Annexin II on human mesangial cells mediates anti-dsDNA antibody binding

Abstract

OBJECTIVES: Cardinal features of lupus nephritis include the production of anti-dsDNA antibodies, mesangial proliferation and renal inflammation. The mechanism through which anti-dsDNA antibodies might interact with resident renal cells remains to be elucidated. We and others have previously demonstrated that anti-dsDNA antibodies could bind to human mesangial cells (HMC) independent of bridging chromatin material. We further characterized the cross-reactive antigen(s) on HMC which bind human polyclonal anti-ds-DNA antibodies. METHODS: HMC were isolated from renal cortical tissue using differential sieving followed by collagenase treatment. Human polyclonal anti-dsDNA antibodies were isolated from patients with lupus nephritis using Protein A-Sepharose followed by DNA-cellulose affinity chromatography. HMC surface proteins were removed by limited trypsin (10μg/ml) treatment and the binding of anti-dsDNA antibodies to cells assessed by flow cytometry and cellular ELISA. HMC plasma membrane fractions were subjected to Western blot analysis, then probed with purified anti-dsDNA antibodies and subjected to MALDI-TOF spectrometry to identify ‘cross-reactive’ membrane proteins. Renal biopsies were assessed by immunohistochemistry. RESULTS: Limited trypsin but not DNAse treatment of HMC significantly reduced antidsDNA antibody binding (p<0.05). Anti-dsDNA antibodies predominantly bound to a cell surface antigen with a molecular weight of 36kDa and 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 (p<0.05), compared with controls and non-lupus kidney diseases. CONCLUSIONS: Our data demonstrated that annexin II on the plasma membrane of HMC mediates anti-dsDNA antibody binding.