Yan S R, Fumagalli L, Berton G
Institute of General Pathology, University of Verona, Italy.
J Inflamm. 1995;45(4):297-311.
Tumor necrosis factor (TNF) stimulates generation of reactive oxygen intermediates, secretion of granule constituents, and rearrangement of the cytoskeleton in neutrophils (PMN); this response requires that PMN be adherent to plasma or extracellular matrix proteins, and is dependent on beta 2 integrins. Tyrosine phosphorylation of distinct proteins [Fuortes et al., J Cell Biol 120:777-784, 1993] and activation of the protein tyrosine kinase p58c-fgr [Berton et al., J Cell Biol 126:1111-1121, 1994] were recently recognized as signals involved in beta 2 integrin-dependent responses of TNF-treated PMN. As the integrin capability to bind their ligands is regulated by divalent cations we investigated whether modulation of PMN adhesion to fibrinogen by divalent cations also affected activation of protein tyrosine kinases. In the absence of divalent cations or in the presence of Ca2+ alone, PMN did not adhere to fibrinogen in response to TNF. However, Mg2+, either alone or together with Ca2+, promoted stimulated adhesion to fibrinogen. We also found that Mn2+ promoted PMN adhesion to fibrinogen without additional stimuli. Analysis of the activity of two src family tyrosine kinases, p58c-fgr and p53/56lyn, showed that their autophosphorylating kinase activity strictly correlated with adhesion. In fact, only in the presence of Mg2+, but not in the absence of divalent cations or in the presence of Ca2+ alone, TNF increased p58c-fgr and p53/56lyn kinase activities; and this was prevented by anti-CD18 antibodies. In addition, Mn2+ strongly promoted activation of p58c-fgr and p53/56lyn without additional stimuli. Analysis of tyrosine phosphorylated proteins with anti-phosphotyrosine immunoblots showed that divalent cations regulated adhesion and protein tyrosine phosphorylation in the same fashion. Detergent extraction of proteins showed that the Mg(2+)-dependent, TNF-stimulated adhesion redistributed p58c-fgr and p53/56lyn to a Triton-insoluble fraction. In addition, analysis of p58c-fgr activity allowed us to demonstrate that the fraction of p58c-fgr which became Triton-insoluble displayed a higher kinase activity. These findings establish that PMN adhesion signals for activation of two different src family tyrosine kinases. The evidence that Mn2+, a strong promoter of integrin function, induces adhesion and activation of tyrosine kinases without additional stimuli suggest the existence of a direct link between beta 2 integrins binding to fibrinogen and activation of tyrosine kinases in neutrophils.
肿瘤坏死因子（TNF）可刺激中性粒细胞（PMN）产生活性氧中间体、分泌颗粒成分并使细胞骨架重排；这种反应要求PMN粘附于血浆或细胞外基质蛋白，且依赖于β2整合素。不同蛋白质的酪氨酸磷酸化[富尔特斯等人，《细胞生物学杂志》120:777 - 784，1993年]以及蛋白酪氨酸激酶p58c - fgr的激活[贝尔顿等人，《细胞生物学杂志》126:1111 - 1121，1994年]最近被认为是参与TNF处理的PMN的β2整合素依赖性反应的信号。由于整合素与配体结合的能力受二价阳离子调节，我们研究了二价阳离子对PMN与纤维蛋白原粘附的调节是否也影响蛋白酪氨酸激酶的激活。在没有二价阳离子或仅存在Ca2 +的情况下，PMN不会因TNF而粘附于纤维蛋白原。然而，Mg2 +单独或与Ca2 +一起可促进对纤维蛋白原的刺激粘附。我们还发现Mn2 +无需额外刺激即可促进PMN与纤维蛋白原的粘附。对两种src家族酪氨酸激酶p58c - fgr和p53/56lyn的活性分析表明，它们的自磷酸化激酶活性与粘附密切相关。事实上，仅在存在Mg2 +时，而不是在没有二价阳离子或仅存在Ca2 +时，TNF会增加p58c - fgr和p53/56lyn激酶活性；而抗CD18抗体可阻止这种情况。此外，Mn2 +无需额外刺激即可强烈促进p58c - fgr和p53/56lyn的激活。用抗磷酸酪氨酸免疫印迹分析酪氨酸磷酸化蛋白表明，二价阳离子以相同方式调节粘附和蛋白酪氨酸磷酸化。蛋白质的去污剂提取表明，Mg(2 +)依赖性的、TNF刺激的粘附将p58c - fgr和p53/56lyn重新分布到Triton不溶性部分。此外，对p58c - fgr活性的分析使我们能够证明变为Triton不溶性的p58c - fgr部分显示出更高的激酶活性。这些发现表明PMN的粘附信号可激活两种不同的src家族酪氨酸激酶。Mn2 +是整合素功能的强促进剂，它无需额外刺激即可诱导粘附和酪氨酸激酶激活，这一证据表明β2整合素与纤维蛋白原的结合和中性粒细胞中酪氨酸激酶的激活之间存在直接联系。
