Purinergic Regulation of Respiratory Diseases

Purinergic Regulation of Respiratory Diseases SUBCELLULAR BIOCHEMISTRY SERIES EDITOR J. ROBIN HARRIS, University of M...

Author: Maryse Picher | Richard C. Boucher

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Purinergic Regulation of Respiratory Diseases

SUBCELLULAR BIOCHEMISTRY SERIES EDITOR J. ROBIN HARRIS, University of Mainz, Mainz, Germany

ASSISTANT EDITORS B.B. BISWAS, University of Calcutta, Calcutta, India P. QUINN, King’s College London, London, UK Recent Volumes in this Series Volume 41

Chromatic and Disease Edited by Tapas K. Kundu and Dipak Dasgupta

Volume 42

Inflammation in the Pathogenesis of Chronic Disease Edited by Randall E. Harris

Volume 43

Subcellular Proteomics Edited by Eric Bertrand and Michel Faupel

Volume 44

Peroxiredoxin Systems Edited by Leopold Folhe´ and J. Robin Harris

Volume 45

Calcium Signalling and Disease Edited by Ernesto Carafoli and Marisa Brini

Volume 46

Creatine and Creatine Kinase in Health and Disease Edited by Gajja S. Salomons and Markus Wyss

Volume 47

Molecular Mechanisms of Parasite Invasion Edited by Barbara A. Burleigh and Dominique Soldati-Favre

Volume 48

The Cronin Family of Proteins Edited by Christoph S. Clemen and Ludwig Eichinger

Volume 49

Lipids in Health and Disease Edited by Peter J. Quinn and Xiaoyuan Wang

Volume 50

Genome Stability and Human Diseases Edited by Heinz-Peter Nasheuer

Volume 51

Choloesterol Binding and Cholesterol Transport Proteins Edited by Robin J. Harris

Volume 52

A Handbook of Transcription Factors Edited by Tim Hughes

Volume 53

Endotoxins: Stricture, Function and Recognition Edited by Xiaoyuan Wang and Peter J. Quinn

Volume 54

Conjugation and Deconjugation of Ubiquitin Family Modifiers Edited by Marcus Groettrup

Maryse Picher Editor-in-Chief

Richard C. Boucher Editor

Purinergic Regulation of Respiratory Diseases

Editor-in-Chief Maryse Picher Cystic Fibrosis Pulmonary Research and Treatment Center University of North Carolina 7011 Thurston-Bowles building Chapel Hill, NC 27599 USA [email protected]

Editor Richard C. Boucher Cystic Fibrosis Pulmonary Research and Treatment Center University of North Carolina 7011 Thurston-Bowles building Chapel Hill, NC 27599 USA [email protected]

ISSN 0306-0225 ISBN 978-94-007-1216-4 e-ISBN 978-94-007-1217-1 DOI 10.1007/978-94-007-1217-1 Springer Dordrecht Heidelberg London New York Library of Congress Control Number: 2011928079 # Springer Science+Business Media B.V. 2011 No part of this work may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording or otherwise, without written permission from the Publisher, with the exception of any material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

INTERNATIONAL ADVISORY EDITORIAL BOARD R. Bittman, Queens College, City University of New York, New York, USA D. Dasgupt, Saha Institute of Nuclear Physics, Calcutta, India A. Holzenburg, Texas A&M University, Texas, USA S. Rottem, The Hebrew University, Jerusalem, Israel M. Wyss, DSM Nutritional Products Ltd., Basel, Switzerland

Nearly half a century has passed since the “Purinergic Nerve Hypothesis” was proposed by Burnstock. The demonstration that ATP is released by nerve endings in a regulated manner and plays a critical role as neurotransmitter raised worldwide controversy and paved the way for the exploration of nucleotides as extracellular messengers. To this date, the number of manuscripts published in the field of “Purinergic Signaling” continues to grow exponentially and has become the main focus of numerous research groups around the world. Families of cell surface receptors named purinoceptors were cloned and identified in most organs and cell types. At the same time, another field of research emerged with the initial studies of Drs. Beaudoin and Zimmermann, who showed that the availability of the purinoceptor agonists is regulated by cell surface enzymes. Twenty years ago, these world leaders launched the “International Symposium on Adenosine and Adenine Nucleotides”, which led to the integration of both fields of purinergic research and groundbreaking discoveries from our basic understanding of cell signaling to therapeutic applications.

The growing list of medical institutions and pharmaceutical companies working together to develop therapeutic approaches based on purinergic signaling is a testimony to the now acclaimed critical importance of these messengers for homeostasis. This book is dedicated to the brave scientists who faced adversity to give birth to this new and exciting era of scientific endeavors.

Preface

Chronic respiratory diseases are complex disorders generally driven by genetic mutations weakening the airways’ ability to respond appropriately to inhaled toxins or pathogens. In the case of cystic fibrosis (CF), functional mutations of the cystic fibrosis transmembrane resistance (CFTR) ion channel impair airway hydration, which fosters the formation of infected mucus plugs requiring mechanical stimulation for clearance. These patients suffer from a progressive and irreversible loss of lung function caused by overwhelming and damaging neutrophilic inflammatory responses. On the other hand, frequent exposure to the toxic components of cigarette smoke is considered the primary cause of chronic obstructive pulmonary disease (COPD). Subjects diagnosed with a1-antitrypsin deficiency are particularly vulnerable to lung destruction by elastases, and to the development of emphysema. Allergic asthma remains a serious challenge, as this disease incorporates multiple genetic factors and environmental stimuli. The patients experience recurrent episodes of breathless, wheezing, coughing and chest tightness triggered by airway hyperresponsiveness (AHR) to allergens. Over the years, the severity of AHR episodes increases, as chronic inflammatory responses to allergens induce extensive airway remodeling and narrowing of the airway passages. Recently, the discovery of significant overlap between the symptoms of these diseases raised serious concerns with respect to our ability to diagnose and treat the patients efficiently. The scientific community has been mandated to open new avenues for the development of discriminative diagnostic tools and customized therapies for these diseases. For decades, the most common diagnostic method used to differentiate asthmatics from COPD patients was AHR induced by inhalation of methacholine. Yet, a third of the COPD patients present significant AHR to this drug. As an alternative, The European Respiratory Society Task Force recently endorsed AHR measurements after inhalation of adenosine monophosphate (AMP) as a more specific diagnosis for asthma than methacholine. This finding spiked the interest of the scientific community for the signaling pathways mediating the effects of AMP in the airways of asthmatic patients. We now know that aerosolized AMP must, first, be dephosphorylated by a cell surface enzyme named ecto 50 -nucleotidase (CD73) in order to

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Preface

generate adenosine, a signaling molecule of the purinergic network. Upon binding to cell surface receptors, adenosine induces histamine release from mast cells, which initiates AHR in asthmatic patients. This narrow window was only the prelude to what would become a major endeavor to expose the purinergic regulation of airway defenses. In the past 15 years, the persistent exponential increase in the number of publications targeting the purinergic regulation of acute lung injury, mucociliary clearance, inflammation, wound healing, remodeling and lung fibrosis is a testimony to the extensive ramifications of this signaling network in chronic respiratory diseases. Clinical and fundamental studies support the existence of disease-specific aberrances in airway concentrations of the signaling molecules, as well as in expression levels of the receptors and related enzymes in lung tissues of asthmatic, COPD and CF patients. This book is a tribute to this exploding field of research, and promises to come for the development of specific diagnostic tools and therapies for the predominant chronic respiratory diseases affecting the general population. The term “Purinome” was recently ascribed to protein network mediating the effects of extracellular purines and pyrimidines. The composition of each “Purinome” is locally refined by different combinations of signaling molecules (ATP, ADP, Ap4A, adenosine), purinergic receptors, cell surface nucleotide-metabolizing enzymes (ectonucleotidases), and nucleoside/nucleotide channels or transporters. These protein clusters mediate tightly concerted actions invested in the maintenance of homeostasis and airway defenses. In chronic disorders, alterations of their global and dynamic equilibrium contribute to the appearance and/or propagation of pathological states. The vast majority of studies conducted on purinergic signaling are devoted to ATP and its metabolite, adenosine. In a nutshell, the local release of ATP constitutes an alarm signal perceived by surrounding cells through interaction with P2 cell surface receptors. This “communique´” informs the cells to take action according to their specific roles in the restoration of homeostasis. The cells’ alertness is maintained by the presence of local ectonucleotidases which promptly eliminate the ATP signal and restore receptiveness. The ingenuity of this communication network resides in the subsequent initiation of a negative feedback messenger from the dephosphorylation of ATP into adenosine. This signaling molecule binds P1 cell surface receptors to assist to restrain ATP-mediated responses and restore baseline activities. This sophisticated machinery works in concerts with other signaling networks, such as those supported by cytokines and growth factors, to maintain healthy lungs free of infection. However, chronic disorders associated with the maintenance of excess ATP or adenosine in the airways recruits surface receptors which induce or aggravate lung complications, including hyperinflammatory responses, tissue damage and airway remodeling leading to the loss of lung function. This book was meticulously designed to systematically introduce the reader to each element of purinergic network, followed by their integration into a mathematical model. Then, evidence is presented for significant aberrances in the regulation of the signaling molecules in chronic respiratory diseases. Three chapters are dedicated to the detailed description of the major respiratory and inflammatory

Preface

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functions regulated by purinergic signaling, and the aspects affected by chronic disorders. Finally, the reader is presented with the animal models and clinical applications currently used for the development of diagnostic and therapeutic approaches chronic respiratory diseases. As editor-in-chief, I wish to thank all contributors for their efforts and the staff of Springer-Verlagh for their professionalism in overseeing this publication. Chapel Hill, NC

Maryse Picher

Contents

1

Nucleotide Release by Airway Epithelia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eduardo R. Lazarowski, Juliana I. Sesma, Lucia Seminario, Charles R. Esther Jr., and Silvia M. Kreda

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2

Mechanisms Regulating Airway Nucleotides. . . . . . . . . . . . . . . . . . . . . . . . . . Maryse Picher

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Computational Model for the Regulation of Extracellular ATP and Adenosine in Airway Epithelia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Guilherme J.M. Garcia, Maryse Picher, Peiying Zuo, Seiko F. Okada, Eduardo R. Lazarowski, Brian Button, Richard C. Boucher, and Tim C. Elston

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4

Regulation of Airway Nucleotides in Chronic Lung Diseases. . . . . . . . Charles R. Esther Jr., Neil E. Alexis, and Maryse Picher

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5

Nucleotide-Mediated Airway Clearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Andreas Schmid, Lucy A. Clunes, Mathias Salathe, Pedro Verdugo, Paul Dietl, C. William Davis, and Robert Tarran

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6

Purinergic Signaling in Wound Healing and Airway Remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Albert van der Vliet and Peter F. Bove

7

Purinergic Regulation of Airway Inflammation . . . . . . . . . . . . . . . . . . . . . . Michael Koeppen, Francesco Di Virgilio, Eric T. Clambey, and Holger K. Eltzschig

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Contents

8

Animal Models of Airway Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Linda F. Thompson, Maryse Picher, and Michael R. Blackburn

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9

Therapeutic Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stephen Tilley, Jon Volmer, and Maryse Picher

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Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors

Neil E. Alexis Center for Environmental Medicine, University of North Carolina, Chapel Hill, NC 27599, USA Michael R. Blackburn Department of Biochemistry and Molecular Biology, University of Texas-Houston Medical School, Houston, TX, USA Richard C. Boucher Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA Peter F. Bove Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA Brian Button Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA Eric T. Clambey Department of Anesthesiology, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA Lucy A. Clunes Department of Pharmacology, School of Medicine, St George’s University, Grenada, West Indies

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Contributors

C. William Davis Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA Paul Dietl Institute of General Physiology, University of Ulm, Ulm 89081, Germany Francesco Di Virgilio Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy Tim C. Elston Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599, USA Holger K. Eltzschig Department of Anesthesiology, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA Charles R. Esther Jr. Pediatric Pulmonology, University of North Carolina, Chapel Hill, NC 27599, USA Guilherme J.M. Garcia Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599, USA Michael Koeppen Department of Anesthesiology, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA Silvia M. Kreda Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA Eduardo R. Lazarowski Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA Seiko F. Okada Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA Maryse Picher Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA

Contributors

Mathias Salathe Division of Pulmonary and Critical Care, University of Miami, Miami, FL 33136, USA Andreas Schmid Division of Pulmonary and Critical Care, University of Miami, Miami, FL 33136, USA Lucia Seminario Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA Juliana I. Sesma Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA Robert Tarran Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA Linda F. Thompson Oklahoma Medical Research Foundation, Oklahoma City, OK, USA Stephen Tilley Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of North Carolina, Chapel Hill, NC 29799, USA Albert van der Vliet Department of Pathology, University of Vermont, Burlington, VT, USA Pedro Verdugo Department of Bioengineering, University of Washington, Friday Harbor, WA 98195, USA Jon Volmer Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA Peiying Zuo Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599, USA

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Chapter 1

Nucleotide Release by Airway Epithelia Eduardo R. Lazarowski, Juliana I. Sesma, Lucia Seminario, Charles R. Esther Jr., and Silvia M. Kreda

Abstract The purinergic events regulating the airways’ innate defenses are initiated by the release of purines from the epithelium, which occurs constitutively and is enhanced by chemical or mechanical stimulation. While the external triggers have been reviewed exhaustively, this chapter focuses on current knowledge of the receptors and signaling cascades mediating nucleotide release. The list of secreted purines now includes ATP, ADP, AMP and nucleotide sugars, and involves at least three distinct mechanisms reflecting the complexity of airway epithelia. First, the constitutive mechanism involves ATP translocation to the ER/Golgi complex as energy source for protein folding, and fusion of Golgi-derived vesicles with the plasma membrane. Second, goblet cells package ATP with mucins into granules, which are discharged in response to P2Y2R activation and Ca2+-dependent signaling pathways. Finally, non-mucous cells support a regulated mechanism of ATP release involving protease activated receptor (PAR)-elicited G12/13 activation, leading to the RhoGEF-mediated exchange of GDP for GTP on RhoA, and cytoskeleton rearrangement. Together, these pathways provide fine tuning of epithelial responses regulated by purinergic signaling events. Keywords ATP release Airway epithelia Ectonucleotidase Thrombin Mucin

E.R. Lazarowski (*), J.I. Sesma, L. Seminario, and S.M. Kreda Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA e-mail: [email protected]; [email protected]; [email protected]; [email protected] C.R. Esther Jr. Pediatric Pulmonology, University of North Carolina, Chapel Hill, NC 27599, USA e-mail: [email protected]

M. Picher and R.C. Boucher (eds.), Purinergic Regulation of Respiratory Diseases, Subcellular Biochemistry 55, DOI 10.1007/978-94-007-1217-1_1, # Springer Science+Business Media B.V. 2011

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1.1 1.1.1

E.R. Lazarowski et al.

Introduction Definitions: The Purinergic Receptors

The actions of extracellular nucleotides are mediated by two families of receptors: P2X receptors (P2XRs) and P2Y receptors (P2YRs) (reviews: [1–3]). The P2XR family includes seven ATP-gated non-selective cation channels modulated by extracellular Ca2+, Na+, Mg2+ and H+. Their activation mobilizes intracellular Ca2+ and causes membrane depolarization. The P2YR family contains eight G protein-coupled receptors activated by ATP, UTP, ADP, UDP, and/or UDP-sugars (Table 1.1). The metabolite of ATP, adenosine, (ADO) mediates cellular responses through four G protein-coupled P1 receptors: A1Rs, A2ARs, A2BRs and A3Rs (reviews: [4–6]). Their agonist selectivity and signaling properties are summarized in Table 1.1. The purinergic network of human airway epithelia accommodates a discrete subset of purinergic receptors (review: [7]). The predominant nucleotide-sensing receptor in the airways is P2Y2R, which is activated to a similar extent and potency by ATP and UTP. Receptor occupation initiates the breakdown of plasma membrane phosphoinositides by phospholipase C, resulting in the formation of two secondary messengers: inositol 1,4,5 tris-phosphate (InsP3) and diacylglycerol (DAG). Whereas InsP3 triggers the Ca2+ release from intracellular stores, DAG activates protein kinase C (PKC)-dependent signaling cascades. These pathways promote the secretion of mucin (review: [8]), inhibition of the epithelial Na+ channel (ENaC) [8–14] and activation of the Ca2+-activated Cl channel (CaCC) recently identified as (ANO)/TMEM16 [15–17]. Human nasal epithelial cells also express P2Y6Rs, Table 1.1 Agonist selectivity and signaling pathways regulated by purinergic receptors Family Natural agonist Signaling P2X receptors ATP ATP-gated cation channel P2X1–P2X7 P2Y receptors P2Y1 P2Y2 P2Y4 P2Y6 P2Y11 P2Y12 P2Y13 P2Y14

ADP ATP ¼ UTP UTP UDP ATP ADP ADP UDP-sugars

Gq/PLC Gq/PLC Gq/PLC Gq/PLC Gq/PLC and Gs/AC Gi/AC inhibition Gi/AC inhibition Gi/AC inhibition and ERK activation

Adenosine receptors A1 A2A A2B A3

Adenosine Adenosine Adenosine Adenosine

Gi/AC inhibition Gs/AC activation Gs/AC activation Gi/AC inhibition

PLC phosphlipase C, AC adenylyl cyclase

1 Nucleotide Release by Airway Epithelia

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which induces CaCC activity with a maximal effect about one-half of P2Y2Rs [18]. A study conducted in human A549 alveolar and BEAS-2B bronchial epithelial cells supports the expression of P2Y14R in human airway epithelial cells, where it participates in highly specific inflammatory responses [19] (see Chap. 7 for details). The presence of P2XRs on airway epithelial surfaces has been documented in various species, including humans (reviews: [20]). Whereas normal bronchial epithelia express P2X4-6 receptors, primary cultures and cell lines from CF patients also express P2X7Rs [21]. The P2X4R was immunolocalized on the apical surface of rabbit tracheal epithelia, on the lower half of the cilia [22]. In the parenchyma, alveolar type 1 cells express P2X4R and P2X7R, the latter being associated with caveolae. The use of selective stable agonists and antagonists suggests that these P2XRs participate in the regulation of ion channels mediating airway hydration (see Chap. 5). However, their contribution remains highly debated because the affinity of the cloned receptors for ATP (EC50 ¼ 10–100 mM) [23] is several orders of magnitude above that of P2Y2Rs regulating the same cellular functions. On the other hand, P2XRs expressed on basolateral epithelial surfaces were found 100-fold more sensitive than those restricted to the apical surface [21]. Furthermore, Rettinger et al. published two detailed studies showing that receptor desensitization masks the nanomolar potency of ATP for P2X1Rs in Xenopus oocytes [24, 25]. These receptors also form heteromers, such as P2X4/6 and P2X4/7 [26], which generally raises their sensitivity to ATP (review: [27]). Clearly, much remains to be uncovered on the properties and roles of P2XRs in the respiratory system. On airway epithelial surfaces, the vast majority of the studies describing ADOmediated responses refer to the A2BR. This P1 receptor has been shown to stimulate the conductance of CFTR [28] and ciliary beat frequency [9]. The A2AR has been identified on human airway epithelia by RT-PCR and using selective agonists and antagonists, where it promotes healing [29, 30] and inflammatory responses [31, 32] (see Chaps. 6 and 7 for details). This summary identifies the purinergic receptors expressed on airway epithelial surfaces, which will facilitate the discussion in the following sections.

1.1.2

Nucleotide and Nucleoside Concentrations on Airway Surfaces

Mucociliary clearance (MCC) represents the first line of defense of the airways against infection, a mechanism known to be tightly regulated by purinergic events taking place within the airway surface liquid (ASL). The signals are initiated by the release of the signaling molecule, ATP, from airway epithelial cells. In vitro studies indicated that resting airway epithelia release ATP at a rate of 300–500 fmolmin1cm2 [33, 34]. The rate of ATP release is counterbalanced by the rate of surface metabolism mediated by ectonucleotidases. This dynamic flow of nucleotides maintains steady-state ASL ATP concentrations around 5–20 nM, which is below the activation threshold of P2Y2Rs [33–35]. However, the metabolism

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of surface ATP continuously generates the signaling molecule ADO, which is maintained at steady-state concentrations capable of activating A2BRs. Indeed, measurements of cyclic AMP production conducted in the presence or absence of ADO-degrading enzymes, revealed that A2BRs are tonically stimulated by endogenous ADO on resting airway epithelial cells in culture [33]. This hypothesis was further validated using sensitive assays for the quantification of ADO within the ASL. Endogenous nucleotides and nucleosides display low intrinsic fluorescence, and UV-based approaches require relatively high concentrations (>0.1 mM) for detection. This limitation was overcome using the chloroacetaldehyde derivatization technique, which quantitatively converts the adenine ring of ADO and its nucleotides into fluorescent 1,N 6-etheno(e)-adenine derivatives, i.e., e-ADO, e-AMP, e-ADP, and e-ATP. The (e)-adenyl purines are separated by HPLC and readily quantified with low nanomolar sensitivity. Using this technique, the concentration of ADO within the ASL of resting epithelial cultures was found in the 100–400 nM range [33], which is above the activation threshold of A2BRs. The recognition that the constitutive1 release of ATP results in physiologically relevant levels of ADO in the ASL suggests a mechanism for the purinergic control of basal MCC activities in resting epithelia, i.e., via A2BR activation. However, the rate of ATP release from airway epithelial cells is enhanced threefold by mechanical stresses, such as rhythmic shear and compression, experienced during tidal breathing or coughing. The resulting transient increase in ASL ATP concentration (100–1,000 nM) is capable of activating P2Y2Rs [36]. Functional studies (e.g. measurement of ASL volume regulation) demonstrated that ATP released from mechanically-stimulated epithelia mediates acute MCC responses via P2Y2R stimulation [36–39]. In summary, airway surfaces initiate purinergic events critical for MCC via ATP release and surface conversion into ADO by ectoenzymes, both inducing specific cell surface receptors and signaling pathways.

1.2

Mechanisms of Nucleotide Release

1.2.1

Constitutive Release of Nucleotides from the Secretory Pathway

1.2.1.1

Lessons Learned from the Yeast

The complex cellular composition of the airways, i.e., ciliated cells and mucinsecreting goblet cells, suggests that several mechanisms and pathways are involved in the release of nucleotides into the airways. Circumstantial evidence supports the involvement of both exocytosis and plasma membrane channels in the cellular 1

We use the term “constitutive” to refer to a release process that occurs in non-stimulated cells.


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Fig. 1.1 Pathways for airway epithelial nucleotide release. Several scenarios account for the constitutive and stimulated release of nucleotides from airway epithelial cells. (i) Nucleotides entering the Golgi lumen via specific transporters are released as a residual cargo product of the constitutive secretory pathway. The fungal venom brefeldin A (BFA) blocks this pathway by disrupting the Golgi apparatus. (ii) Secretory granules (e.g., mucin granules) containing ATP are competent for Ca2+-regulated exocytosis. Bafilomycin Ai (Baf ), an inhibitor of the H+-ATPase that loads ATP into specialized granules in secretory cells, impairs mucin secretion-associated ATP release in goblet cells. (iii) A plasma membrane connenxin/pannenxin-like channel effluxes cytosolic ATP out of the cells

release of nucleotides (Fig. 1.1), but unambiguous support for either vesicular or conductive mechanisms of release in epithelia has only recently begun to surface. Initial evidence for the involvement of the secretory pathway in the release of nucleotides from non-excitatory cells was provided by studies of glucose-dependent ATP release in the yeast Saccharomyces cerevisia [40, 41]. Glucose-dependent ATP release was enhanced in yeast overexpressing Mcd4p, a Golgi-resident transporter postulated to transports ATP to the lumen of the secretory pathway. This mechanism of ATP release was inhibited by the Golgi-disrupting agent brefeldin A [40, 41]. Since ATP release from yeast dramatically decreased when glucose was omitted from the extracellular medium, it was hypothesized that ATP release from these cells reflected an exocytotic mechanism initiated by the activation of a glucosesensing cell surface receptor [40]. The HPLC analysis of adenyl purines released by yeast indicated that, in the absence of glucose, extracellular AMP levels increased robustly as ATP levels decreased, maintaining the net mass of extracellular AMP/ADP/ATP unaffected by glucose [41]. Moreover, short-term removal of glucose from the extracellular medium did not affect the ability of yeast to secrete invertase, a marker of exocytosis [41]. The simplest interpretation of these data is that yeast releases nucleotides constitutively, via vesicle exocytosis, and that the

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energy balance of the cell determines the relative levels of ATP and AMP within the releasable vesicular pool. Additional evidence supporting the involvement of the secretory pathway in the release of nucleotides was generated from the observation that, in most cells, ATP release is accompanied by the release of UDP-sugars. Interest in UDP-sugar release followed the realization that one member of the P2YR family, the P2Y14R, is selectively and potently stimulated by UDP-glucose (UDP-Glc) and other UDPsugars [42]. A series of studies indicated that UDP-Glc is released from a number of cells, including simple cell systems such as yeast and highly differentiated airway epithelial cells [41, 43–45]. The fact that UDP-sugars participate in glycosylation reactions within the secretory pathway suggested that these molecules are released as cargo molecules during the export of glycoconjugates to the plasma membrane, i.e., via the constitutive pathway (Fig. 1.1). This hypothesis has been recently tested at the molecular level. It has been shown that UDP-Glc, UDP-N-acetylglucosamine (UDP-GlcNAc), and other UDP-sugars are synthesized in the cytosol and concentrated in the lumen of the endoplasmic reticulum (ER) and Golgi apparatus to serve as sugar donor substrates for glycosyltransferase reactions [46–48]. UDP, a byproduct of this process, is in turn hydrolyzed to UMP [48]. The entry of UDP-sugars to the ER/Golgi is mediated by ER/Golgi-resident UDP-sugar transporters, which use luminal UMP as antiporter substrate (Fig. 1.1). The UDP-sugar/UMP translocators are multitransmembrane helices that belong to the family of solute carrier SLC35 ER/Golgi nucleotide-sugar transporters [49]. Studies in yeast suggest that the cellular release of UDP-Glc is preceded by its uptake into the ER/Golgi. That is, UDP-Glc release was enhanced in yeast missing the putative nucleotide-sugar transporter encoding gene YMD8 (ymd8D cells), and this release was significantly reduced in ymd8D cells in which Yea4p or HUT1p, the ER-resident UDP-GlcNAc transporter and Golgi UDP-galactose transporter, respectively, were deleted. Thus, UDP-Glc release from yeast is influenced by the rates of ER/Golgi uptake of UDP-GlcNAc and UDP-galactose [41]. Diminished entry of UDP-Glc to the ER/Golgi of YEA4or HUT1-deficient cells likely reflected the decreased availability of the antiporter substrate UMP in the ER/Golgi lumen [48]. However, since Yea4p or HUT1p are not primarily UDP-Glc transporters, the interpretation of these results has remained speculative. The passage of UDP-sugar species through the ER/Golgi before their release was more conclusively established by correlating the amount of UDP-GlcNAc transporter in the ER/Golgi with the cellular release of its substrate, UDP-GlcNAc. By developing an assay which quantifies UDP-GlcNAc concentrations within the low nanomolar range, Sesma et al. demonstrated that yeast, in which the YEA4 gene (yea4D cells) was deleted, display reduced release of UDP-GlcNAc, the natural substrate of Yea4p [45]. Consistent with the role of Yea4p as major ER/Golgi UDP-GlcNAc transporter, the yeaD cells also displayed reduced synthesis of chitin, a glucosamine-rich cell wall component. The reduced UDP-GlcNAc release rate and chitin content of the yeaD cells were returned to normal by complementing the mutant strain with the WT YEA4 gene [45]. The most parsimonious conclusion


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from these studies is that, by facilitating UDP-GlcNAc entry into the ER/Golgi, Yea4p mediates the release of its UDP-sugar substrate from the secretory pathway.

1.2.1.2

Vectorial Release of Nucleotide Sugars from Resting Cells

The above-mentioned studies in yeast paved the way to investigate the role of Golgiderived vesicles in the constitutive release of nucleotide-sugars from airway epithelia. Three gene products, SLC35A3, SLC35B4 and SLC35D2, have been characterized as Golgi-resident UDP-sugar/UMP translocators in humans [49–51]. SLC35D2, also known as HFRC1, is the human homologue to the fruit fly fringe connection (Frc) transporter and may facilitate the transport of UDP-Glc and UDP-GlcNAc into the Golgi [50]. By controlling the entry of UDP-GlcNAc into the Golgi, HFRC1 modulates the surface expression of N-acetylglucosamine-rich glycoconjugates on airway epithelial cells [50]. Consistent with this concept, the human bronchial epithelial cell line 16HBE14o, stably overexpressing HFRC1, displayed enhanced cell surface immunoreactivity towards heparan sulfate and increased apical surface binding of WGA, a lectin that recognizes N-acetylglucosamine and sialic acids in airway epithelia. The overexpression of HFRC1 resulted in an increased rate of mucosal release of UDP-GlcNAc and, to a lesser extent, UDP-Glc release, relative to vectortransformed cells [45]. These studies provide compelling evidence for the contribution of Golgi-derived vesicles to the release of nucleotide-sugars from airway epithelial cells. Measurements of ATP and UDP-sugars release rates in polarized monolayers of human airway epithelial Calu-3 and 16HBE14o cells revealed that the nucleotides accumulate predominantly in the mucosal bath [33, 34, 45], suggesting a membrane specialization for nucleotide release. Similarly to UDP-sugars, ATP is translocated to the ER/Golgi via ATP/AMP antiporters, where it serves as an energy source for protein folding reactions [52, 53]. Therefore, the tonic release of ATP, UDP-sugars, as well as ADP and UDP, likely supports the continuous (“constitutive”) recycling of proteins and glycoconjugates on the apical plasma membrane and the exocytotic release of co-cargo nucleotides (Fig. 1.1).

1.2.2

Calcium-Promoted Release of Nucleotides from Goblet Cells

In addition to the above-described constitutive release, recent studies conducted with goblet-like airway epithelial cells indicated that ATP and UDP-sugars are released concomitantly with MUC5AC, a secretory mucin, during the Ca2+-regulated exocytosis of mucin granules. Electron microscope analysis of polarized Calu-3 cultures indicated that up to 40% of the cells within the monolayer express ~1 mm-diameter electron-translucent granules that resemble the mucin granules of airway mucous

8


goblet cells. Subsequent immunostaining and slot blot analysis revealed the presence of MUC5AC granules in the Calu-3 cultures, which were competent for Ca2+regulated exocytosis in the mucosal compartment. The Ca2+-promoted mucin secretion was accompanied by enhanced ATP release into the mucosal bath [44]. The kinetics of ATP release and mucin-granule secretion were similar and triggered by identical stimuli, suggesting that nucleotides are stored within, and released from, mucin granules in goblet cells [44]. Furthermore, bafilomycin A1, which depletes ATP storage granules [54], markedly impaired ionomycin-promoted ATP release from Calu-3 cells [44]. These data are consistent with the possibility that a vesicular/ granular ATP pool contributes to Ca2+-stimulated ATP release. An important corollary derived from these observations is that ATP/mucin-secreting goblet cells produce paracrine signals for P2Y2R-mediated mucin hydration and MCC activities. The Ca2+-dependent mechanism of ATP release identified in Calu-3 monolayers also stimulated the apical release of UDP-Glc [44], which has pathophysiological implications. High concentrations of UDP-Glc (100–1,000 nM) were recently detected in lung secretions from patients with cystic fibrosis (CF) or other goblet cell hyperplasic lung diseases [44, 45]. These concentrations were demonstrated to activate P2Y14Rs, which are highly expressed on inflammatory cells (lymphocytes and neutrophils) [55–57]. Therefore, the sustained accumulation of UDP-Glc in the ASL in conditions associated with goblet cell hyperplasia and/or mucin hypersecretion (asthma, chronic obstructive pulmonary disease or CF) may amplify inflammatory responses.

1.2.3

Regulated ATP Release from Non-Mucous Cells

1.2.3.1

Rho-Dependent Signaling Participates in ATP Release

The mechanisms supporting nucleotide release from non-mucous lung epithelial cells (i.e. ciliated or alveolar cells) remains unclear due to the scarcity of pharmacological approaches known to promote ATP release. While most studies rely on mechanical or osmotic stimuli, the biochemical signals mediating ATP release remain poorly defined. Recent data suggest that in A549 alveolar cells, hypotonicity-induced ATP release involves the mobilization of intracellular Ca2+ [58–60]. However, in human bronchial epithelial (HBE) cultures dominated by ciliated cells, the chelation of intracellular Ca2+ resulted in only a minor inhibition of hypotonic shockelicited ATP release [34]. However, Ca2+ mobilizing agents, such as ionomycin and UTP, promoted only minor release of ATP relative to hypotonic shock, in both HBE and A549 cells [34, 59–61]. These observations suggest that additional and/or alternative signals to Ca2+ mobilization are required upstream of ATP release in non-mucous cells. This notion appears not to be restricted to epithelial cells. For example, studies in 1321N1 human astrocytoma cells demonstrated that Ca2+ mobilization is not sufficient to reach maximal ATP release in response to


9

pharmacological stimulation. In these cells, the serine protease thrombin promoted a robust Ca2+-dependent nucleotide release response via activation of the proteaseactivated receptor-1 (PAR1). In contrast, carbachol induced a robust Ca2+ mobilization via the muscarinic receptor M3, but modest ATP release from these cells [62, 63]. A more recent study by Dubyak et al. indicated that G protein coupled receptor-elicited ATP release involves the activation of RhoA, consistent with the notion that PAR1 couples to G12/13 in addition to Gq [64]. Studies conducted on human umbilical vein endothelial cells also support a role for Rho in mechanical stress-induced ATP release. In these cells, both hypotonic stress- and lysophosphatidic acid (LPA)-induced ATP release were abolished by the Rho kinase inhibitor Y27632 [65]. Together, these studies suggested that Rho is a central regulator of ATP release in response to physical and pharmacological stimuli. The presence of Rho-stimulating thrombin receptors in lung epithelia [61, 66] provided a physiological approach to investigate the involvement of this GTPase in epithelial ATP release. Consistent with the above-mentioned studies on astrocytoma and endothelial cells [64, 65], thrombin promoted ATP release from A549 cells, which was accompanied by a rapid (1–3 min) activation of RhoA [61]. In contrast, activation of the Gq/PLC/Ca2+-mobilizing signaling pathway by UTP on P2Y2Rs resulted in negligible ATP release from these cells, and a delayed onset (>15 min) of Rho activation [61]. The involvement of Rho was supported by the observation that ATP release from thrombin-stimulated A549 and HBE cultures decreases in the presence of ROCK inhibitors [61]. These observations were further validated by mutational studies. The transfection of A549 cells with the RhoA mutant RhoA(T19N), which tightly binds RhoGEF (guanine exchange factor of Rho) without promoting downstream effector activation, markedly inhibited thrombin-elicited ATP release [61]. Similar results were obtained in cells transfected with p115RGS, a G12/13-inhibitory protein derived from the RGS (regulatory G-protein signaling) domain of p115-RhoGEF [67]. These studies suggest that thrombinpromoted ATP release on non-mucous lung epithelial cells encompasses PARelicited G12/13 activation, leading to the RhoGEF-mediated exchange of GDP for GTP on RhoA. How Rho/ROCK modulates ATP release remains unclear. However, the wellestablished control that ROCK exerts on the phosphorylation status of the myosin regulatory light chain (RLC) supports the attractive hypothesis that, by promoting cytoskeleton rearrangements, Rho facilitates the plasma membrane insertion and/or activation of an ATP channel, such as connexin/pannexin hemichannels.

1.2.3.2

Connexin/Pannexin Hemichannels: A Pathway for ATP Release

A gap junction, also named intercellular channel, allows direct communication between the cytosol of adjacent cells. In mammalian cells, the hemichannel contributed by each cell is formed by connexin or pannexin proteins (review: [68]). Recent evidence suggested that, unlike connexins, pannexins form single plasma membrane hemichannels (pannexons) rather than junctional channels [69].

10


Connexin and pannexin hemichannels have been proposed as diffusion pathways for ATP release under various experimental conditions [70, 71]. First, connexin hemichannels are highly dependent on extracellular Ca2+ ([Ca2+]ex), as they close in the presence of millimolar [Ca2+]ex and open when the [Ca2+]ex is lowered [72–74]. Exposure to low levels of extracellular divalent cations is a well-known procedure to potentiate or trigger the opening of connexin hemichannels leading to ATP release [75, 76]. In contrast, pannexin hemichannels are not gated by [Ca2+]ex [77]. However, they were both reported to conduct ATP [70, 71, 78]. Exposure of A549 cells to thrombin resulted in a robust ATP release response that was inhibited by the non-selective blockers of connexin and pannexin hemichannels, anandamide, flufenamic acid and carbenoxolone [61]. Thrombin-evoked ATP release was accompanied by an increase in cellular uptake of the hemichannel permeable reporter dye, propidium iodide, which was inhibited by the same hemichannel blockers. These results suggest that ATP release from thrombin-stimulated lung epithelial cells occurs via connexin or pannexin hemichannels. A recent study by Ransford and coworkers provides additional support to this hypothesis in airway epithelial cells. These authors reported that ATP release from hypotonic shockstimulated HBE cells was reduced in the presence of non-selective inhibitors of pannexin or after silencing pannexin-1 via shRNA [79]. An important unresolved issue regarding the putative involvement of connexin or pannexin in ATP release is the identification of the signaling pathways that regulate the gating of these hemichannels. Studies in Xenopus oocytes co-expressing pannenxin-1 with P2Y1Rs or P2Y2Rs suggested that pannenxin opens in response to intracellular Ca2+ elevation [80]. This mechanism, however, may not be relevant to airway epithelia since, as explained above, (i) the chelation of intracellular Ca2+ did not inhibit ATP release in hypotonic shock-stimulated HBE cells [34], and (ii) P2Y2R activation does not promote ATP release or propidium iodide uptake in lung epithelial cells [61]. Thus, assuming an involvement of pannexin and/or connexin in ATP release from airway epithelial cells, mechanisms other than intracellular Ca2+ mobilization should regulate the activity of these hemichannels. In this regard, it is noteworthy that the influx of propidium iodide uptake in thrombin-stimulated A549 cells was inhibited by Rho kinase inhibitors [61].

1.3

Conclusions and Future Directions

Nearly two decades have elapsed since the initial observation that extracellular nucleotides and nucleosides promote Cl secretion from airway epithelial cells [81, 82], the initial finding that led to the identification of P2Y2Rs and A2BRs as major purinergic receptors regulating MCC. An interrelated area of active research focuses on the mechanisms of nucleotide release, in particular, the identification of biochemical signals that transduce mechanical forces into ATP release. While clues are emerging, several questions remain unanswered: How do epithelial cells control the constitutive release of nucleotides? To what extent does the Golgi compartment


11

contribute to ATP release in resting cells and what is the contribution of conductive/ transporter mechanisms to this release? What are the molecular sensors for shear and compressive stress? How does mechanical stress impart a secondary messengermediated response? How do non-mucous lung epithelial cells release ATP? Finally, what is the contribution of connexin and pannexin hemichannels to ATP release in response to physiologically relevant stimuli and how are they regulated in epithelial cells? These questions are bound to unravel, yet, new avenues of research for the purinergic regulation of airway defenses. Acknowledgments We would like to thank Lisa Brown for editorial assistance of the manuscript. This work was supported by the National Institute of Health (NIH), National Heart, Lung, and Blood Institute (P01-HL034322) and the Cystic Fibrosis Foundation (CFF-SEMINA08FO).

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Chapter 2

Mechanisms Regulating Airway Nucleotides Maryse Picher

Abstract In the respiratory system, extracellular nucleotides and nucleosides serve as signaling molecules for a wide spectrum of biological functions regulating airway defenses against infection and toxic material. Their concentrations are controlled by a complex network of cell surface enzymes named ectonucleotidases. This highly integrated metabolic network combines the activities of three dephosphorylating ectonucleotidases, namely nucleoside triphosphate diphosphohydrolases (NTPDases), nucleotide pyrophosphatase/phosphodiesterases (NPPs) and alkaline phosphatases (APs). Extracellular nucleotides are also inter-converted by the transphosphorylating activities of ecto adenylate kinase (ectoAK) and nucleoside diphosphokinase (NDPK). Different cell types use specific combinations of ectonucleotidases to regulate local concentrations of P2 receptor agonists (ATP, UTP, ADP and UDP). In addition, they provide AMP for the activity of ecto 50 -nucleotidase (ecto 50 -NT; CD73), which produces the P1 receptor agonist: adenosine (ADO). Finally, mechanisms are in place to prevent the accumulation of airway ADO, namely adenosine deaminases and nucleoside transporters. This chapter reviews the properties of each enzyme and transporter, and the current knowledge on their distribution and regulation in the airways. Keywords Ectonucleotidase CD39 CD73 Ectoenzyme Alkaline phosphatase

2.1

Introduction

Ectoenzymes form a large diverse class of membrane proteins presenting their catalytic site to the extracellular environment. This chapter focuses on ectonucleotidases, which are specialized in the regulation of nucleotides and nucleosides.

M. Picher (*) Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA e-mail: [email protected]


17

18

M. Picher

The original nomenclature based on their biochemical properties (i.e. substrate specificity, optimum pH and selective inhibitors) was recently refined based on cloning studies. For this reason, several ectonucleotidases were given more than one name over the past 50 years, which considerably hindered literature search. This chapter introduces each ectoenzyme family involved in the purinergic regulation of airway defenses, and reconciles the various aliases encountered in the literature. The detailed description of their structure and crystallography has been reviewed elsewhere and is beyond the scope of this discussion. Our intent is to provide discriminative tools for the study of ectonucleotidases, and a detailed description of their distribution and regulation in the airways. There is increasing functional evidence that ectonucleotidases compete with P2 receptors for a limited pool of endogenously released nucleotides [1, 2]. In essence, the dephosphorylation of surface nucleotides terminates or limits the amplitude of the P2 receptor-mediated responses, either through autocrine or paracrine events. For instance, the expression level of NTPDase2 on rat portal myofibroblasts influences the proliferation of adjacent bile duct epithelial cells promoted by P2Y receptors [3]. The modulatory effects of ectonucleotidases will vary locally with the local sources of nucleotides and the substrate selectivity of the P2 receptors.

2.2

Three Families of ATP-Hydrolyzing Ectonucleotidases

The current literature recognizes three families of cell surface enzymes bound to the plasma membrane with their catalytic sites oriented toward the external milieu: the APs, the NTPDases and the NPPs (review: [4]). While APs have been known for >80 years, the other families were more recently defined following the establishment of molecular biology techniques which found associations to previously unrelated genes. This section provides a brief overview of this historical perspective, which will allow readers to search the literature efficiently using the various aliases assigned to each ectonucleotidase over time. This section focuses on the biochemical characteristics of each enzyme, their distribution in the airways and physiological roles. The impact of pathologies on their regulation will be discussed in Chap. 4.

2.2.1

Alkaline Phosphatases

Alkaline phosphatases (APs; EC 3.1.3.1) have been investigated >80 years in human tissues (review: [5]). For decades, most studies focused on the measurement of AP activity released in body fluids as a diagnostic marker of pathologies and disease severity (review: [6]). Their properties emerged in the 1970s as techniques became available for the investigation of plasma membrane glycoproteins. Based on selective inhibitors, sequence mapping and tissue distribution, four different isoenzymes have been identified (Table 2.1): intestinal AP (IAP), tissue non-specific AP

2 Mechanisms Regulating Airway Nucleotides Table 2.1 Mammalian alkaline phosphatases Protein name Aliases Gene (name; accession #) IAP, IALP Human: ALPI; NM_001631 Intestinal alkaline Mouse: Akp3; NM_007432 phosphatase

19

Inhibitors [IC50; mM] [7] Levamisole [6.8] L-p-bromotetramisole [>50] L-phenylalanine [1.0]

Non-specific alkaline phosphatase

NSAP, TNAP, TNSALP Liver-bonekidney AP

Human: ALPL; NM_000478 Mouse: Akp2; NM_007431

Levamisole [0.03] L-p-bromotetramisole [0.01] L-phenylalanine [30.0]

Placental alkaline phosphatase

PLAP, PLALP

Human: ALPP; NM_001632

Levamisole [1.7] L-p-bromotetramisole [0.3] L-phenylalanine [1.0]

Germ cell alkaline phosphatase

GCALP

Human: ALPP2; NM_031313 n.d.

Embryonic alkaline phosphatase

EAP

Mouse: Akp5; NM_007433

n.d.

(TNAP; NSAP), placental AP (PLAP) and germ-like AP (review: [8]). These enzymes are all encoded by chromosome 2, except for TNAP, which is derived from chromosome 1. They form homodimers bound to cell surfaces by a glycosyl phosphatidyl inositol (GPI) linkage limited to the outer leaflet of the bilayer, which confers lateral mobility [9]. It was suggested that GPI-linked proteins are targeted to the apical surface of polarized epithelia to participate in cell-cell interaction and signal transduction through rapid lateral redistribution. The cell surface density of APs is also locally influenced during infection by phosphatidyl inositol-specific phospholipases C or D released from bacteria to cleave the GPI linkage. Alternatively, the activation of bombesin receptors on nasal epithelia stimulated the release of AP [10], thereby supporting the existence of autocrine secretory mechanisms. These highly versatile enzymes catalyze the removal of terminal phosphate groups from a variety of natural substrates, including nucleotides (ATP, ADP, AMP) and glucose-6-phosphate, as well as pyrophosphate and pyridoxal-50 -phosphate both involved in bone mineralization. An aberrant regulation of pyridoxal-50 -phosphate has been shown to provoke epileptic seizures in patients with hypophosphatasia (review: [8]). In addition, APs regulate signaling events through dephosphorylation of the receptor agonists: lysophosphatidate and ceramide 1-phosphate [11]. Synthetic substrates (b-glycerophosphate, p-nitrophenyl phosphate and p-nitrophenyl thymidine 5monophosphate; PNP-TMP) are commonly used to measure AP activity in body fluids. The AP inhibitors classically presented in biochemical studies should be used with caution, as non-specific interactions occur at high concentrations [7, 12–15]. For instance, 10–20 mM levamisole is routinely used to identify TNAP, which is a sufficiently high concentration to recognize other AP isoforms (Table 2.1). On the other hand, many studies use 1 mM levamisole to exclude the activity of all APs in complex enzyme preparations, which is nearly a log lower than the IC50 of IAP.

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M. Picher

Since several APs are often co-expressed, the inhibition conditions should be validated at the mRNA level. The recent development of highly specific inhibitors for TNAP [16, 17] and PLAP [18] may provide the necessary tools to elucidate their respective roles. It is widely believed that APs constitute low affinity enzymes with limited ability to regulate low physiological nucleotide concentrations. This misconception was created because biochemists replicated the assay conditions originally used to quantify AP activity in body fluids. Accordingly, purified TNAP documented under alkaline conditions (pH 9–10) with PNP-TMP or ATP presented a lowaffinity catalytic site, with Km values in the 0.5–10.0 mM [19, 20]. More recently, Fortuna and collaborators compared the kinetic properties of purified TNAP under alkaline (pH 10.0) and physiological (pH 7.5) conditions. They confirmed that TNAP exhibits low affinity for ATP at alkaline pH (Km ¼ 8.3 mM), but also high-affinity at physiological pH (Km ¼ 5 mM) [21]. These data, confirmed by independent groups [22–24], indicate that TNAP has the capacity to regulate endogenous ATP concentrations under physiological conditions. Two members of the AP family have been identified in human airway epithelia: TNAP and PLAP. The surface activity and protein expression of TNAP has been detected on the apical surface of the epithelial barrier, from the trachea down to alveoli [25–27]. While it constitutes 85% of the AP activity measured in bronchoalveolar lavage (BAL) fluid, the soluble enzyme is mainly derived from surfactant secretion [28, 29]. In the alveolar region, immunolocalization and functional assays showed that TNAP is only expressed on the apical membrane and lamellar bodies of Type II pneumocytes [30, 31]. In contrast, PLAP is restricted to Type I pneumocytes [14, 25, 32]. Consequently, these APs are used as cell markers during the purification and culture of specific pneumocytes [33]. The biochemical properties of TNAP support a role in the purinergic regulation of airway defenses [26, 27] (Fig. 2.1). On polarized primary cultures of human bronchial epithelial cells, the enzyme exhibits a low-affinity (KM ¼ 717 mM) and a highaffinity (KM ¼ 6 mM) toward AMP at pH 7.5 [26], as reported for purified APs [23, 34]. However, this enzyme competes with CD73 for the conversion of AMP into ADO, and accounts only for 15% of the overall hydrolytic activity toward physiological ( ADP > ATP) [19]. This intrinsic property suggests that TNAP may play a significant role in the regulation of physiological ATP concentrations on airway surfaces. Other cell types encountered in the airway tissue express TNAP, including fibroblasts, lymphocytes [35], macrophages and neutrophils (reviews: [36, 37]). Ultracytochemical studies revealed that resting neutrophils restrict TNAP to intracellular membranes [38]. Cell stimulation by the bacterial products formylMet-Leu-Phe (fMLP) or lipopolysaccharide (LPS), or phorbol myristate acetate (PMA; protein kinase C activator), induces the mobilization of TNAP to the

2 Mechanisms Regulating Airway Nucleotides

21

Fig. 2.1 The “Purinome” of human airway epithelia. The apical surface concentrates proteins specialized in the regulation of ATP and adenosine levels named ectonucleotidases. Although the enzymatic network is positioned above the surface for clarity, all enzymes are membrane proteins. This network regulates the availability of ATP for P2Y2 receptors and adenosine for A2B receptors, which mediate various epithelial functions involved in airway defenses against infection: ciliary beating activity, airway hydration and inflammation

surface, particularly at sites of cell-to-cell interaction with endothelial or Type I alveolar cells [38, 39]. This focal surface distribution is consistent with the GPI linkage allowing fluent lateral movement within the outer leaflet of the surface membrane. The gram-negative bacteria invading the airways, including Pseudomonas aeruginosa, also exhibit AP activity. However, the enzyme is confined to the periplasmic region [40]. During normal growth, they slough off outer membrane vesicles with a fraction of their periplasmic content, including b-lactamase, hemolytic phospholipase C, AP and Cif (review: [41]). A recent study showed that these vesicles fuse with lipid rafts in the host plasma membrane and these virulence factors enter the cytoplasm via N-WASP–mediated actin trafficking, where they are distributed to specific subcellular locations to affect host cell biology [42]. This vesicular mode of delivery is particularly interesting given the postulated role of APs in the neutralization of LPS. This endotoxin elicits serious inflammatory reactions that may be lethal, particularly when the compound enters the circulation. The toxic moiety of LPS (lipid A) contains two phosphate groups highly conserved among gram-negative bacteria, and essential for their biological actions (review: [43]). The incubation of LPS with AP causes the formation of monophosphoryl lipid A, which is a weak macrophage activator of low toxicity, compared to diphosphoryl lipid A. The first demonstration of endotoxin dephosphorylation by APs was provided by Holst et al. [44], who used commercially available calf IAP to release inorganic phosphate from the Salmonella minnesota and Escherichia coli LPS. Several in vitro and in vivo studies showed that TNAP and IAP have the capacity to reduce the cell

22

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toxicity of LPS and associated inflammatory responses [45–48]. In 1997, Poelstra et al. published two studies showing that endogenous AP neutralizes E. coli LPS at physiological pH [45, 49]. Histochemical analysis of cryostat sections showed that intestinal and renal tissues dephosphorylate E. coli LPS. This enzymatic reaction was blocked by levamisole and the tissue distribution of the released phosphate corresponded to that generated by b-glycerophosphate. In addition, the local intradermal inflammatory reaction (PMN and macrophage infiltration) caused by systemic injection of LPS was strongly attenuated by pre-treatment of the endotoxin with exogenous AP. Since these initial studies, several groups demonstrated the importance of IAP for the maintenance of gut barrier integrity (review: [50]). The therapeutic potential of oral IAP was investigated in colitic rats, which responded by a significant reduction of colonic inflammation in terms of cytokines (TNFa, IL-1b, IL-6) and inflammatory cell influx, and a restoration of normal intestinal wall morphology [51]. A study comparing wild-type and IAP knockout mice showed that IAP also prevents bacterial invasion across the gut mucosal barrier promoted by ischemia-reperfusion [52]. Interestingly, patients with Crohn’s disease and ulcerative colitis exhibit low IAP mRNA levels in intestinal biopsies compared to healthy subjects [51]. These studies motivated the clinical trials currently conducted by AM-Pharma Inc with bovine IAP for the treatment of ulcerative colitis. In the airways, a similar role was proposed for TNAP expressed on airway surfaces against the gram-negative bacteria omnipresent in chronic airway diseases: P. aeruginosa. Commercially available TNAP was found capable of dephosphorylating P. aeruginosa LPS [53]. In addition, pre-treatment of LPS with TNAP markedly reduced the inflammatory reaction of cultured human bronchial epithelial cells, measured by the release of nitric oxide [53]. These data suggest that the uniform expression of TNAP along airway surfaces may constitute a defense mechanism to reduce bacterial toxicity.

2.2.2

Nucleoside Triphosphate Diphosphohydrolases

The existence of cell surface enzymes capable of dephosphorylating ATP and ADP at physiological pH has been documented for over five decades under various aliases: apyrase, ectoATPase, ectoADPase and ATP-diphosphohydrolase (reviews: [4, 54, 55]) (Table 2.2). The identity of these ectonucleotidases was clarified in the 1990s by studies conducted on purified preparations, which revealed that, in many cases, a single enzyme targeted both nucleotides. Given their broad substrate specificity toward purine and pyrimidine nucleotides, the general term nucleoside triphosphate diphosphohydrolase (NTPDase; EC 3.6.1.5) was adopted in 2000 by the scientific community [62]. Since then, the number of publications describing their properties and tissue distribution has grown exponentially. The identification of NTPDase1 as the lymphocyte cell activation antigen, CD39, was undoubtedly the most important event which raised the interest of the scientific community toward this family of ectonucleotidases.

Table 2.2 Mammalian nucleoside triphosphate diphosphohydrolases (NTPDases) Protein Gene HUMAN Gene Locus name Aliases mouse human Accession number NTPDase1 Apyrase, ENTPD1 10q24 NM_001776, U87967 ATPDase, Entpd1 NM_009848 CD39 NTPDase2 CD39L1, ectoENTPD2 9q34 NM_203468, AF144748 ATPase Entpd2 NM_009849, AY376711 NTPDase3 CD39L3, HB6 ENTPD3 3p21.3 NM_001248, AF034840 Entpd3 NM_178676, AY376710 NTPDase4 UDPase, ENTPD4 8p21 NM_004901, AF016032 LALP70 Entpd4 NM_026174 ENTPD5 14q24 NM_001249, AF039918 NTPDase5 CD39L4, ERUDPase, Entpd5 NM_007647, AJ238636 PCPH NTPDase6 CD39L2 ENTPD6 20p11.2 NM_001247, AY327581 Entpd6 NM_172117 NTPDase7 LALP1, ENTPD7 10q24 NM_020354, AF269255 Entpd7 NM_053103, AF288221 NTPDase8 Liver canalicular, ENTPD8 9q34 NM_198585, AY430414 ecto-ATPase, Entpd8 NM_028093, AY364442 hATPDase Km (ATP) mM 10–17

70–400 75

93

ATP/ADP ratio 1.1–1.3

7.2–22 3.6 – 4.2

1.1

[61]

[58]

[58 – 60]

References [56, 57]

2 Mechanisms Regulating Airway Nucleotides 23

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M. Picher

Thus far, eight NTPDases have been cloned and functionally characterized using purified protein preparations and heterologous expression (Table 2.2). They share highly conserved sequence domains named “conserved apyrase regions” which define their catalytic properties. Among them, NTPDase1, 2, 3 and 8 are localized in the plasma membrane with their catalytic site facing the extracellular milieu. They are anchored by two transmembrane domains and readily form homooligomers (dimers, trimers and tetramers), which enhances their activity [63]. Other members of this family are sequestered in the Golgi (NTPDase4) and endoplasmic reticulum (NTPDase7), with their catalytic site facing the lumen of the organelle. In the Golgi, NTPDase4 preferentially uses UDP to assist in the import of nucleotide sugars. Finally, the NTPDase5 and 6 isoforms are secreted as soluble enzymes following heterologous expression. The cell surface NTPDases are functionally differentiated based on substrate preferences. While a single catalytic site supports the hydrolysis of triphosphates and diphosphates, each enzyme hydrolyses these two classes of substrates with distinct efficiencies, which influences the transient accumulation of active intermediates (Table 2.2). For instance, NTPDase1 hydrolyses ATP and ADP at similar rates, resulting in little accumulation of ADP for the activation of P2Y1 or P2Y12 receptors. In contrast, NTPDase2 exhibits a strong preference for ATP, allowing ADP to accumulate for an extended period of time before being converted to AMP. NTPDase3 and 8 generate intermediate metabolic profiles. The readers are referred to the original manuscript of Sevigny et al. [58] for a detailed review of the metabolic properties of human and murine cell surface NTPDases. The physiological consequences of these distinct properties on cell signaling depend on the composition of the local “Purinome” in terms of ectonucleotidases and receptors. A considerable number of compounds are known to inhibit the cell surface NTPDases, although most of them also interfere with P2 receptor activation [64, 65] (Table 2.3). Azide was originally described as an inhibitor of the ATPdiphosphohydrolase [74] later identified as NTPDase1 (CD39) [75, 76]. Recent studies showed that azide inhibits NTPDase1 [66] and NTPDase3 [67], but not NTPDase2 and 8 [68, 69], TNAP [77] or NPPs [78]. However, azide is not suited for animal protocols or prolonged exposures, as the compound induces necrotic cell death [79]. The stable analogue of ATP, 6-N,N-diethyl-D-b,g-dibromomethylene ATP (ARL67156; FPL67156), was the first compound developed to inhibit ectonucleotidases without affecting P2 receptors [80]. In fact, ARL67156 exhibits a weak antagonistic effect against P2X receptors and a weak agonist effect on P2Y receptors. This compounds was used to uncover the purinergic elements involved in complex signaling events, such as neurotransmission in vivo [81]. However, recent studies showed that this weak competitive inhibitor is only effective in the presence of endogenous levels of nucleotides (1,000 – 50% 112.0 Unknown [70] POM-1 2.6 – n.a. 28.8 – n.a. 3.3 – n.a. Unknown [71] a mabNTPDase3 No No 35 ngml1 n.a. – 0% [72] – 70% PSB-06126 No No 1.5 – 75% Unknown [73] PSB-069 15.7 – 100% 18 – 100% 16.4 – 100% Unknown [73] a This value corresponds to the concentration causing 50% inhibition (IC50)

a capillary inlet using membrane preparations of transfected CHO cells, followed by electrophoretic separation of the reaction products. While this method allows for rapid screening of potent inhibitors, the substrate affinities (Michaelis constants; KM) of NTPDase1, 2 and 3 in this expression system were 76, 203, and 311 mM, respectively. These KM values are considerably higher than reported by classical test tube assays with respect to NTPDase1 and NTPDase3 (Table 2.2), suggesting that the inhibitory constants (Ki) of their inhibitors are also overestimated by this method. Nonetheless, this approach remains extremely useful for the identification of selective NTPDase inhibitors. For instance, the P2 receptor antagonists reactive blue 2, suramin and pyridoxalphosphate-6-azophenyl-20 ,40 -disulfonic acid (PPADS) inhibit NTPDase3 at least 15-fold more efficiently than NTPDase1. The capillary method also led to the identification of a novel class of NTPDase inhibitors, the polyoxometalates [71]. Among them, polyoxotungstate (POM-1) is very attractive for animal studies because of its high stability at physiological pH (>14 days). Nonetheless, it is important keep in mind that this compound is equally potent against NTPDase1 and NTPDase3, which are inhibited ten times more efficiently than NTPDase2. The usefulness of POM-1 would be limited to tissues or cell types expressing either NTPDase1 or NTPDase3. While these polyoxometalates were tested on rat NTPDases [71], POM-1 was also found effective in reducing extracellular ATP metabolism in mice [83, 84]. This technique was also used to evaluate the potential of anthraquinone derivatives of reactive blue 2 [73]. This study identified 1-amino-2-sulfo-4-(1-naphthylamino) anthraquinone as a potent and selective inhibitor of NTPDase3 (Ki ¼ 1.5 mM) having no effect on NTPDase1 and 2 [73]. While these drugs were not tested against NTPDase8, this is a minor complication since the distribution of this isoform is limited to the liver [69, 85]. Sevigny and collaborators recently developed the first monoclonal antibody against human NTPDase3, as shown by immunolocalization of the enzyme in the human pancreas [72]. More importantly, this antibody also selectively inhibits the activity of NTPDase3 [72]. The advantage over synthetic drugs is the high

26

M. Picher

specificity and the lack of side-effects. Incidentally, POM-1 was recently found to inhibit central synaptic transmission in cerebral and hippocampal slices by a mechanism which does not involve NTPDase inhibition [86]. Ultimately, the choice of inhibitor will depend on whether the goal is to prevent the degradation of extracellular nucleotides or to target a specific NTPDase. In the latter, the most appropriate inhibitor would be dictated by the NTPDases co-expressed by the target cell or tissue. Unfortunately, in the case of NTPDase2 and NTPDase8, this field of research still relies on silencing protocols and genetically manipulated animal models to identify their physiological roles. In the respiratory system, the presence of NTPDases was initially inferred by Northern blot analysis of human total lung RNA, which only presented a signal for NTPDase1 [75, 87, 88]. Since then, the expression of NTPDase1 in the lung was confirmed by immunohistochemistry in murine [89], bovine [90] and human [91] tissue, specifically on the endothelial and alveolar epithelial cells. More recently, NTPDase1 and 3 were immunolocalized on the epithelial barrier extending the entire respiratory system, except in the alveolar region for NTPDase3 [91]. These enzymes also exhibited unique epithelial polarities which shifted in opposite directions from the proximal to distal airways. In the tracheo-bronchial area, NTPDase1 was limited to the apical surface, whereas NTPDase3 labeled basolateral surfaces and basal cells. In the distal airways, NTPDase1 was detected on the basolateral surface, whereas NTPDase3 expression was bilateral. Such polarity shifts along the airways have been reported for other proteins, including the water channel, aquaporin3 [92]. The mechanisms regulating the epithelial polarities of the NTPDases in the airways have not been elucidated. However, LPS caused the relocation of liver canalicular NTPDase activity from the apical to basolateral surfaces [93], an enzyme recently identified as NTPDase8 [69]. The fact that thousands of LPS particles are inhaled every day and deposited mainly in the distal airways could explain the gradual polarity shifts of the two NTPDases. The functional expression of NTPDase1 and 3 on airway surfaces was demonstrated by selective assays conducted on polarized primary cultures of human bronchial epithelial cells [91]. Fausther and collaborators developed an assay strategy to differentiate the activities of these two azide-sensitive NTPDases [91]. The same culture wells were assayed in the presence of the monoclonal antibodies against NTPDase3 that selectively inhibit this enzyme activity, then assayed in the presence of azide. The use of a wide range of nucleotide concentrations showed that NTPDase1 predominates (40%) under physiological conditions (80% of the total ectoAMPase activity toward physiological ( ADP > ATP) and CF patients (AMP >> ATP > ADO > ADP) [44]. The sputum plugs, collected from the central airways, constitute micro-domains for the regulation of airway purines due to the absence of CD73 activity on the neutrophils. On the other hand, the BAL fluid of CF patients is expected to contain higher ADO levels than the sputum plugs because this collection method samples peripheral airways. The close proximity of epithelial CD73 in small airways, coupled to less densely packed neutrophils, would allow for a more efficient dephosphorylation of AMP into ADO. Incidentally, Esther et al. reported that CF patients maintain ADO levels twofold lower in sputum than in the small airways sampled by BAL procedure [44]. Hence, one should expect differences in purine composition between sputum plugs and BAL fluid in the case of CF patients because of the neutrophilic airway inflammation. In this regard, it is important to mention the extreme variability in CD73 expression among cells recruited to the airways. Whereas macrophages efficiently mediate the dephosphorylation of AMP into ADO, neutrophils and eosinophils depend on the epithelium [45, 46]. Therefore, differences in sputum ADO levels among diseases should correlate positively with the percent macrophages in the order: asthma > COPD > CF (reviews: [42, 47]), unless the expression level of CD73 is affected under pathological conditions. Blackburn et al. demonstrated that chronic respiratory diseases, indeed, modify the expression of CD73 [48]. In COPD and IPF patients, the capacity of lung homogenate to accumulate extracellular ADO increased with CD73 mRNA levels and with disease severity. This up-regulating effect on CD73 was also detected

4 Regulation of Airway Nucleotides in Chronic Lung Diseases

83

in vitro in HBE cultures of CF patients, which exhibited more than threefold higher surface activity and mRNA levels [49]. These authors also documented the impact of these diseases on ADA1, which is responsible for the elimination of lung and airway ADO [50]. In contrast to CD73, the expression of ADA1 was not affected by COPD or IPF, but its activity decreased with disease severity. Unfortunately, they used pentostatin to differentiate ADA1, from ADA2, activity at a concentration (1 mM) that also inhibits ADA2 (Ki ¼ 109 M) [51]. The latter isoform, derived from monocytes/macrophages, has recently been identified as a growth factor with residual ADA activity only detected with high micromolar ADO [52]. Since lung homogenate likely contains ADA2 from monocytes and macrophages, the impact of these diseases on ADA1 remains undetermined. To date, the most selective inhibitor of ADA1 remains EHNA (see Chap. 2 for details). Nonetheless, platelets collected from rats following cigarette smoke exposure exhibited the same metabolic aberrance. This major determinant of COPD accelerated the production of circulating ADO by CD73 activity, while reducing its elimination rate by ADA1 [53]. In this case, an ADA1 inhibitor was not necessary since platelets do not express ADA2. These studies suggest that high airway ADO levels, in COPD patients [27], result from the combined effects of high CD73, and low ADA1, activities in lung tissue.

4.3.2

Epithelial Polarity Shifts and Expression Gradients

A phenomenal breakthrough in our understanding of purinergic regulation in the respiratory system was provided by immunolocalization of CD39 and NTPDase3 [37]. In normal human tissue, close examination of the epithelial barrier revealed a gradual and opposite shift in the epithelial polarity of the two ectonucleotidases toward the alveolar region. Whereas bronchial epithelia confine CD39 activity to their apical surface, the enzyme is expressed on the basolateral of the small airways. In contrast, the polarity of NTPDase3 shifts from basolateral surfaces in upper airways to a bilateral distribution in the distal airways. This behavior is not restricted to the ectonucleotidases, as the water channel, aquaporin3, exhibits a polarity shift in the basolateral-to-apical direction toward the alveoli [54]. This is a powerful revelation, as it modifies our perception of purinergic signaling to account for regional differences in network composition along the barrier. A consequence of these polarity shifts is the existence of expression gradients on the apical surface of the epithelial barrier, which concentrate CD39 in the upper airways and NTPDase3 in the peripheral airways. Interestingly, earlier studies reported similar expression gradients for the ectonucleotidases TNAP and CD73 [55]. From these studies emerges a fascinating pattern in the distribution of the ectonucleotidases, as high-affinity enzymes (CD39 and CD73) are concentrated in the upper airways, whereas low-affinity enzymes (NTPDase3 and TNAP) are concentrated in the distal airways. It was surprising to see NTPDase3 expression end so abruptly before the terminal bronchioles [37]. In the alveoli, high ATP concentrations appear to be regulated by placental alkaline phosphatase (PLAP) on the Type 1 [56–58], and TNAP on the Type 2 (surfactant-secreting) epithelial cells [59, 60].

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C.R. Esther Jr. et al.

This spatial organization predicts that large airways are more efficient at regulating physiological nucleotide levels, whereas small airways are specialized in the removal of excess purines in areas of intense mucin/surfactant and nucleotide release [61–63] (see Chap. 2 for details). The factors maintaining region specific epithelial polarities for ectonucleotidases along airway surfaces were identified by immunolocalization of CD39 and NTPDase3 in airway tissue from CF patients and, in vitro studies designed to reconstitute the disease on airway epithelial cultures [37]. In the chronic respiratory disease, both enzymes adopt the epithelial polarity of normal distal airways throughout the respiratory tract. Whereas HBE cultures from CF patients presented the epithelial polarity of normal HBE cultures, chronic exposure to SMM established the in vivo polarity of CD39 and NTPDase3 typical of the CF diseases in, both, normal and CF cultures. These experiments demonstrated that the relocation of ectonucleotidases is part of the airways’ responses to infection. The fact that distal airways from healthy subjects adopt a similar epithelial polarity of the enzymes as those of CF patients is consistent with the notion that these small diameter airways are particularly susceptible to particle deposition. The hundreds of bacterial products inhaled every day are likely to maintain these areas in state of danger awareness which warrants the positioning of CD39 and NTPDase3 to strategic defense positions. The role of CD39 on the basolateral side of the epithelial barrier could be to reduce neutrophil entry into the airways in response to ATP released locally from leukocytes gathering in the interstitium (see Chap. 7 for details). In CF, the overwhelming neutrophilic airway inflammation [42] may result from a failure to regulate their recruitment due to CD39 down-regulation. On the apical surface, the up-regulation and mobilization of the high-capacity NTPDase3 could protect against the deleterious effects of high micromolar nucleotides in areas of intense bacterial killing or tissue damage, which are known to induce apoptosis through P2X7Rs (review: [64]). Whereas P2X7Rs are not detected in normal HBE cells, they are functionally expressed on CF airway epithelia [65]. It is also noteworthy that NTPDase3 is preferentially expressed on mucous cells, which co-release mucin and nucleotides [61, 62]. Since P2Y2R activation induces mucin secretion (review: [66]), NTPDase3 could locally regulate the amplification cycles of P2Y2R-mediated mucin and ATP release.

4.3.3

Interactive Effects of Hypoxia and Oxidative Stress

Respiratory insufficiency is considered among the most serious complications of patients with obstructive lung diseases. The major causes include lung destruction for COPD or emphysema, airway narrowing and bronchoconstriction for asthma. As for CF, the patients’ inability to clear the viscoelastic mucopurulent material invariably leads to airway obstruction by hypoxic mucus plugs, which often requires mechanical stimulation for clearance (review: [67]). In all diseases, the


85

sporadic and intermittent low oxygen tension experienced by the epithelial surfaces could contribute to the aberrant airway purine concentrations. Oxygen deprivation is well known to induce the coordinated up-regulation of CD39, CD73 and A2BRs (review: [68]). Intestinal epithelial cells react to hypoxia by a fivefold increase in the expression of both enzymes [69]. In blood vessels, the resulting excess ADO reduces inflammatory responses (i.e. leukocyte adhesion) and vascular leakage (review: [68]). Eventually, ADA1 activity increases, as a delayed response, to restore normal ADO levels until CD39 and CD73 expression return to baseline. The fact that COPD and asthmatic patients, but not CF patients, exhibit high airway ADO levels, suggests disease specificity in the impact of hypoxia. Airway epithelial cells possess all the necessary machinery to enhance purinergic signaling in areas of low oxygen tension. Hypoxia influences gene expression through activation of the transcription factor hypoxia-inducible factor-1a (HIF-1a). Low oxygen causes the stabilization of a-subunit and its dimerization with HIF-1, which triggers the translocation of HIF-1a to the nucleus to stimulate the expression of barrier-protective elements, including CD73 [69]. Nitric oxide (NO) is emerging as a positive regulator of HIF-1a-mediated responses. In A549 alveolar epithelial cells, respiratory syncytial virus raised CD73 expression through HIF1a stabilization which required NO secretion [70, 71]. Actually, so many factors activate inducible NO synthase (iNOS) in human airway epithelia (viruses, bacteria and cytokines) that BAL fluid levels of NO are elevated in chronic inflammatory diseases, like asthma [72]. Surprisingly, CF patients exhibit low iNOS and airway NO levels, compared to healthy subjects [73, 74]. In CF, a failure to initiate NO-dependent CD73 up-regulation in response to a pathogen could impair the ADO-mediated mucociliary clearance and allow excessive inflammatory responses (see Chaps. 5 and 7 for details). Colgan et al. demonstrated that hypoxia uses a different transcription factor to up-regulate CD39 [75]. On endothelial cells, both, site-directed mutagenesis and antisense oligonucleotides targeting the SP1 transcription factor caused a complete loss of hypoxia inducibility by CD39 [75]. In human airway epithelia, this transcription factor is being targeted for the suppression of excessive inflammatory responses in CF patients [76]. If SP1 is proven important for CD39 expression in these cells, this therapeutic approach could aggravate its down-regulation in CF patients [37]. The general consensus that hypoxia accelerates the conversion of airway ATP into ADO is consistent with chronic respiratory diseases known to maintain high levels of the signaling molecule, such as COPD and asthma, but not with CF. On the other hand, chronic and intermittent hypoxia is associated with oxidative stress (review: [77]). This airway complication is amplified, in CF, by functional mutations in the Cftr gene which maintain the epithelia under oxidative stress [78]. Airway epithelia respond to pathogens and cytokines by a small oxidative burst to assist in bacterial killing, which is ended by scavengers and antioxidant enzymes (review: [79]). Chronic conditions, like intermittent hypoxia, tilt the “reductionoxidation” (redox) balance toward oxidants, which activate elements of various signaling cascades affecting gene expression. In a murine model of asthma, the

86


antioxidant L-2-oxothiazolidine-4-carboxylic acid inhibited the translocation of NFkB to the nucleus, which reduced the expression of pro-inflammatory cytokines and adhesion molecules [80]. Oxidative stress also impairs the activities of surface proteins through lipid peroxidation, which operates independently from gene expression. Numerous in vivo studies suggest that chronic oxidative stress may be responsible for the suppression of CD39, and up-regulation of CD73, detected in the airways of CF patients [37, 49]. For instance, the glomerular CD39 activity of kidney grafts is reduced in patients with delayed diuresis after renal transplant [81] or renal xenotransplant [82]. In renal biopsies from patients with chronic allograft neuropathy [83], or after ischemia-reperfusion [81], a reduction in CD39 activity occurred concomitant with an increase in CD73 activity. The involvement of oxidative stress was demonstrated using activated neutrophils, which reduced ectoADPase activity in perfused rat kidneys by a mechanism sensitive to antioxidant enzymes [84]. On endothelial cells, CD39 activity was inhibited by an oxidation system (xanthine oxidase/xanthine), which was prevented in the presence of ROS scavengers [85]. Furthermore, the inhibitory effect of TNFa on CD39 activity was prevented by antioxidants [86]. These data suggest that oxidative stress reduces the activity and mRNA expression of CD39 in chronic respiratory diseases. Of note, the intra-tracheal instillation of LPS or influenza was reported to raise the mRNA expression of CD39 in the total lung [87, 88]. However, airway infection recruits immune and inflammatory cells, most of which express CD39. Hence, analysis of gene expression in total lung does not distinguish the responses of recruited and resident cells. In the airways of CF patients, immunolocalization showed that CD39 is down-regulated in the epithelial barrier, but up-regulated in inflammatory cells and fibroblasts [37]. The up-regulation of CD39 on CF airway neutrophils was confirmed at the mRNA level [89]. The selective decrease in CD39 activity on the epithelial barrier of CF patients suffering from the damaging effects of an overwhelming airway neutrophilia supports a protective role against leukocyte adhesion and/or transmigration, as documented extensively for the endothelial barrier (review: [46]) (see Chap. 7 for details).

4.3.4

Amplification Loops for ATP and Adenosine Formation

The discovery that most chronic respiratory diseases are characterized by elevated ATP and/or ADO concentrations in the airway secretions supports the existence of amplification pathways for the production of extracellular ATP and ADO. The first study exploring this signaling mechanism was conducted on skin cancer cells by periodic treatment with exogenous ATP over the course of 2 weeks [90]. Time-course analysis revealed the coordinated up-regulation of ectoATPase, ectoADPase and ectoAMPase activities [90]. More recent studies targeted the response of


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specific ectonucleotidases to stable agonists of the purinergic receptors. Narravula et al. demonstrated that the stable ADO analogue, 5-N-ethylcarboxamidoadenosine (NECA), caused a time- and dose-dependent increase in the expression and surface activity of CD73 [91]. The promoter region of the Cd39 and Cd73 contains a cAMPresponsive element (CRE) which allows A2BR activation to raise gene expression through the formation of the cAMP secondary messenger [92, 93]. Since airway epithelial surfaces express CD73 and A2BRs (review: [66]), the excess ADO detected in the airways of COPD patients and steroid naı¨ve asthmatics [12, 21, 27] could be, in part, maintained by this amplification system. There is also evidence that high airway ATP and ADO levels are maintained by PKC-dependent signaling pathways. A survey of the literature generated over 40 papers which support the widespread up-regulating effect of PKC activation on CD73 in the heart, endothelial and epithelial cells [94–96]. Other studies revealed the additive up-regulating effects of cAMP and PKC on the up-regulation of NPPs and TNAP [97, 98]. Given the fact that P2Y2R activation induces cellular responses through PKC on airway epithelial and inflammatory cells, this signaling cascade could also allow excess ATP to promote ADO accumulation by the coordinated up-regulation of ectonucleotidases. This amplification system for airway ADO would be particularly problematic for respiratory diseases combining excess ATP and ADO, like COPD, since A2BRs and P2Y2Rs appear to amplify each others responses.

4.4

Conclusions

The abovementioned information suggests that chronic respiratory diseases are generally associated with the accumulation of ATP in the airway secretions, likely from bacterial killing and the intense purinergic signaling taking place on the epithelial and inflammatory cells. However, a consensus could not be reached for airway ADO, as high levels are reported in asthmatic and COPD patients, but not in CF patients. Instead, CF is associated with high levels of AMP, the precursor of ADO. This discrepancy raised the possibility that chronic diseases also affect the ectonucleotidases regulating the sequential dephosphorylation of released nucleotides into ADO. Collectively, the scientific evidence provided in this chapter exposes the elegance and receptivity of the enzymatic network regulating ATP and ADO in the respiratory tract. The alarm signal triggered by a pathogen on the epithelial barrier, not only affects the activity and expression of the ectonucleotidases, but causes their relocation to strategic positions to regulate the stimulatory effects of ATP on mucin secretion and inflammatory responses. Chronic respiratory diseases, characterized by severe neutrophilia, may be trapped in an amplification pattern of oxidative stress which reduces CD39 expression and facilitates neutrophil recruitment to the airways.

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Chapter 5

Nucleotide-Mediated Airway Clearance Andreas Schmid, Lucy A. Clunes, Mathias Salathe, Pedro Verdugo, Paul Dietl, C. William Davis, and Robert Tarran

Abstract A thin layer of airway surface liquid (ASL) lines the entire surface of the lung and is the first point of contact between the lung and the environment. Surfactants contained within this layer are secreted in the alveolar region and are required to maintain a low surface tension and to prevent alveolar collapse. Mucins are secreted into the ASL throughout the respiratory tract and serve to intercept inhaled pathogens, allergens and toxins. Their removal by mucociliary clearance (MCC) is facilitated by cilia beating and hydration of the ASL by active ion transport. Throughout the lung, secretion, ion transport and cilia beating are under purinergic control. Pulmonary epithelia release ATP into the ASL which acts in an autocrine fashion on P2Y2 (ATP) receptors. The enzymatic network describes in Chap. 2 then mounts a secondary wave of signaling by surface conversion of ATP into adenosine (ADO), which induces A2B (ADO) receptor-mediated responses. This chapter offers a comprehensive description of MCC and the extensive ramifications of the purinergic signaling network on pulmonary surfaces.

A. Schmid and M. Salathe Division of Pulmonary and Critical Care, University of Miami, Miami, FL 33136, USA e-mail: [email protected]; [email protected] L.A. Clunes Department of Pharmacology, School of Medicine, St George’s University, Grenada, West Indies e-mail: [email protected] P. Verdugo Department of Bioengineering, University of Washington, Friday Harbor, WA 98195, USA e-mail: [email protected] P. Dietl Institute of General Physiology, University of Ulm, Ulm 89081, Germany e-mail: [email protected] C.W. Davis and R. Tarran (*) Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA e-mail: [email protected]; [email protected]


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Keywords CFTR Mucociliary clearance Mucin secretion P2Y2 receptors A2B receptors

5.1

Introduction

The respiratory tract serves as a conduit to bring air to the alveoli, sterilizing and humidifying it in the process, whilst the alveoli act as the primary site for gas exchange. Mucociliary clearance from the respiratory tract constitutes the first line of defense against airway infection (1): Mucus lining the airways traps inhaled pathogens and propels them towards the mouth where they can be expectorated. Transport of this mucus requires regulated mucin secretion, maintenance of cilia beating and adequate hydration of airway surfaces by ion transport processes. Mucin hypersecretion and/or abnormal regulation of ion transport leads to concentration/dehydration of mucus, plugging of the airways and chronic infections. Based on the actions of the airways, the alveolar surfaces are normally free from infection. However, due to their minuteness, alveoli would collapse if their surface tension was not reduced by surfactant, a small, hydrophobic lipid-like molecule. Despite these different functions, all regions of the airways are under purinergic regulation. In section 5.2 we shall discuss how ASL secretion is mediated via CFTR, CaCC and ENaC. In sections 5.3 and 5.4 we shall discuss the regulation of mucin and surfactant secretion respectively and in section 5.5 we shall discuss how ciliary beating is modulated.

5.2 5.2.1

Airway Hydration Definitions

ASL height (volume) is regulated by ion channels that control the mass of salt and water on airway surfaces [1]. The regulation of pulmonary hydration is a highly adaptable mechanism exhibiting dramatic changes before and after birth. In the fetal lung, airway Cl secretion dominates [2, 3] and this fluid secretion drives expansion of the developing lung by providing the distending pressure for air space development [4]. At birth, the lung switches from a secretory to an absorbing organ [5]. The fluid filling the lungs is reabsorbed by the actions of the epithelial sodium channel (ENaC) allowing the lungs to become filled with air [4]. From this point, pulmonary hydration is maintained by a balance between secretive and absorptive mechanisms. The ion channels responsible for fluid secretion and absorption on pulmonary surfaces are under a tight and complex regulation by various mediators, including adenine nucleotides. This section describes each channel contributing to the maintenance of the PCL, and their complex regulation by the purinergic signaling network.

5 Nucleotide-Mediated Airway Clearance

5.2.2

Fluid Secretion

5.2.2.1

The CFTR Chloride Channel

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Evidence that Cl secretion across the apical membrane of airway epithelia provides the driving force for fluid secretion into the lumen emerged from studies on the fetal lamb [2] Cl is taken into the epithelia via the Na+-K+-2Cl co-transporter, which is itself driven by an inwardly-directed Na+ gradient that is maintained by the Na+-K+-ATPase pump (Fig. 5.1). Since the ASL Cl concentration is ~3 fold greater than intracellular Cl, there is no chemical gradient for Cl secretion [5a]. However, inhibition of ENaC or activation of basolateral K+ channels both serve to hyperpolarize the epithelia, providing an electrical driving force for Cl secretion. During Cl secretion, Na+ follows paracellularly to preserve electroneutrality. Water then follows by osmosis causing an increase in ASL height. However, since pulmonary epithelia are highly permeable to water, the net NaCl concentration does not change and remains isotonic with plasma [5b]. Thus, it is the

Fig. 5.1 Chloride secretion provides the driving force for the hydration of pulmonary surfaces. Under basal conditions, the Na+-K+-ATPase pump generates a large electrochemical gradient to facilitate apical Na+ entry via ENaC. The intracellular Cl concentration is maintained above its equilibrium potential by the inward activity of the Na+-K+-2Cl contransporter, with K+ being recycled through basolateral channels [6]. Under these conditions, Cl is at near-electrochemical equilibrium across the apical membrane so there is little net Cl secretion. However, following inhibition of ENaC, the apical membrane is hyperpolarized, generating sufficient electrical gradient for Cl secretion. This hyperpolarization may be further augmented by activation of basolateral K+ channels. Pulmonary ion transport is tightly regulated by purinergic signaling: ATP, released by either mechanical stress or by cell injury stimulates P2Y2Rs. This action simultaneously raises intracellular Ca2+ to activate CaCC and depletes cells of PIP2 to inhibit ENaC. Together, these actions generate net Cl and fluid secretion. Ectonucleotidases then convert ATP into ADO, which binds A2BRs to activate CFTR and inhibit ENaC, again promoting net fluid secretion and pulmonary hydration

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amount, rather than the concentration of NaCl that is altered in the ASL during Cl secretion. Under steady-state conditions, airway fluid secretion is mediated primarily by the cystic fibrosis transmembrane regulator (CFTR). This Cl channel belongs to a family of ATP-binding cassette (ABC) proteins composed of two membrane-spanning domains (MSD1 and 2), two nucleotide-binding domains (NBD1 and NBD2) and a regulatory domain (R) [7]. This multifunctional protein also regulates other ion channels, including the outwardly rectifying chloride channel (ORCC) [8], CaCC [9] and ENaC [10]. The signaling events leading to CFTR activation involve cell surface G protein-coupled receptors (GPCRs) which stimulate adenyl cyclase, leading to cyclic AMP (cAMP) production, and protein kinase A (PKA) activation [11–13]. Two GPCRs are known to mediate CFTR activation on the apical surface of human airway epithelia by this pathway: A2B receptors (A2BRs) and b2 adrenergic receptors. Their endogenous agonists have been shown to stimulate Cl secretion through CFTR and subsequently increase fluid secretion (review: [14]). Nonetheless, biochemical and electrophysiological studies demonstrated that CFTR is activated primarily by A2BRs in human airways. Patch clamp studies on the Calu-3 human bronchial epithelial cell line showed that the Cl channel activity of CFTR is abolished by the non-specific ADO receptor antagonist, 8-(p-sulfophenyl) theophylline (8-spt) [15]. Similar results were obtained by preventing the endogenous formation of ADO using a,b-methyleneADP (AMPCP), an inhibitor of ecto 50 -NT (CD73) [15]. Also, exogenous adenosine deaminase (ADA) reduced cAMP levels in resting primary cultures of human nasal and bronchial epithelial cells [16]. Clancy and collaborators showed that the signaling pathways responsible for ADO-mediated CFTR activation also involve prostanoids [17]. Arachidonic acid stored in cell membranes is released by phospholipase A2 activity and metabolized into the mediators, prostaglandins and leukotrienes, by the actions of cyclooxygenases (COX) (review: [18]). The authors showed that A2BR activation induced the apical secretion of prostaglandins from airway epithelial cultures [17]. While COX inhibition by indomethacin reduced A2BR-mediated CFTR activation, arachidonic acid directly stimulated the channel. This study opens a new realm of possibilities for the purinergic regulation of MCC. Functionally, resting airway epithelial cultures maintain surface ADO levels (350 nM) [16] above the activation threshold (~100 nM) of A2BRs [19]. Whereas airway epithelial cells do not release ADO [20], the ectonucleotidase network metabolizes constitutively released ATP at a rate sufficient to maintain baseline A2BR-mediated CFTR activation. The importance of the A2BR-CFTR axis for airway hydration under steady-state conditions was validated by confocal microscopy studies in which ASL height was monitored on human bronchial epithelial (HBE) cultures before and after addition of 8-spt or ADA [16]. These agents caused severe dehydration the epithelial surfaces, as they depleted the ASL from a height of 7 to 4 mm, which corresponds to the height of folded cilia.


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The concept of protein clusters for the A2BR-CFTR-PKA axis was introduced by a study conducted on patches of apical membranes from Calu-3 cells [21]. First, the co-localization of CFTR and A2BRs within a patch was demonstrated by the effects of exogenous ADO, 8-spt and ADA on the channel activity. Second, cAMP analogues increased the open probability of CFTR, indicating that PKA is also associated to the plasma membrane, in close proximity with the channel. This study also identified the PKA isoform as PKA-II, which is anchored at specific subcellular sites by A kinase anchoring proteins. These observations suggest that CFTR activity is controlled by ADO generated locally from ATP release and metabolism, and that A2BR-mediated cAMP production activates PKA in close proximity to CFTR. An alternative signaling mechanism was proposed for CFTR activation on human airway epithelia. Kunzelmann et al. provided evidence that ATP stimulates CFTR via P2Y2R activation [22]. This signaling cascade involves Gq/11 and phospholipase C (PLC) activation, but not protein kinase C (PKC) or calmodulindependent kinase (CAMK). This mode of activation would not be significant under the baseline conditions provided by static epithelial cultures, which maintain ASL ATP concentrations below the activation threshold for P2Y2Rs. However, stress-induced ATP release, like breathing, would raise the contribution of P2Y2R-mediated CFTR activation for airway hydration.

5.2.2.2

The Calcium-Activated Chloride Channel (CaCC)

In the early 1990s, studies showed that extracellular nucleotides (ATP and UTP) have the capacity to stimulate Cl secretion from airway epithelial cells which lack CFTR [23–25]. Since this response was mediated by an increase in cytosolic Ca2+ concentration [Ca2+]i, these channels were named Ca2+-activated Cl channels (CaCC) [26]. Under steady-state conditions, the [Ca2+]i is insufficient to support CaCC activity. However, several purinoceptors have been shown to induce Ca2+-dependent signaling pathways in human airways, namely P2Y2Rs, P2Y6Rs, and likely P2X4Rs [19, 27, 28] (see Table 1.1 of Chap. 1 for details). However, therapeutic approaches to enhance Cl secretion in airway diseases are focused on P2Y2Rs, which are less susceptible to desensitization by excess agonist [29]. The identity of the CaCC was recently confirmed by three independent groups as TMEM16, member of a family of anion selective channels with eight transmembrane domains named anoctamins [30–32]. The development of specific polyclonal antibodies confirmed the localization of TMEM16 on the apical surface of airway epithelia [33]. Functional studies showed that P2Y2R activation induces Ca2+ mobi lization and (ANO)/TMEM16-associated Cl current [30–32] whilst TMEM16A knockdown with silencing RNA or knockout. Interestingly, this channel was not detected at the mRNA level in alveolar Type 2 cells [34], supporting regional differences in the regulation of fluid secretion.

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While most studies on the purinergic regulation of CaCC address the P2Y2R, the P2Y6 (UDP) receptor present on the apical surface of human nasal epithelial cells [35] has been shown to stimulate Cl secretion through a synergistic increase of both [Ca2+]i and cAMP intracellular levels [36]. Even so, the contribution of this pathway would be rather limited given the relatively small concentrations of uridine nucleotides (3 mM. Moreover, this value is fully

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consistent with the Ca2+ dissociation constant of 18 mM recently determined for synaptotagmin function in chromaffin cells [111]. Notable in the above experiments with Streptolysin-O-permeabilized SPOC1 cells, is that IP3 elicits mucin secretion under conditions in which intracellular stores are the only possible source of Ca2+. Consistent with this observation, we found in intact HBE goblet cells that ATPgS was equally effective in eliciting an intracellular Ca2+ signal when bathed luminally in Ca2+-free (+ EGTA) or Ca2+-replete media. Therefore, Ca2+ mobilization in goblet cells appears to depend exclusively on intracellular stores [89].

5.3.5

Intracellular Ca2+ Stores: ER and/or Mucin Secretory Granules?

The fact that the initial rise in intracellular Ca2+ concentration, following cell activation, is due exclusively to the release from intracellular stores raises an interesting problem regarding the localization of these stores. The general morphology developed for intestinal goblet cells during the 1980s and early 1990s [112] holds that the endoplasmic reticulum (ER) is restricted to the perinuclear area in the basal region of the cell. In general, the more recent fine structural analysis of airway goblet cells has been interpreted similarly [113]. As shown in the upper panel of Fig. 5.3, there would be no obvious source of Ca2+ near the apical membrane exocytic sites, given that the ER, the generally accepted Ca2+ store, is located basally. This structural design suggests that the mucin secretory granules (SGs) themselves would be the only obvious source of Ca2+ in the apical region. There is evidence both for and against this possibility, as detailed below. The electron microprobe analysis of mucin granules in goblet cells of the slug [Ariolimax; [114]] and the frog palate [Rana; [115]] revealed substantial levels of Ca2+, with 2.3 M/kg and 51 mM/kg, respectively. Both levels are well above the generally accepted cytoplasmic and plasma levels of Ca2+, which could be due to inter-species differences or how the standards were reported. A high mucin granule Ca2+ content is consistent with estimates for most other secretory cells, where intracellular Ca2+ appears to be sequestered predominately in SGs [116, 117]. There is also very good evidence for IP3-sensitive Ca2+ release from mucin granules, as ATP applied to intact goblet cells, and IP3 applied to isolated SGs, both cause Ca2+ release from SGs [110, 118]. Hence, the idea of mucin granules acting as IP3-sensitive Ca2+ stores is not unreasonable. On the other hand, whether SGs express IP3 receptors (IP3Rs), and IP3 induces Ca2+ release from granules by direct mechanisms, remains highly controversial [119–123]. The notion that IP3Rs might exist on SGs originates from a study on pancreatic acinar cells [124]. The initial responses to basolateral acetylcholine, or IP3 injected into the basal area, were both visualized within the cell as high Ca2+ concentrations in the apical pole. These observations suggest an apical-to-basolateral gradient of IP3R


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Fig. 5.3 Models of Ca2+ distribution for airway goblet cells. Top: The endoplasmic reticulum (ER) is restricted to the perinuclear region in the basal part of the cell, in which case mucin secretory granules (SGs) act as the primary source of IP3-mediated mucin release. Bottom: The ER is distributed throughout the cell, close to the apical membrane, in which case IP3 releases Ca2+ from the ER. Likely, Ca2+ is also released from SGs by tertiary signaling molecules. Recent data shows IP3 receptors restricted to the ER [74]

sensitivity or density. Blondel et al., first reported in 1995 the immunolocalization of IP3Rs on SGs in rat pancreas using an antibody to a synthetic peptide ([125]; review [120]). This notion was later disputed by the fact that these antibodies bind to rat, not human, insulin SGs [123]. Furthermore, immunogold labeling localized IP3Rs in the lumen of SGs, but not the membrane [126]. Independent studies using affinity purified antibodies and cell fractionation techniques subsequently brought arguments against the presence of IP3Rs at the surface of SGs. First, IP3Rs are associated with the ER fraction of endocrine cells, but are absent from isolated SGs [122]. Second, in the exocrine pancreas, IP3R antibodies stain ER but not SGs, while Western blots associate IP3R with a crude membrane-contaminated SG isolate, but not with Percoll-purified SGs [121]. Third, a study which monitored free Ca2+ inside SGs with a targeted aequorin chimera showed that it was insensitive to IP3, but highly sensitive to cytoplasmic pH, in the PC12 neuronal cell line [127]. Finally, we have shown by immunostaining and confocal microscopy in human airway epithelia that IP3Rs colocalize with ER, but not with mucin SGs [128]. The lack of IP3R in mucin SG membranes was not a mucin-related artifact, since the granule membrane marker,

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Rab3D, was localized to these membranes and appeared to colocalize with MUC5AC in SG lumens. Interestingly, we found that IP3Rs and the ER, rather than being restricted to the perinuclear region, were instead distributed throughout the apical pole of airway goblet cells, right up to the apical membrane [128] (Fig. 5.3; lower panel). It would be surprising that the high Ca2+ concentrations measured in SGs are not exchangeable with the cytosolic Ca2+, especially given the genuinely good quality of some of the data indicating IP3-sensitive Ca2+ release from SGs, including mucin SGs [110, 116, 118, 119, 129, 130]. However, the above evidence for the absence of IP3R in SG membranes is equally strong. Perhaps the strongest negative evidence, however, came from experiments measuring Ca2+ release from isolated pancreas zymogen granules [121]. Crude isolates were shown to release Ca2+ when stimulated with exogenous IP3, whereas granules purified using a Percoll gradient released Ca2+ only upon exposure to ionomycin. Contrary to the purified isolates, the crude isolates were shown by EM to be contaminated by ER and mitochondria [121].

5.3.6

The Growing Complexity of Ca2+ Signaling

Over the past 5 years, studies on exocytic secretion have exposed the complexity of intracellular Ca2+ signaling. For instance, Ca2+ gradients have been demonstrated around zymogen granules stimulated with IP3, cyclic ADP ribose (cADPR), or nicotinic acid adenine dinucleotide phosphate (NAADP) [131]. In addition, b-cell insulin granules were recently shown to contribute to the clearance of cytosolic Ca2+ after cell stimulation [132]. This study identified b-cell insulin granules as the NAADP-sensitive, thapsigargin-insensitive intracellular acidic Ca2+ store of much recent interest [133, 134]. Hence, the Ca2+ release from SGs induced by IP3 is likely mediated indirectly through a sequence of events initiated by the release of Ca2+ from the ER, and terminated by NAADP or other intracellular messengers causing SG Ca2+ release. However, several artifacts have been noted in these studies. For instance, antibodies can be highly specific and effective, yet they are proteins and need access to their target epitope. They are polyelectrolytes that also possess hydrophobic domains which can restrict diffusion and/or access. Hence, positive immunolabeling can provide strong evidence for the presence of a target antigen, but negative labeling can hardly be taken as evidence ruling out the presence of antigen. Similarly, data from organelle separations using Percoll gradients can also be difficult to interpret. The polyvinylpyrrolidone (PVP) coating of the silica nanoparticles of Percoll binds strongly to polar sites. Therefore, the separation media could affect IP3 receptors in functional assays. Lastly, most Ca2+ measurements made in living cells were acquired using widefield fluorescence microscopy, a technique that yields undeniably valuable data. These data, however, always need to be ‘viewed’ cautiously, as the signal recorded is the integral of fluorescence emitted along the full path of excitation, and it is subject to scattering.


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Too few observations of complex cells and tissues have been made under the experimentally optimal conditions of two photon excitation to restrict the fluorescence excitation to the plane being imaged within the cell. One, therefore, expects that progress toward resolution of the question of Ca2+ signaling in and around secretory granules will come as techniques and experimental procedures are improved to eventually circumvent these, and other, artifacts.

5.3.7

Ca2+ Release at the Plasma Membrane: How Close Is ‘Close’?

The absolute requirement of Ca2+ for exocytosis to take place, the absence of Ca2+ entry into goblet cells through apical membrane channels, and the apparent restriction of IP3 to the ER, all require the ER to be localized quite close to the apical membrane. In support of this, immunofluorescence, confocal and electron microscopy studies showed that the ER is distributed in the apical pole, visibly close (50 nm) to the apical membrane (Fig. 5.4; [128]). Second, our recent data using SPOC1 rat airway goblet cells permeabilized with Streptolysin-O also indicate a

Fig. 5.4 Juxtaposition of apical endoplasmic reticulum and mucin secretory granules. (a) Electron micrograph of the apical membrane region of a goblet cell, showing the juxtaposition of a mucin secretory granule (SG) and rough endoplasmic reticulum (rER). The arrays of ribosomes reveal rough ER and the intimate relationships between the plasma membrane, granule, rough ER, and mitochondria (M). (b) Activation pathway for regulated mucin secretion in goblet cells. Extracellular ligands in the airway surface liquid layer (bottom) bind GPCRs in the apical membrane that activate Gq and PLCb1, generating secondary messengers: DAG and IP3. DAG activates the exocytic priming protein Munc13-2 (dotted blue arrow, left fork) and PKC effectors. IP3 induces Ca2+ release from the ER in the vicinity of mucin-containing SGs, activating Syt2 at their surface (dotted blue arrow, right fork) and co-activating Munc13 proteins (Reprint from Ref [74]. With permission)

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remarkably close juxtaposition of local Ca2+ release to exocytic sites [108]. This study took advantage of the 400-fold faster Ca2+-binding kinetics of BAPTA over EGTA, two Ca2+ chelators with essentially equal binding affinities [135]. In cells exposed to EGTA-based intracellular buffer, IP3 was very effective in stimulating mucin release. In contrast, IP3-induced mucin secretion was nearly abolished when the cells were buffered instead with BAPTA. Because of its faster Ca2+ binding kinetics, BAPTA binds Ca2+ closer to the membranes across which Ca2+ is transported. With the buffer conditions used in our experiments, the inhibition of IP3-induced mucin release by BAPTA indicated a juxtaposition distance between IP3R Ca2+ release channels and the SG exocytic apparatus of 10 min later), where the early response coincides with Ca2+ mobilization, whereas the delayed response occurs at almost resting [Ca2+]c values but at times reported for phosphatidic acid generation (P. Dietl, unpublished observation).

5.4.4.2

Purinergic Control of the Post-fusion Phase

What we know about the release mechanism of surfactant from native type 2 cells in the lung still mainly comes from transmission electron microscopy investigations, where tubular myelin was postulated as the extracellular conversion product of released lamellar bodies [230]. There are numerous publications in which snapshots of the exocytotic process were depicted, some of them showing tubular myelin to originate from secreted LBs. Some of these images display fusion pores of various diameters (1 mm), through which surfactant appears to be squeezed out [193]. However, dynamic features of this process within the lung are still largely obscure.


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Advanced microscopy techniques enabled the visualization of the entire exocytotic process (fusion pore formation, fusion pore expansion and surfactant release) in primary cultures of type 2 cells [204, 206, 231, 232] or in situ, however with limited resolution [166]. In these ex vivo experiments, tubular myelin cannot be clearly depicted. Moreover, it appears that the hydrophobic, poorly soluble, complex of surfactant must be actively squeezed through a reluctantly opening fusion pore. Several experimental data are in line with this notion. First, fusion pores in type 2 cells have the tendency to expand very slowly [206]. Second, experiments using laser tweezers as force generators have shown that fusion pores in type 2 cells can act as mechanical barriers of surfactant release [233]. Finally, experiments with green fluorescent protein (GFP)-actin-transfected pneumocytes revealed that an actin coat must be formed around the fused LB before surfactant can be squeezed out, apparently by contraction of the actin coat [232]. This is consistent with earlier observations that actin reorganization accompanies LB exocytosis [234, 235]. In type 2 cells, ATP-induced actin coat formation is inhibited by intracellular Ca2+ immobilization with BABTA-AM, or the phospholipase D inhibitor C2 ceramide [232]. In the absence of an actin coat, the cells were unable to secrete surfactant. The fused LBs remained in a “wait” position as filled bags connected with the plasma membrane [232]. This indicates that Ca2+ and phospholipase D activation in response to ATP significantly control the post-fusion phase (release) of surfactant exocytosis. Currently, one of the most intriguing questions remaining is how actin coat formation is limited to the fused LB and does not affect all LBs in a cell stimulated with ATP (Fig. 5.6). There must be spatially and/or temporarily confined processes that cannot spread throughout the cell. Lipid merger and compartment mixing between the limiting LB membrane and the plasma membrane is the most obvious local phenomenon during exocytosis. Yu and Bement recently disclosed a signalling pathway in oocytes involving PLD-dependent DAG incorporation into the granule membrane after fusion, triggering Cdc42/N-WASP-induced actin assembly [236]. Although this still has to be investigated in type 2 cells, PLD appears to control primarily the post-fusion phase, as neither a primary alcohol which inhibits PLD-induced DAG generation, nor the PLD inhibitor C2 ceramide, can block LB fusion activity [196, 232].

5.5 5.5.1

Ciliary Beating Activity Definition: The Cilium

Ciliated cells are located in the conductive airways proximal to the respiratory bronchioles. One ciliated cell in the large airways carries about 200–250 cilia, each with a diameter of 0.3 mm and a length of 6–7 mm [237]. Along the airways, the cilia

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shorten gradually with airway diameter, with their length approximating 3.5 mm in the seventh generation bronchi [238]. The core of a cilium is composed of two central microtubules surrounded by nine peripheral doublets of microtubules [239]. Besides these motile scaffolding structures made of tubulin, more than 200 proteins were found in a proteomic analysis of human airway cilia [240]. Some of these proteins provide motion to these structures and their scaffolding, namely the inner and outer dynein arms, the radial and circumferential spokes, and the inter-doublet links [241].

5.5.2

Historical Review

Since the discovery of the ATP molecule in 1929 [242], it has become clear that the continuous intracellular availability of ATP is essential for the maintenance of vital cell functions, including ciliary beating. The stimulating effect of intracellular ATP on flagellar movement was originally demonstrated by injection of ATP into the tail of Amoeba discoides [243]. Another study showed the absolute requirement of intracellular ATP by basolateral permeabilization of ciliated airway epithelial cells. Ciliary beating activity slowed down to a complete stand still after only a few minutes of basolateral perfusion with an ATP-free solution, and recovered as soon as the perfusate was replenished in ATP [244]. The underlying mechanism was elucidated by Gibbons and Rowe [245], who showed that intracellular ATP constitutes the energy source for the motor function of the dynein arms. It is interesting to note that the first description of extracellular signaling by purines was also described in 1929, the year of ATP discovery. The ADO was first purified from yeast nucleic acid and injected into animals while its effects on the heart were studied [246]. It has also been known for over 50 years that apical application of ATP to ciliated epithelia stimulates MCC. First, ATP was shown to accelerate the movement of small pieces of aluminum on dissected ciliated pharyngeal mucosa from the frog Rana Pipiens [247]. However, many scientists held the strong belief that ATP is “too precious” as an energy source for extracellular functions, and thus would not be secreted from cells and “abused” as a signal transduction initiator. Nonetheless, the work of many scientists continued, which led to the discovery of purinergic receptors in 1976 [248]. Shortly thereafter, Burnstock proposed the existence of two types of purinergic receptors, namely P1 and P2, preferentially activated by ADO and ATP, respectively [249]. Since then, three major purinoceptor families have been discovered: P1, P2Y and P2X receptors [250]. Among them, the P2Y2R is widely accepted as the most important purinergic receptor regulating ciliary beating and MCC (reviews: [65]). While the P2Y2R is equally stimulated by UTP and ATP, mammalian cells secrete ATP at a considerably higher rate than UTP [37]. Therefore, ATP and its metabolite ADO are considered the most relevant purinergic agonists for the regulation of ciliary beating in the airways.


5.5.3

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Importance of Ca2+ for the Regulation of Ciliary Beating

The stimulating effect of extracellular ATP on ciliary motility was first quantified optically using cultured frog palate epithelial cells [251]. However, the signaling events by which ATP caused an increase in ciliary beating frequency (CBF) were undetermined. In the 1980s, studies conducted in multiple mammalian species revealed the sensitivity of CBF to changes in cytosolic Ca2+concentration [Cac2+] [252–254]. These findings guided future research which led to the discovery that the ATP-induced increase in CBF is Ca2+ dependent [35, 36]. Eventually, the development of methods allowing the simultaneous single cell measurements of Cac2+ and CBF revealed that the temporal correlation between the fast increases in both parameters is within one beat cycle, both for purinergic and cholinergic stimulations [255, 256]. In rabbit tracheal epithelial cells, ATP caused a rapid rise in [Cac2+] and CBF within the 0.1–1,000 mM concentration range [257]. This study also showed no change in CBF upon stimulation with ATP after depletion of Ca2+ intracellular stores using thapsigargin. These, and other, experiments proved that this rapid increase in [Cac2+] and CBF, induced by ATP, is largely due to Ca2+ release from intracellular stores (review: [258]). One has to appreciate that the progress in our understanding of the signaling pathways regulating ciliary beating clearly lagged behind those of other cell functions. For instance, inositol triphosphate (IP3) was found to be responsible for the release of Ca2+ from intracellular stores induced by ATP in rat hepatocytes [259], delineating the pathway from purinergic P2Y receptors to PLC and IP3. This signaling cascade was later identified in ciliated cells [260].

5.5.4

Maintenance of Increased Ciliary Beating After Purinergic Stimulation

As a general rule, the stimulation of a ciliated cell by extracellular ATP induces a rapid increase in CBF, reaching a maximum of approximately twice the baseline value (peak) [19, 257]. During continued exposure to ATP, CBF then decreases to a frequency above baseline and remains at this level for a prolonged period of time (plateau). There is widespread agreement in the response of CBF and [Cac2+] during this initial phase of ATP stimulation in mammalian cells, including human airway epithelia (review: [258]). However, there is significant variability in the behavior in CBF and [Cac2+] after this initial peak response between species, and even between different locations (proximal versus distal airways) within the same species. In rabbit tracheal epithelial cells, prolonged ATP exposure generated a biphasic increase in CBF [257]. The initial increase was explained by Ca2+ release from two distinct stores: a small thapsigargin-insensitive store situated in close proximity to the cilia and a larger thapsigargin-sensitive store located within the cytoplasm.

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The CBF plateau that followed the CBF peak was caused by Ca2+ influx, as it was not maintained within a Ca2+-free perfusate. While Ca2+store-induced Ca2+ influx clearly plays a role here, a new P2X receptor was also found to be gated by ATP in ciliated cells [261]. Influx of Ca2+ through this P2X receptor seemed partially responsible for the CBF plateau seen during prolonged ATP stimulation, at least in rabbit airway epithelial cells. It was proposed that Ca2+ influx through this receptor activates nitric oxide production, which stimulates CBF [260]. Other mechanisms have been described in different species, and they have not been assembled into a unifying paradigm (review: [258]). For instance, prolonged CBF response to ATP in rabbit epithelial cells was proposed to involve the metabolism of ATP to ADO [262]. However, ADO stimulated CBF and MCC in rabbit airway epithelia via the A3Rs [262], which are not expressed in human airway epithelia. A similar biphasic response for CBF was reported in rat tracheal epithelial cells during prolonged perfusion with apical ATP or UTP [263]. Experiments conducted in Ca2+-free perfusate allowed the development of the CBF peak, but prevented the CBF plateau. Therefore, the P2Y2R-PLC signal mobilizing Ca2+ from intracellular stores would be responsible for the initial peak, while the plateau phase was maintained by, both, P2X and P2Y receptors. The surprising finding was that the distal airway epithelial cells did not respond to ATP or UTP by an increase in CBF or [Cac2+] [263]. But the use of thapsigargin and ionomycin to increase [Cac2+], both, stimulated the ciliary beating activity [263]. These data suggest that the difference is not in the composition of the ciliary apparatus, but rather the lack of purinergic receptors in rat distal airways. Species related differences in the purinergic regulation of CBF are even reported between rodents. Using thin slices of mouse distal airways, Delmotte and Sanderson showed that ATP was able to cause an increase in [Cac2+], but had no effect on CBF [264]. This study suggests that the cilia were already maximally stimulated and could no longer respond to the changing Ca2+ concentration. In human airway epithelia, a prolonged exposure to ATP or UTP induced the general biphasic response, as CBF rose quickly to a transient peak at twice baseline, and then relaxed to a plateau above baseline. However, differences in the regulation of the plateau phase were reported for human epithelia, compared to the abovementioned animal preparations. Morse et al. showed that the plateau was significantly lower in the presence of UTP than ATP [19]. Furthermore, this ATP-induced CBF plateau phase was partially inhibited by a broad range P1 antagonist (8-sulfophenyeltheophylline) effective against A1Rs A2ARs and A2BRs, or a transmembrane adenylyl cyclase (tmAC) inhibitor (SQ22536). It was, therefore, postulated that the ATP-induced CBF plateau was, in part, mediated by the metabolism of ATP into ADO, which signals through A2BRs and tAC in human cells (review: [65]), as opposed to A3Rs in rabbits tracheal epithelial cells [262]. However, a recent study showed that even short-term application of ATP or UTP lead to a post-peak CBF plateau in human airway epithelial cells [265]. These prolonged CBF elevations persisted after removal of ATP or UTP, and were not related to nitric oxide production or ADO receptor activation. Instead, they required the activation of a Ca2+-sensitive tmAC to produce cAMP [265, 266]. In fact, the


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potentiating effect of cAMP on CBF is well documented [267–269]. Using real-time in vivo cAMP measurements with a PKA-FRET system, Nlend et al. showed that short-term stimulation by ATP or UTP causes concomitant increases in CBF and cytosolic cAMP concentration [266]. Taken together, these studies suggest that the CBF plateau phase triggered by ATP is maintained by cAMP signals. Cyclic AMP is produced by one of the nine known tmAC or by soluble adenylyl cyclase (sAC). Three tmAC isoforms (AC 1, 3 and 8) are stimulated by Ca2+ in different human tissues [271–273], whereas sAC is stimulated by bicarbonate [274, 275] and Ca2+ [276]. At least two Ca2+-sensitive tmAC isoforms (AC1 and AC8) are localized to the apical membrane of ciliated, human tracheo-bronchial epithelial cells [266]. The role of sAC in the purinergic stimulation of CBF remains poorly understood, but some function can be hypothesized. It is important to mention that sAC is the only AC present in cilia [277]. Since this signaling pathway is stimulated by bicarbonate, pathological conditions leading to an acidification of the airways could affect ciliary beat activity. For instance, cystic fibrosis (CF) is associated with abnormal acidification of airway epithelial cells (review: [278]). Salathe and collaborators [279] recently showed that the stimulation of CBF by sAC-derived cAMP is impaired in the cilia of bronchial epithelial cells from CF patients. While the protein levels of sAC were normal, the decrease in CBF measured during apical exposure to HCO3/CO2 was more pronounced in CF [279]. Whether the disease is characterized by hypo- or hyper-acidification of the intracellular compartments remains controversial (review: [278]). Nonetheless, this study highlights the sensitivity of sAC-mediated ciliary beating activity under pathological conditions.

5.6

Mucociliary Clearance in Airway Diseases

The critical role of MCC for airway defenses is highlighted by chronic obstructive respiratory diseases associated with genetic mutations affecting a single element of this complex signaling network. After 70 years, the “sweat chloride test” remains the gold standard for the diagnosis of CF, which emphasizes the importance of deregulated ion channel activities in this disease (review: [280]). Functional mutations targeting the CFTR gene prevent proper folding and trafficking to the apical membrane, resulting in the inability to secrete Cl into the ASL (review: [281]). The epithelia of CF patients also exhibit Na+ hyperabsorption, raising the possibility that CFTR normally restricts ENaC activity. Numerous studies conducted in heterologous expression systems showed that the surface expression of fully functional CFTR suppresses Na+ absorption by ENaC (review: [282]). The most common CFTR mutation reported in CF patients (DF508) targets the NBD1 region. Suaud et al. reported that NBD1 reduces the surface expression of ENaC by a mechanism independent of Cl transport [283]. This line of thoughts was taken further by Gentzsch et al., who recently demonstrated that physical association

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between CFTR and ENaC prevents the proteolytic activation of ENaC [284]. Incidentally, the DF508 mutation on NBD1 prevented CFTR association with ENaC, resulting in enhanced Na+ and water hyperabsorption. In contrast, the nasal passages of CF patients exhibit enhanced ATP-mediated Cl secretion by CaCC [55]. The fact that this pathway is unable to compensate for the defective CFTR, despite high ATP levels reported in the airway secretions of CF patients [285], is in line with the physical nature of CFTR-mediated ENaC inhibition. Alternatively, CF up-regulates the ectonucleotidases responsible for ATP metabolism on airway surfaces, which locally reduce the availability of P2Y2R agonists for CaCC activities. In this regard, the metabolically-stable P2Y2R agonist, Denufosol, is currently undergoing phase 3 clinical trial for the treatment of airway obstruction in CF patients [56]. For CF patients, these aberrant channel activities translate into severe dehydration of airway surfaces, as defective Cl release via CFTR and excess Na+ absorption via ENaC maintain an inward osmotic gradient for water. The depletion of the ASL causes a domino effect in terms of complications, as dehydrated mucin strands adhere to epithelial cells, forming viscoelastic plugs which are, often, not cleared by coughing but require mechanical intervention [286]. Furthermore, the adhesive mucus plaques provide a nidus for bacterial colonization, leading to chronic infection and the excessive neutrophilic inflammation responsible for irreversible tissue damage and the loss of lung function. Primary ciliary dyskinesia (PCD) illustrates the importance of ciliary beating for MCC as this autosomal recessive disorder is associated with mutations affecting cilia motion. Functional changes vary from differences in CBF coordination to immobility. The term neonates suffer from respiratory distress due to a failure to rapidly and fully transition to air breathing at birth. The mechanism, although not well understood, may involve a role for cilia movement in the removal of fetal lung liquid. Other features of PCD are similar to CF, including airway obstruction, bacterial infection, bronchiectasis and gradual declines in lung function. However, these symptoms appear later in the life, likely because ion transport mechanisms regulating airway hydration are preserved in PCD patients, allowing for effective cough clearance. In the late stage of the disease, PCD patients may experience lung pathology as severe for CF patients and require lung transplantation. For these reasons, their treatment is often inspired from the protocols ascribed to CF patients. From a purinergic point of view, PCD patients with functional mutations allowing for some cilia motion may benefit from treatments with aerosolized A2BR agonists to raise CBF and clear the accumulating mucin secretions. Chronic obstructive pulmonary disease (COPD) is characterized by irreversible airflow limitation due to thickening of the bronchiolar wall by fibrosis and epithelial cell enlargement (review: [287]). Regarding MCC, whereas CF impairs hydration and PCD restricts cilia motion, COPD is associated with excess mucin from submucosal gland enlargement, mucous cell hyperplasia and metaplasia (review: [288]). In addition, the COPD patients with a smoking history target all aspects of MCC, as the toxic particles contained in cigarette smoke inhibit cilia function [289],


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and Cl secretion by CFTR [290] and CaCC [291]. The most compelling evidence that mucus dehydration is a major complication for COPD patients was provided by the study of Dr. Hogg et al., showing mucus adhesion and obstruction of the small airways [292]. Furthermore, since ENaC and CFTR are restricted to ciliated cells on airway epithelia [293], the severe mucus cell metaplasia is expected to weaken the capacity to maintain airway hydration. Eventually, the development of emphysema further hinders MCC as architectural damage allows airway walls to distort and collapse. Consequently, COPD patients also suffer from the inability to clear pathogens, leading to chronic airway infections and inflammation. This disease clearly demonstrates the impact of smoking on airway clearance, and explains the frequent cough reflex of smokers. COPD patients have an advantage over CF patients, which is a chance to improve MCC by giving up smoking, since toxin-related inhibition of ion channel activity appears to be reversible. In contrast, CF patients rely on P2Y2R-dependent CaCC activation, and A2BR-mediated cilia beating activity following the metabolism of ATP into ADO by the local ectonucleotidases. However, experiments conducted on CF airway epithelial cells cultured under static conditions indicated that the ASL does not maintain ATP levels sufficient to activate P2Y2Rs [70]. Exposure of these cultures to cyclic compressive stress mimicking normal tidal breathing induced ATP release and restored mucus transport. This study suggests that the medical devices used to promote MCC through rhythmic mechanical stress function, in part, through ATP release and P2Y2R-induced airway hydration. Hence, physical activity and training exercise are an important part of the clinical management of CF patients to replace the failure of cough clearance (review: [294]). Young CF patients adhering to this program maintain better MCC for extended periods of time, reduced number of exacerbations and delayed loss of lung function. Mucus hypersecretion is a hallmark of asthma and contributes to airway obstruction, wheezing and dyspnea. Nearly 25% of the airway epithelial cells are goblet cells, even in mild disease [295]. In healthy subjects, MUC5AC and MUC5B are secreted by the goblet cells of superficial epithelia and submucosal glands, respectively. As in CF and COPD, asthma is associated with an overexpression of both mucins, and MUC5B appears in the goblet cells (review: [296]). Interestingly, cultured airway epithelial cells were reported to overexpress MUC2 in response to ADO [297]. Yet, mRNA levels of MUC2 remain >20-fold lower than MUC5AC in asthmatic patients. It is, however, intriguing that MUC2 remains undetectable in the airways of CF patients, which maintain normal airway ADO levels [298]. The fact that steroid therapy prevents ATP and ADO from accumulating in the airways of asthmatics [299] supports a protective effect against the development of mucus cell metaplasia and hypersecretion. Likely, similar benefits may be associated with steroid therapy in other chronic airway diseases combining high ATP and/or ADO levels and mucus cell metaplasia, such as COPD [300] and CF [298] (see Chap. 4 for details). Finally, the well documented bronchoconstrictive effects of ADO and AMP in asthmatics are addressed in Chap. 9 from the perspective of a diagnostic application.

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Conclusions

This chapter exposes the widespread ramifications of purinergic signaling in the mechanisms mediating the clearance of airborne pathogens, namely airway hydration, mucin and surfactant secretion, and coordinated ciliary beating activity. The underlying signaling pathways involve the activation of P2Y2Rs and A2BRs following ATP release from epithelial cells, and surface conversion into ADO by local ectonucleotidases. In the absence of stimulation, baseline ASL ADO levels maintain MCC through CFTR activity and cilia beating activity. Normal rhythmic breathing is expected to periodically raise ASL ATP concentrations and activate P2Y2R-dependent pathways to manufacture the mucin sheath. Since mucin strands are released in a densely packed formation, the fluid secreted in response to P2Y2R-CaCC by neighboring ciliated cells may play a critical role in proper mucin hydration and unfolding. Additionally, this excess ATP would provide substrate for ADO formation and enhance A2BR-mediated responses to evacuate excess mucin through coordinated cilia beating activity. Situations calling for an emergency response, such as physical interaction with a pathogen or coughing, would intensify both purinergic routes through robust ATP release responses and metabolism. The importance of this purinergic defense mechanism is outlined by the disastrous consequences of a single mutation on airway clearance. Acknowledgements The authors thank their many colleagues in their respective laboratories whose talents contributed to much of the work described in this review. We also think Edwin Chapman, University of Wisconsin, for permission to redraw Fig. 5.4 and for valuable thoughts on the mechanism of Ca2+/Syt-mediated exocytic fusion. The studies were supported in part by Grant 0120579 to PV from the Biocomplexity Program of the National Science Foundation, Grant HL-063756 to CWD from the National Institutes of Health, and grants to CWD from the North American Cystic Fibrosis Foundation.

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Chapter 6

Purinergic Signaling in Wound Healing and Airway Remodeling Albert van der Vliet and Peter F. Bove

Abstract Airway epithelia are continuously damaged by airborne pollutants, pathogens and allergens, and they rely on intrinsic mechanisms to restore barrier integrity. Epithelial repair is a multi-step process including cell migration into the wounded area, proliferation, differentiation and matrix deposition. Each step requires the secretion of various molecules, including growth factors, integrins and matrix metalloproteinases. Evidence is emerging that purinergic signaling promotes repair in human airway epithelia. An injury induces ATP release, which binds P2Y2 receptors (P2Y2Rs) to initiate protein kinase C (PKC)-dependent oxidative activation of TNFa-converting enzyme (TACE), which then releases the membrane-bound ligands of the epidermal growth factor receptor (EGFR). The P2Y2R- and EGFR-dependent signaling cascades converge to induce mediator release, whereas the latter also induces cytoskeletal rearrangement for cell migration and proliferation. Similar roles for purinergic signaling are reported in pulmonary endothelial cells, smooth muscle cells and fibroblasts. In chronic airway diseases, the aberrant regulation of extracellular purines is implicated in the development of airway remodeling by mucus cell metaplasia and hypersecretion, excess collagen deposition, fibrosis and neovascularization. This chapter describes the crosstalk between these signaling cascades and discusses the impact of deregulated purinergic signaling in chronic lung diseases. Keywords Airway remodeling EGFR P2Y2 receptors Migration Wound healing

A. van der Vliet (*) Department of Pathology, University of Vermont, Burlington, VT, USA e-mail: [email protected] P.F. Bove Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA e-mail: [email protected]


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A. van der Vliet and P.F. Bove

Introduction

The respiratory epithelium forms a continuous lining of the airways that interfaces with the environment, and plays a critical role in maintaining a protective physical barrier against common inhaled pollutants and airborne pathogens (reviews: [1, 2]). To optimally perform these functions, the epithelium has evolved into a complex mixture of cell types working together to support the mucociliary clearance of inhaled material. The epithelium also initiates immune and inflammatory responses to an infection by the secretion of mediators and direct interaction with various cell types. The efficiency of these defense mechanisms depends on the healing capacity of the epithelium. Since the airway epithelium is continuously subjected to injurious insults, it must be capable of responding rapidly to an injury in order to restore barrier integrity and to maintain adequate lung defenses [3, 4]. Studies in animal models have shown that epithelial lesions initially provoke rapid dedifferentiation of epithelial cells near the wound edge, which spread and migrate into the wound to cover the denuded area to reestablish a continuous epithelium. Proliferation of epithelial cells in the repaired area and re-differentiation subsequently contribute to the ultimate restoration of a pseudo-stratified and functional mucociliary epithelium [5]. Microarray analysis of bronchoscopy samples, collected from human subjects 7 days after mechanical (brush) injury, revealed the presence of an epithelial “repair transcriptome” characteristic of actively proliferating cells in the process of re-differentiation [6]. Stem cells or progenitor cells exist in various areas of the lung and are believed to contribute to epithelial self-renewal. These cells may also participate in epithelial repair, although their precise role is not known [7, 8]. Our current understanding of epithelial responses to an injury in human airways has changed drastically over the past decade (reviews: [4, 7, 9]). It is well established that EGFRs are essential to the initiation of epithelial responses to damage, as they activate various signaling cascades leading to cytoskeletal rearrangement and the secretion of potent mediators of cell migration, proliferation and differentiation. In contrast, the importance of surface receptors activated by extracellular nucleotides in EGFR-mediated responses only recently emerged from shear common sense and the convergence of their signaling cascades (review: [9]). The inevitable ATP release from lysed cells supported the notion that massive leakage of this precious source of energy may act as an alarm signal for injury. This chapter provides a comprehensive description of the elegant crosstalk taking place between the purinergic mediators, growth factors and oxidative stress to rapidly restore barrier integrity.

6 Purinergic Signaling in Wound Healing and Airway Remodeling

6.2

6.2.1

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ATP-Dependent Signaling in Epithelial Wound Responses P2YR Regulation of Epithelial Cell Migration and Proliferation

The respiratory epithelium releases ATP during normal tidal breathing, but also in response to various stresses such as mechanical injury, bacterial infection, environmental oxidants, hyperoxia or particulates. This signaling molecule, and its metabolite ADO, are well known to regulate a variety of epithelial functions through activation of purinergic receptors, such as ion transport, cilia beating and the secretion of inflammatory mediators [10–13]. Given that damaged cells release millimolar nucleotides into an injured area, it seems logical that ATP would be involved in repair mechanisms. Indeed, the contribution of purinergic events to epithelial repair has been demonstrated in intestinal and corneal epithelial cultures, where ATP and ADP promote wound closure via P2Y receptors [14–16]. Similar findings were observed in wound repair studies with the BEAS-2B bronchial epithelial cell line and tracheo-bronchial epithelial cells [16, 17]. Airway epithelial monolayers that were wounded, then washed to remove cell debris, exhibited a transient accumulation of extracellular ATP over 40 min, with a peak concentration of 40 nM [17]. Since this protocol excluded the ATP released by cell lysis, it suggests that unwounded epithelial cells release ATP, likely, in response to the mechanical stress caused by the injury. In addition, ATP was measured in condition medium, which underestimates peak concentration reached in a wounded area by several orders of magnitude. Nonetheless, the removal of endogenous ATP with apyrase, or the use of P2Y receptor antagonists, significantly reduced the rate of wound closure. These findings were confirmed by the application of exogenous ATP, or its non-hydrolysable analog ATPgS, which accelerated wound closure [16, 17]. The restoration of barrier integrity is largely mediated by coordinated migration of cell sheets into the wound area. The ability of ATP to promote migration was shown by quantitative Transwell assay, as epithelial cell migration was inhibited by apyrase or P2Y receptor antagonists [17]. Purinergic signaling also regulates the proliferation of human airway epithelial cells. In the A549 human alveolar epithelial cell line, nucleotides stimulated BrdU incorporation with a potency profile of ATP ¼ ADP > UDP ¼ UTP. This response suggests a mixed involvement of P2Y 2Rs and P2Y6Rs [18], which were identified in A549 cells by RT-PCR [19]. In contrast, ATP inhibited the proliferation of an adenocarcinoma-derived cell line from a human lung bronchial tumor (LXF-289) [20]. The potency profile (ATP ¼ ADP ¼ ATPgS >> UTP, UDP) does not support major roles for P2Y2Rs or P2Y6Rs. Nonetheless, this epithelial response was insensitive to P2X receptor agonists. RT-PCR analysis revealed that these cells express the P2 receptors encountered in primary cultures of human airway epithelial cells (P2Y2Rs, P2Y6Rs and P2X4Rs), but also P2Y11Rs (ATP > ADP >> UTP)

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[21] and P2Y13Rs (ADP > ATP) [22]. The aberrant purinergic regulation of wound healing by LXF-289 cells appears to be caused by receptors not normally expressed by human airway epithelia.

6.2.2

P2YR-Dependent Signaling in Pulmonary Vascular Remodeling

Purinergic signaling mediates neovascularization and vascular remodeling under hypoxic conditions associated with pulmonary hypertension [23], a common complication in asthma and chronic obstructive pulmonary disease (COPD) (review: [24]). Using a Transwell migration assay, P2Y2R activation was shown to induce the directional migration of vascular smooth muscle cells [25]. Furthermore, an upregulation of P2Y2Rs was linked to intimal hyperplasia in collared carotid arteries [26]. On isolated endothelial cells from the vasa vasorum microvessels, ATP was shown to dramatically stimulate DNA synthesis, cell migration and Matrigel tube formation, most likely via P2Y1Rs [27]. Interestingly, these purinergic responses were not observed in the pulmonary artery or aortic endothelial cells, suggesting that they are specific for small vessels [27]. In addition, hypoxia-induced ATP release from endothelial cells stimulated proliferative responses in the adventitial fibroblasts through activation of P2X and P2Y receptors [28]. These studies suggest that ATP is an important autocrine and paracrine mediator of vascular remodeling through endothelial cell and fibroblast proliferation, and adventitial thickening. In asthma, neovascularization and angiogenesis are critical events in the development of airway hyperresponsiveness.

6.3

6.3.1

Signaling Mechanisms Involved in P2YR-Mediated Responses Signaling Pathways: Ca2+ Mobilization and MAPK Activation

As mentioned earlier, the effects of extracellular nucleotides on epithelial cell migration and proliferation are primarily mediated by P2YRs, which represent a large family of G protein-coupled receptors (GPCRs) (reviews: [10, 29]). Among them, the P2Y2Rs couple to Gq, which stimulates phospholipase C b (PLCb) activity toward plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), leading to the formation of two secondary messengers: diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) [12, 30] (Fig. 6.1). Whereas DAG propagates cellular responses via novel protein kinase C isoforms (nPKCs), the activation of IP3 receptors on the endoplasmic reticulum triggers Ca2+ release into the cytoplasm


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Fig. 6.1 The P2Y2R-dependent signaling cascade. Injury or mechanical stress induces ATP release from epithelial cells, which binds P2Y2R coupled to the Gq subtype of G protein. Signaling through Gq activates phospholipase C (PLC), which cleaves membrane PIP2 into IP3 and DAG. The IP3 binds endoplasmic reticulum (ER) receptors, leading to Ca2+ mobilization and the activation of conventional protein kinase C isoforms (cPKCs). In contrast, DAG activates novel PKCs (nPKC) associated with the Raf/MEK/ERK1/2 signaling cascade. Other G protein coupled receptors (GPCRs), such as the thrombin receptor, are coupled to G12 and Rho to activate phospholipase D (PLD). This signaling pathway converges on the P2Y2R-dependent signaling cascade via DAG activation

and the activation of conventional PKCs (cPKCs). Other P2YRs couple to Gi, which signal through adenylate cyclase inhibition [30], such as the P2Y14Rs newly discovered on airway epithelia [31]. The complex process of cell migration involves coordinated activations of various signaling pathways in different regions of the cell [32, 33] (Fig. 6.1). At focal adhesions, the arginine-glycine-aspartic acid (RGD) domain of P2Y2Rs binds to clustered integrins (avb3 and avb5) [34]. This interaction allows P2Y2Rs coupled to G0 or G12 to activate ERK1/2-dependent signaling pathways essential for directed chemotaxis [34–36]. In 1321N1 astrocytoma cells, mutations in the RGD domain prevented UTP-induced chemotaxis [35]. On the other hand, RGD does not regulate the Gq-mediated Ca2+ mobilization, suggesting that P2Y2Rs initiate different processes depending on their cellular location. The purinergic regulation of cell migration is supported by physical interaction between P2Y2Rs and filamin A in vascular smooth muscle cells, determined by yeast two-hybrid

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screening [37], and the fact that mutations in this actin-binding protein prevented migration [38]. The proliferative responses induced by ATP in vasa vasorum endothelial cells and adventitial fibroblasts are mediated by multiple parallel signaling pathways, including the activation of ERK1/2 the transcription factor Egr1, and the PI3K-Akt-mTOR-p70 S6K signaling cascade [28, 39]. These signaling cascades provided the first evidence of convergence points between P2Y2R- and EGFR-dependent signaling pathways, which will be described in the following sections.

6.3.2

P2YR-Mediated Gene Regulation and Wound Responses

The localized activation of ERK1/2 by adhesion signals or growth factors has been shown to control organelle structure and cytoskeletal dynamics [32, 40], and to mediate the transcriptional activation of various genes critical in wound responses. Among these genes, MMP-9 is a type IV collagenase typically induced and activated during epithelial injury [41], and an important mediator of epithelial migration and repair [42, 43]. The expression of MMP-9 in airway epithelia is induced by the EGFR-dependent activation of ERK1/2 and PI3K/Akt signaling cascades involving nuclear translocation of the AP-1 and NFkB transcription factors [44, 45] (Fig. 6.2). Recent study shows that the induction and activation of MMP-9, in response to airway epithelial cell injury, is suppressed by P2YR antagonists or apyrase [17]. This study identified MMP-9 activation as one mechanism by which ATP-mediated P2YR activation promotes cell migration and wound repair. This purinergic signal also contributes to the up-regulation of IL-8 [46, 47], potent neutrophil chemoattractant [41]. The humanized airway xenograft model in nude mice reproduces the regeneration dynamics of injured airway epithelia, and exhibits increased epithelial production of MMP-7, MMP-9 and IL-8 [41]. These mediators work in concert to promote epithelial repair by enhancing cell migration and the recruitment of inflammatory cells to protect the wounded area against bacterial invasion [41, 48]. Finally, P2YR activation also promotes the synthesis and release of prostaglandin E2 (PGE2) by the Ca2+-mediated activation of phospholipase A2 (PLA2) [49] (Fig. 6.1), which is also a potent mediator of cell migration [50].

6.3.3

P2YR-Mediated Transactivation of EGFR-Dependent Signaling

Studies conducted with human corneal and airway epithelial cells showed that wound healing and cell migration are initiated through transactivation of the EGFR by the ligands HB-EGF and TGFa [15, 16, 51–53]. Membrane-bound


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Fig. 6.2 The P2Y2R-mediated transactivation of EGFR in the airway epithelium. ATP released from the epithelium initiates a series of events through P2Y2Rs which lead to Ca2+ mobilization and PKC activation, which collectively stimulate the production of hydrogen peroxide (H2O2) from dual oxidase-1 (DUOX1) co-localized apically. Then TACE cleaves transforming growth factor alpha (TGFa) off the apical membrane, which then binds and activates the epidermal growth factor receptor (EGFR). The EGFR-dependent signaling cascades lead to ERK1/2 activation, which induces the expression of healing compounds including, cytokines (IL-8), matrix metalloproteinases (MMP-9) and growth factors. On the other hand, the PI3K/PDK-1/AKT cascade leads to cytoskeletal rearrangement, focal adhesion formation and migration of the cells into the wound in response to the mediators expressed via ERK1/2. For clarity, all components were placed in the same membrane, even through EGFRs are located on the basolateral surface of polarized airway epithelia

TGFa and HB-EGF engage in juxtacrine signaling between neighboring cells, whereas their soluble forms are potent chemoattractants and mitogens. In intact epithelia, the membrane-bound ligands are localized at the apical surface, and are segregated from basolaterally located EGFRs by the tight junctions [54, 55]. An epithelial injury disrupts the tight junctions and allows EGFR transactivation by solubilized ligands, which induces ERK1/2- and PI3K/Akt-dependent repair responses, including proliferation and migration [53–55]. Several mechanisms have been shown to support the GPCR-mediated activation of EGFRs (review: [56, 57]). In 1321N1 astrocytoma cells, a ligand-independent

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mechanism was proposed, whereby P2Y2Rs bind and activate the tyrosine kinase, Src, which phosphorylates EGFR [58]. In these cells, dual immunofluorescence labeling revealed that exogenous UTP induces the clustering of P2Y2Rs and EGFRs to facilitate EGFR activation. In corneal epithelial cultures, Block et al. identified two mechanisms for EGFR activation, depending on the distance from the wound [59]. Time-lapse microscopy analysis revealed that ATP functions as a long range messenger to activate EGFR in cells distant (>0.5 cm) from the wounded area. The signaling pathway involved ATP-mediated activation of phospholipase D (PLD) by an unknown receptor. Near the edge, EGFR activation was insensitive to apyrase, but required phosphorylation by Src kinases. On the airway surfaces, this concept of ATP as a long distance messenger was proposed earlier by Homolya et al. [60]. Their study showed that gentle mechanical stimulation of a single cell induced waves of Ca2+ mobilization even in cells on the opposite side of the wound, which were inhibited by the ATP-hydrolyzing apyrase. Human airway epithelia support a ligand-dependent mechanism for GPCRmediated EGFR activation (Fig. 6.2). This path requires sheddases of the A Disintegin And Metalloproteinase family (ADAM) to solubilize the membrane-bound EGFR ligands (HB-EGF, TGFa) [57, 61]. The injury-related ATP release promotes the P2YR-mediated activation of ADAM-17, also known as TNFa-converting enzyme (TACE), and the subsequent membrane release of EGFR ligands [16, 62]. For this reason, the remaining discussion on the purinergic regulation of EGFRmediated wound healing focuses on this ligand-dependent signaling pathway.

6.3.4

NADPH Oxidase Activation in ATP-Mediated Stress/Wound Responses

The signaling cascades initiated by P2YR activation to induce EGFR transactivation were recently shown to require the oxidation of membrane-bound proteins (reviews: [63, 64]). Airway epithelial cells express two NADPH oxidase homologues, dual oxidase-1 and -2 (DUOX1 and DUOX2). Their principal function is to generate hydrogen peroxide (H2O2), which diffuses into the extracellular milieu to assist in bacterial killing. In addition, H2O2 regulates reduction-oxidation (redox) signaling pathways by the oxidative activation or inactivation of secondary messengers. In human airway epithelia, DUOX1 was immunolocalized to the apical membrane [65] with TACE and the membrane-bound EGFR ligands. While studying the regulation of mucin secretion, Nadel et al. discovered a DUOX1-dependent signaling pathway for EGFR transactivation in human bronchial epithelial cells [66–68]. They showed that neutrophil elastase, cigarette smoke and PKC activation all increase MUC5AC expression by a signaling cascade involving the DUOX1-mediated oxidative activation of TACE, which induces the release of membrane-bound TGFa for EGFR transactivation (Fig. 6.2). Since ATP is amongst the most potent stimuli for mucin secretion, other groups tested the role of P2Y2R-dependent PKC activation for EGFR transactivation. First, Forteza et al. showed that ATP, but not ADP,


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stimulates the apical production of H2O2 in polarized cultures of human airway epithelial cells, which was inhibited by the NADPH oxidase inhibitor, diphenyleneiodonium (DPI) [65]. In these cells, an injury induced H2O2 production, MMP-9 secretion and wound closure, which were all prevented by P2YR antagonists, apyrase or DPI [17]. Through carefully designed stepwise analysis, P2YRs were found to initiate the following signaling cascade on human airway epithelia: PKC/DUOX1/ H2O2/TACE/ pro-TGFa/ TGFa-EGFR [17, 52, 62, 69]. Collectively, these studies provide evidence that nucleotide release, initiated by epithelial damage or infection, initiates wound healing processes through P2YR-induced EGFR transactivation. The association between purinergic signaling and NADPH oxidase activation is not unique to airway epithelial cells, as similar links are emerging for cellular stress responses in macrophages, eosinophils and thyroid tumor cells [70–72]. For example, ATP release and purinergic signaling were recently implicated in neutrophil chemotaxis in response to fMLP, in association with neutrophil NADPH oxidase activation [73] (see Chap. 7 for details). Moreover, injury-induced ATP release and stimulation of P2-like receptors mediate wound responses in plants by mechanisms similarly linked to NADPH oxidase and the expression of wound response genes [74, 75]. Recently, ATP release in plant roots was found associated with regions of active growth and cell expansion, events also critically dependent on NADPH oxidase activation and oxidant generation [76]. In this respect, it is intriguing that the expression levels of P2Y2Rs and DUOX1 follow the same patterns over time in mammalian lung tissue during development [77, 78], both increasing during late gestation. These studies suggest a role for ATP-P2YRs-DUOX1 signaling during alveolar development.

6.4

6.4.1

Adenosine: Mediator of Migration and Neovascularization The ATP Metabolite, Adenosine, Promotes Migration and Angiogenesis

The release of ATP during cell injury or mechanical stress also raises the airway surface concentrations in its breakdown products, ADP, AMP and adenosine (ADO), generated by the activities of ectonucleotidases (see Chap. 2 for details) [79]. Among them, ADO is a signaling molecule which induces various cellular responses by the activation of the P1 family of G protein-coupled receptors (A1Rs, A2ARs, A2BRs, and A3Rs) (reviews: [80–82]). The general misconception that ADO does not contribute significantly to the wound healing mechanisms is based on two major factors. First, studies which compare the effects of exogenous ATP and ADO on wound closure, cell migration or proliferation generally used high micromolar concentrations of these agonists. The use of high (0.1 mM) ADO concentrations yielded no wound healing responses

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on epithelial cells of the airways [16, 18], the cornea [15] or the intestinal wall [14]. In contrast, carefully designed dose–response curves revealed that sub-micromolar levels of ADO (1 nM–1 mM) stimulate migration in human bronchial epithelial cells [83], and proliferation in airway smooth muscle cells [84]. In fact, higher concentrations had no effect on these wound healing responses [83, 84]. Furthermore, the maximal responses generated by ATP and ADO on the migration rates were, both, in the threefold range on BEAS-2B human bronchial epithelial cells [16, 83]. In this respect, the studies reporting a lack of response to apyrase should be revisited, since the metabolic elimination of the P2Y2R signaling would provide additional ADO for A2AR activation. The second argument for the limited role of ADO in the regulation of airway epithelial repair was based on the general notion that these cells normally express mainly (>90%) A2BRs, based on the RT-PCR analysis of mRNA level [85, 86], whereas healing properties are generally ascribed to A2ARs. However, A2ARs were functionally detected on the basolateral surface of human bronchial epithelial Calu3 cells with the selective agonist, CPCA [85]. Furthermore, using selective agonists for each ADO receptor, Allen-Gipson et al. demonstrated that ADO stimulates wound closure via A2AR activation on the BEAS-2B bronchial epithelial culture [87]. This signaling event led to the activation of protein kinase A (PKA), and selective inhibitors of PKA (KT-5720) reduced healing. In contrast, selective agonists of A1Rs and A3Rs induced PKC activation and reduced the wound closure rates [83, 87]. To this effect, it is interesting that PKA activation was shown to induce the migration of bronchial epithelial cells by disruption of the focal adhesions via inactivation of the small GTP binding protein Rho [88]. Collectively, these studies demonstrate that A2AR-mediated PKA activation is responsible for the stimulating effect of ADO on the migration of human airway epithelial cells into an injured area, by a mechanism involving cytoskeletal rearrangement. This signaling pathway also promotes extracellular matrix formation in the alveolar region. In the A549 human alveolar cell line, ADO induced the expression of fibronectin by a PKA-dependent signaling pathway involving cAMP production and the transcription factor cAMP Response Element Binding (CREB) protein [89]. Fibronectin is an essential component of the extracellular matrix also acting as a mediator of adhesion, migration and differentiation. Likewise, fibronectin was shown to induce cell proliferation and inhibit apoptosis in human bronchial epithelial cells [90]. In the vasculature, ADO is also an important mediator of cell migration and proliferation, especially during hypoxia. An emerging concept is that ADO released from hypoxic tissues has an important role in driving angiogenesis (review: [91]). The nucleoside may, either, affect endothelial cells directly, or stimulate the release of vascular endothelial growth factor (VEGF) from adjacent parenchymal cell, which binds VEGF receptor 2 on endothelial cells to stimulate proliferation and migration. In addition, ADO also directly stimulates collagen matrix formation. Collectively, these data are consistent with an in vivo study on external (dorsal excision) and internal (air pouch model) injury repair, indicating that ADO promotes healing, based on the A2AR agonist CGS-21680 and A2AR knockout mice [92].


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Hence, the contribution of ADO to the purinergic regulation of injury repair mechanisms should be investigated further to determine any interaction with EGFR-dependent signaling pathways, and the specific cellular functions, besides migration, which could be modulated by the A2AR-PKA-Rho signaling cascade.

6.4.2

Concerted Wound Responses by ATP and Adenosine Receptors

Since both ATP and ADO appear to be important for epithelial and endothelial cell migration and wound closure, the cell surface regulation of ATP metabolism may be critical for coordinated and directed cell migration, which involves a complex array of signaling pathways in various parts of the cell [33, 93]. The importance of extracellular purine metabolism was recently illustrated in studies demonstrating that cellular migration, angiogenesis or chemotaxis was impaired in cells lacking nucleoside triphosphate diphosphohydrolase 1 (NTPDase1, CD39), a surface enzyme that can hydrolyze ATP and ADP into AMP [94–96] (see Chap. 2 for details). For example, an in vivo study showed that Matrigel plugs containing growth factor implanted in mouse tissue are rapidly populated by monocyte/macrophages, endothelial cells and pericytes, and they also developed new vessels [96]. All these responses were impaired in CD39 knockout mice. In addition, the migration of microglia was stimulated by ATP [94]. Yet, ATP-mediated migration was impaired in microglia from CD39–/– mice and could be restored by addition of ADO [94]. Similarly, studies of neutrophils showed that CD39 localizes to the leading edge of polarized cells during chemotaxis to promote local ATP hydrolysis to ADO. Whereas ATP-P2Y2R signaling maintains the directionality of chemotaxis, ADO-A3R signaling accelerates the migration [73, 95] (see Chap. 7 for details). Based on these studies, it is plausible that regulated ATP hydrolysis may be important for optimal endothelial or epithelial wound responses, which remains to be ascertained.

6.5

Purinergic Receptors in Lung Injury, Mucus Metaplasia and Fibrosis

The integrity of the respiratory epithelium is often compromised in chronic diseases, such as asthma, COPD, idiopathic pulmonary fibrosis (IPF) or cystic fibrosis (CF), due to the repetitive injurious insults, ineffective or excessive repair. Structural changes include the disruption of gap junctions, partial shedding, basal or goblet cell hyperplasia and squamous metaplasia [7, 97, 98]. The process of epithelial regeneration upon injury is complex and involves the dedifferentiation of neighboring cells, which spread and migrate into denuded areas, after which proliferation and

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re-differentiation restore the functional epithelium. In asthma, several of these processes may be dysfunctional and result in a chronic and/or ineffective wound healing (review: [97]). Histological studies of bronchial biopsies typically show increased signs of airway epithelial damage with epithelial shedding, consistent with the presence of clusters of sloughed epithelial cells in the sputum [4, 99]. The large airways of asthmatic subjects also feature goblet cell hyperplasia, which is responsible for the excessive mucus production that contributes to airway obstruction. Goblet cell hyperplasia is also observed in bronchial biopsies from smokers with chronic bronchitis or COPD, although they are typically not associated with the loss of epithelial cells, but with an abnormal epithelial morphology characterized by squamous cell metaplasia (review: [99]). The parenchyma of IPF patients is particularly inflicted by extensive alveolar injury, mucus cell hyperplasia, excess matrix deposition and myofibroblast activation, leading to sub-epithelial fibrosis and the loss of lung function (review: [100]). The transition of differentiated epithelial cells to a mesenchymal phenotype generates myofibroblasts that play an important role in airway remodeling and fibrosis [101, 102]. These studies highlight the major contribution of aberrant epithelial repair in airway diseases. The airways of asthmatic, CF and COPD patients all accumulate abnormally high levels of ATP. Excess airway ADO is also reported in asthmatic and COPD, but not in CF, patients [13, 81, 103–106] (see Chap. 4 for details). Considering the stimulating effects of ATP and ADO on the migration and proliferation of many lung cell types, it is quite feasible that excess purines may be involved in the functional and structural changes gradually taking place in chronic airway diseases. For instance, P2Y2R activation is well known to stimulate mucin secretion in human airway epithelia (review: [107]). Yet, a study conducted on primary cultures of human bronchial epithelial cells does not support a role for these receptors in the development of mucin-related remodeling, as UTP, but not ATP, up-regulated the expression of MUC5B and MUC5AC [108]. In contrast, human nasal epithelial cells up-regulated MUC5AC in response to ATP in a dose-dependent manner [109]. This response required the activation of PLCb3, AKT and ERK1/2, but not PKC, ruling out P2Y2Rs. It is unfortunate that they did not consider the contribution of the ADO generated from ATP metabolism, especially since this epithelial response also involved CREB activation, which requires an increase in cAMP production induced only by A2ARs or A2BRs among the purinergic receptors. Considerable evidence exists for a major role of excess extracellular ADO in the development of epithelial injury and remodeling, as demonstrated by animal models of chronic lung inflammation (review: [80]) (see Chap. 8 for details). Mice lacking adenosine deaminase (ADA), the enzyme responsible for the elimination of intracellular and extracellular ADO, develop severe complications, including the infiltration of macrophages and eosinophils, mucus hypersecretion, fibrosis and airway hyperresponsiveness [110–112]. Interestingly, strong similarities in phenotypes were noted between ADA–/– mice and IL-13 transgenic mice, which were ascribed to an interactive regulation of ADO and IL-13 in lung tissue. While ADA–/– mice exhibit high total lung ADO and IL-13 levels [113], IL-13 transgenic mice maintain high ADO levels and low ADA activity [110, 114]. The existence of an ADO-IL-13


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amplification cycle of production is consistent with the typical airway remodeling of asthmatic patients. Their epithelial cells combine higher expression and activation of EGFR with an increased constitutive activation of Signal Transducer and Activator of Transcription (STAT)-1 and STAT6, which induce IL-13 expression (reviews: [115, 116]). Incidentally, long-term exposure to IL-13 induces mucus cell metaplasia on cultures of human bronchial epithelial cells [117, 118]. In addition, IL-13 dramatically modifies the differentiation process of ciliated cells [117]. Screening of mRNAs differentially expressed during mucociliary differentiation showed that several components involved in cell polarization (RhoA, G25K, bcatenin) were down-regulated in the presence of the cytokine, which functionally translated as lower ciliary beating frequencies. As such, factors which maintain high airway concentrations of IL-13 are expected to prevent ciliated cell differentiation and promote mucus hypersecretion. Incidentally, prolonged exposure to the stable ADO receptor agonist, NECA, raised the expression of IL-13 in primary cultures of human bronchial epithelial cells [119], supporting an indirect mechanism of airway remodeling for chronically-elevated airway ADO. Also, we presented evidence that ATP-P2YR signals activate EGFR, which induces the secretion of pro-fibrotic mediators, such as IL-13 and TGFb1 (review: [120]). In chronic inflammatory lung diseases, the excessive activation of these signaling pathways would induce abnormal or defective epithelial repair and remodeling, and eventually cause the loss of lung function.

6.6

Conclusion

The collective findings summarized in this Chapter establish important roles for ATP and ADO in various injury repair mechanisms for the airways and the vasculature, by the activation of a variety of purinergic receptors. While these mechanisms may be critical for normal wound responses and repair processes, such as epithelial regeneration or neovascularization, inappropriate regulation of purine release and/or metabolism in asthma, CF and COPD appear to precipitate the progression of lung complications. The crosstalk and convergence points identified between the purinergic and EGFR-dependent signaling pathways may eventually lead to the identification of novel targets for the prevention and/or reduction of the inadequate and problematic responses to injury.

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Chapter 7

Purinergic Regulation of Airway Inflammation Michael Koeppen, Francesco Di Virgilio, Eric T. Clambey, and Holger K. Eltzschig

Abstract The immune and inflammatory responses initiated by the interaction of a pathogen with airway surfaces constitute vital mechanisms to eradicate an infection. Sentinel dendritic cells embedded in the mucosa migrate to the lymph nodes to induce immune responses, whereas epithelial cells release chemokines to recruit inflammatory cells engaged in the active destruction of the intruder. All immune and inflammatory cells are regulated by customized purinergic networks of receptors and ectonucleotidases. The general concept is that bacterial products induce ATP release, which activates P2 receptors to initiate an inflammatory response, and is terminated by the conversion of ATP into adenosine (ADO) to initiate P1 receptormediated negative feedback responses. However, this chapter exposes a far more complex purinergic regulation of critical functions, such as the differentiation of naive lymphocytes and the complex maturation and secretion of pro-cytokines (i.e. IL-1b) by the “inflammasome”. This material also reconciles decades of research by exposing the specificity and plasticity of the signaling network expressed by each immune and inflammatory cell, which changes through cell differentiation and in response to infectious or inflammatory mediators. By the end of this chapter, the reader will have a new appreciation for this aspect of airway defenses, and several leads in terms of therapeutic applications for the treatment of chronic respiratory diseases. Keywords Chemotaxis Inflammasome P2X7 Phagocytosis Th17

M. Koeppen, E.T. Clambey, and H.K. Eltzschig Department of Anesthesiology, University of Colorado, Anschutz Medical Campus, Aurora, CO, USA e-mail: [email protected]; [email protected]; [email protected] F. Di Virgilio (*) Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy e-mail: [email protected]


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Introduction

Although this field has witnessed an explosive development over the latest 10 years, the notion that extracellular ATP and ADO are key regulators of immunity and inflammation is still alien to most immunologists. Strong effort is needed to overcome this stubborn and unjustified scepticism surrounding a vital aspect of immune regulation. The reader is referred to several excellent reviews that have been published lately on this topic [1–13] and to the special issue of “Purinergic signaling in the immune system” [14]. Technical advances are an irreplaceable complement of scientific intuition. The study of extracellular purines and pyrimidines is no exception to this rule, as witnessed by the boost provided to the purinergic hypothesis by the recent development of sensitive and reliable methods to measure ADO and ATP levels in whole tissues, and even in living animals. There is no doubt that the prophetic vision of Geoff Burnstock, which led to the groundbreaking discovery that ATP is an extracellular signaling molecule, opened the realm of purinergic signaling to scientific investigation (review: [15]). Yet, it would be difficult to persuade the scientific community of the crucial biological relevance of this notion without the help of novel techniques providing direct proof that extracellular ADO and ATP accumulate in vivo during pathophysiologically relevant responses. In recent years, Nicholas Dale perfectioned a bioelectrode technique for the measurement of ADO and ATP in conscious animals [16–18]. His group measured ATP and ADO concentration in the hypothalamus of rabbits during systemic inflammation [18]. However, these experiments did not address whether ADO and ATP levels change locally at inflammatory sites. Nevertheless, they document a clear temporal relationship between inflammation and purines within the brain, establishing a strong link between extracellular purine in the hypothalamus and fever, a common symptom of inflammation. Di Virgilio et al. developed a luminescence assay that allows in vivo imaging of extracellular ATP [19, 20]. They hypothesized that a cell expressing an “ecto-luciferase” might be used as an in vivo reporter of local extracellular ATP levels. The anticipation was that “ecto-luciferase-engineered” clones, once inoculated into live mice, would retain their ability to sense the ATP concentration of the tissue microenvironment and signal any change. Firefly luciferase is generally used as a reporter of gene expression [21]. A relevant exception is the recent use of luciferase in plants to measure extracellular ATP during root growth [22, 23]. These investigators constructed a chimera by appending a cellulose-binding peptide to firefly luciferase that was targeted to the plant root cell wall, and allowed recording of ATP levels on the root surface. Di Virgilio et al. followed a similar approach to engineer a construct in which luciferase was inserted between the plasma membrane targeting sequence (N-terminus) and the glycosylphosphatidyl inositol anchor (C-terminus) of the human folate receptor [19]. This construct (referred to as plasma membrane luciferase, pmeLUC) localizes to the plasma membrane of mammalian cells to measure ATP in the pericellular space (Fig. 7.1).

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Fig. 7.1 Structure and localization of the plasma membrane luciferase (pmeLUC) construct. (a) The structure comprises full length luciferase inserted in-frame between the N-terminal leader sequence (26 amino acids) and C-terminal glycosyl phosphatidyl inositol (GPI) anchor (28 amino acids) of the folate receptor. (b) Schematic rendering of cell surface localization of pmeLUC, where the enzyme measures ATP concentration near P2 receptors and ectonucleotidases (EctoATPases). (c) Immunofluorescence and (d) fluorescence-activated cell sorting (FACS) analysis of human embryonic kidney (HEK293) cells transfected with pmeLUC (HEK293-pmeLUC) or the empty vector (HEK293-mock) (Reproduced with permission from Pellegatti et al. [19])

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Human embryonic kidney 293 (HEK293) cells were stably transfected with pmeLUC, which measured the in vivo ATP content of interstitium with a detection threshold of 10 mM [19]. In healthy mice, the luminescence is basically undetectable, confirming the long-standing notion that physiological ATP levels in the extracellular space are in the sub-micromolar range. However, in two models of inflammation, cancer (Fig. 7.2a) and experimental colitis (Fig. 7.2b), luciferase-dependent light emission is very bright and long lasting, an unequivocal indication of a high ATP concentration in the microenvironment [20]. While it is obvious that these findings await confirmation in additional models of inflammation, they provide direct proof that ATP accumulates and can be quantitatively measured at sites of tissue injury or inflammation. Calibration of the luminescence signal

Fig. 7.2 Quantification of interstitial ATP concentration using the plasma membrane luciferase (pmeLUC) in murine models of cancer and inflammation. The nude mice were inoculated with HEK293 cells stably transfected with pmeLUC, injected with D-luciferin and imaged by IVISCaliper total body luminometer. (a) Mice bearing a human ovarian carcinoma. Reproduced with permission from Pellegatti et al. [20]. (b) Mice with colitis developed over 6 days by addition of dextran sodium sulphate to the drinking water


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indicates an extracellular ATP concentration in the 100 mM level, i.e. sufficient to activate even the low-affinity P2X7R. On the basis of this evidence, we can safely conclude that intracellular ATP is abundantly released into the interstitium under pathological conditions. Why? A simplistic explanation would be that ATP release is the mere epiphenomenon of cell damage or cell death. Given that the cytoplasm contains millimolar ATP, cell injury or cell death are expected to cause large increases in interstitial ATP concentration. Accruing evidence shows that the immune system has learned to exploit damage-induced ATP release as an early signal of cell damage [3, 24]. Many small molecules, normally sequestered within the cytoplasm, are released to act as damage or distress signals to alert the immune system of an impending danger, such as during trauma or infection [25]. These molecules recruit immune cells, prime them for effector functions, re-direct their differentiation, modulate their interaction with pathogens, and trigger the final steps in cytokine secretion. Extracellular nucleotides, especially ATP, are uniquely suited to perform these functions for the following reasons. First, their extracellular concentration under resting conditions is negligible, while the intracellular concentration is very high, which maintains a high signal-to-noise ratio. Second, nucleotides are water soluble, thus easily diffusible in the watery extracellular milieu. Third, once outside the cell, nucleotides are quickly destroyed by ectonucleotidases to promptly terminate each signal. Fourth, nucleotides interact with a family of highly selective receptors with a wide range of affinities, thus conferring high flexibility to the signaling network. Last but not least, ATP hydrolysis produces ADO, which has a remarkable immunosuppressive activity, and may act as a feedback signal to stop inflammation. A very elegant and definitive demonstration of the undisputable importance of purinergic signaling in pulmonary inflammatory diseases was provided by the elegant study of Idzko et al. [26], who showed that an allergen challenge causes the accumulation of ATP in the bronchoalveolar (BAL) fluid of asthmatic individuals and mice with experimentally-induced asthma. When they lowered (BAL) fluid ATP levels by instillation of the ectonucleotidase named apyrase, they documented a significant reduction of all signs of inflammatory responses to the allergen. A wide range of P2 receptor antagonists also exhibited strong antiinflammatory activities. Finally, they showed that high airway ATP levels initiate a Th2 type of inflammatory response characteristic of asthma, through the recruitment and activation of myeloid dendritic cells. This study unequivocally proves the pivotal role of purinergic signaling in the initiation and regulation of lung inflammation. Numerous groups are currently actively involved in the elucidation of the complex purinergic regulation of immune and inflammatory functions, and it would be unrealistic to cover them all in this section. Our primary goal is merely to highlight the beauty of the dualistic principles behind purinergic signaling, as an integrative mechanism involving ATP release, its conversion to ADO by ectonucleotidases, and the myriad of receptors mediating their actions in a timely fashion.

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General Structure of the Purinergic Network

Extracellular nucleotides regulate immune and inflammatory cells through different combinations of P2Y and P2X surface receptors (review: [1]). The first class is composed of G protein-coupled receptors responding to ATP (P2Y2R, P2Y11R) or ADP (P2Y1R, P2Y12R). Others are activated by pyrimidines, namely P2Y2Rs (ATP ¼ UTP), P2Y4Rs (UTP) and P2Y6Rs (UDP), although these agonists are released at concentrations less than 10% that of the purines. These cells also express members of the P2X family, which are ATP-gated non-selective cation channels. Signal transduction is mediated by rapid influx of Na+ and Ca2+, and efflux of K+, which depolarizes the membrane and raises the intracellular Ca2+ concentration ([Ca2+]i). They are inhibited by oxidized ATP, and activated by benzoylATP (BzATP), which shows little or no activity on P2Y receptors. In the extracellular space, ADO interacts with members of the P1 receptor family which all utilize intracellular cyclic AMP (cAMP) as secondary messenger [27–29]. The baseline ADO levels activate predominantly high-affinity A1Rs and A2ARs, whereas cell lysis or activation will raise ADO levels through ATP release and metabolism, and tilt the balance in favor of the low-affinity A2BRs and A3Rs [30–33] (Table 7.1). An additional level of sophistication is provided by the fact that a member of each subgroup will stimulate (A2ARs and A2BRs) or inhibit (A1Rs and A3Rs) the adenylate cyclase activity responsible for cAMP production [10] (Fig. 7.3). Thus, the amplitude and directionality of cellular responses to ADO are affected by local changes in extracellular concentration and the type of P1 receptor(s) expressed on any given cell [32, 34–40]. It is also important to mention that their expressions are modified under pathological conditions, especially A2ARs and A2BRs, which are highly susceptible to hypoxia, ischemia and lung injury [32, 35, 36, 38–43]. This information emphasizes the need for carefully designed experiments to decipher the role of ADO in the regulation of immune and inflammatory responses. All immune and inflammatory cells co-express several P1 and P2 receptors at their surface, which respond to different concentration ranges of ATP and ADO (Table 7.1). This complex purinergic regulation is amplified by the presence of highly efficient surface enzymes, named ectonucleotidases, which mediate the rapid conversion of P2 receptor agonists (ATP, ADP) into the P1 receptor agonist, ADO. Most immune and inflammatory cells utilize the CD39-CD73 tandem to convert extracellular ATP into ADO. Whereas CD39 dephosphorylates ATP and ADP into AMP, CD73 completes the dephosphorylation of AMP into ADO (see Chap. 2 for details) (review: [44]). Initially, local ATP release may activate, both, low-affinity P2X receptors and high-affinity P2Y receptors, the latter remaining active longer as ATP is gradually eliminated. In contrast, baseline ADO levels are generally sufficient to partially activate high-affinity A1Rs and A2ARs. Following ATP release, the local accumulation ADO would transiently recruit the activities of low-affinity A2BRs and A3Rs. This dynamic view of the purinergic signaling network should be kept in mind while the reader tackles the literature, as studies addressing each receptor individually may distort the overall impact of a signal.


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Table 7.1 Classification of the purinergic receptors in immune and inflammatory cells (References from the review: [81]) Affinity Receptor Ligand (EC50) mM Cell distribution ADO 0.18–0.53 Neutrophils, Monocytes/Macrophages A1 Dendritic Cells A2A ADO 0.56–0.95 Neutrophils, Monocytes/Macrophages Dendritic Cells, T and B Lymphocytes A2B ADO 16.2–64.1 Neutrophils, Monocytes/Macrophages T Lymphocytes ADO 0.18–0.53 Neutrophils, Monocytes/Macrophages A3 Dendritic Cells, T Lymphocytes P2X1 ATP 0.05–1.0 Neutrophils, Monocytes/Macrophages Dendritic Cells, T Lymphocytes, NK cells P2X2 ATP 1–30 ATP 0.3–1.0 P2X3 P2X4 ATP 1–10 Neutrophils, Monocytes/Macrophages, Dendritic Cells, T Lymphocytes, NK cells ATP 1–10 Neutrophils, Monocytes/Macrophages P2X5 Dendritic Cells, T Lymphocytes P2X6 ATP 1–12 ATP 100–780 Neutrophils, Monocytes/Macrophages P2X7 Dendritic Cells, T and B Lymphocytes NK cells P2Y1 ADP 8 Neutrophils, Monocytes/Macrophages Dendritic Cells, T Lymphocytes UTP ¼ ATP 0.14–0.23 Neutrophils, Monocytes/Macrophages P2Y2 Dendritic Cells, T Lymphocytes P2Y4 UTP >> ATP,UDP 2.5 (UTP) Neutrophils, Monocytes/Macrophages Dendritic Cells, T Lymphocytes P2Y6 UDP 0.3 Neutrophils, Monocytes/Macrophages UTP 6.0 Dendritic Cells, T Lymphocytes ATP 17 Neutrophils, Monocytes/Macrophages P2Y11 Dendritic Cells, T and B Lymphocytes P2Y12 ADP 0.07 Monocytes/Macrophages, T Lymphocytes ADP 0.06 Monocytes/Macrophages P2Y13 ATP 0.26 Dendritic Cells, T Lymphocytes P2Y14 UDP-glucose 0.1–0.5 Neutrophils Dendritic Cells, T Lymphocytes

7.3

Bringing Cells to the Scene of the Crime

Nucleotide release being so exquisitely sensitive to cell activation or damage, it would be surprising if airway defenses had not learnt to exploit gradients of extracellular nucleotides to recruit immune and inflammatory cells at sites of infection or tissue injury. The finding that pathogen-derived soluble factors induce ATP release from most of these cells [14, 37, 45–47] supports a critical role in

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Fig. 7.3 Signaling pathways of the adenosine (ADO) receptors. In most cells, the activation of Gi-coupled A1Rs and A3Rs inhibits adenylate cyclase (AC), leading to a reduction of intracellular cyclic AMP (cAMP) concentrations, whereas the Gs-coupled A2ARs and A2BRs cause the opposite effect. In some cell types, receptors coupled to Gq stimulate phospholipase C (PLC), which increases inositol (1,4,5)-triphosphate (IP3) production. The references are from excellent reviews: [27–29]

chemotaxis. This section describes how P2 (ATP) and P1 (ADO) receptors provide fine tuning of the directionality and speed of migration through ATP release and conversion into ADO by the CD39–CD73 tandem.

7.3.1

Resident Dendritic Cells: The Sentinels

As first line of innate defenses, resident immature dendritic cells (imDCs) are embedded within the mucosa, where they monitor airway surfaces for danger signals, such as dying cells and microorganisms (review: [48]). Upon internalization of an antigen, they migrate to the lymph nodes, where they activate naı¨ve T lymphocytes to initiate primary immune responses. The migration of dendritic cells is usually studied in vitro using isolated blood monocytes, which differentiate into imDCs by incubation with granulocyte-macrophage colony-stimulating factor (GM-CSF) [49]. These imDCs will mature in the presence of bacterial products, like lipopolysaccharide (LPS), or a cocktail of pro-inflammatory cytokines. Both, in vitro and in vivo studies demonstrated that the migration of imDCs to the lymph nodes is orchestrated by a complex purinergic network which evolves during maturation. The imDCs express P2Y1Rs, P2Y2Rs, P2Y4Rs and P2X7Rs (review: [1]). Idzko et al. showed that P2Y receptor agonists, including ATP, stimulate the migration of imDCs, but this response is lost during maturation [50]. This group recently identified the P2 receptor responsible for imDCs migration as P2Y2R [51]. The P2Y2R–/– mice exhibited significantly milder allergic airway inflammation, which was explained by a defective migration of blood myeloid imDCs. The stimulating effect of ATP on the chemotaxis of imDCs is rapidly terminated by their CD39-CD73 enzymatic machinery [52]. On the other hand, the resulting


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ADO has also been shown to interact with P1 receptors on DCs, namely A1Rs, A2ARs and A3Rs (review: [1]). On imDCs, ADO stimulates migration through activation of the adenylate cyclase suppressing receptors: A1Rs and A3Rs [53]. Interestingly, the maturation process taking place during chemotaxis causes an up-regulation of A2ARs and a down-regulation of A3Rs, which shifts cellular responses toward cAMP accumulation [49]. Incidentally, Hofer et al. showed that incubation of mouse skin explants with the stable ADO analogue named 50 -(Nethylcarboxamido)-adenosine (NECA) suppresses the emigration of mature DCs [49]. These studies highlight the amazing adaptability of purinergic signaling during the migration of DCs. Whereas ATP and ADO both stimulate chemotaxis of imDCs, the signal for arrival to the lymph nodes would be provided to mature DCs through changes in ADO receptor expression.

7.3.2

Monocytes and Macrophages

The purinergic networks of monocytes and macrophages are among the most extensively studied due to their critical role in the detection and destruction of apoptotic cells and invading pathogens (review: [1]). Elliott et al. demonstrated that the enzymatic removal of extracellular nucleotides, by apyrase (commercial form of CD39), abolished the capacity of supernatant from apoptotic cells to attract monocytes [54]. Likewise, the monocytes of CD39–/– mice showed no migratory response to ATP [55]. These studies showed that extracellular nucleotides are essential for monocytes to reach apoptotic cells. Interestingly, Lambert et al. discovered that their chemotaxis is stimulated by low ATP concentrations, whereas higher concentrations are inhibitory [56]. Therefore, migration rates are expected to gradually decrease as they move through higher ATP concentrations and closer to the site of apoptosis. The concentration-dependent directionality of ATP effects on the recruitment of monocytes is an important point which has clouded the views regarding the role of its metabolite, ADO. It is well established that monocytes and macrophages express CD39 and CD73 at their surface, which effectively dephosphorylate ATP into ADO (review: [1]). Therefore, the use of apyrase to remove ATP and ADP is expected to raise ADO concentrations around the monocytes, due to the highly efficient local CD73 activity. Incidentally, high ADO levels have been shown to inhibit the migration of monocytes [57, 58]. As they move toward the site of apoptosis in an increasing concentration of ATP, the apparent inhibitory effect of ATP would be mediated, indirectly, by surface conversion into ADO, and the increasing concentration of this P1 receptor agonist. In essence, the nucleotides released during apoptosis would act as a “find-me” signal for phagocytes, and the conversion of ATP into ADO as a signal of arrival. Purinergic signaling also stimulates the migration of monocytes toward a site of infection, as pathogens release nucleotides in response to cationic proteins

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released by inflammatory cells [59]. Furthermore, an in vitro study recently showed that monocytes respond to bacterial products by ATP release [60]. The removal of ATP and ADP, using apyrase, impeached on the cells ability to navigate toward the bacterial chemoattractant, C5a [60]. In a mouse model of C5a-induced peritonitis, apyrase was reported to impair monocyte recruitment to the site of infection [60]. This indirect, yet essential, mode of purinergic regulation has been documented in details for neutrophil chemotaxis, as will be discussed in the following section.

7.3.3

Polymorphonuclear Granulocytes

The migration of polymorphonuclear granulocytes (eosinophils, neutrophils and basophils) also falls under the control of purinergic networks. Animal studies have been very useful in identifying cell types targeted by extracellular nucleotides. For instance, P2Y2R–/– mice exhibited an impaired ability to recruit eosinophils to the lungs in response to an ovalbumin sensitization/challenge, routinely used to produce the general features of allergic asthma [51]. Interestingly, the eosinophils of asthmatic patients overexpress P2Y2Rs, which amplifies ATP-induced migration and the production of reactive oxygen species (ROS). On the other hand, asthmatic patients are well known to maintain high airway ADO levels [61–63]. While these cells express A1Rs, A2ARs and A3Rs, the impact of ADO on chemotaxis appears to be mediated by A3Rs (review: [64]). Knight et al. studied the pharmacology of blood eosinophils and determined that chemotaxis, triggered by platelet-activating factor (PAF), is inhibited by antagonists of A3Rs (2Cl-IB-MECA), but not A1Rs (CCPA) or A2ARs (CGS21860) [65]. In addition, tissue implantation of the A3R antagonist, MRS1523, prevented the development of lung eosinophilia in a mouse model of chronically-elevated lung ADO (ADA–/– mice) [66]. These studies are consistent with the pro-inflammatory reputation of A3Rs, and suggest that vascular injection of P1 receptor agonists or antagonists that would tilt the balance toward cAMP production, in eosinophils, could suppress airway inflammatory responses in asthma. The most fascinating discoveries on the purinergic regulation of chemotaxis were made for neutrophils, which offer a comprehensive mechanistic view of long-distance migration. Recent advances in our understanding of extracellular nucleotide metabolism corrected the original belief that neutrophils release AMP [47, 67]. Timecourse analysis of conditioned medium by HPLC demonstrated that neutrophils activated by the bacterial product, N-formyl-methionyl-leucylphenylalanine (fMLP), release ATP, which is rapidly converted into AMP [37, 68]. Mechanisms of ATP release from neutrophils are currently under intense investigation, which is beyond the scope of this chapter (review: [69]). In a very elegant study, Chen et al. studied the purinergic regulation of neutrophil chemotaxis using an imaging technique that monitored extracellular ATP levels around live cells during migration [68]. They observed that ATP is released predominantly from the protruding region at the front end (known as leading edge) of a migrating neutrophil, as it moves toward fMLP. The mechanical stress exerted


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by membrane deformation in this area of the cell membrane would be responsible for the local ATP release. The fact that apyrase nearly abolished chemotaxis supported a critical role for P2 receptors in the promotion of neutrophil migration. Quantitative PCR analysis showed that neutrophils express predominantly P2Y2Rs, A2ARs and A3Rs [68]. Based on this information, Chen et al. conducted a series of experiments to determine their respective roles in neutrophil migration [68]. Immunostaining revealed that P2Y2Rs are distributed uniformly on the surface of latent and activated neutrophils, which would allow them to act as gradient sensors to regulate the directionality of migration. This concept is supported by the lack of orientation of activated neutrophils in the presence apyrase, and neutrophils derived from P2Y2R–/– mice, in an fMLP gradient concentration. A survey of the ectonucleotidases expressed at the surface of human neutrophils suggests that the local conversion of extracellular ATP into ADO is mediated by CD39 and tissue non-specific alkaline phosphatase (TNAP) [70]. The ADO-generating enzyme generally expressed by immune and inflammatory cells, CD73, was not detected at the mRNA level. While the end product of CD39 activity is AMP, TNAP dephosphorylates ATP, ADP and AMP into ADO (review: [71]). The efficiency of TNAP toward ATP is notoriously lower than the other ectonucleotidases over a wide concentration range [72]. In contrast, Picher et al. demonstrated that TNAP dephosphorylates AMP more rapidly than CD73 at the concentrations (0.1 mM) [73] reached at the sites of inflammatory or infection [20] (Figs. 7.2–7.3). The selective expression of a high-capacity ADO-producing enzyme on neutrophils may represent an adaptation to optimize cellular functions in a nucleotide-rich environment. The activation of neutrophils with fMLP recruits CD39 to the leading edge, close to the site of ATP release [70]. A role for ATP metabolism in the purinergic regulation of neutrophil migration was inferred from studies in which CD39 activity was suppressed. The inhibitors, ARL67156 and sodium metatungstate (POM1), reduced migration speed, but not their ability to orient in an fMLP gradient [70]. An in vivo model of neutrophil migration revealed that they migrate 80% more slowly in CD39–/– than in wild-type mice. These data supported a role for ADO as regulator of migration speed during neutrophil chemotaxis. Incidentally, neutrophils express all P1 receptors (review: [1]). The fact that agents stimulating cAMP production reduce the mobility of human neutrophils [74, 75] supports an inhibitory role for A2ARs and/or A2BRs. In contrast, the A3Rs are normally sequestered in the intracellular compartment, but rapidly mobilized to the surface by a chemoattractant [68]. Furthermore, they localize preferentially at the leading edge, with ATP release, CD39 and P2Y2Rs. Antagonists of A3Rs reduced the speed of migration of neutrophils, but not their capacity to align in a chemotactic gradient [68]. In a murine model of sepsis, the lungs of A3R–/– mice accumulated less neutrophils than the wild-type mice [76]. A critical role for lowaffinity A3Rs, instead of high-affinity A1Rs, to suppress cAMP-mediated cellular responses is consistent with the abundance of ADO generated at the leading edge from ATP release and metabolism by the CD39-TNAP tandem. Collectively, these studies demonstrate that an infection triggers a reorganization of the purinergic network at the surface of activated neutrophils, allowing ATP

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release and metabolism to orchestrate two aspects of migration: directionality by ATP (P2Y2Rs) and speed by ADO (A3Rs). Based on the critical role of the P2Y2R as a vasodilator, therapies aiming to reduce neutrophilic airway inflammation using selective antagonists should be administered intraperitoneal to avoid the development of vascular complications, such as hypertension. This case highlights the importance of a broad understanding of purinergic signaling to design appropriate pre-clinical and clinical trials.

7.4

Phagocytosis of Apoptotic Cells and Pathogens

Circulating neutrophils and monocytes are cooperative partners in the phagocytic apparatus of innate immunity (review: [77]). They migrate to sites of trauma or infection to internalize and destroy apoptotic cells and pathogens. Given their common origin, they share many functions, including phagocytosis, antimicrobial and immunomodulatory activities. Yet, they acquire distinct features during differentiation, resulting in different levels of antimicrobial capacities and cytotoxicity, tissue localization and lifespan. Also, an important function of macrophages is the proper clearance of apoptotic neutrophils to avoid massive discharge of cytotoxic molecules on airway surfaces. Neutrophils eliminate pathogens by the processes of phagocytosis, degranulation and oxidative burst. Their cytoplasm contains secretory granules filled with cytotoxic molecules (reactive oxygen species, proteinases, cationic peptides). In most cases, they engulf the pathogen into a phagolysosome, which becomes highly toxic as granules fuse and empty their content. This mechanism maintains the microbicidal molecules confined into the cell until neutrophils are disposed off by macrophages. Occasionally, neutrophils initiate an extracellular killing mechanism for exceedingly large pathogens or unresolved infection. An oxidative burst initiates a cell death program leading to the formation and release of neutrophil extracellular traps (NETs) made of DNA strands and the cytotoxic granule proteins [78, 79]. This groundbreaking discovery was recently summarized in a review which includes a video explaining the methods used to study NETs (review: [80]). In respiratory diseases, characterized by excessive neutrophilic inflammation or oxidative stress, this secondary mechanism may become a precipitating factor in the destruction of the airways by microbicidal agents. This section provides an overview of the purinergic regulation of bacterial killing and phagocytosis. Since hundreds of studies addressed the purinergic regulation of bacterial killing, the readers are referred to an excellent review for most references to lighten the text [81]. Numerous studies have reported that ATP enhances phagocytosis and oxidative burst in neutrophils (review: [81]). Pharmacological characterization of P2Y receptors suggests that only P2Y2Rs are functionally expressed on human neutrophils [82]. The P2Y2R-mediated intracellular Ca2+ mobilization failed to induce cell degranulation, but primed the neutrophils for inflammatory mediator-induced elastase release and oxidative burst. Neutrophils were also shown to functionally


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express P2X7Rs [83]. The selective agonist, BzATP, directly induced an oxidative burst by the production of superoxide. The surface conversion of ATP into ADO amplifies bacterial killing by phagocytosis through high-affinity A1Rs (EC50 ¼ 0.2–0.5 mM) (review: [81]). However, exceedingly high ADO levels would favor extracellular killing, as the low-affinity A2ARs (EC50 ¼ 0.5–1.0 mM) has been shown to suppress phagocytosis, but to induce oxidative burst. Purinergic signaling imposes a similar bimodal regulation of bactericidal activities on macrophages (review: [81]). Whereas low micromolar ATP inhibits the secretion of proteolytic enzymes and stimulates phagocytosis via P2Y2Rs, high micromolar ATP recruits the activities of P2X7Rs to mediate opposite responses and tilt the balance toward extracellular killing. The importance of P2X7Rs is demonstrated by protocols interfering with receptor activation (monoclonal antibody or recombinant P2X7R), which reduced the capacity of human monocytes to phagocytose Staphylococcus aureus and Escherichia coli [84]. Likewise, low ADO levels preferentially bind the high-affinity A1Rs to amplify ATP-mediated phagocytosis. Interestingly, the relatively weak bactericidal capacity of macrophages may result from the distinct behavior of the low-affinity A2ARs, which do not amplify, but rather suppress the oxidative burst induced by P2X7Rs. Purinergic signaling also regulates the initial cell-cell interactions between the phagocytes and apoptotic cells or bacteria. Extracellular nucleotides (ATP, UTP, ADP and UDP) were shown to enhance the adhesion of macrophages to apoptotic cells by an up-regulation of b2 integrin CD11b/CD18 (Mac-1) and the vitronectin receptor (avb3, CD51/CD61) [85], which facilitates phagocytosis. Assays conducted with agonists and antagonists implicate the high-affinity P2X1Rs or P2X3Rs (EC50 ¼ 0.05–1 mM) in these responses [81]. A similar activation of Mac-1 by ATP and ADP has been reported for neutrophils [74, 86], although the mechanism has not been elucidated. Collectively, these studies demonstrate that purinergic signaling normally favors bacterial killing by phagocytosis. In situations of serious infection, excess of ATP and/ or ADO may be perceived as an alarm signal to initiate the more agressive extracellular mode of killing, mediated by pathogen entrapment and destruction within the NETs.

7.5

7.5.1

Complex Roles of P2X7Rs in Dendritic Cells/Macrophages The Regulation of IL-1b Maturation and Secretion

Phagocytosis and killing are only some aspects of the complex chain of events set in motion by pathogen recognition in immune cells. Additional fundamental reactions precede, proceed alongside and follow phagocytosis to optimize pathogen handling and amplify immune responses. In this context, cytokine and chemokine secretion is crucial. Therefore, it is not surprising that extracellular ATP keeps under such

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a tight control the maturation and release of IL-1b, a first-wave cytokine secreted very early to modulate all phases of inflammation [81, 87]. The production of IL-1b by mononuclear phagocytes is controlled at the transcriptional, translational, maturation and secretion levels. Bacterial products induce the transcription of the IL-1b gene and the cytoplasmic accumulation of pro-IL-1b. The maturation of the pro-cytokine occurs within a multi-protein platform named “inflammasome” [88]. Secretion follows soon after maturation, but the route for release is still controversial. As it turns out, extracellular ATP acting at the P2X7R is one of the most potent stimuli for inflammasome activation, and by consequence for IL-1b maturation [89]. The detailed mechanism coupling P2X7Rs to the inflammasome is not entirely uncovered, and might involve a perturbation in intracellular K+ homeostasis [90], and/or the plasma membrane protein pannexin-1 [91, 92]. However, irrespectively of the mechanism of activation, there is no doubt that a functional P2X7R is essential for the activation of the inflammasome by extracellular ATP [93]. Intriguingly, there is a striking difference in requirement for P2X7Rs between extracellular or intracellular pathogens for IL-1b maturation. Extracellular ATP and P2X7Rs are required for S. aureus and E. coli (extracellular pathogens), but not for Salmonella typhimurium or Listeria monocytogenes (intracellular pathogens) [94]. These intracytoplasmic pathogens may get in direct contact with inflammasome components to trigger complex assembly [94, 95]. It is important to mention that extracellular ATP is an incomplete stimulus for IL1b secretion, as it requires priming by bacterial products (typically LPS) [96, 97]. This is due to the inability of ATP to cause IL-1b gene transcription and mRNA translation. Nevertheless, once pro-IL-1b is present in the cytoplasm, ATP is an incredibly efficient stimulus for externalization of the mature cytokine. Two models have been proposed for IL-1b secretion: the modified lysosome pathway [98], and the microvesicle route [99]. The first model proposes that pro-IL-1b and procaspase-1 are targeted to a specialized lysosomal compartment before exocytosis. The second model maintains that pro-IL-1b and IL-1b are loaded into plasma membrane-derived microvesicles simply shed into the extracellular environment. In any case, P2X7R is the trigger for release in both models. Analysis of microvesicles shed from ATP-stimulated DCs showed that inflammasome components are also loaded into this compartment following activation, as if the whole inflammasome is secreted with IL-1b [100], and possibly delivered to the target cell. The immune system has evolved a sophisticated range of checks and controls to prevent undue activation in response to infection. The body is continuously exposed to foreign microorganisms, which are not always harmful (e.g. commensal bacteria), and against which it might even be detrimental to mount an immune response. To prevent inappropriate and potentially harmful reactions, innate immunity has evolved a two-step control mechanism: (a) the detection of the foreign microorganism, (b) and the verification of its actual pathogenicity. In other words, the presence of a pathogen does not induce an inflammatory response without unequivocal proof of its tissue-damaging activity [101]. For this purpose, two classes of molecules have been identified: pathogen-associated molecular patterns (PAMPs)


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signal the presence of the microorganism upon binding to Toll-like receptors (i.e. LPS), and damage-associated molecular patterns (DAMPs) signal cell injury using different receptors (review: [102]). According to this two-step process, only “PAMP-alerted” and “DAMP-stimulated” phagocytes express full effector function. In DCs, the fact that P2X7R activation only influences the late phases of IL-1b regulation fits well with the properties of a DAMP [11, 24]. Likewise, macrophages primed by LPS respond to P2X7R activation by the secretion of IL-1b and IL-6. However, ATP added prior to LPS inhibits IL-12p40 and TNFa secretion [103]. These data highlight the high degree of specificity of this two-step activation process. An interesting, and yet neglected, target for purinergic signaling in respiratory diseases is the eosinophil. These terminally differentiated effector cells are central to airway defenses against parasites, and in the pathogenesis of allergic diseases, as they secrete a wide array of cytotoxic proteins and inflammatory mediators. Extracellular nucleotides are particularly potent agonists for the secretion of IL-8 and eosinophil cationic proteins (ECP) from human eosinophils [104]. This study also revealed the sophistication of the purinergic regulation, as ECP secretion was mediated via P2Y2Rs, while IL-8 secretion was mediated via P2Y6Rs (UDP) and P2X7Rs. Hence, eosinophils express functional P2X7Rs, whose activity may have relevant effects on lung pathophysiology [105]. Pathogen killing has evolved into a complex process targeting common parasites and the so-called “phagocytosis-resistant” pathogens. Extracellular nucleotides extend their modulatory functions well beyond the initial phases of phagocytosis. Converging evidence from several laboratories demonstrated that intra-phagosomal pathogen killing is enhanced by stimulation of the P2X7R [106–108]. This is of relevance as macrophages are often infected by parasites (Mycobacteria, Leishmania, Chlamidia, Trypanosoma) that have evolved strategies to survive within the phagocytic vacuoles. The capacity of P2X7Rs to facilitate pathogen killing is likely an evolutionary adaptation of our innate immune system in the fight against so-called “phagocytosis-resistant” pathogens. This adaptive mechanism is epitomized by mycobacterial and chlamidial infections [106, 108]. Both pathogens survive within an endocytic compartment that avoids fusion with the lysosomes. Stimulation with ATP greatly accelerates the fusion of this endocytic compartment with lysosomes, leading to rapid killing and digestion of the pathogen.

7.5.2

Formation of Multinucleated Giant Cells and Fibrosis

A fascinating feature of alveolar macrophages is their capacity to fuse into multinucleated giant cells (MGCs) (review: [109]). They are reported in lung tissue of asthmatic, COPD, IPF and CF patients experiencing repetitive gastric aspiration. While the role of MGCs remains unclear in these diseases, they were found responsible for the lung complications of hypersensitivity pneumonitis. Also known as extrinsic allergic alveolitis, this granulomatous inflammatory disease is

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caused by repetitive inhalation of antigenic organic particles, and may develop into a debilitating fibrotic lung disease. In this respect, MGCs may contribute to the development of fibrosis in other respiratory diseases, such as IPF, CF and emphysema. Hypersensitivity granuloma formation is induced by T cell-mediated delayedtype hypersensitivity reactions to the antigenic organic particles invading the lung [110]. The circulating antigen-reactive memory CD4+ T cells migrate into the parenchyma, and they specialize into Th1 cells producing IFNg. This cytokine primes alveolar macrophages to secrete greater amounts of TNFa and IL-1b. The latter induce the autocrine secretion of biologically active mediators that attract young macrophages, promote their maturation and fusion into MGCs, which agglomerate with epithelioid cells to form granulomas. Despite the identification of plasma membrane proteins potentially involved in the formation of MGCs, the mechanism still eludes our understanding. Nonetheless, several lines of evidence implicate P2X7R activation. For instance, induction of monocyte fusion by concanavalin A is inhibited by an irreversible blocker of P2X7Rs (oxidized ATP), or a monoclonal antibody against P2X7Rs [111, 112]. Macrophages with high level of P2X7R expression undergo spontaneous fusion [113]. Furthermore, heterologous expression of P2X7Rs confers fusogenic ability [114]. During the phases preceding cell fusion, they relocate at sites of membraneto-membrane juxtaposition between adjacent cells [114]. Efficient MGC formation appears to require the entire purinergic axis supporting the actions of ATP and ADO, as the fusion of human monocytes is enhanced by selective A1R agonists and blocked by a A1R antagonist [115]. The role of ADO is stressed by the peculiar effect of ATP-hydrolyzing enzymes on MGC formation, as apyrase strongly potentiates the fusion process [113, 114]. These studies suggest that P2X7Rs and A1Rs work in concert to induce MGC formation. Therapies aiming to restore normal P2X7R and/or A1R expression may prevent or reduce the formation of pulmonary fibrosis.

7.6 7.6.1

Complex Purinergic Regulation of Immune Responses Dendritic Cells: Tilting the Th1/Th2 Balance

In view of the central role of DCs at the intersection between innate and acquired immunity, it is not surprising that purinergic signaling exerts such a complex modulatory activity on these cells. The imDCs lodged in airway mucosa respond to bacterial products by initiating their migration to the lymph nodes, during which they undergo maturation (review: [48]). Upon arrival, they secrete cytokines which dictate the differentiation of naı¨ve lymphocytes into T cell helper 1 (Th1) or T cell helper 2 (Th2) cells. In particular, DCs produce high levels of IL-12 to promote Th1 immune responses. This directionality influences the inflammatory responses


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to airway infection by the recruitment of distinct cell types, such as Th1-type neutrophils for CF and Th2-type eosinophils for asthma. Di Virgilio et al. showed that the presence of low micromolar ATP distorts the maturation process of LPS-exposed DCs towards a Th2 phenotype, depicted by the typical markers: CD54, CD80, CD83, CD86 and MHC-II [116]. These DCs also exhibited an altered pattern of cytokine secretion, with reduced the release of typical pro-inflammatory cytokines (TNFa, IL-1a, IL-1b, IL-6 and IL-12), and enhanced the release of the immunosuppressive cytokine IL-10 [116, 117]. The immunosuppressive effect of ATP is mediated by P2Y11Rs [118], and is amplified through metabolic conversion into ADO by the CD39–CD73 tandem of DCs (review: [81]). Chronic exposure to micromolar ADO stimulated the secretion of IL-10, but inhibited the secretion of TNFa and IL-12 [53, 81]. This phenotype has important physiological consequences for airway defenses against an infection, as ATP-pulsed DCs will promote the differentiation of lymphocytes into Th2, rather than Th1, cells. This observation is highly relevant for chronic diseases associated with high airway ATP concentrations and a predominant Th2 type of airway inflammatory responses, such as asthma. The purinergic regulation of DC maturation and reactivity is crucially dependent on local ATP concentrations. While low micromolar ATP induces a Th2 response and immunosuppression, higher concentrations have a potent Th1-skewing proinflammatory activity, as demonstrated by stimulation of IL-12 secretion [119]. Evidence suggests that the immunostimulating effects of high micromolar ATP are mediated by P2X7Rs. Murine fetal skin-derived DCs, lacking P2X7Rs, exhibited a reduced ability to secrete IL-1b and to activate T lymphocytes [120]. The phenotype of P2X7R-deficient DCs was partially reproduced by CD39–/– mice, as epidermal Langerhans cells lacking the ability to eliminate excess ATP showed a reduced ability to activate T cells [121]. In summary, the purinergic regulation of DC maturation dictates the directionality of lymphocyte maturation in the lymph nodes toward Th1 or Th2 types of inflammatory profiles, which represents a remote control of airway defenses to infection.

7.6.2

The Lymphocytes and Th17 Immune Responses

Although lymphocytes have been extensively tested for responses to purines, our understanding of purinergic signaling in these cells is still rudimentary (review: [81]). Lymphocytes are known to secrete ATP, express P2 receptors and ATP-metabolizing enzymes. Furthermore, ATP triggers various activities, including the shedding of plasma membrane proteins, and bimodal regulations of cell proliferation and IL-2 secretion [1]. Yet, few responses can be unequivocally traced to a specific P2 receptor and/or signal transduction pathway. For instance, while high micromolar ATP exerts a cytotoxic effect on lymphocytes, low concentrations promote cell

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proliferation, likely, via P2Y receptors [122, 123]. Surprisingly, P2X7R is the main P2 receptor mediating ATP-induced growth stimulation in T lymphocytes and leukemic B lymphocytes [124–126]. In addition, the ATP-mediated stimulation of CD23 and L-selectin shedding has been well documented and is highly reproducible, which has important physiological implications, as it prevents lymphocyteendothelium interaction and recruitment to the inflammatory site [127]. Thanks to their exceptionally high ectonucleotidase activity, lymphocytes are likely to regulate autocrine purinergic events, as well as those of interacting cells, such as DCs [128]. This is well epitomized by Foxp3+ Treg lymphocytes, which exhibit high CD39 and CD73 expressions [129, 130], a situation unique among lymphocytes. Their expression is driven by the specific transcription factor Foxp3, and their catalytic activity is enhanced by T cell receptor (TCR) ligation. Thus, activated Treg cells have at their disposal two highly efficient means to downplay immunostimulation: (1) by lowering extracellular ATP concentration; (2) by raising ADO concentration. Not surprisingly, CD39 null mice show signs of immune deviation towards a Th1 phenotype [131]. The investigation of complex immune responses is often biased by unavoidable simplifications of the experimental approach that makes it difficult to identify the final outcome of an immunomodulatory agent, when it depends on a cascade of cellular interactions. Typically, the “immunoshaping” effect of purines is best investigated within well controlled in vivo models (e.g. [26]). Very often, these experimental settings bring together, in a coherent picture, the scattered and apparently unrelated observations from many different sources. This is elegantly demonstrated by the study of Atarashi et al., reporting the ability of ATP to drive the differentiation of Th17 cells in the gut lamina propria [132]. The Th17 cells are key immunosuppressors concentrated in the intestinal mucosa, where they control bacterial and fungal infection [133] (Fig. 7.4). The process that drives Th17 cell differentiation is however undetermined. It is also well known that ATP is secreted by bacteria under resting conditions or in response to microbicidal peptides [134, 135], but the physiological significance remained elusive. An attractive possibility is that ATP release if one of those pathogen-initiated events shaping immune responses to commensal bacteria. In their study, Atarashi et al. showed that ATP released from gut bacteria activates a subset of CD70highCD11clow lamina propria cells to secrete IL-6, IL-23 and TGF-b, thus driving Th17 cell differentiation (Fig. 7.4). Interestingly, the CD39–CD73 machinery of adjacent Foxp3+ Treg lymphocytes has been shown to suppress their IL-17 production [136]. The mechanism required cell-cell contact and could be reproduced by ADO. This study highlights the importance of conducting in vivo studies to investigate the purinergic shaping of mucosal immunity. Cutting edge research is exposing a similar Th17 type of regulation in the airway mucosa (reviews: [137–139]). The differentiation of Th17 cells is normally restricted by Th1 and Th2 cytokines, including IFNg, which is generally low in chronic inflammatory diseases. Incidentally, high sputum IL-17 concentrations, and high numbers of IL-17F-positive epithelial cells are reported in asthmatic and COPD patients [140]. In mice, intranasal instillation of IL-17 receptor agonists


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Fig. 7.4 Schematic rendition of the role played by ATP in shaping early phases of immune response. (a) Under physiological conditions, at sites of contact with foreign microorganisms (exemplified by the gut lamina propria), baseline ATP release from the commensal bacteria induces a moderate stimulation of dendritic cells (DCs). This results in low level secretion of IL-6, IL-23 and TGF-b, together generating an immunotolerant microenvironment to prevent immuno-mediated tissue damage. (b) If mucosal damage or massive bacterial infection occurs, large quantities of ATP are released, which stimulate DCs to produce higher amounts of these factors (red), resulting in the expansion of the Th17 lymphocyte population, and activation of resident fibroblasts and macrophages

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induces eosinophilia, mucin hypersecretion and hypereactivity, which summarizes the major features of allergic asthma. In addition, IL-17 stimulates the secretion of neutrophil chemoattractants from airway epithelial cells, and thus, may contribute to the severe neutrophilia of airway diseases. Since airway diseases are characterized by high sputum ATP and/or ADO levels (see Chap. 4 for details), the emergence of Th17 signaling in human airways opens a completely new vector of research for purinergic drug development.

7.7 7.7.1

The Respiratory Epithelium Leukocyte Adhesion and Transmigration

Polymorphonuclear granulocytes are critical mediators of airway defenses that readily invade the interstitial space through endothelial barriers to eliminate pathogens. To avoid a catastrophic accumulation of activated granulocytes in lung tissue, their recruitment is normally balanced by efficient clearance mechanisms. Up until recently, phagocytosis was believed to constitute the main elimination mode for lung granulocytes. However, new data suggest a critical role for their transepithelial migration into the airways, where they are expelled by mucociliary clearance (MCC) (review: [141]). The critical role of MCC is highlighted by the overwhelming neutrophilia observed in the airways of cystic fibrosis (CF) patients, which results from an inherited inability to clear mucins and pathogens. On the other hand, granulocytes will also travel to the airways to target inhaled pathogens. Therefore, an efficient gating of endothelial and epithelial barriers is essential to control the extent of inflammatory responses in the lung and the airways, and to prevent tissue damage by excess bactericidal molecules. The purinergic regulation of neutrophil adherence to the endothelial barrier has been reviewed extensively [81]. On endothelial cells, P2X7R activation induces an up-regulation of E-selectins [142], while P2Y2R activation triggers the up-regulation of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) [143, 144]. Neutrophil adhesion to E-selectins and VCAM-1 initiates rolling along the endothelial surface, during which they are primed by ATP to express Mac-1 [145] for firm adhesion via ICAM-1. Simultaneously, ATP is being converted into ADO on the endothelium by the CD39–CD73 tandem. Initially, sub-micromolar ADO activates preferentially the high-affinity A1Rs on neutrophils and endothelial cells, which enhance neutrophil adhesion [146, 147]. Eventually, ADO levels increase and provide a negative feedback through A2AR- and A2BR-mediated cAMP production in the neutrophils, which downregulates Mac-1 (review: [81]). On endothelial cells, these high ADO levels also suppress adhesion by a reduction in VCAM-1 expression.


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Transmigration is facilitated by the activation of endothelial P2Y2Rs, which raise barrier permeability [148]. There is a general consensus that the increasing ADO levels eventually restore barrier function via A2BRs on the endothelial surface [37, 47]. Also, ADO inhibits the secretion of vascular endothelial growth factor (VEGF) from activated neutrophils, which is a potent inducer of endothelial permeability [149]. The adherent leukocytes have been shown to facilitate their passage by suppressing ADO production via CD73 on endothelial cells [150]. In fact, CD73 was initially identified as endothelial Vascular Adhesion Protein 2 (VAP-2) for lymphocyte adhesion [151], suggesting that CD73 inhibition is mediated by direct physical interaction. Following their passage, the restoration of ADO production would essentially close the gate. These studies indicate that ATP constitutes a pro-inflammatory mediator of neutrophil recruitment, whereas high ADO levels constitute a negative feedback to prevent excessive infiltration. Neutrophil transmigration across airway epithelia involves many factors common to the endothelial barrier (Fig. 7.5) (reviews: [152, 153]). Before adhesion to the basolateral surface can proceed, they must replace their L-selectins by Mac-1. Then, adhesion is initiated by binding of Mac-1 to fucosylated proteoglycans and

Fig. 7.5 Purinergic regulation of neutrophil transmigration across epithelia. Their adhesion to the basolateral epithelial membrane is initiated by binding of Mac-1 to fucosylated glycoproteins or JAM-C. They roll along the epithelial surface and the inter-epithelial tunnel by sequential binding to various epithelial molecules, including CD47 and SIRPa. Once at the tight junction, neutrophil JAML binds the epithelial coxsackie and adenovirus receptor (CAR). Once they have traversed the barrier, they adhere to the apical surface, where they resist fluid flow to constitute a defense barrier against invading microorganisms. Their adhesion is mediated by Mac-1 to ICAM1, and DAF to CD97. Purinergic signaling regulates the transmigration of neutrophils. The ATP released by activated neutrophils disrupts barrier properties, whereas conversion to ADO by the CD39–CD73 tandem terminates transmigration by restoration of the barrier properties

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JAM-C on the epithelial surface, which triggers the phosphorylation of myosin light chain (MLC) of tight junction complex to allow their passage. Transmigration follows by adhesive interactions between CD47 on PMNs and the epithelial surface. The JAM-like proteins (JAML) of neutrophils bind the coxsackie and adenovirus receptor (CAR) at the tight junction to complete their transmigration. Once they pass the tight junction, neutrophils transiently bind ICAM-1 and VCAM1 on the apical surface to provide a protective shield, while they eradicate airway pathogens [154]. Eventually, they undergo apoptosis and phagocytosis by macrophages, or they are cleared from the airways by the mucociliary escalator. On airway surfaces, the purinergic stimulation of mucin secretion interferes with neutrophil adhesion. While the bacterial product, fMLP, induced neutrophil adhesion equally well on endothelial and bronchial epithelial cultures, P2Y2R agonists (ATP, UTP and ATPgS) were 50% less efficient at inducing adherence on bronchial surfaces [155]. The role of mucins in the weak association of neutrophils to the airway surfaces was demonstrated by stable transfection of MUC1 in Chinese Hamster Embryo (CHO) cells [155]. Since MUC1 is an intrinsic plasma membrane protein, extending 0.5 mm into the airway surface liquid (ASL) layer, the authors suggested that this anionic mesh prevents neutrophil binding to the epithelial surface. In this case, how could they reconcile the equipotency of neutrophil adherence to endothelial and epithelial surfaces in response to fMLP? More likely, mucins secreted in response to P2Y2R activation (MUC5B and MUC5AC) [156] form a physical shield preventing neutrophils from adhering to the epithelial surface. This notion is consistent with the mucociliary clearance of neutrophils from the airways. Once they pass the tight junction, neutrophils remaining bound to the epithelial surface would conduct their bactericidal activities, before being released and trapped into the mucin layer for evacuation. During airway infection, there is considerable cell-cell communication between bacteria and epithelial cells, which weakens barrier integrity and promotes bacterial invasion into the interstitium. For instance, Pseudomonas aeruginosa has been shown to functionally express a surface adenosine deaminase. This enzyme activity reduces local ADO concentrations, which allows the bacterium to express the potent barrier-disrupting lectin PA-I [157]. This protein causes lethal sepsis in mice by allowing the passage of exotoxin A across the intestinal barrier [158], and is cytotoxic to human nasal epithelial cells in vitro [159]. These studies demonstrate the critical role of airway ADO in the protection of barrier integrity during infection. The purinergic regulation of neutrophil adherence to the basolateral surface of airway epithelia, and their transmigration into the lumen, has not been documented, despite the importance of this mechanism for the endothelium. In airway diseases, like CF, the large clusters of activated neutrophils observed under the barrier [160], likely, maintain high concentrations of ATP close to the epithelial surface, which is known to enhance barrier permeability in other systems [148]. Considering the problematic hyper-inflammatory responses of these patients to airway infection, this observation highlights the urgency of addressing this gap in our understanding of purinergic signaling.


7.7.2

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Epithelial Inflammatory Responses

Innate airway defenses are initiated by physical interaction of the bacterium with pattern recognition molecules on the epithelial surface. The pluripotent ion channel, CFTR, has been shown to bind and internalize P. aeruginosa LPS to initiate NFkBdependent inflammatory responses, including the secretion of chemoattractant cytokines to initiate the recruitment of inflammatory cells (review: [161]). In addition, the flagella interact with Toll-like receptor 5 (TLR5) and asialoGM1 to initiate NFkB- and ERK1/2-dependent signaling cascades which induce and/or require ATP release [162, 163]. This nucleotide is generally perceived as a critical mediator of airway clearance, which is released under stress conditions to eliminate a pathogen or irritant by P2Y2R-mediated flushing out of the airways (see Chap. 5). However, the compelling evidence that ATP initiates inflammatory responses in all immune and inflammatory cells paved the way for similar studies on airway epithelia. Inflammatory responses are generally initiated by the “first wave” cytokines, namely IL-1b and TNFa, locally released by resident DCs and macrophages alerted by the presence of an intruder. In the alveolar cell line A549, both IL-1b and TNFa raised the expression of A2ARs via NFkB activation [164]. Since airway epithelia normally express low levels of A2ARs, the initial response to infection primes the airways for injury repair by the ADO-A2AR axis (see Chap. 6). The most documented response of airway epithelia to “first wave” cytokines is the secretion of the potent neutrophil chemoattractant IL-8. Ribeiro et al. reported that the P2Y2R agonist, UTP, stimulates the secretion of IL-8 from cultures of human bronchial epithelial cells [165, 166]. Numerous studies addressed the identity of the secondary messengers mediating this inflammatory response. We are grateful to Dr. van der Vliet, who recently proposed a unifying mechanism for the purinergic regulation of pathogen-induced IL-8 secretion on human airway epithelia [167]. Their collective work recently culminated into an elegant study giving center stage to the DUOX1/EGFR-dependent signaling cascades. They showed that Toll-like receptor activation by bacterial products causes ATP release, which binds P2Y2Rs to induce H2O2 production by DUOX1. The peroxide activates surface sheddases, which solubilize EGFR ligands. The resulting EGFR activation induces ERK1/2 and NFkB activation, which promote IL-8 secretion. Other routes for the purinergic regulation of IL-8 secretion have been proposed in the recent years. For instance, P2Y14Rs were identified on alveolar A549 and bronchial BEAS-2B epithelial cells at the mRNA level, and by their responsiveness to the selective agonist UDP-glucose [168]. Their activation induced Ca2+ mobilization from intracellular stores and IL-8 secretion [168]. In tracheal epithelial cells, TNFa-induced IL-8 secretion was amplified by the selective P2X receptor agonist, BzATP [169]. This receptor family is generally perceived as a minor player due to their relatively low affinity for ATP. Yet, Di Virgilio has clearly shown that high micromolar ATP concentrations are maintained in areas of inflammation (Figs. 7.2–7.3). Furthermore, P2X7Rs are up-regulated on the airway surfaces of

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CF patients [170]. Thus, studies conducted on aseptic epithelial cultures may significantly underestimate the contribution of P2X receptors to airway defenses. Another well known inflammatory cytokine in the airways is IL-6, which binds the epithelium to induce mucin secretion and neutrophil infiltration (review: [171]). Contrary to IL-8, this cytokine is released on the apical (not basolateral) surface to mediate these autocrine responses [172]. On small airway epithelial cells, nucleotides increased IL-6 expression and stimulated secretion with an order of potency typical of P2Y2Rs (ATPgS > ATP ¼ UTP), and the associated phospholipase C (PLC)/MAP kinase-dependent signaling cascades [173]. The ATP-hydrolyzing apyrase partially reduced the effects of ATP, but not metabolically-stable ATPgS. These data suggested that ADO, generated endogenously from the metabolism of ATP on the airway surfaces [72, 73], participates in the regulation of IL-6. Incidentally, ADO was reported to stimulate IL-6 secretion from Calu-3 bronchial epithelial cells [172]. The use of selective receptor antagonists and RNA interference techniques revealed that the ADO receptor causing IL-6 secretion is A2AR. The common belief is that A2BRs are responsible for airway defenses, as in the case of MCC (review: [174]). This notion has been fueled by the relatively high mRNA levels of this ADO receptor, measured on aseptic cultures of airway epithelial cells [175]. However, we mentioned earlier that airway epithelia overexpress A2ARs in response to “first wave” cytokines, IL-1b and TNFa [164]. Given the importance of A2ARs for the regulation of cytokine secretion in inflammatory cells, this receptor should be revisited on airway epithelia under alarm situations which raise its expression, such as infection. The bactericidal capacity of the epithelial barrier is also enhanced by extracellular nucleotides. Pathogens, including P. aeruginosa [176] and Chlamydophila pneumoniae [177], induce the expression of chemokine ligand 20 (CCL20) in human airway epithelial cells, which is released into the ASL to directly mediate bacterial killing (review: [178]). The fact that P2Y2R (ATPgS, UTP, INS365) or P2Y6R (UDP, INS48823) activation was able to mediate both responses on human airway epithelia [179] supports a role for the pathogen-induced nucleotide release in the induction of bactericidal capacity. In allergic diseases, like asthma, IL-19 has been shown to play a significant role in the secretion of Th2 cytokines. Incidentally, airway epithelia constitute a major source of IL-19, where its secretion is stimulated by IL-4 and IL-13 [180]. In bronchial epithelial cells, the stable ADO receptor agonists, NECA, was reported to induce IL-19 secretion by a mechanism involving A2BRs [181]. The amount of IL-19 released in the conditioned medium was sufficient to induce TNFa secretion from monocytes, which signaled back on the epithelial cells to up-regulate A2BRs. This elegant cross-talk between purinergic and inflammatory mediators may amplify the damaging effects of the Th2 inflammation in asthmatic patients. Prostaglandins and leukotrienes are lipid mediators generated from the metabolism of membrane-derived arachidonic acid (review: [182]). They bind cell surface G protein-coupled receptors to regulate inflammatory processes. For instance, leukotrienes induce the recruitment and activation of various inflammatory cells, fibroblast proliferation and myofibroblast differentiation. On the other hand, the prostaglandin PGE2 enhances cell survival and migration, and suppresses


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pro-fibrotic responses mediated by leukotrienes. An imbalance in the levels of leukotrienes and prostaglandins in BAL fluid is associated with the development of pulmonary fibrosis. Studies showed that purinergic signaling up-regulates eicosanoids in human airway epithelia. In the bronchial cell line BEAS-39, the release of arachidonic acid was stimulated equally by ATP and UTP, supporting a role for P2Y2Rs [183]. In A549 alveolar cells, diadenosine tetraphosphate was implicated in the release of arachidonic acid [184], a dinucleotide known to be converted into ATP on human airway epithelia [185]. Incidentally, P2Y2R agonists (ATP, UTP and INS365) up-regulated by threefold the enzyme responsible for the conversion of arachidonic acid into prostaglandins, cyclooxygenase2 (COX-2), causing an increase in the secretion of PGE2 [186]. The surface metabolism of ATP by ectonucleotidases appears to initiate an amplification wave for this epithelial response. In the human bronchial epithelial cell line, Calu-3, A2BR activation by ADO levels detected in the airways of CF patients [1–10 mM; [63]] induced the apical secretion of arachidonic acid [187] and prostaglandins, but not leukotrienes [188]. Since the A2BR-mediated generation of prostanoids stimulates MCC via CFTR in Calu-3 cells [188], this pathway is expected to participate in innate defenses in diseases not characterized by functional mutations of this ion channel. Furthermore, the purinergic regulation of arachidonic acid metabolism appears to tilt the balance in favor of prostaglandin production, which is expected to enhance the healing capacity and reduce the development of fibrosis. To test this hypothesis, experiments remain to be conducted regarding the impact of ATP on the production and secretion of leukotrienes.

7.8

Conclusion

Our current understanding of the dynamic changes in extracellular ATP and ADO levels, initiated by an infection, allows us to postulate the following chain of events for the purinergic regulation of immune and inflammatory responses. Initially, the accumulation of extracellular ATP, in the presence of near-physiological ADO levels, synergizes with other pro-inflammatory factors to drive immune cell recruitment, cytokine and chemokine release, phagocytosis and the generation of extracellular NETs packed with various bactericidal compounds. An efficient pro-inflammatory action requires ATP levels to rise rapidly above a certain threshold (which in vitro data set at ~50 mM). If this threshold is not reached, yet DCs are exposed to low micromolar ATP for an extended time (12–24 h), they initiate an immunosuppressive response. Eventually, even pro-inflammatory responses, mediated by large releases of ATP, are terminated by the CD39-CD73 metabolic tandem, which also initiates an immunosuppressive response by the resulting ADO. The outcome of inflammatory responses depends on the complex balance between the generation and termination of these crucial events of purinergic regulation. Based on the data presented in this chapter, there is no doubt that understanding this complex interplay will open new avenues for the treatment of inflammatory diseases.

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Acknowledgements This work was supported by United States National Institutes of Health (grants R01-HL092188, R01-DK083385, and R01HL098294) to H.K.E and Deutsche Forschungsgemeinschaft research fellowship to M.K.

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Chapter 8

Animal Models of Airway Diseases Linda F. Thompson, Maryse Picher, and Michael R. Blackburn

Abstract Over the past 20 years, the growing awareness that purinergic signaling events literally shape the immune and inflammatory responses to infection and allergic reactions warranted the development of animal models to assess their importance in vivo in acute lung injury and chronic airway diseases. The pioneer work conducted with the adenosine deaminase (ADA)-deficient mouse provided irrefutable evidence that excess adenosine (ADO) accumulating in the lungs of asthmatic patients, constitutes a powerful mediator of disease severity. These original studies launched the development of murine strains for the two major ectonucleotidases responsible for the generation of airway ADO from ATP release: CD39 and CD73. The dramatic acute lung injury and chronic lung complications, manifested by these knockout mice in response to allergens and endotoxin, demonstrated the critical importance of regulating the availability of ATP and ADO for their receptors. Therapeutic targets are currently evaluated using knockout mice and agonists/antagonists for each ADO receptor (A1R, A2AR, A2BR, and A3R) and the predominant ATP receptors (P2Y2R and P2X7R). This chapter provides an indepth description of each in vivo study, and a critical view of the therapeutic potentials for the treatment of airway diseases. Keywords Adenosine deaminase CD73 CD39 Fibrosis Pulmonary edema

L.F. Thompson (*) Oklahoma Medical Research Foundation, Oklahoma City, OK, USA e-mail: [email protected] M. Picher Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA e-mail: [email protected] M.R. Blackburn Department of Biochemistry and Molecular Biology, University of Texas-Houston Medical School, Houston, TX, USA e-mail: [email protected]


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Introduction: Pulmonary Edema

Pulmonary edema is a life-threatening complication of acute lung injury, acute respiratory distress syndrome and pneumonia, which affects both barriers separating the bloodstream from the airspace: hyper-permeability of the pulmonary endothelial and epithelial barriers to macromolecules and inflammatory cells, and reduction of alveolar fluid clearance capacity mediated by ion channels (review: [1]). This complication is routinely documented in murine models targeting the enzymes and receptors regulating purinergic signaling. Yet, considerable discrepancies are reported between the beneficial or detrimental effects of receptor agonists/antagonists. The following section summarizes the current knowledge on the purinergic regulation of pulmonary edema taking place at the endothelial and epithelial barriers, which reconciles all apparent disagreements. This information will allow the reader to appreciate the impact of excess airway ATP and ADO in respiratory complications in vivo, the usefulness of certain animal models for drug development, and the appropriate administration route for therapeutic compounds.

8.1.1

The Permeability of the Endothelial Barrier

Until recently, our understanding of endothelial barrier regulation was limited to ADO, which plays an essential role in resealing the gate after the transmigration of inflammatory cells (review: [2]). Then again, we have learned through the first seven chapters of this book that purinergic signaling generally initiates an ATPmediated pro-inflammatory response, which is followed by an ADO-mediated antiinflammatory response. Therefore, several groups tested the impact of P2 receptor agonists on the regulation of endothelial barrier permeability. While the use of stable analogues and P2 receptor antagonists clearly demonstrated the sensitivity of the barrier to ATP, these studies provided contradictory conclusions (review: [1]). In this section, we provide a thorough analysis of all data which reconciles the literature under a unifying concept: opposing regulations of barrier permeability by P2Y1Rs (ADP) and P2Y2Rs (ATP), depending on local ATP/ADP concentrations. While these studies were conducted on various endothelia, these receptors are co-localized on pulmonary endothelial cells [3]. In human pulmonary artery and lung microvascular endothelial cultures, the low micromolar ATP concentrations induced a dose-dependent increase in transendothelial electrical resistance (TER) [4]. Likewise, 10 mM ATP reduced the thrombin-mediated barrier permeability of cultured human umbilical vein endothelial cells [5]. Similar data were obtained with non-hydrolysable ATP analogs known to interact with P2Y receptors, ruling out the possibility that these effects were mediated by ADO after ATP metabolism by the CD39-CD73 tandem [4]. This ATP concentration also inhibited the passage of macromolecules across endothelial monolayers, thus labeling this nucleotide as barrier protective [6]. Pharmacological

8 Animal Models of Airway Diseases

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Fig. 8.1 Purinergic regulation of fluid fluxes and barrier permeability in the lung. In the circulation, nucleotides regulate endothelial permeability to fluid and leukocytes by the balancing activities of ATP, ADP and ADO receptors. Whereas P2Y1R activation reduces the transmembrane electrical resistance (TER), P2Y2Rs, A2ARs and A2BRs all protect the lungs against vascular leakage. A second barrier protects the airways against excess fluid accumulation: the epithelial barrier. Alveolar cells express A1Rs and A2ARs which regulate fluid fluxes in and out of the airspace along ion gradients generated by the CFTR chloride (Cl–) channel and the ENaC sodium (Na+) channel. During viral infection, pulmonary edema is caused by the production, release and conversion of UTP into UDP, which activates P2Y6Rs to inhibit Na+ and fluid absorption via ENaC

analysis ascribed this response to P2Y2Rs which initiates a signaling cascade involving the activation of phospholipase C (PLC), but not PKC or Ca2+ mobilization. Incidentally, Kolosova et al. demonstrated that the enhancement of barrier integrity by low micromolar ATP involves reorganization of the intercellular tight junctions, which occurs independently of Ca2+ mobilization or ERK1/2 [4] (Fig. 8.1). The signaling cascade is reminiscent of P2Y2R-mediated induction of cell migration during epithelial repair (see Chap 6 for details). The P2Y2Rs located near tight junctions are coupled to G proteins Gq or Gi2 (not G12 or G13), which induce protein kinase A (PKA) activation, leading to the dephosphorylation of myosin light chain, and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). When phosphorylated, VASP inhibits actin polymerization and the formation of stress fibers that would disrupt the barrier.

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The barrier resistance of human umbilical vein endothelial cells is reduced by selective agonists of P2Y1Rs [7]. Contrary to P2Y2Rs, the signaling mechanism is Ca2+-dependent [7] and requires myosin light chain phosphorylation [8]. Incidentally, the use of ionomycin to trigger Ca2+ entry into the cells facilitated the passage of albumin across aortic endothelial monolayers [6]. On the other hand, this receptor is only activated by ADP, released at high micromolar concentrations by platelets and red blood cells. While CD39 expressed on the endothelial surface generates ADP from ATP, the enzyme does not allow this product to accumulate significantly before further dephosphorylation into AMP. This is an important point, since the affinity of P2Y1Rs for ADP (EC50 ¼ 8 mM) is considerably lower than P2Y2Rs for ATP (EC50 ¼ 0.2 mM) (review: [9]). Under normal conditions, low micromolar ATP would prevent pulmonary edema primarily via P2Y2Rs. On the other hand, massive local ADP release from platelets or red blood cells squeezing into the pulmonary vascular bed would transiently raise barrier permeability via P2Y1Rs, before the nucleotide is dephosphorylated by CD39. In vascular beds, P2Y1Rs have been shown to facilitate the passage of circulating cells through the narrow vessels by inducing nitric oxide-mediated vasodilatation [10]. However, conditions reducing CD39 activity, such as hypoxia/oxidative stress (see Chap. 4), may overwhelm the protective effects of P2Y2Rs and cause pulmonary edema. The highly efficient surface conversion of ATP and ADP into ADO generates a secondary wave of purinergic signaling for the regulation of endothelial barrier integrity. An in vivo study comparing the extent of hypoxia-induced vascular leakage among the knockout mice for each ADO receptor revealed that only the A2BR/ mice respond to hypoxia by significantly worse lung edema than wildtype mice, which was prevented by the A2BR agonist, BAY60-6583 [11]. The capacity of A2BR activation to raise endothelial TER was confirmed on human microvascular endothelial cultures using an antagonist (PSB 1115) and by RNA silencing technique [11]. In fact, Rounds et al. recently showed that A2ARs, A2BRs and ADO transporters work in concert to prevent pulmonary edema through endothelial barrier enhancement [12]. The adenosine deaminase inhibitor, pentostatin, raised surface ADO levels by tenfold, which enhanced the formation of endothelial adherens junction and focal adhesion through Rac1 GTPase activation. The fact that A1Rs and A3Rs were ineffective at regulating endothelial barrier permeability supports the well-established notion that barrier resistance is cyclic AMP (cAMP)-dependent (review: [2]). Under baseline conditions, ADO-mediated TER would be maintained by the high-affinity A2ARs (EC50 ¼ 0.560.95 mM) (review: [9]), whereas crisis situations raising circulating ADO levels (i.e. hypoxia or infection) would recruit the low-affinity A2BRs (EC50 ¼ 16.264.1 mM) (review: [9]) for additional protection against vascular leakage. The therapeutic potential of A2BR agonists for lung edema was demonstrated in a study conducted with endotoxemic pigs [13]. Endotoxin-induced acute lung injury and increase in lung extravascular water were, both, reduced by intravenously infused ADO. A similar response was obtained with the ADO receptor agonist, 2-chloroadenosine, in endotoxemic guinea-pigs [14]. Interestingly, the microvascular endothelial cells express predominantly ADO receptors causing an


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increase in cyclic AMP (cAMP) production (A2ARs and A2BRs), which leads to PKA-dependent VASP phosphorylation, like for the abovementioned P2Y2Rs [15]. These data suggest that circulating nucleotides normally maintain TER by successive waves of ATP- and ADO-mediated signals, which by the same token, would regulate leukocyte migration induced by inflammatory mediators.

8.1.2

Fluid Clearance on Alveolar Epithelia

The respiratory tract is frequently subjected to mechanical stress and infections, which stimulate the release of nucleotides into the airways. Decades of research clearly established that extracellular nucleotides are the major regulators of airway hydration (review: [16]). More importantly, they showed that the epithelial barrier regulates fluid fluxes by purinergic regulation of ion channels. The vectorial movements of Na+ and Cl– ions across the barrier create an osmotic gradient for water to follow. The absorption of Na+ by the epithelial sodium channel (ENaC) mediates fluid clearance, and Cl secretion by the Ca2+-dependent Cl– channels (CaCC) and cystic fibrosis transmembrane regulator (CFTR) hydrates airway surfaces. This subject is covered in details in Chap. 5 for the large airways, where nucleotides are identified as stimulators of fluid accumulation into the airspace. This concept seems to apply to the regulation of alveolar fluid clearance (AFC), as intra-tracheal instillation of ATP caused lung edema in mice [17]. This initial observation launched various initiatives to clarify the involvement of purinergic signaling in the impaired epithelial fluid clearance observed during pulmonary edema. In mice, the respiratory syncytial virus (RSV) causes a transient reduction in AFC, likely to promote clearance by the mucociliary escalator [18]. Using in vivo and in vitro studies, Matalon et al. showed that RSV impairs Na+-driven AFC through ENaC in distal lung and upper airway epithelia [19–21]. In the H441 Clara cells, RSV-mediated ENaC inhibition was partially prevented by blockers of nitric oxide production (1,400 W), or de novo UTP synthesis (A77-1726). During RSV infection, the accumulation of both purines (ATP, ADP) and pyrimidines (UTP, UDP) in the alveolar space [20], and the prevention of fluid accumulation by P2Y receptor antagonists [19], support a role for UTP synthesis and release in the purinergic regulation of AFC. The predominant P2Y receptor capable of mediating pyrimidinergic signals in the respiratory tract is the P2Y2R, which is activated by ATP and UTP, not by ADP or UDP (Chap. 7, Table 7.1). On the other hand, only soluble enzymes degrading pyrimidines (UDP-glucose pyrophosphorylase or apyrase) prevented RSV-induced edema [19]. In addition, intratracheal UTP and UDP, both, reproduced RSVmediated AFC reduction [18]. And, UTP is readily dephosphorylated into UDP by ectonucleotidases on airway surfaces [22, 23]. Contrary to the endothelial surface, the ectonucleotidase population of the epithelial cells allows for significant accumulation of UDP before it is dephosphorylated further into UMP and uridine (see Chap. 2 for details). These results raised the possibility that UTP and UDP,

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both, mediated their effects through P2Y6R activation. This receptor is functionally expressed on the apical surface of airway and alveolar epithelial cells (review: [24]). In A549 human alveolar Type 2 cells, P2Y6Rs activation accounts for the majority of Ca2+-dependent ATP secretion [25]. The identity of this receptor was later confirmed using mice infected with the influenza A virus [26]. Like RSV, this virus induced hypoxemia and inhibited AFC, and was associated with a rapid increase in ATP and UTP concentrations in the BAL fluid. The use of various ion channel blockers and P2Y receptor antagonists confirmed that viral infections induce de novo UTP synthesis and secretion, followed by UTP dephosphorylation into UDP and P2Y6R activation. This signaling cascade inhibits Na+ absorption by ENaC, allowing fluid to accumulate into the alveolar space. This purinergic receptor does not support the ATP-mediated pulmonary edema observed in mice during mechanical ventilation [17]. On the other hand, most purinergic networks accommodate a dual regulation of cellular functions mediated by the balancing effects of nucleotide (P2) receptors and ADO (P1) receptors. Davis et al. demonstrated that CFTR inhibitors, or agents preventing the formation of ADO from ATP metabolism, improved AFC during viral infection [26]. The use of selective P1 receptor antagonists identified the A1R as the culprit responsible for the CFTR-mediated reduction of AFC in infected mice. These findings were quite surprising considering the well established role of A2BRs in the stimulation of fluid secretion by CFTR in human airways (see Chap. 5). As will be described below, the purinergic network of the alveolar wall differs from the airways in terms of ectonucleotidases (see Chap. 2), as well as purinoceptors. We are grateful for Factor et al., who meticulously determined the identity and polarity of the ADO receptors on alveolar epithelial cells [27]. Based on laser capture microdissection and quantitative PCR, they showed that all four receptors are detected on the murine epithelial barrier. Contrary to the upper airways, alveolar cells predominantly express the A2AR, with an mRNA level at least fivefold higher than for the other receptors. Membrane fractionation indicated that A1Rs and A2ARs are concentrated on the apical surface, whereas A2BRs and A3Rs were undetected by Western blot. However, one must keep in mind that in vivo measurements of AFC will include the A2BR-mediated CFTR activation and fluid secretion taking place in the airways. Incidentally, the lung edema induced, in mice, by mechanical ventilation was significantly reduced by intratracheal instillation of an A2BR antagonist (PSB1115) [28]. To specifically study the purinergic regulation of alveolar ion channels, Factor et al. used two approaches: the alveolar cell monolayers and the isolated perfused lungs [27]. Both models revealed that low ADO concentrations (0.0110 nM) and A2AR agonists (CGS 21680) stimulate AFC via ENaC activation. In contrast, low micromolar ADO (1 mM) and A1R agonists (CCPA) reduce AFC via CFTR activation [27], as reported during viral infection [26]. Since both ADO receptors enhance opposing fluid fluxes across the epithelial barrier, the net directionality will depend on the local fluctuations in alveolar ADO concentrations. Collectively, these studies support a complex purinergic regulation of AFC which involves P2Y6Rs (UDP), A2ARs (ADO) and A1Rs (ADO) (Fig. 8.1). In healthy


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lungs, the mechanical stress induced by normal rhythmic breathing induces the release of purines and pyrimidines into the airspace. Under these conditions, only the ADO concentration maintained by ATP release/metabolism reaches the activation thresholds of purinergic receptors, which mediates a balanced ENaC/CFTR activation to optimize AFC. During mechanical ventilation, the excessive ATP release raises ADO levels, tilting the balance toward the CFTR-mediated fluid accumulation into the lung. In addition, mechanical ventilation activates lung phosphoinositides 3-kinase gamma, which degrades cAMP [29], thereby weakening the protective effects of the ADO-A2AR-cAMP pathway against lung edema. Consequently, patients with chronic illnesses associated with high airway ADO levels would be particularly at risk of developing pulmonary edema during mechanical ventilation, combining a dominant A1R-mediated flooding and a weak A2ARmediated AFC. Likewise, bacterial infection causes ATP release from alveolar epithelial cells [30, 31], and thus initiates the same A1R/A2AR imbalance toward flooding. In the case of viral infection, the de novo synthesis and secretion of UTP provides additional UDP for P2Y6R activation, which causes alveolar flooding via ENaC inhibition. In terms of therapeutic application, simultaneous inhibition of CFTR (CFTRinh172), and prevention of ENaC inhibition using a blocker of UTP de novo synthesis (A77-1726), restored normal AFC in infected mice [26]. While this approach seem attractive for the treatment of pulmonary edema, clinical studies reported considerable side-effects during treatments involving A77-1726, or its prodrug leflunomide (review: [32]). Inhibition of UTP synthesis within the cells is expected to impair critical functions, including DNA synthesis. Instead, the development of drugs targeting A1Rs, A2ARs or P2Y6Rs would allow more flexibility in dose optimization with minimal side-effects. Since CFTR and ENaC are primarily regulated by P2Y2Rs and A2BRs in the large airways, targeting these receptors would not compromise the clearance of pathogens by the mucociliary escalator. This section emphasizes the distinct purinergic regulations of barrier permeability on the endothelial and epithelial barriers separating the bloodstream from the alveolar space. Armed with this information, we will begin our description of the murine models which were developed to appreciate the contribution of impaired ATP and ADO regulation in the development of acute and chronic disorders, with a critical eye on the therapeutic strategies proposed to restore lung homeostasis.

8.2

Murine Models of Aberrant Purine Regulation

Murine models were developed to determine the contribution of aberrant ATP and/ or ADO concentrations to the development and progression of complex disorders, including respiratory diseases. Most mammalian cells regulate surface ATP and ADO by the sequential activities of CD39, CD73 and ADA. First, CD39 dephosphorylates ATP and ADP into AMP, and then CD73 converts AMP into ADO.

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Finally, cell surface ADA converts a fraction of the extracellular ADO into inosine, whereas the remaining portion is transported back into the cells through concentrative transporters (see Chap. 2 for details). Consequently, animal models were designed to target each of these ectonucleotidases. In the following section, we compare the phenotype of these mice to chronic respiratory diseases bearing the same metabolic disorders, to gain an appreciation of their role in the lung complications, and determine the potential of purinergic-based therapeutic approaches.

8.2.1

Too Much Adenosine in ADA-Deficient Mice

Humans with mutations in the ada gene resulting in a lack of enzyme activity suffer from severe combined immunodeficiency (ADA-SCID) [33]. Because ADA-deficient patients are extremely rare, considerable effort was expended in the generation of ada gene-targeted mice to better understand this genetic disease. Unfortunately, mice in which the ada gene was deleted in all tissues suffered from perinatal lethality [34, 35]. Surprisingly, the cause of death was not immunodeficiency, as they survive quite well in a clean animal facility, but rather hepatotoxicity. In an attempt to create viable ada/ mice, Blackburn et al. engineered an ada/ mouse strain that expressed an ada mini-gene in the placenta using trophoblast-specific controlling elements in the ada promoter [36]. These mice were viable and had normal liver function. However, they died at about 3 weeks of age from respiratory distress, characterized by severe inflammation, rapid and labored breathing. Their lifespan was extended by treatment with polyethylene glycol-conjugated bovine ADA (PEG-ADA) [37], the same material used for enzyme replacement therapy in ADA-deficient patients [38]. These mice received PEG-ADA intramuscularly every 4 days after birth, which partially suppressed the ADO-mediated lung inflammation. Contrary to intranasal instillation, intramuscular injection of PEG-ADA suppresses early events in the development of lung inflammation, like leukocyte and dendritic cell migration (see Chap. 7 for details). For the purpose of this review, these mice will be referred to as “ADA-deficient mice”. A second strain with partial ADA activity was generated by ectopic expression of a minigene in the gastrointestinal tract of ADA-deficient mice [39]. They developed a milder pulmonary disease and had a lifespan of about 5 months. They will be referred to as “partially ADA-deficient” mice. The ADA-deficient mice appeared normal at birth, but their lungs exhibited larger alveolar spaces by day 5 [36, 37]. There was no inflammatory cell in the lungs at that time, so the defect in alveogenesis preceded the onset of inflammation, which occurred around day 10. By day 18, abnormal alveogenesis was obvious, and was characterized by thickened vascular smooth muscles and marked enlargement of the alveolar spaces. There was also hypertrophy of bronchial epithelia with increased mucus production, and cell debris accumulated in the airways. Most inflammatory cells in bronchoalveolar (BAL) fluid were macrophages, in the form of enlarged and foamy multinucleated giant cells, clustered around bronchioles and


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pulmonary vessels. Few eosinophils accumulated in the interstitium and the luminal space. It was suspected that these structural changes and inflammatory responses were caused by the excess ADO measured in the total lung tissue of ADA-deficient mice. Whereas lung ADO levels were barely detectable in wild-type mice, they rose about 20-fold in ADA-deficient mice, reaching a concentration of 4 nmolmg1 protein. The concentration of the other ADA substrate, deoxyADO was also elevated, but to a lesser extent (80% in P2Y2R/ mice, compared to their wild-type littermates [130]. These studies demonstrate that the “danger signal”, provided by stress-induced ATP release, triggers airway clearance primarily by P2Y2R activation in murine airway epithelia. The beneficial effects of P2Y2R signaling is extended to the mobilization of cells engaged in the destruction of inhaled pathogens, including monocytes/macrophages and leukocytes. In most instances, these actions are essential to resolve airway infection. On the other hand, chronic or exaggerated activation of these functions lead to irreversible damage to the respiratory system. Whether P2Y2Rs are beneficial or detrimental in these two scenarios was examined using models of acute and chronic lung complications. The overall impact of P2Y receptors on the susceptibility to airway infection was investigated using P2Y1R/P2Y2R double knockout mice [131]. When subjected to intra-tracheal instillation of the common airway pathogen, Pseudomonas aeruginosa, all the double knockout mice succumbed within 30 h, whereas 85% of the wild-type mice lived a normal lifespan. After 24 h, the BAL fluid of P2Y1R/ P2Y2R/ mice contained more proteins as a measure of edema, but less proinflammatory mediators (IL-1b, IL-6, GM-GSF and MIP-2). In contrast, the two mouse groups presented similar total cell counts and cell composition, which were primarily neutrophils (>80%). These data suggest that P2Y receptors exert a protective role during lung infection by suppressing pulmonary edema, while promoting the inflammatory responses required to eradicate the infection. The severe pulmonary edema developed by the P2Y1R/P2Y2R/ mice is consistent with our model of fluid regulation (Fig. 8.1), whereby the loss of endothelial P2Y2Rs would prevent ATP from protecting barrier functions. On the other hand, it is intriguing that these mice accumulate BAL neutrophils to the same extent as the wild-type mice, given the critical role of P2Y2Rs for their chemotaxis (see Chap. 7 for details). Unfortunately, double knockout mice are of limited value to discriminate the roles of each receptor. An acute model of airway infection was used to determine the impact of P2Y2Rs localized in the vasculature, on circulating cells or the endothelial surface [132]. Wild-type mice received intravenous saline, or a selective P2Y2R agonist (ATPgS), before intranasal instillation of LPS. The mice which received the P2Y2R agonist exhibited about 50% less inflammatory cells, cytokines and proteins in the BAL fluid, than the saline-treated LPS-exposed mice. The protective effect of ATPgS against vascular leakage was confirmed by Evans blue albumin extravasation into the lung tissue, pointing toward a primary function at the endothelial barrier. This was confirmed in vitro in human lung microvascular endothelial cultures, where ATPgS significantly inhibited the LPS-mediated reduction of transmembrane electrical resistance. These data suggest that intravenous P2Y2R agonists may reduce the airway inflammatory responses and lung injury caused by airborne pathogens. Using this administration route, ATPgS would not promote neutrophil chemotaxis through tissue along a chemotactic gradient [121], which is a major role postulated for this P2Y receptor.

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On inflammatory cells, P2Y2R activation initiates the pro-inflammatory responses required to eradicate microbial pathogens through a series of highly coordinated events culminating in phagocytosis and destruction. In pathological circumstances, like sepsis, they release antimicrobial compounds which cause injury and death of bacteria and host cells (see Chap. 7 for details). The lung damage associated with excessive neutrophilia contributes to a wide array of respiratory complications, including ARDS, COPD and CF (review: [133]). Incidentally, genetic deletion of the P2Y2R improves the survival of mice subjected to sepsis by cecal ligation and puncture [134]. These mice exhibited fewer lung neutrophils and less pulmonary damage than wild-type mice. Since P2Y2R activation provides directionality during neutrophil chemotaxis [121], the leukocytes of P2Y2R/ mice lack the ability to follow an ATP gradient toward the lung. This study suggests that intraperitoneal P2Y2R antagonists may improve the survival of sepsis patients. Chronic respiratory diseases are commonly associated with elevated airway ATP concentrations, as reported in CF, IPF and COPD patients [135–138], and they are raised by allergens in asthmatic patients [68]. The expression of P2Y2Rs is also expected to be elevated in these diseases, as chronic exposure to pathogens or allergens up-regulate the receptor in animal models. For instance, house dust mites caused an increase in P2Y2R expression in eosinophils and dendritic cells from asthmatic patients, which significantly enhanced chemotaxis [139]. It is important to point out that cells from healthy subjects did not up-regulate P2Y2Rs in response to house dust mites, indicating that the purinergic regulation of inflammatory responses is not normally modified by allergens in the general population. On the other hand, rat alveolar Type 2 cells exposed overnight to LPS reacted by raising P2Y2R transcripts by threefold, which resulted in a significant increase in the ATPmediated surfactant secretion [30]. Whereas P2Y2R overexpression may improve MCC, the pro-inflammatory reactions of immune and inflammatory cells may aggravate airway inflammation. Therefore, it is particularly important to understand the costs and benefits of P2Y2Rs in chronic diseases before we can envision therapeutic applications. Asthmatic patients maintain normal airway ATP levels, but these concentrations rise tenfold in response to an allergen [68]. The consequences of this excess ATP were examined in the OVA sensitization/challenge model of allergic asthma [68]. The mice are sensitized by intra-peritoneal injection, and then challenged by nebulization 10 days later. After 24 h, their BAL fluid had accumulated eightfold higher ATP concentrations than in the saline-challenged control animals, as reported in the allergen-challenged asthmatic patients. They also developed all major features of allergic asthma, including eosinophil-dominant lung infiltrates, goblet cell metaplasia, hyperresponsiveness to methacholine and Th2 inflammation in the lymph nodes (IL-4, IL-5 and IL-13). These complications were significantly reduced when the OVA challenges were conducted in the presence of an ATPmetabolizing enzyme (apyrase) or P2Y receptor antagonist (PPADS or suramin). Through a series of elegant in vivo and in vitro protocols, Idzko et al. demonstrated that the excess ATP generated after an allergen challenge triggers airway inflammation by the activation of resident dendritic cells, and their mobilization to the


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lymph nodes to trigger a Th2 type of inflammation. It is interesting that excess ATP in the BAL fluid was not detected 10 min, but rather 24 h, after the challenge in asthmatic patients and in the mice. The mechanisms behind the delayed accumulation of ATP into the airways have not been identified. However, oxidative stress building gradually with airway inflammation has been shown to inhibit CD39 [140], and ATP-hydrolyzing ectonucleotidases expressed on airway epithelial [141], immune and inflammatory [142] cells. Consequently, this study supports the therapeutic potential of P2Y receptor antagonists for the treatment of airway inflammation in asthmatic patients. More recently, P2Y2R/ mice were subjected to this model of allergic asthma to identify the mechanism by which P2Y2Rs promote eosinophilia [139, 143]. The P2Y2R/ mice exhibited a weaker capacity to recruit eosinophils in response to the OVA exposure protocol than the wild-type mice [143]. In vitro assays showed that circulating dendritic cells and bone marrow eosinophils collected from P2Y2R/ mice fail to migrate within an ATP gradient [139]. Three findings support a role for the P2Y2R-mediated up-regulation of VCAM-1 in the purinergic stimulation of lung eosinophilia. This molecule is a potent chemoattractant and adhesion molecule for eosinophils (review: [144]). First, the OVA challenge caused the accumulation of soluble VCAM-1 in the BAL fluid, but to a lower level in P2Y2R/ mice [143]. Second, OVA also induced an up-regulation of membranebound VCAM-1 at the surface of lung endothelial cells, but to a lower extent in P2Y2R/ mice. Finally, adhesion assays confirmed that ATP promotes eosinophil adhesion to the endothelial surface through a P2Y2R-mediated increase in VCAM-1 surface expression. Collectively, these studies suggest that an amplification of the ATP-P2Y2R signaling in asthmatic patients during an allergic reaction, contributes to the development of lung eosinophilia. Accordingly, asthmatic patients may benefit from the intravenous injection of P2Y2R antagonists to suppress eosinophil recruitment to the airways. Chronic obstructive pulmonary disease is another interesting example of P2Y2Rdominant lung pathology. The discovery that the murine model of smoke-induced lung inflammation and emphysema reproduces the high airway ATP concentrations of COPD patients provided the necessary tools to investigate the role of purinergic deregulation in the pathogenesis of this disease [145]. First, the role of ATP in the early events of airway inflammation was investigated using an acute cigarette smoke exposure protocol, which consisted of a daily 20 min session for 4 days. This acute insult caused an eightfold increase in BAL fluid ATP level, the typical macrophage and neutrophil infiltrates, and the accumulation of their cytokines (IL-1b, IL-6, IFNg, MIP-2 and KC). Intratracheal instillation of the ATP-metabolizing apyrase, or a P2Y receptor antagonist (PPADS or suramin), before each smoke inhalation reduced all inflammatory parameters by more than 50%. This acute protocol shows that the excess ATP, accumulating after the first cigarettes, is critical to the early development of smoke-induced airway inflammation. The contribution of ATP-induced inflammatory responses to the development of emphysema was studied using a chronic smoke exposure protocol lasting 4 months [145]. These mice developed foci of emphysema throughout the parenchyma,

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which was essentially prevented by oral administration of a P2Y receptor antagonists, suramin or PPADS. Real-time PCR analysis of P2 receptor expression in the inflammatory cells and lung parenchyma revealed a dramatic (eightfold) and selective up-regulation of P2Y2Rs in the neutrophils of smoke-exposed, compared to air-exposed, animals. The importance of the amplified ATP-P2Y2R signaling axis in the development of smoke-induced airway inflammation and emphysema was clearly demonstrated using P2Y2R/ mice and chimera [137]. While the source of BAL fluid ATP has not been addressed, in vitro experiments showed that human neutrophils release ATP in response to cigarette smoke in a dosedependent manner, and as efficiently as fMLP [137]. Also, cigarette smoke induced an ATP-dependent secretion of neutrophil elastase, which is an important mediator of lung destruction in emphysema (review: [146]). These studies suggest that the initial increase in BAL fluid ATP concentration detected after the first cigarette exposures initiates the development of airway inflammation, which is amplified over time by an up-regulation of P2Y2Rs on immune and inflammatory cells (see Chap. 7 for details). Therefore, the oral prescription of apyrase, or a P2 receptor antagonist, could prevent and reduce the irreversible lung damage and loss of lung function in COPD patients.

8.4.2

The P2X7R in Chronic Lung Diseases

For years, the importance of P2X7Rs for ATP-mediated inflammatory responses has been largely underestimated on the premise that the EC50 of this receptor is orders of magnitude above that of P2Y2Rs (review: [9]). However, the elegant work of Di Virgilio et al. clearly demonstrated that sites of inflammation or tissue injury accumulate ATP in the high micromolar range in vivo, above the activation threshold of P2X7Rs (Chap. 7, Figs. 7.1 and 7.2). The recent finding that major pro-inflammatory cytokines involved in the initiation of airway inflammatory responses (i.e. IL-1b) depend on the P2X7R for their production, maturation and secretion (see Chap. 7 for details) is only one example of the many critical roles emerging for this receptor. Therefore, it is not surprising that the inhibition or deletion of P2X7Rs has such profound implications for airway defenses. The mouse model of COPD, described above by Idzko et al., revealed a remarkable up-regulating effect (10- to 20-fold) of cigarette smoke on the P2X7Rs of neutrophils and macrophages within 4 days [145]. The use of selective antagonists demonstrated that P2X7R activation on these cells promotes all aspects of airway inflammation and fibrosis leading to the development of COPD and emphysema. In wild-type mice subjected to the acute smoke protocol, intratracheal injection of KN62 or A438079, before each exposure, markedly reduced the accumulation of macrophages, neutrophils and their cytokines (IL-1b, IL-6, IFNg, MIP-2 and KC) in the BAL fluid [147]. In mice subjected to the chronic exposure protocol, these agents prevented the development of fibrosis and emphysema. The widespread


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effects of P2X7Rs on airway inflammation are explained by their pivotal role in the secretion of IL-1b from the resident macrophages, which initiate the events leading to leukocyte recruitment. Furthermore, P2X7R-overexpressing neutrophils and macrophages would cause considerable lung destruction by massive discharge of potent cytotoxic molecules which do not discriminate between the bacteria and host cells [146] (see Chap. 7 for details). Since smoke triggers such dramatic increases in BAL fluid ATP level [145] and P2X7R expression, an early treatment with aerosolized antagonists may reduce disease progression and prevent emphysema. Idiopathic pulmonary fibrosis (IPF) is a fatal respiratory disorder characterized by inflammation, tissue fibrosis and destruction of the alveolar architecture. Since the IPF patients maintain high airway ATP concentrations [138], the bleomycininduced murine model of lung fibrosis was used to test the contribution of purinergic deregulation to the pathogenesis of this disease [138]. In mice, the intratracheal administration of bleomycin initiates neutrophil recruitment within 24 h, which normalizes around day 11. First signs of fibrosis appear around day 11, and are reversed within 10 days (review: [65]). Time-course analysis indicated that bleomycin induces a transient increase in BAL fluid ATP level over the first 6 h, which is followed by the initiation of neutrophil recruitment. This time line supports a role for an ATP gradient in neutrophil recruitment to the lungs. Disruption of this ATP gradient by apyrase treatment reduced by >60% the BAL content in neutrophils, myeloperoxidase and metalloproteinases. Similar findings were obtained when comparing wild-type and P2X7R/ mice after bleomycin exposure [138]. This study suggests that the higher availability of ATP for P2X7R activation contributes to the inflammatory processes launching tissue fibrosis and lung destruction. In asthmatic patients, airway ATP concentrations only rise following an allergen challenge [68]. On the other hand, the purinergic network is permanently altered in these patients, as P2X7R expression is up-regulated in BAL fluid macrophages and circulating eosinophils, compared to healthy subjects [148]. A murine model of allergic asthma was used to determine the consequences for airway defenses. Mice sensitized and challenged with OVA summarize the inflammatory responses of asthmatic patients during an allergic reaction [148]. The lung infiltrates contain mainly eosinophils, followed by mast cells, neutrophils and lymphocytes, whereas BAL fluid exhibits the typical Th2 inflammation (IL-4, IL-5 and IL-13). As reported in asthmatic patients, the P2X7R was up-regulated 15-fold in the inflammatory cells and lung tissue of OVA-treated mice. The use of a selective P2X7R antagonist (KN62) on OVA-treated wild-type mice, or OVA-treated P2X7R/ mice, was associated with milder lung inflammation compared to OVA-treated wild-type mice. The mechanism behind the pro-inflammatory role of P2X7Rs in allergic asthma was investigated using bone marrow-derived dendritic cells from wild-type and P2X7R/ mice. The loss of P2X7Rs reduced the capacity of OVA-treated dendritic cells to induce Th2 immunity in vivo. This study highlights another critical role of P2X7Rs in the early events initiating an inflammatory response to an airborne insult, and the potential of selective antagonists for the suppression of Th2 inflammation in asthma.

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8.5


Conclusion

All animal models of acute lung injury and chronic lung diseases are associated with an accumulation of extracellular purines, and an up-regulation of their receptors, except for the anti-inflammatory A2AR. Such massive amplification of purinergic signals in response to hypoxia, ischemia-reperfusion, mechanical ventilation, bacterial products and allergens supports critical and widespread functions in airway defenses. During an acute lung injury, all ADO receptors provide protection against excessive lung inflammation, damage and pulmonary edema. The intravenous administration of agonists yielded the most effective resolution of all lung complications, which identified the microvascular endothelial barrier as primary target. Treatments should focus on agonists of the high-capacity A2ARs and A2BRs to minimize receptor desensitization (Chap. 7, Table 7.1). Whereas P2Y2R agonists may also be considered, possible side-effects may arise from their role in neutrophil and eosinophil recruitment. All models of chronic lung diseases are associated with a down-regulation of the anti-inflammatory A2ARs in inflammatory cells and lung tissue, which tilts the balance toward pro-inflammatory responses to ADO. Whereas A2AR agonists were shown to suppress airway inflammation, they would not address airway hyper-responsiveness, remodeling and fibrosis. Alternatively, the models of asthma, COPD and emphysema suggest that aerosolized antagonists of A2BRs, P2Y2Rs and P2X7Rs resolve most lung complications. However, the A2BR or P2Y2R antagonists must be carefully dosed to avoid any significant impairment of the mucociliary clearance mechanisms. Of particular interest are the P2X7R antagonists, which show tremendous benefits in reducing the development of COPD and emphysema in smokers through a reduction of macrophage activities responsible for the initiation of leukocyte recruitment to the airways. In asthma, aerosolized P2X7R antagonists may prevent resident dendritic cells from initiating the Th2 immunity. One important point to remember is that delivery route will dictate the cost/benefits of a purine drug depending on the local effects of the target receptor, and whether a treatment is corrective or preventive.

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137. Mortaz E, Braber S, Nazary M, Givi ME, Nijkamp FP, Folkerts G (2009) ATP in the pathogenesis of lung emphysema. Eur J Pharmacol 619:92–96 138. Riteau N, Gasse P, Fauconnier L, Gombault A, Couegnat M, Fick L, Kanellopoulos J, Quesniaux VFJ, Marchand-Adam S, Crestani B, Ryffel B, Couillin I (2010) Extracellular ATP is a danger signal activating P2X7 receptor in lung inflammation and fibrosis. Am J Respir Crit Care Med 182:774–783 139. M€uller T, Robaye B, Vieira RP, Ferrari D, Grimm M, Jakob T, Martin SF, Di Virgilio F, Boeynaems JM, Virchow JC, Idzko M (2010) The purinergic receptor P2Y2 receptor mediates chemotaxis of dendritic cells and eosinophils in allergic lung inflammation. Allergy 65:1545–1553 140. Robson SC, Kaczmarek E, Siegel JB, Candinas D, Koziak K, Millan M, Hancock WW, Bach FH (1997) Loss of ATP diphosphohydrolase activity with endothelial cell activation. J Exp Med 185:153–164 141. Fausther M, Pelletier J, Ribeiro CM, Se´vigny J, Picher M (2010) Cystic fibrosis remodels the regulation of purinergic signaling by NTPDase1 (CD39) and NTPDase3. Am J Physiol 298: L804–L818 142. Dwyer KM, Deaglio S, Gao W, Friedman D, Strom TB, Robson SC (2007) CD39 and control of cellular immune responses. Purinergic Signal 3:171–180 143. Vanderstocken G, Bondue B, Horckmans M, Di Pietrantonio L, Robaye B, Boeynaems J-M, Communi D (2010) P2Y2 receptor regulates VCAM-1 membrane and soluble forms and eosinophil accumulation during lung inflammation. J Immunol 185:3702–3707 144. Resnick MB, Weller PF (1993) Mechanisms of eosinophil recruitment. Am J Respir Cell Mol Biol 8:349–355 145. Cicko S, Lucattelli M, M€ uller T, Lommatzsch M, De Cunto G, Cardini S, Sundas W, Grimm M, Zeiser R, D€urk T, Zissel G, Boeynaems JM, Sorichter S, Ferrari D, Di Virgilio F, Virchow JC, Lungarella G, Idzko M (2010) Purinergic receptor inhibition prevents the development of smoke-induced lung injury and emphysema. J Immunol 185:688–697 146. Abboud RT, Vimalanathan S (2008) Pathogenesis of COPD. Part I. the role of proteaseantiprotease imbalance in emphysema. Int J Tuberc Lung Dis 12:361–367 147. Lucattelli M, Cicko S, Muller T, Lommatzsch M, de Cunto G, Cardini S, Sundas W, Grimm M, Zeiser R, Durk T, Zissel G, Sorichter S, Ferrari D, di Virgilio F, Virchow JC, Lungarella G, Idzko M (2011) P2X7 receptor signaling in the pathogenesis of smoke-induced lung inflammation and emphysema. Am J Respir Cell Mol Biol 44:423–429 148. Muller T, Paula Vieira R, Grimm M, Durk T, Cicko S, Zeiser R, Jakob T, Martin SF, Blumenthal B, Sorichter S, Ferrari D, di Virgilio F, Idzko M (2010) A potential role for P2X7R in allergic airway inflammation in mice and humans. Am J Respir Cell Mol Biol (in press)

Chapter 9

Therapeutic Applications Stephen Tilley, Jon Volmer, and Maryse Picher

Abstract The current treatments offered to patients with chronic respiratory diseases are being re-evaluated based on the loss of potency during long-term treatments or because they only provide significant clinical benefits to a subset of the patient population. For instance, glucocorticoids are considered the most effective anti-inflammatory therapies for chronic inflammatory and immune diseases, such as asthma. But they are relatively ineffective in asthmatic smokers, and patients with chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF). As such, the pharmaceutical industry is exploring new therapeutic approaches to address all major respiratory diseases. The previous chapters demonstrated the widespread influence of purinergic signaling on all pulmonary functions and defense mechanisms. In Chap. 8, we described animal studies which highlighted the critical role of aberrant purinergic activities in the development and maintenance of chronic airway diseases. This last chapter covers all clinical and pharmaceutical applications currently developed based on purinergic receptor agonists and antagonists. We use the information acquired in the previous chapters on purinergic signaling and lung functions to scrutinize the preclinical and clinical data, and to realign the efforts of the pharmaceutical industry. Keywords Airway obstruction Bronchoconstriction Ischemia-reperfusion Lung transplant Pulmonary fibrosis

S. Tilley (*) Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of North Carolina, Chapel Hill, NC 29799, USA e-mail: [email protected] J. Volmer and M. Picher Cystic Fibrosis Pulmonary Research and Treatment Center, University of North Carolina, 7011 Thurston-Bowles building, Chapel Hill, NC 27599, USA e-mail: [email protected]; [email protected]


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Introduction

For nearly 30 years, extracellular purines have been suspected to play important roles in the pathogenesis of chronic respiratory diseases. In the 1980s, Cushley et al. reported that inhaled ADO causes bronchoconstriction in asthmatic patients, but not in healthy subjects [1]. Later, it was discovered that asthmatics maintain high airway ADO levels, which is further raised following a challenge [2, 3]. In the past 5 years, the list of respiratory diseases with aberrant airway ADO and/or ATP regulation was extended to CF, COPD and idiopathic pulmonary fibrosis [4–6] (see Chap. 4 for details). Also, the recent finding that ADO induces bronchoconstriction in smokers with COPD supports a role in the “overlapping syndrome” [7–9]. Such widespread occurrence of excess airway ADO motivated scientists and clinicians to investigate the implications of this metabolic aberrance in all aspects of chronic respiratory diseases. The consequences of excess ADO for lung homeostasis were clearly demonstrated by mice lacking adenosine deaminase (ADA) (review: [10]). These ADA/ mice develop bronchial hyperresponsiveness (BHR), severe lung inflammation, mucin hypersecretion, sub-epithelial fibrosis, basement membrane thickening, smooth muscle cell hyperplasia and a disruption of the alveolar network typical of emphysema (see Chap. 8 for details). This stunning discovery launched a series of initiatives to identify which ADO and ATP receptors promote, or attenuate, lung complications. The previous chapters were carefully organized to describe all purinergic receptors, their properties, distribution and roles on the lung resident, immune and inflammatory cells. Chapter 8 described the animal studies that were conducted to compare their impact on lung homeostasis and the potential of selective ligands for the treatment of chronic respiratory diseases. This final chapter describes the progress made, over the past 15 years, in this new field of drug development. First, evidence is provided for the higher diagnostic stringency of ADO-induced BHR for asthma, with respect to histamine and methacholine. Then, we tell the tale of Adagen (Enzon Pharmaceuticals), which was designed for the treatment of ADA-related severe combined immunodeficiency (ADA-SCID), but shows considerable potential for the correction of airway ADO levels in chronic airway diseases. Finally, this chapter covers the ADO and ATP receptor ligands selected by pharmaceutical companies for the treatment of acute lung injury and chronic respiratory diseases, and to improve the outcome of lung transplant. Based on the current understanding of purinergic signaling in the respiratory system, this section offers a critical view on the design of the ligands and the delivery route, which may assist the pharmaceutical industry in future drug discovery.

9.2

AMP Challenges: Diagnostic Tool and Signaling Pathways

Airflow obstruction is a characteristic feature of airway diseases, including asthma and COPD. Adenine nucleotides and nucleosides have been shown to trigger immediate airflow obstruction in asthmatic patients following aerosolized administration.

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Since the bronchoconstriction does not occur in normal subjects, adenosine monophosphate (AMP) has been used worldwide to detect and monitor bronchial hyperresponsiveness (BHR), as a tool to diagnose asthma and guide therapy. In this section, we review the clinical use of AMP challenges, and the current knowledge on the mechanism of action.

9.2.1

Using AMP as a Diagnostic Tool

In 1983, Cushley, Tattersfield and Holgate were the first to report that inhalational exposure to ADO resulted in a concentration-dependent bronchoconstriction in allergic and non-allergic asthmatic patients [1]. In contrast, ADO concentrations up to 30-fold higher had no effect on the airway caliber of normal subjects [11]. Adenine nucleotides, ADP and AMP, also elicited airway narrowing in asthmatics, while the ADO metabolite, inosine, did not induce bronchoconstriction [12]. The airway epithelial surfaces express ectonucleotidases which rapidly dephosphorylate adenine nucleotides into ADO (see Chap. 2 for details). While it is well accepted that the effects of these nucleotides on airway caliber are mediated by ADO, AMP remains the drug of preference for solubility purposes (review: [13]). Prior to the observations of Cushley et al. [1], pharmacological tests for BHR relied on direct airway challenges with methacholine or histamine. These substances activate M3 muscarinic receptors and H1 histamine receptors on airway smooth muscle (ASM) to elicit bronchoconstriction in asthmatics at concentrations having no effect in the airways of normal subjects. While the sensitivity and negative predictive values of methacholine and histamine challenges approach 100%, their specificity and positive predictive values for asthma are very low [14–17]. In fact, adult COPD patients and children with bronchiectasis, bronchiolitis obliterans or CF, typically develop BHR in response to methacholine and histamine [18–22]. In contrast, nonsmoking adults with COPD and children with airway diseases other than asthma are less responsive to an AMP challenge [7–9, 22, 23]. This higher specificity for asthma suggests that aerosol AMP may constitute a powerful diagnostic test to differentiate these airway diseases in children and non-smoking adults. Furthermore, contrary to methacholine, the magnitude of the BHR response to AMP is highly correlated with the degree of underlying airway inflammation in asthmatics [24, 25]. For these reasons, measurement of BHR by AMP challenge has been endorsed by the European Respiratory Society Task Force on indirect airway challenges [26]. It is more specific for asthma than methacholine and histamine challenges [22, 27], correlates more closely with the degree of inflammation in the lower airways [24, 25], and may be used to evaluate the efficacy and potency of inhaled steroids [28–31]. However, the sensitivity of an AMP challenge to detect asthma is limited, the rate of AMP responsiveness varying from 39% to 95.5% in children [32–35] and 50% to 89% in adults [24, 36]. The studies reporting a high degree of sensitivity included large numbers of atopic asthmatics. Accordingly, a study designed to elucidate the determinants of AMP responsiveness revealed that atopic sensitization, assessed by positive skin prick testing, was the major variable affecting AMP

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sensitivity [32]. Compared to non-atopic asthmatics, atopic asthmatics exhibit exaggerated mast cell activation by antigens and allergens. Consequently, these cells have been the focus of intense investigation to better understand the mechanisms of ADO-induced bronchoconstriction.

9.2.2

Signaling Pathways of Bronchoconstriction

The signaling pathways responsible for AMP-induced BHR have been extensively investigated using its active metabolite: ADO. This ubiquitous signaling molecule has been shown to regulate the activities of all mammalian cell types through the activation of cell surface G protein-coupled receptors. Four P1 receptors have been identified and are widely expressed: A1Rs, A2ARs, A2BRs, and A3Rs. The studies conducted in humans and animals support three potential mechanisms for ADO-induced bronchoconstriction targeting different cell types: mast cells, ASM and neurons (Fig. 9.1). The first argument supporting a mast cell-dependent mechanism was provided by a clinical study reporting the accumulation of histamine in the airways of asthmatic patients in response to endobronchial ADO [37]. Accordingly, ADO-induced

Fig. 9.1 Mechanisms of adenosine-induced bronchoconstriction in human airways. Adenosine (ADO; red) can induce muscle contraction directly via A1 receptors (A1R) located on airway smooth muscle (ASM). These receptors are also expressed by the sensory neurons, allowing ADO to induce the release of acetylcholine (Ach; green). This neurotransmitter can induce muscle contraction directly via muscarinic receptors on ASM, or indirectly by the stimulation of histamine release (blue) from mast cells


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BHR was attenuated by mast cell stabilizing agents (i.e. nedocromil) [38–40] and antagonists of the histamine receptor (i.e. terfenadine) [41–43] or leukotriene receptor (i.e. Montelukast) [44]. These studies led many to believe that ADO-induced bronchoconstriction results from mast cell activation. In rodents, the P1 receptor causing mast cell degranulation was identified as the A3R [45–48], whereas this role was ascribed to the A2BR in human mast cells [49–51]. Nonetheless, aerosolized treatment with a dual A2BR/A3R antagonist (QAF805) failed to affect PC20 (provocative concentration required to induce a 20% fall in lung function) in asthmatics [52], suggesting that ADO induces bronchoconstriction by other mechanisms. Human and animal studies suggest that A1Rs located on ASM mediate the ADOinduced bronchoconstriction. On cultures of human ASM, A1R activation stimulated cell contraction by the mobilization of intracellular Ca2+ stores [53]. Their tissue expression was confirmed by immunolocalization in biopsy sections, which also showed that A1Rs are overexpressed in the airway epithelium and ASM of asthmatics [54]. Furthermore, the incubation of normal ASM cultures with serum from asthmatic patients induced the up-regulation of A1Rs [55]. Incidentally, animal studies conducted with A1R agonists and antagonists also support a role for A1Rs located on ASM in ADO-induced BHR [56–58]. The use of knockout mice targeting each P1 receptor revealed that only A1R/ mice fail to react to an ADO challenge [59]. On the other hand, ADO-induced bronchoconstriction was abolished following bilateral vagotomy [59], suggesting a role for sensory neurons. In asthmatic patients, AMP-mediated BHR was attenuated by the anticholinergic drug ipratropium, and by depletion of contractile neuropeptides with bradykinin [60, 61]. These studies support a role for cholinergic neural pathways in the bronchoconstrictor response to ADO. In allergic (not naı¨ve) guinea pigs, the fact that bilateral vagotomy or capsaicin strongly inhibited airway obstruction to AMP (75%), or an A1R agonist (N(6)-cyclopentyladenosine; CPA) (50%), suggests that cholinergic neural input to the ASM is critical for A1R-mediated bronchoconstriction [62]. In human airways, acetylcholine can activate muscarinic receptors on ASM and mast cells (review: [63]). Whether cholinergic mediators induce ASM contraction directly, or through mast cell activation, has not been determined.

9.2.3

Summary

Recent studies on the signaling pathways responsible for ADO-induced BHR have revealed important clues about the airway pathophysiology of asthma, by uncovering a complex interplay between mast cells, neurons and ASM. The continuous investigation of the mechanisms by which nucleotides and nucleosides influence airway caliber in the asthmatic lung may lead to the exploitation of these pathways for therapeutic approaches. For instance, future studies comparing the relative impact of A1R and A2BR antagonists on AMP-mediated BHR may reveal a more prominent role for A2BR-mast cell signaling in the atopic asthmatic patients. Meanwhile, AMP challenges remain an important tool for clinicians and researchers sharing common interests in the diagnosis and treatment of asthma and other respiratory disease.

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Adenosine Deaminase Replacement Therapies Current Clinical Application for ADA-SCID

As part of the purine salvage pathway, intracellular ADA is responsible for the deamination of ADO and deoxyadenosine into inosine and deoxyinosine, respectively [64]. Over 50 mutations spanning the entire Ada gene have been identified, 29 of them resulting in >85% reduction of ADA activity [65, 66]. These functional mutations allow toxic levels of substrates to accumulate and interfere with the development of thymocytes in the thymus and bone marrow, which prevents or weakens the immune responses to infection [67]. Accordingly, inherited ADA deficiency is catalogued as an autosomal recessive immunodeficiency disorder exhibiting a wide range of severity, depending on the level of residual activity: (1) a neonatal and fatal severe combined immunodeficiency disease (SCID) characterized by the absence of cellular and humoral immunity; (2) a delayed onset progressive disease retaining some humoral immunity; (3) a late juvenile onset with progressive attrition of immune defenses; (4) an adult onset disorder [66]. With respect to lung complications, they exhibit recurrent otitis, sinusitis, chronic bronchitis and pneumonia, the severity correlating negatively with ADA activity [68]. Over the years, many therapeutic approaches have been explored for ADA-SCID patients. The best option remains an allogenic bone marrow transplant, which generally leads to complete recovery [69]. In the event that a histocompatible sibling is unavailable, these patients are administered irradiated purified erythrocytes as a source of functional ADA [70]. Unfortunately, this approach only provides a partial and transient recovery of immune functions, and long-term infusions give rise to various complications [71]. In 1981, Stephen Davis proposed that covalent linking of purified bovine ADA to polyethylene glycol (PEG) would prolong its half-life and reduce its antigenicity [71]. This approach proved successful in laboratory animals, extending the half-life of ADA activity in mouse serum from 30 min for the naked protein to 28 h for PEG-ADA. The antigenic properties were virtually eliminated as well, as the serum from mice receiving intravenous PEG-ADA was unable to immunoprecipitate ADA or PEG-ADA, even after repeated injections over a period of several weeks. The circulating PEG-ADA was also able to reduce the intracellular levels of ADO [71] due to the ubiquitous expression of transporters in the plasma membrane of mammalian cells (see Chap. 2 for details). In 1985, Michael Herschfield initiated the therapeutic evaluation of PEG-ADA (Adagen; Enzon Pharmaceuticals) in ADA-SCID patients. The therapy successfully reduced tissue ADO concentrations and dramatically improved immune functions [69]. However, the levels of T, B and natural killer lymphocytes rose over the first few years, but never reached normal values. Also, about 65% of the patients developed antibodies against PEG-ADA within the first year. Their lymphocyte levels began to drop, as they faced a steady decline in immune function. Another major obstacle to this therapy was the cost of biweekly injections of PEG-ADA,


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which rose to $200,000/year [72]. These serious limitations called for alternative approaches for the restoration of ADA. Future directions in the treatment of ADA-SCID include the genetic replacement of ADA function (review: [73]). Over nearly two decades, the gene therapy consisted almost exclusively in the use of retroviral vectors targeting the lymphocytes and hematopoietic progenitors. Patients were successfully colonized by hematopoietic stem cells transfected with the ADA gene, following partial ablation of the bone marrow [74]. However, this groundbreaking approach came with the risk of inspectional mutagenesis. Recent studies suggest that this highly invasive protocol may be replaced by ex vivo lentiviral therapy. In ADA-deficient mice, the intravenous injection of human immunodeficiency virus 1-based lentiviral vector at birth induced the long-term expression of enzymatically active ADA at levels comparable to long-term PEG-ADA treatments [72]. This approach effectively restored immune function and significantly extended survival. As such, this non-invasive method may provide long-term restoration of ADA activity in ADA-SCID patients.

9.3.2

Long-Term Treatments for Pulmonary Diseases

Despite the development of successful treatments for ADA-SCID, the underlying causal link between the metabolic disturbances and associated immunodeficiency were unknown. An animal model of ADA deficiency was developed to investigate this link, but the ADA/ mice suffered from a perinatal lethality due to hepatotoxicity [75]. This complication was circumvented using an ADA minigene possessing only the promoter elements required for expression in the trophoblast, which preserved liver function [76]. These partially ADA-deficient mice were born without ADA activity, as evidenced by a steady accumulation of the ADA substrates: ADO and deoxyadenosine. They displayed typical immunodeficiency in the lymphoid line for ADA-SCID patients [77]. In addition, these mice developed severe pulmonary complications, characterized by BHR to inhaled ADO [78], inflammatory cell infiltration and mucus hypersecretion [77]. The defective alveogenesis resulted in an emphysema-like phenotype by postnatal day 10, and the mice died by postnatal day 21 due to respiratory distress [79] (see Chap. 8 for details). These studies demonstrate that the high systemic ADO levels of SCID patients are responsible for the development of severe lung complications. More importantly, they suggested that the high airway ADO levels of asthmatic [3, 80, 81] and COPD [82] patients promote the inflammation, remodeling and fibrosis leading to BHR and the loss of lung function. The therapeutic potential of PEG-ADA for chronic respiratory diseases was tested in the ADA/ mice. The animals treated with intramuscular PEG-ADA from birth did not develop any lung complication [83]. On the other hand, ADA/ mice which develop lung inflammation over 20 days completely recovered after 3 days of PEGADA treatment at a dose that fully restored normal lung ADO levels [79].

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Interestingly, the microarray analysis of lung tissue revealed that PEG-ADA restored the normal expression of genes critical for inflammatory responses, as well as factors promoting remodeling and fibrosis. This observation supported the therapeutic potential of PEG-ADA for patients with well-established chronic respiratory diseases. This hypothesis was tested using ADA/ mice maintained on a low-dose PEG-ADA regimen from birth, which prolonged their lifespan to 4–5 months [84]. After 3 months, the resulting intermediate ADO level fostered the development of severe pulmonary inflammation, collagen deposition and alteration in airway structure. Their BAL fluid accumulated, both, pro-inflammatory and pro-fibrotic mediators (IL1b, osteopontin, MMP12, TGFb, and PAI-1). All these complications were fully resolved by shifting these mice to high-dose PEG-ADA for 5 weeks. This animal model clearly demonstrated the dramatic consequences of high ADO levels reported in the airways of asthmatic and COPD patients (see Chap. 4 for details), and the therapeutic potential of PEG-ADA for all lung complications, including fibrosis.

9.3.3

Suppression of the ADO/IL-13 Amplification Cycle

Clinical studies showed that the airways of asthmatic, CF and COPD patients also accumulate the cytokine: IL-13 [85–87]. An earlier report of normal IL-13 concentration in COPD sputum [88] may have resulted from the use of enzyme-linked immunoassay, instead of the more sensitive cytometric bead array, as the authors only detected IL-13 in 6/34 patients. In CF patients, a significant negative relationship was established between airway IL-13 levels and lung functions [86]. The significance of IL-13 for chronic airway diseases is supported by the plethora of studies which identified this cytokine as upstream element responsible for the imbalance in metalloproteinase production promoting airway remodeling (reviews: [89, 90]). Incidentally, IL-13 transgenic mice develop mucus cell metaplasia, hypersecretion and sub-epithelial fibrosis [91, 92]. Interestingly, they share many phenotypic traits with the ADA-deficient mice, including eosinophilia, emphysema and fibrosis, and they survive less than 4 months [92]. The development of these lung complications coincides with a steady rise in lung ADO level and reduction in lung ADA activity. One month of PEG-ADA treatment corrected the ADO levels and significantly reduced inflammation, mucin secretion, collagen deposition and emphysema [91]. The existence of an amplification cycle for lung IL-13 and ADO regulation is also supported by the fact that ADA/ mice overexpress IL-13, which was corrected by PEG-ADA treatment [91]. It is proposed that excess ADO, generated in stressed and damaged tissues, induces an up-regulation of IL-13 and recruits inflammatory cells. This cytokine initiates airway remodeling and down-regulates ADA, resulting in further ADO accumulation, which completes the cycle. Consequently, many aspects of chronic airway diseases could be addressed by ADA replacement therapy, by the concomitant reduction of ADO- and IL-13-mediated responses.


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Summary

The current literature highlights the tremendous potential of PEG-ADA to identify the inflammatory, remodeling and fibrotic processes targeted by the excess airway ADO of asthmatic and COPD patients. This remarkably stable compound also has the potential of becoming an effective therapy for a variety of lung diseases dominated by fibrosis and emphysema, that is, if the pharmaceutical industry can reduce the cost of the treatments. At Enzon Pharmaceuticals, PEG-ADA (pegademase bovine) has been prescribed for over 20 years for the treatment of ADA-SCID under the name: Adagen. Patents were recently issued for therapeutic applications in pulmonary diseases, and clinical trials are pending. It is important to note that ADA offers no therapeutic benefit unless pegylated, which provides the metabolic stability. Since this discovery, this stabilizing technique has been applied to a number of bioagents, including interferons (review: [93]).

9.4

Clinical Trials Involving Adenosine Receptors

There are dozens of ADO receptor ligands currently designed by various research laboratories and pharmaceutical companies. This activity has provided pharmacological tools to distinguish the roles of each ADO receptor in the regulation of airway defenses. For details on the strategies behind their structural design, the readers are redirected to excellent reviews for A1Rs [94], A2ARs [95, 96], A2BRs [97, 98] and A3Rs [99]. This chapter focuses on the ligands selected for preclinical and clinical evaluation. In each case, the experimental designs (animal models, delivery routes) chosen to evaluate their therapeutic potential are weighed against the knowledge we have acquired in the first eight chapters of this book, regarding ADO regulation and cell-specific functions.

9.4.1

The A1R Antagonists

The animal models of chronic respiratory diseases predict that A1R antagonists may attenuate BHR, mucin hypersecretion and lung inflammation (see Chap. 8 for details). These bioagents would take advantage of the high A1R expression reported on the airway epithelial and smooth muscle cells of asthmatic patients [54]. On the other hand, smoking was recently reported to down-regulate A1Rs on inflammatory cells [100], suggesting that A1R antagonists may be less effective in smoking asthmatics and most COPD patients. In terms of delivery route, systemic delivery of A1R antagonists in oral capsules would more efficiently prevent the migration of dendritic cell to the lymph nodes, as well as leukocyte adhesion to the endothelial barrier (see Chap. 7 for details). With all this information in mind, we will review the current preclinical and clinical studies with a critical view of the drug development strategies adopted for A1R antagonists.

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Mixed Theophylline/Corticosteroid Treatments

Chemical structure: 1,3-dimethylxanthine The first report of methylxanthines for the treatment of bronchoconstriction was published in 1859 by an asthmatic, Henry Hyde Salter, who described the effects of a strong coffee on his airway symptoms (review: [101]). In the 1920s, Theophylline was demonstrated to relax airway smooth muscle, and was prescribed orally for the treatment of airway hyperresponsiveness [102]. In the 1970s, Theophylline was a popular treatment for stable asthmatic patients. Unfortunately, the dose needed to initiate bronchodilation often induces cardiovascular and gastric side-effects. This drug became less popular as more effective b2 adrenoceptor agonists became available. Over the past decade, Theophylline was revisited using lower doses (0.1–0.5 mg) avoiding most side-effects caused by phosphodiesterase inhibition. Under these conditions, Theophylline exerts potent anti-inflammatory effects, predominantly as an A1R antagonist (review: [103]). In the OVA sensitization/challenge model of allergic asthma, low dose Aminophylline (Theophylline pro-drug) given intraperitoneal prevented early and late phase airway hyperresponsiveness to an OVA challenge, and reduced the inflammatory markers in BAL fluid (IL-4, IL-5, IL-6, IL-10 and TNFa) [104]. In 2006, the anti-inflammatory potential of low-dose Theophylline for asthmatic patients was tested in a clinical study using Aminophylline [105]. A single intravenous injection significantly improved the lung functions, measured as peak expiratory flow (PEF) and peripheral oxygen saturation (SpO2). These clinical benefits were comparable to those of aerosolized Salbutamol, a b2 adrenoceptor agonist. Also, Aminophylline (but not Salbutamol) reduced pulmonary inflammation, measured in BAL fluid as eosinophil cationic protein, histamine, serotonin, thromboxane B2, leukotriene C4. Nowadays, clinicians refine the treatment of asthma and COPD by combining the bronchodilating effect of inhaled corticosteroids with the anti-inflammatory effect of low dose oral Theophylline [106–108] (Table 9.1). In July 2010, the Theophylline extended-release tablet was granted marketing approval by the United States FDA. Table 9.1 Antagonists of the A1R in the pipeline Delivery Proof of Drug Disease route concept Theophylline Asthma and Oral COPD Bamiphylline Asthma and Oral COPD L-97-1

Asthma

Oral

EPI-2010

Asthma

Nebulizer

Clinical trials terminated

Preclinical

Phase Phase 1 2a

Phase 2b

Phase 3


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Bamiphylline Approved in Europe

Chemical structure: 8-benzyl-7,[2-[ethyl(2-hydroxyethyl)amino]-ethyl] Theophylline Trentadil; bamifylline; benzetamophylline, Cloperastine Over the years, more selective A1R antagonists were designed to avoid the sideeffects associated with Theophylline. Several clinical trials, conducted over the past 20 years, reported the potential of Bamiphylline for the treatment of bronchoconstriction and mucin clearance in asthmatic and COPD patients. In 1991, a placebocontrolled study was conducted with COPD patients matched for age [109]. The 7-day oral treatment significantly improved lung functions, based on the breathing pattern, inspiratory muscle strength, neural drive and index of inspiratory neuromuscular coupling. In 1995, a double-blind clinical study assessed the effect of Bamiphylline on mucus clearance in 20 smokers with chronic bronchitis, compared to healthy controls [110]. All subjects received oral Bamiphylline (600 mg per day) or placebo during 15 days. Only the Bamiphylline treated COPD patients exhibited better mucus clearance, clinical score and pulmonary function, with no reported sideeffects. This study suggests that Bamiphylline could improve mucus clearance in other chronic obstructive diseases, such as CF. Oral Bamiphylline has been approved for the treatment of asthma and COPD in Europe, where it shows low incidence of side-effects, such as headaches or gastralgia (1/100,000 patients) [111]. This drug is also sold, as a cough suppressant, by Biocure Pharmaceutical under the name of cloperastine.

9.4.1.3

Is L-97-1 Better than Montelukast?

Chemical structure: [3-[2-(4-aminophenyl)-ethyl]-8-benzyl-7-{2-ethyl-(2-hydroxyethyl)-amino-ethyl}-1-propyl-3,7-dihydro-purine-2,6-dione] Based on the success story of Bamiphylline, Mustafa et al. developed a selective antagonist of this receptor: L-97-1 [57]. Radioligand binding analysis showed a strong affinity for A1Rs (IC50 ¼ 1.42 mM) compared to A2ARs and A2BRs (IC50 > 100 mM). This is a considerable improvement from Bamiphylline, in terms of affinity and selectivity. In the house dust mite sensitization/challenge model of allergic asthma, the oral gavage of L-97-1 prevented the BHR to the final challenge for at least 6 h [57]. This treatment also inhibited late allergic responses, measured after 24 h, by histamine-induced bronchoconstriction. Therefore, L-97-1 has the potential to prevent early and late allergic responses in asthmatic patients. Mustafa et al. also used this animal model to compare the efficiencies of L-97-1 and Montelukast [112], a cysteinyl leukotriene-1 receptor antagonist regularly prescribed to asthmatic patients for the treatment of reversible airflow limitation [113]. First, L-97-1 pretreatment maintained lung dynamic compliance higher than Montelukast over at least 5 h after a house dust mite challenge. Second, L-97-1

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blocked both early and late allergic responses, whereas Montelukast only blocked the late response. Finally, both drugs caused comparable anti-inflammatory effects, in terms of leukocyte accumulation in BAL fluid. In summary, L-97-1 is an antiinflammatory drug as potent as Montelukast, with the added benefit of a more efficient treatment of reversible airflow limitation. The small water-soluble molecule, L-97-1, is under development as oral treatment for asthmatics by Andacea Inc., a company devoted to purinoceptor-based technologies.

9.4.1.4

EPI-2010: A Respiratory Antisense Oligodeoxynucleotide

In 2006, Drs Fire and Mello were awarded the Nobel Prize for discovering the mechanism behind RNA interference in 1998 (review: [114]). The regulation of gene expression through “silencing” was found essential for many cellular processes. The immense impact of this discovery on biomedical research led to the development of a new generation of aerosolized drugs for chronic respiratory diseases. In 1999, Metzger and Nyce designed respirable antisense oligonucleotides (RASONs) as short, single-stranded nucleic acid sequences modified to enhance their stability [58]. They bind the initiation codon of the messenger RNA (mRNA), which prevents translation and targets the message for degradation by RNAses. A clear advantage over traditional drugs is that they are metabolized locally by endogenous nucleases, confining their reactivity to the airways (review: [115]). During a joined venture with Chiesi Pharmaceuticals and Taisho Pharmaceuticals, EpiGenesis Pharmaceutical developed the A1R-specific RASON, named EPI-2010, for the treatment of asthma [116]. A fortuitous homology between the human and rabbit mRNA sequences of the A1R allowed EPI-2010 to be tested in the model of allergic asthma described above for L-97-1. Following house dust mite sensitization, nebulized EPI-2010 caused a dose-dependent attenuation of airway hyperresponsiveness to ADO [58]. The fact that EPI-2010 also reduced airway obstruction caused by house dust mite and histamine challenges supported the existence of an anti-inflammatory effect. In these animals, EPI-2010 was deposited throughout the lung, with no detectable systemic active metabolites, and was excreted primarily in the urine. This original study demonstrated that RASONs can be efficiently delivered to the peripheral lung, where they potently and selectively attenuate the expression of disease-associated genes. In primates, EPI-2010 attenuated the allergen responses to Ascaris lumbricoides for about 7 days, which corresponds to the kinetics of A1R expression. Hence, EPI-2010 may represent the first once-a-week treatment for asthma. In 2003, a Phase 1 clinical trial conducted on asthmatic patients demonstrated that aerosolized EPI-2010 is well-tolerated and shows indications of efficacy [116]. A single dose reduced the need for bronchodilator drugs and the symptom scores during about 1 week. However, because of the disappointing results of a Phase 2 clinical trial, EPI-2010 was discontinued from clinical testing [117]. In this study, 146 patients with persistent airway obstruction (FEV1 ¼ 74.5% predicted;


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12% reversibility), currently receiving inhaled corticosteroids, were administered EPI-2010 (1, 3, or 9 mg) through a nebulizer once or twice weekly. In asthmatic patients under corticosteroid treatment, EPI-2010 caused no significant change in lung function or inflammatory responses over 29 days. Some arguments still support the therapeutic potential of inhaled EPI-2010 for asthmatic patients. In the above clinical trial, the patients had mild-to-moderate asthma, depending on the frequency of symptoms and the variability in peak expiratory flow rate. Despite the apparent stable FEV1 of 74.5% predicted, corticosteroids will raise the FEV1 values to 90–100% of predicted between exacerbations. Consequently, measurements of FEV1 do not constitute a sensitive assessment of asthma severity, compared to the acute changes in airway function caused by bronchoprovocation. This trial should have tested the therapeutic potential of EPI-2010 in asthmatic patients not receiving corticosteroid treatments, and using challenged BHR responses as parameter of clinical benefit. Also, because of safety concerns about the use of antisense oligonucleotides in humans, the doses of EPI-2010 may have been sub-therapeutic. For these reasons, the scientific and clinical communities both support further investigation of aerosolized EPI-2010 for the attenuation of BHR in asthma and COPD. On the other hand, the current information on A1Rs suggests that oral delivery would provide a more potent anti-inflammatory treatment of chronic lung diseases.

9.4.2

The A2AR Agonists

The animal studies and known functions of A2ARs predict that agonists would be prescribed primarily for the treatment of chronic lung inflammation (see Chap. 8 for details). However, this approach faces a serious challenge, as A2AR expression and/or surface affinity are reduced in the lungs of asthmatic and COPD patients [100, 118]. Since these ligands have been shown to cause significant cardiovascular side-effects, including hypotension, strategies have been designed to confine the drugs to the airways. On the other hand, this flaw has been exploited for the benefit of patients undergoing elaborate surgical procedures, such as lung transplant, to prevent the resulting increase in pulmonary arterial blood pressure. This section reviews the A2AR agonists which were selected for preclinical and clinical evaluation of these two major applications. 9.4.2.1

CGS21680 More Efficient than Budesonide

Chemical structure: 2-p-(2-carboxyethyl)phenethylamino-5’-N ethylcarboxamido adenosine hydrochloride The animal studies described in Chap. 8 clearly support the use of A2AR agonists for the treatment of lung inflammation in chronic respiratory diseases. However, the drug should be administered as an aerosol because systemic injection has been

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shown to cause considerable cardiovascular side-effects, including hypotension in human subjects [119]. Therefore, studies were conducted to test the safety and efficacy of the aerosolized A2AR agonists in animal models of airway diseases. The first study evaluated the potential of CGS21680 in an animal model of allergic asthma. In OVA sensitized/challenged Norway rats, intratracheal CGS21680 given 15 min before and after the final challenge reduced eosinophil and neutrophil counts in the BAL fluid [120]. Similar findings were obtained in OVA sensitized/challenged mice [121]. In addition, CGS21680 was found ten times more efficient than Budesonide [121], a corticosteroid currently prescribed to asthmatic patients (review: [122]). On the other hand, CGS21680 did not address OVA-induced bronchoconstriction or mucin secretion, as expected from the pharmacological properties of the A2AR (see Chap. 8 for details). And these doses caused significant hypotension in the anesthetized animals, which may limit the clinical utility (Table 9.2). Aerosolized CGS21680 did not prevent the development of lung inflammation in two models of acute lung injury: intranasal LPS and cigarette smoke inhalation [121]. In Chap. 8, we provided evidence that the microvascular endothelial barrier constitutes the primary target for the treatment of acute lung injury, as A2AR activation inhibits vascular leakage and inflammatory cell infiltration. In the LPSand smoke-challenged mice, the lack of effect of intranasal CGS21680 may reflect a poor choice of delivery route. A study showed that rats subjected to hemorrhagic shock, then given intravenous CGS21680 during resuscitation, developed significantly less acute lung injury than the untreated animals, in terms of vascular leakage and BAL myeloperoxidase activity [123]. This study suggests that the systemic administration of CGS21680 may prevent the acute development of lung injury in situations where blood pressure is monitored. Table 9.2 Agonists of the A2AR in the pipeline Delivery Proof of Drug Disease route concept CGS21680 Asthma Intratracheal Trauma GW328267X Allergic rhinitis

Systemic Intranasal

Asthma

Diskhaler

UK371,104

COPD

Intratracheal

UK432,097

COPD

Inhaled

Cough

Powder

Stedivaze

Sepsis Sickle cell disease

ATL313

Intravenous

Lung Intravenous transplant

Heart/lung Intravenous bypass Clinical trials terminated

Preclinical

Phase Phase 1 2a

Phase 2b

Phase 3


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GW328267X: Flaw in the Design

Chemical structure: (2R,3R,4S,5R)-2-{6-amino-2-[(1-benzyl-2-hydroxyethyl)amino]9H-purin-9-yl}-5-(2-ethyl-2H-tetrazol-5-yl)tetrahydrofuran-3,4- diol The company GlaxoSmithKline identified a new selective agonist for the A2AR: GW328267X [124]. Binding studies conducted in Chinese hamster ovary cells showed that this high-affinity (pKi ¼ 7.8) ligand is a potent agonist (pEC50 ¼ 9.0) of the A2AR. This compound has a lower affinity for A1Rs and A2BRs (pKi 6) and shows relatively weak agonist activity at these receptors. In vitro assays demonstrated that GW328267X inhibits neutrophil and eosinophil activities (oxidative stress and degranulation), which are antagonized by an A2AR antagonist, CGS15943. However, GW328267X also binds A3Rs (pKi ¼ 7.8) as a competitive antagonist. Hence, GW328267X activates A2ARs and blocks A3R-mediated responses equally well. Since A3Rs mediate neutrophil elastase secretion [125], this drug would offer additional protection as a blocker of this receptor. In 2007, the company published results from two clinical studies conducted to evaluate the therapeutic potential of GW328267X for allergic rhinitis [126] and allergic asthma [127]. In a randomized, double-blind, placebo-controlled, threeway balanced, crossover study, 48 men with allergic rhinitis where challenged with house dust mite. Then, they received a nasal spray of GW328267X (50 mg) or Fluticasone (200 mg) twice daily [126]. The corticosteroid is currently prescribed for the treatment of allergic rhinitis. After 7 days, the patients given GW328267X had improved nasal blockage, but not peak nasal inspiratory flow, whereas Fluticasone significantly improved both parameters. Intranasal GW328267X also produced a small, but significant, reduction of inflammatory marker concentrations in the BAL fluid (tryptase and eosinophil cationic protein), while Fluticasone normalized the concentrations of eosinophil cationic protein and neutrophil chemoattractant IL-8. It was concluded that this novel A2AR agonists has limited clinical benefit for the treatment of allergic responses. In the second double-blind, placebo-controlled, three-way balanced crossover study, 15 non-smoking atopic asthmatic patients, not under corticosteroid treatments, were challenged with house dust mite, then inhaled GW328267X (25 mg) or Fluticasone (200 mg) twice daily for 7 days [127]. The A2AR ligand did not protect against early and late asthmatic reactions, or significantly attenuate the inflammatory responses. And higher doses caused considerable side-effects, in terms of hypotension and tachycardia. In contrast, Fluticasone completely inhibited the early and late asthmatic responses, and significantly suppressed inflammation, including the lung eosinophilia. Given such low therapeutic index, GlaxoSmithKline discontinued the evaluation of GW328267X. The failure of GW328267X to provide therapeutic benefits to allergic patients is consistent with the properties of this complex ligand. First, chronic airway diseases, in humans and animal models, are associated with a dramatic down-regulation of A2ARs (see Chap. 8 for details). Second, the antagonistic effect of GW328267X on A3Rs is expected to inhibit the degranulation of eosinophils, but to promote their recruitment to the lungs [128]. As such, this drug could not suppress allergic airway inflammation.

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New UK371,104 Remains in the Lungs

Chemical structure: N-(2,2-diphenylethyl)-2-{[(2-piperidin-1-ylethyl)amino] carbonyl} adenosine In 2008, Pfizer Inc. presented a new selective A2AR agonist carefully designed to minimize side-effects by restricting the molecule to the airways (review: [119]). In rats given an intratracheal dose of 1 mg/kg, the maximum free plasma concentration (Cmax) of UK371,104 was 20 nM, compared to 271 nM for CGS21680. This tenfold difference results from the higher lipophilicity, molecular weight, in vivo clearance and plasma protein binding capacity of UK371,104. The safety and potency of UK371,104 were evaluated in anesthetized guineapigs monitored simultaneously for pulmonary and cardiovascular functions [129]. Whereas intratracheal CGS21680 or UK371,104 prevented capsaicin-induced bronchoconstriction, only CGS21680 provoked hypotension [129]. Furthermore, the protective effect of the new drug against BHR lasted more than tenfold longer than with CGS21680. Overall, UK371,104 offers a sevenfold improvement in potency and 150-fold reduction in side-effect over the lead compound, CGS21680 [119]. These data suggest that aerosolized UK371,104 constitutes the drug of choice for the treatment of chronic airway diseases. The company further refined the structure of UK371,104 to generate UK432,097, [130]. In Phase 1 clinical trial, healthy subjects administered UK432,097 as an inhaled dry powder presented very little dispersal outside of the airways, and no effect on heart rate. In a Phase 2, randomized, double-blind, placebo controlled, parallel group clinical trial designed to evaluate the efficacy and safety of UK432,097 in adults with moderate-to-severe COPD, the patients showed no improvement in lung function over 6 weeks [131]. At the moment, alternative applications are considered for UK432,097, including cough suppression and improving the outcome of mechanically ventilated patients.

9.4.2.4

The ATL Family of A2AR Agonists

Stedivaze was originally developed by Clinical Data Inc. as pharmacological stress agent for myocardial perfusion imaging, to avoid the bronchoconstrictive effects of ADO in patients with asthma and COPD. In summer 2010, a Phase 2 clinical trial showed that a fixed dose, bolus injection, of Stedivaze generates sufficient coronary artery vasodilation for myocardial perfusion imaging, and without causing bronchoconstriction. This study constitutes a milestone toward the goal of developing a safe and well tolerated coronary vasodilator for these patients. Stedivaze is currently in Phase 3 trial for this application. Given the potent anti-inflammatory properties of A2ARs, Stedivaze could represent an asset for the treatment of chronic respiratory diseases, while avoiding bronchospasms. Incidentally, the company Adenosine Therapeutics LLC (acquired by Clinical Data Inc. in 2008) was awarded funding in 2010 by the National Institute of Allergy and Infectious Diseases to explore applications for the treatment of asthma, arthritis and sepsis.


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Stedivaze (Apadenoson; BMS 068645 or ATL146e) Chemical Structure: 4-(3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydrofuran-2-yl)-9H-purin-2-yl]-prop-2-ynyl)-cyclohexanecarboxylic acid methyl ester In 2001, Linden et al. synthesized and characterized Stedivaze as a potent inhibitor of human neutrophil oxidative activity [132]. This compound was found to have 50 times more affinity for the human neutrophil A2AR than CGS21680. Since LPS causes a rapid up-regulation of A2ARs on inflammatory cells [133], a bacterial infection would provide additional binding sites for Stedivaze to suppress inflammation. In 2004, Linden et al. provided evidence that Stedivaze may constitute a potent anti-inflammatory agent for the treatment of sepsis [134]. Human neutrophils primed by LPS responded to the bacterial chemoattractant, formyl-Met-Leu-Phe (fMLP), by an up-regulation of a4/b1 integrin, as reported for the circulating neutrophils of sepsis patients [135]. This sepsis-like neutrophil phenotype has been shown to bind more readily to the vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells, which facilitates their transmigration into the lung tissue. Stedivaze, added before or after priming, was equally effective in inhibiting the up-regulation of a4/b1 integrin, which reduced the neutrophil adhesive properties. In 2010, Linden et al. presented in vivo evidence for the therapeutic potential of Stedivaze in pulmonary diseases. In sickle cell disease, misshapen erythrocytes trigger transient episodes of microvascular ischemia-reperfusion, which reduces the baseline lung functions and maintains lung inflammation. Therefore, they used the murine model of sickle cell disease, NY1DD mice, to test Stedivaze. In these mice, continuous infusion of Stedivaze (10 ng/mg/min) initiated a dose-dependent improvement of the baseline lung functions, which reached maximal efficacy within 3 days [136]. In the NY1DD mice subjected to hypoxia-reperfusion, stedivaze added at the onset of reperfusion prevented further lung injury. This study supports the potential of stedivaze for the treatment of the lung inflammation caused by recurrent ischemia-reperfusion in sickle cell disease. On the other hand, this study did not document the impact the circulating A2AR agonist on blood pressure, which is a major concern for this class of bioagent.

The Next Generation of ATL Derivatives: ATL313 Chemical Structure: 4-(3-(6-amino-9-(5-cyclopropylcarbamoyl-3,4 dihydroxy tetra hydrofuran-2-yl)-9H-purin-2-yl)prop-2-ynyl) piperidine-1- carboxylic acid methyl ester In collaboration with Adenosine Therapeutics Inc., Dr. Linden et al. designed and characterized a series of derivatives of Stedivaze. In 2007, they conducted radioligand binding experiments which yielded the following ranked order of affinity: ATL313 (IC50 ¼ 1.9 nM) > ATL309 and ATL310 > ATL202 [137]. This class of compounds offers an additional improvement from Stedivaze, with 100-fold higher

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affinity for the A2AR than CGS21680. Functional assays conducted with equine neutrophils showed that all these compounds stimulate cAMP formation, and inhibit the production of reactive oxygen species induced by LPS, while respecting this order of potency. Their therapeutic potential for respiratory diseases was first tested with ATL202 in a model of LPS-induced lung injury [138]. Most animal studies, described in Chaps. 8 and 9, were conducted by giving the drug before the challenge. In the present work, the authors compared the efficiency of aerosolized ATL202 given before or after aerosolized LPS. They showed that both strategies significantly decreased the number of neutrophils and cytokine concentrations measured in the BAL fluid. On the other hand, ATL202 only reduced extravasation when administered before a challenge. This experimental design is critical to identify the best delivery route for a specific treatment. The most promising ATL derivative is ATL313, which exhibits a 100-fold higher selectivity for A2ARs than for A1Rs and A3Rs. Since 2007, more than 30 studies have been published on this ligand. This section reviews only those addressing the impact of ATL313 on lung injury and inflammation. Linden’s group conducted a series of ex vivo and in vivo experiments to test whether the vascular administration of ATL313 protects the lungs against ischemia-reperfusion injury, which remains a major complication after organ transplant. In the first study, they used isolated, buffer-perfused murine lungs to determine whether the therapeutic potential of ATL313 targets the resident cells [139]. Their model of ischemiareperfusion significantly impaired pulmonary functions (higher airway resistance and pulmonary artery pressure, lower compliance), caused extensive tissue injury (vascular leakage and edema) and lung inflammation (BAL levels of TNFa, KC, MIP-2 and RANTES). These lung complications were all significantly attenuated by a bolus dose of ATL313 (30 nM) added to the perfusate at the onset of reperfusion. This dose had no significant effect on the lung functions or hemodynamics of control animals. The specificity of the receptor agonist was demonstrated using lung isolated from A2AR/ mice, which showed no reduction in lung complications in response to ATL313. The fact that vascular ATL313 attenuated lung inflammation in the absence of circulating blood reiterates the critical role of the A2AR for the maintenance endothelial barrier integrity (see Chap. 8, Fig. 8.1). As such, ATL313 administered intravenous before the surgical procedure may significantly improve the outcome of lung transplant. In Chap. 8, we provided evidence that all four ADO receptors mediate protection against vascular leakage, edema and inflammation in models of acute lung injury, such as ischemia-reperfusion (Table 9.1). Therefore, experiments were conducted to determine the relative potency of ATL313 using isolated, blood-perfused lungs infused with a selective agonist/antagonist of the A1R (CCPA/DCPCX), the A2AR (ATL313/ZM241385) or the A3R (IB-MECA/MRS1191) during reperfusion [140]. As expected, all receptor agonists significantly improved the lung functions (increased compliance and oxygenation, and decreased pulmonary artery pressure), reduced edema and inflammation (BAL levels of neutrophil


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myeloperoxidase and TNFa), compared to the untreated animals. When each agonist was paired with its antagonist, the protective effects were lost. More importantly, the ATL313 treatment improved lung functions and reduced neutrophil recruitment more efficiently than the A1R and A3R agonists, supporting a superior protective effect during lung transplant. In another study, these authors used the same protocol to show that the addition of ATL313 1 h before ischemia offers the same degree of protection [141]. Together, these studies suggest that ATL-313 infusion before, or at the time of transplant, may protect the lungs against the development of injury and rejection. Incidentally, this drug was reported to improve the outcome of organ islet transplant [142], supporting broad applications for clinical transplantation. Another surgical application currently proposed for ATL313 is the protection of patients against the post-operative trauma caused by cardiopulmonary bypass. In the rats subjected to this procedure, addition of ATL313 to the blood priming solution completely prevented the development of lung edema, tissue injury and inflammation (IL-1b, IL-6, IFNg, and neutrophil myeloperoxidase activity) [143]. This study suggests that ATL313 may prevent acute lung injury caused by cardiopulmonary bypass. The protective anti-inflammatory effect of ATL313 during ischemia-reperfusion extends beyond the lungs’ resident cells [144]. In mice subjected to hilar occlusion, the intravenous injection of ATL313 before the onset of reperfusion significantly preserved lung function and prevented vascular leakage, compared to control animals. Based on BAL neutrophil myeloperoxidase activity and immunohistochemistry, ATL313 was also shown to inhibit the infiltration of neutrophils and CD4+ T cells during reperfusion. It is common knowledge that the number of circulating neutrophils is regulated by IL-17A, a cytokine released by the CD4+ T lymphocytes (review: [145]). The mice subjected to ischemia-reperfusion, after depletion of the CD4+ T cells or neutrophils, had significantly reduced lung injury, but ATL313 did not provide additional protection. In contrast, the BAL fluid levels of IL-17, KC, MCP-1, MIP-1 and RANTES were significantly lower in the neutrophil- and CD4+ T cell-depleted mice, but only further reduced by ATL313 in the neutrophil-depleted mice. This study demonstrates that ATL313 attenuates acute lung injury and inflammation by inhibiting the secretion of IL-17 by CD4+ T cells, and the subsequent neutrophil recruitment to the lungs. This A2AR agonist is currently the focus of negotiations between Clinical Data Inc. and Santen Pharmaceutical Co. regarding ophthalmic diseases, while Clinical Data Inc. is reserving the rights to explore other applications. In this section, all A2AR agonists evaluated for the treatment of chronic respiratory diseases were intensely scrutinized with respect to cardiovascular side-effects, especially hypotension. Yet, in the case of acute insults, such as surgical procedures or trauma, the above studies suggest that short-term vascular administration of an A2AR agonist would essentially normalize the arterial blood pressure raised by ischemia-reperfusion. Thus, a detrimental property for chronic lung diseases may be beneficial for acute lung injury.

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The A2BR Antagonists

The animal models of chronic respiratory diseases revealed that A2BR activation promotes BHR, inflammation, mucin hypersecretion, airway remodeling, fibrosis and emphysema (see Chap. 8 for details). This impressive portfolio suggests that A2BR antagonists may address all major pulmonary complications of patients diagnosed with asthma, COPD and fibrotic pulmonary diseases. In terms of delivery, inhalation should be avoided to preserve the A2BR-mediated airway clearance of mucins and pathogens (see Chap. 5 for details). Pharmaceutical companies have selected three A2BR antagonists, which are now emerging in the pipeline. The report that A2BR expression increases with disease severity in COPD and IPF [146] should be taken into consideration during clinical trials with respect to the targeted population and expected therapeutic index.

9.4.3.1

Comparing CVT-6883 and Montelukast

Chemical Structure: [3-ethyl-1-propyl-8-[1-(3-trifluoromethylbenzyl)-1H-pyrazol4- yl]-3,7-dihydropurine-2,6-dione] Over the past 5 years, chemists at CV Therapeutics synthesized a large number of Theophylline derivatives in search of a selective A2BR antagonist for the treatment of asthma (review: [147]). Based on binding assays, they selected CVT-6883, which has an affinity for A2BRs (Ki ¼ 8 nM) at least 1,000-fold higher than for the other ADO receptors [148]. In various human cell lines, CVT-6883 inhibits cAMP production induced by the stable ADO analog: N-ethyl-carboxamidoadenosine (NECA). The pharmacodynamic and pharmacokinetic properties of CVT-6883 were determined in the rat. An oral dose of 2.0 mg/kg displayed an excellent systemic exposure, a Cmax of 1,100 ng/ml, a dose adjusted under the curve of (dAUC) of 6,500 ng.h/ml, and a half-life of 4 h. The preclinical evaluation of CVT-6883 was conducted in mice using the ragweed sensitization/challenge model of allergic asthma [149]. The intraperitoneal injection of CVT-6883 completely prevented airway hyperresponsiveness to aerosol AMP, the pro-drug of ADO. Interestingly, CVT-6883 was as potent as an optimal dose of Montelukast [112], the cysteinyl leukotriene-1 receptor antagonist currently prescribed to asthmatic patients (review: [113]). The intraperitoneal delivery of CVT-6883 also inhibited the late allergic responses of these mice to ragweed for at least 5 h. On the other hand, the authors chose aerosol delivery to compare the anti-inflammatory properties of CVT-6883 and Theophylline. The drugs were nebulized prior to the last aerosol ragweed challenge, and the BAL fluid analyzed after 5 h. The optimal dose of each drug reduced total cell counts by nearly 50%. While they were similarly efficient in suppressing eosinophil recruitment, only CVT-6883 significantly reduced the lymphocyte counts. Therefore, aerosol CVT6883 shows promises for the treatment of inflammation in allergic asthma.

9 Therapeutic Applications Table 9.3 Antagonists of A2BRs in the pipeline Delivery Proof of Drug Disease route concept CVT-6883 Asthma Oral

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Preclinical

Phase Phase 1 2a

Phase 2b

Phase 3

MRE 2029- Inflammation F20 LAS38096

Asthma

Oral

The potential of long-term treatment with CVT-6883 to resolve all complications of chronic respiratory diseases was addressed using an animal model which develops the airway hyperresponsiveness, inflammation, remodeling, fibrosis and alveolar destruction: ADA-deficient mice [148]. The 14-day treatment consisted of intraperitoneal injections, administered twice daily to animals after they developed significant lung complications. The CVT-6883-treated mice presented significantly reduced airway inflammation (BAL cytokine levels, lymphocytes, eosinophils, neutrophils and macrophages), fibrosis (a1-procollagen and collagen deposition) and alveolar airspace enlargement. These results support substantial benefits for the long-term treatment of chronic lung diseases. In 2008, CV Therapeutics had completed two randomized, single-blind, placebo-controlled, ascending dose clinical trials, in which CVT-6883 was determined to be safe and well tolerated by healthy volunteers [150]. The pharmacokinetic data support the use of one daily oral dose of CVT-6883 for the long-term treatment of chronic respiratory diseases (Table 9.3). This delivery route would also minimize the inhibitory effect of A2BR antagonists on the airway clearance of mucin and pathogens (see Chap. 5 for details). In 2009, Gilead took over CV Therapeutics, which may cause a reorganization of their priorities, with respect to the drugs currently in the pipeline.

9.4.3.2

Anti-Inflammatory MRE 2029-F20

Chemical Structure: N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy] acetamide In 2004, Baraldi et al. announced that they synthesized a large number of xanthine derivatives as potential A2BR antagonists [151]. The most selective compound was named MRE 2029-F20 (KiA2BR ¼ 5.5 nM; KiA1R ¼ 200 nM; KiA2AR, A3R > 1,000). Using isolated human neutrophils, lymphocytes or mast cells, MRE 2029-F20 was shown to inhibit the NECA-mediated cAMP production with IC50 values in the low nanomolar range, which supported a potent anti-inflammatory property. Therefore, MRE 2029-F20 was selected for further evaluation by King Pharmaceuticals [151]. In 2005, Borea et al. developed a radiolabelled MRE 2029-F20 [152, 153], which has been instrumental in the discovery that A2BR expression rises with disease

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severity in the lungs of COPD patients [118]. In recombinant systems, tritiated MRE 2029-F20 binds selectively to A2BRs, with KD values around 2 nM [153]. The same year, they tested the selectivity of MRE 2029-F20 in a human melanoma cell line [154]. The cell proliferation induced by the selective A3R agonist, Cl-IB-MECA, was not inhibited by this A2BR antagonist. Preclinical data in animal models have not yet been reported for this bioagent.

9.4.3.3

LAS38096 for the Treatment of Asthma

Chemical Structure: 4’-(2-furyl)-N-pyridin-3-yl-4,5’-bipyrimidin-2’-amine In 2007, a third compound, LAS38096 was discovered by Almirall Prodesfarma, which exhibits high affinity and selectivity for the A2BR (KiA2BR ¼ 17 nM; KiA1R > 1,000 nM; KiA2AR > 2,500 nM; and KiA3R > 1,000 nM) [155]. In heterologous systems expressing human or murine A2BRs, LAS38096 inhibited NECA-mediated cAMP production with IC50 values in the 320–350 nM range. The capacity of LAS38096 to inhibit inflammatory responses was tested using human and mouse dermal fibroblasts. The bioagent induced a dose-dependent decrease in NECAmediated IL-6 secretion, with IC50 values of 340 and 640 nM, respectively [155]. These data demonstrate that LAS38096 is a functional A2BR antagonist with similar affinity on the human and murine receptor. The pharmacokinetic parameters of an oral dose of LAS38096 were determined in the rat, mouse and dog [155]. The drug exhibited excellent bioavailability in all species. It was absorbed rapidly (tmax < 60 min), with an AUC of 4.0 mM.h and a moderate-to-high plasma clearance. The efficacy of LAS38096 was tested in vivo in the OVA sensitization/challenge model of allergic asthma [156]. Mice treated with the bioagent showed significantly less airway hyperresponsiveness, mucus production, eosinophil infiltration and OVA-specific IgE in the BAL fluid than untreated animals. LAS38096 has been advanced for evaluation of safety and toxicology.

9.4.4

The A3R Agonists

The terms “complex” and “enigmatic” are frequently ascribed to the A3R because this receptor has been shown to induce inflammatory and anti-inflammatory responses (review: [157]). For instance, it stimulates neutrophil chemotaxis, but inhibits eosinophil recruitment to the airways. Nonetheless, A3R-mediated responses are predominantly anti-inflammatory, as demonstrated by the knockout mouse (see Chap. 8 for details). Thus, while agonists and antagonists are available as pharmacological agents, only agonists are currently tested for the treatment of chronic inflammatory disorders.


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Over the last decade, new approaches have been introduced for the treatment of autoimmune diseases (review: [158]). Among the most potent drugs, TNFa antibodies are prescribed for arthritis, psoriasis and Crohn’s diseases. However, to block all TNFa-mediated inflammatory responses compromises host defenses and causes severe adverse effects. Recent studies revealed that the secretion of TNFa from inflammatory cells is inhibited by ADO acting selectively on A3Rs [159, 160]. Since the A3R is not expressed on airway epithelial cells, this approach may reduce lung inflammation by focusing on non-resident cells, with minimal effects on defenses mediated by the epithelial barrier. Major concerns about the use of aerosolized A3R agonists were fueled by reports of BHR in murine models [45–48]. However, it is important to mention that histamine secretion is mediated by A2BRs in canine and human mast cells [49–51], which motivated the use of canine models for the evaluation of A3R agonists as anti-inflammatory agents. Furthermore, our current understanding of ADO-mediated BHR in asthmatics supports an A1R-mediated neural signal upstream from mast cell activation (see Sect. 9.2). As such, the A3R-mediated bronchospasms observed in murine models of asthma are not expected in human subjects. Hopefully, these precisions will stimulate the pharmaceutical industry to develop selective A3R agonists for chronic inflammatory lung diseases.

9.4.4.1

Rebirth of IB-MECA as CF101

Chemical Structure: 1-deoxy-1-[6-[[(iodophenyl)methyl]amino]9H-purine-9-yl]-Nmethyl-(-D-ribofuranuronamide) We owe a dept of gratitude to Dr. Kenneth A. Jacobson, who devoted his career to the development of ADO receptor ligands (review: [99]). In 1993, he designed the first selective A3R agonist, IB-MECA, which has been used in over 300 in vitro and in vivo studies. This metabolically stable molecule binds and activates the human A3R with an affinity (Ki ¼ 0.5 nM) 1,000-fold higher than on the other ADO receptors (review: [157]). In 2000, Dr. Pnina Fishman obtained an exclusive license for IB-MECA, which was renamed CF101, and launched the company Can-Fite Biopharma. The therapeutic potential of CF101 for lung complications was assessed mainly in models of acute lung injury. In isolated, blood-perfused rabbit lungs subjected to 18 h of cold ischemia, the addition of CF101 before reperfusion improved lung compliance and oxygenation, without affecting arterial pressure [140]. With respect to the inflammatory mediators measured in the BAL fluid, the A3R agonist reduced TNFa concentrations by 50% and neutrophil myeloperoxidase activity by 30%. Acute lung injury, quantified in terms of vascular leakage and edema, was also significantly lower in the animals treated with CF101. This study highlights the pleitropic protective effects of intravenous CF101 against acute lung inflammation and injury initiated by episodes of ischemia-reperfusion. Interestingly, Methotrexate was recently shown to enhance the anti-inflammatory

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effects of CF101 by causing an up-regulation of the A3R on monocytes and macrophages [161], which could offer additional clinical benefits for the treatment of acute lung injury. The signaling mechanisms by which CF101 protects the lungs against injury and apoptosis during ischemia-reperfusion were investigated in spontaneously breathing cats [162, 163]. The presence of systemic CF101 during reperfusion significantly reduced the acute lung injury (% injured alveoli, wet/dry weight ratio) and apoptosis (TdT-mediated dUTP nick-end labeling positive cells and caspase 3 activity). Using selective inhibitors, the protective effects of CF101 were found to require ATP-sensitive K+ channels, but not nitric oxide production [163]. The signaling pathways activated by CF101 were identified by monitoring the expression of major protein kinases by SDS-PAGE and Western blot analysis of lung tissue specimens [162]. The A3R agonist induced a gradual up-regulation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2), but not the c-Jun amino-terminal protein kinase (JNK) or p38. Injection of a selective A3R antagonist (MRS1191), before CF101, abolished the response of ERK1/2 without affecting p38 and JNK. These data suggest that the CF101-A3R signals are mediated predominantly by the ERK1/2-dependent pathways. However, A3Rs have been reported to regulate apoptosis via other signaling cascades, such as the phosphoinositide-3 kinase (PI3K)-dependent pathway (review: [157]). Therefore, future studies should verify the impact of selective ERK1/2 inhibitors on the acute lung injury and inflammation caused by ischemia-reperfusion. In two controlled, double-blind, single ascending Phase 1 clinical trials conducted on healthy subjects, oral CF101 was demonstrated to be safe and well tolerated [164]. In oral solution, CF101 was readily absorbed (Tmax ¼ 1–2 h) with a half-life of 9 h. In December 2010, Can-Fite BioPharma announced that CF101 entered Phase 2 or 3 clinical trials for the treatment of cancer, dry eye syndrome, psoriasis and rheumatoid arthritis (Table 9.4). They retained the services of Plexus Ventures for the identification of a partner to support the clinical development and commercialization of CF101 in the United States and Europe, while it is already licensed in Japan and South Korea. Table 9.4 Agonists of A3Rs in the pipeline Drug CF101 (IBMECA)

Disease Acute lung injury

Proof of PrePhase Phase Delivery route concept clinical 1 2a Intravenous

Arthritis Oral psoriasis CF102 (Cl-IBMECA)

Sepsis

Intraperitoneal

Liver Oral diseases

CF502 Acute lung (MRS3558) injury

Intraperitoneal

Phase 2b

Phase 3


9.4.4.2

259

The More Selective Cl-IB-MECA (CF102)

Chemical structure: 2-chloro-N6-(3-lodobenzyl)-adenosine-5’-N-methyluronamide Can-Fite BioPharma is also evaluating the potential of Cl-IB-MECA (CF102), a more selective analogue of IB-MECA (CF101) with binding affinities of 1.4 nM for the A3R, compared to 220 nM for the A1R, and 5,400 nM for the A2AR (review: [157]). Like his close relative, CF102 has been widely used as a pharmacological agent to identify the complex pharmacological properties of the A3R in mammalian tissues. Wagner et al. tested the impact of CF102 on the lung inflammation induced by aerosolized LPS in wild-type and A3R/ mice [165]. The endotoxin induced a linear increase in A3R expression over time in whole lung tissue, reaching sixfold above the baseline after 3 h. In addition, LPS inhalation stimulated leukocyte recruitment into the pulmonary vasculature, lung tissue and airspace. Pretreatment by intraperitoneal injection of CF102 significantly reduced the number of leukocytes in all compartments in the wild-type mice, but not in the A3R/ mice. The CF102 treatment significantly reduced the levels of inflammatory cytokines (TNFa and IL-6) in the BAL fluid of LPS-exposed wild-type mice. In accordance with the barrier protective role of A3Rs during acute lung injury (see Chap. 8 for details), CF102 suppressed the LPS-induced vascular leakage in wild-type mice. One foreseeable side-effect of intraperitoneal CF102 is the enhancement of neutrophil migration speed (review: [166]). Nonetheless, the ligand did not affect the leukocyte counts in the pulmonary vasculature in the control and LPS-exposed animals. In November 2010, Can-Fite Biopharma completed a Phase 1 clinical trial to assess the safety and pharmacokinetic behavior of CF102 in patients with advanced liver cancer. The patients received oral doses of 1, 5 or 25 mg (twice daily) over 28 days. Overall, the drug is safe and well tolerated at doses up to 25 mg, shows good oral bioavailability and linear pharmacokinetic behavior. Also, patient infected with hepatitis virus C experience a significant reduction in virus titer with CF102, consistent with its preclinical anti-viral activity. This A3R agonist hold promises as a novel therapeutic strategy in the treatment of cancer and viral infections.

9.4.4.3

New Generation MRS3558 (CF502)

Chemical Structure: [(10 R,20 R,30 S,40 R,50 S)-4-{2-chloro-6-[(3-chlorophenylmethyl) amino] purin-9-yl}-1-(methylaminocarbonyl)bicyclo [3.1.0] hexane-2,3-diol] A novel A3R agonist, CF502, was recently synthesized at the National Institutes of Health (review: [157]). This molecule displays enhanced selectivity and specificity with >1,000-fold more affinity for the A3R than the A1R, A2AR, and A2BR. Functionally, this molecule represents a significant improvement from the first generation compounds. In the breathing cat model of ischemia-reperfusion, CF502 was found more efficient than CF101 against acute lung injury [162].

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Jacobson et al. used the breathing cat model of ischemia-reperfusion to assess the potential of CF502 for the treatment of acute lung injury [167]. Interestingly, the sedated animals were monitored over 27 h of reperfusion, instead of the usual

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