Lomakina Elena B, Waugh Richard E
Department of Pharmacology and Physiology, University of Rochester, Medical Center, Rochester, NY.
Cell Mol Bioeng. 2010 Jun 1;3(2):106-116. doi: 10.1007/s12195-009-0099-x.
The dynamic response of neutrophils to interleukin-8 (IL-8) is of central interest in inflammation. Chemokine -induced β(2) integrin dependent adhesion can take several minutes after initial contact with IL-8 as evidenced by increased cell adhesion to intracellular adhesion molecule 1 (ICAM-1). The goal of this study is to identify signaling events that are critical for this response. We demonstrate that neither the PI3K inhibitor wortmannin, nor the PKC inhibitor bisindolymaleimide had any effect on IL-8 induced adhesion to ICAM-1. However, inhibition of PLC with U73122 or stopping the release of intracellular calcium by its downstream effector IP3 with caffeine or 2-aminoethoxydiphenyl borate completely blocked the adhesive response. Chelation of intracellular calcium with BAPTA or extracellular calcium with EGTA completely abrogated neutrophil adhesion to ICAM-1. This adhesion is mediated by LFA-1 (α(L)β(2)) within first 300 seconds after chemokine stimulation, followed by Mac-1 (α(M)β(2)) mediated adhesion, beginning 350 seconds after stimulus. Inhibition of p38MAP kinase results in a time course similar to Mac-1 inhibition, consistent with published evidence that Mac-1 mediated adhesion is p38MAP kinase dependent. These findings confirm a PLC dependent, PKC independent pathway from chemokine stimulus to integrin activation previously identified in other cell types, and demonstrate distinct dynamics and different requirements for LFA-1 vs. Mac-1 activation in primary human neutrophils.
中性粒细胞对白介素-8（IL-8）的动态反应是炎症研究的核心关注点。趋化因子诱导的β(2)整合素依赖性黏附在最初接触IL-8后可能需要几分钟，这可通过细胞对细胞间黏附分子1（ICAM-1）的黏附增加得以证明。本研究的目的是确定对此反应至关重要的信号事件。我们证明，磷脂酰肌醇-3激酶（PI3K）抑制剂渥曼青霉素和蛋白激酶C（PKC）抑制剂双吲哚马来酰胺对IL-8诱导的对ICAM-1的黏附均无任何影响。然而，用U73122抑制磷脂酶C（PLC），或用咖啡因或2-氨基乙氧基二苯硼酸通过其下游效应物肌醇-1,4,5-三磷酸（IP3）阻止细胞内钙释放，可完全阻断黏附反应。用1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸（BAPTA）螯合细胞内钙或用乙二醇双四乙酸（EGTA）螯合细胞外钙可完全消除中性粒细胞对ICAM-1的黏附。这种黏附在趋化因子刺激后的最初300秒内由淋巴细胞功能相关抗原-1（LFA-1，α(L)β(2)）介导，随后在刺激350秒后由巨噬细胞抗原-1（Mac-1，α(M)β(2)）介导黏附。抑制p38丝裂原活化蛋白激酶（p38MAP激酶）会导致与抑制Mac-1相似的时间进程，这与已发表的证据一致，即Mac-1介导的黏附依赖于p38MAP激酶。这些发现证实了先前在其他细胞类型中确定的从趋化因子刺激到整合素激活的PLC依赖性、PKC非依赖性途径，并证明了原代人中性粒细胞中LFA-1与Mac-1激活的不同动力学和不同要求。
