Rheumatoid arthritis (RA) is a systemic autoimmune disease which is characterized by a chronic inflammation of multiple synovial joints. Large numbers of leukocytes infiltrate and accumulate within the synovial tissue (ST) and synovial fluid (SF) (1–3). These leukocytes include T cells, especially CXCR6+ memory T cells, monocytes, plasma cells, and granulocytes. While in most patients these cells are dispersed throughout the synovium, in other patients highly organized lymphoid structures resembling germinal centers can be found (4). Although the cause of RA is still unknown, the recruitment and cytokine-induced activation of inflammatory cells is thought to be essential in perpetuation of the inflammatory response and, ultimately, in cartilage and bone destruction (5–7).
The trafficking of leukocytes is regulated through selective expression of an array of chemokines, adhesion molecules, and their corresponding receptors. Chemokines are secreted proteins that attract leukocytes via activation of 7-transmembrane–domain G-protein–coupled receptors (8, 9). Adhesion molecules provide adhesive capacity during cell–extracellular matrix or cell–cell contact, e.g., when leukocytes transmigrate the endothelium (10, 11). In this respect CXCL16 is an exceptional chemokine, because it has the potential to function as a chemoattractant and as an adhesion molecule. While classic chemokines are expressed as small soluble proteins, CXCL16 is first synthesized as a transmembrane protein expressed by macrophages, dendritic cells (DCs), and endothelial cells (12–14). Data from Shimaoka et al (15) have recently suggested that cell surface–expressed CXCL16 can indeed function as an adhesion molecule. However, upon cleavage by proteases, the extracellular domain is released as a soluble chemokine that attracts effector/memory T cells that express CXCR6, the receptor for CXCL16 (12, 14). Furthermore, CXCL16 also acts as a scavenger receptor for oxidized low-density lipoprotein and bacteria (13, 16, 17), confirming that CXCL16 is a multifunctional protein. Concerning structure and mechanism of action, CXCL16 resembles fractalkine, the other transmembrane chemokine. Fractalkine has been shown to mediate adhesion in its transmembrane form, and to mediate chemotaxis as a cleaved protein (18–20).
Kim and colleagues recently reported the accumulation of CXCR6+ T cells in SF of a small number of RA patients (21). As yet, however, nothing is known about the expression of CXCL16, the only known ligand for CXCR6, in RA joints. Therefore, we analyzed the expression of CXCL16, its recently characterized protease ADAM-10, and CXCR6 in vitro and in vivo, within healthy joints and in the joints of RA patients. Our data demonstrate that expression of CXCL16 and ADAM-10 is strongly enhanced in RA synovia, resulting in the recruitment and accumulation of CXCR6+ memory T cells in RA joints. These data imply an important role for CXCL16/CXCR6 in the synovial inflammation that is strongly associated with RA pathogenesis.
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- PATIENTS AND METHODS
Influx of leukocytes, including CXCR6+ leukocytes, into both ST and SF contributes to the pathogenesis of RA. Here, we report that the chemokine CXCL16, the ligand for CXCR6, is normally expressed by macrophages in the thin lining of healthy synovia. In RA synovia, CXCL16 expression is elevated and strongly increased due to the presence of large numbers of synovial macrophages. In addition, we show that these CXCL16+ macrophages strongly express the recently identified CXCL16 protease ADAM-10 in situ. In vitro studies demonstrated that monocyte-derived macrophages express both transmembrane and cleaved CXCL16 and that the expression is enhanced by RA SF and the proinflammatory stimulus TNF. Moreover, only successful anti-TNF therapy is associated with decreased CXCL16 expression in situ. Finally, elevated expression of both CXCL16 and ADAM-10 by RA ST macrophages in situ is associated with high amounts of cleaved CXCL16 in RA SF and with the presence of significantly increased numbers of CXCR6+ T cells in this SF.
CXCL16 is a recently identified transmembrane chemokine expressed by macrophages and DCs (12–14). Upon proteolytic cleavage, the NH2-terminal part of CXCL16 is released and functions as a soluble chemoattractant for CXCR6+ T cells and plasma cells (12, 14). Interestingly, Kim et al reported that CXCR6 is a marker for effector/memory T cells and that large numbers of CXCR6+ T cells were detected in SF from 3 RA patients (21). Here, we extended these observations and added novel data concerning the expression and function of the only ligand for CXCR6, CXCL16. First, we demonstrated that fresh control or RA PBMC contain only small numbers of CXCR6+ T cells (Figures 1A and B). In contrast, up to 80% of the T cells within RA SF expressed CXCR6. Immunohistochemical staining of RA ST for CXCR6 revealed no significant staining of T cells (results not shown). However, PCR analysis demonstrated that low levels of CXCR6 mRNA are also present within ST from RA patients (results not shown). Since chemokine receptors are generally expressed at relatively low levels, and we demonstrated that CXCR6 is rapidly down-regulated upon CXCL16 binding (Figure 6B), these data suggest that CXCR6 is difficult to detect by immunohistochemistry.
Quantitative analysis demonstrated that the expression of CXCL16 mRNA is increased in RA ST compared with control ST (Figure 1D). Immunostaining revealed that in healthy individuals, CXCL16 is expressed by the single layer of cells that make up the synovial lining (Figure 2). In RA patients, however, elevated expression of CXCL16 is detected in the hypercellular synovial lining, while additional CXCL16+ cells are detected in the sublining and within perivascular lymphocyte aggregates. Overall, CXCL16 expression correlated with increased cellularity of the RA synovia. Based on CD68/CXCL16 staining of serial sections and morphology, the CXCL16+ cells in the lining, sublining, and lymphocyte aggregates mainly represent macrophages. Although we did not observe colocalization of CXCL16 with the DC marker CD208/DC-LAMP (results not shown), we cannot exclude the possibility that some DCs or follicular DCs also express CXCL16. Finally, CD31/CXCL16 staining of serial sections implied that some endothelial cells express CXCL16 (Figures 2G and H). These data are consistent with previous results demonstrating that cardiac and umbilical endothelial cells can express CXCL16 (29, 30).
Furthermore, our in vitro studies show that a significantly increased population of macrophages expressed transmembrane CXCL16 upon addition of RA SF or TNF, one of the major cytokines in RA SF (Figure 3). Moreover, TNF-treated macrophages also released increased amounts of cleaved CXCL16. This could be of importance, since TNF is strongly expressed in RA joints and is considered to be a key player in RA pathogenesis (31). In fact, anti-TNF treatment is effectively used to treat RA (32–34). Interestingly, we observed severely reduced CXCL16 expression in ST from RA patients responding to anti-TNF treatment, but not in nonresponding patients (Figure 4). These data suggest that not only in vitro, but also in vivo the expression of CXCL16 is controlled by TNF. We are currently extending these studies with a larger cohort of RA patients receiving anti-TNF therapy.
Interestingly, highly elevated levels of cleaved CXCL16 were present in SF from RA patients, and this coincided with large numbers of ADAM-10+ macrophages (Figures 5 and 6). ADAM-10 has recently been described to be a major protease involved in the cleavage and release of CXCL16 (27, 28). Therefore, our data suggest that in RA, ADAM-10 expression by the thick layer of synovial lining macrophages is involved in the release of large amounts of cleaved CXCL16 in the SF. We note that significant amounts of CXCL16 are also detected in serum of healthy individuals, suggesting that cleavage of CXCL16 also occurs in steady-state conditions. This reasoning is in accordance with our observation that the synovial lining of healthy individuals does express low levels of both CXCL16 and ADAM-10. Since CXCL16 has been suggested to contain multiple restriction sites for proteases (12, 14), other proteases, e.g., matrix metalloproteinases (MMPs) or TNF-converting enzyme, are also likely to be involved in the cleavage of CXCL16. With respect to cleavage of CXCL16 in synovium, MMP-1 could be an interesting candidate since we have recently shown that this protease is abundantly expressed by RA synovial macrophages (35, 36).
Finally, we demonstrate that cleaved CXCL16 indeed activates CXCR6 expressed by RA SF T cells (Figure 6). First, addition of cleaved CXCL16 to these T cells leads to the loss of cell surface CXCR6, suggesting CXCL16-mediated CXCR6 internalization. Ligand-induced activation and subsequent internalization is a common feature of chemokine receptors (37). After being internalized, some chemokine receptors recycle back to the cell membrane, while others are degraded in the lysosomal compartment. As yet, it is not known how CXCR6 behaves after being internalized. However, we have demonstrated that CXCR6+ T cells isolated from RA SF are capable of migrating in response to CXCL16 in vitro (Figure 6C). Therefore, our data imply that CXCL16 and CXCR6 play an important role in the recruitment of activated T cells into RA joints.
Several mouse studies have confirmed the importance of chemokines in RA development in vivo. Administration of a CCL2/monocyte chemoattractant protein 1 (MCP-1) antagonist prevented the onset of arthritis in the MRL-lpr arthritis model (38), and neutralizing CXCL10/inducible protein 10 antibodies prevented adjuvant-induced arthritis (39). Also in RA patients, adhesion molecules and chemokines play important roles in synovial infiltration and disease pathogenesis. For instance, enhanced expression of adhesion molecules, e.g., E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 (32, 40), and various chemokines, including CCL2/MCP-1, CCL5/RANTES, CCL18/DC-CK1, and CXCL8/IL-8, have been detected in RA tissue and/or SF (41–48). Interestingly, therapy with a CCR1 antagonist has recently been shown to be beneficial in RA (49). Oral administration of this antagonist significantly reduced the number of ST macrophages and T cells, and this was correlated with a trend toward clinical improvement compared with placebo-treated controls. Despite the apparent redundancy in the chemokine system, evidence is accumulating that chemokine and chemokine receptor antagonists have strong potential as therapeutic agents for patients with autoimmune disease (37, 50). Our data suggest that the use of either CXCR6 antagonists or protease inhibitors acting on CXCL16 cleavage could be additional novel approaches to treat patients with RA.
Based on the results of this study, the following model for the role of CXCL16/CXCR6 in RA pathogenesis can be envisaged. During inflammation of the joint, locally activated endothelial cells express increased levels of adhesion molecules and chemokines, resulting in enhanced immigration of monocytes. Within the synovial tissue, these monocytes now become attracted by chemokines released by the synovial lining, and they differentiate into macrophages. Differentiating macrophages start to express both CXCL16 and ADAM-10, expression of which is further enhanced by SF from the synovial cavity and/or by TNF released by the macrophages themselves. At the now thickened synovial lining, ADAM-10 cleaves transmembrane CXCL16, resulting in elevated concentrations of cleaved CXCL16 in the SF. Cleaved CXCL16 attracts large numbers of CXCR6+ memory T cells into the RA joint. These memory T cells release cytokines, such as TNF, that can now activate macrophages and other resident cells, thus sustaining the inflammatory cascade contributing to RA pathogenesis.
In conclusion, our data suggest that overexpression of CXCL16 targets CXCR6+ memory T cells to synovia from RA patients. Therefore, CXCL16 and CXCR6 could be intrinsically involved in the inflammation associated with RA pathology.