Impact of caspase-8 and PKA in regulating neutrophil-derived microparticle generation

The morbidity and mortality from sepsis continues to remain high despite extensive research into understanding this complex immunologic process. Further, while source control and antibiotic therapy have improved patient outcomes, many immunologically based therapies have fallen short. Microparticles (MPs) are intact vesicles that serve as mediators of intercellular communication as well as markers of inflammation in various disease processes. We have previously demonstrated that MPs can be produced at the infected foci during sepsis, are predominantly of neutrophil derivation (NDMPs) and can modulate immune cells. In this study, we sought to elucidate the molecular mechanisms underlying NDMP generation. Using thioglycolate (TGA) to recruit and activate neutrophils, we first determined that intra-peritoneal TGA increase NDMP accumulation. We next utilized TGA-elicited neutrophils in vitro to investigate signaling intermediates involved in NDMP production, including the intrinsic and extrinsic caspase pathways, cAMP dependent PKA and Epac activation as well as the role myosin light chain kinase (MLCK) as a final mediator of NDMP release. We observed that NDMP generation was dependent on the extrinsic caspase apoptotic pathway (caspase 3 and caspase 8), cAMP activation of PKA but not of Epac, and on activation of MLCK. Altogether, these data contribute to an overall framework depicting the molecular mechanisms that regulate NDMP generation.