Background
Rheumatoid arthritis (RA) and psoriatic arthritis (PsA) are systemic autoimmune diseases characterized by the chronic inflammation of joints which leads to the destruction of cartilage and bone [
1]. Both T cells and B cells are present in the inflammatory infiltrate of synovial membranes in both diseases [
1‐
3]. However, clinical and experimental evidence indicates that B cells play a dissimilar pathogenic role in RA and PsA; specifically, B lymphocytes are not believed to be a major factor in PsA pathophysiology, as they are in RA [
4‐
6]. Experimental data suggest that B cells ought to migrate and accumulate in the synovial microenvironment in order to exert their pathogenic action in RA patients [
7], through the local production of autoantibodies and proinflammatory soluble factors, as well as by acting as antigen-presenting cells [
6,
8,
9]. However, the driving force responsible for the recruitment of B cells in the synovium of RA and PsA patients is not fully clarified. A better understanding of the mechanisms involved in the recruitment of B cells into inflamed synovial tissue might help identify potential therapeutic targets for the management of joint inflammatory processes such as RA.
A number of chemokines, low molecular weight cytokines, are produced locally in the target sites of leukocyte trafficking and homing [
10,
11]. There are currently described more than 50 chemokines and 20 G-protein-coupled chemokine receptors [
12]. Following the binding of a chemokine to its receptor, intracellular signal pathways lead to functional responses such as chemotaxis, secretion and transcriptional activation [
12,
13]. Most leukocytes express more than one chemokine receptor of varying promiscuity and are, in addition, potentially capable of responding synergistically to concomitant migration signals [
14]. Upon binding to their ligands, chemokine receptors are rapidly internalized following either a degradative or recycling pathway. This mechanism, known as desensitization [
15], limits the magnitude and duration of signaling, thus protecting cells from chemokine overstimulation [
16].
In arthritis, several chemokines and their receptors are involved in the process of leukocyte extravasation and accumulation in the inflamed synovium [
17], as well as in the lymphoid tissue organization of synovial inflammatory infiltrate [
18,
19]. Several chemokines have been identified in the synovial microenvironment of both RA [
17,
20‐
22] and PsA patients [
23‐
25]. While the individual effects of different chemokines are well known, the potential consequences of the concomitant expression of chemokines under pathologic conditions in vivo are still a matter of debate [
26]. With respect to B cells, one study found that the chemokine system influences the development and inflammatory progression of B-cell-mediated autoimmune diseases [
27], and that a rich chemokine environment strongly enhances in-vitro migration by an induced synergism involving this cell type [
28].
The objective of this work was to determine which chemokines play the most relevant roles in the accumulation of B cells in the synovium of patients with RA and PsA. Based on CXCR5 and CCR6 internalization and B-cell migration experiments, our results suggest that CXCL13 and CCL20, the respective ligands of CXCR5 and CCR6, by acting synergistically, might participate in the recruitment of B cells in the synovium in patients with arthritis.
Discussion
The most important findings of this work can be summarized as follows. B cells present in the synovial microenvironment of patients with RA and PsA express the chemokine receptors CXCR5, CCR6 and CXCR7, albeit in significantly lower amounts than those in PB, independent of disease type and their naïve or memory phenotype. Also, this reduction in surface expression is caused by internalization and not due to other forms of downmodulation. Finally, CXCL13 and CCL20, the respective ligands of CXCR5 and CCR6, show a synergistic effect in driving the migration of PB B cells from healthy donors and RA patients. Taken together, these results strongly suggest that CXCL13 and CCL20, acting synergistically, play a key role in the recruitment of B cells in the inflamed synovium.
It has been reported that the accumulation of B cells in the synovium plays an essential role in the pathogenesis of RA [
7]. However, evidence suggests that this cell type does not seem to play a major role in PsA pathogenesis [
4‐
6]. Once in the rheumatoid joint, B cells participate in the pathogenesis of the disease through different mechanisms [
8,
9]. Although a wide variety of chemokines and their receptors are expressed in the synovium of RA [
17,
20,
21] and PsA patients [
23‐
25], which of the chemokines present in the synovial microenvironment play a predominant role in the accumulation of B lymphocytes in the joints of these patients remains to be fully clarified.
In order to investigate which chemokines play a major role in the accumulation of B cells in the inflamed synovium, we first studied the surface expression profile of different chemokine receptors from the CC and CXC family in B cells isolated from PB and SF. Our data showed that the expression of chemokine receptors on B cells present in the inflamed synovia is differentially modulated, either positively or negatively, both in RA and PsA patients. B cells from SF showed significant upregulation of CCR1, CCR2, CCR4, CCR5 and CXCR4, as well as downregulation of CXCR5, CXCR7 and CCR6 surface expression compared to PB B cells in both diseases. Different chemokine receptors have been described on PB B cells from healthy individuals and patients with autoimmune diseases, such as RA and systemic lupus erythematosus (SLE) [
35]. However, only two studies have compared chemokine expression on B cells from PB and SF, one in RA patients [
36] and the other in juvenile idiopathic arthritis (JIA) patients [
37]. As with our own findings, the synovial B cells in these two studies less often expressed CXCR5 and CCR6 compared to PB. Our data for the proportion of B cells positive for CCR5, CCR6, CXCR4 and CXCR5 in both PB and the synovial microenviroment from arthritic patients and healthy controls are compatible with those described previously by Nanki et al. [
36]. However, although the same mAb was used, Nanki et al.’s work show that more than 60% of B cells from patients and controls express CCR7, and ours show that this chemokine receptor is much less expressed on B cells from both patients and controls. As far as we know, a comparative analysis of chemokine expression between B cells in PB and SF from PsA patients has not been studied previously. In addition, this marks the first time that the surface expression levels of CCR1, CCR3, CCR4 and CXCR7 have been analyzed in SF and PB B cells from RA patients.
The expression of certain chemokine receptors is regulated during cell differentiation and maturation [
32,
38]. When B lymphocytes from RA patients were selected based on their memory (CD27
+) or naïve (CD27
–) phenotype, SF B cells expressed consistently lower levels of CXCR5, CXCR7 and CCR6 and higher CXCR4 levels compared to PB regardless of their prior contact with the antigen. The study by Nanki et al. [
36] analyzing the percentage of positive cells, but not MFI, described similar results, except for CXCR4 which was expressed by 100% of B cells from both compartments, as was the case in our own study.
Since chemokine receptors rapidly reduce their surface expression upon ligand binding [
16], it is reasonable to think that the chemokine that causes the downregulation of its receptor in the inflammatory foci should play a relevant biological role; for instance and in our context, in the recruitment of B cells to the joints. The surface expression of CXCR5, CXCR7 and CCR6 were decreased in SF B cells compared to PB. This finding is consistent with a desensitization phenomenon, as a consequence of receptor occupancy by their respective ligands [
16]: CXCL13, CXCL12 and CCL20, chemokines that are present in the synovial microenvironment [
17,
20,
21]. Interestingly, the remaining receptors studied (CCR1, CCR2, CCR4, CCR5 and CXCR4) showed an increased surface expression on SF B cells compared to PB, although many of their respective ligands were also present in the SF [
17]. The explanation for this is elusive since multiple regulatory mechanisms are involved in chemokine receptor signaling and expression [
39,
40]. When we examined the differential expression of CXCR5, CCR6 and CXCR4 on the cell surface and total expression on B cells from the PB and SF of RA patients, we observed that CXCR5 and CCR6 in SF B cells had undergone an internalization process, whereas CXCR4 had not. We interpreted these results as follows: the receptors CXCR5 and CCR6 play a predominant role in the migration of naïve B cells and memory B cells to the inflamed synovium.
CXCL12 is a chemokine abundantly expressed in the synovial tissue of RA patients [
17]. Based on migration experiments using PB B cells from healthy donors, it has been proposed that CXCL12 might play a relevant role in B-cell migration into the rheumatoid synovium [
19,
36]. However, our results tended toward a different direction; CXCR4 is strongly upregulated on the cell surface of SF B cells, a finding counter to the signaling by CXCL12 in these cells. Furthermore, our data on CXCR7 regulation in SF B cells go in the same direction. CXCR7 is an “atypical” chemokine receptor that acts as a decoy receptor for CXCL12 and CXCL11. The expression level of CXCR7 was significantly lower in SF B cells than in PB, suggesting that in the synovial microenvironment this B-cell receptor sequesters CXCL12, consequently reducing the availability of this chemokine for binding with CXCR4.
CXCL13 and CCL20, specific ligands of CXCR5 and CCR6, respectively, have been detected in the synovium of patients with chronic arthritis [
17,
21,
41]. CXCL13 has been described as the most effective chemoattractant for B cells [
42] and CCL20 as a selective chemoattractant factor for lymphocytes [
43] expressing CCR6 receptors, such as naïve B cells [
44]. Migration experiments showed that these two chemokines are chemoattractant for PB B cells from healthy donors. Synergistic effects among several chemokines have been described in vitro, which translates into increased leukocyte migration [
14]. In this study, we present evidence of a novel positive regulatory mechanism of leukocyte migration in which the concomitant presence of adequate concentrations of CXCL13 and CCL20 synergistically increases the chemotaxis of B cells in vitro. We postulate that this type of synergy is mediated by dual receptors in which CXCR5 and CCR6 become activated following simultaneous or sequential binding of their agonists [
14]. However, this issue should be confirmed by additional experimentation. Concurrently with these results, in Transwell experiments B cells from the PB of RA patients migrated to a significantly greater degree in the presence of CXCL13 and CCL20 than at baseline, and synergistically in the presence of both chemoattractants. However, the ability of B cells from SF of these patients to migrate in the presence of these chemokines was significantly lower than that of PB, which suggests that on B cells CXCR5 and CCR6 undergo desensitization in the synovial microenvironment. Conversely, B cells from SF showed very high migration ability in response to CXCL12, even higher than those from PB. This is an interesting finding that suggests additional regulatory mechanisms for CXCR4 expression and function, occurring in B cells migrated to the inflamed synovial tissue. Although the data obtained in both expression of receptors and migration are very consistent among patients, a limitation of this study is that the sample size does not allow the analysis of patients according to the use of steroids or DMARDs.