Apoptosis has been proposed to mediate CD4+ T-cell depletion in human immunodeficiency virus (HIV)-infected individuals. Interaction of Fas ligand (FasL) with Fas (CD95) results in lymphocyte apoptosis, and increased susceptibility to Fas-mediated apoptosis has been demonstrated in lymphocytes from HIV-infected individuals. Cells undergoing apoptosis in lymph nodes from HIV-infected individuals do not harbor virus, and therefore a bystander effect has been postulated to mediate apoptosis of uninfected cells. These data raise the possibility that antigen-presenting cells are a source of FasL and that HIV infection of cells such as macrophages may induce or increase FasL expression. In this report, we demonstrate that HIV infection of monocytic cells not only increases the surface expression of Fas but also results in the de novo expression of FasL. Interference with the FasL-Fas interaction by anti-Fas blocking antibodies abrogates HIV-induced apoptosis of monocytic cells. Human monocyte-derived macrophages from healthy donors contain detectable FasL mRNA, which is further upregulated following HIV infection with monocytotropic strains. HIV-infected human macrophages result in the apoptotic death of Jurkat T cells and peripheral blood T lymphocytes. Interruption of the FasL-Fas interaction abrogates the HIV-infected macrophage-dependent death of T lymphocytes. These results provide evidence that human macrophages can provide a source of FasL, especially following HIV infection, and can thus participate in lymphocyte depletion in HIV-infected individuals.

Fas (also known as CD95), a member of the tumour-necrosis receptor factor family of 'death receptors', can induce apoptosis or, conversely, can deliver growth stimulatory signals. Here we report that crosslinking Fas on primary sensory neurons induces neurite growth through sustained activation of the extracellular-signal regulated kinase (ERK) pathway and the consequent upregulation of p35, a mediator of neurite outgrowth. In addition, functional recovery after sciatic nerve injury is delayed in Fas-deficient lpr mice and accelerated by local administration of antibodies against Fas, which indicates that Fas engagement may contribute to nerve regeneration in vivo. Our findings define a role for Fas as an inducer of both neurite growth in vitro and accelerated recovery after nerve injury in vivo.

Human immunodeficiency virus (HIV) infection leads to numerous perturbations of B cells through mechanisms that remain elusive. We performed DNA microarray, phenotypic, and functional analyses in an effort to elucidate mechanisms of B cell perturbation associated with ongoing HIV replication. 42 genes were up-regulated in B cells of HIV-viremic patients when compared with HIV-aviremic and HIV-negative patients, the majority of which were interferon (IFN)-stimulated or associated with terminal differentiation. Flow cytometry confirmed these increases and indicated that CD21(low) B cells, enhanced in HIV-viremic patients, were largely responsible for the changes. Increased expression of the tumor necrosis factor (TNF) superfamily (TNFSF) receptor CD95 correlated with increased susceptibility to CD95-mediated apoptosis of CD21(low) B cells, which, in turn, correlated with HIV plasma viremia. Increased expression of BCMA, a weak TNFSF receptor for B lymphocyte stimulator (BLyS), on CD21(low) B cells was associated with a concomitant reduction in the expression of the more potent BLyS receptor, BAFF-R, that resulted in reduced BLyS binding and BLyS-mediated survival. These findings demonstrate that altered expression of genes associated with IFN stimulation and terminal differentiation in B cells of HIV-viremic patients lead to an increased propensity to cell death, which may have substantial deleterious effects on B cell responsiveness to antigenic stimulation.

The death domain-associated protein (Daxx) was originally cloned as a CD95 (FAS)-interacting protein and modulator of FAS-induced cell death. Daxx accumulates in both the nucleus and the cytoplasm; in the nucleus, Daxx is found associated with the promyelocytic leukaemia (PML) nuclear body and with alpha-thalassemia/mental retardation syndrome protein (ATRX)-positive heterochromatic regions. In the cytoplasm, Daxx has been reported to interact with various proteins involved in cell death regulation. Despite a significant number of studies attempting to determine Daxx function in apoptotic and non-apoptotic cell death, its precise role in this process is only partially understood. Here, we critically review the current understanding of Daxx function and shed new light on this interesting field.

The epidermis is a multilayered stratified epithelium, continuously regenerated by differentiating keratinocytes, that requires the transcription factor p63 for its development and maintenance. The TP63 gene encodes two major protein isoforms, TAp63 and DeltaNp63, which have both transactivating and transcriptional repressing activities and regulate a wide range of target genes. TAp63 shows clear pro-apoptotic activity, mediated both by death receptors (CD95, TNF, TRAIL) and mitochondrial (bax, puma) pathways. Conversely, DeltaNp63 protects from apoptosis by directly competing for TAp63 target promoters or sequestering it, forming inactive tetramers. Accordingly, p63 is expressed in epithelial tumors, contributing to both tumorigenesis and chemoresistance. However, the predominant physiological role of p63 is in epithelial development, as demonstrated by the lack of epidermis and other epithelia in p63-deficient mice. The specific role of TAp63 and isoforms in epithelial development remains mostly unclear. Nevertheless, recent work utilizing in vivo genetic complementation of TAp63 and/or DeltaNp63 into a p63 null background has shed new light into the specific functions of the two isoforms and allowed the in vivo validation of several p63 transcriptional targets, originally identified by microarray analysis in in vitro systems. However, despite concerted efforts to address the role of p63 isoforms, several questions remain unanswered. The main aim of this review is to critically discuss the data available in the literature and thoroughly analyze the models proposed.

Induction of apoptosis by the cell surface receptor CD95 (APO-1/Fas) has been shown to involve activation of a family of cysteine proteases (caspases). Recently, a new member of this family has been identified, designated FLICE (caspase-8/MACH/Mch5). FLICE is part of the CD95 death-inducing signaling complex and is therefore the most upstream caspase in the CD95 apoptotic pathway. A total of eight different isoforms of FLICE (caspase-8/a-h) have been described. To determine which isoforms are expressed in different cells we have generated a panel of monoclonal antibodies directed against all functional domains of FLICE. Using these antibodies we could show that only two of the FLICE isoforms (caspase-8/a and caspase-8/b) were predominantly expressed in cells of different origin. Both isoforms were recruited to the CD95 death-inducing signaling complex and were activated upon CD95 stimulation with similar kinetics. Taken together, only two of the eight published caspase-8 isoforms could be detected in significant amounts at the protein level.

Tumor progression shares many characteristics with the process of epithelial-to-mesenchymal transition (EMT). Cells that have undergone an EMT are known to have an increased resistance to apoptosis. CD95/Fas is an apoptosis-inducing receptor expressed on many tissues and tumor cells. During tumor progression CD95 is frequently downregulated, and tumor cells lose apoptosis sensitivity. miR-200 microRNAs repress both the EMT-inducing ZEB1 and ZEB2 transcription factors. We now demonstrate that miR-200c sensitizes cells to apoptosis mediated by CD95. We have identified the apoptosis inhibitor FAP-1 as a target for miR-200c. FAP-1 was demonstrated to be responsible for the reduced sensitivity to CD95-mediated apoptosis in cells with inhibited miR-200. The identification of FAP-1 as an miR-200c target provides a molecular mechanism to explain both the downregulation of CD95 expression and the reduction in sensitivity of cells to CD95-mediated apoptosis that is observed in the context of reduced miR-200 expression during tumor progression.

The involvement of the death adaptor protein FADD and the apoptosis-initiating caspase-8 in CD95 and TRAIL death signalling has recently been demonstrated by the analysis of the native death-inducing signalling complex (DISC) that forms upon ligand-induced receptor cross-linking. However, the role of caspase-10, the other death-effector-domain-containing caspase besides caspase-8, in death receptor signalling has been controversial. Here we show that caspase-10 is recruited not only to the native TRAIL DISC but also to the native CD95 DISC, and that FADD is necessary for its recruitment to and activation at these two protein complexes. With respect to the function of caspase-10, we show that it is not required for apoptosis induction. In addition, caspase-10 can not substitute for caspase-8, as the defect in apoptosis induction observed in caspase-8-deficient cells could not be rescued by overexpression of caspase-10. Finally, we demonstrate that caspase-10 is cleaved during CD95-induced apoptosis of activated T cells. These results show that caspase-10 activation occurs in primary cells, but that its function differs from that of caspase-8.

In the colon of ulcerative colitis (UC) patients, apoptotic bodies have been recognized in routine histopathological preparations. To investigate the extent of the apoptosis, colonic biopsies were examined from involved and uninvolved areas of untreated active UC and from normal areas in patients with colonic polyps, utilizing various markers of apoptosis. The markers included DNA breaks detected by TUNEL, Fas (CD95/APO-1) and Fas ligand (Fas-L) localized by immunohistochemistry, electron microscopic features of apoptosis, and laddering of extracted DNA. Apoptosis marker positive cells were found mainly on the luminal epithelium of the normal colon and were present in active UC in crypts of involved and uninvolved areas of the colon, in addition to the luminal epithelium. The DNA extracted from active UC colon electrophoresed as a ladder. These findings suggest that the loss of epithelial cells in active UC occurs mainly by apoptosis in crypts of involved and adjacent uninvolved areas and that the Fas/Fas-L interaction is a mediator of the apoptosis.

Expression of c-Myc sensitizes cells to a wide range of pro-apoptotic stimuli. We here show that this pro-apoptotic effect is mediated through release of mitochondrial holocytochrome c into the cytosol. First, activation of c-Myc triggers release of cytochrome c from mitochondria. This release is caspase-independent and blocked by the survival factor IGF-1. Second, c-Myc-induced apoptosis is blocked by microinjection of anticytochrome c antibody. In addition, we show that microinjection of holocytochrome c mimics the effect of c-Myc activation, sensitizing cells to DNA damage and to the CD95 pathway. Both p53 and CD95/Fas signaling have been implicated in c-Myc-induced apoptosis but neither was required for c-Myc-induced cytochrome c release. Nonetheless, inhibition of CD95 signaling in fibroblasts did prevent c-Myc-induced apoptosis, apparently by obstructing the ability of cytosolic cytochrome c to activate caspases. We conclude that c-Myc promotes apoptosis by causing the release of cytochrome c, but the ability of cytochrome c to activate apoptosis is critically dependent upon other signals.

The possible involvement of Fas/APO-1 (CD95) and TNF in antigen-specific AICD of thymocytes and mature T cells has been investigated. Antigenic stimulation in vivo of influenza hemagglutinin (HA)-specific TCRtg mice was used to demonstrate that the kinetics of thymocyte and peripheral CD4+ T cell deletion are similar in mice with normal (+/+) or defective Fas (lpr/lpr) background, indicating that a Fas-independent pathway(s) is responsible for the deletion of activated T cells. TCRtg-+/+ or TCRtg-lpr/lpr mice injected with murine TNF-blocking MAb (TN3) showed rapid apoptosis of thymocytes after HA stimulation, indicating that death signaling through Fas and TNF receptors is not essential for HA-induced thymocyte deletion. CDC peripheral T cells in TCRtg-lpr/lpr mice did not undergo apoptosis following injection with HA and TN3, indicating that TNF-mediated apoptosis is involved in the deletion of mature T cells after antigenic stimulation. However, apoptosis still occurred in TCRtg-+/+ mice injected with TN3, indicating that both Fas- and TNF-mediated cell death can contribute to the deletion of activated peripheral T cells.

Neoplastic pancreatic epithelial cells are believed to die through caspase 8-dependent apoptotic cell death, and chemotherapy is thought to promote tumour apoptosis. Conversely, cancer cells often disrupt apoptosis to survive. Another type of programmed cell death is necroptosis (programmed necrosis), but its role in pancreatic ductal adenocarcinoma (PDA) is unclear. There are many potential inducers of necroptosis in PDA, including ligation of tumour necrosis factor receptor 1 (TNFR1), CD95, TNF-related apoptosis-inducing ligand (TRAIL) receptors, Toll-like receptors, reactive oxygen species, and chemotherapeutic drugs. Here we report that the principal components of the necrosome, receptor-interacting protein (RIP)1 and RIP3, are highly expressed in PDA and are further upregulated by the chemotherapy drug gemcitabine. Blockade of the necrosome in vitro promoted cancer cell proliferation and induced an aggressive oncogenic phenotype. By contrast, in vivo deletion of RIP3 or inhibition of RIP1 protected against oncogenic progression in mice and was associated with the development of a highly immunogenic myeloid and T cell infiltrate. The immune-suppressive tumour microenvironment associated with intact RIP1/RIP3 signalling depended in part on necroptosis-induced expression of the chemokine attractant CXCL1, and CXCL1 blockade protected against PDA. Moreover, cytoplasmic SAP130 (a subunit of the histone deacetylase complex) was expressed in PDA in a RIP1/RIP3-dependent manner, and Mincle--its cognate receptor--was upregulated in tumour-infiltrating myeloid cells. Ligation of Mincle by SAP130 promoted oncogenesis, whereas deletion of Mincle protected against oncogenesis and phenocopied the immunogenic reprogramming of the tumour microenvironment that was induced by RIP3 deletion. Cellular depletion suggested that whereas inhibitory macrophages promote tumorigenesis in PDA, they lose their immune-suppressive effects when RIP3 or Mincle is deleted. Accordingly, T cells, which are not protective against PDA progression in mice with intact RIP3 or Mincle signalling, are reprogrammed into indispensable mediators of anti-tumour immunity in the absence of RIP3 or Mincle. Our work describes parallel networks of necroptosis-induced CXCL1 and Mincle signalling that promote macrophage-induced adaptive immune suppression and thereby enable PDA progression.

We have identified and characterized ARC, apoptosis repressor with caspase recruitment domain (CARD). Sequence analysis revealed that ARC contains an N-terminal CARD fused to a C-terminal region rich in proline/glutamic acid residues. The CARD domain of ARC exhibited significant homology to the prodomains of apical caspases and the CARDs present in the cell death regulators Apaf-1 and RAIDD. Immunoprecipitation analysis revealed that ARC interacts with caspase-2, -8, and Caenorhabditis elegans CED-3, but not with caspase-1, -3, or -9. ARC inhibited apoptosis induced by caspase-8 and CED-3 but not that mediated by caspase-9. Further analysis showed that the enzymatic activity of caspase-8 was inhibited by ARC in 293T cells. Consistent with the inhibition of caspase-8, ARC attenuated apoptosis induced by FADD and TRADD and that triggered by stimulation of death receptors coupled to caspase-8, including CD95/Fas, tumor necrosis factor-R1, and TRAMP/DR3. Remarkably, the expression of human ARC was primarily restricted to skeletal muscle and cardiac tissue. Thus, ARC represents an inhibitor of apoptosis expressed in muscle that appears to selectively target caspases. Delivery of ARC by gene transfer or enhancement of its endogenous activity may provide a strategy for the treatment of diseases that are characterized by inappropriately increased cell death in muscle tissue.

Correlation studies between cytokines expressed in islets and autoimmune diabetes development in NOD mice and BB rats have demonstrated that beta-cell destructive insulitis is associated with increased expression of proinflammatory cytokines (IL-1, TNF alpha, and IFN alpha) and type 1 cytokines (IFN gamma, TNF beta, IL-2 and IL-12), whereas non-destructive (benign) insulitis is associated with increased expression of type 2 cytokines (IL-4 and IL-10) and the type 3 cytokine (TGF beta). Cytokines (IL-1, TNF alpha, TNF beta and IFN gamma) may be directly cytotoxic to beta-cells by inducing nitric oxide and oxygen free radicals in the beta-cells. In addition, cytokines may sensitize beta-cells to T-cell-mediated cytotoxicity in vivo by upregulating MHC class I expression on the beta-cells (an action of IFN gamma), and inducing Fas (CD95) expression on beta-cells (actions of IL-1, and possibly TNF alpha and IFN gamma). Transgenic expression of cytokines in beta-cells of non-diabetes-prone mice and NOD mice has suggested pathogenic roles for IFN alpha, IFN gamma, IL-2 and IL-10 in insulin-dependent diabetes mellitus (IDDM) development, and protective roles for IL-4, IL-6 and TNF alpha. Systemic administrations of a wide variety of cytokines can prevent IDDM development in NOD mice and/or BB rats; however, a given cytokine may retard or accelerate IDDM development, depending on the dose and frequency of administration, and the age and the diabetes-prone animal model studied (NOD mouse or BB rat). Islet-reactive CD4+ T-cell lines and clones that adoptively transfer IDDM into young NOD mice have a Th1 phenotype (IFN gamma-producing), but other islet-specific Th1 clones that produce TGF beta can adoptively transfer protection against IDDM in NOD mice. NOD mice with targeted deletions of IL-12 and IFN gamma genes still develop IDDM, albeit delayed and slightly less often. In contrast, post-natal deletions of IL-12 and IFN gamma, also IL-1, TNF alpha, IL-2, and IL-6--by systemic administrations of neutralizing antibodies, soluble receptors and receptor antagonists, and receptor-targeted cytotoxic drugs--significantly decrease IDDM incidence in NOD mice and/or BB rats. These cytokine deletion studies have provided the best evidence for pathologic roles for proinflammatory cytokines (IL-1, TNF alpha, and IL-6) and type 1 cytokines (IFN gamma, IL-2 and IL-12) in IDDM development.

This review covers recent advances of CD95 signaling. It focuses on CD95-interacting molecules, formation of the death inducing signaling complex and the role of caspases, particularly caspase-8, and their death substrates. We also discuss the relevance of mitochondria in the CD95-mediated apoptotic process and how viral proteins interfere with crucial steps of this signaling pathway.

Apoptosis occurs in the testis as an important physiological mechanism to limit the number of germ cells in the seminiferous epithelium. Sertoli cells, which tightly regulate germ cell proliferation and differentiation, are implicated in the control of germ cell apoptosis. Fas (APO-1, CD95), a transmembrane receptor protein, transmits an apoptotic signal within cells when bound by Fas ligand (FasL). The Fas system has been implicated in immune regulation, including cytotoxic T cell-mediated cytotoxicity, activation-induced suicide of T cells, and control of immune-privileged sites. Here we propose the Fas system as a key regulator of spermatogenesis. In this model, FasL expressed by Sertoli cells initiates the apoptotic death of germ cells expressing Fas. Using immunohistochemistry, we localized Fas to germ cells and FasL to Sertoli cells. The expression of these genes was dramatically up-regulated after exposure to mono-(2-ethylhexyl) phthalate and 2,5-hexanedione, two widely studied Sertoli cell toxicants known to induce germ cell apoptosis. Mouse germ cells in vitro were susceptible to anti-Fas antibody-induced death, and the survival of rat germ cells was increased after disruption of FasL by antisense oligonucleotide treatment. Unlike its expression in other tissues, testicular expression of Fas in the lpr mouse, a spontaneous mutant of the Fas gene, is similar to that in the normal mouse, arguing for the importance of the Fas system in maintaining testicular homeostasis. These data implicate the Sertoli cell in the paracrine control of germ cell output during spermatogenesis by a Fas-mediated pathway.

The p21 RAS subfamily of small GTPases, including KRAS, HRAS, and NRAS, regulates cell proliferation, cytoskeletal organization, and other signaling networks, and is the most frequent target of activating mutations in cancer. Activating germline mutations of KRAS and HRAS cause severe developmental abnormalities leading to Noonan, cardio-facial-cutaneous, and Costello syndrome, but activating germline mutations of NRAS have not been reported. Autoimmune lymphoproliferative syndrome (ALPS) is the most common genetic disease of lymphocyte apoptosis and causes autoimmunity as well as excessive lymphocyte accumulation, particularly of CD4(-), CD8(-) alphabeta T cells. Mutations in ALPS typically affect CD95 (Fas/APO-1)-mediated apoptosis, one of the extrinsic death pathways involving TNF receptor superfamily proteins, but certain ALPS individuals have no such mutations. We show here that the salient features of ALPS as well as a predisposition to hematological malignancies can be caused by a heterozygous germline Gly13Asp activating mutation of the NRAS oncogene that does not impair CD95-mediated apoptosis. The increase in active, GTP-bound NRAS augments RAF/MEK/ERK signaling, which markedly decreases the proapoptotic protein BIM and attenuates intrinsic, nonreceptor-mediated mitochondrial apoptosis. Thus, germline activating mutations in NRAS differ from other p21 Ras oncoproteins by causing selective immune abnormalities without general developmental defects. Our observations on the effects of NRAS activation indicate that RAS-inactivating drugs, such as farnesyltransferase inhibitors should be examined in human autoimmune and lymphocyte homeostasis disorders.

The nonpolymorphic soluble HLA-G1 (sHLA-G1) isoform has been reported to be secreted by trophoblast cells at the materno-fetal interface, suggesting that it may act as immunomodulator during pregnancy. In this paper, we report that affinity-purified beta2-microglobulin-associated sHLA-G1 triggered apoptosis in activated, but not resting CD8+ peripheral blood cells. We demonstrate by Western blotting that sHLA-G1 enhanced CD95 ligand expression in activated CD8+ cells. Cytotoxicity was inhibited by preincubation of the cells with a CD95 antagonist mAb (ZB4) or a soluble recombinant CD95-Fc, indicating that apoptosis is mediated through the CD95/CD95 ligand pathway. Finally, we show that such sHLA-G1-induced apoptosis depends on the interaction with CD8 molecules, with cell death being blocked by various CD8 mAbs.

Programmed cell death (apoptosis) of activated lymphocytes is critical to immune homeostasis. The cell surface protein Fas (CD95) and its ligand play a pivotal role in regulating lymphocyte apoptosis, and defective expression of either Fas or Fas ligand results in marked over accumulation of mature lymphocytes and autoimmune disease in mice. The results of recent studies suggest that defective lymphocyte apoptosis caused by mutations of the Fas gene can result in a severe autoimmune lymphoproliferative syndrome (ALPS) in humans. To define the clinical, genetic, and immunologic spectrum of ALPS, 9 patients and their families were extensively evaluated with routine clinical studies, lymphocyte phenotyping, genotyping, and in vitro assays for lymphocyte apoptosis. Individual patients were followed up for 3 months to 6 years. ALPS was identified in 9 unrelated children as manifested by moderate to massive splenomegaly and lymphadenopathy, hypergammaglobulinemia, autoimmunity, B-cell lymphocytosis, and the expansion of an unusual population of CD4- CD8- T cells that express the alpha/beta T-cell receptor (TCR). All patients showed defective lymphocyte apoptosis in vitro. Heterozygous mutations of the Fas gene were detected in 8 patients. One ALPS patient lacked a Fas gene mutation. Healthy relatives with Fas mutations were identified in 7 of 8 ALPS kindreds. These relatives also showed in vitro abnormalities of Fas-mediated lymphocyte apoptosis, but clinical features of ALPS were not present in the vast majority of these individuals. ALPS is a unique clinical syndrome in which in vitro abnormalities of lymphocyte apoptosis are associated with abnormal lymphoproliferation and autoimmunity. These findings provide evidence that apoptosis of activated lymphocytes is an important mechanism for maintaining immunologic homeostasis and self-tolerance in humans. Fas gene mutations account for impaired lymphocyte apoptosis in only a subset of patients with ALPS.

Apoptosis or programmed cell death has been reported after CNS trauma. However, the significance of this mechanism in the pathophysiology of spinal cord injury, in particular at the cervical level, requires further investigation. In the present study, we used the extradural clip compression model in the rat to examine the cellular distribution of apoptosis following cervical spinal cord injury, the relationship between glial apoptosis and post-traumatic axonal degeneration and the possible role of apo[apoptosis]-1, CD95 (FAS) and p75 in initiating post-traumatic glial apoptosis. In situ terminal-deoxy-transferase mediated dUTP nick end labeling revealed apoptotic cells, largely oligodendrocytes as identified by cell specific markers, in grey and white matter following spinal cord injury. Apoptotic cell death was confirmed using electron microscopy and by the demonstration of DNA laddering on agarose gel electrophoresis. Beta-amyloid precursor protein was used as a molecular marker of axonal degeneration on western blots and immunohistochemistry. Degeneration of axons was temporally and spatially co-localized with glial apoptosis. FAS and p75 protein expression was seen in astrocytes, oligodendrocytes and microglia, and was also seen in some apoptotic glia after cord injury. Both FAS and p75 increased in expression in a temporal course, which mirrored the development of cellular apoptosis. The downstream caspases 3 and 8, which are linked to FAS and p75, demonstrated activation at times of maximal apoptosis, while FLIP-L an inhibitor of caspase 8, decreased at times of maximal apoptosis. We conclude that axonal degeneration after traumatic spinal cord injury is associated with glial, in particular oligodendroglial, apoptosis. Activation of the FAS and p75 death receptor pathways may be involved in initiating this process.

Although anatomical barriers and soluble mediators have been implicated in immune privilege, it appears that the apoptotic cell death of Fas+ cells by tissue-associated CD95 ligand (Fas ligand, FasL) is an important component. One clinical example of the function of an immune privileged site is the success of human corneal transplants, where a very high percentage of transplants accept without tissue matching or immunosuppressive therapy. Since the mouse cornea expresses abundant Fas ligand and immune privilege has been implicated in the success of these transplants, we examined the role of FasL in corneal transplantation. Our results show that human corneas express functional FasL capable of killing Fas+ lymphoid cells in an in vitro culture system. Using a mouse model for corneal allograft transplantation, FasL+ orthografts were accepted at a rate of 45%, whereas FasL- grafts, or normal grafts transplanted to Fas- mice, were rejected 100% of the time. Histological analysis found that FasL+ grafts contained apoptotic mononuclear cells indicating the induction of apoptosis by the graft, while rejecting FasL- corneas contained numerous inflammatory cells without associated apoptosis. Taken together our results demonstrate that FasL expression on the cornea is a major factor in corneal allograft survival and, thus, we provide an explanation for one of the most successful tissue transplants performed in humans.

Signals from CD4+ T cells induce two opposite fates in B cells: clonal proliferation of B cells that bind specifically to foreign antigens and clonal deletion of equivalent B cells that bind self-antigens. This B cell fate decision is determined by the concerted action of two surface proteins on activated T cells, CD40-and Fas-ligands (CD40L and FasL), whose effects are switched by signals from the B cell antigen receptor (BCR). Foreign antigens that stimulate the BCR acutely cause CD40L and FasL to promote clonal proliferation. CD40L and FasL trigger deletion, however, when the BCRs become desensitized by chronic stimulation with self-antigens or when BCRs have not bound an antigen. The need for both Fas and CD40L to correctly regulate self-reactive B cell fate may explain the severe autoantibody disorders in Fas- or CD40L-deficient children.

Pentraxins are acute-phase proteins produced in vivo during inflammatory reactions. Classical short pentraxins, C-reactive protein, and serum amyloid P component are generated in the liver in response to interleukin (IL)-6. The long pentraxin PTX3 is produced in tissues under the control of primary proinflammatory signals, such as lipopolysaccharide, IL-1 beta, and tumor necrosis factor-alpha, which also promote maturation of dendritic cells (DCs). Cell death commonly occurs during inflammatory reactions. In this study, it is shown that PTX3 specifically binds to dying cells. The binding was dose dependent and saturable. Recognition was restricted to extranuclear membrane domains and to a chronological window after UV irradiation or after CD95 cross-linking-induced or spontaneous cell death in vitro. PTX3 bound to necrotic cells to a lesser extent. Human DCs failed to internalize dying cells in the presence of PTX3, while they took up normally soluble or inert particulate substrates. These results suggest that PTX3 sequesters cell remnants from antigen-presenting cells, possibly contributing to preventing the onset of autoimmune reactions in inflamed tissues. (Blood. 2000;96:4300-4306)

Early events required for induction of apoptosis by CD95 are preassociation of CD95, the formation of the death-inducing signaling complex (DISC) and clustering of CD95 in distinct membrane domains. Here, we identify the molecular ordering of these events and show that the acid sphingomyelinase (ASM) functions upstream of the DISC to mediate CD95 clustering in ceramide-enriched membrane platforms, an event that is required for DISC formation. Experiments in ASM-deficient cells revealed that CD95 ligation, in the absence of ceramide generation, triggers <1% of full caspase 8 activation at the receptor. This event, however, is both necessary and sufficient to trigger translocation of ASM onto the outer leaflet of the plasma membrane, ASM activation and ceramide release, but insufficient for apoptosis induction. Ceramide-mediated CD95 clustering then amplifies the primary CD95 signaling and drives the second step of CD95 signaling, that is, formation of the DISC yielding 100% caspase activity and apoptosis. These studies suggest that the most parsimonious interpretation of the molecular ordering of the earliest events in CD95 signaling, at least in some cells, is: CD95 ligation-->1% of maximum caspase 8 activation->>ASM translocation->>ceramide generation->>CD95 clustering->>DISC formation-->100% of maximum caspase 8 activation->>apoptosis.