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.
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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.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
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.
Acquisition of data. Coelho, Amaral, Sachs, Costa, Rodrigues, Vieira, Silva, Bertini.
Analysis and interpretation of data. Coelho, Pinho, Souza, M. M. Teixeira.
Manuscript preparation. Coelho, A. L. Teixeira, M. M. Teixeira.
Statistical analysis. Coelho, A. L. Teixeira, M. M. Teixeira.