Analysis of phagocytosis by mIgM plus lymphocytes depending on monoclonal antibodies against IgM of largemouth bass (Micropterus salmoides)

The phagocytic actives of B cells in fish have been proven in recent years. In this study, five positive hybridomas secreting monoclonal antibodies (MAbs) against largemouth bass IgM were produced. Indirect immunofluores-cence assay (IFA) demonstrated that five MAbs could specifically recognize membrane-bound IgM (mIgM) molecule of largemouth bass. Indirect ELISA and Western blotting analysis showed that all the five MAbs had no cross-reactions with the other two teleost IgMs. Flow cytometry analysis (FCM) revealed that the percentages of largemouth bass mIgM+ lymphocytes in head kidney, peripheral blood and spleen were 51.66 +/- 0.608%, 16.5 +/- 1.235% and 42.92 +/- 1.091%, respectively. In addition, the phagocytosis rates of mIgM + lymphocytes ingesting Nocardia seriolae from head kidney, peripheral blood and spleen were calculated to be 5.413 +/- 0.274%, 16.6 +/- 0.289% and 26.3 +/- 0.296%, respectively. The qPCR results of sorted cells indicated that most inflammatory cytokines (IFN gamma, IL-1 beta, IL-2, IL-12 beta, IL-34, IL-10), chemokine (CXCL12), chemokines receptors (CXCR2, CXCR4) and genes (Fc gamma RIa, NCF1, CFL, ARP2/3, CD45, Syk, MARCKS) related to Fc gamma R-mediated phagocytic signaling pathway in phagocytic mIgM+ lymphocytes were up-regulated significantly (P < 0.05). Taken together, the results suggested that the MAb (MM06H) produced in this paper could be used as a tool to study mIgM+ lym-phocytes of largemouth bass, and Fc gamma R may participate in the phagocytosis of mIgM+ lymphocytes, which is helpful to further study the role of mIgM+ lymphocytes in innate immunity.

Automated summary

The phagocytic activity of B cells in fish has been confirmed. This study produced five monoclonal antibodies (MAbs) against largemouth bass IgM and demonstrated their ability to specifically recognize the membrane-bound IgM molecule. Flow cytometry analysis revealed the distribution of largemouth bass mIgM+ lymphocytes in different tissues. qPCR analysis showed the upregulation of various cytokines and genes related to the phagocytic signaling pathway in mIgM+ lymphocytes. These findings are important for understanding the role of mIgM+ lymphocytes in largemouth bass and the involvement of Fc gamma R in phagocytosis.