A Study On The Endocytosis And Its Regulatory Mechanism In IgM~+B Cells From Flatfish

Increasing evidence has demonstrated support for the endocytic capacities of teleost B lymphocytes(B cells for shot).It was first investigated in rainbow trout in2006 that particles could be ingested by B cells and endocytic B cells have been demonstrated to have phagolysosome formation and serial downstream degradative activities.Subsequently,endocytic B cells were found in a variety of teleosts,as well as in amphibians,reptiles and mammals.However,only phenomena have been discovered for this process and lack of study on endocytosis mechanism.In this study,the endocytosis efficiency and endocytic pathway of IgM~+B cells in teleost fish were examined,the similarities and differences of endocytosis activity and pathway of IgM~+B cells in different immune organs were analysed,the mechanism in immune activation of B cells and peripheral blood leukocytes by B cell co-recepter CD22 was investigated by using multiple approaches including immunology,cell biology and molecular biology.Flow cytometry,immunofluorescence and electron microscopy were used to identify endocytic Ig M~+B cells in turbot peripheral leukocytes and their morphological characteristics.Moreover,their endocytosis efficiency and endocytic pathway for non-self particles in different sizes were investigated.IgM~+B cells with endocytic capacity have larger nuclears,smaller volumes and less intracellular granularity.IgM~+B cells showed different endocytosis enfficiency for different size fluorescent latex microspheres(in a diameter of 0.1,0.5,1 and 2?m),with highest uptake capacity for 0.5?m beads,moderate for 1?m beads,and low for 0.1?m and 2?m beads.In the presence of macropinocytosis inhibitors,the endocytic activity of turbot peripheral blood leukocytes IgM~+B cells on 0.5?m fluorescence latex microspheres was significantly decreased,while the endocytic activity of IgM~-cells was clathrin-and caveolin-dependent.For the uptake of 1?m beads,IgM~+B cells relied mainly on macropinocytosis and partially on caveolae-mediated pathway,while IgM~-cells utilized the routes similar to that of internalizing 0.5?m beads.Consistently,theinternalizedmicrosphereswereco-localizedwith high-molecular-mass dextran in IgM~+endocytic cells.In addition to latex beads,IgM~+B cells could also ingest inactivated bacteria predominately through macropinocytosis and caveolae-mediated endocytosis.These results collectively indicated that macropinocytosis is principally responsible for particle uptake by turbot IgM~+B cells.The percentage of IgM~+B cells in flounder spleen leukocytes is higher than that in peripheral blood leukocytes,while the endocytic activity is lower than that in PBL IgM~+B cells.Both peripheral blood leukocytes IgM~+B cells and spleen leukocytes IgM~+B cells exhibited endocytic capacities but different endocytosis efficiency.The uptake capacity of PBL IgM~+B cells was higher for 0.5?m beads than that for 1?m beads,while uptake capacity of SL IgM~+B cells showed a contrary result.IgM~+B cells in both peripheral blood leukocytes and spleen leukocytes employed apparently macropinocytosis-dependent endocytic pathway.However,in peripheral blood leukocytes IgM~-cells,the uptake activity was significantly reduced in the presence of macropinocytosis,clathrin-and caveolin-mediated endocytosis inhibitors;in spleen leukocytes IgM~-cells,both clathrin-mediated endocytosis inhibitors and macropinocytosis inhibitors could reduceed the uptake activity.These results suggest that although IgM~+B cells in peripheral blood leukocytes and spleen leukocytes showed different endocytic activity and particle size-dependent endocytosis efficiency,both of them mainly depended on macropinocytosis upon uptake of particles.In mammals,CD22 is a co-receptor on the surface of B cells and plays a crucial role in regulating the immune activity of B cells.However,the function of fish CD22is still unclear.In this study,we characterized for the first time two fish CD22 from Japanese flounder and tongue sole,separately.Sequence alignment and domain analysis showed that PoCD22 and CsCD22 have extracellular immunoglobulin domains and cytoplasmic immunoreceptor tyrosine-based inhibition motifs,which are conserved in CD22 family.Majority of Japanese flounder IgM~+B cells in peripheral blood leukocytes and spleen leukocytes are CD22 positive,and partial IgM~-cells also express CD22 protein.During bacterial infection,the endocytic activity and ROS activity of peripheral blood leukocytes IgM~+B cells was significantly reduced in the presence of rPoCD22 antiserum,while those of spleen leukocytes IgM~+B cells was significantly induced.Results above indicate that CD22 positively regulates the immune activation of peripheral blood leukocytes IgM~+B cells,and negatively regulates that of spleen leukocytes IgM~+B cells.In the presence of rCsCD22antiserum,the proliferation,phagocytosis,and antibacterial activity of peripheral blood leukocytes were significantly increased.These results indicate that CsCD22plays an inhibitory role in PBL activation.To sum up,it was found for the first time that the endocytosis efficiency of teleost IgM~+B cells on non-self particles was size-dependent,and the endocytic capacity had difference in IgM~+B cells of different immune organs(peripheral blood and spleen).Nonetheless,macropinocytosis is principally responsible for particle uptake by teleost IgM~+B cells.B cell co-receptor CD22 played a crucial role in regulating the immune activity of teleost IgM~+B cells as well as peripheral blood leukocytes.These provide us with new ideas for the evolution origin of B lymphocytes and promote our understanding for the prevention of diseases in commercial fish farming.