Interleukin-22 Reduces the Severity of Collagen-Induced Arthritis in Association With Increased Levels of Interleukin-10

Authors


Abstract

Objective

The mechanism of action of interleukin- 22 (IL-22) in inflammatory arthritis remains unknown. IL-22–deficient mice exhibit an intact humoral and cellular immune response to collagen and yet have a reduced incidence of collagen-induced arthritis (CIA). Further, administration of anti–IL-22 does not reduce the severity of clinical arthritis but rather improves only certain aspects of joint inflammation as assessed histologically. This study was undertaken to investigate the mechanism of action and role of systemic IL-22 in modulating target organ inflammation.

Methods

CIA was induced in DBA mice by immunization with collagen and Freund's complete adjuvant. Expression of IL-22 and its receptor (IL-22R) in lymphoid organ and target tissues was determined during various phases of arthritis. The effector functions of IL-22 on induction/regulation of various cytokines in in vitro restimulation cultures were analyzed by enzyme-linked immunosorbent assay (ELISA). Recombinant IL-22 with or without anti–IL-10 antibody was administered to mice following immunization with collagen and prior to the onset of arthritis, and the severity of arthritis was evaluated by clinical scoring and histopathologic assessment. Anticollagen antibodies in mouse sera were analyzed by ELISA.

Results

IL-22 and IL-22R were up-regulated in lymphoid organs and joints during the course of arthritis. IL-22 augmented IL-10, IL-17, and IL-6 in lymphoid tissues in vitro. Administration of recombinant IL-22 was associated with an increase in IL-10 levels in vivo and a significant reduction in the progression of arthritis severity. Anti–IL-10 antibody treatment was associated with the abrogation of this protective effect of IL-22.

Conclusion

Our data demonstrate, for the first time, that IL-22 has a protective role in inflammatory arthritis.

Interleukin-22 (IL-22) belongs to the IL-10 family of cytokines, which also includes IL-19, IL-20, IL-24, and IL-26 (1). IL-22 is produced by a variety of cells, including T cells, natural killer (NK) cells (NK22 cells), γ/δ T cells, and lymphoid tissue inducer cells (2–7). IL-22 receptor (IL-22R) is a heterodimeric complex of IL-22RI and the IL-10R receptor (IL-10RII or IL-10Rβ) shared by IL-10, IL-26, and IL-28/IL-19 (8).

IL-22 has been shown to play a protective role in several types of bacterial infection (9, 10). It is protective in hepatitis, myocarditis, and inflammatory bowel disease (11–14). On the other hand, experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis, was shown to be independent of IL-22 (15). Levels of IL-22 are elevated in psoriatic skin, administration of neutralizing antibody to IL-22 is associated with amelioration of psoriasis in a preclinical model, and IL-22 has been shown to be critical in mediating IL-23–induced dermal acanthosis associated with psoriasis (16–18). Recently, IL-22 was shown to be protective in a T cell–dependent model of autoimmune uveitis and in antigen-induced eosinophilic airways inflammation (19, 20).

Expression of IL-22 and IL-22R is increased on rheumatoid arthritis synovial fibroblasts. IL-22 has been shown to induce the chemokine CCL2 and to induce proliferation of synovial fibroblasts in vitro (21). In a model of spontaneous inflammatory arthritis in IL-1R antagonist–deficient mice, administration of anti–IL-22 was not associated with significant reduction in the clinical severity of joint inflammation. However, histologic examination of the joint tissue showed reductions in discrete parameters of joint inflammation, namely pannus formation and proteoglycan depletion (22). Interestingly, the phenotype of IL-22–deficient mice in the context of inflammatory arthritis is complex. These mice exhibit an increased humoral immune response and an unaltered cellular immune response to type II collagen, and yet have a reduced incidence of arthritis (23). In the same study it was shown that IL-22 induces osteoclastogenesis in vitro and that this effect may be responsible for the reduced incidence of arthritis in IL-22–deficient mice (23). These findings suggest that IL-22 has a pathogenic role in the joint. Its mechanism of action in inflammatory arthritis remains unknown, however. Comprehensive understanding of the mechanism of action of IL-22 is critical to the development of therapeutic agents targeting this pathway.

In this report we provide evidence that IL-22 has pleiotropic effects on a variety of proinflammatory as well as antiinflammatory cytokines during various phases of the systemic immune response leading to inflammatory arthritis. Administration of IL-22 was associated with reduction in the progression of arthritis, an effect that was abrogated upon neutralization of IL-10. These findings are suggestive of an antiinflammatory role of systemic IL-22 in collagen-induced arthritis (CIA).

MATERIALS AND METHODS

Mice and CIA induction.

Male DBA/1LacJ mice (8–10 weeks old; The Jackson Laboratory) were housed under specific pathogen–free conditions at the University of Arizona Animal Care Facility. Preparation of collagen and adjuvant (Freund's complete adjuvant [CFA]), immunization, and clinical scoring of arthritis were performed as previously described (24). All procedures were approved by the University of Arizona Committee for the Use and Care of Animals.

Recombinant IL-22 and anti–IL-10 neutralizing antibody protocol.

Recombinant IL-22 (Insight Genomics) was administered to mice at 5 μg/day. Neutralizing rat antibody to IL-10 (clone JES5-2A5; BioLegend) was administered at 100 μg/day. Rat IgG (100 μg/day) or Hanks' balanced salt solution (HBSS, used to dissolve recombinant IL-22) were used as controls. Recombinant IL-22, anti–IL-10 antibody, rat IgG, or HBSS was injected intraperitoneally for a total of 10–12 days, starting on approximately day 20–22.

Tissue harvest and assays.

Mice were killed by isoflurane inhalation. Blood was collected by cardiac puncture, placed into tubes, and serum was frozen at −80°C for anticollagen antibody assays that were performed at a later date. Spleen and inguinal lymph nodes were collected, and single-cell suspensions were made and used in a variety of restimulation assays. Restimulation was performed by culturing single-cell suspensions of spleen or lymph node cells (both at 5 × 106/ml) with 5 μg/ml of anti-CD3 (clone 1452C11; BioLegend) for 3 days, with 100 μg/ml of chicken collagen for 6 days, or with 2.5 μg/ml Mycobacterium tuberculosis H37Ra (the adjuvant used along with collagen for immunization of mice for CIA induction) (Difco) for 3 days. For some cultures, recombinant IL-22 (100 ng/ml), anti–IL-22 antibody (10 μg/ml), or rat IgG (10 μg/ml; Sigma-Aldrich) was used. Anti–IL-22 antibody (clone 8E11) was a kind gift from Dr. Wenjun Ouyang (Genentech, South San Francisco, CA). Supernatants were collected at the end of the culture period and analyzed for various cytokines. Paws with arthritis were dissected at the fur line, cut into small pieces after removal of overlying skin, and then digested with collagenase to make a single-cell suspension. The paw single-cell suspension (5 × 106/ml) was restimulated in a manner similar to that described above for cells from lymphoid organs, and culture supernatants were frozen for performance of cytokine assays at a later date.

For some experiments, splenocytes were incubated with microbeads coated with anti-CD3, anti-CD4, anti-CD8, anti-CD49b, anti–γ/δ T cell receptor (TCR), anti-CD19, anti-CD11b, or anti-CD11c monoclonal antibody (all from Miltenyi-Biotech). Bead-bound cells were enriched using magnetic separation columns. The purity of the enriched CD3+, CD4+, CD8+, CD49b+, γ/δ TCR+, CD19+, CD11b+, and CD11c+ cells was 95%, 96%, 95%, 95%, 96%, 93%, 94%, and 95%, respectively. Enriched cells were cultured for 3 days at 5 × 106/ml with or without IL-22 or H37Ra, and supernatants were frozen for cytokine assay at a later date. For some experiments, enriched cells (5 × 106/ml) were cultured for 6 hours with phorbol myristate acetate (PMA)/ionomycin (both from Sigma-Aldrich) and supernatants frozen for cytokine assay at a later date.

Cytokine assay.

IL-22, interferon-γ (IFNγ), IL-17, IL-6, tumor necrosis factor α (TNFα), IL-1β, and IL-10 were measured using enzyme-linked immunosorbent assay (ELISA) kits according to the protocols recommended by the manufacturers (R&D Systems for the IL-22 kit; BioLegend for all others). For intracellular flow cytometric analysis of IL-22, splenocytes were stimulated for 6 hours with 5 ng/ml PMA/500 ng/ml ionomycin and brefeldin A (BD Biosciences), followed by surface staining for anti-CD4 or the relevant isotype control. The cells were then treated with Cytofix/Cytoperm according to the instructions of the manufacturer (BD PharMingen). Intracellular cytokine staining was performed with anti–IL-22 antibody and relevant isotype controls. Anti-CD4 antibody and relevant control for flow cytometry were purchased from BioLegend. Anti–IL-22 (clone IL22JOP) and relevant isotype control were purchased from eBioscience. Data were acquired with BD LSR and analyzed using FlowJo software.

Real-time gene expression analysis.

RNA was prepared from spleen or paw specimens obtained from mice with arthritis in various stages, using Mini or Midi RNA isolation columns (Qiagen). RNA was then transcribed to complementary DNA using a reverse transcription kit (Applied Biosystems). Real-time gene expression analysis for IL-22, IL-22R, or IL-10 was performed using inventoried TaqMan primers and probes from Applied Biosystems. GAPDH was used as an internal control. Data were analyzed with SDS software.

Anticollagen antibody assay.

Ninety-six–well ELISA plates were coated overnight with type II collagen and blocked for 1–2 hours with 10% fetal calf serum. Plates were then washed, and diluted serum (1:8,000–1:16,000) was loaded in triplicate, followed by incubation overnight at 4°C. Following this, plates were washed and incubated for 1 hour at room temperature with biotin-conjugated rat anti-mouse IgG1, IgG2a, or IgG2b and streptavidin–horseradish peroxidase. The reaction was developed using tetramethylbenzidine substrate, stopped with acidic solution, and read at 450 nm. As a standard, a representative mouse serum was serially diluted and assigned arbitrary values.

Tissue histologic examination and scoring.

Paws were dissected at the fur line and fixed in neutral buffered formalin. Further tissue processing, sectioning, and hematoxylin and eosin (H&E) staining were performed at the Pathology Services Laboratory, University of Arizona. The tissue sections were scored, using a previously described scoring system (24), by personnel at the Pathology Services Laboratory who were blinded with regard to the treatment protocol.

Statistical analysis.

All ELISAs and real-time assays were performed in triplicate. Data are presented as the mean ± SEM. Statistical significance was determined by Student's t-test or Mann-Whitney test. P values less than 0.05 were considered significant.

RESULTS

Association of arthritis onset with increased expression of IL-22 and IL-22R.

CIA is induced by immunization with collagen and CFA. There is a significant period, lasting 4–5 weeks, following immunization with collagen and prior to the onset of clinical arthritis, which is associated with dynamic changes in proinflammatory as well as antiinflammatory responses. For the purposes of this study we defined the first 2 weeks following immunization with collagen as the initiation phase, days 2–3 after arthritis onset as early arthritis, and days 7–14 after arthritis onset as late arthritis.

Longitudinal analysis of IL-22 expression in lymphoid organs during various phases of arthritis showed that IL-22 expression was induced during early arthritis, with increased levels of IL-22 in arthritic paws (Figure 1A). Such an increase was not seen in paws from naive mice or in paws during the initiation phase. IL-22 was measured in sera from naive mice, and from immunized mice during the initiation phase and early arthritis. It was found that IL-22 was present in the serum in very low amounts, which precluded reliable assessment. Intracellular flow cytometry of splenocytes obtained during various phases of arthritis revealed the expansion of IL-22+ cells during early arthritis (Figure 1B). Since only 0.5% of splenocytes were IL-22+ by flow cytometry, we decided to elucidate the subset of IL-22 producers by enriching for specific subsets, followed by brief stimulation with PMA/ionomycin and measurement of IL-22 in culture supernatants. Among the various subsets assessed, including CD4+ cells, CD8+ cells, γ/δ T cells, CD49b+ cells, CD19+ cells, CD11b+ cells, and CD11c+ cells, IL-22 was produced exclusively by CD4+ and CD49b+ cells (NK cells) (Figure 1C).

Figure 1.

Induction of interleukin-22 (IL-22) responses during arthritis. A and D, Splenocytes and paws from naive mice, mice from the initiation phase (2 weeks following immunization with collagen), and mice with early arthritis were collected for preparation of mRNA followed by real-time polymerase chain reaction analysis for IL-22 (A) and IL-22 receptor type I (IL-22RI) (D). GAPDH was used as an internal control. Data are presented as the fold change over GAPDH expression. B, Levels of IL-22 in sera from naive mice, mice from the initiation phase, and mice with early arthritis were measured by enzyme-linked immunosorbent assay (ELISA), and splenocytes from mice in the 3 groups were stained for CD4 and IL-22 and assessed by flow cytometry. C, Splenocytes from mice with early arthritis were enriched for CD4+ T cells, CD8+ T cells, γ/δ T cells, CD49b+ cells, CD19+ cells, CD11b+ cells, or CD11c+ cells, stimulated for 6 hours with phorbol myristate acetate/ionomycin, and IL-22 levels in the supernatants analyzed by ELISA. Data are representative of 3 independent experiments. Values are the mean ± SEM. P values were determined by Student's t-test. NS = not significant. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.37849/abstract.

Consistent with the increased expression of IL-22, we observed increased expression of IL-22 receptor (IL-22RI) in the lymphoid organs and arthritic paws (Figure 1D). IL-22R is composed of 2 subunits: IL-22RI and IL-10RII. IL-10RII is the subunit shared between IL-10, IL-26, and IL-28/IL-19 receptor complexes. The IL-22RI subunit specifically binds to IL-22 (25). Additionally, naive mice have high levels of IL-22 receptor (IL-22RI) expression in lymphoid organs and detectable levels (IL-22RI) in paws.

Induction of proinflammatory as well as antiinflammatory cytokines by IL-22 in vitro during CIA.

CIA is attenuated in IL-22–knockout mice (23) and, as seen in Figure 1, IL-22 and IL-22RI are up-regulated during arthritis. However, the mechanism of the effector function of IL-22 is unknown. We restimulated single-cell suspensions of draining inguinal lymph node or spleen cells in the presence or absence of IL-22 and evaluated the effect on various T cell– and antigen-presenting cell (APC)–associated cytokines.

Figure 2A shows that addition of IL-22 did not affect IL-17 production in naive mice and led to a significant but modest suppression of IL-17 in splenocytes from mice during the initiation phase. However, in splenocytes from mice with early arthritis, stimulation with collagen and IL-22 resulted in a significant increase in IL-17 production. Since the collagen-specific response is elucidated only following collagen immunization, naive mice were stimulated in a polyclonal manner with anti-CD3 only. IFNγ may be produced by T cells and APCs; hence, anti-CD3, collagen, or H37Ra was used in restimulation cultures to evaluate the effect of IL-22 on IFNγ. In contrast to IL-17, IFNγ production was significantly though modestly suppressed in splenocytes from naive mice and from mice with early arthritis stimulated in a polyclonal manner. There was no change in IFNγ production when splenocytes were stimulated in an antigen-specific manner or with H37Ra in the presence of IL-22.

Figure 2.

Interleukin-22 (IL-22) differentially regulates various cytokines during collagen-induced arthritis. A, Splenocytes (SP) from naive mice, mice from the initiation phase (2 weeks following immunization with collagen [Coll]), and mice with early arthritis were stimulated ex vivo for 3–6 days with anti-CD3 (5 μg/ml), collagen (100 μg/ml), or H37Ra (2.5 μg/ml) in the presence or absence of recombinant IL-22 (100 ng/ml). Levels of IL-17, interferon-γ (IFNγ), IL-6, and IL-10 in supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA). B, IL-22 in culture supernatants of splenocytes from mice with early arthritis was measured by ELISA. C, Splenocytes from mice with early arthritis were stimulated ex vivo for 3–6 days with H37Ra (2.5 μg/ml), anti-CD3 (5 μg/ml), or collagen (100 μg/ml) in the presence or absence of anti–IL-22 antibody (10 μg/ml) or rat IgG (10 μg/ml). Levels of IFNγ, IL-17, IL-6, and IL-10 in supernatants were measured by ELISA. Data are representative of 4 independent experiments. Values are the mean ± SEM. P values were determined by Student's t-test.

IL-6 and TNF are well-studied cytokines in inflammatory arthritis, and therapies targeting these cytokines are used in clinical practice. Stimulation of splenocytes under various conditions in the presence or absence of IL-22 had a dual effect on IL-6 production depending on the phase of arthritis (Figure 2A). IL-22 had no effect on IL-6 production by splenocytes from naive mice. IL-6 production was significantly but modestly suppressed by IL-22 during the initiation phase, and significantly but modestly increased by IL-22 during early arthritis. These effects were observed only when splenocytes were stimulated in a polyclonal manner and were not seen when splenocytes were restimulated with collagen or H37Ra. IL-22 had no effect on the secretion of TNFα from splenocytes under various stimulation conditions, and IL-1β was undetectable in cultures stimulated in the presence or absence of IL-22 under various conditions (data not shown).

Next we evaluated the effect of IL-22 on the regulatory cytokine IL-10, which has a protective role in inflammatory arthritis (26). The data in Figure 2A show that IL-22 induced a significant increase in IL-10 secretion from splenocytes obtained from mice with early arthritis and stimulated with anti-CD3 or H37Ra. This effect did not occur upon stimulation with collagen. There was no change in IL-10 production in stimulation cultures of splenocytes from naive mice or mice from the initiation phase.

Since IL-22 expression was increased in splenocytes from arthritic mice, we measured IL-22 levels in culture supernatants from these mice. Unstimulated splenocytes produced very low amounts of IL-22, and in vitro stimulation with collagen, H37Ra, or anti-CD3 augmented IL-22 production (Figure 2B). We performed additional experiments utilizing anti–IL-22 antibody to evaluate its effector function. Neutralization of IL-22 had no effect on the levels of IL-17, IL-6, TNFα, and IL-1β, significantly induced IFNγ production, and suppressed the production of IL-10 (Figure 2C and data not shown).

We then evaluated the effect of IL-22 on target organs: in this case, arthritic paws. IL-22 did not alter the concentrations of IL-17, TNFα, IL-6, IL-10, or IL-1β in single-cell suspensions from arthritic paws restimulated under various conditions (data not shown). IFNγ was undetectable in restimulation cultures of single-cell suspensions from arthritic joints (data not shown).

Delayed progression of arthritis severity with IL-22 treatment.

Our above-described data showed that IL-22 has pleiotropic effects on a variety of cytokines. These effects were most pronounced in splenocytes from mice with early arthritis, with significant induction of the inflammatory cytokines IL-17 and IL-6 as well as the antiinflammatory cytokine IL-10. Hence, we wished to evaluate the net effect of IL-22 in vivo during the effector phase of arthritis. Recombinant IL-22 was administered daily during the effector phase, starting on approximately day 20–22 after immunization with collagen and CFA, for a total duration of 10–12 days. Control mice received HBSS. As seen in Figure 3A, progression of the severity of arthritis was significantly delayed in mice that received recombinant IL-22. The incidence of arthritis (defined as an arthritis severity score of >1) did not differ between the 2 groups. Figure 3B shows the histopathologic scoring of the paws from the 2 groups at the conclusion of the study. In mice that received recombinant IL-22, scores for inflammation, synovitis, and cartilage and bone damage in paws were significantly reduced. Representative H&E staining of paws from mice receiving IL-22 or HBSS is shown in Figure 3C.

Figure 3.

Delayed progression of arthritis severity in mice treated with interleukin-22 (IL-22). Sixteen DBA mice were immunized with collagen and Freund's complete adjuvant. On approximately day 20–22 following immunization, mice were divided into 2 groups (8 mice per group). The experimental group received daily intraperitoneal injections of 5 μg of recombinant IL-22 in Hanks' balanced salt solution (HBSS), and the control group received HBSS alone intraperitoneally. All mice received these injections for 10–12 days. Clinical arthritis was scored every other day starting on approximately day 21. Mice were killed on approximately day 34, and paws were harvested, fixed with formalin, and stained with hematoxylin and eosin for histologic analysis and scoring. A, Incidence and clinical severity scores of arthritis. B, Histologic scores of inflammation, synovitis, cartilage and bone damage, and overall histologic findings in paws. Data in A and B are representative of 3 independent experiments. Values are the mean ± SEM. P values were determined by Mann-Whitney test. ∗ = P < 0.05 versus IL-22–treated group. C, Representative photomicrographs of paws from mice in the 2 treatment groups. Original magnification × 10.

Antigen-specific restimulation culture of splenocytes showed that there were no differences in the secretion of IL-17 or IFNγ between the HBSS- and IL-22–treated groups (Figure 4A). Anticollagen antibody levels also did not differ between the 2 groups.

Figure 4.

IL-22 does not alter antigen-specific T and B cell responses and augments IL-10 in vivo. Mice were grouped and treated as described in Figure 3 and killed on approximately day 34. Serum was collected for analysis of anticollagen antibodies by ELISA, and spleen and draining inguinal lymph nodes were collected for various assays. A, IL-17 and IFNγ production in single-cell suspensions of splenocytes cultured with collagen or anti-CD3, and anticollagen antibody levels in serum, determined by ELISA. B, IL-10 expression in splenocytes, determined by real-time polymerase chain reaction. C, IL-10 production in splenocytes restimulated with H37Ra, anti-CD3, or collagen, determined by ELISA. Data are representative of 2 independent experiments. Values are the mean ± SEM. P values were determined by Student's t-test. HBSS = Hanks' balanced salt solution (see Figure 2 for other definitions).

Real-time polymerase chain reaction analysis revealed that expression of IL-10 in splenocytes was increased in mice receiving IL-22 compared to mice receiving HBSS (Figure 4B). Furthermore, production of IL-10 was increased in restimulation cultures of splenocytes from mice receiving IL-22, specifically when H37Ra was used (Figure 4C).

Abrogation of the protective effect of IL-22 by anti–IL-10.

In the above-described experiments, IL-22 was shown to have a protective role in arthritis and to augment IL-10 production in vitro and in vivo. We next wished to evaluate whether the protective effect of IL-22 in vivo could be abrogated by neutralizing IL-10. Mice received 1 of 4 treatments: HBSS, recombinant IL-22, recombinant IL-22 plus anti–IL-10, or recombinant IL-22 plus rat IgG. The severity of arthritis was significantly reduced in mice that received daily injections of recombinant IL-22 in comparison to mice receiving HBSS (Figure 5). Administration of anti–IL-10 along with recombinant IL-22 resulted in increased severity of arthritis, similar to that observed in the group receiving HBSS. Mice receiving recombinant IL-22 plus rat IgG had reduced arthritis severity, similar to that in mice receiving IL-22 alone. In accordance with these findings, histopathologic examination of the paws revealed increased inflammatory infiltration, synovitis, cartilage damage, and bone involvement in mice receiving IL-22 plus anti–IL-10 or HBSS in comparison to mice receiving IL-22 alone or IL-22 plus rat IgG (Figure 5B). Representative H&E-stained images of paws from mice in each of the 4 treatment groups are shown in Figure 5C.

Figure 5.

The protective effect of interleukin-22 (IL-22) is abrogated upon administration of anti–IL-10. Twenty-four DBA mice were immunized with collagen and Freund's complete adjuvant and were divided into groups (6 mice per group) receiving 4 different treatments, i.e., Hanks' balanced salt solution (HBSS), recombinant IL-22, recombinant IL-22 plus anti–IL-10, and recombinant IL-22 plus rat IgG. All mice received injections for 12–13 days starting on approximately day 20. Clinical arthritis was scored every other day. Mice were killed on approximately day 33, and paws were harvested, fixed with formalin, and stained with hematoxylin and eosin for histologic analysis and scoring. A, Clinical severity score of arthritis. B, Histologic scores of inflammatory infiltration, synovitis, cartilage and bone damage, and overall histologic findings in paws. Data in A and B are representative of 2 independent experiments. Values are the mean ± SEM. P values were determined by Mann-Whitney test. ∗ = P < 0.05 versus the groups treated with IL-22 alone or IL-22 plus rat IgG. C, Representative photomicrographs of paws from mice in the 4 treatment groups. Original magnification × 10.

IL-22–induced augmentation of IL-10 production in CD11b+ cells.

In order to elucidate which effector cells were producing IL-10 in response to IL-22, we enriched splenocytes from mice with early arthritis for B cells (CD19+), T cells (CD3e+), CD11c+ cells, or CD11b+ cells, followed by in vitro culture in the presence or absence of IL-22. CD11b+ cells produced IL-10, and this production was augmented in the presence of IL-22 (Figure 6A). In accordance with this, expression levels of IL-10 were increased in CD11b+ cells from splenocytes of mice receiving IL-22 in comparison to those from splenocytes of mice receiving HBSS (Figure 6B).

Figure 6.

IL-22 augments IL-10 production in CD11b+ cells. A, Splenocytes from mice with early arthritis were enriched for CD11c+, T, B, or CD11b+ cells, restimulated for 3 days with H37Ra in the presence or absence of IL-22, and supernatants analyzed for IL-10 by ELISA. B, IL-10 expression was analyzed in CD11b+ cells enriched from splenocytes of mice receiving recombinant IL-22 or Hanks' balanced salt solution (HBSS) in vivo as described in Figure 3. C, Splenocytes from mice with early arthritis and from mice with late arthritis were restimulated for 3 days with H37Ra in the presence or absence of IL-22. Supernatants were analyzed for IL-10 production by ELISA. Data are representative of 2 independent experiments in A and B and 3 independent experiments in C. Values are the mean ± SEM. P values were determined by Student's t-test. See Figure 2 for other definitions.

In the present study, administration of IL-22 prior to the onset of arthritis was shown to delay the progression of arthritis and to induce IL-10. It was recently reported that administration of anti–IL-22 antibody after the onset of arthritis reduced bone degradation (22), suggesting that the effect of IL-22 is temporally dependent on the onset of joint inflammation. It is possible that in the presence of prolonged inflammation, IL-22–mediated IL-10 production is blunted. We thus evaluated IL-22–induced IL-10 production in splenocytes from mice with early arthritis and mice with late arthritis. As shown in Figure 6C, induction of IL-10 by IL-22 was attenuated in splenocytes from mice with late arthritis.

DISCUSSION

IL-22 belongs to the IL-10 family of cytokines and has been shown to play a protective or pathogenic role depending on the disease model under study (9–14, 16–20). CIA is associated with dynamic changes in proinflammatory and antiinflammatory immune responses following immunization with collagen, leading to joint inflammation. In this model, a few mice may fail to develop arthritis following immunization with collagen and CFA.

In the present study we have demonstrated up-regulation of the expression of IL-22 and IL-22 receptor (IL-22RI) in lymphoid organs and in arthritic joints during the course of arthritis. Interestingly, modest-to-high levels of IL-22RI expression were observed in splenocytes and paws from naive mice, which may be a strain-specific phenotype. Serum levels of IL-22 were very low during the course of arthritis, which precluded reliable assessment of circulating levels. We found an increased frequency of IL-22+ cells in mice with early arthritis (0.4% of total splenocytes) compared to naive mice (0.03%) or mice from the initiation phase (0.1%). Interestingly, the majority of IL-22+ cells were found to be CD4− by flow cytometry. Further experiments to elucidate the specific cell subset producing IL-22 revealed that CD4+ and CD49b+ cells were the exclusive producers of IL-22, suggesting that even though the numbers of IL-22+CD4+ cells were low (∼0.06%), they may produce high amounts of IL-22. This is consistent with the results of several studies showing that IL-22+ cells may be produced by a variety of cells including CD4+ cells, NK cells, lymphoid tissue inducer cells, or γ/δ cells (2–7).

IL-22–deficient mice have a reduced incidence of arthritis, and administration of anti–IL-22 antibody reduces the levels of histologic markers of inflammation (22, 23); however, the mechanism of action of IL-22 in inflammatory arthritis remains unknown. IL-22 has been reported to mediate a variety of effects. Levels of IL-6, TNFα, and IL-1β are reduced in IL-22–knockout mice (16). In a recent study, IL-22 induced IL-10–secreting regulatory APCs (19). In our study, IL-22 had no effect on IL-17, IL-6, TNFα, IL-1β, or IL-10 production, and reduced IFNγ production in splenocytes from naive mice in vitro. During the initiation phase of arthritis, IL-22 had no effect on IFNγ, IL-1β, IL-10, or TNFα, and suppressed IL-6 and IL-17 in splenocytes. In contrast, during the early arthritis phase, IL-17, IL-6, and IL-10 were up-regulated, and IFNγ was suppressed, by IL-22. The effect of IL-22 on the production of cytokines was most evident in splenocytes from mice with early arthritis in comparison to those from naive mice, even though naive mice had high levels of expression of IL-22RI. It is possible that, despite its lack of effects on levels of IL-10, IL-17, IL-6, or TNF, IL-22 may have other effects on naive splenocytes.

A limited effector function of IL-22 on immune cells has been suggested. Recent studies have demonstrated the expression of IL-22R on CD11b+ cells (19). In a mouse model of psoriasis, administration of IL-22 neutralizing antibody was associated with significant reductions of IL-17A and IL-17F in sera and suppression of IL-17A– and IL-17F–producing CD4+ cells in the cervical lymph nodes as demonstrated by flow cytometry (16). While the expression of IL-22R during inflammation remains to be evaluated, it is possible that IL-22 may have direct and/or indirect effects on immune cells.

Further, IL-22 did not induce any of the above cytokines in single-cell cultures from arthritic paws. IL-22RI expression was, however, induced in arthritic paws (Figure 1D), and IL-22 has been shown to induce CCL2 and proliferation of synovial fibroblasts in vitro (21). Administration of anti–IL-22 has been associated with reduction of discrete parameters of joint inflammation, such as pannus formation and proteoglycan depletion (22). These findings suggest that the effector function of IL-22 in arthritic paws may not be limited to the cytokines measured in this study.

Our data show that IL-22 has pleiotropic effects on a variety of cytokines, with significant induction of the inflammatory cytokines IL-17 and IL-6, as well as the antiinflammatory cytokine IL-10. Further, these effects were most pronounced in splenocytes from mice with early arthritis. CIA is induced following a single injection of collagen and CFA. Using this protocol at our laboratory there is 80% incidence of arthritis, with onset of disease ∼25–28 days after immunization and peak incidence at approximately day 30–32. Since the onset of arthritis is not exactly synchronized, we evaluated the effect of IL-22 treatment around the time of onset of arthritis by administering IL-22 starting on day 20, i.e., 5–7 days prior to arthritis onset. Surprisingly, administration of IL-22 significantly restrained progression of the severity of arthritis. In accordance with the differences in clinical scores, histologic examination of paws revealed significantly reduced scores of inflammation as well as cartilage and bone destruction in mice receiving IL-22. Additionally, administration of IL-22 was associated with increased expression of IL-10 in splenocytes and production of IL-10 from restimulation cultures of splenocytes. Interestingly, there were no changes in anticollagen antibody responses or in IL-17 and IFNγ responses. These findings suggest that, although IL-22 induces a variety of cytokines during early arthritis in vitro, in vivo it has a protective role.

The IL-22–mediated increase in IL-10 levels is limited to CD11b+ cells in vitro. In vivo experiments also showed increased expression of IL-10 in CD11b+ cells from mice receiving IL-22. However, the precise mechanism by which IL-10–producing CD11b+ cells restrict the progression of arthritis remains to be evaluated in further studies. In a recent investigation, Marijnissen et al showed that administration of anti–IL-22 after the onset of arthritis was not associated with a significant reduction in clinical scores (22). However, there was improvement in certain discrete histologic measures of joint inflammation in that study. This may be because Marijnissen and colleagues administered anti–IL-22 antibody after the onset of arthritis while in our study recombinant IL-22 was administered prior to arthritis onset. The finding that the incidence of arthritis is reduced in IL-22–knockout mice suggests that IL-22 may have a distinct role during the induction phase of arthritis, by altering immune pathways (23). Further, IL-22–knockout mice may have immunologic alterations beyond IL-22. Additionally, the increases in IL-10 seen in restimulation cultures of splenocytes were not observed with single-cell suspension cultures of arthritic paws.

The above-described findings suggest that the effector function of IL-22 is nuanced by the temporal and spatial distribution of the inflammatory immune response. This notion is supported by the findings of a recent study of autoimmune uveitis in which administration of IL-22 just prior to the onset of inflammation had a protective effect, in contrast to administration after the onset of eye inflammation (19).

IL-10 levels are elevated in patients with rheumatoid arthritis, and IL-10 has been shown to play a protective role in inflammatory arthritis (26–35). In the present study, IL-22 augmented IL-10 in CD11b+ cells in vitro and in vivo. Further, administration of IL-22 restricted the progression of arthritis. Coadministration of anti–IL-10 and IL-22 abrogated this protective effect. Additionally, IL-22–induced IL-10 production in splenocytes from mice with late arthritis was blunted in comparison to that observed in splenocytes from mice with early arthritis. It is plausible that neutralization of IL-10 is associated with an increased inflammatory load, which may lead to blunting of the protective effect of IL-22. In summary, our current results as well as earlier-described findings support the notion of a dual nature of the effector function of IL-22. In the presence of a sufficiently proinflammatory milieu, an IL-22–mediated antiinflammatory effect may be blunted or possibly switched to a pathogenic effect. In this study we have demonstrated for the first time that IL-22 has a protective function in inflammatory arthritis, when administered prior to the onset of the disease.

AUTHOR CONTRIBUTIONS

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Sarkar 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 conception and design. Sarkar.

Acquisition of data. Sarkar, Zhou, Justa, Bommireddy.

Analysis and interpretation of data. Sarkar, Zhou, Justa.

Acknowledgements

We would like to acknowledge Drs. David A. Fox and Janet Funk for critical review of the manuscript and helpful comments.

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