In this study, using soluble recombinant E-selectin-IgG chimera and E-selectin-transfected CHO cells (CHO-E) we tested the hypothesis that E-selectin promotes 2integrin function in PMN. PMN aggregation or adhesion to CHO-E were evaluated in sheared (1000 rpm) cell suspensions (PMN: CHO-E ratio=5) by optical counting and double color flow cytometry. Soluble E-selectin (1-20 g/ml) dose-dependently induced Mac-1-dependent PMN aggregation, that was accompanied by tyrosine phosphorylation of a protein of 110 kD (PI 10). Genistein (1-100 M) and the specific inhibitor of Src kinases PP1 (1-20 M) dose-dependently inhibited P110 phosphorylation and PMN aggregation. PMN did not adhere to untrasfected CHO cells while 251,5% of PMN adhered to 6010% of CHO-E, at 2 min of shear, by a mechanism 2-integrin dependent. Neuraminidase treatment prevented PMN recruitment. Phosphorylation of PI 10 accompanied PMN adhesion and PP1 blocked both PI 10 phosphorylation and adhesion. Among the selectin ligands, PSGL-1 and L-selectin can trigger 2-integrin activation. In our experimental conditions, neither blockade with specific antibodies (PL 1 and PL2 to PSGL1 and DREG-56 to L-selectin) nor chymotrypsin cleavage of these receptors, significantly modify PMN adhesion to CHO-E. Moreover it was not modified by the combination of PL1 and DREG-56 with Affi-6O, a rabbit policlonal antibody to ESL-1. Unexpectedly, H18/7 and l .2B6, two antibodies against the lectin domain of E-selectin, barely affected PMN adhesion to CHO-E or aggregation induced by soluble E-selectin, suggesting a lectin-site independent effect. PMN did not adhere to resting HUVEC while formed mixed cell conjugates, involving 822% of HUVEC and 761 % of PMN, after 2 min of shear with rIL-1 -stimulated HUVEC; that was accompanied by phosphorylation of PI 10. PMN recruitment was abolished by neuraminidase treatment and was reduced to 152%, 303% and 306,4% of the control by anti-CD 18, a combination of anti-CD 1 la and CD1 Ib antibodies, or PP1 (20 M) respectively . This study indicates that E-selectin binding to sialylated unidentified receptor(s), promotes Src-kinase dependent function of 2 integrins. This mechanism may be relevant for PMN recruitment by the endothelium.
|Issue number||11 PART II|
|Publication status||Published - 2000|
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