Abstract 15870: Neutrophil Akt2 Plays a Critical Role in Regulating AlphaMbeta2 Integrin Function and Heterotypic Platelet-Neutrophil Interactions During Vascular Inflammation
Interaction of activated platelets and neutrophils on the activated endothelium mediates pathological thrombosis and inflammation. However, the regulatory mechanism of heterotypic cell-cell interactions remains to be elucidated. Using real-time intravital microscopy of Akt knockout (KO) mice, we have demonstrated that Akt2, but not Akt1 or Akt3, plays a critical role in neutrophil adhesion to the site of TNF-alpha-induced vascular inflammation. Further, platelet-neutrophil interactions on the activated endothelium were completely inhibited in Akt2 KO mice. Using chimeric mice generated from reciprocal bone marrow transplants on wild-type and Akt2 KO mice, we found that blood cell Akt2, but not endothelial cell Akt2, regulates neutrophil recruitment and platelet-neutrophil interactions during vascular inflammation. As analyzed by flow cytometry, treatment of human and mouse blood with an Akt2 specific inhibitor significantly diminished platelet-neutrophil interactions under shear. To dissect the role of platelet vs neutrophil Akt2 in platelet-neutrophil interactions, platelets or neutrophils were pretreated with an Akt2 specific inhibitor, mixed together, and sheared at 100-500s-1. We found that neutrophil Akt2 plays a central role in platelet-neutrophil interactions. Human neutrophils treated with an Akt2 inhibitor significantly reduced cell adhesion to surfaces coated with ICAM-1 or fibrinogen and diminished activation of alphaMbeta2 integrin - a key receptor for stable interactions of neutrophils with platelets and endothelial cells. Further, neutrophils lacking Akt2 also exhibited decreased adhesion to ICAM-1, and the adhesion was further inhibited by a combination with a blocking anti-beta2 but not anti-alphaM antibody. Compared to wild-type neutrophils, Akt2 KO neutrophils showed decreased phosphorylation of SNARE proteins which are important for exocytosis-mediated alphaMbeta2 translocation during neutrophil activation. These results indicate that neutrophil Akt2 is a crucial regulator for alphaMbeta2 integrin-mediated neutrophil function, thereby playing a key role in heterotypic platelet-neutrophil interactions during thrombo-inflammatory disease.
- © 2012 by American Heart Association, Inc.