Effect Of Immunodominant Peptide-conjugated Spleen Cells On Experimental Autoimmune Glomerulonephritis (EAG) Rats

Objective:In this study,Glomerular basement membrane(GBM)antigen polypeptide pCol(24-38)was conjugated with splenocytes from WKY rats using1-(3-dimethylaminopropyl)-3-ethylcarbodiimide(ECDI),the coupled cells were administered to experimental autoimmune glomerulonephritis(EAG)rat model animals and outcome of the rats was observed.Completion of this study will further elucidate the pathogenesis of anti-glomerular basement membrane disease,and provides a theoretical evidence for the treatment of anti-glomerular basement membrane disease.Methods:EAG animal models were established by subcutaneous immunization of WKY rats with peptide pCol(24-38)containing B,T cell epitope on the non-hinge region of collagen alpha 3 chain(alpha 3(IV)NC1)of rat GBM and irrelevant peptide pCol(38-52)after emulsification with complete Freund's adjuvant.When 24-hour urinary protein levels increased markedly for the first time,peptide pCol(24-38),pCol(38-52)conjugated with WKY rat splenocytes using ECDI separately,and then administered intravenously into EAG animal models for three consecutive days.Six weeks after immunization,rats were sacrificed.Serum creatinine,urea nitrogen and circulating anti-pCol(24-38)antibodies were evaluated.Splenocytes proliferation assay was conducted using EDU incorporation method.At the same time,the remaining splenocytes were analyzed by flow cytometry using antibodies CD45RA-PE,CD4-FITC and CD8a-PECy7.One part of kidney tissue was stained with HE and PAS in paraffin section,and the other part was frozen after OCT embedding and then detected IgG deposition by direct immunofluorescence method.Result:After treatment with spleen cells coupled with peptide pCol(24-38),the24-hour urinary protein content of rats in the experimental group decreased significantly compared with that in the positive control group.The final creatinine and urea nitrogen level decreased significantly,the serum anti-pCol(24-38)antibody level decreased by29%(P<0.05).The immunofluorescence results revealed that the glomerular deposition of IgG in the experimental group was reduced.At the same time,the pathological results of paraffin section showed that glomerular cell necrosis and crescent formation were significantly reduced in the experimental group.The lymphocyte proliferation index of the experimental group decreased by 54%(P<0.001),while the flow cytometry analysis of spleen cells showed that there was no significant difference in the percentage of CD45RA~+and CD8~+T cells except the number of CD4~+T cells decreased by 23.5%(P<0.05).Conclusion:Treatment with the antigen-immunodominant peptide pCol(24-38)conjugated homologous splenocytes can alleviate the progression of anti-GBM disease in EAG animal models to some extent.