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Originally published online as doi:10.1189/jlb.0806486 on January 2, 2007

Published online before print January 2, 2007
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(Journal of Leukocyte Biology. 2007;81:1044-1053.)
© 2007 by Society for Leukocyte Biology

Protective role of IFN-{gamma} in collagen-induced arthritis conferred by inhibition of mycobacteria-induced granulocyte chemotactic protein-2 production

Hilde Kelchtermans*,1, Sofie Struyf{dagger}, Bert De Klerck*, Tania Mitera*, Marijke Alen*, Lies Geboes*, Maarten Van Balen*, Chris Dillen*, Willy Put{dagger}, Conny Gysemans{ddagger}, Alfons Billiau*, Jo Van Damme{dagger} and Patrick Matthys*

* Laboratories of Immunobiology and
{dagger} Molecular Immunology, Rega Institute for Medical Research, and
{ddagger} Laboratory of Experimental Medicine and Endocrinology, Campus Gasthuisberg O and N, University of Leuven, Leuven, Belgium

1 Correspondence: Laboratory of Immunobiology, Rega Institute, University of Leuven, Minderbroederstraat 10, B-3000 Leuven, Belgium. E-mail: hilde.kelchtermans{at}rega.kuleuven.be


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mice with a disrupted IFN-{gamma} system are remarkably susceptible to experimental autoimmune diseases, such as collagen-induced arthritis (CIA), which rely on the use of CFA. The inflammatory lesions of these IFN-{gamma} knockout (KO) mice are characterized by an excessive proportion of neutrophils. Here, we show that the increased severity of CIA in IFN-{gamma}R KO as compared with wild-type mice is accompanied by increased levels of the CXC chemokine granulocyte chemotactic protein-2 (GCP-2), a major neutrophil-attracting chemokine in mice. We demonstrated that the heat-killed mycobacteria present in CFA elicited production of GCP-2 in mouse embryo fibroblast cultures and that this production was inhibited by IFN-{gamma}. Inhibition of GCP-2 production by IFN-{gamma} was STAT-1-dependent. IFN-{gamma} receptor KO mice treated with neutralizing anti-GCP-2 antibodies were protected from CIA, indicating the in vivo importance of GCP-2 in the pathogenesis of CIA. Our data support the notion that one of the mechanisms whereby endogenous IFN-{gamma} mitigates the manifestations of CIA consists of inhibiting production of GCP-2, thereby limiting mobilization and infiltration of neutrophils, which are important actors in joint inflammation. These results may also be applicable to other experimental models of autoimmunity that rely on the use of CFA.

Key Words: mice • neutrophils • chemokines


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many experimental autoimmune diseases rely on immunization with organ-specific autoantigens in CFA, which contains heat-killed mycobacteria. In most of these models, endogenous IFN-{gamma} acts as a disease-protective factor (reviewed in ref. [1 ]). One such model is collagen-induced arthritis (CIA), a polyarthritis inducible in DBA/1 mice by immunization with collagen type II (CII) in CFA and considered to be relevant for the pathogenesis of rheumatoid arthritis (RA) in humans [2 ]. IFN-{gamma} receptor knockout (IFN-{gamma}R KO) DBA/1 mice were found to suffer from an accelerated and more severe form of CIA [3 , 4 ]. The mechanism accounting for the protective role of endogenous IFN-{gamma} in CIA depends on the use of CFA and in particular, on the presence of mycobacteria in the adjuvant. Indeed, if arthritis is induced with CII in IFA, which does not contain mycobacteria, IFN-{gamma}R KO mice are protected instead of being more susceptible [1 , 5 ]. Hence, it appears that IFN-{gamma} somehow counteracts the activity of the mycobacterial adjuvant in the development of CIA. One mechanism of action consists of down-regulation of mycobacteria-induced myelopoiesis, as evident from an increased expansion of immature CD11b+ macrophages and neutrophils in spleens of IFN-{gamma}R KO mice [1 , 5 ]. However, histological examination of the joints of arthritic IFN-{gamma}R KO and arthritic wild-type mice revealed an excessive proportion of neutrophils compared with mononuclear cells in IFN-{gamma}R KO mice. A predominance of neutrophils in the lesions of mice with a disrupted IFN-{gamma} system has also been reported in two other models that rely on the use of CFA, i.e., experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune uveitis (EAU) [6 7 8 ]. Thus, it became relevant to investigate the molecular mechanism underlying the high proportion of neutrophils in CIA in the absence of a functional IFN-{gamma} system.

Neutrophil infiltration in inflammatory lesions is regulated by chemokines. In RA, the development of clinical signs of synovial inflammation correlates with increased local synthesis of the chemokine IL-8 (CXCL8) [9 10 11 12 13 ], the prototype of human CXC chemokines that affect mainly neutrophil migration. IL-8 is produced constitutively by macrophages in the synovial compartment [14 ], and continuous infusion of IL-8 is capable of inducing severe arthritis into rabbit knee joints [15 ]. In mice, IL-8 has not been described, but the major neutrophil-attracting chemokine is granulocyte chemotactic protein-2(GCP-2), generally considered as the functional equivalent of IL-8 in humans [16 , 17 ]. The precursor molecule of GCP-2 has been designated as LPS-induced CXC chemokine (LIX) [18 ].

In the present study, we tested the hypothesis that endogenous IFN-{gamma} exerts its protective effect in CIA by counteracting chemokine-mediated infiltration of neutrophils into the joints. We found higher levels of GCP-2 in synovia of arthritic IFN-{gamma}R KO mice as compared with arthritic wild-type counterparts. Furthermore, we demonstrated that mycobacteria are potent inducers for the production of GCP-2 and that this induction is inhibited profoundly by IFN-{gamma} in a STAT-1-dependent way. Finally, we provided evidence for an important role of GCP-2 in the pathogenesis of CIA in IFN-{gamma}R KO mice by treatment with neutralizing anti-GCP-2 antibodies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies, chemokines, and cytokines
Recombinant mouse (rm)IFN-{gamma} was derived from the supernatant fluid of Mick cells, a Chinese hamster ovary cell line developed in our laboratory [19 ], which carries and expresses an amplified murine (mu)IFN-{gamma} cDNA. This IFN was purified by affinity chromatography to a specific activity of 109.5 units/mg, as described previously [20 ].

Generation of rabbit anti-muGCP-2 antibodies
Immunization of two rabbits was performed using rmuGCP-2(9–78) [21 ]. For the first injection, the antigen (10 µg) was emulsified in 1.5 ml CFA. The rabbits were boosted at least four times at intervals of 4 weeks with 10 µg muGCP-2(9–78) in 1.5 ml IFA. Blood (30 ml) was collected by bleeding the rabbits at the ear vein 10 and 20 days after immunization. Antibodies were purified through protein A/G affinity chromatography (Pharmacia Biotech, Uppsala, Sweden), and endotoxin was removed by the endotrap system (Profos AG, Regensburg, Germany). The efficiency of the endotoxin removal was verified by the Limulus amebocyte lysate assay (Cambrex Bioscience, Verviers, Belgium), and the endotoxin content was less than 1 ng/ml.

The specificity of the anti-GCP-2 antibodies was determined by immunoblot. Equal amounts (150 ng/lane) of recombinant keratinocyte-derived chemokine (rKC; Tebu-bio, Boechout, Belguim), rMIP-2 (Tebu-bio), and rGCP-2(9–78) protein were loaded onto a Tris/tricine gel and subjected to SDS-PAGE under nonreducing conditions. After electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Hybond-P, Amersham Pharmacia Biotech, Piscataway, NJ, USA) by electroblotting. To block nonspecific-binding sites, the blot was immersed in 100 mM NaCl, 10 mM Tris-HCl, pH 7.4 (NT buffer), with 5% (w/v) BSA before incubation with rabbit polyclonal antimouse GCP-2 antibody in NT buffer containing 0.1% (v/v) Tween 20 and 0.5% (w/v) BSA. After three washes in wash buffer [NT buffer with 0.5% (w/v) BSA], the blot was incubated with peroxidase-conjugated goat antirabbit antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA; 1/2000 dilution in wash buffer). Immunocomplexes were visualized with the ECL Plus chemiluminescence kit from Amersham Pharmacia Biotech.

IL-1RI/Fc was obtained from R&D Systems Europe (Abingdon, UK). Human stromal cell-derived factor-1 (SDF-1)/CXCL12 was provided by Dr. Ian Clark-Lewis (University of British Columbia, Vancouver, BC, Canada).

Mice and experimental conditions
The generation and basic characterization of IFN-{gamma}-deficient mice of the 129x BALB/c strain have been described previously [22 ]. These mice were backcrossed for eight generations to the parental BALB/c strain. The generation and basic characteristics of the mutant mouse strain (129/Sv/Ev) with a disruption in the gene encoding for the {alpha}-chain of the IFN-{gamma}R KO have been described [23 ]. These IFN-{gamma}R KO mice were backcrossed with wild-type DBA/1 mice for 10 generations to obtain the IFN-{gamma}R KO DBA/1 mice used in the present study. The homozygous IFN-{gamma}R KO mice were identified by PCR as described [24 ]. IFN-{gamma} ligand KO BALB/c mice and wild-type and IFN-{gamma}R KO DBA/1 mice were bred in the Experimental Animal Centre of the Rega Institute for Medical Research at Leuven (Belgium). Experiments were performed in 8- to 10-week-old mice, but in each experiment, the mutant and wild-type mice were age-matched within 5-day limits.

The STAT-1–/– and IFN-regulatory factor-1 (IRF-1)–/– mice, on a C57BL/6 background, were from Dr. David E. Levy of New York University School of Medicine (NY, USA) and from Dr. Tak Mak of Ontario Cancer Institute (Canada), respectively. The generation and characteristics of STAT-1–/– and IRF–/– mice have been described [25 , 26 ]. C57BL/6 mice (Harlan, Zeist, the Netherlands) were used as wild-type controls. All animal experiments were approved by the local ethical committee (University of Leuven, Belgium).

Antibody administration
IFN-{gamma}R KO DBA/1 mice were immunized with CII in CFA. Mice were treated with 0.25 mg anti-GCP-2 antibodies (in 250 µl PBS), rabbit IgG (Sigma-Aldrich, St. Louis, MO, USA), or PBS, once a week starting from Day –1 (early treatment) or Day 12 (late treatment).

Induction and clinical assessment of arthritis
Native chicken CII (Sigma-Aldrich) was dissolved in 0.05 M acetic acid at 2 mg/ml by stirring overnight at 6°C and emulsified in an equal volume of CFA (Difco Laboratories, Detroit, MI, USA) with added, heat-killed Mycobacterium butyricum (0.5 mg/ml). IFN-{gamma}R KO and wild-type DBA/1 mice were sensitized with a single intradermal injection at the base of the tail with 100 µl of the emulsion on Day 0. From Day 0 postimmunization, mice were examined for signs of arthritis five times a week. The disease severity was recorded following a scoring system for each limb: score 0, normal; score 1, redness and/or swelling in one joint; score 2, redness and/or swelling in more than one joint; score 3, redness and/or swelling in the entire paw; score 4, deformity and/or ankylosis.

Histological examination
Fore limbs, hind limbs, and ankles were fixed in 10% formalin and decalcified with 31.5% (v/v) formic acid and 13% (w/v) sodium citrate. The paraffin sections were stained with H&E. Subsequently, sections were scored for three parameters of arthritis: infiltration of mono- and polymorphonuclear cells, hyperplasia of the synovium, and pannus formation. The severity of each parameter was graded as 0, 1, 2, or 3 (absent, weak, moderate, or severe, respecitvely).

Cell purification
Splenocytes were harvested from 8- to 10-week-old naive mice or immunized mice at the indicated time-point after immunization. Spleens were cut gently into small pieces and passed through cell strainers (Becton Dickinson Labware, Franklin Lakes, NJ, USA). RBCs were lysed by two consecutive incubations (5 and 3 min at 37°C) of the suspension in NH4Cl (0.83% in 0.01 M Tris-HCl, pH 7.2). Remaining cells were washed, resuspended in cold PBS, and counted.

Isolation and induction of mouse embryo fibroblast (MEF)
MEF were isolated from mouse embryos between 16 and 18 days of gestation. The uterine horns were dissected out and placed into a petri dish containing Eagle’s minimal essential medium with Earle’s salts (EMEM; Invitrogen, Paisly, Scotland), supplemented with penicillin (500 IU/ml, Continental Pharma, Brussels, Belgium) and streptomycin (500 µg/ml, Continental Pharma). Each embryo was separated from its placenta and surrounding membranes. Embryos were washed three times with EMEM. Finally, the embryos were minced, and the cells/tissue were suspended in 50 ml trypsin-EDTA and incubated with gentle shaking at 37°C for 10 min. After removal of the trypsin-EDTA, cells were suspended in 50 ml trypsin-EDTA and incubated for 30 min. The resulting cell suspension, free of any larger pieces of tissue, was subjected to low-speed centrifugation, and the resulting cell pellet was washed twice with EMEM containing 10% heat-inactivated FCS (Gibco, Paisley, UK). The remaining cells/tissue were again subjected to trypsinization for 30 min, and the resulting cell pellet was subjected to centrifugation and washed twice with EMEM (10% FCS). The two harvests were pooled, suspended in EMEM (10% FCS), and grown to confluency in a culture flask (75 cm2, BD Falcon, BD Biosciences, San Jose, CA, USA). Cells were passaged every 5–7 days.

For the induction of GCP-2 in MEF, 2 x 105 cells in a total volume of 300 µl EMEM containing 2% heat-inactivated FCS were seeded in chamber slides (Lab-Tek Brand Products, Nalge Nunc International, Naperville, IL, USA). After an incubation of 48 h, cells were stimulated with heat-killed M. butyricum (100 µg/ml) in the presence or absence of IFN-{gamma} (100 units/ml) for 48 h. Supernatants were collected for detection of GCP-2 by ELISA. Cells were harvested using a cell scraper (Lab-Tek Brand Products) to remove the adherent cells from the slide. Cells were washed, and pellets were used for PCR.

Quantitative PCR
Synovial tissues from the ankle joints were isolated carefully under a stereomicroscope. Spleen cells and MEF cells were obtained as described above. Total RNA was extracted using the Micro-to-Midi Total RNA Purification System (Invitrogen Life Technologies, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. cDNA was obtained by RT using Superscript II RT and random primers (Invitrogen Life Technologies), in accordance with the manufacturer’s instructions. For real-time PCR, we used a TaqMan® Assays-on-DemandTM gene expression product from Applied Biosystems (Foster City, CA, USA). Expression levels of GCP-2 (assay ID Mm00436451_g1, Applied Biosystems), MIP-2 (assay ID Mm00436450_m1, Applied Biosystems), KC (assay ID Mm00433859_m1, Applied Biosystems), SDF-1 (assay ID Mm0044552_m1, Applied Biosystems), and IL-1 (Assay ID Mm00434228_m1, Applied Biosystems) were normalized for 18S RNA (Cat. No. 4319413E, Applied Biosystems) expression. Analysis was performed in an ABI Prism 7000 apparatus (Applied Biosystems) under the following conditions: inactivation of possible contaminating amplicons by AmpErase UNG at 50°C for 2 min and initial denaturation at 95°C for 10 min, followed by 40 thermal cycles of 15 s at 95°C and 90 s at 60°C. The relative gene expression was assessed using the 2–{Delta}{Delta}CT method [27 ].

Preparation of synovial fluid
Synovial fluid from the ankle joint was obtained by inserting a 25-gauge needle and injecting 20 µl cold PBS/FCS 2%. Fluid exiting spontaneously from the opening was collected and subjected to ELISA for the measurement of GCP-2 immunoreactivity.

Detection of GCP-2 by ELISA
GCP-2 was detected by an ELISA developed in our laboratory. Wells of 96-well plates were coated overnight at 4°C with 100 µl/well containing purified polyclonal rabbit antimouse GCP-2 (2 µg/ml) in PBS and subsequently washed with 0.05% Tween 20/PBS buffer. Wells were blocked with 200 µl 0.05% Tween 20/PBS containing 0.1% casein. After 1 h incubation at 37°C, wells were washed with 0.05% Tween 20/PBS. Serial dilutions of GCP-2 standards and samples were prepared in 0.05% Tween 20/PBS buffer containing 0.1% casein and incubated in duplicate in the coated wells. After washing with 0.05% Tween 20/PBS, each well received 100 µl detection antibody, i.e., 0.05 µg/ml biotinylated antimouse LIX antibody (R&D Systems Europe). After 1 h incubation at 37°C, the wells were washed and replenished with 100 µl streptavidin-HRP (Jackson Immunoresearch Laboratories) for a 30-min incubation at 37°C. Wells were washed with 0.05% Tween 20/PBS. Finally, 3.3 mg chromogen 3,3'-5,5'-tetramethylbenzidine (Sigma-Aldrich), dissolved in 250 µl DMSO, and 3.3 µl substrate H2O2 were added to 25 ml of the reaction buffer (100 mM sodium acetate/citric acid, pH 4.9). This reaction buffer (100 µl) was added to the wells for a 10-min incubation. Reactions were stopped by adding 100 µl 1 M H2SO4, and color intensity was measured at 450 nm.

The detection limit for GCP-2 was ~8 pg/ml. A ten- to 100-fold dilution of the samples was used. The specificity of the muGCP-2 ELISA was demonstrated by the lack of cross-reactivity with other mouse CXC chemokines: natural MIP-2, natural KC, rSDF-1{alpha}, natural IFN-{gamma}-induced protein-10 (IP-10). In addition to natural and rmuGCP-2 [16 , 21 ], rLIX (R&D Systems Europe), the precursor molecule of GCP-2, was recognized in this immunoassay.

In vitro chemotaxis
Spleen cells of three IFN-{gamma}R KO mice were obtained at Day 21 postimmunization and were pooled. For blocking experiments, rGCP-2 [21 ] (5 ng/ml), natural-purified MIP-2 [17 ] (5 ng/ml), KC (5 ng/ml), or SDF-1/CXCL12 (100 ng/ml) was preincubated for 1.5 h at 37°C with rabbit antimouse GCP-2 antibodies at the indicated concentrations or with irrelevant rabbit IgG (2.7 µg/ml). Transwell filter membranes (5 µm pore, Costar, Boston, MA, USA) were placed in the wells of a 24-well plate, each containing 600 µl buffer, with or without GCP-2, MIP-2, KC, or SDF-1, in the presence or absence of anti-GCP-2 antibodies or rabbit IgG in 600 µl buffer. Cells (106) were loaded on each Transwell filter. The plate was then incubated for 3 h at 37°C, whereupon the filter inserts were removed carefully. The migrated cells were collected, subjected to centrifugation, resuspended in 250 µl PBS (0.37% formaldehyde), counted in a flow cytometer (FACSCalibur, Becton Dickinson, San Jose, CA, USA), and analyzed with Cell Quest® software (Becton Dickinson). The number of cells corresponds with the number of counts registered during a 2-min acquisition.

Measurement of anti-CII antibodies and delayed-type hypersensitivity (DTH) to CII
Blood samples were taken from the orbital sinus and were allowed to clot at room temperature for ~1 h and at 4°C overnight. Individual sera were tested for antibodies directed to chicken CII by ELISA as described earlier [24 ].

For evaluation of DTH reactivity, CII/CFA-immunized mice were s.c.-injected with 20 µg CII/20 µl PBS in the right ear and with 20 µl PBS in the left ear. DTH response was calculated as the percentage swelling (the difference between the increase of thickness of the right ear and the left ear, divided by the thickness of the left ear, multiplied by 100).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Increased levels of GCP-2 in synovia of CII/CFA-immunized IFN-{gamma}R KO mice
IFN-{gamma}R KO mice develop CIA more readily than wild-type controls: When mice are injected with a single immunization of CII in CFA, symptoms of arthritis usually appear in IFN-{gamma}R KO mice from Day 16 onward against Day 30 in wild-type animals [3 , 24 ]. During our previous research, IFN-{gamma}R KO and wild-type mice were subjected to extensive histological examinations at various time-points postimmunization. From the analysis, it became evident that the inflammatory infiltrates in joints of IFN-{gamma}R KO mice contained an excessive proportion of neutrophils compared with mononuclear cells. To find an explanation for the high number of neutrophils present in joints of immunized IFN-{gamma}R KO mice [3 ], synovia of immunized IFN-{gamma}R KO and wild-type mice were isolated at Days 21 and 35, respectively, i.e., a time-point when they reached a similar score of arthritis [3 , 24 ]. The number of mRNA copies for different neutrophil-specific chemokines—GCP-2, KC, and MIP-2—was analyzed by quantitative PCR. Expression of SDF-1, a chemoattractant for T lymphocytes, monocytes, and B cells, was also assessed. Synovia of immunized IFN-{gamma}R KO mice contained levels of GCP-2, which were more than five times higher than those of wild-type mice (Fig. 1A ). Levels of KC and MIP-2 mRNA were more than 15 times higher in synovia of arthritic IFN-{gamma}R KO mice as compared with those of arthritic wild-type mice but were, respectively, five and 20 times lower than those of GCP-2. SDF-1 was found not to be different in synovia of arthritic IFN-{gamma}R KO and wild-type mice. In a next experiment, spleen tissue was also included, and the number of GCP-2 mRNA copies was analyzed by quantitative PCR. Spleen cells of immunized IFN-{gamma}R KO and wild-type mice contained about equal numbers of GCP-2 mRNA copies. Again, synovia of immunized IFN-{gamma}R KO mice contained levels of GCP-2 mRNA, which were more than six times higher than those of immunized, wild-type mice (Fig. 1B) . In additional experiments, we confirmed these findings at the protein level. Thus, ELISA was performed on synovial fluids of immunized IFN-{gamma}R KO and wild-type mice at Days 19 and 43, respectively, when mice reached a clinical score ≥3. GCP-2 was detected in pooled synovial fluid from immunized IFN-{gamma}R KO mice but not in that from immunized wild-type mice (Fig. 1C) . These results provide a possible explanation for the high numbers of neutrophils present in the joints of immunized IFN-{gamma}R KO mice.


Figure 1
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  		Figure 1. Increased GCP-2 levels in synovia of immunized IFN-{gamma}R KO mice. (A) Synovia were isolated from CII/CFA-immunized IFN-{gamma}R KO (n=4) and wild-type mice (n=3) at a time-point when they reached a similar clinical score (Days 20 and 34, respectively). (B) Synovia and spleens were isolated from CII/CFA-immunized IFN-{gamma}R KO (n=4) and wild-type mice (n=3) at Days 21 and 35, respectively. (A, B) cDNA samples were prepared and subjected to quantitative PCR analysis. The relative quantity of GCP-2, KC, MIP-2, and SDF in each sample was normalized to the quantity of 18S RNA. Error bars indicate SEM. (B) The results are representative of two independent experiments. (C) Ankle joints of four immunized IFN-{gamma}R KO and four immunized wild-type mice with a clinical score of arthritis ≥3 were washed at Days 19 and 43, respectively, with PBS/FCS 2% as described in Materials and Methods. Washings were collected, pooled, and subjected to ELISA for the measurement of GCP-2 immunoreactivity. ND, Not detectable.

 
Down-regulation of mycobacteria-induced GCP-2 production by IFN-{gamma}
Because of the high amounts of GCP-2 observed in immunized IFN-{gamma}R KO versus wild-type mice, we tested whether mycobacteria, present in CFA, can induce the production of GCP-2 and if so, whether production is influenced by IFN-{gamma}. In a first experiment, splenocytes were stimulated with mycobacteria, and to exclude any interference from endogenous IFN-{gamma}, cells were isolated from IFN-{gamma} ligand KO BALB/c mice. However, only marginal induction of GCP-2 mRNA could be detected in spleen cultures by quantitative PCR (data not shown). Based on results from Wuyts et al. [28 ], who demonstrated that GCP-2 is produced primarily by fibroblasts, we prepared MEF cultures, which were stimulated with mycobacteria for 48 h, and mRNA levels of GCP-2 were measured using quantitative PCR. As shown in Figure 2A , mycobacteria induced expression of GCP-2 mRNA in these cells. In the presence of IFN-{gamma}, however, expression failed to take place. To confirm these findings at the protein level, we used a GCP-2-specific ELISA. Again, IFN-{gamma} treatment strongly suppressed mycobacteria-induced GCP-2 production (Fig. 2B) . In addition, a dose-dependent inhibition of the production of GCP-2 by increasing amounts of IFN-{gamma} could be shown by ELISA (Fig. 2C) . These experiments demonstrate that IFN-{gamma} can down-regulate the production of GCP-2 in response to the mycobacteria present in CFA and suggest that this action of IFN-{gamma} may help to limit the infiltration of neutrophils at the site of inflammation. These experiments were confirmed in MEF cultures obtained from DBA/1 mice, the strain used to induce CIA (Fig. 2D) . As Wuyts et al. [28 ] demonstrated that in vitro, IL-1 induces GCP-2 expression in human fibroblasts, IL-1 mRNA expression was assessed in MEF cultures stimulated with mycobacteria in the absence or presence of IFN-{gamma} by quantitative PCR. Mycobacteria induced 800-fold expression of IL-1, and this induction was abolished completely in the presence of IFN-{gamma} (data not shown). Therefore, we verified whether induction of GCP-2 by mycobacteria was indirectly a result of induction of IL-1. Addition of rmIL-1RI/Fc (0.5 µg/ml) to the MEF cultures caused an ~50% inhibition of the mycobacteria-induced GCP-2 expression (data not shown). A higher concentration of IL-1R/Fc did not result in a more pronounced inhibition of GCP-2 production, indicating that the mycobacteria-induced GCP-2 production is partly, indirectly a result of induction of IL-1.


Figure 2
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  		Figure 2. IFN-{gamma} inhibits the mycobacteria-induced GCP-2 production in MEF. (A–C) MEF of IFN-{gamma} ligand KO mice were grown to confluency and stimulated for 48 h with mycobacteria (MB; 100 µg/ml) or were left untreated in the absence or presence of IFN-{gamma}. (A) cDNA samples were prepared and subjected to quantitative PCR analysis. The relative quantity of GCP-2 mRNA in each sample was normalized to the quantity of 18S RNA. Results are representative of three independent experiments. (B–D) GCP-2 protein present in the supernatants of stimulated MEF was measured by ELISA. (B) Results represent the mean of two to four cultures ± SEM. (C) Stimulation with mycobacteria (100 µg/ml) in the absence or presence of increasing concentrations of IFN-{gamma}. Results represent the mean of two experiments with duplicate cell cultures per condition ± SEM. (D) MEF of wild-type DBA/1, wild-type C57BL/6, STAT-1–/–, and IRF-1–/– of the C57BL/6 strain were grown to confluency and stimulated for 48 h with mycobacteria (100 µg/ml) in the absence or presence of IFN-{gamma} (100 units/ml). Results represent the mean of three independent experiments (two to four cultures for each condition in each experiment) ± SEM. *, P < 0.05, for comparison with corresponding mycobacterium without IFN-{gamma} (Mann-Whitney U test).

 
STAT-1 is a major mediator of cell activation by IFN-{gamma}. However, IFN-{gamma} also activates STAT-1-independent pathways [29 ], and it has been proposed that these STAT-1-independent pathways may mediate suppressive activities of IFN-{gamma} [30 ]. To investigate whether suppression of GCP-2 production by IFN-{gamma} was STAT-1-dependent, we prepared MEF cultures from wild-type and STAT-1–/– mice of the C57BL/6 strain. At the same time, we investigated whether the inhibition was dependent on IRF-1, a transcription factor acting immediately downstream of STAT-1, by preparing MEF cultures from IRF-1–/– C57BL/6 mice. In MEF from wild-type mice, IFN-{gamma} inhibited the mycobacteria-induced GCP-2 production (Fig. 2D) . Inhibition was also seen in MEF isolated from IRF-1–/– mice but was absent completely in MEF cultures derived from STAT-1–/– mice, indicating that inhibition by IFN-{gamma} is STAT-1-dependent but IRF-1-independent.

Inhibition of in vitro chemotaxis by anti-GCP-2 antibodies and protection in CIA
Anti-GCP-2 antibodies were used to test the in vivo importance of GCP-2 in the pathogenesis of CIA. First, the specificity of the anti-GCP-2 antibodies was verified by immunoblot, as described in Materials and Methods. Specific immunoreactivity could only be observed in the lane where muGCP-2 was loaded (equal amounts of KC and MIP-2 were not detected), confirming the specificity of the rabbit polyclonal antiserum. In vitro chemotaxis assays performed on splenocytes of immunized mice further allowed us to investigate the specificity and the dose-dependency of inhibition of the GCP-2-induced chemotaxis by anti-GCP-2 antibodies. The chemotaxis elicited by GCP-2 was blocked completely by preincubation with anti-GCP-2 antibodies and not by irrelevant rabbit IgG (Fig. 3A and 3B ). Anti-GCP-2 antibody did not inhibit chemotaxis elicited by MIP-2, KC, or SDF-1 (Fig. 3A and 3B) . Dose dependence of inhibition was confirmed in three additional experiments, and the pooled data are represented in Figure 3C .


Figure 3
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  		Figure 3. Anti-GCP-2 antibodies inhibit in vitro chemotaxis induced by GCP-2. (A) Splenocytes were isolated from immunized IFN-{gamma}R KO mice on Day 21 after immunization. Blocking experiments were performed as described in Materials and Methods. Transmigration of splenocytes toward GCP-2 (5 ng/ml), MIP-2 (5 ng/ml), KC (5 ng/ml), and SDF (100 ng/ml) was followed microscopically, and after 3 h of incubation, the membrane inserts were removed, and the cells in the wells were collected and counted by flow cytometry. Forward- and side-scatter (FSC and SSC, respectively) plots of the start population and the various conditions after incubation are shown for a concentration of anti-GCP-2 (aGCP-2) antibodies of 2.7 µg/ml. Figures represent the numbers of migrated cells in the indicated gate. The chemotactic index (C.I.) represents the number of cells migrated to the test sample, divided by the number of cells migrated to the negative control. The results are representative of two (KC, SDF) or four (MIP-2, GCP-2) independent experiments. (B) The mean chemotactic index of the independent experiments described in A was calculated. The mean ± SEM is shown. (C) Dose-response inhibition of GCP-2-induced chemotaxis by anti-GCP-2 antibodies. Experiments were performed as in A, and the percentage inhibition of the GCP-2-induced chemotaxis [100x(chemotactic index without anti-GCP-2–chemotactic index with anti-GCP-2)/chemotactic index without anti-GCP-2] was calculated. The means ± SEM of four independent experiments are shown.

 
In a next set of experiments, we injected anti-GCP-2 antibodies or irrelevant rabbit IgG i.p. in CII/CFA-immunized IFN-{gamma}R KO mice starting from Day –1. The mean arthritic score of irrelevant rabbit IgG-treated IFN-{gamma}R KO mice increased gradually to reach a maximum of 7.6 ± 1.8 on Day 20. In contrast, treatment with anti-GCP-2 antibodies completely protected the mice against clinically apparent arthritis (Fig. 4A ). All mice were killed on Day 22 for histological examination of the joints. Scoring the severity of three parameters of arthritis, as described in Materials and Methods, in all mice showed that the absence or redness and swelling were associated with inhibition of the infiltration on immunocompetent cells and tissue destruction at the histological level. In contrast, sections of arthritic, rabbit IgG-treated mice showed severe inflammatory infiltrate, hyperplasia of the synovium, and bone destruction. Results of quantitative scoring of histological sections are shown in Figure 4B .


Figure 4
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  		Figure 4. Treatment with anti-GCP-2 antibodies prevents CIA in IFN-{gamma}R KO mice, which were immunized on Day 0 with CII in CFA and injected i.p. with 0.25 mg neutralizing anti-GCP-2 antibodies or irrelevant rabbit IgG on Days –1 and 6. (A) Data points represent the mean arthritic score of IFN-{gamma}R KO mice treated with anti-GCP-2 antibodies or rabbit IgG. Error bars indicate SEM. The cumulative incidence (%) of arthritis at the end of the experiment is shown in brackets. (B) Histological analysis of the joints. On Day 22, all mice were killed, and sections of the fore limbs, hind limbs, and ankles were scored for three parameters of arthritis following hemotoxylin staining, as described in Materials and Methods. Histograms represent averages ± SEM. *, P ≤ 0.01, for comparison with mice treated with anti-GCP-2 antibodies (Mann-Whitney U test).

 
This protective effect of anti-GCP-2 antibodies in IFN-{gamma}R KO mice was confirmed in two independent experiments. Moreover, in two additional experiments, anti-GCP-2 antibodies were injected only from Day 12 onward after immunization. Under these conditions, average incidence and severity of arthritis were still lower in treated mice than in controls, although a statistically significant difference could not be ascertained (Table 1 ).


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  		Table 1. Cumulative Incidence and Mean Score of Arthritis in Mice Treated with Anti-GCP-2

 
Reduced humoral and cellular immune responses in IFN-{gamma}R KO mice treated with anti-GCP-2 antibodies
The pathogenesis of CIA is generally considered to depend in part on humoral and cellular immune responses against CII. We wanted to define whether the protection against CIA by anti-GCP-2 antibodies results from modulation of either of these. Total anti-CII IgG was determined in sera of anti-GCP-2-treated and irrelevant rabbit IgG-treated IFN-{gamma}R KO mice on Day 20 after immunization; titers were similar in sera of anti-GCP-2- and rabbit IgG-treated mice (Fig. 5A ). DTH was tested on Day 20 after immunization by injecting 20 µg CII in the left ears and vehicle in the right ears. Bars in Figure 5B represent the percentages of swelling of the CII-challenged ears, normalized to the swelling of the PBS-challenged ears. A significantly lower DTH response to CII was observed in the anti-GCP-2-treated mice as compared with that in control mice. Thus, the absence of clinical and histological symptoms of arthritis in the anti-GCP-2-treated mice appeared to be associated with a significantly decreased cellular immune responsiveness to CII.


Figure 5
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  		Figure 5. Reduced cellular response following treatment of CII/CFA-immunized IFN-{gamma}R KO mice with anti-GCP-2 antibodies. IFN-{gamma}R KO mice were immunized and injected i.p. with 0.25 mg neutralizing anti-GCP-2 antibodies (n=8) or PBS (n=8) on Days –1 and 6. (A) On Day 20, sera of individual mice were analyzed for total anti-CII IgG. Histograms represent averages ± SEM. (B) Twenty days after immunization, mice in each group were challenged with 20 µg CII in the left ear and vehicle in the right ear. DTH responses were measured as the percentage of swelling (i.e., 100x the difference between the increase of thickness of the left and the right ears, divided by the thickness of the right ear) at the indicated times. Histograms represent averages ± SEM. *, P < 0.05, for comparison with control mice (Mann-Whitney U test).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It has been demonstrated previously that IFN-{gamma}-deficient KO mice show an accelerated and more severe form of CIA than their wild-type counterparts [3 , 4 , 31 , 32 ]. IFN-{gamma} acts as an endogenous, protective factor in EAE and EAU [6 7 8 , 33 34 35 36 37 ]. Studies have indicated that IFN-{gamma} exerts this protective effect by suppressing the expansion of activated CD4+ T cells [38 ] or by up-regulating regulatory T cell activity [39 , 40 ].

In the case of CIA, a clue to this protective effect of IFN-{gamma} is the use of CFA in the induction procedure. In the absence of IFN-{gamma}, CFA induces extensive, extramedullary myelopoiesis and an expansion of a CD11b+, doughnut-like population. Cytospin splenocyte preparations showed the presence of a large number of band cells with a ring-shaped nucleus in IFN-{gamma}R KO mice but not in wild-type counterparts [5 ]. These band cells are predominantly immature monocytes/macrophages and neutrophils [41 ].

Neutrophils are considered important in the pathogenesis of arthritis, for example, because of their ability to destroy cartilage [42 43 44 45 ]. In the human system, IL-8 is the most potent, neutrophil-activating chemokine, whereas other Glu-Leu-Arg (ELR)+CXC chemokines, such as GCP-2/CXCL6, are weaker chemoattractants [46 ]. In the mouse, only three ELR+CXC chemokines with neutrophil-attracting properties are known: KC, MIP-2, and GCP-2 [17 ]. The number of mRNA copies for these neutrophil-specific chemokines was found to be higher in synovial tissue from arthritic IFN-{gamma}R KO mice as compared with that of arthritic, wild-type mice. Our results are in line with those of Tran et al. [47 ], who demonstrated that IFN-{gamma} regulates chemokine production. Indeed, they found that in IFN-{gamma} and IFN-{gamma}R-deficient mice with EAE, MIP-2 and T cell activation gene-3 were up-regulated strongly in the CNS. As levels of KC and MIP-2 mRNA were more than five and 20 times lower than those of GCP-2, respectively, and as murine GCP-2 is equally potent to attract murine neutrophils as human IL-8 [16 , 17 , 46 ], we focused on expression of GCP-2 mRNA. In a next set of experiments, analysis by quantitative PCR indicated that the levels of GCP-2 mRNA in synovia of arthritic IFN-{gamma}R KO mice were more than six times higher than those of arthritic, wild-type mice, providing a possible explanation for the high numbers of neutrophils in the joints of IFN-{gamma}R KO mice. In the spleens of IFN-{gamma}R KO and wild-type mice, GCP-2 mRNA levels were not different. It is interesting that in the synovium of IFN-{gamma}R KO mice, the levels of GCP-2 were ~18 times higher than in their spleens, suggesting that a concentration gradient of GCP-2 directs neutrophils toward the joints. GCP-2 measurement performed by ELISA on synovial fluid of immunized IFN-{gamma}R KO and wild-type mice confirmed the higher GCP-2 levels present in the inflamed joints of IFN-{gamma}R KO mice. In humans, IL-8 and GCP-2 are present in synovial fluids and tissues of RA patients [12 , 48 ]. Strong evidence for the importance of IL-8 in the pathogenesis of RA has been provided by Kraan et al. [10 ]: IL-8 was not detectable in synovial extracts of healthy, control individuals, was detectable in clinically uninvolved joints of RA patients, and increased in clinically involved joints of RA patients. Moreover, IL-8 was found capable of inducing synovial inflammation in an animal model [15 ].

As the protective effect of endogenous IFN-{gamma} in CIA depends on the use of mycobacterial adjuvant CFA, we next considered the possibility that the mycobacteria present in CFA are responsible for induction of GCP-2 and that this induction is modulated by IFN-{gamma}. We found that heat-killed mycobacteria induced transcription of GCP-2 mRNA and production of GCP-2 protein in cultured MEF and that addition of IFN-{gamma} to the system inhibited induction in a dose-dependent way. Induction of chemokines by mycobacteria has been demonstrated in several in vitro and in vivo systems. In a human alveolar epithelial cell line (A549), infection with various strains of Mycobacterium tuberculosis induced mRNAs and proteins of MCP-1/CCL2 and IL-8/CXCL8 [49 ]. Stimulation of cultured synovial fibroblasts by the TLR ligands, peptidoglycan, LPS, or dsRNA, induces secretion of various chemokines, including IL-8, GCP-2, IP-10/CXCL10, RANTES/CCL5, and MCP-2/CCL8 [48 , 50 , 51 ]. This induction of chemokines correlated with increased chemokine levels in septic arthritis synovial fluids.

As Wuyts et al. [28 ] demonstrated that IL-1 induces GCP-2 expression in human fibroblasts (which we confirmed in MEF; data not shown), IL-1 mRNA expression was assessed in MEF cultures stimulated with mycobacteria in the absence or presence of IFN-{gamma} by quantitative PCR. Mycobacteria induced 800-fold expression of IL-1, and this induction was abolished completely in the presence of IFN-{gamma}. This inhibition is in line with results of Hu et al. [52 ], who demonstrated that IFN-{gamma} suppresses a broad range of proinflammatory activities of IL-1. Addition of rmIL-1RI/Fc to the MEF cultures caused a 50% inhibition of GCP-2 production, suggesting that mycobacteria-induced GCP-2 production is only elicited partly by induction of IL-1.

Our observation that IFN-{gamma} inhibits GCP-2 production in MEF is in line with that of Wuyts et al. [28 ], who found that IFN-{gamma} down-regulates GCP-2 induction in human fibroblasts by IL-1ß, TNF-{alpha}, LPS, or dsRNA. The major mediator of cell activation by IFN-{gamma} is STAT-1. However, IFN-{gamma} also activates STAT-1-independent pathways [29 ]. Using STAT-1–/– and IRF-1–/– mice, we found that inhibition of GCP-2 production by IFN-{gamma} did depend on activation of STAT-1 but did not require intervention of IRF-1. Hu et al. [52 ] found that in human primary macrophages, Type I and II IFNs inhibited induction of IL-8, MIP-1{alpha}/CCL3, and TNF-{alpha} in a STAT-1-dependent pathway.

Our in vitro data showing that IFN-{gamma} inhibits induction of GCP-2 by mycobacteria support our hypothesis that in CIA, endogenous IFN-{gamma} uses this mechanism to mitigate neutrophil infiltration into inflamed joint tissues. That GCP-2 is a key player in the pathogenesis of CIA was apparent from a last series of experiments in which we showed that treatment with neutralizing anti-GCP-2 antibodies could protect IFN-{gamma}R KO mice completely. Our results are in line with those of Kasama et al. [53 ], who found that neutralization of MIP-2, another potent, neutrophil-attracting chemokine in mice, decreased clinical severity and delayed the onset of CIA.

Joint inflammation in CIA is assumed to depend in part on collagen-specific cellular and/or humoral immune reactivity. Treatment with anti-GCP-2 antibody did not affect antibody production against CII. As we reported earlier [5 ], increased severity of CIA in IFN-{gamma}R KO mice is associated with increased DTH to CII. As to how this relates to GCP-2 production and to neutrophil mobilization, we found in the present study that protection of CII/CFA-immunized IFN-{gamma}R KO mice by early treatment with anti-GCP-2 antibodies was associated with reduced DTH to CII. This supports the notion that neutrophils do play a role in DTH and is in line with reported findings that depletion of neutrophils or neutralization of MIP-2 by specific antibody is associated with reduced DTH reactivity [54 55 56 57 ]. Moreover, Maletto et al. [58 ] demonstrated recently that neutrophils may be important in initiation of disease by their ability to uptake antigens. Indeed, their data suggest that the selection of neutrophils as cells to capture antigens occurs in response to certain inflammatory signals such as bacille Calmette-Guerin vaccines or CFA. By capturing antigen in the periphery, transporting it to lymphoid organs, neutrophils can provide help in sustaining the immune response. These findings may provide an explanation for the fact that the GCP-2 antiserum is effective if applied before immunization with collagen but is less effective if applied 12 days after immunization.

Thus, our data support the notion that one of the mechanisms, whereby endogenous IFN-{gamma} mitigates the manifestations of CIA, consists of inhibiting production of GCP-2, thereby limiting mobilization and infiltration of neutrophils, which are important actors in joint inflammation, by secreting tissue-destructive mediators and by stimulating collagen-specific autoimmunity.

The relevance of our findings for arthritis in humans depends on the presence of a stimulus equivalent to mycobacterial adjuvant. Various ubiquitous bacterial constituents and TLR ligands, more specifically, bacterial cell wall fragments and bacterial DNA containing CpG motifs, have the potential to induce or reactivate experimental arthritis [59 ]. Analysis of synovial samples from RA patients by PCR with pathogen-specific primers has revealed the presence of DNA of mycobacteria species in some of the samples [60 , 61 ]. The presence of bacterial components in the joints of some RA patients was also supported by the demonstration of peptidoglycan/polysaccharide-positive cells in the synovial tissue [62 ]. These bacterial components may lead to enhanced production of GCP-2, resulting in an increased recruitment of inflammatory cells in the synovium and thereby, inducing or exacerbating inflammation in the joints. In clinical trials, IFN-{gamma} administration to RA patients has produced results suggestive of a protective effect [63 64 65 66 ]. Our data indicate that inhibitory effects of IFN-{gamma} on production of neutrophil-specific chemokines may have been involved in these clinical observations.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the Fund of Scientific Research Flanders (FWO Vlaanderen), the Regional Government of Flanders (GOA Program), and the Belgian Federal Government (Interuniversity Attraction Pole Program, IAP5/12). P. M., S. S., and C. G. are postdoctoral research fellows of FWO Vlaanderen, and H. K. holds a fellowship from FWO Vlaanderen. We thank Drs. David Levy and Tak Mak for providing STAT-1–/– and IRF-1–/– mice, Dr. Hubertine Heremans for providing IFN-{gamma}, Dr. Georges Leclercq for help with cell sorting, Dr. Chris De Wolf-Peeters for histological examinations, and Dr. Rik Lories for assistance with isolations of synovia. We are grateful to Dr. Ghislain Opdenakker for critical review of the manuscript.

Received August 1, 2006; revised November 28, 2006; accepted November 30, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Matthys, P., Vermeire, K., Billiau, A. (2001) Mac-1(+) myelopoiesis induced by CFA: a clue to the paradoxical effects of IFN-{gamma} in autoimmune disease models Trends Immunol. 22,367-371[CrossRef][Medline]
  2. Courtenay, J. S., Dallman, M. J., Dayan, A. D., Martin, A., Mosedale, B. (1980) Immunization against heterologous type II collagen induces arthritis in mice Nature 283,666-668[CrossRef][Medline]
  3. Vermeire, K., Heremans, H., Vandeputte, M., Huang, S., Billiau, A., Matthys, P. (1997) Accelerated collagen-induced arthritis in IFN-{gamma} receptor-deficient mice J. Immunol. 158,5507-5513[Abstract]
  4. Manoury-Schwartz, B., Chiocchia, G., Bessis, N., Abehsira-Amar, O., Batteux, F., Muller, S., Huang, S., Boissier, M. C., Fournier, C. (1997) High susceptibility to collagen-induced arthritis in mice lacking IFN-{gamma} receptors J. Immunol. 158,5501-5506[Abstract]
  5. Matthys, P., Vermeire, K., Mitera, T., Heremans, H., Huang, S., Schols, D., De Wolf-Peeters, C., Billiau, A. (1999) Enhanced autoimmune arthritis in IFN-{gamma} receptor-deficient mice is conditioned by mycobacteria in Freund’s adjuvant and by increased expansion of Mac-1+ myeloid cells J. Immunol. 163,3503-3510[Abstract/Free Full Text]
  6. Ferber, I. A., Brocke, S., Taylor-Edwards, C., Ridgway, W., Dinisco, C., Steinman, L., Dalton, D., Fathman, C. G. (1996) Mice with a disrupted IFN-{gamma} gene are susceptible to the induction of experimental autoimmune encephalomyelitis (EAE) J. Immunol. 156,5-7[Abstract]
  7. Willenborg, D. O., Fordham, S., Bernard, C. C., Cowden, W. B., Ramshaw, I. A. (1996) IFN-{gamma} plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis J. Immunol. 157,3223-3227[Abstract]
  8. Jones, L. S., Rizzo, L. V., Agarwal, R. K., Tarrant, T. K., Chan, C. C., Wiggert, B., Caspi, R. R. (1997) IFN-{gamma}-deficient mice develop experimental autoimmune uveitis in the context of a deviant effector response J. Immunol. 158,5997-6005[Abstract]
  9. Peichl, P., Pursch, E., Broll, H., Lindley, I. J. (1999) Anti-IL-8 autoantibodies and complexes in rheumatoid arthritis: polyclonal activation in chronic synovial tissue inflammation Rheumatol. Int. 18,141-145[CrossRef][Medline]
  10. Kraan, M. C., Patel, D. D., Haringman, J. J., Smith, M. D., Weedon, H., Ahern, M. J., Breedveld, F. C., Tak, P. P. (2001) The development of clinical signs of rheumatoid synovial inflammation is associated with increased synthesis of the chemokine CXCL8 (interleukin-8) Arthritis Res. 3,65-71[CrossRef][Medline]
  11. Van den Steen, P. E., Proost, P., Grillet, B., Brand, D. D., Kang, A. H., Van Damme, J., Opdenakker, G. (2002) Cleavage of denatured natural collagen type II by neutrophil gelatinase B reveals enzyme specificity, post-translational modifications in the substrate, and the formation of remnant epitopes in rheumatoid arthritis FASEB J. 16,379-389[Abstract/Free Full Text]
  12. Rampart, M., Herman, A. G., Grillet, B., Opdenakker, G., Van Damme, J. (1992) Development and application of a radioimmunoassay for interleukin-8: detection of interleukin-8 in synovial fluids from patients with inflammatory joint disease Lab. Invest. 66,512-518
  13. Lukacs, N. W., Strieter, R. M., Standiford, T. J., Kunkel, S. L. (1996) Characterization of chemokine function in animal models of diseases Methods 10,158-165[CrossRef][Medline]
  14. Koch, A. E., Kunkel, S. L., Burrows, J. C., Evanoff, H. L., Haines, G. K., Pope, R. M., Strieter, R. M. (1991) Synovial tissue macrophage as a source of the chemotactic cytokine IL-8 J. Immunol. 147,2187-2195[Abstract]
  15. Endo, H., Akahoshi, T., Nishimura, A., Tonegawa, M., Takagishi, K., Kashiwazaki, S., Matsushima, K., Kondo, H. (1994) Experimental arthritis induced by continuous infusion of IL-8 into rabbit knee joints Clin. Exp. Immunol. 96,31-35[Medline]
  16. Wuyts, A., Haelens, A., Proost, P., Lenaerts, J. P., Conings, R., Opdenakker, G., Van Damme, J. (1996) Identification of mouse granulocyte chemotactic protein-2 from fibroblasts and epithelial cells. Functional comparison with natural KC and macrophage inflammatory protein-2 J. Immunol. 157,1736-1743[Abstract]
  17. Wuyts, A., D’Haese, A., Cremers, V., Menten, P., Lenaerts, J. P., De Loof, A., Heremans, H., Proost, P., Van Damme, J. (1999) NH2- and COOH-terminal truncations of murine granulocyte chemotactic protein-2 augment the in vitro and in vivo neutrophil chemotactic potency J. Immunol. 163,6155-6163[Abstract/Free Full Text]
  18. Smith, J. B., Herschman, H. R. (1995) Glucocorticoid-attenuated response genes encode intercellular mediators, including a new C-X-C chemokine J. Biol. Chem. 270,16756-16765[Abstract/Free Full Text]
  19. Dijkmans, R., Volckaert, G., Van Damme, J., De Ley, M., Billiau, A., De Somer, P. (1985) Molecular cloning of murine interferon {gamma} (MuIFN-{gamma}) cDNA and its expression in heterologous mammalian cells J. Interferon Res. 5,511-520[Medline]
  20. Heremans, H., Van Damme, J., Dillen, C., Dijkmans, R., Billiau, A. (1990) Interferon {gamma}, a mediator of lethal lipopolysaccharide-induced Shwartzman-like shock reactions in mice J. Exp. Med. 171,1853-1869[Abstract/Free Full Text]
  21. Van Coillie, E., Van Aelst, I., Wuyts, A., Vercauteren, R., Devos, R., Wolf-Peeters, C., Van Damme, J., Opdenakker, G. (2001) Tumor angiogenesis induced by granulocyte chemotactic protein-2 as a countercurrent principle Am. J. Pathol. 159,1405-1414[Abstract/Free Full Text]
  22. Tagawa, Y., Sekikawa, K., Iwakura, Y. (1997) Suppression of concanavalin A-induced hepatitis in IFN-{gamma}(–/–) mice, but not in TNF-{alpha}(–/–) mice: role for IFN-{gamma} in activating apoptosis of hepatocytes J. Immunol. 159,1418-1428[Abstract]
  23. Huang, S., Hendriks, W., Althage, A., Hemmi, S., Bluethmann, H., Kamijo, R., Vilcek, J., Zinkernagel, R. M., Aguet, M. (1993) Immune response in mice that lack the interferon-{gamma} receptor Science 259,1742-1745[Abstract/Free Full Text]
  24. De Klerck, B., Carpentier, I., Lories, R. J., Habraken, Y., Piette, J., Carmeliet, G., Beyaert, R., Billiau, A., Matthys, P. (2004) Enhanced osteoclast development in collagen-induced arthritis in interferon-{gamma} receptor knock-out mice as related to increased splenic CD11b+ myelopoiesis Arthritis Res. Ther. 6,R220-R231[CrossRef][Medline]
  25. Durbin, J. E., Hackenmiller, R., Simon, M. C., Levy, D. E. (1996) Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease Cell 84,443-450[CrossRef][Medline]
  26. Matsuyama, T., Kimura, T., Kitagawa, M., Pfeffer, K., Kawakami, T., Watanabe, N., Kundig, T. M., Amakawa, R., Kishihara, K., Wakeham, A. (1993) Targeted disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development Cell 75,83-97[CrossRef][Medline]
  27. Livak, K. J., Schmittgen, T. D. (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-{Delta} {Delta} C(T)) method Methods 25,402-408[CrossRef][Medline]
  28. Wuyts, A., Struyf, S., Gijsbers, K., Schutyser, E., Put, W., Conings, R., Lenaerts, J. P., Geboes, K., Opdenakker, G., Menten, P., Proost, P., Van Damme, J. (2003) The CXC chemokine GCP-2/CXCL6 is predominantly induced in mesenchymal cells by interleukin-1ß and is down-regulated by interferon-{gamma}: comparison with interleukin-8/CXCL8 Lab. Invest. 83,23-34
  29. Ramana, C. V., Gil, M. P., Schreiber, R. D., Stark, G. R. (2002) Stat1-dependent and -independent pathways in IFN-{gamma}-dependent signaling Trends Immunol. 23,96-101[CrossRef][Medline]
  30. Ivashkiv, L. B., Hu, X. (2003) The JAK/STAT pathway in rheumatoid arthritis: pathogenic or protective? Arthritis Rheum. 48,2092-2096[CrossRef][Medline]
  31. Chu, C. Q., Song, Z., Mayton, L., Wu, B., Wooley, P. H. (2003) IFN{gamma} deficient C57BL/6 (H-2b) mice develop collagen induced arthritis with predominant usage of T cell receptor Vß6 and Vß8 in arthritic joints Ann. Rheum. Dis. 62,983-990[Abstract/Free Full Text]
  32. Guedez, Y. B., Whittington, K. B., Clayton, J. L., Joosten, L. A., van de Loo, F. A., van den Berg, W. B., Rosloniec, E. F. (2001) Genetic ablation of interferon-{gamma} up-regulates interleukin-1ß expression and enables the elicitation of collagen-induced arthritis in a nonsusceptible mouse strain Arthritis Rheum. 44,2413-2424[CrossRef][Medline]
  33. Billiau, A., Heremans, H., Vandekerckhove, F., Dijkmans, R., Sobis, H., Meulepas, E., Carton, H. (1988) Enhancement of experimental allergic encephalomyelitis in mice by antibodies against IFN-{gamma} J. Immunol. 140,1506-1510[Abstract]
  34. Duong, T. T., Finkelman, F. D., Singh, B., Strejan, G. H. (1994) Effect of anti-interferon-{gamma} monoclonal antibody treatment on the development of experimental allergic encephalomyelitis in resistant mouse strains J. Neuroimmunol. 53,101-107[CrossRef][Medline]
  35. Lublin, F. D., Knobler, R. L., Kalman, B., Goldhaber, M., Marini, J., Perrault, M., D’Imperio, C., Joseph, J., Alkan, S. S., Korngold, R. (1993) Monoclonal anti-{gamma} interferon antibodies enhance experimental allergic encephalomyelitis Autoimmunity 16,267-274[Medline]
  36. Krakowski, M., Owens, T. (1996) Interferon-{gamma} confers resistance to experimental allergic encephalomyelitis Eur. J. Immunol. 26,1641-1646[Medline]
  37. Caspi, R. R., Chan, C. C., Grubbs, B. G., Silver, P. B., Wiggert, B., Parsa, C. F., Bahmanyar, S., Billiau, A., Heremans, H. (1994) Endogenous systemic IFN-{gamma} has a protective role against ocular autoimmunity in mice J. Immunol. 152,890-899[Abstract]
  38. Chu, C. Q., Wittmer, S., Dalton, D. K. (2000) Failure to suppress the expansion of the activated CD4 T cell population in interferon {gamma}-deficient mice leads to exacerbation of experimental autoimmune encephalomyelitis J. Exp. Med. 192,123-128[Abstract/Free Full Text]
  39. Kelchtermans, H., De Klerck, B., Mitera, T., Van Balen, M., Bullens, D., Billiau, A., Leclercq, G., Matthys, P. (2005) Defective CD4+CD25+ regulatory T cell functioning in collagen-induced arthritis: an important factor in pathogenesis, counter-regulated by endogenous IFN-{gamma} Arthritis Res. Ther. 7,R402-R415[CrossRef][Medline]
  40. Wood, K. J., Sawitzki, B. (2006) Interferon {gamma}: a crucial role in the function of induced regulatory T cells in vivo Trends Immunol. 27,183-187[CrossRef][Medline]
  41. Biermann, H., Pietz, B., Dreier, R., Schmid, K. W., Sorg, C., Sunderkotter, C. (1999) Murine leukocytes with ring-shaped nuclei include granulocytes, monocytes, and their precursors J. Leukoc. Biol. 65,217-231[Abstract]
  42. Kitsis, E., Weissmann, G. (1991) The role of the neutrophil in rheumatoid arthritis Clin. Orthop. Relat. Res. Apr.,63-72
  43. De Clerck, L. S., De Gendt, C. M., Bridts, C. H., Van Osselaer, N., Stevens, W. J. (1995) Expression of neutrophil activation markers and neutrophil adhesion to chondrocytes in rheumatoid arthritis patients: relationship with disease activity Res. Immunol. 146,81-87[Medline]
  44. Pillinger, M. H., Abramson, S. B. (1995) The neutrophil in rheumatoid arthritis Rheum. Dis. Clin. North Am. 21,691-714[Medline]
  45. Kunkel, S. L., Lukacs, N., Kasama, T., Strieter, R. M. (1996) The role of chemokines in inflammatory joint disease J. Leukoc. Biol. 59,6-12[Abstract]
  46. Van Damme, J., Wuyts, A., Froyen, G., Van Coillie, E., Struyf, S., Billiau, A., Proost, P., Wang, J. M., Opdenakker, G. (1997) Granulocyte chemotactic protein-2 and related CXC chemokines: from gene regulation to receptor usage J. Leukoc. Biol. 62,563-569[Abstract]
  47. Tran, E. H., Prince, E. N., Owens, T. (2000) IFN-{gamma} shapes immune invasion of the central nervous system via regulation of chemokines J. Immunol. 164,2759-2768[Abstract/Free Full Text]
  48. Pierer, M., Rethage, J., Seibl, R., Lauener, R., Brentano, F., Wagner, U., Hantzschel, H., Michel, B. A., Gay, R. E., Gay, S., Kyburz, D. (2004) Chemokine secretion of rheumatoid arthritis synovial fibroblasts stimulated by Toll-like receptor 2 ligands J. Immunol. 172,1256-1265[Abstract/Free Full Text]
  49. Lin, Y., Zhang, M., Barnes, P. F. (1998) Chemokine production by a human alveolar epithelial cell line in response to Mycobacterium tuberculosis Infect. Immun. 66,1121-1126[Abstract/Free Full Text]
  50. Proost, P., Vynckier, A. K., Mahieu, F., Put, W., Grillet, B., Struyf, S., Wuyts, A., Opdenakker, G., Van Damme, J. (2003) Microbial Toll-like receptor ligands differentially regulate CXCL10/IP-10 expression in fibroblasts and mononuclear leukocytes in synergy with IFN-{gamma} and provide a mechanism for enhanced synovial chemokine levels in septic arthritis Eur. J. Immunol. 33,3146-3153[CrossRef][Medline]
  51. Proost, P., Verpoest, S., Van de Borne, K., Schutyser, E., Struyf, S., Put, W., Ronsse, I., Grillet, B., Opdenakker, G., Van Damme, J. (2004) Synergistic induction of CXCL9 and CXCL11 by Toll-like receptor ligands and interferon-{gamma} in fibroblasts correlates with elevated levels of CXCR3 ligands in septic arthritis synovial fluids J. Leukoc. Biol. 75,777-784[Abstract/Free Full Text]
  52. Hu, X., Ho, H. H., Lou, O., Hidaka, C., Ivashkiv, L. B. (2005) Homeostatic role of interferons conferred by inhibition of IL-1-mediated inflammation and tissue destruction J. Immunol. 175,131-138[Abstract/Free Full Text]
  53. Kasama, T., Strieter, R. M., Lukacs, N. W., Lincoln, P. M., Burdick, M. D., Kunkel, S. L. (1995) Interleukin-10 expression and chemokine regulation during the evolution of murine type II collagen-induced arthritis J. Clin. Invest. 95,2868-2876[Medline]
  54. Tumpey, T. M., Fenton, R., Molesworth-Kenyon, S., Oakes, J. E., Lausch, R. N. (2002) Role for macrophage inflammatory protein 2 (MIP-2), MIP-1{alpha}, and interleukin-1{alpha} in the delayed-type hypersensitivity response to viral antigen J. Virol. 76,8050-8057[Abstract/Free Full Text]
  55. Kudo, C., Yamashita, T., Terashita, M., Sendo, F. (1993) Modulation of in vivo immune response by selective depletion of neutrophils using a monoclonal antibody, RP-3. II. Inhibition by RP-3 treatment of mononuclear leukocyte recruitment in delayed-type hypersensitivity to sheep red blood cells in rats J. Immunol. 150,3739-3746[Abstract]
  56. Kudo, C., Yamashita, T., Araki, A., Terashita, M., Watanabe, T., Atsumi, M., Tamura, M., Sendo, F. (1993) Modulation of in vivo immune response by selective depletion of neutrophils using a monoclonal antibody, RP-3. I. Inhibition by RP-3 treatment of the priming and effector phases of delayed type hypersensitivity to sheep red blood cells in rats J. Immunol. 150,3728-3738[Abstract]
  57. Larsen, C. G., Thomsen, M. K., Gesser, B., Thomsen, P. D., Deleuran, B. W., Nowak, J., Skodt, V., Thomsen, H. K., Deleuran, M., Thestrup-Pedersen, K. (1995) The delayed-type hypersensitivity reaction is dependent on IL-8. Inhibition of a tuberculin skin reaction by an anti-IL-8 monoclonal antibody J. Immunol. 155,2151-2157[Abstract]
  58. Maletto, B. A., Ropolo, A. S., Alignani, D. O., Liscovsky, M. V., Ranocchia, R. P., Moron, V. G., Pistoresi-Palencia, M. C. (2006) Presence of neutrophil-bearing antigen in lymphoid organs of immune mice Blood 108,3094-3102[Abstract/Free Full Text]
  59. Deng, G. M., Nilsson, I. M., Verdrengh, M., Collins, L. V., Tarkowski, A. (1999) Intra-articularly localized bacterial DNA containing CpG motifs induces arthritis Nat. Med. 5,702-705[CrossRef][Medline]
  60. Wilkinson, N. Z., Kingsley, G. H., Sieper, J., Braun, J., Ward, M. E. (1998) Lack of correlation between the detection of Chlamydia trachomatis DNA in synovial fluid from patients with a range of rheumatic diseases and the presence of an antichlamydial immune response Arthritis Rheum. 41,845-854[CrossRef][Medline]
  61. Van der Heijden, I., Wilbrink, B., Schouls, L. M., van Embden, J. D., Breedveld, F. C., Tak, P. P. (1999) Detection of mycobacteria in joint samples from patients with arthritis using a genus-specific polymerase chain reaction and sequence analysis Rheumatology (Oxford) 38,547-553
  62. Van der Heijden, I. M., Wilbrink, B., Tchetverikov, I., Schrijver, I. A., Schouls, L. M., Hazenberg, M. P., Breedveld, F. C., Tak, P. P. (2000) Presence of bacterial DNA and bacterial peptidoglycans in joints of patients with rheumatoid arthritis and other arthritides Arthritis Rheum. 43,593-598[CrossRef]