Tetraspanin interference affects ICAM-1 and VCAM-1 expression and function. (A) Flow cytometry analysis of the expression of adhesion molecules ICAM-1 (HU5/3), VCAM-1 (P8B1), E-Selectin (TEA2/1), and CD44 (HP2/9) in tetraspanin siRNA-treated HUVECs. TNF-α–activated negative siRNA-treated cells are shown in thin lines. Thick lines correspond to the expression in TNF-α–activated tetraspanin siRNA-transfected cells. Expression of the different adhesion molecules in resting cells is shown in dotted lines. The histograms are depicted in a logarithmic scale. (B) Quantitative analysis of the mean fluorescence intensity of ICAM-1, VCAM-1, and E-selectin in TNF-α–activated tetraspanin siRNA-transfected cells. Data represent the mean of 2 independent experiments ±SD as the percentage of the expression referred to that of TNF-α–activated negative control siRNA-transfected cells. (C) Analysis of the adhesion of PBLs to tetraspanin-interfered cells under static conditions. PBLs were loaded with BCECF-AM (2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester) fluorescent probe and allowed to adhere in serum-free medium on confluent TNF-α–activated HUVEC monolayers for 15 minutes at 37°C. After washing, the percentage of adhesion was quantified in a fluorimeter and depicted as the mean ±SD with respect to the adhesion levels of the negative siRNA-transfected cells in 3 different experiments performed in triplicate. (D) siRNA-transfected HUVEC monolayers were seeded onto Transwell insets and activated with 20 ng/mL of TNF-α for 20 hours. Then, human peripheral blood lymphocytes were added to the upper compartment and SDF-1α–containing medium (100 ng/mL) was added to the lower compartment. Cells were allowed to migrate for 2 hours and analyzed by flow cytometry. Data represent the mean ±SD of a representative experiment performed in triplicate. ^**P < .005 in a Student t test. (E) Tetraspanin interference augments PBL detachment under shear stress. The percentage (mean ±SD) of remaining adherent cells is represented for the different flow rates in 4 fields of a representative experiment. ▪ indicates negative siRNA; ▴, CD9 siRNA; and ×, CD151 siRNA. ^*P < .02; ^**P < .005 in a Student t test. (F) Effect of tetraspanin siRNA on lymphocyte adhesion and transmigration under flow conditions. Activated endothelium transfected with specific siRNA oligos for tetraspanins CD9 or CD151 or the negative control oligonucleotide were activated with TNF-α. Thereafter, PBLs were allowed to adhere and transmigrate under physiologic flow conditions (1.8 dyn/cm²) for 10 min. Quantification of rolling, adhesion, transmigration, and detachment events was performed from minute 3.5 to minute 6.5 of perfusion. Values correspond to the arithmetic mean ±SEM of the total number of PBLs interacting with the endothelial monolayer in the 6 different fields analyzed (× 20 objective) from a representative experiment. The total number of PBLs (n) interacting with the HUVEC monolayer in each condition is depicted in the legend of the x-axis. ^*P < .02 in a Student t test.