Src-family kinases mediate an outside-in signal necessary for beta2 integrins to achieve full activation and sustain firm adhesion of polymorphonuclear leucocytes tethered on E-selectin.

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 beta2 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-(beta2 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 beta2-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 beta2-integrin-mediated adhesion to CHO-E. Moreover, the expression of beta2 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 beta2 integrin- and Src-family-kinase-dependent manner. Thus, Src-family kinases, and perhaps Pyk2, mediate a signal necessary for beta2 integrin function in PMN tethered by E-selectin.

SRC-dependent outside-in signalling is a key step in the process of autoregulation of beta2 integrins in polymorphonuclear cells.

In human PMN (polymorphonuclear cells), challenged by P-selectin, the beta2-integrin Mac-1 (macrophage antigen-1) promoted the activation of the SRC (cellular homologue of Rous sarcoma virus oncogenic protein) family members HCK (haematopoietic cell kinase) and LYN (an SRC family protein tyrosine kinase) and phosphorylation of a P-110 (110 kDa protein). SRC kinase activity in turn was necessary for macrophage antigen-1-mediated adhesion [Piccardoni, Sideri, Manarini, Piccoli, Martelli, de Gaetano, Cerletti and Evangelista (2001) Blood 98, 108-116]. This suggested that an SRC-dependent outside-in signalling strengthens the beta2-integrin interaction with the ligand. To support this hypothesis further, in the present study, we used the monoclonal antibody KIM127 or manganese to lock beta2 integrins in a high-affinity state, and homotypic PMN adhesion was analysed to monitor beta2-integrin adhesive function. KIM127 or manganese induced PMN homotypic adhesion and P-110 phosphorylation. Both these processes were abolished by blocking antibodies against the common beta2 chain, by a combination of antibodies against alphaL and alphaM or by inhibitors of SRC activity. Confocal microscopy showed that activation epitopes were expressed by beta2 integrins co-localized with patches of F-actin at the adhesion sites. Blockade of SRC kinases or of actin polymerization prevented clustering of activated integrins as well as F-actin accumulation. FACS analysis showed that SRC inhibitors modified neither basal nor manganese-induced KIM127 binding. An SRC-dependent outside-in signalling initiated by beta2 integrins was also required for adhesion triggered by interleukin-8. These results confirm the hypothesis that an SRC-dependent outside-in signalling triggered by high affinity and ligand binding is necessary to stabilize beta2-integrin-mediated adhesion. Allowing clustering of activated integrins, SRC might link the high-affinity with the high-avidity state. Proline-rich tyrosine kinase-2 appears to be involved in this process.