Gross A, Spiesser S, Terraza A, Rouot B, Caron E, Dornand J
INSERM U431, IFR Eugène Bataillon, Université de Montpellier-II, France.
Infect Immun. 1998 Apr;66(4):1309-16. doi: 10.1128/IAI.66.4.1309-1316.1998.
We examined the expression and activity of inducible nitric oxide synthase (iNOS) in both gamma interferon (IFN-gamma)-treated and untreated murine macrophages infected with the gram-negative bacterium Brucella suis. The bacteria were opsonized with a mouse serum containing specific antibrucella antibodies (ops-Brucella) or with a control nonimmune serum (c-Brucella). The involvement of the produced NO in the killing of intracellular B. suis was evaluated. B. suis survived and replicated within J774A.1 cells. Opsonization with specific antibodies increased the number of phagocytized bacteria but lowered their intramacrophage development. IFN-gamma enhanced the antibrucella activity of phagocytes, with this effect being greater in ops-Brucella infection. Expression of iNOS, interleukin-6, and tumor necrosis factor alpha (TNF-alpha) mRNAs was induced in both c-Brucella- and ops-Brucella-infected cells and was strongly potentiated by IFN-gamma. In contrast to that of cytokine mRNAs, iNOS mRNA expression was independent of opsonization. Similar levels of iNOS mRNAs were expressed in IFN-gamma-treated cells infected with c-Brucella or ops-Brucella; however, expression of iNOS protein and production of NO were detected only in IFN-gamma-treated cells infected with ops-Brucella. These discrepancies between iNOS mRNA and protein levels were not due to differences in TNF-alpha production. The iNOS inhibitor N omega-nitro-L-arginine methyl ester increased B. suis multiplication specifically in IFN-gamma-treated cells infected with ops-Brucella, demonstrating a microbicidal effect of the NO produced. This observation was in agreement with in vitro experiments showing that B. suis was sensitive to NO killing. Together our data indicate that in B. suis-infected murine macrophages, the posttranscriptional regulation of iNOS necessitates an additive signal triggered by macrophage Fcgamma receptors. They also support the possibility that in mice, NO favors the elimination of Brucella, providing that IFN-gamma and antibrucella antibodies are present, i.e., following expression of acquired immunity.
我们检测了经γ干扰素（IFN-γ）处理和未处理的、感染革兰氏阴性菌猪布鲁氏菌的小鼠巨噬细胞中诱导型一氧化氮合酶（iNOS）的表达及活性。细菌分别用含有特异性抗布鲁氏菌抗体的小鼠血清（ops-布鲁氏菌）或对照非免疫血清（c-布鲁氏菌）进行调理。评估了产生的一氧化氮（NO）在杀伤细胞内猪布鲁氏菌中的作用。猪布鲁氏菌在J774A.1细胞内存活并增殖。用特异性抗体进行调理可增加吞噬细菌的数量，但会降低其在巨噬细胞内的生长。IFN-γ增强了吞噬细胞的抗布鲁氏菌活性，在ops-布鲁氏菌感染中这种作用更强。在c-布鲁氏菌和ops-布鲁氏菌感染的细胞中均诱导了iNOS、白细胞介素-6和肿瘤坏死因子α（TNF-α）mRNA的表达，且IFN-γ可使其强烈增强。与细胞因子mRNA不同，iNOS mRNA的表达与调理无关。在经IFN-γ处理、感染c-布鲁氏菌或ops-布鲁氏菌的细胞中，iNOS mRNA的表达水平相似；然而，仅在经IFN-γ处理、感染ops-布鲁氏菌的细胞中检测到iNOS蛋白的表达和NO的产生。iNOS mRNA和蛋白水平之间的这些差异并非由于TNF-α产生的差异所致。iNOS抑制剂Nω-硝基-L-精氨酸甲酯特异性地增加了在经IFN-γ处理、感染ops-布鲁氏菌的细胞中猪布鲁氏菌的增殖，证明了所产生的NO具有杀菌作用。这一观察结果与体外实验一致，体外实验表明猪布鲁氏菌对NO杀伤敏感。我们的数据共同表明，在感染猪布鲁氏菌的小鼠巨噬细胞中，iNOS的转录后调控需要巨噬细胞Fcγ受体触发的附加信号。它们还支持这样一种可能性，即在小鼠中，只要存在IFN-γ和抗布鲁氏菌抗体，即获得性免疫表达后，NO有利于布鲁氏菌的清除。
