CCL20 produced in the cytokine network of rheumatoid arthritis recruits CCR6+ mononuclear cells
and enhances the production of IL-6.
Shimei Tanida; Hiroyuki Yoshitomi; Kohei Nishitani; Masahiro Ishikawa; Toshiyuki Kitaori; Hiromu Ito; Takashi Nakamura
Department of Orthopaedic Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan
Senior author; [email protected],jp
<Introduction>Rheumatoid arthritis (RA) is one of the most popular
chronic inflammatory autoimmune diseases. CD4+ helper T cells are
infiltrated in RA synovium. Among them, IL-17-producing-helper-T
(Th17) cells, which were recently discovered as a novel subset of CD4+
helper T cells, are thought to play a pivotal role in RA. CCL20 is highly
expressed in the synovial fluid of RA compared to that of OA.1) So far,
CCR6, which is expressed by B cells, immature dendritic cells and Tcells,
is the sole receptor of CCL20, and CCL20 is also the sole ligand of CCR6.
Among human CD4+ helper T cells, Th17 cells but neither Th1 cells nor
Th2 cells express CCR6. In this study, we studied the roles of human
CCL20 in the trafficking of lymphocytes and monocytes into the inflamed
joints or in the production of proinflammatory cytokines.
<Material and methods> Human fibroblast like synoviocytes (FLSs)
were prepared from synovium of RA joints.2) Ethical approval was granted
by the institution’s ethics committee, and the written consent of every
patient was obtained. At confluent, the FLSs were stimulated with various
human recombinant cytokines for 48 hours. The supernatants or the cell
lysates were then collected for further analyses. The concentration of TNFα, IL-1β, IL-6 and CCL20 in the supernatants was measured by EnzymeLinked ImmunoSorbent Assay (ELISA). Total RNA was isolated from cell
lysate for quantitative PCR (qPCR).
For chemotaxis assay, mononuclear cells (MNCs) were isolated from
healthy volunteer peripheral blood. Cell migration was evaluated, using the
Transwell system. RPMI 1640 with 0.5% BSA containing human
recombinant CCL20 or 2% conditioned medium was added to the bottom
of the Transwell system and 1x106 MNCs suspended in RPMI 1640 with
0.5% BSA were placed on the top of the Transwell. After 2 h incubation,
the cells migrated into the lower well were collected and counted. The
percentage of CCR6+ cells was measured using a flow cytometer.
All data were reported as the mean ± SD. Student’ s t test was used for
statistical analyses, unless indicated otherwise. P < 0.05 was considered
significant.
<Results and discussions> We measured the production of TNF-α, IL-1β,
IL-6 and CCL20 by FLSs using ELISA. FLSs were stimulated with
designated helper T cell cytokines or chemokine at the concentration of 2
or 10 ng/ml. FLSs require sIL-6R to conduct IL-6 signal because FLSs
produce neither membranous nor soluble IL-6 receptors. We added 100
ng/ml sIL-6R together with IL-6. All the stimulated FLSs failed to produce
a detectable amount of TNF-α or IL-1β. On the other hand, stimulated
FLSs produced a notable amount of IL-6 and CCL20. While IL-6 was
induced by the stimulation of TNF-α, IL-1β, IL-17 and IFN-γ, CCL20 was
induced by TNF-α and IL-1β but not by IL-6. Especially, IL-1β induced
vigorous production of CCL20 (Fig.1).
Fig.1
Anti-IL-1β antibody neutralized the production of CCL20 induced by
TNF-α (Fig.2A). Quantitative RT-PCR showed that TNF-α induced
mRNA of IL-1β in a dose dependent manner (Fig. 2B). These results
indicate that the production of CCL20 induced by TNF-α is partially
attributed to a trace amount of IL-1b induced by TNF-a. To determine the
role of TNF-α-induced IL-6 in the production of CCL20, FLSs were
cultured with both of sIL-6R and TNF-α. Although IL-6 signal alone
failed to induce CCL20, the addition of sIL-6R significantly enhanced the
production of CCL20 induced by TNF-α (Fig. 2C). These results indicate
that IL-6 signal is also involved in the production of CCL20 induced by
TNF-α in an autocrine/paracrine manner.
Fig.2
We examined whether CCL20 preferentially recruits human CCR6+
MNCs. The number of migrated MNCs toward recombinant CCL20 was
increased in a dose dependent manner (Fig. 3A). Furthermore CCR6+ cells
specifically migrated toward recombinant CCL20 (Fig. 3B). To determine
whether IL-1β-stimulated FLSs significantly recruit MNCs, chemotaxis
assays of MNCs toward conditioned medium of IL-1β-stimulated FLSs
were conducted. The percentage of migrated MNCs to the conditioned
medium was significantly increased, and the addition of anti-CCL20
antibody significantly neutralized the migration (Fig. 3C). The percentage
of CCR6+ cells migrating to the conditioned medium was significantly
higher than that to control, and the addition of anti-CCL20 antibody
significantly neutralized the CCR6+-specific migration (Fig. 3D). These
results collectively indicate that among pleiotropic proteins induced by IL1β, CCL20 plays a key role in the trafficking of CCR6+ MNCs including
CCR6+ Th17 cells.
Fig.3
Furthermore, we found the modification of the production of human
CCL20 by the helper-T-cell-derived cytokines such as IL-4, IFN-γ, IL-17
and TGF-β, similarly to that of mice .
We examined whether CCL20 induced proinflammatory cytokines from
FLSs. Although ~10 ng/ml CCL20 failed to induce the production of IL-6
by FLSs, ~50 ng/ml CCL20 induced. In addition to FLSs, Th17 cells are
known to produce CCL20. CCL20 significantly enhanced the production
of IL-6 induced by IL-17, but did not affect that of IL-6 induced by IFN-γ.
These results collectively indicate that CCL20 and IL-17 produced by
Th17 cells coordinately induce the production of IL-6 and are involved in
the cytokine network of RA.
<Conclusions>
・The production of CCL20 is induced and modulated by cytokines from
the macrophages, fibroblasts, and helper T cells.
・The production of CCL20 induced by TNF-α is partially attributed to a
trace amount of IL-1β and enhanced by IL-6 in an autoceine/paracrine
manner.
・CCL20 induced the production of IL-6 and coordinately induced it with
IL-17 from FLSs.
・Among pleiotropic proteins induced by IL-1β from FLSs, CCL20 plays
a crucial role in the trafficking of mononuclear cells.
<References>
1. Hirota K, Yoshitomi H, et al. Preferential recruitment of CCR6expressing Th17 cells to inflamed joints via CCL20 in rheumatoid arthritis
and its animal model. J Exp Med 2007;204: 2803-12.
2. Hiramitsu T, Yasuda T, Ito H, et al. Intercellular adhesion molecule-1
mediates the inhibitory effects of hyaluronan on interleukin-1beta-induced
matrix metalloproteinase production in rheumatoid synovial fibroblasts via
down-regulation of nf-kappab and p38. Rheumatology (Oxford)
2006;45:824-32.
Poster No. 930 • 56th Annual Meeting of the Orthopaedic Research Society