(1/4475) JNK2 is required for efficient T-cell activation and apoptosis but not for normal lymphocyte development.

BACKGROUND: The Jun N-terminal kinase (JNK) signaling pathway has been implicated in cell proliferation and apoptosis, but its function seems to depend on the cell type and inducing signal. In T cells, JNK has been implicated in both antigen-induced activation and apoptosis. RESULTS: We generated mice lacking the JNK2 isozymes. The mutant mice were healthy and fertile but defective in peripheral T-cell activation induced by antibody to the CD3 component of the T-cell receptor (TCR) complex - proliferation and production of interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma) were reduced. The proliferation defect was restored by exogenous IL-2. B-cell activation was normal in the absence of JNK2. Activation-induced peripheral T-cell apoptosis was comparable between mutant and wild-type mice, but immature (CD4(+) CD8(+)) thymocytes lacking JNK2 were resistant to apoptosis induced by administration of anti-CD3 antibody in vivo. The lack of JNK2 also resulted in partial resistance of thymocytes to anti-CD3 antibody in vitro, but had little or no effect on apoptosis induced by anti-Fas antibody, dexamethasone or ultraviolet-C (UVC) radiation. CONCLUSIONS: JNK2 is essential for efficient activation of peripheral T cells but not B cells. Peripheral T-cell activation is probably required indirectly for induction of thymocyte apoptosis resulting from administration of anti-CD3 antibody in vivo. JNK2 functions in a cell-type-specific and stimulus-dependent manner, being required for apoptosis of immature thymocytes induced by anti-CD3 antibody but not for apoptosis induced by anti-Fas antibody, UVC or dexamethasone. JNK2 is not required for activation-induced cell death of mature T cells.  (+info)

(2/4475) Caspase 3 inactivation to suppress Fas-mediated apoptosis: identification of binding domain with p21 and ILP and inactivation machinery by p21.

The death mediator caspase acts as the dominant regulator during cell death induction. The CPP32 subfamily, including caspase 3 (CPP32/Yama/Apopain), is essential for the cell death signaling. We recently reported that activation of caspase 3 is regulated by complex formation with p21 or ILP. In the present study, we investigated the binding domain with p21 and ILP to further characterize the caspase 3 inactivation machinery. Our results show that caspase 3 contains p21 binding domain in the N-terminus and ILP binding domain in the active site. Further, the caspase 3 binding domain in p21 was independent of the Cdk- or PCNA-binding domain. We also found caspase 3 protection by p21 from the p3-site cleavage serineproteinase contributes to the suppression machinery. Here, we propose the caspase 3 inactivation system by p21 and ILP as new essential system in the regulation of cell death.  (+info)

(3/4475) Activation-dependent transcriptional regulation of the human Fas promoter requires NF-kappaB p50-p65 recruitment.

Fas (CD95) and Fas ligand (CD95L) are an interacting receptor-ligand pair required for immune homeostasis. Lymphocyte activation results in the upregulation of Fas expression and the acquisition of sensitivity to FasL-mediated apoptosis. Although Fas upregulation is central to the preservation of immunologic tolerance, little is known about the molecular machinery underlying this process. To investigate the events involved in activation-induced Fas upregulation, we have examined mRNA accumulation, fas promoter activity, and protein expression in the Jurkat T-cell line treated with phorbol myristate acetate and ionomycin (P/I), pharmacological mimics of T-cell receptor activation. Although resting Jurkat cells express Fas, Fas mRNA was induced approximately 10-fold in 2 h upon P/I stimulation. Using sequential deletion mutants of the human fas promoter in transient transfection assays, we identified a 47-bp sequence (positions -306 to -260 relative to the ATG) required for activation-driven fas upregulation. Sequence analysis revealed the presence of a previously unrecognized composite binding site for both the Sp1 and NF-kappaB transcription factors at positions -295 to -286. Electrophoretic mobility shift assay (EMSA) and supershift analyses of this region documented constitutive binding of Sp1 in unactivated nuclear extracts and inducible binding of p50-p65 NF-kappaB heterodimers after P/I activation. Sp1 and NF-kappaB transcription factor binding was shown to be mutually exclusive by EMSA displacement studies with purified recombinant Sp1 and recombinant p50. The functional contribution of the kappaB-Sp1 composite site in P/I-inducible fas promoter activation was verified by using kappaB-Sp1 concatamers (-295 to -286) in a thymidine kinase promoter-driven reporter construct and native promoter constructs in Jurkat cells overexpressing IkappaB-alpha. Site-directed mutagenesis of the critical guanine nucleotides in the kappaB-Sp1 element documented the essential role of this site in activation-dependent fas promoter induction.  (+info)

(4/4475) Antitumor effect of allogenic fibroblasts engineered to express Fas ligand (FasL).

Fas ligand is a type II transmembrane protein which can induce apoptosis in Fas-expressing cells. Recent reports indicate that expression of FasL in transplanted cells may cause graft rejection and, on the other hand, tumor cells may lose their tumorigenicity when they are engineered to express FasL. These effects could be related to recruitment of neutrophils by FasL with activation of their cytotoxic machinery. In this study we investigated the antitumor effect of allogenic fibroblasts engineered to express FasL. Fibroblasts engineered to express FasL (PA317/FasL) did not exert toxic effects on transformed liver cell line (BNL) or colon cancer cell line (CT26) in vitro, but they could abrogate their tumorigenicity in vivo. Histological examination of the site of implantation of BNL cells mixed with PA317/FasL revealed massive infiltration of polymorphonuclear neutrophils and mononuclear cells. A specific immune protective effect was observed in animals primed with a mixture of BNL or CT26 and PA317/FasL cells. Rechallenge with tumor cells 14 or 100 days after priming resulted in protection of 100 or 50% of animals, respectively. This protective effect was due to CD8+ cells since depletion of CD8+ led to tumor formation. In addition, treatment of pre-established BNL tumors with a subcutaneous injection of BNL and PA317/FasL cell mixture at a distant site caused significant inhibition of tumor growth. These data demonstrate that allogenic cells engineered with FasL are able to abolish tumor growth and induce specific protective immunity when they are mixed with neoplastic cells.  (+info)

(5/4475) Fas/Apo [apoptosis]-1 and associated proteins in the differentiating cerebral cortex: induction of caspase-dependent cell death and activation of NF-kappaB.

The developing cerebral cortex undergoes a period of substantial cell death. The present studies examine the role of the suicide receptor Fas/Apo[apoptosis]-1 in cerebral cortical development. Fas mRNA and protein are transiently expressed in subsets of cells within the developing rat cerebral cortex during the peak period of apoptosis. Fas-immunoreactive cells were localized in close proximity to Fas ligand (FasL)-expressing cells. The Fas-associated signaling protein receptor interacting protein (RIP) was expressed by some Fas-expressing cells, whereas Fas-associated death domain (FADD) was undetectable in the early postnatal cerebral cortex. FLICE-inhibitory protein (FLIP), an inhibitor of Fas activation, was also expressed in the postnatal cerebral cortex. Fas expression was more ubiquitous in embryonic cortical neuroblasts in dissociated culture compared to in situ within the developing brain, suggesting that the environmental milieu partly suppresses Fas expression at this developmental stage. Furthermore, FADD, RIP, and FLIP were also expressed by subsets of dissociated cortical neuroblasts in culture. Fas activation by ligand (FasL) or anti-Fas antibody induced caspase-dependent cell death in primary embryonic cortical neuroblast cultures. The activation of Fas was also accompanied by a rapid downregulation of Fas receptor expression, non-cell cycle-related incorporation of nucleic acids and nuclear translocation of the RelA/p65 subunit of the transcription factor NF-kappaB. Together, these data suggest that adult cortical cell number may be established, in part, by an active process of receptor-mediated cell suicide, initiated in situ by killer (FasL-expressing) cells and that Fas may have functions in addition to suicide in the developing brain.  (+info)

(6/4475) Clearance of Chlamydia trachomatis from the murine genital mucosa does not require perforin-mediated cytolysis or Fas-mediated apoptosis.

The molecular mechanisms of resistance to genital infection with the mouse pneumonitis (MoPn) strain of Chlamydia trachomatis are unknown. A role for major histocompatibility complex class II-restricted, interleukin-12-dependent CD4(+) T cells has been established, but the functional activity of these cells does not depend on secretion of gamma interferon. Here we examined the potential contribution of T-cell-mediated cytotoxicity and apoptosis to mucosal clearance of MoPn by using mice deficient in the molecular mediators of target cell lysis. Animals lacking perforin, Fas, Fas ligand, or both perforin and Fas ligand were infected genitally with C. trachomatis MoPn and monitored for expression of immunity to chlamydial antigens and clearance of MoPn from the genital mucosa. In each case, the profile of spleen cytokine production, the magnitude of the host antibody response, and the kinetics of chlamydial clearance were similar to those of genetically intact controls. Compensatory overproduction of tumor necrosis factor alpha, an alternate mediator of apoptosis in certain cell types, did not appear to account for the ability of mutant mice to resolve Chlamydia infections. These results fail to support CD4(+) T-cell-mediated apoptosis or CD8(+) T-cell-mediated cytotoxicity as being critical to the clearance of C. trachomatis MoPn urogenital infections.  (+info)

(7/4475) In vitro induction of activation-induced cell death in lymphocytes from chronic periodontal lesions by exogenous Fas ligand.

Periodontitis is a chronic inflammatory disease which gradually destroys the supporting tissues of the teeth, leading to tooth loss in adults. The lesions are characterized by a persistence of inflammatory cells in gingival and periodontal connective tissues. To understand what mechanisms are involved in the establishment of chronic lesions, we hypothesized that infiltrating lymphocytes might be resistant to apoptosis. However, both Bcl-2 and Bcl-xL were weakly detected in lymphocytes from the lesions, compared with those from peripheral blood, suggesting that these cells are susceptible to apoptosis. Nevertheless, very few apoptotic cells were observed in tissue sections from the lesions. Lymphocytes from the lesions expressed mRNA encoding Fas, whereas Fas-ligand mRNA was very weakly expressed in lymphocytes from the lesions and in periodontal tissues. Since the results indicated that lymphocytes in the lesions might be susceptible to Fas-mediated apoptosis but lack the death signal, we next investigated if these lymphocytes actually undergo apoptosis by the addition of anti-Fas antibodies in vitro. Fas-positive lymphocytes from the lesions underwent apoptosis by these antibodies, but Fas-negative lymphocytes and Fas-positive peripheral lymphocytes did not undergo apoptosis by these antibodies. These results indicate that lymphocytes in the lesions are susceptible to activation-induced cell death and are induced to die by apoptosis after the addition of exogenous Fas ligand.  (+info)

(8/4475) Fas and Fas ligand interaction induces apoptosis in inflammatory myopathies: CD4+ T cells cause muscle cell injury directly in polymyositis.

OBJECTIVE: To investigate the involvement of the Fas/Fas ligand (Fas/FasL) system in the inflammatory myopathies. METHODS: Frozen muscle sections obtained from 7 patients with polymyositis (PM), 4 patients with dermatomyositis (DM), and 3 controls were studied by immunochemistry. Apoptosis was detected by DNA electrophoresis and in situ labeling using the TUNEL method. RESULTS: Fas was detected on muscle fibers and infiltrating mononuclear cells (MNC) in 6 PM patients and 2 DM patients. FasL was expressed mainly on CD4+ T cells and some CD8+ T cells, and on macrophages surrounding Fas-positive muscles in 4 PM patients and 1 DM patient. In 3 of the 5 patients with FasL-positive MNC, the TUNEL method showed that both invaded myonuclei and MNC underwent apoptosis. Chromosomal DNA from the muscle tissue of these patients showed ladder formation. CONCLUSION: Fas/FasL is involved in muscle cell apoptosis in at least 2 of the inflammatory myopathies, PM and DM. Although CD8+-mediated cytotoxicity is thought to be the main mechanism of muscle injury in PM, our data suggest that CD4+ T cells also directly cause muscle cell damage.  (+info)