Arndt Patrick G, Suzuki Naohito, Avdi Natalie J, Malcolm Kenneth C, Worthen G Scott
Department of Medicine and Division of Cell Biology, National Jewish Medical and Research Center, Denver, Colorado, USA.
J Biol Chem. 2004 Mar 19;279(12):10883-91. doi: 10.1074/jbc.M309901200. Epub 2003 Dec 29.
Polymorphonuclear leukocytes (neutrophils) respond to lipopolysaccharide (LPS) through the up-regulation of several pro-inflammatory mediators. We have recently shown that LPS-stimulated neutrophils express monocyte chemoattractant protein 1 (MCP-1), an AP-1-dependent gene, suggesting that LPS activates the c-Jun N-terminal kinase (JNK) pathway in neutrophils. Previously, we have shown the activation of p38 MAPK, but not JNK, in suspended neutrophils stimulated with LPS but have recently shown activation of JNK by TNF-alpha in an adherent neutrophil system. We show here that exposure to LPS activates JNK in non-suspended neutrophils and that LPS-induced MCP-1 expression, but not tumor necrosis factor-alpha (TNF-alpha) or interleukin-8 (IL-8), is dependent on JNK activation. In addition, LPS stimulation of non-suspended neutrophils activates Syk and phosphatidylinositol 3-kinase (PI3K). Inhibition of Syk with piceatannol or PI3K with wortmannin inhibited LPS-induced JNK activation and decreased MCP-1 expression after exposure to LPS, suggesting that both Syk and PI3K reside in a signaling pathway leading to LPS-induced JNK activation in neutrophils. This Syk- and PI3K-dependent pathway leading to JNK activation after LPS exposure in non-suspended neutrophils is specific for JNK, because inhibition of neither Syk nor PI3K decreased p38 activation after LPS stimulation. Furthermore we show that PI3K inhibition decreased LPS-induced Syk activation suggesting that PI3K resides upstream of Syk in this pathway. Finally, we show that Syk associates with Toll-like receptor 4 (TLR4) upon LPS stimulation further implicating Syk in the LPS-induced signaling pathway in neutrophils. Overall our data suggests that LPS induces JNK activation only in non-suspended neutrophils, which proceeds through Syk- and PI3K-dependent pathways, and that JNK activation is important for LPS-induced MCP-1 expression but not for TNF-alpha or IL-8 expression.
多形核白细胞（中性粒细胞）通过上调多种促炎介质来响应脂多糖（LPS）。我们最近发现，LPS刺激的中性粒细胞表达单核细胞趋化蛋白1（MCP-1），这是一个依赖AP-1的基因，提示LPS激活了中性粒细胞中的c-Jun氨基末端激酶（JNK）途径。此前，我们已证明LPS刺激悬浮的中性粒细胞时p38丝裂原活化蛋白激酶（MAPK）被激活，但JNK未被激活，不过最近我们发现在贴壁中性粒细胞系统中TNF-α可激活JNK。我们在此表明，暴露于LPS可激活非悬浮中性粒细胞中的JNK，且LPS诱导的MCP-1表达（而非肿瘤坏死因子-α（TNF-α）或白细胞介素-8（IL-8）的表达）依赖于JNK激活。此外，LPS刺激非悬浮中性粒细胞可激活脾酪氨酸激酶（Syk）和磷脂酰肌醇3激酶（PI3K）。用白皮杉醇抑制Syk或用渥曼青霉素抑制PI3K可抑制LPS诱导的JNK激活，并降低暴露于LPS后的MCP-1表达，提示Syk和PI3K均存在于导致LPS诱导中性粒细胞JNK激活的信号通路中。在非悬浮中性粒细胞中，这种LPS暴露后导致JNK激活的依赖Syk和PI3K的途径对JNK具有特异性，因为抑制Syk或PI3K均不会降低LPS刺激后的p38激活。此外，我们表明PI3K抑制可降低LPS诱导的Syk激活，提示在该途径中PI3K位于Syk的上游。最后，我们表明LPS刺激时Syk与Toll样受体4（TLR4）结合，进一步表明Syk参与了中性粒细胞中LPS诱导的信号通路。总体而言，我们的数据表明LPS仅在非悬浮中性粒细胞中诱导JNK激活，其通过依赖Syk和PI3K的途径进行，且JNK激活对LPS诱导的MCP-1表达很重要，但对TNF-α或IL-8表达不重要。
