IL-17 is a proinflammatory cytokine suspected to be involved in inflammatory and autoimmune diseases such as rheumatoid arthritis. In the present study, we report that IL-17R signaling is required in radiation-resistant cells in the joint for full progression of chronic synovitis and bone erosion. Repeated injections of Gram-positive bacterial cell wall fragments (streptococcal cell wall) directly into the knee joint of naive IL-17R-deficient (IL-17R−/−) mice had no effect on the acute phase of arthritis but prevented progression to chronic destructive synovitis as was noted in wild-type (wt) mice. Microarray analysis revealed significant down-regulation of leukocyte-specific chemokines, selectins, cytokines, and collagenase-3 in the synovium of IL-17R−/− mice. Bone marrow (BM) chimeric mice revealed the need for IL-17R expression on radiation-resistant joint cells for destructive inflammation. Chimeric mice of host wt and donor IL-17R−/− BM cells developed destructive synovitis in this chronic reactivated streptococcal cell wall arthritis model similar to wt→wt chimeras. In contrast, chimeric mice of host IL-17R−/− and donor wt BM cells were protected from chronic destructive arthritis similar as IL-17R−/−→IL-17R−/− chimeras. These data strongly indicate that IL-17R signaling in radiation-resistant cells in the joint is required for turning an acute macrophage-mediated inflammation into a chronic destructive synovitis.

Interleukin-17 is a 17-kDa proinflammatory cytokine that is secreted as a dimer principally by activated T cells and CD4+CD45RO memory T cells (1). IL-17 (also known as IL-17A) is the first discovered member of the IL-17 family that currently consists of six family members (IL-17A–F) (2). IL-17 exhibits pleiotropic biological activities on various types of cells stimulating transcriptional NF-κB activity mediating a broad spectrum of responses, including induction of proinflammatory cytokines, such as IL-6, TNF-α, IL-1β, and chemokines, such as IL-8, MCP-1, and T cell proliferation (1, 3, 4, 5). In addition, IL-17 promotes CSF- and G-CSF-mediated granulopoiesis (6), enhances allorejection via a maturation-inducing effect on dendritic cells (7), and is a strong inducer of neutrophil recruitment through chemokine release (8). Furthermore, studies in IL-17-deficient mice revealed that this T cell cytokine plays an important role in activation of T cells in allergen-specific T cell-mediated immune responses (9).

In contrast to the restricted set of cells that produce IL-17, mRNA of the IL-17R is expressed ubiquitously in many cell types and shares no homology with any other cytokine receptor family (3). Binding of IL-17 to its unique receptor results in activation of the adapter molecule TNFR-associated factor 6 but not TNFR-associated factor 2 (10). IL-17 shares transcriptional pathways with IL-1 and TNF. It can activate NF-κB and all three classes of MAPKs, including ERK1 and ERK2, JNK, and p38 (11, 12).

Overproduction of IL-17 has been associated with several chronic disease conditions, such as lung, gut, and skin inflammation (8, 13, 14, 15, 16, 17) as well as autoimmune diseases, such as rheumatoid arthritis (18, 19, 20), experimental autoimmune encephalomyelitis (21), and patients with systemic sclerosis (22). The expression of IL-17 in Th1 and Th2 cells seems to be different depending upon the conditions. IL-17 is produced by Th1/Th0 cells but not by Th2 cells isolated from RA synovial tissue (23). In contrast, both Th1 and Th2 cell clones from human skin-derived nickel-specific T cells produced IL-17 (24). In mice, IL-17 is produced by T cells expressing TNF-α and IL-6 but not by Th1 or Th2 cells (25).

In arthritis, IL-17 is a proinflammatory cytokine thought to contribute to the joint inflammatory process (18, 26). IL-17 plays a role in the Ag-specific T cell activation phase of collagen-induced arthritis (CIA)4(27). Furthermore, the spontaneous development of destructive arthritis in mice deficient in IL-1R antagonist could completely be prevented after inactivation of IL-17 (28). In addition, inhibition of the IL-17/IL-17R binding by an extracellular domain of IL-17R and Fc fusion protein starting after the immunization protocol of CIA reduced the severity of CIA (29). Moreover, neutralizing endogenous IL-17 using anti-IL-17 sera after onset of CIA still inhibited development of joint pathology (30). These observations strongly implicate a role for IL-17 at various levels in disease pathogenesis of arthritis. IL-17 seems to play a role in T cell immunity and/or propagation of joint inflammation. However, apart from its important role in Ag-specific T cell activation, the requirement of IL-17/IL-17R signaling in local joint resident cells, such as macrophage- and fibroblast-like synoviocytes and endothelial cells in the progression of joint pathology, is unclear.

In this study, we used IL-17R-deficient (IL-17R−/−) mice to investigate the role of IL-17R signaling in the development, as well as progression of chronic arthritis. We used the relapsing streptococcal cell wall (SCW)-induced arthritis model in which T cells are not directly involved in the acute stage of the disease. However, repeated injection of Gram-positive SCW fragments directly in the knee joint turned the acute, macrophage-driven joint inflammation into a chronic destructive arthritis model that becomes T cell dependent with time (31). Our data revealed the critical role for IL-17R signaling in turning the acute macrophage-mediated inflammation into a chronic destructive synovitis. Moreover, we demonstrated using different chimeric mice the requirement of IL-17R signaling in radiation-resistant cells expressing the IL-17R in the joint for full progression of chronic destructive synovitis.

Specified pathogen-free C57BL/6 mice (8–10 wk old, male and female, National Cancer Institute) were used in all experiments and as control (wild-type (wt)) mice for IL-17R−/− mice. IL-17R−/− mice were obtained from Amgen Washington and bred in Louisiana State University Health Sciences Center Animal Care Facilities (13). The IL-17R−/− were backcrossed to C57BL/6 mice for >10 generations. All mice were housed under specific pathogen-free conditions within the Animal Care Facility of Louisiana State University Medical Center Vivarium under an Institutional Animal Care and Use Committee-approved protocol, provided with water and food ad libitum, and housed under 12-h light/dark cycles. Genetic integrity of the colony was monitored by confirming genotype of each individual breeder as previously described by using flow cytometry and a specific PCR (13).

Streptococcus pyogenes T12 organisms were cultured overnight in Todd-Hewitt broth. Cell walls were prepared as described previously (32). The resulting 10,000 × g supernatant was used throughout the experiments. These preparations contained 11% muramic acid. Arthritis was induced in wt and IL-17R−/− mice by intra-articular (i.a.) injection of 25 μg of SCW (Rhamnose content) in 6 μl of PBS into the knee joints. To examine the role of IL-17R signaling in the development and progression of chronic synovitis, five repeated injections of 25 μg of SCW were i.a. injected each 1 wk apart. At different time points, knee joint swelling was measured with an electronic digital caliper (Control). Knee joint thickness is expressed as δ difference in joint swelling (individual knee joint thickness after SCW injection minus normal knee joint thickness measured before the first SCW injection (naive conditions)).

BM cells was harvested from IL-17R−/− and wt mice and transplanted into lethally irradiated wt or IL-17R−/− mice, as described previously (33). Briefly, recipient mice were lethally irradiated with a gamma radiator (GammaCell 40 Exactor; MDS Nordion) in two doses of 500 rad each ∼3 h apart. Donor mice were killed by cervical dislocation, and BM cells were harvested by flushing both femurs and tibias with DMEM (Invitrogen Life Technologies) containing 15% FCS (Atlanta Biologicals) under sterile conditions. Suspended BM cells were washed, and erythrocytes were lysed in 0.15 M NH4Cl lysing solution. Following lethal irradiation, each animal was i.v. injected with 5 million mononucleated BM cells suspended in 200 μl of medium. Four combinations of BM chimeras were created (donor BM→lethally irradiated recipient): wt→wt, IL-17R−/−→IL-17R−/−, wt→IL-17R−/−, and IL-17R−/−→wt. Recipient mice were housed in a barrier facility (individually ventilated cages with high-efficiency particulate air filter) under pathogen-free conditions before and after BM transplantation. Mice were used for experiments after 4 wk of BM reconstitution.

Peripheral blood (100 μl) from the different groups of chimeric mice was collected in heparin tubes. Surface IL-17R expression was determined by flow cytometry using the specific murine anti-IL-17R Ab, clone M177 (rat anti-mouse IL-17R Ig; Amgen Washington), gated on the granulocyte population. Briefly, whole blood was incubated with M177 Ab. Samples were incubated for 30 min on ice. Unlabeled Ab was removed by aspiration after centrifugation. Peripheral blood was resuspended in 150 mM NH4Cl, 10 mM NaHCO3, and 1 mM Na2 EDTA in deionized distilled water to lyse RBC. Samples were incubated with goat anti-rat IgG to Alexa Fluor 488 (Molecular Probes). Data are presented as fluorescence histograms of IL-17R expression gated on the granulocyte population.

Mice were sacrificed by cervical dislocation, and the patella and adjacent synovium were dissected immediately (29). From one patella specimen, two specimens of synovium biopsy tissue with a diameter of 3 mm were punched out with a biopsy punch (Stifle), one from the lateral side and one from the medial side. Six specimens of synovium biopsy tissue (three from the lateral and three from the medial side) were pooled as one sample. In this way, three different samples from wt and IL-17R−/− were isolated. The synovium samples were put immediately in RNAlater (Ambion) and stored in −80°C. Synovium tissue samples were disrupted and homogenized using a rotor-stator homogenization, and RNA were isolated using Qiagen RNeasy mini kit (Qiagen), according to the manufacturer’s directions. Microarray analysis was performed on three separated samples per group.

Each total RNA sample was first examined by spectrophotometric analysis, where the A260:A280 ratio was measured. In addition, the quality and quantity of each RNA sample was verified before hybridization using RNA 6000 Nano Assay (Agilent).

Microarray processing.

U74Av2 chips (Affymetrix) were prehybridized with 200 μl 1× hybridization buffer (100 mM MES, 1 M (Na+), 20 mM EDTA, and 0.01% Tween 20) for 10 min at 45°C in an Affymetrix Genechip Hybridization Oven 640 at 60 rpm. Hybridization was done in a final volume of 300 μl, containing 15 μg of fragmented biotinylated cRNA, 50 pM control oligonucleotide B2 (Affymetrix), eukaryotic hybridization controls (Affymetrix), 0.1 mg/ml herring sperm DNA, and 0.5 mg/ml acetylated BSA in 1× hybridization buffer. The samples were heated to 95°C for 5 min and 45°C for an additional 5 min and then briefly spun down. Two hundred microliters of the hybridization mixture were added to the standard arrays, and hybridizations were conducted for 16 h at 45°C with mixing on a rotisserie at 60 rpm. After hybridization, the solutions were removed, and the arrays were washed on a Fluidics station (Affymetrix). Hybridized arrays were stained for 10 min at 25°C with streptavidin-R PE (Molecular Probes) (10 μg/ml), followed by staining with biotinylated goat anti-streptavidin Ab (Sigma-Aldrich) (3 μg/ml) 10 min at 25°C. Genechips were then stained once again with streptavidin-R PE for 10 min at 25°C. Probe arrays were scanned with a confocal laser scanner (Agilent) at a wavelength of 570 nm. Pixel intensities were then measured, and expression signals were analyzed using a commercial software package (Microarray Suite 5.0; Affymetrix). LIMS 3.0 (Affymetrix) and Data Mining Tools 3.0 (Affymetrix) were used to perform data analysis.

Microarray data were generated using Affymetrix (〈www.affymetrix.com〉) protocols. Absolute expression transcript levels were normalized for each chip by globally scaling all probe sets to a target signal intensity of 500. Three statistical algorithms (detection, change call, and signal log ratio) were then used to identify differential gene expression in experimental and control samples. The detection metric (presence, absence, or marginal) for a particular gene was determined using default parameters in the MAS software (version 5.0; Affymetrix). Transcripts that were absent under both control and experimental conditions were eliminated from further consideration. Statistical significance of signals between the control and experimental conditions (p < 0.05) for individual transcripts was determined using the t test and/or Mann-Whitney U test. Batch analyses was performed in MAS, version 5.0, in which pairwise comparisons between individual experimental and control chips were made to generate a change call and a signal log-ratio value for each transcript. We defined a positive change call as one in which >50% of the change calls for any one transcript had to be increased or marginally increased for up-regulated genes and decreased or marginally decreased for down-regulated genes. Finally, the median value of the signal log ratios from each comparison file was calculated. Only those genes that met the above criteria and had a median signal log ratio of ≥1 for up-regulated transcripts and ≤1 for down-regulated transcripts were kept in the final list of genes. Signal log-ratio values were converted from log2 and expressed as fold changes (34).

Quantitative real-time (Q-PCR) PCR was performed using the ABI/PRISM 7000 Sequence Detection System for quantification with SYBR Green and melting curve analysis (Applied Biosystems). Primer sequences for the reference gene GAPDH and IL-17 were as follows: 5′-GGCAAATTCAACGGCACA-3′ (GAPDH forward), 5′-GTTAGTGGGGTCTCGCTCTG-3′ (GAPDH reverse), 5′-CAGGACGCGCAAACATGA-3′ (IL-17 forward), and 5′-GCAACAGCATCAGAGACACAGAT-3′ (IL-17 reverse). PCR conditions were as follows: 2 min at 50°C and 10 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C, with data collection in the last 30 s. For all PCRs, SYBR Green Master Mix (Applied Biosystems) was used in the reaction. Primer (Biolegio) concentrations were 300 nmol/L. All PCRs were performed in a total volume of 25 μl. Relative quantification of the PCR signals was performed by comparing the cycle threshold value (Ct), in duplicate, of the gene of interest of each sample with the Ct values of the reference gene GAPDH. Q-PCR analysis for each sample was performed in duplicate.

Mice were sacrificed by cervical dislocation. Thereafter, whole knee joints were removed and fixed for 4 days in 10% formalin. After decalcification in 5% formic acid, the specimens were processed for paraffin embedding (35). Tissue sections were stained with H&E. Histopathological changes were scored with the following parameters. Infiltration of cells was scored on a scale of 0–3, depending on the amount of inflammatory cells in the synovial cavity (exudates) and synovial tissue (infiltrate), and is expressed as joint inflammation. Bone destruction was graded on a scale of 0–3, ranging from no damage to complete loss of the bone structure. Histopathological changes in the knee joints were scored in the patella and femur/tibia regions on five semiserial sections of the joint spaced 70-μm apart. Two observers without knowledge of the experimental group, as described earlier (36), performed scoring.

Mice were fixed by cardiac perfusion using freshly prepared 4% paraformaldehyde. Thereafter, whole knee joints were removed before arthritis was developed (naive) and after marked arthritis was present (arthritic). Knee joints were fixed for 4 days in 4% paraformaldehyde. After decalcification in 5% formic acid, the specimens were processed for paraffin embedding (35). Tissues sections (7 μm) were treated with 3% H2O2 for 10 min at room temperature. Sections were incubated for 2 h with 10 mM citrate (pH 6.0) and incubated thereafter for 1 h with a specific murine IL-17R Ab clone M177 (rat anti-mouse IL-17R IgG; Amgen Washington) or F4/80 Ab (rat anti-mouse-IgG MCA497R; Serotec). After rinsing, sections were incubated for 30 min with biotinylated rabbit anti-rat Ab (BA4001; Vector Laboratories). Slides were stained with the ABC-HRP kit (PK6101; Vector Laboratories). Counterstaining was done with Mayer’s hematoxylin. Control sections were stained with an irrelevant primary isotype-specific IgG Ab.

Differences between experimental groups were tested with the Mann-Whitney U test, unless stated otherwise.

To examine the role of the IL-17R signaling pathway in the development of chronic inflammatory synovitis, we used a model which starts as a macrophage-driven process but becomes T cell dependent with time. To this end, mice were repeatedly injected with Gram-positive bacterial cell wall fragments (SCW) directly into the knee joint every week, with a total of five injections.

A single injection of SCW directly into the mouse knee joints induces an acute TLR2, MyD88-dependent joint inflammation, which is predominantly mediated by leukocytes (neutrophils) and macrophages (37). Hardly any up-regulation of IL-17 expression in the synovium was noted during this acute phase of SCW arthritis (Fig. 1). Induction of SCW in IL-17R−/− mice did not lead to significant reduction of acute SCW mediated joint inflammation measured one and seven days after the single injection of SCW (Fig. 2, A and B).

FIGURE 1.

Increase in synovial IL-17 expression after repeated injections of SCW bacterial fragments into the knee joint. At days 0, 7, and 14, 21 wt mice were i.a. injected with SCW fragments directly in the knee joint (arrows). At different time points, knee joint thickness was measured using a caliper, synovial biopsies were taken, and RNA was isolated. IL-17 mRNA expression was measured using Q-PCR technology. These results are expressed as ddCT (SCW vs naive), meaning difference in number of PCR cycles in which IL-17 was first detectable under SCW conditions vs naive conditions (corrected for GAPDH expression). Knee joint thickness is expressed as δ joint thickness (SCW injected vs normal knee joint). At the day of a new i.a. injection, knee joints were first measured and then injected with SCW. Knee joint thickness is expressed as the mean ± SEM of two experiments of at least six mice per group per experiment. For more information regarding Q-PCR, see Materials and Methods.

FIGURE 1.

Increase in synovial IL-17 expression after repeated injections of SCW bacterial fragments into the knee joint. At days 0, 7, and 14, 21 wt mice were i.a. injected with SCW fragments directly in the knee joint (arrows). At different time points, knee joint thickness was measured using a caliper, synovial biopsies were taken, and RNA was isolated. IL-17 mRNA expression was measured using Q-PCR technology. These results are expressed as ddCT (SCW vs naive), meaning difference in number of PCR cycles in which IL-17 was first detectable under SCW conditions vs naive conditions (corrected for GAPDH expression). Knee joint thickness is expressed as δ joint thickness (SCW injected vs normal knee joint). At the day of a new i.a. injection, knee joints were first measured and then injected with SCW. Knee joint thickness is expressed as the mean ± SEM of two experiments of at least six mice per group per experiment. For more information regarding Q-PCR, see Materials and Methods.

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FIGURE 2.

Critical role for IL-17R signaling in turning acute joint inflammation into a chronic destructive synovitis. A and B, At days 0, 7, 14, 21, and 35 (arrows), IL-17R−/− and wt mice were i.a. injected with SCW fragments directly in the knee joint. Every first day (A) and seventh day (B) after the injection of SCW, knee joint thickness was measured using a caliper. At the day of a new i.a. injection, knee joints were first measured and then injected with SCW. Knee joint thickness is expressed as δ joint thickness (SCW injected vs normal knee joint). C, At day 42, the skin of the knee joint was removed, and severity of knee joint inflammation was macroscopically scored (arthritis score) on a scale of 0–2 as described previously (36 ). In addition, knee joints were taken for histology. Synovial infiltrate and loss of bone was scored on a scale of 0–3. Results are expressed as the mean ± SEM of two experiments of at least 10 mice/group/experiment. ∗, p < 0.005; ∗∗, p = 0.001; ∗∗∗, p = 0.0005 vs control wt mice by Mann-Whitney U test.

FIGURE 2.

Critical role for IL-17R signaling in turning acute joint inflammation into a chronic destructive synovitis. A and B, At days 0, 7, 14, 21, and 35 (arrows), IL-17R−/− and wt mice were i.a. injected with SCW fragments directly in the knee joint. Every first day (A) and seventh day (B) after the injection of SCW, knee joint thickness was measured using a caliper. At the day of a new i.a. injection, knee joints were first measured and then injected with SCW. Knee joint thickness is expressed as δ joint thickness (SCW injected vs normal knee joint). C, At day 42, the skin of the knee joint was removed, and severity of knee joint inflammation was macroscopically scored (arthritis score) on a scale of 0–2 as described previously (36 ). In addition, knee joints were taken for histology. Synovial infiltrate and loss of bone was scored on a scale of 0–3. Results are expressed as the mean ± SEM of two experiments of at least 10 mice/group/experiment. ∗, p < 0.005; ∗∗, p = 0.001; ∗∗∗, p = 0.0005 vs control wt mice by Mann-Whitney U test.

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Repeated local injections of SCW fragments directly into the knee joint every week resulted in an increase of IL-17 expression in the synovium (Fig. 1). We next evaluated the role of IL-17R signaling in this model of chronic reactivated arthritis (Fig. 1). As shown in Fig. 2, weekly repeated local injection of SCW in wt mice resulted in joint swelling after every rechallenge (days 1, 8, 15, 22, and 36) but also demonstrated that the remaining smouldering arthritis is brought to a higher level and is getting more and more severe (Fig. 2, A and B). In addition, marked bone erosion was detected after five repeated local injections of SCW in these wt mice (Figs. 2,C and 3). In contrast, IL-17R−/− mice developed an acute joint inflammation after every local injection of SCW (Fig. 2,A); however, in the absence of the IL-17R, the arthritis is not brought to that level and severity as observed in wt mice, indicating that IL-17R deficiency prevented progressive synovitis (Figs. 2 and 3). Moreover, significantly less bone erosion was detected in IL-17R-deficient mice compared with wt mice after five repeated injections of SCW in the knee joint (Figs. 2 and 3). Thus, absence of IL-17R signaling plays a critical role in turning the TLR2-dependent acute joint inflammation into a chronic destructive synovitis.

FIGURE 3.

IL-17R deficiency prevents development of chronic destructive arthritis. A, Section of a knee joint of naive wt control mouse. B, Section of knee joint of naive IL-17R−/− control mouse. C, Section of a knee joint of wt mice taken at day 42 after five repeated injections of SCW fragments. Note the enhanced number of inflammatory cells (↔) and severe bone erosion (∗). D, Section of a knee joint of IL-17R−/− mice taken at day 42 after five repeated injections of SCW fragments. Note the reduced number of inflammatory cells and prevention of bone erosion in the IL-17R−/− compared with wt mice (C and D). H&E staining, original magnification, ×200. F, femur; P, patella; C, cartilage; Cb, cortical bone; S, synovitis; and JC, joint cavity.

FIGURE 3.

IL-17R deficiency prevents development of chronic destructive arthritis. A, Section of a knee joint of naive wt control mouse. B, Section of knee joint of naive IL-17R−/− control mouse. C, Section of a knee joint of wt mice taken at day 42 after five repeated injections of SCW fragments. Note the enhanced number of inflammatory cells (↔) and severe bone erosion (∗). D, Section of a knee joint of IL-17R−/− mice taken at day 42 after five repeated injections of SCW fragments. Note the reduced number of inflammatory cells and prevention of bone erosion in the IL-17R−/− compared with wt mice (C and D). H&E staining, original magnification, ×200. F, femur; P, patella; C, cartilage; Cb, cortical bone; S, synovitis; and JC, joint cavity.

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We next analyzed the mechanism responsible for the suppression of chronic destructive arthritis due to IL-17R signaling deficiency. We examined large gene expression profile in inflamed synovium by microarray analysis by taking synovium biopsies from IL-17R−/− and wt mice after five repeated local injections of SCW. A total of 80 genes met the criteria having a median signal log ratio ≥ 1 by comparing wt vs IL-17R−/− mice. Significantly lower mRNA expression of granulocyte chemotactic protein 2 (LIX), cytokine-induced neutrophil chemoattractant (KC), P-selectin, E-selectin, the proinflammatory cytokine IL-1β, stromal cell-derived factor-1 (SDF-1), and IFN-β was detected in the IL-17R−/− compared with wt mice (Table I). Furthermore, reduced mRNA expression of G-CSF receptor C-X-C-R2, C-C-R1, and the cell activation markers myeloid-related protein (MRP-8) and MRP-14 was detected in IL-17R−/− mice (Table I). This is in line with the histological observations of marked influx of leukocytes (neutrophils) in the synovial infiltrate in the wt mice after five repeated local injections of SCW into the joint. Interestingly, although the IL-17R−/− mice developed acute joint swelling after every rechallenge with SCW, histological analysis revealed markedly less neutrophils in the synovial infiltrate in these knockout mice compared with the wt mice. This indicates that the IL-17R signaling pathway plays a crucial role in recruiting neutrophils to the site of joint inflammation. In addition, the IL-17R signaling is crucial for the persistence of chronic synovitis.

Table I.

Microarray analysis of synovium biopsies from wt vs IL-17R−/− mice after five repeated local injections of SCW in the knee jointa

Up-Regulated Genes
(Fold change ± SD)Mann-Whitney U test
MRP-14 18.9 ± 2.0 p < 0.05 
LIX 17.1 ± 1.4 p < 0.05 
MMP-13 11.5 ± 2.1 p < 0.05 
MRP-8 10.0 ± 1.8 p < 0.05 
IL-1β 6.0 ± 1.9 p < 0.05 
KC (CXCL-1) 4.8 ± 1.3 p < 0.05 
Selectin-platelet 4.4 ± 2.1 p < 0.05 
Selectin-endothelial cell 4.0 ± 1.4 p < 0.05 
G-CSF receptor 3.4 ± 1.7 p < 0.05 
CXCR2 3.2 ± 1.6 p < 0.05 
SDF-1 3.3 ± 1.1 p < 0.05 
CCR1 2.4 ± 1.5 p < 0.05 
IFN-β 2.3 ± 1.4 p < 0.05 
Up-Regulated Genes
(Fold change ± SD)Mann-Whitney U test
MRP-14 18.9 ± 2.0 p < 0.05 
LIX 17.1 ± 1.4 p < 0.05 
MMP-13 11.5 ± 2.1 p < 0.05 
MRP-8 10.0 ± 1.8 p < 0.05 
IL-1β 6.0 ± 1.9 p < 0.05 
KC (CXCL-1) 4.8 ± 1.3 p < 0.05 
Selectin-platelet 4.4 ± 2.1 p < 0.05 
Selectin-endothelial cell 4.0 ± 1.4 p < 0.05 
G-CSF receptor 3.4 ± 1.7 p < 0.05 
CXCR2 3.2 ± 1.6 p < 0.05 
SDF-1 3.3 ± 1.1 p < 0.05 
CCR1 2.4 ± 1.5 p < 0.05 
IFN-β 2.3 ± 1.4 p < 0.05 
a

Data are expressed as the fold change in synovial mRNA expression between wt vs IL-17R−/− mice. Microarray analysis was performed on three separated samples per group. There is a significant (p < 0.05) up-regulation in mRNA expression for all the genes shown.

Significantly lower synovial mRNA expression of matrix metalloproteinase-13 ((MMP)-13, collagenase-3) was detected in the IL-17R-deficient mice compared with wt mice after five locally repeated injections of SCW into the joint (Table I). MMP-13 plays an important role in joint degradation, and these data suggest a direct relation between impaired IL-17 signaling and MMP-13 induction. In addition to microarray analysis, the mRNA expression of the proinflammatory cytokine IL-1β and the metalloproteinase MMP-13 was confirmed by Q-PCR analysis (data not shown).

Immunohistochemistry on whole knee joint sections of normal and arthritic mice using a murine IL-17R mAb revealed IL-17R protein expression on fibroblast-like cells, mononuclear cells, polymorphonuclear cells, endothelial cells, BM cells, and bone lining cells (Fig. 4). Immunohistochemistry revealed IL-17R expression on monocytes/macrophages in the synovial infiltrate and serial sections stained with F4/80 revealed overlap of IL-17R- and F4/80-positive cells, although not all F4/80-positive cells were IL-17R positive (Fig. 4, C–E). Fibroblast cultures derived from mouse synovium were IL-17R positive (Fig. 4, F and G).

FIGURE 4.

IL-17R expression on many different cell types in the joint during arthritis. A and B, Knee joint sections of naive mice showed IL-17R (A)- and F4/80 (B)-positive cells. Note IL-17R expression (A) on synovial lining cells (arrows) as well as on bone lining cells (arrowheads). F4/80-positive cells (B) were detected in the synovial lining as well as in the BM (arrowheads). C–E, Knee joint sections of arthritic mice showed IL-17R-positive cells (C), F4/80-positive cells (D), or control Ab (E). IL-17R expression (C) was detected in the synovial infiltrate on many cells such as on fibroblast-like synoviocytes, monocytes/macrophages, endothelial cells (arrows), as well as on cells along the cortical bone and at sites of focal bone erosion. Areas of F4/80-positive cells (D) revealed overlap with areas of IL-17R-positive cells (C vs D). F and G, IL-17R (F) or isotype specific control Ab (G) staining on cultured fibroblast derived from synovium biopsies of naive mice. Original magnification, A, B, F, and G, ×200; and C–E, ×400. L, synovial lining; Cb, cortical bone; S, synovitis; and Bm, bone marrow.

FIGURE 4.

IL-17R expression on many different cell types in the joint during arthritis. A and B, Knee joint sections of naive mice showed IL-17R (A)- and F4/80 (B)-positive cells. Note IL-17R expression (A) on synovial lining cells (arrows) as well as on bone lining cells (arrowheads). F4/80-positive cells (B) were detected in the synovial lining as well as in the BM (arrowheads). C–E, Knee joint sections of arthritic mice showed IL-17R-positive cells (C), F4/80-positive cells (D), or control Ab (E). IL-17R expression (C) was detected in the synovial infiltrate on many cells such as on fibroblast-like synoviocytes, monocytes/macrophages, endothelial cells (arrows), as well as on cells along the cortical bone and at sites of focal bone erosion. Areas of F4/80-positive cells (D) revealed overlap with areas of IL-17R-positive cells (C vs D). F and G, IL-17R (F) or isotype specific control Ab (G) staining on cultured fibroblast derived from synovium biopsies of naive mice. Original magnification, A, B, F, and G, ×200; and C–E, ×400. L, synovial lining; Cb, cortical bone; S, synovitis; and Bm, bone marrow.

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To investigate the contribution of local synovial IL-17R signaling in the development of chronic synovitis, we created BM chimeric mice. Following lethal irradiation, upon recovery hemopoietic cells will largely be donor derived. Structural tissues, which are less radiosensitive such as myocytes, fibroblasts, and endothelial cells, will not be destroyed and remain host derived.

Four combinations of BM chimeras were created (wt→wt, IL-17R−/−→IL-17R−/−, wt→IL-17R−/−, and IL-17R−/−→wt) and studied. FACS analysis on whole blood using a murine IL-17R mAb revealed IL-17R expression on granulocytes after transplantation of wt BM cells into irradiated wt mice (wt→wt), which was similar compared with IL-17R expression on granulocytes isolated from a naive C57BL/6 mouse (Fig. 5, A and B). The IL-17R was not detectable after BM transplantation of IL-17R−/− mice into irradiated IL-17R−/− mice (IL-17R−/−→IL-17R−/−) (Fig. 5,C). IL-17R was detected by FACS analysis on whole blood after wt BM transplantation into irradiated IL-17R−/− mice (wt→IL-17R−/−) (Fig. 5,D) but not after IL-17R−/− BM transplantation into irradiated wt recipient mice (IL-17R−/−→wt) (Fig. 5 E).

FIGURE 5.

FACS analysis of IL-17R expression in chimeric mice. Four combinations of BM chimeras were created. Lethally irradiated wt or IL-17R−/− mice underwent transplantation with wt or IL-17R−/− BM. Surface IL-17R expression was determined by flow cytometry using a specific murine anti-IL-17R Ab gated on the granulocyte population. Naive wt (C57BL/6) (A); wt→wt (B); IL-17R−/−→IL-17R−/− (C); wt→IL-17R−/− (D); and IL-17R−/−→wt (E).

FIGURE 5.

FACS analysis of IL-17R expression in chimeric mice. Four combinations of BM chimeras were created. Lethally irradiated wt or IL-17R−/− mice underwent transplantation with wt or IL-17R−/− BM. Surface IL-17R expression was determined by flow cytometry using a specific murine anti-IL-17R Ab gated on the granulocyte population. Naive wt (C57BL/6) (A); wt→wt (B); IL-17R−/−→IL-17R−/− (C); wt→IL-17R−/− (D); and IL-17R−/−→wt (E).

Close modal

These different groups of chimeric mice were used to induce the chronic relapsing SCW arthritis model. Similar to the results described earlier, wt→wt chimeric mice developed chronic synovitis. However, significant suppression was seen in the IL-17R−/−→IL-17R−/− chimeric mice (Fig. 6, A and B). Interestingly, chimeric mice expressing the IL-17R on the radiation-resistant population of cells in the joint but not on BM-derived hemopoietic cells (IL-17R−/−→wt) developed a similar pattern and degree of synovitis as the wt→wt chimeras (Fig. 6, A and B). Of high interest, chimeric mice that expressed the IL-17R on BM-derived hemopoietic/lymphoid cells but not on radiation-resistant cells in the joint (wt→IL-17R−/−) developed acute joint inflammation but not progressive synovitis, similarly as IL-17R−/−→IL-17R−/− chimeras (Fig. 6, A and B). Histological analysis confirmed prevention of full-blown synovitis in the wt→IL-17R−/− chimeric mice compared with IL-17R−/−→wt and wt→wt chimeras, although more cell influx was noted compared with IL-17R−/−→IL-17R−/− chimeras (Fig. 6,C). Interestingly, bone erosion was significantly suppressed under conditions where the IL-17R was not expressed on radiation-resistant cells in the joint (Fig. 6 C). This strongly indicates that radiation resistant cells expressing IL-17R in the joint play a critical role in the progression of destructive synovitis.

FIGURE 6.

Requirement of IL-17R signaling on resident synovial cells in development of progressive synovitis. At days 0, 7, 14, 21, and 35 (arrows), four groups of BM chimeric mice were i.a. injected with SCW fragments directly in the knee joints. Every first day (A) and seventh day (B) after injection of SCW, knee joint thickness was measured using a caliper. See Fig. 2 for more details. C, At day 42, the skin of the knee joints was removed, and the severity of knee joint inflammation was macroscopically scored (arthritis score) on a scale of 0–2 as described previously (36 ). In addition, knee joints were taken for histology. Synovial infiltrate and loss of bone was scored on a scale of 0–3. Results are the mean ± SEM of 12 knee joints of 6 chimeras/group. ∗, p < 0.05; ∗∗, p < 0.007; ∗∗∗, p < 0.0002 (IL-17R−/−→wt vs wt→IL-17R−/−) by Mann-Whitney U test.

FIGURE 6.

Requirement of IL-17R signaling on resident synovial cells in development of progressive synovitis. At days 0, 7, 14, 21, and 35 (arrows), four groups of BM chimeric mice were i.a. injected with SCW fragments directly in the knee joints. Every first day (A) and seventh day (B) after injection of SCW, knee joint thickness was measured using a caliper. See Fig. 2 for more details. C, At day 42, the skin of the knee joints was removed, and the severity of knee joint inflammation was macroscopically scored (arthritis score) on a scale of 0–2 as described previously (36 ). In addition, knee joints were taken for histology. Synovial infiltrate and loss of bone was scored on a scale of 0–3. Results are the mean ± SEM of 12 knee joints of 6 chimeras/group. ∗, p < 0.05; ∗∗, p < 0.007; ∗∗∗, p < 0.0002 (IL-17R−/−→wt vs wt→IL-17R−/−) by Mann-Whitney U test.

Close modal

In the present study, we demonstrated that lack of IL-17R signaling prevented full progression and persistence of chronic synovitis. Moreover, IL-17R deficiency prevented bone erosion. In addition, this study revealed the requirement of IL-17R signaling in radiation-resistant cells in the joint for turning an acute macrophage-dependent joint inflammation into a chronic T cell-mediated destructive arthritis.

In the model of murine SCW arthritis, flares can be induced at sites of smoldering arthritis by rechallenge with SCW (31). Acute joint swelling is noted after every rechallenge, but the remaining, smouldering arthritis is brought to a higher level of severity with time. Repeated injections of SCW in the knee joint of RAG-2 deficient mice showed suppression of chronic arthritis indicating a role for T and B cells in the development of chronic synovitis (W. B. Van den Berg et al., manuscript in preparation). Previously, using cytokine-specific inhibitors and TNF-α and IL-1β-deficient mice, it was shown that each flare remained TNF-α sensitive in terms of joint swelling. However, the chronic cellular infiltrate remains under TNF-deficient conditions and joint damage was not reduced significantly (31). Furthermore, IL-1 deficiency results in profound reduction of chronic synovial infiltrate and hardly any erosion was noted. These data suggest that the repeated flare reactions make the cellular process in the synovial tissue progressively an IL-1β-dependent phenomenon (31). In the present study, we clearly show that IL-17R signaling was not required for each acute TNF-α-mediated flare. However, IL-17 deficiency prevented the exacerbation and marked reduction of chronic synovial infiltrate, and bone erosion was noted. This suggests that the lack of IL-17R signaling in joint specific cells had no direct effect on the TNF-α-dependent acute flares. However, the critical role of IL-17R signaling in the development of chronic synovitis overlapped with the IL-1 dependency of the chronic synovial infiltrate characterized in an earlier study (31). Interestingly, significantly lower synovial IL-1 mRNA expression in the IL-17R−/− mice compared with wt mice was detected. This indicates a direct interrelation between synovial IL-17R signaling and synovial IL-1 expression during the chronic phase of arthritis, suggesting IL-17 signaling upstream of synovial IL-1 expression in the progressive chronic joint inflammatory process.

The proinflammatory nature of IL-17 has been implicated in various inflammatory diseases, such as inflammatory arthritis (18), lung inflammation (8, 13), organ allograft rejection (7), and autoimmune diseases (18, 19, 20, 21, 22). IL-17 induces various proinflammatory cytokines, chemokines, and cell adhesion molecules (4, 5). IL-17 supports hematopoiesis and IL-17-induced hematopoiesis results in maturation of cells along the granulocytic pathway (4, 6). Furthermore, IL-17 can specifically and selectively recruit neutrophils into the airways via the release of C-X-C chemokines, and anti-IL-17 Ab treatment inhibited the late-phase neutrophilia following LPS challenge (8, 13, 38). In addition to the chemotactic role of IL-17 in neutrophil accumulation in inflamed tissue, IL-17 is also involved in activation of neutrophils leading to elevated elastase and myeloperoxidase activity (39). In the present study, we demonstrated a critical role for IL-17R signaling in late-phase neutrophilia in the joint during progressive synovitis. In addition, in this study, we created specific chimeras and achieved a high degree of chimerism in myeloid cells with >90% engraftment in peripheral blood. Specific immunohistochemistry studies revealed that all four groups of BM chimeric mice had IL-17-positive cells in the synovium (data not shown), excluding that the suppression of chronic inflammation in chimeric mice was due to lack of IL-17-positive cells in the joint. Furthermore, it suggests that IL-17R signaling between dendritic cells and T cells is not necessary for IL-17 production. Host wt mice transplanted with BM cells of IL-17R−/− mice were still able to develop progressive synovitis. This indicates that IL-17-IL-17R signaling between dendritic cells and T cells that are IL-17R−/− is not critical in the development of progressive joint inflammation. In contrast, these IL-17R−/− activated T cells in the synovium are still able to produce IL-17 that can bind to IL-17R on a radiation-resistant population of cells in the joint contributing to the chronic inflammatory process. Of high interest, host IL-17R−/− mice transplanted with marrow donor cells of wt mice are protected from full-blown synovitis and bone erosion. In these mice, myeloid cells are IL-17R positive; however, IL-17 produced by activated T cells in the synovium cannot bind to joint resident cells, which are IL-17R−/−. Thus, IL-17R signaling in radiation-resistant cells in the joint was required for this leukocyte-mediated chronic inflammatory synovitis to develop.

To speculate about the origin of these radiation-resistant cell types, fibroblast-like synoviocytes, synovial macrophages as well as endothelial cells are potential candidates. In this study, we showed using specific IL-17R immunohistochemistry that many cell types in the joint infiltrate express the IL-17R protein. This is in line with the ubiquitous mRNA expression of IL-17R in many cell types (3). During arthritis, IL-17R expression was found on fibroblast-like cells, chondrocytes (data not shown), mononuclear cells, polymorphonuclear cells, endothelial cells, and bone lining cells. In addition, fibroblast isolated from synovial biopsies of naive mice were IL-17R positive. Under naive conditions many F4/80-positive cells in the synovial lining overlap with areas of IL-17R-positive cells. During arthritis F4/80-positive mononuclear cells in the synovial infiltrate revealed overlap with areas of IL-17R-positive cells, although not all F4/80-positive cells were IL-17R positive. This suggests that repopulation of IL-17R-positive F4/80 cells (predominantly monocytes/macrophages) during the 2-mo period of the chimeric mice experiments may play a role in the slight increase in the infiltrate after five repeated injections with SCW fragments in the wt→IL-17R−/− chimeric group compared with the double IL-17R−/− chimeric mice. Additional studies are needed to fully identify the IL-17 responding cell types that are critical to the chronic inflammatory arthritis process, and this is at present under investigation.

It is hard to distinguish between result and cause from the gene expression profile that was made from synovial tissues after five repeated local injections of SCW in the knee joint of wt and IL-17R−/− mice. There are down-regulated genes, such as IL-1 and MMP-13, that seem likely to be the result of less joint inflammation under IL-17R−/− conditions. In contrast, down-regulation of LIX, KC, selectins E and P, and chemokine receptors may be a direct cause of lack of IL-17R signaling in radiation-resistant cells in the joint. It is known that endothelial cells and fibroblast-like synoviocytes can highly respond to IL-17 by up-regulating cytokines/chemokines such as IL-6, IL-8/KC, and chemokine receptors (18, 26). Interestingly, mediators such as SDF-1 and IFN-β that are predominantly produced by fibroblasts were down-regulated. These data suggest that IL-17/IL-17R signaling in resident joint cells such as fibroblasts, macrophages, and endothelial cells keep up and/or amplify the inflammatory response by producing chemokines/cytokines that are responsible to recruit neutrophils leading to chronic synovitis.

IL-17 plays a potent stimulatory role in osteoclastogenesis. Promotion of type I collagen degradation in synovium and bone by IL-17 has been demonstrated and when combined with IL-1, a marked synergistic release of collagen was noted (40). IL-17, in combination with TNF-α, increased osteoclastic resorption in vitro (41). Furthermore, IL-17 induced the expression of receptor activator of NK-κB ligand (RANKL) in cultures of osteoblasts (19). Regulation of IL-17 and RANKL, as shown by IL-4 gene therapy in collagen arthritis, prevents osteoclastogenesis and bone erosion (36). Early neutralization of IL-17 using a soluble IL-17R fusion protein given systemically starting before arthritis expression in experimental arthritis prevented bone erosion (29, 42). In contrast, localized overexpression of IL-17 in the knee joint of type II collagen-immunized mice resulted in promotion of collagen arthritis and aggravated joint destruction (29). IL-17 promoted bone erosion in collagen arthritis through loss of the RANKL/osteoprotegerin (OPG) balance (43). Systemic OPG treatment prevented joint damage induced by local IL-17 gene transfer in type II collagen-immunized mice, suggesting that IL-17 is a potent inducer of RANKL and that the IL-17-induced promotion of bone erosion is mediated by RANKL. In the present study, the prevention of bone erosion after five repeated local injections of SCW in the joint in the absence of IL-17R signaling was accompanied by a decrease in the RANKL/OPG mRNA balance as was measured by Q-PCR (data not shown). In addition, lack of the IL-17R resulted in a significant reduction of synovial MMP-13 mRNA expression. MMP-1/MMP-13 in mouse is comparable with MMP-13 in humans, and this metalloproteinase plays a key role in the MMP activation cascade and appears to be critical in bone metabolism and homeostasis (44). Furthermore, MMP-13 has a high potential to cleave type I as well as type II collagen. In addition to synovial tissue, osteoblasts produce MMP-13 and under conditions of chronic inflammation, multiple T cell cytokines may synergize to induce MMP-13 (45). Data from the present study revealed that synovial IL-17R signaling is critical in the induction of RANKL and MMP-13 during chronic joint inflammation in vivo.

In conclusion, we have demonstrated the requirement of IL-17R signaling in turning an acute inflammatory arthritis into a chronic synovitis accompanied with tissue destruction. Of high interest, local IL-17R signaling in radiation-resistant joint cells is needed for full progression of destructive synovitis. IL-17R deficiency results in diminished recruitment of leukocytes to the site of joint inflammation. These findings provide further understanding of the role of IL-17-IL-17R signaling in the pathogenesis in chronic joint inflammation and tissue destruction and underscore the importance of interaction of T cell mediators and synoviocytes in the progression of destructive arthritis. Prevention of this interaction warrants consideration as a therapeutic target in chronic, destructive arthritis as well as other chronic inflammatory diseases.

We thank Dr. Patrick Byrne and Terrance Jordan for their technical support with the microarray experiments, Peter Oliver and Connie Porretta for their technical help with the FACS analysis, and Marije Koenders, Birgitte Oppers-Walgreen, and Liduine van den Bersselaar for their technical help with the Q-PCR experiments, preparing histology, and immunohistochemistry.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by Veni Fellowship Grant 906-02-038 (to E.L.) and Talent Stipend S95-384 (to E.L.) from the Netherlands Organization for Scientific Research (NWO).

4

Abbreviations used in this paper: CIA, collagen-induced arthritis; SCW, streptococcal cell wall; wt, wild type; BM, bone marrow; LIX, granulocyte chemotactic protein 2; KC, cytokine-induced neutrophil chemoattractant; MMP, matrix metalloproteinase; i.a., intra-articular; Q-PCR, quantitative real-time PCR; MRP, myeloid-related protein; RANKL, receptor activator of NK-κB ligand; OPG, osteoprotegerin.

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