Umanskiy Konstantin, Robinson Chad, Cave Cindy, Williams Mark A, Lentsch Alex B, Cuschieri Joseph, Solomkin Joseph S
Division of Trauma and Critical Care, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA.
Surgery. 2003 Aug;134(2):378-83. doi: 10.1067/msy.2003.253.
Hydrogen peroxide production by human neutrophils is tightly coupled to integrin ligation. This provides a means for spatial localization of oxidant production. However, integrin specificity and consequent signaling mechanisms for this process remain undefined. In the present study we demonstrate that otherwise unstimulated neutrophils adherent to fibronectin, a beta(1) ligand, but not fibrinogen, a beta(2) ligand, produce hydrogen peroxide in a time-dependent fashion. We hypothesized that signaling proceeded through focal, adhesionlike structures.
Triton-X insoluble (actin cytoskeleton) fractions from suspension phase cells and cells adherent to the beta(1) integrin ligand fibronectin were assessed for the presence of the integrin-associated kinase Pyk2, the scaffolding protein paxillin, and the downstream Src family kinase Lyn. Lysates were subjected to immunoprecipitation with anti-phosphotyrosine and probed for Pyk2, paxillin, and Lyn. Associations between focal adhesion kinase (FAK) and paxillin were determined by immunoprecipitation with anti-FAK and probing with anti-paxillin antibody. Activation of NADPH oxidase was determined by demonstration of redistribution of p47(phox) in Triton-X insoluble fractions.
NADPH oxidase activation, as judged by H(2)O(2) production, occurred with fibronectin-, but not in suspension or fibrinogen-adherent cells. Cells adherent to fibronectin for 20 minutes demonstrated marked increases in Pyk2, paxillin, and Lyn activation in comparison to fibronectin-adherent and suspension phase cells.
This study demonstrates that fibronectin adherence is a significant initiating factor for NADPH oxidase assembly in human neutrophils. This appears to be mediated by beta(1) integrins. We demonstrate formation of focal adhesions containing Pyk2/FAK, paxillin, and Lyn, and translocation of p47(phox).
人类中性粒细胞产生过氧化氢与整合素连接紧密相关。这为氧化剂产生的空间定位提供了一种方式。然而，该过程中整合素的特异性及相应的信号传导机制仍不明确。在本研究中，我们证明了原本未受刺激的中性粒细胞黏附于纤连蛋白（一种β1配体）而非纤维蛋白原（一种β2配体）时，会以时间依赖性方式产生过氧化氢。我们推测信号传导通过局灶性、黏附样结构进行。
对悬浮相细胞以及黏附于β1整合素配体纤连蛋白的细胞的Triton-X不溶性（肌动蛋白细胞骨架）组分进行评估，检测整合素相关激酶Pyk2、支架蛋白桩蛋白以及下游Src家族激酶Lyn的存在情况。裂解物用抗磷酸酪氨酸进行免疫沉淀，然后检测Pyk2、桩蛋白和Lyn。通过用抗黏着斑激酶（FAK）进行免疫沉淀并用抗桩蛋白抗体检测，确定黏着斑激酶（FAK）与桩蛋白之间的关联。通过证明Triton-X不溶性组分中p47(phox)的重新分布来确定NADPH氧化酶的激活情况。
根据过氧化氢的产生判断，NADPH氧化酶的激活发生在黏附于纤连蛋白的细胞中，而在悬浮细胞或黏附于纤维蛋白原的细胞中未发生。与黏附于纤连蛋白的细胞和悬浮相细胞相比，黏附于纤连蛋白20分钟的细胞显示出Pyk2、桩蛋白和Lyn激活的显著增加。
本研究表明，纤连蛋白黏附是人类中性粒细胞中NADPH氧化酶组装的一个重要起始因素。这似乎是由β1整合素介导的。我们证明了含有Pyk2/FAK、桩蛋白和Lyn的黏着斑的形成以及p47(phox)的易位。
