IL-12恢复并促进氟尿嘧啶抑制的T细胞免疫功能

Interleukin-12 Restores and Promotes the T-cell Immune Function Inhibited by 5-Fluorouracil

背景与目的:目前,氟尿嘧啶(5-fluorouracil,5-FU)仍然是临床上广泛应用的肿瘤化疗药物之一。白细胞介素-12(interleukin-12,IL-12)的生物学功能是促进Th1细胞分化以及诱导CD8+T细胞γ干扰素(interferon-γ,IFN-γ)的产生。本研究进一步探讨5-FU对正常人外周血T细胞介导免疫应答的抑制机制,以及IL-12是否能够恢复由5-FU引起的免疫抑制功能。方法:观察5-FU对正常人外周血单核细胞(peripheral blood mononuclear cells,PBMCs)及肝癌细胞株HepG2增殖和活性的影响。另外,PBMCs在anti-CD3或anti-CD3+anti-CD28刺激的条件下,加入不同浓度5-FU(0.20～50.00μg/ml)共同培养,ELISA法检测培养上清中IFN-γ的水平。PBMCs预先加入或不加入5-FU共同培养后,再分别加入anti-CD3+anti-CD28刺激,利用流式细胞术分析T细胞亚群表达细胞因子IFN-γ和IL-2以及细胞活化分子CD25表达的变化。Anti-CD3+anti-CD28、IL-12、5-FU不同配伍组合与PBMCs共培养后收集上清液,ELISA法检测IFN-γ水平,然后利用流式细胞术检测T细胞亚群IFN-γ表达的变化。结果:5-FU抑制PBMCs和HepG2细胞的增殖,同时以浓度依赖方式抑制正常人PBMCsIFN-γ的产生(P=0.024)。T细胞亚群分析的结果表明,加入5-FU组与未加入5-FU组相比,表达IFN-γ的CD4+T细胞百分率从2.1%下降到0.7%,CD8+T细胞百分率从3.9%下降到2.2%;表达IL-2的CD4+T细胞百分率从2.5%下降到0.7%,CD8+T细胞百分率从0.4%下降到0.2%;同时这两种细胞亚群CD25的阳性率和平均荧光密度都明显降低。anti-CD3+anti-CD28组,加入IL-12前表达IFN-γ的CD4+和CD8+T细胞百分率分别为1.1%和3.2%,加入IL-12后分别为1.6%和4.1%;anti-CD3+anti-CD28+5-FU组,加入IL-12前表达IFN-γ的CD4+和CD8+T细胞百分率分别为0.5%和1.1%,加入IL-12后分别为1.0%和2.5%。结论:5-FU抑制肿瘤细胞增殖的同时也抑制正常人的免疫功能,IL-12能够恢复并促进由5-FU抑制的T细胞免疫功能。

BACKGROUND ＆ OBJECTIVE. Currently, 5-fluorouracil （5-FU） is still one of the widely applied chemotherapeutic agents for tumors. Interleukin-12 （IL-12） can promote the differentiation of Thl cells and induce the production of interferon-γ （IFN-γ） by CD8^＋ T cells. This study was to investigate the suppression mechanism of 5-FU on the immune response mediated by T cells from normal human peripheral blood, and to determine the effect of IL-12 on the immune suppression induced by 5-FU. METHODS. The effects of 5-FU on the proliferation of peripheral blood mononuclear cells （PBMCs） and liver cancer cell line HepG2 were examined. PBMCs were stimulated with either anti-CD3 alone or anti-CD3 plus anti-CD28 in the presence or absence of 5-FU at different concentrations （0.20-50.00 μg/ml）. The level of IFN-γ in the culture supernatant was determined by ELISA. PBMCs were pretreated with 5-FU and stimulated with anti-CD3 and anti- CD28 for 2 days. The proportions of CD4^＋IFN-γ^＋, CD8^＋IFN-γ^＋, CD4^＋IL-2^＋ and CD8^＋IL-2^＋ T cells, and the expression of CD25 on CD4^＋ and CD8^＋ T cells were examined by flow cytometry （FCM）. PBMCs were cultured in different combinations with anti-CD3 plus anti-CD28, IL-12 and/or 5-FU for 48 h. IFN-γ level in the supernatant was detected by ELISA. The expression of IFN-γ on CD4^＋ and CD8^＋ T cells were examined by FCM. RESULTS： 5-FU inhibited the proliferation of HepG2 cells and PBMCs, and suppressed INF-γ production in PBMCs in a dose-dependent manner. The proportions of immune T cells were lower in 5-FU-pretreated PBMCs than in control PBMCs （0.7% vs. 2.1% for CD4^＋IFN-γ^＋ T cells, 2.2% vs. 3.9% for CD8^＋IFN-γ^＋ T cells, 0.7% vs. 2.5% for CD4^＋IL-2^＋ T cells, 0.2% vs. 0.4% for CD8^＋IL-2^＋ T cells）. Both the positive rate and mean fluorescence intensity （MFI） of CD25 on CD4^＋ and CD8^＋ T cells were decreased after pretreatment of 5-FU. When stimulated by anti-CD3 and anti-CD28, the proportions of CD4^＋IFN-γ^＋ and CD8^＋IFN-γ^＋ T cells were 1.1% and 3.2% before adding IL-12, and 1.6% and 4.1% after treatment of IL-12. When stimulated by anti-CD3, anti-CD28, and 5-FU, the proportions of CD4^＋IFN-γ^＋ and CD8^＋IFN-γ^＋ T cells were 0.5% and 1.1% before adding IL-12, and 1.0% and 2.5% after treatment of IL-12. CONCLUSIONS; 5-FU could inhibit the proliferation of HepG2 cells and the immune function of PBMCs. IL-12 could restore the T-cell immune function inhibited by 5-FU.