Background Vascular remodeling promotes immune cell infiltration and provides nutrients to hyperplastic tissue in the synovial tissue of patients with rheumatoid arthritis (RA). In solid cancers, angiopoietin (Ang-1 and Ang-2) signaling to Tie2-expressing immunosuppressive monocytes (TEMs) is critical in allowing tumor establishment. A similar role for TEMs in chronic inflammatory disease has not been assessed, but we have recently found that macrophages are the primary cell types in which Tie2 is activated in RA synovial tissue.
Objectives The aim of this study was to examine how complex differentiation stimuli present in the RA synovial microenvironment might regulate expression of Tie2 on macrophages, as well as their effects on expression of macrophage angiogenic factors and macrophage gene expression responses to Ang-1 stimulation.
Methods Human peripheral blood mononuclear cells (PBMCs) were isolated from blood buffy coats and monocytes were differentiated into macrophages in the presence of the pro-inflammatory/classically activating cyokines GM-CSF (5 ng/ml) and interferon- gamma (IFN-γ, 10 ng/ml), in the presence of the anti-inflammatory/alternatively activating cytokines M-CSF (25 ng/ml) or interleukin-10 (IL-10, 10 ng/ml), or in the presence of 20% RA patient synovial fluid (RA SF). At day 6, Tie-2 expression was analyzed by flow cytometry and quantitative PCR. At day 7, differentiated macrophages were stimulated with TNF (10 ng/ml) in the presence or absence of Ang-1 (200 ng/ml) for 4 hours and cells were harvested, and total RNA was isolated and reverse-transcribed to cDNA. Macrophage mRNA expression of 84 angiogenic factors was analyzed using low density quantitative PCR Array.
Results Tie2 expression was maintained following macrophage differentiation in RA SF, and expression levels were similar to those observed in GM-CSF, IFN-γ, M-CSF and IL-10 differentiated macrophages. Gene expression analysis of angiogenic factors demonstrated distinct expression profiles under each polarization condition, although macrophages differentiated in RA SF showed significantly enhanced expression of pro-angiogenic chemokines (C-X-C motif) ligands 3, 5 and 6 (CXCL3, 5 and 6), interleukin-8 (IL-8) and chemokine (C-C motif) ligand 2 (CCL2) and decreased expression of anti-angiogenic chemokines (C-X-C motif) ligands 2, 9, 10 and 11 (CXCL2, 9, 10 and 11) (p values ranging from <0.05 to 0.0005 for each chemokine). TNF-treatment of RA SF-differentiated macrophages enhanced chemokine expression, and synergistic effects of TNF and Ang-1 were observed in promoting CXCL3, 6, and IL-8 expression.
Conclusions Our results suggest that the RA synovial environment promotes pro-angiogenic macrophage differentiation, and these Tie2-expressing synovial macrophages can further potentiate inflammation through the production of chemokines in response to TNF and Ang-1 which perpetuate synovial monocyte influx.
Disclosure of Interest None Declared
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