NF-kappaB activity in endothelial cells is modulated by cell substratum interactions and influences chemokine-mediated adhesion of natural killer cells

Abstract

Because changes in subendothelial matrix composition are associated with alterations of the endothelial immune phenotype, we sought to understand if cytokine-induced NF-kappaB activity and downstream effects depend on substrate adherence of endothelial cells (EC). We compared the upstream phosphorylation cascade, activation of NF-kappaB, and expression/secretion of downstream effects of EC grown on tissue culture polystyrene plates (TCPS) with EC embedded within collagen-based matrices (MEEC). Adhesion of natural killer (NK) cells was quantified in vitro and in vivo. NF-kappaB subunit p65 nuclear levels were significantly lower and p50 significantly higher in cytokine-stimulated MEEC than in EC-TCPS. Despite similar surface expression of TNF-alpha receptors, MEEC had significantly decreased secretion and expression of IL-6, IL-8, MCP-1, VCAM-1, and ICAM-1. Attenuated fractalkine expression and secretion in MEEC (two to threefold lower than in EC-TCPS; p < 0.0002) correlated with 3.7-fold lower NK cell adhesion to EC (6,335 +/- 420 vs. 1,735 +/- 135 cpm; p < 0.0002). Furthermore, NK cell infiltration into sites of EC implantation in vivo was significantly reduced when EC were embedded within matrix. Matrix embedding enables control of EC substratum interaction. This in turn regulates chemokine and surface molecule expression and secretion, in particular of those compounds within NF-kappaB pathways, chemoattraction of NK cells, local inflammation, and tissue repair.

Figure 1

Matrix embedding influences TNF-α-induced NF-κB activation in endothelial cells. (A) Real-time PCR fold change (ΔΔCt) values of IκBα, and REL-A (p65) for MEEC (opened histogram) and EC-TCPS (closed histogram) stimulated for 6 h with 5 ng/ml TNF-α. (B) Protein levels in the EC nucleus of p65 and p50, after 6-h stimulation with 5 ng/ml TNF-α in MEEC (opened histogram) and EC-TCPS (closed histogram). (C) Western blots of phosphorylated IKKα/β in EC-TCPS (levels were not detected in MEEC). (D) Western blots of phosphorylated IκBα over time in EC grown on TCPS. Western blots are representative for three independent experiments; equal loading was verified with β-actin.†p < 0.002, ‡p < 0.02, ≠p < 0.008, *p < 0.0001.

Figure 2

Expression patterns of TNF-α receptors subunits I and II are similar between EC-TCPS and MEEC. EC were stained with antibodies against both subunits of TNF-α receptors. For intracellular staining cells were pretreated with brefeldin A and Triton X-100. Bound primary antibodies were visualized by the addition of specific Alexa Fluor 488-conjugated secondary antibodies and analyzed by confocal laser-scanning microscope (100× magnification).

Figure 3

EC–substratum interaction influences NF-κB-dependent expression and secretion of proinflammtory mediators. (A) Real-time PCR ΔΔCt values for MCP-1, IL-6, and IL-8 following 6-h stimulation with 5 ng/ml TNF-α. (B) Protein levels of MCP-1, IL-6, and IL-8 following 6-h stimulation with 5 ng/ml TNF-α with and without SC514 100 µM, a IKK-2 inhibitor applied to EC-TCPS. §p < 0.0001, fp < 0.02, ‡p < 0.001, *p < 0.05, †p < 0.01.

Figure 4

EC–substratum interaction influences NF-κB-dependent expression of adhesion molecules. Real-time PCR ΔΔCt of (A) VCAM-1 and (B) ICAM in MEEC (opened histogram) and EC-TCPS (closed histogram) stimulated for 6 h with 5 ng/ml TNF-α. Flow cytometry analysis of (C) VCAM and (D) ICAM expression on embedded EC (solid line), TCPS (dashed line), and TCPS with SC514 100 µM (dotted line) following 24-h stimulation with TNF-α. ≠p < 0.008, *p < 0.05.

Figure 5

Matrix embedding attenuates induction of fractalkine mRNA expression in EC. Fractalkine mRNA expression was normalized to GAPDH mRNA expression. (A) The fractalkine/GAPDH ratio for all treatment conditions was expressed as fold induction or repression of the fractalkine/GAPDH ratio determined for stimulation of MEEC with 100 U/ml TNF-α and IFN-γ for 4 h. Data are presented as mean ± SD from eight different experiments. (B) Cytokine stimulation induces significantly lower endothelial fractalkine expression when EC are matrix embedded. Intracellular expression of fractalkine on EC was analyzed by flow cytometry. Ten thousand cells were analyzed. Data are expressed as mean percentage ± SD of EC from eight different experiments at each time point; MEEC secrete lower amounts of soluble fractalkine. (C) Detection of secreted fractalkine levels in supernatants of cytokine-stimulated EC was performed with ELISA. Each value represents mean ± SD from eight different experiments for the denoted sampling period of 4 h. (D) Spearman correlation of soluble fractalkine levels (sCX3CL1) and percentage of EC expressing fractalkine intracellular (CX3CL1⁺-EC). Area of the density ellipse represents the 95% confidence interval.

Figure 6

NK cell binding to EC is reduced when EC are matrix embedded. EC were grown to confluence on polystyrene plates or in Gelfoam matrices, activated with 100 U TNF-α and IFN-γ for 20 h. (A) Attachment of NK cells to cytokine-stimulated EC was visualized via immunofluorescence microscopy (10×) and quantified with attachment of ⁵¹Cr-labeled NK cells (0.5 × 10⁶/well). Labeled cells were resuspended in medium alone or medium containing 20 µg/ml anti-CX3CR1 antibody and added to the EC under gentle rocking conditions (10 cycles/min). After 30 min the medium was decanted and the wells were gently washed. Adherent cells were lysed by treating with 1% Triton in PBS. (B) Total binding was determined by measuring individual well-associated cpm using a gamma counter. Each value represents mean ± SD from eight different experiments.

Figure 7

MEEC chemoattract fewer NK cells to implantation sites of EC. EC were implanted in the subcutaneous dorsal space of Sprague-Dawley rats as either matrix-embedded or saline-suspended cell pellets. On the 28th postoperative day, marked rat NK cell infiltration was noted in and around the injection site of saline-suspended EC pellets. Only few NK cells (blue, black arrows) were found in and around the implantation site of MEEC.

Figure 8

Wet SEM image of monocytes (indicated with black arrows) incubated on a collagen-based matrix for 2 h at 37°C, indicating the lack of a physical barrier for monocyte penetration.