IL-17 mediates articular hypernociception in... : PAIN (original) (raw)

1. Introduction

Rheumatoid arthritis (RA) is a chronic autoimmune disease that presents acute inflammatory episodes. RA is characterized by an increase in the infiltration of immune cells (including neutrophils, T cells, B cells, and macrophage) into the synovial membrane, cavity and periarticular tissues [33]. One of the most prevalent symptoms of RA is the increase in sensitivity to joint pain (hyperalgesia), which causes limitations in movements. Despite its clinical relevance, strategies for the treatment of articular hyperalgesia remain limited. In animal models, hyperalgesia is defined as hypernociception (decrease of nociceptive threshold) [17]. It is broadly accepted that articular hypernociception results mainly from the direct and indirect effects of inflammatory mediators on the sensitization (increase of excitability) of primary nociceptive fibers that innervate the inflamed joints [50,59,60]. Prostaglandins and sympathetic amines are the key mediators of this process and their release is generally stimulated by the release of cytokine cascades (TNF-α, IL-1β and chemokines), a process that appears to be neutrophil-dependent [14,16,20,23,41]. Furthermore, other mediators, such as endothelin-1 (ET-1), acting directly or indirectly, also sensitize primary nociceptive neurons [24,64,66,67].

The pathophysiology of RA is complex and appears to be initiated when the adaptive immune system (cellular or humoral) recognizes self joint antigens as non-self, which triggers a variety of distinct inflammatory effector mechanisms, which include the recruitment of leukocytes [4,34,39,52]. The majority of these mechanisms are mediated by a number of cytokines and chemokines [9]. Several studies have shown that among these cytokines, interleukin (IL)-17 appears to play a crucial role. Mice lacking this cytokine are more resistant to induction of arthritis in several different experimental models [10,49]. Moreover, the levels of IL-17, as well as the number of IL-17 expressing cells, are increased in the synovial fluid of RA patients [11,45]. IL-17 is secreted primarily by CD4+ T cells, traditionally known as Th17 cells, but is also secreted by CD8+ activated memory T cells, NKT cells and γδ lymphocytes [51]. There is convincing data that have demonstrated that IL-17 orchestrates multiple inflammatory events of RA, including recruitment and activation of leukocytes (primarily neutrophils), stimulation of the release of cytokines (TNF-α and IL-1β), chemokines (IL-8, LIX and KC) and cytotoxic mediators (MMPs, superoxide) [1,27,35,57,58,70]. The pro-inflammatory actions of IL-17 are believed to be involved in the lesions of joint tissues observed during RA. Aside from these effects, there is no evidence that IL-17 contributes to the genesis of articular hypernociception. Therefore, we investigated the role of IL-17 in the cascade of events that participate in the genesis of articular hypernociception in a model of antigen (mBSA)-induced arthritis in mice. We focused primarily on the interaction between IL-17 and the classical mediators of inflammatory hypernociception.

2. Materials and methods

2.1. Animals

The experiments were performed on male BALB/C or C57BL/6 WT mice and TNFR1 (C57BL/6 background) deficient mice weighing between 20 and 25g. They were housed in temperature-controlled rooms (22–25°C) and given water and food ad libitum at the animal facility in the Department of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo, Brazil. Animal care and handling procedures were in accordance with the guidelines of the International Association for Study of Pain (IASP) and with the approval of the Animal Ethics Committee of the School of Medicine of Ribeirão Preto, University of São Paulo – USP.

2.2. Drugs

The following materials were obtained from the sources indicated: recombinant murine IL-17 and anti-IL-17 antibody (MAB421) (R&D Systems, Minneapolis, MN); ET-1 (American Peptides); guanethidine, fucoidin, PGE2, dopamine, zymosan, mBSA and CFA (Sigma–Aldrich, St. Louis, MO, USA); indomethacin (Prodome, Campinas, Sao Paulo, Brazil); infliximab (Schering-Plough); IL-1 receptor antagonist (IL-1ra) (National Institute for Biological Standards and Control, South Mimms, Hertfordshire, UK); doxycycline (Pfizer); DF-2156 (Dompe S.p.A., L'Aquila, Italy) was a gift of Prof. Mauro M. Teixeira (Federal University of Minas Gerais); bosentan was kindly donated by Actelion Pharmaceuticals (Allschwil, Switzerland). The drugs were diluted in sterile saline, except bosentan that was diluted in Arabic gum 5% in distilled water.

2.3. Induction of experimental arthritis

2.3.1. Antigen-induced arthritis (AIA)

Mice were immunized as described previously [7,29]. Briefly, mice were sensitized with 500μg of methylated bovine serum albumin (mBSA) in 0.2ml of an emulsion containing 0.1ml saline and 0.1ml complete Freund's adjuvant (CFA; 1mg/ml of Mycobacterium tuberculosis) and given by subcutaneous (s.c.) injection on day 0. The mice were boosted with the same preparation on day 7. Sham-immunized mice were given similar injections but without the antigen (mBSA). Twenty-one days after the initial injection, arthritis was induced in the immunized animals by intraarticular (i.a.) injection of mBSA (10, 30 or 90μg/cavity), IL-17 (3, 10 or 30ng/cavity), PGE2 (30, 100 and 300ng/cavity) or dopamine (10, 30 and 100μg/cavity) dissolved in 10 μL of saline into the right femur–tibial joint.

2.3.2. Zymosan-induced arthritis (ZIA)

ZIA was induced using the protocol described previously [56]. In brief, the joint inflammation was induced by administration i.a. of 30μg of zymosan (from Saccharomyces cerevisiae) diluted in 10μL of saline into the right femur–tibial joint. Control mice were injected with 10μL of saline into the joint.

2.4. Evaluation of articular hypernociception

The articular hypernociception of the femur–tibial joint was evaluated using a previous method [15,30] with modification. In a quiet room, mice were placed in acrylic cages (12×10×17cm high) with a wire grid floor 15–30min before testing for environmental adaptation. Stimulations were performed only when animals were quiet, did not display exploratory movements or defecation, and were not resting on their paws. In these experiments, an electronic pressure-meter was used. It consists of a hand-held force transducer fitted with a polypropylene tip (IITC Inc., Life Science Instruments, Woodland Hills, CA, USA). For this model, a large tip (4.15mm2) was adapted to the probe. An increasing perpendicular force was applied to the central area of the plantar surface of the hind paw to induce flexion of the femur–tibial joint followed by paw withdrawal. A tilted mirror below the grid provided a clear view of the hind paw. The electronic pressure-meter apparatus automatically recorded the intensity of the force applied when the paw was withdrawn. The test was repeated until three subsequently consistent measurements (i.e. the variation among these measurements was less than 1g) were obtained. The flexion-elicited mechanical threshold was expressed in grams (g).

2.5. Experimental protocol

Immunized mice were pre-treated with indomethacin (5mg/kg, i.p. 30min before stimuli injection) [16], a standard cyclooxygenase inhibitor; guanethidine (30mg/kg, s.c. 60min before stimuli injection) [16], a sympathomimetic neuron-blocking agent; bosentan (100mg/kg, p.o. 60min before IL-17 injection) [67], dual endothelin receptor type A (ETA)/endothelin receptor type B (ETB) antagonist; doxycycline (3, 10 and 30mg/kg, p.o. 30min before stimuli injection) [32], a non-selective matrix metalloproteinases (MMPs) inhibitor; fucoidin (20mg/kg, i.v. 15min before and 3.5h after stimuli injection) [69], a leukocyte adhesion inhibitor; DF-2156 (30mg/kg, i.v. 20min before stimuli injection) [18], an allosteric antagonist of CXCR1/2; IL-1ra (50mg/kg, i.v. 30min before and 3.5h after stimuli injection) [68]; infliximab (10mg/kg, i.p. 48h and 60min before IL-17 injection); anti-TNF-α antibody [44]; anti-IL-17 (2.25μg, i.a. co-injection) was administered simultaneously with mBSA. Articular hypernociception was determined at various time-points (see legends) after the i.a. injections of the stimuli.

2.6. In vivo neutrophil migration

Immunized or sham-immunized mice had mBSA, IL-17 or saline injected directly into the articular cavity. At various time-points after the injection of the inflammatory stimuli, the mice were sacrificed. The articular cavities were washed twice with 5μL PBS containing 1mM EDTA, then diluted to a final volume of 100μL with PBS/EDTA to evaluate leukocyte migration at the indicated times. The total number of leukocytes was determined in a Neubauer chamber diluted in Turk's solution. Differential cell counts were determined in cytocentrifuge Rosenfeld stained slices (Cytospin 4; Shandon, Pittsburg, PA). Differential cell counts were performed with a light microscope and the results were expressed as the number (mean±SEM) of neutrophils per cavity [56].

2.7. Cytokine measurements

At indicated times after the i.a. injection of the inflammatory stimuli, animals were terminally anesthetized and the knee joints were removed and homogenized in 500μL of buffer containing protease inhibitors. IL-17, TNF-α, IL-1β and keratinocyte-derived chemokine (KC/CXCL1) concentrations were determined as described previously [16] by ELISA using paired antibodies (R&D Systems). The results are expressed as pg/joint of each cytokine. As a control, the concentrations of these cytokines were determined in immunized animals injected with saline and in sham-immunized mice injected with mBSA.

2.8. MMPs measurements by gelatin zymography

Gelatinolytic activities of MMPs were examined by gelatin zymography as described [32]. The synovial membranes were collected 7h after challenge with IL-17 or saline and homogenized in Tris–CaCl2 buffer containing protease inhibitors. To confirm that the quantified gelatinolytic proteinase activities were specific for MMPs, either _o_-phenanthroline (1mM) was added to incubation buffer, abolishing all gelatinolytic activities. The protein concentrations of the lysate were determined using a BCA Protein Assay Kit (Pierce, Rockford, IL) and 10μg of protein from synovial membrane homogenate were electrophoresed through an 10% polyacrylamide gel copolymerized with gelatin (40mg/mL, type A from porcine skin; Sigma). The gels were washed with 2% Triton X-100 and incubated for 18h at 37°C in activation buffer (50mmol/L Tris–HCl, 150mmol/L sodium chloride, 10mmol/L calcium chloride, and 0.05% sodium azide). After incubation, the gels were stained with 0.05% Coomassie brilliant blue (G-250; Sigma). Zymograms were digitally scanned, and band intensities were quantified using ImageJ Program (NIH- National Institute of Health).

2.9. Determination of PGE2 production

Two and six hours after i.a. injections of IL-17, the knee joints were collected. These samples were collected in a mixture of 1ml extraction solvent (isopropanol/ethyl acetate/0.1N HCl, 3:3:1) and 0.5ml PBS containing indomethacin (20μg/ml). After homogenizing with a polytron, the samples were centrifuged at 2000_g_ for 10min at 4°C. The organic phase was aspirated and evaporated to dryness in a centrifugal evaporator. The pellet was reconstituted in 500μL of 0.1M phosphate buffer (pH 7.4) containing 0.8% sodium azide and 0.1% gelatin. The concentrations of PGE2 were determined by RIA using commercially available reagents [67]. The results are expressed as pg of PGE2 per joint.

2.10. Reverse transcription-polymerase chain reaction (RT-PCR) assays

The COX-2, MMP-2, MMP-9 and prepro-endothelins (PPET-1) mRNA assay were performed by RT-PCR as previously described [68]. Briefly, mice were sacrificed at indicated times after the IL-17 or mBSA injections (i.a.), and the synovial membranes were harvested. Total cellular RNA from synovial membrane was extracted using the Trizol reagent (Invitrogen Life Technologies Corporation, Carlsbad, CA) according to the directions supplied by the manufacturer. The purity of total RNA was measured with a spectrophotometer. The wavelength absorption ratio (260/280nm) was between 1.8 and 2.0 for all preparations. Reverse transcription of total RNA to cDNA was carried out by reverse transcription (Superscript II, Gibco Life Technologies, Grand Island, NY, USA). Real-time quantitative PCR mRNA analysis was performed in an ABI Prism 7500 Sequence Detection System using the SYBR-green fluorescence system (Applied Biosystems, Warrington, UK) for quantification of amplicons. RT-PCR was performed in a 13μL reaction volume and carried out with the following cycling parameters: Initial heating at 95°C (10min) followed by 40 cycles of 94°C (1min), 56°C (1min) and 72°C (2min). Melting curve analysis was performed (65–95°C) to verify the amplification of a single product. Samples with more than one peak were excluded. The data were analyzed according to the comparative cycle threshold (CT) method. Primers pairs for mouse β-actin, COX-2, MMP-2, MMP-9 and PPET-1were as follows:

2.11. Statistical analysis

Data are reported as means±SEM and are representative of two separate experiments. Two-way ANOVA was used to compare the groups and doses at all times when the hypernociceptive responses were measured at different times after the stimulus injection. The means from different treatments were compared by one-way ANOVA with Bonferroni's correction. P values less than 0.05 were considered significant.

3. Results

3.1. IL-17 mediates antigen-induced articular mechanical hypernociception

Previous reports from our laboratory showed that the administration of antigen (mBSA) into the paw or tibio–tarsal joints of immunized mice caused a dose-dependent decrease in the mechanical nociceptive threshold (hypernociception) [19,68]. In this study, we observed that a mBSA challenge into the femur–tibial joint induced articular hypernociception in a time and dose-dependent manner. The i.a. injection of mBSA-induced a significant dose-dependent (10–90μg/joint) decrease in the nociceptive threshold of immunized balb/c mice when compared either to mice that were injected with saline or to mice that were sham-immunized and subsequently injected with mBSA (Fig. 1A). The mBSA-induced articular hypernociception started 3h after injection, which reached a plateau between 7 and 24h and returned to control levels at 72 and 96h after challenge (Fig. 1A).

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Fig. 1.:

Role of IL-17 in mBSA challenge-induced mechanical articular hypernociception. (A) mBSA- immunized or sham-immunized balb/c mice were challenged i.a. with 10–90 μg of mBSA or 10 μL of saline. Articular hypernociception was evaluated 1–96 h following antigen challenge. (B) Articular hypernociception was evaluated 1–24 h after i.a. injection with either mBSA (30 μg) or saline in mBSA-immunized balb/c mice treated with a co-injection of IgG control (α-CTL) or α-IL-17 (2.25 μg/cavity) antibodies. At 24 h, the mice were treated again with a second dose of antibodies. Articular hypernociception was evaluated 3 h following the second administration. (C) mBSA-immunized balb/c mice were injected i.a. either with mBSA (30 μg) or saline and the concentration of IL-17 was determined at 3 and 12 h after challenge. (D) mBSA-immunized balb/c mice were challenged i.a. with either 3–30 ng of IL-17 or 10 μL of saline and articular hypernociception was evaluated over a period of 7 h. Data are means ± SEM (n = 5) and representative of two independent experiments. *p < 0.05 vs. saline group; and # p < 0.05 vs. mBSA group.

In an attempt to evaluate the participation of IL-17 in the genesis of articular hypernociception during mBSA-induced arthritis, we treated mBSA-challenged balb/c mice with a neutralizing antibody against IL-17. Mice that were treated with anti-IL-17 antibody showed significantly reduced articular hypernociception at 5 and 7h after mBSA injection into the knee joints compared to mice that were treated with a control isotype antibody. The anti-nociceptive effect of anti-IL-17 ended 24h after its administration. The mice were treated at this point with the same dose of antibody and the nociceptive response was evaluated 3h later. A significant anti-nociceptive effect was observed with the second dose (Fig. 1B), suggesting that IL-17 continues to be released at this later time-point (27h post mBSA administration) following antigen challenge. Confirming our observations, we also detected increased levels of IL-17 in the joint tissue after the immunized mice were challenged with mBSA (30μg/joint) (Fig. 1C).

The pro-nociceptive role IL-17 in antigen-induced arthritis was further validated when we found that intraarticular (femur/tibial) injection of IL-17 in immunized balb/c mice also produced a dose- and time-dependent decrease in the nociceptive threshold (Fig. 1D). A significant articular nociceptive response was observed 1h after IL-17 injection at doses of 10 and 30ng/joint, which progressively increased 7h following the challenge. A dose of 30ng per joint of IL-17 was used in subsequent experiments. These data clearly suggest the involvement of IL-17 in articular hypernociception during antigen-induced arthritis.

The participation of IL-17 in the genesis of articular hypernociception was also address in zymosan (30μg/joint)-induced arthritis. Differently that was observed in mBSA-induced arthritis, the treatment of balb/c mice with anti-IL-17 antibody did not alter mechanical hypernociception in zymosan-induced arthritis, determined in the intervals of 1–7h after zymosan injection (mechanical threshold in grams: control-saline injection: 9.86±0.38; zymosan: 4.46±0.77*; zymosan+anti-17 antibody: 5.19±04, _n_=5 per experimental group *p<0.05 compared with saline group; one-way ANOVA, followed by Bonferroni's t test. Results refer the hypernociception measured 5h after zymosan injection).

3.2. Cytokines and chemokines mediate the pro-nociceptive role of IL-17

Cytokines (e.g. TNF-α and IL-1β) and chemokines (e.g. KC/CXCL1) play an important role in the genesis of inflammatory hypernociception [17,65]. We investigated the role of these mediators in the IL-17-induced articular hypernociception. IL-17-induced articular hypernociception was reduced in TNFR1−/− mice compared with WT (C57BL/6) mice (Fig. 2A). Additionally, pre-treatment of balb/c mice with infliximab (a clinically used anti-TNF-α antibody) significantly diminished the hypernociceptive effect of IL-17 (Fig. 2B). IL-17-induced articular hypernociception was also inhibited by pre-treatment with an IL-1 receptor antagonist (IL-1Ra) and with DF-2156 (CXCR1/2 antagonist) (Fig. 2B). To further confirm the participation of these cytokines in the pro-nociceptive effect of IL-17, the levels of TNF-α, IL-1β and KC/CXCL1 were evaluated in joint tissue after IL-17 challenge. The administration of IL-17 in the articular cavity increased TNF-α, IL-1β and KC production 1, 2 and 4h after IL-17 injection (Fig. 3A–C, respectively). These findings suggest that TNF-α, IL-1β and CXC chemokines act downstream of IL-17 to mediate mBSA-induced articular hypernociception.

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Fig. 2.:

Role of cytokines and chemokines in IL-17-induced mechanical hypernociception in the knee joint. (A) mBSA-immunized wild type (C57BL/6) or TNFR1−/− mice were challenged i.a. with either IL-17 (30 ng per joint) or saline and articular hypernociception was evaluated 7 h following challenge. (B) IL-17 (30 ng per joint) was injected into balb/c mice that were pre-treated with infliximab (10 mg/kg, i.p. 48 h and 60 min before stimuli injection), IL-1ra (50 mg/kg, i.v. 30 min before and 3.5 h after stimuli injection) or DF-2156 (30 mg/kg, i.v. 20 min before stimuli injection). Articular hypernociception was evaluated 7 h after the challenge. Data are means ± SEM (n = 5), representative of two independent experiments. *p < 0.05, compared with saline group; and # p < 0.05, compared with control/WT groups.

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Fig. 3.:

IL-17 induces pro-inflammatory cytokine production. The concentrations of TNF- α (A), IL-1β (B), and KC (C) in the knee joint injected with either 30 ng of IL-17 or saline in mBSA- immunized balb/c mice were determined at 1, 2, and 4 h after challenge. mBSA-immunized mice were sacrificed and knee joint samples were extracted to determine the levels of cytokines by ELISA. Results are mean ± SEM (n = 4), representative of two independent experiments. *p < 0.05, compared with saline group.

3.3. Neutrophils are involved in IL-17 mediation of antigen-induced arthritis

We had previously shown that the administration of mBSA into the femur–tibial joints of immunized mice induced a massive increase in the number of neutrophils within the joint cavity [29]. Confirming this finding, the mBSA challenge in the joint induced a dose-dependent stimulation of neutrophil migration 7h after the challenge, which returned to basal levels 96h after challenge (Fig. 4A). The treatment of the balb/c mice with a neutralizing antibody against IL-17 also reduced mBSA-induced neutrophil migration when measured at 7h following the challenge (Fig. 4B). Administration of an anti-IL-17 antibody (0 and 24h after mBSA injection) inhibited the neutrophil recruitment to the joint 27h after challenge (Fig. 4C) while an i.a. injection of IL-17 induced a dose-dependent induction of neutrophil recruitment 7h after challenge (Fig. 4D). The interdependence between the pro-nociceptive response and the neutrophil migration that was induced by IL-17 was also evaluated. mBSA-immunized mice were pre-treated with fucoidin (a leukocyte adhesion inhibitor) [47] and subsequently treated with an i.a. injection of IL-17 or mBSA. Seven hours later, the extent of articular neutrophil recruitment and hypernociception were determined. The IL-17- and mBSA-induced migration of articular neutrophils was significantly reduced by fucoidin, which was accompanied by a concomitant reduction in the hypernociceptive response (Fig. 4E and F). These results suggest that the production of IL-17 in the joints during RA trigger a neutrophil-dependent hypernociceptive response.

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Fig. 4.:

IL-17 mediates neutrophil recruitment to the knee joint of mice. Association of hypernociception and neutrophil migration. (A) Neutrophil recruitment from the articular cavity 7 and 96 h after i.a. injection of either mBSA (10–90 μg) or saline. (B and C) mBSA-immunized balb/c mice were challenged i.a. with mBSA (30 μg) or saline and treated with a co-injection of IgG control (α-CTL) or anti-IL-17 (2.25 μg/cavity) antibodies. (B) Neutrophil migration was evaluated 7 h after challenge. (C) Balb/c mice were treated with a second round antibodies and neutrophil recruitment was evaluated 3 h after antibody treatment. (D) Neutrophil recruitment was evaluated 7 h after i.a. injection of either IL-17 (3–30 ng per joint) or saline in mBSA-immunized balb/c mice. Neutrophil migration (E) and articular hypernociception (F) in balb/c mice challenged with IL-17 (30 ng per joint) or mBSA (30 μg per joint) and pre-treated with fucoidin (20 mg/kg, i.v. 15 min before and 3.5 h after stimuli injection) were evaluated 7 h after challenge. Data are means ± SEM (n = 5), representative of two independent experiments. *p < 0.05 vs. saline group; and # p < 0.05 vs. mBSA or IL-17 group.

3.4. MMP-9 mediates the hypernociceptive effect of IL-17

The migrated leukocytes release MMPs, which are enzymes involved in the matrix degradation and production of inflammatory mediators, such as endothelins [22]. MMP also participate in the pro-inflammatory effects of IL-17 [38]. Thus, we investigated the participation of these proteinases in the articular hypernociception during mBSA-induced arthritis. The pre-treatment with a non-specific inhibitor of MMPs, doxycycline reduced the pro-nociceptive effect of mBSA in a dose-dependent manner (Fig. 5A). Furthermore, the joint hypernociceptive effect of IL-17 was also inhibited by pre-treatment with doxycycline (30mg/kg) (Fig. 5B). Consistently, joint injection of IL-17 induced significant increase of MMP-9 mRNA expression in synovial tissue 1.5h after its injection. The MMP-2 mRNA expression was not affected by IL-17 injection (Fig. 5C). Moreover, we investigated whether the increased expression of MMP-9 was related to augment of its enzymatic activity. Gelatin zymography showed increase activity of MMP-9, but not MMP-2, 7h after i.a. administration of IL-17 (Fig. 5D). These findings indicate that MMP-9 (gelatinase B) contributes for the hypernociceptive effect of IL-17.

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Fig. 5.:

Role of MMP-9 and endothelin-1 in articular hypernociception induced by IL-17. (A) mBSA-immunized balb/c mice were pre-treated with doxycycline (3, 10 or 30 mg/kg, p.o. 30 min before stimuli injection) and challenged i.a. with mBSA (30 μg/cavity). Articular hypernociception was determined 7 h after challenge. (B) Articular hypernociception induced by IL-17 (30 ng/cavity) in mBSA-immunized balb/c mice pre-treated with doxycycline (30 mg/kg, p.o. 30 min before stimuli injection). The articular hypernociception was determined 7 h after challenge. (C) mBSA-immunized balb/c mice were challenged i.a. with saline or IL-17 (30 ng). After 1.5 and 5 h, synovial membranes were collected and analyzed for MMP-2 and MMP-9 mRNA expression by PCR. The gene expression was normalized to β-actin expression. (D) A representative SDS-PAGE gelatin zymogram of synovial membranes. Molecular weights of MMP-2 and MMP-9 bands were identified after electrophoresis on 10% SDS–PAGE. Std, standard (2 μL 25% fetal bovine serum). Bottom, intensity of specific MMP bands. The values for 72 kDa MMP-2 and 92 kDa MMP-9. (E) Articular hypernociception in balb/c mice challenged with IL-17 (30 ng per joint) and pre-treated with bosentan (100 mg/kg, p.o. 60 min before stimuli injection) was evaluated 7 h after challenge. (F) The synovial membranes were collected 1.5 after the i.a. injection of IL-17 and analyzed for PPET-1 mRNA expression by PCR. The gene expression was normalized to β-actin expression. (G) Dose– and time–response curve of the articular hypernociception induced by i.a. injection of ET-1 (30–300 pmol/cavity) in mBSA-immunized balb/c mice. The hypernociceptive responses were evaluated 1, 3, 5 and 7 h after the stimuli injection. Results are mean ± SEM (n = 5), representative of two independent experiments. *p < 0.05, compared with saline group; and # p < 0.05, compared with mBSA or IL-17 group.

3.5. Role of ET-1 in the hypernociceptive effect of IL-17

Previous reports of our group demonstrated that ETs are involved in genesis of inflammatory hypernociception in antigen-induced inflammation [69]. Moreover, there is evidence that MMP-9 cleaves Big-endothelin in active endothelin in neutrophils [22]. Therefore, we investigated whether ETs is involved in IL-17-induced hypernociception. The hypernociceptive effect of IL-17 was reduced by pre-treatment of the mice with bosentan (dual ETA/ETB receptor antagonist), 7h after the i.a. injection of IL-17 (Fig. 5E). Moreover, the administration of IL-17 induced increase of PPET-1 mRNA expression at 1.5h after injection (Fig. 5F). According, joint administration of ET-1 in immunized balb/c mice induced a dose- and time-dependent hypernociceptive response (Fig. 5G). Together, these data indicate that endothelins are important mediator involved in IL-17 mediated articular hypernociception.

3.6. IL-17-induced articular hypernociception depends on prostanoids and sympathetic amines

Prostanoids and sympathetic amines are considered the most important final mediators of inflammatory hypernociception since they are released into the inflammatory focus and directly sensitize the primary nociceptive neurons [23,41,53]. We investigated whether PGE2 and sympathetic amines participate in the IL-17-induced articular hypernociceptive response. The articular hypernociception response induced by IL-17 was significantly reduced by pre-treatment with indomethacin or guanethidine, and was more effectively inhibited when the animal were treated by association of indomethacin and guanethidine (Fig. 6A). The i.a. injection of PGE2 and dopamine into immunized balb/c mice produced a time- and dose-dependent decrease in the nociceptive threshold (Fig. 6B and C). Supporting these results, anti-IL-17 treatment inhibited COX-2 mRNA expression induced by mBSA in immunized mice, while i.a. injection of IL-17 promoted an increase of COX-2 mRNA expression at 1.5h after injection (Fig. 6D). Administration of IL-17 also promoted an increase in the PGE2 levels in the joint exudates at 2 and 6h after challenge (Fig 6E). Thus, these findings suggest that prostanoids and sympathetic amines mediate the hypernociceptive actions of IL-17 during antigen-induced arthritis.

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Fig. 6.:

Role of prostanoids and sympathomimetic amines in IL-17-induced mechanical articular hypernociception in the knee joint of mice. (A) Articular hypernociception induced by IL-17 (30 ng/cavity) in mBSA-immunized balb/c mice pre-treated with indomethacin (5 mg/kg, i.p. 30 min before stimuli injection), guanethidine (30 mg/kg, s.c. 60 min before stimuli injection) or indomethacin plus guanethidine. The articular hypernociception was determined 7 h after challenge. (B and C) Dose– and time–response curves of the articular hypernociception induced by i.a. injections of PGE2 (30–300 ng/cavity) (B) and dopamine (10–100 μg/cavity) (C) in mBSA-immunized balb/c mice. The hypernociceptive responses were evaluated 0.5, 1, 3, 5 and 7 h after the stimuli injection. (D) mBSA-immunized balb/c mice were challenged i.a. with either saline, IL-17 (30 ng) or mBSA (30 μg) and treated with co-injection of IgG control (α-CTL) or anti-IL-17 (2.25 μg/cavity) antibodies. After 1.5 h, synovial membranes were collected and analyzed for COX-2 mRNA expression by PCR. The gene expression was normalized to β-actin expression. (E) The knee joints of mBSA-immunized balb/c mice were collected 2 and 6 h after challenge i.a. with IL-17 and assayed for PGE2 levels. Results are mean ± SEM (n = 5), representative of two independent experiments. *p < 0.05, compared with saline group; and # p < 0.05, compared with IL-17 or mBSA group.

4. Discussion

Pain is one of the most important symptoms in RA as it causes a significant impairment in patients [50]. While current therapies exist to reduce this symptom, they are always associated with detrimental side effects. Therefore, the discovery of novel therapeutic targets is necessary. In this study, we demonstrated, for the first time, a pro-nociceptive role of IL-17 in a model of antigen-induced arthritis in mice. Results suggest that IL-17 may be a potential novel therapeutic target for controlling inflammatory pain during RA.

In studying the pain triggering mechanisms in RA, the selection of an experimental model that approximates the physiopathological RA events is important. The mBSA-induced arthritis mice model is a suitable and reproducible experimental system that exhibits several features akin to those observed in human RA, including hypernociception [7]. We had previously shown that plantar or tibio–tarsal joint administration of mBSA produced a dose- and time-dependent decrease of the nociceptive threshold in immunized mice [19,68]. In the present study, the mBSA challenge into femur–tibial joint also produced articular hypernociception with a profile similar to that observed after paw or tibio–tarsal joint injection with the advantage of direct quantification of the cellular influx in the joint.

Cytokines are the most important substances involved in the induction of inflammatory events of RA. The current idea is that the Th17 derived cytokine, IL-17, plays a pivotal role in the physiopathology of RA [11]. This hypothesis is supported by studies that have shown that pharmacological or genetic inhibition of IL-17 production or action attenuated the events of experimental RA, such as leukocyte migration, cartilage and bone erosion [10,48]. These reports have supported the view of IL-17 as potential target in RA. Further supporting this suggestion, we have shown that IL-17 is involved in the cascade of events involved in the genesis of hypernociception during antigen-induced arthritis in mice. Indeed, neutralization of IL-17 with a specific antibody reduced mBSA-induced hypernociception. Moreover, not only was IL-17 found in the mBSA-induced joint exudates, but direct joint administration of recombinant IL-17 decreased the nociceptive threshold of mice. Interestingly, in zymosan-induced arthritis, a model of innate inflammation, IL-17 is not involved in the hypernociception. Zymosan is a polysaccharide from the cell wall of S. cerevisiae that binds to Toll-like receptor 2 inducing a persistent hypernociception, which is mediated by prostaglandin, cytokines and leukotriene B4[28,31]. Thus, the pro-nociceptive effect of IL-17 is mainly observed during Th17-driven immune response. Nevertheless, a recent study demonstrated that an association existed between IL-17 and the establishment of neuropathic pain [43]. Following chronic constriction injury of the sciatic nerve in mice, Th17 cells infiltrate the site of the lesion and release IL-17. Thus, IL-17 targeting drugs could be also an effective strategy in the treatment of neuropathic pain states.

We also evaluated the mechanisms underlying the IL-17-induced articular hypernociceptive effect. Previous reports have demonstrated that the role of IL-17 in the pathogenesis of RA is secondary to the induction of several inflammatory mediators such as TNF-α, IL-1β and CXCR1/2 ligands [3,46,57]. For instance, IL-17 stimulates mRNA expression and synthesis of pro-inflammatory cytokines such as TNF-α and IL-1β in several cell types [37]. Since these cytokines and chemokines also play a crucial role in the genesis of the hypernociceptive response that is triggered by adaptive or innate inflammation, we tested the hypothesis that these cytokines also mediate IL-17-induced articular hypernociception. Supporting our hypothesis, we found that either pharmacological or genetic inhibition of TNF-α, IL-1β or CXCR1/2 ligands prevented the IL-17-induced hypernociceptive effect. In a recent study, we had demonstrated that the mechanical hypernociception induced by mBSA challenge in immunized mice depended on the initial release of TNF-α, which in turn induced the subsequent release of IL-1β and KC [19]. Therefore, in this cascade of hierarchical events of hypernociception during antigen-induced arthritis, IL-17 might play a pivotal role. IL-33 is another cytokine that is involved in the hypernociceptive effect in antigen-induced arthritis [68]. The hypernociceptive action of IL-33 also depends on TNF-α, IL-1β and CXCR1/2 ligands. Thus, it is possible that IL-33 may trigger the IL-17-induced hypernociception. This hypothesis is consistent with recent data showing that the blockade of IL33/ST2 signaling reduced IL-17 axis in experimental arthritis [54,71].

Neutrophils play an important role in inflammatory disorders such as RA, contributing to the tissue damage associated with these diseases [42]. In this study, we observed that the decrease in the nociceptive threshold produced by mBSA-induced arthritis occurs concurrently with an increase in the number of neutrophil that infiltrated the joint, suggesting an association between these two events. Indeed, we found that treatment of mice with fucoidin, which reduced neutrophil accumulation in the joints by inhibiting selectin interactions [47], reduced mBSA-induced hypernociception. These results are consistent with our previous report that showed that inhibition of neutrophil accumulation inhibited the innate or immune inflammation-induced hypernociceptive response [19,20,31,63,69]. The participation of neutrophils in the genesis of hypernociception during mBSA-induced arthritis appears to be triggered by IL-17. This association is supported by the finding that IL-17-induced articular hypernociception was accompanied by neutrophil accumulation in the joints, which was reduced by the inhibition of neutrophil recruitment by fucoidin. It is noteworthy that the mechanisms by which IL-17 mediates neutrophil migration may be secondary to its ability to produce classical chemotatic mediators, such as TNFα and CXCR1/2 ligands, which also mediate IL-17-induced hypernociception [1,46]. Although the role of IL-17-recruited neutrophils in inflammatory hypernociception is not immediately apparent it could result from a variety of mechanism: (a) neutrophils are source of pro-nociceptive cytokines and chemokines [20]; (b) neutrophils play a role in the genesis of inflammatory hypernociception by triggering PGE2 and LTB4 production [31]; (c) neutrophil could also be a source of free radicals (superoxide, peroxynitrite) and MMPs, which contribute to the hypernociception genesis [5].

Concerning MMPs, herein, we demonstrated that MMP-9, but not MMP-2, seems to participate in the pro-nociceptive effect of IL-17. The MMP-2 and MMP-9 (gelatinases A and B, respectively) are overproduced in rheumatoid synovial fluids [62,72]. Recently, it was demonstrated that MMP-2 and MMP-9 are involved in the development of neuropathic pain [40]. Differently of MMP-2, which is constitutively expressed in several cells types, the MMP-9 expression is mainly induced in the leukocytes during inflammation [55]. Moreover, IL-17 induces the production of MMP-9 by human leukocytes [38]. Thus, it is reasonable to suggest that during mBSA-induced arthritis, IL-17 is inducting the release of MMP-9 by emigrated neutrophils that contributes to the hypernociceptive response. Although the mechanisms of MMP-9 mediation of IL-17- hypernociception was not addressed in the present study, there is evidence that MMPs can act as a processing enzyme of different precursors of inflammatory mediators including pro-IL1 and Big ET-1 [21,22,61]. For, instance, the MMP-9 is able to convert Big ET-1 to ET-1 in neutrophils [22]. Based on these evidences and studies showing that endothelins induce hypernociception and mediate the hypernociception induced by antigen and by several cytokines, including IL-15, IL-18 and IL-33 in immunized mice [66–69], the participation of ETs was also addressed in IL-17 hypernociceptive effect. Indeed, we are demonstrating that the hypernociceptive effect of IL-17 is also dependent of endothelins.

In general, the hypernociceptive effects of cytokines (TNF-α, IL-1β) and chemokines and also endothelins do not result from the direct action on primary nociceptive fibers. Instead, these cytokines appear to induce the release of more directly-acting mediators such as prostaglandins and sympathetic amines [23,24,41]. Additionally, the pro-nociceptive role of IL-17 was also mediated by the production and/or release of prostaglandins and sympathetic amines. Out data supports this hypothesis since IL-17 in vivo administration induced an increase in COX-2 mRNA expression and PGE2 production in the synovial membrane. These results corroborate other studies in which IL-17 induces COX-2-dependent PGE2 synthesis in synoviocytes and osteoblasts [27,45] and that prostaglandins were involved in the genesis of antigen-induced hypernociception [19,68]. While it needs to be determined, the production of prostanoids by IL-17 could be secondary to the stimulation of IL-1β, as IL-1β is an important inductor of COX-2 expression and PGE2 production [2,36].

The stimulation of the sympathetic component in IL-17-induced articular hypernociception could be also indirect, in a chemokine-dependent process. Indeed, it was demonstrated that chemokines stimulate the sympathetic component of inflammatory hypernociception [13,16]. Another possibility is that neutrophils may produce sympathetic amines. This hypothesis is based on the findings that neutrophils present the enzymatic machinery to produce these substances, and upon activation by a variety of inflammatory stimuli, they can release sympathetic amines [25,26]. It is important to point out that our results do not exclude the possibility that IL-17 may also acts directly in the sensitive neurons inducing their sensitization. Dual effects (indirectly and direct effect on nociceptive neurons) have been described to other cytokines including IL-6, TNF-α and IL-1β [6,8,12].

In conclusion, our results suggest that IL-17 is a pro-nociceptive cytokine in mBSA-induced arthritis. The IL-17 mechanism of action is dependent on neutrophils, cytokines (TNF-α, IL-1β), CXCR1/2 chemokine ligands, MMP, endothelins, prostaglandins and sympathetic amines. It is therefore reasonable to propose IL-17 as a therapeutic target for the control of pain during RA.

Conflicts of interest

The authors have no conflicts of interest.

Acknowledgments

We thank the excellent technical assistance of Ieda Regina dos Santos Schivo, Sérgio Roberto Rosa, Fabiola Mestriner and Giuliana Bertozi Francisco. This work was supported by grants from CAPES, FAPESP, Pronex and CNPq (Brazil).

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Keywords:

IL-17; Pain; Arthritis; Hyperalgesia; Cytokines; Neutrophil

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