Distribution And Migration Of IgA-secreting Lymphocyte In IgA Nephropathy

Partâ… . Establishment and identification of a mouse IgA nephropathy modelObjective:To establish a mouse model of IgA nephropathy by mucosal immunization, which serves as the basis of study in the pathogenesis, prevention and treatment of IgA nephropathy. Methods:First we establish a mouse model of IgA nephropathy by using oral bovine serum albumin combined tail intravenous injection of staphylococcal enterotoxin B, then urine was collected to count urine red blood cells and to detect 24h urinary protein by Coomassie brilliant blue,and blood was collected to detect serum albumin blood test (ALB), total protein (TP), cholesterol (CHOL) and creatinine (Cr), renal tissues were also collected to detecte the expression of IgA by immunohistochemistry, and to observe the pathological changes by hematoxylin-eosin (HE) staining and periodic acid-Schiff (PAS) staining in the ordinary light microscope. Results:We observed that IgA showed diffuse block deposition in glomerular mesangial area,and the pathological change showed typical mesangial proliferative glomerulonephritis change. In addition, typical proteinuria was also observed in mouse IgA nephropathy model. However, the serum ALB, TP, CHOL and CHOL showed no abnormalities. Conclusion:Oral bovine serum albumin combined tail intravenous injection of staphylococcal enterotoxin B method can successfully induce IgA nephropathy model in mice,which is an ideal method for the current actual situation of domestic. The success of establishment of mouse IgA nephropathy model through mucosal immunization suggests the involvement of mucosal immune system in the development of IgA nephropathyPartâ…¡. Abnormal distribution of and the impact of FTY720 on IgA-secreting lymphocyte in IgA nephropathic miceObjective:To compare the differences of tissue distribution between IgA+ B lymphocytes and IgA+ plasma cells from bone marrow, Peryer's patches, lamina propria of small intestine in IgA nephropathy mouse,and to observe the impact of FTY720 on it. Methods: First we induced mouse IgA nephropathy model, then isolated lymphocytes from bone marrow and Peryer's patches by Ficoll density gradient centrifugation,from intestinal lamina propria by continuous Percoll density gradient centrifugation. Three-color flow cytometry were applied to detect the expression of IgA, B220, CD38 in the surface of lymphocytes from bone marrow, Peryer's patches, lamina propria of small intestine. The differences of IgA+ B lymphocytes and IgA plasma cells distribution among bone marrow, Peryer's patches, lamina propria of small intestine were identified between normal control, IgA nephropathic mice and FTY720 treated IgA nephropathic mice. Results:1.Bone marrow:The ratio between IgA+ lymphocytes and the total lymphocytes significantly increased in IgA nephropathy mice compared with normal mice(P<0.05),and the ratio between IgA+ B220+ lymphocytes and IgA+B lymphocytes decreased in IgA nephropathy mice compared with normal mice(P<0.05). In the end, the ratio between IgA B220- lymphocytes and the total lymphocytes significantly increased in IgA nephropathy mice compared with normal mice(P<0.05). The ratio between IgA+ lymphocytes and total lymphocyte decreased in mice treated with FTY720 compared with mice in model group,and both the ratio between IgA+ B220+lymphocytes and total lymphocyte, and the ratio between IgA+ B220-lymphocytes and total lymphocyte also decreased in mice treated with FTY720 compared with mice in model group.2. Peryer's patches:The ratio between IgA+lymphocytes and total lymphocyte showed no significant differences in IgA nephropathy mice compared with mice in normal control.group,and both the ratio between IgA+B220+lymphocyte and total lymphocyte, and the ratio between IgA+B220- lymphocyte and total lymphocyte also showed no significant differences in IgA nephropathy mice compared with mice in normal control group. The ratio between IgA+lymphocyte and total lymphocyte,and the ratio between IgA+B220-CD38-lymphocyte total lymphocyte both significantly increased in mice treated with FTY720 compared with mice in model groupand mice in normal control group (P<0.05).3. lamina propria of small intestine:Although the ratio between IgA+ lymphocyte and total lymphocyte, and the ratio between IgA+B220+lymphocyte and total lymphocyte, and the ratio between IgA+B220-lymphocyte and total lymphocyte all decreased in IgA nephropathy compared with mice in normal control group and showed no significant differences.The ratio between IgA+ lymphocyte and total lymphocyte,and the ratio between IgA+ B220+ lymphocytes and total lymphocytes, and the ratio between IgA+ B220- lymphocytes and total lymphocytes,all decreased in mice treated with FTY720 compared with mice in model groupand mice in normal control group (P<0.05). Conclusion:the number of IgA+ B lymphocytes and IgA+plasma cells both increased in bone marrow of IgA nephropathy mice compared with mice in normal control group,but the number of IgA+ B lymphocytes and IgA+ plasma cells did not increase in peryer's patches and small intestine lamina propria of IgA nephropathy mice compared with mice in normal control group. furthermore, the number of IgA+ plasma cells did decrease in small intestine lamina propria of IgA nephropathy mice. These results suggest an abnormal migration of IgA+ B cell in Peryer's patches; which migrate to bone marrow instead of small intestine lamina propria and develop to pIgA secreting mature plasmacytes. FTY720 can prevent IgA+ B lymphocytes and IgA+ plasma cells egression from Peryer's patches, thus reduce the numbe of plasma cells in bone marrow. Partâ…¢. CCL28, MadCAM-1, SDF-1 regulate migration of IgA+B lymphocytes and IgA+plasma cells in IgA nephropathic miceObjective:To detect the expression of CCL28, MadCAM-1, SDF-1 in Peryer's patches, lamina propria of small intestine, bone marrow and kidney tissue, and to study the role of lymphocyte homing factor in migration of IgA+B lymphocytes and IgA+plasma cells in IgA nephropathy mice and the relationship with pathogenesis of IgA nephropathy. Methods:Mouse IgA nephropathy model was induced by mucosal immunization, then the expression of CCL28, Madcam-1 in small intestine tissue and the expression of SDF-1 in bone marrow and renal cortex were detected by immunohistochemistry. At the same time, the protein expression of CCL28, MadCAM-1 in small intestinal tissue and Peryer's patches, protein expression of SDF-1 in bone marrow and kidney tissue were all detected by Western blot. In addition, mRNA expression of CCL28, MadCAM-1 in small intestinal tissue and SDF-1 mRNA in bone marrow and kidney tissue were all detected by Real-time quantitative PCR. Results:1. Immunohistochemistry showed that CCL28 expression was strong in lamina propria of small intestine in normal mice, while which was significantly decreased in IgA nephropathy mice and was slightly increased in IgA nephropathy mice treated with FTY720. The expression of MadCAM-1 in small intestine lamina propria was reduced in both IgA nephropathy model mice and mice treated with FTY720 than that in normal mice. SDF-1 expression in bone marrow stroma was not significantly different from each other in those three groups. SDF-1 expression in renal tissue was mainly found in renal tubular epithelial cells, which was weaker in normal mice, and stronger in model group mice and significantly decreased in mice treated with FTY720 when compared with the model mice.2. Western blot showed that both CCL28 and MadCAM-1 protein expression was significantly lower in model mice and mice treated with FTY720 than that in normal mice(P<0.05), but no significant difference was found between model mice and mice treated with FTY720. SDF-1 protein expression in model mice was slightly reduced in bone marrow and slightly increased in renal tissue compared with normal control mice, and slightly increased in bone marrow and renal tissue in mice treated with FTY720 compared with normal control mice, but the differences showed no statistical significance. 3.Real-time quantitative PCR showed that CCL28 mRNA expression decreased in model mice and mice treated with FTY720 compared with the normal mice(P<0.05). MadCAM-1 expression decreased in model mice, and increased in mice treated with FTY720, but the differences showed no statistical significance. The mRNA expression of SDF-1 decreased in bone marrow and increased in renal tissue of IgA nephropathy mice compared with the normal mice(P<0.05). and the SDF-1 mRNA expression showed no statistical difference in mice treated with FTY720 compared with the model mice. Conclusion:The decreased expression of CCL28 and MadCAM-1 in intestinal mucosa tissue reduces the migration of IgA secreting cells into intestinal lamina propria in IgA nephropathy mice.. SDF-1 doesn't play a key role in migrating of IgA secreting cell to bone marrow.. FTY720 does not affect the expression of CCL28 and MadCAM-1, but induces peripheral blood lymphocytes homing to secondary lymphoid organs thus reduces the number of plasma cell in bone marrow. Partâ…£. The effects of FTY720 and cyclosporin A on IgA nephropathy in miceObject:To observe and evaluate the effects of FTY720 on IgA nephropathy in mice. Methods:Mouse IgA nephropathy model were induced by mucosal immunization. FTY720 and cyclosporin A were applied from week 10 to week 14.All the mice were sacrificed after 14 weeks to evaluate the pathology and clinical manifestations between mice treated with FTY720 or with CsA and mice in IgA nephropathy group. Results:The urine protein in IgAN model group significantly increased in week 12, which showed less in FTY720 treated group and CsA treated group. The urine protein significantly decreased in week 14 in both FTY720 treated group and CsA treated group compared with IgAN model group. And there were no significant difference between FTY720 treated mice and nomal mice in week 14.The results of TP, ALB, CHOL, Cr showed no significant differences between all these groups. Glomerular mesangial proliferation and IgA deposition were found in model group and less prominent in FTY720 and CsA treated groups. Conclusion:Both FTY720 and CsA showed obvious therapeutic effect on IgA nephropathy. FTY720 can induce IgA+B lymphocytes and IgA+plasma cells homing to Peryer's patches, reducing the number of plasma cells in bone marrow, thus reducing the production of pathogenic IgA molecules, Therefore, FTY720 is likely to be a effective drugs in cause treatment for IgA nephropathy.