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Departments of
* Molecular Immunology and
Bioregulatory Medicine and Rheumatology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan; and Departments of
Pathology and
Immunology, Juntendo University School of Medicine, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-1
, and IL-6 in the joints was
inhibited by the treatment. Proliferative responses and production of
IFN-
and IL-10 upon restimulation with CII in vitro were
significantly inhibited in LN cells from the anti-B7h mAb-treated
mice. Serum anti-CII IgG1, IgG2a, and IgG2b levels were also
reduced. Our present results showed a beneficial effect of the B7h
blockade on CIA through anti-inflammatory actions and inhibition of
both Th1- and Th2-mediated immune responses, suggesting that the
ICOS-B7h interaction plays an important role in the pathogenesis of CIA
and thus the blockade of this pathway may be beneficial for the
treatment of human rheumatoid arthritis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, new members of the CD28-B7 family have been identified. The
inducible costimulator (ICOS) is one of such molecules expressed on
activated T cells (7, 8, 9, 10). The ICOS ligand, B7 homologous
protein (B7h) (11)/B7-related protein 1 (B7RP-1)
(12)/GL50 (13)/ligand of ICOS
(14), hereafter designated B7h, is
constitutively expressed on B cells and is inducible on
monocytes and dendritic cells at low levels (15, 16). B7h
expression in these APCs and fibroblasts could be induced by
proinflammatory cytokines such as IFN- and TNF-
(11). Ligation of ICOS on activated T cells by mAb or B7h
fusion proteins strongly enhanced the production of multiple cytokines
including IL-4, IL-5, IFN-, and IL-10, whereas IL-2 production was
not clearly enhanced (7, 15, 17). These results suggested
a unique property of the ICOS-mediated costimulation distinct from the
CD28-mediated costimulation. Earlier studies of the ICOS blockade
(10, 18) and ICOS-deficient mice (19, 20, 21)
suggested a predominant role of the ICOS costimulation in Th2-mediated
humoral immune responses. However, recent studies also demonstrated the
involvement of ICOS in Th1- and CD8+ T
cell-mediated cellular immune responses (22, 23, 24, 25). Another
intriguing feature of the ICOS blockade distinct from the CD28 blockade
is the fact that the ICOS blockade was effective at the efferent phase,
but not at the induction phase of both Th1- and Th2-mediated responses
(26, 27). These results suggest that ICOS may be a potent
costimulator for effector T cells.
The collagen-induced arthritis (CIA) model has been extensively used to
elucidate pathogenic mechanisms relevant to human rheumatoid arthritis
and to identify potential targets for therapeutic intervention
(28). A murine model of CIA can be induced in genetically
susceptible mice such as DBA/1 by intradermal injection of type II
collagen (CII) in adjuvant. The development of CIA is known to be
dependent on CD4+ T cell activation and Ab
production against CII (28). In addition to these
Ag-specific immune responses, local production of proinflammatory
cytokines, such as TNF-, IL-1, and IL-6, is involved in the
pathogenesis of CIA (29). In this study, we have examined
the effects of the administration of mAb against B7h in the murine CIA
model and investigated the involvement of the ICOS-B7h costimulatory
pathway in the development and disease progression of CIA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free 6-wk-old male DBA/1J mice were purchased from the Japan Charles River Breeding Laboratories (Kanagawa, Japan) and maintained in the animal facility at the Tokyo Medical and Dental University (Tokyo, Japan). All mice procedures were reviewed and approved by the Animal Care and Use Committee in the Tokyo Medical and Dental University.
Ig fusion protein, transfectants, mAbs, and immunofluorescence
The Ig fusion protein consisting of the extracellular portion of mouse ICOS (aa 1–147) (30) linked to the Fc portion of human IgG1 (ICOS-Ig) and OX40-Ig were prepared as described previously (31). Mouse B7h cDNA was generated by RT-PCR from peritoneal macrophages of BALB/c mice and was subsequently subcloned into a pMKITneo vector (kindly provided by Dr. K. Maruyama, Tokyo Medical and Dental University). Primers used to generate a full-length B7h cDNA were: sense, 5'-GACGAATTCATGCAGCTAAAGTGTCCCTG-3' including an EcoRI cloning site; and antisense, 5'-GACCTCGAGTCAGGCGTGGTCTGTAAGTT-3' including a XhoI cloning site. NRK-52E (NRK) and L cells were transfected with a B7h/pMKITneo expression vector by electroporation, drug selected, and cells expressing B7h were identified by staining with ICOS-Ig. The anti-mouse B7h mAb (HK5.3, rat IgG2a) was generated by immunizing SD rats with mouse B7h-transfected L cells and fusing immune splenocytes with P3U1 myeloma cells and screened for binding to mouse B7h-transfected NRK cells.
For in vitro blocking experiments, anti-mouse CD80 (RM80, rat IgG2a) (32), CD86 (PO3, rat IgG2b) (32), CD154 (MR1, hamster IgG) (33), and CD134L (RM134L, rat IgG2b) (34) mAbs were used. All mAbs were purified from ascites as described previously (3, 32). Rat IgG (Sigma-Aldrich, St. Louis, MO) was used as a control reagent. For immunofluorescent analysis, PerCP-conjugated anti-CD3 mAb (145-2C11, hamster IgG) and FITC-conjugated anti-ICOS mAb (B10.5, rat IgG2a, kindly provided by JT Central Pharmaceutical Research Institute, Osaka, Japan) (35), or appropriate fluorochrome-conjugated control Ig were used. All fluorochrome-conjugated mAbs and control Ig were obtained from BD PharMingen (San Diego, CA), unless otherwise noted. For indirect staining, PE-conjugated anti-human IgG (Caltag Laboratories, Burlingame, CA) and PE-conjugated anti-rat IgG (Caltag Laboratories) were used for the second-step Abs. Immunofluorescence, flow cytometry, and data analysis were performed using FACSCalibur and CellQuest software (BD Biosceinces, San Jose, CA).
Induction of CIA, Ab treatment, and clinical assessment of arthritis
CIA was induced as previously described with minor modifications (36). Briefly, male DBA/1J mice (7–10 wk old) were injected intradermally at the base of the tail with 200 µg of bovine CII (Collagen Research Center, Tokyo, Japan) in 0.05 M acetic acid, emulsified in CFA (Difco, Detroit, MI). Twenty-one days after primary immunization, the mice were boosted in the same way. The day of second immunization (booster) was designated as day 0. The immunized mice were randomly divided and treated with the following regimens. Each group of mice received either control IgG or anti-B7h mAb (50, 100, or 300 µg/body) i.p. on days -1, 1, 3, and 5. In our preliminary experiment, we have observed no obvious differences between the mice treated with three doses of control IgG. We therefore selected a single dose, 100 µg/body for a control group. In the experiments for a delayed treatment, 100 µg/body control IgG or anti-B7h mAb was injected i.p. on days 5, 7, 9, and 11. Mice were examined daily for the onset of CIA. The swelling of four paws was graded from 0 to 4 as follows: grade 0, no swelling; grade 1, swelling of finger joints or focal redness; grade 2, mild swelling of wrist or ankle joints; grade 3, severe swelling of the entire paw; and grade 4, deformity or ankylosis. Each paw was graded, and the four scores were totaled so that the maximal score per mouse was 16. Incidence was expressed as the number of mice that showed paw swelling in the total number of mice examined, and the time of onset was expressed as the mean time when paw swelling was first observed in individual mice.
Histological and radiological assessments of arthritis
CIA mice were killed at day 35. Anteroposterior radiographs of the four limbs were obtained with a cabinet soft x-ray apparatus (CMB-2; Softex, Tokyo, Japan). Then, the hind paws were removed, fixed in Formalin, decalcified in 10% EDTA, embedded in paraffin, sectioned, and stained with H&E.
ICOS, B7h, and proinflammatory cytokine expression in the synovium
In some experiments, the knee joints from CIA mice at day 5 were removed and subjected to immunohistological analysis. Cryostat sections were fixed in acetone and 1% paraformaldehyde. For detection of ICOS, the sections were stained with biotinylated anti-ICOS mAb (B10.5) followed by biotinylated rabbit anti-rat IgG Ab (DAKO-Japan, Kyoto, Japan), Alexa 488-conjugated streptavidin (Molecular Probes, Eugene, OR), normal rat serum for blocking, and then Alexa 594-conjugated rat anti-CD4 (GK1.5) mAb.
For detection of B7h, the sections were stained with biotinylated ICOS-Ig and then HRP-conjugated streptavidin followed by Alexa 488-tyramide (TSA kit 22; Molecular Probes). After blocking with normal rat serum, Alexa 594-conjugated rat anti-CD45R (B220) was further stained. Conjugation of Alexa 594 to anti-CD45R and CD4 mAbs were performed using a Alexa Fluor 594 protein labeling kit (Molecular Probes) according to the protocols recommended by the manufacturer. The staining profiles were obtained with a fluorescence microscope (Olympus BX-50; Olympus, Tokyo, Japan) equipped with a charge-coupled device camera (PXL System; Photometrics, Tucson, AZ) and the image was analyzed by using IPLab Spectrum software (Signal Analytics, Vienna, VA).
For RT-PCR analysis, synovial tissues were isolated from the knee
joints, and total RNA was extracted by using Concert cytoplasmic RNA
reagent (Invitrogen, Tokyo, Japan). First-strand cDNA was synthesized
using oligo(dT) primer and Superscript II reverse transcriptase (Life
Technologies, Gaithersburg, MD). PCR was performed using the following
primers: mouse TNF- (sense, 5'-GCCACCACGCTCTTCTG-3' and antisense,
5'-ATGGGCTCATACCAGGG-3'); mouse IL-1 (sense,
5'-CTGAAAGCTCTCCACCTC-3' and antisense,
5'-GGTGCTGATGTACCAGTTG-3'); mouse IL-6 (sense,
5'-TCCTCTCTGCAAGAGACTT-3' and antisense, 5'-TTCTGCAAGTGCATCATCG-3');
mouse ICOS (sense, 5'-GTACTTCTGCCATGTCTTTG-3' and antisense,
5'-TGAGGTCACACCTGCAAGT-3'); mouse B7h (sense,
5'-GTGTCCCTGTTTTGTGTCC-3' and antisense, 5'-TGAAGTTTGCTGCCACACG-3');
and mouse GAPDH (sense, 5'-GCCAAACGGGTCATCATCTC-3' and antisense,
5'-GACACATTGGGGGTAGGAAC-3'). For quantification of the PCR
products, the amounts of cDNA were preliminarily normalized to produce
the same amount of PCR products for GAPDH based on the intensity of the
ethidium bromide staining of each band measured by the charge-coupled
device imaging system (Densitograph AE-6920 M; Atto, Tokyo, Japan). The
cDNA samples from four individual mice in each group were amplified on
a DNA thermal cycler (PerkinElmer, Norwalk, CT) for 30 cycles except
for 25 cycles of GAPDH. The PCR condition was as follows: 94°C for 1
min, followed by 58°C (cytokines and GAPDH), 55°C (ICOS), or 56°C
(B7h) for 1 min, and 72°C for 2 min with a 15-min extension at 72°C
at the end. The PCR products were electrophoresed on agarose gel,
stained with ethidium bromide, and the image was acquired using the
Densitograph.
CII-specific T cell proliferation and cytokine production
Five days or 10 wk after the second immunization, draining lymph
nodes (LNs) and the spleen were removed. Single-cell suspensions of LN
cells and erythrocyte-depleted splenocytes were prepared in RPMI 1640
medium supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium
pyruvate, 5 x 10-5 M 2-ME, and
antibiotics. For purification of CD4+ T cells,
cells were treated with anti-I-A, anti-CD24, anti-CD45R,
and anti-CD8 mAbs and rabbit complement and the purity of
CD4+ T cells was confirmed to be >95%
CD4+ cells by flow cytometry. Whole splenocytes,
LN cells (5 x 105/well), or
CD4+ splenic T cells (2.5 x
105/well) were seeded in 96-well flat-bottom
microtiter plates and cultured in the presence or absence of the
indicated amounts of denatured (60°C, 30 min) bovine CII (dCII) for
72 h. In the culture with purified CD4+ T
cells, splenocytes from the mice at 5 days after the second
immunization were treated with mitomycin C (50 µg/ml, 37°C, 30 min;
Sigma-Aldrich) and the cells (2.5 x
105/well) were added as APCs. For in vitro mAb
blocking experiments, LN cells from the mice 8 wk after the primary
immunization were used and cultured as described above. The blocking
mAb was added at the start of the assay. All cultures were pulsed with
[3H]thymidine (0.5 µCi/well; DuPont/NEN,
Boston, MA) for the last 16 h, harvested on a Micro 96 Harvester
(Skatron, Lier, Norway), and the incorporated radioactivity was
measured using a microplate beta counter (Micro Beta Plus; Wallac,
Turku, Finland). Supernatants from similar cultures were collected
after 96 h for the assessment of cytokine production by ELISA.
ELISA for mouse IFN-, IL-4, and IL-10 was performed using ELISA kits
(Ready-SET-Go!; eBioscience, San Diego, CA) according to the
protocols recommended by the manufacturer.
Serum anti-CII Ab levels
Serum samples were collected on days 7, 14, and 21, and the titers of anti-CII IgG Abs were measured by ELISA. Bovine CII (1 µg/ml) was coated onto microtiter plates (Maxisorp; Nunc, Roskilde, Denmark) overnight at 4°C. After blocking with 1% BSA in PBS, serially diluted serum samples were added and incubated for 1 h at room temperature. After washing, HRP-conjugated rabbit anti-mouse IgG1, IgG2a, or IgG2b Ab (Zymed Laboratories, San Francisco, CA) was added and incubated for 2 h at 37°C. After washing, Ab binding was visualized using o-phenylenediamine (Sigma-Aldrich). A standard serum composed of a mixture of sera from arthritic mice was added to each plate in serial dilutions and a standard curve was constructed. The standard serum was defined as 1 U and the Ab titers of serum samples were determined by the standard curve.
Statistical analysis
Significant differences between experimental groups were analyzed by the Mann-Whitney U test. Values of p < 0.05 were considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We generated a mAb (HK5.3) against mouse B7h by immunizing SD rats
with B7h transfectants. Similar to the staining with ICOS-Ig, HK5.3
specifically bound to B7h-transfected NRK (B7h/NRK) cells, but not to
parental NRK cells (Fig. 1
A).
Preincubation with HK5.3, but not with control rat IgG (data not shown)
efficiently blocked the ICOS-Ig binding to B7h/NRK cells, indicating
the specific binding of HK5.3 to the ICOS ligand B7h. To evaluate the
functional inhibitory effects of this anti-B7h mAb, we examined the
effect of this mAb on CII-specific proliferative responses in vitro. LN
cells from CII-immunized mice were stimulated with 30 µg/ml dCII and
a panel of mAbs against costimulatory molecules (Fig. 1B)
and the titrated amount of anti-B7h mAb (Fig. 1C) was
added to the culture. The addition of dCII dramatically enhanced
proliferative responses and this enhanced proliferation was clearly
inhibited by anti-B7h mAb as well as by anti-CD80 and CD86
mAbs, anti-CD134L mAb, or anti-CD154 mAb. The inhibitory effect
of anti-B7h mAb occurred in a dose-dependent manner. These results
indicate an inhibitory activity of anti-B7h mAb and a substantial
involvement of B7h in the CII-specific T cell responses in vitro.
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To investigate the role of ICOS-B7h-mediated T cell costimulation
in the development of autoimmune arthritis, we examined the effects of
anti-B7h mAb on the development of CIA. DBA/1J mice were immunized
twice with bovine CII in CFA to elicit CIA. Four groups of mice were
i.p. administrated either 50, 100, and 300 µg of anti-B7h mAb or
100 µg of control IgG every other day from day -1 for four times. As
shown in Fig. 2A, the mice
treated with control IgG developed severe arthritis. In contrast, the
administration of anti-B7h mAb significantly ameliorated the
clinical manifestations of CIA in a dose-dependent manner. As
summarized in Table I, the treatment with
100 µg of anti-B7h mAb significantly delayed the day of onset and
decreased the mean arthritis score and the mean number of arthritic
paws, although the incidence of disease was not affected. The
amelioration of clinical arthritis by the anti-B7h mAb treatment
was confirmed by histopathological examination of the joints. The hind
paw sections from the control IgG-treated mice at day 35 showed
infiltration of mononuclear cells, synovial hyperplasia, pannus
formation, cartilage destruction, and bone erosion that are
characteristic features of arthritis (Fig. 3c). These features were
clearly ameliorated by the anti-B7h mAb treatment (Fig. 3d). Radiological examination also showed the prevention of
bone erosion by the anti-B7h mAb treatment (Fig. 3, a
and b).
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, we started a similar treatment
from day 5 and examined the clinical scores. The delayed treatment was
also effective in reducing the clinical arthritis scores (Fig. 2B). These results demonstrated that the treatment with
anti-B7h mAb either before or after the onset of disease could
inhibit the disease severity of CIA, suggesting the involvement of the
ICOS-B7h pathway in the pathogenesis. Expression of ICOS and B7h in the joints and the draining LNs
To determine the expression of ICOS and B7h in the inflamed joints
and the draining LN cells, immunohistological staining and flow
cytometry were performed using a specific mAb against ICOS and B7h or
ICOS-Ig. ICOS expression was observed on both
CD4+ and CD4- (presumably
CD8+ T) cells in the synovium of the inflamed
joints from the control IgG-treated mice on day 5 (Fig. 4Ab). We first performed
immunohistological staining using anti-B7h mAb; however, the
positive staining was not observed even in the spleen and LN as well as
in the synovial tissue, suggesting that this anti-B7h mAb (HK5.3)
is not applicable to immunohistological staining. We therefore used
ICOS-Ig for detection of B7h expression. Positive cells were observed
within some of the CD45R+ B cells (Fig. 4Ac) and CD11b+ macrophages, and then
weakly on CD11c+ dendritic cells (data not shown)
in the inflamed joints. To further assess the change of ICOS and B7h
expression in the inflamed joints, the mRNA levels for ICOS and B7h
were compared between naive and the control IgG-treated CIA mice. The
mRNA expression for ICOS and B7h in the joints was significantly
enhanced in the CIA mice (Fig. 4B). We next investigated
draining LN cells. The mean total number of LN cells in the control
IgG-treated CIA mice (2.5 ± 0.1 x 107
cells) was clearly enhanced as compared with that in the naive mice and
this enhancement was reduced in the anti-B7h mAb-treated mice
(1.6 ± 0.3 x 107 cells). However, the
ratio of T (CD3+), B
(CD45R+CD3-), or non-T/B
(CD3-CD45R-) LN cells was
not affected by the anti-B7h mAb treatment (data not shown).
ICOS+ T cells were clearly increased in the
control IgG-treated CIA mice (8.2 ± 1.0%) as compared with naive
mice (4.9 ± 0.3%; data not shown), but this increase was
significantly inhibited in the anti-B7h mAb-treated mice (5.3
± 0.3%; Fig. 4C). Consistent with a previous report
(15), a constitutive expression of B7h was observed on B
cells in naive mice and this expression on LN-B cells was not clearly
affected by the CII immunization and the anti-B7h mAb treatment
(data not shown). These results demonstrated a substantial expression
of ICOS and B7h in the local joints as well as in the draining LNs in
CIA mice.
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Since the inflammatory process in the synovium plays a major role
in the development of arthritis (29), we next examined the
change of proinflammatory cytokine expression in the synovium by the
treatment. Consistent with previous reports (29, 37), high
levels of TNF-, IL-1, and IL-6 mRNA were observed in the synovial
tissues from the control IgG-treated mice, but the treatment with
anti-B7h mAb significantly inhibited the expression of these
proinflammatory cytokines (Fig. 5). These
results suggested that the anti-B7h mAb treatment reduced the
arthritic manifestations by down-regulating the expression of
proinflammatory cytokines in the joints.
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The intervention of the ICOS-B7h costimulatory pathway by
anti-B7h mAb might modulate CII-specific T cell responses and might
affect the Th1/Th2 balance. To address these possibilities, splenocytes
and LN cells at day 5 were isolated from the CIA mice, and
proliferative responses and cytokine production against CII were
examined. Splenocytes from the control IgG-treated mice proliferated
well in response to dCII, whereas splenocytes from naive mice did not
proliferate even at a high concentration of dCII (Fig. 6Aa). Splenocytes from the
anti-B7h mAb-treated mice showed significantly reduced
proliferative responses to dCII as compared with those from the control
IgG-treated mice. A similar inhibitory effect of the anti-B7h mAb
treatment was observed when CD4+ splenic T cells
(Fig. 6Ab) or LN cells (Fig. 6Ac) were used as
responder cells. These results suggested that the anti-B7h mAb
treatment at the second immunization inhibited the expansion of
CII-specific T cells in the LN and spleen. We next examined the Th1 and
Th2 cytokine production. LN cells from the control IgG- or anti-B7h
mAb-treated mice were stimulated with 30 µg/ml dCII for 96 h,
and IFN-, IL-4, and IL-10 in the supernatants were measured by
ELISA. LN cells from the control IgG-treated mice produced high levels
of IFN- and IL-10 in response to CII, but the production of these
cytokines by LN cells was greatly reduced in the anti-B7h
mAb-treated mice (Fig. 6B). IL-4 production was undetectable
in both groups of mice in this culture condition (data not shown).
Inhibitory effect by the anti-B7h mAb treatment on IFN-
production was persistently observed even at 10 wk after the second
immunization (Fig. 6C). These results suggested that the
anti-B7h mAb treatment prevented the differentiation and/or
expansion of CII-specific Th1 and Th2 cells and prolonged the
inhibitory effect on Th1-mediated responses.
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It is well known that IgG2a production is mainly induced by the
Th1 cytokine IFN- and other IgG isotypes are regulated by Th2
cytokines such as IL-4, IL-5, IL-6, and IL-10 (38, 39). We
thus investigated the anti-CII IgG1, IgG2a, and IgG2b Ab levels in
the sera from the control IgG- or anti-B7h mAb-treated mice at 7,
14, and 21 days after the second immunization. The serum levels of
anti-CII IgG1, IgG2a, and IgG2b were dramatically increased in
response to the second immunization, but all of these responses were
significantly suppressed by the anti-B7h mAb treatment (Fig. 7). These results indicated that the
anti-B7h mAb treatment inhibited the production of anti-CII Abs
that is dependent on either Th1 or Th2 cells. Since the anti-CII
Abs have been implicated in the pathogenesis of CIA (28),
the reduced production of anti-CII Abs might also be responsible
for the ameliorating effect of the anti-B7h mAb treatment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Various costimulatory molecules, including CD28-CD80/CD86 (6), CD40-CD40 ligand (L) (40), and CD134-CD134L (41), have been implicated in the pathogenesis of CIA. Blockade of the CD28 costimulatory pathway inhibited the development of CIA in mice and rats (6, 42) and CD28-deficient mice were highly resistant to CIA (43), suggesting a critical involvement of CD28 in the induction of CIA. Unlike the constitutive expression of CD28 on naive T cells, ICOS is not expressed on naive T cells but induced after activation (7, 18, 27). In contrast, a considerable level of B7h is expressed on B cells without stimulation (12, 13, 15), whereas CD80 and CD86 are induced by stimulation. Consistent with these reports, we observed a constitutive expression of B7h on B cells in the LNs and the increased number of ICOS+ T cells in the LNs after immunization. In addition to these observations, we first demonstrated the expression of ICOS on T cells and B7h on B cells, macrophages, and dendritic cells in the inflamed joints at protein levels and the increased mRNA expression in the CIA mice. Although further studies are required for the detection of B7h expression on other types of cells in the synovium, our results suggest the possible involvement of ICOS-B7h interactions at the site of joints as well as in the draining LNs.
The actual involvement of the ICOS-B7h interaction in the pathogenesis of CIA was verified by the administration of neutralizing anti-B7h mAb. Blockade of the ICOS costimulatory pathway by anti-B7h mAb resulted in amelioration of inflammatory arthritis as assessed by clinical scoring and histological and radiological examinations. However, this treatment failed to decrease the incidence of disease, while similar treatment with CTLA-4Ig (6), anti-CD154 mAb (40), or anti-CD134L mAb (41) substantially decreased the incidence. Nevertheless, it should be noted that the delayed short-term treatment with anti-B7h mAb was also effective for preventing the progression of disease. In contrast, the treatment with anti-CD4 mAb (44), anti-CD134L mAb (41), or anti-CD80 and CD86 mAbs (6) was not or was less effective after the arthritis had been initiated. These results are consistent with the recent observations in EAE and allergic airway inflammation models, where the blockade of ICOS at the peak of disease, but not during Ag priming, dramatically ameliorated ongoing inflammatory responses (26, 27). Since ICOS is not expressed on naive T cells, it is reasonable that the blockade of ICOS was less effective for preventing the priming of T cells. In contrast, ICOS is expressed on activated T cells and thus the ICOS-B7h interaction may play its primary role in costimulation of already Ag-primed T cells. Consistent with this notion, the expansion of ICOS+ T cells in the draining LN upon second immunization was markedly inhibited by the anti-B7h mAb treatment. In addition, ICOS may also play a costimulatory role in the activation of ICOS+ effector T cells infiltrating in the target tissues as discussed above.
The amelioration of CIA by the anti-B7h mAb treatment appeared to
be correlated with the reduction of CII-specific T and B cell
responses. T cell proliferative responses to CII and both IFN- and
IL-10 production were significantly inhibited by the treatment. In
addition, a marked reduction in all IgG1, IgG2a, and IgG2b subclasses
of serum anti-CII Abs was observed. These results suggested that
both Th1- and Th2-mediated immune responses against CII were comparably
inhibited by the anti-B7h mAb treatment. It has been reported that
the blockade of the ICOS pathway by ICOS-Ig fusion protein or a
neutralizing anti-ICOS mAb exhibited prominent inhibitory effects
in the effector phase of the Th2-mediated immune responses (10, 27, 45). ICOS is preferentially expressed on Th2 cells at higher
levels than on Th1 cells (10, 18, 27) and the blockade of
ICOS in vitro preferentially reduced the production of Th2 cytokines
such as IL-4 and IL-10 (18). Several reports demonstrated
the failure to inhibit Th1-mediated immune responses by the ICOS
blockade (10, 45, 46). On the other hand, a successful
inhibition of Th1-mediated immune responses such as acute allograft
rejection (22) and EAE (23, 26) has been also
demonstrated. Our present results add a new example in which the
blockade of the ICOS-B7h interaction resulted in inhibition of both
Th1- and Th2-mediated immune responses.
In addition to the CII-specific immune responses mediated by T and B
cells, locally produced proinflammatory cytokines also play a critical
role in the development of arthritis. Among such cytokines, TNF-,
IL-1, and IL-6 are crucial and the intervention of their actions
could be a potential strategy for the treatment of arthritis (29, 47, 48, 49, 50). We here showed that the treatment with anti-B7h mAb
efficiently reduced the expression of these proinflammatory cytokines
in the inflamed joints. It has been reported that TNF- induced B7h
expression on fibroblasts and nonlymphoid cells (11, 12)
and that ICOS costimulation strongly enhanced TNF- production by T
cells (51). Therefore, the reciprocal induction of TNF-
and B7h through the ICOS pathway may amplify and perpetuate the local
inflammation. In the acute allograft rejection model, the ICOS blockade
suppressed the production of chemokines as well as IFN- and IL-10
(22). Thus, the ICOS-B7h interaction may be involved in
the regulation of multiple cytokines and chemokines that control local
inflammatory responses.
In conclusion, the blockade of ICOS costimulation by anti-B7h mAb ameliorated CIA through anti-inflammatory actions and suppression of both Th1- and Th2-mediated responses. Intervention of the ICOS costimulatory pathway may be a novel strategy for the treatment of human rheumatoid arthritis and possibly other chronic inflammatory diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Miyuki Azuma, Department of Molecular Immunology, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8549, Japan. E-mail address: miyuki.mim{at}tmd.ac.jp
3 Abbreviations used in this paper: EAE, experimental autoimmune encephalomyelitis; ICOS, inducible costimulator; B7h, B7 homologous protein; B7RP-1, B7-related protein 1; CIA, collagen-induced arthritis; CII, collagen type II; LN, lymph node; dCII, denatured CII.
Received for publication January 23, 2002. Accepted for publication August 12, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Immunity 11:423.[Medline]
and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236:944., anti-IL-1 /, and IL-1Ra. Arthritis Rheum. 39:797.[Medline]
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  N. Takahashi, K. Matsumoto, H. Saito, T. Nanki, N. Miyasaka, T. Kobata, M. Azuma, S.-K. Lee, S. Mizutani, and T. Morio Impaired CD4 and CD8 Effector Function and Decreased Memory T Cell Populations in ICOS-Deficient Patients J. Immunol., May 1, 2009; 182(9): 5515 - 5527. [Abstract] [Full Text] [PDF]
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J. M. Odegard, L. D. DiPlacido, L. Greenwald, M. Kashgarian, D. H. Kono, C. Dong, R. A. Flavell, and J. Craft ICOS Controls Effector Function but Not Trafficking Receptor Expression of Kidney-Infiltrating Effector T Cells in Murine Lupus J. Immunol., April 1, 2009; 182(7): 4076 - 4084. [Abstract] [Full Text] [PDF]
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Y.-L. Hu, D. P. Metz, J. Chung, G. Siu, and M. Zhang B7RP-1 Blockade Ameliorates Autoimmunity through Regulation of Follicular Helper T Cells J. Immunol., February 1, 2009; 182(3): 1421 - 1428. [Abstract] [Full Text] [PDF]
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Q. Chen, J. L. Cannons, J. C. Paton, H. Akiba, P. L. Schwartzberg, and C. M. Snapper A Novel ICOS-Independent, but CD28- and SAP-Dependent, Pathway of T Cell-Dependent, Polysaccharide-Specific Humoral Immunity in Response to Intact Streptococcus pneumoniae versus Pneumococcal Conjugate Vaccine J. Immunol., December 15, 2008; 181(12): 8258 - 8266. [Abstract] [Full Text] [PDF]
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O. Nagashima, N. Harada, Y. Usui, T. Yamazaki, H. Yagita, K. Okumura, K. Takahashi, and H. Akiba B7-H3 Contributes to the Development of Pathogenic Th2 Cells in a Murine Model of Asthma J. Immunol., September 15, 2008; 181(6): 4062 - 4071. [Abstract] [Full Text] [PDF]
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 B. Peng, P. Ye, B. R. Blazar, G. J. Freeman, D. J. Rawlings, H. D. Ochs, and C. H. Miao Transient blockade of the inducible costimulator pathway generates long-term tolerance to factor VIII after nonviral gene transfer into hemophilia A mice Blood, September 1, 2008; 112(5): 1662 - 1672. [Abstract] [Full Text] [PDF]
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M. Watanabe, Y. Takagi, M. Kotani, Y. Hara, A. Inamine, K. Hayashi, S. Ogawa, K. Takeda, K. Tanabe, and R. Abe Down-Regulation of ICOS Ligand by Interaction with ICOS Functions as a Regulatory Mechanism for Immune Responses J. Immunol., April 15, 2008; 180(8): 5222 - 5234. [Abstract] [Full Text] [PDF]
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 J. M. Rojo, E. Pini, G. Ojeda, R. Bello, C. Dong, R. A. Flavell, U. Dianzani, and P. Portoles CD4+ICOS+ T lymphocytes inhibit T cell activation 'in vitro' and attenuate autoimmune encephalitis 'in vivo' Int. Immunol., April 1, 2008; 20(4): 577 - 589. [Abstract] [Full Text] [PDF]
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D. Hawiger, E. Tran, W. Du, C. J. Booth, L. Wen, C. Dong, and R. A. Flavell ICOS Mediates the Development of Insulin-Dependent Diabetes Mellitus in Nonobese Diabetic Mice J. Immunol., March 1, 2008; 180(5): 3140 - 3147. [Abstract] [Full Text] [PDF]
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 A. H.-M. Tan, S. Y.-P. Goh, S.-C. Wong, and K.-P. Lam T Helper Cell-specific Regulation of Inducible Costimulator Expression via Distinct Mechanisms Mediated by T-bet and GATA-3 J. Biol. Chem., January 4, 2008; 283(1): 128 - 136. [Abstract] [Full Text] [PDF]
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S. K. O'Neill, Y. Cao, K. M. Hamel, P. D. Doodes, G. Hutas, and A. Finnegan Expression of CD80/86 on B Cells Is Essential for Autoreactive T Cell Activation and the Development of Arthritis J. Immunol., October 15, 2007; 179(8): 5109 - 5116. [Abstract] [Full Text] [PDF]
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Y. Katsumata, M. Harigai, T. Sugiura, M. Kawamoto, Y. Kawaguchi, Y. Matsumoto, K. Kohyama, M. Soejima, N. Kamatani, and M. Hara Attenuation of Experimental Autoimmune Myositis by Blocking ICOS-ICOS Ligand Interaction J. Immunol., September 15, 2007; 179(6): 3772 - 3779. [Abstract] [Full Text] [PDF]
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K. Kamata, S. Kamijo, A. Nakajima, A. Koyanagi, H. Kurosawa, H. Yagita, and K. Okumura Involvement of TNF-Like Weak Inducer of Apoptosis in the Pathogenesis of Collagen-Induced Arthritis J. Immunol., November 1, 2006; 177(9): 6433 - 6439. [Abstract] [Full Text] [PDF]
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 D. Odobasic, A. R. Kitching, T. J. Semple, and S. R. Holdsworth Inducible Co-Stimulatory Molecule Ligand Is Protective during the Induction and Effector Phases of Crescentic Glomerulonephritis J. Am. Soc. Nephrol., April 1, 2006; 17(4): 1044 - 1053. [Abstract] [Full Text] [PDF]
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V. M. Hubbard, J. M. Eng, T. Ramirez-Montagut, K. H. Tjoe, S. J. Muriglan, A. A. Kochman, T. H. Terwey, L. M. Willis, R. Schiro, G. Heller, et al. Absence of inducible costimulator on alloreactive T cells reduces graft versus host disease and induces Th2 deviation Blood, November 1, 2005; 106(9): 3285 - 3292. [Abstract] [Full Text] [PDF]
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H. Akiba, K. Takeda, Y. Kojima, Y. Usui, N. Harada, T. Yamazaki, J. Ma, K. Tezuka, H. Yagita, and K. Okumura The Role of ICOS in the CXCR5+ Follicular B Helper T Cell Maintenance In Vivo J. Immunol., August 15, 2005; 175(4): 2340 - 2348. [Abstract] [Full Text] [PDF]
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R. Tsukahara, M. Takeuchi, H. Akiba, T. Kezuka, K. Takeda, Y. Usui, M. Usui, H. Yagita, and K. Okumura Critical contribution of CD80 and CD86 to induction of anterior chamber-associated immune deviation Int. Immunol., May 1, 2005; 17(5): 523 - 530. [Abstract] [Full Text] [PDF]
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M. Watanabe, Y. Hara, K. Tanabe, H. Toma, and R. Abe A distinct role for ICOS-mediated co-stimulatory signaling in CD4+ and CD8+ T cell subsets Int. Immunol., March 1, 2005; 17(3): 269 - 278. [Abstract] [Full Text] [PDF]
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 M. Kohyama, D. Sugahara, S. Sugiyama, H. Yagita, K. Okumura, and N. Hozumi Inducible costimulator-dependent IL-10 production by regulatory T cells specific for self-antigen PNAS, March 23, 2004; 101(12): 4192 - 4197. [Abstract] [Full Text] [PDF]
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G. Demirci, F. Amanullah, R. Kewalaramani, H. Yagita, T. B. Strom, M. H. Sayegh, and X. C. Li Critical Role of OX40 in CD28 and CD154-Independent Rejection J. Immunol., February 1, 2004; 172(3): 1691 - 1698. [Abstract] [Full Text] [PDF]
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H. Iwai, M. Abe, S. Hirose, F. Tsushima, K. Tezuka, H. Akiba, H. Yagita, K. Okumura, H. Kohsaka, N. Miyasaka, et al. Involvement of Inducible Costimulator-B7 Homologous Protein Costimulatory Pathway in Murine Lupus Nephritis J. Immunol., September 15, 2003; 171(6): 2848 - 2854. [Abstract] [Full Text] [PDF]
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 K. Vermaelen and R. Pauwels Accelerated Airway Dendritic Cell Maturation, Trafficking, and Elimination in a Mouse Model of Asthma Am. J. Respir. Cell Mol. Biol., September 1, 2003; 29(3): 405 - 409. [Abstract] [Full Text] [PDF]
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 H. Futamatsu, J.-i. Suzuki, H. Kosuge, O. Yokoseki, M. Kamada, H. Ito, M. Inobe, M. Isobe, and T. Uede Attenuation of experimental autoimmune myocarditis by blocking activated T cells through inducible costimulatory molecule pathway Cardiovasc Res, July 1, 2003; 59(1): 95 - 104. [Abstract] [Full Text] [PDF]
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) or 50
(
), 100 (•), or 300 (
) µg of anti-B7h mAb on days -1, 1,
3, and 5. B, Two groups of mice were injected i.p. with
100 µg of control IgG (
) and the control IgG-treated CIA (
)-treated mice are shown. Similar results were
obtained in three independent experiments. *, Statistically different
from the control IgG-treated group (p <
0.05).
