Fas／FasL在超抗原SEA诱导T细胞凋亡中的作用
作者：宋建勋 朱锡华 陈克敏n-zp%, 百拇医药
单位：宋建勋(第三军医大学全军免疫学研究所，重庆 400038)；朱锡华(第三军医大学全军免疫学研究所，重庆 400038)；陈克敏(第三军医大学全军免疫学研究所，重庆 400038)n-zp%, 百拇医药
关键词：Fas系统；T细胞；细胞凋亡；超抗原；Ca2+n-zp%, 百拇医药
免疫学杂志000401 [摘 要] 目的 超抗原能诱导T细胞凋亡，但其作用机制尚未阐明，Fas系统与Ca2+在其中的作用尚待进一步研究。方法 超抗原金黄色葡萄球菌肠毒素A(SEA)诱导人外周血建立的短期SEA反应T细胞系凋亡，1.8%琼脂糖凝胶DNA电泳于不同时间观察凋亡的特征条带；FCM检测Fas、FasL的表达量变化，Fura-2/AM荧光指示剂检测胞浆游离[Ca2+]i的变化。结果 使用FCM特异性检测凋亡亚G0/G1峰大小及1.8%琼脂糖凝胶电泳检测凋亡DNA梯状图谱证明，SEA能诱导短期SEA反应T细胞凋亡，其凋亡的时间与程度与SEA的剂量有关。SEA 1 μg/ml建立的短期SEA反应T细胞对SEA 1 μg/ml的再次刺激，凋亡量随作用时间的延长而增多，在作用的第16 h最高，达58%。FCM间接荧光染色法检测发现，Fas及FasL亦随SEA作用时间的延长而表达增多，16 h时表达最多，分别为92%和60%。用Fura-2/AM荧光指示剂检测，此时细胞胞浆[Ca2+]i已从刺激前的(290±33) nmol/L上升到(680±16) nmol/L。结论 Fas系统与SEA诱导的T细胞凋亡密切相关。换言之，Fas系统在超抗原诱导T细胞凋亡中起作用，而胞浆[Ca2+]i升高与SEA诱导的凋亡T细胞DNA断裂及其它凋亡形态变化有关。n-zp%, 百拇医药
[中图分类号] R392.11； R364 [文献标识码] An-zp%, 百拇医药
The effect of Fas/FasL on T cell apoptosis induced byn-zp%, 百拇医药
staphylococcal enterotoxin A(SEA)n-zp%, 百拇医药
SONG Jian-xun， ZHU Xi-hua， CHEN Ke-min
(Institute of Immunology PLA， the Third Military Medical University， Chongqing 400038，China)i1x[, http://www.100md.com
[Abstract] Objective To investigate the effect of Fas/Fas-ligand (FasL) interactions on the superantigen-induced apoptosis of human peripheral T cell. Methods Apoptosis of SEA-reactive T-cell line， established from human peripheral blood mononuclear cells (PBMC)， was induced by SEA. The features of apoptosis were observed with light microscope， flow cytometry(FCM) and DNA electrophoresis ladder. The Fas/FasL changes and cytosolic Ca2+ concentration were measured with FCM and fluorescent probe Fura-2/AM during the T cell apoptosis. Results A short term SEA reactive T-cell line was established by stimulating PBMC 106 with 1 μg/ml SEA for 3 days and expanding the cells in rIL-2 (200 U/ml). These cells were maintained in rIL-2 for 2 weeks before use. Both FCM and DNA electrophoresis ladder could demonstrate that apoptosis appeared obviously by second stimulation of the SEA-reactive T-cells with SEA 1 μg/ml. Fas/FasL was substantially upregulated with an increase of cytosolic free Ca2+. Conclusion These findings suggest that Fas/FasL plays an important role in the superantigen-induced T cell apoptosis and the increase of cytosolic Ca2+ was related to DNA fragmentation and morphological changes of T cell apoptosis.
[Key words] Fas/FasL；T cell； apoptosis； superantigen； Ca2+t, http://www.100md.com
[Article ID]1000-8861(2000)04-0241-05t, http://www.100md.com
Superantigen(SAg) can cause activation-induced cell death (AICD) after inducing strong proliferative response of specific T cells. It is well known that apoptosis can be induced by superantigen in T cells and probably involves in AICD. However， little is known about the mechanism that mediates the lytic process. As a death molecule， Fas antigen can mediate apoptosis and plays a potential role in AICD of T cells induced by SAg[1～4]. In this experiment， the effect of Fas/FasL-mediated death was investigated on T cell apoptosis induced by SEA (Staphylococcal enterotoxin A， belonging to SAg)， and the changes of [Ca2+]i in apoptosis were observed.t, http://www.100md.com
1 MATERIALS AND METHODSt, http://www.100md.com
1.1 Materialst, http://www.100md.com
1.1.1 Reagents Staphylococcal enterotoxin A (1mg， Military Academy of Medical Science， Beijing)， The blood sample(Southwest Hospital of China， No.971124-1538)，rIL-2(Chongqing). Propidium iodide(PI)， Triton X-100(Sigma).Fura-2/AM，Hepes(Boehringer Mamnheim). λ DNA/EcoR+HindⅢ Marker， (Promega). Purified mouse anti-human CD95 mAb DX2， Mouse anti-human Fas ligand (PharMingen， San Diego，CA). FITC-conjugated goat anti-mouse IgG (Johnson Immuno Research Laboratories， Inc America).
1.1.2 Culture medium RPMI1640(Gibco)inclu-ding 10%NCS， 100 U/ml penicillin， 100 μg/ml streptaquaine， 10 mmol/L HEPES and 2 mmol/L Gln.xun(^7, 百拇医药
1.1.3 Major instruments FACstar plus flow Cytometry (FCM， Becton-Dickinson).TC2323 CO2 incubator(Shell/Jb). MPF-4 fluorescent spectrophotometer (Hitachi).xun(^7, 百拇医药
1.2 Methodsxun(^7, 百拇医药
1.2.1 The establishment of short-term superantigen-specific T cell lines from human peripheral blood T cell lines were established from periphe-ral blood of healthy donors[5]. The human periphe-ral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation and incubated with 0.5～5 μg/ml SEA. After 4 days， the cells were recultured in fresh medium containing interleukin-2(rIL-2， 100 U/ml). The cells were maintained in rIL-2 for 2 weeks before use.xun(^7, 百拇医药
1.2.2 The short-term SEA-specific T cell apoptosis induced by SEA Cell death was induced by 1 μg/ml SEA at various times(0，2，4，8，16，24 and 32 hours)，and also quantitated by FCM[6] for the presence of a sub-G0/G1 DNA peak and analysis by 1.8% agarose gel electrophoresis for DNA ladder.
1.2.3 The expression of Fas and FasL during the short-term SEA-specific T cell apoptosis induced by SEA Cells were washed in PBS containing 0.2% BSA and 0.02% sodium azide(wash buffer). Cells(5×105 cells/ml) were incubated at room temperature in wash buffer containing 2.5 μg/ml normal mouse immunoglobulin for 15 mi-nutes， after which anti-Fas antibody or anti-FasL antibody was added(2.5 μg/ml) and cells were incubated for 30 minutes at 4 °C. Cells were then washed and resuspended in FITC-conjugated goat anti-mouse IgG 2.5 μg/ml for 30 minutes at 4°C. Cells were washed once more and resuspended in PBS containing 1% formaldehyde and analyzed on a FACScan.3, http://www.100md.com
1.2.4 Changes of cytosolic free Ca2+ during the short-term SEA-specific T cell apoptosis induced by SEA Measurement was based on Malgaroli A et al[7]. Briefly， the short-term SEA-specific T cells were induced to apoptosis by SEA and harvested in various time periods (0，2，4，8，16，24 and 32 hours). To these cells， Fura-2/AM was added， then cytosolic free [Ca2+]i measured. [Ca2+]i＝Kd(F-Fmin)/(Fmax-F)，Kd is 224nm. F，Fmax and Fmin were measured by fluorescent spectrophotometer.
2 RESULTS23fqk, 百拇医药
2.1 Apoptosis induced by SEA in short-term SEA-specific T cells The short-term SEA-specific T cells grew together and could be conversed frequently to blastoformation(see figure 1A). Apoptosis appeared obviously in these SEA-specific T cells restimulated with SEA. It could be observed through light microscope that some T cells induced by SEA for 8 hours died (see figure 1B)， and lots of T cells induced for 16 hours by SEA showed clear features of apoptosis(see figure 1C). Analysis of sub G0-G1 peak by flow cytometry showed that apoptotic cell number in the SEA-spe-cific T cells increased with the prolongation of induction time. Apoptotic cell number reached 52% in SEA-specific T cells induced by SEA for 16 hours(see figure 2A). It could also be demonstra-ted that apoptotic cell number in these SEA-specific T cells increased with the rise in concentrations of SEA(see figure 2B).
23fqk, 百拇医药
Fig 1Morphology of apoptosis induced by SEA in SEA-reactive T cells，LM×100
A：SEA-reactive T cells line；B：8 hours treated again by SEA 1 μg/ml；C：16 hours treated again by SEA 1 μg/ml{jd0, 百拇医药
The DNA ladder feature of apoptosis was observed clearly in the SEA-specific T cells induced by SEA again for 16 hours.When induced for 24 hours and 32 hours， DNA ladder was darkened gradually and displayed a clouding lamellar band(see figure 3).
{jd0, 百拇医药
Fig 2Analysis of apoptosis induced by SEA in SEA-reactive T cells by flow cytometry{jd0, 百拇医药
A：dose response of SEA-reactive T cells to addition of SEA for 16 hours；B：kinetics of apoptosis induced by SEA 1 μg/ml in SEA-reactive T cells
{jd0, 百拇医药
Fig 3Analysis of apoptosis induced by SEA in SEA-reactive T cells by 1.8% agarose gel electrophoresis{jd0, 百拇医药
1：DNA marker(λ DNA/EcoR+HindⅢ)；2：0 hour； 3：16 hours； 4：12 hours； 5：8 hours{jd0, 百拇医药
2.2 The expression changes of Fas and FasL during the short-term SEA-specific T cell apopto-sis Fas antigen was higher expressed in the short-term SEA-specific T cells. The mean fluorescence intensity (MFI) reached 85% before induction by SEA again and raised gradually to maximal value of 92% after induced by SEA for 16 hours (see figure 4A).
The MFI of FasL was 8% before induction by SEA again and raised gradually to 60% after induced by SEA for 16 hours， but the raised rate of MFI of FasL was slower than that of Fas antigen(see figure 4B).
-:yqz9, 百拇医药
Fig 4Functional expression of Fas， FasL induced by SEA in SEA-reactive T cells-:yqz9, 百拇医药
A：kinetics of Fas expression induced by SEA 1 μg/ml in SEA-reactive T cells；B：kinetics of FasL expression induced by SEA 1 μg/ml in SEA-reactive T cells-:yqz9, 百拇医药
2.3 Correlation of apoptosis of the SEA-specific T cells induced by SEA and the expression of Fas and FasL When reached the highest expression of Fas(92%) and FasL(60%)， the number of apoptotic cells of SEA-specific T cells induced 16 hours by SEA also increased to maximal value of 58%. It was clear that apoptosis of the SEA-specific T cells induced by SEA correlated with the expression of Fas and FasL.-:yqz9, 百拇医药
2.4 Changes of cytosolic free Ca2+ during the short-term SEA-specific T cell apoptosis The concentration of cytosolic free Ca2+ in the SEA-specific T cells was 290±8 nmol/L before induced by SEA again， and increased obviously to 330±21 nmol/L and 680±16 nmol/L after induction by SEA for 8 and 16 hours， and began to decline when induced longer than 16 hours(see figure 5).
Fig 5Changes of cytosolic free[Ca2+]i during apoptosis induced by SEA(1 μg/ml) in SEA-reactive T cells-\%8r, http://www.100md.com
3 DISCUSSION-\%8r, http://www.100md.com
SAg induced T cell apoptosis can lead to immune tolerance， consistent with clonal deletion or anergy.However， little is known about the mechanism that mediates the lytic process. Fas-mediated killing possibly takes responsibility for T cell apoptosis induced by SAg. Staphylococcal enterotoxin B(SEB) is a bacterial SAg that predominantly interacts with V β 8+ T cells. In vivo treatment of mice with SEB leads to an initial increase in the percentage of V β 8+T cells， followed by a decrease in the numbers of these cells， eventually reaching lower levels than those found before treatment with the SAg. FCM detection shows that SEB-reactive T cells express high level of Fas and FasL. Moreover， in vivo treatment of functional FasL-deficient mutant gld mice with SEB didn’t cause an obvious decrease in the percentage of V β 8+ T cells. Exposure of naive CD4+ T lymphocytes to SEB could induce AICD， but the ability of SEB to induce apoptosis was inhibited by the addition of a Fas-Fc fusion protein. These results suggest the interaction of Fas and FasL brings about specific-T cells’ AICD induced by SEB[8，9]. CD4+ T cells derived from either Fas-defective Lpr or FasL-defective gld mice failed to undergo ACID induced by SEB. These cells were in a condition of proliferation. In other way， the Fas-negative mice obtained by gene knock out didn’t show clonal deletion in peripheral specific T cells affected by endogenous SAg， and just show specific-T cells proliferation complys with infiltration in liver and lung[10]. It is also found that Fas system plays a role in clonal deletion in peripheral specific T cells induced by SEA and endogenous Mtv-7-SAg encoded by mouse mammary tumor virus(MMTV)[4，5]. In this study， we demonstrated that apoptosis appeared obviously in second stimulation of the short-term SEA-reactive T cells from human peripheral blood with SEA 1 μg/ml and Fas/FasL was substantially up-regulated， with increasing cytosolic free[Ca2+]i. Based on these observations we think， SAg induced specific-T cells to express high levels of Fas and FasL in the absence of antigen presenting cells(APC) and other correlated adjuvant molecules. Death signal was delivered by cross-linking Fas with FasL and active intracellular correlated enzymes， causes increase of cytosolic free[Ca2+]i and active Ca2+-dependent endonuclease， which bring about T-cell apoptosis.
In the presence of APC， the interaction of B7 and CD28 molecules on the surface of APC provides a costimulatory signal， and the SAg-specific T cells can up-regulate the Bcl-2 or Bcl-XL gene and restrain the expression of FasL， which lets these cells to override the Fas death signal and allows cells to proliferate[11]. It is well known that anti-CD28 molecule can bind with CD28 on the surface of T cells and accelerate T cell proliferation by imitating B7 molecule.Because high concentration of IL-2 can promote T cells proliferation， and induce the expression of Bcl-2 in SAg-specific T cells to antagonize the T cell apoptosis mediated by Fas antigen， implantation of IL-2-containing osmotic pump can prolong the survival of SAg-reactive T cells expanded in mice injected with bacterial SAg. However， it is not clear whether SAg induces the deprivation of IL-2 after activation of plenty of T cells in vivo[12]. PMA assists SAg’s effect on T cell proliferation by activating PKC pathway and promoting the high expression of IL-2.^5#*], http://www.100md.com
Therefore， SAg-specific T cells can receive two stimulatory signals in the presence of APC and produce strong proliferative response induced by SAg： one is initiation signal by TCR-CD3 complex， in which one end of SAg binds with TCRV β chain and (or no) the other end binds with the negative polymorphic region of MHCⅡ molecules. The other is costimulatory signal by the interactions with B7 molecule in APC and CD28 in T cells. In the absence of APC， SAg-specific T cells can express high Fas and FasL and undergo apoptosis by cross-linking Fas with FasL. Under the condition of high concentration of IL-2 ， SAg-specific T cells will not undergo apoptosis but allow proliferation by up-regulating the expression of Bcl-2 gene in T cells and suppressing apoptosis mediated by Fas antigen.
[Foundation item] This work was supported by the Fund of the “Ninth five” obligatory budget of PLA (96Z038).!l;ih/, 百拇医药
[Biography] Song Jian-xun(1967-)，male， born in Feicheng county， Shangdong， instructor， M.D.，field of research：molecular immunology.!l;ih/, 百拇医药
[REFERENCES]!l;ih/, 百拇医药
[1] STOHL W， ELLIOTT JE， LYNCH DH，et al.CD95 (Fas)-based， superantigen-dependent， CD4+ T cell-mediated down-regulation of human in vitro immunoglobulin responses[J].J Immunol， 1998，160(11)：5 231-5 238.!l;ih/, 百拇医药
[2] CAULEY LS， CAULEY KA， SHUB F， et al.Transferable anergy：superantigen treatment induces CD4+ T cell tolerance that is reversible and requires CD4+ CD8- cells and interferon gamma[J]. J Exp Med， 1997，186(1)：71-81.!l;ih/, 百拇医药
[3] STOHL W， LYNCH DH， STARLING GC， et al. Superantigen-driven， CD8+ T cell-mediated down-regulation：CD95 (Fas)-dependent down-regulation of human Ig responses despite CD95-independent killing of activated B cells[J]. J Immunol， 1998，161(7)：3 292-3 298.!l;ih/, 百拇医药
[4] PAPIERNIK M， PONTOUX C， GOLSTEIN P. Non-exclusive Fas control and age dependence of viral superantigen-induced clonal deletion in lupus-prone mice[J]. Eur J Immunol， 1995，25(6)：1 517-1 523.
[5] ALDERSON M， TOUGH TW， DAVIS-SMITH T， et al. Fas ligand mediates activation-induced cell death in human T lymphocytes[J]. J Exp Med， 1995，181(1)：71-77.!5!/, 百拇医药
[6] NICOLETTI I， MIGLIORATI G， PAGLIACCI MC， et al. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry[J].J Immunol Methods， 1991， 139(2)：271-279.!5!/, 百拇医药
[7] MALGAROLI A， MILANI D， MELDOLESI J， et al. Fura-2 measurement of cytosolic Ca2+ in monolayers and suspensions of various types of animal cells[J]. J Cell Biol， 1987，105(5)：2 145-2 155.!5!/, 百拇医药
[8] BOSHELL M， MCLEOD J， WALKER L， et al. Effects of antigen presentation on superantigen-induced apoptosis mediated by Fas/Fas ligand interactions in human T cells[J]. Immunol， 1996，87(4)：586-592.!5!/, 百拇医药
[9] RENNO T， HAHNE M， TSCHOPP J， et al. Peripheral T cells undergoing superantigen-induced apoptosis in vivo express B220 and upregulate Fas and Fas ligand[J].J Exp Med， 1996，183(2)：431-437.!5!/, 百拇医药
[10] ADACHI M， SUEMATSU S， SUDU T，et al. Enhanced and accelerated lymphoproliferation in Fas-null mice[J]. Proc Natl Acad Sci USA， 1996，93(5)：2 131-2 136.!5!/, 百拇医药
[11] BOISE LH， MINN AJ， NOEL PJ， et al. CD28 costimulation can promote T cell survival by enhancing expression of Bcl-XL [J]. Immunity， 1995，3(1)：87-98.!5!/, 百拇医药
[12] MCLEOD JD， WALKER LS， PATEL YI， et al. Activation of human T cells with superantigen (staphylococcal enterotoxin B) and CD28 confers resistance to apoptosis via CD95 [J]. J Immunol， 1998，160(5)：2 072-2 079.!5!/, 百拇医药
[Received date] 1999-12-24； [Revised date] 2000-04-04(宋建勋 朱锡华 陈克敏)
单位：宋建勋(第三军医大学全军免疫学研究所，重庆 400038)；朱锡华(第三军医大学全军免疫学研究所，重庆 400038)；陈克敏(第三军医大学全军免疫学研究所，重庆 400038)n-zp%, 百拇医药
关键词：Fas系统；T细胞；细胞凋亡；超抗原；Ca2+n-zp%, 百拇医药
免疫学杂志000401 [摘 要] 目的 超抗原能诱导T细胞凋亡，但其作用机制尚未阐明，Fas系统与Ca2+在其中的作用尚待进一步研究。方法 超抗原金黄色葡萄球菌肠毒素A(SEA)诱导人外周血建立的短期SEA反应T细胞系凋亡，1.8%琼脂糖凝胶DNA电泳于不同时间观察凋亡的特征条带；FCM检测Fas、FasL的表达量变化，Fura-2/AM荧光指示剂检测胞浆游离[Ca2+]i的变化。结果 使用FCM特异性检测凋亡亚G0/G1峰大小及1.8%琼脂糖凝胶电泳检测凋亡DNA梯状图谱证明，SEA能诱导短期SEA反应T细胞凋亡，其凋亡的时间与程度与SEA的剂量有关。SEA 1 μg/ml建立的短期SEA反应T细胞对SEA 1 μg/ml的再次刺激，凋亡量随作用时间的延长而增多，在作用的第16 h最高，达58%。FCM间接荧光染色法检测发现，Fas及FasL亦随SEA作用时间的延长而表达增多，16 h时表达最多，分别为92%和60%。用Fura-2/AM荧光指示剂检测，此时细胞胞浆[Ca2+]i已从刺激前的(290±33) nmol/L上升到(680±16) nmol/L。结论 Fas系统与SEA诱导的T细胞凋亡密切相关。换言之，Fas系统在超抗原诱导T细胞凋亡中起作用，而胞浆[Ca2+]i升高与SEA诱导的凋亡T细胞DNA断裂及其它凋亡形态变化有关。n-zp%, 百拇医药
[中图分类号] R392.11； R364 [文献标识码] An-zp%, 百拇医药
The effect of Fas/FasL on T cell apoptosis induced byn-zp%, 百拇医药
staphylococcal enterotoxin A(SEA)n-zp%, 百拇医药
SONG Jian-xun， ZHU Xi-hua， CHEN Ke-min
(Institute of Immunology PLA， the Third Military Medical University， Chongqing 400038，China)i1x[, http://www.100md.com
[Abstract] Objective To investigate the effect of Fas/Fas-ligand (FasL) interactions on the superantigen-induced apoptosis of human peripheral T cell. Methods Apoptosis of SEA-reactive T-cell line， established from human peripheral blood mononuclear cells (PBMC)， was induced by SEA. The features of apoptosis were observed with light microscope， flow cytometry(FCM) and DNA electrophoresis ladder. The Fas/FasL changes and cytosolic Ca2+ concentration were measured with FCM and fluorescent probe Fura-2/AM during the T cell apoptosis. Results A short term SEA reactive T-cell line was established by stimulating PBMC 106 with 1 μg/ml SEA for 3 days and expanding the cells in rIL-2 (200 U/ml). These cells were maintained in rIL-2 for 2 weeks before use. Both FCM and DNA electrophoresis ladder could demonstrate that apoptosis appeared obviously by second stimulation of the SEA-reactive T-cells with SEA 1 μg/ml. Fas/FasL was substantially upregulated with an increase of cytosolic free Ca2+. Conclusion These findings suggest that Fas/FasL plays an important role in the superantigen-induced T cell apoptosis and the increase of cytosolic Ca2+ was related to DNA fragmentation and morphological changes of T cell apoptosis.
[Key words] Fas/FasL；T cell； apoptosis； superantigen； Ca2+t, http://www.100md.com
[Article ID]1000-8861(2000)04-0241-05t, http://www.100md.com
Superantigen(SAg) can cause activation-induced cell death (AICD) after inducing strong proliferative response of specific T cells. It is well known that apoptosis can be induced by superantigen in T cells and probably involves in AICD. However， little is known about the mechanism that mediates the lytic process. As a death molecule， Fas antigen can mediate apoptosis and plays a potential role in AICD of T cells induced by SAg[1～4]. In this experiment， the effect of Fas/FasL-mediated death was investigated on T cell apoptosis induced by SEA (Staphylococcal enterotoxin A， belonging to SAg)， and the changes of [Ca2+]i in apoptosis were observed.t, http://www.100md.com
1 MATERIALS AND METHODSt, http://www.100md.com
1.1 Materialst, http://www.100md.com
1.1.1 Reagents Staphylococcal enterotoxin A (1mg， Military Academy of Medical Science， Beijing)， The blood sample(Southwest Hospital of China， No.971124-1538)，rIL-2(Chongqing). Propidium iodide(PI)， Triton X-100(Sigma).Fura-2/AM，Hepes(Boehringer Mamnheim). λ DNA/EcoR+HindⅢ Marker， (Promega). Purified mouse anti-human CD95 mAb DX2， Mouse anti-human Fas ligand (PharMingen， San Diego，CA). FITC-conjugated goat anti-mouse IgG (Johnson Immuno Research Laboratories， Inc America).
1.1.2 Culture medium RPMI1640(Gibco)inclu-ding 10%NCS， 100 U/ml penicillin， 100 μg/ml streptaquaine， 10 mmol/L HEPES and 2 mmol/L Gln.xun(^7, 百拇医药
1.1.3 Major instruments FACstar plus flow Cytometry (FCM， Becton-Dickinson).TC2323 CO2 incubator(Shell/Jb). MPF-4 fluorescent spectrophotometer (Hitachi).xun(^7, 百拇医药
1.2 Methodsxun(^7, 百拇医药
1.2.1 The establishment of short-term superantigen-specific T cell lines from human peripheral blood T cell lines were established from periphe-ral blood of healthy donors[5]. The human periphe-ral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation and incubated with 0.5～5 μg/ml SEA. After 4 days， the cells were recultured in fresh medium containing interleukin-2(rIL-2， 100 U/ml). The cells were maintained in rIL-2 for 2 weeks before use.xun(^7, 百拇医药
1.2.2 The short-term SEA-specific T cell apoptosis induced by SEA Cell death was induced by 1 μg/ml SEA at various times(0，2，4，8，16，24 and 32 hours)，and also quantitated by FCM[6] for the presence of a sub-G0/G1 DNA peak and analysis by 1.8% agarose gel electrophoresis for DNA ladder.
1.2.3 The expression of Fas and FasL during the short-term SEA-specific T cell apoptosis induced by SEA Cells were washed in PBS containing 0.2% BSA and 0.02% sodium azide(wash buffer). Cells(5×105 cells/ml) were incubated at room temperature in wash buffer containing 2.5 μg/ml normal mouse immunoglobulin for 15 mi-nutes， after which anti-Fas antibody or anti-FasL antibody was added(2.5 μg/ml) and cells were incubated for 30 minutes at 4 °C. Cells were then washed and resuspended in FITC-conjugated goat anti-mouse IgG 2.5 μg/ml for 30 minutes at 4°C. Cells were washed once more and resuspended in PBS containing 1% formaldehyde and analyzed on a FACScan.3, http://www.100md.com
1.2.4 Changes of cytosolic free Ca2+ during the short-term SEA-specific T cell apoptosis induced by SEA Measurement was based on Malgaroli A et al[7]. Briefly， the short-term SEA-specific T cells were induced to apoptosis by SEA and harvested in various time periods (0，2，4，8，16，24 and 32 hours). To these cells， Fura-2/AM was added， then cytosolic free [Ca2+]i measured. [Ca2+]i＝Kd(F-Fmin)/(Fmax-F)，Kd is 224nm. F，Fmax and Fmin were measured by fluorescent spectrophotometer.
2 RESULTS23fqk, 百拇医药
2.1 Apoptosis induced by SEA in short-term SEA-specific T cells The short-term SEA-specific T cells grew together and could be conversed frequently to blastoformation(see figure 1A). Apoptosis appeared obviously in these SEA-specific T cells restimulated with SEA. It could be observed through light microscope that some T cells induced by SEA for 8 hours died (see figure 1B)， and lots of T cells induced for 16 hours by SEA showed clear features of apoptosis(see figure 1C). Analysis of sub G0-G1 peak by flow cytometry showed that apoptotic cell number in the SEA-spe-cific T cells increased with the prolongation of induction time. Apoptotic cell number reached 52% in SEA-specific T cells induced by SEA for 16 hours(see figure 2A). It could also be demonstra-ted that apoptotic cell number in these SEA-specific T cells increased with the rise in concentrations of SEA(see figure 2B).
23fqk, 百拇医药Fig 1Morphology of apoptosis induced by SEA in SEA-reactive T cells，LM×100
A：SEA-reactive T cells line；B：8 hours treated again by SEA 1 μg/ml；C：16 hours treated again by SEA 1 μg/ml{jd0, 百拇医药
The DNA ladder feature of apoptosis was observed clearly in the SEA-specific T cells induced by SEA again for 16 hours.When induced for 24 hours and 32 hours， DNA ladder was darkened gradually and displayed a clouding lamellar band(see figure 3).
{jd0, 百拇医药Fig 2Analysis of apoptosis induced by SEA in SEA-reactive T cells by flow cytometry{jd0, 百拇医药
A：dose response of SEA-reactive T cells to addition of SEA for 16 hours；B：kinetics of apoptosis induced by SEA 1 μg/ml in SEA-reactive T cells
{jd0, 百拇医药Fig 3Analysis of apoptosis induced by SEA in SEA-reactive T cells by 1.8% agarose gel electrophoresis{jd0, 百拇医药
1：DNA marker(λ DNA/EcoR+HindⅢ)；2：0 hour； 3：16 hours； 4：12 hours； 5：8 hours{jd0, 百拇医药
2.2 The expression changes of Fas and FasL during the short-term SEA-specific T cell apopto-sis Fas antigen was higher expressed in the short-term SEA-specific T cells. The mean fluorescence intensity (MFI) reached 85% before induction by SEA again and raised gradually to maximal value of 92% after induced by SEA for 16 hours (see figure 4A).
The MFI of FasL was 8% before induction by SEA again and raised gradually to 60% after induced by SEA for 16 hours， but the raised rate of MFI of FasL was slower than that of Fas antigen(see figure 4B).
-:yqz9, 百拇医药Fig 4Functional expression of Fas， FasL induced by SEA in SEA-reactive T cells-:yqz9, 百拇医药
A：kinetics of Fas expression induced by SEA 1 μg/ml in SEA-reactive T cells；B：kinetics of FasL expression induced by SEA 1 μg/ml in SEA-reactive T cells-:yqz9, 百拇医药
2.3 Correlation of apoptosis of the SEA-specific T cells induced by SEA and the expression of Fas and FasL When reached the highest expression of Fas(92%) and FasL(60%)， the number of apoptotic cells of SEA-specific T cells induced 16 hours by SEA also increased to maximal value of 58%. It was clear that apoptosis of the SEA-specific T cells induced by SEA correlated with the expression of Fas and FasL.-:yqz9, 百拇医药
2.4 Changes of cytosolic free Ca2+ during the short-term SEA-specific T cell apoptosis The concentration of cytosolic free Ca2+ in the SEA-specific T cells was 290±8 nmol/L before induced by SEA again， and increased obviously to 330±21 nmol/L and 680±16 nmol/L after induction by SEA for 8 and 16 hours， and began to decline when induced longer than 16 hours(see figure 5).
Fig 5Changes of cytosolic free[Ca2+]i during apoptosis induced by SEA(1 μg/ml) in SEA-reactive T cells-\%8r, http://www.100md.com
3 DISCUSSION-\%8r, http://www.100md.com
SAg induced T cell apoptosis can lead to immune tolerance， consistent with clonal deletion or anergy.However， little is known about the mechanism that mediates the lytic process. Fas-mediated killing possibly takes responsibility for T cell apoptosis induced by SAg. Staphylococcal enterotoxin B(SEB) is a bacterial SAg that predominantly interacts with V β 8+ T cells. In vivo treatment of mice with SEB leads to an initial increase in the percentage of V β 8+T cells， followed by a decrease in the numbers of these cells， eventually reaching lower levels than those found before treatment with the SAg. FCM detection shows that SEB-reactive T cells express high level of Fas and FasL. Moreover， in vivo treatment of functional FasL-deficient mutant gld mice with SEB didn’t cause an obvious decrease in the percentage of V β 8+ T cells. Exposure of naive CD4+ T lymphocytes to SEB could induce AICD， but the ability of SEB to induce apoptosis was inhibited by the addition of a Fas-Fc fusion protein. These results suggest the interaction of Fas and FasL brings about specific-T cells’ AICD induced by SEB[8，9]. CD4+ T cells derived from either Fas-defective Lpr or FasL-defective gld mice failed to undergo ACID induced by SEB. These cells were in a condition of proliferation. In other way， the Fas-negative mice obtained by gene knock out didn’t show clonal deletion in peripheral specific T cells affected by endogenous SAg， and just show specific-T cells proliferation complys with infiltration in liver and lung[10]. It is also found that Fas system plays a role in clonal deletion in peripheral specific T cells induced by SEA and endogenous Mtv-7-SAg encoded by mouse mammary tumor virus(MMTV)[4，5]. In this study， we demonstrated that apoptosis appeared obviously in second stimulation of the short-term SEA-reactive T cells from human peripheral blood with SEA 1 μg/ml and Fas/FasL was substantially up-regulated， with increasing cytosolic free[Ca2+]i. Based on these observations we think， SAg induced specific-T cells to express high levels of Fas and FasL in the absence of antigen presenting cells(APC) and other correlated adjuvant molecules. Death signal was delivered by cross-linking Fas with FasL and active intracellular correlated enzymes， causes increase of cytosolic free[Ca2+]i and active Ca2+-dependent endonuclease， which bring about T-cell apoptosis.
In the presence of APC， the interaction of B7 and CD28 molecules on the surface of APC provides a costimulatory signal， and the SAg-specific T cells can up-regulate the Bcl-2 or Bcl-XL gene and restrain the expression of FasL， which lets these cells to override the Fas death signal and allows cells to proliferate[11]. It is well known that anti-CD28 molecule can bind with CD28 on the surface of T cells and accelerate T cell proliferation by imitating B7 molecule.Because high concentration of IL-2 can promote T cells proliferation， and induce the expression of Bcl-2 in SAg-specific T cells to antagonize the T cell apoptosis mediated by Fas antigen， implantation of IL-2-containing osmotic pump can prolong the survival of SAg-reactive T cells expanded in mice injected with bacterial SAg. However， it is not clear whether SAg induces the deprivation of IL-2 after activation of plenty of T cells in vivo[12]. PMA assists SAg’s effect on T cell proliferation by activating PKC pathway and promoting the high expression of IL-2.^5#*], http://www.100md.com
Therefore， SAg-specific T cells can receive two stimulatory signals in the presence of APC and produce strong proliferative response induced by SAg： one is initiation signal by TCR-CD3 complex， in which one end of SAg binds with TCRV β chain and (or no) the other end binds with the negative polymorphic region of MHCⅡ molecules. The other is costimulatory signal by the interactions with B7 molecule in APC and CD28 in T cells. In the absence of APC， SAg-specific T cells can express high Fas and FasL and undergo apoptosis by cross-linking Fas with FasL. Under the condition of high concentration of IL-2 ， SAg-specific T cells will not undergo apoptosis but allow proliferation by up-regulating the expression of Bcl-2 gene in T cells and suppressing apoptosis mediated by Fas antigen.
[Foundation item] This work was supported by the Fund of the “Ninth five” obligatory budget of PLA (96Z038).!l;ih/, 百拇医药
[Biography] Song Jian-xun(1967-)，male， born in Feicheng county， Shangdong， instructor， M.D.，field of research：molecular immunology.!l;ih/, 百拇医药
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[Received date] 1999-12-24； [Revised date] 2000-04-04(宋建勋 朱锡华 陈克敏)