自然殺手細胞活化致死之研究
Study of Activation-induced Cell Death of Natural Killer Cell
中文摘要
自然殺手細胞是人類先天免疫系統中重要的作用細胞﹐並可調控後天免疫 反應。目前已知, 在一免疫反應中, 大多數被活化淋巴細胞最終都會死亡
。尤其經由CD3/TCR 複合物的啟動, 再刺激已被活化的 T 細胞, 其後會造成 此細胞進行細胞凋零, 此一過程被稱為活化致死( Activation-induced
cell death: AICD)。 因此, 本論文主要在探討自然殺手細胞活化致死的 現象及調控其活化致死的機制。首先, 利用 PMA/Ionophore, 抗活化型接 受體CD16 抗體, 及抗抑制型接受體 Kp43 抗體, 去刺激細胞。發現 PMA/
Ionophore 或抗 CD16 抗體對細胞刺激 12-15 小時後, 約有 60-70%的細胞死於 細胞凋零, 但抗 Kp43 抗體對細胞的刺激, 則無此現象。我們進一步檢測 TNF 接受體超族群(TNF receptor superfamily)中 Fas 及 Lymphotoxin-b(
LTbR)在上述自然殺手細胞活化致死現象之角色。經由流式細胞分析儀針 對細胞表面分子的分析, 發現剛從健康人血分離出的自然殺手細胞, 可表 達少數Fas 分子和極高量的 LTbR 及 LTb 分子, 但不表達 Fas ligand。 經過 兩禮拜的體外培養後, 則 Fas 的表現量增加﹐其餘三者不變。之後在 PMA/
Ionophore 或抗 CD16 抗體的刺激下, Fas ligand 被表達出來, Fas 的表現量 不變, 而 LTb 和 LTbR 的表達卻降為原本的 30-50%。至於抗 Kp43 抗體的刺激 對於這四種表面分子的表達﹐與刺激前比較﹐並無明顯不同。同時﹐我們 利用桿狀病毒蛋白質表達系統( Baculovirus protein expression
system)製備溶解性 Fas-Fc 融合蛋白及溶解性 LTbR-Fc 融合蛋白後﹐發現溶 解性Fas-Fc 融合蛋白可抑制 PMA/Ionophore 或抗 CD16 抗體所引起的細胞凋 零, 而溶解性 LTbR-Fc 融合蛋白則否。接著,利用介白素-4 號, 發現在一濃 度依存趨勢下(dose dependent ), 也可抑制細胞的活化致死. 綜合以上
資料, 我們證實了自然殺手細胞在經由其表面活化型接受體 CD16 的刺激後
﹐可進行"活化致死"的現象, 且 Fas 所導致的細胞凋零為調控此"活化致 死"的主要機制, 而 LTbR 所導致之細胞凋零則不明顯。相反地, 我們認為 LTbR 可傳遞一抑制細胞凋零的訊號, 因當我們利用溶解性 LTbR-Fc 融合蛋 白去抑制LTb 和 LTbR 的交互作用時, 反而會造成 Fas ligand 的產生並進而 促進細胞凋零. 最後, 我們也證明介白素-4 號有調控此活化致死現象的能 力.
英文摘要
Human natural killer cells (NK) play important roles in innate immune system and have the potential to regulate the adapted immune responses. During a immune response, most activated
lymphocytes eventually died. Especially, restimulating activated T cells by triggering CD3/TCR complex would make them undergo apoptosis, a process termed as activation-induced cell death:
AICD. Thus, we intend to determine the occurrence of AICD on NK cells and mechanisms directing the AICD. First of all, we
stimulated cells by the treatment of PMA/Ionophore or
crosslinking CD16 or Kp43 molecules on the surface of NK cells ,and found that by the treatment of PMA /Ionophore or
crosslinking CD16 molecules for 12-15 hours, 60-70% of short- term cultured NK cells died in the form of apoptosis but not for the crosslinking of Kp43 molecules. We further determine the roles of Fas and LTbR, two members of TNF receptor super-family in the AICD of NK cells. Through analysis toward surface
molecules on NK cells by Flow Cytometer, we have found that freshly isolated NK cells could express low level of Fas
molecules, pretty high amounts of LTb and LTbR molecules but no Fas ligand. Two-week in-vitro culture later, the only difference is the enrichment of Fas molecules. By the treatment of PMA /Ionophore or crosslinking CD16 molecules, Fas ligand molecules were induced, Fas molecules were with same expressive pattern but the expressive amounts of LTb and LTbR molecules were significantly reduced to 30-50% of the original ones.
Stimulation upon crosslinking Kp43 molecules have no significant effects on the expressive patterns of the four molecules. Then we took advantage of Baculovirus protein expression system to synthesize soluble Fas-Fc and soluble LTbR-Fc proteins and found that s-Fas-Fc could prevent cells from apoptosis which resulted from stimulation of PMA/Ionophore or cross- linking CD16 ,but s- LTbR-Fc could not. Finally, the usage of IL-4 has been shown to block the AICD of NK cells in a dose-dependent manner. In summary, we proved that NK cells could undergo " AICD" upon crosslinking the activation-receptor, CD16 and Fas mediated apoptosis played the major roles in regulating AICD, but not for LTbR mediated apoptosis. In contrast, signals delivered from LTbR would inhibit apoptosis and therefor the interactions
betweem LTb and LTbR were essential for survival of human NK cells.
