IL-27 Induces a Th1 Immune Response and Susceptibility to Experimental Arthritis Yanxia Cao, Paul D. Doodes, Tibor T. Glant and Alison Finnegan This information is current as of September 28, 2021. J Immunol 2008; 180:922-930; ; doi: 10.4049/jimmunol.180.2.922 http://www.jimmunol.org/content/180/2/922 Downloaded from

References This article cites 49 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/180/2/922.full#ref-list-1

Why The JI? Submit online. http://www.jimmunol.org/

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on September 28, 2021

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-27 Induces a Th1 Immune Response and Susceptibility to Experimental Arthritis1

Yanxia Cao,† Paul D. Doodes,* Tibor T. Glant,‡ and Alison Finnegan2*†

IL-27 is the newest member of the family comprised of IL-12 and IL-23. IL-27 was originally described as a cytokine that along with IL-12 induces the differentiation of naive precursor T cells into Th1 effector cells. This activity has been called into question based on evidence in infectious disease and autoimmune models in which IL-27 is not absolutely required for the generation of IFN-␥, and IL-27 plays a regulatory role in controlling inflammation. We have previously reported in proteoglycan- induced arthritis (PGIA), a model of rheumatoid arthritis, that severe arthritis is dependent on the production of IFN-␥. In this study, we report that IL-27 was expressed in spleen and joint tissues of arthritic mice. We determined the involvement of IL-27 in PGIA by assessing the progression of arthritis in IL-27R؊/؊ mice. Development of arthritis in IL-27R؊/؊ mice was delayed and ؉/؉ severity reduced in comparison with IL-27R littermate controls. Histology confirmed a reduction in joint cellularity, cartilage Downloaded from destruction, and bone erosion. Diminished arthritis was associated with fewer T cells producing IFN-␥ and decreased IFN-␥ secretion overtime. Moreover, the frequency of IL-4- and IL-17-expressing T cells and the production of IL-4 and IL-17 were similar in IL-27R؊/؊ mice and controls. Our results indicate that IL-27 is critically involved in the induction of inflammation in PGIA. IL-27 functions by inducing the differentiation of IFN-␥-producing T cells in vivo that are essential for the development of arthritis. The Journal of Immunology, 2008, 180: 922–930. http://www.jimmunol.org/ nterleukin-27 is a heterodimeric cytokine composed of EBV- 27R-deficient mice display enhanced accumulation of Th1 effector induced gene 3 (EBI3)3 and p28 that signals through a re- (11) cells and increased production of IFN-␥ during infection with I ceptor complex composed of the unique IL-27R␣ (IL-27R) Toxoplasma gondi, Trypansoma cruzii,orMyobacterium tubercu- (also called WSX-1 or cytokine receptor (TCCR)) and losis (12, 13). Similarly, in Con A-induced hepatitis, experimental gp130. IL-27 belongs to a family of structurally related ligands that autoimmune encephalomyelitis (EAE), and chronic T. gondii in- include IL-12, IL-23, and IL-6 (1–4). IL-27R is expressed on naive fection, absence of IL-27R exacerbates disease and the production T cells, NK cells, monocytes, mast cells, and activated B cells and of IL-17 by T cells (14–16). IL-27 inhibitory activity is related to dendritic cells, whereas IL-27 is produced mainly by activated its ability to suppress several different . IL-27 has been and dendritic cells (1–3, 5–7). Initial studies focused shown to reduce IL-4 production, which in turn may exacerbate by guest on September 28, 2021 on the influence of IL-27 on Th1 responses because of the struc- Th1 responses (17–19). Studies also suggest that IL-27 can limit tural homology between IL-27 and IL-12. These studies showed CD4ϩ T cell IL-2 and IL-17 production (14, 15, 20, 21). Thus, the that similar to IL-12, IL-27 promotes T cell proliferation and the marked suppressor activity of IL-27 in these particular systems production of IFN-␥ by naive T cells (1). Consistent with in vitro may overshadow the role of IL-27 in Th1 differentiation. studies, IL-27R-deficient mice produce less IFN-␥ in vivo and are Proteoglycan (PG)-induced arthritis (PGIA) is a murine more susceptible to infection with Listeria monocytogenes (2). model of rheumatoid arthritis (22). In PGIA, immunization of Moreover, both IL-27 and IL-12 are capable of inducing T-bet, female BALB/c mice with proteoglycan induces a population of IFN-␥, and IL-12R␤2 expression in naive T cells in a STAT1- CD4ϩ T cells to produce IFN-␥ (23). IFN-␥ is a dominant cy- dependent manner (6, 8–10). However, subsequent studies indi- tokine regulating PGIA, as illustrated by the observation that cate that IL-27 can limit several Th1-type immune responses. IL- arthritis onset and severity are reduced under conditions in which IFN-␥ is neutralized with Abs or in mice lacking IFN-␥ (24). Interestingly, PGIA differs from several other autoimmune *Department of Immunology/Microbiology and †Department of Internal Medicine, disease models, specifically collagen-induced arthritis (CIA) Section of Rheumatology, and ‡Department of Orthopedic Surgery, Rush University and “new” EAE, in which autoimmune disease is exacerbated in Medical Center, Chicago, IL 60612 susceptible strains lacking either IFN-␥ or the IFN-␥ receptor Received for publication September 12, 2007. Accepted for publication November 12, 2007. (25–27). Moreover, STAT4-deficient mice are similarly resis- tant to PGIA, indicating that IL-12R signaling is an important The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance component of disease development (24). We were therefore in- with 18 U.S.C. Section 1734 solely to indicate this fact. terested in determining whether IL-27 functioned to enhance or 1 This research was supported by National Institutes of Health Grant AR45652 (to suppress the Th1-mediated response in PGIA. Our results dem- A.F. and T.T.G.). onstrated that IL-27R-deficient mice were more resistant to in- 2 Address correspondence and reprint requests to Dr. Alison Finnegan, Department of duction of arthritis than wild-type mice. The loss of IL-27R Medicine, Section of Rheumatology, Rush University Medical Center, 1735 West ␥ Harrison Avenue, Chicago, IL 60612. E-mail address: [email protected] signaling resulted in reduced IFN- production and fewer IFN- ␥ ϩ 3 Abbreviations used in this paper: EBI3, EBV-induced gene 3; CIA, collagen-in- -producing CD4 T cells. Moreover, production of IL-2, IL-4, duced arthritis; Ct, cycle threshold; EAE, experimental autoimmune encephalomy- and IL-17 was not significantly altered in the absence of IL-27 elitis; PGIA, proteoglycan-induced arthritis; Tg, transgenic; PG, proteoglycan; signaling. These studies demonstrate that IL-27 expression is an TCCR, T cell cytokine receptor. important cytokine for driving the IFN-␥ response that is crit- Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 ical for the induction of PGIA. www.jimmunol.org The Journal of Immunology 923

FIGURE 1. Activation of primary PG-specific T cells by IL-27. CD4ϩ T cells from PG-specific TCR Tg mice were cultured with naive irradiated spleen cells in presence or absence of PG (5 ␮g/ml), IL-27 (1 ng/ml), or the combination of PG and IL-27. T cell proliferation (A) was measured on day 4 by the incorporation of [3H]thymidine. T cell IFN-␥ production (B) was measured in day 4 supernatants by ELISA. T cell IFN-␥ mRNA transcripts (C) were measured at4hbyPCR. T cell RNA from nonimmune mice was used to calculate the relative fold increase in IFN-␥ RNA. Values are the mean Ϯ SD .p Ͻ 0.05. Data are representative of two separate experiments ,ء .of quadruplicate cultures Downloaded from

Materials and Methods nology Associates). Data represent the mean Ϯ SEM of Abs from six to Mice eight individual mice. T cell cytokine receptor-deficient mice (designated in this study as IL- Assessment of T cell activation by proliferation 27RϪ/Ϫ) were provided by F. de Sauvage (Genentech, South San Fran- cisco, CA) (2). IL-27RϪ/Ϫ mice were backcrossed to BALB/c (Charles CD4ϩ (2.5 ϫ 105 cells/well) purified by AutoMACS selection (Miltenyi http://www.jimmunol.org/ River Laboratories) for seven generations and then intercrossed to obtained Biotec) were stimulated with PG (10 ␮g/ml) and irradiated (2500 rad) IL-27Rϩ/ϩ and IL-27RϪ/Ϫ littermates. Female BALB/c age-matched IL- spleen cells (2.5 ϫ 105 cells/well) from naive IL-27Rϩ/ϩ mice. Cells were 27Rϩ/ϩ and IL-27RϪ/Ϫ littermates 12–14 wk of age were used in all ex- cultured in quadruplicate in 96-well Falcon plates (Fisher Scientific) in periments. TCR transgenic (Tg) mice expressing a TCR specific for a dom- RPMI 1640 medium containing 10% FCS, 100 ␮g/ml penicillin, 100 inant epitope of human PG were used to test primary responses to IL-27 ␮g/ml streptomycin, and 2 mM L-glutamine (complete medium). Cultures (28, 29). All animal experiments were approved by the Institutional Animal were pulsed on day 4 with [3H]thymidine overnight and examined for Care and Use Committee at Rush University Medical Center. proliferation. In other experiments, as indicated in figure legends, CD4ϩ T cells from naive PG-specific TCR Tg were stimulated with irradiated naive Induction and assessment of arthritis BALB/c mice spleens under suboptimal concentrations of PG (5 ␮g/ml)

in the presence or absence of IL-27 (1 ng/ml; eBiosciences). Cells were by guest on September 28, 2021 Human cartilage was obtained from patients undergoing joint replacement cultured in quadruplicate in 96-well Falcon plates (Fisher Scientific) in surgery, and was provided through the Orthopedic Tissue, Transplant, and serum-free HL-1 medium containing 100 ␮g/ml penicillin, 100 ␮g/ml Implant Repository of Rush University Medical Center, with the approval streptomycin, and 2 mM L-glutamine. Cultures were pulsed on day 2 with of the Institutional Review Board. PG (aggregan) was extracted from pooled [3H]thymidine overnight and examined for proliferation. cartilage samples, as previously described (23). Female mice were immunized i.p. with 150 ␮g of human PG measured as protein, emulsified in dimethyl- dioctadecylammonium bromide (DDA) (Sigma-Aldrich), as previously de- Assessment of cytokines scribed (30, 31), and boosted with 100 ␮g of PG/DDA at 3 and 6 wk. For cytokine production, nylon wool-purified T cells (2.5 ϫ 105 cells/well) Mice were monitored for arthritis and scored in a blinded manner. Paws ϩ from peritoneal exudates (pooled from four to six mice) or CD4 T cells were scored for arthritis on a scale from 1 to 4, as follows: 0, normal; 1, (2.5 ϫ 105 cells/well) selected by AutoMACS from spleen of PG-immu- mild swelling affecting several digits or mild swelling of the paw; 2, mod- nized mice were stimulated with PG (10 ␮g/ml) and irradiated (2500 rad) erate erythema and swelling of the paw; 3, more intense erythema and ϩ ϩ spleen cells (2.5 ϫ 105 cells/well) from naive IL-27R / mice. Cells were swelling affecting a greater proportion of the paw; 4, severe swelling af- cultured in quadruplicate in 96-well Falcon plates (Fisher Scientific) in fecting the entire paw and with hardening of the articular tissue. Each complete medium. Supernatants were harvested at day 4 for the detection animal received a cumulative score ranging from 0 to 16, based on indi- of IFN-␥, IL-4, and IL-17 cytokines, and 24 h for the detection of IL-2 by vidual paw scores of 0–4. The same investigator inspected and scored the conventional sandwich ELISA (BD Biosciences). For spleen cytokine pro- paws three times per week. Histology was performed to determine the duction, spleen cells from PG-immunized mice were isolated and cultured extent of joint inflammation and damage. Hind limbs were dissected, de- (2 ϫ 106/ml) in complete medium in 24-well Falcon plates (Fisher Scien- calcified, embedded in paraffin, and sectioned at 6 ␮m. Sagittal sections tific) in medium alone or PG (20 ␮g/ml). Supernatants were harvested and were stained with H&E, and cellular infiltration was measured on a scale assayed by ELISA for TNF-␣, IL-6 (BD Biosciences), and IL-27 (R&D from 0 to 4, with 0 being no infiltrating cells and 4 being massive ϩ Systems). The number of CD4 T cells secreting cytokines was assessed infiltration. by ELISPOT. Capture Ab was incubated overnight at 4°C on sterile Mul- Detection of anti-proteoglycan Ab by ELISA tiscreen plates (Millipore), and then blocked for 1 h before addition of CD4ϩ T cells (1.5 ϫ 105 cells/well) selected by AutoMACS from spleen Mice immunized with human PG were bled from the orbital plexus, and of PG-immunized mice and stimulated with PG (2 ␮g/ml) and irradiated ϩ ϩ serum was obtained and examined for Abs against mouse proteoglycan and (2500 rad) spleen cells (2.0 ϫ 105 cells/well) from naive IL-27R / mice. human proteoglycan by ELISA. Cells were incubated for 48 h (IFN-␥) and 72 h (IL-17 and IL-4), and Enzyme immunoassay tissue culture 96 half-area plates (Costar) were cytokines were detected with cytokine-specific Abs. Plates were dried and coated overnight at 4°C with 0.5 ␮g of chondroitinase ABC-digested hu- read using ELISPOT reader and software (ImmunoSpot Analyzer and Im- man PG, or 0.75 ␮g of native mouse proteoglycan in carbonate buffer. munoSpot software; CTL Analyzers). Serum was serially diluted in buffer (PBS/0.5% Tween 20). Serially diluted serum samples and internal standards samples (pooled sera from arthritic Quantitative RT-PCR mice) were incubated with immobilized PG. Abs were detected with HRP- labeled rabbit anti-mouse IgG1 and IgG2a (Zymed Laboratories), which RNA was isolated from spleen and joint tissue using Tri-Reagent (Mo- were then detected with the substrate o-phenylenediamine. The relative lecular Research Center). RNA was further subjected to DNase I (In- concentration of Ab was determined from a standard curve of known con- vitrogen Life Technologies) digestion before reverse transcription. Re- centrations of unlabeled murine IgG1 and IgG2a Ab (Southern Biotech- verse transcription was performed with SuperScript III (Invitrogen Life 924 IL-27 INDUCES A Th1 RESPONSE IN VIVO Downloaded from http://www.jimmunol.org/

FIGURE 2. IL-27 expression in PGIA. IL-27Rϩ/ϩ were immunized i.p. with human PG in adjuvant three times at 3-wk intervals and monitored for the incidence and severity of PGIA. A, IL-27 and IFN-␥ production from arthritic mice spleen cells stimulated with PG. B, Ebi3 and Il-27 mRNA transcripts from arthritic joints and spleen cells stimulated with PG. Joint tissue and spleen from nonimmune mice were used to calculate the relative fold increase in cytokine RNA. Suppression of PGIA onset and severity in IL-27RϪ/Ϫ mice. IL-27Rϩ/ϩ and IL-27RϪ/Ϫ mice were immunized and monitored for the incidence, arthritic score, and severity of PGIA at given time points. Incidence (C) denotes the percentage of mice that develop PGIA. Arthritic score (D) by guest on September 28, 2021 is the sum of all paws inflamed and noninflamed in individual mice divided by the total number of mice. Severity (E) (cumulative arthritis score) is the p Ͻ 0.05 for IL-27Rϩ/ϩ ,ء .sum of paw inflammation scores in individual mice divided by the total number of arthritic mice. Values are the mean Ϯ SEM (n ϭ 10) vs IL-27RϪ/Ϫ (n ϭ 11). Data are representative of four separate experiments.

Technologies). Real-time PCR was performed with 1 ␮l of reverse- is crossed. PCR product quality was monitored using post-PCR melting transcription product in an IQ5 real-time PCR detection system (Bio- curve analysis. Controls were from naive nonimmunized and target Rad) by using IQ SYBR Green PCR Supermix (Bio-Rad), according to genes from PG-immunized IL-27Rϩ/ϩ and IL-27RϪ/Ϫ joint and spleen the manufacturer’s guidelines. The PCR cycling conditions were as tissues. follows: 50 cycles of 15 s at 95°C, 20 s at 60°C, and 30 s at 72°C. All samples were run in triplicate. To verify that equivalent amounts of RNA were added to each PCR, PCR amplification of the murine ␤-actin Statistical analysis was performed for each sample. Relative fold induction was calculated Ϫ(⌬⌬Ct) ⌬⌬ ⌬ Ϫ⌬ using the formula 2 , where Ctis Ct(treatment) Ct(control), The nonparametric Mann-Whitney U test was used to compare data for ⌬ Ϫ Ͻ CtisCt(target gene) Ct(actin), and Ct is the cycle at which the threshold statistical significance. Significance is based on a p value 0.05.

FIGURE 3. Suppression of histopa- thology in the joint of IL-27RϪ/Ϫ mice. Hind limbs of mice were dissected, fixed in formalin, decalcified, and em- bedded in paraffin. The tissue sections were stained with H&E. Sections are from representative ankle joints from IL-27Rϩ/ϩ (A and C) and IL-27RϪ/Ϫ mice (B and D). Magnification ϫ4(A and B) and ϫ10 (C and D). Thick arrow indicates an area of cartilage destruc- tion, and thin arrow indicates an area of bone erosion. Cellular infiltration (E) was measured on a scale of 0–4. Val- p Ͻ 0.05 ,ء .ues are the mean Ϯ SEM for IL-27Rϩ/ϩ (n ϭ 12) vs IL-27RϪ/Ϫ (n ϭ 17) for two separate experiments. The Journal of Immunology 925

a suboptimal concentration of PG (Fig. 1A). Similarly, IL-27 stimulated IFN-␥ production and IFN-␥ mRNA expression sig- nificantly higher than either PG or IL-27 alone (Fig. 1, B and C). These data confirm previous reports on the ability of IL-27 to induce proliferation and IFN-␥ production in naive T cells (1, 8).

IL-27 is required for the development of PGIA We next assessed whether IL-27 was produced in PGIA. Spleen cells from arthritic mice were restimulated in vitro with PG, and supernatants were examined for IL-27 and IFN-␥ production by ELISA. IFN-␥ and IL-27 were produced in spleen cell cultures activated with PG (Fig. 2A). We used real-time PCR to quantify FIGURE 4. Suppression of cytokines in joint tissues of IL-27RϪ/Ϫ the relative transcript abundance of IL-27 in spleen and arthritic mice. TNF-␣, IL-6, and IL-1␤ (A) and IFN-␥ and IL-17 (B) mRNA tran- joint tissues of PG-immunized mice. Relative to nonimmune mice, scripts using RNA isolated from joint tissues of IL-27Rϩ/ϩ and IL-27RϪ/Ϫ a 5- to 6-fold increase in Ebi3 (which encodes EBI3) and Il27 mice immunized with PG 8 wk after immunization. Joint tissue from (which encodes for p28) in joint tissues of arthritic mice was ob- nonimmune mice was used to calculate the relative fold increase in cyto-

served. Similarly, EBI3 and Il27 mRNA transcripts were increased Downloaded from ϩ/ϩ -p Ͻ 0.05 for IL-27R (n ϭ in spleen cells of arthritic mice (Fig. 2B). Macrophages and den ,ء .kine RNA. Values are the mean Ϯ SD Ϫ/Ϫ ϭ 3–6) vs IL-27R (n 3–6). Data are representative of four separate dritic cells are the primary source of IL-27, suggesting that the experiments. increase in Ebi3 and Il27 transcripts in the joint tissues of arthritic mice could be due to infiltration of migrating cells into the joint or to the activation of resident synoviocytes. Results Although studies have indicated that IL-27 is important for http://www.jimmunol.org/ IL-27 activates primary proteoglycan-specific T cells to the differentiation of naive CD4ϩ T cells into Th1 effector cells, ␥ proliferate and produce IFN- in several infectious and noninfectious disease models, IL-27R- Several reports indicate that IL-27 augments T cell proliferation deficient mice generate robust Th1 responses characterized by and IFN-␥ production (1, 2). We were therefore interested in enhanced IFN-␥ and IL-2 production. Given the importance of determining whether IL-27 contributes to the Th1 phenotype in IFN-␥ in susceptibility to arthritis in PGIA, we assessed the PGIA. We initially examined whether IL-27 augmented CD4ϩ requirement for IL-27 signaling in PGIA. Wild-type (IL- T cell proliferation and IFN-␥ production in PG-specific TCR 27Rϩ/ϩ) and IL-27R-deficient (IL-27Ϫ/Ϫ) mice were immu- Tg mice. IL-27 enhanced PG-specific T cell proliferation under nized with proteoglycan and evaluated for arthritis incidence by guest on September 28, 2021

FIGURE 5. Similarity in proteoglycan-specific Ab response, T cell proliferation, and IL-2 production in IL-27Rϩ/ϩ and IL-27RϪ/Ϫ mice. Serum proteoglycan- specific human IgG1 (hIgG1) and human IgG2a (hIgG2a) (A) and mouse IgG1 (mIgG1) and mouse IgG2a (mIgG2a) (B) were measured by ELISA. C,T cells from proteoglycan-immunized mice were cultured with naive irradiated spleen cells in presence or absence of proteoglycan, and proliferation was measured on day 4 by the incorporation of [3H]thymidine. D, IL-2 pro- duction was measured at 24 h after stimulation of CD4ϩ T cells with naive irradiated spleen cells in the presence or absence of proteoglycan. Values are the mean Ϯ SD. p Ͻ 0.05 for IL-27Rϩ/ϩ (n ϭ 7–10) vs IL-27RϪ/Ϫ ,ء (n ϭ 7–10). Data are representative of three separate experiments. 926 IL-27 INDUCES A Th1 RESPONSE IN VIVO Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 6. Substantial reduction in the production and frequency of T cells secreting IFN-␥ in IL-27RϪ/Ϫ mice. IL-27Rϩ/ϩ and IL-27RϪ/Ϫ mice were immunized, as described in Fig. 2. Peritoneal exudate cells and spleens from three to four mice were obtained at 4, 8, and 11 wk after immunization. T cells from peritoneal exudates and CD4ϩ T cells from spleen were stimulated with proteoglycan in the presence of irradiated naive spleen cells and assayed for IFN-␥, IL-4, and IL-17 production by ELISA and the frequency of T cells producing IFN-␥, IL-4, and IL-17 by ELISPOT. Peritoneal exudate T cells (A, D, and G), CD4ϩ spleen T cells (B, E, and H), and CD4ϩ spleen T cells ELISPOT (C, F, and I). Values are the mean Ϯ SEM of quadruplicate cultures. .p Ͻ 0.05 ,ء and severity (Fig. 2, C–E). IL-27Rϩ/ϩ mice developed arthritis synovial hyperplasia. Cartilage erosion and disintegrating chon- with 100% incidence, whereas the incidence of arthritis in IL- drocytes were also seen in the remaining layer of the articular 27RϪ/Ϫ mice was significantly inhibited over many weeks. In surface. Conversely, cellular infiltrate and histopathological signs the small percentage of IL-27RϪ/Ϫ mice that developed arthri- of arthritis were significantly reduced in IL-27RϪ/Ϫ (Fig. 3, B, D, tis, in general disease was less severe than IL-27Rϩ/ϩ mice and E). In the IL-27RϪ/Ϫ mice, the lack of paw erythema and (Fig. 2, C–E). Similar results were observed in four separate swelling correlated with the absence of cellular infiltrate and car- experiments. These data demonstrate that IL-27 signaling was tilage destruction. critical for the development of PGIA. Proinflammatory cytokines play a major role in driving inflam- To determine the extent of inflammation, we examined ankle mation in arthritis. We therefore measured mRNA transcripts for joint histology in IL-27Rϩ/ϩ and IL-27RϪ/Ϫ mice, as depicted in TNF-␣, IL-6, and IL-1␤ using RNA isolated from joint tissues of Fig. 3, A–D. The histologic picture in IL-27Rϩ/ϩ was character- IL-27Rϩ/ϩ and IL-27RϪ/Ϫ mice immunized with PG. There was a istic of acute arthritis. Mononuclear and polymorphonuclear cell significant decrease in TNF-␣, IL-6, and IL-1␤ transcripts in IL- infiltration was abundant in the tissues and joint spaces. There was 27RϪ/Ϫ mice in comparison with IL-27Rϩ/ϩ mice (Fig. 4A). In edema of the synovial and periarticular tissues accompanied by addition, T cell-derived cytokine transcripts for IFN-␥ and IL-17 The Journal of Immunology 927

FIGURE 7. Substantial reduction in the production of cytokines and cytokine transcripts produced systemically in proteoglycan-immunized IL-27RϪ/Ϫ mice. IL-27Rϩ/ϩ and IL-27RϪ/Ϫ mice were immunized, as described in Fig. 2. Spleen (n ϭ 4) was obtained at 8 wk. Spleen cells were stimulated with proteoglycan, and 24 h later supernatants were harvested from cytokine ELISA and RNA was isolated for detection of mRNA transcripts by PCR. A, Expression of RNA transcripts relative to RNA transcripts from nonimmune IL-27Rϩ/ϩ and IL-27RϪ/Ϫ mice was used to calculate relative fold increase Ϯ in cytokine transcripts. B, Cytokine concentrations in the absence (Media) and presence of proteoglycan (PG). Values are the mean SEM of quadruplicate Downloaded from .p Ͻ 0.05. Data are representative of two separate experiments ,ء .cultures

were also significantly reduced in IL-27RϪ/Ϫ mice (Fig. 4B). The can-specific IL-2 production between IL-27Rϩ/ϩ and IL- reduction in cytokines reflects the lack of inflammation in the IL- 27RϪ/Ϫ mice (Fig. 5D). Ϫ/Ϫ

27R mice. These results demonstrate a necessity for IL-27 in http://www.jimmunol.org/ ␥ the development of inflammation in PGIA. IL-27 is required for the production of IFN- IFN-␥ is a critical cytokine that controls the onset and severity of PG-specific Ab production and T cell activation in PGIA. We next examined whether the production of IFN-␥ cor- IL-27R-deficient mice related with inhibition of arthritis in IL-27RϪ/Ϫ mice. T cell IFN-␥ Proteoglycan-specific CD4ϩ T cells and autoantibodies are neces- production was examined in several different ways. Because PGIA sary for the development of PGIA; neither T cells nor serum Abs is induced by injection of PG into the peritoneal cavity, we isolated are capable of inducing arthritis on their own (30, 32). It has re- the peritoneal exudate. We surmised that there may be a higher cently been shown that IL-27 inhibits IgG1 and induces IgG2a frequency of PG-specific T cells that migrate to the site of Ag. At class switch in B cells (33). Therefore, we next tested whether in 4 and 8 wk, T cells were purified and restimulated in culture with by guest on September 28, 2021 vivo IL-27 signaling alters PG-specific IgG1 and IgG2a isotype PG in the presence of irradiated naive spleen cells. At the same expression. PG-immunized mice were bled periodically over the time points, spleen CD4ϩ T cells were restimulated with PG and course of disease development. The concentration of PG-specific naive APCs. Supernatants were assayed for the production of Abs increased over time, but the differences between IL-27Rϩ/ϩ IFN-␥ by ELISA. At 4, 8, and 11 wk, CD4ϩ T cells were assessed and IL-27RϪ/Ϫ mice remained constant. The data in Fig. 5, A and for the frequency of IFN-␥-producing T cells by ELISPOT. From B, represent Ab concentrations at 11 wk after immunization with the same cultures, IL-4 and IL-17 were measured. T cells isolated PG. There was no significant difference in the IgG1 Abs specific from the peritoneal cavity produced substantially more IFN-␥ than for human or mouse PG (Fig. 5, A and B). For the PG-specific spleen T cells, suggesting that PG-specific T cells migrate to the IgG2a isotype, Abs specific for human were always significantly peritoneal cavity. Furthermore, T cells from IL-27RϪ/Ϫ mice pro- reduced in IL-27RϪ/Ϫ in comparison with IL-27Rϩ/ϩ mice, duced significantly less IFN-␥ whether from peritoneal exudates or whereas Abs specific for mouse PG were reduced, but the data did spleen. The frequency of IFN-␥-positive CD4ϩ T cells from IL- not reach significance (Fig. 5, A and B). 27RϪ/Ϫ mice was similarly reduced in comparison with IL- In Fig. 1, we showed that primary PG-specific T cell prolifer- 27Rϩ/ϩ mice (Fig. 6, A–C). ation was augmented in the presence of IL-27. We assessed PG- We have previously reported that IL-4 functions to reduce the specific CD4ϩ T cell proliferation at 4, 6, 8, and 11 wk after severity of PGIA (23). Because it has been suggested that IL-27 immunization with PG. CD4ϩ T cells were purified from spleen inhibits Th2 responses in infectious and noninfectious re- and stimulated in vitro with irradiated naive spleen cells plus Ag. sponses, we next tested whether an increase in IL-4 production Data in Fig. 5C are representative of T cell proliferation at 4 wk in IL-27RϪ/Ϫ mice might be the explanation for reduction of after immunization. There was no difference in proteoglycan-spe- arthritis in the absence of IL-27 signaling. Our results indicate cific T cell proliferation between IL-27Rϩ/ϩ and IL-27RϪ/Ϫ mice. that IL-27RϪ/Ϫ and IL-27Rϩ/ϩ cells were generally equivalent T cell proliferation at later time points, weeks 6, 8, and 11, also with respect to IL-4 production when evaluated either by the demonstrated no difference between mice sufficient and deficient number of cells expressing IL-4 or by the secretion of IL-4 over in IL-27R expression (data not shown). These data suggest that time. IL-4 was only transiently inhibited at 4 wk in the T cell although IL-27 may be important for primary T cell proliferation, peritoneal exudate cultures (Fig. 6, D–F). Thus, suppression of by 4 wk after T cell sensitization to PG, IL-27R-deficient T cells PGIA in IL-27RϪ/Ϫ mice was not due to an increase in IL-4 were able to overcome the early deficit in IL-27. production. Recent data indicate IL-27 limits the production of IL-2 (12, It has recently been found that IL-27 plays an essential role in 20, 21). Therefore, we examined the production of IL-2 in the inhibition of IL-17 production (14, 15). Both groups show that spleen T cells from IL-27ϩ/ϩ and IL-27Ϫ/Ϫ mice immunized neuroinflammation is exacerbated in the absence of IL-27R due to with proteoglycan. There was no difference between proteogly- an increase in IL-17 expression. Because we found arthritis was 928 IL-27 INDUCES A Th1 RESPONSE IN VIVO inhibited in the absence of IL-27R, IL-17 is unlikely to play a data show that in IL-27RϪ/Ϫ mice there was a reduction in the major role in PGIA. Similar to reported findings, we confirmed in frequency of IFN-␥ secretion CD4ϩ T cell and in the production of vitro that IL-27 inhibited IL-17 production in anti-CD3-activated IFN-␥. Similar to IL-12, IL-27 can also activate STAT4, a signal- CD4ϩ T cells (data not shown). Assessment of IL-17 levels from ing pathway necessary for polarization of Th1 cells into effector PG-activated T cells obtained from peritoneal exudates or spleen cells (9). In PGIA, STAT4 is required because STAT4Ϫ/Ϫ mice indicated that very little IL-17 was produced. However, there was are resistant to arthritis induction (24). In addition, we observed a transient rise in IL-17 production in IL-27RϪ/Ϫ in comparison that IL-12-deficient mice (p40Ϫ/Ϫ and p35Ϫ/Ϫ) are less susceptible with IL-27Rϩ/ϩ mice at 4 wk in spleen CD4ϩ T cells that was not to arthritis than wild-type mice (data not shown). Thus, IL-12 and sustained at later time points (Fig. 6, G–I). These data indicate in IL-27 may both contribute to the activation of STAT4 in PGIA. In vivo levels of IL-17 were not affected by a deficiency in IL-27R addition to the role of STAT4 in IFN-␥ transcription, STAT4 reg- and that there was no correlation between IL-17 expression and ulates the expression of a number of genes involved in inflamma- suppression of PGIA in the IL-27RϪ/Ϫ mice. Overall, the results tion and Th1 responses that probably contribute to induction of described in this study lead to the conclusion that IL-27RϪ/Ϫ cells arthritis (34–37). produce less IFN-␥ and that the reduction in IFN-␥ contributes to PG-specific Abs are necessary for the development of arthritis. resistance in the development of PGIA. Thus, the reduction in PG-specific IgG2a isotype could contribute

Ϫ/Ϫ to the suppression of arthritis in IL-27R-deficient mice. There are Reduced production of proinflammatory cytokines in IL-27R two possible pathways by which IL-27 regulates IgG2a class mice in PGIA switch. IL-27 induces IgG2a expression directly through the acti-

Because of the critical importance of IFN-␥ in the pathogenesis of vation of T-bet (33) and indirectly through activation of IFN-␥ Downloaded from PGIA, we determined whether the decrease in the propensity to (38). Interruption of the IL-27 signaling through either of these produce IFN-␥ in the IL-27RϪ/Ϫ mice led to a decrease systemic pathways would result in reduction of IgG2a isotype switch ob- expression of inflammatory cytokines. We therefore measured served in PG-sensitized IL-27R-deficient mice. mRNA transcripts for TNF-␣, IL-1␤, IL-6, Ebi3, and IL-27 and In agreement with our results, IL-27R-deficient mice infected TNF-␣, IL-1␤, IL-6, and IL-27 production from spleen cultures with either L. monocytogenes or Leishmania major have an im- ϩ/ϩ Ϫ/Ϫ stimulated with proteoglycan from IL-27R and IL-27R paired IFN-␥ response and are less resistant to infection (2, 3). http://www.jimmunol.org/ mice. There was a significant decrease in TNF-␣, IL-1␤, Ebi3, and However, in L. major infection, a reduction in IFN-␥ correlates IL-27 transcripts and PG-stimulated TNF-␣, IL-6, and IL-27R se- with a rise in IL-4 production, and disease resistance could be cretion in IL-27RϪ/Ϫ mice in comparison with IL-27Rϩ/ϩ mice restored by neutralization of IL-4. IL-27 is known to inhibit (Fig. 7). These proinflammatory cytokines are most likely pro- GATA-3, a critical transcription factor for Th2 differentiation (9). duced by activated macrophages and dendritic cells in the spleen. Thus, the ability of IL-27 to inhibit GATA-3 expression, rather The reduced production of IFN-␥ by CD4ϩ T cells may result in than its requirement for Th1 differentiation, may be crucial to con- decrease in and activation and in turn trolling L. major infection. Similarly, IL-27 controls T. cruzi par- cytokine production. Thus, the reduction in arthritis in the IL- asitemia by suppressing Th2 responses (13). We have previously 27RϪ/Ϫ mice could be a consequence of inhibition of several reported that PGIA is exacerbated in IL-4- and STAT6-deficient by guest on September 28, 2021 proinflammatory pathways. mice, indicating that IL-4 regulates the severity of arthritis (24). However, despite the regulatory role of IL-4 in PGIA, we found Discussion that the lack of IL-27 signaling has no effect on the number of The present study was designed to define the role of IL-27R ex- IL-4-secreting cells or on the amount of IL-4 produced. Hence, an pression in autoimmune arthritis and to determine whether the ex- increase in IL-4 in IL-27RϪ/Ϫ is not involved in suppression pression of IL-27R is necessary for development of arthritis and of PGIA. Th1 cell activation. Previous studies indicate that IL-27R signaling Recently, several anti-inflammatory properties of IL-27 have enhances primary T cell activation and IFN-␥ production. We been uncovered that may provide an explanation for some of the showed in the PGIA model that primary PG-specific T cell pro- conflicting outcomes in IL-27R signaling. This is illustrated in liferation and the production of IFN-␥ are augmented in the pres- IL-27R-deficient mice, in which exposure to T. cruzi, T. gondii,or ence of IL-27, confirming that IL-27 is an important differentiation M. tuberculosis results in an excess production of proinflammatory factor for Th1 cells. We have previously reported that in the ab- cytokines, including IFN-␥ (11–13). These findings indicate IL-27 sence of IFN-␥ or STAT4, the onset and severity of PGIA are is not absolutely required for the generation of IFN-␥ and reveal a substantially reduced, indicating that PGIA is mediated by Th1 regulatory role of IL-27 in controlling inflammation. The robust cells (24). The presence of IL-27 in the spleen and IL-27 mRNA Th1 response in IL-27R-deficient mice infected with T. gondii con- transcripts in joint tissues of arthritic mice suggested that IL-27 trols parasite replication, but is unable to down-regulate the pro- was involved in driving the Th1-type inflammatory response in tective inflammatory responses, which results in the lethality of PGIA. To directly demonstrate the contribution of IL-27 signaling disease (12, 13). Similarly, IL-27R-deficient mice infected with T. to the development of PGIA, we used mice deficient in IL-27R cruzi develop liver necrosis as well as enhanced sensitivity to Con expression. The consequence of IL-27R deficiency was a signifi- A-induced hepatitis (13, 16). Because liver disease improves with cant delay in the onset and severity of arthritis. The reduction in neutralization of IFN-␥, IL-27 appears to function in these models erythema and swelling of paws was reflected in a decrease in cel- of infectious disease as a regulator of Th1-mediated inflammation lular infiltration in the joint, and reduced synovial hyperplasia and (13). However, this is clearly not the case in PGIA, in which IFN-␥ cartilage destruction. Moreover, there was a marked decrease in and several proinflammatory cytokines are down-regulated in the inflammatory cytokine mRNA transcript expression in joint tissues absence of IL-27R signaling. Why might IL-27 function to pro- of IL-27RϪ/Ϫ in comparison with IL-27ϩ/ϩ mice. mote Th1 responses in PGIA, but suppress Th1 responses in T. IL-27R signaling supports Th1 responses through the induction cruzi or T. gondi infection? One possibility is that the threshold of of several essential components necessary for Th1 cell differenti- Th1 activation determines whether IL-27 promotes or suppresses ation. IL-27 induces the transcription factors STAT1 and T-bet, Th1 responses (39). Infection with T. gondii elicits high levels of which in turn activate IL-12R␤2 and IFN-␥ genes (6, 8, 9). Our IL-12 production (12). This strong Th1-polarizing condition could The Journal of Immunology 929 result in IL-27 delivering a negative signal to suppress IFN-␥ pro- kine composed of EBI3 and p28 protein, induces proliferation of naive CD4ϩ T duction. In PGIA, the susceptible strain BALB/c is bias toward a cells. Immunity 16: 779–790. 2. Chen, Q., N. Ghilardi, H. Wang, T. Baker, M. H. Xie, A. Gurney, I. S. Grewal, Th2 response due to a premature loss of IL-12 responsiveness and F. J. de Sauvage. 2000. Development of Th1-type immune responses requires because of reduced IL-12R expression (40–42). Under these sub- the TCCR. Nature 407: 916–920. optimal polarizing conditions, IL-27 primary activity is to support 3. Yoshida, H., S. Hamano, G. Senaldi, T. Covey, R. Faggioni, S. Mu, M. Xia, A. C. Wakeham, H. Nishina, J. Potter, et al. 2001. WSX-1 is required for the differentiation of Th1. initiation of Th1 responses and resistance to L. major infection. Immunity 15: Two recent reports indicate IL-27 is involved in the regulation 569–578. 4. Moriguchi, M., N. Chauhan, A. Finnegan, W. Strober, D. M. Frucht, M. Gadina, of Th17 cell activity (14, 15). EAE is dependent on IL-17-produc- and J. J. O’Shea. 2002. -12, interleukin-23, and interleukin-27. In Tar- Ϫ/Ϫ ing T cells, and in IL-27R mice exacerbation of EAE is asso- geted Therapies in Rheumatology. S. J. Smolen and P. Lipsky, eds. Martin Dunitz ciated with an increase in IL-17 expression in CD4 T cells (14, 43). Ltd., London, pp. 243–257. 5. Trinchieri, G., S. Pflanz, and R. A. Kastelein. 2003. The IL-12 family of het- Similarly, chronic infection with T. gondii in mice lacking IL-27R erodimeric cytokines: new players in the regulation of T cell responses. Immunity leads to lethal CD4-mediated neuroinflammation associated with 19: 641–644. an aggravated IL-17 response despite control of parasite replica- 6. Hibbert, L., S. Pflanz, R. De Waal Malefyt, and R. A. Kastelein. 2003. IL-27 and ␥ IFN-␣ signal via Stat1 and Stat3 and induce T-Bet and IL-12R␤2 in naive T cells. tion (14). Because IFN- regulates IL-17 production (44, 45), aug- J. Cytokine Res. 23: 513–522. mented IL-17 expression could be due to a failure to produce 7. Devergne, O., M. Hummel, H. Koeppen, M. M. Le Beau, E. C. Nathanson, IFN-␥ in the IL-27R-deficient mice. In support of this, absence of E. Kieff, and M. Birkenbach. 1996. A novel interleukin-12 p40-related protein ␥ induced by latent Epstein-Barr virus infection in B lymphocytes. J. Virol. 70: IFN- in EAE leads to an increase in IL-17 production and exac- 1143–1153. erbation in disease symptoms (43, 45), and in M. tuberculosis- 8. Takeda, A., S. Hamano, A. Yamanaka, T. Hanada, T. Ishibashi, T. W. Mak, infected mice lacking IFN-␥ there is an increase in the number of A. Yoshimura, and H. Yoshida. 2003. Cutting edge: role of IL-27/WSX-1 sig- Downloaded from naling for induction of T-bet through activation of STAT1 during initial Th1 IL-17-producing T cells (46). In PGIA, we clearly show that IL-27 commitment. J. Immunol. 170: 4886–4890. does not play a major role in suppressing IL-17 production. In 9. Lucas, S., N. Ghilardi, J. Li, and F. J. de Sauvage. 2003. IL-27 regulates IL-12 ϩ IL-27RϪ/Ϫ, there was a transient rise in IL-17 secretion at 4 wk responsiveness of naive CD4 T cells through Stat1-dependent and -independent mechanisms. Proc. Natl. Acad. Sci. USA 100: 15047–15052. after immunization with PG, but this was not sustained at 8 wk and 10. Owaki, T., M. Asakawa, N. Morishima, K. Hata, F. Fukai, M. Matsui, ϩ there was no difference in the number of CD4 T cells secreting J. Mizuguchi, and T. Yoshimoto. 2005. A role for IL-27 in early regulation of Th1 differentiation. J. Immunol. 175: 2191–2200. IL-17 at 4, 8, or 11 wk. http://www.jimmunol.org/ 11. Holscher, C., A. Holscher, D. Ruckerl, T. Yoshimoto, H. Yoshida, T. Mak, How does PGIA compare with another well-studied model of C. Saris, and S. Ehlers. 2005. The IL-27 receptor chain WSX-1 differentially arthritis, CIA? In PGIA, a deficiency in IFN-␥ results in resistance regulates antibacterial immunity and survival during experimental tuberculosis. to development of arthritis, indicating that PGIA model of arthritis J. Immunol. 174: 3534–3544. 12. Villarino, A., L. Hibbert, L. Lieberman, E. Wilson, T. Mak, H. Yoshida, is Th1 mediated. However, in CIA, a deficiency in IFN-␥R results in R. A. Kastelein, C. Saris, and C. A. Hunter. 2003. The IL-27R (WSX-1) is exacerbation of disease (26, 27). The recent findings that IFN-␥ is an required to suppress T cell hyperactivity during infection. Immunity 19: 645–655. important inhibitor of IL-17 and that a deficiency in IL-17 or inhibi- 13. Hamano, S., K. Himeno, Y. Miyazaki, K. Ishii, A. Yamanaka, A. Takeda, M. Zhang, H. Hisaeda, T. W. Mak, A. Yoshimura, and H. Yoshida. 2003. WSX-1 tion of IL-17 suppresses CIA indicate that CIA is an IL-17-medi- is required for resistance to Trypanosoma cruzi infection by regulation of proin- ated disease (44, 45, 47, 48). Because Th1 and Th17 cells are flammatory cytokine production. Immunity 19: 657–667.

14. Batten, M., J. Li, S. Yi, N. M. Kljavin, D. M. Danilenko, S. Lucas, J. Lee, by guest on September 28, 2021 differentiated by different sets of cytokines, it is likely that the F. J. de Sauvage, and N. Ghilardi. 2006. Interleukin 27 limits autoimmune en- cytokine milieu in which PGIA and CIA are initiated determines cephalomyelitis by suppressing the development of -producing T the T cell phenotype of the disease (49). cells. Nat. Immunol. 7: 929–936. 15. Stumhofer, J. S., A. Laurence, E. H. Wilson, E. Huang, C. M. Tato, In addition to the MHC and non-MHC gene differences between L. M. Johnson, A. V. Villarino, Q. Huang, A. Yoshimura, D. Sehy, et al. 2006. the two models, there are other differences. The development of Interleukin 27 negatively regulates the development of interleukin 17-producing PGIA is recessive, whereas CIA is a dominant disease. Another T helper cells during chronic inflammation of the central nervous system. Nat. Immunol. 7: 937–945. difference is the higher incidence of arthritis in female mice, 16. Yamanaka, A., S. Hamano, Y. Miyazaki, K. Ishii, A. Takeda, T. W. Mak, whereas the reciprocal is true in CIA (22, 50). How these dissim- K. Himeno, A. Yoshimura, and H. Yoshida. 2004. Hyperproduction of proin- ilarities contribute to the T cell cytokine dependence is presently flammatory cytokines by WSX-1-deficient NKT cells in concanavalin A-induced hepatitis. J. Immunol. 172: 3590–3596. unknown. It is important to point out that whereas no animal model 17. Artis, D., L. M. Johnson, K. Joyce, C. Saris, A. Villarino, C. A. Hunter, and is identical with rheumatoid arthritis, they remain highly relevant P. Scott. 2004. Cutting edge: early IL-4 production governs the requirement for to human disease because they provide important information on IL-27-WSX-1 signaling in the development of protective Th1 cytokine responses following Leishmania major infection. J. Immunol. 172: 4672–4675. the mechanisms underlying human disease and they provide in- 18. Artis, D., A. Villarino, M. Silverman, W. He, E. M. Thornton, S. Mu, S. Summer, sight for the development of novel therapeutic agents. T. M. Covey, E. Huang, H. Yoshida, et al. 2004. The IL-27 receptor (WSX-1) is In summary, IL-27 regulation of T cell immune responses is an inhibitor of innate and adaptive elements of type 2 immunity. J. Immunol. 173: 5626–5634. complex. The circumstances under which IL-27 is required for Th1 19. Shimizu, S., N. Sugiyama, K. Masutani, A. Sadanaga, Y. Miyazaki, Y. Inoue, cell differentiation are not obvious. However, for PGIA, in which M. Akahoshi, R. Katafuchi, H. Hirakata, M. Harada, et al. 2005. Membranous IFN-␥ unambiguously regulates the severity of arthritis, IL-27 is glomerulonephritis development with Th2-type immune deviations in MRL/lpr mice deficient for IL-27 receptor (WSX-1). J. Immunol. 175: 7185–7192. an important cytokine for driving the Th1 response. 20. Villarino, A. V., J. S. Stumhofer, C. J. Saris, R. A. Kastelein, F. J. de Sauvage, and C. A. Hunter. 2006. IL-27 limits IL-2 production during Th1 differentiation. J. Immunol. 176: 237–247. Acknowledgments 21. Owaki, T., M. Asakawa, S. Kamiya, K. Takeda, F. Fukai, J. Mizuguchi, and We thank Keith Hamel for critically reviewing this manuscript, and T. Yoshimoto. 2006. IL-27 suppresses CD28-mediated [correction of medicated] Dr. Jeffrey Oswald and all the staff of the Comparative Research Center for IL-2 production through suppressor of cytokine signaling 3. J. Immunol. 176: 2773–2780. their expert technical assistance. 22. Glant, T. T., A. Finnegan, and K. Mikecz. 2003. Proteoglycan-induced arthritis: immune regulation, cellular mechanisms, and genetics. Crit. Rev. Immunol. 23: 199–250. Disclosures 23. Finnegan, A., K. Mikecz, P. Tao, and T. T. Glant. 1999. Proteoglycan (aggrecan)- The authors have no financial conflict of interest. induced arthritis in BALB/c mice is a Th1-type disease regulated by Th2 cyto- kines. J. Immunol. 163: 5383–5390. 24. Finnegan, A., M. J. Grusby, C. D. Kaplan, S. K. O’Neill, H. Eibel, T. Koreny, References M. Czipri, K. Mikecz, and J. Zhang. 2002. IL-4 and IL-12 regulate proteoglycan- 1. Pflanz, S., J. C. Timans, J. Cheung, R. Rosales, H. Kanzler, J. Gilbert, L. Hibbert, induced arthritis through Stat-dependent mechanisms. J. Immunol. 169: T. Churakova, M. Travis, E. Vaisberg, et al. 2002. IL-27, a heterodimeric cyto- 3345–3352. 930 IL-27 INDUCES A Th1 RESPONSE IN VIVO

25. Ferber, I. A., S. Brocke, C. Taylor-Edwards, W. Ridgway, C. Dinisco, 38. Finkelman, F. D., I. M. Katona, T. R. Mosmann, and R. L. Coffman. 1988. IFN-␥ L. Steinman, D. Dalton, and C. G. Fathman. 1996. Mice with a disrupted IFN-␥ regulates the isotypes of Ig secreted during in vivo humoral immune responses. gene are susceptible to the induction of experimental autoimmune encephalomy- J. Immunol. 140: 1022–1027. elitis (EAE). J. Immunol. 156: 5–7. 39. Villarino, A. V., E. Huang, and C. A. Hunter. 2004. Understanding the pro- and 26. Manoury-Schwartz, B., G. Chiocchia, N. Bessis, O. Abehsira-Amar, F. Batteux, anti-inflammatory properties of IL-27. J. Immunol. 173: 715–720. S. Muller, S. Huang, M. C. Boissier, and C. Fournier. 1997. High susceptibility 40. Hsieh, C. S., S. E. Macatonia, A. O’Garra, and K. M. Murphy. 1995. T cell ␥ to collagen-induced arthritis in mice lacking IFN- receptors. J. Immunol. 158: genetic background determines default T helper phenotype development in vitro. 5501–5506. J. Exp. Med. 181: 713–721. 27. Vermeire, K., H. Heremans, M. Vandeputte, S. Huang, A. Billiau, and 41. Guler, M. L., N. G. Jacobson, U. Gubler, and K. M. Murphy. 1997. T cell genetic P. Matthys. 1997. Accelerated collagen-induced arthritis in IFN-␥ receptor-defi- background determines maintenance of IL-12 signaling: effects on BALB/c and cient mice. J. Immunol. 158: 5507–5513. B10.D2 T helper cell type 1 phenotype development. J. Immunol. 159: 28. Berlo, S. E., P. J. van Kooten, C. B. Ten Brink, F. Hauet-Broere, 1767–1774. M. A. Oosterwegel, T. T. Glant, W. Van Eden, and C. P. Broeren. 2005. Naive 42. Bix, M., Z. E. Wang, B. Thiel, N. J. Schork, and R. M. Locksley. 1998. Genetic transgenic T cells expressing cartilage proteoglycan-specific TCR induce arthritis ϩ upon in vivo activation. J. Autoimmun. 25: 172–180. regulation of commitment to production by a CD4 T cell-intrinsic 29. Berlo, S. E., T. Guichelaar, C. B. Ten Brink, P. J. van Kooten, F. Hauet-Broeren, mechanism. J. Exp. Med. 188: 2289–2299. K. Ludanyi, W. van Eden, C. P. Broeren, and T. T. Glant. 2006. Increased ar- 43. Komiyama, Y., S. Nakae, T. Matsuki, A. Nambu, H. Ishigame, S. Kakuta, thritis susceptibility in cartilage proteoglycan-specific T cell receptor-transgenic K. Sudo, and Y. Iwakura. 2006. IL-17 plays an important role in the development mice. Arthritis Rheum. 54: 2423–2433. of experimental autoimmune encephalomyelitis. J. Immunol. 177: 566–573. 30. O’Neill, S. K., M. J. Shlomchik, T. T. Glant, Y. Cao, P. D. Doodes, and 44. Harrington, L. E., R. D. Hatton, P. R. Mangan, H. Turner, T. L. Murphy, A. Finnegan. 2005. Antigen-specific B cells are required as APCs and autoanti- K. M. Murphy, and C. T. Weaver. 2005. Interleukin 17-producing CD4ϩ effector body-producing cells for induction of severe autoimmune arthritis. J. Immunol. T cells develop via a lineage distinct from the T helper type 1 and 2 lineages. Nat. 174: 3781–3788. Immunol. 6: 1123–1132. 31. Hanyecz, A., S. E. Berlo, S. Szanto, C. P. Broeren, K. Mikecz, and T. T. Glant. 45. Park, H., Z. Li, X. O. Yang, S. H. Chang, R. Nurieva, Y. H. Wang, Y. Wang, 2004. Achievement of a synergistic adjuvant effect on arthritis induction by ac- L. Hood, Z. Zhu, Q. Tian, and C. Dong. 2005. A distinct lineage of CD4 T cells tivation of innate immunity and forcing the immune response toward the Th1 regulates tissue inflammation by producing interleukin 17. Nat. Immunol. 6: Downloaded from phenotype. Arthritis Rheum. 50: 1665–1676. 1133–1141. 32. Glant, T. T., and K. Mikecz. 2004. Proteoglycan aggrecan-induced arthritis: a 46. Cruz, A., S. A. Khader, E. Torrado, A. Fraga, J. E. Pearl, J. Pedrosa, murine autoimmune model of rheumatoid arthritis. Methods Mol. Med. 102: A. M. Cooper, and A. G. Castro. 2006. Cutting edge: IFN-␥ regulates the induc- 313–338. tion and expansion of IL-17-producing CD4 T cells during mycobacterial infec- 33. Yoshimoto, T., K. Okada, N. Morishima, S. Kamiya, T. Owaki, M. Asakawa, tion. J. Immunol. 177: 1416–1420. Y. Iwakura, F. Fukai, and J. Mizuguchi. 2004. Induction of IgG2a class switching 47. Lubberts, E., L. A. Joosten, B. Oppers, L. van den Bersselaar, in B cells by IL-27. J. Immunol. 173: 2479–2485. C. J. Coenen-de Roo, J. K. Kolls, P. Schwarzenberger, F. A. van de Loo, and

34. Lawless, V. A., S. Zhang, O. N. Ozes, H. A. Bruns, I. Oldham, T. Hoey, http://www.jimmunol.org/ W. B. van den Berg. 2001. IL-1-independent role of IL-17 in synovial inflam- M. J. Grusby, and M. H. Kaplan. 2000. Stat4 regulates multiple components of mation and joint destruction during collagen-induced arthritis. J. Immunol. 167: IFN-␥-inducing signaling pathways. J. Immunol. 165: 6803–6808. 1004–1013. 35. Lund, R. J., Z. Chen, J. Scheinin, and R. Lahesmaa. 2004. Early target genes of IL-12 and STAT4 signaling in Th cells. J. Immunol. 172: 6775–6782. 48. Nakae, S., A. Nambu, K. Sudo, and Y. Iwakura. 2003. Suppression of immune 36. White, S. J., G. H. Underhill, M. H. Kaplan, and G. S. Kansas. 2001. Cutting induction of collagen-induced arthritis in IL-17-deficient mice. J. Immunol. 171: edge: differential requirements for Stat4 in expression of glycosyltransferases 6173–6177. responsible for selectin ligand formation in Th1 cells. J. Immunol. 167: 628–631. 49. Harrington, L. E., P. R. Mangan, and C. T. Weaver. 2006. Expanding the effector 37. Nakahira, M., H. J. Ahn, W. R. Park, P. Gao, M. Tomura, C. S. Park, CD4 T-cell repertoire: the Th17 lineage. Curr. Opin. Immunol. 18: 349–356. T. Hamaoka, T. Ohta, M. Kurimoto, and H. Fujiwara. 2002. Synergy of IL-12 and 50. Cremer, A. A., E. F. Rosloniec, and A. H. Kang. 1998. The cartilage collagens: IL-18 for IFN-␥ gene expression: IL-12-induced STAT4 contributes to IFN-␥ a review of their structure, organization, and role in the pathogenesis of experi- promoter activation by up-regulating the binding activity of IL-18-induced acti- mental arthritis in animals and in human rheumatic disease. J. Mol. Med. 76:

vator protein 1. J. Immunol. 168: 1146–1153. 275–288. by guest on September 28, 2021