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To clarify whether mouse monoclonal antibodies (mAb) against human thrombomodulin (TM), which react with human TM present on the endothelial cell (EC) surface, have anti–endothelial cell antibody (AECA) activity and influence antiinflammatory properties of human TM expressed on the EC surface in vitro.Three preparations of mouse mAb against human TM that react with different sites of the human TM epidermal growth factor–like domain were tested for their ability to 1) bind to ECs, 2) modulate cytokine secretion from ECs, EC adhesion molecule expression, and neutrophil adhesion to ECs, and 3) stimulate nuclear translocation of NF-κB through the degradation of cytoplasmic IκB in ECs. Recombinant human interleukin-1β (IL-1β) was used as a positive control, and mouse IgG1 and mouse IgG2a were used as negative controls.The 3 preparations of mouse mAb against human TM that bind to unfixed EC monolayers enhanced IL-6 and IL-8 secretion from ECs, up-regulated expression of endothelial leukocyte adhesion molecule 1, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 on EC monolayers, and enhanced neutrophil adhesion to ECs to a degree similar to that observed with IL-1β stimulation, but they did not induce the secretion of tumor necrosis factor α or IL-1β from ECs throughout the incubation period. The 3 preparations stimulated nuclear translocation of NF-κB through the degradation of cytoplasmic IκB. Mouse IgG1 and mouse IgG2a did not exhibit such effects.These results suggest the possibility that AECA can react with antigens such as TM that are present on the EC surface and activate ECs. Such events on ECs may lead to vascular inflammation and damage in patients with connective tissue diseases and vasculitis in which AECA are present.