Dose-dependent inhibition of leukocyte adhesion to endothelial cells.

Confluent monolayer cultures of HUVECs were treated with indicated drugs at different concentrations for 2 hours before labeled adhesion-indicator cells were added. Percentages of adherent Jurkat cells (A) were calculated in 4 independent experiments as average of triplicate determinations for each treatment condition when compared to untreated cultures after 16 hours of adhesion. Data points in panel A represent mean percentage ± SD of adherent Jurkat cells from the 4 experiments. CsA indicates cyclosporin A; keto, ketoconazole; lacta, lactacystin; rito, ritonavir. The treatment groups differed from each other P = .0018. Kruskal-Wallis test was performed for analysis of the means. Inhibition of adhesion of HL60 and U937 cells to HUVECs by ritonavir (B) was determined in 8 wells per treatment condition. All adherent cell HUVEC monolayers were activated with TNF-α (5 ng/mL) and subsequently treated with ritonavir at indicated concentrations for 4 hours. Calcein AM-labeled HL60 and U937 cells were dispensed on top of the HUVECs in fresh culture media and allowed to adhere for 30 minutes. Cells that remained adherent after exposure to minimal shear force were then read as fluorescence intensity and cell numbers were calculated based on standard curves for each cell type (mean values and SD are illustrated). Adhesion of U937 and HL60 in the treated samples differed from the untreated samples (P = .010 and P = .015, respectively). Wilcoxon rank-sum tests were performed for all comparisons of the means.