Monoclonal anti-endothelial cell antibodies from a patient with Takayasu arteritis activate endothelial cells from large vessels

Objective. To create monoclonal anti-endothelial cell antibodies (mAECA) fr om a patient with Takayasu arteritis to evaluate their ability to activate human umbilical vein endothelial cells (HUVEC), and to characterize the mec hanism of EC activation. Methods, A panel of mAECA was generated from peripheral blood lymphocytes o f a patient with Takayasu arteritis, using Epstein-Barr virus transformatio n. Activity against macrovascular EC (HUVEC) and microvascular EC (human bo ne marrow EC immortalized by SV40) antigens was detected by enzyme-linked i mmunosorbent assay. Inhibition studies were used to select the monoclonal a ntibodies (mAECA) which share the same EC epitope binding specificity as th e total IgG-AECA from the Takayasu arteritis patient. The binding of the mA ECA to human aortic EC was studied by immunohistochemistry. The secretion l evels of interleukin-6 (IL-6) and von Willebrand factor (vWF) were determin ed, to serve as markers for EC activation. The activated EC were examined f or the adherence of a monocytic cell line (U937), as well as for expression of vascular cell adhesion molecule 1, intercellular adhesion molecule 1, a nd E-selectin, In addition, nuclear extracts of the mAECA-treated EC were a nalyzed for the induction of translocation of nuclear factor kappa B (NF-ka ppa B), using a specific NF-kappa B oligoprobe in an electrophoretic mobili ty shift assay. Results. Six mAECA were selected, the mixture of which produced 100% inhibi tion of binding of the original IgG (from the patient with Takayasu arterit is) to HUVEC. All mAECA possessed high activity against macrovascular EC, b ut none had significant anti-microvascular EC activity. The mAECA, but not normal human IgG, had anti-human aortic EC activity. Four of the 6 mAECA ac tivated EC, manifested by increased IL-6 and vWF secretion. The 4 mAECA ind uced EC expression of adhesion molecules and increased adhesion of U937 mon ocytic cells to EC, In addition, these mAECA stimulated the nuclear translo cation of the NF-kappa B transcription factor. Conclusion. Our findings suggest that AECA may directly stimulate EC in Tak ayasu arteritis through elevation of adhesion molecule expression associate d with NF-kappa B activation and adhesion of monocytes, and may therefore p lay a pathogenic role in the development of the vasculopathy in Takayasu ar teritis.