Cross-reaction of anti-DNA autoantibodies with membrane proteins of human glomerular mesangial cells in sera from patients with lupus nephritis

Abstract

Anti-DNA autoantibodies were thought to play a major role in the pathogenesis of lupus nephritis (LN). A recent study revealed that affinity-purified anti-DNA antibodies had a cross-reaction with human glomerular mesangial cells (HMC). However, whether the cross-reaction was antigen-antibody-mediated was unclear. The aim of the current study was to investigate the binding of anti-DNA antibodies to HMC membrane proteins and to characterize the target antigens. Affinity-purified IgG anti-DNA antibodies were purified by DNA-cellulose chromatography in sera from nine patients with biopsy-proven active lupus nephritis. In vitro cultured primary HMCs were disrupted by sonication and HMC membranes were obtained by differential centrifugation. The membranes of human umbilical vein endothelial cells (HUVEC), human proximal renal tubular epithelial cell line (HK2) and peripheral mononuclear cells (PMC) were obtained as controls. Binding of anti-DNA antibodies to the membrane proteins was investigated by Western blot analysis using soluble membrane proteins as antigens. Both HMC membrane and affinity-purified anti-DNA antibodies were treated with DNase I to exclude DNA bridging. All nine affinity-purified anti-DNA antibodies could blot the HMC membrane proteins, and there were at least three bands at 74 kDa, 63 kDa and 42 kDa that could be blotted. Among the nine IgG preparations, all nine (100%) could blot the 74 kDa band; eight (88.9%) could recognize 63 kDa and 42 kDa protein bands separately. After DNase treatment, the same bands could still be blotted by most affinity-purified anti-DNA antibodies. Affinity-purified anti-DNA antibodies could also blot similar bands on membrane proteins of other cells, but some bands were different. In conclusion, anti-DNA autoantibodies could cross-react directly with cell membrane proteins of human glomerular mesangial cells and might play an important role in the pathogenetic mechanism in lupus nephritis.

Fig. 1

Western blot analysis to reveal the reactivity of IgG anti-DNA antibodies from sera of the patients with lupus nephritis with membrane proteins of human mesangial cells; the membrane proteins were not predigested with DNase I. Lane 1: blank control; lane 2: normal control; lanes 3–11: IgG anti-DNA antibodies from sera of the patients with lupus nephritis.

Fig. 2

Western blot analysis to reveal the reactivity of IgG anti-DNA antibodies from sera of the patients with lupus nephritis with membrane proteins of human mesangial cells predigested with DNase I. Lane 1: blank control; lane 2: normal control; lanes 3–11: IgG anti-DNA antibodies from sera of the patients with lupus nephritis

Fig. 3

Western blot analysis to reveal the specificity of IgG anti-DNA antibodies from serum of one patient with lupus nephritis. Membrane proteins of human mesangial cells were used as antigens. Lane 1: blank control; lane 2: normal control; lane 3: affinity-purified IgG anti-DNA antibodies; lane 4: affinity-purified IgG anti-DNA antibodies as lane 3 but preincubated with 50 µg/ml dsDNA.

Fig. 4

Western blot analysis to reveal the reactivity of IgG anti-DNA antibodies with membrane proteins of four different cells. The membrane proteins of human glomerular mesangial cells (HMC) (lanes 1, 2), human umbilical vein endothelial cell (HUVEC) (lanes 3, 4), human proximal renal tubular epithelial cell line (HK₂) (lanes 5, 6) and peripheral mononuclear cells (PMC) (lanes 7, 8) were used as antigens. Lanes 1, 3, 5, 7: affinity-purified IgG from a healthy volunteer as negative control; lanes 2, 4, 6, 8: affinity-purified IgG anti-DNA antibodies from a patient with lupus nephritis.