CCL20 produced in the cytokine network of rheumatoid arthritis recruits CCR6+ mononuclear cells and enhances the production of IL-6
Introduction
Rheumatoid arthritis (RA) is one of the most popular chronic inflammatory autoimmune diseases. Synovitis of RA leads to the degeneration of cartilages and ligaments, the induction of osteoclasts and the destruction of the articular structures in multiple joints. Classical autocrine and paracrine networks of proinflammatory cytokines in synovial fibroblast/macrophage axis have been thought to be the main pathogenesis of RA. Notable clinical effects of TNF, IL-1 and IL-6 inhibitors strongly support the importance of the classic cytokine network in human RA [1]. On the other hand, migration of leukocytes including granulocytes, monocytes and lymphocytes contributes to the pathogenesis of the synovitis in RA [2], [3], [4], [5], [6], [7]. Among them, CD4+ T cells occupy approximately 40% of all the cells infiltrating in RA synovium at some point [8], and are thought to play a pivotal role in the initiation and perpetuation of the inflammation in RA [9].
In the process of leukocyte trafficking, selectins, chemoattractants and integrins have fundamental roles. Chemokines are chemoattractants and comprise a family of small 8–10 kDa proteins regulating differentiation, proliferation, morphology and trafficking via the interaction with G-protein-coupled chemokine receptors. Chemokines are subdivided into four families (CXC, CC CX3C and C) according to the position and structure of the first two cysteines [10]. Chemokines including CXCL8 (IL-8), CCL2, CCL3, CCL5 (RANTES), CX3CL1 (Fractalkine), CXCL12 (SDF-1) and CCL20 are thought to be involved in the trafficking of leukocytes to the inflamed sites of RA [11], [12], [13], [14].
CCL20, which is also called macrophage inflammatory protein 3α (MIP-3α), liver and activation-regulated chemokine (LARC), or Exodus-1 [15], is highly expressed in the synovial fluid of RA compared to that of OA [14], [16]. So far, CCR6 is the sole receptor of CCL20 and CCL20 is also the sole ligand of CCR6. CCR6 is expressed by B cells, immature dendritic cells and T cells [15]. Among human CD4+ helper T cells, IL-17-producing-helper-T (Th17) cells but neither Th1 cells nor Th2 cells express CCR6 [6]. Th17 cells were recently discovered as a novel subset of CD4+ helper T cells and are thought to play a pivotal role in autoimmune diseases including RA and multiple sclerosis [14], [17], [18]. However, the roles of human CCL20 in the trafficking of lymphocytes and monocytes into the inflamed joints or in the production of proinflammatory cytokines remain unclear.
The production of CCL20 is known to be up-regulated in synovium explants or FLSs of RA with the stimulation of TNF-α, IL-1β and IL-17 [16], [19], [20]. In this study, we further investigated the relationship of TNF-α, IL-1β and IL-6 in the cascade of the CCL20 production, the modulations of CCL20 production by helper-T-cell-producing cytokines including IFN-γ, and the influence of CCL20 in the induction of proinflammatory cytokines. Moreover, we showed that IL-1β-stimulated human FLSs recruit CCR6+ mononuclear cells (MNCs) including CCR6+ Th17 cells in a CCL20 dependent manner.
Section snippets
Cytokines and reagents
Human recombinant TNF-α, human recombinant IL-1β, human recombinant IL-4, human recombinant IL-6, human recombinant soluble IL-6 receptor (sIL-6R), human recombinant IL-12, human recombinant IFN-γ, human recombinant MIP-3α/CCL20, human recombinant IL-17A were purchased from PeproTech USA (Rocky Hill, NJ, USA). Human recombinant TGF-β1 was purchased from Cosmo bio USA, Inc. (Carlsbad, CA, USA). The following reagents were purchased from R&D Systems (Minneapolis, MN, USA): human recombinant
IL-1β and TNF-α induced a notable amount of CCL20 from human RA FLSs but IL-6 did not
A notable amount of CCL20 (∼1000 pg/ml) is detectable in the synovial fluid of human RA [14]. We measured the production of TNF-α, IL-1β, IL-6 and CCL20 by collagen-digested FLSs from human RA patients using ELISA. Unstimulated FLSs failed to produce a detectable amount of TNF-α, IL-1β and CCL20 (Fig. 1A–D). Furthermore, FLSs were stimulated with individual TNF-α, IL-6, IL-1β, IL-17, IL-4, IFN-γ, IL-12, IL-23, or CCL20 at the concentration of 2 or 10 ng/ml. FLSs require sIL-6R to conduct IL-6
Discussion
In this study, we have shown that FLSs stimulated by IL-1β recruited MNCs in a CCL20/CCR6 dependent manner, implying that CCL20 acts as a bridge between native synovial fibroblasts/macrophages and infiltrating leukocytes in the pathogenesis of RA. Furthermore, we have shown that cytokines derived from CD4+ helper T cells modulate the production of CCL20 by FLSs: IL-17 from CCR6+ Th17 cells enhanced as previously reported [19]. The effect of IFN-γ was somehow complicate: IFN-γ enhanced the
Funding
This work was supported by Grants-in-Aid for Scientific Research [Grant No. 20791032].
Acknowledgments
We thank J. Toguchida for technical supports, and M. Kobayashi, S. Nakamura and R. Arai for the collection of samples. The authors have no competing interests.
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