IL-4, a well-recognized modulator of macrophage activation, is perceived as an anti-inflammatory cytokine; however, under certain circumstances IL-4 may function as a proinflammatory cytokine. We have previously demonstrated that IL-4 treatment of mice with proteoglycan-induced arthritis (PGIA) inhibited the development of disease. To determine whether the capacity of IL-4 to inhibit disease is dependent on IL-4-mediated regulation of IL-12, we assessed the requirement for IL-4 in modulating development of PGIA. Immunization of mice, lacking IL-4 and Stat6, with proteoglycan results in a significant increase in arthritis severity in comparison to wild-type controls, suggesting that arthritis severity is regulated by IL-4 through a Stat6-dependent mechanism. Concomitant with exacerbated disease in IL-4−/− mice, there is a significant increase in the systemic production of proinflammatory cytokines IL-12, TNF-α, and IFN-γ and in levels of mRNA transcripts for proinflammatory cytokines and chemokines in joints. Disease is suppressed in Stat4−/− mice indicating that elevated levels of IL-12 contribute to exacerbation of arthritis and that suppression is accompanied by reduced levels of IFN-γ production. In support of this, IFN-γ−/− mice are protected from PGIA and the degree of inflammation is similar to Stat4−/− mice. The decrease in disease severity in IFN-γ−/− and Stat4−/− mice correlates with diminished TNF-α levels but there is no switch to a Th2-type response. Taken together, these results suggest that IL-4 regulates the severity of disease in PGIA by controlling IL-12 production, which in turn regulates the magnitude of IFN-γ expression through a Stat4-dependent pathway.

Interleukin-4 is a pleiotropic cytokine dictating the nature of the immune response to infectious Ags and self Ags through its effect on a variety of cell populations. IL-4 plays an essential role in T and B cell differentiation where it is critical for initiating type 2 immune responses (1, 2). In addition, IL-4 is recognized as an anti-inflammatory cytokine that modulates macrophage activity through the suppression of proinflammatory cytokine expression (3, 4, 5, 6). Macrophage-induced cytokines are important for differentiation of Th1-type cells and as such IL-4 may be essential for the regulation of Th1-type responses (2, 7). Moreover, the activity of Th1-type cells can be directly inhibited by IL-4 (8, 9).

The ability of Th2-associated cytokines to inhibit the development of deleterious Th1-mediated responses suggested that they might be used as therapeutics in autoimmune disease. Previously, we and others have demonstrated that in vivo treatment with IL-4 was effective in various autoimmune models including proteoglycan-induced arthritis (PGIA),3 collagen-induced arthritis (CIA), diabetes in nonobese diabetic mice and experimental autoimmune encephalomyelitis (EAE) (10, 11, 12). However, there is also evidence suggesting that IL-4 may potentiate Th1-mediated responses. In experimental autoimmune uveoretinitis in rats and a Th1 cell transfer model of colitis, IL-4 treatment augmented disease that was associated with an increase in IFN-γ, TNF-α, and NO (13, 14).

Because IL-12 is critical for Th1 cell differentiation (15, 16), the ability of IL-4 to either inhibit or promote Th1-mediated responses may depend on the capacity of IL-4 to regulate IL-12 production. However, the inhibitory activity of IL-4 on IL-12 production is controversial. Several reports demonstrate that IL-4 inhibits the production of IL-12 by dendritic cells or macrophages in vitro (17, 18, 19, 20), whereas others have reported that IL-4 augments the induction of IL-12 (21, 22, 23). Studies on the effect of IL-4 on IL-12 production have generally been performed in vitro and the interrelationship between IL-4 and IL-12 in vivo is not clearly understood. To solve this discrepancy and investigate the interrelationship between IL-4 and IL-12 in vivo, we used a model of autoimmune arthritis in which BALB/c mice, immunized with human cartilage proteoglycan, develop disease (24). This model shows many similarities with rheumatoid arthritis and is Th1-mediated (10). Furthermore, the predisposition of BALB/c mice toward a Th2 phenotype makes this an ideal model for studying IL-4 regulation of Th1 responses in vivo (25, 26). We were interested in determining whether IL-4 plays a key role in susceptibility to disease and examining how IL-12 contributes to this process. In mice lacking the IL-4 gene, disease severity is significantly increased in comparison to wild-type (WT) mice. This increase in disease severity in IL-4−/− mice is Stat6-dependent and correlates with elevated levels of IL-12 and IFN-γ in IL-4−/− mice. Furthermore, Stat4−/− and IFN-γ−/− mice are protected from developing arthritis. Our results indicate that IL-4 and IL-12 regulate susceptibility to PGIA by controlling IFN-γ expression.

Breeding pairs of IL-4−/− mice were obtained from H. Eibel (Klinische Forschergruppe fur Rheumatologie, Freiburg, Germany). Stat4−/− and Stat6−/− mice were obtained from M. J. Grusby (Harvard School of Public Health, Boston, MA). IFN-γ−/− mice were purchased from The Jackson Laboratory (Bar Harbor, ME). To match the original BALB/c background, WT BALB/c mice were purchased from The Jackson Laboratory for IL-4−/− and IFN-γ−/− mice and from Taconic Farms (Germantown, NY) for Stat-deficient mice. Of note, some of the variability in arthritis severity may be due to our previous observation that different BALB/c substrains exhibit differences in disease severity (27).

Human cartilage tissue was obtained at the time of joint replacement surgery and prepared as previously described (28, 29). Female BALB/c mice, WT, or mice with targeted gene disruptions were injected i.p. with 100 μg of cartilage proteoglycans (measured as protein) on days 0, 21, and 42. The first and third injections of proteoglycan were given in CFA (Difco, Detroit, MI) and the second injection of proteoglycan was given in IFA (Difco) as previously described (10, 24, 28, 29).

A standard scoring system, based upon swelling and redness of each paw, was used for the assessment of arthritis severity. The first clinical symptom of swelling was recorded as the time of disease arthritis. Joint swelling was scored (ranging from 0 to 4 of each paw) and expressed as acute cumulative arthritis score. The severity of arthritis in each paw was graded according to an established scoring system as follows: 0 = normal, 1 = mild swelling, 2 = moderate swelling, 3 = pronounced swelling, and 4 = severe swelling affecting the entire paw, with a maximum cumulative score of 16 per animal (27, 29). To control for variation between groups of mice, arthritis was induced in WT and gene-deficient mice (8–12 mice) on two separate occasions. The arthritis data presented represent results from one of the two experiments.

Spleens were obtained 4 wk after the last injection with proteoglycan. Single cell suspensions were prepared as previously described (10). Splenocytes (2.0 × 106 cells/ml) were incubated in 24-well Falcon plates (Fisher Scientific, Pittsburgh, PA) in HEPES-buffered RPMI 1640 medium as described (10). Cells were cultured in the absence or presence of proteoglycan (25 μg/ml). IFN-γ was measured in supernatants on day 5, IL-4 on day 3, and TNF-α and IL-12 on day 1. Cytokine concentrations, IL-4, IFN-γ, and TNF-α and IL-12, were measured by OptEIA kits (BD PharMingen, San Diego, CA). Inhibition of IFN-γ production by anti-IL-12 Abs (BD PharMingen) and recombinant IL-4 was performed with splenocytes harvested from proteoglycan-immunized mice. Splenocytes (5 × 105/ml) in triplicate were incubated with titrated concentration of anti-IL-12 or rIL-4 in the presence of proteoglycan (25 μg/ml). rIL-4 was a gift from Schering-Plough Research Institute (Kenilworth, NJ).

Hind paws were homogenized with a polytron homogenizer (KRI Works, Cincinnati, OH) on ice. Homogenate was centrifuged to remove large debris, and RNA was extracted with TRI Reagent (Molecular Research Center, Cincinnati, OH). RNase protection assays (RPA) were performed on 10 μg of RNA using the Riboquant MultiProbe RPA System (BD PharMingen) according to the manufacturer’s instructions. Templates were used to detect a set of cytokine and chemokine transcripts (mCK-3b (TNF-β, lymphotoxin-β, TNF-α, IL-6, IFN-γ, IFN-β, TGF-β1, TGF-β2, TGF-β3, and macrophage inflammatory factor); mCK-2b (IL-12p35, IL-12p40, IL-10, IL-1α, IL-1β, IL-1 receptor antagonist (IL-1ra), IL-18, IL-6, IFN-γ, and macrophage inflammatory factor); and mCK-5 (lymphotactin, RANTES, eotaxin, macrophage inflammatory protein (MIP)-1β, MIP-1α, MIP-2, IFN-γ-inducible protein-10, monocyte chemotactic protein (MCP)-1, TCA-3) as well as the housekeeping gene GAPDH and L3. Labeled ([α-32P]UTP) antisense RNA was synthesized by in vitro transcription from a cDNA template provided in the kit. Antisense RNA probe was purified by phenol/chloroform extraction and ethanol precipitation and then hybridized with mRNA samples at 56°C overnight. RNase was used to digest ssRNA. Protected dsRNA was purified by phenol/chloroform extraction and ethanol precipitation. The samples were electrophoresed on a 5% denaturing polyacrylamide gel. The gel was dried and exposed to a PhosphorImager screen. Radioactivity of the samples was measured and analyzed by scanning densitometry on a STORM PhosphorImager (Molecular Dynamics, Sunnyvale, CA). The level of mRNA for each cytokine was expressed as the ratio (in units of density) of each cytokine to GAPDH.

The Mann-Whitney U test was used to analyze nonparametric data for statistical significance. Values of p < 0.05 were considered significant.

Naive BALB/c mice have a propensity toward a Th2 phenotype (25, 26). However, once immunized with proteoglycan, they develop a dominant Th1-type IFN-γ response while still producing significant quantities of IL-4 (10). IL-4 is a potent inhibitor of inflammation in PGIA and effectively suppresses acute inflammation (10). We were interested in ascertaining whether levels of endogenous IL-4 play a critical role in regulating inflammation. WT and IL-4−/− mice were immunized with proteoglycan and the development of arthritis was monitored over time by evaluating the onset and severity of inflammation. IL-4−/− mice exhibited an increase in the frequency of arthritic animals, 100% incidence in IL-4−/− mice (9 of 9) vs 75% in WT mice (9 of 12) (Fig. 1,A). In IL-4−/− mice, joint swelling and redness were first observed 20 days after the last immunization with proteoglycan, and reached a maximum severity score of 9.5 ± 1.2 (Fig. 1,B). By comparison, in WT mice arthritis was delayed until 27 days and the arthritis score was milder reaching a maximum of 3.6 ± 1.0 between days 36 and 40 (Fig. 1 B).

FIGURE 1.

Proteoglycan-induced arthritis is enhanced in IL-4−/− mice in comparison to WT controls. Mice were immunized with proteoglycan on days 0, 21, and 42 as described in Materials and Methods. “Days after Immunization” indicates the days after the third immunization with proteoglycan. A, Incidence of PGIA, expressed as the percentage of arthritic animals in the WT and IL-4−/− groups. B, Disease severity, expressed as the cumulative arthritis score in affected animals. Values are the mean and SEM of WT (n = 12), and IL-4−/− (n = 9) mice and represent one of two experiments performed. ∗, Data that are significantly different (p < 0.05) between WT and IL-4−/− mice.

FIGURE 1.

Proteoglycan-induced arthritis is enhanced in IL-4−/− mice in comparison to WT controls. Mice were immunized with proteoglycan on days 0, 21, and 42 as described in Materials and Methods. “Days after Immunization” indicates the days after the third immunization with proteoglycan. A, Incidence of PGIA, expressed as the percentage of arthritic animals in the WT and IL-4−/− groups. B, Disease severity, expressed as the cumulative arthritis score in affected animals. Values are the mean and SEM of WT (n = 12), and IL-4−/− (n = 9) mice and represent one of two experiments performed. ∗, Data that are significantly different (p < 0.05) between WT and IL-4−/− mice.

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To determine whether an increase in joint inflammation correlated with an earlier expression of cytokine and chemokine mRNA transcripts in the joints of IL-4−/− mice, RPA were performed. We harvested hind paws of mice before any sign of redness or swelling was evident. These time points were chosen based on our observation that cytokine and chemokine transcripts are expressed shortly before the onset of joint inflammation. We found that the relative levels of mRNA for IL-1β, IL-1ra, IL-6, IFN-γ, lymphotoxin-β, TNF-α, TGF-β1, and TGF-β3 as well as the chemokine mRNA for MIP-1β, MIP-1α, MIP-2, and TCA-3 were increased in IL-4−/− mice in comparison to control mice 9 wk after the initial immunization with proteoglycan (Fig. 2). However, at an earlier time point, 7 wk after the initial immunization, neither the IL-4−/− nor WT mice expressed an increase in cytokine or chemokine transcripts in comparison to nonimmunized animals (Fig. 2). These results demonstrate that IL-4 is important in controlling initiation of inflammation in the joint.

FIGURE 2.

Joint cytokine and chemokine mRNA levels are increased in IL-4−/− mice. A and B, Cytokine mRNA transcripts. C, Chemokine mRNA transcripts. RNA was obtained from ankle joints of WT and IL-4−/− mice 7 and 9 wk after the initial immunization with proteoglycan. Joints were homogenized, and RNA was extracted. RPAs were performed using RPA as described in Materials and Methods. Values are the mean and SD of the ratio of cytokines or chemokines to GAPDH mRNA of four RNA samples from WT and IL-4−/− mice. ∗, Statistically significant differences between WT and IL-4−/− mice.

FIGURE 2.

Joint cytokine and chemokine mRNA levels are increased in IL-4−/− mice. A and B, Cytokine mRNA transcripts. C, Chemokine mRNA transcripts. RNA was obtained from ankle joints of WT and IL-4−/− mice 7 and 9 wk after the initial immunization with proteoglycan. Joints were homogenized, and RNA was extracted. RPAs were performed using RPA as described in Materials and Methods. Values are the mean and SD of the ratio of cytokines or chemokines to GAPDH mRNA of four RNA samples from WT and IL-4−/− mice. ∗, Statistically significant differences between WT and IL-4−/− mice.

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Binding of IL-4 to the IL-4R initiates intracellular signaling through several pathways but primarily through the Stat6 and the insulin receptor substrate pathways (30, 31, 32, 33). To determine whether the effect of IL-4 on the severity of arthritis was mediated through Stat6, we examined the development of arthritis in Stat6−/− mice. There was no difference in the incidence of arthritis between the Stat6−/− and WT mice; all mice developed disease. However, arthritis developed more rapidly in the Stat6−/− mice (Fig. 3,A); moreover, arthritis was very severe with a clinical score of 9.5 ± 1.71. By comparison, arthritis in WT mice was significantly less severe, with a score of 6.0 ± 1.11(Fig. 3 B). Based on these data, the primary pathway by which IL-4 regulates inflammation is through Stat6.

FIGURE 3.

Proteoglycan-induced arthritis is exacerbated in Stat6−/− mice. PGIA was induced as described in Fig. 1. “Days after Immunization” indicates the days after the third immunization with proteoglycan. A, Incidence of PGIA, expressed as the percentage of arthritic animals in the WT and Stat6−/− groups. B, Disease severity, expressed as the cumulative arthritis score in affected animals. Values are the mean and SEM of WT (n = 8), and IL-4−/− (n = 8) mice and represent one of two experiments performed. ∗, Data that are significantly different (p < 0.05) between WT and Stat6 −/− mice.

FIGURE 3.

Proteoglycan-induced arthritis is exacerbated in Stat6−/− mice. PGIA was induced as described in Fig. 1. “Days after Immunization” indicates the days after the third immunization with proteoglycan. A, Incidence of PGIA, expressed as the percentage of arthritic animals in the WT and Stat6−/− groups. B, Disease severity, expressed as the cumulative arthritis score in affected animals. Values are the mean and SEM of WT (n = 8), and IL-4−/− (n = 8) mice and represent one of two experiments performed. ∗, Data that are significantly different (p < 0.05) between WT and Stat6 −/− mice.

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One potential mechanism by which IL-4 might control inflammation is inhibiting proinflammatory cytokine production. Therefore, we examined whether secretion of IL-12 and TNF-α was increased in IL-4−/− and Stat6−/− mice. To test this, splenocytes harvested from proteoglycan-immunized mice were cultured in the presence or absence of proteoglycan. Analysis of cytokine expression revealed that IL-12 and TNF-α levels released in response to proteoglycan challenge in vitro were significantly increased in cells obtained from IL- 4−/− and Stat6−/− mice in comparison to cells from WT animals (Fig. 4, A and B). These results suggest that endogenous IL-4 exerts an inhibitory effect in vivo on TNF-α- and IL-12-producing cells and that the enhanced production of proinflammatory cytokines may contribute to aggravated disease in IL-4−/− mice.

FIGURE 4.

TNF-α, IL-12, and IFN-γ production is substantially increased in IL-4−/− mice. WT (n = 5), IL-4−/− (n = 5), and Stat6−/− (n = 5) mice were immunized with proteoglycan, and spleens were harvested 2 wk after the second injection with Ag. Spleen cells were cultured in medium alone (control) or were stimulated with proteoglycan (20 μg/ml). Supernatants were harvested for A, IL-12, B, TNF-α at 24 h, and C, IFN-γ at 5 days and assayed using ELISA. Data represent the mean and SEM of individual mice and represent one of two experiments. D, IL-4 inhibits IFN-γ produced by IL-4−/−, but not Stat6−/−, mice. Splenocytes from proteoglycan-immunized IL-4−/− and Stat4−/− mice were activated with proteoglycan in vitro in the presence of rIL-4 at various concentrations ranging from 0.015 to 0.060 μg/ml. Supernatants were harvested for measurement of IFN-γ by ELISA. Values represent the mean and ± SEM of cytokine levels. ∗, Statistically significant differences (p < 0.05) between WT and IL-4−/− and Stat6−/− mice.

FIGURE 4.

TNF-α, IL-12, and IFN-γ production is substantially increased in IL-4−/− mice. WT (n = 5), IL-4−/− (n = 5), and Stat6−/− (n = 5) mice were immunized with proteoglycan, and spleens were harvested 2 wk after the second injection with Ag. Spleen cells were cultured in medium alone (control) or were stimulated with proteoglycan (20 μg/ml). Supernatants were harvested for A, IL-12, B, TNF-α at 24 h, and C, IFN-γ at 5 days and assayed using ELISA. Data represent the mean and SEM of individual mice and represent one of two experiments. D, IL-4 inhibits IFN-γ produced by IL-4−/−, but not Stat6−/−, mice. Splenocytes from proteoglycan-immunized IL-4−/− and Stat4−/− mice were activated with proteoglycan in vitro in the presence of rIL-4 at various concentrations ranging from 0.015 to 0.060 μg/ml. Supernatants were harvested for measurement of IFN-γ by ELISA. Values represent the mean and ± SEM of cytokine levels. ∗, Statistically significant differences (p < 0.05) between WT and IL-4−/− and Stat6−/− mice.

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Because IL-12 is a potent activator of IFN-γ (16), and IL-12 is up-regulated in the IL-4−/− arthritic mice, one possible pathway by which IL-4 may enhance disease is through an increase in IFN-γ production. To investigate this possibility, we assessed whether there was an increase in the level of IFN-γ in IL-4−/− and Stat6−/− arthritic mice. Splenocytes harvested from IL-4−/− and Stat6−/− mice secreted significantly more IFN-γ in response to proteoglycan than splenocytes from WT mice (Fig. 4,C). These results suggest that IL-4 regulates levels of IFN-γ in proteoglycan-immunized mice. To confirm that IL-4 has the capacity to regulate IFN-γ production, we tested whether IL-4 would inhibit the proteoglycan-induced IFN-γ production from IL-4−/− and Stat4−/− mice. Splenocytes from proteoglycan-immunized IL-4−/− and Stat6−/− mice were activated with proteoglycan in vitro and rIL-4 was used at various concentrations ranging from 15 to 60 ng/ml. As shown in Fig. 4 D, IL-4 inhibited the production of proteoglycan-induced IFN-γ from IL-4−/−, but not Stat6−/−, cells. These results demonstrate that IL-4 may inhibit IFN-γ production through an IL-12-dependent pathway.

We next assessed whether IL-12 could contribute to the exacerbation of arthritis observed in the IL-4−/− and Stat6−/− mice. IL-12-mediated activation of Stat4 is required for the induction of Th1-mediated immune responses (16). Therefore, we examined whether induction of arthritis was suppressed in Stat4−/− mice. WT mice progressively developed arthritis over a number of weeks after the last immunization with proteoglycan. Arthritis developed in 80% of WT mice (12 arthritic of 15 immunized) with an arthritic score of 5.23 ± 1.26. In Stat4−/− mice, development of arthritis was significantly reduced with an incidence of 28.5% (4 arthritic of 14 immunized) and a maximum arthritic score of 1.86 ± 0.95 (Fig. 5, A and B). These data demonstrate that in the absence of the Stat4-signaling pathway the severity of disease was significantly reduced suggesting that IL-12 was required for induction of disease.

FIGURE 5.

Arthritis is suppressed and IFN-γ production is reduced in Stat4−/− mice. Incidence (A) and arthritis score (B) were monitored in WT (n = 17) and Stat4−/− (n = 14) mice. C, IFN-γ production is significantly reduced in Stat4−/− mice. IFN-γ was measured in the medium of unstimulated (control) and proteoglycan-stimulated splenocytes harvested at 52 days postimmunization from WT and Stat4−/− animals. D, Abs specific for IL-12 inhibited IFN-γ production by splenocytes from proteoglycan-immunized WT and IL-4−/− mice in a dose-dependent manner. Data represent the mean ± SEM of individual mice. ∗, Statistically significant differences (p < 0.05) between WT and IFN-γ or Stat4-deficient mice.

FIGURE 5.

Arthritis is suppressed and IFN-γ production is reduced in Stat4−/− mice. Incidence (A) and arthritis score (B) were monitored in WT (n = 17) and Stat4−/− (n = 14) mice. C, IFN-γ production is significantly reduced in Stat4−/− mice. IFN-γ was measured in the medium of unstimulated (control) and proteoglycan-stimulated splenocytes harvested at 52 days postimmunization from WT and Stat4−/− animals. D, Abs specific for IL-12 inhibited IFN-γ production by splenocytes from proteoglycan-immunized WT and IL-4−/− mice in a dose-dependent manner. Data represent the mean ± SEM of individual mice. ∗, Statistically significant differences (p < 0.05) between WT and IFN-γ or Stat4-deficient mice.

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We have previously shown that immunization with proteoglycan induces high levels of IFN-γ (10). Because production of IFN-γ by CD4+ T cells can occur by Stat4-dependent and -independent mechanisms, we assessed the levels of proteoglycan-induced IFN-γ production in Stat4−/− mice (16, 34). Splenocytes obtained from proteoglycan-immunized WT and Stat4−/− mice were cultured in the presence and absence of proteoglycan. Production of spontaneous and proteoglycan-induced IFN-γ was significantly reduced in Stat4−/− mice in comparison to WT mice (Fig. 5,C). To further confirm that IL-12 is involved in the production of IFN-γ, we used a neutralizing anti-IL-12 Ab (Fig. 5 D). IFN-γ production by splenocytes of both WT and IL-4−/− mice was blocked in the presence of anti-IL-12 Abs. Taken together these data suggest that IL-12, through a Stat4-dependent mechanism, is essential for the induction of PGIA. These data further suggest that IFN-γ may be necessary for development of arthritis.

The hallmark of a Th1-mediated disease is the production of IFN-γ; however, in a number of Th1-mediated autoimmune diseases, elimination of IFN-γ either by neutralizing Abs or in IFN-γ−/− and IFN-γ receptor−/− mice leads to disease exacerbation (35, 36, 37, 38, 39, 40). Our results in the Stat4−/− mice in which reduction in disease correlated with decreased levels of IFN-γ pointed to a critical role of IFN-γ in the development of PGIA. Therefore, we were interested in directly testing whether elimination of IFN-γ inhibited disease. We first assessed whether neutralizing Abs specific for IFN-γ administered before the development of disease but after the third immunization with proteoglycan could suppress disease development. WT mice were treated with either 500 μg of rat Ig or anti-IFN-γ Ab every other day for 30 days. Development of arthritis was monitored over time. Control mice exhibited symptoms of inflammation at 17 days after the last immunization with proteoglycan and 100% of the mice became arthritic by day 28 (Fig. 6,A). However, in the anti-IFN-γ-treated group, disease was suppressed during the 30 days of treatment and continued to be suppressed until day 44; it was not until day 50 that all mice were arthritic (Fig. 6,A). The severity of disease was similar in the control and the anti-IFN-γ-treated mice at day 50 postimmunization (Fig. 6 B). These data demonstrate that IFN-γ is important for the induction of PGIA.

FIGURE 6.

PGIA is reduced in mice treated with IFN-γ neutralizing Abs and in IFN-γ−/− mice. WT mice were treated with 500 μg anti-IFN-γ mAb (n = 6) or rat Ig (n = 6) every other day for 30 days starting after the third immunization with proteoglycan. Incidence (A) and arthritic score (B) are indicated for the treated mice. Incidence (C) and arthritis score (D) are indicated in WT (n = 14) and IFN-γ−/− (n = 14) for up to 42 days after the last injection with Ag. E, TNF-α was reduced and IL-4 was unchanged in IFN-γ −/− and Stat4 −/− mice in comparison to WT. Production of TNF-α and IL-4 was measured by splenocytes harvested from WT (n = 5), IFN-γ−/− (n = 5), and Stat4−/− (n = 5) ∼50 days after the last immunization with Ag. Data represent the mean ± SEM of individual mice. ∗, Data are statistically significant different (p < 0.05) between WT and IFN-γ- or Stat4-deficient mice.

FIGURE 6.

PGIA is reduced in mice treated with IFN-γ neutralizing Abs and in IFN-γ−/− mice. WT mice were treated with 500 μg anti-IFN-γ mAb (n = 6) or rat Ig (n = 6) every other day for 30 days starting after the third immunization with proteoglycan. Incidence (A) and arthritic score (B) are indicated for the treated mice. Incidence (C) and arthritis score (D) are indicated in WT (n = 14) and IFN-γ−/− (n = 14) for up to 42 days after the last injection with Ag. E, TNF-α was reduced and IL-4 was unchanged in IFN-γ −/− and Stat4 −/− mice in comparison to WT. Production of TNF-α and IL-4 was measured by splenocytes harvested from WT (n = 5), IFN-γ−/− (n = 5), and Stat4−/− (n = 5) ∼50 days after the last immunization with Ag. Data represent the mean ± SEM of individual mice. ∗, Data are statistically significant different (p < 0.05) between WT and IFN-γ- or Stat4-deficient mice.

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To further confirm the requirement for IFN-γ in the induction of PGIA, we used IFN-γ−/− mice. WT and IFN-γ−/− mice were immunized with proteoglycan and the development of arthritis was monitored. Consistent with the data obtained form anti-IFN-γ treatment, in IFN-γ−/− mice, disease onset was significantly delayed. At day 27 after the last immunization with proteoglycan, only 21% (3 arthritic of 14 immunized) of the IFN-γ−/− mice were arthritic whereas 92% (13 of 14) of WT mice were arthritic (Fig. 6,C). Arthritis was significantly more severe (severity score 6.70 ± 1.13) in WT than IFN-γ−/− animals (severity score 1.24 ± 0.33) (Fig. 6 D). However, over an extended period of time, up to 80–100 days after the last immunization with proteoglycan, additional IFN-γ−/− mice (36%, 13 of 36) became mildly arthritic (severity score 2.43 ± 0.64). These results suggest that IFN-γ plays a significant role in the development of inflammation by promoting disease in PGIA.

Inhibition of arthritis could occur through the reduction in proinflammatory cytokine production or enhanced Th2-mediated responses. We observed that TNF-α production was significantly suppressed in splenocytes of Stat4−/− and IFN-γ−/− mice in comparison to WT mice (Fig. 6 F). However, when IL-4 production was examined there was no difference in IL-4 production between WT and either Stat4−/− or IFN-γ−/−. Taken together, these results suggest that in PGIA, IFN-γ regulates the degree of disease severity whereas IL-12 controls the magnitude of IFN-γ expression through a Stat4-dependent pathway.

Because Ig isotypes are regulated by Th1 and Th2 cytokines, we examined whether the increase in IFN-γ production in the IL-4−/− and Stat6−/− mice led to an increase in the IgG2a isotype which is tightly associated with Th1-biased help and IFN-γ activities. Mice were bled 2 wk after the last immunization with proteoglycan and murine proteoglycan-specific autoantibody isotypes were assayed by ELISA. In WT mice, the IgG1 and the IgG2a isotype were similar, however, in IL-4−/− and Stat6−/− mice there was a 6-fold increase in the IgG2a isotype (Fig. 7). In the absence of IFN-γ, there was a significant reduction in IgG2a, demonstrating the dependency of the IgG2a response on IFN-γ. These data further confirm that the enhanced IFN-γ production observed from splenocytes of IL-4−/− and Stat6−/− is mirrored in an enhanced secretion of IgG2a autoantibody isotypes. These IgG2a autoantibodies may be pathogenic and contribute to the aggravated disease in the IL-4−/− mice.

FIGURE 7.

Proteoglycan-specific autoantibody IgG2a isotype is increased in IL-4−/− and Stat6−/− mice. WT (n = 30), IL-4−/− (n = 8), Stat6−/− (n = 12), IFN-γ−/− (n = 12), and Stat4−/− (n = 12) were immunized with proteoglycan and serum autoantibody isotypes, IgG1 and IgG2a, were measured by ELISA. Values represent the mean and SD of Ab isotype levels. ∗, Data are statistically significant different (p < 0.05) between WT and cytokine-deficient mice.

FIGURE 7.

Proteoglycan-specific autoantibody IgG2a isotype is increased in IL-4−/− and Stat6−/− mice. WT (n = 30), IL-4−/− (n = 8), Stat6−/− (n = 12), IFN-γ−/− (n = 12), and Stat4−/− (n = 12) were immunized with proteoglycan and serum autoantibody isotypes, IgG1 and IgG2a, were measured by ELISA. Values represent the mean and SD of Ab isotype levels. ∗, Data are statistically significant different (p < 0.05) between WT and cytokine-deficient mice.

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The intent of this study was to address the regulatory role of endogenous IL-4 in autoimmune arthritis and to explore mechanisms by which arthritis is controlled. We used mice with targeted deletion of IL-4, Stat6, Stat4, and IFN-γ genes and characterized the proinflammatory response in PGIA that leads to the development of disease. Our first important observation is that endogenous IL-4 plays a critical role in controlling disease severity. In IL-4−/− mice, joint inflammation is much more severe than in WT animals. To begin to understand how endogenous IL-4 regulates disease, we first examined the IL-4 signaling mechanism. IL-4 activates a number of different signaling pathways that could lead to IL-4-dependent gene activation. These include activation of Stat6 through the activation of Janus kinases 1 and 3 (30, 41), phosphorylation of insulin receptor 1 and 2 (33, 42), activation of Ras/mitogen-activated protein kinase (43, 44), and activation of phosphatidylinositol-3 kinase (45). The Stat6 pathway appears to dominate in PGIA because there was very little difference between IL-4−/− and Stat6−/− mice in either the degree of arthritis or in the development of inflammatory cytokines. An increase in disease severity correlates with an increase in proinflammatory cytokines IL-12, TNF-α, and IFN-γ produced by spleen cells of IL-4−/− mice as well as with mRNA transcripts for proinflammatory cytokines and chemokines in the joint. Although IL-4 is a recognized inhibitor of proinflammatory genes such as IL-12 and TNF-α (15, 35, 38, 39, 40, 43), there are instances in which IL-4 promotes IL-12 and TNF-α production (21, 22, 23). We observed an increase in TNF-α and IL-12 in IL-4−/− and Stat6−/− mice, suggesting that the dominant activity of IL-4 in vivo is anti-inflammatory. We observed only a modest increase in TNF-α in the absence of IL-4, which may be due to other anti-inflammatory pathways that are still active in the IL-4−/− mice, such as those related to IL-10 (46). It is interesting to note that the increase in IL-12, TNF-α, and IFN-γ was proteoglycan-specific, indicating that T cells play a major role in either production of cytokines or in stimulating macrophages to secrete enhanced levels of these inflammatory cytokines.

IL-4 regulation of proinflammatoy cytokines in vivo is likely to be dependent on the interaction between macrophage/dendritic cells and T cells. Macrophage-derived IL-12 directly induces IFN-γ expression in Th1 cells and macrophages and these cytokines amplify each other’s function. There are several mechanisms by which IL-4 could regulate expression of IFN-γ. IL-4 has been shown to inhibit Stat4 induction (47, 48, 49), IL-12Rβ2 expression (9, 50), and the transcription factor, T-bet (51, 52). In our studies, we cannot distinguish between an increase in IL-12 or IFN-γ due to loss of IL-4 as a direct effect on macrophages or an indirect effect through an increase in IFN-γ produced by Th1 cells. Thus, the level of endogenous IL-4 is critical in controlling inflammatory responses instigated by macrophages, dendritic cells, or Th1 cells.

Our second important observation is that elevated levels of IL-12 and IFN-γ contribute to the exacerbation of disease in the IL-4−/− mice. We found that arthritis is suppressed in the absence of IL-12 signaling through Stat4 and the reduction in disease in Stat4−/− mice is associated with a lower level of IFN-γ production. The reduced levels of IFN-γ may be one explanation for the reduction in PGIA in Stat4−/− mice. However, Stat4 is also involved in the induction of other genes that are characteristic of Th1 cells, in particular, IL-18R, CCR5, and the ligand for the adhesion molecule P-selectin (53, 54, 55). When we assessed the development of arthritis in IFN-γ−/− mice, we observed that they are also protected from PGIA and the degree of reduction in disease is very similar to Stat4−/− mice. These data suggest IL-4 regulates arthritis through a pathway that involves IL-12 signaling through Stat4, which controls IFN-γ production. It is interesting to note that over an extended period of time, 80 days after the last immunization with Ag, additional mice (36%) develop mild inflammation (severity score 2.43 ± 0.64) which suggests that while IFN-γ is required for severe disease other genetic factors contribute to the susceptibility to disease.

There are several possible explanations for the reduction in arthritis in IFN-γ−/− mice. Because T cells are required for the development of PGIA, lack of T cell priming would suppress disease. However, IFN-γ−/− and WT T cells proliferated to challenge with proteoglycan to a similar degree (data not shown), which does not support this possibility. Another possibility is the importance of IFN-γ in the activation of macrophages and the production of proinflammatory cytokines. Our data strongly support this concept because the reduction in arthritis in IFN-γ −/− and Stat4−/− mice correlate with lower levels of TNF-α. Alternatively, a reduction in IFN-γ could lead to a switch from Th1-type cells to Th2-type cells; however, we were unable to detect an increase in IL-4-secreting cells in IFN-γ−/− or Stat4−/−mice.

In contrast to our results, previous studies demonstrated that IFN-γ plays a dual role in autoimmune inflammatory diseases such as CIA and EAE, functioning as both a disease-limiting (56, 57, 58, 59) and a disease-promoting factor (60, 61). CIA and EAE are exacerbated in mice deficient in either IFN-γor IFN-γ receptor genes, suggesting that the suppressive effects of IFN-γ dominate (35, 37, 38, 40). The differences between these models and PGIA with respect to the requirement for IFN-γ is currently unknown.

The third major observation we have made is that the expression of proteoglycan autoantibodies of the IgG2a isotype strongly correlate with the level of endogenous IL-4 and IFN-γ. We observed a dramatic increase in the IgG2a response in IL-4−/− and Stat6−/−mice and reciprocally a decrease in the IgG2a response in IFN-γ−/− mice. These findings demonstrate that IL-4 and IFN-γ play a critical role in regulating the proteoglycan-specific IgG2a response. The mechanism of IL-4 suppression of IgG2a in vivo has not been well understood. It has been observed that treatment in vivo with anti-IL-4 Abs minimally affect the IgG2a responses (62), whereas in IL-4-deficient mice immunized with OVA the IgG2a response was increased (63). There is no evidence that IL-4 directly inhibits IgG2a transcription in B cells. Our data is consistent with studies showing that IFN-γ stimulates IgG2a secretion and extend these findings to show that IL-4 regulates IFN-γ production and thus IgG2a secretion.

In summary, we demonstrate that IL-4 regulates arthritis through control of proinflammatory cytokine expression, IL-12, TNF-α, and IFN-γ. These cytokines in turn are capable of amplifying macrophage and T cell activity and Ab production all of which may contribute to disease severity in PGIA.

We thank Dr. T. T. Glant for providing human and mouse proteoglycans and Yanxia Cao for technical assistance. We thank Dr. Satwant Narula (Schering-Plough Research Institute) for the generous gift of rIL-4.

1

This work was supported by National Institutes of Health Grants AR45652 (to A.F. and K.M.), AR47412 (to J.Z.), and AI40171 (to M.J.G.). M.J.G. is a Scholar of the Leukemia and Lymphoma Society.

3

Abbreviations used in this paper: PGIA, proteoglycan-induced arthritis; CIA, collagen-induced arthritis; EAE, experimental autoimmune encephalomyelitis; WT, wild type; RPA, RNase protection assay; IL-1ra, IL-1 receptor antagonist; MIP, macrophage inflammatory protein; MCP, monocyte chemotactic protein.

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