Interleukin-17–induced proliferation of fibroblast-like synovial cells is mTOR dependent
Article first published online: 27 APR 2011
Copyright © 2011 by the American College of Rheumatology
Arthritis & Rheumatism
Volume 63, Issue 5, pages 1465–1466, May 2011
How to Cite
Saxena, A., Raychaudhuri, S. K. and Raychaudhuri, S. P. (2011), Interleukin-17–induced proliferation of fibroblast-like synovial cells is mTOR dependent. Arthritis & Rheumatism, 63: 1465–1466. doi: 10.1002/art.30278
- Issue published online: 27 APR 2011
- Article first published online: 27 APR 2011
- Accepted manuscript online: 4 FEB 2011 01:53PM EST
Although interleukin-17 (IL-17) is primarily associated with induction of tissue inflammation, there is increasing evidence that IL-17 may have other functions, such as induction of human mesenchymal stem cell proliferation (1). In a recent report, Zhang and colleagues made a critical observation that IL-17 induces the proliferation of fibroblast-like synoviocytes (FLS) derived from patients with rheumatoid arthritis (RA), by up-regulating Cyr61 expression (2). They have also analyzed the signaling mechanism for this pathway and have observed that IL-17 induces Cyr61 through the p38 MAPK and NF-κB pathways.
In light of these findings, we want to share our observations that IL-17 can induce proliferation of RA FLS in an mTOR signal kinase–dependent manner. This study was approved by the Institutional Review Board at the Department of Veterans Affairs Medical Center, Sacramento. Synovial tissue was obtained from RA patients who had had knee surgery. FLS from 5 RA patients were isolated and cultured in the presence of recombinant IL-17 (10 ng/ml) for 5 days, as per our established protocol (3), and FLS proliferation was measured by MTT assay. We observed that IL-17 significantly increased FLS proliferation (P < 0.01 by t-test) (Figure 1a). Furthermore, we noted that IL-17–induced proliferation of FLS was reduced in the presence of an IL-17 receptor (IL-17R) monoclonal antibody (100 ng/ml) (P < 0.01 by t-test). Based on these results, we investigated the mechanisms of the mitogenic effect of IL-17.
The mTOR signaling pathway is a central regulator of cell proliferation (4). To determine whether IL-17–induced proliferation of FLS is mediated through the mTOR pathway, we cultured FLS with IL-17 in the presence of rapamycin (10 nm/ml) or without rapamycin for 5 days. Rapamycin is a potent specific inhibitor of the mTOR signaling system, and we found that it significantly inhibited IL-17–induced proliferation (P < 0.01 by t-test) (Figure 1a), which indicates that IL-17–induced proliferation of FLS is mediated through the mTOR signal kinase activation pathway. To obtain further confirmation of these results, we evaluated IL-17–induced FLS proliferation with the 5,6-carboxyfluorescein succinimidyl ester (CFSE) dilution assay. FLS from RA patients were stained with CFSE (Molecular Probes Invitrogen) and were cultured with recombinant IL-17 (10 ng/ml) or with IL-17 with rapamycin (10 nM) for 5 days and CFSE dilution was assessed by flow cytometry. Data were analyzed using FlowJo software (TreeStar). Similar to the results of the MTT assay, we observed a marked decrease of IL-17–induced FLS proliferation in the presence of rapamycin (Figures 1b and c). In the CFSE dilution assay, it was clear that there was minimal proliferation in the second- and third-generation cells in the presence of rapamycin (Figure 1c). Although Zhang et al used a concentration of IL-17 as high as 100 ng/ml, we have found that a dose of 10 ng/ml is adequate for proliferation of FLS.
Based on our observation that rapamycin, an mTOR inhibitor, significantly reduced IL-17–induced proliferation of FLS, we further investigated the effect of IL-17 on the mTOR signaling pathway. FLS were cultured in serum-free Dulbecco's modified Eagle's medium (DMEM) for 48 hours. Cells were then exposed to recombinant IL-17 for 2 hours. FLS that had been cultured in serum-free DMEM without addition of IL-17 were used as a control. Cell lysates were collected and analyzed for expression of mTOR and its activated counterpart phosphorylated mTOR (phosphor ser 2448), using Western blotting. We found that IL-17–treated FLS had a higher amount of phosphorylated mTOR, in comparison to the untreated cells (Figure 1d). Band density was measured using ImageJ software (NIH; online at: http://rsbweb.nih.gov/ij/).
The contributing role of Th17 cells in autoimmunity has provided a new dimension of understanding the disease processes of human autoimmune disease. Considerable evidence has been presented that supports the notion that IL-17 and its receptor system have a critical role in the pathogenesis of inflammatory arthritis, such as RA and psoriatic arthritis (5, 6). IL-17 is a proinflammatory cytokine and its effect at the disease site is regulated by the induction of tumor necrosis factor α, IL-1β, IL-6, IL-8, and matrix metalloproteinases in FLS. Our findings suggest that, in addition to its proinflammatory role, the IL-17/IL-17R system has the potential to directly influence the RA disease process by inducing FLS proliferation, which is a key component of pathologic hypertrophic synovial tissue in RA. The mechanism for FLS proliferation could occur via the activation of mTOR pathway or the Cyr61 loop. Further investigation is necessary to elucidate the IL-17–induced interactions between the Cyr61 loop and the mTOR pathway.