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Abstract
Objecti
Interleukin-15 (IL-15) recruits and activates synovial T cells, and IL-15 plays an important role in amplifying and perpetuating inflammation in the pathogenesis of rheumatoid arthritis (RA). Stromal cell-derived factor-1 (SDF-1) is a potent chemoattractant for memory T cells in the inflamed RA synovium. This study investigated the effect of IL-15 on SDF-1 production in RA fibroblast-like synoviocytes (FLS).
Methods
The expressions of IL-15 and SDF-1 were determined from the synovium of patients with RA and osteoarthritis (OA) by performing immunohistochemistry. The expressions of SDF-1 was measured from the RA FLS that were cultured with IL-15 and IL-17 by real-time RT-PCR and ELISA. The SDF-1 expression was also measured, via ELISA, from the RA FLS stimulated by IL-15 together with the inhibitors of such intracellular signal molecules as phosphatidylinositol 3-kinase (PI 3-kinase, LY294002), STAT3 (AG490), MAP Kinase (PD98059), NF-κB (parthenolide) and activator protein 1 (AP-1, curcumin).
Results
IL-15 and SDF-1 were mainly expressed in the RA synovium compared to that of the OA synovium. IL-15 increased the SDF-1 expressions and it, and had an additive effect with IL-17 on the SDF-1 expressions in the cultured RA FLS. The IL-15 induced increase of the SDF-1 expression in the cultured RA FLS was blocked by the inhibitors of PI 3-kinase, NF-κB and AP-1.
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Fig. 1.
Immunohistochemical staining for stromal-cell derived factor-1 (SDF-1) and interleukin-15 (IL-15) on the synovium of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). IL-15 and SDF-1 were significantly more expressed in the RA synovium compared to that of the OA synovium (original magnification, ×400).
Fig. 2.
The expressions of SDF-1 from the RA synovial fibroblasts stimulated with various concentrations of IL-15 (0, 0.1, 1, 10 and 50 ng/mL) were determined by real-time RT-PCR (A) and ELISA (B). IL-15 increased the SDF-1 expression in a dose-dependent manner.
Fig. 3.
The productions of SDF-1 from the RA FLS stimulated with IL-15 and IL-17 were determined by ELISA. IL-15 and IL-17 up-regulated SDF-1 production. IL-17 had an additive effect with IL-15 on the SDF-1 production. ∗p<0.01 compared to nil, †0.05 compared to IL-15 1 ng/mL, ‡<0.01 compared to IL-15 10 ng/mL
Fig. 4.
(A) The IL-15 induced increase of SDF-1 was blocked by the inhibitors of PI3-kinase, NF-kB and AP-1. RA FLS that were, pre-treated with the inhibitors of PI3-kinase (LY294002), STAT3 (AG490), MAPK (PD98059), NF-kB (parthenolide) and AP-1 (curcumin), were cultured with IL-15; the production of SDF-1 was determined by ELISA. Up-regulation of SDF-1 by IL-15 was blocked by the inhibitors of PI3-kinase, NF-kB and AP-1. (B) MTT assay was done to confirm that the inhibitors did not have cell toxicity. This assay showed that there was no difference in cell viability after all the stimulations and/or inhibitions. ∗p<0.05 compared to IL-15 10 ng/mL, †<0.01 compared to IL-15 10 ng/mL.
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