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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Objective

To investigate the mechanism of interleukin-6 (IL-6) blockade in autoimmune arthritis, by comparing the effect of anti–IL-6 receptor (anti–IL-6R) monoclonal antibody (mAb) treatment with the effect of soluble tumor necrosis factor (sTNFR)–Fc fusion protein treatment on T helper cell differentiation in collagen-induced arthritis (CIA).

Methods

DBA/1 mice were immunized with type II collagen (CII) to induce arthritis and were left untreated or were treated with anti-IL-6R mAb or TNFR-Fc. T helper cell differentiation and cytokine expression during the development of arthritis in these mice were analyzed.

Results

Immunization with CII predominantly increased the frequency of Th17 cells rather than Th1 cells. The frequency of FoxP3+ Treg cells was also increased after immunization. Treatment of mice with CIA with anti–IL-6R mAb on day 0 markedly suppressed the induction of Th17 cells and arthritis development, but treatment with this antibody on day 14 failed to suppress both Th17 differentiation and arthritis. In contrast, treatment of mice with CIA with TNFR-Fc from day 0 to day 14 suppressed neither Th17 differentiation nor arthritis, but treatment from day 21 to day 35 successfully ameliorated arthritis without inhibiting Th17 induction. Neither antibody treatment increased the frequency of Treg cells.

Conclusion

Our results indicate that the protective effect of IL-6 blockade, but not tumor necrosis factor (TNF) blockade, in CIA correlates with the inhibition of Th17 differentiation. Our findings suggest that IL-6 blockade in rheumatoid arthritis in human is also likely to involve a therapeutic mechanism distinct from that of TNF blockade and thus may represent an alternative therapy for patients in whom the disease is refractory to TNF blockade.

Although the etiology of rheumatoid arthritis (RA) is currently unknown, proinflammatory cytokines, such as interleukin-6 (IL-6), tumor necrosis factor α (TNFα), and interleukin-1 (IL-1), have been demonstrated to play key roles in the pathogenesis of RA (1). Furthermore, anti-TNFα therapy and therapies that inhibit IL-1 or IL-6 signaling have been shown to be effective in the treatment of RA. However, it is unknown whether these anticytokine therapies have modes of action distinct from each other. Importantly, a substantial proportion of patients show insufficient responses to a given anticytokine therapy. Thus, a clearer understanding of the protective mechanism of these therapies is important both in understanding the pathogenesis of autoimmune arthritis and in predicting an effective treatment for an individual patient.

Collagen-induced arthritis (CIA) is the most widely studied animal model of RA and has been useful not only in aiding understanding of the pathogenesis of this disease, but also in the development of new therapies (2). In mice with CIA, CD4 T cells are important in disease induction, and Th1 cells have been considered to be the major mediator of the disease. However, the notion that CIA is a Th1-mediated disorder has been challenged by studies using Th1-defective mice. Mice lacking interferon-γ (IFNγ), IFNγ receptor, or IL-12p35 develop accelerated arthritis after induction of CIA (1). Furthermore, recent studies have suggested that highly proinflammatory IL-17–producing Th17 cells, rather than Th1 cells, are central to the pathology of autoimmune arthritis (1, 3, 4). Consistent with the findings of these studies, neutralization (5) or genetic deletion (6) of IL-17 in mice inhibits the development of CIA. In patients with RA, IL-17 has been detected in synovial fluid, while IFNγ is rarely detectable (1).

To date, in vitro experiments using cultured murine CD4 T cells have revealed that the inflammatory cytokine milieu in the presence of transforming growth factor β (TGFβ) strongly induces Th17 differentiation (7). While some studies have demonstrated a role for IL-1 and TNFα in the development of Th17 cells in vitro (7, 8), the presence or absence of IL-6 is likely to be of particular importance, because stimulation of CD4 T cells in vitro with IL-6 plus TGFβ potently induces Th17 differentiation, whereas stimulation with TGFβ alone triggers the development of FoxP3+ immunosuppressive Treg cells (7, 9, 10). In humans, although the mechanism of Th17 differentiation is still poorly understood compared with that in mice, IL-6 has been shown to promote Th17 differentiation in vitro (11, 12). However, an IL-6–independent pathway of Th17 differentiation has also been reported (13, 14). Thus, the in vivo role of IL-6 in the development of Th17 cells remains to be established.

In this study, we investigated the protective effect of IL-6 blockade in vivo with respect to T helper cell development in CIA. We also examined the effect of TNFα blockade on Th17 differentiation in this model and compared it with that of IL-6 blockade.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Mice and induction of CIA.

Male DBA/1J mice were purchased from Charles River Japan (Yokohama, Japan). Mice were kept under specific pathogen–free conditions. Experiments were conducted in accordance with institutional ethics guidelines. CIA was induced as described previously (15), by intradermal immunization with bovine type II collagen (CII) (Cosmo Bio, Tokyo, Japan) and complete adjuvant H37Ra (Difco, Detroit, MI) on day 0 and day 21.

Anti-mouse IL-6 receptor (IL-6R) monoclonal antibody (mAb) and TNF receptor (TNFR)–Fc.

Rat anti-mouse IL-6R mAb (MR16-1) has been described previously (15). An optimal dose of MR16-1 (8 mg/mouse) was injected intraperitoneally on day 0 or day 14 after induction of CIA. As a control, rat IgG or phosphate buffered saline was injected in the same manner. TNFR-Fc (etanercept; 1 mg/mouse) purchased from Takeda Pharmaceutical (Osaka, Japan) was injected intraperitoneally 3 times per week.

Evaluation of arthritis severity in mice with CIA.

Clinical symptoms of arthritis were evaluated visually in each limb and graded on a scale of 0–4, where 0 = no erythema or swelling, 0.5 = swelling of 1 or more digits, 1 = erythema and mild swelling of the ankle joint, 2 = mild erythema and mild swelling involving the entire paw, 3 = erythema and moderate swelling involving the entire paw, and 4 = erythema and severe swelling involving the entire paw. The clinical score for each mouse was the sum of the scores for the 4 limbs (maximum score 16).

Anti-mouse IL-2 antibody.

Anti-mouse IL-2 antibody (S4B6) was purchased from BD Biosciences (San Diego, CA). To reduce the number of Treg cells, 600 μg of antibody was administered intraperitoneally to each mouse 5 days before CIA induction.

Cell preparations.

Single-cell suspensions were obtained from murine lymphoid organs as previously described (16). To obtain naive CD4 T cells, CD4 T cells were first enriched using MACS CD4 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Following surface staining as described below, CD4+CD62L+CD25− populations were further selected using the FACSAria cell sorter (BD Biosciences, Tokyo, Japan). The purity of these naive CD4 T cells was routinely >95%.

Flow cytometric analysis.

Surface antigens were stained as previously described (16) with the following antibodies: anti-CD4, anti-CD8, anti-CD25, and anti-CD62L (BD Biosciences). For the detection of FoxP3, cells were stained using a FoxP3 staining kit (eBioscience, San Diego, CA). To detect cytokine production by flow cytometry, cells were harvested after stimulation with 50 ng/ml of phorbol myristate acetate (Sigma, St. Louis, MO) and 750 ng/ml of ionomycin (Calbiochem, San Diego, CA) for 3 hours in the presence of 3 μM of monensin (Sigma). Intracellular cytokine staining was performed using the FoxP3 staining kit to detect FoxP3 simultaneously. Antibodies used were anti-IFNγ, anti–IL-4 (BioLegend, San Diego, CA), and anti–IL-17 (BD Biosciences). Stained cells were analyzed with FACSCanto (BD Biosciences). Each fluorescence-activated cell sorting (FACS) profile was obtained using cells from 1 mouse.

Cell cultures.

Cells were cultured in RPMI 1640 (Wako, Osaka, Japan) supplemented with 10% fetal calf serum (Hyclone, Irvine, CA), 2-mercaptoethanol (Nacalai Tesque, Kyoto, Japan), penicillin G, and streptomycin. Splenocytes and lymph node cells were cultured in the presence of CII (100 μg/ml; Chondrex, Redmond, WA). Naive CD4 T cells were stimulated with plate-bound anti-CD3e (1 μg/ml; BD Biosciences) and anti-CD28 (5 μg/ml; BD Biosciences). Conditioned medium (described below), human TGFβ (1 ng/ml; PeproTech, London, UK), anti–IL-6R (10 μg/ml; MR16-1), TNFR-Fc (10 μg/ml), anti–IL-21 (10 μg/ml; R&D Systems, Minneapolis, MN), anti–IL-23 (10 μg/ml; eBioscience), and/or mouse TNFα (10 ng/ml; PeproTech) was added to the culture. After 3 days, cells were harvested and analyzed.

Conditioned medium from bone marrow–derived dendritic cells (DCs).

Bone marrow cells from wild-type mice were cultured in the presence of murine granulocyte–macrophage colony-stimulating factor (20 ng/ml; R&D Systems) as described previously (13). Nine days later, nonadherent cells were harvested and restimulated with lipopolysaccharide (LPS) (10 μg/ml; Sigma). After a further 2 days, culture supernatants were harvested.

5,6-carboxyfluorescein succinimidyl ester (CFSE) assays.

Splenocytes and lymph node cells were labeled in 3 μM CFSE (Molecular Probes, Eugene, OR). These cells were then cultured with various factors (indicated in the figures) for 3 days, harvested, stained with antibodies against CD4, IL-17, and FoxP3 as described above, and analyzed with FACSCanto.

Cytokine measurements.

Serum concentrations of murine IL-17 were measured using Bio-Plex (Bio-Rad, Tokyo, Japan). IL-17 levels in culture supernatants were determined by enzyme-linked immunosorbent assay (R&D Systems).

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Collagen-induced arthritis (CIA) is a Th17-related disease and its severity is restrained by Treg cells.

First, we investigated T helper cell differentiation in response to CII immunization. Typically, DBA/1 mice developed signs of arthritis after the second immunization (given ∼21 days after the first CII immunization) and showed maximum arthritis around day 35 (data not shown) (15). As shown in Figure 1A, immunization of DBA/1 mice once with CII significantly increased the frequency of IL-17–producing CD4 T cells (Th17) in inguinal lymph nodes before the onset of arthritis (day 10) compared with that in nonimmunized mice (P < 0.05). The second CII immunization further increased the frequency of Th17 cells in lymph nodes at the onset of arthritis (day 26) (Figure 1A). Although IFNγ–producing Th1 cells were also induced by CII immunization in vivo, their frequency was lower than that of Th17 cells.

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Figure 1. Increased frequency of Th17 cells in mice with collagen-induced arthritis (CIA). A, Cytokine production in CD4 T cell populations in inguinal lymph node cells from nonimmunized mice and from type II collagen (CII)–immunized mice harvested on day 10 or day 26 after immunization. Cytokine production was examined by intracellular staining of interleukin-17 (IL-17) and interferon-γ (IFNγ). Values are the mean ± SD (n = 5 mice per group); values in boldface are representative data obtained from 1 mouse per group. ∗ = P < 0.05 versus nonimmunized controls, by Student's t-test. B, Cytokine production in inguinal lymph node cells from nonimmunized mice and from CII-immunized mice harvested on day 29 after immunization and cultured for 4 days in the presence of CII (100 μg/ml). Cytokine production was examined by intracellular staining, gating on CD4 T cells. Values are the mean ± SD (n = 3–4 mice per group) and are representative of 3 independent experiments with similar results; values in boldface are representative data obtained from 1 mouse per group. ∗ = P < 0.05 versus nonimmunized controls, by Student's t-test. C, Cytokine production in CD4 cells from the spleen and inflamed ankle joints of mice with CIA harvested on day 31 after immunization. Cytokine production was examined by staining with antibodies against CD4, IFNγ, and IL-17. Values are the mean ± SD (n = 2 mice per group) and are representative of 4 independent experiments with similar results; values at the top are representative data obtained from 1 mouse per group.

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Inguinal lymph node cells from mice with CIA were subsequently cultured in the presence of CII, and CII-responsive T helper subsets were analyzed. As shown in Figure 1B, the frequencies of Th17 and Th1 cells were significantly increased in cultured cells from immunized mice compared with those from nonimmunized mice (P < 0.05). In addition, the frequency of Th17 cells was higher than that of Th1 cells. Interestingly, while its significance is currently unknown, a fraction of CD4 T cells that produced both IFNγ and IL-17 was noted in immunized mice, as reported previously in human studies (17). An analysis of T helper cell differentiation in lymphocytes collected from inflamed ankle joints was subsequently performed. As shown in Figure 1C, CD4 T cells in inflamed joints produced effector cytokines at higher levels than did splenic CD4 T cells. In addition, these cells preferentially produced IL-17 rather than IFNγ. Taken together, these results indicate that CD4 T cells of DBA/1 mice preferentially differentiate into Th17 cells in response to CII immunization and support the notion that CIA is a Th17-related, rather than a Th1-related, disease.

We then investigated the development of FoxP3+ Treg cells in CIA. Interestingly, we found that the frequency of FoxP3+ Treg cells was significantly increased after 1 or 2 rounds of CII immunization (P < 0.05) (Figure 2A). The up-regulation of Treg cells may reflect a physiologic countermechanism that regulates harmful autoimmune responses. To address this issue, we used an anti–IL-2 neutralizing antibody that, upon intraperitoneal injection, selectively reduces natural CD4+CD25+ Treg cells in mice (18). Anti–IL-2 treatment before CIA induction significantly reduced the frequency of CD4+CD25+ and CD4+FoxP3+ T cells in the peripheral blood (P < 0.05) (Figures 2B and C). Unexpectedly, the onset of arthritis was delayed in mice pretreated with anti–IL-2 (Figure 2D). This finding is most likely due to the loss of IL-2 itself and is consistent with the previously reported observation that administration of recombinant human IL-2 at the onset of disease exacerbates CIA (19). However, following the onset of disease, the severity of arthritis is markedly enhanced in mice pretreated with anti–IL-2. These results support the notion that Treg cells play an important role in the amelioration of CIA (20).

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Figure 2. Increased frequency of FoxP3+ Treg cells in mice with CIA. A, CD4 and FoxP3 in inguinal lymph node cells from nonimmunized mice and from CII-immunized mice harvested on day 10 or day 26 after immunization and examined by flow cytometry. Values are the mean ± SD (n = 5–6 mice per group); values in boldface are representative data obtained from 1 mouse per group. ∗ = P < 0.05 versus nonimmunized controls, by Student's t-test. B and C, Frequency of CD4+CD25+ cells (B and left panel in C) and CD4+FoxP3+ cells (right panel in C) in mice treated with phosphate buffered saline (control) and mice treated with anti–IL-2 antibody 5 days before the first immunization with CII. The effect of anti–IL-2 antibody on the frequency of peripheral Treg cells was monitored by fluorescence-activated cell sorting of blood cells 2 days before the first immunization. Bars in C show the mean and SD. ∗ = P < 0.05 versus controls, by Student's t-test. D, Arthritis scores in mice treated with anti–IL-2 antibody or control vehicle and visually evaluated after the first immunization with CII. Values are the mean and SEM (n = 5 mice per group). See Figure 1 for definitions.

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Inhibition of Th17 differentiation and promotion of Treg cell differentiation in vitro by anti–IL-6R antibody (MR16-1).

Previous studies have demonstrated that inflammatory cytokines derived from activated DCs are potent promoters of Th17 differentiation (7). Among these cytokines, IL-6 can potently induce Th17 differentiation and can reciprocally inhibit Treg cell differentiation in vitro (4). To test the effect of rat anti–IL-6R mAb on T helper cell differentiation in vitro, we first prepared conditioned medium comprising culture supernatants of LPS-stimulated bone marrow–derived DCs, which mimic an inflammatory cytokine milieu. When sorted CD62LhighCD25− naive CD4 T cells were stimulated with anti-CD3 plus anti-CD28 in the presence of the conditioned medium, the differentiation of Th17 cells was slightly enhanced and that of FoxP3+ Treg cells was suppressed (Figure 3A). Consistent with the results of a previous study (7), the addition of TGFβ to the conditioned medium dramatically increased the frequency of Th17 cells (Figure 3A).

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Figure 3. Effects of anti–interleukin-6 receptor (anti–IL-6R) antibody (MR16-1) and tumor necrosis factor receptor (TNFR)–Fc on T helper cell differentiation in vitro. CD4+CD62LhighCD25− naive T cells were stimulated with plate-bound anti-CD3 and anti-CD28. A, IL-17 and FoxP3 expression in CD4+ cells cultured in the presence of conditioned medium from bone marrow–derived dendritic cells (DCCM) alone or conditioned medium and human transforming growth factor β (hTGFβ). B, IL-17 and FoxP3 expression in CD4+ cells cultured in the presence of conditioned medium and human TGFβ, with either anti–IL-6R monoclonal antibody (mAb) (MR16-1), TNFR-Fc, tumor necrosis factor α (TNFα), anti–IL-21 mAb, or anti–IL-23 mAb. After 3 days of culture, intracellular staining of IL-17 and FoxP3 was performed. Gated CD4 T cell populations are shown. Values are the mean ± SD (n = 2 mice per group) and are representative of 3 independent experiments with similar results; values in boldface are representative data obtained from 1 mouse per group.

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We then investigated the effects of anticytokine treatment on Th cell differentiation. As shown in Figure 3B, the addition of MR16-1 to the cell culture potently inhibited Th17 differentiation in response to TGFβ and conditioned medium and partially restored Treg cell induction. Neither TNF blockade with TNFR-Fc nor the addition of TNFα to the culture inhibited Th17 cell induction or restored Treg cell generation. Treatment with other anticytokine antibodies showed slight inhibitory effects on the induction of Th17 cells, but their effects were much less marked than that observed with MR16-1 treatment. In addition, an increase in the frequency of Treg cells was observed in MR16-1–treated cultures only (Figure 3B).

Taken together, these results indicate that IL-6 is a key inflammatory cytokine that regulates Th17 and Treg cell development in vitro. In addition, IL-6 blockade by MR16-1 within an inflammatory cytokine milieu is effective in inhibiting the development of Th17 cells in vitro.

Inhibition of CII induction of Th17 cells in DBA/1 mice by in vivo treatment with MR16-1 on day 0 but not on day 14.

We then tested the effect of MR16-1 on T helper cell differentiation in vivo. DBA/1 mice were immunized once with CII, and MR16-1 was administered intraperitoneally on the same day. Eight days after the first CII immunization, the cytokine profile of CD4 T cells in inguinal lymph node cells was determined using flow cytometry. As shown in Figure 4A, Th17 cells were significantly less frequent in mice treated with MR16-1 than in control mice (P < 0.05), suggesting that MR16-1 inhibited Th17 differentiation in vivo. In contrast, MR16-1 treatment did not affect the frequency of Th1 cells. Interestingly, the frequency of Treg cells tended to be slightly lower in mice treated with MR16-1 than in control mice (Figure 4A). Thus, these results did not demonstrate the promoting effect of MR16-1 on Treg cell differentiation.

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Figure 4. Effect of MR16-1 treatment in vivo on arthritis development and T helper cell differentiation in CIA. A, IL-17, IFNγ, and FoxP3 in CD4+ inguinal lymph node cells from mice treated intraperitoneally with phosphate buffered saline (control) or MR16-1 on day 0 and harvested on day 8. Intracellular staining of IFNγ and IL-17 or FoxP3 was performed. Gated CD4 T cell populations are shown. Values are the mean ± SD (n = 3 mice per group) and are representative of 3 independent experiments with similar results; values in boldface are representative data obtained from 1 mouse per group. ∗ = P < 0.05 versus controls, by Student's t-test. B, Gross appearance of the hind limbs from a control mouse treated intraperitoneally with rat IgG on day 0 (d0), a mouse treated intraperitoneally with MR16-1 on day 0, and a mouse treated intraperitoneally with MR16-1 on day 14 (day 35 after the first CII immunization). C, IL-17, IFNγ, and FoxP3 in CD4+ inguinal lymph node cells from mice treated with rat IgG on day 0, MR16-1 on day 0, rat IgG on day 14, or MR16-1 on day 14 and harvested on days 26–28. Intracellular staining of IFNγ and IL-17 or FoxP3 was performed. Values are the mean ± SD (n = 4 mice per group) and are representative of 3 independent experiments with similar results; values in boldface are representative data obtained from 1 mouse per group.∗ = P < 0.05 versus controls, by Student's t-test. D, Levels of IL-17 in culture supernatants of inguinal lymph node cells (left) and serum (right). Inguinal lymph node cells were stimulated in vitro with CII for 3 days and culture supernatants were harvested. IL-17 concentration in supernatants was determined by enzyme-linked immunosorbent assay. Serum IL-17 concentrations were determined by Bio-Plex. Bars show the mean and SD for inguinal lymph nodes and the mean and SEM for serum (n = 4 mice per group). Ab = antibody (see Figure 1 for other definitions).

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As we reported previously (15), a single injection of MR16-1 on day 0 (on the same day as the first CII immunization) was sufficient to ameliorate joint inflammation in CIA (Figure 4B). We investigated Th17 cell development after the second CII immunization in MR16-1–treated mice. Similar to findings in mice after the first CII immunization, the frequency of Th17 cells in lymph nodes was significantly lower in MR16-1–treated mice than in control mice with CIA on day 28 (P < 0.05). Th1 differentiation was only marginally inhibited by MR16-1 treatment, and the frequency of Treg cells was not increased by MR16-1 treatment (Figure 4C). These results suggest that MR16-1 treatment on day 0 was sufficient to suppress the induction of Th17 cells that occurred in mice following the second immunization with CII. In contrast to administration of MR16-1 on day 0, the delayed administration of MR16-1 on day 14 failed to suppress the development of CIA (Figure 4B), which was consistent with the results of our previous study (15). In accordance with these findings, we found that on day 28, the frequency of Th17 cells and Treg cells in lymph nodes of mice that received MR16-1 on day 14 was comparable with that in control mice with CIA (Figure 4C). This result suggests that delayed treatment with MR16-1 is ineffective in the suppression of Th17 induction in CIA.

We then evaluated CII-specific IL-17 production from lymph node cells in vitro. As shown in Figure 4D, lymph node cells from control mice with CIA produced high amounts of IL-17 in response to CII stimulation. In contrast, when lymph node cells from mice treated with MR16-1 on day 0 were stimulated with CII, they produced dramatically reduced amounts of IL-17. In addition, lymph node cells from mice treated with MR16-1 on day 14 produced levels of IL-17 similar to those in mice treated with control antibody (Figure 4D, left). These results indicated that in vivo administration of MR16-1 on day 0, but not on day 14, effectively inhibited CII-induced Th17 cell development in mice. Similar results were observed when we evaluated serum levels of IL-17 in mice treated with MR16-1 (Figure 4D, right panel). Thus, MR16-1 must be administered at the same time as the initial immunization with CII to prevent the development of CII-specific Th17 cells. The inhibition of Th17 development by MR16-1 is likely to represent an important mechanism of action in the suppression of CIA.

In vivo treatment with MR16-1 on day 0 inhibits the expansion of CII-responsive Th17 cells, but not CII-responsive Treg cells, upon second stimulation with CII in vitro.

It is of interest that a single injection of MR16-1 on day 0 is sufficient to inhibit the induction of Th17 cells even after a second immunization with CII. We therefore obtained splenocytes and lymph node cells on day 8 from untreated mice immunized once with CII and from mice treated with MR16-1 (on day 0) immunized once with CII, and then stimulated these cells with CII in vitro, thereby mimicking a second in vivo CII immunization. At the same time, we labeled these cells with CFSE to monitor the CII-responsive CD4 cell population (CFSElowCD4+ cells) using FACS. As shown in Figure 5A, secondary stimulation in vitro with CII induced the expansion of splenic CD4 T cells from both control CII-immunized mice and MR16-1–treated mice, but the response was slightly reduced in the latter.

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Figure 5. Analysis of CII-responsive CD4 T cells by 5,6-carboxyfluorescein succinimidyl ester (CFSE) dilution assay in splenocytes (A) and inguinal lymph node cells (B) from mice treated intraperitoneally with phosphate buffered saline on day 0 (control) and mice treated intraperitoneally with MR16-1 on day 0 (MR16-1) and in splenocytes from control mice, with MR16-1 added to the culture as indicated (C), on day 8 after the first CII immunization. Cells were labeled with CFSE and cultured in the presence of CII. After 3 days, cells were harvested, stained with CD4, FoxP3, and IL-17, and analyzed by flow cytometry. Gated populations are shown. Values are the mean ± SD (n = 3 mice per group) and are representative of 2 independent experiments with similar results; values in boldface are representative data obtained from 1 mouse per group. ∗ = P < 0.05 versus controls, by Student's t-test. Ab = antibody (see Figure 1 for other definitions).

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We further investigated the frequency of CII-responsive Th17 cells and Treg cells by means of gating on CFSElowCD4 T cells. As shown in Figure 5A, CII-responsive splenic CD4 T cells from control mice contained a high frequency of Treg cells as well as Th17 cells, albeit at a lower frequency. In contrast, CII-responsive CD4 T cells from MR16-1–treated mice contained a significantly reduced fraction of Th17 cells compared with control mice (P < 0.05) but had a comparable or somewhat increased fraction of Treg cells.

Similar findings were obtained when we examined lymph node cells from control mice with CIA and from MR16-1–treated mice (Figure 5B). These results suggest that MR16-1 treatment on day 0 modulates T helper cell differentiation following the first CII immunization and promotes the expansion of protective Treg cells without enhancing the expansion of Th17 cells after the second CII immunization.

We then obtained spleen cells from immunized mice and stimulated them with CII in the presence of MR16-1, thus mimicking delayed MR16-1 treatment. As shown in Figure 5C, while MR16-1 in vitro marginally inhibited the proliferation of CD4 T cells, it had no inhibitory effect on the expansion of Th17 cells. This result suggests that delayed administration of MR16-1 in CIA is ineffective in inhibiting the expansion of pathogenetic Th17 cells.

Lack of association between CIA suppression by TNFR-Fc and Th17 inhibition.

TNFα blockade is effective in the treatment of CIA and has been used as therapy for RA in humans (1). To investigate the action of TNFα blockade on Th17 development in CIA, we immunized mice with CII and treated them with sTNFR-Fc from day 0 to day 14 (3 times per week). Interestingly, the administration of TNFR-Fc during the induction of CIA failed to inhibit the development of arthritis (Figure 6A).

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Figure 6. Effect of tumor necrosis factor receptor (TNFR)–Fc treatment in vivo on arthritis development and T helper cell differentiation. A, Arthritis scores in mice treated with phosphate buffered saline (control), mice treated with TNFR-Fc from day 0 (d0) to day 14, and mice treated with TNFR-Fc from day 21 to day 35. Values are the mean and SEM (n = 6–8 mice per group). B, IL-17, IFNγ, and FoxP3 in CD4+ inguinal lymph node cells from control mice, mice treated with TNFR-Fc from day 0 to 14, and mice treated with TNFR-Fc from day 21 to day 35 and harvested on days 26–27. Intracellular staining of IFNγ and IL-17 or FoxP3 was performed. Gated CD4 T cell populations are shown. Values are the mean ± SD (n = 3 mice per group) and are representative of 3 independent experiments with similar results; values in boldface are representative data obtained from 1 mouse per group. C, Serum levels of IL-17 in control mice, mice treated with TNFR-Fc from day 0 to day 14, and mice treated with TNFR-Fc from day 21 to day 35, as determined by Bio-Plex. Bars show the mean and SEM (n = 3 mice per group). See Figure 1 for other definitions.

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We subsequently treated mice with CIA with TNFR-Fc at later time points, beginning on day 21. In sharp contrast to administration during the induction phase, TNF blockade beginning on day 21 potently suppressed the development of arthritis (Figures 6A and B). However, analysis of lymph node cells revealed that Th17 cells in mice treated with TNFR-Fc beginning on day 21 were increased to levels similar to those in control mice and mice treated with TNFR-Fc on day 0 (Figure 6B). We observed a slight decrease in Treg cells in mice treated with TNFR-Fc beginning on day 21 (Figure 6B). In addition, serum levels of IL-17 in both TNFR-Fc–treated groups were similar to those in control mice (Figure 6C). These results indicate that TNFα plays a minor role in the induction of Th17 cells in CIA, and thus strongly suggests that the protective mechanism of TNF blockade in CIA is distinct from that of anti–IL-6R therapy.

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

In this study, we demonstrated that Th17 cells represent the dominant effector T helper cell subset in CIA. Our findings are consistent with the crucial role of IL-17 in the pathology of CIA demonstrated by studies of IL-17–deficient mice or mice treated with anti–IL-17 antibody (5, 6). We also showed that partial depletion of Treg cells in CIA following administration of a neutralizing anti–IL-2 antibody results in delayed but enhanced arthritis, indicating that Treg cells play a significant protective role in reducing the severity of CIA. Thus, since the development of these 2 T helper subsets in vitro are modulated by proinflammatory cytokines, including IL-6 and TNFα, we compared the protective effect in vivo of IL-6 blockade with that of TNFα blockade in CIA, with respect to T helper cell development.

MR16-1 treatment on day 0 inhibited the induction of Th17 cells, indicating that IL-6 is a critical cytokine regulating Th17 development not only in vitro, but also in vivo, during the development of CIA. Moreover, the inhibitory effect of MR16-1 treatment on day 0 on Th17 development was also observed after the second CII immunization, and these mice developed less severe arthritis than did control mice with CIA. In contrast, when MR16-1 was administered on day 14, neither Th17 cell induction nor arthritis development was inhibited. These results indicate that the protective efficacy of MR16-1 is correlated with the inhibition of initial Th17 differentiation. These results also may explain why MR16-1 treatment at later points is much less effective than that at the early stage of CIA (15).

We found that MR16-1 treatment in vivo did not increase, but instead reduced, the frequency of Treg cells in CIA. Since the induction of CIA results in an increase in the frequency of Treg cells, possibly as a counteracting mechanism to limit inflammation, the reduced severity of arthritis we observed in MR16-1–treated mice may in turn lead to a reduction in Treg cell frequency in these mice. Although some evidence indicates that IL-6 inhibits TGFβ-induced generation of adoptive Treg cells in vitro, it remains unclear whether IL-6 interferes with the development of thymus-derived natural FoxP3+ Treg cells, a major population of Treg cells in vivo. In this context, we recently found that the frequency of FoxP3+ Treg cells is not reduced in IL-6–transgenic mice (Fujimoto M, et al: unpublished observations). Thus, it is likely that IL-6 plays a minor or limited role in the regulation of natural Treg cell development in vivo.

However, our analyses left open the possibility that MR16-1 may contribute to the induction of adoptive Treg cells in CIA. In CII-responsive CD4 T cells identified by CFSE dilution assay, in which CII-responsive adoptive Treg cells were presumably enriched, we found that MR16-1 treatment on day 0 slightly, although not significantly, increased the frequency of FoxP3+ Treg cells. However, even in this CII-responsive population, we observed a stronger effect of MR16-1 on Th17 cells than that on Treg cells. Thus, while MR16-1 may have an antiinflammatory effect by shifting the balance of Th17 and Treg cells, we believe at present that the major inhibitory action of MR16-1 on CIA is the suppression of Th17 development.

The present study did not reveal any protective mechanisms of IL-6 blockade other than the regulation of initial T helper responses. IL-6 is a pleiotropic cytokine involved in a variety of inflammatory responses. For example, IL-6 induces mononuclear cell recruitment at the inflammatory site. IL-6 can function as a growth factor for T cells and stimulate B cells to induce antibody production (21, 22). IL-6 can promote bone destruction by inducing synovial cell proliferation and osteoclast differentiation (23). These functions of IL-6 are believed to play a pathologic role in the effector phase of autoimmune arthritis and, presumably, are attenuated by IL-6 blockade. A clear limitation of MR16-1 (rat IgG1) compared with TNFR-Fc in CIA is that repeated injection of this antibody in mice is not possible because it induces an anaphylactoid reaction (Mihara M, et al: unpublished observations). However, in a previous study of glucose-6-phosphate isomerase–induced arthritis, another model of human RA, a single administration of MR16-1 either before or after disease onset showed protective efficacy (24), suggesting an effect of MR16-1 that is distinct from that on T helper cell differentiation. Nevertheless, the results of the present study suggest that the modulation of T helper responses, and particularly the inhibition of Th17 cell development, by the early administration of MR16-1 is the key protective mechanism for the observed inhibition of CIA.

As a comparison with MR16-1 treatment, mice with CIA were treated with TNFR-Fc, an established therapy for experimental arthritis models (25) and human RA. In accordance with the results of our in vitro experiments, TNFR-Fc treatment from day 0 to day 14 failed to inhibit Th17 development in CIA. Moreover, TNFR-Fc treatment at this phase did not suppress arthritis. However, unlike MR16-1 treatment, TNFR-Fc treatment after the second immunization successfully inhibited CIA without affecting the development of Th17 cells. These results clearly indicate that TNFα blockade by TNFR-Fc in vivo in CIA has no inhibitory effect on the induction of Th17 cells and, thus, suggest a critical role of TNFα in the effector phase of CIA. The effect of TNFα blockade cannot be attributed to the induction of Treg cells, since their frequency in mice treated with TNF-Fc beginning on day 21 was not increased, but rather, was slightly reduced, presumably reflecting the amelioration of arthritis in these mice. Taken together, these results suggest that there are considerable differences in the protective mechanisms of action between IL-6 blockade and TNFα blockade in vivo. Thus, the blockade of IL-6 may represent an alternative treatment for RA patients whose disease has previously failed to respond to TNFα blockade.

Clinical trials of anti–IL-6R antibody (tocilizumab) demonstrated that IL-6 blockade is highly efficacious in the treatment of established RA (26–28), and tocilizumab has been approved as a therapy for RA in Japan. It is likely, however, that antirheumatic drugs generally work better in early disease than in established RA (29). In addition, there is the hypothesis of “window of opportunity,” a concept that early diagnosis coupled with early therapy might induce clinical remission (29). In this context, anti–IL-6R antibody, as a modulator of initial T helper responses, appears to be an attractive therapy for early RA to induce clinical remission. A more aggressive therapy such as the combination of anti–IL-6R antibody and anti-TNF agents might be more advantageous, since these agents appear to have nonoverlapping mechanisms.

Future studies are needed to establish the effect of anti–IL-6R therapy on T helper responses in human RA and to assess its clinical impact on the outcome and prognosis of the disease.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Dr. Naka had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Fujimoto, Kishimoto, Naka.

Acquisition of data. Fujimoto, Serada, Mihara, Uchiyama, Yoshida, Koike, Ohsugi, Nishikawa, Kimura, Naka.

Analysis and interpretation of data. Fujimoto, Naka.

Manuscript preparation. Fujimoto, Ripley, Naka.

Statistical analysis. Fujimoto.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES