Phagocytosis Of Murine B Lymphocytes

As an important mechanism linking innate immune and adaptive immune,phagocytosis is characterized by internalization of particulate antigen over0.5μm,participation of special receptors, actin reorgnization, and phagolysosomeformation, et al. According to classic immune theory, phagocytosis is commonlymanaged by professional phagocytes such as macrophage, monocytes andneutrophils, et al or unprofessional phagocytes such as fibroblasts and epithelialcells. As a kind of classic adaptive lymphocytes, B cells were traditionally takenas professional antibody secreting cells and had no phagocytic and microbicidalabilities. However, within recent years, phagocytosis of B cells were found inearly vertebrates such as teleost fish like rainbow trout, amphibian like Xenopuslaevis, and even reptiles like red-eared slider (Trachemys scripta).In former research, our group constructed TgVH3B4transgenic mice, whoseB cells express BCR recognizing Staphylococcus aureus. Flow cytometer assaydisclosed the co-localization of B cells and Staphylococcus aureus in theperitoneal cavity. B cells in peritoneal cavity are majorly consisted of B-1cells, which are a group of B lymphocytes different from traditional B-2cell. B-1cellsare majorly localized in peritoneal cavity, pleural cavity and cerebral spinal cordmembrane cavity, et al; and express CD11b which were thought to be expressedon professional phagocytes; and grow adherently in vitro; and even share similarprofile with macrophage; and have close development relationship withprofessional phagocytes. As a primitive subset of B cells, B-1cells might reservethe phagocytic ability. In order to further disclose the evolutionary pathway andenrich the role of B cell, especially B-1cells, in innate and adaptive immune, thisstudy went deep into the verification and mechanism exploration of B cellphagocytosis.Major Methods and Results:1) Visualization assay: FITC labeled60Co sterilized Staphylococcus aureus,Escherichia coli and fluorescent microspheres were intraperitoneallyadministrated to C57/BL6mice, and peritoneal cavity lavage were collected30min later (in vivo phagocytosis assay). B cells were magnetic sorted, andanalyzed by TEM, or labeled fluorescently by anti-CD19antibodies and analyzedby laser co-focal microscopy and flow cytometry. These observations verifiedthat murine peritoneal cavity and spleen B cells internalized Staphylococcusaureus, Escherichia coli and fluorescent microspheres.2) Actin reorganization analysis: Peritoneal B cells from wild type micewere inhabited in vitro with FITC labeled60Co sterilized Staphylococcus aureus(in vitro phagocytosis assay), under interference of cochicine and cytochalasin Bin different concentrations, for8hours. Then phagocytic indexes were analyzedby flow cytometry after labeling fluorescently by anti-CD19antibodies et al.Results showed that actin inhibitants such as cochicine and cytochalasin B could inhibit B cells’ phagocytosis, which indicated the actin reorganization is neededin B cells’ phagocytic process.3) Phagolysosome formation: After in vivo phagocytosis assay usingfluorescent microspheres, peritoneal B cell were prepared with cytospin. Then thelysosomes/phagolysosomes were visualized by acid phosphatase staining, andwere analyzed using fluorescent microscopy. We found internalized fluorescentmicrospheres were all in confusion with lysosomes, so phagolysosome formationis normal in phagocytic B cells.4) Intracellular killing assay: Peritoneal B cells were inhabited with viableStaphylococcus aureus, and gentamycin protection assay was used to analyzesurvival amount of intracellular bacteria after different length of time. Resultsshowed that although phagocytic volume of B cells is relatively low, but thekilling rate is similar with other phagocytes.5) Phenotypic analysis: After in vivo phagocytosis assay usingStaphylococcus aureus, peritoneal B cells were labeled by anti-CD19, CD11b,CD5, B220(CD45Ro) antibodies, and then analyzed by flow cytometry.Phenotypic characters of phagocytic B cells were disclosed to be CD11b+B220lowB-1cells, and within which, CD5B-1cells, namely B-1b cells, share strongerphagocytic abilities than CD5~+B cells, namely B-1a cells.6) Receptors analysis:①Antibodies anti-CD11b, TLR2, IgM et al were used to block potentialreceptors, and within which, CD11b or TLR2blocking showed no apparenteffects on B cells’ phagocytic rate, only combination usage of anti-IgM andanti-Igκ antibodies could effectively inhibit B cells’ phagocytic rate, whileneither single one could do.②Comparison between phagocytic rates of peritoneal B cells from wild type and TgVH3B4transgenic mice showed that the later ones showed muchhigher phagocytic rate.③Actin, as a cross-recognized antigen of TgV_H3B4BCR, was used tocompete with Staphylococcus aureus in combination to B cells from TgV_H3B4transgenic mice in in vitro phagocytosis assay. Results showed that actinsuccessfully inhibited the TgV_H3B4B cells’ phagocytosis.All results above indicated that B cell receptor is shown to take part in theprogress of phagocytosis as a phagocytic receptor.7) Migration assay: At different time points after intraperitoneallyadministration of Staphylococcus aureus, phagocytic peritoneal B cells ratio werecompared, which disclosed that phagocytic peritoneal B cells populationdecreased fast within1.5hours. Peritoneal B cells experienced in vitrophagocytosis were adoptive transfer to gender and age matched mice peritonealcavity, then the omenta and spleen were dissected and observed under fluorescentmicroscopy after immunofluorescence assay, and B cells containingStaphylococcus aureus were found aggregating to the milk spots on omenta, andStaphylococcus aureus were found in the spleen with1day. Phagocytic B cellswere labeled by anti-CD11b antibodies and analyzed by flow cytometry, andCD11b expression was found to be related with higher migration ability.8) T cell proliferation assay: B cells containing sterilized Staphylococcusaureus were coculured with CFSE labeled spleen T cells.5days later, cells wereharvested and labeled with anti-CD4antibody before flow cytometry analysis.Result showed that B cells containing Staphylococcus aureus induced strongerspleen CD4~+T cell proliferation from mice after intraperitoneally administrationof Staphylococcus aureus5weeks before than from mice without suchpre-interference. Major Conclusions:This research for the first time disclosed B cell receptor mediatedphagocytosis of mammal B lymphocytes, which share classic phagocytosischaracters, and could kill phagocytosed bacteria by classic microbicidalmechanism like phagolysosome formation. Phagocytic B cells showed activeCD11b related migration and induced antigen specific proliferation of CD4+Tcells. Phagocytic ability further supports the close evolutionary relationshipbetween B cells and phagocytes, especially that B-1cells evolutionarily occurredmore early. Phagocytic process also supplements the particulate antigenpresentation mechanism of B cells. And the immune initiation role of B cells toparticulate antigens was further enlightened according to our research, and thismight facilitate the exploration of new immune therapy to infections andautoimmune related diseases.