Polyclonal IgG Anti-dsDNA Antibodies Exert Cytotoxic EfFect on Cultured Rat Mesangial Cells by Binding to Cell Membrane and Augmenting Apoptosis

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IgG anti-double stranded DNA antibodies (anti-dsDNA) purified from serum of patients with active systemic lupus erythematosus (SLE), have been found to be cytotoxic to the cultured rat mesangial cells (MC). In the present study, by use of immunofluorescent staining, immunoblotting, radioimmunoprecipitation, and cell cycle analysis, we showed that IgG anti-dsDNA could bind to the membrane of MC. The bound epitope was a 28 kDa protein, which would disappear if the cells were treated in advance with proteinase K (100 micrograms/ml). In addition, binding of MC by 20 micrograms/ml of anti-dsDNA IgG F(ab')2 activated plasma membrane (equivalent to 80 IU/ml of calf thymus double-stranded DNA binding activity) resulted in release of much more 3H-arachidonic acid than binding by 20 micrograms/ml of human IgG F(ab')2 (26.71 +/- 3.75% in the case of anti-dsDNA vs. 4.73 +/- 2.86% in the case of IgG). To understand further the cytotoxic mechanism of anti-dsDNA, we incubated MC with anti-dsDNA, for a variety of periods (from 10 minutes to 24 hours). After incubation, the cells were fixed and stained with hematoxylin-eosin for morphologic observation. Simultaneously, the genomic DNA was extracted and analyzed in 1.8% agarose gel electrophoresis. We found that cell death caused anti-dsDNA followed a process of apoptosis rather than necrosis. These results suggest that binding of anti-dsDNA with MC membrane may activate endonuclease which will fracture the DNA and lead to programmed cell death.