Introduction
Rheumatoid arthritis (RA) is a complex autoimmune disease of unknown etiology. It is characterized by chronic inflammation of the synovial membrane and the formation of a pannus, which leads to swollen joints and finally to joint destruction. Inflammatory cells such as monocytes and neutrophils, together with T and B cells, infiltrate the synovial membrane [
1]. Migration of lymphocytes from the blood to the synovial tissue is a multi-step process controlled in part by interactions between chemokines and their receptors [
2,
3].
About 50 chemokines have been identified in humans and are divided into four groups according to their cysteine motifs [
4]. After activation and differentiation, cells of the lymphoid lineages dynamically change their expression profiles of chemokine receptors, which results in specific migration in response to chemokines [
5,
6].
Under pathological conditions, such as RA, chemokines direct lymphocytes into the chronically inflamed synovial tissue [
7,
8]. Both the residual synovial-lining cells and infiltrating leukocytes are the source of pro-inflammatory chemokines such as CCL2, CCL3, CCL5, CCL20, CXCL9 and CXCL10, as well as chemokines important for the homeostasis of lymphocytes, such as CXCL12 and CXCL13 [
2,
9‐
13]. As a consequence of the synovial inflammation, these chemokines are also found in the synovial fluid.
Contradictory results have been reported for chemokine receptor expression on peripheral blood T cells of patients with RA [
13,
14] and even less is known about the expression of chemokine receptors on B cells. The classical chemokine receptor on B cells is CXCR5. Its ligand CXCL13 is a potent B cell chemo-attractant molecule directing B cells into the follicles of secondary lymphoid organs [
15]. In addition to CXCR5, the chemokine receptors CXCR4 and CCR7 have been implicated in B cell migration into the follicular structures, and especially CXCR4 in directing plasma cells into the bone marrow [
6]. These chemokines and their corresponding chemokine receptors are also expressed in the inflamed synovial tissue of patients with RA. In particular, high expression of CXCL13 was found when the synovial tissue contained large aggregates of B cells, resembling the structure of germinal centers [
11,
17]. These findings suggested that the chemokine CXCL13 is involved in B cell trafficking into the inflamed tissue and has a role in the formation of ectopic lymphoid tissue [
18,
19].
CXCR3 is a chemokine receptor for the inflammatory chemokines CXCL9, CXCL10 and CXCL11. It has been described as a marker for malignant B cells and is absent from the majority of normal peripheral blood B cells [
20,
21]. The analysis of patients with multiple sclerosis showed that CXCR3 becomes upregulated on infiltration of the inflamed cerebrospinal fluid [
20].
To address the question of B cell migration, we analyzed chemokine receptor expression on peripheral blood B cells. Expression profiles of patients with RA were compared with those from patients with systemic lupus erythematosus (SLE), who – in contrast to patients with RA – usually show no or very little B cell accumulation at the site of chronic inflammation [
22]. In addition, patients with osteoarthritis (OA) were included into the study as a control cohort. OA is a non-inflammatory degenerative joint disease. Our data suggest that the chronic inflammation in patients with RA and patients with SLE leads to changes in the chemokine receptor expression pattern on peripheral B cells; however, the expression patterns of chemokine receptors in RA and SLE are differentially regulated.
Methods
Patients and controls
Heparinized whole blood (9 ml) from patients with RA, patients with SLE and patients with OA was obtained from the Departments of Rheumatology and Orthopedics (Charité, Humboldt University, Berlin, Germany). Patients with RA were diagnosed in accordance with the American College of Rheumatology criteria [
23]. As control, blood samples from healthy blood donors were analyzed. The demographic data and the treatment of patients are summarized in Table
1. The scientific ethics committee of Charité approved the study, and informed consent was obtained.
Table 1
Demographics and medications of patients in the study
Total numbers | 26 | 11 | 13 | 21 |
Age (years) | | | | |
Mean | 55.1 | 42.5 | 71.2 | 42.6 |
Range | 25–76 | 27–78 | 62–79 | 25–64 |
Sex (n) | | | | |
Female | 21 | 10 | 8 | 15 |
Male | 5 | 1 | 5 | 6 |
Duration of disease (years) | | | | |
Mean | 8.1 | 9.5 | 3.3 | - |
Range | 0.1–37 | 2–25 | 0.5–10 | - |
Clinical parameter (mean ± SD) | | | | |
Leukocytes (109/l) | 9.5 ± 2.7 | 4.3 ± 1.1 | 7.8 ± 2.7 | nd |
CRP (mg/dl) | 3.1 ± 3.0 | 1.1 ± 0.9 | 0.5 ± 0.6 | nd |
ESR (mm/h) | 37.5 ± 30.5 | 34.9 ± 16.7 | 28.5 ± 19.4 | nd |
Treatment (n) | | | | |
None | 6 | - | - | 21 |
Corticosteroids and/or NSAID | 7 | - | 8a | - |
Anti-TNF-α therapy | 13b | - | - | - |
Predisolone | - | 11c | - | - |
Cyclophosphamide (i.v.) | - | 6 | - | - |
Cell isolation and flow cytometry
Peripheral blood mononuclear cells (PBMC) were isolated by gradient centrifugation with Ficoll (Amersham Biosciences) and then stained (30 min at 4°C) with a biotin-coupled B cell-specific mAb against CD19 (clone SJ25-C1; Southern Biotechnology Associates), with the Cy5-labeled mAb against CD27 (clone 2E4; gift from RA van Lier, Academic Medical Center, Amsterdam) and with fluorescein isothiocyanate (FITC)-labeled mAb specific for one of the chemokine receptors CXCR3 (clone 49801.111; R&D Systems), CXCR5 (clone 51505.111; R&D Systems), CCR5 (clone 45523.111; R&D Systems), CCR6 (clone 53103.111; R&D Systems), CCR7 (clone 3D12; a gift from M Lipp, Max Delbrück Center, Berlin) or CCR9 (clone 112509.111; R&D Systems). Antibody against CXCR4 was labeled with phycoerythrin (PE; clone 12G5; BD Pharmingen). Before incubation with streptavidin-PE or streptavidin-FITC (for CXCR4) (0.5 μg/ml; Pharmingen), cells were washed twice in phosphate-buffered saline/2% BSA/4 mM EDTA.
To determine the frequency of CD5+ B cells, PBMC were stained as described above with biotin-CD19, Cy5-CD27 and PE-labeled mAb against CD5 (clone UCHT2; BD Pharmingen). For the analysis of chemokine receptor expression, CD5+ B cells were purified by magnetic cell sorting with CD19-specific beads (Miltenyi). Isolated B cells were stained as described above with PE-CD5 and Cy5-CD27 together with the FITC-labeled mAb against the chemokine receptors CXCR3 or CXCR5.
Propidium iodide (1 μg/ml; Sigma) was added immediately before cytometric analysis for the exclusion of dead cells. Flow cytometric analyses were performed by fluorescence-activated cell sorting (FACS) software (FACSCalibur and CellQuest; Becton Dickinson).
Measurement of CXCL10 concentrations
For further analysis, sera of controls and those of patients analyzed for chemokine receptor expression were stored at -20°C. The concentration of CXCL10 was determined by using a sensitive ELISA test kit (HyCult Biotechnology). Samples were tested in duplicate and values were compared with a standard curve.
Transmigration assay
Cell migration was examined in Transwell™ inserts (Corning Costar) with a diameter of 6.5 mm and 5 μm pores, by using fibronectin-precoated membranes as described previously [
24]. In brief, 5 × 10
5 PBMC were suspended in RPMI 1640 medium (Life Technologies) without methyl red, supplemented with 0.5% BSA. Cells were added to the upper well and chemokine dilution or assay medium to the lower compartment; they were incubated for 90 min at 37°C under CO
2-buffered conditions. Migrated cells from triplicate wells were pooled and analyzed by flow cytometry. Optimal chemokine concentrations for migration were 50 nM for CXCL12 (R&D Systems) and 100 nM for CXCL10 (R&D Systems).
Statistical analyses
Statistical analyses were performed with GraphPad Prism software (Prism 3.0 software for windows; GraphPad). Frequencies of B cells were calculated with CellQuest software (Becton Dickinson) and variations in chemokine receptor expression on B cells within the analyzed group were compared in a hierarchic statistic analysis, using Kruskal–Wallis and the nonparametric Mann–Whitney U test. Correlations were determined by Spearman's product-moment correlation for interval data (GraphPad software). P < 0.05 was considered significant.
Discussion
The analysis of peripheral blood B cells from patients with RA and patients with SLE showed significant differences in their chemokine receptor expression when compared with B cells from healthy individuals and also from patients with OA. In patients with RA and patients with SLE a fraction of B cells showed decreased expression of CXCR5, CXCR4 and CCR6, chemokine receptors that have been associated with B cell homing into follicles [
26,
27]. In contrast, the expression of CXCR3, a receptor reactive to inflammatory chemokines [
4,
10], was increased. These changes in chemokine receptor expression seem to be associated with chronic inflammation, because they were not observed when B cells from patients with OA were analyzed. Importantly, a comparison of B cells from patients with RA and patients with SLE showed distinct disease signatures. A negative correlation of CXCR5 and CXCR3 expression in B cells was seen only in patients with RA.
CXCR5 was previously shown to be expressed on most mature circulating B cells [
8]. This receptor is the main chemokine receptor responsible for the controlled migration of B cells into secondary lymphoid organs [
28]. The analysis of blood samples from healthy individuals showed that after activation of B cells and their differentiation into plasma cells and to some extent into memory cells, CXCR5 becomes downregulated (Fig.
3). In line with these results are data from an experiment
in vitro in which stimulation with anti-CD40 antibodies led to a downregulation of CXCR5 [
29]. The finding of a significant decrease in the fraction of CXCR5
+ B cells in patients with RA and also in patients with SLE by our study may reflect the chronic activation of B cells as reported for these groups of patients [
30].
Lower levels of chemokine receptor CXCR5 and higher levels of CXCR3 on peripheral blood B cells may represent a generalized change in the profile and might be seen on all of the activated B cells in the systemic compartment or these changes in chemokine receptor expression might serve in selective recruitment into the inflamed tissue. In line with this interpretation we observed CXCR3 expression on synovial tissue B cells. However, comprehensive studies are under way to further delineate the expression of chemokine receptors and their function in migration into the effected tissue.
CXCR3 was described as a marker for malignant B cells, and its expression on normal peripheral blood B cells has been controversial [
20,
21]. Our results show that practically all B cells express CXCR3, although with a low mean fluorescence intensity. After differentiation, the level of CXCR3 expression seems to be upregulated because the frequency of CXCR3
hi B cells increases from naive to memory B cells. Activation of human B cells using a culture system
in vitro showed that after stimulation with cytokines the expression of CXCR3 is upregulated [
31]. Similarly, it was shown for T lymphocytes that after activation with interleukin-2 the expression of CXCR3 is upregulated [
32]. The observed increase in the frequency of CXCR3
hi B cells may result from the chronic activation and differentiation of B cells in patients with RA.
A significant increase in the fraction of CXCR3
hi B cells was observed only when blood samples from patients with RA were analyzed (Fig.
6). In sera of patients with SLE but not in those from patients with RA, Narumi and colleagues [
33] described high titers of CXCL10. Because CXCR3 expression may be influenced by the level of CXCL10, sera from healthy controls and from the different patient groups were tested for the presence of this chemokine. However, we did not find elevated titers of CXCL10 in our patients with SLE (Fig.
9). These results exclude the possibility that a ligand-induced receptor internalization might underlie the lower frequency of CXCR3
hi-expressing B cells in patients with SLE.
Using a transmigration assay we were able to show that low levels of CXCR3 expression are sufficient to permit a response to the migrational stimulus of the chemokine CXCL10. However, these chemotaxis results
in vitro are not necessarily predictive of their lymphocyte-recruiting activity
in vivo. The different levels of chemokine receptor CXCR3 on peripheral blood B cells may still affect B cell migration. Elevated levels of the interferon-γ-inducible chemokines CXCL9 and CXCL10, both ligands for CXCR3, have been found in chronically inflamed synovial tissue [
10]. These chemokines, which are normally involved in the chemotaxis of neutrophils, T cells and mast cells, might also influence the migration of CXCR3
+ B cells. Further experiments will be required to show whether the significant upregulation of CXCR3 on peripheral blood B cells supports their accumulation in the inflamed synovial tissue.
Whereas B cells from healthy controls and patients with OA showed little inter-individual variation in the expression of chemokine receptors, individual patients with RA and patients with SLE gave a rather heterogeneous picture (Fig.
7). For each of the chemokine receptors analyzed, the fractions of negative, low and highly positive B cells varied tremendously and were seen on both B cells and memory cells.
Little is known about the mechanisms controlling chemokine receptor expression and what might cause the variability in their expression level on peripheral blood B cells. One possibility might be that the modulation of chemokine receptor expression is associated with rheumatoid factor (RF) antibody titers. The majority of patients with RA analyzed were positive for RF. A correlation of chemokine receptor expression and the level of RF was therefore not seen.
An attempt to correlate the level of chemokine receptor expression with age or sex of the patients, with disease duration or with disease activity failed. There was no clear-cut correlation to be seen. Furthermore, from our data it seems unlikely that the variability in chemokine receptor expression results from the different treatment regimes of individual patients with RA (Fig.
10). Individual variability of chemokine receptor expression on B cells was as great in recently diagnosed, yet untreated, patients with RA as in those receiving anti-TNF-α therapies, which suggests that TNF-α itself is unlikely to be the cause of receptor modulation.
RA and SLE are chronic inflammatory diseases, and both are characterized by a continuous activation of B cells. Whereas SLE is a more systemic inflammatory disease, in most patients with RA the inflammation is localized primarily to the synovial membrane. To what extent the described differences in chemokine receptor expression between B cells from patients with RA and patients with SLE might influence the migrational pattern of B cells needs to be delineated by continuing studies, potentially permitting new therapeutic avenues.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
MH made acquisition of data and their interpretation, performed the statistical analysis and drafted the article. TD was responsible for assessment of patients and revising the article critically. G-RB was involved in the analysis and careful discussion of the data. CB coordinated the study, was involved in the critical discussion of results and their interpretation and helped to draft the article. All authors read and approved the final manuscript.