Molecular biology on murine macrophage activated by bacterial infection or bacterial components

(1)分离纯化了经细菌脂多糖(LPS)刺激的小鼠(C3H/HEN)巨噬细胞内最主要的磷酸化胞浆蛋白--65 kDa蛋白(Pp65)。它是丝氨酸激酶的底物，具有与人L-纤溶酶相似的氨基酸序列。在骨髓细胞、肝细胞(可能是Kupffer细胞)和脾细胞中也发现了这种蛋白，而在红细胞和胸腺细胞中没有发现这种蛋白。在低应答C3H/HeJ小鼠的腹腔巨噬细胞中检测到相同的蛋白。而C3H/HeJ巨噬细胞的蛋白在脂多糖刺激下不能被磷酸化。蛋白激酶C(PKC)或钙调蛋白依赖的蛋白激酶抑制剂(IH)对pp65蛋白的磷酸化有影响，而酪氨酸酶的IH不影响pp65蛋白的磷酸化。PKC IH影响TNF-α和IL-1βmRNA的表达及其活性分子的产生。但钙调素依赖的IH抑制IL-1的mRNA表达和产生，但不抑制肿瘤坏死因子的表达和产生。此外，用非致热类脂A类似物处理巨噬细胞可诱导pp65蛋白磷酸化对内毒素刺激的不应态。(2)嗜肺军团菌或鼠伤寒沙门氏菌感染小鼠腹膜巨噬细胞后，pp76(L.P.)或pp85和72(S.t.)对这些微生物的感染是特异的。(3)细胞因子如肿瘤坏死因子α、肿瘤坏死因子β、白介素1β和白介素6可增强小鼠腹腔巨噬细胞对沙门氏菌或铜绿假单胞菌的杀灭作用。

(1) The most dominantly phosphorylated cytosolic protein, 65kDa protein (pp65), in the murine (C3H/HeN) peritoneal macrophages after stimulation with bacterial lipopolysaccharide (LPS) was isolated and purified. It was a substrate of serine kinase and possessed the amino acid sequences similar to human L-plastin. This protein was also found in the bone marrow cells, liver cells (presumably Kupffer cells) and spleen cells, while it could not be seen in the red blood cells and thymocytes. Identical protein was detected in the peritoneal macrophages of LPS-low responder C3H/HeJ mice. However, the protein in C3H/HeJ macrophages was unable to be phosphorylated by the stimulation with LPS. The posphorylation of pp65 was affected by inhibitors (IH) of protein kinase C (PKC) or calmodulin-dependent kinase, though it was not affected by IH of tyrosine kinase. PKC IH affected the expression of TNF-alpha and IL-1beta mRNA and the production their active molecules. However, calmodulin-dependent IH inhibited mRNA expression and production of IL-1, but did not inhibit those of TNF. Furthermore, the previous treatment of macrophages with nonpyrogenic lipid A analogues induced refractory states on pp65 phosporylation to LPS stimulation.(2) Murine peritoneal macrophages that had been infected with virulent strain of Legionella pneumophila or Salmonella typhimurium exhibited the protein phosphorylation of pp76(L. p.) or pp85 and 72 (S. t.) which was specific to the infection of these organisms. Live organisms themselves or Heat-killed organisms of these strains did not cause the phosphorylation.(3) It was demonstrated that cytokines such as TNF-alpha, TNF-beta, IL-1beta and IL-6 enhanced the killing of the organisms of Salmonella or Pseudomonas aeruginosa by the peritoneal macrophages of mice.