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

细菌感染或细菌成分激活小鼠巨噬细胞的分子生物学

基本信息

批准号：
03454184
负责人：
NAKANO Masayasu
金额：
$ 3.84万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

项目摘要

(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.

(1)分离纯化了细菌脂多糖（LPS）刺激后小鼠（C3H/HeN）腹膜巨噬细胞中磷酸化最显著的胞质蛋白65kDa （pp65）。它是丝氨酸激酶的底物，具有与人l -活蛋白相似的氨基酸序列。骨髓细胞、肝细胞（可能是库普弗细胞）和脾脏细胞中也发现了这种蛋白质，而红细胞和胸腺细胞中却没有发现这种蛋白质。在脂多糖低反应C3H/HeJ小鼠的腹腔巨噬细胞中检测到相同的蛋白。然而，C3H/HeJ巨噬细胞中的蛋白在LPS刺激下不能被磷酸化。pp65的磷酸化受到蛋白激酶C （PKC）或钙调素依赖性激酶抑制剂（IH）的影响，而不受酪氨酸激酶IH的影响。PKC IH影响tnf - α和il -1 β mRNA的表达及其活性分子的产生。然而，钙调素依赖性IH抑制mRNA的表达和IL-1的产生，但不抑制TNF的产生。此外，先前用非热原性脂质A类似物治疗巨噬细胞诱导pp65磷酸化对LPS刺激的不耐状态。(2)小鼠腹腔巨噬细胞感染嗜肺军团菌或鼠伤寒沙门菌后，pp76(L)蛋白磷酸化。p.)或pp85和72 (S. t.)，这是对这些有机体感染的特异性。这些菌株的活生物体本身或热灭活生物体都没有引起磷酸化。(3)实验证明，tnf - α、tnf - β、il -1 β和IL-6等细胞因子对小鼠腹腔巨噬细胞对沙门氏菌或铜绿假单胞菌的杀伤作用增强。