A2.15 SRC-family kinases independent integrin signalling is required for elevated extracellular vesicle production of neutrophils

Background Beta-2 integrin (CD18) and the Src-family kinases (Hck, Fgr, Lyn) are required for development of autoimmune arthritis in vivo. Immune cell or platelet derived extracellular vesicles (EV) were shown to play a crucial role in synovial inflammation and local joint destruction by activating multiple signalling pathways in synovial fibroblasts including NF-κB, activator protein-1, p38 mitogen-activated protein kinases and c-Jun N-terminal kinase pathways. Upon activation, synovial fibroblasts upregulated cyclooxygenase 2 and prostaglandin E synthase, induced the release of matrix metalloproteases, and increased the production of proangiogenic chemokines. In this present study we aimed to reveal the role of integrin signalling and Src-family kinases in neutrophilic granulocyte EV production. Methods CD18, CD11a, CD11b, Src-family knockout and wild-type neutrophils were separated from murine bone marrow and then stimulated with opsonized yeast. Extracellular vesicles were separated by two step centrifugation and filtration. The separated vesicles were counted and analysed with EV optimised flow cytometry. The structure of EVs was analysed with electron microscopy. Phagocytosis of opsonized bacteria by knockout neutrophils was also determined. Results Strong phagocytosis of bacteria was seen in case of wild-type cells. CD18, CD11b or Src-family kinases deficient cells showed a strongly impaired phagocytosis however it remained intact if CD11a integrin was knocked out. All knockout and the wild-type cells produced right side out annexin V positive EVs spontaneously. After opsonized yeast activation EV production by wild-type cells increased two-fold. This elevated EV production was completely or partially inhibited in the absence of CD18 or CD11b resp. The elevated EV production was not affected if CD11a was missing and surprisingly also if all the Src-family members were knocked down. Conclusion Murine bone marrow derived neutrophils are able to increase EV production after a complex biological activation. This activation signal needs functioning Mac-1 complex but not the Src-family kinases and the massive cytoskeletal reorganisation induced by the phagocytosis.