Interleukin-4 inhibits interleukin-11 production by rheumatoid synovial cells
H. Taki,
E. Sugiyama,
A. Kuroda,
T. Mino and
M. Kobayashi
First Department of Internal Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan
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Abstract
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Objective. To examine the effect of interleukin-4 (IL-4) on IL-11 production by rheumatoid synovial cells.
Methods. Freshly isolated rheumatoid synovial cells (FRS) were obtained by collagenase digestion of rheumatoid arthritis (RA) synovial tissue specimens taken at the time of operation. Rheumatoid synovial cells at four to eight passages were used as cultured rheumatoid synovial fibroblasts (RSF). IL-11 concentration was measured by ELISA.
Results. IL-4 inhibited the production of IL-11 by FRS in a dose-dependent manner. This inhibition was observed in FRS obtained from six patients, and the mean inhibition was 46.5%. The inhibitory effect of IL-4 on IL-11 production was cancelled by the addition of anti-IL-4 antibody. IL-4 also inhibited IL-11 production by IL-1
stimulated cultured RSF.
Conclusion. IL-4 inhibited IL-11 production by rheumatoid synovial cells. IL-4 has a protective effect on bone resorption. On the contrary, IL-11 participates in bone resorption via osteoclastogenesis. Therefore, IL-4 may exert its protective effect on bone resorption, at least in part, via inhibition of IL-11 production in rheumatoid joints.
KEY WORDS: Rheumatoid arthritis, Synoviocytes, Interleukin-4, Interleukin-11.
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Introduction
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Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by persistent synovitis that leads to joint destruction [1]. Proinflammatory cytokines such as tumour necrosis factor-
(TNF
), interleukin (IL)-1 and IL-6 are produced by the inflamed synovia in RA [2], and are thought to participate in the pathophysiology of RA.
In contrast, IL-4 was hardly detected in RA synovium [3, 4]. IL-4 inhibits the production of TNF
, IL-1 [59], IL-6 [811] and prostaglandin E2 (PGE2) [5, 7, 12]. IL-4 does not merely inhibit many proinflammatory mediators; it also up-regulates the expression of anti-inflammatory mediators such as IL-1 receptor antagonist [1317] and the IL-1 type II receptor [18], indicating that IL-4 is an anti-inflammatory cytokine. In addition, IL-4 inhibits bone resorption [19, 20].
On the contrary, IL-11 induces osteoclastogenesis [21, 22], leading to accelerated bone resorption [23, 24]. Recently, we and others have found that rheumatoid synovial cells produce large amounts of IL-11 [2527]. IL-11 production by rheumatoid synovial cells was stimulated by IL-1 [27, 28], TNF
[29] and PGE2 [27, 29].
IL-4 inhibits IL-6 production by rheumatoid synoviocytes [911]. IL-11 is an IL-6-type cytokine, and many of its properties overlap with those of IL-6 [30, 31]. However, little is known about the effect of IL-4 on the production of IL-11. In this study, we examined the effect of IL-4 on IL-11 production by rheumatoid synovial cells.
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Materials and methods
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Synovial cell culture
Rheumatoid synovia were obtained from six patients defined as having RA according to the classification of the American College of Rheumatology [32]. Synovial tissues were dispersed with 1 mg/ml of collagenase (Wako Pure Chemical Industries, Osaka, Japan) and 5 µg/ml of DNase I (Sigma-Aldrich Japan, Tokyo, Japan) in serum-free Dulbecco's modified Eagle medium (DMEM) containing penicillin and streptomycin at 37°C. Cells were cultured overnight in plastic dishes containing DMEM supplemented with 10% heat-inactivated fetal calf serum (DMEM/FCS). Non-adherent cells were removed, and the freshly isolated rheumatoid synovial cells (FRS) were incubated in 24-well dishes at a density of 5 x 104 cells per well containing 500 µl of DMEM/FCS in the absence or presence of IL-4 (specific activity 1 x 106 U/mg protein), kindly provided by Dainippon Pharmaceutical (Osaka, Japan), for 6 h. Media were exchanged with fresh DMEM/FCS and the cells were further incubated for 24 h in the absence or presence of IL-4.
FRS were further grown to be confluent and were transferred to 10-cm culture dishes after trypsinization. Rheumatoid synovial cells at four to eight passages were used as cultured rheumatoid synovial fibroblasts (RSF). RSF were seeded in 24-well dishes at 5 x 104 cells per well with 500 µl of DMEM/FCS. RSF were preincubated with IL-4 for 6 h. Media were exchanged with fresh DMEM/FCS, and cultured RSF were stimulated with IL-1
, kindly provided by Dainippon Pharmaceutical, in the absence or presence of IL-4.
IL-11 measurement
The concentration of human IL-11 was measured by ELISA using Quantikine Human IL-11 Immunoassay Kit (R & D Systems, Minneapolis, MN) according to the manufacturer's instructions.
Statistical analysis
Values are presented as mean ± S.D. Data were analysed by Wilcoxon's paired non-parametric test. Differences were considered significant at P < 0.05.
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Results and discussion
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We first examined whether IL-4 inhibited the spontaneous production of IL-11 by FRS. IL-4 inhibited IL-11 production in a dose-dependent manner with a maximal effect at 100 U/ml of IL-4 (Fig. 1a
). This inhibitory effect was consistently observed in FRS obtained from six patients, resulting in a mean inhibition of 46.5% (Fig. 1b
). Simultaneous addition of anti-IL-4 antibody to the culture medium prevented the inhibitory effect of IL-4 on IL-11 production (data not shown).

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FIG. 1. (a) Inhibition by IL-4 of IL-11 production by freshly-isolated FRS. The FRS were incubated with 0, 1, 10 or 100 U/ml of IL-4 for 6 h. After exchange of medium, FRS were further incubated under the same condition for 24 h. The amounts of IL-11 in cell-free culture supernatants were measured by ELISA. Values are mean + S.D. of triplicate determinations. (b) Inhibitory effect of IL-4 on IL-11 production by FRS from six patients. FRS from six patients were treated with 100 U/ml of IL-4 for 24 h, and the amounts of IL-11 in cell-free culture supernatants were measured by ELISA. ast;P < 0.05.
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Although FRS produced a large amount of IL-11, cultured RSF produced only a small amount. As reported previously [2729], IL-1
can stimulate IL-11 production by RSF. As shown in Fig. 2
, IL-4 inhibited IL-11 production by IL-1
-stimulated RSF.
This is the first report demonstrating that IL-4 inhibits the production of IL-11 by rheumatoid synovial cells. The inhibition was specific because it was concentration-dependent and was completely abolished in the presence of anti-IL-4 antibody. However, we did not obtain a consistent result regarding the effect of IL-4 on IL-11 gene expression. This might have been due to partial suppression of IL-11.
In contrast to our study, Elias et al. [33] reported that IL-4 is a weak agonist for IL-11 production. This discrepancy might be explained either by (1) cell-specificity of the effect of IL-4 on IL-11 production, or (2) opposite effects of IL-4 in unstimulated and IL-1
-stimulated cells.
The role of IL-11 in the pathogenesis of RA has been a matter of controversy. Recent work has demonstrated that IL-11 ameliorates experimental arthritis in mice [34]. Although IL-11 was effective during the early phase, it did not affect disease activity during the late phase. Thus, the effect of IL-11 on RA may depend on the phase of inflammation.
Furthermore, IL-11 has been reported to inhibit TNF
production in human monocytes [35]. However, the effect was achieved only if the specific IL-11 receptor was provided. At present, little information is available about the expression of IL-11 receptors on monocytes and the existence of soluble IL-11 receptors in RA synovial fluid in vivo. The effect of IL-11 on TNF
production in RA synovia in vivo needs further elucidation.
It has also been reported that IL-11 is abundant in RA synovia and synovial fluid [26, 35]. Nevertheless, IL-11 present at the loci of inflammation does not restrict inflammation efficiently. IL-11 may protect RA synovia by inducing tissue inhibitors of metalloproteinases [28, 35] or inhibiting metalloproteinase production [35]. On the other hand, IL-11 promotes osteoclastogenesis [21, 22]. Therefore, we speculate that IL-11 is produced primarily to terminate inflammation in RA synovium, but that the effect of IL-11 may not be sufficient to terminate the local inflammation, and the persistence and excess of IL-11 production in the loci of inflammation may lead to bone resorption. Further studies are needed to elucidate the effect of IL-11 in RA.
The fact that IL-11 induced osteoclastogenesis [21] indicates that IL-11 might be involved in rheumatoid osteoporosis. On the contrary, IL-4 was reported to inhibit bone resorption [19, 20]. IL-4, scarcely detected in rheumatoid synovium [3, 4], inhibited IL-11 production in rheumatoid synovial cells. On the other hand, IL-1, which is abundant in rheumatoid synovium [1], induced IL-11 production. The imbalance between IL-4 and IL-1 might further explain the increased level of IL-11 in RA synovia, which might lead to inflammatory osteoporosis in patients with RA. Modulation of the cytokine network in inflamed synovium may reduce osteoporosis at the site.
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Notes
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Correspondence to: H. Taki. 
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Submitted 6 April 1999;
revised version accepted 10 January 2000.