Oxygen radical production in neutrophils interacting with platelets and surface-immobilized plasma proteins: role of tyrosine phosphorylation

The interaction between neutrophil granulocytes and platelets is considered to play an important role in the inflammatory process induced by an implanted foreign material. However, the cellular mechanisms involved remain incompletely understood. We used a luminol-dependent chemiluminescence (CL) technique to analyze the generation of reactive oxygen species (ROS) in human neutrophils interacting with different plasma protein-coated surfaces in the presence or absence of unstimulated or stimulated platelets. The role of tyrosine phosphorylation in the regulation of NADPH oxidase activity was evaluated with quantitative fluorescence microscopy and the specific tyrosine kinase inhibitor genistein. We found that the ROS-production is 2 to 3 times higher in neutrophils on immunoglobulin G (IgG)-coated surfaces than in cells interacting with albumin- or fibrinogen-coated surfaces. Incubation with superoxide dismutase and catalase revealed that about 45% of the ROS was released extracellularly on IgG surfaces whereas corresponding values were 90% and 85% in neutrophils interacting with albumin and fibrinogen, respectively. The presence of platelets markedly increased the extracellular generation of ROS, mainly in neutrophils interacting with IgG- or fibrinogen-coated surfaces whereas the intracellular production was only modestly affected. Quantitative fluorescence microscopy of neutrophils stained with FITC-conjugated anti-phosphotyrosine antibodies showed a correlation between tyrosine phosphorylation, cell spreading, and ROS production. Platelets markedly amplified the anti-phosphotyrosine staining on both fibrinogen- and IgG-coated surfaces whereas the low level of tyrosine phosphorylation in neutrophils on albumin-coated surfaces was not further elevated by platelets. Furthermore, the tyrosine kinase inhibitor genistein inhibited both extra- and intracellular ROS production in neutrophils regardless of the presence of platelets. We demonstrate that plasma protein coating and the presence of platelets are crucial for the inflammatory response of adhering neutrophils and that the oxidative response correlates with the extent of tyrosine phosphorylation of proteins in focal contacts.