Expression of apoptotic regulatory molecules in renal cell carcinoma: elevated expression of Fas ligand.
(25/2716)
Renal cell carcinoma (RCC) is the most common renal neoplasm. Despite being infiltrated by tumour infiltrating lymphocytes (TIL), these TIL are unable to control tumour growth in vivo, suggesting that the cytotoxic capacity of TIL against RCC is impaired, or that the tumour cells are resistant to killing and therefore escape detection by the immune system. It is postulated that the expression of apoptotic regulatory molecules in RCC favours tumour cell survival. The present study has therefore determined the expression of Fas (APO-1/CD95), Fas ligand (Fas L) and bcl-2 in these tumours. The expression of Fas, Fas L and bcl-2 mRNA transcripts was determined in RCC, normal kidney and peripheral blood by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), following RNA extraction and cDNA synthesis from tissues and cell samples. Transcript levels were measured by densitometry after Southern blot hybridization of PCR products with internal radio-labelled oligonucleotide probes; a densitometry score was assigned to each hybridizing DNA band and expressed as a ratio of the glyceraldehyde-3-phosphate dehydrogenase content. In peripheral blood, the expression of Fas L and bcl-2 transcripts was similar between patients and normal healthy individuals; however, Fas transcript expression was significantly down-regulated in the patients' versus normal peripheral blood (P = 0.026). Most interestingly, significantly up-regulated Fas L expression was observed in RCC compared to normal kidney (P = 0.041). In contrast, bcl-2 transcripts were well represented in normal kidney but markedly decreased in RCC (P = 0.021). The expression of Fas transcripts in normal kidney and RCC was variable. These data demonstrate elevated expression of Fas L transcripts in RCC, but the functional relevance of this remains to be investigated. (+info)
Does HIV cause depletion of CD4+ T cells in vivo by the induction of apoptosis?
(26/2716)
The central pathogenic feature of AIDS is the dramatic loss of CD4+ lymphocytes. Despite more than a decade of intense research, the exact mechanism by which HIV causes this is still not understood. A major model for T cell depletion, proposed originally by Ameison and Capron in a report published in 1991, is that HIV sensitizes CD4+ T cells for activation-induced apoptosis. The apoptotic model of T cell depletion is discussed, and experiments that address the questions of whether apoptosis is restricted to infected cells or 'bystander' T cells, and whether T cell apoptosis requires participation of separate HIV-infected haematopoietic cell populations, are reviewed. (+info)
Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.
(27/2716)
Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/ threonine kinase Akt, which then phosphorylates and inactivates components of the apoptotic machinery, including BAD and Caspase 9. In this study, we demonstrate that Akt also regulates the activity of FKHRL1, a member of the Forkhead family of transcription factors. In the presence of survival factors, Akt phosphorylates FKHRL1, leading to FKHRL1's association with 14-3-3 proteins and FKHRL1's retention in the cytoplasm. Survival factor withdrawal leads to FKHRL1 dephosphorylation, nuclear translocation, and target gene activation. Within the nucleus, FKHRL1 triggers apoptosis most likely by inducing the expression of genes that are critical for cell death, such as the Fas ligand gene. (+info)
The extracellular signal-regulated kinase pathway is required for activation-induced cell death of T cells.
(28/2716)
T cells can undergo activation-induced cell death (AICD) upon stimulation of the T cell receptor-CD3 complex. We found that the extracellular signal-regulated kinase (ERK) pathway is activated during AICD. Transient transfection of a dominant interfering mutant of mitogen-activated/extracellular signal-regulated receptor protein kinase kinase (MEK1) demonstrated that down-regulation of the ERK pathway inhibited FasL expression during AICD, whereas activation of the ERK pathway with a constitutively active MEK1 resulted in increased expression of FasL. We also found that pretreatment with the specific MEK1 inhibitor PD98059 prevented the induction of FasL expression during AICD and inhibited AICD. However, PD98059 had no effect on other apoptotic stimuli. We found only very weak ERK activity during Fas-mediated apoptosis (induced by Fas cross-linking). Furthermore, preincubation with the MEK1 inhibitor did not inhibit Fas-mediated apoptosis. Finally, we also demonstrated that pretreatment with the MEK1 inhibitor could delay and decrease the expression of the orphan nuclear steroid receptor Nur77, which has been shown to be essential for AICD. In conclusion, this study demonstrates that the ERK pathway is required for AICD of T cells and appears to regulate the induction of Nur77 and FasL expression during AICD. (+info)
Bystander target cell lysis and cytokine production by dengue virus-specific human CD4(+) cytotoxic T-lymphocyte clones.
(29/2716)
Dengue hemorrhagic fever, the severe form of dengue virus infection, is believed to be an immunopathological response to a secondary infection with a heterologous serotype of dengue virus. Dengue virus capsid protein-specific CD4(+) cytotoxic T-lymphocyte (CTL) clones were shown to be capable of mediating bystander lysis of non-antigen-presenting target cells. After activation by anti-CD3 or in the presence of unlabeled antigen-presenting target cells, these clones could lyse both Jurkat cells and HepG2 cells as bystander targets. Lysis of HepG2 cells suggests a potential role for CD4(+) CTL in the liver involvement observed during dengue virus infection. Three CD4(+) CTL clones were demonstrated to lyse cognate, antigen-presenting target cells by a mechanism that primarily involves perforin, while bystander lysis occurred through Fas/Fas ligand interactions. In contrast, one clone used a Fas/Fas ligand mechanism to lyse both cognate and bystander targets. Cytokine production by the CTL clones was also examined. In response to stimulation with D2 antigen, CD4(+) T-cell clones produced gamma interferon, tumor necrosis factor alpha (TNF-alpha) and TNF-beta. The data suggest that CD4(+) CTL clones may contribute to the immunopathology observed upon secondary dengue virus infections through direct cytolysis and/or cytokine production. (+info)
Antiretroviral cytolytic T-lymphocyte nonresponsiveness: FasL/Fas-mediated inhibition of CD4(+) and CD8(+) antiviral T cells by viral antigen-positive veto cells.
(30/2716)
C57BL/6 (H-2(b)) mice generate type-specific cytolytic T-lymphocyte (CTL) responses to an immunodominant Kb-restricted epitope, KSPWFTTL located in the membrane-spanning domain of p15TM of AKR/Gross murine leukemia viruses (MuLV). AKR.H-2(b) congenic mice, although carrying the responder H-2(b) major histocompatibility complex (MHC) haplotype, are low responders or nonresponders for AKR/Gross MuLV-specific CTL, apparently due to the presence of inhibitory AKR. H-2(b) cells. Despite their expression of viral antigens and Kb, untreated viable AKR.H-2(b) spleen cells cause dramatic inhibition of the C57BL/6 (B6) antiviral CTL response to in vitro stimulation with AKR/Gross MuLV-induced tumor cells. This inhibition is specific (AKR.H-2(b) modulator spleen cells do not inhibit allogeneic MHC or minor histocompatibility antigen-specific CTL production), dependent on direct contact of AKR.H-2(b) cells in a dose-dependent manner with the responder cell population, and not due to soluble factors. Here, the mechanism of inhibition of the antiviral CTL response is shown to depend on Fas/Fas-ligand interactions, implying an apoptotic effect on B6 responder cells. Although B6.gld (FasL-) responders were as sensitive to inhibition by AKR.H-2(b) modulator cells as were B6 responders, B6.lpr (Fas-) responders were largely insensitive to inhibition, indicating that the responder cells needed to express Fas. A Fas-Ig fusion protein, when added to the in vitro CTL stimulation cultures, relieved the inhibition caused by the AKR.H-2(b) cells if the primed responders were from either B6 or B6.gld mice, indicating that the inhibitory AKR.H-2(b) cells express FasL. Because of the antigen specificity of the inhibition, these results collectively implicate a FasL/Fas interaction mechanism: viral antigen-positive AKR.H-2(b) cells expressing FasL inhibit antiviral T cells ("veto" them) when the AKR.H-2(b) cells are recognized. Consistent with this model, inhibition by AKR.H-2(b) modulator cells was MHC restricted, and resulted in approximately a 10- to 70-fold decrease in the in vitro expansion of pCTL/CTL. Both CD8(+) CTL and CD4(+) Th responder cells were susceptible to inhibition by FasL+ AKR.H-2(b) inhibitory cells as the basis for inhibition. The CTL response in the presence of inhibitory cells could be restored by several cytokines or agents that have been shown by others to interfere with activation-induced cell death (e.g. , interleukin-2 [IL-2], IL-15, transforming growth factor beta, lipopolysaccharide, 9-cis-retinoic acid) but not others (e.g., tumor necrosis factor alpha). These results raise the possibility that this type of inhibitory mechanism is generalized as a common strategy for retrovirus infected cells to evade immune T-cell recognition. (+info)
Adenovirus-mediated delivery of fas ligand inhibits intimal hyperplasia after balloon injury in immunologically primed animals.
(31/2716)
BACKGROUND: Adenoviral constructs have been used for studies of injury-induced vascular hyperplasia in immunologically naive laboratory animals, but their usefulness for intra-arterial gene therapy may be limited by the prevalence of preexisting immunity to adenovirus in the patient population. Here, we explored the efficacy of adenovirus-mediated transfer of Fas ligand, a cytotoxic gene with immunomodulatory properties, in inhibiting injury-induced vascular lesion formation in both naive and immunologically primed animals. METHODS AND RESULTS: Lesion formation was evaluated in balloon-injured carotid arteries of naive and adenovirus-immunized rats that were infected with adenoviral constructs expressing Fas ligand (Ad-FasL), the cyclin-dependent kinase inhibitor p21 (Ad-p21), or beta-galactosidase (Ad-betagal). In naive rats, Ad-FasL induced apoptosis in medial vascular smooth muscle cells and inhibited intimal hyperplasia by 60% relative to Ad-betagal-treated vessels (P<0.05), whereas the cytostatic agent Ad-p21 decreased lesion size by 58% (P<0.05). In animals preimmunized with an adenoviral vector containing no transgene, Ad-FasL significantly inhibited neointima formation (73% reduction, P<0.05), but Ad-p21 failed to inhibit neointima formation relative to controls. Immunologically primed rats displayed robust T-cell infiltration in Ad-p21- and Ad-betagal-treated vessels, but T-cell infiltration was markedly attenuated in Ad-FasL-treated vessels. CONCLUSIONS: Our data demonstrate that adenovirus-mediated Fas ligand delivery can inhibit intimal hyperplasia in both immunologically primed and naive animals, whereas the efficacy of an adenovirus-mediated p21 delivery is limited to immunologically naive animals. This study documents, for the first time, the therapeutic efficacy of intravascular adenoviral gene transfer in animals with preexisting immunity to adenovirus. (+info)
Co-expression of Fas and Fas-ligand on the surface of influenza virus-infected cells.
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Influenza virus-infected cultured cells undergo apoptosis after an increment of Fas (APO-1/CD95) on the cell surface. By flow cytometry, cell surface Fas-ligand was detected in virus-infected cells with a time course similar to that of Fas. Moreover, Fas and Fas-ligand were co-expressed in those cells. The mode of induction, however, appeared to be distinct for the two proteins. Influenza virus infection induced the externalization of phosphatidylserine on the cell surface at the early stage of apoptosis, an event that has been observed in cells undergoing Fas-mediated apoptosis. In fact, apoptosis of the virus-infected cells was inhibited in the presence of an antagonistic anti-Fas-ligand monoclonal antibody. These results suggest that influenza virus infection causes augmented expression of both Fas and Fas-ligand and apoptosis is induced when the infected cells come into contact with each other. (+info)