Deficiency of Src family kinases p59/61hck and p58c-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. Tyrosin 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 p59/61hck and p58c-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 O2- 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 mic, 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 PMN 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 2 integrins by surface-bound mAbs was elicited O2- 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 O2- 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 58c-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.
Lowell
C A
CA
Department of Laboratory Medicine, University of California, San Francisco 94143, USA.
Fumagalli
L
L
Berton
G
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015703:Antigens, CD / Q000276:immunology; D001853:Bone Marrow / Q000276:immunology; D001854:Bone Marrow Cells; D018845:CD11 Antigens / Q000276:immunology; D018821:CD18 Antigens / Q000276:immunology / Q000494:pharmacology; D002448:Cell Adhesion; D002478:Cells, Cultured; D005340:Fibrinogen; D006801:Humans; D039661:Integrin beta3; D016023:Integrins / Q000276:immunology; D018799:Intercellular Adhesion Molecule-1 / Q000276:immunology; D051379:Mice; D018345:Mice, Knockout; D008817:Mice, Mutant Strains; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000276:immunology*; D010980:Platelet Membrane Glycoproteins / Q000276:immunology; D011505:Protein-Tyrosine Kinases / Q000172:deficiency / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000172:deficiency / Q000502:physiology*; D051572:Proto-Oncogene Proteins c-hck; D016897:Respiratory Burst; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D019061:src-Family Kinases / Q000502:physiology*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018845:CD11 Antigens; D018821:CD18 Antigens; D039661:Integrin beta3; D016023:Integrins; D010980:Platelet Membrane Glycoproteins; D011518:Proto-Oncogene Proteins; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D018799:Intercellular Adhesion Molecule-1; D009240:N-Formylmethionine Leucyl-Phenylalanine; D005340:Fibrinogen; D011505:Protein-Tyrosine Kinases; C496631:HCK protein, human; C496632:Hck protein, mouse; D051572:Proto-Oncogene Proteins c-hck; C053102:proto-oncogene proteins c-fgr; D019061:src-Family Kinases