Abstract 25: Neutrophil Akt2 Regulates Heterotypic Cell-Cell Interactions During Vascular Inflammation

Abstract

Introduction: Interactions between platelets, leukocytes and endothelial cells are important during microvascular occlusion. Although the major receptors and counter receptors have been well identified, it remains unclear how heterotypic cell-cell interactions are regulated under inflammatory conditions. Hypothesis: We assessed the hypothesis that AKT, a Ser/Thr protein kinase, plays a regulatory role in platelet-neutrophil-endothelial cell interactions during vascular inflammation. Methods and Results: Using real-time fluorescence intravital microscopy to evaluate mice lacking specific Akt isoform, we demonstrated that Akt2, but not Akt1 and Akt3, regulates neutrophil adhesion and crawling, and platelet-neutrophil interactions on the activated endothelium during TNF-α-induced cremaster venular inflammation. Further, using chimeric mice generated from bone marrow transplants on wild-type (WT) and Akt2 knockout (KO) mice, we found that hematopoietic cell-associated AKT2 is important for the regulatory effect in vivo. Studies with an AKT2-specific inhibitor and cells isolated from WT and Akt KO mice revealed that platelet- and neutrophil-associated AKT2 regulates heterotypic neutrophil-platelet aggregation under conditions of shear. In particular, neutrophil AKT2 was critical for intracellular calcium mobilization, β2-talin1 interaction, and membrane translocation of αMβ2 integrin - a crucial receptor for neutrophil-platelet interactions. We observed that the basal phosphorylation levels of AKT isoforms are markedly increased in neutrophils and platelets from patients with sickle cell disease (SCD), an inherited hematological disorder associated with vascular inflammation and occlusion. AKT2 inhibition significantly reduced heterotypic aggregation of neutrophils and platelets isolated from SCD patients and diminished neutrophil adhesion and neutrophil-platelet aggregation in venules of Berkeley (SCD) mice, thereby improving blood flow rates. Conclusion: Our results provide genetic and pharmacologic evidence that neutrophil AKT2 regulates αMβ2 integrin function and is important for neutrophil recruitment and neutrophil-platelet interactions under thrombo-inflammatory conditions such as SCD.