Figure 2.
Figure 2. Effect of CD40 gene silencing by siRNA-2 on HUVEC leukocyte adhesion. CD40 gene silencing by siRNA-2 prevents CD154-mediated induction of leukocyte adhesion in human endothelial cells. (A) RNAi-mediated inhibition of CAM expression on CD154-activated endothelial cells. HUVECs, either untransfected or transfected for 2 hours with 100 nM siRNA-2 or msiRNA-2 as indicated in “Study Design,” were activated by coincubation with Jurkat D1.1 (CD154+) cells (ATCC CRL 1095; a Jurkat T cell subclone that constitutively expresses CD154) at a T/EC ratio of 5:1 at 6 hours (E-selectin) or 16 hours (ICAM-1 and VCAM-1) prior to analysis. At 48 hours after transfection, the cocultures were washed extensively with warm phosphate-buffered saline (PBS) to release the attached Jurkat D1.1 cells from the HUVEC monolayer. The endothelial cells were analyzed for CAM expression in the MoFlo flow cytometer (DakoCytomation, Fort Collins, CO) and were readily distinguished from residual T cells by light scatter. In i-iii, solid-line histograms represent basal CAM expression from untreated HUVECs, and filled histograms represent CAM expression from D1.1 (CD154+)–stimulated, untreated HUVECs. In iv-vi, solid-line histograms represent CAM expression from D1.1 (CD154+)–stimulated, siRNA-2–transfected HUVECs, and filled histograms represent CAM expression from D1.1 (CD154+)–stimulated, msiRNA-2–transfected HUVECs. Dotted-line histograms display the corresponding immunoglobulin G (IgG) isotype-matching controls in each panel. Bar diagrams quantify the percentage of CAM expression after siRNA treatment, calculated as follows: ((CAM expression for siRNA–treated, D1.1 (CD154+)–induced cells) - (basal CAM expression)) / ((D1.1 (CD154+)–induced CAM expression) - (basal CAM expression)) × 100. CAM expression data are the mean fluorescence intensity (MFI) values of the corresponding histograms from 3 independent experiments (*P < .05, versus msiRNA-2). (B) RNAi-mediated inhibition of leukocyte adhesion on CD154-activated, but not in TNF-α–activated, endothelial cells. HUVECs, either untransfected or transfected for 2 hours with 100 nM siRNA-2 or msiRNA-2 as indicated in “Study Design,” were activated either by coculture with Jurkat D1.1 (CD154+) cells at a T/EC ratio of 5:1 (iv-vi), or by incubation with TNF-α (300 U/mL) (vii-ix) at 16 hours prior to the assay. At 48 hours after transfection, the cocultures were washed extensively with warm PBS to release the attached Jurkat D1.1 cells from the HUVEC monolayer, which was further coincubated with HL-60 cells for 30 minutes (2 × 106 cells per well from 6-well plate, in 2 mL endothelial cell culture medium). Supernatant aspiration plus 2 rounds of brief washing with PBS removed nonadherent cells. Shown are phase-contrast microscopic images of PBS-washed, adherent cells taken at × 40 magnification. The images were acquired as monochromatic JPEG files using a SPOT camera and SPOT 3.2.4 software (Diagnostic Instruments, Sterling Heights, MI) mounted on an Olympus IX-70 inverted microscope (Olympus America, Melville, NY) and linked to an iMac G4 Apple computer (Apple Computers, Cupertino, CA). Controls refer to untreated HUVEC monolayer (i); untreated HUVECs coincubated with HL-60 cells (ii); untreated Jurkat D1.1 (CD154+)–activated HUVECs (iii). (C) To quantify the percentage of leukocyte adhesion to siRNA-2–transfected HUVECs, the same assay as in panel B was performed with the use of calcein AM–loaded HL-60 cells (5 μM calcein AM for 60 minutes at 37°C). Finally, attached cells were harvested by mild trypsinization and the ratio of adherent, fluorescence-labeled HL-60 cells to unlabeled endothelial cells was determined by flow cytometry after counting 10 000 events per sample. Shown are density plots corresponding to the following: unlabeled HL-60 cells (i); calcein AM–loaded HL-60 cells (ii); calcein AM–loaded HL-60 cells plus siRNA-2–transfected HUVEC mix (iii); calcein AM–loaded HL-60 cells plus msiRNA-2–transfected HUVEC mix (iv), all from one representative assay. The mean ratio obtained from msiRNA-2–treated HUVECs was assigned a 100% adhesion. Data are from 5 independent experiments (*P < .05, versus msiRNA-2).

Effect of CD40 gene silencing by siRNA-2 on HUVEC leukocyte adhesion. CD40 gene silencing by siRNA-2 prevents CD154-mediated induction of leukocyte adhesion in human endothelial cells. (A) RNAi-mediated inhibition of CAM expression on CD154-activated endothelial cells. HUVECs, either untransfected or transfected for 2 hours with 100 nM siRNA-2 or msiRNA-2 as indicated in “Study Design,” were activated by coincubation with Jurkat D1.1 (CD154+) cells (ATCC CRL 1095; a Jurkat T cell subclone that constitutively expresses CD154) at a T/EC ratio of 5:1 at 6 hours (E-selectin) or 16 hours (ICAM-1 and VCAM-1) prior to analysis. At 48 hours after transfection, the cocultures were washed extensively with warm phosphate-buffered saline (PBS) to release the attached Jurkat D1.1 cells from the HUVEC monolayer. The endothelial cells were analyzed for CAM expression in the MoFlo flow cytometer (DakoCytomation, Fort Collins, CO) and were readily distinguished from residual T cells by light scatter. In i-iii, solid-line histograms represent basal CAM expression from untreated HUVECs, and filled histograms represent CAM expression from D1.1 (CD154+)–stimulated, untreated HUVECs. In iv-vi, solid-line histograms represent CAM expression from D1.1 (CD154+)–stimulated, siRNA-2–transfected HUVECs, and filled histograms represent CAM expression from D1.1 (CD154+)–stimulated, msiRNA-2–transfected HUVECs. Dotted-line histograms display the corresponding immunoglobulin G (IgG) isotype-matching controls in each panel. Bar diagrams quantify the percentage of CAM expression after siRNA treatment, calculated as follows: ((CAM expression for siRNA–treated, D1.1 (CD154+)–induced cells) - (basal CAM expression)) / ((D1.1 (CD154+)–induced CAM expression) - (basal CAM expression)) × 100. CAM expression data are the mean fluorescence intensity (MFI) values of the corresponding histograms from 3 independent experiments (*P < .05, versus msiRNA-2). (B) RNAi-mediated inhibition of leukocyte adhesion on CD154-activated, but not in TNF-α–activated, endothelial cells. HUVECs, either untransfected or transfected for 2 hours with 100 nM siRNA-2 or msiRNA-2 as indicated in “Study Design,” were activated either by coculture with Jurkat D1.1 (CD154+) cells at a T/EC ratio of 5:1 (iv-vi), or by incubation with TNF-α (300 U/mL) (vii-ix) at 16 hours prior to the assay. At 48 hours after transfection, the cocultures were washed extensively with warm PBS to release the attached Jurkat D1.1 cells from the HUVEC monolayer, which was further coincubated with HL-60 cells for 30 minutes (2 × 106 cells per well from 6-well plate, in 2 mL endothelial cell culture medium). Supernatant aspiration plus 2 rounds of brief washing with PBS removed nonadherent cells. Shown are phase-contrast microscopic images of PBS-washed, adherent cells taken at × 40 magnification. The images were acquired as monochromatic JPEG files using a SPOT camera and SPOT 3.2.4 software (Diagnostic Instruments, Sterling Heights, MI) mounted on an Olympus IX-70 inverted microscope (Olympus America, Melville, NY) and linked to an iMac G4 Apple computer (Apple Computers, Cupertino, CA). Controls refer to untreated HUVEC monolayer (i); untreated HUVECs coincubated with HL-60 cells (ii); untreated Jurkat D1.1 (CD154+)–activated HUVECs (iii). (C) To quantify the percentage of leukocyte adhesion to siRNA-2–transfected HUVECs, the same assay as in panel B was performed with the use of calcein AM–loaded HL-60 cells (5 μM calcein AM for 60 minutes at 37°C). Finally, attached cells were harvested by mild trypsinization and the ratio of adherent, fluorescence-labeled HL-60 cells to unlabeled endothelial cells was determined by flow cytometry after counting 10 000 events per sample. Shown are density plots corresponding to the following: unlabeled HL-60 cells (i); calcein AM–loaded HL-60 cells (ii); calcein AM–loaded HL-60 cells plus siRNA-2–transfected HUVEC mix (iii); calcein AM–loaded HL-60 cells plus msiRNA-2–transfected HUVEC mix (iv), all from one representative assay. The mean ratio obtained from msiRNA-2–treated HUVECs was assigned a 100% adhesion. Data are from 5 independent experiments (*P < .05, versus msiRNA-2).

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