Mycophenolic acid reduces anti-DNA antibody binding and cytokine secretion in renal proximal tubular epithelial cells - implications in lupus nephritis

Abstract

PURPOSE: Lupus nephritis is a severe organ manifestation of systemic lupus erythematosus. Cardinal features of lupus nephritis include elevated levels of circulating anti-DNA antibodies, their deposition in the mesangium and tubular basement membrane, mesangial proliferation and inflammation. Subsets of anti-DNA antibodies have been shown to be internalized by various cells and transferred to the nucleus where they elicit alterations in cellular functions. Proximal tubular epithelial cells (PTEC) constitute the predominant cell type within the tubulo-interstitium, and there is compelling evidence to suggest that they play a pivotal role in the immunopathogenesis of various renal parenchymal diseases including lupus nephritis. Mycophenolate mofetil (MMF) is a new immunosuppressant used for the treatment of lupus nephritis. Although its action on lymphocyte proliferation is well documented, its effect on non-immune cells has not been fully elucidated. This study aims to investigate the modulatory role of mycophenolic acid (MPA), the active metabolite of MMF, on anti-DNA antibody binding and internalization in PTEC and their subsequent effect on cytokine secretion. METHODS: Polyclonal double-stranded anti-DNA antibodies were isolated from serum samples of 10 patients with lupus nephritis using DNA-cellulose and protein A-Sepharose affinity chromatography. HK-2 cells were used in this study, which are normal PTEC immortalized by the transduction with the human papilloma virus 16 E6/E7 genes. Growth-arrested HK-2 cells were stimulated with normal IgG or anti-DNA antibodies (final concentration 10 g/ml) in the presence or absence of MPA (5 g/ml) for periods up to 48 h, after which time the supernatant was collected to measure IL-6 and IL-8 secretion. Binding and internalization of anti-DNA antibodies to HK-2 cells under control and experimental conditions was determined by cellular ELISA and immunohistochemistry, respectively. RESULTS: Anti-DNA antibody binding to HK-2 cells correlated with SLE disease activity. Anti-DNA antibodies bound to the cell surface of HK-2 cells and were localized to the nucleus within 30 min of administration. This was accompanied by a significant induction of IL-6 and IL-8 secretion when compared to normal IgG (P 0.05 for both). Such effects were more prominent in cells stimulated with anti-DNA antibodies derived from active patients. Co-incubation of anti-DNA antibodies with MPA significantly reduced anti-DNA antibody binding and cytokine secretion (P 0.05). CONCLUSION: Our data demonstrated that MPA reduced anti-DNA antibody binding to HK-2 cells and subsequent induction of cytokine secretion, suggestive that MPA may reduce inflammatory responses in the tubulo-interstitium during pathogenesis of lupus nephritis.