Walzog B, Seifert R, Zakrzewicz A, Gaehtgens P, Ley K
Department of Physiology, Freie Universität Berlin, Germany.
J Leukoc Biol. 1994 Nov;56(5):625-35. doi: 10.1002/jlb.56.5.625.
Polymorphonuclear leukocytes (PMNs) exert most of their physiological functions while adherent to surfaces rather than in suspension. PMN adhesion is largely dependent on the function of the beta 2 integrins, CD11a,b,c/CD18. We mimicked engagement of beta 2 integrins by antibody cross-linking of CD18 on isolated human PMNs using both intact monoclonal antibody and F(ab')2 fragments. Within seconds of CD18 cross-linking, we observed a significant, transient rise of intracellular free Ca2+ concentration by 200-300 nM, which was largely due to Ca2+ mobilization from intracellular stores. The Ca2+ signal was blocked after pretreatment with phorbol myristate acetate, an activator of protein kinase C, but not with herbimycin A, a potent inhibitor of tyrosine kinases. In addition to the rise of intracellular free Ca2+ concentration, CD18 cross-linking induced exocytosis of azurophilic granules (release of 26% of total PMN elastase), which was significantly inhibited by herbimycin A. Moreover, 2.2-fold up-regulation of CD18 antigen and significant down-regulation of surface expression of the granulocyte adhesion molecule L-selectin were induced. Granulocyte F-actin content as measured by nitrobenzoxadiazole-phallacidin increased significantly 1 min after CD18 cross-linking. By contrast, CD18 cross-linking by soluble antibodies did not induce superoxide production, but PMNs bound to immobilized monoclonal antibodies against CD18 released significant amounts of superoxide. Initial signaling through beta 2 integrins does not appear to be mediated by a phospholipase C isoform activated through tyrosine phosphorylation, because the Ca2+ signal was not altered by herbimycin A. However, more complex cellular responses including exocytosis were found to require tyrosine phosphorylation. We show that engagement of beta 2 integrins provides an important stimulatory signal to PMNs inducing degranulation, modulation of L-selectin, and cytoskeletal changes.
多形核白细胞（PMN）在附着于表面而非悬浮状态时发挥其大部分生理功能。PMN的黏附在很大程度上依赖于β2整合素CD11a、b、c/CD18的功能。我们使用完整的单克隆抗体和F(ab')2片段，通过抗体交联分离的人PMN上的CD18来模拟β2整合素的结合。在CD18交联后的几秒钟内，我们观察到细胞内游离Ca2+浓度显著、短暂地升高200 - 300 nM，这主要是由于细胞内储存的Ca2+动员所致。在用蛋白激酶C激活剂佛波酯肉豆蔻酸酯预处理后，Ca2+信号被阻断，但用酪氨酸激酶的强效抑制剂赫曲霉素A预处理则无此效果。除了细胞内游离Ca2+浓度升高外，CD18交联还诱导了嗜天青颗粒的胞吐作用（释放PMN总弹性蛋白酶的26%），这被赫曲霉素A显著抑制。此外还诱导了CD18抗原2.2倍的上调以及粒细胞黏附分子L - 选择素表面表达的显著下调。用硝基苯并恶二唑 - 鬼笔环肽测量的粒细胞F - 肌动蛋白含量在CD18交联后1分钟显著增加。相比之下，可溶性抗体介导的CD18交联未诱导超氧化物产生，但与固定化抗CD18单克隆抗体结合的PMN释放了大量超氧化物。通过β2整合素的初始信号传导似乎不是由通过酪氨酸磷酸化激活的磷脂酶C同工型介导的，因为Ca2+信号未被赫曲霉素A改变。然而，发现包括胞吐作用在内的更复杂的细胞反应需要酪氨酸磷酸化。我们表明，β2整合素的结合为PMN提供了一个重要的刺激信号，诱导脱颗粒、L - 选择素的调节和细胞骨架变化。
