Chemokines are a family of >50 small chemotactic proteins, divided into 4 structural subfamilies (CC, CXC, C, and CX3C). They have wide-ranging roles in immunity, including the organization of lymphoid organ architecture; homeostatic trafficking of naive lymphocytes; migration of T and B cells to functional niches within lymphoid organs (such as germinal centers) during adaptive immune responses; constitutive trafficking of mast cells, monocyte/macrophages, and memory T cells to uninflamed tissues; and leukocyte recruitment to sites of inflammation (1). Chemokines act by increasing integrin-mediated leukocyte adhesion to endothelium, thereby facilitating leukocyte extravasation, and by establishing ordered gradients that direct leukocyte movement within tissues. Certain chemokines have additional functions such as promoting angiogenesis, inducing leukocyte degranulation, and activating cells to secrete mediators of inflammation. Chemokines exert their biologic activity via binding to G protein–coupled receptors; >20 are known, classified according to their ligand subfamily (CCR, CXCR, XCR, and CX3CR). Considerable redundancy exists in chemokine–chemokine receptor pairings, with most chemokine receptors binding multiple chemokines and many chemokines binding to more than one receptor.
There has been extensive interest in characterizing the function of chemokines and their receptors in rheumatoid arthritis (RA). Numerous chemokines have been identified in the synovium and synovial fluid (SF) of RA patients, including, but not limited to, CCL2–5, CCL8, CCL19–21, CXCL1, CXCL5–10, CXCL12, CXCL13, CXCL16, CX3CL1, and XCL1 (2, 3). Synovial macrophages and fibroblast-like synoviocytes are thought to be the primary sources of chemokines in RA tissue, although other cells such as neutrophils, mast cells, lymphocytes, and endothelial cells also contribute to the chemokine milieu. Most chemokine receptors are expressed in RA synovium, generally on multiple cell types; these include CCR1–7, CXCR1–6, CX3CR1, and XCR1 (2, 3). There are only limited data on chemokine and chemokine receptor inhibition in RA. These consist of results from small clinical trials of CCR1, CCR2, and CCL2 inhibitors, with only a nonsignificant trend toward improvement in the trial of the CCR1 antagonist (3–5).
Multiple chemokines and chemokine receptors have been implicated in animal models of arthritis on the basis of experiments employing inhibitory antibodies, pharmacologic antagonists, and/or gene-targeted mice, although we are unaware of any model that has been comprehensively examined. For collagen-induced arthritis (CIA), blockade of CCL2, CCL3, CXCL2, CXCL13, CXCL16, CCR1, or CXCR4 inhibited disease, whereas genetic deficiency of CCR2 exacerbated it (2, 6–10). Adjuvant-induced arthritis was ameliorated by blockade of CCL2, CCL5, CXCL1, CXCL5, CXCL10, CCR2, CXCR2, or CXCR3 (2, 11–14). Antigen-induced arthritis was dampened by blockade of CXCR2 or genetic deficiency of CCR7 or CXCR5 (15, 16). These models are dependent on both an initial adaptive immune response and a downstream inflammatory cascade involving innate immune cells and cytokines such as interleukin-1 (IL-1) and tumor necrosis factor α (17). Hence, studies of these models leave unclear whether the primary role of chemokines is to orchestrate T and B cell responses during the initial adaptive immune response or to mediate inflammatory cell recruitment to arthritic joints during the effector phase.
In the K/BxN mouse model of arthritis, the upstream adaptive immune response required to generate arthritogenic autoantibodies can be conveniently separated from the downstream autoantibody-mediated effector phase. The initial transgenic model was discovered after NOD mice were fortuitously crossed with C57BL/6 (B6) mice carrying the KRN mouse T cell receptor transgenes (18). This receptor recognizes a peptide from a ubiquitous glycolytic enzyme, glucose-6-phosphate isomerase (GPI), presented by the NOD mouse–derived Ag7 major histocompatibility complex molecule (17). In K/BxN mice, an autoimmune response develops, with production of pathogenic anti-GPI antibodies that induce arthritis when transferred into normal recipients. These antibodies deposit on joint surfaces and mediate arthritis by activating the alternative complement pathway and triggering Fcγ receptors (19). Neutrophils and mast cells are both required for serum-transferred arthritis, and recruitment of neutrophils is critically dependent on leukotriene B4 (LTB4) and its receptor, BLT1, such that disease does not develop in their absence (20–22).
Here we present a comprehensive analysis of chemokines and their receptors in K/BxN serum–transferred arthritis, coupling gene expression profiling and knockout mice. Our results highlight the contribution of chemokines that trigger CXCR2, in particular in the recruitment of neutrophils.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
Focusing on the roles of chemokines and their receptors in the effector phase of antibody-induced arthritis, we demonstrated that CXCR2 is necessary to sustain joint inflammation. CXCR2 recognizes CXC chemokines containing the ELR motif; in mice, such chemokines include CXCL1, CXCL2, CXCL3, CXCL5, and CXCL7 (2). Of these, CXCL1, CXCL2, and CXCL5 were up-regulated in inflamed joints during K/BxN serum–transferred arthritis. In addition, it was recently reported that CXCR2 also recognizes extracellular matrix degradation products produced by matrix metalloproteases (MMPs), and these proteases had expression kinetics similar to those of the CXC chemokines (36) (Table 1). MMPs also can enhance the potency of CXC ligands of CXCR2 through proteolytic processing, as has been shown, for instance, for MMP-9 and MMP-13 acting on CXCL2 (37). The driving force for joint expression of CXCR2 ligands may be IL-1, a cytokine expressed in parallel with disease activity that is critical for serum-transferred arthritis and that among its many activities can stimulate CXC chemokine production (38, 39) (Table 1). CXCR2 ligands may mediate the neutrophil recruitment that is observed when IL-1 is introduced into tissues such as joints (40). In support of this hypothesis, neutrophil infiltration into the hippocampus triggered by transgenic IL-1 expression has been shown to be CXCR2 dependent (41).
BM cell transfer experiments revealed that CXCR2 needed to be displayed principally on hematopoietic cells. This receptor can mediate trafficking of multiple hematopoietic cell lineages involved in inflammatory arthritis, including neutrophils, monocyte/macrophages, and mast cells (33, 34, 36). Mast cell CXCR2 is unlikely to account for the dependence on CXCR2, since this cell type is required only prior to the onset of measurable arthritis in this system (20). It is also unlikely that defective monocyte/macrophage recruitment explains the importance of CXCR2, since only a limited number of SF macrophages expressed detectable CXCR2.
Instead, our data support a role for CXCR2 in recruitment of neutrophils to inflamed joints. This conclusion is consistent with findings in the antigen-induced arthritis model of reduced numbers of rolling, arrested, and total neutrophils in synovial tissue after CXCR2 inhibition (42, 43). Those studies could not determine whether reduced neutrophil recruitment was due to a cell-autonomous requirement for CXCR2 on neutrophils themselves, or whether it was an indirect effect of decreased disease severity from CXCR2 blockade. We demonstrated that CXCR2 does indeed have intrinsic activity on neutrophils, since CXCR2-deficient neutrophils had a reduced capacity to migrate into inflamed joints when compared side-by-side with CXCR2-expressing neutrophils. A similar requirement for neutrophil CXCR2 has been demonstrated in experimental autoimmune encephalitis (44). Of note, the mild reduction in disease activity in mice lacking CXCR2 on nonhematopoietic cells may reflect a small secondary contribution of endothelial CXCR2, which can facilitate the transendothelial movement of neutrophils (35).
Although CXCR2 conferred an advantage in neutrophil migration to inflamed joints, large numbers of CXCR2-deficient neutrophils were still found in arthritic joints in the competition experiments (Figure 5D). Their presence likely reflected the activity of other neutrophil chemoattractants including LTB4, which, along with its receptor BLT1, is required for serum-transferred arthritis and plays a sustained role after disease onset (21, 22). BLT1-deficient neutrophils were abundant in the SF of BLT1-knockout mice that had received wild-type neutrophils to reconstitute arthritis susceptibility, similar to our observations on CXCR2-deficient neutrophils in the competition experiment (22). We suspect that CXCR2 and BLT1 can each mediate neutrophil recruitment in the absence of the other, but that neither mediator can fully compensate for the other. Since neutrophils are the critical source of LTB4 in serum-transferred arthritis, CXCR2-mediated neutrophil recruitment may be amplified by LTB4 released from the newly arrived neutrophils (21). In accord with this scenario, neutrophil migration to joints in response to intraarticular injection of CXCL1 and CXCL5 was greatly reduced by coadministration of an LTB4 inhibitor (43). The complement receptor C5aR is also capable of mediating neutrophil chemotaxis and is required for serum-transferred arthritis (19). It may act in concert with CXCR2 and BLT1 to drive recruitment of neutrophils to inflamed joints. In human RA, CXC chemokines, LTB4, and C5a are all present in SF, although clinical trials of BLT1 and C5aR inhibitors have yielded disappointing results (45–47). We are unaware of any reported clinical trials of a CXCR2 inhibitor in RA.
Perhaps our study's most striking finding was the surprising dearth of chemokine receptors required for the effector phase of autoantibody-induced arthritis. Like both human RA and other animal models of arthritis, the K/BxN serum–transferred disease was accompanied by up-regulation of signal chemokines, with the caveat that mRNA levels might not always correlate with protein levels. While the increased expression of ELR+ CXC chemokines in ankle tissue was reflected by reduced arthritis in the absence of their receptor, CXCR2, this correlation of induction level with disease severity did not hold either for the receptors of other up-regulated CC chemokines or for CCL2. It is possible that receptor and/or ligand redundancy permits other receptors to take the place of any single chemokine receptor targeted. Results for certain of the receptors tested, in particular CX3CR1, fell just below statistical significance, so we cannot exclude the possibility that they have a moderate contribution to disease that might be confirmed by further testing. Moreover, some chemokine receptors were not included in this study for technical reasons, most notably CXCR4 (the receptor for CXCL12), which could not be tested since deficiency in this molecule is lethal during embryogenesis.
However, these caveats do not explain why inhibition or deficiency of CCR1, CCR2, CCR7, CXCR3, CXCR5, CCL2, or CCL3 altered disease severity in other arthritis models but not in serum-transferred arthritis (6, 9, 10, 14, 16, 32). These disparate results cannot simply be attributed to inadequate genetic targeting, since studies in other animal models used the same CCR7-, CXCR5-, and CCL3-knockout lines, and the other lines used here have previously been clearly demonstrated to lack expression of the targeted gene (16, 23, 24, 32, 48, 49). The discrepancy may reflect the contributions of these chemokines and chemokine receptors to adaptive immunity, consistent with evidence of altered T or B cell activity reported in many of those studies. In CIA, increased disease severity with CCR2 deficiency was associated with enhanced titers of anticollagen antibodies, formation of rheumatoid factor (not detected in controls), and decreased activation-induced cell death (10). In a study demonstrating improvement in adjuvant-induced arthritis with anti-CXCR3 monoclonal antibody therapy, CXCR3−/− mouse T cells had a reduced capacity to migrate to inflamed joints in comparison with wild-type mouse T cells (14). CCR7−/− and CXCR5−/− mice with antigen-induced arthritis had reduced titers of antibodies to the inciting antigen, reduced T cell proliferation in response to antigen, and aberrant or absent lymphoid follicle formation within chronically inflamed synovial tissue (16).
CCL3 represents a special case, since it has been shown to have a role in anticollagen antibody–induced arthritis, which, like serum-transferred arthritis, is induced by passive antibody transfer (32). The primary difference between the 2 models besides their autoantibody specificity is the requirement for lipopolysaccharide (LPS) in anticollagen antibody–induced arthritis. LPS induces CCL3 and promotes neutrophil accumulation in a CCL3-dependent manner (50). It may be that, in the absence of CCL3, LPS is unable to provide enough additional proinflammatory activity to induce disease upon transfer of anticollagen antibodies, whereas antibodies alone are sufficient to induce disease in K/BxN serum–transferred arthritis.
This study demonstrates a necessary role for CXCR2 in sustaining autoantibody-induced arthritis and identifies this receptor as a second critical element by which neutrophils can be recruited to inflamed joints, alongside BLT1. Our findings highlight CXCR2 and potentially also CXCR1 (a closely related human receptor without a known murine homolog that binds CXCL6 and CXCL8; also known as IL-8) as attractive therapeutic targets for RA (2). The importance of CXCR2 in the arthritis effector phase, the stage when most patients present with disease, enhances its appeal.