Src-family kinases mediate an outside-in signal necessary for β2 integrins to achieve full activation and sustain firm adhesion of polymorphonuclear leucocytes tethered on E-selectin

• Published in: Biochemical journal Vol. 396; no. 1; pp. 89 - 98
• Main Authors: Totani, Licia, Piccoli, Antonio, Manarini, Stefano, Federico, Lorenzo, Pecce, Romina, Martelli, Nicola, Cerletti, Chiara, Piccardoni, Paola, Lowell, Clifford A., Smyth, Susan S., Berton, Giorgio, Evangelista, Virgilio
• Format: Journal Article
• Language: English
• Published: Portland Press Ltd 15.05.2006
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/BJ20051924

In cell suspensions subjected to high-shear rotatory motion, human PMN (polymorphonuclear cells) adhered to E-selectin-expressing CHO (Chinese-hamster ovary) cells (CHO-E), and formed homotypic aggregates when challenged by E-selectin–IgG fusion protein, by a mechanism that involved β2 integrins. Both heterotypic and homotypic PMN adhesion was accompanied by tyrosine phosphorylation of a 110 kDa protein (P110). This event was prevented by blocking anti-(β2 integrin) antibodies and by inhibitors of Src-family kinases, suggesting that it was part of an ‘outside-in’ signalling that was initiated by integrin engagement. Interestingly, Src-family kinase inhibitors prevented β2-integrin-mediated (i) homotypic PMN adhesion triggered by E-selectin–IgG, (ii) heterotypic CHO-E/PMN adhesion in mixed-cell suspensions, and (iii) firm adhesion of PMN to CHO-E monolayers under physiological flow. Similarly to PMN treated with Src-family kinase inhibitors, PMN from hck−/−fgr−/− and hck−/−fgr−/−lyn−/− mice showed significant impairment of β2-integrin-mediated adhesion to CHO-E. Moreover, the expression of β2 integrin activation epitopes at the sites of cell–cell contact in CHO-E/PMN conjugates was abolished by Src-family kinase inhibitors. One component of P110 was identified as the FAK (focal adhesion kinase) Pyk2 (proline-rich tyrosine kinase 2), which was phosphorylated in a β2 integrin- and Src-family-kinase-dependent manner. Thus, Src-family kinases, and perhaps Pyk2, mediate a signal necessary for β2 integrin function in PMN tethered by E-selectin.