Anti-proteinase-3 (PR3) antibodies (C-ANCA) recognize various targets on the human umbilical vein endothelial cell (HUVEC) membrane

Numerous studies suggest that C-ANCA are directly pathogenic in vasculitis by activating leucocytes (oxidative burst, enzyme release, endothelial cyto toxicity, etc.). We and others have shown that C-ANCA can also directly act ivate HUVEC, but the precise target on HUVEC is unknown. We show in this st udy that C-ANCA recognize various targets on the HUVEC membrane (different from PR3 in our model), leading to secondary cell activation. Polyclonal af finity-purified C-ANCA recognized targets on the unfixed endothelial membra ne in fluorescent ELISA, flow cytometry, and immunoprecipitation studies. C -ANCA did not react with Fc gamma receptors. Reverse transcriptase-polymera se chain reaction (RT-PCR) experiments showed that HUVEC did not express PR 3. The targets of polyclonal and monoclonal anti-PR3 antibodies on the endo thelial membrane were not the same. Some epitopes were lost after trypsin-E DTA digestion and formaldehyde fixation of cells, whereas anti-PR3 targeted unfixed HUVEC. This suggests that anti-PR3 react with the endothelial memb rane and recognize conformational epitopes shared with PR3. Endothelial cel ls may thus participate in the inflammation associated with Wegener's granu lomatosis and contribute to the emergence of clinical manifestations.