Immunoparalysis after multiple trauma

Injury, Int. J. Care Injured (2007) 38, 1346—1357

www.elsevier.com/locate/injury

Immunoparalysis after multiple trauma Sven K. Tschoeke *, Wolfgang Ertel ´-University Hospitals Berlin, Department of Trauma and Reconstructive Surgery, Charite Campus Benjamin Franklin, 12203 Berlin, Germany Accepted 15 August 2007

KEYWORDS Immunosuppression; HO-1; Th17; T-regulatory cells; Apoptosis

Summary The immunological sequelae following multiple trauma constitute an ongoing challenge in critical care management. The overall immune response to multiple trauma is a multilevel complex interdependently involving neurohormonal, cellular and haemodynamic factors. Immunoparalysis is characterised by a reduced capacity to present antigens via downregulated HLA-DR and an unbalanced monocyte—T cell interaction. Trauma-induced death of functionally conducive immune cells in the early recovery phase is significant in the emergence of posttraumatic multiple organ dysfunction or failure. Novel findings may contribute to more appropriate immunomonitoring and improved treatment. We must consider the preservation and support of immune function as the ultimate therapeutic goal, which may override the current strategy of simply antagonising excessive pro- or anti-inflammatory immune responses of the severely injured person. This review focuses on the injury-induced conduct of key immune effector cells and associated effects promoting immunoparalysis after multiple trauma. # 2007 Elsevier Ltd. All rights reserved.

Introduction The immunological response to severe injury and multiple trauma remains a serious challenge in critical care management. Subsequent life-threatening posttraumatic complications are associated with overproduction of proinflammatory mediators (e.g. cytokines, chemokines) and the critical imbalance of cell-regulated innate immunity. In prolonged deregulated immune cell homeostasis, the immu* Corresponding author. E-mail address: [email protected] (S.K. Tschoeke).

nological sequelae commonly evolve a state of hyperinflammation or immunosuppression or both, ultimately leading to multiple organ dysfunction (MODS) or lethal failure (MOF).49 Although the hyperinflammatory response within the innate immune reaction to multiple trauma is not inevitable, it characteristically involves exaggerated release of proinflammatory mediators. These include interleukin (IL)-1, IL-6, IL-8 and IL-18, tumour necrosis factor (TNF)-a, neutrophil activation, microvascular adherence and an uncontrolled polymorphonuclear (PMN) and macrophage oxidative burst. Continuous IL-6 release induces an acute-phase response and, more importantly,

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Immunoparalysis after multiple trauma

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accounts for the upregulation of other major antiinflammatory mediators and cytokine inhibitors, such as prostaglandin (PG) E2, IL-1ra, IL-10 and transforming growth factor (TGF)-b.63 This possibly reflects a protective mechanism limiting further endogenous tissue damage. In immunosuppression, the production of anti-inflammatory mediators (IL4, IL-10) prevails and anergic immune cells subside.13,49 Data from both experimental and clinical research have repeatedly demonstrated that this dynamic process is further influenced by a variety of comorbid parameters, including injury-induced activation of the neuroendocrine system (e.g. glucocorticoids, catecholamines) and subsequent changes in microcirculation, coagulation and diverse metabolic sequences. These, in turn, not only network within the innate immune response, but are in themselves capable of critically affecting the prevailing inflammatory profile and consequently disease progression. The merging immunological influence on these events appears to be a polarisation of the primarily proinflammatory T helper-1 (Th1) immune response towards an

Table 1 Effects of multiple injury on immune cell activity Type of cell and action T and B lymphocytes Myelodepression Lymphopenia CD4+/CD8+ ratio TGF-b IL-17 producing Th17 phenotype Immunosuppressive Treg phenotype Monocytes/macrophages Immunocompetence and activation HLA-DR expression Antigen-presenting capacity PGE-2-mediated depression of immune function IL-12 production IFN-g

Effect on activity

Decrease Increase Increase Increase Decrease Decrease Decrease Increase Decrease Decrease

Common characteristics of T cells and monocytes Immune profile shift Th1 ! Th2 Trauma-induced apoptosis Increase Polymorphonuclear neutrophils Chemotaxis Phagocytic capacity Release of elastase Release of O2 radicals b-integrin expression Leukotrien B4 production Apoptosis

Decrease Decrease Increase Increase Decrease Decrease Delay

increased and counterinflammatory Th2 phenotype. The immune cell interaction and cytokine response are critically impaired, with subsequent immunoparalysis representing one of the main causes of posttraumatic complications. This review aims to elucidate the basic underlying mechanism of immunoparalysis after multiple trauma and the functional contribution of its key players (Table 1), focusing on a few crucial aspects which might be targeted in distinct therapeutic approaches.

The innate immune response to injury In the past decade, enormous research efforts have identified broadly distributed immune sensors recognising numerous antigenic stimuli, including those from viruses, bacteria, fungi and parasites, with considerable specificity. Among the key components striving to control any noxious agent capable of activating the host’s innate immune system are the antigen-presenting cells (APC), consisting of the immunocompetent T cells (CD4+ and CD8+), neutrophils, monocytes and macrophages, natural killer (NK) and dendritic cells (DC). However, activation of the immune system is not an inevitable response to pathogen-related stimuli. In severe trauma, APC are activated by a variety of endogenous danger signals, including oxygen free radicals, heat shock proteins (HSP), DNA adducts, diverse cellular damage-related molecules and inflammatory proteins (cytokines, chemokines). Upon recognition of and binding to a specific receptor, these APC initiated antigen-processing mechanisms ranging from phagocytosis or endocytosis of specific noxia to various downstream signalling events which stimulate the production of inflammatory mediators (Fig. 1). Cytokine production, however, has been shown to follow divergent pathways when triggered by a particular antigen.85 The signalling mechanisms underlying this diversity remain unclear, but the majority of receptors involved have been identified and investigated. IL-1 receptors and the transmembrane Toll-like receptors (TLRs) have both been proposed as critically important sensors on the surface of all major immune cells.2,3 In particular, TLR have been shown to recognise specific pathogenic antigens, including prokaryote-derived lipoproteins, glycolipids, flagellin, CpG DNA and lipopolysaccharides.3 Binding of TLR agonists to immune cells has not only been proven to induce a cellspecific cytokine response through numerous routes of ultimate nuclear factor-kB (NF-kB) activation, but also to affect the immune cells’ behaviour,

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Figure 1 Activated immunocompetent cells respond to multiple trauma with production of numerous inflammatory mediators. Stress-induced neurohormonal signalling, hepatogenic acute-phase proteins and complement activation impact on overall immune regulation, changing behaviour of immune cells. Unbalanced immune complex phenotype shifts towards predominant immunosuppressive periphery, evolving to immunoparalysis.

including migrating potential, differentiation and maturation, as well as their activation status.60,100 Severe trauma and associated blood loss have been shown to lead to decreased endotheliallyderived nitric oxide (NO), resulting in increased platelet aggregation,111 deregulated vasorelaxation33 and greater neutrophil infiltration.62 The consecutive increase of microvascular permeability and ultimate loss of endothelial integrity are exaggerated by a rise in adhesion molecules, including intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, P selectin and E selectin.36 Thus, systemic and compartmentalised innate immune effects may well be distinct and significantly dependent upon a number of physiological parameters, including haemodynamics,

oxygen saturation, lactate acidosis, composition of immune and inflammatory mediators, immune cellular interactions and subsequent tissue damage, hence determining the overall posttraumatic outcome.

T cell impairment and the role of T-regulatory cells after severe injury The specific nature of the T cell response is largely determined by the antigens expressed on the relevant cell surface, subsequent cellular interactions being controlled by signalling mechanisms and biochemical reactions. The naive CD4+ T cell (Th0) is a multipotential precursor with defined antigen

Immunoparalysis after multiple trauma recognition specificity but substantial plasticity for development down distinct effector or regulatory lineages, contingent upon signals from cells of the innate immune system. These non-committed Th0 cells are driven to further differentiation, depending on the predominant promoting cytokine composition, either into Th1-type cells by IL-12 or Th2type cells by IL-4.71 Th1-type cells have been shown to initiate and augment the delayed immune response by activating macrophages and neutrophils, promoting the production of opsonising antibodies and advancing the development of cytotoxic CD8+ T effector cells. The inflammatory mediators secreted include mostly IL-2, interferon (IFN)-g and TNF-a. In contrast, Th2-type cells promote the immediate immune response by inducing the production of non-opsonising antibodies such as immunoglobulin (Ig) E through the activation of eosinophil leukocytes and mast cells. Th1 cells represent the classical proinflammatory immune response, and Th2-type cells produce oppositional anti-inflammatory mediators, including IL-4, IL-5, IL-10, IL-13 and granulocyte macrophage colony-stimulating factor (GM-CSF).50 Interestingly, early studies investigating the effects of T cell signalling were able to demonstrate the reciprocal regulatory functions of these many mediators in respect of Th1 versus Th2 cell activity. It has since been acknowledged that IFNg produced by Th1 cells suppresses Th2 activity, and that IL-4 and IL-10 increase the Th1-immune response. In the setting of severe injury and related blood loss, suggested alterations of T cell immune functions range from changes in their subset proportions and level of activity to decreased proliferation. Hence, several observations of multiply injured patients have described the prominent immunocompromised state of deregulated T cell functions. This may be a result of cellular exhaustion and desensitisation promoted by changes of the CD4+/CD8+ T cell ratio and increased activity of immunosuppressive cytokines reflecting a Th2-type inflammatory profile.1,30,64 In this context, the concept of polarisation from an initial Th1-type to a dominant Th2-type innate immune response has become a major focus of the attempt to elucidate the diverse mechanisms underlying posttraumatic immunosuppression. This notion has provided the framework for understanding Tcell biology and the interplay between innate and adaptive immunity for almost two decades. Most recently, a CD4+ T cell subpopulation capable of producing IL-17 has been reported to amend the lineage of proinflammatory Th-cells, bringing new aspects to the concept of T cell-related immunosuppression. Although these cells appeared to

1349 express characteristic Th1-type properties, particular expressions appeared to be significantly distinct. These included CTLA-1, ICOS, programmed death ligand 1, CD153, Fas and TNF-related activationinduced cytokine, thus demonstrating Th17 to exhibit particular phenotypic and developmental differences from both Th1 and Th2-type lineages.72 Findings from mice depleted of IL-23, a functionally dispensable but essential inducing factor in Th17 cell differentiation, demonstrated a strong resistance to autoimmune encephalomyelitis (EAE) or collagen-induced arthritis (CIA) in correlation with the absence of IL-17-producing Th17 cells.34 In addition, Nakae et al. demonstrated that IL-23 or IL-17 was capable of suppressing Th1 cell differentiation in the presence of exogenous IL-12 in vitro.72 In further analyses, Th17 development and differentiation were found to be dependent upon IL-1, IL-6 and TGF-b.61,107 Moreover, TGF-b was shown to upregulate IL-23R expression, thereby conferring responsiveness to IL-23.61 In this context TGF-b, as a pivotal molecule within innate and adaptive immunity, has gained particular attention. Although beneficial in initiating and controlling immune responses and maintaining immune homeostasis, immunosuppressive pathways mediated by TGF-b may obscure immune surveillance mechanisms, resulting in failure to recognise or respond adequately to host, foreign or tumour-associated antigens. Recent data on immunoregulatory effects induced by TGF-b have thus revealed an interesting link between the CD4+ T cell and an additional immunosuppressive subset, the CD4+ CD25+ T regulatory lineage,8,19 as shown in Fig. 2. Tregs are distinguished from the conventional CD4+ CD25 T cells by the expression of CD45RBlow, L-selectin (CD62L)+, CD69, glucocorticoidinduced TNFR-like protein (GITR)-6, TGF-b1+, TLR4+ and the forkhead transcription factor (Foxp3) surface phenotype. Tregs have been shown to suppress inflammatory responses in vivo through mechanisms mediated by IL-10 and TGF-b. Previous reports demonstrated a marked elevation of human circulating CD4+ CD25+ Treg cells among immunoparalysed infected patients.69,103 Moreover, animal experiments investigating the inflammatory and immune responses to burns demonstrated the ability of mice with depleted CD4+ CD25+ Treg cells to execute a normal Th1 immune response, in contrast to non-depleted mice; this clearly demonstrated suppressed Th1 activity.77 These data were later confirmed in a group of severely injured patients, among whom Treg-mediated suppression of CD4+ proliferation and protective Th1 cytokine production was significantly disrupted by enhanced Treg activity.58 Although the engagement of TGF-b in the

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Figure 2 The trauma-induced monocyte—T cell interaction Immune response to multiple injury activates monocytes to communicate with naı¨ve T cells via antigen presentation. Excess production of inflammatory mediators enhances regulatory activity of immune-suppressing Treg and Th17-phenotype T cells. Monocyte deactivation supplements predominant immunosuppressive events and risk of posttraumatic complications.

Treg cells was discounted, an emerging body of data has established a close link between Treg cells and TGF-b. Meanwhile, TGF-b was confirmed as inducing the expression of Foxp3, further regulating the development, expansion and suppressive capacity of Treg cells.45,74,116 In a similar context, Choi and colleagues demonstrated that inhibition of heme oxygenase (HO)-1, the rate-limiting heme-degrading enzyme in the heme breakdown pathway, influenced both Foxp3 and HO-1 expression in peripheral CD4+ CD25+ Tregs, with subsequent amelioration of immunosuppressive cell function and impaired proliferation and production of cytokines.17 Furthermore, findings from an animal model suggested that the antiinflammatory effects of HO-1 were driven via enhancement of the secretion of IL-10 and promotion of the percentage of CD4+ CD25 (high) Treg.115 Conversely, findings from these studies suggested that Foxp3 induced heme oxygenase-1 (HO-1) expression and subsequently such conformable regulatory phenotypes in a cell—cell contact-dependent manner. The vital functions of heme oxygenase (HO)-1, which is also known as heat shock protein (HSP) 32, have been described in the maintenance of immune cell homeostasis, by virtue of its anti-

inflammatory, anti-apoptotic and anti-oxidative properties.15,48 Although the protective aspects of HO-1 are generally considered beneficial in some models of inflammation and injury, including inflammatory bowel diseases (IBD),102 acute pacreatitis,73 ischaemic spinal cord injury57 and hyperoxic acute lung injury (HALI),95 recent investigations of its effect in toxic liver injury27 and also in ischaemic/reperfusion injury of the kidney46 have reported that HO-1 inhibition reveals organ function and tissue preservation.25 Thus, in the aftermath of multiple injury, the sustained upregulation of such immunotolerant mechanisms may display a paradox by promoting a detrimental clinical course in states of recovery where active and adequate immune responses are indispensable.

Dysfunction of monocyte and macrophage networking in multiple trauma Monocytes and their mature phagocytotic phenotype, macrophages, play a central role in antigen presentation and are the fundamental component of the innate type-1 T cell regulated immune response

Immunoparalysis after multiple trauma to injury. Their key functions include the recognition, uptake and killing of invading organisms in order to initiate an adequate immune response. Prolonged impairment of monocyte function, with the characteristically decreased antigen-presenting capacity and disrupted monocyte—T cell interaction, has been associated with the development of septic complications after severe and multiple tissue trauma. As one of the essential MHC II antigen complex components, the human leukocyte antigen (HLA)-DR, presented on most immune effector cells, has been shown to be both positively and negatively regulated during inflammation by a variety of cytokines and stress mediators, including IL-1, IL-10, TNF-a, IFN-g, glucocorticoids and catecholamines.5,52,91,106 Furthermore, activation of the essential immunocompetent functions is enhanced by immunostimulatory cytokines such as IFN-g or GM-CSF. In response to an appropriate stimulus, HLA-DR expression has been frequently shown correlation with the monocytic antigen-presenting function, together with the ability to produce the appropriate inflammatory mediators and ensure an effective immune response to injury. Moreover, sustained decreased expression of HLA-DR on monocytes is associated with a negative outcome in septic cases and has been identified as a major underlying cause of posttraumatic complications after severe injury. Previous in vitro studies by Wolk et al. suggested the persistent downregulation of specific MHC class II molecules (e.g. HLA-DR, HLA-DP and HLA-DQ) to be associated with an impaired capacity to induce antigen-specific T cell proliferation and IFN-g production.113 Preceding experiments by Kox and Docke were able to demonstrate a beneficial effect of administering proinflammatory IFN-g 1b to recover depressed HLA-DR expression on monocytes from septic patients.23,53 Restitution of monocytic function was reflected by the significant increase of IL-6 and TNF-a after a temporary state of predominant anti-inflammatory production of IL-4 and IL-10 by T-helper (Th2) cells, respectively. Other adverse effects of antigen presentation have been related to the increased production of PGE2, a small lipid member of the prostanoid family, synthesised by cyclooxygenases COX-1 and COX-2; COX-2 has been described as related to inflammatory regulation. It has been suggested that, in respect of antigen presentation by monocytes and macrophages, increased PGE2 depresses Ia antigen expression (murine MHC class II equivalent) and further exerts a downregulatory effect on TNF-a and membrane IL-1 expression by macrophages via the accumulation of intracellular cAMP.54 Numerous animal models of inflammation have demonstrated that PGE2 represses macrophage activation, inhibits

1351 maturation of DC in lymph nodes and inhibits proliferation and stimulation of T cells by promoting a biased cytokine production towards a Th2 profile. This induces the increased expression of IL-10 and, in turn, inhibits the potential of several immune effector cells to secrete biologically active IL12.37,47,93,98,105 Antigen presentation and production of certain regulatory monocyte-derived cytokines, functional deregulation and paralysis with consecutive impairment of IL-12 production are further key components in the imbalance of the monocyte-regulated and T cell-mediated immune response. Studies including severely traumatised patients have demonstrated depressed IL-12 production by the mononuclear phagocyte system, ultimately promoting T cell commitment towards a Th2 pattern early after trauma. The appearance of the Th2 pattern was defined as the result of elevated numbers of cells producing IL-4 without major alterations in T cell capacity for producing IFN-g. Furthermore, the development of adverse clinical outcomes and the subsequent duration of the inflammatory response were closely correlated with the degree of alterations in monocyte and T cell responses.97 In concordance with findings from the functional analyses of Treg cells described earlier, it was additionally suggested that heme oxygenase (HO)-1 was involved in monocyte-mediated depression of the immune response.66 Studies examining the monocytic cell line U937 assessed characteristic properties of monocytes and macrophages, such as the activation and migration driven by monocyte chemoattractant protein (MIP)-1, being derogated through inhibition of MIP-1 production induced by HO-1.94 It was proposed that, with respect to the dysfunctional monocyte and macrophage response after multiple trauma, macrophage activation with uncontrolled phagocytosis of blood cells and their precursors contributed to the unexplained thrombocytopenia of some patients with sepsis.89 Recent reports have supported the hypothesis that the apparently futile process of erythrophagocytosis and subsequent heme catabolism by activated macrophages, via the macrophage haemoglobin scavenger receptor CD163,29 constitutes a negative regulatory pathway in systemic inflammation. This is otherwise considered a specific feature of haemophagocytic syndromes, characterised by an inappropriate cellular immune response among people with defective cytotoxic activity of T and NKcells.28 Interestingly, CD163 surface expression, exclusive to the monocyte lineage, is upregulated by glucocorticoids, IL-6, the anti-inflammatory cytokine IL-10 and cell surface TLR activation.109 In contrast, monocytic cells treated with GM-CSF

1352 and IL-4 for dendritic differentiation demonstrated downregulation of this antigen. This effect was increased by other proinflammatory mediators such as lipopolysaccharide, IFN-g, and TNF-a and TGF-b, all capable of suppressing CD 163 expression.12,84 Moreover, studies of people with bacteraemia have suggested that the soluble form of CD163 (sCD163), shed from stimulated monocytes upon selective cell surface TLR activation,108 positively correlates with a fatal outcome.68 It may therefore be possible to suggest, that in addition to the trauma-promoted inactivation of otherwise viable monocytes and macrophages, the enhancement of CD163+ monocytes could potentiate the acquired defect of cellmediated immune competence.

Premature apoptosis of immune effector cells after multiple trauma A number of research advances have focused on the mechanisms of apoptosis of various immune cells by suggesting that premature programmed cell death plays a determining role in the immunosuppressed response to a severe stimulus (e.g. injury, ischaemia/reperfusion, burn, infection) and subsequent clinical outcome.44,110 Direct apoptotic organ injury, and the immune suppression secondary to apoptotic losses in monocyte, macrophage, T cell, B cell, NK and dendritic cell populations may significantly contribute to the risk of consecutive opportunistic infections. Moreover increased apoptosis, particularly in lymphoid tissues and potentially in selected parenchymal tissues of solid organs, may contribute to the sepsis-associated MODS, and have been considered potential therapeutic targets for interventional treatments. Studies investigating the underlying pathophysiology of such diverse complications as sepsis and ischaemia/reperfusion injury have suggested that programmed cell death (apoptosis) is a major contributor to the traumainduced derangement of cellular homeostasis and the host’s subsequent inappropriate immune response.78,79,110 Characterised by irreversible cell shrinkage, chromatin condensation, DNA fragmentation and formation of apoptotic bodies, apoptosis plays an important role in normal development of mammalian tissue and cellular turnover. Apoptosis proceeds via auto-activation of cytosolic and mitochondrial caspases (cystein-containing aspartate-specific proteases), which cleave various proteins, resulting in cell destruction.20 The extensively investigated downstream signalling pathways have been described as predominantly caspase-dependent, following either the extrinsic receptor-mediated

S.K. Tschoeke, W. Ertel activation of caspase-3/7 via binding to members of the TNF-receptor superfamily (e.g. Fas receptor, TNF receptor I), or the mitochondrially induced intrinsic pathway. The latter involves the mitochondrial release of cytochrome c with subsequent activation of caspase-9, and finally the cleaved and active form of the cell death-promoting enzyme, caspase-3.118 This particular mitochondrially induced apoptotic pathway is, in part, dependent on the balance of Bcl family members (Bax, Bcl-xL and Bcl-2) bound to the mitochondrial membrane as either pro- (Bax) or anti-apoptotic (Bcl-xL, Bcl-2) proteins.96 Previous studies have demonstrated that antibody-mediated inhibition of Bcl-2 increases mitochondrial permeability, thus leading to the emission of intramitochondrial proteins into the cytosol, such as cytochrome c, otherwise essential for mitochondrial survival.18 Numerous clinical studies and experimental models of traumatic neuronal injury, sepsis, ischaemia/ reperfusion injury and burn have demonstrated that the increased production of endogenous inflammatory mediators (HSPs, oxygen free radicals, NO, TNF-a, IL-1 and IL-6) may activate apoptotic signalling pathways in various innate immune cells.4,6,87,90 However, data from multiple trauma cases are extremely limited and have not yet yielded an appropriate explanation for the underlying pathobiological mechanism of posttraumatic immunosuppression.35 In particular, it has been suspected that the immunosuppressive effects of NO are associated with APC-related regulatory mechanisms. For example, studies in an animal model of thermal injury demonstrated that the addition of the NO donor, S-nitroso-N-acetyl-penicillamine, induced apoptosis, attenuated mitochondrial oxidative metabolism and induced mitochondrial membrane depolarisation. This conclusively supported the concept that NO induces suppression of cell-mediated immune responses by selective action on Th1 T cells, thereby promoting a Th2 response.16,21 Other progressive findings, established predominantly from local tissue analyses (e.g. tissue-specific cells in nerve, gut, spleen and liver tissue) and their correlation with MODS or sepsis, postulated that the deregulation of Fas expression and its associated ligand (FasL) on T cells and mononuclear cells (PBMC) was positively correlated with the likelihood among critically ill patients of developing MODS.43,82,86,110,117 In line with these and other observations among severely injured patients, studies investigating those undergoing major surgical trauma have described similar increases of Fas and FasL in T cells within 24 h postoperatively. This suggests that the upregulation of Fas and FasL may be strongly associated with the apoptosis-induced

Immunoparalysis after multiple trauma lymphocytopenia leading to an increased risk of post-surgical infections.22 It has also been postulated that particularly activated T cells positively affect cytokine and chemokine production via the Fas system, thus promoting injury in respective local tissue compartments.32,38,65,76 Furthermore, studies investigating the cellular interactions of human Treg in septic shock demonstrated the inhibition of LPS-induced retention of monocyte CD14 to be, at least in part, mediated via the extrinsic Fas/FasL pathway.104 Because loss of CD14 is a hallmark of monocyte apoptosis, the reported findings suggest a critical link to Treg-induced immunosuppression, subsequent to the inhibition of monocyte survival through a pro-apoptotic, yet unidentified, mechanism.

Neurohormonal regulation of cell-mediated immunosuppression The physiological host reaction to severe or multiple trauma implies an excessive activation of the neuroendocrine stress response. It has been proposed that catecholamines and glucocorticoids present another important component in the initiation and amplification of the characteristic posttraumatic immune derangements described so far.10 Furthermore, it has been suggested that neurohormonal signalling, via binding of glucocorticoids, catecholamines or adrenergic agonists to the corresponding receptors on immune cells, suppresses cytokine production and thus impairs a competent immune regulatory cell—cell interaction.14,59 In 1986, Blazar and colleagues demonstrated that, for example, the immunological activity of NK cells of volunteers was reduced by an average of 66% following the infusion of cortisone, epinephrine and glucagon in amounts known to reproduce serum levels seen after injury of moderate severity.9 Experimental and clinical studies involving posttraumatic multiple organ failure were able to demonstrate the consecutive release of these neurotransmitters and hormones into the extracellular milieu of immune cells. This would promote Th2type T cell development, together with the development of an anti-inflammatory macrophage phenotype via attenuation of IL-12 and enhancement of IL-10 production.42,56,81 Other studies have impressively demonstrated the ability of the central nervous system to influence and orchestrate diverse local and systemic immune regulatory mechanisms and responses.83,101 For example, it was reported that peripheral blood lymphocytes from people with malignant brain tumours responded poorly to mitogens or presented

1353 antigens in association with reduced IL-2 production and expression of the IL-2 receptor (IL-2R).11,88 It is therefore possible to suggest that concomitant head injury, as a frequent component of the multiply injured patient, may critically impair the systemic immune response in addition to other traumarelated local and systemic immune cell responses. Results from a rat model of haemorrhagic shock demonstrated the suppressive effect of noradrenergic innervation on the haemorrhage-induced increase in tissue TNF-a content. The authors concluded that these effects of norepinephrine might be protective in tissue injury but would be more likely to contribute to generalised trauma-induced immunosuppression.67 Other in vitro studies in a rat model of acute brain injury showed that, within minutes of applying catecholamines, secretion of interleukin-10 from unstimulated monocytes was triggered through a b-adrenoreceptor-mediated, cAMP/protein kinase A-dependent pathway. Furthermore, these authors reported that the badrenoreceptor antagonist propranolol prevented the increase in IL-10 plasma levels.112 Similarly, a study investigating the effects of b-2-adrenergic receptor (b-2-AR) agonists on the expression of co-stimulatory molecules on lipopolysaccharide-stimulated human peripheral blood mononuclear cells clearly demonstrated that endogenous catecholamines elicited immunosuppressive effects through b-2-AR stimulation, possibly due to downregulation of the expression of ICAM-1, CD40 and CD14 on monocytes.55 As above, these results suggested that the sympathetic nervous system might regulate the T-helper cell balance via the peripheral end-effectors of the stress system. In contrast, studies investigating the effects of dopamine reported that the systemic injection of dopamine or an agonist of its 1 receptors significantly enhanced protection against neuronal death after mechanical and biochemical injury to the central nervous system via activation of the extracellular signal-regulated kinase (ERK) 1/2 signalling pathway in a T celldependent manner.51 In correlation with these observations, the suppressive activity and the adhesive and migratory abilities of Treg cells were significantly reduced. Recent work by Palmer and colleagues suggested that both CD1d expression by APC and CD1drestricted NKT cells, a specific subset of the T cell lineage known to regulate T cell immunity,70 were required for immune suppression after injury.80 Furthermore, preceding studies by the same authors demonstrated that, early after injury, IL-4 was almost exclusively produced by the NKT cell population, in addition to the functional loss of production of proinflammatory IFN-g.31 In this context, findings

1354 from adrenalectomised mice subjected to restraint stress suggested that NKT cells are resistant to endogenous steroid hormones, thus emphasising their importance in stress-associated immunosuppression.92 Other reports from models investigating lipopolysaccharide-related hepatotoxicity inversely complemented these findings by demonstrating that low norepinephrine activity increased hepatic NKT cell apoptosis. Subsequent polarisation of the hepatic cytokine production sensitized the liver to lipopolysaccharide toxicity. In line with these observations, it has been recognised that the sympathetic purinergic stress hormone adenosine plays a prominent role in traumarelated immunosuppression.39 Early studies investigating the effects of adenosine receptor stimulation were able to demonstrate reduced IL-2 production and decreased lymphocyte proliferation,24 in addition to inducing lymphoctye apoptosis in the presence of adenosine.99 More particularly, it has been reported that both macrophage and DC functions were directly impaired by binding adenosine to its respective receptors, thus decreasing their phagocytic activity by suppressing the production of superoxide26 and nitric oxide.41 Finally, MHC II antigen presentation was significantly reduced in IFN-g-primed macrophages exposed to adenosine in vitro, resulting in decreased production of TNF-a and IL-12 via adenosine A2 and A3 receptors and, conversely, enhanced production of antiinflammatory IL-10.40

Summary and conclusion Immunosuppression, as the result of a deregulated immune response to multiple trauma, remains a debatable issue within the appropriate management of critically injured patients. Although numerous mediators responsible for the imbalance of immune functions and cellular interplay have been recognised and described in great detail, we are far from a curative ‘magic bullet’ that might prevent the detrimental outcome associated with posttraumatic immunoparalysis. Driven by a complex of inflammatory mediators (e.g. TGF-b, PGE2, IL-4, IL-10), the shift to a predominant Th2-type immune response and the induction of diverse cell deathsignalling mechanisms promote profound repression of immune effector cell activation and function, which is primarily characterised by reduced antigen recognition and cellular anergy. In turn, the antiinflammatory profile favours the activation of other predominantly immune-suppressing cells, such as the CD4+ CD24+ Foxp3 Treg cells. Although many inflammatory mediators, in excess, exert rather

S.K. Tschoeke, W. Ertel detrimental effects on the host, their presence is often essential for initiating the appropriate immune response to injury. Thus, as we progress in understanding the intracellular signalling pathways and consecutive changes in intercellular communication and immune function, we must acknowledge that the immune response to multiple injury is a dynamic event involving highly complex and multilevel regulatory physiological, particularly neurohormonal and cell-mediated, processes. This highlights the importance of immunomonitoring when attempting to redeem trauma-induced immunoparalysis and to modulate the cell-specific or overall apoptotic response of involved immune cells to multiple injury. Therefore, current therapeutic efforts promise exciting new strategies, including cell-specific targeting of selected signalling pathways, to adjust or re-establish the appropriate function of essential immune effector cells.7,75,114

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