Trypsin inhibits lipopolysaccharide signaling in macrophages via toll-like receptor 4 accessory molecules
Trypsin inhibits lipopolysaccharide signaling in macrophages via toll-like receptor 4 accessory molecules
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DOI：
10.1016/j.lfs.2012.06.030
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发表时间：
2012-08-21
期刊：
影响因子：
6.1
通讯作者：
Sato, Koichi
中科院分区：
文献类型：
--
作者：
Komatsu, Hiroyuki;Shimose, Akihiro;Sato, Koichi
目的：为了研究胰蛋白酶在巨噬细胞免疫应答中的作用，并确定蛋白酶激活受体（PARS）是否参与胰蛋白酶的作用，主要方法：我们使用从C57 BL/6野生型小鼠、PAR 2敲除小鼠和ddY小鼠分离的RAW 264.7细胞和腹腔巨噬细胞。在存在或不存在胰蛋白酶、凝血酶和PAR亚型特异性激动剂（PARs-AP）的情况下，用脂多糖（LPS）刺激巨噬细胞。巨噬细胞的活化通过一氧化氮的产生和炎症介质的表达来定量，所述炎症介质例如诱导型一氧化氮合酶、白细胞介素-1 β和白细胞介素-6。为了阐明胰蛋白酶对LPS受体的影响，我们还研究了toll样受体4（TLR 4）、可溶性MD-2（sMD-2）、膜结合MD-2（mMD-2）、可溶性CD 14（sCD 14）和膜结合CD 14（mCD 14）的表达。为了直接研究胰蛋白酶对CD 14蛋白的影响，我们表达了重组CD 14蛋白。关键发现：胰蛋白酶抑制LPS诱导的一氧化氮产生和诱导型一氧化氮合酶，白细胞介素-1 β和白细胞介素-6的表达。在野生型巨噬细胞和PAR 2敲除巨噬细胞中观察到胰蛋白酶的相同抑制作用。此外，其他PAR激动剂凝血酶、PAR 1-AP、PAR 2-AP和PAR 4-AP不能模拟胰蛋白酶的作用。尽管胰蛋白酶不影响TLR 4或mMD-2的表达，但胰蛋白酶可降低sCD 14、mCD 14和5 MD-2的表达。意义：胰蛋白酶通过降解TLR 4辅助分子，非依赖性地抑制LPS信号转导。这一观察结果提供了一个更好的理解复杂的免疫反应在急性胰腺炎。(C)2012 Elsevier Inc. All rights reserved.
Aims: To examine the role of trypsin in the immune response of macrophages and to determine whether protease-activated receptors (PARS) are involved in the effects of trypsin.Main methods: We used RAW264.7 cells and peritoneal macrophages isolated from C57BL/6 wild-type mice, PAR2 knockout mice, and ddY mice. Macrophages were stimulated with lipopolysaccharide (LPS) in the presence or absence of trypsin, thrombin, and PAR subtype-specific agonists (PARs-AP). Activation of macrophages was quantified by nitric oxide production and expression of inflammatory mediators, such as inducible nitric oxide synthase, interleukin-1 beta, and interleukin-6. To clarify the effect of trypsin on LPS receptors, we also investigated the expression of toll-like receptor 4 (TLR4), soluble MD-2 (sMD-2), membrane-bound MD-2 (mMD-2), soluble CD14 (sCD14), and membrane-bound CD14 (mCD14). To directly investigate the effect of trypsin on CD14 protein, we expressed recombinant CD14 protein.Key findings: Trypsin inhibited LPS-induced nitric oxide production and expression of inducible nitric oxide synthase, interleukin-1 beta, and interleukin-6. The same inhibitory effects of trypsin were observed in wild-type macrophages and in PAR2 knockout macrophages. Furthermore, the other PAR agonists, thrombin, PAR1-AP, PAR2-AP, and PAR4-AP, did not mimic the effect of trypsin. Although trypsin did not affect TLR4 or mMD-2 expression, sCD14, mCD14, and 5MD-2 expressions were decreased by trypsin. Furthermore, trypsin also degraded recombinant CD14 protein.Significance: Trypsin inhibited LPS signaling PAR-independently via degradation of TLR4 accessory molecules. This observation provides a better understanding of the complicated immune response in acute pancreatitis. (C) 2012 Elsevier Inc. All rights reserved.