NADPH oxidase activation in fibronectin adherent human neutrophils: A potential role for beta1 integrin ligation.
BACKGROUND
Hydrogen peroxide production by human neutrophils is tightly coupled to integrin ligation. This provides a means for spatial localization of oxidant production. However, integrin specificity and consequent signaling mechanisms for this process remain undefined. In the present study we demonstrate that otherwise unstimulated neutrophils adherent to fibronectin, a beta(1) ligand, but not fibrinogen, a beta(2) ligand, produce hydrogen peroxide in a time-dependent fashion. We hypothesized that signaling proceeded through focal, adhesionlike structures.


METHODS
Triton-X insoluble (actin cytoskeleton) fractions from suspension phase cells and cells adherent to the beta(1) integrin ligand fibronectin were assessed for the presence of the integrin-associated kinase Pyk2, the scaffolding protein paxillin, and the downstream Src family kinase Lyn. Lysates were subjected to immunoprecipitation with anti-phosphotyrosine and probed for Pyk2, paxillin, and Lyn. Associations between focal adhesion kinase (FAK) and paxillin were determined by immunoprecipitation with anti-FAK and probing with anti-paxillin antibody. Activation of NADPH oxidase was determined by demonstration of redistribution of p47(phox) in Triton-X insoluble fractions.


RESULTS
NADPH oxidase activation, as judged by H(2)O(2) production, occurred with fibronectin-, but not in suspension or fibrinogen-adherent cells. Cells adherent to fibronectin for 20 minutes demonstrated marked increases in Pyk2, paxillin, and Lyn activation in comparison to fibronectin-adherent and suspension phase cells.


CONCLUSIONS
This study demonstrates that fibronectin adherence is a significant initiating factor for NADPH oxidase assembly in human neutrophils. This appears to be mediated by beta(1) integrins. We demonstrate formation of focal adhesions containing Pyk2/FAK, paxillin, and Lyn, and translocation of p47(phox).
Umanskiy
Konstantin
K
Division of Trauma and Critical Care, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, USA.
Robinson
Chad
C
Cave
Cindy
C
Williams
Mark A
MA
Lentsch
Alex B
AB
Cuschieri
Joseph
J
Solomkin
Joseph S
JS
D002448:Cell Adhesion; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004789:Enzyme Activation; D005353:Fibronectins / Q000378:metabolism*; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; D022001:Focal Adhesions / Q000378:metabolism; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism*; D008024:Ligands; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism / Q000502:physiology*; D051419:Paxillin; D010545:Peroxides / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D013997:Time Factors; D019061:src-Family Kinases / Q000378:metabolism
D003598:Cytoskeletal Proteins; D005353:Fibronectins; D019012:Integrin beta1; D008024:Ligands; C496061:PXN protein, human; D051419:Paxillin; D010545:Peroxides; D010750:Phosphoproteins; D011506:Proteins; D019255:NADPH Oxidases; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases