Apoptosis in the Resolution of Systemic Inflammation

J. c. Marshall and R. W. G. Watson

Introduction

Admission to a contemporary leU is precipitated by a heterogeneous group of dis• ease processes, yet the final common pathway to death is strikingly similar [I). Fully 80% of all leu deaths occur in association with the multiple organ dysfunction syn• drome (MODS) [2), a poorly understood process characterized by the development of progressive physiologic dysfunction in organ systems initially unaffected by the primary disease process. Activation of a host inflammatory response by infection, injury, or ischemia, invariably proceeds the development of MODS [3). The proxi• mate causes of organ injury are not bacterial products, but the same endogenous host-derived mediators of inflammation that are responsible for containing an in• fectious challenge and initiating the process of tissue repair. MODS can be concep• tualized as the consequence of an overly activated or inappropriately prolonged systemic inflammatory response, therefore an understanding of the mechanisms underlying the resolution of inflammation is important to the development of clini• cal strategies to prevent the syndrome.

The Resolution of Inflammation

Mechanisms

As important to the host as the ability to activate an efficient response to an exoge• nous threat, is the ability to terminate that response once the threat has passed. Both humoral and cellular counter-inflammatory mechanisms have been identified. Monocyte-mediated non-specific responses are regulated through autocrine and paracrine pathways involving the release of counter-inflammatory mediators such as lL-IO [4) or lL-lra [5) that restore the cell to a resting state. For the neutrophil, however, the expression of an inflammatory response results both in the extravasa• tion of large numbers of cells into an inflammatory focus, and in the activation of these cells for antimicrobial activity. Lacking a mechanism to retreat from an in• flammatory focus, cell death through the controlled process of is the dom• inant process leading to the termination of a neutrophil-mediated response [6).

J.-L. Vincent (ed.), Yearbook of Intensive Care and Emergency Medicine 1997 © Springer-Verlag Berlin Heidelberg 1997 Apoptosis in the Resolution of Systemic Inflammation 101

Apoptosis

Apoptosis is a form of cellular death, effected through the expression of an endoge• nous, genetically-regulated program, that results in the rapid elimination of the cell without evoking a significant inflammatory response. First recognized more than a century ago, apoptosis is now appreciated to be central to the normal development of multi-cellular organisms, mediating such phenomena as tissue remodeling dur• ing embryogenesis, the deletion of auto reactive T cell clones during immune matu• ration, and the elimination of transformed cells by natural killer cells [7]. It is also the mechanism through which cells with a fixed life span, such as epithelial cells and neutrophils, die. Early features of apoptosis include cytoplasmic blebbing, loss of normal cell-cell adhesion, and the elimination of specialized structures such as microvilli {Fig. I). In contrast to the cell dying a necrotic death, the apoptotic cell shrinks, its chromatin condenses, and the nucleus fragments as a result of internucleosomal cleavage of DNA. The cellular contents become condensed into membrane-bound apoptotic bodies that are rapidly phagocytosed, principally by fixed tissue macrophages. Apoptosis occurs rapidly. Once triggered, it is complete within 45 min, with the result that static tissue sections may fail to reflect the dynamic process occuring in vivo. Neutrophil apoptosis is readily recognized morphologically by either light (Fig. 2) or electron microscopy. Apoptosis results in a characteristic pattern of DNA injury generating fragments that are multiples of 180 base pairs, and producing a characteristic latter pattern on DNA gel electrophoresis. Injury to DNA can also be detected by flow cytometry as altered binding of propidium iodide [8].

Expression and Regulation of Apoptosis

Apoptosis is effected through the activation of intracellular proteases that degrade key structural and nuclear [9]. It is dependent on the transcription of pro• apoptotic genes, and can be blocked by inhibitors of synthesis. However, its expression can be modulated by signals from the exterior of the cell, by alterations in the transduction of these signals to the nucleus, and by the activation of anti• apoptotic genes.

Genetic Regulation

An understanding of the genetic regulation of apoptosis has been facilitated by systematic study of the nematode, C. elegans, where genes promoting (ced-3 and ced- 4) and inhibiting (ced-9) apoptosis have been sequenced and cloned. The interleukin(IL)-I~ converting (ICE), initially described as an enzyme that converts pro IL-l ~ to its active form, was the first identified mammalian homo• log of ced-3 [10]. To date, nine members of this family have been described in human cells [11]. All are cysteine proteases with a common pentapeptide sequence - QACRG - that constitutes the active site of the molecule [12],and all cleave their tar• get proteins at aspartic acid residues. ICE homologs target a number or proteins fun- 102 J. c. Marshall and R. W. G. Watson

Necrosis Apoptosis

Swelling of the cell / " Nuclear chromatin compaction " Cell Shrinkage ~

I Nuclear fragmentation and Rupture of cell t Budding of the cell into membrane and release apoptotic bodies of intracellular contents o

Toxic to surrounding cells resulting on the activation an inflammatory response ,---~~ Ingestion of apoptotic bodies by '" local phagocytes with no in duction of an inflammatory

Fig. 1. Necrotic cell death is associated with loss of membrane integrity, and leakage of cytoplasmic constituents into the surrounding tissues, evoking an inflammatory response. In contrast, cells dying a controlled apoptotic death condense to form apoptotic bodies that are phagocytosed by local fIxed tissue macrophages without eliciting inflammation. Necrosis is readily evident by histologic exam• ination. Because of both the rapidity of the process and the absence of a local response, apoptosis is less evident histologically damental to cell survival including nuclear involved in DNA repair (poly• (ADP-ribose) polymerase (PARP) , and DNA-dependent protein kinase), compo• nents of the cytoskeleton including , and other enzymes of the ced-3 family [11]. Mammalian homologs of ced-4 have not yet been identified. Apoptosis in the Resolution of Systemic Inflammation 103

Fig. 2. Photomicrograph of neutrophil apoptosis. Apoptotic neutrophils can be recognized by virtue of their condensed nuclear chromatin (arrows)

In contrast, ced-9 homologs, typified by bel-2, initially identified as a protein over• expressed in a non-Hodgkin B cell lymphoma, serve to protect the cell against a wide variety of apoptotic stimuli [13]. While some members of the bel-2 family including bel-x and bel-xL are anti-apoptotic, others actually increase rates of apoptosis.

Signalling Pathways

Signalling pathways for apoptosis are incompletely defined. Generation of ceramide from membrane sphingomyelin constitutes a key second messenger system leading to apoptosis [14]. Ceramide-mediated signalling appears to be intimately related to intracellular signalling cascades dependent on sequential protein phosphorylations. Ceramide has also been reported to activate MAP kinase (MAPK) [15], and to medi• ate Fas- and tumor necrosis factor (TNF)-induced apoptosis through activation of SAP kinase (SAPK) [16]. Similarly, studies with inhibitors of tyrosine kinases show their activation to be necessary for apoptosis [17, 18]. In contrast, activation of pro• tein kinase C [19] or elevation of intracellular levels of cyclic monophos• ph ate (cAMP) [20] inhibit apoptosis.

Neutrophil Apoptosis

Apoptosis in the neutrophil is a constitutive process that is activated when the cell leaves the bone marrow, with the result that circulating neutrophils survive no more 104 J. C. Marshall and R. W. G. Watson than a day before they die an apoptotic death, and are removed by fixed tissue mac• rophages of the liver and spleen [6]. The mechanism of spontaneous neutrophil apoptosis is not known. Mature neutrophils express the pro-apoptotic gene, ICE but not the anti-apoptotic bcl-2, suggesting that they lack an intrinsic means of rescue from programed cell death. However, the expression of other anti-apoptotic genes in the neutrophil has not been studied.

The FasITNFr Family of Death Receptors

Fas (CD95/ABO-l) is a widely distributed cell surface protein and a member of a family of proteins, including the p55 component of the for TNF, the nerve receptor, and CD40 [21]. Receptors of the Fas family are intimately as• sociated with a family of membrane bound proteins which can activate ICE or mem• bers of the ICE family and thus trigger apoptotic cell death (Fig. 3). Activation of Fas in vivo occurs as a consequence of its interaction with Fas a 40 kD protein with a more limited cellular expression, first identified on cytotoxic T cells [22]. Hu• man neutrophils express both Fas and Fas ligand, and the autocrine interaction of these two molecules may mediate constitutive apoptosis in the neutrophil [23]. Fas expression plays a critical role in normal cellular homeostasis. A mouse strain lacking functional Fas, the MRLllpr strain, develops an autoimmune disorder that is similar to systemic lupus erythematosus and that results in death from nephritis and

FasL TNF

ICE proteases!/! ? N!,"-~ activation ~ Apoptosis

Fig. 3. Fas and TNF are two members of a family of transmembrane death receptors. Their cytoplas• mic tails associate with additional signalling proteins that lead to activation of enzymes of the ICE family and induce apoptotic cell death. Signalling through TNFrl (and possibly Fas) can also lead to cell activation by activation of the latent cytoplasmic , NF-KB. It is possible that costimulatory signals delivered by other membrane receptors such as the p2 , regulate sub• sequent cellular responses following receptor engagement Apoptosis in the Resolution of Systemic Inflammation 105 arthritis [24]. These mice also demonstrate enhanced susceptibility to septic shock induced by Staphylococcal enterotoxin B [25]. In contrast, administration of an ago• nistic antibody to Fas induces lethal hepatitis in mice, suggesting that activation of Fas resulting in hepatocyte death may be pathogenically involved in some cases of fulminant hepatic failure [26]. The cytoplasmic tail of the TNF receptor contains an amino acid sequence that shows 28% homology to the death domain of Fas, and its engagement by soluble or cell associated TNF-a also triggers apoptosis [27]. The induction of apoptosis in tar• get cells is an important mechanism ofTNF-mediated cytotoxicity [28]. TNF recep• tor engagement, however, can also lead to cellular activation through activation of the cytoplasmic transcription factor, NF-KB [29]. How a signal receptor can trigger the diametrically opposite responses of cellular activation and programed cell death is unknown.

Modulation of Neutrophil Apoptosis by Environmental Signals

The expression of apoptosis in the neutrophil is exquisitely sensitive to a variety of environmental signals arising from soluble regulatory mediators in the inflammato• ry microenvironment and events intrinsically linked to the localization of the neu• trophil to an inflammatory focus, or its activation for antimicrobial activity (Table 1). Several of these merit further description. Bacterial products such as endotoxin or formyl peptides retard the expression of neutrophil apoptosis in vitro, an effect that favors prolonged cellular survival in the face of an infectious challenge. However, retardation of apoptosis by viruses may prove detrimental to the host. Certain viral gene products including the product of the cowpox CrmA gene, an inhibitor ofICE [30, 31],and the baculovirus p35 protein [32], are capable of inhibiting apoptosis in mammalian cells, and thus of preventing the normal elimination of virally-infected cells. It has recently been appreciated that an homologous family of genes, designated inhibitor of apoptosis proteins (lAP),

Table 1. Factors that modulate neutrophil apoptosis

Retard Accelerate

Soluble mediators IL-l~ IL-6 IL-2 TNF-a IL-6 Fas ligand G-CSF GM-CSF Interferon-y CSa LPS FMLp Glucocorticoids

Cellular processes ~2 engagement Phagocytosis of E. coli Elevated levels L-selectin engagement Elevated cAMP Reduced intracellular thiollevels 106 J. c. Marshall and R. W. G. Watson

exists in human cells [33,34]. Whether they exert an important biologic role in the normal regulation of apoptosis is unknown. In contrast to the effects of bacterial products, the phagocytosis of whole bacteria by the neutrophil results in accelerated rates of apoptosis [35]. Neutrophils isolated from the peritoneal cavity of experimental animals in whom a cellular influx has been stimulated by proteose peptone showed delayed rates of apoptosis [36]. Similarly, we have found [37] delayed apoptotic rates in neutrophils isolated from the lungs of rats following intratracheal challenge with endotoxin. Im• paired apoptosis is a consequence of the process of neutrophil transmigration into the lung. It occurs when the inflammatory is TNF, a known to trig• ger apoptosis in vitro, and the apoptotic delay can be reproduced in vitro by trans• migration of lipopolysaccharide (LPS)-primed neutrophils across an endothelial monolayer. Moreover, the effect can be mimicked by cross-linking of ~2 integrins on the neutrophil surface, while cross-linking of L-selectin triggers apoptosis [37]. An apoptotic delay is associated with prolonged functional survival, reflected in aug• mented respiratory burst activity. Thus neutrophil adhesion molecules may playa role in the physiologic regulation of apoptosis in the inflammatory microenviron• ment, and so in the persistence of neutrophil activation at the site of an inflammato• ry stimulus.

Apoptosis in Critical Illness

The systemic inflammatory response syndrome (SIRS) denotes the generalized ex• pression of a process that is normally localized to the site of an acute challenge [38]. Although apoptosis in critically ill patients has not been extensively studied, the available data show intriguing abnormalities. Lymphopenia is a common concomitant of trauma in critical illness. Studies in patients with major thermal injury show increased rates of apoptosis in peripheral blood lymphocytes [39]. On the other hand, neutrophils harvested from burn pa• tients show increased survival secondary to retarded neutrophil apoptosis; the ap• optotic delay is mediated by a soluble serum factor whose activity can be blocked with a blocking antibody to granulocyte macrophage-colony stimulating factor (GM-CSF) [40], suggesting that the release of this endogenous mediator also mod• ulate cell survival in the systemic circulation. In preliminary studies in a group of patients with SIRS, we have found that rates of spontaneous neutrophil apoptosis in vitro are reduced to approximately one quarter those of control cells, and that the al• tered responsiveness can be attributed to both cellular changes in patients' neu• trophils, and to soluble factors in the serum.

Conclusion

Control of regulation of cell death is as important to the survival of living organisms as is the regulation of growth and development. Derangements of the normal pro• cess of apoptotic cell death are now recognized to playa critical role in a variety of disease processes including autoimmunity, degenerative disorders, and cancer, and Apoptosis in the Resolution of Systemic Inflammation 107 this recognition has opened the door to novel therapeutic possibilities directed at manipulation of apoptosis. An evolving understanding of the role of inflammatory cell apoptosis in the expression and termination of the host response to an acute life• threatening stimulus should similarly provide the intensivist with new tools to mod• ulate the inflammatory response for the ultimate benefit of the critically ill patient.

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