NADPH Oxidase 2 Is Critical for Heterotypic Neutrophil-Platelet Interactions during Vascular Inflammation

Intravital microscopic studies have shown that neutrophil-platelet interactions on activated endothelial cells are the major determinant of vascular occlusion under thromboinflammatory conditions. Heterotypic neutrophil-platelet interactions are mainly mediated by two crucial receptors and counter receptors, P-selectin-P-selectin glycoprotein ligand-1 and glycoprotein Ibalpha-alphaMbeta2 integrin. Although the major receptors and counter receptors are well identified, it remains unclear how heterotypic cell-cell interactions are regulated during vascular disease. Recently, we demonstrated that neutrophil AKT2 is a key regulator for neutrophil-platelet interactions during vascular inflammation. Since it is known that AKT2 is important for NADPH oxidase 2 (NOX2) activity and reactive oxygen species (ROS) generation during neutrophil activation, we investigated whether NOX2 regulates the heterotypic neutrophil-platelet interaction during vascular inflammation. Using real-time fluorescence intravital microscopy, we found that platelet-neutrophil interactions are nearly completely inhibited during TNF-alpha-induced cremaster venular inflammation in NOX2 KO mice, compared with WT mice. Studies with bone marrow chimera further demonstrated that both hematopoietic and endothelial cell NOX2 are crucial for neutrophil-platelet interactions on the inflamed venules. In vitro studies with neutrophils and platelets isolated from WT and NOX2-deficient mice indicated that both neutrophil and platelet NOX2 are required for neutrophil-platelet aggregation under shear conditions. Using ROS scavengers and NOX2 KO cells, we observed that neutrophil NOX2-generated ROS regulate the activation and ligand-binding activity of alphaMbeta2 integrin during neutrophil activation and that platelet NOX2-produced ROS are important for P-selectin exposure upon agonist stimulation and the ligand-binding function of glycoprotein Ibalpha. Interestingly, neutrophil NOX2 was crucial for store-operated calcium entry (SOCE), but not calcium release from the endoplasmic reticulum, whereas platelet NOX2 modulated intracellular calcium release, but not SOCE. We further demonstrated that the differential regulation of platelet and neutrophil NOX2 in calcium signaling correlates with the differences in the phosphorylation of AKT, ERK, and p38MAPK, suggesting the importance of AKT and its downstream molecules for NOX2-mediated calcium signaling. Our results indicate that neutrophil and platelet NOX2-produced ROS play critical roles in regulating the function of surface receptors required for heterotypic neutrophil-platelet interactions during vascular inflammation.