Anti-dsDNA antibodies from patients with lupus nephritis bind to annexin II on human mesangial cells

Abstract

Lupus nephritis is characterized by the production of anti-dsDNA antibodies and proliferative glomerulonephritis. The mechanism through which anti-dsDNA antibodies interact with resident renal cells remains to be elucidated. We and others have previously demonstrated that human anti-dsDNA antibodies can bind to human mesangial cells (HMC) without the need of bridging chromatin material. In this study, we characterized the cross-reactive antigen(s) on HMC that mediate the binding. Human polyclonal anti-dsDNA antibodies were isolated from patients with lupus nephritis using Protein A-Sepharose followed by DNA-cellulose affinity chromatography. The binding of anti-dsDNA antibodies to HMC was assessed by flow cytometry and cellular ELISA. HMC plasma membrane fractions were subjected to Western blotting, probed with purified anti-dsDNA antibodies, and analyzed with MALDI-TOF spectrometry to identify ‘cross-reactive’ membrane proteins. Renal biopsies were examined with immunohistochemistry. Limited trypsin but not DNase treatment of HMC significantly reduced anti-dsDNA antibody binding (P<0.05). Anti-dsDNA antibodies predominantly bound to a cell surface antigen with molecular weight of ∼36kDa. This band was identified as annexin II by MALDI-TOF spectrometry. The binding activity between anti-dsDNA antibodies and annexin II correlated with disease activity. Glomerular annexin II expression was significantly increased in active lupus nephritis compared with controls and non-lupus kidney diseases (P<0.05), and co-localized with IgG and C3 deposition. Our data demonstrate that annexin II on the plasma membrane of HMC mediates anti-dsDNA antibody binding.