Green S J, Crawford R M, Hockmeyer J T, Meltzer M S, Nacy C A
Department of Cellular Immunology, Walter Reed Army Institute of Research, Washington, DC 20307-5100.
J Immunol. 1990 Dec 15;145(12):4290-7.
Macrophages exposed to IFN-gamma and infected with amastigotes of Leishmania major develop the capacity to eliminate the intracellular pathogen. This antimicrobial activity of activated macrophages correlates with the initiation of nitrogen oxidation of L-arginine, yet other reports suggest that two signals are required for induction of this biochemical pathway for effector activity. In the present studies, macrophages treated with up to 100 U/ml IFN-gamma, or 100 ng LPS, or 10(7) amastigotes produced minimal quantities (less than 9 microM) of NO2- and failed to develop cytotoxic effector activities. In contrast, the combination of IFN-gamma and either LPS (greater than 0.1 ng) or amastigotes (10(6) induced high concentrations (much greater than 30 microM) of NO2- and macrophage cytotoxicity against intra- and extracellular targets. The induction of nitrogen oxidation by amastigotes could be dissociated from LPS-induced events by 1) performing the assays in the presence of polymyxin B (which blocked LPS effects, but not amastigote effects), 2) determining the threshold of IFN-gamma required to prime cells for subsequent trigger (1 U/ml for LPS trigger effects; 10-fold higher for amastigotes), and 3) determining the heat sensitivity of the two trigger agents (amastigote effects abolished at 100 degrees C; LPS effects unaffected at this temperature). Further, culture fluids from amastigote-infected macrophages did not contain detectable LPS (less than 6 pg/ml). Possible parasite and cell-associated factors that could contribute to the induction of nitrogen oxidation and cytotoxic activity of IFN-gamma treated macrophages were examined: only certain intact microorganisms, LPS from a variety of bacteria, and the cytokine TNF alpha were effective. Both NO2- production and intracellular killing were abolished by the addition of anti-TNF-alpha mAb in the assay. TNF-alpha was produced by amastigote-infected macrophages and IFN-gamma dramatically enhanced secretion of this cytokine; IFN-gamma alone had no effect. Endogenous TNF-alpha produced during infection of macrophages with L. major acted in an autocrine fashion to trigger the production of L-arginine-derived toxic nitrogen intermediates that killed the intracellular parasites.
暴露于γ干扰素并感染了硕大利什曼原虫无鞭毛体的巨噬细胞，会产生清除细胞内病原体的能力。活化巨噬细胞的这种抗菌活性与L-精氨酸的氮氧化起始相关，但其他报告表明，诱导这种效应活性的生化途径需要两个信号。在本研究中，用高达100 U/ml的γ干扰素、或100 ng脂多糖（LPS）、或10⁷个无鞭毛体处理的巨噬细胞产生的亚硝酸盐（NO₂⁻）量极少（小于9 μM），且未产生细胞毒性效应活性。相比之下，γ干扰素与LPS（大于0.1 ng）或无鞭毛体（10⁶）的组合可诱导高浓度（远大于30 μM）的NO₂⁻以及巨噬细胞对细胞内和细胞外靶标的细胞毒性。无鞭毛体诱导的氮氧化可通过以下方式与LPS诱导的事件分离：1）在多粘菌素B存在下进行测定（多粘菌素B可阻断LPS的作用，但不阻断无鞭毛体的作用）；2）确定引发细胞后续触发所需的γ干扰素阈值（LPS触发效应为1 U/ml；无鞭毛体则高10倍）；3）确定两种触发剂的热敏感性（无鞭毛体效应在100℃时消除；此温度下LPS效应不受影响）。此外，无鞭毛体感染的巨噬细胞的培养液中未检测到可检测到的LPS（小于6 pg/ml）。研究了可能有助于诱导γ干扰素处理的巨噬细胞的氮氧化和细胞毒性活性的寄生虫和细胞相关因子：只有某些完整的微生物、来自多种细菌的LPS以及细胞因子肿瘤坏死因子α（TNF-α）是有效的。在测定中加入抗TNF-α单克隆抗体可消除NO₂⁻的产生和细胞内杀伤。TNF-α由无鞭毛体感染的巨噬细胞产生，γ干扰素可显著增强这种细胞因子的分泌；单独的γ干扰素则无作用。巨噬细胞被硕大利什曼原虫感染期间产生的内源性TNF-α以自分泌方式作用，触发L-精氨酸衍生的有毒氮中间体的产生，从而杀死细胞内寄生虫。
