Project Summary/Abstract Increasing evidence suggests that activated neutrophils play a critical role in promoting vaso-occlusion and morbidity in sickle cell disease (SCD). Multiple factors are thought to contribute to neutrophil activation in SCD, including products of red cell (RBC) hemolysis and cellular interactions between neutrophils and the endothelium or platelets. Once neutrophils are activated, stimulus-dependent effects on neutrophil phenotype and functional capacity have been reported, resulting in dysfunctional neutrophils which propagate inflammation. Unlike prior studies in SCD which largely suggest that neutrophil activation occurs via indirect mediators, our preliminary data support the hypothesis that sickle RBCs (SS RBCs) directly activate neutrophils, resulting in enhanced adhesiveness and increased degranulation responses. We show that these 2 effector functions are further enhanced when SS RBC phosphatidylserine (PS) exposure is increased and when SS RBC adhesion receptors are activated. Furthermore, our preliminary studies show that in steady-state, exchange transfusion reduces neutrophil activation. Based on the preliminary data presented in this application, we will test the hypothesis that intact SS RBCs induce a specific neutrophil activation profile which recapitulates neutrophil dysfunction in SCD. We also hypothesize that therapies that reduce the RBC characteristics enabling this effect might both reduce neutrophil activation and decrease the frequency and severity of vaso-occlusive events. In Aims 1 and 2, we will delineate upstream characteristics of RBCs contributing to neutrophil activation as well as downstream consequences of SS RBC-induced neutrophil interaction in vitro. In Aim 1, we will establish the effect of SS RBCs on neutrophil phenotype, function, and gene expression. In Aim 2, we will establish the requirements needed for SS RBCs to activate neutrophils by examining physical requirements such as concentration thresholds and need for contact. We will also establish the role of PS exposure and the known activatable RBC membrane adhesion receptors (BCAM/Lu, ICAM4, CD47, and CD44) on neutrophil activation. In Aim 3, we will determine if the SS RBC-neutrophil interactions and measures of neutrophil activation we have described in vitro reflect those seen in patients during acute chest syndrome (ACS). In addition, we will determine the effect of transfusion, which we have shown affects neutrophil activation in steady-state, on in vivo neutrophil activation during acute illness. Together, these studies build on our novel observation that SS RBCs are able to directly induce neutrophil activation. The work outlined in this proposal will systematically define the characteristics of SS RBCs which cause neutrophil activation and the functional/phenotypic/transcriptional changes seen in neutrophils after activation by SS RBCs. These results will potentially identify new therapeutic targets and will provide signi...