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 monoclona l antibodies (mAb) stimulates cytoskeletal rearrangement leading to ce ll spreading and respiratory burst. Tyrosine phosphorylation of a vari ety of proteins and activation of the Src family kinases within polymo rphonuclear leukocytes (PMN) have recently been implicated in the intr acellular signaling pathways generated by leukocyte integrins (Yan, S. R., L. Fumagalli, and G. Berton. 1995. J. Inflammation. 45:217-311.) T o directly test whether these functional responses are dependent on th e 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 wil d-type mice released significant amounts of O-2(-) when adherent to fi brinogen-, fibronectin-, or collagen-coated surfaces, in the presence of activating agents such as tumor necrosis factor (TNF) or formyl-met hionyl-leucyl-phenylalanine, as described for human PMNs. PMNs from hc k(-/-)fgr(-/-) double-mutant mice, however, failed to respond. This de fect 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 mol ecule-1 (ICAM-1), while hck(-/-)fgr(-/-) PMNs, failed to respond. Dire ct cross-linking of the subunits of beta(2) and beta(3) integrins by s urface-bound mAbs also elicited O-2(-) production by wild-type PMNs, w hile the double-mutant hck(-/-)fgr(-/-) cells failed to respond. Photo microscopy and cell adhesion assays revealed that the impaired functio nal responses of hck(-/-)fgr(-/-) PMNs were caused by defective spread ing 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 p 58(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 kina ses in integrin signaling within leukocytes, and it is also the best e xample of overlapping function between members of this gene family wit hin a defined signal transduction pathway.