New recognition of Leishmania major by infected macrophages

(A)当L.major在体内感染IRF-4缺陷巨噬细胞时，与野生型相比，早期（感染后2-5周）足垫厚度增加。(B) IRF-4缺陷型巨噬细胞与野生型细胞感染L.major的比例相同。然而，当L.major在体外感染IRF-4缺陷巨噬细胞48小时后，感染的巨噬细胞比例明显低于野生型，这表明IRF-4负向调节L.major从感染的巨噬细胞中被排斥。IRF-4缺陷的l - major感染巨噬细胞产生的TNF-α和NO明显高于野生型。有趣的是，IRF-4缺陷和L.major感染的巨噬细胞产生的IL-12远低于野生型。我们预计识别L.major的信号与TLR信号不同，因为我们已经报道过在IRF-4缺陷的巨噬细胞中，通过TLR产生的IL-12和TNF-α显著增强。IRF-4 KO巨噬细胞感染L.major后细胞因子产生的转录水平调控制备了多种变性L.major抗原，用于鉴定L.major分子。热变性或蛋白酶K处理的l .主抗原不影响IRF-4 KO巨噬细胞TNF-α或IL-12的细胞因子产生模式。l .主要分子为不溶性部分，为蛋白酶K抗性分子。提示主要抗原不是蛋白质。而离子交换色谱（如Bio-Cad）是不可能的。(C)对于l .主抗原的分离，采用凝胶过滤法，因为凝胶过滤法可以根据分子量对每个分子进行分离。

(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.