Nitric oxide protects cultured rheumatoid synovial cells from Fas-induced apoptosis by inhibiting caspase-3

Nitric oxide (NO) is elevated in the synovial fluids and sera of patients with rheumatoid arthritis (RA) and is thought to be an important proinflammatory mediator in the rheumatoid synovium. To test the hypothesis that NO might modulate the apoptosis-inducing signal pathway, we investigated the effects of NO on rheumatoid synovial-cell apoptosis induced by Fas ligation with anti-Fas antibody. Pretreatment of synovial cells with the NO donor S-nitro-N-acetylpenicillamine (SNAP) prevented the Fas-mediated induction of apoptosis. The activation of caspase-3 was required to mediate Fas-induced synovial cell apoptosis. The NO donor SNAP inhibited Fas-induced caspase-3 activation in rheumatoid synovial cells. However, NO did not interrupt Fas-induced caspase-8 cleavage or subsequent cytochrome c release into the cytosol in rheumatoid synovial cells. These data indicate that NO prevents apoptosis in rheumatoid synovial cells by directly inhibiting caspase-3 activation. Thus, we propose that NO interferes with cell death signal transduction and may contribute to rheumatoid synovial cell proliferation by inhibiting induction of apoptosis.     

CD4+ T-cell-mediated cytotoxicity against staphylococcal enterotoxin B-pulsed synovial cells.

Apoptosis of synovial cells in rheumatoid arthritis (RA) synovium determined in vivo is suggested to counteract the overgrowth of synovium. Immunohistological examination has revealed the infiltration of activated CD4+ T cells, which express Fas ligand (FasL), in RA synovium. The presence of a putative antigen (Ag) of autoimmune disorders in a target organ may induce the activation of specific T cells in the inflammatory region such as RA synovium. We examined the possible role of CD4+ T cells activated by synovial cells in a staphylococcal enterotoxin B (SEB)-dependent manner, inducing synovial cell apoptosis. Synovial cells were cultured with or without interferon-gamma (IFN-gamma) and further incubated with CD4+ T cells in the presence of SEB. After the cocultivation, both the cytotoxicity and FasL expression of CD4+ T cells were investigated. Constitutive Fas expression was detected on both unstimulated and IFN-gamma-stimulated synovial cells. CD4+ T cells did not kill SEB-pulsed unstimulated synovial cells efficiently. In contrast, when CD4+ T cells were incubated with IFN-gamma-stimulated synovial cells with SEB whose human leucocyte antigen (HLA)-DR and -DQ expression was markedly induced, significant cytotoxicity by these cells against synovial cells was detected. The addition of anti-HLA-DR and -DQ monoclonal antibodies (mAbs) or human Fas chimeric protein (hFas-Fc) reduced this cytotoxicity. FasL expression of CD4+ T cells cocultured with IFN-gamma-stimulated synovial cells with SEB was significantly induced. Furthermore, the addition of mAbs against CD54, CD58 and CD106 inhibited both the cytotoxicity and FasL expression of CD4+ T cells induced by IFN-gamma-stimulated synovial cells in the presence of SEB, indicating the importance of costimulatory molecules on synovial cells in activating CD4+ T cells. Our results suggest that CD4+ T cells are activated by synovial cells by an SEB-dependent manner and express FasL, inducing Fas-mediated apoptosis of the latter cells. These phenomena may regulate the overgrowth of synovial cells in RA synovium.     

p38 MAPK在顺铂诱导肾小管上皮细胞凋亡中的作用

目的:探讨p38 MAPK在顺铂诱导大鼠近端肾小管上皮细胞(RPTC)凋亡中的作用。方法:首先采用Western blot实验检测0、5、10和20μmol/L顺铂处理24 h对细胞凋亡的影响,确定最佳处理剂量;而后采用20μmol/L顺铂联合50 mg/L p38 MAPK抑制剂SB203580刺激RPTC,实验分为对照组、顺铂组及顺铂+SB203580(加入SB203580处理RPTC 1 h后再给予顺铂处理24 h)。采用相差荧光显微镜观察和流式细胞术分析顺铂处理后RPTC的凋亡情况;采用Ac-DEVD-AFC试剂盒检测RPTC裂解液中的caspase活性;Western blot实验检测p38、磷酸化p38、cleaved PARP和cleaved caspase-3等的蛋白水平;pH计检测顺铂处理后RPTC外环境pH值改变。结果:20μmol/L顺铂处理RPTC 24 h,可以明显诱导细胞凋亡;顺铂处理15 min后RPTC中p38 MAPK开始磷酸化并达到高峰。顺铂处理后12.08%的RPTC呈凋亡形态,具有增强的caspase活性,并且cleaved PARP和cleaved caspase-3水平明显升高(P0.05);p38 MAPK抑制剂SB203580可抑制p38的磷酸化,降低RPTC的凋亡率和caspase活性,并减少cleaved PARP和cleaved caspase-3的蛋白水平。同时,SB203580可逆转顺铂诱导的RPTC培养基pH值的改变。结论:p38 MAPK的磷酸化在顺铂诱导的RPTC凋亡中发挥作用。顺铂诱导RPTC凋亡后,可改变细胞外酸性环境,并可被p38 MAPK抑制剂SB203580所抑制。     

MAPK和caspase-3在异基因CD8+T淋巴细胞诱导血管内皮细胞凋亡中的作用

目的： 探讨MAPK和caspase-3在异基因CD8+T细胞诱导血管内皮细胞凋亡中的作用。方法：免疫磁珠阳性分选异基因CD8+T细胞,AnnexinⅤ/FITC试剂盒检测异基因CD8+T细胞诱导的HUVECs和HDMECs凋亡率,Western blotting检测血管内皮细胞内caspase-3、MAPK表达。观察SB203580 (p38MAPK抑制剂)、SP600125 (JNK抑制剂)、PD98059（ERK抑制剂）、Z-DEVD-FMK（caspase-3抑制剂）对内皮细胞凋亡的影响。结果：异基因CD8+T细胞作用24 h和48 h后,HUVECs凋亡率分别为41.7％±10.1％和29.4％±8.3％,HDMECs凋亡率分别为28.9％±7.2％和15.2％±4.8％,与对照组相比均具有显著差异（P<0.01）。异基因CD8+T细胞作用后,HUVECs和HDMECs内磷酸化p38MAPK表达、caspase-3裂解增强,而磷酸化JNK、ERK无明显变化。Z-DEVD-FMK和SB203580可显著抑制异基因CD8+T细胞诱导的HUVECs和HEMEC凋亡,并降低内皮细胞caspase-3表达。结论：p38MAPK和caspase-3介导了异基因CD8+T细胞诱导的血管内皮细胞凋亡。     

Butyric Acid-Induced T-Cell Apoptosis Is Mediated by Caspase-8 and -9 Activation in a Fas-Independent Manner

Our previous study demonstrated that butyric acid, an extracellular metabolite of periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat cells. In this study, we examined whether CD95 ligand-receptor interaction is involved in butyric acid-induced T-cell apoptosis. Flow cytometry analysis indicated that expression of Fas in Jurkat and T cells from peripheral blood mononuclear cells was not affected by butyric acid treatment. Furthermore, the expression of Fas and FasL protein in Western blotting was not affected by butyric acid treatment. Coincubation with blocking anti-Fas antibodies prevented Fas-induced apoptosis but not butyric acid-induced apoptosis. Anti-FasL antibodies also did not prevent butyric acid-induced apoptosis at any dose examined. Although cytotoxic anti-Fas antibody affected butyric acid-induced apoptosis, a synergistic effect was not seen. Time-dependent activation of caspase-8 and -9 was recognized in butyric acid- as well as Fas-mediated apoptosis. IETD-CHO and LEHD-CHO, specific inhibitors of caspase-8 and -9, respectively, completely blocked Fas-mediated apoptosis and partially prevented butyric acid-induced apoptosis. These results suggest that the Fas-FasL interaction is not involved in butyric acid-induced apoptosis and that caspase-8 and -9-dependent apoptosis plays an important role in butyric acid-induced apoptosis, as well as Fas-induced apoptosis.     

Butyric acid-induced T-cell apoptosis is mediated by caspase-8 and -9 activation in a Fas-independent manner

Our previous study demonstrated that butyric acid, an extracellular metabolite of periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat cells. In this study, we examined whether CD95 ligand-receptor interaction is involved in butyric acid-induced T-cell apoptosis. Flow cytometry analysis indicated that expression of Fas in Jurkat and T cells from peripheral blood mononuclear cells was not affected by butyric acid treatment. Furthermore, the expression of Fas and FasL protein in Western blotting was not affected by butyric acid treatment. Coincubation with blocking anti-Fas antibodies prevented Fas-induced apoptosis but not butyric acid-induced apoptosis. Anti-FasL antibodies also did not prevent butyric acid-induced apoptosis at any dose examined. Although cytotoxic anti-Fas antibody affected butyric acid-induced apoptosis, a synergistic effect was not seen. Time-dependent activation of caspase-8 and -9 was recognized in butyric acid- as well as Fas-mediated apoptosis. IETD-CHO and LEHD-CHO, specific inhibitors of caspase-8 and -9, respectively, completely blocked Fas-mediated apoptosis and partially prevented butyric acid-induced apoptosis. These results suggest that the Fas-FasL interaction is not involved in butyric acid-induced apoptosis and that caspase-8 and -9-dependent apoptosis plays an important role in butyric acid-induced apoptosis, as well as Fas-induced apoptosis.     

p38 MAPK as a negative regulator of VEGF/VEGFR2 signaling pathway in serum deprived human SK-N-SH neuroblastoma cells

Evidence suggests that vascular endothelial growth factor (VEGF) mediates neuroprotection to prevent an apoptotic cell death. The p38 mitogen-activated protein kinase (MAPK) pathway is implicated as an important mediator of neuronal apoptosis but its role in VEGF-mediated neuroprotection is unclear. Herein, we show that treatments with the p38 MAPK inhibitor, SB202190, enhanced VEGF-mediated survival in serum deprived SK-N-SH neuroblastoma cells by decreasing caspase-3/7 activation while increasing the phosphorylation of the extracellular signal-regulated kinase (ERK1/2) and Akt signaled through the VEGF receptor, VEGFR2. A blockade of VEGFR2 signaling with a selective inhibitor, SU1498 or gene silencing with VEGFR2 siRNA in SB202190 treated cells abrogated this prosurvival response and induced high activation levels of caspase-3/7. These findings suggested that the protection elicited by p38 MAPK inhibition in serum starved cells was dependent on a functional VEGF/VEGFR2 pathway. However, p38 MAPK inhibition attenuated caspase-3 cleavage in SU1498/SB202190 treated cells, indicating that p38 MAPK and caspase-3 only contributed in part to the total levels of caspase-3/7 induced by VEGFR2 inhibition. Pretreatments with the pan caspase inhibitor, z-VAD-fmk, prevented the apoptosis induced by VEGFR2 inhibition and promoted survival in serum starved cells irrespective of p38 MAPK inhibition. Collectively, our findings suggest that p38 MAPK exerts a negative effect on VEGF-mediated signaling through VEGFR2 in serum starved neuroblastoma cells. Furthermore, VEGF signals protection against a caspase-mediated cell death that is regulated by p38 MAPK-dependent and -independent mechanisms.     

抗癌药SN38通过脂筏促进Fas介导的细胞凋亡

目的:探讨抗癌药SN38对Fas介导凋亡的影响机制,并进一步探讨脂筏在该过程的作用。方法:单独或者联合应用SN38和CH11(Fas抗体)作用于脂筏阴性和脂筏阳性的细胞株WR/FAS-SM(-)细胞和WR/FAS-SMS1细胞,通过Western blot的方法检测细胞内部DNA损伤的信号分子以及caspase分子的活化情况,并分析它们的活化强度和作用特点。结果:在SN38和CH11的共同作用下,WR/FAS-SMS1细胞ATM-Chk1-p53途径及caspase-3.8活化,p21活化水平降低,而WR/FAS-SM(-)细胞的Chk1-p53途径以及caspase-3.8活化,而p21活化水平则未见降低。结论:SN38和CH11的共同应用与SN38单独应用相比,通过活化ATM-Chk1-p53途径,导致p21活化水平降低,进而活化caspase-3,8,从而促进WR/Fas-SMS1细胞的凋亡;该过程中脂筏可能通过调控p21的不同活化状态最终导致了WR/FAS-SM(-)细胞和WR/FAS-SMS1细胞间凋亡率的差异。     

IFN-gamma reverses IL-2- and IL-4-mediated T-cell steroid resistance

Corticosteroids are the most common therapeutic approach for control of tissue inflammation. Combination IL-2/IL-4 is known to induce T-cell steroid resistance. This can be reversed with IFN-gamma; however, the mechanism by which this occurs is unknown. In the current study, we found that treatment of peripheral blood mononuclear cells with combination IL-2/IL-4 for 48 hours, but not with IL-2 or IL-4 alone, abrogated dexamethasone (DEX)-induced glucocorticoid receptor (GCR)-alpha nuclear translocation in both CD4(+) and CD8(+) T cells. The presence of IL-4 significantly down-regulated IFN-gamma production by IL-2-stimulated cells. Importantly, addition of IFN-gamma to the IL-2/IL-4 combination restored GCRalpha nuclear translocation in response to DEX. Furthermore, DEX-induced mitogen-activated protein kinase (MAPK) phosphatase-1 induction, used as a readout for corticosteroid-induced transactivation, was significantly greater (P < 0.05) in media and IL-2/IL-4/IFN-gamma-treated conditions compared with IL-2/IL-4-treated cells. The combination of IL-2/IL-4 induced p38 MAPK activation in CD3(+) cells (30.5 +/- 5.7% cells expressed phospho-p38 MAPK versus no phospho-p38 MAPK expression after media treatment). The presence of the p38 MAPK inhibitor, SB203580, or IFN-gamma inhibited p38 MAPK phosphorylation and enhanced GCRalpha nuclear translocation in response to DEX. These data indicate that combination IL-2/IL-4 inhibits GCRalpha nuclear translocation in human T cells, and this effect is reversed by IFN-gamma via inhibition of p38 MAPK activation.     

The role of p38 MAPK in valproic acid induced microglia apoptosis

Valproic acid (VPA), a widely prescribed drug for seizures and bipolar disorder, induces apoptosis in microglia, but the underlying mechanism by which microglia apoptosis in response to VPA is not yet known. In this study, we found that the mitochondrial pathway played an important role in VPA-induced apoptosis in both BV-2 microglia and mouse primary microglial cells. In addition, VPA increased the level of phospho-p38 mitogen-activated protein kinase (MAPK), but had no effects on phospho-ERK and phospho-JNK MAPKs. Moreover, p38 inhibitor SB203580 strongly inhibited VPA-induced apoptosis and caspase-3 activation. Taken together, our results clearly demonstrated that VPA could induce apoptosis of microglia via p38 MAPK and mitochondrial apoptosis pathway.     

Rapid up-regulation of c-FLIP expression by BCR signaling through the PI3K/Akt pathway inhibits simultaneously induced Fas-mediated apoptosis in murine B lymphocytes

Cross-linking of BCR rapidly induces protection of B cells from Fas-mediated apoptosis, which has been assumed one of the important survival mechanisms of B cells during antigen stimulation. In the mouse B cell line A20, which is sensitive to Fas-mediated apoptosis, stimulation of BCR inhibited apoptosis induced via Fas upstream of caspase-8 activation with an associated rapid increase in the expression of both short and long forms of cellular caspase-8/FLICE-inhibitory protein (c-FLIP). The c-FLIP competitively inhibited the recruitment of caspase-8 to the death-inducing signaling complex (DISC), which took as long as 3h to form after the stimulation of Fas in A20 cells. Knockdown of c-FLIP by a short hairpin RNA-expressing method rendered BCR-stimulated A20 cells sensitive to Fas-mediated apoptosis. The BCR-induced rapid expression of c-FLIP was not affected by inactivation of NF-kappaB, but was inhibited by either treatment with a PI3K inhibitor, LY294002, or expression of a dominant negative PI3K p85 subunit, both of which suppressed phosphorylation of Akt and sensitized BCR-stimulated A20 cells to Fas-mediated apoptosis. Overexpression of constitutively active Akt was shown not only to up-regulate c-FLIP expression but also to render A20 cells resistant to Fas-mediated apoptosis. Moreover, treatment with LY294002 also suppressed BCR-induced up-regulation of c-FLIP expression in spleen B cells. Taken together, BCR-stimulation was shown to rapidly trigger a survival signal against simultaneously or ongoingly stimulated Fas-mediated apoptosis by promoting a PI3K/Akt signaling pathway-mediated up-regulation of c-FLIP expression.     

Mechanisms Underlying Production And Externalization of Oxidized Phosphatidylserine in Apoptosis: Involvement of Mitochondria

The present study was performed by using selective inhibitors of caspase-8 and caspase-3 functioning upstream and downstream from mitochondria, respectively to determine whether mitochondria are involved in the mechanisms underlying production and externalization of oxidized phosphatidylserine (PSox) during Fas-mediated apoptosis. Treatment with anti-Fas antibody induced caspase-3 activation, chromatin condensation, release of cytochrome c (cyt c) from mitochondria into the cytosol as well as production of PSox and its exposure to the cell surface in Jurkat cells. Inhibition of caspase-8 by pretreatment with Z-IETD-FMK, a membrane permeable selective caspase-8 inhibitor reduced mitochondrial cyt c release, the amount of PSox not only within but also on the surface of Jurkat cells, caspase-3 activation, and apoptotic cell number after treatment with anti-Fas antibody. In contrast, Z-DEVD-FMK, a membrane permeable selective caspase-3 inhibitor was unable to inhibit cyt c release, and the amount of PSox both within and on the surface of the cells after anti-Fas antibody, although it suppressed caspase-3 activation and apoptosis. Thus, these results strongly suggest that mitochondria play an important role in production of PSox and subsequent its externalization during apoptosis.     

p38有丝分裂原激酶活化参与继发性脊髓损伤后神经细胞凋亡的实验研究

探索脊髓损伤(SCI)后p38有丝分裂原激酶(p38MAPK)的表达及其与神经细胞凋亡的关系。采用Allen’s法建立大鼠急性SCI动物模型,用蛋白印迹法和免疫组织化学染色方法检测SCI后p38MAPK和caspase-3表达的变化;采用实时定量PCR法检测SCI后caspase-3 mRNA的表达;用原位末端标记法(TUNEL)检测SCI后神经细胞凋亡。结果表明SCI后总p38MAPK表达无变化,但p38MAPK磷酸化明显增多,于伤后6h达高峰;伤后24hcaspase-3表达明显增加。TUNEL检测显示,伤后6h受损伤的脊髓组织有少许凋亡细胞出现,24h细胞凋亡最明显。鞘内注射p38MAPK抑制剂SB203580,明显减少caspase-3的表达,并减少细胞凋亡。以上结果显示SCI后损伤局部p38MAPK激活诱导了caspase-3基因表达,导致神经细胞凋亡;抑制p38MAPK可以阻止SCI后继发的神经细胞凋亡。     

Low-Intensity Pulsed Ultrasound Activates Integrin-Mediated Mechanotransduction Pathway in Synovial Cells

Low-intensity pulsed ultrasound (LIPUS) suppresses synovial hyperplasia and synovial cell proliferation characterized for rheumatoid arthritis, but the molecular mechanisms remain unknown. The purpose of this study was to examine the mechanotransduction pathway via the integrin/mitogen-activated protein kinase (MAPK) pathway in LIPUS exposure on the synovial membrane cells. Rabbit knee synovial membrane cell line, HIG-82, was cultured with or without FAK phosphorylation inhibitor, PF-573228. One hour after stimulation with PF-573228, the cells exposed to LIPUS for 20 min or sham exposure. A possible integrin/MAPK pathway was examined by immunofluorescence and Western blotting analysis with antibodies targeting specific phosphorylation sites on intracellular signaling proteins. LIPUS exposure increased phosphorylation of FAK, JNK, ERK, and p38, but the phosphorylation was inhibited by PF-573228. In conclusion, LIPUS exposure might be involved in cell apoptosis and survival of synovial membrane cells via integrin/FAK/MAPK pathway.     

Inhibition of human rheumatoid arthritis synovial cell survival by hecogenin and tigogenin is associated with increased apoptosis, p38 mitogen-activated protein kinase activity and upregulation of cyclooxygenase-2

We conducted our study to assess the antiproliferative and proapoptotic potential of hecogenin and tigogenin, two saponins which are structurally similar to diosgenin. We particularly focused our attention on mitogen-activated protein kinase (MAPK) activation in relation to apoptosis but also with the COX-2 expression and activity. Rheumatoid arthritis (RA) synoviocytes were isolated from fresh synovial biopsies obtained from five RA patients undergoing hip arthroplasty. Measurement of cell proliferation was determined using the MTT assay. Apoptosis was evaluated by studying caspase-8, caspase-9 and caspase-3 activities but also by quantification of DNA fragmentation. Quantification of human phospho-MAPKs was realized by ELISA. COX-2 expression was demonstrated by Western blot analysis and COX-2 activity by assay of endogenous prostaglandin E2 (PGE2) production. Tigogenin was more effective than hecogenin in inducing apoptosis in human RA fibroblast-like synoviocytes (FLS) which was caspase dependent but poly(ADP-ribose) polymerase independent and characterized by DNA fragmentation. Our results demonstrated hecogenin- and tigogenin-induced apoptosis through activation of p38 without affecting the JNK and ERK pathways. Indeed, pretreatment with a p38 inhibitor decreased saponin-induced apoptosis with a significant decrease in DNA fragmentation. Furthermore, the rate of apoptosis induced by hecogenin or tigogenin was associated with overexpression of COX-2 correlated with overproduction of endogenous PGE2. These new results provide strong evidence that a family of structurally similar plant steroids is capable of inducing apoptosis in human RA FLS with different rates and different signalling pathways. This study also confirms the discussed appearance of the downregulation or upregulation of COX-2 in cell apoptosis as a function of cell type.     

An involvement of SR-B1 mediated p38 MAPK signaling pathway in serum amyloid A-induced angiogenesis in rheumatoid arthritis

Serum amyloid A (SAA) has been reported high expression in autoimmune diseases, such as rheumatoid arthritis (RA). However, detailed molecular mechanisms induced by SAA in the pathogenesis of RA are still unclear. Herein, we focused on the role of SAA–SR-B1 mediated p38 MAPK signaling pathway in the process of RA angiogenesis. Our results showed that both SAA and SR-B1 predominantly localized to vascular endothelial cells, lining and sublining layers in RA synovium. In a series of in vitro experiments with human umbilical vein endothelial cells (HUVECs), SAA induced the endothelial cells (ECs) proliferation, migration and tube formation. However, blockage of SR-B1 and p38 MAPK inhibited SAA-induced cells proliferation, migration and tube formation. In conclusion, our data showed a possible molecular mechanism for SAA–SR-B1 induced angiogenesis events via p38 MAPK signaling pathway.     

Role of GADD45 beta in the regulation of synovial fluid T cell apoptosis in rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by persistent Th1 cell infiltration and production of inflammatory cytokines in the location of joint lesion. It is known that infiltrated Th1 cells in the synovial fluid (SF) of RA patients are resistant to apoptosis. Here we demonstrate that Th1 cells accumulated in patient SF expressed a high level of GADD45β (Growth Arrest and DNA Damage-inducible 45β) which further inhibited Th1 cell apoptosis. Interestingly, in vitro culture of T cells with SF from RA patients increased GADD45β expression in Th1 cells and inhibited their apoptosis. Silencing of GADD45β by RNAi abolished the anti-apoptotic effect of RA SF, which was accompanied by down-regulation of Bcl-2 and up-regulation of Bax. Further analysis showed that TNF-α and IL-12 in RA SF could stimulate GADD45β expression in Th1 cells and inhibit their apoptosis. Taken together, our results suggest a novel mechanism by which specific cytokines in the RA SF elevate GADD45β expression in local Th1 cells and subsequently leading to the enhanced T cell survival.     

Induction of apoptosis in human lymphocytes by the herbicide 2,4-dichlorophenoxyacetic acid

Dimethylammonium salt of 2,4-dichlorophenoxyacetic acid (DMA-2,4-D) is a widely used herbicide that is considered moderately toxic. In the present study we found that DMA-2,4-D is able to cause apoptosis in peripheral blood lymphocytes of healthy individuals and Jurkat T cells. Apoptosis induced by DMA-2,4-D was dose and time dependent, independent of Fas, TNF receptor 1 or the aromatic hydrocarbon receptor, and involved disruption of the mitochondrial transmembrane potential and activation of caspase-9. ZVAD-FMK, a broad-spectrum inhibitor of caspases, blocked DMA-2,4-D-induced apoptosis completely. While an inhibitor of caspase-9, as well as caspase-9 and caspase-3 inhibitors in combination, strongly blocked DMA-2,4-D-induced apoptosis, an inhibitor of caspase-3 had a moderate inhibitory effect. Unlike Fas-mediated apoptosis, the initiator caspase, caspase-8, was not involved in DMA-2,4-D-induced apoptosis. Transfection of Jurkat cells with Bcl-2 prevented DMA-2,4-D-induced disruption of the mitochondrial transmembrane potential and led to a complete blockage of apoptosis. Our data indicate that DMA-2,4-D kills human lymphocytes by initiating apoptosis via a direct effect on mitochondria. The activation of caspases occurs downstream of mitochondrial damage, and the dysfunction of mitochondria appears to be sufficient for triggering all downstream events leading to apoptosis.     

Regulation of GRB2 and FLICE2 expression by TNF-alpha in rheumatoid synovium

Chronic rheumatoid arthritis (RA) is characterized by the hyperplasia of synovial tissue, which results from dysregulation of proliferative and antiapoptotic signals transduced in the synovial cells by unknown mechanisms. To identify candidate factors involved in the regulation of synovial hyperplasia, the expression profile of 205 apoptosis-related genes was compared between tissues from patients with RA and osteoarthritis (OA) using a cDNA microarray. Upregulated genes in the RA synovium included TNFR2, FLICE2, and signaling molecules involved in a MAP kinase pathway (GRB2, MAPK p38). In contrast, genes encoding SARP1 and various cell cycle regulators were down-regulated in the RA synovium relative to OA. Importantly, the expression levels of GRB2 and FLICE2 genes were remarkably enhanced in RA synoviocytes but not in OA synoviocytes in response to tumor necrosis factor (TNF)-alpha treatment. Thus, these results suggest that over-expression of GRB2 and FLICE2 in RA synovium is caused by TNF-alpha inducibility differentially regulated in RA synoviocytes and provide potential pathogenic roles of these genes in the hyperplasia of the RA synovium.     

Lipopolysaccharide Stimulates Butyric Acid-Induced Apoptosis in Human Peripheral Blood Mononuclear Cells

We previously reported that butyric acid, an extracellular metabolite from periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat T cells. In this study, we examined the ability of butyric acid to induce apoptosis in peripheral blood mononuclear cells (PBMC) and the effect of bacterial lipopolysaccharide (LPS) on this apoptosis. Butyric acid significantly inhibited the anti-CD3 monoclonal antibody- and concanavalin A-induced proliferative responses in a dose-dependent fashion. This inhibition of PBMC growth by butyric acid depended on apoptosis in vitro. It was characterized by internucleosomal DNA digestion and revealed by gel electrophoresis followed by a colorimetric DNA fragmentation assay to occur in a concentration-dependent fashion. Butyric acid-induced PBMC apoptosis was accompanied by caspase-3 protease activity but not by caspase-1 protease activity. LPS potentiated butyric acid-induced PBMC apoptosis in a dose-dependent manner. Flow-cytometric analysis revealed that LPS increased the proportion of sub-G₁ cells and the number of late-stage apoptotic cells induced by butyric acid. Annexin V binding experiments with fractionated subpopulations of PBMC in flow cytometory revealed that LPS accelerated the butyric acid-induced CD3⁺-T-cell apoptosis followed by similar levels of both CD4⁺- and CD8⁺-T-cell apoptosis. The addition of LPS to PBMC cultures did not cause DNA fragmentation, suggesting that LPS was unable to induce PBMC apoptosis directly. These data suggest that LPS, in combination with butyric acid, potentiates CD3⁺ PBMC T-cell apoptosis and plays a role in the apoptotic depletion of CD4⁺ and CD8⁺ cells.