Adipokines and Inflammation Alter the Interaction Between Rheumatoid Arthritis Synovial Fibroblasts and Endothelial Cells

Abstract

Objective: The long-distance migration of rheumatoid arthritis synovial fibroblasts (RASFs) in the severe combined immunodeficiency (SCID) mouse model of rheumatoid arthritis (RA) suggests that an interaction between RASFs and endothelial cells (EC) is critical in this process. Our objective was to assess whether immunomodulatory factors such as adipokines and antirheumatic drugs affect the adhesion of RASFs to ECs or the expression of surface molecules. Methods: Primary ECs or human umbilical vein endothelial cell (HUVEC) and primary RASFs were stimulated with adiponectin (10 μg/mL), visfatin (100 ng/mL), and resistin (20 ng/mL) or treated with methotrexate (1.5 and 1,000 μM) and the glucocorticoids prednisolone (1 μM) and dexamethasone (1 μM), respectively. The expression of adhesion molecules was analyzed by real-time polymerase chain reaction. The interaction of both cell types was analyzed under static (cell-to-cell binding assay) and dynamic conditions (flow-adhesion assay). Results: Under static conditions, adipokines increased mostly binding of RASFs to EC (adiponectin: 40%, visfatin: 28%, tumor necrosis factor α: 49%). Under flow conditions, visfatin increased RASF adhesion to HUVEC (e.g., 0.5 dyn/cm²: 75.2%). Reduced adhesion of RASFs to E-selectin was observed after treatment with dexamethasone (e.g., 0.9 dyn/cm²: -40%). In ECs, tumor necrosis factor α (TNF-α) increased expression of intercellular adhesion molecule 1 (20-fold) and vascular cell adhesion molecule 1 (77-fold), whereas P-selectin was downregulated after stimulation with TNF-α (-6-fold). Conclusion: The adhesion of RASFs to EC was increased by visfatin under static and flow conditions, whereas glucocorticoids were able to decrease adhesion to E-selectin. The process of migration and adhesion of RASFs to ECs could be enhanced by adipokines via adhesion molecules and seems to be targeted by therapeutic intervention with glucocorticoids.

Keywords: adipokines; endocrine; endothelium; fibroblast; inflammation; rheumatoid arthritis.

Figure 1

Experimental setup of the flow-adhesion assay. Capillaries (E) were monitored microscopically (A). Flow rates of RASF-containing suspensions were regulated by a syringe pump (B). The pump was connected to the capillaries by a tube. Another tube was connected to a collection vessel (D) after passing through the capillaries. Synovial fibroblast migration was evaluated by three video sequences per setting (C).

Figure 2

VCAM-1 and ICAM-1 expression by RASFs after stimulation with selected adipokines and therapeutics. Results were compared to non-stimulated controls. (A) mRNA expression of VCAM-1 by RASFs after stimulation with adiponectin (n = 5), visfatin (n = 10), resistin (n = 10), TNF-α (n = 9), or therapeutics (n = 8 each). Adipokines and methotrexate did not have any effect on expression of VCAM-1, whereas TNF-α upregulated expression of VCAM-1 [16.4-fold (rhombus), 95% CI = 4.9–55], but not significantly. Dexamethasone (−5.09-fold, 95% CI = 0.095–0.408) and prednisolone [−3.2-fold (rhombus), 95% CI = 0.14–0.717] downregulated expression of VCAM-1. (B) mRNA expression of ICAM-1 by RASFs after stimulation with adiponectin (n = 4), visfatin (n = 7), resistin (n = 7), TNF-α (n = 5), and therapeutics (each n = 5). Adipokines and therapeutics did not have any effect on expression of ICAM-1. Tumor necrosis factor α significantly upregulated expression of ICAM-1 [20.4-fold (rhombus), 95% CI = 6.07–68].

Figure 3

VCAM-1, ICAM-1, and P-selectin expression by EC after stimulation with selected adipokines and therapeutics. Results were compared to non-stimulated controls. (A) mRNA expression of VCAM-1 by primary EC after stimulation with adiponectin (n = 5), visfatin (n = 10), resistin (n = 10), TNF-α (n = 9), or therapeutics (n = 8 each). Adipokines and therapeutics did not have any effect on expression of VCAM-1, whereas TNF-α significantly upregulated expression of VCAM-1 [76.8-fold (rhombus), 95% CI = 11.8–499]. (B) mRNA expression of ICAM-1 by primary EC after stimulation with adiponectin (n = 6), visfatin (n = 9), resistin (n = 9), TNF-α (n = 8), glucocorticoids (n = 4 each), or methotrexate (n = 5 each). Adiponectin led to a significant increased expression of ICAM-1 [−2.5-fold (rhombus), 95% CI = 0.26–0.6]. Resistin visfatin and therapeutics did not have any effect on expression of ICAM-1, whereas TNF-α significantly upregulated expression of ICAM-1 [37.02-fold (rhombus), 95% CI = 15–91.4]. (C) mRNA expression of P-selectin by primary EC after stimulation with adiponectin (n = 5), visfatin (n = 8), resistin (n = 8), TNF-α (n = 7), glucocorticoids (each n = 3), or methotrexate (each n = 4). Adipokines and therapeutics did not have any effect on expression of P-selectin. Tumor necrosis factor α significantly downregulated expression of P-selectin [−6.3-fold (rhombus), 95% CI = 0.069–0.37].

Figure 4

Evaluation of RASFs binding to EC. A cell-to-cell binding assay using primary EC was used to evaluate the effect of the adipokines adiponectin, visfatin, resistin, and TNF-α (n = 8 each), glucocorticoids (prednisolone, dexamethasone, n = 7 each), and methotrexate (n = 6 each dosage). Unstimulated RASFs served as control. The percentage of change in adherent RASFs compared to unstimulated RASFs was calculated. Data in figure are shown in percentages as dot plots with estimated marginal means. Stimulation with selected adipokines increased and prednisolone decreased adhesion to EC in most of the cell samples (adiponectin: 26%, visfatin 19%, resistin 17%, prednisolone: −15%, NS).

Figure 5

Rheumatoid arthritis synovial fibroblast adhesion to E-selectin and HUVEC under flow conditions. A flow adhesion assay was used to evaluate the effect of the selected adipokines visfatin, resistin, TNF-α (A & C) and therapeutics prednisolone, dexamethasone and methotrexate (B & D) to E-selectin and HUVEC (each n = 6). Unstimulated RASF served as control. The percentage of change in adherent RASF compared to unstimulated RASF was calculated. (A) Visfatin increased adhesion to E-Selectin in most of the samples (NS). (B) Stimulation with dexamethasone significantly (p = 0.043) decreased adhesion to E-selectin coated capillaries (8.4 ml/h: −40.9%, 30.5 ml/h: −40%, 60.5 ml/h: −29.7%). (C) Significant increase (p = 0.002) of adhesion to HUVEC could be observed after stimulation with visfatin (18.4 ml/h: 75.2%, 30.5 ml/h: 37.9%, 60.5 ml/h: 49.8%). (D) Stimulation with therapeutics did not reach any significant change in adhesion.