The Study On The Role Of OBF-1 For Antibody-Secreting Cells And Autoimmunity In MRL-lpr Mice

OBF-1(Oct binding factor 1), also known as OCA-B or Bob-1, is a B cells-specific transcriptional co-activator. It can regulate the development and function of B cells. OBF-1 deficient mice involve in partially blocking B cell maturation at multiply stage and completely disrupting germinal center formation, leading to humoral immune deficiency. MRL/MpJ-Fas^lpr/lpr (MRL-lpr) mice is one of the models for studying human systemic lupus erythematosus which is characterized by autoantibody formation and immunocomplex-mediated glomerulonephritis. In the study, OBF-1 deficient MRL-lpr mice and OBF-1 wild-type MRL-lpr mice were bred. Based On the two kinds of animal models, the impact of OBF-1 deficiency on the antibody-secreting cells and the autoimmunity was investigated.1 To establish the models of OBF-1 deficient MRL-lpr mice and OBF-1 wild-type MRL-lpr mice.Crossing OBF-1 null mice with MRL-lpr mice to create first filial generation (F1). F2 was generated by intercrossing the F1 hybrids. Genotypes of OBF-1 and Fas from the F2 hybrids were screened by PCR. OBF-1 homozygous deficient MRL-lpr mice was chosed to backcross with the MRL-lpr strain for six to eight generations to minimize the genetic background differences. Among the offsprings with nearly same genetic background of MRL-lpr strain, which of genotype of OBF-1^+/+Fas^-/- and OBF-1^-/-Fas^-/- were selected for experiments.2 The impact of OBF-1 deficiency in MRL-lpr mice on the antibody-secreting cells and the autoimmunity.2.1 The impact of OBF-1 on the autoimmunity in MRL-lpr miceAll kinds of Immunoglobulin titers, anti-double-strand DNA (dsDNA) antibody and anti-nuclear antibody (ANA) were measured by sandwich ELISA in the sera from two groups of aged 3⁴-month-old mice with genotype of OBF-1^+/+MRL-lpr and OBF-1^-/-MRL-lpr, respectively, each genotype with 6 mice. The results showed that the levels of total immunoglobulins were significantly higher, especially the production of immunoglobulinG (IgG) subclass (IgG1, IgG2a, IgG2b and IgG3) in OBF-1 wild-type MRL-lpr mice, with the lowest level of 5000μg/mL for IgG2a, compared to OBF-1 deficient MRL-lpr with the highst level of 200μg/mL for IgG2a. Autoantibody (anti-dsDNA Ab and anti-nuclear Ab) were also detected in OBF-1 wild-type MRL-lpr mice, but undetected in OBF-1deficiency MRL-lpr. The above results obtained may demonstrate that the levels of all immunoglobulins were reduced significantly and that autoantibodies were eliminated in OBF-1 deficiency MRL-lpr mice.Both IgG and complement C3 deposits were found in the kidney sections from the glomeruli of OBF-1^+/+MRL-lpr mice by immunofluorescence staining. In contrast, no significant depositions were detected in the kidney of OBF-1^-/-MRL-lpr mice. PAS staining of the kidney showed crescent formation and enlarged glomeruli in the OBF-1^+/+MRL-lpr; however, the OBF-1^-/-MRL-lpr mice had no sign described above in their kidney. The results may demonstrate that OBF-1 deficiency protected against the development of immune complex deposits and glomerulonephritis in the MRL-lpr mice.2.2 The mechanism of defective autoantibody production in OBF-1 deficiency MRL-lpr mice.To study the impact of OBF-1 deficiency on antibody-secreting cells (ASCs), by ELISPOT assay, OBF-1^+/+MRL-lpr mice developed a high level of IgG-secreting cells of about 850 spots in 105 splenic cells, meanwhile, dramatically decreased in the OBF-1^-/-MRL-lpr mice with few spots. In agreement with the results in the spleen, ASCs from bone marrow in OBF-1^-/-MRL-lpr mice was markedly reduced. The results from ELISPOT assay may domonstrate that OBF-1 was essential for the IgG-secreting cells formation in the MRL-lpr mice.Semi-quantitative RT-PCR were used to define gene expression levels of Blimp-1, Xbp-1, Irf-4, Bcl-6 and J chain, which are very important for the plasma cell information during the B-cell differentiation and activation. The result showed that no significant difference for Irf-4, xbp-1, Blimp-1 and Bcl-6 in the mRNA level between OBF-1^-/-MRL-lpr and OBF-1^+/+MRL-lpr mice, but the J chain gene expression was markedly reduced in the former mice. To further know whether transcription of the J chain depends on OBF-1 or not, the expression of the J chain in separated splenic B cells from OBF-1 null and OBF-1 wild- type mice were checked, no significant difference was observed. Combined above the results may have conclusion that the transcription inhibition of the J chain in OBF-1^-/-MRL-lpr mice appeared to be the consequence of interaction between lpr and OBF-1. The results may demonstrate that loss of OBF-1 impacted on the ASCs in mRNA levels of MRL-lpr mice.By flow cytometry detect and analysis, the number of both CD19⁺IgM⁺ immature and CD19⁺IgD⁺ mature B cells were not reduced in B cells from spleen or bone marrow of the OBF-1^-/-MRL-lpr mice, compared to that of the OBF-1^+/+MRL-lpr mice. These results indicated that loss of OBF-1 did not impair development of immature or mature B cells in the MRL-lpr mice. Although the development of CD3⁺, CD4⁺ and CD8⁺ T cells were not impaired in spleen and lymph node of the OBF-1^-/-MRL-lpr mice, the number of CD3⁺B220⁺ T cells was markedly reduced. The data lead to the conclusion that the autoantibody production was likely involved in this group of T cells, unlikely in B cells.In summary, the loss of OBF-1 can abolish autoantibody and prevent immune complex deposits and glomerulonephritis in the MRL-lpr mice, indicating that OBF-1 is essential for the formation of autoantibody and plays a critical role in autoimmunity development in MRL-lpr mice, which could be used as a valuable target for developing drugs against systemic lupus erythematosus.