Cytokines and the Abdominal Surgeon

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MEDICAL INTELLIGENCE UNIT

3

Moshe Schein • Leslie Wise

Cytokines and the Abdominal Surgeon

R.G. LANDES C OM PA N Y

MEDICAL INTELLIGENCE UNIT 3

Cytokines and the Abdominal Surgeon Moshe Schein, M.D., F.C.S. (SA) Associate Professor of Surgery, Cornell University College of Medicine Brooklyn, NY

Leslie Wise, M.D., F.R.C.S. (Eng) Professor of Surgery, Cornell University College of Medicine Brooklyn, NY

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

MEDICAL INTELLIGENCE UNIT Cytokines and the Abdominal Surgeon R.G. LANDES COMPANY Austin, Texas, U.S.A. Copyright © 1998 R.G. Landes Company All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the U.S.A. Please address all inquiries to the Publishers: R.G. Landes Company, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081

ISBN: 1-57059-536-4

While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data

CIP information applied for but not received at time of publication.

MEDICAL INTELLIGENCE UNIT 3 PUBLISHER’S NOTE


Landes Bioscience produces books in six Intelligence Unit series: Medical, Molecular Biology, Neuroscience, Tissue Engineering, Biotechnology and Environmental. The authors of our books are acknowledged leaders in their fields. Topics are unique; almost without exception, no similar books exist on these topics. Our goal is to publish books in important and rapidly changing areas of bioscience for sophisticated researchers and clinicians. To achieve this goal, we have accelerated our publishing program to conform to the fast pace at which information grows in bioscience. Most of our books are published within to 120 days of receipt of Associate Professor of90 Surgery, the manuscript. WeUniversity would likeCollege to thank our readers for their Cornell of Medicine continuing interest and welcome any comments or suggestions they Brooklyn, NY may have for future books.

Moshe Schein, M.D., F.C.S. (SA)

Judith Kemper Leslie Wise, M.D., F.R.C.S. (Eng)

Professor of Surgery, Production Manager Landes Company Cornell University College of R.G. Medicine Brooklyn, NY

R.G. LANDES COMPANY AUSTIN, TEXAS U.S.A.

CONTENTS 1. Introduction—Cytokines and the Abdominal Surgeon ......................... 1 Moshe Schein and Leslie Wise 2. Relevance of Molecular Biology for the Abdominal Surgeon ................ 3 Philip S. Barie and Steven M. Cohen Introduction ............................................................................................. 3 Structure and Function of DNA and Chromosomes ............................ 4 Human Genome Project ......................................................................... 4 Molecular Diagnostic Tests ..................................................................... 5 Protein Hybridization ............................................................................. 6 Programmed Cell Death ......................................................................... 8 Genetic Predisposition to Disease: Preparing for the Genetic Revolution .............................................. 10 Gene Therapy in Surgery ...................................................................... 11 Antisense Oligonucleotides as Potential Gene Therapy ...................... 13 3. Cytokine Response in Abdominal Surgery ............................................ 17 Edward Lin, Steve E. Calvano, and Stephen F. Lowry Introduction ........................................................................................... 17 Injury and Principal Cytokine Responses ............................................ 19 Clinical Considerations ......................................................................... 25 Conclusions ........................................................................................... 30 4. Cytokines and the Abdominal Operative Wound ................................. 35 Frank J. Thornton and Adrian Barbul Introduction ........................................................................................... 35 Overview of the Repair Process ............................................................ 35 Platelet Degranulation ........................................................................... 36 Immune Cell Influx ............................................................................... 37 Transforming Growth Factor ! .......................................................... 37 Interleukins ............................................................................................ 38 Interferon-∀ ................................................................................................... 38 Tumor Necrosis Factor # ................................................................... 39 Nitric Oxide ........................................................................................... 39 Extracellular Matrix Production ........................................................... 39 Angiogenesis .......................................................................................... 40 Wound Contraction .............................................................................. 40 Epithelialization ..................................................................................... 42 Cytokines in Surgical Site Infection ...................................................... 43 Conclusion ............................................................................................. 44

5. Cytokines and Postoperative Abdominal Adhesions ............................ 53 Shaun G. Appleton and Jeremy N.Thompson Introduction ........................................................................................... 53 Adhesion Formation ............................................................................. 54 Interleukin-1 (IL-1) ............................................................................... 55 Tumor Necrosis Factor-alpha (TNF-#) ............................................... 56 Interleukin-6 (IL-6) ............................................................................... 57 Transforming Growth Factor-beta (TGF-!) ........................................ 57 Other Cytokines ..................................................................................... 58 Conclusions ........................................................................................... 58 6. Cytokines in Blunt Abdominal Trauma ................................................ 63 Matthias W. Wichmann and Eugen Faist Introduction ........................................................................................... 63 Introduction to the Cytokines Studied in Trauma Research .............. 63 Pro- and Anti-Inflammatory Mediators .............................................. 66 Experimental Trauma Research ............................................................ 66 Experimental Immunomodulation Following Trauma-Hemorrhage ...................................................... 67 Clinical Trauma Research ..................................................................... 67 Conclusions ........................................................................................... 68 7. Cytokines in Acute Pancreatitis .............................................................. 73 James Norman and Larry C. Carey Clinical Pancreatitis and the Immune Response ................................. 73 Amplification of the Pancreatitis-Associated Inflammatory Cascade ...................................................................... 75 Local Cytokine Production During Pancreatitis.................................. 76 The Effects of Local Cytokine Production on Pancreatitis Severity ... 76 Cytokine-Induced Acinar Cell Death During Pancreatitis ................. 77 Acute Pancreatitis Induces Systemic Hyperinflammation .................. 77 Antagonism of Cytokines During Experimental Acute Pancreatitis .............................................................................. 79 Is Anti-Cytokine Therapy for Acute Pancreatitis a Clinical Possibility? ......................................................................... 79 8. Cytokines and Perioperative Nutrition ................................................. 83 Hideaki Saito and Lin Ming-Tsan Introduction ........................................................................................... 83 Nutritional Route and Cytokine Biology ............................................. 83 Nonstressed Stable Conditions ............................................................. 84 Tissue Cytokine Biology ........................................................................ 84 Stressed Conditions ............................................................................... 86 Cytokine Biology at Local Inflammatory Sites..................................... 87 Specific Nutrients and Cytokine Biology ............................................. 88 Glutamine .............................................................................................. 89

Arginine .................................................................................................. 90 Nucleotides ............................................................................................ 91 Immune-Enhancing Diets ..................................................................... 91 Anabolic Hormones And Cytokine Biology ........................................ 91 Summary ................................................................................................ 92 9. Cytokine Response to Laparoscopic Surgery ......................................... 97 Harry van Goor and R. Jan A. Goris Introduction ........................................................................................... 97 Rationale for Less Tissue Trauma and Stress Response in Laparoscopy ................................................ 97 Cytokines and Tissue Trauma .............................................................. 98 Cytokines in Laparoscopic Cholecystectomy ....................................... 99 Cytokines in Other Laparoscopic Procedures ...................................... 99 Do Circulating Cytokines Accurately Reflect Tissue Trauma? ......... 100 Cytokines and Outcome After Laparoscopic Surgery ....................... 101 Conclusions ......................................................................................... 103 10. Cytokines in Experimental Peritonitis ................................................. 107 Martin K. Angele, Alfred Ayala, and Irshad H. Chaudry Introduction ......................................................................................... 107 Choice of Models ................................................................................. 108 The Response of Cytokines in Experimental Models of Sepsis and Clinical Sepsis ............................................................ 109 Summary .............................................................................................. 113 11. Hematopoietic Cytokines, G-CSF and Abdominal Surgery ............... 117 Artur Bauhofer, Wilfried Lorenz, Ilhan Celik, Benno Stinner, José Solovera, and Ronald Lorijn Introduction ......................................................................................... 117 Preclinical Biology: Hematopoietic Growth Factors (HGFS) in Infectious Disease ........................................................................ 119 Clinical Reality: The Overall Complexity of Abdominal Infections ................................................................. 123 New Concepts: Demonstrating Effectiveness of Drugs in Clinical Trials and Daily Practice for Abdominal Infections .... 127 A Convincing Example for Increasing Clinical Complexity in Preclinical Trials: Experience with rhg-CSF (Filgrastim) ......... 130 First Explanations for rhg-CSF Positive and Negative Trials: Relevance for Designing and Conducting Definitive Clinical Trials ................................................................................... 130 Perspectives: Filgrastim in Abdominal Surgery ................................. 133 Conclusion ........................................................................................... 133 List of Abbreviations ........................................................................... 134

12. Cytokines and Spontaneous Bacterial Peritonitis ............................... 143 Theresa Propst and A. Propst Introduction ......................................................................................... 143 Pathogenesis ......................................................................................... 143 Diagnosis .............................................................................................. 145 Symptoms ............................................................................................ 145 Differential Diagnosis .......................................................................... 146 Cytokines in Chronic Liver Disease .................................................... 146 Cytokines in SBP ................................................................................. 147 Conclusion ........................................................................................... 149 13. Secondary Peritonitis and Cytokines ................................................... 151 Réne G. Holzheimer Introduction ......................................................................................... 151 Peritonitis and Cytokines in Experimental Models ........................... 151 Dosage of Cytokines ............................................................................ 152 Function of Peritoneal Cells ................................................................ 152 Local Response ..................................................................................... 152 Effect of Cytokines and Growth Factors in Peritonitis ...................... 153 Anti-Endotoxin-Antibodies ................................................................ 153 Pathogens ............................................................................................. 154 Anti-Inflammatory Cytokines ............................................................ 154 Second Hit ............................................................................................ 154 Therapy ................................................................................................ 155 Secondary Peritonitis and Cytokines in Clinical Studies .................. 155 Conclusions ......................................................................................... 156 14. Cytokines and Combination Treatment of Intra-Abdominal Infections ............................................................. 163 William G. Cheadle and Hiram C. Polk, Jr. Introduction ......................................................................................... 163 Overview of Our Current Research .................................................... 163 The Macrophage .................................................................................. 164 The Role of Endotoxin ........................................................................ 165 The Neutrophil .................................................................................... 165 Lymphocytes ........................................................................................ 166 Therapy of Intra-Abdominal Infection .............................................. 166 15. Cytokines, Abdominal Trauma and the “Second Hit” Phenomenon ..................................................... 171 Roger Saadia and Jeffrey Lippman Mutiple Organ Failure After Trauma ................................................. 171 Cytokines and the Postinjury Inflammatory State ............................ 171 “One-Hit” and “Two-Hit” Models ..................................................... 172 Priming and Activation ....................................................................... 173 Clinical Observations .......................................................................... 173 “First Hit” and Cytokine Patterns ...................................................... 174

“Second Hit” and the Inflammatory Response .................................. 175 Conclusion ........................................................................................... 176 16. Cytokines and Mesenteric Ischemia ..................................................... 179 David A. Partrick, Ernest E. Moore and Walter L. Biffl Introduction ......................................................................................... 179 Pathogenesis of Multiple Organ Failure ............................................. 179 Mesenteric Ischemia as a Model of Systemic Inflammation ............. 180 Gut-Derived Mediators of Neutrophil Priming ................................ 181 Interleukin-1b ...................................................................................... 184 Interleukin-6 ........................................................................................ 185 Interleukin-8 ........................................................................................ 188 Clinical Implications ........................................................................... 188 17. The Significance of Intestinal Cytokines ............................................. 197 Per-Olof Hasselgren Introduction ......................................................................................... 197 Cytokines in Inflammatory Bowel Disease ........................................ 197 Studies in Experimental Animals ........................................................ 202 Intestinal Cytokines During Sepsis and Endotoxemia ...................... 202 The Significance of Intestinal Cytokines ............................................ 208 18. Cytokines in Abdominal Vascular Surgery .......................................... 215 C.V. Soong and B.J. Rowlands Introduction ......................................................................................... 215 Evidence of Endotoxemia and Cytokine Production in Abdominal Vascular Surgery ...................................................... 216 The Mechanism of Production of Cytokines in Aortic Surgery ....... 217 The Effects of Elevated Cytokines ....................................................... 221 Potential Therapeutic Manipulation .................................................. 222 Conclusion ........................................................................................... 223 19. Cytokines and Abdominal Organ Transplantation ............................ 233 Stephen W. Chung, Charles H. Scudamore and Reginald Gorczynski Introduction ......................................................................................... 233 The Cytokine Response in Acute Allograft Rejection ........................ 234 Experimental Models of Acute Rejection ........................................... 236 Conclusion ........................................................................................... 240 20. Anti-Cytokine Strategies in Peritonitis: Is Local, Intra-Abdominal, Therapy Possible? .................................................................................. 245 Alfred Ayala and Irshad H. Chaudry Introduction ......................................................................................... 245 Models of Sepsis/Peritonitis ................................................................ 246 Differential Effects of Sepsis/Peritonitis of Immune Cells ................ 247 Potential Intra-Abdominal Pro- or Anti-Inflammatory Cytokine Therapies? ........................................................................ 256 Summary .............................................................................................. 263

21. The Antibiotic-Induced Cytokine Response ....................................... 273 Timothy G. Canty Jr., Edward Boyle Jr., and E. Patchen Dellinger Introduction ......................................................................................... 273 Antibiotics, Bacterial Modulins, and Cytokines ................................ 273 Antibiotic Induced Release of Bacterial Modulins: History .............. 274 Background .......................................................................................... 275 Antibiotic Induced Release of Bacterial Modulins: Endotoxin ......... 275 Antibiotics and Endotoxin (In Vitro) ................................................ 276 Antibiotics and Endotoxin (Animal Studies) .................................... 276 Antibiotics and Endotoxin (Clinical Studies) .................................... 277 Do Antibiotics Differ in Potential for Endotoxin Release? ............... 277 Cytokine Stimulating Microbial Components Other Than LPS ...... 279 Cytokine Synthesis and Antibiotics .................................................... 281 Conclusion ........................................................................................... 281 22. Cytokines and Pentoxifylline in Surgical Sepsis .................................. 285 J. Schröder, K.H. Staubach, F. Stüber and P. Zabel Introduction ......................................................................................... 285 Long-Term Administration of Pentoxifyllin in Surgical Sepsis ........ 286 Effects of Pentoxifylline on Multiple Organ Dysfunction ................. 288 Cytokines and Pentoxifylline .............................................................. 290 Perspectives .......................................................................................... 291 23. Epilogue ................................................................................................. 295 Moshe Schein and Leslie Wise Introduction ......................................................................................... 295 Infection vs Inflammation and Sepsis ................................................ 295 Current Terminology in Clinical Sepsis ............................................. 296 Local Inflammation vs Contamination, Infection or Sepsis ............. 297 Contamination, Infection, Sepsis: A Continuum .............................. 298 Clinical Relevance ................................................................................ 298 Index ....................................................................................................... 303

EDITORS Moshe Schein, MD, FCS (SA) Associate Professor of Surgery, Cornell University College of Medicine New York Methodist Hospital Brooklyn, NY Chapters 1, 23 Leslie Wise, MD, FRCS (Eng) Professor of Surgery, Cornell University College of Medicine and Albert Einstein College of Medicine Chairman, Department of Surgery Brooklyn, NY Chapters 1, 23

CONTRIBUTORS Shaun G. Appleton, FRCS Department of Surgery Chelsea and Westminster Hospital London, UK Chapter 5

Philiph Barrie, MD Professor of Surgery Cornell University College of Medicine New York, NY Chapter 2

Martin K. Angele, MD Research Associate Center for Surgical Research and Department of Surgery Brown University School of Medicine and Rhode Island Hospital Providence, RI Chapter 10

Artur Bauhofer, PhD Institute of Theoretical Surgery, University Marburg Marburg, Germany Chapter 11

Alfred Ayala, PhD Associate Professor Center for Surgical Research and Department of Surgery Brown University School of Medicine and Rhode Island Hospital Providence, RI Chapters 10, 20 Adrian Barbul MD Professor of Surgery, The Johns Hopkins University School of Medicine Baltimore, MD Chapter 4

Walter L. Biffl, MD Department of Surgery University of Colorado Health Sciences Center Denver, CO Chapter 16 Edward Boyle, Jr. MD Department of Surgery, University of Washington Seattle, WA Chapter 21 Steve E. Calvano, PhD UMDNJ-Robert Wood Johnson Medical School Department of Surgery New Brunswick, NJ Chapter 3

Timothy G. Canty, Jr. MD Department of Surgery University of Washington Seattle, WA Chapter 21

Steven M. Cohen, DO Research Fellow Department of Surgery, Cornell University Medical College Chapter 2

Larry C. Carey, MD Professor and Chairman Department of Surgery University of South Florida Tampa, Florida Chapter 7

E. Patchen Dellinger, MD Professor of Surgery Department of Surgery University of Washington Seattle, WA Chapter 21

Ilhan Celik, MD Institute of Theoretical Surgery University of Marburg Marburg, Germany Chapter 11

Eugen Faist, MD Professor or Surgery Ludwig-Maximillians-Universitat, Klinikum Grosshardern Munich, Germany Chapter 6

Irshad H. Chaudry, PhD Professor of Surgery, Molecular Pharmacology, Physiology and Biotechnology Director, Center of Surgical Research Brown University School of Medicine and Rhode Island Hospital Providence, RI Chapters 10, 20 William G. Cheadle, MD Associate Professor of Surgery Veterans Affairs Medical Center, Price Institute for Surgical Research, and the Department of Surgery, University of Louisville School of Medicine Louisville, KY Chapter 14 Stephen W. Chung, MD, PhD Department of Surgery Vancouver Hospital and Health Sciences Centre Vancouver, BC, Canada Chapter 19

R. Jan A. Goris, MD, PhD Professor and Chairman Department of Surgery, University Hospital Nijmegen, Nijmegen, The Netherlands Chapter 9 Reginald Gorczynski, PhD, MD Departments of Immunology University of Toronto Toronto, Canada Chapter 19 Per-Olof Hasselgren, MD Professor of Surgery University of Cincinnati Medical Center Cincinnati, OH Chapter 17 Réne G. Holzheimer, MD, PhD Department of Surgery Martin-Luther-Universität HalleWittenberg Halle, Germany Chapter 13

Edward Lin, DO Department of Surgery The New York Hospital Queens Flushing, New York Chapter 3 Jeffrey Lippman, MB BCh, FFA (SA) Associate Professor-Division of Anesthesiology and Intensive Care The Royal Brisbane Hospital and University of Queensland Brisbane, Australia Chapter 15 Wilfried Lorenz, MD Professor and Director Institute of Theoretical Surgery University Marburg Marburg, Germany Chapter 11 Ronald Lorijn MD, PhD AMGEN-Europe Lucerne, Switzerland Chapter 11 Stephen F. Lowry, MD Professor and Chairman, Department of Surgery UMDNJ-Robert Wood Johnson Medical School New Brunswick, NJ Chapter 3 Lin Ming-Tsan, MD Department of Surgery The University of Tokyo Tokyo, Japan Chapter 8 Ernest E. Moore, MD Professor of Surgery Department of Surgery University of Colorado Health Sciences Center Denver, CO Chapter 16

James Norman, MD Associate Professor of Surgery and Medicine Department of Surgery University of South Florida Tampa, Florida Chapter 7 David A. Partrick, MD Department of Surgery University of Colorado Health Sciences Center Denver, CO Chapter 16 Hiram C. Polk, Jr, MD Professor and Chairman Veterans Affairs Medical Center, Price Institute for Surgical Research, and the Department of Surgery, University of Louisville School of Medicine Louisville, KY Chapter 14 Theresa Propst, MD Department of Internal Medicine University of Innsbruck, Austria Chapter 13 Albert Propst, MD Department of Internal Medicine University of Innsbruck, Austria Chapter 13 Brian J. Rowlands, MD, FRCS Professor of Surgery Queen’s Medical Centre Nottingham, UK Chapter 18 Roger Saadia, MD, FRCS Professor of Surgery, University of the Witwatersrand Chief Surgeon-Baragwanath Hospital Johannesburg, South Africa Chapter 15

Charles H. Scudamore, MD, Msc Department of Surgery Vancouver Hospital and Health Sciences Centre Vancouver, BC, Canada Chapter 19 Hideaki Saito, MD Associate Professor, Department of Surgery The University of Tokyo Tokyo, Japan Chapter 8 J. Schröder, MD Department of General and Thoracic Surgery The Christian-Albrechts-University Hospital Kiel, Germany Chapter 22 José Solovera MD, PhD AMGEN-Europe Lucerne, Switzerland Chapter 11 C.V. Soong, MD, FRCS Senior Registrar Department of Surgery Queen’s University of Belfast Belfast, UK Chapter 18 F. Stüber, MD Department of General and Thoracic Surgery The Christian-Albrechts-University Hospital Kiel, Germany Chapter 22

K.H. Staubach, MD Department of General and Thoracic Surgery The Christian-Albrechts-University Hospital Kiel, Germany Chapter 22 Benno Stinner, MD Department of General Surgery University Marburg Marburg, Germany Chapter 11 Jeremy N.Thompson, MChir, FRCS Department of Surgery Chelsea & Westminster Hospital London Chapter 5 Frank J. Thornton, MB, FRCS(I) Department of Surgical Research, Sinai Hospital of Baltimore and the Johns Hopkins Medical Institutions Baltimore, MD Chapter 4 Harry van Goor, MD, PhD Department of Surgery, University Hospital Nijmegen The Netherlands Chapter 9 Matthias W. Wichmann, MD Ludwig-Maximillians-Universitat Klinikum Grosshardern Munich, Germany Chapter 6 P. Zabel, MD Department of General and Thoracic Surgery The Christian-Albrechts-University Hospital Kiel, Germany Chapter 22

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CHAPTER 1

Introduction—Cytokines and the Abdominal Surgeon Moshe Schein and Leslie Wise

R

ecent advances in molecular biology permitted the identification of various cytokines as the key “proximal” links in the infectious-inflammatory cascades, mediating local and systemic inflammation, sepsis, and tissue injury. A huge body of data concerning cytokines is available-a MEDLINE search today (December 12, 1997) generated 31897 citations mentioning the word cytokines. We, clinicians find ourselves overwhelmed by such abundant information, understanding less about more. This book was conceived to epitomize and simplify the most complex and confused known facts which imply that cytokines have an important role in many conditions which are commonly encountered by the abdominal surgeon. The authors for the various chapters were chosen based on their previous contributions to the specific topic to be discussed. Both basic researchers and clinicians were selected in order to provide a balanced picture, “tolerated” by scientists and attractive to clinical surgeons. Some repetition and overlapping in the chapters were unavoidable as this is a multi-author book. In chapter 2, Drs. Barie and Cohen from New York, discuss the relevance of molecular biology to the surgeon. Drs. Lin, Calvano and Lowry from New Jersey offer, in chapter 3, a detailed overview on the cytokine-response following abdominal operations. They emphasize that the extent and complexity of surgery, together with the adequacy of the peri-operative care, are important determinants of the ensuing inflammatory response. The role of cytokines in the healing of surgical and nonsurgical wounds is discussed, in chapter 4, by Drs. Thornton and Barboul from Baltimore, who also highlight possible future clinical implications in complicated or compromised wounds. In chapter 5, Messers Appelton and Thompson, from London, deal with the role played by cytokines in the formation of postoperative peritoneal adhesions; cytokine-manipulation to control adhesions appears an attractive future option. The next chapter—no. 6—by Dr. Wichmann and Professor Faist from Munich review the data concerning the effects of trauma on the cytokine network. In chapter 7, Drs. Norman and Carey from Tampa tell us all what is known about cytokines in acute pancreatitis, predicting exciting future therapeutic implications. Drs. Saito and Ming-Tsan from Tokyo (chapter 8) discuss how the different peri-operative nutrition modalities modulate the cytokine-response. In chapter 9, Dr. Van Goor and Professor Goris from the Netherlands summarize the (reduced) cytokine response associated with laparoscopic-minimally invasive surgery. Drs. Angele, Ayala and Chaudry Cytokines and the Abdominal Surgeon, edited by Moshe Schein and Leslie Wise. © 1998 R.G. Landes Company.

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from Rhode Island (chapter 10) synthesize what is known about cytokines in experimental peritonitis-offering clinical relevance. In chapter 11, Drs. Bauhofer, Celik, Stinner, Solovera and Lorijn, and Professor Lorenz from Marburg, discuss the potentials of hematopoietic cytokines and G-CSF in the modulation of “surgical sepsis”, emphasizing the complexity of clinical scenarios as opposed to experimental settings. Drs. Propst and Propst from Innsbruck— our only “nonsurgeon” contributors—discuss in chapter 12 the role of cytokines in spontaneous bacterial peritonitis; practical diagnostic and prognostic issues are included. In chapter 13, Dr. Holzheimer from Halle (Germany) focuses on the role of cytokines in secondary bacterial peritonitis, emphasizing the intraperitoneal compartmentalization of the inflammatory response. In chapter 14, Drs. Cheadle and Polk from Louisville sum up their research on the future “combination treatment” of intra-abdominal infections in which anticytokine regimens would be included. Professors Saadia (Johannesburg) and Lippman (Brisbane, Australia) discuss in chapter 15 the issue of cytokines related to the “second hit” phenomenon, best studied hitherto in the settings of multiple trauma. Drs. Patrick, Moore and Biffl from Denver write in chapter 16 about cytokine-related events occurring during mesenteric ischemia. Dr. Hasselgren from Cincinnati (chapter 17) details the significance of intestinal cytokines-locally and systemically. In chapter 18, Mr. Soong from Belfast and Professor Rowlands from Nottingham encapsulate the complex inflammatory events that follow major abdominal aortic surgery, emphasizing the many remaining uncertainties. Chapter 19, by Drs. Chung, Scudamore (Vancouver), and Gorczynski (Toronto), is dedicated to the role of cytokine—be it surveillance of rejection or its prevention—in the field of transplantation. Drs. Ayala and Chaudry, in chapter 20, return to the topic of peritonitis, asking whether local, intra-abdominal anticytokine therapy is possible. In chapter 21, Drs. Canty, Boyle and Dellinger from Seattle consider the significance of the antibioticinduced cytokine response. The last contribution, by Drs. Schroder, Staubach, Stuber and Zabel from Kiel (Germany) discuss their experiments in modulating cytokineresponse in “surgical sepsis” with pentoxifylline. And finally, in chapter 23 the editors “conclude” the discussion, emphasizing the current terminology of local and systemic inflammation versus infection. It is hoped that this book will provide the surgeon with a compact source to learn how cytokines apply to his or her practice and what may be their current and future clinical relevance.

Relevance of Molecular Biology for the Abdominal Surgeon

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CHAPTER 2

Relevance of Molecular Biology for the Abdominal Surgeon Philip S. Barie and Steven M. Cohen

Introduction

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ecent advances in cellular and molecular biology, especially the ability to study cell-cell and ligand-receptor interactions, have increased our understanding of the pathophysiology of disease. Important advances have occurred in sepsis and shock, ischemia/reperfusion injury, transplantation immunology, wound healing, atherogenesis, thrombosis and hemostasis, and carcinogenesis. At the same time, the practice of surgery is changing fundamentally. Minimally-invasive abdominal surgery is now routine for many procedures, and developing rapidly for others. This evolution is part of a long-term trend that has seen many procedures become the province of nonsurgeons (e.g., therapeutic endoscopy by gastroenterologists, percutaneous drainage and endovascular prosthetics by interventional radiologists). By extension, it is plausible that the surgery of the future may be a minimally-invasive procedure for gene transfer. The very nature of such procedures makes it likely that nonsurgeons will be very interested in performing such procedures. Molecular biology and clinical medicine are merging (the discipline is already called molecular medicine by some). To continue to provide patient care in an era of gene therapy and biotechnology, the surgeon must understand the principles of molecular biology in order to retain a fundamental understanding of biological processes. This is not a fundamental change in philosophy, but rather it is true to the heritage of surgical education and that has made “surgical basic science” the cornerstone of training and practice for generations. Surgeons who are knowledgeable and prepared will have ample opportunity to become involved in molecular medicine and gene therapy. The surgeon can make a unique contribution to the field. Direct access to diseased tissue provides understanding of the physiologic and anatomic relationships in disease which are essential prerequisites to structural and molecular analysis.1,2 Some diseases, such as oncology, cardiovascular disease, and digestive diseases are areas where specialty “turf ” is becoming anachronistic and the surgeon has evolved to a vital role in a collaborative practice model. Certain disorders are truly surgical diseases, and probably will remain so. Many aspects of infection and the host response; metabolism, nutrition, and wound healing; trauma and burns; organ transplantation; and repair of congenital anomalies: Cytokines and the Abdominal Surgeon, edited by Moshe Schein and Leslie Wise. © 1998 R.G. Landes Company.

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Each are examples where surgical investigation and therapy remains paramount. Because of the morbidity and mortality associated with these disorders, and since no other specialty so often encounters these problems in a clinical setting, it behooves surgeons to pioneer the investigation of these surgical disorders by a detailed molecular analysis which will allow development of more effective therapies including strategies based on gene therapy. Many researchers have argued convincingly that the reason for failure of many of the “promising” therapeutic trials for disease processes such as sepsis, is due to an insufficient understanding of the complexity on a molecular basis.1

Structure and Function of DNA and Chromosomes For the practicing surgeon, knowledge of the molecular mechanisms by which a differentiated cell develops and maintains its specialized function is imperative to a more in-depth understanding of human disease and potential treatment. The surgeon’s understanding of the field of molecular biology must have a fundamental basis. It is imperative to understand how inherited information is encoded within DNA, and how this information is regulated and expressed. A variety of new molecular techniques, such as the polymerase chain reaction (PCR), have simplified the investigative work by identifying genes for sequencing and characterizing individual regions of the chromosome.2 Likewise, recombinant DNA technology has made available a number of naturally occurring substances, such as cytokines, which are being studied extensively as potential pharmacotherapeutic agents due to their ability to modulate a variety of host responses.3 Deoxyribonucleic acid (DNA) is the material that encodes all inherited information in normal eukaryotic cells. In the chromosomes, DNA consists of a double helical structure, which allows for the direct copying or replication of genetic instructions.4 During replication, the intertwined strands of DNA are unwound, separated, and then copied by an enzyme known as DNA polymerase, which uses one strand as a template for the synthesis of its complementary partner. The copying is always in a 5' to 3' direction. The only variable part of the DNA strand is the series of four bases along the deoxyribose-phosphate backbone. The sequence of bases in the gene encodes instructions for synthesis of a specific peptide chain. A certain codon (three nucleotide triplets) denotes which regions of genes are to be translated.5 A gene must contain the instructions about the amino acid chain (protein) to be made (the codon or coding region), and it must also include sequences that regulate transcription of a gene which will control the expression of the protein product and ensure that the protein is produced at the correct time in cellular development and differentiation, and that it is made in the correct tissues and in the right amount. Examples of these gene regulatory sites are promoters, or upstream promoter elements, which are regions of DNA to which RNA polymerases or transcription factors bind and initiate RNA synthesis.6 The central dogma of molecular biology describes the flow of genetic information within the cells from DNA to RNA (transcription), and RNA to protein (translation). The completed protein that is released following translation usually must undergo a considerable amount of posttranslational modification before it becomes functional. These modifications include phosphorylation, glycosylation, and cleavage.7

Human Genome Project

Substantial efforts are underway to characterize the human genome.8 The human genome is believed to harbor up to 100,000 genes, of which about one-half have


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been sampled to date in the form of expressed sequence tags.6,8,9 More than 16,000 human genes have been mapped relative to a framework map that contains about 1000 polymorphic genetic markers.10 Only a small fraction of the total DNA in the human genome (probably less than 10%) consists of coding information for proteins.11 Recombinant deoxyribonucleic acid (DNA) technology and molecular cloning allow the recovery of multiple copies of a segment of DNA, producing large amounts of material for analysis.9 The gene map will unify existing genetic and physical maps with the nucleotide and protein sequence databases in a fashion that should speed the discovery of genes underlying inherited human disease.10 The human genome project will complete the Herculean task of sequencing the entire human genome early in the 21st century. The human genome contains more than 3 billion nucleotides; the structural detail of their organization will be known precisely. Once known, it is planned that the human neurobiology project will define the function of the approximately 70,000 genes that are expressed in the human brain. There are currently at least 68,000 genes in the brain about which little or nothing is known.11 That is just the beginning, and just the brain. Considering what little we know about cancer genetics and how it already has profound implications for patient care, the enormity and potential of molecular medicine is awesome.

Molecular Diagnostic Tests Classical diagnostic tests (from the clinical laboratory of virtually any facility) target many different ions and macromolecules (enzymes, substrates, metabolites, etc.) at various levels (from cellular to the intact organism). In contrast, molecular diagnostic tests essentially target only two moieties, epitomes and nucleic acid sequences.12 An epitope is a unique space/change occupying region within a parent molecule, for binding by a monoclonal antibody. The epitope may occur singly or repetitively within the same molecule. Because epitopes may change conformation and hence antibody specificity, antibodies can be used not only to identify molecules, cell types, or secretory products, but may also define functional regions or even activate the cell. Information about nucleic acid sequences relates to the precise order of the ribose or deoxyribose sugar bases. Such information can be obtained through one of three methodologies: Annealing (hybridization between two strands of DNA and/or RNA; interactions between nucleic acids and recognition enzymes (restriction/hydrolysis enzymes); or nucleic acid sequencing methodologies (which are cumbersome, expensive, and seldom used). Restriction endonucleases are enzymes that occur naturally, mainly in bacteria, and that cleave foreign DNA. These restriction enzymes, of which more than 400 (with 100 different specificities) have been isolated, are in regular use for recombinant technology. Additionally, the insertion of human DNA into bacterial plasmids or bacteriophages is a keystone of recombinant DNA technology.13 Molecular cloning is the isolation of a specific segment of DNA (such as a gene or part of a gene) and the generation of many identical copies, or clones, of that segment of DNA. Because a given segment of DNA cannot replicate itself, the DNA segment is joined to a vector (or carrier) DNA molecule which is able to replicate itself and can replicate the DNA segment that has been joined to it. The DNA segment, with its vector, is a recombinant DNA molecule.5 In order to provide the necessary milieu for DNA replication, recombinant DNA molecules are inserted into host cells, usually bacteria such as E. coli, which become factories to reproduce large quantities of the recombinant DNA molecule.9

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Once a particular DNA sequence is isolated, a set of recombinant DNA molecules is constructed from a source that contains the sequence of interest. This set of clones of complimentary DNA (called a cDNA library) should contain all sequences contained in the original source (genomic DNA or messenger RNA). Once a library is constructed, it is screened to identify the specific clone containing the DNA sequence of interest. The most commonly used screening method of a library is nucleic acid hybridization. Once a gene is cloned and sequenced, dysregulation can be studied by a number of techniques. Genes cloned from infectious pathogens can be used for rapid diagnosis of infection. Tumor-specific genes such as oncogenes can be developed into reagents for identification of tumor cells. Cloned genes can be introduced, deleted, or expressed variably in microorganisms, cells, tissues, or intact animals. Recombinant human proteins are already in use therapeutically (e.g., insulin, erythropoietin), and can be used, via hybridoma techniques, to develop monoclonal antibodies for diagnosis.

Protein Hybridization The first recombinant DNA technology was the Southern blot, which can be used for the detection and characterization of a single gene at a concentration ratio of 1:2x107. High molecular weight genomic DNA is purified and then digested with a specific restriction enzyme.14,15 Each lane of a Southern gel (see below) consists of identical DNA digested with several different enzymes, whereas for comparison purposes several different samples are digested with the same enzyme. The DNA fragments are separated on agarose (a molecular sieving gel), denatured into single strands with NaOH, and transferred (blotted) to an immobilizing membrane (nitrocellulose or nylon). The hybridization probes are either cloned gene fragments, their RNA transcripts, or short sequence-specific synthetic oligonucleotides, labeled radioactively after denaturation. Hybridizations are carried out under exacting conditions of anion concentrations, temperature, and time, to favor reassociation of only complementary DNA. The Southern analysis forms the basis of the restriction fragment length polymorphism (RLFP) analysis. Genetic mutations lead to new, abnormally sized restriction fragments. A point mutation that creates a new restriction site between two others causes the disappearance of the parent restriction fragment and the appearance of two new, smaller fragments. The converse is also true. To perform RLFP analysis, test genomic DNA is digested, Southern blotted, and hybridized. Polymorphisms in restriction fragment length become diagnostic indicators of genetic polymorphism. Although the Southern and RLFP methodologies have been largely supplanted by the polymerase chain reaction, they remain useful for the identification of immunologic diseases, infectious disease diagnosis, tissue typing for transplantation, and innumerable inherited disorders. The Northern blot is a simple test that allows identification of the mRNA transcripts of a single gene.14 An aliquot of RNA is purified from a tissue source, separated and denatured by an agarose gel migration, and blotted on to nitrocellulose. Specific species are hybridized to labeled probes as described above. A control blot is performed to detect a gene that is expressed constitutively, such as !-actin, so as to control for undegraded RNA loaded on to the gel. Abnormalities of mRNA production characteristic of several neurologic and infectious diseases can be identified. Northern blots are limited by the fact that relatively large amounts of RNA are re-


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quired for detection of single gene transcripts. Some tissue samples (i.e., needle biopsies) may be inadequate for the purpose. Weakly expressed genes are difficult to detect. Northern analysis has largely been supplanted by nuclease protection analysis or the reverse transcriptase polymerase chain reaction (RT-PCR). The PCR is a rapid procedure for rapid in vitro enzymatic amplification of a specific segment of DNA.6 The technology relies heavily on the unique thermodynamic properties of AT and CG base pairs. Armed with even limited information about the sequence information of a protein or gene, very short DNA oligonucleotides complimentary to sequences on the ends of opposite DNA strands of the gene of interest are synthesized, incubated with DNA, free deoxyribonucleotide triphosphates, and purified heat-stable DNA polymerase (Taq). Heat denaturation anneals the oligonucleotide primers perfectly to the complementary sequences on singlestranded DNA. Then, Taq catalyzes the synthesis and elongation of both strands of the gene of interest in opposing directions. Repetitive heating and cooling allow the reaction to repeat indefinitely with automated equipment. When sufficient replication has occurred, direct visual identification with ultraviolet light is possible. As with molecular cloning, PCR has spawned a multitude of experiments that were previously impossible. The number of applications of PCR continues to grow, including direct cloning from genomic DNA or cDNA, in vitro mutagenesis and engineering of DNA, genetic fingerprinting of forensic samples, assays for the presence of infectious agents, prenatal diagnosis of genetic diseases, analysis of allelic sequence variations, analysis of RNA transcript structure, genomic footprinting, and direct nucleotide sequencing of genomic DNA and cDNA.16,17 The sensitivity and power of PCR has allowed for its use in medical genetics, cancer detection, organ transplantation, virology, and specific diagnosis of infectious diseases.18 In particular, PCR has revolutionized the approach to the study of cancer. Direct examination of genomic changes that occur during tumor initiation, progression, and metastasis is now possible.8 The main drawback of PCR technology is its extraordinary sensitivity; falsepositives are possible due to heterologous annealing of primers, or laboratory contamination of even minuscule amounts of DNA. The sensitivity of PCR can be applied to high-resolution identification and quantitation of gene expression by incorporation of a “reverse transcription” step early in the reaction (RT-PCR). Reverse transcription is so named because it is the transcription of DNA from an RNA template. A natural process in the life cycle of retroviruses, it is catalyzed by a family of retroviral enzymes known as reverse transcriptases. In RT-PCR, total RNA from a cell or tissue sample is extracted by one of several standard approaches, then reverse-transcribed into cDNA using primer oligonucleotides. Under ideal conditions, a single cDNA molecule is transcribed off of every mRNA molecule so that quantitative analysis by subsequent PCR is possible. The sensitivity of RT-PCR for detection of infinitesimally low levels of normal gene expression allows for the ready detection of upregulation of pathologic gene expression in disease. The RT-PCR-based estimation of cytokine gene expression has become an important diagnostic tool for the monitoring of chronic inflammatory conditions and the response to therapy.19,20 The enzyme-linked immunoabsorbent assay (ELISA) is the most-used diagnostic application for monoclonal antibodies, being a simple, rapid, and relatively inexpensive analysis for almost any antigenic epitope. There is a high degree of reproducibility, and the technology lends itself well to automation. The radioimmunoassay (RIA), which is a more sensitive and specific but also more resource-intensive assay,

8


is essentially an ELISA with a radiolabeled reagent. There are two steps in the performance of ELISA, antigen immobilization and subsequent identification and quantitation. Immobilizing monoclonal antibodies are coated on to polystyrene plates, while any remaining protein-binding sites on the plates are blocked with albumin. Serial dilutions of the test sample are then allowed to react with the antibody on the plate, and compared to a standard curve of highly-purified test antigens developed in parallel. After washing to remove any unbound antigen, a second monoclonal antibody that is usually directed against a different epitope on the test antigen is added. The second antibody is linked covalently to an enzyme, such as peroxidase or alkaline phosphatase, which then catalyzes a colorimetric reaction once its own substrate and reagents are added. The color reaction is thus directly proportional to the amount of enzyme product present. Thus, ELISA was the first molecular test to use products of hybridoma (monoclonal antibodies) and recombinant DNA (purified reagents) technology; these tests are now in the mainstream of the clinical laboratory.12 The Western blot is even more informative than the ELISA, because the target molecules are first separated on the basis of size.21,22 Size separation is accomplished by electrophoresis in polyacrylamide gels after denaturation with heat and sodium dodecyl sulfate (SDS), which places a negative charge on the protein.21 Treated proteins then migrate through an electrified gel purely on the basis of molecular weight. Reduction with 2-mercaptoethanol, which hydrolyzes disulfide bonds, unfolds proteins and facilitates migration. Separation of proteins based on their isoelectric point in a pH-gradient gel, wherein migration ceases at the isoelectric point when the negative charge is neutralized, is also possible. Isoelectric focusing in one direction, combined with denaturation electrophoresis in the opposite direction, provides for a two-dimensional separation that is virtually unique for most proteins. Once the protein is separated, immunologic identification is carried out. Protein is transferred either electrophoretically or osmotically from the separation gel to a nitrocellulose or nylon membrane, which adsorbs protein avidly. After nonspecific protein blocking, a monoclonal antibody or antiserum is introduced that is specific for the protein in question. An enzyme-labeled second antibody that recognizes the first reagent is then introduced, and the target protein band can be visualized. Incorporation of molecular weight markers and test substrate in parallel electrophoretic and transfer steps makes for positive identification of the test protein. However, some protein epitopes cannot be identified by Western blotting because integrity of the specific epitope is dependent on the native protein configuration. Protein disruption by SDS may make antibody recognition impossible. The demanding technology means that Western blotting is usually performed as a confirmatory test after an initial screening ELISA is positive. This sequence of tests is used clinically for testing for the human immunodeficiency virus.

Programmed Cell Death With the possible exception of cancer genetics, programmed cell death (apoptosis) has generated more interest in biological investigation than any other phenomenon. Apoptosis has been implicated in a host of physiologic processes and disease states, and its potential for regulation as a therapeutic strategy is enormous. Apoptosis is central to the regulated death of cells during normal embryonic development. In adults, physiologic cell death figures prominently in cyclically-stimulated or hormone-dependent tissues such as the endometrium, prostate, adrenal, and mammary gland, as well as in “steady-state” turnover in many tissues, such as small bowel


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enterocytes.23 Regulated cell death is fundamental to the development, regulation, and function of the immune system. Cell death in viral infection, the pro-inflammatory host response to infection or major tissue injury, AIDS, neurodegenerative diseases, myocardial ischemia, and a host of malignant tumors. Apoptosis is an innate process by which cells systematically inactivate disassemble, and degrade their own structural and functional components. It can be activated intracellularly by constituitive processes, or activated by extracellular stimuli such as proteins, cytokines, hormones, radiation, oxidative stress, or hypoxia. Control of cell death programs is physiologically stringent, and dysregulated apoptosis may result in excessive, untimely, or inadequate responses. Upregulated apoptosis of neuronal cells may lead to neurodegenerative disease, or severe immunodeficiency syndromes where T-lymphocytes are the target cell. Conversely, suppressed apoptosis may account for chronic viral infections, autoimmune diseases, lympho-proliferative disorders, and neoplastic transformation. From the perspective of the gastrointestinal tract, apoptosis has been implicated in a long list of pathologic entities.24 Apoptosis has been associated with peptic ulcer disease and atrophic gastritis. Numerous hepatocyte diseases have been related to apoptosis, including cholestasis, toxicity from ethanol and other poisons, and viral infections, whereas cholangiocyte-related pathology includes sclerosing cholangitis and primary biliary cirrhosis. Apoptosis has been implicated in the pathogenesis of both acute and chronic pancreatitis, malabsorption syndromes, inflammatory bowel disease, and gastroesophageal reflux disease. Carcinoma of the esophagus, stomach, pancreas, colon, liver, and bile duct have been implicated. Failed apoptosis of sloughed tumor cells may be central to the pathogenesis of metastasis, and for unresponsiveness to antitumor chemotherapy. Apoptosis occurs in distinct stages.23,25 In commitment, the cell becomes irreversibly committed to death after a lethal stimulus has been received. The execution phase is when recognizable structural changes occur. Nuclear chromatin condenses and aggregates as DNA is fragmented progressively. Mitochondrial transmembrane potential is decreased, the electron transport mechanism is uncoupled, and oxygen radical production is increased. The nuclear structure is altered, and mitochondrial function is impaired. In the cytoplasm, protein crosslinking occurs, cytoskeletal filaments aggregate, and the endoplasmic reticulum fuses with the plasma membrane, compromising cellular integrity. The cell becomes spherical, loses contact with its neighbors, shrinks, and projects protruberances known as apoptotic bodies. Phagocytic cells engulf apoptotic cells avidly in the clearance phase. The entire process takes only a few hours to complete. In many ways, apoptosis is controlled by cytokines.26 Two members of the tumor necrosis factor (TNF) family of cell surface signaling molecules, Fas/APO-1/CD95 and the soluble TNF receptor TNFR-1, are potent inducers of apoptosis upon stimulation of their respective ligands, Fas ligand and TNF.27 Fas signaling, which is of critical importance in apoptotic regulation of the immune system, is in turn mediated by known effectors of the death pathway known as interleukin-1b converting enzyme (ICE)-like cysteine proteases. The ICE family proteases are several, including CPP32/Yama/Apopain, which is a mediator of the heat-stress or heat-shock protein response. Proteolysis of several vital cellular proteins, such as those involved in DNA repair, cytoskeletal integrity, and cell signaling, is mediated by ICE-like proteases. Proteolytic cleavage may activate nascent protein functions necessary to execute the death pathway, inactivate countervailing growth signals, and facilitate cellular destruction.23

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There appears to be a critical integration between regulation of apoptosis and neoplastic transformation. Perhaps best-studied is the apoptosis-promoting function of p53, which exerts its growth-suppressing activity by function as a transcription factor. The most intriguing p53-responsive apoptosis-promoting gene product identified thus far is the Bax protein. The Bax protein was identified for its ability to antagonize the apoptosis-suppressive effects of the Bcl-2 oncogene, which was first identified at the chromosomal breakpoint of certain follicular B-cell lymphomas. Bcl-2 is only one of many apoptosis-regulating genes, but it is unique in that it exerts its effects to promote cell survival by blocking apoptosis rather than promoting proliferation, probably through an antioxidant mechanism of its protein product. Cell death or survival may depend on the ratio of Bcl-2 to Bax gene product. Loss of apoptotic potential, by mutation of p53 or abnormal expression of genes such as Bcl-2, is becoming recognized as an important factor in the development of tumors, by permitting the survival of DNA-damaged cells. Bcl-2 is expressed at the base of colonic crypts.24 Expression is common in colonic dysplasia, adenomas, and carcinomas, suggesting that Bcl-2 alterations occur early in colonic carcinogenesis. Interestingly, Bcl-2 is not expressed in the small intestine epithelium whereas Bax is, which may account for the paucity of small intestine adenocarcinomas. Moreover, cyclooxygenase-2 (COX-2), an isoform that suppresses apoptosis when overexpressed, can be suppressed by sulindac, a nonsteroidal antiinflammatory drug. Sulindac therapy leads to regression of adenomatous polyps in patients with familial adenomatous polyposis, making it possible that drug therapy can restore or promote apoptosis in a therapeutically beneficial way.

Genetic Predisposition to Disease: Preparing for the Genetic Revolution In cancer genetics, most individual genes, once mutated, predispose patients to neoplastic transformation rather than acting as the direct cause. Several mutations may be necessary for neoplastic transformation; some may be acquired through life as somatic mutations, whereas others may be inherited, as is the case with a tumor suppressor gene. The National Cancer Institute has embarked on the Cancer Genome Anatomy Project to define all genes relevant to all malignant tumors.28 Methods for identification of persons at risk to develop cancer are an expected result.29 Examinations of several genes have already had profound implications.30,31 In the case of BRCA1 gene mutations, which have been associated with increased susceptibility to breast and ovarian carcinoma,30 such susceptibility will no longer be determined on the basis of family history, which cannot distinguish between sporadic clustering or inherited predisposition. Risk can be determined with precision; what could be more reassuring than the knowledge that one does not carry inherited increased susceptibility to breast or ovarian cancer? On the other hand, what could be more disconcerting than the knowledge that one does? The availability of such genetic testing raises issues of counseling and confidentiality. Counseling is critical for issues of screening, prophylaxis of disease, and even family planning. Confidentiality is critical in an era where the electronic dispersion of information has made security of medical records of concern. With the possibility of a person’s genome on a microchip already being discussed,32 confidentiality is a major issue. Imagine a scenario in which an employer, about to make a long-term employment commitment for a position of great responsibility, decides that the prospective employee’s medical risk is too great to justify the hiring.


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Screening is an integral part of cancer management for the surgeon, and all surgeons involved in oncologic care must be prepared to discuss screening and risk factors with their patients. Referral to a genetic counselor is not always possible, because the number of trained counselors and medical geneticists is inadequate to meet anticipated needs.33 Evidence suggests that without continuing education, the likelihood of misinterpretation of currently-available genetic tests is high.34 Nearly onethird of physicians who received results of a genetic test that detects mutations that cause familial adenomatous polyposis (a known predisposition to colon carcinoma) misinterpreted the test results.34 Equally of concern, only 19% of the patients being tested had received genetic counseling before the test, and only 17% provided written informed consent, implying that many patients were unaware of the discriminatory potential of testing. The elements of patient preparation for genetic testing have been described.35 The personal and family history remain essential, and a physical examination is performed to identify stigmata of already-present disease. The natural history of the suspected disease is reviewed with the patient. The predictive value of the test must be discussed, as well as any other genetic or nongenetic conditions that might influence the onset or severity of the condition. Given that the testing allows the patient and the physician to glimpse the future in the medical sense, the risks, benefits, and limitations of testing must be disclosed. The medical, behavioral, and social implications of positive, negative, and indeterminate tests must be discussed. Available tests and strategies for ongoing surveillance in test-positive subjects should be outlined. Confidentiality is paramount. Persons need to know beforehand to whom (physicians, relatives, insurers, etc.) the results will be disclosed. The logistics of the testing procedure must be disclosed, including cost and expectations for payment. The mechanism for communication of results should be understood. The alternatives to genetic testing and the option of declining to be tested must be disclosed. Written disclosure should be provided and written consent for testing should be obtained. The test itself must be done by a laboratory that is in compliance with Federal Clinical Laboratory Improvement Amendments (1988). Research laboratories must have their results verified by a compliant laboratory before clinical use. Test results are best disclosed in person by a medical professional (e.g., the surgeon) who is knowledgeable about the implications and limitations of testing. The professional who discloses the results should be involved in the testing process in the context of an established relationship with the test subject. The approach to testing must be centered on the individual to be tested. Information must be presented clearly and in a nonjudgmental fashion. The motives behind the request for testing should be understood. Sensitive and empathic support should be provided throughout. How should the surgeon prepare him- or herself for the genetic revolution? Knowledge of basic biological alterations offers the best hope for prevention or therapy.36 Health professionals must make genetics education a priority. The Internet has numerous resources for continuing medical education in medical genetics, and more are becoming available.36

Gene Therapy in Surgery Gene therapy will revolutionize medicine, and surgeons should be part of the revolution. Not only will historically “surgical” diseases be treated, effectively, with

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gene therapy, but surgically-created access to discrete locations may be a critical component of therapy. Although genetic manipulation of germ cells has raised a major ethical debate—witness the recent furor over the possible cloning of human beings— the genetic manipulation of somatic cells for therapeutic benefit is being pursued vigorously. It is also possible that gene therapy may portend major advances in the prophylaxis of disease. Several factors play a role in gene therapy. First and foremost, the appropriate gene must be identified, sequenced, and replicated. The selected gene must be delivered to the target cell, incorporated into the cell, expressed by the cell (the protein encoded by the inserted DNA must be produced), and expression must occur without toxicity to the host. Toxicity to the target cell may be the goal, however, as in the case with gene therapy for cancer. Incorporation may be accomplished by any of several vectors, including bacterial plasmids (for ex vivo production of human proteins by bacteria), or any of several viruses for in vivo therapy, including retroviruses, adeno-associated viruses or parvoviruses, herpesviruses, and most commonly adenoviruses. There are at least 49 serotypes of adenoviruses, of which Types 2 and 5 are most often exploited. The therapeutic gene is incorporated into the viral genome and after administration to the test subject or patient, the adenovirus infects the target cell. An analogy may be drawn to a “Trojan horse”; the vector deposits the new gene and then either becomes moot or self-destructs. The fact that viral vectors are used to deliver genetic material raises relevant issues related to host defense.37 Viral vectors stimulate host defenses, and could destroy the vector before incorporation takes place. For some diseases, investigators are trying to make the vectors “stealthy”, so that tolerance or nonrecognition occurs long enough (a few hours is long enough) for incorporation. In the case of gene therapy for cancer this is less of an issue, because cytotoxicity is usually the goal of therapy. The host is usually already immunosuppressed by virtue of disease or conventional adjuvant therapy, and the gene may need to be expressed only briefly for lethal cytotoxicity to occur. Several strategies for suppression of tumor growth are being studied, and initial results have been promising.38,39 A converting enzyme may be administered along with a pro-drug, to convert the nontoxic precursor to a cytotoxin. The gene in such a strategy is referred to as a “suicide gene”, because the cell is induced to self-destruct. In one notable example, incorporation of the cytosine deaminase gene from E. coli has been administered via adenovirus vector to patients with metastatic colon cancer.40 Oral 5-fluorocytosine, a clinically-available, relatively nontoxic antifungal agent serves as substrate for cytosine deaminase, which converts it to 5-fluorouracil (5-FU). 5-fluorouracil has known efficacy against colon carcinoma and is a mainstay of conventional adjuvant therapy, but its toxicity to normal mucosal cells is high and sometimes dose-limiting. Regional delivery of 5-FU can be accomplished by an infusion port placed into the hepatic artery, but morbidity is high from a major operative procedure, and systemic toxicity can occur. By direct injection of the adenoviral vector into the metastatic lesions, high local drug concentrations are achieved where they are needed, but systemic toxicity is minimized. In the pilot study, preoperative CT-guided adenoviral vector injection directly into metastatic lesions, followed by surgery one week later, demonstrated more CD8+ (T-helper) lymphocytes in treated as compared to untreated lesions, and there was more apoptosis after treatment as assayed by DNA fragmentation.40


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Gene therapy will likely lend itself to many other “surgical” diseases, including cardiovascular disease41,42 and peripheral vascular disease.42-44 Growth-promoting genes may induce the conversion of fibroblasts to cardiac myocytes to improve pump function.41 Gene therapy may stimulate the formation of collateral vessels in areas of ischemic myocardium by introduction of angiogenic growth factors. Coronary collateral flow has been increased after administration of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF).42 Paradoxically, the VEGF pathway has become an important target of cancer researchers who hope to inhibit angiogenesis and render tumors ischemic.45 Other potential strategies of interest to the vascular surgeon include gene therapy of hypercholesterolemia and atherosclerosis, or direct manipulation of disease peripheral vasculature. Already, genetic material can be delivered directly to an experimental lesion using endovascular insertion techniques. Transplant surgeons are interested in gene therapy both from the standpoint of tissue growth or replacement, and from the standpoint of the regulation and control of the immune response.46 Genetic antiinflammatory therapies will also be of intense interest to the surgical intensivist.47

Antisense Oligonucleotides as Potential Gene Therapy So-called “antisense oligonucleotides” may be of critical value and importance as therapy in the future.47-50 In double-stranded DNA, the nucleotide sequence that contains the information to be translated is called the sense strand. Its complimentary strand is thus the antisense strand. The antisense approach uses short oligonucleotides designed to hybridize to a target mRNA, forming a heteroduplex that inactivates the mRNA and brings protein synthesis to a halt. An attraction of this approach is its potential applicability to any gene product, but much development needs to be done. Antisense therapy could be delivered either as synthetic oligonucleotides delivered exogenously (liposomes, naked DNA), or via the more complex endogenous production of antisense RNA transcripts generated by expression vectors transfected into the cell. Multiple points in the protein synthetic sequence could theoretically be targeted. Active development issues include stability, the length of the sequence (15-20 base pairs is the minimun to ensure sufficient specificity), cellular uptake, target specificity, appropriate negative controls, the potential for oligonucleotide:protein interactions, and cost. Antisense technology has already been used to block cytokine production in vivo, and against intercellular adhesion molecules. In the latter case, there is therapeutic potential for transplant immunosuppression as well as the host response to bacterial infection. In oncology, a number of clinical trials have been initiated against molecular targets including p53, and Bcl-2.51

References 1. Tzeng E, Shears LL, Lotze MT, Billiar TR. Gene therapy. Curr Prob Surg 1996; 33:1032-34. 2. Smith MP. Exploring molecular biology. Arch Surg 1995; 130:811-816. 3. Hebert JC, O’Reilly M, Bednar MM. Modifying the host response to injury. Surg Clin North Am 1995; 75:335-349. 4. Watson GD, Crick FHC. Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid. Nature 1974; 248:765. 5. Lodish H, Baltimore D, Berk A et al. Molecular Cell Biology. 3rd ed. New York: Scientific American Books, Inc. 1995:10-594.

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6. Lyerly HK, Sullenger BA. Molecular biology in surgery. In: Sabiston DC, Lyerly HK, eds. The Biological Basis of Modern Surgical Practice. 10th ed. Philadelphia: WB Saunders, 1997:16-35. 7. Watson JD, Hopkins NH, Roberts JW, Steitz JA, Weiner AM. Molecular Biology of the Gene. 4th ed. Menlo Park, CA: Benjamin/Cummings Publishing Co., 1987:282-309. 8. Hall CC, Herring JA, Hall TJ. Molecular oncology and the surgeon. Am Surg 1995; 61:156-160. 9. Shore EM, Kaplan FS. Molecular biology for the clinician. Part II: Tools of molecular biology. Clin Orthop Rel Res 1995; 320:247-278. 10. Schuler GD, Boguski MS, Stewart EA, Stein LD et al. A gene map of the human genome. Science 1996; 274:540-546. 11. Rosenberg RN. Molecular neurogenetics. The genome is settling the issue. JAMA 1997; 278:1282-1283. 12. Hardy KJ, Young HA, Lagoo AS. Molecular diagnostics. Immunol Allergy Clin North Am 1994; 14:199-223. 13. Rosenthal N. Tools of the trade. Recombinant DNA. New Engl J Med 1994; 331:599-602. 14. Kroczek RA. Southern and Northern analysis. J Chromatogr 1993; 618:133-145. 15. Borst M, Miller DM. DNA isolation and Southern analysis: A clinician’s view. Am J Med Sci 1990; 299:356-360. 16. Ausubel FM, Brent R, Kingston RE et al. The polymerase chain reaction. In: Current Protocols in Molecular Biology. Massachusetts: John Wiley & Sons, Inc. 1997; (suppl)37:15.1.1-15.1.5. 17. Abbas AK, Lichtman AH, Pober JS. Cellular and Molecular Immunology. Philadelphia, PA: WB Saunders Company, 1994:56-72,109-111. 18. Mariani BD, Martin DS, Levine MJ et al. Polymerase chain reaction detection of bacterial infection in total knee arthroplasty. 1996; 331:11-22. 19. Lagoo-Deenadalayan S, Lagoo S, Barber WH et al. A standardized approach to PCRbased semiquantitation of multiple cytokine gene transcripts from small cell samples. Lymphokine Cytokine Res 1993; 12:59-66. 20. Dallman MJ, Montgomery RA, Larsen CP et al. Cytokine gene expression: Analysis using northern blotting, polymerase chain reaction, and in situ hybridization. Immunol Rev 1991; 119:163-179. 21. Burnette WN. Western blotting. Electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 1981; 112:195-208. 22. Fido RJ, Tatham AS, Shewry PR. Western blotting analysis. Methods Molec Biol 1995; 49:423-437. 23. Webb SJ, Harrison DJ, Wyllie AH. Apoptosis: An overview of the process and its relevance to disease. In: Kaufmann SH, ed. Apoptosis. Pharmacological Implications and Therapeutic Opportunities. Adv Pharmacol 1997; 41:1-34. 24. Que F, Gores GJ. Apoptosis and the gastrointestinal system. In: Kaufmann SH, ed. Apoptosis. Pharmacological Implications and Therapeutic Opportunities. Adv Pharmacol 1997; 41:409-428. 25. Allen RT, Hunter WJ III, Agrawal DK. Morphological and biochemical characterization and analysis of apoptosis. J Pharmacol Toxicol Methods 1997; 37:215-228. 26. May WS Jr. Control of apoptosis by cytokines. In: Kaufmann SH, ed. Apoptosis. Pharmacological Implications and Therapeutic Opportunities. Adv Pharmacol 1997; 41:219-246.


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27. Eischen CM, Leibson PJ. The Fas pathway in apoptosis. In: Kaufmann SH, ed. Apoptosis. Pharmacological implications and therapeutic opportunities. Adv Pharmacol 1997; 41:107-132. 28. Strausberg RL, Dahl CA, Kuaisner RD. New opportunities for uncovering the molecular basis of cancer. Nat Genet 1997; 15:415-416. 29. Pyeritz RE. Family history and genetic risk factors. Forward to the future. JAMA 1997; 278:1284-1285. 30. Shatuck-Eidens D, Oliphant A, McClure M et al. BRCA1 sequence analysis in women at high risk for susceptibility mutations: Risk factor analysis and implications for genetic testing. JAMA 1997; 278:1242-1250. 31. Gronberg H, Isaacs SD, Smith JR et al. Characteristics of prostate cancer in families potentially linked to the heredity prostate cancer 1 (HPC1) locus. JAMA 1997; 278:1251-1255. 32. DeRisi J, Penland L, Brown PO et al. Use of a cDNA micro array to analyze gene expression patterns in human cancer. Nat Genet 1996; 14:457-460. 33. Stephenson J. As discoveries unfold, a new urgency to bring genetic literacy to physicians. JAMA 1997;278:1251-1255. 34. Predisposition genetic testing for late-onset disorders in adults. A position paper of the National Society of Genetic Counselors. JAMA 1997; 278:1217-1220. 35. Collins FS. Preparing health professionals for the genetic revolution. JAMA 1997; 278:1285-1286. 36. Sikorski R, Peters R. Genomic medicine. Internet resources for medical genetics. JAMA 1997; 278:1212-1213. 37. Wolff G, Worgall S, vanRooijen N et al. Enhancement of in vivo adenovirus-mediated gene transfer and expression by prior depletion of tissue macrophages in the target organ. J Virol 1997; 71:624-629. 38. Lairmore T, Norton JA. Advances in molecular genetics. Am J Surg 1997; 173:37-41. 39. Evoy D, Hirschowitz E, Naama HA et al. In vivo adenoviral-mediated gene transfer in the treatment of pancreatic cancer. J Surg Res 1997; 69:226-231. 40. Crystal RG, Hirschowitz E, Lieberman M et al. Phase I study of direct administration of a replication-deficient adenovirus vector containing the E. coli cytosine deaminase gene in metastatic colon carcinoma of the liver in association with the oral administration of the pro-drug 5-fluorocytosine. Hum Gene Ther 1997; 8:985-1001. 41. Tam SKC, Gu W, Nadal-Ginard B et al. Molecular cardiomyoplasty: Potential cardiac gene therapy for chronic heart failure. J Thorac Cardiovasc Surg 1995; 109:918-924. 42. Rowland RT, Cleveland JC Jr, Meng X et al. Potential gene therapy strategies in the treatment of cardiovascular disease. Ann Thorac Surg 1995; 60:721-728. 43. Clowes AW. Vascular gene therapy in the 21st century. Thromb Hemostasis 1997; 78:605-610. 44. Schwartz LB, Moawad J. Gene therapy for vascular disease. Ann Vasc Surg 1997; 11:189-199. 45. Harris AL. Antiangiogenesis for cancer therapy. Lancet 1997; 349:13-15. 46. Knechtle SJ. Gene therapy and transplantation-a brief review. Transpl Proc 1996; 28(Suppl 1):19-23. 47. Liu M, Slutsky AS. Anti-inflammatory therapies: Application of molecular biology techniques in intensive care medicine. Intensive Care Med 1997; 23:718-731. 48. Aoki M, Morishita R, Higaki J et al. In vivo transfer of antisense oligonucleotides in to the myocardium using HVJ-liposome method. Biochem Biophys Res Comm 1997; 231:540-545.

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49. Branch A. A hitchhiker’s guide to antisense and nonantisense biochemical pathways. Hepatology 1996; 24:1517-1529. 50. Crooke ST. Advances in understanding the pharmacological properties of antisense oligonucleotides. Adv Pharmacol 1997; 40:1-49.51. 51. Ho PT, Parkinson DR. Antisense oligonucleotides as therapeutics for malignant diseases. Semin Oncol 1997; 24:187-202.

Cytokine Response in Abdominal Surgery

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CHAPTER 3

Cytokine Response in Abdominal Surgery Edward Lin, Steve E. Calvano, and Stephen F. Lowry

Introduction

A

side from cutaneous procedures, operations on the abdominal viscera remain one of the most frequently performed elective surgeries in the United States. While remarkable perioperative and technical advances have been made in the management of patients with abdominal pathology, the significance of circulating as well as localized mediator release leading to tissue inflammation in abdominal surgery remains relatively unknown. Classically, the stress response comprises activation of the hypothalamic-pituitary-adrenal axis, the acute phase response and the autonomic nervous system.1 Each such response functions, at least acutely, to restore homeostasis to the injured patient. Contemporary investigations of the injury response, mediated by inflammatory molecules referred to as cytokines, have identified their role as a critical mechanism(s) that induce many of the metabolic and physiologic derangements characteristic of surgical or traumatic injury. Cytokines are produced by diverse cell populations and are capable of exerting multiple (pleiotropic) proinflammatory or anti-inflammatory influences.2 These are essential components of the inflammatory response mounted against local injuries and infections (Table 3.1). These responses may be elicited locally via autocrine or paracrine activities or systemically in an endocrine fashion. Our current understanding related to the pathophysiology of these inflammatory mediators have been largely derived from the study of patients with endotoxemia and/or sepsis. The cytokine response to infections or injury is qualitatively similar, but generally magnified in comparison to controlled, elective surgery.3 The clinical manifestation of presumably excessive proinflammatory cytokine activation is often referred to as the systemic inflammatory response syndrome (SIRS).4 Such a response necessitates an intact immunologically responsive host and is not, by definition, obligatorily the result of an infectious process. In clinical practice, the criteria for SIRS, which reflects abnormalities in leukocyte count, respiratory rate, heart rate and body temperature, might also be anticipated in many patients undergoing abdominal surgery. Dysregulation of this normal inflammatory cytokine response, as might occur with secondary traumatic or infectious insults, may augment the state of inflammation leading to shock, tissue injury, and multiple organ failure (MOF).1 Certainly, Cytokines and the Abdominal Surgeon, edited by Moshe Schein and Leslie Wise. © 1998 R.G. Landes Company.

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Table 3.1. Catalog of selected cytokines released during controlled surgical injury and their potential beneficial effects TNF-!

∀ PMN release from bone marrow ∀ PMN activation, migration to injured site, degranulation

and superoxide production PMN cytotoxicity against mycotic infections differentiation (activation) of M# M# antiviral/antiparasite activities hepatocyte acute phase reactant (APR) production through IL-6 induction ∀ wound healing/remodeling ∀ endothelial procoagulant activity and leukocyte adhesion ∀ vascular endothelial permeability ∀ neovascularization in wounds ∀ collagen synthesis/fibroblast proliferation ∀ osteoclast activity in bone healing ∀ T-lymphocyte activation and proliferation ∀ PMN release from bone marrow ∀ PMN migration to injured site ∀ differentiation (activation) of M# ∀ granulocyte/macrophage colony-stimulating factor (GM-CSF) ∀ functional PMN restoration ∃ pain perception ∀ %-endorphin release ∀ brain opiate-like receptors ∀ hepatocyte acute phase reactant (APR) production through IL-6 induction ∀ wound healing/remodeling ∀ osteoclast activity in bone healing ∀ overall immunocompetence ∀ lymphokine-activated killers (LAK) production ∀ cytotoxic T-lymphocyte proliferation ∀ reticuloendothelial system (RES) activity ∀ gut barrier immunity ∀ fibroblast antiviral activity ∀ lymphocyte differentiation ∀ B-lymphocyte immunoglobulin production ∀ acute phase reactant (APR) production (e.g., C-reactive protein, fibrinogen, amyloid A, !-1-antitrypsin, haptoglobin) ∀ prostaglandin production ? wound healing properties ∀ chemotaxis of PMN, lymphocytes, M#s to sites of injury and inflammation ∃ cytokine synthesis by lymphocytes and M#s Modulates inflammatory activities of TNF-!, IL-1, IL-6, IL-8, INF-& ∀ ∀ ∀ ∀

IL-1

IL-2

IL-6

IL-8 IL-10

continued...


19

(continued..)

IL-12

IFN-&

∀ ∀ ∀ ∀ ∀ ∀ ∀ ∀ ∀ ∀

Stimulates CD4+ and CD8+ T cells lymphocyte and NK cell proliferation B-lymphocyte immunoglobulin production hematopoiesis IL-2 and IFN-& production M# and PMN activation against invading organisms (including viral) expression of MHC class I and II surface antigens and tumor-associated antigens M# oxidative and cytotoxic activity overall lymphocyte proliferation B-lymphocyte immunoglobulin production IL-1 and TNF-! activity

GM-CSF ∀ myeloproliferation (M#s, PMNs, eosinophils) Partial stimulation of megakaryocyte progenitors ∀ chemotaxis of PMNs and M#s ∀ cytokine production by M#s

the clinical implications of unregulated proinflammatory mediator activities cannot be ignored, as mortalities from MOF in surgical intensive care units remain high and resistant to improvement based upon currently available therapies.2 A proper perspective of the immunobiologic sequelae of cytokine response arising from injury or inflammation may have important clinical applications, particularly in the approach towards the patient undergoing complex abdominal surgery. The catalogue of known cytokines presently approximate 30, and is steadily expanding.3 This chapter will highlight the cytokines that have been more extensively investigated as well as address their potential clinical relevance to the acute response and outcome in major abdominal surgeries.

Injury and Principal Cytokine Responses Tumor Necrosis Factor-! The inflammatory response to severe cross-sectional tissue injury and/or infectious agents evokes a complex cascade of proinflammatory cytokines. Of these, tumor necrosis factor (TNF-!) and interleukin-1 (IL-1) appear to be the earliest and perhaps most potent mediators of the subsequent host response.3 The release of TNF-! in response to acute injury is both rapid and short-lived. Experiments simulating an acute inflammatory response by means of endotoxin challenge have invariably demonstrated a monophasic TNF appearance curve, peaking at approximately 90 minutes.4 This is followed by undetectable levels of bioactive protein within 4 hours (Fig. 3.1). Despite a circulating half-life of less than 20 minutes, the brief appearance of TNF-! seems sufficient to induce the characteristic metabolic and severe hemodynamic changes.9 In addition, the signaling for both secondary cytokines distal in the cascade as well as of intracellular toxins appears to occur within this time frame. This brief appearance of TNF-! and of absent biologic activity over subsequent periods suggests the presence of effective endogenous modulators, which might avert further deleterious consequences from excessive TNF-!

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Fig. 3.1. Representative circulating TNF-! release in normal adult volunteers upon brief lipopolysaccharide (LPS, endotoxin) challenge at time = 0. A rapid peak is reached at 90 minutes, followed by undetectable levels at 4 hours after challenge.

500

450

400

350

300

250 TNF (pg/mL)

200

150

100

50

0 0

LPS

0.5

1

1.5

2

2.5

3

3.5

4

5

6

7

8

TIME (hour)

activity. Indeed, this has proven to be the case, as several natural antagonist mechanisms to TNF-! production and/or activity have been identified.5 From experimental models of endotoxemia and in severe sepsis, endogenous inhibitors in the form of cleaved extracellular domains of the transmembrane TNF-! receptors (soluble TNF receptors, sTNFR) are readily detectable in the circulation.6 These receptors may serve a protective role by competitively sequestering excess circulating TNF, but are probably only capable of doing so against low levels of TNF-! activity and for relatively brief periods.7,8 The sources of TNF-! synthesis include monocytes/macrophages (M) and T cells, all of which are abundant within the peritoneum and splanchnic tissues9 (Table 3.2). Moreover, with hepatic Kupffer cells representing the single largest concentrated population of M#s in the body,10 peritoneal injury, liver surgery and procedures upon the splanchnic viscera may have profound influences on the generation of inflammatory mediators and homeostatic responses such as acute-phase protein production (Fig. 3.2).

Interleukin-1 TNF-! also induces the biosynthesis and release of interleukin-1 (IL-1) within Ms and endothelial cells.13 The potency and effects of IL-1 reflect that of TNF-! and they exert several similar physiologic derangements.11 At high doses, both IL-1 and TNF-! induce a state of hemodynamic decompensation reminiscent of septic shock.12 The circulating half-life of IL-1 is less than 10 minutes, a fact which makes its detectability in acute injury or illness less likely than that of TNF-!.13 Among its multiple effects, IL-1 induces the classic inflammatory febrile response to injury by stimulating local prostaglandin activity in the anterior hypothalamus. It also serves as a mediator for attenuating pain perception from surgery by promoting the release of endorphins and increasing the number of central opiate-like receptors.14


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Table 3.2. Principal sources of selected cytokines TNF-! IL-1

IL-2 IL-6

IL-8

IL-10 IL-12

INF-&

GM-CSF

M# T cells M# B and T cells NK cells Endothelial cells Epithelial cells Keratinocytes Fibroblasts Dendritic cells TH1 cells T cells M# Fibroblasts Endothelial cells M# Endothelial cells T cells Platelet B cells TH2 cells M# PMN Keratinocytes Dendritic cells TH1 cells NK cells M# T cells Fibroblasts Endothelial cells Stromal cells

Endogenous inhibitors of IL-1, known as IL-1 receptor antagonists (IL-1ra), are also released in response to sepsis or endotoxemia.13,14 However, to date, the administration of exogenous sTNFR or IL-1ra have not resulted in any appreciable outcome improvements in clinical sepsis. Distal cytokine mediators, released as part of the inflammatory cascade initiated by TNF-! and IL-1 include IL-2, IL-4, IL-6, IL-8, granulocyte-macrophage colonystimulating factor (GM-CSF) and interferon (IFN).14

Interleukin-2 Although necessary as an inflammatory mediator in promoting immunoglobulin activity and gut barrier integrity, IL-2 is not detected in acute injury.2 Indeed, IL-2 secretion by lymphocytes is impaired following acute injury and several disease states, notably cancer and acquired immunodeficiency syndrome (AIDS).15,16

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Fig. 3.2. Hypothetical schema for the hepatic acute phase response following injury. Sources of stress and subsequent antigen/endotoxin release may be derived from the splanchnic circulation or from other injury foci (e.g., traumatic, tumor load). The antigen/endotoxin enters the portal circulation and is cleared by hepatic Kupffer cells (M#). Some antigen/endotoxin, particularly when in excess or in liver failure, may escape clearance and enter the systemic circulation. The activated M# is capable of inducing cytokine release that serves as signals for hepatocyte synthesis of acute phase proteins (e.g., haptoglobin, C-reactive protein, ceruloplasmin, complement factors, fibrinogen, -1-antitrypsin, 2-macroglobulin). However, cytokines may also “spill” into the systemic circulation and contribute to the systemic inflammatory response.

Perioperative hemorrhage and transfusions have also been demonstrated to reduce IL-2 production.17,18 Attenuated IL-2 expression following injury partially defines the transient immunocompromised state of the surgical patient. The clinical significance of this depressed IL-2 activity remains unclear. Nevertheless, a nadir in gut barrier IL-2 activity resulting from surgery is hypothesized to predispose the patient to enteric organism activation of the inflammatory cytokine cascade in the gut, with potential inflammation systemically.14 Recent evidence has demonstrated accelerated lymphocyte programmed cell death (apoptosis), in association with diminished IL-2 activity, mediated by the proapoptotic Fas/CD95 cell receptor in the early postoperative period.19 The combined diminution of lymphocyte survival and IL-2 activity may further contribute to the immunocompromised phenotype of the surgical patient. Other studies have demonstrated that following surgical stress, a lymphocyte population density shift from type-1 T-helper cells (TH1, cell-mediated immune re-


23

Fig. 3.3. Specific immunity related to the magnitude of injury as depicted by cholecystectomy, laparoscopically (lesser magnitude) vs. open cholecystectomy (greater magnitude). A type-1 T-helper cell response (TH1) is favored in lesser injuries, with intact cell-mediated immunity against microbial infections. A TH1 response can be activated by the cytokines IFN-&, TNF and IL-1, but IL-12 appears to be the most potent stimulant. A shift toward type-2 T-helper cell response (TH2) is associated with greater injuries and is predominantly immunosuppressive. By nature of its antibody-mediated immunity, it is ineffective against microbial invasions. TH2 cells activate B-lymphocyte IgG4 and IgE production, which are poor complement activators and among the least efficient opsonizing antibodies. Among the cytokines capable of inducing a TH2 response, IL-4 appears to be the most potent. Although not a cytokine, glucocorticoids are potent upregulators of IL-4 production and are effective stimulants of TH2 immunity, explaining in part the immunosuppressive effects of this agent. Although predominantly immunosuppressive, TH2 response to injury is necessary in regulating the excessive inflammatory response mediated by such cytokines as TNF and IL-1. (Concept derived from references 17, 21, 23, 25).

sponse including IL-2, IL-12 and interferon-! production) to type-2 T-helper cells (TH2, antibody-mediated immune response including IL-4, IL-6, IL-10, IL-13 production) is observed.20 This population shift may partially explain the diminished IL-2 activity seen following surgery. Furthermore, the predominant effects of TH2 response are immunosuppressive, which accentuate the risks for postoperative infections17 (Fig. 3.3).

Interleukin-4 The importance of IL-4 is steadily gaining recognition. This glycoprotein molecule is produced by activated TH2 cells with diverse biologic effects on hematopietic cells.21 Of particular importance in its role in antibody-mediated immunity is the capacity to enhance M MHC class II (HLA-DR and HLA-DP) expression and adhesion molecules, making them efficient antigen-presenting cells.22 As a potent antiinflammatory cytokine, it can downregulate several functions associated with activated human Ms, namely the effects of IL-1, TNF-!, IL-6, IL-8, and superoxide production. However, these antiinflammatory effects of IL-4 are not seen with resting monocytes.27 The importance of this cytokine is therefore in its capacity to downregulate the response of inflammatory M’s that are exposed to stimuli

24


such as bacterial endotoxin or inflammatory cytokines (e.g., IL-1 or TNF-!). Several lines of experiments have demonstrated that IL-4 is a potent inducer of programmed cell death in inflammatory Ms, but this effect can be abrogated by IFN.23 Indeed, IL-4 and IFN have been extensively demonstrated to antagonize the effects of each other on B cells.28 IL-4 also appears to increase M susceptibility to the antiinflammatory effects of glucocorticoids.27 Although studied to a lesser extent, IL-13 has been found to share several important properties with IL-4.26

Interleukin-6 There is evidence to suggest that interleukin-6 (IL-6) exerts both pro-inflammatory and anti-inflammatory influences.24 The high frequency for detection of circulating IL-6 during acute injury or stress and the correlation of such levels to surgery made it an indicator of systemic inflammatory response as well as a predictor of surgical morbidity.25 TNF-! and IL-1 are major inducers of IL-6, which can be expressed from virtually all cell types including the intestines. Upon injury, IL-6 levels in the circulation can peak between 4 to 6 hours and persist for as long as 10 days.29 Clinically and experimentally, IL-6 levels appear to be roughly proportional to the extent of tissue injury related to surgery rather than the duration of the surgical procedure itself.26,27 IL-6 is the primary mediator of the hepatic acute phase protein response during injury.31,28,29 Furthermore, it induces polymorphonuclear cell (PMN) activation during injury and inflammation.30 However, IL-6 also delays the phagocytic disposal of senescent or dysfunctional PMNs during injury, thereby potentially perpetuating the inflammatory response.31 The persistence of activated PMNs following surgical injury can also have distant contributions to cardiovascular and pulmonary compromise.29 IL-6 exhibits antiinflammatory properties via attenuation of TNF and IL-1 activity and promotion of soluble TNFR (sTNFR) and IL-1 receptor antagonist (IL-1ra) release.32 However, prolonged and persistent expression of IL-6 has also been associated with immunosuppression and postoperative infectious morbidity.

Interleukin-8 The appearance of interleukin-8 (IL-8) activity is temporally associated with IL-6 following injury and has been proposed as an additional biomarker for the risk of MOF following injury.33 Like IL-6, IL-8 does not produce the hemodynamic instability characteristic of TNF-! and IL-1, but rather serves as a PMN activator and a potent chemoattractant.34 Hence, IL-8 is being increasingly established as a major contributor to organ injury such as acute lung injury not infrequently seen following major surgeries.

Interleukin-10 Interleukin-10 (IL-10) serves as an important endogenous regulatory protein during the inflammatory response. The activity of this cytokine is identified with modulation of TNF-! activity.35 Its appearance in the circulation during endotoxemia, for example, follows that of TNF-!. Supporting experiments have demonstrated that deletion of IL-10 during endotoxemia increases monocyte TNF-! production, but restitution of IL-10 reduces TNF-!.36 IL-10 may also have additional protective roles following injury-induced inflammation by promoting IL-1ra and sTNFR production. In animal experiments, the sustained systemic production of IL-10 during septic peritonitis appears to modulate the systemic inflammatory response. Murine ex-


25

periments have demonstrated rapid induction of IL-10 messenger RNA (mRNA) activity following cecal ligation and puncture (CLP).37 Blocking this activity with anti-IL-10 enhanced mortality following CLP. However, this immunomodulatory effect may also abrogate the necessary proinflammatory response necessary for local clearance of invading organisms.

Interleukin-12 The role of interleukin-12 (IL-12) in the immune response to injury and inflammatory events has gained significant interest. Its capacity to promote the differentiation of TH1 cells and the production of IFN makes it a pivotal molecule in cell-mediated immunity following injury or infection (bacterial, viral and parasitic).38 IL-12 is also implicated in preventing programmed cell death (apoptosis) in selected T-lymphocyte populations following their activation.39

Interferon (IFN) Much of IL-12 biology is mediated through the production and activities of interferon (IFN).40 Stimulated human T helper (TH) cells, through bacterial antigens, IL-2 and IL-12 induce IFN. Upon release from activated T cells, IFN is detectable in vivo within 6 hours following stimulation and has a half-life of approximately 30 minutes. IFN levels peak at 48 to 72 hours and may persist for 7 to 8 days after surgery.14,17 Surgical wounds also demonstrate the presence of IFN production 5 to 7 days following injury.41 IFN has important roles in activating circulating and tissue M#s. Its activation of peritoneal Ms and Kupffer cells has potential immunologic implications in abdominal surgery.42 Furthermore, alveolar M# activation mediated by IFN may be synergistic with IL-8 in the induction of acute lung injury following major surgery.23,25

Clinical Considerations The abdominal surgical patient often presents with a diverse array of preoperative disease states that may influence the postoperative inflammatory response. Such preoperative conditions may include malnutrition, cancer, sepsis, bowel obstruction, peritonitis, critical illness and an element of acquired or induced immunodeficiency. Surgically induced factors that may influence the postoperative inflammation include the extent of cross-sectional tissue injury/resection, wound size, anesthetic regimen, and hemorrhage. In essence, patients with abdominal pathology may exhibit preexisting activation of cytokine expression. Consequently, an interpretation of the postoperative inflammatory response must account for these factors as well as the magnitude of trauma incurred during surgery.

Exploratory Laparotomy Elective exploratory laparotomy would seemingly represent a model system by which to judge the inflammatory response. Murine experiments have demonstrated an impairment in peritoneal M microbicidal activity and antigen presentation following laparotomy.47 However, recovery of normal function in this M population normally occurs on postoperative day 3. This recovery can be accelerated with IFNadministration although such treatment may also augment local inflammatory responses mediated by TNF and IL-1. The local inflammatory response mediated by these M populations through IL-2 and IL-6 activity is also markedly diminished following laparotomy.43

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The impaired immunocyte and cytokine response following laparotomy in some patients is purported to increase postoperative infectious complications. Systemically, IL-6 levels are found to be elevated following surgical stress, although a clear association with the magnitude of surgical therapy have yet to be established. It has been suggested that the impaired antigen-presentation ability observed in circulating Ms following major abdominal surgery may be mediated, in part, by IL-10 induced downregulation of major histocompatibility II (MHC-II) expression on these cell populations.44

Length of Surgery and Injury Magnitude The correlation between inflammatory cytokine levels, after major abdominal procedures (e.g., hepatic, gastric or pancreatic resections), with the duration of surgery further demonstrate the complexity of this response. Several experimental and clinical studies imply that the type of surgery or the extent of tissue injury are the primary determinants of cytokine responses and that the crossover of these inflammatory mediators into the systemic circulation portends the adverse outcome in certain patients.45 Utilizing IL-6 levels as a potential marker of cytokine responses to elective abdominal surgery, the peak levels are highest in aortic surgery immediately following aortic cross-clamping.46 Levels of IL-6 are lower in colorectal surgery of similar duration.31 Open cholecystectomy had the lowest levels of cytokine activity among the procedures evaluated. In a rat model comparing sham laparotomy to laparotomy plus splanchnic artery occlusion, significant increases in TNF and IL-6 activity were observed in the latter group.32 These attest to the greater contribution of tissue injury to the inflammatory cytokine response than the actual duration of surgery. Cytokine expression following tissue injury must also account for the acuity of this response. While the degree of tissue injury correlates with elevated cytokine levels, the slope of the appearance of cytokine activity during the early postoperative phase also reflects the degree of surgical injury.32 In surgeries of similar duration, an acute and “excessive” rise in IL-1 and IL-6 levels rather than gradual elevations, have been predictive of postoperative complications (e.g., MOF, ARDS) and poorer clinical outcome.30,47

Intestinal Surgery The intestines serve as major sources of inflammatory cytokine production, particularly in response to injurious stimuli or stresses.48 While this inflammatory cytokine response is probably essential for local homeostasis, excessive production can spill into the systemic circulation as demonstrated by hepatic vein cannulation in healthy human subjects.49 The concept of gut barrier dysfunction with resultant bacterial translocation has been widely promoted as a mechanism for induction of cytokine activity.50,51 Only limited data exists to address this concept in humans. In a classic study,52 mesenteric lymph node cultures of patients with bowel obstruction demonstrated positive bacterial cultures in approximately 60% of patients, while only 4% of nonobstructed patients had such findings. Similarly, patients with inflammatory bowel disease (IBD) had higher incidence of bacterial translocation and local cytokine response than patients without IBD.53,54 Should bacterial translocation or regional antigen stimulation exist in a surgical patient, this might serve as a mechanism for enhancing cytokine activity during the early postoperative phase. Unfortunately, necessary studies which


27

integrate translocation, local and systemic mediator responses, and clinical outcome have not been conducted. Brief periods of splanchnic shock and/or hypoperfusion followed by reperfusion might also disrupt normal intestinal barrier function.56 This has implications for procedures where cross-clamping of arterial vessels and splanchnic/organ hypoperfusion may accentuate the local or systemic inflammatory cytokine response. Simple traction of the bowel during surgery has been demonstrated to increase endotoxin and microbial permeability through the gut, leading to rises in IL-6 activities.55 This strongly suggests that extensive visceral manipulation might also contribute to the exaggerated local and systemic cytokine response in abdominal surgery.

Hepatobiliary Surgery The liver Kupffer cells are essential for the clearance of endotoxins derived from the intestinal tract, as well as for the neutralization and/or clearance of inflammatory cytokine mediators.56 Perioperative hepatic impairment may contribute to the excessive systemic inflammatory response and attenuate the acute phase protein response following injuries such as burns, trauma, circulatory shock, anesthesia and surgery.57,58 Obstructive jaundice or liver resection can further impair Kupffer cell function. Bile serves to transport immunoglobulin A (IgA), which binds gut-derived bacterial products for excretion into the feces.59 Bile acids are also capable of neutralizing endotoxin. Hepatic dysfunction preoperatively or from hepatic resections may impair normal bile secretion, which has been associated with increased intestinal endotoxin translocation.60 This may permit endotoxin and inflammatory mediators to bypass the regulatory mechanisms of the liver into the systemic circulation. Indeed, patients undergoing hepatectomy have elevated systemic endotoxin levels compared to their preoperative states.61 The clinical implications of such findings have been suggested, but not yet demonstrated, to correlate with postoperative morbidity or mortality. Surgical injury to the hepatic parenchyma also induces local release, as well as systemic elevations of TNF-! and IL-6.61,62 The local release of inflammatory mediators such as IL-1 may also inhibit hepatocyte proliferation, an effect that can be partially abrogated with the administration of a specific IL-1 antagonist (IL-1ra).62 Experimental evidence has further demonstrated the role of Fas/CD95 ligand (FasL) in promoting hepatocyte apoptosis during systemic inflammatory response.63 Clinically, this may explain a mechanism for liver failure commonly observed after hepatic resections as well as in sepsis.64 Furthermore, the systemic spillage of inflammatory cytokines may explain the lung injury, cardiac complications and other organ dysfunction often observed following major hepatic resections or hepatic cryoablation.65

Nutritional Considerations In addition to maintaining the metabolic and physiologic requirements of the surgical patient, adequate nutrition is also essential for immune competence. Malnutrition is prevalent among patients with abdominal pathology such as cancer, obstruction, and peritonitis. In these patients, a major cause of immune deficit might be an impairment of the normal intestinal barrier.56 Hypothetically, this increases the susceptibility to bacterial and endotoxin translocation and potential activation of local inflammatory mediator activity. Local or systemic release of proinflammatory cytokines may have both acute and indolent sequelae, such as immune dysfunction,

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increased metabolic demands, and cachexia.56 Indeed, studies have demonstrated that bowel rest and antecedent total parenteral nutrition can exaggerate the inflammatory cytokine response to any subsequent endotoxin challenge.66 Clinically, these preexisting conditions have the potential to adversely affect the outcome of patients undergoing any major surgery that might serve as an additional trigger for cytokine release. It is hypothesized that efforts to maintain or restore intestinal barrier function might minimize further metabolic derangements, restore local immunity and thereby serve to optimize surgical outcomes.67 It has been demonstrated that some forms of cachexia may result from prolonged activity of TNF-!, IL-1 and IL-6, and these mediators can promote peripheral muscle protein mobilization for splanchnic and hepatic use.68 Although cytokine activity may not be either predominant or uniform in this effect, muscle wasting in malnourished surgical patients predisposes them to morbidities such as wound complications, poor healing, weakness, and delayed weaning from ventilator support. The normal inflammatory response can also be markedly altered or exaggerated as a result of infections, surgical stress or injury. In the worst scenario, it may predispose the patient to systemic organ injury (e.g., liver, lung, kidney) and increase late mortality.73 The potential for nutritional modulation of the inflammatory response is controversial. Several purported immunonutritional agents such as glutamine, arginine, omega-3 fatty acids, and fish oil are under intense investigation.69-71 While the capacity of these agents to minimize postoperative morbidities and modulate the cytokine response are reported, these agents are not without adverse effects. Furthermore, clinical outcomes following the use of these agents remain unclear.

Cancer Patients Patients with solid and hematologic malignancies may exhibit proinflammatory cytokine responses that result in significant physiologic and metabolic derangements.72 These cytokine mediators are likely contribute to cancer cachexia, anorexia, anemia, leukocytosis, acute phase responses and coagulopathy.73 These responses may originate from the tumors or from host immunocytes. Several studies in tumor-bearing hosts have demonstrated an exaggerated inflammatory response to insult such as sepsis.48,74 One would expect that surgical injury to patients with abdominal tumor can augment the existing inflammatory cytokine response. In clinical practice, patients undergoing major abdominal cancer operations are frequently critically ill postoperatively, requiring intensive support. The tumor load, malnutrition, and antitumor therapies may all potentially contribute to an ongoing inflammatory response in patients with malignancy. It is yet to be documented that these patients uniformly have an alteration of the cytokine response.

Peritonitis Peritonitis is a frequent indication for urgent surgical intervention. Some reports have demonstrated improved survival in patients capable of mounting adequate local cytokine responses during episodes of intraabdominal sepsis.75 Patients undergoing surgery for peritonitis exhibit an enhanced systemic inflammatory response, even after the initial pathology is properly managed.76,77 It is hypothesized that the response to surgery is additive to the ongoing cytokine response mounted in peritonitis.78,79 Whether this intact inflammatory response correlates to improved outcomes can only be inferred.


29

Hemorrhage Blood loss of varying degrees often accompanies abdominal surgery. While small volume loss is generally well tolerated, greater blood loss and resuscitation perioperatively appears to augment the inflammatory cytokine response.80 In addition, any element of ischemia or shock likely predisposes to antigen translocation across the gut barrier, and this further serves to activate the cytokine cascade. Moreover, an insult such as gut ischemia and reperfusion during surgery generate cytokines such as IL-6, IL-8 that “prime” circulating Ms and PMNs to enhance immunoactivity.81,82 These primed immunocytes, when subjected to a subsequent insult such as endotoxin, may precipitate remote organ injury (e.g., renal failure, ARDS, cardiotoxicity).88 The implications of these responses are applicable in abdominal vascular surgery, major hepatic and pancreatic surgeries, and preoperative gastrointestinal bleeding.

Anesthesia Important advances have been made relating cytokine response to various anesthetic agents. While it is well-documented that effective local/spinal anesthesia attenuates the metabolic and stress response for some abdominal surgeries, it remains to be determined that this similarly influences cytokine activity.83 Studies comparing epidural-general anesthesia to general anesthesia alone demonstrated no change in cytokine response. However, one study reporting humoral inhibition by adding intravenous prednisolone to epidural-general anesthesia was effective in decreasing the global stress response to surgery, including diminished IL-6 secretion.84 These data may suggest that the cytokine response during abdominal surgery is not the result of nociceptive (afferent neural or pain impulse) stimulus. Ketamine has often been advocated in the septic patient for its role in attenuating endotoxin-induced TNF-! production as well as for its cardiovascular stabilizing effects.85 This also requires confirmation by outcome analysis. Anesthesia using propofol and alfentanil have been shown to diminish the expression of IL-6 postoperatively.86 Whereas surgical injury favors the predominance of immunosuppressive TH2 cell population, the use of propofol may partially counter this effect.87

Minimally-Invasive Surgery The paradigm of minimally-invasive surgery is the laparoscopic cholecystectomy. In general, laparoscopic surgery for cholecystectomy is associated with a lesser inflammatory cytokine response when compared to an open technique.88-91 The primary marker for these observations has been IL-6, implying that wound size and the amount of visceral manipulation may be important determinants of the cytokine response.92-94 The diminished inflammatory cytokine response has been hypothesized to be a function of altered peritoneal milieu, which impairs immunocyte activity.95-97 One study has demonstrated that carbon dioxide (CO2) insufflation in humans impairs lymphocyte function for a period of two days secondary to an acidified peritoneal environment.98 The conversion from laparoscopic to open cholecystectomy has also been linked with a shift from TH1 to TH2 immunity and an associated alteration in the pattern of cytokine release.25 Data on cytokine responses to other laparoscopic procedures is scarce. Therefore, no studies are available as yet to address the cytokine response in technically

30


more complex surgery, such as laparoscopic splenectomy, gastric, vascular or endocrine procedures.

Conclusions It is evident that the inflammatory cytokine response is an integral facet of abdominal surgery. The cytokine response results not only from the surgical injury, but is also influenced by the patient’s preoperative status. Cytokine responses following surgery in some immunosuppressed populations and AIDS patients have yet to be addressed. The cytokine mediator response to minimally invasive surgeries remains to be explored from a mechanistic and clinical outcome perspective. Preoperative preparation is perhaps equally important to surgical technique in the optimization of a patient’s surgical outcome. This may include the use of antibiotics, proper nutritional and hemodynamic support. An enhanced understanding of cytokine response in abdominal surgery with respect to certain clinical conditions may serve to minimize any excessive systemic inflammation and improve the surgical outcome. As yet, there is no evidence to suggest that manipulations of the proinflammatory cytokine cascade will provide any clinical benefit to the surgical patient. Supported in part by National Institutes of Health grant GM 34695. Dr. Lin is also supported by The Surgical Society of The New York Hospital Queens.

References 1. Lin E, Calvano SE, Lowry SF. Disordered apoptosis as a mechanism for adverse outcome. In: Vincent JL, ed. Yearbook of Intensive Care and Emergency Medicine, 1997:91-99. 2. Marshall JC, Cook DJ, Christou NV et al. Multiple organ dysfunction score: a reliable descriptor of a complex clinical outcome. Crit Care Med 1995; 23:1638-1652. 3. Damas P, Canivet JL, Groote DD et al. Sepsis and serum cytokine concentrations. Crit Care Med 1997; 25:405-412. 4. Van der Poll T, Lowry SF. Tumor necrosis factor in sepsis: Mediator of multiple organ failure or essential part of host defense? Shock 1995; 3(1):1-12. 5. Lowry SF. Cytokines and their soluble antagonists in sepsis and critical illness. In: Tellado JM, Forse RA, Solomkin JS, eds. Prog Surg 1995; 20:115-124. 6. Rogy MA, Coyle SM, Oldenburg HSA et al. Persistently elevated soluble tumor necrosis factor receptor and interleukin-1 receptor antagonist levels in critically ill patients. J Am Coll Surg 1994; 178:132-138. 7. Van Zee KJ, Kohno T, Fischer E et al. Tumor necrosis factor soluble receptors circulate during experimental and clinical inflammation and can protect against excessive tumor necrosis factor in vitro and in vivo. Proc Natl Acad Sci USA 1992; 89:4845-4849. 8. Van der Poll T, Lowry SF. Endogenous mechanisms regulating TNF and IL-1 during sepsis. In: Vincent JL, ed. Yearbook of Intensive Care and Emergency Medicine. 1995:385-397. 9. Fong Y, Moldawer LL, Shires GT et al. The biologic characteristics of cytokines and their implications in surgical injury. Surg Gyn Obst 1990; 170:363-378. 10. Keogh C, Fong Y, Marano MA et al. Identification of a novel tumor necrosis factor/Cachectin from the livers of burned and infected rats. Arch Surg 1990; 125:79-85. 11. Fischer E, Marano MA, Barber AE et al. Comparison between effects of interleukin-1 administration and sublethal endotoxemia in primates. Am J Physiol 1991; 261:R442R448.


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12. Lowry SF, Calvano SE. Soluble cytokine and hormonal mediators of immunity and inflammation. In: Howard RJ, Simmons RL, eds. Surgical Infectious Diseases. 3rd ed. Appleton and Lange, 1995; 313-326. 13. Fischer E, Van Zee KJ, Marano M et al. Interleukin-1 receptor antagonist circulates in experimental inflammation and in human disease. Blood 1992; 79: 2196-2200. 14. Van der Poll T, van Deventer SJH, ten Cate H et al. Tumor necrosis factor is involved in the appearance of interleukin 1 receptor antagonist in endotoxemia. J Infect Dis 1994; 169:665-667. 15. Ertel W, Faist E, Nestle C. Kinetics of interleukin-2 and interleukin-6 synthesis following major mechanical trauma. J Surg Res 1990; 48:622-628. 16. Faist E, Schinkel C, Zimmer S. Update on the mechanisms of immune suppression of injury and immune modulation. World J Surg 1996; 20:454-459. 17. Abraham E, Regan RF. The effects of hemorrhage and trauma on interleukin 2 production. Arch Surg 1983; 120:1341-1346. 18 Mack VE, McCarter MD, Naama HA et al. Dominance of T-helper 2-type cytokines after severe injury. Arch Surg 1996; 131:1303-1309. 19. Oka M, Hirazawa K, Yamamoto K et al. Induction of Fas-mediated apoptosis on circulating lymphocytes by surgical stress. Ann Surg 1996; 223(4):434-440. 20. Decker D, Schondorf M, Bidlingmaier F et al. Surgical stress induces a shift in the type-1/type-2 T-helper cell balance, suggesting down-regulation of cell-mediated and up-regulation of antibody-mediated immunity commensurate to trauma. Surgery 1996; 326-325. 21. Keegan AD, Ryan JJ, Paul WE. IL-4 regulates growth and differentiation by distinct mechanisms. The Immunologist 1996; 4(6):194-198. 22. Mangan DF, Baldwin R, Wahl SM. IL-4 enhances programmed cell death (apoptosis) in stimulated human monocytes. J Immunol 1992; 148(6):1812-1816. 23. Mangan DF, Wahl SM. Differential regulation of human monocyte programmed cell death (apoptosis) by chemotactic factors and proinflammatory cytokines. J Immunol 1991; 147(10):3408-3412. 24. Biffl WL, Moore EE, Moore FA. Interleukin-6 in the injured patient. Ann Surg 1996; 224(5):647-664. 25. Baigrie RJ, Lamont PM, Kwiatkowski D. Systemic cytokine response after major surgery. Br J Surg 1992; 79:757-760. 26. Cruickshank AM, Fraser WD, Burns HJG et al. Response of serum interleukin-6 in patients undergoing elective surgery of varying severity. Clin Sci 1990; 79:161-165. 27. Bitterman H, Kinarty A, Lazarovich H et al. Acute release of cytokines is proportional to tissue injury induced by surgical trauma and shock in rats. J Clin Immunol 1991; 11(4):184-192. 28. Oldenburg HSA, Rogy MA, Lazarus DD et al. Cachexia and the acute-phase protein response in inflammation are regulated by interleukin-6. Eur J Immunol 1993; 23:1889-1894. 29. Berger D, Bolke E, Seidelmann M et al. Time-scale of interleukin-6, myeloid related proteins (MRP), C reactive protein (CRP), and endotoxin plasma levels during the postoperative acute phase reaction. Shock 1997; 7(6):422-426. 30. Lee A, Whyte KB, Haslett C. Inhibition of apoptosis and prolongation of neutrophil functional longevity by inflammatory mediators. J Leukocyte Biol 1993; 54:283-288. 31. Biffl WL, Moore EE, Moore FA et al. Interleukin-6 delays neutrophil apoptosis. Arch Surg 1996; 131:24-30. 32. Tilg H, Trehu E, Atkins MB. Interleukin-6 (IL-6) as an antiinflammatory cytokine: induction of circulating IL-1 receptor antagonist and soluble tumor necrosis factor receptor p55. Blood 1994; 83:113-118.

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33. Patrick DA, Moore FA, Moore EE et al. The inflammatory profile of interleukin-6, interleukin-8, and soluble intercellular adhesion molecule-1 in postinjury multiple organ failure. Am J Surg 1996; 172:425-431. 34. Van Zee KJ, Fischer E, Hawes AS et al. Effects of intravenous IL-8 administration in nonhuman primates. J Immunol 1992; 148(6):1746-1752. 35. Gerard C, Bruyns C, Marchant A et al. Interleukin 10 reduces the release of tumor necrosis factor and prevents lethality in experimental endotoxemia. J Exp Med 1993; 177:547-550. 36. Van der Poll T, Marchant A, Keogh CV et al. Interleukin-10 impairs host defense in murine pneumococcal pneumonia. J Infect Dis 1996; 174:994-1000. 37. Van der Poll T, Marchant A, Buurman WA et al. Endogenous IL-10 protects mice from death during septic peritonitis. J Immunol 1995; 155:5397-5401. 38. Trinchieri G. Interleukin-12: a cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type 1 and cytotoxic lymphocytes. Blood 1994; 84:4008-4027. 39. Jelinek DF, Braaten JK. Role of IL-12 in human B lymphocyte proliferation and differentiation. J Immunol 1995; 154:1606-1613. 40. Heinzel FP, Rerko DM, Ling P et al. Interleukin 12 is produced in vivo during endotoxemia and stimulates synthesis of gamma interferon. Infect Immunol 1994; 62:4244-4249. 41. Barbul A, Regan MB. The regulatory role of T lymphocytes in wound healing. J Trauma 1990; 30:S97-S102. 42. Redmond HP, Hofmann K, Shou J et al. Effects of laparotomy on systemic macrophage function. Surgery 1992; 111:647-655. 43. Zellweger R, Ayala A, Zhu XL et al. Effect of surgical trauma on splenocyte and peritoneal macrophage immune function. J Trauma 1995; 39(4):645-650. 44. Klava A, Windsor ACJ, Farmery SM et al. Interleukin-10: a role in the development of postoperative immunosuppression. Arch Surg 1997; 132:425-429. 45. Roumen RH, Hendriks T, van der Ven-Jongekrijg J et al. Cytokine patterns in patients after major vascular surgery, hemorrhagic shock, and severe blunt trauma. Ann Surg 1993; 218(6):769-776. 46. Barry MC, Kelly C, Burke P et al. Immunological and physiological responses to aortic surgery: effect of reperfusion on neutrophil and monocyte activation and pulmonary function. Br J Surg 1997; 84:513-519. 47. Meduri GU, Headley S, Kohler G et al. Persistent elevation of inflammatory cytokines predicts a poor outcome in ARDS. Chest 1995; 107:1062-1073. 48. Ogle CK, Guo X, Hasselgren PO et al. The gut as a source of inflammatory cytokines after stimulation with endotoxin. Eur J Surg 1997; 163:45-51. 49. Fong Y, Marano MA, Moldawer LL et al. The acute splanchnic and peripheral tissue metabolic response to endotoxin in humans. J Clin Invest 1990; 85:1896-1904. 50. Mowat AM, Viney JL. The anatomical basis of intestinal immunity. Immunol Rev 1997; 156:145-166. 51. Reynolds JV, O’Farrelly C, Feighery C et al. Impaired gut barrier function in malnourished patients. Br J Surg 1996; 83:1288-1291. 52. Deitch EA. Simple intestinal obstruction causes bacterial translocation in man. Arch Surg 1989; 207:549-554. 53. Palmer HR, Duerden BI, Holdsworth CD. Bacteriological and endotoxin studies in cases of ulcerative colitis submitted to surgery. Gut 1980; 21:851-854. 54. Sher ME, D’Angelo AJ, Stein TA et al. Cytokines in Crohn’s colitis. Am J Surg 1995; 169(1):133-136. 55. Brinkmann A, Wolf CF, Berger D et al. Perioperative endotoxemia and bacterial translocation during major abdominal surgery: evidence for the protective effect of endogenous prostacyclin? Crit Care Med 1996; 24(8):1293-1301.


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56. Enayati P, Brennan MF, Fong Y. Systemic and liver cytokine activation. Arch Surg 1994; 129:1159-1164. 57. Kimura F, Miyazaki M, Suwa T et al. Reduced hepatic acute-phase response after simultaneous resection for gastrointestinal cancer with synchronous liver metastases. Br J Surg 1996; 83:1002-1006. 58. Ferri M, Gabriel S, Gavelli A et al. Bacterial translocation during portal clamping for liver resection. Arch Surg 1997; 132:162-165. 59. Fujita T, Kobayashi S, Tomoyuki S et al. Relationship between circulating secretory immunoglobulin A levels and portal blood cytokine levels during major abdominal surgery. Arch Surg 1997; 132:124-127. 60. Bailey ME. Endotoxin, bile salts and renal function in obstructive jaundice. Br J Surg 1976; 63:774-778. 61. Van Leeuwen PAM, Hong RW, Rounds JD et al. Hepatic failure and coma after liver resection is reversed by manipulation of gut contents: The role of endotoxin. Surgery 1991; 110:169-175. 62. Boermeester MA, Straatsburg IH, Houdijk APJ et al. Endotoxin and interleukin-1 related hepatic inflammatory response promotes liver failure following partial hepatectomy. In: Boermeester MA (ed). Pathogenesis and Modulation of the Systemic Inflammatory Response in Endotoxemia and Sepsis. 1994; 63-84. 63. Kondo T, Suda T, Fukuyama H et al. Essential roles of the Fas ligand in the development of hepatitis. Nature Med 1997; 3(4):409-413. 64. Wang P, Ba ZF, Chaudry IH. Mechanism of hepatocellular dysfunction during early sepsis. Arch Surg 1997; 132:364-370. 65. Yanaga K, Yamamoto TK, Nishizaki T et al. Cardiac complications after hepatic resection. Br J Surg 1996; 83:1448-1451. 66. Braxton CC, Coyle SM, Montegut WJ et al. Parenteral nutrition alters monocyte TNF receptor activity. J Surg Res 1995; 59:23-28. 67. Windsor ACJ, Klava A, Somers SS et al. Manipulation of local and systemic host defence in the prevention of perioperative sepsis. Br J Surg 1995; 82:1460-1467. 68. Fong Y, Marano M, Barber A et al. Total parenteral nutrition and bowel rest modify the metabolic response to endotoxin in humans. Ann Surg 1989; 210(4):449-457. 69. Wigmore SJ, Fearon KCH, Ross JA. Modulation of human hepatocyte acute phase protein production in vitro by n-3 and n-6 polyunsaturated fatty acids. Ann Surg 1997; 225(1):103-111. 70. Li J, Kudsk KA, Janu P et al. Effect of glutamine-enriched total parenteral nutrition on small intestinal gut-associated lymphoid tissue and upper respiratory tract immunity. Surgery 1997; 121:542-549. 71. Parry-Billings M, Baigrie RJ, Lamont PM et al. Effects of major and minor surgery on plasma glutamine and cytokine levels. Arch Surg 1992; 127:1237-1240. 72. Ng EH, Lowry SF. Nutritional support and cancer cachexia. Hem Onc Clin N Am 1991; 5(1):161-184. 73. Mealy K. Proinflammatory cytokines and the host response to cancer. In: Vincent JL ed. Yearbook of Intensive Care and Emergency Medicine 1995; 398-412. 74. Sherry B, Gelin J, Fong Y et al. Anticachectin/tumor necrosis factor-alpha antibodies attenuate the development of cachexia in two murine transplantable tumor models. FASEB J 1989; 3:1556-1562. 75. Riche F, Panis Y, Laisne MJ et al. High tumor necrosis factor serum level is associated with increased survival in patients with abdominal septic shock: A prospective study in 59 patients. Surgery 1996; 120:801-807. 76. Sautner T, Gotzinger P, Redl-Wenzl EM et al. Does reoperation for abdominal sepsis enhance the inflammatory host response? Arch Surg 1997; 132:250-255. 77. Schein M, Wittmann DH, Holzheimer R et al. Hypothesis: Compartmentalization of cytokines in intraabdominal infection. Surgery 1996; 119:694-700.

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78. Hammond JMJ, Potgieter PD. The influence of surgery on cytokines in patients with intraabdominal sepsis. Anaesth Intens Care 1996; 24:430-434. 79. Tang GJ, Kuo CD, Yen TC et al. Perioperative plasma concentrations of tumor necrosis factor and interleukin-6 in infected patients. Crit Care Med 1996; 24:423-428. 80. Ayala A, Lehman DL, Herdon CD et al. Mechanism of enhanced susceptibility to sepsis following hemorrhage. Arch Surg 1994; 129:1172-1178. 81. Ayala A, Wang P, Ba ZF et al. Differential alterations in plasma IL-6 and TNF levels after trauma and hemorrhage. Am J Physiol 1991; 260:R167-R171. 82. Botha AJ, Moore FA, Moore EE et al. Sequential systemic platelet-activating factor and interleukin-8 primes neutrophils in patients with trauma at risk of multiple organ failure. Br J Surg 1996; 83:1407-1412. 83. Norman JG, Fink GW. The effects of epidural anesthesia on the neuroendocrine response to major surgical stress: A randomized prospective trial. Am Surgeon 1997; 63:75-80. 84. Schulze S, Sommer P, Bigler D et al. Effect of combined prednisolone, epidural analgesia, and indomethacin on the systemic response after colonic surgery. Arch Surg 1992; 127:325-331. 85. Schmidt H, Ebeling D, Bauer H et al. Ketamine attenuates endotoxin-induced leukocyte adherence in rate mesenteric venules. Crit Care Med 1995; 23:2008-2014. 86. Crozier TA, Muller JE, Quittkat D et al. Effect of anaesthesia on the cytokine responses to abdominal surgery. Br J Anaesth 1994; 72:280-285. 87. Salo M, Pirttikangas CO, Pulkki K. Effects of propofol emulsion and thiopentone on T helper cell type-1/type-2 balance in vitro. Anaesth 1997; 52:341-344. 88. Chambrier C, Chassard D, Bienvenu J et al. Cytokine and hormonal changes after cholecystectomy. Ann Surg 1996; 224(2):178-182. 89. Jakeways MSR, Mitchell V, Hashim IA et al. Metabolic and inflammatory responses after open or laparoscopic cholecystectomy. Br J Surg 1994; 81:127-131. 90. Deuss U, Dietrich J, Kaulen D et al. The stress response to laparoscopic cholecystectomy: investigation of endocrine parameters. Endoscopy 1994; 26:235-238. 91. Mealy K, Gallagher H, Barry M et al. Physiological and metabolic responses to open and laparoscopic cholecystectomy. Br J Surg 1992; 79:1061-1064. 92. Glaser F, Sannwald GA, Buhr HJ et al. General stress response to conventional and laparoscopic cholecystectomy. Ann Surg 1995; 221:372-380. 93. Joris J, Cigarini I, Legrand M et al. Metabolic and respiratory changes after cholecystectomy performed via laparotomy or laparoscopy. Br J Anaesth 1992; 69:341-345. 94. Iwanaka T, Arkovitz M, Arya G et al. Evaluation of operative stress and peritoneal macrophage function in minimally invasive operations. J Am Coll Surg 1997; 184:357-363. 95. Targarona E, Pons MJ, Balague C et al. Acute phase is the only significantly reduced component of the injury response after laparoscopic cholecystectomy. World J Surg 1996; 20:528-534. 96. Redmond HP, Watson WG, Houghton T et al. Immune function in patients undergoing open vs laparoscopic cholecystectomy. Arch Surg 1994; 129:1240-1246. 97. Kobayashi E, Yoshida T, Yamauchi H et al. Immune function in patients undergoing open vs laparoscopic cholecystectomy. (letter) Arch Surg 1995; 130:676. 98. Evrard S, Falkenrodt A, Park A et al. Influence of CO2 pneumoperitoneum on systemic and peritoneal cell-mediated immunity. World J Surg 1997; 21:353-357.

Cytokines and the Abdominal Operative Wound

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CHAPTER 4

Cytokines and the Abdominal Operative Wound Frank J. Thornton and Adrian Barbul

Introduction

P

rofound advances in our understanding of the healing process have occurred in the past decade.1,2 The concept of endogenous cytokine release which synchronizes the complex processes of cellular proliferation and matrix deposition within wounds has brought us closer to a therapeutic approach to wound healing.3-5 Laparotomy wounds include several layers: skin, subcutaneous tissue, fascia, muscle and peritoneum. In the past it was considered essential to carry out closure of the abdominal wall in individual layers, with particular emphasis on closure of the peritoneal layer.6-8 More recent animal and clinical studies have shown that healing of a midline incision occurs through formation of a dense fibrous scar that juxtaposes the two surfaces of the wound en masse.9

Overview of the Repair Process The normal repair process is a complex series of interactive processes including inflammation, fibroplasia with collagen deposition and maturation.2 Sequentially, repair begins with exposure of thrombocytes to exposed collagen, followed rapidly by chemotaxis of inflammatory cells and fibroblasts into the wound site. Next, activation of macrophages and fibroblasts results in matrix production and continued proliferation of fibroblasts until wound healing is complete (Fig. 4.1.). Cytokines have the potential to initiate, sustain and terminate these complex biological events resulting in successful wound repair.5,10-15 This review describes the temporal sequence of cellular events occuring in the abdominal surgical wound and the role cytokines play in orchestrating the process. The cytokines known to contribute to the healing process include platelet-derived growth factor (PDGF),16,17 platelet factor 4,18-21 transforming growth factors-! (TGF- !)22 and ∀ (TGF-∀), 23 fibroblast growth factors (basicFGF/FGF-2 and acidicFGF/FGF-1),24-26 vascular endothelial growth factor (VEGF),27,28 keratinocyte growth factor (KGF),29 IFN-#,30 interleukins 1, 2, 4, 6, 8,10, and 1231-35 and tumor necrosis factor-! (TNF-!).36-38 These peptides mediate their effect via specific receptors located on target cells. They can act locally on adjacent cells (paracrine), travel systemically (endocrine) or exert their effect on the cell of origin (autocrine or intracrine). Cytokines and the Abdominal Surgeon, edited by Moshe Schein and Leslie Wise. © 1998 R.G. Landes Company.

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Fig. 4.1. Scheme of cellular involvement in the wound healing process.

Platelet Degranulation Performance of a laparotomy wound is followed immediately by bleeding at the tissue edges. Platelet contact with the exposed collagen of the damaged tissue results in adherence and aggregation of these thrombocytes as well as a release reaction during which !-granules within the platelets release platelet factor-4 (PF-4), ∀ -thromboglobulin, PDGF, and TGF- ∀ , as well as other prostaglandins and leukotrienes. PDGF is derived predominantly from platelets and activated macrophages but also from keratinocytes, smooth muscle cells, activated fibroblasts and endothelial cells.17 PDGF is chemotactic to monocytes, neutrophils, fibroblasts and smooth muscle cells.39 It activates inflammatory cells further, promoting the inflammatory response of healing and stimulates collagenase production by fibroblasts which help in debriding the wound edges of nonviable tissue. Collagenase production may in turn initiate the early decrease in wound breaking strength experienced in uncomplicated repair.40 PDGF is also strongly mitogenic to mesenchymal cells, such as fibroblasts and smooth muscle cells, inducing resident fibroblasts around the wound to proliferate and modulate their phenotype as repair proceeds.41 PF-4 is also strongly chemotactic to inflammatory cells18 and fibroblasts as is ∀-thromboglobulin and TGF-∀.42 PDGF has also been implicated in inducing transcription of genes encoding cytokines necessary for directing cell activation, migration and communication. In rodents, the JE gene has been shown to play a critical role in the wound healing cascades and has a human homolog which encodes the monocyte chemotactic factor-1 (MCF-1).16 PDGF induces transcription of this gene, as well as others including several platelet !-granule proteins, together named the superfamily of small in-


37

ducible genes (SIG). IL-8, an activator and chemotactic factor for neutrophils and T lymphocytes43,44 is another member of the SIG family and protects endothelial cells from neutrophil contact thus protecting the endothelial surface against neutrophilmediated damage.45 Macrophage inflammatory proteins (MIP-1 and 2) are chemotactic to, and induce degranulation of polymorphonuclear cells further stimulating the inflammatory reaction46 as well as recruiting monocytes to the wound site.47 Platelet basic protein, ∀-thromboglobulin, connective tissue-activating peptide III (CTAP-III) and neutrophil-activating peptide-2 (NAP-2) are variant forms derived from a common gene product. CTAP-III is mitogenic for human dermis48 while monocyte derived NAP-2 stimulates the release of elastase from human neutrophils.49

Immune Cell Influx After haemostasis is achieved the inflammatory process continues up until the third postoperative day. Cytokine induced chemotactic forces attract neutrophils into the wound with subsequent phagocytosis of bacteria. However, in the absence of significant surgical site infection, these cells are not essential for wound healing to occur.50 Monocytes appear in the abdominal wound within 48-96 hours of surgery to become the wound macrophages51 and are essential to the healing process.52-54 Their influx into the wound and eventual role in the repair process depends largely on the generation of a diverse range of mononuclear phagocyte chemoattractants in the wound site.55 PDGF and TGF-∀ are nonspecific chemoattractants produced during the platelet release reaction39 recruiting monocytes, neutrophils and many other cells to the wound site. Macrophage Chemotactic Protein (MCP-1) is produced by macrophages themselves as well as fibroblasts and endothelial cells in response to stimulation with IFN-#, TNF-!, IL-1∀ and LPS.56,57 Therefore, not only do monocytes respond to specific chemoattractants but they themselves, once established in the wound, secrete a repertoire of cytokines to further amplify the inflammatory process and selectively recruit cells to the wound. As previously stated PDGF and TGF-∀ while primarily derived from platelet a-granules are also secreted later in the repair process by macrophages to function as coordinators of matrix remodeling and angiogenesis as well as chemotactic factors. IL-8, produced by macrophages, also participates in angiogenesis.58 Many of the cytokines and growth factors secreted by fibroblasts, macrophages and the other wound cells display pleitrophic effects being influential in cell migration and mitosis, matrix deposition and remodelling as well as angiogenesis. PDGF and TGF-∀ are among these growth factors as well as insulinlike growth factor (IGF-1), FGF and the interleukin family of cytokines. The central role of lymphocyte in the wound is now well recognised.59-61 Lymphocytes like macrophages appear to regulate wound healing through cytokine release.62

Transforming Growth Factor (TG-∀) TGF-∀ is released from platelet a-granules in a latent form which is transformed to its active form by plasmin, a product of the coagulation cascade.63 It exists in five isoforms with the relative abundance of these subtypes determining the degree of scarring seen in the mature wound. TGF-∀1 and ∀2 are implicated in scarring, while TGF-∀3 is a potential antiscarring agent.64 TGF-∀1 is the predominant isoform in wound fluid65 and affects almost all pathways in the wound repair process. In the active form TGF-∀ is strongly chemotactic for inflammatory cells and fibroblasts, with this effect being lost above or below an “optimum” concentration.66-68 The effect of TGF-∀ on fibroblast proliferation is still not completely understood. In vitro

38


studies have demonstrated inhibition of proliferation.69 TGF-∀ also induces PDGF production by fibroblasts and induces fibronectin, procollagen type I and glycosaminoglycan synthesis in these cells. It exerts chemoattractant forces on cells required to potentiate repair in the wound and subsequently stimulates granulation tissue formation by these cells. Probably its greatest influence is on matrix formation through its action on the fibroblast cell. TGF-∀ regulates genetic transcription of many matrix proteins including collagen, fibronectin, glycosaminoglycans.70 Also, some of the negative effects of antineoplastic agents on the healing process are mediated through changes in TGF-∀ expression either at a transcriptional or a post-transcriptional level e.g., mRNA expression for TGF-∀ is reduced in adriamicin-impaired skin wounds71 and exogenous TGF-∀ reverses this effect in experimental wound chambers.72 The collagen deposition promoted by TGF-∀ has been shown in experimental models to be paralleled by increased breaking strength of the wound.73 TGF-∀ is also inhibitory to endothelial cell proliferation despite its ability to stimulate angiogenesis when injected in vivo.74

Interleukins In the wound site IL-1 is predominantly a product of the monocyte population. It regulates connective tissue regeneration by stimulating fibroblast proliferation and fibroblast synthesis of collagen (in absence of serum),75,76 fibronectin,77 hyaluronic acid78 and elastin.79 It acts as a costimulus with TNF-! to produce plasminogen activator80 and collagenase33,81 both of which help in the digestion of pericellular ground substance and thus promote increased cell motility which is especially important in epithelialization. IL-2 also participates in wound healing by increasing collagen synthesis.32 The effect of blood transfusion on the repair process has been ascribed to diminution of wound IL-2 levels.82,83 IL-6 has been implicated in suppressing fibroblast proliferation.35 Recent evidence also suggests that reoperative trauma for cases of abdominal sepsis induces an early postoperative increase in IL-6 levels.84 This surge in IL-6 production has been associated with postoperative hypotension often seen in these septic patients and may contribute to the resultant hypoxia in the wound and to compromised healing. IL-4 and IL-10 are inhibitors of macrophage metalloproteinases, they also suppress iNOS production while simultaneously inducing arginase.85 TGF-∀ similarly downregulates iNOS expression while incresing arginase activity. IFN-# has the opposite effect.86 Shearer et al have interpreted this as a regulatory phenomenon mediated by inflammatory cytokines, creating an NO regulated cytotoxic environment early in the wound healing process. This is followed by an increase in arginase activity later in the repair process which produces an environment more favorable to fibroblast proliferation and collagen synthesis. IL-12 is a potent inhibitor of angiogenesis87 possibly as a result of its induction of IFN-#.88

Interferon-# IFN-# is another major participant in the repair process. It selectively decreases collagen production in fibroblasts and type I and III procollagen mRNA levels while inducing fibronectin at a transcriptional level.30,89,90 Similar down-regulation of collagen synthesis has been shown in vivo.91 Along with other lymphokines, IL-4 and IL-10, it inhibits matrix metalloproteinase production by macrophages but has little effect on enzyme activity in other wound cells.34,92,93 It also participates in the induc-


39

tion of the inducible isoform of nitric oxide synthase (iNOS) and may exert its effects in the wound through the free radical NO, the by-product of iNOS activity. This latter pathway may also be responsible for the activation and increased cytotoxicity seen in macrophages stimulated by IFN-#.94

Tumor Necrosis Factor-! TNF-! in the wound serves to regulate macrophage proliferation while differentiation occurs,95 inhibits collagen synthesis38,96 and at higher concentrations increases collagenase production97-99 (Fig. 4.2). Other activities of TNF-! include chemotaxis and invasive migration of fibroblasts into the wound,36,100 induction of other proinflammatory cytokines, including IL-1 and specialized cytokines IL-6 and IL-8 thus promoting the early inflammatory response essential to normal repair.13,101

Nitric Oxide The precise role of the free radical, nitric oxide (NO) in wound healing still defies full explanation. NO synthesis is inducible by cytokines and lipopolysaccharide, in particular IL-1, TNF-! and IFN-#.102 It is synthesised in wounds by the inducible isoform of the enzyme nitric oxide synthase (iNOS). Hypoxia, which is inevitably present in laparotomy wounds is a costimulus with inflammatory cytokines for the production of NO in macrophages.103 Macrophage activation in an anoxic environment has also been shown to increase TNF-! and IL-6 production.104 Our laboratory has demonstrated decreased wound healing in murine cutaneous wounds after systemic inhibition of inducible nitric oxide synthase (iNOS).105 Collagen synthesis by wound fibroblasts also varies with NO concentration, collagen synthesis increasing at low concentrations of NO and decreasing at higher concentrations.106 This finding may reflect the in vivo scenario seen in systemic sepsis where we have shown decreased collagen synthesis in colonic anastomoses paralleled by a large increase in iNOS activity.107 Overall, these results suggest that there exists a level of NO at which wound healing is maximized but above or below which the process is suboptimal. More recently Schäffer et al have demonstrated that wound fibroblasts are phenotypically altered during the healing process to synthesize NO which in turn, regulates their collagen synthetic and contractile activities.108 In human keratinocytes iNOS activity is induced by IFN-# while IFN-! and TGF-∀ antagonize this effect.109 NO inhibits endothelial cell proliferation and bFGF-induced phenotypic modulations.110 Also, TGF-∀ down-regulates iNOS expression in vivo.111 These findings further support the view that an intricate web of interactions exists between cytokines and NO in the healing process. Further work is necessary to clarify the biological and clinical relevance of these interactions.

Extracellular Matrix Production Fibroblasts, the primary source of extracellular matrix, take on a range of functions as they evolve with the healing wound. During the first 3 days after wounding, fibroblasts refrain from migrating into the wound site. Instead they proliferate at the wound edge under the potent mitogenic influence of PDGF.17 The failure of these cells to migrate into the wound site during this time is not fully understood. However, the presence of fibronectin surface receptors and a noncompliant cytoskeletal framework have been proposed as causative factors.112 A phenotypic change occurs after day 3 resulting in fibroblast invasion into the wound site. The primary difference in these cells from the proliferating phenotype is the expression of vitronectin receptors, which has been shown by in vitro studies to

40


be induced by FGF.113,114 The time for adequate FGF accumulation and subsequent induction of this receptor to occur may explain the 3-4 day delay in fibroblast migration. The wound fibroblasts begin immediately to secrete matrix proteins. Wound cytokines influence the fibroblast rate of synthesis of these proteins which constitute the ECM (Table 4.1). Unfortunately most information concerning the regulation of fibroblast metabolism and collagen secretion is derived from in vitro work, with findings dependent on the presence or absence of serum. Much less evidence exists for their role in vivo. Interferon-# decreases collagen type I and III synthesis in fibroblasts at a transcriptional level30 with little effect on other proteins. TNF-! also reduces collagen synthesis while simultaneously inducing collagenase production.38 Its synergistic effect on collagen production with #-interferon has been suggested as a possible down-regulatory mechanism for matrix production towards the end of the repair process37,115 (Fig. 4.2). TGF-∀ and IL-1 have been found to enhance collagen synthesis in fibroblasts70,116,117 and furthermore have increased wound healing when applied topically. 118-120 In addition TGF- ∀ induces tissue inhibitor of metalloproteinase and blocks collagenase induction by other cytokines.121 The balance of cytokines in the wound during the first three days of healing may determine the collagenase activity and therefore the suture holding capacity of the wound. This is especially relevant to laparotomy wounds where the breaking strength in rats has been shown to decrease by 47% during the first 48 hours.40

Angiogenesis Due to vascular damage at the time of surgery the centre of a wound is hypoxic. New blood vessel formation or angiogenesis is a prerequisite to successful completion of wound repair.4,122 The soluble factors responsible for neo-vascularization remain incompletely defined. FGF-2 was the first angiogenesis factor to be isolated and acts as an endothelial cell mitogen, stimulating tube formation and proteinase secretion by endothelial cells as a prelimenary step in neo-capillary formation.26,123 The proteinases cause breakdown of the vascular basement membrane permitting vascular endothelial cell invasion during new vessel formation.124 Fibronectin is also essential to this process as a substrate which modulates endothelial cell shape fascilitating its growth.125 FGF is synthesised by many cells, including macrophages, keratinocytes and neutrophils. It has a high affinity for heparin as well as the syndecan family of cell surface receptors, both of which may influence the mobilization of bFGF. These syndecan receptors may also determine upon which cells in the wound site it exerts its regulatory influence.126-128 The mechanism by which FGF is secreted by the cell is incompletely elucidated.129 One theory suggests that heparin sulphate plays a role, and provides a mechanism of storage for FGF in the basement membrane.130 FGF possesses mitogenic properties when applied to keratinocytes with a potential role in re-epithelialization of the wound.131 In vivo studies have demonstrated more rapid re-epithelialization with topical application of FGF-2 while in combination with PDGF it facilitated earlier capillary ingrowth in wounds of diabetics.132,133 Other potential cytokine mediators of angiogenesis are IL-1, TGF-!, EGF, G-CSF, GM-CSF and TGF-∀74,134-136 while platelet factor 4 and IL-12 inhibit angiogenesis.87,137

Wound Contraction By day 7 fibroblasts have become the main cellular component of granulation tissue. At about this time fibroblasts take on characteristics normally seen in smooth


41

Table 4.1. Cytokines-sources and functions in the wound Cytokine

Cellular Source in Wound Function in Repair Process

PDGF

Platelet, activated macrophage, activated fibroblast, keratinocyte, smooth muscle cell and endothelial cell

Chemotactic to monocytes, neutrophils, smooth muscle cells and fibroblasts. Activates inflammatory cells. Mitogenic to mesenchymal cells. Weakly angiogenic. Phenotypically alters fibroblast to myofibroblast. Stimulates collagenase synthesis by fibroblasts. Induction of the SIG family of genes.

TGF-∀

Platelet, lymphocyte, macrophage, keratinocyte, fibroblast, endothelial cell, smooth muscle cell

Strongly chemotactic to macrophages, neutrophils, lymphocytes and fibroblasts. Induces fibroblast proliferation and phenotypic alteration to myofibroblast form. Stimulates angiogenesis. Induces PDGF, procollagen, glycosaminoglycan and fibronectin synthesis by the fibroblast. Upregulates TIMPs production while blocking collagenase induction.

FGF -1 and -2 Basement membrane storage. Macrophage, neutrophil, keratinocyte, lymphocyte.

Strongly angiogenic: endothelial cell mitosis, tube formation and proteinase secretion. Mitogenic to keratinocytes.

IFN-#

Lymphocyte, macrophage

Enhances neutrophil and macrophage function. Antagonizes phenotypic change in fibroblast to its contractile counterpart. Decreases proliferation of fibroblasts and collagen synthesis. Induces fibronection transcription. Induces other cytokines and NO.

IL-1

Macrophage, keratinocyte, Lymphocyte differentiation and fibroblast, endothelial cell, activation. Increases collagenase and smooth muscle cell. plasminogen activator synthesis. Induces collagen synthesis in fibroblasts and their proliferation. Decreases synthesis of TIMPs. Induces keratinocyte migration.

IL-6

Keratinocyte, fibroblast, macrophage,endothelial cell, lymphocyte

continued...

Lymphocyte proliferation. Suppresses fibroblast proliferation. Has been implicated in causing wound hypoxia in sepsis (see text).

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Table 4.1. (continued) Cytokine

Cellular Source in Wound Function in Repair Process

TNF-!

Macrophage, keratinocyte

Decreases collagen synthesis and increases collagenase activity. Stimulates cytokine production and NO.

EGF

Macrophage, platelet, keratinocyte

Stimulates proliferation and migration of epithelial cells.

TGF-!

Macrophage, keratinocyte Angiogenesis. (40% homology with EGF) Growth of epithelial cells, endothelial cells and fibroblasts.

GM-CSF

Keratinocyte,fibroblast, lymphocyte, endothelial cell

Indirectly induces phenotypic change of fibroblast to myofibroblast.

Nitric Oxide

Most mammalian cells when stimulated with LPS or inflammatory cytokines

Still poorly understood. Fibroblast collagen synthesis appears to vary with NO concentration as does fibroblast proliferation. Inhibits endothelial proliferation.

muscle cells, including !-smooth muscle actin expression, and have been named myofibroblasts.138-140 PDGF and TGF-∀ have been implicated in phenotypically changing the fibroblast into its myofibroblastic counterpart.41,141,142 GM-CSF also promote cell metamorphosis by accumulating macrophages in the wound site which in turn release cytokines that induce !-smooth muscle actin expression.143,144 Through their regulation of cytokine release ECM components may also control fibroblast phenotypic change.145,146 IFN-# was found to antagonize this myofibroblastic change by decreasing !-smooth muscle actin mRNA and protein147 which correlates well with this cytokines antiproliferative activity148 and decrease of collagen synthesis149,150 all of which appear to act as “quenching” factors in the repair process. Interferon-! and -∀ appear to have similar inhibitory effects on wound contraction.62

Epithelialization Epithelialization plays a minor role in the primary healing of uncomplicated laparotomy wounds because of the small surface area to be traversed by migrating epithelial cells. Nevertheless its successful completion establishes a barrier to contamination of the deeper wound layers.151 Reepithelialization commences within several hours of the injury and involves early migration of keratinocytes from the wound edge.152,153 These cells undergo phenotypic changes as they migrate over the underlying wound matrix, becoming flattened and elongated,154,155 expressing actin and myosin156 and loosing their junctional attachment to adjacent cells and basement membrane.153 Cytokines control many of these events and have been shown in clini-


43

Fig 4.2. Inflammatory cytokines influence the net collagen production in the wound site through their effect on fibroblast proliferation, collagen synthesis and collagen degradation.

cal studies to augment reepithelialization. Epidermal growth factor (EGF), one of the first growth factors to be discovered, stimulates the proliferation and migration of epithelial cells157 as does TGF-!, a structurally similar cytokine.158 These cytokines exert their effect by increasing expresion of the collagen integrin !2∀1.159 IL-1 also induces keratinocyte migration but through a different pathway.119,160 TNF-! and IL-1 also upregulate keratinocyte secretion of a urokinase-type plasminogen activator (uPA) and expression of its cell-surface associated receptor uPA-R. Plasminogen that is also bound to a membrane binding site is converted by uPA to plasmin which is then available for proteolysis of pericellular glycoproteins.80 IL-1 also increases collagenase activity which also contributes to increased cell motility.81

Cytokines in Surgical Site Infection Surgical site infection increases the morbidity and mortality for the surgical patient especially when it develops into abdominal wound dehiscence. Knowledge of cytokine behaviour in the infected wound is incomplete. It is known that the bacterial presence in a wound induces thrombocytopenia.161,162 Since the primary source of wound cytokines in early repair is the platelet we can postulate that a temporal imbalance occurs in wound cytokine concentrations as a result of infection. Increased numbers of neutrophils and macrophages are also seen in infected wounds which further alter the balance of cytokines in the wound site. Hypoxia of the wound has been demonstrated to promote surgical site infection.163 Macrophage activation under hypoxic conditions induces production of increased quantities of the cytokines TNF-! and IL-1b and for prolonged periods.104 These cytokines act synergistically to increase the production of matrix metalloproteinases, while simultaneously decreasing the endogenous production of tissue inhibitor of metalloproteinases (TIMPs).164 The alterations in cytokine behaviour in surgical site sepsis needs to be properly elucidated before the application of exogenous recombinant cytokines as a therapeutic answer to the problem.

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Conclusion In the context of wound healing endogenous cytokines act as multifunctional signaling peptides regulating a wide spectrum of activities. Their role as mediator between such divergent cell populations as the immune cells, fibroblasts, endothelial cells and keratinocytes establishes a harmonious environment which eliminates contaminant organisms and nonviable tissue, promotes matrix deposition, angiogenesis and re-epithelialization, with the ultimate goal of ensuring a rapid and secure wound closure especially in the case of laparotomy wounds where dehiscence is associated with high morbidity and mortality. Our ever increasing knowledge of cytokines and our ability to manufacture recombinant forms is bringing us to a new era in the management of the complicated or compromised wound.3,5 Factors to be remembered, however, before widespread application of topical cytokine therapy are firstly the appropriate timing and quantity applied since some cytokines work in a narrow dosage and time range and secondly, that administration of combinations of growth factors may be more therapeutic than single cytokine application. Also difficulties encountered in translating in vitro data to in vivo situations need to be overcome.165 Nevertheless, the future of advances in wound healing appear to lie in the domain of cytokines. As a contemporary, Ambroise Paré might rephrase—“I dressed the wound; God healed it—with cytokines”.166

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117. Postlethwaite AE, Raghow R, Stricklin GP et al. Modulation of fibroblast functions by interleukin 1: Increased steady-state accumulation of type I procollagen messenger RNAs and stimulation of other functions but not chemotaxis by human recombinant interleukin 1 alpha and beta. J Cell Biol 1988; 106:311-8. 118. Beck LS, Chen TL, Mikalauski P, Ammann AJ. Recombinant human transforming growth factor-beta 1 (rhTGF-beta 1) enhances healing and strength of granulation skin wounds. Growth Factors 1990; 3:267-75. 119. Mertz PM, Sauder DL, Davis SC et al. IL-1 as a potent inducer of wound re-epithelization. Prog Clin Biol Res 1991; 365:473-80. 120. Sauder DN, Kilian PL, McLane JA et al. Interleukin-1 enhances epidermal wound healing. Lymphokine Res 1990; 9:465-73. 121. Edwards DR, Murphy G, Reynolds JJ et al. Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor. Embo J 1987; 6:1899-904. 122. Arnold F, West DC. Angiogenesis in wound healing. [Review]. Pharmacol Ther 1991; 52:407-22. 123. Mignatti P, Mazzieri R, Rifkin DB. Expression of the urokinase receptor in vascular endothelial cells is stimulated by basic fibroblast growth factor. J Cell Biol 1991; 113:1193-201. 124. Mignatti P, Tsuboi R, Robbins E et al. In vitro angiogenesis on the human amniotic membrane: requirement for basic fibroblast growth factor-induced proteinases. J Cell Biol 1989; 108:671-82. 125. Ingber DE. Fibronectin controls capillary endothelial cell growth by modulating cell shape. Proc Natl Acad Sci USA 1990; 87:3579-83. 126. Klagsbrun M, D’Amore PA. Regulators of angiogenesis. [Review]. Annu Rev Physiol 1991; 53:217-39. 127. Bernfield M, Kokenyesi R, Kato M et al. Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans. [Review]. Annu Rev Cell Biol 1992; 8:365-93. 128. Tekotte H, Engel M, Margolis RU, Margolis RK. Disaccharide composition of heparan sulfates: brain, nervous tissue storage organelles, kidney, and lung. J Neurochem 1994; 62:1126-30. 129. Mignatti P, Rifkin DB. Release of basic fibroblast growth factor, an angiogenic factor devoid of secretory signal sequence: A trivial phenomenon or a novel secretion mechanism? [Review] J Cell Biochem 1991; 47:201-7. 130. D’Amore PA. Modes of FGF release in vivo and in vitro. [Review]. Cancer Metastasis Rev 1990; 9:227-38. 131. O’Keefe EJ, Chiu ML, Payne RJ. Stimulation of growth of keratinocytes by basic fibroblast growth factor. J Invest Dermatol 1990; 90:767-9. 132. Hebda PA, Klingbeil CK, Abraham JA, Fiddes JC. Basic fibroblast growth factor stimulation of epidermal wound healing in pigs. J Invest Dermatol 1990; 95:626-31. 133. Greenhalgh DG, Sprugel KH, Murray MJ, Ross R. PDGF and FGF stimulate wound healing in the genetically diabetic mouse. Am J Pathol 1990; 136:1235-46. 134. Mahadevan V, Hart IR, Lewis GP. Factors influencing blood supply in wound granuloma quantitated by a new in vivo technique. Cancer Res 1989; 49:415-9. 135. Schreiber AB, Winkler ME, Derynck R. Transforming growth factor-alpha: A more potent angiogenic mediator than epidermal growth factor. Science 1986; 232:1250-3. 136. Bussolino F, Mantovani A. Effect of granulocyte-macrophage colony-stimulating factor on endothelial cells. Blood 1991; 78:2475-6. 137. Maione TE, Gray GS, Petro J et al. Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides. Science 1990; 247:77-9.


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138. Sappino AP, Schurch W, Gabbiani G. Differentiation repertoire of fibroblastic cells: expression of cytoskeletal proteins as marker of phenotypic modulations. [Review]. Lab Invest 1990; 63:144-61. 139. Finesmith TH, Broadley KN, Davidson JM. Fibroblasts from wounds of different stages of repair vary in their ability to contract a collagen gel in response to growth factors. J Cell Physiol 1990; 144:99-107. 140. Schmitt-Graff A, Desmouliere A, Gabbiani G. Heterogeneity of myofibroblast phenotypic features: an example of fibroblastic cell plasticity. [Review]. Virchows Arch 1994; 425:3-24. 141. Desmouliere A, Geinoz A, Gabbiani F, Gabbiani G. Transforming growth factorbeta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol 1993; 122:103-11. 142. Montesano R, Orci L. Transforming growth factor beta stimulates collagen-matrix contraction by fibroblasts: implications for wound healing. Proc Natl Acad Sci U S A 1988; 85:4894-7. 143. Vyalov S, Desmouliere A, Gabbiani G. GM-CSF-induced granulation tissue formation: relationships between macrophage and myofibroblast accumulation. Virchows Arch B Cell Pathol Incl Mol Pathol 63:231-9. 144. Rubbia-Brandt L, Sappino AP, Gabbiani G. Locally applied GM-CSF induces the accumulation of alpha-smooth muscle actin containing myofibroblasts. Virchows Arch B Cell Pathol Incl Mol Pathol 1991; 60:73-82. 145. Desmouliere A, Gabbiani G. Modulation of fibroblastic cytoskeletal features during pathological situations: the role of extracellular matrix and cytokines. [Review]. Cell Motil Cytoskeleton 1994; 29:195-203. 146. Streuli CH, Schmidhauser C, Kobrin M et al. Extracellular matrix regulates expression of the TGF-beta 1 gene. J Cell Biol 1993; 120:253-60. 147. Desmouliere A, Rubbia-Brandt L, Abdiu A et al. Alpha-smooth muscle actin is expressed in a subpopulation of cultured and cloned fibroblasts and is modulated by gamma-interferon. Exp Cell Res 1992; 201:64-73. 148. Duncan MR, Berman B. Gamma interferon is the lymphokine and beta interferon the monokine responsible for inhibition of fibroblast collagen production and late but not early fibroblast proliferation. J Exp Med 1985; 162:516-27. 149. Granstein RD, Deak MR, Jacques SL et al. The systemic administration of gamma interferon inhibits collagen synthesis and acute inflammation in a murine skin wounding model. J Invest Dermatol 1989; 93:18-27. 150. Granstein RD, Flotte TJ, Amento EP. Interferons and collagen production. [Review]. J Invest Dermatol 95:75S-80S. 151. Woodley DT, Chen JD, Kim JP et al. Re-epithelialization. Human keratinocyte locomotion. [Review]. Dermatol Clin 1993; 11:641-6. 152. Winter GD. Formation of the scab and the rate of epithelisation of superficial wounds in the skin of the young domestic pig. 1962. J Wound Care 1995; 4:366-7. 153. Stenn K Sa. Re-epithelialization. In: Clark PM, ed. The Molecular and Cellular Biology of Wound Repair. New York: Plenum Press 1988; 321-335. 154. Odland G, Ross R. Human wound repair. I. Epidermal regeneration. J Cell Biol 1968; 39:135-51. 155. Kim JP, Zhang K, Chen JD et al. Mechanism of human keratinocyte migration on fibronectin: unique roles of RGD site and integrins. J Cell Physiol 1992; 151:443-50. 156. Bereiter-Hahn J, Strohmeier R, Kunzenbacher I et al. Locomotion of Xenopus epidermis cells in primary culture. J Cell Sci 1981; 52:289-311. 157. Cohen S. The epidermal growth factor (EGF). [Review]. Cancer 1987; 51:1787-91.

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158. Derynck R. Transforming growth factor-alpha: Structure and biological activities. [Review]. J Cell Biochem 1986; 32:293-304. 159. Chen JD, Kim JP, Zhang K et al. Epidermal growth factor (EGF) promotes human keratinocyte locomotion on collagen by increasing the alpha 2 integrin subunit. Exp Cell Res 1993; 209:216-23. 160. Chen JD, Lapiere JC, Sauder DN et al. Interleukin-1 alpha stimulates keratinocyte migration through an epidermal growth factor/transforming growth factor-alphaindependent pathway. J Invest Dermatol 1995; 104:729-33. 161. Dudgeon LS, Goadby HK. The examination of the tissues and some observations on the blood platelets of rabbits at intervals of five minutes, and later, after intravenous inoculation of Staphylococcus aureus and Indian ink. J Hyg 1931; 31:247. 162. Weil MH, Spink WW. A comparison of shock due to endotoxin with anaphylactic shock. J Lab Clin Med 1957; 50:502. 163. Hunt TK, Niinikoski J, Zederfeldt B. Role of oxygen in repair processes. Acta Chir Scand 1972; 138:109-10. 164. Mauviel A. Cytokine regulation of metalloproteinase gene expression. [Review] J Cell Biochem 1993; 53:288-95. 165. Postlethwaite AE, Kang AH. Advantages and limitations of in vitro models of wound healing and tissue repair. Prog Clin Biol Res 1988; 266:237-42. 166. Pare A. The classic. Compound fracture of leg, Pare’s personal care (MII, 328). Clin Orthop 1983; 178:3.

Cytokines and Postoperative Abdominal Adhesions

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CHAPTER 5

Cytokines and Postoperative Abdominal Adhesions Shaun G. Appleton and Jeremy N.Thompson

Introduction

A

dhesions are the fibrinous deposits that form between mesothelial surfaces following injury or inflammation. Following abdominal surgery, over 90% of people will develop peritoneal adhesions.1 As the number of laparotomies performed has increased so has the incidence of adhesions and their complications, notably bowel obstruction, infertility and pelvic pain. Acquired adhesions result from peritoneal injury which may be caused by infection, ischemia, a reaction to foreign materials such as sutures, gauze particles and glove powder, or from the coaption of two denuded peritoneal surfaces.2,3 In developed countries adhesions are the leading cause of bowel obstruction accounting for more than 40% of all cases of intestinal obstruction4 and 60-70% of those involving the small bowel.5 In developing countries the commonest cause of small bowel obstruction is still strangulated hernia because elective abdominal surgery and early hernia repair are relatively rare. About 1% of patients will develop intestinal obstruction due to postoperative adhesions within one year of surgery and 0.5% within four weeks of their operation.1 A survey of 1200 hospitals in Germany estimated that 2.6% laparotomies were performed for adhesional bowel obstruction,6 while a review of British surgeons revealed that on average, adhesive obstruction accounted for three to four laparotomies and another seven to eight admissions for conservative management per general surgeon per year.7 In women the most common causes of adhesions are prior surgery, infection and endometriosis.8 The relationship between pelvic adhesions and infertility is well established9-11 and adhesiolysis can increase pregnancy rates among previously infertile women. The relationship between adhesions and pelvic pain is less clear-cut though nearly 80% of patients report a disappearance or substantial reduction in pelvic pain after adhesiolysis.12 As well as the surgical workload, adhesions impose a huge financial burden; in 1988 in the United States there were over 282,000 hospitalizations for adhesiolysis costing an estimated 1.18 billion US dollars.13

Cytokines and the Abdominal Surgeon, edited by Moshe Schein and Leslie Wise. © 1998 R.G. Landes Company.

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Adhesion Formation In order to appreciate the complexities of adhesion formation, an understanding of the anatomy and pathophysiology of the peritoneum must be considered. The peritoneum is a unique organ covering an area of 10,000 cm2 in an adult, almost as much as the skin.14 It consists of a single layer of mesothelial cells resting on a highly vascularised, but loosely packed, connective tissue framework. A small volume of fluid within the peritoneal cavity provides lubrication between the parietal and visceral surfaces. This peritoneal fluid contains a variable number of resident peritoneal macrophages that exhibit minimal biological activity at rest. However, when stimulated by peritoneal injury or infection they release large quantities of interleukin-1 (IL-1! and IL-1∀), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-!).15 These proinflammatory and chemotactic mediators effect the production of an inflammatory exudate and a change in the peritoneal white cell population. Large numbers of polymorphonuclear neutrophils (PMNs) appear and, in the absence of infection disappear within three to four days. Macrophages follow and reach a peak about the fifth day. It is these postsurgical macrophages that play a vital role in the modulation of inflammation by secreting a range of substances, including cyclooxygenase and lipoxygenase metabolites, plasminogen activators, plasminogen activator inhibitors, collagenase, elastase, IL-1 and IL-6, TNF and other cytokines.16 Peritoneal macrophages also recruit new mesothelial cells onto the surface of the injured peritoneum. These cells are thought to be derived from the primitive mesenchymal cells supporting the mesothelial layer and differentiate in response to cytokines and other macrophage-secreted mediators.5 Islands of new mesothelial cells appear throughout the injured area and rapidly proliferate accomplishing re-epithelialization within eight days.17 It is a unique feature of the mesothelium that, unlike dermal defects, both large and small peritoneal defects heal at the same speed.18 Mesothelial cells themselves are an important source of inflammatory and fibrogenic mediators secreting IL-1, IL-6 and IL-8 when appropriately stimulated.19-21 This stimulus is mediated by IL-1∀ and TNF-! released from resident and infiltrated macrophages.20 Several groups have investigated the cytokine levels in peritoneal fluid and plasma in response to surgery.40-47 They have shown an initial rise in TNF-! within 3 hours of surgery followed by rises in IL-1∀ and IL-6 within 6 hours of surgery. IL-6 reached the highest and most sustained concentrations locally and could also be detected in the plasma23 (Fig. 5.1). The inflammatory peritoneal exudate formed in response to injury is rich in fibrin and lysis of fibrin deposits is a physiological property of normal mesothelial surfaces.24 Mesothelial cells possess plasminogen-activating activity;25 tissue plasminogen activator (tPA) is the major plasminogen activator in human peritoneum.26 This property is important in preventing fibrinous adhesions developing between tissue surfaces within body cavities. Peritoneal injury results in rapid synthesis and release of plasminogen activator inhibitors-1 and -2 by mesothelial, endothelial and inflammatory cells, with loss of plasminogen-activating activity.27 Studies of peritoneal fluid after elective operation have demonstrated that this loss occurs over six to twelve hours and is associated with marked increases in the concentrations of plasminogen activator inhibitors-1 and -2. The production of plasminogen activator inhibitors appears to be mediated directly by the inflammatory cytokines in the peritoneal fluid following injury. TNF-!, IL-1 and IL-6 have all been shown both individually and synergistically to stimulate plasminogen activator-1 production by human mesothelial cells in culture.28


55 Fig. 5.1. Concentrations of cytokines in the peritoneal fluid following surgery (mean ± SEM).

The duration of the reduction in plasminogen-activating activity on the peritoneal surface probably determines the fate of the initial fibrinous adhesions. Prolonged depression of fibrinolytic activity allows the organisation and permanent formation of fibrous adhesions. Recovery of plasminogen-activating activity within three to four days leads to lysis of fibrin and adhesion-free tissue healing (Fig. 5.2). Much of the experimental work on adhesions and cytokines has investigated the role of the early inflammatory mediators; IL-1, IL-6 and TNF-! and the fibrogenic cytokine TGF-∀.

Interleukin-1 (IL-1) IL-1 consists of two subtypes ! and ∀ with a similar spectrum of biological activity. IL-1 is produced by all forms of antigen-presenting cells and is an important and ubiquitous early mediator of inflammation. In vivo effects of IL-1 include expression of adhesion molecules on vascular endothelium, regulation of fibrin deposition

56

Cytokines and the Abdominal Surgeon Release of cytokines, arachidonic acid metabolites, oxygen free radicals and growth factors

TNF-! IL-1 IL-6

TNF-∀

Plasminogen Activator Inhibitors

INJURY

INFLAMMATION

FIBRIN DEPOSITION

Plasminogen activators

FIBRIN REMNANTS

LYSIS

INGROWTH OF FIBROBLASTS AND CAPILLARIES

RESOLUTION

PERMANENT ADHESION

Fig. 5.2. Pathogenesis of adhesions.

and lysis, collagen deposition by fibroblasts, stimulation of fibroblast proliferation, increase in collagenase production by fibroblasts and modulation of granulocyte production.29 IL-1 and TNF-! overlap in many of their biological activities. In a clinical setting levels of IL-1 in peritoneal fluid rise within six hours of operation and reach a peak at 12 hours.40 Levels of IL-1 (! and ∀) in the plasma are low or undetectable following surgical operations,40,44,47 but one study has shown a significant and persistent rise in levels of IL-1 receptor antagonist in the plasma following minor surgical trauma.47 In a rat adhesion model, recombinant IL-1! given intraperitoneally at operation and for five days afterwards resulted in increased adhesion formation compared to controls.30 However, this effect was not seen in rats treated with IL-1! at two weeks after surgery30 highlighting the importance of the cytokine in the initial inflammatory response. Intravenous administration of antibodies to IL-1 immediately after surgery reduced adhesion formation in a rat model and this effect was potentiated in rats given both anti-IL-1 and anti-TNF-! antibodies.31 Histologically these adhesions also demonstrated lower levels of collagen deposition and fibroblast invasion.

Tumor Necrosis Factor-alpha (TNF-!) Much interest has been focused on the effects of the pro-inflammatory cytokine TNF-! on wound healing and fibroblast function.32 The effects of TNF-! are similar to IL-1 in stimulating the production of prostaglandins by different types of fibroblasts and TNF-! induces the synthesis and release of IL-1 from monocytes, fibroblasts and endothelial cells. TNF-! is the earliest cytokine to be produced in the peritoneal cavity after injury reaching peak concentrations between three and eight hours after surgery and remaining elevated for 24 hours.40 The rise in concentration of TNF-! in the perito-


57

neal fluid is proportional to both the length of surgery and degree of peritoneal bacterial contamination.42 Increased levels of TNF-! have also been found in the peritoneal fluid of women with endometriosis and infertility.38 Women with endometriosis are said to be more prone to postoperative adhesions,33 presumably due to the localized sterile inflammatory process and the large numbers of activated peritoneal macrophages. One group of investigators has shown TNF-! to be a good biological marker for intra-abdominal adhesion formation.34 They subjected three groups of rats to laparotomy: the first group acted as a control and had only saline irrigation of the peritoneal cavity, the second group underwent caecal abrasion while the third group had a limited small bowel resection and anastomosis. Levels of TNF-! were measured in the blood and peritoneal fluid at 30, 90 and 180 minutes after the operation. At 3 weeks the rats were killed and the adhesions graded. There were more adhesions in the groups which had caecal abrasion and small bowel resection compared to the control but there was also a significant correlation between the grade of adhesions and the concentration of TNF-! in the peritoneal fluid and plasma. Rats given intravenous antibodies to TNF-! preoperatively showed no significant reduction in adhesion formation unless also given antibodies to IL-1 when there was a synergistic reduction in adhesion formation.31 Peritoneal fibrinolytic activity plays a pivotal role in the pathophysiology of adhesions35 and TNF has been shown to mediate the release of plasminogen activator inhibitor-1 by human peritoneal mesothelial cells.36

Interleukin-6 (IL-6) This multifunctional cytokine is secreted by T cells, fibroblasts and macrophages in response to injury. IL-6 is a normal constituent of peritoneal fluid and several investigators have found elevated levels of IL-6 in the peritoneal fluid of women with adhesions, endometriosis and pelvic inflammatory disease.37-39 Following surgical injury there is a marked rise in IL-6 levels in both peritoneal fluid40-43 and plasma.40,44-47 This rise starts within six hours of the beginning of the operation, reaches a peak at 12 hours40 and is proportional to the duration of the operation.48 Peak concentrations of IL-6 are 300-fold greater in the peritoneal fluid than in the blood. This difference may be explained by a number of factors, including incomplete peritoneal absorption, dilution and first-pass hepatic metabolism. However it seems likely that the systemic IL-6 response is a secondary reflection of events within the peritoneal cavity. The rise in IL-6 levels in peritoneal fluid is more sustained than those of IL-1∀ and TNF-! reaching a plateau between 24 and 72 hours.46 The induction of IL-6 synthesis is probably stimulated by TNF-! and IL-1∀ released by resident mononuclear phagocytes following surgical injury.48,49 Intravenous injection of IL-6 prior to adhesion formation in a rat model resulted in a slight but significant increase in the grade of intraperitoneal adhesions, while treatment with anti-IL-6 antibody preoperatively showed a decrease in adhesion grade.50

Transforming Growth Factor-beta (TGF-∀) The central role of TGF-∀ in wound healing and tissue fibrosis is well recognized.51,52 Released initially by platelets and subsequently by macrophages, activated T cells and neutrophils, TGF-∀ is a potent regulator of inflammation and fibrosis. It

58


has a chemotactic action on fibroblasts and inflammatory cells and promotes angiogenesis, cell proliferation, differentiation and deposition of extracellular matrix.53,54 The evidence for the role of TGF-∀ in the formation and regulation of adhesions is based on (1) the role of TGF-∀ in fibrosis in other tissues; (2) the presence and (3) activity of TGF-∀ and/or its receptor in the peritoneal cavity and in adhesions; (4) the effect of exogenous TGF-∀ or its neutralizing antibody on the formation of adhesions in experimental models.52 Excessive or sustained production of TGF-∀1 is a key molecular mediator of tissue fibrosis in many organs. Topical application of TGF-∀ enhances experimental wound healing55 and intravenous administration leads to liver and kidney fibrosis in rats. 56 All three isoforms of TGF- ∀ ( TGF- ∀1 , TGF- ∀ 2, TGF- ∀3) are present immunohistochemically in surgically induced adhesions in a rat model. Fibroblasts, inflammatory cells and the endothelial and smooth-muscle cells of arterioles in the fibrous tissue all stain for TGF-∀.57 The intact peritoneum demonstrates minimal staining for TGF-∀3, but once injured by surgery or inflammation, levels of all three isoforms rise substantially.58 TGF-∀1 is also found in the peritoneal fluid of patients who have developed adhesions following intraperitoneal immunotherapy with interleukin-2 (IL-2) and IL-2 activated killer cells.58 Rats given TGF-∀ following surgery develop more severe adhesions than controls and histologically their adhesions have a greater density of inflammatory cells and fibroblasts.59 However administration of neutralizing antibody to TGF-∀ (all isoforms) is not effective in reducing the level of fibrous adhesions though it does reduce the cellularity of the fibrous tissue.59,60 More specific blockade by giving antibodies to the different TGF-∀ isoforms has shown a significant reduction in adhesion formation in a rat model when using anti-TGF-∀1 but not anti-TGF-∀2.60

Other Cytokines Epidermal growth factor (EGF) is known to stimulate fibroblast proliferation and collagen deposition.61 EGF, transforming growth factor-! (TGF-!) and the EGF receptor have been identified immunohistochemically in fibrous adhesions from both rats and patients.62 The highest immunostaining for EGF was associated with the inflammatory cells infiltrated into the fibrous adhesion. Interleukin-2 (IL-2) is produced by activated T lymphocytes and is known to stimulate the production of fibrogenic mediators from peripheral blood leucocytes.58 In rat peritoneal macrophage culture IL-2 induces the expression of platelet-derived growth factor (PDGF-! and ∀ chains) within two hours of treatment.63 As PDGF is a fibrogenic cytokine this suggests that it may play a role in the well-recognized development of adhesions in patients receiving intraperitoneal immunotherapy with IL-2-activated killer cells and IL-2.

Conclusions The healing of injured mesothelial surfaces is a complex process integrating both the coagulation cascade and the immune system in an effort to arrest bleeding, prevent infection and restore tissue integrity and function. Any imbalance in the control mechanisms of this process will lead to an impairment of wound healing or excess tissue formation—fibrosis; or adhesion formation. Cytokines have been shown to play a key role in adhesion formation following operation or other injury to the peritoneum. Adhesions form soon after surgery and


59

much attention has been focused on the cytokines that mediate the early inflammatory reaction. By experimentally augmenting or blocking the action of these cytokines the degree of adhesion formation can be altered. Cytokine manipulation to control adhesion formation is an attractive option. However further research is necessary into the complex interactions that occur between mediators during fibrin deposition and fibrinolysis. In addition, the localization of cytokine modification to the areas of adhesion formation, minimizing the systemic effects of therapy and preventing adhesions without obviating the beneficial aspects of the inflammatory process are all potential problems that remain to be addressed.

References 1. Menzies D, Ellis H. Intestinal obstruction from adhesions-how big is the problem? Ann R Coll Surg Eng 1990; 72:60-63. 2. Ellis H. The causes and prevention of intestinal adhesions. Br J Surg 1982; 69:241-243. 3. Ellis H. Prevention and treatment of adhesions. Infect Surg 1983:803-807. 4. Menzies D. Postoperative adhesions: Their treatment and relevance in clinical practice. Ann R Coll Surg Engl 1993; 75:147-153. 5. Menzies D. Peritoneal adhesions: incidence, cause and prevention. Ann Surg 1992; 24(Pt I):29-45. 6. Treutner K-H, Bertram P, Loser S et al. Prevention and treatment of peritoneal adhesions - a questionnaire survey on 1200 hospitals in Germany. Der Chirurg 1995; 66:398-403. 7. Scott-Coombes DM, Vipond M, Thompson JN. General surgeons attitudes to the treatment and prevention of abdominal adhesions. Ann R Coll Surg Eng 1993; 75:123-128. 8. Pijlman BM, Dorr PJ, Brommer EJP et al. Prevention of adhesions: Review. Eur J Obstet Gynecol Reprod Biol 1994; 53:155-163. 9. Trimbos-Kemper TCM, Trimbos JB, van Hall EV. Adhesion formation after tubal surgery: results of the eighth-day laparoscopy in 188 patients. Fertil Steril 1985; 43:395-400. 10. Diamond E. Lysis of postoperative pelvic adhesions in infertility. Fertil Steril 1979; 31:287-295. 11. Frantzen C, Schlösser HW. Microsurgery and postinfectious tubal infertility. Fertil Steril 1982; 38:397420. 12. diZerega GS. Biochemical events in peritoneal tissue repair. Eur J Surg 1997; Suppl 577:10-16. 13. Fox RN, Larsen JW, Stillman RJ et al. Economic impact of hospitalizations for lower abdominal adhesiolysis in the United States in 1988. Surg Gynecol Obstet 1993; 176:271-276. 14. Esperanza MJ, Collins DL. Peritoneal dialysis efficiency in relation to body weight. J Paediatr Surg 1966; 1:162-169. 15. Topley N, Mackenzie R, Jörres A et al. Cytokine networks in CAPD: interactions of resident cells during inflammation in the peritoneal cavity. Perit Dial Int 1993; 13:282-285. 16. Rodgers KE, diZerega GS. Function of peritoneal exudate cells after abdominal surgery. J Invest Surg 1993; 6:9-23. 17. Raftery AT. Regeneration of parietal and visceral peritoneum: An electron microscopical study. J Anat 1973; 115:321-328.

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18. Ellis H, Harrison W, Hugh TB. The healing of the peritoneum under normal and pathological conditions. Br J Surg 1965; 52:471-476. 19. Topley N, Brown Z, Jörres A et al. Human peritoneal mesothelial cells synthesize IL-8: synergistic induction by interleukin-1∀ and tumour necrosis factor-!. Am J Pathol 1993; 142:1876-1886. 20. Betjes MGH, Tuk CW, Struijk DG et al. Interleukin-8 production by human peritoneal mesothelial cells in response to tumour necrosis factor a, interleukin-1 and medium conditioned by macrophages co-cultured with Staphylococcus epidermidis. J Infect Dis 1993; 168:1202-1210. 21. Douvdevani A, Rapaport A, Konforty A et al. Human peritoneal mesothelial cells synthesize IL-1a and b. Kidney Int 1994; 46:993-1001. 22. Topley N, Williams JD. The role of the peritoneal membrane in the control of inflammation in the peritoneal cavity. Kidney Int 1994; 46:S71-78. 23. Scott-Coombes DM, Whawell SA, Thompson JN. Peritoneal cytokine response in surgery. Br J Surg 1994; 81:756. 24. Porter JM, McGregor FH Jr, Mullen DC et al. Fibrinolytic activity of mesothelial surfaces. Surg Forum 1969; 20:80-82. 25. Raftery AT. Regeneration of peritoneum: A fibrinolytic study. J Anat 1979; 129:659-664. 26. Vipond MN, Whawell SA, Thompson JN et al. Peritoneal fibrinolytic activity and intra-abdominal adhesions. Lancet 1990; 335:1120-1122. 27. Whawell SA, Wang Y, Fleming KA et al. Localisation of plasminogen activator inhibitor-1 production in inflamed appendix by in situ mRNA hybridisation. J Pathol 1993; 169:67-71. 28. Whawell SA, Thompson JN. Cytokine induced release of plasminogen activator inhibitor-1 by human mesothelial cells. Eur J Surg 1995; 161:315-317. 29. Dinarello CA, Mier JW. Lymphokines. N Engl J Med 1987;317:940-5. 30. Hershlag A, Otterness IG, Bliven ML et al. The effect of interleukin-1 on adhesion formation in the rat. Am J Obstet Gynecol 1991; 165:771-774. 31. Kaidi A, Nazzal M, Gurchumelidze T et al. Preoperative administration of antibodies against tumour necrosis factor-Alpha (TNF-!) and Interleukin-1 (IL-1) and their impact on peritoneal adhesion formation. Am Surg 1995; 58:302-306. 32. Rapala K. The effect of tumour necrosis factor-! on wound healing. Ann Chirurg Gynae 1996; 85 (Suppl.211):1-53. 33. Haney AF. Endometriosis, macrophages, and adhesions. Prog Clin Biol Res 1993; 381:19-44. 34. Kaidi AA, Gurchmelidze T, Nazzal M et al. Tumour necrosis factor-alpha: a marker for peritoneal adhesion formation. J Surg Res 1995; 58:516-518. 35. Vipond MN, Whawell SA, Thompson JN et al. Peritoneal fibrinolytic activity and intra-abdominal adhesions. Lancet 1990; 335:1120-1122. 36. Whawell SA, Scott-Coombes DM, Vipond MN et al. Tumour necrosis factor-mediated release of plasminogen activator inhibitor 1 by human peritoneal mesothelial cells. Br J Surg 1994; 81:214-216. 37. Buyalos RP, Watson JM, Funari VA et al. Elevated interleukin-6 levels in peritoneal fluid of patients with pelvic pathology. Fertil Steril 1992; 58:302-306. 38. Overton C, Fernandez-Shaw S, Hicks B et al. Peritoneal fluid cytokines and the relationship with endometriosis and pain. Hum Reprod 1996; 11:380-386. 39. Rier SE, Parsons AK, Becker JL. Altered interleukin-6 production by peritoneal leukocytes from patients with endometriosis. Fertil Steril 1994; 61:294-299. 40. Badia JM, Whawell SA, Scott-Coombes DM et al. Peritoneal and systemic cytokine response to laparotomy. Br J Surg 1996; 83(3):347-348.


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41. Tokunaga A, Onda M, Fujita I et al. Sequential changes in the cell mediators of peritoneal and wound fluids after surgery. Jpn J Surg 1993; 23:841-844. 42. Tsukada K, Katoh H, Shiojima M et al. Concentrations of cytokines in peritoneal fluid after abdominal surgery. Eur J Surg 1993; 159:475-479. 43. Fujita I, Onda M, Tokunaga A. Expression of interleukin-6 and tumour necrosis factor in peritoneal mononuclear cells after gastrectomy. Wound Rep Regen 1995; 3:168-175. 44. Baigre RJ, Lamont PM, Kwiatkowski D et al. Systemic cytokine response after major surgery. Br J Surg 1992; 79:757-760. 45. Ohzato H, Yoshizaki K, Nishimoto N et al. Interleukin-6 as a new indicator of inflammatory status: detection of serum levels of interleukin-6 and C-reactive protein after surgery. Surgery 1992; 111:201-209. 46. Sakamoto K, Arakawa H, Mita S et al. Elevation of circulating interleukin-6 after surgery: factors influencing the serum level. Cytokine 1994; 6:181-186. 47. Grzelak I, Olszewski WL, Zaleska M et al. Blood cytokine levels rise even after minor surgical trauma. J Clin Immunol 1996; 16(3):159-164. 48. Van Deuren M, Dofferhoff ASM, van der Meer JWM. Cytokines and the response to infection. J Pathol 1992; 168:349-356. 49. Topley N, Jörres A, Luttmann W et al. Human peritoneal mesothelial cells synthesize IL-6: Induction by IL-1∀ and TNF-!. Kidney Int 1993; 43:226-233. 50. Saba A, Kaidi A, Godziachvili, V et al. Effects of Interleukin-6 and its neutralising antibodies on peritoneal adhesion formation and wound healing. Am Surg 1996; 62:569-572. 51. Border WA, Noble NA. Transforming growth factor ∀ in tissue fibrosis. N Engl J Med 1994; 331(19):1286-1292. 52. Chegini N. The role of growth factors in peritoneal healing: transforming growth factor-beta. Eur J Surg (Suppl.) 1997; (577):17-23. 53. Wahl SM, Hunt DA, Wakefield LM et al. Transforming growth factor type ∀ induces monocyte chemotaxis and growth factor production. Proc Natl Acad Sci USA 1987; 84:5788-5792. 54. Postlethwaite AE, Keski-Oja J, Moses HL et al. Stimulation of the chemotactic migration of human fibroblasts by transforming growth factor ∀. J Exp Med 1987; 165:251-256. 55. Roberts AB, Sporn MB. Physiological actions and clinical applications of transforming growth factor ∀ (TGF-∀). Growth Factors 1993; 8:1-9. 56. Terrell TG, Working PK, Chow CP et al. Pathology of recombinant human transforming growth factor-b1 in rats and rabbits. Int Rev Exp Pathol 1993; 34:43-67. 57. Chegini N, Gold LI, Williams RS et al. Localization of transforming growth factor beta isoforms (TGF-beta1, TGF-beta2, TGF-beta3) in surgically induced pelvic adhesions in the rat. Obstet Gynecol 1994; 83:449-454. 58. Kovacs EJ, Brock B, Silber IE et al. Production of fibrogenic cytokines by interleukin2-treated peripheral blood leukocytes: Expression of transforming growth factorbeta and platelet-derived growth factor B chain genes. Obstet Gynecol 1993; 82:29-36. 59. Williams RS, Rossi AM, Chegini N et al. Effect of transforming growth factor b on postoperative adhesion formation and intact peritoneum. J Surg Research 1991; 52:65-70. 60. Lucas PA, Warejcka DJ, Young HE et al. Formation of abdominal adhesions is inhibited by antibodies to transforming growth factor-beta1. J Surg Res 1996; 65:135-138.

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61. Laato M, Niinikoski J, Lundberg C et al. Effect of epidermal growth factor on experimental granulation tissue. J Surg Res 1986; 41:252-255. 62. Chegini N, Simms J, Williams RS et al. Identification of epidermal growth factor, transforming growth factor-alpha and epidermal growth factor receptor in surgically induced pelvic adhesions in the rat and intraperitoneal adhesions in the human. Am J Obs Gyn 1994; 171:321-327. 63. Kovacs EJ, Van Stedum S, Neuman JE et al. Selective induction of PDGF gene expression in peritoneal macrophages by interleukin-2. Immunobiology 1994; 190(3):263-274.

Cytokines in Blunt Abdominal Trauma

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CHAPTER 6

Cytokines in Blunt Abdominal Trauma Matthias W. Wichmann and Eugen Faist

Introduction

B

lunt abdominal trauma may become a life-threatening situation for the trauma victim. A recent study by Brenneman et al1 showed that 73% of nonsurvivors following blunt trauma suffered from abdominal injuries. Furthermore, 57% of the surviving trauma victims presented with abdominal injuries.1 In total 65% (50/76) of all patients with severe blunt trauma (ISS 50) suffered from abdominal injuries.1 A study of 433 patients with multiple injuries by Faist et al2 showed abdominal involvement in 29% of all cases. Severe blunt abdominal trauma is not only associated with soft-tissue injury, but also occurs frequently in conjunction with some form of blood loss (hemorrhage). This can be concluded from the observation that most patients with blunt abdominal trauma present with lacerations of the liver and/or spleen as well as retroperitoneal hematoma.1 In this context it is well recognized that liver and/or spleen are the most frequently injured organs following blunt abdominal trauma.3 Laceration of the liver or the spleen is associated with blood loss into the organ itself or into the abdominal cavity once the organ-capsule is ruptured. Studying the effects of blunt abdominal trauma on the cytokine system should, therefore, focus on the effects of soft-tissue trauma and/or hemorrhage on this system of cellular mediators. With regard to this, the term cytokines includes lymphokines, interferons, colony stimulating factors and tumor necrosis factors.4 A number of experimental animal studies as well as clinical investigations have been carried out focusing on the effects of soft-tissue trauma and/or hemorrhage on the cytokine system. These studies mainly involved the following parameters: Interleukin- (IL-) 1, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, Tumor-Necrosis-Factor (TNF), Transforming-Growth-Factor (TGF), and Interferon (IFN). This chapter will provide an account of the results obtained in experimental animals, as well as in clinical studies on trauma patients.

Introduction to the Cytokines Studied in Trauma Research Cytokines are proteins of relatively low molecular mass which regulate all important biological processes, i.e., cell growth, cell activation, inflammation, immunity, Cytokines and the Abdominal Surgeon, edited by Moshe Schein and Leslie Wise. © 1998 R.G. Landes Company.

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tissue repair, fibrosis and morphogenesis.5 These proteins are effective at very low concentrations (pg/ml), which is due to their mode of action involving high-affinity receptors on the cell surface.5

Interleukin-1 IL-1 is a cytokine produced by activated mononuclear phagocytes and is a mediator of inflammation. It shares many properties in common with tumor necrosis factor. IL-1 is comprised of two principal polypeptides of 17 kDa which have the same biological activities and bind to the same receptor on cell surfaces. While the one IL-1 acts as a membrane-associated substance the other IL-1 is found in the circulation. The effects of IL-1 differ according to whether it is produced in lower or in higher concentrations. At low concentrations, the effects are mainly immunoregulatory, at higher levels IL-1 produces endocrine effects (fever, release of acutephase proteins).6 Virtually all cells of the body have receptors for IL-1 and can respond to it. In the brain IL-1 induces fever and it causes increased release of corticosteroids. Furthermore, the production of prostaglandins and degradative enzymes is induced in B and T lymphocytes.5

Interleukin-2

IL-2 is a 15.5 kDa glycoprotein which is synthesized by CD4+ T helper lymphocytes; a small amount is also produced by CD8+ T cells. The amount of IL-2 produced by CD4+ T helper cells is a principal marker of the strength of an immune response. Inadequate IL-2 formation can, therefore, lead to antigen-specific T lymphocyte anergy. High levels of IL-2 are required for stimulation of natural killer cells.6 IL-2 is used in experimental cancer therapy, especially for renal cancer; its benefit may be related to the activation of cells that can produce cytotoxic anticancer effects.5

Interleukin-3

IL-3 is a 20 kDa lymphokine which is synthesized by activated CD4+ T helper lymphocytes and acts as a colony-stimulating factor. It promotes proliferation and differentiation of other lymphocytes as well as mast cell proliferation.6 IL-3 stimulates the growth of precursors of all the hemopoietic lineages.5

Interleukin-4

IL-4 is a 20 kDa cytokine which is synthesized mainly by CD4+ T helper cells and by activated mast cells. IL-4 induces switching of B lymphocytes to produce IgE and it may have some role in cell-mediated immunity. In mice IL-4, furthermore, serves as a growth and differentiation factor for B cells.6 In macrophages IL-4 induces MHC class II expression and inhibits cytokine release (TNF, IL-1, IL-6).7

Interleukin-6 IL-6 is a 26 kDa cytokine produced by endothelial cells, mononuclear phagocytes, fibroblasts, activated T lymphocytes, and a number of neoplasms. IL-6 is secreted in response to IL-1, IFN-! or TNF and mainly acts on hepatocytes and B cells. Hepatocytes form acute-phase proteins and B lymphocytes differentiate into antibody forming cells in response to IL-6.6 IL-6 is a central regulatory protein involved in the control of acute phase protein synthesis in hepatocytes.7


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Interleukin-8 IL-8 is an 8 kDa protein produced by macrophages and endothelial cells. It has a strong chemotactic effect on T lymphocytes and neutrophils and it has antiviral, immunomodulatory, as well as antiproliferative properties. Furthermore, IL-8 blocks the neutrophil-mediated injury; on the other hand it facilitates neutrophil adherence to endothelial cells. Moreover, it is involved in inflammation and cell-migration.6

Interleukin-10 IL-10 is an 18 kDa polypeptide which acts as a cytokine synthesis inhibitory factor. It is synthesized by CD4+ and CD8+ T lymphocytes, monocytes, macrophages, activated B cells, and keratinocytes. IL-10 inhibits cytokine synthesis (TNF, IL-1, IL-6) by TH1 cells, blocks antigen presentation, and the formation of interferon. IL-10 also inhibits antigen presentation by macrophages, thereby blocking the formation of IL-1, IL-6 and TNF.6

Interleukin-12 IL-12 is a heterodimeric molecule comprised of 35 and 40 kDa chains. It is a growth factor for activated CD4+ and CD8+ T cells and for natural killer cells. IL-12 may have future importance for the therapeutic use in the treatment of tumors or infections.6

Interleukin-13 IL-13 has a molecular weight of 10 kDa and is synthesized by activated T cells and inhibits inflammatory cytokine production in human peripheral blood lymphocytes.8 It can be considered a modulator of B cell responses and, furthermore, suppresses the cytotoxic functions of monocytes/macrophages and the generation of proinflammatory cytokines.6

Tumor Necrosis Factor-∀ TNF-∀ is a 50 kDa cytotoxic monokine which participates in inflammation, wound healing, and remodeling of tissue. It can, furthermore, induce septic shock and cachexia. TNF-∀ is synthesized by a number of cells including monocytes, macrophages, T lymphocytes, B lymphocytes, and natural killer cells.6 TNF stimulates the release of IL-1, IL-6, IL-8, platelet activating factor, leukotrienes, thromboxane A2, and prostaglandins.7

Transforming Growth Factor TGF is a 25 kDa multi functional cytokine which plays a role in immunosuppression following trauma and supports wound healing as well as collagen synthesis. TGF, furthermore, counteracts the activation of T and B lymphocytes.9 It has been described as an anti-inflammatory cytokine which inhibits the synthesis of monocyte/macrophage-derived proinflammatory cytokines.10 TGF decreases TNF-∀ and IL-6 synthesis on a transcriptional level, while reducing IL-1 release on a posttranscriptional level.7

Interferon-! IFN-! is a glycoprotein that is a 21 to 24 kDa homodimer synthesized by activated T lymphocytes and natural killer cells. It has antiproliferative and antiviral properties and is a strong activator of mononuclear phagocytes as well as natural killer cells. IFN-! induces expression of class II MHC molecules on a number of

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different cells and contributes to the differentiation of B and T lymphocytes.6 A decrease of immune activation leads to increased production of IFN-! and an increase in antigen-presenting-cell function. Excessive production of IFN-! may play a part in the induction of autoimmune diseases.5

Pro- and Anti-Inflammatory Mediators Despite the magnitude of effects and target tissues/cells of the above mentioned mediators, some of these cytokines are also referred to as pro- or anti-inflammatory mediators. So-called proinflammatory mediators are: Tumor Necrosis Factor, IL-1, IL-6 and IL-8. Anti-inflammatory mediators are: IL-4, IL-10, IL-13, Transforming Growth Factor.7

Experimental Trauma Research A number of different animals, such as rats, mice, pigs, rabbits and baboons, have been used to study the effects of blood loss and/or soft-tissue trauma on immune responses. Nonetheless, most of the work performed in this area of research was carried out using rodents. This is due to the fact that these animals are cheaper than larger animals, easier to handle and, furthermore, standardized immunological assays exist for these animals, especially for mice.11 Blunt abdominal trauma is associated with some form of tissue injury. In this regard it is well known that tissue trauma per se induces significant depression of cell-mediated and humoral immunity.12-14 Moreover, it was observed that hemorrhagic shock without soft-tissue trauma also results in significant depression of humoral and cell-mediated immunity.15-17 In addition it was observed that simple hemorrhage results in increased susceptibility to sepsis.18 Nonetheless, a recent study demonstrated that tissue trauma or hemorrhagic shock alone, does not produce a long-lasting depression of cell-mediated and humoral immunity.19 In their study Zellweger et al19 compared the effects of laparotomy (soft-tissue trauma) alone, hemorrhagic shock alone and of the combination of soft-tissue trauma with hemorrhage on macrophage and splenocyte function at 5 days after the experiment. In this study a significant depression of splenocyte and macrophage immune function at 120 hrs after trauma was only observed in animals subjected to soft-tissue trauma in conjunction with hemorrhagic shock.19 These findings indicate that only severe blunt abdominal trauma which results in soft-tissue trauma and (major) blood loss should be considered for studies of the cytokine system. With regard to this, it has been observed that soft-tissue trauma in conjunction with hemorrhagic shock resulted in a long-lasting depression of cellmediated immunity as indicated by depressed splenocyte proliferative capacity, depressed IL-2 and IL-3 release as well as depressed macrophage IL-6 release at 5 days after onset of the experiment.19 This observation of markedly depressed splenocyte and macrophage function was confirmed in a number of subsequent studies.20-22 It is of interest, that in one of these studies it was shown that closed bone fracture alone did not produce any deleterious effects on macrophage and splenocyte immune function, as opposed to the effects of soft-tissue trauma alone.22 In additional physiological studies using rats instead of mice it was observed that trauma-hemorrhage results in endothelial cell dysfunction, impaired hepatocellular function, depressed cardiac output and impaired microcirculation.23-25


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Experimental Immunomodulation Following Trauma-Hemorrhage Several studies were carried out to determine the potentially beneficial effects of different drugs and hormones on the depressed cell-mediated and humoral immunity following trauma-hemorrhage. In this respect beneficial effects of pharmacological interventions are defined as the return of cell-mediated and humoral immune functions to levels comparable to those observed in sham-operated animals. With regard to this, beneficial effects were observed following the administration of interferon-!,26,27 ATP-MgCl2,28-30 n-3 polyunsaturated fatty acids,31 ibuprofen,32 chloroquine,33-35 anti-TNF antibodies,36 diltiazem,37 chemically modified/nonanticoagulant heparin,25,38 pentoxifylline,39 PAF-antagonists,40 prolactin41,42 and melatonin.21,43 While a number of these interventions were not as effective in the clinical setting as in the laboratory, some of these drugs still have to be clinically tested, e.g., ATPMgCl2, chemically modified heparin, n-3 polyunsaturated fatty acids, prolactin and melatonin.

Clinical Trauma Research The response to trauma begins in the immune system at the moment of injury and all patterns of trauma (sepsis, hemorrhage, ischemia, ischemia-reperfusion, softtissue trauma, burns) share the ability to activate macrophages and induce the release of proinflammatory cytokines.44,45 The production and release of all cytokines so far examined is altered by physiologic stress and it is well recognized that changes in the release of various cytokines have an important role in mediating functional changes of the immune as well as the circulatory system.46 During recent years a large number of clinical investigators have studied the effects of trauma on immunological parameters as indicated by circulating cytokine levels and/or the in vitro release of cytokines by peripheral blood mononuclear cells. Circulating levels of IL-6 and TNF were studied following elective surgery/trauma and while no changes of circulating TNF levels following surgery/trauma were observed, the authors reported a rise in acute phase protein levels (C-reactive protein, CRP) following the peak circulating levels of IL-6.47 Nonetheless, the observed changes in plasma IL-6 levels showed only poor correlation with blood loss, fever, white cell count or duration of surgery.47 Another study confirmed the observed correlation between circulating CRP and IL-6 levels.48 These investigators, however, reported a close relationship between the duration of surgery and the circulating levels of IL-6, an observation which is supported by another, more recent, study.48,49 Moreover, this correlation between trauma, rise in circulating IL-6 and CRP levels was subsequently confirmed in a study involving patients undergoing pancreaticoduodenectomy.50 In a different study involving patients with major surgery, TNF∀ and IFN-! were not detected in the circulation, while an early and short-lived IL-1 response to trauma could be observed by the authors.51 Again a rise in circulating IL-6 was observed following surgical trauma in this study.51 A study on trauma patients by Fabian et al52 did not show any overproduction of TNF in whole blood following trauma, which makes the therapeutic targeting of TNF in exaggerated inflammatory response difficult to justify. On the other hand, an in vitro study by Kim et al8 showed a strong immunomodulatory potential of IL-13 on the increased production of TNF-∀, IL-1, IL-6 and IL-8 by peripheral monocytes obtained from trauma patients.

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An analysis of serial postresuscitation patient samples by Hoch et al53 demonstrated rapid increases in IL-6 and IL-8 within 12 hours after injury. Increased levels of IL-6 and IL-8 were observed for more than 5 days in patients with an ISS 25, while no significant elevation of TNF-∀ could be observed at any time-point.53 Surprisingly, in a recent study by Hauser et al54 no increased gene expression of the proinflammatory cytokines TNF-∀, IL-1 and IL-6 was observed in peripheral blood mononuclear cells from trauma patients. Furthermore, these authors reported a significant increase in IL-10 expression, while no change in IL-4 could be detected.54 This observation, which is inconsistent with the occurrence of systemic endotoxemia and subsequent global immunocyte activation after trauma may be due to the fact that the patterns of gene expression do not always correlate well with the detected levels of cytokine in the circulation and/or the culture supernatant of immunocytes. In a recent study by Schinkel et al45 it was shown that massive trauma causes the immediate activation of chemotactic cytokines (IL-8, ENA-78) and soluble adhesion molecules (sE-selectin, sP-selectin) within minutes after trauma which subsequently persist for a number of weeks (depending on the trauma mechanism). Nonetheless, no correlation between serum concentrations of these mediators and the prediction of infectious complications or outcome could be observed in this study.45 Despite the above mentioned studies and the so far inconsistent information which was collected in clinical studies it appears that increased generation of IL-1 and IL-6 (and maybe TNF) contribute to the acute phase response and hypermetabolic phase which accompanies trauma.46 Furthermore, severe immunosuppression which involves T- and B-cell function may result from the various effects of stress-related alterations in cytokine release.46 In this respect it has been proposed that traumatic stress causes a severe disintegration of the intact monocyte-T cell interaction, which is associated with profound changes in macrophage forward-regulatory capacities and substantial depression of T cell function.55 Prostaglandin E2 (PGE2) is considered as a major mediator of this stress/trauma induced immune suppression, since increased PGE2 synthesis and release results in decreased T cell mitogenesis, decreased IL-2 release and IL-2 receptor expression.55 Furthermore, PGE2 causes a shift of T-helper activities towards the TH2 direction which results in increased release of the immunosuppressive cytokines IL-4 and IL-10.55 Whether or not this proposed central role for PGE2 in the severe immune dysfunction following trauma holds true in the clinical situation certainly needs further thorough investigation. As far as cytokine alterations following blunt abdominal trauma in patients are concerned, it needs to be emphasized that blunt abdominal trauma alone, without blood loss and hypotension and/or without major tissue destruction probably does not induce significant changes of circulating cytokines. We, therefore, focused in this chapter on the known effects of hemorrhagic shock and/or soft-tissue injury as a consequence of blunt abdominal trauma on the cytokine network.

Conclusions To date a large body of information and data exists concerning the effects of trauma on the cytokine network. This data, however, was collected mainly in experimental animal research. Clinical studies did not always support the experimental laboratory results since patients and clinical trauma settings usually do not compare very well with the laboratory. Nevertheless, a clear cause and effect relationships between trauma, the acute phase response and the cytokine network (represented by IL-6) has been established. This indicates that the detection methods for the cytokines


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possibly involved in the host reaction to trauma may not always be as accurate as necessary and, furthermore, it is very well possible that researchers are not always looking for the correct cytokine in the correct body fluid at the correct point of time following trauma. Finally, clinical researchers are limited in their access to certain body fluids/tissues (e.g., cerebrospinal fluid, liver tissue, splenic tissue). All of these limitations make clinical research of the cytokine system much more difficult than laboratory research and a lot of clinical research still remains to be carried out before routine determination of cytokines following trauma will be established as a means of clinical work-up of trauma-patients. Moreover, a long way is ahead of clinical researchers before the observed immunological changes following traumatic insult can be beneficially influenced by immunomodulatory therapeutic interventions. These immune system derived interventions should protect all cellular host defense compartments from hyper-activation as well as exhaustion and we are convinced that only a combination of drugs and therapeutical efforts can effectively control this posttraumatic dyshomeostasis of the various cell systems.55

References 1. Brenneman FD, Boulanger BR, McLellan BA et al. Acute and Long-Term Outcomes of Extremely Injured Blunt Trauma Victims. J Trauma 1995; 39:320-324. 2. Faist E, Baue AE, Dittmer H. Multiple organ failure in polytrauma patients. J Trauma 1983; 23:775-787. 3. Davis KA, Brody JM, Cioffi WG. Computed tomography in blunt hepatic trauma. Arch Surg 1996; 131:255-260. 4. Rook G. Cel-Mediated Immune Reactions. In: Roitt I, Brostoff J, Male D, eds. Immunology. 3rd ed. St. Louis; Baltimore; Boston: Mosby, 1993:8.1-8.16. 5. Feldman M. Cell Cooperation in the Antibody Response. In: Roitt I, Brostoff J, Male D, eds. Immunology. 3rd ed. St. Louis; Baltimore; Boston: Mosby, 1993: 7.1-7.16. 6. Cruse JM, Lewis RE. Illustrated Dictionary of Immunology. Boca Raton; New York; London: CRC Press, 1995. 7. Ertel W, Scholl FA, Trentz O. The role of anti-inflammatory mediators for the control of systemic inflammation following severe injury. In: Faist E, Baue AE, Schildberg FW, eds. The Immune Consequences of Trauma, Shock and Sepsis. Mechanisms and Therapeutic Approaches. Lengerich; Berlin; Duesseldorf: Pabst Science Publishers, 1996:453-470. 8. Kim C, Schinkel C, Fuchs D et al. Interleukin-13 Effectively Down-regulates the Monocyte Inflammatory Potential During Traumatic Stress. Arch Surg 1995; 130:1330-1336. 9. Bank U, Reinhold D, Kunz D et al. Regulation of neutrophil degranulation by interleukin-6 and transforming growth factor. In: Faist E, Baue AE, Schildberg FW, eds. The Immune Consequences of Trauma, Shock and Sepsis. Mechanisms and Therapeutic Approaches. Lengerich; Berlin; Duesseldorf: Pabst Science Publishers, 1996:335-345. 10. Cavaillon J, Marie C, Pitton C et al. Regulation of neutrophil derived IL-8 production by anti-inflammatory cytokines. In: Faist E, Baue AE, Schildberg FW, eds. The Immune Consequences of Trauma, Shock and Sepsis. Mechanisms and Therapeutic Approaches. Lengerich; Berlin; Duesseldorf: Pabst Science Publishers, 1996:327-334. 11. Chaudry IH, Ayala A. Models of hemorrhage and hemorrhagic shock. In: Chaudry IH, Ayala A, eds. Immunological Aspects of Hemorrhage. Austin; Georgetown: R.G. Landes Company, 1992:35-40.

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12. McRitchie DI, Girotti MJ, Rotstein OD et al. Impaired antibody production in blunt trauma. Arch Surg 1990; 125:91-96. 13. Faist E, Ertel W, Mewes A et al. Trauma-induced alterations of the lymphokine cascade. In: Faist E, Ninnemann J, Green D, eds. Immune Consequences of Trauma, Shock, and Sepsis: Mechanisms and Therapeutic Approaches. Berlin: Springer Verlag, 1989:79-94. 14. Stephan RN, Mitsuyoski S, Conrad PJ et al. Depressed antigen presentation function and membrane interleukin 1 activity of peritoneal macrophages after laparotomy. Surgery 1987; 102:147-154. 15. Stephan RN, Kupper TS, Geha AS et al. Hemorrhage without tissue trauma produces immunosuppression and enhances susceptibility to sepsis. Arch Surg 1987; 122:62-68. 16. Chaudry IH, Ayala A, Ertel W et al. Editorial Review. Hemorrhage and resuscitation: Immunological Aspects. Am J Physiol 1990; 259:R663-R678. 17. Schmand JF, Ayala A, Chaudry IH. Effects of trauma, duration of hypotension, and resuscitation regimen on cellular immunity following hemorrhagic shock. Crit Care Med 1994; 22:1076-1083. 18. Ayala A, Perrin MM, Wagner MA et al. Enhanced susceptibility to sepsis following simple hemorrhage: Depression of Fc and C3b receptor mediated phagocytosis. Arch Surg 1990; 125:70-75. 19. Zellweger R, Ayala A, Zhu X et al. Effect of surgical trauma on splenocyte and peritoneal macrophage immune function. J Trauma 1995; 39:645-650. 20. Wichmann MW, Zellweger R, DeMaso CM et al. Mechanisms of immunosuppression in males following trauma hemorrhage: Critical role of testosterone. Arch Surg 1996; 131:1186-1192. 21. Wichmann MW, Zellweger R, DeMaso CM et al. Melatonin administration attenuates depressed immune functions after trauma-hemorrhage. J Surg Res 1996; 63:256-262. 22. Wichmann MW, Zellweger R, Williams C et al. Immune function is more compromised following closed bone fracture and hemorrhagic shock than hemorrhage alone. Arch Surg 1996; 995-1000. 23. Wang P, Ba ZF, Chaudry IH. Endothelial cell dysfunction occurs very early following trauma-hemorrhage and persists despite fluid resuscitation. Am J Physiol 1993; 265:H973-H979. 24. Wang P, Ba ZF, Lu MC et al. Measurement of circulating blood volume in vivo after trauma-hemorrhage and hemodilution. Am J Physiol 1994; 266:R368-R374. 25. Wang P, Ba ZF, Chaudry IH. Chemically modified heparin improves hepatocellular function, cardiac ouput, and microcirculation after trauma-hemorrhage and resuscitation. Surgery 1994; 116:169-176. 26. Ayala A, Wang P, Chaudry IH. Insights into the mechanism by which interferongamma improves macrophage function following hemorrhage and resuscitation. J Surg Res 1993; 54:322-327. 27. Ertel W, Morrison MH, Ayala A et al. Interferon-gamma attenuates hemorrhageinduced suppression of macrophage and splenocyte functions and decreases susceptibility to sepsis. Surgery 1992; 111:177-187. 28. Chaudry IH, Ohkawa M, Clemens MG. Improved mitochondrial function following ischemia and reflow by ATP-MgCl2. Am J Physiol 1984; 246:R799-R804. 29. Chaudry IH. Use of ATP following shock and ischemia. Ann N Y Acad Sci 1990; 603:130-141. 30. Wang P, Ba ZF, Morrison MH et al. Mechanism of the beneficial effects of ATPMgCl2 following trauma-hemorrhage and resuscitation: Downregulation of inflammatory cytokine (TNF, IL-6) release. J Surg Res 1992; 52:364-371.


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31. Ertel W, Morrison MH, Ayala A et al. Modulation of macrophage membrane phospholipids by n-3 polyunsaturated fatty acids increases interleukin-1 release and prevents suppression of cellular immunity following hemorrhagic shock. Arch Surg 1993; 128:15-21. 32. Ertel W, Morrison MH, Meldrum DR et al. Ibuprofen restores cellular immunity and decreases susceptibility to sepsis following hemorrhage. J Surg Res 1992; 53:55-61. 33. Ertel W, Morrison MH, Ayala A et al. Chloroquine attenuates hemorrhagic shockinduced immunosuppression and decreases susceptibility to sepsis. Arch Surg 1992; 127:70-76. 34. Ertel W, Morrison MH, Ayala A et al. Chloroquine attenuates hemorrhagic shock induced suppression of Kupffer cell antigen presentation and MHC class II antigen expression through blockade of tumor necrosis factor and prostaglandin release. Blood 1991; 78:1781-1788. 35. Zhu X, Ertel W, Ayala A et al. Chloroquine inhibits macrophage tumour necrosis factor- mRNA transcription. Immunology 1993; 80:122-126. 36. Ertel W, Morrison MH, Ayala A et al. Anti-TNF monoclonal antibodies prevent haemorrhage induced suppression of Kupffer cell antigen presentation and MHC class II antigen expression. Immunology 1991; 74:290-297. 37. Meldrum DR, Ayala A, Chaudry IH. Mechanism of diltiazem’s immunomodulatory effects after hemorrhage and resuscitation. Am J Physiol 1993; 265:C412-C421. 38. Zellweger R, Ayala A, Zhu X et al. A novel nonanticoagulant heparin improves splenocyte and peritoneal macrophage immune function after trauma-hemorrhage and resuscitation. J Surg Res 1995; 59:211-218. 39. Wang P, Ba ZF, Zhou M et al. Pentoxifylline restores cardiac output and tissue perfusion following trauma-hemorrhage and decreases susceptibility to sepsis. Surgery 1993; 114:352-359. 40. Zellweger R, Ayala A, Schmand JF et al. PAF-antagonist administration after hemorrhage-resuscitation prevents splenocyte immunodepression. J Surg Res 1995; 59:366-370. 41. Zellweger R, Wichmann MW, Ayala A et al. Prolactin: a novel and safe immunomodulating hormone for the treatment of immunodepression following severe hemorrhage. J Surg Res 1996; 63:53-58. 42. Zhu X, Zellweger R, Ayala A et al. Prolactin inhibits the increased cytokine gene expression in Kupffer cells following haemorrhage. Cytokine 1996; 8:134-140. 43. Wichmann MW, Haisken JM, Ayala A et al. Melatonin administration following hemorrhagic shock decreases mortality from subsequent septic challenge. J Surg Res 1996; 65:109-114. 44. Harris BH, Gelfand JA. The immune response to trauma. Semin Pediatr Surg 1995; 4:77-82. 45. Schinkel C, Faist E, Zimmer S et al. Kinetics of circulating adhesion molecules and chemokines after mechanical trauma and burns. Eur J Surg 1996; 162:763-768. 46. Abraham E. Effects of stress on cytokine production. Methods Achiev Exp Pathol 1991; 14:45-62. 47. Pullicino EA, Carli F, Poole S et al. The relationship between the circulating concentrations of interleukin 6 (IL-6), tumor necrosis factor (TNF) and the acute phase response to elective surgery and accidental injury. Lymphokine Res 1990; 9:231-238. 48. Yoshizaki K. Clinical significance of cytokines-interleukin 6 in disease. Rinsho Byori 1990; 38:375-379. 49. Sakamoto K, Arakawa H, Mita S et al. Elevation of circulating interleukin 6 after surgery: factors influencing the serum level. Cytokine 1994; 6:181-186.

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50. Wortel CH, van Deventer SJ, Aarden LA et al. Interleukin-6 mediates host defense responses induced by abdominal surgery. Surgery 1993; 114:564-570. 51. Baigrie RJ, Lamont PM, Dallman M et al. The release of interleukin-1 beta (IL-1) precedes that of interleukin 6 (IL-6) in patients undergoing major surgery. Lymphokine Cytokine Res 1991; 10:253-256. 52. Fabian TC, Croce MA, Fabian MJ et al. Reduced tumor necrosis factor production in endotoxin-spiked whole blood after trauma: Experimental results and clinical correlation. Surgery 1995; 118:63-72. 53. Hoch RC, Rodriguez R, Manning T et al. Effects of accidental trauma on cytokine and endotoxin production [see comments]. Crit Care Med 1993; 21:839-845. 54. Hauser CJ, Lagoo S, Lagoo A et al. Human peripheral mononuclear cells do not show proinflammatory patterns of cytokine transcription in early trauma: a preliminary report. Shock 1995; 4:247-250. 55. Faist E, Schinkel C, Zimmer S. Update on the Mechanisms of Immune Suppression of Injury and Immune Modulation. World J Surg 1996; 20:454-459.

Cytokines in Acute Pancreatitis

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CHAPTER 7

Cytokines in Acute Pancreatitis James Norman and Larry C. Carey

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cute pancreatitis is a serious clinical entity for which no directed therapy exists beyond simple supportive care. More than 250,000 people are hospitalized with a bout of acute pancreatitis every year in the United States, a rate which is similar in most European countries. Although the causative etiology differs from report to report depending on the type of hospital and the country from which the study originated, most experts now believe the cause is fairly uniform world wide. Alcohol and gallstones combine as etiologic factors for approximately 90% of all cases, with the remainder being secondary to bile duct instrumentation, hyperlipidemia, hypercalcemia, trauma, and a few other rare causes. The actual inciting event leading to the development of pancreatic inflammation is still poorly understood. Most investigators believe that the normal process of proenzyme production, packaging, and secretion is impeded leading to one enzyme activating another, a process termed colocalization. The prematurely activated enzymes are then preferentially secreted at the basolateral rather than apical aspect of the acinar cell. The end result is the presence of activated digestive enzymes within the pancreatic interstitium. Many treatments have been proposed and tested which aim to attenuate this autodigestive process, yet a number of clinical trial have failed to show a benefit.1

Clinical Pancreatitis and the Immune Response Regardless of whether the initiating event is alcohol, gallstones or some other less common cause, acute pancreatitis progresses in a predictable manner through several pathologic stages (Fig. 7.1). The first is the intracellular disturbances described above which lead to activation of digestive enzymes. Very quickly, a local inflammatory process is initiated which results in the local production of inflammatory mediators. Virtually all patients with acute pancreatitis will experience these two phases, with some resolving completely at this point. Most patients, however, will go on to enter the third phase which is characterized by a systemic hyperinflammatory state expressed as the development of fever, tachycardia, tachypnea, and mild acid-base disturbances.2 Although this systemic hyperinflammatory state is usually mild, occasionally it may be very severe resulting in overt distant organ failure. The forth and final stage of pancreatitis is relatively uncommon and occurs a number of days or even weeks following the initial inflammatory stages and is characterized by the development of local complications such as infected necrosis, pancreatic abscess, or pseudocyst formation. Cytokines and the Abdominal Surgeon, edited by Moshe Schein and Leslie Wise. © 1998 R.G. Landes Company.

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Fig. 7.1. The four pathologic stages of acute pancreatitis. Pancreatitis progresses through four rather predictable states beginning with the activation of intrapancreatic enzymes leading to the rapid development of local inflammation. Activated enzymes as well as products of local inflammation can have dramatic effects on acinar cell viability. The third phase of acute pancreatitis begins shortly after the second and is characterized by the signs and symptoms associated with the systemic production of inflammatory mediators. The trigger for this systemic hyperinflammatory response is still unclear but the mediators produced are ultimately responsible for the distant organ dysfunction, hypoperfusion, tachycardia, and fluid shifts known collectively as the systemic inflammatory response syndrome (SIRS). The fourth phase occurs in a minority of acute pancreatitis patients a week or more into the disease and is characterized by the development of local complications such as infected necrosis and pseudocyst formation.

The key to understanding the complex pathophysiology of acute pancreatitis lies in determining why some patients fail to develop systemic hyper-inflammation and the systemic inflammatory response syndrome (SIRS) associated with it. These patients have a quick, uncomplicated recovery. This response is contrasted by others who rapidly progress into wide-spread inflammatory mediator over-production leading to distant organ dysfunction. Significant strides were taken in understanding this process in the early 1990s with the discovery that acute pancreatitis was associated with the appearance of dramatic levels of circulating inflammatory cytokines. A number of investigators followed the appearance of cytokines in patients hospitalized with pancreatitis hoping to find a marker predictive of disease severity. Each has shown that circulating levels of IL-6 and IL-8 (and often IL-1 and TNF) were higher in patients with complicated pancreatitis than they were in those with simple uncomplicated disease. When compared to Ranson’s and APACHE scoring, serum cytokine concentrations were usually found to be more specific in predicting the severity of acute pancreatitis, end-organ failure, overall mortality and even the duration of hospital stay. These close correlations provided the first direct evidence that cytokines play a major role in determining just how far a patient progresses down the path from local to systemic illness.3 IL-6 is a member of the inflammatory cytokine family which is well characterized as the primary inducer of the acute phase protein response during all types of


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insults in direct correlation to their severity.4 It is not surprising, therefore, that the severity of pancreatitis would be manifest by the degree and duration of IL-6 elevation. Patients with complicated and lethal pancreatitis have significantly higher serum levels of IL-6 than those with simple uncomplicated disease. This correlation is so high that a number of authors advocate measurement of IL-6 during the first 24 hours of acute pancreatitis onset to help decide the need for more aggressive resuscitation and monitoring techniques including whether to put a patient in the ICU and initiate invasive hemodynamic monitoring. IL-8 is also recognized as an inflammatory cytokine whose primary target cell is the neutrophil, where it causes degranulation and the release of enzymes (e.g., elastase) capable of tissue destruction when overproduced5 (Fig. 7.2). The relationship between pancreatitis severity and circulating levels of IL-8 parallels IL-6 production closely. Results from recent clinical trials of a platelet activating factor antagonist have confirmed the ability of these two cytokines to predict outcome and the occurrence of organ dysfunction,6,7 yet the lack of rapid, reproducible means to determine IL-6 or IL-8 levels by most hospitals has thus far kept these mediators from becoming more commonly used in the clinical setting. As such tests become available, it is likely that measurement of IL-6 and/or IL-8 early in the course of pancreatitis will become routine. Although IL-6 and IL-8 are reliable measures of systemic stress, their production is not felt to be the driving force behind pancreatitis-associated organ dysfunction and therefore they are not potential targets for antagonism at this time. In contrast, IL-1 and TNF are the primary members of the inflammatory cytokine family, are primary inducers of IL-6 and IL-8 production, and are believed to initiate and/or propagate nearly all of the detrimental consequences of severe pancreatitis, such as fever, hypoperfusion, circulatory collapse, shock, metabolic acidosis, cardiac dysfunction, and the production of ARDS. The presence of both in the serum and ascites of patients with severe pancreatitis has been documented by a number of investigators, but this is an inconsistent finding. Production of both of these cytokines is closely regulated and occurs only briefly in most circumstances, and as during sepsis, their presence in the systemic circulation during pancreatitis portends a very poor prognosis. As will be discussed subsequently, nearly all experimental models of pancreatitis have implicated IL-1 and TNF as the major pathologic cytokines associated with local and systemic tissue destruction. It must be remembered that the presence of inflammatory cytokines in the serum is not a prerequisite for them to be responsible for the development of organ dysfunction and SIRS.

Amplification of the Pancreatitis-Associated Inflammatory Cascade Although there has been a recent emphasis on the importance of cytokine activity during pancreatitis, a number of other inflammatory mediators are also believed to play a major role in the progression of pancreatitis into a systemic process.8 Those which contribute significantly include platelet activating factor (PAF), nitric oxide (NO), and reactive oxygen intermediates, with bradykinin and compliment likely playing a less important role. Figure 7.2 demonstrates the close relationships and means for amplification which exist between each of the aforementioned compounds produced during severe acute pancreatitis. It is the cumulative effect of these mediators which eventually leads to vascular leakage, hypovolemia, ARDS, shock, and MOSF. As illustrated, essentially all are produced under the influence of inflammatory cytokines or play an active role in initiating or amplifying the cytokine cascade. For

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example, PAF is a very potent vasodilator and leukocyte activator which has been implicated in the development of SIRS during acute pancreatitis. The production of PAF is so intimately tied to that of IL-1 and TNF that inhibition of PAF attenuates IL-1 and TNF production just as inhibition of either of these cytokines attenuates PAF production. Although our emphasis has been placed on the role of cytokines, their production influences the appearance of other mediators which also play a large role in determining pancreatitis outcome.

Local Cytokine Production During Pancreatitis Concomitant with the clinical observations of cytokine and PAF activity during acute pancreatitis was the finding that all animal models of pancreatitis demonstrate the same phenomenon. Inflammatory cytokines are produced in the pancreas itself within 30 minutes of experimental pancreatitis induction, which is often prior to appreciable changes in pancreatic histology.9,10 Although it has been suggested that the acinar cell may produce IL-1 and TNF, the majority of evidence to date suggests that leukocytes which invade the pancreas in great number during pancreatitis are the predominant source. One of the most important observations made during these experiments was that the concentration of TNF and IL-1 within pancreatic tissues was several orders of magnitude higher than they were in the serum, levels which are known to be toxic to many cell types. These findings were substantiated by showing high levels of TNF in the portal vein of animals with pancreatitis which was significantly higher than corresponding systemic levels. It is believed that the liver serves to clear TNF produced in the pancreas thereby preventing it from reaching the general circulation. These findings also help explain the difficulty in isolating TNF routinely from the serum of patients with pancreatitis.

The Effects of Local Cytokine Production on Pancreatitis Severity Since inflammatory cytokines are present in high concentrations within the pancreatic parenchyma during acute pancreatitis, a number of experiments have been conducted to determine if they were capable of initiating pancreatitis. Studies on isolated human pancreata perfused with large amounts of IL-1 or TNF found little or no evidence that these mediators alone could propagate pancreatitis. Further, in vitro studies showed that IL-1 and TNF could not induce co-localization or the activation or release of enzymes from acinar cells, but exposure to TNF in concentrations found within the pancreas during pancreatitis was capable of killing the cells. Good evidence has also been found that IL-6 plays a regulatory role within the acinar cell through induction of regulatory genes including CCK receptor expression.11 These findings may help explain why cytokine blockade is capable of decreasing pancreatitis severity while all cytokines tested to date lack the ability to initiate enzyme activation or the development of pancreatitis. The use of knockout animals has also helped investigate whether IL-1 and/or TNF are necessary for the initiation and development of pancreatitis by showing that pancreatitis does develop in the absence of receptors for either of these two cytokines, yet its severity/lethality never reaches that of wild-type mice12,13 (Fig. 7.3). Combined, these studies have helped establish that pancreatitis is not triggered or induced by IL-1 and/or TNF, yet both play an important and very detrimental role to its progression.


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Fig. 7.2. Amplification of the Pancreatitis-Associated Inflammatory Cascade. Acute pancreatitis incites an inflammatory cascade within the pancreas as well as in multiple tissues of the body. The systemic activation of leukocytes results in the production of a number of powerful mediators such as interleukin (IL)-1, tumor necrosis factor (TNF), platelet activating factor (PAF), IL-6, IL-8, and nitric oxide (NO). Each of these mediators possess the ability to feed back and amplify the production of the others. Pancreatitis has the propensity to incite this process beyond the point of normal control mechanisms. The end result is a cascade of events leading to the release of multiple mediators in tissues such as the lungs, liver, pancreas, and spleen.

Cytokine-Induced Acinar Cell Death During Pancreatitis Another mechanism by which TNF (and possibly IL-1) may contribute to the severity of pancreatitis is through the induction of apoptosis of acinar cells. Apoptosis is a well orchestrated process by which nucleated cells are induced to kill themselves.14 This process is distinctly different from simple necrosis which is not energy dependant and does not follow a preset cascade of events. Several investigators have recently demonstrated that acute pancreatitis is associated with the induction of acinar cell apoptosis, the degree of which mimics the severity of pancreatitis. These studies suggested that a portion of pancreatic cell death which was previously believed to be due to simple necrosis mediated by autodigestion, may in fact be apoptotic cell death mediated by TNF produced within the inflamed gland.15,16 Although it is still unclear whether apoptosis is harmful or even protective during acute pancreatitis, it seems apparent that intra-pancreatic cytokines play a significant regulatory role in acinar cell viability.

Acute Pancreatitis Induces Systemic Hyperinflammation Further insight into the role of IL-1 and TNF in the propagation of distant organ failure was given by the observation that both cytokines are produced systemically

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Fig. 7.3. The importance of IL-1 and TNF activity on pancreatitis severity. The importance of IL-1 and TNF to the progression of pancreatitis is demonstrated by the use of transgenic knockout animals which lack active receptors for either IL-1 or TNF. Although the loss of cytokine activity has no effect on the initiation of pancreatitis, the end result is a much less severe hyperinflammatory response and a significant reduction in mortality.

during pancreatitis and not just within the pancreas. Regardless of the animal model used, IL-1 and TNF are produced in the spleen, lung, and liver with pancreatic production always preceding that in distant sites, by hours or even days, depending on the rapidity with which pancreatitis develops.17 Patients with severe pancreatitis possess circulating leukocytes which produce significantly more TNF, IL-6, IL-8 (and to a lesser degree, IL-1) while possessing circulating monocytes, lymphocytes, and PMNs which are hyperstimulated when compared to those with mild uncomplicated pancreatitis. Just as was seen in various animal models, these mediators are overproduced without regard to the etiology of clinical pancreatitis. Inflammatory cytokine production (along with PAF activity) within the pulmonary parenchyma is now believed to be the direct cause of ARDS whether during sepsis or acute pancreatitis. What remains a mystery, however, is the signal responsible for causing distant tissues to respond to pancreatic inflammation in this manner. As shown in Figure 7.1, local pancreatic cytokine production is likely to contribute to systemic cytokine gene induction, but this is probably not the primary mechanism. High levels of activated trypsin and other pancreatic enzymes can induce cytokine production from macrophages in vitro, but it is still unclear if activated enzymes in concentrations typically seen during clinical pancreatitis can induce this reaction, or if some other as of yet unknown mediator is responsible. Together, the activation of leukocytes systemically and their production of inflammatory cytokines in multiple tissues during acute pancreatitis resembles that seen in


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severe sepsis and undoubtedly plays a central role in the development of the clinical syndrome we know as acute pancreatitis.

Antagonism of Cytokines During Experimental Acute Pancreatitis As it became clear in the early 1990s that pancreatitis was associated with the appearance of inflammatory cytokines, a number of investigators postulated that their antagonism would have beneficial effects. The first studies utilized anti-TNF antibodies, soluble TNF receptors, or IL-1 receptor antagonists. Regardless of the approach, these studies showed that proximal blockade of the cytokine cascade would significantly decrease pancreatic edema, necrosis, and inflammation while decreasing mortality by more than half. Delayed (therapeutic) cytokine antagonism was shown to be effective as well, becoming the first pancreatitis experiment to demonstrate this critical correlation to clinical pancreatitis. It is now fairly well established that interruption of the cytokine cascade at any point between gene induction and receptor binding will attenuate experimental pancreatitis as long as the blockade takes place prior to the onset of severe organ dysfunction. It is less clear how effective delaying therapy beyond this point will be, but the efficacy of these agents is likely to fall off just as was seen in a number of sepsis trials.

Is Anticytokine Therapy for Acute Pancreatitis a Clinical Possibility? A number of treatments are now available to treat specific aspects of the clinical syndrome of acute pancreatitis. Ventilator management has progressed to the point that ARDS is a less common cause of pancreatitis associated mortality, yet it continues to be the major cause of morbidity leading to the need for intensive care monitoring.2 Similarly, the judicious use of broad spectrum antibiotics such as imipenem and cefuroxime has been shown to reduce the number of septic complications and improve survival in a subgroup of patients with acute necrotizing pancreatitis.3 The need for a specific therapy for all patients with this disease aimed at preventing distant organ dysfunction and the sequelae of pancreatitis associated systemic hyperinflammation, however, still remains. A great deal has been learned over the past few years about the production of inflammatory cytokines during acute pancreatitis. At the same time, the dominant role these few mediators play in the pathogenesis of SIRS and organ dysfunction is now becoming much better understood. There is little doubt that preventing the effect of IL-1, TNF, or PAF dramatically alters the expected course of experimental pancreatitis. The question remaining is whether similar antagonism during clinical pancreatitis would benefit patients with this disease. One of the major considerations to be examined is the suitability of acute pancreatitis to cytokine antagonism in the clinical setting and whether a therapeutic window for such antagonism exists.3 Figure 7.4 details the typical presentation and subsequent development of organ dysfunction in patients with pancreatitis. Although some patients present a day or two after the onset of pain, the vast majority seek medical attention within 8 to 12 hours. Similarly, few patients presenting shortly after the onset of pain will exhibit organ dysfunction at that time. By the second and third day, however, the incidence of organ dysfunction rises rapidly to distinguish between patients likely to have a protracted and complicated course from those with simple uncomplicated pancreatitis. It is now known that cytokine production during clinical pancreatitis begins shortly after pain onset but does not peak until 36 to

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Fig. 7.4. Time course of acute pancreatitis progression demonstrating a therapeutic window for inflammatory mediator antagonism. The majority of patients with acute pancreatitis will present within 18 hours after the onset of pain. This is followed closely by inflammatory cytokine production usually lasting several days. Although distant organ dysfunction is occasionally manifest at the time of presentation, the vast majority of patients develop severe systemic manifestations of pancreatitis 2 to 4 days later. This type of presentation allows for an interventional window during which time specific inflammatory mediator antagonists could be administered to attenuate or block the development of distant organ dysfunction/failure.

48 hours later. If the goal is to prevent distant organ dysfunction (the major cause of pancreatitis morbidity and mortality) this clinical scenario provides a theoretical therapeutic window beginning at hospital presentation and lasting for two to three days (Fig. 7.4). This has been supported by the results of the European phase III trial which showed a more beneficial effect when a PAF antagonist was given within 48 hours of pain onset when compared to those receiving the antagonist between 48 and 72 hours after pain onset or even later. Directed therapy for acute pancreatitis has eluded physicians for many years. This clinical goal has taken many different forms, some of which worked poorly even in experimental animals. Cytokine antagonism has been shown to be effective in virtually all experimental animal models of acute pancreatitis even when implemented in a delayed fashion. Anticytokine therapy in patients with acute pancreatitis has distinct advantages over similar therapies during sepsis in that pancreatitis patients will declare the onset of their disease and therefore the initiation of the cytokine cascade. In contrast, septic patients typically declare themselves only with the onset of systemic organ dysfunction, usually well after the cytokine cascade has been initiated or even peaked. The therapeutic window provided by pain onset during acute pancre-


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atitis may allow antagonism of specific mediators prior to the initiation of dramatic cascades which eventually lead to inflammatory mediator overproduction. Since the majority of the morbidity and mortality of this disease is due to this cascade of events, acute pancreatitis may be ideally suited for this type of antagonism. As we learn more about these powerful mediators, a scenario is likely to develop which will allow specific therapies during different phases of pancreatitis to prevent progression of gland destruction and the development of systemic complications.

References 1. Leach SD, Gorelick FS, Modlin IM. New perspectives on acute pancreatitis. Scand J Gastroenterol 1992; 27:Suppl 192:29-38. 2. Steinberg W, Tenner S. Acute pancreatitis. New England J Med 1994;330:1198-1220. 3. Norman J. The role of cytokines in the pathogenisis of acute pancreatitis. Amer J Sur 1997 in press. 4. Kishimoto T, Akira S, Narazaki M et al. Interleukin-6 family of cytokines and gp130. Blood 1995; 86:1243-1254. 5. Baggiolini M, Loetscher P, Moser B. Interleukin-8 and the chemokine family. Int J Immunopharmac 1995;17:103-108. 6. Kingsnorth AN, Galloway SW, Formela LJ. Randomized, double-blind phase II trial of Lexipafant, a platelet-activating factor antagonist, in human acute pancreatitis. British J Surg (1995); 82:1414-1420. 7. ingsnorth AN. Early treatment with lexipafant, a platelet activating factor antagonist reduces mortality in acute pancreatitis: A double blind, randomized, placebo controlled study. Gastroenterology; 112:A453. 8. Kinsgnorth AN. The role of cytokines and their inhibitors in acute pancreatitis. Gut 1997; 40:1-4. 9. Norman J, Fink G, Franz M. Acute pancreatitis induces intrapancreatic tumor necrosis factor gene expression. Arch of Surg 1995; 130:966-970. 10. Fink G, Norman J. Specific changes in the pancreatic expression of the interleukin1 family of genes during experimental acute pancreatitis. Cytokine. In Press. 11. Dusetti NJ, Ortiz EM, Mallo GV et al. Pancreatitis-associated protein I (PAP I), an acute phase protein induced by cytokines. Identification of two functional interleukin-6 response elements in the rat PAP I promoter region. J Biol Chem 1995; 270:22417-22421, 12. Norman JG, Fink G, Franz M et al. Active interleukin-1 receptor required for maximal progression of acute pancreatitis. Ann of Surg 1996; 223:163-169. 13. Denham W, Fink G, Norman J. Transgenic animals demonstrate modest additive detrimental effects of IL-1 and TNF during acute pancreatitis. Gastroenterology 1997. In Press. 14. Nagata, S. Apoptosis by death factor. Cell 1997; 69:355-365. 15. Gukoyskaya AS, Sandoval D, Zaninovich V et al. Tumor necrosis factor regulates cell death in caerulein-induced pancreatitis. Gastroenterology 1996; 111:A2791. 16. Kaiser AM, Saluja AK, Sengupta A et al. Relationship between severity, necrosis, and apoptosis in five models of experimental acute pancreatitis. Am J Physiol 1995; 269:C1295-C1304. 17. Norman J, Fink G, Denham W et al. Tissue specific cytokine production during experimental acute pancreatitis: A problem mechanism for distant organ dysfunction. Dig Dis and Sciences 1997; 42:1783-1788.

Cytokines and Peri-Operative Nutrition

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CHAPTER 8

Cytokines and Peri-Operative Nutrition Hideaki Saito and Lin Ming-Tsan

Introduction urgical stress triggers the production of a variety of cytokines by immune cells.1,2 These cytokines initiate immunological, hematological and metabolic alterations in association with surgical stress. Appropriate synthesis and release of cytokines, such as TNF, IL-1, IL-6 and IFN-! are essential for normal host defense. In contrast, exaggerated systemic cytokine responses may be harmful to the host. Recent studies have demonstrated that the production of cytokines following surgical insult is influenced by nutritional support. This production may be influenced by nutritional status, nutritional routes, specific nutrients such as glutamine and fatty acids, and anabolic hormones (Table 8.1). These nutritional modalities may exert beneficial biological effects in response to surgical insult through the modulation of cytokine production. Understanding the role of nutrients in cytokine production in normal states, as well as under surgical stress, facilitates choosing the most appropriate nutritional support for preventing complications and improving outcomes of postoperative patients. This article focuses on the role of nutrition in the modulation of cytokine responses in surgical stress, especially in the perioperative period.

S

Nutritional Route and Cytokine Biology Previous investigations have suggested the advantages of enteral nutrition over parenteral nutrition following surgical insult. These advantages include maintenance of intestinal barriers,3 improvement of systemic and local protein metabolism,3,4 and better stress hormone responses.3,5 Antecedent or early enteral feeding has been shown to improve outcomes in various animal models and in trauma patients, as compared to parenteral feeding.6-9 However, the precise mechanisms by which enteral feeding maintains immunity and improves outcomes, under conditions of surgical stress, are unclear. Recent investigations have demonstrated that part of the effectiveness of the enteral route may lie in appropriate cytokine production by the immune cells in response to inflammatory stimuli.

Cytokines and the Abdominal Surgeon, edited by Moshe Schein and Leslie Wise. © 1998 R.G. Landes Company.

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Table 8.1. Nutritional modalities that modulate cytokine biology 1. Nutritional routes a. Parenteral b. Enteral 2. Specific nutrients a. Polyunsaturated fatty acids b. Glutamine c. Arginine d. Nucleotides e. Vitamins E and D f. Combinations of specific diets 3. Anabolic hormones a. Growth hormone b. Insulin-like growth factor 1

Nonstressed Stable Conditions Circulating Cytokines

Lowry et al10 measured circulating TNF, IL-1 and IL-6 levels in normal volunteers and patients before and after one week of parenteral nutrition. Systemic TNF, IL-1 and IL-6 levels were undetectable in healthy volunteers before and after 1 week of TPN. Both TNF and IL-6 were detected in the systemic circulation in some patients both before and after TPN, but post-TPN levels were not significantly increased. Fong et al5 also reported that circulating TNF levels in nonstressed subjects receiving TPN were indistinguishable from those in subjects on enteral feeding. These results suggest that parenteral nutrition has little effect on circulating cytokines without an associated overt stress. In contrast, Santos et al11 demonstrated higher plasma levels of TNF and IL-6 in volunteers receiving enteral feeding than in those receiving TPN. We investigated the effect of preoperative nutritional route on the cytokine response in patients with colorectal cancer (Fig. 8.1).12 The levels of IL-6 and IL-8 were higher in the TPN patients than in the orally fed patients, indicating that effects stimulating the production of systemic cytokines may have been present in the TPN patients before surgery. Thus, human data concerning the effect of nutritional route on circulating cytokines in an unstressed condition are contradictory. Animal studies have demonstrated that the route of nutritional supply does not influence serum cytokine levels under nonstressed stable conditions.7,13

Tissue Cytokine Biology Evidence is accumulating that the gut is a cytokine-producing organ. Gut associated lymphoid tissues include lymphocytes, mast cells, macrophages, Paneth cells and M cells. These cells are a rich source of cytokines such as IL-1,2, 4, 5, 6, TNF, and IFN-!.14 In addition, enterocytes15 produce cytokines. Ogle et al16 have demonstrated messenger RNA expression for TNF-∀, IL-1 and IL-6 to be increased in the jejunum of parenterally fed rats, as compared with chow-fed controls, suggesting local stimulation of intestinal cytokine-producing cells by bacteria and endotoxin following parenteral nutrition.

Cytokines and Peri-Operative Nutrition

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Fig. 8.1. Systemic IL-6 responses before and after colon surgery with different nutritional routes (left). The IL-6 levels were higher in the TPN than in the oral group before the operation (*p=0.09 vs the oral group). Plasma IL-6 levels were significantly higher in the TPN group than in the oral group on postoperative day 1(**p

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