Baseline clinical and demographics features
A total of 96 consecutive patients with RA were recruited, from which 40 serum samples (35 women and 5 men, mean age 51.80 ±13.37 years) and 56 synovial samples (43 women and 13 men, mean age 48.85±13.39 years) were obtained. Patients characteristics are displayed in Table 1, below.
Cytokines and chemokines levels in patients with RA
At baseline, there were no differences in IL-33, MIG and IL-6 concentrations between the synovial and serum samples of RA patients (Figure 1 A, B and D). IP-10 concentration was significantly higher in synovial fluid than in serum (Figure 1 C). IL-8, TNF-α and IL-β concentrations were significantly higher in serum than in synovial fluid (Figure 1 E, F and G).
IL-33 concentration follows an inverted-U-shaped curve in response to cytokines and chemokines
IL-33 concentration exhibited an inverted-U-shaped curve in response to IL-6, IL-1β, CXCL8 (IL-8), CXCL9 (MIG) and CXCL10 (IP-10) but not TNF-α in synovial fluid of RA patients (Figure 2). The same IL-33 inverted-U-shaped correlation was observed after treatment with CXCL9 (MIG) but not CXCL10 (IP-10), TNF-α, IL-6, IL-1β or CXCL8 (IL-8) in the serum of RA patients (Figure 3).
mRNA and protein expression levels of IL-6, CXCL 8, CXCL9, CXCL10, IL-1β and TNF-α in FLS stimulated by IL-33
IL-33 affected the mRNA expression of IL-6, CXCL8, CXCL9, CXCL10, IL-1β and TNF-α in FLS. However, only protein expression of IL-6, IL-8, CXCL10 and TNF-α was detectable in the supernatants of the FLS cell culture. CXCL9 and IL-1β protein expression was not detected in the supernatants of FLSs, even at a low level. IL-33 affected both mRNA and proteins expression of IL-6 in FLS. A high response of FLS at a concentration of 50ng/ml IL-33 suggests that it has a narrow working concentration on FLSs (Figure 4 A). A greater response at 4h than 24h suggests that IL-33 is effective over a precise time frame in FLSs. Over time, the effect cof IL-33 on IL-6 secretion by FLS changed (Figure 4 B). Different to IL-6, the expression of IL-8 reached a peak after 24h at a concentration of 50ng/ml of IL-33 (Figures 4 C, D). CXCL9 mRNA expression was up-regulated in 50ng and 100ng after 4h, but only 50ng after 24h, while CXCL9 protein could not be detected in the FLS culture supernatant (Figure 4 E). IL-6, IL-8, and IL-1β mRNA were detected at high levels in both 50ng and 100ng concentrations of IL-33 at both time points, but protein expression (Figure 4 F). CXCL10 mRNA expression was down-regulated as the concentration of IL-33 increased. The ELISA results also demonstrated that CXCL10 protein expression decreased as the concentration of IL-33 increased after 24h. Conversely, TNF-α and IL-17 increased CXCL10 mRNA expression in FLS (Figure 4 H). TNF-α was not affected by different concentrations of IL-33, or by IL-17 (Figures 4 I, J).