Protein kinase C beta mediates CD40 ligand-induced adhesion of monocytes to endothelial cells

Abstract

Accumulating evidence supports the early involvement of monocyte/macrophage recruitment to activated endothelial cells by leukocyte adhesion molecules during atherogenesis. CD40 and its ligand CD40L are highly expressed in vascular endothelial cells, but its impact on monocyte adhesion and the related molecular mechanisms are not fully understood. The present study was designed to evaluate the direct effect of CD40L on monocytic cell adhesion and gain mechanistic insight into the signaling coupling CD40L function to the proinflammatory response. Exposure of cultured human aortic endothelial cells (HAECs) to clinically relevant concentrations of CD40L (20 to 80 ng/mL) dose-dependently increased human monocytic THP-1 cells to adhere to them under static condition. CD40L treatment induced the expression of vascular cell adhesion molecule-1 (VCAM-1) mRNA and protein expression in HAECs. Furthermore, exposure of HAECs to CD40L robustly increased the activation of protein kinase C beta (PKCβ) in ECs. A selective inhibitor of PKCβ prevented the rise in VCAM-1 and THP-1 cell adhesion to ECs. Moreover, stimulation of ECs to CD40L induced nuclear factor-κB (NF-κB) activation. PKCβ inhibition abolished CD40L-induced NF-κB activation, and NF-κB inhibition reduced expression of VCAM-1, each resulting in reduced THP-1 cell adhesion. Our findings provide the evidence that CD40L increases VCAM-1 expression in ECs by activating PKCβ and NF-κB, suggesting a novel mechanism for EC activation. Finally, administration of CD40L resulted in PKCβ activation, increased VCAM-1 expression and activated monocytes adhesiveness to HAECs, processes attenuated by PKCβ inhibitor. Therefore, CD40L may contribute directly to atherogenesis by activating ECs and recruiting monocytes to them.

Figure 1. CD40L induces the adhesion of THP-1 cells or human peripheral monocytes to ECs.

(A) HAECs were incubated with the indicated concentrations of CD40L for 24 h, and static adhesion assays were performed as detailed in Methods. Attached THP-1 cells were visualized and counted on an inverted fluorescent microscopy. Magnification, ×20. (B) Quantification of fluorescence density expressed as means ± SEM. ^* P<0.05 vs 0 ng/mL. (C) HAECs were incubated in the presence of (40 ng/mL) for the indicated hours, and then static adhesion assays were performed. ^*P<0.05 vs 0 h. (D) HAECs were incubated in the presence of PBS (control) or CD40L (80 ng/mL) for 24 h, and static adhesion assays were performed with the use of human peripheral monocytes. ^* P<0.05 vs control. (D) Platelets were activated as described and incubated with HAECs, then THP-1 cells adhesion was analyzed by static adhesion assays. ^* P<0.05 vs resting platelets.

Figure 2. CD40L induces expression of VCAM-1 in ECs.

(A and B) HAECs were incubated in the presence of indicated concentrations of for 24 hours. A, Total RNA was isolated and subjected to quantitative RT-PCR to analyze VCAM-1 mRNA levels. ^* P<0.05 or ^** P<0.01 vs 0 ng/mL. (B) VCAM-1 protein expression was determined by Western blot. Total cell lysates were subjected to SDS-PAGE and immunoblotting. Blots represent 4 independent experiments with similar results. (D) HAECs were pretreated with antibodies (50 μg/mL) for 30 minutes and then incubated in the presence of PBS (control) or CD40L (80 ng/mL) for 24 h, and static adhesion assays were performed. ^* P<0.05 vs CD40L. Data are representative of 4 independent experiments with similar results.

Figure 3. CD40L induces the accumulation of THP-1 cells on ECs under flow conditions.

(A and B) HAECs were incubated in the presence of CD40L (80 ng/mL) or PBS (control) for 24 h, and flow adhesion assays were performed at 37°C. In some experiments, HAECs were pretreated with anti–VCAM-1 antibody (50 μg/mL) for 30 minutes before assay. ^* P<0.05 vs CD40L. Photographs captured from microscope represent 3 independent experiments with similar results. ×20. (B) Quantification of attached THP-1 cells. ^* P<0.05 vs CD40L. Data are representative of 4 independent experiments with similar results.

Figure 4. Interaction of CD40 with CD40L mediates CD40L-enhanced VCAM-1 expression and THP-1 adhesion.

HAECs were transfected with vectors encoding the CD40 siRNA or control siRNA and then incubated with CD40L (80 ng/mL) for 24 h. (A) Representative blot showing CD40 protein expression in HAECs transfected with control or CD40 siRNA. (B) VCAM-1 protein expression and (C) THP-1 adhesion were determined as indicated. Data are shown as representative blots or are expressed as the means ± SEM by three independent assays from 4 independent experiments. ^* P<0.05 vs CD40L plus Control siRNA.

Figure 5. CD40L induces NF-κB activation.

(A) HAECs were transfected with NF-κB reporter for 24 h and then incubated with the indicated concentrations of CD40L for another 8 h. The luciferase activity was determined using β-gal as the control. Results of three independent experiments are expressed as fold of control. ^* P<0.05, ^** P<0.01 vs 0 ng/mL. (B and C) HAECs were stimulated with the indicated concentrations of CD40L for 2 h. Cells were lysed and the protein extracts were assayed for p65 DNA-binding activity. (C) IκB-α protein expression was measured by immunoblotting. (D) HAECs were infected with Ad-IκB, or Ad-GFP for 24 h and then incubated with CD40L (80 ng/ml) for another 24 h. VCAM-1 protein expression was determined by Western blot. The results were reproducible in 4 independent experiments. ^* P<0.05.

Figure 6. Effect of CD40L on NF-κB activation and IκBα degradation in ECs.

(A) HAECs were pretreated with PKCβ inhibitor (5 nM) for 30 minutes and then incubated in the presence of PBS (control) or CD40L (80 ng/mL) for 2 h. Cytosol and nuclear fractions were prepared to measure NF-κB p65 and IκB-α protein expression by immunoblotting. (B) HAECs were pretreated with PKCβ inhibitor (5 nM) for 30 minutes and then incubated in the presence of PBS (control) or CD40L (80 ng/mL) for 24 h. Total cell lysates were prepared and subjected to immunoblotting. Blots represent 4 independent experiments with similar results.

Figure 7. CD40L increases PKCβ phosphorylation and the translocation of PKCβ from cytosol to the membrane.

Confluent HAEC were exposed to CD40L (80 ng/mL, 1 h), and the translocation of PKCβ and PKCβ phosphorylation was assayed as described in Methods. (A) CD40L increased the phosphorylation of PKCβ in HAEC. The blot is a representative of 3 blots obtained from 3 independent experiments. (B) PKCβ activity was determined as described in Methods (n = 4). ^* P<0.05 vs CD40L plus GFP. (C) CD40L increases the translocation of PKCβ from cytosol into the nucleus. The blot is a representative of 3 blots from 3 individual experiments. (D) Analysis of the purity of subcellular fractions. The subcellular fractions were prepared as described in Methods. Marker enzymes were detected by Western blot with the use of specific antibodies.

Figure 8. PKCβ mediates CD40L induced mice monocyte activation.

C57BL/6J mice were injected IV with CD40L (1.5 mg/kg/d) from femoral veins. After 3 days, monocytes were isolated from plasma. In some experiments, monocytes were isolated from plasma. (A) Representative blots showing PKCβ activation in the aortas. (B) VCAM-1 expression. (C) Monocytes were isolated from plasma and adhesion was assayed as indicated. The results are representative of 6 mice in each group.