Development of monoclonal antibodies against IgM of common carp (Cyprinus carpio) and analysis of phagocytosis by mIgM+ B lymphocytes.

Phagocytosis is a specific function of phagocytes, but recent studies have revealed that B cells, such as those of mammals, amphibians, and teleost, also exhibit phagocytic activity. However, the phagocytic capacity of B cells in common carp (Cyprinus carpio), an economically important freshwater fish, remains unexplored. In this paper, we generated a mouse anti-carp IgM monoclonal antibody (mAb) and confirmed its specificity by demonstrating no cross-reactivity with IgMs from three other teleost species by Western blotting and ELISA. Indirect fluorescence assay (IFA) showed that common carp mIgM+ B lymphocytes could be classified into two distinct subpopulations: IgMlo and IgMhi. The IgMhi subpopulation predominated in peripheral blood, accounting for 60.5% of mIgM+ B lymphocytes, whereas the IgMlo subpopulation was more abundant in the head kidney, representing 63.7% of mIgM+ B lymphocytes. Flow cytometry analysis revealed that phagocytosis rates of 1-μm fluorescent microspheres were significantly higher than those of 3-μm microspheres by peripheral blood (54.4% vs. 29.8%), spleen (49.6% vs. 28.6%), and head kidney (49.9% vs. 29.4%) mIgM+ B lymphocytes. Additionally, intracellular reactive oxygen species (ROS) levels in mIgM+ B lymphocytes were significantly elevated after phagocytosis of 1-μm microspheres (34.5% in peripheral blood, 32.6% in spleen, and 30.5% in head kidney) compared to those of 3-μm microspheres (19.9%, 18.2%, and 18.9%, respectively). Quantitative RT-PCR analysis further demonstrated that after phagocytosis of 1-μm microspheres, the expression levels of phagocytosis- and antigen presentation-related genes in mIgM+ B lymphocytes were higher than those of the 3-μm microspheres group, whereas inflammatory factors expression remained unchanged. These findings provide the evidence that teleost B cells exhibit tissue-specific differences in phagocytic capacity, offering new insights into the innate immune functions of teleost B lymphocytes.