Galley - Inflammation and Immunity

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Critical Care Focus10: Inflammation and

Immunity

EDITOR

DR. HELEN F. GALLEY

BMJ Books


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Critical Care Focus

10: Inflammation and Immunity


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Critical Care Focus

10: Inflammation and Immunity

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


DR PAUL G P LAWLER

Clinical Director of Intensive Care


DR NEIL SONI

Consultant in Anaesthesia and Intensive Care

Chelsea and Westminster Hospital

DR MERVYN SINGER

Reader in Intensive Care

University College Hospital, London


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BMJ Publishing Group 2003BMJ Books is an imprint of the BMJ Publishing Group

All rights reserved. No part of this publication may be reproduced, stored in aretrieval system, or transmitted, in any form or by any means, electronic,

mechanical, photocopying, recording and/or otherwise, without the prior writtenpermission of the publishers.

First published in 2003by BMJ Books, BMA House,Tavistock Square,

London WC1H 9JR

www.bmjbooks.comwww.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


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v

Contents

Contributors vii

Preface viii

Introduction ix

1 Immunoparalysis 1

JEAN-MARC CAVAILLON, HELEN F GALLEY

2 Apoptosis and the inflammatory process 18NIGEL R WEBSTER

3 Virus interaction with host immunity 33

LAWRENCE S YOUNG

4 The double-edged role of the neutrophil in inflammatory

responses 47

PAUL G HELLEWELL

5 T cell immunity and sepsis 65

EGBERT PRAVINKUMAR

6 Metalloproteinases and inflammation 77

ANDREW J GEARING

7 Glucocorticoid therapy in septic shock 90

PIERRE-EDOUARD BOLLAERT

Index 99


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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.


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Contributors

Pierre-Edouard BollaertProfesseur 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 PravinkumarLecturer 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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viii

Preface to the Critical Care

Focus series

The Critical Care Focus series aims to provide a snapshot of currentthoughts 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


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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 paralysisled 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 theplasma 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 inthe 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 thekiller 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 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.

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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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 andnecrosis. 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 cleaveprotein 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 combinationof 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.

INTRODUCTION


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1: ImmunoparalysisJEAN-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 under-

stood. 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, andbacterial 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 anti-

inflammatory 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


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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).


Figure 1.2 Approaches to monitoring immune status resulting from trauma, haemorrhage, burns,surgery, or sepsis.

IMMUNE STATUS

Immune suppression

Lymphocyte

proliferation

Anergy to skin

test antigens

Natural killer

cell activity Cytokineproduction

Lymphocyte/monocyte

population changes

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.


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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 wereretrospectively 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.Thisstudy 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

IMMUNOPARALYSIS

uneventful recovery

major sepsis

death

80

60

40

%HLA-DRpo

sitivemonocytes

20

Days after injury

1 3 6 9 12 15 18

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 Met al.5


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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 andmitogens, 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 andtrauma 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 stress-

induced 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 activityin 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 patientswith 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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colleagues19 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 significantlyhigher 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.

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 non-

septic 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 undergoingabdominal 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

IMMUNOPARALYSIS


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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).


ng/ml

TNF IL-1

10

8

6

4

2

0

ng/ml

15

12

9

6

3

0pre post 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 withpermission 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 anti-

inflammatory cytokines. Cytokines are commonly classified in one or theother 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,


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IFN, and transforming growth factor- (TGF) are recognised as anti-

inflammatory 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 influencecytokine 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 Monocyteproduction 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 reflectpotential 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 incritically ill patients. IL-12 enhanced the secretion of IFN in healthy

subjects, but was ineffective in critically ill patients. Although the anti-

inflammatory 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

IMMUNOPARALYSIS


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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 severelyinjured 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 non-

infectious inflammation (patients undergoing cardiac surgery with

cardiopulmonary bypass).30

Cytokine release after activation of lymphocyteswith 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 vitroexperiments 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



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blood assays in meningococcal infection,33 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.

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 productionby 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

IMMUNOPARALYSIS


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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 reflectionof 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 pro-

inflammatory 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 endotoxin-

tolerant macrophages,50,51 it was of interest to investigate the expression ofthis 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 LPS-

induced IL-1ra and IL-10. This latter observation suggests that the defect

may occur at the different signalling pathways within the cell rather than atthe 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 (NFB) 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 tightlycontrolled by a group of inhibitory proteins (IB), which maintain NFB in

an inhibited state in the cytoplasm of effector cells.The sequence of events

leading to NFB activation involves phosphorylation, ubiquitination, and

proteolysis of IB, allowing exposure of a nuclear recognition site. NFB

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). NFB,

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 NFB is the p65 unit, whereas the p50 unit has

shown no or minimal activation capacity.

To investigate the role of NFB 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 NFB 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

NFB available for activation and inhibit transcription of NFB-dependent

genes. On the other hand, Ziegler-Heitbrock and colleagues demonstratedthat 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 NFB 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. Subsequentin vitro stimulation of mononuclear cells with LPS did not induce further

NFB 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,

P 0.04). The reduced expression of nuclear NFB was not due to its

inhibition by IB since very low expression of IB as well as low levels ofp65 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 NFB expression that

resemble those reported during LPS-tolerance: global downregulation of

NFB 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 heterodimersand 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 IB was higher. Although no direct

correlation was found between levels of IL-10 or TGF and NFB, these

immunosuppressive cytokines were significantly elevated in the trauma

patients by 10 days after admission. This long-term low basal and LPS-

induced activation of NFB 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 anda 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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IL-2 production and proliferation,57 whilst activation of MAPK p38 was

associated with T cell immune dysfunction.58

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 initialvalues. 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 NFB,

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 NFB in alveolar

macrophages observed in patients with ARDS had increased compared with

control patients, but there was no increase in the activation of the othertranscription 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

NFB 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, theproduction 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

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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 subsequentnosocomial 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, NFB activity within leukocytes reflects cellular

hyporeactivity. Thus the immunoparalysis reported in sepsis and SIRS

patients, often revealed by a diminished capacity of leukocytes to respondto LPS, is not a generalised phenomenon, and SIRS is associated with a

compartmentalised responsiveness involving either anergic or primed cells.

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21 Cavaillon J-M. Possibilities and problems of cytokine measurements. In RedlH, 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. AnnSurg1984;200:31120.

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25 Munoz C, Carlet J, Fitting C, Misset B, Bleriot JP, Cavaillon J-M. Dysregulationof in vitro cytokine production by monocytes during sepsis. J Clin Invest1991;88:174754.

26 Ertel W, Keel M, Neidhardt Ret al. Inhibition of the defense system stimulating

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27 OSullivan ST, Lederer, JA, Horgan AF, Chin DHL, Mannick JA, Rodrick ML.Major injury leads to predominance of the T helper-2 lymphocyte phenotypeand diminished interleukin-12 production associated with decreased resistanceto infection.Ann Surg1995;222:48292.

28 Mack VE, McCarter MD, Naana HA, Calvano SE, Daly J-M. Dominance of

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30 Muret J, Marie C, Fitting C, Payen D, Cavaillon J-M. Ex vivo T-lymphocytederived cytokine production in SIRS patients is influenced by experimentalprocedures. 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 Invest1993;91:85361.

32 Marie C, Muret J, Fitting C, Losser MR, Payen D, Cavaillon J-M. Reducedex vivo interleukin-8 production by neutrophils in septic and nonseptic systemicinflammatory response syndrome. Blood1998;91:343946.

33 Van Deuren M,Van Der Ven-Jongekrijg H, Demacker PNM et al. Differentialexpression of proinflammatory cytokines and their inhibitors during the courseof meningococcal infections.J Infect Dis 1994;169:15761.

34 Marie C, Muret J, Fitting C, Payen D, Cavaillon J-M. Interleukin-1 receptorantagonist production during infectious and non-infectious systemicinflammatory response syndrome. Crit Care Med2000;28:227783.

35 Brandtzaeg P, Osnes L, vsteb R, Jo GB, Westwik AB, Kierulf P. Netinflammatory capacity of human septic shock plasma evaluated by a monocyte-based target cell assay: identification of interleukin-10 as a major functionaldeactivator of human monocytes.J Exp Med1996;184:5160.

36 Ayala A, Meldrum DR, Perrin MM, Chaudry IH.The release of transforming

growth factor-beta following haemorrhage: its role as a mediator of hostimmunosuppression. Immunology 1993;79:47984.

37 Ayala A, Knotts JB, Ertel W et al. Role of interleukin 6 and transforming growthfactor-beta in the induction of depressed splenocyte responses following sepsis.

Arch Surg1993;128:8994.38 Jones S, Romano F. Dose- and time-dependent changes in plasma catecholamines

in response to endotoxin in conscious rats. Circ Shock 1989;28:5968.39 Severn A, Rapson NT, Hunter CA, Liew FY. Regulation of tumor necrosis

factor production by adrenaline and by -adrenergic agonists. J Immunol1992;148:34415.

40 van der Poll T, Coyle SM, Barbosa K, Braxton CC, Lowry SF. Epinephrine

inhibits tumor necrosis factor-alpha and potentiates interleukin-10 productionduring human endotoxemia.J Clin Invest1996;97:71319.

41 Luger TA, Kalden DH, Scholzen TE, Brzoska T. -melanocyte-stimulatinghormone as a mediator of tolerance induction. Pathobiology 1999;67:31821.

42 Delgado M, Pozo D, Martinez C et al.Vasoactive intestinal peptide and pituitaryadenylate cyclase-activating polypeptide inhibit endotoxin-induced TNFproduction by macrophages: in vitro and in vivo studies. J Immunol1999;162:235867.

43 Chrousos GP. The hypothalamicpituitaryadrenal axis and immune-mediatedinflammation.N Engl J Med1995;332:135162.

44 Choudhry MA, Ahmad S, Ahmed Z, Sayeed MM. Prostaglandin E2 down-

regulation of T cell IL-2 production is independent of IL-10 during Gramnegative sepsis. Immunol Lett1999;67:12530.

45 Zweigner J, Gramm HJ, Singer OC, Wegscheider K, Schumann RR. Highconcentration of lipopolysaccharide-binding protein in serum of patients withsevere sepsis or septic shock inhibit the lipopolysaccharide response in humanmonocytes. Blood2001;98:38008.

46 Vreugdenhil ACE, Snoeck AMP, vant Veer C, Greve JWM, Buurman WA.LPS-binding protein circulates in association with apoB-containing lipoproteinsand enhances endotoxin-LDL/VLDL interaction. J Clin Invest 2001;107:22534.

47 Cavaillon J-M, Fitting C, Haeffner-Cavaillon N, Kirsch J, Warren HS.

Cytokine response by monocytes/macrophages to free and lipoprotein-bound lipopolysaccharide. Infect Immun 1990;58:237582.

48 Kitchens RL,Thompson PA,Viriyakosol S, OKeefe GE, Munford RS. PlasmaCD14 decreases monocyte responses to LPS by transferring cell-bound LPS toplasma lipoproteins.J Clin Invest2001;108:48593.



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49 Cavaillon J-M.The non specific nature of endotoxin tolerance. Trends Microbiol1995;3:3204.

50 Nomura F, Akashi S, Sakao Y et al. Endotoxin tolerance in mouse peritonealmacrophages correlates with down-regulation of surface Toll-like receptor 4expression.J Immunol2000;164:34769.

51 Medvedev AE, Kopydlowski KM, Vogel SN. Inhibition of lipopolysaccharide-induced signal transduction in endotoxin-tolerized mouse macrophages:dysregulation of cytokine, chemokine, and Toll-like receptor 2 and 4 geneexpression.J Immunol2000;164:556474.

52 Blackwell TS, Blackwell TR, Christman JW. Induction of endotoxin tolerancedepletes nuclear factor-kappaB and suppresses its activation in rat alveolar

macrophages.J Leukocyte Biol1997;62:88591.53 Ziegler-Heitbrock L, Wedel A, Schraut W et al. Tolerance to lipopolysaccharide

involves mobilization of nuclear factorB with predominance of p50homodimers.J Biol Chem 1994;269:170014.

54 Adib-Conquy M, Adrie C, Moine P et al. Nuclear factorB expression in

mononuclear cells of septic patients resembles that observed in LPS-tolerance.Am J Respir Crit Care Med2000;162:187783.

55 Adib-Conquy M, Asehnoune K, Moine P, Cavaillon J-M. Long term impaired

expression of nuclear factorB and IB in peripheral blood mononuclearcells of patients with major trauma.J Leukocyte Biol2001;70:308.

56 Learn CA, Boger MS, Li L, McCall CE. The phosphatidylinositol 3 kinasepathway selectively controls sIL-1ra not interleukin-1 production in the septicleukocytes.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 Immunol1998;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 Physiol2001;281:C6629.

59 Schwartz MD, Moore EE, Moore FA et al. Nuclear factor kappa B is activatedin alveolar macrophages from patients with acute respiratory distress syndrome.Crit Care Med1996;24:128592.

60 Moine P, McIntyre R, Schwartz MD et al. NF-kappaB regulatory mechanismsin 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 Invest1990;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 macrophagesfrom inflammatory bowel disease. Gastroenterol1997;112:1493505.

63 Rana N, Braun DP, House R, Gebel H, Rotman C, Dmowski WP. Basal andstimulated secretion of cytokines by peritoneal macrophages in women withendometriosis. Fertil Steril1996;65:92530.

64 Wu MY, Ho HN, Chen SU, Chao KH, Chen CD, Yang YS. Increase in theproduction of IL-6, IL-10 and IL-12 by LPS stimulated peritoneal macrophagesfrom women with endometriosis.Am J Reprod Immunol1999;41:10611.

65 Nssler NC, Stange B, Nussler AK et al. Upregulation of intraepithelial

lymphocyte function in the small intestinal mucosa in sepsis. Shock2001;16:4548.

66 Munford RS, Pugin J. Normal responses to injury prevent systemicinflammation and can be immunosuppressive. Am J Respir Crit Care Med2001;163:31621.

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2: Apoptosis and the

inflammatory processNIGEL 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 oftumours, 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


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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 andtherefore 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

APOPTOSIS AND THE INFLAMMATORY PROCESS

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


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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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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 necroticcells. 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.


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

A B

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.


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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 ofthe 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 whichcytotoxic 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 sometypes 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



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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 triggerapoptosis: 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-4Anti-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 continuedsurvival), 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 9activates 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).


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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 signalthe 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 CD4T helper lymphocytes results in expression of Fas-L.

During ablation of the immune repertoire in the fetal/neonatal period, self-

antigen 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. Sometumours 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-3homologue). Recruitment into the complex results in dimerisation of pro-

caspase-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.



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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 signalsto the nuclear factor B (NFB) 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

Death adaptor

ICE/

Caspase 1 Caspase 8

Caspase 3m

Bad, Bax

Cytochrome c

APOPTOSIS Nucleus

Bcl2/Bcl-XL

FADD TRADD TRAF 1

FLIPNFB

activation

FasL Apo3L TNF

DR4/

DR5

Fas/

CD95

DR3 TNF R1Free radicals

NO Steroids

+/

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 pro-

domain, 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 caspaserecruitment 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


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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

cytochrome c

Apaf-1

pro-caspase 9

pro-caspase 3 caspase 3

APOPTOSIS

caspase 9

(ced-4 equivalent)

mitochondrion

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 pro-

caspase 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 cytochromec

and AIF is downstream of BCL-2function 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 andinactivates 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, especiallylung 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 CD4T cells. However, fewer

than 1 in 100 000 CD4T 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 lymphnode), 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 fromhigh 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. Theimmune/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).


Hypermetabolic

Hyperdynamic

Hypometabolic

Hypodynamic

Exaggerated inflammatory

response

SEPSIS

Immune hyporesponsiveness

Ischaemic/

necrotic

Apoptotic

cell death

Documents

Galley - Inflammation and Immunity