OBJECTIVE

We investigated the potential relationship between T-cell phenotype, inflammation, endotoxemia, and atherosclerosis evaluated by carotid intima-media thickness (IMT) in a cohort of HIV-positive patients undergoing long-term virologically suppressive combination antiretroviral therapy (cART).

METHODS

We studied 163 patients receiving virologically suppressive cART.

METHODS

We measured IMT (carotid ultrasound); CD4+/CD8+ T-cell activation (CD38, CD45R0), differentiation (CD127), apoptosis (CD95), and senescence (CD28, CD57) (flow cytometry); plasma sCD14, IL-6, TNF- α, sVCAM-1, hs-CRP, anti-CMV IgG (ELISA); LPS (LAL). The results were compared by Mann-Whitney, Kruskal-Wallis or Chi-square tests, and factors associated with IMT were evaluated by multivariable logistic regression.

RESULTS

Of 163 patients, 112 demonstrated normal IMT (nIMT), whereas 51 (31.3%) had pathological IMT (pIMT: ≥1 mm). Of the patients with pIMT, 22 demonstrated an increased IMT (iIMT), and 29 were shown to have plaques. These patient groups had comparable nadir and current CD4+, VLs and total length of time on cART. Despite similar proportions of CD38-expressing CD8+ cells (p = .95), pIMT patients exhibited higher activated memory CD8+CD38+CD45R0+ cells (p = .038) and apoptotic CD4+CD95+ (p = .01) and CD8+CD95+ cells (p = .003). In comparison to nIMT patients, iIMT patients tended to have lower numbers of early differentiated CD28+CD57- memory CD4+ (p = .048) and CD28-CD57-CD8+ cells (p = .006), both of which are associated with a higher proliferative potential. Despite no differences in plasma LPS levels, pIMT patients showed significantly higher circulating levels of sCD14 than did nIMT patients (p = .046). No differences in anti-CMV IgG was shown. Although circulating levels of sCD14 seemed to be associated with a risk of ATS in an unadjusted analysis, this effect was lost after adjusting for classical cardiovascular predictors.

CONCLUSIONS

Despite the provision of full viral suppression by cART, a hyperactivated, pro-apoptotic T-cell profile characterizes HIV-infected patients with early vascular damage, for whom the potential contribution of subclinical endotoxemia and anti-CMV immunity should be investigated further.

BACKGROUND

Loss of CD95 expression in tumour cells occurs frequently in colon carcinoma and may be associated with disease progression. On the other hand, neo-expression of CD95L in tumour cells may contribute to immune evasion.

OBJECTIVE

We aimed at further exploring the functional role and prognostic significance of the CD95/CD95L death inducing system in colon carcinomas.

METHODS

CD95 and CD95L expression was examined by immunohistochemistry in 128 R0 resected UICC (International Union against Cancer) stage II/III colon carcinomas and correlated with disease free survival.

RESULTS

CD95 expression in tumour cells was observed in only 30 carcinomas (23.4%) whereas the others had at least a minor subpopulation of CD95 negative cells. Loss of CD95 in tumour cells was related to adverse prognosis in uni- and multivariate analysis (p = 0.046 and p = 0.036, respectively). Tumour infiltrating lymphocytes (TIL) were the major source of CD95L in colon carcinomas. CD95L+TIL were present in 83% of cases whereas CD95L was found in tumour cells in only 12% of cases. Moreover, a high rate of CD95L+TIL correlated with prolonged disease free survival in patients with UICC stage II (p = 0.05) but not in those with stage III.

CONCLUSIONS

Loss of CD95 in tumour cells may be an independent prognostic factor in colon carcinomas. The CD95L counterattack is not a relevant feature in colon carcinoma but CD95L+TIL may contribute to tumour control in the early stages of the disease, exerting a concurrent selection pressure in the direction of CD95 abrogation/resistance.

The extrinsic apoptosis pathway is activated when certain members of the tumor necrosis factor (TNF) receptor superfamily (TNFRSF) are oligomerized by their cognate ligands that are members of the TNF superfamily (TNFSF). The apoptosis-inducing capacity of a member of the TNFRSF relies on the presence of a death domain (DD) in the intracellular portion of the receptor protein. Such receptors are also referred to as death receptors. Binding of a TNFSF ligand to a TNFRSF receptor that is expressed on the surface of a cell results in the formation of a receptor proximal protein complex. This protein complex is the platform for further signaling events within the cell. In case of death receptors like TNF-related apoptosis-inducing ligand receptor 1 (TRAIL-R1/DR4), TRAIL-R2 (KILLER/APO-2/DR5/TRICK), CD95 (Fas, APO-1), or TNF receptor 1 (TNF-R1), this complex is termed death-inducing signaling complex (DISC). The compositions of the various DISCs have been intensively studied in the last 12 years. For the CD95 and the TRAIL-R1/R2 DISCs, it is now clear that the adaptor protein Fas-associated DD protein (FADD) forms part of these complexes and is necessary for recruitment of the proapoptotic signaling molecules caspase-8 and caspase-10. Recruitment of these proteases allows for their activation at the DISC and subsequent induction of apoptosis. The caspase-8 homologous cellular FLICE-like inhibitory protein (cFLIP) can also be recruited to the DISC. cFLIP acts as an anti-apoptotic regulator by interfering with activation of caspases 8 and 10 at the DISC. Interestingly, treatment of TRAIL-resistant tumor cells with conventional chemotherapeutic drugs or with proteasome inhibitors renders these cells sensitive for TRAIL-induced apoptosis. By applying the methodology of the biochemical analysis of the TRAIL DISC described here, we were able to show that this sensitization is mainly due to changes in the biochemical composition of the DISC as the apoptosis-initiating protein complex of the extrinsic pathway.

Members of the tumour necrosis factor (TNF) receptor family exert pleiotropic effects and can trigger both apoptosis and proliferation [1]. In their cytoplasmic region, some of these receptors share a conserved sequence motif - the 'death domain' - which is required for transduction of the apoptotic signal by recruiting other death-domain-containing adaptor molecules like the Fas-associated protein FADD/MORT1 or the TNF receptor-associated protein TRADD [2-4]. FADD links the receptor signal to the activation of the caspase family of cysteine proteases [5,6]. Functional inactivation of individual receptor family members often fails to exhibit a distinctive phenotype, probably because of redundancy [7-9]. To circumvent this problem, we used a dominant-negative mutant of FADD (FADD-DN) which should block all TNF receptor family members that use FADD as an adaptor. We established transgenic mice expressing FADD-DN under the influence of the lck promoter and investigated the consequences of its expression in T cells. As expected, FADD-DN thymocytes were protected from death induced by CD95 (Fas/Apo1), whereas apoptosis induced by ultraviolet (UV) irradiation, anti-CD3 antibody treatment or dexamethasone was unaffected, as was spontaneous cell death. Surprisingly, however, we also observed profound inhibition of thymocyte proliferation in vivo and of activation-induced proliferation of thymocytes and mature T cells in vitro. This inhibition of proliferation was not due to increased cell death and appeared to be p53 dependent.

UVB-induced DNA damage is a crucial event in UVB-mediated apoptosis. On the other hand, UVB directly activates death receptors on the cell surface including CD95, implying that UVB-induced apoptosis can be initiated at the cell membrane through death receptor clustering. This study was performed to measure the relative contribution of nuclear and membrane effects in UVB-induced apoptosis of the human epithelial cell line HeLa. UVB-mediated DNA damage can be reduced by treating cells with liposomes containing the repair enzyme photolyase followed by exposure to photoreactivating light. Addition of photolyase followed by photoreactivation after UVB reduced the apoptosis rate significantly, whereas empty liposomes had no effect. Likewise, photoreactivating treatment did not affect apoptosis induced by the ligand of CD95, CD95L. UVB exposure at 4 degrees C, which prevents CD95 clustering, also reduced the apoptosis rate, but to a lesser extent. When cells were exposed to UVB at 4 degrees C and treated with photolyase plus photoreactivating light, UVB-induced apoptosis was almost completely prevented. Inhibition of caspase-3, a downstream protease in the CD95 signaling pathway, blocked both CD95L and UVB-induced apoptosis, whereas blockage of caspase-8, the most proximal caspase, inhibited CD95L-mediated apoptosis completely, but UVB-induced apoptosis only partially. Although according to these data nuclear effects seem to be slightly more effective in mediating UVB-induced apoptosis than membrane events, both are necessary for the complete apoptotic response. Thus, this study shows that nuclear and membrane effects are not mutually exclusive and that both components contribute independently to a complete response to UVB.

Although ischemia-reperfusion of mouse kidney is known to cause severe renal failure due to tubular cell death, the exact cellular mechanism responsible for this phenomenon is not clear. To investigate the spatial and temporal development of renal cell death and the role of Fas/APO-1/CD95 (Fas) in this process, the left renal vessels were occluded in a group of mice for 30, 60, or 120 min followed by reperfusion for 24 h (n = 4 for each group). Analysis of the isolated DNA in agarose-gel electrophoresis revealed a typical ladder pattern of bands consisting of multiples of 180 to 200 bp, considered the hallmark of apoptosis. The intensity of the bands increased proportionately with the duration of ischemia. Histochemical analysis using terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling showed the presence of nuclei with DNA double-strand breaks specifically in distal renal tubules of the outer medulla. The presence of apoptosis was also confirmed by electron microscopy. Analysis of total RNA by Northern blotting revealed one appropriate-sized band for Fas mRNA in the normal kidney, which intensified in the ischemia-reperfused kidney. Moreover, nonradioactive in situ hybridization revealed that distal renal tubular epithelial cells were positive for Fas mRNA in the outer medulla. Fas antigen was also localized to the renal tubular epithelial cells of the outer medulla by immunohistochemistry. The number of apoptotic cells in the ischemia-reperfusion kidney of the lpr/lpr mouse was low. These findings strongly indicate that ischemia-reperfusion of the kidney induces apoptosis of a specific area of tubular epithelial cells in the outer medulla through the Fas system.

Fas/CD95/Apo-1 is a cell surface receptor that transduces apoptotic death signals following activation and has been implicated in triggering apoptosis in infected or damaged cells in disease states. Apoptosis is a major mechanism of neuronal loss following hypoxic-ischemic injury to the developing brain, although the role of Fas in this process has not been studied in detail. In the present study, we have investigated the expression and function of Fas in neuronal cells in vitro and in vivo. Fas was found to be expressed in the 14 day old rat brain, with strongest expression in the cortex, hippocampus and cerebellum. Cross-linking of Fas induced neuronal apoptosis both in neuronal PC12 cells in culture and following intracerebral injection in vivo, indicating that neuronal Fas was functional as a death receptor. This death was shown to be caspase dependent in primary neuronal cultures and was blocked by the selective caspase 8 inhibitor IETD. Finally, cerebral hypoxia-ischemia resulted in a strong lateralised upregulation of Fas in the hippocampus, that peaked six to twelve hours after the insult and was greater on the side of injury. These results suggest that Fas may be involved in neuronal apoptosis following hypoxic-ischemic injury to the developing brain.

Vitamin E derivative, RRR-alpha-tocopheryl succinate (vitamin E succinate, VES), is a potent pro-apoptotic agent, inducing apoptosis by restoring both transforming growth factor-beta (TGF-beta) and Fas (CD95) apoptotic signaling pathways that contribute to the activation of c-Jun N-terminal kinase (JNK)-mediated apoptosis. Objectives of these studies were to characterize signaling events involved in the pro-apoptotic actions of a naturally occurring form of vitamin E, delta-tocotrienol, and a novel vitamin E analog, alpha-tocopherol ether acetic acid analog [alpha-TEA; 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxyacetic acid]. Like VES, alpha-TEA and delta-tocotrienol induced estrogen-nonresponsive MDA-MB-435 and estrogen-responsive MCF-7 human breast cancer cells to undergo high levels of apoptosis in a concentration- and time-dependent fashion. Like VES, the two compounds induced either no or lower levels of apoptosis in normal human mammary epithelial cells and immortalized but nontumorigenic human MCF-10A cells. The pro-apoptotic mechanisms triggered by the structurally distinct alpha-TEA and delta-tocotrienol were identical to those previously reported for VES, that is, alpha-TEA- and delta-tocotrienol-induced apoptosis involved up-regulation of TGF-beta receptor II expression and TGF-beta-, Fas- and JNK-signaling pathways. These data provide a better understanding of the anticancer actions of a dietary form of vitamin E (delta-tocotrienol) and a novel nonhydrolyzable vitamin E analog (alpha-TEA).

Central nervous system (CNS) destruction in spinal cord injury (SCI) is caused by a complex series of cellular and molecular events. Recent studies have concentrated on signaling by receptors in the tumor necrosis factor receptor (TNFR) superfamily that mediate diverse biological outcomes ranging from inflammation to apoptosis. From the perspective of basic science research, understanding how receptor signaling mediates these divergent responses is critical in clarifying events underlying irreversible cell injury in clinically relevant models of SCI. From a clinical perspective, this work also provides novel targets for the development of therapeutic agents that have the potential to protect the spinal cord from irreversible damage and promote functional recovery. In this review, we discuss how the formation of alternate signaling complexes and receptor membrane localization after SCI can influence life and death decisions of cells stimulated through two members of the TNFR superfamily, Fas/CD95 and TNFR1.

IFN-gamma inhibits the growth and differentiation of erythroid precursor cells and mediates hemopoietic suppression through mechanisms that are not completely understood. We found that treatment of human erythroid precursor cells with IFN-gamma up-regulates the expression of multiple members of the TNF family, including TRAIL and the recently characterized protein TWEAK. TWEAK and its receptor fibroblast growth factor-inducible 14 (Fn14) were expressed by purified erythroblasts at all the stages of maturation. Exposure to recombinant TWEAK or agonist anti-Fn14 Abs was able to inhibit erythroid cell growth and differentiation through caspase activation. Because other members of the TNF family such as TRAIL and CD95 ligand (CD95L) are known to interfere with erythroblast growth and differentiation, we investigated the role of different TNF/TNFR family proteins as potential effectors of IFN-gamma in the immature hemopoietic compartment. Treatment of erythroid precursor cells with agents that blocked either TRAIL, CD95L, or TWEAK activity was partially able to revert the effect of IFN-gamma on erythroid proliferation and differentiation. However, the simultaneous inhibition of TRAIL, TWEAK, and CD95L resulted in a complete abrogation of IFN-gamma inhibitory effects, indicating the requirement of different receptor-mediated signals in IFN-gamma-mediated hemopoietic suppression. These results establish a new role for TWEAK and its receptor in normal and IFN-gamma-mediated regulation of hematopoiesis and show that the effects of IFN-gamma on immature erythroid cells depend on multiple interactions between TNF family members and their receptors.

CD8+ T cells are effective mediators of immunity against Listeria monocytogenes, but the mechanisms by which they provide antilisterial immunity are poorly understood. CD8+ T cells efficiently lyse target cells in vitro by at least two independent pathways. To test the hypothesis that CD8+ T cell-mediated immunity to L. monocytogenes is dependent on perforin or CD95 (Fas, Apo-1), we used C57BI/6 (B6) and perforin-deficient (PO) mice to generate CD8+ T cell lines specific for the L. mono cytogenes-encoded Ag listeriolysin O (LLO). Both lines specifically produce IFN-gamma and TNF-alpha, and mediate target cell lysis in vitro. Cytolysis mediated by the PO-derived CD8+ T cell line is delayed relative to the B6-derived line and is completely inhibited by anti-CD95 Abs. In vivo, PO-derived CD8+ T cells provide specific antilisterial immunity in B6 hosts, CD95-deficient hosts, and IFN-gamma-depleted hosts. However, PO-derived CD8+ T cells fail to provide antilisterial immunity in hosts depleted of TNF-alpha. These results indicate that single Ag-specific CD8+ T cells derived from PO mice can mediate antilisterial immunity by a mechanism that is independent of CD95 or IFN-gamma, but requires TNF-alpha.

Autoantigens found on pancreatic islets can move to draining lymph nodes, where they are able to cause the activation and consequent deletion of autoreactive T cells by a mechanism termed cross-tolerance. This deletion depends on signalling through CD95 (also known as Fas), a member of the superfamily of tumour-necrosis-factor receptors. Here we describe a new mechanism that protects against autoimmunity: this mechanism involves another member of this superfamily, CD30, whose function was largely unknown. CD30-deficient islet-specific CD8-positive T cells are roughly 6,000-fold more autoaggressive than wild-type cells, with the transfer of as few as 160 CD30-deficient T cells leading to the complete destruction of pancreatic islets and the rapid onset of diabetes. We show that, in the absence of CD30 signalling, cells activated but not yet deleted by the CD95-dependent cross-tolerance mechanism gain the ability to proliferate extensively upon secondary encounter with antigen on parenchymal tissues, such as the pancreatic islets. Thus, CD30 signalling limits the proliferative potential of autoreactive CD8 effector T cells and protects the body against autoimmunity.

The expression and function of Fas (CD95/APO-1), a cell surface receptor directly responsible for triggering cell death by apoptosis, was investigated on human T lymphocytes resident within the intestinal lamina propria, a major site of antigen challenge and persistent lymphocyte activation. Three color immunofluorescence and FACS analysis indicated that virtually all freshly isolated human gut lamina propria T lymphocytes (T-LPL) express Fas, together with the marker of progress activation CD45R0. A discrete fraction of freshly isolated T-LPL also constitutively expressed Fas ligand (FasL), perhaps as a result of recent in vivo activation. Importantly, whereas Fas cross-linking did not result in apoptosis induction in peripheral blood T lymphocytes (T-PBL), Fas was found to be fully effective in generating the apoptotic signal in T-LPL. This was associated with the activation of an acidic sphingomyelinase and with ceramide generation, early events known to be involved in Fas-mediated apoptotic signaling. By contrast, acidic sphingomyelinase activation and ceramide production were not detectable in T-PBL after Fas cross-linking. However C2-ceramide, a cell permeant synthetic analog of ceramide, could efficiently induce apoptosis in T-LPL and T-PBL when added exogenously. These data indicate that T-LPL constitutively express both Fas and FasL and that Fas cross-linking generates signals resulting in sphingomyelin hydrolysis and apoptosis, outlining a potential mechanism involved in intestinal tolerance. Moreover, they provide the first evidence of a role for ceramide-mediated pathways in normal immunoregulation.

Phytohemagglutinin-activated peripheral CD95+ T cells (day 1 T cells) are resistant to CD95-mediated apoptosis. After prolonged interleukin-2 treatment, these T cells become CD95-mediated apoptosis-sensitive (day 6 T cells). To elucidate the molecular mechanism of apoptosis resistance, day 1 and day 6 T cells were tested for formation of the CD95 death-inducing signaling complex (DISC). DISC-associated active Fas-associated DD protein (FADD)-like interleukin-1 beta-converting enzyme-like protease (FLICE) also referred to as MACH/caspase 8 was only found in apoptosis-sensitive day 6 T cells. Further-analysis of mRNA and protein expression levels of apoptosis-signaling molecules FADD, receptor interacting protein, hematopoietic cell protein tyrosine phosphatase, Fas-associated phosphatase-1, FLICE, bel-2, bcl-xL, and, bax-alpha showed that only the expression level of bcl-xL correlated with T cell resistance to CD95-mediated apoptosis (day 1 T cells: bcl-xhiL; day 6 T cells: bcl-XloL). In T cells activated in vitro, up-regulation of bcl-xL, has previously been correlated with general apoptosis resistance. However, the experiments presented suggest that resistance to CD95-mediated apoptosis in T cells can also be regulated at the level of recruitment of FLICE to the DISC.

To evaluate the role of ceramide (Cer) in apoptosis signaling, we examined Cer formation induced by CD95, etoposide, or gamma-radiation (IR) in relation to caspase activation and mitochondrial changes in Jurkat T cells. The Cer response to all three stimuli was mapped in between caspases sensitive to benzoyloxycarbonyl-VAD-fluoromethylketone (zVAD-fmk) and acetyl-DEVD-aldehyde (DEVD-CHO). Cer production was independent of nuclear fragmentation but associated with the occurrence of other aspects of the apoptotic morphology. Caspase-8 inhibition abrogated Cer formation and apoptosis induced by CD95 but did not affect the response to etoposide or IR, placing CD95-induced Cer formation downstream from caspase-8 and excluding a role for caspase-8 in the DNA damage pathways. CD95 signaling to the mitochondria required caspase-8, whereas cytochrome c release in response to DNA damage was caspase-independent. These results indicate that the caspases required for the Cer response to etoposide and IR reside at or downstream from the mitochondria. Bcl-2 overexpression abrogated the Cer response to etoposide and IR and reduced CD95-induced Cer accumulation. We conclude that the Cer response to DNA damage fully depends on mitochondrion-dependent caspases, whereas the response to CD95 partially relies on these caspases. Our data imply that Cer is not instrumental in the activation of inducer caspases or signaling to the mitochondria. Rather, Cer formation is associated with the execution phase of apoptosis.

Ceramide has been shown to be capable to trigger apoptosis in almost any cell, including tumor cells. Ceramide is generated by a de novo pathway or by sphingomyelinases. Sphingomyelinases hydrolyze sphingomyelin in biological membranes to release ceramide and they are named acid, neutral and alkaline sphingomyelinase depending on their maximum activity at acid, neutral and alkaline pH values, respectively. Stimuli that trigger a release of ceramide to mediate apoptosis include CD95, TNF-receptor, DR5, gamma-irradiation, cytotoxic drugs, UV-light, bacteria, viruses, some forms of developmental death, anti-CD20 and disruption of the cell's contact with its matrix, to name a few. Here, we will focus on the role of acid sphingomyelinase in malignant tumors, which function in apoptosis is best characterized and documented by genetic models. We will discuss concepts that unify the biological actions of ceramide and describe the role of ceramide in important anti-tumor treatment modalities, such as gamma-irradiation and chemotherapy.

The CD95 signaling pathway comprises proteins that contain one or two death effector domains (DED), such as FADD/Mort1 or caspase-8. Here we describe a novel 37 kDa protein, DEDD, that contains an N-terminal DED. DEDD is highly conserved between human and mouse (98. 7% identity) and is ubiquitously expressed. Overexpression of DEDD in 293T cells induced weak apoptosis, mainly through its DED by which it interacts with FADD and caspase-8. Endogenous DEDD was found in the cytoplasm and translocated into the nucleus upon stimulation of CD95. Immunocytological studies revealed that overexpressed DEDD directly translocated into the nucleus, where it co-localizes in the nucleolus with UBF, a basal factor required for RNA polymerase I transcription. Consistent with its nuclear localization, DEDD contains two nuclear localization signals and the C-terminal part shares sequence homology with histones. Recombinant DEDD binds to both DNA and reconstituted mononucleosomes and inhibits transcription in a reconstituted in vitro system. The results suggest that DEDD is a final target of a chain of events by which the CD95-induced apoptotic signal is transferred into the nucleolus to shut off cellular biosynthetic activities.

We prepared mAbs specific for the mouse Fas Ag (CD95) and used them to analyze the expression and apoptosis-inducing activity of the Fas Ag on murine immunocytes. Cytofluorometry of mouse bone marrow, thymus, and splenocytes using the mAbs indicated that cells of the T lineage, except for bone marrow cells, expressed Fas Ag on the surface. CD4-CD8- undifferentiated thymocytes expressed low levels of Fas Ag. Immature CD4+CD8+ thymocytes and mature CD4+CD8- and CD4-CD8+ thymocytes were highly positive for Fas Ag. CD4+CD8+ thymocytes were specifically sensitive to the apoptosis-inducing activity of anti-Fas, although CD4-CD8-, CD4+CD8-, and CD4-CD8+ thymocytes were resistant. Spleen T cells were resistant to anti-Fas, whereas they expressed Fas Ag. The superantigen, staphylococcal enterotoxin B (SEB) administered to BALB/c mice, induced clonal expansion and successive clonal deletion of spleen T cells bearing the V beta 8 TCR, which specifically reacts to SEB. Such clonal deletion of V beta 8 T cells was highly suppressed in lpr mice, which have defects in the Fas Ag gene. In SEB-administrated BALB/c mice, expression of Fas Ag was significantly enhanced on V beta 8, but not on V beta 6 T cells, which cannot react to SEB. Moreover, V beta 8 T cells in SEB-primed mice were sensitive to the cell-killing activity of anti-Fas, although V beta 6 T cells were resistant. These findings show that the expression level and apoptosis-inducing activity of Fas Ag on peripheral T cells are directly up-regulated by stimulation through the TCR in vivo.

The tumor suppressor p53 protein supports growth arrest and is able to induce apoptosis, a signaling cascade regulated by sequential activation of caspases. Mechanisms that lead from p53 to activation of individual initiator caspases are still unclear. The present model for caspase-2 activation includes PIDDosome complex formation. However, in certain experimental models, elimination of complex constituents PIDD or RAIDD did not significantly influence caspase-2 activation, suggesting the existence of an alternative activation platform for caspase-2. Here we have investigated the link between p53 and caspase-2 in further detail and report that the latter is able to utilize the CD95 DISC as an activation platform. The recruitment of caspase-8 to this complex is required for activation of caspase-2. In the experimental system used, the DISC is formed through a distinct, p53-dependent upregulation of CD95. Moreover, we show that caspase-2 and -8 cleave Bid, and that both act simultaneously upstream of mitochondrial cytochrome c release. Finally, a direct interaction between the two caspases and the ability of caspase-8 to cleave caspase-2 are demonstrated. Thus, the observed functional link between caspase-8 and -2 within the DISC represents an alternative mechanism to the PIDDosome for caspase-2 activation in response to DNA damage.

BACKGROUND

Polychromatic flow cytometry (PFC) allows the simultaneous determination of multiple antigens in the same cell, resulting in the generation of a high number of subsets. As a consequence, data analysis is the main difficulty with this technology. Here we show the use of cluster analysis (CA) and principal component analyses (PCA) to simplify multicolor data visualization and to allow subjects' classification.

METHODS

By eight-colour cytofluorimetric analysis, we investigated the T cell compartment in donors of different age (young, middle-aged, and centenarians). T cell subsets were identified by combining positive and negative expression of antigens. The resulting data set was organized into a matrix and subjected to CA and PCA.

RESULTS

CA clustered people of different ages on the basis of cytofluorimetric profile. PCA of the cellular subsets identified centenarians within a different cluster from young donors, while middle-aged donors were scattered between these groups. These approaches identified T cell phenotypes that changed with increasing age. In young donors, memory T cell subsets tended to be CD127+ and CD95- whereas CD127-, CD95+ phenotypes were found at higher frequencies in people with advanced age.

CONCLUSIONS

Our data suggest the use of bioinformatic approaches to analyze large data-sets generated by PFC and to obtain the rapid identification of key populations that best characterize a group of subjects.

METHODS

We identified BetA as a new cytotoxic agent active against neuroectodermal tumor cells including neuroblastoma, medulloblastoma, glioblastoma and Ewing sarcoma cells, representing the most common solid tumors of childhood.

RESULTS

BetA induced apoptosis by a direct effect on mitochondria independent of accumulation of wild-type p53 protein and independent of death-inducing ligand/receptor systems such as CD95. Mitochondrial perturbations on treatment with BetA resulted in the release of soluble apoptogenic factors such as cytochrome c or AIF from mitochondria into the cytosol, where they induced activation of caspases. Overexpression of the anti-apoptotic proteins Bcl-2 or Bcl-X(L) that blocked loss of the mitochondrial membrane potential and cytochrome c release from mitochondria also conferred resistance to BetA. Most importantly, BetA exhibited potent antitumor activity on neuroblastoma cells resistant to CD95- or doxorubicin-triggered apoptosis and on primary tumor cells from patients with neuroectodermal tumors.

CONCLUSIONS

Thus, BetA may be a promising new agent in the treatment of neuroectodermal tumors including neuroblastoma in vivo.

Fas (CD95) is a membrane surface receptor, which, in the lungs, is expressed in macrophages, neutrophils, and epithelial cells. In mice, Fas activation leads to a form of lung injury characterized by increased alveolar permeability. We investigated whether Fas-mediated lung injury occurs primarily as a result of Fas activation in myeloid cells (such as macrophages) or in nonmyeloid cells (such as epithelial cells). Chimeric mice lacking Fas in either myeloid or nonmyeloid cells were generated by transplanting marrow cells from lpr mice (which lack Fas) into lethally irradiated C57BL/6 mice (MyFas(-) group) or vice versa (MyFas(+) group). Additional mice transplanted with marrow cells from their same strain served as controls (Fas(+) ctr and Fas(-) ctr groups). Sixty days after transplantation, the mice received intratracheal instillations of the Fas-activating mAb Jo2 (n = 10/group), or an isotype control Ab (n = 10/group), and were euthanized 24-h later. Only animals expressing Fas in nonmyeloid cells (Fas(+) ctr and MyFas(-)) showed significant increases in lung neutrophil content and in alveolar permeability. These same mice showed tissue evidence of lung injury and caspase-3 activation in cells of the alveolar walls. Despite differences in the neutrophilic response and lung injury, there was no statistical difference in the lung cytokine concentrations (KC and MIP-2) among groups. We conclude that Fas-mediated lung injury requires expression of Fas on nonmyeloid cells of the lungs. These findings suggest that the alveolar epithelium is the primary target of Fas-mediated acute lung injury, and demonstrate that apoptotic processes may be associated with neutrophilic inflammation.

Ultraviolet irradiation is a major environmental cause of skin cancers, whereas ultraviolet-induced DNA repair and apoptosis are defense mechanisms that rescue and/or protect keratinocytes from this risk. Multiple pathways are involved in ultraviolet-induced keratinocyte apoptosis, including activation of p38-mitogen-activated protein kinase, protein kinase C, and CD95, each of which are associated with caspase activation. Alternatively, ceramides could serve as ultraviolet-induced, second messenger lipids, because they induce cell cycle arrest and apoptosis in a variety of cell types, including keratinocytes. We investigated the role of ceramide versus caspase, and the responsible pathway for ceramide generation in ultraviolet B-induced apoptosis of cultured normal human keratinocytes maintained in low calcium (0.07 mm) medium. Ultraviolet B (40 mJ per cm2) significantly inhibited cultured normal human keratinocyte proliferation, assessed as [3H-methyl]thymidine-thymidine incorporation into DNA, 2 h after irradiation. Terminal nick deoxynucleotide end-labeling-positive apoptotic cells (14.8% at 24 h and 34.4% at 48 h) and trypan blue-positive apoptotic cells (8.4% at 24 h and 28.6% at 48 h) became evident in a time-dependent manner after ultraviolet B irradiation, in parallel with activation of caspase-3. The ceramide content of irradiated cultured normal human keratinocytes increased significantly by 8 h, whereas glucosylceramide only modestly increased, and sphingomyelin content remained unaltered. Metabolic studies with radiolabeled serine, palmitic acid, and phosphorylcholine revealed that the ultraviolet B-induced increase in ceramide results primarily from increased de novo synthesis rather than accelerated sphingomyelin hydrolysis. Increased ceramide synthesis, in turn, could be attributed to increased activity of ceramide synthase (i.e., 1.7-fold increase 8 h after ultraviolet B irradiation), whereas serine palmitoyltransferase activity did not change. Both fumonisin B1, an inhibitor of ceramide synthase, and ISP-1, myriocin an inhibitor of serine palmitoyltransferase, significantly attenuated the ultraviolet B-induced apoptosis in a caspase-3-independent fashion, whereas co-incubation with a caspase-3 inhibitor (Ac-DEVD-chloromethyl-ketone) further attenuated the ultraviolet B-induced apoptosis. Thus, increased de novo ceramide synthesis signals ultraviolet B-induced apoptosis, by a pathway independent of, but in concert with, caspase-3 activation.

Two pathways have been implicated in the induction of apoptosis by cytotoxic T cells: the granule exocytosis pathway and a pathway using CD95 (Fas/APO-1). To test whether apoptosis induced by either of these pathways could be blocked by Bcl-2, we exposed bcl-2-transfected cells to CTL derived from normal, perforin-deficient, or CD95 ligand mutant (gld) mice. Although the levels of Bcl-2 expression achieved were able to protect FDC-P1 and Yac-1 transfectants from a variety of apoptotic stimuli, the cells were not protected from cytolysis mediated by CTL from any of these sources, by NK cells, or granules isolated from CTL. However, Bcl-2 expression significantly inhibited apoptosis induced by purified granzyme B and perforin. These results suggest that while Bcl-2 is capable of inhibiting the apoptotic pathway utilized by perforin and granzyme B, other granule components can bypass this block. We conclude that CTL harbor potent killing mechanism(s) in addition to those provided by CD95 ligand or perforin and granzyme B that cannot be overcome by Bcl-2.