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,1,1
* Dipartimento Clinico e Sperimentale di Medicina e Farmacologia, Torre Biologica, Policlinico Universitario, Messina, Italy; and
Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, Tossicologia e Chemioterapia, Università di Perugia, Perugia, Italy
2Correspondence: C. R., E-mail: riccardi{at}unipg.it and S.C., Dipartimento Clinico e Sperimentale di Medicina e Farmacologia, Torre Biologica, Policlinico Universitario, 98123 Messina, Italy. E-mail: salvator{at}unime.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, IL-6, TNF
, MIP-1, and MIP-2, inducible NOS (iNOS), COX-2, and nitrotyrosine poly-ADP-ribose (PAR) were also less in CII-treated GITR–/– mice. Although CD4+CD25+ Treg cells from GITR+/+ and GITR–/– CII-challenged mice exerted similar suppressor activity in vitro, GITR triggering abrogated GITR+/+ Treg suppressor activity and costimulated CD4+CD25– GITR+/+ effector cells. Furthermore, Treg cells from GITR–/– protected more than Treg cells from GITR+/+ mice against CIA when cotransferred with Treg-depleted splenocytes from arthritic GITR+/+ animals into severe combined immunodeficient (SCID) mice. In conclusion, GITR plays a critical role in the immunological response against CII and in the development of CIA.—Cuzzocrea, S., Ayroldi, E., Di Paola, R., Agostini, M., Mazzon, E., Bruscoli, S., Genovese, T., Ronchetti, S., Caputi, A. P., Riccardi, C. Role of glucocorticoid-induced TNF receptor family gene (GITR) in collagen-induced arthritis.
Key Words: rheumatoid arthritis • T lymphocyte activity • chemokines
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Moreover, abnormalities in several of these factors, such as TNF/TNFR, 4-1BB/4-1BBL, CD40L, and FasL, may contribute to a breakdown in immune tolerance and to the development of autoimmune/inflammatory diseases (1
2
3
4)
.
The most common autoimmune synovial joint disease worldwide is rheumatoid arthritis (RA) (5)
, which is characterized by acute and chronic joint inflammation, cell proliferation, apoptosis, tissue destruction/fibrosis, and positivity of several inflammation markers such as nitrotyrosine, poly-ADP-ribose (PAR), inducible NOS (iNOS), COX-2, and myeloperoxidase (MPO) (6
7
8
9)
. Despite much research, the pathogenesis of RA is not entirely clear; nevertheless, the OX40/OX40L interaction is reported to make a contribution (4)
and abnormalities in 4-1BB/4-1BBL, CD27, and CD40 function have been associated with increased severity of disease (3
, 4
, 10)
. Furthermore, TRAIL–/– mice are hypersensitive to collagen (CII) -induced arthritis (CIA) and, in a RA model, the CD30/CD30L interaction counters the Th1-driven disease, T lymphocyte infiltration, at the site of inflammatory response, IFN production, and tissue damage (11
12
13)
.
We had previously identified a 22 kDa protein belonging to the TNFR superfamily (TNFRSF) (14)
, which we termed GITR (glucocorticoid-induced TNFR family-related gene), and cloned it in the dexamethasone-treated 3DO T cell line. Like other members of the family, GITR contains repeats of cysteine-rich extracellular motif, a trans-membrane domain, and a cytoplasm tail. The cytoplasm domain in particular has similarities with the intracellular regions of other superfamily members such as 4-1BB, OX-40, CD27, and CD40, which, as coactivating molecules, are involved in regulating T lymphocyte activity (15
, 16)
. GITR is expressed at different levels in normal T lymphocytes, including single positive CD4+ and CD8+ thymocytes, spleen, and lymph node cells (17
, 18)
. Again, like all members of the TNFRSF, GITR is overexpressed upon T lymphocyte activation, suggesting it plays a role in the control of T cell activation and immune response development (15)
. GITR is constitutively expressed on CD4+CD25+ regulatory T (Treg) lymphocytes, which exert suppressor activity and are consequently involved in control of several autoimmune/inflammatory diseases (19
, 20)
. When activated, GITR functions as a coaccessory stimulus to the TCR/CD3 complex and increases activation of CD4+CD25– effectors, but down-regulates CD4+CD25+ Treg cell suppressor activity (17
18
19
20
21)
. In GITR-deficient mice (GITR–/–) we recently generated, TCR/CD3-induced activation of T lymphocytes is deregulated, suggesting GITR contributes to T lymphocyte regulation (22)
.
Many of the pathological cellular and humoral modifications in human RA are observed in the murine model of CIA (23)
. The autoimmune/inflammatory response involves autoreactive CII-specific CD4+ effector T cells and specific antibodies against CII (24
25
26)
. Activated CD4+CD25– effector T lymphocytes play a primary role in disease induction and contribute to increases in IFN, macrophage activation, IL-6, and TNF (27
28
29
30)
. T cells, which are often found in the infiltrate in the joint tissues, stimulate MIP-1, MIP-2, and cytokine release. At advanced stage disease, TNF is also detectable in the plasma, thus contributing to inflammatory reactions involving macrophages, neutrophils, and mast cells in the joints (31
32
33
34
35)
.
Although CD4+CD25+ Treg cells are reported to be involved in arthritis development, as they inhibit CII-induced disease (36
, 37)
, this finding has not been confirmed in some arthritis models such as proteoglycan-induced arthritis (38)
.
In the present study, we investigated the role of GITR in the pathogenesis and development of RA using the CIA model and observed that GITR–/– mice are less sensitive to disease induction than GITR+/+ mice. The mechanism underlying reduced susceptibility involves GITR modulation of Treg and effector T cell activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Severe combined immunodeficient (SCID) mice (C57BL/6J SCID, H-2b), 8–10 wk old, were purchased from Charles River (Calco, Milan, Italy). Animal care was in compliance with regulations in Italy (D.M. 116192), Europe (O.J. of E.C. L 358/1 12/18/1986), and USA (Animal Welfare Assurance No A5594-01, Department of Health and Human Services, USA).
Induction of CIA
Chicken CII (Sigma-Aldrich Company, Milan, Italy) was dissolved in 0.01 M acetic acid at a concentration of 2 mg/mL by overnight stirring at 4°C and frozen at –70°C until used. Complete Freund’s adjuvant (CFA) was prepared by adding Mycobacterium tuberculosis H37Ra at a concentration of 5 mg/mL to incomplete Freund’s adjuvant. (Sigma-Aldrich Company). Before injection, to induce CIA as described elsewhere (23)
, CII was emulsified with an equal volume of CFA. On day 1, mice were injected (primary injection) intradermally at the base of the tail with 100 µL of the emulsion (containing 100 µg of CII). On day 21, a second injection (booster injection) of CII in CFA was administered. All stock solutions were prepared in nonpyrogenic saline (0.9% NaCl; Baxter Healthcare Ltd., Thetford, Norfolk, UK).
Clinical assessment of CIA
Mice were evaluated daily for arthritis by using a macroscopic scoring system: 0 = no signs of arthritis; 1 = swelling and/or redness of the paw or one digit; 2 = two joints involved; 3 = more than two joints involved; 4 = severe arthritis of the entire paw and digits (39)
. The arthritic index for each mouse was calculated by adding the four scores of individual paws. Clinical severity was determined by quantifying the change in the paw volume using plethysmometry (model 7140; Ugo Basile, Comerio, Italy).
Radiography
Mice were anesthetized with sodium pentobarbital (45 mg/kg, i.p.) and placed on a radiographic box 90 cm from the X-ray source. Radiographic analysis of normal and arthritic mouse hind paws (Philips X12, Germany) with a 40 kW exposition for 0.01 s was performed by a blinded investigator. The following radiographical criteria were considered: score 0, no bone damage; score 1, tissue swelling and edema; score 2 joint erosion; 3, bone erosion and osteophyte formation.
Histology
On day +35 animals were killed under anesthesia, and paws and knees were removed and fixed for histological examination, done by a blind investigator. The following morphological criteria were considered: score 0, no damage; score 1, edema; score 2, inflammatory cell presence; score 3, bone erosion. For microscopic histological evaluation, both hind paws and knees were removed and fixed in 10% formalin. The paws were then trimmed, placed in decalcifying solution for 24 h, embedded in paraffin, sectioned at 5 µm, stained with hematoxylin/eosin, and studied using light microscopy (Dialux 22 Leitz).
TUNEL assay
TUNEL assay was conducted according to the manufacturer’s instructions (Apotag, HRP kit DBA, Milan, Italy). Briefly, sections were incubated with 15 µg/mL proteinase K for 15 min at room temperature, then washed with PBS. Endogenous peroxidase was inactivated by 3% H2O2 for 5 min at room temperature and washed with PBS. Sections were immersed in terminal deoxynucleotidyltransferase (TdT) buffer containing TdT and biotinylated dUTP, incubated in a humid atmosphere at 37°C for 90 min, then washed with PBS. The sections were incubated at room temperature for 30 min with anti-FITC horseradish peroxidase (HRP) -conjugated antibody and the signals were visualized with diaminobenzidine. The number of TUNEL-positive cells/high-power field was counted in 5 to 10 fields for each coded slide, as described previously (40)
.
Western blot analysis for iNOS and COX-2
iNOS and COX-2 expression was detected, as described (40)
, in joint extracts by Western blot analysis. At a specified time, joint tissues from left hind paws were collected and frozen until use. The frozen joint tissues were pulverized, and protein was extracted using RIPA buffer (50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate). Protein concentration was estimated by the DC Protein Assay kit (Bio-Rad, Milan, Italy). Equal amounts of protein samples (80 µg/lane) from pooled joint extracts were fractionated by 8% sodium dodecyl sulfate-PAGE and transferred onto a nitrocellulose membrane in transfer buffer (39 mmol/L glycine, 48 mmol/L Tris base, 0.037% sodium dodecyl sulfate, 20% methanol) at 100 mA for 2 h. The membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 and 5% dry milk for 1 h at room temperature, and subsequently probed overnight at 4°C with mouse monoclonal anti-iNOS (1:10.000) or anti-COX-2 (1:500) Abs (in PBS, 5% w/v nonfat milk and 0.1% Tween-20). Anti-ß-tubulin mAb (Calbiochem, San Diego, CA, USA) was used as control. Immunoreactive protein bands were visualized using HRP-conjugated goat anti-mouse IgG (1:5000) followed by enhanced chemioluminescence. The protein bands of iNOS and COX2 on X Omat films were quantified by scanning densitometry (Imaging Densitometer GS-700 BIO-RAD U.S.A.).
Immunohistochemical localization of iNOS, nitrotyrosine, PAR, and COX-2
Tyrosine nitration was determined by immunohistochemistry as described (41)
. After sacrifice, at day +35, murine joints were trimmed, placed in decalcifying solution for 24 h, and 8 µm sections were prepared from paraffin embedded tissues. After deparaffinization, endogenous peroxidase was quenched with 0.3% H2O2 in 60% methanol for 30 min. Sections were permeabilized with 0.1% Triton X-100 in PBS for 20 min. Nonspecific adsorption was minimized by incubating sections in 2% normal goat serum in PBS for 20 min. Endogenous biotin or avidin binding sites were blocked by sequential incubation for 15 min with avidin and biotin. Sections were then incubated overnight with anti-iNOS (1:500; Santa Cruz; DBA, Milan, Italy), anti-nitrotyrosine (1:1000; Upstate, DBA, Milan Italy), anti-COX-2 (1:500 Santa Cruz; DBA, Milan, Italy), anti-PAR antibody (1:500; Alexis, Milan Italy) Abs, or control solutions. Controls included buffer alone or nonspecific purified rabbit IgG. Specific labeling was detected with a biotin-conjugated goat anti-rabbit IgG (for nitrotyrosine) or a biotin-conjugated goat anti-mouse IgG (for PAR and for COX-2) and avidin-biotin peroxidase complex (Vector, DBA, Milan, Italy).
Chemokine assessment
Murine chemokines (MIP-1 and MIP-2) were evaluated in the aqueous joint extracts. Briefly, after removing the skin and separating the limb from the foot below the ankle joint, joint tissues were prepared and homogenized on ice in 3 mL lysis buffer (PBS containing 2 mM PMSF and 1 mg/mL of aprotinin, antipain, leupeptin, and pepstatin A) using Polytron (Brinkinarm Instruments, Westbury, NY, USA). Homogenized tissues were then centrifuged at 2000 g for 10 min. Supernatants were sterilized with a 0.2 µm millipore filter and stored at –80°C until analyzed. The protein content was measured by a protein assay kit (Pierce Chemical Co., Rockford, IL, USA). MIP-1 and MIP-2 were quantified using a modified a double ligand method as described elsewhere (42)
. Briefly, flat-bottomed 96-well microtiter plates were coated with 50 µL/well rabbit anti-chemokine antibodies (1 µg/mL in 0.6 mol/L NaCl, 0.26 mol/L H3BO4, and 0.08 N NaOH, pH 9.6) for 16 h at 4°C, then washed with PBS, pH 7.5, 0.05% Tween 20 (wash buffer). Nonspecific binding sites on microtiter plates were blocked with 2% BSA in PBS and incubated for 90 min at 37°C. Plates were rinsed four times with wash buffer. Diluted aqueous joint samples (50 µL) were added and incubated for 1 h at 37°C. After washing plates, a chromogen substrate was added before incubation at room temperature. The reaction was terminated with 50 µL/well of 3M H2SO4 solution and the colorimetric reaction was read at 490 nm in an ELISA reader. This ELISA method had a consistent sensitivity limit of 
30 pg/mL.
Serum anti-CII antibody determination
The serum antibodies to CII were quantitated by ELISA using biotin-labeled goat anti-mouse IgG (Southern Biotechnology Associates, Inc., Birmingham, AL, USA) according to the method of Watson et al. (43)
. Serum was prepared from the blood of control and arthritic mice 35 days post-CII immunization.
Plasma TNF
TNF levels were evaluated in the plasma from CIA mice as described previously (41)
. The assay was carried out using a colorimetric commercial ELISA kit (Calbiochem-Novabiochem Corporation, Milan, Italy) with a lower detection limit of 10 pg/mL.
MPO assay
Neutrophil infiltration to the inflamed joints was indirectly quantified using an MPO assay, as described for neutrophil elicitation (42)
. Tissue was prepared as described above and placed in a 50 mM phosphate buffer (pH = 6.0) with 5% hexadecyltrimethyl ammonium bromide (Sigma Chemical Co.). Joint tissues were homogenized, sonicated, and centrifuged at 12,000 g for 15 min at 4°C. Supernatants were assayed for MPO activity using a spectrophotometric reaction with O-dianisidine hydrochloride (Sigma Chemical Co.) at 460 nm.
In vitro cytokine production by draining regional lymph nodes
To study regional lymph nodes response to CII, GITR+/+ and GITR–/– mice were immunized at footpad with 150 µg of CII emulsified with an equal volume of complete CFA. Five days after immunization, regional (poplyteal plus inguinal) lymph nodes were removed and cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS, streptomycin (100 µg/mL), 10 mM HEPES, 0.1% nonessential amino acids, 1 mM sodium pyruvate, and 50 µM 2-ME. Cells (1.25x106/well) were plated in vitro on a layer of irradiated splenocytes (5x106/mL) and cultured in the presence or absence of 100 µg/mL CII for 3 days. The supernatants were used for ELISA assay of IFN-, IL-6, IL-10, as described (17)
. The antibodies were respectively purified rat anti-mouse IL-6 mAb (capture antibody) or biotin rat anti-mouse IL-6 mAb (detection antibody), purified rat anti-mouse IL-10 mAb or biotin rat anti-mouse IL-10 mAb, and rat anti-mouse IFN- mAb or biotin rat anti-mouse IFN- mAb. All antibodies were purchased from PharMingen (San Diego, CA, USA).
CD4+CD25+ cell purification
CD4+CD25+ cells were purified from lymph nodes using CD4+CD25+ T cell isolation kit (Miltenyi Biotec, Bologna, Italy) following the manufacturer’s instructions. Briefly, after isolating CD4+ cells, the CD25+ PE-labeled cells were magnetically labeled with anti-PE microbeads as described elsewhere (17)
. The cell suspension was loaded onto a column, which was placed in the magnetic field of a MACS Separator. The flow-through cells were CD4+CD25– cells, while the retained cells eluted from the column were CD4+CD25+ cells. To achieve highest purities, two consecutive column runs were performed. Flow cytometry showed that >98% of magnetically retained cells were CD25+ and >98% of flow-through cells were CD25–.
Suppressor assay
CD4+CD25– and CD4+CD25+ cells were cocultured for 72 h, as described (17
, 20)
, by adding 5 x 104 cells/well for each subpopulation (ratio 1:1) or 5 x 104 effector and 2.5 x 104 Treg cells (ratio 2:1) on a layer of irradiated splenocytes (5x104 cells/well). Soluble anti-CD3, 0.5 µg/mL (PharMingen), and/or anti-GITR, 2 µg/mL (R&D System, Minneapolis, MN, USA) Abs were added. Proliferation was evaluated by thymidine incorporation. 2.5 µCi [3H] thymidine (Amersham International, Amersham, UK) per well were added to the cultures 10 h before harvesting with a multiple suction-filtration apparatus (Mash II) on a fiberglass filter (Whittaker Co.) Radioactivity was count in a ß-counter (Packard). The supernatants were used for ELISA assay of IFN- and IL-2, as described (17)
.
RT-PCR
Total RNA was extracted using TRIzol (Invitrogen). RNA samples were treated with RNase-free DNase I (Invitrogen) and total RNA was reverse transcribed using Superscript III reverse transcriptase and oligo(dT) primer (Invitrogen) in a final volume of 20 µL.
The PCR was performed using the follows primer sequences: Foxp3: 5'-CAGCTGCCTACAGTG CCCCTAG-3' and 5'-CATTTGCCAGCAGTGGGTAG-3'; HPRT: 5'-GTTGGATACAGGCC AGACTTTGTTG-3' and 5'-GAAGGGTAGGCTGGCCTATAGGCT-3'; IFN-: 5'-TACTGCCAC GGCACAGTCATTGAA-3' and 5'-GCAGCGACTCCTTTTCCGCTTCCTT-3'; IL-2: 5'-GTCACA GCGCACCCACTTCAAG-3' and 5'-GCTTGTTGAGATGATGCTTTGACA-3'; ß-actin: 5'-GTGGGCCGCTCTAGGCACCAA-3' and 5'-CTCTTTGATGTCACGCACGATTTC-3'.
Bands were quantified using KODAK 1D Image Analysis Software.
Flow cytometry
Harvested cells were spun down and washed with PBS before addition of Abs. Either anti-GITR or control isotype Ab was added undiluted to the cells (10 µL/sample) and kept at +4°C for 30’. The secondary anti-goat FITC-conjugated Ab (Sigma, St. Louis, MO, USA) was added diluted 1:100 (10 µL/sample) and kept at +4°C for 30’. Flow cytometric analysis was conducted on a Becton Dickinson FACScan running LYSIS II software.
Transfer of arthritis into SCID mice
Spleen cells (1x107) and spleen cells depleted of CD25+ cells (1x107) were isolated on day +35 from GITR+/+ and GITR–/– arthritic mice and injected intraperitoneally into SCID mice.
Spleen cells were depleted of CD25+ by negative selection. Briefly, spleen cells were incubated with PE-labeled anti-CD25 and the stained cells were incubated with anti-PE microbeads (Miltenyi Biotech). CD25+ depletion was controlled by flow cytometry (<0.1% of recovered cells expressed CD25).
The CD4+CD25+ Treg cells, purified from nonarthritic mice as described above, were injected intraperitoneally (5x105) along with CD25+-depleted spleen cells (1x107). All donor cells were injected together with 100 µg CII.
Data analysis
Data are expressed as mean ± SE of n observations (i.e., the number of animals for the in vivo studies). In the experiments involving histology or immunohistochemistry, the figures shown are representative of at least three experiments. Data sets were examined by 1- or 2-way ANOVA, and individual group means were then compared with Student’s unpaired t test. For the arthritis studies, the Mann-Whitney U test (2-tailed, independent) was used to compare medians of the arthritic indices (42)
. P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. To assess the role of GITR in CIA development, we characterized the disease in GITR+/+ and GITR–/– Sv129 mice by evaluating several inflammatory parameters that are typical of CIA. CIA developed rapidly in Sv129 mice immunized with CII plus adjuvant with an inflammatory response similar to that of DBA1 mice. In GITR+/+ mice, clinical signs (periarticular erythema and edema) first appeared in the hind paws 24 to 26 days postchallenge (Fig. 1
A) with a 100% incidence at day +30. Hind paw erythema and swelling increased in frequency and severity in a time-dependent mode with maximum arthritis indices of 8 being observed 29 to 35 days after primary immunization (Fig. 1B
). GITR–/– mice had significantly less joint inflammation, as shown by a significantly lower incidence of arthritis (P<0.01) and lower arthritis indices over time, than wild-type GITR+/+ mice (Fig. 1A, B
).
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No macroscopic evidence of either hind paw erythema or edema in the sham groups of GITR+/+ or GITR–/– mice (adjuvant alone-treated controls) was detected (data not shown).
Figure 1C
illustrates the time-dependent increase in hind paw volume (each value is the mean of both hind paws). In GITR–/– mice paw edema was significantly less (P<0.01) than in GITR+/+ mice starting from day +28 (Fig. 1C
). No increase in hind paw volume over time was observed in the sham mice (data not shown).
The rate and the absolute gain in body weight were similar in CII-immunized GITR–/– and GITR+/+ mice for the first 3 wk. From day +26 to day +35, GITR+/+ mice gained significantly (P<0.01) less weight than GITR–/– mice (Fig. 1D
). Weight gain of sham control mice was similar to that of CII-challenged GITR–/– mice (data not shown).
On day +35, histological examination of GITR+/+ paws revealed signs of severe arthritis, with bone erosion and severe or moderate necrosis (Fig. 2
Aa, c). Bone erosion and necrosis were significantly less in joints from GITR–/– mice (Fig. 2A
b, d). No evidence of disease was observed in the sham groups (Fig. 2A
e shows the GITR+/+ sham control). Radiography of hind paws confirmed bone erosion in GITR+/+ mice (Fig. 2B
a), with less being observed in GITR–/– mice (Fig. 2B
b) and no evidence of disease in the sham mice (Fig. 2B
c shows GITR+/+ sham control). Significant differences emerged in histological and radiography scores in sham controls and CII-treated mice and between CII-treated GITR+/+ and CII-treated GITR–/– mice (P<0.01).
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Chondrocyte apoptosis, a measure of joint tissue damage characteristic of CIA (5)
, was observed in the joints of GITR+/+ mice (TUNEL+ cells were 2.01±0.23/field, Fig. 2C
a). No signs of chondrocyte apoptosis were found GITR–/– mice (TUNEL+ cells were 0.50±0.1/field, Fig. 2C
b). No apoptosis was observed in the joints of sham controls (data not shown).
These results indicate that CII-treated Sv129 GITR–/– mice are less susceptible to CIA-induced tissue damage than GITR+/+ mice.
Less chemokine expression and neutrophil infiltration in CII-treated GITR–/– mice
In CIA, as the histological pattern of joint pathology correlates with the influx of leukocytes into the joint, the joint space, and the surrounding tissue (5)
, we assessed the role of GITR in chemokine expression. Figure 3
A, B shows the MIP-1 and MIP-2 expression patterns, as assessed by ELISA, correlated with the development of arthritis. MIP-l and MIP-2 were significantly (P<0.01) increased at day +35 in joints from GITR+/+ mice (Fig. 3A, B
). MIP-1 and MIP-2 levels were increased in GITR–/– mice at day +35, compared with sham controls, but were significantly lower (P<0.01) than in GITR+/+ mice (Fig. 3A, B
).
|
Neutrophil infiltration into inflamed joint tissue was assessed by measuring MPO activity, which directly correlates with neutrophil count. MPO activity was markedly increased, on day +35, in CII-immunized GITR+/+ mice compared with sham controls (Fig. 3C
). In GITR–/– mice, MPO activity was greater than in the sham group but significantly less (P<0.01) than in the GITR+/+ animals (Fig. 3C
).
Augmented TNF production has been described in CIA and plasma levels are detected in animals with active disease (34
, 35)
. To test whether GITR modulates the inflammatory process by regulating proinflammatory cytokine secretion, we analyzed plasma levels of TNF in GITR–/– and GITR+/+ mice. Substantially more TNF production was found in GITR+/+ mice on day +35 than in the sham group (Fig. 3D
). TNF levels were significantly (P<0.01) lower in GITR–/– than in GITR+/+ mice (Fig. 3D
).
Serum anti-CII specific antibody titers were significantly increased in GITR+/+ and GITR–/– mice on day +35 days compared with sham controls, which had negligible anti-CII antibody titers. Anti-CII Ab titers increased significantly less in GITR–/– mice (P<0.01, vs. GITR+/+) (Fig. 3E
). These results indicate that in GITR–/– mice, lower chemokine and anti-CII specific Ab levels are associated with less neutrophil infiltration than in GITR+/+ mice.
Less nitrotyrosine formation, PAR activation, iNOS and COX-2 expression in CII-treated GITR–/– mice
Release of free radicals and oxidant molecules during chronic inflammation may contribute significantly to tissue injury (7)
. On day +35, positive staining for nitrotyrosine, a marker of nitrosative injury, was found in the joints of GITR+/+ mice (Fig. 4
Aa). Less nitrotyrosine formed in GITR–/– mice (Fig. 4A
b), indicating less severe oxidant-induced damage. Immunohistochemical analysis of joint sections from GITR+/+ mice revealed positive staining for PAR (Fig. 4A
c). PAR positivity was less in the joints of CII-treated GITR–/– mice (Fig. 4A
d). No staining for nitrotyrosine or PAR was observed in joints of the sham controls (Fig. 4A
e, f show the GITR+/+ sham control).
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iNOS and COX-2, two enzymes involved in NO and PGs production, have been implicated in inflammatory processes, including RA (44)
. Immunohistochemical analysis of joint sections from GITR+/+ mice treated with CII revealed positive staining for iNOS (Fig. 4B
a) and COX-2 (Fig. 4B
c). Less positive staining for iNOS (Fig. 4B
b) and COX-2 (Fig. 4B
d) was found in the joints of GITR–/– mice. There was no staining for iNOS or COX-2 in sham controls (Fig. 4B
e, f show the GITR+/+ sham control). In addition, Western blot analysis of joint extracts revealed less COX-2 and iNOS expression in CII-treated GITR–/– mice than CII-treated GITR+/+ (Fig. 4C, D
). These results indicate that CIA is induced in Sv129 mice and that GITR contributes to the production of inflammatory molecules.
Less in vitro cytokine production by draining regional lymph nodes of GITR–/– mice
Many experimental models have reported activation of effector CD4+ T lymphocytes and production of IFN and IL-6 in the early stages of CIA (24
, 28
29
30)
. We evaluated IFN and IL-6 production in CII-challenged GITR+/+ and GITR–/– mice by assessing in vitro production by draining regional lymph nodes after CII restimulation (see Materials and Methods). Figure 5
shows that although in vitro restimulation with CII increased IFN and IL-6 production in GITR+/+ and GITR–/– lymph node cells (Fig. 5A, B
: column 4 vs. column 3; column 8 vs. column 7, respectively), significantly (P<0.01) lower amounts of IFN (Fig. 5A
: column 8 vs. column 4) and IL-6 (Fig. 5B
: column 8 vs. column 4) were observed in GITR–/– mice. No significant differences in IL-10 production were detected (Fig. 5C
). These results confirm that CII significantly increases Th-1-like cytokine production in lymph node cells (27)
, with significantly lower levels being induced in GITR–/– mice.
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Reduced susceptibility to CIA in GITR–/– mice is due to GITR modulation of effector and Treg cell functions
CD4+CD25+ Treg cells play a role in CIA because they act as suppressors of CD4+CD25– effector T lymphocytes (36
, 37)
. GITR triggering, in vivo and in vitro, reverses Treg cell inhibitory activity (19
, 20)
.
To assess the involvement of Treg cells in the reduced susceptibility of GITR–/– mice to CIA, we first examined the number and the in vitro function of Treg cells from CII-challenged GITR+/+ and GITR–/–. For this purpose, purified CD4+CD25+cells (purity >98%), obtained from lymph nodes of GITR+/+ and GITR–/– CII-treated or sham control mice were counted and analyzed by PCR for Foxp3 expression, as marker of Treg cells, on days +5, +24, and +35. No significant differences in cell counts and Foxp3 expression were found at any time point for as long as they were monitored (Fig. 6
A, B shows day +24 as a representative example). No differences emerged in Foxp3 expression, on total CD4+ populations, in GITR+/+ and GITR–/– groups (not shown).
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To analyze Treg function with no GITR interference on effector cells, CD4+CD25+ Treg cells from sham and CII-treated mice were cocultured with untreated GITR–/– CD4+CD25– effectors in a suppression assay, as described elsewhere (17
, 19)
. 72 h after stimulation with anti-CD3 Abs, proliferation, IFN, and IL2 mRNA production were evaluated. Figure 6C
shows suppressor activity overlapped in Treg cells from sham controls, CII-treated GITR+/+ and GITR–/– mice as measured as 3H-thimidyne uptake. The anti-GITR Ab abrogated Treg suppressor activity from CII-stimulated GITR+/+ mice but not from CII-stimulated GITR–/– mice (Fig. 6C
). Suppression of cell proliferation occurs at effector/Treg ratio 1:1 and 2:1. When the suppression assay was performed using effector cells from GITR+/+ mice, the anti-GITR Ab not only suppressed GITR+/+ Treg cell activity but costimulated GITR+/+ effector cells (not shown). These results suggest that although GITR expression is not essential for Treg activity in the development of CIA, GITR triggering abrogates Treg activity (17
, 19
, 20)
and increases CD4+CD25– effector cell proliferation.
Using CD4+CD25– effector cells from GITR+/+ untreated mice, suppression was evaluated as IFN and IL-2 mRNA expression. Treg cells GITR+/+ and GITR–/– from CII-treated mice suppressed IL-2 and IFN production to the same extent (Fig. 6D
). Similar results were obtained when IL-2 and IFN were measured in the supernatants of cell cultures by ELISA assays (Fig. 6E
). Suppression of IL-2 and IFN production was detectable at 1:1 effector/Treg ratio in RT-PCR and at effector/Treg ratio 1:1 and 2:1 in ELISA assay.
Next, we compared the INF production of total or CD25+-depleted CD4+ cells from lymph nodes of CII-challenged GITR+/+ and GITR–/– mice 24 days after immunization. Figure 7
A indicates that, upon in vitro CII restimulation, GITR–/– CD4+ cells produce less INF than GITR+/+ CD4+ cells and that CD25+ depletion increased IFN production in both groups, with a greater increase in GITR–/– (Fig. 7A
). Ratios (CD4+ vs. CD25+-depleted CD4+) in IFN production are 3.2 in nonstimulated GITR+/+ cells (column 5 vs. column 1) and 7 in nonstimulated GITR–/– cells (column 6 vs. column 2) (P<0.01); 1.7 in CII restimulated GITR+/+ (column 7 vs. column 3) and 2.5 in CII restimulated GITR–/– (column 8 vs. column 4) (P<0.05).
|
GITR triggering provided costimulatory signals to CD4+CD25– responder T cells by increasing anti-CD3-induced IFN production (17)
. We measured IFN production by effector CD4+ T cells from lymph nodes from CII-treated GITR+/+ and GITR–/– mice after in vitro restimulation with CII and/or anti-GITR Ab. Figure 7A
shows anti-GITR Ab increased the CII-induced IFN production in GITR+/+ (column 7 vs. column 9) but not in GITR–/– cells. Moreover CII induced GITR up-regulation on CD4+CD25– effector cells and on CD4+CD25+ Treg cells of GITR+/+ mice (Fig. 7B
).
Transfer of arthritis into SCID mice
Spleen cells from GITR–/– or GITR+/+ arthritic mice, with and without CD25+ T cell depletion, were injected intraperitoneally into SCID mice together with 100 µg CII. Other groups received GITR+/+ CD25+-depleted arthritogenic spleen cells plus Treg cells from GITR–/– or GITR+/+ untreated mice. Fourteen days after transfer, hind paws were histologically scored. GITR–/– spleen cells transferred CIA into SCID mice significantly less effectively than GITR+/+ spleen cells (Fig. 8
, a vs. b). Paws of SCID mice receiving GITR+/+ spleen cells revealed signs of severe arthritis, with bone erosion and inflammatory infiltration. Bone erosion and necrosis were significantly less in joints of SCID mice receiving arthritogenic GITR–/– spleen cells (Fig. 8)
. Injection of CD25+-depleted, GITR+/+ and GITR–/– arthritogenic spleen cells was associated with more severe disease but mice receiving CD25+-depleted GITR–/– developed a less severe paw lesion (Fig. 8
, c vs. d). The ratio between the arthritic scores of SCID mice receiving GITR–/– CD25+-depleted spleen cells and mice receiving GITR–/– spleen cells (Fig. 8
, column d vs. column b) is 2.7 vs. the 1.5, which emerged as the ratio between the arthritic scores of SCID mice receiving GITR+/+ CD25+-depleted spleen cells and mice receiving GITR+/+ spleen cells (Fig. 8
, column c vs. column a). Furthermore, CD4+CD25+ Treg cells from GITR–/– mice more effectively prevented CIA, induced by arthritogen GITR+/+ CD25+-depleted spleen cells, than those from GITR+/+ mice (Fig. 8
, compare e with f). Altogether, these data indicate that GITR triggering suppresses Treg cell activity and delivers costimulatory stimuli to effector T cells, which may contribute to the development of the immune response against CII.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the pathogenesis of RA is still not entirely clear, disease progression is characterized by inflammatory cell infiltrate into the granuloma-like pannus and the joint fluid, cartilage destruction, and bone erosion. Activated macrophages produce cytokines, including IL-6 and TNF, which in turn stimulate synovial cell proliferation, pannus formation, chondrocyte apoptosis, bone degradation, and joint destruction (5
, 28
, 31
32
33
34
35)
. The active inflammatory stage of RA has several histological features in common with chronic inflammation, including the organized focal accumulation of mononuclear cells in the developing pannus, proliferation of fibroblast-like synovial cells, and injury to the surrounding tissue. While synovial cell proliferation and leukocyte infiltration are fundamental in the ongoing process of joint inflammation, identifying the mediators that trigger and maintain the pathological cascade is arduous (5)
. Well-characterized experimental animal models have been used to assess these cellular and molecular events; murine CIA, for example, possesses many of the cellular and humoral modifications found in human RA (23
, 45)
.
Costimulatory molecules have recently been reported to be involved in T lymphocyte activation and CIA induction (46
, 47)
; supporting this hypothesis, we found that GITR, a T cell costimulatory molecule, played a role in the onset of CIA because GITR–/– mice were less susceptible to experimental CIA than GITR+/+ mice.
Chemotactic factors and lymphocyte, neutrophil, and macrophage recruitment into joint tissue are hallmarks of CIA and human RA. MIP-1 and MIP-2 have been found in tissue from the inflamed joints of human RA (32
, 33)
, and the present study confirms they are expressed at sites of inflamed joints in CIA. MIP-1 and MIP-2 levels were significantly lower in GITR–/– than in GITR+/+ mice, and the little neutrophil infiltration found in GITR–/– mice correlated well with less joint tissue damage as shown by histological and radiographic findings and chondrocyte apoptosis.
Other inflammatory mediators are known to participate in RA: iNOS levels increase significantly (48)
and COX-2-deficient mice are less susceptible to CIA (49)
. At the site of inflammation, the production of oxygen free radical scavengers such as NO, hydrogen peroxide, superoxide and hydroxyl radicals contribute to tissue damage. As inhibitors of NOS activity reduce the development of arthritis, NO appears to be implicated in the pathophysiology of CIA (50)
. Nitrotyrosine is also generated in CIA and contributes to cellular injury and cell death via several mechanisms, including peroxidation of membrane lipids, protein denaturation and DNA damage. Furthermore, recent evidence suggests that PAR polymerase (PARP) activation plays a major role in arthritis (51)
.
This study provides evidence of less COX-2 and iNOS expression in the joint tissue of GITR–/– than in GITR+/+ mice, with significantly less nitrotyrosine and PARP expression, suggesting the GITR-related modulation of the inflammatory process is due, at least in part, to inhibition of PARP activation. Furthermore, our data provide the first evidence that GITR may regulate the oxidative stress and the PARS pathway, which is activated during experimental arthritis.
T lymphocyte activation and antibody responses against CII immunization are crucial events in the immune response and CIA development, and although the roles of B cells and Ab production are clear (26)
, the part played by T lymphocytes is still controversial. Furthermore, cytokines such as IFN- (51)
, IL-6 (52)
, and TNF (34
, 35)
are claimed to possess a pathogenetic and prognostic value. Increased IFN, for example, has been observed in the early/inductive phase of disease, a few days after CII injection (30)
, and treatments inhibiting IFN are reported to have therapeutic potential (53)
. Similarly, a significant increase in IL-6 production has been found at the onset of disease in CII-treated animals (28
, 52)
.
Our results indicate decreased in vitro IFN and IL-6 production by CII-stimulated T lymphocytes from GITR–/– mice, suggesting major T cell activation in the GITR+/+ mice. As no significant changes were detected in IL-10 production, Th1 activity appears increased without a concomitant shift toward Th2 (27)
. Moreover, significantly lower plasma TNF concentrations were observed in GITR–/– mice. All this evidence implicates GITR in the triggering of CIA.
Injection of CII-stimulated CD4+ cells into SCID mice transfers CIA (24)
, and the early activation of effector CD4+ T cell populations and cytokine production is followed by an inflammatory cascade resulting in tissue damage and disease. Recently, Treg cells have been implicated in an inhibitory setting in several autoimmune/inflammatory diseases, including CIA (36
, 37)
. GITR effects on Treg and effector T cells are aimed at increasing the T cell response. Stimulation by GITRL or cross-linked anti-GITR Abs increases anti-CD3-driven CD4+CD25– effector T lymphocyte activation and IFN production (17
, 18)
. On the other hand, GITR is constitutively expressed on Treg cells and its stimulation abrogates their suppressive activity (19
, 20)
. The balance between effector and Treg cell activities is one determinant of the final outcome of immune response, so that upsetting this balance could result in a breakdown of immunological tolerance and autoimmune response.
In this study, we established the relative effects of GITR triggering on CD4+CD25– effector cells and on CD4+CD25+ Treg cells in the development of CIA. Although depletion of CD4+CD25+ from CD4+ cells of lymph nodes increased CII-induced IFN production in GITR+/+ and GITR–/– cells, the increase is greater in GITR–/– cells, suggesting major suppressor activity by GITR–/– CD4+CD25+ cells. Adding CD4+CD25+ cells from GITR+/+ and GITR–/– CII-challenged mice to CD4+CD25– effector cells inhibited proliferation and IFN production to the same extent, but triggering GITR by an anti-GITR Ab resulted in abrogation of GITR+/+ Treg cell activity with increased effector cell proliferation, suggesting that GITR needs to be triggered to deliver inhibitory signals to the Treg population.
Several hypotheses could feasibly account for the minor IFN response in the absence of GITR triggering after depletion of CD4+CD25+ from CD4+ GITR+/+ cell population. It is worth noting that adding Treg cells to effectors gives a high, perhaps nonphysiological, Treg/effector cell ratio, while removal of CD25+ may more appropriately reveal the actual Treg cell suppression activity. It could also be speculated that GITR–/– Treg cells display a more efficient Treg activity because in vivo chronic engagement of GITR by its ligand provides a continuous brake to Treg activity in GITR+/+ mice. Furthermore, continuous GITR triggering could render CD4+CD25+ effectors less susceptible to Treg-mediated suppressor activity, as previously suggested (54)
. Alternatively, the recently identified CD25– GITR+ CD4+ population with suppressor activity (55)
may exert a residual suppressor activity in the CD4+CD25+-depleted population of GITR+/+ mice.
In addition, the present results demonstrate that GITR stimulation increases CII-driven IFN production, confirming earlier reports that GITR triggering costimulates CD4+CD25– effector cells (17
, 18)
. Therefore, the protective effect of GITR deletion on the CIA experimental model may be sustained by GITR’s double function: costimulation of effector cells and inhibition of suppressive activity of Treg cells. The in vivo transfer experiments in SCID mice supports this hypothesis. Mice receiving GITR–/– arthritogen spleen cells developed less severe paw lesions than SCID mice receiving GITR+/+ arthritogen spleen cells. Depletion of CD25+ Treg cells is associated with worsening in both groups, but always less severe disease in mice receiving GITR–/– CD25+-depleted spleen cells. These results indicate that GITR activity, not only on Treg cells but on effector cells, is involved in regulating disease onset and progression. Furthermore, as GITR–/– Treg cells are more effective in preventing disease, both GITR effects (i.e., the major effector response and less Treg efficiency) potentiate the immune response.
What emerges overall from this investigation into the role of GITR in the pathogenesis and development of autoimmune/inflammatory diseases is that GITR triggering is required for the development of the immune response against CII and that GITR deletion protects from CIA development. Future studies will address the role of GITR in the pathogenesis of autoimmune/inflammatory responses in human RA.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication January 26, 2005. Accepted for publication March 30, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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