HRS cell-derived LTα-induced upregulation of adhesion molecule expression in HUVECs is dependent on NF-κB activation. (A) Incubation of HUVECs with C/S from KM-H2, L1236, and L428 cells but not L540 cells for 4 hours induced upregulation of ICAM-1, VCAM-1, and E-selectin to levels comparable to that seen with TNF-α–stimulated HUVECs. C/S-activated HUVECs incubated with purified mouse IgG (MuIgG) and anti-MHC class I mAb were used, respectively, as negative and positive controls. (B) (Upper) Enhanced p65 nuclear translocation in C/S-stimulated HUVECs is completely inhibited by pretreatment of the HUVEC monolayer with 20 μM Bay11-7085 for 30 minutes before C/S stimulation. (Lower) Bay11-7085 pretreatment also prevented the upregulation of ICAM-1, VCAM-1, and E-selectin expression on C/S-stimulated HUVECs. (C) Although C/S derived from KM-H2, L1236, and L428 exhibits significant cytotoxicity in L929 cells, L540-derived C/S did not induce any L929 cell death. (D) (Left) Neutralization of TNF-α in KM-H2 C/S did not improve L929 cell viability, (right) although the dosage used can effectively neutralize 10 ng/mL of recombinant TNF-α in vitro. (E) Neutralization of LTα in the various HRS cell-derived C/S successfully reduced their cytotoxic effects in L929 cells compared with untreated C/S and MuIgG-treated C/S. (F) The highest concentration of LTα was detected in L1236 C/S, whereas negligible amounts of LTα were present in L540-derived C/S. Values shown are mean ± standard error of the mean (SEM) from 3 experiments. *P ≤ .05 compared with unstimulated control or C/S-stimulated HUVECs for A and B; *P ≤ .05 compared with untreated C/S for C-F. Western blot is representative of 3 independent experiments.