Inhibition of B cell receptor-mediated apoptosis by IFN.
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IFNs are a family of cytokines that are involved in the regulation of immune and inflammatory responses. Clinical use of IFN-alpha/beta encompasses treatment for a variety of diseases; however, prolonged exposure to IFN-alpha/beta results in elevated levels of autoreactive Abs. In this study, we investigated the potential of IFNs to modulate apoptotic signals in B cells. We demonstrate that IFN-alpha or IFN-beta inhibit Ag receptor-mediated apoptosis in a dose-dependent manner. Inhibition of phosphatidylinositol 3' (PI3)-kinase did not abolish the effect of IFN, indicating that the antiapoptotic mechanism is PI3-kinase- and protein kinase B/Akt-independent. Instead, IFN-alpha and IFN-beta, but not IFN-gamma, significantly increase the levels of the survival protein Bcl-2, and to a lesser extent, Bcl-xL expression. Thus, IFN-alpha/beta-mediated inhibition of B cell Ag receptor-triggered apoptosis may offer a model for the process that leads to the escape of self-reactive B cells from negative selection and consequently results in autoantibody production. (+info)
Tumor necrosis factor-alpha and lipopolysaccharide induce apoptotic cell death in bovine glomerular endothelial cells.
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BACKGROUND: The glomerular endothelial cell is a specialized microvascular cell type involved in the regulation of glomerular ultrafiltration. During gram-negative sepsis, glomerulonephritis, and acute renal failure, bacterial lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha) may cause severe cell damage. Our aim was to study and compare the direct effects of TNF-alpha and LPS on the induction of apoptosis in bovine glomerular endothelial cells. METHODS: Primary bovine glomerular endothelial cells were stimulated with TNF-alpha or LPS, and apoptotic cell death was investigated by DNA fragmentation analysis, morphological studies, measurement of cytochrome c efflux and mitochondrial permeability transition, Bak, Bad, Bax, Bcl-2, Bcl-xL protein expression, and caspase-3-like protease activity. RESULTS: TNF-alpha, as well as LPS, elicited apoptotic cell death both time and concentration dependently. Along with DNA ladder formation, we detected the formation of 50 kbp high molecular weight DNA fragments, nuclear condensation, and mitochondrial permeability transition. Concerning all parameters, LPS signaling proved to be more rapid than TNF-alpha. Mechanistically, TNF-alpha-induced cell death was preceded by an efflux of mitochondrial cytochrome c into the cytosol and, subsequently, by a marked increase in the proapoptotic protein Bak and a decrease in the anti-apoptotic Bcl-xL protein content. Comparable but more pronounced effects were seen with LPS. Later, caspase-3-like protease activity was first detectable after 10 hours and was continuously increased up to 24 hours in both TNF-alpha- and LPS-stimulated cells. Correspondingly, we detected an extended cleavage of the nuclear enzyme poly(ADP-ribose) polymerase. Caspase inhibitors Z-Asp-CH2-DCB and Z-VAD-fmk blocked both TNF-alpha- and LPS-induced apoptosis in a comparable manner. Only Z-Asp-CH2-DCB was able to block apoptotic cell death completely. CONCLUSION: Both bacterial LPS and TNF-alpha potently induced apoptotic cell death in glomerular endothelial cells. Direct endotoxin-induced apoptosis may therefore be relevant in the progression of acute renal failure, which is a frequent complication of gram-negative sepsis. (+info)
Induction of cell death by tumour necrosis factor (TNF) receptor 2, CD40 and CD30: a role for TNF-R1 activation by endogenous membrane-anchored TNF.
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Several members of the tumour necrosis factor receptor (TNF-R) superfamily can induce cell death. For TNF-R1, Fas/APO-1, DR3, DR6, TRAIL-R1 and TRAIL-R2, a conserved 'death domain' in the intracellular region couples these receptors to activation of caspases. However, it is not yet known how TNF receptor family members lacking a death domain, such as TNF-R2, CD40, LT-betaR, CD27 or CD30, execute their death-inducing capability. Here we demonstrate in different cellular systems that cytotoxic effects induced by TNF-R2, CD40 and CD30 are mediated by endogenous production of TNF and autotropic or paratropic activation of TNF-R1. In addition, stimulation of TNF-R2 and CD40 synergistically enhances TNF-R1-induced cytotoxicity. These findings describe a novel pro-apoptotic mechanism induced by some members of the TNF-R family. (+info)
Human fetal retinal pigment epithelial cells induce apoptosis in the T-cell line Jurkat.
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PURPOSE: To investigate the mechanism(s) involved in human fetal retinal pigment epithelium (HFRPE)-mediated T-cell death. METHODS: Pure HFRPE cells were isolated and cultured. Normal and interferon (IFN)-gamma-activated HFRPE from early and late in vitro passages were incubated with cells from the human T-cell leukemia line Jurkat (Jkt). Cultures were pulsed with [3H]-thymidine to measure Jkt cell proliferation. Jkt cells were evaluated for apoptosis either by staining with an ethidium bromide/acridine orange mixture (AO/EB) or with Annexin V-phycoerythrin. The role of Fas ligand (FasL) molecule in HFRPE-mediated apoptosis was assessed by using a mutant Jkt cell line (DD3), which is deficient in Fas-mediated signaling. The involvement of the antiapoptotic human gene bcl-xL was determined by using Jkt cells that were stably transfected with bcl-x(L). The role of cell- cell contact in the induction of apoptosis was evaluated in a transwell system in the presence or absence of neutralizing antibodies against IFN-gamma and tumor necrosis factor (TNF)-alpha. RESULTS: HFRPE cells inhibited the proliferation of Jkt cells by inducing apoptosis through a FasL-independent pathway. Passaging and IFN-gamma activation strengthened the inhibitory effect of HFRPE cells on the proliferation of Jkt cells. At lower HFRPE passages (P2), bcl-alphaL, overexpression rescued the HFRPE cell-mediated apoptosis. The separation of the cells by the transwell system did not affect the HFRPE cell-mediated suppression. This suppressive effect was not mediated by the secretion of IFN-gamma or TNF-alpha molecules. CONCLUSIONS: HFRPE cells suppressed the proliferation of Jkt cells by inducing apoptosis. HFRPE cells induced a stronger inhibitory effect on Jkt cells at higher in vitro passages. The HFRPE-induced apoptosis was not mediated through the FasL/Fas pathway or through the secretion of the apoptosis-inducing cytokines IFN-gamma and TNF-alpha. The bcl-xL gene may play a role in preventing HFRPE cell-induced apoptosis in Jkt cells. These combined results suggest that the HFRPE-mediated suppression of primary T cells may also be mediated by the induction of apoptosis. Therefore, the retinal pigment epithelium may play a role in the induction of immune privilege in the subretinal space. (+info)
bcl-x prevents apoptotic cell death of both primitive and definitive erythrocytes at the end of maturation.
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bcl-x is a member of the bcl-2 gene family, which regulates apoptotic cell death in various cell lineages. There is circumstantial evidence suggesting that bcl-x might play a role in the apoptosis of erythroid lineage cells, although there is no direct evidence. In this study, we used Bcl-X null mouse embryonic stem (ES) cells, and showed that Bcl-X is indispensable for the production of both embryonic primitive erythrocytes (EryP) and adult definitive erythrocytes (EryD) at the end of their maturation. In vivo, bcl-x-/- ES cells did not contribute to circulating EryD in adult chimeric mice that were produced by blastocyst microinjection of the bcl-x-/- ES cells. bcl-x-/- EryP and EryD were produced by in vitro differentiation induction of ES cells on macrophage colony-stimulating factor-deficient stromal cell line OP9, and further analysis was carried out. The emergence of immature EryP and EryD from bcl-x-/- ES cells was similar to that from bcl-x+/+ ES cells. However, prominent cell death of bcl-x-/- EryP and EryD occurred when the cells matured. The data show that the antiapoptotic function of bcl-x acts at the very end of erythroid maturation. (+info)
Death signals from the B cell antigen receptor target mitochondria, activating necrotic and apoptotic death cascades in a murine B cell line, WEHI-231.
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B cell antigen receptor (BCR)-mediated cell death has been proposed as a mechanism for purging the immune repertoire of anti-self specificities during B cell differentiation in bone marrow. Mitochondrial alterations and activation of caspases are required for certain aspects of apoptotic cell death, but how the mitochondria and caspases contribute to BCR-mediated cell death is not well understood. In the present study, we used the mouse WEHI-231 B cell line to demonstrate that mitochondrial alterations and activation of caspases are indeed participants in BCR-mediated cell death. The peptide inhibitor of caspases, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), blocked cleavage of poly(ADP-ribose) polymerase and various manifestation of nuclear apoptosis such as nuclear fragmentation, hypodiploidy and DNA fragmentation, indicating that signals from the BCR induced the activation of caspases. In addition, z-VAD-fmk delayed apoptosis-associated changes in cellular reduction-oxidation potentials as determined by hypergeneration of superoxide anion, as well as exposure of phosphatidylserine residues in the outer plasma membrane. By contrast, although z-VAD-fmk retarded cytolysis, it was incapable of preventing disruption of the plasma membrane even under the same condition in which it completely blocked nuclear apoptosis. Mitochondrial membrane potential loss was also not blocked by z-VAD-fmk. Bongkrekic acid, a specific inhibitor of mitochondrial permeability transition pores, suppressed not only the mitochondrial membrane potential but also the change of plasma membrane permeability. Overexpression of Bcl-xL prevented mitochondrial dysfunction, nuclear apoptosis and membrane permeability cell death triggered by BCR signal transduction. These observations indicate that death signals from BCR may first cause mitochondrial alterations followed by activation of both necrotic and apoptotic cascades. (+info)
Caspases induce cytochrome c release from mitochondria by activating cytosolic factors.
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We investigated the ability of caspases (cysteine proteases with aspartic acid specificity) to induce cytochrome c release from mitochondria. When Jurkat cells were induced to undergo apoptosis by Fas receptor ligation, cytochrome c was released from mitochondria, an event that was prevented by the caspase inhibitor, zVAD-fmk (zVal-Ala-Asp-CH2F). Purified caspase-8 triggered rapid cytochrome c release from isolated mitochondria in vitro. The effect was indirect, as the presence of cytosol was required, suggesting that caspase-8 cleaves and activates a cytosolic substrate, which in turn is able to induce cytochrome c release from mitochondria. The cytochrome c releasing activity was not blocked by caspase inhibition, but was antagonized by Bcl-2 or Bcl-xL. Caspase-8 and caspase-3 cleaved Bid, a proapoptotic Bcl-2 family member, which gains cytochrome c releasing activity in response to caspase cleavage. However, caspase-6 and caspase-7 did not cleave Bid, although they initiated cytochrome c release from mitochondria in the presence of cytosol. Thus, effector caspases may cleave and activate another cytosolic substrate (other than Bid), which then promotes cytochrome c release from mitochondria. Mitochondria significantly amplified the caspase-8 initiated DEVD-specific cleavage activity. Our data suggest that cytochrome c release, initiated by the action of caspases on a cytosolic substrates, may act to amplify a caspase cascade during apoptosis. (+info)
GATA-1 and erythropoietin cooperate to promote erythroid cell survival by regulating bcl-xL expression.
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The transcription factor GATA-1 is essential for normal erythropoiesis. By examining in vitro-differentiated embryonic stem cells, we showed previously that in the absence of GATA-1, committed erythroid precursors fail to complete maturation and instead undergo apoptosis. The mechanisms by which GATA-1 controls cell survival are unknown. Here we report that in erythroid cells, GATA-1 strongly induces the expression of the anti-apoptotic protein bcl-xL, but not the related proteins bcl-2 and mcl-1. Consistent with a role for bcl-xL in mediating GATA-1-induced erythroid cell survival, in vitro-differentiated bcl-xL-/- embryonic stem cells fail to generate viable mature definitive erythroid cells, a phenotype resembling that of GATA-1 gene disruption. In addition, we show that erythropoietin, which is also required for erythroid cell survival, cooperates with GATA-1 to stimulate bcl-xL gene expression and to maintain erythroid cell viability during terminal maturation. Together, our data show that bcl-xL is essential for normal erythroid development and suggest a regulatory hierarchy in which bcl-xL is a critical downstream effector of GATA-1 and erythropoietin-mediated signals. (+info)