［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Ca^2＋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［Ca^2＋］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 1μg／ml建立的短期SEA反应T细胞对SEA 1μg／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16h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，16h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［Ca^2＋］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［Ca^2＋］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。

［中图分类号］R392．11；R364［文献标识码］A

The effect of Fas／FasL on T cell apoptosis induced by

staphylococcal enterotoxin A（SEA）

SONG Jian-xun， ZHU Xi-hua， CHEN Ke-min

（Institute of Immunology PLA， the Third Military Medical University， Chongqing 400038，China）

［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 Ca^2＋ 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 Ca^2＋． Conclusion These findings suggest that Fas／FasL plays an important role in the superantigen-induced T cell apoptosis and the increase of cytosolic Ca^2＋ was related to DNA fragmentation and morphological changes of T cell apoptosis．

［Key words］ Fas／FasL；T cell； apoptosis； superantigen； Ca^2＋

［Article ID］1000-8861（2000）04-0241-05

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 ［Ca^2＋］i in apoptosis were observed．

1 MATERIALS AND METHODS

1．1 Materials

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．

1.1.3 Major instruments FACstar plus flow Cytometry （FCM， Becton-Dickinson）．TC2323 CO₂ incubator（Shell／Jb）． MPF-4 fluorescent spectrophotometer （Hitachi）．

1．2 Methods

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．

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．

1.2.4 Changes of cytosolic free Ca^2＋ 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 ［Ca^2＋］i measured． ［Ca^2＋］i＝Kd（F-F_min）／（F_max-F），Kd is 224nm． F，F_max and F_min were measured by fluorescent spectrophotometer．

2 RESULTS

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