Walker D H, Popov V L, Crocquet-Valdes P A, Welsh C J, Feng H M
Department of Pathology, University of Texas, Medical Branch, Galveston 77555-0609, USA.
Lab Invest. 1997 Jan;76(1):129-38.
In a murine model of rickettsial disease in which, as in human rickettsioses, endothelial cells are the major target of infection, depletion of IFN-gamma or TNF-alpha converts a sublethal infection into a uniformly fatal disease with overwhelming rickettsial growth and decreased nitric oxide (NO) synthesis. The kinetics of NO production and rickettsial survival and growth were examined on Days 1, 2, and 3 after inoculation of endothelial cells with Rickettsia conorii under four different experimental conditions: (a) no cytokine treatment, (b) treatment with IFN-gamma and TNF-alpha, (c) treatment with cytokines and NG monomethyl-L-arginine, a competitive inhibitor of NO synthesis, and (d) treatment with sodium nitroprusside, a source of NO. Endothelial cells were examined for the presence of inducible nitric oxide synthase mRNA by specific reverse transcriptase-PCR after stimulation with IFN-gamma and TNF-alpha. Cytokine-stimulated and unstimulated rickettsiae-infected endothelial cells were examined by electron microscopy to observe the cellular and rickettsial events. Transformed and diploid mouse endothelial cells stimulated by the combination of recombinant murine IFN-gamma and TNF-alpha killed intracellular Rickettsia conorii by a mechanism that required the synthesis of NO. The antirickettsial effect and NO synthesis were inhibited by treatment of endothelial cells with NG monomethyl-L-arginine. Addition of nitroprusside, which released NO, also exerted a strong antirickettsial effect in the absence of IFN-gamma and TNF-alpha. Endothelial inducible nitric oxide synthase mRNA was detected 4 hours after cytokine stimulation, increased substantially at 8 hours, and decreased to low levels by 72 hours. Ultrastructural evaluation revealed that endothelial cells effected rickettsial killing in association with autophagy. Double membranes of endothelial cell granular endoplasmic reticulum surrounded rickettsiae, which were also observed being destroyed within phagolysosomes. This study demonstrated for the first time that endothelial cells are capable of killing rickettsiae. When stimulated by the combination of IFN-gamma and TNF-alpha, mouse endothelial cells kill Rickettsia conorii by an NO-dependent mechanism. Within the endothelium, NO exerts a rickettsicidal effect.
在立克次体病的鼠模型中，如同人类立克次体病一样，内皮细胞是主要感染靶标，γ干扰素（IFN-γ）或肿瘤坏死因子-α（TNF-α）的缺失会将亚致死性感染转变为一致致命的疾病，伴有立克次体大量增殖以及一氧化氮（NO）合成减少。在四种不同实验条件下，于接种康氏立克次体后的第1、2和3天，检测内皮细胞中NO产生、立克次体存活及生长的动力学情况：（a）无细胞因子处理；（b）用IFN-γ和TNF-α处理；（c）用细胞因子和NG-单甲基-L-精氨酸（一种NO合成的竞争性抑制剂）处理；（d）用硝普钠（一种NO来源）处理。在用IFN-γ和TNF-α刺激后，通过特异性逆转录聚合酶链反应检测内皮细胞中诱导型一氧化氮合酶mRNA的存在情况。通过电子显微镜检查细胞因子刺激的和未刺激的立克次体感染的内皮细胞，以观察细胞和立克次体相关事件。重组鼠IFN-γ和TNF-α联合刺激转化的和二倍体小鼠内皮细胞，通过一种需要NO合成的机制杀死细胞内的康氏立克次体。用NG-单甲基-L-精氨酸处理内皮细胞可抑制抗立克次体作用和NO合成。添加释放NO的硝普钠，在无IFN-γ和TNF-α时也发挥强大的抗立克次体作用。细胞因子刺激4小时后可检测到内皮诱导型一氧化氮合酶mRNA，8小时时大幅增加，72小时时降至低水平。超微结构评估显示内皮细胞通过自噬作用实现立克次体杀伤。内皮细胞颗粒内质网的双层膜围绕着立克次体，也观察到立克次体在吞噬溶酶体内被破坏。本研究首次证明内皮细胞能够杀死立克次体。当受到IFN-γ和TNF-α联合刺激时，小鼠内皮细胞通过NO依赖性机制杀死康氏立克次体。在内皮细胞内，NO发挥立克次体杀伤作用。
