IL-17B and IL-17C Are Associated with TNF-α Production and Contribute to the Exacerbation of Inflammatory Arthritis

Yumi Yamaguchi, Keishi Fujio, Hirofumi Shoda, Akiko Okamoto, Nelson H. Tsuno, Koki Takahashi and Kazuhiko Yamamoto
J Immunol November 15, 2007, 179 (10) 7128-7136; DOI: https://doi.org/10.4049/jimmunol.179.10.7128

Abstract

IL-17A is a T cell-derived proinflammatory cytokine that contributes to the pathogenesis of rheumatoid arthritis. Recently, six related molecules have been identified to form the IL-17 family, as follows: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. Whereas IL-17A and IL-17F up-regulate IL-6 in synovial fibroblasts, IL-17B and IL-17C are reported to stimulate the release of TNF-α and IL-1β from the monocytic cell line, THP-1 cell. However, their detailed function remains to be elucidated. We report in this study the effects of IL-17 family on the collagen-induced arthritis (CIA) progression by T cell gene transfer and bone marrow chimeric mice. The mRNA expressions of IL-17 family (IL-17A, IL-17B, IL-17C, and IL-17F) and their receptor (IL-17R and IL-17Rh1) genes in the arthritic paws of CIA mice were elevated compared with controls. Although IL-17A and IL-17F were expressed in CD4+ T cells, IL-17B and IL-17C were expressed in the cartilage and in various cell populations in the CIA arthritic paws, respectively. In vitro, IL-17A, IL-17B, IL-17C, and IL-17F induced TNF-α production in mouse peritoneal exudate cells. In vivo, adoptive transfer of IL-17B- and IL-17C-transduced CD4+ T cells evidently exacerbated arthritis. Bone marrow chimeric mice of IL-17B and IL-17C exhibited elevated serum TNF-α concentration and the high arthritis score upon CIA induction. Moreover, neutralization of IL-17B significantly suppressed the progression of arthritis and bone destruction in CIA mice. Therefore, not only IL-17A, but also IL-17B and IL-17C play an important role in the pathogenesis of inflammatory arthritis.

Interleukin-17A is a T cell-derived proinflammatory cytokine that is involved in the development of rheumatoid arthritis (RA).3 IL-17A was originally named CTLA-8 after being cloned from activated T cells, and shares 57% homology to the protein encoded by the open reading frame 13 gene of the T lymphotropic herpesvirus saimiri (1). IL-17A is present at significant levels in the synovium and synovial fluid of patients with RA (2, 3). IL-17A is a potent inducer of various cytokines such as IL-1, TNF-α, and IL-6. T cell IL-17A stimulates the production of IL-1 and TNF-α from human PBMC-derived macrophages in vitro (4). IL-17A also enhances IL-1-mediated IL-6 production by RA synoviocytes in vitro as well as TNF-α-induced synthesis of IL-1, IL-6, and IL-8 (5, 6). These results indicate that IL-17A synergizes with IL-1 and TNF-α and contributes to inflammation of RA.

In in vivo studies, systemic as well as local overexpression of IL-17A in collagen-induced arthritis (CIA) has been shown to accelerate the onset of CIA and to aggravate the joint pathology (7). Moreover, treatment with anti-IL-17A Abs after the onset of CIA reduces the joint inflammation and histologic destruction of cartilage (8). IL-17A deficiency protects IL-1R antagonist-deficient mice from spontaneous development of destructive arthritis (9). Therefore, IL-17A plays a crucial role in the pathogenesis of arthritis through synergistic effects with IL-1 and TNF-α. However, IL-17A can directly induce joint destruction in an IL-1-independent manner and can bypass TNF-dependent arthritis (7, 10). This suggests that there is an IL-17A-dependent pathway to the destructive arthritis and anti-IL-17A cytokine therapy is an additional new antirheumatic strategy for RA besides anti-TNF/anti-IL-1 therapy.

Recently, the IL-17 family was determined to consist of six related molecules, as follows: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. These molecules have a molecular mass of 20–30 kDa and consist of 163–202 aa that bear 20–50% homology to IL-17A, especially within the C-terminal region. They share four conserved cysteine residues that may participate in the formation of intermolecular disulfide linkages (11, 12). The different IL-17 family members seem to have very distinct expression patterns, suggesting distinct biological roles.

Interestingly, IL-17F has the highest homology with IL-17A and is also expressed by activated T cells in response to IL-23 stimulation (13, 14, 15). However, the precise effect of IL-17F on arthritis has not been clarified. In contrast to the restricted expression of IL-17A and IL-17F, IL-17B mRNA can be detected in a wide range of tissues, including the spinal cord, testis, stomach, small intestine, pancreas, prostate, and ovary (16, 17). It has been recently reported that IL-17B is highly expressed in chondrocytes that are located at the mid and deep zones of normal bovine articular cartilage (11). In contrast, IL-17C expression has been confined only to rare expression sequence tags in adult prostate and fetal kidney libraries (17). However, the cell sources of IL-17B and IL-17C have not been identified in the development of inflammatory arthritis.

A common feature of IL-17 family members is the induction of neutrophil migration. IL-17A and IL-17F both mobilize neutrophils partly through granulopoiesis and CXC chemokine induction (12). Intranasal administration of adenovirus expressing IL-17A, IL-17C, or IL-17F resulted in neutrophilia in the bronchoalveolar lavage (18). Moreover, i.p. injection of human rIL-17B caused marked neutrophil migration in normal mice (17). In contrast, the members can be divided into two groups according to the induction of cytokine production. Although IL-17A and IL-17F up-regulate IL-6 and IL-8 in human fibroblasts (19, 20), IL-17B and IL-17C are reported to stimulate the release of TNF-α and IL-1β from the monocytic cell line THP-1 (17). Taken together, these results indicate that IL-17 family members induce inflammatory cytokines not only through activated T cells, but also through activated monocytes/macrophages.

Based on the structural and functional similarities among IL-17 family members, we speculated that not only IL-17A, but also other IL-17 family members are involved in the pathogenesis of many inflammatory and autoimmune disorders, especially in the development of RA. We focused on IL-17A, IL-17B, IL-17C, and IL-17F, which can affect inflammatory cytokine production of fibroblasts and macrophages. Recently, IL-17C expression in synovial fluid mononuclear cells and PBMCs of RA patients was reported (21). However, the biological effect of IL-17 family members in arthritis has not been analyzed.

In the present study, we investigated the expression and effect of IL-17 family members in arthritis. In vitro, not only IL-17A, but also IL-17B and IL-17C induced the mRNA expression of inflammatory cytokines such as IL-1β, IL-6, and IL-23 in the 3T3 cell line and peritoneal exudate cells (PECs). The supernatant of the PECs stimulated with each IL-17 family member all increased TNF-α production significantly compared with controls. In vivo, CD4+ T cells transduced with each of IL-17B, IL-17C, or IL-17F exacerbated CIA in mice to the same degree as CD4+ T cells transduced with IL-17A. Mice reconstituted with bone marrow (BM) cells transduced with each of IL-17B, IL-17C, or IL-17F suffered from severe CIA. Moreover, neutralization of IL-17B significantly suppressed the progression of arthritis and bone destruction in CIA mice. Our results suggest that not only IL-17A, but also the other IL-17 family members (IL-17B, IL-17C, and IL-17F) are associated with inflammatory cytokines such as IL-1 and TNF-α and contribute to the exacerbation of autoimmune arthritis.

Materials and Methods

Animals

DBA/1J mice were purchased from Japan SLC. All mice were used at 6–8 wk of age. All animal experiments were conducted in accordance with the institutional and national guidelines.

Collagen-induced arthritis

CIA was induced, as described previously (22, 23, 24). In brief, bovine type II collagen (BCII) (Chondrex) was emulsified with an equal volume of CFA (Chondrex). DBA/1J mice were immunized intradermally at the base of the tail with 100 μg of BCII emulsified with CFA. On day 21, the mice were boosted by intradermal injection with 100 μg of BCII emulsified with IFA (Difco). The arthritis score was determined by erythema, swelling, or ankylosis per paw, as described previously (25, 26). The clinical arthritis score was defined as the sum of the scores of all four paws of each mouse.

Cytokines and cell lines

Recombinant murine IL (mIL)-17A, mIL-17B, mIL-17C, and mIL-17F were obtained from R&D Systems. The mouse fibroblast cell line 3T3 was obtained from American Type Culture Collection. This cell line was cultured with RPMI 1640 (Invitrogen Life Technologies) medium supplemented with 10% FCS, 2 mM γ-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5 × 10−5 M 2-ME. Ba/F3 cells were maintained in RPMI 1640 medium supplemented with 10% FCS, 2 mM γ-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 ng/ml rmIL-3 (R&D Systems).

Murine PECs

Murine PECs were isolated after i.p. injection of 3 ml of 5% sterile fluid Brewer’s thioglycolate broth (Sigma-Aldrich) into 8-wk-old DBA/1J mice (27). After culture of the PECs in a 6-well plate for 2 h, floating cells were removed by extensive washing, and attached cells were maintained in the medium described above for 3 days. More than 80% of the cultured cells were macrophages as determined by flow cytometric analysis of CD11b-positive cells. The following recombinant murine cytokines were added to the culture medium and incubated for 24 h: 50 ng/ml mIL-17A, mIL-17B, mIL-17C, or mIL-17F.

Preparation of retroviral constructs of mIL-17 family cDNAs

mIL-17A, mIL-17B, mIL-17C, and mIL-17F were isolated from the murine T lymphocyte cDNA library according to the reported nucleotide sequence from National Center for Biotechnology Information (mIL-17A NM_010552; mIL-17B NM_019508; mIL-17C NM_145834; mIL-17F NM_145856). The full-length fragments were subcloned into retrovirus vector murine stem cell virus/internal ribosome entry site/GFP (pMIG), as described previously (28).

Production of retroviral supernatants and retroviral transduction

Retroviral supernatants were obtained by transfection of pMIG carrying each of the IL-17 family genes into PLAT-E packaging cell lines using FuGENE 6 transfection reagent (Roche Diagnostic System), as described previously (29). For the detection of GFP-positive cells, we used an EPICS XL flow cytometer (Beckman Coulter).

Gene transduction to mouse splenocytes and adoptive transfer

Total splenocytes were cultured for 48 h in the presence of Con A (10 μg/ml) (Sigma-Aldrich) and mIL-2 (50 ng/ml) (R&D Systems). Retroviral gene transduction was performed, as described previously (30, 31). A CD4+ T cell population was prepared by negative selection by MACS with anti-CD19 mAb, anti-CD11c mAb, and anti-CD8a mAb (BD Pharmingen). The gene-transduced CD4+ T cells were suspended in PBS and injected i.v. (1 × 107) at 23 days after the first immunization of BCII.

BM precursor cell isolation, infection, and transfer

BM precursor cell isolation, retrovirus infection, and transfer were performed, as described previously (32). In brief, DBA/1J mice were treated with 5 mg/body 5-fluorouracil (Sigma-Aldrich) dissolved in PBS. After 5 days, BM cells were harvested and cultured with 50 ng/ml mIL-3, mIL-6, and murine stem cell factor (R&D Systems) for 48 h. Then the BM cells were spin infected with the retrovirus supernatants with 16 μg/ml polybrene (Sigma-Aldrich) for 90 min at 2400 rpm and 25°C. Recipient mice were treated by 700 rad of whole-body radiation and were injected with 1 × 106 of the BM cells i.v. Recipient mice were maintained for 6 wk until analysis or immunization.

RNA isolation, cDNA synthesis, and quantitative real-time PCR

RNA of the cells was extracted using an RNeasy Micro Kit and RNeasy Mini Kit (Qiagen). RNA from the tissues was isolated by the acid guanidinium thiocyanate-phenol-chloroform extraction method using ISOGEN (Nippon Gene). RNA was reverse transcribed to cDNA with random primers (Invitrogen Life Technologies) and Superscript III, according to the manufacturer’s protocol (Invitrogen Life Technologies). To determine the cellular expression of each protein, quantitative real-time PCR analysis was performed using an iCycler (Bio-Rad). The PCR mixture consisted of 25 μl of SYBR Green Master Mix (Qiagen), 15 pmol of forward and reverse primers, and the cDNA samples, in a total volume of 50 μl. We calculated the quantitative PCR data with δ cycle threshold method, and relative RNA abundance was determined based on control β-actin abundance. To measure the relative efficiency, amplifications were performed on the serial diluted cDNA samples using primers for the target and the reference (β-actin) genes. We made plots of the log cDNA dilution vs δ cycle threshold, and confirmed that the efficiencies of the target and the reference genes were similar because the absolute value of the slope was close to zero (data not shown) (33, 34). The primer pairs used in the quantitative real-time PCR were as follows: mouse IL-17A, sense 5′-GCTCCAGAAGGCCCTCAGA-3′, antisense 5′-AGCTTTCCCTCCGCATTGA-3′; mouse IL-17B, sense 5′-CGGTGCCTATGTTTGGGTTGC-3′, antisense 5′-GGGTTGGTGGTTGGCTCAGAA-3′; mouse IL-17C, sense 5′-CACAGATGAGAACCGCTACCC-3′, antisense 5′-GCGGATGAACTCGGTGTGGAA-3′; mouse IL-17F, sense 5′-CAACGCTGCATACAAAAATCA-3′, antisense 5′-TTAAGTGAGGCATTGGGAACA-3′; mouse IL-17R, sense 5′-CCACTCTGTAGCACCCCAATG-3′, antisense 5′-CCTGGAGATGTAGCCCTGGTC-3′; mouse IL-17Rh1, sense 5′-GCAAGGAAGGAGCACGAAGAC-3′, antisense 5′-CTCGGCGATTTTCTTTTTCTG-3′; mouse TNF-α, sense 5′-CATCTTCTCAAAATTCGAGTGACAA-3′, antisense 5′-TGGGAGTAGACAAGGTACAACCC-3′; mouse IL-1β, sense 5′-CAACCAACAAGTGATATTCTCCATG-3′, antisense 5′-GATCCACACTCTCCAGCTGCA-3′; mouse IL-6, sense 5′-CACTTCACAAGTCGGAGGCTTA-3′, antisense 5′-GCAAGTGCATCATCGTTGTTC-3′; mouse IL-23, sense 5′-TGGCATCGAGAAACTGTGAGA-3′, antisense 5′-TCAGTTCGTATTGGTAGTCCTGTTA-3′; and mouse β-actin, sense AGAGGGAAATCGTGCGTGAC-3′, antisense 5′-CAATAGTGATGACCTGGCCGT-3′.

Immunoassays of cytokines and anti-type II collagen Ab

The concentrations of mIL-6, mTNF-α, and mIL-17A in mouse sera and culture supernatants were measured by sandwich ELISA, according to the manufacturer’s protocol (BD Pharmingen). An automatic microplate reader (Bio-Rad 550) was used to measure the OD. Mouse serum IgG anti-type II collagen Ab titer was measured, as previously described (35).

Isolation of cartilage

Murine articular cartilage was isolated from patellae, as described previously (36). In brief, patellae were decalcified in 3.5% EDTA for 4 h at 4°C, when the whole cartilage layer was stripped off. Because old cartilage is more calcified, decalcification of the patellae of old mice (>3 mo) was performed overnight at 4°C.

Cell purification

Briefly, the arthritic paws of the CIA mice were cut into pieces, digested with collagenase type IV (Sigma-Aldrich), and stained with mAbs (Fc blocking with anti-mouse CD16/CD32 mAb, and staining with anti- mouse CD3-PE mAb, anti-mouse CD4-allophycocyanin mAb, anti-mouse CD11b-FITC mAb, anti-mouse CD11c-FITC mAb, anti-mouse CD19-FITC mAb, biotinylated anti-mouse I-A/I-E (MHC class II) mAb, and streptavidin PE Ab that were obtained from BD Pharmingen). Cell sorting of a specific cell population was performed with a FACSVantage flow cytometer (BD Biosciences).

Intracellular cytokine staining and flow cytometry

IL-17 family expressions of Ba/F3 cells transduced with each of IL-17 family members were examined using intracellular cytokine staining. Ba/F3 cells were infected with the retroviral supernatants in the presence of 10 μg/ml polybrene (Sigma-Aldrich) for 120 min. These cells were stained with anti-mouse IL-17A mAb conjugated to PE (BD Pharmingen), biotinylated anti-mouse IL-17B polyclonal Ab (R&D Systems), anti-mouse IL-17C polyclonal Ab (R&D Systems), and anti-mouse IL-17F mAb (R&D Systems), respectively. Bovine anti-goat IgG-PE (Santa Cruz Biotechnology) and F(ab′)2 goat anti-rat IgG PE (Serotec) were used as secondary reagents for IL-17C and IL-17F staining, respectively. Cell fixation and permeabilization were performed using Cytofix/Cytoperm reagent (BD Pharmingen), according to the manufacturer’s protocol (BD Pharmingen), and analyzed by flow cytometry. Splenocytes isolated from BM chimeric mice of IL-17A were also stained with anti-mouse IL-17A mAb in the same way.

Anti-IL-17B Ab treatment in CIA mice

CIA was induced in DBA/1J mice, as described above. Mice exhibited the first clinical signs of arthritis (arthritis score between 1 and 2) and were injected i.p. with 100 μg of polyclonal anti-mouse IL-17B Abs (R&D Systems). PBS was i.p. injected as a control. Arthritis was assessed using a scoring system, as described above. Mice were sacrificed at 10 days after the onset of arthritis, and the paws were removed. Joint pathology was evaluated on decalcified H&E-stained sections.

Histopathology

The tarsal joints of sacrificed CIA mice were embedded in paraffin wax after 10% formaldehyde fixation and decalcification. The sections were stained with H&E. Synovial tissues were graded by mononuclear cell infiltration and pannus invasion, as described previously (37).

Statistical analysis

Data are expressed as the means ± SD. All results were obtained by at least three independent experiments. Statistical significance was determined by the Mann-Whitney U test and unpaired Student’s t tests. A value of p < 0.05 was considered statistically significant.

Results

IL-17 family genes (IL-17A, IL-17B, IL-17C, and IL-17F) were highly expressed in the arthritic paws of CIA mice

First, we examined the expressions of IL-17 family members and IL-17Rs in the arthritic paws of CIA mice by quantitative PCR. The mRNA expressions of all IL-17 family genes examined (IL-17A, IL-17B, IL-17C, and IL-17F) were highly elevated in the arthritic paws compared with the controls. In accordance with previous report of high in vivo expression of IL-17R in RA (38), mRNA expressions of IL-17Rs (IL-17R and IL-17Rh1) were also elevated (Fig. 1⇓A). As expected, the mRNA expressions of inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-23) were also elevated in the arthritic paws compared with controls (Fig. 1⇓B).

FIGURE 1.
FIGURE 1.

The expression of IL-17 family members and IL-17R genes in the arthritic paws of CIA mice. A, The expressions of IL-17 family genes and IL-17R genes were examined in the arthritic paws of CIA mice (▪; n = 3) and in control mice (□; n = 3) by quantitative PCR. B, The expressions of inflammatory cytokines. C, The expressions of IL-17 family members in the sorted cell populations of the arthritic paws of CIA mice. The data are representative of three independent experiments.

We next examined cell populations in the arthritic paws of CIA mice that express IL-17 family members. Subpopulations of the cells were sorted with various cell surface markers using a flow cytometer. As expected, CD4+ T cells expressed IL-17A and IL-17F significantly. IL-17B was expressed exclusively in the inflammatory cartilage of CIA mice. In contrast, IL-17C was expressed in a broad range of cells, i.e., CD4+ T cells, CD11b+ MHC class II+ macrophages, and CD11c+ MHC class II+ dendritic cells (Fig. 1⇑C). These results suggested that CD4+ T cells mainly express IL-17 family members, especially IL-17A, IL-17C, and IL-17F, at the inflammatory site.

IL-17 family induced several proinflammatory cytokines

We next investigated whether IL-17 family members have an influence on mouse fibroblast cell lines and mouse peritoneal macrophages. Cells of the mouse fibroblast line 3T3 were cultured with each of the IL-17 family members (50 ng/ml), and cytokine expression was examined after 24 h of incubation. IL-17A induced IL-1β and IL-6 expressions, as previously reported (2). Moreover, IL-17B, IL-17C, and IL-17F also induced IL-1β expression in 3T3 (Fig. 2⇓A).

FIGURE 2.
FIGURE 2.

The proinflammatory effects of IL-17 family members on mouse fibroblasts and macrophages. A, Relative expression of the cytokine genes in 3T3 cell. The mouse fibroblast cell line 3T3 was cultured with each of mIL-17A, mIL-17B, mIL-17C, or mIL-17F for 24 h, and the expressions of inflammatory cytokines were measured by quantitative PCR. B, Relative expression of the cytokine genes in mouse thioglycolate-elicited PECs. PECs were cultured with each of mIL-17A, mIL-17B, mIL-17C, or mIL-17F for 24 h, and the expressions of inflammatory cytokines were measured by quantitative PCR. C, The secreted IL-6 and TNF-α levels in the supernatants of 3T3 and PECs were measured by ELISA. Error bars indicate ± SD. The data are representative of three independent experiments. Significance of differences between control (medium) and each IL-17 family was determined; ∗, p < 0.05.

To examine the effects of IL-17 family members on mouse macrophages, thioglycolate-elicited PECs were isolated and cultured with each of the IL-17 family members (50 ng/ml). IL-17A induced IL-1β, IL-6, and IL-23 expressions in PECs. Interestingly, IL-17B also induced IL-1β, IL-6, and IL-23 expressions. Moreover, IL-17C induced IL-1β and IL-23 expressions in PECs (Fig. 2⇑B). In addition, PECs stimulated with every IL-17 family member produced significantly increased amount of TNF-α protein compared with the control, and PECs stimulated with IL-17A and IL-17B produced significantly increased amount of IL-6 protein (Fig. 2⇑C). These results suggested that IL-17A, IL-17B, IL-17C, and IL-17F stimulate fibroblasts and macrophages to produce inflammatory cytokines.

Exacerbation of CIA by transfer of IL-17 family-transduced CD4+ T cells

Because IL-17B and IL-17C induce the expression of inflammatory cytokines in fibroblasts and macrophages, we hypothesized that IL-17B and IL-17C have an effect on the process of arthritis. We subcloned cDNA fragment of mIL-17A, mIL-17B, mIL-17C, or mIL-17F to pMIG retrovirus vector. These vectors were retrovirally transduced to Ba/F3 cells, and protein expressions of IL-17 family members were confirmed with intracellular staining of each IL-17 family cytokine (Fig. 3⇓A).

FIGURE 3.
FIGURE 3.

The effects of transfer of IL-17 family-transduced CD4+ T cells on CIA. A, Intracellular IL-17 family expressions in Ba/F3 cells retrovirally transduced with each IL-17 family member. GFP-gated IL-17 family-transduced (mIL-17A, mIL-17B, mIL-17C, or mIL-17F) Ba/F3 cells were analyzed for IL-17A, IL-17B, IL-17C, or IL-17F expression compared with GFP-gated empty vector (pMIG)-transduced Ba/F3 cells. B, Representative FACS analysis of IL-17 family-transduced CD4+ T cells was shown. Numbers in dot plots indicate the percentage of GFP+ CD4+ and GFP− CD4+ cells, and the percentages of the GFP+ cells within total CD4+ cells were shown below. C and D, CD4+ T cells transduced with each of IL-17 family genes were transferred to collagen-immunized mice before the onset of arthritis (day 23). The incidence of arthritis (C) and the progression of arthritis scores (D) are shown. Values are the mean of arthritis score (n = 20 mice per group). Significance of differences between control (pMIG) and each IL-17 family-transduced mice was determined; ∗∗, p < 0.005; ∗, p < 0.05.

To examine the proinflammatory effects of the IL-17 family in vivo, we retrovirally transduced the IL-17 family genes to CD4+ T cells. The transduction efficiencies were ∼30% on average (Fig. 3⇑B). These IL-17 family-transduced CD4+ T cells were adoptively transferred to BCII-immunized DBA1 mice before the onset of arthritis. They exacerbated the progression of arthritis, as observed by the arthritis score (Fig. 3⇑, C and D). The IL-17 family member-transduced CD4+ T cells had no significant effect on the serum levels of anti-BCII IgG Abs at 14 days after the onset of CIA (data not shown). These results indicated that the effect of IL-17 family members on the progression of arthritis was not associated with the elevations of anti-BCII Abs.

IL-17 family BM chimeric mice exhibited high arthritis scores upon CIA induction

To examine the proinflammatory effect of constitutively expressed IL-17 family members, we established IL-17 family BM chimeric mice by transfer of gene-transduced BM cells to lethally irradiated mice. In a previous study, the attempt to generate IL-17A-overexpressing mice with a conventional transgenic approach was unsuccessful because these mice were embryonic lethal (39). In accordance with the previous report, mice that expressed IL-17A with high efficiency (i.e., for which the percentage of GFP+ cells in the spleen was >50%) became gaunt and died within 1 mo after BM transplantation (data not shown). When the percentage of GFP+ cells in the spleen was 5–15%, the mice appeared to be healthy for several months. We therefore used BM chimeric mice that expressed IL-17 family genes in ∼5–15% of spleen cells. Eight weeks after the BM transplantation, mIL-17A was readily detected by intracellular cytokine staining (Fig. 4⇓A). Moreover, the serum concentration of mIL-17A was significantly elevated in these mice (Fig. 4⇓B). Therefore, the BM chimeric mice were successfully allowed to express the transduced cytokines systemically. Then we immunized these mice with BCII 8 wk after BM transplantation. BM chimeric mice of IL-17A and IL-17F exhibited early onset and high arthritis scores upon CIA induction (Fig. 5⇓, A and B). BM chimeric mice of IL-17B and IL-17C clearly exacerbated arthritis, as assessed by the arthritis score. In contrast, BM chimeric mice of IL-17B and IL-17C did not result in significant differences in the onset of disease (Fig. 5⇓, C and D). BM expression of IL-17 family member did not affect the anti-BCII Ab responses at 14 days after the onset of CIA (data not shown). These results indicated that the effect of IL-17 family members on the exacerbation of arthritis was not associated with the responses of anti-BCII Abs.

FIGURE 4.
FIGURE 4.

Generation of IL-17 family chimeric mice by BM transplantation of gene-transduced BM cells. Each of IL-17 family genes was transduced to BM cells with retrovirus vector and transferred to lethally irradiated mice. A, The intracellular expression of IL-17A protein in the spleen of IL-17A BM chimeric mice 8 wk after BM transplantation. The percentage of GFP+ cells expressing IL-17A is indicated. The data are representative of three independent experiments. B, The concentration of IL-17A protein in the serum of IL-17A BM chimeric mice (n = 6) and control mice (pMIG BM chimeric mice) (n = 6). The levels of IL-17A were measured by ELISA.

FIGURE 5.
FIGURE 5.

Incidence of CIA and arthritis scores in IL-17 family BM chimeric mice. Incidence of CIA and arthritis scores in IL-17A and IL-17F BM chimeric mice (A and B), and in IL-17B and IL-17C BM chimeric mice (C and D). Mice were immunized with BCII 8 wk after the BM transplantation. Values are the mean of experiments for IL-17A and IL-17F BM chimeric mice (n = 20 per group) and experiments for IL-17B and IL-17C BM chimeric mice (n = 30 per group). Significance of differences between control (pMIG) and each IL-17 family BM chimeric mice was determined; ∗∗, p < 0.005; ∗, p < 0.05. E, The mRNA expression of inflammatory cytokines in the spleen of BM chimeric mice of IL-17B and IL-17C, which were immunized with BCII (▪; n = 15 per group) or nonimmunized controls (□; n = 6 per group). Significance of differences between control (pMIG) and each IL-17 family BM chimeric mice was determined; ∗∗, p < 0.005; ∗, p < 0.05. F, The secreted TNF-α and IL-6 levels in the serum of IL-17 family BM chimeric mice that were immunized with BCII (▪; n = 15) or nonimmunized controls (□; n = 6). Significance of differences between control (pMIG) and each IL-17 family BM chimeric mice was determined; ∗∗, p < 0.005; ∗, p < 0.05.

We next examined the alterations of inflammatory cytokine production in these BM chimeric mice. Interestingly, nonimmunized IL-17C BM chimeric mice showed increased mRNA expression of TNF-α in the spleen compared with controls (Fig. 5⇑E). Moreover, in the spleen of BCII-immunized IL-17C BM chimeric mice, the mRNA expressions of TNF-α, IL-6, and IL-23 were up-regulated. In contrast, BCII-immunized IL-17B BM chimeric mice showed increased mRNA expression of IL-6 in the spleen compared with controls (Fig. 5⇑E). When we examined the concentrations of TNF-α and IL-6 protein in the sera of IL-17 family BM chimeric mice, the BCII-immunized IL-17B and IL-17C BM chimeric mice showed increased TNF-α concentration in the sera. And the BCII-immunized IL-17A and IL-17B BM chimeric mice showed increased IL-6 production in the sera (Fig. 5⇑F). These results suggested that IL-17B and IL-17C enhanced inflammation in this mouse model of arthritis by increased inflammatory cytokine production.

Neutralization of IL-17B significantly suppressed the progression of arthritis

As shown in Fig. 5⇑, we found that IL-17B exacerbated the progression of CIA as well as IL-17A with the method of retrovirus-mediated BM chimeric mice. Regarding IL-17A, neutralizing Abs against IL-17A have been previously shown to be effective in the treatment of CIA (8). We examined the effect of IL-17B blockade in CIA mice. CIA mice were systemically treated with polyclonal anti-mouse IL-17B Abs immediately after the first signs of arthritis. Neutralization of IL-17B significantly suppressed the progression of CIA compared with the controls (Fig. 6⇓A). Moreover, histological analysis revealed significant reduction of cell infiltration and pannus invasion in the anti-IL-17B Ab-treated mice (Fig. 6⇓B). These results indicated that IL-17B was associated with the progression of arthritis in CIA mice.

FIGURE 6.
FIGURE 6.

Effect of anti-IL-17B Ab treatment in CIA mice. A, CIA mice received i.p. injection of anti-mouse IL-17B Abs after the first clinical signs of arthritis (arthritis score between 1 and 2). As a control, PBS was injected. The arthritis score was shown. B, Histological score of the inflammatory joints of CIA mice treated with anti-IL-17B Abs was evaluated at 10 days after the onset of arthritis. Cellular infiltration and pannus invasion were graded in all four paws of the mice. Values are the mean of arthritis scores for anti-IL-17B Ab-treated mice and control mice (n = 5 per group). Significance of differences between control and anti-IL-17B Ab-treated mice was shown.

Discussion

RA is considered to be an autoimmune disease, and is characterized by sustained inflammation of the joints and destruction of cartilage and bone. Several inflammatory cytokines are known to mediate the pathogenesis of arthritis, and TNF-α and IL-6 are the most important cytokines in the pathogenesis of RA. IL-17A, IL-17B, IL-17C, and IL-17F have the capacity to induce TNF-α production in PECs in vitro. In vivo, the mRNA expression of TNF-α was spontaneously increased in the spleen of IL-17C BM chimeric mice. Moreover, TNF-α productions in the sera of BCII-immunized IL-17B and IL-17C BM chimeric mice were up-regulated. Although IL-17A induced TNF-α production in PECs, IL-17A BM chimeric mice did not show up-regulated production of TNF-α. This result is consistent with previous observation in THP-1 cell line that IL-17B and IL-17C stimulated the release of TNF-α, whereas IL-17A has only a weak effect on TNF-α (17). In contrast to IL-17B and IL-17C, IL-17A may not be directly associated with TNF-α production in vivo. Moreover, the mRNA expression in the spleen and serum concentration of IL-6 were significantly up-regulated in IL-17B BM chimeric mice that were immunized with BCII. These results showed the close association of IL-17B and IL-17C with TNF-α and IL-6 in vivo and clearly suggested the importance of IL-17B and IL-17C in the pathogenesis of RA.

To date, the cell sources of IL-17B and IL-17C have not been identified. In this study, we showed that IL-17B was expressed in the inflammatory cartilage of CIA mice, whereas IL-17C was expressed in a broad range of cells, i.e., CD4+ T cells, CD11b+ MHC class II+ macrophages, and CD11c+ MHC class II+ dendritic cells. IL-17A and IL-17F were expressed in CD4+ T cells, as expected. These results suggested that CD4+ T cells are involved in the expression of IL-17 family members, especially IL-17A, IL-17C, and IL-17F, at the inflammatory site. Although we did not detect a unique cell source of IL-17C, the arthritis-promoting effect of IL-17C-transduced CD4+ T cells suggests the importance of IL-17C expressed in CD4+ T cells.

In our in vivo analysis, we observed arthritis-promoting effects of the IL-17 family members. As shown in Fig. 3⇑, the transfer of mIL-17A-, mIL-17B-, mIL-17C-, and mIL-17F-transduced CD4+ T cells evidently exacerbated arthritis as assessed by the arthritis score. This effect was also confirmed in the CIA of the mIL-17A, mIL-17B, mIL-17C, and mIL-17F BM chimeric mice. The arthritis-promoting effect of IL-17A was previously reported in a study using adenovirus vector (5, 40). In contrast to IL-17A, which hastened the onset of arthritis, IL-17B and IL-17C did not affect the onset of arthritis evidently. This fact suggests that IL-17B and IL-17C affect arthritis rather in the effector phase. To our knowledge, this is the first observation of an in vivo arthritis-promoting effect of IL-17B and IL-17C.

Blockade of IL-17A has recently been shown to be effective in the treatment of CIA (8). In the present study, we have demonstrated the therapeutic potential of IL-17B blockade after the onset of CIA. Because blockade of TNF-α or IL-1β is not always effective in RA patients, blockade of additional cytokine might be a useful therapeutic option. Therefore, our data strongly suggest that IL-17B as well as IL-17A could be an important target for the treatment of inflammatory arthritis.

In a recent study, the combination of IL-6 and TGF-β was reported to strongly induce IL-17A production in Th17 cells (41). Moreover, it was recently recognized that IL-23 contributes to the expansion of autoreactive IL-17A-producing T cells and promotes chronic inflammation dominated by IL-17A, IL-6, IL-8, and TNF-α (14, 42). Thus, IL-17B and IL-17C may exacerbate arthritis via IL-6- and IL-23-mediated promotion of IL-17A production. However, the possibility that IL-17B and IL-17C exert a cooperative proinflammatory response together with IL-17A and IL-17F in arthritis by regulating the release of cytokines such as IL-6, IL-1β, and IL-23 still remains to be examined.

IL-17F has the highest homology with IL-17A and, like IL-17A, is produced by activated T cells. IL-17F appears to have an effect similar to that of IL-17A on cartilage proteoglycan release and inhibition of new cartilage matrix synthesis (11). Although IL-17F is thought to contribute to the pathology of inflammatory disorders such as RA, the in vivo effect of IL-17F on arthritis was not elucidated. In this study, we found that transduction of BM-expressed IL-17F resulted in both an earlier onset and a subsequent aggravation of arthritis.

We also found that the mRNA expression of all IL-17 family and IL-17R genes examined (mIL-17A, mIL-17B, mIL-17C, mIL-17F, mIL-17R, and mIL-17Rh1) was elevated in the arthritic paws of CIA mice compared with the paws of the control mice. The receptor for IL-17A is IL-17R (also named IL-17AR), which is extensively expressed in various tissues or cells tested, in contrast to the exclusive expression of IL-17A in activated T cells. Recently, IL-17R signaling has been suggested to play a crucial role in driving the synovial expression of proinflammatory and catabolic mediators, such as IL-1, IL-6, matrix metalloproteinase (MMP)-3, MMP-9, and MMP-13, in streptococcal cell wall-induced arthritis (43). IL-17R-deficient (IL-17R−/−) mice that were locally injected five times with streptococcal cell wall fragments into the knee joints showed a significant reduction of joint thickness and cartilage damage that was accompanied by reduced synovial expression of IL-1, IL-6, and the MMPs 3, 9, and 13 compared with arthritic wild-type mice. Therefore, these results indicate the critical role of IL-17R signaling during progression from an acute, macrophage-driven joint inflammation to a chronic, cartilage-destructive, T cell-mediated synovitis. There are four additional receptor-like molecules that share homology to IL-17R, i.e., IL-17Rh1 (also named IL-17RB or IL-17BR), IL-17RL (also named IL-17RC), IL-17RD, and IL-17RE. IL-17Rh1 was shown to bind to IL-17B, but with higher affinity to IL-17E (11, 12).

Although IL-17A transgenic mice have been reported to be embryonic lethal (39), we established BM-overexpressing mice that constitutively expressed IL-17A. The adequate control of the expression level was critically important. In our experiment, the serum concentration of IL-17A was elevated to ∼600 pg/ml in IL-17A BM chimeric mice. This serum concentration of IL-17A was similar to those in patients with inflammatory diseases such as RA, inflammatory bowel diseases, familial Mediterranean fever, and the acute stage of Kawasaki disease (3, 44, 45, 46). Therefore, our BM chimeric mice approach may be useful to elucidate the physiological role of inflammatory cytokines that show lethal phenotypes in the conventional gene-transgenic technique.

In conclusion, we found that IL-17 family genes were up-regulated in association with their receptors in CIA. Each of the IL-17 family members clearly exacerbated the progression of CIA with the method of retrovirus-mediated BM chimeric mice. IL-17B and IL-17C have the capacity to exacerbate inflammatory arthritis in association with increased TNF-α and IL-6 productions from macrophages. Moreover, neutralization of IL-17B significantly suppressed the progression of arthritis and bone destruction in CIA mice. Therefore, our results suggest that not only IL-17A, but also the IL-17 family members IL-17B, IL-17C, and IL-17F play an important role in the pathogenesis of inflammatory arthritis and should be a new therapeutic target of arthritis.

Acknowledgments

We are grateful to Yayoi Tsukahara and Kayako Watada for their excellent technical assistance.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • 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 with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This study was supported by Program and Project Grant funding from Japan Society for the Promotion of Science; Ministry of Health, Labour and Welfare; and Ministry of Education, Culture, Sports, Science and Technology.

  • ↵2 Address correspondence and reprint requests to Dr. Keishi Fujio, Department of Allergy and Rheumatology, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8655, Japan. E-mail address: kfujio-tky{at}umin.ac.jp

  • ↵3 Abbreviations used in this paper: RA, rheumatoid arthritis; BCII, bovine type II collagen; BM, bone marrow; CIA, collagen-induced arthritis; mIL, murine IL; MMP, matrix metalloproteinase; PEC, peritoneal exudate cell; pMIG, murine stem cell virus/internal ribosome entry site/GFP.

  • Received April 12, 2007.
  • Accepted September 5, 2007.

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