Galley - Inflammation and Immunity

Critical Care Focus 10: Inflammation and Immunity
EDITOR DR. HELEN F. GALLEY
BMJ Books
EDITOR DR HELEN F GALLEY Senior Lecturer in Anaesthesia and Intensive Care University of Aberdeen EDITORIAL BOARD PROFESSOR NIGEL R WEBSTER Professor of Anaesthesia and Intensive Care University of Aberdeen DR PAUL G P LAWLER Clinical Director of Intensive Care University of Aberdeen DR NEIL SONI Consultant in Anaesthesia and Intensive Care Chelsea and Westminster Hospital DR MERVYN SINGER Reader in Intensive Care University College Hospital, London
BMJ Publishing Group 2003 BMJ Books is an imprint of the BMJ Publishing Group All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording and/or otherwise, without the prior written permission of the publishers. First published in 2003 by BMJ Books, BMA House, Tavistock Square, London WC1H 9JR www.bmjbooks.com www.ics.ac.uk British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN 0-7279-1689-0 Typeset by Newgen Imaging Systems (P) Ltd, Chennai. Printed and bound in Spain by GraphyCems, Navarra
Contents
Contributors Preface Introduction 1 Immunoparalysis JEAN-MARC CAVAILLON, HELEN
vii viii ix 1
F GALLEY
2 Apoptosis and the inflammatory process

NIGEL R WEBSTER
18
3 Virus interaction with host immunity

LAWRENCE S YOUNG
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4 The double-edged role of the neutrophil in inflammatory responses

PAUL G HELLEWELL
47
5 T cell immunity and sepsis

EGBERT PRAVINKUMAR
65
6 Metalloproteinases and inflammation

ANDREW J GEARING
77
7 Glucocorticoid therapy in septic shock PIERRE-EDOUARD BOLLAERT Index
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Critical Care Focus series

Also available: H F Galley (ed) Critical Care Focus 1: Renal Failure, 1999. H F Galley (ed) Critical Care Focus 2: Respiratory Failure, 1999. H F Galley (ed) Critical Care Focus 3: Neurological Injury, 2000. H F Galley (ed) Critical Care Focus 4: Endocrine Disturbance, 2000. H F Galley (ed) Critical Care Focus 5: Antibiotic Resistance and Infection Control, 2001. H F Galley (ed) Critical Care Focus 6: Cardiology in Critical Illness, 2001. H F Galley (ed) Critical Care Focus 7: Nutritional Issues, 2001. H F Galley (ed) Critical Care Focus 8: Blood and Blood Transfusion, 2002. H F Galley (ed) Critical Care Focus 9: The Gut, 2002.
Contributors
Pierre-Edouard Bollaert Professeur des Universits, Service de Ranimation Mdicale, Centre Hospitalier Universitaire, Nancy, France Jean-Marc Cavaillon Unit dImmunology-Allergie, Institute Pasteur, Paris, France Helen F Galley Senior Lecturer in Anaesthesia and Intensive Care, University of Aberdeen, UK Andrew J Gearing Chief Executive Officer, Biocomm International, Melbourne, Victoria, Australia Paul G Hellewell Professor of Vascular Biology, University of Sheffield, UK Egbert Pravinkumar Lecturer in Intensive Care Medicine, University of Aberdeen, UK Nigel R Webster Professor of Anaesthesia and Intensive Care and Honorary Consultant, University of Aberdeen, UK Lawrence S Young Director of Institute and Head of Division of Cancer Research, UK Institute for Cancer Studies, University of Birmingham, UK
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Preface to the Critical Care Focus series
The Critical Care Focus series aims to provide a snapshot of current thoughts and practice, by renowned experts. The complete series should provide a comprehensive guide for all health professionals on key issues in todays field of critical care. The volumes are deliberately concise and easy to read, designed to inform and provoke. Most chapters are produced from transcriptions of lectures given at the Intensive Care Society meetings and represent the views of world leaders in their fields. Helen F Galley
viii
Introduction
Immunoparalysis Jean-Marc Cavaillon and Helen F Galley Several studies indicate that depression of immune function induced by traumatic injury is aetiologically involved in the development of infection or sepsis. Nevertheless, the mechanisms behind the maintenance of the sustained suppression of immune function remain incompletely understood. Alterations of immune responses have been regularly reported in patients with systemic inflammatory responses syndrome (SIRS). The observation that some patients have apparent immune paralysis led to the concept of compensatory anti-inflammatory response syndrome or CARS. In this article, we describe this phenomenon of immunoparalysis but, although alterations in immune response are probably associated with an enhanced sensitivity to nosocomial infections, there is no clear demonstration that they are directly responsible for poor outcome in sepsis. Apoptosis and the inflammatory process Nigel R Webster Cells that are damaged by injury undergo swelling and leakage of cell contents, leading to inflammation of surrounding tissues. This process is called necrosis. Cells that are induced to commit suicide, in contrast, shrink, and the mitochondrial membrane becomes breached such that release of cytochrome c occurs. Chromatin (DNA and protein) in the nucleus becomes degraded into small, membrane-wrapped fragments, and the phospholipid phosphatidylserine, which is normally hidden within the plasma membrane, is exposed on the surface. This is then bound by receptors on phagocytic cells, such as macrophages, which engulf the cell fragments, leading to a quiet orderly removal of dead cells. This pattern
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CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY
of events is called programmed cell death or apoptosis. The cellular machinery of programmed cell death is as intrinsic to the cell as, for example, mitosis. This article will describe the regulation and process of apoptosis and its relevance to disease, including the inflammatory response in patients with sepsis. Virus interaction with host immunity Lawrence S Young Resistance to and recovery from viral infections depends on the interactions between virus and host. The defences mounted by the host may act directly on the virus or indirectly on virus replication by altering or killing the infected cell. The non-specific host defences function early in the encounter with virus to prevent or limit infection, while the specific host defences function after infection in initiation of immune responses to subsequent challenges. Viruses have evolved complex strategies to manipulate host immune defences to their advantage, permitting viral replication without massive inflammatory responses integrating their needs with that of their host man. However, some persistent and latent viral infections can lead to serious disease and malignancy. This article will describe the hostvirus interactions and particularly focus on the role of latent infection with the EpsteinBarr virus in tumour development the killer within. The double-edged role of the neutrophil in inflammatory responses Paul G Hellewell Accumulation of leucocytes in tissues is essential for effective host defence. The major role of neutrophils is to phagocytose and destroy infectious agents but they can also cause host damage, and neutrophilmediated injury has been implicated in several inflammatory conditions seen on the Intensive Care Unit (ICU). This article provides an overview of the vital role of neutrophils in host defence, and the consequences of host damage. An increased understanding of neutrophil biology is likely to result in intelligent intervention strategies. T cell immunity and sepsis Egbert Pravinkumar Immune responses essential for defeating systemic microbial infections depend on intact innate and acquired immune responses. Recognition
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INTRODUCTION
molecules, inflammatory cells, and the cytokines allow host tissues to recognise invading microbes and to initiate intercellular communication between the innate and acquired immune systems. However, activation of innate immunity may also occur in the absence of microbial recognition, through expression of internal signals produced by tissue ischaemia and necrosis. Induction of the innate immune system can have catastrophic effects on patients with sepsis. Exaggerated production of cytokines and the induction of mediators such as nitric oxide, platelet activation factor, and prostaglandins have been implicated in the endothelial changes and induction of a procoagulant state, leading to hypotension, inadequate organ perfusion, and necrotic cell death, associated with multiorgan dysfunction syndrome. This article provides an overview of the T cell immune system, its regulation, and the influence of sepsis.
Metalloproteinases and inflammation Andrew J Gearing Matrix metalloproteinases (MMPs) are a large family of zinc-containing endoproteinases, which have similar structures but differ in substrate specificity, cellular sources, and inducibility. MMP activity is controlled at the transcriptional level and by a family of at least four endogenous natural inhibitors (tissue inhibitors) of MMPs called TIMPs. MMPs cleave protein components of the extracellular matrix, membrane receptors, and cytokines, and have a role in cell extravasation. This article describes the action, regulation, and roles of MMPs in inflammatory and immune responses.
Glucocorticoid therapy in septic shock Pierre-Edouard Bollaert Recent findings highlighting the role of the ability of the hypothalamic pituitaryadrenal axis to respond appropriately to a septic insult have led to a reappraisal of the use of steroids in septic shock. Recent work has suggested that physiological doses of corticosteroids given for a longer duration may be beneficial in catecholamine-dependent septic shock leading to a more rapid withdrawal of vasopressor therapy and a trend toward improved survival. A recent multicentre study of patients in septic shock has suggested a reduction in mortality in patients with relative adrenal insufficiency receiving replacement therapy with a combination of hydrocortisone and fludrocortisone. This article describes studies of corticosteroid therapy in patients with septic shock and comments on the possible benefits of corticosteroid therapy in this population.
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1: Immunoparalysis
JEAN-MARC CAVAILLON, HELEN F GALLEY
Introduction
Several studies indicate that depression of immune function induced by traumatic injury is aetiologically involved in the development of infection or sepsis. Nevertheless, the mechanisms behind the maintenance of the sustained suppression of immune function remain incompletely understood. Alterations of immune responses have been regularly reported in patients with systemic inflammatory response syndrome (SIRS). Trauma, haemorrhage, burns, surgery, or sepsis are associated with events such as tissue injury, blood loss, hypoxia, transfusion, microbial infection, and bacterial translocation, which contribute to an inflammatory response and affect the quality of the immune response. Drugs (for example, anaesthetics, opioids) also influence immune responses (Figure 1.1). Depressed immune status including decreased blood cell counts, low expression of surface markers (for example, MHC Class II antigen), altered natural killer (NK) cell activity, reduced cellular cytotoxicity and antigen presentation, poor proliferation in response to mitogens, and depressed cytokine production, are seen in vitro, and illustrated in vivo by anergy to skin test antigens. These observations led Roger Bone to coin the concept of compensatory antiinflammatory response syndrome or CARS.1 Bone postulated that when the SIRS response predominated it was associated with an organ dysfunction and cardiovascular compromise leading to shock; in contrast, when CARS predominated it was characterised by anti-inflammatory responses associated with a suppression of the immune system termed immunoparalysis.1 It was initially accepted that the SIRS response occurred first and was followed in some patients by the CARS response. However, the two syndromes most probably occur concomitantly.2 In this article, we will describe the phenomenon of immunoparalysis in SIRS patients. Although alterations in immune response are probably associated with an enhanced sensitivity to nosocomial infections, there is no clear demonstration that they are directly responsible for poor outcome in sepsis. Indeed, since the investigations of
1
CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY Anaesthesia Opioids Blood loss Transfusion Hypoxia Tissue injury
Bacterial translocation
Inflammatory response
IMMUNE STATUS Figure 1.1 Contributory factors to altered immune status, resulting from trauma, haemorrhage, burns, surgery, or sepsis.
immune function may depend upon numerous parameters (for example, nature of the activators, cell types used, initial compartment of the cells, the cytokine investigated), interpretation of findings is not easy.
Measures of immune dysfunction

How can this modification of immune status be monitored? Lymphocyte and monocyte population changes and also HLA-DR expression are simple examples. Immune suppression can be assessed in vitro as poor lymphocyte proliferation in response to mitogens, reduced NK cell activity, reduced neutrophil function, reduced cytokine production, and in vivo anergy to skin test antigens (Figure 1.2).
IMMUNE STATUS
Lymphocyte/monocyte population changes Immune suppression Lymphocyte proliferation Natural killer cell activity Cytokine production Anergy to skin test antigens
Figure 1.2 Approaches to monitoring immune status resulting from trauma, haemorrhage, burns, surgery, or sepsis. 2
IMMUNOPARALYSIS
HLA-DR expression Abnormal antigen presentation has been observed in SIRS patients,3 and decreased HLA-DR expression on monocytes may contribute to this defect.4 In a study by Hershman and colleagues,5 60 trauma patients were retrospectively divided into three groups: those with an uneventful recovery (n 17), those with recovery after major sepsis (n 27), and those who did not survive (n 16). HLA-DR expression on peripheral blood monocytes was compared with that of 77 healthy volunteers. After the initial injury, there was a significant decrease from normal in the three groups of trauma patients, and this returned to normal after one week in the group of patients who recovered uneventfully. In those who developed sepsis, HLA-DR expression took three weeks to return to normal and in the patients who did not survive, expression never returned to normal.This study demonstrated that monocyte HLA-DR antigen expression was able to distinguish those patients who survived severe trauma, from those who died (Figure 1.3). HLA-DR antigen expression correlated directly with the clinical course and identified a group of patients at high risk of infection and death following trauma.5 Low HLA-DR expression is now widely recognised as a good marker of the intensity of the immune depression and of increased risk of bacterial infection.6,7
80 uneventful recovery %HLA-DR positive monocytes major sepsis 60
40
death 20 1 3 6 9 12 15 18
Days after injury Figure 1.3 Percentage of monocytes expressing HLA-DR expression in 60 trauma patients, 17 of whom had an uneventful recovery, 27 developed sepsis and 16 died. Dotted line represents mean HLA-DR expression in 77 healthy controls. Reproduced with permission from Hershman M et al.5 3
Lymphocyte proliferation Impaired lymphocyte transformation in SIRS patients was reported more than three decades ago.8 The impairment was proportional to the severity of the injury. In vitro lymphocyte proliferative response to antigens and mitogens, and in mixed lymphocyte reactions, are all significantly decreased in trauma patients.8,9 The length of depressed lymphocyte responses may exceed two weeks, and lower responses and longer depression have been observed in patients who become infected.9,10 Natural killer cell activity The ability of the cell to mount an NK cell response provides another means to monitor immune status. NK cell activity was studied in burn and trauma patients11 and was shown to be significantly depressed over a very long period of time for the more severely burned patients. Patients with lesser burns and traumatically injured patients had an altered NK activity for a shorter period. Interestingly, Blazar et al. further showed that stressinduced mediators (cortisol, epinephrine, glucagon) had the capacity to reduce NK activity in healthy volunteers.11 In the study by Maturana et al.,12 patients with septic shock (n 11) had also a markedly lower NK activity than healthy controls (n 10), independently of the effector:target cell ratio in the experimental system. In another study, NK cell activity in patients with septic shock (n 20) was also lower than in healthy volunteers (n 15). Pre-incubation of peripheral blood lymphocytes with either interferon- (IFN ) or interleukin-2 (IL-2) enhanced NK cell activity in healthy controls but not in patients with sepsis indicating the difficulty in reversing the depressed immune responsiveness.13 Neutrophil functions Although apoptosis of circulating neutrophils (PMN) is delayed in patients with SIRS or sepsis,14 function of the cells is altered. This is the case of phagocytosis and bactericidal activity15 and of migration.16 The reduced responsiveness of PMN to chemoattractant agents may reflect the action of nitric oxide,16 the decreased expression of certain chemokine receptors,17 or a deactivation occurring in the bloodstream after interacting with large amounts of circulating chemokines as indicated, for example, by the huge amounts of IL-8 found associated to PMN in septic patients.18 Delayed hypersensitivity The in vitro evidence of immune depression is also reflected in vivo by tests of delayed type hypersensitivity. Several years ago, Christou and
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IMMUNOPARALYSIS
colleagues skin tested 727 surgical patients with recall antigens prior to operation. Patients who had normal skin test responses were of similar age and had equal degrees of surgical procedures performed compared with those patients who were anergic (that is, had depressed skin test responses). Postoperatively, sepsis, mortality, and death from sepsis were significantly higher in the anergic population, reconfirming the hypothesis that skin test anergy in patients preoperatively is a signal of increased risk for septic complications and death in such patients. These authors20 also reported that surgical patients who were anergic to a battery of five skin test antigens had a two-fold higher rate of postoperative infection than those who reacted to only two antigens, and were more than five times more likely to die in the postoperative period.
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Cytokine production The analysis of sepsis and SIRS patients reveals a paradoxical situation: an overwhelming production of cytokines as assessed by their concentrations within the bloodstream21 and a profound reduction of the capacity of circulating cells to produce cytokines upon in vitro activation. Among pioneering work is the study by Wood and co-workers.22 They studied the production of IL-1 and IL-2 by peripheral blood mononuclear cells from 23 burn patients and 23 matched controls. Serial measurements were made of IL-1 production by monocytes after stimulation with lipopolysaccharide (LPS), and of IL-2 production by lymphocytes after stimulation with the mitogen phytohaemagglutinin (PHA). Lymphocyte IL-2 production from 12 patients with more than 30% body surface area burns revealed lower IL-2 production compared with patients with less than 30% burns. Patients with systemic sepsis also had lower IL-2 production than nonseptic patients. IL-1 production was increased compared with controls early after injury, but was subsequently within the normal range regardless of burn size. The percentage of circulating helper T lymphocytes, the principal source of IL-2, was also reduced, although this did not always correlate with IL-2 production, which remained depressed after recovery of the T cell population. This study indicated that failure to produce IL-2, which is a powerful mediator of cellular immune responses, is an important mechanism underlying the defective cell-mediated immunity seen in burn patients.22 Surgery also leads to significant modulation of the immune system, and cytokine release in particular. Cabie et al. investigated the consequences of surgery on in vitro cytokine production by human monocytes stimulated by LPS.23 The responsiveness of cells obtained the day before, during and after surgery was compared in patients undergoing abdominal aortic surgery (n 9), carotid surgery (n 4), and spinal surgery (n 4). A significant decrease in monocyte tumour necrosis factor- (TNF ), interleukin-1 (IL-1 ), and IL-1 production during
5
surgery was reported, whereas IL-6 production remained unchanged. By day 2 following surgery, a significant increase in monocyte responsiveness was observed and levels of cytokine production were similar to initial values (Figure 1.4).
TNF ng/ml 10 ng/ml 15 IL-1
12
0 pre post
0 pre post
Figure 1.4 Influence of surgery on in vitro monocyte tumour necrosis factor (TNF ) and interleukin-1 (IL-1 ) production in nine patients undergoing aortic surgery, four patients undergoing surgery for atheromatous lesions of the carotid artery, and four patients undergoing spinal surgery. Isolated cells were stimulated for 24 hours with 2 g/ml lipopolysaccharide (LPS). Pre one day before surgery and post 3 hours into the surgical procedure. Reproduced with permission from Cabie A et al.23
Characterisation of the ex vivo cytokine production in sepsis and SIRS

Inflammation is characterised by an interplay between pro- and antiinflammatory cytokines. Cytokines are commonly classified in one or the other category: IL-1, TNF , interferon- (IFN ), IL-12, IL-18, and granulocyte-macrophage colony stimulating factor (GM-CSF) are well characterised as pro-inflammatory cytokines whereas IL-4, IL-10, IL-13,
6
IMMUNOPARALYSIS
IFN , and transforming growth factor- (TGF ) are recognised as antiinflammatory cytokines. However, this dichotomy may be too simplistic and it should be remembered that the amount of cytokine produced, the target cell, the activating signal, the timing and sequence of cytokine action, and even the experimental model, are parameters that greatly influence cytokine properties.24 There have been several studies investigating in vitro stimulated cytokine release in patients with sepsis, in stimulated whole blood or isolated cell preparations. Monocyte-derived cytokines Several years ago, Munoz et al. studied the capacity of monocytes from septic patients to produce cytokines in response to LPS.25 Monocyte production of IL-1 , IL-1 , IL-6 and TNF in patients with sepsis syndrome (n 23) or non-infectious shock (n 6) was measured at admission and at regular intervals during intensive care unit (ICU) stay. Reduced LPS-induced production of cytokines was most pronounced in patients with Gram-negative infections. Recovery of cytokine production was observed among surviving patients but not in non-surviving patients. The data suggest that complex regulatory mechanisms can downregulate the production of cytokines by monocytes during severe infections. The suppression of lymphocyte and monocyte responses may reflect potential defects in the upregulation of the IL-12 and IFN pathway. These cytokines exert protective effects during experimental endotoxaemia through upregulation of cellular immunity and phagocytic functions and are part of a positive regulatory feedback loop that enhances the production of the other. In a study by Ertel et al.,26 LPS-stimulated whole blood from 25 critically ill patients and 12 healthy individuals was incubated with either recombinant human (rh) IL-12 or rhIFN .They found that, although IFN increased the release of IL-12 from LPS-stimulated whole blood from healthy subjects in a dose-dependent manner, this effect was not seen in critically ill patients. IL-12 enhanced the secretion of IFN in healthy subjects, but was ineffective in critically ill patients. Although the antiinflammatory cytokine, IL-10, but not IL-4, mimicked suppression of the IL-12-IFN pathway similar to that observed during critical illness, the release of anti-inflammatory cytokines (IL-4, IL-10, TGF ) was decreased in LPS-stimulated blood from critically ill patients. This study suggested that deactivation of IL-12 and IFN -producing leukocytes occurred in vivo. Lymphocyte-derived cytokines The T lymphocyte population comprises both T helper 1 (Th1) and T helper 2 (Th2) subsets. Th1 cells produce cytokines predominantly concerned with
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pro-inflammatory responses (IFN , TNF , IL-2) and Th2 produce cytokines concerned with anti-inflammatory responses (IL-4, IL-10, and IL-13). It has been regularly reported that the production of Th1 cytokines was mainly altered in SIRS patients, whereas this was not the case for the Th2 cytokines.27,28 However, the study of purified T cells from 37 severely injured trauma patients showed that T cell anergy was a global depression of both Th1 and Th2 cytokine profile. Interestingly, depressed T cell proliferation and depressed cytokine production correlate to poor clinical outcome.29 Muret et al. further illustrated that infectious and non-infectious SIRS exert a more subtle modulation on circulating cell reactivity. They investigated the production of IL-2, IL-4, IL-5, and IL-10 by peripheral blood mononuclear cells in 13 patients with sepsis and 13 patients with noninfectious inflammation (patients undergoing cardiac surgery with cardiopulmonary bypass).30 Cytokine release after activation of lymphocytes with either concanavalin A (ConA), PHA, or anti-CD3 antibodies was studied. ConA-induced IL-10 was reduced in both groups of patients compared with healthy controls. In sepsis patients, ConA-induced IL-2, IL-5, and IL-10 production was decreased but not that released in response to PHA or anti-CD3. In cardiac patients, only anti-CD3-induced IL-10 production was reduced.These data indicate that subtle modifications of the reactivity of circulating cells occur during infectious and non-infectious inflammation, dependent on the cell stimulus. This suggests that regulation of both Th1 and Th2 responses is occurring in patients with sepsis and SIRS, and that the cell stimulant used determines the results achieved.
Neutrophil-derived cytokines McCall and co-workers31 reported that neutrophils from patients with the sepsis syndrome were consistently resistant to LPS stimulation such that synthesis of IL-1 was depressed. This downregulation occurred concomitant with an upregulation in expression of the type 2 IL-1 receptor (IL-1r2). Similar findings were not seen in uninfected patients with severe trauma or shock from other causes. In another study,32 Marie et al. reported depressed IL-8 release from neutrophils in patients who had undergone cardiac surgery with cardiopulmonary bypass and patients with sepsis. Cells were activated with either LPS or heat-killed streptococci. Compared with healthy controls, the release of IL-8 in both groups of patients was significantly reduced whether activated by LPS or by heat-killed streptococci. These observations suggest that stressful conditions related to inflammation, independently of infection, resulted in hyporeactivity of circulating neutrophils, suggestive of LPS tolerance. However, in vitro experiments suggested that neutrophils from healthy controls (in contrast to monocytes) could not be rendered tolerant to LPS. Interestingly, while IL-1 receptor antagonist (IL-1ra) was shown to be enhanced in whole
8
IMMUNOPARALYSIS
blood assays in meningococcal infection, Marie et al. found that the release of IL-1ra by isolated PMN was diminished in SIRS patients.34 Not a universal defect It is noteworthy that the diminished capacity of leukocytes from SIRS patients to produce cytokines as compared with healthy donors is not obtained with all activating signals. For example, McCall et al.,31 in their studies of patients with sepsis, failed to observe a decreased capacity of PMN to release IL-1 when they used heat-killed staphylococci, while immune-depression was revealed with LPS. In sepsis, IL-2, IL-5, and IL-10 production in response to conA was reduced, but not when phytohaemagglutinin or anti-CD3 were used.30 More recently, in cardiac arrest and resuscitated patients, hyporeactivity, assessed in terms of TNF production, was observed with LPS stimulation, but not with heat-killed staphylococci (Adrie et al., personal communication). A similar dissociation between stimuli that reveal hyporeactivity (LPS, CpG, IL-1, TNF) and those that do not (for example, staphylococci, streptococci) has also been observed in trauma patients (Adib-Conquy et al., personal communication). These observations suggest that differential alteration of signalling pathways may occur within the cells of SIRS patients, depending upon the nature of the activating agent and the nature of the cytokine being analysed.
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Mechanisms of hyporesponsiveness of monocytes

Desensitising agents The presence of deactivating or immunosuppressive agents within the bloodstream may contribute to the hyporeactivity of circulating leukocytes. IL-10 has been identified as a major functional deactivator of monocytes in human septic shock plasma,35 and TGF was shown in animal models of haemorrhagic shock and of sepsis to be the causative agent of the depressed splenocyte responsiveness.36,37 Furthermore, there is accumulating evidence for a strong interaction between components of the nervous and the immune systems, and numerous neuromediators have been shown to behave as immunosuppressors. Catecholamines suppress the activity of immunocompetent cells and are found at higher concentrations in stressful situations.38 Catecholamines are known to inhibit TNF production39 and to favour IL-10 release.40 Similarly, alpha-melanocyte-stimulating hormone contributes to immunosuppression by inducing IL-10 production by human monocytes.41 In addition, vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide directly inhibit endotoxin induced pro-inflammatory cytokine secretion.42 SIRS is also associated
9
with an activation of the hypothalamuspituitaryadrenal axis, which leads to the release of glucocorticoids, well known for their potent ability to limit cytokine production.43 Finally, prostaglandins are produced during sepsis and can also contribute to the downregulation of cytokine production.44 Endotoxin neutralising molecules As mentioned previously, the reduced capacity of monocytes to produce inflammatory cytokines has been established, particularly in experimental systems using LPS as a triggering agent. Since numerous recent studies have analysed the hyporeactivity phenomenon using whole blood cultures, it is possible that endotoxin-neutralising molecules have interfered in these studies. Indeed, Adrie et al. have shown that the hyporeactivity to LPS was both an intrinsic property of circulating monocytes, as well as the reflection of a specific neutralising activity within the plasma of SIRS patients (personal communication). It has been reported that plasma of septic patients contains large amounts of LPS binding protein (LBP), which can either inhibit the LPS molecules,45 or transfer LPS to lipoproteins,46 known for their inhibitory activity towards LPS.47 Furthermore, sera from septic patients contain amounts of soluble CD14, which also favours the shuttle of LPS towards lipoproteins.48 Toll-like receptors Toll-like receptors (TLR) are a family of receptors that recognise components of bacteria, virus, parasites, and fungi, and induce a proinflammatory response by several cell types. So far, 10 human TLRs differing in their specificity for microbial components have been cloned, which respond to various components, including LPS from Gram-negative bacteria, lipopeptides of Gram-positive cell walls, bacterial DNA, and flagella. TLR4 was identified as the receptor for LPS and requires the presence of an extracellular accessory protein called MD-2. CD14 physically associates with LPS complexed with LBP and transfers the endotoxin to the TLR4 and MD-2 dimer; each component of this complex is required for efficient LPS-induced signalling. Many parameters of immunoparalysis observed in SIRS patients are reminiscent of the endotoxin tolerance phenomenon, which characterises the refractoriness of cells or the inability of whole animals to respond to a second endotoxin challenge shortly after a first encounter.49 Because recent studies reported a downregulation of surface expression of TLR4 in endotoxintolerant macrophages,50,51 it was of interest to investigate the expression of this molecule on the surface of monocytes from SIRS patients. A decreased expression of TLR4, but not TLR2, on CD14 positive cells was found in 11 trauma patients compared with 6 healthy subjects (Adib-Conquy et al.,
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personal communication). However, this lower expression of TLR4 is not sufficient to explain the decreased capacity of the cells to respond to stimuli. Indeed, while LPS-induced TNF is decreased, this was not the case of LPSinduced IL-1ra and IL-10. This latter observation suggests that the defect may occur at the different signalling pathways within the cell rather than at the initiation of the signalling cascade on the cell surface.
Nuclear factor kappa B Transcription factors are DNA-binding proteins which regulate gene expression. Nuclear factor kappa B (NF B) is one such transcription factor which is critical for maximal expression of many cytokines involved in the pathogenesis of inflammation. Activation and regulation of NF B is tightly controlled by a group of inhibitory proteins (I B), which maintain NF B in an inhibited state in the cytoplasm of effector cells. The sequence of events leading to NF B activation involves phosphorylation, ubiquitination, and proteolysis of I B, allowing exposure of a nuclear recognition site. NF B then migrates to the nucleus, binds to specific promoter sites, and activates transcription of target genes (for example, TNF, IL-1, IL-6, IL-8). NF B, part of the Rel/NF-B family of transcription factors, is involved in the regulation of immune and acute-phase responses at the transcriptional level. Rel proteins can be divided into two groups based on their structures, functions, and modes of synthesis. The first group of Rel proteins consists of p65 (also known as RelA), c-Rel, and RelB, each of which contains one or more transcriptional-activation domain necessary for gene induction.The second group consists of p105 and p100, which, upon proteolytic processing, give rise to p50 and p52, respectively. Members of both groups of Rel proteins can form homo- or heterodimers. Studies have shown that the transactivator form of NF B is the p65 unit, whereas the p50 unit has shown no or minimal activation capacity. To investigate the role of NF B in the mechanism of endotoxin tolerance in macrophages, Blackwell and co-workers52 used a rat alveolar macrophage cell line made endotoxin tolerant by exposure to low concentrations of LPS for 48 hours. This treatment induced a state of tolerance such that subsequent exposure to high-dose LPS resulted in decreased production of cytokines compared with LPS-sensitive cells. This decreased cytokine production was associated with impaired activation of NF B with depletion of both RelA and p50. This study suggested endotoxin tolerance might be mediated by depletion of RelA/p50, which could limit the amount of NF B available for activation and inhibit transcription of NF B-dependent genes. On the other hand, Ziegler-Heitbrock and colleagues demonstrated that endotoxin tolerance of monocytes was associated with an increase of the inactive p50 homodimer and a decrease of the p50/p65 active heterodimer.53
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Accordingly, Adib-Conquy et al. studied NF B expression and dimer characteristics in mononuclear cells of patients with sepsis and major trauma and healthy controls.54,55 The expression of p50/p65 heterodimer was significantly reduced in all patients as compared with controls. The p50/p50 homodimer was reduced in the survivors of sepsis. Subsequent in vitro stimulation of mononuclear cells with LPS did not induce further NF B nuclear translocation: the survivors of sepsis showed low expression of both p50/p65 and p50/p50, while non-survivors of sepsis showed a predominance of the inactive homodimer and a low p50/p65:p50/p50 ratio when compared with controls. In the latter group of patients there was a negative correlation between plasma IL-10 levels and the p50/p65:p50/p50 ratio after in vitro LPS stimulation (r 0.8, .04). The reduced expression of nuclear NF B was not due to its P 0 inhibition by I B since very low expression of I B as well as low levels of p65 and p50, were found in the cytoplasm of mononuclear cells from sepsis patients when compared with controls. These results demonstrate that upon LPS activation, mononuclear cells of systemic inflammatory response syndrome patients show patterns of NF B expression that resemble those reported during LPS-tolerance: global downregulation of NF B in survivors of sepsis and presence of large amounts of the inactive homodimer in the non-survivors of sepsis.54 In trauma patients, after 1, 3, 5, and 10 days following admission of patients in the intensive care units, expression of both p50/p65 heterodimers and p50/p50 homodimers was significantly reduced compared with controls. After LPS stimulation in vitro, the p50/p65:p50/p50 ratio was significantly lower in cells from trauma patients than from healthy controls and the ex vivo expression of I B was higher. Although no direct correlation was found between levels of IL-10 or TGF and NF B, these immunosuppressive cytokines were significantly elevated in the trauma patients by 10 days after admission. This long-term low basal and LPSinduced activation of NF B might be linked to immunoparalysis.55
Signalling pathways There are still very few studies in humans addressing whether some alterations of the signalling pathways might explain part of the immunodepression seen in circulating cells in SIRS patients. Most interestingly, Learn et al.56 reported that in septic patients the repressed production of IL-1 and the selective elevation of the secreted form of IL-1ra in response to LPS, was linked to a probable alteration in the interleukin-1 receptor-associated kinase (IRAK) signalling pathway and a maintained efficient phosphatidylinositol 3-kinase-dependent signalling pathway. In murine model of sepsis, it was reported that inhibition of p59fyn phosphorylation and kinase activity was associated with T lymphocyte
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IMMUNOPARALYSIS
IL-2 production and proliferation, whilst activation of MAPK p38 was associated with T cell immune dysfunction.58
57
Cells derived from inflammatory foci

The hyporesponsiveness of peripherally derived cells in terms of the capacity of the cells to produce cytokines is not a generalised phenomenon, and cells derived from inflammatory foci are, in contrast, activated. In a baboon model of inflammation, the capacity of alveolar macrophages after unilateral lung irradiation was studied.2 Over a 1 month period, bronchoalveolar lavage was undertaken in five baboons, macrophages were recovered and production of TNF was measured.This study showed a significant increase of spontaneous, LPS-, staphylococci- and streptococci-induced TNF release in macrophages from the inflamed lung compared with the initial values. Macrophages from lungs of patients with acute respiratory distress syndrome (ARDS) also clearly demonstrated no deactivation of the cells. Schwartz et al.59 investigated activation of the transcription factors NF B, nuclear factor-IL-6 (NF-IL-6), cyclic adenosine monophosphate (cAMP)responsive element binding protein, serum protein-1 (SP-1), and activating protein-1 (AP-1) in alveolar macrophages from six patients with ARDS and from six control patients without lung injury. Activation of NF B in alveolar macrophages observed in patients with ARDS had increased compared with control patients, but there was no increase in the activation of the other transcription factors. Moine and colleagues60 subsequently showed decreased cytoplasmic levels of p50, p65 and c-Rel in alveolar macrophages from patients with ARDS, consistent with enhanced migration of liberated NF B dimers from the cytoplasm to the nucleus. When leukocytes are derived from the peritoneal cavity or the gut of patients suffering peritonitis,61 inflammatory bowel diseases,62 or endometriosis,63,64 LPS-induced cytokine production by peritoneal macrophages or mononuclear cells from the lamina propria was enhanced as compared with healthy controls. Following injection of endotoxin, the production of IFN by intra-epithelial lymphocytes was enhanced upon stimulation as compared with control animals.65 A similar enhanced activity of intra-epithelial T lymphocytes after endotoxaemia was observed when cellular cytotoxicity and proliferation were monitored. Altogether, these examples illustrate that local inflammation is associated with an enhanced activity of resident and/or infiltrating leukocytes, whilst systemic inflammation is associated with a reduced activity of circulating leukocytes.
Conclusion
Sepsis and non-infectious SIRS are paradoxically associated with an exacerbated production of cytokines, as assessed by their presence in
13
biological fluids, and a diminished ability of circulating cells to produce cytokine upon in vitro activation.This might represent a protective response against an overwhelming dysregulation of the pro-inflammatory process, but on the other hand it may induce a state of immune paralysis (endogenous immunosuppression) leading to an increased risk of subsequent nosocomial infections.66 However, cellular hyporeactivity is not a global phenomenon and some signalling pathways are unaltered and allow the cells to respond normally to certain stimuli. Furthermore, during sepsis and SIRS, cells derived from tissues or inflammatory foci are either fully responsive to ex vivo stimuli or even primed, in contrast to cells derived from haematopoietic compartments (blood), which are hyporeactive. In addition to cytokine production, NF B activity within leukocytes reflects cellular hyporeactivity. Thus the immunoparalysis reported in sepsis and SIRS patients, often revealed by a diminished capacity of leukocytes to respond to LPS, is not a generalised phenomenon, and SIRS is associated with a compartmentalised responsiveness involving either anergic or primed cells.
References
1 Bone RC, Grodzin CJ, Balk RA. Sepsis: a new hypothesis for pathogenesis of the disease process. Chest 1997;112:23543. 2 Cavaillon J-M, Adib-Conquy M, Cloz-Tayarani I, Fitting C. Immunodepression in sepsis and SIRS assessed by ex vivo cytokine production is not a generalized phenomenon: a review. J Endotoxin Res 2001;7:8593. 3 Polk HC Jr, George CD,Wellhausen SR et al. A systematic study of host defense processes in badly injured patients. Ann Surg 1986;204:28299. 4 Livingston DH, Appel SH, Wellhausen SR, Sonnenfeld G, Polk HC. Depressed interferon gamma production and monocyte HLA DR expression after severe injury. Arch Surg 1988;123:130912. 5 Hershman MJ, Cheadle WG, Wellhausen SR, Davidson PF, Polk HC Jr. Monocyte HLA-DR antigen expression characterizes clinical outcome in the trauma patient. Br J Surg 1990;77:2047. 6 Cheadle WG, Hershman MJ, Wellhausen SR, Polk HC Jr. HLA-DR antigen expression on peripheral blood monocytes correlates with surgical infection. Am J Surg 1991;161:63945. 7 Van den Berk JMM, Oldenburger RHJ, van den Berg AP et al. Low HLA-DR expression on monocytes as a prognostic marker for bacterial sepsis after liver transplantation. Transplantation 1997;63:18468. 8 Salo M, Merikanto J, Eskola J, Nieminen S, Aho AJ. Impaired lymphocyte transformation after accidental trauma. Acta Chir Scand 1970;145:36772. 9 Keane RM, Birmingham W, Shatney CM, Winchurch RA, Munster AM. Prediction of sepsis in the multitraumatic patients by assays of lymphocyte responsiveness. Surg Gynecol Obst 1983;156:1637. 10 Levy EM, Alharbi SA, Grindlinger G, Black PH. Changes in mitogen responsiveness lymphocyte subsets after traumatic injury: relation to development of sepsis. Clin Immunol Immunopathol 1984;32:22433. 11 Blazar BA, Rodrick ML, OMahony JB et al. Suppresion of natural killer-cell function in humans following thermal and traumatic injury. J Clin Immunol 1986;6:2636.
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12 Maturana P, Puente J, Miranda D, Sepulveda C, Wolf ME, Mosnaim AD. Natural killer cell activity in patients with septic shock. J Crit Care 1991;6:425. 13 Puente J, Carvajal T, Parra S et al. In vitro studies of natural killer cell activity in septic shock patients. Response to a challenge with alpha-interferon and interleukin-2. Int J Clin Pharmacol Ther Toxicol 1993;31:2715. 14 Jimenez MF, Watson WG, Parodo J et al. Dysregulated expression of neutrophil apoptosis in the systemic inflammatory response syndrome. Arch Surg 1997;132:126370. 15 van Dijk WC, Verbrugh HA, van der Tol ME et al. Interactions of phagocytic and bacterial cells in patients with bacteremia caused by Gram-negative rods. J Infect Dis 1980;141:4419. 16 Benjamin CF, Ferreira SH, Cunha FDQ. Role of nitric oxide in the failure of neutrophil migration in sepsis. J Infect Dis 2000;182:21423. 17 Cummings CJ, Martin TR, Frevert CW et al. Expression and function of the chemokine receptor CXCR1 and CXCR2 in sepsis. J Immunol 1999;162: 23416. 18 Marie C, Fitting C, Cheval C et al. High levels of leukocyte-associated interleukin-8 upon cell-activation and in patients with sepsis syndrome. Infect Immun 1997;65:86571. 19 Christou NV. Host-defence mechanism in surgical patients: a correlative study of the delayed hypersensitivity skin-test response, granulocyte function and sepsis. Can J Surg 1985;28:3946. 20 Christou NV, Meakins JL, MacLean LD. The predictive role of delayed hypersensitivity in preoperative patients. Surg Gynecol Obstet 1981;152:297301. 21 Cavaillon J-M. Possibilities and problems of cytokine measurements. In Redl H, Schlag G, eds. Cytokines in Severe Sepsis and Septic Shock, Prog. Inflam. Res., Basel: Birkhuser Publishing Ltd. 1998, 95119. 22 Wood JJ, Rodrick ML, OMahony JB et al. Inadequate interleukin 2 production. A fundamental immunological deficiency in patients with major burns. Ann Surg 1984;200:31120. 23 Cabie A, Fitting C, Farkas JC et al. Influence of surgery on in-vitro cytokine production by human monocytes. Cytokine 1992;4:57680. 24 Cavaillon J-M. Pro- versus anti-inflammatory cytokines: myth or reality. Cell Mol Biol 2001;47:695702. 25 Munoz C, Carlet J, Fitting C, Misset B, Bleriot JP, Cavaillon J-M. Dysregulation of in vitro cytokine production by monocytes during sepsis. J Clin Invest 1991;88:174754. 26 Ertel W, Keel M, Neidhardt R et al. Inhibition of the defense system stimulating interleukin-12 interferon-gamma pathway during critical Illness. Blood 1997;89:161220. 27 OSullivan ST, Lederer, JA, Horgan AF, Chin DHL, Mannick JA, Rodrick ML. Major injury leads to predominance of the T helper-2 lymphocyte phenotype and diminished interleukin-12 production associated with decreased resistance to infection. Ann Surg 1995;222:48292. 28 Mack VE, McCarter MD, Naana HA, Calvano SE, Daly J-M. Dominance of T helper 2 type cytokine after severe injury. Arch Surg 1996;131:13039. 29 Puyana JC, Pellegrini JD, De AK, Kodys K, Silva WE, Miller CL. Both T-helper-1- and T-helper-2-type lymphokines are depressed in posttrauma anergy. J Trauma 1998;44:103745. 30 Muret J, Marie C, Fitting C, Payen D, Cavaillon J-M. Ex vivo T-lymphocyte derived cytokine production in SIRS patients is influenced by experimental procedures. Shock 2000;13:16974. 31 McCall CE, Grosso-Wilmoth LM, LaRue K, Guzman RN, Cousart SL. Tolerance to endotoxin-induced expression of the interleukin-1 beta gene in
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blood neutrophils of humans with the sepsis syndrome. J Clin Invest 1993;91:85361. Marie C, Muret J, Fitting C, Losser MR, Payen D, Cavaillon J-M. Reduced ex vivo interleukin-8 production by neutrophils in septic and nonseptic systemic inflammatory response syndrome. Blood 1998;91:343946. Van Deuren M, Van Der Ven-Jongekrijg H, Demacker PNM et al. Differential expression of proinflammatory cytokines and their inhibitors during the course of meningococcal infections. J Infect Dis 1994;169:15761. Marie C, Muret J, Fitting C, Payen D, Cavaillon J-M. Interleukin-1 receptor antagonist production during infectious and non-infectious systemic inflammatory response syndrome. Crit Care Med 2000;28:227783. Brandtzaeg P, Osnes L, vsteb R, Jo GB, Westwik AB, Kierulf P. Net inflammatory capacity of human septic shock plasma evaluated by a monocytebased target cell assay: identification of interleukin-10 as a major functional deactivator of human monocytes. J Exp Med 1996;184:5160. Ayala A, Meldrum DR, Perrin MM, Chaudry IH. The release of transforming growth factor-beta following haemorrhage: its role as a mediator of host immunosuppression. Immunology 1993;79:47984. Ayala A, Knotts JB, Ertel W et al. Role of interleukin 6 and transforming growth factor-beta in the induction of depressed splenocyte responses following sepsis. Arch Surg 1993;128:8994. Jones S, Romano F. Dose- and time-dependent changes in plasma catecholamines in response to endotoxin in conscious rats. Circ Shock 1989;28:5968. Severn A, Rapson NT, Hunter CA, Liew FY. Regulation of tumor necrosis factor production by adrenaline and by -adrenergic agonists. J Immunol 1992;148:34415. van der Poll T, Coyle SM, Barbosa K, Braxton CC, Lowry SF. Epinephrine inhibits tumor necrosis factor-alpha and potentiates interleukin-10 production during human endotoxemia. J Clin Invest 1996;97:71319. Luger TA, Kalden DH, Scholzen TE, Brzoska T. -melanocyte-stimulating hormone as a mediator of tolerance induction. Pathobiology 1999;67:31821. Delgado M, Pozo D, Martinez C et al.Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit endotoxin-induced TNF production by macrophages: in vitro and in vivo studies. J Immunol 1999;162:235867. Chrousos GP. The hypothalamicpituitaryadrenal axis and immune-mediated inflammation. N Engl J Med 1995;332:135162. Choudhry MA, Ahmad S, Ahmed Z, Sayeed MM. Prostaglandin E2 downregulation of T cell IL-2 production is independent of IL-10 during Gram negative sepsis. Immunol Lett 1999;67:12530. Zweigner J, Gramm HJ, Singer OC, Wegscheider K, Schumann RR. High concentration of lipopolysaccharide-binding protein in serum of patients with severe sepsis or septic shock inhibit the lipopolysaccharide response in human monocytes. Blood 2001;98:38008. Vreugdenhil ACE, Snoeck AMP, vant Veer C, Greve JWM, Buurman WA. LPS-binding protein circulates in association with apoB-containing lipoproteins and enhances endotoxin-LDL/VLDL interaction. J Clin Invest 2001;107: 22534. Cavaillon J-M, Fitting C, Haeffner-Cavaillon N, Kirsch J, Warren HS. Cytokine response by monocytes/macrophages to free and lipoproteinbound lipopolysaccharide. Infect Immun 1990;58:237582. Kitchens RL, Thompson PA, Viriyakosol S, OKeefe GE, Munford RS. Plasma CD14 decreases monocyte responses to LPS by transferring cell-bound LPS to plasma lipoproteins. J Clin Invest 2001;108:48593.
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49 Cavaillon J-M. The non specific nature of endotoxin tolerance. Trends Microbiol 1995;3:3204. 50 Nomura F, Akashi S, Sakao Y et al. Endotoxin tolerance in mouse peritoneal macrophages correlates with down-regulation of surface Toll-like receptor 4 expression. J Immunol 2000;164:34769. 51 Medvedev AE, Kopydlowski KM, Vogel SN. Inhibition of lipopolysaccharideinduced signal transduction in endotoxin-tolerized mouse macrophages: dysregulation of cytokine, chemokine, and Toll-like receptor 2 and 4 gene expression. J Immunol 2000;164:556474. 52 Blackwell TS, Blackwell TR, Christman JW. Induction of endotoxin tolerance depletes nuclear factor-kappaB and suppresses its activation in rat alveolar macrophages. J Leukocyte Biol 1997;62:88591. 53 Ziegler-Heitbrock L, Wedel A, Schraut W et al. Tolerance to lipopolysaccharide involves mobilization of nuclear factor B with predominance of p50 homodimers. J Biol Chem 1994;269:170014. 54 Adib-Conquy M, Adrie C, Moine P et al. Nuclear factor B expression in mononuclear cells of septic patients resembles that observed in LPS-tolerance. Am J Respir Crit Care Med 2000;162:187783. 55 Adib-Conquy M, Asehnoune K, Moine P, Cavaillon J-M. Long term impaired expression of nuclear factor B and I B in peripheral blood mononuclear cells of patients with major trauma. J Leukocyte Biol 2001;70:308. 56 Learn CA, Boger MS, Li L, McCall CE. The phosphatidylinositol 3 kinase pathway selectively controls sIL-1ra not interleukin-1 production in the septic leukocytes. J Biol Chem 2001;276:202349. 57 Choudhry MA, Uddin S, Sayeed MM. Prostaglandin E2 modulation of p59fyn tyrosine kinase in T lymphocytes during sepsis. J Immunol 1998;160:92935. 58 Song GY, Chung CS, Chaudry IH, Ayala A. MAPK p38 antagonism as a novel method of inhibiting lymphoid immune suppression in polymicrobial sepsis. Am J Physiol Cell Physiol 2001;281:C6629. 59 Schwartz MD, Moore EE, Moore FA et al. Nuclear factor kappa B is activated in alveolar macrophages from patients with acute respiratory distress syndrome. Crit Care Med 1996;24:128592. 60 Moine P, McIntyre R, Schwartz MD et al. NF-kappaB regulatory mechanisms in alveolar macrophages from patients with acute respiratory distress syndrome. Shock 2000;13:8591. 61 Fieren MWJ, Van Den Bemd GJ, Bonta IL. Endotoxin-stimulated peritoneal macrophages obtained from continuous ambulatory peritoneal dialysis patients show an increased capacity to release interleukin-1 in vitro during infectious peritonitis. Eur J Clin Invest 1990;B4537. 62 Rugtveit J, Nilsen EM, Bakka A, Carlsen H, Brandtzaeg P, Scott H. Cytokine profiles differ in newly recruited and resident subsets of mucosal macrophages from inflammatory bowel disease. Gastroenterol 1997;112:1493505. 63 Rana N, Braun DP, House R, Gebel H, Rotman C, Dmowski WP. Basal and stimulated secretion of cytokines by peritoneal macrophages in women with endometriosis. Fertil Steril 1996;65:92530. 64 Wu MY, Ho HN, Chen SU, Chao KH, Chen CD, Yang YS. Increase in the production of IL-6, IL-10 and IL-12 by LPS stimulated peritoneal macrophages from women with endometriosis. Am J Reprod Immunol 1999;41:10611. 65 Nssler NC, Stange B, Nussler AK et al. Upregulation of intraepithelial lymphocyte function in the small intestinal mucosa in sepsis. Shock 2001;16:4548. 66 Munford RS, Pugin J. Normal responses to injury prevent systemic inflammation and can be immunosuppressive. Am J Respir Crit Care Med 2001;163:31621.
17
2: Apoptosis and the inflammatory process

NIGEL R WEBSTER
Introduction
Cells that are damaged by injury, such as by mechanical damage or exposure to toxic chemicals, undergo swelling, from disruption of the ability of the plasma membrane to control the passage of ions and water, with consequent leakage of cell contents, leading to inflammation of surrounding tissues.This process is called necrosis. Cells which are induced to commit suicide, in contrast, shrink, and the mitochondrial membrane becomes breached, such that release of cytochrome c occurs. Chromatin (DNA and protein) in the nucleus becomes degraded into small, membrane-wrapped fragments, and the phospholipid phosphatidylserine, which is normally hidden within the plasma membrane, is exposed on the surface. This is then bound by receptors on phagocytic cells such as macrophages, which engulf the cell fragments, leading to a quiet orderly removal of dead cells. This pattern of events is called programmed cell death or apoptosis. The cellular machinery of programmed cell death is as intrinsic to the cell as, for example, mitosis. This article will describe the regulation and process of apoptosis and its relevance to disease, including the inflammatory response in patients with sepsis.
Identification of apoptosis
A series of careful observational studies in the 1950s and 1960s demonstrated the importance of physiological cell death in development. By the 1970s, a process of cell death, characterised by a rigid set of structural changes, was also observed in a wide variety of physiological circumstances: negative selection in the immune system, cytotoxic T cell killing, atrophy induced by hormones and other stimuli, the growth and regression of tumours, and tissue development after exposure to teratogens. These distinctive structural changes characterising cell death were identical to those found in cell death during normal development and
18
APOPTOSIS AND THE INFLAMMATORY PROCESS
raised the possibility that a programmed death pathway, similar to that in development, might also occur in adult tissues in response to a variety of stimuli. This type of death was called apoptosis a term derived from the Greek word meaning the dropping of leaves from the trees. The word apoptosis is often mispronounced it derives from apo- and -ptosis and therefore the second p is silent. This term is applied to a group of characteristic structural and molecular events which separate this type of cell deletion from necrosis (Box 2.1). In contrast to necrosis, which involves a group of cells simultaneously, apoptosis may occur in a single cell surrounded by a group of viable cells. Apoptosis is a selective process for deletion of cells in various biological systems and, in a similar manner to proliferation, is tightly regulated, with both processes playing essential roles in the homeostasis of renewable tissues.13
Box 2.1 Key facts about apoptosis Normal process modelling in vertebrate development cell loss accompanying atrophy in adult tissues deletion of B and T lymphocytes occurs widely in tumours Characteristic morphological changes Characteristic biochemical changes
The process of apoptosis

Structurally, the dying cell loses contact with its neighbours, undergoes a dramatic process of bubbling, blebbing, and shrinkage, and disintegrates into a cluster of membrane-bound fragments. Inside, there are compacted organelles and prominent and characteristic chromatin condensation. Apoptotic cells in tissues are rapidly recognised and phagocytosed by their neighbours, or by specialised phagocytes, in whose phagosomes they are safely destroyed within a few hours.Tissues can shrink to half their original cell number in a day or two, with little disturbance in structure, no inflammatory process, and few accumulating dead cells.
Apoptosis versus necrosis

All of this is very different from the changes in cells exposed to severe toxicological injury or major degrees of hypoxia, where damage to
19
Box 2.2 Morphological features of apoptosis versus necrosis Necrosis Loss of membrane integrity Swelling of cytoplasm and mitochondria Ends with total cell lysis No vesicle formation, complete lysis Disintegration of organelles Apoptosis Mitochondria becomes leaky Membrane blebbing, no loss of integrity Aggregation of chromatin at the nuclear membrane Shrinking of cytoplasm and condensation of nucleus Ends with fragmentation of cell into smaller bodies Formation of membrane bound vesicles (apoptotic bodies)
Box 2.3 Biochemical features of apoptosis versus necrosis Necrosis Loss of regulation of ion homeostasis No energy requirement Random digestion of DNA Postlytic DNA fragmentation (late event of death) Apoptosis Tightly regulated process Energy (ATP)-dependent Non-random fragmentation of DNA (ladder pattern) Pre-lytic DNA fragmentation Release of cytochrome c into cytoplasm by mitochondria Activation of caspase cascade Translocation of membrane phosphatidyl-serine
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Box 2.4 Physiological significance of apoptosis versus necrosis Necrosis Affects groups of contiguous cells Evoked by non-physiological disturbances Phagocytosis by macrophages Significant inflammatory response Apoptosis Affects individual cells Induced by physiological stimuli (lack of growth factors, hormonal environment) Phagocytosis by adjacent cells or macrophages No inflammatory response
energy-dependent membrane ion pumps leads to progressive cellular swelling and rupture necrosis. When this occurs there is usually an acute inflammatory reaction, apparently stimulated by neutrophil chemotactic factors originating from intracellular proteins lost from the necrotic cells. The characteristic morphological and biochemical features and physiological significance of necrosis versus apoptosis are given in Boxes 2.2, 2.3, and 2.4. Figure 2.1 shows a normal eosinophil and one undergoing apoptosis.
Figure 2.1 A transmission electron photomicrograph of (A) a normal eosinophil and (B) an eosinophil undergoing apoptosis showing aggregation of chromatin, blebbing, and shedding of intracytoplasmic granules. 21
Why do we need apoptosis?

There are two reasons why apoptosis is physiologically vital. The first is the role it plays in fetal development and other key processes. This is most eloquently seen in the change from a tadpole to a frog. The resorption of the tadpole tail at the time of its metamorphosis into a frog occurs by apoptosis. In man the formation of the fingers and toes of the fetus requires the removal, by apoptosis, of the tissue between them. The formation of synapses between neurones requires that surplus cells be eliminated by apoptosis, and the sloughing off of the endometrium at the start of menstruation also occurs by apoptosis. The second reason for apoptosis is the need to destroy cells that represent a threat to the integrity of the organism. This might include, for example, cells infected with viruses one of the methods by which cytotoxic T (Tc) lymphocytes kill virus-infected cells is by inducing apoptosis and some viruses are able to mount countermeasures (see Chapter 3 in this volume). Apoptosis is also important in cells with DNA damage where disruption of proper embryonic development leading to birth defects can occur, or the cell can become cancerous. Cells respond to DNA damage by increasing their production of p53 a potent inducer of apoptosis. Mutations in the p53 gene an oncogene producing a defective protein, are often found in cancer cells, and radiation and chemotherapeutic agents used in cancer therapy induce apoptosis in some types of cancer cells. Mice with both copies of p53 deleted develop multiple malignancies, and p53 mutation is associated with many human cancers. Following DNA damage, for example, by radiation, p53 levels rise, and proliferating cells arrest in the G1 phase of mitosis. This allows time for DNA repair prior to the next round of replication. This arrest is mediated by stimulation of expression of p21CIP1, a cyclin kinase inhibitor.
Why can apoptosis be a problem?

Although the process of apoptosis is physiologically essential for both development and removal of dangerous cells, initiation of the sequence of events leading to cell death through apoptosis can lead to both unwanted removal of healthy cells, and propagation of inflammatory responses through release of cytokines (see description of the actions of caspase enzymes below).
Caenorhabditis elegans and the genetics of apoptosis

C. elegans is a hermaphrodite nematode worm with a life cycle from egg to sexual maturity of about 3 days. The genome of this organism has been
22
fully sequenced and contains 19 099 genes. The adult hermaphrodite consists of exactly 959 somatic cells of very precisely determined lineage and function. Development control occurs through apoptotic removal of exactly 131 cells, and thus C. elegans is an ideal organism to study the genetics of apoptosis. It has been found that three key genes trigger apoptosis: ced-3 and ced-4 (C. elegans cell death genes) and egl-1 (C. elegans egg laying defective gene) (Figure 2.2). Ced-3 has a mammalian counterpart, originally known as interleukin 1 converting enzyme or ICE, but now termed caspase 1 (Cys catalytic Asp targeting protease). Thirteen caspases are known in mammalian systems and have conserved sequence and subunit structure; of these four play key effector roles in apoptosis and four are initiators in the activation process.4 Ced-4 acts as an adapter for caspase activation in C. elegans; the mammalian counterpart is called apoptosis activating factor (Apaf-1). A fourth gene in C. elegans promotes survival, that is, it acts as a negative regulator of apoptosis, ced-9. The mammalian equivalent is the BCL-2 family of genes. Bcl-2 and ced-9 proteins are 23% identical, and bcl-2 can substitute for ced-9 in C. elegans. However, in higher animals, bcl-2 is a member of a large family of closely related proteins, some of which promote survival and some death (Box 2.5). The similarities between ced-3 and ICE, ced-4 and Apaf-1, and between ced-9 and bcl-2 strongly suggest that programmed cell death in C. elegans parallels apoptosis in higher animals, but in a much more simplified form. Figure 2.2 shows the C. elegans apoptosis genes and their human equivalents.5
Pro-apoptotic ced-3 ced-4 Anti-apoptotic ced-9
caspase-1(ICE) Apaf-1 BCL-2 gene family
Figure 2.2 A transmission electron micrograph of the nematode worm Caenorhabditis elegans. Its apoptosis genes ( left) with the mammalian equivalents (right) are indicated.
Signals for apoptosis

The cascade of events leading to apoptosis takes place as a result of either the withdrawal of positive signals (that is, signals needed for continued survival), or the initiation of negative signals (that is, those which instigate cell death). Signals can arise within the cell or from so-called death activators binding to receptors at the cell surface.6
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Box 2.5 The bcl-2 protein family Pro-apoptotic Bad Bak Bax Bcl-Xs Bid Bik Anti-apoptotic AL Bcl-2 Bcl-W Bcl-XL Mcl-1
Withdrawal of positive signals The continued survival of most cells requires that they receive continuous stimulation from other cells and, for many, continued adhesion to the surface on which they are growing. Some examples of positive signals include specific growth factors for neurones, and interleukin-2 (IL-2), an essential factor for the mitosis of lymphocytes. Receipt of negative signals Internal signals In a healthy cell, the outer membranes of mitochondria express the protein bcl-2 on their surface. The role of the mitochondrion in apoptosis is discussed further below. Bcl-2 is bound to a molecule of the protein Apaf-1, and internal damage in the cell causes bcl-2 to release Apaf-1.This results in leakage of cytochrome c from mitochondria into the cytoplasm. The released cytochrome c and Apaf-1 bind to molecules of caspase 9. The resulting complex of cytochrome c, Apaf-1 and caspase 9 (with ATP) is called an apoptosome and these aggregate in the cytosol. Caspase 9 activates other caspases, which leads to digestion of structural proteins in the cytoplasm, degradation of chromosomal DNA, and ultimately phagocytosis of the cell (see caspases below).
24
External signals External signals which initiate apoptosis include increased levels of oxidants within the cell or damage to DNA by these oxidants or other agents, such as ultraviolet light, x rays, and chemotherapeutic drugs. In addition there are several molecules that bind to specific receptors on the cell surface and signal the start of apoptosis. Signalling pathways link key receptors to the caspase proteolytic cascade. These death activators include tumour necrosis factor (TNF ) which binds to the TNF receptor; lymphotoxin (also known as TNF ) which also binds to the TNF receptor; and Fas ligand (FasL), a molecule that binds to the Fas cell-surface receptor (also called CD95). Fas expression and immune function Fas is a transmembrane receptor protein which is very widely distributed, and is constitutively expressed in some cells for example, liver, or induced in cells such as lymphocytes on activation. The activating ligand, Fas-L, is expressed in a narrower range of cells, including antigen-activated T lymphocytes, testis, eye, and central nervous system. Persistent and strong stimulation of CD4 T helper lymphocytes results in expression of Fas-L. During ablation of the immune repertoire in the fetal/neonatal period, selfantigen stimulation causes killing of self-reactive B lymphocytes and self or mutual killing of the Fas-L expressing T lymphocytes. The development of tolerance against a potential antibody represents a similar process. Some tumours also express Fas-L constitutively for example, melanoma and lung tumours. This renders them resistant to immune intervention any visiting lymphocytes are induced to commit suicide through apoptosis. Expression of Fas-L in testis, eye, and central nervous system gives rise to the phenomenon of immune privilege, in which these tissues do not reject allografts, but kill invading lymphocytes instead. Mechanism of Fas-L and TNF-induced apoptosis When Fas binds Fas-L, changes occur in the cell membrane; its cytoplasmic domain contains a sequence called the death domain (DD), a sequence critically required for stimulated apoptosis.The changes in the cell membrane result in incorporation of an intracellular DD-containing protein called Fas-associated death domain (FADD) protein (synonymous with a protein previously named MORT1). The N-terminal region of FADD contains another domain referred to as death effector domain or DED.This associates with another DED at the N-terminus of pro-caspase-8 (the pro-caspase formerly known as pro-FLICE or MACH1, or Mort-associated ced-3 homologue). Recruitment into the complex results in dimerisation of procaspase-8, releasing the active caspase-8. Caspase-8 then initiates the whole effector protease cascade by acting on pro-caspase-3 to liberate caspase 3.
25
A similar sequence involves the TNF receptor (TNFR), signalled by TNF, and DR3 or Apo3 signalled by Apo3L. TNF binds to TNFR, recruiting the DD protein TRADD, and acts as an intermediary for binding FADD, activating pro-caspase 8 in the same way as FAS. However, TRADD can also be occupied by another DD protein RIP, which signals to the nuclear factor B (NF B) and Jun pathways, which are inhibitory to apoptosis. TNF is expressed mainly in activated lymphocytes and macrophages, while TNFR is expressed ubiquitously. DR3 is expressed by spleen and thymus cells, and Apo3L by T cells. This complex pathway is represented in a simplified form in Figure 2.3.
Apo2L TRAIL Free radicals DR4/ Steroids NO DR5 Death adaptor Bcl2/Bcl-XL FasL Fas/ CD95 FADD Apo3L DR3 TNF TNF R1
TRADD
TRAF 1
ICE/ Caspase 1
Caspase 8 FLIP NFB activation
m Cytochrome c Bad, Bax
+/ Caspase 3
APOPTOSIS
Nucleus
Figure 2.3 The complex pathway involved in the signalling of apoptosis events. For explanation and abbreviations/acronyms see text.
Caspases
Caspases are a group of proteases, named because they cleave proteins mostly each other at aspartic acid (Asp) residues. Caspases initially exist as immature pro-caspases (zymogens) and require processing to be activated, in a similar way to the proteins of the clotting or complement cascades. There are three basic domains in the immature form: the prodomain, the large subunit, and the small subunit. Some caspases are initiators that is, their targets are downstream effector caspases. The initiator caspases have a large pro-domain, since these are regulated by proteins other than caspases.The effector caspases have small pro-domains since they are directly regulated by other caspases. In other words, the pro-domain is important for proteinprotein interactions. The large pro-domain interacts with other proteins in the cell, containing caspase recruitment domains (CARD domains). These interactions lead to the cleavage and activation of the inhibitor caspases, which then go on to cleave the effector caspases. Once activated the long pro-domain caspases
26
may then cleave caspases with short pro-domains to achieve their activation.7 The full process of apoptosis in mammalian cells involves several caspases, some of which have specialised purposes; for example a whole subgroup is involved in cytokine activation rather than apoptosis (Figure 2.4).
m cytochrome c
mitochondrion
cytochrome c Apaf-1
(ced-4 equivalent)
pro-caspase 9
caspase 9
pro-caspase 3
caspase 3
APOPTOSIS Figure 2.4 A simplified representation of the role of caspases in apoptosis. For abbreviations/acronyms see text.
The initiator caspases include caspases 2, 8, 9, and 10, and act on procaspase 3. The effector caspases are 3, 6, and 7, which act on proteins involved in cell structure. The inflammatory caspases mentioned earlier in this chapter are 1, 4, 5, 11, 12, and 13. Substrates for these enzymes are pro-IL-1 and pro-IL-18. IL-18 is a relatively recently elucidated cytokine, which has similar effects to IL-1 . Thus the signals for apoptosis can also result in propagation of inflammatory events via pro-inflammatory cytokine release. The net effects of caspases are outlined in Box 2.6.
Box 2.6 Net effect of caspase activity Halts cell cycle progression Disables homeostatic and repair mechanisms Initiates detachment of the cell from surrounding tissue structure Disassembles structured components Marks dying cell for engulfment by other cells such as macrophages
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Redundancy of caspases Caspase-1 or ICE is not a single enzyme, but one of a family of related proteases, which are co-expressed. This appears to provide redundancy for an important function and circumvents what was once an embarrassment, that caspase-1 knockout mice still undergo apoptosis.
The role of mitochondria in apoptosis

Recently, the mitochondrion has been identified as playing a central role in apoptosis. Several findings support this idea: members of the BCL-2 gene family localise into the mitochondrial membrane and inhibit apoptosis and a mutant BCL-2, which cannot insert into the mitochondrial membrane, is a less potent inhibitor of apoptosis. BCL-2/-XL can also recruit Apaf-1 into the mitochondrial membrane and may prevent Apaf-1 from activating caspases, thereby inhibiting apoptosis. In addition some mitochondrial proteins induce apoptosis when leaked into the cytosol: during apoptosis, cytochrome c and apoptosis inducing factor (AIF) are released from the mitochondria and, with other factors, such as Apaf-1 and Apaf-3, lead to caspase activation and apoptosis. Increased levels of BCL-2 can prevent the release of these molecules, whereas caspase inhibitors cannot. This indicates the release of cytochrome c and AIF is downstream of BCL-2 function but upstream of the caspases. Apoptosis is also associated with a change in the mitochondrial membrane potential, a phenomenon known as permeability transition. The permeability transition can be blocked by excess BCL-2 but not by inhibitors of caspases, indicating that the permeability transition is downstream of BCL-2 but upstream of caspase activation. BCL-2, bcl-XL, and another apoptosis-related gene, BAX, are capable of forming selective ion pores in membranes, thus forming channels in the mitochondrial membrane that could regulate permeability transition and the release of molecules such as cytochrome c and AIF.
Apoptosis and disease

Apoptosis and cancer Cancer cells are known to use inhibition of apoptosis.8 For example, one of the two human papilloma viruses (HPV) that have been implicated in causing cervical cancer produces a protein (E6) which binds and inactivates the apoptosis promoter p53. EpsteinBarr Virus (EBV), the cause of mononucleosis and a cause of Burkitts lymphoma, secretes a protein that stimulates cells to increase endogenous production of bcl-2.
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Both these actions make the cell more resistant to apoptosis and therefore enables cancer cells to continue to proliferate. Some B-cell leukaemias and lymphomas express high levels of bcl-2, thus blocking apoptotic signals that they may receive. Melanoma cells avoid apoptosis by inhibiting the expression of the gene encoding Apaf-1, and some cancer cells, especially lung and colon cancer cells, secrete elevated levels of a soluble decoy molecule that binds to FasL, preventing binding to Fas and blocking cytotoxic T cell killing mechanisms. Apoptosis and AIDS The hallmark of acquired immunodeficiency syndrome (AIDS) is the decline in CD4 T cell numbers. These cells are responsible, directly or indirectly (as T helper cells), for all immune responses. Human immunodeficiency virus (HIV) invades CD4 and one might assume that it is this infection by HIV that causes loss of CD4 T cells. However, fewer than 1 in 100 000 CD4 T cells in the blood of AIDS patients are actually infected with the virus and, although the mechanism is not clear, apoptosis appears to be involved. Since all T cells, both infected and uninfected, express Fas, expression of an HIV gene (termed Nef) in a HIV-infected cell causes the cell to express high levels of FasL at its surface while preventing an interaction with self-Fas preventing self-elimination. However, when the infected T cell encounters an uninfected cell (for example, in a lymph node), the interaction of FasL with Fas on the uninfected cell kills it by apoptosis. Apoptosis and organ transplants For many years it has been known that certain tissues including the anterior chamber of the eye and the testes are immunologically privileged sites such that antigens within these sites fail to elicit an immune response from high constitutive expression of high levels of FasL. This finding raises the possibility of a new way of preventing graft rejection. However, animal studies have produced mixed results. Allografts that have been genetically modified to express FasL have shown increased survival for kidneys but not for hearts or islets of Langerhans. Apoptosis and sepsis Apoptosis is also relevant to inflammatory responses in sepsis. The immune/inflammatory balance in sepsis reflects the balance between pro- and anti-inflammatory responses, and the balance between hypermetabolic/ hyperdynamic versus hypometabolic/hypodynamic responses. There is also
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probably a balance between ischaemic or necrotic cell death and apoptotic cell death. Ultimately, the balance between all these components probably then governs death or survival of the patient (Figure 2.5).
SEPSIS
Hypermetabolic Hyperdynamic
Exaggerated inflammatory response
Ischaemic/ necrotic versus Apoptotic cell death
Hypometabolic Hypodynamic
Immune hyporesponsiveness
Death/survival
Figure 2.5 A representation of the balance phenomenon in sepsis.
There is some evidence of apoptosis in sepsis. There is evidence of mitochondrial injury in animal models of sepsis in terms of ultrastructural mitochondrial injury, altered oxygen delivery, and maximal oxygen extraction, and impaired mitochondrial function related to organ failure.9,10 In ex vivo studies, monocyte mitochondrial membrane potential was shown to be decreased in septic patients, particularly in nonsurvivors,11 and deranged mitochondrial redox state in critically ill patients has been reported.12 Cells are exposed to oxygen-derived radicals as a by-product of electron transport within mitochondria. If antioxidant protection is inadequate, this results in oxidative stress, causing altered mitochondrial membrane potential and triggering release of cytochrome c, activation of pro-caspases and apoptosis. Endotoxin-induced oxidative stress causes mitochondrial damage,9,10 triggers apoptotic pathways,13,14 and results in caspase activation.15,16 Apoptosis associated with caspase-3 activation has been shown in patients with sepsis.17,18 Oxidative stress in such patients, including:
increased levels of lipid peroxides and direct detection of circulating radicals using electron paramagnetic resonance; decreased antioxidant capacity associated with non-survival; decreased concentrations of individual protective antioxidants; detectable circulating redox-reactive iron; ischaemiareperfusion leading to xanthine oxidase activation; and evidence of abnormal handling of exogenous antioxidants has been shown.1924
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Conclusion
Apoptosis is a mechanism of programmed cell death and it is essential for survival of the total organism. For example, without selective destruction of non-self T cells, an animal would lack immunity. It is also vital for normal development, such that there is programmed destruction of cells during embryogenesis. Later in life, apoptosis has a protective role in removing damaged cells, such as after viral infection or exposure to UV light causing sunburn (peeling). Without apoptosis tumour development would be much more common, and consequently defects in control of apoptosis may result in cancer. Apoptosis occurs in patients with sepsis and hence organ cell mass will decrease. However, in other cells, for example neutrophils, apoptosis is inhibited. The contribution of apoptosis to the outcome of patients with sepsis and multi-organ failure is as yet unknown.
References
1 Hengartner MO. The biochemistry of apoptosis. Nature 2000;407:7706. 2 Roy S, Nicholson DW. Cross-talk in cell death signaling. J Exp Med 2000;192:F215. 3 Slee EA, Harte MT, Kluck RM et al. Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol 1999;144:28192. 4 Nicholson DW. From bench to clinic with apoptosis-based therapeutic agents. Nature 2000;407:81016. 5 Alnemri ES, Livingston DJ, Nicholson DW et al. Human ICE/CED-3 protease nomenclature. Cell 1996;87:171. 6 Finkel E. The mitochondrion: is it central to apoptosis? Science 2001;292:6246. 7 Srinivasula SM, Saleh A, Ahmad M, Fernandes-Alnemri T, Alnemri ES. Isolation and assay of caspases. Methods Cell Biol 2001;66:127. 8 Wyllie AH, Bellamy CO, Bubb VJ et al. Apoptosis and carcinogenesis. Br J Cancer 1999;80(Suppl. 1):347. 9 Crouser ED, Julian MW, Dorinsky PM. Ileal VO2-DO2 alterations induced by endotoxin correlate with severity of mitochondrial injury. Am J Respir Crit Care Med 1999; 160:134753. 10 Trumbeckaite S, Opalka JR, Neuhof C, Zierz S, Gellerich FN. Different sensitivity of rabbit heart and skeletal muscle to endotoxin-induced impairment of mitochondrial function. Eur J Biochem 2001;268:14229. 11 Adrie C, Bachelet M, Vayssier-Taussat M et al. Mitochondrial membrane potential and apoptosis peripheral blood monocytes in severe human sepsis. Am J Respir Crit Care Med 2001;164:38995. 12 Yassen K, Galley HF, Lee A, Webster NR. Mitochondrial redox state in the critically ill. Br J Anaesth 1999;83:3257. 13 McDonald TE, Grinman MN, Carthy CM, Walley KR. Endotoxin infusion in rats induces apoptotic and survival pathways in hearts. Am J Physiol Heart Circ Physiol 2000;279:H205361. 14 Ayala A, Herdon CD, Lehman DL, Chaudry IH. Differential induction of apoptosis in lymphoid tissues during sepsis: variation in onset, frequency, and nature of the mediators. Blood 1996;87:426175.
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15 Panaretakis T, Shabalina IG, Grander D, Shoshan MC, DePierre JW. Reactive oxygen species and mitochondria mediate the induction of apoptosis in human hepatoma HepG2 cells by the rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoic acid. Toxicol Appl Pharmacol 2001;173: 5664. 16 Kajiwara K, Ikeda K, Kuroi R et al. Hydrogen peroxide and hydroxyl radical involvement in the activation of caspase-3 in chemically induced apoptosis of HL-60 cells. Cell Mol Life Sci 2001;58:485-91. 17 Hotchkiss RS, Swanson PE, Freeman BD et al. Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction. Crit Care Med 1999; 27:123051. 18 Schroeder S, Lindemann C, Decker D et al. Increased susceptibility to apoptosis in circulating lymphocytes of critically ill patients. Langenbecks Arch Surg 2001; 386:426. 19 Goode HF, Cowley HC, Walker BE, Webster NR. Decreased antioxidant status and increased lipid peroxidation in patients with sepsis and secondary organ dysfunction. Crit Care Med 1995;23:64651. 20 Galley HF, Davies MJ, Webster NR. Ascorbyl radical formation in patients with sepsis: effect of ascorbate loading. Free Rad Biol Med 1995;20:13943. 21 Galley HF, Davies MJ, Webster NR. Xanthine oxidase activity and free radical generation in patients with sepsis syndrome. Crit Care Med 1996;24:164953. 22 Galley HF, Webster NR. Elevated serum bleomycin-detectable iron concentrations in patients with sepsis syndrome. Intensive Care Med 1996;22: 2269. 23 Cowley HC, Bacon PJ, Goode HF, Webster NR, Jones JG, Menon DK. Plasma antioxidant potential in severe sepsis: A comparison of survivors and nonsurvivors. Crit Care Med 1996;24:117983. 24 Galley HF, Howdle PD, Walker BE, Webster NR. The effects of intravenous antioxidants in patients with septic shock. Free Rad Biol Med 1997;23:76874.
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3: Virus interaction with host immunity

LAWRENCE S YOUNG
Introduction
Resistance to and recovery from viral infections depends on the interactions between virus and host. The defences mounted by the host may act directly on the virus or indirectly on virus replication by altering or killing the infected cell. The non-specific host defences function early in the encounter with a virus to prevent or limit infection while the specific host defences function after infection in the initiation of immune responses to subsequent challenges. Viruses have evolved complex strategies to manipulate host immune defences to their advantage, permitting viral replication without massive inflammatory responses, integrating their needs with that of their host man. However, some persistent and latent viral infections can lead to serious disease and malignancy. This article will describe the hostvirus interactions and particularly focus on the role of latent infection with EpsteinBarr virus in tumour development the killer within.
Viral spread in host

Some viral infections remain at the site of primary infection (localised) while others spread to other tissues and systems (systemic).
Polarised infection of epithelial cells Some viruses preferentially bud out of epithelial cells to the lumen (for example, in gastrointestinal tract or in bronchioles or alveoli (as is the case with influenza)) while other viruses preferentially bud to the basal side, inward toward the bodys other systems (for example, vaccinia, the virus used to vaccinate against smallpox).
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Dissemination via blood/lymph Once the virus leaves the primary site of infection, it may be carried in blood or lymph (for example, measles). Alternatively, direct inoculation of blood can occur via transfusion or intravenous drug use or by bites. Secondary infection may result when virions pass out of blood or lymph via gaps in capillaries or specific transport mechanisms. Dissemination via nerves Some viruses reside in and are transported via nerves following primary infection, for example, herpes simplex virus and varicella-zoster virus. Replication inside neurones is not necessary.Transport is often intra-axonal. Virions may be latent in nerve cell bodies in ganglia and be transported back to pass back to epithelial cells upon reactivation.
Virulence host versus viral factors

Differences that individuals might exhibit in susceptibility and disease for a given virus can be attributable to strain variation among viral populations and sometimes to differences in the hosts genetic, behavioural, or environmental background.
Host defence against viral infection

The immune response can be considered as several overlapping systems. A very simplified view includes the humoral immune system, which consists of antibodies (the product of plasma cells, which in turn are derived from B lymphocytes) and complement; the non-specific or innate immune system, including monocytes/macrophages and natural killer (NK) cells; the cytotoxic T lymphocyte (CTL) system, which recognises the major histocompatibility complex class I (MHC class I) and disposes of infected cells; and the T helper cell system, whose function is to release cytokines and thereby induce and activate other components of the various responses. These are summarised in Table 3.1. Non-specific or innate immunity Physical barriers The host has several inherent barriers to prevent or limit infection.The skin acts as an impenetrable barrier to most viruses and only if this barrier is breached will viruses be able to infect the host. Viruses gain entry into host
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VIRUS INTERACTION WITH HOST IMMUNITY
Table 3.1 Host defences against viral infections. Host defence Early non-specific responses Effector Fever Phagocytosis Inflammation NK cell activity Interferon Cytotoxic T cells Macrophages Lymphokines ADCC Humoral immune responses Antibody Antibody plus complement Target(s) Virus replication Virus Virus replication Virus-infected cell Virus replication, immunomodulation Virus-infected cell Virus, virus-infected cell Virus-infected cells, immunomodulation Virus-infected cell Virus, virus-infected cell Virus, virus-infected cell
Cell-mediated immune responses
Abbreviations: ADCC, antibody-dependent cellular cytotoxicity; NK cell, natural killer cell. Table 3.2 Some common viruses and their immunological targets. Virus HIV EpsteinBarr virus Influenza A Rhinovirus Receptor CD4 CR2 Glycophorin A ICAM-1 Cell type infected Th cells B cells Many cell types Many cell types
Abbreviations: HIV, human immunodeficiency virus; CD4, cluster of differentiation antigen 4; Th, T helper lymphocytes; CR2, complement receptor type 2; ICAM-1, intercellular adhesion molecule-1.
cells by first binding to specific receptors on cells (Table 3.2) and thus host expression of these receptors will limit the host range of the virus. If a host lacks the receptor for a virus or if the host cells lack some component necessary for the replication of a virus, the host will be resistant to that virus. For example, mice lack receptors for polio viruses and are therefore resistant to polio virus. Mucus also acts as a barrier and can prevent viral infection by competing with virus receptors on cells. For example, orthomyxo- and paramyxovirus families bind to sialic acid receptors. Sialic acid-containing glycoproteins in mucus can compete with the sialic acid receptors on cells and limit binding of virus to the cells. The ciliated epithelium, which drives the mucociliary elevator, can help diminish infectivity of certain viruses, particularly respiratory infections. The low pH
35
of gastric secretions also inactivates most viruses, although enteroviruses are resistant and can survive and replicate in the gut. The pH of inflammatory infiltrates is also low and can help limit viral infections by inactivating viruses. The replication of some viruses is reduced at temperatures above 37 C and therefore fever can help to inhibit virus replication. Macrophages Since macrophages migrate to a variety of locations in tissues they are often one of the first types of cells to encounter infecting viruses. Macrophages contribute to antiviral defences in several ways. Intrinsic antiviral activity is the ability of macrophages to resist infection by a virus, and extrinsic antiviral activity is the ability of macrophages to kill other cells infected with virus. Although they can be infected with viruses, many viruses are incapable of replicating in macrophages. However, some viruses are able to survive and even replicate and can thus be spread by macrophages. Virusinfected cells that become coated with IgG antibodies can be killed by macrophages by antibody-dependent cell-mediated cytotoxicity (ADCC) and macrophages are also a source of interferon (IFN) (see below). Natural killer cells Natural killer (NK) cells are large granular lymphocytes and also play a role in resistance to viral infection. They act by recognising and killing virusinfected cells through a mechanism that is not MHC-restricted or antigenspecific, such that NK cells will kill cells infected with many different viruses. NK cells can also mediate ADCC and can kill virus-infected cells by this mechanism; NK cell activity is enhanced by IFN. Interferons Interferon (IFN) was discovered over 40 years ago when it was found that supernatant fractions from virus-infected cells contained a protein that was able to confer resistance to other cells by acting on the cells to make them resistant to infection. There are three types of interferon, IFN , IFN , and IFN . IFN and IFN are also referred to as Type I interferon and IFN as Type II. There are approximately 20 subtypes of IFN but only one IFN and IFN . The interferons are not constitutively expressed but are tightly regulated at the transcriptional level by a number of mediators; constitutive gene expression is repressed by a labile repressor protein, which binds to the promoter region upstream of the gene and inhibits transcription. Gene transcription also requires activator proteins to bind to the promoter region. Inducers of IFN either inhibit repressor or enhance activator protein expression. IFN and IFN are induced by virus infection, double-stranded RNA, and bacterial cell wall components such as lipopolysaccharide (LPS). RNA viruses are the most potent inducers of IFN while DNA viruses are poor IFN inducers, with the
36
exception of poxviruses. IFN is induced by mediators that activate lymphocytes such as mitogens and antigen. Secreted IFN protein binds to IFN receptors on adjacent cells and induces an antiviral state.This is achieved through transcription of a group of genes that code for proteins preventing intracellular viral replication. In addition, synthesis of two enzymes, which results in the inhibition of protein synthesis, occurs. One enzyme indirectly affects protein synthesis by breaking down viral mRNA and the other directly affects protein synthesis by inhibiting elongation. Although the infected cell may die as a consequence of the inhibition of host protein synthesis, the progress of the infection is stopped. Uninfected cells are not killed by IFN since activation of the two enzymes requires double-stranded RNA, which is not produced in uninfected cells. Some viruses are able to inhibit the antiviral effects of IFN: for example, the adenoviruses produce an RNA, which prevents the activation of the protein kinase by double-stranded RNA. The mechanisms of the antiviral effects of IFN are shown in Figure 3.1.
Virus RIP Infected cell Dead infected cell
Interferon-alpha (leukocytes, etc.) and interferon-beta (fibroblasts) IFN receptor Influenza virus +
Antiviral state
2 5 oligoadenylate synthase, protein kinase, etc.
MHC 1
mRNA degradation, inhibition of protein synthesis
Figure 3.1 The mode of action of interferon (IFN). IFN binds to receptor on neighbouring cells, initiating new gene expression through interaction with the IFN responsive element (IRE). An antiviral state is induced and viral growth is prevented. 37
IFN not only induces the production of antiviral proteins, it also has other effects on cells, some of which indirectly contribute to the ability of the host to resist or recover from a viral infection. IFN can help modulate immune responses by its effects on Class I and Class II MHC molecules. IFN , IFN , and IFN increase expression of Class I molecules on all cells thereby promoting recognition by cytotoxic T (Tc) cells, which can destroy virus-infected cells. IFN can also increase expression of Class II MHC molecules on antigen-presenting cells resulting in better presentation of viral antigens to CD4 T helper cells. Also IFN can activate NK cells and the intrinsic and extrinsic antiviral activities of macrophages. IFNs have been used in the treatment of a number of viral and other conditions; the antiproliferative activity of IFNs makes them useful in the treatment of some malignancies, although the side effects of IFN therapy limit their casual use in clinical medicine. Complement The interaction of a complement-fixing antibody with a virus-infected cell or with an enveloped virus can result in the lysis of the cell or virus. Thus, by interfacing with the specific immune system, complement also plays a role in resistance to viral infections. Cytokines Cytokines other than IFN also may play a role in resistance to virus infection. Tumour necrosis factor alpha (TNF ), interleukin-1 (IL-1), and IL-6 have been shown to have antiviral activities in vitro. These cytokines are produced by activated macrophages but their contribution to resistance in vivo has not been fully elucidated. Specific immunity Antibodies Antibodies produced by the specific immune system are involved mainly in resistance to subsequent infection with the virus. IgG, IgM, and IgA antibodies can all play a role in immunity to virus infection, but the relative contributions of the different classes depend on the virus and the route of entry. For example, IgA will be more important in viruses that infect the mucosa while IgG antibodies will be more important in infections that occur through blood-borne infection. Antibody can directly neutralise virus infectivity by preventing receptor binding or entry of the virus into the cell. Complement-fixing antibodies can assist in the lysis of virus-infected cells or enveloped viruses. Antibodies can also act as opsonins and augment phagocytosis of viruses either by promoting their uptake or by agglutinating them to make them more easily phagocytosed. Antibody-coated virusinfected cells can be killed by NK cells. Antibodies can have both beneficial
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and harmful effects for the host: although the host has a variety of defences to protect against viral infections, sometimes it is the immune response to the infection that is the direct cause of tissue injury. Immunopathological damage Some viruses, such as lymphocytic choriomeningitis virus, produce large amounts of immune complexes in the circulation, which accumulate in the vasculature and the kidneys where they fix complement and result in tissue damage through release of vasoactive amines and recruitment of inflammatory cells. Other examples of viruses that cause these effects are: measles, respiratory syncytial virus, dengue, and serum hepatitis virus. Infants infected with cytomegalovirus have circulating immune complexes that are deposited in the kidneys and joints resulting in arthritis and glomerular nephritis. Fatal haemorrhagic shock syndrome associated with dengue virus infection is due to fixation of complement by circulating immune complexes with release of products of the complement cascade, leading to sudden increased vascular permeability, shock, and death. Immune adherence Coating of viruses with antibody can enhance their uptake by phagocytic cells but if the virus is able to survive in the phagocyte, spread of virus infection occurs. Dengue and human immunodeficiency virus (HIV) can survive in macrophages, for example. T cells T cells play a major role in recovery from viral infections. Cytotoxic T cells (CTLs) generated in response to viral antigens on infected cells can kill the infected cells and prevent the spread of infection. Helper T (Th) cells are involved in generation of CTLs and in assisting B cells to make antibody. In addition, cytokines secreted by T cells can recruit and activate macrophages and NK cells, thereby mobilising a concerted attack on the virus (Figure 3.2). Relative contributions of host defence mechanisms The relative contribution of the various host defence mechanisms depends on the nature of the virus and the route of entry. Antibodies, for example, will be more important in infections in which viraemia occurs and may not be helpful in infections with herpes or paramyxoviruses, in which the virus can be passed from cell to cell by cell fusion. In this instance, cell-mediated immunity is more important. If a virus only infects cells in the mucosal surface, IgA antibodies may be important. An understanding of the host defence mechanisms is important for vaccine development. If IgA antibodies are important for protection against a particular virus, then a vaccine must
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CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY Th1 cytokines
CTL Killing Target cell
Th2 cytokines
T Antigen presentation
B Antibody production
Antigen-presenting cell Figure 3.2 How T cells mediate both cytotoxicity and antibody production (Th, T helper cell; CTL cytotoxic T lymphocyte).
be able to stimulate production of IgA antibodies on the appropriate mucosal surface. Alternatively, if CTLs are important, then the vaccine must be able to stimulate CTL production; live vaccines are often preferred since they usually lead to the generation of CTLs, unlike killed vaccines.
Immunosuppression
Many viruses are able to suppress immune responses and thereby overcome or minimise host defences. The best example is HIV, which infects the CD4 cells, thereby destroying the specific immune system. Other viruses (for example, measles virus) can also infect lymphocytes and affect their replication and differentiation.Virus-induced immunosuppression is a major concern in vaccine development.
Immune evasion by viruses

Viruses are able to evade the host immune response through four main mechanisms. Some of the mechanisms by which viruses can evade host defences are illustrated in Box 3.1. Latent or persistent infection Herpes simplex virus (HSV) establishes a lifelong latent infection in neurones, punctuated by bouts of recurrent disease in some people.1 The latent form of the virus is not a good target for cytotoxic T lymphocytes, owing to both low viral protein expression and also because neurones are
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Box 3.1 Viral immune evasion strategies Latent or persistent infection, for example HSV1 in dorsal root ganglia Mutation, for example HIV, influenza virus A express non-immunogenic antigens Preventing recognition of infected cells target cells with low HLA expression, for example neurones reduce HLA class 1 surface expression, for example CMV inhibit antigen processing, for example HSV Target host cytokines inhibit or mimic pro-inflammatory cytokines, for example IL-6 produce anti-inflammatory homologues, for example EBV/IL-10 Abbreviations: CMV, cytomegalovirus; EBV, EpsteinBarr virus; HIV, human immunodeficiency virus; HLA, human leucocyte antigen; HSV1, herpes simplex 1; IL-6, interleukin-6; IL-10, interleukin-10. not well recognised by CTL. However, infection recurs in mucosal and other superficial tissues, and, although recurrent disease is often associated with mild immune suppression, normal or near-normal humoral response and cellular immune responses are generally present; in spite of this, HSV is able to produce a productive infection for several days. This is because the virus is able to block antigen presentation in human cells by blocking transport mechanisms involved in antigen presentation.2,3 EpsteinBarr virus (EBV) is another virus that establishes a persistent infection this time in human lymphocytes. In the latent state, EBV expresses only a small number of proteins. One of these proteins (EBNA-1) does not seem to induce a cytotoxic response, although other proteins expressed during the latent phase, as well as proteins expressed during the replicative stage, are recognised by CTL. There are other peculiarities of the CTL response to EBV-infected cells that hint that other immune evasion mechanisms might occur, but this is still not well understood. Evasion of antibody and complement mediated killing Antibodies and complement affect viruses at two points in their replication cycles: during their extracellular phase, antibodies can bind and neutralise the virus directly, and during the viral intracellular phase, antibody and complement can interact with exposed (membrane-associated) viral proteins, leading to ADCC or complement-mediated cytolysis. Antibody binding during the extracellular phase seems to be relatively difficult
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to evade and few viruses are resistant to this antibody-mediated neutralisation. Many viruses, however, encode proteins that provide protection against attack during the intracellular phase. For example, HSV expresses both a complement receptor and a receptor for the Fc region of immunoglobulin.1 Poxviruses are also relatively resistant to complement, but through different mechanisms. A secreted viral protein, now known as vaccinia virus complement-control protein (VCP), is homologous to the endogenous complement-control protein and, like the endogenous protein, inhibits the complement cascade.4 A second viral protein, similar to VCP, is membrane anchored and may prevent generation of the complement cascade at the cell surface, protecting the virus during the intracellular phase.
Inhibition of immune recognition Cytotoxic T lymphocytes recognise virus-infected cells through the MHC class I antigen-presentation system. CTL can recognise MHC class I complexes containing foreign peptides at the cell surface, and respond by lysing the affected cell. This pathway is important in controlling viral infections, and it is not surprising that many viruses have developed systems to deal with it. There are several mechanisms that have arisen; no two viruses seem to target the same component of the pathway. Human adenoviruses can downregulate MHC class I surface expression, through at least two general methods. Most subgroups of adenovirus express a small glycoprotein that binds to many haplotypes of human MHC class I, causing it to remain in the endoplasmic reticulum. By preventing cell surface expression, CTLs are prevented from recognising and eliminating adenovirus-infected cells.5 A second mechanism is used by some other adenoviruses. These viruses block MHC class I expression at the transcriptional level apparently by preventing the normal processing of the ubiquitous transcription factor nuclear factor kappa B (NF B).6 Cells infected with other adenovirus serotypes also show transcriptional downregulation of the peptide transporter genes which are essential for antigen processing leading to a defect in MHC class I cell surface expression in infected cells. Transcriptional downregulation of MHC class I genes is also seen in many other types of viral infections. Human and cytomegaloviruses also alter MHC I surface expression, but through different mechanisms to adenoviruses. In cells infected with CMV, part of the MHC class I molecule becomes unstable and is rapidly degraded. At least two of the poxviruses downregulate cell-surface MHC class I through yet another unknown mechanism. In cells infected with these viruses, the cell-surface MHC class I is turned over much more rapidly than normal and is not replaced. A less potent inhibition of MHC class I antigen presentation is also seen in cells infected with vaccinia virus, which occurs at least partly through an inhibition of cellular protein degradation.7
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Targeting of host cytokines Poxviruses are able to effectively block cytokines. Myxoma virus, for example, expresses truncated receptors for TNF and IFN .8,9 These proteins lack the transmembrane protein of the normal receptor, and are secreted extracellularly, but since they still possess the cytokine-binding domain, they block the action of the cytokine. Several other cytokines are targeted by poxviruses.10 Adenoviruses also can block some of the effects of IFN , through a very different mechanism. IFN mediates some of its effects by activating a specific protein kinase, which initiates a series of events resulting in an inhibition of protein synthesis in the affected cell; this affects viral as well as cellular proteins (see above). Adenoviruses express a small RNA (VA1 RNA) which binds to this kinase and prevents the initiation of the protein synthesis inhibition pathway.11 Adenoviruses are also resistant to some, though not all, of the effects of TNF. At least four viral proteins are involved in this, with considerable redundancy: there are three sets of proteins involved, not all of which are effective in all cell types. These proteins are very different both structurally and in their subcellular location: one is associated with the nuclear membrane; one is a non-membrane-associated nuclear and cytoplasmic protein; and two form a complex associated with the plasma membrane. However, all block signalling from the TNF receptor.12 As well as blocking cytokine activity, some viruses have developed cytokine homologues to cause long-term skewing of the immune response. EBV (see below) expresses a homologue of the cytokine IL-10 termed BCRF1. IL-10 is a marked anti-inflammatory cytokine with multiple effects. It can inhibit the expression of IFN and other cytokines in some cell types, inhibit T cell differentiation, and bias the immune response to a Th2-type response, which favours viral replication. The EBV version of IL-10 has similar, but not identical, effects. The full effects of BCRF1 are not clear, but in general it seems that it helps subdue the antiviral response, preventing elimination of EBV. The role of this virus in various diseases deserves particular mention.
EpsteinBarr virus
After producing an initial infection, many viruses go on to establish dormant or latent infections, which may or may not be associated with serious disease. One of the most common persistent infections is that caused by EpsteinBarr virus, a herpes virus that infects over 90% of the worlds adults. The primary infection is mild and usually occurs in childhood. Lifelong carrier status then ensues and periodic release of infectious virus into saliva is how infection is spread. EBV is associated with
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two benign conditions, glandular fever and oral hairy leukoplakia in acquired human deficiency syndrome (AIDS). However, the virus has also been linked to the development of a variety of human malignancies, including Burkitts lymphoma, Hodgkins disease, nasopharyngeal carcinoma, some T cell lymphomas, post-transplant lymphoproliferative disease, and, more recently, certain cancers of the stomach and smooth muscle. The viral latent genes play an important role in the transformation process. EBV was originally discovered in the tumour cells of Burkitts lymphoma, an undifferentiated B cell tumour, and is also implicated in the development of immunoblastic B cell lymphomas in immunocompromised patients. In vitro EBV (Figure 3.3) is able to transform normal B cells into immortal lymphoblastoid cells. The lymphoblastoid cells express latent proteins: six nuclear proteins EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C, and EBNA leader protein (EBNA-LP) and three membrane proteins LMP1, LMP2A, and LMP2B. The latent viral proteins appear to be key effectors of the immortalisation process. The expression of the latent proteins varies between different types of tumours. Immunoblastic lymphomas which can develop in immunosuppressed patients (for example, after transplantation) resemble the phenotype of the lymphoblastoid cells.
Epithelial cell
EBV
B lymphocyte
LMPs EBNAs
Virus latency Virus replication

Figure 3.3 The interaction between EpsteinBarr virus, B lymphocytes, and epithelial cells in virus latency and replication (EBNA, nuclear latency protein; LMP membrane-associated latency protein). , 44
The B cell transformation induced by EBV can be suppressed by cytotoxic T cells, and infusion of CTLs in transplant recipients can be used to prevent and treat EBV-induced lymphoma. Epithelial tumours have been associated with EBV and Figure 3.3 illustrates how EBV may persist in basal epithelial cells and be reactivated as the cells replicate. However, current evidence suggests that primary EBV infection and virus persistence are mediated through B cells.13 Although a role for epithelial cells in EBV persistence and replication cannot be excluded, it is assumed that EBV infection of epithelial cells is probably an accidental event resulting from local reactivation of latently infected B cells. The contribution EBV to the process of tumour development requires further understanding of EBV latent gene function and interaction between host and virus. The new evidence firmly suggests that B cells are implicated in EBV persistence and highlights the complex strategies that viruses have evolved to integrate with their hosts. The evidence also suggests that therapeutic approaches aimed at preventing EBV latency in B cells is likely to be effective in preventing and treating virus-associated tumours.
Summary
Viruses have evolved complex strategies to evade and even manipulate host immune defences to their advantage, permitting viral replication without massive inflammatory responses. After producing the initial symptoms of acute viral infection, many viruses go on to establish latent infections. Some latent infections are harmonious and there are no ill effects for the host. However, some persistent infections are associated with serious disease (for example, AIDS and hepatitis) and malignancy. An understanding of these infections is crucial for the development of antiviral strategies for the treatment and prevention of virus-associated disease.
References
1 Dubin G, Fishman NO, Eisenberg RJ, Cohen GH, Friedman HM. The role of herpes simplex virus glycoproteins in immune evasion. Curr Top Microbiol Immunol 1992;179:11120. 2 Hill A, Jugovic P, York I et al. Herpes simplex virus turns off the TAP to evade host immunity. Nature 1995;375:41115. 3 Frh K, Ahn K, Djaballah H et al. A viral inhibitor of peptide transporters for antigen presentation. Nature 1995;375:41518. 4 Kotwal GJ, Isaacs SN, McKenzie R, Frank MM, Moss B. Inhibition of the complement cascade by the major secretory protein of vaccinia virus. Science 1990;250:82730. 5 Cox J, Bennink JR,Yewdell JW. Retention of adenovirus E19 glycoprotein in the endoplasmic reticulum is essential to its ability to block antigen presentation. J Exp Med 1991;174:16297.
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6 Schouten GJ, Van der Eb JR, Zantema A. Downregulation of MHC class I expression due to interference with p105-NFkB1 processing by Ad12E1A. EMBO J 1995;14:1498507. 7 Townsend A, Bastin J, Gould K et al. Defective presentation to class I-restricted cytotoxic T lymphocytes in vaccinia-infected cells is overcome by enhanced degradation of antigen. J Exp Med 1988;168:121124. 8 Upton C, Macen JL, Schreiber M, McFadden G. Myxoma virus expresses a secreted protein with homology to the tumor necrosis factor receptor gene family that contributes to viral virulence. Virology 1991;184:37082. 9 Upton C, Mossman K, McFadden G. Encoding of a homolog of the IFN receptor by myxoma virus. Science 1992;258:136972. 10 Spriggs MK. Cytokine and cytokine receptor genes captured by viruses. Curr Opin Immunol 1994;6:5269. 11 Katze MG, DeCorato D, Safer B, Galabru J, Hovanessian AG. Adenovirus VAI RNA complexes with the 68 000 Mr protein kinase to regulate its autophosphorylation and activity. EMBO J 1987;6:68997. 12 Wold WSM, Tollefson AE, Hermiston TW. Strategies of immune modulation by adenoviruses. In McFadden G, ed. Viroceptors, Virokines and Related Immune Modulators Encoded by DNA Viruses. Austin, TX: R.G. Landes Co., 1995, 14786. 13 Young LS. EpsteinBarr virus infection and persistence: a B cell marriage in sickness and in health. Lancet 1999;354:11412.
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4: The double-edged role of the neutrophil in inflammatory responses

PAUL G HELLEWELL
Introduction
Accumulation of leukocytes in tissues is essential for effective host defence. The major role of neutrophils is to phagocytose and destroy infectious agents but they can also cause host damage and neutrophil-mediated injury has been implicated in several inflammatory conditions seen on the Intensive Care Unit (ICU). This article provides an overview of the vital role of neutrophils in host defence and the consequences of host damage. An increased understanding of neutrophil biology is likely to result in intelligent intervention strategies.
Neutrophils
Neutrophils are leukocytes with lobulated nuclei, containing primary and secondary cytoplasmic granules, and comprising 5060% of the total circulating leukocytes. They provide the first line of defence against invasion by bacteria that have penetrated physical innate immune barriers. Once an inflammatory response is initiated, neutrophils migrate rapidly to sites of infection or injury under the influence of chemotactic agents and adhesion molecules.Their targets include bacteria, fungi, protozoa, viruses, virally infected cells, and tumour cells. The development of a neutrophil in the bone marrow involves several maturational stages: myeloblast, promyeloblast, myelocyte, metamyelocyte, non-segmented (band) neutrophil, and finally the functional segmented neutrophil. In a normal healthy adult around 1011 neutrophils per day are produced and this increases during acute inflammation. When mature neutrophils are released from the bone marrow into the circulation they are able to survive for only around 6 hours before either being removed from the circulation or accumulating in tissues in inflammation, where they can survive for several days. Blood neutrophils are in equilibrium between the
47
circulating and intravascular tissue pools (for example, in the pulmonary circulation). Senescent neutrophils undergo apoptosis (programmed cell death see Chapter 2) prior to removal through phagocytosis by macrophages. The half-life of neutrophils decreases markedly during acute inflammation, since the tissue requirement for newly recruited neutrophils increases considerably.
Neutrophils in host defence

Phagocytosis Neutrophils must move from the circulation to the site where phagocytosable material occurs.This is regulated by the leukocyte adhesion cascade and involves leukocyte, endothelial cell adhesion molecules, and chemotactic factors generated by the infectious agents or by tissue cells (for example, macrophages) as a result of their initial contact with components of the immune system. Phagocytosis is a complex process involving several morphological and biochemical steps. After recognition and particle binding to the phagocyte
Receptors CD11b, FMLP-R Oxidants O2 ,H2O2, OH ONOO, HOCI
Antimicrobial peptides myeloperoxidase, lactoferrin, Iysozyme, defensins, bacterial permeability-inducing protein
Proteases elastase, cathepsins, matrix metalloproteinases
Figure 4.1 The different array of products from neutrophils that contribute to cytotoxicity and host defence. Abbreviations: O , superoxide anion; H2O2, hydrogen peroxide; OH , hydroxyl radical; 2 ONOO , peroxynitrite; HOCl, hypochlorous acid; FMLP-R, formyl methyl leucine phenylalanine receptor. 48
THE DOUBLE-EDGED ROLE OF THE NEUTROPHIL
surface, ingestion or engulfment, phagosome origination, phagolysosome formation (fusion of the phagosome with lysosomes), killing and degradation of ingested cells or other material proceeds. A marked increase in oxygen consumption called the respiratory burst leads to the release of superoxide and other oxygen radicals, and also the secretion of a variety of enzymes and biologically active substances controlling inflammatory and cytotoxic reactions (Figure 4.1). This process is usually well controlled and avoids release of the toxic components into the extracellular milieu. Tissue damage can occur, however, when neutrophil microbicidal products are released extracellularly to such an extent that host defences such as antioxidants and protease inhibitors in the immediate vicinity are overwhelmed (Figure 4.2).
Antioxidants and radical scavengers
Reactive oxygen species
Proteases
Protease inhibitors
Adhesion molecule Figure 4.2 The interaction of the neutrophil with vascular endothelium results in the creation of a microenvironment, which can result in local microvascular injury.
The importance of neutrophils in fighting bacterial and fungal infections is well recognised. Recently, it has been shown that neutrophils also bind to viruses and virally infected cells via antibody and complement receptors. Viruses such as influenza can be inactivated by neutrophils through damage to viral proteins (for example, haemagglutinin and neuraminidase) mediated by the myeloperoxidase released during degranulation.
Regulation of neutrophil function

Adhesion molecule expression To fulfil the role of phagocytosis, neutrophils must interact with and penetrate the vascular cell wall and migrate in the tissue. Adhesion molecules
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play a crucial role in orchestrating these processes. Adhesion molecules are cell surface proteins involved in the binding of cells, usually leukocytes to each other, to endothelial cells, or to the extracellular matrix. These molecules become expressed and activated in response to inflammatory signals. The interactions and responses which are initiated by binding of adhesion molecules to their receptors or ligands have vital roles in host defence. A number of families of adhesion molecules mediate the interaction between circulating leukocytes and vascular endothelial cells, including the integrins, the selectins, and the immunoglobulin (Ig) superfamily. These adhesion molecules and their ligands are shown in Box 4.1.
Box 4.1 Adhesion molecules involved in leukocyte emigration from vessels during inflammation Rolling sialyl-Lewisx L-selectin P-selectin E-selectin Stopping 2 integrins VLA-4 ( 4 1) ICAM-1 VCAM-1 4 7 integrin MadCAM-1 Aggregation and shape change CR3 (CD11b/CD18) LFA-1 (CD11a/CD18) P-selectin Migration through vessel wall 2 integrins ICAM-1 VCAM-1 PECAM-1 See text for abbreviations
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The movement of circulating leukocytes from the circulation into inflamed tissues (leukocyte adhesion cascade) comprises three stages: 1 rolling of leukocytes along the vascular cell wall (mediated through transient interactions between selectin proteins and their carbohydratebearing ligands), followed by 2 exposure of the rolling leukocyte to endothelial expressed chemoattractants, leading to activation of both neutrophils and high affinity binding between integrins and glycoproteins of the Ig superfamily to mediate firm adhesion, leading to 3 extravasation (crawling along the endothelium, diapedesis, and migration into tissue) in response to a tissue-generated chemoattractant gradient (Figure 4.3).
CD11b
ATTACHMENT (Rolling) ADHESION
CD62L CD15b GlyCAM
CD11a
CD54 CD62P CD31 CD62E
IL-8
DIAPEDESIS
Fibronectin Collagen
Laminin Extracellular matrix
Figure 4.3 The stages involved in neutrophil extravasation in postcapillary venules. (See text for explanation.)
Rolling leukocytes can be defined as those which move through vessels at a rate that is lower than that of red blood cells. Rolling leukocytes may not be committed to either firmly adhering to the vessel wall or rolling along the entire vessel length they frequently detach and return to the flowing blood. Leukocyte rolling has also been shown to occur under normal physiological conditions in tissues that are continually exposed to external inflammatory stimuli for example, in skin and the gastrointestinal tract. In inflamed tissue, leukocyte rolling frequently but not always leads to the point where the leukocyte stops and becomes firmly attached to the endothelial cell surface. This strong, high-affinity, adhesive interaction is referred to as firm adhesion, denoting the absence of movement of the leukocyte.
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Selectins The selectin family has three members named according to the cells in which they were originally discovered.
L-selectin is constitutively expressed on leukocytes and its target cells

are activated endothelial cells.
E-selectin is produced exclusively by endothelial cells after cytokine

activation and its counter-receptors are on neutrophils, monocytes, eosinophils, lymphocyte subsets, and some tumour cells. P-selectin is preformed and stored for rapid release in the granules of platelets. In the WeibelPalade bodies of endothelial cells, P-selectin is expressed constitutively and after cytokine activation. Its target cells are the same as those for E-selectin.
The selectin family members, L-selectin, P-selectin, and E-selectin, are involved in the initial capture and mediate leukocyte rolling along the surface of activated endothelium. P-selectin and L-selectin can mediate leukocyte capture, P-selectin mediates fast leukocyte rolling, while interaction with E-selectin results in slow rolling. L-selectin is shed by proteolytic cleavage from the surfaces of lymphocytes and neutrophils in vitro following activation by a variety of agents, and in vivo from neutrophils during inflammation. It has been suggested that loss of surface L-selectin might be necessary to allow leukocytes to migrate through the endothelium. Soluble L-selectin retains bioactivity and at high concentrations can inhibit binding of cells to endothelium, suggesting a possible role for soluble L-selectin in modulating this binding in vivo. High levels of soluble L-selectin have been found in plasma from apparently normal individuals and several studies have reported that levels of circulating L-selectin are increased in the critically ill.12 Three ligands for L-selectin on endothelial cells have been identified.
The first ligand, GlyCAM-1, is expressed almost exclusively in

peripheral and mesenteric lymph node high endothelial venules.
The second L-selectin ligand, originally called sgp90, has now been
shown to be a cluster of differentiation (CD) antigen CD34.
The third ligand for L-selectin is MadCAM 1, a mucin-like

glycoprotein found on mucosal lymph node in high endothelial venules. P-selectin is mobilised to the endothelial cell surface within minutes in response to a variety of inflammatory or thrombogenic agents and is rapidly recycled to intracellular compartments. Transcription of P-selectin mRNA can be activated in endothelial cells by inflammatory mediators. Although P-selectin is primarily involved in the adhesion of myeloid, B cells, and a subset of T cells to activated endothelium, it is also involved in the adhesion of platelets to monocytes and neutrophils, playing a central role in
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neutrophil accumulation within thrombi. P-selectin is found in the plasma of normal individuals and, like L-selectin, a number of studies have reported that levels of soluble P-selectin in biological fluids may be elevated in a variety of pathological conditions.3,4 The carbohydrate ligand sialyl Lewisx (sLex) has been identified as a ligand for both P- and E-selectin. P-selectin also reportedly binds selectively to a glycoprotein present on myeloid cells, blood neutrophils, monocytes, and lymphocytes, termed P-selectin glycoprotein ligand-1 (PSGL-1), a ligand that also can bind E-selectin.5 O-linked glycans at the N-terminus of PSGL-1 (the region that binds to P-selectin) are capped with sLex. P-selectin-mediated rolling of leukocytes is absent in mice deficient in PSGL-1. Integrins The integrins are a large family of glycoproteins which can be subdivided according to the particular subunit they possess. The family comprises a heterodimer of an and integrin chain. The 2 integrins are expressed particularly by leukocytes, giving rise to their alternative name, the leukocyte integrins, whereas the others, in general, are more widely distributed. 2 integrins are represented by three molecules each containing two different subunits: lymphocyte function-associated antigen-1 (LFA-1), complement receptor type 3 (CR3 or Mac-1), and complement receptor type 4 (CR4). Each of them contains the same 2 integrin subunit, called CD18, plus another integrin subunit: CD11a ( L), CD11b ( M), or CD11c ( X). LFA-1 comprises CD11a plus CD18 and is expressed by lymphocytes (including T cells), myeloid cells (monocytes, macrophages, and granulocytes), and a variety of other cell types. CR3 comprises CD11b plus CD18, and CR4 is CD11c and CD18. Both CR3 and CR4 are expressed by myeloid cells. This integrin family of adhesion molecules expressed on endothelial cells acts as receptors for intercellular and vascular cell adhesion molecules (ICAMs and VCAMs) see below in the section on the Ig superfamily. Different sets of integrins are expressed by different populations of leukocytes to provide specificity for binding to different types of adhesion molecules expressed along the vascular endothelium. ICAM-1 and ICAM-2 are the two main ligands for LFA-1. CR3 can bind fragments of complement components and it mediates phagocytosis of complement-coated particles by professional phagocytes. Ig superfamily Along with ICAM-1 and ICAM-2, plateletendothelial cell adhesion molecule (PECAM-1) is a member of the large Ig superfamily (IgSF). Both ICAM-1 and ICAM-2 are expressed by endothelial cells and PECAM-1 has been identified on neutrophils, monocytes, and platelets and is present
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in large amounts on endothelial cells where it is concentrated at cellcell junctions. The Ig superfamily of adhesion molecules binds to integrins on leukocytes and mediates their firm adhesion to the blood vessel wall and extravasation into the surrounding tissue. Chemokines, such as monocyte chemoattractant protein 1 (MCP-1) and interleukin-8 (IL-8), cause a conformational change in integrins to permit ligand binding. Chemokines Chemokines have selective chemoattractant activities for different types of leukocytes and play an important role in the process of transmigration.The involvement of chemokine in leukocyte trafficking begins with presentation of a chemokine on the endothelial cell surface, as a signal to trap a specific type of leukocyte as the cell is undergoing selectin-mediated rolling along the endothelium.The leukocyte then becomes selectively activated by the chemokine so that the cell stops rolling and becomes firmly adhered. The leukocyte then moves along the chemotactic gradient formed by the chemokines on the endothelium, undergoes diapedesis, and migrates into the tissue space, while still responding to a chemotactic gradient. Cytokines Cytokines produced by neutrophils themselves and/or lymphocytes, monocytes/macrophages, or endothelial cells, regulate many aspects of neutrophil function. This regulation can occur at the transcriptional levels, through effects on mRNA stability or on neutrophil priming. Several of the adhesion molecules are regulated through nuclear factor kappa B (NF B). In the initial phase of an acute inflammatory response, circulating neutrophils are activated by exposure to activating inflammatory mediators including complement fragments (C5a) and to priming agents including interleukin-1 (IL-1), tumour necrosis factor (TNF ), and lipopolysaccharide (LPS). Endothelial cells can be activated by TNF , IL-1, and LPS leading to expression of several adhesion molecules. Platelet activating factor (PAF) produced by endothelial cells may also act on nearby neutrophils to increase their adhesion to the endothelium.6 Neutrophils also synthesise and secrete cytokines themselves in either an autocrine or paracrine manner. These include IL-1, IL-6, IL-8, TNF , and granulocyte/macrophage colony stimulating factor (GM-CSF). The pyrogenic cytokines, IL-1, TNF , and IL-6 all prime various pathways that contribute to the activation of NADPH oxidase. IL-8 is also a potent chemoattractant; it synergises with interferon (IFN ), TNF , GM-CSF, and granulocyte colony stimulating factor (G-CSF) to amplify neutrophil cytotoxic functions. Cytokines also increase the microbiostatic and killing capacities of neutrophils against bacteria,
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protozoa, and fungi. IFN and GM-CSF independently amplify neutrophil antibody-dependent cytotoxicity. The anti-inflammatory cytokines, IL-4, IL-13, and IL-10 inhibit the production of TNF , IL-1, and IL-8 which dampens neutrophil activation. Some cytokines prolong neutrophil survival. The acute inflammatory response can be terminated by the secretion of macrophage inflammatory protein-1 (MIP-1) from neutrophils, which attracts macrophage recruitment to clear accumulated neutrophils. Along with cytokines, other mediators, including bioactive lipids, neuroendocrine hormones, histamine, and adenosine, are also involved in the regulation of neutrophil activation. Bioactive lipids Bioactive lipids originate mainly from arachidonic acid which is metabolised to prostaglandins, leukotrienes, and lipoxins. Leukotriene B4 (LTB4) is a strong neutrophil chemoattractant and vasoactive leukotrienes (LTC, LTD, and LTE) increase microvascular permeability and may contribute to ischaemiareperfusion injury. In contrast to leukotrienes, prostaglandins suppress most neutrophil functions, possibly through their ability to elevate intracellular cAMP. Lipoxins LXA and LXB are potent inhibitors of neutrophil microbicidal activity. In many inflammatory conditions, the levels of PAF rise in the affected tissues, and injury can be attenuated by PAF antagonists. PAF directly primes neutrophils for superoxide generation and elastase release. Hormones The major stress hormones are involved in the regulation of inflammation at both the systemic and local levels. There are bidirectional interactions between cytokines and neurotransmitters, which provide a means of indirect chemical communication between the neuroendocrine and immune systems. Growth hormone, prolactin, -endorphin, glucocorticoids, and catecholamines are all involved in neutrophil regulation. Growth hormone primes the oxidative burst of human neutrophils through a process initiated by growth hormone interaction with the prolactin receptor on neutrophils. The growth-promoting effects of growth hormone are mediated through insulin-like growth factor 1, which is also a strong neutrophil-priming agent. Prolactin, which shares considerable functional and structural similarities with growth hormone, primes the oxidative burst of neutrophils. Glucocorticoids and opioids are generally considered to be immunosuppressive. Glucocorticoids impair the phagocytic and cytotoxic activities of neutrophils and reduce the capacity to produce superoxide and nitric oxide, and secrete lysosomal enzymes in response to activation. The oxidative burst of phagocytes is also inhibited by epinephrine and
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-endorphin, mediated via non-opioid receptors. Glucocorticoids can downregulate cytokine and adhesion molecule expression through effects on nuclear factor B (NF B). Corticosteroids are able to diffuse through the cell membrane and bind to their cytosolic glucocorticoid receptor (GR). This complex subsequently undergoes nuclear translocation and is then able to modulate transcriptional activation through association with promoter elements and glucocorticoid response elements (GRE). Depending upon the target gene, activated GRs may stimulate or inhibit gene transcription. Other evidence points to activated GRs antagonising transcription factor activity through proteinprotein interactions at a number of sites. Competition between GR and transcription factors for nuclear co-activators (which promote transcription) has also been suggested. Other mediators Adenosine is a potential anti-inflammatory agent released from damaged host cells. The neutrophil respiratory burst is inhibited by adenosine occupancy of receptors, although only if it is added before the triggering agent. It has no effect on the initiation or progress of degranulation. The interactions between platelets and neutrophils are essential for both cell types. Activated platelets can bind to neutrophils and stimulate the oxidative burst while they themselves synthesise vasoconstrictor leukotrienes. Like prostaglandins, many immunosuppressive mediators use cAMP as a second messenger. Increased intracellular cAMP in neutrophils is associated with decreases in a number of microbicidal functions. Phagocyte priming and activation may, in fact, be controlled by shifts in the intracellular ratio of cGMP to cAMP, since cGMP is stimulatory.
Disorders of adhesion
The importance of leukocyte adhesion molecules is evidenced by the inherited disorder called leukocyte adhesion deficiency (LAD). Leukocytes of patients with LAD-1 syndrome lack 2 integrin expression. It occurs in two forms, with severe or moderate clinical manifestations. In the severe form, 2 integrin is completely absent from leukocytes and presents with severe, life-threatening infections with high mortality, such that patients rarely survive beyond 2 years of age. The moderate deficiency is accompanied by partial integrin expression; patients express 2.56.0% of normal LFA-1, CR3, and CR4 levels, and have only recurrent skin infections. Leukocytes from patients with LAD-2 syndrome do not express sLex antigen owing to disordered synthesis of fucose, an important component of sLe, and cannot bind E-selectin and P-selectin, such that leukocyte rolling is defective. LAD-2 patients suffer from recurrent bacterial infections but can survive into childhood. Mental retardation is present because of the deranged fucose metabolism.
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Neutrophils and host tissue damage

Although neutrophils are essential to host defence (Box 4.2), they have also been implicated in the pathology of many acute and chronic inflammatory conditions and ischaemiareperfusion injury. Hydrolytic enzymes of neutrophil origin and oxidatively inactivated protease inhibitors can be detected in fluid isolated from inflammatory sites. Under normal conditions, neutrophils can migrate to sites of infection without damage to host tissues. However, damage may occur through several independent mechanisms. These include premature activation during the migration process, extracellular release of toxic products during bacterial killing, removal of infected or damaged host cells and debris as a first step in tissue remodelling, or failure to terminate acute inflammatory responses.
Box 4.2 The double-edged role of neutrophils Benefits First line in host defence Regulates plasma leakage Phagocytosis Initiates repair Implied contribution to disease Myocardium Gut Skin/soft tissue CNS Septic shock Trauma Meningitis Burns Acute rejection Abbreviations: CNS, central nervous system.
Ischaemia and reperfusion Ischaemiareperfusion injury is associated with an influx of neutrophils into the affected tissue and subsequent activation. This may be triggered
57
by substances released from damaged host cells or as a consequence of superoxide generation through xanthine oxidase. Early leukocyte interaction with endothelium may contribute to injury to blood vessels and surrounding brain tissue following acute ischaemic stroke. P-selectin is rapidly expressed on ischaemic endothelium in the brain vasculature. Ruehl et al. undertook a study using a selectin inhibitor, fucoidin, to block leukocyte adhesion in an attempt to limit the inflammatory component of reperfusion injury in the brain.7 The study determined the effects of fucoidin on cerebral infarction size and neurological function after 4 hours of middle cerebral artery occlusion and reperfusion, and 24 hours of reperfusion. It was found that selectin blockade reduced cerebral infarction size by 50% and improved neurological function. This study confirms the involvement of neutrophil adhesion in ischaemic stroke, and suggests that inhibition of selected adhesion molecules may be beneficial. Outcome from ischaemic renal injury in rats has also been shown to be improved by selectin inhibition.8
Neutrophils and inflammatory disease

Under normal conditions, blood may contain a mixture of normal, primed, activated, and spent neutrophils. In the inflammatory site, mainly activated and spent neutrophils are present. Endothelial activation and damage occur early during sepsis and play a major role in the pathophysiology of systemic inflammation. Various markers of endothelial activation are increased during sepsis and systemic inflammation, and in most studies, the level of markers such as ICAM, VCAM, and E-selectin correlate well with the severity of inflammation and the course of the disease. In patients with sepsis and organ failure, a pronounced and persistent increase in plasma VCAM-1 and ICAM-1, and E- and Lselectins has been demonstrated.9,10 However, decreased responsiveness of 2 integrin expression to TNF activation in critically ill patients has also been described.11 A good example of the neutrophil paradox is seen in acute respiratory distress syndrome (ARDS) neutrophils have been implicated in the pathology of this condition because of the large influx of these cells into the lung and the associated tissue damage caused by oxidants and hydrolytic enzymes released from the activated neutrophils. The impairment of neutrophil microbicidal activity that occurs as the ARDS worsens may be a protective response on the part of the host, which is induced locally by inflammatory products. This downregulation of neutrophil function may explain why many of these patients eventually die from overwhelming pulmonary infections. The acute phase of thermal injury is also associated with neutrophil activation, and this is followed by a general impairment in various
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neutrophil functions. In patients suffering from severe burns, a strong correlation has been established between the onset of bacteraemic infection and reduction in the proportion and absolute numbers of neutrophils positive for antibody and complement receptors. Activation of neutrophils by immune complexes in synovial fluid contributes to the pathology of rheumatoid arthritis. Chronic activation of neutrophils may also initiate tumour development because some reactive oxygen species generated by neutrophils damage DNA and proteases promote tumour cell migration. Oxidants of neutrophil origin have also been shown to oxidise lowdensity lipoproteins (LDL), which are then more effectively bound to the plasma membrane of macrophages through specific scavenger receptors. Uptake of these oxidised LDL by macrophages is thought to initiate atherosclerosis. In addition, primed neutrophils have been found in people with essential hypertension, Hodgkins disease, inflammatory bowel disease, psoriasis, sarcoidosis, and sepsis. Hydrolytic damage to host tissue and therefore chronic inflammatory conditions may occur only when antioxidant and antiprotease screens are overwhelmed. Antiprotease deficiency is thought to be involved in the pathology of emphysema. Many antiproteases are members of the serine protease inhibitor (SERPIN) family. Although the circulation is rich in antiproteases, these large proteins may be selectively excluded at sites of inflammation because neutrophils adhere tightly to their targets. Oxidative stress may initiate tissue damage by reducing the concentration of extracellular antiproteases to below the level required to inhibit released proteases. Chlorinated oxidants can inactivate antiproteases such as protease inhibitor and macroglobulin (which are endogenous inhibitors of elastase) but, surprisingly, simultaneously activate latent metalloproteases such as collagenases and gelatinase, which contribute to the further inactivation of antiproteases.
Antineutrophil cytoplasmic antibodies

Cytoplasmic constituents of neutrophils can give rise to the formation of specific antineutrophil cytoplasmic antibodies (ANCA), which have been linked to the development of systemic vasculitis and glomerulonephritis. ANCA are antibodies directed against enzymes found mainly within the primary (azurophil) granules of neutrophils. There are three types of ANCA, which can be distinguished by the patterns they produce by indirect immunofluorescence when tested on normal ethanol-fixed neutrophils.
Diffuse fine granular cytoplasmic fluorescence (cANCA) is typically

found in Wegeners granulomatosis, in some cases of microscopic
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polyarteritis and ChurgStrauss syndrome, and in some cases of crescentic and segmental necrotising glomerulonephritis, but is rare in other conditions. The target antigen is usually proteinase 3. Perinuclear fluorescence (pANCA) is found in many cases of microscopic polyarteritis and glomerulonephritis. These antibodies are often directed against myeloperoxidase but other targets include elastase, cathepsin G, lactoferrin, and lysozyme. The third group designated atypical ANCA includes neutrophil nuclear fluorescence and some unusual cytoplasmic patterns and, while a few of the target antigens are shared with pANCA, the others have not been identified yet.
Anti-adhesion molecule therapy in inflammation

There are a number of approaches possible for the modulation of adhesion molecule expression and function (Box 4.3). There are several antibodies and small-molecule antagonists against adhesion molecules available. Using antisense nucleotides is highly specific and an antisense oligonucleotide to ICAM-1 has been shown to reduce inflammation in vivo in patients with chronic inflammatory conditions such as Crohns disease.12 Blocking adhesion molecules directly with monoclonal antibodies has been extensively tested both in vitro and in vivo. In a study using a baboon Escherichia coli model of sepsis, it was found that a
Box 4.3 Therapeutic strategies to target adhesion molecules Modulate expression Cytokine and cytokine receptor antagonists Transcriptional inhibitors Antisense oligonucleotides Fucosyl transfersase inhibitors Metalloproteinase inhibitors (to reduce L-selectin shedding) Block function Monoclonal antibodies Soluble adhesion molecules/immunoadhesins Oligosaccharides Glycomimetics Small molecule inhibitors Integrin inhibitors
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monoclonal antibody to E- and L-selectin did not improve gas exchange or protect against lung injury, and was associated with decreased survival time.13 In another study, administration of either the selectin antibody or an ICAM-1 antibody resulted in increased and prolonged levels of IL-6, IL-8, and TNF receptor-1 (TNFR-1).14 Anti-ICAM-1 was found to reduce eosinophil migration and airway hyperresponsiveness in a primate model of asthma15 but there have been no studies of asthma in humans. Soluble adhesion moleculeIgG constructs (immunoadhesins) are effective as inhibitors of leukocyte trafficking in animal models of lung inflammation but have not been tested clinically. Although blocking antibodies directed against integrins have been used successfully in animal models of inflammatory disease, there are problems associated with repeated antibody administration in terms of their use in chronic conditions. Integrin inhibitors such as neutrophil inhibitory factor (NIF) may be more useful. NIF binds to the cation binding site in the A domain of CD11b to inhibit neutrophil adhesion to endothelial cells in vitro, and overexpression in endothelial cells in vivo prevents LPS-induced lung injury in the mouse.16 Other small molecule integrin inhibitors prevent 4 integrin binding to fibronectin. Inhalation of such inhibitors has anti-inflammatory activity and this effect appears to be independent of an action on circulating leukocytes. A likely target is therefore 4 integrins on intrapulmonary mast cells or macrophages. Metalloproteinase inhibitors may also be useful by preventing leukocyte L-selectin shedding, thus impairing endothelial transmigration. Oligosaccharides such as sLex and sLea are weak inhibitors of E-selectindependent leukocyte rolling in vivo (requiring over 30 mg/kg to inhibit established rolling). They have no effect on P- or L-selectin-dependent rolling. They have, however, been reported to attenuate leukocyte accumulation at inflammatory sites when given as pretreatment at low mg/kg doses. This suggests that pretreatment may confer some additional activity of the oligosaccharide. sLex has been tested in clinical trials and was noted for its lack of anti-inflammatory activity. The major ligand for P-selectin is P-selectin glycoprotein ligand-1 (PSGL-1) and this ligand is expressed on the surface of monocyte, lymphocyte, and neutrophils. A truncated form of recombinant human PSGL-1 has been covalently linked to IgG (rPSGL-Ig) to make a fusion peptide that competitively inhibits PSGL-1. This fusion peptide has been developed as an inhibitor of neutrophilendothelial cell adherence for the treatment of ischaemia reperfusion injury. To determine the potential for deleterious effects from inhibition in P-selectin-mediated neutrophil attachment in the presence of bacterial infection, the effects of therapeutic doses of rPSGL-Ig were tested in three standard laboratory sepsis models by Opal and co-workers.17 The experimental models were the murine systemic Listeria monocytogenes infection model, the Pseudomonas aeruginosa
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bacteraemia model in neutropenic rats, and the caecal ligation and puncture-induced peritonitis model in rats. Recombinant human PSGL-Ig had no adverse effects on mortality or immune clearance in systemic bacterial infection in any of the three infection models. The PSGL-1 inhibitor did significantly decrease local neutrophil infiltration and bacterial clearance but did not increase the systemic levels of proinflammatory cytokines. The results indicate that rPSGL-Ig did not exacerbate infection in these experimental sepsis models and may therefore be useful in sepsis. In view of the critical role of fucosyltransferases in conferring function on selectin ligands such as PSGL-1, specific inhibitors of these enzymes are also potential therapeutic agents. Strategies to reduce transcriptional factor activation (for example, NF B) may also be used to abrogate adhesion molecule expression and hence neutrophil mediated host injury. It is generally accepted that activation of NF B involves an oxidative step, and the antioxidant N-acetylcysteine and other antioxidants have been shown to reduce NF B activation. Such antioxidant therapy has been shown to result in decreased expression of chemokines (IL-8) and adhesion molecules (ICAM-1) expression in healthy subjects,18 animal models of sepsis or inflammation,19,20 and patients with sepsis.21,22 The use of the antioxidant superoxide dismutase in preserving organs for transplant also reduces adhesion molecule expression.23 The primary role of neutrophil extravasation is protective and adhesion molecules are critical in regulating leukocyte extravasation. Despite the progress made in the adhesion molecule field and the proven efficacy of blocking the function of these glycoproteins in disease models, the role of each of them in human disease has not been established and awaits the outcome of clinical studies. Complete blockade may be detrimental to host defence and strategies that compete to reduce rather than block adhesion molecule expression, particularly at the local level, may be more helpful.
Summary
Neutrophils provide a crucial role in host defence against infection through extravasation to sites of infection or inflammation, and phagocytosis. The accumulation of neutrophils in inflamed tissue results from adhesive interactions between neutrophils and endothelial cells. These adhesive interactions are largely confined to the postcapillary venules in the microvasculature. This process involves several steps: capture and rolling, firm adhesion, diapedesis, and migration. The nature and magnitude of the leukocyteendothelial cell adhesive interactions are determined by a variety of factors, including expression of adhesion molecules on leukocytes and/or endothelial cells, signalling by cytokines and chemotactic factors, products
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of leukocyte and endothelial cell activation, and other mediators. Despite the vital role of neutrophils in host defence, many chronic, acute, and autoimmune inflammatory conditions are associated with neutrophilmediated injury to host tissue, resulting in the concept of dampening adhesive actions as a therapeutic strategy in inflammatory diseases.
References
1 Giannoudis PV, Smith RM. The effects of trauma and sepsis on soluble L-selectin and cell surface expression on L-selectin and CD11b on leukocytes. J Trauma 1999;46:984. 2 Kayal S, Jais JP, Aguini N, Chaudiere J, Labrousse J. Elevated circulating E-selectin, intercellular adhesion molecule 1, and von Willebrand factor in patients with severe infection. Am J Respir Crit Care Med 1998;157:77684. 3 Cummings CJ, Sessler CN, Beall LD, Fisher BJ, Best AM, Fowler AA III. Soluble E-selectin levels in sepsis and critical illness. Correlation with infection and hemodynamic dysfunction. Am J Respir Crit Care Med 1997;156:4317. 4 Sakamaki F, Ishizaka A, Handa M et al. Soluble form of P-selectin in plasma is elevated in acute lung injury. Am J Respir Crit Care Med 1995;151:18216. 5 Norman KE, Katopodis AG, Thoma G et al. P-selectin glycoprotein ligand-1 supports rolling on E- and P-selectin in vivo. Blood 2000;96:358591. 6 Miotla JM, Jeffery PK, Hellewell PG. Platelet-activating factor plays a pivotal role in the induction of experimental lung injury. Am J Respir Cell Mol Biol 1998;18:197204. 7 Ruehl ML, Orozco JA, Stoker MB, McDonagh PF, Coull BM, Ritter LS. Protective effects of inhibiting both blood and vascular selectins after stroke and reperfusion. Neurol Res 2002;24:22632. 8 Nemoto T, Burne MJ, Daniels F et al. Small molecule selectin ligand inhibition improves outcome in ischemic acute renal failure. Kidney Int 2001;60:220514. 9 Whalen MJ, Doughty LA, Carlos TM, Wisniewski SR, Kochanek PM, Carcillo JA. Intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 are increased in the plasma of children with sepsis-induced multiple organ failure. Crit Care Med 2000;28:26007. 10 Maekawa K, Futami S, Nishida M et al. Effects of trauma and sepsis on soluble L-selectin and cell surface expression of L-selectin and CD11b. J Trauma 1998;44:4608. 11 Rosenbloom AJ, Pinsky MR, Napolitano C et al. Suppression of cytokinemediated beta2-integrin activation on circulating neutrophils in critically ill patients. J Leukocyte Biol 1999;66:839. 12 Yacyshyn BR, Bowen-Yacyshyn MB, Jewell L et al. A placebo-controlled trial of ICAM-1 antisense oligonucleotide in the treatment of Crohns disease. Gastroenterology 1998;114:113342. 13 Carraway MS, Welty-Wolf KE, Kantrow SP et al. Antibody to E- and L-selectin does not prevent lung injury or mortality in septic baboons. Am J Respir Crit Care Med 1998;157:93844. 14 Welty-Wolf KE, Carraway MS, Ghio A, Kantrow SP, Huang YC, Piantadosi CA. Pro-inflammatory cytokines increase in sepsis after anti-adhesion molecule therapy. Shock 2000;13:4049. 15 Wegner CD, Gundel RH, Reilly P, Haynes N, Letts LG, Rothlein R. Intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of asthma. Science 1990;247:4569.
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16 Zhou MY, Lo SK, Bergenfeldt M et al. In vivo expression of neutrophil inhibitory factor via gene transfer prevents lipopolysaccharide-induced lung neutrophil infiltration and injury by a beta2 integrin-dependent mechanism. J Clin Invest 1998;101:242737. 17 Opal SM, Sypek JP, Keith JC Jr, Schaub RG, Palardy JE, Parejo NA. Evaluation of the safety of recombinant P-selectin glycoprotein ligand-immunoglobulin G fusion protein in experimental models of localized and systemic infection. Shock 2001;15:28590. 18 Desideri G, Croce G, Marinucci MC et al. Prolonged, low dose alphatocopherol therapy counteracts intercellular cell adhesion molecule-1 activation. Clin Chim Acta 2002;320:59. 19 Altavilla D, Deodato B, Campo GM et al. IRFI 042, a novel dual vitamin E-like antioxidant, inhibits activation of nuclear factor-kappaB and reduces the inflammatory response in myocardial ischemia-reperfusion injury. Cardiovasc Res 2000;47:51528. 20 McGilvray ID, Rotstein OD. Antioxidant modulation of skin inflammation: preventing inflammatory progression by inhibiting neutrophil influx. Can J Surg 1999;42:10915. 21 Spapen H, Zhang H, Demanet C, Vleminckx W, Vincent JL, Huyghens L. Does N-acetyl-L-cysteine influence cytokine response during early human septic shock? Chest 1998;113:161624. 22 Paterson RL, Galley HF,Webster NR. The effect of N-acetylcysteine on nuclear factor B activation, interleukin-6, interleukin-8 and intercellular adhesion molecule-1 expression in patients with sepsis. Crit Care Med 2002;(in press). 23 Koo DD, Welsh KI, West NE et al. Endothelial cell protection against ischemia/reperfusion injury by lecithinized superoxide dismutase. Kidney Int 2001;60:78696.
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5: T cell immunity and sepsis

EGBERT PRAVINKUMAR
Introduction
Immune responses essential for defeating systemic microbial infections depend on intact innate and acquired immune responses. Recognition molecules, inflammatory cells, and the cytokines allow host tissues to recognise invading microbes and to initiate intercellular communication between the innate and acquired immune systems. However, activation of innate immunity might also occur in the absence of microbial recognition, through expression of internal signals produced by tissue ischaemia and necrosis. Induction of the innate immune system can have catastrophic effects on patients with sepsis. Exaggerated production of cytokines and the induction of mediators, such as nitric oxide, platelet activation factor, and prostaglandins, have been implicated in the endothelial changes and induction of a procoagulant state, leading to hypotension, inadequate organ perfusion, and necrotic cell death, associated with multiorgan dysfunction syndrome. This chapter provides an overview of the T cell immune system, its regulation, and the influence of sepsis.
Sepsis
The pro-inflammatory state has been defined as being a systemic inflammatory response syndrome (SIRS).1,2 However, most patients survive this initial event, and the pro-inflammatory state ultimately resolves. The cytokines and humoral mediators responsible for the induction of the innate immune response and SIRS also contribute to the development of acquired or specific immune defects. The patient may then enter an immunological state characterised by T cell hyporesponsiveness, anergy, and a defect in antigen presentation that has been recently termed compensatory anti-inflammatory response syndrome (CARS).
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Immunity
The immune system has a complicated array of cells, which have developed to recognise a wide variety of micro-organisms. The T and B cells form the basis of adaptive immunity, the hallmark of the immune system in higher animals. This is characterised by antigen-specific receptors on T and B cells, which produce targeted effector responses. This occurs in two stages: antigen recognition by the T and B cells resulting in cell priming, activation, and differentiation, and the effector response owing to the movement of the activated and differentiated T cells from the lymphoid tissue to the disease site, or to the release of antibody from activated B cells or plasma cells into the blood and tissue fluids and to the infective foci3,4 (Figure 5.1).
B cells Development T-cell dependent response B cell encounters Th2 antigen helper cell Processes and expresses antigen (1) in MHC class II Nave mature molecules B cell Plasma cell B cell T-cell in dependent response Ti antigen antigen specific (2)
PERIPHERY
Secretes antibody
PreB cell
Gene rearrangement
Bone marrow
Secretes antibody Plasma cell
T cells Development PERIPHERY T-cell priming Armed effector cells (1) CD4 Th1 inflammatory cells activate macrophages
(2) CD4 Th2 cells Bone Nave CD4 or help antibody responses Antigen presented T-cell proliferation marrow Thymocytes CD8 T-cell with MHC and differentiation undergo positive antigen specific (3) CD8 cytotoxic cell and negative selection
Figure 5.1 Development and maturation of B and T lymphocytes. Adapted with permission from Parkin and Cohen.4
T cells
Development The T lymphocytes develop from the progenitor cells within the bone marrow and migrate to the thymus as thymocytes. Gene arrangement occurs early in the development of the T cells, even before antigen exposure, which results in the production of over 10 T cell receptors adequate to cover the range of pathogens likely to be encountered in life. The process of random rearrangement and splicing together of multiple DNA segments which code for the antigen-binding areas of the receptors leads to the production of antigen-specific T cell receptors.5 T cells that have undergone gene rearrangement and emerge from the thymus are called nave T cells, as they have not yet faced an immune
66
T CELL IMMUNITY AND SEPSIS
challenge with their specific antigen. These nave cells bear receptors and populate the secondary lymphoid tissue. The lymphoid organs communicate with tissues using lymphatics and blood vessels and provide an excellent environment for an immune challenge, since they contain the antigen-presenting cells and cytokines necessary for the maintenance of T lymphocytes. Although about 95% of T lymphocytes are sequestered within lymphoid tissue, they are not static but move continuously from one lymphoid tissue to another, via the blood or lymph, travelling around the whole body in 12 days. When these nave T cells meet the antigenpresenting cells bearing their particular antigen, activation occurs over a period of 23 days.4 T cell antigen-specific receptors (TCR) have two forms. The and the chain are the most common form of the TCR.The and chains are less common and the function of the T lymphocyte bearing such TCRs is uncertain. T and B cells recognise antigens separately despite the similarity in the gene rearrangement processes. The TCR binds linear peptides usually of eight to nine amino acids this generally means antigen that has been broken down by intracellular processing. Antibody recognises the conformational structure (shape) of epitopes, and hence such antigens do not require processing.4 The main peripheral pool of lymphocytes which express TCRs respond to antigen in the form of short peptides bound to major histocompatible complex (MHC) class I or class II molecules. MHC class I ligands alert the immune system to active intracellular infection and target infected cells for destruction, whereas MHC class II ligands guide intercellular cooperation between haematopoietic cells in an immune response and help to combat extracellular infections.
T cell subsets Based on the expression of MHC binding protein, T lymphocytes can be divided into two types of effector T cells: T helper cells (helper or regulatory T cells Th cells), which express the pan-MHC-class II binding protein, CD4, and T cytotoxic cells (cytotoxic or killer T cells Tc cells), which express the pan-MHC-class I binding protein, CD8.
Tc cells mediate lysis of autologous cells infected by intracellular pathogens (cell-mediated immunity), and Th cells induce B cell production of antibodies (humoral immunity). Their stimulation of antibody responses has prompted investigation into the common regulation of humoral and cell-mediated immunity by T helper cells (Box 5.1).6
67
Box 5.1 Key features of helper and cytotoxic T cells Helper T cells CD4 surface protein Express MHC class II binding protein Activated by exogenous antigens Non-targeted response Induce B cell production of antibodies Characterised by cytokine release Th1 and Th2 subtypes Cytotoxic T cells CD8 surface protein Express MHC class I binding protein Activated by endogenous antigens Highly targeted response No humoral immunity
There are two ways in which antigen loading onto MHC can occur. The antigen may have been produced endogenously within the cell (such as viral or tumour proteins) and is complexed with MHC class I through intracellular processing pathways. Alternatively, specialised professional antigen-presenting cells might have taken up exogenous antigen by endocytosis and these are complexed with MHC class II. The recognition of antigen by the TCR is different for CD4 and CD8 cells. CD4 lymphocytes only recognise antigen presented with MHC class II, and CD8 cells only recognise antigen presented with MHC class I. Since CD4 and CD8 cells have very different functions, the MHC that presents antigen will determine the type of effector response generated. Endogenous antigens complexed with MHC class I molecules activate CD8 Tc cells and CD8 responses are highly targeted to the cell that they recognise. Because all nucleated cells express MHC class I, this means that any such cell infected with a virus or other intracellular pathogen, or producing abnormal tumour antigens, can present these antigens with class I and be removed by cytotoxic attack. Exogenous antigens complexed to MHC class II molecules activate CD4 helper T cells, which leads to production of cytokines, in turn activating a wide range of cells around them. The reaction clearly needs to be tightly regulated, achieved by only a small number of class II antigen-presenting cells being able to drive the response. Dendritic cells (DC) play an important role in the activation and maturation of the primary nave T cells. They form close contacts with antigen-specific Th and Tc cells, and these interactions influence both the
68
cell types. The DClymphocyte collaboration results in: 1 T cell activation and proliferation with the enhancement of DC maturation; 2 T cell killing of DC resulting in the switching off of the immune response; 3 reciprocal activation of DC and natural killer cell (NK). This DCNK interaction is fully capable of stimulating nave T cells in vitro.7
T helper cell subsets

Mosmann et al.8 first described the existence of subpopulations of CD4 cells known as Th type 1 (Th1) and Th type 2 (Th2) lymphocytes. Initial studies in mice showed that Th1 cells produced interleukin-2 (IL-2), interferon (IFN , and tumour necrosis factor (TNF , also known as lymphotoxin ). Th2 cells produce IL-4 as well as IL-5, IL-9, IL-10, and IL-13. Th1 and Th2 cells are now known to exist in man. All T helper lymphocytes start out as nave Th0 cells, which, after being activated, are capable of differentiating into either Th1 or Th2 effector cells.9 Th1/Th2 cells and cytokines Both Th1 and Th2 cells produce a range of cytokines (Table 5.1), low molecular weight proteins that mediate inflammation and influence the process and direction of many immunological reactions. The cytokines are nominally functionally divided into pro-inflammatory and antiinflammatory cytokines.4,10 It should be noted that, although Th1 type immune responses are characterised by high production of the Th1 associated cytokines IL-2, IFN , and TNF , this should not imply that these cytokines are exclusively produced by Th cells.
Table 5.1 Subtypes of CD4 T-helper (Th) cells and their characteristic cytokines and effects. Subtype Nave Th0 Th1 Defining cytokines IL-2 IL-2 IFN TNF IFN , IL-4, others IL-4, IL-5, IL-9, IL-10, IL-13 Effects Proliferate and differentiate to effector cells Cell-mediated immunity, opsonising antibody
Mature Th0 Th2
Unclear Humoral immunity, inhibits inflammation
Th1 cells are important for the eradication of intracellular pathogens, including bacteria, parasites, yeasts, and viruses. The hallmark
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pro-inflammatory cytokines of Th1 cells include IFN , and TNF , which can activate microbicidal activity as well as cytokine production in macrophages.11 IFN stimulates phagocytosis, the oxidative burst, and intracellular killing of microbes. A Th1 response is often accompanied by the production of complement-fixing antibodies of the immunoglobulin (Ig) G2a isotype, as well as the activation of NK cells and cytotoxic CD8 T cells expressing IFN and perforin.11 If uncontrolled, Th1 cells can mediate immunopathology and have also been implicated in autoimmune diseases such as type 1 diabetes and multiple sclerosis. IFN release following Th1 activation upregulates expression of class I and class II MHC molecules on a variety of cells, thereby stimulating antigen presentation to T cells. Both IFN and TNF induce other cell types, including endothelial cells, keratinocytes, and fibroblasts, to secrete pro-inflammatory cytokines, such as TNF and chemotactic cytokines chemokines. They also stimulate adhesion molecule expression on endothelial cells and induce endothelial cell retraction and vascular smooth muscle relaxation. The result is accumulation of blood in dilated, leaky vessels, causing migration of leukocytes into areas of danger and allowing recruitment of innate immune cells and opsonins into the interstitium. Th2 cells produce the cytokines IL-4, IL-5, IL-9, IL-10, and IL-13 and stimulate antibody production. Th2 cells activate mast cells and eosinophils, thereby eradicating helminths and other extracellular parasites. It is interesting that IL-4, IL-5, IL-9, IL-10, and IL-13 have been strongly implicated in allergic and atopic reactions, as well as in causing the airway inflammation seen in asthma and reactive airway disease. This is in keeping with findings that Th2-derived cytokines can induce airway hyperreactivity as well as the production of IgE. IL-4, IL-10, and IL-13 activate B cell proliferation, antibody production, and immunoglobulin class-switching. Class-switching from IgG to IgE cannot occur without IL-4 or IL-13, making the production of IgE a perfect bioassay for the presence of Th2 cells in vivo. IL-10 inhibits the secretion of pro-inflammatory cytokines, suppresses phagocytosis, the oxidative burst, and intracellular killing, and inhibits antigen presentation to T cells, causing T cell anergy. Like IL-10, IL-4 and IL-13 also inhibit phagocytosis and intracellular killing, suppress inflammatory cytokine production, and may induce T cell anergy. IL-5 is a potent haematopoietic cytokine, which stimulates bone marrow production of eosinophils, as well as activation and chemotaxis of eosinophils and basophils, whereas IL-9 is the equivalent haematopoietic and stimulatory factor for mast cells.
Th1 and Th2 cell regulation Both Th1 and Th2-specific cytokines can promote growth or differentiation of their own respective T cell subset, but additionally might inhibit the
70
development of the opposing subset. This might explain why Th1 and Th2 responses are often mutually exclusive.12 The IFN secreted by Th1 cells directly suppresses IL-4 secretion and thus inhibits differentiation of nave Th0 cells into Th2 cells. Conversely, IL-4 and IL-10 inhibit the secretion of IL-12 and IFN , blocking the ability of Th0 cells to polarise into Th1 cells (Figure 5.2).
IL-12
Th0 Cell
IL-4
Po la
DHEA, IFN-/ HSP/LPS/LTA/CpG
Po
n io at ris la
r is
at
io
Epi/NE, cortisol, PG estgn/pgstn, frust. phag.
Th1 Cell
Th2 Cell
IL-2, IFN-, LT- (TNF-) Strong CMI, weak humoral, antibody class switch to IgG1 and IgG3
IL-4 , IL-5, IL-9, IL-10, IL-13 Suppress CMI, strong humoral, antibody class switch to IgG2,4 and IgE
Figure 5.2 Summary of Th1/Th2 induction. Cytokines, hormones, and microbial antigens stimulate the innate immune system to produce either IL-12 or IL-4 around a newly activated T cell. IL-12 induces Th0 polarisation to the Th1 phenotype and inhibits polarisation to the Th2 phenotype, whereas IL-4 acts reciprocally. Abbreviations: CMI, cell-mediated immunity; CpG, purine-purine-C-Gpyrimidine-pyrimidine DNA hexamer; DHEA, dehydroepiandrosterone; Epi/NE, epinephrine/ norepinephrine; HSP heat shock protein; LTA, lipotechoic acid; LPS, lipopolysaccharide; PG, , prostaglandin; estgn/pgstn, estrogen/progesterone; frust. phag., frustrated phagocytosis. Adapted with permission from Spellberg and Edwards.6
There are also differences in proliferation requirements between Th1 and Th2 cells. Nave T cells and Th1 cells absolutely require IL-2 for activation and proliferation. However, Th2 cells are perfectly capable of proliferating without IL-2 if IL-4 and/or IL-1 is present, which is convenient for them since they secrete large quantities of IL-4. Therefore, in clinical situations where IL-2 is in limited supply, for example in patients taking cyclosporine or high dose glucocorticoids, only Th2 cells will be able to proliferate in response to antigenic exposure. The factors which determine whether newly activated nave T cells become mature Th1 or Th2 cells are: local cytokine environment (IL-12 for Th1 and IL-4 for Th2) presence of immunologically active hormones
71
antigen dose and route type of antigen-presenting cell affinity of the TCRs for MHC-antigen complex costimulatory interactions between cell surface molecules.
Polarisation into Th1 or Th2 may not occur until an activated T cell arrives at the site of danger. T cells exposed to IL-12 during this time differentiate to become Th1 cells, and T cells exposed to IL-4 differentiate to become Th2 cells. When the immune system is in doubt about whether Th1 or Th2 cells should be generated, Th2 outcomes are favoured. For instance, if both IL-12 and IL-4 are present at the time of T cell activation, the effect of the IL-4 dominates, and the T lymphocytes polarise to become Th2 effectors, resulting in activation of mast cells and eosinophils. Molecular mechanisms of polarisation Investigators have found that ligation of the IL-12 receptor on Th0 cells activates the transcription factor signal transducer and activator of transcription 4 (STAT4), which triggers regulatory sequences leading to Th1 polarisation (Figure 5.3). Conversely, ligation of the IL-4 receptor on Th0 cells triggers activation of STAT6, which suppresses Th1 polarisation
IL-4R
STAT 6 GATA-3 C-maf Th2
IL-4, IL-5
+T-bet IFN- IL-4, IL-5
IL-4 CD4+ T Cell IL-12 STAT 4 IL-8R IL-12R erm T-bet Th1 IFN-
IL-4, IL-5 IFN-
Figure 5.3 Interleukin 12 (IL-12) and IL-4 direct the development of Th1 and Th2 subsets producing IFN or IL-4, through STAT4- and STAT6-dependent pathways, respectively. Th2 cells express the transcription factors c-maf and GATA-3. Ectopic expression of GATA-3 induces the expression of Th2-specific cytokines and inhibits IFN production in developing and committed Th1 cells. T-bet, a newly described Th1-specific T-box transcription factor, induces IFN and represses IL-4 and IL-5 in both developing and committed Th2 cells. Adapted with permission from OGarra and Arai.3 72
and leads to Th2 polarisation. Indeed, mice that do possess STAT4 or STAT6 genes are unable to mount type 1 or type 2 responses respectively. Even more recently, downstream targets of STAT4 and STAT6 have been identified. The transcription factor GATA-3 directly induces Th2 polarisation and inhibits Th1 polarisation, acting with STAT6.12 Conversely, activation of the transcription factor ERM by STAT4 induces IFN expression. Recently the penultimate effector of the STAT4-induced Th1 polarisation, the transcription factor T-bet, was identified. T-bet exerts direct control over IFN gene expression, and ectopic expression of T-bet in already polarised Th2 cells redirects the lymphocytes to adopt a Th1 profile. Th1 and Th2 cells in sepsis The predominant T helper cell subset in patients with sepsis has been shown to be Th2, compared with critically ill non-septic patients. This is due to a relatively larger number of Th2 cells and smaller number of Th1 cells without a change in the number of total T helper cells. The predominance of Th2 cells in sepsis would lead to increased IgE synthesis and B cell activation.13 Markedly increased circulating concentrations of IL-10 have been reported in patients with sepsis particularly in patients who do not survive. Therefore, in severe sepsis IL-10 mediated suppression of Th1 cells leading to predominance of Th2 responses and preferential release of IL-4, IL-5, IL-10, and IL-13 along with B cell proliferation could occur.
Cytotoxic T cells
These are directly cytotoxic to cells bearing their specific antigen. Cytotoxic cells of the immune system are of two main types: cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Over the past several years, it has become increasingly apparent that these two types of cell have common effector mechanisms. CD8 CTL and NK cells are known to cause damage to their target cells by inducing granule exocytosis of granzymes or via the FasL system. However in sepsis there is strong evidence that Tc cells kill their target cells by programming them to undergo apoptosis.4,14 Both NK cells and CTLs induce cell death by means of FAS-mediated and granule-mediated pathways, which trigger the intrinsic apoptotic response. The FAS pathway has provided a model for how apoptosis is triggered through death receptors. Similar signalling pathways through FASassociated death domain protein (FADD) and caspase activation, occur for other death receptors, such as TNF-related apoptosis-inducing ligand (TRAIL). The granule pathway involves the granules of the cytolytic cells,
73
which contain various proteins, some of which are now known to have a defined role in inducing target-cell death, such as the pore-forming protein perforin and a family of serine proteinases known as granzymes. After binding to the target cell, CD8 T cells insert perforins into the cell membrane, in the same way as NK cells do. These activate caspase enzymes which induce DNA fragmentation and cell apoptosis. Cytoplasmic granules containing granzymes pass through the pores from the T cell into the target cytoplasm. Although perforin alone has been shown to mediate membrane damage in vitro, it is the combined action of perforin and granzymes that is necessary for the induction of apoptosis in vivo. Cytolytic cells from perforin-deficient mice have obvious defects in the ability to induce both membrane and apoptosis. Also, mice that do not have the gene for perforin (knockout mice) are more susceptible to tumours and infection.15 So far, a total of eleven granzymes have been found in human and rodent cytolytic cells. Of this extensive family of granzymes, only four granzymes A, B, H, and K are present ubiquitously in human cytolytic cells. Of these, granzymes A and B are the most abundant and, at present, the best characterised. Recently, however, granzymes A, B, and H have been shown to enter target cells in the absence of perforin.14
Apoptosis of T cell in sepsis

Apoptosis of mature T lymphocytes occurs through at least two distinct processes: active apoptosis antigen-driven (TNF and FasL), and passive apoptosis lymphokine withdrawal (lack of IL-2 production) (Figure 5.4) as described in Chapter 2. Antigen-driven T cell apoptosis takes place indirectly either by increased glucocorticoid release or by the antigen-induced expression of death cytokines, like FasL and TNF . These death cytokines engage specific receptors that assemble caspase-activating protein complexes. Fas-deficient T cells exhibit reduced but clearly evident TCR-induced death, and the residual apoptosis is blocked by inhibiting TNF . In resting T cells, the genes for FasL and TNF are weakly induced by TCR stimulation, but in IL-2-stimulated T cells these death cytokines are induced more strongly. This difference can explain in part why TCR engagement kills cycling but not resting T cells, and thereby might explain lymphopenia in septic patients. A high concentration of the growth factor IL-2 enhances the expression of FasL on antigen-stimulated T cells and the development of sensitivity to Fas-mediated apoptosis. Thus, IL-2 is both a growth factor for T cells and a feedback regulator of T cell responses. In some cell populations, such as CD8 T lymphocytes, activation-induced cell death is apparently triggered not through Fas, but through TNF receptor signalling. Conversely, apoptosis of CD4 T cells is usually a
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T CELL IMMUNITY AND SEPSIS Antigen + T cell
No stimulus
FasL TNF IL-2
FasL TNF IL-2
Active apoptosis antigen driven
Passive apoptosis lymphokine withdrawal
Figure 5.4 Apoptosis in T cells occurs in two pathways an antigen-driven active pathway and a passive pathway initiated by lymphokine withdrawal.
result of the interaction of two co-expressed molecules on activated cells Fas and FasL. Lymphocytes are programmed to die unless protected by growth factors. Cell death from growth factor withdrawal may result from the cytoplasmic activation of caspases regulated partly by mitochondria and the bcl-2 protein. This type of apoptosis does not involve the Fas/TNF receptor family. The fate of cycling T cells is thus linked to the prevailing state of the immune response. Without continuous antigen stimulation, the expression of IL-2 and its receptor falls, and passive apoptosis ensues. Therefore, passive apoptosis decreases the expanded population of the T cells at the end of an immune response.15 Impairment of the adaptive immune response after trauma or burns is characterised by failure of IL-2 production from Th1 cells. When major injury leads to a predominance of Th2 cells because expression of IL-2 is decreased, passive apoptosis of T cells occurs. It is presumed that this increased apoptotic loss of T lymphocytes further increases the susceptibility to sepsis that is manifested in severely injured patients. Furthermore, it has been shown that increased lymphocyte apoptosis in peripheral blood T cells from burned patients appears to contribute to decreased lymphocyte immune responsiveness.16
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Summary
The T cell immune system has a delicate and coordinated arrangement of cells which undergo specific regulation and polarisation to induce pro-inflammatory and anti-inflammatory cytokines. Manipulation of the genetic pathways controlling Th1/Th2 polarisation in the future may allow clinicians to intervene to promote type 1 or type 2 immune responses.
References
1 Oberholzer A, Oberholzer C, Moldawer LL. Sepsis syndromes: understanding the role of innate and acquired immunity. Shock 2001;16:8396. 2 Bone RC. Sir Isaac Newton, sepsis, SIRS, and CARS. Crit Care Med 1996; 24:11258. 3 OGarra A, Arai N. The molecular basis of T helper 1 and T helper 2 cell differentiation. Trends Cell Biol 2000;10:54250. 4 Parkin J, Cohen B. An overview of the immune system. Lancet 2001;357:177789. 5 Muzio L, Polentarutti N, Bosisio D et al. Toll-like receptors: a growing family of immune receptors that are differentially expressed and regulated by different leukocytes. J Leukocyte Biol 2000;67:4506. 6 Spellberg B, Edwards JE. Type1/type 2 immunity in infectious diseases. Clin Infect Dis 2001;32:76102. 7 Gerosa F, Baldani-Guerra B, Nisii C et al. Reciprocal activating interaction between natural killer cells and dendritic cells. J Exp Med 2002;195:32733. 8 Mosmann TR, Cherwinski H, Bond MW et al.Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986;136:234857. 9 Sad S, Mosmann TR. Single IL-2-secreting precursor CD4 T cell can develop into either Th1 or Th2 cytokine secretion phenotype. J Immunol 1994;153: 351422. 10 Townsend MJ, McKenzie AN. Unravelling the net? cytokines and diseases. J Cell Sci 2000;113:354950. 11 OGarra A. Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 1998;8:27583. 12 Ouyang W, Lhning M, Gao Z et al. Stat6-independent GATA-3 autoactivation directs IL-4-independent Th2 development and commitment. Immunity 2000; 12:2737. 13 Ferguson NR, Galley HF, Webster NR. T helper cell subset ratios in patients with severe sepsis. Intensive Care Med 1999;25:1069. 14 Kagi D, Vignaux F, Ledermann B et al. Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science 1994;265:52830. 15 Lenardo M, Chan KM, Hornung F et al. Mature T lymphocyte apoptosisimmune regulation in a dynamic and unpredictable antigenic environment. Ann Rev Immunol 1999;17:22153. 16 Teodorczyk-Injeyan JA, Cembrzynska-Nowak M, Lalani S et al. Immune deficiency following thermal trauma is associated with apoptotic cell death. J Clin Immunol 1995;15:31828.
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6: Metalloproteinases and inflammation

ANDREW J GEARING
Introduction
Matrix metalloproteinases (MMPs) are a large family of zinc-containing endoproteinases, which have similar structures but differ in substrate specificity, cellular sources, and inducibility. MMP activity is controlled at the transcriptional level and by a family of at least four endogenous natural inhibitors (tissue inhibitors) of MMPs called TIMPs. MMPs cleave protein components of the extracellular matrix, membrane receptors, and cytokines, and have a role in cell extravasation. This article describes the action, regulation, and roles of MMPs in inflammatory and immune responses.
The action of matrix metalloproteinases

The MMPs are a distinct subgroup of metalloproteinases a family of zinc-dependent endopeptidases, which collectively are able to degrade all extracellular matrix proteins (Table 6.1). At present, 25 vertebrate MMPs and 22 human homologues have been identified. MMPs are involved in regulating normal tissue turnover and repair; they include the collagenases, gelatinases, stromelysins, membrane-type metalloproteinases, matrilysin, and metalloelastase.1 The MMPs generally have broad, but not necessarily overlapping, substrate specificities. The MMP substrates include matrix proteins such as collagen, elastin, and fibronectin. However, MMPs can also cleave the proforms (zymogens) of MMPs, enzyme inhibitors, and even the cell-bound precursors of cytokines and cytokine receptors. In addition to their matrix protein substrates, MMPs also cleave cell surface molecules and other pericellular non-matrix proteins, thereby regulating cell behaviour in several ways. Thus like the many proteins they modify, the MMPs influence diverse physiological and pathological processes, including aspects of embryonic development, tissue morphogenesis, wound repair, inflammatory diseases, and cancer.
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Table 6.1 The main substrates of the human matrix metalloproteinases. MMP number MMP-1 Alternative nomenclature Collagenase Main substrates Collagens I, II, III, VII, VIII, X, XI, gelatin, laminin, fibrin, fibrinogen, fibronectin, IL-1 , pro-TNF Collagens I, III, IV, V, VII, X, XI, elastin, fibrin, fibrinogen, fibronectin, gelatin, laminin, IL-1 , plasminogen, pro-TGF , pro-TNF , substance P Collagens III, IV, V, VII, IX, X, XI, E-cadherin, elastin, fibrin, fibrinogen, fibronectin, gelatin, laminin, IL-1 , plasminogen, pro-TNF Collagens I, IV, E-cadherin, elastin, fibrinogen, fibronectin, gelatin, laminin, plasminogen, pro-TNF Collagens I, II, III, fibrinogen, substance P Collagens IV, V, XI, XIV, elastin, gelatin, fibrin, fibrinogen, IL-1 , laminin, plasminogen, pro-TGF , substance P Collagens III, IV, V, elastin, fibrinogen, fibronectin, gelatin Serpin Collagens I, IV, elastin, factor XII, fibronectin, gelatin, plasminogen, pro-TNF Collagens I, II, III, VI, IX, X, XIV, factor XII, fibrinogen, fibronectin, gelatin Collagens I, II, III, factor XII, fibronectin, gelatin, laminin, pro-MMP-2, pro-TNF Collagen III, fibronectin, pro-MMP-2 Collagen I Collagens I, IV, fibronectin, gelatin
MMP-2
Gelatinase A, 72 kDa gelatinase
MMP-3
Stromelysin 1
MMP-7
Matrilysin
MMP-8 MMP-9
Neutrophil collagenase Gelatinase B, 92 kDa gelatinase
MMP-10 MMP-11 MMP-12
Stromelysin 2 Stromelysin 3 Metalloelastase
MMP-13
Collagenase-3
MMP-14
MT1-MMP
MMP-15 MMP-16 MMP-17 MMP-18 MMP-19 MMP-20
MT2-MMP MT3-MMP MT4-MMP Collagenase-4 RASI-1 Enamelysin
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METALLOPROTEINASES AND INFLAMMATION
Table 6.1 Continued MMP number MMP-24 MMP-25 MMP-26 Alternative nomenclature MT5-MMP MT6-MMP Endometase, Matrilysin-2 Main substrates
Collagen IV, fibrinogen, fibronectin, gelatin
Abbreviations: MT-MMP, membrane type metalloproteinase.
MMPs can be identified by the following:
they are proteinases, which degrade at least one component of the

extracellular matrix;
they contain a zinc ion and are therefore inhibited by chelating agents; they are secreted as zymogens; they are inhibited by TIMPs and share common amino acid sequences
(Box 6.1).
Box 6.1 Summarised properties of matrix metalloproteinases Structural homology Proteolytic activation of zymogens Varying substrate specificities Distinctive cellular expression patterns Roles in cytokine and other mediator release Roles in cellular migration Important for immune and inflammatory processes
However, the enzymes MMP-11 (stromelysin 3) and membrane-type MMPs (MT-MMPs) do not slot into these definitions quite so easily. MT-MMPs are membrane-associated rather than secreted and are mainly expressed by tumour cells.
Cellular production of MMPs

MMPs are expressed by many cell types, including T and B lymphocytes, macrophages, and neutrophils, and are activated at sites of inflammation, tissue destruction, and in tumours both in animal models and human disease.1 Macrophage production and secretion of large quantities of many
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MMPs, after contact with matrix proteins, is enhanced by surface determinants on activated T cells and suppressed by cytokines from T helper type I (Th1) and type 2 (Th2) cells. T cells predominantly secrete MMP-2 and MMP-9, after 1-integrin- or vascular cell adhesion molecule (VCAM)-1-dependent stimulation by cytokines and inflammatory mediators. MMPs from both T cells and macrophages facilitate secretion of tumour necrosis factor (TNF ) by cleavage of the membrane-bound form, and T cell MMPs prepare connective tissue matrices for T cell chemotaxis across basement membranes and through tissues. The greater amounts of diverse MMPs from macrophages are capable of degrading connective tissues, which may release stored growth factors. Thus MMPs have a major role in the inflammatory response through cytokine and receptor processing and cellular extravasation.
MMP genes IL-1 TNF PDGF LPS TGF heparin steroids
Transcription
degradation phorbol ester mRNA
Zymogens Activation of TNF MT-MMP LPS Activation uPA plasmin Positive feedback secretion localisation Active enzymes TIMP-1 and 2 tetracyclines synthetic inhibitors proteolytic processing Positive effects Negative effects PAI-1 PAI-2 TIMP-1, -2, -3, -4
Proteolysis
Figure 6.1 Regulation mechanisms for matrix metalloproteinases. Abbreviations: IL-1, interleukin-1; LPS, lipolysaccharide; MMP matrix metalloproteinase; MT-MMP membrane type MMP; PAI, , , plasminogen activator inhibitor; PDGF, platelet derived growth factor; TGF, transforming growth factor; TIMP tissue inhibitor of matrix metalloproteinases; TNF, tumour necrosis factor; uPA, , urokinase plasminogen activator. 80
Regulation of MMPs
Since MMPs have the potential for massive tissue destruction, expression and activity are necessarily tightly regulated (Figure 6.1). Regulation of the MMPs is achieved at various levels (Box 6.2).
Box 6.2 Regulation of MMPs Inductive and suppressive signalling Intracellular signal transduction Transcriptional activation and repression Post-transcriptional mRNA processing mRNA degradation Intracellular activation of susceptible MMPs Constitutive secretion Regulated secretion Cell surface expression Proteolytic activation Proteolytic processing and inactivation Protein inhibition Extracellular matrix and cell surface localisation Endocytosis and intracellular degradation
Most MMPs are closely regulated at the level of transcription, with the notable exception of MMP-2, which is often constitutively expressed and controlled through a unique mechanism of enzyme activation and some degree of post-transcriptional mRNA stabilisation. At the transcriptional level, MMPs are regulated by cytokines such as TNF , interleukin-1 (IL-1 ), platelet derived growth factor (PDGF), phorbol ester, transforming growth factor (TGF ), corticosteroids, extracellular matrix proteins, cell stress, and changes in cell shape. Lipopolysaccharide (LPS) also upregulates the pro-MMP-9 gene and is able to activate pro-MMP-9 via LPS-associated proteinases.2 In malignant tissues, the tight control of MMP expression is lost and high levels of MMP mRNA may be induced by growth factors and by oncogene activation. Secretion in a latent form MMPs are secreted as zymogens, which require processing to expose the active catalytic site.1 This processing step can be achieved by the action of
81
the MMPs themselves or other enzymes such as plasmin, urokinase plasminogen activator (uPA), and MT-MMPs. All MMPs can be activated in vitro with organomercurial compounds. Endogenous inhibitors There are at least four members of the TIMP family, TIMP-1 to TIMP-4, which inhibit MMPs by binding in a one-to-one molar ratio with the pro-MMP to prevent activation.3 Individual TIMPs differ in their ability to inhibit various MMPs. For example, TIMP-2 and TIMP-3 inhibit MT1-MMP, whereas TIMP-1 does not. Likewise, TIMP-1 is a relatively poor inhibitor of MT3-MMP, and TIMP-3 appears to be a more potent inhibitor of MMP-9 than other TIMPs. TIMP-3 is also unique in its ability to inhibit ADAMs-10 and -17, ADAMTS-4, and ADAMTS-5, whereas TIMP-1 can inhibit ADAMTS-1 (see section on ADAMS below). In addition, the TIMPs differ in terms of their gene regulation and tissuespecific patterns of gene expression. TIMP-3 also has the unique ability to bind to heparan sulphate proteoglycans within the extracellular matrix, thereby concentrating it to specific regions within tissues and basement membranes. TIMPs are important in establishing a balance between matrix synthesis and matrix degradation caused by the MMPs and are therefore usually co-regulated.4 However, some studies have shown that MMPs and TIMPs can be regulated independently and sometimes reciprocally, mainly in cancer, but also during acute inflammation.57 TNF increases MMP-9 gene expression and hence release of pro-MMP-9, but does not upregulate expression of TIMP-1.7 TIMP-1 and -2, tetracyclines, and synthetic inhibitors can also directly inhibit proteolysis by activated MMPs.1 TIMPs are not the only endogenous MMP inhibitors. Alpha-2macroglobulin is a major endogenous inhibitor of the MMPs and may be more important than originally appreciated, because it is an abundant plasma protein. It is therefore the major inhibitor of MMPs in tissue fluids, in contrast to TIMPs, which may act locally. Also since 2-macroglobulin/MMP complexes are removed by receptor-mediated endocytosis, 2-macroglobulin plays an important role in the irreversible clearance of MMPs, whereas TIMPs inhibit MMPs in a reversible manner.
The ADAM family

The adamalysins are a family of proteins in the metzincin superfamily of metalloproteases, which also includes the MMPs. There are two subfamilies of adamalysins: the snake venom metalloproteases (SVMPs) and the ADAMs (proteins containing a disintegrin and metalloprotease
82
domain). Around 20 ADAMs have been identified to date.8 The ADAMs are expressed by a wide variety of cell types, and are involved in functions as diverse as sperm-egg binding, myotube formation, neurogenesis, and proteolytic processing of cell surface proteins. ADAMTS are ADAMs with thrombospondin motifs. This family has at least nine members and their structural domains are extracellular matrix proteins distinct from members of the ADAM family, which are largely anchored on the cell surface. The expression of mRNA for ADAMTS-1 in various cells is upregulated by stimulation with IL-1 and LPS, suggesting an involvement in immune reactions.
MMPs and inflammation

MMPs participate in and promote inflammatory processes, but they may also blunt such processes. The cell surface molecules whose shedding is prevented by MMP inhibitors include several cytokines such as TNF , Fas ligand and TGF , cytokine receptors such as TNFR1, TNFR2, and IL-6R, adhesion molecules, and amyloid precursor protein.914 Recently, processing of chemokines by MMPs has also been reported.15,16 Thus metalloproteases have important roles in immunity and inflammation. TNF Mature active TNF is released from a cell membrane-anchored precursor by proteolytic cleavage. Broad spectrum synthetic inhibitors of MMPs prevent the processing of the TNF precursor but do not inhibit the release of other cytokines.9,14 Purified MMPs, stromelysin, matrilysin, collagenase, and the gelatinases can all cleave a recombinant pro-TNF substrate to yield mature TNF. TNF converting enzyme (TACE) is a unique member of the ADAM family, which cleaves membrane-bound TNF to generate soluble TNF .17 Several studies have shown that TACE is the major TNF -converting enzyme in vivo, although other MMPs are also able to cleave membranebound pro-TNF . TACE-deficient cells are also compromised in the generation of other soluble proteins that are produced as the result of cleavage of a membrane precursor form, suggesting that TACE is involved in several different ways. The action of TNF is mediated through TNF receptors 1 and 2 (TNFR1 and TNFR2). TNFR1 (55 kDa; CD120a) transduces the signal for apoptosis and the transcription factor, nuclear factor B (NF B) activation, whereas TNFR2 (75 kDa; CD120b) transduces the signal for thymocyte proliferation. The soluble forms of TNFR1 and TNFR2 are released by a metalloprotease and act as antagonists by competing with
83
surface receptors for binding of TNF . Another TNFR family member, CD30, which is capable of transducing an apoptotic signal, is also released from the surface of cells by a metalloprotease.11 Fas-ligand is a 40 kDa transmembrane protein of the TNF receptor (TNFR) family, which binds to Fas ligand from activated T cells. Ligation of Fas induces apoptosis, which is involved not only in immune homeostasis but also in cytotoxic reactions. Soluble Fas-ligand generated by MMP activity can kill Fas-positive activated T cells.13 IL-6 receptors The majority of soluble IL-6 receptors (IL-6R) are generated by proteolytic cleavage at the cell surface, rather than by the de novo synthesis. Soluble IL-6R can act as agonists and upregulate IL-6 effects on the production of acute phase proteins. Chemokines Chemokines provide directional cues for leukocyte migration and activation, which are essential for normal leukocyte trafficking and for host responses during inflammation and infection. MMPs modulate the activity of monocyte chemoattractant protein-3 by selective proteolysis.15,16 MMP-2 can cleave monocyte chemoattractant protein-3, thereby inactivating it and generating a chemokine receptor-binding antagonist that further impedes inflammation. This suggests that MMPs are important in regulating chemokines and therefore regulate leukocyte activity in vivo and that MMPs are both effectors and regulators of the inflammatory response.
Metalloproteinase inhibitors as therapeutic tools

Once activated, MMPs are subject to inhibition by TIMPs.3,4 Despite these controls, excessive MMP production and activation is thought to be a key feature of the pathology of many inflammatory and malignant diseases. MMPs have been a target for the pharmaceutical industry for many years and a number of substrate-based pseudopeptide inhibitors have been described (reviewed by Michaelides).18 Demyelinating neuroinflammatory disease Multiple sclerosis (MS) is characterised by the presence of demyelinated plaques or lesions, which can disrupt the bloodbrain barrier causing leakage of plasma proteins. These plaques contain inflammatory cells
84
(T lymphocytes and monocytes) in addition to activated glial cells. It is thought that an initial influx of inflammatory cells drives the subsequent development of the lesions. Indeed, in animal models of MS, agents which prevent T cell or macrophage infiltration or activation are effective in reducing disease severity. The destructive nature of the MS lesions led to an early interest in proteases and mediators of tissue breakdown, particularly the role of MMPs. Expression of MMPs in the central nervous system (CNS) can mediate leukocyte recruitment, bloodbrain barrier leakage, and myelin destruction, and can perpetuate the immunoinflammatory response by generating immunogenic peptides and releasing the pro-inflammatory cytokine TNF . The cellular components of an MS plaque, including activated T cells, macrophages, microglia, and astrocytes, are capable of expressing a wide range of MMPs in vitro. MMP-2 and MMP-9, stromelysin-1, and collagenase have also been detected in patients with MS and MMP-9 increases during relapse of MS.19,20 Indirect evidence for expression of TACE activation is provided by the fact that TNF is elevated in patients with MS during clinical relapse. There is good evidence that MMP expression in the CNS can contribute to the pathology observed in MS. Injection or induction of MMPs in the brains of rats causes breakdown of the bloodbrain barrier and tissue destruction, and lymphocytes may use MMPs for transmigration through vascular endothelium. MMPs have been shown to degrade myelin basic protein in the myelin sheath, which is destroyed in MS, liberating immunogenic peptides; these may propagate the autoimmune response that drives MS.21 TNF and Fas ligand have been implicated in the pathogenesis of MS and can be blocked by MMP inhibitors. In a murine model for MS, experimental autoimmune encephalomyelitis (EAE), a potent MMP inhibitor, was effective in blocking and reversing acute disease and reducing the number of relapses in chronically relapsing animals.22
GuillainBarr syndrome GuillainBarr syndrome (GBS) is an acute inflammatory demyelinating neuropathy associated with long-lasting morbidity and substantial risk of mortality, and is particularly relevant to the intensive care unit. GBS is associated with an increase of pro-inflammatory cytokines including TNF , a decrease of anti-inflammatory cytokines (for example TGF ), and increased MMP-9 expression. MMPs destroy basement membranes and other matrix components, promoting transmigration of inflammatory cells from the circulation to nerve tissue, a key phenomenon in GBS. In a study of sciatic nerve from rats with EAN, quantitative polymerase chain reaction (PCR) analysis revealed upregulation of MMP-9 mRNA
85
associated with enhanced enzyme activity, with peak levels concurrent with maximal disease severity.23 Nerve biopsies from GBS patients also have increased MMP-9 expression in comparison with non-inflammatory neuropathies.23 An MMP-inhibitor, BB-1101, prevented the development of EAN or significantly reduced disease in the rat model depending on the timing of administration.24 These findings indicate that MMP-9 may contribute to the pathogenesis of inflammatory demyelination of the peripheral nervous system. In a study by Creange et al.,25 circulating MMP-9 concentrations correlated with disease severity in GBS. MMP-9 plasma levels were increased in 67% of patients on admission and decreased during recovery. During the course of GBS, MMP-9 expression was progressively balanced by its inhibitor TIMP-1, as assessed by the MMP-9/TIMP-1 ratio. MMP-9 and TIMP-1 plasma levels and the MMP-9:TIMP-1 ratio correlated positively with disability. MMP-9 therefore appears to have an important role in the pathogenesis of GBS and therefore could represent an interesting therapeutic target. In contrast to patients with inflammatory diseases, MMP-9 levels are not elevated in patients with amyotrophic lateral sclerosis (motor neurone disease).26 Elevated matrix metalloproteinase-9 concentrations in plasma have been shown in other chronic inflammatory illnesses including rheumatoid arthritis, asthma, and chronic bronchitis.27,28
Bacterial meningitis High concentrations of MMP-9 and TIMP-1 in the cerebrospinal fluid (CSF) are also reported in adult patients with bacterial meningitis.29 In a rat model of meningococcal meningitis, intracisternal injection of heat-killed meningococci caused a disruption of the bloodbrain barrier, an increase in intracranial pressure, and detectable MMP-9 activity in the CSF 6 hours after meningococcal challenge. The MMP inhibitor batimastat (BB-94) significantly reduced the bloodbrain barrier disruption and the increase in intracranial pressure irrespective of the time of batimastat administration (15 minutes before and 3 hours after meningococcal challenge).These results suggest that MMPs are involved in the alterations of bloodbrain barrier permeability during experimental meningococcal meningitis.29 In time-course studies of pneumococcal meningitis in rats, MMP-8 and -9 were 100- to 1000-fold transcriptionally upregulated, both in CSF cells and in brain tissue. Concentrations of TNF and MMP-9 in CSF peaked 12 hours after infection and were closely correlated. Treatment with BB-1101, a hydroxamic acid-based inhibitor of MMP and TACE, downregulated the CSF concentration of TNF and decreased the incidences of seizures and mortality. Therapy with BB-1101, together with antibiotics, attenuated neuronal necrosis in
86
the cortex and apoptosis in the hippocampus when given as a pretreatment at the time of infection and also when administration was started 18 hours after infection. Functionally, the neuroprotective effect of BB-1101 preserved learning performance of rats assessed 3 weeks after the disease had been cured. Thus, combined inhibition of MMP and TACE appeared to prevent brain injury and neurological sequelae in bacterial meningitis.30 Acute inflammation MMP activity may also have a role in the acute inflammatory response. In a small study of 10 critically ill patients with severe sepsis and 12 critically ill non-septic control patients, MMP-9 concentrations were significantly elevated in both groups of patients compared with healthy subjects, but there was no difference between patients with and without sepsis, suggesting that MMP-9 represents a non-specific marker of systemic inflammation.31 There has been one other report of elevated MMP-9 concentrations in patients with septic shock, highest in the patients who died, and which were decreased by treatment to reduce circulating endotoxin levels.32 TNF and MMP-9 concentrations also increase in patients undergoing cardiac surgery with cardiopulmonary bypass.6 TIMP-1 levels, in contrast, decrease, suggesting independent regulation.
Summary
The MMPs are a large family of enzymes which cleave matrix proteins, proforms of MMPs, enzyme inhibitors, and the cell bound precursors of cytokines and cytokine receptors, and regulation therefore have a role in inflammation. MMP expression is increased during inflammation and may be independent to that of TIMPs, the endogenous inhibitors of MMPs.There is considerable evidence that MMPs are expressed in multiple sclerosis and GuillainBarr syndrome where they contribute to leukocyte invasion, bloodbrain barrier breakdown, myelin destruction, and TNF release, and initial studies of exogenous MMP inhibitors have been promising.
References
1 Goetzl EJ, Banda MJ, Leppert D. Matrix metalloproteinases in immunity. J Immunol 1996;156:14. 2 Min D, Moore AG, Bain MA, Breit SN, Lyons JG. Activation of macrophage promatrix metalloproteinase-9 by lipopolysaccharide-associated proteinases. J Immunol 2002;168:244955. 3 Murphy G, Willenbrock F. Tissue inhibitors of matrix metalloendopeptidases. Methods Enzymol 1995;248:496510.
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4 Overall CM. Regulation of tissue inhibitor of matrix metalloproteinase expression. Ann NY Acad Sci 1994;732:5164. 5 Sadowski T, Steinmeyer J. Effects of non-steroidal anti-inflammatory drugs and dexamethasone on the activity and expression of matrix metalloproteinase-1, matrix metalloproteinase-3 and tissue inhibitor of metalloproteinases-1 by bovine articular chondrocytes. Osteoarthritis Cartilage 2001;9:40715. 6 Galley HF, MaCaulay GD, Webster NR. Matrix metalloproteinase-9 and its inhibitor and tumour necrosis factor during cardiopulmonary bypass. Anaesthesia 2002;57:65862. 7 Bramhall SR, Neoptolemos JP, Stamp GW, Lemoine NR. Imbalance of expression of matrix metalloproteinases (MMPs) and tissue inhibitors of the matrix metalloproteinases (TIMPs) in human pancreatic carcinoma. J Pathol 1997;182:34755. 8 Killar L, White J, Black R, Peschon J. Adamalysins. A family of metzincins including TNF-alpha converting enzyme (TACE). Ann N Y Acad Sci 1999; 878:44252. 9 Gearing AJ, Beckett P, Christodoulou M et al. Processing of tumour necrosis factor-alpha precursor by metalloproteinases. Nature 1994;370:5557. 10 Arribas J, Coodly L, Vollmer P, Kishimoto TK, Rose-John S, Massague J. Diverse cell surface protein ectodomains are shed by a system sensitive to metalloprotease inhibitors. J Biol Chem 1996;271:1137682. 11 Crowe PD, Walter BN, Mohler KM, Otten-Evans C, Black RA, Ware CF. A metalloprotease inhibitor blocks shedding of the 80 kDa TNF receptor and TNF processing in T lymphocytes. J Exp Med 1995;181:120510. 12 Preece G, Murphy G, Ager A. Metalloproteinase-mediated regulation of -selectin levels on leucocytes. J Biol Chem 1996;271: 1163440. 13 Tanaka M, Suda T, Haze K et al. Fas ligand in serum. Nature Med 1996;2: 31722. 14 Gearing AJ, Beckett P, Christodoulou M et al. Matrix metalloproteinases and processing of pro-TNF-alpha. J Leukocyte Biol 1995;57:7747. 15 McQuibban GA, Gong JH, Tam EM, McCulloch CA, Clark-Lewis I, Overall CM. Inflammation dampened by gelatinase A cleavage of monocyte chemoattractant protein-3. Science 2000;289:12026. 16 McQuibban GA, Butler GS, Gong JH et al. Matrix metalloproteinase activity inactivates the CXC chemokine stromal cell-derived factor-1. J Biol Chem 2001; 276: 435038. 17 Black RA, Rauch CT, Kozlosky CJ et al. A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature 1997;385:72933. 18 Michaelides MR, Curtin ML. Recent advances in matrix metalloproteinase inhibitors research. Curr Pharm Des 1999;5:787819. 19 Chandler S, Miller KM, Clements JM et al. Matrix metalloproteinases, tumor necrosis factor and multiple sclerosis: an overview. J Neuroimmunol 1997; 72:15561. 20 Lee MA, Palace J, Stabler G, Ford J, Gearing A, Miller K. Serum gelatinase B, TIMP-1 and TIMP-2 levels in multiple sclerosis. A longitudinal clinical and MRI study. Brain 1999;122:1917. 21 Opdenakker G, Van Damme J. Cytokine-regulated proteases in autoimmune diseases. Immunol Today 1994;15:1037. 22 Liedtke W, Cannella B, Mazzaccaro RJ et al. Effective treatment of models of multiple sclerosis by matrix metalloproteinase inhibitors. Ann Neurol 1998; 44:3546. 23 Kieseier BC, Clements JM, Pischel HB et al. Matrix metalloproteinases MMP-9 and MMP-7 are expressed in experimental autoimmune neuritis and the GuillainBarr syndrome. Ann Neurol 1998;43:42734.
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24 Redford EJ, Smith KJ, Gregson NA et al. A combined inhibitor of matrix metalloproteinase activity and tumour necrosis factor-alpha processing attenuates experimental autoimmune neuritis. Brain 1997;120:1895905. 25 Creange A, Sharshar T, Planchenault T et al. Matrix metalloproteinase-9 is increased and correlates with severity in GuillainBarr syndrome. Neurology 1999;53:168391. 26 Beuche W, Yushchenko M, Mader M, Maliszewska M, Felgenhauer K,Weber F. Matrix metalloproteinase-9 is elevated in serum of patients with amyotrophic lateral sclerosis. Neuroreport 2000;11:341922. 27 Gruber BL, Sorbi D, French DL et al. Markedly elevated serum MMP-9 (gelatinase B) levels in rheumatoid arthritis: a potentially useful laboratory marker. Clin Immunol Immunopathol 1996;78:16171. 28 Cawston T. Matrix metalloproteinases and TIMPs: properties and implications for the rheumatic diseases. Molec Med Today 1998;4:1307. 29 Paul R, Lorenzl S, Koedel U et al. Matrix metalloproteinases contribute to the bloodbrain barrier disruption during bacterial meningitis. Ann Neurol 1998;44: 592600. 30 Leib SL, Clements JM, Lindberg RL et al. Inhibition of matrix metalloproteinases and tumour necrosis factor alpha converting enzyme as adjuvant therapy in pneumococcal meningitis. Brain 2001;124:173442. 31 Yassen KA, Galley HF,Webster NR. Matrix metalloproteinase-9 concentrations in critically ill patients. Anaesthesia 2001;56:72932. 32 Nakamura T, Ebihara I, Shimada N, Shoji H, Koide H. Modulation of plasma metalloproteinase-9 concentrations and peripheral blood monocyte mRNA levels in patients with septic shock: effect of fiber-immobilized polymyxin B treatment. Am J Med Sci 1998;316:35560.
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7: Glucocorticoid therapy in septic shock

PIERRE-EDOUARD BOLLAERT
Introduction
Recent findings highlighting the role of the ability of the hypothalamic pituitaryadrenal axis to respond appropriately to a septic insult have led to a reappraisal of the use of steroids in septic shock. Recent work has suggested that physiological doses of corticosteroids given for a longer duration may be beneficial in catecholamine-dependent septic shock, leading to a more rapid withdrawal of vasopressor therapy and a trend toward improved survival. A recent multicentre study of patients in septic shock has suggested a reduction in mortality in patients with relative adrenal insufficiency receiving replacement therapy with a combination of hydrocortisone and fludrocortisone. This article will describe studies of corticosteroid therapy in patients with septic shock and comment on the benefits of corticosteroid therapy in this population.
Rationale for steroid therapy in sepsis

Septic shock is one of the leading causes of death in intensive care units (ICUs) worldwide. Scientists have made great improvements in understanding mechanisms of inflammation, and the sequence of activation of the various pro- and anti-inflammatory markers is now well known. In contrast, physicians have failed to improve survival from septic shock despite the development of specific targets considered to have a key role in host survival in sepsis. Corticosteroids were the first antiinflammatory drugs tested in septic patients. However, the use of corticosteroids in patients with sepsis or septic shock has been controversial for many decades, since clinical studies have reported beneficial, as well as negative, results. Two meta-analyses published in 1995 assessed corticosteroid therapy in sepsis.1,2 Lefering et al. investigated 49 studies of corticosteroids in patients with sepsis and septic shock but only 10 of the publications were
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GLUCOCORTICOID THERAPY IN SEPTIC SHOCK
prospective, randomised, controlled trials with an exact description of dosage and regimen. Only one study showed a significantly positive effect of steroid treatment and, overall, no positive effect was observed. Another meta-analysis by Cronin and colleagues2 determined the effect of corticosteroid therapy on morbidity and mortality in patients with sepsis and identified nine relevant, randomised, controlled trials of corticosteroid therapy in sepsis and septic shock in critically ill adults. These authors reported that corticosteroids appeared to increase mortality in patients with overwhelming infection and had no beneficial effect in the subgroup of patients with septic shock. Thus it was concluded that there was no supporting evidence for the use of corticosteroids in patients with sepsis or septic shock, and their use may in fact be harmful. However, a few small studies in the early 1990s of low doses of hydrocortisone in catecholamine-dependent septic shock led to a reappraisal of the use of steroids specifically in these patients.3,4 The role of corticosteroids in the inflammatory response The systemic inflammatory response to infection leads to release of cytokines which in turn act on the central nervous system (CNS), resulting in stimulation of the hypothalamuspituitaryadrenal axis (Figure 7.1). The release of glucocorticoid dampens the pro-inflammatory cytokine release, probably via transcriptional effects. There are a number of
T IFN Superantigens Virus IL-12 M Microbial products LT TNF IL-1
IL-6 IL-8 Glucocorticoid MIF
TARGET CELLS
ADRENALS
ACTH
CNS
Figure 7.1 Activation of the hypothalamuspituitaryadrenal axis in the inflammatory response. For abbreviations see text. 91
proposed molecular mechanisms by which corticosteroids can regulate and antagonise transcription factor activity, thereby inhibiting inflammatory mediator expression. Corticosteroids are able to diffuse through the cell membrane and bind to their cytosolic glucocorticoid receptor (GR). This complex subsequently undergoes nuclear translocation and is then able to modulate transcriptional activation through association with promoter elements and glucocorticoid response elements (GRE). Depending upon the target gene, activated GRs may stimulate or inhibit gene transcription (Table 7.1).
Table 7.1 Effects of glucocorticoids on gene transcription.
Increased transcription
Lipocortin-1 -adrenoreceptors -adrenoreceptors Interleukin-1 receptor antagonist Interleukin-1 receptor 2 Inhibitory B protein
Decreased transcription
Interleukin-1 Interleukin-2 Tumour necrosis factor Granulocyte-macrophage colony stimulating factor Interleukin-8 Macrophage inhibitory protein-1 Type II nitric oxide synthase Cyclo-oxygenase-2 Cytoplasmic phospholipase A2 Nuclear factor B inducible kinase-1-receptors Nuclear factor B inducible kinase-2-receptors Intercellular adhesion molecule-1 E-selectin
It remains to be elucidated whether the glucocorticoid effect on a transcription factor is a direct or an indirect event, such as the consequence of an earlier effect in the activation sequence. It has been shown that glucocorticoids can induce the gene transcription and synthesis of the inhibitory protein I B which maintains nuclear factor kappa B (NF B) in an unactivated state in the cell cytoplasm. Other evidence points to activated GR antagonising transcription factor activity through protein protein interactions at a number of sites. Competition between GR and transcription factors for nuclear co-activators (which promote transcription) has also been suggested. The concept of relative adrenal insufficiency in septic shock Rothwell et al. used corticotrophin stimulation tests to assess adrenocortical function in patients with septic shock.5 Thirteen of 32
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patients had a poor cortisol response (rise less than 250 nmol/l) to corticotrophin and all of these patients died. However, there were only six deaths among the 19 patients with adequate responses (Figure 7.2). These results led to the concept that some patients with septic shock might have relative adrenocortical insufficiency. The mortality rate in patients with septic shock who were nonresponders to a short corticotrophin test was higher than responders in five separate studies.59 In addition Annane and colleagues investigated the pressor response to norepinephrine (noradrenaline) according to the response to the corticotrophin test.9 Basal cortisol level, norepinephrine mean arterial pressure (MAP) dose-response curve, and cortisol response to intravenous corticotrophin bolus were obtained in nine patients with septic shock and in six normal volunteers. In patients with septic shock, the dose-response curve to noradrenaline was determined a second time, 60 min after a 50 mg intravenous hydrocortisone bolus. Patients with septic shock had increased basal cortisol levels and a blunted cortisol response compared with healthy controls. Five patients had impaired adrenal function reserve and showed a significant decrease in pressor sensitivity to norepinephrine compared with those patients with an adequate adrenal response (Figure 7.3). In septic patients, hydrocortisone improved the pressor response to norepinephrine particularly in patients with impaired adrenal functional reserve. The authors concluded that in septic shock, impaired adrenal functional reserve may partly be accounted for by the depressed pressor sensitivity to norepinephrine, which may be substantially improved by physiological doses of hydrocortisone.
1000 800 600 400 200 0 200 0 500 1000 Basal cortisol (nmol/litre) 1500
Non-survival Survival
Cortisol response (nmol/ litre)
2000
Figure 7.2 Response to the corticotrophin test and survival in sepsis. For details see text. Reproduced with permission from Rothwell et al.5 93
CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY 40 Increase in mean arterial pressure (mmHg)
30
20
10
10 001
01
10
Noradrenaline titration (g/kg/min) Figure 7.3 Pressor response to norepinephrine according to the response to the corticotrophin test. For details see text. Reproduced with permission from Annane et al.9
The mechanism of this depressed cardiovascular response may be related to nitric oxide.10 In studies using endotoxin-treated rats, a substantial increase in the activity of type II (inducible) nitric oxide synthase was associated with hyporeactivity to contractile responses elicited by norepinephrine. Reduced induction of type II nitric oxide synthase was found to be due to the elevation of endogenous glucocorticoid levels. Barber et al. undertook a study in experimental human endotoxaemia, which has increased the understanding of glucocorticoid regulation of endotoxin-elicited cytokine production.11 A total of 23 normal human subjects were given endotoxin (lipopolysaccharide, LPS) alone or combined with pretreatment with hydrocortisone infusion for 6 hours immediately before or at the same time as LPS administration. Some subjects were rendered hypercortisolaemic for a 6-hour period for a further 6, 12, or 144 hours prior to LPS administration. LPS administration was followed by significant elevations in temperature, pulse, and resting energy expenditure, as well as in epinephrine, cortisol, and C-reactive protein. Levels of the cytokines tumour necrosis factor (TNF ) and interleukin-6 (IL-6) were undetectable before LPS administration but increased with peak concentrations, at 90 and 120 min after LPS respectively (Figure 7.4). Glucocorticoids significantly attenuated the temperature and pulse rate response to LPS alone when
94
GLUCOCORTICOID THERAPY IN SEPTIC SHOCK 900 800 700 600 (pg/ml) 500 400 300 200 100 0 TNF IL-6 LPS HC and LPS simultaneously HC 12 h before LPS HC 144 h before LPS
Figure 7.4 Effects of hydrocortisone on cytokines in human experimental endotoxaemia. For details see text. Reproduced with permission from Barber et al.11
given immediately before and at the same time as LPS. Peak levels of epinephrine and C-reactive protein were also suppressed.TNF levels were undetectable in this group but the IL-6 response was unchanged. Such effects were not seen in subjects who had a 6-hour interval between hydrocortisone infusion and LPS challenge. It was concluded that hypercortisolaemia participates in regulation of the haemodynamic, hormonal, and cytokine responses to endotoxin, and that a complex temporal relationship between hypercortisolaemia and LPS-induced cytokine and systemic responses exists.
Effects of prolonged steroid replacement in septic shock

Thus preliminary studies suggested that low doses of corticosteroids might improve haemodynamics in patients with septic shock. In a study by this author, the effect of hydrocortisone on shock reversal, haemodynamics, and survival was examined in a prospective, randomised, double-blind, placebo-controlled study of 41 patients with septic shock requiring catecholamine for longer than 48 hours.8 Patients were randomly assigned either hydrocortisone (100 mg intravenously three times daily for 5 days) or placebo therapy. Reversal of shock was defined by a stable systolic arterial pressure (over 90 mmHg) for 24 hours or more without catecholamine or fluid infusion. Of the 22 hydrocortisone-treated patients and 19 placebo-treated patients, 68% and 21% achieved 7-day shock
95
reversal, respectively. At 28-day follow-up, reversal of shock was higher in the hydrocortisone group (Figure 7.5). Crude 28-day mortality was 32% in hydrocortisone treated patients and 63% in placebo patients and shock reversal within 7 days after the onset of corticosteroid therapy was a very strong predictor of survival. There were no significant differences in outcome in responders and non-responders to a short corticotrophin test. The rates of gastrointestinal bleeding and secondary infections did not differ in the two groups. A similar study was undertaken by Briegel and co-workers, who investigated the effects of stress doses of hydrocortisone on the duration of vasopressor therapy in human septic shock in a prospective, randomised, double-blind study in 40 consecutive patients with septic shock.12 Patients were prospectively randomised to receive either stress doses of hydrocortisone or placebo. Hydrocortisone was started with a loading dose of 100 mg given within 30 minutes and followed by a continuous infusion of 0.18 mg/kg/h. When septic shock had been reversed, the dose of hydrocortisone was reduced to 0.08 mg/kg/h. This dose was kept constant for 6 days. The hydrocortisone infusion was tapered in steps of 24 mg/day as soon as the infection was resolved. Shock reversal was achieved in 18 of the 20 patients treated with hydrocortisone compared with 16 of the 20 patients treated with placebo. Hydrocortisone significantly reduced the time to cessation of vasopressor support. The median time of vasopressor support was 2 days in the hydrocortisone-treated group and 7 days in the placebo group. Infusion of stress doses of hydrocortisone reduced the time to cessation of vasopressor therapy in human septic shock. Thus
100 Probability of shock reversal (%) 90 80 70 60 50 40 30 20 10 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 Days P = 0.005 Placebo HC
Figure 7.5 Probability of shock reversal in patients treated with hydrocortisone. For details see text. Reproduced with permission from Bollaert et al.8 96
administration of modest doses of hydrocortisone in pressor-dependent septic shock for 96 hours can improve haemodynamics and may have a beneficial effect on survival. A recent multicentre study of 299 patients in septic shock suggested a reduction in mortality in patients with relative adrenal insufficiency receiving replacement therapy with a combination of hydrocortisone and fludrocortisone.13 Currently a large multinational, prospective, doubleblind, randomised, placebo-controlled study (Corticus) has commenced. This study aims to evaluate adrenal function in patients with septic shock and to assess the benefits of corticosteroid therapy in this population.
Summary
A short course of high-dose corticosteroid therapy is probably not useful in human sepsis. Prolonged moderate-dose corticosteroid therapy is probably beneficial in septic shock requiring catecholamines, reducing duration of catecholamine support and increasing survival. Optimal timing and dosage of steroids remain to be further assessed.
References
1 Lefering R, Neugebauer EA. Steroid controversy in sepsis and septic shock: a meta-analysis. Crit Care Med 1995;23:1294303. 2 Cronin L, Cook DJ, Carlet J et al. Corticosteroid treatment for sepsis: a critical appraisal and meta-analysis of the literature. Crit Care Med 1995;23: 14309. 3 Briegel J, Forst H, Hellinger H, Haller M. Contribution of cortisol deficiency to septic shock. Lancet 1991;338:5078. 4 Schneider AJ,Voerman HJ. Abrupt hemodynamic improvement in late septic shock with physiological doses of glucocorticoids. Intens Care Med 1991;17:4367. 5 Rothwell PM, Udwadia ZF, Lawler PG. Cortisol response to corticotropin and survival in septic shock. Lancet 1991;337:5823. 6 Sibbald W, Short A, Cohen MP, Wilson RF. Variations in adrenocortical responsiveness during severe bacterial infections. Ann Surg 1977;186:2933. 7 Bouachour G, Roy PM, Guiraud MP. The repetitive short corticotropin stimulation test in patients with septic shock. Ann Intern Med 1995;123:9623. 8 Bollaert PE, Charpentier C, Levy B, Debouverie M, Audibert G, Larcan A. Reversal of late septic shock with supraphysiologic doses of hydrocortisone. Crit Care Med 1998;26:64550. 9 Annane D, Bellissant E, Sebille V et al. Impaired pressor sensitivity to noradrenaline in septic shock patients with and without impaired adrenal function reserve. Br J Clin Pharmacol 1998;46:58997. 10 Szabo C, Thiemermann C, Wu CC, Perretti M, Vane JR. Attenuation of the induction of nitric oxide synthase by endogenous glucocorticoids accounts for endotoxin tolerance in vivo. Proc Natl Acad Sci USA 1994;91:2715. 11 Barber AE, Coyle SM, Marano MA et al. Glucocorticoid therapy alters hormonal and cytokine responses to endotoxin in man. J Immunol 1993;150:19992006.
97
12 Briegel J, Forst H, Haller M et al. Stress doses of hydrocortisone reverse hyperdynamic septic shock: a prospective, randomized, double-blind, single-center study. Crit Care Med 1999;27:72332. 13 Annane D, Sebille V, Charpentier C et al. The effect of a seven-day treatment with low doses of hydrocortisone and fludrocortisone on mortality in patients with septic shock. J Am Med Assoc 2002;288:86271.
98
Index
Page numbers in bold type refer to figures; those in italic refer to tables or boxed material. 2-macroglobulin, MMP inhibitor 82 acquired immune response 65 activation-induced cell death 74 active apoptosis 745 acute inflammatory response 87 acute respiratory distress syndrome (ARDS) 13, 58 adamalysins 82 ADAM family 823 ADAMTS, ADAMs with thrombospondin 83 adaptive immunity 66 adenosine, anti-inflammatory agent 56 adenoviruses 37, 42 adhesion molecules blockade of 602 disorders of 56 expression down-regulated 56 expression stimulated 54, 70 neutrophils and 4951, 62 see also ICAM; Ig superfamily (IGSF); integrins; VCAMs adrenal insufficiency (relative) 935, 97 AIDS 29, 44 AIF (apoptosis-inducing factor) 28 airway disease 70 allergic reactions 70 alpha-melanocyte stimulating hormone 9 ANCAs (antineutrophil cytoplasmic antibodies) 5960 anti-adhesion molecule therapy 602 anti-apoptopic genes 23 antibodies antineutrophil cytoplasmic 5960 antiviral immunity 389, 412 host defence mechanism 34, 35, 3940 monoclonal 601 production 66, 70 antibody-dependent cell-mediated cytotoxicity (ADCC) 36 anti-CD3 antibodies 8, 9 antigen-driven apoptosis 745 antigen recognition 42, 668 antigen skin test anergy 5 anti-inflammatory cytokines 43, 55 antineutrophil cytoplasmic antibodies (ANCAs) 5960 antioxidant therapy 62 antiprotease deficiency 59 anti-sense nucleotides 60 Apaf-1 (apoptosis activating factor) 234, 25, 28, 29 Apaf-3 (apoptosis activating factor) 28 apoptosis active 745 antigen-driven 745 cell death activation-induced cell death 74 programmed, apoptosis 1831 genetics of 223 inflammation and 1831 necrosis compared 1921 of neutrophils 4 passive 745 in sepsis 735 signalling pathways 246, 834 apoptosis activating factors (Apafs) 223, 25, 28, 29 apoptosis-inducing factor (AIF) 28 apoptosome 24 asthma 61, 70 atherosclerosis 59 atopic reactions 70 atypical ANCA 60 autoimmune response 70, 85 2 integrin 58 bacteria 7, 49, 57, 69 bacterial meningitis 867 batimastat (BB-94) 86 BAX gene 29 BAX protein 24
99
INDEX
BB-94 (batimastat) 86 BB-1101 867 B cell malignancies 29, 44 B cells 445, 66, 70, 72 antibody production 34, 39 antigen recognition 67 BCL-2 genes 23, 24, 28 Bcl-XL protein 24, 28 BCRF1, IL-10 homologue 43 blood-borne infection 34, 38 bloodbrain barrier disruption 847 B lymphocytes see B cells brain tissue injury 58 Burkitts lymphoma 28, 44 burns 1, 5, 589, 75 Caenorhabditis elegans, genetics of apoptosis 223 cAMP, with immunosuppressive mediators 56 cancer apoptosis inhibition in 29 B cell malignancies 28, 44 EpsteinBarr virus-induced 28, 44 IFN therapy 37 MMPs in 82, 847 primed neutrophils in 59 cardiovascular response to hydrocortisone 934 CARS (compensatory antiinflammatory response syndrome) 1, 65 caspases 223, 248, 734 caspase 1 (interleukin 1- converting enzyme ICE) 23 caspase 3 30 caspase 8 256 caspase 9 24 catecholamine-dependent septic shock 907 catecholamines, endogenous 9 CD4+ Th cells 25, 38, 678 in HIV/AIDS 29, 40 CD8+ Tc cells 678, 74 CD34 (cluster of differentiation) 52 CD95 (cell-surface receptor) 25 ced genes 23 cell adhesion molecules 4953, 58 cell death genes 223 see also apoptosis; death cell-mediated immunity 5, 7, 3940, 679
100
cell membrane death domain 256 central nervous system (CNS) 847, 91 see also neuromediators; neurones cervical cancer 289 chemokines 4, 54, 84 chlorinated oxidants 59 chronic inflammatory conditions 601 ChurgStrauss syndrome 60 cluster of differentiation (CD34) 52 colon cancer 29 compensatory anti-inflammatory response syndrome (CARS) 1, 65 complement, antiviral defence 34, 35, 38, 412, 53 complement cascade 39, 42 complement-fixing antibodies 38, 70 complement fragments (C5a) 54 complement receptors 53 concanavalin A (ConA) 8, 9 corticosteroids endogenous 56, 79, 80 therapy 957 corticotrophin stimulation tests 93, 94 Crohns disease 60 CTLs see Tc (cytotoxic) cells cyclosporine 71 cytochrome c 18, 20, 24, 28 cytokine homologues, viral 43 cytokines 56, 6970 anti-inflammatory 68, 43, 55 antiviral defences 34, 35, 38, 39, 43 death cytokines 74 glucocorticoids and 556, 945 MMPs regulated by 7980 neutrophils regulated by 546 pro-inflammatory 6, 78, 6970, 91 pyrogenic 54 cytomegaloviruses 39, 42, 43 cytoplasmic constituents of neutrophils 5960 cytosol, apoptosome in 24 cytotoxic activity of neutrophils 556 cytotoxic chemotherapy 22 cytotoxic T cells see Tc (cytotoxic) cells death, see cell death death activators 234 death cytokines 74 death domain (DD) 256 death effector domain (DED) 26 death receptors 73 delayed hypersensitivity 45 demyelinating diseases 846
INDEX
dendritic cells (DC) 689 dengue virus 39 desensitising agents, in monocyte hyporesponsiveness 910 diabetes (type 1), autoimmune 70 diapedesis, leukocytes in 51, 54, 62 diffuse fine granular cytoplasmic fluorescence (cANCA) 59 disease, apoptosis in 2930 DNA damage, apoptosis-induced by 22, 245 DNA viruses 36 E6 protein 28 effector caspases 23, 256 effector protease cascade 25 effector response 66 elastase release 55 emphysema 59 endogenous inhibitors of MMPs 82 endoproteinases, zinc-containing see matrix metalloproteinases (MMPs) endothelial cells 53, 54, 58 endotoxin-elicited cytoxines 94 endotoxin neutralising molecules 10 endotoxin tolerance phenomenon 1011 epithelial cells, viral infections 33, 35, 44, 45 EpsteinBarr virus (EBV) 28, 35, 41, 435 ERM, transcription factor 72 E-selectin 50, 523, 58 experimental autoimmune encephalomyelitis (EAE) 85, 86 Fas 256, 734 Fas-associated death domain (FADD) 256, 73 Fas cell-surface receptor (CD95) 25 Fas-L in apoptosis 25, 26, 745, 84 binding (inactivation) 29 in immune privilege 29 in MS 85 fever, antiviral defence 356 firm adhesion, for leukocytes 51, 62 fucoidin 58 fucose, disordered synthesis of 56 fucosyl transferase inhibitors 60, 62 gastric secretions in antiviral defence 356
GATA-3 transcription factor 72, 73 genes apoptosis regulation by 223 arrangement in T cell development 66 transcription, glucocorticoids in 92 gene transcription factors in inflammation 1112, 92 in nave T cell polarisation 712 gene transcription inhibitors 60, 62 glandular fever 44 glomerulonephritis 59, 60 glucocorticoid receptors (GRs) 56, 92 glucocorticoid response elements (GREs) 56, 92 glucocorticoids endogenous 10, 556, 74 therapeutic 71, 907 GlyCAM-1, cell adhesion molecules 52 glycomimetics 60, 612 glycoproteins 35 see also integrins granule signalling pathway 734 granulocyte/macrophage colony stimulating factor (GM-CSF) 54 granzymes 734 growth factors 55, 75 see also PDGF; TGFGuillainBarr syndrome (GBS) 856 haemorrhagic shock 9 helper T cells see Th (helper) cells hematopoietic cytokine 70 hemorrhagic shock syndrome 39 heparan sulphate proteoglycans binding 82 herpes simplex virus (HSV) 34, 39, 401, 42 HIV 35, 40 HLA-DR expression 2, 3 Hodgkins disease 44, 59 hormones, in inflammation 556 host cytokines, viral targeting of 43 host defence mechanism 3940, 489 host immunity interaction 3345 host tissue damage 578 HSV (herpes simplex virus) 34, 39, 401, 42 human papilloma viruses (HPV) 28 humoral immune system 34, 35 hypersensitivity, delayed in immune depression 45
101
INDEX
hypertension, primed neutrophils in 59 hyporeactivity phenomenon 114 hypothalamus-pituitary-adrenal axis 10, 91 I B, NF B inhibitory proteins 11, 12 ICAMs (intercellular adhesion molecules) 53, 58 ICAM-1 50, 601 ICAM-1 antibodies 61 IFN antiviral defences 368 therapy 37 IFN- 7, 545, 6970, 71 IFN- receptors 43 IFN responsive element 37 Ig class-switching 70 complement-fixing antibodies 70 superfamily (IGSF) 534 IgA antibodies 3840 IgE synthesis 72 IgG antibodies 36, 389 IgM antibodies 389 IL-1 in antiviral defence 38 production 59, 54 IL-1 5, 7 IL-1 MMPs regulated by 79, 80 production depressed 5, 6, 7, 89 IL-1 converting enzyme (ICE) 23 IL-1 receptor antagonist (IL-1ra) 89 IL-1 receptor-associated kinase (IRAK) pathway 12 2 IL-1 receptor (IL-1r2) 8 IL-2 in apoptosis 745 in nave T cell activation 71 production 5, 8, 9, 12, 6970 IL-4 6970, 712 IL-5 8, 9, 6970 IL-6 in antiviral defence 38 production 6, 7, 54 response to LPS 945 IL-6 receptors 84 IL-8 8, 54 IL-9 6970 IL-10 monocyte deactivator 9 production 8, 9, 6970, 72 IL-10 homologue 43 IL-12 7
102
IL-12IFN- pathway 7 IL-13 6970 IL-18 27 immune adherence 38, 39 immune dysfunction, measures of 29 immune evasion 403 immune privilege 25, 29 immune recognition, inhibition of 42 immune response acquired 65 ADAMTS in 83 innate 65 in viral infections 3345 immune system Fas expression and 25 humoral 34, 35 innate 34 T cell 6576 immunity adaptive 66 apoptosis in 1831 cellular 7 depression of 12 specific 389 immunocompromised patients 445 see also AIDS; HIV; transplant patients immunoglobulin see Ig immunoparalysis 114 immunopathological damage 39 immunosuppression endogenous (immune paralysis) 114 virus-induced 40 immunosuppressive mediators 556 infection latent or persistent 401 leukocyte migration in 84 inflammation anti-adhesion molecule therapy 602 apoptosis and 1831 MMPs 79, 834 neutrophils 8, 57 non-infectious 8 steroid therapy 90, 913 inflammatory caspases 223, 26 inflammatory diseases 589, 601, 847 inflammatory foci 13 inflammatory responses 12, 4763, 87 inflammatory signals 4950 influenza virus 33, 35, 49 inhibitor caspases 26 inhibitory proteins 1112, 92 initiator caspases 223, 256
INDEX
innate immunity 34, 65 insulin-like growth factor (IGF) 55 integrin inhibitors 60, 61 integrins 53, 54, 58 intercellular cell adhesion molecules (ICAMs) 50, 53 interferon see IFN interleukin see IL ischaemia-reperfusion injury 55, 578, 61 ischaemic stroke 58 killer T cells see Tc (cytotoxic) cells kinase activity 12 latent infection 401, 435 latent proteins 44 leukaemia, B-cell 29 leukocyte adhesion cascade 48 leukocyte adhesion deficiency (LAD) 56 leukocytes blood vessel injury by 58, 62 hyporeactivity 7, 9, 13, 14 migration 84 rolling 50, 51, 523, 62 see also neutrophils leukotrienes 55, 56 lipid peroxides 30 lipids, bioactive 55 lipopolysaccharide see LPS lipoproteins 10 low density (LPL) 59 lipoxins 55 LPS cytokine production reduced in sepsis 7, 89 stimulation 5, 6, 13 IFN induction by 36 MMPs regulation by 80, 81 priming agent 54 tolerance to 10, 1112 glucocorticoid in 945 LPS binding protein (LBP) 10 LPS-induced signalling 10 L-selectin 50, 523, 58 lung cancer 29 lymph, viral dissemination 34 lymphocyte-derived cytokines 78 lymphocyte function-associated antigen-1 (LFA-1) 53 lymphocytes granular 36 IL-2 production 5
MMPs expressed by 7980 proliferation in immune dysfunction 2, 4 self-killing 25 viral infection 40, 41 see also B cells; T cells lymphocytic choriomeningitis virus 39 lymphoid tissue, nave T cells in 67 lymphokine withdrawal 745 lymphomas 289, 44 lymphopenia 74 macrophage inflammatory protein-1 (MIP-1) 55 macrophages antiviral defences 34, 35, 36, 37, 38 endotoxin tolerance 10, 1112 inflammatory foci 13 LDL uptake 59 MMPs expressed by 7980 MadCAM (mucin-like glycoprotein) 50, 52 major histocompatability complex see MHC malignancy see cancer MAPK p38 12 matrix metalloproteinases see MMPs matrix proteins 77, 789 measles 39, 40 melanoma 29 membrane proteins, virus infection 43 metalloproteinase inhibitors 60, 61 metzincin superfamily, adamalysins 82 MHC class I 34, 38, 42, 678 MHC class I ligands 67 MHC class II 38, 678 MHC class II ligands 67 microvascular see vascular mitochondria 24, 28, 30 mitochondrial membrane 18, 20, 28, 30 MMP-2 79, 80 MMP-9 79, 80 in acute inflammatory response 87 in bacterial meningitis 867 in demyelinating diseases 86 MMPs cellular production 7980 in inflammation 7787 regulation 812 therapeutic tools 847 monoclonal antibodies 601 monocyte chemoatractant protein 1 (MCP-1) 54
103
INDEX
monocytes cytokine-derived 5, 7 hyporesponsiveness 912 immune dysfunction and 2, 3 in innate immune system 34 mucous, anti-virus barrier 35 multiorgan dysfunction syndrome 65 multiple sclerosis 70, 845 myxoma virus 43 natural killer (NK) cells 4, 346, 38, 69, 73 nave T cells 667, 689, 71 necrosis 1819 apoptosis compared 1921 necrotic cell death 30 nerves, viral dissemination 34 neuroendocrine system, and immune response 556 neuromediators, as immunosuppressors 9 neurones HSV latent infection 401 see also central nervous system (CNS) neutrophil inhibitory factor (NIF), anti-inflammation therapy 61 neutrophils 8, 478, 546 adhesion, ischaemic stroke 58 benefits and harm 578 cytotoxic activity 556 function altered in immune dysfunction 4 regulation of 4956 host tissue damage 578 inflammatory response 4763 hyporeactivity 8 inflammatory diseases 589 MMPs expressed by 79 NF B (nuclear factor B) 1112, 13, 42, 54, 56 NK (natural killer) cells 4, 346, 38, 69, 73 norepinephrine 934 nosocomial infections 1, 14 nuclear factor B (NF B) 1112, 13, 42, 54, 56 nuclear proteins 44 oligosaccharides 60, 61 opioids, immunosuppressive 556 opsonins 38 oral hairy leukoplakia 44 organ transplantation, apoptosis in 29
104
orthomyxovirus 35 oxidative burst 556, 70 oxidative stress 30, 59 oxygen consumption, in phagocytosis 49 oxygen delivery in sepsis 19, 30 oxygen-derived radicals 30, 49 p53, apoptosis promoter 22, 28 p59fyn phosphorylation 12 PAF (platelet activating factor) 54, 55 paramyxoviruses 35, 39 parasites, intracellular pathogens 69 passive apoptosis 745 PDGF (platelet-derived growth factor) 80, 81 perforin, membrane damage by 70, 74 perinuclear fluorescence (pANCA) 60 peritonitis 13 permeability transition 28 pH, low in gastric secretions 356 phagocytes, in apoptosis 18, 19, 21 phagocytosis cytokines in, in sepsis 7 IFN- in 70 neutrophils in 489, 53, 556, 62 of viruses 38, 39 phorbol ester 80, 81 phosphatidylinositol 3kinasedependent signalling pathway 12 phytohaemagglutinin (PHA) 8, 9 pituitary adenylate cyclase-activating polypeptide 9 plasma membrane, in apoptosis 18, 20 platelet activating factor (PAF) 54, 55 platelet-derived growth factor (PDGF) 80, 81 platelet-endothelial cell adhesion molecule (PECAM-1) 50, 534 platelets, adhesion to neutrophils 52, 56 polyarteritis 60 poxviruses 37, 42, 43 pro-apoptopic genes 23 programmed cell death 1831 see also apoptosis pro-inflammatory cytokines 6, 78, 6970, 91 pro-inflammatory state see systemic inflammatory response syndrome (SIRS) prolactin 55 prostaglandin 10
INDEX
proteases effector cascade 25 inhibitors 57 see also caspases protein synthesis inhibition pathway 43 P-selectin 50, 523, 58 P-selectin glycoprotein ligand-1 (PSGL-1) 60, 612 pyrogenic cytokines 54 reactive oxygen species 49, 59 regulatory T cells see Th (helper) cells Rel proteins 1112 respiratory burst 49 respiratory syncytial virus 39 rheumatoid arthritis 59 rhinovirus 35 RNA viruses 367, 43 double-stranded 36 rolling leukocytes 50, 51, 523, 62 selectins 50, 523, 58, 61 antibodies to 61 inhibitor 58 ligands for 50, 523 sepsis anti-inflammation therapy 62 antioxidant therapy 62 apoptosis in 2930, 735 inflammatory response 1, 3, 69, 10, 1112 lymphopenia in 74 neutrophils in 58, 59 T cell immune system and 6576 sepsis syndrome 7, 8 septic shock 4, 9, 87 glucocorticoid therapy 907 serine protease inhibitor (SERPIN) family 59 serum hepatitis virus 39 shock haemorrhagic 9 non-infectious 7 see also septic shock sialic acid-containing glycoproteins 35 sialyl Lewisx (sLex) 50, 53 signalling pathways altered in immunodepression 9, 12 in apoptosis 234, 256 for death receptors 73 signal transducer and activator of transcriptions (STATs) 712 SIRS (systemic inflammatory response syndrome) 114, 65
small molecule inhibitors 60, 61 snake venom metalloproteases (SVMPs) 82 steroid therapy, in sepsis 907 stress-induced mediators 4 superoxide generation 55, 58 surgery, inflammatory response 1, 56, 8 synovial fluid 59 systemic inflammatory response syndrome (SIRS) 114, 65 systemic vasculitis 59 T-bet, transcription factor 72 T cells 669 apoptosis of 745 immune system dysfunction 8, 12, 65 sepsis 6576 specific immunity 39 MMPs regulated by 7980 nave 667, 689, 71 Tc (cytotoxic) cells 734 apoptosis induced by 22 blocked in cancer 29 immune system cell-mediated 679 specific immunity 39, 40 virus recognition 22, 34, 35, 38, 41, 42 T cell antigen-specific receptors (TCR) 667 T cell receptors 66 TGF (transforming growth factor ) 9, 80, 81, 856 Th (helper) cells 6973 AIDS-induced loss 29 IL-2 produced by 5 immune system CTL system 34, 35, 39 humoral immunity 679 specific immunity 39 Th1 (helper) cells 78, 6972 Th2 (helper) cells 78, 6972 thermal injury neutrophils in 589 see also burns thrombospondin, with ADAM (ADAMTS) 83 thymocyte proliferation 83 tissue inhibitors of MMPs (TIMPs) 77, 79, 80, 82, 867 TLR4, LPS receptor 10 TNF, apoptosis induced by 26
105
INDEX
TNF 734 antiviral defence 38 in apoptosis 73, 745 death activator 25 endotoxin-elicited production 945 in inflammation 13, 58, 856 MMP regulator 7980, 834 neutrophil production of 54 priming agent 54 sepsis-induced changes 7 surgery-induced changes 5, 6 TNF converting enzyme (TACE) 83 TNF receptors 43 TNF 6970 TNF receptor-associated death domain (TRADD) 26 TNF receptors (TNFRs) 834 TNF-related apoptosis-inducing ligand (TRAIL) 73 tolerance against potential antibody 25 endotoxin tolerance phenomenon 1011 toll-like receptors (TLR) 1011 transcription see gene transcription transforming growth factor (TGF ) 9, 80, 856 transmembrane receptor protein see Fas transplant patients, viral infection 445 trauma inflammatory response 1, 3, 4, 8 NF B expression in 1112
passive apoptosis in 75 see also surgery tumour necrosis factor see TNF tumours 59, 67, 79, 80 vaccine development 3940 vaccinia virus 33, 42 vaccinia virus complement control protein (VCP) 42 varicella-zoster virus 34 vascular cell adhesion molecules (VCAMs) 50, 53, 58 vascular system inflammation 4951, 62 microvascular leakage 55, 70 therapeutic blockade 58 vasoactive intestinal peptide 9 vasoconstrictor leukotrienes 56 vasopressor therapy 967 VCAMs, vascular cell adhesion molecules 50, 53, 58 viraemia 39 virulence, host v viral factors 34 viruses dissemination 34 host immunity 22, 3345, 49 intracellular pathogens 412, 68, 69 Wegeners granulomatosis 59 yeasts, pathogens 69 zinc-containing endoproteinases see MMPs zymogens, MMP proforms 77, 79, 81
106

Galley - Inflammation and Immunity

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Galley - Inflammation and Immunity

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Critical Care Focus 10: Inflammation and Immunity

EDITOR DR. HELEN F. GALLEY

Critical Care Focus 10: Inflammation and Immunity

Critical Care Focus 10: Inflammation and Immunity

Contributors Preface Introduction 1 Immunoparalysis JEAN-MARC CAVAILLON, HELEN

2 Apoptosis and the inflammatory process

3 Virus interaction with host immunity

4 The double-edged role of the neutrophil in inflammatory responses

5 T cell immunity and sepsis

6 Metalloproteinases and inflammation

7 Glucocorticoid therapy in septic shock PIERRE-EDOUARD BOLLAERT Index

Critical Care Focus series

Preface to the Critical Care Focus series

CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY

Measures of immune dysfunction

80 uneventful recovery %HLA-DR positive monocytes major sepsis 60

CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY

CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY

Characterisation of the ex vivo cytokine production in sepsis and SIRS

CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY

Mechanisms of hyporesponsiveness of monocytes

CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY

CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY

Cells derived from inflammatory foci

CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY

CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY

2: Apoptosis and the inflammatory process

APOPTOSIS AND THE INFLAMMATORY PROCESS

The process of apoptosis

Apoptosis versus necrosis

CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY

APOPTOSIS AND THE INFLAMMATORY PROCESS

CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY

Why do we need apoptosis?

Why can apoptosis be a problem?

Caenorhabditis elegans and the genetics of apoptosis

APOPTOSIS AND THE INFLAMMATORY PROCESS

Pro-apoptotic ced-3 ced-4 Anti-apoptotic ced-9

caspase-1(ICE) Apaf-1 BCL-2 gene family

Signals for apoptosis

CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY

APOPTOSIS AND THE INFLAMMATORY PROCESS

CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY

Caspase 8 FLIP NFB activation

m Cytochrome c Bad, Bax

APOPTOSIS AND THE INFLAMMATORY PROCESS

CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY

The role of mitochondria in apoptosis

Apoptosis and disease

APOPTOSIS AND THE INFLAMMATORY PROCESS

CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY

Exaggerated inflammatory response

Ischaemic/ necrotic versus Apoptotic cell death

APOPTOSIS AND THE INFLAMMATORY PROCESS

CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY

3: Virus interaction with host immunity

Viral spread in host

CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY

Virulence host versus viral factors

Host defence against viral infection

VIRUS INTERACTION WITH HOST IMMUNITY

Cell-mediated immune responses

CRITICAL CARE FOCUS 10: INFLAMMATION AND IMMUNITY