The new IL-1 family member IL-1F8 stimulates production of inflammatory mediators by synovial fibroblasts and articular chondrocytes

Abstract

Six novel members of the IL-1 family of cytokines were recently identified, primarily through the use of DNA database searches for IL-1 homologues, and were named IL-1F5 to IL-1F10. In the present study, we investigated the effect of IL-1F8 on primary human joint cells, and examined the expression of the new IL-1 family members in human and mouse joints. Human synovial fibroblasts (hSFs) and human articular chondrocytes (hACs) expressed the IL-1F8 receptor (IL-1Rrp2) and produced pro-inflammatory mediators in response to recombinant IL-1F8. IL-1F8 mRNA expression was increased in hSFs upon stimulation with proinflammatory cytokines, whereas in hACs IL-1F8 mRNA expression was constitutive. However, IL-1F8 protein was undetectable in hSF and hAC culture supernatants. Furthermore, although IL-1beta protein levels were increased in inflamed human and mouse joint tissue, IL-1F8 protein levels were not. IL-1F8 levels in synovial fluids were similar to or lower than those in matched serum samples, suggesting that the joint itself is not a major source of IL-1F8. Serum levels of IL-1F8 were similar in healthy donors, and patients with rheumatoid arthritis, osteoarthritis and septic shock, and did not correlate with inflammatory status. Interestingly however, we observed high IL-1F8 levels in several serum samples in all groups. In conclusion, IL-1F8 exerts proinflammatory effects in primary human joint cells. Joint and serum IL-1F8 protein levels did not correlate with inflammation, but they were high in some human serum samples tested, including samples from patients with rheumatoid arthritis. It remains to be determined whether circulating IL-1F8 can contribute to joint inflammation in rheumatoid arthritis.

Figure 1

IL-1Rrp2 expression by hSFs and hACs. The left panels show a RT-PCR analyses of IL-1Rrp2 expression by (a) hSFs and (b) hACs treated or not treated for 8 hours by IL-1β (1 ng/ml) and/or TNF-α (10 ng/ml), as detailed under Materials and method and in Table 1. The images show representative agarose gel electrophoresis of PCR products. The right panels show real-time PCR analysis of IL-1Rrp2 mRNA levels in hSFs and hACs stimulated (black columns) or not stimulated (white columns) for 8 hours with IL-1β (1 ng/ml) and TNF-α (10 ng/ml). The amount of 28S rRNA was monitored as an internal control. The expression of IL-1Rrp2 mRNA was corrected for 28S rRNA levels and the IL-1Rrp2/28S ratios were normalized to the maximal value observed in each experiment, which was set to 100%. The results shown represent the mean ± standard error of data obtained with samples from three (hSFs) or four (hACs) independent cultures. IL, interleukin; IL-1Rrp2, IL-1 receptor related protein 2; hAC, human articular chondrocyte; hSF, human synovial fibroblast; RT-PCR, reverse transcriptase polymerase chain reaction; TNF, tumour necrosis factor.

Figure 2

Production of IL-6, IL-8 and nitric oxide by hSFs and hACs: effects of IL-1F8 and IL-1β. Shown is an analysis of the effects of IL-1F8 and IL-1β on production of (a) IL-6 and (c) IL-8 by hSFs, and of (b) IL-6, (d) IL-8 and (e) nitric oxide by hACs. Cells were treated with the indicated cytokine concentrations for 48 hours, as detailed under Materials and method. *P < 0.05 versus control; ^#P < 0.05 versus 0.1 ng/ml IL-1β; ^&P < 0.05 versus 500 ng/ml IL-1F8, determined using analysis of variance. IL, interleukin; hAC, human articular chondrocyte; hSF, human synovial fibroblast.

Figure 3

Production of IL-6 by hACs: effects of IL-1β, IL-1F8, heat-inactivated IL-1F8 and anti-IL-1Rrp2 antibodies. (a) Analysis of the effects of IL-1β (1 ng/ml), IL-1F8 and heat-inactivated IL-1F8 (5 mg/ml) on IL-6 production by hACs after 48 hours treatment, as detailed under Materials and method. (b) Analysis of the effects of anti-IL-1Rrp2 antibodies on IL-6 production by hACs. Cells were stimulated or not (control) for 48 hours with IL-1F8 (5 mg/ml) or IL-1b (1 ng/ml), as indicated, in the presence (black columns) or absence (white columns) of blocking anti-IL-1Rrp2 antibodies (10 mg/ml). P < 0.05 versus control; ^#P < 0.05 versus 5 μg/ml IL-1F8, as determined by analysis of variance. hAC, human articular chondrocyte; IL, interleukin; IL-1Rrp2, IL-1 receptor related protein 2.

Figure 4

Kinetics of IL-1β and IL-1F8 mRNA production by HSFs and hCAs in response to IL-1 and/or TNF-α. (a) Analysis of IL-1F8 mRNA levels in hSFs treated or not treated for 8 hours with IL-1β (1 ng/ml) and/or TNF-α (10 ng/ml), as detailed under Materials and method. A representative agarose gel electrophoresis of PCR products is shown. (b) Real-time PCR analysis of IL-1F8 mRNA levels in hSFs stimulated (black columns) or not stimulated (white columns) for 8 hours with IL-1β (1 ng/ml) alone, IL-1β (1 ng/ml) plus TNF-α (10 ng/ml), or IL-1α (1 ng/ml) alone, as indicated. The amount of 28S rRNA was monitored as an internal control. The expression of IL-1F8 mRNA was corrected for 28S rRNA levels and the IL-1F8/28S ratios were normalized to the maximal value observed in each experiment, which was set to 100%. The results shown represent the mean ± standard error of data obtained with samples from three independent cultures. *P < 0.05 versus, as determined by analysis of variance. (c) Fold increase (after correction for 28S RNA levels) in IL-1β (dashed line) and IL-1F8 (solid line) mRNA levels after treatment of hSFs with 1 ng/ml IL-1β for the indicated times, as revealed by real-time PCR analysis. Basal IL-1F8/28S and IL-1β/28S levels were respectively 6.4 and 20 (arbitrary units). (d) Real-time PCR analysis of IL-1F8 and IL-1β mRNA levels in hACs stimulated (black columns) or not stimulated (white columns) with IL-1β (1 ng/ml) and TNF-α (10 ng/ml) for 8 hours. The amount of 28S rRNA was monitored as an internal control. The expression of IL-1F8 and IL-1β mRNA was corrected for 28S rRNA levels and the IL/28S ratios were normalized to the maximal value observed in each experiment, which was set to 100%. The results shown represent the mean ± standard error of data obtained with samples from six independent cultures. *P < 0.05 versus, as determined by analysis of variance. hAC, human articular chondrocyte; hSF, human synovial fibroblast; IL, interleukin; RT-PCR, reverse transcriptase polymerase chain reaction; TNF, tumour necrosis factor.

Figure 5

IL-1F8 protein levels in control individuals, patients with RA, OA and septic shock. Shown are serum IL-1F8 protein levels in healthy donors (n = 16), patients with RA (n = 28) or OA (n = 16) patients, and patients with septic shock (n = 12), as determined by ELISA. Individual values (grey dots) and mean (stippled lines) ± standard error (black lines) are shown. Differences between the groups were not significant. ELISA, enzyme-linked immunosorbent assay; IL, interleukin; OA, osteoarthritis; RA, rheumatoid arthritis.