Elevated Circulating Levels of Thrombospondin-1 in Plasma Are Associated with Increased Circulating Levels of TGFb and Pro-Inflammatory Cytokines in Patients Afflicted with Rheumatoid Arthritis: Utilization of the Multiplexed Protein Profiling on Microarray by Rolling-Circle Amplification Technique.

Rheumatoid Arthritis (RA) is a chronic, autoimmune disease that affects a vast population worldwide with women being afflicted three times more than men. There is evidence for an increased risk of cardiovascular events in RA patients compared to the general population. These cardiovascular events may be associated with the chronic inflammatory state in which activation of coagulation leads to thrombin generation. Our laboratory has evidence that thrombospondin-1 (TSP1), an adhesive molecule that plays a major role in RA, promotes thrombin generation on the surface of a monocytic cell line (Isordia-Salas et al, Thromb Res.2005;116(6)). We also have documented that disrupting the TSP1 interaction on human neutrophils prevents the development of erosive arthritis in an experimental animal model (Manns et al, Arthritis and Rheumatism54(8), 2006). This observation is mediated by a novel pathway whereby TSP1 induces the up-regulation of Connective Tissue Growth Factor (CTGF). Therefore, to assess whether our in vitro and in vivo observations can be extrapolated to human disease, blood samples were collected from 20 patients afflicted with rheumatoid arthritis and 13 from healthy donors which served as the negative control. Plasma samples were separated and analyzed by Enzyme-Linked Immunosorbent Assay (ELISA) to determine the levels of transforming growth factor beta (TGF-β), protrombin F1+2 fragments (F1+2) and thrombospondin-1, and by multiplexed cytokine protein profiling on microarray by rolling-circle amplification (RCA) to determine cytokine levels. The F1+2 plasma levels showed an elevated trend in the RA group (p=0.06). TSP1 plasma levels were significantly increased in the RA group compared to the normal control (p=0.0004). Pro-inflammatory cytokine levels including IL-1β (p=0.0365), IL-6 (p=0.0029), TNF-α (p=0.0339), interferon-inducible protein 10 (p=0.0003) and macrophage inflammatory protein -1α (p=0.0012) were found elevated in the RA group compared to the normal control group. Some regulatory cytokines such as transforming growth factor-β (0.06), interferon-gamma (p=0.06) and IL-13 (p=0.22) showed no statistically significance between groups, but all of them showed a trend for higher circulation levels in plasma in the RA group. In summary, comparison between normal individuals and RA patients showed an increase in the levels of cytokines in the RA afflicted patients confirming what has been reported in the literature. We were able to correlate an increase in proteolytic factors and TSP1 levels in the RA patients with an increase of pro-inflammatory cytokines. Further studies are needed to elucidate why TSP1 acts as a pro-inflammatory molecule on the neutrophil surface of the RA patients.