A role for DNA in anti-DNA antibodies binding to endothelial cells.
Vascular injury and microvascular thrombosis are prominent features of systemic lupus erythematosus, as are circulating DNA-binding antibodies (DNAb). Experimental glomerulonephritis can be induced by anti-endothelial cell antibodies, and polyreactive DNAb might be pathogenetic by binding to endothelial cells, perhaps influencing their non-thrombogenic nature. To test this hypothesis, eight monoclonal antibodies (mAb) that bind to DNA derived from (NZB x NZW)F1 or MRL/Mp-lpr/lpr mice, were tested for their ability to bind to human umbilical vein endothelial cells (HUVEC). Binding was assessed using flow cytometry, fluorescence microscopy and cellular ELISA. Three of the eight mAb, at concentrations employed in this study, bound to HUVEC and dermal fibroblasts. Of these three mAb, one bound also to platelets. Two of the three demonstrated strong binding to (1) freshly isolated, collagenase-digested HUVEC, (2) 2nd passage HUVEC in suspension after trypsinization and, (3) 2nd passage HUVEC growing on plastic plates. To determine whether DNA itself acted as a ligand in this binding, prior treatment with DNAase was studied. Treatment of the endothelial cells with DNAase had no effect on the binding of one mAb, but DNAase treatment of this monoclonal itself resulted in a 60% reduction in binding to HUVEC, suggesting that the binding might be mediated through DNA in the form of a DNA/anti-DNA immune complex. In contrast, DNAase digestion of the endothelial cells caused a 40% reduction in the binding of the other two monoclonal antibodies. Furthermore, one of the two mAb bound 30% more to HUVEC after themselves being subjected to DNAase treatment. These two monoclonals may therefore be binding directly to HUVEC, possibly to DNA associated with the membrane. Prior DNAase digestion of dermal fibroblasts had a more profound effect on the binding of all three autoantibodies compared to HUVEC after similar treatment. Therefore, DNA can bind independently to either antibody or cell, thus supporting build up of complexes and capture of preformed complexes. Functionally, the binding of mAb to HUVEC did not influence thrombin-induced prostacyclin synthesis, in contrast to a control monoclonal anti-endothelial cell antibody EN4, which did.
Frampton
G
G
Renal Unit, UMDS, London, UK.
Hobby
P
P
Morgan
A
A
Staines
N A
NA
Cameron
J S
JS
D000818:Animals; D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D003429:Cross Reactions; D004247:DNA / Q000276:immunology*; D003851:Deoxyribonucleases; D004730:Endothelium, Vascular / Q000276:immunology* / Q000378:metabolism; D011464:Epoprostenol / Q000096:biosynthesis; D006497:Heparan Sulfate / Q000276:immunology; D006801:Humans; D008181:Lupus Nephritis / Q000276:immunology; D051379:Mice
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D004247:DNA; D003851:Deoxyribonucleases; D011464:Epoprostenol; D006497:Heparan Sulfate