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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC Feb 1, 2011.
Published in final edited form as:
PMCID: PMC2846113

IFN-γ Regulates the Requirement for IL-17 in Proteoglycan-Induced Arthritis


The contribution of the pro-inflammatory cytokines IFN-γ and IL-17 to the pathogenesis of experimental arthritis is controversial. In proteoglycan-induced arthritis (PGIA) severe arthritis is dependent on the production of IFN-γ whereas IL-17 is dispensable. In collagen-induced arthritis (CIA) and antigen-induced arthritis (AIA), although high levels of IFN-γ are secreted, disease is exacerbated in IFN-γ or IFN-γ receptor deficient mice due to the ability of IFN-γ to suppress IL-17 expression. In the present study, we investigated the effect of IFN-γ on the IL-17 response and its consequences in PGIA. In PG-immunized IFN-γ−/− mice, despite reduction in arthritis, the PG-specific CD4+ T cell IL-17 response was significantly increased. Elevated IL-17 contributed to development of arthritis as disease in IFN-γ /IL-17−/− was significantly reduced in comparison to either IFN-γ−/− or IL-17−/− mice. A contribution of IFN-γ and IL-17 to the development of arthritis was also identified in T-bet−/− mice. PG-specific CD4+ T cells from T-bet−/− mice produced reduced IFN-γ and elevated concentrations of IL-17. Both IFN-γ and IL-17 contribute to arthritic as T-bet−/− mice lacking IL-17 (T-bet/IL-17−/−) were resistant whereas WT, T-bet−/−, and IL-17−/− mice were susceptible to PGIA. T cell proliferation and autoantibody production did not correlate with development of disease, however, expression of cytokines and chemokines in joint tissues demonstrate that IFN-γ and IL-17 cooperatively contribute to inflammation. These results demonstrate that both IFN-γ and IL-17 have the potential to induce PGIA but it is the strength of the IFN-γ response that regulates the contribution of each of these T helper effector cytokines to disease.

Keywords: T cells, Cytokines, Chemokines, Rodents, Autoimmunity, Rheumatoid Arthritis


Rheumatoid arthritis (RA) is a chronic, progressive autoimmune disease primarily affecting the synovial joints and causing both significant morbidity and increased mortality (1). The etiology of the disease is unclear; however RA is strongly linked to particular MHC alleles, implying that some aspect of the CD4+ T cell response to self antigen is crucial to the autoimmune process. Pro-inflammatory CD4+ T cells have been divided into Th1, Th2, and Th17 subsets based on their production of IFN-γ, IL-4 and IL-17 respectively. Murine models of arthritis, PGIA and CIA, were originally classified as Th1-mediated diseases based on abundant IFN-γ production (24). IFN-γ has several pro-inflammatory properties that contribute to inflammation in arthritis. Activation of macrophages by IFN-γ results in induction of cytokines, nitric oxide and superoxide production and expression of MHC class I and class II molecules (59). In PGIA, neutralization of IFN-γ inhibits arthritis and IFN-γ−/− mice developed arthritis with delayed onset and reduced severity in comparison to WT mice. However, IFN-γ−/− mice eventually succumb to arthritis in some cases as severe as WT mice. These findings indicate that IFN-γ is an important pro-inflammatory cytokine promoting disease severity in PGIA (3, 4). In CIA, the role for IFN-γ is more complex. Complete elimination of IFN-γ or IFN-γ receptor signaling leads to exacerbation of disease (1012). On the other hand, neutralization of IFN-γ at an early stage of disease inhibits arthritis (13). It was originally thought that a failure to suppress T cell expansion and induce apoptosis and/or an increase in IL-1β was responsible for enhanced arthritis (14, 15). More recently, it was found that IFN-γ inhibits IL-17 production. IL-17 has emerged as an important pro-inflammatory T cell cytokine in several models of arthritis (1618). Thus, the ability of IFN-γ to suppress Th17 cells appears to account for augmented disease in IFN-γ−/− or IFN-γ receptor deficient mice in CIA and AIA as inhibition of IL-17 with neutralizing antibodies suppressed arthritis (19, 20). Contrary to a dependence on IL-17 in CIA and AIA, in PGIA, IL-17 deficiency mice develop arthritis similar to wildtype (WT). To begin to resolve the controversy between these different models of arthritis, we investigated the IFN-γ regulated IL-17 response in PGIA

In this study, using several cytokine- and transcription factor-deficient mice we show that a deficiency in IFN-γ converts PGIA from an IL-17-independent to an IL-17-dependent arthritis. In addition, we show that the transcription factor T-bet regulates IL-17 production. Moreover, T-bet−/− mice are susceptible to PGIA due to the combined effects of low IFN-γ and high IL-17 expression. Taken together these results show that the strength of the IFN-γ response regulates whether IL-17 is expression and its consequences in PGIA.

Materials and Methods


IFN-γ−/−, T-bet−/−, IL-17−/− mice were backcrossed to BALB/c for ≥10 generations. Double knockout mice were generated by intercrossing and selected for by PCR. WT, IFN-γ−/−, and T-bet−/− mice were from the Jackson Laboratory (Bar Harbor, ME) and maintained at the Rush University Medical Center facility. Female WT and gene-deficient mice age matched, 12–14 weeks, were used in all experiments. All animal experiments were approved by the Institutional Animal Care and Use Committee at Rush University Medical Center (Chicago, IL).

Induction and Assessment of Arthritis

Human cartilage was obtained from The Orthopedic Tissue, Transplant, and Implant Repository of Rush University Medical Center, with the approval of the Institutional Review Board. PG was isolated as previously described (21). Mice were immunized with 150 µg human PG i.p. as measured by protein in dimethyldioctadecyl ammonium bromide (DDA) (Sigma Aldrich, St. Louis, MO) and boosted with 100 µg PG in DDA at weeks three and six (22). Mice were monitored for arthritis twice weekly and scored in a blinded manner. Arthritis severity was scored for each paw on a scale from one to four as described (22).

Assessment of T cell activation by proliferation

MACS purified CD4+ T cells (Miltenyi Biotech, Auburn, CA) (2.5×105 cells/ml) and irradiated (2500 rad) naïve spleen cells (2.5×105 cells/ml) were cultured in the presence or absence of PG (10 µg/ml) in RPMI 1640 complete media. Cells were incubated at 37°C in 5% CO2 for 5 days and were pulsed with [3H] thymidine (0.5 µCi/well) for the last 18 hours. Cell cultures were harvested and incorporated [3H] thymidine was measured (MicroBeta Trilux, PerkinElmer, CA).

Detection of serum antibody titers by ELISA

Sera from immunized mice were serially diluted and incubated with the immobilized human and mouse PG, and plate-bound human PG- or mouse PG-specific antibody was detected using peroxidase-conjugated rabbit IgG against mouse IgG1 and IgG2a (Zymed, San Francisco, CA). Data compared to a standard curve of unlabeled murine IgG1 and IgG2a (Southern Biotechnology Associates, Birmingham, AL).

Assessment of intracellular cytokine production

Spleen cells were incubated with ionomycin (500ng/ml) and PMA (50ng/ml) in the presence of GolgiSTOP (BD Biosciences, San Jose, CA) for 4 hours. Cells were stained for extracellular CD4 then permeabilized with the Cytofix/Cytoperm Plus Kit (BD Biosciences) and stained for IFN-γ and IL-17 using specific antibodies or isotype control (BD Biosciences). Data was acquired using FACSCanto II and analyzed using FACSDiva software (BD Biosciences).

Assessment of cytokines

Purified CD4+ T cells (Miltenyi Biotech) (2.5 × 105 cells/ml) were incubated in the presence or absence of PG (20 µg/ml) and naïve irradiated (2500 rad) spleen cells (2.5 × 105 cells/ml) in RPMI 1640 complete (4). Cytokines were measured from day 4 culture supernatants by both IFN-γ ELISA (BD Biosciences) and IL-17 ELISA kits (R&D Systems, Minneapolis, MN).


Hind joints were taken at week 12 and fixed in formalin, decalcified, embedded in paraffin and stained with hematoxylin and eosin. Cellular infiltration was assessed by a blinded observer using a scale of 0–4. Values represent mean ± SEM of (n = 5–10) sections.

Quantitative reverse transcription-polymerase chain reaction (RT-PCR)

RNA was isolated from spleen and joint tissue using Tri-Reagent (Molecular Research Center, Cincinnati, OH). RNA was then treated with DNase I (Invitrogen, Carlsbad, CA) digestion before reverse transcription. Reverse transcription was performed with SuperScript III (Invitrogen). Real-time PCR was performed with 1 µl reverse transcription product in an IQ5 real-time PCR detection system (Bio-Rad, Hercules, CA) by using QuantiFast SYBR Green PCR Supermix (QIAGEN, Valencia, CA) according to the manufacturer's guidelines. The PCR cycling conditions were as follows: 50 cycles of 15 seconds at 95°C, 20 seconds at 60°C, and 30 seconds at 72°C. All samples were run in triplicate. To verify that equivalent amounts of RNA were added to each PCR reaction, PCR amplification of the murine β actin was performed for each sample. Relative fold induction was calculated using the formula 2−(ΔΔCt), where ΔΔCt is ΔCt(treatment) − ΔCt(control), ΔCt is Ct(target gene)Ct(actin), and Ct is the cycle at which the threshold is crossed. PCR product quality was monitored using post-PCR melting curve analysis. Controls were from naïve non-immunized and target genes from PG-immunized joint spleen tissues.

Statistical analysis

The Mann-Whitney U test was used to compare non-parametric data for statistical significance. P < 0.05 was considered significant.


IFN-γ deficiency inhibits arthritis and enhances IL-17 production

We have demonstrated a requirement for IFN-γ in PGIA (4) and in the present study reconfirm these findings (Fig.1A). WT and IFN-γ−/− mice were immunized with PG and the onset and severity of arthritis monitored over time. IFN-γ−/− mice developed arthritis with a delay in onset and reduced severity in comparison to WT mice; however, IFN-γ−/− mice often succumb to arthritis in some cases as severely as WT mice. These findings indicate that IFN-γ is an important pathogenic factor governing disease severity in PGIA. Recent studies in murine models of arthritis CIA and AIA show that IFN-γ suppresses IL-17 production leading to enhanced disease in IFN-γ deficient mice (19, 20). Since arthritis is not exacerbated in IFN-γ−/− in PGIA the regulation of IL-17 in vivo may be different. Therefore, we examined expression of cytokines from splenic CD4+ T cells. Purified CD4+ T cells from PG-immunized WT and IFN-γ−/− mice were re-stimulated in vitro with PG and irradiated naïve spleen cells. CD4+ T cells from WT mice secreted very little IL-17, whereas CD4+ T cells from IFN-γ−/− mice produced abundant levels of IL-17 (Fig. 1B). Intracellular FACS analysis of ex-vivo spleen cells gated on CD4+ T cells from PG-immunized mice also revealed marked increase in the frequency of intracellular IL-17 positive CD4+ T cells in IFN-γ−/− mice (Fig.1C, D). These data confirm reports by others that IFN-γ−/− suppresses IL-17 in vivo and demonstrate that IFN-γ regulates the balance between Th1 and Th17 cells in vivo in PGIA. Thus, it was possible that the PG-induced arthritis that develops late in the IFN-γ−/− mice was dependent on IL-17.

Figure 1
IFN-γ deficiency inhibits PGIA and enhances IL-17 production. Groups of age matched WT (closed circles)(n=15) and IFN-γ−/− (open circles)(n=15) mice were immunized with PG in adjuvant three times at 3-wk intervals and monitored. ...

Late onset arthritis in IFN-γ−/− mice is IL-17-dependent

To determine whether elevated IL-17 in the IFN-γ−/− mice was responsible for late onset arthritis observed in IFN-γ−/− mice, we bred IFN-γ−/− and IL-17−/− mice to generate double deficient mice (IFN-γ/IL-17−/−) and compared their development of arthritis to groups of age matched WT, IFN-γ−/−, and IL-17−/− mice. As we have previously reported, WT and IL-17−/− mice are similarly susceptible to PGIA (22). However, the onset and severity of arthritis in IFN-γ/IL-17−/− mice was significantly reduced in comparison to WT, IFN-γ−/− and IL-17−/− mice (Fig. 2A). Histological examination of hind limb joints showed similar characteristic of acute arthritis in WT and IL-17−/− mice as previously reported (22). There was predominant polymorphnuclear and mononuclear cell infiltration in tissues and joint spaces with edema of the synovial and periarticular tissues accompanied by synovial hyperplasia. Cartilage destruction and disintegrating chondrocytes were also seen in the remaining articular surface in addition to extensive bone erosion as previously described (22, 23). Conversely, the cellular infiltration and histopathology were significantly reduced in IFN-γ−/− and further reduced in IFN-γ/IL-17−/− mice (Fig. 2 B, C).

Figure 2
Late onset arthritis in IFN-γ−/− mice is IL-17 dependent. Groups of age matched WT (closed circles) (n=14), IFN-γ−/− (open circles) (n=8), IL-17−/− (closed triangles) (n=8) and IFN-γ/IL-17 ...

Experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune uveitis (EAU) can be induced by either Th1 and Th17 cells, however, in EAE the composition of the infiltrating cell populations are distinct indicating that different populations of cells can trigger a clinically indistinguishable sign of disease (24, 25). In PGIA, despite a difference in the requirement for IFN-γ and IL-17, the composition of the cell populations infiltrating the synovial cavity in WT, IFN-γ−/−, and IL-17−/− strains was similar. Approximately 90% of the infiltrating cells were Gr-1/CD11b+ neutrophils [(22) and data not shown]. There was insufficient synovial fluid to make this determination in the IFN-γ /IL-17−/− mice. These data demonstrate that IFN-γ and IL-17 can both contribute to the development of PGIA but that IL-17 comes into play only under conditions where IFN-γ levels are reduced.

Reduced cytokine and chemokine transcripes in joints of IFN-γ−/− and IFN-γ/IL-17−/− mice

PG-specific T cells and autoantibody are required for the development of PGIA; neither serum antibodies nor T cells alone are able to transfer arthritis (26, 27). PG-specific T-cell proliferation was not reduced in IL-17−/−, IFN-γ−/− and IFN-γ/IL-17−/− mice in comparison to WT (data not shown) and did not correlate with the development of arthritis. There was no difference in PG-specific IgG1 response between WT, IFN-γ−/−, and IL-17−/−; however, a significantly lower IgG1 response was observed in IFN-γ/IL-17−/− mice (data not shown) and the PG-specific IgG2a response was reduced in the IFN-γ−/− and IFN-γ/ IL-17−/− mice as expected as IFN-γ is responsible for IgG2a isotype switch (data not shown).

To directly assess how a deficiency in IFN-γ and IL-17 affected the microenvironment favorable for cellular infiltration into the synovial tissue, we evaluated the expression of several inflammatory mediators in the hind paws of PG-immunized mice. We selected several cytokines (IL-1β, IL-6, TNF-α, and GM-CSF), chemokines (CXCL1, CXCL2, CXCL9, CXCL10, CXCL11, CXCL12, CCL2, CCL3, CCL8, and CCL20) and NOS based on their known activity in RA, their induction by IFN-γ or IL-17 and our previous identification in prearthritic and arthritic joints in PGIA (2832). We obtained RNA from ankle joints of PG-immunized WT, IL-17−/−, and IFN-γ−/− mice at a time point when joint swelling was on the rise, with the exception of joints from IFN-γ/IL-17−/− mice which were not swollen. Although measuring protein levels for the cytokines and chemokines would have been ideal, synovial fluid could not be obtained from IFN-γ/IL-17−/− mice. Transcripts of several cytokines and chemokines were not consistently increased in immunized WT mice, these include TNF-α, GM-CSF, CXCL9, CXCL10, CXCL11, and CXCL12 (data not shown). In contrast, joint tissues from WT and IL-17−/− mice expressed reproducibly elevated transcripts for IL-1β, IL-6, NOS, CXCL1, CXCL2, CCL2, CCL3, CCL8, and CCL20 (Fig. 3C). In IFN-γ−/− and IFN-γ/IL-17−/− joint tissues two patterns of cytokine and chemokine expression emerged. In IFN-γ−/− mice, IL-1β, IL-6, NOS, CXCL1, CXCL2, CCL2, CCL3, CCL8, and CCL20 transcripts were significantly decreased in comparison to either WT or IL-17−/− mice whereas in IFN-γ /IL-17−/− mice IL-6, CCL2, CCL8, and CCL20 were further reduced in comparison to IFN-γ−/− mice. These data demonstrate that IFN-γ either directly or indirectly plays a major role in regulating expression of cytokines and chemokines that are important for inflammation and cellular infiltration in the joint. In addition, these data show that in the absence IFN-γ, IL-17 was pro-inflammatory and further contributed to the expression of cytokines/chemokines in joint tissue.

Figure 3
Reduced cytokine and chemokine transcripts in joint tissue of IFN-γ−/− and IFN-γ/IL-17−/− mice. IL-β, IL-6, NOS, CXCL1, CXCL2, CCL2, CCL3, CCL8, CCL20 mRNA transcripts using RNA isolated from joint ...

T-bet−/− mice are susceptible to PGIA

T-bet is a major transcription factor regulating the production of IFN-γ in Th1 cells (33). Given the importance of IFN-γ in PGIA, we anticipated that T-bet would be essential for induction of arthritis. However, the kinetics and severity of arthritis development in T-bet−/− mice was similar to WT mice (Fig. 4A, B). We speculated that sufficient pro-inflammatory cytokines might be produced that permit the development of arthritis in the absence of T-bet as IFN-γ can be expressed through T cell receptor signaling or IL-12 activation of T cells (34). Also, T-bet is important for regulation of IL-17 as T-bet−/− T cells increase production of IL-17 (35). Thus, the combination of low IFN-γ and high IL-17 might be sufficient to induce arthritis.

Figure 4
T-bet deficient mice are susceptible to PGIA and IL-17 expression is enhanced. WT and T-bet−/− mice were immunized with PG as described. (A) Arthritis score (left panel) and incidence (right panel) in WT (closed circles) (n=10) and T-bet ...

To begin to examine this possibility, we assessed CD4+ T cell cytokine production from PG-immunized WT and T-bet−/− mice. CD4+ T cells from T-bet−/− mice secreted significantly less IFN-γ than CD4+ T cells from WT mice (Fig. 4B). In contrast, CD4+ T cells from WT mice secreted very little IL-17 in comparison to abundant production by T-bet−/− T cells (Fig. 4B). Intracellular FACS analysis of ex-vivo spleen cells gated on CD4+ T cells from PG-immunized mice revealed a similar pattern; the frequency of intracellular IL-17 positive CD4+ T cells in T-bet−/− was significantly higher than in WT. (Fig. 4C, D). These data demonstrate that T-bet is important in the production of IFN-γ and in the inhibition of IL-17 by CD4+ T cells in PGIA. In addition, since IFN-γ was not completely suppressed, the residual IFN-γ and the increase in IL-17 might account for the susceptibility to PGIA in T-bet−/− mice.

Development of arthritis in T-bet−/− mice is dependent on IL-17

To determine whether the elevated levels of IL-17 in the T-bet−/− mice promoted arthritis, we bred mice deficient in T-bet and IL-17 to generate T-bet/IL-17−/− mice and compared the development of disease in age matched WT, T-bet−/−, and IL-17−/− mice. Arthritis onset and severity were significantly reduced in T-bet/IL-17−/− mice compared to either WT, T-bet−/− or IL-17−/− mice (Fig. 5A). The histological picture in T-bet−/− mice was similar to WT and IL-17−/− mice with polymorphonuclear and mononuclear infiltration accompanied by edema of the synovium and periarticular tissue. Cellular infiltration coincided with cartilage destruction and bone erosion. This histological picture was substantially reduced in T-bet/IL-17−/− mice compared to the WT, T-bet−/− and IL-17−/− controls (Fig. 5B, C). Thus, in T-bet−/− mice the increase in IL-17 together with the T-bet-independent IFN-γ response may account for PGIA susceptibility.

Figure 5
IL-17 and IFNγ contribute to arthritis in T-bet−/− mice. Groups of age mated mice were immunized with PG. (A) Arthritis score (left panel) and incidence (right panel) in WT (closed circles) (n=14), T-bet−/− (open ...

Reduced cytokine and chemokine transcribes in joints of T-bet−/− and T-bet/IL-17−/− mice

As described above PG-specific T cell activation and autoantibody production did not correlate with the development of arthritis (data not shown). We showed that loss of IFN-γ substantially reduced expression of inflammatory mediators in the joint. However, in T-bet−/− mice IFN-γ expression was reduced but not ablated. We assessed the express of inflammatory mediators in joints of PG-immunized mice. Transcripts from WT and IL-17−/− mice were compared to transcripts from T-bet−/− and T-bet/IL-17−/− mice (Fig. 6C). In T-bet−/− mice, only NOS, CXCL1, CCL2, and CCL20 were inhibited in comparison to WT and IL-17−/− mice suggesting that the residual IFN-γ produced in T-bet−/− mice was inflammatory and responsible for elevated expression of IL-1β, IL-6, CXCL2 CCL3, and CCL8 transcripts. Similar to the IFN-γ/IL-17−/− mice, in the T-bet/IL-17−/− mice IL-6, CCL2, CCL8, and CCL20 were reduced in comparison to IL-17−/− and in some cases T-bet−/− mice, indicating that T-bet is protective.

Figure 6
Reduced cytokine and chemokine transcripts in joint tissue of T-bet−/− and T-bet/IL-17−/− mice. IL-β, IL-6, NOS, CXCL1, CXCL2, CCL2, CCL3, CCL8, CCL20 mRNA transcripts using RNA isolated from joint tissue of PG ...


This study was designed to determine how the pathogenic and protective properties of IFN-γ control chronic autoimmune disease. We have previously shown that in mice deficient in IFN-γ, PGIA is suppressed but not eliminated; demonstrating that IFN-γ is pathogenic in this disease model (3, 4). In the present study, we found that there was an increase in expression of IL-17 by CD4+ T cells in the absence IFN-γ despite reduced onset of disease demonstrating that IFN-γ regulates IL-17 expression in PGIA. In several autoimmune disease models, ablation of IFN-γ leads to exacerbated disease due to an increase in IL-17 expression (19, 20). We found that failure to limit inflammation late in PGIA is due to the increase in IL-17 as a deficiency in both IFN-γ and IL-17 completely inhibited PGIA. Although production of cytokines either IFN-γ or IL-17 from CD4+ T cells was consistently associated with disease these cytokines are expressed by several cell populations. Since CD4+ T cells are insufficient to induce PGIA on transfer into naïve recipients and in the absence of mice where cytokines can be conditionally deleted in T cells, IFN-γ and IL-17 produced by other cell populations may also participate in arthritis. It is important to note, in PGIA, that IL-17 is pathogenic only if IFN-γ is ablated or reduced indicating that IL-17 can function as a pro-inflammatory cytokine in PGIA if it is released from suppression by IFN-γ. Our data indicate that in PGIA, IFN-γ is pathogenic but also is protective as there is minimal expression of IL-17 in WT mice. On the basis of these data, we speculate that the dominance of IL-17 in other autoimmune diseases such as CIA and AIA may be to a reduced or contracted IFN-γ response that diminishes the pathogenic response while simultaneously decreases IL-17 regulation. An alternative possibility is that the mechanism by which IFN-γ regulates IL-17 is defective in models where IL-17 is dominant.

T-bet is a major transcription factor for the production of IFN-γ and IFN-γ itself acts through STAT1 to activate T-bet (36, 37). Similar to a deficiency in IFN-γ, a deficiency in T-bet leads to an increase in CD4+ T cells IL-17 production in PGIA. Other studies have similarly identified a requirement for T-bet in IL-17 regulation (35, 38). There are at least two possible mechanisms by which T-bet ablation leads to an increase in IL-17 expression. Since T-bet is important for IFN-γ production, reduction in IFN-γ either directly or indirectly releases IL-17 from suppression. Alternatively, since IFN-γ inhibits IL-17 expression by signaling through the STAT-1/T-bet pathway, loss of T-bet would disrupt this pathway and block suppression. We were initially surprised that T-bet−/− mice were susceptible to arthritis since PGIA is dependent on IFN-γ. We hypothesized that because IFN-γ was present at low levels and IL-17 was dramatically increased the combined pro-inflammatory effects of IFN-γ and IL-17 results in arthritis susceptibility. Indeed, a deficiency in both T-bet and IL-17 substantially reduced PGIA. These data suggest that the increase in IL-17 in T-bet−/− mice together with the remaining IFN-γ contribute to inflammation in PGIA. Although our data correlate CD4+ T cell IFN-γ and IL-17 with the development of PGIA, recent data in EAE suggest that T-bet-dependent development of disease is independent of IFN-γ and IL-17 (38). In EAE, therapeutic administration of small interfering RNA specific for T-bet (39) or a deficiency in T-bet inhibits disease (40). In addition, T-bet is essential for the development of CIA induced with mAbs against type II collagen (41). Several studies have highlighted the role of T-bet in B cell, NK cells, NKT cells, DC, and CD8+ T cells and it is an important transcription factor for functions other than IFN-γ production thus the difference in the requirement for T-bet may be model specific (4246). This is specifically illustrated in models of colitis where in oxazolone-induced colitis T bet is protective whereas in colitis induced by transfer of CD4+ CD62L+ T cells into an immunodeficient host, T-bet is pathogenic (47).

What is the mechanism by which IFN-γ and IL-17 contribute to arthritis in PGIA? Because there was no clear correlate in T and B cell responses with the development of PGIA, we assessed the effect of IFN-γ and IL-17 on inflammatory microenvironment of the joint by examining the expression of joint tissue cytokine and chemokine RNA transcripts. The RNA transcripts for CXCL1, CXCL2, CCL2, CCL3, CCL8, and CCL20 are upregulated in arthritic joints of WT and IL-17−/− mice. These chemokines are chemotaxic for macrophages, neutrophils, lymphocytes, and dendritic cells. The role of these CXCL1 and CXCL2 in neutrophil/macrophage recruitment correlates with the dominance of these cell populations in the synovial tissue and fluid in PGIA. In IFN-γ−/− mice, there was a significant reduction in CXCL1, CXCL2, CCL2, CCL3, CCL8, and CL20 with a further reduction in CCL2, CCL8, and CCL20 in the IFN-γ/IL-17−/− mice. The reduction in chemokine RNA expression correlates with a reduction in cellular infiltration and histopathology in IFN-γ and IFN-γ/IL-17−/− mice.

IFN-γ is documented to both induce and suppress chemokine expression and neutrophil recruitment which maybe important in the progression of a normal inflammatory response. For example, in a model of peritonitis induced with Staphylococcus epidermidis, a deficiency in IFN-γ reduces the influx of neutrophils into the peritoneal cavity and the expression of neutrophil chemokine ELR+ CXC chemokines (CXCL1 and CXCL2) through a mechanism that involves IL-1 and IL-6. As infection proceeds IFN-γ regulates neutrophil removal initiated by suppression of ELR+ CXC chemokines (4850). Thus, IFN-γ can act sequentially as pro- and then as anti-inflammatory in regulating leukocyte influx. The pro-inflammatory properties of IFN-γ are also evident in mice infected with pneumonia virus where IFN-γ is important for CCL3 expression (51). In addition, IFN-γ activates CCL2 expression in IL-1β activated RA synovial fibroblast (52). In CIA, IFN-γ is shown to inhibit neutrophil recruitment by suppressing CXCL5 expression (53). In addition, in RA synovial fibroblast, IFN-γ inhibits IL-1β induced CXCL-8 expression (52).

The chemokines CCL20 is recognized by CCR6 which is expressed by Th17 cells and has been reported to preferentially recruit Th17 cells to the inflamed joint (31). Also it has been found that IL-17 synergizes with TNF-α to up-regulate the expression of CXCL-1 and CCL20 (54), 51). Our data show that several chemokines which are reported to be involved in the recruitment of IL-17 and in IL-17 synergistic activity are activated by IFN-γ. Importantly, these chemokines are decreased despite the increase in IL-17 expression that occurs in the IFN-γ−/− mice. It is clear that for some of these chemokines, CCL2, CCL8, and CCL20, IL-17 is also important for their expression. Thus, these data suggest that under circumstances where IFN-γ is unable to regulate IL-17 production, IFN-γ and IL-17 can cooperate in the activation of chemokine expression and the recruitment of cell to the joint.

We observed a similar regulation of chemokine expression in T-bet−/− mice where IFN-γ was reduced but not eliminated. Expression of chemokine RNA transcripts in the joint tissue reflects the presence of residual IFN-γ as some chemokines; CXCL2, CCL3, and CCL8 were no longer reduced in T-bet−/− mice. However, elimination of IL-17 resulted in suppression of several chemokine. These data further support the cooperation of IFN-γ and IL-17 in chemokine expression in joint tissue although whether this effect is directly or indirect is not known.

Cytokine transcripts for IL-1β IL-6, and NOS were consistently detected in WT and IL-17−/− mice. Our data show that similar to chemokine expression these cytokines were suppressed in IFN-γ deficient mice and further inhibited in IFN-γ/IL-17−/− mice. In T-bet−/− mice only NOS was reduced and IL-6 and NOS further reduced in T-bet/IL-17−/− mice. IL-1β and IL-6 activate chemokine expression so that the regulation of chemokine expression by IFN-γ and IL-17 may be indirectly through cytokine expression. Taken together, both the pathogenic and protective properties of IFN-γ are evident in cytokines and chemokines expression in the joint tissue although the mechanism by which this occurs is unclear.

The respective role of Th1 and Th17 cells in RA remains unclear. Elevated levels of IFN-γ protein and RNA transcripts have been identified in synovial tissues and fluid in RA patients (5557), and a clinical trial involving IFN-γ blockade showed significant improvement (58). IL-17 is also expressed in some but not all RA patients in sera, synovial fluid and synovial biopsies (31, 59, 60). The overlapping inflammatory roles of T helper phenotypes demonstrated here suggest a mechanism by which patients may be refractory to therapeutic interventions focused on unique components of one inflammatory pathway, i.e. anti-IFN-γ antibody, while blockade of common downstream effectors, i.e. anti-TNF antibody, showed greater success. The highly heterogeneous nature of human disease suggests that animal models may represent different subtypes of RA, each of which may respond differently to a given therapeutic intervention. It is possible that the subtypes of RA, and therefore perhaps the nature of T helper involvement, are a consequence of different etiologies that result in a similar disease phenotype. If this is the case, the efficacy of biological therapeutics needs to be tailored for the specific nature of inflammation of an individual or to focus on elements common to both inflammatory pathways. It is important to clarify how the pro-inflammatory and anti-inflammatory properties control the outcome of autoimmune disease as therapeutic interventions may disrupt a balance that could inadvertently lead to increased disease severity.

Recent data demonstrate that inflammation in EAU and EAE models are not solely dependant upon the Th17 subset, as had been suggested, but rather either Th1 or Th17 subsets can mediate pathology (24, 25). We confirm and extended these findings in a Th1-mediated model of RA demonstrating that a robust IFN-γ response in WT mice readily suppresses the IL-17 response; making the contribution of IL-17 to inflammation negligible. Uncovering a role for Th17 effector cells in PGIA required relief from IFN-γ mediated suppression of IL-17. However, even under these circumstances IL-17 alone was insufficient to drive severe arthritis. These data support the notion that in Th17-mediated diseases, the IFN-γ response may be ineffective thereby permitting Th17 effector cells to predominate.


The authors thank Dr. Jeffrey Oswald and all the staff of the Comparative Research Center for their expert assistance.

This work is supported by National Institutes of Health Grant AR 47652 ( to A.F., T.T.G., and K.M.) and AR 56999 (to A.F.)

Abbreviations used in this paper

rheumatoid arthritis
proteoglycan-induced arthritis.


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