Background Tie2 is a tyrosine kinase receptor with an essential role in blood vessel remodelling and angiogenesis, through its activation by angiopoietin (Ang)-1 and Ang-2. We have found that Tie2 is mainly expressed and activated in RA synovial macrophages, that Ang-2 plays a crucial role in the murine arthritis and that Tie2 signalling promotes the inflammatory activation of human macrophages. However, the effect of Tie2 signalling on macrophages in the RA synovial environment has not been assessed yet. The aim of this study was examine how differentiation stimuli present in the RA and psoriatic arthritis (PsA) synovial microenvironment regulate Tie2 expression and signalling on macrophages, and the role of Tie2 signalling in macrophages from RA and PsA patients.
Material and methods Monocytes were isolated from blood buffy coats (HD) and from RA and PsA patients and differentiated into macrophages with IFN-γ (10 ng/ml), IL-10 (10 ng/ml), or with 20% synovial fluid from RA or PsA patients (RA SF and PsA SF). Tie2 expression was analysed by flow cytometry and quantitative PCR. Macrophages were stimulated with TNF (10 ng/ml) in the presence or absence of Ang-1 or Ang-2 (200 ng/ml) for 4 h, the RNA was isolated and reverse-transcribed to cDNA. Macrophage mRNA expression of inflammatory mediators and angiogenic factors was analysed by qPCR and qPCR Array.
Results Tie2 protein and mRNA expression was observed in RA and PsA SF macrophages and expression levels where similar those found in IL-10 and IFN-g HD macrophages. Gene expression analysis of angiogenic factors demonstrated distinct expression profiles under each condition, although unsupervised clustergram analysis demonstrated a high relationship between IL-10, RA SF and PsA SF macrophages. TNF stimulation of RA SF and PsA SF macrophages enhanced expression of the pro-inflammatory cytokines IL-6, IL-8 and TNF and the chemokines CCL-2, CXCL-2, -3, -5, -6 and -10. Ang-1 and Ang-2 stimulation significantly synergized with TNF to promote IL-6, IL-8, CCL-2, CXCL-2, -3, and -6 expression. In RA and PsA macrophages differentiated in IL-10 and IFN-g, Ang-1 and Ang-2 stimulation significantly synergized with TNF to enhance the expression of IL-6, IL-12B and IL-8 and the chemokines CCL-2 and CXCL-6.
Conclusion Tie2 is functionally expressed by both RA and PsA macrophages. In RA and PsA SF-differentiated macrophages, and IL-10 and IFN-g -differentiated macrophages from RA and PsA patients, Angiopoietin stimulation enhances TNF induced pro-inflammatory cytokine and chemokine expression. These results suggest that Tie2 signalling, in combination with TNF, induces a pro-inflammatory profile in arthritic macrophages, and provides a molecular basis for the role of Tie2 signalling in promoting disease progression in both early and established RA.