Deficiency of Src family kinases p59/61(hck) and p58(c-fgr) results in defective adhesion-dependent neutrophil functions

Cross-linking of the neutrophil beta(2)- or beta(3)-related leukocyte response integrins by extracellular matrix (ECM) proteins or monoclonal antibodies (mAb) stimulates cytoskeletal rearrangement leading to cell spreading and respiratory burst. Tyrosine phosphorylation of a variety of proteins and activation of the Src family kinases within polymorphonuclear leukocytes (PMN) have recently been implicated in the intracellular signaling pathways generated by leukocyte integrins (Yan, S.R., L. Fumagalli, and G. Berton. 1995. J. Inflammation. 45:217-311.) To directly test whether these functional responses are dependent on the Src family kinases p59/61(hck) and p58(c-fgr), we examined adhesion-dependent respiratory burst in PMNs isolated from hck(-/-), fgr(-/-), and hck(-/-)fgr(-/-) knockout mice. Purified bone marrow PMNs from wild-type mice released significant amounts of O-2(-) when adherent to fibrinogen-, fibronectin-, or collagen-coated surfaces, in the presence of activating agents such as tumor necrosis factor (TNF) or formyl-methionyl-leucyl-phenylalanine, as described for human PMNs. PMNs from hck(-/-)fgr(-/-) double-mutant mice, however, failed to respond. This defect was specific for integrin signaling, since respiratory burst was normal in hck(-/-)fgr(-/-) PMNs stimulated by immune complexes or PMA. Stimulation of respiratory burst was observed in TNF-primed wild-type PMNs plated on surfaces coated with murine intracellular adhesion molecule-1 (ICAM-1), while hck(-/-)fgr(-/-) PMNs, failed to respond. Direct cross-linking of the subunits of beta(2) and beta(3) integrins by surface-bound mAbs also elicited O-2(-) production by wild-type PMNs, while the double-mutant hck(-/-)fgr(-/-) cells failed to respond. Photomicroscopy and cell adhesion assays revealed that the impaired functional responses of hck(-/-)fgr(-/-) PMNs were caused by defective spreading and tight adhesion on either ECM protein- or mAb-coated surfaces. In contrast, hck(-/-) or fgr(-/-) single mutant cells produced O-2(-) at levels equivalent to wild-type cells on ECM protein, murine ICAM-1, and antiintegrin mAb-coated surfaces. Hence, either p59/61(hck) and p58(c-fgr) is required for signaling through leukocyte beta(2) and beta(3) integrins leading to PMN spreading and respiratory burst. This is the first direct genetic evidence of the importance of Src family kinases in integrin signaling within leukocytes, and it is also the best example of overlapping function between members of this gene family within a defined signal transduction pathway.