New recognition of Leishmania major by infected macrophages

(1)体内感染IRF-4基因缺陷巨噬细胞后，早期(感染后2-5周)大鼠足垫厚度较野生型增加。(2)IRF-4基因缺陷巨噬细胞与野生型细胞感染L型的比例相同。但体外感染IRF-4基因缺陷的巨噬细胞48h后，感染巨噬细胞的比例明显低于野生型，提示IRF-4负性调节巨噬细胞排斥感染的巨噬细胞。IL-4缺乏的巨噬细胞产生的肿瘤坏死因子-α和NO显著高于野生型巨噬细胞。有趣的是，IRF-4缺乏的巨噬细胞和主要感染L的巨噬细胞产生的IL-12远低于野生型。我们认为识别L.Major的信号不同于Tlr信号，因为我们已经报道，在Irf-4缺乏的巨噬细胞中，通过Tlr产生的IL-12和肿瘤坏死因子-α显著增强。IRF-4 KO巨噬细胞感染L.main后细胞因子产生的变化在转录水平上的调节。制备各种变性乳杆菌主要抗原，鉴定主要乳杆菌主要分子。热变性或蛋白水解酶K处理的L-主要抗原不影响IRF-4KO巨噬细胞产生肿瘤坏死因子-α或IL-12的模式。L主要分子为不溶组分，为抗蛋白酶K分子。提示主要抗原不是蛋白质。而用Bio-Cad等离子交换层析法是不可能的。(C)主要抗原的分离采用凝胶过滤的方法，因为它可以按分子量分离每个分子。

(A)When L.major infected to IRF-4 deficient macrophages in vivo, footpad thickness increased at the early stage (2-5 weeks after infection)compared to wild type.(B)The rate of the L.major infection was same between IRF-4 deficient macrophages and wild-type cells. However, the proportion of the infected macrophages remarkably decreased compared to wild type when L.major infected to IRF-4 deficient macrophages for 48 hrs in vitro, suggested that IRF-4 negatively regulated exclusion of L.major from the infected macrophages. TNF-α and NO from IRF-4 deficient, L.major-infected macrophages produced much higher than wild-type. Interestingly, IL-12 from IRF-4 deficient, and L.major infected macrophages produced much lower than wild-type. We expected that signaling to recognize L.major was different from TLR signaling because we have already reported that both IL-12 and TNF-α production through TLR remarkably enhanced in IRF-4 deficient macrophages. Alternation of cytokine production in IRF-4 KO macrophages to infect with L.major regulated at the transcriptional level. Various kind of denatured L.major antigens were made to identify major L.major molecule. Heat denatured or Proteinase K treated L.major antigen did not affect cytokine production pattern of TNF-α or IL-12 in IRF-4 KO macrophages. L.major molecule contained in insoluble fraction and was Proteinase K resistant molecule. It suggested that major antigen was not protein. And it was impossible to use ion exchange chromatography such as Bio-Cad.(C)To separate L.major antigen, gel filtration method have been performed because it was able to separate each molecules by molecular weight.