Apoptosis induced by DNA damage and other stresses can proceed via expression of Fas ligand (FasL) and ligation of its receptor, Fas (CD95). We report that activation of the two transcription factors NF-kappa B and AP-1 is crucially involved in FasL expression induced by etoposide, teniposide, and UV irradiation. A nondegradable mutant of I kappa B blocked both FasL expression and apoptosis induced by DNA damage but not Fas ligation. These stimuli also induced the stress-activated kinase pathway (SAPK/JNK), which was required for the maximal induction of apoptosis. A 1.2 kb FasL promoter responded to DNA damage, as well as coexpression with p65 Rel or Fos/Jun. Mutations in the relevant NF-kappa B and AP-1 binding sites eliminated these responses. Thus, activation of NF-kappa B and AP-1 contributes to stress-induced apoptosis via the expression of FasL.
The folate antagonist methotrexate (MTX) is extensively used in graft-versus-host disease, rheumatoid arthritis, and other chronic inflammatory disorders. In addition to its antiinflammatory activity associated with increased release of adenosine, MTX exerts antiproliferative properties by inhibition of dihydrofolate reductase and other folate-dependent enzymes. However, the mechanisms of immunosuppressive properties associated with low-dose MTX treatments are still elusive. We report here that MTX (0.1-10 M) induces apoptosis of in vitro activated T cells from human peripheral blood. PBL exposed to MTX for 8 h, then activated in drug-free medium, underwent apoptosis, which was completely abrogated by addition of folinic acid or thymidine. Apoptosis of activated T cells did not require interaction between CD95 (Fas, APO-1) and its ligand, and adenosine release accounted for only a small part of this MTX activity. Apoptosis required progression of activated T cells to the S phase of the cell cycle, as it was prevented by drugs or anti-
Naive murine B cells are known to proliferate and differentiate in response to LPS or CpG, which bind to TLR4 and TLR9, respectively. However, the naive murine B cell compartment is heterogeneous and comprises four different B cell subsets: B-1a, B-1b, marginal zone (MZ), and follicular (FO) B cells. B-1a, B-1b, and MZ B cells are specialized in the response to thymus-independent Ag, and FO B cells are involved in the response to thymus-dependent Ag. This study was undertaken to compare those four naive B cell subsets for their responses to TLR agonists. Quantitative RT-PCR analysis revealed that expression of TLR transcripts differs quantitatively but not qualitatively from one subset to the other. All TLR agonists, with the exception of flagellin and poly(I:C), stimulate B cell proliferation whatever the subset considered. However, TLR ligation leads to massive differentiation of B-1 and MZ B cells into mature plasma cells (PC) but only marginally promotes PC differentiation of FO B cells. Moreover, TLR stimulation strongly up-regulates expression of Blimp-1 and XBP-1S, two transcription factors known to be instrumental in PC differentiation, in B-1 and MZ B cells but not in FO B cells. Altogether, our findings suggest that B-1 and MZ B cells are poised to PC differentiation in response to the microbial environment and that TLR agonists can be instrumental in stimulating Ab-mediated innate immune protection during microbial infections.
Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus that has recently been associated with necrotizing pneumonia. In the present study, we report that in vitro, PVL induces polymorphonuclear cell death by necrosis or by apoptosis, depending on the PVL concentration. PVL-induced apoptosis was associated with a rapid disruption of mitochondrial homeostasis and activation of caspase-9 and caspase-3, suggesting that PVL-induced apoptosis is preferentially mediated by the mitochondrial pathway. Polymorphonuclear cell exposure to PVL leads to mitochondrial localization of the toxin, whereas Bax, 1 of the 2 essential proapoptotic members of the Bcl-2 family, was still localized in the cytosol. Addition of PVL to isolated mitochondria induced the release of the apoptogenic proteins cytochrome c and Smac/ DIABLO. Therefore, we suggest that PVL, which belongs to the pore-forming toxin family, could act at the mitochondrion level by creating pores in the mitochondrial outer membrane. Furthermore, LukS-PV, 1 of the 2 components of PVL, was detected in lung sections of patients with necrotizing pneumonia together with DNA fragmentation, suggesting that PVL induces apoptosis in vivo and thereby is directly involved in the pathophysiology of necrotizing pneumonia.
T cell receptor engagement activates transcription factors important for cytokine gene regulation. Additionally, this signaling pathway also leads to activationinduced apoptosis in T lymphocytes that is dependent on FasL transcription and expression. Here we demonstrate that nuclear factor B (NF-B), which is involved in the transcriptional regulation of many cytokine genes expressed in activated lymphocytes, also plays a role in T cell activation-induced FasL expression. Inhibition of NF-B activity in a T cell hybridoma leads to decreased FasL expression and apoptosis upon T cell receptor stimulation. We identified the NF-B site in the FasL promoter that contributes to such regulation. Co-expression of p65 (Rel A) with the FasL promoter enhanced its activity, and co-expression of IB dramatically inhibited the inducible promoter activity. In contrast, the transcription factor AP-1 is not required for activation-induced FasL promoter activity. These results define a role for NF-B in mediating FasL expression during T cell activation. Activation-induced cell death (AICD)1 is a major mechanism to maintain immune homeostasis. AICD occurs in mature T lymphocytes to limit antigen-specific responses. Upon clearing antigens and/or pathogens from the host, activated T cells are deleted via the activation of apoptosis, which we and others have shown to be dependent upon Fas ligand (FasL, CD95 ligand) expression and ligation of its receptor, Fas (CD95) (1-5). However, despite our understanding of the importance of FasL in controlling homeostasis, little is known about its transcriptional regulation.T cell receptor ligation activates a cascade of signaling events which leads to the activation of several transcription factors that regulate cytokine gene expression including interleukin-2 (IL-2) (6). Signaling events post-TCR ligation and phospholipase C-␥1 lead to two distinct signals: activation of the protein kinase C pathway and release of intracellular Ca 2ϩ stores, subsequent Ca 2ϩ influx, and the activation of the serine phosphatase, calcineurin (7). These two signals can be mimicked by treatment of cells with the pharmacological agents phorbol myristate acetate and ionomycin. Calcineurin-mediated activation of the nuclear factor of activated T cells (NF-AT) and its role in cytokine gene expression is well established (7-9), and more recently it was shown that NF-AT sites in the FasL promoter are important for T cell activation-induced FasL expression (10).Like NF-AT, nuclear factor B (NF-B) is also involved in the transcriptional regulation of the interleukin 2␣ receptor (11) and the IL-2 genes (12). NF-B is present in the cytoplasm as two major precursor forms either as a RelA⅐p50 complex with the inhibiting IB or as a heterodimeric RelA⅐p105 complex (13). Release of the RelA-p50 from its inhibitor, IB, is dependent on the phosphorylation of IB (14), and recently, the kinases that phosphorylate IB have been identified (15). NF-B is then translocated into the nucleus as active heterodimers consisting of Rel A (p65), Re...
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