Ballieux B E, Zondervan K T, Kievit P, Hagen E C, van Es L A, van der Woude F J, Daha M R
Department of Nephrology, University Hospital, Leiden, The Netherlands.
Clin Exp Immunol. 1994 Jul;97(1):52-60. doi: 10.1111/j.1365-2249.1994.tb06579.x.
Binding of both proteinase 3 (PR3) and myeloperoxidase (MPO) to endothelial cells (EC) has been suggested to be involved in the vascular damage seen in patients with Wegener's granulomatosis or microscopic polyangiitis. In the present study we investigated in detail the interaction of MPO and PR3 with cultured human umbilical vein endothelial cells (HUVEC) and its matrix products. In addition, we investigated whether interaction of PR3 or MPO with HUVEC monolayers also resulted in antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by anti-neutrophil cytoplasmic antibody (ANCA)-positive patient sera or rabbit IgG anti-PR3 or anti-MPO. Preincubation of HUVEC monolayers with PR3 or MPO resulted in a dose-dependent binding of both PR3 and MPO. However, HUVEC, preincubated with PR3 or MPO, followed by ANCA or by rabbit anti-PR3 or anti-MPO, were not susceptible to ADCC. Detailed analysis of the binding of PR3 to HUVEC monolayers showed that PR3 binds primarily to the extracellular matrix of endothelial cells, and to a very limited extent to the cells themselves. For MPO it was shown that it binds both to the extracellular matrix and to the endothelial cells themselves. However, after binding to HUVEC cultures, MPO was not detectable by polyclonal rabbit or human antibodies specific for MPO, probably because MPO is bound to sites not accessible for immunoglobulins. Binding of PR3 to HUVEC cultures (cells + matrix) was inhibited by fetal calf serum and by alpha 1-antitrypsin, but inactivation of enzymatic activity of PR3 by PMSF did not influence binding of PR3 to HUVEC cultures. Binding of MPO to HUVEC cultures was not influenced by fetal calf serum.
蛋白酶3（PR3）和髓过氧化物酶（MPO）与内皮细胞（EC）的结合被认为与韦格纳肉芽肿或显微镜下多血管炎患者的血管损伤有关。在本研究中，我们详细研究了MPO和PR3与培养的人脐静脉内皮细胞（HUVEC）及其基质产物的相互作用。此外，我们还研究了PR3或MPO与HUVEC单层的相互作用是否也会导致抗中性粒细胞胞浆抗体（ANCA）阳性患者血清或兔IgG抗PR3或抗MPO介导的抗体依赖性细胞介导的细胞毒性（ADCC）。用PR3或MPO预孵育HUVEC单层会导致PR3和MPO的剂量依赖性结合。然而，用PR3或MPO预孵育后的HUVEC，再用ANCA或兔抗PR3或抗MPO处理，对ADCC不敏感。对PR3与HUVEC单层结合的详细分析表明，PR3主要结合在内皮细胞的细胞外基质上，与细胞本身的结合非常有限。对于MPO，研究表明它既结合细胞外基质，也结合内皮细胞本身。然而，在与HUVEC培养物结合后，用对MPO特异的多克隆兔或人抗体无法检测到MPO，可能是因为MPO结合到了免疫球蛋白无法接近的位点。PR3与HUVEC培养物（细胞+基质）的结合受到胎牛血清和α1-抗胰蛋白酶的抑制，但苯甲基磺酰氟（PMSF）使PR3的酶活性失活并不影响PR3与HUVEC培养物的结合。MPO与HUVEC培养物的结合不受胎牛血清的影响。
