Particle-Lung Interactions, Second Edition

Particle-Lung Interactions LUNG BIOLOGY IN HEALTH AND DISEASE Executive Editor Claude Lenfant Former Director, Nation...

Author: Peter Gehr | Christian Mühlfeld | Barbara Rothen-Rutishauser | Fabian Blank

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Particle-Lung Interactions

LUNG BIOLOGY IN HEALTH AND DISEASE

Executive Editor Claude Lenfant Former Director, National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Immunologic and Infectious Reactions in the Lung, edited by C. H. Kirkpatrick and H. Y. Reynolds The Biochemical Basis of Pulmonary Function, edited by R. G. Crystal Bioengineering Aspects of the Lung, edited by J. B. West Metabolic Functions of the Lung, edited by Y. S. Bakhle and J. R. Vane Respiratory Defense Mechanisms (in two parts), edited by J. D. Brain, D. F. Proctor, and L. M. Reid Development of the Lung, edited by W. A. Hodson Lung Water and Solute Exchange, edited by N. C. Staub Extrapulmonary Manifestations of Respiratory Disease, edited by E. D. Robin Chronic Obstructive Pulmonary Disease, edited by T. L. Petty Pathogenesis and Therapy of Lung Cancer, edited by C. C. Harris Genetic Determinants of Pulmonary Disease, edited by S. D. Litwin The Lung in the Transition Between Health and Disease, edited by P. T. Macklem and S. Permutt Evolution of Respiratory Processes: A Comparative Approach, edited by S. C. Wood and C. Lenfant Pulmonary Vascular Diseases, edited by K. M. Moser Physiology and Pharmacology of the Airways, edited by J. A. Nadel Diagnostic Techniques in Pulmonary Disease (in two parts), edited by M. A. Sackner Regulation of Breathing (in two parts), edited by T. F. Hornbein Occupational Lung Diseases: Research Approaches and Methods, edited by H. Weill and M. Turner-Warwick Immunopharmacology of the Lung, edited by H. H. Newball Sarcoidosis and Other Granulomatous Diseases of the Lung, edited by B. L. Fanburg Sleep and Breathing, edited by N. A. Saunders and C. E. Sullivan Pneumocystis carinii Pneumonia: Pathogenesis, Diagnosis, and Treatment, edited by L. S. Young Pulmonary Nuclear Medicine: Techniques in Diagnosis of Lung Disease, edited by H. L. Atkins Acute Respiratory Failure, edited by W. M. Zapol and K. J. FaIke

For information on volumes 25–188 in the Lung Biology in Health and Disease series, please visit www.informahealthcare.com 189. 190. 191. 192. 193. 194. 195. 196. 197. 198. 199. 200. 201. 202. 203. 204. 205. 206. 207. 208. 209. 210. 211. 212. 213. 214. 215. 216. 217.

Interventional Pulmonary Medicine, edited by J. F. Beamis, Jr., P. N. Mathur, and A. C. Mehta Lung Development and Regeneration, edited by D. J. Massaro, G. Massaro, and P. Chambon Long-Term Intervention in Chronic Obstructive Pulmonary Disease, edited by R. Pauwels, D. S. Postma, and S. T. Weiss Sleep Deprivation: Basic Science, Physiology, and Behavior, edited by Clete A. Kushida Sleep Deprivation: Clinical Issues, Pharmacology, and Sleep Loss Effects, edited by Clete A. Kushida Pneumocystis Pneumonia: Third Edition, Revised and Expanded, edited by P. D. Walzer and M. Cushion Asthma Prevention, edited by William W. Busse and Robert F. Lemanske, Jr. Lung Injury: Mechanisms, Pathophysiology, and Therapy, edited by Robert H. Notter, Jacob Finkelstein, and Bruce Holm Ion Channels in the Pulmonary Vasculature, edited by Jason X.-J. Yuan Chronic Obstructive Pulmonary Disease: Cellular and Molecular Mechanisms, edited by Peter J. Barnes Pediatric Nasal and Sinus Disorders, edited by Tania Sih and Peter A. R. Clement Functional Lung Imaging, edited by David Lipson and Edwin van Beek Lung Surfactant Function and Disorder, edited by Kaushik Nag Pharmacology and Pathophysiology of the Control of Breathing, edited by Denham S. Ward, Albert Dahan, and Luc J. Teppema Molecular Imaging of the Lungs, edited by Daniel Schuster and Timothy Blackwell Air Pollutants and the Respiratory Tract: Second Edition, edited by W. Michael Foster and Daniel L. Costa Acute and Chronic Cough, edited by Anthony E. Redington and Alyn H. Morice Severe Pneumonia, edited by Michael S. Niederman Monitoring Asthma, edited by Peter G. Gibson Dyspnea: Mechanisms, Measurement, and Management, Second Edition, edited by Donald A. Mahler and Denis E. O’Donnell Childhood Asthma, edited by Stanley J. Szefler and Sfren Pedersen Sarcoidosis, edited by Robert Baughman Tropical Lung Disease, Second Edition, edited by Om Sharma Pharmacotherapy of Asthma, edited by James T. Li Practical Pulmonary and Critical Care Medicine: Respiratory Failure, edited by Zab Mosenifar and Guy W. Soo Hoo Practical Pulmonary and Critical Care Medicine: Disease Management, edited by Zab Mosenifar and Guy W. Soo Hoo Ventilator-Induced Lung Injury, edited by Didier Dreyfuss, Georges Saumon, and Rolf D. Hubmayr Bronchial Vascular Remodeling in Asthma and COPD, edited by Aili Lazaar Lung and Heart–Lung Transplantation, edited by Joseph P. Lynch III and David J. Ross

218. 219. 220. 221. 222.

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225. 226. 227. 228. 229. 230. 231. 232. 233. 234. 235. 236. 237. 238. 239. 240. 241.

Genetics of Asthma and Chronic Obstructive Pulmonary Disease, edited by Dirkje S. Postma and Scott T. Weiss Reichman and Hershfield’s Tuberculosis: A Comprehensive, International Approach, Third Edition (in two parts), edited by Mario C. Raviglione Narcolepsy and Hypersomnia, edited by Claudio Bassetti, Michel Billiard, and Emmanuel Mignot Inhalation Aerosols: Physical and Biological Basis for Therapy, Second Edition, edited by Anthony J. Hickey Clinical Management of Chronic Obstructive Pulmonary Disease, Second Edition, edited by Stephen I. Rennard, Roberto Rodriguez-Roisin, Ge´rard Huchon, and Nicolas Roche Sleep in Children, Second Edition: Developmental Changes in Sleep Patterns, edited by Carole L. Marcus, John L. Carroll, David F. Donnelly, and Gerald M. Loughlin Sleep and Breathing in Children, Second Edition: Developmental Changes in Breathing During Sleep, edited by Carole L. Marcus, John L. Carroll, David F. Donnelly, and Gerald M. Loughlin Ventilatory Support for Chronic Respiratory Failure, edited by Nicolino Ambrosino and Roger S. Goldstein Diagnostic Pulmonary Pathology, Second Edition, edited by Philip T. Cagle, Timothy C. Allen, and Mary Beth Beasley Interstitial Pulmonary and Bronchiolar Disorders, edited by Joseph P. Lynch III Chronic Obstructive Pulmonary Disease Exacerbations, edited by Jadwiga A. Wedzicha and Fernando J. Martinez Pleural Disease, Second Edition, edited by Demosthenes Bouros Interventional Pulmonary Medicine, Second Edition, edited by John F. Beamis, Jr., Praveen Mathur, and Atul C. Mehta Sleep Apnea: Implications in Cardiovascular and Cerebrovascular Disease, Second Edition, edited by Douglas T. Bradley and John Floras Respiratory Infections, edited by Sanjay Sethi Acute Respiratory Distress Syndrome, edited by Augustine M. K. Choi Pharmacology and Therapeutics of Airway Disease, Second Edition, edited by Kian Fan Chung and Peter J. Barnes Sleep Apnea: Pathogenesis, Diagnosis, and Treatment, Second Edition, edited by Allan I. Pack Pulmonary Hypertension, edited by Marc Humbert and Joseph P. Lynch III Tuberculosis: The Essentials, edited by Mario C. Raviglione Asthma Infections, edited by Richard Martin and Rand E. Sutherland Chronic Obstructive Pulmonary Disease: Outcomes and Biomarkers, edited by Mario Cazzola, Fernando J. Martinez, and Clive P. Page Bronchopulmonary Dysplasia, edited by Steven H. Abman Particle-Lung Interactions, Second Edition, edited by Peter Gehr, Christian M€ uhlfeld, Barbara Rothen-Rutishauser, and Fabian Blank

The opinions expressed in these volumes do not necessarily represent the views of the National Institutes of Health.

Particle-Lung Interactions Second Edition

edited by

Peter Gehr University of Bern Bern, Switzerland

Christian Mühlfeld University of Giessen Giessen, Germany

Barbara Rothen-Rutishauser University of Bern Bern, Switzerland

Fabian Blank University of Bern Bern, Switzerland

Informa Healthcare USA, Inc. 52 Vanderbilt Avenue New York, NY 10017 # 2010 by Informa Healthcare USA, Inc. Informa Healthcare is an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 1-4200-7256-0 (Hardcover) International Standard Book Number-13: 978-1-4200-7256-3 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequence of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Particle-lung interactions / edited by Peter Gehr . . . [et al.]. — 2nd ed. p. ; cm. — (Lung biology in health and disease ; 241) Includes bibliographical references and index. ISBN-13: 978-1-4200-7256-3 (hardcover : alk. paper) ISBN-10: 1-4200-7256-0 (hardcover : alk. paper) 1. Pulmonary toxicology. 2. Particles. 3. Lungs—Dust diseases. 4. Pulmonary endothelium. 5. Pulmonary surfactant. I. Gehr, Peter. II. Series: Lung biology in health and disease ; v. 241. [DNLM: 1. Lung–metabolism. 2. Aerosols. 3. Air Pollutants. 4. Air Pollution—adverse effects. 5. Allergens. 6. Inhalation Exposure. 7. Lung—immunology. W1 LU62 v.241 2009 / WF 600 P273 2009] RC720.P37 2009 616.2’00471—dc22 2009023883 For Corporate Sales and Reprint Permissions call 212-520-2700 or write to: Sales Department, 52 Vanderbilt Avenue, 7th floor, New York, NY 10017. Visit the Informa Web site at www.informa.com and the Informa Healthcare Web site at www.informahealthcare.com

Introduction

The current and rightfully ever increasing concerns for air pollution associated with climate changes, and its health effects, make this second edition of Particle-Lung Interactions extremely timely. Years back, the interest in pollution, the associate particles, and irritant gases focused on indoor situation, but soon the interest was extended to the air that we breathe, wherever we are. In fact, it seems that the concerns about outside air particles soon became greater than those about indoor air particles, primarily because of the public health implications. Unquestionably, the interest in this matter has considerably increased during the last 10 years or so, especially since the publication of the first edition of Particle-Lung Interactions. Furthermore, as the editors of this new edition— Drs. Peter Gehr, Christian Mühlfeld, Barbara Rothen-Rutishauser, and Fabian Blank—point out in their Preface, two new “things” have appeared. First, the increased knowledge and interest in nanoparticles, which are now well-defined materials. The focus on their effect on health and conversely their use to improve drug delivery have become a very productive research area. Second, the interactions of these particles with organs, especially the respiratory tract and the lung, have shifted the focus of their impact to the cellular level. The scientific community is working hard to determine the mechanisms of these interactions and their impact at the subcellular and molecular levels. It is also recognized that although the lung is the port of entry of these nanoparticles, the particles will also impact on other organs, such as the cardiovascular system, if absorbed. The inclusion of this monograph in the series Lung Biology in Health and Disease will inform its readers about a dynamic field, but it will also stimulate basic and clinical research on an important, even critical, area. As the executive editor of this series, I express my thanks to the editors and authors of this volume for the opportunity to present it to our readership. Claude Lenfant, MD Vancouver, Washington, U.S.A.

vii

Preface

Leonardo da Vinci (1452–1519), prototype of the universal genius, was a painter, musician, architect, and an engineer. He was also a sculptor and an anatomist. In his Anatomical Atlas he added the short comment above the trachea, “dust is harmful.” Today we know that Leonardo da Vinci was right. During the past decades, a growing body of epidemiological and experimental literature has provided evidence that inhalation of “dust,” that is, airborne particles in the widest sense, substantially contributes to creating adverse health effects. The characteristics of the particles that may be inhaled with every breath depend on many factors, like season, climate, and environment. Similarly diverse are the health effects of particles, ranging from respiratory to cardiovascular diseases, from asthma over increased susceptibility to viral infections to myocardial infarction. In recent years, there has been evidence that the fraction of nano-sized particles, that is, particles smaller than 100 nm (0.1 mm) are of particular concern. When the first edition of this book entered the market, the editors anticipated that the field of particle-lung interactions was about to enter a new era. Cautiously, the editors mentioned the likely changes in the preface: Since this monograph first took shape, the field of particle-lung interactions has expanded considerably. The “bad” particles are currently ultrafine particles released into the environment from combustion processes. The “good” particles are those carrying insulin into the lungs. The treatment of diabetes via the inhalation route will most likely become the first approved aerosol treatment for a systemic disease. Nevertheless, we both hope that established knowledge and controversial issues are sufficiently reflected in the volume on particle-lung interactions.

In 2007, Joachim Heyder and Peter Gehr were asked by the publisher of Claude Lenfant’s series on Lung Biology in Health and Disease, Informa Healthcare, whether they would be interested in publishing a second edition of their volume, number 143, on Particle-Lung Interactions. Due to various circumstances Joachim Heyder was not able to work on the second edition. Therefore, Peter Gehr asked three young researchers around him to co-edit the next edition. This gave rise to the new editor team for this second edition on Particle-Lung Interactions. Although Joachim Heyder’s wisdom, editorial skills and fundamental and comprehensive knowledge cannot be overrated, the composition of the new editorial team provided different advantages.

ix

x

Preface

Throughout all the work of editing, we experienced a very close and respectful collaboration. Two things have changed, since the appearance of the last book. Whereas nano-sized particles were briefly mentioned by only a few authors earlier, nanomaterials are now a major aspect in this second edition. A totally new field dealt with in many chapters, which is rapidly gaining significance, is manufactured nanoparticles. Publications in this field are still rather sparse but rapidly growing in number. A lot of experimental work is still needed, and this is particularly true for the good particles as they were called in the preface of the first edition. The hope expressed almost 10 years ago that the treatment of diabetes via the inhalation route will most likely become the first approved aerosol treatment for a systemic disease was abruptly destroyed when Pfizer stopped the production of insulin, apparently due to poor sales. Since then, Lilly and Novo Nordisk have also dropped their plans to produce inhalable insulin. The lung is a major target of ambient air pollution. The relationship between increased concentrations of air pollution and adverse health effects in children, subjects with asthma and COPD, and vulnerable adults is well documented. Major adverse health effects include not only increased respiratory symptoms, decreased lung function, and increased hospitalization but also altered mucociliary clearance, COPD, asthma, and increased mortality. Several chapters in this book deal with the various diseases associated with particle inhalation. A number of in vitro studies have been performed to investigate how fine and nano-sized particles enter tissues and cells of the lungs and to determine what effect they may have. In contrast to fine particles that are taken up by cells by phagocytosis, nanoparticles may enter cells by an endocytic or by another, yet to be defined mechanism. This circumstance could be a major cause for the enhanced adverse health effects of nano-sized particles. Many publications have shown that the main cellular effect of exposure to combustion-derived nanoparticles is the production of reactive oxygen species, which is a major factor in inflammation and toxicity. There is increasing evidence that these nanoparticles may pose a particular danger because of their high content of organic chemicals and the pro-oxidative potential. The key properties associated with these particles are their large surface area and the presence of metals and organics, all of which have the potential to produce oxidative stress. Moreover, the same particles may also have genotoxic effects. These aspects are discussed in this book. Studies investigating the interactions of the lungs with these minute particles have also been stimulated by advances in nanotechnology. This technological branch has been overwhelming us with newly designed materials that due to different physicochemical behaviors offer great advantages in many applications, ranging from antibacterial clothing to car lacquers and to medicinal particles. The latter have the potential not only of drug delivery via

Preface

xi

inhalation for systemic treatment but also of targeting drugs to specific cell types and even organelles. This might significantly reduce the side effects of current therapeutics or lead to new developments in diagnostics and therapy. However, due to their minute size, potential toxicity should be considered for manufactured nanoparticles as well. In most studies, investigators found that these artificially produced nanoparticles were associated with the generation of reactive oxygen species by cells exposed to the particles. A lot of work is still needed to determine whether the conclusions drawn for combustion-derived particles can be extrapolated to manufactured nanoparticles. Since both nanotechnology in its widest sense and nanotoxicology have been recognized to have a great future impact, governmental programs in the whole world are supporting both research fields financially. The concern about possible hazardous effects of manufactured nanoparticles crosses the borders of scientific curiosity quickly and enters broad public awareness. Some of the fears associated with nanoparticles are related to the lessons learned from the deleterious effects of inhaled asbestos particles. This time particle researchers are working hard to assess the risks associated with manufactured nanoparticles well in advance to avoid the health consequences and financial burdens that may follow after exposure. While the exposure of combustion-derived nanoparticles affects everyone, it is obvious that the exposure to manufactured nanoparticles may primarily affect people working in the production of nanoparticles, their processing, or application. These important concerns have caused the development of a whole new scientific field known as “nanotoxicology.” In January 2006, the first international congress on nanotoxicology took place in Miami and a second one was held in September 2008 in Zurich. Both journals and books entirely devoted to the subject have entered the market. Nevertheless, a great amount of work still needs to be done. In the light of the new developments, the second edition of this book has to face the dilemma that the established knowledge from the first edition needs to be combined with the vastly growing knowledge gained during the past few years, a process that will surely continue. This dilemma is aggravated by the publishers’ request to shorten the book compared to the first edition. Therefore, the second edition of this book has been rearranged completely. Fundamental aspects on pulmonary structure and function as well as different kinds of particles have been shifted into the first and second part of this book to avoid repetition of these aspects in different chapters. The third part deals with the inhalation of particles, their deposition and retention in the respiratory tract as well as their clearance and translocation. The two following parts are devoted to the interaction of particles with biological structures, starting at the systemic and organ level (part 4) and finishing with the cellular and molecular effects inhalation of particles may have (part 5). We provide the reader with a

xii

Preface

combination of both established knowledge and, hopefully, exciting new observations. We are well pleased with the result of the second edition and we warmly thank our colleagues for their extraordinary support. Without their special effort, it would have never been possible to finish this publication. We are grateful to the staff of Informa Healthcare for their invaluable and professional assistance in editing this book. The interactions with the authors as well as with the publisher were always stimulating. We do hope that the result will serve its purpose and that this book will take its place among the well-received monographs in the series in Lung Biology in Health and Disease. Peter Gehr Christian M€ uhlfeld Barbara Rothen-Rutishauser Fabian Blank

Contributors

Matthias Amrein

University of Bern, Bern, Switzerland

Fabian Blank Michael Bur

University of Calgary, Calgary, Alberta, Canada

Saarland University, Saarbrücken, Germany

Christopher Carosino

University of California, Davis, California, U.S.A.

Daniel L. Costa U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, U.S.A. Ken Donaldson

University of Edinburgh, Edinburgh, U.K.

Mark W. Frampton New York, U.S.A. Peter Gehr

University of Rochester Medical Center, Rochester,


Peter J. Gerber

University of Berne, Berne, Switzerland

Beatriz Gonzalez-Flecha Massachusetts, U.S.A.

Harvard School of Public Health, Boston,

Robert N. Grass Institute for Chemical and Bioengineering, ETH Zurich, Zurich, Switzerland Francis H. Y. Green Stephanie Hein Andreas Henning


Saarland University, Saarbrücken, Germany Saarland University, Saarbrücken, Germany

Jens M. Hohlfeld Fraunhofer Institute of Toxicology and Experimental Medicine, and Hannover Medical School, Hannover, Germany

xiii

xiv

Contributors

Katherine Horvath University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. Ilona Jaspers University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, U.S.A. Malcolm King

University of Alberta, Edmonton, Alberta, Canada

Wolfgang G. Kreyling Helmholtz Zentrum München—German Research Center for Environmental Health, Institute for Lung Biology and Disease (iLBD) and Focus Network Nanoparticles and Health, Neuherberg, Germany Claus-Michael Lehr Zoya Leonenko

Saarland University, Saarbrücken, Germany

University of Waterloo, Waterloo, Ontario, Canada

Ludwig K. Limbach Zurich, Switzerland

Institute for Chemical and Bioengineering, ETH Zurich,

Winfried Mo¨ller Helmholtz Zentrum München—German Research Center for Environmental Health, Institute for Lung Biology and Disease (iLBD) and Clinical Cooperation Group Inflammatory Lung Diseases, Gauting, Germany Amy K. Madl University of California, Davis, and ChemRisk, LLP, San Francisco, California, U.S.A. Christian Mu¨hlfeld* Matthias Ochs{



Kent E. Pinkerton

University of California, Davis, California, U.S.A.

Barbara Rothen-Rutishauser


Jonathan M. Samet Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, U.S.A. Samuel Schu¨rch


Current affiliations: * University of Giessen, Giessen, Germany. { Hannover Medical School, Hannover, Germany.

Contributors

xv

Carsten Schleh Fraunhofer Institute of Toxicology and Experimental Medicine, and Hannover Medical School, Hannover, Germany Otmar Schmid Helmholtz Zentrum München—German Research Center for Environmental Health, Institute for Lung Biology and Disease (iLBD) and Focus Network Nanoparticles and Health, Neuherberg, Germany Saarland University, Saarbrücken, Germany

Marc Schneider

Holger Schulz Helmholtz Zentrum München—German Research Center for Environmental Health, Institute for Lung Biology and Disease (iLBD) and Focus Network Nanoparticles and Health, Neuherberg, Germany Joel Schwartz U.S.A.

Harvard School of Public Health, Boston, Massachusetts,

Manuela Semmler-Behnke Helmholtz Zentrum München—German Research Center for Environmental Health, Institute for Lung Biology and Disease (iLBD) and Focus Network Nanoparticles and Health, Neuherberg, Germany Paul A. Solomon Nevada, U.S.A.

U.S. Environmental Protection Agency, Las Vegas,

Wendelin J. Stark Institute for Chemical and Bioengineering, ETH Zurich, Zurich, Switzerland Tobias Stoeger Helmholtz Zentrum München—German Research Center for Environmental Health, Institute for Lung Biology and Disease (iLBD) and Focus Network Nanoparticles and Health, Neuherberg, Germany Vicki Stone Mark J. Utell U.S.A.

Edinburgh Napier University, Edinburgh, U.K. University of Rochester Medical Center, Rochester, New York,

Contents

Introduction Claude Lenfant . . . . . . . vii Preface . . . . . . . . . . . . . . . . . . . . . . . . ix Contributors . . . . . . . . . . . . . . . . . . . . xiii Part 1: Structure and Function of the Lung

1. Functional Aspects of Lung Structure as Related to Interaction with Particles . . . . . . . . . . . . . . . . . . . . . . . . . Christian M€ uhlfeld and Matthias Ochs

1

Part 2: Particle Characteristics

2. Ambient Tropospheric Particles . . . . . . . . . . . . . . . . . . . . . Paul A. Solomon and Daniel L. Costa

17

3. Manufactured Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . Robert N. Grass, Ludwig K. Limbach, and Wendelin J. Stark

38

4. Particulate Carriers for Pulmonary Drug Delivery . . . . . . . Stephanie Hein, Andreas Henning, Michael Bur, Marc Schneider, and Claus-Michael Lehr

60

Part 3: Inhalation of Particles

5. Deposition, Retention and Clearance, and Translocation of Inhaled Fine and Nano-Sized Particles in the Respiratory Tract . . . . . . . . . . . . . . . . . . . . . . . . . . Winfried M€ oller, Wolfgang G. Kreyling, Otmar Schmid, Manuela Semmler-Behnke, and Holger Schulz

79

Part 4: Health Effects of Particle Inhalation

6. The Epidemiology of Particle Health Effects . . . . . . . . . . . . Joel Schwartz xvii

108

xviii

Contents

7. Cardiovascular Consequences of Particles . . . . . . . . . . . . . . Mark W. Frampton, Mark J. Utell, and Jonathan M. Samet 8. Autonomic Mediation of the Cardiac Responses to Particle Exposure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Beatriz Gonzalez-Flecha

120

137

9. Effects of Airborne Particles on Respiratory Viral Infection Ilona Jaspers and Katherine Horvath

151

10. Airborne Particles and Structural Remodeling of the Lungs Amy K. Madl, Christopher Carosino, and Kent E. Pinkerton

167

Part 5: Cellular and Molecular Mechanisms of Particle Health Effects

11. Effect of Particles on Mucus and Mucociliary Clearance . . . Malcolm King

193

12. The Role of Surfactant in Particle Exposure . . . . . . . . . . . . Francis H. Y. Green, Samuel Sch€ urch, Matthias Amrein, Peter J. Gerber, and Zoya Leonenko

204

13. Nanoparticle–Cell Membrane Interactions . . . . . . . . . . . . . Barbara Rothen-Rutishauser, Fabian Blank, Christian M€ uhlfeld, and Peter Gehr

226

14. Mechanisms and Processes Underlying Toxicological Responses to Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vicki Stone and Ken Donaldson

243

15. Aeroallergen-Lung Interactions . . . . . . . . . . . . . . . . . . . . . Carsten Schleh and Jens M. Hohlfeld

266

16. Dose-Response Relationships . . . . . . . . . . . . . . . . . . . . . . . Tobias Stoeger and Otmar Schmid

288

Index

....

305

1 Functional Aspects of Lung Structure as Related to Interaction with Particles ¨ HLFELD* and MATTHIAS OCHS{ CHRISTIAN MU University of Bern, Bern, Switzerland

I.

Organization of the Lung

The organization of the lung, that is, the arrangement of the composing structures, differs from that of other human organs as, at the end of a normal breath, the lung consists of about 80% air, 10% blood, and only 10% tissue. In general, the latter is composed of structures lining the airspaces and the blood vessels and, in between, interstitial structures providing mechanical and metabolic features of lung function. In addition, the lung contains a second circulation that provides blood to the tissue itself, a neuroendocrine system, an immune system, and a covering layer at the “outer” surface of the lung—the visceral pleura. Functionally, the pulmonary tissue needs not only to be strong enough to separate air and blood effectively but also to provide a large surface area and a thin tissue barrier for gas diffusion between air and blood. To meet these functional demands, the lung possesses an air-conducting zone (airways) and a gasexchange region (alveolar region), the two of which differ significantly with respect to their qualitative composition and quantitative contribution to lung structure. Therefore, despite its small volume, pulmonary tissue has a high degree of complexity with over 40 different cell types and highly specialized noncellular components. Obviously, a small chapter like this cannot cover all aspects of lung structure, and the reader may refer to more comprehensive articles for further reading (1). However, as part of a well-focused book, this chapter provides the possibility of highlighting certain aspects of lung structure in greater detail than it is usually done in more comprehensive articles. It aims at describing those structures that are most relevant to the focus of this book on nano-sized particles (NSP). In contrast to larger-sized particles, NSP are deposited effectively in the gasexchange region of the lung (see chap. 5), which is why we chose to describe the structures of the alveoli as a portal of entry in the beginning. This includes the interaction between particles and surfactant (chap. 12) and the alveolar epithelium (chaps. 13 and 14). NSP have been shown to be translocated to the circulation, which provides a possible explanation for cardiovascular particle effects (chap. 7); however, parts of these effects have also been related to the activation of the nervous system of Current affiliations: * University of Giessen, Giessen, Germany. { Hannover Medical School, Hannover, Germany.

1

2

M€ uhlfeld and Ochs

the lung (chap. 8). Furthermore, there is a large body of evidence for interactions between particles and the pulmonary and systemic immune system (chaps. 9 and 15). We have therefore decided to focus the second part of this chapter on the structures known to be affected by particle exposure and constituting possible portals of exit or effect, namely, the capillary endothelium, the nervous system, and the immune system.

II.

General Design of the Air-Blood Barrier

At the level of alveoli, we find the barrier separating the airspaces from the vasculature—the air-blood barrier across which gas exchange takes place and along which NSP may enter the systemic circulation. The air-blood barrier is made of three tissue compartments: the alveolar epithelium (with the alveolar lining layer containing surfactant on top), the interstitium (including cellular elements as well as the connective tissue fiber scaffold in the extracellular matrix), and the capillary endothelium. The total surface area available for gas exchange in the human is about 140 m2, nearly the size of a singles tennis court. At the same time, the arithmetic mean thickness of the air-blood barrier is only about 2 mm (2). Over about half of this surface, we find the so-called thin parts of the air-blood barrier where the basement membranes of alveolar epithelium and capillary endothelium are fused, thus minimizing the interstitium. At the other half, termed the thick parts of the air-blood barrier, the two basement membranes are separated, thereby leaving room for interstitial cells (mainly fibroblasts, but also mobile cells like macrophages), elastic fibers, and bundles of collagen fibrils (Fig. 1). These different parts of the air-blood barrier are likely to behave differently with respect to the fate of inhaled particles.

Figure 1 Schematic drawing of the air-blood barrier. Surfactant forms a surface film (SF) at the

air-liquid interface. Under the watery hypophase, the alveolar epithelium consists of the large alveolar epithelial type I cells (AECI) with their thin processes and the cuboidal alveolar epithelial type II cells (AECII). Inside the septum, capillaries (C) are embedded in a network of collagen fibrils (co) and elastic fibers (el) and interstitial cells (IC).

Functional Aspects of Lung Structure as Related to Particles

III.

3

Surface Structures Lining the Portal of Entry for Particles

A. Surfactant

Once particles have entered alveoli, the first structure they come in contact with is the pulmonary surfactant layer at the air-liquid interface. The pulmonary surfactant system protects alveolar integrity in several ways. (i) It lowers surface tension at the air-liquid interface, thus keeping alveoli open. (ii) It prevents fluid fluxes into the alveolar lumen by lowering alveolar surface tension, thus keeping alveoli dry. (iii) It contributes several important components to the lung’s innate immune system, thus keeping alveoli clean. These biophysical (i and ii) and immunomodulatory (iii) functions are performed by a sophisticated system of remarkable biochemical and morphological complexity (for review, see Refs. 3–5). The morphological complexity (and beauty) of the surfactant system can only be revealed by electron microscopy of carefully prepared tissue samples. Type II alveolar epithelial cells (see below) synthesize, store, secrete, and to a large extent, recycle all components of surfactant. Therefore, an intracellular surfactant compartment within type II cells [in specialized lysosome-related organelles termed “lamellar bodies” (6)] and an intra-alveolar surfactant compartment can be distinguished. The alveolar lining layer that covers the alveolar epithelium as a thin and continuous film consists of the surface film and an aqueous hypophase underneath. Secreted surfactant components can be found in the surface film as well as within the hypophase. The intracellular and intraalveolar metabolism of surfactant is schematically summarized in Figure 2. Biochemically, surfactant is a mixture of lipids, mainly saturated phospholipids, and proteins, among them are the surfactant apoproteins SP-A, -B, -C, and -D. The surfactant proteins have important functions in surfactant subtype assembly, surfactant biophysics, surfactant homeostasis, and innate immunity. While the hydrophobic SP-B and SP-C are tightly associated with surfactant lipids, thereby fundamentally influencing the biophysical characteristics of surfactant (integral surfactant proteins), the hydrophilic SP-A and SP-D belong to the collectin protein family and are mainly involved in intraalveolar surfactant subtype assembly and innate immunity (associated surfactant proteins). In the alveolar lumen, SP-A is mainly found in the corners of tubular myelin figures whereas SP-D is mainly present as a free protein in the hypophase. Together with SP-A, SP-B is important for tubular myelin formation, thus stabilizing active surfactant forms (the “large aggregates” fraction of surfactant obtained by bronchoalveolar lavage), while the hydrophobic proteins SP-B and SP-C and, in conjunction, SP-A can enhance the adsorption of phospholipids into the surface film. SP-A might also inhibit surfactant secretion and stimulate surfactant reuptake of “spent” surfactant (the “small aggregates” fraction of surfactant obtained by bronchoalveolar lavage), which enters the type II cells as small unilamellar vesicles (Table 1). Interestingly, the lung collectins SP-D and, to a somewhat lesser degree, SP-A have also been localized outside the respiratory system, for example, in the gastrointestinal, urinary, and genital tract; mesothelium; synovial membrane; middle ear; lacrimal apparatus; and skin (7–9), indicating that they can exert their functions at a variety of mucosal surfaces. Accordingly, their expression at these extrapulmonary sites is increased under inflammatory conditions.

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Figure 2 Schematic drawing of the current view of surfactant metabolism. The complex mixture

of surfactant lipids and proteins is synthesized in alveolar epithelial type II cells via nuclear transcription, translation at the rough endoplasmic reticulum (rER), posttranslational processing in the Golgi apparatus (Golgi), and further processing and transport in MVB. Some surfactant components might bypass the Golgi apparatus and MVB. Surfactant lipid and the hydrophobic proteins SP-B and -C are assembled in so-called CB showing structural characteristics of MVB and LB. Surfactant is stored in LB and secreted via this organelle by exocytosis. An alternative route by direct exocytosis of small vesicles and/or MVB is known for SP-A but may also be valid for other components, for example, SP-D. After exocytosis to the HY, LB are unpacked and, upon addition of SP-A, form a lattice of surfactant membrane, named tubular myelin (TM), from which the SF consisting of a monolayer and, in places, a surface-associated surfactant reservoir can be generated. HY and SF are termed the alveolar lining layer (ALL). Surfactant that is removed from the airliquid interface is either reused or degraded. “Spent” surfactant has the morphological appearance of UV and can be taken up and degraded by alveolar macrophages (AM), be cleared via the airways, or be taken up by alveolar epithelial type II cells. There, the surfactant components may be degraded, enter de novo synthesis, or be transported to CB or LB for resecretion. The surfaceactive “large aggregates” (LA) fraction of intra-alveolar surfactant obtained by bronchioalveolar lavage largely corresponds to TM whereas the inactive “small aggregates” (SA) fraction largely corresponds to UV. Thus, the functional status of the surfactant system is directly reflected in intraalveolar surfactant subtype composition. Abbreviations: MVB, multivesicular bodies; CB, composite bodies; HY, hypophase; LB, lamellar bodies; SF, surface film; UV, unilamellar vesicles.

B. Alveolar Epithelium

All along the lower airway tree, from the trachea to the about 480 million alveoli in the human (10), there is an uninterrupted layer of epithelial cells. The structural properties, however, differ considerably between the conducting zone and the gas-exchange zone. The pseudostratified epithelium of trachea and bronchi becomes a simple columnar epithelium in bronchioli. At the beginning of the gas-exchange region, there is an


5

Table 1 Characteristics and Functions of SP-A, -B, -C, and -D

Protein

Characteristics

Functions

SP-A

Hydrophilic; collectin; 26–38 kD (monomer); usually octadecamer (six triplet monomers)

SP-B

Hydrophobic; 8–9 kD

SP-C

Hydrophobic; 3.5–4.2 kD

SP-D

Hydrophilic; collectin; 39–46 kD (monomer); usually dodecamer (four triplet monomers)

Surfactant homeostasis (surfactant secretion inhibition, tubular myelin formation, large aggregate stability, surfactant recycling); immunomodulation (interaction with pathogens, interaction with immune cells, antioxidative functions, antimicrobial functions) Surfactant homeostasis (tubular myelin formation, surface film formation and stability) Surfactant homeostasis (surface film formation and stability) Surfactant homeostasis (small aggregate structure and reuptake); immunomodulation (interaction with pathogens, interaction with immune cells, antioxidative functions, antimicrobial functions)

Abbreviation: SP, surfactant-associated protein.

abrupt transition to a thin squamous epithelium. At all levels, the epithelium consists of more than one cell type. Usually, we find at least a mosaic of lining cells and secretory cells. At the level of alveoli, we find two cell types: the squamous type I cells that form the actual lining and the cuboidal type II cells that have secretory functions. Although type II cells are more numerous than type I cells (16% vs. 8% of all alveolar cells), type I cells cover about 95% of the alveolar surface (11,12). The alveolar epithelium is “sealed” by tight occluding junctions between epithelial cells (13). With their thin extensions, type I cells spread out broadly over the epithelial basement membrane. They have long been considered as the “silent” alveolar epithelial cell type, providing only passive barrier functions. However, recent studies strongly suggest that type I cells are actively involved in alveolar ion and fluid homeostasis (14,15). Type II cells serve two main functions: they produce and secrete surfactant (see above) and they act as progenitor cells for the regeneration of the alveolar epithelium under physiological and pathological (cuboidal metaplasia in acute lung injury) conditions, being able to differentiate into both type I and type II cells. They have thus been referred to as “defender of the alveolus” (16,17). Besides surfactant components with immunomodulatory functions, type II cells are also capable of secreting a variety of proinflammatory mediators, including a broad profile of eicosanoid metabolites, cytokines, and chemokines, thereby actively contributing to the modulation of the inflammatory status in the alveolus (17). C. Surface Structures and Particles

There is a growing body of literature on the interaction between particles and surfactant as well as the alveolar epithelium. Several chapters of this book deal with diverse functions of these structures as related to particles. Interactions between particles and

6


surfactant are addressed by Green et al. (chap. 12). In addition, the chapters on aeroallergen-lung interactions by Schleh and Hohlfeld (chap. 15) as well as Jaspers and Horvath (chap. 9) refer to the immunomodulatory functions of surfactant proteins A and D. Translocation across the alveolar epithelium is addressed by Mo¨ller et al. (chap. 5) and Rothen-Rutishauser et al. (chap. 13). Toxicological responses and the pharmaceutical significance of the alveolar epithelium are discussed by Stone and Donaldson (chap. 14) as well as Hein and colleagues (chap. 4), respectively.

IV.

Pulmonary Structures Related to the Portals of Exit of Inhaled Particles

A. Capillary Endothelium

Like the alveolar epithelium, the endothelium lining alveolar capillaries is made of a thin but uninterrupted squamous cell layer. The alveolar capillary endothelium is of the continuous type, that is, in contrast to, for example, the kidney or the liver, there are no pores in the endothelial cells. Compared to the alveolar epithelium, the occluding junctions between capillary endothelial cells are rather leaky, resulting in a higher permeability (13). Like type I cells of the alveolar epithelium, capillary endothelial cells contain numerous caveolae, plasma membrane invaginations that can be used for endo- and transcytosis. Thus, the lining cells of both the alveolar and the capillary side of the air-blood barrier are equipped for exerting caveolae-mediated transport functions. The Capillary Endothelium and Particles

Although the barrier function of the capillary endothelium is often in the focus, its metabolic function needs to be stressed both for local pulmonary reactions to particle exposure and as a source for the release of mediators to the systemic circulation. B. Nervous System

The innervation of the lungs consists of efferent and afferent neurons whose axons reach the extra- and intrathoracic airways mainly by the vagal nerve or by spinal nerve branches. The efferent innervation of the lungs can be divided into sympathetic nerves, cholinergic parasympathetic nerves (18), nonadrenergic noncholinergic parasympathetic nerves (19), and the so-called efferent function of sensory nerves (20). The afferent innervation of the lungs can be structured into slowly adapting stretch receptors (SAR), rapidly adapting stretch receptors (RAR), C fibers, and sensory receptors associated with neuroepithelial bodies (NEB). A unifying classification of the afferent neurons is difficult, if not impossible, due to the complexity and the plasticity of the pulmonary innervation. For example, useful ways of characterizing the afferent nerve phenotypes include conduction velocity and cell diameter, ganglion origin, modality, neurotransmitters, immunoreactivity for certain cellular markers, and morphology (for review, see Refs. 21 and 22). In addition, the phenotype of the neurons with respect to the expression of neurotransmitters may be altered by changes in the pulmonary environment (23,24). Stimulation of afferent nerves has a variety of possible effects including


7

Figure 3 Peribronchiolar nerve in the mouse lung. (A) Low-power electron micrograph of the bronchiolar epithelium with Clara cells and ciliated cells as well as the underlying interstitium. The incomplete rectangle indicates the region from which the high-power micrograph (B) is taken. A small nerve consisting of unmyelinated axon bundles (thin arrow), a myelinated axon (thick arrow), and a vas nervorum (asterisk) is shown. Due to incomplete fixation, the myelin sheath (arrow head) shows an artificial enlargement of the distance between the membranes, particularly on the left side of the myelin sheath.

aspiration reflex, apnea, changes in blood pressure, lower-airway mucosal secretion, bronchoconstriction, and cough (25). A peribronchiolar nerve is shown in Figure 3. Efferent Innervation

The preganglionic sympathetic nerves arise from the upper six thoracic segments of the spinal cord and project on the postganglionic neurons in the paravertebral sympathetic ganglia. The main function of the sympathetic innervation is the innervation of the blood vessels and the submucosal glands with only limited innervation of human airway smooth muscle (26). The preganglionic cholinergic parasympathetic nerves start in the vagal nuclei of the brainstem and reach intramural airway ganglia with the vagus nerves where they synapse with postganglionic neurons that innervate blood vessels, submucosal glands, and airway smooth muscle. The cholinergic parasympathetic innervation is abundant in proximal airways and decreases to the periphery (27). Its main effects are bronchoconstriction, mucus secretion, and bronchial vasodilation. While the “efferent” axon reflexes of sensory neurons also lead to bronchoconstriction, the nonadrenergic noncholinergic parasympathetic nerves have bronchodilatory effects. There is evidence that these effects are mediated by nitric oxide (19,28). Interestingly, autoradiographic studies of the human lung revealed that muscarinic receptors are abundant in submucosal glands and airway ganglia, but are also moderately present in intrapulmonary bronchi, airway smooth muscle of large and small airways, and also the alveolar wall (29). Similarly, b-adrenoreceptors were found abundantly in the airway epithelium, alveolar walls, and submucosal glands, and to a lesser extent, over airway and vascular smooth muscle (30).

8

M€ uhlfeld and Ochs Afferent Innervation

The respiratory tract has a trigeminal (nasal mucosa innervation), vagal (tracheobronchial innervation), and spinal (parts of bronchopulmonary innervation) afferent innervation. Accordingly, the cell bodies of the respective neurons lie within the trigeminal ganglia, the nodose (inferior), and jugular (superior) vagal ganglia or the dorsal root ganglia, respectively. While the nodose ganglia are derived from epibranchial placodes, the jugular ganglia are of neural crest origin (31,32). The different embryonic origin is also related to a different phenotypic modality of the associated fibers (21,33). According to conduction velocity and cell diameter, three main afferent fiber types innervating the lungs can be identified, namely, Ab and Ad as well as C fibers. Ab fibers are myelinated with relatively high conduction velocity. Ad fibers are also myelinated but thinner, and exhibit a slower conduction velocity. C fibers are unmyelinated and possess the slowest conduction velocity of the three subtypes (21). In the dorsal root ganglia, and presumably also in the vagal ganglia, the size of the cell bodies in the ganglia is positively correlated with conduction velocity (34). The mechanosensors SAR and RAR are myelinated fibers, mainly of the type Ad, in contrast to the C fibers, which are thought to be involved in the detection of thermal, inflammatory, and nociceptive stimuli. The differentiation of the low threshold mechanosensors SAR and RAR is based on their adaptation to prolonged deep inspiration. SAR typically discharge with changes in lung volume (tidal ventilation) and respond to sustained lung inflation in a nonadapting manner. In contrast, RAR also discharge during tidal breathing but react with a discharge burst upon maintained inflation and rapidly adapt to preinflation values. C fibers are relatively insensitive to mechanical stimuli but very sensitive to chemical stimuli, for example, to the experimentally applied capsaicin (35). According to the accessibility to chemicals injected into the bronchial or pulmonary arteries, they are classified into bronchial or pulmonary C fibers. Most of the afferent fibers in the airways are either glutamatergic or tachykinergic fibers (36,37). However, other transmitters and neuropeptides are expressed by subsets of afferent nerves, for example, calcitonin gene-related peptide (38), vasoactive intestinal peptide (39), or neuropeptide Y (40). The receptors of RAR, SAR, and C fibers are the free nerve endings, and the only receptor of higher order known in the airways so far are NEB. These highly specialized pulmonary endocrine cells are normal components of the intrapulmonary airways and become more frequent to the peripheral airways (22). The cells of NEB contain secretory granules that store ATP, serotonin, and several neuropeptides, including bombesin, calcitonin gene-related peptide, enkephalin, among others (41). Although the function of NEB has been speculative for many years, it has now become clear that their role in lung physiology changes during ontogeny. In the fetal mammal, they influence lung growth; in the postnatal lung, they have an oxygen-sensing function; and in adults, NEB have important functions in airway regeneration but are also related to the development of various types of lung cancer (42,43). The Nervous System and Particles

The methodological difficulties in studying the nervous system of the lung and, in particular, the neuronal plasticity explains why there is so little information on interactions between particles and the pulmonary nervous system. However, in a later


9

Figure 4 Flow chart of the immune system of the lung. This schematic drawing provides an

overview on the cellular and noncellular components of the immune system.

chapter of this book, Gonzalez-Flecha (chap. 8) deals with the neural origin of particleassociated reactive oxygen species. It will be interesting to understand which parts of the pulmonary nervous system are sensitive to particle exposure and what mechanisms drive the responses described in chapter 8. C. Immune System

The whole respiratory tract, from the nares or lips to the alveoli, contains a set of defense mechanisms that compensates for the entering of particles and microorganisms during respiration. The effectiveness of this defense system is illustrated by the fact that the large alveolar surface area of normal lungs is free of infectious agents. The main function of the respiratory tract defense system is to recognize and eliminate potentially harmful particles in an appropriate, non-overreacting way. The characteristics of the defense system change according to the anatomical location and consist of cellular and noncellular mechanisms, the latter including mechanical properties and soluble proteins. Most of the immune cells of the respiratory tract can be subdivided into groups according to the compartments in which they reside and the functional and phenotypic specializations associated with the anatomical location (Fig. 4). Noncellular Components

The noncellular components of the pulmonary defense system include sneezing and coughing, mucociliary clearance, and the barrier properties of the airway and alveolar epithelia. Sneezing and coughing bring about turbulence and shear forces strong enough to remove free or liquid-bound material such as debris, mucus, and other particles from

10


the nose and the airways, respectively (44). The airways are covered by a mucus blanket that is moved to the throat by the beat of the cilia of ciliated airway epithelial cells. Thus, particles that are deposited in the airways become trapped in the mucus layer and are transported with the mucus. This is termed the “mucociliary clearance” or “mucociliary escalator” (45). All air-conducting and gas-exchanging surfaces of the respiratory tract are covered by an epithelium, the characteristics of which depend on the localization. As mentioned above, the epithelium of the alveolar region is very thin and consists of two cell types, whereas the pseudostratified epithelium of the airways is composed of many different cell types. In addition to these mechanical properties, there are various liquid compartments in the airspaces, which contain protective proteins, such as surfactant proteins A and D (see above), components of the complement system, antimicrobial peptides, and immunoglobulins. Many of these proteins opsonize inhaled particles but some also have direct antimicrobial functions (46). Airway epithelial cells synthesize a large number of antimicrobial peptides including lysozyme, defensins, and lactoferrin (47). Cellular Components

According to the compartment, there are four types of macrophages in the lung: alveolar, airway, interstitial, and intravascular macrophages. Pulmonary macrophages are derived from blood monocytes and therefore originate from the bone marrow. This was originally proven for alveolar macrophages by Thomas and coworkers (48). In contrast to airway and alveolar macrophages, which can be isolated by bronchoalveolar lavage, intravascular and interstitial macrophages are not easy to obtain and therefore less well investigated. Intravascular macrophages are thought to act as sentinels of inflammatory proteins and antigens reaching the lung via the bloodstream. They can bind to and ingest microparticles, endotoxins, and particles circulating in the blood (49). Interstitial macrophages reside underneath the alveolar epithelium in regions where lymphatic vessels begin, that is, in connective tissue sleeves at the periphery or the center of acini. They are thought to migrate through the epithelium and replace alveolar macrophages, but may also take up antigens that have translocated over the alveolar epithelium. After antigen uptake, they may either migrate to the lymph vessels or become permanent residents of the interstitial space (50). Airway and alveolar macrophages are the first cellular defense line against inhaled particles. They are found within the mucus blanket of the airways or the watery hypophase of the alveoli and have a high capacity of phagocytosis (51). Airway macrophages usually exit the lungs through the mucociliary clearance, whereas alveolar macrophages may move from their alveolar localization either to the interstitial space or to the airways from where they take the same route as airway macrophages (52). From the interstitial space, they may move to the lymphatics (53) or become permanent residents of the interstitium. Alveolar macrophages possess the ability to move in an amoeboid manner along the alveolar epithelium by transient connections of their pseudopodia to the epithelial cells. Thus, they may move from one alveolus to another through pores of Kohn (Fig. 5). Uptake of antigens and pathogens by alveolar macrophages is enhanced by opsonization or inflammatory stimuli (e.g., interferon g) and is facilitated by scavenger receptors (e.g., MARCO) or mannose receptors and toll-like receptors (e.g., TLR-2), the importance of which is best


11

Figure 5 Alveolar macrophage in the mouse lung. This electron micrograph shows an alveolar macrophage, probably on his way from one alveolus to another through a pore of Kohn. Intracellularly, a large volume of phagolysosomes with lamellar body–like appearance is seen. The small nuclear profile on the right upper side of the macrophage indicates that this macrophage is not sectioned centrally.

documented by the detrimental effects of deficiency of these receptors (54–58). Above that, alveolar macrophages express a variety of other receptors, which enable their response to inflammatory stimuli and interaction with other cells of the defense system (58). While earlier data indicated that the life span of alveolar macrophages is in the range of a few months (59,60), recent studies in a mouse model have suggested that alveolar macrophages are quite long-lived cells with a life span not much shorter than the animal itself (61). Dendritic and Langerhans cells are antigen-presenting cells that are mainly found in the epithelium of the larger airways with their frequency becoming lower to the periphery, although other localizations, including alveolar septa, subpleural tissue, and alveoli, have been described as well (62–64). They have long, branched dendritic cell processes and an irregular, folded nucleus. In the tissues, they form a network the density of which may be increased by a variety of stimuli, including exposure to interferon g (65), cigarette smoking (66), and allergen inhalation (67). After antigen uptake, dendritic cells may either move to regional lymph nodes to activate lymphatic T cells (68) or they may stay in the lung and present antigen to local T lymphocytes (69). As shown in Figure 4, pulmonary lymphocytes can be classified into lymphocytes associated with the epithelial surface or lymphoid aggregates, interstitial/intraepithelial

12


lymphocytes, and intravascular lymphocytes (70). Lymphocytes in lymphoid aggregates (bronchus-associated lymphatic tissue, BALT) are not prominent in human lungs, except for infection and chronic inflammation. The functions of pulmonary lymphocytes are cytotoxic actions against cells infected with viruses, antibody and cytokine production, and immune tolerance (71). Plasma cells, which produce antibodies, are relatively abundant not only around the acini of the seromucous glands of bronchi but also in the interstitium around bronchioles. In the airways, the main immunoglobulin is IgA, whereas in the alveoli, IgG predominates (72). Granulocytes (neutrophils, eosinophils, and basophils) are relatively rare in the normal human lung, but their frequency may increase due to certain stimuli or as a characteristic feature of a disease such as asthma. Mast cells contain metachromatic secretory granules that contain heparin and histamine and have a scroll-like appearance in the electron microscope. Mast cells are present in normal lungs but their number may increase in various diseases (44). The different cells of the pulmonary defense system are not functionally distinct players, but they communicate with each other by a great amount of cytokines and receptors and there is an increasing body of literature on this topic (73–77). The Immune System and Particles

As complex as the immune system of the lung are the interactions between inhaled particles and the pulmonary immune system. Alveolar macrophages, for example, have long been thought of as mere removers of the alveoli. However, their ability to interact with various cell types of the immune system as well as alveolar epithelial cells highlights the complex nature of the system as a whole. In this book, two different but clinically very important aspects of interactions between inhaled particles and the pulmonary immune system are addressed by Schleh and Hohlfeld (chap. 15) as well as Jaspers and Horvath (chap. 9).

V. Summary The present chapter has highlighted various aspects of lung structure as far as they are related to particle exposure. We are convinced that a broad knowledge of the normal structural and functional properties of the lung is essential for a scientific examination of particle-lung interactions. In a short chapter like this, we had to be very selective and focus on those aspects that we have nominated as the “portal of entry” and the “portals of exit” of inhaled particles. Both these terms should be reflected upon in a general way, as pointed out in the small passages about particles and the respective structural compartment. Within this book, we hope that this uncommon portrait of the lung helps the reader find her or his way through the subsequent chapters. Looked at from a broader perspective, this image may also stimulate the meeting of diverse areas in the field of particle-lung interactions and promote the investigation of the cross talk between the different structures and help in establishing structure-function relationships, thus providing a more integrated picture of the diverse interactions between particles and the lung.


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2 Ambient Tropospheric Particles PAUL A. SOLOMON U.S. Environmental Protection Agency, Las Vegas, Nevada, U.S.A.

DANIEL L. COSTA U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, U.S.A.

I.

Introduction

Atmospheric particulate matter (PM) is a complex mixture of solid and liquid particles suspended in ambient air (also known as the atmospheric aerosol). Ambient PM arises from a wide range of sources and/or processes, and consists of particles of different shapes, sizes, and compositions, with an array of physicochemical properties (1,2). Because particle size best relates to particle aerodynamics, diameter is most often used when describing ambient PM, most notably with regard to its atmospheric transport, lung deposition, and sampling for scientific or regulatory purposes. Particle number concentration, surface area, and volume (where particle volume particle density ¼ mass) are typically used to describe the size distributions of particles comprising ambient PM (Fig. 1). When coupled with chemical composition and PM optical properties (and sometimes charge), one obtains a more complete appreciation of the complex physicochemical nature of ambient PM. PM of most interest to health scientists is that which is considered inhalable, that is, it enters human airways, including but especially beyond the nose and mouth (4–7). Such PM encompasses a size-range from a few nanometers to *10 mm aerodynamic diameter (AD). AD is a function of particle density and shape and is the prime descriptive parameter for PM that is collected or segregated by inertial measurement methods, including the respiratory tract. With standardization of individual particles as unit-density spheres with the same settling velocity as irregularly shaped particles, as described as an “aerodynamic diameter,” widespread application and comparisons of ambient particles can be achieved (for additional detail see Refs. 2 and 8). Further, it is common to find ambient PM described by its MMAD (mass median AD), which is the normalized particle AD with half the total mass of the particles in the air residing above that number and half the mass below (2). The geometric standard deviation (GSD), also referred to as sigma g (sg), describes the statistical distribution associated with MMAD and defines the heterogeneity of the particle sizes around that median value. MMAD (or its volume equivalent as is sometimes used) is important since it is widely utilized by both atmospheric and health scientists; in the latter case, to predict size-related deposition patterns in the respiratory tract.

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Figure 1 Typical number and volume distributions of atmospheric particles with the different

modes. Source: Adapted from Figure 3-2 in Ref. 3.

Historically, airborne particles have been categorized into three major size fractions or modes based on their mass (volume) or number concentration distributions (Fig. 1). These empirically defined size ranges are termed ultrafine (2 mm) and ultrafine ( 0.05), respectively, whereas the surfaces were wetted at 3.8 mJ/m2 (SE ¼ 0.89, n ¼ 20) and 5.8 mJ/m2 (SE ¼ 0.52, n ¼ 20) (p < 0.05), respectively. Further experiments with pollen of malvaceae and maize (spiky and fine knobbly surfaces) were wetted at 10.0 mJ/m2 (SE ¼ 0.52, n ¼ 10) and 22.75 mJ/m2 (SE ¼ 0.81, n ¼ 10), respectively (p < 0.05). These results show that resistance to spreading of a DPPC film on various surfaces is dependent on the extent these surfaces are curved. This is seen with cubic sapphire plates, which have at their corners, a radius of curvature of about 0.75 mm, spiky malvaceae pollen with an even smaller radius on top of their spikes, or talc with various highly curved surfaces. These highly curved surfaces resisted wetting by the DPPC film to a higher degree than more moderately curved surfaces such as those of cylindrical sapphire plates, maize pollens, or polystyrene spheres, which have a surface-free energy similar to that of talc but a smooth surface. The macroscopic plane surfaces of the particles demonstrated the greatest resistance to spreading. This was explained by the extremely fine grooves in the nanometer range, as revealed by electron microscopy. In summary, to understand the effects of airborne particles retained on the surfaces of the respiratory tract, and ultimately their pathological potential, not only the particle size and surface chemistry but also the particle shape should be taken in consideration. After crossing the surfactant film, the particles may cross the epithelium and exert effects on cell biology. Ultrafine particles may reside in the interstitium (82) or travel into the lung tissue and blood stream (83–87) and account for the well established extrapulmonary effects (85). Fine particles settling in the alveolar lung abide on type I alveolar epithelial cells 95% of the time, because these thin cells cover most of the alveolar surface. A majority of respirable particles (98%) are cleared from the lung over a period of time. Ultrafine particles, on the other hand, do not elicit phagocytosis by macrophages (83), inhibit phagocytosis (88,89), and have the potential to penetrate into the bloodstream. In an animal model, Geiser et al. (90) found no evidence of significant clearance of inhaled TiO2 nanoparticles in rats by lung surface macrophages. There is accumulating evidence that nanoparticles are internalized by passive mechanisms. Red blood cells have been shown to incorporate fine particles (0.2 mm) (91,92) even though they are unable to phagocytose and are devoid of clathrin and caveolin, associated with active uptake. Passive uptake of particles in lung epithelial culture models has been demonstrated (82,91,93). Irrespective of whether ultrafine particles enter cells passively or actively, uptake efficiency should depend on the adhesive interaction and be governed by the particles’ surface properties. However, red blood cells take up a range of different particles (TiO, gold, or polystyrene) equally well (93). This may be explained, by our recent observation, using force spectroscopy, that particles exposed to culture media or serum immediately become coated by proteins and so lose their specific binding properties (94). Thermodynamically, any specific or nonspecific interaction between a particle and a cell will be reflected in a global way by the adhesive interaction. We recently established the measurement of the adhesive interaction (work of adhesion) between the apex of an AFM sensor tip (acting as the particle) and a primary type II cell culture (95). We found that the work of adhesion developed over the first 100 seconds after contact. During this time, the AFM tip appeared to be taken up by and moved into the cell. This

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study demonstrates that the thermodynamic parameters governing the first steps of particle uptake and their time course can be studied quantitatively with force spectroscopy.

V. Role of Surfactant in Particle Clearance Changes in surface tension caused by expansion and contraction of the surface film during respiration and surface tension gradients along the airways may enhance particle clearance (56,65,66). Surfactant also enhances particle clearance by accelerating ciliary beat frequency (96–98) and by conditioning the viscosity of the mucus (99). These latter effects might be mediated by the stimulatory effects of surfactant on chloride ion transport across airway epithelium (100).

VI.

Role of Surfactant in Particle Toxicity

Upon the initial contact between inhaled particles and the host at the air-liquid interface of the conducting airways and alveoli, the particles may become coated with surfactant. This process may change their toxicology or pharmacokinetics or even affect surfactant function directly (101). The amount of a surfactant adsorbed to the surface of particles of quartz or kaolin after mild saline rinsing amounted to the equivalent of a bilayer in which one monolayer coats the particle with the zwitterionic head groups toward the dust’s surface and the ionic phosphorylcholine heads of the second layer orientate toward the aqueous phase (102). These in vitro observations are in keeping with the observations of deposited particles studied by electron microscopy, which show coating with a bilamellar film (2). In vitro studies with DPPC, a primary component of surfactant, adsorbed to mineral and organic particles, have shown a marked suppression of their cytotoxic activity (103,104). This effect was demonstrated as long ago as 1957 (105), when it was demonstrated that quartz cytotoxicity was reduced following the adsorption of surfactant. In vivo, most inhaled mineral dust particles are rapidly phagocytosed into macrophages and incorporated into secondary lysosomes; surfactant coating enhances this process (106). In the intracellular environment, particles are exposed to hydrolytic lysosomal enzymes, including phospholipase A2. In vitro studies modeling this process have shown that removal of the lipid coating by phospholipase A2 restores the cytotoxicity of mineral dusts (102). The rate of removal of the surfactant coating by macrophages is different for kaolin and quartz; the coating being removed more slowly from kaolin than from quartz (107). Thus, the strength of adsorption of surfactant to the particle surface and the kinetics of desorption in the lysosome may account for the differences in cytotoxicity between these dusts (108). These models are consistent with the prompt neutralization of particle toxicity by adsorbed surfactant and a gradual retoxification within the macrophage (109). The reduced cytotoxicity of surfactant-coated particles may be related to a suppression of surface-free radical activity (110) by surfactant. Natural surfactant is capable of scavenging hydrogen peroxide (H2O2), the oxygen free radicals (O˙ and OH˙), and radical species derived from peroxynitrite (111). In the process, peroxidation of

The Role of Surfactant in Particle Exposure

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unsaturated lipids in surfactant and degradation of SPs may occur with impairment of surfactant function (112–114) and type II cell metabolism (115). Alveolar lining fluid (112,116,117) and airway mucus (118,119) also contain potent antioxidants, including catalase, superoxide dismutase, peroxidases, and glutathione. These enzymes are physically associated with airway and alveolar surfactant and may serve to protect surfactant from oxidative injury. Prolonged inhalation of dusts by humans (120), rodents (121,122), and other species (123,124) is associated with an increase in the number of type II cells and increased secretion of surfactant. The stimulation of surfactant appears to be directly related to the toxicity of the dust. It may be so florid, as in acute silicosis, that flooding of the alveolar spaces with surfactant lipids and associated proteins may occur, a condition known as alveolar lipoproteinosis (120). In experimental lipoproteinosis in the rat, the major lipid component is disaturated phosphatidylcholine (121), but all lipid fractions are increased. In the sheep model of experimental silicosis, phosphatidylglycerol, phosphatidylethanolamine, and phosphatidylinositol showed the greatest increases following silica exposure (123). The excess production of surfactant in response to silica dust may be an adaptive response, perhaps to reduce particle cytotoxicity or to compensate for oxidant-induced lipid peroxidation (125,126). The adsorption of surfactant to particles could also affect the surface tension in the conducting airways or alveoli (127–129). A significant biological effect would probably be seen in vivo only for fine and ultrafine particles, such as fumes and smoke (130), which have high surface areas relative to their mass. Abnormalities of surfactant function have also been reported in bronchoalveolar lavage (BAL) fluid from cigarette smokers (130–135). The adsorption of surfactant to porous particles, such as diesel exhaust soot containing absorbed mutagens (136), might affect their genotoxic activity. Particle-free surfactant extracts of diesel soot are not mutagenic in vitro (136,137).

VII.

Implications for Surfactant in Disease States

A. Particle-Induced Acute Lung Injury

Adverse effects on pulmonary surfactant have been demonstrated after experimental exposures to wood smoke (130); hydrochloric acid (138); hypochlorous acid (139); and toxic gases such as hydrogen sulfide (140), nitrogen dioxide (141), nitric oxide (125), ozone (142), and other particles (143). The impaired ability of surfactant to lower surface tension may be a direct consequence of the particle-surfactant interaction (101) or could be mediated by inflammatory cells or their products. Inhaled particles, such as asbestos and silica, stimulate macrophages to release reactive oxygen metabolites (144,145). Activated neutrophils also release proteolytic enzymes, such as elastase and lysozyme (146,147). Activated polymorphonuclear leukocytes impair surfactant function through a process that involves proteolysis of SPs (114,148). SP-A also has antioxidant properties of its own (148,149). Karagiorga et al. (150) reported an increase of cholesterol in surfactant from acute respiratory distress syndrome (ARDS) patients over control (8.5 5% w/w) from 15.6 2% w/w up to 43 22% w/w. Markart et al. (151) also found cholesterol levels

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enhanced in clinical ARDS samples. Cholesterol in surfactant is also increased to 20% w/w in animal models of lung injury (152). We recently analyzed the effect of an elevated level of cholesterol in surfactant. At cholesterol levels of 20%, the lowest surface tension observed in vitro using captive bubble surfactometry (CBS) was 16 mN/m (35). In our more recent studies using atomic force microscopy, we show the mechanism by which cholesterol inhibits surfactant (48,153). A comparison of the currently proposed mechanisms of surfactant inhibition in our laboratory singles out increased levels of cholesterol as having the most pronounced inhibitory effect (154). Hence, particle-induced lung injury may impair surfactant by causing inflammation, which in turn results in increased cholesterol levels in surfactant. Many of the adverse effects of surfactant deficiency can be reversed in the experimental model by intratracheal instillations of natural and artificial surfactants (155–157) and surfactant-associated proteins (158); it may be appropriate to treat acute lung injury with these preparations. The effect of particle-induced injury on airway surfactant has been studied in the guinea pig. Animals were exposed to sulfuric acid aerosol at high concentration (43 mg/m3) for four hours (14). The aqueous lining of the airways was preserved using an osmium FC fixative (13). Following exposure, the surface of the mucous layer was covered with a granular protein-like material. TEM revealed irregular thickening of the osmiophilic film at the air-mucus interface. The surface tension of the acid-treated tracheas (~32 mN/m) was similar to that of the control animals, and the ability of the mucus to submerge particles was not compromised, indicating that the surface film was functionally normal. It is possible that the known inhibitory effects of tissue injury and protein leakage on surfactant activity were mitigated by an excess of surfactant flowing from the alveolus. The morphological data were consistent with this, for the osmiophilic film at the air-mucus interface was greatly thickened in the acid-exposed animals (14). B. Asthma

By definition, asthma is a recurrent reversible obstruction of the airways accompanied by bronchial hyperreactivity and inflammation. It may be triggered by exogenous or endogenous stimuli. Asthma has increased during the past decades, mainly in industrialized countries and, in epidemiological studies, is strongly associated with exposure to fine (PM2.5) airborne particles (159) and specific allergens. Immune mechanisms as well as nonspecific stimuli can trigger an asthma attack. How inhaled allergens and irritant particulates are processed following deposition in the airways may have profound implications for the genesis of asthma and for the perpetuation of the asthmatic response. There are many reasons to consider that surfactant may be important in the pathogenesis of asthma. Surfactant’s role in promoting displacement and retention of particles in the airways may be important in determining the site and amount of antigen presentation. Alveolar and airway surfactant, by lowering surface tension, are important for maintaining small airway patency (160,161) and prevent liquid accumulation in the lumen (56). Surfactant dysfunction has been demonstrated in a guinea pig model of allergic asthma (162) and, in this model, the airway narrowing can be alleviated by surfactant inhalation (163). SP-A is deficient in the bronchoalveolar fluid of some asthmatics (164) and surfactant inhalation may alleviate the symptoms of asthma (165). Enhanced oxygen


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free radical release by alveolar macrophages from asthmatic patients (166) might be influenced by the hydrophilic proteins in surfactant (149,167,168) or be suppressed by their lipid components (116,146,156). The immunosuppressive effects of the lipid component of surfactant (169,170), particularly its effects on interleukin (IL)-2 release and adhesion molecules (171), may also be important in dampening inflammatory reactions that occur at the surface of the airways. Finally, a surfactant layer may have important implications for airway responses to inhaled drugs (172,173).

VIII.

Summary

Retention of particles by surface, line tension, and electrostatic forces exerted on inhaled particles by surfactant situated at the aqueous phase–air phase interface is the initial step of a complex cascade of defense mechanisms in the lungs. Surfactant coating of particles renders them more palatable to phagocytic cells, reduces their cytotoxicity, and enhances mucociliary clearance. Surfactant has important antimicrobial and antioxidant properties. Its role in particle-associated toxicity appears to be related to the surface properties of the particle. Given the complexity of the system and the multitude of interactions between inhaled particles and surfactant, it is necessary to examine the role of surfactant in diseases associated with particle exposures, such as some forms of acute alveolar injury and asthma. A knowledge of the mechanisms involved and, in particular, the role of surfactant will greatly enhance our ability to understand these diseases and to develop appropriate therapeutic strategies.

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13 Nanoparticle–Cell Membrane Interactions ¨ HLFELD*, BARBARA ROTHEN-RUTISHAUSER, FABIAN BLANK, CHRISTIAN MU and PETER GEHR University of Bern, Bern, Switzerland

I.

Introduction

During the past years, there has been a substantial increase in the debate on the potential harmful effects of nanomaterials (1–4), defined as materials with a diameter below 100 nm, and nanotubes, which have two dimensions below 100 nm while the third dimension can be much larger (5). Among these nanomaterials, specific concern is expressed about adverse health effects of nanoparticles (NP), since recent studies indicate a specific toxicological effect of inhaled combustion-derived ultrafine particles (UFP) (6–11). Although there are obvious differences between UFP, which are polydispersed and have a chemically complex nature, and NP, which are in contrast monodispersed with precise chemically engineered characteristics, the same toxicological principles have been assumed (3). An important basis for the current concerns about the possible adverse health effects of NP and nanotubes has been provided by research in the field of inhalation toxicology (12). Rodent inhalation studies provided evidence that NP induce considerably stronger pulmonary toxicity when compared at equal mass dose with larger particles. For instance, inhaled or intratracheally instilled poorly soluble particles of low toxicity such as carbon black and titanium dioxide induce pulmonary inflammation in proportion to their surface area (3,13,14). These findings indicate that a large surface area deposited in the lung may be sufficient to initiate inflammation. The in vivo studies are supported by in vitro studies where UFP show stronger inflammatory or toxic responses than larger-sized particles of the same chemical composition (15). In vitro and in vivo studies with UFP, such as diesel exhaust particles, carbon black, and welding fumes, have also strongly contributed to the findings that UFP are the most harmful constituent of environmental particulate air pollution (1). Support for the relevance of environmental particles in the nano-size range also comes from selected human studies showing associations between ambient exposure to UFP and adverse health effects (7,16). Various aspects of NP toxicity have been described in a number of recent reviews (1,3,5,17,18). Such studies also include the issues of the material composition and exposure sources, routes of exposure and administration (e.g., inhalation, oral, dermal, intravenous application), or aspects of distribution and translocation [e.g., air-blood *

Current affiliation: University of Giessen, Giessen, Germany.

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tissue barrier, blood-brain barrier, olfactory nerve], and clearance from the body. Most important however, detailed mechanism whereby NP may interact with their target cells and how this can impact on toxicity are still poorly understood. The principal interaction of NP with their biological environment occurs at the cellular level, that is, by interaction with structural and functional cell compartments (nucleus, organelles) (18). Reactive oxygen species generation is described in many studies as a hallmark of the biological effects of NP that have entered the cells (17,19). Besides the cell membrane, interactions with and effects in mitochondria and in components and compartments of the cell nucleus are considered as in view of their potential relevance for NP-induced toxicity. In this article, we discuss, on the basis of existing literature, the potential uptake and entering mechanism of NP across tissue and cell membranes as well as their possible interaction with intracellular compartments.

II.

Membranes

A. Tissue Membranes

Despite the existence of structural and functional barriers in the lung, respiratory diseases are frequent and increasing (7,9,20), and more attention has been directed toward elucidating how and when the antigens overcome these barriers. Insoluble particles deposited in the airways are largely cleared by the mucociliary action but not all deposited particles are removed by this mechanism. The lung barriers may not be effective to protect the body from particles less than 100 nm in size. Deposition as well as the subsequent fate of inhaled UFP and NP is different from larger-sized particles. Diffusion is the main mechanism for deposition of UFP and NP in the respiratory tract. On the basis of predictive mathematical models, it has been shown that of all deposited particles, 90% of inhaled 1 nm particles are deposited in the nasopharyngeal compartment whereas 20 nm particles have the highest deposition rate in the alveolar region (50%) (3). Most of the translocation studies described have been done for ambient UFP. Once deposited, a fraction of UFP appear to move rapidly to extrapulmonary sites via blood vessels and reach other organs like the heart or the brain, where they may penetrate into or through cells (12,21–23). The structural barriers protect the respiratory system against harmful and innocuous particulate material (24). This is important as the internal surface area of the lungs is vast [alveoli plus airways approximately 150 m2 (25)], facilitating easy access to the lung tissue, including cells of the defense system, such as dendritic cells and macrophages. The series of structural barriers are summarized in Table 1. The fate of inhaled and deposited particles depends on their physical-chemical characteristics (e.g., chemical composition, size/geometry, surface charge, coating, and aggregation status), the exposed cell type (professional phagocytes (marcophages), professional antigen-presenting cells (dendritic cells), epithelial or endothelial cells), as well as on their microenvironment (like the liquid-lining layer or the aqueous hypophase, the lung surfactant at the interface of those microcompartments with the air; the blood plasma; the basement membrane). A first line of structural barriers includes the surfactant system (26–28). All inhaled particles (PM10: particulate matter with a diameter equal or smaller than 10 mm) deposited in the airways are displaced into the subphase below the surfactant film by

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Rothen-Rutishauser et al.

Table 1 The Structural Barriers of the Tissue Membranes

Structural barriers

References

The surfactant film The aqueous surface lining layers including the mucociliary escalator A population of macrophages (professional phagocytes) in the airways and in the alveoli The epithelial cell layer endowed with tight junctions between the cells The dendritic cells that are located within the epithelium making small cytplasmic processes toward the luminal side The basal lamina and extracellular matrix The capillary endothelium

26–28 29 30,31 32,33 34–38 39,40 41,42

surface- and line-tension forces exerted on it by the surfactant film and may be modified by surfactant components or coated with surfactant or surfactant components during the displacement process (26,28,43,44). As a result of the displacement, particles come into contact with the lung epithelium endowed with tight junctions (32), whose effectiveness is greatly enhanced by the aqueous surface lining layer and the mucociliary escalator (29). A second line of defense comprises cells of the innate and the adaptive immune system positioned throughout the respiratory tree. These cells are located above and below the respiratory epithelium and their activities are tightly regulated to protect the integrity of the airways and the vital gas exchange region (45). The innate response is largely governed by macrophages (46,47) whereas respiratory tract dendritic cells are responsible for the adaptive immune response (48,49). In vivo, alveolar macrophages occupy the luminal aspect of the epithelium (50,51) while immature dendritic cells occupy the basal aspect of the epithelium (52,53). In an in vitro study, it has been shown that macrophages form small cytoplasmic processes through the epithelium toward the basal side (34). Dendritic cells are located in the epithelium and the lamina propria. Those in the epithelium access the luminal side with fine cytoplasmic processes pushed between the epithelial cells (35,54,55). Transport of the particles to the dendritic cells presupposes their passage across the epithelium either through the epithelial cells or between the epithelial cells, through the tight junctions. It has been shown in gut mucosa that subepithelial dendritic cells were capable of capturing antigens outside the epithelium by extending fine cytoplasmic processes through the tight junctions (35–38). We have obtained evidence from in vitro studies that dendritic cells collect particles (1 mm in diameter) on the luminal side of the epithelium (Fig. 1) and that dendritic cells and macrophages collaborate as sentinels against fine particles by building a transepithelial interdigitating network of cell processes (34). In further studies, by using a triple-cell coculture model simulating an airway epithelial barrier, the entering and translocation of fine particles and NP into cells was compared. The model included epithelial cells, macrophages, and dendritic cells. The interaction of fluorescently labeled polystyrene particles of two different sizes (1 mm, and 50 nm) with the cells of the triple-cell coculture system was studied by laser scanning microscopy in combination with immunofluorescence methods and digital image restoration (56). After 24 hours, macrophages and dendritic cells were found filled with 1 mm particles and only few of these particles were detected in epithelial cells (Fig. 2). By applying a


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Figure 1 Laser scanning micrograph of a particle exposed triple cell coculture. A dendritic cell

residing underneath the insert membrane pushed processes between the epithelial cells upward into the “luminal space” to take up 1 mm particles (arrow).

deconvolution algorithm, even NP, that is, 50 nm polystyrene particles, could be visualized and many particles were found in macrophages as well as in dendritic cells, and again, only few particles in epithelial cells (Fig. 2). By a quantitative analysis, using a contingency table analysis (57) to compare the localization of fine particles with NP in different cell types, we found evidence for different translocation characteristics (56). Although we only found few particles of all sizes in epithelial cells in the triple-cell coculture system, an important role of the epithelial cells for particle translocation in vivo has to be considered. Particulate antigens less than 0.1 mm are able to cross the air-blood barrier of the lung and thus can enter the circulatory system (58). In the human lung, however, there is only one study that describes a rapid and significant translocation of inhaled carbonaceous NP to the systemic circulation and other extrapulmonary organs (58), whereas most other studies only detected a low degree of translocation for iridium (21) or carbonaceous NP (59,60). The studies of Nemmar et al. (58) and Mills et al. (59) had a very similar design, and Mills et al. have provided a convincing discussion that the strong translocation observed by Nemmar et al. (58) was mainly related to the translocation of soluble pertechnate, which was cleaved from the carbonaceous particles. It is therefore currently accepted that the degree to which inhaled UFP and NP translocate to

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Figure 2 Intracellular particle localization in triple-cell cocultures visualized by laser scanning microscopy. Fluorescent labeled polystyrene particles (1 mm, and 50 nm) were added to the cells for 24 hours. After fixation cells at the upper side of the insert (upper row) were stained for CD14 (macrophages) and F-actin (all cells); cells at the lower side (lower row) for CD86 (dendritic cells) and F-actin (all cells). Macrophages and dendritic cells were filled with particles (arrows), considerably fewer particles were found in epithelial cells (arrowhead). All images represent xzprojections. Source: Modified from Ref. 56.

the circulation is rather small, however, knowledge about cumulative effects of this translocation are lacking so far. After antigens have passed through the epithelial barrier, they may pass through the basement membrane and subsequently through the subepithelial connective tissue layer, and eventually come into contact with endothelial cells lining the capillaries. Since endothelial cells play an important role in inflammation processes (61), particles might affect endothelial cell function and viability inducing proinflammatory stimuli. It has been proposed that the permeability of the lung tissue barrier to NP is controlled at the epithelial and the endothelial levels (62). Antigens that have passed through the epithelial and endothelial barrier are transported by the blood circulation and reach other organs like the liver or the heart (21,63). There is also evidence that inhaled UFP can reach the brain (64) by translocation along or in neurons from the nasal epithelium to the brain. Recently it has also been shown that ultrafine manganese oxide particles are able to translocate to the olfactory bulb and other regions of the central nervous system in a whole-body exposure model (65), although the discussion whether entire particles were translocated or solubilized manganese was transported to the central nervous system (66) needs further research. B. Cellular Membranes

The surfactant film at the air-liquid interface in airways and alveoli and the phospholipid bilayers of cells with different types of proteins shaping the cellular membranes are considered actual membranes although their composition is completely different. The


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membranes also include the nuclear membrane and the membranes of the different organelles. Pulmonary surfactant is produced in the lung to decrease surface tension of the fluid lining. It is a complex mixture of phospholipids, neutral lipids (particularly cholesterol), and proteins. So far, four surfactant-associated proteins have been described, surfactant proteins-A, -B, -C, and -D, which are essential for the formation and structural integrity of surfactant components (67). Upon deposition, the first structure in the alveolus NP are interacting with is surfactant; however, there is only very limited knowledge about this interaction. For larger-sized particles, it has been shown that once the particles get into contact with the surfactant film, they are displaced from the airspace to the hypophase due to wetting forces (26,28,68), where they may be interacting with proteins of the hypophase (e.g., surfactant protein-A and -D or glycoproteins) (69). For NP, this interaction with surfactant has not been shown so far but it is likely to be similar. Surfactant lipids and proteins may adsorb to the surface of PM2.5 particles, thereby eventually modulating the function of pulmonary surfactant (70). Similarly, Bakshi and colleagues have demonstrated that gold NP sequester lung surfactant and may interfere with its normal function (71). After displacement of NP into the hypophase, the particles come into close contact with the membranes of the cells. All cell membranes have a common structure: They consist of a very thin film of lipid and protein molecules, held together mainly by noncovalent interactions (70,72). The lipid molecules are arranged as a continuous double layer about 5 nm thick in cell membranes. This lipid bilayer serves as a relatively impermeable barrier to the passage of most water-soluble molecules. The transmembrane protein molecules mediate specific functions as transporting proteins across the bilayer or catalyzing membrane-associated reactions. Some proteins serve as structural links that connect the cytoskeleton through the lipid bilayer to the extracellular matrix or an adjacent cell by integrins and cadherins, while others serve as receptors to detect and transduce chemical signals into the cell’s environment (73). How NP may enter cells across the cell membrane is described in detail in the next section “Mechanisms of nanoparticle penetration.” All eukaryotic cells contain functionally distinct, membrane-enclosed compartments. The main types are the nucleus and the organelles mitochondria, endoplasmic reticulum, Golgi apparatus, peroxisomes, lysosomes, and endosomes. Nucleus and organelles are enclosed by a double lipid bilayer containing distinct proteins (74). NP can cross the membranes of organelles since they have been localized in lysosomes, mitochondria, and the nucleus. This is described in the section “Nanoparticle localizations inside cells.”

III.

Mechanisms of Nanoparticle Penetration

The plasma membrane of the cells is a dynamic structure and segregates the chemically distinct intracellular milieu (the cytoplasm) from the extracellular environment by coordinating the entry and exit of small and large molecules. While essentially small molecules are able to traverse the plasma membrane through the action of integral membrane protein pumps or channels, macromolecules must be carried into the cells in membrane-bound vesicles derived from the invagination and pinching-off

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of pieces of the plasma membrane to form endocytic vesicles. This process is termed endocytosis and two types of endocytosis are distinguished on the basis of the size of endocytic vesicles formed: pinocytosis (“cellular drinking”) involves the ingestion of fluid and molecules via small vesicles (0.25 mm in diameter). Whereas all eukaryotic cells are continuously ingesting fluid and molecules by pinocytosis, large particles are ingested mainly by specialized phagocytic cells. The mechanistic complexities that govern endocytosis suggest that great evolutionary effort has been expended to control entry into the cell, and thereby to control cellular responses to the environment (75). The different mechanisms of cellular entering and intracellular trafficking have been discussed in Ref. 76 and are summarized in Figure 3.

Figure 3 Summary of possible entering mechanisms of nano-sized particles. Particles may

actively be taken up by cells via phagocytosis (A), macropinocytosis (B), clathrin-mediated endocytosis (C), clathrin- and caveolae-independent endocytosis (D) or by caveolae-mediated endocytosis (E). In most cases of active uptake, particles will be transported via vesicular structures to form phagolysosomes or endosomes (A–D) but they may also be transported to the endoplasmic reticulum, cytosol or through the cell as part of transcytotic processes (E). Apart from these mechanisms, a passive movement through the plasma membrane with subsequent access to all subcellular compartments, including nucleus and mitochondria, has been proposed (F). The significance of particular intracellular localizations and entering mechanisms for specific cellular responses awaits further study. Source: Modified from Refs. 17 and 76.


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A. Phagocytosis

By contrast to pinocytosis, phagocytosis involves the uptake of large particles (>0.25 mm) into cells. It is an actin-based, mostly receptor-mediated mechanism, and is usually independent of clathrin (77). Phagocytosis in mammals is conducted primarily by specialized cells, including macrophages, dendritic cells, monocytes, and neutrophils that function mainly to clear large pathogens, such as bacteria, or large debris such as dead cells or the remnants of dead cells. Monocytes/macrophages and neutrophils have been referred to as professional phagocytes. They are very efficient at internalizing particles. On the other hand, most cells have some phagocytic capacity. For example, thyroid and bladder epithelial cells phagocytose erythrocytes in vivo (77), and numerous cell types have been induced to phagocytose particles in culture. In particular, there are a number of studies demonstrating the uptake of fine and UFP by lung epithelial cells in vitro (78–80) and in vivo (81). Particle internalization is initiated by the interaction of specific receptors on the surface of the phagocyte with ligands on the surface of the particle. This leads to the polymerization of actin at the site of ingestion and the internalization of the particle via an actin-based mechanism. After internalization, the phagosome matures by a series of fusion and fission events with components of the endocytic pathway, culminating in the formation of the mature phagolysosome. Since endosome-lysosome trafficking occurs primarily in association with microtubules, phagosome maturation requires the coordinated interaction of the actin- and tubulin-based cytoskeletons (75). Particle uptake by professional phagocytes serves as defense mechanism to remove foreign material from the body. However, for inhaled NP it is discussed that they are recognized to a lesser extent by alveolar macrophages than fine particles (82), leading to an increased contact with epithelial cells, penetration into deeper regions of the lung, and eventually, translocation into the circulation (19). In cultured macrophages, the entering of NP (0.078 mm) after cytochalasin D treatment could not be blocked completely, contrary to fine particles (1 mm in size), therefore particle uptake by any actin-based mechanism can be excluded (83). The kinetics of such processes are considered to depend largely on NP surface properties as well as in in vivo surface modifications, for example, by interactions with endogenous proteins (3). Contrasting uptake mechanism of NP in professional phagocytes versus other cell types in relation to different behavior with the cell membrane structures (e.g., receptors) are indicated in a number of studies. For instance, silver NP were observed on cellular membrane surfaces and free in the cytoplasm but also trapped by phagocytic or endocytic vesicles. This suggests that internalization of silver NP occurred through two distinct mechanisms, that is, by phagocyosis and/or endocytosis and perhaps by diffusion (3,84). B. Macropinocytosis

Like phagocytosis, the signaling cascades that induce macropinocytosis involve an actin-driven formation of membrane protrusions. However, instead of invaginating a ligand-coated particle, they collapse onto and fuse with the plasma membrane to generate large endocytic vesicles called macropinosomes that sample large volumes of extracellular milieu. Macropinocytosis fulfils diverse functions. It can be transiently induced in most cells and might play a role in down regulation of activated signaling

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molecules (75). Activation of antigen-presenting dendritic cells triggers extensive and prolonged macropinocytotic activity, enabling these cellular sentinels to sample large volumes of the extracellular milieu and to fulfill their role in immune surveillance (85). However, little is known about the nature of the whole uptake process (75) and there is no evidence so far that NP are taken up by macropinocytosis. C. Caveolae-Mediated Endocytosis

Caveolae are flask-shaped invaginations of the plasma membrane of a diameter of 50 to 100 nm observed in several cell types including capillary endothelium, type I alveolar epithelial cells, smooth muscle cells, and fibroblasts. They were also identified in macrophages (86,87) and dendritic cells (88). They are known to demarcate cholesterol and sphingolipid-rich microdomains of the plasma membrane in which many diverse signaling molecules and membrane transporters are concentrated (89). The shape and structural organization of caveolae are conferred by caveolin, a dimeric protein that binds cholesterol, inserts as a loop into the inner leaflet of the plasma membrane (90), and self associates to form a striated caveolin coat on the surface of the membrane invaginations. In most cells, even after activation, caveolae are only slowly internalized (half time:>20minutes) and the small vesicles carry little fluid volume. Thus, it is unlikely that this process contributes significantly to bulk fluid uptake (75). Whether this process is important for the endocytosis of NP needs further research. D. Clathrin-Mediated Endocytosis

This uptake mechanism is very well studied and constitutively occurs in all mammalian cells and carries out the continuous uptake of essential nutrients such as the cholesterolladen low-density lipoprotein particles that bind to the low-density lipoprotein receptor, and iron-laden transferrin that binds to transferrin receptors (91,92). Therefore it is, like most pinocytic pathways, a receptor-mediated endocytosis. Clathrin-mediated endocytosis involves the concentration of high-affinity transmembrane receptors and their bound ligands into “coated pits” on the plasma membrane, which are formed by the assembly of cytosolic proteins, the main assembly protein being clathrin. Coated pits invaginate and pinch of to form endocytic vesicles, clathrin-coated vesicles, which are encapsulated by a polygonal clathrin coat and carry concentrated receptor-ligand complexes into the cell (75). Clathrin is a three-legged structure, called triskelion, formed by three clathrin heavy chains, each with a tightly associated clathrin light chain (92,93). Again, whether clathrin-mediated endocytosis is important for NP needs to be studied in more detail. E.

Clathrin- and Caveolae-Independent Endocytosis

This mechanism is just poorly understood. It is generally referred to as cholesterol-rich microdomains, called rafts, with a diameter of 40 to 50 nm. Their unique lipid composition provides a physical basis for specific sorting of membrane proteins, glycoproteins, and/or glycolipids based on their transmembrane regions (94,95). These small rafts can presumably be captured by and internalized within any endocytic vesicle.

Nanoparticle–Cell Membrane Interactions F.

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Other Possible Penetrating Mechanisms

All of the previously presented endocytic pathways have in common, that the particle, which is taken up, is finally located in an intracellular vesicle. However, there are studies that reported the intracellular localization of UFP and NP of different materials, which were not membrane bound, indicating alternative pathways for particles to enter the cells (83,84,96). Furthermore, the localization of NP in cells that were treated with cytochalasin D in these studies excluded the possibility that the particles were taken up by an actin-driven process (83). In another study, red blood cells that do not have any phagocytic receptors on their surface nor the necessary intracellular structures for phagocytosis or endocytosis were used, and 1 mm polystyrene particles were found attached to the cell surface whereas 0.078 mm nanoparticles could be detected inside the cells, thus they must have entered the cells by nonphagocytic or nonendocytic mechanisms (83,96). Besides other possible mechanisms, passive and active (receptor-mediated) diffusion through membrane pores and passive uptake by Van der Waals or steric interactions (subsumed as adhesive interactions) (97) are suggested by the authors of these studies. However, it remains to be determined which chemical and physical properties of membranes and particles are responsible for the translocation of UFP/NP into cells, the nucleus, and organelles in vitro and in vivo.

IV.

Nanoparticle Localization in Cells

In many studies, NP have been found as aggregates in vesicles. In vivo inhaled gold NP have been detected only as aggregates inside small vesicles in macrophages of the rat lung (98). Also, in in vitro studies, vesicles containing NP aggregates have been described, a finding that might be explained by the fact that NP in suspensions have the tendency to aggregate. Polystyrene particles with a diameter of 50 nm were also phagocytosed by cells when they formed bigger agglomerates in media, and these aggregates were found to be membrane-bound inside the cells. Additionally, single polystyrene particles were identified and they could be detected freely inside the cytoplasm (data not shown). Other studies have shown vesicles containing mostly large aggregates of titanium dioxide in A549 cells (80) or membrane-bound ceria NP agglomerates in human lung fibroblasts (99). However, in all these studies, only conventional transmission electron microscopy was used to detect the particles, and it might well be that single NP or small aggregates of few particles could not be identified. Sophisticated microscopic methods are needed to detect and localize different NP inside cells or tissues (100). One example was the application of electron energy loss spectroscopy to identify titanium dioxide or silverenhanced gold NP inside cells. The particles were localized inside different cell types, for example, epithelial cells, macrophages, and dendritic cells as membrane-bound larger aggregates and free as smaller aggregates or individual particles in the cytoplasm (56) (Fig. 4). NP penetration in vitro into cells does not exclusively occur by any of the expected endocytic processes but also by other yet unknown mechanisms that had been called adhesive interactions, as for instance, electrostatic, Van der Waals, or steric interactions (83,97,101). Particles within cells have been found not to be membranebound (56,83,102) (Fig. 4). Hence, they have direct access to cytoplasmic proteins,

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Figure 4 Micrographs of energy-filtering transmission electron microscopy by means of electron

energy loss spectroscopy. They show cells of the triple-cell cocultures containing titanium dioxide and silver-enhanced gold particles. Titanium dioxide particles (A) were found inside cells as aggregates in vesicles (A, left panel), and as single particles or as small aggregates free in the cytoplasm (A, right panel). Silver-enhanced gold particles (B) were found in cells as single particles or as small aggregates, always free in the cytoplasm and also in the nucleus (B, arrow). The circles mark the region where the element analysis was performed. Source: Modified from Ref. 56.

important biochemical molecules in organelles like the respiratory chain in the mitochondria and the DNA in the nucleus, which may greatly enhance their hazardous potential. It is generally accepted that besides the cell membrane, mitochondria and cell nucleus are the major cell compartments relevant for possible nanoparticle-induced toxicity (17). A recent study shows that carbon-based NP, such as Buckminster fullerene (C60), can enter macrophages and is distributed within the cytoplasm and found in lysosomes as well as in the nucleus (103). Other in vitro experiments revealed penetration of UFP into mitochondria of macrophages and epithelial cells, associated with oxidative stress and mitochondrial damage (104). This penetration of UFP led to a loss of cristae in the mitochondria. The inner mitochondrial membrane was destroyed by UFP. Regardless of the mechanisms causing this mitochondrial damage, there is evidence that it is the organic substances attached to the particle that are responsible for this


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(105). The inner mitochondrial membrane is the structure carrying the respiratory chain, which converts molecular oxygen into energy stored as ATP. Penetration of particles into the nucleus has been shown in a number of in vivo studies for inhaled titanium dioxide NP in rats (83,106) and in vitro studies in different cell types for nano wires (107), gold NP (56) (Fig. 4), polystyrene NP, and C60 (103). Tsoli and colleagues (108) have shown that Au55 clusters interact with DNA of different cell lines in a way that may be the reason for the strong toxicity of these tiny 1.4 nm particles.

V. Conclusion Besides a series of structural and functional barriers, which protect the respiratory system from inhaled particulate antigen, respiratory diseases are frequent and increasing. Many epidemiologic studies have shown a direct relationship between ambient air pollution and increased health effects. Additionally, recent studies indicate that engineered NP are specifically toxic. It is well known that environmental UFP and artificial NP have the capacity to enter different cell types. After particle deposition in the lung, NP may cross the tissue and cellular barriers and reach different tissue compartments with various cell types. They may also enter capillaries and ultimately end up in many other organs. NP of various materials can cross any cellular membrane, although the mechanisms are just poorly understood. Once inside the cells, they may move freely in the cytoplasm or enter organelles like the mitochondria or nuclei, causing severe problems for the cells, the tissue, and the organ, which may have consequences for health. The distribution of the particles (particle trafficking) inside the cells as single particles or as aggregates is of interest to correlate the penetration mechanism and the intracellular translocation with the induction of cellular responses.

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14 Mechanisms and Processes Underlying Toxicological Responses to Particles VICKI STONE Edinburgh Napier University, Edinburgh, U.K.

KEN DONALDSON University of Edinburgh, Edinburgh, U.K.

I.

Overview of Particle Toxicity

The aim of this chapter is to provide an overview of the multiple mechanisms of particle-induced toxicity leading to a number of diverse disease endpoints. It lays out the notion of dose as it relates to particle effects—the principal pathogenic processes, cellular events, and diseases caused by particles—and then summarizes the toxic mechanisms for a range of common pathogenic particles. Figure 1 provides an overview of those processes and diseases that arise from exposure to a range of particle types. In addition, in writing this chapter it became apparent that the literature currently available and reviewed here provides the basis for initiating a matrix to relate physicochemical characteristics to biologically effective dose (BED) and toxicity of all respirable particles. A. Adverse and Pathogenic Effects Caused by Particles

Table 1 provides a list of the more common diseases caused by particles and the setting in which they commonly arise—occupational or environmental. Occupational Exposure

The history of occupational health documents that exposure to particles is associated with disease development. The highest exposures to particles occur in occupational settings, and the best-documented particle-related diseases have historically been the pneumoconioses. The pneumoconioses are fibrosing lung diseases specifically caused by particle exposure and include silicosis, asbestosis, and coal workers’ pneumoconiosis (1). There are many documented pneumoconioses arising from other types of dust but the above three formed the main occupational disease legacy of the industrial revolution. All these are still prevalent diseases and have been studied in detail by particle toxicologists. Classically, the pneumoconioses arise in manual workers (e.g., miners, quarriers) as a result of high exposures to airborne particles. The disease is found in the lungs, when there is a high lung burden (dose) of particles, often measurable at autopsy,

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Figure 1 A general diagram of the pathogenic processes induced by relatively low concentrations

of respirable air pollution particles in the general population (top half) and to relatively high concentrations of industrial dusts in occupational groups (lower half).

Table 1 Diseases Associated with Particle Exposure in Occupational (Occ) and Environmental

(Env) Settings Site

Setting

Disease

Exemplar particles

Airways

Occ/Env

Bronchitis

Occ Occ Occ/Env

Small airways disease Sensitization/asthma Lung cancer

Env

Exacerbations of airways disease—COPD and asthma Emphysema Silicosis (pneumoconiosis)— nodular fibrosis Asbestosis (pneumoconiosis)— interstitial fibrosis Progressive massive fibrosis Silicosis (pneumoconiosis)— nodular fibrosis Mesothelioma Stroke/heart attack

Coalmine dust, organic dust, PM10 Coalmine dust, quartz Metals, organic dusts Quartz, metals, asbestos, PM10 PM2.5

Parenchyma

Occ Occ

Airways

Occ Occ

Pleura, peritoneum Cardiovascular system

Occ Env

Coalmine dust, quartz Quartz, coalmine dust Fibers Coalmine dust

Fibers PM2.5

Toxicological Responses to Particles

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due to a high workplace exposure. There is no known susceptibility in these workers, except for occasional unusual proclivities such as Caplan’s syndrome (2), and indeed most workers in such industries would normally be anticipated to have good pulmonary health at the outset of their employment, as they were employed in physically demanding manual jobs. Because of the expansion of nanotechnology, exposure to a variety of engineered nanoparticles generated in large volumes (e.g., nanotubes, fullerenes, nanoclays, silicates) is also anticipated (3). Almost no data are currently available to quantify exposure in these work environments, and no data exist that attempt to quantify health effects due to the short history of these exposures. Whether or not these exposures will result in adverse health effects (acute or chronic) remains to be seen, and forms the driving force behind the expanding field of nanotoxicology. Environmental Exposure

There is also a public burden of ill health due to environmental/ambient particles. The epidemics of ill health associated with sulfurous smog episodes resulting from domestic coal burning in the early parts of the 20th century (4) are now a thing of the past. However, there remains a consistently present burden of ill health as a consequence of ambient particles (5). Particulate matter (PM) represents a part of the air pollution cocktail present in ambient air, which also comprises gases such as ozone, nitrogen dioxide, and so on. PM is measured as the mass of particles collected using the PM10 or PM2.5 sampling conventions (6). The adverse health effects of PM10 are seen at the levels found in modern cities today and there is often no threshold (5). The diverse adverse effects that PM cause are shown in Table 1 marked with “Env” in the second column and include deaths and exacerbations of airways disease, and deaths from, and exacerbations of cardiovascular disease. These adverse health effects of air pollution have been measured in epidemiological studies, and there is good coherence between the acute effects seen in time series and panel studies and the chronic effects seen in environmental studies. The exposure to PM is very low—in the low tens of mg/m3 compared to workplace exposures that are in the mg/m3 region. The toxicity caused by such low doses of PM may be explained by the fact that the populations that are affected are susceptible and already have diseases characterized by inflammation and oxidative stress, the same effects that are caused by particles, so providing the opportunity for interaction and worsening of their disease. B. Toxic and Pathogenic Processes

A wide spectrum of pathobiological processes are seen in cells and tissues exposed to particles, and these can be understood in terms of the properties of the particles, for example, ability to cause oxidative stress and cell death, fiber length exceeding the phagocytic ability of the cells, and so on, and these are discussed below. Inflammation is a common finding following particle exposure and there is a clear link between inflammation and fibrosis, cancer, cardiovascular disease, and airways disease, the pathological outcomes associated with particle exposures. Central to understanding toxic response is defining and understanding the “dose” for different particles, and this is a unifying theme throughout this review.

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II.

Dose

As in all toxicology, defining the dose is central to understanding the biological response to particles.

A. Defining the Biologically Effective Dose for Particles and a General Scheme for Particle Responses at the Cellular Level

The toxicological responses to particles are diverse, occurring primarily in the lungs but there is also increasing data showing that other tissues can be affected by particle inhalation exposures. In toxicology, response follows dose and the dose-response relationship is key to understanding the outcome of any exposure. The concept of the BED is a key one in mechanistic toxicology. Rather than the total dose expressed as mass of toxin per unit mass of tissue, the common expression of dose, the BED, is the fraction of the mass dose that actually delivers a specific toxic damage. Thus, while the mass of a classical toxin like dimethylbenzanthracene (DMBA) in tissue constitutes the total dose, it is only the fraction of DMBA that is biotransformed to the diol epoxide that is adductforming; therefore, the BED for adduct formation is the dose of diol epoxide, which is much less than the total DMBA mass. To date, for inhaled particles there is little evidence for any biotransformation, and the BED of any particle is in fact some physicochemical entity or combination of physicochemical entities for example, surface area, surface area times surface reactivity, fiber length, and so on (Table 2). Exposure is frequently used as a surrogate for dose, but exposure measured by the common metric of mass is often not closely related to the BED, since there has been no modification by entry and residence in the target tissue. In the case of particles, this complexity increases, since the elutriating effect of negotiating the airways prior to deposition “size-fractionates” the exposure and macrophage clearance further operates on the particles.

Table 2 BED for Different Particles and the Mismatch Between the Current Metric and the BED

Particle

Biologically effective dose

Current metric

Quartz

Area of reactive (unblocked or unpassivated) surface Biopersistent fibers longer than 2.5 mm

Respirable mass

Asbestos

Number of fibers >5 mm length, 3 Mass by PM10 convention Respirable mass Nuisance dust standard of respirable mass l l

PM10 Welding fume (NP) Carbon black (NP)

Organics/metals/surfaces Soluble transition metals Surface area

Abbreviation: BED, biologically effective dose.


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Figure 2 Diagram to demonstrate the relationship between factors involved in the response to

particle deposition in lung tissue.

Furthermore, even the total deposited mass of particles does not represent the BED as research has shown that, for all particle studied so far, the BED is a physicochemical characteristic such as surface area, transition metal content, fiber length, etc. (Table 2). The BED then acts on cells and tissues to produce a stress of some sort (Fig. 2), typically oxidative stress but also stress caused by macrophage production of proteases or complement activation, for example. This causes the induction of defenses such as antioxidant enzymes, anti-proteases, etc. If the dose and therefore the stress is low level, then the threshold may not be exceeded, and the stress is ameliorated by the stress-defense response, for example, antioxidants are able to deal with the oxidative stress and return the tissue to normal redox balance. However, if the BED is large and the stress is severe enough to exceed the threshold, then damage ensues with oxidation of proteins, induction of inflammation, and generation of oxidative adducts; while at very high levels of stress, cell death may be provoked (7) (Fig. 2). Being able to define the BED for particles is important as the BED could ideally be used as the exposure metric, which would produce optimal risk management; additionally, in epidemiological studies relating endpoints to the BED would greatly improve correlation of exposure to response. Defining the BED for particles also enhances our understanding of how adverse effects are produced at the cellular/molecular level since the BED, by definition, is the particle characteristic(s) that “drives toxicity.” Conversely, interpreting the cellular/molecular events caused in cells by particles and their variants and subfractions can also provide the data that allows the BED to be defined. Recently, the authors have suggested that oxidative stress potential (see below) might be considered as an improved metric for PM10 exposure, since toxicological studies have identified it as a key BED for PM10 (8). Considerable particle toxicology research has gone into defining the BED for the principal pathogenic particles, and these are shown in

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Table 2. Currently, for the main pathological particles, there is a considerable mismatch between the exposure metric and the BED as determined from research (9) (Table 2). The toxicological mechanism, including the BED, is described below for some categories of particles.

III.

Common Processes and Mechanisms Involved in Particle-Induced Responses

A number of processes and mechanisms are outlined below, which are relevant to particle-induced responses. The cardiovascular effects of particles, although relevant, are not included as they form the basis of chapter 7. In addition, the mechanisms by which particles might cross epithelial barriers could also be considered under this heading, so the reader is referred to chapter 13. A. Oxidative Stress

Most organisms contain antioxidants that protect the cells and molecules of that organism from the damaging effects of oxidants. Oxidants include free radicals and reactive oxygen species (ROS) that are made naturally (e.g., by normal metabolism), and also from endogenous sources (e.g., UV radiation, xenobiotic metabolism). ROS are oxygen-containing molecules that possess an unpaired electron, making them highly reactive and damaging to endogenous molecules. Oxidative stress occurs when there is an imbalance between oxidants and antioxidants, favoring the presence of oxidants either due to excessive production of oxidants or depletion of antioxidants (10). Because of the depletion of antioxidants, the potential for ROS-induced oxidation of DNA, proteins, and lipids are enhanced, which usually leads to loss of function and, ultimately, cytotoxicity (11). Depletion of antioxidants can be caused by excessive exposure to ROS but is also more likely in an individual who is compromised, for example, due to poor diet or inflammatory disease. It is also very important to note that sublethal levels of oxidative stress can activate cellular signaling processes including calcium-signaling, transcription factor activation leading to either protective mechanisms (e.g., upregulation of antioxidant gene defense mechanisms), or proinflammatory signaling (e.g., cytokine gene expression). The latter can be associated with disease (see below). There is much evidence to demonstrate that inhaled particles result in at least two potential sources of ROS. The first is generated by phagocytic cells, which on ingestion of particles generate ROS and reactive nitrogen species as part of their antimicrobial activities. This is particularly important for high aspect ratio (fiber-like) particles such as asbestos, which due to complete uptake by macrophages result in frustrated phagocytosis and continual superoxide anion production (12,13). The second source includes ROS and free radicals generated directly at the particle surface (10). Many pathogenic particles have been shown to generate ROS, and this oxidative ability has been considered a key mechanism of toxicity. For example, Fubini (14) identified a variety of radical types including superoxide anions and the peroxyradical at the surface of a number of standard quartz samples. The production of ROS by phagocytic cells exposed to quartz particles was demonstrated by Castranova et al. (15), while Vallyathan et al. (16)


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demonstrated that quartz was able to induce oxidative stress in rats (10 mg/rat), associated with upregulation of the antioxidant enzymes superoxide dismutase and glutathione peroxidase, two and eight days following instillation. Oxidative damage was evidenced by lipid peroxidation, which increased with time. Albrecht et al. (17) also investigated the ability of quartz particles to induce oxidative damage in the rat lung. This study indicated that pulmonary oxidants such as hydrogen peroxide and nitrite were elevated seven days following silica exposure, which were also associated with increased antioxidant levels of the bronchoalveolar lavage fluid, suggesting that antioxidant defenses had been switched on in response to the particle exposure. Coating the particle surface prevented this oxidant generation and antioxidant response, demonstrating the importance of the quartz surface in generating ROS. Interestingly, the oxidant effects of the silica particles were not associated with DNA damage detected as 8-hydroxy-20 -deoxyguanosine at day 7. Asbestos-induced disease has also been linked to ROS and oxidative stress. Gilmour et al. (18) demonstrated that asbestos and synthetic vitreous fibers were able to generate free radicals using the cell-free DNA plasmid assay in which free radicals cause scission. The ability of fibers to induce ROS production was found to be associated with their ability to activate the transcription factor nuclear factor kappa B (NF-kB) in epithelial cells in vitro; NF-kB is a key transcription factor in the control of genes that regulate inflammation (19,20). In vivo, Brown et al. (21) also demonstrated that pathogenic fibers can induce oxidative stress in the rat lung, as indicated by glutathione and ascorbate depletion. In addition to the results with asbestos, the plasmid assay has also been used to demonstrate the ability of PM10 (22), carbon nanoparticles (23), and a panel of metal nanoparticles (24) to induce ROS production. Additional support for the production of ROS by nanoparticle carbon black (25) and polystyrene beads (26) has been published using the dye 2,7-dichlorofluorescin-diacetate that fluoresces under oxidizing conditions (27). Dichlorofluorescin diacetate (DCFH-DA) can also be used to measure intracellular ROS (28); for example, Wilson et al. (25) demonstrated that carbon nanoparticles (14 nm diameter), but not larger carbon particles (260 nm diameter), stimulated an increase in intracellular ROS in the macrophage cell line Monomac6. The role of ROS production by nanoparticles in inducing cytotoxicity was demonstrated by Stone et al. (23) when the carbon black nanoparticle caused depletion of glutathione in epithelial cells, and that decreased cell viability could be prevented by antioxidants. More recently, Sayes et al. (29) have also demonstrated that C60 NPs elicit oxidative stressmediated toxicity in vitro. Instillation of nanoparticle carbon black into the rat lung is also associated with a depletion of the antioxidant glutathione (11). These studies therefore provide evidence that nanoparticles induce oxidative stress, suggesting that the particle-generated ROS impact upon cells in the body. B. Inflammation

The role of the immune system is to defend the body against foreign material, including infectious agents and particles. With respect to inhaled particles, the defensive response is led by the macrophage, as this cell is responsible for identification and clearance of foreign particles that deposit on the surface of the airspaces. The influx of inflammatory

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cells such as macrophages and neutrophils into a tissue following some sort of insult is the hallmark of inflammation. In the case of particle deposition beyond the ciliated airways, clearance revolves around uptake of the particles by these professional phagocytes (30). Macrophages can take up relatively large materials (>0.5 mm) such as bacteria or cell debris, but the natural elutriation that is caused as the airstream negotiates the bends and bifurcations on the bronchial tree means that only small particles, less than about 5 mm, reach centriacinar regions, where the alveoli are present (31). During phagocytosis, the cell recognizes ligands via cell surface receptors, but the receptors involved in particle uptake are ill understood. Receptor binding then triggers the polymerization and rearrangement of the actin cytoskeleton to form membrane extensions so that the plasma membrane surrounds the material to be internalized (31–33). The phagosome that is formed then fuses with lysosomes, so that the cargo can be degraded if possible (33). This process appears to be relatively successful for finesized (>100 nm, but

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