The chemokine receptors CXCR1 and CXCR2 play a role in mediating neutrophil recruitment and neutrophil-dependent injury in several models of inflammation. We undertook this study to investigate the role of these receptors in mediating neutrophil adhesion, subsequent migration, and neutrophil-dependent hypernociception in a murine model of monarticular antigen-induced arthritis (AIA).
AIA was induced by administration of antigen into the knee joint of previously immunized mice. Intravital microscopy studies were performed to assess leukocyte rolling and adhesion. Mechanical hypernociception was investigated using an electronic pressure meter. Neutrophil accumulation in the tissue was measured by counting neutrophils in the synovial cavity and assaying myeloperoxidase activity. Levels of tumor necrosis factor α (TNFα) and the chemokines CXCL1 and CXCL2 were quantified by enzyme-linked immunosorbent assay. Histologic analysis was performed to evaluate the severity of arthritis and leukocyte infiltration.
Antigen challenge in immunized mice induced production of TNFα, CXCL1, and CXCL2 and also resulted in neutrophil recruitment, leukocyte rolling and adhesion, and hypernociception. Treatment with reparixin or DF2162 (allosteric inhibitors of CXCR1/CXCR2) decreased neutrophil recruitment, an effect that was associated with marked inhibition of neutrophil adhesion. Drug treatment also inhibited TNFα production, hypernociception, and the overall severity of the disease in the tissue.
Blockade of CXCR1/CXCR2 receptors inhibits neutrophil recruitment by inhibiting the adhesion of neutrophils to synovial microvessels. As a consequence, there is decreased local cytokine production and reduced hypernociception, as well as ameloriation of overall disease in the tissue. These studies suggest a potential therapeutic role for the modulation of CXCR1/CXCR2 receptor signaling in the treatment of arthritis.
Rheumatoid arthritis (RA) is a chronic inflammatory disease of the joints that affects 0.5–1.0% of the adult population worldwide and is associated with significant morbidity (1, 2). Cytokines are directly implicated in many of the immune processes that are associated with the pathogenesis of RA. In recent years, the targeted blockade of these cytokines has been a major therapeutic advance in the management of RA. Nevertheless, cytokine-based therapy must be administered via a systemic route, current treatments are costly, and many patients fail to respond to blockade of either tumor necrosis factor α (TNFα) (3) or interleukin-1β (4). Moreover, minor adverse events, including injection-site reactions, are common (5), and susceptibility to serious infection is a commonly reported risk (6, 7). Thus, new therapeutic options for the treatment of arthritis are clearly needed.
The neutrophil is the most abundant of all leukocytes in the joints of patients with active RA (8, 9) and yet is a relatively understudied cell in this disease. Neutrophils are attracted into diseased joints by the chemoattractants commonly detected in rheumatoid synovial fluid (8, 9). More direct evidence for the involvement of neutrophils in the pathogenesis of RA has come from studies of animal models of disease (9–11). Indeed, recent evidence has shown a key role for neutrophils in both the initiation and progression of the disease in the K/BxN mouse model (9).
Among the mediators of inflammation that have been shown to activate neutrophils and induce their recruitment in vivo, much interest has been placed on the role of CXC chemokines (12). Previous studies demonstrated that blockade of the action of Glu-Leu-Arg motif–positive CXC chemokines or their receptors, CXCR1 and CXCR2, appears to be a valid strategy for the treatment of neutrophil-associated injuries in several models of inflammation (10, 12–15).
We hypothesized that CXCR1/CXCR2 would be a major inducer of neutrophil adhesion to the synovial microvascular endothelium and hence would mediate the migration of these cells into the joints, in an antigen-induced arthritis (AIA) mouse model. As a corollary of this hypothesis, blockade of CXCR1/CXCR2 would be accompanied by inhibition of neutrophil accumulation in the joints, leading to prevention of neutrophil-dependent joint injury in this mouse model. To test our hypothesis, we investigated the effect of treatment with the CXCR1/CXCR2 allosteric inhibitor reparixin (12, 13) and its long-acting derivative, DF2162 (10), in mice with AIA.
MATERIALS AND METHODS
Eight-to-10–week-old male C57BL/6J (wild-type) mice were obtained from the Centro de Bioterismo of the Universidade Federal de Minas Gerais (UFMG) in Brazil and kept in the animal facilities of the Laboratório de Imunofarmacologia, Department of Biochemistry and Immunology at UFMG. The mice were maintained with filtered water and food ad libitum in a controlled environment (stable temperature and humidity). All animal care and handling procedures were in accordance with the guidelines of the International Association for the Study of Pain (16), and all experiments received prior approval from the UFMG ethics committee (certificate 166/2006).
The mice were immunized intradermally at the base of the tail with 500 μg of methylated bovine serum albumin (mBSA; Sigma, St. Louis, MO) in 100 μl of an emulsion of saline and an equal volume of Freund's complete adjuvant (CFA; Sigma) on day 0 (17). Fourteen days later, antigen challenge was performed in the mice. Each mouse received an injection of 10 μg of mBSA (10 μg mBSA in 10 μl sterile saline) in the left knee joint. At different time points (6 hours, 24 hours, 48 hours, and 7 days) after antigen challenge, the mice were killed. The knee cavity was washed with phosphate buffered saline (PBS) (2 × 5 μl), and the periarticular tissue was removed from the joint for evaluation of cytokines, chemokines, and myeloperoxidase (MPO) activity.
The total number of leukocytes in the tissue was determined by counting the leukocytes in a Neubauer chamber after staining tissue samples with Turk's solution. Differential counts were obtained from cytospin preparations (Shandon III; Thermo Shandon, Frankfurt, Germany) by evaluating the percentage of each leukocyte on a slide stained with May-Grünwald-Giemsa stain.
Initial experiments evaluated the kinetics of neutrophil influx, using intravital microscopy, and also evaluated cytokine production after intraarticular antigen challenge in immunized mice. To evaluate the role of CXCR1/CXCR2, mice were treated with reparixin (30 mg/kg subcutaneously [SC]; Dompé Pharma, L'Aquila, Italy) 40 minutes before and 3 and 6 hours after challenge with antigen. Control mice received saline (200 μl SC). Preliminary experiments in mice (13) have shown that a dose of 30 mg/kg reparixin is the maximally effect dose for preventing neutrophil influx.
The levels of cytokines and chemokines and the number of neutrophils were evaluated at 24 hours after antigen challenge. Hypernociception related to joint inflammation was evaluated 6 and 24 hours after antigen challenge. In order to evaluate the role of CXCR1/CXCR2 in mediating neutrophil rolling and adhesion to the synovial microvasculature, intravital microscopy was carried out 24 hours after challenge, preceded 30 minutes prior to the procedure by treatment with reparixin (30 mg/kg intravenously [IV]) or saline (100 μl IV, as control).
Further experiments were carried out using the long-acting reparixin analog DF2162 (10). DF2162 was resuspended in 0.05% carboxymethylcellulose and administrated orally at a dose of 15 mg/kg, using an administration schedule similar to that for reparixin. All parameters in mice treated with DF2162 were evaluated 24 hours after antigen challenge.
Intravital microscopy of the knee joint.
Intravital microscopy was performed in the synovial microcirculation of the mouse knee, as described previously (18). Briefly, the left hind limb was placed on a stage, with the knee slightly flexed and the patellar tendon mobilized and partly ressected. The intraarticular synovial tissue of the knee joint was then visualized for the determination of leukocyte rolling and adhesion.
A 20-fold objective was used to select 2–4 regions of interest in each mouse. To measure the leukocyte–endothelial cell interactions, the fluorescent marker rhodamine 6G (Sigma) was injected IV as a single bolus of 0.15 mg/kg immediately before the measurements. Rhodamine epiilumination was achieved with a 150W variable HBO mercury lamp in conjunction with a Zeiss filter set 15 (546/12-nm band-pass filter, 580-nm Fourier transforms, 590-nm late potentials; Zeiss, Wetzlar, Germany). The microscopic images were captured with a video camera (5100 HS; Panasonic, Secaucus, NJ) and recorded on an S-VHS videotape, using both filter blocks consecutively. Data analysis was performed off-line.
Rolling leukocytes were defined as those cells moving slower than the cells moving at a regular flux in a given vessel. The flux of rolling cells was measured as the number of rolling cells passing by a given point in the venule per minute, with results expressed as cells/minute. A leukocyte was considered to be adherent if it remained stationary for at least 30 seconds, and total leukocyte adhesion was quantified as the number of adherent cells within a 100-μm length of venule, with results expressed as cells/mm2.
Evaluation of hypernociception.
Mice were placed in a quiet room in acrylic cages (12 × 10 × 17 cm in height) with a wire-grid floor for 15–30 minutes, before testing for environmental adaptation. Stimulations were performed only when the mice were in quiet conditions, i.e., without exploratory movements or defecation and not resting on their paws. In these experiments, an electronic pressure meter was used. This apparatus consisted of a hand-held force transducer fitted with a polypropylene tip (Insight Instruments, Ribeirão Preto, San Paulo, Brazil). For the present model, a large tip (4.15 mm2) was adapted to the probe (19, 20). An increasing perpendicular force was applied to the central area of the plantar surface of the hind paw to induce dorsal flexion of the femorotibial joint, followed by withdrawal of the paw. A tilted mirror below the grid provided a clear view of each animal's hind paw. The electronic pressure meter automatically recorded the intensity of the force applied when the paw was withdrawn, with results expressed as the flexion-elicited withdrawal threshold (in grams). The test was repeated until 3 measurements yielded consistent results (i.e., the variation among these measurements was lower than 0.5 gm).
Hypernociception was tested before and after injection of saline or antigen, with results expressed as the change in the withdrawal threshold. This was calculated by subtracting the zero-time mean measurements from the time-interval mean measurements. The mean ± SEM withdrawal threshold was 12.8 ± 0.5 gm (n = 30) at the zero-time measurement (i.e., before injection of hypernociceptive agents)
The knee joint was removed and fixed for 12 hours with 8% paraformaldehyde (pH 7.2). The joints were then incubated in 20% EDTA at pH 7.2 for 3 days at room temperature to decalcify the bone. Samples were washed with PBS and dehydrated. After being embedded in paraffin, the joints were sliced into 3-μm–thick sections that were stained with hematoxylin and eosin (H&E). To eliminate potential bias, the slides were scored by 2 independent observers (FMC and VP). The sections were graded subjectively using various parameters, as follows: severity of synovial hyperplasia (pannus formation), cellular exudate, and cartilage depletion/bone erosion (each scored 0 [normal] to 3 [severe]), and extent of synovial infiltrate (scored 0–5, with higher scores indicating greater infiltration). The grades for all parameters were subsequently summed to obtain an arthritis index, with results expressed as the median arthritis score (21).
Quantification of neutrophil accumulation in the tissue.
The extent of neutrophil accumulation in the mouse tissue was measured by assaying MPO activity, using a technique routinely performed in our laboratory (10, 22). Briefly, the knee joint was removed and frozen at −70°C. Upon thawing of the sample, the tissue (0.1 gm of tissue per 1.9 ml of buffer) was homogenized and processed for determination of MPO activity. The assay included 25 μl of 3,3′-5,5′-tetramethylbenzidine (Sigma) in PBS (pH 5.4) as the color reagent. The number of neutrophils in each sample was calculated with reference to a standard curve of the number of neutrophils obtained from the peritoneal cavity of 5% casein–treated mice processed in the same manner, with results in the synovial tissue expressed as the relative number of neutrophils per milligram of tissue wet weight. Using this method allows the test to be specific for neutrophils, as opposed to macrophages and lymphocytes (results not shown).
Measurement of cytokines and chemokines in periarticular tissue.
The concentrations of TNFα and the chemokines CXCL1 (also known as keratinocyte-derived chemokine) and CXCL2 (also known as macrophage inflammatory protein 2) were measured in the periarticular tissue using a commercially available enzyme-linked immunosorbent assay (ELISA), following the instructions supplied by the manufacturer (DuoSet kits; R&D Systems, Minneapolis, MN). Briefly, 100 mg of tissue was homogenized in 1 ml of PBS (0.4M NaCl and 10 mM NaPO4) containing antiproteases (0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM benzethonium chloride, 10 mM EDTA, and 20 kallikrein inhibitor units of aprotinin A) and 0.05% Tween 20. The samples were then centrifuged for 10 minutes at 3,000g, and the supernatant was immediately assessed by ELISA at a 1:3 dilution in PBS. All samples were assayed in duplicate.
Reparixin (R-2-[4-isobutyl-phenyl]propionyl methylsulfonamide) and DF2162 were synthesized in the Department of Chemistry at Dompé Pharma. Methylated BSA and CFA were purchased from Sigma. Reparixin was dissolved in saline, and DF2162 was dissolved in 0.05% carboxymethylcellulose.
Results are expressed as the mean ± SEM. Differences between groups were evaluated by analysis of variance followed by Student's t-tests and Newman-Keuls post hoc tests. P values less than 0.05 were considered significant.
Kinetics of joint inflammation in the AIA model.
Initial experiments evaluated the kinetics of joint inflammation after intraarticular challenge with antigen in immunized mice. There was an increase in the number of neutrophils in the synovial cavity (Figure 1A) and in the relative number of neutrophils in the periarticular tissue (Figure 1B), which was first detected 6 hours after challenge and peaked at 24 hours after challenge. Neutrophil accumulation in the joint was still observed after 48 hours and subsided by 7 days after antigen challenge (Figure 1A). In the periarticular tissue, neutrophil accumulation returned to baseline levels by 48 hours after challenge (Figure 1B).
Neutrophil influx was preceded by an increase in the levels of CXCL1 in the periarticular tissue (Figure 1C). Levels of CXCL1 peaked at 6 hours and dropped to baseline levels by 48 hours after challenge. Levels of CXCL2 in the periarticular tissue followed a similar expression pattern (mean ± SEM 343 ± 44 pg per 100 mg tissue after PBS challenge versus 1,107 ± 207 pg per 100 mg tissue at 6 hours, 774 ± 42 pg per 100 mg tissue at 24 hours, 831 ± 203 pg per 100 mg tissue at 48 hours, and 862 ± 188 pg per 100 mg tissue at 7 days after antigen challenge [n = 5 per group]; P < 0.05). The concentration of TNFα in the periarticular tissue peaked at 48 hours and was still detectable by 7 days after antigen challenge (Figure 1D).
Leukocyte–endothelial cell interactions in the synovial microcirculation were also investigated. As seen in Figures 2A and B, intraarticular antigen challenge in immunized mice was accompanied by an increase in leukocyte rolling and adhesion, which was mostly seen within the first 48 hours after challenge. Histologic analysis of H&E-stained joint sections from the mice with AIA demonstrated a dense infiltration of neutrophils in the synovium, as well as synovial hyperplasia at 24 hours after challenge (results not shown). Further experiments evaluating the effects of CXCR2 receptor antagonists were conducted in the joints primarily at 24 hours after administration of antigen.
Inhibition of joint inflammation by CXCR2 antagonists in the AIA model.
Treatment with reparixin greatly reduced antigen-induced recruitment of neutrophils into the synovial cavity (Figure 3A) and into the periarticular tissue (Figure 3B). Results of quantification of the histologic features concurred with the qualitative aspects of the synovial tissue, and there was a significant reduction in the arthritis score in reparixin-treated animals (Figure 3C). Histopathologic assessment of the tissue sections showed that treatment with reparixin reduced the inflammatory exudate, especially in perivascular regions (Figure 3D).
Treatment with reparixin did not modify the levels of antigen-induced CXCL1 in the joints of mice with AIA (Figure 4A) but did decrease the levels of CXCL2 (mean ± SEM 645 ± 108 pg per 100 mg tissue in control [untreated] mice versus 386 ± 58 pg per 100 mg tissue in vehicle [PBS]–treated mice and 398 ± 38 pg per 100 mg tissue in reparixin-treated mice [n = 5 animals per group]; P < 0.05). The compound also reduced the antigen-induced release of TNFα in the periarticular tissue at 24 hours after challenge (Figure 4B).
In addition to the local production of cytokines and influx of neutrophils, there was significant hypernociception after intraarticular antigen challenge in immunized mice (Figure 4C). Inflammation-related hypernociception was first noticeable at 3 hours after challenge, became more intense by 6 hours after challenge, peaked at 24 hours after challenge, and subsided by 7 days after challenge. Treatment with reparixin diminished the extent of inflammation-related hypernociception observed at 6 hours and 24 hours after antigen challenge in immunized mice (Figure 4D).
In order to confirm the relevance of CXCR1/CXCR2 in this mouse model of AIA, further experiments were conducted with an orally active derivative of reparixin, DF2162 (10). DF2162 was administered by oral gavage at a dose of 15 mg/kg. At this dose, treatment with DF2162 was associated with a significant reduction in neutrophil recruitment into the synovial cavity and the periarticular tissue at 24 hours after AIA induction (Table 1). In addition, this dose of the compound also reduced local production of TNFα (Table 1) and the inflammation-related hypernociception (Figure 4D) observed at 24 hours in this model of AIA.
Table 1. Effects of treatment with DF2162 on neutrophil recruitment and on the levels of TNFα, CXCL1, and CXCL2 after initiation of antigen-induced arthritis in mice*
Values are the mean ± SEM resuts from 5–6 mice per group. DF2162 (15 mg/kg) or vehicle (0.05% carboxymethylcellulose) was administered orally 40 minutes before and 3 and 6 hours after arthritis induction. The number of neutrophils in the synovial cavity was assessed 24 hours after injection of 10 μg of methylated bovine serum albumin or 10 μl sterile saline (control) into the knee joint of immunized mice. The number of neutrophils in the periarticular tissue was assessed at 24 hours using a myeloperoxidase assay. The concentrations of CXCL1, CXCL2, and tumor necrosis factor α (TNFα) in the periarticular tissue were assessed by enzyme-linked immunosorbent assay at 24 hours after arthritis induction.
Inhibition of leukocyte–endothelial cell interactions by reparixin in the AIA model.
A series of experiments were then conducted using intravital microscopy to evaluate whether the effects of reparixin in AIA correlated with its ability to prevent interactions between leukocytes and synovial microvessels. In order to avoid any possible confounding effect of the compound in the local production of cytokines (see Figure 4B), reparixin was administered only 30 minutes prior to the intravital microscopy procedure. As seen in Figure 5, treatment of the mice with reparixin reduced leukocyte rolling (60% inhibition) and abrogated leukocyte adhesion (100% inhibition) to the synovial microvessels at 24 hours after challenge in immunized mice. The majority of leukocytes interacting with endothelial cells, as assessed in histologic sections of the tissue, were neutrophils (Figure 3D and results not shown).
In the present study, we identified a significant role for CXCR1/CXCR2 in modulating tissue inflammation in a mouse model of experimental arthritis. Of note, treatment with CXCR1/CXCR2 allosteric inhibitors prevented local neutrophil influx, reduced local production of TNFα, and diminished hypernociception, an index of pain. Inhibition of leukocyte–endothelial cell interactions, especially the effects on firm adhesion of leukocytes, appeared to be a major mechanism of action of the inhibitors in this model of antigen-induced inflammation in the knee joint.
Neutrophils are thought to play an important role in the development of inflammatory joint disease, as has been evidenced in several studies involving experimental models of arthritis (9–11). Treatment with reparixin or DF2162 greatly decreased the influx of neutrophils into the knee joint and the periarticular tissue after antigen challenge in immunized mice. Inhibition of neutrophil influx was associated with significant amelioration of overall disease in the tissue. These results are consistent with the known role of CXCR2 in mediating the influx of neutrophils in several models of inflammation in vivo, including a model of AIA in rats (for example, see refs. 10, 12–15, and23).
More recently, it has become clear that, in addition to CXCR2, murine leukocytes, especially neutrophils, also possess CXCR1 (24). The role of this receptor in mediating neutrophil recruitment in vivo is not known, and the tools to investigate the receptor are not, as yet, readily available. Of note, the compounds used in the present experiments, reparixin and DF2162, inhibited the function of both CXCR1 and CXCR2 (10, 13), and therefore any speculation regarding the relative roles of each receptor in this system is not possible. The compounds had no significant effect on the expression of CXCR2 on circulating neutrophils in our experiments (results not shown). Together with the findings from other published studies (10, 15, 25), our results demonstrate that inhibition of CXCR1/CXCR2 appears to be an effective means of preventing neutrophil influx in models of arthritis.
We also evaluated the mechanisms by which reparixin could be preventing neutrophil influx in the system. To this end, we used intravital microscopy to study the interaction between leukocytes and endothelial cells in the synovial microvasculature (18). Our results showed that firm adhesion of leukocytes to endothelial cells was suppressed in reparixin-treated mice. This is consistent with the role of CXCR2 in triggering integrin-dependent adhesion of neutrophils to endothelial cells (26, 27). There was also partial inhibition of rolling of leukocytes. This was surprising, inasmuch as leukocyte rolling is dependent on selectins and their ligands but not on chemoattractant receptors such as CXCR1/CXCR2 (27).
Expression of selectins or selectin ligands on endothelial cells in a particular site of inflammation is modulated by the expression of cytokines such as TNFα (27, 28). Our results showed that reparixin treatment prevented TNFα production when the treatment was administered before antigen challenge. However, in the intravital microscopy experiments, reparixin was administered just prior to the microscopy procedure and did not modify local levels of cytokines (results not shown). Hence, prevention of the expression of cell adhesion molecules that mediate rolling does not appear to be a mechanism that would explain the short-term effects of reparixin treatment.
One alternative explanation for the effects of reparixin is that adhered neutrophils could be relevant in the rolling of further leukocytes, as has been shown in other systems (29). Although this was a phenomenon that was observed in our system, there was not enough resolution to quantify rolling on adhered cells. Taken together, our data suggest that short-term inhibition of neutrophil–endothelial cell interactions is a major mechanism by which reparixin may interfere with AIA in mice.
Cytokine-based therapies, especially strategies that block or antagonize TNFα, have been used for the treatment of RA and found to be useful in preventing progression of disease in groups of patients (30). In the present study, blockade of CXCR1/CXCR2 with reparixin or DF2162 significantly inhibited the local production of TNFα. Although a role of TNFα in facilitating the influx of neutrophils in vivo is well known, many studies have also clearly demonstrated that the influx of neutrophils facilitates the local production of TNFα (31, 32). Therefore, inhibition of neutrophil influx could potentially explain the lower levels of TNFα in the joints of immunized mice after antigen challenge and subsequent treatment with reparixin.
It is not clear whether the neutrophils themselves produce TNFα or whether these cells release other intermediate mediators that induce the release of TNFα by resident cells, including macrophages. Regardless of the TNFα-producing cell type, the present results and those from other studies are consistent with the notion of a positive cooperative loop between TNFα and neutrophil influx, which appears to be relevant in joint inflammation and injury. The ability of reparixin to prevent neutrophil adhesion and the consequent influx of neutrophils may lead to suppression of this positive loop and have beneficial effects in arthritis.
Pain is the most frequent and disabling symptom in patients with arthritis. In experimental models, pain related to joint inflammation is better described as hypernociception (33). Several effects of reparixin could explain its ability to ameliorate inflammation-related hypernociception induced by AIA. Our previous study (34) and those from other investigators (35, 36) have clearly shown an essential role for neutrophils in mediating inflammation-related hypernociception induced by antigen challenge in mice. Moreover, blockade of TNFα prevents inflammation-related hypernociception induced by a range of stimuli, including antigen injection (for review, see ref.33). Thus, inhibition of both neutrophil influx and local TNFα production could account for the inhibitory effects of reparixin in AIA-associated hypernociception related to inflammation.
In conclusion, this study shows that treatment with allosteric inhibitors of CXCR1/CXCR2 prevented 3 major aspects of arthritis, namely neutrophil recruitment, TNFα production, and inflammation-related hypernociception. Importantly, this study shows that the compounds appear to act via blockade of leukocyte adhesion, with consequent inhibition of neutrophil migration to the site of joint inflammation. These beneficial effects of allosteric inhibitors of CXCR1/CXCR2 suggest that these compounds deserve further evaluation for the treatment of arthritis.
Dr. Mauro Teixeira had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Coelho, Pinho, Bertini, A. L. Teixeira, M. M. Teixeira.