FORMATION OF EOSINOPHIL EXTRACELLULAR TRAP INDUCED BY LYSOPHOSPHATIDYLSERINE AND ACTIVATED PLATELETS

Abstract

<Abstract> Eosinophils are crucial effector cells that play a role in host defense against parasitic infection and allergic inflammatory diseases, largely through release of cytotoxic granule proteins and interaction with other immune and nonimmune cells. Recent evidence demonstrates that activated eosinophils undergo eosinophil extracellular trap (EET), the extracellular release of threads of decondensed chromatin decorated with histones and cell free granules, which is also accompanied by loss of membrane integrity and cell viability. EET has been implicated in inflammatory immune responses in asthmatic airways. Platelets are tiny fragments of cells that are essential for normal blood clotting. Beyond central role in hemostasis, platelets have increasingly been appreciated as immune cells that have a role in inflammation through interactions with other leukocytes and through release of soluble mediators and membrane-bound small particles, including platelet-derived microparticles (PMPs). Mounting evidence have propose bidirectional interactions between eosinophils and platelets in response to inflammatory responses. We recently demonstrated that human eosinophils selectively and abundantly express P2Y10, a G protein-coupled receptor, which binds a bioactive lipid mediator, lysophosphatidylserine (LysoPS). In the first series of experiments, we aimed to probe a function of LysoPS in eosinophils. we demonstrated that LysoPS induced EET formation accompanied by both lytic cell death and degranulation. LysoPS-induced EET was independent to reactive oxygen species production and irrelevant to several signaling pathways examined, but dependent on protein arginine deiminase 4. Eosinophils from severe asthmatics exhibited greater degranulation, but not EET formation, in response to LysoPS, than those from nonsevere asthmatics, along with great expression of surface P2Y10. These results suggest that LysoPS-dependent EET or degranulation plays a potential role in eosinophilic inflammation of severe asthma. In the second series of experiments, we examined whether activated platelets induced EET formation. To this end, platelets were stimulated with a calcium ionophore A23187 and platelet agonists thrombin and ADP and then fractionated to conditioned medium (CM) and pellet. Our results showed that both CM and cell pellet from the A23187-, but not thrombin or ADP-activated platelet cultures induced EET formation, suggesting that fully activated platelets are capable of eliciting EET formation. The EET-inducing activities by CM and pellet from the A23187-activated platelet cultures were proportional to the number of activated platelets. Confocal microscopic analysis demonstrated that PMPs, as assessed by CD41 expression, were entrapped in EET and aggregated around EET-undergoing eosinophils, when platelets were activated with pellet from the A23187-activated platelet cultures. Thus, platelet-eosinophil cross-talk modulates inflammatory responses through EET formation. Collectively, our results imply two important facets in EET, a newly emerging eosinophil activity involved in host defense and inflammation. First, our identification of LysoPS as a potent inducer of EET expands a repertoire of known inducers that is currently very limited. Second, we for the first time show that fully activated platelets induce an appreciable amount of EET, translating activation of platelets to the notable eosinophil activity, EET