IL-17B and IL-17C Are Associated with TNF-?
Production and Contribute to the Exacerbation of
Yumi Yamaguchi,* Keishi Fujio,2* Hirofumi Shoda,* Akiko Okamoto,* Nelson H. Tsuno,†
Koki Takahashi,†and Kazuhiko Yamamoto*
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-? and IL-1? 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-? 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-? 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. The Journal of Immunology, 2007, 179: 7128–7136.
cloned from activated T cells, and shares 57% homology to the
protein encoded by the open reading frame 13 gene of the T lym-
photropic 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-?, and IL-6. T cell IL-17A stimulates the production of IL-1
and TNF-? from human PBMC-derived macrophages in vitro (4).
IL-17A also enhances IL-1-mediated IL-6 production by RA sy-
noviocytes in vitro as well as TNF-?-induced synthesis of IL-1,
IL-6, and IL-8 (5, 6). These results indicate that IL-17A synergizes
with IL-1 and TNF-? and contributes to inflammation of RA.
nterleukin-17A is a T cell-derived proinflammatory cytokine
that is involved in the development of rheumatoid arthritis
(RA).3IL-17A was originally named CTLA-8 after being
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 destruc-
tion of cartilage (8). IL-17A deficiency protects IL-1R antago-
nist-deficient mice from spontaneous development of destruc-
tive arthritis (9). Therefore, IL-17A plays a crucial role in the
pathogenesis of arthritis through synergistic effects with IL-1
and TNF-?. However, IL-17A can directly induce joint destruc-
tion in an IL-1-independent manner and can bypass TNF-de-
pendent 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 for-
mation of intermolecular disulfide linkages (11, 12). The different
IL-17 family members seem to have very distinct expression pat-
terns, 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 stimu-
lation (13–15). 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 re-
cently reported that IL-17B is highly expressed in chondrocytes
*Department of Allergy and Rheumatology, Graduate School of Medicine, University
of Tokyo, Tokyo, Japan; and†Department of Transfusion Medicine and Immunohe-
matology, University of Tokyo, Tokyo, Japan
Received for publication April 12, 2007. Accepted for publication September 5, 2007.
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.
1This 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.
2Address 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@
3Abbreviations 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.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
that are located at the mid and deep zones of normal bovine ar-
ticular cartilage (11). In contrast, IL-17C expression has been con-
fined 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 inflam-
A common feature of IL-17 family members is the induction of
neutrophil migration. IL-17A and IL-17F both mobilize neutro-
phils 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 induc-
tion of cytokine production. Although IL-17A and IL-17F up-reg-
ulate IL-6 and IL-8 in human fibroblasts (19, 20), IL-17B and
IL-17C are reported to stimulate the release of TNF-? and IL-1?
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 ac-
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 fi-
broblasts and macrophages. Recently, IL-17C expression in syno-
vial fluid mononuclear cells and PBMCs of RA patients was re-
ported (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 inflam-
matory cytokines such as IL-1?, 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-? 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 sig-
nificantly suppressed the progression of arthritis and bone destruc-
tion 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-? and contribute to the exacerbation of autoimmune arthritis.
Materials and Methods
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.
CIA was induced, as described previously (22–24). 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 ?g of BCII emulsified with CFA. On day 21, the mice were
boosted by intradermal injection with 100 ?g of BCII emulsified with IFA
(Difco). The arthritis score was determined by erythema, swelling, or an-
kylosis 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 cul-
tured with RPMI 1640 (Invitrogen Life Technologies) medium supple-
mented with 10% FCS, 2 mM ?-glutamine, 100 U/ml penicillin, 100 ?g/ml
streptomycin, and 5 ? 10?5M 2-ME. Ba/F3 cells were maintained in
RPMI 1640 medium supplemented with 10% FCS, 2 mM ?-glutamine, 100
U/ml penicillin, 100 ?g/ml streptomycin, and 1 ng/ml rmIL-3 (R&D
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 mu-
rine T lymphocyte cDNA library according to the reported nucleotide se-
quence 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
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
?g/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 per-
formed, 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 ?g/ml poly-
brene (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 ? 106of 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 Technolo-
gies). 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 ?l of SYBR Green Master Mix (Qiagen),
15 pmol of forward and reverse primers, and the cDNA samples, in a
total volume of 50 ?l. We calculated the quantitative PCR data with ?
cycle threshold method, and relative RNA abundance was determined
based on control ?-actin abundance. To measure the relative efficiency,
7129The Journal of Immunology
amplifications were performed on the serial diluted cDNA samples us-
ing primers for the target and the reference (?-actin) genes. We made
plots of the log cDNA dilution vs ? cycle threshold, and confirmed that
the efficiencies of the target and the reference genes were similar be-
cause 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?-GGGTTG
GTGGTTGGCTCAGAA-3?; mouse IL-17C, sense 5?-CACAGATGAG
AACCGCTACCC-3?, antisense 5?-GCGGATGAACTCGGTGTGGA
A-3?; mouse IL-17F, sense 5?-CAACGCTGCATACAAAAATCA-3?,
antisense 5?-TTAAGTGAGGCATTGGGAACA-3?; mouse IL-17R,
sense 5?-CCACTCTGTAGCACCCCAATG-3?, antisense 5?-CCTGGA
GATGTAGCCCTGGTC-3?; mouse IL-17Rh1, sense 5?-GCAAGGAA
GGAGCACGAAGAC-3?, antisense 5?-CTCGGCGATTTTCTTTTTCT
G-3?; mouse TNF-?, sense 5?-CATCTTCTCAAAATTCGAGTGACA
A-3?, antisense 5?-TGGGAGTAGACAAGGTACAACCC-3?; mouse
IL-1?, sense 5?-CAACCAACAAGTGATATTCTCCATG-3?, antisense
5?-GATCCACACTCTCCAGCTGCA-3?; mouse IL-6, sense 5?-CACT
TCACAAGTCGGAGGCTTA-3?, antisense 5?-GCAAGTGCATCATC
GTTGTTC-3?; mouse IL-23, sense 5?-TGGCATCGAGAAACTGTGAG
A-3?, antisense 5?-TCAGTTCGTATTGGTAGTCCTGTTA-3?; and
mouse ?-actin, sense AGAGGGAAATCGTGCGTGAC-3?, antisense
Immunoassays of cytokines and anti-type II collagen Ab
The concentrations of mIL-6, mTNF-?, 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 previ-
ously (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.
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 pres-
ence of 10 ?g/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?)2goat anti-rat IgG PE
(Serotec) were used as secondary reagents for IL-17C and IL-17F stain-
ing, 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 cy-
tometry. 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 ?g of polyclonal anti-mouse IL-17B Abs
(R&D Systems). PBS was i.p. injected as a control. Arthritis was as-
sessed 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.
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 in-
filtration and pannus invasion, as described previously (37).
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.
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 exam-
ined (IL-17A, IL-17B, IL-17C, and IL-17F) were highly ele-
vated in the arthritic paws compared with the controls. In ac-
cordance 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. 1A). As expected, the
mRNA expressions of inflammatory cytokines (TNF-?, IL-1?,
IL-6, and IL-23) were also elevated in the arthritic paws com-
pared with controls (Fig. 1B).
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 inflam-
matory 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.
1C). 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 in-
fluence on mouse fibroblast cell lines and mouse peritoneal mac-
rophages. Cells of the mouse fibroblast line 3T3 were cultured with
each of the IL-17 family members (50 ng/ml), and cytokine ex-
pression was examined after 24 h of incubation. IL-17A induced
IL-1? and IL-6 expressions, as previously reported (2). Moreover,
IL-17B, IL-17C, and IL-17F also induced IL-1? expression in 3T3
To examine the effects of IL-17 family members on mouse mac-
rophages, thioglycolate-elicited PECs were isolated and cultured
with each of the IL-17 family members (50 ng/ml). IL-17A in-
duced IL-1?, IL-6, and IL-23 expressions in PECs. Interestingly,
IL-17B also induced IL-1?, IL-6, and IL-23 expressions. More-
over, IL-17C induced IL-1? and IL-23 expressions in PECs (Fig.
2B). In addition, PECs stimulated with every IL-17 family member
produced significantly increased amount of TNF-? protein com-
pared with the control, and PECs stimulated with IL-17A and IL-
17B produced significantly increased amount of IL-6 protein (Fig.
2C). These results suggested that IL-17A, IL-17B, IL-17C, and
IL-17F stimulate fibroblasts and macrophages to produce inflam-
7130EFFECTS OF THE IL-17 FAMILY ON ARTHRITIS PROGRESSION
Exacerbation of CIA by transfer of IL-17 family-transduced
Because IL-17B and IL-17C induce the expression of inflam-
matory cytokines in fibroblasts and macrophages, we hypothe-
sized 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 expres-
sions of IL-17 family members were confirmed with intracel-
lular staining of each IL-17 family cytokine (Fig. 3A).
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 av-
erage (Fig. 3B). These IL-17 family-transduced CD4?T cells
were adoptively transferred to BCII-immunized DBA1 mice be-
fore 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 sig-
nificant effect on the serum levels of anti-BCII IgG Abs at 14
days after the onset of CIA (data not shown). These results
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 (f; n ? 3) and in control mice
(?; n ? 3) by quantitative PCR. B,
The expressions of inflammatory cy-
tokines. 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.
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-? 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.
The proinflammatory effects of IL-17 family members on mouse fibroblasts and macrophages. A, Relative expression of the cytokine genes
7131The Journal of Immunology
indicated that the effect of IL-17 family members on the pro-
gression of arthritis was not associated with the elevations of
IL-17 family BM chimeric mice exhibited high arthritis scores
upon CIA induction
To examine the proinflammatory effect of constitutively ex-
pressed 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 gen-
erate IL-17A-overexpressing mice with a conventional trans-
genic approach was unsuccessful because these mice were em-
bryonic 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. 4A). Moreover, the serum concentration
of mIL-17A was significantly elevated in these mice (Fig. 4B).
Therefore, the BM chimeric mice were successfully allowed to
express the transduced cytokines systemically. Then we immu-
nized 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 ar-
thritis, as assessed by the arthritis score. In contrast, BM chi-
meric mice of IL-17B and IL-17C did not result in significant
differences in the onset of disease (Fig. 5, C and D). BM ex-
pression 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 re-
sponses of anti-BCII Abs.
of IL-17 family-transduced CD4?T
cells on CIA. A, Intracellular IL-17
family expressions in Ba/F3 cells ret-
rovirally 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
cells. B, Representative FACS analy-
sis 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
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 ar-
thritis score (n ? 20 mice per group).
Significance of differences between
control (pMIG) and each IL-17 fam-
ily-transduced mice was determined;
??, p ? 0.005; ?, p ? 0.05.
The effects of transfer
plantation 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.
Generation of IL-17 family chimeric mice by BM trans-
7132EFFECTS OF THE IL-17 FAMILY ON ARTHRITIS PROGRESSION
We next examined the alterations of inflammatory cytokine
production in these BM chimeric mice. Interestingly, nonim-
munized IL-17C BM chimeric mice showed increased mRNA
expression of TNF-? in the spleen compared with controls (Fig.
5E). Moreover, in the spleen of BCII-immunized IL-17C BM
chimeric mice, the mRNA expressions of TNF-?, 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. 5E). When we ex-
amined the concentrations of TNF-? 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-? concentration in the sera. And the BCII-immunized IL-
17A and IL-17B BM chimeric mice showed increased IL-6 pro-
duction in the sera (Fig. 5F). 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 pro-
gression 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 arthri-
tis. Neutralization of IL-17B significantly suppressed the progres-
sion of CIA compared with the controls (Fig. 6A). Moreover, his-
tological analysis revealed significant reduction of cell infiltration
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 immu-
nized with BCII 8 wk after the BM
transplantation. Values are the mean
IL-17F BM chimeric mice (n ? 20
IL-17B and IL-17C BM chimeric
mice (n ? 30 per group). Significance
(pMIG) and each IL-17 family BM
??, p ? 0.005; ?, p ? 0.05. E, The
mRNA expression of inflammatory
cytokines in the spleen of BM chi-
meric mice of IL-17B and IL-17C,
which were immunized with BCII (f;
n ? 15 per group) or nonimmunized
controls (?; n ? 6 per group). Sig-
nificance of differences between con-
trol (pMIG) and each IL-17 family
BM chimeric mice was determined;
??, p ? 0.005; ?, p ? 0.05. F, The
secreted TNF-? and IL-6 levels in the
serum of IL-17 family BM chimeric
mice that were immunized with BCII
(f; n ? 15) or nonimmunized con-
trols (?; n ? 6). Significance of dif-
ferences between control (pMIG) and
each IL-17 family BM chimeric mice
was determined; ??, p ? 0.005;
?, p ? 0.05.
Incidence of CIA and
7133 The Journal of Immunology
and pannus invasion in the anti-IL-17B Ab-treated mice (Fig. 6B).
These results indicated that IL-17B was associated with the pro-
gression of arthritis in CIA mice.
RA is considered to be an autoimmune disease, and is character-
ized 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-? 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-? pro-
duction in PECs in vitro. In vivo, the mRNA expression of TNF-?
was spontaneously increased in the spleen of IL-17C BM chimeric
mice. Moreover, TNF-? productions in the sera of BCII-immu-
nized IL-17B and IL-17C BM chimeric mice were up-regulated.
Although IL-17A induced TNF-? production in PECs, IL-17A BM
chimeric mice did not show up-regulated production of TNF-?.
This result is consistent with previous observation in THP-1 cell
line that IL-17B and IL-17C stimulated the release of TNF-?,
whereas IL-17A has only a weak effect on TNF-? (17). In contrast
to IL-17B and IL-17C, IL-17A may not be directly associated with
TNF-? production in vivo. Moreover, the mRNA expression in the
spleen and serum concentration of IL-6 were significantly up-reg-
ulated 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-? 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 ex-
pressed in a broad range of cells, i.e., CD4?T cells, CD11b?
MHC class II?macrophages, and CD11c?MHC class II?den-
dritic cells. IL-17A and IL-17F were expressed in CD4?T cells,
as expected. These results suggested that CD4?T cells are in-
volved 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-promot-
ing effect of IL-17C-transduced CD4?T cells suggests the impor-
tance 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 arthri-
tis-promoting effect of IL-17A was previously reported in a study
using adenovirus vector (5, 40). In contrast to IL-17A, which has-
tened 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 knowl-
edge, 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 demon-
strated the therapeutic potential of IL-17B blockade after the onset
of CIA. Because blockade of TNF-? or IL-1? is not always ef-
fective 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-? was re-
ported 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-? (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 cooper-
ative proinflammatory response together with IL-17A and IL-17F
in arthritis by regulating the release of cytokines such as IL-6,
IL-1?, 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 elu-
cidated. In this study, we found that transduction of BM-expressed
IL-17F resulted in both an earlier onset and a subsequent aggra-
vation 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. Re-
cently, IL-17R signaling has been suggested to play a crucial role
in driving the synovial expression of proinflammatory and cata-
bolic mediators, such as IL-1, IL-6, matrix metalloproteinase
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.
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
7134 EFFECTS OF THE IL-17 FAMILY ON ARTHRITIS PROGRESSION
(MMP)-3, MMP-9, and MMP-13, in streptococcal cell wall-in-
duced 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 thick-
ness and cartilage damage that was accompanied by reduced sy-
novial expression of IL-1, IL-6, and the MMPs 3, 9, and 13 com-
pared 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 ad-
ditional 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 em-
bryonic lethal (39), we established BM-overexpressing mice that
constitutively expressed IL-17A. The adequate control of the ex-
pression level was critically important. In our experiment, the se-
rum 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, 44–46). Therefore, our
BM chimeric mice approach may be useful to elucidate the phys-
iological role of inflammatory cytokines that show lethal pheno-
types in the conventional gene-transgenic technique.
In conclusion, we found that IL-17 family genes were up-reg-
ulated 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-? and IL-6 productions from mac-
rophages. Moreover, neutralization of IL-17B significantly sup-
pressed 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.
We are grateful to Yayoi Tsukahara and Kayako Watada for their excellent
The authors have no financial conflict of interest.
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7136 EFFECTS OF THE IL-17 FAMILY ON ARTHRITIS PROGRESSION