nondiagnostic bronchoscopy with standard TBNA. Endobronchial ultrasound is another important modality used to help improve accuracy of TBNA. With the development of a miniaturized 20 MHz transducer that can be inserted via a 2.8 mm working channel, lymph nodes and masses adjacent to the airway can now be visualized (Figure 3). Like CT fluoro, EBUS has also been shown to improve the yield of TBNA. Recently, a bronchoscope with a dedicated ultrasound probe and distinct working TM channel has been developed (Puncturescope , Olympus Corporation, Tokyo, Japan), and has the benefit

Figure 1 CT fluoroscopy showing the biopsy forceps in the target.


Several studies have shown that the use of autofluorescence increases the detection of early stage lung cancer by up to sixfold when compared to white light bronchoscopy. The definitive role of AF bronchoscopy in the early detection of lung cancer remains to be defined. Diffuse Parenchymal Lung Disease

Figure 2 CT fluoroscopy showing the biopsy forceps anterior to the target, near the pleura. Note that without rotation of the patient/C-arm, the biopsy forceps may appear to be within the target on standard fluoroscopy.

Figure 3 Endobronchial ultrasound image showing tumor invading airway wall from the 12:00–2:00 position.

of providing real-time guidance for TBNA of mediastinal and hilar lymph nodes, with excellent results. EBUS has been shown to better differentiate airway invasion versus compression by adjacent tumor when compared to CT, and can suggest the histology of a solitary pulmonary nodule based on the ultrasound morphology. Autofluorescence (AF) bronchoscopy is an increasingly popular tool used for the early detection of cancer in the central airways, primarily carcinoma in situ (CIS), and squamous cell carcinoma. When exposed to light in the violet–blue spectrum (400–450 nm), the normal airway fluoresces green. As submucosal disease progresses from normal, to metaplasia, to dysplasia, to CIS, there is a progressive loss of the green AF, causing a red–brown appearance of the airway wall.

Diffuse parenchymal lung disease describes a group of infectious, inflammatory, and fibrotic disorders which may involve the interstitial, alveolar, bronchial, and vascular structures of the tracheobronchial tree. The most commonly used sampling techniques for patients with diffuse disease include BAL, bronchial brushing, transbronchial biopsy (TBBx), and occasionally endobronchial biopsy (EBBx) and TBNA. Though associated with a low morbidity, transbronchial biopsy should be used only when the potential results will impact on treatment decisions. Diseases in which transbronchial biopsy can prove diagnostic or has been shown to significantly increase the diagnostic yield as compared to less invasive means include lymphangitic carcinomatosus, sarcoidosis, rejection after lung transplantation, hypersensitivity pneumonitis, and sometimes, invasive fungal infection. The overall diagnostic yield for transbronchial biopsy in this category depends on the disease entity. For example, the yield for sarcoidosis can approach 90%, but is much lower for patients with vasculitis or cryptogenic organizing pneumonia. Aside from providing a specific diagnosis in cases of cancer or infection, the results of BAL can serve to limit the differential diagnosis considerably. For example, a BAL with lymphocyte predominance suggests granulomatous disease such as sarcoidosis, berylliosis, or a lymphoproliferative disorder. Neutrophil predominance suggests bacterial infection, acute interstitial pneumonia, and can be seen in patients with asbestosis or usual interstitial pneumonitis. Eosinophils are seen in patients with eosinophilic pneumonias, hypereosinophilic syndromes, or Churg–Strauss syndrome. Patients with pulmonary alveolar proteinosis (PAP), have a unique appearance to the BAL fluid that is described as milky or opaque. The alveolar macrophages are filled with PAS-positive material, and lamellar bodies can be seen under electron microscopy. Infectious Diseases

Community acquired pneumonia The role of bronchoscopy in community acquired pneumonia (CAP) remains controversial. When used, BAL and protected brush are the main diagnostic procedures, and the specimens should ideally be sent for


respiratory tract culture prior to the initiation of antibiotics, the use of semiquantitative or quantitative culture data, and the use of negative culture data to discontinue antibiotics in patients who have not had changes in their antibiotic regimen within the last 72 h. Additionally, the use of a bronchoscopic strategy was supported as a way to reduce 14-day mortality.

Figure 4 (a) Protected specimen brush; (b) protected specimen brush with the protective wax cap expelled.

quantitative culture, with a threshold of 104 colonyforming units (CFU) for the BAL and 103 CFU for the protected brush (Figure 4). In addition to providing a microbiologic diagnosis, another important indication for bronchoscopy in patients with CAP is to rule out an obstructing endobronchial lesion in the right clinical setting. Obviously, it is crucial to avoid contamination with upper airway secretions when performing bronchoscopy in patients with pneumonia. Key procedural aspects include minimizing suctioning, as well as minimizing the instillation of lidocaine, as secretions in the working channel will be flushed back into the airways, and high concentrations can be bacteriostatic. Healthcare and ventilator associated pneumonia The American Thoracic Society and Infectious Disease Society have recently reviewed these topics in great detail, and used the techniques of evidence based medicine to guide their recommendations. Some major points included the collection of a lower

Immunocompromised host The early diagnosis and initiation of the appropriate antibiotic is the cornerstone of successful treatment of the immunocompromised patient with pneumonia. Additionally, it is important to note that multiple diagnoses can often be present simultaneously in these patients, and noninfectious conditions may have a similar presentation of cough, dyspnea, fever, and an infiltrate on imaging. Bronchoscopy is an excellent method of evaluating these patients as less invasive techniques can miss the diagnosis in approximately 30% of patients, and earlier diagnosis may improve mortality. BAL is the most commonly used bronchoscopic technique used to obtain a diagnosis in immunocompromised patients, with an overall diagnostic yield of approximately 60–70%. In comparative studies, the sensitivity of TBBx has been shown to be roughly the same, though the combined use of both techniques may increase the yield. The results of BAL have been shown to change management in up to 84% of cases. Even in the immunocompromised host, bronchoscopy with BAL remains a safe procedure. Brushing and TBBx, however, have been associated with a higher incidence of bleeding complications in patients who are thrombocytopenic. The ‘gold standard’ for tissue diagnosis remains open lung biopsy, which can yield a specific diagnosis in 62% of patients, and result in a significant increase in survival. There are no data suggesting that bronchoscopy reduces mortality in this patient population. Hemoptysis There are many causes of hemoptysis including infectious, inflammatory, vascular, and neoplastic processes. Though one would think that bronchoscopy can often make the diagnosis of a radiographic occult neoplasm in a patient with hemoptysis, a bronchoscopic diagnosis of malignancy is made in o5% of patients. Indications for bronchoscopy in patients with normal chest imaging include age 440 years, male gender, and a 440 pack-year smoking history. Though the appropriate timing for bronchoscopy is controversial, there is a greater likelihood of identifying the bleeding source when performed within the first 48 h of symptoms. The combined use of bronchoscopy and CT is also recommended. If patients are clinically stable, we prefer


to obtain the CT first, as it can serve as a ‘road map’ to guide bronchoscopy.

Therapeutic Bronchoscopy Bronchoscopy in Hemoptysis

The definition of massive hemoptysis has ranged from 100–1000 cm3 expectorated in a 24 h period. As the majority of patients with massive hemoptysis die from asphyxia, and not exanguination, and the anatomic dead-space is approximately 150 cm3, we consider any amount over 100 cm3 in 24 h as massive. In addition to identifying the source and cause of bleeding, bronchoscopy clearly plays an important therapeutic role in patients with massive hemoptysis. If available, we strongly recommend rigid bronchoscopy as the procedure of choice in these patients. In addition to securing an airway, and providing oxygenation and ventilation, the rigid bronchoscope allows the passage of large-bore suction catheters as well as a variety of other tools that can help stop the bleeding including cryotherapy, electrocautery, argon plasma coagulation (APC), and Nd:YAG laser, as well as bronchial blockers. If rigid bronchoscopy is not available, we recommend tracheal intubation with the largest endotrachcal tube available, with selective right or left-mainstem intubation to protect the non-bleeding lung as needed. Double-lumen endotracheal tubes, or specialized tubes that come with an endobronchial blocker (e.g., Univent, Vitaid, Williamsville, NY), can be more difficult to place, especially in the setting of massive hemoptysis. The main role of flexible bronchoscopy in the patient with massive hemoptysis lies in obtaining lung isolation, and ‘protecting the good lung’, as the suction channel of a flexible scope is relatively small. All bronchoscopists should become familiar with bronchial blockers (e.g., Arndt Bronchial Blocker, Cook Critical Care, Bloomington, IN), which can be passed in parallel to the flexible scope, and some, even inserted through the working channel. These catheters are guided to the culprit segmental, lobar, or mainstem bronchus and the balloon is inflated to the recommended volume/pressure. After a maximum of 24 h, the balloon should be deflated under bronchoscopic visualization. Other bronchoscopic techniques used to control hemoptysis include the topical application of iced saline, epinephrine (1 : 20 000), thrombin/thrombinfibrinogen, or cyanoacrylate solutions. Other Therapeutic Bronchoscopic Techniques

Argon plasma coagulation Argon plasma coagulation (APC) is a noncontact method using ionized

argon gas (plasma) to achieve tissue coagulation and hemostasis. As the plasma is directed to the closest grounded source, APC has the ability to treat lesions lateral to the probe, or around a bend, that would not be suitable for laser therapy. The depth of penetration for APC is approximately 2–3 mm, and hence the risk of airway perforation is also less when compared to lasers. Laser therapy The Nd:YAG laser is the most widely used laser in the lower respiratory system, and has been used for both benign and malignant disease. The primary advantage of laser photoresection includes rapid destruction/vaporization of tissue. Lesions most amenable to laser therapy are central, intrinsic, and short (o4 cm), with a visible distal endobronchial lumen. When lesions meet these criteria, patency can be re-established in more than 90% of cases. Care must be taken however, as the depth of penetration can approach 10 mm and airway perforation with resultant pneumothorax, pneumomediastinum, and vascular injury have been reported. In view of this, we encourage its use only by experienced interventional bronchoscopists. Nevertheless, the safety record of laser bronchoscopy is excellent, with an overall complication rate of o1%. Cryotherapy Cryotherapy is a safe and effective tool for a variety of airway problems. By releasing nitrous oxide stored under pressure, the tip of the cryoprobe rapidly cools to 891C. We primarily use cryotherapy for the removal of organic foreign bodies with high water content such as grapes and vegetable matter, in addition to facilitating the removal of tenacious mucus or blood clots when performing flexible bronchoscopy. Compared to the other techniques described in this chapter, cryotherapy results in delayed tumor destruction, requiring a repeat bronchoscopy to remove the necrotic tumor. The distinct advantage of cryotherapy lies in the fact that the normal cartilage and fibrous tissue of the airway are relatively cryoresistant, in addition to the lack of risk of airway fires. Electrocautery Electrocautery uses alternating current at high frequency to generate heat, which cuts, vaporizes, or coagulates tissue depending on the power. Electrocautery is a contact mode of tissue destruction and a variety of cautery probes are available including blunt tip probes, knifes, and snares. We favor the use of the cautery snare for pedunculated lesions of the airway as the stalk can be cut and coagulated while preserving the majority of the tissue for pathologic interpretation. As with laser therapy,


the risks of electrocautery include airway perforation, airway fires, and damage to the bronchoscope. Photodynamic therapy Photodynamic therapy (PDT) involves the intravenous injection of a photosensitizing agent, then activating the drug with a nonthermal laser to produce a phototoxic reaction and cell death. As tumor cells retain the drug longer than other tissues, waiting approximately 48 h after drug injection will lead to preferential tumor cell death as compared to normal tissue injury. As with cryotherapy, maximal effects are delayed, and a repeat, ‘clean-out’ bronchoscopy should be performed 24–48 h after drug activation. The primary side effect from PDT is systemic phototoxicity, which can last up to 6 weeks after injection. Newer drugs are being developed with the hopes of increasing tumor selectivity and reducing the duration of skin phototoxicity. As laser activation uses a nonthermal laser source, airway fires are not an issue. PDT has been shown to be curative for early stage lung cancer of the airways and is an especially attractive option for patients with endobronchial CIS who are not surgical candidates due to other comorbidities. Brachytherapy Brachytherapy refers to endobronchial radiation, primarily used for the treatment of malignant airway obstruction. The most commonly used source of radiation is iridium-192 (192Ir), which is inserted bronchoscopically via a catheter. Brachytherapy may be delivered by either low-dose rate (LDR), intermediate-dose rate (IDR), or high-dose rate (HDR) methods, with most authors currently recommending the afterloading HDR technique. This allows the bronchoscopist to place the catheter in the desired location and the radiation oncologist to deliver the radiation in a protected environment. The main advantage of HDR is patient convenience, as each session lasts o30 min; however, multiple bronchoscopies are required to achieve the total 1500 cGy dose that is currently recommended. The LDR technique may be appropriate for patients who live far from the hospital or who are otherwise hospitalized as it only requires one bronchoscopy, but the catheter has to stay in place for 20–60 h. The main advantage of brachytherapy as compared with external-beam radiation is the fact that less normal tissue is exposed to the toxic effects of radiation. The most common side effects include intolerance of the catheter, radiation bronchitis, airway perforation, and, occasionally, massive hemorrhage. Treatment of tumors in the right and left upper lobes has the highest incidence of hemorrhage, as these are located near the great vessels.

Airway stents Montgomery is credited as initiating the widespread use of airway stents after his development of a silicone T-tube in 1965 for use in patients with tracheal stenosis. Dumon, however, introduced the first completely endoluminal airway stent in 1990. Airway stents are the only technology that can alleviate extrinsic airway compression. They are commonly used in conjunction with the other modalities for patients with intrinsic or mixed disease. Over the last 15 years, there has been an explosion in both stent design and the number of endoscopists who place airway stents. As with any procedure, it is crucial to understand the indications and contraindications of the procedure as well as be able to anticipate, prevent and manage the associated complications. Unfortunately, the ideal stent has

Figure 5 Metal stent in the trachea.

Figure 6 Some available silicone stents ex vivo.


not yet been developed. This stent would be easy to insert and remove, yet would not migrate; would be of sufficient strength to support the airway, yet be flexible enough to mimic normal airway physiology and promote secretion clearance; biologically inert to minimize the formation of granulation tissue; and available in a variety of sizes. There are currently two main types of stents: metal, generally Nitinol (Figure 5), and silicone (Figure 6). Though metal stents are easily placed, they can be extremely difficult to extract, and may cause excessive granulation tissue formation (Figure 7). They are

Figure 7 Metal stent removal.

available in covered and uncovered varieties. For malignant airway obstruction, the only appropriate metal stents are covered models, which minimize tumor ingrowth. Some authors feel that there is no indication for an uncovered metal stent. The main advantage of metal stents is their larger internal : external diameter ratio as compared to that of silicone stents. Though silicone stents require rigid bronchoscopy for placement, they are more easily removed and are significantly less expensive. The future of airway stenting likely lies in the creation of bioabsorbable stents, made out of materials such as vicryl filaments or poly-L-lactic acid (SR-PLLA). In addition to malignant airway obstruction, airway stents can be helpful in patients with tracheobronchomalacia and tracheoesophageal fistula. In patients with tracheoesophageal fistula, doublestenting of the esophagus and airway is recommended to maximally prevent aspiration.

Powered instrumentation The microdebrider is a tool consisting of a hollow metal tube with a rotating blade coupled with suction. We have had excellent results with this technology in the treatment of both benign and malignant central airway obstruction. A primary advantage of the microdebrider is the rapidity of obtaining a patent airway and the lack of risk of airway fires as compared to modalities using heat.

Figure 8 Super dimension bronchoscopy showing the locatable guide in the target in the axial, coronal, and sagittal planes on CT, as well as the ‘fighter-pilot’ view in the lower right, with the target 0.2 cm directly ahead.


Future Directions in Bronchoscopy In the appropriate patient, lung volume reduction surgery has been shown to improve both quality and quantity of life. The major drawback to this surgery is the associated morbidity and cost of the procedure. Recent studies have suggested that lung volume reduction can be obtained bronchoscopically, either by creating channels in the distal airways/parenchyma, or by the placement of one-way endobronchial valves to promote deflation of hyperinflated lung that does not significantly contribute to gas exchange. The results of large-scale, multicenter trials will become available within the next several years, but the preliminary data are promising. The use of electromagnetic navigation is a novel technology that has been shown to allow accurate sampling of peripheral solitary pulmonary nodules o1 cm in diameter. Briefly, a virtual bronchoscopy is generated from the patients CT scan. Anatomic landmarks that are easily identified, such as the carinae, are marked, as is the target. At the time of bronchoscopy, the patient lies in an electromagnetic field and a steerable, ‘locatable guide’ is placed through an ‘extended working channel’ of the bronchoscope. The location of the guide in the electromagnetic field is accurate to o5 mm in the x, y, and z axes, as well as yaw, pitch, and roll. The points previously identified in the virtual bronchoscopy are then marked with the locatable guide, which in essence, marries the CT scan with the bronchoscopic image. Navigation is then performed by looking at the CT in the axial, sagittal, and coronal planes, and the guide is steered toward the target (Figure 8). Once found, the guide is removed, and standard bronchoscopic tools such as TBNA needles and forceps are placed through the extended working channel. This technology not only has the potential to revolutionize diagnostic bronchoscopy,

but therapeutic bronchoscopy as well. For example, if a patient with a 2.5 cm, stage Ia, nonsmall cell cancer is not an operative candidate, this technology may allow for bronchoscopic treatment by either radiofrequency ablation or the implantation of fiducials to allow stereotactic radiosurgery. See also: Alveolar Hemorrhage. Bronchiectasis. Bronchiolitis. Bronchomalacia and Tracheomalacia. Drug-Induced Pulmonary Disease. Interstitial Lung Disease: Overview. Lung Anatomy (Including the Aging Lung). Mediastinal Masses. Panbronchiolitis. Pneumonia: Overview and Epidemiology; The Immunocompromised Host. Tumors, Malignant: Overview. Upper Airway Obstruction. Upper Respiratory Tract Infection.

Further Reading Becker HD (1991) Atlas of Bronchoscopy: Technique, Diagnosis, Differential Diagnosis, Therapy. Philadelphia: BC Decker. Bolliger CT and Mathur PN (2000) Interventional Bronchoscopy. Basel: Karger. Detterbeck FC, DeCamp MM Jr, Kohman LJ, and Silvestri GA (2003) Invasive staging: the guidelines. Chest 123(90010): 167S–175S. Ernst A, Feller-Kopman D, Becker HD, and Mehta AC (2004) Central airway obstruction. American Journal of Respiratory and Critical Care Medicine 169(12): 1278–1297. Ernst A, Silvestri GA, and Johnstone D (2003) Interventional pulmonary procedures: guidelines from the American College of Chest Physicians. Chest 123(5): 1693. Fagon JY, Chastre J, Wolff M, et al. (2000) Invasive and noninvasive strategies for management of suspected ventilator-associated pneumonia: a randomized trial. Annals of Internal Medicine 132(8): 621–630. Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-associated pneumonia. (2005) American Journal of Respiratory and Critical Care Medicine 171(4): 388–416. Prakash USB (1994) Bronchoscopy. New York: Raven Press.

C Calcium Channels

see Ion Transport: Calcium Channels.

CAPSAICIN M G Belvisi and D J Hele, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract The pungent ingredient in red pepper fruits of the genus Capsicum, which includes paprika, jalapenõ, and cayenne, is called capsaicin. Capsaicin is known to stimulate sensory nerves leading to the activation of nociceptive and protective reflex responses (e.g., cough, bronchospasm) and the release of neurotransmitters from both peripheral and central nerve endings. This latter effect causes a collection of inflammatory responses often referred to as ‘neurogenic inflammation’, which in the airways results in bronchoconstriction, plasma extravasation, and mucus hypersecretion. The capsaicin receptor has recently been identified and has been named the type 1 vanilloid receptor (VR1; TRPV1). It has previously been suggested that there is an upregulation of TRPV1 expression in inflammatory diseases and that inappropriate activation of this receptor may lead to sensory nerve hyperresponsiveness. Thus, it would appear that airway inflammatory diseases (e.g., asthma and chronic obstructive pulmonary disease) may respond to treatment with an effective and selective inhibitor of TRPV1 and to this end much work is being carried out to develop novel inhibitors.

via the peripheral release of neuropeptides, a phenomenon known as ‘neurogenic inflammation’. A characteristic feature of many nociceptive sensory fibers is their sensitivity to capsaicin. However, until recently the molecular mechanisms involved in activation of sensory nociceptive fibers were unknown. Pharmacological evidence for the presence of a ‘capsaicin receptor’ in sensory nerves was provided by the discovery of two capsaicin analogs, resiniferatoxin (potent agonist) and capsazepine (an antagonist). First, specific binding sites for resiniferatoxin were demonstrated on dorsal root ganglion membranes and second, capsazepine has been found to inhibit numerous capsaicin-evoked neuronal responses including those in the airways. The capsaicin receptor has recently been identified and has been named the type 1 vanilloid receptor (VR1; transient receptor potential vanilloid 1 (TRPV1)).

Capsaicin and Functional Responses in the Airways Cough

Introduction Sensory nerves in the airways regulate central and local reflex events such as bronchoconstriction, airway plasma leakage, and cough. Sensory nerve activity may be enhanced during inflammation such that these protective reflexes become exacerbated and deleterious. Sensory nerve reflexes are under the control of at least two different classes of sensory fiber, the myelinated rapidly adapting stretch receptors (RARs) and nonmyelinated capsaicin-sensitive C fibers. In the airways, activation of RARs and C fibers elicits cough, bronchoconstriction, and mucus secretion via an afferent central reflex pathway. Activation of C fibers in the airways also mediates efferent excitatory nonadrenergic noncholinergic (e-NANC) responses such as bronchoconstriction, mucus secretion, plasma exudation, and vasodilatation

Inhalation challenge with capsaicin and low pH (e.g., citric acid) has been shown to evoke cough and has been used as a model for the last 50 years to investigate the action of potential antitussive therapies in clinical trials. In fact, agonists at TRPV1 such as capsaicin and resiniferatoxin are the most potent stimulants of the cough reflex so far described in man. Therefore, it has been suggested that TRPV1 activation may be one of the primary sensory mechanisms in cough. However, if activation of TRPV1 is an important initiating factor for the cough reflex then an endogenous capsaicin-like ligand must be present. A number of putative endogenous ligands that are known to activate TRPV1 have been demonstrated to cause cough. TRPV1 has been shown to be sensitive to a fall in the extracellular pH, and H þ ions not only stimulate the receptor directly but also

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increase the sensitivity of the receptor to capsaicin and low pH solutions, which are known to elicit cough in animals and humans. Interestingly, the TRPV1 antagonists capsazepine and iodo-resiniferatoxin have been shown to inhibit capsaicin, citric acid, and anandamide-induced cough in conscious guinea pigs suggesting that these agents are inducing cough via a common mechanism, that is, activation of TRPV1 (Figure 1). However, it is unlikely that TRPV1 activation is the only stimulus for cough since some tussigenic agents such as hypertonic saline are not inhibited by TRPV1 antagonism.

Neurogenic Inflammation Capsaicin activates sensory nerves leading to the activation of nociceptive and protective reflex responses and the release of neurotransmitters from both peripheral and central nerve endings. This latter effect causes a collection of inflammatory responses 2

Coughs min–1

Capsazepine (10 µM) 1.5

Control

1

0.5

0

30 µM

80 µM

Capsaicin

Citric acid (0.25 M)

Figure 1 Representation of data demonstrating that the TRPV1 antagonist capsazepine inhibits capsaicin and citric acid-induced cough in conscious guinea pigs suggesting that these agents are inducing cough via a common mechanism, i.e., activation of TRPV1.

often referred to as ‘neurogenic inflammation’, which in the airways results in bronchoconstriction, plasma extravasation, and mucus hypersecretion (Figure 2). Bronchoconstriction

Early experiments using the relatively weak TRPV1 antagonist capsazepine provided pharmacological validation that capsaicin-induced bronchospasm in guinea pig bronchi did indeed involve the activation of TRPV1. Contractile responses to resiniferatoxin and capsaicin were unaffected by the neurokinin (NK)-1 antagonist CP 96345, partially inhibited by the NK-2 antagonist SR 48968, but nearly abolished by a combination of the antagonists. These data suggest that resiniferatoxin and capsaicin both release tachykinins that act on both NK-1 and NK-2 receptor subtypes in a TRPV1-dependent manner. More recently, a more potent and selective agent has been used to confirm these observations. Interestingly, contractile and relaxant responses to capsaicin and resiniferatoxin have also been examined in human isolated bronchus (5–12 mm outside diameter). Bronchi isolated from 10 of 16 lungs contracted in response to capsaicin. The capsaicin-induced contractions were mimicked by resiniferatoxin and inhibited by capsazepine. The contractile response to capsaicin was not affected by the potent NK-2 selective antagonist SR 48968, whereas responses to concentrations of NKA, NKB, substance P, neuropeptide g, and neuropeptide K, which produced contractions of a similar size, were almost abolished by SR 48968. These results suggest that capsaicin and resiniferatoxin can alter smooth muscle tone in a TRPV1-dependent manner, but this response does not appear to involve substance P or related neurokinins. Airway hyperresponsiveness (AHR) to bronchoconstrictor agents is recognized as a critical feature of

Capsaicin

Vasodilatation

Mucus secretion

Plasma exudation

C fibers

Central reflexes, e.g., cough

Neuropeptide release SP, NKA, CGRP…

Bronchoconstriction Figure 2 Activation of sensory nerves by capsaicin. SP, substance P; CGRP, calcitonin gene-related peptide; NKA, neurokinin A. Adapted from Barnes PJ (2001) Neurogenic inflammation in the airways. Respiration Physiology 125: 145–154.

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bronchial asthma and sensory nerves in the airway are strongly implicated in the hyperresponsiveness. Several studies have demonstrated that pretreatment with capsaicin (which depletes sensory neuropeptides) significantly inhibited the late bronchial response that was observed after ovalbumin inhalation, AHR, and eosinophil accumulation in an allergic guinea pig model, and AHR to histamine in a rabbit model. It remains to be seen whether TRPV1 antagonists are effective in this regard. Plasma Extravasation

Activation of C fibers in the airways also mediates efferent excitatory nonadrenergic noncholinergic (e-NANC) responses such as plasma exudation and vasodilatation via the peripheral release of neuropeptides. Plasma extravasation has been shown to be induced in rats or guinea pigs by intravenous injections of substance P (SP) and capsaicin. The effect of intravenous capsaicin was absent in capsaicin-desensitized animals and in those pretreated with capsazepine. Capsaicin-induced plasma extravasation was also markedly inhibited by CP 96345, a nonpeptide antagonist of tachykinin NK-1 receptors. These data suggest that capsaicin-induced plasma leakage is mediated by the release of neuropeptides and the activation of NK-1 receptors via a TRPV1dependent mechanism. Mucus Secretion

When stimulated capsaicin-sensitive C fibers (afferents) containing the neuropeptides SP, NKA, and calcitonin gene-related peptide have also been shown to evoke neurogenic secretion from airway mucussecreting cells. However, although capsaicin has been shown to elicit mucus secretion, there is no data available describing the effect of TRPV1 antagonists on this response.

Molecular Mechanism of Action of Capsaicin

Capsazepine Airway C fiber

Capsaicin, RTx − H+ (citric acid, low pH), heat +

Cholinergic reflex activation

TRPV1

Cough Chest tightness

AHR

Mucus secretion

Figure 3 Events mediated via TRPV1 following the activation of nociceptive airway C fibers by noxious stimuli.

(PMg/PNaB5). TRPV1 is activated by heat (4431C), but is effectively dormant at normal body temperature and low pH (o5.9) and may act as an integrator of chemical and physical pain-eliciting stimuli. When activated, TRPV1 produces depolarization through the influx of Na þ , but the high Ca2 þ permeability of the channel is also important for mediating the response to pain. Gating by heat is direct but the receptor can be opened by ligands or stimuli such as mild acidosis, which also reduces the threshold for temperature activation and potentiates the response to capsaicin. To summarize, TRPV1 mediates nociception and contributes to the detection and integration of diverse chemical and thermal stimuli. The expected role for TRPV1 is in pain pathways and recent data from a study with TRPV1 knockouts showed impaired inflammatory thermal hyperalgesia. This has led to a growing interest in developing small molecule antagonists for this target. Recent preclinical data demonstrating efficacy in rodent models of both thermal and mechanical neuropathic or inflammatory pain has fuelled this enthusiasm. The established role of sensory nerve activation in the cough reflex and the role of TRPV1 in inflammatory pain has also alerted the respiratory community to the therapeutic potential of TRPV1 antagonists as antitussives and as therapy for airway inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD).

The Type 1 Vanilloid Receptor

TRPV1 is a membrane-associated vanilloid receptor. It is a nociceptor-specific ligand-gated ion channel expressed on the neuronal plasma membrane of nociceptive C fibers and is required for the activation of sensory nerves by vanilloids such as capsaicin, the pungent extract from plants in the genus Capsicum (e.g., hot chilli peppers) (Figure 3). TRPV1 also mediates the response to painful heat, extracellular acidosis, protons, and tissue injury. TRPV1 is an outwardly rectifying cation-selective ion channel with a preference for calcium (PCa/PNaB10) and magnesium

Ligands for TRPV1

Exogenous agonists of TRPV1 include capsaicin and resiniferatoxin. Endogenous agonists include the cannabinoid receptor agonist anandamide, N-arachidonoyl-dopamine, inflammatory mediators (bradykinin, 5-HT, and prostaglandin E2 (PGE2)), hydrogen ions, heat, arachidonic acid, lipoxin A4, prostacyclin, ethanol, and several eicosanoid products of lipoxygenases including 12-(S)- and 15-(S)-hydroperoxyeicosatetraenoic acids, 5-(S)-hydroxyeicosatetraenoic acid and leukotriene B4. Interestingly, the effect of bradykinin

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may be indirect given that data exists suggesting that it appears to activate bradykinin B2 receptors on afferent neurons leading to the generation of lipoxygenase metabolites that have agonist activity at TRPV1. The evidence to support the suggestion that TRPV1 can be gated not only by vanilloids such as capsaicin, but also by protons, heat, agonists at certain G-protein-coupled receptors, ethanol, cannabinoids, and lipoxygenase products of arachidonic acid has come from electrophysiological patch-clamp recording studies on the cell bodies of TRPV1-expressing cells. Furthermore, pharmacological antagonism of TRPV1 has been shown to inhibit action potential discharge evoked by each of these stimuli. However, due to the nonselective effects of several of these tool compounds, this pharmacological approach has its limitations. Furthermore, if the antagonist inhibits but does not completely block a given response, it is not clear whether activation of the TRPV1 is obligatory for the response or merely contributes to the end response. In studies using TRPV1–/– mice it was concluded that whereas TRPV1 is required for action potential discharge of C fiber terminals evoked by capsaicin and anandamide, it plays a contributory role in responses evoked by low pH of bradykinin. These data suggest that TRPV1 is one of multiple ion channels responsible for the bradykinin-evoked generator potentials (i.e., membrane depolarization) in C fiber terminals. TRPV1 Receptor Antagonists

Antagonists of TRPV1 include ruthenium red, a dye that exhibits properties of noncompetitive antagonism for the TRPV1, and has a poorly defined mechanism of action and limited selectivity. Capsazepine is another agent characterized as a weak but relatively selective, competitive TRPV1 receptor antagonist (over other TRPVs). However, at the concentrations required to antagonize the TRPV1 (approximately 10 mM), capsazepine has demonstrated non-specific effects such as inhibition of voltage-gated calcium channels and nicotinic receptors. Furthermore, this ligand has poor metabolic and pharmacokinetic properties in rodents, where it undergoes extensive firstpass metabolism when given orally. Recently, a number of antagonists with improved potency and/or selectivity have been described and these include antagonists that are structurally related to agonists such as iodo-resiniferatoxin, which is 100-fold more potent than capsazepine. Previous evidence has indicated that iodo-resiniferatoxin is a potent antagonist at the TRPV1 both in vitro and in vivo. Since then several studies have used this

antagonist as a pharmacological tool in order to explore the role for TRPV1 in various physiological settings. Recently, however, although the in vitro antagonistic activity of iodo-resiniferatoxin has been confirmed, in vivo studies have demonstrated some agonist activity of this agent at high doses. Therefore, there is a possibility that this agent retains some agonistic activity and that an agonist-dependent desensitization effect on sensory nerves may well contribute to some of the antagonistic activity observed. The GlaxoSmithKline lead antagonist SB-366791, which has a good selectivity profile in a series of assays, and potent vanilloid agonist AM-404 should provide useful tools for probing the physiology and pharmacology of TRPV1. Several TRPV1 receptor antagonists that share no obvious structural resemblance to any class of vanilloid agonist have also been developed. These compounds have been discovered by high-throughput random screening projects. At present this group of antagonists includes: (1) N-(haloanilino)carbonyl-N-alkyl-N-arylethylendiamines discovered at SKB; (2) N-diphenyl-Nnapthylureas discovered at Bayer; and (3) pyridol [2,3-d]-pyrimidin-4-ones discovered at Novartis.

Localization of TRPV1 The capsaicin-sensitive vanilloid receptor is expressed mainly in sensory nerves including those emanating from the dorsal root ganglia and afferent fibers that innervate the airway, which originate from the vagal ganglia. In the dorsal root and trigeminal ganglion, TRPV1 is localized to small and mediumsized neurons. Somatic sensory neurons of the vagus nerve are located in the jugular ganglion, whereas visceral sensory neurons of the nerve are located in the nodose ganglion. Previous studies have demonstrated that TRPV1-containing neurons are abundant in these ganglia. The traditional view that TRPV1 is simply a marker of primary sensory nerves is now being challenged. The TRPV1 receptor-has been detected in guinea pig and human airways by receptor-binding assays and has been identified in nonneuronal cell types such as mast cells, fibroblasts, and smooth muscle.

Role of TRPV1 in Airway Inflammatory Disease A role for TRPV1 in airway inflammatory disease would depend on the presence of endogenous activators of this channel under physiological and pathophysiological conditions. In fact, it is quite possible that this situation does, in fact, exist in the ‘disease’ scenario given that TRPV1 activation can be initiated

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and responses to other activators potentiated in an acidic environment, which has been shown to be present in diseases such as asthma and COPD. Interestingly, it has previously been established that the airway response to TRPV1 activation is enhanced in certain disease conditions. In particular, it has been reported that the cough response elicited in response to capsaicin is exaggerated in diseases such as asthma and COPD. Anandamide is also an endogenous ligand known to be produced in central neurons. However, more recent studies have suggested that anandamide is synthesized in lung tissue as well as a result of calcium stimulation suggesting that this mediator could also be involved in the activation of TRPV1 under normal and disease conditions. Inflammatory agents (e.g., bradykinin, ATP, PGE2, and nerve growth factor (NGF)) can indirectly sensitize TRPV1 to cause hyperalgesia. Thus, bradykinin and NGF, which activate phospholipase C (PLC), release TRPV1 from phosphatidylinositol 4,5-biphosphate (PIP2)-mediated inhibition. However, PLC also regulates TRPV1 by diacylglycerol (DAG) formation and subsequent activation of protein kinase C (PKC), which in turn phosphorylates and sensitizes TRPV1. Inflammatory stimuli, including prostaglandins and NGF among others, have also been shown to upregulate the expression and function of TRPV1 via the activation of p38 mitogen-activated protein-kinase (MAPK)- and protein kinase A (PKA)-dependent pathways. Previous data has suggested that there is an upregulation of TRPV1 in inflammatory diseases. For example, TRPV1 immunoreactivity is greatly increased in colonic nerve fibers of patients with active inflammatory bowel disease. Furthermore, a recent study has found an increase in TRPV1 expression in sensory nerves found in the airway epithelial layer in biopsy specimens from patients with chronic cough compared to noncoughing, healthy volunteers. There was a significant correlation between the tussive response to capsaicin and the number of TRPV1positive nerves in the patients with cough. Therefore, it has been postulated that TRPV1 expression may be one of the determinants of the enhanced cough reflex found in patients with chronic cough and that the recently described TRPV1 antagonists could be effective in the treatment of chronic persistent cough due to diverse causes.

Conclusions Thus, it would appear that airway inflammatory diseases (e.g., asthma and COPD) may respond to treatment with an effective and selective inhibitor of the ‘capsaicin receptor’ TRPV1 and to this end much

work is being carried out to develop novel inhibitors. Interestingly, capsaicin-sensitive nerve stimulation in subjects with active allergic rhinitis produces reproducible and dose-dependent leukocyte influx, albumin leakage, and glandular secretion. These results provide in vivo evidence for the occurrence of neurogenic inflammation in the human upper airway with active allergic disease and may therefore suggest the therapeutic utility of TRPV1 antagonists in the management of this disease. In addition, the treatment of persistent cough is a facet of airway diseases that is sorely in need of effective treatment and a TRPV1 inhibitor may prove extremely effective against cough induced by gastroesophageal acid reflux, for example, as well as that associated with asthma and other diseases of the airways described above. See also: Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Kinins and Neuropeptides: Bradykinin; Neuropeptides and Neurotransmission; Tachykinins. Neurophysiology: Neural Control of Airway Smooth Muscle; Neuroanatomy.

Further Reading Barnes PJ (2001) Neurogenic inflammation in the airways. Respiratory Physiology 125: 145–154. Belvisi MG (2003) Airway sensory innervation as a target for novel therapies: an outdated concept? Current Opinion in Pharmacology 3: 239–243. Belvisi MG (2003) Sensory nerves and airway inflammation: role of A delta and C-fibres. Pulmonary Pharmacology and Therapeutics 16: 1–7. Belvisi MG and Geppetti P (2004) Cough * 7: current and future drugs for the treatment of chronic cough. Thorax 59: 438–440. Caterina MJ and Julius D (2001) The vanilloid receptor: a molecular gateway to the pain pathway. Annual Review of Neuroscience 24: 487–517. Coleridge HM and Coleridge JC (1994) Pulmonary reflexes: neural mechanisms of pulmonary defence. Annual Review of Physiology 56: 69–91. Holzer P (1991) Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacological Reviews 43: 143–201. Hwang SW and Oh U (2002) Hot channels in airways: pharmacology of the vanilloid receptor. Current Opinion in Pharmacology 2: 235–242. Karlsson JA (1993) A role for capsaicin sensitive, tachykinin containing nerves in chronic coughing and sneezing but not in asthma: a hypothesis. Thorax 48: 396–400. Maggi CA and Meli A (1988) The sensory-efferent function of capsaicin-sensitive sensory neurons. General Pharmacology 19: 1–43. Morice AH and Geppetti P (2004) Cough 5: the type 1 vanilloid receptor: a sensory receptor for cough. Thorax 59: 257–258. Rogers DF (2002) Pharmacological regulation of the neuronal control of airway mucus secretion. Current Opinion in Pharmacology 2: 249–255. Szallasi A and Blumberg PM (1999) Vanilloid (Capsaicin) receptors and mechanisms. Pharmacological Reviews 51: 159–212. Szolcsanyi J (2004) Forty years in capsaicin research for sensory pharmacology and physiology. Neuropeptides 38(6): 377–384.

320 CARBON DIOXIDE

CARBON DIOXIDE R A Klocke, University at Buffalo, Buffalo, NY, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Large quantities of CO2 produced in tissues must be efficiently transferred into blood and transported to the lungs where CO2 is excreted. Transport of CO2 dissolved in solution alone is not adequate to meet these requirements. Carbon dioxide is transported as three different, but interrelated, entities in blood. A small portion of total CO2 content in blood exists as dissolved carbon dioxide. Although small in absolute quantity, dissolved CO2 has a key role in exchange because it is the only form that rapidly crosses membranes separating blood from tissues and alveolar gas. The largest quantity of CO2 exists in blood as bicarbonate ion. This ion is a product of the dissociation of carbonic acid, a compound formed when CO2 combines with water. A modest amount of carbon dioxide is transported as carbamate, a compound formed by binding of CO2 to amino groups of the hemoglobin molecule. Exchange of oxygen augments simultaneous CO2 exchange through changes in the molecular configuration of hemoglobin. This structural change increases buffering of hydrogen ions and binding of CO2 as carbamate. The time required for completion of these interrelated processes is critical because blood remains in the pulmonary capillaries for less than 1 s. Two processes overcome this limitation. The natural rate of conversion between CO2 and carbonic acid is quite slow, but is catalyzed in vivo by carbonic anhydrase. In addition, a specialized transporter protein facilitates bicarbonate transfer across the erythrocyte membrane. Reactions of CO2 play a major compensatory role in diseases that cause metabolic acidosis. Hydrogen ions combine with bicarbonate ions and are converted into CO2, which is subsequently excreted in the lungs. This process reduces circulating hydrogen ions and is an important factor in defense of acid–base homeostasis. Although the rare congenital absence of isomers of carbonic anhydrase have been previously associated with abnormalities in the brain, bones, and kidneys, recent evidence raises the possibility that CO2 excretion may also be impaired in these patients.

Introduction Carbon dioxide (CO2) and water are the end products of oxidative metabolism that generates the energy required for maintenance of body homeostasis. Large quantities of CO2 produced in tissues must be efficiently transferred into blood and transported to the lungs where it is excreted. Transport of CO2 dissolved in solution alone is not adequate to meet these requirements.

CO2 Transport in Blood Carbon dioxide is transported in three forms: as dissolved CO2, as bicarbonate ion, and bound to hemoglobin as a carbamate compound.

Table 1 Relative contributions of dissolved CO2, bicarbonate ion, and carbamate compounds to carbon dioxide transport in arterial blood and excretion in the lungs

Dissolved CO2 Bicarbonate ion Carbamate compounds

Transport (%)

Excretion (%)

5 88 7

8 79 13

Dissolved CO2

At normal CO2 tensions present in blood, dissolved CO2 has an approximate concentration of 1.2 mM. The difference between arterial and venous blood is small and accounts for only 8% of the total CO2 excreted in the lungs (Table 1). However, dissolved CO2 is crucial to gas exchange since it is the only form that can rapidly traverse the capillary membranes separating circulating blood from tissues and alveolar gas. Bicarbonate Ion

Carbon dioxide combines with water to form carbonic acid, a weak acid with a pH of 3.5. At the pH range present in the body, this acid almost completely dissociates into hydrogen ions and bicarbonate ions: CA

H2 O þ CO2 2 H2 CO3 2Hþ þ HCO 3

½1

The natural rate of formation of carbonic acid from CO2 is very slow and requires 60–90 s to reach completion. However, erythrocytes contain large quantities of carbonic anhydrase (CA), an enzyme that catalyzes this reaction. The hydrogen ions formed in this reaction are buffered effectively by hemoglobin, which is the dominant nonbicarbonate buffer in blood. Release of oxygen from the hemoglobin molecule alters the quaternary structure of hemoglobin, thereby increasing the affinity of binding sites for hydrogen ions (the Bohr effect). This increases the buffering capacity of deoxygenated hemoglobin. Figure 1 illustrates the buffering curves of oxygenated and reduced hemoglobin. As hemoglobin is reduced in the tissues, its buffering curve shifts upward and significant amounts of hydrogen ion can be buffered without any change in pH. It is estimated that approximately one half of the hydrogen ions released in aerobic metabolism are buffered in this manner. The majority of CO2 in blood is carried as bicarbonate (Table 1).

CARBON DIOXIDE 321

H+ added (mEq)

3.0

Ar

Ve

ter

no

ial

us

Actual ∆pH

2.0 ∆H+ from CO2

1.0 0.0 ∆pH without H+ binding 7.30

7.35 pH

7.40

Figure 1 Buffering curves of hemoglobin in arterial and venous blood. With release of oxygen bound to hemoglobin, buffering sites on hemoglobin are able to bind more hydrogen ions (the Bohr effect). The increased binding of hydrogen ions occurs as blood–acid base status shifts from the arterial to venous buffering curve. Hydrogen ions generated from CO2 exchange in the tissues are buffered with less change in pH than would occur if buffering took place along the arterial curve.

Carbamate Compounds

Amino groups of proteins can reversibly bind hydrogen ions: þ R-NHþ 3 2H þ R-NH2

½2

where R represents the remainder of the protein moiety. Carbon dioxide can bind to the uncharged amino groups: R-NH2 þ CO2 2R-NHCOOH 2R-NHCOO þ Hþ

½3

to form carbamic acids that dissociate into carbamate and hydrogen ions at normal blood pH. The quantity of CO2 bound to serum proteins and the e-amino groups of hemoglobin is minimal and does not change significantly between arterial and venous CO2 tensions. However, the a-amino groups of the N-termini of the hemoglobin molecule contribute substantially to CO2 transport. The changes in molecular configuration accompanying reduction of hemoglobin alter the equilibrium constants of reactions [2] and [3], favoring binding of CO2 as carbamate. As a result, the relative contribution of carbamate to the exchange of CO2 is approximately twice as great as its relative concentration in blood (Table 1). The a-amino groups of the b-chains of hemoglobin are affected to a greater extent by conformational changes of the molecule and are responsible for three-quarters of the carbamate contribution to exchange. The concentration of 2,3-diphosphoglycerate (2,3DPG) within the erythrocyte influences the contribution of carbamate compounds to CO2 exchange. The

two N-terminal portions of the b-chains on the surface of hemoglobin are more widely separated in the reduced state as a result of the accompanying change in the quaternary structure. Positive charges on the b-chains induce the negatively charged 2,3DPG molecule to enter the enlarged cavity between the two separated b-chains of reduced hemoglobin. The positioning of 2,3-DPG near the N-terminal a-amino groups alters the equilibrium constants of reactions [2] and [3], and reduces the quantity of the uncharged form of the a-amino groups that binds CO2. Haldane Effect

The carbon dioxide dissociation curve of normal human blood is illustrated in Figure 2. The total concentration of CO2 in all forms is plotted as a function of the carbon dioxide partial pressure in blood. As seen in the figure, at any given PCO2 the total CO2 content is greater in blood with a reduced oxygen content compared to fully oxygenated blood. This is known as the Haldane effect, named after one of the investigators who first described this phenomenon. The difference in CO2 content between the two curves at the same PCO2 is termed oxylabile CO2. It is produced by the changes in the quaternary structure of hemoglobin that accompany oxygenation and reduction. Oxylabile CO2 has two components. The first is an increase in bicarbonate concentration resulting from greater buffering of hydrogen ions by reduced hemoglobin. The second component is the result of increased carbamate formation associated with reduced hemoglobin. The Haldane effect enhances the transport of carbon dioxide. The shift of the CO2 dissociation curve caused by release of oxygen allows for transport of CO2 with a lower CO2 tension in venous blood than would occur if there were no shift in the position of the dissociation curve (Figure 2). The synergism between oxygen and carbon dioxide exchange facilitates transport of CO2 to a much greater extent than the transport of O2.

Time-Dependent Processes in CO2 Exchange The actual exchange of CO2 in the pulmonary capillaries involves a series of complicated processes. Exchange of each form in which CO2 is transported is discussed separately, but the processes occur simultaneously (Figure 3). The reactions involved in conversion of bound CO2 to dissolved CO2 are complex and require finite time. These reactions probably reach completion in 0.3–0.4 s, which is

322 CARBON DIOXIDE 60

70% HbO2

CO2 content (ml dl−1)

40

54

∆PCO2

v

v′

52 20 97.5% HbO2

50 a

∆PCO2 without Haldane effect

48 40

45

50

0 0

20

40

60

PCO (mmHg) 2 Figure 2 Carbon dioxide dissociation curves of blood. The two curves represent the relationship between CO2 content and PCO2 of arterial (oxygen saturation of 97.5%) and venous (oxygen saturation of 70%) blood. Binding of oxygen to hemoglobin causes a shift from the venous to arterial CO2 dissociation curve. The inset in the figure illustrates the effect of this shift on blood PCO2 . The difference in PCO2 between arterial (a) and venous blood (v ) is substantially less than would occur if the Haldane effect were not operative and CO2 exchange took place along a single dissociation curve (a and v 0 ). Data used to construct the dissociation curves taken from Forster RE, DuBois AB, Briscoe WA, and Fisher AB (1986) The Lung, p. 238. Chicago: Yearbook Medical Publishers.

sufficient for excretion of CO2 to occur during the time the blood remains in the pulmonary capillary (0.7 s at rest, 0.5 s during exercise). Although the process of diffusion of CO2 across the alveolar–capillary membrane is quite rapid (B0.01 s), the chemical reactions and other transport processes necessary for CO2 exchange appreciably slow the actual rate of exchange. In peripheral tissues, these same processes occur in an opposite manner.

Dissolved CO2

When blood reaches the pulmonary capillaries, dissolved CO2 diffuses from the plasma and the interior of the erythrocyte across the alveolar–capillary membrane into the alveoli. The reduction in dissolved CO2 rapidly lowers capillary PCO2, and disturbs the equilibrium of carbamate and bicarbonate reactions. This promotes conversion of the latter two compounds into CO2 which, in turn, diffuses out of the capillary into the alveoli.

Bicarbonate Ion

As blood PCO2 falls, equation [1] is reversed and bicarbonate is converted first into carbonic acid and then into free CO2. Carbonic anhydrase inside the erythrocyte catalyzes this reaction by a factor of 13 000–15 000. Hydrogen ions required for conversion of bicarbonate to CO2 are largely supplied by hemoglobin. The simultaneous binding of oxygen to hemoglobin causes the release of hydrogen ions. In the plasma, the conversion of bicarbonate ion to CO2 occurs at a much slower rate. Plasma proteins are much less effective buffers than hemoglobin and are not affected by simultaneous oxygen transfer. Therefore, fewer hydrogen ions are available for bicarbonate conversion. There is a modest amount of carbonic anhydrase attached to the capillary endothelium, but this is sufficient to provide catalysis that is only 1/100 of that inside the red cell. As a result of the differences in buffering power and catalysis, the concentration of bicarbonate inside the erythrocyte decreases much more rapidly than that in plasma.

CARBON DIOXIDE 323 Carbonic Anhydrase

CO2 Alveolus CA

CA

Plasma

CA

Cl − HCO− + H+ 3

CA

CA

CA

CA

CO2 + H2O

H2CO3

3 Cl−

HCO3− + H+

HHb

Hb− + H+ R−NHCOO− + H+

CA

CO2 + H2O + H+ + R−NH2 R−NH+3

H2CO3

R−NHCOOH

Figure 3 Carbon dioxide exchange in the lung. Solid lines indicate reactions that take place rapidly. The dashed line represents the slower reaction of formation of CO2 from H2CO3 as catalyzed by carbonic anhydrase (CA) localized to the capillary endothelium. The dotted lines indicate buffering reactions of hemoglobin. The number 3 indicates the band 3 anion transporter protein that exchanges bicarbonate and chloride ions between the erythrocyte and plasma. Reproduced from Klocke RA (1997) Carbon dioxide transport. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientific Foundation, 2nd edn., pp. 1633–1642. New York: Raven Press, with permission from Lippincott Williams & Wilkins.

This difference in bicarbonate concentration leads to transfer of bicarbonate from the plasma into the erythrocyte. Bicarbonate ions cannot enter the red cell independently because they are negatively charged. They exchange simultaneously in the opposite direction with intracellular chloride ions. This paired bicarbonate–chloride exchange is accomplished in an electrically neutral fashion by a transmembrane protein known as the band 3 anion transporter protein. Through this process of anionic exchange, more than one-half of the total bicarbonate converted to CO2 in the lung originates in plasma, but enters the erythrocyte to be converted into CO2 before being excreted. Carbamate Compounds

As PCO2 decreases within the erythrocyte, through reactions [2] and [3] carbon dioxide bound to the a-amino groups is released as free CO2 and diffuses into the alveoli. The hydrogen ions required for these reactions are supplied through the buffering reactions of hemoglobin. Simultaneous binding of oxygen facilitates the release of CO2 bound to hemoglobin. Oxygenation reduces the affinity of the amino groups for CO2 and promotes CO2 release. Because this portion of CO2 exchange is dependent on oxygenation of hemoglobin, this reaction is delayed until oxygen is first bound to hemoglobin.

Five of the more than one dozen identified isomers of the enzyme carbonic anhydrase (CA) are potentially involved in CO2 exchange in the lungs and tissues. Both CA I and II are present in large quantities within the erythrocytes of humans and many mammals. This enzymatic activity is much greater than that thought necessary for efficient CO2 exchange, but the reason for the excess enzyme is not apparent. CA I is a less potent enzyme and its activity is partially inhibited by chloride ion. Although CA I comprises 85–90% of the total enzyme inside human erythrocytes, CA II is responsible for the majority of total enzymatic activity. Some erythrocytic CA II is bound to intracellular portions of the band 3 anion transporter protein, providing a highly efficient complex for interconversion of CO2 and HCO3 and transmembrane exchange of the bicarbonate ion. CA IV is a high-potency isomer that is localized to membranes, especially the sarcolemma of muscle tissue and the capillary endothelium of most tissues. Its impact on gas exchange in the lung is probably limited by its low concentration and the lack of substantial plasma buffering compared to that inside the red cell. Mathematical models of CO2 exchange in muscle suggest sarcolemmal CA IV may play a role in maintaining acid-base balance when large quantities of lactic acid are produced during exercise. CA III and V isomers also are present in some striated muscle, but their role, if any, in CO2 exchange is unclear.

Carbon Dioxide in Disease Carbon dioxide elimination can be impaired in multiple disease states. However, this derangement is rarely caused by abnormal transport, but is usually the result of reduced ventilation or mismatching of ventilation and blood flow within the lung. Genetic deficiencies of CA II are accompanied by renal tubular acidosis, osteopetrosis, and cerebral calcifications. Most studies have failed to detect abnormalities in CO2 exchange in patients at rest, presumably because CA I present in erythrocytes is sufficient to support CO2 exchange. However, studies have not been conducted during exercise when abnormalities would be more likely to occur. A recent description of patients with carbonic anhydrase deficiencies suggests that even CO2 exchange at rest may not be completely normal. The difference between end-tidal gas (a surrogate for alveolar gas) and arterial blood CO2 tensions was increased, implying that CO2 exchange did not reach equilibrium during transit through the pulmonary capillary bed. Further studies are required to validate this possible impairment.

324 CARBON MONOXIDE

The carbon dioxide dissociation curve of blood is markedly affected by acid–base alterations. Abnormalities that increase blood bicarbonate concentration, i.e., metabolic alkalosis and compensated respiratory acidosis, increase the total quantity of carbon dioxide in all forms at any given CO2 tension. The opposite is true in metabolic acidosis, which is characterized by a marked reduction in bicarbonate, and therefore total CO2 content, at any give PCO2 . The ratio of the arterial–venous difference in CO2 content divided by the arterial–venous difference in PCO2 provides an index of the efficiency of carbon dioxide transport. Although this ratio has varied in studies of patients with acid–base abnormalities, it is significantly reduced only in metabolic acidosis. This decreased efficiency of CO2 transport is caused by a reduction in the Haldane effect in metabolic acidosis. This results in elevation of venous, and therefore tissue, PCO2 . However, it is important to note that there are few studies of CO2 transport in clinical circumstances and much more data is needed to characterize fully the effects of acid–base disturbances on CO2 exchange. Reactions of carbon dioxide have a prominent role in acid–base balance that is particularly emphasized in metabolic acidosis. This common condition occurs most frequently when oxidation of glucose is impaired and energy is generated via biochemical pathways that produce strong acids. Two common conditions are impairment of oxygen delivery to tissues leading to production of lactic acid and diabetic ketoacidosis that results in generation of acetoacetic and b-hydroxybutyric acids. In both situations, these strong acids ionize completely and release large quantities of hydrogen ions. With the resultant increase in hydrogen ion concentration, bicarbonate ion binds to hydrogen ion to form carbonic acid, which is further converted into CO2 and water. The CO2 formed in this buffering reaction is excreted in

the lungs, completing the removal of free hydrogen ions from the circulating blood. Thus, the reactions of carbon dioxide play a major role in defending acid–base balance in these circumstances. See also: Acid–Base Balance. Hemoglobin. Ventilation, Perfusion Matching. Ventilation: Overview.

Further Reading Forster RE, DuBois AB, Briscoe WA, and Fisher AB (1986) The Lung, pp. 235–242. Chicago: Yearbook Medical Publishers. Geers C and Gros G (2000) Carbon dioxide transport and carbonic anhydrase in blood and muscle. Physiological Reviews 80(2): 681–715. Henry RP and Swenson ER (2000) The distribution and physiological significance of carbonic anhydrase in vertebrate gas exchange organs. Respiratory Physiology 121(1): 1–12. Klocke RA (1987) Carbon dioxide transport. In: Farhi LE and Tenney SM (eds.) Handbook of Physiology: The Respiratory System, pp. 173–197. Bethesda: American Physiological Society. Klocke RA (1997) Carbon dioxide transport. In: Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) The Lung: Scientific Foundation, 2nd edn., pp. 1633–1642. New York: Raven Press. Lindskog S and Silberman DN (2000) The catalytic mechanism of mammalian carbonic anhydrases. In: Chegwidden WR, Carter ND, and Edwards YH (eds.) The Carbonic Anhydrases: New Horizons, pp. 175–195. Basel: Birkhauser. Swenson ER (2000) Respiratory and renal roles of carbonic anhydrase in gas exchange and acid–base regulation. In: Chegwidden WR, Carter ND, and Edwards YH (eds.) The Carbonic Anhydrases: New Horizons, pp. 281–341. Basel: Birkhauser. Tanner MJA (2002) Band 3 anion exchanger and its involvement in erythrocyte and kidney disorders. Current Opinion in Hematology 9(2): 133–139. Venta PJ (2000) Inherited deficiencies and activity variants of the mammalian carbonic anhydrases. In: Chegwidden WR, Carter ND, and Edwards YH (eds.) The Carbonic Anhydrases: New Horizons, pp. 403–412. Basel: Birkhauser. Wetzel P and Gros G (2000) Carbonic anhydrases in striated muscle. In: Chegwidden WR, Carter ND, and Edwards YH (eds.) The Carbonic Anhydrases: New Horizons, pp. 375–399. Basel: Birkhauser.

CARBON MONOXIDE D Morse, University of Pittsburgh, Pittsburgh, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Carbon monoxide (CO) is generated in the human body by the catabolism of heme. This reaction is catalyzed by an enzyme known as heme oxygenase, which has both an inducible (heme oxygenase-1) and constitutive (heme oxygenase-2) form. CO was once believed to be a mere byproduct of heme breakdown, but is now known to modulate a number of cellular functions

including inflammation, cellular proliferation, and apoptotic cell death. This article offers a broad overview of our current understanding of the role of CO in respiratory disease.

Introduction Carbon monoxide (CO) is known to most pulmonologists as an air pollutant arising from the partial combustion of organic molecules and as a potentially lethal gas when inhaled in high concentrations. The avid binding of CO to heme iron results

CARBON MONOXIDE 325 CO

Heme

Heme oxygenase

Biliverdin

Biliverdin reductase

Bilirubin

Fe2+ Ferritin Figure 1 The enzymatic reaction catalyzed by heme oxygenase results in the release of CO.

in displacement of oxygen from hemoglobin, thus reducing the oxygen carrying capacity of blood. This phenomenon in turn leads to tissue ischemia and the familiar symptoms of CO poisoning. It is less widely appreciated that CO, in addition to being an environmental pollutant, is a biological product of ordinary metabolism. CO is generated in the human body by the catabolism of heme. This endogenously produced CO results in the normal baseline human carboxyhemoglobin level of 0.4–1%, and CO can be measured in the breath as it is excreted. The enzyme that releases CO from the breakdown of heme is known as heme oxygenase. There is a constitutively expressed form of this enzyme (heme oxygenase-2 (HO-2)) and an inducible form (heme oxygenase-1 (HO-1)). As one of the three byproducts of heme degradation (Figure 1), CO was initially considered a catabolic waste product. In fact, it was suggested in 1969 that CO production from humankind could contribute significantly to air pollution. We now know that this concern was misplaced, and it has more recently become evident that CO production serves a number of biological purposes. This article focuses on the functions of CO that relate most closely to pulmonary medicine, but it should be noted that a large body of literature exists describing functions for CO such as neurotransmission and vasodilation that cannot be discussed in detail here.

Structure CO is a colorless, odorless diatomic gas composed of a single carbon atom that is triply bonded to a single oxygen atom. It is an inert gas that does not readily engage in chemical reactions, but its affinity for heme is approximately 200 times that of oxygen. CO is slightly soluble in water (and therefore plasma).

Regulation of Activity The rate-limiting step in the generation of CO is heme catalysis by heme oxygenase. The inducible

form of heme oxygenase, HO-1, is widely distributed throughout the body and is highly evolutionarily conserved. Its activity is transcriptionally regulated, and it is induced by more stimuli than any other known gene. The inducers of HO-1 include mainly conditions that would cause stress to cells, such as increased oxidant burden, radiation, heavy metals, and hypoxia. Not surprisingly, there are a number of cis-acting DNA sequence elements that serve as potential binding sites for transcription factors in the proximal promoter region and at distal enhancer sites. The dominant sequence element in the distal enhancer regions of mouse and human ho-1 is the stress-responsive element (StRE). The StREs represent targets of multiple dimeric proteins generated by intrafamily homodimerization or intra- and interfamily heterodimerization of individual members of the Jun, Fos, CREB, ATF, Maf, and the Cap’n’collar/ basic-leucine zipper subclasses of the basic-leucine zipper superfamily of transcription factors.

Biological Functions Anti-Inflammatory Effects

Several lines of evidence suggest that one of the reasons CO may be generated under conditions of stress is for its ability to dampen inflammation. There has been one reported case of human HO-1 deficiency, and the affected child exhibited signs of chronic inflammation and was highly vulnerable to oxidative stress. Experimental studies have shown that when mice or macrophages are stimulated with lipopolysaccharide, a component of bacterial cell walls, the production of proinflammatory cytokines (such as tumor necrosis factor alpha, interleukin-6 and interleukin-1b) is greatly increased. This model is frequently used to simulate human sepsis, a condition that is associated with variable elevation of these same cytokines. When these same mice or cells are administered low concentrations of CO (250 ppm) along with lipopolysaccharide, the production of the proinflammatory cytokines is inhibited. Furthermore, the production of the anti-inflammatory cytokine interleukin-10 is augmented by CO treatment. CO has also been shown to affect the expression of granulocytemacrophage colony-stimulating factor (GM-CSF), a glycoprotein that promotes the proliferation and the differentiation of hematopoietic progenitor cells into neutrophils and macrophages. A number of chronic inflammatory pulmonary diseases such as chronic obstructive pulmonary disease (COPD), asthma, and sarcoidosis are associated with elevated levels of GM-CSF. The anti-inflammatory effects of CO are mediated primarily via the mitogen-activated proteinkinase (MAPK) signaling pathways.

326 CARBON MONOXIDE Antiapoptotic Effects

Programmed cell death, or apoptosis, must be finely balanced for the maintenance of health and resolution of disease. Under conditions of stress, excessive apoptosis may lead to worsening organ dysfunction, and one could postulate that a stress-induced antiapoptotic molecule could provide protection. This thinking led to the examination of an antiapoptotic role for CO. The first experiments performed in vitro demonstrated that applying CO to fibroblasts or endothelial cells could prevent cell death in response to tumor necrosis factor alpha. This effect was confirmed in animal models of disease, including ischemia/reperfusion injury and transplantation. The antiapoptotic effect of CO is not universal, however. In Jurkat T cells, CO has been shown to increase Fas/ CD95-induced apoptosis. Additionally, high concentrations of CO lead to apoptosis of tissue in rodents, associated with CO poisoning and tissue injury. As with the anti-inflammatory effects, the antiapoptotic effects of CO are primarily mediated by the MAPK signaling pathways. Antiproliferative Effects

Regulation of cell proliferation is important not only for the maintenance of homeostasis, but in the body’s response to disease. It appears likely that stress-induced production of CO modulates cell growth in disease states, thereby potentially altering the progression of a variety of illnesses. The antiproliferative function of CO has been established in vitro in a number of different cell types. CO applied directly to smooth muscle cells causes growth arrest, and endogenous CO released from vascular smooth muscle cells as a consequence of hypoxiainduced HO-1 expression has the same effect. This endogenously produced CO also results in increased production of guanosine 30 ,50 -(cyclic)phosphate (cyclic GMP) in co-cultured endothelial cells, which in turn leads to downregulation of the expression of endothelial-derived mitogens such as platelet-derived growth factor and endothelin-1. The antiproliferative effect of CO has been implicated in the protection conferred by CO against vascular stenosis associated with balloon catheter injury. CO has also been shown to suppress the proliferation of human airway smooth muscle cells, which may be relevant to the pathogenesis of asthma, where inflammatory cell-derived mediators stimulate smooth muscle proliferation. CO has been further shown to arrest the growth of cultured T lymphocytes, macrophages, and fibroblasts, which may account in part for the influence of CO on the progression of immune-mediated and fibrotic lung diseases. The

antiproliferative effects of CO are generally mediated by increased intracellular cyclic GMP levels, although in some cell types the MAPK pathways may participate as well.

Carbon Monoxide in Respiratory Diseases The lung is an organ with a particular vulnerability to oxidative injury as the interface between the atmosphere and the rest of the body. Oxidative stress may also be caused by pathologic conditions such as infection, inflammation, and ischemia reperfusion. Lungs therefore require potent defense mechanisms and possess high levels of antioxidant enzymes. HO-1 is one of the few inducible molecules that can protect the lungs from an increased oxidant burden under stressful circumstances. In the lungs, HO-1 is highly expressed in alveolar macrophages, but is also found in epithelial cells, fibroblasts, and endothelial cells. The CO produced by HO-1 activity is primarily excreted in the breath, and the level of exhaled CO has been shown to increase in disease states and varies with therapy or exacerbations. Our current knowledge about the role of CO in lung disease is derived mainly from preclinical animal and cell culture studies; highlights of this work are summarized below and in Figure 2. Increasingly, the focus of CO research has been directed at potential therapeutic applications, such as using inhaled CO to limit lung injury due to a variety of insults. The CO animal and cell studies alluded to in the sections that follow generally use a concentration range of 50–250 ppm. For reference, this inhaled concentration would result in carboxyhemoglobin levels ranging from 8% to 25% at equilibrium (after 8 h of exposure) in humans. Acute Lung Injury

Many of the initial studies done to characterize the location and function of HO-1 in the lung used rodent models of acute lung injury such as hyperoxia. In rodents, high concentrations of inhaled oxygen result in lung edema and death within several days. This oxidant injury results in increased expression of HO-1 in the lung, and overexpression of HO-1 in the bronchiolar epithelium by adenoviral gene transfer results in enhanced protection from the lethal effects of hyperoxia. Inhaled CO confers similar protection against hyperoxic lung injury. In vitro studies suggest that a part of the mechanism of protection by CO is due to prevention of cell death in response to increased oxygen tension. Ventilator-induced lung injury may complicate acute lung injury in humans, and recent rodent studies suggest that inhaled CO also exerts anti-inflammatory effects in the setting of high tidal volume ventilation.

ell p

roli fe

rati o

n

CARBON MONOXIDE 327

le c

CO

Bron

onst

rictio

n

Sm

oot hm

us c

choc

Pulmonary Fibrosis

CO

Apoptosis

CO

Inflammation

thought to be major contributors to the pathogenesis of COPD. Patients with COPD have lower HO-1 expression in alveolar macrophages than controls, and a microsatellite polymorphism in the promoter for HO-1 has been associated with the development of emphysema. This suggests that an abnormal response of HO-1 to oxidative stress may be linked to the development of COPD.

CO

Immunohistochemical analysis of lung tissue from patients with various forms of interstitial lung disease reveals increased expression of HO-1, primarily in alveolar macrophages. Increased expression of HO-1 by adenoviral transfer has been shown to suppress lung fibrosis in a murine bleomycin model, and inhaled CO has similar effects. The mechanism by which HO-1 and CO confer protection against fibrosis has not been fully elucidated, but CO has been shown to alter the cytokine milieu, decrease matrix production by fibroblasts, slow fibroblast proliferation, and inhibit epithelial apoptosis, any of which could contribute to a modulatory effect on fibrosis.

CO

Pulmonary Vascular Disease

Fibroproliferation

Figure 2 Schematic summary of CO effects in lung disease. CO has been shown to suppress cell proliferation, bronchoconstriction, apoptosis, inflammation, and fibroproliferation.

Obstructive Lung Disease

Asthma is a disease characterized by inflammation, and so it is not surprising that exhaled CO levels have been shown to increase during asthma exacerbations. Studies in mice and cell culture suggest that the CO generated in the lung of asthmatics may affect the course of disease. Low-concentration inhaled CO has been shown to attenuate aeroallergeninduced inflammation in mice, resulting in decreased interleukin-5 and eicosanoid mediator levels, as well as decreased bronchoalveolar lavage inflammatory cell counts. Exogenously administered CO has also been shown to decrease airway hyperresponsiveness in mice, and as previously noted, CO may modulate the lung remodeling in asthma due to its inhibitory effect on airway smooth muscle cell proliferation. Exposure to reactive oxygen species and an imbalance in oxidant/antioxidant status are also

Following the discovery that nitric oxide could mediate vascular relaxation by increasing intracellular levels of cyclic GMP, it was natural to postulate that CO would have a similar effect, as CO was also known to induce cyclic GMP. We now know that CO causes vasodilation directly via activation of soluble guanylate cyclase in smooth muscle cells and indirectly through inhibition of the vasoconstrictors endothelin-1 and platelet-derived growth factor-B. In addition to these vasodilatory effects, CO may also prevent vascular remodeling by inhibiting smooth muscle cell proliferation. Enhanced activity of HO-1 in the lung has been shown to prevent the development of hypoxic pulmonary hypertension and inhibit structural remodeling of the pulmonary vessels in rats. Inhaled CO has also been shown to attenuate experimental pulmonary hypertension in rats. CO has also been strongly implicated in the development of hepatopulmonary syndrome in experimental models, and patients with hepatopulmonary syndrome have been shown to have higher carboxyhemoglobin levels than control subjects. In summary, CO production under conditions of physiological stress results in a wide range of adaptive responses that are relevant to pulmonary disease. Improving our understanding of the biological activities of CO will continue to enhance our knowledge of pulmonary pathophysiology. The goals of current research include such things as monitoring disease

328 CAVEOLINS

activity via measurement of exhaled CO, or even manipulating levels of CO in the body therapeutically. If successful, such research could bring CO fully into the day-to-day practice of pulmonary medicine. See also: Acute Respiratory Distress Syndrome. Apoptosis. Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview. Pulmonary Fibrosis.

Further Reading Ameredes BT, Otterbein LE, Kohut LK, et al. (2003) Low-dose carbon monoxide reduces airway hyperresponsiveness in mice. American Journal of Physiology: Lung Cellular and Molecular Physiology 285(6): L1270–L1276. Brouard S, Otterbein LE, Anrather J, et al. (2000) Carbon monoxide generated by heme oxygenase-1 suppresses endothelial cell apoptosis. The Journal of Experimental Medicine 192(7): 1015– 1026. Dolinay T, Szilasi M, Liu M, and Choi AM (2004) Inhaled carbon monoxide confers antiinflammatory effects against ventilatorinduced lung injury. American Journal of Respiratory and Critical Care Medicine 170(6): 613–620. Fujita T, Toda K, Karimova A, et al. (2001) Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis. Nature Medicine 7(5): 598–604. Horvath I, Donnelly LE, Kiss A, et al. (1998) Raised levels of exhaled carbon monoxide are associated with an increased expression of heme oxygenase-1 in airway macrophages in asthma: a new marker of oxidative stress. Thorax 53: 668–672.

Carcinoma

Maines MD (1993) Carbon monoxide: an emerging regulator of cGMP in the brain. Molecular and Cellular Neurosciences 4: 389–397. Morita T and Kourembanas S (1995) Endothelial cell expression of vasoconstrictors and growth factors is regulated by smooth muscle cell-derived carbon monoxide. The Journal of Clinical Investigation 96: 2676–2682. Morita T, Mitsialis SA, Hoike H, Liu Y, and Kourembanas S (1997) Carbon monoxide controls the proliferation of hypoxic smooth muscle cells. Journal of Biological Chemistry 272: 32804–32809. Otterbein LE, Bach FH, Alam J, et al. (2000) Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nature Medicine 6: 422–428. Otterbein LE, Haga M, Zuckerbaun BS, et al. (2003) Carbon monoxide suppresses arteriosclerotic lesions associated with chronic graft rejection and with balloon injury. Nature Medicine 9: 183–190. Otterbein LE, Mantell LL, and Choi AM (1999) Carbon monoxide provides protection against hyperoxic lung injury. American Journal of Physiology: Lung Cellular and Molecular Physiology 276: L688–L694. Yachie A, Niida Y, Wada T, et al. (1999) Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. The Journal of Clinical Investigation 103: 129–135. Yet SF, Perrella MA, Layne MD, et al. (1999) Hypoxia induces severe right ventricular dilatation and infarction in heme oxygenase-1 null mice. The Journal of Clinical Investigation 103: R23–R29. Zhou Z, Song R, Fattman CL, et al. (2005) Carbon monoxide suppresses bleomycin-induced lung fibrosis. American Journal of Pathology 166(1): 27–37.

see Tumors, Malignant: Bronchogenic Carcinoma; Bronchial Gland and Carcinoid Tumor;

Carcinoma, Lymph Node Involvement.

CAVEOLINS M P Lisanti and J-F Jasmin, Albert Einstein College of Medicine, New York, NY, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Caveolae are small invaginations of the plasma membrane that are implicated in endocytosis, vesicular trafficking, and signal transduction. Caveolin proteins (Cav), the principal structural proteins of caveolae, consist of three distinct genes, namely Cav1, Cav-2, and Cav-3. Cav-1 and Cav-2 are usually coexpressed and are particularly abundant in endothelial cells, fibroblasts, smooth muscle cells, and epithelial cells. In contrast, Cav-3 is muscle-specific and is solely expressed in smooth, cardiac, and skeletal muscle cells. Caveolin proteins appear to play a role as key regulators of pulmonary structure and function. Homozygous deletion of the Cav-1 gene (Cav-1 ( / )) results in numerous pulmonary abnormalities, characterized by a loss

of Cav-1 and Cav-2 protein expression, a loss of caveolae formation, thickened alveolar septa, lung hypercellularity, pulmonary endothelial cell proliferation, fibrosis, and exercise intolerance. Cav-2 ( / ) mice show similar pulmonary abnormalities to the Cav-1 ( / ) mice, but without a loss of Cav-1 expression and normal caveolae formation. Furthermore, Cav-1 ( / ) mice develop pulmonary hypertension and right ventricular hypertrophy. Interestingly, the endogenous levels of caveolin proteins appear to be altered in numerous pulmonary disorders, such as pulmonary hypertension, lung cancers, pulmonary fibrosis, and pulmonary interstitial edema.

Introduction Caveolae, which were first identified in the 1950s as ‘smooth’ uncoated pits, are now defined as 50– 100 mm flask-shaped invaginations of the plasma

CAVEOLINS 329

membrane; they are implicated in endocytosis, vesicular trafficking, and signal transduction (Figure 1). Caveolae can either be observed as curved U-shaped invaginations of the plasma membrane, fully invaginated caveolae, grape-like clusters of interconnected caveolae (caveosome), or as a transcellular channel (as a consequence of the fusion of individual caveolae) (Figure 2). These caveolar domains are a subset of lipid rafts that contain the essential coat protein, named caveolin (Cav). As shown in Figure 3, caveolins appear as membrane-bound proteins that form homo-oligomers and show an unusual topology, with both their NH2 and COOH termini facing the cytoplasm. The caveolin gene family consists of

three distinct genes, namely Cav-1, Cav-2, and Cav3. Cav-1 and Cav-2 are usually coexpressed and are particularly abundant in endothelial cells, adipocytes, fibroblasts, and smooth muscle cells. On the other hand, Cav-3 appears to be muscle-specific and is thus solely expressed in smooth, cardiac, and skeletal muscle cells. Cav-1 further encodes two isoforms, namely Cav-1a and Cav-1b. These two Cav-1 isoforms derive from two distinct Cav-1 mRNAs generated by alternative transcription initiation sites.

Expression of Caveolin Proteins in the Lungs Although all three caveolin isoforms are expressed in the lungs, Cav-1 still remains the most extensively studied isoform, with wide expression in endothelial cells, fibroblasts, smooth muscle cells, bronchial epithelial cells, and alveolar type I cells. However, Cav-1 is poorly expressed in alveolar type II cells. Cav-1 is also present in the developing lung, suggesting important roles for caveolin proteins in lung development. Interestingly, in the developing lung, the expression of the two Cav-1 isoforms appears to be cell-type specific, with Cav-1a being expressed in endothelial cells and Cav-1b being expressed in alveolar type I cells. This cell-type specific expression could be ascribed to differential transcriptional regulation of Cav-1 in lung epithelial and endothelial cells. Whether this cell-type specific expression of Cav-1 isoforms also occurs in adult mouse lung remains to be clarified.

RBC

Figure 1 Electron micrograph (EM) depicting endothelial cell caveolae (arrows) from a mouse lung endothelial cell. RBC, red blood cell. Scale ¼ 200 nm. Adapted from Razani B, Wang XB, Engelman JA, et al. (2002) Caveolin-2-deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae. Molecular and Cellular Biology 22(7): 2329–2344, with permission of the American Society for Microbiology.

6

Caveolin Proteins and Pulmonary Endocytosis and Transcytosis Based on their appearance and membrane localization, caveolae were initially proposed as transport vesicles. These vesicles were then thought to either

1

2 5

3 4

Nucleus

Figure 2 Schematic representation of a cell illustrating morphological variants of caveolae. These include: 1, traditional caveolae; 2, fully invaginated caveolae; 3, cavicles (internalized caveolae not associated with the plasma membrane); 4, caveosome or rosette-like structures; 5, grape-like clusters of interconnected caveolae; 6, transcellular channels.

330 CAVEOLINS

internalize macromolecules (endocytosis) or transport them across the cell (transcytosis). The presence of several proteins implicated in vesicle formation, docking, and fusion in lung caveolae strongly supports a role for caveolae in endocytosis and transcytosis. In accordance with this idea, several viruses, bacteria, and toxins, such as simian virus 40 and cholera toxin, are thought to be preferentially internalized by caveolae. Furthermore, transcytosis of gold-conjugated albumin appears to be restricted to caveolae in the lungs. The caveolar localization of gp60, a 60 kDa albumin-binding protein, supports the role of caveolar domains in the transcytosis of albumin. The recent generation of caveolin-deficient mice elegantly confirmed the implications of lung caveolae in albumin endocytosis. Indeed, lung endothelial cells of Cav-1 ( / )-deficient mice show defects in the uptake and transport of gold-conjugated albumin.

Caveolin Proteins and Pulmonary Signal Transduction Besides their roles as transport vesicles, caveolae microdomains also function in signal transduction via their compartmentalization of signal transduction

C-terminal

cascades. Indeed, lung caveolae are known to compartmentalize several signaling molecules, such as protein kinase C (PKC), phosphatidylinositol-3 (PI3) kinase, heterotrimeric G-protein subunits, extracellular signal-related kinase (ERK), endothelial nitric oxide synthase (eNOS), signal transducer and activator of transcription-3 (STAT3), and Ras-related GTPases. In accordance with a role in signal transduction, lung caveolae are also enriched in the calcium ATPase and inositol triphosphate receptors. Most of the proteins sequestered within caveolar domains possess caveolin-binding motifs and consequently interact with Cav-1. These protein–protein interactions are mediated by the interaction of a caveolin-binding motif (within specific target proteins) with a specific region of Cav-1 (residues 82–101), named the scaffolding domain (Figure 3). Interestingly, Cav-1 also functions as a negative regulator of many of these signaling molecules (Figure 4). The generation of Cav-1 ( / )-deficient mice supports the Cav-1mediated ‘negative regulation’ of several proteins, such as eNOS, ERK1/2, and STAT3. For example, lungs of Cav-1 ( / )-deficient mice show increased NO production, as well as microvascular hyperpermeability. Interestingly, administration of an NOS inhibitor

Transmembrane domain Scaffolding domain Oligomerization domain

N-terminal Figure 3 Schematic representation of the topology of caveolins as the principal structural proteins of caveolae. Some important domains of Cav-1, such as the oligomerization, scaffolding, and transmembrane domains, are illustrated.

CAVEOLINS 331 Caveolin-1

82

1

101

178

DGIWKASFTTFTVTKYWFYR

(Scaffolding domain of Cav-1)

ΦXΦXXXXΦXXΦ

(Caveolin-binding sequence motif)

Inactivation

PI3 Kinase

eNOS

STAT3

ERK1/2

G-protein subunits

PKC

Modulation of pulmonary signal transduction Figure 4 Schematic representation of the interaction of the Cav-1 scaffolding domain with the caveolin-binding sequence motifs of PI3 kinase, eNOS, STAT3, ERK1/2, G-protein subunits, and PKC. Reproduced from Okamoto T, Schlegel A, Scherer PE, and Lisanti MP (1998) Caveolins, a family of scaffolding proteins for organizing ‘preassembled signaling complexes’ at the plasma membrane. Journal of Biological Chemistry 273: 5419–5422, copyright & 1998 by the American Society for Biochemistry and Molecular Biology.

(L-NAME) to Cav-1 ( / )-deficient mice reverses their microvascular hyperpermeability, thus supporting Cav-1-mediated inhibition of eNOS activity. Similarly, Cav-1 ( / )-deficient mice show hyperactivation of cardiac ERK1/2 and pulmonary STAT3 signaling.

Pulmonary Phenotypes of Caveolin-Deficient Mice Surprisingly, mice with homozygous deletion of the different caveolin genes are viable and fertile. Yet, Cav-1 ( / )-deficient mice show numerous abnormal phenotypes, such as a reduction in life span, loss of caveolae formation in tissues that usually express Cav-1, loss of Cav-2 expression, resistance to dietinduced obesity, decreased vascular tone secondary to eNOS hyperactivation, and the development of cardiomyopathy. On the other hand, Cav-3 ( / )deficient mice show a loss of caveolae formation in their cardiac and skeletal muscles and develop progressive cardiomyopathy. Importantly, the generation of caveolin-deficient mice identified caveolin proteins as key regulators of pulmonary structure and function. Indeed, Cav-1 ( / )-deficient mice demonstrate pulmonary abnormalities, characterized by thickened alveolar septa, hypercellularity, fibrosis, endothelial cell

proliferation, and exercise intolerance. Importantly, selective homozygous deletion of the Cav-2 gene results in similar thickening of the alveolar septa, hypercellularity, and endothelial cell proliferation as in Cav-1 ( / )-deficient mice, but without any defects in caveolae formation or Cav-1 expression. These findings suggest a selective role for Cav-2 in lung structure and function. Interestingly, Cav-1 ( / )deficient mice also develop pulmonary hypertension and right ventricular hypertrophy, as shown by increases in the right ventricular systolic pressures and the ratio of the right ventricular weight over the left ventricular weight, respectively. Whether Cav-2 ( / )-deficient mice also develop pulmonary hypertension and right ventricular hypertrophy still remains to be determined. Similarly, although Cav-3 is highly expressed in the smooth muscle cells of the lungs, the potential pulmonary phenotypes of Cav-3 ( / )-deficient mice still remain to be assessed.

Pathological Implications of Caveolin Proteins The endogenous expression of caveolin proteins appears to be altered in numerous lung diseases, such as pulmonary hypertension, lung cancers, pulmonary fibrosis, and pulmonary interstitial edema.

332 CAVEOLINS Pulmonary Hypertension

Given the pulmonary phenotype of caveolin-deficient mice and their roles as negative regulators of several pro-proliferative signaling pathways, caveolin proteins could be important regulators of pulmonary hypertension development. Accordingly, both Cav-1 and Cav-2 protein levels appear to be decreased in the lungs of rats subjected to myocardial infarction (MI)-induced pulmonary hypertension. The downmodulation of Cav-1 and Cav-2 expression is associated with the increased tyrosine-phosphorylation of STAT3, as well as the upregulation of Cyclin D1 and D3 in the lungs of MI rats. The decreased expression of caveolin proteins could thus represent an initiating mechanism leading to the activation of several proproliferative pathways, such as the STAT3/cyclinsignaling cascades and consequently leading to the development of pulmonary hypertension. This hypothesis is supported by the findings of increased STAT3 phosphorylation and increased cyclin D1/D3 expression in the lungs of Cav-1 ( / )- and Cav-2 ( / )-deficient mice. Furthermore, pulmonary Cav-1a expression was shown to be decreased in rats with monocrotaline-induced pulmonary hypertension as early as 48 h after monocrotaline treatment. Accordingly, monocrotaline treatment of cultured pulmonary arterial endothelial cells also results in decreased Cav-1a expression, increased STAT3, and ERK1/2 phosphorylation, and the stimulation of DNA synthesis within 48 h. Lung Cancers

Caveolin proteins are often perceived as tumor-suppressor genes. Accordingly, the Cav-1 gene is localized to a suspected tumor-suppressor locus on human-chromosome 7 (7q31.1/D7S522). Caveolin1’s well-known negative regulation of several proproliferative proteins, such as ERK1/2, H-Ras, c-Src, and cyclin D1, strongly supports the tumor-suppressor role of Cav-1. Accordingly, a reduction of Cav-1 expression was reported in numerous lung carcinoma cell lines. Caveolin-1 gene transcriptional activity also appears to be downregulated in human lung adenocarcinomas. The generation of caveolin-deficient mice strongly supports the tumor-suppressor roles of Cav-1. Indeed, Cav-1 ( / )-deficient mice interbred with tumor-prone transgenic mice show accelerated development of multifocal dysplastic mammary lesions, as well as enhanced lung metastasis. Although Cav-1 definitely appears to act as a tumor-suppressor gene in numerous cancers, its precise role in the development of metastasis remains obscure. Indeed, the expression of Cav-1 conversely increases in ipsilateral hilar/peribronchial lymph node

metastases isolated from patients with lung carcinoma. The upregulation of Cav-1 even correlates with the cancer stage and mortality rate in patients with lung adenocarcinoma and pulmonary squamous cell carcinoma. Although the increased expression of Cav-1 might promote the inhibition of apoptosis, the induction of filopodia formation, or even the stimulation of angiogenic processes, the exact mechanisms underlying the prometastatic effects of Cav-1 remain puzzling and may well be cell-type specific. Pulmonary Fibrosis

Given the pulmonary phenotype of caveolin-deficient mice, caveolin proteins could also be implicated in the development of lung fibrosis. Accordingly, the expression of Cav-1 is altered in several models of lung fibrosis, such as irradiation, CdCl2/TGF-b1, and bleomycin. Western blot analysis of total lung homogenates indicates an overall decrease in Cav-1 expression in mice with irradiation- and bleomycininduced fibrosis. This decrease in pulmonary Cav-1 expression is associated with hyperactivation of the Ras-p42/44 MAP kinase pathway (ERK signaling), which is thought to favor collagen deposition. Importantly, lung fibroblasts isolated from scleroderma patients with pulmonary fibrosis also show a marked decrease in Cav-1 expression and hyperactivation of ERK signaling. Interestingly, the expression of Cav-1 appears to be differentially regulated in alveolar type I cells and endothelial cells. Indeed, although the expression of Cav-1 appears to decrease in lung epithelial cells, it conversely might increase in lung endothelial cells. Whereas an upregulation of endothelial Cav-1 expression could indicate an increase in endothelial caveolar transcytosis, the downregulation of epithelial Cav-1 expression might impair the metabolic function and reduce the signal transduction capacity of alveolar type I cells. Thus, the loss of lung epithelial Cav-1 expression appears as an indicator of subcellular alterations in lung fibrogenesis. Pulmonary Interstitial Edema

Caveolin proteins might also be implicated in the development of pulmonary interstitial edema. Indeed, slight increases (B5%) in extravascular water are sufficient to drive the recruitment of endothelial caveolae and the upregulation of Cav-1 protein levels at the air–blood barrier plasma membrane. These effects could be subsequent to increased hydraulic interstitial pressures and shear stress. The recruitment of endothelial caveolae might thus represent an important mechanotransduction response to flow increases.

CD1 333

Conclusions Caveolin proteins represent novel regulators of pulmonary structure and function, which may play essential roles in the development of a number of pulmonary diseases. Indeed, caveolin-deficient mice show numerous abnormalities in pulmonary structure and function. Furthermore, caveolin protein expression appears to be altered in several pulmonary disorders such as pulmonary hypertension, lung cancers, pulmonary fibrosis, and pulmonary interstitial edema. Thus, tight regulation of caveolin protein expression appears fundamental for normal pulmonary functioning. Whether in vivo modulation of the caveolin protein levels could have therapeutic effects on pulmonary diseases remains to be determined. Interestingly, in vivo administration of a cell-permeable Cav-1 mimetic peptide was previously shown to reduce microvascular hyperpermeability, inflammation, and tumor progression in mice. Whether the in vivo administration of such a cell-permeable Cav-1 peptide could complement the decreased expression of endogenous Cav-1 and prevent the development of pulmonary hypertension, pulmonary fibrosis, and some lung cancers remains to be investigated. See also: Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Pulmonary Edema. Signal Transduction. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Tumors, Malignant: Overview. Vascular Disease.

Further Reading Cohen AW, Hnasko R, Schubert W, and Lisanti MP (2004) Role of caveolae and caveolins in health and disease. Physiological Reviews 84: 1341–1379. Gratton JP, Lin MI, Yu J, et al. (2003) Selective inhibition of tumor microvascular permeability by cavtratin blocks tumor progression in mice. Cancer Cell 4: 31–39. Ho CC, Huang PH, Huang HY, et al. (2002) Up-regulated caveolin-1 accentuates the metastasis capability of lung

adenocarcinoma by inducing filopodia formation. American Journal of Pathology 161: 1647–1656. Jasmin JF, Frank PG, and Lisanti MP (2005) Caveolin proteins in cardiopulmonary disease and lung cancers. Advances in Molecular and Cell Biology 36: 211–233. Jasmin JF, Mercier I, Hnasko R, et al. (2004) Lung remodeling and pulmonary hypertension after myocardial infarction: pathogenic role of reduced caveolin expression. Cardiovascular Research 63: 747–755. Mathew R, Huang J, Shah M, Patel K, Gewitz M, and Sehgal PB (2004) Disruption of endothelial-cell caveolin-1alpha/raft scaffolding during development of monocrotaline-induced pulmonary hypertension. Circulation 110: 1499–1506. Okamoto T, Schlegel A, Scherer PE, and Lisanti MP (1998) Caveolins, a family of scaffolding proteins for organizing ‘‘preassembled signaling complexes’’ at the plasma membrane. Journal of Biological Chemistry 273: 5419–5422. Razani B, Engelman JA, Wang XB, et al. (2001) Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities. Journal of Biological Chemistry 276: 38121–38138. Razani B, Wang XB, Engelman JA, et al. (2002) Caveolin-2deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae. Molecular and Cellular Biology 22: 2329–2344. Razani B, Woodman SE, and Lisanti MP (2002) Caveolae: from cell biology to animal physiology. Pharmacological Reviews 54: 431–467. Schubert W, Frank PG, Razani B, et al. (2001) Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo. Journal of Biological Chemistry 276: 48619– 48622. Schubert W, Frank PG, Woodman SE, et al. (2002) Microvascular hyperpermeability in caveolin 1 ( / ) knock-out mice. Journal of Biological Chemistry 277: 40091–40098. Tourkina E, Gooz P, Pannu J, et al. (2005) Opposing effects of protein kinase C alpha and protein kinase C epsilon on collagen expression by human lung fibroblasts are mediated via MEK/ ERK and caveolin-1 signaling. Journal of Biological Chemistry 280: 13879–13887. Williams TM, Medina F, Badano I, et al. (2004) Caveolin-1 gene disruption promotes mammary tumorigenesis and dramatically enhances lung metastasis in vivo. Role of Cav-1 in cell invasiveness and matrix metalloproteinase (MMP-2/9) secretion. Journal of Biological Chemistry 279: 51630–51646. Zhao YY, Liu Y, Stan RV, et al. (2002) Defects in caveolin-1 cause dilated cardiomyopathy and pulmonary hypertension in knockout mice. Proceedings of the National Academy of Sciences, USA 99: 11375–11380.

CD1 S M Behar, Brigham and Women’s Hospital at Harvard Medical School, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract CD1 proteins are a distinct lineage of antigen-presenting molecules that evolved to present lipid and glycolipid antigens to T

cells. The human CD1 locus contains five distinct genes, CD1A– E, which have an overall structure similar to class I major histocompatibility complex (MHC) including their association with b2 microglobulin. However, unlike class I MHC, the CD1 antigen-binding groove is larger, deeper, and lined almost exclusively with hydrophobic amino acids. The ability of CD1 to present lipid antigens to T cells significantly expands the universe of antigens that can be recognized by T cells. CD1 proteins are divided into two groups: group 1 CD1 (CD1a, -b, and -c) and

334 CD1 group 2 CD1 (CD1d). Group 1 CD1-restricted human T cells can recognize mycobacterial cell wall lipids and are elicited as part of the immune response to Mycobacterium tuberculosis. Some CD1d-restricted T cells, also known as natural killer T (NKT) cells, recognize endogenous self-antigens while others recognize foreign microbial antigens. CD1d-restricted NKT cells rapidly secrete cytokines following activation and are thought to be an important link between innate and adaptive immunity. NKT cells appear to enhance pulmonary immunity against several pulmonary pathogens. Conversely, NKT cells are required for the development of airway hyperreactivity and inflammation in rodent models of allergic pulmonary hypersensitivity. Thus, CD1-restricted T cells are an important component of the T cell response to infection and tumors, and have an important role in the regulation of many diverse immune responses.

Introduction CD1 was first recognized as a human cell surface antigen frequently expressed by thymocytes and certain neoplasms of T cell origin. Later, it was found to be highly expressed by Langerhans’ cells and dendritic cells (DCs). Even today, CD1 remains a useful phenotypic marker of these cells. However, the finding that CD1 was a target of certain autoreactive human T cells led to the insight that CD1 may be an antigen-presenting molecule. We now understand that while certain CD1-restricted T cell clones are autoreactive, presumably because they recognize endogenous self-lipid antigens, other CD1-restricted T cells recognize foreign antigens. In a series of landmark studies, Porcelli, Beckman, and Brenner demonstrated that CD1b and CD1c present microbial antigens to human T cells. Eventually, the antigens presented by CD1 began to be identified. The first antigen to be purified was mycolic acid, a unique lipid molecule found in the cell wall of Mycobacterium tuberculosis, which was recognized by a CD1brestricted T cell clone. Mycolic acid, which is responsible for the acid-fastness of M. tuberculosis and is thought to be a bacterial virulence factor, belongs to a family of a-branched b-hydroxy fatty acids and its two acyl chains are approximately 90 carbons long. This discovery shattered the paradigm that T cells could only recognize short peptide sequences and was the first evidence that CD1 presented microbial lipid antigens to T cells. These findings have generated considerable interest in determining whether CD1-restricted T cells contribute to host defense against infection, as the lipid antigens recognized by them may be potential vaccine candidates. Microbial pathogens are unable to rapidly vary lipid antigen structure when subjected to immune selective pressure because lipids are products of multistep biosynthetic pathways. CD1-presented antigens are also attractive vaccine candidates because the limited degree of polymorphisms in the

CD1 locus makes it more likely that such antigens would elicit a consistent immune response, even in genetically diverse human populations.

Structure The human CD1 genetic locus is located on chromosome 1 and is unlinked to the major histocompatibility complex (MHC) locus, which is found on chromosome 6. Five distinct genes are encoded within the human CD1 locus, CD1A, -B, -C, -D, and -E, and in contrast to MHC, there is only limited polymorphism between unrelated individuals. CD1A, -B, and -C have been designated group I CD1 based on their primary sequence homology. Genes encoding CD1 proteins have been detected in all mammals studied to date, and these studies have observed tremendous variation in the number and type of CD1 genes found in different species. Importantly, mice and rats only have orthologs of CD1D (group II CD1). The murine CD1 genes, known as CD1d1 and CD1d2, are more than 95% homologous to one another and may have arisen by gene duplication. The homology between human and mouse CD1d is striking as human CD1d-restricted T cells recognize murine CD1d, and vice versa. Thus, rodents are a useful experimental model to ascertain the function of CD1d and CD1d-restricted T cells. Small animal models that are suitable for the study of group I CD1 also exist and include the rabbit and the guinea pig. Thus, the CD1 genetic locus is evolutionarily conserved in mammals and is distinct from the MHC locus. While CD1 and MHC may share a common ancestor, CD1 has evolved into a distinct lineage of antigen-presenting molecules specialized for the presentation of lipid antigens to T cells. Despite only limited amino acid sequence homology to MHC, the CD1a, -b, -c, -d, and -e polypeptides all associate with b2 microglobulin and form a tertiary structure similar to class I MHC (Figure 1). However, unlike class I MHC, the CD1 antigenbinding groove is larger, deeper, and lined almost exclusively with hydrophobic amino acids. These properties make CD1 particularly well suited to bind lipid and glycolipid molecules. Nearly all of the lipid antigens presented by CD1 have a polar head group and one or two lipid tails. Combining crystallographic and structure-function data, a model emerges in which the hydrophobic acyl chains of the lipid antigen are buried deep within the antigenbinding groove, while the polar head group is closer to the molecular surface and just emerges from the antigen-binding groove. The T cell receptor (TCR) recognizes the combination of the polar head group and the surface of the CD1 protein. The crystal structures of human CD1a and CD1b, and murine

CD1 335

(a)

(b)

(c)

Figure 1 Crystal structure of CD1b. In 2002, Gadola et al. solved the three-dimensional structure of CD1b with phosphatidylinositol in the antigen-binding groove. The CD1b heavy chain is displayed in blue and b2-microglobulin is displayed in yellow. The phosphatidylinositol phospholipid ligand is colored according to the CPK scheme. In (a) and (b), CD1b is observed as it might appear on the cell surface, looking either parallel (a) or perpendicular (b) to the two a-helices formed by the a1 and a2 domains that create the antigen-binding pocket. In (c), the perspective is looking down at the ‘top’ of CD1b, which is the portion of the molecule that interacts with the T cell receptor. The view in (c) is enlarged compared to (a) and (b), and the a3 domain and b2 microglobulin have been removed for clarity.

CD1d, have all been solved and reveal some interesting differences. The antigen-binding groove of CD1a is shallower and less complex than that observed for CD1b or CD1d. CD1b has the most complicated antigen-binding pocket consisting of a maze of channels, giving it the capacity to bind lipid antigens with very long acyl chains. Thus, the antigenbinding groove of the different CD1 molecules may affect the binding and selection of the antigens that are presented to CD1-restricted T cells.

Regulation of Production and Activity Group I CD1 is strongly expressed on cortical thymocytes and on a variety of APC subtypes in several different tissues. For example, Langerhans’ cells in the skin express very high levels of CD1a, and other DCs, particularly in inflamed tissue, express CD1a, -b, and -c. In addition, CD1c and CD1d are found on certain B cell subsets. Murine CD1d is more widely expressed, and is detected on nearly all lymphocytes and myeloid-lineage cells. Although little is known about CD1 regulation, cell surface expression of group I CD1 increases during cytokine-induced differentiation and maturation of monocyte-derived dendritic cells. Similarly, CD1d is upregulated during macrophage activation by cytokines and microbial products. Modulation of CD1 surface expression may be one way that the immune system regulates activation of CD1-restricted T cells. This could be particularly important for the regulation of immune responses by CD1d-restricted natural killer (NK) cells. Following protein translation, CD1 associates with b2m in the endoplasmic reticulum. Processing and loading of lipid antigens requires a unique set of accessory molecules, which are beginning to be

delineated. These include glycosidases to trim sugars from glycolipid antigens and microsomal triglyceride transfer protein and saponins that facilitate lipid transfer between membranes and CD1. Following transportation to the cell surface, CD1 proteins undergo extensive recycling through the endocytic pathway, which is dependent on a tyrosine-containing motif found in the cytoplasmic tail of CD1. For example, CD1a, which has a truncated cytoplasmic tail lacking the tyrosine-based motif, is primarily expressed on the cell surface although it can also be detected in the recycling endosome. In contrast, CD1b is expressed on the cell surface but also has an extensive distribution in the endocytic compartment and colocalizes with class II MHC. Such differences are likely to affect the source and type of lipid each CD1 molecule encounters. For example, CD1b may have evolved to sample lipids from the late endocytic compartment while CD1a may have evolved to sample lipids from the extracellular milieu. Thus, each CD1 protein follows a unique recycling pathway and has a different steady state distribution.

Biological Function The main function of both group I and group II CD1 is antigen presentation of lipids and glycolipids to T cells. Although there are likely to be certain constraints on the type of lipids presented by CD1, a surprisingly diverse number of lipids have been shown to bind or be presented by CD1 to T cells. Group I CD1 presents a number of lipids derived from the cell wall of M. tuberculosis to human T cells including the lipopeptide didehydroxymycobactin (CD1a), mycolic acid and glucose monomycolate (both CD1b), and isoprenoid glycolipids (CD1c).

336 CD1

Both self and foreign antigens have been identified that are presented by CD1d. CD1d-restricted T cells can recognize phospholipids (including phosphatidylinositol and phosphatidylethanolamine) and certain ceramides, although whether these antigens are important in immunoregulation or autoimmune disease is not yet certain. In addition, certain lipids from Leishmania donovani and M. tuberculosis bind to CD1d and activate NKT cells. Finally, synthetic lipid antigens are presented by CD1d and activate CD1drestricted T cells. The prototype of these antigens is a-galactosylceramide. Although their physiological relevance is unknown, these synthetic antigens have been extremely useful tools. Because these antigens are potent and specific activators of CD1d-restricted T cells, they have been used in vitro and in vivo to study the function of CD1d-restricted T cells, particularly in whole animal models, and may ultimately be useful pharmacologically as a way to exploit the therapeutic potential of these T cells. As more CD1presented antigens are discovered, their apparent diversity substantiates the hypothesis that antigen presentation by CD1 to T cells significantly expands the number of microbial antigens that can be recognized by T cells and enhances their capacity to mediate antimicrobial immunity.

CD1-Restricted T Cells Similar to the MHC/peptide complex, recognition of the CD1/lipid antigen complex is mediated by the T cell receptors (TCRs) (Figure 2). Both the ab and the gd TCRs can recognize the CD1/antigen complex, and although the initial descriptions of CD1-restricted T cells were distinguished by their lack of CD4 or CD8 accessory molecules, examples of CD4 þ and CD8 þ CD1-restricted T cells have been reported. Functional differences among these various CD1-restricted T cell subsets exist. For example, human CD4 þ and CD4 8 CD1d-restricted T cells differ in the spectrum of cytokines produced following activation. Another difference is that while CD1restricted M. tuberculosis antigen-specific T cells can kill infected cells, CD8 þ T cells accomplish this using cytotoxic granules, whereas killing by CD4 8 T cell-mediated killing is CD95/CD95L dependent (Figure 2). Like other T cells, CD1-restricted T cells require costimulation for their proliferation. However, they are typically CD28 negative and their costimulatory requirement appears to be independent of the CD28-CD80/CD86 axis. The V, D, and J gene segments encoding group 1 CD1-restricted TCRs are similar to those used by MHC-restricted T cells. Interestingly, many CD1-restricted TCRs have an increased frequency of basic residues in the

CDR3 region. These positively charged residues are hypothesized to interact with the polar head group of lipid antigens, which usually carry a negative charge. An important advance in the biology of CD1d-restricted T cells was the finding that many NKT cells are CD1d-restricted. NKT cells are a unique subset of lymphocytes found in humans and other species that not only express the T cell antigen receptor (i.e., TCR), but also cell surface antigens that are characteristic of NK cells. NKT cells are not abundant in lymphoid organs but are increased in frequency in the bone marrow and certain peripheral organs such as the liver. Two distinct subsets of CD1d-restricted NKT cells have been described. The best-described subset is referred to as invariant NKT cells (iNKT cells), because they express an invariant TCR alpha (a) chain. Murine iNKT cells use a canonical Va14Ja281 TCRa chain, which preferentially pairs with TCR beta (b) chains encoded by Vb2, -7, or -9. Human iNKT cells are similar and are encoded by an invariant Va24-JaQ TCRa chain. Thus, the TCR repertoire of iNKT cells has only limited diversity. A second subset of CD1d-restricted T cells uses a more diverse repertoire, although it may be less varied than MHC-restricted T cells. Although NKT cells have become synonymous with CD1d-restricted T cells, caution is required as conventional T cells can also express NK cell markers, particularly when activated. Thus, more precise methods are required to identify CD1-restricted T cells. The specificity of most iNKT cells for the synthetic antigen a-galactosylceramide provides one way to identify these T cells. Thus, flow cytometry has been used to detect binding of CD1d multimers loaded with a-galactosylceramide to iNKT cells. When identified this way, the phenotype of iNKT cells resembles memory T cells. Even their pattern of chemokine receptor expression is typical of T cells that home to peripheral tissues rather than lymphoid organs. Consistent with their activated phenotype, CD1d-restricted iNKT cells rapidly produce a burst of IL-4 within hours of stimulation, followed secretion of large amounts of interferon gamma (IFN-g). Not surprisingly, iNKT cells can influence whether CD4 þ T cell responses acquire a Th1- or Th2-like phenotype. Thus, iNKT cells are increasingly viewed as a link between innate and adaptive immunity, because of the kinetics of their response, as well as their ability to influence conventional T cell immunity (Figure 2).

CD1 in Respiratory Diseases How CD1 and CD1-restricted T cells affect the expression of respiratory diseases is still being defined. As antigen-presenting molecules, CD1 and

CD1 337 Perforin Granzyme Granulysin

IL - 12 T cell Activation of adaptive immunity

Fas - FasL T cell

CD1b TCR

IFN - TNF -

(a)

T cell

APC TCR CD1b

IFN - TNF -

NK cell

Activation of innate immunity

IFN - TNF -

Effector functions

M

Immunoregulation

Perforin Granzyme Granulysin

IL - 12 T cell

Fas - FasL NKT cell

NKT cell

APC TCR CD1d

IFN - TNF -

(b)

Activation of adaptive immunity

CD40–CD40L

IFN - TNF-

CD1d TCR

M

Activation of innate immunity

IFN -

Effector functions

NK cell

Immunoregulation

Figure 2 The effector and immunoregulatory functions of CD1-restricted T cells. (a) Presentation of microbial or self-lipid antigens by CD1a, -b, or -c expressed on DCs leads to activation of group I CD1-restricted T cells. When stimulated, these T cells secrete IFN-g and TNF-a, which can lead to activation of macrophages (Mf). CD1-restricted T cells are also potent cytolytic T cells and kill target cells either by granule-dependent mechanisms or by Fas-mediated killing. CD1-restricted T cells also stimulate DC maturation and IL-12 production. Thus, by inducing DC maturation and secreting cytokines, group 1 CD1-restricted T cells can modulate other innate and adaptive immune responses. (b) CD1d presentation of exogenous antigens such as aGalCer or endogenous ligands leads to the activation of CD1d-restricted NKT cells. IL-12 is an important costimulatory signal for NKT cell activation, which is produced by DCs after CD40–CD40L interaction or after TLR activation by microbial ligands. Activated NKT cells secrete IFN-g, TNF-a, and IL-4, which can stimulate other cell types and influence the polarization of CD4 þ T cells. In addition, NKT cells are potent cytolytic cells, and when activated can kill target cells by the mechanisms shown. Adapted from Sko¨ld M and Behar SM (2005) The role of group 1 and group 2 CD1-restricted T cells in microbial immunity. Microbes and Infection 7: 544–551, with permission from Elsevier.

CD1-restricted T cells potentially play a role in immunity to pathogens, tumors, and a variety of immunologically mediated diseases. Lower numbers of circulating CD1d-restricted iNKT cells are found in patients with several types of cancers, including multiple melanomas and prostate cancer, and in patients with autoimmune conditions such as systemic sclerosis and rheumatoid arthritis. Patients with sarcoidosis and autoimmune diabetes not only have fewer iNKT cells, but those that are present are dysfunctional. These findings support a role for NKT cells in natural tumor immunosurveillance and in

preventing the emergence of autoimmune disease, but do not delineate how NKT cells exert their protective role. The ability of CD1 to present microbial antigens to T cells has strongly implicated CD1restricted T cells in microbial immunity. Not only can CD1-restricted T cells recognize mycobacterial lipids, but these lipid antigens are processed and presented by M. tuberculosis infected cells, demonstrating that the CD1 antigen-presentation pathway is physiologically relevant during infection. Furthermore, recognition of infected cells leads to activation of CD1-restricted T cells, which induces IFN-g

338 CD11/18

and tumor necrosis factor alpha secretion and target cell lysis. Thus, CD1-restricted T cells express effector functions that are beneficial during infection. Finally, to address the relevance of these observations during human tuberculosis, it should be noted that CD1-restricted responses to mycobacterial lipid antigens are detected in the peripheral blood of people infected with M. tuberculosis, indicating that CD1restricted T cell responses are physiologically primed following infection. Mouse models for human diseases are powerful tools to identify the mechanisms by which the immune system combats disease. As CD1d orthologs exist in mice, more information has been obtained on the role of CD1d and CD1d-restricted NKT cells in murine models of human disease. For example, it has been shown that iNKT cells contribute to natural tumor immunosurveillance. Although NKT cells prevent the induction of certain autoimmune diseases, they are an important source of Th2 cytokines during the induction of allergen-induced airway hyperreactivity. Lastly, CD1d-restricted NKT cells are important for host protection against pathogenic viruses, bacteria, fungi, and parasites. Mice lacking CD1d are more susceptible to certain pulmonary infections including Pseudomonas aeruginosa, Pneumococcus pneumoniae, and Cryptococcus neoformans. In other models, specific activation of CD1d-restricted NKT cells can enhance immunity and prolong survival of mice infected with virulent M. tuberculosis. NKT cells are thought to make an early contribution to host immunity, which is not surprising given their rapid activation and production of various cytokines. Less is known about the in vivo role of group 1 CD1-restricted T cells but more information is becoming available with the development of alternative small animal models such as the guinea pig and the

potential to make transgenic mice expressing human group I CD1 genes. See also: Interferons. Leukocytes: T cells; Pulmonary Macrophages. Systemic Disease: Sarcoidosis.

Further Reading Bollyky PL and Wilson SB (2004) CD1d-restricted T-cell subsets and dendritic cell function in autoimmunity. Immunology and Cell Biology 82: 307–314. Brigl M and Brenner MB (2004) CD1: antigen presentation and T cell function. Annual Review of Immunology 22: 817–890. Dascher CC and Brenner MB (2003) Evolutionary constraints on CD1 structure: insights from comparative genomic analysis. Trends in Immunology 24: 412–418. Gadola SD, Zaccai NR, Harlos K, et al. (2002) Structure of human CD1b with bound ligands at 2.3 A, a maze for alkyl chains. Nature Immunology 3: 721–726. Godfrey DI and Kronenberg M (2004) Going both ways: immune regulation via CD1d-dependent NKT cells. Journal of Clinical Investigation 114: 1379–1388. Kronenberg M and Gapin L (2002) The unconventional lifestyle of NKT cells. Nature Reviews: Immunology 2: 557–568. Moody DB and Porcelli SA (2003) Intracellular pathways of CD1 antigen presentation. Nature Reviews Immunology 3: 11–22. Porcelli SA (1995) The CD1 family: a third lineage of antigen presenting molecules. Advances in Immunology 59: 1–98. Shinkai K and Locksley RM (2000) CD1, tuberculosis, and the evolution of major histocompatibility complex molecules. Journal of Experimental Medicine 191: 907–914. Sko¨ld M and Behar SM (2003) Role of CD1d-restricted NKT cells in microbial immunity. Infection and Immunity 71: 5447–5455. Sko¨ld M and Behar SM (2005) The role of group 1 and group 2 CD1-restricted T cells in microbial immunity. Microbes and Infection 7: 544–551. van der Vliet HJ, Molling JW, von Blomberg BM, et al. (2004) The immunoregulatory role of CD1d-restricted natural killer T cells in disease. Clinical Immunology 112: 8–23. Watts C (2004) The exogenous pathway for antigen presentation on major histocompatibility complex class II and CD1 molecules. Nature Immunology 5: 685–692.

CD11/18 D S Wilkes and T J Webb, Indiana University School of Medicine, Indianapolis, IN, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Integrins are cell membrane receptors that assemble as heterodimers by the noncovalent association of the a and b subunits. The b2 family of leukocyte integrins comprises four a subunits (CD11a–d) with a common b2 subunit (CD18). These adhesion molecules have been shown to play an important role in host defense. The critical role of the CD11/CD18 receptors in

leukocyte adherence has been shown using mAb blocking experiments, knockout animal models, and studies of individuals with a deficiency in these glycoproteins. The use of mAbs against these adhesion receptors results in defective adherence, which suggests that this approach may have therapeutic potential in treating certain pulmonary diseases.

Introduction Lymphocyte migration from the bloodstream into secondary lymphoid organs is essential for maintaining homeostasis and for providing efficient defense

CD11/18 339

globular head of an integrin. Inserted into the b propeller fold is the ‘inserted’ or I domain. The I domain is present in 9 out of 18 a subunits and is homologous to the A domain of the von Willebrand factor protein. Consequently, this region is frequently referred to as the I/A domain. A key feature of the I/A domain is the metal ion-dependent adhesion site or the MIDAS motif, which is composed of D-X-S-X-S. It is the MIDAS motif plus surrounding residues that form the actual ligand binding site. The b2 subunit (CD18) also contains a MIDAS motif, but it has not been directly shown to bind metal ions. The short and highly conserved cytoplasmic region of CD18 contains tyrosine and several serine and threonine residues, which account for stimulus-induced phosphorylation (Figure 2). An interesting feature of the extracellular region of CD18 is a highly conserved cysteine-rich region (CRR) consisting of four tandem repeats of an eight-cysteine motif. Notably, a point mutation in this region prevents cell surface expression of CD18. The a subunits of CD11/CD18 are highly homologous but distinct from the common b subunit. Mac-1 and p150, 95 are more related to each other (63% identity at the amino acid level) than to LFA-1 (36% homology), and this is reflected by their close functional similarity. Polymorphisms in LFA-1 and Mac-1 have been reported. Accordingly, monoclonal antibodies (mAbs) have been shown to recognize specific activation or conformation states of CD11/CD18 receptors.

against pathogens. This migration occurs mostly through high endothelial venules and is partially regulated by signaling cascades involving adhesion and activation. Integrins mediate the firm adhesion of leukocytes to the endothelium that allows for the transmigration of cells into tissue. The CD11/CD18 leukocyte adhesion molecules consist of four cell surface membrane glycoproteins, namely LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), p150, 95 (CD11c/CD18), and adb2 (CD11d/CD18). The a subunits form a noncovalent association with a common b subunit (CD18) of 94 kDa to form a1b2 heterodimers. The divalent cations Ca2 þ and Mg2 þ are essential for the stabilization and function of the a1b2 complex. Signaling events through the b2 integrins lead to full activation status of the molecules. The active form of integrin is usually characterized by lateral clustering of the proteins on the cell membrane and by a change in structural conformation.

Cellular Location and Structure The four distinct genes encoding for the a subunits occur in a cluster on chromosome 16, band p11– p13.1. This region also contains the gene encoding sialophorin (CD43), a leukocyte adhesion receptor, and the b isoform protein kinase C (PKC), suggesting the presence of a gene cluster involved in leukocyte adhesion and activation. The gene encoding CD18 is on chromosome 21, band q22. The major structural features of CD11/CD18 are illustrated in Figure 1. CD11a–d and CD18 are transmembrane proteins spanning the plasma membrane once, with a short C-terminal cytoplasmic region and a large N-terminal extracellular domain. In the a subunit there are seven repeating homologous sequences at the N terminus. This part of the a subunit has been modeled as a b propeller fold and is a major contributor to the

Biosynthesis The a and b subunits of CD11/CD18 are synthesized in leukocytes from independent precursors. The two subunits associate as precursors prior to further carbohydrate processing in the Golgi. Association of ab

Signal sequence Cysteine rich repeats

I/A domain MIDAS motif D-X-S-X-S

2 1 22

1

2

MIDAS motif D-X-S-X-S

3

4

110

1 361

M

M

M

5

6

7

449

2

3

4 628 700 723 769 TM C

Figure 1 Schematic diagram of the primary structure of CD11/CD18. The main structural features of the a (CD11) and b (CD18) subunits are illustrated. In the b subunit, the red regions 1–4 outline the four cysteine-rich repeats. The yellow region represents the transmembrane (TM) site, and C represents the cytoplasmic end. In the a subunit, the seven tandem repeats in the extracellular portion are shown in teal, and repeats 5–7 contain a metal binding site (M).

340 CD11/18

CD18 2-integrins

Mac-1

LPS

CD11b

Tyrosine kinases Cytoskeleton rearrangement & cell motility

Ca2+ influx

Monocyte Lymphocyte CD11a LFA-1

CD18

ICAM-1

Endothelial cells Figure 2 Overview of b2 integrin signaling. b2 integrin engagement induces phosphorylation of tyrosine kinases. This can result in cytoskeletal rearrangement, an event that controls cell motility. Also, Ca2 þ signals are generated as described in detail in the legend to Figure 3.

Table 1 b2 adhesion molecules and their ligands Name

Structure

Ligand

Cell distribution

CD11a/CD18 (LFA-1) CD11b/CD18 (Mac-1)

aLb2 aMb2

ICAM-1 (CD54), ICAM-2 (CD106) iC3b, factor w, fibrinogen, ICAM-1?, LPS, Leishmania gp63

CD11c/CD18 (p150,95)

axb2

iC3b

CD11d/CD18

adb2

All leukocytes Monocytes, macrophages, some lymphocytes, natural killer cells, neutrophils, granulocytes Monocytes, granulocytes, macrophages, natural killer cells, certain lymphocytic tumor cell lines Monocytes, macrophage foam cells, splenic red pulp macrophages, lymphocytes

LFA-1, lymphocyte function-associated antigen; Mac-1, macrophage-1 antigen; ICAM, intercellular adhesion molecules; iC3b, proteolytic fragment of complement protein C3; factor w, coagulation factor w.

precursors appears to be important for further carbohydrate processing and cell surface expression of the heterodimers. Following association, the CD11 and CD18 subunit precursors undergo an increase in their molecular mass that is accompanied by a conversion on N-linked carbohydrates to their complex form. As expected, more CD18 precursors are synthesized in lymphocytes and monocytes than each of the three a subunits. Interestingly, the type of a subunit affects the glycosylation pattern of CD18.

Expression and Biological Function The CD11/CD18 glycoproteins are differentially expressed (Table 1). The level of CD11/CD18

expression is dependent on the cell type and the state of cell activation and differentiation. The CD11/ CD18 glycoproteins are only expressed in leukocytes. Whereas CD11a/CD18 is present on all leukocytes, CD11b, c/CD18 is normally restricted to expression on monocytes, macrophages, polymorphonuclear lymphocytes (PMNs), and natural killer (NK) cells. However, p150, 95 and the recently described adb2 are mainly expressed by tissue macrophages. In B and T cells, all CD11/CD18-dependent functions – such as mitogen, antigen, and alloantigeninduced proliferation, T cell-mediated cytotoxicity, B cell aggregation, and Ig production – are inhibited by anti-LFA-1 mAbs. This is consistent with LFA-1 being the only heterodimer normally expressed on these

CD11/18 341

Whereas MAC-1 is a phagocytic receptor, used by neutrophils to engulf bacteria, yeast, and other microorganisms, the roles of p150, 95 and adb2 have not been well documented. All CD11/CD18 receptors contribute to chemotaxis and adhesion to cytokine-activated endothelium by neutrophils and monocytes and to antibody-dependent cellular cytotoxicity (ADCC).

cells. Granulocytes, monocytes, and NK cells express all three CD11/CD18 heterodimers, which accounts for the wider range of CD11/CD18-dependent functions mediated by these cells. LFA-1 seems to mediate an early Mg2 þ -dependent adhesion step in cell–cell interaction. Studies have shown that LFA-1 is primarily responsible for adhesion and signaling at the immunological synapse, involved in the clustering of proliferating lymphocytes, and found between CTLs and their targets. Anti-LFA-1 mAbs inhibit T-cell proliferation in response to stimuli that require cell–cell contact. Notably, APC-independent T-cell responses do not require LFA-1. Also, at high effector:target ratios or during secondary stimulation, the role of LFA-1 is significantly diminished, perhaps as a result of increased expression of several other adhesion molecules. Mac-1 is essential for binding iC3b and other ligands, spreading, homotypic adhesion, and phagocytosis of opsonized particles in neutrophils, a role that may also be fulfilled by p150, 95 in resident macrophages. Following activation, Mac-1 can initiate signaling via its linkage to the actin cytoskeleton and associated signaling proteins. Also, Mac-1 is thought to act as a signaling partner for other leukocyte receptors, including lipopolysaccharide (LPS)/LPS binding protein receptors (CD14), formyl-methionylleucyl-phenylalanine (FMLP) receptors, urokinase plasminogen activator receptors (CD87), and FcRs.

Ligands Several ligands have been shown to interact with CD11/CD18 molecules in a divalent cation-dependent manner, as shown in Table 1. The first wellcharacterized ligand was iC3b, a component of the complement system. iC3b binds to Mac-1 and p150, 95 at 37oC in resting cells, but it is greatly enhanced in cells activated by phorbol esters. LFA-1 binds to the two N-terminal domains of intracellular adhesion molecules (ICAM)-1 and -2. ICAM-1 is inducible and expressed on leukocytes and endothelial and epithelial cells in response to inflammatory cytokines (Figure 3). In contrast to ICAM-1, ICAM-2 is constitutively expressed on endothelial cells and a variety of B and T lymphoblastoid cell lines, and its expression is not enhanced by inflammatory stimuli. However, unlike ICAM-2, ICAM-1 was uniquely found to be involved in migration of lymphocytes into inflamed tissue and in trapping lymphocytes in the lung. Concurring with

Endothelial cells

ICAM-1 CD18

CD11a

Lymphocyte

Tyrosine kinases

PLC- 1/2

PLC- 1/2

PIP2 DAG PKC

Ins(1,4,5)P3 Ca2+ release

Figure 3 b2 integrins can induce calcium signaling in leukocytes. Ligand binding of b2 integrins causes activation of tyrosine kinases. The tyrosine kinases are phosphorylated, which increases the catalytic activity of PLC-g. Then, PLC-g catalyzes the hydrolysis of phosphatidylinositol-4,5-biphosphate (PIP2) into both diacylglycerol (DAG) and inositol triphosphate (Ins(1,4,5)P3). Next, Ins(1,4,5)P3 stimulates the release of Ca2 þ from internal stores, which is followed by an influx of Ca2 þ .

342 CD11/18

those data, studies using a murine model of asthma found that ICAM-1 is important in the migration of lymphocytes to the lungs during an allergic inflammatory response. Also, in another study examining the homing mechanisms of cytotoxic T cells, LFA-1 was shown to be critical for the retention of effector CD8 T cells in the lungs. Moreover, in an adoptive transfer model involving alloreactive CD4 T cells, adherence of T cells in the lungs was shown to be partially dependent on LFA-1 and its receptor ICAM-1. Conflicting data exist regarding whether Mac-1 also binds to ICAM-1; however, binding of two other ligands, coagulation factor w and fibrinogen, to an activated form of Mac-1 has been shown. All three heterodimers (CD11a–c/CD18) bind to LPS, as determined by blocking studies using mAbs, suggesting that binding is mediated by homologous regions in the CD11 subunits or by the common CD18 subunit.

CD11/CD18 in Respiratory Disease Leukocyte adhesion deficiency (LAD-1) is a disorder caused by the lack of surface expression of CD11/ CD18, which is due to heterogeneous defects in the CD18 subunit. The classical disease is characterized by recurring necrotic soft tissue infections, impaired wound healing with a lack of pus, and severe gingivitis. Also, phagocytes from patients with this disorder have functional defects in aggregation, chemotaxis, phagocytosis, endothelial cells, and complement iC3b binding and ADCC. Two phenotypes of LAD-1 have been reported based on the level of CD18 expressed. In the severe form, CD11/CD18 is not detectable, and death from bacterial infections usually occurs during the first few years of life. However, in the moderate phenotype cells express 2–5% of the normal level of CD18 and the clinical course is much milder. In CD18deficient mice, as with LAD-1 patients, marked neutrophilia was found. However, in contrast to LAD-1 or CD18-deficient mice, CD11b-deficient mice do not exhibit any leukocytosis or display a significant increase in bacterial infections. Nevertheless, in vivo studies have shown that these mice have defective firm adhesion, and in vitro studies have shown that neutrophils from these mice have defects in adhesion, iC3b-mediated phagocytosis, phagocytosis-induced respiratory burst, and homotypic aggregation. Remarkably, although CD11a-deficient mice displayed normal CTL responses to a systemic virus infection, they failed to develop a DTH response to 2,4-dinitrofluorobenzene sensitization and challenge. Because ICAM-1 is a major ligand for LFA-1 and perhaps Mac-1, ICAM-1-deficient mice are

particularly interesting. Two groups have reported ICAM-1 knockout mice. Although both ICAM-1deficient murine lines develop normally and have moderate leukocytosis, the mice displayed multiple abnormalities in their inflammatory responses, specifically impaired neutrophil emigration in response to chemical peritonitis, resistance to septic shock, and decreased contact hypersensitivity reaction. Intriguingly, it has been shown that during allergic lung inflammation, there is a delayed increase in eosinophils in the airway lumen and a prolonged presence of eosinophil infiltrates in lung tissue in ICAM-2deficient mice. In contrast, the importance of ICAM2 in binding T cells has been demonstrated in frozen section assays of chronically inflamed human airway epithelium; however, inflammation of the lungs in ICAM-2-deficient mice was due to an accumulation of eosinophils, not lymphocytes. Overall, it has been suggested that ICAM-2 expressed in vascular endothelium, alveolar walls, or large airway epithelium participates in the traffic of eosinophils from the bloodstream to the airway lumen.

Conclusion Lymphocyte homing is tightly regulated; however, the molecular basis of lymphocyte migration to the lungs is not well understood. Because the lung is the portal of entry for airborne pathogens, lymphocytes must constantly recirculate. Circulating lymphocytes become transiently trapped within the lung. This has been shown for normal lymphocytes and even more so for activated/memory lymphocytes. Notably, retention is mediated, at least in part, by LFA-1. Interestingly, studies have shown that effector and memory cytotoxic T lymphocytes traverse to the lungs and reside in the lung parenchyma for extended periods. In addition, it has been suggested that normal lung microvessels can constitutively support the retention of activated CD8 T cells. Moreover, murine studies investigating the role of LFA-1 in an acute RSV infection showed that treatment of mice with neutralizing Abs to LFA-1 resulted in diminished illness and delayed viral clearance. These data further support the hypothesis that using blocking mAbs against b2 integrins could be an immunotherapeutic strategy. Many diseases that affect the lungs involve inflammation. Following sequestration and initial adhesion, neutrophils migrate along the capillary endothelium and into the interstitium and alveoli. This emigration can occur through either a CD11/CD18-dependent or -independent pathway. The adhesion pathway used depends on the stimulus. Stimuli that have been shown to induce CD18-dependent emigration include Escherichia coli, E. coli lipopolysaccharide,

CD14 343

Pseudomonas aeruginosa, IgG immune complexes, interleukin-1, and phorbol myristate acetate. These stimuli appear to act by inducing the translocation of NF-kB, resulting in the production of inflammatory cytokines and ICAM-1 on the pulmonary capillary endothelial cells. Stimuli that have been shown to induce the CD11/CD18-independent neutrophil emigration include Streptococcus pneumoniae, group B Streptococcus, Staphylococcus aureus, hydrochloric acid, hyperoxia, and C5a. Previous studies have shown that the absence of CD11/CD18 in granulocytes and monocytes attenuates the acute cellular inflammatory responses in vivo; therefore, these data suggest that blocking CD11/CD18 may ablate the tissue damage induced by these cells. Consequently, this was shown to be the case in several ischemia–reperfusion injury animal models. In fact, giving anti-Mac-1 mAbs before or soon after the induction of ischemia–reperfusion injury significantly reduced tissue injury, microvascular permeability, and acidosis. However, the lack of protection of the lung tissue in some of these models may be an indication of the importance of other adhesion pathways mediating PMN migration. Lastly, detailed investigations of the LAD-1 syndrome in humans and studies involving adhesion molecule-deficient mice have provided novel insights into the cell and molecular biology of leukocyte emigration. However, further studies must be done to address the discrepancies observed between deficient animals and wild-type animals treated with blocking mAbs.

Acknowledgments This work was supported by National Institutes of Health grants HL60797 and HL/AI67177 to D S Wilkes. See also: Adhesion, Cell–Matrix: Integrins. Coagulation Cascade: Fibrinogen and Fibrin. Complement. Dendritic Cells. Leukocytes: Eosinophils; Neutrophils; Monocytes; Pulmonary Macrophages.

Further Reading Arnaout MA (1990) Structure and function of the leukocyte adhesion molecules CD11/CD18. Blood 75: 1037–1050. Buyon JP, Slade SG, Reibman J, et al. (1990) Constitutive and induced phosphorylation of the a- and b-chains of the CD11/CD18 leukocyte integrin family: relationship to adhesion-dependent functions. Journal of Immunology 144: 191–197. Dayer JM, Isler P, and Nicod LP (1993) Adhesion molecules and cytokine production. American Review of Respiratory Disease 148: S70–S74. Doerschuk CM (2000) leukocyte trafficking in alveoli and airway passages. Respiratory Research 1: 136–140. Doerschuk CM, Mizgerd JP, Kubo K, et al. (1999) Adhesion molecules and cellular biomechanical changes in acute lung injury. Chest 116: 37S–43S. Etzioni A, Doerschuk CM, and Harlan JM (1999) Of man and mouse: leukocyte and endothelial adhesion molecule deficiencies. Blood 94: 3281–3288. Graham IL, Gresham HD, and Brown EJ (1989) An immobile subset of plasma membrane CD11b/CD18 (Mac-1) is involved in phagocytosis of targets recognized by multiple receptors. Journal of Immunology 142: 2352–2358. Hogg N and Bates PA (2000) Genetic analysis of integrin function in man: LAD-1 and other syndromes. Matrix Biology 19: 211– 222. Lehmann JCU, Jablonski-Westrich D, Haubold U, et al. (2003) Overlapping and selective roles of endothelial intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 in lymphocyte trafficking. Journal of Immunology 171: 2588–2593. Pilewski JM and Albelda SM (1993) Adhesion molecules in the lung: an overview. American Review of Respiratory Disease 148: 531–537. Thatte J, Dabak V, Williams MD, et al. (2003) LFA-1 is required for retention of effector CD8 T cells in mouse lungs. Blood 101: 4916–4922. van Spriel AB, Keusen JHW, van Egmond M, et al. (2001) Mac-1 (CD11b/CD18) is essential for Fc receptor-mediated neutrophil cytotoxicity and immunologic synapse formation. Blood 97: 2478–2486. van Spriel AB, van Ojik HH, Bakker A, et al. (2003) Mac-1 (CD11b/CD18) is crucial for effective Fc-receptor mediated immunity to melanoma. Blood 101: 253–258. Xu B, Wagner N, Pham LN, et al. (2003) Lymphocyte homing to bronchus-associated lymphoid tissue (BALT) is mediated by L-selectin/PNAd, a4b1 integrin/VCAM, and LFA-1 adhesion pathways. Journal of Experimental Medicine 197: 1255– 1267.

CD14 Y Tesfaigzi and M Daheshia, Lovelace Respiratory Research Institute, Albuquerque, NM, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract CD14 is a glycolipid-anchored membrane glycoprotein expressed on cells of the myelomonocyte lineage including

monocytes, macrophages, and some granulocytes. CD14 is a key molecule in the activation of innate immune cells and exists as a membrane-anchored or soluble form. It is encoded by a gene on human chromosome 5q31.1, a region where several genes implicated in asthma pathogenesis are localized. CD14 is expressed in a variety of hematopoietic and parenchymal cells and it has a range of biological activity including cell differentiation, immune response, and host-pathogen interactions. While CD14 is an essential part of the lipopolysaccharide

344 CD14 (LPS) receptor complex, it requires interaction with long-terminal repeat 4 to successfully transmit LPS-induced signals to the cell. In addition, CD14 is an integral part of the mechanism by which macrophages interact and engulf apoptotic cells. A functional single nucleotide polymorphism within the promoter region of CD14 that affects expression levels is associated with a higher risk of developing atopy. The interactions of CD14 with other receptors are important for the normal signaling of LPS and host-pathogen interactions and affect the susceptibility or resistance to a variety of diseases including atopy and septic shock.

Introduction CD14 was originally discovered through reactivity of monoclonal antibodies on human peripheral blood monocytes and was cloned in 1988. Subsequently, the protein was purified, and our understanding of its role in inflammatory responses has been increasing steadily. While this protein is best known for its ability to act as a receptor for bacterial endotoxin, lipopolysaccharides (LPS), and to elicit proinflammatory host responses, other functions for CD14 such as those related to the recognition of apoptotic cells and clearance by phagocytosis have also gained importance. This latter role involves a process that suppresses inflammation and appears to contradict its proinflammatory function, making this protein highly interesting to study.

Structure The CD14 protein is encoded by a gene localized on the human chromosome 5q31.1 or on the syntenic mouse chromosome 18. In humans and mice, CD14 has an amino acid length of 375 and 366, respectively, that is transcribed by a 1367-nucleotide mRNA. CD14 exists in two different forms: a membrane-anchored and a soluble (sCD14) form. The membranebound CD14 is a glycoprotein with a molecular weight of 53–55 kDa, attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. The sCD14 is generated when the membrane-bound CD14 is shed and released into the bloodstream.

levels of sCD14 have been associated with inflammatory/infectious diseases such as dermatitis, septic shock, malaria infection, human immunodeficiency virus (HIV), and severe burns.

Biological Functions CD14 and LPS Binding

While LPS is the main ligand for CD14, several other molecules such as peptidoglycans, phospholipids, and dextrans, can interact with CD14. Several lines of evidence suggest a crucial role for CD14 in the biological effects of LPS: (1) The neutralizing antibody to CD14 inhibits the interaction of LPS with the receptor and protects primates from endotoxininduced shock; (2) CD14-deficient mice are 10–100 times less sensitive to LPS than their wild-type counterparts because they produce very low amounts of proinflammatory cytokines and are resistant to a lethal dose of Gram-negative bacteria; (3) overexpression of CD14 renders mice more susceptible to LPS-induced septic shock or to Gram-negative bacteria-induced death. The susceptibility of CD14-deficient mice is specific to Gram-negative bacteria because responses to infection by Gram-positive bacteria such as Staphylococcus aureus are not affected by the absence of CD14. The interaction between LPS and CD14 is facilitated by the soluble LPS-binding protein (LBP), which increases the affinity of LPS to CD14 by more than 100- to 1000-fold. Therefore, LBP-deficient mice are more resistant to LPS than wild-type mice and are defective in their ability to mount appropriate inflammatory responses to bacterial infection. For example, LBP-deficient mice are incapable of controlling infection by Gram-negative bacteria such as Salmonella and succumb to a dose of pathogen, which is well tolerated by their heterozygous or wild-type littermates. It has also been reported that LBP may be incorporated as a transmembrane protein in the cytoplasmic membrane of mononuclear cells to mediate activation of these cells by LPS, revealing that LBP has a different mode of action in LPS signaling.

Regulation of Expression

CD14 as a Component of LPS Receptor Complex

The regulatory sequence of CD14 contains multiple consensus-binding sites for CAAT/enhancing and binding protein (C/EBP) and Sp transcription factors. The gene expression is induced by transforming growth factor beta (TGF-b) and vitamin D and is downregulated by interleukin (IL)-4. Membranebound CD14 is expressed by a variety of cells, including monocytes/macrophages, neutrophils, granulocytes, and hepatocytes. Increased serum

Although CD14 is the major membrane receptor for binding LPS, it is incapable of inducing intracellular signaling because it lacks an intracellular domain. CD14 must form a complex with Toll-like receptor 4 (TLR4), which is bound to myeloid differentiation protein-2 (MD-2) to transmit intracellular signaling in response to LPS binding (Figure 1). The mature human MD-2 and TLR4 proteins consist of 160 and 839 amino acid residues, respectively. After LPS

CD14 345

LPS LPS LBP

MD-2

LBP

TLR4

LPS

CD14

TIR

of NF-kB, but it does so via an alternative pathway that does not require MyD88 and TRAF6. Additionally, TRIF/TICAM can activate the interferon related factor 3 (IRF3) pathway and increase interferon beta (IFN-b) to turn on the JAK/STAT pathway. LPS signaling is decreased in the absence of CD14 or TLR4, revealing the primordial importance of these molecules for LPS effects. Although MyD88 ( / ) mice showed a significant reduction in tumor necrosis factor alpha (TNF-a) production, these mice still exhibit NF-kB and MAPK activation and IFN-b release in response to LPS. Additionally, TRIF/TICAM-1 ( / ) mice exhibit suppressed expression of IFN-b as well as lower IFN-g production; however, the MyD88-dependent activation of IRAK-1, NF-kB, and MAPK are not affected. Complete loss of LPS-induced responses are only observed in mice deficient for both MyD88 and IRIF/TICAM-1 genes, suggesting that LPS acts entirely through these two signaling pathways. CD14 and Phagocytosis

Figure 1 Interaction of CD14 with TLR-4 for signaling through the intracellular TIR domain. LPS, lipopolysaccharide; LBP, LPSbinding protein; TLR4, Toll-like receptor 4; MD-2, myeloid differentiation protein-2.

interacts with the LBP, the heterodimer binds to CD14, which subsequently activates TLR4/MD-2 and initiates signaling through the Toll/IL-1R (TIR) intracellular domain. However, airway epithelial, endothelial, and dendritic cells respond to LPS even though they lack the membrane-bound CD14 because the soluble CD14 is believed to form the LPS complex and activate TLR4. Activation of LPS receptor complex (LPSRC) causes many cellular changes, including increased transcription of multiple genes, release of a variety of cytokines, cell proliferation, and cell death. LPSRC activation results in the recruitment of at least two major cytoplasmic proteins to TLR4: the myeloid differentiation factor 88 (MyD88) and the TIR-containing adapter molecule (TRIF, also known as TICAM). The MyD88-dependent pathway leads to the recruitment of the MyD88 adaptor-like protein (Mal, also known as TIRAP), then the IL-1R-associated kinase (IRAK) and TRAF6, which can either activate mitogen-activated protein kinase kinase (MAPKK) with the ultimate activation of activation protein 1 (AP-1), or can activate IKK and phosphorylate IKba, causing the rapid ubiquitin-dependent proteolysis of the IKK and translocation of free nuclear factor kappa B (NF-kB) to the nucleus. The TRIF/ TICAM pathway can also activate IKK and induction

Cells undergoing apoptosis are cleared rapidly by phagocytosing macrophages, thus preventing the release of intracellular material from dying cells, which would cause inflammation and tissue damage. The removal of apoptotic cells in the body involves a complex array of macrophage receptors, including CD14. CD14 is upregulated in migratory macrophages infiltrating Burkitt’s lymphoma tumors to engage in clearance of apoptotic cells. The monoclonal antibody 61D3 that binds to CD14 on the surface of human macrophages markedly inhibits the capacity of macrophages to interact with and engulf cells undergoing apoptosis. The CD14-mediated interaction of cells with macrophages is specific for apoptotic cells because interaction of viable cells with macrophages is not affected by the monoclonal antibody 61D3. When transiently expressed on the surface of COS cells (African green monkey kidney fibroblast-like cell line), CD14 was found to impart an increase in both of apoptotic lymphocytes by these cells, and both were inhibited by the monoclonal antibody against CD14. Quantitative histological studies indicated that CD14-deficient mice have defective clearance capacity leading to persistence of apoptotic cells in all tissues studied. Additionally, TL-9 mouse macrophages deficient in CD14 expression were completely unable to phagocytose apoptotic thymocytes, and removal of CD14 from J774 macrophages significantly reduced the phagocytic ability of these cells. These studies show that CD14 is an integral part of the phagocytosis process. There is growing evidence that CD14 is a crucial component of the mechanism by which apoptotic

346 CD14

cells are recognized. CD14 interacts with phosphatidylserine, whose exposure to the outer leaflet of the cell membrane is a universal feature of apoptotic cells. CD14 acts as a tethering receptor for apoptotic cells, and when CD14 was removed from macrophages by cleaving its glycosylphosphatidylinositol tether, the phagocytosis of apoptotic lymphocytes by macrophages was substantially reduced. The intracellular adhesion molecule-3 (ICAM-3) is one of many molecules on the surface of apoptotic cells that supports the essential recognition by macrophages. Interestingly, the ICAM-3-mediated interaction of apoptotic leukocytes involves CD14 but is independent of macrophage lymphocyte function-associated antigen-1 (LFA-1), a2b2, and avb3. Several models have suggested that the engagement of CD14 and apoptotic cells would not induce generation of inflammatory responses, as is the case following CD14 and LPS interaction. The size and structural differences between the two ligands could explain why the distinct biological outcome of CD14 activation and the interaction of CD14 and the apoptotic cells do not need the presence of LBP. It is possible that when CD14 binds to apoptotic cells, it does not engage TLR4 or if TLR4 is activated, CD14 interaction with the dying cells, may interact with yet unknown receptor(s) to block the TLR4 responses. CD14 and Respiratory Disease

Several lines of evidence have led to the hypothesis that the CD14 gene may be associated with asthma. Engagement of CD14 by LPS and other bacterial cell wall components enhances IL-12 secretion from dendritic cells, which is believed to be an obligatory signal for the differentiation of naive T cells into Th1-like cells (Figure 2). Activation of CD14 by LPS leads to the release of IL-12, IL-18, and IFN-g, which can deviate the Th2 immune response and decrease the production of immunoglobulin E (IgE) by the B cells. It is also conceivable that upregulation of sCD14 could induce apoptosis in the pathogenic cells involved in asthma and/or induce the induction of suppressive factors such as IL-10 or TGF-b, leading to a decreased severity of the disease. These hypotheses are supported by reports in several British cohorts that reduced sCD14 levels in amniotic fluid and breast milk are associated with the subsequent development of atopy and that high levels of sCD14 are protective against development of recurrent wheezing in hospitalized children with respiratory syncytial virus infection. Furthermore, rhinovirus infection, the most common trigger of

T

sCD14

Regulatory sequence

Coding sequence

CD14 locus

+ DC

+ Macrophage

IL-12 Th0

IFN- B cells

+ Th1

− −

IgE

IFN-

Figure 2 A T allele within the regulator promoter region of CD14 is associated with increased sCD14 levels, which is known to mediate interleukin-12 (IL-12) and interferon gamma (IFN-g) production by dendritic cells (DC) and macrophages, respectively. These cytokines cause a Th1 differentiation of T lymphocytes and suppress the production of IgE by B cells.

acute asthma exacerbations, significantly reduces CD14 levels. The CD14 gene is located in a chromosomal locus proximal to several genes implicated in the pathogenesis of asthma, including IL-3, IL-4, IL-5, IL-9, IL-13, and granulocyte-macrophage colonystimulating factor (GM-CSF). C-159 T, a common single nucleotide polymorphism (SNP) in the proximal promoter region of CD14, has been associated with different levels of sCD14 expression, and homozygous carriers of the T allele have significantly increased sCD14 and decreased total serum IgE. Increased sCD14 levels are associated with low IL-4 and high IFN-g production. The underlying mechanism is believed to be the decreased binding of transcription factor Sp3 to the GC box that includes the polymorphic nucleotide, resulting in enhanced transcriptional activity. Many population-based studies have found associations of the functional promoter polymorphism C159 T with a severity or susceptibility to atopy and serum IgE levels. However, different studies have reported conflicting results as to whether the C or the T allele is associated with atopy, and two studies in Germany involving a large cohort of children found no association with asthma. It is still unclear whether these differences are caused by other SNPs within the same gene or differences in LPS concentrations within various studies. A role for conferring increased sensitivity of this SNP with LPS exposure was supported by the finding in a study of farmers that those homozygous for the -159 T allele have significantly lower lung function and an increased prevalence of wheezing compared to those with the C allele.

CELL CYCLE AND CELL-CYCLE CHECKPOINTS 347 See also: Apoptosis. Asthma: Overview. Dendritic Cells. Endotoxins. Interferons. Leukocytes: Monocytes; Pulmonary Macrophages. Toll-Like Receptors.

Further Reading Baldini M, Vercelli D, and Martinez FD (2002) CD14: an example of gene by environment interaction in allergic disease. Allergy 57(3): 188–192. Cook DN, Pisetsky DS, and Schwartz DA (2004) Toll-like receptors in the pathogenesis of human disease. Nature Immunology 5(10): 975–979. Gregory CD (2000) CD14-dependent clearance of apoptotic cells: relevance to the immune system. Current Opinion in Immunology 12(1): 27–34. Koppelman GH and Postma DS (2003) The genetics of CD14 in allergic disease. Current Opinion in Allergy and Clinical Immunology 3(5): 347–352. Savill J, Dransfield I, Gregory C, and Haslett C (2002) A blast from the past: clearance of apoptotic cells regulates

immune responses. National Review of Immunology 2(12): 965–975. Schmitz G and Orso E (2002) CD14 signalling in lipid rafts: new ligands and co-receptors. Current Opinion in Lipidology 13(5): 513–521. Takeda K and Akira S (2004) TLR signaling pathways. Seminars in Immunology 16(1): 3–9. Triantafilou M and Triantafilou K (2002) Lipopolysaccharide recognition: CD14, TLRs and the LPS-activation cluster. Trends in Immunology 23(6): 301–304. Ulevitch RJ, Mathison JC, and da Silva Correia J (2004) Innate immune responses during infection. Nature Immunology 5(10): 987–995. Ulevitch RJ and Tobias PS (1999) Recognition of Gram-negative bacteria and endotoxin by the innate immune system. Current Opinion in Immunology 11(1): 19–22. Wright SD (1995) CD14 and innate recognition of bacteria. Journal of Immunology 155(1): 6–8. Zhang G and Ghosh S (2001) Toll-like receptor-mediated NFkappaB activation: a phylogenetically conserved paradigm in innate immunity. Journal of Clinical Investigation 107(1): 13–19.

CELL CYCLE AND CELL-CYCLE CHECKPOINTS A Carnero, Centro Nacional de Investigaciones Oncologicas, Madrid, Spain & 2006 Elsevier Ltd. All rights reserved.

Abstract Completion of the cell cycle requires the precise coordination of a variety of macromolecular synthesis, assembly, and movement events. The DNA must be replicated and the chromosomes condensed, segregated, and decondensed. The spindle pole must duplicate, separate, and migrate, and changes in the cell and nuclear membrane must ensure a complete and correct duplication. Checkpoint controls are superimposed to ensure correct cell-cycle transition by monitoring the execution of specific events and coupling them to further progression. Cell-cycle checkpoints ensure that the system only proceeds to the next stage when the previous stage has been completed. Correct control of the cell cycle results from the coordinated and sequential activation–inactivation of a family of key regulators known as cyclin-dependent kinases. In this article, the mechanisms that ultimately regulate the checkpoints and their contribution to the cell cycle are discussed.

Checkpoints and the Cell Cycle In 1970, Lee Hartwell and colleagues recognized the first cell division cycle (cdc) mutants among a large collection of temperature-sensitive lethal mutants of Saccharomyces cerevisiae. These clones were further identified as mutants that arrested division at a unique stage of the cell cycle regardless of their stage at the time they were shifted to restrictive temperature. The

phenotypes of the cdc mutants revealed that the execution of late events in the cell cycle depended on the prior completion of early events. Cell division occurs by an ordered series of metabolic and morphogenic changes that are collectively called the cell cycle. The cell cycle is divided into four distinct phases (Figure 1): *

*

*

*

Synthetic (S) phase, in which the replication of DNA takes place Mitotic (M) phase, in which equal distribution of duplicated DNA and cellular division take place Gap 1 (G1) phase: the gap between completion of the M phase and the beginning of the S phase Gap 2 (G2) phase: the gap between completion of the S phase and the beginning of the M phase

Completion of the cell cycle requires the precise coordination of a variety of macromolecular synthesis, assembly, and movement events. The DNA must be replicated and the chromosomes condensed, segregated, and decondensed. The spindle pole must duplicate, separate, and migrate, and changes in cell and nuclear membrane must ensure a complete and correct duplication. Checkpoint controls are superimposed to ensure correct cell-cycle transition by monitoring the execution of specific events and coupling them to further progression. Cell cycle checkpoints ensure that the system only proceeds to the next stage when the

348 CELL CYCLE AND CELL-CYCLE CHECKPOINTS Spindle checkpoint

G2/M checkpoint

G2

CDK4

CDK2 CDK2

Cyclin D

G1 CDK1

Cyc lin E

Cyc

lin B

M

G1 checkpoint

S Cyclin A DNA damage checkpoint Figure 1 Cell cycle and cell-cycle checkpoints.

previous stage has been completed. The key checkpoints appear to be as follows: * *

*

*

G2/M: No entry to M without completion of S. Spindle checkpoint: Prevents anaphase onset until completion of mitotic spindle assembly. Start or restriction (R) point: Blocks progress for nonproliferating or differentiated cells; integrates external signals to proceed with the cell cycle. DNA damage: monitoring of DNA damage and block of cell-cycle progress; allows repair mechanisms to proceed; if DNA repair fails, the cell may be directed into apoptosis.

The coordination of these complex processes is achieved by a series of coordinated and sequential phase transitions of key regulators known as cyclin-dependent kinases (CDKs), which are one of the major targets detected in the cdc mutants (Figure 1).

Mammalian Cell Cycle Regulators The CDKs are a family of closely related Ser/Thr kinases that are activated by association with a regulatory subunit called cyclin (Table 1). The typical catalytic subunit contains a 300-amino acid catalytic core that is inactive when monomeric and unphosphorylated at certain positions. Cyclins are members of structurally related proteins whose levels usually oscillate during the cell cycle. The homology among cyclins is mostly limited to the cyclin box, the domain responsible for

CDK binding and activation. Mutations in this region inhibit both binding and activation. Each CDK interacts with a specific subset of cyclins, although the size of the subset varies (Table 1). The cyclin function is primarily controlled by changes in cyclin levels. In mammalian cells, sequential oscillation in the levels of major cyclins (E, A, and D) reflects levels of messenger RNA and specific proteolysis. Whereas cyclins are essential to activate CDKs, the specific destruction of cyclins is central to the proper regulation of the cell cycle. In terms of proteolysis, the cyclins can be roughly divided into two classes – those that are constitutively unstable through the cell cycle and whose level is therefore determined by the ratio of transcription (cyclins D and E) and those that are unstable in only one phase of the cell cycle. The G2 cyclins (A and B type) are stable throughout interphase and specifically destroyed at mitosis. Cyclin degradation involves the ubiquitin-dependent degradation machinery. However, the details of the conjugation cyclin–proteasome differ for the different cyclins. E and D cyclins are ubiquitinated by the SCF complex, whereas G2 cyclins are ubiquitinated by the anaphase-promoting complex (APC). The SCF complex is active throughout the cell cycle, and the destruction of its substrates depends on their phosphorylation, with different phosphate-binding proteins (F box proteins) guiding different sets of substrates to destruction. The APC is activated at the onset of anaphase and degrades its substrates as the cell exits mitosis.

CELL CYCLE AND CELL-CYCLE CHECKPOINTS 349 Table 1 Complexes of cyclin-dependent kinases (CDKs) Kinase

Regulatory subunit

Substrate

Function

CDK1 CDK2 CDK3 CDK4 CDK5 CDK6 CDK7 CDK8 CDK9 CDK10 CDK11

Cyclin A and B Cyclin A, E, and D Cyclin E Cyclin D p35, cyclin D Cyclin D Cyclin H Cyclin C Cyclin T

pRb, NF, Histona H1 pRb, p27 E2F1/DP1 pRb, Smad3 NF, Tau pRb CDK1/2/4/6 RNA pol II pRb, MBP Ets2 RNA pol II

G2/M G1/S, S G1/S G1/S Neuronal differentiation G1/S CAK Transcriptional regulation G1/S Transcription Transcription

Cyclin L

Degradation Cyclin synthesis

P P P pRb

CKIs Cyclin

CDK

Inactive

Active

Cyclin

Cyclin

CDK

CDK

thr161

thr14

P P P pRb E2F

tyr15 thr161

E2F

thr161 thr14 tyr15

Cyclin H

CDC25 WEE 1

CDK7

DPI

E2F

Transcription

thr161 Figure 2 CDK regulation at the G1 checkpoint.

In addition to cyclin-binding, complete CDK activation requires phosphorylation at a conserved 160/ 161 threonine residue. This phosphorylation may affect the cyclin-binding site because it enhances the binding of some CDK/cyclin complexes. This activating phosphorylation is performed by another CDK/ cyclin complex termed CAK, which consists of CDK7 bound to cyclin H. Mammalian CDK7/cyclin H complexes can phosphorylate CDK1 and CDK2 complexed with various cyclins as well as CDK4 (Figure 2). CDK/cyclin complexes can also be inhibited by phosphorylation of the CDK subunit at the amino terminus (Thr14 and Tyr15 residues in human CDK1 and CDK2). Inactivation via tyrosine 15 phosphorylation is important in the control of the initiation of mitosis (Figure 2). The phosphate in tyrosine 15 is removed by a specific phosphatase, CDC25. There are at least three CDC25 family members in

mammalian cells. CDC25C is more likely to activate cyclin B/CDC2 at mitosis, whereas CDC25A is phosphorylated and activated by cyclin E/CDK2 at the initiation of DNA synthesis. Finally, numerous studies have identified additional regulatory subunits for the CDKs, the cyclindependent kinase inhibitory proteins (CKIs). These proteins bind and inhibit CDK activity. There are two families of CKIs: the CIP/KIP family and the INK4 family (Table 2). The CIP/KIP family includes p21waf1/cip1/sdi1(p21waf1), p27kip1, and p57kip2 and seems to be central in differentiation processes. The members of this family are general inhibitors of all the cyclin/CDK complexes and accumulate in response to a broad range of antiproliferative stimuli. These proteins preferentially associate with the cyclin/CDK complex rather than with the individual CDK subunits. The overexpression of these proteins

350 CELL CYCLE AND CELL-CYCLE CHECKPOINTS Table 2 Involvement of cell-cycle regulatory elements in human cancer Protein

Alteration

Tumor

CDK4

Mutation

Melanoma

Overexpression Overexpression Overexpression Overexpression

Breast, prostate, gastric, parathyroid carcinoma, lung cancer Colorectal carcinoma Breast, ovarian, and gastric carcinoma Hepatocellular carcinoma, lung cancer

CDC25A CDC25B p27KIP1 p57KIP2

Activation Activation Inactivation/degradation Deletion/inactivation

Head and neck cancer, lung cancer Breast cancer, lymphoma, head and neck cancer, lung cancer Breast, prostate, and colorectal carcinoma, lung cancer Beekwith–Wiedemann syndrome

p16INK4a p15INK4b

Deletion/inactivation, mutation Deletion/inactivation

Melanoma, lymphoma, lung cancer, pancreatic carcinoma Leukemia, lymphoma

Plk1 Aurora A Aurora B

Overexpression Overexpression Overexpression

Lung cancer, head and neck squamous carcinoma Several tumors, lung cancer Several tumors, lung cancer

pRb

Deletion/inactivation

Retinoblastoma, lung cancer

p53

Mutation, deletion

Many tumors, lung cancer

Cyclin Cyclin Cyclin Cyclin

D1 D2 E A

arrests cells in different states of the cell cycle according to their biochemical function. The INK4 family of CKIs consists of four related proteins: p16ink4a, p15ink4b, p18ink4c, and p19ink4d. These proteins specifically bind and inactivate CDK4 and CDK6 kinases, inducing G1 arrest. These proteins appear to bind the CDK subunit alone, competing with their activating partner, cyclin D. It is possible that these proteins inhibit CDK activity by reducing the affinity for cyclin D or by competitively preventing the binding of cyclin D to the CDK subunit.

The Cell Cycle Progression through the cell cycle results from the coordinated and sequential activation–inactivation of the previously described elements. Restriction Point

After the proliferative stimuli (growth factors and oncogenes) induce cell proliferation, a first checkpoint (the R point) at late G1 integrates both positive and negative external and internal signals before the cell commits to another round of DNA replication (Figure 1). In mammalian cells the R point is regulated mainly by the CDKs bound to the D type of cyclins, and its activation is mainly driven by transcriptional activation of cyclin D synthesis. Most growth factors and oncogenes trigger cyclin D transcription. There are three types of D cyclins, and they are in part cell-type specific, with most cells expressing D3 and either D1 or D2. The D type of cyclins

associate and activate CDK4 and, in some cells, also CDK6. These cyclins have a very short half-life and their expression is highly growth factor inducible. Dtype cyclins are fundamental in cell-cycle regulation of the retinoblastoma protein (pRb; the major regulator of the R checkpoint). The phosphorylation of pRb is controlled by two opposing reactions: phosphorylation by CDKs in G1, which converts pRb into its inactive form in terms of ligand-binding activity, and dephosphorylation in late M by phosphatases, which reconstitute active pRb. As a result of this interplay of enzymatic reactions, the pRb phosphorylation state fluctuates as the cell passes through the cell cycle. In cycling cells, pRb is found in its active, underphosphorylated form only during the early period of the G1 phase. Although changes in the array of phosphorylation events may occur at other phases and inactivating phosphorylation of newly synthesized pRb may occur throughout the cycle, pRb phosphorylation in late G1 phase and its dephosphorylation in late M phase are the two critical events regulating pRb growth-restraining activities. In vitro, cyclin E- and cyclin D-activated kinases phosphorylate pRb differentially producing an overlapping subset of sites. This has led to the development of three different models of how different phosphorylation events may contribute to pRb control in cells. The first possibility is that both CDK complexes have identical effects, inactivating pRb by phosphorylating in different positions. Alternatively, phosphorylation by different complexes could have different effects by regulating the binding to a different subset of effectors. Finally, combined phosphorylation of both kinases may be necessary to

CELL CYCLE AND CELL-CYCLE CHECKPOINTS 351

completely inactivate pRb. It is difficult to determine which model is correct. Cyclin D1 and pRb functions seem to be interdependent. When cyclin D1 is ectopically expressed in G1 phase, pRb is phosphorylated earlier than in the normal cycle and G1 progression is accelerated. Antibodies to cyclin D1 arrest most cell types before S phase; however, they do not arrest if they lack functional pRb. The effect of the INK4 family of CKIs also seems to be dependent on the presence of functional pRb since the absence of pRb makes the cells insensitive to INK4induced G1 arrest. Unlike cyclin D, cyclin E is both rate-limiting and required for the G1/S transition in both pRb-positive and -negative cells. It has been shown that combined expression of c-myc and oncogenic ras allows entry of growth factor-deprived cells into S phase without pRb phosphorylation. Activation of cyclin E, but not cyclin D, was seen in these cells. A possible target of this pRb-independent pathway is the other members of the pRb family of pocket proteins, p107 and p130, that form stable complexes with cyclin E/CDK2 and cyclin A/CDK2 and whose expression is dramatically regulated at the end of G1 or S phase, respectively. Sequential phosphorylation of particular sites may result in the orchestrated disruption of pocket protein complexes containing different transcription factors, leading to the sequential succession of events required to promote progression through the cell cycle. However, CDK4 is dispensable in most cell types, probably due to a compensatory role by CDK6. Both CDK4 and CDK6 null mice develop normally with minor problems, such as diabetes or some impaired hematopoiesis, respectively. Double CDK4/CDK6 null mice die in early stages of embryogenesis due to severe anemia, but they show normal organogenesis. Double null MEFS proliferate normally and become immortal upon serial passage. These results indicate that in certain circumstances, D-type CDKs are not essential for cell-cycle entry. However, it still has to be confirmed whether a true redundancy for the CDK4/6 activity exists or whether it is an effect of molecular plasticity in early embryogenesis that will never take place in normal developing embryos. One of the consequences (in addition to chromatin remodeling) of pRb phosphorylation is the release of sequestered E2F proteins. The E2F proteins heterodimerize with DP1, forming the active transcription factor. The activity of E2F/DP1 induces the transcription of a number of genes involved in DNA synthesis and cell proliferation. High levels of INK4 proteins lead to inhibition of CDK4/6 kinases and keep pRb unphosphorylated and bound to the E2F transcription factor. The sequestered factor is thus inactivated, inducing a block in G1. This scheme is

much more complex in its details. First, there are six members of the E2F transcription factor family (five activators and a negative regulator, E2F6), and only three of them (E2F1, -2, and -3) are under direct control of pRb. Hypophosphorylated pRb binds the other two members only weakly. E2F4 and E2F5 appear to bind preferentially to p107 and p130, which have a weak affinity for the other three. This suggests the possible existence of two parallel pathways: pRb/E2F1, -2, or -3 and p107/p130/E2F4 or -5. Also, pRb may regulate a number of downstream effectors besides E2Fs, including Elf-1, Myo-D, PU.1, ATF-2, and c-Abl proteins; however, the effector functions of most of these are not well-known. S Phase

Once external signals have been integrated at the R checkpoint and the cells are committed to duplication, the cells initiate the S phase, during which other CDKs take control of DNA replication. CDK2 is considered to be most directly involved in DNA replication. Cyclins E and A sequentially activate CDK2, and both are thought to be essential for initiating DNA replication. Ectopic overexpression of cyclin E in mammalian fibroblasts causes premature entry into S phase, and microinjection of antibodies against cyclin E prevents S phase initiation. Cyclin A activates CDK2 soon after cyclin E does, concomitant with DNA synthesis. Ablation of cyclin A synthesis blocks entry into S phase. Cyclin A co-localizes with sites of DNA replication in S phase nuclei, suggesting that it may be directly involved in the assembly, activation, or regulation of replication structures. In vitro studies using SV40 as a model system have demonstrated that CDKs are necessary for the assembly of replication origin initiation complexes containing unwound DNA. Multiple experiments demonstrate the essential role of CDK2 in the cell cycle. Overexpression of p27 or dominant-negative CDK2 mutants inhibit proliferation. Furthermore, injection of antibodies against CDK2 subunit or treatment of the cells with CDK2 inhibitors also block proliferation. Moreover, a central role for CDK2/cyclin E has been indicated from observations of mice engineered to express cyclin E in place of cyclin D1. Animals lacking cyclin D1 have proliferative defects and fail to activate CDK2 in a subset of tissues. These phenotypes were suppressed in a knock-in animal that expresses cyclin E from the cyclin D1 promoter, suggesting that loss of CDK/cyclin D phosphorylation of pRb has no effect if cyclin E is no longer dependent on pRb inactivation.

352 CELL CYCLE AND CELL-CYCLE CHECKPOINTS

However, recent findings challenge this view. The inhibition of CDK2 by several ways has no effect on proliferation in some cell lines. High levels of CDK4 may compensate for the absence of CDK2. Moreover, both CDK4 and CDK2 activities may be unnecessary for proliferation in pRb-negative cells. Similar conclusions were derived using CDK2 null mice, which develop normally except for some defects in meiosis. DNA Damage Checkpoint

The cells will arrest at the G1/S checkpoint in response to DNA damage to prevent the replication of mutated DNA. p53 tumor suppressor plays a critical role during this damage-induced checkpoint because p53-deficient cells fail to undergo G1/S arrest after genotoxic stress. After exposure to genotoxic insults, p53 is activated and transcriptionally regulates many targets (e.g., p21 waf1, GADD45, reprimo, and 143-3 d) that directly provoke cell-cycle arrest. G2/M Checkpoint and Early Mitosis

The orchestration of mitotic events requires exquisite regulation, and the penalty for errors in chromosome segregation is severe. Chromosomal imbalances are responsible for many cases of abortions, birth defects, and cancer. Once S phase is complete, the cell duplicates cell structures and separates the chromosomes. For this process, the cell must establish another checkpoint at the onset of G2, before the initiation of mitosis. The key component is a mitotic regulator composed of CDK1 and cyclin B. Together, these proteins form the mitosis-promoting factor (MPF), the activity of which initiates mitosis. MPF is regulated by periodic accumulation and destruction of cyclins, although additional controls regulate the complex. The mitotic phase can be defined by three transitions that involve cyclin B. First, cyclin B activates MPF and initiates prophase. CDK1 bound to cyclin B must also be phosphorylated by CAK in threonine 161 and dephosphorylated in tyrosine 15 and threonine 14. This activation will induce the specific phosphorylation of mitotic targets triggering downstream events. Nuclear lamins, kinesin-related motors and other microtubule-binding proteins, condensins, and Golgi matrix components are substrates of CDK1/cyclin B important for nuclear envelope breakdown (NEBD), chromosome separation, spindle assembly, chromosome condensation, and Golgi fragmentation. Second, MPF activates a ubiquitin–proteolytic system that degrades cyclin B and initiates anaphase. Finally, the cyclin B destruction machinery is turned off and the cycle is reset.

Centrosomes are the major microtubule-organizing center of animal cells. In most cell types, duplicated centrosomes remain closely paired and continue functioning as single microtubule organizing centers during G2. After G2, however, they separate and migrate apart. The separation of centrosomes seems to be regulated by several kinases. Nek2, a member of the NIMA family, is thought to phosphorylate the centrosomal protein c-Nap1 causing the dissolution of a dynamic structure that tethers duplicate centrosomes to each other. An abrupt increase in c-Nap1 phosphorylation at the G2/M transition is caused by the cell-cycle-regulated inhibition of a type 1 phosphatase. At a later step, several kinesinrelated motor proteins and cytoplasmic dynein are required for centrosome separation. In organisms undergoing open mitosis, NEBD occurs soon after centrosome separation. During interphase, the nuclear envelope is stabilized by the nuclear lamina, but at the onset of mitosis, this structure disassembles as a consequence of lamin hyperphosphorylation. The predominant kinase triggering mitotic lamina depolymerization in vivo is CDK1/cyclin B. PLKs are key regulators of mitosis and cytokinesis because they are subject to complex temporal and spatial control. PLK1 seems to be involved in mitotic entry, centrosome maturation, spindle-assembly, and APC regulation. PLK1 is involved in the recruitment of a-tubulin during centrosome maturation, a process by which centrosomes increase their microtubule nucleation activity required for spindle formation. The association of PLKs with the centrosome also has implications for the regulation of mitotic entry. The initial activation of CDK1/cyclin B occurs at the centrosome, probably through phosphorylation of both CDC25 and Myt1. In mitotic cells, PLK1 also associates with the kinetochores. These structures play an important role in the spindle checkpoint. This checkpoint operates to ensure that all chromosomes have undergone bipolar attachment to the spindle before sister chromatids are separated. Another family of kinases that plays a critical role in chromosomal segregation and cell division is the Aurora family. Aurora kinases are implicated in the centrosome cycle, spindle assembly, chromosome condensation, chromosome–microtubule attachment, the spindle checkpoint, and cytokinesis. Aurora kinases are regulated through phosphorylation, the binding to specific partners, and proteolysis. In mammals, there are three Aurora members that differ in expression patterns, localization, and timing of activity. Aurora A is upregulated at the onset of mitosis. It localizes at centrosomes during interphase and at both spindle poles and microtubules during early mitosis. Aurora B


reaches maximal levels later in mitosis, first associated with the centromeres/kinetochores, then relocalizes to the midzone of the central spindle and finally concentrates at the midbody between dividing cells. Aurora A is implicated primarily in centrosome maturation and spindle assembly, whereas Aurora B is thought to regulate chromosome condensation and cohesion, kinetochore assembly, the spindle checkpoint, and the coordination between chromosome segregation and cytokinesis. Aurora B constitutes a key link between structural aspects of the microtubule–kinetochore interactions and checkpoint signaling. The third member of the family, Aurora C, has a more restricted expression pattern and its functions are mostly unknown. Spindle Checkpoint

Proper distribution of chromosomes between daughter cells is controlled by the spindle-assembly checkpoint that separates metaphase from anaphase, two clearly defined stages within mitosis. At this stage (metaphase), sister chromatids (pairs of identical chromosomes to be segregated between the daughter cells) are aligned at the midzone of the mitotic spindle. The spindle checkpoint, a safeguard mechanism that prevents defects in chromosome segregation, inhibits anaphase onset until the mitotic spindle is correctly assembled. Metaphase–anaphase transition

At the start of anaphase, cohesins, the molecules responsible for sister chromatid cohesion, are degraded by a protease known as separase. In turn, separase is activated by degradation of a specific inhibitor, securin, by APC, a ubiquitin–ligase multiprotein complex (Figure 3). APC is the ultimate gatekeeper of the spindle-assembly checkpoint, ensuring that sister chromatids do not segregate unless an identical set of chromosomes are properly aligned at the spindle midzone. APC activity is regulated primarily by Mad2, a sensor of microtubule attachment to the kinetochores that sequesters Cdc20, a molecule required for APC activation. Mad2 binds to Bub3 and BubR1. The Mad/Bub complex is recruited to unattached kinetochores and inhibits the activation of the APC/Cdc20 complex, which is necessary for sister chromatid separation. This results in metaphase arrest until all kinetochores are correctly aligned. Completion of kinetochore attachment leads to the dissociation of Mad2 from Cdc20 (Figure 3). The spindle checkpoint requires mitotic CDK activity. Inhibiting CDK activity overrides checkpointdependent arrest. Following inhibition, the interaction between APC and Cdc20 transiently increases while the inhibitory checkpoint protein Mad2 dissociates from Cdc20. CDK inhibition also overcomes Mad2-induced mitotic arrest. In addition, in vitro

Improper spindle Aur Plk

Normal spindle

Bub

Cdc20

Cdc20

APC

APC Separase Cohesin cleaved Sister chromatid separation

Securin

UbUbUbUb

Cohesin intact

Metaphase arrest

Figure 3 Spindle checkpoint.

Mad2

354 CELL CYCLE AND CELL-CYCLE CHECKPOINTS

CDK1-phosphorylated Cdc20 interacts with Mad2 rather than APC. Thus, CDK1 activity seems to be required to restrain APC/Cdc20 activation until completion of spindle assembly. In addition, evidence indicates that CDK1 activity is required for the efficient function of the spindle checkpoint protein Bub1. Phosphorylation of key elements may play a role in the timing of the events that lead to mitosis exit and in regulating the spindle checkpoint function. Phosphorylation of APC subunits by PLKs appears to help APC activity. PLK-1 recruits spindle checkpoint proteins to centromeres and creates the tension-sensing phosphoepitope on mitotic kinetochores and APC. Aurora B, Survivin, and INCENP physically interact and form a complex that localizes to the kinetochores in prometaphase, to the cell equator during metaphase, and to the midbody during cytokinesis. The absence of function of these proteins has revealed their involvement in the spindle checkpoint. These proteins are also required to establish bipolar attachment of chromosomes, probably by destabilizing kinetochore–microtubule attachments that lack tension. Aurora communicates with the other downstream components probably through direct phosphorylation of BubR1 and Mps1.

Cell Cycle and Cell-Cycle Checkpoints in Respiratory Diseases Hanahan and Weinberg suggested that all cancer genotypes are the manifestation of six essential physiological alterations shown by most, and perhaps all, types of human tumors. Although breaching of these six physiological barriers seems to be necessary for most tumors to achieve full malignancy, cancer is increasingly viewed as a cell-cycle disease. This view reflects evidence that the vast majority of tumors, and likely all tumors, have suffered one or more defects that derail the cell-cycle machinery (Table 2). Such defects can target either components of the cell cycle, including checkpoint mechanisms, or elements of the upstream signaling cascades, whose events eventually converge to trigger cell-cycle events. Lung cancer remains a worldwide major health challenge. Despite improvements in treatment, the 5year survival rate for individuals with lung cancer is only approximately 15%. Histologically, 80% of lung cancers are non-small cell lung cancer (NSCLC), whereas the remaining 20% of cases are small cell lung cancer (SCLC). On the basis of cell morphology, adenocarcinoma and squamous cell carcinoma are the most common types of NSCLC.

The pRb protein is inactivated in more than 90% of SCLCs as a result of different mechanisms, including point mutations and abnormal mRNA expression. Changes in the other Rb family members (p107 and p130) have been detected in only a few cases. In contrast to SCLC, the majority of NSCLC cases exhibit abnormalities in the upstream regulators of the pRb pathway, including inactivation of p16ink4a, reduced levels of p27Kip, and enhanced expression of cyclin D1. p16ink4a abnormalities are frequently found in NSCLCs but are rare in SCLCs. Perhaps 30–50% of early stage primary NSCLCs do not express p16ink4a (Table 2). The overexpression of cyclin A has been consistently shown to be a negative prognostic indicator of lung cancer. Also, cdc25A, one of the rate-limiting mechanisms for G1 progression into S phase, is frequently overexpressed in NSCLC. The gene cdk10 is essential for cellular proliferation, and its effect is also exerted in the G2/M transition. Only recently was this gene found to be overexpressed in lung adenocarcinomas. Also, cyclin B1 and CDK1, regulators of the G2/M transition, are overexpressed in lung cancer. Following DNA damage, a series of events is initiated that ends with p53 activation and the transcriptional regulation of many cell-cycle regulators. The fundamental importance of p53 in lung cancer is highlighted by the frequency of its mutations—80% in SCLC and 50% in NSCLC. Once the p53 gene is deleted or mutated, cells become susceptible to DNA damage and deregulated cell growth.

Ceramide and the Cell Cycle The sphingolipid ceramide is generated from the membrane-associated sphingomyelin by the activation of neutral sphingomyelinase in response to a variety of extracellular inducers. Ceramide acts as a second messenger to mediate many of the effects of these inducers. Ceramide is an important molecule that regulates diverse signaling pathways involving apoptosis, cell senescence, cell cycle, and differentiation. For the most part, ceramide’s effects are antagonistic to growth and survival. Interestingly, ceramide and the progrowth agonist, diacylglycerol (DAG), appear to be regulated simultaneously but in opposite directions in the sphingomyelin cycle. Whereas ceramide stimulates signal transduction pathways that are associated with cell death or at least are inhibitory to cell growth (e.g., stress-activated protein kinase or mitogen-activated protein kinase pathways), DAG activates the classical and novel isoforms of the protein kinase C (PKC) family. These PKC isoforms are associated with cell growth


and cell survival. Thus, ceramide and DAG generation may serve to monitor cellular homeostasis by inducing prodeath or progrowth pathways, respectively. Ceramide levels are elevated in response to diverse stress challenges, including chemotherapeutic drug treatment, irradiation, or treatment with prodeath ligands such as tumor necrosis factor-a. Consistent with this notion, ceramide is a potent apoptogenic agent. Ceramide activates stress-activated protein kinases such as c-jun N-terminal kinase and thus affects transcription pathways involving c-jun. Ceramide activates protein phosphatases such as protein phosphatase-1 and protein phosphatase-2. Ceramide activation of protein phosphatases has been shown to promote inactivation of a number of progrowth cellular regulators, including the kinases PKC-a and Akt, Bcl-2, and the retinoblastoma protein. Ceramide levels remain relatively constant throughout the life span of fibroblasts but increase with the onset of cellular senescence (a terminally arrested state that somatic cells acquire after a limited round of replication), increasing severalfold compared to the levels of young cells. Whereas in differentiation and apoptosis ceramide levels increase transiently, in senescence the increase in ceramide levels is permanent. The addition of a cell-permeable ceramide to young fibroblasts to concentrations usually seen in senescent cells mimics the senescent phenotype, with many of its markers. Higher concentrations of ceramide induce apoptosis. Cells treated with ceramide are unable to produce the AP-1 transcription factor and progressively inhibit pRb phosphorylation. It has been found that ceramide levels increase as cells exit mitosis to G1 phase. This increase precedes the dephosphorylation of pRb that occurs as the cells exit G2/M. In contrast, no changes in ceramide have been observed during other cell-cycle phase transitions, suggesting a role for the endogenous ceramide in the transition from G2/M to G1. In agreement with this, microinjection of ceramide or sphingosine was sufficient to reinitiate the cell cycle by reactivating G2/M transition in Xenopus oocytes. Ceramide also inhibits phospholipase D and PKC. Phospholipase D and PKC transduce mitogenic signals, and their progressive inactivation has been observed during cellular senescence, probably due to the increased in ceramide.

Acknowledgments This work was supported by grant BIO-01-0069 from the Spanish Ministry of Science and Technology and grant PI020126 from the Spanish Ministry of Health. See also: DNA: Repair; Structure and Function. Tumor Necrosis Factor Alpha (TNF-a). Tumors, Malignant: Overview.

Further Reading Adams PD (2001) Regulation of the retinoblastoma tumor suppressor protein by cyclin/cdks. Biochimia et Biophysica Acta 1471(3): M123–M133. Bell SP and Dutta A (2002) DNA replication in eukaryotic cells. Annual Review of Biochemistry 71: 333–374. Hanahan D and Weinberg RA (2000) The hallmarks of cancer. Cell 100: 57–70. Harris SL and Levine AJ (2005) The p53 pathway: positive and negative feedback loops. Oncogene 24: 2899–2908. Hartwell LH, Culotty J, Pringle JR, and Reid BJ (1974) Genetic control of the cell division cycle in yeast. V. Genetic analysis of mutants. Genetics 74: 267–286. Hartwell LH, Culotty J, and Reid BJ (1970) Genetic control of the cell division cycle in yeast. I. Detection of mutants. Proceedings of the National Academy of Sciences USA 66: 352–359. Hartwell LH and Kastan MB (1994) Cell cycle control and cancer. Science 266: 1821–1828. Jeffrey PD, Russo AA, Polyak K, et al. (1995) Mechanism of CDK activation revealed by the structure of a cyclin A–CDK2 complex. Nature 376: 313–320. Kelly TJ and Brown GW (2000) Regulation of chromosome replication. Annual Review of Biochemistry 69: 829–880. Malumbres M and Carnero A (2003) Cell cycle deregulation: a common motif in cancer. Progress in Cell Cycle Research 5: 5–18. Meijer L, Jezequel A, and Ducommun B (2000) Progress in Cell Cycle Research, vol. 4. Norwell, MA: Kluwer. Murray AW (2004) Recycling the cell cycle: cyclins revisited. Cell 116: 221–234. Murray AW and Hunt T (1993) The Cell Cycle: An Introduction. Oxford: Oxford University Press. Osada H and Takahasi T (2002) Genetic alterations of multiple tumor suppressors and oncogenes in the carcinogenesis and progression of lung cancer. Oncogene 21: 7421–7434. Peters JM (2002) The anaphase-promoting complex: proteolysis in mitosis and beyond. Molecular Cell 9: 931–943. Sherr CJ (2004) Principles of tumor suppression. Cell 116(2): 235–246. Sherr CJ and Roberts JM (1999) CDK inhibitors: positive and negative regulators of G1-phase progression. Genes & Development 13: 1501–1512. Stein GS, Baserga R, Giordano A, and Denhardt DT (eds.) (1999) The Molecular Basis of Cell Cycle and Growth Control. New York: Wiley–Liss.

356 CHEMOKINES

CHEMOKINES O Morteau, Children’s Hospital, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Chemokines are an extended family of small size chemoattractant proteins that mediate leukocyte migration and positioning. They have been classified into the C, CC, CXC, and CX3C subfamilies based on the position of a cysteine residue in their primary structure. Chemokines interact with structurally related G-protein-coupled receptors located on the surface of leukocytes, and trigger a cascade of intracellular events involving several distinct signaling pathways. The biological functions of chemokines include chemoattraction, integrin activation, leukocyte degranulation and mediator release, and angiogenesis, or angiostasis. They are also involved in various ways in the establishment and maintenance of the innate and adaptive immune systems. The importance of chemokines and their receptors in the pathogenesis of respiratory diseases is exemplified in conditions as diverse as airway allergy (asthma), bacterial and viral infections (tuberculosis, respiratory syncytial virus infection), allograft complications (acute lung allograft rejection, graft-versus-host disease), lung cancer, and other pathologies including chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis, and acute respiratory distress syndrome.

1996, some chemokine receptors were found to act as co-receptors for human immunodeficiency virus (HIV)-1, and in 1998, viral chemokines were identified and characterized. The role of chemokines and their receptors (including CCR4, CCR9, and CCR10) in tissue-specific homing was confirmed in 1999. The fast-expanding chemokine family was designated by various successive terms, including ‘the platelet factor-4 family’, ‘the small inducible cytokine family’, and ‘the intercrines’. The standard term ‘chemokines’ (a neologism for ‘chemotactic cytokines’) was eventually coined in 1992 at the Third International Symposium on Chemotactic Cytokines in Baden. In 1995, the International Union of Pharmacology Committee on Receptor Nomenclature and Classification (NC-IUPHAR) created a subcommittee on chemokine receptor nomenclature. In 1999, a chemokine nomenclature system that parallels the chemokine receptor nomenclature was proposed at the Keystone Symposium on Chemokines and Chemokine Receptors.

Introduction

Chemokine Structure and Nomenclature

Chemokines are an extended family of small size chemoattractant cytokines that facilitate leukocyte migration and positioning, and exert their effects via binding to receptors located at the target cell surface. To date, 43 chemokines have been identified, binding to 19 different receptors. The first chemokine was discovered by Walz and co-workers in 1977. It was a procoagulant and angiostatic factor called platelet factor 4, now renamed CXCL4. From 1984 to 1989, several investigators cloned the cDNAs for structurally related proteins, including IP-10 (IP10 – 10 kDa IFN (interferon)-ginducible protein), JE, Mig, RANTES (RANTES – regulated on activation normal T cell expressed and secreted), I-309, KC, and MIP-1 a (MIP – macrophage inflammatory protein). At the time a function had yet to be attributed to this growing gene family, and it is only with the characterization of the neutrophil chemoattractant interleukin 8 (IL-8), also called CXCL8, that chemokines were recognized as chemotactic molecules. Between 1989 and 1994, interest in the chemokine field grew dramatically as MCP-1 (MCP – monocyte chemoattractant protein), RANTES, and eotaxin were reported to target monocytes, T cells, and eosinophils, respectively. In

Most chemokines are secreted proteins of 67 to 127 amino acids (only CXCL16 and CX3CL1 are membrane-bound), and contain a conserved tetra-cysteine motif located at the NH2 terminus. Chemokine nomenclature is based on the relative position of the first two of these four consensus cysteines (Table 1). The separation of the cysteines by a nonconserved amino acid defines the CXC chemokine subclass, while the presence of two adjacent cysteines defines the CC chemokine subclass. Two minor chemokine subclasses (C and CX3C) either lack two out of four canonical cysteines (XCL1 and XCL2 chemokines), or contain three intercalated nonconsensus amino acids (CX3CL1 chemokine). CXC chemokines can be further discriminated into two structural subgroups (ELR þ and ELR–), based on the presence or absence of the tripeptide motif glutamic acid–leucine–arginine (ELR) N-terminal to the first cysteine. One structure/function correlate is the specificity of ELR þ CXC chemokines for neutrophils. Another one is the ability of ELR þ CXC chemokines to promote the neoformation of blood vessels (angiogenesis), while ELR CXC chemokines exert the opposite effect (angiostasis), as we will see later. The CXC, CC, CX3C, and C chemokine subclasses are

CHEMOKINES 357 Table 1 Chemokines and their receptors Class

Systemic name

Common synonyms

Receptor

CXC (a)

CXCL1 CXCL2 CXCL3 CXCL4 CXCL5 CXCL6 CXCL7 CXCL8 CXCL9 CXCL10 CXCL11 CXCL12 CXCL13 CXCL14 CXCL15 CXCL16

GROa, MGSA, KC (mouse), MIP-2 (mouse) GROb, MIP-2a GROg, MIP-2b Platelet factor 4 ENA-78 GCP-2 PBP, CTAP-III, b-TG, NAP-2 IL-8, MDNCF, GCP, LIF, NAP-1, LYNAP Mig, CGR-10 (mouse) IP-10, CGR2 (mouse) I-TAC, b-R1, IP9, H174 SDF-1a, SDF-1b, PBSF, TLSF, TPAR1 BCA-1, BLC BRAK, Bolekine

CXCR2 CXCR2 CXCR2 Unknown CXCR2 CXCR1, CXCR2 CXCR2 CXCR1, CXCR2 CXCR3 CXCR3 CXCR3 CXCR4 CXCR5 Unknown Unknown CXCR6

CC(b)

CCL1 CCL2 CCL3 CCL4 CCL5 CCL6 CCL7 CCL8 CCL9 CCL10 CCL11 CCL12 CCL13 CCL14 CCL15 CCL16 CCL17 CCL18 CCL19 CCL20 CCL21 CCL22 CCL23 CCL24 CCL25 CCL26 CCL27 CCL28

1-309, TCA-3 (mouse), SIS-f (mouse) MCP-1, MCAF, HC11, JE (mouse) MIP-1a, LD-78a, PAT464, GOS19, hSISa MIP-1b, ACT-2, HC21, MAD-5, LAG-1 RANTES, SIS-d C10 (mouse), MRP-1 (mouse) MCP-3, NC28, FIC, MARC (mouse) MCP-2, HC14 MRP-2 (mouse), MIP-1g (mouse) Eotaxin MCP-5 (mouse) MCP-4, ckb10, NCC-1 CC-1, HCC-1, NCC-2, CCCK-1, Ckb1 HCC-2, Lkn-1, MIP-5, CC-2, NCC-3 HCC-4, LEC, NCC-4, LMC, Mtn-1, LCC-1 TARC DC-CK-1, PARC, MIP-4, AMAC-1, ckb7 MIP-3b, ELC, exodus-3, ckb11 MIP-3a, LARC, exodus-1, ST38 (mouse) 6Ckine, SLC, exodus-2, TCA4, ckb9 MDC, STCP-1, dc/bck (mouse) MPIF-1, MIP-3, ckb8-1 Eotaxin-2, MPIF-2, ckb6 TECK, ckb15 Eotaxin-3, MIP-4a ESkine, CTACK, skinkine, ILC (mouse) MEC

CCR8 CCR2 CCR1, CCR5 CCR5 CCR1, CCR3, CCR5 Unknown CCR1, CCR2, CCR3 CCR3 Unknown Unknown CCR3 CCR2 CCR2, CCR3 CCR1 CCR1, CCR3 CCR1 CCR4 Unknown CCR7 CCR6 CCR7 CCR4 CCR1 CCR3 CCR9 CCR3 CCR10 CCR10

C (g)

XCL1 XCL2

Lymphotactin a, SCM-1a Lymphotactin b, SCM-1b

XCR1 XCR2

CX3C (d)

CX3CL1

Fractalkine, neurotactin (mouse)

CX3CR1

sometimes referred to as a, b, g, and d chemokines, respectively. Aside from the official nomenclature, chemokines can be divided on the basis of their biological functions into inflammatory chemokines, homeostatic chemokines, and dual chemokines (Table 2). Inflammatory/inducible chemokines are expressed in inflamed tissues in response to proinflammatory

cytokines or pathogens. They orchestrate innate and adaptive immune responses via recruitment of effector leukocytes in infection, inflammation, tissue injury, and tumors. By contrast, homeostatic/ constitutive chemokines are involved in immune surveillance through regulation of lymphocyte and dendritic cell trafficking. They guide leukocytes during hematopoiesis in the bone marrow and thymus,

358 CHEMOKINES Table 2 Inflammatory, homeostatic, and dual chemokines Chemokines

Receptors

Functions

Inflammatory CXCL6 CXCL8 CXCL1 CXCL2 CXCL3 CXCL5 CX3CL1 CCL2 CCL3 CCL5 CCL7 CCL8 CCL11 CCL13 CCL14 CCL15 CCL24 CCL26 CCL27 CCL28 XCL1 XCL2

CXCR1, CXCR2 CXCR1, CXCR2 CXCR2 CXCR2 CXCR2 CXCR2 CX3CR1 CCR2 CCR1, CCR5 CCR1, CCR3, CCR5 CCR1, CCR2, CCR3 CCR3 CCR3 CCR2, CCR3 CCR1 CCR1, CCR3 CCR3 CCR3 CCR10 CCR10 XCR1 XCR1

Innate immunity

Homeostatic CXCL12 CXCL13 CXCL14 CCL18 CCL19 CCL21

CXCR4 CXCR5 Unknown Unknown CCR7 CCR7

Hematopoiesis Homeostasis of leukocytes Development of antigen-presenting cells T cell–dendritic cell interaction (spleen, lymph node) T lymphopoiesis Spleen and lymph node T cell homing

Dual function CXCL9 CXCL10 CXCL11 CXCL16 CCL1 CCL17 CCL20 CCL22 CCL25

CXCR3 CXCR3 CXCR3 CXCR6 CCR8 CCR4 CCR6 CCR4 CCR9

T lymphopoiesis Adaptive immunity (Th1 responses)

Extravasation Innate and adaptive immunity

T lymphopoiesis, extravasation T lymphopoiesis, adaptive immunity T lymphopoiesis Development of dendritic cells, adaptive immunity Adaptive immunity (cutaneous T cells) T lymphopoiesis, adaptive immunity, T cell and B cell trafficking in small intestine

Adapted from Moser B, Wolf M, Walz A, and Loetscher P (2004) Chemokines: multiple levels of leukocyte migration control. Trends in Immunology 25: 75–84.

during initiation of adaptative immune responses in the spleen, lymph nodes, and Peyer’s patches, and in immune surveillance of healthy peripheral tissues. Sharing both functions are the ‘dual chemokines’, which participate in immune defense functions and also target noneffector leukocytes, such as precursor and resting mature leukocytes, at sites of leukocyte development and immune surveillance. Genes encoding inflammatory chemokines are found in two major clusters on human chromosomes 4 (CXC chemokines) and 17 (CC chemokines), while genes for homeostatic chemokines are located alone

or in small clusters on chromosomes 1, 2, 5, 7, 9, 10, and 16.

Chemokine Receptors Human formyl-peptide receptor was the first chemokine receptor cloned in 1990. Two human receptors for CXCL8 (IL-8) were subsequently cloned in 1991: CXC chemokine receptor 1 (CXCR1) and CXCR2. As of today, 19 receptors have been identified (Table 1). The protein sequences of chemokine receptors share 25% to 80% amino acid identity,

CHEMOKINES 359

which suggests a common ancestor. All chemokine receptors are G-protein-coupled receptors (GPCRs) with seven transmembrane domains. However, chemokine receptors share a few features that are less commonly found in other GPCRs. They include a length of 340 to 370 amino acids; an acidic N-terminal segment; the sequence DRYLAIVHA (or a variation of it) in the second intracellular loop; a short basic third intracellular loop; and a cysteine in each of the four extracellular domains. An additional characteristic is the presence of a tyrosine sulfation in the N-terminus, which is critical for CCR5 activity as an HIV co-receptor. Even though the three-dimensional structure of chemokine receptors is unknown, the presence of seven transmembrane domains was inferred from the structure of rhodopsine. However, the presence of extracellular and intracellular loops is more speculative. It is commonly accepted that chemokine receptors contain seven transmembrane domains separated by amino acid loops directed toward the outside (extracellular loops) and inside (intracellular loops) of the cell (Figure 1). While extracellular domains serve as binding sites for the chemokine ligands, intracellular loops interact with G proteins and scaffolding proteins to trigger a cascade of intracellular changes (see chemokine–chemokine receptor interactions). The ligand-binding site of chemokine receptors is highly complex. It is composed of multiple noncontiguous domains and at least two distinct subtypes: one for docking and the other for triggering.

Extracellular space NH2

Cell membrane

IV

III II

Cytoplasm

V

I

VI VII

The classification systems of chemokines and chemokine receptors parallel each other. As illustrated in Table 1, human CC and CXC chemokine receptor names consist of the CCR and CXCR roots (where R stands for receptor) followed by numbers. Similarly, CC and CXC chemokine names consist of the CCL and CXCL roots (where L stands for ligand) followed by numbers. The lymphotactin and fractalkine receptors are named XCR1 and CX3CR1, respectively. A chemokine receptor has a distinct chemokine and leukocyte specificity, but may bind multiple chemokines. Reciprocally, a chemokine may bind multiple receptor subtypes. However, a number of chemokine–chemokine receptor relationships are monogamous (SDF-1/CXCR4, TECK/CCR9, BCA1/CXCR5, MIP-3a/CCR6, lymphotactin/XCR1, fractalkine/CX3CR1) (where SDF represents stromal cell-derived factor, TECK represents thymus-expressed chemokine, BCA represents B-cell attracting chemokine). Importantly, distinct receptor subtypes specific for the same chemokine and same function can be coexpressed on the same cell. In addition, some nonchemokine ligands (including several HIV proteins) can bind chemokine receptors as well. For example, CCR3 and mainly CCR5 and CXCR4, serve as co-receptors for HIV infection of CD4 þ cells.

Chemokine–Chemokine Receptor Interaction Chemokine Binding

Chemokine receptors are allosteric molecules, which means that their conformation can be modified by the interaction with another molecule (ligand), allowing further binding to a third molecule (G-protein). More specifically, chemokine binding to the extracellular domains of the chemokine receptor triggers a change in tertiary structure of the receptor. This modification allows the intracellular domains to bind and activate heterotrimeric G-proteins. In response, the activated G-proteins exchange guanosine diphosphate for guanosine triphosphate (GTP), and dissociate into a- and bg-subunits. Chemokine receptors can couple to several Ga isotypes. For example, chemotaxis, the main function of most chemokines, requires coupling of the receptor to Gai, a Ga isotype sensitive to pertussis toxin. This was demonstrated by the complete inhibition of cell migration following cell treatment with pertussis toxin.

COOH

Figure 1 Schematic structure of a chemokine receptor. The seven transmembrane domains and the extracellular and intracellular loops are represented.

Intracellular Signaling

The bg-subunits that dissociate from Gai mediate chemokine-induced signals by activating the

360 CHEMOKINES

phosphoinositide-3 kinases (PI3K), which produce phosphoinositol-3, 4, 5-triphosphate (PIP3). Chemokine stimulation triggers a change in leukocyte shape associated with the formation of an elongated edge, or pseudopod. PIP3K and its product PIP3 translocate to the pseudopod, where they colocalize with GTPase Rac. PIP3 not only activates Rac via specific guanine nucleotide exchange factors (GEFs), but also serves as a docking site for protein kinase B, which translocates to the pseudopod and can induce actin polymerization. Rac, a protein required for leukocyte migration, activates downstream effectors (p21-activated kinase, and WAVE), which stimulate actin-related protein (Arp) 2/3. The actin polymerization induced by Arp allows further pseudopod extension. In moving leukocytes, formyl peptide activates a different set of mediators. Parallel to the sequence of events triggering Rac activation, another pathway leads from the pertussis toxin-sensitive Ga12/13 to the activation of Rho by Rho GEFs at the uropod (extended pole of the leukocyte opposite to the pseudopod). Activated Rho induces focal activation of myosin II and contraction of actin–myosin complexes via its effector p160ROCK, a serine/threonine kinase. This process triggers the retraction of the pseudopod. The Rac and Rho pathways, which occur at opposite edges of the leukocyte, inhibit each other. This mutual inhibition is critical to induce and maintain functional and morphological cell polarity, and drive locomotion. At least one additional signaling pathway remains to be characterized. It is the one leading to the rapid integrin activation involved in leukocyte–endothelial cell adhesion. Chemokine Receptor Silencing

Chemokine receptor signaling is a transient process, due to the presence of GTPase activity in the Ga subunits. GTP hydrolyzation catalyzed by GTPase induces the reunion of Ga with Gbg, allowing the return to the initial conformation of inactive heterotrimers. Molecules called regulators of G-protein signaling can, depending on the Ga isotype, either stimulate or inhibit the GTPase activity of the Ga subunits. Chemokine receptor silencing can also be mediated by desensitization triggered by receptor phosphorylation by G-protein-coupled receptor kinases, and by downregulation induced by receptor sequestration and internalization. Chemokine Receptor-Independent Pathways

Alternative GPCR-independent (and chemokine receptor-independent) signaling pathways result from

the binding of sulfated sugars of the glycosamine family to a few chemokines, such as CCL5 and CXCL4.

Biological Functions of Chemokines At least four major biological functions can be attributed to chemokines: leukocyte chemoattraction, integrin activation, leukocyte degranulation, and angiogenesis or angiostasis. Chemoattraction

Chemokines attract leukocytes that bear the appropriate chemokine receptors via formation of a chemokine gradient. This gradient acts as a road map for the migrating cells, guiding them toward their final destination. As described earlier (see chemokine– chemokine receptor interactions), the signaling of cell movement by chemokine receptors is a complex phenomenon and is not yet fully elucidated. Integrin Activation

Leukocyte migration involves passage from the tissues to the blood and lymphatic vessels and from the vessels to the tissues (extravasation). Cells undergo a multistep process to bind the vessel endothelium. They tether to vessel walls via activation of selectins, then bind the endothelium via activation of integrins, which are able to regulate their affinity through rapid conformational change. This conformational change in the integrin structure leads to cell arrest, a mechanism that allows cells to subsequently cross the endothelium. Chemokines can activate integrins, and are involved in the multistep process for neutrophils, T cells, eosinophils, and monocytes. Leukocyte Degranulation and Mediator Release

Chemokines can stimulate leukocyte degranulation or release of inflammatory mediators, which can be features of allergic reactions. For example, CCL2 (MCP-1) stimulates histamine release by basophils and mast cells, and CXCL8 (IL-8) stimulates neutrophil granule exocytosis. In addition, chemokines stimulate the macrophage respiratory burst leading to the release of reactive oxygen intermediates. Angiogenesis or Angiostasis

The formation of new blood vessels (angiogenesis) is a physiological feature associated with embryonic development, wound healing, chronic inflammation, and the growth of malignant tumors. Some chemokines (mostly CXC chemokines) promote angiogenesis, while others mediate its inhibition (angiostasis).

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As stated earlier (see section ‘Chemokine structure and nomenclature’), the angiogenic property of the CXC chemokines correlates with the presence of the tripeptide motif glutamic acid–leucine–arginine (ELR). Proangiogenic chemokines include the ELR þ CXC chemokines CXCL1, 2, 3, 5, 6, 7, 8, and 12, and CCL2 (MCP-1). Antiangiogenic chemokines include the ELR– CXC chemokines CXCL 4, 9, 10, 11, and 13, and CCL21 (SLC)(SLC – secondary lymphoid-tissue chemokine). As we will see later, the angiogenic and angiostatic properties of chemokines are of major relevance in lung cancer and inflammation. The four biological functions reviewed above are used in combination for various biological responses. For example, tumor rejection involves leukocyte chemotaxis to the tumor and angiostasis; allergic reactions involve leukocyte migration and degranulation.

Chemokines in Respiratory Diseases Asthma and Airway Allergy

Asthma is characterized by airway hyperresponsiveness (AHR) and T-helper-2 (Th2) cell, eosinophil, and mast cell infiltration (see Asthma: Overview; Extrinsic/Intrinsic). A number of chemokines and receptors are associated with the development of asthma (Table 3). It is important to bear in mind that most studies on chemokines in asthma are based on the use of mouse models. Although these murine models are useful tools, their relevance to the human disease is not absolute, due partly to species differences. Moreover, mouse models are characterized by acute allergic reactions, whereas human asthma is a chronic disease. Finally, differences may arise between the various murine models, in terms of the cell populations and chemokines involved in the allergic response, and the relevance of a clearance mechanism. Eotaxins are a family of chemokines that attract eosinophils. Three forms (CCL11 or eotaxin, CCL24 or eotaxin-2, and CCL26 or eotaxin-3) were identified in humans, whereas two forms only were identified in mice. CCL11 (eotaxin) is a potent chemoattractant of eosinophils. It binds exclusively to the CCR3 receptor, which is expressed on the surface of eosinophils, mast cells, and basophils. Neutralization of CCL11 reduces both airways inflammation and AHR, via reduction of eosinophil and Th2 trafficking. However, mice genetically deficient in CCL11 were only partially protected in models of allergic airway inflammation, which supports the idea of functional redundancy between chemokines. Although the CCL11 receptor CCR3 has been regarded as a potential therapeutic target, a model of

CCR3-deficient (knockout) mouse gave new insights on the complexity of the mechanisms involved. CCR3 knockouts exhibited a dramatic reduction of eosinophil recruitment to the lung following allergen challenge. Unexpectedly though, AHR was increased in these mice, which was later attributed to an increased accumulation of mast cells in the trachea. An additional study showed a protection against allergen-induced AHR in CCR3-deficient mice in a model that did not involve mast cell recruitment. CCL17 (TARC) (TARC – thymus- and activationregulated chemokine) (see Chemokines, CC: TARC (CCL17)) CCL22 are chemokines that induce the selective migration of Th2 cells via binding to CCR4. Neutralization of either CCL17 or CCL22 leads to abrogation of lung eosinophilia and AHR in mice. Additional studies have confirmed that CCL22 and CCR4 contribute to the recruitment of Th2 cells to the lung during allergen-driven inflammation in mice and humans. CCL1 is the ligand to the CCR8 chemokine receptor expressed on Th2 cells, and the CCL1/CCR8 tandem has been suspected to mediate the asthmatic reaction. However, studies using CCL1 and CCR8 knockout mice gave controversial results. It is suggested that CCL1/CCR8 might be involved in eosinophil recruitment in some models, but might not be critical to Th2 recruitment to the lung in vivo. CXCR4 expression on leukocytes is widespread, and this receptor is involved in B and T lymphopoiesis. In addition, CXCR4 mediates Th2-cell trafficking during allergic reactions in mice, as shown in studies where CXCR4 blockade with either an antibody or a receptor antagonist resulted in abrogation of both airway inflammation and AHR. Increased levels of CCL2 (MCP-1), CCL7 (MCP3), and CCL13 (MCP-4) were observed in bronchoalveolar lavage (BAL) cells, and bronchial biopsies of asthmatic patients, but the role of these chemokines in asthma is still controversial (see Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12)). Allergic diseases caused by the fungus Aspergillus fumigatus are collectively referred to as allergic bronchopulmonary aspergillosis (ABPA) in humans, and allergic aspergillosis or fungal asthma in rodents. ABPA is characterized by severe lung eosinophilia, increased mucous secretion, enhanced serum IgE production, and chronic lung fibrosis. Specific chemokines are associated with ABPA. CCR2 and its major ligand CCL2 (MCP-1) (see Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12)) are the key factors in the clearance of A. fumigatus spores in mice. Genetic deficiency in either CCR2 or CCL12 leads to exacerbated allergic disease in mice. By contrast, the absence of the CXCL8 (IL-8) receptor

362 CHEMOKINES Table 3 Chemokines and chemokine receptors in respiratory diseases Clinical condition

Chemokines potentially involved

Chemokine receptors potentially involved

Asthma

CCL11 (eotaxin) CCL17 (TARC) CCL22 CCL1 CCL2 (MCP-1) CCL7 (MCP-3) CCL13 (MCP-4) CCL5 (RANTES) CXCL8 (IL-8)

CCR3 CCR4 CCR4 CCR8 CXCR4 CCR2 CCR2 CCR2 CCR5 CXCR2

Chronic obstructive pulmonary disease

CXCL8 (IL-8) CCL2 (MCP-1) CXCL10 (IP-10)

CXCR2 CCR2 CXCR3

Acute respiratory distress syndrome

CCL2 (MCP-1) CXCL8 (IL-8) CXCL1 (GROa) CXCL2 (GROb) CXCL5 (ENA-78) KC (mouse CXCL1) CCL5 (RANTES)

CCR2 CXCR2 CXCR2 CXCR2 CXCR2 CXCR2 CCR1, CCR3, CCR5

Idiopathic pulmonary fibrosis

CCL2 (MCP-1) CCL3 (MIP-1a) CCL4 (MIP-1b) CXCL2 (GROb)

CCR2 CCR5 CCR5 CXCR2

Acute lung allograft rejection

CCL5 (RANTES) CCL2 (MCP-1) CXCL9 (Mig)

CCR1, CCR3, CCR5 CCR2 CXCR3

Graft-versus-host disease

CCL2 (MCP-1) CCL3 (MIP-1a) CCL5 (RANTES) CXCL9 (Mig) CXCL10 (IP-10)

CCR2 CCR5 CCR5 CXCR3 CXCR3

Tuberculosis

CCL2 (MCP-1) CCL3 (MIP-1a) CCL5 (RANTES) CCL7 (MCP-3) CCL12 (MCP-5, mouse) CXCL5 (ENA-78) CXCL8 (IL-8) CXCL10 (IP-10)

CCR2 CCR1, CCR5 CCR1, CCR3, CCR5 CCR1, CCR2, CCR3 CCR2 CXCR2 CXCR1, CXCR2 CXCR3

Respiratory syncytial virus infection

CCL5 (RANTES) CCL2 (MCP-1) CCL3 (MIP-1a) CCL4 (MIP-1b) CXCL8 (IL-8) CX3CL1 (fractalkine)

CCR1, CCR3, CCR5 CCR2 CCR1, CCR5 CCR5 CXCR1, CXCR2 CXCR1

Lung cancer

CXCL5 (ENA-78) CXCL9 (Mig) CXCL10 (IP-10) CXCL11 (I-TAC) CXCL12 (SDF-1)

CXCR2 CXCR3 CXCR3 CXCR3 CXCR4

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CXCR2 or of CCR4 in mice enhances fungal clearance dramatically. CCL5 (RANTES) and its receptor CCR5 inhibit the innate response of alveolar macrophages against A. fumigatus. Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) (see Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General; and Smoking Cessation) is characterized by neutrophil, macrophage, and CD8 þ T-cell infiltration. CXCL8 is found in very high concentrations in the sputum of COPD patients, and these levels correlate with disease severity. In addition, CXCL8 neutralization by blocking antibodies reduces the chemotactic response of neutrophils to sputum from COPD patients. CXCL8 activates neutrophils via the CXCR1 and CXCR2 receptors. CXCR2 is also expressed on monocytes, which makes it a more attractive therapeutic target than CXCR1. Smallmolecule inhibitors of CXCR2 are currently being tested in clinical trials. The expressions of CCL2 (MCP-1) and its receptor CCR2 are increased in macrophages and epithelial cells of COPD patients. The CCL2/CCR2 couple might play a role in the recruitment of blood monocytes to the lung. Similarly, the expressions of some CXCR3 ligands, such as CXCL10 (IP-10), are elevated in CD8 þ T cells in peripheral airways of COPD patients.

levels correlate with neutrophil concentration in BAL, but not with the severity of lung injury or subsequent clinical course. CCL2 is detectable in BAL of ARDS patients at the onset of ARDS, and persists in the lungs of patients with sustained ARDS. CCL2 concentrations are increased in ARDS patients with sepsis and shock, and this augmentation correlates with increased survival. No correlation was found between CCL2 levels and leukocyte numbers, which suggests that CCL2 may play another function than promoting monocyte recruitment, in ARDS. A number of animal models have been used to mimic ARDS, resulting in a short list of chemokines potentially involved in the disease. In a model of ischemia–reperfusion injury, CXCL5 production correlated with the occurrence of neutrophil-dependent lung injury, and treatment with anti-CXCL5 antibodies were able to attenuate lung damage. In a model of bacterial pneumonia, depletion of CXCL2 was associated with a higher mortality, suggesting a protective role for that chemokine in ARDS. Similarly, lung-specific transgenic expression of KC, the rodent homolog of human CXCL1, was shown to enhance resistance to Klebsiella-induced pneumonia in mice. CCL5 (RANTES) mediates T cell and monocyte chemotaxis via the CCR1, CCR3, and CCR5 receptors (see Chemokines, CC: RANTES (CCL5) for review). In a murine model of pancreatitis-associated lung injury, treatment with the CCL5 (RANTES) receptor antagonist Met-RANTES protected against lung damage but provided little or no protection against pancreatitis.

Acute Respiratory Distress Syndrome

Acute (or adult) respiratory distress syndrome (ARDS) is characterized by the rapid onset of severe respiratory failure due to injury of the lung alveoli and the surrounding capillaries. ARDS can be triggered by clinical events as diverse as major surgery, trauma, multiple transfusions, severe burns, pancreatitis, and sepsis. Several chemokines have been found in increased concentrations in the BAL fluid from patients with established, or at risk for, ARDS. They include CCL2 (see Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12)), and the CXCR2 ligands CXCL8 (which also bind CXCR1), CXCL1 (GROa) (GRO – growth-related oncogene), CXCL2 (GROb) (see Chemometrics, CXC: CXCL1 (GRO1)–CXCL3 (GRO3)) and CXCL5 (ENA-78) (ENA – epithelial neutrophil-activating protein). Levels in CXCL8 were reportedly higher in patients with ARDS plus pneumonia, than in those with ARDS or pneumonia alone. In patients with established ARDS, CXCL8

Idiopathic Pulmonary Fibrosis

The BAL levels of CCL2 (see Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12)), CCL3, and CCL4 were increased in patients with idiopathic pulmonary fibrosis (IPF), although the expression of CCR5 (which binds CCL3 and CCL4) was markedly reduced in lymphocytes from IPF patients. This is in agreement with the hypothesis of a downregulation of the Th1 response in IPF. CXCL2 concentration was also elevated in the BAL of IPF patients. Acute Lung Allograft Rejection

Acute lung allograft rejection is a major complication of lung transplantation and can lead to bronchiolitis obliterans syndrome (BOS), which is characterized by the progressive obliteration of the small airways. Several chemokines were detected in elevated concentrations in the BAL of patients with acute lung

364 CHEMOKINES

allograft rejection or BOS. They include CCL5 (RANTES), CCL2, CXCL9 (see Chemokines, CXC: CXCL9 (MIG)), and CXCL8. In a rat model, in vivo neutralization of either CCL5 or CXCR9 attenuated acute lung allograft rejection via a decrease in mononuclear cell recruitment, and (in the case of CXCL9) a reduction in the expression of the CXCL9 receptor CXCR3. In a murine model, loss of CCL2/CCR2 signaling reduced the recruitment of mononuclear phagocytes following tracheal transplantation and led to attenuation of BOS (see Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12)). Graft-versus-Host Disease

Graft-versus-host disease (GVHD) is a complication of allogeneic bone marrow transplantation (allo-BMT), in which the immune cells of the donor’s bone marrow attack the recipient’s organs and tissues, particularly the skin, eyes, stomach, intestines, liver, and lung. In the lung, GVHD can be associated with idiopathic pneumonia syndrome (IPS), an inflammatory disease characterized by diffuse interstitial pneumonitis and alveolitis leading to interstitial fibrosis. Host macrophages are recruited to the lung during the peritransplantation period, and thought to contribute to alloantigen presentation to donor T cells infiltrating the lung, and to lung damage via cytokine production. Several studies using mouse models have focused on the role of chemokines in the recruitment of effector cells to the lung during GVHD and IPS. The chemokines expressed in the lung after allo-BMT include CCL2, CCL3, CCL4, CCL5, CCL11, and the CXCR3 ligands CXCL9 (see Chemokines, CXC: CXCL9 (MIG)), CXCL10, and CXCL11. The roles of these chemokines differ, depending on when they are evaluated after allo-BMT. For example, CCL2, CXCL10, and CXCL11 are expressed prior to CCL3 and CCL4. Neutralization of CXCL9 and CXCL10 with antibodies reduced IPS severity and CD8 þ T-cell infiltrates. Further inhibition occurred with the elimination of CXCR3 expression from the donor T cells, showing that CXCR3 is a key component of T-cell recruitment to the lung after allo-BMT. The CCL3/CCR5 couple also influences the recruitment of donor T cells to the lung, and a differential role was established for CCR5, depending on the conditioning (lethal irradiation versus no irradiation) of the recipient. The elimination of CCL5 expression from donor T cells inhibited their recruitment to the lung at late time points after transplantation, probably via a decrease in CCR5 expression.

The production of the CCR2 ligand CCL2 (MCP1) in the lung correlated with host macrophage recruitment, and elimination of CCR2 but not of CCL2 expression decreased IPS severity. Tuberculosis

Mycobacterium tuberculosis is a strong inducer of chemokine expression. Human macrophages produce CCL2, CCL3, CCL4, and CCL5 in response to virulent strains of M. tuberculosis. Additional in vitro studies suggest that the chemokines interacting with CCR1, CCR2, and CCR5 may mediate leukocyte recruitment to the site of M. tuberculosis infection. However, direct evidence for a role of these chemokines in tuberculosis is still missing. Expression of CXCL8 and CCL2, but not CCL3, CCL4, and CCL5 was detected in a human alveolar epithelial cell line in response to virulent strains of M. tuberculosis. Granulocytes from the blood of tuberculosis patients produced CXCL8 and CXCL1 in response to a M. tuberculosis antigen. CXCL10 concentration was elevated in the bronchial epithelium of tuberculosis patients, and BAL levels of CCL2, CCL5, CCL7, CCL12, CXCL8, and CXCL10 were increased in tuberculosis patients (see Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12)). It was hypothesized that the increase in expression of the HIV co-receptors CCR5 and CXCL4 on M. tuberculosis-infected monocytes may result in increased productivity of HIV infection. Expression of CCL2, CCL3, CCL7, CCL12, CXCL5, and CXCL10 was observed in response to M. tuberculosis infection in mice. However, CCL2deficient mice were able to clear M. tuberculosis infection, despite an initial increase in bacterial burdens in the lung and spleen. Yet, mice deficient in the CCL2 receptor CCR2 were unable to control infection induced by high M. tuberculosis doses and succumbed. CCR5-deficient mice controlled M. tuberculosis aerosol and intravenous infection and formed granulomas. It is possible that CCR1 compensates for the absence of CCR5 in those mice. Finally, expression of chemokines by macrophages in response to M. tuberculosis appears to be influenced by the cytokine tumor necrosis factor (TNF)-a, which is a major product of M. tuberculosis infection in macrophages. Respiratory Syncytial Virus Infection

Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis and pneumonia among infants and children under one year of age. Some RSV-infected lung epithelial cells were shown to express increased amounts of CC and

CHEMOKINES 365

CXC chemokines, including CCL1 (I-309), CCL20 (Exodus-1), CCL17 (TARC), CCL5 (RANTES), CCL2 (MCP-1), CCL22 (MDC) (MDC – macrophage-derived chemokine), CCL3 (MIP-1a), CCL4 (MIP-1b), CXCL1 (GROa), CXCL2 (GROb), CXCL3 (GROg), CXCL5 (ENA-78), CXCL8, CXCL11 (I-TAC) (I-TAC – IFN-g-inducible T-cell a-chemoattractant), and CX3CL1 (fractalkine). The expression of CCL5, which mediates eosinophil and monocyte recruitment to the lung, is frequently enhanced during RSV infection in patients and murine models. Productions of CCL2, CCL3, and CCL4 are elevated in monocytes and eosinophils exposed to RSV. Infection of eosinophils with RSV leads to the release of CCL5, and CCL3, which is a chemoattractant for human blood eosinophils. CCL3 levels were found highly elevated in the respiratory tract of children with RSV-associated severe bronchiolitis. CXCL8 is a powerful chemoattractant for neutrophils, the main inflammatory cell type to migrate to the lung during RSV infection. CXCL8 expression is markedly increased during RSV infection. A surface protein of RSV exhibits mimicry with the chemokine CX3CL1, and binds the CX3CR1 receptor, which induces leukocyte migration and facilitates RSV infection of cells. Lung Cancer

Cell transformation from preneoplastic to neoplastic, tumor growth, invasion, and metastases are events that depend on the formation of new blood vessels (angiogenesis). Angiogenesis is the product of an imbalance in the expression of angiogenic versus angiostatic factors. As noted earlier, ELR þ CXC chemokines promote angiogenesis, while ELR CXC chemokines mediate angiostasis. ELR þ CXC chemokines are important mediators of angiogenesis during tumorigenesis of non-small cell lung cancer (NSCLC), and their presence correlates with patient prognosis. In a murine model, CXCL5 showed a high degree of correlation with NSCLC-derived angiogenesis. By contrast, the ELR– CXC chemokines CXCL4, CXCL9, CXCL10, and CXCL11 are angiostatic factors. The angiostatic action of CXCL9, CXCL10, and CXCL11 in NSCLC is mediated via CXCR3. In addition, CXCL12 plays important roles in lung cancer invasion and metastasis, and in the spread of

lung metastases to other organs (see Chemokines, CXC: CXCL12 (SDF-1)). See also: Asthma: Overview; Extrinsic/Intrinsic. Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12); RANTES (CCL5); TARC (CCL17). Chemokines, CXC: CXCL12 (SDF-1); CXCL9 (MIG); CXCL1 (GRO1)–CXCL3 (GRO3). Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General; Smoking Cessation.

Further Reading Algood HMS, Chan J, and Flynn JAL (2003) Chemokines and tuberculosis. Cytokine & Growth Factor Reviews 14: 467–477. Gerard C and Rollins BJ (2001) Chemokines and disease. Nature Immunology 2: 108–115. Hoffman SJ, Laham FR, and Polack FP (2004) Mechanisms of illness during respiratory syncytial virus infection: the lungs, the virus and the immune response. Microbes and Infection 6: 767– 772. Humbles AA, Lu B, Friend DS, et al. (2002) The murine CCR3 receptor regulates both the role of eosinophils and mast cells in allergen-induced airway inflammation and hyperresponsiveness. Proceedings of the National Academy of Science, USA 99: 1479–1484. Lloyd CM and Rankin SM (2003) Chemokines in allergic airway disease. Current Opinion in Pharmacology 3: 443–448. Mackay CR (2001) Chemokines: immunology’s high impact factor. Nature Immunology 2: 95–101. Moser B, Wolf M, Walz A, and Loetscher P (2004) Chemokines: multiple levels of leukocyte migration control. Trends in Immunology 25: 75–84. Murphy PM (2002) International union of pharmacology. XXX. Update on chemokine receptor nomenclature. Pharmacological Reviews 54: 227–229. Puneet P, Moochhala S, and Bathia M (2005) Chemokines in acute respiratory distress syndrome. American Journal of Physiology Lung Cellular and Molecular Physiology 288: L3–L15. Rosenkilde ME and Schwartz TW (2004) The chemokine system – a major regulator of angiogenesis in health and disease. APMIS 112: 481–495. Rot A and von Andrian UH (2004) Chemokines in innate and adaptive host defense: basic chemokine grammar for immune cells. Annual Reviews in Immunology 22: 891–928. Schuh JM, Blease K, Kunkel SL, and Hogaboam CM (2003) Chemokines and cytokines: axis and allies in asthma and allergy 14: 503–510. Strieter RM, Belperio JA, Burdick MD, et al. (2004) CXC chemokines: angiogenesis, immunoangiostasis and metastases in lung cancer. Annals of the New York Academy of Science 1028: 351–360. Thelen M (2001) Dancing to the tune of chemokines. Nature Immunology 2: 129–134. Wysocki CA, Panoskaltsis-Mortari A, Blazar BR, and Serody JS (2005) Leukocyte migration and graft-versus-host disease. Blood 105: 4191–4199.

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CHEMOKINES, CC Contents

C10 (CCL6) MCP-1 (CCL2)–MCP-5 (CCL12) RANTES (CCL5) TARC (CCL17) TECK (CCL25)

C10 (CCL6) R M Strieter and M P Keane, The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

IL-3, IL-4, IL-13, GM-CSF, SCF Pulmonary fibrosis +


CCL6

Abstract CCL6 is a CC chemokine that was cloned in the mouse in 1991. Although a human homolog has not been described to date, this novel chemokine shares significant homology with the human CC chemokine, CCL15. In contrast to the other members of the CC chemokine family, CCL6 has four instead of three exons. CCL6 is produced by macrophages, fibroblasts, keratinocytes, vascular smooth muscle cells, and skeletal muscle. CCL6 is chemotactic for monocytes, macrophages, and T cells. CCL6 has been shown to be present in a variety of chronic inflammatory disorders. High expression of CCL6 has been found in the cerebellum and spinal cord of mice with experimental autoimmune encephalitis. Furthermore, intracerebroventricular injection of CCL6 protein promotes the recruitment of large number of macrophages. CCL6 has an important role in the development of pulmonary fibrosis and it mediates some of the pro-fibrotic effects of interleukin-13 (IL-13). CCL6 also has an important role in the airway remodeling seen in animal models of asthma. Interestingly, CCL6 has a protective effect in animal models of peritoneal sepsis and it improves macrophage phagocytic functions. Furthermore, CCL6 has been shown to have a role in tumor growth and invasion.

M, T cells

− IFN-

Airway remodeling Bronchial hyperreactivity

Figure 1 Regulation and function of CCL6 in respiratory disease.

CCL6 is a CC chemokine that was cloned in the mouse in 1991. It was identified along with a series of other hematopoietic specific mRNAs in mouse bone marrow cultures stimulated with granulocytemacrophase colon-stimulating factor (GM-CSF). Although a human homolog has not been described to date, this novel chemokine shares significant homology with the human CC chemokine, CCL15. Both of these chemokines are chemotactic for monocytes and T cells.

heparin-binding proteins. The chemokines display four highly conserved cysteine amino acid residues. The CC chemokine family has the first two NH2-terminal cysteines in juxtaposition, and designated the CC cysteine motif. In contrast to the other members of the CC chemokine family, CCL6 has a novel second exon. With the other members of the CC family, the second exon contains the first three of four highly conserved cysteine residues and the third exon contains the fourth. In contrast, exons 3 and 4 of CCL6 contain the four conserved cysteines distributed in a similar manner to exons 2 and 3 of the other CC chemokines. The novel second exon of CCL6 codes for a large number of charged amino acids. The CCL6 gene (Scya6) is closely lined to the CCL2 gene (Scya2) locus on mouse chromosome 11. The genes for all of the other murine CC chemokines are also located on chromosome 11, suggesting that they all evolved from the same ancestral gene. The primary translation product of CCL6 contains 116 amino acids including an amino terminal signal peptide. CCL6 has an amino terminal region of 28 amino acids, which is in contrast to the other CC chemokines that have an amino terminal region length of 9–10 amino acids.

Structure

Regulation of Production and Activity

Chemokines in their monomeric form range from 7 to 10 kDa and are characteristically basic

CCL6 is expressed in hematopoietic cells, fibroblasts, keratinocytes, vascular smooth muscle cells, and

Introduction

CHEMOKINES, CC / C10 (CCL6) 367

skeletal muscle and is stimulated by exposure to interleukin-3 (IL-3), IL-4, IL-13, and GM-CSF (see Figure 1). Furthermore, C10 is differentially regulated by T-helper-1 (Th1) and T-helper-2 (Th2) cytokines. Bone-marrow-derived macrophages produce CCL6 in response to IL-4, IL-10, and IL-13 in a dose-dependent manner. In contrast, interferon gamma (IFN-g) inhibits IL-3- and GM-CSF-induced expression of CCL6. Stem cell factor has been shown to induce CCL6 from eosinophils.

Biological Function CCL6 is chemotactic for monocytes, macrophages, and T cells. CCL6 has been shown to be present in a variety of chronic inflammatory disorders. High expression of CCL6 has been found in the cerebellum and spinal cord of mice with experimental autoimmune encephalitis. Furthermore, intra-cerebroventricular injection of CCL6 protein promotes the recruitment of large number of macrophages. In a model of chronic peritoneal inflammation, there is delayed (24 h) but sustained elevation in CCL6 levels in peritoneal fluid. These findings demonstrate an important role for CCL6 in chronic inflammation and specifically macrophage recruitment. Interestingly, despite its proinflammatory actions, CCL6 has been shown to improve survival in a murine model of septic peritonitis. Following cecal ligation and puncture, there is a significant increase in levels of CCL6 over baseline, and neutralization of CCL6 leads to increased mortality. In contrast, administration of systemic CCL6 immediately after surgery leads to a significant improvement in survival. This is associated with an increase in TNF-a and CCL2. CCL6 and IL-1 can enhance TNF-a secretion from peritoneal macrophages and IL-13 induces CCL6 from these peritoneal macrophages. CCL6 also enhances the bacterial phagocytic capability of peritoneal macrophages and leads to a significant decrease in bacteremia. In similar murine models of septic peritonitis Stat6 / mice are protected from lethality, and this is associated with increased peritoneal lavage levels of CCL6, CCL22, IL-12, and TNF-a. This in turn leads to enhanced bacterial clearance. Further support for the important role in mononuclear cell recruitment is seen in a model of skin healing. CCL6 has been shown to be strongly expressed in full-thickness excisional skin wounds as early as day 1 and to be localized to macrophages of the granulation tissue and the keratinocytes at the wound edge, suggesting an important role in the strong macrophage influx that is seen in healing skin. Furthermore, CCL6 has been shown to be expressed

in skeletal muscle and may contribute to the mononuclear cell influx in muscle tissue that is associated with muscular dystrophy. In addition to its chemotactic properties, CCL6 also promotes local tissue apoptosis, thereby facilitating tumor invasion in a murine cancer model. CCL6 also directly stimulates tumor growth and the leukemogenic phenotype in 32D cells.

Receptors The receptor for CCL6 is not known. Interestingly, deletion of CCR1 leads to a similar effect on IL-13induced inflammation as neutralization of CCL6, suggesting that CCL6 may signal through CCR1.

CCL6 in Respiratory Disease CCL6 is elevated in the pathogenesis of bleomycininduced pulmonary fibrosis. Similarly both IL-4 and IL-13, which are potent inducers of CCL6, are elevated during the pathogenesis of bleomycin-induced pulmonary fibrosis. Neutralization of IL-13, but not IL-4, attenuates the development of fibrosis and levels of CCL6. This is consistent with the known important role of IL-13 in the development of pulmonary fibrosis. Furthermore, neutralization of CCL6 also attenuates bleomycin-induced pulmonary fibrosis and intrapulmonary macrophage numbers. This suggests that in addition to its direct effect on fibroblasts, IL-13 has a role in the development of pulmonary fibrosis via the induction of CCL6 and the subsequent recruitment of macrophages. Further support for the role of CCL6 in lung remodeling and repair is seen in studies using IL-13 transgenic mice. The treatment of IL-13 þ / þ mice with anti-CCL6 leads to decreased levels of mRNA for matrix metalloproteinase-2 (MMP-2), MMP-9, and tissue inhibitor of metalloproteinase-4 (TIMP-4). Furthermore, neutralization of CCL6 leads to a decrease in the ability of IL-13 to stimulate production of CCL2, CCL3, MMP-2, MMP-9, and cathepsins K, L, and S. Interestingly, deletion of CCR1 leads to a similar effect on IL-13-induced inflammation as neutralization of CCL6 suggesting that CCL6 may signal through CCR1. In a murine model of fungal-induced asthma, there is a significant increase in CCL6 in bronchoalveolar lavage (BAL), and this is significantly higher than levels of CCL2, CCL3, or CCL11. In this model the major source of CCL6 is alveolar macrophages, fibroblasts, and vascular smooth muscle cells. Neutralization of CCL6 prior to intratracheal challenge with Aspergillus fumigates leads to decreased airway inflammation and bronchial hyperresponsiveness although it had no effect on IL-10 or

368 CHEMOKINES, CC / MCP-1 (CCL2)–MCP-5 (CCL12)

IgE levels. In a chronic model of fungal allergic airway disease, CCR1 / mice have significantly lower levels of CCL6, CCL11, CCL22, and IL-4 and IL-13 at 30 days as compared to CCR1 þ / þ mice. This is associated with a decrease in subepithelial fibrosis and fewer numbers of goblets cells. Interestingly, there is no difference in airway hyperresponsiveness at 30 days, suggesting that the Th2-inducible chemokines are most important in the airway remodeling of asthma rather than the bronchial hyperreactivity. See also: Asthma: Overview; Allergic Bronchopulmonary Aspergillosis. Chemokines. Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12). Extracellular Matrix: Basement Membranes; Degradation by Proteases. Interleukins: IL-6. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Eosinophils; Monocytes; T cells. Matrix Metalloproteinases. Pulmonary Fibrosis. Transgenic Models. Tumors, Malignant: Overview.

Further Reading

Orlofsky A, Wu Y, and Prystowsky MB (2000) Divergent regulation of the murine CC chemokine C10 by Th(1) and Th(2) cytokines. Cytokine 12: 220. Steinhauser ML, Hogaboam CM, Matsukawa A, et al. (2000) Chemokine C10 promotes disease resolution and survival in an experimental model of bacterial sepsis. Infection and Immunity 68: 6108. Wang W, Bacon KB, Oldham ER, and Schall TJ (1998) Molecular cloning and functional characterization of human MIP-1 delta, a new C–C chemokine related to mouse CCF-18 and C10. Journal of Clinical Immunology 18: 214. Yi F, Jaffe R, and Prochownik EV (2003) The CCL6 chemokine is differentially regulated by c-Myc and L-Myc, and promotes tumorigenesis and metastasis. Cancer Research 63: 2923. Zhu Z, Ma B, Zheng T, et al. (2002) IL-13-induced chemokine responses in the lung: role of CCR2 in the pathogenesis of IL-13induced inflammation and remodeling. Journal of Immunology 168: 2953.

MCP-1 (CCL2)–MCP-5 (CCL12) A D Luster, W K Hart, and A M Tager, Harvard Medical School, Boston, MA, USA & 2006 Elsevier Ltd. All rights reserved.

Asensio VC, Lassmann S, Pagenstecher A, et al. (1999) C10 is a novel chemokine expressed in experimental inflammatory demyelinating disorders that promotes recruitment of macrophages to the central nervous system. American Journal of Pathology 154: 1181. Berger MS, Kozak CA, Gabriel A, and Prystowsky MB (1993) The gene for C10, a member of the beta-chemokine family, is located on mouse chromosome 11 and contains a novel second exon not found in other chemokines. DNA and Cell Biology 12: 839. Berger MS, Taub DD, Orlofsky A, et al. (1996) The chemokine C10: immunological and functional analysis of the sequence encoded by the novel second exon. Cytokine 8: 439. Hogaboam CM, Gallinat CS, Taub DD, et al. (1999) Immunomodulatory role of C10 chemokine in a murine model of allergic bronchopulmonary aspergillosis. Journal of Immunology 162: 6071. Keane MP, Belperio JA, Henson PM, and Strieter RM (2005) Inflammation, injury and repair. In: Nadel M, et al. (eds.) Textbook of Respiratory Medicine, 4th edn. Philadelphia: Elsevier/ Saunders. Keane MP, Belperio JA, and Strieter RM (2003) Chemokines in pulmonary fibrosis. In: Strieter RM, Kunkel SL, Standiford TJ, and Lenfant C (eds.) Chemokines in the Lung, Lung Biology in Health and Disease, vol. 172. New York: Decker. Keane MP, Belperio JA, and Strieter RM (2003) Cytokine biology and the pathogenesis of interstitial lung disease. In: Schwarz MI and King TE (eds.) Interstitial Lung Disease, 4th edn. Hamilton, ON: BC Decker. Ma B, Zhu Z, Homer RJ, et al. (2004) The C10/CCL6 chemokine and CCR1 play critical roles in the pathogenesis of IL-13-induced inflammation and remodeling. Journal of Immunology 172: 1872. Orlofsky A, Berger MS, and Prystowsky MB (1991) Novel expression pattern of a new member of the MIP-1 family of cytokine-like genes. Cell Regulation 2: 403. Orlofsky A, Lin EY, and Prystowsky MB (1994) Selective induction of the beta chemokine C10 by IL-4 in mouse macrophages. Journal of Immunology 152: 5084.

Abstract The attraction of leukocytes into tissues is essential for inflammation and host response to infection. This process is controlled by chemotactic cytokines known as chemokines. The monocyte chemoattractant proteins (MCP) are an important subfamily of chemokines that share structural as well as functional features. By activating various G-protein-coupled chemokine receptors, the MCPs induce the recruitment and activation of multiple subsets of leukocytes, including monocytes and macrophages, effector CD4 þ and CD8 þ T cells, B cells, dendritic cells, basophils, mast cells, natural killer cells, and eosinophils. Increased expression of MCP chemokines has been detected in a variety of respiratory diseases involving the recruitment of these leukocytes into the lung, including both inflammatory diseases such as asthma, in which leukocyte recruitment participates in disease pathogenesis, as well as infectious diseases such as tuberculosis, in which leukocyte recruitment participates in host immune responses. Functional roles for the MCP chemokines have been demonstrated in several rodent models of respiratory diseases, in which disease severity can be modulated by inhibition of MCP signaling. The MCPs and their receptors therefore are attractive targets for rational development of drugs that will have therapeutic potential in multiple respiratory diseases.

Introduction The monocyte chemoattractant proteins (MCP) constitute an important subfamily of chemotactic cytokine (CC) chemokines that share structural features and have overlapping functions. By activating G-protein-coupled CC chemokine receptors present on the surface of different classes of leukocytes, the MCPs participate in inflammatory responses by

CHEMOKINES, CC / MCP-1 (CCL2)–MCP-5 (CCL12) 369

directing the recruitment and activation of these cells. Four human MCP (hMCP) proteins, hMCP-1 (CCL2), hMCP-2 (CCL8), hMCP-3 (CCL7), and hMCP-4 (CCL13), and four mouse MCP (mMCP) proteins, mMCP-1, mMCP-2, mMCP-3, and mMCP5 (CCL12), have been identified that share substantial amino acid identity. The cross-species assignments of orthologs among these genes is not entirely clear, but it is generally accepted that mMCP-1, mMCP-2, and mMCP-3 are orthologs of hMCP-1, hMCP-2, and hMCP-3, respectively. However, no mouse ortholog has been described for human MCP-4 to date, and mMCP-5 has no ortholog in the human genome, likely reflecting divergence of the human and mouse MCP gene clusters after speciation.

The secondary and tertiary structure of MCP-1 has been solved by both nuclear magnetic resonance (NMR) and crystal analyses. As is characteristic of the chemokine family, the N-terminal cysteines are followed by a long loop that leads into three

hMCP-2

hMCP-1

hMCP-3

hMCP-4

Structure As is characteristic of chemokines, the MCPs are small basic proteins. All the MCPs are members of the CC, or b-chemokine family, whose primary structures contain four conserved cysteine residues, of which the first two are adjacent to each other. Substantial homology exists between all members of the MCP family, with amino acid identity ranging from 74% between hMCP-1 and hMCP-3, to 38% between mMCP-1 (excluding a unique C-terminal extension) and mMCP-2 (Figure 1). Phylogenetic analysis of the protein sequences of the MCP family (Figure 2) suggests that the human and mouse MCP gene clusters diverge by gene duplication after speciation.

-23

Hu Hu Hu Hu Mu Mu Mu Mu

MCP-1 MCP-2 MCP-3 MCP-4 MCP-1 MCP-2 MCP-3 MCP-5

Hu Hu Hu Hu Mu Mu Mu Mu

MCP-1 MCP-2 MCP-3 MCP-4 MCP-1 MCP-2 MCP-3 MCP-5

mMCP-1

mMCP-3

mMCP-2

mMCP-5 Figure 2 Phylogenetic analysis of protein sequences of human and mouse MCPs. Analysis was performed using the neighborjoining method of ClustalW.

+1

+25

MK VSAALLCLLLIAATFIPQGLAQPDAINAPVTCCYNFTNRKISVQRLAS MK VSAALLCLLLMAATFSPQGLAQPDSVSIPITCCFNVINRKIPIQRLES MK ASAALLCLLLTAAAFSPQGLAQPVGINTSTTCCYRFINKKIPKQRLES MK VSAVLLCLLLMTAAFNPQGLAQPDALNVPSTCCFTFSSKKISLQRLKS MQ VPVMLLGLLFTVAGWSIHVLAQPDAVNAPLTCCYSFTSKMIPMSRLES MK IYAVLLCLLLIAVPVSPEKLTGPD--KAPVTCCFHVLKLKIPLRVLKS MR ISATLLCLLLIAAAFSIQVWAQPDGPNA-STCCY-VKKQKIPKRNLKS MK IS-TLLCLLLIATTISPQVLAGPDAVSTPVTCCYNVVKQKIHVRKLKS +26

+75

YRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT YT RITNIQCPKEAVIFKTQRGKEVCADPKERWVRDSMKHLDQIFQNLKP YRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTPKL YV-ITTSRCPQKAVIFRTKLGKEICADPKEKWVQNYMKHLGRKAHTLKT Y K R I T S S R C P K E A V V F V T K L K R E V C A D P K K E W V Q T Y I K N L D R N Q M R S E P>> YE R I N N I Q C P M E A V V F Q T K Q G M S L C V D P T Q K W V S E Y M E I L D Q K S Q I L Q P YR R I T S S R C P W E A V I F K T K K G M E V C A E A H Q K W V E E A I A Y L D M K T P T P K P YR R I T S S Q C P R E A V I F R T I L D K E I C A D P K E K W V K N S I N H L D K T S Q T F I L>>

Figure 1 Alignment of protein sequences of human and mouse MCPs. For comparison to the other MCPs, the unique C-terminal extension of mMCP-1 is not included, and the 98 amino acid N-terminal sequence is presented. Amino acids conserved between different MCPs are boxed.


antiparallel b-pleated sheets in what is referred to as a ‘Greek key’ motif. The protein then terminates in an a-helix that overlies the b-pleated sheets. With regard to quaternary structure, all MCPs exist as dimers under conditions required for NMR and Xray crystallographic structural analyses. However, the physiologic quaternary structures of the MCPs, for example, whether they function physiologically as monomers, dimers, or higher-order multimers, remains to be determined.

members of the CC chemokine family, therefore only bind CC chemokine receptors (CCRs). All of the MCP proteins described to date, with the exception of mMCP-2, activate CCR2 to varying degrees. In addition, hMCP-2, hMCP-3, hMCP-4, and mMCP-3 activate CCR3, hMCP-2 and hMCP-3 activate CCR1, and hMCP-2 activates CCR5 (Figure 3). The CC chemokine receptor(s) activated by mMCP-2 have not yet been determined.

Biological Function Regulation of Production and Activity MCPs are generally considered to be ‘inflammatory’ chemokines, whose inducible expression directs the migration of leukocytes participating in inflammatory responses, as opposed to ‘homeostatic’ chemokines, whose constitutive expression directs the positioning of leukocytes under basal conditions. During inflammatory responses, the MCPs are highly induced in multiple cell types and tissues by a variety of stimuli, such as lipopolysaccharide, tumor necrosis factor alpha, interleukin (IL)-1b, interferon gamma (IFN-g), and platelet-derived growth factor (Table 1). Conversely, expression of MCP family members can be suppressed by glucocorticoids or immunusuppressive cytokines such as transforming growth factor beta and IL-10. In addition to transcriptional regulation of MCP expression, MCP activity can also be regulated by posttranslational modification. For example, glycosylation of hMCP-1 reduces its ability to direct the migration of monocytes in vitro. Posttranslational truncation of the first four or five N-terminal amino acids of hMCP-1, or the first five amino acids of hMCP-2, abrogates their chemoattractant activity. Additionally, the truncated form of MCP-2 acts as an inhibitor of other chemokines, including MCP-1, MCP-2, MCP-3, and RANTES (regulated upon activation normally T cell expressed and secreted).

Receptors The MCPs stimulate cells by binding to specific surface chemokine receptors, which belong to the G-protein-coupled seven transmembrane domain receptor superfamily. Binding of MCPs to these receptors activates multiple signaling pathways that regulate the cellular machinery that propels cells during MCP-directed migration. Each of the major chemokine families has its own specific set of chemokine receptors. Although many chemokines bind to more than one chemokine receptor, the members of each chemokine family bind only to the chemokine receptors specific for that family. The MCPs, as

MCPs stimulate the directed migration, or chemotaxis, and activation of leukocytes that express receptors specific for these chemokines. The induction of MCP expression in tissue sites of inflammation consequently directs the recruitment of activated leukocytes into these sites as a critical feature of host inflammatory responses to infection, injury, or allergy. The classes of leukocytes recruited by the MCPs can be predicted by the chemokine receptors expressed by these cells (Figure 3). As noted, all of the MCP proteins except mMCP-2 are active on CCR2, which is expressed on monocytes and macrophages, antigen-experienced (CD45RO þ ) T cells, B cells, immature dendritic cells, basophils, mast cells, and natural killer (NK) cells. hMCP-2, hMCP-3, hMCP-4, and mMCP-3 are active on CCR3, which is expressed by eosinophils, basophils, and TH2polarized CD4 þ T cells. hMCP-2 and hMCP-3 are active on CCR1, which is expressed by monocytes, T cells, dendritic cells, and eosinophils. hMCP-2 is also active on CCR5, which is expressed by TH1-polarized CD4 þ T cells and antigen-experienced (CD45RO þ ) CD8 þ T cells, monocytes and macrophages, and dendritic cells. Mice transgenically overexpressing MCP-1 driven by various different tissue-specific promoters have been generated. Consistent with MCP-1 directing the migration of monocytes and macrophages, these transgenic mice generally have increased accumulation of these cells specifically in those tissues in which there is increased MCP-1 expression. For example, mice overexpressing MCP-1 driven by the surfactant protein C promoter, in which MCP-1 is constitutively secreted into the airspaces, have increased numbers of monocytes, as well as lymphocytes, recovered in bronchoalveolar lavage (BAL) fluid. In contrast, normal numbers of monocytes and lymphocytes are present in the lung parenchyma of these mice. Mice genetically deficient for MCP-1 have also been generated. These mice are viable, develop normally, and have normal numbers of monocytes in their circulation and macrophages in their tissues. These mice have reduced monocyte recruitment in models of

CHEMOKINES, CC / MCP-1 (CCL2)–MCP-5 (CCL12) 371 Table 1 MCP production MCP

Cell type/organ

Inducer

MCP-1

Fibroblasts Endothelial cells Vascular smooth muscle cells Monocytes/macrophages (including cell lines HL60, U937, THP1) Neutrophils Keratinocytes Synovial cells Type II pneumocyte cell line Mesangial cells Retinal pigmented epithelial cells Malignant cell lines (glioma, sarcoma, melanoma, hepatoma) Luteal cells Secondary lymphoid organs Lung (epithelium, alveolar macrophages) Brain (astrocytes) Spinal cord Seminal vesicles Kidney

PDGF / IL-1 / TNF-a / viruses / dsRNA / LPS / cholera toxin IL-1 / TNF / IFN-g / IL-4 / MM-LDL / stretch PDGF / MM-LDL / stretch LPS / IFN-g / PMA

MCP-2

MCP-3

MCP-4

MCP-5

Fibroblasts Neutrophils Osteosarcoma cell line Astrocytes Organs: small intestine, peripheral blood, heart, placenta, lung, skeletal muscle, ovary, colon, spinal cord, pancreas, thymus Porcine luteal cells Fibroblasts Monocytes Platelets Bronchial epithelium Kidney Astrocytes Skin Endothelial cells Dermal fibroblasts Bronchoalveolar lavage cells Bronchial epithelial cell lines (A549, BEAS-2B) PBMC Nasal epithelium Arterial plaques (endothelial cells/macrophages) Organs: small intestine, thymus, colon, heart, placenta Macrophage cell line (RAW 264.7) Lung (alveolar macrophages/smooth muscle cells) Spinal cord Lymph node stromal cells Thymic stromal cells

TNF IFN-g IL-1 IL-1 / TNF IL-1 / TNF / IFN-g / basic FGF / LIF / IL-6 IL-1 / TNF / LPS

Asthma and granuolma models EAE models of multiple sclerosis / seizure Contusion injury Inflammation (e.g., glomerulonephritis)/hypoxia/transplant rejection IL-1 / IFN-g / dsRNA / measles virus IL-1 / IFN-g EAE / multiple sclerosis

PDGF TNF / IL-1 / IFN-g / LPS / lipoarabinomannan Asthma models glomerulonephritis EAE / multiple sclerosis Atopy IL-1 / TNF Asthma IL-1 / TNF / IFN-g PHA / IL-2 Sinusitis Atherosclerosis

IFN-g / LPS Asthma models Spinal cord contusion injury

dsRNA, double stranded RNA; EAE, experimental allergic encephalomyelitis; IFN-g, interferon gamma; IL, interleukin; LIF, leukemia inhibitory factor; LPS, lipopolysaccharide; MM-LDL, minimally modified-low density lipoprotein, PDGF, platelet-derived growth factor; PHA, phytohemagglutanin; PMA, phorbol myristate acetate; PBMC, peripheral blood mononuclear cells. Reproduced from Rollins BJ (2000) MCP-1, MCP-2, MCP-3, MCP-4, and MCP-5. In: Oppenheim JJ and Feldman M (eds.) Cytokine Reference, pp. 1145–1160. San Diego: Academic Press, with permission from Elsevier.

372 CHEMOKINES, CC / MCP-1 (CCL2)–MCP-5 (CCL12) MCPs activating CCR2:

hMCP-1 mMCP-1 hMCP-2 mMCP-3 hMCP-3 mMCP-5 hMCP-4

Cells expressing CCR2: Monocytes/ TH1 CD4+ macrophages T cells

TH2 CD4+ T cells

Biologic activity:

CD8+ T cells

B cells

Dendritic cells

Basophils/ mast cells

NK cells

Cell recruitment and activation

(a) MCPs activating CCR3:

hMCP-2 hMCP-3

MCPs activating CCR1:

hMCP-4 mMCP-3

Cells expressing CCR3:

Cells expressing CCR1: TH2 CD4+ T cells

Biologic activity:

hMCP-2 hMCP-3

Monocytes/ macrophages

Basophils/ Eosinophils mast cells


(b)

Biologic activity:

Dendritic cells

Eosinophils


(c)

MCPs activating CCR5:

hMCP-2

Cells expressing CCR5: Monocytes/ macrophages

Biologic activity:

TH1 CD4+ T cells

CD8+ T cells

Dendritic cells


(d) Figure 3 Key biological functions of MCPs. By signaling through the chemokine receptors (a) CCR2, (b) CCR3, (c) CCR1, and (d) CCR5, the MCPs induce the recruitment and activation of multiple classes of leukocytes, including monocytes and macrophages, effector CD4þ and CD8þ T cells, B cells, dendritic cells, basophils and mast cells, NK cells, and eosinophils.

tissue inflammation, however, including models of peritonitis and delayed hypersensitivity reactions, indicating that MCP-1 has a nonredundant role in directing monocytes into inflamed tissues.

Role of MCPs in Respiratory Diseases As would be expected from their biologic activity, increased expression of MCP chemokines has been

CHEMOKINES, CC / MCP-1 (CCL2)–MCP-5 (CCL12) 373

detected in a variety of respiratory diseases involving the recruitment of leukocytes into the lung, including inflammatory diseases such as asthma, in which leukocyte recruitment participates in disease pathogenesis, as well as infectious diseases such as tuberculosis, in which leukocyte recruitment participates in host immune responses. Functional roles for the MCP chemokines have been demonstrated in a variety of respiratory disease models in rodents, in which disease severity has been modulated by targeting MCP/CCR2 signaling. Asthma

Increased levels of MCP-1, MCP-3, and MCP-4 have been noted in the airways of asthmatic subjects, and gene polymorphisms of MCP-1 have been associated both with asthma susceptibility and severity. Further, treatment resulting in improvements in forced expiratory volume has been associated with reductions in airway expression of MCP-3 and MCP4. Increased airway expression of the MCPs, including MCP-1, MCP-3, and MCP-5, has also been noted in mouse models of allergic pulmonary inflammation. Inhibition or deletion of the MCPs, or one of their receptors, CCR2, has produced conflicting results in these models, however, and consequently the role of the MCPs in asthma remains to be definitively established.

Other Pulmonary Infections

Elevated expression of MCP-1 in the lung has been demonstrated in mouse models of infection with Streptococcus pneumoniae, Pseudomonas aeruginosa, Mycoplasma pneumoniae, influenza A, Aspergillus fumigatus, Cryptococcus neoformans, and Pneumocystis carinii. MCP-1 has been demonstrated to participate in the recruitment of both macrophages and NK cells in a mouse model of pneumococcal pneumonia, and to direct the recruitment of macrophages responsible for the phagocytosis and removal of dying neutrophils in a mouse model of pseudomonas pneumonia. In so doing, MCP-1 expression is thought to attenuate the development of lung injury in the pseudomonas model. In a model of influenza pneumonia, CCR2-deficient mice are protected from the early pathological manifestations of infection due to reduced macrophage recruitment. In this model, however, the delay in macrophage accumulation in the CCR2-deficient mice caused a subsequent delay in T cell recruitment, and an increase in pulmonary viral titers at early time points following infection. Finally, in a model of invasive aspergillus pneumonia performed in neutropenic mice, treatment of infected mice with neutralizing antiMCP-1 antibody reduced NK cell recruitment at early time points, and led to a greater than threefold increase in pathogen burden in lungs and twofold greater mortality.

Tuberculosis Lung Allograft Rejection

Increased levels of MCP-1, MCP-3, and MCP-4 have been noted in the lungs of patients with tuberculosis. A requirement for CCR2, and presumably its MCP ligands, for control of Mycobacterium tuberculosis infection has been dramatically demonstrated in mouse models. Mice genetically deficient for CCR2 have a rapidly progressive and fatal course following high- or moderate-dose M. tuberculosis infection, developing lung mycobacterial burdens 100-fold greater than wild-type mice controls, which control infection and survive. CCR2-deficient mice demonstrated multiple defects in leukocyte trafficking following infection, including defects in early macrophage recruitment to the lung and subsequent defects in macrophage and dendritic cell recruitment to the mediastinal lymph nodes. T cell activation in the mediastinal lymph nodes was consequently delayed, resulting in reduced accumulation of T cells primed to produce IFN-g in the lungs of infected CCR2-deficient mice. Though this remains to be demonstrated in humans, the cellular responses mediated by the activation of CCR2 by its MCP ligands appear essential for immune control of M. tuberculosis infection.

MCP-1 levels are elevated in the lungs of transplant recipients experiencing acute rejection, as well as those experiencing chronic rejection, that is, bronchiolitis obliterans syndrome (BOS), compared with healthy transplant recipients. Elevated BAL levels of MCP-1 further have been shown to predict the development of BOS. Additionally, in allograft recipients shifted from cyclosporine- to tracrolimus-based immunosuppression due to refractory acute rejection, clinical and functional stabilization was accompanied by significant and sustained reductions in lavage MCP-1 levels. Induction of MCP-1 expression has also been noted in mouse and rat models of allograft rejection and BOS. In a mouse model of tracheal transplantation, both the recruitment of mononuclear phagocytes and the severity of BOS were significantly reduced by the interruption of MCP-1/CCR2 signaling. Idiopathic Pulmonary Fibrosis

Elevated levels of MCP-1 have been noted in the lungs of idiopathic pulmonary fibrosis patients. Similarly, the expression of MCP-1 is increased in the


lungs in rodent models of pulmonary fibrosis. In the mouse model of pulmonary fibrosis induced by bleomycin, inhibition of endogenous MCP-1 function by the transgenic expression of a dominant negative N-terminal deletion MCP-1 mutant reduced pulmonary fibrosis, although interestingly, without affecting macrophage or lymphocyte recruitment into the lung. A similar result has been reported in experiments using CCR2-deficient mice, which were protected from fibrosis despite having leukocyte recruitment in the lung equivalent to that of wild-type controls. However, another group of investigators has recently reported that in their experiments performing the bleomycin model of pulmonary fibrosis in CCR2-deficient mice, lung macrophage recruitment was reduced. MCP-1/CCR2 signaling thus may contribute to the development of pulmonary fibrosis through both leukocyte-dependent and nonleukocyte-dependent effects. Acute Lung Injury/Acute Respiratory Distress Syndrome

Elevated MCP-1 levels are present in BAL fluid of acute respiratory distress syndrome patients, and have been correlated both with alveolar monocyte recruitment and lung injury score. Elevated MCP-1 expression has also been noted in several rodent models of acute lung injury, including a model of gastric aspiration-induced pneumonitis. In this model, MCP-1-deficient mice had decreased survival compared with wild-type controls. Wild-type mice in this model demonstrated areas of compartmentalized inflammation in the lung with prominent granuloma formation following acid aspiration, whereas MCP1-deficient mice demonstrated severe diffuse pneumonitis without granulomas, suggesting that MCP-1 protects uninjured lung regions by promoting the isolation and compartmentalization of tissue with active inflammation. Pulmonary Hypertension

Elevated levels of MCP-1 have been noted in the circulation of patients with both primary pulmonary hypertension (PPH) and chronic thromboembolic pulmonary hypertension (CTEPH). In patients with PPH, treatment with epoprostenol has been associated with significant reductions in circulating MCP-1 levels. In patients with CTEPH, circulating MCP-1 levels were significantly correlated with pulmonary vascular resistance. Elevated levels of MCP-1 in the circulation as well as in BAL fluid have also been demonstrated in a rat model of pulmonary hypertension induced by monocrotaline. Inhibition of MCP-1 signaling in this model reduced mononuclear cell

infiltration into the lung, as well as right ventricular systolic pressure, right ventricular hypertrophy, and medial hypertrophy of the pulmonary arterioles. Other Respiratory Diseases

Elevated expression of the MCPs has also been noted in several other respiratory diseases, including chronic obstructive pulmonary disease, pulmonary alveolar proteinosis, sarcoidosis, hypersensitivity pneumonitis, eosinophilic pneumonia, and coalworker’s pneumoconiosis.

Concluding Remarks By virtue of their ability to direct the migration of multiple classes of leukocytes into the lungs, the MCPs are importantly involved in multiple respiratory diseases. In inflammatory pathologies in which leukocyte recruitment participates in disease pathogenesis, such as asthma, allograft rejection, or ARDS, inhibition of MCP signaling is an attractive strategy for therapeutic intervention. In infectious pathologies in which leukocyte recruitment participates in host immune responses, such as tuberculosis or other pneumonias, augmentation of MCP signaling may be beneficial. The MCPs and their receptors therefore represent attractive targets for rational development of drugs that will have therapeutic potential in multiple respiratory diseases. See also: Acute Respiratory Distress Syndrome. Asthma: Overview. Dendritic Cells. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Eosinophils; Monocytes; T cells; Pulmonary Macrophages. Vascular Disease.

Further Reading Belperio JA, Keane MP, Burdick MD, et al. (2001) Critical role for the chemokine MCP-1/CCR2 in the pathogenesis of bronchiolitis obliterans syndrome. Journal of Clinical Investigation 108: 547–556. Goodman RB, Strieter RM, Martin DP, et al. (1996) Inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome. American Journal of Respiratory and Critical Care Medicine 154: 602–611. Gu L, Tseng S, Horner RM, et al. (2000) Control of TH2 polarization by the chemokine monocyte chemoattractant protein-1. Nature 404: 407–411. Gunn MD, Nelken NA, Liao X, and Williams LT (1997) Monocyte chemoattractant protein-1 is sufficient for the chemotaxis of monocytes and lymphocytes in transgenic mice but requires an additional stimulus for inflammatory activation. Journal of Immunology 158: 376–383. Ikeda Y, Yonemitsu Y, Kataoka C, et al. (2002) Anti-monocyte chemoattractant protein-1 gene therapy attenuates pulmonary hypertension in rats. American Journal of Physiology: Heart and Circulatory Physiology 283: H2021–H2028.

CHEMOKINES, CC / RANTES (CCL5) 375 Katsushi H, Kazufumi N, Hideki F, et al. (2004) Epoprostenol therapy decreases elevated circulating levels of monocyte chemoattractant protein-1 in patients with primary pulmonary hypertension. Circulation Journal 68: 227–231. Lu B, Rutledge BJ, Gu L, et al. (1998) Abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice. Journal of Experimental Medicine 187: 601–608. Miotto D, Christodoulopoulos P, Olivenstein R, et al. (2001) Expression of IFN-gamma-inducible protein; monocyte chemotactic proteins 1, 3, and 4; and eotaxin in TH1- and TH2-mediated lung diseases. Journal of Allergy and Clinical Immunology 107: 664–670. Moore BB, Paine R III, Christensen PJ, et al. (2001) Protection from pulmonary fibrosis in the absence of CCR2 signaling. Journal of Immunology 167: 4368–4377. Nomiyama H, Egami K, Tanase S, et al. (2003) Comparative DNA sequence analysis of mouse and human CC chemokine gene clusters. Journal of Interferon Cytokine Research 23: 37–45. Peters W, Scott HM, Chambers HF, et al. (2001) Chemokine receptor 2 serves an early and essential role in resistance to Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences of the United States of America 98: 7958–7963. Reynaud-Gaubert M, Marin V, Thirion X, et al. (2002) Upregulation of chemokines in bronchoalveolar lavage fluid as a predictive marker of post-transplant airway obliteration. Journal of Heart and Lung Transplantation 21: 712–730. Rollins BJ (2000) MCP-1, MCP-2, MCP-3, MCP-4, and MCP-5. In: Openheim JJ and Feldman M (eds.) Cytokine Reference, pp. 1145–1160. San Diego: Academic Press. Rosseau S, Hammerl P, Maus U, et al. (2000) Phenotypic characterization of alveolar monocyte recruitment in acute respiratory distress syndrome. American Journal of Physiology: Lung Cellular and Molecular Physiology 279: L25–L35. Szalai C, Kozma GT, Nagy A, et al. (2001) Polymorphism in the gene regulatory region of MCP-1 is associated with asthma susceptibility and severity. Journal of Allergy and Clinical Immunology 108: 375–381.

RANTES (CCL5) A E I Proudfoot, C A Power, and Z Johnson, Serono Pharmaceutical Research Institute, Geneva, Switzerland & 2006 Elsevier Ltd. All rights reserved.

Abstract RANTES (regulated upon activation normally T cell expressed and secreted) is a member of the subfamily of cytokines called chemokines, the name given to chemoattractant cytokines in 1994. The family consists of small basic proteins whose role is to provide the directional signal for leukocyte migration. The chemokine family are distinct from other cytokines in that they act on G-protein-coupled heptahelical transmembrane spanning receptors. Initially, chemokines were named according to their function but due to the confusion in the nomenclature which arose from the simultaneous identification by different laboratories of many chemokines in the mid to late 1990s, a systematic nomenclature was introduced in 2001. In the new nomenclature the chemokines were named according to their genomic localization, and RANTES was renamed CCL5. RANTES probably

has the broadest range of target cells of known chemokines due to its multiple receptor usage and thus is implicated in the recruitment of memory T cells, monocytes, eosinophils, and basophils, thus making it one of the key players in respiratory disease.

Introduction Chemokines are structurally classified into subfamilies based on their pattern of disulfide bridging, which allows their division into four subclasses as defined by the distribution of the four highly conserved cysteines. Thus the a-chemokines have the first Cys pair close to the N-terminus and separated by an amino acid (CXC chemokines) whereas the b-chemokines have the first two Cys residues adjacent (CC chemokines). These two subclasses comprise the majority of chemokines, but there are also two minor subclasses, which each have only one or two members: CX3C chemokines have three amino acids separating the amino terminal Cys residues and C chemokines possess only one disulfide bridge. Regulated upon activation normally T-cell expressed and secreted (RANTES)/CCL5 belongs to the b-chemokine or CC-chemokine subclass. RANTES was initially isolated from a T cell cDNA library although it was subsequently found to be induced in other cell types upon induction with proinflammatory cytokines.

Structure RANTES has the canonical monomeric fold of the chemokine family (Figure 1(c)) and the canonical dimeric form of the CC subfamily (Figure 1(b)) consisting of a flexible N-terminal loop, followed by three antiparallel b-sheets, which are connected by short flexible turns called the 30s and 50s loop and an a-helical C-terminus (Figure 1(a)). RANTES has an extremely high molecular weight, 4600 kDa, as determined by techniques such as size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC). This high-molecular-weight quaternary structure is not only found in vitro, but the protein has been observed to be secreted from granules of T cells as a complex with soluble heparin, also greater than 600 kDa. Such a higher-order quaternary structure is not limited to RANTES but has also been described for MIP-1a and MIP-1b, both of which share receptor usage with RANTES. Since RANTES interacts with several receptors, mutagenesis has been used to identify the epitopes conferring receptor specificity. One common feature has emerged in that the N-terminus is critical for receptor activation. Both truncation by total protein synthesis, and extension by either retention of the initiating methionine of the recombinant protein in

376 CHEMOKINES, CC / RANTES (CCL5)

(a)

(b)

Arg47 Lys45

Arg44

(c)

(d)

Figure 1 Structure of RANTES. (a) Monomer; (b) dimer. (c) RANTES (red) is overlaid with six other chemokines (gray) from the CC (MCP-1 and MIP-1b), CXC (IL-8, NAP-2, and PF4) and CX3C (fractalkine) classes. (d) The BBXB motif forming the GAG binding site of RANTES on the 40s loop.

procaryotic expression systems, or by chemical coupling techniques, have shown that this significantly affects receptor activation capacity. Truncation of the first eight amino acids produced a protein that had acquired the ability to bind to CCR2, and extension of the N-terminal produced antagonists (see below). The one exception was the deletion of the first two amino acids producing the variant 3-68-RANTES; this molecule had enhanced activity for CCR5 whilst inhibiting CCR1-mediated events. RANTES has a second essential interaction, which is low-affinity binding to glycosaminoglycans (GAGs), a common feature amongst the chemokine family. The binding site for GAG has been identified for RANTES as being a BBXB motif on the 40s loop (Figure 1(d)). Furthermore the variant in which the basic residues in this motif were replaced with Ala residues was shown to have lost its ability to recruit cells in vivo, thereby proving conclusively that GAG binding is essential for chemokine activity in vivo.

Regulation of Production and Activity Genomic Localization

The RANTES locus has been mapped to chromosome 17q11.2–q12 using somatic cell hybrids and by in situ hybridization of the cDNA probe. Several other CC chemokine genes have also been mapped to this locus.

The RANTES gene spans 7.1 kb and comprises three exons of 133, 112, and 1075 bases respectively and two introns of approximately 1.4 and 4.4 kb. This exon–intron organization is similar to that reported for other CC chemokine family members. The locus has been sequenced from nine mammalian species which code for proteins with striking homology, where the identity ranges from 71% to 100%, with similarities ranging from 79% to 100% (Figure 1). Expression Pattern

RANTES expression was initially described as being restricted to antigen- or mitogen-activated T cells. However it turns out that RANTES is also expressed by a broad range of cell types after cellular activation by tumor necrosis factor alpha (TNF-a) and interleukin (IL)-1b including epithelial cells and fibroblasts. In addition RANTES is very strongly expressed by platelets. RANTES expression is a hallmark of inflammatory diseases and RANTES mRNA or protein has been detected in pathological specimens (disease tissue or fluids) from bronchoalveolar lavage (BAL) from asthmatics as well as many other inflammatory diseases (Table 1).

Receptors RANTES is one of the most promiscuous chemokines in that it binds to several receptors (Figure 2). It

CHEMOKINES, CC / RANTES (CCL5) 377

binds to three CC receptors with high affinity (CCR1, CCR3, and CCR5 through which it induces cellular recruitment) and to CCR4 with low affinity. In addition, it binds to the promiscuous nonsignaling receptors DARC and D6, which are thought to act as chemokine sinks. RANTES has the highest affinity for CCR1 and CCR5, binding with low nanomolar affinity, whilst the affinity for CCR3 has been reported to range from 1 to 100 nM, and that for CCR4 is micromolar. CCR1 and CCR5 are expressed

Table 1 Disease association for RANTES receptors Receptor

Disease indication

CCR1

Multiple sclerosis Rheutmatoid arthritis Organ transplant Asthma, rhinitis Nephritis

CCR3

Asthma Allergy

CCR5

Multiple sclerosis Rheumatoid arthritis Transplant Asthma Nephritis IBD Acquired immune deficiency syndrome (AIDS)

on T cells, immature dendritic cells, and monocyte/ macrophages; in the case of the latter, the expression of CCR1 is predominant on circulating monocytes, whereas CCR5 is upregulated when monocytes acquire the macrophage phenotype. Using specific small molecule inhibitors of CCR1 and CCR5, it has been shown that whilst both receptors can mediate transendothelial migration induced by RANTES, RANTES activation of CCR1 mediates cellular arrest whilst activation of CCR5 mediates cellular spreading. Its role on CCR3 and CCR4 is less clear, although high levels of RANTES expression in samples from asthmatic patients (see below) could imply that it assists in the recruitment of eosinophils through CCR3. RANTES signals through the classical chemoattractant receptor signaling pathway initiated by ligand-induced receptor activation, which results in the formation of a heterotrimeric G-protein complex at its cytoplasmic tail by recruitment of a membraneanchored Gbg heterodimer to the receptor-bound Ga subunit. The Gbg complex regulates the activation of phosphatidyl inositol-phospholipase Cb, catalyzing the breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG), which act synergistically in the mobilization of intracellular Ca2 þ . DAG and Ca2 þ activate various isoforms of protein kinase C, which

CXCL16 CXC13 CXCL12

D6

CCR6 XCL1 XCL2 CX3CL1

Constitutive: basal trafficking/homing

DARC

CCL27 CCL28

CXCL20

XCR1

CCL11 CCL7 CCL13

CCR8

CXCR5

CXCR4

CCL5 CCL5

CXCR6

CCL1

CX3CR1 CXCR1

CCR10

CCL25

Inducible: inflammatory

CCR9 CCR7

CCL19 CCL21

CXCR3

CCR5 CCL3 CCL4

CCL5 CCL8

CXCR2

CCR4 CCL17 CCL22

CCR1 CCR3 CCL5 CCL7 CCL11 CCL13

CCR2 CCL2 CCL8 CCL7 CCL13

CCL5

CXCL8 CXCL6 CXCL8 CXCL5 CXCL1 CXCL2 CXCL3 CXCL7

CXCL9 CXCL10 CXCL11

CCL3 CCL7

Figure 2 Chemokine receptors and their ligands. The high-affinity receptors to which RANTES/CCL5 binds are indicated as those that signal (red) and those that do not signal (black). Since RANTES has only been reported to interact with CCR4 with low (micromolar) activity it is not depicted as a CCR4 ligand in this figure.

378 CHEMOKINES, CC / RANTES (CCL5)

can phosphorylate many other downstream targets. The two major signaling pathways activated through the binding of chemokines are: (1) the phosphoinositide 3 kinase (PI3K) pathway, and studies with PI3Kg / mice have demonstrated that RANTES activity is severely hampered in the absence of this enzyme and (2) the mitogen-activated protein-kinase (MAPK) pathway, which is responsible for phosphorylating and activating downstream transcription factors that in turn regulate the synthesis of other inflammatory proteins, including chemokines.

Biological Function Since their identification, the importance of chemokines in cell migration has been demonstrated in both basal cell trafficking and in inflammation. An interesting contrast in the chemokine system exists between the receptors involved in basal trafficking (controlled by homeostatic chemokines), which tend to be specific, whilst those involved in inflammatory cell trafficking are shared by several chemokines (Figure 2). In inflammatory situations, expression of inducible or inflammatory chemokines is upregulated. Control of expression of these chemokines is under the temporal control of proinflammatory cytokines. Proinflammatory cytokines such as IL-1b, TNF-a, and interferon gamma (IFN-g) alone or in combination induce chemokine expression at sites of inflammation in nonlymphoid tissue. RANTES is an example of an inducible/inflammatory chemokine. Inflammatory chemokine receptors (including those

to which RANTES binds, that is, CCR1, CCR3, and CCR5) are upregulated on inflammatory cells following priming, which are then attracted to sites of inflammation. RANTES may potentially mediate the activation and trafficking of a range of immune cells including T cells, monocytes, basophils, eosinophils, natural killer cells, dendritic cells, and mast cells, all of which play a role in respiratory diseases (Figure 3). RANTES is often associated with inflammatory exudates and is predominantly secreted by CD8 þ T cells, epithelial cells, fibroblasts, and platelets, in which it is stored in alpha granules as densely packed aggregates. Much understanding of the chemokine system has come from studies using knockout mice. Despite its being one of the earliest identified and most widely studied chemokines, there are few reports in the literature describing the RANTES-deficient mouse. Published work indicates a role for RANTES in both normal T-cell function and in monocyte and T-cell recruitment in the contact hypersensitivity response. However no data with the RANTES / mice in airway inflammation are available. On the other hand there are numerous publications on studies of knockout mice for the various RANTES receptors, but again, none for CCR1 / or CCR5 / mice in airways inflammation. A role for CCR1 has been demonstrated in the murine model of multiple sclerosis, experimental allergic encephalomyelitis (EAE), CCR5-deleted mice are fully susceptible to EAE, surprisingly, as high levels of CCR5 expression are seen in multiple sclerosis patients. However, deletion of

Resting T cell

B cell

Activated T cell CCR1,3,(4),5

Eosinophil CCR1,3

NK cell CCR5

Basophil mast CCR3,(4)

Dendritic cell CCR1,5

Monocyte CCR1(5) Macrophage CCR5(1)

Neutrophil Figure 3 Leukocyte subsets. Leukocytes recruited by RANTES that play a role in respiratory disease are shown in red whilst those that are not recruited by RANTES are shown in black. The RANTES receptors found on each cell type are indicated.

CHEMOKINES, CC / RANTES (CCL5) 379

CCR3, which is the predominant chemokine receptor expressed by eosinophils, results in a 50–70% reduction in eosinophil accumulation in the airway lumen and the lung tissue after challenge. These data confirm the role of CCR3 in eosinophil recruitment to the lung during allergic airways inflammation. However, no protection was seen in the bronchial hyperreactivity to methacholine, which could be explained by the increase in lung tissue mast cells seen in this model and highlights the controversial role of the eosinophil in respiratory disease.

Role of RANTES in Respiratory Disease Increased RANTES expression, in combination with the presence of RANTES receptor expressing cells, has been associated with a variety of human inflammatory diseases. RANTES produced by lung epithelial cells has been identified as a major eosinophil chemoattractant in the BAL fluid of asthmatics. Additionally bronchial biopsies from patients with asthma show elevated RANTES mRNA expression, and levels of RANTES have been shown to reach a peak expression level approximately 4 h after allergen challenge in human subjects. Furthermore RANTES is a potent lymphocyte and monocyte chemoattractant acting via CCR1 and CCR5, and therefore contributes toward the recruitment of T cells and macrophages into the lungs of asthmatic patients. In line with its activity as a chemoattractant for T cells and eosinophils, RANTES has been observed at high levels in polyps from patients suffering from nasal polyposis in chronic hyperplastic sinusitis. Therapeutic Potential

The therapeutic potential of RANTES as an anti-inflammatory and anti-infective target has been shown by several approaches in animal models of disease. The use of neutralizing antibodies has clearly demonstrated the importance of this chemokine in several pulmonary pathologies. Airway obstruction in an experimental model of obliterative bronchiolitis and airway hyperreactivity in a respiratory syncytial viral infection model were both considerably ameliorated by treatment with neutralizing RANTES antibodies. Beneficial effects were also observed with anti-RANTES antibodies in acute lung allograft rejection and allograft transplant-induced fibrous airway obliteration as well as in cardiac allograft rejection. As mentioned above, the N-terminal region of RANTES is critical for receptor activation. In fact modification of this region has produced several variants with receptor antagonist properties. The most

widely studied is Met-RANTES, which is produced by the extension of the N-terminus by the initiating methionine when the human protein is produced recombinantly in Escherichia coli. The protein retains high affinity for human CCR1, CCR3, and CCR5 but is unable to cause receptor activation except in the case of CCR5, on which it has partial agonist activity. Pharmacological studies on the murine receptors surprisingly showed that whilst it also retains high affinity for murine CCR1 and CCR5, it is no longer able to compete for binding of Eotaxin/ CCL11 from murine CCR3. This variant has shown the beneficial effects of inhibiting RANTES receptors in many models, in the lung as well as other organs. In fact Met-RANTES was shown to be more effective than individual antichemokine antibody treatment in the ovalbumininduced airway inflammation model. This was interesting since it does not bind to murine CCR3 and yet still reduces eosinophil accumulation in the lungs, indicating that blockade of CCR1 and/or CCR5 may also be potential targets for asthma. In an extensive dose–response study in this model, the inhibition observed was inversely bell-shaped, for which a definitive explanation remains to be elucidated. However the lack of efficacy at high doses could be one of the reasons why it has not been developed for the clinic, in addition to the residual partial agonist activity, which was shown not to be the reason for the inverse bell-shaped dose–response. CCR1 has been shown to be crucial in the inflammatory response to the murine pneumonia virus, which has been used as a model for severe human respiratory syncytial virus disease. This model has highlighted the importance of inflammation to the pathogenesis of chronic disease, demonstrating that the inflammatory response remains active and acute even when virus replication ceases in response to appropriate antiviral therapy. Met-RANTES treatment prevented the inflammatory response to the virus and resulted in reduced morbidity and mortality when administered in conjunction with the antiviral agent ribavirin. These results highlight the interesting possibility of using chemokine antagonists in dual therapies. This had been previously demonstrated by using Met-RANTES in synergy with cyclosporin to prevent organ transplant rejection, at suboptimal doses of both agents. Another N-terminally modified RANTES variant, AOP-RANTES, provided several important insights into the inhibition of human immunodeficiency virus (HIV) infectivity. This variant was produced by chemical modification of the N-terminal in an attempt to improve the characteristics of Met-RANTES. Unexpectedly, this variant was even more potent on CCR5 than the parent WT-RANTES protein,

380 CHEMOKINES, CC / TARC (CCL17)

whilst retaining antagonistic properties on CCR1. It was found to be an extremely potent inhibitor of HIV infectivity, not due to its antagonistic properties on CCR5 but on the contrary due to its ability to drive internalization of CCR5, and moreover, to prevent the recycling of functional receptors to the cell surface, thereby removing the essential co-receptor. Furthermore, it provided evidence that infection of primary macrophages could be prevented, an issue that was much debated in the chemokine–HIV field since there were reports both of inhibition and enhancement of infection by R5 HIV strains (those that require CCR5 for cell entry). Despite a different mode of action, AOP-RANTES showed the same profile of inhibition of airways inflammation in mice. Yet another RANTES variant has provided novel insights into strategies that can effectively inhibit inflammation. As discussed above, mutation of the heparin binding site in RANTES abrogates its ability to recruit cells in vivo, but what was unexpected is that it was capable of inhibiting RANTES-mediated recruitment, even by the non-specific inflammatory agent thioglycollate. This inhibitory effect translated into a potent inhibitor of clinical symptoms in the murine model of multiple sclerosis, experimental autoimmune encephalomyelitis, EAE. Whilst heparin has been shown to reduce inflammatory symptoms both in human and in rodent disease models, including pulmonary inflammation, the effect was attributed to an effect on proinflammatory cytokines. Given the fact that the GAG mutant 44AANA47RANTES had anti-inflammatory properties, together with the fact that heparin could inhibit both RANTES- and thioglycollate-mediated cellular recruitment in vivo, its anti-inflammatory effect could also be attributed to the interference with the establishment of chemokine gradients. Again this variant was able to reduce the cellular infiltrate into inflamed lungs in mice. In summary, this single chemokine provides many therapeutic targets since it acts with high affinity on three receptors, all implicated in respiratory disease. Moreover, in view of the extensive knowledge of the protein, it provides several different approaches to inhibit its activity. See also: Chemokines. Interleukins: IL-5; IL-16. Platelets.

Further Reading Chvatchko Y, Proudfoot AE, Buser R, et al. (2003) Inhibition of airway inflammation by amino-terminally modified RANTES/ CC chemokine ligand 5 analogues is not mediated through CCR3. Journal of Immunology 171: 5498–5506.

Gerard C and Rollins BJ (2001) Chemokines and disease. Nature Immunology 2: 108–115. Handel TM, Johnson Z, Crown SE, Lau EK, and Proudfoot AEI (2005) Regulation of protein function by glycosaminoglycans as exemplified by chemokines. Annual Review of Biochemistry 74: 385–410. Hebert CA (ed.) (1999) Chemokines in Disease. Totowa, NJ: Humana Press. Johnson Z, Kosco-Vilbois MH, Herren S, et al. (2004) Interference with heparin binding and oligomerization creates a novel antiinflammatory strategy targeting the chemokine system. Journal of Immunology 173: 5776–5785. Johnson Z, Schwarz M, Power CA, Wells TN, and Proudfoot AEI (2005) Multi-faceted strategies to combat disease by interference with the chemokine system. Trends in Immunology 26: 268–274. Luster AD (1998) Chemokines: chemotactic cytokines that mediate inflammation. New England Journal of Medicine 338: 436–445. Mack M, Luckow B, Nelson PJ, et al. (1998) AminooxypentaneRANTES induces CCR5 internalization but inhibits recycling: a novel inhibitory mechanism of HIV infectivity. Journal of Experimental Medicine 187: 1215–1224. Power CA (2002) Knock out models to dissect chemokine receptor function in vivo. Journal of Immunological Methods. Proudfoot AEI (2002) Chemokine receptors: multifaceted therapeutic targets. Nature Reviews: Immunology 2: 106–115. Proudfoot AEI, Handel TM, Johnson Z, et al. (2003) Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. Proceedings of the National Academy of Sciences, USA 100: 1885–1890. Rothenberg ME (ed.) (2000) Chemokines in Allergic Disease. New York: Dekker. Schall TJ, Bacon K, Toy KJ, and Goeddel DV (1990) Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES. Nature 347: 669–671. Schall TJ, Jongstra J, Dyer BJ, et al. (1988) A human T cell-specific molecule is a member of a new gene family. Journal of Immunology 141: 1018–1025. Simmons G, Clapham PR, Picard L, et al. (1997) Potent inhibition of HIV-1 infectivity in macrophages and lymphocytes by a novel CCR5 antagonist. Science 276: 276–279.

TARC (CCL17) T L Ness, C M Hogaboam, and S L Kunkel, University of Michigan, Ann Arbor, MI, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Thymus and activation-regulated chemokine (TARC) was the first CC chemokine identified as a T-cell chemoattractant. It is known as CC chemokine ligand 17 (CCL17) according to the new nomenclature and shares most amino acid identity with CCL22. CCL17 expression is highest in the thymus, but can also be detected in other tissues such as the small intestine, colon, and lung. CCL17 is completely absent from the spleen. Dendritic cells are the predominant source of constitutive CCL17 production, whereas most immune cells and structural

CHEMOKINES, CC / TARC (CCL17) 381 cells are capable of inducible expression. CCL17 primarily acts through its receptor CCR4 as a chemoattractant of T-helper-2 (Th2) cells. However, CCR4 is also found on Th1 cells, regulatory T cells, natural killer (NK) T cells, NK cells, platelets, and macrophages. CCL17 inhibits classical macrophage activation and type 1 responses, which further drive type 2 immune responses. CCL17 is associated with several Th2-mediated respiratory diseases such as bronchial asthma, allergic rhinitis, pulmonary fibrosis, and eosinophilic pneumonia, and represents a potential target for treatment of these pathologic disorders.

Introduction Thymus and activation-regulated chemokine (TARC) was first identified and cloned from phytohemagglutinin-stimulated peripheral blood mononuclear cells by Imai and colleagues in 1996. TARC was one of four novel sequences isolated using an Epstein-Barr virus vector signal sequence trap. As its name indicates, TARC is constitutively produced in the thymus, but can also be detected in other tissues such as the colon, small intestine, and lung. It was the first CC chemokine identified that specifically interacted with a high-affinity receptor found predominantly on T cells, to a lesser degree on monocytes, but not on granulocytes. TARC acts as a powerful T cell chemoattractant and has been implicated in the pathogenesis of several Th2-mediated diseases. TARC was renamed CC chemokine ligand 17 (CCL17) according to the new nomenclature system for chemokines and chemokine receptors.

Structure While most CC chemokines are found on human chromosome 17 (mouse chromosome 11), CCL17 is

tightly linked to CX3CL1 and CCL22 on human chromosome 16q13 (mouse chromosome 8q). The CCL17 gene (2176 bp) comprises 4 exons and encodes a 94 amino acid preprotein with a 23 amino acid signal sequence. The CCL17 protein contains the four cysteine residues characteristic of all CC chemokines and shares the highest degree of homology with CCL22 (32% amino acid identity) (Figure 1). The secreted CCL17 protein is approximately 8 kDa and has a theoretical isoelectric point of 9.7. The tertiary monomeric structure of CCL17 is a threestranded antiparallel b-sheet flanked by a C-terminal helix, like other CC chemokines, although dimerization results in a more compact molecule than other CC chemokines. CCL17 has a greater distribution of basic charge on its surface, allowing it to bind more tightly to glycosoaminoglycans than most CC chemokines. This contributes to its ability to form a gradient within the extracellular matrix of endothelial cells to attract and immobilize receptorexpressing cells. Polymorphism screening of the human CCL17 gene identified two rare single nucleotide changes within exon 3 (2134C4T and 2037G4A) and a polymorphism in the promoter region (–431C4T). The promoter region contains nuclear factor kappa B (NF-kB) and signal transducer and activator of transcription 6 (STAT-6) recognition sites as well as the regulatory elements required for interleukin (IL)-4 and tumor necrosis factor alpha (TNF-a)-mediated induction. The –431C4T change alters the putative binding site for the transcription factor activator protein 4 and results in increased promoter activity in vitro. Individuals homozygous for the –431C4T

2176 bp

C>T

SP

(a)

CC

CX3CL1

CCL17

FracTARC (b) Figure 1 Sequence structure of CCL17. (a) The genomic sequence of human CCL17 (2176 bp) is comprised of four exons. Untranslated regions are depicted as hatched boxes. The locations of the signal peptide (SP), CC motif, and 431C4T polymorphism are indicated. (b) In the mouse, tissue-specific (brain and kidney) alternative splicing results in production of fracTARC, a transcript containing the signal peptide sequence of CX3CL1/fractalkine (red) and the entire coding region of CCL17 (blue) minus the first three N-terminal amino acids.


polymorphism have elevated systemic concentrations of CCL17 as well as an increased incidence of asthma. Murine CCL17 is unique in that its transcription is regulated not only from its own promoter, but also from the promoter of the closely linked CX3CL1 (fractalkine) gene. Alternative splicing results in expression of murine fracTARC, a protein containing the CX3CL1 signal sequence and the mature CCL17 protein. Processing of the signal sequence results in loss of the first three amino acids from the N-terminus, normally found in CCL17. Transcriptional regulation of CCL17 is tissue specific, as CCL17 is expressed in the thymus and lung while fracTARC can only be found in the brain and kidney. fracTARC has not been detected in human tissues thus far.

Regulation of Production and Activity Dendritic cells (DCs), specifically myeloid-related DCs (CD11c þ CD11b þ ), are responsible for constitutive production of CCL17 in most peripheral lymphoid and nonlymphoid organs. Immature monocyte-derived DCs (moDCs) express high levels of CCL17 that are increased several-fold upon maturation. CCL17 is inducible in most leukocytes including DCs, macrophages, mast cells, B cells, and naive T cells after exposure to various cytokines (IL-3, IL-4, IL-13, and granulocyte-macrophage colonystimulating factor (GM-CSF)), phytohemagglutinin, ovalbumin, or stimulation with Toll-like receptor agonists (i.e., lipopolysaccharide). Transforming growth factor beta (TGF-b) has been shown to act as a negative regulator of CCL17 production. Structural cells such as bronchial, epithelial, and endothelial cells, keratinocytes, fibroblasts, and smooth muscle cells are also a significant source of inducible CCL17. CCL17 is not found in the spleen, even after systemic bacterial infection or in vitro stimulation of cells from the spleen. The expression pattern of this chemokine accentuates its importance in mediating the attraction of effector cells to sites commonly exposed to infection and/or stimulation by foreign antigens, i.e., mucosa and lymph nodes (LNs).

Biological Function DCs fulfill a fundamental regulatory role linking both innate and adaptive immune responses. DCs act as professional antigen-presenting cells, transporting antigens to the draining lymph nodes (LNs), where they prime T-cells and initiate antigen-specific responses. DCs produce several constitutive and inducible cytokines/chemokines that are responsible for activating and recruiting specific T-cell

populations. CCL18 and CCL19 direct naive T cells into the T-cell zone, while CCL2, CCL3, CCL17, and CCL22 attract activated and memory T cells. CCL17 is a highly selective chemoattractant for Th2 cells and CLA þ skin-homing memory T cells (Figure 2). CCL17 expression is upregulated in the skin of patients with psoriasis, cutaneous delayed type hypersensitivity, and atopic dermatitis (AD) as well as in the epithelium of mice with AD-like disease. Serum levels of CCL17 are increased in AD patients and correlate with disease severity, eosinophil number, and soluble E-selectin. Intradermal injection of CCL17 in mice results in dose-dependent recruitment of CD4 þ lymphocytes into the skin. CCL17 regulates its own production from keratinocytes and stimulates transcription of IL-4 mRNA, further highlighting its importance in driving Th2 responses. When CCL17 is neutralized (genetic deletion or antibody neutralization), mice are more resistant to allograft rejection, contact hypersensitivity, bacteriainduced fulminant hepatitis, and antigen-specific asthmatic reactions. All improvements were associated with reduced effector T-cell recruitment and decreased Th2 cytokine production. Although CCL17 is foremost recognized as a Th2 cell chemoattractant, its receptor is also found on DCs, Th1 cells, CD25 þ T cells, natural killer (NK) cells, NK T cells, platelets, and macrophages. Th1 cells and CD4 þ NK T cells migrate in response to CCL17. Receptor expression on CD25 þ regulatory T cells suggests that CCL17 may serve as an important immunomodulatory mediator of innate and acquired immune responses. In vitro treatment with CCL17 induces Ca2 þ mobilization, aggregation, and granule release from platelets. Recently, the effects of CCL17 on macrophages has been the subject of increasing interest. Macrophages undergo differentiation comparable to T-cell maturation (Th1/Tc1 vs. Th2/Tc2). Resident macrophages that are classically activated (caMf or M1 macrophages) by stimulation with microbial products exhibit strong antimicrobial innate immune responses such as production of inflammatory cytokines/chemokines, expression of inducible nitric oxide synthase, efficient killing activity, and induction of a Th1 response. In contrast, IL-4 and IL-13 drive the development of alternatively activated macrophages (aaMf), characterized by expression of anti-inflammatory/immunoregulatory cytokines/ chemokines, increased mannose receptor expression, arginase production, and decreased killing activity. Individuals with a predominance of aaMf are more susceptible to infection. AaMf inhibit the generation of caMf via production of IL-10 and CCL17. This

CHEMOKINES, CC / TARC (CCL17) 383 Adaptive immune response Memory T cells

Innate immune response NK cells NK T cells

CD45RO

CCR8

Regulatory T cells Th2 cells

CCL17

CD25 CCR4

CLA

Skin homing T cells

DCs

Platelets

caM /M1

Figure 2 CCL17 regulates both innate and adaptive immune responses. CCL17 is centrally involved in the adaptive immune response (shown in blue circle) as a potent chemoattractant of CD4 þ Th2 cells (CLA þ skin homing, CD25 þ regulatory, and CD45RO þ memory T cells). CCR4 is the receptor for CCL17. CCR4 is also expressed on several innate immune cells (shown in yellow circle). Natural killer (NK) cells and dendritic cells (DCs) chemotax in response to CCL17. Evidence suggests that CCL17 utilizes both CCR4 and CCR8 on NK cells. CCL17 triggers granule release and aggregation of platelets in a CCR4-dependent manner. CCL17 inhibits classical activation of macrophages (caMf), thereby suppressing type 1 innate immune responses. Natural killer T cells (NK T cells) and DCs play a fundamental role linking both innate and adaptive immune responses (shown in the overlapping green region). DCs produce several cytokines/chemokines that activate and attract innate cells while also serving as professional antigen-presenting cells priming antigenspecific responses.

has been demonstrated in murine models of thermal injury and sepsis. Thus, CCL17 functions as an effective promoter of type 2 responses primarily through its chemoattraction of Th2 cells while modulating type 1 responses by suppressing caMf.

Receptors Initial studies demonstrated high-affinity binding of radiolabeled CCL17 to Jurkat cells; CXCL8 or other CC chemokines could not compete with this binding. CCL17-induced T-cell chemotaxis was pertussis toxin sensitive suggesting involvement of a G-protein-coupled receptor. In a series of transfection studies, CCR4 was identified as the primary receptor for CCL17. CC chemokine receptor 4 (CCR4) is found on a variety of cells (Figure 2). CCL17 and CCR4 have been implicated in the pathogenesis of many allergic diseases as they are central mediators involved in the homing of T cells to the skin. In addition to CCL17, CCL22 is the only other ligand known to bind CCR4. Despite the fact that they bind the same receptor, the different binding affinities, expression patterns, and kinetics of these two chemokines suggests important nonredundant roles in vivo. CCL22 binds CCR4 with higher affinity and is a more potent inducer of integrin-dependent

T-cell adhesion. CCR4 internalization is rapid following CCL22 binding, but delayed after exposure to CCL17. CCL22 desensitizes CCR4 þ T cells to CCL17, while CCL17 has no effect on CCL22 signaling. CCL22 is subject to serine protease degradation whereas CCL17 is a much more stable ligand. Considering the abundant expression of CCL17 (and not CCL22) by structural cells, it is very likely that CCL17 acts to specifically arrest CCR4high cells (i.e., Th2 cells) in circulation while CCL22 subsequently directs these cells within the affected tissue. CCR8 plays a role in activation, migration, and proliferation of lymphoid cells. Early binding and chemotaxis studies also identified CCR8 as a potential receptor for CCL1, CCL4, and CCL17. However, no intracellular Ca2 þ changes were induced upon ligation of CCL4 or CCL17. CCL17-induced chemotaxis was attributed to low levels of CCR4 expression, as at physiologically relevant concentrations, CCL17 was unable to bind, inhibit CCL1-binding, or chemoattract CCR8high-expressing cells. However, the receptor usage of CCL17 may be cell type dependent as CCL17 is able to compete with CCL1 for binding in IL-2-activated adherent NK cells. CCL1 and CCL22 only partially inhibit CCL17-induced responses suggesting that CCL17 is able to act through both CCR4 and CCR8 in these cells.


CCL17 also interacts with both chemokine clearance receptors, the Duffy antigen/DARC and the D6 receptor. The Duffy antigen, found on red blood cells, binds both CC and CXC chemokines, while D6 is expressed on lymphatic endothelial cells and is specific for inflammatory CC chemokines. While ligand binding does not produce a functional signal, it does induce rapid internalization of these ligand/receptor complexes, facilitating regulation of inflammatory responses and return to homeostatic levels.

CCL17 in Th2-Type Respiratory Diseases CD4 þ T cells play a fundamental role in the pathogenesis of bronchial asthma and other allergic respiratory diseases. Cytokines/chemokines are pivotal mediators of inflammation, Th2 cytokine production, eosinophil and T-cell recruitment, and airway hyperresponsiveness (AHR) observed in asthmatic patients. When moDCs are treated in vitro, cells from allergic patients upregulate CCL17 expression compared to similarly treated cells from nonallergic patients. CCR4 is detected on virtually all T cells from endobronchial biopsies of asthmatic patients following allergen challenge. CCL17 concentrations are higher in the serum, airway epithelium, and bronchoalveolar lavage (BAL) fluid from asthmatics after antigen challenge. Children suffering from acute and chronic asthma exhibit high levels of plasma CCL17, which directly correlate with the severity of the disease. Individuals homozygous for the – 431C4T CCL17 polymorphism have more peripheral eosinophilia, are more susceptible to antigen challenge, produce higher levels of allergen-specific immunoglobulin E (IgE), and have a higher incidence of atopic asthma. Blocking CCL17 with a neutralizing antibody attenuates several aspects of pulmonary inflammation (eosinophilia, T-cell recruitment, and Th2 cytokine production) and AHR in a murine model of ovalbumin-induced asthma. While mice deficient for the CCL17 receptor showed no protection in this model, the CCR4 / mice were protected against allergic airway inflammation initiated by chronic fungal challenge. These data clearly indicate that CCL17 plays a significant role in the inflammation and pathogenesis observed in asthmatic patients. In addition to asthma, CCL17 is centrally involved in the pathogenesis of several respiratory diseases. Allergic rhinitis is a condition characterized by eosinophil, mast cell, and Th2 cellular infiltration into the nasal mucosa. The epithelium and mononuclear cells in the nasal mucosa of allergic patients express increased levels of CCL17, compared to nonallergy patients. CCL17 is significantly increased in BAL

fluid of patients with eosinophilic pneumonia, an inflammatory lung disorder often caused by fungal or helminth infections or drug reactions. Concentrations of CCL17 directly parallel expression of the Th2 cytokines IL-5 and IL-13 and the number of CCR4 þ CD4 þ T cells in the BAL fluid. Inflammatory cell recruitment is central to the pathogenesis of interstitial lung disease. In murine bleomycin-induced pulmonary fibrosis and rat radiation pneumonitis models, CCL17 and CCR4 are upregulated in the lung during the disease. CCL17 was localized to epithelial cells (bleomycin model) or alveolar macrophages (radiation model) in the lung and CCR4 was predominantly found on lung macrophages as well as lymphocytes (only in the radiation model). Epithelial cells were also a prominent source of CCL17 in idiopathic pulmonary fibrosis patients. Neutralization of CCL17 resulted in significantly fewer T cells, macrophages, NK cells, and neutrophils recruited into the BAL fluid, as well as reduced fibrosis. In a murine Th2-mediated lung granuloma model, CCL17 neutralization decreased granuloma size and CCR4 þ cell recruitment and increased overall cytokine/ chemokine production. No CCL17- or CCR4-specific compounds have been developed for use in human trials. In Th2mediated respiratory diseases, anti-allergic and antiinflammatory agents result in decreased CCL17 expression, Th2 cell recruitment, CCR4 expression, eosinophilia, IgE production, and type 2 cytokine production, further highlighting the importance of CCL17 in Th2-mediated pathogenesis. See also: Allergy: Overview; Allergic Reactions; Allergic Rhinitis. Asthma: Overview; Acute Exacerbations. Bronchoalveolar Lavage. Chemokines. Dendritic Cells. Dust Mite. Endothelial Cells and Endothelium. Epithelial Cells: Type I Cells; Type II Cells. Fibroblasts. G-Protein-Coupled Receptors. Interleukins: IL-4; IL-10; IL-13. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Monocytes; T cells; Pulmonary Macrophages. Platelets. Tumor Necrosis Factor Alpha (TNF-a).

Further Reading Alferink J, Lieberam I, Reindl W, et al. (2003) Compartmentalized production of CCL17 in vivo: strong inducibility in peripheral dendritic cells contrasts selective absence from the spleen. Journal of Experimental Medicine 197(5): 585–599. Belperio JA, Dy M, Murray L, et al. (2004) The role of the Th2 CC chemokine ligand CCL17 in pulmonary fibrosis. Journal of Immunology 173(7): 4692–4698. Chvatchko Y, Hoogewerf AJ, Meyer A, et al. (2000) A key role for CC chemokine receptor 4 in lipopolysaccharide-induced endotoxic shock. Journal of Experimental Medicine 191(10): 1755– 1764.

CHEMOKINES, CC / TECK (CCL25) 385 D’Ambrosio D, Albanesi C, Lang R, et al. (2002) Quantitative differences in chemokine receptor engagement generate diversity in integrin-dependent lymphocyte adhesion. Journal of Immunology 169(5): 2303–2312. Imai T, Yoshida T, Baba M, et al. (1996) Molecular cloning of a novel T cell-directed CC chemokine expressed in thymus by signal sequence trap using Epstein–Barr virus vector. Journal of Biological Chemistry 271(35): 21514–21521. Inngjerdingen M, Damaj B, and Maghazachi AA (2000) Human NK cells express CC chemokine receptors 4 and 8 and respond to thymus and activation-regulated chemokine, macrophage-derived chemokine, and I-309. Journal of Immunology 164(8): 4048–4054. Jakubzick C, Wen H, Matsukawa A, et al. (2004) Role of CCR4 ligands, CCL17 and CCL22, during Schistosoma mansoni egginduced pulmonary granuloma formation in mice. American Journal of Pathology 165(4): 1211–1221. Katakura T, Miyazaki M, Kobayashi M, Herndon DN, and Suzuki F (2004) CCL17 and IL-10 as effectors that enable alternatively activated macrophages to inhibit the generation of classically activated macrophages. Journal of Immunology 172(3): 1407– 1413. Katoh S, Fukushima K, Matsumoto N, et al. (2003) Accumulation of CCR4-expressing CD4 þ T cells and high concentration of its ligands (TARC and MDC) in bronchoalveolar lavage fluid of patients with eosinophilic pneumonia. Allergy 58(6): 518–523. Kawasaki S, Takizawa H, Yoneyama H, et al. (2001) Intervention of thymus and activation-regulated chemokine attenuates the development of allergic airway inflammation and hyperresponsiveness in mice. Journal of Immunology 166(3): 2055–2062. Mariani M, Lang R, Binda E, Panina-Bordignon P, and D’Ambrosio D (2004) Dominance of CCL22 over CCL17 in induction of chemokine receptor CCR4 desensitization and internalization on human Th2 cells. European Journal of Immunology 34(1): 231–240. Schuh JM, Power CA, Proudfoot AE, et al. (2002) Airway hyperresponsiveness, but not airway remodeling, is attenuated during chronic pulmonary allergic responses to Aspergillus in CCR4 / mice. FASEB Journal 16(10): 1313–1315. Vestergaard C, Deleuran M, Gesser B, and Larsen CG (2004) Thymus- and activation-regulated chemokine (TARC/CCL17) induces a Th2-dominated inflammatory reaction on intradermal injection in mice. Experimental Dermatology 13(4): 265–271.

TECK (CCL25) K F Buckland and C M Hogaboam, University of Michigan, Ann Arbor, MI, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Thymus-expressed chemokine (TECK) is a CC chemokine first identified in 1997. The sequence of TECK/CCL25 was selected by comparison of random sequencing products to previously described molecular sequences of murine CC chemokines. CCL25 is expressed predominantly in the thymus and its function is associated with T-cell development. In addition, CCL25 is highly expressed in the small intestine and has a pivotal role in the selective recruitment of lymphocytes to the epithelial lining of the small intestine. The function of CCL25 is comparable to

that of the lymphoid chemokines, CCL17, CCL19, CCL21, and CXCL12. CCL25 mRNA was also detected in murine pro-Tcells, a population of noncommitted intrathymic T-cell progenitor cells. Detection of mCCL25 mRNA in murine fetal thymus further supported early hypotheses that the role of CCL25 was early in thymic development. This lymphoid tissue chemokine is important in the migration, localization, and maturation of immature double positive thymocytes. In addition, CCL25 participates in the development and gut-homing of T-cell subsets and localization of immunoglobulin A (IgA)-secreting plasma cells within the gut associated lymphoid tissue. CCL25 exerts these functions primarily via binding of the CC chemokine receptor CCR9.

Introduction Thymus-expressed chemokine (TECK) is a CC chemokine that was first identified by Vicari and colleagues in 1997. This discovery was achieved by analysis of a murine cDNA library from RAG-1 deficient mice in experiments designed to identify novel genes involved in T-lymphocyte development. Initial analysis of the expression of the murine TECK transcript revealed expression predominantly in the thymus, hence thymus-expressed chemokine or TECK. It has since been designated as CCL25 according to the novel chemokine and chemokine receptor nomenclature.

Structure Following identification of murine CCL25, homologous genes were identified in human, rat, and hamster genomes. The nucleotide homology between human (hCCL25) and murine (mCCL25) transcripts of CCL25 is 71%. The amino acid sequence of hCCL25 is described in Figure 1. The unprocessed precursor is 150 amino acids long and has a molecular weight of 16 339 Da. Alternative splicing can yield two isoforms of hCCL25. Isoform 2 is an antagonist of isoform 1. Although CCL25 contains the conserved cysteine motif, which grants membership of the CC chemokine family, the structural relationship of CCL25 to other CC chemokines is distant and its homology low. Greatest amino acid identity is shared with CCL17. The percentage protein identity shared between CCL25 and some of the most closely related chemokines is outlined in Table 1. CCL25 displays differences from other chemokines even at the gene level. The hCCL25 gene is located on chromosome 19p13.2, not chromosome 17 as would be expected. This region is syntenic with mouse chromosome 8, where the mCCL25 gene has been mapped, while the genes encoding most chemokines are clustered on mouse chromosome 11.

386 CHEMOKINES, CC / TECK (CCL25) MNLWLLACLVAGFLGAWAPAVHTQ∗GVFEDCCLAYHYPIGWAVLRRAWTY IQEVSGSCNLPAAIFYLPKRHRKVCGNPKSREVQRAMKLLDARNKVFAKLHH NMQTFQAGPHAVKKLSSGNSKLSSSKFSNPISSSKRNVSLLISANSGL Figure 1 The amino acid sequence structure of CCL25 is given using the accepted standard lettering for amino acids. The asterisk ( * ) denotes the putative cleavage site for the signal peptide. Conserved double cysteines are present at positions 30 and 31. Disulfide bonds are predicted between cysteines at positions 30–58 and 31–75, highlighted in blue. Alternative splicing can occur at position 65 or 150, highlighted in red. Amino acids 1–23, highlighted in green, from a potential FT chain, a site for posttranslational modification. The sequence consisting of amino acids 24–150 is known to yield small inducible protein A25, black text.

Table 1 Percentage amino acid identity between CCL25 and other chemokines mCCL25 hCCL25 hCCL17 mCCL3 mCCL4 mCCL7 mCCL12 mCCL11

hCCL25

49.3 28.7 23.9 23.9 22.7 23.1 22.7

Regulation of Production and Activity CCL25 synthesis is highly regulated, as only a few specific cell populations are able to secrete this chemokine. Murine CCL25 mRNA is expressed at significant levels only in the thymus and small intestine, although low levels of mRNA were also detected in liver, brain, testis, and pro-T cells. The high thymic expression was revealed to be care of major histocompatibility complex (MHC) II þ CD11c þ thymic dendritic cells (DCs). In contrast, CCL25 expression is absent from CD11c thymic DCs and neither bone marrow nor naive splenic DCs express CCL25. Thymic endothelium also expresses CCL25 at low levels, while endothelial cells are a major site of CCL25 expression in the small intestine. Both the production and the responses to CCL25 can be enhanced by antigen stimulation. Lipopolysaccharide stimulation can induce CCL25 expression in the spleen in vivo, while responsiveness of thymocytes to CCL25 is enhanced following T-cell receptor (TCR) stimulation and in CD69 þ thymocyte subsets. Thus costimulatory conditions may be required for CD8 þ T cells to retain CCL25 receptor expression and effectively migrate to the intestinal lamina propria.

Biological Function Chemokines such as CCL25 are a key component of the system of tissue-restricted recirculation of memory and effector lymphocytes. CCL25 expression in

human and mouse thymus was localized to thymic DCs. In contrast, stimulated and unstimulated human monocyte-derived DCs and bone marrowderived cells all lacked CCL25 expression. CCL25 mRNA was also detected in murine pro-T-cells, a population of noncommitted intrathymic T-cell progenitor cells. Alternatively these cells can deviate from T-cell lineage to become natural killer (NK) cells or DCs. Detection of mCCL25 mRNA in murine fetal thymus supported early hypotheses that the role of CCL25 was early in thymic development. mCCL25 is chemotactic for murine activated monocytes, DCs, thymocytes, and a human lymphocyte cell line, THP-1, following their activation with interferon gamma (IFN-g). In contrast CCL25 was inactive on neutrophils, bone marrow cells, splenic, and peripheral blood lymphocytes or NK cell-enriched populations in vitro. Further none of these was recruited following intraperitoneal injection of CCL25 in vivo. Chemokines such as CXCL12, CCL3, CCL21, and CCL25 are involved in the selection processes allowing T-cell progenitors (pro-T cells) to progress into functional single positive T cells. In the developing thymus maturing thymocytes migrate from the cortex to the medulla. Early in embryogenesis CCL25, CXCL12, and CCL21 are expressed but not the related chemokines CCL17, CCL19, or CCL22. CCL25 expression has been localized to MHC II þ N418 þ DCs in the thymic medullary stroma. This region of the thymus is where single positive thymocytes emigrate from, into the bloodstream. CCL25 induces chemotaxis of immature CD4 þ CD8 þ double positive and mature single positive CD4 þ or CD8 þ human thymocytes. Thus CCL25 is thought to induce chemotaxis of double positive thymocytes from the cortex to the medullary region of the thymus and also to regulate localization of single positive thymocytes within the medulla. Through the chemoattraction of thymocytes and macrophages, CCL25 produced by thymic DCs may be the driving chemokine in achieving the colocalization of DCs with thymocytes and macrophages required for clonal deletion of self-reactive thymocytes. However, CCL25 is not solely responsible

CHEMOKINES, CC / TECK (CCL25) 387

for chemotaxis of thymocytes, which can also respond to other chemokines including CXCL12. The specificity of CCL25 for particular thymocyte subsets is of great interest in further elucidating its function. Lymphocyte recirculation is tightly regulated by the coordinated expression of chemoattractant receptors and adhesion molecules. This ensures that lymphocytes, which have previously encountered antigen, home to the appropriate site in which antigen exposure may reoccur. CCL25 is highly expressed in the epithelial lining of the small intestine and strongly associated with intestinal homing of particular lymphocyte populations to this antigenrich site. Importantly, CCL25 expression is weak or absent in other segments of the intestinal tract including colon and stomach, which supports the high specificity of lymphocyte trafficking. This restricted expression profile also separates the function of CCL25 from CCL17, another CC chemokine that is highly expressed in the thymus and small intestine but in contrast to CCL25, is also expressed in lung, colon, and activated mononuclear leukocytes. The CCL25 receptor is expressed only by discreet subsets of memory CD4 þ and CD8 þ lymphocytes that also express the intestinal homing adhesion molecule a4b7 but not by other systemic memory populations. In the intestine CCL25 is expressed in CD11c þ dendritic stromal cells, and c-kit þ Lin bone marrowderived cells chemotax toward CCL25. Thus CCL25 has a role in the formation of gut cryptopatches, an essential process in the generation of thymus-independent intraepithelial lymphocytes. CCL25 also has an important role in B-cell homing to the gut. CCL25 is abundantly expressed in epithelial cells lining the small intestine and is a potent and selective chemoattractant for immunoglobulin A (IgA)-secreting plasma cells. Oral antigens induce effector/memory cells that express essential receptors for intestinal homing, namely the integrin a4b7 and CCR9, the receptor for CCL25. This imprinting of gut tropism is mediated by DCs from Peyer’s patches in a positive feedback mechanism. Similar to T-cell development, bone marrow pre-pro B cells also migrate toward CCL25 while more mature bone marrow-derived B cells do not. Further, CCR9 / mice had a reduction in pre-pro B-cell numbers. CCR9/CCL25 interaction provides a cell survival signal in certain cell populations. CCL25 selectively rescues T cells but not thymocytes from tumor necrosis factor alpha (TNF-a)-induced apoptosis via activation of livin, a member of the inhibitor of apoptosis (IAP) family. However, antibody neutralization of CCL25 did not prevent repopulation of fetal thymus with T-cell precursors. Anti-CCL25

antibody treatment in 2–4-week-old mice led to a significant reduction in the total number of CD8 þ gd and ab in the intraepithelial lymphocyte compartment demonstrating an important role for CCL25 in the development of these cell populations. Further, a reduction in TCRgd þ numbers in the intestinal mucosal epithelium correlated with an increased number present in lymph nodes and spleen in CCR9 / mice supporting the requirement for CCL25/CCL25 receptor for homing of TCRgd þ intraepithelial lymphocytes. Emergence of double positive thymocytes and TCRab þ cells during embryogenesis was also delayed by approximately 1 day and pre-pro B-lymphocyte numbers were reduced. Therefore CCL25 has differential functions across separate cell types. Immature double positive thymocytes use CCL25 for differentiation, homing, development, maturation, and selection. Mature single positive thymocytes, T and B cells, use CCL25 for localization and cell homeostasis, and to resist apoptosis. In addition malignant cells have been shown to use CCL25 for inappropriate proliferation and resistance to apoptosis as well as infiltration.

Receptors CC chemokine receptor 9 (CCR9) is a receptor for CCL25. This G-protein-coupled receptor was additionally identified and named as CCR10, D6, and GPR-9-6 but these are in fact the same receptor and are henceforth known as CCR9. CCL25 selectively and efficaciously binds CCR9 but CCR9 is not selective for CCL25 but also binds CCL2–5, CCL7–8, and CCL12–13. CCR9 is highly expressed in the thymus and by T-lymphocyte subsets found in the small intestine. Expression has been localized to thymocytes and DCs in the thymus, and intraepithelial lymphocytes and lamina propria lymphocytes in the small intestine. Upregulation of CCR9 occurs during intraepithelial lymphocyte precursor differentiation allowing responsiveness to CCL25. CCR9 expression is at least several-fold greater in thymus than in spleen, bone marrow, lymph node, liver, or peripheral blood leukocytes. Activation of CCR9 leads to phosphorylation of glycogen synthase kinase 3b and forkhead transcription factor. This provides a cell survival signal. CCR9 also signals via activation of pkb/Akt, PI3-K, and mitogen-activated protein kinase (MAPK) but does not require MAPK for activation of chemotaxis. CCR9 is highly expressed by intestinal melanoma cells and abhorrent expression of CCR9 is associated with metastasis to the small intestine. CCR9/CCL25 play an important role in T-cell progenitor migration within the thymus. Immature

388 CHEMOKINES, CC / TECK (CCL25) CCR9 DN3

= GPCR

Subcapsular zone

DN4 pDP

= Thymocyte

DN2 DP CCL25 Thymocyte generation

DN1

Cortex 69+ SP

DC

Medulla SP

SP

Thymus Migration/ circulation

CCR9

CCX-CKR?

CCR11 TCR

Homing and expansion

EC CCL17

CD4 SP

CCR4

Immunity balance & regulation

DC

CCL25 CCR9 DC

Lung

SP

TCR CD8 SP

TCR SP

Small intestine

Figure 2 CCL25 is important in the migration, localization, and maturation of. immature CD4 þ CD8 þ double positive thymocytes and TCRgd þ T cells. In addition, CCL25/CCR9 participate in the migration and development of intraepithelial lymphocytes of the small intestine. The role of an additional receptor for CCL25, CCX-CKR, is as yet unknown but it is highly expressed in the lung. DN, double negative thymocyte; DP, double positive thymocyte; GPCR, G-protein-coupled receptor; SP, single positive thymocyte; TCR, T-cell receptor.

double negative thymocytes do not express CCR9 but CCR9 is highly expressed in double positive thymocytes. Single positive CD8 þ thymocytes continue to express CCR9 as they mature into naive T cells and migrate out of the thymus. CCL25 induces migration of immature double positive and mature single positive thymocytes via CCR9. CCL25 is also involved in the development of ab and gd T-cell lineages. Approximately 50% of TCRgd þ thymocytes express CCR9 and are responsive to CCL25 compared to 3% of TCRgd double negative cells. In TCRgd þ cells CCL25 response predominates over CXCL12. Circulating TCR gd þ T cells and CD8 þ T cells from lymph node, spleen, Peyer’s patches, and small intestine express CCR9. Conversely CD4 þ thymocytes have lost expression of CCR9 and this correlates with a lack of CCR9 on CD4 þ T cells in secondary lymphoid organs. The expression of chemokine receptors selective for intestinal trafficking is further supported by

upregulation of expression of a4b7, an intestinal homing integrin. Only Peyer’s patch DCs are able to induce high-level expression of a4b7 and response to CCL25 sufficient for gut homing of T cells. In correlation, CCR9 expression is maintained following activation of CD8 þ ab lymphocytes, with ovalbumin and lipopolysaccharide, in mesenteric lymph nodes but is rapidly downregulated on the same lymphocyte subsets if activated in the peripheral lymph nodes. Circulating CCR9 þ T cells that lack a4b7 are likely to have only recently exited the thymus. Surprisingly, gene deletion of CCR9 had no major effect on intrathymic T-cell development. Functional redundancy revealed by CCR9 / mice may be due to CCL25 binding by CCX-CKR.

CCL25 in Respiratory Diseases CCL25 expression is high in the thymus where its function is associated with T-cell development, and

CHEMOKINES, CC / TECK (CCL25) 389

in the small intestine where it has a pivotal role in the selective recruitment of lymphocytes to the epithelial lining. The functional requisite for CCL25/CCR9 in T-cell trafficking is not fully understood as mCCL25 also binds mCCX-CKR, a receptor expressed throughout the body and abundantly in the lung. The affinity of this receptor for CCL25 is high but the efficacy of this interaction is low. As in the mouse, binding of human CCL25 to CCX-CKR also fails to elicit functional responses. However significant diversity between human and murine CCX-CKR biology has hampered investigation of the in vivo function of this ligand–receptor interaction. CCL25 mRNA has been detected in the tonsils of humans, mice, and pigs. Mucosal sites such as the intestinal and respiratory epithelium present highly specialized challenges to the immune system as they are constantly exposed to potential pathogens. The palatine and nasopharangeal tonsils produce IgA and may play a similar role in aerodigestive tract as the Peyer’s patches of the gastrointestinal tract. Leukocytes are central in the pathophysiology of infectious and allergic respiratory diseases. Chemokines mediate the migration and activation of lymphocytes in normal and inflammatory conditions. CCL25 is known to have roles in both primary and secondary lymphoid organs, the thymus and small intestine, respectively. These different lymphoid organs and other immunological sites such as the thymus, spleen, small intestine, lymph nodes, and intestinal and respiratory epithelium can be discussed as separate compartments. Indeed, each has a specific role in lymphocyte trafficking. However, therapeutic strategies must consider that the immune system functions as a highly integrated system in vivo. Any imbalance or dysregulation, of for instance a chemokine, for example, CCL25, in one apparently isolated tissue site can be cause or effect (or exacerbation) of disease at a distant site. Despite the lack of expression of CCL25 in the lung this chemokine may still have an important contribution to the immunological component of certain respiratory diseases due to its pivotal role in thymocyte development and trafficking of these T-lymphocyte progenitor cells (Figure 2). CCL25- or CCR9-selective compounds are unlikely to be useful acute therapy in respiratory

diseases. However, CCR9/CCL25 expression or responsiveness may prove a good phenotypic marker perhaps for postimmunological therapy assessment of thymic involution and atrophy. Indeed immunodeficient states caused by chemotherapy or following respiratory infection may be managed better for the long term by stimulating thymic reactivation, perhaps involving increased CCL25/CCR9 activity. See also: Chemokines. Chemokines, CC: TARC (CCL17). Chemokines, CXC: CXCL12 (SDF-1). Dendritic Cells. Endothelial Cells and Endothelium. G-Protein-Coupled Receptors. Leukocytes: T cells.

Further Reading Bowman EP, Kuklin NA, Youngman KR, et al. (2002) The intestinal chemokine thymus-expressed chemokine (CCL25) attracts IgA antibody-secreting cells. Journal of Experimental Medicine 195: 269–275. Gosling J, Dairaghi DJ, Wang Y, et al. (2000) Cutting edge: identification of a novel chemokine receptor that binds dendritic celland T cell-active chemokines including ELC, SLC, and TECK. Journal of Immunology 164: 2851–2856. IUIS/WHO Subcommittee on Chemokine Nomenclature (2003) Chemokine/chemokine receptor nomenclature. Cytokine 21(1): 48–49. Kunkel EJ, Campbell DJ, and Butcher EC (2003) Chemokines in lymphocyte trafficking and intestinal immunity. Microcirculation 10: 313–323. Mora JR, Bono MR, Manjunath N, et al. (2003) Selective imprinting of gut-homing T cells by Peyer’s patch dendritic cells. Nature 424: 88–93. Nomiyama H, Amano K, Kusuda J, et al. (1998) The human CC chemokine TECK (SCYA25) maps to chromosome 19p13.2. Genomics 51: 311–312. Uehara S, Song K, Farber JM, et al. (2002) Characterization of CCR9 expression and CCL25/thymus-expressed chemokine responsiveness during T cell development: CD3(high)CD69 þ thymocytes and gammadeltaTCR þ thymocytes preferentially respond to CCL25. Journal of Immunology 168: 134–142. Vicari AP, Figueroa DJ, Hedrick JA, et al. (1997) TECK: a novel CC chemokine specifically expressed by thymic dendritic cells and potentially involved in T cell development. Immunity 7: 291–301. Youn BS, Kim CH, Smith FO, et al. (1999) TECK an efficacious chemoattractant for human thymocytes, uses GPR-9-6/CCR9 as a specific receptor. Blood 94: 2533–2536. Zaballos A, Gutierrez J, Varona R, et al. (1999) Cutting edge: identification of the orphan chemokine receptor GPR-9-6 as CCR9, the receptor for the chemokine TECK. Journal of Immunology 162: 5671–5675.

390 CHEMOKINES, CXC / CXCL12 (SDF-1)

CHEMOKINES, CXC Contents

CXCL12 (SDF-1) IL-8 CXCL9 (MIG) CXCL10 (IP-10) CXCL1 (GRO1)–CXCL3 (GRO3)

CXCL12 (SDF-1) R M Strieter and B N Gomperts, The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA

sequence is highly conserved among species with 99% homology between mouse and humans. This highlights the importance of this molecule in development and regeneration.


Abstract SDF-1 (stromal cell-derived factor-1) is also known as CXCL12, and is a CXC chemokine that binds to the CXC chemokine receptor, CXCR4. It is widely expressed and highly conserved having 99% homology with lower vertebrates, suggesting that it is important in primordial development. Two splice variants, SDF-1a and SDF-1b, have been identified and determined to have different regulation and functions. The SDF-1/CXCR4 complex results in activation of a variety of signal transduction pathways. SDF-1 plays a role in several functions, including trafficking of hematopoietic and nonhematopoietic adult stem cells, development of the central nervous system, vasculature and hematopoietic cells, cell motility, chemotaxis and adhesion, and cell proliferation and survival. It also inhibits lymphotropic strains of HIV-1 to enter into CXCR4 þ cell lines. SDF-1 plays a critical role in lung repair after injury through recruitment of circulating mesenchymal progenitor cells such as fibrocytes and progenitor epithelial cells. SDF-1 is also an important molecule in invasion and organ-specific metastasis of cancer; hence, SDF-1 is one of the most important known chemokines.

Introduction Stromal cell-derived factor-1 (SDF-1) was discovered independently by three different groups and has been known as pre-B-cell growth-stimulating factor/ stromal cell-derived factor-1 (PBSF/SDF-1) or TPA repressed gene 1 (TPAR1). SDF-1 is referred to as CXCL12 according to the consensus chemokine nomenclature. There are two alternatively spliced variants of CXCL12, CXCL12-a (68 residues) and CXCL12-b (72 residues), which have been shown to have different functions. A new isoform, CXCL12-g, was recently described. Subsequent studies show CXCL12 to be a very efficient chemoattractant for lymphocytes and monocytes. CXCL12 is constitutively expressed in a wide range of tissues and the CXCL12

Structure Although genes encoding other members of the chemokine family are localized on chromosome 4q or 17q, the human CXCL12 gene maps to chromosome 10q. cDNA of CXCL12-a and CXCL12-b encode proteins comprised of 89 and 93 amino acids, respectively. The nucleotide sequence contains a single open reading frame of 267 nucleotides encoding the 89 amino acid polypeptide. CXCL12 contains four conserved cysteine residues of the chemokine family. It does not contain the ELR motif, although its role in angiogenesis is controversial. The genomic structure of the CXCL12 gene reveals that human CXCL12-a and CXCL12-b are encoded by a single gene that arises by alternative splicing. The strong evolutionary conservation and unique chromosomal localization of the CXCL12 gene suggest that CXCL12-a and CXCL12-b may have important functions distinct from those of other members of the chemokine family. The N-terminus appears to be critical for the activity of CXCL12, in that peptides corresponding to the N-terminal 9 residues of CXCL12 have been shown to bind to CXCR4 and to induce intracellular calcium and chemotaxis in T lymphocytes. The individual peptides have similar activities to CXCL12 but are less potent. A dimer of CXCL12 amino acid residues 1–9 demonstrates enhanced activity. The Nterminal peptides can act as CXCR4 agonists or antagonists. Nuclear magnetic resonance spectroscopy demonstrates that CXCL12 is a monomer with a disordered N-terminal region (residues 1 8), and differs from other chemokines in the packing of the hydrophobic core and surface charge distribution (Figure 1).

CHEMOKINES, CXC / CXCL12 (SDF-1) 391

5′ flanking region

Exon 1

Exon 2

Exon 3

Exon 4

GCGCG 5′

3′ CXCL12- CXCL12-

(a)

∗∗

Residues 123456789

89 amino acids

Isoform splice site

93 amino acids

Refresh motif 12-17 C-terminus

N-terminus

9 amino acid binding site

Loop region docking site

Serum cleavage 67

Residues 3 N-terminus

C-terminus

CXCL12-

72

Residues 3 N-terminus

CXCL12-

C-terminus

(b) Figure 1 (a) Schematic cartoon of the CXCL12 gene demonstrating the approximate splice site for CXCL12-a and CXCL12-b. (b) Line diagram of CXCL12 structure with binding and docking sites and sites of cleavage to create CXCL12-a and CXCL12-b isoforms. *Only Lys-1 and Pro-2 are directly involved in receptor activation.

Regulation of Production and Activity Synthesis

CXCL12 is produced by a wide variety of cell types. mRNA for CXCL12 has been found in lung, brain, heart, liver, kidney, spleen, lymph nodes, stomach, gastrointestinal tract, muscle, lung-derived fibroblasts, and especially high levels are found in bone marrow stromal cells, especially those that support B cell lymphopoiesis. CXCL12 is regulated through complicated interactions among cytokines/growth factors, chemokines, and other regulatory factors. For example, CXCL12 gene expression is regulated by the transcription factor hypoxia-inducible factor1 (HIF-1) in endothelial cells, resulting in selective in vivo expression of CXCL12 in ischemic tissues in direct proportion to reduced oxygen tension. Interferon gamma significantly reduces CXCR4 expression and SDF-1-induced cell migration and proliferation of CXCR4-positive cells. Processing

CXCL12-a and CXCL12-b are secreted as full-length molecules. When exposed to human serum, fulllength CXCL12-a (1–68) undergoes processing first at the C-terminus to produce CXCL12-a (1–67) and

then at the N-terminus to produce CXCL12-a (3–67). By contrast, full-length CXCL12-b (1–72) is processed only at the N-terminus to produce CXCL12-b (3–72). Serum processing of CXCL12-a at the C-terminus reduces the ability of the polypeptide to bind to heparin and to stimulate B cell proliferation and chemotaxis. The additional processing at the N-terminus renders both forms of CXCL12 unable to bind to heparin and activate cells. The differential processing of CXCL12-a and CXCL12-b allows precise regulation of CXCL12’s biologic activity (Figure 1). The processing of CXCL12-g has not been determined. Activation

The binding of CXCL12 to its receptor, CXCR4, results in the activation of several signaling pathways. It has been suggested that after binding to CXCR4, CXCL12 initially triggers dimerization of the receptor. In addition, there is evidence that CXCR4 has to be included in membrane lipid rafts in order to more efficiently interact with other surface receptors, downstream proteins of signal transduction pathways, and therefore to induce chemotaxis. CXCR4 activation leads to dissociation of the heterotrimeric protein complex (Gabg) to a and bg subunits that


CXCL12 Plasma membrane Integrins

CXCR4 Src ptk

JAKS P

Gi Gi

Fak

P13Ky

Pax

src PDK1

PIP3

Ras mTOR

Akt

PTEN SHIP

Rho

Rac

cdc42

Raf 1B PKC

NF-B

Actin polymerization Cytoskeletal rearrangements Chemotaxis

mek Erk1/2

Transcriptional activation Cell growth/survival Figure 2 Simplified version of signal transduction pathways activated by the CXCL12/CXCR4 axis that are relevant to cell growth, proliferation, and chemotaxis. Activation of these pathways varies among cell types.

results in phosphorylation of the tyrosine residues of the C-terminus via JAK2 and JAK3 kinases. The phosphorylated CXCR4/CXCL12 complex is then rapidly internalized through a mechanism involving G-protein-coupled receptor kinases followed by the binding of b-arrestin. This process terminates CXCR4 receptor signaling; and activates a number of signaling pathways, including calcium flux, and focal adhesion components, such as proline-rich kinase-2 (Pyk-2), p130Cas, focal adhesion kinase, paxilin, Crk, Crk-L, protein kinase C, phospholipase C-g, mitogen-activated protein kinase (MAPK) p42/ 44-ELK-1, PI3-kinase, AKT, and nuclear factor kappa B (NF-kB) cascades. PI3-kinase ultimately signals through Rho/Rac/cdc42 to upregulate downstream effector proteins involved in actin polymerization and cytoskeletal rearrangements resulting in chemotaxis and adhesion molecule upregulation. CXCL12 activation of CXCR4 under conditions in which the receptor is stimulated with less than saturable concentrations results in movement of CXCR4 into clathrin-coated pits and from there into endosomes, for trafficking back to the plasma membrane to be re-expressed on the cell surface. However, if CXCR4 is exposed to prolonged saturating concentrations of CXCL12 a significant proportion of CXCR4 from endosomes moves to the lysosome for degradation.

Hematopoietic cells lacking protein tyrosine phosphatases (SHIP1 and SHIP2) have altered chemotaxis to CXCL12, implying that they are important in modulating activation by CXCR4 and the absence of membrane-expressed hematopoietic phosphatase, CD45, also results in reduced chemotaxis in lymphocytes. After CXCL12 stimulation, CD45 has also been shown to interact with CXCR4 in the membrane lipid rafts (Figure 2).

Biological Functions Development

Targeted disruption of the CXCL12 gene in mice results in perinatal mortality at around e15.5. The numbers of B cell progenitors in mutant embryos are severely reduced in fetal liver and bone marrow, but myeloid progenitors are only reduced in the bone marrow and not in the fetal liver, indicating that CXCL12 is responsible for B cell lymphopoiesis and bone marrow myelopoiesis. Similarly, mice deficient for CXCR4 die perinatally and display profound defects in the hematopoietic and nervous systems. CXCR4-deficient mice have severely reduced B cell lymphopoiesis, reduced myelopoiesis in fetal liver, and a virtual absence of myelopoiesis in bone marrow. However, T-cell lymphopoiesis is unaffected.

CHEMOKINES, CXC / CXCL12 (SDF-1) 393

Furthermore, CXCR4 is expressed in developing vascular endothelial cells, and mice lacking CXCR4 or CXCL12 have defective formation of the large vessels supplying the gastrointestinal tract. They are also defective in cardiogenesis and have abnormal cerebellar development. The similarities between the CXCL12- and CXCR4-deficient mice indicate that CXCR4 is the primary physiological receptor for CXCL12, suggesting a monogamous relationship between CXCR4 and CXCL12. This receptor ligand selectivity is unusual among chemokines and their receptors, as it is common to have several ligands that bind to one receptor.

Trafficking

Varying SDF-1 expression levels in different cells and tissues create a chemokine gradient that regulates trafficking of cells that express the CXCR4 receptor namely: hematopoietic stem/progenitor cells such as CD34 þ hematopoietic stem cells, pre-B cells, and T cells (in particular naive T cells). In addition, more recently, CXCR4 has been found on primordial germ cells, neural stem cells, liver stem cells, muscle stem cells, retinal pigment epithelial progenitor cells, intestinal epithelial cells, fibrocytes/circulating mesenchymal stem cells, and circulating progenitor epithelial cells in regeneration of tissue after injury, implying that the CXCL12/CXCR4 biological axis is also important in trafficking of nonhematopoietic stem/progenitor cells.

Cell Motility and Chemotaxis

CXCL12 has been shown to induce cell motility through rearrangement of F-actin bundles. CXCL12 may accumulate in tissues after binding to heparin sulfate proteoglycans, which then provides a directional CXCL12 gradient for chemotaxis. CXCL12induced cell movement can be inhibited by blocking the PI3Kinase-AKT pathway in many cell lines, including A549 and H157 non-small cell lung cancer (NSCLC) cell lines. Interestingly, metastases of human NSCLC cells in severe combined immunodeficiency (SCID) mice are 99% positive for CXCR4 expression, whereas significantly fewer cells of the primary tumor express CXCR4. Under hypoxic conditions, nuclear HIF-1a induces increased levels of CXCR4 expression on these cancer cell lines, which leads to increased ability of these tumor cells to invade and metastasize. PI3-kinase inhibitors and overexpressing PTEN inhibit activation of HIF-1a and CXCR4 expression.

Cell Adhesion

CXCL12 via CXCR4 increases adhesion of cells to fibrinogen, fibronectin, stroma, and endothelial cells, through activation of adhesion molecules, such as integrins. Cell Secretion

After stimulation by CXCL12, cells secrete more matrix metalloproteinases (e.g., MMP-9), nitric oxide, and vascular endothelial growth factor (VEGF), probably through activation of the NF-kB pathway. Cell Proliferation and Survival

In some experimental conditions, CXCL12 was found to stimulate proliferation and survival of hematopoietic cells, astrocytes, and some tumor cell lines through activation of the PI3-kinase-AKT and MAPK p42/44 pathways. CXCL12 has also been found to be a survival factor for glioblastoma cells and induces proliferation in a dose-dependent manner. This proliferation correlates with phosphorylation and activation of both ERK 1/2 and AKT, and these kinases are independently involved in glioblastoma cell proliferation.

Receptor CXCR4 is a G-protein-coupled seven transmembrane receptor that was originally cloned as an orphan chemokine receptor and was known as LESTR or fusin. CXCR4 is expressed on the cell surface of most leukocytes, including all B cells, and monocytes and most T lymphocyte subsets, but just weakly on NK cells. It is also expressed on nonhematopoietic cells such as endothelial cells and epithelial cells, and adult stem cells such as fibrocytes and circulating progenitor epithelial cells. CXCR4 is also an essential cofactor for T-tropic HIV-1 and HIV-2 env-mediated fusion and entry into CD4 þ lymphocytes. Recently, a second alternatively spliced CXCR4 receptor was cloned and named CXCR4-Lo. CXCR4Lo has lower gene expression in most tissues than CXCR4 except in the spleen and lung; its function is not yet known.

CXCL12 in Respiratory Diseases Lung Injury and Repair

Circulating mesenchymal progenitor cells, fibrocytes, express CXCR4 and traffic to the lungs in response to CXCL12 and mediate fibrosis in a murine model of bleomycin-induced pulmonary fibrosis. Specific


neutralizing antibodies to CXCL12 inhibit pulmonary recruitment of these circulating fibrocytes and attenuate lung fibrosis. In addition, CXCL12 expression is increased in the submucosal glands, ducts, and airway epithelium in a mouse tracheal transplant model, which provides a chemokine gradient for CXCR4 þ circulating progenitor epithelial cells to traffick and contribute to repair of the injured airway. The mucus in the submucosal gland lumen provides proteoglycans to depot CXCL12 and create the CXCL12 gradient for chemotaxis. The CXCL12 expression in epithelial cells moves apically to the airway surface during regeneration of the airway. Moreover, CXCL12 can induce neovascularization, which is critical in acute and chronic lung injury and repair. For example, bronchial biopsy specimens from patients with asthma show a significant increase in the number of blood vessels and colocalization of CXCL12-positive endothelial cells together with CXCL12-positive macrophages and T lymphocytes in the submucosa compared with control subjects. These findings suggest that CXCL12 may play a role in remodeling of asthmatic airways via angiogenesis. Lung Cancer

CXCL12 is an important molecule in lung cancer invasion and metastasis and is also important in the development of lung metastases from tumors from other organs (e.g., breast, renal cell carcinoma, rhabdomyosarcoma, etc.). Both NSCLC tumor specimens resected from patients and NSCLC cell lines express CXCR4, but not CXCL12. NSCLC cell lines undergo chemotaxis in response to CXCL12. CXCL12–CXCR4 activation of NSCLC cell lines show PI3 kinase activation, intracellular calcium mobilization and MAPK activation with enhanced ERK-1/2 phosphorylation without change in either proliferation or apoptosis. Target organs in a murine model that are the preferred destination of human NSCLC metastases produce higher levels of CXCL12 than the primary tumor, thereby creating a chemotactic gradient. The administration of specific neutralizing anti-CXCL12 antibodies to SCID mice with human NSCLC abrogates organ metastases, without affecting primary tumor-derived angiogenesis. The CXCL12–CXCR4 biological axis is therefore involved in regulating the metastasis of NSCLC. In addition, the ability of NSCLC cells to metastasize correlates with their expression of CXCR4, which is upregulated under conditions of hypoxia through HIF-1a. Inhibition of PI3-kinase or overexpression of PTEN reduces CXCR4 expression and their metastatic potential.

Small cell lung cancer (SCLC) cells express high levels of functional CXCR4 receptors for CXCL12. CXCL12 induces integrin activation, which results in increased adhesion of SCLC cells to fibronectin and collagen. This is mediated by activation of integrins and CXCR4, and is inhibited by CXCR4 antagonists. Stromal cells protect SCLC cells from chemotherapy-induced apoptosis, and this protection can also be antagonized by CXCR4 inhibitors. Renal cell carcinoma commonly metastasizes to other organs such as the adrenal gland and lung, both of which express high levels of CXCL12. Both heterotopic and orthotopic mouse models of tumors from a renal carcinoma cell line develop lung metastases, which can be abrogated by treating the mice with neutralizing antibodies to CXCL12. In summary, CXCL12 is a pleiotropic chemokine with a variety of functions beyond that of leukocyte trafficking that are relevant to the lung. See also: Adhesion, Cell–Cell: Epithelial. Angiogenesis, Angiogenic Growth Factors and Development Factors. Chemokines. Endothelial Cells and Endothelium. Epithelial Cells: Type I Cells. Extracellular Matrix: Basement Membranes; Collagens; Matrix Proteoglycans; Surface Proteoglycans. Fibroblasts. G-Protein-Coupled Receptors. Human Immunodeficiency Virus. Hypoxia and Hypoxemia. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Eosinophils; Neutrophils; T cells. Matrix Metalloproteinases. Myofibroblasts. Pulmonary Fibrosis. Signal Transduction. Stem Cells. Vesicular Trafficking.

Further Reading Gazitt Y (2004) Homing and mobilization of hematopoietic stem cells and hematopoietic cancer cells are mirror image processes, utilizing similar signaling pathways and occurring concurrently: circulating cancer cells constitute an ideal target for concurrent treatment with chemotherapy and antilineage-specific antibodies. Leukemia 18: 1–10. Kucia M, Jankowski K, Reca R, et al. (2004) CXCR4-SDF-1 signaling, locomotion, chemotaxis and adhesion. Journal of Molecular Histology 35: 233–245. Luster AD (1998) Chemokines–chemotactic cytokines that mediate inflammation. New England Journal of Medicine 338: 436– 445. Mackay CR (2001) SDF-1. In: Oppenheim JJ and Feldman M (eds.) Cytokine Reference, pp. 1119–1127. San Diego: Academic Press. Murdoch C (2000) CXCR4: chemokine receptor extraordinaire. Immunology Reviews 177: 175–184. Murphy PM (2002) International Union of Pharmacology. XXX. Update on chemokine receptor nomenclature. Pharmacological Reviews 54: 227–229. Phillips RJ, Burdick MD, Hong K, et al. (2004) Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. Journal of Clinical Investigation 114: 438–446.

CHEMOKINES, CXC / IL-8

IL-8 R M Strieter, M P Keane, and J A Belperio, The David Geffen School of Medicine at UCLA, Los Angeles, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract CXCL8/IL-8 is a member of the CXC chemokine family. The CXC chemokines can be further divided into two groups on the basis of a structure/function domain consisting of the presence or absence of three amino acid residues (Glu–Leu–Arg; ‘ELR’ motif) that precede the first cysteine amino acid residue in the primary structure of these cytokines. CXCL8/IL-8 and the other ELR þ CXC chemokines are chemoattractants for neutrophils and act as potent angiogenic factors. CXCL8/IL-8 binds to the CXCR1 and CXCR2 receptors, which are found on neutrophils, T lymphocytes, monocytes/macrophages, eosinophils, basophils, keratinocytes, and mast cells and endothelial cells. CXCL8/IL-8 has been shown to have an important role in the recruitment of neutrophils in bacterial pneumonia and to correlate with the development and mortality of adult respiratory distress syndrome. Furthermore, CXCL8/IL-8 also has a key role in the vascular remodeling that is seen in pulmonary fibrosis.

Introduction CXCL8 is a member of the CXC chemokine family and is also known as IL-8. The CXC chemokines can further be divided into two groups on the basis of a structure/function domain consisting of the presence or absence of three amino acid residues (Glu–Leu– Arg; ‘ELR’ motif) that precede the first cysteine amino acid residue in the primary structure of these cytokines. CXCL8/IL-8 and the other ELR þ CXC chemokines are chemoattractants for neutrophils and act as potent angiogenic factors.

Structure The precursor molecule of CXCL8/IL-8 consists of 99 amino acids with an associated 20 amino acid signal sequence. Several mature forms of CXCL8/IL8 have been identified, that are a result of repeated NH2-terminal amino acid cleavage, including the major 72 amino acid mature form. The 72 amino acid form of CXCL8/IL-8 binds to neutrophils twofold more than the 77 amino acid form, and is twoto threefold more potent in inducing cytochalasin B-treated neutrophil degranulation. Interestingly, the 77 amino acid form can induce apoptosis in leukemic cells, whereas the 72 amino acid form does not. The NH2-terminus of CXCL8/IL-8, similar to other CXC chemokines that bind and activate neutrophils, contains three highly conserved amino acid residues consisting of Glu–Leu–Arg (the ELR motif). The COOH-terminus of CXCL8/IL-8 contains a

395

heparin-binding domain, which may be important in binding to glycosaminoglycans.

Regulation of Production and Activity The gene for CXCL8/IL-8 is found on human chromosome 4, q12–21, and consists of four exons and three introns. The 50 -flanking region of CXCL8/IL-8 contains the usual ‘CCAAT’ and ‘TATA’ box-like structures. In addition, this region has a number of potential binding sites for several nuclear factors. The CXCL8/IL-8 promoter region is regulated in a cell-specific fashion requiring a nuclear factor kappa B (NF-kB) element plus either activating protein 1 (AP-1) or a C/EBP (NF-IL-6) element under conditions of transcriptional induction with tumor necrosis factor alpha (TNF-a) or interleukin-1 (IL-1). Although the CXCL8/IL-8 promoter has two C/EBP (50 and 30 ) cis-elements with NF-kB nested between them, the 50 -C/EBP element appears to be the only C/ EBP element involved in transcriptional regulation of CXCL8/IL-8. In specific cell lines, the AP-1 along with NF-kB or C/EBP and NF-kB cis-elements is sufficient for full transcriptional activation of the CXCL8/IL-8 promoter. Members of the NF-kB/rel and C/EBP families of transcriptional factors can interact on a protein:protein level via the Rel:basic leucine zipper (bZIP) domain of these proteins, respectively. In order for full transcriptional activation of the promoter, there is a cooperative interaction between all three major cis-elements (AP-1, 50 -C/EBP and NF-kB). While the promoter region of CXCL8/ IL-8 gene has been extensively studied, the mechanisms related to signal transduction and optimal transactivation of the CXCL8/IL-8 gene have been less studied. Mitogen-activated protein (MAP) kinases are necessary to optimally induce the gene expression and protein production of CXCL8/IL-8 from airway epithelial cells in response to TNF-a. TNF-a activation of the MAP kinases is able to achieve the induction of CXCL8/IL-8 gene expression and protein production by NF-kB-dependent, -independent, and posttranscriptional mechanisms. Direct inhibition of the MAP kinases (extracellular regulated kinase (ERK) and c-jun N-terminal protein kinase (JNK)) and NF-kB, but not p38, decreases TNF-a-induced transcription of the CXCL8/IL-8 promoter. Inhibition of JNK signaling reduces TNFa-induced transcription of the CXCL8/IL-8 promoter in an NF-kB-dependent manner, whereas inhibition of ERK impairs TNF-a-induced transcription of the CXCL8/IL-8 promoter in an NF-kB-independent and AP-1-dependent manner. Activation of the p38 MAP kinase is important for promoting TNFa-induced CXCL8/IL-8 protein production in a

396 CHEMOKINES, CXC / IL-8

posttranscriptional manner. These findings would also be relevant to downstream signal transduction events related to activation of Toll-like receptors and IL-1 type I receptor for the induction of CXCL8/IL-8 gene expression and protein production.

Biological Function CXCL8/IL-8 is a potent recruiter of neutrophils and induces angiogenesis. It is also chemotactic for eosinophils. CXCL8/IL-8 has dose-dependent effects on basophils, which can lead to either stimulation or inhibition of release of both LTB4 and histamine. CXCL8/IL-8 also leads to activation of basophils with enhanced binding of basophils to endothelial cells. CXCL8/IL-8 is not chemotactic for monocytes; however, stimulation of monocytes with CXCL8/ IL-8 leads to a rise in cytosolic calcium and generation of reactive oxygen species. CXCL8/IL-8 can selectively inhibit the IL-4-induced IgE and IgG4 production from B cells. CXCL8/IL-8 can induce migration and proliferation of keratinocytes (Figure 1).

Receptors Chemokine activities are mediated through pertussissensitive G-protein-coupled receptors. CXCL8/IL-8 binds to the CXCR1 and CXCR2 receptors, which are found on neutrophils, T lymphocytes, monocytes/macrophages, eosinophils, basophils, keratinocytes, and mast cells and endothelial cells. While the transmembrane and the second and third intracellular/cytoplasmic domains of these receptors are well conserved, the NH2- and COOH-terminal ends of these receptors are variable. The intracellular COOH-terminus of these receptors is rich in serine and threonine amino

acid residues that may be important in phosphorylation and signal coupling via G proteins.

CXCL8/IL-8 in Respiratory Disease CXC chemokines have been found to play a significant role in mediating neutrophil infiltration in the lung parenchyma and pleural space in response to endotoxin and bacterial challenge. Passive immunization with neutralizing CXCL8/IL-8 antibodies blocked 77% of endotoxin-induced neutrophil influx in the pleura of rabbits. However, in the context of microorganism invasion, depletion of a CXC chemokine and reduction of infiltrating neutrophils may have a major impact on the host (Figure 2). CXCL8/IL-8 has been implicated in mediating neutrophil sequestration in the lungs of patients with pneumonia. CXCL8/IL-8 has been found in the bronchoalveolar lavage of patients with community acquired pneumonia and nosocomial pneumonia. While there is no murine structural homolog for CXCL8/IL-8, both CXCL1 and CXCL2/3 have been found in murine models of Klebsiella pneumoniae, Pseudomonas aeruginosa, Nocardia asteroides, and Host defense/pneumonia Acute lung injury/ARDS CXCL8

Angiogenesis Tumor growth Figure 2 Role of CXCL8/IL-8 in respiratory disease.

Monocyte/macrophage Lymphocytes

Epithelial cells

CXCL8

Chemotaxis

Endothelial cells

Neutrophils Eosinophils

Fibroblasts Neutrophils

Chemotaxis

Keratinocytes Basophils

Endothelial cells

Angiogenesis Figure 1 Source and biological functions of CXCL8/IL-8.

Pulmonary fibrosis

CHEMOKINES, CXC / IL-8

Aspergillus fumigatus pneumonia. In a model of A. fumigatus pneumonia, neutralization of TNF resulted in marked attenuation of the expression of CXCL1 and CXCL2/3 that was paralleled by a reduction in the infiltration of neutrophils and associated with increased mortality. In addition, Laichalk and associates administered a TNF agonist peptide consisting of the 11 amino acid TNF binding site (TNF70–80) to animals intratracheally inoculated with K. pneumoniae and found markedly elevated levels of CXCL2/3 associated with increased neutrophil infiltration. Depletion of CXCL2/3 during the pathogenesis of murine K. pneumoniae pneumonia resulted in a marked reduction in the recruitment of neutrophils to the lung that was paralleled by increased bacteremia and reduced bacterial clearance in the lung. Since ELR þ CXC chemokine ligands in the mouse use the CXC chemokine receptor, CXCR2, Standiford and associates used specific neutralizing antibodies to CXCR2 and demonstrated that blocking CXCR2 results in markedly reduced neutrophil infiltration in response to P. aeruginosa, N. asteroides, and A. fumigatus pneumonias. The reduction in neutrophil elicitation was directly related to reduced clearance of the microorganisms and increased mortality in these model systems. These studies have established the critical importance of ELR þ CXC chemokines in acute inflammation and the innate immune response to a variety of microorganisms. Sekido and associates demonstrated that CXCL8/ IL-8 significantly contributed to reperfusion lung injury in a rabbit model of lung ischemia-reperfusion injury. Reperfusion of the ischemic lung resulted in the production of CXCL8/IL-8, which correlated with maximal pulmonary neutrophil infiltration. Passive immunization of the animals with neutralizing antibodies to CXCL8/IL-8 prior to reperfusion of the ischemic lung prevented neutrophil extravasation and tissue injury, suggesting a causal role for CXCL8/IL-8 in this model. Ventilator-induced lung injury in a murine model is associated with increased expression of CXCL1 and CXCL2 that parallels lung injury and neutrophil recruitment. Furthermore, these levels correlated with NF-kB activation. CXCR2–/– mice were protected from ventilatorinduced lung injury. Several studies have demonstrated that CXCL8/ IL-8 levels correlate with the development and mortality of the acute respiratory distress syndrome. Of particular interest is the study of Donnelly and colleagues, which correlated early increases in CXCL8/IL-8 in bronchoalveolar lavage fluid (BALF) with an increased risk of subsequent development of adult respiratory distress syndrome (ARDS), and also

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demonstrated that alveolar macrophages were an important source of CXCL8/IL-8 prior to neutrophil influx. High concentrations of CXCL8/IL-8 were found in BALF from trauma patients, some within 1 h of injury and prior to any evidence of significant neutrophil influx. Patients who progressed to ARDS had significantly greater BALF levels of CXCL8/IL-8 than those who failed to develop this condition. Levels of CXCL8/IL-8 in plasma, as opposed to lavage, were not found to be significantly different between patients who did or did not develop ARDS. Furthermore, there is an imbalance in the expression of ELR þ (including CXCL8/IL-8) as compared to ELR– CXC chemokines from BALF of patients with ARDS as compared to controls. This imbalance correlated with angiogenic activity and both procollagen I and procollagen III levels in BALF. These findings suggest that CXCL8/IL-8 and other CXC chemokines have an important role in the fibroproliferative phase of ARDS via the regulation of angiogenesis. Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology that is characterized by the accumulation of neutrophils within the airspace and mononuclear cells within the interstitium, followed by the progressive deposition of collagen within the interstitium and subsequent destruction of lung tissue. While the mechanisms of cellular injury and the role of classic inflammatory cells remain unclear, increases in neutrophils in BALF and in lung tissue have been demonstrated from patients with IPF. While the number or proportion of neutrophils in BALF does not correlate with activity of alveolitis and has limited prognostic value, declines in BALF neutrophils typically occur among patients exhibiting favorable responses to therapy. Neutrophilic alveolitis has been described in humans with IPF, collagen vascular diseases with associated ILD, as well as diverse animal models of pulmonary fibrosis. CXCL8/IL-8 is significantly elevated in IPF, as compared to either normal or sarcoidosis patients, and correlates with BALF presence of neutrophils. The alveolar macrophage is an important cellular source of CXCL8/IL-8 in IPF. In addition, these studies have suggested that levels of CXCL8/IL-8 in IPF may correlate with a worse prognosis. While studies have suggested an important role for CXCL8/IL-8 in mediating neutrophil recruitment, CXC chemokines have been found to exert disparate effects in regulating angiogenesis. This latter issue is relevant to IPF, as the pathology of IPF demonstrates features of dysregulated and abnormal repair with exaggerated angiogenesis, fibroproliferation, and deposition of extracellular matrix, leading to progressive fibrosis and loss of lung function. There have been limited investigations to delineate factors that

398 CHEMOKINES, CXC / CXCL9 (MIG)

may be involved in the regulation of this angiogenic activity during pulmonary fibrosis. In IPF lung tissue there is an imbalance in the presence of CXC chemokines that behave as either promoters of angiogenesis CXCL8/IL-8 or inhibitors of angiogenesis CXCL10. This imbalance favors augmented net angiogenic activity. Lung tissue from IPF patients has elevated levels of CXCL8/IL-8, as compared to control lung tissue, and demonstrates in vivo angiogenic activity that can be significantly attributed to CXCL8/IL-8. Immunolocalization of CXCL8/IL-8 demonstrated that the pulmonary fibroblast was the predominant interstitial cellular source of this chemokine, and areas of CXCL8/IL-8 expression were essentially devoid of neutrophil infiltration. This supports an alternative biological role for CXCL8/IL-8 or other ELR þ CXC chemokines in the interstitium of IPF lung tissue. The pulmonary fibroblast is the predominant cellular source of CXCL8/IL-8 in the interstitium of IPF, supporting the notion that the pulmonary fibroblast has a pivotal role in mediating the angiogenic activity during the pathogenesis of IPF. Indeed, the pulmonary fibroblast has received increasing attention as a pivotal cell in the pathogenesis of IPF. Relative levels of CXCL8/IL-8 and CXCL10 from IPF pulmonary fibroblast conditioned media demonstrated a significant imbalance favoring CXCL8/IL-8-induced angiogenic activity. In contrast, normal pulmonary fibroblasts had greater levels of CXCL10 that favored a net inhibition of angiogenesis. The difference in expression of CXCL8/IL-8 and CXCL10 between IPF and control pulmonary fibroblasts lends further support to the notion of a phenotypic difference between IPF and normal pulmonary fibroblasts, which has been well described. See also: Acute Respiratory Distress Syndrome. Angiogenesis, Angiogenic Growth Factors and Development Factors. Chemokines. Chemokines, CXC: CXCL1 (GRO1)–CXCL3 (GRO3). Extracellular Matrix: Basement Membranes. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Leukocytes: Eosinophils; Neutrophils; Monocytes; T cells. Pneumonia: Overview and Epidemiology. Pulmonary Fibrosis. Transgenic Models. Tumors, Malignant: Overview.

pneumonia and adult respiratory distress syndrome. Infection and Immunity 61: 4553. Donnelly SC, Strieter RM, Kunkel SL, et al. (1993) Interleukin-8 and development of adult respiratory distress syndrome in atrisk patient groups. Lancet 341: 643–647. Greenberger MJ, Strieter RM, Kunkel SL, et al. (1996) Neutralization of macrophage inflammatory protein-2 attenuates neutrophil recruitment and bacterial clearance in murine Klebsiella pneumonia. Journal of Infectious Disease 173: 159. Jordana M, Schulman J, McSharry C, et al. (1988) Heterogeneous proliferative characteristics of human adult lung fibroblast lines and clonally derived fibroblasts from control and fibrotic tissue. American Review of Respiratory Disease 137: 579. Keane MP, Arenberg DA, Lynch JP, et al. (1997) The CXC chemokines, IL-8 and IP-10, regulate angiogenic activity in idiopathic pulmonary fibrosis. Journal of Immunology 159: 1437. Keane MP, Donnelly SC, Belperio JA, et al. (2002) Imbalance in the expression of CXC chemokines correlates with bronchoalveolar lavage fluid angiogenic activity and procollagen levels in acute respiratory distress syndrome. Journal of Immunology 169: 6515. Koch AE, Polverini PJ, Kunkel SL, et al. (1992) Interleukin-8 (IL8) as a macrophage-derived mediator of angiogenesis. Science 258: 1798. Laichalk LL, Bucknell KA, Huffnagle GB, et al. (1998) Intrapulmonary delivery of tumor necrosis factor agonist peptide augments host defense in murine Gram-negative bacterial pneumonia. Infection and Immunity 66: 2822. Lynch JP, Standiford TJ, Kunkel SL, Rolfe MW, and Strieter RM (1992) Neutrophilic alveolitis in idiopathic pulmonary fibrosis: the role of interleukin-8. American Review of Respiratory Disease 145: 1433. Sheppard D (2001) Pulmonary fibrosis: a cellular overreaction or a failure of communication? Journal of Clinical Investigation 107: 1501. Southcott AM, Jones KP, Li D, et al. (1995) Interleukin-8, differential expression in lone fibrosing alveolitis and systemic sclerosis. American Journal of Respiratory and Critical Care Medicine 151: 1604. Strieter RM (2002) Interleukin-8: a very important chemokine of the human airway epithelium. American Journal of Physiology: Lung Cellular and Molecular Physiology 283: L688. Strieter RM, Kunkel SL, Elner VM, et al. (1992) Interleukin-8: a corneal factor that induces neovascularization. American Journal of Pathology 141: 1279. Strieter RM, Kunkel SL, Showell H, et al. (1989) Endothelial cell gene expression of a neutrophil chemotactic factor by TNF-a, LPS, and IL-1b. Science 243: 1467. Strieter RM, Polverini PJ, Kunkel SL, et al. (1995) The functional role of the ‘ELR’ motif in CXC chemokine-mediated angiogenesis. Journal of Biological Chemistry 270: 27348.

CXCL9 (MIG) Further Reading Belperio JA, Keane MP, Burdick MD, et al. (2002) Critical role for CXCR2 and CXCR2 ligands during the pathogenesis of ventilatorinduced lung injury. Journal of Clinical Investigation 110: 1703. Broaddus VC, Boylan AM, Hoeffel JM, et al. (1994) Neutralization of IL-8 inhibits neutrophil influx in a rabbit model of endotoxin-induced pleurisy. Journal of Immunology 152: 2960. Chollet-Martin S, Montravers P, Gibert C, et al. (1993) High levels of interleukin-8 in the blood and alveolar spaces of patients with

P C Fulkerson and M E Rothenberg, Cincinnati Children’s Hospital, Cincinnati, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Human CXCL9 was identified in 1993 by a differential hybridization screening technique, comparing cDNA libraries

CHEMOKINES, CXC / CXCL9 (MIG) 399 obtained from an unstimulated and interferon gamma (IFN-g)activated macrophage cell line. CXCL9 is a member of the alpha or CXC subfamily of chemokines. The CXC chemokines can be subdivided based on the presence or absence of a tripeptide motif Glu–Leu–Arg (ELR) N-terminal to the conserved CXC region. CXCL9, a non-ELR-containing CXC chemokine, mediates most of its biological function through binding to CXCR3, a seven-transmembrane-domain receptor coupled to G proteins. CXCL9 primarily attracts activated T lymphocytes, preferentially of the Th1 phenotype, which express high levels of CXCR3. The expression of CXCL9 is induced primarily by the Th1-associated cytokine IFN-g and correlates with tissue infiltration of T lymphocytes in a number of Th1-associated diseases, suggesting that CXCL9 plays an important role in the regulation of effector cell recruitment to sites of inflammation. Remarkably, CXCL9 also regulates eosinophil accumulation in experimental asthma, a Th2-associated lung disease. In addition, CXCL9 has potent angiostatic activity, inhibiting blood vessel growth in wound repair and inhibiting tumor growth and tumor-associated vessel expansion.

preferentially of the Th1 phenotype, which express high levels of CXCR3.

Structure CXCL9 is a member of the alpha or CXC subfamily of chemokines. CXC chemokines have a single amino acid that separates invariant cysteines one and two (of four). The CXC chemokines can be subdivided based on the presence or absence of a tripeptide motif Glu–Leu–Arg (ELR) N-terminal to the conserved CXC region. The gene coding for CXCL9, a nonELR-containing CXC chemokine, maps to the CXC gene cluster on human chromosome 4 (4q21) and murine chromosome 5 (5 53 cM). The gene is organized into four exons and the arrangement is highly conserved between humans and mice (Figure 1). The 2.5 kb human mRNA transcript and 1.3 kb murine transcript is translated into a 125 and 126 amino acid protein, respectively. The human and murine CXCL9 proteins are 68% homologous. Despite differences in primary sequences, chemokines have a remarkably similar three-dimensional structure, comprised of a short N-terminal region, a large core, which is stabilized by disulfide bonds and hydrophobic interactions, and a C-terminal a-helix. The most highly conserved sequence among chemokines is the four cysteine residues. The first and third cysteines and the second and fourth cysteines are linked by conserved disulfide bonds. While chemokines share four common secondary structural elements, three b-strands arranged in a single antiparallel b-sheet and one a-helix, most of the variation in chemokine amino acid sequence occurs in the Nand C-terminal regions. Structure–function studies have shown that chemokines have two main sites of interaction with their receptors, one in the N-terminus and the other in the N-loop region after the second cysteine. Deletion of just a few of the N-terminal

Introduction Cytokines play a central role in macrophage physiology, both as macrophage activators (e.g., interferons (IFN)) and mediators of macrophage activity (e.g., chemokines). Monokine induced by interferon gamma (IFN-g) (MIG, CXCL9) was discovered in an effort to identify genes induced during macrophage activation. A differential hybridization screening technique, comparing cDNA libraries obtained from an unstimulated and IFN-g-activated mouse macrophage cell line, was utilized to initially identify the murine CXCL9 gene. The murine CXCL9 cDNA was then used to screen a cDNA library made from an IFN-g-activated human macrophage cell line. This led to the identification of a new member of the chemokine gene family, human MIG/CXCL9, in 1993. CXCL9 mediates most of its biological function through binding to CXCR3, a seven-transmembrane-domain receptor coupled to G proteins. CXCL9 primarily attracts activated T lymphocytes, Human CXCL9/SCYB9 gene Exon 1

Exon 2

Exon 3

Exon 4

(a)

Murine CXCL9/Scyb9 gene Exon 1

Exon 2

Exon 3

Exon 4

(b) CDS Figure 1 Human and murine CXCL9 gene organization. The gene coding for CXCL9 maps to the CXC gene cluster on human chromosome 4 (4q21) and murine chromosome 5 (5 53 cM). The gene is organized into four exons and the arrangement is highly conserved between humans and mice. CDS, coding sequence.

400 CHEMOKINES, CXC / CXCL9 (MIG)

residues of CXCL9 results in a loss of receptor binding capacity. In addition, the non-ELR CXC chemokines contain a C-terminal segment rich in positively charged amino acids. Notably, CXCL9 has a unique extended basic C-terminal region, which when truncated results in a dramatic decrease in its biological activity.

Regulation of CXCL9 Production and Activity The IFNs are an important group of cytokines that mediate some of their biological effects through regulation of specific RNA and protein expression in a responding cell. The expression of CXCL9 is primarily dependent on the Th1 cytokine IFN-g, but in its absence, IFN-a or IFN-b is able to induce CXCL9 expression. The IFN-g-induced transcriptional activation of CXCL9 depends upon responsive elements in the promoter region of the gene, which are recognized by signal transducers and activators of transcription (STAT)1 or STAT1-containing factors. The promoter of CXCL9 also contains regions that mediate transcriptional activation in response to tumor necrosis factor alpha (TNF-a). IFN-g and TNF-a synergize to induce the expression of several genes, including CXCL9. In addition to inducing expression, transcription factors modulate inflammatory responses by negatively regulating chemokine expression. The Th2associated transcription factor STAT6 and the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR-g) have been shown to inhibit IFN-g-induced expression of CXCL9. The N-terminal region of most chemokines is crucial for receptor binding and signaling activities.

Amino peptidases or endopeptidases that process chemokines at the N-terminus play an important role in the regulation of chemokine activity. The membrane-associated protease dipeptidyl peptidase IV/CD26 cleaves two amino acids from polypeptides with a proline, alanine, or hydroxyproline at the second position. The removal of the N-terminal dipeptide from CXCL9 results in decreased receptor binding capacity and a dramatic decrease in chemotactic activity. In addition, matrix metalloproteinase (MMP) 9, another class of chemokine-processing proteases, cleaves CXCL9 within its extended C-terminus. C-terminal truncation of CXCL9 results in a significant decrease in its biological activity. Since the highly basic C-terminal segment of CXCL9 may associate with extracellular matrixpoteoglycans, localization and establishment of chemotactic gradients may also be affected by protease processing of CXCL9.

Biological Function Chemokines are chemotactic cytokines that orchestrate the migration and activation of leukocyte populations under baseline (homeostatic) and inflammatory conditions. The predominant function of CXCL9 is the recruitment of primed T lymphocytes to the sites of inflammation (Figure 2). CXCL9 has been associated with the infiltration and retention of activated T lymphocytes in inflammatory diseases of the skin, joints, and central nervous system. Furthermore, studies have implicated CXCL9 as a critical mediator of primed T-lymphocyte trafficking in transplant models. In addition to promoting leukocyte recruitment, chemokines can also function as natural

Angiostasis inhibits capillary growth R3

CXC

CXCR3 Promotes T and B lymphocyte recruitment and function

CCR3 Inhibits eosinophil recruitment

Figure 2 CXCL9 regulates lymphocyte and eosinophil chemoattraction and inhibits angiogenesis. The predominant function of CXCL9 is the recruitment of primed T lymphocytes expressing high levels of CXCR3 to the sites of inflammation. In addition to promoting leukocyte recruitment, chemokines can also function as natural antagonists to prevent the recruitment or activation of a different cellular population. In animal models, CXCL9 has been shown to function as a potent inhibitor of eosinophil recruitment toward diverse stimuli. In addition, CXCL9 inhibits blood vessel growth in wound repair and tumor models.

CHEMOKINES, CXC / CXCL9 (MIG) 401

antagonists to prevent the recruitment or activation of a different cellular population. In animal models, CXCL9 has been shown to function as a potent inhibitor of eosinophil recruitment toward diverse stimuli. Although cell trafficking is considered the central task of chemokines, there is increasing evidence that a variety of other leukocyte functions may also be modulated by chemokines. CXCL9 has also been shown to stimulate effector functions, such as cytokine production (by recruited T lymphocytes) and promote T-cell proliferation. In a host defense model using gene-targeted mice, murine CXCL9 contributed to the humoral response to a bacterial pathogen, suggesting that CXCL9 not only recruits T cells to sites of inflammation, but also maximizes interactions among activated T cells, B cells, and dendritic cells within lymphoid organs. Angiogenesis, the growth of new capillaries from either pre-existing vessels or from progenitor endothelial cells, is an important biological event that is essential to several physiologic and pathologic processes, including development, wound repair, and tumor growth. CXC chemokines have pivotal, yet opposing, roles in the control of inflammation and angiogenesis, as a result of the shared expression of their specific receptors by both leukocytes and endothelial cells. The ELR-containing CXC chemokines are potent promoters of angiogenesis. In contrast, members that lack the ELR motif, such as CXCL9, are potent inhibitors of angiogenesis. It is proposed that the angiostatic properties of CXCL9 are important to prevent unlimited vessel growth in wound repair.

Receptors Chemokines mediate their functions through binding to seven-transmembrane-domain receptors coupled to G proteins. The receptors often recognize more than one chemokine, and alternatively, several chemokines can bind to multiple receptors. CXCL9 shares its primary receptor, CXCR3, with two other non-ELR-containing CXC chemokines, CXCL10 and CXCL11. CXCR3 is expressed on a small subset of circulating blood T cells, B cells, and natural killer (NK) cells, but T-cell activation greatly enhances CXCR3 expression. CXCR3 is preferentially expressed on Th1 lymphocytes, suggesting that CXCR3 and its ligands are more active in the setting of Th1driven inflammatory responses. Besides being an agonist for CXCR3, CXCL9 has been shown to act as a natural antagonist for CCR3, a Th2-associated chemokine receptor highly expressed on eosinophils. Studies have demonstrated that CXCL9 inhibits CCR3-mediated functional responses in both human

and mouse eosinophils. This suggests that the polarization of an inflammatory response may be enhanced with the expression of CXCR3 ligands, including CXCL9, that attract Th1 cells, but also concomitantly block the migration of Th2 cells in response to CCR3 ligands.

CXCL9 in Respiratory Diseases Lung transplantation is a therapeutic option for many patients with end-stage pulmonary disorders. Acute and chronic lung rejection is a common complication. Acute rejection is characterized by perivascular and peribronchiolar leukocyte infiltration and is the major risk factor for the development of chronic lung allograft rejection, or bronchiolitis obliterans syndrome (BOS), the leading cause of morbidity and mortality post-lung transplantation. BOS is a chronic inflammatory process characterized by persistent peribronchiolar leukocyte infiltration that eventually invades and disrupts the basement membrane, submucosa, and airway epithelium. The persistent leukocyte infiltration is followed by an aberrant repair process, with increased matrix deposition and granulation tissue formation, ultimately leading to obliteration of airways. Elevated levels of CXCL9 (and the other CXCR3 ligands) have been found in the bronchoalveolar lavage (BAL) fluid from patients with acute and chronic lung allograft rejection, as compared with healthy lung transplant recipients. In a murine model of BOS, neutralization of CXCL9, or its receptor CXCR3, resulted in inhibition in the recruitment of mononuclear cells and a marked reduction in extracellular matrix deposition and airway obliteration. Similarly, CXCL9 neutralization resulted in attenuation of intragraft mononuclear cells and decreased parameters in an animal model of acute lung allograft rejection. These studies demonstrate the importance of CXCL9/CXCR3 interactions during both acute and chronic allograft rejection, suggesting that CXCL9 may be an important ligand in promoting the continuum of acute to chronic rejection. Diffuse lung injury is a frequent complication of allogeneic stem cell transplantation (SCT). Idiopathic pneumonia syndrome (IPS), a noninfectious diffuse lung injury, is associated with a high degree of mortality. The pathophysiology of IPS involves toxic damage due to irradiation and chemotherapy, a donor T-cell response, and the production of inflammatory cytokines. IPS is characterized by a diffuse pneumonitis involving both alveolar and interstitial space, as well as mononuclear cell infiltrates around vascular and bronchial structures. These pathologic changes are further associated with decreased

402 CHEMOKINES, CXC / CXCL10 (IP-10)

pulmonary function. In a murine SCT model, increased BAL fluid level of CXCL9 is associated with recruitment of CXCR3 expressing donor CD8 þ T cells to the lung after SCT. Neutralization of CXCL9 reduces the IPS severity and the recruitment of donor T cells to the lung after SCT. Together with the lung rejection studies, the significant decrease in lung injury and pathology in these models has been predominantly attributed to reduced effector cell recruitment and underscores the importance of the CXCL9/CXCR3 interaction in inflammatory diseases in the lung. In addition to being associated with the chemoattraction of Th1-associated effector cells into the lung, CXCL9 has been shown to inhibit the accumulation of cells in Th2-mediated asthma models. One of the hallmarks of allergic airway disease is the accumulation of an abnormally large number of inflammatory cells, such as eosinophils and T lymphocytes, into the lung. In experimental asthma models, CXCL9 has been shown to negatively regulate eosinophil recruitment into the lung. With no detectable CXCR3 on the surface of eosinophils, CXCL9 inhibited eosinophil chemoattraction in vitro. Moreover, intravenous administration of low doses of CXCL9 (B10– 30 mg kg 1) induced strong and specific inhibition of eosinophil recruitment into the lung in response to a variety of stimuli. Importantly, CXCL9 also inhibited a CCR3-mediated functional response, superoxide anion formation, in eosinophils. Regulation of eosinophil accumulation and activation in the lung is important since recent studies have demonstrated dramatic protection from experimental asthma in eosinophil-deficient animal models. Tumor cells have evolved the ability to regulate the angiogenic process through an imbalance in the regulatory factors resulting in an environment favoring angiogenesis. Studies have shown that the balance in expression between the angiogenic and the angiostatic CXC chemokines is correlated with the aggressiveness and metastic potential of human nonsmall cell lung carcinoma (NSCLC). Overexpression of CXCL9, an angiostatic CXC chemokine, at the tumor site resulted in inhibition of NSCLC tumor growth and metastasis via a decrease in tumor-derived vessel density. These findings support the importance of CXCL9 in inhibiting NSCLC tumor growth by attenuation of tumor-derived angiogenesis and demonstrate the potential of delivery of potent angiostatic CXC chemokines as a therapeutic option for NSCLC. See also: Allergy: Overview. Asthma: Overview. Chemokines, CXC: CXCL10 (IP-10). G-Protein-Coupled Receptors. Leukocytes: Eosinophils; T cells.

Further Reading Baggiolini M (1998) Chemokines and leukocyte traffic. Nature 392: 565–568. Baggiolini M, Dewald B, and Moser B (1997) Human chemokines: an update. Annual Review of Immunology 15: 675–705. Cascieri MA and Springer MS (2000) The chemokine/chemokinereceptor family: potential and progress for therapeutic intervention. Current Opinion in Chemical Biology 4: 420–427. Chada S, Ramesh R, and Mhashilkar AM (2003) Cytokine- and chemokine-based gene therapy for cancer. Current Opinion in Molecular Therapeutics 5: 463–474. Farber JM (1997) Mig and IP-10: CXC chemokines that target lymphocytes. Journal of Leukocyte Biology 61: 246–257. Liao F, Rabin RL, Yannelli JR, et al. (1995) Human mig chemokine: biochemical and functional characterization. Journal of Experimental Medicine 182: 1301–1314. Luster AD (1998) Chemokines: chemotactic cytokines that mediate inflammation. New England Journal of Medicine 338: 436– 445. Moser B and Loetscher P (2001) Lymphocyte traffic control by chemokines. Nature Immunology 2: 123–128. Moser B, Wolf M, Walz A, and Loetscher P (2004) Chemokines: multiple levels of leukocyte migration control. Trends in Immunology 25: 75–84. Park MK, Amichay D, Love P, et al. (2002) The CXC chemokine murine monokine induced by IFN-g (CXC chemokine ligand 9) is made by APCs, targets lymphocytes including activated B cells, and supports antibody responses to a bacterial pathogen in vivo. Journal of Immunology 169: 1433–1443. Romagnani P, Lasagni L, Annunziato F, Serio M, and Romagnani S (2004) CXC chemokines: the regulatory link between inflammation and angiogenesis. Trends in Immunology 25: 201–209. Rothenberg ME (2000) Basic science of chemokines and their receptors. In: Rothenberg ME (ed.) Chemokines in Allergic Disease, pp. 1–67. New York: Dekker. Schwarz MK and Wells TN (2002) New therapeutics that modulate chemokine networks. Nature Reviews: Drug Discovery 1: 347–358. Zlotnik A and Yoshie O (2000) Chemokines: a new classification system and their role in immunity. Immunity 12: 121–127.

CXCL10 (IP-10) F Kheradmand and D B Corry, Baylor College of Medicine, Houston, TX, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Interferon-inducible protein of 10 kDa (IP-10, CXCL10) is the tenth member of the CXC family of small chemotactic cytokines and plays an essential role in the recruitment of T-helper-1 (Th1) cells, natural killer (NK) cells, macrophages, and dendritic cells into sites of tissue inflammation. In addition to leukocyte trafficking, CXCL10 binds to several G-protein-coupled receptors and induces a variety of cellular effects such as inhibition of endothelial cell proliferation, inhibition of growth factor-dependent hematopoiesis, and tumor necrosis. In humans, diverse cellular responses to CXCL10 are mediated through its two cell surface receptor isoforms that have distinct tissue expression patterns. CXCL10 acts as an antagonist of CCR3, a T-helper-2

CHEMOKINES, CXC / CXCL10 (IP-10) (Th2) cell-specific chemokine receptor through competition with eotaxin (CCL11), a functional ligand for this receptor. Modulation of CXCL10 is implicated as a mediator of several pathological conditions of the lung such as sarcoidosis, pulmonary fibrosis, emphysema, and asthma. In mice, overexpression of CXCL10 results in exaggerated Th2 immune responses to antigen in a murine model of allergic lung disease, whereas deletion of this gene results in worsening of pulmonary fibrosis in a bleomycin-based model of acute lung injury. Excessive production of CXCL10 by human lung T cells is associated with smoking-induced emphysema. Activated human macrophages obtained from the lungs of patients with emphysema showed high expression of CXCR3, a functional CXCL10 receptor, ligation of which induced secretion of macrophage metalloelastase (matrix metalloproteinase 12; MMP-12), an elastolytic proteinase implicated in lung destruction in emphysema.

Introduction As part of a directed search for cytokine-responsive genes, a gene with a predicted polypeptide size of 10 kDa (interferon-inducible protein of 10 kDa, IP10, CXCL10) was discovered. Subsequently, a variety of cells including T cells, endothelial cells, monocytes, fibroblasts, and keratinocytes were shown to release CXCL10 in response to interferon gamma (IFN-g) and lipopolysaccharide (LPS). CXCL10 peptide shows approximately 40% homology to the other members of the CXC family, in particular interleukin-8 (IL-8) and platelet factor 4 (PF4). Further characterization of the amino acid sequence of this protein showed that the first two of four conserved cysteines are separated by one amino acid (CXC, where X could be any amino acid). Thus, the common name IP-10 was changed to CXCL10 using the chemokine systematic nomenclature. Consistent with its anti-angiogenic effects, this cytokine is shown to play a prominent role in T-cell-dependent tumor immunity and, most recently, upregulation of this chemokine is reported in chronic and acute inflammatory diseases such as acute allograft rejection, central nervous system inflammation in multiple sclerosis, psoriasis, and emphysema (see Figure 1).

Structure The human CXCL10 gene is located on chromosome 4 (q12–21) in a cluster that contains many other members of the CXC family such as IL-8 and PF4. Mouse and human CXCL10 have 67% sequence identity while the two other mouse chemokines that bind to the same CXCR3 receptor, CXCL9, and CXCL11, are less than 30% identical to CXCL10. Nuclear magnetic resonance (NMR) spectroscopy further showed that CXCL10 and CCL11 bind in a similar manner to both CCR3 and CXCR3. A ‘twostep’ model describes binding of CXCL10 to its receptor, where in the first step, the docking domain or the region between the first pair of conserved cysteines and the first b-strand binds to a region of the N-terminus of CXCR3. In the second or activation step, residues near the N-terminus of CXCL10 bind to a second region on the receptor that can, only in the case of CXCL10, induce an active conformation. This second step is not fulfilled upon binding of CXCL10 to CCR3, providing an atomic basis for the inhibition of this receptor by CXCL10.

Regulation of Production and Activity Transient and rapid increase in CXCL10 mRNA and protein synthesis in response to IFN-g and LPS was first reported in a monocytic cell line, but was later reported in a variety of primary cells. Additionally, stimulation of cells with double-stranded RNA has been shown to induce the transcription of CXCL10 in a manner dependent on intact interferon-stimulated response element (ISRE) and nuclear factor kappa B (NF-kB) binding sites within its promoter. Thus, in addition to IFN-g, multiple Toll-like receptors (TLRs) such as TLR-3 ligand or double-stranded RNAs can also induce upregulation of this chemokine. Immunohistochemical analysis has shown that bronchial epithelium is an important source of

Protective functions

Tumor immunity Lung fibrosis T-cell host defense

403

Pathological diseases

CXCL10

Emphysema Allograft rejection Sarcoidosis Asthma

LPS Figure 1 Biological functions of CXCL10. Many cells of the lung including epithelial cells, macrophages, and T cells express CXCL10 upon activation by IFN-g or LPS. CXCL10 plays a protective role in T-cell-dependent tumor immunity and host defense. Under pathological conditions, CXCL10 is associated with emphysema, allograft rejection, sarcoidosis, and exaggerated allergic lung inflammation.


CXCL10, which serves to recruit activated T-helper1 (Th1) cells for normal host defense against intracellular pathogens of the lung. Induction of CXCL10 mRNA in response to IFN-g is seen as early as 30 min, with peak levels seen after 5 h. Signaling intermediates requisite to CXCL10 gene expression include Jak-2 and STAT1. Interestingly, prostaglandin E2, histamine, and vasoactive intestinal peptide, which all inhibit Jak-2 and STAT1, also inhibit IFNg-dependent induction of CXCL10. Peroxisome proliferator-activated receptor-g (PPAR-g), a member of the nuclear hormone receptor superfamily, originally shown to play an important role in adipocyte differentiation and glucose homeostasis, also regulates inflammatory responses by inhibiting IFN-g-induced mRNA and protein expression of CXCL10 in endothelial cells, although the molecular mechanism for this inhibition is not understood.

Biological Function The most notable biological activity of CXCL10 is its ability to direct the recruitment of Th1 and related effector cells in acute and chronic inflammatory conditions. Along with the other CXCL chemokine members, CXCL10 must bind to cell surface heparan sulfate proteoglycans to establish the necessary chemokine gradients required for extravasation of these critical cellular mediators of host defense and tissue transplant rejection. As with other chemokines, the functions of CXCL10 are redundant with respect to other class-specific chemokines. In experimental models of transplantation, anti-CXCL10 monoclonal antibodies prolonged allograft survival but, surprisingly, CXCL10-deficient mice acutely rejected the same allografts. This can be partly explained by redundancy in the activity of two other members of the CXC family, CXCL9 (Mig) and CXCL11 (I-TAC), which share the same receptor as CXCL10. Nonetheless, in support of an important role for CXCL10 in allograft rejection, compared with wild-type donors, use of CXCL10 null donor hearts grafted into wild-type mice reduced intragraft expression of cytokines, chemokines, and their receptors, and associated leukocyte infiltration, and graft injury. These findings support the notion that tissue-specific generation of CXCL10 in response to inflammation is sufficient for the progressive infiltration and amplification of multiple effector pathways, and that targeting of CXCL10 can prevent acute rejection under some circumstances. Further, CXCL10 blockade impeded the expansion and migration of peripheral antigen-specific T cells in a mouse model of virus-induced organ-specific autoimmune disease. Finally, the role that CXCL10 plays

in many pathological conditions is complicated by its dual regulatory activity since it can act as an agonist for Th1 responses by binding to its own receptor, and as an antagonist for CCR3 by competing with CCR3 ligands for binding to this receptor.

Receptors CXCL10 binds to and activates two distinct receptors, CXCR3 and CXCR3B, as do monokine induced by IFN-g (Mig/CXCL9) and IFN-inducible T-cell alpha chemoattractant (ITAC/CXCL11). These receptors were cloned from a human CD4 þ T-cell library and were found to have significant sequence homology to the two IL-8 receptors. Both the human and mouse CXCR3 genes were mapped to the X chromosome. Receptor structure function analysis revealed that similar to the other chemokine receptor family members, CXCR3 is a seven transmembrane G-protein-coupled receptor, which mediates calcium mobilization and chemotaxis in response to the ligands CXCL10 and CXCL9. Initially, expression of CXCR3 was thought to be restricted to IL-2-activated T lymphocytes and not in resting T and B lymphocytes, monocytes, or granulocytes. Subsequently, CXCR3 was found on other activated cells of the immune system including natural killer (NK) cells, granulocytes, and macrophages. In addition to chemotaxis, the known CXCR3 ligands, CXCL9, CXCL10, and CXCL11, induce secretion of macrophage metalloelastase (matrix metalloproteinase 12; MMP-12) from activated human lung macrophages. Mutational analysis showed that binding of CXCL10 to its receptor is through the N-terminal residue Arg-8, preceding the first cysteine. CXCR3expressing, GAG-deficient Chinese hamster ovary cells remained responsive to CXCL10, suggesting that the CXCR3 and heparin binding sites of CXCL10 are only partially overlapping an interaction thought to be important for its sequestration on endothelial and other cells.

CXCL10 in Respiratory Diseases CXCL10 has been associated with a wide spectrum of lung inflammatory diseases through both pro- and antifibrotic effects and its ability to recruit cells that secrete or respond to IFN-g. For example, CXCL10 mRNA and protein are markedly increased in the lungs of mice challenged with bleomycin, a potent inducer of lung fibrosis. However, CXCL10 is thought to be protective in this setting as mice deficient in CXCL10 showed increased susceptibility to fibrosis while transgenic CXCL10 overexpressing mice showed reduced mortality and less severe

CHEMOKINES, CXC / CXCL10 (IP-10)

can contribute to Th2-type inflammation in experimental asthma. Contrary to this study, adenovirusmediated overexpression of CXCL10 in the airways in a mouse model of asthma resulted in significant inhibition of eosinophils in the BAL fluid accompanied by enhanced IFN-g, and reduced IL-4 secretion that was dependent on IFN-g. These findings are more in line with the above-mentioned multifunctional activity of CXCL10 regarding its binding to CXCR3 and acting as a potent antagonist of many Th2 chemokines. Collectively, these contradictory data illustrate that local expression of the chemokine CXCL10 can exert diverse effector responses depending on the exact immunological context. Smoking-induced emphysema is associated with chronic activation of Th1 type inflammation and, in particular, upregulation of CXCL9 and CXCL10, and their shared receptor, CXCR3 on lung airway epithelial cells, T cells, and macrophages. Expression of CXCL10 has also been demonstrated in the lung macrophages and in T cells isolated in the lungs of patients with smoking-induced emphysema. The accumulation of T cells and monocytes at sites of ongoing inflammation represents the earliest step in a series of events that lead to granuloma formation in sarcoidosis and destruction of lung elastin fibers in emphysema. High levels of a CXCL10 protein have been demonstrated in the BAL fluid of patients with pulmonary sarcoidosis and lymphocytic alveolitis as compared with patients with inactive disease or control subjects. Similarly, lung CD4 þ T cells secreted a higher concentration of CXCL10 in emphysema subjects as compared to controls. In addition, more recently it has been shown that beyond their historic role as chemoattractants, members of the CXCL

bleomycin-induced pulmonary fibrosis. The mechanism for CXCL10-dependent survival in this model was inhibition of recruitment of lung fibroblasts, which are largely responsible for synthesis of the matrix proteins that underlie most fibrosing conditions. Paradoxically, however, elevated levels of CXCL10 have been found in bronchoalveolar lavage (BAL) fluids of humans who have undergone lung transplantation with acute and chronic rejection, a complication that usually leads to widespread fibrosis of the transplanted lung. However, in vivo neutralization of CXCR3 or CXCL10 decreased intragraft recruitment of CXCR3-expressing mononuclear cells and attenuated chronic lung rejection and bronchiolitis obliterans syndrome in mice. Furthermore, overexpression of CXCL10 was associated with worsening of lung fibrosis in the same transplantation model. Thus, CXCL10 appears to suppress lung fibrosis due to bleomycin, but is a primary mediator of lung transplant rejection. Although CXCL10 is primarily associated with Th1, or type I, immunity, its upregulation in type II inflammatory conditions such as asthma has also been reported. Mice that overexpress CXCL10 in the lung were shown to exhibit significantly increased eosinophilia, IL-4 levels, and CD8 ( þ ) lymphocyte recruitment compared with wild-type controls that received the same allergen. In addition, there was an increase in the percentage of IL-4-secreting T lymphocytes in the lungs of CXCL10 transgenic mice. In contrast, mice deficient in CXCL10 demonstrated the opposite results compared with wild-type controls, with a significant reduction in these measures of Th2-type allergic airway inflammation. These findings suggest that CXCL10, a Th1-type chemokine, CXCR3

405

Neutrophil Neutrophil elastase

A1AT IFN-

Elastin degradation Airway obstruction Emphysema

Th1 cell CXCL10

MMP-12

CXCR3 Macrophages Figure 2 CXCL10 in emphysema. Th1 effector cells of the lung secrete CXCL10, which, through its receptor CXCR3, recruits additional lung T cells, thus amplifying the inflammatory response to tobacco smoke. CXCL10 also recruits activated macrophages and neutrophils and induces macrophages to secrete MMP-12, an elastolytic enzyme that inhibits a1-antiproteinase (A1AT), which in turn is required for protection against human emphysema.


family (in particular CXCL10) actively induce production of MMP-12; this is a potent inhibitor of a1-antitrypsin, which is linked to the development of smoke-induced emphysema in humans (see Figure 2). Taken together, these data suggest that CXCL10 plays an important role in regulating recruitment of Th1 inflammation and may play a role in lung destruction that is associated with the chronic inflammatory lung diseases such as emphysema and sarcoidosis. See also: Chemokines. Chemokines, CXC: CXCL9 (MIG). Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General; Smoking Cessation. Drug-Induced Pulmonary Disease. Eotaxins. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens; Matricellular Proteins; Matrix Proteoglycans; Surface Proteoglycans; Degradation by Proteases. G-Protein-Coupled Receptors. Leukocytes: Monocytes; T cells; Pulmonary Macrophages. Lipid Mediators: Leukotrienes. Matrix Metalloproteinases. Serine Proteinases.

Luster AD, Greenberg SM, Leder P, et al. (1985) Gamma-interferon transcriptionally regulates an early-response gene containing homology to platelet proteins. Nature 315: 672–676. Luster AD and Leder P (1993) IP-10, a -C-X-C- chemokine, elicits a potent thymus-dependent antitumor response in vivo. Journal of Experimental Medicine 178: 1057–1065. Marx N, Mach F, Sauty A, et al. (2000) Peroxisome proliferatoractivated receptor-gamma activators inhibit IFN-gamma-induced expression of the T cell-active CXC chemokines IP-10, Mig, and I-TAC in human endothelial cells. Journal of Immunology 164: 6503–6508. Medoff BD, Sauty A, Tager AM, et al. (2002) IFN-gamma-inducible protein 10 (CXCL10) contributes to airway hyperreactivity and airway inflammation in a mouse model of asthma. Journal of Immunology 168: 5278–5286. Moser B and Willimann K (2004) Chemokines: role in inflammation and immune surveillance. Annals of the Rheumatic Diseases 63: ii84–ii89. Saetta M, Mariani M, Panina-Bordignon P, et al. (2002) Increased expression of the chemokine receptor CXCR3 and its ligand CXCL10 in peripheral airways of smokers with chronic obstructive pulmonary disease. American Journal of Respiratory and Critical Care Medicine 165: 1404–1409. Wiley R, Palmer K, Gajewska B, et al. (2001) Expression of the Th1 chemokine IFN-gamma-inducible protein 10 in the airway alters mucosal allergic sensitization in mice. Journal of Immunology 166: 2750–2759.

Glossary Further Reading Agostini C, Cassatella M, Zambello R, et al. (1998) Involvement of the IP-10 chemokine in sarcoid granulomatous reactions. Journal of Immunology 161: 6413–6420. Belperio JA, Keane MP, Burdick MD, et al. (2003) Role of CXCL9/CXCR3 chemokine biology during pathogenesis of acute lung allograft rejection. Journal of Immunology 171: 4844–4852. Campanella GS, Lee EM, Sun J, and Luster AD (2003) CXCR3 and heparin binding sites of the chemokine IP-10 (CXCL10). Journal of Biological Chemistry 278: 17066–17074. Dufour JH, Dziejman M, Liu MT, et al. (2002) IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking. Journal of Immunology 168: 3195–3204. Gasperini S, Perin A, Piazza F, et al. (1995) The IP-10 chemokine binds to a specific cell surface heparan sulfate site shared with platelet factor 4 and inhibits endothelial cell proliferation. Involvement of the IP-10 chemokine in sarcoid granulomatous reactions. Journal of Experimental Medicine 182: 219–231. Grumelli S, Corry D, Song L, et al. (2004) An immune basis for lung parenchymal destruction in chronic obstructive pulmonary disease and emphysema. Public Library of Science Medicine 1: 74–83. Hancock WW, Gao W, Csizmadia V, et al. (2001) Donor-derived IP-10 initiates development of acute allograft rejection. Journal of Experimental Medicine 193: 975–980. Loetscher M, Gerber B, Loetscher P, et al. (1996) Chemokine receptor specific for IP10 and mig: structure, function, and expression in activated T-lymphocytes. Journal of Experimental Medicine 184: 963–969. Loetscher P, Pellegrino A, Gong JH, et al. (2001) The ligands of CXC chemokine receptor 3, I-TAC, Mig, and IP10, are natural antagonists for CCR3. Journal of Biological Chemistry 276: 2986–2991.

Adipocytes – fat cells Allograft – graft of tissue from non-self donor of the same species Bleomycin – a chemotheraputic agent Chemokine – small chemoattractant proteins that stimulate the migration and activation of cells Cytokines – proteins made by cells that affect the function of another cell Dendritic cells – cells found in T-cell regions of lymph node and the most potent stimulators of T-cell responses Eotaxins – CC chemokines that act predominantly on eosinophils Jak-2 – a Janus-family tyrosine kinase (JAK) signaling molecules that are activated by aggregation of cytokine receptors Lipopolysaccharide – a cell wall antigen present in Gram-negative bacteria Matrix metalloproteinases – a large family of zincdependent endopeptidases Multiple sclerosis – an autoimmune-mediated neurodegenerative disorder Proteoglycans – cell surface proteins with carbohydrate side chains

CHEMOKINES, CXC / CXCL1 (GRO1)–CXCL3 (GRO3) 407

Psoriasis – an inflammatory skin disorder STAT1 – signal transducer and activator of transcription, a transcription factor, that is phosphorylated by JAKs T-helper cell type 1 – a subset of CD4 T cells that are characterized by the cytokines they produce. They are mainly involved in activating macrophages Toll-like receptors – cell surface receptors that are initiated by Toll pathway that activates the transcription factor NF-kB by degrading its inhibitor IkB

nuclear factor kappa B (NF-kB) transcription factor dependent. Probing against leukocyte gene library revealed two additional GRO-like molecules, designated GROb and GROg; these had respective 90% and 86% amino acid identity with GROa. Compared to GROa, there are 11 amino acid substitutions in GROb and 15 in GROg of which some confer conformational changes in protein structure. The GROb and GROg molecules were found to be identical to independently identified human MIP-2a and MIP-2b which are chemokine genes homologous to the MIP-2 genes of mice. It is now accepted that the GROa, GROb, and GROg are members of the CXCL groups of chemokines and in current standardized nomenclature are designated CXCL1–3.

CXCL1 (GRO1)–CXCL3 (GRO3)

Structure

S W Chensue, VA Ann Arbor Healthcare System, Ann Arbor, MI, USA

Genes consisting of approximately 1100 nucleotide base pairs for CXCL1–3 are encoded on long arm of chromosome 4 in or near the CXC chemokine locus (4q21). The protein structure of CXCL1 closely resembles other members of the CXCL group. In solution, CXCL1 is a dimeric chemokine consisting of monomers of 73 amino acid residues. The dimer contains a six-stranded antiparallel b-sheet packed against two C-terminal antiparallel a-helices (Figure 1). The CXC designation derives from characteristic conserved double-cysteine motif separated by single amino acid in the N-terminal portions of the molecule. CXCL1–3 are also classed as ELR chemokines based on the presence of a glutamate–leucine–arginine sequence adjacent and N-terminal to the CXC motif.


Abstract CXCL1–3 are members of the CXCL class of chemokines with neutrophil chemotactic and angiogenic biologic properties. These molecules exist as dimers and interact principally with the guanosine nucleotide-protein-coupled, CXCR2 chemokine receptor expressed by neutrophils as well as by other cell types. CXCL1–3 are elicited by microbial products and cytokines and likely contribute to inflammatory and repair responses as part of a broad spectrum of chemotactic mediators. Evidence to date indicates that these molecules participate in multiple pulmonary diseases including infections, adult respiratory distress syndrome, chronic obstructive pulmonary disease, hyperoxia-induced lung injury, fibrosis, and neoplasm.

Introduction The GROa molecule was first identified in the 1980s as an early response gene produced by cultured melanoma cells with autocrine growth promoting function. GROa also known as melanoma growth stimulating activity (MGSA) was expressed constitutively by serum-cultured transformed fibroblasts and could be induced in normal cells by the inflammatory cytokines, interleukin-1 (IL-1), and tumor necrosis factor alpha (TNF-a). While initially thought to be a growth-related oncogene due to its growthpromoting effects on melanoma cells, it was quickly apparent that GROa was structurally related to the supergene family of small 8–15 kDa inflammatory neutrophil chemotactic proteins or chemokines that included IL-8, neutrophil activating protein-2, and macrophage inflammatory protein-2 (MIP-2). Like these molecules, GROa was mapped to the chemokine locus on chromosome 4 and its expression was

Figure 1 Secondary molecular structure of CXCL1 (GROa) dimer. Note six antiparallel b-pleated sheets (red) positioned against two antiparallel a-helices (blue).

408 CHEMOKINES, CXC / CXCL1 (GRO1)–CXCL3 (GRO3)

Regulation of Production While CXCL1 is produced constitutively in some transformed cell lines, under physiologic conditions it is likely induced in normal cells by exogenous stimuli such as microbial products and/or inflammatory cytokines. In particular, bacterial endotoxin, IL-1, and TNF-a are potent inducers of CXCL chemokines in mononuclear phagocytes, epithelial cells, and structural mesenchymal cells. However, it should be noted that numerous infectious agents have been described as inducers of CXCL gene expression. The gene loci for CXCL1–3 have NF-kB binding sites and as such, the NF-kB transcription pathway is considered critical in regulating the expression of these genes. It is likely that during infection microbial products interact with Toll-like receptors on host cells to initiate phosphorylation events leading to release of active NF-kB which translocates to the nucleus to bind to gene promoter sites. The CXCL genes represent only a portion of the expressed gene profile. IL-1 can be among those induced genes and there is evidence indicating that IL-1 may regulate CXCL1–3 gene expressions by stabilizing transcript levels through inhibition of transcript degradation. The restoration of transcription regulatory pathways results in eventual arrest of gene expression. Neoplastic cells can display dysregulated CXCL gene expression which may allow them to function as autocrine growth or oncogenic factors.

Biological Function Originally, CXCL1 (GROa) was considered to be an oncogenic growth factor since it appeared to have the capacity to promote growth and malignant transformation of cultured melanocytes. Since that time a number of other functions have been attributed to this group of molecules. These include regulation of tissue repair-related mesenchymal cell function. Two examples should be mentioned. First, CXCL1 caused a dose-dependent decrease in the expression of interstitial collagens by cultured rheumatoid synovial fibroblasts. The effect was specific, as there was no demonstrable effect on other products. Unlike its mitogenic effect on melanoma cells, it had no effect on the proliferation rate of fibroblasts. Thus, CXCL1 may limit or temper the scarring process following injury. Second, similar to other ELRþ chemokines CXCL1 is implicated as a proangiogenic factor, that is, stimulating the growth of new small vessels at sites of injury or tumor growth. This contrasts with ELR-CXCL chemokines which inhibit angiogenesis. These opposing activities suggest the presence of

carefully orchestrated signals that are required to shape events involved in the tissue repair response. The best-characterized function of CXCL1–3 is their chemoattractant activity. Like other ELRþ CXCL chemokines, these molecules have potent effects on neutrophil migration and activation. This activity appears to be further amplified by natural enzymatic truncation of the amino terminal portion of the molecules; this modification causes a 30-fold increase in biologic potency. Thus, under conditions of inflammation, proteolytic enzymes may boost the chemotactic function of these molecules. Numerous studies have provided circumstantial evidence for an in vivo association of neutrophil accumulation with production of CXCL1–3. However, it has been difficult to provide direct evidence by neutralization approaches due to the redundancy of CXC ligands. Some direct evidence for in vivo biologic activity has been derived from CXCR2 receptor knockout mice which display impaired neutrophil recruitment and delayed wound healing. In addition, engineered chemical antagonists of the CXCR1 and CXCR2 receptors likewise cause impaired neutrophil mobilization. Taken together, the functional activities of CXCL1–3 would appear to be teleologically appropriate for the acute inflammatory response to infection. Following epithelial injury and infection, these molecules would simultaneously promote epithelial healing, leukocyte recruitment, and neovascularization, representing a coordinated effort to prevent further infestation, initiate microbial elimination, and optimize delivery of additional leukocytes and antimicrobial humoral factors.

Receptors The functional high-affinity receptor for CXCL1–3 is well characterized and is designated by standardized nomenclature as CXCR2, formerly known as IL-8B or IL-8 type 2 receptor. CXCR2 is promiscuous and binds multiple ELRþ CXC ligands. CXCR2 is an archetypical guanosine nucleotide-protein-coupled receptor (GPCR) with seven transmembrane hydrophobic domains having three intracellular and three extracellular hydrophilic loops. Ligation of the extracellular amino-terminal portion of the receptors triggers calcium influx and intracellular activation of transduction factors such as protein kinase C and MAP kinase. These events stimulate downstream functions such as cell movement, degranulation, adhesion, and respiratory burst. CXCR2 receptors have been detected on neutrophils, monocytes, basophils, mast cells, endothelial cells, T lymphocytes, natural killer, and neuroendocrine cells. However, neutrophils appear to be the dominant expressing cells.

CHEMOKINES, CXC / CXCL1 (GRO1)–CXCL3 (GRO3) 409

Due to the promiscuity of the receptor, it is difficult to assess the contribution of individual CXC ligands in experimental animals with targeted deletion of CXCR2. Another promiscuous receptor for CXCL1– 3 is the Duffy antigen/chemokine receptor. Duffy binds both CXC and CC chemokines and is expressed by erythrocytes in Duffy-positive individuals, endothelial cells of postcapillary venules, and Purkinje cells of the cerebellum. The normal physiological function of Duffy remains unclear. The transduction pathways activated by Duffy are also unknown and the receptor has not been shown to act through a G protein. Reportedly, Duffy facilitates CXCL1 distribution and enhances neutrophil movement across endothelial cell monolayers.

Role of CXCL1–3 in Respiratory Diseases In addition to beneficial functions, CXCL1–3 have been implicated to participate in multiple pulmonary diseases ranging from infectious to neoplastic and usually are part of a broader spectrum of chemokine and cytokine mediators (Figure 2). In the lung, alveolar macrophages are critical sentinel cells and are likely an important source of CXCL1–3 but type II alveolar epithelial cells are known to be a potential source. The roles of these chemokines in different lung diseases are discussed separately below.

Infections In view of their neutrophil chemotactic properties, it might be expected that CXCL1, 2, or 3 would be detected during lung bacterial infections that elicit

Inflammation and repair Neutrophil recruitment

significant neutrophil influx. Indeed, these chemokines are readily detectable in bronchoalveolar lavage fluids of patients with infectious and inflammatory lung disease but at low levels in those of normal volunteers. Experimental studies of Pseudomonas aeruginosa, an important Gram-negative bacterium causing fatal necrotizing pneumonias, have shown this agent to be capable of inducing CXCL chemokines in alveolar macrophages and epithelial cells. Depletion of alveolar macrophages impairs both the production of CXCL1 and the recruitment of neutrophils. Other Gram-negative bacterial causes of pneumonia such as Escherichia coli are similarly capable of eliciting CXCL chemokines and innate recognition of bacterial endotoxin appears to be the common stimulatory molecule. However, Grampositive bacteria and Pneumocystis are also capable of eliciting CXCL1. In general, viruses such as respiratory syncitial virus (RSV) appear to elicit strong CC chemokine responses in the lung, but epithelial damage and secondary infection will elicit CXCL chemokines.

Adult Respiratory Distress Syndrome It is well known that critically ill patients are at increased risk for acute lung injury which is clinically manifest as adult respiratory distress syndrome (ARDS). CXCL1 is notably elevated in lung lavage fluids obtained from ARDS patients. Accordingly, neutrophil influx correlates well with levels of CXCL chemokines. The alveolar epithelial damage observed in ARDS appears to be related to the inflammation and as such the CXCL chemokines might be contributing to the disease. Due to extensive redundancy in neutrophil recruitment, CXCL1–3 likely only contribute partially and there is evidence to indicate that other chemokines such as CXCL8 may be more important.

Tissue repair

Angiogenesis

Chronic Obstructive Pulmonary Disease

CXCL1,2,3

Tumor growth

ARDS Hyperoxia injury

Fibrosis COPD Lung diseases

Figure 2 Potential areas of functional involvement of CXCL1–3 in physiology and lung diseases.

There is strong evidence to suggest that inflammatory leukocytes including neutrophils contribute to the destruction of structural matrix observed in chronic obstructive pulmonary disease (COPD). Circumstantial evidence suggests that CXCL chemokines might contribute to neutrophil recruitment and activation during COPD. CXCL chemokines including CXCL1 are present in the sputum and lung lavage fluid of COPD patients. Cigarette smoke, the major cause of COPD, has been shown to elicit CXCL1 and neutrophil influx in rat lungs. Moreover, the neutrophil mobilization could be blocked with CXCL2 receptor antagonists. This receptor also appears to show

410 CHEMOKINES, CXC / CXCL1 (GRO1)–CXCL3 (GRO3)

enhanced expression on monocytes from smokers. These cells likewise migrate to lungs where they can potentially produce matrix destructive enzymes.

Fibrosing Alveolitis Cryptogenic fibrosing alveolitis (CFA) is a progressive fibrotic disease of the lung characterized by significant local recruitment of neutrophils which can comprise 10–20% of the cells in the lung lavage of affected patients. These cells are thought to contribute to the disease. Patients with CFA have increased levels of CXCL1 in their plasma and their neutrophils are hyperresponsive to the chemokine. Thus, chemokine-targeted therapies may have potential benefit in controlling the disease.

Hyperoxia-Induced Lung Injury Hyperoxia-induced lung injury is a potential serious side effect of oxygen therapy in hospitalized patients. The disease is characterized by infiltration of neutrophils with associated endothelial and epithelial cell injury, followed by interstitial fibrosis. CXCL1–3 may be important contributors to this condition. Animal experiments demonstrate that oxygen treatment induces expression of CXCL1–3, which correlates with the influx of neutrophils and mortality. Genetic deletion of the CXCR2 receptor blocked neutrophil recruitment and resulted in reduced oxygen-induced mortality.

Lung Cancer The role of CXCL1–3 in neoplastic disease of the lung remains controversial and studies are very limited. Due to their angiogenic and mitogenic properties, these chemokines are generally considered to support the growth of neoplastic tissue. Growth can potentially be enhanced by direct stimulation of chemokine receptors expressed by tumor cells especially melanocytic and neuroendocrine types. In addition, angiogenic activity would help to provide vascular support to growing tumors. In contrast to this notion, at least one study has provided evidence that CXCL2 is angiostatic and could inhibit the growth of Lewis lung carcinoma in mice. Clearly,

more studies would be needed to help clarify the contribution of CXCL1–3 in lung carcinoma. See also: Acute Respiratory Distress Syndrome. Chemokines. Chronic Obstructive Pulmonary Disease: Overview. Interleukins: IL-1 and IL-18. Pneumonia: Community Acquired Pneumonia, Bacterial and Other Common Pathogens. Tumor Necrosis Factor Alpha (TNF-a). Tumors, Malignant: Overview.

Further Reading Belperio JA, Keane MP, Arenberg DA, et al. (2000) CXC chemokines in angiogenesis. Journal of Leukocyte Biology 68: 1–8. Cao Y, Chen C, Weatherbee JA, Tsang M, and Folkman J (1995) gro-beta, a –C–X–C– chemokine, is an angiogenesis inhibitor that suppresses the growth of Lewis lung carcinoma in mice. Journal of Experimental Medicine 182: 2069–2077. Glynn PC, Henney EM, and Hall IP (2001) Peripheral blood neutrophils are hyperresponsive to IL-8 and Gro-alpha in cryptogenic fibrosing alveolitis. European Respiratory Journal 18: 522–529. Goodman RB, Pugin J, Lee JS, and Matthay MA (2003) Cytokinemediated inflammation in acute lung injury. Cytokine & Growth Factor Reviews 14: 523–535. Hashimoto S, Pittet JF, Hong K, et al. (1996) Depletion of alveolar macrophages decreases neutrophil chemotaxis to Pseudomonas airspace infections. American Journal of Physiology 270: L819– L828. Sager R, Haskill S, Anisowicz A, Trask D, and Pike MC (1991) GRO: a novel chemotactic cytokine. Advances in Experimental Medicine and Biology 305: 73–77. Stoeckle MY (1991) Post-transcriptional regulation of gro alpha, beta, gamma, and IL-8 mRNAS by IL-1 beta. Nucleic Acids Research 19: 917–920. Sue RD, Belperio JA, Burdick MD, et al. (2004) CXCR2 is critical to hyperoxia-induced lung injury. Journal of Immunology 172: 3860–3868. Traves SL, Smith SJ, Barnes PJ, and Donnelly LE (2004) Specific CXC but not CC chemokines cause elevated monocyte migration in COPD: a role for CXCR2. Journal of Leukocyte Biology 76: 441–450. Vanderbilt JN, Mager EM, Allen L, et al. (2003) CXC chemokines and their receptors are expressed in type II cells and upregulated following lung injury. American Journal of Respiratory Cell and Molecular Biology 29: 661–668. Villard J, Dayer-Pastore F, Hamacher J, Aubert JD, Schlegel-Haueter S, and Nicod LP (1995) GRO alpha and interleukin-8 in pneumocystis carinii or bacterial pneumonia and adult respiratory distress syndrome. American Journal of Respiratory and Critical Care Medicine 152: 1549–1554. Wuyts A, Govaerts C, Struyf S, et al. (1999) Isolation of the CXC chemokines ENA-78 GRO alpha and GRO gamma from tumor cells and leukocytes reveals NH2-terminal heterogeneity. Functional comparison of different natural isoforms. European Journal of Biochemistry 260: 421–429.

CHEMORECEPTORS / Central 411

CHEMORECEPTORS Contents

Central Arterial

Central

PaCO2

N S Cherniack, UMDNJ-New Jersey Medical School, Newark, NJ, USA M D Altose, Case Western Reserve University School of Medicine, Cleveland, OH, USA

Chemoreceptors Chemical drives


Controller Ventilatory demand

Abstract The powerful stimulation of ventilation by carbon dioxide inhalation and the near-constancy of arterial PCO2 during rest and exercise indicate the importance of CO2/H þ receptors in the regulation of breathing. There are both peripheral and central chemoreceptors with the central ones accounting for about 70% of the CO2 effect. These sensors operate in feedback control systems that help maintain levels of CO2 in the body within narrow limits. Techniques that allow individual cells to be studied have provided much new information on central chemoreceptors. Groups of neurons that increase their activity in response to changes in acidity in their external environment are widely scattered in the medulla. It is generally believed that the proximal stimulus to these central receptors is intracellular acidity. These neuronal groups when stimulated by the changes in local hydrogen ion concentration excite breathing, but each group separately accounts for a rather small part of the increase in ventilation that can be elicited by CO2 inhalation. There are no data yet on whether the various sensory groups have different thresholds though some seem to be excited only during wakefulness, which might imply a high threshold, and others only during sleep, which is not easily explained by threshold differences. Furthermore, it is not known whether the various groups differ in their operative ranges or sensitivity. It is also possible that some of these groups are parts of other functional systems involved, for example, in producing arousal. Otherwise healthy humans who do not respond to breathing CO2 can maintain normal levels of arterial CO2 when awake. But in patients with thoracic diseases, low sensitivity to CO2 may contribute to CO2 retention. During sleep, a threshold level of CO2/H þ is required to prevent apnea. In the absence of a response to CO2, abnormal elevation of PCO2 occurs during sleep. Depressed chemosensitivity may be involved in the sudden infant death syndrome. In addition, depressed chemosensitivity may predispose to respiratory failure in patents with thoracic diseases.

Introduction Description of Central Chemoreceptor Effects on Ventilation

Respiration is regulated by a negative feedback control system that serves to hold PCO2 in the arterial

PaO2 PbrainCO2

Respiratory muscles, lung

Ventilation O2, CO2 body stores PaCO2

PaO2 PbrainCO2

Figure 1 Block diagram showing components and pathways involved in the chemical control of breathing.

blood and in the brain within narrow limits and to maintain acid–base homeostasis (see Figure 1). This system consists of a controller made up of respiratory neurons and chemoreceptors and a plant or controlled system consisting of the body tissues in which CO2 is generated by metabolic processes. The system operates to maintain PCO2 (or H þ ) at some desired level called the operating point. The two components are linked via the circulation, which carries information about levels of PCO2 and PO2 in the blood. When CO2 levels in the body rise as a result of increases in metabolic CO2 production or disturbances that decrease ventilation, central chemoreceptors in the brain and peripheral chemoreceptors in the carotid and aortic bodies are stimulated. Impulses from these chemoreceptors, in turn, act on respiratory motor neurons in the medulla to produce compensatory increases in ventilation to return PCO2 to its normal level. Similarly, disturbances that increase ventilation and reduce levels of PCO2 and H þ ion cause compensatory decreases in breathing to help restore PCO2 /H þ levels. When CO2 is breathed, the chemoreceptors augment ventilation, minimizing the rise in PCO2 that would otherwise occur. The increases in ventilation

412 CHEMORECEPTORS / Central

PCO

2

A

B

Ventilation Figure 2 Effect of ventilation on PCO2 . At low ventilation and high PCO2 (A, slope highlighted in red) the effect of ventilation on PCO2 is greater than at (B) where ventilation is higher and PCO2 is lower. Slope is an index of plant gain.

are proportional to the deviation in PCO2 when CO2 is inhaled but the compensation is never sufficient to restore PCO2 to its original levels. The sensitivity or gain of the chemical controller is usually expressed as the change in ventilation produced by a given change in PCO2 in the arterial blood and averages about 2 l for each mmHg change in PCO2 in adult humans. In general, the higher the gain the less is the rise in PCO2 . However, the efficacy of the control system also depends on the effect of ventilation on PCO2 . The ratio of the change in PCO2 to the change in ventilation is sometimes called plant gain. As shown in Figure 2, because the effects of ventilation on PCO2 are less at lower than at higher levels of PCO2 , plant gain increases as resting arterial PCO2 rises (when breathing CO2 free gas). Increases in plant gain and controller gain (more precisely their product or loop gain) will shorten the time needed for the control system to reach equilibrium after a disturbance. However, too high a loop gain can lead to control-system instability and oscillations in minute ventilation. All animals that extract oxygen from the atmosphere have CO2/H þ responsive chemoreceptors. CO2 receptors have been found on the ventral surface of the caudal and rostral medulla of the isolated bullfrog brainstem. Fish have CO2 levels of only a few mmHg and receptors for CO2/H þ sample the ambient water rather than changes within the body. In anuran amphibians, CO2 chemoreceptors account for 70% of the ventilatory response to CO2. In cold-blooded species the normal PCO2 varies considerably with body temperature becoming lower as temperature drops. In mammals, on the other hand, there is relatively little direct effect of temperature on PCO2 levels that roughly average 40 mmHg in the arterial blood of humans at rest. Temperature elevations, if anything, increase ventilation, particularly in panting animals.

Carbon dioxide chemoreceptors have been most extensively studied in mammals. Approximately 70% of the ventilatory response to CO2 results from the activation of central chemoreceptors in the brain. Only about 30% of the increase in ventilation following the inhalation of a CO2 enriched gas mixture is attributable to the activity of peripheral chemoreceptors, mainly the carotid body chemoreceptors. Because of the rapidity of the effects of CO2 inhalation on the arterial blood as compared to the brain, the peripheral chemoreceptors respond far more quickly to changes in CO2 than do the central ones. Both central and peripheral chemoreceptors respond to PCO2 only when it reaches a threshold value. The peripheral chemoreceptors are believed to respond to lower levels of PCO2 than the central chemoreceptors. Drives from central and peripheral chemoreceptors have an additive effect. While the peripheral chemoreceptors respond to changes in arterial PCO2 , the central chemoreceptors respond to changes in PCO2 in the brain. Levels of CO2 in the brain depend on rates of cerebral perfusion in relation to metabolism as well as ventilation. Since changes in PCO2 occurring naturally are always associated with changes in H þ , it is now generally considered that the proximate stimulus for central chemoreception is intra- and extracellular brain H þ , rather than the H þ concentration of the cerebrospinal fluid (CSF), previously thought to be the main factor determining ventilation. The importance of chemical control in mammals in determining ventilation depends on behavioral states and higher brain center influences may modify the characteristics of the chemical controller. It is generally agreed that chemical stimuli are the main factors regulating breathing during nonrapid eye movement (nonREM) sleep when decreases in PCO2 of only a few mmHg can produce apnea. In the waking state, neural drives emanating from sensory receptors in the body and from the environment constitute a greater portion of the ventilatory drive as shown in Figure 3. As a result breathing can be maintained at near-normal levels in the absence of any discernible response to inhaled CO2. A discernible response to CO2 is also not necessary for normal PCO2 levels to be maintained during moderate exercise, but in its absence PCO2 rises considerably rather than decreasing when the anaerobic threshold is exceeded.

Structure and Function Location and Morphology of Central Chemoreceptors

The precise location and distribution of central respiratory-related chemoreceptors in the brain has

Ventilation


Awake

Asleep

0 PaCO2

Figure 3 Effect of changes in arterial PCO2 on ventilation during sleep and wakefulness. As PaCO2 is decreased, apnea occurs during sleep but not in the awake state.

been a matter for investigation for decades and has not yet been fully established. It is generally thought that the central chemoreceptors are grouped in clusters in several different locations in the medulla and even in other areas of the brain. The characteristics of the different clusters in terms of their thresholds, sensitivities, and mechanisms of sensing CO2 seem to vary. The study of central chemoreceptors is further complicated because CO2 centrally affects more than ventilation. It increases sympathetic activity, causes the release of catecholamines, helps produce arousal, increases cerebral blood flow, increases anxiety, and can produce a sense of air hunger. Thus, it is possible that different central chemoreceptors may also differ in their functions. In studies in intact animals, central chemoreceptors were defined as cells that, when stimulated by acid or CO2, lead to an increase in ventilation or respiratory motor activity and/or when inhibited, decrease the respiratory stimulating actions of acid or CO2 without affecting respiratory activity excited by other types of respiratory stimuli. For some time it was believed that respiratory motor neurons themselves had properties of chemosensitivity. Animal studies by Loeschke and by Mitchell and others, using the direct application of acidic or basic fluids, demonstrated discrete clusters of chemoresponsive neurons on the ventrolateral surface of the medulla. These include a rostral chemosensitive area (Mitchell’s area) and a caudal chemosensitive area (Loeschke’s area). Stimulation of the Loeschke and Mitchell areas on the ventral surface of the medulla increases breathing in anesthetized animals. Correspondingly, ablation by cooling of the intermediate area between them, presumably by blocking afferent nerve fibers from the rostal and caudal areas, produces apnea. It has been observed that patients with brain lesions around

Mitchell’s area show depressed ventilatory responses to CO2 during sleep. This supports the presumption that the ventrolateral medulla is an important site of central chemoreceptors. With time, more precise ways of stimulating or blocking specific areas of the brain using microinjection or dialysis with various agents such as carbonic anhydrase inhibitors, solutions equilibrated with CO2, neuronal stimulants and inhibitors, and neurotoxins were developed. Using these techniques, additional chemoreceptive areas were identified farther from the ventral surface and in the dorsal areas of the medulla, in the pons, in the midbrain, and even in higher brain structures. However, stimulation and ablation interventions in vivo are imperfect techniques for identifying the precise location of central chemoreceptors because the penetration of a local intervention to deeper and more distant structures cannot be totally controlled. Even quite localized stimulation and ablation can produce reactions in more distant areas through neural connections that can be either excitatory or inhibitory to breathing. These interventions can affect systems other than respiration, such as circulation, metabolism, autonomic nervous system, and arousal, which may indirectly alter breathing. Techniques that use fluorescent voltage-sensitive dyes and those that measure increased activity of early immediate genes such as c-fos and c-jun to signal neural responses following exposure to high CO2 levels have identified neurons activated by CO2. However, such responses may also not be specific and lead to increases in ventilation. Studies of isolated brain and spinal cord preparations of neonatal animals and in brain slices and cell cultures have identified additional chemoreceptive areas and more importantly have elucidated critical cellular and molecular mechanisms that are involved in sensing changes in CO2/H þ . However, in these studies the criteria for chemoresponsiveness have been broadened to include changes in transmembrane potentials. The relationship of these membrane changes to respiratory activity is indirect. Groups of cells that depolarize and increase their excitability with hypercapnia and with local changes in acidity have also been found in nonrespiratory areas of the brain but the magnitude of the responses is less than in cells from areas involved with respiration. It is currently believed that central chemoreceptors away from sites near the ventral medullary surface (like the retrotrapezoid nucleus) are located in the dorsal medullary regions including the nucleus of the solitary tract, the locus ceruleus, the median raphe, the pre-Boetzinger complex, and the fastigial nucleus


of the cerebellum. Chemosensitive cells that respond to CO2 by increasing respiratory frequency have also been found in the spinal cord of brainstem–spinal cord preparations from newborn mice. Almost all of the areas in which CO2/H þ responsive cells have been found are concerned with other physiological functions besides respiration. For example, neurons in the raphe are also involved in blood pressure control, thermoregulation, and the sleep– waking cycle making it difficult to distinguish direct from indirect effects on respiration. The effect of CO2 in producing arousal seem to be mediated in particular by receptors in the cerebellum, midbrain, posterior thalamus, and basal ganglia, in areas where chemosensitive cells have been identified. How the different chemoreceptors contribute to the overall ventilatory response to CO2/H þ is not clear. One possibility is that there is a hierarchical arrangement of central chemoreceptors with various groups differing in their threshold and sensitivity to CO2/H þ . This is suggested by the observation that direct stimulation of a single group of chemoreceptors produces only 20–30% of the overall ventilatory response to the inhalation of 7% CO2. Furthermore, the threshold and sensitivity of some chemoreceptors may be influenced by higher brain centers. For example, when artificial CSF equilibrated with 25% CO2 is used to focally stimulate chemoreceptors in different locations in the rat brain, the results vary during naturally occurring cycles of sleep and wakefulness. Stimulation of the median raphe increased breathing 15–20% (by increasing frequency) in sleeping rats but had no effect during wakefulness. On the other hand, stimulation of the retrotrapezoid nucleus increased tidal volume and ventilation by 24% but only during wakefulness. Stimulation with the same fluid in the nucleus of the solitary tract increased breathing both during sleep and wakefulness. Other factors that can affect the responsiveness of any one group of central chemoreceptors include the blood flow to the site and excitatory or inhibitory inputs from other chemosensitive areas. Moreover, since few experiments report effects of focal stimulations on ventilation/CO2/H þ response slopes it is difficult to distinguish effects on the chemoreceptive process itself from effects that add to or subtract from ventilation without necessarily affecting the sensing of CO2. There are no anatomical features that distinguish chemosensitive cells that are excited by acid changes from nonchemosensitive cells. In fact, not all such cells have the same morphology. In general, they are located in close proximity to blood vessels in the brain and are situated so as to sense changes in cerebrospinal H þ concentration.

Chemoreceptor cells on the ventral surface of the medulla are located in the marginal glial layer and are surrounded by fine blood vessels. The technique of c-fos immunohistochemical staining has identified CO2-responsive chemoreceptor cells in both the ventral and dorsal medulla with dendrites directed toward the surface of the brain, suggesting that the dendrite may in fact be the chemosensitive portion of the cell. Cellular Mechanisms of Chemoreception

It has been observed that for a given change in extracellular fluid pH at the medullary surface, the ventilatory response is greater with CO2 inhalation than with the intravenous infusion of a fixed acid. One possible explanation is that CO2 and H þ have separate and distinct stimulatory effects on ventilation. Alternatively, since CO2 penetrates the cell membrane much more easily than H þ it may be that hydrogen ion changes at some site like the cell interior, more accessible to the freely diffusible CO2 than to fixed acid, is the true stimulus and that the change in internal cellular pH is the proximate signal to the central chemoreceptors. Nonchemosensitive cells rapidly restore internal pH after hypercapnic acidosis but this process is slowed in chemosensitive cells allowing them to signal H þ changes. Internal pH is restored through an Na þ /H þ exchange mechanism in response to acidosis and by Cl–/HCO–3 exchange following an alkaline challenge. An inhibition or an absence of the Na þ /H þ subtype 3 exchanger may be important in allowing chemoreceptor cells to respond to cellular acidity. Calcium channels may also be involved in the cellular mechanisms of chemoreception. L-type calcium channels in chemosensitive cells in the locus ceruleus are activated by intracellular acidity leading to membrane depolarization. There is the possibility that some chemoreceptors are stimulated by changes in the intracellular–extracellular pH gradient rather than by intracellular pH alone. Some central chemosensitive cells are also thought to contain pH-sensitive TASK-1 potassium channels as well as calcium-activated potassium channels. These potassium channels are inhibited by extracellular acidosis resulting in a reduction in the intracellular–extracellular potassium gradient and a lowering of the depolarization threshold. It may be that different types of central chemoreceptors utilize different sensing processes. In ectotherms, extracellular fluid H þ varies inversely with temperature without any effect on ventilation. This indicates that at least in cold-blooded animals H þ per se may not be the sole or main stimulus to the chemoreceptors. Rather, it has been


proposed that ventilation is controlled so as to maintain a constant ratio of hydroxyl to hydrogen ion (H þ /OH–) by an amino acid whose dissociation with temperature is like that of water. The fractional dissociation of imidazole (alpha-imidazole) like water remains constant with changes in temperature and temperature-related changes in H þ . However, such a protein would be affected by changes in pH under isothermal conditions. Based on these findings, the alpha-stat theory proposes that central chemoreceptors regulate ventilation as a function of the fractional dissociation (alpha) of the imidazole group of histadine, part of a protein considered to be the pH-sensitive molecule in the central chemoreceptor. This mechanism in cold-blooded animals may in turn regulate the patency of membrane potassium or calcium channels so that intracellular pH also remains constant as the ambient temperature changes. Neurotransmitters are also considered to play a role in mediating chemoresponses. There is considerable evidence that acetylcholine through a muscarinic receptor type is involved in the transmission of both peripheral and central chemoreceptor signals. Other neurotransmitters have also been implicated in central chemoreceptor function. Suramin, a P2 purinoceptor antagonist, when injected into the retrofacial area of the medulla (Boetzinger complex) of anesthetized rats decreases phrenic nerve discharge and reduces the respiratory response to CO2. Cells in the rat medullary raphe that are stimulated by acidosis are serotonergic and contained tryptophan hydroxylase, the rate-limiting enzyme for serotonin synthesis. On the other hand, cells that are inhibited by acidosis lack this enzyme. Other studies indicate that glutamate receptors, neurokinin, and gamma-aminobutyric acid (GABA) receptors may also play a role in mediating central chemoreceptor responses. The transmission of chemoreceptor signal to respiratory motor pathways is only partially affected by synaptic blockade. This suggests the signal transmission also involves metabolic and/or electrical coupling via intracellular channels that form gap junctions. Gap junctions have been demonstrated in CO2-sensitive cells in the nucleus of the solitary tract/ dorsal motor nucleus of the vagus, the medullary raphe, and locus ceruleus. While these gap junctions seem to be involved in the sensing of CO2, how they operate to facilitate or modify chemoresponses is unknown. Cardiovascular Effects of Central Chemoreceptors

Hypercapnia in addition to its effect on respiration also stimulates the autonomic nervous system.

Increased levels of CO2 heighten sympathetic discharge. About half of this increase is attributable to central and the rest to peripheral chemoreceptors. Neurons that mediate increases in sympathetic activity are found on the rostal ventrolateral medulla, in the same vicinity as respiratory chemoreceptors. However, it is not clear whether the same neurons excite ventilation and stimulate sympathetic activity. The increased sympathetic activity that is produced by both central and peripheral chemoreceptors is offset by vagal stretch receptor activity that increases as ventilation is stimulated by hypercapnia. Increases in blood pressure have been reported with cyanide injections into the ventral medulla suggesting that there are oxygen-responsive cells that can stimulate sympathetic activity. Depolarization occurs when cyanide is injected into cells cultured from the rostral ventral medullary surface but this response is absent when heme-oxygenase activity is blocked. There are also thought to be hypoxia receptors in the pre-Boetzinger complex that have a respiratory stimulating action. It has been suggested that these oxygen-sensitive neurons are involved in gasping. The effects of hypoxia on the brain are complex since hypoxia also exerts an important depressive effect on ventilation perhaps by increasing levels of GABA. Hypoxia can also increase lactate levels simulating central CO2 receptors and augment cerebral blood flow.

Central Chemoreceptors in Health and Disease Measurement of Chemoreceptor Sensitivity

As CO2 increases, ventilation rises linearly until it reaches about two-thirds of the maximum breathing capacity. Tidal volume and frequency also increase linearly until tidal volumes of 1.5–2.0 l are attained. Tidal volumes thereafter tend to plateau and ventilation increases primarily through increases in breathing frequency. The frequency of breathing increases more as a result of a shortening of expiratory rather than inspiratory duration. CO2 sensitivity is measured by steady state and rebreathing techniques. Steady-state methods require long periods for PCO2 in both the arterial blood and the brain to reach equilibrium. In addition, hypercapnia produces a near-linear increase in cerebral blood flow so that brain PCO2 rises less than arterial PCO2 as higher concentrations of CO2 are inhaled. Consequently, changes in brain PCO2 are not simply related to arterial PCO2 . The rebreathing technique is a simpler and more rapid method for assessing chemosensitivity. After


30–45 s of rebreathing a gas mixture with an initial concentration of 7% CO2 and 30% or more oxygen, differences in alveolar, arterial, and cerebral venous PCO2 narrow greatly and with continued rebreathing increase with time at essentially the same rate. Steady-state and rebreathing methods for measuring ventilatory responses to CO2 provide comparable results in normal humans. It has been suggested that a period of hyperventilation preceding rebreathing to lower starting levels of PCO2 allows threshold levels of PCO2 to be discerned. The large contribution of neural drives from higher brain centers, independent of blood gas levels in the wakefulness state, complicates the interpretation of measurements of CO2 response in conscious subjects. Probably the most accurate measurements of CO2 sensitivity may be obtained in nonREM sleep, when breathing is almost exclusively under chemical control, provided that the test can be carried out without affecting sleep stage. The more rapid response to CO2 of peripheral versus central chemoreceptors has been used to separate peripheral from central receptor contributions. Techniques have included devolution of the steady response or by measuring the effects of a single breath of gas containing high concentrations of CO2. In animals the direct effects of CO2 or acids on peripheral chemoreceptor activity can be assessed but there is no direct way of measuring central chemoreceptor neural responses. Since diseases of the lung or thorax, by changing their mechanical properties, can alter the ventilatory response to CO2 even if chemoreceptor function is normal, more direct ways of assessing respiratory output have been proposed. These include measuring the force generated by the inspiratory muscles contracting nearly isometrically against an occluded airway (occlusion pressure) and measurement of the electrical activity of the diaphragm via surface or esophageal electrodes. Factors Affecting CO2 Responsiveness

Ventilatory responses to CO2 are quite variable in humans. Unlike the response to hypoxia, which increases substantially soon after birth, the central response to CO2 is nearly fully developed in the newborn. The ventilatory effect of CO2 on peripheral chemoreceptors does increase somewhat in the early newborn period as evidenced by an enhanced transient response. The ventilatory responses to CO2 increase as a function of body size but otherwise do not change appreciably with advancing age. Even accounting for body size the ventilatory response to CO2 in women is less than in men. Hypercapnic

ventilatory responses in women also vary through the menstrual cycle and are greatest in the luteal phase as a result of the respiratory stimulant effect of the higher progesterone levels. High progesterone/ estrodiol levels seem to explain the hyperventilation of pregnancy. A variety of other endocrine and metabolic factors can influence CO2 responsiveness. Hyperthyroidism increases hypercapnic ventilatory responses and the augmentation of chemosensitivity is closely linked to the increase in metabolic rate. Conversely, hypothyroidism blunts chemosensitivity and when severe, can lead to hypoventilation and hypercapnia. The acid–base status of the body also has a significant effect on CO2 responsiveness. Elevated bicarbonate ion concentration increases the buffering of H þ and decreases the ventilatory response to CO2. There are important behavioral influences on breathing and on the ventilatory responses to CO2. The removal of wakefulness effects during sleep and anesthesia blunts CO2 responsiveness. Relaxation techniques such as yoga can also reduce the ventilatory response to CO2. In contrast, emotional states of fear and anxiety increase resting breathing and lower the resting PCO2 level and may also increase CO2 sensitivity. It is still not clear whether these behavioral effects originating from higher brain centers act to enhance the sensitivity of brainstem chemical control mechanisms or whether they constitute a separate parallel pathway. The hyperventilation syndrome is a condition characterized by persistent hyperventilation and hypocapnia and a variety of symptoms including shortness of breath, chest pain, palpitations, dizziness, and parasthesias. This syndrome is often associated with agoraphobia and panic attacks. The observation that panic attack can be precipitated in these patients by breathing CO2-enriched gas mixtures has led to the suggestion that the disorder is the result of chemoreceptor hyperactivity. However, ventilatory responses to CO2 are generally no greater than normal in the hyperventilation syndrome. The marked variability in respiratory chemosensitivity is due in part to genetic influences. There is a familial correspondence in ventilatory responsiveness and the correspondence is closer in identical as compared to fraternal twins. Genetic influences have also been shown in animal studies: mutant mice with abnormalities in RET, MASH-1, and PHOX2B genes have depressed CO2 responses. Disorders of Central Respiratory Chemical Control

During sleep and with the loss of wakefulness drives, central chemoreceptors play a particularly important


role in maintaining regular breathing. Normally during sleep, PCO2 rises by a few mmHg and ventilatory responses to CO2 are somewhat reduced. If CO2 sensitivity during sleep is heightened, loop gain is increased. Under these conditions a reduction in PCO2 of only a few mmHg during nonREM sleep may destabilize respiratory control and lead to the development of periodic apneas. This is the basis for Cheyne–Stokes respiration, commonly seen in patients with heart failure, particularly during sleep. An accompanying decrease in CO2 drive to upper airway muscles can result in a loss of upper airway patency in individuals with predisposing anatomic abnormalities, setting the stage for obstructive sleep apnea. As PCO2 rises during the course of an apneic period, ventilatory drive increases or there may be a direct arousal effect so that the apnea terminates and breathing resumes only to begin the entire cycle over again. Persistent and severe periodic apneas during sleep can lead to chronic CO2 retention and hypercapnia even during wakefulness. This can be reversed by treatment of the sleep apnea with continuous positive airway pressure that serves to tether open the upper airways. Chronic hypercapnia often accompanies severe respiratory diseases that markedly impair the function of the ventilatory pump. However, not all individuals with comparable levels of ventilatory dysfunction develop hypercapnia. The propensity for hypercapnia is thought to result only when severe diseases of the ventilatory pump are associated with low CO2 responsiveness. That some individuals are intrinsically predisposed to the development of hypercapnia is suggested by the observations that normal relatives of hypercapnic patients with chronic obstructive pulmonary disease have lower CO2 responses than do relatives of normocapnic patients with chronic obstructive pulmonary disease. Other studies have failed to find abnormal chemoresponses in patients with lung disease and hypercapnia and have attributed the elevated PCO2 levels to disordered gas exchange in the lungs and a rapid-shallow breathing pattern that minimizes alveolar ventilation. Impaired chemosensitivity is also the critical underpinning of the obesity-hypoventilation (Pickwickian) syndrome. These patients who suffer from severe obesity that imposes a mass load on the chest wall also demonstrate chronic hypercapnia and experience obstructive sleep apnea leading to severe daytime somnolence. If the impairment in chemosensitivity is sufficiently severe, hypercapnia can result even in the absence of diseases of the ventilatory pump. Central alveolar hypoventilation is characterized by absent ventilatory responses to hypercapnia and hypoxia. Often

near-adequate levels of ventilation can be maintained during wakefulness but hypoventilation can become profound during sleep. It is thought that congenital central alveolar hypoventilation, also termed Ondine’s curse, is caused by maldevelopment of neural crest cells; it is often associated with Hirschsprung’s disease in which varying lengths of the colon are aganglionic. Patients often have a family history of the disorder that is associated with several different gene mutations especially of the PHOX2B gene, the receptor tyrosine kinase, the RET gene and endothelin1 and 3 genes, and brain-derived neurotrophic factor. Artificial mechanical ventilatory support is often required to maintain adequate levels of ventilation and a normal PCO2 . See also: Hypoxia and Hypoxemia. Kinins and Neuropeptides: Neuropeptides and Neurotransmission; Other Important Neuropeptides.

Further Reading Bradley SR, Pieribone VA, Wang W, et al. (2002) Chemosensitive serotonergic neurons are closely associated with large medullary arteries. Nature Neuroscience 5: 401–402. Bruce EN and Cherniack NS (1987) Central chemoreceptors. Journal of Applied Physiology 62: 389–402. Cherniack NS, Dempsey J, Fencl V, et al. (1977) Workshop on assessment of respiratory control in humans. I. Methods of measurement of ventilatory responses to hypoxia and hypercapnia. American Review of Respiratory Diseases 115: 177– 181. Dean JB, Ballantyne D, Cardone DL, Erlichman JS, and Solomon IC (2002) Role of gap junctions in CO2 chemoreception and respiratory control. American Journal of Physiology. Lung Cellular and Molecular Physiology 283: L665–L670. Dreshaj IA, Haxhiu MA, and Martin RJ (1998) Role of the medullary raphe nuclei in the respiratory response to CO2. Respiratory Physiology 111: 15–23. Erlichman JS, Cook A, Schwab MC, Budd TW, and Leiter JC (2004) Heterogeneous patterns of pH regulation in glial cells in the dorsal and ventral medulla. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 286: R289–R302. Kiwull-Schone H, Wiemann M, Frede S, Bingmann D, and Kiwull P (2003) Tentative role of the Na þ /H þ exchanger type 3 in central chemosensitivity of respiration. Advances in Experimental and Medical Biology 536: 415–421. Morrell MJ, Heywood P, Moosavi SH, Guz A, and Stevens J (1999) Unilateral focal lesions in the rostrolateral medulla influence chemosensitivity and breathing measured during wakefulness, sleep and exercise. Journal of Neurology, Neurosurgery and Psychiatry 67: 637–645. Nattie EE (1999) CO2, brainstem chemoreceptors and breathing. Progress in Neurobiology 59: 299–331. Nattie EE (2001) Central chemosensitivity, sleep, and wakefulness. Respiratory Physiology 129: 257–268. Neubauer JA and Sunderram J (2004) Oxygen-sensing neurons in the central nervous system. Journal of Applied Physiology 96: 367–374.

418 CHEMORECEPTORS / Arterial

F L Powell, University of California – San Diego, La Jolla, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Arterial chemoreceptors include the carotid and aortic bodies, which are small organs with a very high blood flow. Glomus cells in the arterial chemoreceptors sense changes in arterial PO2 , PCO2 , and pH by mechanisms involving ion channels and hemecontaining molecules. Decreased PO2 and pH, and increased PCO2 , depolarize glomus cells and trigger the release of neurotransmitters. An unidentified neurotransmitter depolarizes sensory nerve endings in the carotid and aortic body so the intensity of the arterial stimulus is coded as the frequency of action potentials in the afferent nerves going to respiratory and cardiovascular centers in the medulla. Hypoxia and hypercapnia potentiate the effect of each other on the carotid body and the effect of PCO2 is mediated by changes in intracellular pH. The response to changes in arterial blood gases is extremely rapid (in seconds). Arterial chemoreceptors exhibit plasticity during chronic hypoxia by increasing their O2-sensitivity.

Introduction Arterial chemoreceptor is a generic term for both the carotid body chemoreceptors and aortic body chemoreceptors. Arterial chemoreceptors respond to changes in arterial PO2 , PCO2 , and pH and evoke negative feedback reflexes in the respiratory and cardiovascular systems to maintain blood gas homeostasis. These are the most important chemoreceptors that respond to PO2 , making them essential for a normal hypoxic ventilatory response. Most of our knowledge about arterial chemoreceptors is based on studies of the carotid body, and the aortic bodies will be considered similar unless noted otherwise.

Structure The carotid bodies are small (E2 mm diameter in humans) sensory organs located near the carotid

Cell Types

Carotid bodies include several different types of cells (Figure 1). Glomus, or type I, cells are round or ovoid, 10–12 mm in diameter, and thought to be the primary chemoreceptor cell in the carotid body. These cells develop from the neural crest and are neurosecretory. The ultrastructure of glomus cells is typical of cells that actively synthesize and secrete O2, CO2, H+ Blood SN

Arterial

sinus at the bifurcation of the common carotid artery at the base of the skull. The aortic bodies are on the aortic arch near the aortic arch baroreceptors. Afferent nerves travel from the carotid bodies to the central nervous system (CNS) in the glossopharyngeal (IX cranial) nerve, and from the aortic bodies to the CNS in the vagus (X cranial) nerve. Efferent nerves to the carotid bodies include sympathetic and parasympathetic innervation of blood vessels, as well as sympathetic innervation of chemoreceptor cells. The carotid body is organized into lobules or glomoids that are surrounded by dense capillary networks and penetrated by the branches of the carotid sinus nerve. Blood supply to the carotid body is from the 1– 2 mm long carotid body artery, which branches off the external carotid artery and continues through the carotid body to perfuse the superior cervical and nodose ganglia. Blood supply to the aortic bodies is from short vessels branching off the aortic arch. Carotid bodies have an extremely high blood flow for their size, for example, 1.5 l per 100 g per min in a cat or E15 times blood flow to the brain tissue. There may be some shunt pathways for blood flow past the capillary networks in carotid body glomoids.

C

Okada Y, Chen Z, and Kuwana S (2001) Cytoarchitecture of central chemoreceptors in the mammalian ventral medulla. Respiratory Physiology 129: 13–23. Patrick W, Webster K, Puddy A, Sanii R, and Younes M (1995) Respiratory response to CO2 in the hypocapnic range in awake humans. Journal of Applied Physiology 79: 2058–2068. Sun MK and Spyer KM (1991) Responses of rostroventrolateral medulla spinal vasomotor neurones to chemoreceptor stimulation in rats. Journal of the Autonomic Nervous System 33: 79–84. Wiemann M and Bingmann D (2001) Ventrolateral neurons of medullary organotypic cultures: intracellular pH regulation and bioelectric activity. Respiratory Physiology 129: 57–70.

GC DA NE ACh SP

SC

Figure 1 Schematic of carotid body. PO2 , PCO2 , and pH in arterial blood stimulate glomus cells (GC) to release an unidentified neurotransmitter that excites the carotid sinus nerve (CSN) and sends action potentials to the medulla. Clear- and dense-cored vesicles contain dopamine (DA), norepinephrine (NE), acetylcholine (ACh), and substance P (SP). Sheath cells (SC) surround glomus cells.

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substances, including both clear and dense-cored vesicles. Glomus cells are connected to each other by gap junctions and make presynaptic and postsynaptic connections with afferent nerves in the carotid sinus nerve. Glomus cells contain several neurotransmitters and neuromodulators, as well as the enzymes to synthesize these substances (Figure 1). Opiates (e.g., met-enkephaline) and catecholamines are packaged in dense-cored vesicles in glomus cells. Dopamine is the most abundant catecholamine in the carotid body but norepinephrine is found as well. Clear-cored vesicles are thought to contain acetylcholine. Other neuroactive agents in glomus cells include serotonin (5-hydroxytryptamine), substance P, neurokinin A, bombesin, and atrial natriuretic peptide (ANP). Sheath cells, also known as type II or sustentacular cells, resemble glial or Schwann cells. Sheath cell bodies are located near the periphery of the carotid body and their processes reach into the organ to cover the surfaces of glomus cells that are not in contact with nerves or other glomus cells. Sheath cells generally prevent direct connections between glomus cells and capillary endothelial cells; however, they do not act as a diffusion barrier for relatively large (40 000 molecular weight) substances from the blood. Carotid bodies also include capillary endothelial cells and autonomic ganglia cells.

potassium channel conductance, which depolarizes glomus cells. Similarly, heme-oxygenase (HO) may act as an O2-sensor because hypoxia inhibits HO-2 and the production of carbon monoxide (CO); glomus cells are inhibited by low concentrations of CO. Nitric oxide synthases (NOS-1 and NOS-3) may sense O2 because hypoxia inhibits NOS and nitric oxide (NO) inhibits carotid body activity. However, NOS is only in the carotid sinus nerve afferents so it is not essential for O2-sensing by glomus cells. Finally, there is evidence for mitochondrial cytochromes with low oxygen affinity in glomus cells that may act as O2-sensors, although it is not clear how these depolarize glomus cells. Several kinds of potassium (K þ ) channels also contribute to O2-sensing in glomus cells. Background K þ channels (also known as leak currents) are sensitive to physiological levels of hypoxia, which decreases their open probability and depolarizes the glomus cell. Glomus cells also have a unique inward rectifying K þ channel responsible for an HERG-like K þ current that is sensitive to O2. However, it is not known if this channel responds to physiological levels of hypoxia. Glomus cells also have large conductance Ca2 þ -activated K þ channels (BK channels) that may be physically linked with HO-2 in the glomus cell membrane. BK channels are O2-sensitive and opened by CO so they represent a mechanism of oxygen sensing that involves oxidases and K þ channels.

Oxygen Sensing

CO2 and H þ Sensitivity

Oxygen sensing in the carotid body has been localized to the glomus cells but the precise mechanism is still unknown. Decreased PaO2 depolarizes the glomus cell, which increases intracellular calcium Ca2 þ to cause exocytosis of a neurotransmitter and excitation of the carotid sinus nerve. In carotid bodies in vivo, PO2 at the glomus cells is less than the arterial PO2 but it is greater than tissue PO2 in most other organs. This is because blood flow is relatively high and diffusion distances are small in carotid bodies. Hence, molecular mechanisms of O2-sensing in the carotid bodies are sensitive to PO2 in the physiological range. Candidates for molecular sensors providing the initial event in oxygen sensing fall into two general classes: heme-containing enzymes and O2sensitive ion channels. Multiple O2-sensors that are sensitive to different ranges of PO2 may be involved in glomus cell chemotransduction. Heme-containing enzymes that could sense O2 include NADP(H) oxidase in glomus cells. NADP(H) oxidase generates reactive oxygen species (ROS) such as H2O2 in the presence of oxygen. ROS increases potassium channel conductance so hypoxia decreases

The mechanism of PCO2 and pH chemoreception in glomus cells appears to be a common response to changes in intracellular pH. Intracellular acidity inhibits Ca2 þ -activated K þ currents in glomus cells, leading to depolarization and increased current through voltage-gated Ca2 þ channels. The increase in intracellular Ca2 þ promotes exocytosis of an excitatory neurotransmitter that stimulates the carotid sinus nerve. The increase in intracellular Ca2 þ from hypoxia is potentiated by hypercapnia, contributing to the multiplicative effect of O2 and CO2 on carotid body activity. PCO2 can diffuse into glomus cells and cause large changes in intracellular pH. Extracellular pH changes in blood cause smaller changes in intracellular pH of glomus cells. Hence, carotid body chemoreceptors are less sensitive to metabolic changes than respiratory changes in pH.

Sensory Coding Figure 2 shows the effect of arterial stimuli on the frequency of action potentials in the carotid sinus

420 CHEMORECEPTORS / Arterial P aCO

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Action potential frequency (impulses/s)

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carotid sinus nerve. Arterial chemoreceptor activity increases when PaO2 falls below 100 mmHg if PaCO2 is maintained at normal levels. However, if PaCO2 decreases, then PaO2 must decrease further to stimulate chemoreceptor activity to the same level. This latter scenario would occur, for example, when hypoxia stimulates ventilation and decreases PaCO2 . Conversely, carotid body chemoreceptors can be stimulated at higher PO2 levels if PaCO2 is increased. Hence, CO2 potentiates carotid body O2-sensitivity and vice versa. This synergistic, or multiplicative, effect of hypoxia and hypercapnia on carotid body chemoreceptors is important because it explains the multiplicative effect of PaO2 and PaCO2 on ventilation. CO2/pH

10 7.45 5 0 0 (b)

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Figure 2 Carotid body chemoreceptor response (action potential frequency) to PaO2 and PaCO2 (a) and PaCO2 and pHa (b) for two different fibers. Increasing PCO2 increases the response to PO2 , and the effect of the two stimuli together is more than the sum of the individual effects, as explained in the text. Decreasing pH increases the response at any PCO2 , but PCO2 still has an effect when pH is held constant. Adapted from Biscoe TJ, Purves MJ, and Sampson SR (1970) Journal of Physiology 208: 121 and Hornbein TF (1968) In: Torrance RW (ed.) Arterial Chemoreceptors. Blackwell Scientific Publications.

nerve. The response to O2 is hyperbolic while the response to CO2 is essentially linear. As explained above, hypoxia, hypercapnia, and acidity cause glomus cells to depolarize, increase intracellular Ca2 þ , and release an excitatory neurotransmitter. These are graded responses to stimuli so the frequency of action potentials in the carotid sinus nerve codes the stimulus intensity. Hypoxia

The physiological stimulus for carotid bodies is O2 partial pressure; they do not respond to changes in O2 content or hemoglobin saturation if PO2 remains constant. This explains the lack of ventilatory response in clinical conditions such as anemia or carbon monoxide poisoning. There is some evidence that aortic bodies may respond to changes in O2 content. Carotid bodies are sensitive to PO2 over the entire physiological range, although they are more sensitive to hypoxia. At normal PaO2 (100 mmHg) and PaCO2 (40 mmHg), there is a low, tonic level of activity in the

Figure 2(b) shows the effect of pH and PCO2 on carotid body chemoreceptors. PaCO2 changes can affect chemoreceptor activity even if pHa is held constant, and vice versa. Aortic bodies in humans do not respond to pHa changes, and this is one exception to remember about the aortic bodies being qualitatively different from the carotid bodies. Therefore, the carotid bodies are the only chemoreceptors that respond to metabolic changes in pHa when PaCO2 is constant in humans. Not shown in Figure 2 is the increase of slope of the carotid body response to PCO2 caused by hypoxia. This results from the multiplicative effect of O2 and CO2 on the carotid body described above. Pattern and Speed of Response

Arterial chemoreceptors respond rapidly (within seconds) to changes in PaO2 , PaCO2 , and pHa. Changes in arterial blood gases that occur in phase with breathing, especially during conditions such as exercise, can be sensed by arterial chemoreceptors and may stimulate ventilation. This rapid response explains how ventilation can be altered within a single breath when arterial blood gases change. Carotid body chemoreceptors contain carbonic anhydrase, which will increase the speed of response to PaCO2 according to the intracellular pH-sensing mechanisms described above. Although different patterns of action potential frequency (e.g., bursting) can occur with different stimuli, the CNS only responds to the average discharge frequency from carotid bodies. There is no evidence that stimulating carotid body chemoreceptors with O2 versus CO2, for example, can result in different reflex effects. Neurotransmitters

Despite the identification of several neuroactive substances being released from carotid bodies during

CHEMORECEPTORS / Arterial

stimulation, the excitatory neurotransmitter between glomus cells and the carotid sinus nerve is not known. Acetylcholine is the most likely candidate. Acetylcholine stimulates carotid body activity via nicotinic receptors although chemoreception is not blocked by nicotinic or muscarinic receptor blockers. Substance P stimulates carotid body activity but it is not present in the glomus cells of all species. Norepinephrine and serotonin excite carotid sinus nerve activity but they do not appear to be the primary neurotransmitters from glomus cells and act as neuromodulators. Hypoxia stimulates release of dopamine from glomus cells in a graded fashion but dopamine is mainly inhibitory in the carotid body. Dopamine acts primarily on dopamine-2 autoreceptors on glomus cells to limit excitability by negative feedback. Met- and leuenkephalins also depress carotid sinus nerve activity. Multiple inhibitory neurotransmitter systems suggest that limiting and modulating chemoreceptor activity is physiologically significant. Autonomic Modulation

Stimulating the peripheral end of the transected carotid sinus nerve inhibits carotid body chemoreceptor activity in experimental animals. This involves at least two mechanisms. Parasympathetic nerves release NO to cause vasodilation, which increases local PO2 and decreases hypoxic stimulation. Sensory fibers may also release NO, which increases cyclic guanosine monophosphate (GMP) in glomus cells and inhibits their activity. It is not known if the autonomic effect is physiologically significant in vivo.

Arterial Chemoreceptors and Respiratory Disease Diseases of the arterial chemoreceptors per se are relatively rare in populations living at sea level but chemodectomas (carotid body tumors) are more common in populations found at high altitude. This may contribute to the blunted hypoxic ventilatory response observed in some high-altitude natives and people suffering from chronic mountain sickness. The carotid bodies and their innervation may also be damaged during surgical procedures in the region, such as carotid endarterectomy. However, the consequences of this for respiration are relatively unstudied. More commonly, arterial chemoreceptors are important in respiratory disease in terms of the physiological responses they elicit with, for example, hypoxemia or CO2 retention. The basic responses to changes in arterial blood gases have been described above. It is known that these responses can be altered by chronic hypoxia in healthy subjects, for example

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during acclimatization to hypoxia at high altitude. It is reasonable to suspect that similar adaptations may take place in patients with chronic hypoxemia from respiratory disease, although arterial CO2 levels are higher than in normal subjects acclimatized to altitude. However, it has been impossible to study the arterial chemoreceptors in patients with respiratory disease to date so it is not clear if the adaptations described for normal subjects occur in patients. Similarly, it is not clear if some of the difference in the progress of respiratory diseases in different patients depends on individual differences in the premorbid arterial chemoreceptor sensitivities and reflex response, or in the ability of these processes to adapt to chronic hypoxia. The effects of chronic hypoxia on arterial chemoreceptors in healthy subjects are described next. Plasticity in Chronic Hypoxia

Chronic hypoxia (days to weeks) increases O2-sensitivity of carotid body chemoreceptors. This increases afferent input from carotid bodies to respiratory centers in the brain and stimulates ventilation. This contributes to the time-dependent increase in ventilation during acclimatization to hypoxia. The effect is specific to hypoxia because it occurs even when arterial pH and PCO2 are held at normal levels during hypoxia and it does not occur with chronic application of other stimuli such as CO2. Changes in ion channels with chronic hypoxia tend to depolarize glomus cells and increase O2-sensitivity. Chronic hypoxia downregulates K þ conductance, increases the density of Na þ and Ca2 þ channels, and preferentially increases Ca2 þ influx through L-type Ca2 þ channels in glomus cells. Chronic hypoxia also increases the density of O2-sensitive Ca2 þ -activated K þ channels on glomus cells. Chronic hypoxia also affects neurotransmitters in the carotid body, although it does not cause a coordinated increase in excitatory, or decrease in inhibitory neurotransmitters. Substance P, an excitatory neurotransmitter in the carotid body, decreases in carotid bodies during chronic hypoxia. Dopamine, an inhibitory neurotransmitter in the carotid body, increases in carotid bodies during chronic hypoxia. Chronic hypoxia increases nicotinic receptors on afferent nerve endings in the carotid body but the effects of acetylcholine on O2-sensitivity do not change. The expression of new neuromodulators in the carotid body can increase O2-sensitivity. Endothelin1 (ET1) is not observed in carotid bodies under normoxic control conditions but it stimulates carotid bodies. Chronic hypoxia increases ET1 and blocking the ETA receptor for ET1 eliminates the increase in

422 CHEST WALL ABNORMALITIES

O2-sensitivity with chronic hypoxia in experimental animals. Chronic hypoxia also causes significant morphological changes in the carotid body but these cannot explain the functional changes. Carotid bodies show hypertrophy, hyperplasia of glomus cells, and increased capillarity after exposure to 2 or more weeks of hypoxia. These changes are reversible. Neovascularization is stimulated by endothelial growth factor (VEGF), which is controlled by hypoxic inducible factor 1a (HIF-1a), and endothelin-1 (ET1). See also: Acid–Base Balance. Arterial Blood Gases. Chemoreceptors: Central. High Altitude, Physiology and Diseases. Ventilation: Control.

Further Reading Biscoe TJ, Purves MJ, and Sampson SR (1970) Journal of Physiology 208: 121.

Dinger B, He L, Chen J, Stensaas L, and Fidone S (2003) Mechanisms of morphological and functional plasticity in the chronically hypoxic carotid body. In: Lahiri S, Semenza G, and Prabhakar N (eds.) Oxygen Sensing: Responses and Adaptation to Hypoxia, pp. 439–465. New York: Dekker. Fidone S, Gonzales C, Almaraz L, and Dinger B (1997) Cellular mechanisms of peripheral chemoreceptor function. In: Crystal RG and West JB (eds.) The Lung: Scientific Foundations, 2nd edn., pp. 1725–1737. Philadelphia: Lippincott-Raven. Gonzalez C, Almaraz L, Obeso A, and Rigual R (1994) Carotid body chemoreceptors: from natural stimuli to sensory discharges. Physiological Reviews 74: 829–898. Heath D and Williams DR (1995) High-Altitude Medicine and Pathology, 4th edn. Oxford: Oxford University Press. Hornbein TF (1968) In: Torrance RW (ed.) Arterial Chemoreceptors. Blackwell Scientific Publications. Prabhakar N (2000) Oxygen sensing by the carotid body chemoreceptors. Journal of Applied Physiology 88: 2287– 2295. Zak FG and Lawson W (1982) The Paraganglionic Chemoreceptor System: Physiology, Pathology, and Clinical Medicine. New York: Springer.

CHEST WALL ABNORMALITIES J L Allen and O H Mayer, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA K W Gripp, A.I. DuPont Hospital for Children, Wilmington, DE, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Chest wall abnormalities can substantially impact the quality of breathing. Chest wall dysfunction can occur as a primary disorder due to congenital (e.g., VATER syndrome), genetic (e.g., Jarcho–Levin syndrome), or acquired (e.g., scoliosis) causes, or can occur as a secondary disorder due to conditions such as obesity, neuromuscular disease, or chronic obstructive pulmonary disease. Recently, much has been learned about the genetic mechanisms producing chest wall disorders. These mechanisms include dominant negative action (e.g., Marfan syndrome), gain-of-function mutation (e.g., achondroplasia), haploinsufficiency (e.g., Campomelic dysplasia), and loss-of-function mutations (e.g., Jarcho–Levin syndrome). Clinical features common to all these disorders include, in severe cases, respiratory insufficiency due to pulmonary hypoplasia or respiratory pump failure. There are a variety of noninvasive interventions to help counteract the effects of chest wall dysfunction; these improve airway clearance (mechanical percussion devices and cough assist devices) and improve ventilation (noninvasive or invasive nocturnal or continuous mechanical ventilation). Surgical interventions to correct or prevent further progression of the disorder include bracing, spinal fusion, and, more recently, introduction of the vertical expandable prosthetic titanium rib (VEPTR), which has the advantage of allowing for future spinal growth.

Introduction The chest wall, including the ribcage and respiratory muscles, comprises the ‘pump’ component of the respiratory system. Respiratory loads, primarily elastic and resistive, arise from the lung and the chest wall itself. Disorders of the thoracic cage can affect both the pump and the load components. This chapter describes some of the basic mechanisms by which congenital abnormalities of the chest wall are produced, and the evaluation and treatment of chest wall disorders.

Etiology Chest wall abnormalities can be categorized as primary or secondary. Primary chest wall disorders can be congenital or acquired. There are two types of congenital abnormalities. Those with a genetic basis such as achondroplasia or Jarcho–Levin syndrome have a predictable recurrence risk, while those that arise embryologically or secondary to intrauterine environmental causes, such as VATER (vertebral, anal, tracheo-esophageal fistula, radial, and renal anomalies) syndrome, are without a known genetic basis or recurrence risk. Acquired chest wall disorders, such as idiopathic scoliosis or pectus excavatum become apparent later in childhood or adolescence; while they may be familial, there is no certain genetic or environmental cause.

CHEST WALL ABNORMALITIES 423

Secondary chest wall disorders lead to dysfunction caused by other illnesses or mechanical limitations. Examples include chest wall muscle weakness due to neuromuscular disorders and malnutrition, restrictive chest wall disease due to pleural effusions or obesity, and mechanical inefficiencies of the chest wall imposed by diaphragmatic flattening and loss of the area of apposition seen in the pulmonary hyperinflation of obstructive lung diseases such as cystic fibrosis, asthma, and chronic obstructive pulmonary disease. Only primary chest wall disorders are discussed in this article. Secondary chest wall disorders are discussed in the articles listed in ‘See also’.

Pathology Skeletal dysplasias may lead to severe pulmonary complications, due either to pulmonary hypoplasia or to respiratory pump failure. The underlying defect in skeletal dysplasia may affect either the extracellular matrix or the intracellular metabolism of cells forming skeletal structures. Though the ribs and cartilage provide the bulk of the support of the thoracic cavity, muscular abnormalities can also have substantial impact on thoracic mechanics and function. Congenital Disorders, Genetic Basis Known

Intrinsic skeletal dysplasias vary greatly in severity and in their effects on the respiratory system, ranging from lethality due to pulmonary hypoplasia to respiratory pump failure and restrictive lung disease. The most common form of skeletal dysplasia (frequency 1/10 000) is achondroplasia, characterized by

rhizomelic short stature and macrocephaly. Patients with achondroplasia have short flared ribs, forming a shallow thorax with decreased chest wall circumference. In addition, patients often have severe midfacial hypoplasia causing obstructive apnea, or central apnea due to brainstem compression secondary to a small foramen magnum and mild hydrocephalus. The rare patients with homozygous achondroplasia show a much more severe phenotype and die in early infancy, often from respiratory complications. In Jarcho–Levin syndrome, or spondylothoracic dysplasia, rib and spinal abnormalities produce a short thorax with a fan-like configuration of ribs (Figure 1) due to their fusion at the costovertebral junctions. This also presents in infancy and causes severe lung restriction and respiratory insufficiency for which some patients require mechanical ventilation. The characteristic findings of cleidocranial dysplasia are wide, late-closing fontanels, hypoplastic or absent clavicles, and supernumerary teeth. More subtle abnormalities include moderate short stature, a narrow thorax with short ribs, hypoplastic pubic bones, and brachydactyly. Respiratory distress may occur but is relatively rare. Other clinical malformation syndromes affecting the thorax and their genetic bases (where known) are listed in Table 1. Congenital Disorders, Environmental or Genetic Basis Unknown

Jeune syndrome, or asphyxiating thoracic dystrophy, is a disorder of rib development in which the cross-sectional area of the ribcage does not increase

Figure 1 10-year-old girl with Jarcho–Levin syndrome and the fan-like arrangement of ribs with bilateral vertical expandable prosthetic ribs in place.

Table 1 Malformation syndromes affecting the thorax Disorder

Physical findings

Thoracic involvement

Respiratory complications

Inheritance mode

Gene

Pathomechanism

VATER association

Anal atresia; TE fistula; radial and renal anomalies

None

Embryologic in origin

Hemifacial microsomia with microtia and small mandible; may be bilateral; preauricular skin tags; renal anomalies Aortic dilatation; lens dislocation; arachnodactyly; joint laxity Multiple fractures, joint laxity; blue sclera; dentinogenesis imperfecta Macrocephaly, short limb dwarfism; narrow foramen magnum Bowed long bones; male to female sex reversal; cleft palate

Rare, due to small thoracic cage and/or scoliosis Rare, due to small thoracic cage and/or scoliosis

Sporadic

Facioauriculovertebral spectrum; Goldenhar syndrome Marfan syndrome

Vertebral segmentation anomalies; structural rib anomalies; scoliosis Hemivertebrae; structural rib anomalies; scoliosis

Sporadic

None

Embryologic in origin

Scoliosis; pectus excavatum or carinatum; pneumothorax Rib fractures

Rarely due to abnormal thorax or pneumothorax Unstable thorax with multiple rib fractures

Autosomal dominant

FBN1

Dominant negative action

Autosomal dominant

COLIA1 or COLIA2

Dominant negative action

Small thoracic cage

Restrictive lung disease; central and obstructive apnea Respiratory insufficiency may lead to death in infancy Rare

Autosomal dominant

FGFR3

Gain-of-function

Autosomal dominant

SOX9

Haploinsufficiency

Autosomal dominant

CBFA1

Haploinsufficiency

Autosomal recessive (most cases of Puerto Rican ancestry)

DLL3

Loss of function

Autosomal recessive

DTDST

Loss of function

Osteogenesis imperfecta Achondroplasia

Campomelic dysplasia

Cleidocranial dysplasia Spondylothoracic dysplasia; Jarcho–Levin syndrome Diastrophic dysplasia

Wide anterior fontanel; supernumerary teeth; short stature Vertebral and rib segmentation defects; short neck; long fingers

Short limb dwarfism, clubfeet; cleft palate; cervical kyphosis

Small thoracic cage; kyphocoliosis

Partially or completely absent clavicle Short thorax due to vertebral and rib defects

Progressive kyphoscoliosis

Respiratory insufficiency due to small thoracic volume, may be lethal Respiratory compromise due to kyphosis

Category refers to the presumed genetic mechanism by which the mutations cause the phenotype (see text). Mutations in the listed genes have been identified in the respective disorders. In some disorders, there is a strict correlation between the genotype (i.e., a specific mutation or a mutation in a specific gene), while in others, heterogeneity is suspected or proven (e.g., not all patients with Jarcho–Levin syndrome have mutations in DLL3). The genetic basis of the disorders are discussed in detail in ‘Pathogenesis’ section.


Figure 2 Anterior computed tomography (CT) reconstruction of Jeune syndrome with the narrow and long thorax also showing the short ribs.

Figure 3 CT image of Jeune syndrome at the carina that demonstrates the short ribs and anterior chest wall deformity.

normally and the thorax becomes long but narrow or constricted (Figures 2 and 3). It often presents in early infancy and results in severe pulmonary compromise, often requiring invasive mechanical ventilation and aggressive airway clearance. Acquired Disorders

The most frequent chest wall disorder in children is pectus excavatum. Though pectus excavatum and

pectus carinatum may be cosmetically disturbing, they usually have little or no impact on cardiopulmonary function. Pectus excavatum, while typically acquired as an isolated deformation, can occur with connective tissue disorders like Marfan syndrome, or in subjects with substantial respiratory muscle weakness like spinal muscular atrophy type 1. Scoliosis is most commonly idiopathic, but can also be congenital or secondary. Idiopathic scoliosis is the most common form of scoliosis, accounting for 80–85% of all lateral spinal curvature. The frequency of onset increases with age, with the most common age of onset being adolescence (11 and older); idiopathic scoliosis is very uncommon under 4 years of age. Though it commonly presents with the relatively benign finding of spinal asymmetry on forward bending, if the curve is greater than 351, there is a greater than 40% chance of significant lung restriction and of moderate to severe gas trapping likely due to airway compression. The risk for progression is related to both the degree of curve and the age of the patient, with curves of 301 rarely progressing in adulthood. Scoliosis is much more likely to progress in prepubertal children. Congenital scoliosis is less common than idiopathic scoliosis and differs in having a causative spinal abnormality. Abnormalities include hemivertebrae, as in Goldenhar syndrome or the facioauriculovertebral spectrum, abnormal vertebral segmentation, as in the VATER association, and inadequate ossification. In congenital scoliosis the curve may not be clinically apparent at birth, but with longitudinal growth the spine will grow asymmetrically creating a curve. Because of the many different causes, the rate of progression and prognosis is very variable. Secondary scoliosis can occur due to inadequate truncal support, such as in neuromuscular disease like Duchenne muscular dystrophy and spinal muscular atrophy (Figure 4). The curve occurs after progressive loss of muscle tone and truncal support with the spine curving to one side and, in some situations, rotating. This can be further worsened with loss of ambulation and the different forces that act on the ribcage when a child is in the sitting position as opposed to upright. Isolated rib fusion can occur and can also lead to progressive scoliosis as the lateral edge of the thorax is fixed while the spine continues to grow, leading to a poorly compliant chest wall. Conversely, absence of ribs can also cause progressive scoliosis due to inadequate support with a highly compliant or flaccid thoracic cavity.


embryologic anomaly without a known genetic cause and no significant recurrence risk, presents with structural vertebral and rib anomalies. A second example is the facioauriculovertebral spectrum, also known as Goldenhar syndrome or hemifacial microsomia. In addition to structural vertebral and rib anomalies, eye and ear findings and a small jaw may affect one side of the face, or rarely, both sides. Gene Mutations: Autosomal Dominant, Dominant Negative Action

Figure 4 Chest radiograph in a 2-year-old male with spinal muscular atrophy type-1 demonstrating significant scoliosis.

Clinical Features Though there are many different causes of chest wall abnormalities, the clinical manifestations are often quite similar. They can vary from primarily cosmetic disorders, as in pectus excavatum, carinatum, and mild idiopathic scoliosis, to severe restrictive lung disease as in the severe genetic skeletal dysplasias, progressive congenital scoliosis, and neuromuscular disease. With an increasing scoliotic curve, the lung on the concave side becomes more compressed and the range of motion of the diaphragm will decrease. This decreases vital capacity and, with further worsening, can lead to significant nocturnal hypoventilation or to more continuous hypoventilation and the need for noninvasive ventilation due to eventual respiratory muscle fatigue and failure. Pulmonary compression may also lead to poor airway clearance in the affected lung due to inability to fully expand the lung and airway narrowing that may occur from the lung compression or airway bending. In this environment, poor airway clearance can lead to retained secretions or ‘mucus plugging’ and segmental atelectasis, persistent or chronic pneumonia, and bronchiectasis.

Pathogenesis Embryologic Abnormalities

Congenital anomalies of the chest wall can occur due to embryological anomalies, or due to underlying gene mutations. The VATER association, an

In contrast to embryologic abnormalities, disorders caused by gene mutations may have a recurrence risk, and may be present in other family members in addition to the patient. Autosomal dominant conditions include the connective tissue disorders, Marfan syndrome and osteogenesis imperfecta (Table 1). Marfan syndrome is caused by heterozygous mutations in the fibrillin 1 gene (FBN1) resulting in an abnormal gene product. Fibrillin is the major constitutive element of extracellular microfibrils. Incorporation of a structurally abnormal protein disrupts the macromolecular structure of the extracellular microfibrils, despite the fact that the patient has one wild-type allele encoding structurally and functionally normal fibrillin. This mechanism of action, by which the structurally abnormal gene product is disease-causing regardless of the presence of normal gene product, is called dominant negative. Another example for this mechanism is osteogenesis imperfecta, resulting from a single mutation in one of the genes encoding a procollagen chain. Mature collagen molecules consist of three procollagen chains forming a triple helical structure. Incorporation of a single structurally abnormal component disrupts this complex structure and leads to the abnormal connective tissue properties including brittle bones. Autosomal Dominant, Gain-of-Function Mutations

Achondroplasia is caused by a heterozygous point mutation in the fibroblast growth factor receptor 3 gene (FGFR3), resulting in a glycine to arginine amino acid substitution at position 380 of the protein product. This membrane-bound receptor has an intracellular tyrosine kinase domain that is activated upon ligand binding and ultimately regulates cell proliferation. The function of the normal receptor is negative regulation of enchondral growth, as demonstrated by the skeletal overgrowth seen in mice lacking both functional copies of the gene. The pathogenesis of the mutation involves constitutive activation of FGFR3, inhibiting proliferation of growth plate chondrocytes and thus causing short limb dwarfism. This gain-of-function mechanism is


further enhanced by diversion of the abnormal gene product from the normal lysosomal degradation process to increased recycling with prolonged survival. Patients whose parents both have achondroplasia are at 25% risk for homozygosity of the mutation, resulting in extreme short limb dwarfism and lethal respiratory insufficiency. A second lethal skeletal dysplasia, thanatophoric dysplasia, is also caused by mutations in FGFR3. Autosomal Dominant, Haploinsufficiency

A third pathogenic mechanism causing autosomal dominant skeletal disorders is haploinsufficiency for the functional gene product. This mechanism most often affects protein products acting as transcription factor regulating the expression of other genes. A 50% decrease of the functional protein is diseasecausing in dosage sensitive pathways. Campomelic dysplasia and cleidocranial dysostosis are examples of skeletal dysplasias due to gene mutations resulting in haploinsufficiency for the respective transcription factors (see Table 1). Autosomal Recessive Conditions

Diastrophic dwarfism (achondrogenesis type IB) and Jarcho–Levin syndrome are due to mutations in both alleles of the respective gene, causing loss-of-function of protein products with enzymatic or transport function (Table 1). The pattern of malformation seen in Jarcho–Levin syndrome is suggestive of abnormal segmentation in early embryological development. In contrast to haploinsufficiency, loss-of-function in both gene alleles is necessary to cause disease. Achondrogenesis type IB causes a clinical syndrome of extreme short stature, poor ossification of the skull and vertebral bodies, severe micromelia of the limbs, extremely short ribs, and stellate long bones. The mutation resides in the diastrophic dysplasia sulfate transporter (DTDST) gene, leading to decreased or absent sulfate transport and abnormalities in cartilage proteoglycans sulfation.

Management and Current Therapy Nonsurgical

For subjects with hypoventilation or respiratory failure, intermittent nocturnal noninvasive ventilation can be initiated using a nasal interface. Continuous mechanical ventilation that allows both mobility and full use of both the mouth and nose can be achieved using rhythmic abdominal compression using a Pneumo-Belts or having a tracheostomy placed to allow for invasive mechanical ventilation. In situations in which mobility is not necessary, patients may also use negative pressure ventilation, using a device that applies negative pressure directly to the thorax (cuirass or ‘chest shell’) or to the body below the neck (PortaLungs or poncho/jacket). Maximizing airway clearance with manual or mechanical chest percussion and postural drainage augmented with nebulized b2-adrenergic agonists is also very important. For subjects with segmental or lobar compression, the application of distending pressure via nasal mask or tracheostomy can help to open the airway and maintain patency during breathing and coughing. Applying cyclic positive pressure to inflate the lungs rapidly followed by negative pressure using

Other Disorders

Other disorders with structural abnormalities of the chest wall are genetic in nature but the abnormal genes have not yet been identified. Examples include Jeune’s syndrome, the cerebrocostomandibular syndrome (gaps in the posterior aspects of the ribs), and the Fryns syndrome (diaphragmatic hernias in combination with multiple other anomalies). Fryns syndrome is inherited as an autosomal recessive trait, and the multiple affected organ systems are suggestive of underlying mutations in a gene widely expressed during early development.

Figure 5 Postoperative lateral view of chest CT reconstruction in a 4-year-old girl with scoliosis and rib fusion, with VEPTR in place between the 4th and 5th ribs (black arrow) and between the iliac crest and 5th rib posteriorly (white arrow). Note the expanded intercostal space between the 4th and 5th ribs.


Figure 6 Postoperative posterior view of chest CT reconstruction in a 4-year-old girl with scoliosis and rib fusion, with VEPTR in place between the 4th and 5th ribs (white arrow) and between the iliac crest and 5th rib (black arrow) posteriorly.

a device such as the Cough Assists device can replace or augment coughing, especially in patients with significant muscle weakness or cognitive disability. Surgical

Though it is not necessary to correct pectus excavatum in many situations, a ratio of the transverse to the anterioposterior diameter of greater than 3.5 is felt by many to be an indication for surgery. Severe pectus excavatum can impair cardiac output in the upright position and increase the work of breathing at high workloads. The Nuss procedure can correct an abnormality by applying internal, anterior pressure at the apex of the pectus curve. A rigid curved rod is inserted along the inner anterior chest wall and is then rotated up and sutured in place. The correction can be quite substantial and often the bar can be removed after a year without the defect returning. Correction for scoliosis has traditionally involved straightening the spine either with traction alone or permanently with posterior spinal fusion. For idiopathic scoliosis with curves of 451, surgical repair is often performed after a trial of bracing. While scoliosis surgery may not result in an immediate gain in lung volume, it helps minimize the future loss of vital capacity. However, spinal fusion reduces spinal growth in the fused segments and limits future thoracic growth. Alternatives include spinal growing

rods or the vertical expandable prosthetic titanium rib (VEPTR), both of which allow thoracic expansion. For more substantial chest wall abnormalities such as congenital scoliosis with fused ribs, absent ribs, or chest wall constriction from a variety of causes, reconstruction using the VEPTR allows for both lateral stabilization and vertical expansion of the thorax with growth (Figures 5 and 6). The devices can be arranged in a variety of orientations with expansion of the ribs laterally, or from the spine or iliac crest. See also: Chronic Obstructive Pulmonary Disease: Overview. Cystic Fibrosis: Overview. Lung Development: Overview; Congenital Parenchymal Disorders; Congenital Vascular Disorders. Nutrition in Respiratory Disease. Obesity. Pectus Excavatum. Pediatric Pulmonary Diseases. Pleural Effusions: Overview. Respiratory Muscles, Chest Wall, Diaphragm, and Other. Trauma, Chest: Bronchopleural Fistula; Postpneumonectomy Syndrome; Subcutaneous Emphysema. Ventilation, Mechanical: Negative Pressure Ventilation; Noninvasive Ventilation; Positive Pressure Ventilation; Ventilator-Associated Pneumonia.

Further Reading Beiser G, Epstein S, Stampfer M, et al. (1972) Impairment of cardiac function in patients with pectus excavatum, with improvement after operative correction. New England Journal of Medicine 287(6): 267–272.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview 429 Boyer J, Amin N, Taddonio R, and Dozor A (1996) Evidence of airway obstruction in children with idiopathic scoliosis. Chest 109: 1532–1535. Byers P and Steiner R (1992) Osteogenesis imperfecta. Annual Review of Medicine 43: 269–282. Campbell R and Hell-Vocke A (2003) Growth of the thoracic spine in congenital scoliosis after expansion thoracoplasty. Journal of Bone and Joint Surgery America 85-A(3): 409–420. Castile R, Staats B, and Westbrook P (1982) Symptomatic pectus deformities of the chest. American Review of Respiratory Diseases 126(3): 564–568. Cho J, Guo C, Torello M, et al. (2004) Defective lysosomal targeting of activated fibroblast growth factor receptor 3 in achondroplasia. Proceedings of the National Academy of Sciences, USA 101(2): 609–614. Cornier A, Ramirez N, Arroyo S, et al. (2004) Phenotype characterization and natural history of spondylothoracic dysplasia syndrome: a series of 27 new cases. American Journal of Medical Genetics 128A: 120–126. Francomano C (1995) The genetic basis of dwarfism. New England Journal of Medicine 332: 58–59.

Chloride Channels

Hunter A, Reid C, Pauli R, and Scott C (1996) Standard curves of chest circumference in achondroplasia and the relationship of chest circumference to respiratory problems. American Journal of Medical Genetics 62: 91–97. Kose N and Campbell R (2004) Congenital scoliosis. Medical Science Monitor 10(5): RA104–RA110. Lyons-Jones K (1988) Smith’s Recognizable Patterns of Human Malformation. Philadelphia: Saunders. McMaster M (2001) Congenital scoliosis. In: Weinstein S (ed.) The Pediatric Spine: Principles and Practice, 2nd edn, pp. 161–177. Philadelphia: Lipincott Williams & Wilkins. Park H, Lee S, Lee C, et al. (2004) The Nuss procedure for pectus excavatum: evolution of techniques and early results on 322 patients. Annals of Thoracic Surgery 77: 289–295. Stacey A, Bateman J, Choi T, et al. (1988) Perinatal lethal osteogenesis imperfecta in transgenic mice bearing engineered mutant pro-alpha 1(I) collagen. Nature 332: 131–136. Tosi L (1997) Osteogenesis imperfecta. Current Opinion in Pediatrics 9(1): 94–99. Wong K-S, Hung I, Wang C, and Lien R (2004) Thoracic wall lesions in children. Pediatric Pulmonology 37: 257–263.

see Ion Transport: Chloride Channels.

CHRONIC OBSTRUCTIVE PULMONARY DISEASE Contents

Overview Acute Exacerbations Emphysema, Alpha-1-Antitrypsin Deficiency Emphysema, General Smoking Cessation

Overview P J Barnes, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Chronic obstructive pulmonary disease (COPD) is characterized by progressive and largely irreversible airflow limitation due to narrowing and fibrosis of small airways and loss of airway alveolar attachments as a result of emphysema. Cigarette smoking is the most important risk factor, but other noxious gases are important too in developing countries. Genes may determine which smokers are susceptible to the development of airflow obstruction. There is a specific pattern of inflammation characterized by increased numbers of macrophages, neutrophils, and T lymphocytes (particularly CD8 þ cells), which is an amplification of the inflammation seen in normal cigarette smokers and increases with disease progression. Proteinases, particularly MMP-9, result in degradation of alveolar wall elastin resulting in emphysema. The major symptom is exertional dyspnea

caused by air trapping and hyperinflation as small airways close, but cough and sputum production are common due to mucus hypersecretion. Management of COPD involves smoking cessation. Bronchodilator therapy reduces hyperinflation; regular long-acting inhaled b2-agonists and anticholinergics are preferred to short-acting drugs. Inhaled corticosteroids do not reduce inflammation but reduce exacerbations and are usually given to patients with severe disease with frequent exacerbations. Long-term oxygen is indicated for patients with respiratory failure who have evidence of pulmonary hypertension. Nonpharmacological treatments include pulmonary rehabilitation, noninvasive ventilation, and lung volume reduction.

Introduction Chronic obstructive pulmonary disease (COPD) is characterized by progressive development of airflow limitation that is not fully reversible. The term COPD encompasses chronic obstructive bronchiolitis with obstruction of small airways and emphysema

430 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview

with enlargement of airspaces and destruction of lung parenchyma, loss of lung elasticity, and closure of small airways. Chronic bronchitis, by contrast, is defined by a productive cough of more than 3 months duration for more than 2 successive years; this reflects mucus hypersecretion and is not necessarily associated with airflow limitation. COPD is common and is increasing globally. It is now the fourth leading cause of death in the US and the only common cause of death that is increasing; this is thought to be an underestimate as COPD is likely to be contributory to other common causes of death. It is predicted to become the third commonest cause of death and the fifth commonest cause of disability worldwide in the next few years. It currently affects more than 5% of the adult population and is underdiagnosed in the community. COPD consumes an increasing proportion of healthcare resources, which currently exceed those devoted to asthma by threefold.

Etiology Several environmental and endogenous factors, including genes, increase the risk of developing COPD (Table 1). Environmental Factors

In industrialized countries, cigarette smoking accounts for most cases of COPD, but in developing countries, other environmental pollutants, such as particulates associated with cooking in confined spaces, are important causes. It is likely that there are important interactions between environmental factors and a genetic predisposition to develop the disease. Air pollution (particularly sulfur dioxide and particulates), exposure to certain occupational chemicals such as cadmium, and passive smoking may all Table 1 Risk factors for COPD Environmental factors

Endogenous (host) factors

Cigarette smoking Active Passive Maternal

a1-Antitrypsin deficiency Other genetic factors Ethnic factors Airway hyperresponsiveness? Low birth weight

Air pollution Outdoor Indoor: biomass fuels Occupational exposure Dietary factors High salt Low antioxidant vitamins Low unsaturated fatty acids Infections

be additional risk factors. The role of airway hyperresponsiveness and allergy as risk factors for COPD is still uncertain. Atopy, serum IgE, and blood eosinophilia are not important risk factors. However, this is not necessarily the same type of abnormal airway responsiveness that is seen in asthma. Low birth weight is also a risk factor for COPD, probably because poor nutrition in fetal life results in small lungs, so that decline in lung function with age starts from a lower peak value. Genetic Factors

Longitudinal monitoring of lung function in cigarette smokers reveals that only a minority (15–40% depending on definition) develop significant airflow obstruction due to an accelerated decline in lung function (two- to fivefold higher than the normal decline of 15–30 ml forced expiratory volume in 1s (FEV1) year 1) compared to the normal population and the remainder of smokers who have consumed an equivalent number of cigarettes (Figure 1). This strongly suggests that genetic factors may determine which smokers are susceptible and develop airflow limitation. Further evidence that genetic factors are important is the familial clustering of patients with early onset COPD and the differences in COPD prevalence among different ethnic groups. Patients with a1-antitrypsin deficiency (proteinase inhibitor (Pi)ZZ phenotype with a1-antitrypsin levels o10% of normal values) develop early emphysema that is exacerbated by smoking, indicating a clear genetic predisposition to COPD. However, a1-antitrypsin deficiency accounts for o1% of patients with COPD and many other genetic variants of a1-antitrypsin that are associated with lower than normal serum levels of this Pi have not been clearly associated with an increased risk of COPD. This has led to a search for associations between COPD and polymorphisms of other genes that may be involved in its pathophysiology. So far, few significant associations have been detected and even those reported have not been replicated in other studies. A 10-fold increased risk of COPD in individuals who have a polymorphism in the promoter region of the gene for tumor necrosis factor alpha (TNF-a) that is associated with increased TNF-a production has been reported in a Taiwanese population but not confirmed in Caucasian populations. Several other genes have been implicated in COPD, but few have been replicated in different populations (Table 2). Techniques such as DNA microarray (gene chips) to detect single nucleotide polymorphisms, proteomics to detect novel proteins, and gene expression profiling to measure which known and novel genes are expressed are now

CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview 431

FEV1 (% predicted at age 25)

100 Nonsmoker or nonsusceptible smoker 75 Susceptible smoker Stopped smoking aged 50 years

50 Disability 25

Stopped smoking aged 60 years

Death 0 25

50 Age (years)

75

Figure 1 Natural history of COPD. Annual decline in airway function showing accelerated decline in susceptible smokers and effects of smoking cessation. Patients with COPD usually show an accelerated annual decline in forced expiratory volume in 1 s (FEV1), often greater than 50 ml year 1, compared to the normal decline of approximately 20 ml year 1, although this is variable between patients. The classic studies of Fletcher and Peto established that 10–20% of cigarette smokers are susceptible to this rapid decline. However, with longer follow-up more smokers may develop COPD. The propensity to develop COPD amongst smokers is only weakly related to the amount of cigarettes smoked and this suggests that other factors play an important role in determining susceptibility. Most evidence points towards genetic factors, although the genes determining susceptibility have not yet been determined.

Table 2 Some of the genes associated with COPD susceptibility Candidate genes

Risk

a1-Antitrypsin

ZZ genotype high risk MZ, SZ genotypes small risk Associated in some populations Associated in some studies

a1-Chymptrypsin Matrix metalloproteinase-1, -2, -9, -12 Microsomal epoxide hydrolase Glutathione S-transferase Heme oxygenase-1 Vitamin D binding protein TNF-a promoter

Increased risk Increased risk Small risk Inconsistent Inconsistent

Interleukin-13

Small risk

being used to study cigarette smokers who develop COPD compared with matched smokers who do not. This may identify markers of risk, but may also reveal novel molecular targets for the development of treatments of the future.

Pathology The term COPD includes chronic obstructive bronchiolitis with fibrosis and obstruction of small airways and emphysema with enlargement of airspaces and destruction of lung parenchyma, loss of lung elasticity, and closure of small airways. Chronic bronchitis, by contrast, is defined by a productive cough of more than 3 months duration for more than 2

successive years; this reflects hypersecretion of mucus and is not necessarily associated with airflow limitation. Most patients with COPD have all three pathological mechanisms (chronic obstructive bronchitis, emphysema, and mucus plugging) as all are induced by smoking, but may differ in the proportion of emphysema and obstructive bronchitis (Figure 2). There has been debate about the predominant mechanism of progressive airflow limitation and recent pathological studies suggest that it is closely related to the degree of inflammation, narrowing, and fibrosis in small airways. Emphysema may contribute to the airway narrowing in the more advanced stages of the disease. There is inflammation in small airways with an increase in macrophages and neutrophils in early stages of the disease indicating innate immune response, but in more advanced stages of the disease there is an increase in lymphocytes (particularly cytotoxic CD8 þ T cells) including lymphoid follicles, indicating acquired immunity. Emphysema describes loss of alveolar walls due to destruction of matrix proteins (predominantly elastin) and loss of type 1 pneumocytes as a result of apoptosis. Several patterns of emphysema are recognized: centriacinar emphysema radiates from the terminal bronchiole, panacinar emphysema involves more widespread destruction, and bullae are large airspaces. Emphysema results in airway obstruction by loss of elastic recoil so that intrapulmonary airways close more readily during expiration. Chronic hypoxia may lead to hypoxic vasoconstriction, with structural changes in pulmonary

432 CHRONIC OBSTRUCTIVE PULMONARY DISEASE / Overview COPD

Normal

Disrupted alveolar attachments (emphysema) Mucosal inflammation, fibrosis

Emphysema + small airway obstruction

↑

Lung hyperinflation trapped gas Dyspnea

Mucus hypersecretion Airway held open by alveolar attachments

Airway obstructed by


Cough and sputum

↓ Exercise tolerance

• Loss of attachments • Mucosal inflammation + fibrosis

Deconditioning

• Obstruction of lumen by mucus

Figure 2 Mechanisms of airflow limitation in COPD. The airway in normal subjects is distended by alveolar attachments during expiration, allowing alveolar emptying and lung deflation. In COPD, these attachments are disrupted because of emphysema thus contributing to airway closure during expiration, trapping gas in the alveoli, and resulting in hyperinflation. Peripheral airway are also obstructed and distorted by airway inflammation and fibrosis (chronic obstructive bronchiolitis) and by occlusion of the airway lumen by mucus secretions which may be trapped in the airway because of poor mucociliary clearance.

Chronic hypoxia

Pulmonary vasoconstriction Muscularization Intimal hyperplasia

Pulmonary hypertension

Fibrosis Obliteration

Cor pulmonale Edema Death

Figure 3 Vascular changes in COPD. Chronic hypoxia results in hypoxic pulmonary vasoconstriction and leads to structural changes, resulting in secondary pulmonary hypertension. Over time, this may lead to right heart failure (cor pulmonale), which has a poor prognosis, most patients only surviving for 6–12 months.

vessels that eventually lead to secondary pulmonary hypertension (Figure 3). Inflammatory changes similar to those seen in small airways are also seen in pulmonary arterioles.

Clinical Features The clinical features of COPD are usually straightforward and care should be taken to evaluate those features of the illness that are not typical. Symptoms

The symptoms of COPD are slowly progressive over many years, in contrast to the episodic and variable

Poor health status Figure 4 Symptoms of COPD. The most prominent symptom of COPD is dyspnea, which is largely due to hyperinflation of the lungs as a result of small airways’ collapse due to emphysema and narrowing due to fibrosis, so that the alveoli are not able to empty. Hyperinflation induces an uncomfortable sensation and reduces exercise tolerance. This leads to immobility and deconditioning and results in poor health status. Other common symptoms of COPD are cough and sputum production as a result of mucus hypersecretion, but not all patients have these symptoms and many smokers with these symptoms do not have airflow obstruction (simple chronic bronchitis).

symptoms of asthma. Patients have usually lost a considerable amount of their lung volume by the time they present to a doctor, with FEV1 values of o50%. There is usually a history of heavy smoking for many years, often 425 pack years (1 pack year ¼ 20 cigarettes daily for 1 year). Progressive shortness of breath on exertion is the predominant symptom and compensatory behavior of the patient may delay diagnosis. Dyspnea results from the hyperinflation secondary to small airway narrowing resulting in poor quality of life which is further exacerbated by the deconditioning due to reduced physical activity (Figure 4). Cough and sputum production are common symptoms but are also found in cigarette smokers without airflow limitation (chronic bronchitis). A change in the character of the cough may indicate lung carcinoma. Wheezing may occur during exacerbations and periods of breathlessness. Ankle swelling may occur when there is cor pulmonale. Weight loss often occurs in advanced disease, but the mechanism is uncertain. Loss of skeletal muscle bulk may be a response to systemic inflammation. Signs

When FEV1450% predicted, there may be no abnormal signs. The typical patient with more severe COPD shows a large, barrel-shaped chest due to hyperinflation, diminished breath sounds, and


distant heart sounds due to emphysema, and prolonged expiration with generalized wheezing on expiration. Diagnosis

Diagnosis is commonly made from the history of progressive dyspnea in a chronic smoker and is confirmed by spirometry, which shows an FEV1/vital capacity (VC) ratio of o70% and FEV1 o80% predicted. Staging of severity is made on the basis of FEV1 (Table 3), but exercise capacity and the presence of systemic features may be more important determinants of clinical outcome. Measurement of lung volumes by body plethysmography shows an increase in total lung capacity, residual volume, and functional residual capacity, with consequent reduction in inspiratory capacity, representing hyperinflation as a result of small airway closure (Table 4). This results in dyspnea which may be measured by Table 3 Staging of COPD by severity of airflow limitation Stage 0 (at risk) Stage I (mild)

Stage II (moderate)

Stage III (severe)

Stage IV (very severe)

Chronic symptoms (cough, sputum) Normal spirometry 7Chronic symptoms (cough, sputum) FEV1/FVC o70%, FEV1 480% predicted Chronic symptoms (cough, sputum, dyspnea) FEV1/FVC o70%, FEV1 o80–50% predicted Chronic symptoms (cough, sputum, dyspnea) FEV1/FVC o70%, FEV1 450–30% predicted Chronic symptoms (cough, sputum, dyspnea) FEV1 o30% predicted or respiratory insufficiency/right heart failure

dyspnea scales and reduced exercise tolerance, which may be measured by a 6 min or shuttle walking test. Carbon monoxide diffusion is reduced in proportion to the extent of emphysema. A chest X-ray is rarely useful but may show hyperinflation of the lungs and the presence of bullae. High-resolution computerized tomography demonstrates emphysema but is not used as a routing diagnostic test. Blood tests are rarely useful; a normocytic normochromic anemia is more commonly seen in patients with severe disease than polycythemia due to chronic hypoxia. Arterial blood gases demonstrate hypoxia and in some patients hypercapnia. Natural History

COPD is slowly progressive with an accelerated decline in FEV1, leading to slowly increasing symptoms, decreasing lung function, and eventually respiratory failure (Figure 1). The only strategy to reduce disease progression is smoking cessation, although this is relatively ineffective once FEV1 has fallen below 50% predicted. Patients with more severe COPD develop acute exacerbations, which have a prolonged effect on quality of life for many months. Most of the medical costs associated with COPD are linked to acute exacerbations that lead to hospital admission. There is still debate about the role of acute exacerbations in disease progression, but the decline in lung function may be accelerated further following an acute exacerbation.

Pathogenesis Recently, there have been important new insights into the pathogenesis of COPD. Chronic Inflammatory Mechanisms

Table 4 Lung function test abnormalities in COPD Lung function test

Result

Forced expiratory volume in 1 s (FEV1, liters) Forced vital capacity (FVC, liters) FEV1/FVC (%) Peak expiratory flow (PEF, liters/min) Total lung capacity (TLC, liters) Inspiratory capacity (IC, liters) Functional residual capacity (FRC, liters) Residual volume (RV, liters) Specific airway conductance (sGaw, cmH2O 1 s 1) Transfer factor for carbon monoxide (TLCO, ml/min/ mmHg) Transfer coefficient corrected for alveolar volume (KCO (TLCO/VA), ml/min/mmHg/l)

k k k k m k m m k k k

There is a specific pattern of chronic inflammation, particularly affecting small airways and lung parenchyma. This leads to fibrosis and narrowing of small airways and to destruction of the lung parenchyma, resulting in abnormalities in gas exchange and collapse of small airways on expiration. The inflammation of COPD is characterized by increased numbers of neutrophils, macrophages, and T lymphocytes (particularly CD8 þ cytotoxic T cells) and the degree of inflammation increases with disease severity (Figure 5). The pattern of inflammation is markedly different from that of asthma (Table 5). Many inflammatory mediators are now implicated in COPD, including lipid mediators (such as leukotriene B4), chemokines (such as interleukin (IL)-8), and proinflammatory cytokines (such as tumor necrosis factor


Cigarette smoke (and other irritants) Alveolar macrophage

Epithelial cells TGF- CTG

Fibroblast

Chemotactic factors IL-8, CXC chemokines LTB4

CD 8+ lymphocyte

Neutrophil

Proteases

Emphysema

Fibrosis


Figure 5 Inflammatory mechanisms in COPD. Cigarette smoke (and other irritants) activate macrophages in the respiratory tract that release neutrophil chemotactic factors, including interleukin-8 (IL-8) and leukotriene B4 (LTB4). These cells then release proteases that break down connective tissue in the lung parenchyma, resulting in emphysema, and also stimulate mucus hypersecretion. These enzymes are normally counteracted by protease inhibitors, including a1-antitrypsin, secretory leukoprotease inhibitor (SLPI), and tissue inhibitor of matrix metalloproteinases (TIMP). Cytotoxic T cells (CD8 þ ) may also be recruited and involved in alveolar wall destruction.

Table 5 Differences between inflammation in COPD and asthma Inflammation

COPD

Asthma

Inflammatory cells

Neutrophils CD8 þ T cells þþþ CD4 þ cells þ Macrophages þþþ LTB4 TNF-a IL-8, GRO-a Oxidative stress þþþ Epithelial metaplasia Fibrosis þþ Mucus secretion þþþ AHR 7 Peripheral airways Predominantly parenchymal destruction 7

Eosinophils Mast cells CD4 þ T cells Macrophages þ LTD4, histamine IL-4, IL-5, IL-13 Eotaxin Oxidative stress þ Epithelial shedding Fibrosis þ Mucus secretion þ AHR þþþþ All airways No parenchymal effects


Inflammatory effects

Location

Response to corticosteroids

þþþ

LT, leukotriene; TNF, tumor necrosis factor; IL, interleukin; GRO, growth-related oncogene; AHR, airway hyperresponsiveness.

alpha (TNF-a) and IL-6. Oxidative stress is markedly increased in COPD and increases with disease severity; it is due to both the effects of cigarette smoke and the release of reactive oxygen species from activated inflammatory cells. There is also an increase in proteases, particularly enzymes that degrade elastin, such as neutrophil elastase (derived predominantly from neutrophils) and matrix metalloproteinase-9 (MMP-9, derived predominantly from macrophages). MMP-9 may be the predominantly elastolytic enzyme causing emphysema; it also activates transforming growth factor beta (TGF-b), a cytokine that

is expressed particularly in small airways that may result in the characteristic peribronchiolar fibrosis (Figure 6). MMP-12 may also contribute to elastolysis and is prominent in smoke exposure models of COPD in mice, where it plays a critical role in activating TNF-a. Amplifying Mechanisms

In normal cigarette smokers, there is a similar inflammatory response to that seen in COPD patients, but COPD represents an amplification of the normal


Chemotactic peptides

Macrophage

Neutrophils Neutrophil elastase

↓1-AT Pro-MMP-9

↑MMP-9 Elastolysis

Latent TGF-

Emphysema

Active TGF-

Small airway fibrosis (chronic obstructive bronchiolitis) Figure 6 Possible interrelationship between small airway fibrosis and emphysema in COPD. Transforming growth factor (TGF)-b is activated by matrix metalloproteinase-9 (MMP-9) and is in turn activated by MMP-9.

inflammatory response of the respiratory tract to inhaled noxious agents. Acute exacerbations represent a further amplification of the inflammatory response. The molecular and genetic mechanisms of this amplification remain to be determined. However, recently it has been shown that histone deacetylase activity (particularly HDAC2) is markedly reduced in the lungs, airways, and macrophages of COPD patients and that this is correlated with increased expression of inflammatory genes, such as IL-8. This amplifying mechanism may be induced by oxidative stress, which impairs the function of HDAC2 in particular. This also accounts for the steroid resistance of COPD since HDAC2 is required for switching off of activated inflammatory genes. Adenovirus infection also amplifies inflammation in cells in vitro and in experimental animals in vivo and its effects may also be mediated through a reduction in HDAC activity in lungs. In COPD patients, there is evidence for latent adenovirus infection in lungs of many COPD patients. Systemic Effects

There is increasing evidence for systemic (nonpulmonary) effects in patients with COPD, particularly in patients with severe disease and these might have an important negative impact on the quality of life (Table 6). The commonest systemic effect is loss of lean body mass and atrophy of skeletal muscles. This may reflect poor mobility, but may also be due to systemic effects of inflammatory mediators such as TNF-a and IL-6, which may induce apoptosis of skeletal muscles. Other systemic effects include osteoporosis, depression, and normocytic normochromic anemia. Circulating levels of acute phase proteins, such as

Table 6 Systemic abnormalities in COPD Systemic feature

Possible mechanisms

Cachexia Muscle wasting

TNF-a, IL-6, leptin Apoptosis of skeletal muscle due to TNF-a? Inactivity Chronic hypoxia, erythropoetin TNF-a? TNF-a, IL-6 CRP, fibrinogen?

Polycythemia Normocytic anemia Depression Cardiovascular abnormalities Osteoporosis

Inactivity, corticosteroids, cytokines?

TNF, tumor necrosis factor; IL, interleukin; CRP, C-reactive protein.

C-reactive protein and IL-6, are increased even in the stable state, but are further increased during exacerbations and this may be a factor predisposing to the markedly increased incidence of ischemic heart disease.

Management COPD is managed according to the severity of the disease, with a progressive escalation of therapy as the disease progresses (Figure 7). Antismoking Measures

Smoking cessation is the only measure so far shown to slow the progression of COPD, but in advanced disease, stopping smoking has little effect and the chronic inflammation persists. Nicotine replacement therapy (gum, transdermal patch, inhaler) helps in quitting smoking, but bupropion, a noradrenergic antidepressant, is more effective. More effective


0: at risk FEV1 ≥80%

I: mild FEV1 ≥80%

II: moderate FEV1 79–50%

III: severe FEV1 49−30%

IV: very severe FEV1 3 cm from lung apex 1. HIgh flow O2 2. Tube thoracostomy with pleural drainage or Heimlich valve or 3. Catheter aspiration

1. High flow O2 2. Tube thoracostomy with pleural drainage or Heimlich valve 3. Surgery + talc/abrasions after 1st episode

Tube thoracostomy with pleural drainage unit Hemopneumothorax requires large bore (36 Fr) chest tube

Surgery with stapling of blebs > 2 cm + talc/abrasion (pneumothorax prevention is performed by some experts after 1st episode of PSP)

Figure 2 Management algorithm for the various forms of pneumothorax.

settings, large air leaks are often present and may exceed the capacity of small-caliber tubes to effectively drain the space and prevent the risk of tension. Moreover, the risk of a hemopneumothorax is high in trauma, requiring larger chest tubes to drain and monitor intrapleural bleeding. For hospitalized patients, the chest tube is connected to an underwater drainage unit to establish a one-way seal. These drainage units contain three elements connected in series. These elements are a suction chamber that allows the adjustment of any negative pressure applied to pleural space, an underwater seal that prevents atmospheric air from entering the pleural space and allows continuous monitoring of an air leak, and a fluid collection chamber. Underwater drainage units are indicated for patients with a pneumothorax with large air leak,

or with significant volumes of pleural fluid that would clog a Heimlich valve. A chest tube should initially be placed to water seal without suction. The application of suction has been shown to perpetuate a bronchopleural fistula and delay removal of the tube thoracostomy. Chest tube suction is indicated when the lung does not fully expand on imaging. The chest tube can be safely pulled after the resolution of the air leak and radiographic demonstration of normal lung re-expansion. Surgery and Pleurodesis

Surgery for patients with a pneumothorax has several objectives: managing a persistent air leak that fails to resolve with chest tube drainage and performing pleurodesis with or without resection of blebs to

POLYMYOSITIS AND DERMATOMYOSITIS 479

prevent pneumothorax recurrence. Several techniques have been developed to accomplish these objectives. Open thoracotomy allows for the resection or stapling of apical blebs followed by a mechanical pleural abrasion or parietal pleurectomy to create a pleural symphysis and prevent pneumothorax recurrence. Pneumothorax recurrence rates are less than 1%. However, with the significant postoperative morbidity associated with an open thoracotomy, less invasive techniques have been developed. The application of video-assisted thoracic surgery (VATS) allows for an endoscopic approach in the management of pneumothorax. This technique allows for the endoscopic stapling of apical blebs and partial pleurectomy. Pleurodesis can be accomplished by either pleural abrasion or insufflation of talc. VATS is an acceptable technique in preventing pneumothorax recurrence for patients with PSP and SSP. Currently, VATS is the surgical procedure of choice in the management of SSP. Although there is no current data to indicate that bullectomy must be performed when talc is administered by poudrage, less successful procedures may be performed in patients at prohibitive risk for general anesthesia. The timing for VATS in the management of pneumothorax remains a matter of debate. Most authors agree that pneumothorax prevention is cost justified in SSP after the first pneumothorax occurrence. However, patient preferences and underlying lung diseases will influence this recommendation. In the management of PSP, most experts recommend VATS only after ipsilateral recurrence. Pleurodesis should not be withheld in patients who might need lung transplantation. Although surgery is technically more difficult after pleurodesis, the consequences of pneumothorax in this fragile subset of patients can be catastrophic. The time that is required from the pleurodesis procedure until an effective pleural symphysis has occurred remains unknown but is estimated at approximately 2 weeks. Therefore, the time from pneumothorax occurrence to safe resumption of air travel remains controversial

and depends on whether pleurodesis was performed and type of lung disease. In general, individuals with previous pneumothoraces should be discouraged from diving. See also: Asthma: Acute Exacerbations. Hypoxia and Hypoxemia. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis; Lymphangioleiomyomatosis. Pleural Space. Signs of Respiratory Disease: Lung Sounds. Trauma, Chest: Bronchopleural Fistula; Subcutaneous Emphysema.

Further Reading Baumann MH, Strange C, Heffner JE, et al. (2001) Management of spontaneous pneumothorax: an American College of Chest Physicians Delphi consensus statement. Chest 119: 590–602. Boutin C, Astoul P, Rey F, and Mathur PN (1995) Thoracoscopy in the diagnosis and treatment of spontaneous pneumothorax. Clinics in Chest Medicine 16: 497–503. Dines DE, Clagett OT, and Payne WS (1970) Spontaneous pneumothorax in emphysema. Mayo Clinic Proceedings 45: 481–487. Dulchavsky SA, Schwartz KL, Kirkpatrick AW, et al. (2001) Prospective evaluation of thoracic ultrasound in the detection of pneumothorax. Journal of Trauma 50: 201–205. Gilbert TB and McGarth GJ (1994) Tension pneumothorax: aetiology, diagnosis, pathophysiology, and management. Journal of Intensive Care Medicine 9: 139–150. Henry M, Arnold T, and Harvey J (2003) BTS guidelines for the management of spontaneous pneumothorax. Thorax 58(supplement 11): 39–52. Miller AC (2003) Spontaneous pneumothorax. In: Light RW and Lee YCG (eds.) Textbook of Pleural Diseases, pp. 445–463. London: Arnold. Sahn SA and Heffner JE (2000) Spontaneous pneumothorax. New England Journal of Medicine 342: 868–874. Schramel FM, Postmus PE, and Vanderschueren RG (1997) Current aspects of spontaneous pneumothorax. European Respiratory Journal 10: 1372–1379. Schramel FM, Sutedja TG, Braber JC, van Mourik JC, and Postmus PE (1996) Cost-effectiveness of video-assisted thoracoscopic surgery versus conservative treatment for first time of recurrent spontaneous pneumothorax. European Respiratory Journal 9: 1821–1825. Strange C and Jantz MA (2004) Pneumothorax. In: Demosthenes B (ed.) Pleural Disease (Lung Biology in Health and Disease), vol. 186, pp. 661–667. New York: Dekker.

POLYMYOSITIS AND DERMATOMYOSITIS J H Ryu, Mayo Clinic, Rochester, MN, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Polymyositis (PM) and dermatomyositis (DM) are idiopathic inflammatory myopathies of autoimmune origin involving the

skeletal muscles. Pulmonary involvement occurs and is a source of morbidity and mortality. The main forms of pulmonary involvement include interstitial lung disease, respiratory muscle weakness, and aspiration pneumonia. Non-specific interstitial pneumonia, organizing pneumonia, usual interstitial pneumonia, diffuse alveolar damage, and lymphocytic interstitial pneumonia are the main histologic patterns found. Anti-Jo-1 antibody (anti-histidyl-tRNA synthetase) is commonly found

480 POLYMYOSITIS AND DERMATOMYOSITIS in patients with PM/DM-associated interstitial lung disease. Progressive interstitial lung disease and respiratory muscle weakness can both cause respiratory failure. Aspiration pneumonia is related to weakness of the pharyngeal and upper esophageal muscles. Uncommon pulmonary manifestations include pleural effusion and pleuritis, pulmonary hypertension, and vasculitis. Corticosteroid therapy is the first line treatment and is generally effective in controlling both the myositis and the pulmonary involvement. However, in some patients PM/DM deteriorates despite corticosteroids and immunosuppressive therapy. Better understanding of the pathogenetic mechanisms in PM/DM will lead to more specific, target-directed therapy.

Diagnosis

Idiopathic inflammatory myopathies represent a heterogeneous group of autoimmune syndromes involving the skeletal muscle. The common traits of these disorders include muscle weakness and inflammation. Three major forms of idiopathic inflammatory myopathy include polymyositis (PM), dermatomyositis (DM), and inclusion-body myositis. Each of these entities has distinctive clinical and histopathologic features but the cause remains unknown. Idiopathic inflammatory myopathies are associated with systemic complications including involvement of the lungs. Pulmonary complications in idiopathic inflammatory myopathies are common causes of morbidity and mortality. This article focuses on lung involvement that occurs in patients with PM and DM. The prevalence of inflammatory myopathies has been estimated to range from 0.6 to 6 per 100 000 and estimated incidence has varied from 0.1 to 0.9 per 100 000 per year. There is meager data on the relative incidence of the different forms of inflammatory myopathies. DM may occur in children and adults, whereas PM occurs mainly after the second decade of life. Both DM and PM are more common in females. In all age groups, DM is more common than PM.

The clinical diagnosis of PM and DM is confirmed by three tests: serum muscle enzyme level, electromyography, and muscle biopsy. The most sensitive muscle enzyme test is the serum creatine kinase (CK) level which is usually elevated in patients with active PM or DM, but may occasionally be normal. Electromyographic results are usually abnormal, but the findings are non-specific and may be patchy. The definitive diagnosis of inflammatory myositis requires a muscle biopsy obtained for conventional light microscopic examination as well as immunohistochemical and electron microscopic studies. Pathologic features common to both disorders include necrosis as well as various stages of regeneration involving the muscle fibers. These changes are accompanied by inflammation involving predominantly mononuclear cells and an increased amount of connective tissue. However, there are some microscopic, immunohistochemical, and ultrastructural findings in the muscle biopsy that distinguish PM from DM. Magnetic resonance imaging or computed tomographic scanning can sometimes be helpful, for example, selecting an appropriate biopsy site, but their diagnostic accuracy has not been adequately evaluated. The most common autoantibodies in PM/DM are antisynthetase antibodies directed against various aminoacyl t-RNA synthetases. The best-known antisynthetase antibody is anti-Jo-1 (anti-histidyl tRNA synthetase), which is the most common type detected in patients with PM/DM-associated interstitial lung disease (ILD). The diagnostic and prognostic value of anti-Jo-1 in patients with PM/DM has not been adequately evaluated. Serum level of KL-6, a mucinlike high-molecular-weight glycoprotein, tends to be elevated in patients with PM/DM-associated ILD and may be a useful marker for assessing the activity of ILD.

Etiology

Pathogenesis

The cause of PM/DM is unknown, but these diseases are likely multifactorial in origin involving both genetic predisposition and acquired factors. The role of genetic factors is suggested by occasional familial occurrences, a higher frequency of other autoimmune disorders in the first-degree relatives of these patients, and linkage to certain human leukocyte antigens (HLA). Multiple genes, for example, immune response genes, likely contribute to susceptibility to these disorders. Exposure to certain environmental toxins, drugs, or infectious agents may play a role in some cases. PM-like syndrome has been described in chronic graft-versus-host disease.

Pathogenesis of PM and DM likely involves autoimmune responses, but target antigens have not been identified. In DM, the primary target antigen may be the endothelium of the endomysial capillaries. A high percentage of CD4 þ T cells and B cells are seen in the perivascular inflammation. In PM, certain CD8 þ T cells are clonally expanded in the muscle, possibly driven by an autoantigen. The autoimmune nature of this process is suggested by the association with autoantibodies, other autoimmune disorders, histocompatibility genes, and various T-cell products. A variety of inflammatory chemokines and proinflammatory cytokines including interleukin (IL)-1, IL-2,

Introduction


IL-6, IL-10, tumor necrosis factor alpha (TNF-a), and transforming growth factor beta have been described to be upregulated in PM and DM. Although antisynthetase antibodies are more commonly found in patients with PM/DM-associated ILD compared to those without ILD, the pathogenesis of ILD in these patients remains unknown. The development of ILD in these patients does not appear to correlate with the extent and severity of the muscle or skin disease.

Animal Models A completely satisfactory animal model of PM or DM has yet to be developed. Some animal models for PM have been produced experimentally in guinea pigs, rats, and mice by immunization with homogenates of muscle or muscle protein preparations such as skeletal myosin or C protein. This process has been shown to produce experimental autoimmune myositis with infiltration of T cells into muscle and appearance of necrotic as well as regenerating muscle fibers.

Clinical Features Muscle Weakness

Patients with PM and DM usually present with varying degrees of muscle weakness that is progressive over several weeks to months. Muscle weakness tends to be more severe in the shoulder and pelvic girdle muscles resulting in difficulty performing certain tasks, such as rising from a chair, climbing stairs, or combing hair. Myalgias are relatively uncommon. As the disease advances, dysphagia and respiratory muscle weakness may occur, but sensation remains normal. Skin Involvement

DM is recognized by the characteristic skin rash that often precedes muscle weakness. These skin manifestations include the heliotrope rash on the upper eyelids, Gottron rash on the knuckles, and an erythematous rash on the face, neck, and upper chest and shoulders (Shawl sign). In some patients, the skin rash may be the dominant manifestation and muscle strength may appear normal (amyopathic dermatomyositis). Subcutaneous calcinosis can occasionally be seen, particularly in juvenile DM, and may result in pain, ulcerations, and infections. Other organs including the gastrointestinal tract, joints, heart, and lungs can be involved in patients with PM/DM. In addition, features of PM/DM can

overlap with other autoimmune and connective tissue diseases in some patients. Lung Involvement in PM/DM

The main forms of pulmonary involvement in PM/ DM include ILD, respiratory muscle weakness, and aspiration pneumonia (Table 1). The reported prevalence of pulmonary involvement in PM/DM has varied from 5% to 80%, depending largely on the method of detection, the diagnostic criteria, and the study population. For example, the prevalence of ILD in retrospective studies has ranged from 5% to 9%. However, the use of high-resolution computed tomography (HRCT) in prospective surveys has yielded a prevalence as high as 80%. Women with PM/DM are at a higher risk of developing ILD and the mean age at presentation is 50 years. Less common respiratory manifestations of PM/DM include pulmonary hypertension, vasculitis, and pleural involvement. The clinician needs to keep in mind that a respiratory problem occurring in a patient with a known systemic disease may not only be a manifestation related to the underlying disease, but could also be a complication resulting from treatment, or an unrelated separate disease process. A broad perspective needs to be maintained at the outset of this diagnostic evaluation. In those patients with suspected ILD, the clinician needs to consider the need for lung biopsy to identify the underlying histologic pattern. Assessment of the tempo of the pulmonary disease, radiologic pattern seen on HRCT, and the clinical context will be helpful in reaching this decision. For example, bronchoscopy with bronchoalveolar lavage may suffice if the main concern is possible opportunistic infection. On the other hand, distinguishing different forms of interstitial pneumonias that can occur in patients with PM/DM generally requires a surgical lung biopsy. In many cases, however, identifying the specific histologic pattern of interstitial pneumonia may not necessarily alter treatment decisions since corticosteroid therapy is usually Table 1 Respiratory manifestations of PM and DM Interstitial lung disease Non-specific interstitial pneumonia Organizing pneumonia Usual interstitial pneumonia Lymphocytic interstitial pneumonia Diffuse alveolar damage Aspiration pneumonia Respiratory muscle weakness Others Pleuritis and pleural effusion Pulmonary vasculitis Pulmonary hypertension

482 POLYMYOSITIS AND DERMATOMYOSITIS

the primary mode of treatment for patients with PM/DM. Interstitial Lung Disease

ILD is probably the most common form of pulmonary involvement in PM/DM. ILD is more common in patients with detectable autoantibodies to tRNA synthetases or a mucin-like glycoprotein (KL-6) in the serum. In some patients the ILD precedes the muscle or skin manifestation. There appears to be no correlation between the extent and severity of the muscle or skin disease and the development of ILD. Typical presenting manifestations in patients with PM/ DM-associated ILD include progressive exertional dyspnea, nonproductive cough, and bibasilar crackles. Occasionally, the respiratory symptoms may be abrupt in onset and severe, resembling acute respiratory distress syndrome. Digital clubbing is rare. Anti-Jo-1 antibody (anti-histidyl-tRNA synthetase), the most common type of antisynthetase autoantibody in patients with PM/DM, is detected more commonly in patients with associated ILD (50–70%) than in those without (10–15%). Some patients may present with ILD associated with anti-Jo-1 antibody in the absence of muscle involvement. In patients with PM/DM-associated ILD, the most common histologic pattern seen in the lung is non-specific interstitial pneumonia (NSIP). Histologic features of NSIP are characterized by a uniform-appearing, cellular interstitial pneumonia with a lymphoplasmacytic infiltrate within alveolar septa. Varying amounts of fibrosis are admixed with the chronic inflammation. In contrast to usual interstitial pneumonia (UIP), in NSIP, fibroblastic foci are rare and microscopic honeycombing is absent. The NSIP pattern of lung injury can be seen in patients with other connective tissue diseases as well as in many other clinical contexts. Less common forms of ILD seen in PM/DM include organizing pneumonia, UIP, diffuse alveolar damage (DAD), and lymphocytic interstitial pneumonia (LIP). Subclassification of ILD into these histologic patterns is valuable in predicting response to therapy and prognostication. Pulmonary vascular inflammation is uncommonly seen and includes cases of pulmonary capillaritis. Pleuritis is also relatively uncommon. Chest radiography on patients with PM/DM-associated ILD usually reveals reduced lung volumes and bilateral interstitial opacities, more prominent in the lower lung fields. Consolidation is less commonly seen and honeycombing is unusual. Findings on HRCT will generally reflect the underlying histopathologic pattern of ILD. Main findings on HRCT for those patients with NSIP pattern are reticular

Figure 1 High-resolution computed tomography of the chest of a 38-year-old woman with dermatomyositis and non-specific interstitial pneumonia. Ground-glass opacities and reticular densities with peripheral predominance are seen as well as associated bronchiolectasis. Non-specific interstitial pneumonia was diagnosed by a surgical lung biopsy prior to the diagnosis of dermatomyositis.

Figure 2 Computed tomography of the chest in a 67-year-old woman who presented with patchy areas of consolidation, ground-glass opacities, and pleural effusions. Lung biopsy demonstrated organizing pneumonia and polymyositis was eventually diagnosed.

and/or ground-glass opacities present bilaterally with or without consolidation (Figure 1). Traction bronchiectasis may also be present but honeycombing is usually absent. Patients with organizing pneumonia (Figure 2) or DAD (especially during organizing phase) will have predominantly consolidative opacities on HRCT. Those with the UIP pattern of lung injury will characteristically manifest subpleural peripheral reticular opacities with or without honeycombing. Lymphocytic interstitial pneumonia is characterized by poorly defined centrilobular nodules, ground-glass opacities, and scattered thinwalled cysts. Pleural effusions may be seen in up to 20% of patients with PM/DM-associated ILD and are generally small in size.


Pulmonary function testing in patients with PM/ DM-associated ILD usually demonstrates restrictive abnormalities with reduced lung volumes and diffusing capacity as well as evidence of abnormal gas exchange. Bronchoscopy is generally nondiagnostic in patients with PM/DM-associated ILD. Transbronchial biopsy may yield evidence of interstitial inflammation and fibrosis but is unlikely to characterize fully the underlying histologic pattern. Bronchoalveolar lavage generally demonstrates lymphocytosis in the bronchoalveolar lavage fluid for those with NSIP and organizing pneumonia but can yield variable findings in those patients with other histologic patterns. Respiratory Muscle Weakness

Involvement of respiratory muscles by progressive inflammatory myopathy can result in hypoventilation and occasionally respiratory failure. Isolated diaphragmatic weakness without peripheral muscle involvement has been described as well. Rarely, laryngeal involvement may occur. In patients with respiratory muscle weakness, chest radiography reveals reduced lung volumes with diaphragmatic elevation and discoid basilar atelectasis. Pulmonary function testing demonstrates changes of a restrictive abnormality with reduced maximal respiratory pressures. Aspiration Pneumonia

Dysphagia and reflux resulting from myopathy of the striated muscles of the hypopharynx and upper esophagus can predispose to aspiration. Impaired cough from respiratory muscle weakness may contribute to increased risk of aspiration for patients with PM/DM. Aspiration pneumonia has been reported to occur in 15–20% of patients with PM/DM. The diagnosis of aspiration pneumonia is usually made based on the radiologic findings and the clinical context. Respiratory muscle weakness is usually suggested by clinical and radiographic findings. Reduced maximal respiratory pressures will confirm this diagnosis. Chest radiography and CT typically demonstrate patchy consolidation in the dependent portions of the lungs. Biopsy confirmation is sought only if presenting clinical or radiologic features are atypical. Other Respiratory Manifestations

Several other respiratory manifestations have been associated with PM/DM. Pulmonary hypertension can rarely be a direct manifestation of PM/DM but is seen more commonly secondary to progressive

ILD or chronic ventilatory insufficiency. Few cases of pulmonary vasculitis including pulmonary capillaritis with associated diffuse alveolar hemorrhage have been reported in patients with PM/DM. The use of immunosuppressive agents such as methotrexate and cyclophosphamide can be associated not only with opportunistic pneumonias due to impairment of host defenses, but also drug-induced lung diseases. Pleural manifestations have included pleural effusions, pleuritis, and occasionally spontaneous pneumothorax. DM, more so than PM, can be associated with cancer including lung cancer.

Current Therapy The goal of therapy in PM and DM is to improve muscle strength and to ameliorate extramuscular manifestations. There have been very few controlled clinical trials. The treatment of inflammatory myopathies remains largely empirical, using agents that are nonselective in their effects on the immune system. Prednisone is the first-line drug and early initiation of therapy leads to better outcome. The initial dose of prednisone is usually 1 mg kg 1 day 1 with slow tapering to an alternate-day dosing over the following several months. Approximately twothirds of patients will demonstrate a response to initial corticosteroid therapy. Large doses of intravenous corticosteroids, for example, 0.5–1 g methylprednisolone or equivalent given daily for 3 days, have been used in the initial management of severe cases. Addition of another immunosuppressive drug is commonly needed for steroid-sparing effect or for additive therapeutic effects in controlling the myositis. Azathioprine (2–3 mg kg 1 day 1) or methotrexate (up to 25 mg weekly) are commonly used for this purpose. In patients with aggressive disease, a combination of prednisone and another immunosuppressive drug can be started from the outset. Other treatment options include cyclophosphamide, cyclosporine, chlorambucil, mycophenolate mofetil, leflunomide, tacrolimus, anti-TNF agents (etanercept, infliximab), immune modulators (eculizumab, rituximab), plasmapheresis, total lymphoid irradiation, intravenous immunoglobulins, and autologous hemopoietic stem cell transplantation. None of these treatment modalities has been adequately tested in controlled clinical trials. In addition, relative efficacy of these agents used alone or in various combinations has not been clarified. Each patient needs to be individually assessed in regard to presenting manifestations, comorbid factors, and potential risks associated with the use of these agents. Rehabilitative measures including a regular exercise program may help to prevent or reverse impaired muscle function

484 POLYMYOSITIS AND DERMATOMYOSITIS

and exercise tolerance. Better understanding of pathogenetic mechanisms will lead to more specific, target-directed therapy in the future. Management of respiratory involvement in patients with PM/DM depends on the type of pulmonary complication, the activity of the underlying PM/ DM, and comorbid factors. In many cases, treatment of PM/DM itself, as outlined above, is the most important component in managing the pulmonary complication. For example, treatment of ILD associated with PM/DM involves corticosteroid and immunosuppressive therapy used to treat the underlying disease. The most common regimens used in managing patients with PM/DM-associated ILD are prednisone alone or in combination with azathioprine. The improvement in response to treatment is seen over the course of 4–6 months. This response to treatment in patients with PM/DM-associated ILD partly depends on the histologic pattern of lung injury. Patients with NSIP, organizing pneumonia, or LIP respond better to corticosteroid therapy and have a more favorable prognosis compared to those with UIP. Those patients with the DAD pattern have the worst prognosis. Thus, subclassification of histologic findings in patients with PM/DM-associated ILD appears to be clinically useful in gauging expected response to treatment and prognosis. The serum CK level usually parallels the disease activity in PM/DM and should be monitored at regular intervals. In addition, reliable functional measures of muscle strength and endurance can help monitor the activity of PM/DM and response to therapy. Pulmonary function studies, chest radiographs, and HRCT of the chest provide objective reassessment of pulmonary involvement in PM/DM. Other management options to be considered for patients with PM/DM and pulmonary involvement include ventilatory support and lung transplantation. In patients with respiratory failure from severe ILD or respiratory muscle weakness, invasive or noninvasive ventilatory support may be needed to survive an episode of exacerbation in their disease. Lung transplantation may be an option for those patients with severe pulmonary fibrosis resulting from PM/ DM-associated ILD, in the absence of significant comorbid factors that may contraindicate this procedure and if the underlying PM/DM is under control.

Prognosis The 5-year survival rate after diagnosis for patients with PM/DM overall is approximately 80%. The most common causes of death in these patients are cancer and pulmonary complications. At least a third of the patients with PM/DM are left with mild to

severe muscle weakness. Pulmonary causes of death in patients with PM/DM include progressive ILD, ventilatory failure due to respiratory muscle weakness, recurrent aspiration pneumonia, pneumonias related to immunosuppressive therapy, and occasionally progressive pulmonary hypertension. The natural history of untreated ILD in PM/DM is not entirely known, but 5-year survival rates have been reported to be 50–60% for patients with PM/DM-associated ILD. Incomplete resolution of pulmonary opacities with residual bibasilar linear opacities is commonly observed in these patients. DM, more so than PM, is associated with an increased risk of cancer. This excess risk of cancer appears to be highest around the time of diagnosis. Malignancies of the lungs, ovaries, breasts, and stomach are reported most frequently. See also: Interstitial Lung Disease: Cryptogenic Organizing Pneumonia. Pulmonary Effects of Systemic Disease. Pulmonary Fibrosis. Respiratory Muscles, Chest Wall, Diaphragm, and Other.

Further Reading American Thoracic Society/European Respiratory Society (2002) International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias. American Journal of Respiratory and Critical Care Medicine 165: 277–304. Bandoh S, Fujita J, Ohtsuki Y, et al. (2000) Sequential changes of KL-6 in sera of patients with interstitial pneumonia associated with polymyositis/dermatomyositis. Annals of the Rheumatic Diseases 59: 257–262. Bonnefoy O, Ferretti G, Calaque O, et al. (2004) Serial chest ct findings in interstitial lung disease associated with polymyositis– dermatomyositis. European Journal of Radiology 49: 235–244. Braun NM, Arora NS, and Rochester DF (1983) Respiratory muscle and pulmonary function in polymyositis and other proximal myopathies. Thorax 38: 616–623. Cottin V, Thivolet-Bejui F, Reynaud-Gaubert M, et al. (2003) Interstitial lung disease in amyopathic dermatomyositis, dermatomyositis, and polymyositis. European Respiratory Journal 22: 245–250. Dalakas MC and Hohlfeld R (2003) Polymyositis and dermatomyositis. Lancet 362: 971–982. Douglas WW, Tazelaar HD, Hartman TE, et al. (2001) Polymyositis–dermatomyositis-associated interstitial lung disease. American Journal of Respiratory and Critical Care Medicine 164: 1182–1185. Engel AG and Hohlfeld R (2004) Inflammatory myopathies. In: Engel AG and Franzini-Armstrong C (eds.) Myology: Basic and Clinical, 3rd edn., pp. 1321–1366. New York: McGrawHill. Fathi M, Dastmalchi M, Rasmussen E, et al. (2004) Interstitial lung disease, a common manifestation of newly diagnosed polymyositis and dermatomyositis. Annals of the Rheumatic Diseases 63: 297–301. Hill CL, Zhang Y, Sigurgeirsson B, et al. (2001) Frequency of specific cancer types in dermatomyositis and polymyositis: a population-based study. Lancet 357: 96–100.

PRIMARY CILIARY DYSKINESIA 485 Ikezoe J, Johkoh T, Kohno N, et al. (1996) High-resolution CT findings of lung disease in patients with polymyositis and dermatomyositis. Journal of Thoracic Imaging 11: 250–259. Marie I, Hatron PY, Hachulla E, et al. (1998) Pulmonary involvement in polymyositis and in dermatomyositis. Journal of Rheumatology 25: 1336–1343. Mastaglia FL, Garlepp MJ, Phillips BA, and Zilko PJ (2003) Inflammatory myopathies: clinical, diagnostic and therapeutic aspects. Muscle & Nerve 27: 407–425.

Potassium Channels

Schwarz MI, Sutarik JM, Nick JA, et al. (1995) Pulmonary capillaritis and diffuse alveolar hemorrhage: a primary manifestation of polymyositis. American Journal of Respiratory and Critical Care Medicine 151: 2037–2040. Tazelaar HD, Viggiano RW, Pickersgill J, and Colby TV (1990) Interstitial lung disease in polymyositis and dermatomyositis: clinical features and prognosis as correlated with histologic findings. American Review of Respiratory Diseases 141: 727–733.

see Ion Transport: Potassium Channels.

PRIMARY CILIARY DYSKINESIA H Mitchison, The Royal Free Hospital, London, UK M Salathe, University of Miami, Miami, FL, USA M Leigh and J L Carson, University of North Carolina, Chapel Hill, NC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Primary ciliary dyskinesia (PCD) is caused by ultrastructural ciliary defects that lead to abnormal ciliary beating and, subsequently, mucociliary dysfunction. PCD presents clinically with bronchiectasis, sinusitis, and, in up to 50% of cases, situs inversus. The ultrastructural defects of cilia are diverse but include in many cases outer and/or inner dynein arms. Recent advances have shown that ciliary defects in the embryonic node during development are responsible for the random right–left axis determination in these patients. Genetic approaches have elucidated at least some of the heterogeneous molecular defects underlying PCD. This article summarizes the current knowledge about this disease with respect to clinical manifestations, laboratory diagnosis and pathogenesis, situs inversus, genetics, and therapeutic considerations.

A report of a patient with the seemingly disparate symptoms of bronchiectasis and situs inversus 100 years ago is likely the first account of primary ciliary dyskinesia (PCD). Kartagener refined the description of the syndrome to include chronic sinusitis. However, only approximately 30 years ago, Afzelius and co-workers identified absent axonemal dynein arms in motile cilia with the ‘9 þ 2’ microtubular arrangement of the airway epithelium and in sperm flagella as the cellular defect leading to what had come to be known as Kartagener’s syndrome or immotile cilia syndrome. Recent studies have demonstrated considerable heterogeneity of dynein arm morphology at the ultrastructural level among patients with this syndrome. Moreover, half of patients with clinical

symptoms and ciliary ultrastructural defects do not exhibit situs inversus. Thus, the term PCD is currently used to describe individuals with congenital abnormalities of cilia and flagella and the clinical symptoms of bronchiectasis and chronic sinusitis. Kartagener’s syndrome, which in addition to bronchiectasis and sinusitis includes situs inversus, is thus considered a subset of PCD. The overall incidence of PCD is 1 in 20 000, with enrichment in certain populations. PCD is usually an autosomal recessive disorder, but unusual cases of PCD with apparent dominant or X-linked inheritance pattern have also been reported.

Clinical Manifestations Many clinical features of PCD reflect abnormal ciliary beating leading to impaired mucociliary clearance. Symptoms of mucociliary dysfunction in the nose, sinuses, and middle ear are recurrent or persistent rhinitis, sinusitis, and otitis media. Chronic productive cough is the major symptom of mucociliary dysfunction in the lower airways, and this chronic bronchitis can lead to bronchiectasis. Neonatal respiratory problems, situs inversus, and male infertility, common in PCD, are most likely linked to ciliary or flagellar dysfunction. Chronic nasal congestion is common and often present from early infancy, with little or no seasonal variation. Almost all PCD patients have chronic sinusitis, radiographically demonstrated by mucosal thickening, cloudiness, and/or opacification of all paranasal sinuses. Nasal polyps occur in approximately one-third of patients and may be apparent in early childhood. Almost all patients have chronic otitis media that is much more prominent in early childhood. At the time of diagnosis, most patients

486 PRIMARY CILIARY DYSKINESIA

have either chronic tympanic membrane perforations or have had multiple tympanostomies with insertion of ventilation tubes. Many have conductive hearing loss. Chronic productive cough is an almost universal feature of PCD. The cough is most apparent in the early morning but may occur at night or in association with exercise. In many children, exercise tolerance is normal but becomes impaired with advancing obstructive airway disease. Findings on chest examination are variable, with localized crackles that may or may not clear following forceful cough. Wheezing is relatively uncommon. Chronic airway infection ultimately results in bronchiectasis. Cultures of sputum or bronchoscopic aspirates may yield Hemophilus influenzae, Staphylococcus aureus, Streptococcus viridans, and Streptococcus pneumoniae. Patients with long-standing disease may have chronic infection with Pseudomonas aeruginosa. Pulmonary function tests may be normal early but typically demonstrate mild to moderate obstruction that becomes more severe in adulthood. Bronchodilator responsiveness is variable. Longitudinal analyses of children with PCD suggest that lung function may remain stable over relatively long periods of time. Most patients have a history of transient respiratory problems in the first days of life, with tachypnea, cough, increased secretions, and hypoxemia. The etiology for these respiratory problems is often unexplained or attributed to aspiration pneumonitis or neonatal pneumonia. The possibility of PCD is rarely considered except in cases with situs inversus, persistent atelectasis, persistent pneumonia, or a family history of PCD. The high occurrence of transient neonatal respiratory distress in PCD patients suggests that ciliary activity may be important for clearing fluid during the transition from a fluid-filled fetal lung to an air-filled neonatal lung. Situs inversus occurs in up to 50% of patients with PCD. Typically, all viscera in the chest and abdomen are transposed (situs inversus totalis). Male infertility is common and attributable to impaired sperm motility since ultrastructural and functional ciliary defects are mirrored in sperm flagella. However, some patients with PCD may have absent dynein arms in their cilia but normal dynein arms in sperm with normal motility. The occurrence of ciliary defects in cells lining the fallopian tubes has led to speculation that infertility and ectopic pregnancies could be increased in women with PCD, but this area has not been examined systematically. Hydrocephalus or dilated ventricles have been reported in a few patients with PCD. Ultrastructural and functional defects in ventricular ependymal cilia

provide a theoretical basis for this association, a hypothesis supported by hydrocephalus observed in knockout mouse models.

Laboratory Diagnosis and Pathogenesis Although a variety of heterogeneous ciliary ultrastructural lesions have been described among patients with PCD, a few studies have suggested that clinical symptoms suggestive of PCD are not always accompanied by evidence of an ultrastructural ciliary defect. This could be due to specimen processing or as yet uncharacterized or poorly understood ciliary defects not amenable to conventional electron microscopic inspection. Despite such reports, electron microscopic documentation of ciliary defects remains the standard for diagnosing PCD. In a recent study, 43% of patients exhibited abnormalities (absence or dysmorphology) of the outer dynein arm, 29% of the inner dynein arm, and 24% of both arms. The remaining 4% exhibited anomalies of the central microtubular pairs, radial spokes, or nexin links. A valid diagnosis of PCD thus may be indicated by the absence of both dynein arms, by the absence of either the inner or the outer dynein arm, or by consistent evidence of dynein arm dysmorphology (Figure 1). PCD in association with an absence of radial spokes or the transposition of an outer ODA IDA

(a)

(b)

(c)

(d)

0.15 µm

Figure 1 (a) Cross-section of ciliary axoneme from a healthy human subject. ODA, outer dynein arm; IDA, inner dynein arm. (b) Cross-section of ciliary axoneme from a PCD patient with an ODA defect. (c) Cross-section of ciliary axoneme from a PCD patient with an IDA defect. (d) Cross-section of ciliary axoneme from a PCD patient with absent IDA and ODA.

PRIMARY CILIARY DYSKINESIA 487

peripheral microtubular doublet to occupy the locus of a central microtubular doublet yielding an 8 þ 2 axonemal configuration has also been described. In the normal cilium, the bending motility of the axoneme mediated by ATPase-driven microtubular sliding is a highly ordered event. In contrast, the disorganization of the PCD axoneme yields a beat pattern that ranges from complete immotility to a vigorous motility that may appear virtually normal to an untrained observer. Thus, assessment of motility to aid the laboratory diagnosis and pathogenesis of PCD has been problematic, although it has been used successfully in specialized centers. Efforts to simply evaluate ciliary clearance by assessing the time required for a patient to taste a drop of saccharin placed on the nasal turbinate have fallen into disrepute. To date, the only definitive relationship between ciliary motility and ultrastructural integrity in PCD has been the evidence that cilia with no axonemal dynein arms reveal no motility. Other phenotypes seem to exhibit some, albeit dysfunctional, motion. Ultrastructural evidence has shown that the central microtubular pairs of cilia serve as vectors indicating the direction of beat (airway cilia with absent central pair beat in a circular pattern), and that in PCD the beating direction of adjacent cilia is disoriented relative to one another. Although this feature may help to identify patients with PCD, it also has been observed, at least focally, among individuals with transient upper respiratory infections and thus should not be considered an index lesion.

Situs Inversus The reasons for situs inversus totalis in up to half of the patients with PCD remained uncertain until recently (and continues to be controversial). The first clear indication that situs inversus was related to abnormal ciliary function was the cloning of an axonemal dynein heavy-chain gene, left–right dynein, found to be mutated in a strain of mice with a 50% incidence of situs inversus. This murine gene was expressed in the embryonic node at embryonic day 7.5 – the location and time of right–left axis determination. Cilia were found in the embryonic node at this developmental time as well, but ultrastructural analysis revealed them to be of the ‘9 þ 0’, usually nonmotile, variety. Thus, it came as a surprise when it was shown that embryonic node cilia are in fact motile, despite their ‘9 þ 0’ configuration. With an unusual circular motion, their motility was different from that of ‘9 þ 2’ cilia, but their beating was critical for correct left–right axis determination. Immotile embryonic node cilia were associated with a 50% chance of developing situs inversus. The flow across

the embryonic node created by motile cilia seems to be sensed by nonmotile, flow-sensing cilia, localized in the periphery of the embryonic node. The bending of these cilia elicits a calcium signal on only one side of the node. This signal could cause the appropriate, unilateral expression of genes that ultimately determine the correct left–right axis. Abnormalities in motile or nonmotile cilia of the embryonic node will lead to random axis determination and thus to situs inversus in 50% of cases. Several mouse models of random left–right axis determination are used for further investigation of these issues, but their detailed description is beyond the scope of this article. Ciliary defects can also affect primary or nonmotile cilia (with the ‘9 þ 0’ microtubule arrangement). Since many cell types in the body express nonmotile cilia, PCD can also be associated with disorders that reflect nonmotile ciliary dysfunction other than situs inversus, such as cystic kidney disorder and retinitis pigmentosa.

Genetics Genetic approaches to investigate the heterogeneous molecular defects underlying PCD have focused on identification of disease-causing genes using genetic linkage analysis (positional cloning) and candidate gene analysis. Although a genetic linkage of PCD families to the HLA locus on chromosome 6 was identified, candidate gene analysis in this region has not revealed the causative gene. Linkage analysis using inbred families and homozygosity mapping has identified four PCD loci: DNAH5 on chromosome 5p15, CILD2 on 19q, and additional loci on 16p12 and 15q13–15. Selection of candidate genes for mutational analysis has also proven successful, with identification of mutations in DNAI1 on chromosome 9p13–p21 and DNAH11 on 7p15. However, genetical heterogeneity can even be found within groups of families with the same ultrastructural defect. Mutations in genes encoding two different axonemal outer dynein arm components (DNAI1 and DNAH5) have been shown to cause PCD in patients lacking outer dynein arms. Mutations in the DNAI1 and DNAH5 genes are proposed to account for approximately 24% of PCD cases overall and presumably for a larger percentage of the subgroup of PCD cases associated with outer dynein arm deficiencies. Six DNAI1 mutations have been reported, and these account for mutations in 6 of 47 PCD families screened so far. One mutation, a T insertion predicted to cause a splice-site mutation (219 þ 3insT), occurs more frequently. The relative frequency of this allele may indicate a mutation hotspot in the DNAI1 gene or a population founder effect in PCD.

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Ten DNAH5 mutations have been reported. Of 25 PCD families compatible for linkage to the DNAH5 gene, mutations were found in 8. These are mostly premature truncation mutations predicting loss of motor- and microtubule-binding sites from the protein. Two missense mutations also occur, both located in functionally conserved amino acids. No apparent phenotype differences occur between patients with combinations of truncation or missense mutations. However, a patient homozygous for a splice-site mutation (IVS74-1G4C) has been reported with a partial outer dynein arm deficiency and 54% shortened outer dynein arms, contrasting with patients homozygous for two truncation mutations who displayed the complete absence of outer dynein arms, suggesting a genotype–phenotype correlation. Defects in another axonemal heavy-chain dynein, DNAH11, were identified in a patient with Kartagener’s syndrome and normal cilia ultrastructure. DNAH11 is the human homolog of the mouse left– right dynein gene. DNAH11 mutations are therefore associated with situs inversus and possibly a minority of PCD cases. Mutational analysis on candidate genes encoding other axonemal structural components has excluded the dynein genes for the heavy chain DNAH9, the intermediate chain DNAI2, the light chain TCTEX2, and the gene encoding the central complex protein, hPF20 as major causes of PCD. In one report, an absence of DNAH7 protein in cilia from a PCD patient was shown but no coding mutations were found, suggesting the likely involvement of another gene as the primary defect. Other candidate genes involved in ciliary function have also been investigated, including the genes for the transcription factor FOXJ1 and DNA polymerase lambda (DPCD). Genetic work is aided by the selection of candidate disease genes with a conserved homolog in Chlamydomonas reinhardtii, a biflagellated eukaryotic unicellular alga and an established ciliary model organism. Dysmotile Chlamydomonas strains with axonemal defects similar to those of PCD patients have been and will be valuable in identifying genes associated with PCD. Furthermore, advances in proteomics and comparative bioinformatics provide comprehensive sets of ciliary genes and proteins, which comprise excellent new PCD candidates.

Therapeutic Considerations No specific therapeutic modalities are available to correct ciliary dysfunction in PCD. Management should include aggressive measures to enhance clearance of mucus, prevent respiratory infections, and treat bacterial infections in the airways, sinuses, and

middle ear. Few clinical trials have been conducted because PCD is rare and most centers follow only a few PCD patients. Approaches to enhance mucus clearance from the lung in PCD include chest percussion with postural drainage, mechanical oscillatory Vest percussion, vigorous aerobic exercise, and other maneuvers to encourage cough and deep breathing. Bronchodilators such as albuterol may aid mucus clearance in patients who are bronchodilator responsive. Inhaled corticosteroids have been used, but the role of antiinflammatory agents has not been defined. Measures to prevent respiratory tract infection and irritation should be considered, including routine immunizations (for pertussis, measles, H. influenzae type b, S. pneumoniae, and influenza) and preventive counseling to avoid exposure to respiratory pathogens, tobacco smoke, and other irritants. Prompt institution of antibiotic therapy for bacterial infections (bronchitis, sinusitis, and otitis media) can prevent or delay irreversible damage. Sputum culture results can direct appropriate antimicrobial therapy. In some patients, symptoms recur within days to weeks after completing a course of antibiotics. This subgroup may benefit from extended use of a broad-spectrum antibiotic. If detected and treated early, P. aeruginosa colonization of the airways can be eradicated; however, long-standing pseudomonal infection of bronchiectatic airway is unlikely to clear, even with long-term intravenous antibiotic therapy. Use of tympanostomy with ventilation tube placement may benefit children with chronic ear infections and conductive hearing impairment. Nasal polypectomy and/or sinus drainage may provide short-term relief of symptoms in severe sinusitis without response to antibiotic therapy. However, long-term benefits are unclear. Lobectomy should be considered only in special cases. In patients with end-stage lung disease, lung transplantation has been performed successfully.

Prognosis Chronic lung disease with bronchiectasis may progress to severe disability and eventually respiratory failure. The rate of disease progression is variable. A number of individuals have experienced a normal or near-normal life span. Better diagnostic tools are needed to facilitate earlier diagnosis and institution of therapy, thereby improving the overall health and prognosis for these patients. See also: Bronchiectasis. Symptoms of Respiratory Disease: Cough and Other Symptoms.

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Further Reading Afzelius BA (1976) A human syndrome caused by immotile cilia. Science 193: 317–319. Avidor-Reiss T, Maer AM, Koundakjian E, et al. (2004) Decoding cilia function: defining specialized genes required for compartmentalized cilia biogenesis. Cell 117: 527–539. Bush A and O’Callaghan C (2002) Primary ciliary dyskinesia. Archives of Disease in Childhood 87: 363–365. Dutcher SK (1995) Flagellar assembly in two hundred and fifty easy-to-follow steps. Trends in Genetics 11: 398–404. El Zein L, Omran H, and Bouvagnet P (2003) Lateralization defects and ciliary dyskinesia: lessons from algae. Trends in Genetics 19: 162–167. Ibanez-Tallon I, Heintz N, and Omran H (2003) To beat or not to beat: roles of cilia in development and disease. Human Molecular Genetics 12(special issue no. 1): R27–R35. Kartagener M (1933) Zur Pathogenese der Bronchiektasien. Beitra¨ge zur Klinik der Tuberkulose 83: 489–501. Leigh MW (1998) Primary ciliary dyskinesia. In: Chernick V and Boat TF (eds.) Kendig’s Disorders of the Respiratory Tract in Children, pp. 819–825. Philadelphia: Saunders.

McGrath J, Somlo S, Makova S, Tian X, and Brueckner M (2003) Two populations of node monocilia initiate left–right asymmetry in the mouse. Cell 114: 61–73. Meeks M and Bush A (2000) Primary ciliary dyskinesia (PCD). Pediatric Pulmonology 29: 307–316. Nonaka S, Tanaka Y, Okada Y, et al. (1998) Randomization of left–right asymmetry due to loss of nodal cilia generating leftward flow of extraembryonic fluid in mice lacking KIF3B motor protein. Cell 95: 829–837. Noone PG, Leigh MW, Sannuti A, et al. (2004) Primary ciliary dyskinesia: diagnostic and phenotypic features. American Journal of Respiratory and Critical Care Medicine 169: 459– 467. ¨ ber einen Fall von Bronchiektasie bei einem Siewert A (1904) U Patienten mit situs inversus viscerum. Berliner klinische Wochenschrift 41: 139–141. Sleigh MA (1981) Primary ciliary dyskinesia. Lancet 2: 476. Spiden S and Mitchison HM (2001) Homozygosity mapping as an approach for identifying genes involved in primary ciliary dyskinesia. In: Salathe M (ed.) Cilia and Mucus: From Deve lopment to Respiratory Defense, vol. 10, pp. 109–117. New York: Dekker.

PRIMARY MYELOFIBROSIS C J McNamara, The Royal Free Hospital, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Chronic idiopathic myelofibrosis refers to an acquired clonal hematopoietic stem cell disorder that is characterized by hyperplasia of progenitor cells in the bone marrow, reactive bone marrow fibrosis, characteristic changes in the blood and extramedullary hematopoiesis (EMH). Complications include thrombotic disease, bleeding and leukemic transformation. Respiratory complications are less common and include thromboembolic disease, pulmonary hypertension and EMH in the lung. The long-term prognosis is poor. Current treatments are largely palliative; however, experimental treatments targeted at the underlying pathogenesis are likely to become available shortly.

Introduction Chronic idiopathic myelofibrosis (CIMF) is an acquired clonal hematopoietic stem cell disorder that is characterized by hyperplasia of progenitor cells in the marrow, including dysplastic megakaryocytes and clonal monocytes, which secrete growth factors that lead to varying degrees of fibrosis, osteosclerosis, and new vessel formation in the bone marrow. Extramedullary hematopoiesis (EMH), also known as agnogenic myeloid metaplasia, is usually present and refers to ectopic hematopoiesis that occurs most often in the liver and spleen but may be seen in any organ, including the lungs and pleura. This is most likely a

consequence of the replacement of normal hematopoietic tissue by collagen fibrosis, the abnormal mobilization of hematopoietic progenitors into the peripheral blood and their localization in other organs.

Pathology The diagnosis of CIMF is based on morphologic findings and the exclusion of other pathologies known to cause fibrosis of the marrow (see Table 1). It is important to be rigorous in the application of diagnostic criteria as marrow fibrosis and EMH are not specific for CIMF. Bone marrow fibrosis may also be seen in other myeloproliferative disorders, such as polycythemia vera and essential thrombocythemia, as well as in association with metastatic bone marrow tumors. Examination of the blood film frequently suggests the diagnosis of CIMF. Patients in the fibrotic phase of the illness are usually anemic and have characteristic changes on the blood film, such as leukoerythroblastosis (a left-shift in the granulocyte count and nucleated red cells, which are normally absent in the blood) and marked red cell anisopoikilocytosis that includes tear drop cells (see Figure 1). These findings are suggestive but not diagnostic of CIMF. They may also be seen in conditions that replace normal bone marrow that may or may not have associated marrow fibrosis.

490 PRIMARY MYELOFIBROSIS Table 1 Morphologic findings Prefibrotic stage

Fibrotic stage

Bone marrow Minimal/absent reticulin fibrosis Cellularity increased Abundant atypical megakaryocytes with morphologic atypia

Bone marrow Established reticulin fibrosis Cellularity reduced Abundant atypical megakaryocytes with Morphologic atypia Osteosclerosis

Peripheral blood No or mild leukoerythroblastosis Mild red cell anisopoikilocytosis with few tear drop poikilocytes

Peripheral blood Leukoerythroblastosis Marked red cell anisopoikilocytosis with prominent tear drop poikilocytosis

Figure 1 Leukoerythroblastic blood film from a patient with CIMF demonstrating tear drop red cells and a nucleated red blood cell (Wright’s stain, 400).

Figure 2 Silver staining of trephine biopsy demonstrating increased marrow reticulin (200).

The prefibrotic phase of CIMF is typically associated with a leukocytosis (median white cell count 15 109 l 1 range (1.6–88.3) 109 l 1) and the platelet count is elevated in half of the patients.

Figure 3 End-stage CIMF marrow demonstrating replacement of normal hematopoiesis by fibrous tissue and fibroblasts (Hematoxylin–eosin stain, 200).

The median number of CD34 þ hematopoietic progenitor cells circulating in the periphery is increased in the majority of patients. The bone marrow may be difficult to aspirate because of the fibrotic changes, resulting in a ‘dry tap’. A trephine biopsy is required in all cases to establish the diagnosis. Silver or trichrome staining of the marrow trephine identifies an increase in type III collagen in all patients in the fibrotic phase of the disease (Figure 2). Collagen fibrosis is not present in the prefibrotic or cellular phase, making diagnosis difficult. The end stage, densely fibrotic marrow typically has little granulopoiesis or erythropoiesis but numerous dysplastic megakaryocytes distributed throughout the areas of fibrosis (Figure 3). Immunostaining of the marrow identifies increased marrow vascularity only in the advanced fibrotic phase.

Clinical Features The annual incidence of CIMF is 0.5–1.5 per 100 000. The median age at diagnosis is more than 60 years of age with an equal sex distribution.

PRIMARY MYELOFIBROSIS 491 Table 2 Clinical and laboratory findings Prefibrotic stage

Fibrotic stage

Splenomegaly and/or hepatomegaly: absent or only mild enlargement Mild anemia Mild leukocytosis Mild thrombocytosis

Splenomegaly (90%) and/or hepatomegaly (50%) Moderate to severe anemia Leukocytosis or leukopenia Thrombocytosis or thrombocytopenia

Clinical and laboratory features depend largely on whether the patient is in the early/prefibrotic (20–30%) or the fibrotic phase (70–80%) of the illness at the time of presentation (see Table 2). Up to one-third of patients are detected while asymptomatic, either on a routine blood film or after the finding of hepatomegaly or splenomegaly on physical examination. Splenomegaly is present in most cases. Patients may develop abdominal pain and distension due to splenic enlargement, which can be immense. Impingement of the spleen on adjacent structures may cause local symptoms. Splenic infarction may produce severe left upper quadrant pain that may be confused for respiratory or other abdominal pathologies. The fibrotic phase is characterized by progressive pancytopenia secondary to bone marrow failure. Symptoms related to cytopenias appear, particularly anemia requiring transfusion. Ineffective erythropoiesis and sequestration of red cells and platelets in an enlarged spleen exacerbate the cytopenias. Constitutional symptoms are a common feature in CIMF and include fatigue, weight loss, fever, and sweats. Along with symptomatic organomegaly these symptoms account for the poor quality of life of many CIMF patients. Hematologic Complications

Thrombotic complications are the most important causes of morbidity and mortality in CIMF with a combined rate of 45.6 complications per 100 patient years. This may manifest as deep venous thrombosis (DVT) or pulmonary embolism (PE). In addition, thrombosis at unusual sites, including the hepatic and portal veins as well as the cerebral venous sinuses, may occur, producing distinct clinical syndromes. Patients are at equal risk of arterial thrombosis, the most common sites being the vessels of the limbs or the central nervous system and, less commonly, the coronary or mesenteric arteries. The cause of the prothrombotic state seen in CIMF is not entirely understood. Paradoxically, bleeding may also complicate CIMF, but this is less often fatal. Qualitative platelet

function abnormalities are well documented and hemorrhage may occur even when the platelet count is elevated. However, these abnormalities are usually of little clinical significance. When bleeding does occur, it is most often of the type affecting small vessels in the skin and mucosal sufaces. Antiplatelet agents used to ameliorate the thrombotic tendency may contribute to the bleeding tendency. Leukemic transformation of CIMF occurs in up to 20% of patients within 10 years of diagnosis and portends a very poor prognosis. This event is often heralded by worsening cytopenias, constitutional symptoms, and the appearance of myeloblasts in the peripheral blood. Changes in hepatic vascular compliance can lead to portal hypertension and gastroesophageal varices. Iron overload may develop in patients requiring multiple transfusions because of anemia. Hyperuricemia may also occur due to ineffective hematopoiesis and increased cell turnover. Respiratory Complications

These occur infrequently as the main clinical feature of CIMF. Pulmonary infections may occur, secondary to immunosuppression related either to bone marrow failure or from treatment of the disease. EMH can involve any organ, including the lung and pleura. Consolidation of lung parenchyma and pleural effusions may occur as a result. Leukemic infiltration of the lung has also been documented. There are reports of pulmonary hypertension (PHT) occurring in CIMF patients. Potential causes include occlusion of pulmonary arteries by circulating megakaryocytes, thromboembolic phenomenon, extramedullary hematopoiesis infiltrating pulmonary parenchyma, and in situ pulmonary thrombosis secondary to chronic disseminated intravascular coagulation. Portal hypertension, which is a well-recognized complication of CIMF, may contribute to PHT. Thrombocytosis and accelerated EMH may complicate splenectomy and increase the risk of pulmonary complications. However, whether splenectomy predisposes CIMF patients to PHT remains controversial. Pulmonary edema may occur as congestive cardiac failure and other cardiovascular complications are seen with increased frequency in CIMF. Chemotherapeutic agents used in CIMF may also be associated with lung injury.

Pathogenesis The underlying molecular basis for CIMF is unknown. The clonal nature of the proliferating progenitor cells is demonstrated by cytogenetic analysis and

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X-chromosome studies. Cytogenetic abnormalities occur in less than 60% of patients and none of these are specific for CIMF. Gene expression studies have identified abnormalities in cell signaling pathways, such as the JAK/STAT pathway, offering hope of novel, targeted therapeutic strategies in the future. The observed bone marrow fibrosis is a reactive phenomenon, mediated by fibrogenic cytokines secreted by abnormal, dysplastic megakaryocytes or monocytes. Specific cytokines and growth factors, such as transforming growth factor beta 1 (TGF-b1), basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF), are likely to have an important role in the development and progression of CIMF by stimulating polyclonal fibroblasts to synthesize extracellular matrix and cell adhesion proteins. They also promote the transcription of proteases that inhibit enzymes involved in extracellular matrix degradation. The importance of the megakaryocyte in disease pathogenesis is highlighted by murine experiments where mice given marrow grafts containing cells infected with the megakaryocyte growth factor, thrombopoietin, develop myelofibrosis.

Management and Current Therapy Current therapies for CIMF are unsatisfactory and largely palliative. Allogeneic hematopoietic stem cell transplantation offers the only possibility of cure but is restricted to a small number of patients because of advanced age and comorbidity in the majority of patients. Only 10% of patients are less than 45 years of age. The increasing use of nonmyeloablative conditioning regimens may make this treatment more accessible to CIMF patients. More than one-half of patients require therapy to control elevated blood counts and reduce extramedullary hematopoiesis. Currently, the most frequently used treatment is the ribonucleotide reductase inhibitor hydroxyurea. Treatment of anemia includes the use of erythropoietin, corticosteroids, and androgen preparations. Splenectomy may be helpful in patients with symptoms from organ enlargement or those with cytopenias due to splenic sequestration blood components. The success of this procedure varies according to the indication and is associated with the potential for significant morbidity and mortality. This is mainly related to postsplenectomy thrombocytosis, hepatic enlargement, and thrombosis. In those patients who are not fit for a surgical procedure, splenic irradiation may alleviate symptoms for a short period. Radiotherapy may also be useful in the management of extramedullary hematopoiesis that produces symptoms in strategic locations.

Prophylaxis of thrombotic disease is a cornerstone of management in CIMF. Aspirin is used as primary prophylaxis against thrombotic episodes in patients with elevated platelet counts. Warfarin anticoagulation is reserved for the treatment and secondary prophylaxis of thrombosis. Managing PHT is similar for other patients without CIMF. There may, however, be additional benefit in reducing the platelet count by plateletpheresis or cytoreductive therapy. Antiplatelet therapy may not be effective in preventing PHT. Among the novel investigational agents presently available, thalidomide has been shown to produce a demonstrable improvement in splenomegaly and blood counts in a variable proportion of patients. Thalidomide inhibits the activity of fibrogenic cytokines in vitro. Newer agents targeting dysregulated cytokine production are likely to become available shortly.

Prognosis The median survival for CIMF patients is approximately 5 years. However, the rate of marrow fibrosis and the natural history of the disease are quite variable. CIMF most often pursues a chronic progressive course. Causes of death include fatal thrombosis, hemorrhage, leukemic transformation, bone marrow failure, and cardiac failure. The prognosis for patients with PHT is poor, with a median survival of only 18 months. Other adverse prognostic factors include advanced age, anemia, and male gender. There is intense interest in prognostic scoring models to predict outcome and facilitate decision making particularly when aggressive therapies with appreciable morbidity and mortality, such as bone marrow transplantation, are being considered. See also: Fibroblasts. Pulmonary Thromboembolism: Deep Venous Thrombosis; Pulmonary Emboli and Pulmonary Infarcts. Tumors, Malignant: Overview. Vascular Disease.

Further Reading Arora B, Sirhan S, Hoyer JD, Mesa RA, and Tefferi A (2005) Peripheral blood CD34 count in myelofibrosis with myeloid metaplasia: a prospective evaluation of prognostic value in 94 patients. British Journal of Haematology 128: 42–48. Barbui T, Cortellazo S, Viero P, et al. (1983) Thrombohaemorrhagic complications in 101 cases of myeloproliferative disorders: relationship to platelet number and function. European Journal of Cancer and Clinical Oncology 19: 1593–1599. Barosi G, Viarengo G, Pecci A, et al. (2001) Diagnostic and clinical relevance of the number of circulatory CD34-positive cells in myelofibrosis with myeloid metaplasia. Blood 98: 3249–3255.

PROGRESSIVE SYSTEMIC SCLEROSIS 493 Cervantes F, Barosi G, Demory JL, et al. (1998) Myelofibrosis with myeloid metaplasia in young individuals: disease characteristics, prognosis factors and identification of risk groups. British Journal of Haematology 102: 684–690. Chagraioui H, Kamura H, Tulliez M, Vainchenker W, and Wendling F (2002) Prominent role of TGF-b1 secreted by hematopoietic cells in bone marrow fibrosis induction. Blood 100: 3495–3503. Garcia-Manero G, Schuster S, Patrick H, and Martinez J (1999) Pulmonary hypertension in patients with myelofibrosis secondary to myeloproliferative diseases. American Journal of Hematology 60: 130–135. Jaffe ES, Harris NL, Stein H, and Hardiman JW (eds.) (2001) The World Health Organization Classification of Neoplasms of the Hematopoietic and Lymphoid Tissues. Lyon: IARC Press. Kvasnicka HM, Thiele J, Werden C, Zankovich R, Diehl V, and Fischer R (1997) Prognostic factors in idiopathic osteomyelofibrosis. Cancer 80: 708–719. Le Basse-Kerdiles MC and Martyre MC (1999) Dual implication of fibrogenic cytokines in the pathogenesis of fibrosis and myeloproliferation in myeloid metaplasia with myelofibrosis. Annals of Hematology 78: 437–444. Mesa RA, Li CY, Ketterling RP, Schroeder GS, Knudson RA, and Tefferi A (2005) Leukemic transformation in myelofibrosis with

myeloid metaplasia: a single-institution experience with 91 cases. Blood 105: 973–977. Tefferi A, Mesa RA, Schroeder G, et al. (2001) Cytogenetic findings and their clinical relevance in myelofibrosis with myeloid metaplasia. British Journal of Haematology 113: 763–771. Tefferi A, Silverstein MN, and Noel P (1995) Agnogenic myeloid metaplasia. Seminars in Oncology 22: 327–333. Thiele J, Kvasnicka HM, Boeltken B, et al. (1999) Initial (prefibrotic) stages of idiopathic (primary) myelofibrosis (IMF) – a clinicopathological study. Leukemia 13(11): 1741–1748. Thiele J, Zankovich R, Steinberg T, Fischer R, and Diehl V (1989) Agnogenic myeloid metaplasia (AMM) – correlation of bone marrow lesions with laboratory data: a longitudinal clinicopathological study on 114 patients. Hematological Oncology 7: 327–343. Visani G, Finelli C, Castelli U, et al. (1990) Myelofibrosis with myeloid metaplasia; clinical haematological parameters, predicting survival in a series of 133 patients. British Journal of Haematology 75(1): 4–9. Ward HP and Block MH (1971) The natural history of agnogenic myeloid metaplasia in a critical evaluation of its relationship with the myeloproliferative syndrome. Medicine 50: 357–420.

PROGRESSIVE SYSTEMIC SCLEROSIS M M Freemer, University of California, San Francisco, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The pulmonary manifestations of progressive systemic sclerosis (PSS) are important determinants of a patient’s prognosis. Although the etiology and pathogenesis of PSS are unknown, it is recognized that this is a complex disease; it is likely that different genetic susceptibilities and environmental exposures lead to the disease in different individuals. Individuals with diffuse PSS tend to develop interstitial pneumonia, most commonly fibrotic non-specific interstitial pneumonia. Isolated pulmonary hypertension is frequently seen in individuals with limited scleroderma. Screening for these forms of lung disease with high-resolution computed tomography, pulmonary function tests, and echocardiography is important, given the availability of therapeutic options.

Scleroderma, literally hard (sclero) skin (derma), is a systemic disease with the potential for destructive visceral involvement in addition to disfiguring cutaneous lesions. Descriptions of the skin changes in systemic sclerosis date back to Hippocrates. However, it was not until the twentieth century that Goetz described the visceral manifestations of the disease, providing the term ‘systemic sclerosis’. Progressive systemic sclerosis (PSS) has been reported throughout the world, but the prevalence of the disease remains

unknown. In the United States, the prevalence is estimated to be 300 000. PSS affects women more commonly than men at a ratio of approximately 4:1. Although people of any age may be affected, the onset of disease usually occurs in the fourth through sixth decades of life. Studies vary in the reported prevalence of lung disease in PSS (given differing definitions of pulmonary involvement); however, using the most sensitive diagnostic modalities available, as well as evidence from autopsy studies, most (B70%) PSS patients have lung disease. The pulmonary manifestations have been shown to vary with ethnicity; in blacks and Asians, there is a higher prevalence of lung disease which is also more severe and accelerated than in Caucasians. The two primary forms of lung disease, fibrotic interstitial pneumonia and pulmonary hypertension, play a critical role in PSS patients’ prognosis. In fact, lung disease increases the risk of death at least twofold.

Etiology Genetic Contribution

The cause of PSS is unknown but likely involves both genetic predisposition and environmental exposures. The strongest evidence for a genetic contribution to the development of PSS is found in the Choctaw

494 PROGRESSIVE SYSTEMIC SCLEROSIS

Native Americans in Oklahoma, in whom the prevalence of the disease is more than double that in the general population. The first-degree relatives of any individual with systemic sclerosis have a 13-fold increased relative risk of disease. However, given the disease is so rare, familial cases account for less than 2% of all PSS cases. Environmental Contribution

Environmental exposures that have been considered possible etiologic agents in systemic sclerosis include retroviruses, cytomegalovirus (CMV), organic solvents, vinyl chlorine, silica dust, L-tryptophan, toxic oil, silicone, pesticides, and drugs (e.g., appetite suppressants, cocaine, and carbidopa).

Pathology Interstitial Pneumonia

The hallmark pathologic features of PSS are fibrosis and vascular disease. The lung is no exception, with fibrotic interstitial pneumonias accounting for the vast majority of the primary parenchymal lung disease in PSS. Studies on multiple ethnic groups that applied the current American Thoracic Society and European Respiratory Society classification system for interstitial pneumonias demonstrated that although the overwhelming majority of cases are fibrotic non-specific interstitial pneumonia (NSIP), approximately 20% of the NSIP cases were cellular. In addition to the cases of NSIP, a usual interstitial pneumonia (UIP) pattern was also seen in other PSS patients. There is insufficient evidence to determine whether the prognosis of patients varies with the histopathologic pattern (UIP or NSIP). Vascular Disease

The other primary lung disease in PSS is a vasculopathy. Although it remains contested, there is evidence that the pulmonary circulation in PSS undergoes locally mediated arterial vasoconstriction as visibly occurs in the peripheral circulation with Raynaud’s phenomenon (a characteristic feature of PSS).

PSS patients with isolated pulmonary hypertension develop vascular changes that resemble those in other forms of secondary pulmonary hypertension: arteriolar thickening, medial hypertrophy, and concentric intimal proliferation (onion skinning). In addition to isolated pulmonary hypertension (in the absence of interstitial pneumonia), other PSS patients develop interstitial lung disease with secondary pulmonary hypertension.

Clinical Features The criteria for the diagnosis of PSS were developed in 1980; however, 8 years later an expert panel proposed subclassification into two patterns of disease, limited and diffuse (Table 1). Although the utility of the distinction between limited and diffuse disease remains controversial, it is nevertheless relevant to the consideration of the anticipated pulmonary manifestations in different patient subgroups. Diffuse Progressive Systemic Sclerosis

Disease manifestations In patients with diffuse disease, both the skin and the viscera are involved; the former includes the trunk and acral regions, whereas the latter may include the lungs, kidneys, gastrointestinal tract, and heart. In patients with diffuse disease, PSS progresses relatively rapidly. For example, Raynaud’s phenomenon occurs within 1 year of the cutaneous lesions. Within the first 3 years of disease onset, most PSS patients who will develop interstitial lung disease have already done so. The symptoms of pulmonary disease in these patients are non-specific, such as cough and dyspnea. On serologic evaluation, patients with diffuse disease may be positive for antitopoisomerase antibodies. The pathologic pattern seen most frequently in diffuse PSS patients is fibrotic interstitial pneumonia (NSIP or UIP). Pulmonary hypertension may develop as a complication of progressive lung disease. Disease evaluation In diffuse PSS patients with cough or dyspnea, high-resolution computed tomography (HRCT) is the most appropriate imaging

Table 1 Comparison of limited and diffuse PSS features Feature

Limited systemic sclerosis

Diffuse systemic sclerosis

Pulmonary disease Time of lung disease onset

Primary pulmonary hypertension Late in PSS; more than 5 years of disease duration Anticentromere (70% of patients) Decreased or diminishing diffusing capacity

Fibrotic interstitial lung disease Early in PSS; usually before 5 years of disease duration Antitopoisomerase (30% of patients) Decreased forced vital capacity

Associated antibodies Physiologic predictors of disease progression

PROGRESSIVE SYSTEMIC SCLEROSIS 495

modality, given the reduced sensitivity of chest radiography (Figure 1). In patients with respiratory symptoms, areas of ground-glass opacification on HRCT, and restrictive or diffusion deficits on physiologic testing, bronchoalveolar lavage (BAL) may serve three purposes. The first purpose is diagnostic – to assess the presence of ‘alveolitis’ (an elevated percentage of neutrophils or eosinophils in comparison to standardized normal values), which is an indicator of active disease that will progress in the absence of therapy. In addition, BAL eosinophilia has been shown to be associated with diminished survival. Finally, if clinically indicated, BAL may help to exclude infection as the cause of the patient’s symptoms and radiographic abnormalities. Further evaluation with pulmonary function testing is also useful prognostically; an abnormal forced vital capacity at initial evaluation is associated with a higher mortality rate. The utility of lung biopsy in patients with PSS has not been adequately examined.

can progress to right heart failure, with dyspnea as their only respiratory symptom. They are very commonly anticentromere positive. Given the high mortality associated with pulmonary hypertension, PSS patients should be screened regularly with echocardiograms to assess pulmonary artery pressures. Diminished diffusing capacity on pulmonary function testing predicts the development of pulmonary hypertension.

Limited Progressive Systemic Sclerosis

Pathogenesis

In patients with limited disease, the cutaneous involvement remains distal to the elbows and knees, although it may involve the face. In these patients, PSS progresses relatively slowly. For example, Raynaud’s phenomenon has usually been present for years prior to the cutaneous lesions. Isolated pulmonary hypertension occurs late in those with limited disease. Some patients with limited disease have CREST, with characteristic systemic findings (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasias). Patients with limited disease (including those with CREST)

The pathogenesis of PSS remains unclear, and the explanation for pulmonary involvement has also not been elucidated. A central question in the pathogenesis of PSS (as well as other autoimmune diseases) is why autoantibodies are formed. Endogenous proteins for which autoantibodies have been found in systemic sclerosis patients include nucleolar antigens, RNA polymerases, centromeres, and fibrillin. With respect to lung disease, two specific autoantibodies are of interest. Anticentromere antibody has been associated with limited PSS and may be considered

Figure 1 Typical HRCT findings in PSS interstitial lung disease. Bilateral, basilar predominant areas of ground-glass opacification associated with reticulations are typical HRCT findings in PSS.

Other Disease Manifestations that Affect the Lung

Aspiration pneumonia is a common finding in PSS patients due to the presence of esophageal dysmotility (Figure 2). A relatively rare primary pulmonary manifestation of PSS is vasculitis with pulmonary hemorrhage. Similarly, restrictive disease secondary to thoracic skin tightening is also quite infrequent. Notably, the incidence of lung cancer is elevated in PSS patients.

Figure 2 Typical pathologic findings in non-specific interstitial pneumonia. This lung biopsy demonstrates a temporally and spatially homogeneous pattern of interstitial thickening and inflammation. In many areas, aggregates of lymphocytes can be seen, and germinal centers may be present.

496 PROGRESSIVE SYSTEMIC SCLEROSIS

‘protective’ against interstitial lung disease. Although antitopoisomerase can be seen in those with diffuse disease and interstitial lung disease, this autoantibody is less common (occurring in approximately 30% of patients with diffuse PSS). Autoantibody Formation

Although definitive evidence for the mechanisms by which these autoantibodies develop is not available, several hypotheses exist. The importance of microchimerism for the development of autoimmunity is an area of active investigation. Microchimerism is the concept that the presence of HLA-disparate cells within an individual results in the formation of antibodies. The source of HLA-disparate cells in individuals with autoimmune disease may include fetal cells (in women who have been pregnant), maternal cells, twin sibling cells, or transfused or transplanted cells. Molecular mimicry is an alternative concept that an antibody formed against an exogenous antigen cross-reacts with an endogenous (self-) antigen with resultant autoantibodies. Similarly, it is possible that autoantibodies develop when proteins are changed in some way. Such alteration may include damage caused by the environmental exposures noted previously. Immune System Dysfunction

In addition to autoantibody formation, immune system dysfunction also likely contributes to the development of PSS. In the skin, T-cell infiltration, predominantly CD4 þ T cells, precedes fibrosis. In the lung, as assessed by BAL, CD8 þ T cells predominate, with an elevated CD8:CD4 ratio. The generally accepted hypothesis is that these cells produce inflammatory cytokines that lead to increased collagen deposition by fibroblasts. In PSS, it remains controversial whether the predominant abnormality is in B-cell or T-cell function, and the T-cell response does not appear to be a purely Th1 or Th2 response. Vasculopathy

In addition to the importance of the immune system, PSS patients also have a vasculopathy. The cause of vascular injury has not been established. Although the vascular damage may result from the deposition of immune complexes, other potential contributing factors include dysregulation of endothelial cell apoptosis, endothelial cell proliferation, and release of von Willebrand factor. Genetic Polymorphisms

Interestingly, several genetic association studies in PSS patients indicate that specific genes may

influence the pathogenetic pathway of PSS at many levels, including the development of autoimmunity, tissue fibrosis, and vasculopathy. For example, a variety of HLA regions, which may influence autoantibody formation, have been shown to be associated with PSS in different ethnic groups. Genomewide screening in the Choctaw population revealed genetic polymorphisms associated with systemic sclerosis, including fibrillin-1; secreted protein, acidic and rich in cysteine (SPARC); and topoisomerase-1. Alterations in the proteins encoded by these polymorphic genes may be involved in the development of antibodies to these proteins. Other genes that have been associated with PSS include polymorphisms of the genes for transforming growth factor beta, tumor necrosis factor, interleukin-1A (IL-1A), and IL-4. Such polymorphisms may affect an individual’s response to antigens. Finally, evidence that PSS patients can have alterations in transcriptional factors for collagen and fibronectin may explain the aberrant amount of protein production. The manner in which environmental exposures, individuals’ genetic susceptibility, T- and B-cell activity, as well as vascular disease interact to produce the heterogeneous disease that we call PSS is unknown. An attempt to relate these possible components of the pathogenesis of PSS is represented in Figure 3.

Animal Models There are several animal models for PSS. One genetic model is the tight-skinned mouse (TSK1) that carries a duplication of exons in the fibrillin gene. A second tight-skinned mouse (TSK2) has also been described that develops skin fibrosis. The TSK2 mutation has been mapped to chromosome 1. Unfortunately, although TSK mice have lung disease, it resembles emphysema rather than the interstitial disease seen in PSS patients. Another animal model was discovered at the University of California at Davis in chickens (UCD L200) that are born with missing combs. These animals rapidly develop severe systemic disease, including pulmonary fibrosis, which is often present at 1 week of age. Other animal models of PSS pulmonary disease are those used in many models of pulmonary fibrosis, such as bleomycin.

Management and Current Therapy Although the patterns of disease manifestations in PSS patients with diffuse versus limited disease can be useful in anticipating an individual’s course, many patients may be difficult to classify and any PSS patient can develop any of the pulmonary disease

PROGRESSIVE SYSTEMIC SCLEROSIS 497

Genetic predisposition: (polymorphisms of) Fibrillin-1 (FBN1) Secreted protein, acidic and rich in cysteine (SPARC) Topoisomerase-1 Transforming growth factor beta (TGF-)* Tumor necrosis factor (TNF)* Interleukin 1A (IL-1A)* Interleukin 4 (IL-4)* HLA* Transcription of fibronectin and collagen*

B cells

Fibroblast proliferation

Microchimerism Molecular mimicry

Vascular injury: ? Endothelial cell proliferation ? Dysregulation of apoptosis ? von Willebrand's factor

Autoantibody formation

Environmental exposures: Retroviruses Cytomegalovirus (CMV) Organic solvents Vinyl chlorine Silica dust L-tryptophan Toxic oil Silicone Pesticides Drugs (e.g., appetite suppressants, cocaine, carbidopa)

Cytokine release ?Th1 vs. Th2

T cells ? Role of CD8 cells in lung ? Role of CD4 cells in skin

Figure 3 Proposed pathogenesis of systemic sclerosis. The pathogenesis of scleroderma is unknown. Any proposed pathway must explain both the development of fibrosis and the vascular disease, although in an individual patient the pulmonary involvement is usually either fibrotic or vascular disease. There are many proposed mechanisms for the development of autoantibodies, including genetic susceptibility, environmental exposures, molecular mimicry, and microchimerism. Although B cells play a role in antibody production, it remains unclear whether scleroderma is primarily a B- or T-lymphocyte-mediated disease. In either case, it appears that the cytokines that are released favor fibroblast proliferation. Cytokines may also have a role in endothelial cell proliferation that results in vascular injury or abnormal endothelial cell apoptosis. *Alterations in these genes may cause alterations in the resultant proteins and, therefore, lead to antibody production. However, alterations in these genes (or their expression) may also be more important in other steps of the proposed pathogenetic pathway, such as cytokine or T-cell function.

manifestations. Moreover, given the availability of efficacious treatment options for PSS lung disease, screening is appropriate. Therefore, the management of PSS patients should involve screening for pulmonary disease including HRCT, pulmonary function tests, and echocardiography. Serial screening with echocardiography, for example, has been shown to be useful to detect incident disease. In addition, an evaluation for esophageal dysmotility is important for the prevention of aspiration pneumonia. Treatment for Interstitial Pneumonia

PSS patients with interstitial pneumonia with demonstrated physiologic deficits, abnormal HRCT findings (ground-glass and reticulations), symptoms, and BAL evidence of alveolitis should receive therapy. There is only retrospective evidence that the combination of cyclophosphamide and prednisone can increase survival in these individuals. The results

of a multicenter randomized, placebo-controlled trial of this combination therapy are anticipated. Additional investigations are needed to determine the necessary duration of therapy, the efficacy of cyclophosphamide in comparison to other agents, and the potential benefit of combinations of therapeutic agents. Treatment for Pulmonary Hypertension

Treatment for pulmonary hypertension has been revolutionized with the advent of outpatient-based pulmonary vasodilators. Patients with PSS and pulmonary hypertension benefit from (parenteral) prostacyclin therapy as well as (oral) endothelin inhibitors in terms of their functional status. Patients now have alternative choices of treatment, including phosphodiesterase inhibitors such as sildenafil. Trials comparing these agents and/or using them in combination with cytotoxic agents remain to be performed.

498 PROTEASOMES AND UBIQUITIN See also: Alveolar Hemorrhage. Interstitial Lung Disease: Overview; Idiopathic Pulmonary Fibrosis. Pulmonary Effects of Systemic Disease. Vascular Disease. Vasculitis: Overview.

Further Reading Black CM and Du Bois R (1996) Organ involvement: pulmonary. In: Clements PJ and Furst DE (eds.) Scleroderma, pp. 299–331. Philadelphia: Williams & Wilkins. Bolster MB and Silver RM (1999) Assessment and management of scleroderma lung disease. Current Opinion in Rheumatology 11(6): 508–517. Bouros D, Wells AU, Nicholson AG, et al. (2002) Histopathologic subsets of fibrosing alveolitis in patients with systemic sclerosis and their relationship to outcome. American Journal of Respiratory and Critical Care Medicine 165: 1581–1586. Bryan C, Knight C, Black CM, and Silman AJ (1999) Prediction of five-year survival following presentation with scleroderma. Arthritis and Rheumatism 42(12): 2660–2665. D’Angelo WA, Fries JF, Masi AT, and Schulman LE (1969) Pathologic observations in systemic sclerosis (scleroderma): a study of fifty-eight autopsy cases and fifty-eight matched controls. American Journal of Medicine 46: 428–440.

Freemer MM and King TE (2003) Connective tissue diseases. In: Schwarz MI and King TE (eds.) Interstitial Lung Disease, pp. 553–562. New York: Decker. Kim EA, Lee KS, Johkoh T, et al. (2002) Interstitial lung diseases associated with collagen vascular diseases: radiologic and histopathologic findings. Radiographics 22: S151–S165. Latsi PI and Wells AU (2003) Evaluation and management of alveolitis and interstitial lung disease in scleroderma. Current Opinion in Rheumatology 15(6): 748–755. Steen V (2003) Predictors of end stage lung disease in systemic sclerosis. Annals of Rheumatic Disease 62: 97–99. Steen V and Medsger TA (2003) Predictors of isolated pulmonary hypertension in patients with systemic sclerosis and limited cutaneous involvement. Arthritis and Rheumatism 48(2): 516–522. Steen VD and Medsger TA (2000) Severe organ involvement in systemic sclerosis with diffuse scleroderma. Arthritis and Rheumatism 43(11): 2437–2444. Subcommittee for Systemic Sclerosis Criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee (1980) Preliminary criteria for the classification of systemic sclerosis (systemic sclerosis). Arthritis and Rheumatism 23: 581–590. White B, Moore WC, Wigley FM, Xiao HQ, and Wise RA (2000) Cyclophosphamide is associated with pulmonary function and survival benefit in patients with scleroderma and alveolitis. Annals of Internal Medicine 132(12): 947–954.

PROTEASOMES AND UBIQUITIN D Attaix, Institut National de la Recherche Agronomique, Ceyrat, France & 2006 Elsevier Ltd. All rights reserved.

Abstract The ubiquitin–proteasome-dependent pathway degrades most cell proteins and is involved in the control of many major biological functions. The ubiquitination/deubiquitination system is a complex machinery responsible for the specific tagging and proofreading of substrates degraded by the 26S proteasome, but ubiquitination itself also serves other functions. The formation of a polyubiquitin degradation signal is usually required for proteasome-dependent proteolysis. Hierarchical families of enzymes, which may comprise hundreds of members to achieve high selectivity, control this process. Polyubiquitinated substrates are recognized by the 26S proteasome and degraded into peptides. The 26S proteasome also recognizes and degrades some nonubiquitinated proteins, and several proteasome populations participate in protein breakdown. In fact, mammalian cells contain multiple ubiquitin- and/or proteasome-dependent pathways. The role of these systems in respiratory diseases is still largely unknown. The proteasome is activated in mechanical ventilation-associated diaphragmatic atrophy and possibly in patients with chronic obstructive pulmonary diseases who exhibit muscle wasting. More importantly, the ubiquitin–proteasome system is responsible for the breakdown of the hypoxia-inducible factor-1a subunits, a transcription factor that may regulate the expression of up to 5% of the human genome.

Introduction A large amount of information on the characterization and regulation of proteolysis has been obtained over the last two decades. In particular, we now know that the ubiquitin–proteasome pathway is the major nonlysosomal process responsible for the breakdown of most short- and long-lived proteins in mammalian cells. In addition, the pathway controls various major biological events (transcriptional control, cell-cycle progression, oncogenesis, etc.) via the breakdown of specific protein substrates. There are two main steps in the pathway: (1) covalent attachment of a polyubiquitin chain to the substrate (i.e., polyubiquitination); and (2) specific recognition of this signal, and degradation of the tagged protein into peptides by the 26S proteasome (Figure 1).

Ubiquitination The 76 amino acid polypeptide ubiquitin was first characterized in 1980 as the critical component for the ATP-dependent proteolysis in reticulocytes. Ubiquitin prevails in all eukaryotes and is highly conserved among species. Ubiquitination is defined

PROTEASOMES AND UBIQUITIN 499

Free Ub

Ub

ATP

AMP

Ub

E1

DUBs

E2

Ub

E2-interacting domain

E3

PolyUb substrate

Substrate-binding site

Ub Ub Ub

Lid: PolyUb recognition Base: PolyUb recognition 19S and ATP hydrolysis

Ub

26S proteasome

20S

Peptidase activities

19S

Peptides Figure 1 Ubiquitin (Ub) is first activated by the ubiquitin-activating enzyme (E1) and transferred on one ubiquitin-conjugating enzyme (E2). The E2 with or without a ubiquitin–protein ligase (E3), which possesses a substrate-specific binding site, polyubiquitinates the target protein. The polyubiquitin degradation signal is then recognized by several subunits of the 19S complex of the 26S proteasome and recycled by the deubiquitinating enzymes (DUBs) into free ubiquitin, while the substrate is unfolded and injected into 20S proteolytic core of the 26S proteasome and cut into peptides.

as the covalent attachment of ubiquitin to a protein substrate. This is a multiple step process. In brief, ubiquitin is initially activated in the presence of ATP to a high-energy thiol ester intermediate by the ubiquitin-activating enzyme (E1) (Figure 1). E1 then transfers ubiquitin to one of the ubiquitinconjugating enzymes (E2s), which also forms a thiol ester linkage between the active site cysteine and

ubiquitin. E2s and/or ubiquitin-protein ligases (E3s), which play a role in the selection of proteins for conjugation, bind the first ubiquitin molecule to protein substrates via an isopeptide bond between the activated C-terminal glycine residue of ubiquitin and the e-amino group of a lysine residue of the substrate. The resulting monoubiquitinated protein is usually not targeted for degradation by the proteasome.

500 PROTEASOMES AND UBIQUITIN

Typical monoubiquitinated conjugates are receptors or basic proteins such as histones. Alternatively, E2s and/or E3s catalyze the formation of polyubiquitinated conjugates. This is usually achieved by transfer of additional activated ubiquitin moieties to Lys48 of the preceding conjugated ubiquitin molecule. The ubiquitin-conjugating system is hierarchical. In mammals, there is a single E1, at least 30–40 E2s, and several hundred E3s. Ubiquitination Machinery

The enzymes of the ubiquitin system are present in both the cytosol and nucleus. Only E1 and some E2s can form a polyubiquitin degradation signal, but usually this process also requires one E3. A given E2 interacts with a limited number of E3s (and vice versa), which in turn recognize their specific protein substrates (Figure 1). In addition, several substrates are known to be ubiquitinated by different combinations of E2s and E3s. This results in a wide range of ubiquitination pathways, which are specific for a given protein or a class of substrates. E3s are responsible for the selective recognition of protein substrates and therefore play a key role in the ubiquitin pathway. To date, all known E3s are described as HECT (homologous to E6-AP C-terminus) domain E3s, RING (really interesting new genes) finger E3s, and U-box-containing E3s. The first major group of E3s corresponds to enzymes of the HECT domain family. The N-terminus region of HECT E3s is responsible for substrate recognition, and the HECT domain itself in the C-terminus region mediates E2 binding and ubiquitination of the target protein via thiol ester linkage formation with ubiquitin. Mammalian genome sequencing projects have identified numerous potential uncharacterized HECT E3s. Most E3s are RING finger E3s. The RING finger structure is defined by eight cysteine and histidine residues that coordinate two zinc ions. There are several hundred cDNAs encoding RING finger proteins in the GenBank database. RING finger E3s are either monomeric proteins (i.e., the N-end rule enzyme E3a that binds to proteins bearing basic or bulky hydrophobic N-terminal amino acid residues) or multiple subunit complexes. These complexes form at least three distinct E3 families called the cyclosome or APC (anaphase-promoting complex), the SCF (Skp1-Cdc53-F-box protein family), and the VCB-like (Von Hippel–Lindau tumor suppressorElonginC/B) E3s. All these complexes contain a catalytic core and substrate-specific adapter proteins. For example, in the huge SCF E3 family the catalytic core is formed by three subunits that include the

RING finger subunit and an E2. The adapter protein Skp1 recruits F-box proteins, which themselves recruit specific protein substrates through protein– protein interaction domains such as leucine-rich repeats or WD-40 domains. Only a couple of U-box E3s have been characterized so far. The U-box domain has a three-dimensional structure close to that of the RING finger.

Signals that Target Substrates for Ubiquitination and Proteolysis Very importantly, ubiquitination is not only a degradation signal, but also directs proteins to a variety of fates, which include roles in ribosomal function, DNA repair, protein translocation, and modulation of structure or activity of the target proteins. For example, many monoubiquitinated proteins are targeted for endocytosis. By contrast, and in order to be efficiently degraded by the 26S proteasome, most substrates must be bound to a polyubiquitin degradation signal that comprises at least 4 (and up to 20–30) ubiquitin moieties. The formation of the polyubiquitin degradation signal is determined by short regions in the primary sequence of the targeted protein. This includes the nature of the N-terminal amino acid of the substrate (N-end rule pathway), phosphorylation and/or dephosphorylation events, and a very degenerate 9 amino acid motif called the ‘destruction box’, which is a crucial signal for the ubiquitination and breakdown of mitotic cyclins and other cell-cycle regulators. The 26S proteasome, however, is not an absolute ubiquitin-dependent proteolytic enzyme, as it also degrades a growing number of nonubiquitinated substrates. The precise mechanisms by which such nonubiquitinated substrates can be recognized by the 26S proteasome are unclear.

Deubiquitination Eukaryotic cells also contain DUBs (deubiquitinating enzymes), which are encoded by the UCH (ubiquitin C-terminal hydrolase) and the UBP (ubiquitin-specific processing protease) gene families. UCHs are relatively small proteins (o40 kDa) and only four isoforms have been characterized in the human genome. UCHs mainly hydrolyze small amides and esters at the C-terminus of ubiquitin. In contrast, UBPs are 50–250 kDa proteins and constitute a large family; there are at least 63 distinct human UBPs. UBPs are involved in several biological processes, including the control of growth, differentiation, and genome integrity. In proteasome-dependent proteolysis, the

PROTEASOMES AND UBIQUITIN 501

putative major roles of DUBs are to maintain free ubiquitin levels by processing the products of ubiquitin genes, which all encode fusion proteins, and to recycle polyubiquitin degradation signals into free monomers. DUBs also deubiquitinate substrates erroneously tagged for degradation (proofreading), and keep 26S proteasomes free of polyubiquitin chains that can interfere with the binding of another substrate.

Proteasomes The second major step in the ubiquitin–proteasome pathways is the degradation of polyubiquitinated proteins by the 26S proteasome, which is formed by the binding of two 19S regulatory complexes with the 20S proteasome (Figure 1). Proteasomes are particles responsible for the major neutral proteolytic activity in mammalian cells. They represent up to 1% of soluble proteins, but their abundance varies among cell types. Usually the proteasome concentration is proportional to the rate of tissue-protein turnover. Accordingly, proteasomes are rather abundant in lungs. Proteasomes are present in both the nucleus and the cytosol, but some particles are also associated with the endoplasmic reticulum and the cytoskeleton. Proteasomes were first discovered in liver cells and reticulocytes in 1979; since then, they have been identified by numerous groups in various tissues. The term ‘proteasome’ has only prevailed since 1988; prior to that, over 21 different names were used for this structure. 20S Proteasome

The mammalian 20S proteasome is a cylindrical particle composed of four stacked rings of subunits, with each ring containing seven different subunits (Figure 2). The outer rings are composed of a-subunits, and the two inner rings of b-subunits, which contain the catalytic sites inside the particle. Thus, the 20S proteasome is a self-compartmentalizing protease, as substrates must enter the catalytic chamber in order to be degraded into peptides. In eukaryotes, the 20S proteasome contains at least two chymotrypsin-like, two trypsin-like, and two caspase-like active sites, which are allosterically regulated. Proteasomes hydrolyze most peptide bonds and generate peptides that are typically 3–22 amino acids long and do not conserve biological properties, except for antigen presentation. Very importantly the 20S proteasome is the proteolytic core of a modular system in which peptidase activities can be modulated by the binding of regulatory complexes (see below). The binding of these

Figure 2 The structure of the mammalian 20S proteasome at 2.75 A˚ resolution. This lateral view shows the four rings of a external and b internal subunits.Reproduced from Unno M, Mizushima T, Morimoto Y, et al. (2002) MMDB: Entrez’s 3D-structure database. Structure (Cambridge) 10: 609–618.

complexes induces conformational changes in asubunits that open the gate separating the catalytic chamber of the 20S proteasome from the intracellular environment. Furthermore, there are immunoproteasomes in which three catalytic b subunits are replaced by three distinct b subunits, so that catalytic properties are altered to generate more efficiently antigenic peptides. Thus, there are different populations and subtypes of proteasomes in a given tissue that differ by their catalytic properties. The 19S Complex

The 19S complex (or PA700) stimulates both peptidase and proteolytic activities of the 20S proteasome. This complex contains about 18 different subunits, including at least two polyubiquitin chain receptors, and can be topologically defined by two subcomplexes called the base and the lid (Figure 1). In humans the base contains six ATPases and three non-ATPase subunits, while the lid only contains non-ATPase subunits. The ATPases provide energy for the assembly of the 26S proteasome, the recognition of the polyubiquitin degradation signal, the breakdown of ubiquitinated proteins into peptides, the gating of the proteasome channel, the unfolding of protein substrates and their injection into the catalytic chamber of the proteasome, and finally for peptide release. Other Proteasome Activators

The 11S regulator (or PA28ab) modulates only 20S proteasome peptidase activities. PA28 particles play a role in antigen presentation, by generating peptides

502 PROTEASOMES AND UBIQUITIN

for MHC class I molecules. Other proteasome activators called PA28g and PA200 play a role in growth control and DNA repair, respectively. Finally, there are hybrid proteasomes, in which one 11S regulator and one 19S complex bind simultaneously to a 20S proteasome. Such complexes are induced by interferon-g and play a role both in antigen presentation and in the breakdown of some proteins.

Regulation of Activity The activity of the ubiquitin system is first regulated by the equilibrium between ubiquitination and deubiquitination rates of protein substrates. Proteasome activities are controlled by a variety of subtle mechanisms that include the synthesis and processing of proteasome subunits, the assembly of 20S and 26S proteasomes, posttranslational modifications (e.g., phosphorylation of some 20S and 19S subunits), the binding of proteasome activators and inhibitors, the replacement of catalytic subunits, etc. These processes are influenced by complex signaling pathways, which may implicate mediators such as hormones and cytokines.

transduction, receptor downregulation, and the control of inflammation. In particular, recent studies have shown that hypoxia-inducible factor-1a (HIF-1a) subunits are normally degraded in a ubiquitin- and proteasome-dependent fashion in the presence of oxygen. In hypoxia, HIF-1a proteolysis is blocked and this transcription factor becomes a major player in a widespread oxygen-sensing and signal transduction mechanism. The HIF-1 activation cascade includes protein phosphorylation, nuclear translocation, DNA binding, aryl hydrocarbon receptor nuclear translocator (ARNT) dimerization, recruitment of general and tissue-specific transcriptional cofactors, and target gene transactivation. Indeed, it has been estimated that HIF-1 may regulate the expression of up to 5% of the human genome. Finally, the activation of the ubiquitin–proteasome system plays a key role in muscle wasting. Accordingly, the proteasome is activated in mechanical ventilation-associated diaphragmatic atrophy. Patients with chronic obstructive pulmonary diseases (COPDs) who exhibit muscle wasting may have similar adaptations.

Forthcoming Applications Biological Roles of the Ubiquitin–Proteasome System in the Respiratory Tract The role of the ubiquitin–proteasome pathways in the respiratory tract and respiratory diseases is still largely unknown. However, a number of functions can be assigned to the pathway based on the known roles of the system in other cells and tissues (Table 1). First, the pathway should be responsible for housekeeping functions (basal protein turnover, elimination of abnormal (miscoded, misfolded, oxidized) proteins, etc). Second, the system should play a critical immunological role by generating peptides for class I antigen presentation. Third, the system is involved in many key biological functions such as transcriptional control, cell-cycle progression, oncogenesis, development and differentiation, signal

Not surprisingly, there has been a growing number of studies on the ubiquitin–proteasome system in the respiratory tract and related topics since 1999. In addition, studies on HIF-1 are leading to surprisingly broad applications since drugs that target this system may lead to better and new therapies for cancer, heart attack, stroke, and inflammation. Furthermore, clinical trials in this area are beginning to emerge. For example, Bortezomib is a novel proteasome inhibitor produced by Millennium Pharmaceuticals (Cambridge, MA) that inhibits the growth of lung cancer cell lines in vitro and in vivo in athymic nude mouse xenografts. Bortezomib is currently in Phase II trials in lung cancer patients. The proteasome system also plays a vital role in controlling inflammation (e.g., via the breakdown of inhibitor kB)/ nuclear factor kB), which is key to many respiratory diseases (e.g., asthma and COPD). Thus, it has been

Table 1 Major roles of the ubiquitin–proteasome system in the respiratory tract Housekeeping functions

Immunological role

Regulatory functions

Production of energy

Basal protein turnover Elimination of abnormal proteins

Class I antigen presentation

Cell cycle Transcription Development and differentiation Oncogenesis Signal transduction Control of inflammation

Muscle wasting in COPD patients?

PROTEINASE INHIBITORS / Alpha-2 Antiplasmin 503

suggested that proteasome inhibitors could also be useful in the treatment of such diseases, but there are still no clinical trials in this area. See also: Cell Cycle and Cell-Cycle Checkpoints. Chronic Obstructive Pulmonary Disease: Overview. Gene Regulation. Hypoxia and Hypoxemia. Proteinase Inhibitors: Antichymotrypsin; Cystatins; Secretory Leukoprotease Inhibitor and Elafin. Transcription Factors: Overview. Tumors, Malignant: Overview.

Further Reading Attaix D and Briand Y (2003) The proteasome in the regulation of cell function. International Journal of Biochemistry and Cell Biology 35/5: 545–755. Attaix D, Combaret L, Kee AJ, and Taillandier D (2003) Mechanisms of ubiquitination and proteasome-dependent proteolysis in skeletal muscle. In: Zempleni J and Daniel H (eds.) Molecular Nutrition, pp. 219–235. Oxon: CAB International. Bunn PA Jr (2004) The potential role of proteasome inhibitors in the treatment of lung cancer. Clinical Cancer Research 10: 4263s–4265s.

Coux O, Tanaka K, and Goldberg AL (1996) Structure and functions of the 20S and 26S proteasomes. Annual Review of Biochemistry 65: 801–847. Glickman MH and Ciechanover A (2002) The ubiquitin–proteasome proteolytic pathway: destruction for the sake of construction. Physiological Reviews 82: 373–428. Hilt W and Wolf D (eds.) (2001) Proteasomes: The World of Regulatory Proteolysis. Austin: RG Landes & Co. Marx J (2004) Cell biology. How cells endure low oxygen. Science 303: 1454–1456. (This paper nicely summarizes recent findings on HIF-1 leading to possible new strategies for treating various diseases.) Peters JM, Harris JR, and Finley D (eds.) (1998) Ubiquitin and the Biology of the Cell, p. 472. New York: Plenum. Pickart CM (2001) Mechanisms underlying ubiquitination. Annual Review of Biochemistry 70: 503–533. Unno M, Mizushima T, Morimoto Y, et al. (2002) MMDB: Entrez’s 3D-structure database. Structure (Cambridge) 10: 609– 618. Voges D, Zwickl P, and Baumeister W (1999) The 26S proteasome: a molecular machine designed for controlled proteolysis. Annual Review of Biochemistry 68: 1015–1068. Zwickl P and Baumeister W (eds.) (2002) The Proteasome-Ubiquitin Protein Degradation Pathway, p. 213. Berlin: Springer.

PROTEINASE INHIBITORS Contents

Alpha-2 Antiplasmin Antichymotrypsin Cystatins Secretory Leukoprotease Inhibitor and Elafin

Alpha-2 Antiplasmin H R Lijnen, University of Leuven, Leuven, Belgium & 2006 Elsevier Ltd. All rights reserved.

Abstract Alpha-2 antiplasmin (AP), the main physiologic plasmin inhibitor in mammalian plasma, is a 70 kDa single chain serpin (serine proteinase inhibitor) with reactive site peptide bond Arg–Met. It inhibits plasmin very rapidly following formation of an inactive 1:1 stoichiometric complex. The high reaction rate requires the presence of a free active site and free lysine-binding sites(s) in plasmin. The pathophysiologic relevance of AP is evidenced by the finding that homozygous-deficient patients show a bleeding tendency; heterozygotes, in contrast, frequently have no or only mild bleeding complications. Homozygous AP-deficient mice have been generated which display normal fertility, viability, and development. Several in vivo studies confirmed that these mice have an enhanced endogenous fibrinolytic capacity without however showing overt bleeding. These findings suggest that the main role of AP is in regulating plasmin activity in the circulating blood and in controlling intravascular fibrinolysis.

In patients with respiratory diseases, including the adult respiratory distress syndrome, depressed fibrinolytic activity, partially as a result of enhanced AP levels, is a consistent finding, leading to disturbance of the hemostatic balance favouring fibrin deposition.

Introduction The fibrinolytic (plasminogen/plasmin) system contains a proenzyme, plasminogen, that is converted to the active enzyme plasmin, which degrades fibrin, by tissue-type (tPA) or urokinase-type (uPA) plasminogen activator. Inhibition of the fibrinolytic system may occur at the level of plasmin (by a2-antiplasmin, AP) or at the level of plasminogen activators (by plasminogen activator inhibitors) (Figure 1). For a long time, it was accepted that there were two functionally important plasmin inhibitors in human plasma: an immediately reacting and a slower reacting one identical to a2-macroglobulin and a1-antitrypsin, respectively. About 30 years ago, another

504 PROTEINASE INHIBITORS / Alpha-2 Antiplasmin Tissue-type plasminogen activator Urokinase-type plasminogen activator

Plasminogen activator inhibitor-1 Plasminogen activator inhibitor-2 Plasminogen

Plasmin 2-Antiplasmin Fibrin

Fibrin degradation products

Figure 1 Schematic representation of the fibrinolytic system.

A-chain of plasmin S

S 561 Arg–OH Plg. act.

791 HO–Asn

S

Lys–H 77

S

Val–H 562 B-chain of plasmin

LBS 741 Ser HO–Lys 464

Lys 448

Lys 441

Complementary LBS

Met – Arg Cys 377 376 Reactive site H-Met Asn Gln Cys 1 13 14

Cys 2-Antiplasmin Cys

Fibrin-binding site Figure 2 Schematic representation of the plasmin-a2-antiplasmin complex. LBS, high-affinity lysine-binding site in plasmin; Plg. act., site of cleavage in plasminogen for plasminogen activators.

plasmin inhibitor, AP or a2-plasmin inhibitor, was identified. On activation of plasminogen in plasma, this inhibitor preferentially binds plasmin. Complete activation of plasminogen (concentration about 2 mM), resulting in saturation of AP (concentration about 1 mM), must occur before excess plasmin is neutralized by the other inhibitors. AP, like many other plasma proteinase inhibitors, has a broad in vitro inhibitory spectrum, but its physiologic role as an inhibitor of proteinases other than plasmin seems negligible.

Structure Protein Structure

AP is a single-chain protein of 70 kDa containing about 13% carbohydrate (attached at Asn99, Asn268, Asn282, and Asn289). The molecule consists of 464 amino acids and contains one Cys43–Cys116 disulfide bond and possibly two unpaired cysteines (Cys76 and Cys125) (Figure 2). It belongs to the serine proteinase inhibitor family (serpins). The method of choice for the purification of AP consists of chromatography on

PROTEINASE INHIBITORS / Alpha-2 Antiplasmin 505

insolubilized plasminogen fragments that contain kringles 1–3 with a high-affinity lysine-binding site (LBS; see below). Two molecular forms are present in about equal amounts in purified preparations: a native 464-residue-long inhibitor with N-terminal methionine (Met1–AP) and a 12-amino acid shorter form with N-terminal asparagine (Asn13–AP). It is not known whether Asn13–AP is present in the circulating blood or whether it is generated in vitro. The N-terminal Gln14-residue of AP can cross-link to Lys303 in the Aa-chains of fibrin(ogen) in a process that requires Ca2 þ and that is catalyzed by activated coagulation factor XIII. Asn13–AP is more efficiently cross-linked to fibrin than Met1–AP. Mechanism of Interaction with Plasmin

The time course of the inhibition of plasmin by AP is compatible with a kinetic model composed of two successive reactions: a fast, reversible second-order reaction followed by a slower, irreversible first-order transition. The model can be represented by the following scheme: k1

k2

P þ AP $ PAP - PAP0 k1

where P is plasmin, AP is a2-antiplasmin, PAP is a reversible inactive 1:1 stoichiometric complex, and PAP0 is an irreversible inactive complex. The second-order rate constant (k1 ¼ 2 to 4 107 M 1 s 1) of this inhibition is among the fastest protein–protein reactions described. The first step of the process depends on the presence of a free LBS and active site in the plasmin molecule and can be divided into two parts: a reaction between the LBS (mainly kringles 1–3) in the plasmin A chain and the plasmin(ogen)-binding site in the C-terminal part of AP (complimentary LBS), and subsequently a reaction between the active site Ser741 in the B-chain of plasmin with the reactive site peptide bond in AP (Arg376–Met377). A covalent bond is formed between the active site seryl residue in plasmin and the reactive site arginyl residue in the inhibitor (Figure 2). Plasmin and AP thus form a stoichiometric 1:1 complex of about 140 kDa. A nondisulfide-bonded peptide (8 kDa) is released concomitantly with complex formation. Upon disulfide bond reduction, the complex is dissociated in two parts: an intact plasmin A chain (60 kDa), and a stable complex between the plasmin B chain and cleaved AP (80 kDa). In plasma, the reaction rate between plasmin and AP may be reduced by the presence of proteins such as histidine-rich glycoprotein or fibrinogen, which interact with the LBS of plasmin(ogen). The half-life of plasmin molecules on the fibrin surface, which are

protected from inhibition by AP because their LBS and active site are occupied, is two to three orders of magnitude longer than that of free plasmin. Gene Structure

The gene for human AP, located on chromosome 17p13, is approximately 16 kb and contains 10 exons. The N-terminal region of the protein, comprising the fibrin cross-linking site is encoded by exon IV, whereas both the reactive site and the plasminogen-binding site in the C-terminal region are encoded by exon X. A ‘TATA box’ sequence is located 17 nucleotides upstream from the proposed transcription initiation site. The 50 -flanking region contains multiple ‘GC box’ and ‘CCAAT box’-like sequences.

Regulation of Production and Activity The concentration of AP in normal human plasma is about 7 mg dl 1 (about 1 mM); it is produced in the liver and kidney. Its in vivo half-life is 2.6 days, whereas the plasmin–AP complex disappears from plasma with a half-life of about half a day; both are cleared via the liver. The inhibitor in normal plasma is heterogeneous and consists of functionally active and inactive material. Complete activation of the plasminogen present in normal plasma converts only about 70% of the AP antigen into a complex with plasmin; 30% of the inhibitor is functionally inactive or slower reacting. These two forms of the inhibitor differ in their binding to plasminogen. The form that does not bind remains an active plasmin inhibitor but reacts much more slowly with plasmin; it lacks a 26-residue peptide from the C-terminal end that contains the plasminogen-binding site. The plasminogen-binding form of AP is primarily synthesized and becomes partly converted to the nonplasminogen-binding form in the circulating blood.

Biological Functions Phenotypic analysis of AP gene-deficient mice, generated by homologous recombination in embryonic stem cells, revealed a functional role for AP in several biological processes. In vivo studies revealed that these mice have an enhanced endogenous fibrinolytic capacity without overt bleeding. This is reflected by a higher spontaneous lysis rate of experimental pulmonary emboli, by a reduced fibrin deposition in the kidneys following challenge with endotoxin, by more limited photochemically induced arterial thrombosis, and by reduced infarct size following induction of focal cerebral ischemia by ligation of the left middle cerebral artery. In a vascular injury restenosis model,

506 PROTEINASE INHIBITORS / Alpha-2 Antiplasmin

AP deficiency has no significant effect on smooth muscle cell migration and neointima formation. The absence of a bleeding phenotype in these mice, in contrast to man, may reflect the fact that the coagulation system adequately prevents bleeding if the fibrinolytic system is not dramatically challenged. In man, rare cases of congenital AP deficiency and dysfunctional AP as well as acquired deficiencies have been reported. The first case of congenital homozygous AP deficiency was described in a patient who presented with a hemorrhagic diathesis. Several cases of heterozygosity have been described with no or only mild bleeding symptoms. The AP levels in all heterozygotes described thus far are consistently between 40% and 60% of normal. Antigen and activity levels usually correspond well, suggesting that the deficiency is due to decreased synthesis of a normal AP molecule. The bleeding tendency in these patients may be due to premature lysis of hemostatic plugs, because in the absence of AP, the half-life of plasmin molecules generated on the fibrin surface is considerably prolonged. Different molecular defects have been identified in AP, such as a trinucleotide deletion in exon VII leading to deletion of Glu137 (AP Okinawa), or an insertion of a cytidine nucleotide in exon X leading to a shift in the reading frame of the mRNA resulting in deletion of the C-terminal 12 amino acids of native AP and replacement with 178 unrelated amino acids (AP Nara). These mutations may lead to the deficiency by affecting the folding of the protein into the native configuration and thereby blocking its intracellular transport from the endoplasmatic reticulum to the Golgi complex. Plasma levels of AP in a normal population range between 80% and 120% of that in pooled human reference plasma. The level is significantly decreased in liver cirrhosis and in several other liver diseases, which may be an important factor in the increased fibrinolytic activity observed in liver cirrhosis. Decreased levels of AP have also been observed in patients with disseminated intravascular coagulation or with some forms of renal disease. AP levels may be significantly reduced in patients undergoing thrombolytic therapy as a result of systemic activation of the fibrinolytic system, in particular associated with the use of non-fibrin-specific thrombolytic agents. An abnormal AP (AP Enschede) associated with a serious bleeding tendency was found in two siblings in a Dutch family. These individuals had 3% of normal functional activity and 100% of normal antigen levels. The ability of the abnormal AP to reversibly bind plasmin or plasminogen is not affected, but it is converted from an inhibitor of plasmin to a substrate.

The molecular defect consists of the insertion of an extra alanine residue (GCG insertion) 7 to 10 positions on the N-terminal side of the P1 residue (Arg376).

a2-Antiplasmin in Respiratory Diseases Studies in the 1970s and 1980s indicated that microemboli in the pulmonary circulation sometimes persist longer than normal, resulting in the delayed microembolism syndrome with characteristic pathophysiological changes in the lungs. This delayed dissolution of microemboli may be caused by inhibition of the fibrinolytic system, mediated by AP. This is confirmed by a later finding that injection of AP into rats with intravascular coagulation in the lungs delayed the elimination of fibrin. Intra-alveolar fibrin deposition (hyaline membrane formation) is a hallmark of many acute inflammatory lung diseases. This may be beneficial in the gas exchange area by sealing leakage sites (e.g., at sites of denuded alveolar epithelium) and by providing a matrix for wound repair. However, it may also be harmful if abundant and persistent, by compromising endothelial monolayer integrity. In addition, surfactant components may be incorporated into polymerizing fibrin with loss of surface activity and alveolar instability, as well as altered mechanical properties and reduced fibrinolytic capacity. Besides favoring alveolar collapse and shunt-flow these events may play a pathogenetic role in the rapid onset of lung fibrosis in acute and widespread inflammatory lung injury. Abundant deposition of bronchoalveolar fibrin and fibronectin occurs during the exudative phase of the adult respiratory distress syndrome (ARDS), promoting hyaline membrane formation and alveolar fibrosis. Several studies in ARDS patients have reported depressed fibrinolytic activities due to decreased uPA activity and enhanced PAI-1 and AP levels in the bronchoalveolar compartments. In addition, procoagulant activity is enhanced. Pronounced disturbances of the alveolar hemostatic balance also occur in patients with severe pneumonia, comparable to those seen in ARDS triggered by nonpulmonary underlying events. A marked increase in procoagulant activity (mainly through the tissue factor pathway) is accompanied by decreased fibrinolytic activity in the alveolar lining layer with decreased uPA (patients with pneumonia) and increased PAI-1 and AP levels (patients with pneumonia receiving respiratory therapy). This shift in hemostatic balance, favoring fibrin formation, is a consistent feature of inflammatory lung injury, whether triggered by nonpulmonary systemic events or by primary lung infection.

PROTEINASE INHIBITORS / Antichymotrypsin

It has not been investigated whether reduction of AP levels may represent a treatment modality for patients with respiratory diseases. To our knowledge no pharmacologic agents have been described that allow specific inhibition of AP in vivo. The most rational approach to reduce AP activity would be to infuse plasmin moieties. However, in view of the high plasma concentration of AP, this would require large amounts of plasmin, a protease with broad substrate specificity. Whereas high-dose plasmin activity may be hazardous in itself, its use would also require stringent monitoring of AP activity levels, since excess plasmin may compromise the hemostatic system by degrading several plasma proteins. Therefore, it seems unlikely that this will represent a potential treatment option. See also: Acute Respiratory Distress Syndrome. Fibrinolysis: Overview; Plasminogen Activator and Plasmin.

Further Reading Aoki N (1990) Molecular genetics of alpha 2 plasmin inhibitor. Advances in Experimental Medicine and Biology 281: 195–200. Aoki N, Sumi Y, Miura O, and Hirosawa S (1993) Human alpha 2-plasmin inhibitor. Methods in Enzymology 223: 185–197. Bertozzi P, Astedt B, Zenzius L, et al. (1990) Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome. New England Journal of Medicine 322: 890–897. Favier R, Aoki N, and de Moerloose P (2001) Congenital alpha(2)plasmin inhibitor deficiencies: a review. British Journal of Haematology 114: 4–10. Gu¨nther A, Mosavi P, Heinemann S, et al. (2000) Alveolar fibrin formation caused by enhanced procoagulant and depressed fibrinolytic capacities in severe pneumonia. Comparison with the acute respiratory distress syndrome. American Journal of Respiratory and Critical Care Medicine 161: 454–462. Hirosawa S, Nakamura Y, Miura O, et al. (1988) Organization of the human alpha2-plasmin inhibitor gene. Proceedings of the National Academy of Sciences USA 85: 6836–6840. Holmes WE, Nelles L, Lijnen HR, and Collen D (1987) Primary structure of human alpha2-antiplasmin, a serine protease inhibitor (serpin). Journal of Biological Chemistry 262: 1659–1664. Idell S, Koenig KB, Fair DS, et al. (1991) Serial abnormalities of fibrin turnover in evolving adult respiratory distress syndrome. American Journal of Physiology 261: L240–L248. Koie K, Ogata K, Kamiya T, et al. (1978) Alpha2-plasmin-inhibitor deficiency (Miyasato disease). Lancet 2: 1334–1336. Lijnen HR and Collen D (1989) Congenital and acquired deficiencies of components of the fibrinolytic system and their relation to bleeding or thrombosis. Fibrinolysis 3: 67–77. Lijnen HR and Collen D (1995) Mechanisms of physiological fibrinolysis. Baillie`re’s Clinical Haematology 8: 277–290. Lijnen HR, Okada K, Matsuo O, et al. (1999) Alpha2-antiplasmin gene deficiency is associated with enhanced fibrinolytic potential without overt bleeding. Blood 93: 2274–2281. Wiman B and Collen D (1977) Purification and characterization of human antiplasmin, the fast-acting plasmin inhibitor in plasma. European Journal of Biochemistry 8: 19–26.

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Wiman B and Collen D (1978) Molecular mechanism of physiological fibrinolysis. Nature 272: 549–550. Wiman B and Collen D (1979) On the mechanism of the reaction between human alpha2-antiplasmin and plasmin. Journal of Biological Chemistry 254: 9291–9297.

Antichymotrypsin N Kalsheker, K Morgan, and S Chappell, University of Nottingham, Nottingham, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Alpha-1-antichymotrypsin, also referred to as SERPIN A3, is a major plasma serine proteinase inhibitor. Its main physiological target is neutrophil cathepsin G, an enzyme that plays a key role in the killing of bacteria by neutrophils. The protein also plays a key role as a molecular chaperone for Ab peptides, derived from amyloid precursor protein in the brain and is a constituent of the senile plaques found in the brains of patients with Alzheimer’s disease. The protein behaves as an acute-phase reactant and plasma concentrations can increase about fourfold during inflammation. Genetic deficiency of alpha-1-antichymotrypsin has been reported and may result in altered lung function. The protein provides a natural defense against uninhibited proteolytic enzymes and this may be particularly important in the lung, an organ that is vulnerable to proteolytic damage. Alpha-1-antichymotrypsin also has an important role in controlling the inflammatory process in the lung where several serine proteinases are known to be both proinflammatory and can disturb the structural integrity of the lung.

Introduction Alpha-1-antichymotrypsin (ACT) is a plasma serine proteinase inhibitor (also referred to as SERPIN A3). It is a glycoprotein and its main target is chymotrypsin-like serine proteinases. It is particularly effective against the neutrophil enzyme cathepsin G, and its physiological role is to control the activity of this proteinase.

Protein Structure and Biological Function ACT has a molecular mass of between 55 and 66 kDa. The three-dimensional structure is in keeping with that of other serpins. It consists of three beta-sheets and nine alpha-helices. The interaction with target protease occurs at the reactive center loop (RCL) at the C-terminus. The RCL acts as bait for a target proteinase. The proteinase docks onto the RCL, which corresponds to residues 342 to 367, denoted P17-P90 , following the nomenclature of Schechter and Berger, and cleavage of the leucineserine bond, located at position 358–359, results in a

508 PROTEINASE INHIBITORS / Antichymotrypsin

major conformational change. The loop inserts into a space in the A-sheet as a strand, and the proteinase then moves to the opposite pole of the molecule. During this translocation process, the catalytic site of the serine proteinase is distorted so that it is rendered inactive. The inhibitor-proteinase complex is then rapidly removed from the circulation by a receptor system that belongs to the low-density lipoprotein receptor family and the tightest binding has been shown for low-density lipoprotein receptor associated protein 2 (Figure 1). A number of other proteinases are inhibited by ACT, including mast cell chymases and lymphocyte cell surface proteins; ACT has been shown to modulate lymphocyte activity by loss of both killing and antibody-dependent cytolytic activity. Cathepsin G, the main target of ACT, has been shown to be involved in the conversion of angiotensin I to angiotensin II, although the main role of the enzyme is related to bacterial killing and tissue remodeling. Another unusual property of ACT is its ability to bind DNA. Three basic lysine residues at positions 212–214 are critical for this and probably relate to the electronic charge provided by basic amino acid residues. The RCL is susceptible to proteolysis by a variety of microbial and mammalian proteinases. Most of these enzymes cause rapid inactivation of ACT as an inhibitor by cleavage of single sites, often between P5 lysine and P0 leucine. After cleavage at these sites, the molecule becomes a potent chemoattractant. Furthermore, the cleaved ACT has been shown to have a slow clearance underscoring its role in chemotaxis. A key function of neutrophils is to provide a defense against bacterial infection by a variety of mechanisms including superoxide generation and release of powerful proteinases. It has also been shown that ACT inhibits neutrophil superoxide generation and may play an important part in modulating neutrophil function. Also, cathepsin G is particularly effective against Gram-negative bacteria and it also stimulates airway gland secretion. In 1988, Abraham and coworkers reported that ACT was a significant component of the senile plaques associated with Alzheimer’s disease. The protein binds Ab1-42 peptide, comparable in specificity and stability to a protease–proteinase inhibitor interaction. The peptide is derived from amyloid precursor protein and ACT acts as a molecular chaperone that can stimulate amyloid fibril formation. The ACT-Ab complex is also probably cleared by the same receptor system that handles the inhibitor–protease complex described above. In 2002, Parmar and colleagues suggested that polymers have chemotactic activity. Thus, the ability of serpins to polymerize may have important implications for respiratory disease.

Figure 1 Model of the covalent protease. The protease is shown in gray and ACT is shown in yellow. Initial docking of the protease is followed by partial insertion of the protease and the P1–P10 bond remains intact. This is followed by an acylenzyme complex and full insertion of the protease. This is accompanied by movement of the protease to the opposite pole of ACT and distortion of the active site of the protease. Reproduced from O’Malley KM, Barry S, and Cooperman BS (2001) Formation of the covalent chymotrypsin–antichymotrypsin complex involves no large-scale movement of the enzyme. Journal of Biological Chemistry 276: 6631–6637, with permission from The American Society for Biochemistry & Molecular Biology.

The ACT Gene and Regulation The ACT gene is located on chromosome 14q32.1, is approximately 12 000 bp in length, and is organized into five exons and four introns. It is about 250 kb away from the alpha-1-antitrypsin gene and is in a cluster of genes that have arisen by gene duplication. The major site of synthesis is the liver, but other tissues also express the protein, including the heart, lung, kidney, brain, and prostate. In the lung, both

PROTEINASE INHIBITORS / Antichymotrypsin 57 bp

Exon size Intron size

649 bp 2.2 kb

I

509

268 bp 151 bp 442 bp 3.4 kb 1.6 kb 0.7 kb

II

III

IV

V

TSS (+1)

−125 STAT A

−117 −95

−87 −29 STAT B

−23 Promoter

Proximal enhancer region

13213

−13202

NF-B

−12985

−12979

AP-1

−12831 NF-B

Distal enhancer region Figure 2 Diagrammatic representation of the ACT gene structure. Exons are shown as boxes. The transcription start size (TSS) is designated þ 1. Regulatory regions are numbered relative to the TSS (not to scale). The elements marked STAT A and STAT B are on the sites at which oncostatin-M exerts its effects. The transcription factors that bind to the distal enhancer are shown.

alveolar macrophages and airway epithelia have been shown to synthesize ACT. The normal reference range for plasma concentrations of ACT is between 0.3 and 0.6 g l 1. During inflammation the concentrations can increase up to fourfold in about 8 h. This increase is mediated by cytokines such as interleukin-6 (IL-6) and oncostatin-M (OSM). The regions of the gene that contribute to expression are highlighted in Figure 2. In lung epithelial cells, OSM, IL-1, and dexamethasone have been shown to stimulate ACT production, both individually and, more dramatically, in combination between about three- to sixfold, depending on the cell type. OSM is the most potent stimulus in bronchial epithelial cells and induces expression by activating pathways that involve signal transducer and activator of transcription (STAT) elements, located at 125 to 127 bp and 96 to 87 bp away from the transcription initiation site identified by Kordula et al. in 1998. An additional element, located in a 413 bp long fragment, approximately 13 kb upstream from the transcription initiation site, has been shown to be involved in the IL-1 and tumor necrosis factor (TNF) alpha response. Located within this fragment are three transcription factor-binding sites, two nuclear factor kappa B (NF-kB) sites at 13 213 to 13 202 bp

and 12 831 to 12 820 bp, and an activating protein A (AP-1) site at 12 985 to 12 979 bp. In the liver cell line, Hep G2, IL-1, and TNF-a had a minimal effect, suggesting that there may be significant tissue-specific differences in response to cytokines.

Association with Disease The most important risk factor for the development of chronic obstructive pulmonary disease (COPD) is tobacco smoking. However, only 10–20% of heavy smokers develop symptomatic COPD. Based on the observations of alpha-1-antitrypsin deficiency, and its association with COPD, this has led to the protease–antiprotease imbalance theory of the causation of disease where inhibited proteinase contributes to lung damage. In 1990, Lindmark et al. described in a Swedish population heterozygotes for partial ACT deficiency occurring at a frequency of about 1 in 200–300 of the population. Some of these individuals had evidence of high residual volumes and total lung capacity, though these did not appear to give rise to serious pulmonary disease. This is analogous to the situation occurring in heterozygotes for alpha-1-antitrypsin deficiency. In 1993, Poller et al. reported an association of leucine 55-to-proline substitution, and a proline 229-to-alanine substitution in ACT with

510 PROTEINASE INHIBITORS / Antichymotrypsin

Proinflammatory proteinases

Anti-inflammatory inhibitors

Neutrophil cathepsin G

Alpha-1-antichymotrypsin

Chymase (activates interleukin-1)

Alpha-1-antichymotrypsin

Neutrophil elastase

Alpha-1-antitrypsin

Neutrophil proteinase 3 (activates interleukin-1 and tumor necrosis factor)

Modified inhibitors (chemotaxis) Recruitment of neutrophils

Figure 3 Balance of inflammation in the lung.

COPD. This association has been challenged. Other polymorphisms have been reported, including a polymorphism at 15 of the ACT signal peptide, where either alanine or threonine occurs at this residue. This polymorphism is in linkage disequilibrium with a promoter polymorphism that has been shown to exert a modest functional effect in the liver and in astrocytes. However, there are marked cell-specific differences in the expression of the promoter polymorphism, certainly in neuronal tissue, suggesting that expression can vary markedly, depending on the tissues being studied. One small study by Ishii et al. in 2000 reported that homozygotes for the alanine in the signal sequence have an odds ratio of 2.7 (1.2– 6.2) for COPD. This study should be interpreted with caution as only a small sample size was used, and was probably underpowered to detect significant differences. To assess the role of ACT in COPD, larger sample sizes are required and a more thorough analysis of polymorphic variants of the gene needs to be undertaken.

This may influence the half-life of the protein. Serine proteinases such as cathepsin G have broad specificity, and are capable of degrading structural elements such as elastin and collagen type III and IV under physiological conditions. They play a key role in tissue remodeling in addition to their role in bacterial killing. In addition, such enzymes have the potential to generate proinflammatory signals. Therefore, inhibition proteolytic activity is essential for both maintenance of structural integrity and control of inflammation. This is particularly important in the lung, an organ that is susceptible to the harmful effects of proteolysis (Figure 3).

Biological Role of ACT in the Lung

See also: Chronic Obstructive Pulmonary Disease: Overview. Interleukins: IL-6. Oncostatin M. Transcription Factors: NF-kB and Ikb.

ACT is synthesized locally in the lung and both alveolar macrophages and human lung epithelial cells have been shown to produce it. It has been suggested that ACT in lung secretions is not effective as an inhibitor of chymotrypsin-like enzymes as it only retains about 15% of its inhibitory activity. This may reflect cleaved or alternative forms of the molecule in which the RCL has been lost. It is likely that local production controls the activity of target proteinases in the lung and probably contributes to a local anti-inflammatory effect. It has been suggested that there is a modified pattern of glycosylation of ACT produced in bronchial epithelial cells compared with that produced by the liver and the unglycosylated form is identical to that produced in the liver.

Conclusion ACT is an important modulator of proteinase activity and more is being learnt about its role at specific sites – from its role as a molecular chaperone for Ab peptides in the brain to its anti-inflammatory role in the lung. These insights should identify pathological mechanisms for disease causation.

Further Reading Abraham CR, Selkoe DJ, and Potter H (1988) Immunochemical identification of the serine protease inhibitor alpha-1-antichymotrypsin in the brain amyloid deposits of Alzheimer’s disease. Cell 52: 487–501. Baumann H and Gauldie J (1994) The acute phase response. Immunology Today 15: 74–80. Berman G, Afford SC, Burnett D, and Stockley RA (1986) Alpha1-antichymotrypsin in lung secretions is not an effective proteinase inhibitor. Journal of Biological Chemistry 261: 14095– 14099. Cichy J, Potempa J, Chawla RK, and Travis J (1995) Regulation of a-1-antichymotrypsin synthesis in cells of epithelial origin. FEBS Letters 359: 262–266.

PROTEINASE INHIBITORS / Cystatins 511 Eriksson S, Janclauskene S, and Lannfelt L (1995) Alpha-1-antichymotrypsin regulates Alzheimer b-amyloid peptide fibril formation. Proceedings of the National Academy of Sciences, USA 92: 2313–2317. Hudig D, Haverty T, Fulcher C, and Redelman J (1981) Inhibition of human natural cytotoxicity by macromolecular antiproteases. Journal of Immunology 126: 1569–1574. Ishii T, Matsuse T, Teramoto S, et al. (2000) Association between alpha-1-antichymotrypsin polymorphism and chronic obstructive pulmonary disease. European Journal of Clinical Investigation 30: 543–548. Kalsheker NA (1996) Alpha-1-antichymotrypsin. International Journal of Biochemistry and Cell Biology 28: 961–964. Kordula T, Rydel RE, Brigham EF, et al. (1998) Oncostatin-M and the interleukin-6 and soluble interleukin-6 receptor complex regulates alpha-antichymotrypsin express in human cortical astrocytes. Journal of Biological Chemistry 273: 4112–4118. Lindmark BE, Arborelius M, and Eriksson S (1990) Pulmonary function in middle-aged women with heterozygous deficiency of the serine protease inhibitor a-1-antichymotrypsin. American Review of Respiratory Diseases 141: 884–888. O’Malley KM, Barry S, and Cooperman BS (2001) Formation of the covalent chymotrypsin–antichymotrypsin complex involves no large-scale movement of the enzyme. Journal of Biological Chemistry 276: 6631–6637. Parmar JS, Mahadeva R, Reed BJ, et al. (2002) Polymers of alpha1-antichymotrypsin are chemotactic for human neutrophils – a new paradigm for the pathogenesis of emphysema. American Journal of Respiratory Cell and Molecular Biology 26: 723–730. Poller W, Faber J-P, Weidinger S, et al. (1993) A leucine to proline substitution causes a defective a-1-antichymotrypsin allele associated with familial obstructive lung disease. Genomics 17: 740–743. Poller W, Willnow TE, Hilpert J, and Herz J (1995) Differential recognition of a-1-antichymotrypsin–Cathepsin G complexes by the low density lipoprotein receptor-related protein. Journal of Biological Chemistry 270: 2841–2845. Reeves EP, Lu H, Jacobs HL, et al. (2002) Killing activity in neutrophils is mediated through activation of proteases by kt flux. Nature 416: 291–297. Rollini P and Fournier K (1997) A 370 kb cosmid configuration of the serpin gene cluster on human chromosome 14q32.1: molecular linkage of the genes encoding alpha-1-antichymotrypsin, protein C inhibitor, kallistatin, alpha-1-antitrypsin and corticosteroid binding globulin. Genomics 46: 409–415.

Cystatins P A Pemberton, Arriva Pharmaceuticals, Inc., Alameda, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract The cystatins comprise a superfamily of proteins related primarily by virtue of DNA and amino acid sequence homology. The superfamily consists so far of four distinct types of molecules ranging from the simpler low-molecular-weight type I and II cystatins, which function primarily to inhibit lysosomal cysteine proteinases (CPs), to the higher-molecular-weight type III and IV cystatins, which possess additional latent functions expressed only during episodes of injury and inflammation, or have evolved entirely novel inhibitory functions.

The role cystatins play in respiratory diseases such as asthma and COPD is poorly understood. However, they do modulate the immune response by acting directly on neutrophils, macrophages, and antigen presenting cells. It is also clear that they do not function independently of other proteolytic pathways involved in remodeling of the lung. Limited proteolysis inactivates cystatins allowing lysosomal CP activity to directly contribute to lung tissue degradation and also liberates kinins which signal through G-protein-coupled receptors to cause both constriction and dilation of the bronchioles, pain via stimulation of sensory nerves, mucus secretion, cough, and edema.

Introduction The human cystatins comprise a superfamily of potent protein-based inhibitors of cysteine proteinases (CPs). It is one of the many protein proteinase inhibitor superfamilies that are involved in regulating mammalian homeostasis, including the serpins (e.g., a1-antitrypsin; AAT) and low-molecular-weight kunitz (e.g., tissue factor pathway inhibitor; TFPI), and kazal-type (e.g., pancreatic secretory trypsin inhibitory; PSTI) inhibitors. Current knowledge of their function suggests that they primarily serve to inhibit the activity of lysosomal proteinases that may be released during normal or pathological cellular or tissue remodeling events. The first cystatin described was isolated from chicken egg white in 1963 and found to exhibit potent inhibitory properties against the CPs papain and ficin. The name ‘cystatin’ was proposed by Barrett et al. in 1963 and later used to describe homologous proteins in the same superfamily. The first full sequence of a human cystatin was that of cystatin C. There are more than a dozen human cystatins all with different properties, unique distribution patterns, and functions. These have been grouped into four main cystatin types on the basis of DNA and protein sequence homology and over the last few years the superfamily has expanded to include additional CP inhibitors, molecules that have no CP inhibitory activity, and yet others that have evolved functions unrelated to CP inhibition.

The Cystatin Superfamily Type I Cystatins

Type I cystatins are intracellular and present in the cytosol of many different cell types. They are typically 100 amino acids long and lack disulfide bonds. There are two human cystatins called ‘stefins’ A and B to stress their difference from other cystatin superfamily members, but they do contain a general structure similar to the ‘cystatin-fold’ of other cystatins and similar CP inhibitory activity. In evolutionary

512 PROTEINASE INHIBITORS / Cystatins

terms, stefins A and B are closely related and form a distinct subgroup. Type II Cystatins

Type II cystatins are typically 120–125 residues long and contain two disulfide bonds. They are translated with a secretory peptide leader sequence and are considered extracellular but can also be found intracellularly. They are broadly distributed and can be found in most body fluids. Mammalian type II cystatins all present two disulfide bridges at the C-terminal end of the sequence with 10–20 residues between the cysteines. Significant diversity in the type II superfamily members arises from the existence of multigene families encoding many different proteins (Table 1) and by polymorphisms affecting the coding sequence and function of the protein. Several diseases are associated with functional deficiencies or aggregation states of certain type II cystatins. The ‘classical’ type II cystatins C, D, S, SA, and SN are 450% identical at the protein sequence level. In addition, several posttranslational modifications are found in the members of this family. They may also be glycosylated or phosphorylated. Examples of these are cystatins E/M (glycosylated on N108) and cystatins S and SN (consensus phosphorylation sites at S2 and S98, respectively). Cystatin S has been isolated from nasal and bronchoalveolar (BAL) fluids with varying states of phosphorylation, but the significance of this is currently unknown.

that possesses both direct and indirect actions in the airways including bronchoconstriction and bronchodilation, stimulation of cholinergic and sensory nerves, increased mucus secretion, and cough and edema resulting from promotion of microvascular leakage. Its activities are mediated primarily via the B1 and B2 G-protein-coupled receptors. Type IV Cystatins

The type IV cystatins are a small family of abundant fetal and bone glycoproteins known as the fetuins. The two related human members of this family are a2 Heremans Schmid glycoprotein (a2-HS glycoprotein) and histidine-rich glycoprotein (HRG). The fetuins are N- and O-glycosylated and phoshorylated. The N-terminal region consists of two tandem type II cystatin domains followed by a C-terminal region comprised of a histidine-rich domain between two proline-rich domains. The N- and C-terminal regions are linked by a disulfide bond. a2-HS has a structure similar to HRG except that it lacks the histidine-rich tandem repeat. Unlike the cystatin domains in the kininogens, the fetuins are devoid of CP inhibitor activity and consistent with this, they lack the conserved structural motifs responsible for CP inhibition. Surprisingly, orthologs have been found in snake venom that lack CP inhibitory activity but possess metalloproteinase inhibitory functions.

Structure: Function of Cystatins Type III Cystatins

These are multidomain proteins first described as kinin precursor proteins or kininogens. There are two types of human kininogens: high- and low-molecular-weight kininogens. These proteins are of high-molecular mass (60–120 kDa) and present three tandemly repeated type-2 like cystatin domains (D1, D2, and D3) with a total of eight disulfide bridges (six conserved and two additional at the beginning of cystatin domains D2 and D3). The D2 and D3 domains possess CP inhibitory activity similar to the type II cystatins. They are glycosylated proteins but the glycosylation sites are not present in the cystatin domains. Kininogens are expressed intravascularly and are found in blood plasma. However, in addition to their function as CP inhibitors, the kininogens serve as substrates for a diverse group of serine proteinases collectively termed the kininogenases. The kininogenases liberate the kinin family of inflammatory peptides from the parent molecules. The kinins consist primarily of bradykinin (BK) and kallidin (lysyl bradykinin). BK is a nine amino acid peptide released during inflammation and tissue injury

The alignment of cystatin sequences has identified three functional regions that have been conserved for more than a billion years of evolution and are responsible for the CP inhibitory activity of the superfamily: a glycine (G) residue in the N-terminal region of the molecule, a glutamine (Q) – X – valine (V) – X – glycine (G) motif (QXVXG: the ‘cystatin motif’) in one hairpin loop (see later), and a proline (P) – tryptophan (W) motif in a second hairpin loop. These regions form a surface on the cystatin molecule that can dock and bind into the enzymatically active site(s) of papain-like CPs. The classic example of a type II cystatin is cystatin C, for which the X-ray crystallographic structure of the chicken protein has been solved (Figure 1). The main feature of the structure is a five-stranded b-sheet wrapped around a five turn a-helix commonly referred to as the ‘cystatin fold’. The N-terminal 10 residues are disordered and flexible and the conserved G11 residue is present at the N-terminus of the five turn a-helix. The QXVXG motif is found on a hairpin loop located between bstrands B and C and the PW motif on a hairpin loop between b-strands D and E. Collectively, these three

Table 1 Cystatin genes, selected family members, and functions Cystatin type

Members

Common name(s)

Chromosomal Location/ (gene name)

Location

Function

Disease association

I

A

3cen q21

Intracellular, skin, blood

B

Stefin A, (epidermal SH-protease inhibitor) Stefin B, (CPI-B)

21

Intracellular, broad tissue distribution

Cysteine proteinase inhibitor (CPI) CPI

Progressive myoclonus epilepsy

C

Post-g-globulin

20p11.21 (CST3)

Widespread tissue/body fluids Saliva, tears Epidermal keratinocytes, sweat glands Intra- and extra-cellular, hematopoietic cells Saliva, tears, urine

II

D E/M F

20p11.21 (CST5 ) 11q13 (CST6) Leukocystatin

S SN SA III

IV

20p11.21 (CST7 ) 20p11.21 (CST4 ) 20p11.21 (CST1) 20p11.21 (CST2)

L-kininogen

a2-CPI

H-kininogen

a1-CPI

Fetuins

a2-HS glycoprotein HRG

3q26-qter

Blood, body fluids

3q27

Fetal, bone

CPI CPI CPI

Hereditary cystatin C amyloid angiopathy (HCCAA) Type 2 harlequin icthyosis Inflammatory lung disorders

CPI, regulators of vascular permeability, bronchoconstriction

Inflammatory lung disorders


are some of the most potent naturally occurring mammalian inflammatory agents. All the three functions are critical for the maintenance of respiratory health and, in the settings of respiratory diseases such as asthma and chronic obstructive pulmonary disease (COPD), all the three contribute to the acute and chronic nature of these diseases. However, with the exception of the proinflammatory functions of the type III cystatins (kininogens), the contribution of cystatin superfamily members to pulmonary health and disease is poorly understood. Cystatins: Inhibitory Functions and Interaction with Other Proteolytic Systems

Figure 1 Three-dimensional structure of chicken cystatin C. A five-turn a-helix (green) is in the center of the structure. The conserved G11 residue is highlighted in yellow at the aminoteminal end of the a-helix. The conserved QXVXG motif is on a hairpin loop between b-sheet strands B and C. The conserved PW motif is on a hairpin loop between b-sheet strands D and E.

regions form a wedge-shaped edge complementary to the active site of the CP with many side-chain interactions. Each domain interacts with the target proteinase independently and binding of cystatins to CPs may or may not result in conformational changes in either protein. For example, cystatin binding to papain does not induce any conformational change in either protein whereas binding to cathepsin B involves the initial displacement of a loop occluding the active site to allow subsequent tight binding to occur. Cystatins differ considerably in their ability to displace the loop of cathepsin B. In most cases, the affinity of the cystatins for their respective target CPs is very high with inhibitory constants (Ki’s) in the nanomolar (nM) range but in several instances, where values may be in the micromolar (mM) range, effective inhibition may result from high local concentrations of the cystatins. Examples of this are the S-like cystatins which exist in high concentrations in saliva and tears.

Cystatins and Respiratory Health Cystatins serve at least three functions in respiratory health and disease: they can directly inhibit endogenous or exogenous CPs; they can modulate the activity of the immune system against inhaled bacteria and viruses and their degradation products (kinins)

A well-established mechanism for the loss of lung function observed during the progression of emphysema is the enlargement of the pulmonary airspaces due to an imbalance in degradative proteases and their respective inhibitors (see Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General). The CPs almost certainly contribute either directly or indirectly to this degradation, in particular, lysosomal cathepsins (cat) B, H, K, L, and S (see Cysteine Proteases, Cathepsins). Increased concentrations of cat L have been detected in the BAL fluids of patients with emphysema and alveolar macrophages from patients with COPD secrete more CP activity than macrophages from normal smokers or nonsmokers despite the fact that cystatin C concentrations are also increased. Direct inhibition of CP activity by the cystatins (primarily type II) in normal lung tissue likely contributes to the protection of the elastic lung tissue by directly inhibiting these CPs. The cystatins and CPs do not operate independently of other degradative systems (and their respective inhibitors) but participate in feedback systems that can produce profound changes in proteolytic activity referred to as the proteolytic burst (Figure 2). For example, several matrix metalloelastases, produced by inflammatory cells or activated airway epithelium, can inactivate AAT. Bronchial epithelial cells secrete procathepsin B and cystatin C, both of which are substrates for neutrophil elastase. Procathepsin B is activated by neutrophil elastase while human cystatin C is inactivated by cleavage between amino acids G11 and G12. The latter observation may explain why active cat L has been found in the BAL fluid of COPD patients despite the presence of elevated cystatin C levels. Cystatin C is also produced by monocytes and macrophages and its release is downregulated by proinflammatory lipopolysaccharide (LPS) and interferon gamma (IFN-g), which coincidentally increase

PROTEINASE INHIBITORS / Cystatins 515

Inflammatory stimulus (genetic/environmental (e.g., pollution))

Activated airway epithelium

Cytokines (e.g., IFN-, IL-8)

Neutrophil

↑ Neutrophil elastase

α1AT

SLPI

Monocyte/macrophage

↑ Matrix metalloproteases

–

–

TIMPs

↑ Cathepsins

–

Cystatins

Proteolysis of lung tissue (loss of lung function) Figure 2 Interactions of inflammatory pathway proteases and inhibitors. IFN-g, interferon gamma; IL-8, interleukin 8; a1AT, alpha 1-antitrypsin; TIMPS, tissue inhibitors of metalloproteases; SLPI, secretory leukocyte protease inhibitor.

the expression of cat B, D, H, L, and S. In addition, cat B, L, and S can inactivate secretory leukocyte protease inhibitor (SLPI). Endogenous cystatins are not able to inhibit elastolytic CPs produced by bacterial pathogens such as Staphylococcus aureus or Clostridium histolyticum, but other proteases, such as V8 protease produced by S. aureus, may contribute to an increased elastolytic burden in the lung by inactivating AAT directly. In addition, some potent inhaled allergens possess cystatin inhibitory activity (e.g., cat Fel D3) or CP activity (e.g., dust mite Der P1) and these may have direct effects on lung epithelium and immune surveillance cells of the lung. Immunomodulatory Functions of Cystatins

Cystatins have a wide range of effects in and on the immune cells present in the pulmonary space. Cystatin C is chemotactic for neutrophils, yet inhibits superoxide production and neutrophil-mediated phagocytosis. It also modulates macrophage responses to

IFN-g by increasing production of nitric oxide sixto eightfold via a mechanism independent of its CP inhibitory activity; however, it also increases the production of TNF-a and IL-10. CPs have essential functions in antigen presenting cells (APCs) and cystatin C also plays an important role in modulating major histocompatibility complex (MHC) class II-mediated antigen presentation in peripheral dendritic cells by controlling cat S-mediated degradation of the invariant chain (Ii). This processing prevents targeting of the MHC class II molecules to the lysosomes for degradation. During maturation of APCs in the lymphoid tissue, endosomal cat S activity increases due to a decrease in the levels of cystatin C. Cathepsins K and F can also degrade Ii and cat K is found in bronchial epithelial cells that can serve as nonprofessional APCs. In contrast, cat F’s expression is restricted to hematopoietic cells making it a prime candidate for a role in immunomodulation in these cells. Finally, some members of the human cystatin superfamily (e.g., cystatin S) have potent bactericidal


Environmental antigens/allergens

Respiratory glandular epithelium

Monocytes Neutrophils macrophages

IgE-mediated mast cell activation

Cytokines

Kininogenases (tKal, pKal, cat L, tryptase/neutrophil elastase)

Histamine Proteases: (e.g., tryptase, chymase)

Type III cystatins (HK/LK) Bronchoconstriction Kinins (bradykinin/kallidin)

Mucus secretion Pain Cough Edema

Figure 3 Generation of proinflammatory kinins from type III cystatins. tKal, tissue kallikrein; pKal, plasma kallikrein; cat L, cathepsin L; HK, high-molecular-weight kininogen; LK, low-molecular-weight kininogen.

activity unrelated to CP inhibitory activity which resides in specific peptide sequences present in the structure. Others (e.g.,C, D, and S) are able to block the replication of certain viruses. Cystatin C is a potent inhibitor of herpes simplex virus (HSV)-1, whereas cystatins C and D both inhibit coronavirus replication in human lung cells. The likely mechanism of action involves cellular uptake followed by inhibition of the host or viral CPs required for viral replication. Generation and Function of Proinflammatory Kinins from Type III Cystatins

Perhaps the best understood role for cystatins in respiratory health comes from our current understanding of how BK and kallidin are released from precursor kininogens (type III cystatins) and the multiple direct and indirect effects they have on the respiratory system. Under inflammatory conditions, there are multiple kininogenases that could contribute to kinin generation in the lung (Figure 3). Plasma kallikrein (pKal), tissue kallikrein (tKal), cat L, and a mixture of neutrophil elastase and mast cell tryptase are all able to liberate kinins from kininogens. In the case of highmolecular kininogen, degradation by pKal creates a kinin-free two-chain disulfide-linked molecule containing a heavy chain and a light chain that retains CP inhibitory activity.

tKal has been identified as the major kininogenase of the airway and cleaves both HK and low-mole cular-weight kininogen to yield lysyl-bradykinin (kallidin). In asthmatic airways, the underlying glandular epithelium releases tKal that contributes to the intial phase of kinin generation but the recruitment of activated monocytes, neutrophils, and alveolar macrophages contributes to the late increases in tKal that are associated with the development of airway hyper-responsiveness (AHR). Activated mast cells, macrophages, and neutrophils also release the tryptase, cat L, and elastase that contribute to kinin generation. It has been suggested that this mechanism also contributes to the etiology of other chronic inflammatory conditions such as chronic bronchitis. The generation of kinins by pKal may represent a more acute inflammatory response such as observed during acute pneumonia. Kinins cause bronchoconstriction in asthmatic subjects when given by inhalation or intravenously. Their effects and mechanism of action are described in more detail in Kinins and Neuropeptides: Bradykinin.

Conclusions The cystatins comprise a superfamily of proteins related primarily by virtue of DNA and amino acid

PROTEINASE INHIBITORS / Secretory Leukoprotease Inhibitor and Elafin 517

sequence homology. The superfamily consists so far, of four distinct types of molecules ranging from the simpler low- molecular-weight type I and II cystatins, which function primarily to inhibit lysosomal CPs, to the higher-molecular-weight type III and IV cystatins, which possess additional latent functions expressed only during episodes of injury and inflammation, or have evolved entirely novel inhibitory functions. The role cystatins play in respiratory diseases such as asthma and COPD is poorly understood. However, they do modulate the immune response by acting directly on neutrophils, macrophages, and APCs. It is also clear that they do not function independently of other proteolytic pathways involved in remodeling of the lung. Limited proteolysis inactivates cystatins allowing lysosomal CP activity to directly contribute to lung tissue degradation and also liberates kinins which signal through G-protein-coupled receptors to cause both constriction and dilation of the bronchioles, pain via stimulation of sensory nerves, mucus secretion, cough, and edema. See also: Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deficiency; Emphysema, General. Cysteine Proteases, Cathepsins. Kinins and Neuropeptides: Bradykinin.

Kaufman HF (2003) Interaction of environmental allergens with airway epithelium as a key component of asthma. Current Allergy and Asthma Reports 3(2): 101–118. Leung-Tack J, Tavera C, Gensac MC, Martinez J, and Colle A (1990) Modulation of phagocytosis-associated respiratory burst by human cystatin C: role of the N-terminal tetra-peptide Lys–Pro–Pro–Arg. Experimental Cell Research 188: 16–22. Leung-Tack J, Tavera C, Martinez J, and Colle A (1990) Neutrophil chemotactic activity is modulated by human cystatin C, an inhibitor of cysteine proteinases. Inflammation 14: 247–258. Margis R, Reis EM, and Villeret V (1998) Structural and phylogenetic relationships among plant and animal cystatins. Archives of Biochemistry and Biophysics 359: 24–30. Pierre P and Mellman I (1998) Developmental regulation of invariant chain proteolysis controls MHC class II trafficking in mouse dendritic cells. Cell 93: 1135–1145. Rawlings ND and Barrett AJ (1990) Evolution of proteins of the cystatin superfamily. Journal of Molecular Evolution 30: 60–71.

Secretory Leukoprotease Inhibitor and Elafin T D Tetley, Imperial College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Further Reading Barrett AJ, Rawlings ND, Davies ME, et al. (1986) Cysteine proteinase inhibitors of the cystatin superfamily. In: Barrett AJ and Salvesen G (eds.) Proteinase Inhibitors, pp. 515–569. New York: Elsevier. Bobek LA and Levine MJ (1992) Cystatins – inhibitors of cysteine proteinases. Critical Reviews in Oral Biology and Medicine 3: 307–332. Burnett D, Abrahamson M, Devalia JL, et al. (1995) Synthesis and secretion of procathepsin B and cystatin C by human bronchial epithelial cells in vitro: modulation of cathepsin B activity by neutrophil elastase. Archives of Biochemistry and Biophysics 317: 305–310. Buttle DJ, Burnett D, and Abrahamson M (1990) Levels of neutrophil elastase and cathepsin B activities, and cystatins in human sputum: relationship to inflammation. Scandinavian Journal of Clinical and Laboratory Investigation 50: 509–516. Churg A and Wright JL (2005) Proteases and emphysema. Current Opinion in Pulmonary Medicine 11: 153–159. Collins AR and Grubb A (1991) Inhibitory effects of recombinant human cystatin C on human coronaviruses. Antimicrobial Agents and Chemotherapy 35: 2444–2446. Dickinson DP (2002) Cysteine peptidases of mammals: their biological roles and potential effects in the oral cavity and other tissues in health and disease. Critical Reviews in Oral Biology and Medicine 13: 238–275. Dickinson DP (2002) Salivary (SD-type) cystatins: over one billion years in the making but to what purpose? Critical Reviews in Oral Biology and Medicine 13(6): 485–508. Hall JM (1992) Bradykinin receptors: pharmacological properties and biological roles. Pharmacology and Therapeutics 56: 131–190.

Secretory leukoprotease inhibitor (SLPI) and elafin are acidstable, low-molecular-weight antiproteinases that are produced by the goblet cells, Clara cells, and alveolar type II cells in the pulmonary epithelium, and are also present in macrophages, while neutrophils contain SLPI. SLPI inhibits neutrophil elastase, cathepsin G, trypsin, chymotrypsin, and chymase. Elafin inhibits neutrophil elastase and proteinase-3. SLPI is 11.7 kDa; elafin (6 kDa) is a cleavage product of preelafin (also called trappin-2), which is 9.9 kDa. The inhibitory site of both inhibitors resides in the C-terminal four disulfide whey acidic protein (WAP) domain, which has 40% homology, and they belong to the WAP family of proteins located on chromosome 20q12–13. The N-terminal WAP domain of pre-elafin contains a transglutaminase substrate domain that enables the molecule to become tethered to cell surfaces and matrix proteins; proteolytic cleavage releases the C-terminal 6 kDa inhibitory domain. SLPI is also found in close association with elastic tissue, possibly reflecting its cationic properties. SLPI and elafin have antimicrobial activity against Gram-positive and Gram-negative bacteria, while SLPI has also been shown to have antiviral and antifungal properties. In addition, SLPI and elafin are immunomodulatory, interacting directly with lipopolysaccharide to subdue its inflammatory activity, and inhibiting release of mediators from inflammatory cells. SLPI also plays a significant role in wound healing, partly by preventing proteolytic activation of proinflammatory mediators. SLPI and elafin levels and activity change during lung diseases (e.g., chronic obstructive pulmonary disease, acute respiratory distress syndrome, and pneumonia), reflecting the degree of inflammation, proteolytic load, and oxidative stress. The possibility of SLPI and elafin therapy is under active investigation.

518 PROTEINASE INHIBITORS / Secretory Leukoprotease Inhibitor and Elafin

Introduction Secretory leukoproteinase inhibitor (SLPI; also known as antileukoprotease or mucus proteinase inhibitor) and elafin (also known as elastase-specific inhibitor and skin-derived antileukoprotease, SKALP) are low-molecular-weight, cationic, nonglycosylated, acid-stable, serine proteinase inhibitors that are synthesized and released at mucosal surfaces and within the skin, and are also important in host defense and tissue repair (Figure 1). Both antiproteases are potent, reversible inhibitors of neutrophil elastase; in addition, SLPI inhibits cathepsin G, trypsin, chymotrypsin and chymase, whereas elafin inhibits proteinase-3.

Structure and Function SLPI

SLPI consists of 107 amino acids with a molecular weight of 11.7 kDa and has two highly homologous cysteine-rich domains, each of which contains eight cysteine residues that form four disulfide bridges. These four disulfide core whey acidic protein (WAP) domains are so named after the original observation of the WAP motif in whey acidic protein in milk whey of lactating mice. SLPI is one of a series of small proteins which contain the same WAP motif, or repeated WAP domains, that have been located on chromosome 20q12–13. The protease inhibitory activity of SLPI is located in the C-terminal region, at residues Leu72-Met73. Within the lung, SLPI is synthesized and secreted by epithelial secretory cells throughout the lower respiratory tract, including the tracheobronchial goblet cells, Clara cells, and

alveolar epithelial type II cells, as well as the serous cells of the submucosal glands. SLPI is produced constitutively and is released apically into the epithelial lining liquid at high concentrations; it is also released basally and has been found in close association with elastic tissue, possibly reflecting its cationic properties, and, unlike alpha-1 proteinase inhibitor, is a very effective inhibitor of neutrophil elastase bound to elastin. In addition, SLPI is present in monocytes, macrophages, and neutrophils. Elafin

Elafin was originally found to have a molecular weight of 6.0 kDa, following isolation from skin and lung secretions. It consists of 57 amino acids, with a four disulfide core WAP domain which also has 40% homology with the C-terminal WAP region of SLPI. Like other WAP proteins, the elafin gene is located on chromosome 20q12–13. When the gene was subsequently cloned, the protein was found to be larger: B12 kDa and 117 amino acids inclusive of the signal peptide, 95 amino acids and 9.9 kDa exclusive of the signal peptide, due to the presence of an N-terminal domain that is unlike that of SLPI, or any other members of the WAP family. Instead, it contains a transglutaminase substrate domain. Proteins containing both the WAP domain and the transglutaminase substrate domain have now been brought together under the acronym trappin (transglutaminase substrate and WAP domain containing protein) for the trappin gene family. Elafin is the C-terminal region of trappin-2, being the second trappin to be discovered. Trappin-2 refers to the 117 amino acid molecule and is also called pre-elafin. The transglutaminase domain contains five glutamine and

Interstitial pre-elafin Elafin Antiproteolytic TNF Microbes LPS

Pre-elafin Antimicrobial

IL-1

Elafin SLPI

NE

Immunomodulatory Wound healing

SLPI Interstitial SLPI Figure 1 The sources of SLPI and elafin: the epithelium, macrophages, and neutrophils. Interleukin-1 beta (IL-1b) and tumor necrosis factor (TNF) (released by macrophages), serine proteinases (released by neutrophils; NE, neutrophil elastase), and microbial agents, such as lipopolysaccharide (LPS), stimulate SLPI and elafin release into the airway lumen and into the interstitium, where they exhibit antiproteolytic, antimicrobial, immunomodulatory, and wound healing properties.


lysine residue repeats (GQDPVK) which act as acyl % donor and acceptor sites,% rendering the protein susceptible to cross-linking by tissue transglutaminase; this property has resulted in the use of the term cementoin for the N-terminal region of pre-elafin. Thus, once secreted, the molecule can become tethered to extracellular matrix and cell surfaces and operate as an anchored protein. This then restricts the activity of pre-elafin to specific tissue sites and is likely to be an important mechanism in controlling site-specific activity of elastin. However, proteolytic release of the 57 amino acid C-terminal elafin molecule allows the protease inhibitory region to transude into other tissue compartments. Again, this may occur under specific circumstances to enable elafin to function in alternative lung compartments. Elafin is synthesized and secreted by tracheobronchial epithelium, Clara cells, and alveolar type II epithelial cells, and is present in macrophages. It is also present in lung-lining liquid, but usually at much lower concentrations than SLPI (B10% of SLPI levels). Unlike SLPI, it is not produced at high levels constitutively.

Regulation of SLPI and Elafin Synthesis SLPI is synthesized by lung epithelial cell lines and primary human lung epithelial cells in vitro at relatively high levels; in contrast, constitutive levels of elafin mRNA and protein are generally much lower. The expression of both SLPI and elafin can be stimulated in pulmonary epithelial cell lines and human primary bronchial and alveolar epithelial cells by early response proinflammatory stimuli such as tumor necrosis factor alpha (TNF-a) and interleukin 1 beta (IL-1b) via nuclear factor kappa B (NF-kB). Furthermore, while corticosteroids alone have little effect on SLPI and elafin, they synergize with TNF-a and IL-1b to further stimulate SLPI and elafin synthesis. Bacterial LPS and inactivated Pseudomonas aeruginosa can also induce SLPI and elafin synthesis by epithelial cells in vitro. Thus, exposure of pulmonary epithelial cells in vivo to proinflammatory cytokines and bacterial components likely upregulates pulmonary antiproteinase defense, possibly in preparation for the increased release of inflammatory cell proteases. Interestingly, neutrophil elastase has been shown to stimulate elafin and SLPI mRNA expression in airway epithelial cells in vitro, suggesting feedback control on excessive release of neutrophil elastase. Such feedback control mechanisms may be common during neutrophilic lung inflammation; both proteins have been shown to be altered in bronchoalveolar lavage (BAL) fluid during inflammation and may be differentially expressed, thus altering the efficacy of the antiproteinase screen

(see below). Few studies have been performed on the regulation of elafin expression by primary cells.

Antiproteinase Function SLPI and elafin were originally named for their ability to inhibit neutrophil elastase, although they are also important inhibitors of mast cell proteinases. Neutrophil elastase is a potent enzyme that impacts on numerous processes. For example, it degrades elastin and other connective tissue proteins, it is ciliostatic and a secretagogue and likely contributes to excessive mucus production and retention in chronic obstructive pulmonary disease (COPD), it can stimulate cytokine synthesis as well as activating and inactivating cytokines, it cleaves cell-surface receptors, such as CD14, and it activates complement. Many of the cleavage products have chemotactic activity and so can amplify inflammation. Furthermore, neutrophil elastase can amplify proteolytic potential by activating latent matrix metalloproteinases (MMPs) and inactivating their inhibitors. Lung injury following intratracheal neutrophil elastase into experimental animals is prevented by pretreatment either with pre-elafin (but not elafin) or with the antiproteolytic region of SLPI, illustrating the importance of these proteins in controlling neutrophil elastase-induced inflammation. Thus, neutrophil elastase activity is normally strictly regulated to achieve optimal activity in response to infection. Because SLPI, pre-elafin, and elafin can access substrate-bound and free enzyme, in the interstitium and lumen, as well as being reversible, they compliment the activity of alpha-1 antitrypsin (54 kDa, glycosylated, the major circulating serine proteinase inhibitor), which irreversibly binds to and inactivates free elastase prior to clearance via the blood. Thus, SLPI and elafin work in concert with alpha-1 antitrypsin to ensure complete inhibition and clearance of neutrophil elastase and other serine proteinases from inflamed tissue.

Antimicrobial Activity SLPI and elafin proteins have antimicrobial properties similar to defensins, which appear to be unrelated to their antiprotease activity, and which have been related to the N-terminal domain of SLPI and to both the N- and C-terminals of elafin. In vitro, SLPI has antibacterial activity towards Gram-positive (e.g., Staphylococcus aureus, Listeria monocytogenes) and Gram-negative (e.g., Escherichia coli) bacteria. Elafin is also active in vitro against S. aureus and P. aeruginosa. SLPI has been shown in vitro to block the infection of peripheral blood monocytes, primary T cells, and macrophages by


human immunodeficiency virus-1 (HIV-1), possibly by binding to annexin II at the cell surface, recently proposed to be a receptor for HIV-1, or by blocking chemokine receptors CXCR4 or CCR5. Interestingly, SLPI is not very active against other retroviruses, but has activity against other types of virus, including Sendai virus and influenza virus. SLPI also has antifungal properties, inhibiting the ability of Aspergillus fumigatus and Candida albicans to trigger release of proinflammatory cytokines from human epithelial cell lines. Overexpression of elafin reduces lung injury following P. aeruginosa infection in a mouse model, which may reflect specific microbicidal activity and/or be related to other anti-inflammatory mechanisms (see below).

Role of SLPI and Elafin in Immunity The role of SLPI and elafin in the immune response is currently under intensive investigation. In an endotoxic shock model, SLPI-deficient (SLPI / ) mice were more susceptible to lipopolysaccharide (LPS) exposure, which led to higher mortality than that in wild-type mice, and also resulted in higher macrophage IL-6 and increased NF-kB expression. In vivo and in vitro investigations following augmentation of SLPI indicate that one mechanism of action is downregulation of NF-kB, by inhibiting IkappaBalpha/beta degradation and upregulation of IkappaBalpha. SLPI also prevents activation of IL-1 receptor associated kinase (IRAK), a Toll-like receptor 4 (TLR4) adaptor molecule required for cellular signaling following TLR4 activation by LPS. Thus, downregulation of NF-kB and other intracellular molecules known to be involved in the inflammatory response is likely one of the mechanisms by which SLPI inhibits release of proinflammatory mediators (e.g., prostanoids, MMPs, and TNF) from lung cells in vivo. Another anti-inflammatory mechanism may be to prevent the interaction and biological reactivity of the infective agent with cellular targets. For example, SLPI binds to LPS. Interestingly, LPS triggers release of SLPI from pulmonary epithelial cells and macrophages, possibly to control excessive LPS activity (see below). These, and similar studies, illustrate an anti-inflammatory and immunomodulatory role for SLPI. Intranasal administration of engineered human pre-elafin to mice prior to intranasal LPS inhibited inflammatory neutrophil influx, two neutrophil chemokines (macrophage inflammatory protein-2 (MIP-2) and KC, murine homologs of IL-8), as well as downregulating expression of IL-1, suggesting significant anti-inflammatory mechanisms for elafin. However, adenovirus-mediated overexpression of the full-length human pre-elafin gene in mice (who do

not have an elafin gene equivalent) resulted in increased pulmonary airway inflammatory cell influx and elevated TNF-a and MIP-2 in BAL following LPS challenge. It is suggested that this apparent proinflammatory response may be an important acute defense mechanism, triggering the innate immune response system to combat bacterial infection, as has been proposed for some defensins. Since mice do not normally express elafin, it is difficult to appreciate the influence this itself may have, particularly in the context of cellular responses following elafin binding to LPS (see below). Another putative mechanism of action of SLPI and elafin in host defense involves binding to LPS and intercepting its interaction with target cells. As LPS is a rapid, potent activator of the innate immune system, its activity needs to be carefully modulated to elicit an appropriate response. The presence, in respiratory secretions, of constitutively high quantities of SLPI, and not insignificant levels of elafin, release of which can be further stimulated by endogenous and exogenous agents, is probably crucial during microbial infection with Gram-negative bacteria as both proteins bind LPS. Very small quantities of LPS evoke strong systemic responses which need to be tightly regulated. Macrophages transfected with SLPI that overexpress the inhibitor respond to LPS, but are less sensitive, showing a subdued response. SLPI has been shown to bind to LPS and CD14–LPS to prevent cell-surface TLR4 binding and macrophage activation. This is thought to be an important feedback control mechanism to modulate the intensity of the cellular response. More recently, elafin has also been demonstrated to bind to the lipid A core of LPS. Production of TNF-a by macrophages was inhibited by LPS–elafin complexes only in the presence of serum. However, if the experiment was carried out in a serum-free environment, then production of TNF-a increased. This fits with the findings using transgenic elafin mouse models and it is postulated that, within a serum-free environment, such as the lung lumen, elafin facilitates LPS to initiate an immune response in the absence of large quantities of LPS-binding protein. However, systemically, binding of LPS by elafin (which, if present at high levels, likely reflects acute tissue injury) would subdue an overt immune response.

Role of SLPI in Wound Healing The expression of SLPI is elevated during cutaneous wound healing in humans. Absence of SLPI leads to delayed healing due to reduced matrix production and exaggerated inflammatory response. Wound


healing in SLPI-deficient mice is impaired. In this model, there is increased inflammatory cell influx with elevated elastase activity, increased NK-kB activation, as well as an increase in levels of active transforming growth factor beta (TGF-b). It is suggested that in the absence of SLPI, there is greater macrophage activation, release of proteolytic enzymes, and cleavage and activation of latent TGF-b. Together, activated TGF-b and chemotactic peptides, generated during matrix turnover, stimulate a cycle of leukocyte recruitment during the remodeling process and delay the healing process. Thus, there appears to be a complex interaction between SLPI and the growth factor TGF-b that closely regulates connective tissue synthesis and repair processes. In addition, proepithelins are growth factors, released by epithelial cells, that stimulate re-epithelialization and inhibit proinflammatory chemokine release from epithelium; they are processed to generate epithelin which has opposite effects, stimulating IL-8 release and inhibiting epithelial growth. SLPI forms complexes with proepithelins which prevents elastolytic conversion of anti-inflammatory proepithelins to proinflammatory epithelin. In SLPI-null mice, application of proepithelins reverses the defect in wound healing, supporting the suggestion that SLPI enables re-epithelialization during wound healing by preventing proteolytic degradation of proepithelins. In in vitro studies of human lung fibroblasts, SLPI stimulates hepatocyte growth factor (HGF; also called scatter factor), which regulates mitogenesis and morphogenesis and is therefore relevant to normal tissue development and remodeling. SLPI has also been shown to inhibit fibroblast-mediated scar formation in a collagen gel model in vitro. The ability of SLPI to promote wound healing has been ascribed to a number of qualities, including its antiproteolytic activity and autocrine growth factor activity. Together with its antimicrobial and antiinflammatory properties, SLPI is clearly an important multifunctional mediator of wound healing.

SLPI and Elafin in Respiratory Disease Uncontrolled/elevated serine proteinases have been implicated in the pathology of a number of pulmonary diseases, including COPD, cystic fibrosis, bronchiectasis, acute respiratory distress syndrome (ARDS), bronchiolitis obliterans, bacterial pneumonia, asthma, and fibrosis. Abnormal SLPI and elafin expression and/or activity leading to uncontrolled serine proteinase activity may be a factor. SLPI is the major serine proteinase inhibitor in conducting airway secretions (between 50% and 90% of the total), reflecting synthesis and release by goblet cells and

serous cells in the bronchial glands. In the small airways, bronchioles, and alveolae, the Clara cells and type II epithelial cells also produce SLPI, although it forms relatively less of the total serine proteinase inhibitory screen (approximately 20% of the total). Elafin is synthesized by the same cells, but its contribution to the antiproteinase screen is usually relatively low since it is not released at high levels constitutively, but production may be triggered by external and endogenous acute stimuli, such as bacteria and IL-1b. Clearly, the levels and activity of SLPI and elafin during pulmonary disease will reflect both the changes in secretory cell profile (e.g., cell death, proliferation, and migration) and regulation of inhibitor expression by local mediators. The relative contribution of leukocyte-derived SLPI and elafin to lung antiprotease defense is less clear. Most studies of the role of SLPI and elafin in lung disease have measured levels in BAL fluid and sputum and there are no data regarding interstitial levels or upregulation of mRNA expression in individual cells. Secretions from subjects with COPD contain high levels of SLPI, probably reflecting increased numbers of SLPI-expressing epithelial cells in large and small airways, and possibly also because of increased release by leukocytes. However, during exacerbations, if uninhibited, active neutrophil elastase is present in lung secretions, SLPI is reduced. Furthermore, epithelial SLPI expression is reduced in areas of epithelial damage or epithelial metaplasia. Although a number of studies show that there is an apparent compensatory increase in pulmonary SLPI in alpha-1 antitrypsin deficiency, in alpha-1 antitrypsin-deficient subjects with COPD, SLPI has been shown to be compromised, possibly for the same reasons as alpha-1 antitrypsin-sufficient subjects, that is, increased neutrophil elastase and epithelial damage and/ or metaplasia. There is little information regarding elafin expression during COPD. There are fewer studies of SLPI and elafin in other lung diseases. In respiratory distress situations, SLPI and elafin are significantly elevated in BAL fluid from those at risk of developing ARDS compared to healthy subjects. In those who go on to develop ARDS, SLPI, but not elafin, levels increase further, to above the levels for those at risk of developing ARDS. Furthermore, SLPI concentrations in BAL fluid correlate with multiorgan failure score in patients with acute lung injury. SLPI is also increased in BAL fluid during pneumonia. However, despite increased levels of these locally produced inhibitors, antiprotease activity in lung secretions is usually reduced, particularly during severe disease, due to oxidation, complexing with elastase or degradation. In contrast, the level of SLPI is reduced in BAL fluid


collected from patients with bronchiolitis obliterans syndrome (after heart–lung transplantation). Although elafin has not been measured in lung secretions from subjects with pneumonia and bronchiolitis obliterans, it has been shown to be markedly elevated in BAL fluid obtained from subjects with active farmer’s lung, supporting its role as an acuteresponse antiprotease, but, interestingly, remained elevated in those who had recovered.

Alpha-1-Antitrypsin Deficiency; Emphysema, General. Clara Cells. Defensins. Endotoxins. Epithelial Cells: Type II Cells. Extracellular Matrix: Basement Membranes; Elastin and Microfibrils; Collagens. Gene Regulation. Human Immunodeficiency Virus. Interleukins: IL-1 and IL-18. Interstitial Lung Disease: Cryptogenic Organizing Pneumonia. Leukocytes: Neutrophils; Pulmonary Macrophages. Serine Proteinases. Toll-Like Receptors. Transcription Factors: NF-kB and Ikb. Transforming Growth Factor Beta (TGF-b) Family of Molecules. Viruses of the Lung.

SLPI and Elafin Augmentation Therapy SLPI and pre-elafin are not glycosylated, and recombinant forms are identical to the native proteins and therefore have therapeutic potential. As outlined above, the advantage of these inhibitors over alpha-1 antitrypsin is that their physicochemical properties enhance localization to the interstitium, where they can inhibit matrix-bound proteases. Whether SLPI is administered via inhalation, intratracheally, intravenously, or intraperitoneally, it is very effective in animal models, including acute lung injury, bleomycininduced fibrosis, and protease- and allergen-induced airway constriction and hyperreactivity. Furthermore, in an ovine model, aerosolized SLPI caused an increase in glutathione levels in lung lining liquid, enhancing antioxidant protection. There is minimal metabolism of SLPI in the lung and it remains active following inhalation of the aerosolized recombinant protein. However, studies of healthy subjects and cystic fibrosis patients show that its half-life in lunglining liquid is short, requiring treatment twice daily. Nevertheless, the antiproteinase activity increases, while IL-8 levels decrease in BAL fluid from SLPI-treated individuals. A difficulty with inhalation therapy is delivery of the drug to diseased/affected regions. This problem is particularly relevant for conditions involving mucous hypersecretion and airway remodeling, such as cystic fibrosis and emphysema. There are no similar studies of elafin. An alternative to inhalation therapy is gene therapy. In animal models, overexpression of intratracheally administered adenoviral elafin gene confers protection against P. aeruginosa-induced lung injury and S. aureus-induced inflammation. Although these studies suggest that this approach to augmenting pulmonary antiproteases might be useful, it is still in its infancy. There are no studies in humans. Furthermore, in the face of ongoing pulmonary disease, the problems associated with optimal delivery and expression of the protein are significant. See also: Acute Respiratory Distress Syndrome. Antiviral Agents. Asthma: Overview. Chronic Obstructive Pulmonary Disease: Overview; Emphysema,

Further Reading Aarbiou J, van Schadewijk A, Stolk J, et al. (2004) Human neutrophil defensins and secretory leukocyte proteinase inhibitor in squamous metaplastic epithelium of bronchial airways. Inflammation Research 53: 230–238. Ashcroft GS, Lei K, Jin W, Longenecker G, et al. (2000) Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing. Nature Medicine 6: 1147– 1153. Chughtai B and O’Riordan TG (2004) Potential role of inhibitors of neutrophil elastase in treating diseases of the airway. Journal of Aerosol Medicine 17: 289–298. Clauss A, Lilja H, and Lundwall A (2002) A locus on human chromosome 20 contains several genes expressing protease inhibitor domains with homology to whey acidic protein. Biochemical Journal 368: 233–242. Doumas S, Kolokotronis A, and Stefanopoulos P (2005) Antiinflammatory and antimicrobial roles of secretory leukocyte protease inhibitor. Infection and Immunity 73: 1271–1274. Greene C, Taggart C, Lowe G, et al. (2003) Local impairment of anti-neutrophil elastase capacity in community-acquired pneumonia. Journal of Infectious Diseases 188: 769–776. Henriksen PA, Hitt M, Xing Z, et al. (2004) Adenoviral gene delivery of elafin and secretory leukocyte protease inhibitor attenuates NF-kappa B-dependent inflammatory responses of human endothelial cells and macrophages to atherogenic stimuli. Journal of Immunology 172: 4535–4544. Hiemstra PS (2002) Novel roles of protease inhibitors in infection and inflammation. Biochemical Society Transactions 30: 116– 120. McMicheal JW, Roghanian A, Jiang L, Ramage R, and Sallenave JM (2005) The antimicrobial antiproteinase elafin binds to lipolysaccharide and modulates macrophage responses. American Journal of Respiratory Cell and Molecular Biology 32: 443–452. Molhuizen HOF, Alkemade HAC, Zeeuwen PLJM, et al. (1993) SKALP/elafin: an elastase inhibitor from cultured human keratinocytes: purification, cDNA sequence, and evidence for transglutaminase cross-linking. Journal of Biological Chemistry 268: 12028–12032. Sallenave JM (2002) Antimicrobial activity of antiproteinases. Biochemical Society Transactions 30: 111–115. Schalkwijk J, Wiedow O, and Hirose XY (1999) The trappin gene family: proteins defined by an N-terminal transglutaminase substrate domain and a C-terminal four-disulphide core. Biochemical Journal 340: 569–577. Taggart CC, Greene CM, McElvaney NG, and O’Neill S (2002) Secretory leucoprotease inhibitor prevents lipopolysaccharideinduced IkappaBalpha degradation without affecting phosphorylation or ubiquitination. Journal of Biological Chemistry 277: 33648–33653.

PROTEINASE-ACTIVATED RECEPTORS 523

PROTEINASE-ACTIVATED RECEPTORS R C Chambers, University College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract The discovery of the proteinase-activated receptors (PARs) resulted in the establishment of a new paradigm for ligand– receptor interaction mechanisms leading to cellular signaling in that proteolysis rather than classical ligand binding activates these receptors. The PARs were originally regarded as cellular signaling receptors for the coagulation serine proteinase thrombin. However, the cloning of additional PARs led to the identification of other proteinase activators, including trypsin, mast cell tryptase, and more recently nonserine proteinases such as MMP-1. PARs are widely distributed in the human lung, and recent evidence indicates a role for PARs in the pathophysiology of lung inflammatory and fibrotic responses.

Introduction Proteinase-activated receptors (PARs) belong to the family of seven transmembrane domain G-proteincoupled receptors and derive their name from their unique mechanism of activation. Unlike other Gprotein-coupled receptors, which are activated by direct ligand binding, activation of the PARs involves the proteolytic unmasking of a cryptic ligand that is already tethered to the receptor. Proteolysis is mediated by certain proteinases, of which thrombin and upstream proteinases of the extrinsic pathway of coagulation, as well as trypsin and tryptase are the most well recognized. The human gene for PAR1 (formerly known as the thrombin receptor) was discovered in 1991 by Shaun Coughlin and colleagues at the University of California–San Francisco at the end of an intense research effort aimed at identifying the cellular mechanism by which thrombin mediates platelet aggregation. The same year also saw the cloning of the hamster PAR1 gene by Van Obberghen-Schilling and coworkers in Strasbourg. Following the discovery of the thrombin receptor and its unique activation mechanism, it appeared possible that other receptors with a similar mechanism of activation might exist. For example, platelets obtained from PAR1 null mice surviving to adulthood still responded strongly to thrombin. The search for additional thrombin receptors culminated in the discovery of two further thrombin-sensitive receptors, PAR3 and PAR4. Genomic screening led to the identification of a further receptor, PAR2, with a similar mechanism of activation by the more broadspectrum proteinase trypsin but not thrombin.

Structure The four PAR genes are between 3.5 and 3.7 kb long and share a similar two-exon structure encoding around 400 amino acids (Table 1). Human PAR1, PAR2, and PAR3 cluster together on band q13 on chromosome 5, suggesting that they may have arisen by gene duplication from a single ancestral gene. In contrast, PAR4 is located separately at position p12 on chromosome 19. The sequence homology between human PARs is between 27% and 33%; with the greatest difference noted between PAR4 and the other three PARs in terms of both the N- and C-termini and the cleavage site. Comparison of amino acid sequence alignment between species revealed that the PARs are highly conserved between humans and mice and that homologs of these genes are present in amphibians. The predicted protein structure of the PARs share several features with classical seven transmembrane G-protein domain-linked receptors with their signature configuration consisting of seven helical hydrophobic transmembrane regions that in turn give rise to three intra- and three extracellular loops, a C-terminal intracellular tail, and a long N-terminal extracellular domain (Figure 1).

Regulation of Production and Activity Activation of the PARs is mediated via limited proteolysis of the N-terminus resulting in the cleavage of around 40 amino acids (Figure 1 and Table 1). This leads to the unmasking of a tethered ligand sequence that interacts with the second extracellular loop of the receptor to induce a conformational change allowing the receptor to interact and signal via heterotrimeric G-proteins, which in turn trigger a variety of downstream signal transduction pathways. There is little doubt that thrombin is an important physiological activator of PAR1, PAR3, and PAR4. The upstream coagulation proteinase, factor Xa, as well as the more potent tissue factor–factor VIIa–factor Xa complexes, activate both PAR1 and PAR2, depending on cell type. PAR1, PAR2, and PAR4 can also be activated by trypsin, whereas mast cell tryptase is thought to be a physiological activator of PAR2. A number of other proteinase activators have been described and are listed in Table 1. These include nonserine proteinases, such as the matrix metalloproteinase MMP-1. Recently, nonendogenous proteinases, including proteinases released by house dust mites and certain bacteria, have also been

Table 1 Human proteinase-activated receptor expression and pharmacology No. of amino acids

High-affinityactivating proteinases

Low-affinityactivating proteinases

Tethered ligand sequence

Activating peptides

Nonpeptide antagonists

Inactivating proteinases

Tissue expression

Cell types

PAR1

425

Thrombin

Trypsin, TF/FVIIa/ FXa, granzyme A, plasmin, trypsin IV, MMP-1

R41mSFLLRN

SFLLRNNH2, TFLLRNH2

RWJ56110, RWJ58259

Cathepsin G, neutrophil proteinase-3, elastase, chymase, Der p1

Airways, blood, brain, bone, breast, cardiovascular system, endometrium, immune system, intestine, lung parenchyma, lymph node, nervous system, skin

PAR2

397

Trypsin, tryptase, trypsin II, trypsin IV

Matriptase/ MTSP1, TF/FVIIa/ FXa, proteinase-3, Der p1, Der p3, Der p9

R34mSLIGKV

SLIGKVNH2, SFLLRNNH2

None to date

Elastase, chymase

Airways, blood, brain, cardiovascular system, GI tract, immune system, intestine, nervous system, pancreas, skin, testes, urogenital tract, eye

PAR3

374

Thrombin

Trypsin, Factor Xa

K38mTFRGAP

None known

–

Cathepsin G

Immune system

PAR4

385

Thrombin, trypsin

Cathepsin G,

R47mGYPGQV

GYPGQVNH2, AYPGKFNH2

YD-3

Unknown

Airway, blood, cardiovascular system

Astrocytes, epithelial cells, endothelial cells, fibroblasts, hematopoietic progenitor cells, keratinocytes, macrophages, mast cells, natural killer cells, neuronal cells, platelets, smooth muscle cells, T cells Endothelial cells, eosinophils, epithelial cells, fibroblasts, keratinocytes, mast cells, macrophages, monocytes, neuronal cells, neutrophils, platelets, smooth muscle cells, T cells Megakaryocytes of the bone marrow, platelets, T cells Epithelial cells, smooth muscle cells, endothelial cells, platelets, fibroblasts

Der p1, 3, and 9, house dust mite Dermatophagoides pteronyssinus proteinase 1, 3, and 9. MMP-1, matrix metalloproteinase-1. MT-SP1, Membrane-type serine protease 1. NH2, amide. Letters denote amino acid sequences in one letter code; arrow denotes cleavage site.

PROTEINASE-ACTIVATED RECEPTORS 525 Thrombin

Cleavage

Tethered ligand

G

PAR-1 Cell signaling Figure 1 Mechanism of activation of proteinase-activated receptor by thrombin. (1) The cleavage of an extracellular fragment of the receptor by thrombin unmasks a tethered ligand that binds to the body of the receptor. (2) The resulting conformational change induces cell signaling via activation of heterotrimeric G-proteins. PAR, proteinase-activated receptor; Ga, b, g, G-protein subunits.

recently shown to activate PARs in vitro. However, confirmation for the importance of these enzymes in activating PARs in vivo is still lacking. Delineating the signaling pathways and cellular responses elicited by PAR activators has been greatly aided by the use of peptide agonists, which mimic the tethered ligand sequence unmasked following receptor cleavage. Current evidence suggests that PAR1, PAR2, and PAR4 act as signaling receptors, whereas PAR3 is thought to act as a thrombin-docking receptor for efficient presentation of the proteinase to PAR4 at low concentrations. Following PAR activation, signal transduction is mediated via heterotrimeric G-proteins. PAR1 is relatively promiscuous in its ability to couple to multiple G-proteins, including Go/i, Gq, and G12/13. This enables the receptor to mediate its pluripotent effects via various signaling pathways, including amongst others phospholipase c/protein kinase c (PLC/PKC), mitogen-activated protein-kinase (MAPK), c-Jun NH2-terminal kinase (JNK), and nuclear factor kappa B (NF-kB) pathways. Fewer signaling studies have been performed for the other three PARs. Current evidence suggests that PAR2 interacts with Gq/11 and possibly Go/i. As mentioned above, PAR3 does not appear to signal, whereas PAR4 has been shown to activate Gi, G12/13, and Gq pathways. In terms of the regulation of PAR signaling, the mechanisms involved are similar to those employed by other members of the seven transmembrane domain G-protein-coupled receptors. This includes classical receptor desensitization by phosphorylation at the C-terminus followed by endocytosis and degradation. Cell responsiveness is re-established via the appearance of new receptors cycled to the cell surface from pre-existing intracellular stores and de novo protein synthesis. Several proinflammatory cytokines, as well as the activating proteinases themselves (e.g., thrombin), have been reported to induce PAR gene expression. Finally, PAR signaling may also be controlled by inactivation by certain proteinases as a result of proteolysis at nonactivating sites (Table 1).

Biological Function The clearest physiological role for the PARs is in the activation of platelets by thrombin, one of the key events involved in blood clotting. In humans, this response is mediated by PAR1 and PAR4, whereas murine platelet responses appear to involve binding to PAR3 and subsequent cleavage of PAR4. Therefore, there are important differences in receptor utilization between humans and mice and this needs to be borne in mind when extrapolating results obtained in experimental models to human physiology and pathophysiology. Intense research efforts into the role of PARs (in particular PAR1 and PAR2) in other cell types has revealed that these receptors influence a wide range of cellular responses via both direct effects and their ability to induce the synthesis and release of a host of secondary mediators, including growth factors, chemokines, lipid mediators, and potent proinflammatory cytokines. It is therefore not surprising that these receptors are capable of influencing a wide range of cellular responses, including promoting cellular proliferation, differentiation, cytoskeletal reorganization, apoptosis, migration, and extracellular matrix production. In vivo evidence for the importance of these responses in the regulation of inflammation, vascular tone, angiogenesis, tissue and blood vessel repair, immune responses, as well as cell invasion in both pathological and nonpathological conditions is rapidly accumulating.

PARs in Respiratory Disease Fibroproliferative Lung Disease

Interest in the role of PARs in fibrotic lung disease was fuelled by the observation that activation of the coagulation cascade is a characteristic feature of both chronic and acute lung injury. In the normal uninjured lung, the alveolar hemostatic balance is generally antithrombotic and profibrinolytic. However, in both acute lung injury and chronic fibrotic lung disease, there is good evidence that this balance is shifted in

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favor of increased procoagulant activity and decreased fibrinolytic capability. Extravascular intra-alveolar accumulation of fibrin, often evident as hyaline membranes, is commonly observed in the lungs of patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and chronic fibrotic lung disease. Levels of tissue factor, the initiator of the extrinsic coagulation pathway is highly upregulated in the lungs of patients with idiopathic pulmonary fibrosis, interstitial pneumonia associated with systemic sclerosis and in idiopathic bronchiolitis obliterans with organizing pneumonia. Bronchoalveolar lavage fluid (BALF) from patients with ARDS also contains tissue factor/factor VII/VIIa complexes and levels of active thrombin have been shown to be increased in the lungs of patients with pulmonary fibrosis associated with systemic sclerosis and in pulmonary fibrosis associated with chronic lung disease of prematurity. Thrombin BALF levels are also elevated in animal models of lung injury (e.g., bleomycin) and pharmacological inhibition of the coagulation cascade has been shown to be protective against lung collagen accumulation in these models. There is extensive in vitro evidence that activation of PAR1 by thrombin exerts both proinflammatory and profibrotic effects on a number of cell types present in the lung. For example, activation of PAR1 by either thrombin or factor Xa leads to the release of a host of secondary proinflammatory mediators, increases endothelial cell permeability, influences inflammatory cell trafficking, and promotes fibroblast proliferation and transformation into activated myofibroblasts, the main cell type responsible for extracellular matrix deposition in the fibrotic lung. PAR1 immunoreactivity is increased both in patients with pulmonary fibrosis and in the bleomycin model of this condition. This raises the possibility that the coagulation proteinases may contribute to the pathogenesis of these disorders, at least in part via PAR1-dependent pathways. Support for a pivotal role for PAR1 in this model was recently obtained for both the inflammatory and fibrotic phases of this injury model. Lung collagen accumulation in response to bleomycin injury is attenuated by up to 60% and is preceded by similar reductions in inflammatory cell recruitment and microvascular leak. This protection is associated with a reduction in a number of PAR1inducible genes, including the chemokine monocyte chemotactic protein factor 1 (MCP-1) and the profibrotic mediators connective tissue growth factor and transforming growth factor beta (TGF-b1). Antagonists are currently being developed as potent antithrombotic agents and may therefore prove useful for interfering with the cellular effects associated with excessive activation or recurrent

activation of the coagulation cascade in response to both acute and chronic lung injury. Asthma and Airway Remodeling

Thrombin levels are increased in BALF from patients with asthma and have recently been shown to correlate with levels of interleukin-5 (IL-5), TGF-b, and inflammatory cell numbers after segmental challenge in asthmatic patients. Several functional responses elicited by thrombin observed in in vitro and ex vivo studies might contribute to the pathology of this condition. Thrombin acts as a bronchoconstrictor, exerts mitogenic effects for airway smooth muscle cells and fibroblasts, and, as mentioned above, releases a host of proinflammatory cytokines by a number of cell types, including airway and alveolar epithelial cells. Current in vitro and ex vivo evidence suggests an important role for PAR1 in mediating a number of these effects but confirmation of the importance of this receptor in this disease setting in vivo is still lacking. In contrast, evidence for a role for PAR2 in this syndrome is rapidly accumulating. Mast cell tryptase, a known PAR2 activator, has been used as a marker of allergic inflammation for many years. Tryptase inhibitors have been shown to be effective in animal models and, albeit to a lesser extent, in clinical trials. PAR2 protein expression by epithelial cells is further increased in asthmatic patients. Experimental evidence suggests several mechanisms by which activation of PAR2 might contribute to this syndrome. Similar to PAR1, activation of PAR2 in vitro leads to the release of potent proinflammatory mediators from a number of cell types present in the airway, and may therefore contribute to airway inflammation. This is consistent with the observation that the early inflammatory response to allergen inhalation is attenuated in PAR2-deficient mice. These mice also produce lower serum IgE levels, so it is possible that PAR2 may also play a role in the sensitization process. Of interest, PAR2 has been reported to trigger dendritic cell development suggesting a mechanism by which this receptor may influence this process in mice. The recent finding that the house dust mite allergens Der p1 (cysteine proteinase), Der p3 (serine proteinase), and Der p9 (serine proteinase) induce the release of the proinflammatory cytokines IL-6 and IL-8 from respiratory epithelial cells in a PAR2-dependent manner suggests another mechanism by which this receptor may contribute to the asthma syndrome. Finally, trypsin and PAR2 activators also influence airway smooth muscle contraction and relaxation in vitro but current in vivo evidence suggests that this receptor exerts a predominantly bronchoprotective/bronchodilator effect. PAR2 is

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therefore emerging as a receptor that may exert deleterious effects by contributing to airway inflammatory responses but that may also play important roles in airway homeostatic mechanisms. This will need to be borne in mind when targeting PAR2 for therapeutic intervention in any respiratory condition. Chronic Obstructive Pulmonary Disease

Tryptase levels are detectable in patients with COPD and in smokers, and intratracheal administration of trypsin to rats reproduces some of the cardinal features of COPD. Expression of PAR2 is increased in airway vessels of patients with bronchitis, raising the possibility that PAR2 may also be important in this disease setting. See also: Asthma: Overview. Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12). Chronic Obstructive Pulmonary Disease: Overview. Coagulation Cascade: Overview; Factor VII; Factor X; Thrombin. Connective Tissue Growth Factor. G-Protein-Coupled Receptors. Interstitial Lung Disease: Overview. Pulmonary Effects of Systemic Disease. Signal Transduction. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading Asokananthan N, Graham PT, Stewart DJ, et al. (2002) House dust mite allergens induce proinflammatory cytokines from respiratory epithelial cells: the cysteine protease allergen, Der p 1, activates protease-activated receptor (PAR)-2 and inactivates PAR-1. Journal of Immunology 169(8): 4572–4578. Chambers RC (2003) Proteinase-activated receptors and the pathophysiology of pulmonary fibrosis. Drug Development Research 60: 29–35.

Proteoglycans

Cocks TM, Fong B, Chow JM, et al. (1999) A protective role for protease-activated receptors in the airways. Nature 398: 156–160. Hernandez-Rodriguez NA, Cambrey AD, Harrison NK, et al. (1995) Role of thrombin in pulmonary fibrosis. Lancet 346: 1071–1073. Howell DCJ, Goldsack NR, Marshall RP, et al. (2001) Direct thrombin inhibition reduces lung collagen accumulation and connective tissue growth factor mRNA levels in bleomycin-induced pulmonary fibrosis. American Journal of Pathology 159: 1383–1395. Howell DCJ, Johns RH, Lasky JA, et al. Absence of proteinaseactivated receptor-1 signaling affords protection from bleomycin-induced lung inflammation and fibrosis. American Journal of Pathology 166: 1353–1365. Idell S (2003) Coagulation, fibrinolysis, and fibrin deposition in acute lung injury. Critical Care Medicine 31(supplement 4): S213–S220. Idell S, Gonzalez K, Bradford H, et al. (1987) Procoagulant activity in bronchoalveolar lavage in the adult respiratory distress syndrome. Contribution of tissue factor associated with factor VII. American Review of Respiratory Diseases 136: 1466–1474. Imokawa S, Sato A, Hayakawa H, et al. (1997) Tissue factor expression and fibrin deposition in the lungs of patients with idiopathic pulmonary fibrosis and systemic sclerosis. American Journal of Respiratory and Critical Care Medicine 156: 631–636. Knight DA, Lim S, Scaffidi AK, Roche N, Chung KF, Stewart GA, and Thompson PJ (2001) Protease-activated receptors in human airways: upregulation of PAR-2 in respiratory epithelium from patients with asthma. Journal of Allergy and Clinical Immunology 108: 797–803. Ossovskaya VS and Bunnett NW (2004) Protease-activated receptors: contribution to physiology and disease. Physiological Reviews 84: 579–621. Rasmussen UB, Vouret-Craviari V, Jallat S, et al. (1991) cDNA cloning and expression of a hamster alpha-thrombin receptor coupled to Ca2 þ mobilization. FEBS Letters 288(1 2): 123–128. Schmidlin F, Amadesi S, Dabbagh K, et al. (2002) Protease-activated receptor 2 mediates eosinophil infiltration and hyperreactivity in allergic inflammation of the airway. Journal of Immunology 169: 5315–5321. Vu TK, Hung DT, Wheaton VI, and Coughlin SR (1991) Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell 64(6): 1057–1068.

see Extracellular Matrix: Matrix Proteoglycans; Surface Proteoglycans.

PROTEOME G Westergren-Thorsson and G Marko-Varga, Lund University, Lund, Sweden J Malmstro¨m, Institute of Systems Biology, Seattle, WA, USA K Larsen, Lund University, Lund, Sweden & 2006 Elsevier Ltd. All rights reserved.

Abstract Proteomics can be defined as the studies of protein properties on a large scale to obtain a global view of biological processes at

the protein level. Essentially, proteomics requires protein separation and identification, and in many cases quantification. The cornerstones in proteomics are protein/peptide separation by gel electrophoresis and/or different chromatographic techniques, and identification by mass spectrometry followed by bioinformatic and biological interpretation of data. By combining these different separation techniques and mass spectrometry it is now possible to identify low abundant proteins with 10–1000 copies per cell. Today, substantial research efforts in proteome studies of the lung are focused on obtaining new diagnostic markers, as well as fingerprinting disease mechanisms.

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Introduction During the past 10 years, several new techniques such as cDNA microarray, yeast two-hybrid analysis, and mass spectrometry (MS) have been introduced that allow simultaneous high-throughput analysis of multiple mRNAs and proteins within the same sample. These technologies have received a great deal of attention and gradually begun to infiltrate biochemistry and cell biology laboratories. The term proteome was introduced for the first time in 1994 at the first Proteome meeting in Siena, Italy, and was used to describe the protein complement of a genome. Proteomics can be defined as ‘‘a large-scale study of protein properties, e.g., expression level, posttranscriptional modification

Proteomics

Separation

Bioinformatics

MS

Data analysis

Biological readouts Figure 1 The main components essential for proteome analysis.

and protein interaction, in order to obtain a global view of disease processes or cellular processes at the protein level.’’ Three strategies have had a strong impact in the field of biology: (1) the generation of protein–protein linkage maps; (2) the annotation of genomic DNA sequences by generation of MS/MS peptide sequences; and (3) the measurement of protein expression by quantitative methods. The data output from a typical proteomics experiment is huge and therefore computer-based data storage and analysis is required. Essentially, proteomics is based on protein separation, identification, and data analysis followed by biological readouts (Figure 1). Proteomics have a great potential to give rise to novel discoveries and to generate new testable hypotheses by choosing the appropriate study design. Therefore, different types of separation techniques as well as MS will be discussed in more detail below along with some recent proteome findings relevant for the lung.

Protein/Peptide Separation Originally, protein separation was performed by twodimensional gel electrophoresis (2-DE) as described by O’Farrel in 1976 and Klose in 1975, and was for many years the most efficient way of separating proteins in complex mixtures. 2-DE is dependent on protein separation by isoelectric focusing and molecular weight (Figure 2(a)). Spots of interest are excised and identified with peptide mass fingerprinting by matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI–TOF MS). By

5−100 kDa

pH 4−7 pI 3−10 NL Mol.wt 90−95 kDa A

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Control (a)

TGF- (b)

Figure 2 Example of 2D gel electrophoresis on cell lysate from control and TGF-b-treated fibroblast cultures. (a) Solubilized cells were applied on 4.7–7 NL pI strips and separated on 14% acrylamide gels. The spots were visualized by silver staining. The enlarged area from the lower part of the gel shows two spots, which were affected by TGF-b treatment compared to the control. (b) The separation of proteins was compared between different pI intervals. Proteins of specific interest in a certain pH interval were better separated in a narrower pI interval such as 4.5–5.5 compared to 3–10.

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using image analysis, it is possible to quantify the relative protein expression and thereby enable monitoring of protein expression over time or allow comparison between cellular or clinical material (Figure 2(a)). By using zoom gel electrophoresis with a narrower pI interval such as 4.5–5.5 compared to 3–10, it is possible to get a better separation of proteins of specific interest (Figure 2(b)). Although 2-DE has been the method of choice for many years, it has a number of drawbacks regarding difficulties to automatize sensitivity and separation of large proteins, glycosylated proteins, and membrane proteins. Several of these limitations are avoided when chromatographic-based separation techniques are used. The coupling of high-pressure liquid chromatography (HPLC) with MS has proven to be an alternative method to 2-D gels. Examples of such separation techniques are two-dimensional (strong-cationic exchange (SCR)/reverse phase (RP)) or three-dimensional (SCR/avidin affinity/RP) chromatographic separations of peptide mixtures generated by tryptic digestion of protein samples.

Sample plate

Detector Laser

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Mass Spectrometry (MS) Important technological advances have occurred in the field of mass spectrometry. Research on the ionization principles used today was awarded the Nobel Prize in Chemistry in 2002. A mass spectrometer consists of an ion source, a mass analyzer that measures the mass-to-charge ratio (m/z) of the ionized analytes, and a detector that registers the number of ions at each m/z value. Today, the ionization methods of choice are electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). Both methods are commonly used to volatize and ionize the protein or peptides for mass spectrometric analysis (Figures 3(a) and 3(b)). In MALDI, the peptides are embedded in a crystal matrix and ionized by a laser pulse. In ESI, the ionization occurs in solution and therefore is mainly used when coupled to chromatographic liquid-based separation. There are four basic types of mass analyzers currently used in proteomics research: ion-trap, time of flight (TOF), quadruple, and Fourier transform ion cyclotron (FT-MS) analyzers. In proteome analysis, it is of particular importance that the mass analyzers are sensitive, have high resolution and mass accuracy, and that they can generate ion-rich mass spectra from peptide fragments (tandem MS/MS spectra). In tandem MS/MS, the charged peptides are separated in the first MS according to their m/z ratio to create a list of the most intense peptide peaks. In the second analysis, the instrument is adjusted to select only a specific m/z and direct this peptide into the collision cell. By

(c) Figure 3 Schematic pictures of two types of mass spectrometer. (a) A matrix-assisted laser desorption/ionization–time of flight (MALDI–TOF) mass spectrometer; (b) a quadruple (Q) TOF instrument with electrospray ionization (ESI).

using the appropriate collision energy, fragmentation occurs predominantly at the peptide bond, generating daughter ions representing a ladder of fragments, each of which differs by the mass of a single amino acid. MALDI-TOF-TOF instruments characterize the sequences of peptides by using a collision cell between the two mass analyzers (Figure 3(c)).

Combined Strategies of Protein Separation and Quantitative MS The developments in the field of mass spectrometry over the past years have resulted in the identification of more than 2000 expressed proteins and the mapping of posttranslational modifications. An important step was when Hunt and colleagues pioneered the use of LC-MS/MS for the analysis of complex peptide mixtures, which is today at the core of MS-based proteomics. The combination of several protein/peptide separation steps based on different analytical properties is capable of detecting proteins of very low abundance, although considerable effort is required and a sufficient amount of sample material must be available. Relative quantification is possible by introducing stable isotopes via metabolic labeling of amino acids

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through enzymatic transfer of 18O from water to peptides or via chemical reactions using isotopecoded affinity tags or similar reagents. The most promising and well-developed method is based on pairs of isotope-coded affinity tags (ICAT), developed by Gygi and co-workers in 1999. The ICAT reagent contains a biotin moiety and a linker region with nine deuterium or hydrogen atoms. The reagent labels cysteine-containing peptides, which are enriched by binding the biotin tag to streptavidin. The tag introduces a mass shift in one of the samples by 9 Da, which can then be analyzed by dual mass spectrometry in MS mode for quantification and in MS/MS mode for protein identification. The ratio of the signal intensities can then accurately indicate the abundance ratio for the two peptides. Stable isotope dilution LC-MS/MS is commonly used to detect changes in quantitative protein profiles accurately.

Bioinformatics A common denominator of all proteomic approaches is the large amount of data that needs to be collected, making data analysis a difficult task. To enable analysis of these data, a bioinformatics approach is necessary, the main problems of which are the management, storage, and visualization of data and to draw accurate biological conclusions from the data. Computer-based storage, organization, and annotation are therefore essential to process and analyze these large datasets, especially when combining multiple experiments. To extract and compile the data into a list of identified proteins there is a need to electronically store and search within the data and to develop the data using statistical principles. Storage and compilation of these redundant data sets can be accomplished by relational databases that store the independent data in separate tables and, upon request, combine the data at desired levels.

Gene Ontology Once the data is made electronically searchable, the next daunting task becomes apparent – to be able to draw biological conclusions from the data. Even though protein sequences are easily stored and analyzed in computers, the same is not true for protein function and protein structure. To alleviate this problem, a number of classifications have been developed. One classification scheme that has recently received much attention is the Gene Ontology Project in which biological function is described by a set of defined terms organized in a hierarchical structure. Of primary importance is to reduce the complexity of the data by narrowing the number of entries

through intelligent clustering of the data in hierarchical levels. The use of public databases and software tools is essential in this process. This enables correct categorizing of the dataset, highlighting protein groups that seem to give similar matches to the perturbed state. From this, new hypotheses can be postulated and addressed, either by additional proteomic experiments or by functional validation.

Application of Proteomics in Respiratory Diseases To date, most studies related to diseases of the lung have used different body fluids such as blood, urine, and nasal lavage samples, but the most interesting results have been obtained with broncheoalveolar lavage fluid (BALF) and pulmonary edema fluid. Using animal experiments where animals are subjected to various challenges also gives relevant information. In addition, specific cell systems linked to respiratory diseases have been analyzed with and without specific perturbations. A general approach on plasma and urine proteomes has given promising results comparing controls and patients with different lung cancers. Using 1-D and 2-D mLC-MS/MS on albumin and IgG-depleted serum did enable the reliable identification of more than 100 proteins, where 16 were specific for lung adenocarcinoma. However, to reach clinical significance a greater number of patients must be analyzed. In urine, general markers for lung cancer using 2-D gels and MALDI-TOF-MS/MS indicated that proteins such as CD59 glycoprotein precursor and transthyretin may be useful markers for cancer. The use of BALF is very informative concerning several disorders of the lung such as fibrosing interstitial lung diseases, asthma, and chronic obstructive pulmonary disease (COPD). BALF has a very complex structure as proteins originate from many different cells and tissue compartments. It is very important to optimize the sample preparation regarding concentration and removal of albumin. Most identified proteins obtained by 2D-gel electrophoreses and various MS-methods are serum proteins. Interestingly, a large group of identified nonserum proteins contains those involved in immunological responses and inflammation. Other groups contain tissue repair and proliferation proteins and another contains cytoskeletal proteins. The group consisting of antioxidant proteins is also prominent. This field of research is very promising and is expected to give a lot of new basic information concerning processes of the epithelium, underlying extracellular matrix, mucus-producing cells, macrophages, and other inflammatory cells in the bronchial trees. However, the complexity

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is so great that it is unlikely that individual proteins will be suitable as markers for disease processes. Rather, sets of proteins will be useful for providing information of physiological, pathophysiological, and clinical concern. All these studies indicate that the generation of adequate results with proteomics requires well-designed experiments and well-characterized sample materials. This is especially important with clinical material where the biological diversity is large. By combining various separation techniques and MS approaches different scientific questions can be answered. A good example of study design to obtain biomarkers of specific common cell types or differentiation of cells involved in the inflammatory process is to use a global system with 2-DE followed by MALDI-TOF; this is particularly successful in identifying highly abundant proteins (10 000 copies per cell) such as cytoskeletal, scavenger, metabolic, and adhesion proteins. This approach has been used by several groups, where factors such as endothelin, transforming growth factor beta (TGF-b), and platelet-derived growth factor (PDGF) have been added to human fibroblast cell cultures to mimic remodeling processes that occur in the lung in several respiratory diseases. Related identifications as previously described have also been made in studies on BALF or cell cultures established from lung biopsies from control patients with different lung disorders like COPD, idiopathic pulmonary fibrosis (IPF), and asthma. By using nongel-based quantitative proteomic techniques involving liquid chromatography and MS/MS and protein labeling with ICAT, it is possible to identify proteins present at low levels (a few copies per cell) such as transcription and splicing factors. By using this technology it has been possible to gain insights into the role of TGF-b in splicing of mRNA. TGF-b specifically regulates splicing factors that are involved in exon inclusion of EDA in fibronectin, which is required for the differentiation of a fibroblast into a more activated type of fibroblast called a myofibroblast. Another important protein that has been associated with remodeling processes of extracellular matrix in asthma is ADAM33, a disintegrin metalloprotease that is also spliced; TGF-b might have a regulatory role in the splicing process. Thanks to the methods described above, today we have over 2000 identities of the human pulmonary fibroblast proteome, of both constituently and regulatory type. The future task is to determine the proteome directly on small biopsies taken after bronchoscopy or surgery of patients with respiratory disorders. This could be achieved with laser capture

microdissection (LCM), where the proteome could be determined from histological preparations taken from biopsies by using lasers to cut out specific regions. However, only very abundant proteins have been identified by this approach so far. The most challenging part of proteomics may be to answer scientific questions that have not been asked as yet, which gives these methods a high potential to generate new discoveries and hypotheses. Studies of the proteome so far have expanded our view of protein expression. In view of the rapid development of the techniques described above, it is likely that in the future there will be new insights into the lung proteome and pathobiology of respiratory diseases, provided well-controlled clinical material is available. See also: Bronchoalveolar Lavage. Fibroblasts.

Further Reading Aebersold R and Goodlett DR (2001) Mass spectrometry in proteomics. Chemical Review 101: 269–295. Fehninger TE, Sato-Folatre JG, Malmstro¨m J, et al. (2004) Exploring the context of the lung protoeome within the airway mucosa following allergen challenge. Journal of the Proteome Research 3: 307–320. Fujii K, Nakano T, Kanazawa M, et al. Clinical-scale highthroughput human plasma proteome analysis: lung adenocarcinoma. Proteomics (in press). Gygi SP, Rist B, Gerber SA, et al. (1999) Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nature Biotechnology 17: 994–999. Hirsch J, Hansen KC, Burlingame AL, and Matthay MA (2004) Proteomics: current techniques and potential applications to lung disease. American Journal of Physiology. Lung Cellular and Molecular Physiology 287: L1–L23. Krutchinsky AN, Kalkum M, and Chait BT (2001) Automatic identification of proteins with a MALDI–quadrupole ion trap mass spectrometer. Analytical Chemistry 73: 5066–5077. Malmstro¨m J, Larsen K, Hansson L, et al. (2002) Proteoglycan and proteome profiling of human pulmonary fibrotic tissue utilizing miniaturized sample preparation: a feasibility study. Proteomics 2(4): 394–404. Malmstro¨m J, Larsen K, Malmstro¨m L, et al. (2004) Proteome annotations and identifications of the human pulmonary fibroblast. Journal of Proteome Research 3: 525–537. Malmstro¨m J, Tufvesson E, Bjermer L, Marko-Varga G, and Westergren-Thorsson G (2004) Remodelling of connective tissue during pathological conditions in the lung. Respiratory Medicine 18: 1–8. Malmstro¨m L, Malmstro¨m J, Marko-Varga G, and WestergrenThorsson G (2002) Proteomic 2DE database for spot selection, automated annotation, and data analysis. Journal of Proteome Research 1: 135–138. Pandey A and Mann M (2000) Proteomics to study genes and genomes. Nature 405: 837–846. Plymoth A, Lofdahl CG, Ekberg-Jansson A, et al. (2003) Human bronchoalveolar lavage: biofluid analysis with special emphasis on sample preparation. Proteomics 3: 962–972.

532 PULMONARY ALVEOLAR MICROLITHIASIS Shiio Y, Donohoe S, Yi EC, et al. (2002) Quantitative proteomic analysis of Myc oncoprotein function. EMBO Journal 21: 5088–5096. Wattiez R and Falmagne P (2005) Proteomics of bronchoalveolar lavage fluid. Journal of Chromatography B. Analytical and Technological Biomedical Life Sciences 815: 169–178.

Relevant Website http://www.geneontology.org – Gene Ontology Project website where biological function is described by a set of defined terms organized in a hierarchical structure.

PULMONARY ALVEOLAR MICROLITHIASIS G Castellana and V Lamorgese, Unita` Operativa di Pneumologia, Bari, Italy & 2006 Elsevier Ltd. All rights reserved.

Abstract Pulmonary alveolar microlithiasis (PAM) is a rare chronic disease of unknown etiology and pathogenesis, characterized by the widespread presence in the alveoli of minute calcific deposits known as microliths. The deposits are probably due to an inherited defect with autosomal recessive transmission. The disease is present worldwide and a recent review of the literature counted 576 cases. It has been most frequently reported in Europe, followed by Asia. The nation with the highest number of reported cases is Turkey, followed by Italy in second place. Cases of PAM are defined as (1) ‘familial’ when two or three, or in exceptional cases even four or five siblings are affected, and (2) ‘sporadic’ when screening of the rest of the family yields negative results. In most cases patients have mild clinical symptoms, contrasting with the severe radiographic appearance: these are characteristic features that should raise the suspicion of PAM. High-resolution computed tomography (HRCT) has made it possible to define the extent and severity of the disease more precisely. Bronchoalveolar lavage (BAL) and transbronchial biopsy (TBB) confirm the presence of microliths in the alveoli. Symptomatic cases generally feature the presence of effort dyspnea, a cough, chest pain, asthenia, finger clubbing, crepitations, and a restrictive defect revealed by lung function assessment. Macroscopic findings include markedly increased lung weight. The disease is initially endoalveolar, then involvement of the interalveolar tissue begins to appear and subsequently progression is observed, with the development of pleural calcifications, emphysema bubbles, and ossification nodules. Treatment is at present only palliative. Steroids have proved to be inefficacious while the results of administration of sodium etidronate and the BAL technique are controversial. Affected individuals may undergo progression to end-stage disease requiring lung transplantation.

Introduction Pulmonary alveolar microlithiasis (PAM) is a rare idiopathic disease characterized by the diffusion in the alveoli of innumerable minute calculi called microliths (see Figure 1). It comes under the general heading of dysmetabolic elective thesaurotic pneumoalveolitis.

Figure 1 Chest radiograph of a patient affected by PAM, showing fine microliths with a diffuse, uniform spread obscuring the cardiac and diaphragmatic borders (sandstorm lung).

The first to draw a concise, precise macroscopic description of the disease was an Italian researcher, Malpighi, in 1686, who wrote: ‘‘In vesciculis pulmonum innumeri lapilli sunt.’’ Much later, in 1918, a Norwegian, Harbitz, provided an accurate autoptic and radiological description of a second case. The third case was reported by a German, Schildknecht, in 1932, and in 1933 a Hungarian pathologist, Puhr, coined the name of PAM for the disease. Another Italian, Mariani, highlighted the clinical, functional, and radiological features in 1947. The first to report multiple cases in a single family was Mickailov from Bulgaria, in 1954. Since then, numerous reports have been made of individual and familial case histories or of small series of patients with the disease. Sosman, a radiologist, published a definitive description of the disease in 1957 and the first world review of 45 cases of PAM, extended by Perosa and Ramunni to 74 cases in 1959. The most recent worldwide review counted 576 cases. PAM is present in all continents with no clear geographic or racial distribution, although it is most prevalent in Europe, followed by Asia, particularly

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civilian purposes (military service) or health reasons (a higher frequency of diseases with respiratory involvement). The exceptional recent finding of analogous microliths outside the lungs, in the seminal vesicles in two patients and in the testicles in another one, raises the suspicion that the underlying metabolic disorder could also be localized in the male genital apparatus. Although cases of PAM have been described in all age groups, they are most frequently discovered in patients under 40 years of age. The youngest reported cases were premature twins and two newborns, while the most aged elderly case involved an 80-year old. Figure 2 shows the percentage of world cases of PAM subdivided by continents and Figure 3 by age at diagnosis.

Asia Minor. The nation with the highest number of recorded cases is Turkey, followed in order of prevalence by Italy, the US, India, Russia, Germany, Spain, Japan. The percentage of males versus females affected by the disease appears to be practically identical. Cases of PAM are defined as (1) ‘familial’ when two, three, or exceptionally even four, five, or six family members are affected and (2) ‘sporadic’ when screening of the family yields negative results. Sporadic cases have been found to be predominant in the male sex, whereas familial cases seem to develop more commonly in the female members. However, the higher male incidence in sporadic cases could perhaps be ascribed to more frequent X-ray examinations performed in male patients for

40 35 30 25 20 15 10 5 0 Europe

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South America

Oceania

Figure 2 Percentage of world cases of PAM subdivided by continent.

18 16 14 12 10 8 6 4 2 0 0–12

13–20

21–30

31–40

41–50

51–60

Figure 3 Percentage of world cases of PAM subdivided by age at diagnosis.

61–70

71–80 Unspecified

534 PULMONARY ALVEOLAR MICROLITHIASIS

The disease does not affect only humans and has been described in orangutans, dogs, sheep, cats, and nude mice.

Etiology and Pathogenesis The etiology and pathogenesis are still unknown. Among those postulated, the most generally accepted etiology is an inherited metabolic abnormality, limited to the alveolar surface, involving the enzyme carbonic anhydrase. This promotes alkalinity of the alveolar surface, and hence a precipitation of calcareous salts. Research has also been made to ascertain whether a defect in calcium regulation could be responsible for the disease, but the few studies of calcium metabolism conducted in patients with PAM were unable to provide confirmation of this hypothesis. In the past, a controversy arose between the ‘nature’ and ‘nurture’ theories. One school of thought supported a hereditary disease with autosomal recessive transmission, since in familial cases the heredity is of horizontal type, in the sense that it affects members of the same sibship. The other school supported an environmental etiology, according to which the disease might be due to inhalation of given substances, as has been clearly documented in some cases. Nowadays, the consensus is that the disease is of inherited type but that exogenous factors can facilitate the formation of the microliths in the lungs. In fact, numerous family histories have been documented: family cases account for 36.56% of the total PAM cases. Horizontal sibling affection is the norm, although in two family groups first cousins were involved. Exceptionally, horizontal and vertical sibship was reported in two family groups, one featuring five patients – two sisters and three children of one of them – and the other, a woman, her brother and his child. In several family cases the parents were cousins. Two important reports supporting the hereditary hypothesis have recently been published, describing two particular family groups, one in Italy and one in Turkey, in which cases of PAM were present in several generations.

Pathology At autopsy, macroscopic findings include markedly increased lung weight, reaching maximum values of about 5 kg. The lungs are described as dark red, with a rigid consistency and granular surface, or with whitish striations. They always feature a much greater density than normal, and sometimes have a wood-hard or stone-like consistency. They are

Figure 4 Electron microscopy image of a microlith.

difficult to incise, and the operation provokes a strident noise. The cut edge is dark red and shows a uniform dissemination of grayish, shiny granules the size of pinheads: it has the appearance of sandpaper or a surface covered by a fine layer of sand. Histological findings consist of the presence of circular calcific formations (microliths), with a diameter ranging from 30 to 500 mm, inside the alveoli. Their lamellar, concentric surface features a compact central portion surrounded by successive layers arranged concentrically like an onion skin. As much as 4/5 of the alveolar surface may be involved in the microlithic process (see Figure 4). Generally, there is one microlith per alveolus that takes the shape of the cavity without deforming it. Instead, larger microliths deform the alveolus and compress the interalveolar septa, which become atrophied. The size of the microliths depends on the time since formation; smaller elements at the start of their evolution do not contain calcium deposits. Larger microliths have a very irregular, striated shape, probably resulting from fusion of smaller elements. Initial cases show a surprisingly intact lung parenchyma: in fact, the disease is initially endoalveolar and then progression is observed, with the development of pleural calcifications, emphysema bubbles, and ossification nodules. Microliths have also been observed in the interalveolar tissue, bronchioles, and lymph nodes or encapsulated in a bone tissue mass. The interalveolar septa are described as normal in initial cases but thickened in advanced cases. Several reports of more complete histochemical studies have been made, attempting to explain the pathogenesis of the disease: they have shown that the central nucleus consists of protein, neutral

PULMONARY ALVEOLAR MICROLITHIASIS 535

mucoprotein, and acid mucopolysaccharides. Contrasting results on the chemical composition of the lamellae have been obtained in different studies. Instead, there is a general consensus that the calcium content accounts for 70% of the inorganic component, while the calcium salt is hydroxyapatite. Some traces of aluminum, magnesium, iron, and silicon have also been reported.

Radiology The radiological aspects of the disease have been amply described. With the most recent technologies (e.g., computed tomography (CT), high-resolution computed tomography (HRCT), and magnetic resonance imaging (MRI)), these aspects can now be studied in greater detail. Superficial assessment of the radiological findings in patients with PAM can elicit difficulties in differential diagnosis with other diseases associated with miliary dissemination such as tuberculosis, mycosis, sarcoidosis, hemosiderosis, pneumoconiosis, and amyloidosis, which can present with diffuse opacifications, albeit with more severe symptoms. Due to the intense calcifications present in PAM, 3–5 times stronger X-ray exposure than normal is needed to obtain good radiograms demonstrating the typical picture of the disease. The radiological aspects and their evolution can be subdivided into four phases. In the early stage of the disease, known as precalcific, the radiological aspects are not yet typical due to the small number and lesser calcification of the microliths. This PAM model may fail to be recognized, also because it is occasionally observed in asymptomatic children. The second phase already shows the typical radiological picture: the lungs appear ‘sandy’, featuring diffuse, scattered micronodules with a diameter less than 1 mm and the typical calcific opacity. The microliths are typically clear-cut, shiny, with a uniform size and distributed throughout the pulmonary zones, although there tends to be greater density in the medial and inferior regions. The overall appearance resembles that of sandpaper, but the outlines of the heart and diaphragm are still clearly visible. This second calcification model is generally present in cases discovered in childhood or adolescence. By the third phase the number and volume of the opacifications have increased. The picture has become more granular, nodular, and confused due to initial thickening of the interstitial weave, that partly masks the micronodules. In the medial and inferior fields, superimposed opacifications hide the outlines of the heart and diaphragm. This third radiological model is more often seen in young adults.

By the fourth phase, the number and size of the calcific deposits have grown still more, and there is intense calcification of the interstices and sometimes pleural serosa, making the lungs appear almost entirely opaque; there is an overall aspect of ‘white lungs’ due to the diffuse presence of calcification, although the apical regions may be partially spared. Apart from interstitial calcific fibrosis, there may be paraseptal emphysema, large bubbles, or air cysts in the superior lobes, as well as pneumothorax and zones of ossification. This fourth radiological model is generally seen in older adults, or in any case in advanced cases. The extent and severity of PAM generally depend on the patient’s age and the speed of progression of the disease. Serial controls do not necessarily reveal constant deterioration of the radiological picture and the disease may remain stationary for many years. HRCT is best able to reveal the sandpaper aspect, expressing alveolitis, and smaller calcific deposits. It can also show interstitial calcification along the bronchovascular fascia, in the interlobular septa and subpleural site, especially in the dorsal and inferior regions of the parenchyma. The diffuse presence of calcification in the different pulmonary structures can be visualized by HRCT, which can demonstrate the characteristic overall appearance of ‘shiny lung’ or ‘stony lung’ (see Figure 5). As the microliths become confluent, the typical alveolar aspect of the disease with patterns of pervious bronchi surrounded by microliths becomes more and more unmistakable. HRCT is a more powerful tool than radiography and conventional CT for assessing the extent, degree of evolution and severity of the disease, and especially involvement of the secondary lobule,

Figure 5 CT scan with diffuse, ground-glass opacities (stony lung) in all pulmonary fields, and faint calcific densities, sometimes confluent at the posterior and inferior subpleural regions.

536 PULMONARY ALVEOLAR MICROLITHIASIS

pulmonary fibrosis, and the presence of pleural calcification. Moreover, HRCT can help to identify initial cases, can monitor evolution of the disease, and can replace lung biopsy because the morphological appearance delineated by this examination is sufficient for a firm diagnosis of PAM to be made.

Symptoms and Clinical Course In the cases reported between 1950 and 1960, patients affected by PAM were observed only when conditions of respiratory insufficiency had already developed, and diagnosis was most often made at autopsy. Today, approximately 50% of cases are identified when few or no symptoms have yet developed, and the findings come as a surprise in subjects undergoing chest X-rays for other reasons: checkups for military service or employment, or to exclude tuberculosis, or else for preoperative assessment or work-up for other organ disease. The disease is then confirmed by the presence of microliths in the bronchoalveolar lavage (BAL), by transbronchial biopsy (TBB), CT, bone scintigraphy with Tc99. The best diagnostic schedule for PAM is the association of BAL and HRCT; the former can document the diagnosis, whereas the latter provides further information about the degree of inflammation and/or fibrosis or calcification of the interstices. This association avoids the need for TBB, a more invasive investigation burdened by a higher complication rate. Nevertheless, if there is already a known case in the family, standard radiography of the chest and HRCT is quite sufficient in any other family member with the suspicion of the disease. In view of the rarity of the disease, after discovery of a single case, it would be helpful to screen the whole family, in the hope that in very early cases the use of therapeutic BAL could yield more promising results, analogous to those obtained in alveolar proteinosis. Regular follow-up can monitor the clinical course, demonstrating the speed of progression of the disease. In symptomatic patients the most common complaints are dyspnea, followed by cough without sputum, chest pain, and asthenia. In advanced disease there is chronic cor pulmonale, respiratory failure, and finger clubbing. Owing to the discrepancy between the severe radiological picture, showing diffuse multiple lesions in both lungs, and the relatively minor clinical symptoms, it is often difficult to pinpoint the chronological order characterizing the first phases of accumulation of the calcific deposits in the alveoli, and the clinical picture at onset and evolution of the disease. Most of the descriptions of this disease have

been case reports or case series, or some isolated reports underlining particular features. However, in five cases reported in the literature it was possible to reconstruct the time of onset of the disease, thanks to the available documentation consisting of X-rays taken some years before onset, demonstrating absence of any radiological abnormalities. Thus, the disease was not present in these patients from birth, although there have been exceptional reports of PAM in newborns and fetuses. Nevertheless, in most cases it was not possible to establish how long before, in terms of months or years, the disease started to develop. Functional assessment has most frequently shown a restrictive syndrome, whereas in the few reported initial cases the spirometric findings were generally normal.

Therapy In many cases prognosis is good: a number of reports describe patients followed up for many years during which the clinical and radiographic pictures progressed very slowly. The longest-lived patient was followed up for 50 years after diagnosis. However, less frequently the disease can evolve rapidly leading to cor pulmonale and respiratory failure. Several attempts have been made to treat this disorder, but with unsatisfactory results. Steroids were ineffective while BAL removes microliths with smaller diameters than the caliber of the alveolar ducta, but is inefficacious against intra-alveolar microliths just one size larger. Conflicting results have also been described for sodium etidronate administration. Affected individuals may progress to endstage lung disease requiring lung transplantation. There are no data currently available as to whether the disease ever recurs in patients who underwent lung transplant for this condition, as follow-up of these patients has never been reported. We believe more research is needed to find a parallel between a calcium–phosphorus metabolism disorder of the lung and the etiology of the disease. Clearly, only after its etiology and pathogenesis have been fully established may effective treatment strategies be devised. See also: Bronchoalveolar Lavage. Extracellular Matrix: Collagens. Signs of Respiratory Disease: Clubbing and Hypertrophic Osteoarthropathy.

Further Reading Arslan A, Yalin T, Akan H, and Belet U (1996) Pulmonary alveolar microlithiasis associated with calcifications in the seminal vesicles. Journal of Belge Radiologie 79: 118–119.

PULMONARY CIRCULATION 537 Castellana G, Castellana R, Fanelli C, Lamorgese V, and Florio C (2003) La microlitiasi alveolare polmonare: decorso clinico e radiologico, convenzionale e HRCT, in tre casi. Ipotesi di classificazione radiologica della malattia. La Radiologia Medica 106: 160–168. Castellana G, Gentile M, Castellana R, Fiorente F, and Lamorgese V (2002) Pulmonary alveolar microlithiasis: clinical features, evolution of the phenotype, and review of the literature. American Journal of Medical Genetics 111: 220–224. Castellana G and Lamorgese V (1997) La microlitiasi endoalveolare polmonare. Caso clinico a sostegno dell’ipotesi ereditaria. Rassegna Di Patologia Dell’Apparato Respiratorio 12: 247–251. Castellana G and Lamorgese V (1998) La microlitiasi alveolare polmonare: rivisitazione della casistica italiana. Rassegna Di Patologia Dell’Apparato Respiratorio 13: 405–407. Castellana G and Lamorgese V (2003) Pulmonary alveolar microlithiasis. World cases and review of the literature. Respiration 70: 549–555. Chang YC, Yang PC, Luh KT, Tsang YM, and Su CT (1999) High-resolution computed tomography of pulmonary alveolar microlithiasis. Journal of Formosan Medical Association 98: 440–443. Chinachoti N and Tangchai P (1957) Pulmonary alveolar microlithiasis associated with inhalation of snuff in Thailand. Diseases of the Chest 32: 687–689.

Edelman JD, Bavaria J, Kiaser LR, et al. (1997) Bilateral sequential lung transplantation for pulmonary alveolar microlithiasis. Chest 112: 1140–1144. Mariotta S, Ricci A, Papale M, et al. (2004) Pulmonary alveolar microlithiasis: report on 576 cases published in the literature. Sarcoidosis, Vasculitis, and Diffuse Lung Diseases 21: 173–181. Moran CA, Hochholzer L, Hasleton PS, Johnson FB, and Koss MN (1997) Pulmonary alveolar microlithiasis. A clinicopathologic and chemical analysis of seven cases. Archives of Pathology & Laboratory Medicine 121: 607–611. Perosa L and Ramunni M (1959) La microlitiasi endoalveolare del polmone. Recenti Progressi in Medicina 26: 353–429. Senyigit A, Yaramis A, Gurkan F, et al. (2001) Pulmonary alveolar microlithiasis: a rare familial inheritance with report of six cases in a family. Respiration 68: 204–209. Sosman MC, Dodd GD, Jones WD, and Pillmore GU (1957) The familial occurrence of pulmonary alveolar microlithiasis. American Journal of Roentgenology 77: 947–1012. Sosman MC, Dodd GD, Jones WD, and Pillmore GU (2004) The familial occurrence of pulmonary alveolar microlithiasis. Lung Disease 21: 173–181. Ucan ES, Keyf AI, Aydilek R, et al. (1993) Pulmonary alveolar microlithiasis: review of Turkish reports. Thorax 48: 171–173.

PULMONARY CIRCULATION L A Shimoda, Johns Hopkins School of Medicine, Baltimore, MD, USA

Anatomy, Structure, Histology of the Pulmonary Circulation


Abstract The pulmonary vasculature is unique, both in volume and function. The pulmonary circulation, a low-pressure vascular bed that accommodates the entire cardiac output, carries the mixed venous blood to the alveoli, where gas exchange occurs, and then back to the left heart for distribution of oxygenated blood to the rest of the tissues in the body. As compared with the systemic circulation, the pulmonary arteries have thinner walls with much less vascular smooth muscle. Moreover, in order to maintain the low pulmonary arterial pressure, normal pulmonary vascular resistance is approximately one-tenth that of the systemic circulation. Factors that control pulmonary blood flow include vascular structure, gravity, mechanical effects of breathing, and the influence of neural and humoral factors. A unique aspect of the pulmonary circulation is the pressor response to hypoxia, as the systemic circulation dilates in response to decreased oxygen concentrations. In addition to gas exchange, the pulmonary circulation also serves to filter the blood, removing microemboli, and participates in the metabolic regulation of a variety of vasoactive hormones. Several diseases can affect the function of the pulmonary circulation, including primary and secondary pulmonary hypertension, arteriovenous malformation, embolism, and fibrotic lung disease.

The pulmonary circulation encompasses the circuit by which deoxygenated blood from the right heart enters the lungs via the pulmonary arteries, is channeled through the alveolar/capillary units where the blood is oxygenated, and is returned to the left side of the heart by the pulmonary veins for distribution to the systemic circulation. The anatomy of the pulmonary circulation differs in several respects from that of the systemic circulation. Blood exits the right ventricle into the main pulmonary artery, the diameter of which is similar to that of the aorta, but it has thinner walls. During gestation and immediately following birth, the pulmonary artery is nearly identical to that of the aorta, but, postnatally, the elastic tissue gradually diminishes. The main pulmonary artery divides into the right and left main branches. Each main branch further divides to supply each lobe before entering the lung. Within the lung, each lobar artery subdivides into rather irregular branches corresponding to the bronchial tree. The close proximity of the pulmonary arteries and airways underscores the relationship

538 PULMONARY CIRCULATION

between ventilation and perfusion that defines the normal function of the lung. The large pulmonary arteries (43000 mm in diameter) are classified as elastic, with the media comprised primarily of elastic fibers and some smooth muscle. As vascular diameter decreases these elastic arteries gradually give rise to vessels with increased smooth muscle content. In general, arteries between 3000 and 150 mm in diameter can be considered muscular arteries, but are still more thin walled than systemic arteries of the same diameter. Pulmonary arteries also exhibit a thin intima comprised of endothelial cells, collagen, and fibroblasts and a longitudinal elastic lamina, which allows for expansion during inspiration. The small arterioles have a nonuniform smooth muscle cell layer, giving way to the small nonmuscular preacinar arterioles, which are located proximal to terminal bronchi. At the alveoli, the terminal arterioles break into a network of pulmonary capillaries within the alveolar walls. The capillaries have a very thin wall (approximately 2 mm) consisting of a single layer of endothelial cells and contain most of the surface area of the pulmonary vasculature. Indeed, the maximal surface area of the capillary network is approximately 20 times that of the rest of the pulmonary circulation. Gas exchange between the alveolar gas and blood takes place within the pulmonary capillary bed after which the blood flows into venules, which are indistinguishable in structure from arterioles. However, while each small arteriole supplies a specific unit of respiratory tissue, the venules drain several portions of the lung. Venules do not follow the bronchial tree and unite to form the pulmonary veins, which conduct the oxygenated blood into the left ventricle. Although 99% of the lung blood flow passes through the pulmonary circulation, 1% is carried by the bronchial circulation, which supplies oxygenated blood from the systemic circulation to the lung. Similar to the pulmonary circulation, branching of the intrapulmonary bronchial arteries along the length of the bronchial tree results in a vast network of capillaries, which form extensive anastomoses with the pulmonary vasculature. Owing to these multiple connections with the pulmonary circulation, the deoxygenated bronchial venous blood drains via pulmonary veins to the left heart, producing an anatomic right-to-left shunt. The small pulmonary vessels, including the capillaries, can be subdivided into extra-alveolar or alveolar based on the pressures to which they are subjected. In addition to the intravascular pressure, vessels in the lung are exposed to alveolar pressure and pressure exerted by lung tissue connections. Both of these forces increase with lung inflation; however,

they act in opposite directions, with alveolar pressure directed inward and tissue pressure directed outward. The alveolar vessels are surrounded by alveolar pressure, due to localization within the septal wall. The extra-alveolar vessels are surrounded by septa and are typically contained in a sheath of connective tissue. In addition to the effects of alveolar pressure, the connection of these vessels to lung parenchyma subjects them to substantial tissue forces. These vessels include a subset of the pulmonary capillaries called the corner vessels, which are located in the alveolar parenchyma at the alveolar corners.

Pulmonary Circulation in Normal Lung Function Physiological Function

The pulmonary vasculature is unique, both in capacity and function. Responsible for several physiological functions, the primary function of the pulmonary circulation is exchange of gases, adding oxygen and removing carbon dioxide from mixed venous blood. Under normal conditions, gas exchange occurs primarily in the alveolar capillaries, where blood flow rapidly equilibrates with the alveolar air. The average transit time for red blood cells within the capillary network is approximately 0.5–1 s. Since the volume of the capillary bed is roughly equal to stroke volume, the entire capillary volume is exchanged with each heartbeat. In addition to gas exchange, the lung also filters the blood, preventing thrombi and other microemboli from entering the systemic circulation. Moreover, since the entire circulating blood flow passes through the lungs, the pulmonary circulation plays an important role in performing metabolic functions. For example, the pulmonary endothelium contains an abundance of angiotensin-converting enzyme, and is the major site in the body for conversion of angiotensin I to the active vasopressor angiotensin II (ANG II) and, conversely, for inactivation of the vasodilator bradykinin. Other functions include supplying nutrients to the alveoli and acting as a blood reservoir to transiently support left ventricle output during periods of decreased right ventricular output. Regulation of Blood Flow

Vascular resistance The pulmonary circulation is the only circulation in the body to conduct the entire cardiac output, and the only one in which the arteries carry oxygen poor blood. In utero, oxygen is delivered to the fetus via the placenta and the pulmonary circulation is a high-resistance circuit, with little blood flow. Perinatally, with the commencement of

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ventilation, pulmonary vascular resistance falls, and blood flow increases 10-fold. In the adult, normal pulmonary artery pressures are approximately 25 mmHg systolic and 10 mmHg diastolic, while pulmonary venous pressure is approximately 9 mmHg. Given the relationship between blood flow, pressure, and resistance, which by Ohm’s law is defined as: pulmonary arterial venous pressure ¼ pulmonary blood flow=pulmonary vascular resistance and the large volume of blood flow that must be accommodated (approximately 5 l min 1 at rest), the low pulmonary arterial pressure required to maximize the efficiency of the right ventricle work load must be maintained by low pulmonary vascular resistance, which is approximately one-tenth that of the systemic circulation. In the normal lung, significant increases in cardiac output have very little effect on pulmonary artery pressure. Indeed, a rapid 30% change in volume in moving from lying to standing, or a doubling of blood flow during exercise, can be accommodated with little rise in pulmonary arterial pressure. In order to maintain this low-pressure system, increases in cardiac output must be balanced by a reduction in pulmonary vascular resistance (Figure 1). This is possible because the pulmonary vasculature is highly distensible and possesses a significant reserve capacity. Decreases in pulmonary vascular resistance in response to increases in blood flow are not mediated by alterations in vascular tone, but are due to two passive processes: recruitment and distension (Figure 2). With an increase in blood flow, pressure rises transiently and opens or recruits capillaries and other small vessels that had been closed during resting

conditions due to insufficient intravascular pressure. This increase in vascular pressure also causes distension or expansion of individual capillaries. Both of these processes reduce pulmonary vascular resistance and minimize any flow-induced increase in pulmonary vascular pressure. Lung volume The resistance of both the alveolar and extra-alveolar vessels is affected by alveolar volume. Since these vessels are exposed to different surrounding pressures, their resistance is differentially regulated by changes in lung volume, leading to a complex relationship between lung volume and pulmonary vascular resistance (Figure 3). The extraalveolar vessels are positioned such that they become enlarged with increased lung volume (i.e., during inspiration) due to the fall in pleural pressure, which results in increased transmural pressure, and the pull exerted by the alveolar septa. Conversely, the increase in alveolar volume during inspiration results in greater air pressure compared to vascular pressure

Distension

Recruitment


Figure 2 Schematic demonstrating the effects of recruitment and distension on capillary flow.


Total

Alveolar

Extra-alveolar

Mean pulmonary arterial pressure Figure 1 Diagram illustrating the inverse relationship between pulmonary arterial pressure and resistance.

Lung volume Figure 3 Variation in pulmonary vascular resistance as a function of lung volume.

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and stretches the alveolar walls, causing the pulmonary capillaries to be compressed and elongated, thus increasing the resistance of these vessels. During expiration, lung volume falls and the process is reversed. Below functional residual capacity (FRC), extra-alveolar resistance is high due to lack of tension in the parenchyma, while above FRC the alveolar vessels collapse and the net effect of alveolar and extra-alveolar changes is that pulmonary vascular resistance increases with lung inflation. Thus, pulmonary vascular resistance is lowest near FRC and progressively rises with both increasing and decreasing lung volume. Blood flow heterogeneity Within the lung, blood flow is not uniform. The distensibility, compressibility, and low intravascular pressure that characterize the pulmonary circulation cause pulmonary blood flow and pulmonary vascular resistance to be influenced by factors that are independent of vascular smooth muscle tone, including both gravitational and structural factors. For example, within a column of fluid, the weight of the fluid exerts a higher pressure at the bottom than near the top. Similarly, in upright individuals, the vertical height of the lung is approximately 24 cm (at FRC), and while alveolar pressure is fairly uniform throughout the lung, intravascular pressure increases in the bottom (dependent) portions of the lungs due to gravity-dependent hydrostatic effects. This pressure gradient causes progressive vascular distension, decreased resistance, and increased flow at the bottom (base) of the lung.

Based on the relationship between pulmonary arterial pressure (PPA), pulmonary venous pressure (PV), and alveolar pressure (Palv), the lung can be divided into three zones (Figure 4). In zone 1, hydrostatic effects cause both arterial and venous pressures to fall below alveolar pressure such that Palv4PPA4PV and the alveolar vessels are completely collapsed, restricting blood flow to only the corner vessels. Under zone 2 conditions, PPA4 Palv4PV and the alveolar vessels are partially collapsed and the driving force for flow is the pressure gradient between arterial and alveolar pressures (PPA–Palv). In zone 3, PPA4PV4Palv, all of the alveolar blood vessels are fully open, and blood flow is driven by the difference between pulmonary arterial and venous pressure (PPA PV). In a seated or standing normal subject, zone 1 conditions are typically absent but, if present, will be found at the apex of the lung. Conversely, at the bottom of the lung, blood flow is greatest and zone 3 conditions are present, while zone 2 conditions can be found in the mid-lung. In addition to gravitational forces, the extent and location of the lung zones vary with body position. In a supine subject, zone 1, if present, will be in the ventral portion of the lungs while flow increases to the anatomically dependent (dorsal) regions of the lungs, resulting in zone 3. When a subject lies on their side, blood flow increases to the dependent lung. In certain cases, alveolar pressure may exceed vascular pressure in the nondependent portions of the lung. For example, zone 1 and 2 regions can be created and/or increased during hemorrhage or hypovolemia,

Zone 1 Palv>PPA>PV

PPA>Palv>PV

Height

Zone 2

Zone 3 PPA>PV>Palv

Blood flow Figure 4 Diagram illustrating the zone 3 model describing regional variation in pulmonary blood flow. PPA, arterial pressure; PV, venous pressure; Palv, alveolar pressure. Adapted from West JB, Dollery CT, and Naimark A (1964) Distribution of blood flow in isolated lung: relation to vascular and alveolar pressures. Journal of Applied Physiology 19: 713–724.


which result in low intravascular pressures, or during positive pressure ventilation or forced expiration, where alveolar pressure is increased. Regional blood flow differences can exist even between alveoli with similar vertical position, indicating that blood flow heterogeneity is not entirely dependent on the effects of gravity. At the microvascular level, the branching nature of the pulmonary vascular tree results in structural heterogeneities within isogravitational planes, producing variations in local driving pressures and resistances. This heterogeneity in driving pressure creates gravity-independent differences in regional pulmonary blood flow. Regulation of Pulmonary Vascular Tone

Factors that influence vascular smooth muscle cell tone, including neural and circulating factors and oxygen concentration, are potent regulators of both pulmonary vasomotor responses and vascular caliber. Nervous control The pulmonary circulation is supplied with both sympathetic and parasympathetic innervation. In general, increased sympathetic activity leads to release of catecholamines (e.g., dopamine, norepinephrine, epinephrine, and neuropeptide Y) that cause vasoconstriction and an increase in pulmonary vascular resistance. Pulmonary arteries contain fewer cholinergic than adrenergic nerve fibers. Parasympathetic stimulation causes the release of acetylcholine and vasoactive intestinal polypeptide, which mediate vascular dilation and a decrease in pulmonary vascular resistance. The lung also contains nonadrenergic, noncholinergic nerves that can be excitatory (e-NANC) or inhibitory (i-NANC). Release of vasoactive intestinal peptide, calcitonin generelated peptide, substance P, and nitric oxide from i-NANC nerves mediates vasodilation, while the e-NANC nerves mediate vasoconstriction, although the neurotransmitter involved remains unclear. Curiously, in contrast to the systemic vasculature, there appears to be minimal nervous control in the pulmonary circulation with respect to basal vascular caliber. Moreover, while the existence and activity of e-NANC and i-NANC nerves have been demonstrated in vitro, regulation of tone in vivo by these nerves has not been demonstrated. However, stimulation of adrenergic nerves may modulate pulmonary vascular resistance and blood flow during exercise and cold exposure and may increase in regulatory contribution during pathological states, particularly during pulmonary edema and embolism. Humoral factors A number of humoral factors can participate in active regulation of pulmonary

vascular tone. Some of these factors are endogenous, derived from the vascular endothelium, while others are produced by circulating cells or in other vascular beds. If the changes in vasomotor tone induced by these factors are not uniform, significant redistribution of blood flow can occur. Vasodilators Factors that induce pulmonary vasodilation include nitric oxide and nitrites, adenosine, bradykinin, atrial natriuric factor (ANP), and the eicosanoids prostaglandin E1 (PGE1) and PGI2 (prostacyclin). Nitric oxide and PGI2 are produced by the endothelium and are the most studied of the pulmonary vasodilators. Both have a short half-life and are quickly metabolized. Thus, neither is suited for action as a circulating factor and instead, once released by endothelial cells, quickly diffuse to the underlying smooth muscle and cause relaxation via stimulation of cGMP and cAMP. Bradykinin, a product of the renin–angiotensin system, exerts its dilatory influence by stimulating nitric oxide release. Indeed, in vitro, removal of the endothelium results in a loss of dilation in response to bradykinin, with vasoconstriction observed in some cases due to activation of receptors on the smooth muscle. ANP is a peptide produced primarily by stretch of the right heart, as occurs with an increase in pulmonary artery pressure. Given that the pulmonary circulation is the first vascular bed to see ANP, it is not surprising that plasma levels of ANP are 30% greater in the pulmonary than systemic circulation. Additionally, pulmonary arteries appear to be significantly more sensitive to the vasodilatory effects of ANP than arteries from other vascular beds. Although all of the aforementioned factors have been demonstrated to produce changes in vasomotor tone, only PGI2 and nitric oxide appear to regulate basal pulmonary vascular tone in the normal lung. Under pathological conditions, however, modulation of tone may be more pronounced. Vasoconstrictors Pulmonary vasoconstriction is caused by serotonin, endothelin-1 (ET-1), ANG II, histamine, and prostaglandins. Several of these factors are derived from the vascular endothelium. For example, arachidonic acid, which is readily taken up in the pulmonary circulation, is metabolized into a number of vasoactive eicosanoids, including PGE2, PGF2a, thromboxane, and leukotrienes, all of which diffuse to the smooth muscle and cause contraction. The lung endothelium is also the primary site of metabolism of angiotensin I, with approximately 60– 80% of plasma angiotensin I converted to ANG II, the vasoactive form of the peptide, in a single pass through the pulmonary circulation. ET-1 is perhaps

542 PULMONARY CIRCULATION 2% O2

PPA (mmHg)

30 25 20 15 10

15

PPA

10 Pt

5 0

Hypoxia One of the most unique aspects of the pulmonary circulation is the hypoxic pressor response. Unlike the systemic vasculature, which dilates in response to hypoxia in order to increase blood flow and oxygen delivery to tissues, alveolar hypoxia causes profound pulmonary vasoconstriction. Pulmonary vascular resistance rapidly increases as oxygen tension decreases, beginning within 1– 2 min after a drop in oxygen levels and reaches maximal response within 5 min (Figure 5). Hypoxic pulmonary vasoconstriction is maintained for the duration of hypoxia, and rapidly reverses (within 1 min) with a return to normoxia. When hypoxia is localized, this mechanism is thought to divert blood flow from regions of the lung that are poorly ventilated, helping to maintain arterial oxygen tension. However, in chronic lung disease, where alveolar hypoxia is global and prolonged, pulmonary hypertension develops. The exact mechanism underlying the hypoxiainduced increase in pulmonary vascular tone is unknown. Several factors have been shown to modulate the response, including prostaglandins, ANG II, serotonin, and leukotrienes, although these have all been ruled out as mediators of the response. Hypoxia has a direct effect on pulmonary vascular smooth muscle, although the maximal contractile response requires alterations in the release of the endothelial cell-derived mediators nitric oxide (decreased) and ET-1 (increased). Hypoxic pulmonary vasoconstriction is not mediated through the autonomic nervous system, as the response can be observed in isolated, perfused lungs that lack nervous input. Although the large pulmonary arteries are capable of responding to hypoxia, it is generally accepted

5 min

(a)

P (mmHg)

the most potent endogenous vasoconstrictor in the lung. While ET-1 can elicit nitric oxide production and transient dilation when receptors on the endothelial cells are activated, its main action is vasoconstriction mediated by receptors on the smooth muscle cells. Synthesized in the pulmonary endothelium, ET-1 secretion has been shown to increase in response to shear stress and hypoxia. Indeed, enhanced ET-1 levels are believed to contribute to the development of pulmonary hypertension. Circulating cells are also an important source of vasoconstrictors that can act on the pulmonary circulation, including serotonin, a primary product of activated platelets, and histamine, produced by mast cell granules. While these factors do not appear to be involved in basal regulation of tone, vasoconstrictors can modulate tone under pathological conditions, such as in pulmonary hypertension induced by anorexic agents, where serotonin uptake is impaired.

10 s

(b) Figure 5 (a) Effect of hypoxia on pulmonary arterial pressure in an isolated perfused rat lung model. In this preparation, left atrial pressure is negative and changes in pulmonary arterial pressure reflect changes in pulmonary vascular resistance. (b) Expanded scale showing the effect of ventilation on pulmonary arterial pressure. PPA, arterial pressure; Pt, tracheal pressure. Courtesy of J T Sylvester.

that the main site of increased resistance during hypoxic pulmonary vasoconstriction is in the small, muscular arterioles. There is evidence that postcapillary hypoxic venoconstriction may also occur, although the magnitude of the contribution of these vessels to the increase in total pulmonary vascular resistance is uncertain. Our understanding of the pulmonary circulation has been greatly aided by evaluation of pulmonary function in a variety of species, including sheep, rats, dogs, cats, ferrets, mice, cattle, guinea pigs, goats, and rabbits. Despite the obvious size differences, the pulmonary circulation across species is quite similar. For example, all mammals respond to hypoxic challenge with HPV, although the degree of the response varies from minor (rabbits) to robust (cattle). In developing animal models of human disease, the most widely used is the mouse. Although the mouse appears to lack a bronchial circulation, major advantages of this model include small size and ease of maintenance, quick availability, and reduced genetic variability. Perhaps the most important advantage of murine models is the ability to alter gene expression, providing a powerful tool to explore the functional role of specific proteins in physiological and pathophysiological conditions.


Pulmonary Circulation in Respiratory Diseases

obstructive sleep apnea, may also result in pulmonary hypertension.

Primary Pulmonary Hypertension

Interstitial Lung Disease

Pulmonary vascular disease may be primary or secondary to other disorders of the lung or other organs. Primary pulmonary hypertension (PPH), also known as idiopathic pulmonary arterial hypertension, is a disease of unknown etiology, although certain cases have been linked to appetite suppressants. PPH is characterized by an elevated resting pulmonary artery pressure that increases dramatically during exercise. Increased pulmonary vascular resistance, due in part to pulmonary arteriolar obstruction with hypertrophy of wall elements, necrotizing arteritis, and/or endothelial cell lesions increases pulmonary arterial pressure, causing right ventricular hypertrophy in the absence of any other cardiac abnormality. Although the underlying cause of PPH remains unclear, dysfunction of the pulmonary endothelium, which is a rich source of both vasodilators and vasoconstrictors, may contribute. In the normal lung, the balance of vasodilators to vasoconstrictors favors low pulmonary vascular resistance. However, with endothelial cell dysfunction, release of the vasodilators nitric oxide and PGI2 is impaired while release of the vasoconstrictors ET-1 and thromboxane may be augmented, resulting in a net vasoconstriction. In addition, excessive endothelial cell proliferation may obliterate the lumen of small arteries and alveolar vessels, further increasing vascular resistance.

Interstitial lung disease refers to a diverse group of diseases with the common feature of alterations in the alveolar interstitial space, most commonly septal destruction and fibrosis. While interstitial lung disease will produce fibrosis of the vessels, early in the disease process only a small part of the pulmonary arterial tree is involved, and hypoxemia during exercise, due to reduced diffusion capacity and/or ventilation–perfusion mismatch, is commonly the only alteration in lung function. As the disease progresses to later stages, resting hypoxemia may be observed along with subsequent pulmonary hypertension.

Secondary Pulmonary Hypertension

Secondary pulmonary hypertension and right heart failure are common complications of chronic lung diseases, including emphysema, chronic bronchitis, cystic fibrosis, and severe chronic asthma. In this case, elevated pulmonary arterial pressure is caused by a combination of hypoxic pulmonary vasoconstriction, polycythemia, alveolar hypercapnia and acidosis, and raised intra-alveolar pressure during expiration. Global hypoxemia is also a consequence of residence at high altitude. The decrease in vascular caliber associated with prolonged hypoxia is comprised of both a reversible and fixed component. The reversible component is due to sustained active contraction of the pulmonary vascular smooth muscle while the fixed component is due to structural remodeling of the pulmonary circulation, primarily increased muscularization of the small pulmonary arteries. In addition to chronic lung diseases that produce continuous alveolar hypoxia, in recent years it has become clear that prolonged exposure to intermittent hypoxic episodes, as occurs in

Pulmonary Edema

Pulmonary edema can occur as a consequence of heart failure or lung microvascular injury and leads to an increase in vascular resistance. With left ventricular failure, elevated diastolic pressure results in elevated pulmonary venous pressure, which interferes with normal capillary blood flow and increases capillary pressure. The accumulation of fluid in the interstitial compartment around the vessels can lead to impaired gas exchange or, more commonly, can exaggerate the effects of lung volume on vascular resistance and affect blood flow distribution. In more severe cases, when the interstitial spaces are filled, or when the endothelial lining of the lung microvasculature is injured, alveolar flooding can occur. In either case, excess fluid in the alveoli results in marked deterioration in gas exchange. In some cases, alterations in vasomotor tone can cause edema, independent of microvascular injury or cardiac function. For example, excessive vasoconstriction in response to hypoxia is believed to be the underlying cause of high-altitude pulmonary edema. Arteriovenous Malformation and Stenosis

A pulmonary arteriovenous malformation is a direct communication between a pulmonary artery and a pulmonary vein producing a right-to-left shunt. These malformations can occur as a consequence of liver dysfunction, genetic abnormalities, as with Osler– Weber–Rendu disease, or are idiopathic. Pulmonary arteriovenous malformations are not uncommon, and in one-third of cases, are multiple. In addition to pulmonary hypotension, capillary dilation and extensive shunting results in impaired gas exchange and hypoxemia. Pulmonary vascular stenoses, although rare, occur most commonly at the bifurcation of the main

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pulmonary artery. The increase in pulmonary vascular resistance caused by the narrowing of the artery results in right ventricular hypertension. Stenoses in the left heart valves lead to passive pulmonary venous hypertension. The elevation in venous pressure results in elevated capillary and arterial pressures. Pulmonary Embolism

Pulmonary embolism is perhaps the most common pulmonary vascular disease. Clots originate in the systemic veins, often in the deep veins of the lower extremities, and formation is augmented following injury, venous stress, and hypercoagulable states. The thrombi detach and become lodged in the pulmonary arterial circulation. Occasionally, the right side of the heart is a source of a pulmonary embolus. Given the large reserve capacity of the pulmonary capillaries, many thromboemboli can go undiagnosed, and resolve quickly. Massive blockages produce a functional decrease in cross-sectional area of the pulmonary circulation, resulting in a significant increase in pulmonary vascular resistance and elevated pulmonary arterial pressure. Subsequent right ventricular strain decreases cardiac output and, if severe, can result in death. In approximately one-tenth of patients, large thromboemboli cause local cessation of flow and ischemic necrosis of the lung parenchyma (pulmonary infarction). See also: Bronchial Circulation. Diffusion of Gases. Endothelial Cells and Endothelium. High Altitude, Physiology and Diseases. Hypoxia and Hypoxemia. Oxygen–Hemoglobin Dissociation Curve. Peripheral

Gas Exchange. Pulmonary Vascular Remodeling. Smooth Muscle Cells: Vascular. Ventilation: Uneven. Ventilation, Perfusion Matching.

Further Reading Bakhle YS and Vane JR (1974) Pharmacokinetic function of the pulmonary circulation. Physiological Reviews 54(4): 1007–1045. Crofton J and Douglas A (eds.) (1975) The pulmonary circulation. In: Respiratory Diseases, 2nd edn., pp. 34–37. Philadelphia: Lippincott Co. Crystal RG, West JB, Weibel ER, and Barnes PJ (eds.) (1997) The Lung: Scientific Foundations, 2nd edn., sect. 5. New York: Lippincott-Raven. Dawson CA (1984) Role of pulmonary vasomotion in physiology of the lung. Physiological Reviews 64(2): 544–616. Hlastala MP and Glenny RW (1999) Vascular structure determines pulmonary blood flow distribution. News in Physiological Sciences 14: 182–186. Keith IM (2000) The role of endogenous lung neuropeptides in regulation of the pulmonary circulation. Physiological Research 49(5): 519–537. McMurtry IF (1986) Humoral control. In: Bergofsky EH (ed.) Abnormal Pulmonary Circulation, pp. 83–125. New York: Churchill-Livingstone. Nadel JF and Nadel JA (eds.) (1988) Textbook of Respiratory Medicine. Philadelphia: WB Saunders. Peacock AJ (ed.) (1966) Pulmonary Circulation. London: Chapman and Hall Medical. Sylvester JT and Brower RG (1990) Pulmonary blood flow. In: Stein JH (ed.) Internal Medicine, 3rd edn., pp. 583–586. Boston: Little, Brown and Co. Ward JPT and Aaronson PI (1999) Mechanisms of hypoxic pulmonary vasoconstriction: can anyone be right? Respiratory Physiology 115(3): 261–271. West JB, Dollery CT, and Naimark A (1964) Distribution of blood flow in isolated lung: relation to vascular and alveolar pressures. Journal of Applied Physiology 19: 713–724.

PULMONARY EDEMA M A Matthay and T E Quinn, University of California, San Francisco, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Pulmonary edema refers to the abnormal collection of fluid in the extravascular spaces of the lung such as the interstitium and the alveoli. Its two main pathophysiologic mechanisms are increased hydrostatic forces within the lung microvasculature and increased microvascular permeability. Understanding the pathophysiology of pulmonary edema requires a firm understanding of normal lung fluid balance. The Starling equation, which describes the net flow of fluid across a semipermeable membrane, applies to the filtration of fluid from the pulmonary microvasculature into the pulmonary interstitium. Interstitial fluid is primarily removed by the lung lymphatic vessels, and alveolar

fluid is removed via active transport mechanisms. Pulmonary edema occurs because of either increased hydrostatic forces or increased vascular permeability which then causes an increase in fluid filtration sufficient to overwhelm fluid removal mechanisms. The treatment of hydrostatic pulmonary edema targets a reduction in pulmonary microvascular pressure with diuretics, vasodilators, and sometimes inotropic agents. The treatment of increased permeability pulmonary edema is mainly supportive. Mechanical ventilation of patients with increased permeability pulmonary edema should be performed with a low tidal volume, lung-protective strategy.

Introduction Pulmonary edema refers to the abnormal collection of fluid in the extravascular spaces of the lung such as the interstitium and the alveoli. Its two main

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pathophysiologic mechanisms are increased hydrostatic forces within the lung microvasculature and increased microvascular permeability. Pulmonary edema from either mechanism may lead to profoundly diminished lung function, respiratory distress, and even death.

Normal Lung Fluid Balance Starling Forces

Understanding the mechanisms of pulmonary edema formation requires a firm understanding of normal lung fluid balance. The Starling equation describes the net flow of fluid across a semipermeable membrane. Applied to the lung microvasculature, it can be written as follows: Qf ¼ K½ðPmv Pi Þ sðpmv pi Þ where Qf is the net filtration of fluid from the lung microvasculature to the interstitium, K is the filtration coefficient or leakiness of the endothelium to water, Pmv is the pulmonary microvascular pressure, Pi is the pulmonary interstitial pressure, s is the protein reflection coefficient, pmv is the protein osmotic pressure in the microvasculature, and pi is the interstitial protein osmotic pressure. Under normal conditions, the microvascular endothelium is somewhat leaky to fluid, and the pulmonary interstitial pressure is slightly negative. On the other hand, the endothelium is quite impermeable to proteins with a reflection coefficient near 1. Thus, the fluid filtered from the micro vessels is low in protein compared to the fluid in the vascular space. On balance, there is normally a small net flow of fluid from the lung microvasculature to the interstitium. Lung Fluid Clearance and Protective Mechanisms

Interstitial fluid clearance As described above, fluid is filtered into the lung interstitium from the microvasculature under normal conditions. This fluid must be constantly removed to maintain homeostasis. The most important mechanism for lung fluid removal is the lung lymphatic system. The pulmonary interstitium is rich with lymphatic channels which absorb filtered fluid and protein and convey it along channels in the interlobular septa to the mediastinal lymphatic vessels. Another mechanism of fluid removal from the lung under normal conditions is the direct reabsorbtion of fluid into the pulmonary venules. Since the normal filtered fluid is low in protein (low pi) and the intravascular fluid is protein rich (high pmv), Starling forces along the venules, where intravascular hydrostatic pressure (Pmv) is normally low, will favor reabsorbing fluid. Two other pathways

of lung fluid clearance, which are less important under normal conditions, include filtration through the visceral pleura into the pleural space, and movement of fluid via the loose peribronchovascular connective tissue into the mediastinum. Under pathological conditions in which excess fluid is filtered into the pulmonary interstitium, the above described mechanisms become safety factors against the development of pulmonary edema and alveolar flooding. The lymphatic system’s rate of liquid clearance can increase up to 10 times normal when hydrostatic forces increase. Second, as increased fluid is filtered into the perimicrovascular space, pressure in that space (Pmv) increases and protein concentration falls (decreased pi). These changes in the perimicrovascular space oppose further fluid accumulation. Third, the loose peribronchovascular connective tissue has a high capacitance, allowing a large amount of fluid accumulation (up to 500 ml) before tissue pressure rises. Finally, if fluid accumulation in the pulmonary interstitium continues, pressure in that space will increase enough to allow filtration across the visceral pleura and the formation of a pleural effusion. A large amount of fluid can sequester in the pleural space before lung function is sufficiently diminished to impact gas exchange. Alveolar fluid clearance In the presence of interstitial pulmonary edema, the alveoli are protected from flooding by two main mechanisms. In contrast to the relatively leaky microvascular endothelium, the alveolar epithelium has tight junctions which render it quite impermeable to interstitial fluid and protein. However, if edema fluid accumulation exceeds the capacity of the interstitial clearance mechanisms for long enough, the alveolar epithelial barrier changes, and alveolar flooding occurs. If the edema is formed from increased hydrostatic pressure, the alveoli are flooded with edema fluid that has a low protein concentration relative to plasma. If the edema is caused by increased permeability (acute respiratory distress syndrome (ARDS)), the alveoli are flooded with protein-rich fluid. In either case, alveolar fluid clearance then becomes vital to the restoration of lung function. Alveolar fluid clearance occurs primarily by active sodium transport. The alveolar type II cells are probably responsible for most of the active sodium transport out of the alveolus. Sodium is transported into the type II cell via an amiloride-sensitive epithelial sodium channel (ENaC) on the apical surface. The oubain-sensitive Na–K ATPase on the basolateral membrane surface then transports the sodium out of the cell. As an osmotic gradient forms, water flows out of the alveolus, possibly via water channels

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called aquaporins. In this fashion, water is rapidly cleared from flooded alveoli.

Pulmonary Edema Pathogenesis Pulmonary edema forms from two main mechanisms, increased microvascular pressure (Pmv) and increased microvascular permeability. The most common reason for increased Pmv is elevated left atrial pressure (cardiogenic pulmonary edema). The most common reasons for elevated left atrial pressure include severe volume overload, left ventricular systolic dysfunction, left ventricular diastolic dysfunction, mitral valve disease, and aortic valve disease. A full discussion of all of the etiologies of cardiogenic pulmonary edema is beyond the scope of this article. Nevertheless, all of these disorders cause pulmonary edema via the same pathophysiologic mechanism, that is, increased microvascular pressure causing increased fluid filtration into the interstitium. If the resultant edema fluid accumulation is confined to the interstitium by the safety factors described above, then only mild, if any, symptoms will result. However, if increased microvascular pressure is severe or persists, then the defense mechanisms are overwhelmed, alveolar flooding ensues, and gas exchange is impaired. Because the capillary endothelium and alveolar epithelium remain intact, the edema fluid suctioned out of the airway has a low protein content compared with plasma (ratio of p0.65). Increased vascular permeability (increased K and decreased s from the Starling equation) is caused by damage to the endothelium and alveolar epithelium. When this occurs, both the interstitium and alveoli are flooded with protein-rich fluid (edema fluid to plasma protein concentration ratio 40.65). Because of the damage to the alveolar epithelium, alveolar fluid clearance is usually impaired. Gas exchange is usually significantly diminished. Increased permeability pulmonary edema is also known as acute lung injury, or, in its severest form, ARDS (see Acute Respiratory Distress Syndrome). There are numerous causes of acute lung injury, the most common of which are listed in Table 1. These causes of acute lung injury may be grouped into those that constitute a direct insult to the alveolar epithelium such as aspiration of gastric contents or pneumonia, and those that are an indirect insult to the alveolar epithelium such as sepsis. Some less common types of pulmonary edema include postobstructive pulmonary edema (see Upper Airway Obstruction), neurogenic pulmonary edema, and high-altitude pulmonary edema (HAPES) (see High Altitude, Physiology and Diseases). Postobstructive pulmonary edema occurs in the setting of

Table 1 Clinical disorders associated with acute lung injury Direct

Indirect

Pneumonia Aspiration Thoracic trauma Inhalation injury Fat emboli Near drowning Reperfusion after transplant or embolectomy

Sepsis Nonthoracic trauma Acute pancreatitis Drug overdose Transfusion associated (TRALI) Cardiopulmonary bypass Burns

an acute upper airway obstruction such as postanesthesia laryngospasm. A patient with an acutely obstructed upper airway will make very vigorous respiratory effort leading to markedly negative intrathoracic pressures, which are transmitted to the perimicrovascular interstitium (decreased Pi from the Starling equation). Also, large negative intrathoracic pressures increase both preload and afterload on the heart. The increased preload and afterload probably increase pulmonary microvascular pressure (Pmv). Thus, there are increased net hydrostatic forces, and edema forms. Neurogenic pulmonary edema is thought to occur because of the massive catecholamine surge produced after a neurologic event. This surge causes marked systemic vasoconstriction and redistribution of blood from the systemic to pulmonary circulation. The resulting increased pulmonary vascular pressures may produce a hydrostatic pulmonary edema (low protein) or lead to physical damage to the vascular walls. This latter scenario causes an increased permeability edema. Similarly, HAPES may result from severe, nonhomogeneous pulmonary vasoconstriction. The vasoconstricted areas shunt blood flow to other areas of the lung leading to increased Pmv in the perfused areas. Again, a low-protein hydrostatic edema may result, or there may be damage to the endothelium from the increased pressure causing high-protein, increased-permeability edema.

Diagnosis Hydrostatic Pulmonary Edema

The vast majority of cases of hydrostatic pulmonary edema are of cardiac origin. The diagnosis rests heavily on the history, physical examination, and chest radiography. Most patients with acute pulmonary edema of any cause will present with dyspnea in which case the history of present illness should focus on dyspnea severity, time of onset, pace of onset, and associated symptoms. Patients with acute cardiogenic pulmonary edema may have sudden, severe dyspnea. Because a significant number of these severely affected patients have pulmonary

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edema secondary to an acute coronary event, one should thoroughly question the patient or family about chest pain or angina equivalents. Patients may also give a history of recently worsening chronic congestive heart failure symptoms such as worsening dependent edema, orthopnea, and paroxysmal nocturnal dyspnea. A history of dietary indiscretion is common in patients with an acute exacerbation of chronic congestive heart failure. The past medical history should focus on prior history of coronary artery disease, valvular heart disease, hypertension, or cardiomyopathy. On physical examination, patients with acute cardiogenic pulmonary edema may be very anxious and sitting ‘bolt upright’ in bed. In the most severe cases, patients may develop cyanosis, the development of which signifies severe respiratory failure and impending death if not corrected quickly. Other signs include jugular venous distension, an S3 gallop on heart examination, pitting edema, a palpable liver edge, and ascites. The respiratory examination is characterized by the presence of wet rales, possible extending up to the apices of the lung. Patients may also exhibit the use of accessory respiratory muscles. Chest radiography is valuable in diagnosing pulmonary edema. In cardiogenic pulmonary edema, the heart silhouette is often enlarged. If only interstitial edema is present, there may be evidence of apical vascular engorgement (so-called vascular redistribution), septal or Kerley’s lines, and decreased definition of smaller blood vessels and bronchial structures (perivascular and peribronchial cuffing). When alveolar flooding occurs, confluent parenchymal opacities develop. In hydrostatic edema, the radiographic opacities often develop centrally first. In severe cases, there may be complete opacification bilaterally with air bronchograms. Chest radiography cannot reliably distinguish between hydrostatic pulmonary edema and increased permeability pulmonary edema (acute lung injury (ALI)). Several other diagnostic tests may be useful in patients with dyspnea or respiratory distress and suspected cardiogenic pulmonary edema. As mentioned

above, acute pulmonary edema is often associated with an acute coronary event, so an electrocardiogram should be performed in all patients with suspected acute cardiogenic pulmonary edema. Particular attention should be paid to electrocardiographic signs of ischemia or infarction such as ST segment elevation, severe ST segment depression, new Q waves, or a new left bundle branch block. Likewise, creatine phosphokinase-MB (CPK-MB) and troponin levels are useful in patients with suspected cardiogenic pulmonary edema to rule out myocardial infarction. Blood levels of B-type natriuretic peptide (BNP) are useful in emergency department patients with dyspnea and suspected cardiogenic pulmonary edema, however, their diagnostic accuracy in inpatients is unproven. Arterial blood gases are useful in assessing the severity of respiratory compromise. Interstitial pulmonary edema may be associated with normal or slightly reduced oxygenation (decreased PaO2) with a reduced PaCO2 from tachypnea. With alveolar flooding, significant intrapulmonary shunt develops, and a markedly reduced PaO2 will result if untreated. Echocardiography may be very helpful in determining the etiology of pulmonary edema. Normal echocardiographic structure and function argue strongly against pulmonary edema of cardiac origin. Finally, pulmonary artery catheterization may provide valuable information in patients with pulmonary edema and shock. Not only can normal pulmonary artery occlusion pressures exclude cardiogenic pulmonary edema, but the clinician can follow trends in the pulmonary artery catheter data to help guide fluid and vasopressor management. Increased Permeability Pulmonary Edema

Increased permeability pulmonary edema is also known as ALI or ARDS in its severest form. Currently, its diagnosis is based on a set of criteria as set forth by the American–European Consensus Conference on Acute Respiratory Distress Syndrome (see Table 2) (see Acute Respiratory Distress Syndrome). These criteria identify a patient population with

Table 2 Diagnostic criteria for acute lung injury (ALI) acute respiratory distress syndrome (ARDS) Oxygenation PaO2 =FiO2 p300 for acute lung injury

a

a

Chest radiograph

Left atrial pressure

Bilateral infiltrates on frontal chest radiograph

Pulmonary artery occlusion pressure p18 mmHg when measured or no clinical evidence of left atrial hypertension

For the diagnosis of acute respiratory distress syndrome, use PaO2 =FiO2 p200 with the same chest radiograph and left atrial pressure criteria. Adapted from Bernard GB, Artigas A, Brigham K, et al. (1994) The American–European Consensus Conference on ARDS: definitions, mechanisms, relevant outcomes, and clinical trial coordination. American Journal of Respiratory and Critical Care Medicine 149: 818–824.

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hypoxemia and bilateral infiltrates on chest radiograph whose condition cannot be explained by increased left atrial pressure (noncardiogenic). Based on these criteria, the most useful data in the diagnosis of acute lung injury are the history, chest radiograph, and arterial blood gases. The history in suspected ALI should focus on eliciting the presence of one of the common causative conditions (see Table 1). In postoperative patients, a thorough examination of the anesthesia record for blood products transfused or witnessed aspiration during induction or recovery is helpful. On lung examination, patients with ALI may have bilateral rales or evidence of consolidation, but these findings are non-specific. Thus, the physical examination in suspected ALI patients should be directed toward determining whether the patient’s edema can be explained by elevated left atrial pressure and whether the patient has one of the potential causes of ALI. The absence of any history or physical examination evidence for volume overload or congestive heart failure in a patient with pulmonary edema strongly suggests ALI. In addition, the patient’s abdomen, rectum, and skin should be meticulously examined for a potential source of sepsis. As indicated by the diagnostic criteria, the chest radiograph and arterial blood gases are the most useful diagnostic tests in ALI. The chest radiograph may show only bilateral interstitial edema, but most likely it will demonstrate areas of alveolar filling. More severe cases may show extensive consolidation of both lungs. Because pneumonia is the most common cause of ALI, there also may be focal consolidation with air bronchograms. By definition, arterial blood gas analysis will demonstrate significant hypoxia and intrapulmonary shunt. A respiratory alkalosis may be present early in the course of ALI due to hypoxic respiratory drive and/or sepsis, but later respiratory acidosis may develop from worsening lung compliance and increased dead space. In addition, hypoxia and sepsis may cause a metabolic acidosis. Other laboratory tests should be directed at potential causes of ALI. Because sepsis and pneumonia are the most common causes of ALI, cultures of blood, sputum (or airway aspirate), urine, wounds, and, if appropriate, cerebrospinal fluid should be obtained. Abdominal tenderness on examination should be evaluated with imaging studies and amylase and lipase levels. Other possible diagnostic studies in ALI include pulmonary artery catheterization and echocardiography. Both of these modalities can be useful in determining whether the pulmonary edema is due to a cardiogenic source. Pulmonary artery catheterization may also provide valuable diagnostic information about the etiology of shock states which frequently

accompany ALI. Another potential advantage of pulmonary artery catheterization is that the hemodynamic data may be useful in guiding fluid and vasopressor therapy. However, the benefit of routine use of pulmonary artery catheters in ALI patients is not well established, and this issue is the subject of an ongoing multicenter, randomized, controlled trial.

Management Cardiogenic Pulmonary Edema

Some patients with mild cardiogenic pulmonary edema can be treated and released from the emergency department with close follow-up. However, patients with new onset or severe symptoms, hypoxia, evidence of acute ischemia, or poor follow-up should be admitted to the hospital for treatment and monitoring. Indications for intensive care unit admission will vary with each facility’s capability, but patients with profound hypoxia, need for ventilatory support, or need for invasive monitoring should be admitted to a critical care unit. Pulse oximetry should be monitored early in all patients who present with dyspnea, and patients with significant hypoxia or at risk for worsening should have continuous pulse oximetry. Patients with a suspected ischemic cause of their pulmonary edema should have continuous electrocardiographic monitoring also. An arterial catheter should be placed in patients receiving nitroprusside for severe cardiogenic pulmonary edema with hypertension and patients receiving vasopressors for shock. Also, mechanically ventilated patients requiring frequent arterial blood gases may benefit from an arterial catheter. Pulmonary artery catheterization for hemodynamic monitoring may be useful in patients with severe cardiogenic pulmonary edema and shock, but its use is controversial. Supportive care for patients with cardiogenic pulmonary edema includes oxygen supplementation and support of ventilation if necessary. Noninvasive ventilation initiated early in patients with severe hypoxia or respiratory distress may prevent the need for mechanical ventilation. This modality may not be appropriate for patients with an acute coronary syndrome, since it may not reduce the work of breathing and myocardial oxygen demand as much as intubation and mechanical ventilation. Intubation and mechanical ventilation is required in patients whose hypoxia or respiratory distress is refractory to noninvasive ventilation or who require rapid reduction in work of breathing. Pharmacotherapy in cardiogenic pulmonary edema is aimed at reducing pulmonary microvascular pressure (Pmv from the Starling equation) and reducing

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dyspnea. If there is an underlying cause of the acute pulmonary edema such as acute myocardial infarction, it must be treated aggressively. The reduction in pulmonary microvascular pressure is primarily achieved by vasodilating medications and diuretics. Loop diuretics such as furosemide are the most commonly used diuretics in acute cardiogenic pulmonary edema because of their rapid onset and effectiveness in producing intravascular volume reduction. They even have a small, venodilating effect which may slightly decrease pulmonary microvascular pressure immediately. However, even with the rapid onset of diuresis provided by furosemide, patients with severe acute cardiogenic pulmonary edema will need other medications that work more quickly to reduce pulmonary microvascular pressure, namely vasodilating agents. In addition to its usefulness in relieving dyspnea, morphine has venodilating effects which act to increase peripheral blood pooling. This pooling reduces left ventricular end diastolic pressure, and thereby pulmonary microvascular pressure is reduced. Nitroglycerin and nitroprusside are two agents that provide immediate vasodilation and can be given as a titratable intravenous infusion. Nitroglycerin causes both arterial and venous dilation, although venodilation is the more profound effect. Its dilation of epicardial coronary arteries may also play a role in its antianginal effect. Nitroprusside has more prominent arterial dilating effects. Both of these agents produce a rapid reduction in cardiac preload and afterload with consequent reduction in pulmonary microvascular pressure. Finally, nesiritide is recombinant human B-type natriuretic peptide. Despite its name, its dominant clinical action seems to be vasodilation instead of natriuresis. Nesiritide has been evaluated for use in acute cardiogenic pulmonary edema, but there is little evidence that its increased cost compared with nitroglycerin is justified by increased benefit. When cardiogenic pulmonary edema is refractory to the usual measures described above, or when it is accompanied by cardiogenic shock, inotropic agents may provide short-term improvement. The currently available inotropes fall into two categories, b-adrenergic agonists and phosphodiesterase inhibitors. The most commonly used adrenergic agonist in cardiogenic pulmonary edema is dobutamine. It stimulates cardiac b1-adrenergic receptors causing increased inotropy and chronotropy. The chief side effects are tachycardia and increased ventricular ectopy. In addition, dobutamine may cause vasodilation and hypotension because of its stimulation of b2 receptors in vascular smooth muscle. Phosphodiesterase inhibitors, such as milrinone, produce their inotropic effect by increasing cardiac intracellular

cyclic AMP, which increases intracellular calcium. These agents also cause peripheral vasodilation and reduction in blood pressure, so blood pressure should be closely monitored upon initiation of milrinone. Like the adrenergic agonists, milrinone has been associated with increased ventricular ectopy. Levosimendan is a novel inotropic agent currently undergoing clinical trials. Its mechanism of action, calcium sensitization of cardiac myocytes, may avoid some of the adverse effects of adrenergic agonists and phosphodiesterase inhibitors. Overall, the use of inotropic agents in acute cardiogenic pulmonary edema has not been shown to improve mortality, and they are associated with potentially serious adverse effects. Inotropic agents should therefore not be routinely used in patients with acute cardiogenic pulmonary edema. Permeability Pulmonary Edema

For practical purposes, permeability pulmonary edema and ALI are the same. ARDS is the most severe form of ALI. The large majority of cases of ALI will be treated in intensive care units either because of the severity of the hypoxia and respiratory distress or because of the severity of the causative disorder such as sepsis. Pulse oximetry should be monitored frequently or continuously in ALI patients, because they are at high risk of respiratory decompensation. An arterial catheter for frequent blood gas analysis is helpful, especially in intubated patients or patients at high risk of intubation. The relative benefit of a central venous catheter versus a pulmonary artery catheter for central venous pressure monitoring is currently under study in a multicenter, randomized, clinical trial. Since there is no specific pharmacotherapy for ALI, the mainstays of management are treatment for the underlying condition and supportive care. Unless there is a clear noninfectious cause of ALI, most patients should be treated with broad-spectrum antibiotics intitially. Source control, such as drainage of abscesses, should be performed urgently. Supportive care usually includes intubation and mechanical ventilation. Importantly, the method of mechanical ventilation is the only specific treatment for ALI. A mechanical ventilation regimen with low tidal volume (6 ml kg 1 based on ideal body weight), plateau pressures of less than 30 cm water, and a moderate level of positive end-expiratory pressure has been shown to significantly reduce mortality in ALI patients (see Table 3). In attempting to reach these goals, a pH as low as 7.25 and PaO2 as low as 55 mmHg are usually tolerated. Thus, unless there is a contraindication to the permissive hypercapnea (such as elevated intracranial pressure) or permissive

550 PULMONARY EFFECTS OF SYSTEMIC DISEASE Table 3 Protocol for low tidal volume ventilation for acute lung injury Variables

Protocol

Ventilator mode Tidal volume

Volume assist control p6 ml kg 1 body weight predicted by height; may decrease to 4 ml kg 1 if needed to achieve plateau pressure goal p30 cmH2O 6–35 breath min 1 Adjust ventilator rate to achieve arterial pHX7:30 if possible. Adjust flow to achieve I : E 1 : 1.0–1.3. 55pPaO2 p80 mmHg or 88%pSpO2 p95% 0:3 0:4 0:4 0:5 0:5 0:6 0:7 0:7 0:7 0:8 0:9 0:9 0:9 1:0 1:0 1:0 5 5 8 8 10 10 10 12 14 14 14 16 18 18 22 24

Plateau pressure Ventilator set rate Arterial pH Inspiratory flow, I:E Oxygenation FiO2/PEEP (cmH2O)

Adapted with permission from Acute Respiratory Distress Syndrome Network (2000) Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. New England Journal of Medicine 342(18): 1302–1308. Copyright & 2000 Massachusetts Medical Society. All rights reserved.

hypoxemia (such as an acute coronary syndrome), all patients who meet the diagnostic criteria for ALI should be ventilated with the low tidal volume strategy. Nutritional support should be considered early, because many ALI patients will have a prolonged ventilator course. Current clinical trials in patients with ALI are investigating possible pharmacologic agents such as activated protein C and granulocyte– macrophage colon stimulating factor (GM-CSF). The value of reducing pulmonary microvascular pressure by fluid restriction and diuresis is also being investigated in a randomized clinical trial. See also: Acute Respiratory Distress Syndrome. High Altitude, Physiology and Diseases. Upper Airway Obstruction.

Further Reading Acute Respiratory Distress Syndrome Network (2000) Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. New England Journal of Medicine 342(18): 1302–1308. Bernard GB, Artigas A, Brigham K, et al. (1994) The American– European Consensus Conference on ARDS: definitions, mechanisms, relevant outcomes, and clinical trial coordination. American Journal of Respiratory and Critical Care Medicine 149: 818–824.

Brower RG, Lanken PN, MacIntyre N, et al. (2004) Higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome. New England Journal of Medicine 351(4): 327–336. Gehlbach BK and Geppert E (2004) The pulmonary manifestations of left heart failure. Chest 125: 669–682. Givertz MM, Colucci WS, and Braunwald E (2001) Clinical aspects of heart failure: high output heart failure; pulmonary edema. In: Braunwald E, Zipes DP, and Libby P (eds.) Heart Disease: A Textbook of Cardiovascular Medicine, pp. 534–561. Philadelphia: Saunders. Guyton AC and Lindsey AW (1959) Effect of left atrial pressure and decreased plasma protein concentration on the development of pulmonary edema. Circulation Research 7: 649–657. Matthay MA, Folkesson HG, and Clerici C (2002) Lung epithelial fluid transport and the resolution of pulmonary edema. Physiological Reviews 82(3): 569–600. Matthay MA and Sakuma T (2001) Pulmonary edema: formation and reabsorption. In: Scharf SM, Pinsky MR, and Magder S (eds.) Respiratory–Circulatory Interactions in Health and Disease, pp. 361–387. New York: Dekker. Sharma M and Teerlink JR (2004) A rational approach for the treatment of acute heart failure: current strategies and future options. Current Opinion in Cardiology 19: 254–263. Staub NC (1988) Lung liquid and protein exchange. Applied Cardiopulmonary Pathophysiology 2: 117–123. Taylor AE (1981) Capillary fluid filtration: starling forces and lymph flow. Circulation Research 49: 557–575. Ware LB and Matthay MA (2000) The acute respiratory distress syndrome. New England Journal of Medicine 342(18): 1334– 1349.

PULMONARY EFFECTS OF SYSTEMIC DISEASE O P Sharma, University of Southern California School of Medicine, Los Angeles, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Pulmonary manifestations of systemic disorders are common. Almost all systemic diseases can involve the lung. In many illnesses, lung complications remain relatively benign, producing

few or no symptoms; such is the case with pleural effusions related to renal or hepatic failure. In others diseases, lung involvement becomes a nuisance but is manageable; this occurs in gastroesophageal reflux disease and arteriovenous malformations in Osler–Rendau–Weber disease. On the other hand, in certain diseases lung involvement progresses relentlessly, causing death or serious injury that influences the course of the primary illness; such is the case in severe pneumonia in a diabetic patient or portopulmonary hypertension in chronic liver failure.

PULMONARY EFFECTS OF SYSTEMIC DISEASE 551

Introduction It is difficult to assemble all that is known of the pulmonary manifestations of the systemic disorders that afflict the human body. The normal lung is essential for the rest of the body, and almost all organs influence lung behavior. These interrelationships are often complex and difficult to understand. Nevertheless, the interactions need to be recognized, explored, and diagnosed. Only a select few pulmonary complications of systemic diseases are described here. This chapter provides an overview of the most common and frequent changes observed in chronic hepatic, gastrointestinal, renal, metabolic and endocrine, hematological, and cutaneous disorders. The occurrence of lung disease in multisystem connective tissue diseases and vasculitides is widely recognized and described elsewhere in this encyclopedia.

The Liver–Lung Interface The interests of the pulmonologist and the hepatologist overlap in many ways. Both acute and chronic liver diseases affect pulmonary circulation. The lungs and liver can be simultaneously involved in many infections, such as tuberculosis, brucellosis, Q fever, and histoplasmosis; granulomatous disease, such as sarcoidosis; and genetic anomalies, such as a1-antirypsin deficiency. Autoimmune diseases may involve both organs. A disease may start in the lung but be diagnosed because of hepatic involvement. For instance, cancer from the lung or the illness may start in the liver but be recognized because of the pleuropulmonary expression (e.g., amebiasis). Some of the common liver–lung relationships are described here (Table 1). Hyperventilation and Cirrhosis

Hyperventilation is a common respiratory manifestation of cirrhosis. Arterial hypoxemia may be the Table 1 Pulmonary complications of liver diseases Structure

Illness

Pulmonary circulation

Hyperventilation Hepatopulmonary syndrome Portopulmonary hypertension Bronchitis, bronchiectasis BOOP, LIP, NSIP, UIP Basal atelectasis Chest X-ray infiltrate Raised diaphragms Hydrothorax Chylothorax Thoracobiliary fistula

Lung parenchyma

Pleura

BOOP, bronchiolitis obliterans obstructive pneumonitis; LIP, lymphocytic interstitial pneumonitis; NSIP, non-specific interstitial pneumonitis; UIP, usual interstitial pneumonitis.

cause in some patients, but often the degree of hyperventilation is greater than one would expect based on the degree of arterial hypoxemia. Elevated ammonia levels may be responsible, but studies have been unable to demonstrate a consistent correlation between ammonia levels and arterial carbon dioxide tension in patients who are hyperventilating. The speculation here is that hyperventilation is caused by chronic interstitial lung edema related to decreased intravascular oncotic pressure. Hepatopulmonary Syndrome

Hepatopulmonary syndrome is a clinical triad of advanced liver disease, intrapulmonary vascular dilatations in the absence of a primary cardiopulmonary disease, and hypoxemia. Hypoxemia results from low ventilation–perfusion ratios in areas of precapillary/capillary dilatations and anatomic shunting through microvascular arteriovenous communications. A reduction in transfer factor is related to the thickening of the small veins and capillaries by a layer of collagen. The vasoactive substances that induce pulmonary vasodilatation are not precisely known, but the list includes nitric oxide (NO), endothelin-1, and arachidonic acid and its metabolites. Finger clubbing is frequent, and platypnea and orthodeoxia are common. Cyanosis and clubbing are common in patients with autoimmune hepatitis and long-standing cirrhosis. Portopulmonary Hypertension

As many as 20% of patients with advanced liver disease have a mean pulmonary artery pressure of more than 25 mmHg. Causes include hyperdynamic circulation, increased blood volume, and nonembolic pulmonary vasoconstriction/obliteration. The latter process is termed portopulmonary hypertension. Histological findings include medial and intimal hypertrophy, endothelial proliferation, and plexogenic/ fibrotic changes. These changes are indistinguishable from histological changes seen in patients with primary pulmonary hypertension. However, patients with portopulmonary hypertension have increased cardiac output (Table 2). Pleural Effusions

Pleural effusions, unilateral or bilateral, occur in approximately 10% of patients with advanced liver disease. These transudative effusions are frequently right-sided and rarely occur in the absence of ascites. Nevertheless, pleural effusion may develop in the patient without clinically evident ascites. Negative pleural space pressure is generated with inspiration draws in the ascitic fluid through small diaphragmatic

552 PULMONARY EFFECTS OF SYSTEMIC DISEASE Table 2 Diagnosis and management of hepatopulmonary syndrome and portopulmonary hypertension Feature

Hepatopulmonary syndrome

Portopulmonary hypertension

Diagnostic criteria

Chronic liver dysfunction Intrapulmonary vascular dilatation Hypoxemia No existing lung disease Diffusion–perfusion abnormality Spider angiomata

Mean PAP 425 PCWP o15 Portal hypertension No other cause of pulmonary hypertension Vasoconstriction Vascular thickening Vascular obliteration Vascular remodeling Dyspnea, syncope, chest pain Loud P2, TR murmur, hypoxemia Echocardiography, cardiac catheterization Treat pulmonary hypertension

Pathophysiology

Symptoms Signs Diagnostic studies Treatment

Dyspnea, platypnea Cyanosis, clubbing, orthodeoxia Echocardiography ( þ ) bubble study Liver transplant

Figure 1 Posteroanterior and lateral views of the chest showing extensive bullous emphysema in a 29-year-old Caucasian male with a1-antitrypsin deficiency.

defects called Lieberman’s pores. Patients with a mild to moderate amount of fluid have no symptoms, but dyspnea and hypoxemia are present in patients with massive pleural effusion. Aggressive diuretic therapy and salt restriction to reduce the volume of ascites constitute the medical treatment. Thoracentesis is usually ineffective. Pleurodesis is helpful in select cases but is not without risk. Transjugular intrahepatic portosystemic shunt greatly reduces the need for repeat thoracentases in symptomatic patients. Refractory hepatic hydrothorax due to uncontrollable ascites is an indication for liver transplantation. a1-Antitrypsin Deficiency

a1-Antitrypsin deficiency is synthesized in the rough endoplasmic reticulum of the liver. It comprises 80–90% of the serum a1-globulin and is an inhibitor

of trypsin and other proteases. The abnormal a1 synthesis evolves from a single-point gene mutation located on chromosome 14 (more than 75 abnormal a1 alleles exist), which is codominantly expressed in the hepatocytes. The spectrum of a1-antitrypsindeficient liver disease extends from liver failure and need for transplantation in childhood to asymptomatic individuals with no evidence of disease. Emphysema, the most common pulmonary manifestation of a1 deficiency, affects predominantly the lung bases (Figure 1). It is caused by the imbalance between the protective a1 protein and neutrophil elastase derived from leukocytes. Smoking accelerates the destructive process. Combined liver and lung manifestations of a1 deficiency in the same patient are rare. In select patients with progressive lung disease, replacement therapy with weekly intravenous a1 protein from pooled donors may slow the decline.

PULMONARY EFFECTS OF SYSTEMIC DISEASE 553 Table 3 Sarcoidosis and primary biliary cirrhosis: a comparison Feature

Sarcoidosis

Primary biliary cirrhosis

Sex Age (years) Pulmonary symptoms Chest X-ray Pruritus Jaundice Serum alkaline phosphatase Serum angiotensin-converting enzyme Mitochondrial antibody Kveim–Siltzbach test Bronchoalveolar lavage Liver biopsy Lung biopsy Prognosis

Almost equal 20–45 Yes Abnormal No No Raised Raised Normal Positive Lymphocytosis Solid, discrete granulomas Noncaseating granulomas Generally good

80% women Middle age No Normal Yes Yes Raised Raised Raised (98%) Negative Lymphocytosis Poorly formed granulomas No granulomas, lymphocytes Generally poor

The replacement therapy has no role in the treatment of liver disease. Liver transplantation for a1-deficient childhood liver disease has a 3-year survival rate of 83–100%.

usually asymptomatic. Occasionally, they may result in an increase in alkaline phosphatase. Rarely, massive granulomatous inflammation may lead to liver fibrosis.

Primary Biliary Cirrhosis and Interstitial Lung Disease

Pulmonary Complications of Hepatic Transplantation

Primary biliary cirrhosis (PBC) is a disease of unknown origin in which intrahepatic bile ducts are progressively destroyed. T helper cells and cytotoxic T cells play an important role in the pathogenesis. The true incidence of lung disease in PBC is very low. However, many observations point to the existence of subclinical or asymptomatic lung inflammation in these patients. In early stages of PBC, bronchoalveolar lavage may reveal lymphocytic alveolitis similar to that seen in patients with sarcoidosis and Crohn’s disease. Gas transfer studies are abnormal, particularly in patients who have Sjogren’s or CREST syndrome in association with PBC. Nevertheless, severe restrictive abnormalities in pure PBC patients are rare. Chest X-ray abnormalities include hilar adenopathy, nodules, and interstitial reticular opacities. Lung biopsy specimens in patients with associated Sjogren’s syndrome have shown giant cells. Occasionally, noncaseating granulomas resembling sarcoidosis have been described; however, there are many differences between PBC and sarcoidosis (Table 3).

Chronic pulmonary disease may be a contraindication to liver transplantation. Severe hypoxemia due to right-to-left shunting with an arterial oxygen pressure of less than 50 mmHg is an absolute contraindication. In the early postoperative days after transplantation, the right diaphragm is paralyzed and right lower lobe atelectasis is common. Pleural effusions occur in almost all patients, but only 18% require aspiration. Pulmonary edema can occur from excess fluids and blood products during the operation. Later in the course, complications include pneumonias caused by bacteria, fungi, viruses, as well as Legionella and Pneumocystis carinii. Many of these infections occur after discharge from the hospital in patients with well-functioning livers. Azathioprine and cyclosporine can cause interstitial pneumonitis and acute respiratory distress syndrome (ARDS), respectively.

Gastrointestinal System and the Lungs Gastroesophageal Reflux Disease

The Liver in Sarcoidosis

Granulomas are found in 4–10% of needle biopsies, and in 10% of these no cause is found even after extensive investigation. Hepatic granulomas are always part of a generalized disease process. Approximately two-thirds of patients with systemic sarcoidosis have granulomas in the liver. Thus, liver biopsy may offer the diagnosis when other less invasive techniques have failed. Hepatic granulomas in sarcoidosis are

Cough can be the sole presenting symptom of gastroesophageal reflux. It is caused by one of three mechanisms. Reflux of stomach contents may irritate the esophageal mucosa and initiate the cough reflex through vagal sensory pathways. Aspirated gastric material may irritate sensory receptors of the tracheobronchial tree. Lastly, stomach contents may reach the hypopharynx and larynx, irritating the afferent limb of the cough reflex without aspiration.

554 PULMONARY EFFECTS OF SYSTEMIC DISEASE

Diagnosis is certain only when the cough stops in response to antireflux therapy. Pulmonary Complications of Esophageal Sclerotherapy

Esophageal varices are injected acutely at the time of bleeding; the remaining varices are obliterated later in the course of the illness. Fever, chest pain, usually retrosternal and nonpleuritic, and dysphagia are frequent complications. Pleural effusions – right, left, or bilateral – are frequent in patients who have chest pain and have a large volume of sclerosant injected. The fluid is usually an exudate. Aspiration pneumonia is another complication. Rarely, ARDS may complicate esophageal sclerotherapy with sodium morrhuate. Bronchoesophageal fistula formation has been described after endoscopic sclerotherapy.

bronchiectasis. Biopsies of the lung tissue usually show thickening of the epithelium and basement membrane with inflammatory cell infiltration of the underlying connective tissue. On the other hand, in Crohn’s disease subclinical inflammatory lung disease is frequent. Bronchoalveolar lavage fluid from patients with Crohn’s disease has shown that lymphocytes predominate with increased T4 subset during active disease. Despite the presence of alveolar lymphocytosis, there have been only a few reported cases of otherwise unexplained lung disease in these patients. The mechanisms leading to alveolar lymphocytosis in Crohn’s disease and its relationship with sarcoidosis remain uncertain.

Endocrine and Metabolic Disease Diabetes Mellitus

Pancreatitis

The lungs may be involved in 50–70% of patients with acute pancreatitis. Abnormalities include asymptomatic reduction in arterial oxygenation, significant hypoxemia with a normal chest radiograph, non-specific opacities, pleural effusion, and ARDS. The latter occurs in approximately 15% of the patients with acute pancreatitis and carries a poor prognosis. The onset of pulmonary symptoms in the patient with acute pancreatitis portends a poor prognosis. Sixty percent of deaths from acute pancreatitis that occur during the first week are associated with respiratory failure. Only 25% of those who require mechanical ventilation survive. During and after the second week, pulmonary complications are usually the result of pancreatic infection or pseudocyst formation. Pleural effusions and ascites reflect the severity of the illness. Inflammatory Bowel Disease

Chronic airway inflammation, both bronchiectasis and purulent bronchitis, is common in inflammatory bowel disease, particularly in ulcerative colitis. Involvement of the small airways is unusual. Organizing pneumonia, non-specific interstitial pneumonitis, and eosinophilic pneumonias have been described in ulcerative colitis. The pulmonary manifestations may appear at any time during the course of the bowel disease, but they follow the clinical onset of inflammatory bowel disease in 80% of cases. The airway inflammation is unrelated to the activity of the bowel disease or therapy. Dyspnea, fever, cough, expectoration, and chest pain are common and non-specific symptoms. In early stages, the chest radiograph may be normal and pulmonary function testing may reveal no consistent abnormality. Effective therapy of ulcerative colitis may prevent the worsening of

Patients with diabetes mellitus have an increased incidence of acute and chronic pulmonary infections. Pneumococcal pneumonia occurs with higher frequency, but Staphylococcus aureus and Escherichia coli are also frequent. The mechanisms behind the increased susceptibility of a diabetic patient to these infections include impaired chemotactic, phagocytic, and bactericidal activities of the neutrophils. Hyperglycemia decreases intracellular bactericidal activity of lymphocytes. The diminished phagocytic ability of monocytes increases the risk of fungal infections, particularly mucormycosis. Tuberculosis reactivation is two or three times more common in malnourished diabetic patients. Tuberculosis in these patients tends to affect the lower lobes. Sixty percent of nonsmoking, non-insulin-dependent diabetics have a mild reduction of the elastic recoil of the lung and diffusing capacity. Abnormalities of airflow or gas distribution are uncommon; however, the vagus-mediated basal airway tone is altered. Ventilatory responses to hypoxia and hypercapnia are abnormal due to altered function of the peripheral and central chemoreceptors. Hyperthyroidism

Dyspnea is common in thyrotoxicosis. Its cause is unclear, and its severity varies. Increased oxygen consumption, increased carbon dioxide production, increased minute ventilation, decreased vital capacity, impaired diffusion capacity, low lung compliance, respiratory muscle weakness, and increased central ventilatory drive have all been implicated in causes of dyspnea. Hypoxic and hypercapnic ventilatory drives are increased in patients with thyrotoxicosis compared to healthy controls; treatment of the disease normalizes both. Thyrotoxicosis patients tend to have high-output left ventricular failure. An

PULMONARY EFFECTS OF SYSTEMIC DISEASE 555

enlarged gland can compress the trachea and cause wheezing and strider. Hyperactivity of the airways has also been observed. Hypothyroidism

Hypothyroidism decreases central ventilatory sensitivity to hypoxia and hypercapnia. These abnormalities return to normal after successful treatment of hypothyroidism. Severe hypothyroidism can lead to respiratory failure and coma. Unsuspected hypothyroidism is one of the frequently overlooked causes of failure to wean from mechanical ventilation. Myxedema severe enough to cause respiratory failure and sleep apnea is associated with a decrease in tendon reflexes. The airway obstruction may be worsened by an enlarged tongue or oropharyngeal narrowing due to mucopolysaccharide and protein deposits in the soft tissue. Myopathic changes in the muscles may rarely occur. Transudative pleural effusions, unilateral or bilateral, have been observed. Parathyroid Disease

Both hyperparathyroid and hypoparathyroid patients can have sufficient neuromuscular weakness causing restrictive pulmonary impairment. Chest X-ray in hyperparathyroid patients may reveal osteopenia and osteitis fibrocystica, particularly in the clavicles and scapula. Occasionally, soft tissue calcifications are present. Pituitary and Adrenal Glands

Patients with acromegaly, particularly men, have increased lung volumes without airway obstruction or air trapping. The diffusing capacity remains normal, indicating that lung enlargement results from an increase in size rather than in the number of alveoli. There is an increase in the frequency of sleep apnea in patients with excessive growth hormone secretion. It can be central or obstructive in origin. Reproductive System

The triad of pleural effusion, ascites, and ovarian fibroma is called Meigs–Salmon syndrome. The syndrome also occurs in association with other ovarian and pelvic neoplasms, including fibroma, coma, granulosa cell tumor, Brenner tumors, adenocarcinoma, and fibroma of the uterus. Ascites is due to fluid secretion by the tumor. The pleural effusion, predominantly right-sided, results from ascitic fluid seeping through pores in the diaphragm. The effusion is frequently a transudate. Occasionally, the effusion may be left-sided or bilateral. Removal of the pelvic tumor causes the disappearance of hydrothorax as well as ascites.

Renal Disorders and the Lungs Chronic Renal Disease

A number of pleuropulmonary abnormalities complicate the course of chronic renal disease. Pulmonary edema, pleural effusion, pleural thickening and calcification, and pulmonary artery atherosclerosis and hypertension are well-described manifestations of renal disease. Urinothorax is an unusual cause of pleural effusion. The pleural fluid is a transudate with a creatinine level higher than that of serum creatinine and a pHo7.30. It is caused by extravasation of urine from the retroperitoneal space into the thorax. Pulmonary Complications of Renal Dialysis

Transient hypoxemia can occur within 15 min of initiation of hemodialysis. It can sometimes be severe enough to exacerbate myocardial ischemia. The decrease in PaO2 is related to acetate in the bath that causes hypoventilation and increased oxygen consumption. Dialysate of carbon dioxide and bicarbonate causes hypocapnia with a lag in bicarbonate regeneration from acetate metabolism. High-bicarbonate baths cause metabolic alkalosis. The compensating hypoventilation results in hypercapnia, which can be prevented by decreasing the bicarbonate concentration in the bath. Hypoxia can also result from complement activation by a bioincompatible dialyzer membrane causing leukostasis and plugging of the pulmonary capillaries. Using synthetic membranes and a citrate anticoagulant can reduce this effect. In some cases, hypoxemia may persist after dialysis has ended or develop in the postdialysis period.

Hematological Diseases and Lung Disease Sickle Cell Anemia

The combination of pleuritic chest pain, fever, cough, and parenchymal infiltrates on chest radiograph constitutes the acute chest syndrome. Infectious pneumonia and sickling of the abnormal red blood cells, either singly or in combination, are usually responsible. Rib infarctions, which are commonly observed radiologically, may also play a role. In situ thrombosis leading to pulmonary infarction may also occur. Infants and children commonly present with symptoms of infection, whereas adults usually have chest pain. The involvement of other organs – causing hemiplegia, an altered mental status, renal failure, and petechiae – suggests fat embolism. Recurrent episodes of acute chest syndrome can cause chronic lung disease. Hypoxemia is frequent in acute chest syndrome. Although oxygen may reduce the red cell

556 PULMONARY EFFECTS OF SYSTEMIC DISEASE

sickling, it does not alter the duration of the event, presumably because the damage due to vaso-occlusion occurred before oxygen administration. High oxygen concentration, in excess of that necessary to maintain a safe level of saturation, has not been recommended because it may suppress erythropoiesis. However, the suppression of erythropoiesis in this situation is poorly defined. The hemoglobin level and reticulocyte count can decline in patients on oxygen therapy. Monitoring is advised, although it is believed that oxygen concentrations of less than 50% do no harm; however, discomfort can be associated with the administration of oxygen therapy. Bone Marrow Transplantation

Patients undergoing bone marrow transplantation after aggressive salvage chemoradiation therapy are at high risk to develop pulmonary complications. Within the first 100 days after transplantation, pulmonary edema from fluid overload or myocardial injury, diffuse alveolar hemorrhage from cyclosporine toxicity, and transfusion reactions are seen. ARDS may be caused by pneumonia and sepsis due to bacterial and fungal organisms. Interstitial pneumonitis due to Pneumocystis carinii or cytomegalovirus, chemotherapy, or pulmonary embolism and veno-occlusive disease can occur during this period. Late complications are bronchopneumonia from the aforementioned infections, fat embolism, venoocclusive disease, interstitial pneumonitis of a nonspecific nature, bronchiolitis obliterans, organizing pneumonia, lymphocytic interstitial pneumonitis, and graft-versus-host disease. Severe supraglottic airway obstruction due to Epstein–Barr virus lymphoproliferative disease has been reported in children.

Dermatological Disorders and Lung Disease There are many multisystem disorders that affect the skin and lungs. This section includes clinical syndromes in which skin involvement is frequent, familiar, and easily recognizable, whereas the associated pulmonary disease is relatively infrequent, often overlooked, and not uncommonly misdiagnosed. Telangiectasia

Hereditary hemorrhagic telangiectasia (Osler–Weber– Rendau disease) is an inherited autosomal-dominant familial disorder with mutations localized to chromosome 9q3. This gene encodes endoglin, the transforming growth factor beta binding protein. The classical triad of hereditary hemorrhagic telangiectasia consists of epistaxis, telangiectasia, and a positive family history. Symptoms and signs due to pulmonary

arteriovenous malformations (PAVMs) occur between the fourth and sixth decades of life and include dyspnea, cyanosis, hemoptysis, clubbing, and a bruit. Because most of the PAVMs are located in the lower lung fields, orthodeoxia is a feature in some patients. Platypnea or improvement in dyspnea in the flat position is due to decreased blood flow through the pulmonary arteriovenous communications. On chest radiograph, this malformation can appear as a small compact nodule or a ‘comma-like density’ with vascular connections to hilar structures (Figure 2(a)). Conventional tomography, magnetic resonance imaging, and angiography are helpful in delineating the atrioventricular malformation and its vascular connections (Figure 2(b)). Occasionally, a vascular tumor may have an appearance on computed tomography suggestive of a vascular malformation. The definitive therapy in symptomatic patients includes embolotherapy and resection. Ataxia–telangiectasia syndrome is a recessively transmitted triad of sinopulmonary infections, telangiectasia, and cerebellar atrophy. The illness appears in childhood with ataxia and lower respiratory infections; telangiectasias follow. Hypoplasia of the thymus causes in vivo and in vitro impairment of delayed-type hypersensitivity. Immunoglobulin synthesis is stunted. Death results from either respiratory failure or lymphoma. Nodules, Albinism, and Yellow Nails

The lungs are affected in approximately 10% of neurofibromatosis (Recklinghausen syndrome) patients. The pulmonary changes usually appear after adolescence and consist of intrathoracic neurofibromas, interstitial lung disease, and honeycombing. Dyspnea is the main symptom; crackles and finger clubbing are rare. Tuberous sclerosis results from a genetic abnormality in the development of mesodermal tissue. The classic triad of epilepsy, mental retardation, and adenoma sebaceum is diagnostic. Lung involvement consists of interstitial lung disease, honeycombing, and spontaneous pneumothorax. Women are more commonly affected than men, and the average age of onset of symptoms is 34 years. The average life expectancy after the appearance of symptoms is 5 years. Cor pulmonale and spontaneous pneumothorax are the causes of death. Albinism, pulmonary fibrosis, and platelet dysfunction occur in Hermansky–Pudlak and related syndromes. Ceroid lipofuschin inclusions, a sine qua non of the disease, are found in the reticuloendothelial system. The incidence of pulmonary involvement is twice as high in women as in men. Dry cough and

PULMONARY FIBROSIS 557

Figure 2 (a) Posteroanterior view of the chest showing nodular densities in both lungs in a 30-year-old woman with a history of hemoptysis. (b) Pulmonary angiogram in the same patient showing multiple arteriovenous malformations.

dyspnea first appear in the third and fourth decades. The disease may remain stable, progress slowly, or progress rapidly to end-stage fibrosis and death. Yellow nail syndrome is a triad of yellow nails, lymphedema, and pleural effusion. Other pulmonary complications are bronchiectasis, sinusitis, and lower respiratory infections. Impaired lymphatic drainage is the underlying abnormality. See also: Chronic Obstructive Pulmonary Disease: Emphysema, Alpha-1-Antitrypsin Deficiency. Gastroesophageal Reflux. Interstitial Lung Disease: Overview. Neurofibromatosis. Pleural Effusions: Overview. Pulmonary Fibrosis. Sleep Apnea: Overview. Systemic Disease: Sarcoidosis; Sickle Cell Disease. Vascular Disease.

Further Reading Eikenberry M, Bartakova H, Defor T, et al. (2005) Natural history of pulmonary complications in children after bone marrow

transplantation. Biology of Blood & Marrow Transplantation 11(1): 56–64. Fuchizaki U, Miyamori H, Kitagawa S, et al. (2003) Hereditary haemorrhagic telangiectasia (Rendu–Osler–Weber disease). Lancet 362: 1490. Hattori H, Hattori C, Yonekura A, and Nishimura T (2003) Two cases of sleep apnea syndrome caused by primary hypothyroidism. Acta Oto-Laryngologica. Supplementum 550: 59–64. Ingbar DH (2000) The pulmonary system in thyrotoxicosis. In: Braverman LE and Utiger RD (eds.) Werner and Ingbar’s the Thyroid. A Fundamental and Clinical Text, 8th edn. Philadelphia: Lippincott Williams & Wilkins. Murray J (1992) Pulmonary Complications of Systemic Disease, Lung Biology in Health Disease, vol. 59. New York: Dekker. Nassar A, Ghobrial G, Romero C, et al. (2004) Culture of Mycobacterium avium subspecies paratuberculosis from the blood of patients with Crohn’s disease. Lancet 363: 1039–1044. Saner F, Lang H, Fruhauf N, et al. (2003) Postoperative ICU management of liver transplantation patients. European Journal of Medical Research 8: 511–516. Sharma O (1988) Pulmonary manifestations of systemic disease. Seminars in Respiratory Medicine 9(3). Taylor C, Carter F, Poulose J, et al. (2004) Clinical presentation of acute chest syndrome in sickle cell disease. Postgraduate Medicine Journal 80(944): 346–349.

PULMONARY FIBROSIS D G Morris, Yale School of Medicine, New Haven, CT, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Fibrosis of the lung results from relentless replacement of normal elastic lung with inflexible scar. This is caused by environmental exposures, systemic inflammatory diseases, and by other

as yet unknown cellular events. It complicates a range of diseases including connective tissue diseases, sarcoidosis, and chronic hypersensitivity pneumonitis. It occurs without known cause in the disease idiopathic pulmonary fibrosis. Idiopathic pulmonary fibrosis is largely untreatable, progressive, and causes substantial morbidity with an approximately 50% 3year mortality rate. The fundamental biological processes that have been implicated in the pathogenesis of pulmonary fibrosis are: (1) altered regulation of inflammation and cytokine imbalance; (2) altered regulation of apoptosis; (3) oxidant-mediated

558 PULMONARY FIBROSIS lung injury; (4) dysregulated wound repair and epithelial to mesenchymal transformation; (5) protease/antiprotease imbalance; and (6) altered eicosanoid homeostasis. This manuscript will review our current biological understanding of pulmonary fibrosis in the context of these six fundamental processes and provide a framework for the interpretation of future studies in both animal models and patients with disease. Continued focused scientific and therapeutic investigations are critical to progress in the treatment of these vexatious lung diseases.

Historical Overview Progressive scarring in the lung, or pulmonary fibrosis, has plagued mankind since antiquity (see Interstitial Lung Disease: Hypersensitivity Pneumonitis. Occupational Diseases: Hard Metal Diseases – Berylliosis and Others; Silicosis. Pneumonia: Fungal (Including Pathogens); Mycobacterial). Beginning in the late nineteenth century, the concept of chronic fibrotic lung disease emerged. As Sir William Osler presciently and succinctly noted in the first (1892) edition of his classic textbook The Principles and Practice of Medicine: ‘‘So diverse are the different forms [of chronic interstitial pneumonia or cirrhosis of the lung] and so varied the conditions under which this change occurs that a proper classification is extremely difficult.’’ This situation remains little changed today.

Biological Paradigms of Pulmonary Fibrosis Chronic alveolitis, or inflammation of the distal bronchoalveolar gas exchange unit, presages fibrosis in many clinical conditions. Inflammation associated with lung injury is also a major feature of the principal experimental model of pulmonary fibrosis, bleomycin-induced lung injury. Bleomycin, which is derived from the bacteria Streptomyces verticillus, is a chemotherapeutic agent that is used to treat a range of human malignancies. It causes pulmonary fibrosis, particularly after high cumulative dosing. In mice, when administered directly through the trachea, by prolonged subcutaneous infusion, or through repeated injections, bleomycin also causes lung injury followed by a stereotypical, strain-dependent pulmonary fibrosis. Although bleomycin-induced pulmonary fibrosis is the dominant experimental model for human pulmonary fibrosis, many investigators question its validity. This skepticism is based on the model’s dependence on both the development of lung injury and the necessity for inflammation prior to the development of fibrosis. These do not occur in all forms of human fibrotic lung disease. Also, the fibrosis of bleomycin-induced lung injury is centered on distal airways and within alveolar spaces whereas idiopathic pulmonary fibrosis in humans is peripheral,

subpleural, and interstitial. Nevertheless, the effects of drugs, biological agents such as antibodies, and genetic manipulations on the development of pulmonary fibrosis are almost universally tested in this experimental system because of its ease, reproducibility, and broad scientific acceptance. Indeed, the past decade has witnessed an explosion of genetic and pharmacologic data on bleomycininduced pulmonary fibrosis in rodent models principally in genetically manipulated mice. The molecular pathways implicated are summarized in Table 1. An overview of the data suggests six general biological pathways that are pivotal to the development, maintenance, or resolution of experimental pulmonary fibrosis. These are: (1) cytokine imbalance and altered regulation of inflammation; (2) abnormal regulation of apoptosis; (3) oxidantmediated lung injury; (4) dysregulated wound repair and epithelial mesenchymal transformation; (5) protease/antiprotease imbalance, including altered homeostasis of the coagulation/anticoagulation system; and (6) altered eicosanoid metabolism. Genetic and pharmacologic studies in mice suggest that each of the components of these pathways is critical to the fibrotic phenotype that follows bleomycin administration because the alteration of any one is generally sufficient to prevent or exacerbate fibrosis and/or inflammation. The interactions among these pathways are complex, cell-type specific, and very poorly understood. Persistent Inflammation and Cytokine Imbalance

The response of the lung to acute injury apparently follows a stereotypical pattern. In acute lung injury produced by infectious, mechanical, or systemic inflammatory responses, normal endothelial and epithelial barriers to protein and solute flow are lost. This causes alveolar flooding, the formation of an acellular fibrin clot, and the elaboration of a provisional extracellular matrix. The injury causes both epithelial apoptosis and necrosis. Recent studies from genetically manipulated mice suggest that epithelial apoptosis may be both proinflammatory and profibrotic. This contrasts with the anti-inflammatory effect of lymphocyte apoptosis. Key cytokines, chemokines, and cell adhesion molecules, such as interleukin-1 beta, macrophage inflammatory protein (CXCL2), interferon gamma, and the leukocyte adhesion molecules L-selectin, and inflammatory cell adhesion molecule (ICAM), recruit macrophages, circulating monocytes, lymphocytes, and neutrophils. These cells constitute the acute inflammatory response at the site of injury. Following closely on the heels of this injury, the lung initiates the resolution

PULMONARY FIBROSIS 559 Table 1 Molecule

Functional class

Inferred fibrotic/inflammatory role

5-Lipoxygenase Angiotensin 1 receptor Bleomycin hydrolase Cathepsin K CCL6 (C10) CD44 (hyalurinic acid receptor) CD28 Cox2 CXCL2 (MIP2) CXCL10/IP10 Ccr2 CXCR3 Connective tissue growth factor (CTGF) Cytosolic phospholipase A (2) Decorin Endothelin 1 Endothelin receptor A, B Egr 1 Granulocyte-macrophage colony stimulating factor ICAM IL1b IL-10 IL-13 Integrin avb6 IFN-g

Paracrine mediator synthesis Cell signaling, vasoconstriction Cysteine protease Cysteine protease Chemokine ECM receptor Cell surface receptor, co-stimulatory Paracrine factor enzyme Chemokine Chemokine Chemokine receptor Chemokine receptor Cytokine Paracrine mediator synthesis Extracellular protein Cell signaling, ECM synthesis Cell signaling, ECM synthesis Cell signaling, apoptosis Cytokine

Proinflammatory/profibrotic Profibrotic Anti-inflammatory/antifibrotic Antifibrotic Proinflammatory/profibrotic Anti-inflammatory Proinflammatory/profibrotic Proinflammatory/antifibrotic Proinflammatory/profibrotic Antifibrotic Proinflammatory/profibrotic Antifibrotic Profibrotic Proinflammatory Antifibrotic Profibrotic Profibrotic Profibrotic Antifibrotic

Adhesion molecule Cytokine Cytokine Cytokine Cell surface receptor, TGF-b activation Cytokine

L-selectin Leukotriene C4 Leukotriene E4 Mast Cell Chymase MMP-7 (matrilysin) Relaxin TNF-a TGF-a Thrombin Tissue Plasminogen Activator (tPA) p38 MAPK PAI-1 PDGF Plasminogen Prostaglandin D2 synthase Prostaglandin E2 Smad 3 Smad 7 Superoxide dismutase Tissue inhibitor of metalloprotease TGF-b1 Urokinase-type plasminogen activator

Adhesion molecule Paracrine factor Paracrine factor Extracellular protease Metalloprotease Paracrine factor Cytokine Cytokine Clotting factor, paracrine factor Extracellular protease activator Cell signaling Extracellular protease inhibitor Cytokine Extracellular protease Paracrine factor synthesis Paracrine factor Intracellular signaling Intracellular signaling inhibitor Extracellular enzyme Protease inhibitor Cytokine Extracellular protease activator

Proinflammatory Proinflammatory Anti-inflammatory Proinflammatory/profibrotic Anti-inflammatory/profibrotic Proinflammatory/profibrotic (by LOF), antifibrotic (by GOF) Proinflammatory/profibrotic Profibrotic Profibrotic Antifibrotic Proinflammatory/profibrotic Antifibrotic Antifibrotic ( þ / ) Profibrotic Profibrotic Antifibrotic Profibrotic Profibrotic Profibrotic Antifibrotic Anti-inflammatory/antifibrotic Antifibrotic Profibrotic Antifibrotic Antifibrotic Antifibrotic Anti-inflammatory/profibrotic Antifibrotic

phase during which the amplification of the inflammatory response is arrested, wound repair processes are initiated and, ideally, lung structure is returned to normal. The pleiotropic cytokine transforming growth factor beta 1 (TGF-b1) is critical to the development of lung injury and the switch from inflammation to resolution. Failure to resolve fully lung inflammation and complete normal repair causes fibrosis. In humans

this is clearly demonstrated in patients with underlying connective tissue diseases or chronic lung infections with fungal or mycobacterial pathogens. Indeed, the pneumoconioses, sarcoidosis, chronic hypersensitivity pneumonitis, and connective tissue disease associated interstitial lung diseases are fibrotic lung diseases in which foci of fibrosis are closely associated with accumulated chronic inflammatory cells. In these states, interstitial lung

560 PULMONARY FIBROSIS

inflammation does not resolve normally but is evidently still sufficient to trigger a healing or fibrotic phase of repair. This progression of inflammation to fibrosis is also seen following acute lung injury in the late, or the so-called fibroproliferative, stage of acute respiratory distress syndrome. However, the role of inflammation as the primary cause of all fibrotic lung disease remains a matter of spirited debate. The principal evidence weighing against inflammation as the sole mover of fibrosis is the disease idiopathic pulmonary fibrosis, in which interstitial inflammation is sparse or even absent, and in which intense immunosuppressive therapy has been tried without substantial benefit (see Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis). As in humans, bleomycin causes intense inflammation in susceptible strains of mice prior to the development of pulmonary fibrosis. Therefore, the fact that many inflammatory mediators have been implicated in fibrotic lung disease in mice is perhaps not surprising (Table 1). Of all the cytokines and chemokines associated with pulmonary fibrosis, the most consistently identified and best validated target in mice is TGF-b1 (see Transforming Growth Factor Beta (TGF-b) Family of Molecules). TGF-b1 is a pleuripotent cytokine that, broadly speaking, has four principal functions in the postnatal lung: inhibiting epithelial growth and inducing epithelial apoptosis, promoting fibroblast proliferation, enhancing the synthesis of multiple collagen isoforms and elastin, and inducing an anti-inflammatory state by downregulating both activated macrophages and lymphocytes. Excessive TGF-b1 activity has been conclusively shown to produce lung fibrosis in a multitude of models, and the prevention of the activation of latent TGF-b1 prevents both acute lung injury and fibrosis in response to bleomycin. Interestingly, pharmacologic blockade of TGF-b1 activity after the initiation of fibrosis, while limiting damage, has not yet been shown to cause complete resolution of fibrosis and return to normal lung architecture. Whether this is due to persistent downstream activity in other signaling pathways that prevent resolution or incomplete blockade of TGF-b1 activity in vivo remains undetermined. TGF-b1 (as well as TGF-b2 and TGF-b3) is constitutively secreted in inactive form. TGF-b1 is kept inactive and closely associated with the extracellular matrix through a noncovalent association with latency-associated protein and latent TGF-b1 binding protein. The principal regulatory step controlling the in vivo effects of TGF-b1 is its activation. Genetic manipulations in mice that block TGF-b1 activation in vivo have been shown to be sufficient to block the development of pulmonary fibrosis following administration of bleomycin (see

Transforming Growth Factor Beta (TGF-b) Family of Molecules). Altered Cellular Apoptosis

The role of apoptosis in lung disease – particularly fibrotic lung disease – is complex and cell-type specific. Epithelial apoptosis is prominent in a number of lung diseases including acute lung injury, pulmonary emphysema, and lung cancer. It is gaining ascendance in pulmonary fibrosis as well. The regulation of fibroblast apoptosis, or more specifically apoptosis of the more differentiated and contractile fibroblast subtype, the myofibroblast, is also the focus of increased scrutiny. Recent data from genetically manipulated mice suggest that both Fas/Fas Ligand-mediated and non-Fas-mediated apoptotic pathways play important regulatory roles. Data from TGF-b1 transgenic mice have shown not only that overexpression of active TGF-b1 induces pulmonary fibrosis, but that blockade of the striking epithelial apoptosis induced by overexpression of this cytokine largely ameliorates the fibrotic phenotype. Immunohistochemical evidence of increased epithelial apoptosis has also been noted in the lungs of patients with pulmonary fibrosis. Other studies from primary fibroblasts isolated from patients with fibrotic lung disease suggest intrinsic differences in the apoptotic machinery in these cells leading to decreased apoptosis. Isolation of fibroblasts from these patients shows increased fibroblast survival in response to inflammatory stimuli (interleukin-6) rather than the more normal increase in fibroblast apoptosis after such stimulation. The apoptosis story differs too among lymphocytes. Interestingly, selective deletion of Fas expression in T lymphocytes leads to increased surface Fas Ligand (FasL) on activated T cells. Among other things, this results in increased peripheral apoptosis of lymphocytes and an accumulation of leukocytes within the lung, eventually leading to pulmonary fibrosis. There have been no systematic studies of lymphocyte homeostasis in the lungs of patients with pulmonary fibrosis. In summary, the predominance of current data suggests that increased epithelial apoptosis and decreased fibroblast apoptosis are characteristic of fibrotic lung disease. These may both be the result of increased cytokine activity, particularly TGF-b1, or the result of the alteration of a combination of cellspecific intra- and extracellular signaling events, perhaps as a response to alterations in the mechanics of the extracellular environment. The role of lymphocyte apoptosis in human disease is uncertain. Moreover, the role of apoptosis in general as the primary


initiating event or the consequence of a more proximal derangement remains unknown. No agents specifically targeting apoptosis are in clinical trials for pulmonary fibrosis. Oxidant-Mediated Lung Injury

Oxidant-mediated lung injury has been implicated in a wide variety of lung diseases including pulmonary emphysema, lung injury, and fibrosis. The activities of a number of extracellular proteins, proteases, and antiproteases are sensitive to the redox state in the extracellular space. These proteins transduce alterations in the redox state into cellular events such as altered intracellular signaling, activation or inactivation of cytokines, and matrix remodeling. The instruments of oxidant-mediated lung injury include superoxide anion, nitric oxide, hydrogen peroxide, and hypohalides. Many of these agents are produced endogenously as part of innate host defenses – particularly by the membrane-bound NADPH system of phagocytes, and by the actions of nitric oxide synthase. Inducible nitric oxide synthase 2 (NOS2A), in particular, is abundant in the lung and several resident lung cells dramatically induce its expression after stimulation by proinflammatory cytokines or endotoxin exposure. The lung has a well-developed mechanism for buffering oxidant stress under normal conditions. These chemical buffers include reduced glutathione, several serum proteins including albumin, cerruloplasmin, transferrin, vitamins C and E, as well as the enzymes superoxide dismutase and catalase. Glutathione is by far the most important antioxidant in the lung with concentrations in alveolar lining fluid that are over 100 times higher than circulating levels. The level of reduced glutathione, which is capable of scavenging oxidant molecules, is decreased by chronic ethanol ingestion, and chronic inflammation. However, the level of glutathione is also tightly regulated in vivo with much of the regulation presumed to occur in lung epithelial cells themselves. Substrate availability – particularly cysteine and intracellular levels of the rate-limiting first synthetic enzyme glutamate cysteine ligase (GCL) are critical regulatory factors. GCL is a heterodimeric enzyme composed of a 73 kDa catalytic heavy-chain subunit (GCLC) and a 31 kDa modifier light-chain subunit (GCLM). Interestingly, TGF-b1 inhibits expression of the heavy-chain gene in immortalized respiratory epithelial cells and expression of both the heavy- and light-chain proteins is reduced in lung tissue from patients with idiopathic pulmonary fibrosis. Many studies have documented substantially lower levels of glutathione in the alveolar lining fluid

recovered from patients with pulmonary fibrosis. Some studies have suggested that use of oral N-acetylcysteine (NAC) effectively raises the levels of glutathione in the alveolar lining fluid and may also stabilize or improve lung function in idiopathic pulmonary fibrosis. In a recently completed and as yet unpublished trial in Europe (the Idiopathic Pulmonary Fibrosis International Group Exploring NAC I Annual; IFIGENIA), 1800 mg of NAC (600 mg po t.i.d.) reportedly improved vital capacity by 9% (po0.05) and the diffusing capacity for carbon monoxide by 24% (po0.005) compared to placebo after 1 year of treatment. Of note, all 155 patients in this randomized, placebo-controlled trial also received prednisone and azathioprine. Dysregulated Wound Repair and Epithelial Mesenchymal Transformation

Many authors have drawn an analogy between idiopathic pulmonary fibrosis and a nonhealing wound. The roles of appropriate epithelialization and epithelial-fibroblast interactions in limiting the progression of fibrosis have been of particular interest. Others have suggested that, under the particular and currently unknown tissue microenvironment that promotes ongoing fibrosis, epithelial cells undergo a fundamental phenotypic transition to become fibroblastic cells – a so-called epithelial to mesenchymal transformation. Such trans-differentiation of epithelial cells to mesenchymal cells has been well documented in chronic renal and liver fibrosis. Recently, recruitment of bone marrow-derived circulating collagen-producing CD34 þ cells called fibrocytes has also been implicated in experimental pulmonary fibrosis. In a series of elegant experiments, investigators fluorescently labeled bone marrow cells, which were engrafted into mice that did not express the tag. Following bleomycin injury, substantial numbers of fluorescently labeled fibroblasts were localized in areas of injury indicating the cells derived from transplanted marrow developed into scar tissue in the lung. Other investigators have shown that fibrocytes, circulating cells that express both the leukocyte marker CD34 and Col1, traffick to the lung following bleomycin-induced lung injury in response to CXCL12 (SDF-1), which is produced in the lung following injury. The role of these cells in chronic fibrotic lung disease or repair of acute lung injury in humans is unknown. Two molecular signaling pathways that have been implicated in this process of epithelial-mesenchymal cross-talk are sonic hedgehog, a secreted factor that is critical to early lung morphogenesis and that is also induced in response to lung injury, and Wnt

562 PULMONARY FIBROSIS

(pronounced ‘wint’), another secreted factor that is important in both epithelial migration and lung development. Expression of the protease matrilysin (MMP7) is induced by Wnt signaling. Matrilysin is also among the most induced genes in the lungs of patients with idiopathic pulmonary fibrosis based on global gene expression analysis. Matrilysin also plays a critical role in the regulation of the inflammatory response through its cleavage of syndecan 1 and the release of the neutrophil chemoattractant KC. Matrilysin-deficient mice have reduced neutrophil accumulation in the alveolar airspace and a delay in the development of pulmonary fibrosis following lung injury with bleomycin. This illustrates the tight interplay between parenchymal and inflammatory cell biology in vivo in response to injury and the complexity of dissecting the molecular unpinning experimentally. Another molecular pathway that has been implicated in mesenchymal cell proliferation and potentially epithelial to mesenchymal transformation in vivo is the endothelin-1 pathway. Endothelins are a family of three proteins (Edn1, Edn2, Edn3), which vary in size from 160 to 214 amino acids, that are broadly expressed and mediate contraction of vascular and intestinal smooth muscle through their actions on two G-protein-coupled receptors, endothelin receptors A and B. Endothelins play a critical role in the maintenance of vascular tone, cardiac remodeling, intestinal motility, and the maintenance of glomerular function. In humans, endothelin 1 – the best characterized of the endothelins – is most highly expressed in the lungs and cardiac myocytes. Constitutive transgenic overexpression of Edn1 in the lungs and kidneys of mice induces pulmonary fibrosis and glomerulosclerosis. As a result of these findings, a dual endothelin receptor blocker, bosentan, which is used in primary pulmonary hypertension, is now also used in clinical trials to assess for efficacy in pulmonary fibrosis in humans. Protease/Antiprotease Imbalance

Genes encoding proteases are among the most common in the human genome. They play critical regulatory roles in inflammation, tissue remodeling, cell death, and migration. In the extracellular space, proteases are particularly important in regulating the deposition and maintenance of extracellular matrix. Proteases are expressed by a wide range of cells and both their expression and secretion are tightly regulated. Beyond intracellular control mechanisms, protease activity is buffered by antiproteases. These molecules bind to and inhibit the proteolytic activity of proteases. Among the most important of these

protease inhibitors are alpha-1 antiprotease (alpha-1 antitrypsin), which is secreted by the liver and bathes the entire extracellular space; the tissue inhibitors of metalloproteases (TIMPs), which are produced locally and have particular substrate specificities; and the cystatins, which are inhibitors of cysteine proteases. Of these, the locally produced inhibitors TIMPs have attracted the most interest in the tissue level regulation of collagen and elastin deposition and removal. In general, deficiencies of these protease inhibitors are thought to result in a ‘profibrotic’ environment. Interestingly, another protease/antiprotease system, the thrombin/plasmin/plasmin-activator-inhibitor pathway, has also been implicated in pulmonary fibrosis in mice. The details of this pathway are outlined in Coagulation Cascade: Thrombin. In this system, an excessive amount of the TGF-b1inducible protease inhibitor plasminogen activator inhibitor-1 (PAI-1) inhibits the dissolution of provisional matrix by plasmin and therefore promotes fibrosis. Consistent with this hypothesis, mice deficient in the gene for PAI have reduced pulmonary fibrosis in response to bleomycin. Altered Eicosinoid Homeostasis

Eicosinoids are biologically active derivatives of arachidonic acid, which are liberated from cell membrane phospholipids by the action of phospholipase A2. Eicosinoids are produced in one form or another by every cell and are rapidly mobilized following calcium influx. There are three broad categories of eicosinoids: prostenoids, leukotrienes, and lipoxins. The principal prostanoids PGD2, PGF2a, PGE2, PGI2, and TxA2 (thromboxane) are derived from arachidonic acid through the enzymatic activity of cyclooxygenase-1 and -2. Leukotrienes, which are produced principally by inflammatory cells, result from the enzymatic activity of 5-lipoxygenase. Lipoxins are produced via 5-, 12-, and 15-lipoxygenases and are generally considered to be anti-inflammatory. All of the eicosinoids have a rapid onset of action and are quickly metabolized making them both ideal mediators of cell–cell communication. The topic of eicosinoid biology is more fully reviewed elsewhere (see Lipid Mediators: Overview; Leukotrienes; Prostanoids; Platelet-Activating Factors; Lipoxins). The role of eicosinoids in pulmonary fibrosis is complex and controversial. In general, experimental data in genetically manipulated mice support the notion that leukotrienes promote the development of fibrosis (since deletion of 5-LO, a key regulatory enzyme, is protective) and prostenoids prevent fibrosis (since deletion of cyclooxygenase-2 exacerbates


fibrosis). Elevated levels of 5-lipoxygenase metabolites have been demonstrated in both lavage fluid and tissue from patients with idiopathic pulmonary fibrosis, and alveolar macrophages are probably the principal source. The stimulus for this excessive 5-lipoxygenase activity, if any, is unknown. The principal prostenoid implicated in pulmonary fibrosis is PGE2. Reduced levels have been documented both in humans with fibrotic lung disease and in susceptible mouse strains following bleomycin-induced fibrosis – consistent with its presumed protective role. Substantial work remains to be done in this area, as the key regulatory events, cellular substrate, and postsignaling events – many of which may be celltype and cell-state dependent – remain poorly understood. Notably, both pharmacologic and dietary interventions alter the activity of this pathway making an improved understanding of this signaling

Normal alveolar structure

pathway in disease pathogenesis of particular immediate appeal. A single-center clinical trial is currently underway to assess the efficacy of a 5-LO inhibitor in human pulmonary fibrosis.

Moving Models from Mice to Man Despite the dramatic progress over the last 10 years in the understanding of critical molecular events underlying scarring in the lungs of mice following bleomycin, there has been frustratingly little progress in the area of therapeutics. Fortunately, if the initiation of clinical trials is any indication, pulmonary fibrosis has now caught the attention of the pharmaceutical industry and many new compounds are now in various stages of clinical development. Translating basic molecular insight into clinically useful pharmaceuticals has been hampered by several factors. These

Fibrosis

Air Pulmonary arterial blood

Pulmonary venous blood

Dense fibrosis

CXCR3 CCL6 Leukotrienes Cox2 PGE2

Type II alveolar epithelial cell

Alveolar capillaries

Collagen Fibronectin Vitronectin Hyaluronan

Repopulating stem cells

Alveolar macrophage

(a)

IL-1, IL-13 IFN-, IP-10 Alveolar O3 macrophage SOD GSH Latent TGF- Mra7 v6 Decorin Active TGF-

CD44

Epithelial to mesenchymal transformation AT1 ICAM thrmb ET1 L-selectin UPA tPA Myofibroblasts, PAI-1 fibrocytes Disruption of Egr 1 basement membrane Epithelial apoptosis CTGF PDGF

Type I alveolar epithelial cell

Mature collagen Basement membrane

Normal lung

(b)

Figure 1 Pulmonary fibrosis requires a complex, multicellular environment. (a) Normal lung architecture and function. The healthy lung is a delicate, reticular structure with close apposition of epithelial cells lining the airway and alveolar space. These cells are separated from underlying stroma by tight junctions between cells, and a continuous basement membrane composed of laminin, type IV collagen, entactin, and proteoglycans. The structural support of the lung, or stroma, contains blood vessels, structural cells such as fibroblasts that secrete elastin, collagen (fibrillar, nonfibrillar, and low molecular weight) and proteoglycans, and migratory leukocytes such as dendritic cells, lymphocytes and interstitial macrophages. (b) Molecular processes implicated in the fibrotic process; the role of epithelial denudation, disruption of the epithelial basement membrane, and activation of inflammatory and noninflammatory cells is emphasized. The net result of these processes is loss of functional gas exchange units through capillary drop-out, decreased alveolar compliance, and interstitial thickening (see Table 1 and text for abbreviations). Reproduced from Journal of Clinical Investigation, 2004, with permission.

564 PULMONARY FUNCTION TESTING IN INFANTS

include the relative infrequency of pulmonary fibrosis in comparison to other lung diseases such as asthma, lung cancer, and pulmonary infections. Further, the relatively indolent clinical course and lack of good surrogate markers of disease progression has led to a need for both prolonged trials and large patient numbers. Finally, idiopathic pulmonary fibrosis has only recently been clearly separated from the other myriad diseases that have fibrosis as a final common pathway. This separation of idiopathic pulmonary fibrosis from the other idiopathic interstitial pneumonias has had a sobering effect by highlighting both the grim survival (50% mortality on average at 3 years) and the poor response to conventional immunosuppressive therapies. Fortunately, as fundamental molecular insights have identified key molecular pathways, the pharmaceutical industry has developed more directed therapies. These include small molecule inhibitors, humanized monoclonal antibodies, and other targeted therapeutics. Progress in the future will depend on the ability to capitalize on these initial forays and to maintain the focus of both the scientific and private sectors on this devastating human disease (Figure 1). See also: Interstitial Lung Disease: Hypersensitivity Pneumonitis; Idiopathic Pulmonary Fibrosis. Lipid Mediators: Overview; Leukotrienes; Prostanoids; PlateletActivating Factors; Lipoxins. Occupational Diseases: Hard Metal Diseases – Berylliosis and Others; Silicosis. Pneumonia: Fungal (Including Pathogens); Mycobacterial. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading Borzone G, Moreno R, Urrea R, et al. (2001) Bleomycin-induced chronic lung damage does not resemble human idiopathic pulmonary fibrosis. American Journal of Respiratory and Critical Care Medicine 163(7): 1648–1653.

Chapman HA (2004) Disorders of lung matrix remodeling. Journal of Clinical Investigation 113(2): 148–157. Hashimoto N, Jin H, Liu T, Chensue SW, and Phan SH (2004) Bone marrow-derived progenitor cells in pulmonary fibrosis. Journal of Clinical Investigation 113(2): 243–252. Jiang D, Liang J, Hodge J, et al. (2004) Regulation of pulmonary fibrosis by chemokine receptor CXCR3. Journal of Clinical Investigation 114(2): 291–299. Kaminski N, Allard JD, Pittet JF, et al. (2000) Global analysis of gene expression in pulmonary fibrosis reveals distinct programs regulating lung inflammation and fibrosis. Proceedings of the National Academy of Sciences 97(4): 1778– 1783. Kolb M, Margetts PJ, Anthony DC, Pitossi F, and Gauldie J (2001) Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis. Journal of Clinical Investigation 107(12): 1529–1536. Kuhn C III, Boldt J, King TE Jr, et al. (1989) An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis. American Review of Respiratory Diseases 140: 1693–1703. Lee CG, Cho SJ, Kang MJ, et al. (2004) Early growth response gene 1-mediated apoptosis is essential for transforming growth factor beta1-induced pulmonary fibrosis. Journal of Experimental Medicine 200(3): 377–389. Lynch JP III (2004) Idiopathic Pulmonary Fibrosis. New York: Dekker. Morris DG, Huang X, Kaminski N, et al. (2003) Loss of avb6 integrin-mediated TGFb activation causes Mmp12 dependent pulmonary emphysema. Nature 422(6928): 169–173. Phillips RJ, Burdick MD, Hong K, et al. (2004) Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. Journal of Clinical Investigation 114(3): 438–446. Selman M, King TE Jr, and Pardo A (2001) Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy. Annals of Internal Medicine 134: 136–151. Teder P, Vandivier RW, Jiang D, et al. (2002) Resolution of lung inflammation by CD44. Science 296(5565): 155–158. Thannickal VJ, Toews GB, White ES, Lynch JP, and Martinez FJ (2004) Mechanisms of pulmonary fibrosis. Annual Review of Medicine 55: 395–417. Zuo F, Kaminski N, Eugui E, et al. (2002) Gene expression analysis reveals matrilysin as a key regulator of pulmonary fibrosis in mice and humans. Proceedings of the National Academy of Sciences 99(9): 6292–6297.

PULMONARY FUNCTION TESTING IN INFANTS J Stocks, Institute of Child Health, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Infant pulmonary function tests (IPFTs) can be used to assess lung volumes, forced expiratory maneuvers, and respiratory mechanics in healthy infants and those with respiratory disease. International guidelines for standardizing equipment and measurements have been published. Such measurements are more

complex and time consuming than in children and adults, requiring specially trained personnel and facilities. IPFTs are generally performed during sleep (which, beyond the neonatal period, generally requires light sedation), in the supine position, and while breathing through a face mask. There are marked differences in developmental respiratory physiology that must be considered when performing and interpreting results from infants, including the rapid lung and airway growth and the fact that infants are preferential nose breathers and have a highly compliant chest wall. Although IPFTs have occasionally been used in clinical management, their primary role has been in

PULMONARY FUNCTION TESTING IN INFANTS 565 epidemiological and clinical research, where they have been used to investigate early determinants of lung function (including the detrimental effects of maternal smoking, intrauterine growth retardation, and preterm delivery), the nature and severity of various respiratory diseases, airway reactivity, and response to therapeutic interventions, including different types of ventilatory management.

Introduction Measurement of pulmonary function is an integral component of respiratory physiology and clinical assessment of lung diseases in school-aged children and adults. Despite their heightened vulnerability to respiratory diseases, similar assessments in infants and young children were, until recently, restricted to specialized research establishments. This was largely due to the lack of suitable equipment and difficulties in performing such measurements in small, potentially uncooperative subjects. The realization that insults to the developing lung may have lifelong effects, and that much of the burden of respiratory disease in childhood and later life has its origins in infancy and fetal life, emphasized the need for sensitive and reliable methods of assessing respiratory function in this age group. During the past 10 years, there has been increasing commercial availability of equipment for assessing respiratory function of infants that, together with the publication of international guidelines for standardized methods of data collection and analysis, has facilitated more widespread use of these tests.

The Differences in Assessing Lung Function in Infants Compared to Older Subjects Developmental differences in respiratory physiology that impact both on measurements of infant lung function and on interpretation of results are summarized in Table 1. These include factors such as the highly compliant chest wall, the tendency of infants to modulate expiratory flows and timing to dynamically elevate functional residual capacity (FRC) above that determined by passive mechanisms, and preferential nose breathing during early life. Major differences in performing pulmonary function tests (PFTs) in children younger than 2 years of age are summarized in Table 2 and relate to the following: *

*

*

* *

Sleep state. Tests should generally be confined to periods of quiet sleep, with a regular breathing pattern. Sedation. Usually necessary beyond 1 month postnatal age, and most commonly achieved using an oral dose of chloral hydrate (50–100 mg kg 1). Ethical issues, including the need for fully informed (written) parental consent. Posture Duration of tests. Given the need for explanation of procedures to parents prior to assent, clinical examination, sedation, and subsequent interval

Table 1 Major differences in respiratory physiology in the infant compared to the adult Chest wall Configuration (horizontally placed ribs – decreased efficiency) High chest wall compliance resulting in (i) Low outward recoil of chest wall with limited distending pressure (ii) Reduced ‘passive’ FRC and tendency to atelectasis (iii) Airway closure during tidal breathing with increased tendency to peripheral airway obstruction (iv) Impaired gas exchange (ventilation: perfusion imbalance) (v) Increased work of breathing due to chest wall recession (paradoxical ribcage–abdominal movement) Preferential nose breathers Assessments of resistance in infants include that of the nasal passages, with nasal resistance (Rn) comprising B50% total resistance (i) Diminished sensitivity for detecting lower airway disease or response to therapeutic interventions (ii) Infant PFTs must be deferred for at least 3 weeks following an upper respiratory infection (iii) Pharmacological challenges by aerosol (bronchodilators or constrictors) may be preferentially deposited in the nose Dynamic elevation of FRC Expiratory flows and timing are modulated to maintain end expiratory lung volume (FRC) above that passively determined by the outward recoil of chest wall and inward recoil of lung, thereby partially counteracting effects of high chest wall compliance This ‘dynamic elevation’ of FRC is achieved by combining a relatively short expiratory time (rapid respiratory rate) with prolongation of the expiratory time constant (achieved by laryngeal braking or postinspiratory diaphragmatic activity) Transition to a more relaxed pattern of expiration occurs between 6 and 12 months of age Respiratory reflexes Stronger vagally mediated, stretch receptor activity than in adults; infants have an active Hering–Breuer inflation reflex during tidal breathing, such that airway occlusion at end tidal inspiration evokes a brief expiratory pause; this allows measurements of passive respiratory mechanics to be obtained in infants

566 PULMONARY FUNCTION TESTING IN INFANTS Table 2 Major differences in measuring pulmonary function in infants compared to older subjects

Consciousness Sedation Cooperation Duration of test Equipment Airway attachment Airflow Posture Used in clinical management Personnel Facilities

Informed consent

Infant

Child or adult

Sleeping Yes (if 41 month) Passive Up to 3 h Limited commercial availability Face mask Nose breathing Supine Rarely Two highly trained staff, at least one with clinical (medical/nursing) qualifications Additional facilities for feeding, changing, settling to sleep required, in addition to full resuscitation equipment From parents

Awake None Active o1 h Widely available Mouthpiece Mouth breathing Seated or standing Frequently Respiratory technician or therapist

From subject (and/or parent)

(vi) incorporate suitable software for optimizing data collection, analysis, and quality control. *

*

Figure 1 Use of face mask with ring of silicone putty to effect airtight seal during infant lung function tests.

*

Safety issues. Infant PFTs require two fully trained staff with basic life-support skills, together with a hygienic and safe testing environment with full resuscitation facilities. Pulse oximetry is used for continuous monitoring throughout the testing session. Anthropometry. Although essential for all lung function tests, accurate measurements using a stadiometer for length are particularly crucial during infancy due to rapid somatic growth.

Tests Used to Assess Pulmonary Function in Infants

until onset of sleep, the entire testing session may take 3 or 4 h per infant, only 30–60 min of which will actually be spent on data collection. A similar amount of time per infant may be required when recruiting subjects for research studies. Equipment, which must

A wide range of techniques have been adapted for use in infants, the applicability of which according to age, clinical status, and measurement conditions is summarized in Table 3.

(i)

Many of the techniques for measuring lung volumes in older children and adults have been successfully adapted for use in infants and young children. Since infants cannot be instructed to perform special breathing maneuvers, lung volume measurements in this age group have primarily been those that can be undertaken during tidal breathing (i.e., functional residual capacity (FRC) using either plethysmographic or gas dilution/washout techniques). In recent years, it has become possible to obtain measurements over an extended volume range in specialized centers using what has become known as the ‘raised volume technique’.

(ii) (iii) (iv)

(v)

be miniaturized, with minimization of both dead space and resistance; be easily disinfected between every subject; provide rapid access to the infant at all times; be adapted for nose-breathing subjects, a mask with or without silicone putty generally being used to effect an airtight seal between the apparatus and the infant (Figure 1). Any air leak will invalidate all measurements; have appropriate frequency response and resolution characteristics in the presence of low signal:noise ratio and rapid respiratory rate; and

Assessment of Lung Volumes and Ventilation

PULMONARY FUNCTION TESTING IN INFANTS 567 Table 3 Applicability of infant respiratory function tests in specific situationsa Diagnosis/ situation

Neonatal ICU Ventilatedb Spontaneous breathing Unsedated: o1 month Sedated 1–24 months

Tidal breathing

RIP

Esophageal Occlusion Forced Forced MBW or gas Plethysmography manometry ðFRC þ Raw Þ techniques oscillation expirations dilution (RL , CL ) (RRS , CRS ) (HIT or LFOT) (tidal or (FRC þ VI) RVRTC)

þ þ

(þ) þ

þ þ

þ þ

(þ) (þ)

# (þ)

(þ) þ

NA (þ)

þ

þ

þ

þ

(þ)

þ

þ

þ

þ

þ

Rarely applicable

þ

(þ)

þ

þ

þ

a The choice of tests depends not only on available equipment and what is feasible but also on the underlying clinical or research question. b Leaks around tracheal tube with or without interactions between ventilated and spontaneous breaths may invalidate measurements in intubated infants. þ , useful; ( þ ), limited to specialized research application; NA, not applicable; #, only by negative forced deflation; RIP, respiratory inductance plethysmography; RL and CL, lung resistance and compliance, respectively; RRS and CRS, respiratory resistance and compliance, respectively; ICU, intensive care unit; HIT, high interrupter technique (airway wall mechanics); LFOT, low-frequency forced oscillation (partitioned tissue mechanics); RVRTC, raised volume rapid thoracoabdominal compression; MBW, multiple breath inert gas washout; FRC, functional residual capacity; VI, ventilation inhomogeneity; Raw, airway resistance.

Tidal Breathing Parameters

Tidal breathing parameters are measured while the infant breathes through a face mask and flowmeter. They have been used in clinical and research settings *

* *

*

* *

to determine tidal volume, breathing frequency, and minute ventilation; to investigate the control of breathing; as an integral part of sleep staging and to establish regularity of breathing pattern prior to assessment of lung volumes and mechanics; in combination with CO2, O2, exhaled NO, and SaO2 monitoring and other clinical information; as an indirect measure of airway mechanics; and to trigger equipment.

Attempts to quantify patterns of tidal flow–volume loops have resulted in numerical descriptors of the tidal flow pattern, such as the time to peak tidal expiratory flow as a ratio of total expiratory time (tPTEF:tE) (Figure 2). This index may be reduced in the presence of airway obstruction and has been shown to be a useful outcome measure in various epidemiological studies investigating early determinants of airway function. However, tPTEF:tE is only distantly related to airway function and, as with most tidal breathing parameters, conveys mixed information on the interaction between control of breathing and airway mechanics, thereby requiring cautious interpretation, especially within individuals. Assessments of Functional Residual Capacity

FRC can be measured using either plethysmography, which measures all gas within the thorax, including

that trapped behind closed airways, or a gas dilution or washout technique, wherein only the gas that communicates readily with the airway opening is measured. Both techniques have been used widely to assess lung volume in infancy during clinical and epidemiological research. The principles of plethysmography are identical for infants and older subjects (Figures 3 and 4) with the major difference being that measurements are made in the sleeping, supine infant and that the airway occlusion is held for approximately 8–10 s to allow the infant to make at least two complete respiratory efforts against the occlusion. Equipment that meets most of the published international specifications is now commercially available. The main advantages of this technique are the rapidity and repeatability with which measurements can be made (five repeat measures within o5 min) and the fact that it can detect hyperinflation and gas trapping in infants with airway obstruction. However, plethysmography is not suitable for bedside measurements in sick or small infants due to the relatively bulky equipment. Simultaneous measurements of airway resistance can be obtained if the respired gas can be maintained under body temperature and pressure saturated conditions using a heated rebreathing bag, although this additional application is usually limited to specialist physiology departments. Methods of assessing FRC by gas dilution or washout are also essentially the same in infants as in older subjects, and guidelines for standardized equipment and measurements have been published. Although closed-circuit helium dilution has been widely used in the past, there are difficulties in reducing circuit size sufficiently for use in infants, as

568 PULMONARY FUNCTION TESTING IN INFANTS Insp

Exp

Insp

Exp

Flow

Volume

PTIF VT

tPTIF

PTEF

tE

tI

tPTEF

t tot Time

(a)

(b)

Time

V PTEF

PTEF

TEF50

Flow

Exp Insp PTIF

0.75 (c)

TIF50

0.5 VT

0.25

0.1

Volume

Figure 2 Tidal breathing parameters: (a) and (b) time-based trace of tidal volume and (c) tidal flow–volume loop.

Vent

Tidal flow & volume

3-way valve

Rebreathing bag with servo-controlled heater and thermometer

Heated pneumotachometer

Calibration syringe

∆ V pleth

Pressure at airway opening

Controlled mechanical leak Reference chamber

Figure 3 Infant plethysmography.

well as challenges posed when attempting to maintain a constant volume in the face of varying oxygen consumption and CO2 production. Consequently, there has been increasing emphasis on the use of

washout techniques in infants and young children, using either the bias flow nitrogen (N2) washout technique, which is based on a mixing chamber technique, or, more recently, the multiple breath inert gas

PULMONARY FUNCTION TESTING IN INFANTS 569 Flow (ml s–1)

150

Volume (ml)

69

−150

−69

Pao (kPa)

0.5

−0.5 0

5

10

15

20

25

30

Time (s)

(a)

2.0 1.5

Pao (kPa)

1.0 0.5 0.0 −0.5

FRCp 0 = 221.6 ml FRCp 1 = 221.7 ml

−1.0

FRCp 2 = 235.8 ml −1.5 −2.0

Mean

−50

FRCp

−40

(b)

= 226.3 ml

−30

−20

−10

0

10

20

30

40

50

Box volume change (ml)

Figure 4 Plethysmographic assessments of FRC in an infant. (a) Time-based recording; (b) x–y plot of box volume vs. Pao during airway occlusion.

washout (MBW) technique. The latter measures breath-to-breath changes in the concentration of an inert gas during the washout process and provides information on both lung volume and ventilation efficiency. Gas Mixing Efficiency

Although described many years ago, in the past the MBW technique for assessing gas mixing efficiency or ventilation inhomogeneity was only used

intermittently in infants. During recent years, technological advances combined with increasing awareness that conventional measures of airway function may not detect early changes in peripheral airway function until lung disease is well established have led to a resurgence of interest in this field. The MBW is applicable to subjects of all ages, including bedside measurements in unsedated infants, because measurements are performed during spontaneous tidal breathing. The lung clearance index, a measure of ventilation inhomogeneity that is calculated from the


cumulative expired volume required to clear a tracer gas from the lungs, is a sensitive indicator of early airway disease in infants with cystic fibrosis.

Respiratory Mechanics: Resistance and Compliance Assessments of respiratory resistance and, particularly, compliance are performed relatively frequently in infants and young children compared to older subjects. This probably reflects the relative ease with which such measurements can be performed in early life as well as the fact that, in contrast to older subjects, changes in the elastic properties of the lung often dominate the underlying pathophysiology of lung disease in newborn infants. These techniques are performed during tidal breathing and require little cooperation from the infant other than breathing through a face mask. Esophageal manometry was once commonplace during assessments of lung function in infants, but it has generally been replaced by the less invasive assessments of passive respiratory mechanics using one of the occlusion techniques. Plethysmographic assessments of airway resistance have been used to study normal airway growth and development in relation to lung volume during the first year of life and to discriminate between healthy infants and those with respiratory disease or prior wheezing. Passive Respiratory Mechanics

Measurements of passive respiratory mechanics (compliance, resistance, and expiratory time constant) require complete relaxation of the respiratory muscles. Although this is only feasible in highly trained adults, such relaxation can be routinely induced in infants in whom the vagally mediated Hering–Breuer inflation reflex (HBIR) is active within the tidal volume range. The ‘occlusion technique’ for measuring passive respiratory mechanics is based on the ability to invoke the HBIR by performing brief intermittent airway occlusions during spontaneous tidal breathing (Figure 5). Activation of vagally mediated pulmonary stretch receptors when the airway is occluded above FRC leads to inhibition of inspiration and prolongation of expiratory time. Provided there is no respiratory muscle activity and rapid equilibration of pressures across the respiratory system during the occlusion (as shown by the presence of a pressure plateau), alveolar pressure, and hence the elastic recoil of the respiratory system, can be measured at the airway opening (Figure 6). By relating this recoil pressure either to the volume above the passively determined end expiratory volume at which the airway occlusion was performed or to the airflow occurring on release of the occlusion, the compliance and resistance of the respiratory system

Flow and volume

Pneumotachometer Shutter

Airway opening pressure

Face mask

Figure 5 The occlusion technique.

P

Flow

∆P

Pressure A Time ∆F

B

Compliance = ∆V/ ∆P Resistance = ∆P / ∆Flow Vx Volume

∆V Figure 6 Calculation of passive respiratory mechanisms.

can be measured. The major limitation of this technique, as with all methods that utilize intermittent airway occlusions, is that in the presence of severe airway obstruction, pressures may not equilibrate rapidly enough to allow accurate measurements at the airway opening. Furthermore, it assumes that the time constant of the respiratory system (which is the product of resistance and compliance) can be described by a single value, which is unlikely to be true in the presence of respiratory disease. Forced Oscillation Technique

The forced oscillation technique (FOT), in which the impedance of the respiratory system is measured by superimposing small-amplitude pressure oscillations on the respiratory system and measuring the resultant oscillatory flow, is another technique that has been adapted for use in infants and preschool children. Respiratory impedance describes the spectral (frequency domain) relationship between pressure and airflow throughout the respiratory cycle, providing a global measure of resistive, elastic (viscous), and inertial forces and the opportunity to investigate the frequency dependence of respiratory mechanics. In recent years, there have been two interesting modifications to the FOT that, although remaining

PULMONARY FUNCTION TESTING IN INFANTS 571

strictly in the research domain, are of potential future clinical significance. Depending on the frequency of the applied pressure wave, the resultant impedance contains different mechanical information. The response to very slow pressure oscillations (o2 Hz), during what has been referred to as the low-frequency forced oscillation technique, allows the noninvasive partitioning of lung function into airway and tissue parameters. This approach may be of particular interest for studies in preterm infants, in whom parenchymal disease is a major component of acute respiratory illness. In contrast, at very high frequencies, as are utilized in the high-frequency interrupter technique, the information derived is primarily related to airway wall mechanics, which is particularly important in wheezing disorders.

commercially available infant lung function equipment, however, has yet to be established. V0 maxFRC is significantly lower (by approximately 15%) in boys than in girls throughout infancy and in infants whose mothers smoked during pregnancy. Forced Expiration from Raised Lung Volume

Despite the popularity of the tidal RTC, its value when assessing either baseline airway function or bronchial responsiveness may be limited by the dependence of reported values of V0 maxFRC on the resting lung volume. The latter may not be stable in infants, particularly in the presence of disease or during intervention studies. During the 1990s, the tidal RTC

Forced Expiratory Maneuvers Partial Forced Expiratory Maneuvers

Large-bore tubing & valve Flowmeter

Inflatable jacket

Figure 8 Measurements of partial forced expiratory maneuvers in an infant.

500

400

V ′maxFRC = 300 ml s−1

300

Flow (ml s−1)

Infants cannot be instructed to perform forced expiratory maneuvers, but partial expiratory flow volume curves can be produced by wrapping a jacket around the chest and abdomen that is inflated at the end of a tidal inspiration to force expiration. The resultant changes in airflow (and hence volume) are recorded through a flowmeter attached to a face mask, through which the child breathes (Figures 7 and 8). This technique is often referred to as the ‘squeeze’ or tidal rapid thoracoabdominal compression (RTC) technique. Maximal expiratory flow at FRC (V0 maxFRC), which measures forced expiratory flows at low lung volumes (similar to FEF75 or FEF85 in older children), is the most commonly reported parameter derived from this technique (Figure 9). In the presence of airway disease, not only is V0 maxFRC reduced but also there are characteristic changes in the shape of the flow volume curve (Figure 10). Guidelines regarding data collection and analysis for the tidal RTC have been published by the European Respiratory Society/American Thoracic Society task force, as have sex-specific collated reference data. The validity of such reference equations for interpreting data collected with the new generation of

200

100 Pressure relief valve Compressed air 50 l tank

Figure 7 The tidal RTC (squeeze) technique for measuring partial forced expiratory maneuvers.

F=0

0

−100 40

20

0

−20

−40

Volume (ml) Figure 9 Calculation of V 0 maxFRC from a partial RTC maneuver.

572 PULMONARY FUNCTION TESTING IN INFANTS 200

200

V ′max FRC

100 Flow (ml s−1)

Flow (ml s−1)

100

0

−100

0

−100

40

20

0

40

−20

Volume (ml)

(a)

(b)

20

0

−20

Volume (ml)

Figure 10 Comparison of partial flow volume loops in: (a) healthy infant and (b) airway obstruction.

Pressure relief valve

Fresh gas flow

Insp Exp

Pneumotachometer Face mask

Pressure relief valve Compressed air

Y-piece Pressure port

Large-bore 3-way tap

Inflatable bladder Jacket

100 l tank

Figure 11 Forced expiratory maneuvers from raised lung volume.

technique was modified so that forced expiratory flows and volumes could be measured over an extended volume range in what became known as the ‘raised volume RTC or RVRTC technique’ (Figure 11). Similar to spirometric assessments in older subjects, the RVRTC technique allows the infant’s lungs to be inflated toward total lung capacity (TLC) before rapid inflation of the jacket initiates forced expiration from this elevated lung volume, with the maneuver ending when the infant reaches residual volume (Figure 12). Application of three–five augmented breaths to induce a respiratory pause before forcing expiration encourages the child to breathe out toward residual volume. The relationship between a tidal and raised volume forced expiratory flow volume curve is shown in Figure 13. The rapidity of lung emptying during early life generally precludes measurement of forced expired volume in 1 s (FEV1), with results of FEV0.5 being more commonly reported in infants.

Use in Clinical Practice Although there is little doubt about the potential value of infant lung function tests as a means of providing objective outcome measures in clinical or epidemiological research studies, their potential usefulness with respect to influencing clinical management within an individual infant remains far more debatable. Lung function tests at any age are rarely performed for diagnostic purposes but rather to monitor the nature and severity of respiratory disease or to assess the response to treatment. ‘Typical’ changes in selected lung function parameters according to diagnostic group are summarized in Table 4. It is generally agreed that no single lung function test will provide the answer and that a combination of tests is required, the results of which should be interpreted in light of other information regarding current clinical status and prior medical and social

PULMONARY FUNCTION TESTING IN INFANTS 573 Apnea Flow 500

Forced expiration Volume 250

Tidal breathing

Pj

Passive lung inflations Jacket inflation

2.5

Pao 2.5

10 s Figure 12 Time-based recording of forced expiratory maneuvers using the raised volume technique.

900

Flow (ml s−1)

600

300

0

–300 150

F=0

100

50

0

–50

Volume (ml) Figure 13 Overlay of tidal and raised volume FEFV curves.

history. Choice of technique also depends on the setting, with tests such as the raised volume technique requiring highly trained staff and sophisticated equipment (Table 3). The clinical usefulness of any technique depends not only on its ability to measure parameters that are relevant to the underlying pathophysiology and to discriminate between health and disease but also on within-subject repeatability both within and between test sessions. Although highly reproducible measurements of lung function can be made in infants during

the same test session, little is known about the ‘between-test repeatability’. Similar problems arise with respect to distinguishing the effects of disease from those of growth and development since the timeconsuming nature of the tests and need for sedation limit the numbers of healthy infants who can be studied. If appropriate equipment were to be miniaturized and/or incorporated into the ventilatory circuit so that continuous online monitoring of appropriate parameters could be undertaken, the major area in which infant lung function tests could influence clinical management in the future is probably the neonatal and pediatric intensive care unit. Interpretation of results will depend on knowledge of growth and development of the lung, particularly after extremely preterm delivery. There may also be a role for infant lung function testing with respect to longitudinal measurements from birth and throughout the preschool years in high-risk groups, such as those with persistent wheezing, cystic fibrosis, and chronic lung disease. The potential prognostic value of such tests within individual subjects is unknown since it is only during the past few years that more routine assessments of lung function have been possible throughout the preschool years, but such studies are currently under way.

Uses and Applications in Respiratory Research The major role for infant lung function testing remains firmly within the research arena, in which it has been extensively used to examine the early determinants of airway function and to investigate underlying pathophysiology and response to therapeutic interventions in a variety of respiratory diseases during

Table 4 Typical changes in pulmonary function in various respiratory diseases during infancya Diagnosis

Respiratory rate

Forced expiratory maneuver V 0 maxFRC (partial FEFV)

FEV0.5, FEF% (RVRTC)

Resistance

Compliance

FRCpleth

FRCgas

Comments

Typically small, stiff lungs during acute phases Airway obstruction as evidenced by m resistance and k flows becomes apparent with onset of chronic changes Gas trapping and hyperinflation rare unless tested during acute attack Marked hyperinflation with or without gas trapping; (FRCpleth FRCgas); despite marked airway obstruction, V 0 maxFRC may appear normal due to elevated FRC Marked m in inspiratory resistance and flattening of inspiratory flow– volume loop, with k inspiratory flows. Nb sedation may be contraindicated if upper airway obstruction occurs Diminished FRC not always evident if dynamic elevation is within normal limits

Acute RDS

m

NA

NA

N

k

NA

k

CLDI (BPD)

m or N

k or N

k

m

k or N

N

k or N

Wheezing

m or N

k or N

k

m or N

N

m or N

N

Cystic fibrosis

m

k or N

k

m

N

mm or N

m or N

Upper airway obstruction

N

N

N

mm (especially during inspiration)

N

N

N

Pulmonary hypoplasia

mm

N

N

N or k

N for lung size, k for body size

k

k

a

Due to the wide range of normative data, values from individual infants with respiratory disease may fall within ‘normal’ limits, with the changes indicated here only becoming significant when groups of infants with similar conditions are studied. V 0 maxFRC, maximum expiratory flow at FRC; FEFV, forced expiratory flow volume curve; RVRTC, raised volume rapid thoracoabdominal compression technique; FEV0.5, forced expired volume in 0.5 s; FRCgas, FRC by gas dilution or washout; FRCpleth, plethysmographic FRC; N, normal; m, increased; k, decreased; NA, not applicable; CLDI, chronic lung disease of infancy; BPD, bronchopulmonary dysplasia; RDS, respiratory distress syndrome.

PULMONARY FUNCTION TESTING IN INFANTS 575 Table 5 Reasons for assessing pulmonary function in infants Epidemiological research * Influence of early life events (e.g., maternal smoking, preterm delivery, intrauterine growth retardation) or genotype (e.g., family history of asthma/atopy) on lung development and relative risk of developing respiratory disease * Determinants of lung function: body size, age, maturity, sex, ethnic group * Longitudinal studies from infancy to school age: tracking of lung function Methodological research * Development and validation of methods and equipment for assessing respiratory function in infants * Establishment of reference data and prediction equations * Assessment of within- and between-subject repeatability Clinical research * Early recognition of lung disease (e.g., cystic fibrosis) * Determination of type and severity of pulmonary defect * Long-term effects of neonatal illness or respiratory support * Assessment of bronchial responsiveness Clinical assessment Serial measures to monitor disease progression or response to therapy may be justified (i) In infants who present with unexplained tachypnea, hypoxia, cough, or respiratory distress in whom a definitive diagnosis is not apparent from physical examination (ii) In those with known respiratory disease of uncertain severity in whom there is need to justify management decisions (iii) In infants with severe, chronic obstructive lung disease who do not respond to an adequate clinical trial of combined corticosteroid and bronchodilator therapy (iv) To optimize ventilatory support in the intensive care unit (only feasible if appropriate expertise, equipment, and techniques available)

early life (Table 5). It is now realized that much of the burden of respiratory disease in childhood and later life has its origins in infancy and early childhood and that remarkable ‘tracking’ of lung function occurs from infancy throughout life. This emphasizes the need to prevent lung injury both before and after delivery, when lung growth is so rapid. In addition, despite advances in molecular biology, the effects of new diagnostic and therapeutic advances still need to be evaluated in vivo by employing objective physiological outcome measures such as those provided by infant PFTs. Furthermore, the increased survival of extremely preterm infants without a reduction in the prevalence of chronic lung disease has increased awareness of the need for a better understanding, of both lung growth and development, and the effect of different ventilatory strategies in order to minimize lung injury during this critical period. Clinical Research Applications

In addition to population-based studies, in which they have been used to investigate the effects of factors such as preterm delivery, sex, intrauterine growth retardation, family history of asthma, and maternal smoking in pregnancy, infant PFTs have been widely used in clinical research. V0 maxFRC has been shown to be lower in infants with recurrent wheeze, bronchiolitis, tracheomalacia, and those with a history of life-threatening events. Airway

function is reduced at an early stage in infants with cystic fibrosis, even in the absence of clinically recognized lower respiratory illness (LRI), which may have important potential implications for early interventions in cystic fibrosis. Although several reports have suggested that the raised volume technique may be a more sensitive means of discriminating changes in lung function in infants with respiratory disease than either tidal breathing parameters or V0 maxFRC, it should be noted that this technique is more complex to apply and that guidelines to standardize data collection and analysis are only just beginning to emerge. Bronchial Responsiveness

One of the most extensively studied clinical research areas in which infant lung function tests have been applied is that of bronchial responsiveness using both bronchial challenge and bronchodilation. There is no standardized approach to performing either type of intervention with respect to agent used, dosage, mode of delivery, outcome measures, or methods of analyzing and reporting results. This has resulted in considerable difficulties when attempting to elucidate age-related changes in airway responsiveness during early life. Interpretation of these studies is further complicated by the lack of information regarding intersubject, between-test repeatability in the same test session.


Despite these difficulties, and the generally poor response to bronchodilators during early life, there is convincing evidence that the airways are fully innervated and capable of responding to a range of challenges during both fetal and early postnatal life. The effectiveness of bronchodilators in wheezy infants remains controversial, reflecting the fact that in many infants who wheeze, the reduction in baseline airway function is not due to reversible bronchoconstriction but, rather, transient conditions associated with diminished airway patency. Applications during and Following Intensive Care

Numerous studies have attempted to use parameters derived from infant lung function tests to assess the effects of preterm delivery, neonatal lung disease, and ventilatory support. The most commonly used approaches in recent years have been assessments of passive respiratory mechanics and lung volumes. Specific difficulties in undertaking and interpreting measurements of infant lung function during intensive care include the *

*

*

* *

*

*

relative invasiveness of many techniques in clinically unstable infants; lack of sensitivity of respiratory mechanics to detect subtle changes within individuals due to the magnitude of tracheal tube resistance; confounding of results due to interactions between the ventilator and spontaneous breathing activity; leaks around the tracheal tube; heterogeneous nature of the population with respect to maturity, body size, and clinical severity; multitude of possible treatment modalities that infants may be exposed to; and inappropriate correction for body size.

It is becoming increasingly evident that preterm delivery, even in the absence of any initial respiratory disease, may have an adverse effect on subsequent lung growth and development that persists and may even worsen throughout the first years of life. Most studies of infants with chronic lung disease of infancy have suggested that lung volumes are low early in infancy but subsequently become normal or elevated. Such infants may also have reduced compliance and increased resistance during the first year of life. Infant PFTs have also been used as objective outcome measures to assess the effect of different types of ventilatory support, including extracorporeal membrane oxygenation and high-frequency oscillation during the neonatal period on subsequent lung growth and development.

Summary During the past 20 years, there has been enormous progress in the field of infant lung function testing with respect to the range of tests and equipment now available, their applications in research and clinical studies, and the degree of national and international collaboration. The major role for lung function testing in infants remains firmly within the research arena, in which it has been extensively used to examine the early determinants of airway function and to investigate underlying pathophysiology and response to therapeutic interventions in a variety of respiratory diseases during early life. In recent years, there has been increasing emphasis on developing techniques that can be used in unsedated infants and for those requiring ventilatory support. Future strategies need to encompass a multicenter, multidisciplinary, collaborative approach, with results from infant pulmonary function tests being integrated with those from other disciplines, including imaging, genetics, inflammation, and immunology. See also: Arterial Blood Gases. Asthma: Overview. Breathing: Breathing in the Newborn. Bronchiolitis. Bronchodilators: Beta Agonists. Bronchopulmonary Dysplasia. Cystic Fibrosis: Overview. Environmental Pollutants: Overview. Genetics: Overview. Infant Respiratory Distress Syndrome. Lung Development: Overview; Congenital Parenchymal Disorders; Congenital Vascular Disorders. Pediatric Pulmonary Diseases. Signs of Respiratory Disease: Breathing Patterns. Ventilation, Mechanical: Positive Pressure Ventilation.

Further Reading Frey U (2001) Clinical applications of infant lung function testing: does it contribute to clinical decision making? Paediatric Respiratory Reviews 2: 126–130. Frey U, Stocks J, Coates A, Sly P, and Bates J (2000) Standards for infant respiratory function testing: specifications for equipment used for infant pulmonary function testing. European Respiratory Journal 16: 731–740. Gappa M, Ranganathan S, and Stocks J (2001) Lung function testing in infants with cystic fibrosis: lessons from the past and future directions. Pediatric Pulmonology 32: 228–245. Godfrey S, Bar-Yishay E, Avital A, and Springer C (2003) What is the role of tests of lung function in the management of infants with lung disease? Pediatric Pulmonology 36(1): 1–9. Goldstein AB, Castile RG, Davis SD, et al. (2001) Bronchodilator responsiveness in normal infants and young children. American Journal of Respiratory and Critical Care Medicine 164(3): 447–454. Hoo AF, Stocks J, Lum S, et al. (2004) Development of lung function in early life: influence of birth weight in infants of nonsmokers. American Journal of Respiratory and Critical Care Medicine 170: 527–533.

PULMONARY THROMBOEMBOLISM / Deep Venous Thrombosis 577 Jones MH, Howard J, Davis S, Kisling J, and Tepper RS (2003) Sensitivity of spirometric measurements to detect airway obstruction in infants. American Journal of Respiratory and Critical Care Medicine 167(9): 1283–1286. Lum S, Stocks J, Castile R, et al. (2005) Raised volume forced expirations in infants: recommendations for current practice. European Respiratory Society/American Thoracic Society consensus statement. American Journal of Respiratory and Critical Care Medicine (in press). Ranganathan S, Stocks J, Dezateux C, and the London Collaborative Cystic Fibrosis Group (2004) The evolution of airway function in infancy and early childhood following clinical diagnosis of cystic fibrosis. American Journal of Respiratory and Critical Care Medicine 169: 928–933. Schibler A and Frey U (2002) Role of lung function testing in the management of mechanically ventilated infants. Archives of Disease in Childhood: Fetal and Neonatal Edition 87(1): F7–F10.

Pulmonary Hypertension

Stocks J (2005) Pulmonary function tests in infants and young children. In: Chernick V, et al. (eds.) Kendig’s Disorders of the Respiratory Tract in Children, 7th edn. New York: Elsevier. Stocks J and Dezateux C (2003) The effect of parental smoking on lung function and development during infancy. Respirology 8: 266–285. Stocks J and Hislop AA (2002) Structure and function of the respiratory system: developmental aspects and their relevance to aerosol therapy. In: Bisgaard H, O’Callaghan C, and Smaldone GC (eds.) Drug Delivery to the Lung: Clinical Aspects, pp. 47– 104. New York: Dekker. Stocks J, Sly PD, Tepper RS, and Morgan WJ (1996) Infant Respiratory Function Testing. New York: Wiley. Turner SW, Palmer LJ, Rye PJ, et al. (2004) The relationship between infant airway function, childhood airway responsiveness, and asthma. American Journal of Respiratory and Critical Care Medicine 169(8): 921–927.

see Vascular Disease.

PULMONARY THROMBOEMBOLISM Contents

Deep Venous Thrombosis Pulmonary Emboli and Pulmonary Infarcts

Deep Venous Thrombosis V F Tapson, Duke University Medical Center, Durham, NC, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Deep venous thrombosis (DVT) is a common and under-recognized disorder that places patients at risk for potentially fatal pulmonary embolism as well as chronic, painful postthrombotic syndrome. Thrombosis can affect essentially any vein in the body, depending on the underlying injury or condition and can involve superficial or deep veins. Stasis, venous injury, and hypercoagulability (thrombophila) are the classic basic risk factors from which all other risks are derived. The history and physical examinations are notoriously insensitive and non-specific for both DVT and pulmonary embolism. Leg swelling, erythema, warmth, pain, and/or tenderness may be present, but these symptoms may represent other entities such as cellulitis or trauma, and clinically distinguishing these can be difficult, requiring further evaluation. Compression ultrasound is the firstline diagnostic test, although when clinical suspicion is low, a negative D-dimer is generally adequate to rule out DVT. Treatment options for acute DVT include anticoagulation with low molecular weight or standard heparin. Larger, more symptomatic DVT may be better served by catheter-directed thrombolytic therapy with careful attention to the risk. Inferior vena cava filter placement is indicated if there is bleeding or

substantial bleeding risk, or recurrence on adequate therapy. DVT is often but not always preventable but prophylaxis must be considered for all at-risk patients. Preventive methods include low molecular weight heparin, standard heparin, or pneumatic compression. Low molecular weight heparin has distinct advantages over standard heparin.

Background and Incidence Deep venous thrombosis (DVT) is a common and under-recognized disorder that places patients at risk for potentially fatal pulmonary embolism as well as chronic, painful postthrombotic syndrome. Venous thromboembolism (VTE) includes the spectrum of thrombosis and pulmonary embolism (PE). Venous thrombosis can affect essentially any vein in the body, depending on the underlying injury or condition. Occlusion of veins of the arms, legs, and pelvis as well as more central veins including the superior and inferior vena cava can be symptomatic or asymptomatic. Thrombosis in the deep veins of the legs can involve superficial veins or can be DVT. The most devastating complication of acute DVT is acute, fatal PE. This article focuses primarily on thrombosis of the extremities, especially the legs, for this reason. The disease is more common in the setting of specific predipositions,

578 PULMONARY THROMBOEMBOLISM / Deep Venous Thrombosis

but can be idiopathic. It will address the risk factors, diagnostic approach, prophylaxis for medical patients at risk, and therapeutic options for DVT. The incidence of DVT is unknown. Population studies of the overall age- and sex-adjusted annual incidence of VTE is 1 to 2 per 1000 people. A significant minority of these cases represent recurrent disease. However, the precise incidence is difficult to be certain of because DVT is often asymptomatic(even when it precedes fatal PE), and because the number of autopsies per year is so low. Thus, as many as two million per year may develop this disease in US with approximately 60 000 to 200 000 patients dying from acute PE.

Table 1 Risk factors for venous thromboembolisma

Pathophysiology

Genetic/molecular factors Antithrombin III deficiency Factor V Leiden (activated protein C resistance) Protein C deficiency Protein S deficiency Prothrombin gene (G20210A) defect Heparin cofactor 2 deficiency Dysfibrinogenemia Disorders of plasminogen Elevated factor VIII levels Elevated factor XI levels Hyperhomocysteinemia

Venous thrombi typically develop within a deep vein at a site of trauma and/or in the presence of sluggish blood flow, particularly in the valve cusps in the venous sinuses of the calf veins. Fibrin and platelets accumulate with propagation of the clot in the direction of blood flow. Endogenous fibrinolysis may result in a partial or complete thrombolysis. Recanalization of residual thrombus will render the vein narrow and irregular, with venous valvular incompetency. Collateral vessels may develop.

Risk Factors for DVT Recognizing the risk factors for DVT offers several potential benefits. Their presence, together with compatible symptoms and signs, helps one suspect DVT or PE as well as being critical in determining appropriate prophylaxis in patients at risk (Table 1). The role of their presence in lowering the diagnostic suspicion for DVT is our primary reason for emphasizing them here. Nearly 150 years ago, Rudolf Virchow, the German pathologist, described the triad of stasis, venous injury, and hypercoagulability and its association with the development of venous thrombosis. This simple concept is perhaps one of the most enduring themes in medicine today, with essentially every risk factor for DVT being derived from this triad. The risk of DVT is significant in both medical and surgical patients with most surgical scenarios entailing at least some risk. One of the most easily recognized DVT (and thus, PE) risk factors is major surgery, particularly orthopedic surgery. Based on venographic studies, performed on patients not receiving prophylaxis, the prevalence of DVT at 7–14 days after total hip replacement, total knee replacement, and hip fracture surgery is about 50–60%, with proximal DVT rates of about 25%, 15–20%, and 30%, respectively. The leg undergoing the procedure is most commonly

Clinical factors Age greater than 40b Prior history of venous thromboembolism Major surgical procedure/trauma Hip fracture Immobilization/paralysis Varicose veins Congestive heart failure Myocardial infarction Obesity Pregnancy/postpartum Oral contraceptive therapy Cerebrovascular accident Cancer Paroxysmal nocturnal hemoglobinemia Acute medical illness with restricted mobility

Acquired factors Antiphospholipid antibody syndrome Lupus anticoagulant Anticardiolipin antibodies Myeloproliferative disease Hyperhomocysteinemia a

The presence of risk factors may lower the threshold for considering the diagnosis of DVT or PE. b While the risk of VTE begins to increase at approximately age 40, a significant increase is generally not recognized until approximately age 70.

affected but in some cases the other leg may be too. Patients undergoing other major surgery are also at risk, and associated underlying disease such as cancer may compound this risk. The risk of DVT appears to be approximately 24% in those not receiving antithrombotic treatment, and about 55% in patients with lower limb paresis or paralysis. Hereditary and acquired thrombophilias increase the risk, but are often not known to the clinician when a patient presents with symptoms. Pregnancy and the postpartum period are the most common settings in which women below age 40 develop DVT or PE, and are associated with a fivefold increase in risk. In patients admitted for treatment for respiratory disease, congestive heart failure, or other medical illnesses such as infection or rheumatic disease, the incidence of VTE without prophylaxis may be 15% or higher.

PULMONARY THROMBOEMBOLISM / Deep Venous Thrombosis 579

Serious illness and multiple risk factors increase the incidence of DVT. The risk is high in critically ill patients because of underlying disease, immobility, and veno-invasive catheters and devices. Thus, the reduced mobility associated with medical illness imparts a significant risk for DVT. In addition, the relative immobilization associated with prolonged air travel places individuals at risk with the travel distance correlating with the risk of DVT. Appropriate preventive approaches will be discussed subsequently in the section on prophylaxis.

Clinical Manifestations The history and physical examination are notoriously insensitive and non-specific for both DVT and PE. While the symptomatic presentation of DVT depends to some degree on the extent of thrombosis, it is clear that very large thrombi may evolve and never result in DVT symptoms but present first as symptomatic or even fatal PE. Leg swelling, erythema, warmth, pain, and/or tenderness may be present, but may be symptoms of other entities such as cellulitis or trauma, and clinically distinguishing these can be difficult, requiring further evaluation (Table 2). Pain with dorsiflexion of the foot (Homans’ sign) may be present in the setting of DVT, but this finding is neither sensitive nor specific. The differential diagnosis includes cellulitis, musculoskeletal pain, trauma, a ruptured Baker’s cyst, or asymmetric edema unrelated to DVT. Symptoms and signs of PE may be present, with the most common being dyspnea, pleuritic chest pain, hemoptysis, tachypnea, and tachcardia (see Pulmonary Thromboembolism: Deep Venous Thrombosis).

Diagnosis Blood Tests

If DVT is accompanied by acute PE, hypoxemia may be present. Some individuals, particularly young patients without underlying lung disease, may have a Table 2 Common symptoms and signs of acute deep venous thrombosisa Pain Cramping Tenderness Swelling Warmth Homans’ sign Erythema Palpable venous cord a

Symptoms and signs, including Homans’ sign, are notoriously non-sensitive and non-specific. Deep venous thrombosis can be present and even extensive without symptoms or signs. Symptoms of acute PE may be simultaneously present.

normal PaO2, and in rare cases, a normal alveolar– arterial oxygen difference. Plasma tests of circulating D-dimer (a specific derivative of cross-linked fibrin), in patients with acute PE have been extensively evaluated. A normal enzyme-linked immunosorbent assay (ELISA) appears sensitive in excluding PE, particularly when the clinical suspicion is relatively low. A number of D-dimer assays are available, and the sensitivity and specificity of these assays vary. A positive D-dimer test means that DVT or PE is possible, but the lack of specificity dramatically limits this result. This tenet makes the use of D-dimer very limited in hospitalized patients in whom infection, cancer, trauma, and other disease states are common, and frequently associated with a positive assay. These tests can now be done more quickly with very high sensitivity. A positive Ddimer is not proof of DVT and/or PE, and while the sensitivity of these tests are quite good, a strong clinical suspicion should not be ignored. Radiographic Studies

Venography has been the gold-standard test for DVT for decades, and while it is still deemed the most sensitive test, it is rarely done now. With the advent of ultrasound, a diagnostic test that is 490% sensitive in the setting of symptomatic DVT, the use of venography has become extremely uncommon. Ultrasonography is the first-line diagnostic test for suspected DVT in most settings. While color Doppler signals are examined, and a clot may be visualized, the most sensitive portion of the examination is compression of the vessels. If they do not compress, regardless of whether clot is seen, it is highly likely that there is venous thrombosis. The non-compressibility of leg vessels is the most specific sign for DVT (Figure 1). One drawback of ultrasound is that lack of compressibility by itself, does not distinguish new from old DVT. Magnetic resonance imaging (MRI) has proven extremely sensitive for both acute and chronic DVT, although it is generally not necessary (Figure 2). Above the knee, it is likely as sensitive or even more sensitive than venography. It is suitable to consider MRI in the setting of suspected DVT when severe edema, trauma, or a plaster cast or other device prevents the effective use of ultrasound. A major limitation of ultrasound is its reduced sensitivity in the setting of asymptomatic DVT. Thus, it is not generally used as a screening test. Diagnostic testing for suspected acute DVT is presented in Table 3.

Treatment Treatment options for acute DVT are the same as for acute PE and include anticoagulation with


low-molecular-weight heparin (LMWH) or standard heparin, thrombolytic therapy, and inferior vena cava (IVC) filter placement. Each approach has specific indications as well as advantages and disadvantages. Thrombolytic therapy has traditionally been used less often for acute DVT than for PE but increased interest in local thrombolytic delivery (directly into the involved vein) has led to increased research and application in the clinical setting. Heparin and LMWH

Figure 1 Ultrasound image of deep venous thrombosis of the common femoral vein. Compression is being applied and the vein does not flatten. There are no echoes suggesting thrombosis; it is lack of compression that is diagnostic.

Figure 2 Magnetic resonance image of deep venous thrombosis involving the left femoral vein.

The primary anticoagulants used to treat acute DVT and/or PE include unfractionated heparin and LMWH. These substances exert a prompt antithrombotic effect by accelerating the action of antithrombin III, preventing thrombus extension. Although they do not directly dissolve thrombus or emboli, they allow the fibrinolytic system to proceed unopposed and more readily reduce the size of the thromboembolic burden. While thrombus growth can be prevented, early recurrence can sometimes develop even in the setting of therapeutic anticoagulation. When DVT is diagnosed (or strongly suspected), anticoagulation should be immediately instituted unless contraindications are present. Confirmatory diagnostic testing should be arranged as soon as possible. LMWH should be considered first, based on the clear advantages (Table 4). Among the advantages of LMWH over standard heparin are the greater bioavailability of the LMWHs and more predictable dosing. In addition, the latter anticoagulants can be subcutaneously administered once or twice daily even at therapeutic doses and do not require monitoring of the activated partial thromboplastin time (APTT). Intravenous LMWH is never required

Table 3 Diagnostic testing for acute deep venous thrombosis Procedure

Major advantages

Major disadvantages

Compression ultrasound

Rapid, sensitive, noninvasive Least expensive imaging test Portable

Contrast venography

Very sensitive/specific Most accurate for calf DVT Very sensitive/specific Can combine with PE evaluation

Not sensitive for asymptomatic DVT Less sensitive for new vs. old clot Less sensitive for calf, pelvic clot Casts, severe obesity/edema may lower sensitivity Invasive Can (rarely) cause phlebitis More confining, claustrophobia Metal may contraindicate Much slower than CT More expensive than ultrasound Contrast/radiation

a

Magnetic resonance imaging

Computed tomography (CT) D-dimer

a

Sensitive, less data Easily combined with PE evaluation Sensitive (if ELISA) Inexpensive

Currently is first-line test in most settings.

Non-specific Should be used only if clinical suspicion relatively low

PULMONARY THROMBOEMBOLISM / Deep Venous Thrombosis 581 Table 4 Potential advantages of low-molecular-weight heparins over unfractionated heparin

Table 5 Initiation of LMWH for therapy of acute DVT (and/or PE)

Similar or superior efficacy Similar or superior safety Superior bioavailability Subcutaneous administrationa Once or twice daily dosing No laboratory monitoringb Less phlebotomy Lower incidence of heparin-induced thrombocytopenia Earlier ambulation Home therapy in certain patient subsets

Determine appropriateness of outpatient therapya Begin LMWH by subcutaneous administrationb Determine whether monitoring needed (extremes of weight, renal insufficiency, pregnancy) Warfarin from day 1; initial dose 5–10 mg, adjust according to INR Check platelet count between days 3 and 5; HIT is unusual but can occurc Stop LMWH after X5 days of combined therapy, and when INR is X2.0 for two consecutive days Anticoagulate with warfarin for X3 months (goal INR 2.0–3.0)d

a

a

For either prophylaxis or treatment. No monitoring needed for either prophylaxis or treatment. In a few specific ‘treatment’ settings such a renal insufficiency if renal functions changing or is severely abnormal, or if the body weight o40 kg, or 4150 kg, measuring antifactor Xa levels is recommended to aid in dosing.

b

in VTE. In addition, LMWHs have a more profound effect in inhibiting clotting factor Xa relative to thrombin. The reduced frequency of heparin-induced thrombocytopenia (HIT) with LMWH relative to unfractionated heparin is a very compelling reason to use LMWH instead of the latter whenever possible. Because of efficacy, safety, and convenience compared with standard heparin, these drugs are replacing it in many settings. A major advance in therapy over the last decade has been the move to outpatient therapy made feasible by the LMWH preparations (Table 5). When standard, unfractionated, intravenous heparin is initiated, the APTT should be followed at 6 h intervals until it is consistently in the therapeutic range of 1.5–2.0 times control values. Heparin is administered as an intravenous bolus of 5000 units followed by a maintenance dose of at least 30 000– 40 000 units per 24 h by continuous infusion. The lower dose is administered if the patient is considered at high risk for bleeding. Another recommended option has been an 80 units kg 1 bolus followed by 18 units kg 1 hour 1. This aggressive approach decreases the risk of subtherapeutic anticoagulation. It is possible that early initiation of warfarin, before therapeutic heparin or LMWH may intensify hypercoagulability and increase the clot burden due to the short half-life of anticoagulation factors that are also inhibited by warfarin. Adequately depleting clotting factors take approximately 4 to 5 days. Thus, at least five days of intravenous heparin or LMWH is generally recommended. Heparin should be maintained at a therapeutic level until two consecutive therapeutic international normalized ratio (INR) values of 2.0– 3.0 have been documented at least 24 h apart. Documented proximal DVT or PE should be treated for

Potential outpatients should be medically stable without severely symptomatic DVT. They should be compliant, capable of self-administration (or have a family member or visiting nurse for administration), at low risk of bleeding, and reimbursement should be addressed. b Enoxaparin (Lovenox) and tinzaparin (Innohep) are the two LMWHs that are FDA-approved for treatment of DVT. Data/indication for outpatient therapy is with enoxaparin. While LMWH preparations are sometimes used for patients presenting with PE in United States, and while clinical trials support this use, the FDA-approvals read ‘established DVT with or without PE’. c Heparin-induced thrombocytopenia. d Duration of warfarin therapy should be at least 6–12 months in patients with idiopathic DVT. LMWH, low-molecular-weight heparin, DVT, deep venous thrombosis, PE, pulmonary embolism, INR, international normalized ratio.

3–6 months. Treatment over a more extended interval is appropriate when significant risk factors persist, when thromboembolism is idiopathic, or when previous episodes of VTE have been documented. Oral Therapy

Warfarin remains the only FDA-approved oral drug for DVT. It inhibits the vitamin K-dependent coagulation factors II, VII, IX, and X. Although a prolongation of the prothrombin time (PT) can begin 5–7 h after drug administration due to the short halflife of factor VII, warfarin’s ability to fully exert its effect can take more than 72 h. Warfarin and LMWH or heparin can be started on the same day, but concomitant administration of these drugs for 4 or 5 days is recommended. Bleeding related to warfarin therapy increases with intensity and duration of therapy. Warfarin-induced skin necrosis is a rare but serious complication mandating immediate cessation of the drug. It is related, at least in some patients, to protein C or S deficiency. Warfarin crosses the placenta and may cause fetal malformations if used during pregnancy. The INR should be kept between 2.0 and 3.0. Newer oral drugs with rapid onset, and lack of significant drug and food interactions are being investigated. While heparin and LMWH work indirectly, requiring antithrombin III as a cofactor,


direct thrombin inhibitors have several advantages over heparin, including efficacy against fibrin clotbound thrombin. Lack of need for INR monitoring is among the advantages over warfarin. Complications of Anticoagulation

Bleeding is the major complication of anticoagulation. The rates of major bleeding in recent trials using heparin by continuous infusion or high-dose subcutaneous injection are o5%. HIT typically develops five or more days after the initiation of heparin therapy, occurring in about 5% of patients. The platelet count is usually o100 000 but may be higher. A significant drop without the latter criterion may still be seen in HIT; absolute platelet counts may be deceiving. The syndrome is caused by heparin-dependent IgG antibodies that activate platelets via their Fc receptors. The formation of heparin-dependent IgG antibodies and the risk of thrombocytopenia is lower with LMWH than with standard heparin. Testing for HIT (platelet aggregation and/or ELISA) should be performed in suspected HIT but heparin should be stopped without waiting for results. Both argatroban and lepirudin have been FDA-approved for use in the setting of VTE with HIT. Vena Cava Interruption

If a patient cannot be anticoagulated, IVC filter placement can be performed to prevent lower extremity thrombi from embolizing to the lungs. The primary indications for filter placement include contraindications to anticoagulation, significant bleeding complications during anticoagulation, and recurrent DVT or PE while on adequate therapy. A number of filter designs exist. These devices are effective and complications including insertion-related problems and migration are unusual. Temporary filters are being placed increasingly in patients in whom the risk of bleeding appears short term. Most of these devices can be removed up to 2 weeks later, and some may remain in place even longer, with subsequent removal.

commonly used systemically for acute massive PE. Tenecteplase has been studied extensively in acute myocardial infarction, and would be expected to be effective in VTE, but far less data are available about this agent. Catheter-directed techniques are being used increasingly for acute DVT. Such aggressive therapy with thrombolytics may reduce the frequency of postphlebitic syndrome. As with anticoagulants, hemorrhage is the primary adverse effect associated with thrombolytic therapy. Invasive procedures should be minimized as much as possible. The most devastating complication associated with systemic thrombolytic therapy is the development of intracranial hemorrhage which occurs in o1% of patients. Retroperitoneal hemorrhage may result from a vascular puncture above the inguinal ligament and may be life-threatening. The primary contraindications to thrombolytic therapy include active bleeding, surgery within the previous one to two weeks (depending on specific procedure), or any intracranial pathology or previous intracranial surgery. Catheter-directed thrombolysis appears to be safer, faster, and more effective in removing an acute iliofemoral venous thrombus than previous thrombolytic methods. This technique may result in a reduction in frequency of postthrombotic syndrome.

Postthrombotic Syndrome Postthrombotic (postphlebitic) syndrome is characterized by leg pain, edema, and other signs of venous insufficiency. Leg ulceration may eventually develop as a result of prolonged venous hypertension. Approximately 30% of patients with VTE develop this often debilitating disease. The risk of developing this problem depends on the speed of vein recanalization and development of ipsilateral recurrent events, but not necessarily the extent of thrombosis. Fitted compression stockings are the mainstay of therapy, which may decrease the risk of postthrombotic syndrome by 50%.

Thrombolytic Therapy

Prevention

Thrombolytic agents activate plasminogen to form plasmin which then results in fibrinolysis as well as fibrinogenolysis. These agents can dramatically accelerate clot lysis in acute PE and DVT. The use of thrombolytic therapy in patients with proximal occlusive DVT associated with significant swelling and symptoms is increasing. Clinical trials have culminated in the approval of streptokinase, urokinase, and recombinant tissue-type plasminogen activator (tPA) for the treatment of VTE but these are more

Measures to prevent VTE appear to be grossly underutilized. A substantial reduction in the incidence of DVT can be achieved when patients at risk receive appropriate prophylaxis. For example, the risk of DVT after total hip or knee replacement is 50% or greater without prophylaxis. The superiority of LMWH over unfractionated heparin is evidenced in the clinical trials evaluating exceptionally high-risk patients such as those above, as well as spinal cord injury and trauma patients. Unfractionated heparin

PULMONARY THROMBOEMBOLISM / Pulmonary Emboli and Pulmonary Infarcts 583

is clearly less effective in these settings. In hospitalized general medical patients, anticoagulant prophylaxis should always be strongly considered as the rate of DVT, based upon a venographic endpoint, is as high as 15% in patients receiving placebo. The rate of DVT, including proximal DVT is statistically significantly lower when enoxaparin is administered compared with placebo. It appears that either LMWH (enoxaparin 40 mg subcutaneously or dalteparin at 5000 units once daily) or subcutaneous heparin (5000 units every 8 h) is adequate for medical patient prophylaxis; again, LMWH has clear advantages. Pneumatic compression should be used when anticoagulant prophylaxis is contraindicated. See also: Anticoagulants. Coagulation Cascade: Overview; Fibrinogen and Fibrin; Thrombin. Fibrinolysis: Overview. Pulmonary Thromboembolism: Pulmonary Emboli and Pulmonary Infarcts. Thrombolytic Therapy.

Further Reading Bates SM and Ginsberg JS (2004) Clinical practice. Treatment of deep vein thrombosis. New England Journal of Medicine 351: 268–277. Geerts WH, Pineo GF, Heit JA, et al. (2004) Prevention of venous thromboembolism. The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 126: 338S– 400S. Gelfand EV, Piazza G, and Goldhaber SZ (2003) Venous thromboembolism guidebook, 4th edn. Critical Pathways in Cardiology: A Journal of Evidence-Based Medicine 2(4): 247–265. Goldhaber SZ and Tapson VF (2004) A prospective registry of 5,451 patients with ultrasound confirmed deep vein thrombosis. American Journal of Cardiology 93: 259–262. Heit JA, Silverstein MD, Mohr DN, et al. (1999) Predictors of survival after deep vein thrombosis and pulmonary embolism: a population-based, cohort study. Archives of Internal Medicine 159: 445–453. Kearon C (2003) Natural history of venous thromboembolism. Circulation 107: I22–I30. Kearon C (2004) Long term management of patients after venous thromboembolism. Circulation. Silverstein MD, Heit JA, Mohr DN, et al. (1998) Trends in the incidence of deep vein thrombosis and pulmonary embolism: a 25-year population-based study. Archives of Internal Medicine 158: 585–593. Tapson VF (2002) Venous thromboembolism. In: Topol E (ed.) Textbook of Cardiovascular Medicine, pp. 667–684. Philadelphia, PA: Lippincott–Raven. Tapson VF, Carroll BA, Davidson BL, et al. (1999) The diagnostic approach to acute venous thromboembolism. Clinical Practice Guideline. American Thoracic Society. American Journal of Respiratory and Critical Care Medicine 160: 1043–1066. von Virchow R (1846) Weitere Untersuchungen ueber die Verstopfung der Lungenarterien und ihre Folge. Traube’s Beitraege exp path u Physiol, Berlin 2: 21–31. Watson LI and Armon MP (2004) Thrombolysis for acute deep vein thrombosis. Cochrane Database of Systematic Reviews 4: CD002783.

Pulmonary Emboli and Pulmonary Infarcts V F Tapson, Duke University Medical Center, Durham, NC, USA & 2006 Elsevier Ltd. All rights reserved.

Background and Incidence Pulmonary embolism (PE) occurs when blood clots (thrombi), usually from the deep veins of the legs, travel to the lungs, causing a spectrum of potential consequences, including dyspnea, chest pain, hypoxemia, and sometimes, death. Patients may present with symptoms of either deep vein thrombosis (DVT) or PE, or both, and these two entities represent a continuum of one disease known as pulmonary (or venous) thromboembolism. Pulmonary embolism probably accounts for 100 000 to 200 000 deaths per year in the United States. While some patients who die from acute PE may have an underlying terminal illness, it is clear that this disease is responsible for death in a considerable number of patients with an otherwise good prognosis. Autopsy studies have repeatedly documented the high frequency with which PE has gone unsuspected and thus, undetected. Pulmonary thromboembolism occurs worldwide, and is usually associated with specific risk factors although in many circumstances the disease appears idiopathic (Table 1). A crucial point is that DVT, and therefore PE, are often preventable. Furthermore, prophylaxis continues to be underutilized. The incidence of venous thromboembolism (VTE) is high in hospitalized patients and both surgical as well as medical patients are at risk. Our discussion will be limited to pulmonary thromboembolism and will not include nonthrombotic entities such as fat embolism.

Pathophysiology One or more components of Virchow’s triad (stasis, hypercoagulability, and venous injury), described more than 150 years ago, are present in nearly all patients. The risk increases with age. Idiopathic thromboembolism likely involves an underlying prothrombotic state that has not been characterized. Deep vein thrombi frequently originate in the calf veins and propagate proximally to the popliteal vein or above before embolizing. Thrombosis developing in the axillary–subclavian veins due to the presence of a central venous catheter, particularly in patients with malignant disease, as well as in those with effort-induced upper extremity thrombosis may result in PE as well.

584 PULMONARY THROMBOEMBOLISM / Pulmonary Emboli and Pulmonary Infarcts Table 1 Risk factors for pulmonary thromboembolism Clinical factors Age greater than 40 (more significant after age 70) Prior history of pulmonary thromboembolism Major surgical procedure/trauma Hip fracture Immobilization/paralysis Varicose veins Congestive heart failure Myocardial infarction Other acute medical illnesses Obesity Pregnancy/postpartum Oral contraceptive therapy Cerebrovascular accident Cancer Antiphospholipid antibody syndrome (including lupus anticoagulant) Genetic/molecular factors Anti-thrombin III deficiency Factor V Leiden (activated protein C resistance) Protein C deficiency Protein S deficiency Prothrombin gene (G20210A) defect Dysfibrinogenemia Disorders of plasminogen Elevated factor VIII levels Elevated factor XI levels Hyperhomocysteinemia Combined genetic mutations

The impact of an embolic event depends upon the extent of reduction of the cross-sectional area of the pulmonary arterial bed as well as upon the presence or absence of concomitant cardiopulmonary disease. In the setting of massive PE, cardiac output is diminished but may be sustained as the mean right atrial pressure increases. In the absence of underlying cardiopulmonary disease, obstruction of 25% to 30% of the vascular bed by emboli is associated with a rise in pulmonary artery pressure; hypoxemia also contributes to vasoconstriction and a further rise in pulmonary artery pressure. More than 50% obstruction of the pulmonary arterial bed is usually present before there is substantial elevation of the mean pulmonary artery pressure. Because the right ventricle is not accustomed to pumping against a high pressure, it may ultimately fail resulting in hypotension and often death, if not treated. Patients with underlying cardiopulmonary disease often experience a more substantial deterioration in cardiac output than normal individuals in the setting of massive PE.

Clinical Manifestations Unfortunately, the history and physical examination are notoriously insensitive and non-specific for both

Table 2 Symptoms of acute pulmonary embolism

Dyspnea Pleuritic chest pain Cough Leg pain Hemoptysis Palpitations Wheezing Angina-like pain

All patients (N ¼ 383)

No previous cardiopulmonary disease (N ¼ 117)

78% 59% 43% 27% 16% 13% 14% 6%

73% 66% 37% 26% 13% 10% 9% 4%

The symptoms listed above were based upon data from the Prospective Investigation of Pulmonary Embolism (PIOPED) study and modified from tables presented in Stein PD (ed.) (1996) Pulmonary Embolism. Baltimore: Williams and Wilkins.

DVT and PE. Patients with DVT often have no erythema, warmth, pain, swelling, or tenderness. When these are present, they may represent other entities such as cellulitis, though further evaluation may be appropriate. Pain with dorsiflexion of the foot (Homans’ sign) may be present in the setting of DVT but this finding is neither sensitive nor specific. The most common symptom of acute PE is a sudden onset of dyspnea. Pleuritic chest pain and hemoptysis occur more commonly when small, peripheral emboli obstruct very distal vessels, causing pulmonary infarction. Palpitations, anxiety, cough, and lightheadedness are among the non-specific symptoms of acute PE but these may have a number of other potential causes, contributing to diagnostic confusion. Syncope indicates more massive embolism. Pulmonary embolism should be considered whenever unexplained dyspnea, syncope, hypotension, or hypoxemia are present. The first indication of acute PE may be sudden death. Tachypnea and tachycardia are the most common signs of PE but are also nonspecific. Other possible physical findings include fever, wheezing, rales, a pleural rub, a loud pulmonic component of the second heart sound, a right-sided fourth heart sound, and a right ventricular lift. Symptoms and signs consistent with PE (Tables 2 and 3) should be particularly heeded in the setting of risk factors for VTE such as concomitant malignancy, immobility, or the postoperative state.

Diagnosis Patients with pulmonary thromboembolism may present with symptoms or signs of DVT or PE or both. When patients present with suspected DVT, (e.g., calf pain and/or swelling) a leg study, generally compression ultrasound, should be pursued unless

PULMONARY THROMBOEMBOLISM / Pulmonary Emboli and Pulmonary Infarcts 585 Table 3 Signs of acute pulmonary embolism

Tachypnea (20/min) Crackles Tachycardia (4100/min) Leg swelling Loud P2a DVTb Wheezes Diaphoresis Temperature (X38.5) Pleural rub Fourth heart sound Third heart sound Cyanosis Homans’ sign Right ventricular lift

All patients (N ¼ 383)

No previous cardiopulmonary disease (N ¼ 117)

73%

70%

55% 30%

51% 30%

31% 23% 15% 11% 10% 7%

28% 23% 11% 5% 11% 7%

4%

3% 24% 3% 1% 4% 4%

5% 3% 3%

a

P2, pulmonic component of second heart sound. DVT, deep venous thrombosis. The clinical signs listed above were based upon data from the Prospective Investigation of Pulmonary Embolism (PIOPED) study and modified from tables presented in Stein PD (ed.) (1996) Pulmonary Embolism. Baltimore: Williams and Wilkins. b

there is a clear, alternative explanation. In the setting of dyspnea and/or chest pain, the differential diagnosis is broad, and may include a flare of asthma or chronic obstructive lung disease, pneumothorax, pneumonia, anxiety with hyperventilation, heart failure, angina or myocardial infarction, musculoskeletal pain, rib fracture, pericarditis, pleuritis from connective tissue disease, herpes zoster, intrathoracic cancer, and occasionally intra-abdominal process. The presence of obvious risk factors for VTE, such as prolonged immobility, trauma, recent surgery, medical illness with reduced mobility, cancer, pregnancy, myocardial infarction, recent prolonged travel, or previous thromboembolism in the setting of compatible symptoms and signs should prompt consideration of this entity. It is important to realize that acute PE can be superimposed upon another underlying cardiopulmonary disease, upon which new or worsening symptoms are sometimes blamed. The use of clinical probability scores based upon simple clinical parameters such as presence of tachycardia, hemoptysis, and the presence or absence of risk factors such as cancer have been used to help exclude PE. While these scoring systems have been used in clinical trials, they have not been widely employed in clinical practice.

Blood Tests

Hypoxemia is common in acute PE. Some individuals, particularly young patients without underlying lung disease, may have a normal arterial oxygen tension (PaO2) and even rarely a normal alveolar– arterial difference. A sudden decrease in the PaO2 or oxygen saturation in a patient unable to communicate an accurate history (e.g., a demented or mechanically ventilated patient) suggests the possibility of acute PE. The diagnostic utility of plasma measurements of circulating D-dimer (a specific derivative of crosslinked fibrin) in patients with acute DVT and PE has been extensively evaluated. A normal enzymelinked immunosorbent assay (ELISA) appears sensitive in excluding PE, particularly when the clinical suspicion is relatively low. There are a number of D-dimer assays available, and the sensitivity and specificity of these assays vary. A positive D-dimer test means that DVT or PE is possible, but it is by no means proof. While both cardiac troponin T and troponin I levels have been found to be elevated in acute PE, they are not sensitive enough to rule out PE without additional diagnostic testing. Brain natriuretic peptide levels have also been found to be elevated in acute PE but may be elevated in other settings in which right or left ventricular stretch occurs.

Electrocardiography and Chest Radiography

Electrocardiographic findings, which are present in the majority of patients with acute PE, are nonspecific but these abnormalities, including ST-segment abnormalities, T-wave changes, and left or right axis deviation are common. Only one-third of patients with massive or submassive emboli have manifestations of acute cor pulmonale such as the S1 Q3 T3 pattern, right bundle branch block, P-wave pulmonale, or right axis deviation. The utility of electrocardiography in suspected acute PE is best characterized by its ability to establish or exclude alternative diagnoses, such as acute myocardial infarction. The chest radiograph is often abnormal in patients with acute PE, but as with electrocardiography, it is nearly always non-specific. Common radiographic findings include atelectasis, pleural effusion, pulmonary infiltrates, and mild elevation of a hemidiaphragm. Classic findings of pulmonary infarction such as Hampton’s hump or decreased vascularity (Westermark’s sign) are suggestive of the diagnosis, but are infrequent.

586 PULMONARY THROMBOEMBOLISM / Pulmonary Emboli and Pulmonary Infarcts Specific Imaging Studies

Specific imaging studies are required for the definitive diagnosis of acute PE. A diagnostic algorithm for suspected acute PE is presented in Figure 1. While pulmonary angiography remains the gold-standard test for suspected acute PE, it is rarely necessary. The ventilation–perfusion (VQ) scan was previously the most common diagnostic test utilized when PE was suspected, but over the past decade, spiral (helical) computed tomography (CT) scanning has replaced it at many centers. A normal VQ scan (or perfusion scan alone) rules out the diagnosis with a high enough degree of certainty so that further diagnostic

evaluation is almost never necessary. Unfortunately, low or intermediate probability (nondiagnostic) scans are commonly found with PE which requires further evaluation. The diagnosis of PE should be aggressively pursued even when the lung scan is low or intermediate probability if the clinical setting suggests the diagnosis, particularly when clinical probability is high. Stable patients with suspected acute PE, nondiagnostic lung scans and adequate cardiopulmonary reserve (absence of hypotension or severe hypoxemia), may undergo noninvasive lower extremity testing in an attempt to diagnose DVT. A positive compression

Suspected acute pulmonary embolisma

Clinical suspicion moderate to high

VQ normal; PE ruled out

Clinical suspicion low

(+) CT scan or VQ

scanb

D-dimer (−)

CT normala VQ scan HPc or CT (+) No further testing needed; PE proven US of legs

US (+) (treat)

No further studies (PE ruled out)

CT or VQ nondiagnosticd

US (−)

Pulmonary arteriogram (+) = treat (−) = no treatmenta

Abbreviations: PE, pulmonary embolism; VQ, ventilation-perfusion; CT, computed tomography; US, compression ultrasound; HP, high probability. a Level

of clinical suspicion may affect algorithm. For example, based upon outcome studies, additional studies after a normal CT may not always be necessary.

b The

optimal scenario for a VQ scan is patient with clear chest radiograph and no underlying cardiopulmonary disease. Patients with significant renal insufficiency should undergo a VQ scan instead of CT. Chest CT can be performed together with CT venography which may enhance sensitivity. c In

setting of prior PE, a HP VQ scan may reflect the previous PE. In this case, prior VQ scans should be reviewed whenever possible. d If

VQ scan nondiagnostic, a CT scan can also be considered, instead of angiography.

Figure 1 The algorithm for evaluating suspected acute pulmonary embolism depends to some degree on the particular resources available. A primary difference among algorithms is whether computed tomography or ventilation–perfusion scanning is utilized. In some institutions, ultrasound is utilized prior to a chest imaging study and, if positive, eliminates the need for the latter.

PULMONARY THROMBOEMBOLISM / Pulmonary Emboli and Pulmonary Infarcts 587

ultrasound may present the opportunity to treat without further testing. If the ultrasound is negative, additional testing is needed. Magnetic resonance imaging of the lower extremities and/or lungs may also be useful after a nondiagnostic lung scan if the medical facility has experience with this technique. Spiral CT scanning has been found to be approximately 70–80% sensitive, and greater than 90% specific. This test has the greatest sensitivity for emboli in the main, lobar, or segmental pulmonary arteries. The specificity for clot in these vessels is excellent. For subsegmental emboli, spiral CT appears less accurate, although the importance of emboli this size has been questioned. An advantage of spiral CT over VQ scanning and arteriography includes the ability to define nonvascular structures such as lymphadenopathy, lung tumors, emphysema, and other parenchymal abnormalities as well as pleural and pericardial disease. Another advantage of spiral CT over other diagnostic methods is the rapidity with which a study can be performed. A computed tomography scan demonstrating a very large PE is shown in Figure 2. Potential disadvantages of CT include the fact that it is not portable at present, and because of the need for intravenous contrast, patients with significant renal insufficiency cannot be scanned without risk of renal failure. In summary, with suspected PE, if the clinical probability is deemed low, and a sensitive D-dimer test is negative, no further testing is needed. If the clinical probability is not low, either a chest CT scan or VQ scan is performed. Additional testing such as with

Figure 2 This patient presented with sudden onset severe dyspnea 4 days after undergoing total knee replacement. Spiral computed tomography imaging reveals a large filling defect in the left main pulmonary artery (arrow), diagnostic of acute pulmonary embolism.

compression ultrasound may be needed. Finally, the use of CT venography performed at the same time as chest CT and with the same intravenous contrast bolus, may enhance the sensitivity by identifying DVT even if PE cannot be seen. Echocardiography

Echocardiography, which can often be obtained more rapidly than either lung scanning or pulmonary arteriography may reveal findings which strongly support hemodynamically significant pulmonary embolism. Imaging or Doppler abnormalities of right ventricular size or function may suggest the diagnosis. Unfortunately, because these patients often have underlying cardiopulmonary disease such as chronic obstructive lung disease, neither right ventricular dilation nor hypokinesis can be reliably used even as indirect evidence of PE in such settings.

Treatment Options for treatment of acute DVT and PE include anticoagulation with low-molecular-weight heparin (LMWH) or standard heparin, thrombolytic therapy, and inferior vena cava filter placement. Massive PE is occasionally treated with surgical embolectomy. Each approach has specific indications as well as advantages and disadvantages. Heparin and LMWH

The primary anticoagulants used to treat acute DVT and/or PE include unfractionated heparin and LMWH. These substances exert a prompt antithrombotic effect by accelerating the action of antithrombin III, preventing thrombus extension. Although they do not directly dissolve thrombus or emboli, they allow the fibrinolytic system to proceed unopposed and more readily reduce the size of the thromboembolic burden. There are substantial advantages of LMWH preparations over unfractionated heparin. When DVT or PE are diagnosed (or strongly suspected), anticoagulation should be immediately instituted unless contraindications are present. Confirmatory diagnostic testing should be undertaken as soon as possible. When standard, unfractionated, intravenous heparin is initiated, the activated partial thromboplastin time (APTT) should be followed at 6 h intervals until it is consistently in the therapeutic range of 1.5–2.0 times control values. Achieving a therapeutic APTT within 24 h after the onset of treatment of PE has been shown to reduce the recurrence rate. Heparin is administered as an intravenous bolus of 5000 units followed by a maintenance dose

588 PULMONARY THROMBOEMBOLISM / Pulmonary Emboli and Pulmonary Infarcts

by continuous infusion of at least 30 000 units per 24 h. Alternatively, a weight-adjusted regimen of 80 units/kg bolus followed by 18 units/kg/h is administered. This aggressive approach decreases the risk of subtherapeutic anticoagulation. At least five days of heparin or LMWH is generally recommended. Warfarin is initiated after the parenteral anticoagulant has been started (generally within 24 h). The parenteral drug should be maintained at a therapeutic level until two consecutive therapeutic international normalized ratio (INR) values of 2.0–3.0 have been documented at least 24 h apart. The LMWH preparations have tremendous advantages over unfractionated heparin and have dramatically changed treatment of thromboembolic disease. Among the differences between these two substances is the greater bioavailability of the LMWHs and more predictable dosing. The latter anticoagulants are subcutaneously administered once or twice daily even at therapeutic doses and do not require monitoring of the APTT. Intravenous LMWH is never required in VTE. In addition, LMWHs have a more profound effect in inhibiting clotting factor Xa relative to thrombin. The reduced frequency of heparin-induced thrombocytopenia with LMWH relative to unfractionated heparin is a very compelling reason to use LMWH instead of the latter whenever possible. Because of efficacy, safety, and convenience, compared with standard heparin, these drugs are replacing standard heparin in most settings. Anti-factor Xa levels appear reasonable to monitor in certain settings such as in morbidly obese patients, very small patients (o40 kg), pregnant patients, and those with renal insufficiency. Because these drugs are renally metabolized, the dose must be changed when the creatinine clearance is less than 30 ml per min. With severe renal insufficiency, standard heparin should be considered. There is no clear agreement on a weight limit above which LMWH should not be used, but some feel that an upper limit of approximately 150 kg is reasonable, with intravenous standard heparin being used in larger patients. It is unnecessary to monitor other patients with antifactor Xa levels. In the United States, at the present time, two LMWH preparations (enoxaparin and tinzaparin) are FDA-approved for use to treat patients presenting with DVT with or without acute PE. Finally, an ‘ultra-LMWH’, fondaparinux, has been studied in patients with acute DVT and PE and has proven effective for these indications. It should be noted that the prophylactic doses of these agents differ from the doses used for treating active disease. The characteristics of LMWHs compared with standard

Table 4 A comparison of LMWH with unfractionated heparin Characteristic

UFH

LMWH

Mean molecular weight Protein binding Anti-Xa activity Anti-IIa activity Administration (treatment) Subcutaneous Administration (prophylaxis) Subcutaneous Monitoring during treatment Outpatient therapy Incidence of HIT Reversibility with protamine

12 000–15 000 Substantial Substantial Substantial Intravenous

4000–6000 Minimala Substantial Minimal

Subcutaneous

APTT every 6 h Difficult 3–5% Complete

None in most settingsb Simplified o1% Partial

a

This implies significantly superior bioavailability of LMWH relative to UFH. b LMWH requires a dose change and sometimes monitoring in renal insufficiency (creatinine clearance o30 ml min 1), significant obesity (4150 kg), very small patients (o40 kg), and pregnant patients. Anti-Xa levels are followed, and not the activated partial thromboplastin time. For one LMWH (enoxaparin), the therapeutic dose is reduced from the usual recommended dose of 1 mg kg 1 q12 h, to 1 mg kg 1 qd when the creatinine clearance is o30 ml min 1, rendering monitoring unnecessary unless there are dynamic changes in renal function. Similarly, in this setting, the prophylactic dose of 40 mg qd is reduced to 30 mg qd. No such recommendations can be made for the other preparations. LMWH, low-molecular-weight heparin; UFH, unfractionated heparin; APTT, activated partial thromboplastin time; HIT, heparininduced thrombocytopenia.

unfractionated heparin are shown in Table 4. The approach to therapy utilizing these drugs is shown in Table 5. Documented proximal DVT or PE should be treated for 3–6 months. Treatment over a more extended interval is appropriate when significant risk factors persist, when thromboembolism is idiopathic, or when previous episodes of VTE have been documented. Bleeding is the major complication of anticoagulation. The rates of major bleeding in recent trials using heparin by continuous infusion or highdose subcutaneous injection are less than 5%. Direct Thrombin Inhibitors

Clinical studies suggest that more than 90% of patients with clinical heprin-induced thrombocytopenia have a platelet count fall of more than 50% during their heparin treatment. The syndrome is caused by heparin-dependent IgG antibodies that activate platelets via their Fc receptors. If a patient is placed on heparin for VTE and the platelet count progressively decreases to at least 50% of

PULMONARY THROMBOEMBOLISM / Pulmonary Emboli and Pulmonary Infarcts 589 Table 5 Initiation of LMWH for therapy of acute pulmonary thromboembolism Begin LMWH by subcutaneous administrationa Determine whether monitoring needed (extremes of weight, renal insufficiency, pregnancy) Consider outpatient therapy in appropriateb Warfarin from day 1; initial dose 5–10 mg, adjust according to INR Check platelet count between days 3 and 5c Stop LMWH after X5 days of combined therapy, and when INR is X2.0 for two consecutive days Anticoagulate with warfarin for X3 months (goal INR 2.0–3.0)d a

Enoxaparin, tinzaparin, and fondaparinux, are the preparations that are FDA-approved for treatment of DVT. Enoxaparin is approved for outpatient therapy in appropriate settings. Fondaparinux is approved for both inpatient DVT and PE. While enoxaparin and tinzaparin are sometimes used for patients presenting with PE in the United States, and clinical trials too support this use, the FDA-approvals read ‘‘established DVT with or without PE.’’ b Potential outpatients should be medically stable without severely symptomatic DVT. They should be compliant, capable of self-administration (or have a family member or visiting nurse for administration), at low risk of bleeding, and reimbursement should be addressed. c Heparin-induced thrombocytopenia occurs more commonly with unfractionated heparin than with LMWH. d The duration of warfarin therapy should be at least 6–12 months in patients with idiopathic venous thromboembolism or in those with persisting risk factors. When deemed safe, indefinite anticoagulation should be considered in these patients. LMWH, low-molecular-weight heparin; DVT, deep venous thrombosis; PE, pulmonary embolism; INR, international normalized ratio.

the baseline value or to less than 150 000 mm 3 heparin therapy should be discontinued. The formation of heparin-dependent IgG antibodies and the risk of thrombocytopenia is lower with LMWH than with standard heparin. Both argatroban and lepirudin have been FDAapproved for use in the setting of VTE with heparininduced thrombocytopenia. The half-life of argatroban is 45 min, but is prolonged in patients with hepatic dysfunction. Lepirudin is excreted by the kidneys so the dosage must be reduced in renal insufficiency. There is no antidote for either drug at present. Warfarin-induced skin necrosis is a rare but serious complication occurring in the setting of heparin-induced thrombocytopenia. Vena Cava Interruption

If a patient cannot be anticoagulated, inferior vena cava (IVC) filter placement can be performed to prevent lower extremity thrombi from embolizing to the lungs. The primary indications for filter placement include contraindications to anticoagulation, recurrent embolism while on adequate therapy, and

significant bleeding complications during anticoagulation. Filters are sometimes placed in the setting of massive PE when it is believed that any further emboli might be lethal, particularly if thrombolytic therapy is contraindicated. These devices are effective and complications, including insertion-related problems and migration, are unusual. More recently, retrievable filters are being placed in patients in whom the risk of bleeding appears short-term. Most of these devices can be removed up to two weeks later, and some may remain in place even longer with subsequent removal. Thrombolytic Therapy and Pulmonary Embolectomy

Thrombolytic agents activate plasminogen to form plasmin which then results in fibrinolysis as well as fibrinogenolysis. These agents can dramatically accelerate clot lysis in acute PE (and DVT) and such an approach was first documented more than several decades ago. Clinical trials have culminated in the approval of streptokinase, urokinase, and recombinant tissue-type plasminogen activator (tPA) for the treatment of massive PE. Urokinase is no longer available. For the past several decades, the clearly accepted recommendation to use intravenous thrombolytic therapy has been for PE with hemodynamic instability (hypotension). Other potential indications include PE associated with severely compromised oxygenation, and/or with echocardiographic evidence of right ventricular dysfunction without hypotension. More direct techniques, such as catheter-directed administration of intraembolic thrombolytic therapy, have been utilized in small clinical studies but the data is inadequate to formulate recommendations. The use of catheter-directed thrombolytic therapy in patients with proximal occlusive DVT associated with significant swelling and symptoms is increasing. Hemorrhage is the primary adverse effect associated with thrombolytic therapy with the most devastating complication being intracranial hemorrhage which occurs in approximately 1% of patients in clinical trials. The primary contraindications to thrombolytic therapy include active bleeding, surgery within the previous one to two weeks (depending on the specific procedure), or intracranial pathology. When patients appear to be at extraordinary risk of rapid death from PE, clinical judgment should be individualized with regard to contraindications. Pulmonary embolectomy may be appropriate in patients with massive embolism who cannot receive thrombolytic therapy.

590 PULMONARY THROMBOEMBOLISM / Pulmonary Emboli and Pulmonary Infarcts

Prognosis In the International Cooperative Pulmonary Embolism Registry of 2454 patients, all consecutive patients with a diagnosis of PE were included. PE was found to be the principal cause of death. The 3month mortality was 17.5%. Mean 1-month mortality rates of treated and untreated PE have been estimated at 5–8% and 30%, respectively. While a small percentage of patients with acute PE will ultimately develop chronic dyspnea and hypoxemia due to chronic thromboembolic pulmonary hypertension, most patients who survive the acute episode have no long-term pulmonary sequelae. However, chronic leg pain and swelling from DVT (postphlebitic syndrome) may result in significant morbidity.

Prevention Measures to prevent VTE appear to be grossly underutilized. A substantial reduction in the incidence of DVT can be achieved when patients at risk receive appropriate prophylaxis. For example, the risk of DVT after total hip or knee replacement is 50% or greater without prophylaxis. The superiority of LMWH over unfractionated heparin has been demonstrated in these settings, as well as in other highrisk settings such as acute spinal cord injury. In hospitalized general medical patients, anticoagulant prophylaxis should always be strongly considered as the rate of DVT, based upon a venographic endpoint, is as high as 15% in patients receiving placebo. Two LMWHs (enoxaparin and dalteparin) are FDA-approved for medical patient prophylaxis in the United States. Subcutaneous heparin administered as 5000 units every 8 h has been studied more recently and extensively than 5000 units every 12 h in the medical patient setting, and if standard heparin is used, this regimen may be the more efficacious one. The advantages of LMWH, including a reduction in heparin-induced thrombocytopenia, have resulted in increased use of these agents. Intermittent pneumatic compression devices should be utilized when pharmacologic prophylaxis is contraindicated. Both methods combined would be reasonable in patients deemed to be at exceptionally high risk, but an additional reduction in risk in such patients has not been well substantiated. Each hospitalized patient should be assessed for the need for such prophylactic measures and all hospitals should strongly consider formulating their own written guidelines for each particular clinical setting, based upon the available medical literature.

See also: Anticoagulants. Coagulation Cascade: Overview; Fibrinogen and Fibrin; Thrombin. Pulmonary Thromboembolism: Deep Venous Thrombosis.

Further Reading Ahearn GS and Bounameaux H (2000) The role of the D-dimer in the diagnosis of venous thromboembolism. Seminars in Respiratory and Critical Care Medicine 21: 521–536. Bratzler DW, Raskob GE, Murray CK, Bumpus LJ, and Piatt DS (1998) Underuse of venous thromboembolism prophylaxis for general surgery patients. Physician practices in the community hospital setting. Archives of Internal Medicine 158: 1909–1912. Dolovich LR, Ginsberg JS, Douketis JD, Holbrook AM, and Gillian C (2000) A meta-analysis comparing low molecular weight heparins with unfractionated heparin in the treatment of venous thromboembolism. Archives of Internal Medicine 160: 181–188. Geerts WH, Pineo GF, Heit JA, et al. (2001) Prevention of venous thromboembolism. Seventh American College of Chest Physicians Consensus Conference on Antithrombotic Therapy. Chest 126: 338S–400S. Goldhaber SZ and Tapson VF (2004) A prospective registry of 5,451 patients with ultrasound confirmed deep vein thrombosis. American Journal of Cardiology 93: 259–262. Kakkar VV, Howe CT, Nicolaides AN, et al. (1970) Deep vein thrombosis of the legs: is there a ‘high risk’ group? American Journal of Surgery 120: 527–530. Konstantinides S, Geibel A, Heusel G, Heinrich F, and Kasper W (2002) Heparin plus alteplase compared with heparin alone in patients with submassive pulmonary embolism. The New England Journal of Medicine 347: 1143–1150. Levine M, Gent M, Hirsh J, et al. (1996) A comparison of low molecular-weight-heparin administered primarily at home with unfractionated heparin administered in the hospital for proximal deep vein thrombosis. The New England Journal of Medicine 334: 677–681. Merli G, Spiro T, Olsson C-G, et al. (2001) Subcutaneous enoxaparin once or twice daily compared with intravenous unfractionated heparin for treatment of venous thromboembolic disease. Annals of Internal Medicine 134: 191–202. Perrier A, Roy PM, Sanchez O, et al. (2005) Multidetector-row computed tomography in suspected pulmonary embolism. New England Journal of Medicine 352: 1760–1768. Samama MM, Cohen AT, Darmon J-Y, et al. (1999) A comparison of enoxaparin with placebo for the prevention of venous thromboembolism in acutely ill medical patients. New England Journal of Medicine 341: 793–800. Stein PD (ed.) (1996) Pulmonary Embolism. Baltimore: Williams and Wilkins. Tapson VF, Carroll BA, Davidson BL, et al. (1999) The diagnostic approach to acute venous thromboembolism. Clinical practice guideline. American Thoracic Society. American Journal of Respiratory and Critical Care Medicine 160: 1043–1066. The PIOPED investigators (1990) Value of the ventilation/perfusion scan in acute pulmonary embolism. Results of the prospective investigation of pulmonary embolism diagnosis. Journal of the American Medical Association 263: 2753–2759. Wells PS, Anderson DR, Rodger M, et al. (2001) Excluding pulmonary embolism at the bedside without diagnostic imaging: management of patients with suspected pulmonary embolism presenting to the emergency department by using a simple clinical model and D-dimer. Annals of Internal Medicine 135: 98–107.

PULMONARY VASCULAR REMODELING 591

PULMONARY VASCULAR REMODELING I F McMurtry and S A Gebb, University of Colorado Health Sciences Center, Denver, CO, USA P L Jones, University of Pennsylvania, Philadelphia, PA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Sustained vasoconstriction and vascular remodeling, comprised of adventitial and medial thickening of conduit and muscular pulmonary arteries, as well as muscularization of pulmonary arterioles, occurs in various forms of moderate pulmonary hypertension. By comparison, the severe idiopathic, familial, or secondary pulmonary arterial hypertension (PAH) that causes significant morbidity and mortality via right heart failure also involves a spectrum of constrictive and obliterative neointimal lesions. The pathogenesis of these neointimal lesions is poorly understood. This article addresses the controversy of vasoconstriction versus cell proliferation and matrix remodeling in the pathogenesis of PAH, recent studies of new rat models of severe PAH, the potential role of hemodynamic forces in the pulmonary arteriopathy, and possible links between genetic disruption of bone morphogenetic protein receptor type II signaling and development of PAH.

Introduction The normal adult pulmonary circulation is a lowresistance, low-pressure, high-flow system, and the pulmonary arteries (PAs) are correspondingly thin walled and highly distensible. However, there are several distinct clinical disorders and at least three genetic mutations that are associated with the development of pulmonary arterial hypertension (PAH), accompanied by pulmonary vascular wall remodeling and reduced vascular distensibility (Table 1). Despite differences in the underlying pathogenic mechanisms, pulmonary vascular remodeling, comprising cell- and matrix protein-dependent reorganization and thickening of the adventitial and medial layers of the vascular wall, is common to most forms of pulmonary hypertension. Whether idiopathic (primary), familial, or secondary to some other disease, severe PAH is characterized by adventitial and medial hypertrophy, neointimal hyperplasia and fibrosis, and complex neointimal lesions that constrict and obliterate the lumen of medium and small pulmonary arteries (Figure 1). The resultant increases in pulmonary vascular resistance and PA stiffness cause pressure and volume overloading of the right ventricle, which can lead to right heart failure and death.

Table 1 Diagnostic classification of pulmonary hypertension Pulmonary arterial hypertension (PAH) Idiopathic PAH Familial PAH PAH Related to: Collagen vascular disease Congenital systemic to pulmonary shunts Portal hypertension HIV infection Drugs and toxins Other (glycogen storage disease, Gaucher’s disease, hereditary hemorrhagic telangiectasia, hemoglobinopathies, myeloproliferative disorders, splenectomy) PAH associated with significant venous or capillary involvement Pulmonary veno-occlusive disease Pulmonary capillary hemagiomatosis Persistent pulmonary hypertension of the newborn Pulmonary venous hypertension Left-sided atrial or ventricular heart disease Left-sided valvular heart disease Pulmonary hypertension associated with lung disease and/or hypoxemia Chronic obstructive pulmonary disease Intersitial lung disease Sleep disordered breathing Alveolar hypoventilation disorders Chronic exposure to high altitude Developmental abnormalities Pulmonary hypertension due to chronic thrombotic and/or embolic disease Thromboembolic obstruction of proximal pulmonary arteries Thromboembolic obstruction of distal pulmonary arteries Pulmonary embolism (tumor, parasites, foreign material) Miscellaneous Sarcoidosis, histiocytosis X, lymphangiomatosis, compression of pulmonary vessels (adenopathy, tumor, fibrosing mediastinitis) Modified from Simonneau G, Galie N, Rubin LJ, et al. (2004) Clinical classification of pulmonary hypertension. Journal of the American College of Cardiology 43: 5S–12S.

The minority of adult PAH patients who show significant pulmonary vascular responsiveness to acute vasodilator testing, that is, a reduction of both mean PA pressure and pulmonary vascular resistance of at least 20% are treated with anticoagulants and Ca2 þ channel blockers, while those who appear to have a nonreactive or ‘fixed’ pulmonary vascular bed receive anticoagulants and prostacyclin (PGI2) analogs or endothelin 1 (ET-1) receptor blockers. These treatments improve exercise capacity and prolong

592 PULMONARY VASCULAR REMODELING

(a)

(b)

(c)

(d)

(e)

(f) ∗

(g)

(i)

(h)

(j)

(k)

Figure 1 Main histopathological features of arteriopathy in severe PAH (see original paper for details, sections were stained with Verhoeff–van Gieson unless specified). Medial hypertrophy: (a) pre-acinar pulmonary artery, 80; (b) intra-acinar artery, 600. Concentric laminar intimal thickening: (c) intra-acinar artery, 500; (d) pre-acinar artery. The vessel was decorated with anti-smooth muscle actin antibodies (SMA), revealing the intimal thickening (white arrow) to be composed of SMA-positive cells, 150. Eccentric (e) 100 and concentric nonlaminar (f) 100 intimal thickening of pre-acinar arteries. Plexiform lesions: (g) intra-acinar artery decorated with SMA showing SMA-negative endothelial cells (arrow) surrounded by a rim of SMA-positive cells (rusty color), 380; (h) pre-acinar artery adjacent to a plexiform lesion (arrow) and dilation lesions (shown by asterisk) 60. (i) Dilation lesions (arrows), 40; (j) colanderlike lesion, HE 400; (k) lymphomonocytic arteritis, HE, 300. Reproduced from Pietra GG, Capron F, Stewart S, et al. (2004) Pathologic assessment of vasculopathies in pulmonary hypertension. Journal of the American College of Cardiology 43: 25S–32S, with permission from The American College of Cardiology Foundation.

life, but do not cure PAH. Thus, new insights into the pathogenesis of the arteriopathy are needed. This article highlights the controversy of vasoconstriction versus vascular cell proliferation and

structural remodeling in the pathogenesis of PAH, recent studies describing new rodent models of severe PAH, the potential role of hemodynamic forces in the pulmonary arteriopathy, and the possible


mechanistic links between genetic disruption of bone morphogenetic protein receptor type II (BMPRII) signaling and development of PAH.

Vasoconstriction versus Proliferation Sustained pulmonary vasoconstriction has historically been considered a key component of the pathogenesis of PAH. Classic histopathological studies suggested that pulmonary vasoconstriction, medial hypertrophy of muscular arteries, and muscularization of arterioles preceded the development of neointimal thickening and plexiform lesions. Subsequent biochemical and molecular evidence in hypertensive lungs and PAs of an imbalance in vasoconstrictor signals (increased ET-1, thromboxane, serotonin, and angiotensin II, and decreased smooth muscle cell (SMC) hyperpolarizing K þ channel activity) over vasodilator signals (deficient nitric oxide (NO), PGI2, and vasoactive intestinal peptide, and increased phosphodiesterase activity) supported the concept that vasoconstriction was an important pathogenic factor in PAH, especially in the early stages of the disease. Yet, in addition to regulating vascular tone, these same vasoactive signals also regulate vascular cell growth, and the current view of many investigators of adult PAH is that the increased pulmonary vascular resistance is due largely, if not solely, to structural luminal narrowing and obliteration, and the disease should be considered one of uncontrolled vascular cell proliferation, deficient apoptosis, and excessive matrix protein deposition, rather than of inappropriate vasoconstriction. This argument, and the evidence for it, can be found in several recent papers, commentaries, and reviews. Because most adult PAH patients respond poorly, if at all, to acute administration of pulmonary vasodilators, such as PGI2 analogs, adenosine, and NO, it can be argued that by the time they are diagnosed and treated the vasoconstrictor component has disappeared (or that it was never involved), and the increased vascular resistance is due to structural luminal narrowing and obliteration of small distal PAs. Another possibility, however, is that the most effective vasodilator has not yet been tested. For example, while acute administration of a Ca2 þ channel blocker does not reduce sustained pulmonary hypertension in chronically hypoxic rats, a Rhokinase inhibitor, which can ‘activate’ myosin light chain phosphatase and thereby reverse increased Ca2 þ sensitivity of the contractile apparatus of hypertensive vascular smooth muscle (Figure 2), nearly normalizes the pressure and resistance. RhoA/Rho kinase signaling, which is increased in hypertensive arteries, is a convergence point in the signaling

pathways of several different G-protein-coupled receptors, and can theoretically mediate the response to multiple simultaneous vasoconstrictor stimuli. Thus, not all vasodilators are created equal, and it would be interesting to investigate if an inhaled Rhokinase inhibitor is any more effective than inhaled PGI2 or NO in eliciting selective pulmonary vasodilation in severe PAH. Whether or not vasoconstriction initiates the process in all cases, it is clearly involved in the pathogenesis of PAH in some patients, and appears to be more prevalent in children than adults. Moreover, even adult patients whose PAH has been successfully reversed by long-term intravenous epoprostenol (PGI2 analog) have to be maintained on oral vasodilators to prevent recurrence of the disease. Thus, it is premature to dismiss the importance of vasoconstriction, and perhaps a more accurate view of the pathogenesis of most forms of PAH is that it involves both vasoconstriction and an imbalance in vascular cell proliferation/apoptosis and matrix catabolism and deposition. Regardless, the key clinical question is whether simply lowering pulmonary arterial pressure with an effective vasodilator will reverse the obliterative neointimal and plexiform lesions of severe PAH, or does the therapy also have to more directly disrupt the autocrine or paracrine production of growth factors and inflammatory cytokines and/or the activation of intracellular signaling pathways responsible for the increased cell proliferation, survival, migration, and matrix protein deposition? In this regard, it is noteworthy that inhibition of Rho-kinase activity prevents induction of SMC tenascin-C (TN-C), a pro-proliferative matrix protein that is induced in experimental and clinical PA lesions.

Animal Models Most studies of the mechanisms of hypertensive pulmonary vascular remodeling are in animal models. The two most commonly used are hypoxia- and monocrotaline-induced pulmonary hypertension in rats, although transgenic mice are increasingly being used. While the rat models have provided important groundwork for the current use of Ca2 þ channel blockers, PGI2 analogs, ET-1 receptor antagonists, inhaled NO, and phosphodiesterase inhibitors in the clinical treatment of PAH, and continue to provide new insights into the myriad cellular and molecular mechanisms involved in regulation of pulmonary vascular tone and structure, it should be emphasized that neither develops the obstructive neointimal lesions found in severe PAH. In fact, if the hypertensive lungs of chronically hypoxic or monocrotaline-injected rats

594 PULMONARY VASCULAR REMODELING Stimulus (ET-1, 5-HT, TXA2, thrombin, S1P, etc.) PLC

GEF

GPCR

Ca2+

Rho-GT

No, statins

Active

GTP Rho-GDP Inactive IP3 Relaxation MLCK

Ca2+ Ca2+

Ca2+

Actin

P MBS

Ca2+ Ca2+

Ca2+

P Ca CaM MLCK Ca

P CPI-17

MLCP

P MLCP

Active

Inactive

P Myosin

P Myosin Ca2+

Y-27632 fasudil

Rho-kinase

Myosin

Ca2+ CaM Ca2+

Ca2+

SR

Myosin

Actin Contraction

Figure 2 RhoA/Rho kinase-mediated Ca2 þ sensitization of vascular smooth muscle cell contraction. Numerous G-protein-coupled receptor (GPCR)-dependent vasoconstrictors, including endothelin-1 (ET-1), serotonin (5-HT), thromboxane (TXA2), thrombin, and sphingosine 1-phosphate (S1P), lead to stimulation of guanine nucleotide exchange factors (GEF) and activation of the small GTPase RhoA, which binds to and activates Rho-kinase. Activated Rho-kinase, in turn, phosphorylates the myosin-binding subunit (MBS) of myosin light chain phosphatase (MLCP) and/or the MLCP inhibitory protein CPI-17, which inhibits MLCP and thereby increases phosphorylation of MLC and augments contraction at a given level of cytosolic [Ca2 þ ] and Ca2 þ -calmodulin (Ca2 þ -CaM)-mediated activation of MLC kinase (MLCK). Sustained RhoA/Rho kinase-mediated vasoconstriction is reversed by the Rho-kinase inhibitors Y27632 and fasudil (HA-1077) and by inhibition of RhoA activation by nitric oxide (NO) or statins. (PLC-b, phospholipase C beta; IP3, inositol-1, 4, 5-trisphosphate; SR, sarcoplasmic reticulum; GAP, GTPase activating protein).

are vasodilated and fixed under positive intravascular pressure, it is observed that the adventitial and medial thickening of small muscular PAs causes little, if any, reduction in lumen area. Thus, it appears that much of the muscular PA medial hypertrophy and luminal narrowing typically reported in hypoxia- and monocrotaline-induced hypertensive rat lungs fixed without maximal vasodilation is due to vasoconstriction (vasospasm), rather than to structural inward remodeling of the vascular wall. Recent studies showing marked reversal of established pulmonary hypertension in either chronically hypoxic or monocrotaline-injected rats by acute administration of Rho-kinase inhibitors, such as Y27632 and fasudil, supports the idea that sustained vasoconstriction, rather than structural medial thickening of either muscular PAs or neomuscularized pulmonary arterioles, is a major cause of increased pulmonary vascular resistance in these models. This is not to say that the remodeling plays no role in the

increased resistance, but rather that its apparent contribution is exaggerated in PAs that are not vasodilated before fixation. It is noteworthy that in studies of vascular remodeling in systemic hypertension, it has long been appreciated that the direct contribution of vascular structure to luminal narrowing needs to be determined under conditions of complete lack of vascular tone, that is, under maximal vasodilation. Variations of the standard monocrotaline-injected and chronically hypoxic rat models have recently been developed as an attempt to more closely mimic the obstructive multicell-type intimal lesions found in patients with severe PAH. One such model is the leftlung pneumonectomized plus monocrotaline-injected rat that develops smooth muscle a-actin-positive, but endothelial cell (EC) CD31-negative, neointimal lesions in the small peripheral PAs/arterioles of the right lung. It has been speculated that the combination of monocrotaline pyrrole-induced EC injury


and increased blood flow (shear stress) is responsible for the lesions, but the role of increased blood flow and shear stress is unclear (see below). Histological examination has suggested more than 50% occlusion of some arteries by medial hypertrophy and neointimal hyperplasia. Treatment of these rats with the pleiotrophic agent simvastatin reverses the severity of PA medial hypertrophy, neointimal lesions, and hypertension. This was associated with downregulation of lung tissue expression of various inflammatory genes and upregulation of eNOS and the cell-cycle inhibitor p27Kip1, and with decreased proliferation and increased apoptosis of cells in the media and neointima. Monocrotaline has also recently been found to cause severe PAH and development of neointimal lesions in the small distal PAs of ET-1 type B (ETB) receptor-deficient rats. The neointimal lesions express high levels of the proangiogenic factor vascular endothelial growth factor (VEGF) and the proliferative matrix protein TN-C, and also contain proliferating cells that express EC and SMC markers. Cardiac output was very low in the pulmonary hypertensive ETB receptor-deficient rats; so increased blood flow was apparently not a factor in sustaining the expression of pro-proliferative molecular signals and the neointimal hyperplasia. Another recent rat model combines VEGFR-II inhibition by the receptor tyrosine kinase inhibitor SU5416 (semaxinib) with exposure to chronic hypoxia to cause severe PAH associated with development of occlusive PA neointimal lesions, which resemble plexiform lesions. The development of pulmonary arteriopathy is accompanied by markers of increased EC death and attenuated by treatment with a nonspecific caspase inhibitor. Thus, it has been proposed the neointimal lesions are initiated by EC death followed by selection and exuberant overgrowth of an apoptosis-resistant EC phenotype, as has also been suggested to occur in some PAH patients. While all three rat models of severe PAH develop occlusive neointimal lesions in small distal PAs, there are apparent differences in the proliferative cell phenotypes involved, that is, SMCs in the pneumonectomy plus monocrotaline model, cells expressing smooth muscle and EC markers in the ETB receptordeficient plus monocrotaline model, and ECs in the SU5416 plus hypoxia model. Explanations of these differences in cell phenotype are not obvious, but possibly relate to inconsistencies in detection methods for the chosen cell-specific antigens, or to differences in the way in which the lung injury and hypertension are induced. Interestingly, formation of neointimal lesions in all three models apparently requires a combination of severe EC injury

(monocrotaline pyrrole or SU5416) and some other abnormality (pneumonectomy, ETB receptor deficiency, or hypoxia). It is intriguing that the differences in neointimal cell phenotype in these rat models are reminiscent of the varied cell phenotypes, for example, ECs, modified SMCs, fibroblasts, and myofibroblasts, that have been described in the neointimal and plexiform lesions of human PAH. If these phenotypic differences are real, instead of being artifacts of tissue preparation or antibody specificity, they could reflect differences in the combination of pathogenic factors causing the PAH in different patients. Whereas the presence of PA neointimal lesions in the three rat models is certain, the relative roles of vasoconstriction and structural encroachment in the apparent luminal occlusion are not. In these studies none of the lungs was maximally vasodilated during fixation. Preliminary experiments by M Oka and colleagues in the CVP research laboratory show significant pulmonary vasodilation by acute intravenous administration of the Rho-kinase inhibitor fasudil in the pneumonectomy plus monocrotaline and SU5416 plus hypoxia models (the ETB receptor model has not yet been tested). This suggests the severe PAH is not due solely to vascular structural remodeling but also importantly involves sustained Rho-kinase-mediated pulmonary vasoconstriction (Figure 2). Interestingly, in addition to its anti-inflammatory and antiproliferative effects, simvastatin, which reverses the pneumonectomy plus monocrotaline PAH, might also inhibit vasoconstriction by both inhibiting the activation of RhoA/Rho-kinase signaling and increasing the bioavailability of NO. Despite the drawbacks of using animal models to study the mechanisms and potential therapies of human diseases, studies of these rat models will hopefully provide new insights into the pathogenesis and treatment of severe PAH in humans. Possibly, these systems can be used to establish the properties of the neointimal cells and determine if and by what mechanisms they originate from the adventitia, media, or intima of the arterial wall, or even from circulating progenitor cells. Chronic treatment of rats with Rho-kinase inhibitors attenuates development of hypoxic pulmonary hypertension and reverses established monocrotaline-induced pulmonary hypertension, and it will be important to determine their effectiveness in the neointimal lesion models.

Hemodynamic Forces Hemodynamic forces, that is, pulsatile blood pressure (circumferential wall stress), hydrostatic pressure, and blood flow (wall shear stress), are considered


major regulators of EC and SMC function and arterial wall thickness and luminal diameter. These biomechanical forces interact in complex ways with biophysical elements and numerous circulating and locally generated chemical signals to induce various types of adaptive and maladaptive arterial wall remodeling, including hypertrophic, eutrophic, or hypotrophic changes in medial mass, inward or outward changes in structural luminal diameter, and neointima formation. Interestingly, there is evidence in systemic arteries that hypertensive remodeling of the media that reduces luminal area does not necessarily involve cell growth or an increase in wall material, that is, the inward remodeling can be eutrophic as a consequence of rearrangement of existing cells and matrix proteins. In addition, studies of systemic resistance arteries in organ culture show that ET-1induced SMC contraction can, independently of changes in pressure and flow, cause eutrophic inward structural remodeling of the vascular wall, a response that may involve signals transmitted directly from activated SMCs to the matrix via b3-integrins. There is a lively debate among investigators in this area as to whether or not these structural changes have functional consequences with respect to the hypertension, but it is apparent that the contractile phenotype of the SMCs is well maintained, which differs from the dedifferentiation that occurs at sites of vascular injury and neointimal formation. Similarly, sustained vasoconstriction and an adaptive vascular remodeling leading to increased medial muscularization may occur in moderate pulmonary hypertension, but what then triggers and sustains development of severe PAH and the maladaptive neointimal pathology? It is well established in the systemic circulation that whereas arteries exposed to normal or high levels of blood flow and laminar shear stress are relatively free of disease, neointimal and atherosclerotic lesions tend to develop in areas where shear stress is low or disturbed (turbulent, oscillatory), such as at arterial branch ostia and bifurcations. So, despite universal exposure of arteries to environmentally induced risk factors, local hemodynamic forces affect neointimal pathology. Numerous in vitro and in vivo studies indicate that the protective effect of laminar shear stress is mediated largely by EC function. Laminar fluid shear stress maintains normal EC function by stimulating production of NO and PGI2 and expression of several other antioxidant, anti-inflammatory, antithrombotic, antiproliferative, and antiapoptotic proteins. In contrast, low or disturbed shear stress has the opposite effect and promotes endothelial oxidative stress and dysfunction, which can lead to increased vascular permeability to bloodborne protein and lipid mediators, adhesion and

activation of platelets, and infiltration of inflammatory cells. These events, in turn, generate a multitude of factors (reactive oxygen species, cytokines, chemokines, and growth factors) that increase EC turnover, activate matrix metalloproteinases, disrupt the elastic lamina, and stimulate medial and/or adventitial SMC/myofibroblast proliferation, migration, and matrix protein production resulting in neointimal formation. Compelling studies by Passerini and colleagues comparing gene expression in porcine aortic ECs isolated from disease-free areas of laminar versus disturbed flow suggest that while disturbed flow alone does not cause neointimal pathology, it apparently induces a proinflammatory phenotype that ‘primes’ the ECs for adverse responses to additional risk factors. Conversely, in mouse models of no or low flow, such as that created by ligation of an upstream artery, neointimal thickening occurs in the absence of other perturbation. Thus, low or disturbed flow-promoted neointimal thickening is a complex, multifactorial process that appears to be initiated by endothelial dysfunction, plus recent studies in various strains of rats and mice indicate that the severity of vascular remodeling is genetically determined. An important unresolved question concerning the pathogenesis of PAH is what roles are played by blood pressure and flow. Are the neointimal thickening and plexiform lesions causes or consequences of the pulmonary hypertension, or both? Medial and intimal thickening can apparently occur independently of pulmonary hypertension in localized PAs, but does development of the diffuse neointimal thickening of PAH depend on increases in PA pressure and/or flow? Congenital left to right cardiac shunts can lead to the development of severe PAH and neointimal lesions. While the hypertension and arteriopathy are generally ascribed to effects of increased blood flow and shear stress, greater consideration needs to be given to the effects of the high PA pressure. This is especially true in cases of large ventricular septal defects or a large patent ductus arteriosus where systemic arterial pressure can be transmitted into the pulmonary circulation. Animal studies indicate that systemic artery to PA shunts, which increase both pressure and flow in the downstream PAs, can induce severe PAH and formation of neointimal lesions, whereas large arteriovenous shunts, which increase cardiac output and pulmonary blood flow, but not pressure, cause no or only modest pulmonary hypertension and vascular remodeling. In young pigs, for example, anastomosis of left lower lung lobe artery to aorta exposes PAs to high flow and pressure and leads to medial thickening and


intimal fibrosis with subsequent development of plexiform and dilatation lesions. Although the cause-and-effect relationship is unclear, it is interesting that at the time neointimal lesions are most severe, shunt flow, but not pressure, is markedly reduced. Similarly, in calves with a systemic to left PA shunt, those with the lowest shunt flow at 10 weeks had the most severe small PA obliteration. These observations are similar to those in the rat models of PA neointimal formation discussed above in which the neointimal lesions coexist with low pulmonary blood flow as an apparent consequence of cardiac failure. Thus, whatever the role of increased blood flow in initiating the hypertensive process, the neointimal lesions are evidently sustained during low flow. In fact, recent work indicates that increased pulmonary blood flow via an aortocaval fistula actually attenuates development of pulmonary hypertension and neointimal hyperplasia in the pneumonectomy plus monocrotaline rat. Collectively, these observations suggest that high pulmonary arterial pressure is a major factor in inducing formation of PA neointimal lesions. High flow alone is apparently not sufficient, and based on the inverse relationship between flow and neointimal formation observed in systemic arteries, the question arises as to whether low flow might actually promote initiation or progression of the lesions. The role of shear stress in the arteriopathy of PAH is poorly understood. However, it can be speculated that adverse effects of disturbed flow on EC function might explain the tendency of obliterative PA neointimal lesions to develop at sites distal to vessel bifurcations (Figure 3) and the ostia of supernumerary arteries. If high blood pressure is, in fact, both cause and consequence of neointimal thickening in PAH, then an interesting possible pathogenic scenario is that it promotes the neointimal pathology by inducing vascular inflammation. Inflammatory-induced endothelial dysfunction is believed to play an important role in vascular diseases, including PAH, but a key question is what induces the inflammation? In some cases, the inflammation is due to systemic immunological disorders or infections, such as those that occur in connective tissue diseases or HIV infection. However, some patients with idiopathic PAH show evidence of autoimmunity and/or systemic inflammation with no identifiable cause. Similar observations have been made in patients with systemic hypertension, and a current hypothesis is that high systolic pulse pressure causes a chronic low-grade oxidative stress and inflammation of the endothelium, possibly via increased cyclic wall stress, activation of local EC angiotensin II and

Figure 3 In severe PAH, concentric-obliterative and/or plexiform lesions show a tendency to develop at sites just distal to bifurcations of small- to medium-sized muscular PAs. In this section stained with FVIII (see original paper for details), the cells lining the multiple lumina of the plexiform lesions stained positive for FVIII. Dilatation lesions can be seen within and adjacent to the plexiform lesions (arrows). Reprinted from American Journal of Pathology, 1999, 155: 411–419, with permission from the American Society for Investigative Pathology.

ET-1 signaling, and increased NAD(P)H oxidasedependent production of reactive oxygen species. Perhaps similar mechanisms are operative in the pathogenesis of PAH.

BMPRII Signaling: A Missing Link? The recent discovery that heterozygous germline mutations in the BMPRII gene (BMPR2) are associated with B50% of familial and B20% of idiopathic PAH may provide important clues to the relative roles of vasoconstriction and vascular remodeling in PAH. Before considering this possibility, it is important to outline bone morphogenetic protein (BMP) signaling in normal cells. BMPs are developmentally regulated, TGF-b-related polypeptides that exert diverse cellular effects, depending upon cell type, context, and differentiation state. Briefly, BMPs (including BMP2, 4, 6, and 7) stimulate heterodimerization and activation of two types of BMP receptors (i.e., I and II) and initiate phosphorylation of the Smad proteins (1, 5, and 8), which combine with the common mediator Smad4. Upon translocation to the nucleus, the Smad complex binds target genes to activate or repress their transcription. Thus, it is tenable to hypothesize that whereas wild type BMPRII signaling suppresses pulmonary vascular cell proliferation and PA remodeling, haploinsufficiency generated by heterozygous mutations impairs this type of control. Indeed, BMP2, 4, and 7 do suppress normal main PA SMC proliferation,


and this response is lost in cells isolated from idiopathic PAH patients. Nevertheless, this ‘one-hit’ hypothesis is clearly insufficient, since it fails to explain why subpopulations of normal cultured peripheral PA SMCs actually proliferate in response to BMPs, and why PAH in individuals with BMPR2 mutations may take many years to develop, or never develop at all. A possible explanation is suggested by the recent finding that heterozygous BMPR2-mutant mice exhibit pulmonary hypertension and muscularization of distal PAs only after challenged with a leukotriene-induced inflammatory stress. Therefore, BMPR2 mutations may predispose PA SMCs to react abnormally to secondary genetic or extrinsic factors that are experienced over time in a cell-type and site-specific manner. In other words, development of PAH, even in patients with BMPR2 mutations, requires multiple ‘hits’. In addition, whereas the development of pulmonary hypertension in the inflammatory-stressed heterozygous BMPR2-mutant mice was immediate, the muscularization of PAs was delayed. This emphasizes that increased pulmonary vasoconstriction should not be ignored as a possible initiating factor in the PAH associated with BMPR2 haploinsufficiency. In terms of gene structure and function, a variety of heterozygous, inactivating mutations have been found in all 13 coding exons and the flanking intronic sequences of BMPR2. It is believed that these diverse mutations may generate a modified receptor that is deficient in ligand binding (due to mutations in the extracellular domain) or kinase activity (due to mutations in cytoplasmic domains), thereby subverting the normal ability of wild type BMPRII to promote EC regeneration and survival on the one hand, while suppressing SMC proliferation on the other. These functions appear to be mediated by Smad signaling, at least in SMCs. Although it is conceivable that any one of the mutations could result in deficient BMPRII signaling, it is unclear whether different mutations stimulate or repress the same or different signal transduction pathways and target genes in PAH. Recent work by K Ihida–Stansbury in our group shows that PA SMCs harboring ligandbinding domain BMPR2 mutations express higher levels of extracellular signal-regulated kinase (ERK)1/2 activity than those bearing kinase domain mutations, despite the fact that both cell types overexpress similar levels of the SMC mitogen, TN-C. Thus, it is likely that specific signaling pathways are fine-tuned by distinct BMPRII domains. In support of this, there is evidence that the wild type BMPRII cytoplasmic tail domain binds to and, depending on cell type, either inhibits or activates LIM kinase 1, which is a key regulator of the actin cytoskeleton via

the F-actin depolymerizing factor cofilin. Since a subset of familial PAH patients possess BMPR2 mutations that truncate the LIM kinase 1-interacting tail domain, and because cytoskeletal actin dynamics control MAP kinase activity, gene expression, and contraction in SMCs and ECs, it is conceivable that ligand binding and cytoplasmic domain mutations activate or repress different downstream signaling pathways that converge to control common mediators associated with pulmonary vasoconstriction and/or vascular remodeling. Additional studies on BMPRII signaling are clearly needed, and these will hopefully answer several outstanding questions, including: why do BMPR2 mutations affect pulmonary vascular cells, whereas systemic vascular cells bearing the same genetic abnormality are seemingly unaffected, why do normal ECs and SMCs isolated from different PA segments respond differently to BMPs, which target genes or signaling molecules are activated or inhibited by BMPR2 mutations, if one or more of the mutations promotes vasoconstriction, do they do so by increasing SMC contractility or the balance of vasoconstrictors to vasodilators, and do pulmonary hemodynamic forces modulate the function of wild type and mutated BMPRIIs? Eagerly awaited answers to these questions will eventually provide new insights into how BMPRII signaling influences pulmonary vascular tone and structure. See also: Neonatal Circulation. Smooth Muscle Cells: Vascular. Vascular Disease.

Further Reading Boudreau NJ and Jones PL (1999) Extracellular matrix and integrin signalling: the shape of things to come. Biochemical Journal 339: 481–488. Bund SJ and Lee RMKW (2003) Arterial structural changes in hypertension: a consideration of methodology, terminology and functional consequence. Journal of Vascular Research 40: 547–557. Cool CD, Stewart JS, Werahera P, et al. (1999) Three-dimensional reconstruction of pulmonary arteries in plexiform pulmonary hypertension using cell-specific markers. Evidence for a dynamic and heterogeneous process of pulmonary endothelial cell growth. American Journal of Pathology 155: 411–419. Dorfmuller P, Perros F, Balabanian K, and Humbert M (2003) Inflammation in pulmonary arterial hypertension. European Respiratory Journal 22: 358–363. Humbert M, Morrell NW, Archer SL, et al. (2004) Cellular and molecular pathobiology of pulmonary arterial hypertension. Journal of the American College of Cardiology 43: 13S–24S. Jeffery TK and Morrell NW (2002) Molecular and cellular basis of pulmonary vascular remodeling in pulmonary hypertension. Progress in Cardiovascular Diseases 45: 173–202. Malek AM, Alper SL, and Izumo S (1999) Hemodynamic shear stress and its role in atherosclerosis. Journal of the American Medical Association 282: 2035–2042.

PULMONARY VASCULAR REMODELING 599 Mandegar M, Fung YCB, Huang W, et al. (2004) Cellular and molecular mechanisms of pulmonary vascular remodeling: role in the development of pulmonary hypertension. Microvascular Research 68: 75–103. Newman JH, Fanburg BL, Archer SL, et al. (2004) Pulmonary arterial hypertension: future directions: report of a National Heart, Lung and Blood Institute/Office of Rare Diseases workshop. Circulation 109: 2947–2952. Newman JH, Trembath RC, Morse JA, et al. (2004) Genetic basis of pulmonary arterial hypertension: current understanding and future directions. Journal of the American College of Cardiology 43: 33S–39S. Pietra GG, Capron F, Stewart S, et al. (2004) Pathologic assessment of vasculopathies in pulmonary hypertension. Journal of the American College of Cardiology 43: 25S–32S. Shimokawa H and Takeshita A (2005) Rho-kinase is an important therapeutic target in cardiovascular medicine. Arteriosclerosis, Thrombosis, and Vascular Biology 25: 1767–1775.

Simonneau G, Galie N, Rubin LJ, et al. (2004) Clinical classification of pulmonary hypertension. Journal of the American College of Cardiology 43: 5S–12S. Stenmark KR, Davie NJ, Reeves JT, and Frid MG (2005) Hypoxia, leukocytes, and the pulmonary circulation. Journal of Applied Physiology 98: 715–721. Stewart DJ (2005) Bone morphogenetic protein receptor-2 and pulmonary arterial hypertension: unraveling a riddle inside an enigma? Circulation Research 96: 1033–1035. van Suylen RJ, Smits JFM, and Daemen MJAP (1998) Pulmonary artery remodeling differs in hypoxia- and monocrotaline-induced pulmonary hypertension. American Journal of Respiratory and Critical Care Medicine 157: 1423–1428. Voelkel NF and Cool C (2004) Pathology of pulmonary hypertension. Cardiology Clinics 22: 343–351. Widlitz A and Barst RJ (2003) Pulmonary arterial hypertension in children. European Respiratory Journal 21: 155–176.

R RADIATION-INDUCED PULMONARY DISEASE G D Rosen and D S Dube, Stanford University Medical Center, Stanford, CA, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract This article discusses the natural history, clinical epidemiology, diagnosis, and treatment of radiation-induced lung diseases (RILDs). Furthermore, we review current experimental developments and assess potential areas for future investigation. Radiation therapy (RT) is a key component of the approach to the treatment of thoracic neoplasms. The use of radiation for the treatment of cancer exploits the preferential cytotoxicity of radiation to cells with high mitotic index. By the same principle, non-neoplastic cells with high mitotic index are also vulnerable to the deleterious effects of radiation. As a consequence, RT to the lung culminates in significant injury to normal cells with high mitotic index such as vascular endothelial cells and alveolar type II pneumocytes. The clinical manifestations of radiation-induced lung injury fall into two categories, acute radiation pneumonitis (RP) and radiation-induced lung fibrosis (RIF) which is a late sequelae of RT. Epidemiological analyses reveal that radiationmediated lung injury is amplified by many factors, such as radiation dose–lung volume relationships, concomitant use of chemotherapy, and the abrupt withdrawal of previously instituted steroid therapy. The cornerstone of the management of RILDs is prevention that is accomplished through surveillance of lung function, prompt evaluation of respiratory symptoms, and the early institution of steroids upon making a diagnosis of RP.

Introduction The discovery of X-rays by Wilhelm Roentgen in 1895 heralded the origin of radiation oncology. Shortly after the discovery of X-ray radiation, the first use of radiation for the treatment of cancer was reported. The development of the cathode ray tube and subsequently cobalt-60 teletherapy and linear accelerators revolutionalized the generation of Xrays for cancer treatment. Groover and co-workers first reported in 1922 that side effects of radiation therapy (RT) were radiation pneumonitis (RP) and pulmonary fibrosis (see Pulmonary Fibrosis). In 1928, Antoine Lacassagne observed that a short duration of the interval between radiation treatments, high radiation doses, low voltage, and opposing fields were associated with radiation injury. A rise in the number of cases of radiation-induced lung diseases (RILDs) can result from several

factors: (1) an increase in the incidence and prevalence of neoplasms as a result of the growing number of elderly patients; (2) use of RT as part of the conditioning regimen prior to bone marrow transplantation; and (3) increase in the number of immunocompromised hosts, in whom malignancies are more prevalent (see Table 1 for a list of RILDs). Both, the demand to improve the efficacy of RT and the need to limit lung injury, have spurred technical modifications in the delivery of RT. At present, the use of RT is preceded by rigorous planning that encompasses accurate tumor localization through the use of ultrasound (US), magnetic resonance imaging (MRI), and computer tomography (CT) (see Lung Imaging). The future of RT will, in part, focus on the reduction of side effects through targeting of RT to tumor but not normal cells and enhancing cytocidal effects of lower doses of RT through the use of radio-sensitizers and genetic engineering as well as the antagonism of endogenous mediators of RILDs.

Etiology Genetic Factors

The lack of stereotyped uniformity in the incidence of RILD among recipients of RT suggests that genetic factors may be crucial to the development of radiation lung injury. Investigators have identified that the loss of heterozygosity at the mannose 6-phosphate insulinlike growth factor-2 receptor (M6P/IGF2R) leads to elevated transforming growth factor beta (TGF-b) levels (see Transforming Growth Factor Beta (TGF-b) Family of Molecules). They proposed that the elevated TGF-b levels enhance the susceptibility of the surrounding tissues toward the development of RP. The repertoire of potential genes that are likely to be involved in the immune response to radiation injury is immense. Moreover, the interaction of potential genes is likely to be very complex but at present the precise role of genetic factors is not well understood. Host Factors

A variety of patient factors – age, sex, tobacco use, tumor site, and pre-RT pulmonary function tests

602 RADIATION-INDUCED PULMONARY DISEASE Table 1 Radiation-induced lung diseases

Table 2 Risk factors for radiation-induced lung disease

Parenchymal Acute Radiation-induced pneumonitis Diffuse alveolar damage Infectious sequel due to immunosuppression Late effects Pulmonary fibrosis Propensity to infection due to anatomical distorting effects of radiation

Patient factors Age Female sex (small lung volumes) Genetic susceptibility

Vascular Pulmonary hypertension Superior vena caval syndrome Secondary pulmonary venous hypertension from cardiomyopathy Pulmonary vessel thrombosis Fatal pulmonary hemorrhage (FPH) Airway disease Radiation-induced bronchitis (RIB) Tracheal stenosis Bronchiolitis obliterans Bronchiectasis Airway hyperreactivity Pleural diseases Pleural fibrosis Pneumothorax Broncho-pleural fistula Chylothorax Thoracic cage disease Radionecrosis of vertebrae, ribs Thoracic cage cutaneous contracture Neurological Phrenic nerve palsy Chronic pain syndromes Spinal cord damage Neoplasms Secondary malignancy Miscellaneous Recurrent cough Exercise-induced dyspnea Mediastinal fibrosis

(PFTs) as well as the Karnofsky performance status (Table 2) – have been proposed to impact the emergence of RP. The role of each factor alone is unclear because some of the studies exploring their significance reveal different dosing regimens which is the most important factor in the development of RP. It is clear that underlying pretreatment patient conditions that reduce total lung volume favor the development of RP. A retrospective study of 84 patients with small or non-small cell lung cancer (NSCLC) evaluated the role of gender, age, surgery, chemotherapy, chronic obstructive pulmonary disease (COPD), and performance status. The study found only COPD and the concomitant use of mitomycin as independent risk factors which are associated with the emergence of

Dosimetric variables V20, MLD Radiation fraction 42.67 Gy Medication Concurrent use of chemotherapy during cancer therapy Withdraw of corticosteroids

RP. Another study that assessed the influence of patient-specific factors on RT-induced reduction in pulmonary perfusion employed a multivariate analysis to analyze the effect of tobacco history, pre-RT diffusion capacity, TGF-b, chemotherapy exposure, disease type, and mean lung dose. The study found a trend but it was not statistically significant toward increased radiation sensitivity among patients who were nonsmokers and receiving radiation doses greater than 40 Gy. The authors concluded that patient-specific factors have a minimal effect on RTinduced reduction in regional lung perfusion. A study using CT scans among patients with limited small cell lung cancer (SCLC) that was designed to evaluate the magnitude of individual variation in the severity of lung fibrosis following treatment with chemoradiation, found significant patient-to-patient heterogeneity in patients at risk for developing radiationinduced fibrosis. The role of age in the development of RP is conflicting: some studies have failed to identify an association between age and the emergence of RP while others have reported an association. As alluded to previously, the propensity to develop RT may be more related to pretreatment lung volume than age. Dosimetric Factors

Several investigators have shown that dosimetric factors are the best predictors of clinically manifest RP following external-beam RT for a variety of neoplasms (see Table 2 and Radiotherapy). The dosimetric factors that have been evaluated include mean lung dose (MLD), V20, V30 (defined as the percentage of the total lung volume receiving greater than 20 or 30 Gy of radiation, respectively), and radiation dose per fraction. A prospective study of 96 patients who underwent three-dimensional conformal radiotherapy (3D-CRT) for NSCLC observed a threshold effect for the emergence of RP at radiation doses of 20–40 Gy. The study also noted that the MLD, V20, V30, and age were predictive of RP following RT.

RADIATION-INDUCED PULMONARY DISEASE 603

71 patients chemoradiation

382 patients RT alone

100 6/7

Incidence of RP (%)

80

2/2

60 6/12 40

6/29

20

11/50

14/82

2/23 10/95

2/14

0 0

20

40

60

V20 (%) Figure 1 Incidence of radiation pneumonitis (RP) in relation to V20 and effects of concurrent chemoradiation. Reproduced from Tsujino K, Hirota S, Endo M, et al. (2003) Predictive value of dose–volume histogram parameters for predicting radiation pneumonitis after concurrent chemoradiation for lung cancer. International Journal of Radiation Oncology, Biology, Physics 55(1): 110–115, with permission from Elsevier.

A univariate and multivariate analysis of patient- and treatment-related factors among lung cancer patients treated with conventional fractionated radiotherapy and concurrent chemoradiation identified V20 as the best predictor of the incidence and grade of the ensuing RP post-RT. The study also demonstrated that concurrent use of chemotherapy increases the incidence of RP (Figure 1). A study designed to correlate dose–volume histogram factors to clinically manifest RP reported that all dosimetric factors (V30, MLD, normal tissue complication probability (NTCD)) correlated with the emergence of RP. However, an analysis of factors such as age, irradiation of the lower lung field, and low pre-RT pulmonary function demonstrated no correlation with the emergence of RP. An intriguing observation is that the incidence of RP may be lower among smokers. Chemotherapy

The concurrent use of chemotherapy with radiation in regimes that exploit the radiosensitizing effects of drugs such as taxanes has been associated with the potentiation of the pneumotoxic effects of radiation. The compounding effects of chemotherapy on pneumonitis appear to be independent of the mechanisms of action of individual chemotherapeutic agents although some agents such as bleomycin are known to be radiosensitizers. Moreover, there is no clear dose–effect relationship between chemotherapy

Table 3 Selected chemotherapy regimes that potentiate radiation lung toxicity Doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) Bleomycin, adriamycin, cyclophosphamide, and vincristine Paclitaxel

and the emergence and extent of RP. A study of 13 SCLC cases treated with bleomycin, adriamycin, cyclophosphamide, and vincristine in addition to radiotherapy noted severe pulmonary fibrosis in five cases, two of which were fatal. In a study of 41 patients with breast cancer who were treated with RT concurrently with chemotherapy, the regimes that included paclitaxel showed that RP developed at a rate of 14.6%. In comparison, patients treated with RT and chemotherapy without paclitaxel developed RP at the rate of 1.1% (95% confidence interval ¼ 0.2–2.3%, po0.0001). Additional chemotherapeutic drugs that have been associated with RP are shown in Table 3. Pathology

Murine models of RP strongly suggest that injury to pulmonary capillary endothelial cells and type II pneumocytes mediates the pathogenesis of RP (Figure 2) (see Endothelial Cells and Endothelium). Within an hour of RT, quantitative and qualitative

604 RADIATION-INDUCED PULMONARY DISEASE

Radiation therapy with 137Cs or 60Co collimated to the appropriate lung sections

Mouse strain, e.g., C 57BL/6 with or without genetic manipulation and fed ad libitum and housed in germ free environment

Histopathological analysis Early RP (0–8 weeks) • type II pneumocyte injury followed by proliferation • inflammation and endothelial cell damage • fibrosis Late RP (after 12 weeks) • persistent free radical damage of endothelium and fibrosis

Analysis of outcomes • respiratory distress • survival

Bronchoalveolar lavage for cytokines, e.g., IL-6, TGF-, and surfactant proteins

Analysis of gene expression • supra oxide dismutase • TGF- and other cytokine genes • surfactant genes

Mathematical modeling of the spatial effects of radiation, e.g., dose–volume relationships

Figure 2 Murine radiation pneumonitis (RP) model. Reproduced from Hynes RO (2002) Integrins: bi-directional, allosteric, signaling machines. Cell 110(6): 673–687, with permission from Elsevier.

changes are observed in type II pneumocytes that include reduced intracytoplasmic lamellar bodies followed by the release of surfactant. These changes are followed by a loss of type II cells and the synthesis of less viscous surfactant. A compensatory lamellar body repletion and type II pneumocyte proliferation are observed 4–12 weeks after RT. These changes occur simultaneously with a florid inflammatory cellular infiltrate into the alveolar septae. Inflammatory cells such as mast cells, plasma cells, fibroblasts, macrophages, and polymorphonuclear cells populate the infiltrating cells. Overall, the inflammatory picture usually resolves by 6–9 months although continued type II pneumocyte and arterial smooth muscle proliferation, and free-radical-induced endothelial and type II pneumocyte damage can lead to extensive collagen deposition, that is, radiationinduced lung fibrosis (RIF). The histopathological features of RP and RIF are shown in Figure 2.

Clinical Features Incidence and Prevalence

The observation that not all radiologically evident RP is associated with symptomatic disease leads to higher prevalence and survival statistics when radiological criteria are used in isolation to diagnose RP. In contrast, clinical criteria for diagnosing RP yield lower prevalence rates. The incidence of RP following external-beam RT varies from 1% to 34%. The incidence of RP depends on the type of tumor owing to technical variation in irradiation strategies, but the variability in dosimetry is still the largest factor that determines the risk of developing RP (Table 3). RP is uncommon in breast cancer when RT is restricted to the thoracic wall following mastectomy or after breast-conserving surgery. However, the inclusion of the supraclavicular fossa, axilla, and lung apex increases the incidence of pneumonitis from 1.4% to 3.9%. Incorporation of all


lymph nodes above the diaphragm, lung apices, and axilla bilaterally in the irradiation field for the treatment of Hodgkin’s lymphoma is associated with less than 5% risk of RP. Compared with RT for lung cancer, smaller areas of lung are irradiated in Hodgkin’s lymphoma. As a result, long-term follow-up of patients treated with RT for lymphoma shows only clinically insignificant pulmonary function changes in diffusing capacity of the lung for carbon monoxide (DLCO) and total lung capacity. In a study of 590 patients with stage 1A-IIIB Hodgkin’s disease, RP was observed in 3% of the patients receiving radiation alone while whole lung irradiation was associated with a 15% risk; the concurrent use of chemotherapy elevated the risk to 11%. A prospective evaluation of 96 patients with NSCLC who were treated with 3D-CRT reported an incidence of grade 1 RP to be 44% at 6 weeks while grade 2 or greater RP was evident in 7.8%. Mean lung dose, V20, V40, and age greater than 60 were predictive of RP. As we discussed previously, estimates of the prevalence of RIF are challenging owing to the variability in the diagnostic criteria, diagnostic methods, and the difficulty in excluding pre-existing lung injury. A prospective study, using CT scans, of 24 patients with advanced NSCLC treated with chemoradiation was undertaken to evaluate the incidence of RIF. Using the European Organization for Research and Treatment of CancerRadiation Therapy Oncology Group Scale, the investigators found the prevalence of RIF grades 1, 2, and 3 to be 14%, 33%, and 19%, respectively. Japanese Clinical Oncology Group trials for lung cancer observed 29 deaths out of 1176 (2.5%) that resulted from RT after a follow-up of four and a half years. Clinical Features of Radiation Pneumonitis

An inconstant relationship has been reported between radiological RILDs and clinically manifest RP. On occasion, clinically evident RP may precede radiographic changes. The spectrum of RP ranges from selflimiting radiological disease or evanescent dyspnea to respiratory failure that culminates in mechanical ventilatory support or rapid demise. RP manifests itself clinically 4–12 weeks after lung irradiation. Dyspnea is the most prominent and frequently the initial manifestation of RP and can be quantified using dyspnea scores (DSs) such as the Abratt or Borg scale. The Abratt scale defines a DS of 1 as dyspnea during brisk ambulation on a flat surface or while walking up an incline. A DS of 2 is dyspnea on ambulating distances greater than 100 m while a DS of 3 denotes failure to ambulate distances greater than 100 m. Patients with a DS of 4 are dyspneic at rest. A prospective

evaluation of lung function among patients irradiated for lung cancer observed a DS of 1 in 50% of the cases, while the remaining cases had a score of 2. At a 6-month follow-up, the DS increased from 1 to 2 in half of the cases. A DS of 3 was observed in approximately 5% of the cases. Cough is the second commonest manifestation of RP. Characteristically, the cough is nonproductive. In some cases, the cough becomes productive with pink frothy sputum but frank hemoptysis is rare in RP. Occasionally, systemic symptoms with ill-defined global malaise and fever occur together with dyspnea. A study of 29 patients with RP revealed the prevalence of symptoms to be dyspnea (93%), cough (58%), and fever (7%). Physical evaluation may be unrevealing. Classic auscultatory findings of RP include rales in the irradiated zone. However, RP has been reported in pulmonary locations outside the zone of irradiation and these findings may thus exist outside the irradiated lung zones. Regional RP may manifest as a focal consolidation. Also, pleural rubs may be evident in cases of radiation-induced pleuritis. A variety of conditions with a similar temporal appearance to RIP may mimic pneumonitis in the peri-irradiation period and include infectious pneumonitis, drug-induced pneumonitis, pulmonary alveolar hemorrhage, and less commonly, residual tumor. Also, underlying lung disease may confound an accurate diagnosis of RP. Clinical Features of Radiation-Induced Lung Fibrosis

RIF is characterized by an incipient pleuroparenchymal interstitial injury that ensues 6–24 months after RT. All patients who have received RT are at risk for developing RIF irrespective of whether they had evidence of RP. The clinical manifestations of RIF are diverse and patients with mild focal fibrosis may be asymptomatic. In contrast, patients with large areas of lung irradiation may present with a DS greater than 2 that can evolve into dyspnea at rest. Late cardiovascular and pulmonary complications accrue from the effects of RT on the vasculature. Advanced cases of RIF have been associated with delayed effects on the vasculature including thrombo-embolic disease, superior vena cava syndrome, and secondary pulmonary hypertension and cor pulmonale. The physical evaluation often reveals varying degrees of dyspnea and cutaneous effects of RT on the thoracic cavity may be evident on visual inspection. For example, the patient may show a deviated trachea and radiation-induced skeletal deformities. Chest excursion may be diminished along with inspiratory rales which is associated with a restrictive


process. Regional air flow may also be diminished in some settings so PFTs may reveal a mixed obstructive and restrictive picture. Pulmonary hypertension is characterized by a loud pulmonic heart sound, pulmonic and tricuspid regurgitation, elevated jugular venous pressure, engorged liver, and bilateral leg edema. Superimposed complications accruing from fibrosis and the altered lung function and anatomy include bronchiectasis and recurrent pneumonia.

Diagnostic Evaluation Chest X-Rays

The chest X-ray (CXR) is useful in the surveillance of patients following RT. It also allows the speedy diagnosis of RP, in addition to assisting in the establishment of alternative diagnoses. The clinical utility is enhanced by the fact that it is inexpensive and can be deployed rapidly. Classic radiographic features of RP include a diffuse hazy opacification that evolves into a patchy appearance. In some settings, the abnormalities coalesce over time into a uniformly radiodense appearance that mirrors the configuration of the irradiated field. More frequently, these changes are transient but in other cases they progress to frank RIF. RIF may be detected by serial radiographs beginning at 6 months after RT and initial manifestations include patchy and linear opacities. These changes culminate in reticular parenchymal opacities, the progression of which leads to lung-volume reduction. Pleural adhesions to the thoracic wall, pericardium, or the diaphragm can occur. Radiographic appearances vary depending on the method and location of irradiation. The employment of oblique radiation beams in the treatment of breast cancer, for example, is associated with atypical distribution of interstitial opacities. Apical opacities are encountered with the use of supraclavicular portals. Peripheral reticular opacities result from tangential beam portals (as observed in breast cancer) and paramediastinal opacities are encountered following irradiation of a mediastinal tumor. Computer Tomography

CT permits the early detection of subtle parenchymal changes that may not be evident on routine chest radiographs. Moreover, the superior parenchymal definition seen with high-resolution computer tomography (HRCT) enables the earlier detection of consolidation, subtle pulmonary parenchymal changes, and endobronchial occlusion that may herald tumor recrudescence. Characteristic CT appearances of RP include nonhomogeneous opacities and alveolar filling defects. Homogeneous reticular ground glass

opacities are encountered more often in RIF. Ground glass opacities are observed in both RP and RIF and can progress into reticular opacities. Over time there is associated volume loss, and pleural and diaphragmatic thickening. In three-dimensional confocal radiotherapy, lung injury may adopt unusual radiographic configuration which reflects the field and mode of radiation delivery. A study looking at 87 CT scans in 17 patients noted CT abnormalities in 15 of the 17 cases within 16 weeks of RT. In three out of the 15 cases, there were no changes indicative of RP evident at 16 weeks. In three cases, CXR changes became apparent much later than 1, 5, and 8 weeks respectively. In nine cases, the radiological features of lung injury were detected simultaneously by either CT or CXR. This same study reported air bronchograms (25%), loss of lung volume (15%), and pleural thickening as changes complicating RT. Adjuncts to CT scans have included gallium scintigraphy but its value is limited by low specificity and sensitivity. Bronchoscopy

The use of bronchoscopy in the evaluation of RP is limited. Most patients receiving RT are also immunocompromised; consequently, the emergence of parenchymal opacities on CXR necessitates bronchoscopy to exclude alternative etiologies such as infections, or diffuse alveolar damage. In RP, inspection of the airways may show hyperemia and mucosal engorgement. Bronchoalveolar lavage fluid (BALF) from irradiated patients compared to that of nonirradiated patients with lung cancer showed that total lymphocyte and eosinophil counts were higher in irradiated lungs. Moreover, human leukocyte antigen DR (HLADR)-positive CD4 þ T cells and HLADR-positive CD8 þ T cells as well as intercellular adhesion molecule (ICAM)-1-positive T cells are markedly increased. An interesting observation from this study was that the emergence of ICAM-1 positive cells correlated with the interval between initiation of RT and emergence of RP. However, other investigators have found no difference in the lymphocytic content between irradiated breast cancer cases that develop RP and those that do not. Pulmonary Function Tests

PFTs in lung cancer patients treated with RT and followed for a mean of 38 months noted that RT caused a decline of forced vital capacity (FVC) (89% of subjects), forced expiratory volume in 1 s (FEV1)(89%), and DLCO (90%). These variables returned to baseline at 1 year (105%, 100%, and 90% of subjects, respectively). Nevertheless, the study further observed that a progressive decline


followed the initial improvement phase. The study was however limited by its size. De Jaeger and coworkers evaluated the effect of RT on pulmonary function post-RT in 82 cases of NSCLC and correlated it to radiation dose, tumor regression, and changes in lung perfusion as measured by single photon emission CT (SPECT) lung perfusion scan. They estimated the post-RT perfusion using dose distribution and a dose–effect relation for loco-regional lung perfusion to calculate a predicted perfusion reduction (PPR). Reperfusion was defined as the difference between the baseline perfusion and the PPR. Tumor regression was associated with an improvement in FEV1 but a decline in DLCO, while the PPR did not correlate with improvement of FEV1 and FVC. Overall, RT causes restrictive ventilatory defects with diminution in FVC and FEV1 due to reduced compliance caused by fibrosis. The changes in FVC and FEV1 are variable and rather limited in their utility to follow the effects of RT. In contrast, the assessment of the alveolar functional integrity by DLCO is more sensitive for detecting RILDs. Serological Markers

No single serologic marker is a consistent predictor of risk for development of RP. Consequently, a search for a reliable marker for RP is ongoing. Markers that have been described as showing a positive correlation with RP include TGF-b, interleukin (IL)-1-a and IL-6. The clinical significance of these findings has, however, been questioned by others. The cytokeratin 19

fragment, a marker of epithelial cell damage, mucinlike high molecular weight glycoprotein (KL-6/MUC1), intercellular adhesion molecule-1 (ICAM-1), type III procollagen N-terminal peptide (P-III-P), laminin P1, and surfactant protein A and D have been shown to significantly correlate with RF. However, the clinical utility of these markers remains to be validated. Pathogenesis

The leading theories of the mechanism of RILDs invoke the role of free radicals and cytokines. Figure 3 shows a schematic representation of the putative mechanisms of RP and RIF. Free Radicals

Radiation causes structural modification of DNA, lipids, and proteins through peroxidation, aldehyde reactions, reactive oxygen species, and activates cytokine cascades that mediate processes that impair the structural integrity of pneumocytes and endothelial cells. Also, Cottier and co-workers showed that desferroxamine levels are elevated in patients who are likely to develop RP following RT. One group of investigators has observed that smokers undergoing RT for esophageal and breast cancer are less prone to RP possibly because glutathione (GSH) levels are known to be elevated in smokers compared to nonsmokers. Since GSH functions as a scavenger for singlet oxygen and superoxides, it may abrogate the proinflammatory effects of RT (76). Hashimura and co-workers have observed that LTC4 and LTD4 as

Radiation

DNA damage

Free radicals Reactive oxygen species Hydroxyperoxides


Radiation pneumonitis

Endogenous antioxidant mechanisms counteract the deleterious effects of free radicals Figure 3 Hypothetical mechanisms of radiation-induced pneumonitis.

Protein and lipid peroxidation


well as glutathione peroxidase are acutely upregulated in the irradiated mouse lung and remain persistently elevated 24 h postradiation and that the second generation antihistamine, azelastine, and coenzyme – Q, both free radical scavengers ameliorated these changes. Further evidence supporting the theory that antioxidants protect against RP has been provided by experiments that demonstrate that the overexpression of manganese superoxide dismutase (MnSOD) in a transgenic murine model or the exogenous administration of the iNOS inhibitor L-NAME ameliorates the deleterious effects of RT. Cytokines

Both in vitro and clinical observations of the irradiated lung support the immunological basis of RILDs. As reviewed above, RT induces the generation of reactive oxygen species that is thought to initiate and propagate the pathological manifestations of RP. It has been shown that irradiated mice upregulate the chemokine macrophage chemoattractant protein 1 (CCL2), macrophage-derived chemokines (CCL22), thymus-derived and activation-derived chemokines (CCL17), and the CCR-4 receptors on alveolar lymphocytes and macrophages (see Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12)) (Figure 4). Both CCL22 and CCL17 are the natural ligands for CCR-4 and assist in the recruitment of proinflammatory cells and also regulate the proportion of Th2/Th1 lymphocytes. An analysis of the BAL from irradiated lungs reveals elevated TGF-b, IL-6, and IL-1 whose levels appear to correlate with the extent of RP (see Interleukins: IL-1 and IL-18; IL-6). TGF-b plays a pivotal role in the pathogenesis of other fibrotic lung diseases such as idiopathic pulmonary fibrosis where TGF-b mediates the differentiation of lung fibroblasts into myofibroblasts (see Transforming Growth Factor Beta (TGF-b) Family of Molecules. Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis) (Figure 5). The myofibroblast elaborates cellular matrix, for example, elastin and collagen, and produces TGF-b. IL-6 is pleiotropic cytokine that regulates multiple pro-inflammatory cytokines and is crucial for lymphocyte development. The role of TGF-b in RP is supported by the observation that the administration of a recombinant TGF-b1 type II receptor (TGF-bRII) gene, which inhibits TGF-b activation, in a mouse model of RP attenuates histological evidence of radiation-induced lung injury. The administration of halofuginone, an inhibitor of TGFb, was shown to reduce the colocalization of TGF-b with the tight junction protein Zo-1 and also reduces radiation-induced lung injury. This is interesting because the localization of TGF-b to Zo-1 is essential

Figure 4 Acute radiation pneumonitis showing alveolar injury with edematous thickened alveolar septa, atypical alveolar lining cells, and loose fibroblastic foci (H&E 200 ). Permission of Gerald Berry, M.D., Department of Pathology, Stanford University Medical School.

Figure 5 Chronic radiation pneumonitis characterized by mixed interstitial fibrosis and fibroblastic foci, patchy mononuclear inflammatory cell pneumonitis, foamy macrophages within distorted airway lumens, and fibrointimal thickening of muscular pulmonary arteries (H&E 200 ). Permission of Gerald Berry, M.D., Department of Pathology, Stanford University Medical School.

for the epithelial to mesenchymal transition suggesting that TGF-b-mediated epithelial–mesenchymal transition may play a role in radiation-induced lung injury. In summary, radiation induces chemokines and cytokines which promote the expression of proinflammatory and profibrotic mediators in RP.

Management and Current Therapy Steroids

Steroids are the mainstay for the treatment of RP (see Corticosteroids: Therapy). Up to 80% of RP cases have a dramatic clinical and radiological response to steroids. Supportive evidence for the use of steroids


accrues from clinical experience and in vitro data. The employment of steroids in the treatment of experimental mouse models of RP demonstrates that steroids delay the emergence of RP, attenuate the endothelial damage, and reduce the inflammatory cellular infiltrate. Notably, the effects of steroids on RP do not affect the emergence of RIF but the discontinuation of steroids in mice with experimental RP leads to accelerated mortality. Human data supporting the use of steroids are lacking. On the whole, the discontinuation of steroids in human subjects leads to worsening of RP but there is no data to support the prophylactic use of corticosteroids to prevent the disease. The duration of steroid therapy is subjective but is on the order of several weeks accompanied by a slow taper. There are also sporadic reports showing cyclosporine is of benefit in steroid-refractory radiation pneumonitis. Angiotensin converting enzyme (ACE) inhibition has been suggested as a protective strategy against RP based on limited animal data. However, there is no sufficient human data to support the use of ACE inhibitors for protection against RP. In summary, these observations support the use of steroids in clinically symptomatic RP, although the empiric use of steroids in asymptomatic patients’ post-RT is not recommended. Novel Therapeutic Targets

Ongoing research to identify novel methods to mitigate the adverse effects of RT focuses on reducing total radiation doses and blocking molecular mediators of RP. Technological innovations have dominated the impetus to reduce the unintended lung injury. However, there is promise that targeting RT specifically to the tumor may be improved with DNAbased technology to activate proapoptotic genes such as tumor necrosis factor-related apoptosis-inducing ligand (Apo-2L) (TRAIL), which preferentially activates apoptotic pathways in tumor versus normal cells. Additional strategies are focusing on limiting damage from RT-induced free radicals by increasing scavenger activity through activation of manganese superoxide dismutase or administration of antioxidants (see Oxidants and Antioxidants: Antioxidants, Enzymatic; Antioxidants, Nonenzymatic; Oxidants). Other potential strategies include the antagonism of mediators of inflammation such as blocking TGF-b, chemokines, and IL-6. See also: Chemokines, CC: MCP-1 (CCL2)–MCP-5 (CCL12). Corticosteroids: Therapy. Endothelial Cells and Endothelium. Interleukins: IL-1 and IL-18; IL-6.

Interstitial Lung Disease: Idiopathic Pulmonary Fibrosis. Lung Imaging. Oxidants and Antioxidants: Antioxidants, Enzymatic; Antioxidants, Nonenzymatic; Oxidants. Pulmonary Fibrosis. Radiotherapy. Transforming Growth Factor Beta (TGF-b) Family of Molecules.

Further Reading Abratt RP, Morgan GW, Silvestri G, and Willcox P (2004) Pulmonary complications of radiation therapy. Clinics in Chest Medicine 25(1): 167–177. Chen Y, Williams J, Ding I, et al. (2002) Radiation pneumonitis and early circulatory cytokine markers. Seminars in Radiation Oncology 12(supplement 1): 26–33. Epperly M, Bray J, Kraeger S, et al. (1998) Prevention of late effects of irradiation lung damage by manganese superoxide dismutase gene therapy. Gene Therapy 5: 196–208. Garipagaoglu M, Munley MT, Hollis D, et al. (1999) The effect of patient-specific factors on radiation-induced regional lung injury. International Journal of Radiation Oncology, Biology, Physics 45(2): 331–338. Geara FB, Komaki R, Tucker SL, Travis EL, and Cox JD (1998) Factors influencing the development of lung fibrosis after chemoradiation for small cell carcinoma of the lung: evidence for inherent interindividual variation. International Journal of Radiation Oncology, Biology, Physics 41(2): 279–286. Groover TA, Christie AC, and Merrit EA (1922) Observations on the use of the copper filter in the treatment of deep-seated malignancies. Southern Medical Journal 15: 440–444. Gross NJ (1977) Pulmonary effects of radiation therapy. Annals of Internal Medicine 86: 81–92. Hynes RO (2002) Integrins: bi-directional, allosteric, signaling machines. Cell 110(6): 673–687. Kong FM, Anscher MS, Sporn TA, et al. (2001) Loss of heterozygosity at the mannose 6-phosphate insulin-like growth factor 2 receptor (M6P/IGF2R) locus predisposes patients to radiation-induced lung injury. International Journal of Radiation Oncology, Biology, Physics 4991: 35–41. Movsas B, Raffin TA, Epstein AH, and Link CJ Jr (1997) Pulmonary radiation injury. Chest 111: 1061–1076. Muraoka T, Bandoh S, Fujita J, et al. (2002) Corticosteroid refractory radiation pneumonitis that remarkably responded to cyclosporin A. Internal Medicine 41(9): 730–733. Park KJ, Chung JY, Chun MS, and Suh JH (2000) Radiationinduced lung disease and the impact of radiation methods on imaging features. Radiographics 20(1): 83–98. Rancati T, Ceresoli GL, Gagliardi G, Schipani S, and Cattaneo GM (2003) Factors predicting radiation pneumonitis in lung cancer patients: a retrospective study. Radiotherapy and Oncology 67(3): 275–283. Rosiello RA and Merrill WW (1990) Radiation-induced lung injury. Clinics in Chest Medicine 11(1): 65–71. Tsujino K, Hirota S, Endo M, et al. (2003) Predictive value of dose–volume histogram parameters for predicting radiation pneumonitis after concurrent chemoradiation for lung cancer. International Journal of Radiation Oncology, Biology, Physics 55(1): 110–115. Wang LW, Fu XL, Clough R, et al. (2000) Can angiotensin-converting enzyme inhibitors protect against symptomatic radiation pneumonitis? Radiation Research 153(4): 405–410.

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RADIOTHERAPY P Van Houtte, Institut Jules Bordet, Brussels, Belgium & 2006 Elsevier Ltd. All rights reserved.

Abstract Radiotherapy uses the properties of ionizing radiation to treat mainly cancers. It was introduced soon after the discovery of Xrays by Roentgen and radium by Marie Curie at the end of the nineteenth century. Over the years, the technique has evolved due to major technical developments, due to better machines, powerful computers, and a better knowledge of the interaction between radiation and matter. Nowadays, it remains a major component in the treatment of lung cancer and is a major part in the multidisciplinary management of this disease.

History and General Principles Very soon after their discovery, X-rays (discovered by Roentgen in 1895) and radium (discovered by Marie Curie in 1898) were used to treat cancer; the first report was published as early as 1898. Nowadays, the practice of radiotherapy requires a knowledge of the disease treated, the physics of the radiation, and the effect of radiation on normal tissues and tumors. The progress made during the last decades in radiation oncology results from major improvements in technical equipment and from a better understanding of the interaction between radiation and tumors which have led to the development of new treatment modalities. The type of radiation used in clinical practice is called ionizing radiation. During absorption in tissues, such radiation produces the ejection of an orbital electron, creating an ion. This will be the starting point of a cascade of events leading ultimately to the destruction of the cells either by inducing apoptosis or by causing a loss of their proliferative capacity (the cells will die during a subsequent intermitotic phase). This explains the delay between the radiation and the later clinical signs and symptoms occurring days, weeks, or months after the treatment. Different types of radiation are available: electromagnetic waves characterized by their wavelength or frequency (their energy is directly proportional to the frequency and inversely proportional to the wavelength) and particle beams, mainly electrons (protons and carbon ions are only used in a few places worldwide). The following considerations apply to radiotherapy practice: the dose, the volume, and the time effects. There is a relation between the total dose and the volume. This applies to both the tumor and

normal tissues. The radiation dose is necessary to control an increase of a tumor with respect to its size, volume, or the number of cells present. To control with a high probability a 3 cm epithelial tumor, doses in excess of 65 Gy are necessary. Larger tumors need higher total doses. In contrast, normal tissue tolerance is inversely proportional to the volume irradiated. Timing is another issue: increasing the duration of the treatment will lead to a loss of efficacy due to the risk of inducing an active repopulation within the tumor: several studies of lung cancers have demonstrated a negative impact of treatment breaks or prolonging the duration of the treatment. Before 1951, most treatment units available were X-ray machines producing only photon beams with limited penetration; the orthovoltage units operated in the range of 200 to 300 kVp. In the 1950s, the development of supervoltage units (60Co and 137Cs teletherapy machines, betatron, and later linear accelerators) completely modified the radiation practice by offering machines more efficient both in terms of beam energy available and in accuracy. The linear accelerator uses high-frequency electromagnetic waves to accelerate electrons to high energy through a microwave accelerator structure; it can produce photon or electron beams of different energies. The introduction of computed treatment planning based on data generated by computed tomography (CT) has dramatically changed the practice since the end of the 1970s, allowing a dose distribution to be established first in two and later in three dimensions: there is an optimization process to deliver the doses to the tumor while protecting the normal tissue. The modern radiation treatment is a complex multistep procedure requiring the cooperation and skill of different professionals (physicians involved in the different imaging procedures, nurses, technologists, physicists, dosimetrists). The initial step requires a precise clinical evaluation of the patient and the extent and nature of the disease. The clinical evaluation is based on all sources of information available: clinical examination, imaging procedures such as CT, magnetic resonance imaging (MRI), and radiographs. This must provide a precise staging of the tumor and sufficient information to take the decision to treat or not the patient. After the treatment decision, the different target volumes must be defined: the palpable or visible extent of the tumor represents the gross tumor volume. A margin has to be added around this volume to include direct

RADIOTHERAPY

subclinical spread representing the clinical target volume. Additional volume may be added to cover the possible regional lymph nodes. For radiation treatment planning, margins have to be added to the clinical target volume to take into account the variation in size and position of tissues during treatment (patient movement, breathing during treatment) and the possible variation in the daily set-up; this represents the planning target volume. Furthermore, the radiation oncologist must define the different critical structures. The next step is the treatment planning. The goal is to individualize the treatment to the patient needs and to obtain ideally a complete description of the radiation (Figures 1 and 2). Another important step is to provide a proper immobilization which can be made with foam, plastic, or plaster and a marking technique to ensure the daily reproducibility of the treatment.

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The daily radiation treatment aims to deliver the radiation in the condition defined by the treatment planning and to reproduce it several times: this includes treatment parameters (field sizes, beam angles, beam energy, wedge filters), the patient’s position, and the quality of the radiotherapy unit. Several tools are useful to reduce the risk of errors and improve the accuracy of the treatment: a routine machine calibration, a system verifying the individual parameters for each patient, a portal imaging system giving online the position of the beam inside the patient. Nowadays, intensity modulated radiation therapy is available: modern linear accelerators are equipped with multileaf collimators which allow shaping of the beam while the leaf may be modified during the radiation leading to a difference in radiation intensity. The last technological development is fourdimensional radiation taking into account the time factor: for lung cancer, there is always a possible displacement of the tumor due to the patient’s breathing or heart movements; the gating technique allows irradiation during a specific phase of the breathing cycle while the tracking technique follows the displacement of the tumor.

Uses in Respiratory Medicine

Figure 1 Dose distribution for an inoperable stage I non-small cell lung cancer treated with a fields technique delivering 65 Gy to the tumor while limiting the dose to the lungs, the spinal cord, and the heart.

Indications for radiotherapy for lung cancer depend on several factors related to the tumor (extension, pathology, symptoms) and the host (the patient’s performance status and other diseases, and his attitude). Several textbooks are available and only a short summary will be presented on the principal indications as well as their limits and new approaches. Furthermore, management of lung cancer is more and more a multidisciplinary effort. Non-Small Cell Lung Cancer

Figure 2 PET CT of right lung tumor with partial atelectasia and lymph node involvement. The PET image allowed to modify the radiation field to include only the area of FDG-uptake and differentiating the tumor from the simple atelectasia.

Surgery remains the cornerstone in terms of cure for early lung cancer, but only fewer than one-third of patients are candidates for a surgical resection, usually presenting with state I to IIIa disease. Patients with more extensive but still localized disease within the chest (some stage IIIa and mainly stage IIIb) and those not candidates for a surgical resection due to medical contraindication are often treated with radiation. What have we learned over the last three decades? Local control and survival is closely related not only to the tumor extent and the patient’s status (weight loss, performance status) but also on the radiation treatment (total dose, fractionation, and quality). By increasing the dose from 40 to 60–65 Gy, doses used in most trials, the local control and survival was improved but the results were still very dismal with

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Figure 3 Simulation film for a left lung tumor with positive lymph nodes within the aortopulmonary arc and with the position of the different leafs from a multileaf collimator (a) and a portal image taken before treatment to check the accuracy of the beam position before treatment (b).

figures as low as 20% for local control. This is easily explained by the relation between tumor size and dose: most tumors are larger than 3 cm requiring higher total doses. Several approaches were and are under investigation: combined modalities, modifying the fractionation, and increasing the physical dose using the three- or four-dimensional approaches (Figure 3). In the 1980s, it was strongly believed that the best way of combining drugs and radiation was to start with the drugs: amongst the theoretical advantages, induction chemotherapy might also improve the results of chest irradiation due to a better oxygenation of the tumor or to a possible protection of normal tissue in case of response. Indeed, a trial observed an improvement in long-term survival after two cycles of cisplatine and vinblastine followed by chest irradiation (60 Gy with daily 2 Gy fractions). This was confirmed by additional trials and a meta-analysis. Nevertheless, this survival benefit was mainly due to a reduction in distant metastases and not to a better locoregional control. A concurrent approach is becoming more popular. The main concern is certainly an increase in acute hematological and nonhematological toxicity especially esophagitis but it is possible to take advantage of the radiosensitizing properties of drugs. Several reported trials all point in the same direction, with an improved 2 year survival rate, around 35% compared to less than 30% for the sequential approach. Fewer local relapses were observed. The problem is certainly the increase in acute esophagitis, a temporary side effect which leads very rarely to permanent esophageal stenosis. There are also many unsolved questions: the best sequence (induction followed by a concurrent approach, a concurrent approach followed by an adjuvant schedule), the place of a

maintenance chemotherapy, the drugs to be associated with cisplatine or carboplatine, the drug administration (single or multiple administration), and of course the radiation technique itself. In an attempt to improve the efficacy of radiation, classical or accelerated hyperfractionated schedules have been developed to increase the biological dose and to overcome the issue of repopulation. Indeed, for epithelial tumors, there is a risk of inducing a tumor proliferation after 3 to 4 weeks leading to a loss of efficacy. Furthermore, the tolerance of normal tissue is directly influenced by the dose delivered by each fraction which is not the case for the tumor: bigger fractions increase the risk of late effects. The CHART schedule (54 Gy delivered with three daily fractions of 1.5 Gy over 12 consecutive days including the weekend) was clearly superior to 60 Gy in 6 weeks with daily 2 Gy per fraction; the survival benefit was due to an improvement in local control. Another approach is certainly to use the modern tools available which allow to escalate the dose above 70 Gy by reducing the irradiated volume; studies are on going. Combining radiation or chemoradiotherapy with surgery is another alternative. Preoperative radiotherapy is currently only advocated for superior sulcus tumor. Postoperative radiotherapy has no place in case of a complete resection with clear margins except for some selected stage IIIa (nodal extracapsular spread) or stage IIIb. The place of surgery for a clinical proved stage IIIa disease (with positive mediastinal lymph node) remains controversial after an induction program: survival is in the same range for patients operated after chemoradiotherapy or not. Small endobronchial lesions may be treated with endobronchial brachytherapy. The technique requires the introduction of a thin flexible catheter during a

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flexible fiberbronchoscopy. Then the bronchial volume to be treated is determined as well as the dose required. After the dose distribution study, the treatment is performed by inserting the radiation source in the right configuration using an afterloading machine. The radiation takes usually 10–20 min while the whole procedure takes less than 2 h. Treatment is well tolerated and widely applicable even on outpatient basis. Small Cell Lung Cancer

This disease is characterized by a rapid proliferation and early dissemination. Chemotherapy is the basis of the treatment. In the curative treatment of small cell lung cancer, there are two indications for radiation: chest radiotherapy and prophylactic cranial irradiation. For limited disease, locoregional failures after only chemotherapy were very common and chest irradiation was added to the drugs. The classical approach includes the delivery of cisplatine etoposide concurrently with chest irradiation using either one or two fractions per day. Early administration, during the first cycles of chemotherapy, is usually preferable rather than delaying the radiation to the end of the chemotherapy. This can lead to a 5 year survival of around 20% to 25% in selected groups. Nowadays, studies are evaluating new drugs as well as higher total radiation dose similar to the one used for nonsmall cell lung cancer due to the still high rate of local failure (between 30% and 50%). Brain relapse is a common pattern of failure with a figure as high as 50% at 2 years. Prophylactic cranial irradiation has been showed to decrease the relapse rate by a factor of 2 with dose between 20 and 30 Gy. This has led to some improvement in survival for patients in complete remission. The process is nowadays advocated for patients with limited disease in complete remission but should not be given concurrently with chemotherapy to avoid the risk of toxicity. Palliative Radiotherapy

Radiation is often used to relieve the patients of symptoms due to the primary tumors (superior vena cava syndrome, chest pain, hemoptysis) or due to distant metastases (spinal cord compression, bone or brain metastases). Usually, the treatment delivers a few fractions with larger doses with the aim of only relieving the symptoms, which is achieved in more than two-thirds of patients. Brain metastases require special care: in some selected cases, radiosurgery may be advocated for single or a few small lesions. If the primary lesion may be or is under control and there is no other distant metastasis, this treatment may even

Figure 4 The ciberknife allowing to treat in stereotactic condition.

be curative with 5 year survival (Figure 4). Endobronchial brachytherapy may also be an alternative in case of endobronchial disease or compression. See also: Bronchoscopy, General and Interventional. Tumors, Malignant: Overview; Bronchogenic Carcinoma; Chemotherapeutic Agents.

Further Reading Arriagada R, Le Pećhoux C, and Pignon JP (2003) Resected nonsmall cell lung cancer: need for adjuvant lymph node treatment? From hope to reality. Lung Cancer 42: S57–S64. Auperin A, Arriagada R, Pignon JP, et al. (1999) Prophylactic cranial irradiation for patients with small-cell lung cancer in complete remission. New England Journal of Medicine 341: 476–484. Bradley J, Graham MV, Winter K, et al. (2005) Toxicity and outcome results RTOG 9311: a phase I-II dose-escalation study using three-dimensional conformal radiotherapy in patients with inoperable non-small-cell lung carcinoma. International Journal of Radiation Oncology, Biology, Physics 61: 318–328. Chang JY, Liu HH, and Komaki R (2005) Intensity modulated radiation therapy and proton radiotherapy for non-small cell lung cancer. Current Oncology Reports 7: 255–259. Farray D, Mirkovic N, and Albain K (2005) Multimodality therapy for stage III non-small cell lung cancer. Journal of Clinical Oncology 23: 3257–3269.

614 REACTIVE AIRWAYS DYSFUNCTION SYNDROME Grills IS, Yan D, Martinez AA, et al. (2003) Potential for reduced toxicity and dose escalation in the treatment of inoperable nonsmall-cell lung cancer: a comparison of intensity-modulated radiation therapy (IMRT) 3D conformal radiation, and elective nodal irradiation. International Journal of Radiation Oncology, Biology, Physics 57: 875–890. Hazard LJ and Sause WT (2004) Combined modality therapy for unresectable non-small cell lung cancer. In: Sculier JP and Fry WA (eds.) Malignant Tumors of the Lung, pp. 237–252. New York: Springer. Lee SW, Choi EK, Park HJ, et al. (2003) Stereotactic body frame based fractionated radiosurgery on consecutive days for primary or metastic tumors in the lung. Lung Ca 40: 309–315. Mornex F (2004) Non-small cell lung cancer. Seminars in Radiation Oncology 14(4). Saunders M, Dische S, Barrett A, et al. (1999) Continuous hyperfractionated, accelerated radiotherapy (CHART) versus conventional radiotherapy in non-small cell lung cancer: mature data from the randomized multicentre trial. Radiotherapy and Oncology 52: 137–148.

Steel G (2002) Basic Clinical Radiobiology. London: Arnold. Taulelle M, Vincent P, Chauvet B, Garcia R, and Reboul F (1999) Endobronchial brachytherapy. In: Brambilla C and Brambilla E (eds.) Lung Tumors: Fundamental Biology and Clinical Management, pp. 537–552. New York: Dekker. Van Houtte P (2001) The role of radiotherapy and the value of combined treatment in lung cancer. European Journal of Cancer 37(supplement 7): 91–98. Van Houtte P and Mornex F (2001) Radiotherapy of nonsmall cell and small cell lung cancer. In: Spiro SG (ed.) Lung Cancer, pp. 190–218. Hudders Field: European Respiratory Society. Van Houtte P, Roelandts M, Cappello M, and Mornex F (2004) Small cell lung cancer: limited disease. In: Sculier JP and Fry WA (eds.) Malignant Tumors of the Lung, pp. 277–286. New York: Springer. Van Limbergen E and Po¨tter R (2002) Bronchus cancer. In: Gerbaulet A, Po¨tter R, Mazeron JJ, Meertens H, and Van Limbergen E (eds.) The GEC ESTRO Handbook of Brachytherapy, pp. 545–560. Leuven: ESTRO.

REACTIVE AIRWAYS DYSFUNCTION SYNDROME S M Tarlo, University of Toronto, Toronto, ON, Canada C A Redlich, Yale University, New Haven, CT, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Reactive airways dysfunction syndrome (RADS) refers to asthma that begins after exposure to a very high level of respiratory irritant (usually accidental). The initial criteria included the onset of asthma symptoms within 24 h of a single such exposure, absence of preceding lung disease, objective evidence of asthma (spirometry showing a significant bronchodilator response, or methacholine or histamine challenge showing significant airway hyperresponsiveness), and continuation of asthma symptoms for at least three months. The criteria for this syndrome have been variably expanded to include multiple irritant exposures, less massive levels of exposure, a more delayed onset of symptoms after exposure, or a shorter duration of symptoms, termed ‘irritant-induced asthma’. RADS is considered a subset of this more inclusive, but less well defined entity. The diagnosis of RADS is largely circumstantial, relying on the history of exposure, absence of documented preceding airway disease, and objective documentation of asthma. Airway pathology does not clearly distinguish this syndrome from other asthma. Therapy is the same as for other types of asthma including corticosteroids and bronchodilators, as well as evaluation of workplace exposures. The outcome varies: symptoms and pulmonary function changes can clear within months but may persist for years.

Introduction The term reactive airways dysfunction syndrome (RADS) was initially used by Brooks and colleagues in 1985 to describe asthma that begins after exposure to a very high level of respiratory irritant (usually

accidental). The initial criteria (Table 1) included the onset of asthma symptoms within 24 h of such exposure, absence of preceding lung disease, objective evidence of asthma (spirometry showing a significant bronchodilator response, or methacholine or histamine challenge showing significant airway hyperresponsiveness), and continuation of asthma symptoms for at least three months. Subsequently, the criteria for this syndrome have been expanded to include multiple and less extreme exposures, a more delayed onset of symptoms after exposure, or a shorter Table 1 Criteria for diagnosis of RADSa *

* *

*

Onset of asthma symptoms within 24 h of exposure to a high level of a respiratory irritant agent (usually severe enough to require medical attention at the time) Persistence of symptoms for at least 12 weeks Objective evidence of asthma: airway hyperresponsiveness on histamine or methacholine challenge, or airflow limitation with significant bronchodilator responsiveness (at least 12% increase in FEV1) No previously documented evidence of asthma or other chronic lung disease

a Criteria for irritant-induced asthma are less stringent and may include those with symptom onset up to 7 days or longer after the acute exposure, duration of symptoms less than 12 weeks after onset, more than one single massive exposure, or less than a massive exposure. FEV, forced expiratory volume. Adapted from Tarlo SM and Chan-Yeung M (2005) Occupational asthma. In: Rosenstock L, Cullen MR, Redlich CA, and Brodkin D (eds.) Clinical Occupational and Environmental Medicine, 2nd edn. Philadelphia, PA: Saunders, with permission from Elsevier.

REACTIVE AIRWAYS DYSFUNCTION SYNDROME 615 Table 2 Some causes of irritant-induced occupational asthma Agents (with high-level exposures)

Exposure example

Volatile di-isocyanates, e.g., TDI

Spills in polurethane foam manufacture and other diisocyanate manufacturing/ using companies Paper mills Hospital workers, metal platers Accidental mixing of bleach and ammonia while cleaning Industrial settings, firefighters World Trade Center collapse with exposed firefighters, other aid workers, and building occupants Silo gas in farm workers Welders Police officers Paint sprayers Pesticide use

Chlorine spills (puffs) Acid spills, e.g., acetic acid Hypochlorite fumes Chemical fires Calcium oxide

Nitrogen oxides Welding fumes Tear gas Spray paint Metam sodium

Adapted from Tarlo SM and Chan-Yeung M (2005) Occupational asthma. In: Rosenstock L, Cullen MR, Redlich CA, and Brodkin D (eds.) Clinical Occupational and Environmental Medicine, 2nd edn. Philadelphia, PA: Saunders, with permission from Elsevier.

duration of symptoms, termed ‘irritant-induced asthma’. Irritant-induced asthma has been used to include both RADS and patients who fit this broader more inclusive description. Since the diagnosis of RADS is largely circumstantial (relying on the exposure assessment, absence of documented preceding airway disease, and objective documentation of asthma), the certainty of diagnosis is greatest when the strictest criteria are used. Irritant-induced asthma has also been termed ‘occupational asthma without latency’ or ‘nonsensitizer asthma’ to differentiate it from immune-mediated or sensitizer-induced occupational asthma. Irritant-induced asthma, or the more narrowly defined RADS, has been reported following high-level exposure to various respiratory irritant gases, fumes, and chemicals including sulfur dioxide, di-isocyanates perchloroethylene, chlorine, phosphoric acid, welding fumes diethylaminoethanol, nitrogen tetroxide, and alkaline dust exposure from the World Trade Center collapse (Table 2).

Epidemiology The incidence of RADS or irritant-induced asthma in the general population is unknown. In different studies, in groups with accidental exposure to a high concentration of a respiratory irritant agent, irritant-induced asthma has been reported in variable percentages of those exposed (1–20%), likely related to the diagnostic criteria used, extent and nature of the exposure, as well as host factors. Among those who develop RADS-like

symptoms following a high acute exposure, a variable but substantial number (up to 60%) can have persistent airway hyperresponsiveness. Limited compensation or clinical data, which is influenced by referral, reporting and other factors, have found a variable percentage of occupational asthma cases attributed to irritants (o5% to B25%), with a lower prevalence if more restrictive RADS criteria are used. Of great interest is the possibility that chronic or recurrent exposure to lower concentrations of respiratory irritants might also lead to asthma, as this type of exposure is much more common than acute high exposures. It is accepted that such exposures can exacerbate asthma in those with pre-existing asthma. Whether such exposures can cause new-onset asthma remains unclear. However, increasingly, epidemiologic associations are reported between chronic exposure to respiratory irritants and increased risk of asthma in several settings including textile workers, cleaners, janitors, and pulp workers exposed to chlorine or ozone. Risk factors for RADS or irritant-induced asthma have not been well defined, although the dose of exposure is clearly important. For example, a doseresponse relationship was found in hospital laboratory workers accidentally exposed to acetic acid, the greatest prevalence of airway hyperreactivity and symptoms being in the highest exposure group. Host susceptibility factors are also involved, as the syndrome occurs in only a minority of those with similar exposure. Several reports have included a large proportion of smokers and atopics, which may be risk factors. Although the identified presence of underlying airway disease is an exclusion criterion for RADS, it is possible that those with RADS and a previous smoking or atopic history may have had underlying subclinical airway disease, or there may be unidentified genetic or other predisposing factors, perhaps analogous to the genetic predisposition demonstrated to endotoxin. The airway responsiveness to moderate or relatively low concentrations of inhaled irritants (as illustrated by challenges with methacholine, histamine, mannitol, and other agents) is greater in asthmatics, atopic individuals without asthma, those with chronic obstructive lung disease, and in normal subjects following a respiratory viral infection. However, the possible role of these as predisposing factors for RADS or irritant-induced asthma is unknown. While it is clear clinically that respiratory irritant exposure will aggravate asthma, there is clear information as to the effect in asthmatics of exposures such as those that would cause RADS in nonasthmatics. There is no information to indicate whether the airway response under the same exposure conditions would be the same or greater, or whether the concurrent use

616 REACTIVE AIRWAYS DYSFUNCTION SYNDROME

of medications such as inhaled corticosteroids by those with previous asthma may ameliorate some of the effects. It may be expected that those with asthma might have earlier symptoms in an accidental exposure area and might therefore leave the area sooner; no study has clearly addressed this.

Pathogenesis and Pathology The underlying mechanisms of RADS are not well understood. The airway injury and inflammatory response following an acute irritant respiratory exposure most commonly gets resolved. However, in some cases the airway injury may result in persistent changes such as airway remodeling and chronic inflammation as with other asthma. It has been postulated that inhalation of a high concentration of an irritant substance injures the bronchial epithelium, with subsequent sloughing of the epithelium, neurogenic inflammation, airway edema, release of inflammatory mediators, growth factors and chemokines, recruitment and/or activation of mast and other inflammatory cells, and myofibroblast activation, leading to chronic airway inflammation, subepithelial fibrosis, airway hyperresponsiveness, and asthma symptoms. The mechanisms, pathways, and responses likely resemble those involved in the pathogenesis of chronic asthma due to other causes, i.e., epithelial injury, activation of the epithelial-mesenchymal trophic unit, airway remodeling, and chronic inflammation, as described by Holgate and others (Figure 1). Airway pathology does not clearly distinguish RADS from other causes of asthma. Reported biopsies show denuded epithelium, airway inflammation, and subepithelial fibrosis. In some reports, subepithelial fibrosis is more prominent with less eosinophilia and inflammatory infiltrate than in atopic asthma. However, findings have been limited

to small numbers of patients at variable time periods following the acute exposure and have not been consistent. Therefore, there is only limited information about the pathology of RADS to date.

Clinical Features and Diagnosis The typical RADS presentation is the development of new asthmatic symptoms such as cough, wheezing, and shortness of breath following acute exposure to a high level of irritant accidentally released in the workplace, which persist for at least three months after the initial exposure. Objective support for asthma is documented by a significant bronchodilator response on spirometry or a positive histamine or methacholine challenge. Once RADS exists, nonspecific environmental stimuli such as cigarette smoke, household cleaners, diesel exhaust, or cold air can precipitate symptoms. Acute irritant exposures capable of causing RADS can exacerbate preexisting asthma, and are also associated with several nonasthmatic disorders that can have overlapping symptoms including rhinitis, vocal cord dysfunction, and increased cough sensitivity, complicating diagnosis and management. These nonasthmatic conditions can be distinguished from RADS as they do not cause airway hyperresponsiveness, but they can coexist with RADS. The differential diagnosis of RADS or irritant-induced asthma includes: 1. Induction of new-onset asthma by a high level of respiratory irritant that also has the capacity to be a respiratory sensitizer (e.g., di-isocyanates), producing irritant-induced occupational asthma and concurrent sensitization to that agent. 2. Underlying airway hyperresponsiveness or underlying asthma with aggravation by the irritant exposure, triggering symptoms. This is the main differential diagnosis of RADS/irritant-induced

Irritant stimulus Epithelial and neuronal damage + inflammatory mediators Release profibrogenic growth factors, e.g., TGF-

Inflammation: activation of neutrophils, ± eosinophils, ± mast cells, ± T lymphocytes; release IL-4, IL-13, other mediators

Repair epithelium via EGF, other mediators − symptoms resolve

Chronic inflammation, myofibroblast activation and airway remodeling Airway hyperresponsiveness − chronic asthma manifestations Figure 1 Theoretical model of pathogenesis of RADS/Irritant-induced asthma. TGF, transforming growth factor; EGF, epithelial growth factor; IL, interleukin.

REACTIVE AIRWAYS DYSFUNCTION SYNDROME 617

asthma. This diagnosis may be evident in a patient who has known underlying asthma, but can be hard to distinguish from new-onset asthma in a patient who has a history of previous occasional mild asthma-like symptoms but no previous diagnosis of asthma. 3. Irritant effects mimicking asthma in a patient with coincidental underlying airway hyperresponsiveness, e.g., following an accidental high level of exposure to an irritant, there may be recurrent episodes of vocal cord dysfunction, hyperventilation/panic attacks, or rhinitis, which may mimic asthma. Distinguishing between these possible diagnoses is assisted by detailing the history, obtaining as much objective information as possible about the exposure agents and levels of exposure (obtained from the workplace), and reviewing previous medical records relating to respiratory status prior to the exposure and following the implicated exposure, including emergency medical treatment records. Industrial hygiene data documenting the high exposure typically are not available, as the events are frequently sporadic or accidental. Further investigations may be needed to assess the likelihood of other diagnoses such as vocal cord dysfunction or sinusitis. There is no gold standard or specific test to confirm the diagnosis of RADS or irritant-induced asthma, and thus the diagnosis depends largely on the history and ruling out other possible disorders.

Animal Models Several attempts to develop an animal model of RADS/IIA have been unsatisfactory to date, although Demnati and co-workers have reported an increase in lung resistance and methacholine responsiveness for up to one month in rats exposed to very high levels of chlorine (1500 ppm for 5 min), but no airway inflammation. Conversely, Klonne and coworkers showed no adverse response to chlorine in a 1-year inhalational toxicity study in rhesus monkeys. A report of chlorine-induced changes in airways of mice found airway epithelial cell loss as well as alveolar changes. There was associated airway hyperresponsiveness, which was prevented by inhibiting inducible NO synthase.

Management, Current Therapy, and Outcome Therapy is as for other asthma: treatment for acute exacerbations includes systemic corticosteroids and bronchodilators; chronic management includes

environmental control measures, inhaled corticosteroids, and bronchodilators. Treatment of acute high irritant exposures preceding the onset of RADS is the same as for other acute inhalational exposures, including general supportive care and evaluation of the airways and lung parenchyma. Corticosteroids are frequently given in an acute setting, with the hope of reducing airway inflammation and promoting resolution, but there is little data demonstrating beneficial effect. When an accidental exposure occurs in a workplace setting, occupational hygiene investigation of the conditions leading to the exposure is necessary to prevent recurrence. The affected workers may then be able to return to the same work if their asthma is adequately controlled. However, with some asthmatics, even moderate irritant exposures in the workplace may be sufficient to aggravate asthma. Subjects with non-specific airway hyperresponsiveness can develop reactions to levels of irritants that do not provoke symptoms in healthy subjects. Respiratory protective devices may be helpful for short-term exposures or the worker may need to relocate to a cleaner environment. When the initial exposure has been to an agent, which can also be a respiratory sensitizer (e.g., a di-isocyanate spill), the worker may have been sensitized at the time of the accidental exposure; subsequent exposures to moderate or low concentrations of that agent would then trigger asthma exacerbations. This should be assessed by careful monitoring of such workers on return to work, with serial peak flow or spirometry monitoring and serial measures of airway responsiveness. There can be discordance between symptoms and lung function tests, especially since high irritant exposures can also result in disorders such as rhinitis, vocal cord dysfunction, increased cough sensitivity, and chemical sensitivity or panic disorders, which may symptomatically be difficult to distinguish from asthma, and which may not respond to asthma treatment. For example, some firefighters developed disabling cough without airway hyperresponsiveness following exposure to World Trade Center dust. The natural history of RADS or irritant-induced asthma is not well defined. Limited case series and reports of subjects with RADS have shown that airway hyperreactivity can persist for years after an acute exposure in a substantial but variable proportion of subjects. Socioeconomic impact can be significant and may not correlate with pulmonary function measures. The determinants of resolution or progression are poorly defined. See also: Asthma: Overview; Extrinsic/Intrinsic. Occupational Diseases: Inhalation Injury, Chemical.

618 REFLEXES FROM THE LUNGS AND CHEST WALL

Further Reading Alberts WM and do Pico GA (1996) Reactive airways dysfunction syndrome. Chest 109: 1618–1626. Arbour NC, Lorenz E, Schutte BC, et al. (2000) TLR4 mutations are associated with endotoxin hyporesponsiveness in humans. Nature Genetics 25: 187–191. Balmes JR (2002) Occupational airways diseases from chronic low-level exposures to irritants. Clinics in Chest Medicine 23: 727–735. Balmes J, Becklake M, Blanc P, et al. (2003) American Thoracic Society Statement: Occupational contribution to the burden of airway disease. American Journal of Respiratory and Critical Care Medicine 167: 787–797. Banouch GI, Alleyne D, Sanchez R, et al. (2003) Persistent hyperreactivity and reactive airways dysfunction in firefighters at the World Trade Centre. American Journal of Respiratory and Critical Care Medicine 168: 54–62. Brooks SM, Hammad Y, Richards I, Giovinco-Barbas J, and Jenkins K (1998) The spectrum of irritant-induced asthma: sudden and not-so-sudden onset and the role of allergy. Chest 113: 42–49. Brooks SM, Weiss MA, and Bernstein IL (1985) Reactive airways dysfunction syndrome (RADS). Persistent asthma syndrome after high level irritant exposures. Chest 88: 376–384. Cullinen P and Newman Taylor AJ (1998) Acute toxic chemical exposures. In: Banks DE and Parker JE (eds.) Occupational

Lung Diseases: An International Perspective, pp. 453–463. London: Chapman and Hall Medical. Holgate ST, Holloway J, Wilson S, et al. (2004) Epithelial-mesenchymal communication in the pathogenesis of chronic asthma. Proceedings of the American Thoracic Society 1: 93–98. Kern DG (1991) Outbreak of the reactive airways dysfunction syndrome after a spill of glacial acetic acid. American Review of Respiratory Diseases 144: 1058–1064. Tarlo SM (1989) Broder I. Irritant-induced occupational asthma. Chest 96: 297–300. Tarlo SM (2000) Workplace respiratory irritants and asthma. Occupational Medicine: State of the Art Reviews 15: 471–483. Tarlo SM (2003) Workplace irritant exposures: do they produce true occupational asthma? Annals of Allergy, Asthma and Immunology 90(supplement 2): 19–23. Tarlo SM and Chan-Yeung M (2005) Occupational asthma. In: Rosenstock L, Cullen MR, Redlich CA, and Brodkin D (eds.) Clinical Occupational and Environmental Medicine, 2nd edn. Philadelphia: Elsevier, W.B. Saunders. Vandenplas O, Toren K, and Blanc PD (2003) Health and socioeconomic impact of work-related asthma. European Respiratory Journal 22: 689–697. Zock JP, Kogevinas M, Sunyer J, et al. (2001) Asthma risk, cleaning activities and use of specific cleaning products among Spanish indoor cleaners. Scandinavian Journal of Work and Environmental Health 27: 76–81.

REFLEXES FROM THE LUNGS AND CHEST WALL D R McCrimmon and G F Alheid, Northwestern University, Chicago, IL, USA E J Zuperku, Medical College of Wisconsin, Milwaukee, WI, USA

secretion. Chest wall receptors can reflexively alter motor drive to the diaphragm but have relatively little influence on respiratory muscle activation during eupnea.


Introduction Abstract A variety of mechanical and chemical sensory receptors in the lungs and chest wall regulate breathing pattern and defend the respiratory system. Ascribing specific reflex responses to activation of particular receptors has been complicated by the difficulty in accessing and selectively activating receptor subtypes. Receptors within the lower airways and lungs are classified based on whether the sensory afferent fibers are myelinated or unmyelinated. Their axons primarily course in the vagus nerves. Receptors with myelinated axons include the slowly and rapidly adapting pulmonary stretch receptors (SARs and RARs). SARs respond to lung inflation and mediate the Breuer–Hering inspiratory inhibiting and expiratory facilitating reflexes. Activation of RARs by inhaled irritants evokes airway protective reflexes including a rapid breathing frequency, bronchoconstriction, and mucus secretion. These receptors also elicit sighs in response to a decrease in airway compliance or a reduction in lung volume below the normal resting volume. Cough is produced by a subset of receptors with small myelinated axons. Activation of receptors with unmyelinated fibers (C-fibers) also produces airway protective reflexes consisting of a shallow rapid breathing pattern, bronchoconstriction, and mucus

The airways, lungs, and chest wall are endowed with a variety of mechanical and chemical sensory receptors that regulate breathing pattern and defend the respiratory system. The receptors of the lower airways and lungs have been classified into two main types based on whether the sensory afferent fibers are myelinated or unmyelinated. For both types of fibers the vagus is the primary pathway to the central nervous system where they terminate in the nucleus of the solitary tract (NTS) within the medulla. Receptors are further subtyped according to their stimulus response characteristics and anatomical locations. Receptors with myelinated axons are the slowly and rapidly adapting pulmonary stretch receptors (SARs and RARs) while those with unmyelinated axons include pulmonary and bronchial C-fiber endings and neuroepithelial bodies (NEBs). Each of the receptor types can contribute to more than one type of reflex response. This results

REFLEXES FROM THE LUNGS AND CHEST WALL 619

in part from the varied location of the receptive fields (e.g., intrapulmonary, extrapulmonary, or extrathoracic airways) and from coactivation of multiple receptor types. In addition, ascribing specific reflex responses to activation of particular receptors has been complicated by the difficulty in accessing and selectively activating receptors deep within the airways.

NAmb

Slowly Adapting Pulmonary Stretch Receptors SARs play a role in controlling breathing pattern, airway smooth muscle tone, systemic vascular resistance, and heart rate. They are located in airway smooth muscle and are stimulated by stretch during lung inflation (Figure 1(a)). SARs behave as if they are

RAR

NTS

NAmb

Glu

VRC

VRC

SAR

C-fiber SP, CGRP, NKA Calbindin

NTS

Ach VIP

Ach VIP

Glu Phr

IML

Glu

DRG

PG

JG

JG

Ach Phr n.

A

(To diaphragm)

SM (a)

NG

NEB

NE

V

V

A VIP NO

SP CGRP

Ach

NG

AG

AG

VIP NO SM

Ach

Ach

(b)

Figure 1 Schematic diagram of pulmonary reflex pathways. (a) Myelinated slowly (SAR, orange) and rapidly (RAR, red) adapting receptor afferent pathways are depicted relative to a section of bronchiole pseudostratified columnar epithelium. SARs are located in airway smooth muscle (SM) and are stimulated by stretch during lung inflation. Sensory endings of RARs are located in airway epithelium where they respond to the inhalation of effective irritants. They are also postulated to be located in proximity to bronchial venules where they may be activated by edema. SAR and RAR neurons are found in the nodose (NG) and jugular ganglia (JG) of the vagus nerve and synapse in the nucleus of the solitary tract (NTS). Airway efferents originate mainly from cholinergic (Ach, blue) parasympathetic motoneurons in the nucleus ambiguus (NAmb). These synapse on cholinergic (blue) and noncholinergic neurons (magenta) in local airway ganglia (AG) which in turn target airway smooth muscle. Additional efferent targets include mucus secreting glands (at the level of the trachea), mucus secreting epithelial cells (e.g., goblet cells; not shown). Sensory information reaching the NTS alters activity of parasympathetic vagal motoneurons in the nucleus ambiguus and premotor neurons in the ventral respiratory column (VRC) of the medulla. Alterations in respiratory rate and pattern are effected by projections of inspiratory premotor neurons to phrenic motoneurons (Phr) in the cervical spinal cord. The latter innervate the diaphragm via the phrenic nerve (Phr n.). Expiratory premotor neurons in the caudal ventrolateral medulla also project to abdominal and intercostal motoneurons in the thoracic spinal cord (not shown). A, arteriole; Glu, glutamate; V, venule. (b) Unmyelinated C-fiber pulmonary afferents (green) and afferents to neuroepithelial bodies (NEB) (red, green) are depicted on the same diagram as in (a). C-fibers project diffusely throughout the parenchyma of the lung including the airway epithelial (mucosal) and submucosal layers, and in the vicinity of pulmonary blood vessels. Damage or irritation of the C-fibers and/or the surrounding lung tissue may lead to (antidromic) local release of neuropeptides from C-fiber sensory ending (i.e., the ‘axon reflex’); this appears to include extravasation from small venules. The axon reflex has been postulated to contribute to airway hyperresponsivity. Vagal calbindin-positive myelinated axons (red) and unmyelinated spinal afferents (green) contribute to the innervation of neuroepithelial bodies (NEB) which are potential pulmonary oxygen sensors. Vasoactive intestinal peptide (VIP) and nitric oxide synthase (NO) positive efferent axons originating in airway ganglia also target NEBs (connection not shown). CGRP, calcitonin gene related peptide; DRG, dorsal root ganglion; NE, norepinephrine; NKA, neurokinin A; PG, paravertebral ganglia; SP, substance P.


organized in series with airway smooth muscle as they are also stimulated by smooth muscle contraction. SAR Response Characteristics

While thought to relay information about lung volume, the discharge patterns of SARs are more closely related to transpulmonary pressure. Their discharge frequency displays a modest dynamic component but only partially adapts (o30% after 2 s) when lung inflation is held constant (Figure 2). A considerable proportion of SARs (between 27% and 60% in the different species studied) are active at end expiratory volume (i.e., at the functional residual capacity (FRC)), whereas others are recruited only during inspiration.

B–H inspiratory inhibitory reflex. SAR activity during the expiratory phase lengthens expiratory duration (TE) and is known as the B–H expiratory facilitatory reflex. The resulting combination of breathing frequency and VT results in a breathing pattern that tends to minimize the work of breathing while maintaining appropriate alveolar ventilation. Removal of SAR input produces a slow deep pattern accompanied with a higher amount of elastic work. The B–H reflex is active at normal resting tidal volumes in all mammals that have been examined, with the exception of humans where it is only activated when tidal volume exceeds 2–3 times normal. However, the B–H reflex in preterm and neonatal infants is very robust, especially during active sleep (REM), where end inspiratory occlusion of the airways prolongs TE by over 400%.

Breuer–Hering Reflexes SARs mediate the Breuer–Hering (B–H) inhibitory reflexes, which modify the breathing pattern. During inspiration, lung inflation increases SAR discharge frequency, which reflexively shortens the duration of the inspiratory phase (TI) and thereby reduces tidal volume (VT). This aspect of the reflex is known as the

Control of Expiratory Airflow and End Expired Volume

SARs appear to play a role in the control of expiratory flow rate. One aspect of this control involves the combined input of SARs located in the lower airways and those in the trachea. Together these

PT

SAR

PT

RAR

Figure 2 Responses to sustained inflation of the lungs in an anesthetized cat of individual slowly adapting (upper pair of traces) and rapidly adapting (lower pair of traces) afferent fibers teased from the vagus nerve. The ventilatory pump was stopped in expiration just before start of each record. The upper traces in each pair indicate intratracheal pressure (increased pressure upwards), lower traces show action potentials. The line at the bottom marks time in 100 and 500 ms intervals. Reproduced from Knowlton GC and Larrabee G (1946) A unitary analysis of pulmonary volume receptors. American Journal of Physiology 147: 100–114, used with permission from The American Physiological Society.


receptors mediate a compensatory motor control process that retards expiratory airflow by neurally mediated increases in laryngeal resistance and in diaphragm activity during the initial portion of expiration, along with decreases in abdominal muscle activity. This ‘expiratory braking’ reduces the rate of lung deflation, thereby increasing the duration of pulmonary stretch receptor discharge with a resultant increase in TE. SARs also act to increase the efficiency of breathing by controlling the expiratory musculature. This is mediated by modulating the activity of expiratory premotor neurons in the medulla. Starting at transpulmonary pressure levels well below those at FRC, SARs excite expiratory premotor neurons. However, as transpulmonary pressure increases above FRC levels, the expiratory premotor neurons are progressively inhibited, and their discharge is abolished at high transpulmonary pressure levels. This volume/ pressure-dependent biphasic activation–inhibition pattern is relayed via spinal motoneurons to expiratory internal intercostal and abdominal muscles. At end inspiration, lung volume and lung recoil force are high and the requirement for expiratory muscle activity is less. However, as lung volume and SAR activity decrease, the gradual reduction of inhibition allows greater expiratory muscle activity, thereby tending to preserve a constant expulsive force in the face of the declining lung recoil (Figure 3).

relationship between dead space and airway resistance. Smooth muscle control of local airflow resistance is important for the proper distribution of airflow to the various lung compartments. For example, body position alters vertical gradients in regional lung volume, ventilation, and perfusion. The resulting mismatches in ventilation and perfusion can be reduced by the reflex control of airway smooth muscle. Respiratory Sinus Arrhythmia

SARs also play a role in the production of respiratory sinus arrhythmia. Heart rate increases during inspiration and decreases during expiration. The exact relationship between heart rate and the phase of respiration depends on respiratory frequency and tidal volume. The greatest effect occurs at respiratory frequencies of 5–6 breaths min 1 and diminishes as the frequency is increased. While the central nervous system produces sinus arrhythmia, activation of SARs contributes to its magnitude. Within the medulla, the effect is to increase heart rate by inhibiting cardiac vagal motoneurons that innervate the atrial sinus node and produce slowing. This reflex helps stabilize cardiac output by allowing heart rate to increase during the reduction in left ventricular stroke volume that occurs during inspiration from a reduction in left ventricular filling.

Rapidly Adapting Pulmonary Stretch Receptors

An increase in SAR activity also relaxes airway smooth muscle by reducing parasympathetic tone to the airway. This negative feedback optimizes the reciprocal

Rapidly adapting receptors (RARs) adapt rapidly to a maintained mechanical stimulus, yet their response to a chemical one is often long lasting. They

PT (mmHg)

Control of Airway Smooth Muscle

20

Increased expiratory airflow resistance

0

Neuron (spikes s–1)

Phrenic

5s

100 0

Figure 3 Activation of slowly adapting receptors by increases in airway pressure (PT), secondary to an increase in expiratory airway resistance (indicated by horizontal bar) in a dog. Increased resistance slowed the centrally generated breathing frequency as indicated by a reduction in burst frequency on motor output to the diaphragm (phrenic nerve) and increased the firing rate (spike s 1) of an expiratory bulbospinal premotor neuron. Reproduced from Bajic J, Zuperku EJ, Tonkovic-Capin M, and Hopp FA (1992) Expiratory bulbospinal neurons of dogs. I. Control of discharge patterns by pulmonary stretch receptors. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 262: R1075–R1086, used with permission from The American Physiological Society.


are responsible for a range of powerful respiratory reflexes, especially cough, augmented breaths, hyperpnoea, and constriction of the trachea and bronchi. Other reflexes from lung RARs include airway mucus secretion and closure of the larynx. Location

RARs are located in airways from the nose to smaller bronchi. However, only a few RARs have been found in the smaller bronchi and none are in alveoli. The greater concentration of RARs near the carina and in the hilar airways may be optimized for defense mechanisms against inhaled irritants. RAR sensory terminals form branching arrays of nonmyelinated fibers arranged circumferentially in the airway epithelium and submucosa (Figure 1(a)). Response Characteristics

During quiet breathing, RARs are not very active, but when active their discharge is irregular both in duration and in its timing within the respiratory cycle (Figures 2 and 4). However, RAR activity is readily recruited by inhalation of effective irritants, rapid changes in airway volume, and intraluminal mechanical stimuli, or by the release of inflammatory or immunological mediators. RARs are heterogeneous. Transpulmonary pressure is an important factor in their activation. In addition, RARs are stimulated by pulmonary edema as well as a variety of irritants

such as cigarette smoke, nicotine, and ammonia, or by intravenous injections of other chemicals such as bradykinin, substance P, histamine, and serotonin. They are sensitized by airway smooth muscle contraction and by decreases in lung compliance. The reflexes induced by RAR activation depend on the characteristics of individual receptors and their site in the airways. Activation of epipharyngeal RARs elicits a series of augmented inspirations (the aspiration reflex) while those in the bronchi can produce an increase in respiratory rate or an augmented expiratory effort or a full cough (see below). Stimulation of a subset of laryngeal RARs produces a cough. The removal of inhaled irritants is further facilitated by RAR-induced bronchoconstriction and increased mucus secretion. The resulting greater airflow velocity and turbulence increase the probability of inhaled irritants becoming entrapped in the mucus and transported back toward the mouth. Cough Reflex

The cough is an airway defense mechanism consisting of a large inspiratory effort followed by a forced, rapid expiratory effort and bronchoconstriction. Together these create high gas velocities that expel airway mucus in which foreign particles are entrapped. Reflex increases in mucus production aid this process. Coughs are evoked in response to a variety of mechanical or chemical stimuli that are likely to activate different sensory pathways. While a primary role in

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Figure 4 Activation of an initially inactive rapidly adapting receptor by progressive reduction in dynamic lung compliance (Cdyn; b–d) in a dog. (a) Initially with compliance maximal, the receptor was silent. (b–d) Progressively lowered compliance after positive end expiratory pressure (PEEP) was removed for 5, 10, and 20 ventilatory cycles, respectively; in each case activity was recorded 1 min after PEEP restored. (b) Cdyn reduced by 23%, receptor stimulated but did not fire in all ventilation cycles. (c) Cdyn reduced by 35%. (d) Cdyn reduced by 41%. AP, action potentials; PT, tracheal pressure. Reproduced from Jonzon A, Pisarri TE, Coleridge JC, and Coleridge HM (1986) Rapidly adapting receptor activity in dogs is inversely related to lung compliance. Journal of Applied Physiology 61: 1980–1987, used with permission from The American Physiological Society.


the triggering of cough has usually been assigned to RARs, recent research has suggested that coughs may be triggered by a subset of polymodal Ad-fibers (cough receptors). Like RARs in general, these Adfibers have cell bodies in the superior or nodose ganglia of the vagus nerve. Also like RARs, they are responsive to mechanical and acid stimuli but are unresponsive to capsaicin, bradykinin, or airway stretch. C-fibers have also been ascribed a role in the production or facilitation of cough. This is considered below with C-fiber reflexes.

respond to a variety of inflammatory agents (e.g., capsaicin, histamine, and bradykinin) (Figure 5) as well as inhaled irritants (e.g., cigarette smoke, ozone, sulfur dioxide, ammonia, acrolein). However, not all receptors are stimulated by any given agent, thus suggesting the presence of functional subgroups. C-fibers with axons in the vagus nerve synthesize a number of neuroactive substances including substance P, neurokinin A, and CGRP. In response to prolonged or intense stimulation, these neuropeptides are released from sensory terminals in a local or

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RARs are responsible for the occasional deep spontaneous inspirations shown by all mammals at rest. As lung compliance decreases during quiet breathing, the sensitivity of RARs increases until a central nervous threshold is reached and an augmented breath results that reverses the decrease in lung compliance and prevents atelectasis. Sighs can also be provoked by lung deflations below FRC, by intravenous injections of substance P, serotonin or histamine, or by inhalation of ammonia, all of which are known to stimulate both RARs and C-fiber receptors; however, selective stimulants of C-fiber receptors do not cause augmented breaths.

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Unmyelinated (C-) fibers constitute the most abundant airway afferents with endings distributed throughout the entire respiratory tract, from large airways to the lung parenchyma. Their endings are located superficially in airway mucosa where they form a network of substance P and calcitonin gene related peptide (CGRP) containing fibers (Figure 1(b)). Most pulmonary C-fiber afferents reach the central nervous system through the vagus nerves from cell bodies in the jugular and nodose ganglia. Others, however, have cell bodies in dorsal root ganglia with fibers coursing through sympathetic nerves to reach the spinal cord. Lung C-fibers are difficult to selectively activate and record, and this has impeded the task of unequivocally establishing the pathophysiological roles of these afferents. C-fibers are frequently divided into two populations based on their accessibility to neuroactive agents administered into the bronchial (bronchial C-fibers) or pulmonary (pulmonary C-fibers) circulations. The two groups differ with respect to their apparent sensitivities to several chemicals including bradykinin and prostaglandins. Nevertheless, the reflex changes in breathing pattern elicited by their activation appear to be the same. Both C-fiber groups

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Figure 5 C-fiber afferent and arterial pressure responses to injection of the C-fiber stimulant capsaicin into the pulmonary circulation (upper traces). While not as sensitive as SAR and RAR to lung inflation C-fibers can be activated by larger lung inflations (lower traces). Top traces: pulmonary C-fiber with an ending in the right upper lobe of an anesthetized open-chest rat. Middle traces: airway pressure (PT). Lower traces: arterial blood pressure (BP). Reproduced from Ho C-Y, Gu Q, Lin YS, and Lee L-Y (2001) Sensitivity of vagal afferent endings to chemical irritants in the rat lung. Respiratory Physiology 127: 113–124, with permission from Elsevier.


‘axon’ reflex. The resulting actions on a variety of tissues and cells (airway and vascular smooth muscles, cholinergic ganglia, mast cells, and mucous glands) produce pronounced local effects including bronchoconstriction, extravasation of macromolecules, and airway edema. Stimulation of bronchopulmonary C-fibers activates several reflex responses with similarities to those elicited by RAR activation. These include a rapid, shallow breathing pattern. Although C-fibers usually demonstrate little spontaneous activity, removing their input to the central nervous system results in a slower, deeper breathing pattern, consistent with a small tonic influence on breathing pattern. When activation is produced by an intravenous injection of, for example, capsaicin there is frequently an initial apnea preceding the rapid, shallow pattern. However, the apnea is likely a response to the initial experimentally induced massive C-fiber discharge, with the shallow rapid respiratory pattern representing the more usual response to a more slowly developing pathophysiological activation. A role in cough production has also been ascribed to C-fibers because several stimuli that stimulate cough (e.g., citric acid, capsaicin, and bradykinin) are also known to activate C-fibers. Furthermore, depletion of airway C-fibers by capsaicin pretreatment abolishes citric acid-induced cough in unanesthetized animals while cough in response to mechanical stimulation of the airway persists. Pharmacological blockade of tachykinin receptors also reduces cough in response to a number of stimuli, including cigarette smoke and bronchospasm in guinea pigs. However, since direct C-fiber stimulation does not induce cough and can even inhibit it, the role of these fibers in cough remains unclear. C-fiber stimulation may contribute to cough by an indirect effect through a local axon reflex where there is C-fiber retrograde release of neuropeptides at the sensory endings in response to the various irritants described above. These activate receptors on nearby RAR (polymodal Ad) fibers as well as produce edema and inflammation. C-fiber reflexes also include broncho- and laryngoconstriction. In dogs and cats bronchoconstriction appears to be reflex mediated via pathways within the central nervous system, as blockade of ganglionic transmission with atropine prevents the reflex, while in guinea pigs, the reflex is atropine insensitive but attenuated by antagonists of neurokinin receptors, suggesting a contribution by the local axon reflex. In humans, capsaicin inhalation results in a relatively small centrally mediated bronchoconstriction which may be masked by concomitant activation of a nonadrenergic, noncholinergic (NANC) bronchodilator pathway.

C-fiber activation also produces edema, mucus secretion, airway mucosal vasodilation, bradycardia, and hypotension. Contributing to the C-fiber-induced edema is a reflex increase in bronchial circulation that occurs despite a vagally mediated systemic hypotension. The increase in blood flow is focused on the irritated region containing the activated C-fibers and is largely due to an inhibition of sympathetic outflow. The reflex response appears to be centrally mediated and includes activation of vagal efferents. Activation of C-fibers has also been associated with burning and choking sensations in the airways and upper chest.

Neuroepithelial Bodies Pulmonary neuroendocrine cells are scattered throughout the epithelium of the upper and lower airways. Within intrapulmonary airways neuroendocrine cells are often organized into clusters termed neuroepithelial bodies (NEBs). NEBs receive extensive innervation from vagal fibers as well as from dorsal root ganglion neurons and from local neurons with cell bodies in airway ganglia (Figure 1(b)). As with other neuroendocrine cells, they contain dense core vesicles that enclose a variety of neuroactive molecules including biogenic amines, especially serotonin, and several peptides including gastrin-releasing peptide, CGRP, enkephalin, somatostatin, cholecystokinin, and substance P. The contributions of NEBs to pulmonary homeostasis have yet to be established; based in large part on their resemblance to carotid body chemoreceptors, they are postulated to function as airway chemoreceptors. Whatever their functional role, it may vary with developmental age. They are among the first cells to mature in the fetal lung and they may consequently contribute to lung development. Immediately postnatally, a time when carotid chemoreceptors are relatively insensitive to hypoxia, it is suggested they provide chemosensory drive to breathing. In maturity NEBs may become more relevant for control of airway and/or pulmonary arteriolar tone.

Reflexes Originating from the Chest Wall Afferent activity from muscle spindles and tendon organs in intercostal and abdominal muscles influences the motor activity of neighboring synergist and antagonistic muscles as well as the diaphragm via spinal segmental pathways. In addition, intercostal muscle vibration, chest compression, or intercostal stretch can reduce or terminate inspiratory motor activity via supraspinal pathways. This latter effect is due to the activation of the tendon organs. These


afferents do not influence the quiet breathing pattern but when the respiratory system is loaded or stressed, for example, during exercise, tendon organ afferents can modulate rate and depth of breathing. However, this effect is much smaller than those mediated by the lung receptors. The diaphragm contains few muscle spindles. Tendon organs, although relatively sparse, outnumber the spindles. Their afferent fibers course in the phrenic nerve and electrical stimulation of phrenic afferents inhibits phrenic efferent activity via segmental and supraspinal connections. It is generally accepted that phrenic afferents have little influence on respiratory muscle activation during eupneic breathing. However, there is emerging evidence that when these afferents are activated by mechanical stimulation they can reflexively alter efferent drive to the diaphragm. This may be due to sudden changes in lung volume, as occurs in going from the supine to the upright posture, or in response to chemical stimuli.

Lung Reflexes in Disease Since discharge patterns of the SARs are greatly influenced by their anatomical location within the tracheobronchial tree and the orientation of their receptor endings in airway wall structures, disease processes that alter airway caliber and tone and/or lung mechanics affect the SARs and the reflexes they mediate. The sensitivity of SARs to normal stimuli can be increased by bronchoconstriction, airway obstruction, or decreases in lung compliance. For example, an increase in lung collagen associated with pulmonary fibrosis results in a decrease in lung volume and compliance, and a reduction in diffusing capacity that can result in hypoxia and hypercapnia. These alterations are usually associated with a rapid, shallow breathing at rest and during exercise, and the sensation of dyspnea. The volume sensitivity of SARs in an experimentally induced pulmonary fibrosis animal model is significantly increased during inspiration, and SAR activity during the deflation phase of expiration is significantly reduced. These two effects acting via the B–H reflex would shorten inspiratory and expiratory durations, and account, at least in part, for the observed rapid, shallow breathing patterns. The activities of RARs and C-fiber afferents also appear to be altered in various pulmonary disease states, including pulmonary fibrosis. Pulmonary C-fibers are activated in a variety of pathologic conditions including pulmonary congestion and edema. These receptors have been referred to as ‘juxtapulmonary capillary receptors’ because of their sensitivity to an increase in interstitial fluid volume or pressure resulting from an increase in

pulmonary vascular pressure or fluid filtration across pulmonary capillaries. Pulmonary embolism and inflammation also strongly excite C-fibers as a result of the local release of chemical mediators such as serotonin and cyclooxygenase metabolites. RAR and C-fiber receptor sensitivity can be increased in airway disease as well as by exposure to ozone, histamine, and prostaglandins. It has been suggested that this sensitization may occur in disease states and underlie bronchial hyperresponsiveness. Interestingly, viral infection of the airways can lead to a transformation in RAR phenotype in which substance P and neurokinin A are detected within RAR cell bodies in the nodose ganglion where they are not normally present. Assuming these peptides are transported to the sensory terminals in the airways they may contribute to neurogenic inflammation of the airways and hypersensitive cough through the axon reflex. See also: Mucus. Respiratory Muscles, Chest Wall, Diaphragm, and Other. Symptoms of Respiratory Disease: Cough and Other Symptoms. Ventilation: Overview; Control.

Further Reading Adriaensen D and Brouns I (2003) Functional morphology of pulmonary neuroepithelial bodies: extremely complex airway receptors. Anatomical Record A 270A: 25–40. Bajic J, Zuperku EJ, Tonkovic-Capin M, and Hopp FA (1992) Expiratory bulbospinal neurons of dogs. 1. Control of discharge patterns by pulmonary stretch receptors. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 262: R1075–R1086. Barnes PJ (2001) Neurogenic inflammation in the airways. Respiratory Physiology 125: 145–154. Bolser DC, Lindsey BG, and Shannon R (1987) Medullary inspiratory activity: influence of intercostal tendon organs and muscle spindle endings. Journal of Applied Physiology 62: 1046–1056. Canning BJ, Mazzone SB, Meeker SN, et al. (2004) Identification of the tracheal and laryngeal afferent neurones mediating cough in anaesthetized guinea-pigs. Journal of Physiology (London) 557: 543–558. Coleridge HM and Coleridge JCG (1986) Reflexes evoked from tracheobronchial tree and lungs. In: Fishman AP (ed.) Handbook of Physiology: The Respiratory System, pp. 395–429. Bethesda: American Physiological Society. Daly MB (1997) Effects of respiration on the cardiovascular system. In: Huang CL-H, Dolphin AC, Green R, and Spyer KM (eds.) Peripheral Arterial Chemoreceptors and Respiratory– Cardiovascular Integration, pp. 182–224. New York: Oxford University Press. Ho C-Y, Gu Q, Lin YS, and Lee L-Y (2001) Sensitivity of vagal afferent endings to chemical irritants in the rat lung. Respiratory Physiology 127: 113–124. Iscoe S (1998) Control of abdominal muscles. Progress in Neurobiology 56: 433–506. Jonzon A, Pisarri TE, Coleridge JC, and Coleridge HM (1986) Rapidly adapting receptor activity in dogs is inversely related

626 RELAPSING POLYCHONDRITIS to lung compliance. Journal of Applied Physiology 61: 1980–1987. Jordan D (2001) Central nervous pathways and control of the airways. Respiratory Physiology 125: 67–81. Knowlton GC and Larrabee G (1946) A unitary analysis of pulmonary volume receptors. American Journal of Physiology 147: 100–114. Lee L-Y, Lin YS, Gu Q, Chung E, and Ho C-Y (2003) Functional morphology and physiological properties of bronchopulmonary C-fiber afferents. Anatomical Record A 270A: 17–24. Lee L-Y and Pisarri TE (2001) Afferent properties and reflex functions of bronchopulmonary C-fibers. Respiratoy Physiology 125: 47–65. Reynolds SM, Mackenzie AJ, Spina D, and Page CP (2004) The pharmacology of cough. Trends in Pharmacological Sciences 25: 569–576. Sant’Ambrogio G and Sant’Ambrogio FB (1991) Reflexes from the airway, lung, chest wall, and limbs. In: Crystal RG, West JB, et al. (eds.) The Lung: Scientific Foundations, pp. 1383–1395. New York: Raven Press. Schelegle ES and Green JF (2001) An overview of the anatomy and physiology of slowly adapting pulmonary stretch receptors. Respiratory Physiology 125: 17–31. Tonkovic-Capin M, Zuperku EJ, Stuth EA, et al. (2000) Effect of central CO2 drive on lung inflation responses of expiratory bulbospinal neurons in dogs. American Journal of Physiology 279: R1606–R1618. Widdicombe J (2003) Functional morphology and physiology of pulmonary rapidly adapting receptors (RARs). Anatomical Record A 270: 2–10.

Glossary Axon reflex – activation of a sensory receptor results in orthodromic conduction of the action potential toward the central nervous system, and antidromic conduction along peripheral branches of the

axon. The action potential can produce release of neuroactive agents from retrogradely invaded terminals Breuer–Hering reflex (B–H) – reflex changes in breathing pattern elicited by activation of SAR. Activation during inspiration shortens inspiratory duration and reduces tidal volume. Activation by maintained inflation during expiration results in expiratory prolongation Functional residual capacity (FRC) – volume of air in the lungs at the end of a normal expiration Non-adrenergic, non-cholinergic (NANC) – airway nerve fibers that release any of a variety of transmitter substances but do not release either norepinephrine or acetylcholine Neuroepithelial bodies (NEBs) – collection of endocrine cells in airway epithelium that contain dense core vesicles and a variety of neuroactive substances. They are extensively innervated by vagal fibers and fibers arising in dorsal root ganglia. They are responsive to hypoxia but their physiological role requires clarification Rapidly adapting pulmonary stretch receptor (RAR) – receptor in epithelium and subepithelial layers of airways with myelinated axon responsive to inhaled irritants, large lung inflation Slowly adapting pulmonary stretch receptor (SAR) – receptor in airway smooth muscle with myelinated axon that is responsive to lung inflation

RELAPSING POLYCHONDRITIS A Mehta and J C Yataco, Cleveland Clinic Foundation, Cleveland, OH, USA & 2006 Elsevier Ltd. All rights reserved.

Abstract Relapsing polychondritis is a rare, chronic, episodic, and progressive inflammatory disease of unknown cause. It is characterized by inflammation and destruction of cartilaginous structures found in ears, nose, joints, and the tracheobronchial tree. Relapsing polychondritis can also affect other proteoglycan-rich structures, such as the eye, kidneys, heart, blood vessels, and inner ear, or cause systemic symptoms such as fever, lethargy, and weight loss. The exact etiology and pathogenesis of relapsing polychondritis remains unknown, but evidence suggest that it is an immunologically mediated disease. The most common manifestations include auricular and nasal chondritis, arthritis, episcleritis, and inflammation of cartilaginous tissues along the

laryngotracheal tree. The correct diagnosis is usually delayed in most patients due to its rarity and its multiple clinical features. No exact data exist on the incidence, prevalence, and mortality of relapsing polychondritis. The most common natural course of relapsing polychondritis is fluctuating but progressive with episodic flare-ups of inflammation that can eventually lead to significant dysfunction of the involved organs. Corticosteroids have been shown to be the most effective treatment, but their side effects prevent any long-term therapy. Other immunosuppressing agents, such as dapsone, methotrexate, azathioprine, and cyclophosphamide, have also been used.

Introduction Relapsing polychondritis is a rare, chronic, episodic, and progressive inflammatory disease in which different cartilaginous structures found in ears, nose,

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joints, and the tracheobronchial tree are mainly affected. Relapsing polychondritis can also affect other proteoglycan-rich structures, such as the eye, kidneys, heart, blood vessels, and inner ear, or cause systemic symptoms such as fever, lethargy, and weight loss. The correct diagnosis is usually delayed in most patients due to its rarity and its multiple clinical features. No exact data exist on the incidence, prevalence, and mortality of relapsing polychondritis. The incidence in Rochester, Minnesota, is estimated to be 3.5 per 1 million population per year. The most common natural course of relapsing polychondritis is fluctuating but progressive with episodic flare-ups of inflammation that can eventually lead to significant dysfunction of the involved organs. Corticosteroids have been shown to be the most effective treatment, but their side effects prevent any long-term therapy. Other drugs, such as dapsone, methotrexate, azathioprine, and cyclophosphamide, have also been used.

Etiology and Pathogenesis The exact etiology and pathogenesis of relapsing polychondritis remains unknown. The role of the immune system in the pathogenesis of relapsing polychondritis has been supported by different lines of evidence: 1. Serum autoantibodies against collagen type II (present in cartilage in large amounts), as well as types IX and XI, have been found in some patients with relapsing polychondritis. 2. Cell-mediated immune responses directed toward cartilage components have been demonstrated by some investigators. 3. Immunofluorescence studies of cartilage have shown granular IgG, IgA, IgM, and C3 deposits at the junction of fibrous and cartilaginous tissue, suggesting the presence of immune complexes. 4. An association with HLA-DR4 antigen has been reported more frequently in patients with relapsing polychondritis compared to controls. 5. Tissue infiltration composed of plasma cells, lymphocytes, and polymorphonuclear leukocytes has been well described. 6. The frequent association of relapsing polychondritis with other autoimmune diseases and the response to immunosuppressive agents support the theory of autoimmune pathogenesis.

Pathology The initial pathologic change is a loss of basophilic staining, followed by cellular infiltrates of

lymphocytes, plasma cells, and neutrophils at the chondrodermal junction in the affected cartilaginous sites. The lymphocytosis is a CD4 helper T cells dominate process. IgG and C3 may now be seen throughout the matrix. Finally, the chondrocytes become vacuolated and pyknotic, and they are replaced by fibroblastic granulation tissue that completely disrupts the tissue architecture. Focal regions of calcification and bone formation may be present. The ocular histopathology in patients with relapsing polychondritis includes mononuclear inflammatory cells, plasma cells, and even true vasculitis.

Clinical Features Relapsing polychondritis usually develops between the ages of 40 and 60 years, but the disease has been described in almost all ages. Both genders are affected with the same frequency, but most cases involve white patients. Since relapsing polychondritis is uncommon and can affect multiple organs and systems at different times, there is usually a significant mean delay of 2.9 years until diagnosis is established. Relapsing polychondritis should be considered a syndrome that can be primary or secondary, depending on its association with other entities. Up to 37% of patients have an associated condition, such as a hematologic disorder, connective tissue disease, vasculitis, dermatologic disorder, or other autoimmune disorder (Table 1). Auricular Chondritis, Vestibular Dysfunction, and Nasal Chondritis

Auricular chondritis is the most frequent presenting manifestation in patients with relapsing polychondritis. Patients present with external ear pain and redness and swelling of the cartilaginous portion of the ear with sparing of the lobule (Figure 1). The involvement of the external ear usually occurs in a relapsing pattern persisting for several weeks or resolving spontaneously after a few days. After several episodes or after a severe single one, the cartilaginous pinna becomes floppy or deformed. The external auditory canal and Eustachian tube are frequently involved also, with edema and collapse causing subsequent conductive hearing loss and otitis media. Sensorineural hearing loss with or without symptoms of vestibular dysfunction (dizziness, ataxia, nausea, and vomiting) can occur presumably from vasculitis in branches of the internal auditory artery. Nasal chondritis develops suddenly, is very painful, and the inflammation can destroy the distal part of the nasal cartilage leading to a saddle nose deformity (Figure 2).

628 RELAPSING POLYCHONDRITIS Table 1 Diseases associated with relapsing polychondritis Hematologic diseases Myelodysplastic syndromes Pernicious anemia Hematologic malignancies Collagen vascular diseases Rheumatoid arthritis Systemic lupus erythematosus Reiter’s syndrome Seronegative spondylarthropathies Scleroderma Sjo¨gren syndrome Systemic vasculitides Wegener’s granulomatosis Polyarteritis nodosa Behçet disease Churg–Strauss syndrome Other diseases Autoimmune thyroid disease Myasthenia gravis Inflammatory bowel disease Primary biliary cirrhosis Vitiligo Lichen planus Panniculitis Mixed cryoglobulinemia

Figure 1 Significant deformity of the external ear due to severe cartilaginous inflammation.

Otorhinolaryngeal Manifestations

More than 50% of patients develop chondritis of the laryngotracheal cartilages and may present with anterior neck pain, cough, hoarseness, dyspnea, wheezing, or choking. Acute upper airway obstruction may develop due to inflammation and edema of the glottic, laryngeal, or subglottic area. Some patients may require an emergency tracheostomy even before there is an established diagnosis.

Pulmonary Manifestations

Involvement of the respiratory tract (trachea and firstto-second order bronchi) is the presenting feature in up to 26% of patients with relapsing polychondritis. Acute swelling of the tracheobronchial tree can lead to localized or diffuse obstruction of the airway tract. The damaged tracheal and bronchial rings may lose their rigidity and collapse, allowing the development of dynamic obstruction. Thus, pulmonary function tests with flow volume loops should be performed in all patients, even in those without respiratory symptoms. Spirometry shows obstructive ventilatory impairment with reduction in expiratory flow and maximal voluntary ventilation. Other findings seen in patients with relapsing polychondritis include tracheal/bronchial stenosis due to granulation tissue,

Figure 2 Saddle nose deformity due to severe cartilaginous inflammation.

calcification of the airway walls, and obstructive bronchiectasis. Chest radiographic findings are usually not sensitive or specific but include pneumonia, atelectasis, calcifications in the airway wall, and prominence of


proliferation, but other pathologies, such as IgA nephropathy, segmental necrotizing crescentic glomerulonephritis, and tubulointerstitial nephritis, have also been described. Cardiovascular Manifestations

Figure 3 Inflammatory destruction of the distal tracheal rings. Reproduced from Becker H and Kayser K (1991) Atlas of Bronchoscopy, p. 27. St Louis: Mosby, with permission.

the thoracic aorta. Computed tomography (CT) of the chest is more useful in revealing details of the tracheobronchial tree (e.g., narrowing of the lumen and calcifications or thickening of the tracheal/bronchial wall). Bronchoscopic exam of the airway confirms the diagnosis, showing erythema, edema, deformation, and collapse of the tracheobronchial tree (Figure 3). In addition, biopsies of the cartilage can be obtained through bronchoscopy. It is important to remember that bronchoscopy is less accurate in evaluating the functional status of the airway compared to pulmonary function testing. Bronchoscopy may also precipitate further respiratory distress since it can induce trauma in an airway suffering active inflammation. Musculoskeletal Manifestations

Arthritis is the second most common presenting symptom and is present in up to 85% of patients with relapsing polychondritis. When relapsing polychondritis is not associated with any other collagen vascular disease, the arthritis is nonerosive, asymmetric, seronegative, and can affect all synovial joints. Metacarpophalangeal, interphalangeal, and knee joints are the most commonly affected. Renal Manifestations

Renal disease occurs in 10–22% of patients with relapsing polychondritis, especially in cases associated with vasculitis or other collagen vascular diseases. The most common renal pathology is mesangial

Aortic regurgitation is the most frequent form of cardiovascular involvement in relapsing polychondritis, occurring in approximately 4–10% of patients. Ascending aortitis extending to the valve ring is responsible for aortic regurgitation and explains the common association with aortic aneurysms. Other forms of cardiovascular involvement include abdominal aortic aneurysms, myocarditis, pericarditis, coronary vasculitis, impairment of the conduction system, thrombophlebitis, and arterial thrombosis. These thrombotic complications have been explained by the presence of vasculitis or an associated antiphospholipid syndrome. Dermatologic Manifestations

Skin involvement in the absence of an associated condition occurs in approximately 35% of cases. The most common manifestations are oral aphthous ulcers, followed by macules, papules, nodules, and livedo reticularis. The most common histologic diagnosis in these lesions is leukocytoclastic vasculitis, with other histologic patterns including neutrophilic dermatosis, thrombotic occlusion of dermal vessels, and even granulomatous vasculitis. Patients with an associated myelodysplastic syndrome commonly have dermatologic involvement. Elderly patients with relapsing polychondritis and skin involvement should be monitored for the development of a myelodysplastic syndrome. Neurologic Manifestations

Central and peripheral nervous system involvement occurs in only 3% of patients. The cranial nerves are most commonly affected, but seizures, headache, hemiplegia, encephalopathy, and cerebral aneurysms have been described. Ocular Manifestations

Ocular disease occurs eventually in up to 60% of patients with relapsing polychondritis. Most of these patients tend to develop multiple systemic manifestations. Episcleritis and scleritis are the most common manifestations, usually in parallel with inflammation of the nose and joints (Figure 4). Other types of ocular involvement are uveitis, keratitis, keratoconjunctivitis sicca, central retinal vein occlusion, and ischemic optic neuropathy.

630 RELAPSING POLYCHONDRITIS Table 2 Diagnostic criteria for relapsing polychondritis McAdam et al. (1976) criteriaa Bilateral auricular chondritis Nasal chondritis Nonerosive, seronegative inflammatory polyarthritis Ocular inflammation (conjunctivitis, keratitis, scleritis and/or episcleritis, uveitis) Respiratory tract chondritis (laryngeal and/or tracheal cartilage) Cochlear and/or vestibular dysfunction (neurosensory hearing loss, tinnitus, and vertigo)

Figure 4 Episcleritis is a common manifestation of relapsing polychondritis. Reproduced from Forbes and Jackson (2002) Color Atlas and Text of Clinical Medicine, p. 128. St Louis: Mosby, with permission.

Michet et al. (1986) criteriab Proven inflammation in two of three cartilaginous sites: auricular, nasal, or laryngotracheal Proven inflammation in one of three cartilaginous sites: auricular, nasal, or laryngotracheal; plus two other clinical signs, including ocular inflammation, vestibular dysfunction, seronegative arthritis, and hearing loss a

Three criteria are the minimum required to confirm the diagnosis. b One diagnostic criteria is enough to confirm the diagnosis.

Diagnosis Although relapsing polychondritis seems to be easy to diagnose when it presents with its most typical symptoms, the correct diagnosis in many cases is delayed due to its rarity and multiple possible presenting features. McAdam and co-workers were the first to propose diagnostic criteria using the most common clinical features, requiring at least three out of six features to confirm the diagnosis (Table 2). Michet and colleagues modified these diagnostic criteria, requiring chondritis in two of three sites (auricular, nasal, or laryngotracheal) or one of those sites and two other signs, including ocular inflammation, vestibular dysfunction, seronegative arthritis, and hearing loss. In most patients, it is not necessary to biopsy the affected cartilaginous sites. No specific laboratory test exists for the diagnosis of relapsing polychondritis. During acute exacerbations, it is possible to observe elevated erythrocyte sedimentation rate, anemia of chronic disease, leukocytosis, thrombocytosis, and hypergammaglobulinemia. Serum antibodies to type II collagen have been detected in 20–50% of patients. However, the use of these antibodies is limited due to a relatively low specificity. Other serologic tests, such as rheumatoid factor, antinuclear antibodies, antineutrophil cytoplasmic antibodies, and complement levels, are helpful if other associated conditions are present simultaneously. Other ancillary tests, such as urinalysis, creatinine, echocardiography, CT chest, pulmonary function tests, and skin biopsy, can be helpful to assess the organs and systems affected by the disease. Some autoimmune disorders can overlap clinical features or involve multiple organs, as is the case for relapsing polychondritis. Wegener’s granulomatosis

and polyarteritis nodosa can cause ocular inflammation, polyarthitis, and cochlear and vestibular symptoms. However, these two entities have not been reported to cause chondritis and usually affect the lung parenchyma, which is not seen in relapsing polychondritis. Rheumatoid arthritis can also cause ocular inflammation, vasculitis, and multiple organ involvement. However, the joint involvement of rheumatoid arthritis is usually symmetric and erosive as opposed to the nonerosive arthritis seen in relapsing polychondritis. Vasculitis of medium and large vessels, such as the polyarteritis nodosa and Takayasu’s arteritis, may mimic the cardiovascular involvement seen in relapsing polychondritis with development of aneurysms or arteritis with or without thrombosis.

Management and Therapy There is no standard medical therapy for relapsing polychondritis due to its rarity, wide variety of presentations, and unpredictable course. Therapeutic guidelines are based on reports of treatment success from small series of patients or isolated cases. Arthritis or mild chondritis of nose and ears can be treated with the nonsteroidal anti-inflammatory drugs dapsone or colchicine. However, systemic corticosteroids are the traditional agents used in acute exacerbations. Prednisone at a high dose of 1 mg kg 1 day 1 is used for cases with ocular involvement, laryngeal inflammation, sensorineural hearing loss, vestibular symptoms, and vascular and renal involvement. Although steroids may resolve acute inflammation of the cartilage and decrease the frequency and


degree of the exacerbations, they have no effect on the overall progression of the disease and do not alter the risk of multisystem organ involvement or the structural damage to cartilaginous organs. When an acute exacerbation goes into remission, the high doses of steroids can be gradually tapered to moderate maintenance doses. In patients with chronic active inflammation that requires high-dose steroids, the use of methotrexate or azathioprine may allow a reduction of the corticosteroid dose. In cases of severe end-organ damage, such as ocular, pulmonary, cardiac, or renal involvement, pulses of oral or intravenous cyclophosphamide can be used. Cyclosporine A has also been used with success in refractory cases to other agents. Infliximab has been tried in resistant relapsing polychondritis and associated scleritis. The respiratory problems caused by relapsing polychondritis can present suddenly and become very difficult to manage. Adliff and colleagues reported the use of nasal CPAP as a pneumatic splint for the tracheobronchial tree, preventing airway collapse and obtaining temporary control of airway patency. For sudden and severe upper airway obstruction, a tracheostomy can be life-saving. Despite a significant risk, endotracheal intubation may be needed, preferably with a small endotracheal tube because of the reduced glottic diameter. Airway stents, montgomery T tubes, and self-expanding metallic stents have been used in patients with collapse or refractory stenosis of the airway. They are able to gain airway patency but are associated with many potential problems, including erosion of the stent through the trachea, sudden asphyxia from stent displacement, aspiration pneumonia, development of granulation tissue or mucosal ulceration, and retention of airway secretions. Most patients with relapsing polychondritis have a fluctuating but slowly progressive course with unpredictable severity and site of involvement by the inflammatory process. In a 6-year follow-up study from the Mayo Clinic, 86% of patients were reported to have intermittent manifestations with a median of five episodes. The majority of patients develop some disability, depending on the organs involved and the severity of the disease (deafness, impaired vision, speech problems, and cardiopulmonary problems). In terms of mortality, Trentham and co-workers in 1998 reported a 94% survival rate with an average disease duration of 8 years. This number represents an improvement compared to the 55% survival rate at 10 years reported by McAdam et al. in 1976. The improved survival has been explained by improved medical and surgical management of the respiratory and cardiovascular complications. The most common

causes of death are infections, vasculitis, and malignancies. Pulmonary infections are of particular importance. They are severe and complicated due to the use of corticosteroid therapy and airway stricture/collapse, which impair the defense and clearing mechanisms of the lung. See also: Bronchomalacia and Tracheomalacia. Bronchoscopy, General and Interventional. Corticosteroids: Therapy. Extracellular Matrix: Collagens. Immunoglobulins. Leukocytes: T cells. Pulmonary Effects of Systemic Disease. Upper Airway Obstruction.

Further Reading Becker H and Kayser K (1991) Atlas of Bronchoscopy, p. 27. St Louis: Mosby. Crockford MP and Kerr IH (1988) Relapsing polychondritis. Clinical Radiology 39: 386–390. Del Rosso A, Petix NR, Pratesi M, and Bini A (1997) Cardiovascular involvement in relapsing polychondritis. Seminars in Arthritis and Rheumatism 26: 840–844. Ebringer R, Rook G, Swana GT, Bottazzo GF, and Doniach D (1981) Autoantibodies to cartilage and type II collagen in relapsing polychondritis and other rheumatic diseases. Annals of the Rheumatic Diseases 40: 473–479. Eng J and Sabanathan S (1991) Airway complications in relapsing polychondritis. Annals of Thoracic Surgery 51: 686–692. Forbes and Jackson, Color Atlas and Text of Clinical Medicine, p. 128. St Louis: Mosby. Hebbar M, Brouillard M, Wattel E, et al. (1995) Association of myelodysplastic syndrome and relapsing polychondritis: further evidence. Leukemia 9: 731–733. Kent PD, Michet CJ Jr, and Luthra HS (2004) Relapsing polychondritis. Current Opinion in Rheumatology 16: 56–61. Krell WS, Staats BA, and Hyatt RE (1986) Pulmonary function in relapsing polychondritis. American Review of Respiratory Disease 133: 1120–1123. Lee-Chiong TL Jr (1998) Pulmonary manifestations of ankylosing spondylitis and relapsing polychondritis. Clinics in Chest Medicine 19: 747–757. Letko E, Zafirakis P, Baltatzis S, et al. (2002) Relapsing polychondritis: a clinical review. Seminars in Arthritis and Rheumatism 31: 384–395. McAdam LP, O’Hanlan MA, Bluestone R, and Pearson CM (1976) Relapsing polychondritis: prospective study of 23 patients and a review of the literature. Medicine (Baltimore) 55: 193–215. Michet CJ Jr, McKenna CH, Luthra HS, and O’Fallon WM (1986) Relapsing polychondritis. Survival and predictive role of early disease manifestations. Annals of Internal Medicine 104: 74–78. Molina JF and Espinoza LR (2000) Relapsing polychondritis. Bailliere’s Best Practice & Research: Clinical Rheumatology 14: 97–109. Park J, Gowin KM, and Schumacher HR Jr (1996) Steroid sparing effect of methotrexate in relapsing polychondritis. Journal of Rheumatology 23: 937–938. Tillie-Leblond I, Wallaert B, Leblond D, et al. (1998) Respiratory involvement in relapsing polychondritis. Clinical, functional, endoscopic, and radiographic evaluations. Medicine (Baltimore) 77: 168–176. Trentham DE and Le CH (1998) Relapsing polychondritis. Annals of Internal Medicine 129: 114–122.

632 RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER

RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER C J Jolley and J Moxham, King’s College London, London, UK & 2006 Elsevier Ltd. All rights reserved.

Abstract Respiratory muscles are skeletal muscles whose function is to pump air in and out of the lungs. The chest wall muscles, diaphragm, and other muscles, including abdominal muscles, neck muscles, and upper limb muscles, may be broadly divided into inspiratory or expiratory muscles, according to whether their actions increase or decrease the capacity of the thoracic cage. The recruitment of individual muscles also depends on whether ventilatory requirements are high or low. Respiratory muscles share the common structural, physiological, and biochemical features of skeletal muscles, but are adapted to meet their unique task of being able to be active throughout life. These features determine their ability to generate contractile force, which is translated into inspiratory or expiratory pressure, and their resistance to fatigue. An imbalance between the load on the respiratory muscles and their capacity, resulting in breathlessness and ultimately respiratory failure, is a consequence of obstructive and restrictive respiratory disorders, and neuromuscular disease.

Anatomy, Histology and Structure Respiratory muscles are skeletal muscles whose function is to move air in and out of the lungs, through their actions on the thoracic cage. Respiratory movements have been studied throughout medical history, from Hippocrates to the present day. Advances in respiratory physiology, histochemical techniques, genetics, and cell physiology have recently provided further insights into the functional anatomy of the respiratory muscle pump. Anatomy

The chest wall is made up of the vertebral column posteriorly, sternum and costal cartilages anteriorly, and the ribs and intercostal spaces laterally. It comprises the lateral boundaries of the thoracic cavity, which communicates superiorly with the neck through the thoracic inlet, and is separated from the abdomen inferiorly by the diaphragm muscle. Although the respiratory muscles are conventionally divided into inspiratory and expiratory groups, some overlap exists. Generally, muscles that elevate the ribs increase both the lateral and dorsoventral diameters of the ribcage, reduce intrathoracic pressure, and are inspiratory. Muscles that lower the ribs are usually expiratory.

Inspiratory muscles Diaphragm The diaphragm is a dome-shaped structure. The muscular portions of the left and right hemidiaphragm are inserted into a mobile central tendon. Direct and indirect evidence, from observations made in humans, that the muscular portion of each hemidiaphragm is made up of two embryologically and anatomically distinct components has been supported by electrophysiological studies in mammalian quadrupeds (Figure 1). 1. The costal diaphragm is derived from myoblasts in the body wall. It arises from the deep surfaces of the lower six ribs and costal cartilages, and runs directly upwards, parallel to and against the inner surface of the ribcage, forming the zone of apposition (ZAP) before inserting into the central tendon. The ZAP contributes one-third of the area of the ribcage at end expiration, less during inspiration. 2. The crural (lumbar) component of the diaphragm is derived from the mesentery of the esophagus. The left and right crura arise from the sides of the bodies of the first two (left), and first three (right), lumbar vertebrae and intervertebral discs, and from the medial and lateral arcuate ligaments. These are thickened upper margins of fascia, which extend from the second to the first lumbar vertebra, and from the first lumbar vertebra to the lower border of the twelfth rib, respectively. The median arcuate ligament connects their fibrous medial borders, crossing over the anterior border of the surface of the aorta. Some fibers of the right crus pass up to the left, forming a sphincteric sling around the esophagus. Innervation Phrenic nerves, arising from the third, fourth, and fifth cervical nerve roots supply motor innervation to each hemidiaphragm. Despite earlier electrophysiological studies suggesting that the costal and crural parts had separate segmental innervation, topographical tracer studies now suggest that this is mixed at both a spinal level and within the phrenic motor nucleus. Supraspinal control of the diaphragm is a combination of phasic automatic brainstem activity and cortical input. Transcranial magnetic stimulation of hemiplegic stroke patients has demonstrated that each hemidiaphragm is separately represented in the contralateral motor cortex.

RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER 633

Last rib

h arc al t os

La t. l um bo

c

Xiphoid process

Opening for lesser splanchnic nerve

Figure 1 Diaphragm as viewed from below, showing the origins of the costal and crural muscular components, and the central tendon. Reproduced from Gray H (1918) Anatomy of the Human Body. Philadelphia: Lea & Febiger.

Chest wall muscles Intercostal muscles The external intercostal and parasternal muscles are inspiratory. The internal intercostal and transversus thoracis are expiratory muscles, but are described here together with other muscles in the anatomical group. The thin muscle layers span the intercostal spaces, overlapping, from superficial to deep (Figures 2 and 3). 1. Muscle fibers of the external intercostals run downward and forward, from the tubercles of the rib above to the costal cartilage of the rib below. Anteriorly, they are replaced by a fibrous aponeurosis, the anterior intercostal membrane, which continues to the sternum. Muscle mass also decreases caudally. 2. Muscle fibers of the internal intercostals run downward and backward, from the sternocostal junction of the rib above, to near the tubercle of

the rib below, replaced posteriorly by a fibrous aponeurosis, the posterior intercostal membrane. Muscle mass in humans shows no definite rostrocaudal gradient. Internal intercostal muscles between the costochondral junction and the sternum are called the parasternal intercostals. They do not extend below the fifth interspace in humans. 3. Transversus thoracis arises from either side of the lower third of the posterior surface of the body of the sternum, posterior surface of the xiphoid process, and the sternal ends of the costal cartilages of the lower three or four ribs. Its fibers diverge upward and laterally, inserting into the lower borders and inner surfaces of the costal cartilages of the second, third, fourth, fifth, and sixth ribs. The lowest fibers of this muscle are directed horizontally, continuous with those of the transversus abdominis muscle, the intermediate fibers obliquely, and the highest almost vertically (Figure 3).


on nd Te

Sternum

ct. majo r of pe

Ulna

Radius

Figure 2 Chest wall, pectoral, and upper limb muscles. Muscle fibers of the external intercostals run downward and forward, and muscle fibers of the internal intercostals run downward and backward. Reproduced from Gray H (1918) Anatomy of the Human Body. Philadelphia: Lea & Febiger.

Levatores costarum There are 12 pairs of muscles. They arise from the tip of the transverse process of C7 and T1–T11 vertebrae, and insert into the rib below. Serrati muscles Serratus posterior superior arises from a thin and broad aponeurosis from the lower part of the ligamentum nuchae, the spinous processes of the seventh cervical and upper two or three thoracic vertebrae, and the supraspinal ligament. Directed downward and laterally, it is inserted into the upper borders of the second, third, fourth, and fifth ribs, a little beyond their angles. Serratus posterior inferior arises from a thin aponeurosis from the spinous processes of the lower two thoracic and upper two or three lumbar vertebrae, and the supraspinal ligament. It passes obliquely

upward and laterally, dividing into four flat digitations, which are inserted just beyond the angles of the lower four ribs, along their lower borders. It lowers and fixes the ribs. Innervation The intercostal muscles and serrati are supplied by the intercostal nerves, which are the anterior rami of the first eleven thoracic spinal nerves. Levatores costarum muscles are supplied by the posterior rami of the thoracic spinal nerves. Abdominal muscles In functional terms, the abdominal muscles are also an important part of the chest wall, but are mostly expiratory, and are described below. Neck muscles The scalenes comprise three muscle heads that run from the transverse processes of the

RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER 635 1

2

3

4

5

6

Internal intercostal muscle

Sternal origin of diaphragm Figure 3 Inner (posterior) surface of the sternum and ribcage, showing the anatomical relationship between the internal intercostals, transversus thoracis, transversus abdominis, and diaphragm. The lowest fibers of transversus thoracis are continuous with those of transversus abdominis. Reproduced from Gray H (1918) Anatomy of the Human Body. Philadelphia: Lea & Febiger.

lower five cervical vertebrae to the upper surface of the first two ribs. The sternomastoids run from the mastoid process to the ventral surface of the manubrium sterni and the medial third of the clavicle (Figure 4). Innervation The scalenes are supplied by branches from the second to the seventh cervical nerves. The sternomastoids are supplied by the spinal accessory nerve and the anterior rami of the second and third cervical nerves. The latter are believed to be proprioceptive. Expiratory muscles Muscles of the ventrolateral abdominal wall These are the most important expiratory muscles (Figure 5). The rectus abdominis arises from the sternum, and the fifth, sixth, and seventh costal cartilages, running caudally to insert into the pubis. It is enclosed in sheaths formed by the aponeuroses of the three muscles situated laterally. These are, from superficial to deep: 1. the external oblique muscle, extending from the external surface of the lower eight ribs to the iliac crest, the inguinal ligament, and the linea alba;

2. the internal oblique muscle, extending from the iliac crest and inguinal ligament, diverging superiorly to insert into the costal margin and an aponeurosis contributing to the rectus sheath; and 3. the transversus abdominis muscle that arises from the inner surface of the lower six ribs, and runs circumferentially to terminate in the rectus sheath. Innervation The abdominal muscles are supplied by the lower six thoracic nerves. The internal and external obliques, and transversus abdominis muscles are also supplied by the iliohypogastric and ilioinguinal nerves. Internal intercostal and transversus thoracis muscles See ‘Intercostal muscles’. Serratus posterior inferior See ‘Serrati muscles’. This may assist forced expiration. Muscles connecting the upper limb and chest wall Pectoral muscles may act as expiratory muscles


Mastoid process

Levator scapulae Splenius capitis Sternomastoid Scalenus anterior Scalenus medius 1st rib

Clavicle

Sternal origin of sternomastoid Clavicular origin of sternomastoid

Figure 4 Neck muscles. The scalenes and sternomastoid muscles act as inspiratory muscles by elevating the upper ribcage. Scalenus posterior (not shown) may be absent, or blended with scalenus medius. Reproduced from Gray H (1918) Anatomy of the Human Body. Philadelphia: Lea & Febiger.

during forced expiration if the shoulder girdle is fixed (Figure 2). Histology and Structure

Overview of skeletal muscle structure The fundamental unit of skeletal muscle is the motor unit, which is a single motor neuron plus the group of muscle fibers it supplies. Respiratory muscles are therefore made up of thousands of skeletal muscle fibers, bound together with nerves and blood vessels by connective tissue. Motor neuron cell bodies are located in the anterior horn of the spinal cord. Muscle fibers Skeletal muscle fibers are multinucleate cells containing striated, thread-like myofibrils, which run the entire length of the muscle fiber and can be seen by light microscopy. They also contain mitochondria, and have an extensive sarcoplasmic reticulum (SR) which stores calcium. The outer cell membrane (sarcolemma) has extensions that project deep into the fiber to the SR, which surrounds the myofibrils. These are the transverse (t)-tubules.

Voltage-sensitive calcium release channels dihydropyridine receptor (DHPR) in t-tubular membranes, and isoforms of the calcium-sensitive calcium release channels, or ryanodine receptors, (RyR1 and RyR3), mediate calcium release from the SR during excitation–contraction coupling in skeletal muscle (Figure 6). Sarcomere structure Sarcomeres are the functional units of the muscle fiber. They contain thick (15 nm diameter) filaments made of the protein myosin, which hydrolyzes adenosine triphosphate (ATP), and thin (5 nm diameter) filaments, made mostly of the protein actin, in addition to the regulatory protein troponin, which binds Ca2 þ , and tropomyosin. Sarcomere shortening, described by the sliding filament model, causes muscle contraction. Fiber type classification Muscle fibers are classified on the basis of their physiological behavior as fast (or fatigable) or slow (fatigue-resistant) (largely an expression of aerobic enzyme activity and calcium metabolism) and by myosin heavy chain isoform into

RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER 637

External intercostals

Internal intercostals

Rectus abdominis External oblique

Internal oblique

Transversus abdominis

Figure 5 Attachments of the intercostal muscles and abdominal muscles to the thoracic cage. In functional terms, the abdominal muscles are an important part of the chest wall. Reproduced from Zemlin E and Zemlin WR (1997) Study Guide/ Workbook to Accompany Speech and Hearing Science, 4th edn. Stipes Publishing Co., with permission.

Diaphragm muscle fibers have a smaller cross-sectional area than those in limb muscles but a similar number of capillary vessels. There is also an inverse relationship between aerobic enzyme activity and cross-sectional area. These functional adaptations likely improve oxygen diffusion and contribute to fatigue resistance. Muscle-fiber changes due to training and inactivity do not appear to happen in the same way in respiratory muscles as in limb muscles. General increases in the aerobic capacity of all respiratory muscle fiber types have been observed following whole-body endurance training in rats. Metabolic enzymes and myofibrillar proteins are also not strictly coupled as they are in limb muscles, and the response of aerobic metabolism to endurance training is less marked. Changes in respiratory muscle fiber characteristics are also responsible for increases in contractile strength during perinatal development, as embryonic and neonatal myosin are replaced by adult isoforms. Increases in fatigue-resistant (type 1) myosin from 10% in premature infants, to 25% at term, compared to 55% in adults, have been observed. Diaphragm contractility decreases with age. This is likely to be due to changes other than fiber type composition.

Respiratory Muscle Function in Normal Subjects Inspiration

types 1, 2A, 2B, and 2X. Type 2B is not expressed in human skeletal muscle. With only a few exceptions, each motor unit is composed of fibers of one type, determined mainly by the function of the muscle. Contractile activity can stimulate phenotype shifts between fiber types, depending on whether the stimulus is endurance- or resistance-training. In limb muscles, both endurance- and resistance-training result in transformation of fiber type from 2X to 2A. Transformation from type 1 to 2A has been observed following sprint training, and from 2A to 1 after endurance training. Immobilization shifts muscle fibers to the fast phenotype (Table 1). Fiber types in respiratory muscles The normal adult human diaphragm contains approximately 55% type 1 (slow) fibers, 21% fast oxidative (type 2A) fibers, and 24% fast glycolytic (type 2X) fibers. Fast-fiber types are larger, hence overall o50% is ‘slow’ myosin, and 40% is the 2A isoform. Human intercostal muscles contain slightly more slow-fibers than the diaphragm, approximately 60% in both internal and external intercostal muscles.

Action of the diaphragm accounts for about 70% of minute ventilation in normal humans. The pistonlike downward displacement of the central dome of the diaphragm increases the axial diameter of the thoracic cavity. Most of the diaphragm’s ability to expand the lower ribcage during inspiration is however due to the ‘insertional component’ of the costal diaphragm. The abdominal contents provide a fulcrum, which opposes further downward displacement of the central tendon, allowing elevation of the lower rib. Thirdly, downward displacement of the diaphragm also increases intra-abdominal pressure, causes the anterior abdominal wall to move outwards. This abdominal pressure is also transmitted to, and so expands, the lower ribcage through the ZAP. The effect size of this ‘appositional component’ of costal diaphragm action depends on the area of the ZAP and on the magnitude of abdominal pressure change, that in turn depends on abdominal compliance. The external intercostals, parasternal, and scalene muscles assist the diaphragm, and are active during resting breathing. Comparative studies with the dog suggest that the action of the scalenes, together with


(a)

Mitochondria

Z line

(b)

Z line

Sarcomere

Thick filament Thin filament Z line

H zone I band

I band (c)

A band

Figure 6 (a) Light micrograph of a single human diaphragm muscle fiber. Magnification 40. (b) Electron micrograph of human diaphragm muscle. Magnification 10 000. Sarcomeres, the functional units of skeletal muscle, extend from Z line to Z line. (c) Schematic diagram of the main contractile elements of a single sarcomere. The striated appearance seen in (a) is created by a pattern of alternating dark A bands (thick filaments containing myosin), and light I bands (containing actin, tropomyosin, and troponin) which are bisected by dense Z lines. Z lines anchor the sarcomeres, which extend from Z line to Z line. The H zone is the portion of the A band where the thick and thin bands do not overlap. Light and electron micrographs courtesy of A Moore and A Stubbings, Imperial College, London.

RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER 639 Table 1 Skeletal muscle fiber classification according to myosin heavy chain (MyHC) isoform and functional properties MyHC isoform Maximum shortening velocity Myofibrillar ATPase activity Ca2 þ uptake in the sarcoplasmic reticulum Time course of muscle twitch Fatigue resistance Metabolism

Type 1 Slow Low Slow Slow High Oxidative

ribcage muscles, reduces ribcage distortion and therefore elastic work. The external and parasternal intercostals elevate the ribs, and so are inspiratory, and the internal intercostals lower the ribs, and are expiratory. As in the diaphragm, inspiratory drive is matched spatially to respiratory advantage. The inspiratory effect of the external intercostals is greatest in the dorsal portion of the rostral interspace, decreasing downwards and anteriorly, and also decreasing toward the lowest interspaces in the parasternals. During resting breathing, activity appears to be limited to the parasternal intercostals and the external intercostals in the dorsal portion of the more rostral interspaces. In humans, inspiratory drive to the external intercostals appears to be greater than that to the parasternals in the same (rostral) interspace. In forced inspiration, serratus posterior superior elevates the ribs, and serratus posterior inferior draws the lower ribs downward and backward, thus elongating the thorax; and fixes the lower ribs. The sternomastoids act by elevating the upper ribcage. Abdominal muscle recruitment also assists the diaphragm through the ZAP (see above). Expiration

In normal subjects at rest, expiration is passive, but when ventilatory requirements rise, as in exercise, or with disease, the expiratory muscles become active. The abdominal muscles are the most important expiratory muscles. They increase intra-abdominal pressure, displacing the diaphragm into the thorax, and increasing intrathoracic pressure. Expiratory electromyogram (EMG) activity of the internal intercostals has been recorded during resting breathing, but only in the ventrolateral portions of the caudal interspaces, where they have the greatest expiratory mechanical advantage. The transversus thoracis muscle is only active during forced expiration. Its theoretical expiratory effect is limited by its small muscle mass. The pectoral muscles can be recruited to aid forced expiration if the shoulder girdle is fixed. Physiological Determinants of Force (Pressure) Generation

Contraction results in either increased tension or muscle shortening, depending on whether the ends of

Type 2A Fast High High Fast Intermediate Oxidative gycolytic

Type 2X Very fast Very high Very high Fast Low Gycolytic

the muscle are fixed, or not. The force generated by muscle contraction is also related to the number of fibers stimulated, the frequency of stimulation, and length of the muscle at the time of stimulation. Force generation therefore depends on the force–length, force–frequency, and force–velocity relationships, and all are relevant to respiratory muscle function and ventilation. The force produced by the inspiratory muscles is greatest at low lung volumes, and that of the expiratory muscles at high lung volumes. Mean twitch transdiaphragmatic pressure decreases at 3.5 cmH2O l 1 at volumes above normal predicted thoracic gas volume in chronic obstructive pulmonary disease (COPD). Muscle shortening causes a disproportionately large reduction in force generated by stimulation at low frequencies, resulting in the force-generating capacity of the diaphragm being disproportionately diminished in response to physiologic firing frequencies (10–20 Hz). As inspiratory flow rises, the capacity of the respiratory muscles to generate tension is reduced, reflecting the force–velocity relationship. Measurement of Respiratory Muscle Activity

Volitional and nonvolitional tests of respiratory muscle strength are summarized in Figure 7. Pressure swings per unit time (pressure–time product) and normalized to maximum volitional efforts (tension– time index) during respiration are used to assess respiratory muscle workload, relative to capacity. Quantification of spontaneous EMG activity is also of value in the assessment of neural drive, and compound muscle action potential (CMAP) responses to electrical or magnetic phrenic nerve stimulation aid diagnosis of neuromuscular disorders.

Respiratory Muscles in Respiratory Disease Load–Capacity Balance

The clinical significance of respiratory muscle weakness depends on the degree of weakness and the load imposed on the respiratory system. When the respiratory system is otherwise normal, muscle weakness must be severe before breathlessness or respiratory

640 RESPIRATORY MUSCLES, CHEST WALL, DIAPHRAGM, AND OTHER Inspiratory muscle tests

Units

Normal values

Nonvolitional Bilateral TwPdi Right unilateral TwPdi Left unilateral TwPdi

cmH2O cmH2O cmH2O

>18 >7 >8

Volitional Sniff pdi Sniff poes Sniff nasal pressure Mouth inspiratory pressure

cmH2O cmH2O cmH2O cmH2O

>80 >60 >60 (F) or >70 (M) >60 (F) or >80 (M)

cmH2O

>15

Expiratory muscle tests Nonvolitional TwT10 Volitional Mouth expiratory pressure Cough gastric pressure

cmH2O cmH2O

>60 (F) or >80 (M) >100 (F) or >120 (M)

Diaphragm EMG (esophageal) Right phrenic nerve latency Right CMAP amplitude Left phrenic nerve latency Left CMAP amplitude

ms mV ms mV

6–8.0 ms >1.0 mV 6.5–8.5 ms >1.0 mV

Pulmonary function Dynamic compliance Vital capacity (lying) Vital capacity (seated) Vital capacity postural drop KCO PaO2 PaCO2 Bicarbonate pH

l cmH2O −1 l l %

0.1–0.3

kPa kPa HCO3

>11 4.5–6.0 21–28 7.35–7.45

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