International Immunology

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International Immunology, Vol. 12, No. 2, 187-193, February 2000
© 2000 Japanese Society for Immunology

IL-6 inhibits the proliferation of fibroblastic synovial cells from rheumatoid arthritis patients in the presence of soluble IL-6 receptor

Norihiro Nishimoto, Aie Ito, Mika Ono, Hiromi Tagoh, Tomoshige Matsumoto, Tetsuya Tomita¹, Takahiro Ochi¹ and Kazuyuki Yoshizaki

Department of Medical Science I, School of Health and Sport Sciences, Osaka University, 2-1 Yamadaoka, Suita-city, Osaka 565-0871, Japan
¹ Department of Orthopedics, Osaka University Medical School, Osaka 565-0871, Japan

Correspondence to: K. Yoshizaki

	Abstract

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IL-6 and tumor necrosis factor (TNF)- have been proven to play an important role in the development of rheumatoid arthritis (RA). It is well known that TNF- induces IL-6 production from synovial cells as well as their proliferation. The effect of IL-6 on synovial cells, however, is not clear. An in vitrostudy was performed to determine the effect of IL-6 on the proliferation of synovial cells. Fibroblastic synovial cells isolated from the synovial tissues of eight RA patients were employed after the third to sixth passages. IL-6 in the presence of soluble IL-6 receptor (sIL-6R) inhibited the proliferation of synovial cells in a dose-dependent manner in seven cases without increasing the number of necrotic or apoptotic cells, while TNF- increased synovial cell proliferation in all cases. The inhibitory effect of IL-6 was observed only in the presence of sIL-6R although small amounts of IL-6R were detected in these cells by RT-PCR analysis. However, anti-IL-6R or anti-gp130 mAb treatment increased spontaneous growth of synovial cells in all eight cases, suggesting that endogenous IL-6 and a small amount of IL-6R expressed in synovial cells suppressed their growth without exogenous IL-6 or sIL-6R. In addition, the IL-6–sIL-6R complex reduced the TNF--induced proliferation of synovial cells while TNF- induced their IL-6 production. These data suggest that IL-6 may act as a negative feedback factor for TNF--induced synovial cell growth.

Keywords: IL-6, rheumatoid arthritis, soluble IL-6 receptor, synovial cell, tumor necrosis factor-

	Introduction

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Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by persistent synovitis accompanied by synovial cell proliferation, progressive destruction of cartilage and bone, presence of rheumatoid factor, elevation of acute phase proteins, hyper--globulinemia, and thrombocytosis. These clinical abnormalities may be explained by the uncontrolled hyperproduction of IL-6 found in both serum and synovial fluid of RA patients (1–4). This hypothesis is supported by the evidence that anti-IL-6 mAb treatment was therapeutically effective in RA patients (5). However, the direct effect of IL-6 on the proliferation of synovial cells which is a characteristic feature of RA has remained unclear.

IL-6 is a pleiotropic cytokine with a wide range of biological activities such as regulation of immune response and bone remodeling, generation of inflammatory responses, and support of hematopoiesis (6). IL-6 also acts as a growth factor for various cells such as epidermal keratinocytes (7), mesangial cells (8), renal carcinoma cells (9) and human multiple myeloma cells (10). On the other hand, IL-6 has a growth inhibitory effect on dermal fibroblasts (11), acute myeloid leukemia cells (12) and breast carcinoma cells (13,14). The receptor system of IL-6 consists of a ligand binding receptor (IL-6R) and its signal transducer, glycoprotein 130 (gp130), on the cell surface (15). The soluble form of IL-6R (sIL-6R) has been identified in vivo and the IL-6–sIL-6R complex can also induce homodimerization of gp130 and mediate the signal into cells which express not IL-6R but gp130 on their surface (16). Therefore, the increased quantity of sIL-6R found in synovial fluid of RA patients (3,4) may also play a role in the development of RA. To determine the functions of IL-6 and sIL-6R in RA synovitis, we investigated their effect on the proliferation of fibroblastic synovial cells.

	Methods

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Fibroblastic synovial cells
Fibroblastic synovial cells were isolated from the synovial tissues of RA patients with their informed consent and in accordance with the guidelines of our institution's ethical committee, and RA-6 synovial cells were kindly provided by Dr Mihara. The clinical features and medications of these patients are listed in Table 1. The synovial tissues were minced and treated with 0.2 mg/ml of DNase (Nacalai Tesque, Kyoto, Japan) and 2 mg/ml of collagenase (Sigma, St Louis, MO) for 1 h at 37°C as previously reported (17). Cell suspensions were passed through a mesh and synovial cells were isolated with the aid of Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden). The cells were then cultured in DMEM with 10% FCS, 2 mmol/l L-glutamine, 50 nmol/ml 2-mercaptoethanol, 100 U/ml penicillin and 100 µg/ml streptomycin in a culture flask, and the non-adherent cells were removed. Adherent fibroblastic cells were considered to be synovial cells and we used cells which had been passaged 3–6 times for this experiment. Flow cytometric analysis characterized the cell-surface markers of these cells as CD14^–, HLA-DR^–, CD3^–, CD19^–, CD106 (vascular cell adhesion molecule-1)⁺ and CD54 (intercellular adhesion molecule-1)⁺. This profile is consistent with that previously reported (18).

View this table: [in this window] [in a new window]   Table 1. Clinical features and medication of patients with RA

 
Cytokines and antibodies
IL-6 was provided by Ajinomoto (Kawasaki, Japan), sIL-6R by Tosoh (Kanagawa, Japan), TNF- by Hayashibara Biochemical Laboratory (Okayama, Japan), humanized anti-IL-6R mAb (rhPM-1) (19) by Chugai Pharmaceutical (Shizuoka, Japan) and mouse anti-gp130 mAb (gpx22) (20) by Tosoh. Anti-TNF- mAb was purchased from R & D Systems (Minneapolis, MN), irrelevant polyclonal human IgG from Midorijuji (Osaka, Japan) and purified mouse IgG1 (MOPC21) from ICN (Costa Mesa, CA).

Cell proliferation assay
Cells were cultured hexacately (3x10³ cells/200 µl/well) with or without TNF- (1–10 ng/ml), IL-6 (1–100 ng/ml) and/or sIL-6R (1–100 ng/ml) in 96-well flat-bottomed culture plates (Costar, Cambridge, MA) for 5 days. DNA synthesis was measured by liquid scintillation counting after the last 2 day label with 18.5 kBq/well of [³H]thymidine of a 5 day culture as previously described (11,21). For the kinetic study, DNA synthesis of the cells was examined after 3, 5, 7 and 9 days of culture. The inhibitory effect of IL-6–sIL-6R on DNA synthesis of the synovial cells was confirmed by the addition of anti-IL-6R mAb (rhPM-1) and/or anti-gp130 mAb (gpx22) at a dose of 25 µg/ml in in vitro culture system. Cell viability was measured by Trypan blue exclusion assay and apoptosis by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method after a 5 day culture.

Cell-surface IL-6R expression
Cell-surface IL-6R and gp130 were identified by using biotinylated anti-IL-6R mAb (PM-1, 1) and anti-gp130 mAb (gpx22, 1) respectively, in conjunction with phycoerythrin-labeled streptavidin as described before (22). Cells (2x10⁵) from single-cell suspensions were incubated with 500 ng of mAb for 20 min on ice, washed in PBS with 10% FCS and 0.1% NaN₃, and incubated with the appropriate second-step reagents. Isotype-matched 1 mAb with irrelevant specificity was used as negative control. Flow cytometric analysis was performed on a FACSCalibur (Becton Dickinson, Mountain View, CA) calibrated with fluorescent CaliBRITE beads using AutoCOMP software (Becton Dickinson). Data were obtained with CellQuest software (Becton Dickinson).

RT-PCR assay
RNA was prepared by using TRIzol (Life Technologies, Grand Island, NY) according to the manufacturer's instructions. Briefly, 1 µg of total RNA was reverse transcribed with 200 U of MMLV reverse transcriptase (Promega, Madison, WI), 0.5 µg of oligo(dT) primer, 10 nmol of each dNTP and 20 U of RNase inhibitor in 20 µl at 37°C for 60 min and denatured at 90°C for 5 min. PCR was performed in 50 µl of reaction solution containing cDNA derived from 200 ng of total RNA, 1.25 U of rTaq DNA polymerase (Toyobo, Osaka, Japan), 10 nmol of each dNTP, and 0.5 nmol of sense and antisense primers pairs shown below with a DNA thermal cycler, Gene Amp PCR system 2400 (Perkin-Elmer, Norwalk, CT). PCR cycles were operated on a regimen of 30 s of denaturation at 94°C, 30 s of primer annealing at 60°C and 30 s of extension/synthesis at 72°C, for 25 cycles for IL-6R and for 18 cycles for GAPDH. The nucleotide sequence of the sense primer for IL-6R was 5'-CATTGCCATTGTTCTGAGGTTC-3' (1580–1601 nucleotides of the cDNA) for membrane binding IL-6R or 5'-CCAGGAGGAGTTCGGGCAAGG-3' (1319–1339 nucleotides of the cDNA) for sIL-6R, that of the antisense primer for IL-6R was 5'-AGTAGTCTGTATTGCTGATGTC-3' (1809–1830 nucleotides of the cDNA), that of the sense primer for GAPDH was 5'-GTCATCATCTCTGCCCCCTCTGCT-3' (415–438 nucleotides of the cDNA) and that of the antisense primer for GAPDH was 5'-GACGCCTGCTTCACCACCTTCTTG-3' (834–857 nucleotides of the cDNA). PCR products were separated by electrophoresis on 2.5% agarose gels containing ethidium bromide, visualized by UV light and photographed.

Measurement of culture supernatant IL-6 and sIL-6R
Cells were cultured (1.5x10⁴ cells/500 µl/well) with or without TNF- (10 ng/ml) in a 48-well flat-bottomed culture plate (Costar) for 1 or 3 days. Culture supernatants were collected and filtered by a 0.22 µm filter unit (Millipore, Bedford, MA), and IL-6 concentration was measured by means of a chemiluminescence enzyme immunoassay system (Lumipulse 1200, sensitivity 0.2 pg/ml; Fujirebio, Tokyo, Japan). sIL-6R concentration was measured by ELISA (sensitivity 3.5 pg/ml; R & D Systems).

Statistical analysis
Statistical analysis was performed by Student's t-test for the inhibitory effect of IL-6–sIL-6R complex on the proliferation of synovial cells which were stimulated with or without TNF- and for the elimination of the inhibitory effect by anti-IL-6R antibody or anti-gp130 antibody.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Growth inhibitory effect of IL-6–sIL-6R complex on fibroblastic synovial cells
The effects of IL-6 and sIL-6R on the proliferation of synovial cells were first examined. Representative data from RA-1 are shown in Fig. 1. Although IL-6 alone had no effect on the proliferation of synovial cells, a combination of IL-6 and sIL-6R inhibited it in a dose-dependent manner at a concentration of >1 ng/ml of IL-6 (Fig. 1A). This inhibitory effect was also dependent on the concentration of sIL-6R (1–100 ng/ml), and maximum effect (76% inhibition) was obtained with the combination of 100 ng/ml each of IL-6 and sIL-6R (Fig. 1B). Of the eight RA cases we examined, a combination of IL-6 and sIL-6R inhibited the synovial cell growth in seven (63 ± 24% inhibition, mean ± SD, n = 7) (Table 2). There is no difference in cell viability and apoptosis between groups with and without IL-6–sIL-6 examined by Trypan blue exclusion and the TUNEL method (data not shown). Since medications, clinical characteristics and disease activities might have influenced the synovial cell proliferation, we examined the relation between them. However, there was no correlation between the inhibitory effect and medication, clinical characteristics or disease activities of the patients (Tables 1 and 2).

View larger version (34K): [in this window] [in a new window]   Fig. 1. The effect of IL-6 and/or sIL-6R on the proliferation of RA synovial cells. (A) IL-6 combined with sIL-6R inhibited the proliferation of synovial cells in a dose-dependent manner. Synovial cells (3x103 cells/200 µl/well) were cultured with 1–100 ng/ml of IL-6 in the presence (closed bar) or absence of sIL-6R (100 ng/ml) (dotted bar) for 5 days and DNA synthesis was determined by measuring the [3H]thymidine uptake. Each bar indicates the value of [3H]thymidine uptake (c.p.m.) averaged from six wells and error bars represent the SD. (B) sIL-6R also inhibited proliferation. Cells were cultured as described in (A), but with 1–100 ng/ml of sIL-6R in the presence () or absence of IL-6 (100 ng/ml) () and DNA synthesis was determined in the same manner. Representative data from RA-1 are shown in these figures. Comparisons of mean values were performed using Student's t-tests. *P < 0.001; **P < 0.0001.

 

View this table: [in this window] [in a new window]   Table 2. Proliferation of synovial cells of patients with RA

 
Next, we examined the effect of the IL-6–sIL-6R complex on the TNF--induced growth of synovial cells, since TNF- reportedly increases the proliferation of synovial cells (23), and also since a large quantity of TNF- as well as of IL-6 and sIL-6R is present in synovial fluid from the affected joints of RA patients (24–26). We were able to confirm that 1–10 ng/ml of TNF- augmented the proliferation of synovial cells in all cases as was previously reported (Fig. 2 and Table 2), while IL-6 reduced the TNF--induced proliferation of synovial cells in a dose-dependent manner in the presence of sIL-6R (Fig. 2A). Moreover, kinetic study confirmed that this reduction by IL-6 with sIL-6R was also observed for different culture periods for up to 9 days (Fig. 2B). Similar results were obtained with cells from all patients (data not shown). In addition, sIL-6R alone also showed some inhibitory effect on TNF--induced synovial cell growth but not without the presence of TNF-. This phenomenon was observed at least in RA-1, -3, -5, -6 and -8. This is probably because the inhibitory signal was introduced by exogenously added sIL-6R in combination with endogenously produced IL-6 which was strongly induced by the addition of TNF- (Table 3).

View larger version (79K): [in this window] [in a new window]   Fig. 2. IL-6 and TNF- compete in the growth stimulatory effect on RA synovial cells. (A) DNA synthesis in the cells cultured with or without TNF- (1–10 ng/ml), IL-6 (1–100 ng/ml) and/or sIL-6R (100 ng/ml) was determined by measuring [3H]thymidine uptake. Representative data from RA-4 are shown in these figures. Comparison of mean values were performed using Student's t-tests. NS, not significant; *P < 0.001 Each bar indicates the value of [3H]thymidine uptake (c.p.m.) on day 5 averaged from six wells and error bars represent the SD. TNF--induced synovial cell growth was inhibited by the IL-6–sIL-6R complex at every TNF- concentration. sIL-6R alone also showed some inhibitory effect on TNF--induced synovial cell growth. (B) Kinetic study for the DNA synthesis of RA synovial cells was performed on day 3, 5, 7 and 9 with (1) TNF- (10 ng/ml) (), (2) TNF- (10 ng/ml) and IL-6 (100 ng/ml) (), (3) TNF- (10 ng/ml) and sIL-6R (100 ng/ml) (), (4) TNF- (10 ng/ml), IL-6 (10 ng/ml) and sIL-6R (100 ng/ml) (), (5) TNF- (10 ng/ml), IL-6 (100 ng/ml) and sIL-6R (100 ng/ml) (), (6) IL-6 (100 ng/ml), sIL-6R (100 ng/ml) (), and (7) without reagents (). Each bar indicates the value of [3H]thymidine uptake (c.p.m.) averaged from six wells and error bars represent the SD. Comparisons of mean values were performed using Student's t-tests. NS, not significant; *P < 0.0001.

 

View this table: [in this window] [in a new window]   Table 3. IL-6 secretion by synovial cells of patients with RA

 
Elimination by anti-IL-6R antibody and anti-gp130 antibody of IL-6–sIL-6R complex-mediated synovial cell growth inhibition
Anti-IL-6R mAb and anti-gp130 mAb were used to confirm whether the IL-6–sIL-6R complex indeed introduces the growth inhibitory signal to synovial cells and that such a signal is mediated by gp130. In this experiment, humanized anti-IL-6R mAb was used for possible therapeutic application. Both anti-IL-6R mAb (25 µg/ml) and anti-gp130 mAb (25 µg/ml) could clearly eliminate the growth inhibition of synovial cells induced by the IL-6–sIL-6R complex (Fig. 3). Similar results were obtained with cells from all patients (Table 2). Furthermore, treatment with these two antibodies tended to increase the DNA synthesis of synovial cells in the absence of IL-6 or sIL-6R as shown in Fig. 3. These phenomena were observed in all cases including RA-2 which did not respond to the exogenous IL-6–sIL-6R. (stimulation index = 1.66 ± 0.92, mean ± SD, n = 8) (Table 2).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Anti-IL-6R mAb and anti-gp130 mAb eliminate the RA synovial cell growth inhibited by the IL-6–sIL-6R complex. Synovial cells (3x103 cells/200 µl/well) were cultured with humanized anti-IL-6R mAb (rhPM-1) (25 µg/ml) or mouse anti-gp130 mAb (gpx22) (25 µg/ml) in the presence or absence of IL-6(100 ng/ml) and sIL-6R (100 ng/ml). Each bar indicates the value of [3H]thymidine uptake (c.p.m.) averaged from six wells and error bars represent the SD. Comparisons of mean values were performed using Student's t-tests. *P < 0.0001; **P < 0.001.

 
Expression of IL-6R on synovial cells
In order to identify the IL-6R and gp130 expression by the synovial cells, we first performed flow cytometric analysis using mAb specific to these receptor components. Cell-surface IL-6R was not detectable in any cases, while gp130 was always detected on the synovial cells, although both IL-6R and gp130 were strongly detected on the human myeloma cell line, U266, as a positive control (Fig. 4). Similar FACS profiles of IL-6R and gp130 expression were obtained from all eight cases. Next, we used ELISA to examine whether sIL-6R was secreted in the culture supernatant of synovial cells, but no significant amount of sIL-6R was detected. Thirdly, RT-PCR analysis, however, revealed that most of the synovial cells expressed only a small amount of IL-6R including the transmembrane region which was ~100th of that expressed by U266 (Fig. 5), which contain a large quantity of IL-6R (11,000 sites per cell) (27) and show an autocrine growth mechanism mediated by IL-6 (28). At the same time, a very small quantity of truncated transcript, which had lost a membrane portion as a result of alternative splicing and was to be translated to sIL-6R, was observed when we used an appropriate combination of primer pairs (data not shown). These findings indicate that synovial cells express gp130, but not much IL-6R or sIL-6R so that they require exogenous sIL-6R to transduce the IL-6 signal. In RA-2, whose synovial cell growth was not inhibited by exogenous IL-6 and sIL-6R but increased by anti-IL-6R antibody treatment, a relatively larger amount of IL-6R was detected by RT-PCR, while no significant relation was observed between the amount of IL-6R and the sensitivity to the IL-6–sIL-6R complex.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Flow cytometric profile of IL-6R and gp130 expression on RA synovial cells. Cells (2x105) were stained with 500 ng of biotinylated anti-IL-6R mAb (PM-1) and anti-gp130 mAb (gpx22), in conjunction with phycoerythrin-labeled streptavidin. Flow cytometric analysis was performed on a FACSCalibur. IL-6R was not detectable on the surface of RA synovial cells by flow cytometric analysis (A), while gp130 was detected (B). The antibody-specific staining patterns are shown as shadowing area. Representative data from RA-1 are shown in this figure. Essentially the same flow cytometric profiles were obtained in all eight cases. Both IL-6R (C) and gp130 (D) were detected on human myeloma cell line, U266, as a positive control.

 

View larger version (51K): [in this window] [in a new window]   Fig. 5. Expression of IL-6R transcript in RA synovial cells. RT-PCR analysis for IL-6R and GAPDH transcript was performed as described in Methods. The amount of IL-6R transcript in most RA synovial cells was ~100th of that of U266 myeloma cell line. A relatively larger amount of IL-6R was expressed in the synovial cells from RA-2 whose synovial cell growth was not inhibited by the addition of IL-6 and sIL-6R.

 
IL-6 production by synovial cells
To determine the possible contribution of endogenous IL-6 to growth inhibition, production of IL-6 in culture supernatant was examined. Synovial cells produced considerable amounts of IL-6 (2.02 ± 1.31 ng/ml on day 3, n = 8) without any stimulation. Furthermore, TNF- (10 ng/ml) augmented IL-6 secretion (34.4 ± 29.8 ng/ml on day 3, n = 8) from synovial cells as was previously reported (29,30). The enhanced production of endogenous IL-6 by TNF- can explain the observation that additional sIL-6R alone showed some inhibitory effect on synovial cell growth in the presence of TNF- (Fig. 2). Therefore, endogenous IL-6 can also contribute to the growth inhibition of synovial cells.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
It was demonstrated in our study that IL-6 in the presence of sIL-6R inhibited the proliferation of synovial cells without any increase in cell death in seven out of eight RA patients. Furthermore, endogenous IL-6 produced by the synovial cells seemed to inhibit their own growth because anti-IL-6R mAb treatment increased their DNA synthesis in all cases. Our observation contradicts the one previously reported by Mihara et al. (21), i.e. that IL-6 induced synovial cell growth. It is not clear why there was such a different response. Since the stimulatory activity of IL-6 reported was observed in only one of three cases they examined (C. Mihara, pers. commun.), it might have been an exception. Furthermore, when we re-examined the same synovial cell sample provided by Mihara, IL-6 with sIL-6R actually inhibited the growth of the cells (Table 2; RA-6). The same author found that growth of dermal fibroblasts was inhibited by the IL-6–sIL-6R complex (11). We also observed that the IL-6–sIL-6R complex inhibited the growth of fibroblastic synovial cells from an osteoarthritis (OA) patient (data not shown). Thus growth inhibition by IL-6 may be a common phenomenon among fibroblastic cells.

The levels of IL-6 and sIL-6R are known to be higher in RA than in OA synovial fluid, and 2–40 ng/ml of IL-6 and 20–80 ng/ml of sIL-6R are reportedly present in the synovial fluid of active RA cases (1–4). Since similar concentrations of IL-6 and sIL-6R exerted a growth inhibitory effect in our experiment, our in vitro observation may well reflect the in vivo phenomenon observed in patients.

We confirmed that a significant quantity of IL-6 was secreted into the culture supernatant of synovial cells and that production of IL-6 was increased in the presence of TNF-. On the other hand, flow cytometric analysis could not detect any cell-surface IL-6R but its signal transducer, gp130. sIL-6R was not detectable by ELISA in the culture supernatant, too. Only RT-PCR analysis could demonstrate a small amount of IL-6R transcript in synovial cells. Therefore, exogenous sIL-6R was required to transduce the optimum IL-6 signal into fibroblastic synovial cells. At the same time, however, the small amount of IL-6R expressed by synovial cells may also partially contribute to transduction of the growth inhibitory signal because anti-IL-6R mAb treatment increased the DNA synthesis.

To investigate the inhibitory mechanism, we examined whether the growth inhibition was caused by IL-6-induced programmed cell death. However, no loss of cell viability or increase in apoptosis was found after IL-6–sIL-6R treatment. An intracellular signal of IL-6 may cause cell cycle arrest. Quite recently, Taniguchi et al. have demonstrated the expression of p16^INK4a, which induced the cell cycle arrest in RA synovial fibroblast (31). IL-6 may induce p16^INK4a expression. The intracellular mechanism of IL-6–sIL-6R to inhibit the synovial cell proliferation should be elucidated in future experiments.

Next, we wish to address the relation between IL-6 and TNF- in the pathophysiology of RA. It was demonstrated that murine anti-IL-6 mAb treatment reduced the activity of RA (5). TNF- induced IL-6 secretion from synovial cells in vitro (29,30) and chimeric anti-TNF- antibody (cA2) was also found to be therapeutically effective for RA, resulting in the normalization of serum IL-6 levels during treatment with cA2 (32,33). Therefore, Feldmann et al. hypothesized a cytokine cascade in which TNF- was placed upstream from IL-6 in RA pathogenesis and caused abnormalities through IL-6 (34). However, we demonstrated that IL-6 and TNF- had an opposite effect at least on the proliferation of fibroblastic synovial cells. In addition, Alonzi et al. reported DBA/1J, IL-6^–/– mice were completely protected from collagen-induced arthritis by inactivation of the IL-6 gene, while arthritis in TNF- transgenic mice was not affected (35). This demonstrates that IL-6 does not always act as a mediator of TNF- in the cytokine cascade of RA development. Since TNF- induces the secretion of IL-6 from synovial cells and IL-6 in turn negatively controls the TNF- function which augments the proliferation of synovial cells, IL-6 may act as a negative feedback factor in TNF--induced synovial cell growth. This feedback mechanism can partly explain the in vivo phenomenon that synovial tissue grows slowly in RA affected joints. Then, the question of how blockade of the IL-6 signal could demonstrate a therapeutic effect needs to be addressed. Recently, we reported the histological improvement by the administration of the humanized anti-IL-6R mAb in a RA animal model established by an engraft of RA synovial tissue into severe combined immunodeficiency mice (36). Anti-IL-6R mAb treatment markedly reduced the number of inflammatory cells especially in the pannus portion. These findings, together with the data presented here, suggest that the therapeutic effect of the blockade of the IL-6 signal is achieved through modulating the function of inflammatory cells rather than through direct regulation of synovial cell growth. Elimination of inflammatory cells may then cause a reduction of TNF- production in the synovium and may consequently decrease synovial cell proliferation. Immunological responses in RA are regulated by a complex network of many factors such as cytokines, the soluble form of cytokine receptors, chemokines, proteases and prostaglandins. Further study is required to understand the interactions of these factors which result in failure to appropriately regulate immunological responses in RA.

	Acknowledgments

 
We thank Mrs Chieko Aoki, Mrs Kaori Nakahara, Mr Noriyuki Danno and Mrs Ritsuko Okamoto for their excellent technical assistance, and Ms Takako Nakao for her outstanding secretarial assistance.

	Abbreviations

 

gp130 glycoprotein 130

IL-6R IL-6 receptor

OA osteoarthritis

RA rheumatoid arthritis

rhPM-1 recombinant humanized anti-IL-6R mAb

sIL-6R soluble form of IL-6 receptor

TNF tumor necrosis factor

TUNEL TdT-mediated dUTP-biotin nick end-labeling

	Notes

 
Transmitting editor: K. Okumura

Received 13 May 1999, accepted 18 October 1999.

	References

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