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Blood, 15 May 2005, Vol. 105, No. 10, pp. 3965-3971. Prepublished online as a Blood First Edition Paper on February 1, 2005; DOI 10.1182/blood-2004-08-2992.
IMMUNOBIOLOGY Regulation of CXCR3 and CXCR4 expression during terminal differentiation of memory B cells into plasma cellsFrom the Department for Humoral Immunology, German Rheumatism Research Center, Berlin, Germany; Department of Medicine, Rheumatology and Clinical Immunology, Charité University Hospital, Berlin, Germany; Department of Microbiology and Immunology, University of Buffalo, Buffalo, NY; Department for Research and Development, Miltenyi Biotec, Bergisch Gladbach, Germany; and MRC Centre for Immune Regulation, Division for Immunity and Infection, University of Birmingham, Birmingham, United Kingdom.
C-X-C motif chemokine receptor 3 (CXCR3) and CXCR4 expressed on immunoglobulin G (IgG)–plasma-cell precursors formed in memory immune responses are crucial modulators of the homing of these cells. Here, we studied the regulation of the expression of these chemokine receptors during the differentiation of human memory B cells into plasma cells. We show that CXCR3 is absent on CD27- naive B cells but is expressed on a fraction of memory B cells, preferentially on those coexpressing IgG1. On differentiation into plasma-cell precursors, CXCR3+ memory B cells maintain the expression of this chemokine receptor. CXCR3- memory B cells up-regulate CXCR3 and migrate toward concentration gradients of its ligands only when costimulated with interferon  (IFN-), but not interleukin 4 (IL-4), IL-1 , IL-6, IFN- , IFN-, or tumor necrosis factor (TNF-). In contrast, the differentiation of CXCR4- B cells into plasma cells is generally accompanied by the induction of CXCR4 expression. These results show that lack of CXCR4 expression on plasma-cell precursors is not a limiting factor for plasma-cell homing and that the expression of CXCR3 on memory B cells and plasma-cell precursors is induced by IFN-, provided in human T helper type 1 (Th1)–biased immune responses. Once induced in memory B cells, CXCR3 expression remains part of the individual cellular memory.
Antibodies secreted by terminal differentiated B cells are important in immune defense,1 but also can contribute to the pathogenesis of autoimmune diseases.2 On stimulation by antigen and CD40 signaling by T cells, naive B cells can form germinal centers yielding memory B cells expressing antibodies of higher affinities.3 On a second contact with antigen, memory B cells can differentiate into proliferating antibody-secreting plasmablasts found in the extrafollicular areas of secondary lymphoid tissues.4 These cells further differentiate into mature nondividing plasma cells either remaining within the tissues of their origin or accumulating in mucosa-associated tissues, bone marrow, and sites of inflammation.5,6 Plasma-cell homing to different tissues is a crucial regulator of the strength and duration of humoral immune responses. Maintained antibody responses are associated with plasma-cell homing to the bone marrow.7 Under pathologic conditions, longterm survival of plasma cells is also supported in chronically inflamed tissues.8,9 The local production of antibodies by plasma cells resident at such sites is likely to be important in clearing pathogens. In individuals suffering from systemic inflammatory autoimmune diseases, disturbed plasma-cell localization is associated with the production of autoantibody titers refractory to conventional therapy.10 During differentiation into plasma cells, B cells change their chemokine receptor expression profile.5,6,11 Expression of C-C chemokine receptor type 9 (CCR9) and CCR10 allows homing of immunoglobulin A (IgA) plasma cells to mucosal tissues.12,13 IgG+ plasma-cell precursors formed in a secondary immune response against systemic antigenic challenge migrate toward ligands for C-X-C motif chemokine receptor 3 (CXCR3) and CXCR4, likely allowing them to migrate to inflamed tissue and bone marrow, respectively.14,15 Additionally, both chemokine receptors and their cognate ligands likely also play a role for the localization of activated B cells, plasmablasts, and plasma cells within secondary lymphoid tissues. Plasma cells surrounding the germinal centers are colocalized with CXCL12, the ligand for CXCR4.16,17 In the draining lymph nodes, CXCL9 is produced by a subset of high endothelial venules (HEVs) surrounding the B-cell follicles.18
Here, we show that in vivo, CXCR3 is expressed only on a fraction of human memory B cells, preferentially those expressing IgG1. Once induced, the expression of this chemokine receptor is maintained during plasma-cell differentiation. Thus, the information whether this chemokine receptor is used during an immune response seems to be part of the B-cell memory. On CXCR3- B cells, interferon In contrast, the majority of memory B cells express CXCR4. On B cells lacking this receptor, on activation with various stimuli, plasma-cell differentiation is generally accompanied by induction of CXCR4 expression. Thus, lack of CXCR4 expression on plasma-cell precursors is not a limiting factor for plasma-cell homing to the bone marrow.
Media and cytokines
Cell culture medium RPMI 1640 (Life Technologies, Paisley, United Kingdom) was used, supplemented with 10% fetal calf serum (FCS), 20 µM Cell culture
B cells were purified from human blood of healthy donors. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated by a standard Ficoll-Hypaque PLUS (Amersham Biosciences, Uppsala, Sweden) gradient method. B cells were enriched from PBMCs to more than 98% purity (data not shown) by magnetic-associated cell sorting (MACS). PBMCs were incubated 15 minutes at 4°C with antihuman CD19 microbeads as indicated by the manufacturer (Miltenyi Biotec; Bergisch Gladbach, Germany), washed, and loaded on 2 succeeding light-sensitive (LS) columns (Milteny Biotec). For some cultures, CXCR3- or CXCR4- cells were isolated by fluorescence-activated cell sorting (FACS DIVA, Becton Dickinson, Heidelberg, Germany). The isolated cells were mainly cultured in a 2-step culture system. B cells were initially cocultured with the murine thymoma cell line EL4-B5 (50 Gy irradiated)19, constitutively expressing CD40L with the addition of recombinant IL-2 (50 U/mL) and IL-10 (50 U/mL) for a B cell/T cell ratio of 1:10. After 3 days of culture, B cells were isolated by MACS by using antihuman CD19 beads as described. Subsequently, they were cultured for another 5 days in the presence of recombinant IL-2 (50 U/mL) and IL-10 (50 U/mL). In some cultures IL-4 (100 U/mL), IL-1 Staining and flow cytometry Isolated PBMCs were stained with antihuman CXCR3-phycoerythrin (PE), CXCR4-allophycocyanin (APC), CD19-biotinylated, IgM-fluorescein isothiocyanate (FITC) purchased from BD Pharmingen (Le Pont de Claix, France), and IgA-FITC (Cymbus Biotechnology, Hampshire, United Kingdom). The following antibodies were produced and coupled in the institute to fluorochromes or biotin: CD19-FITC (clone BU 12), CD27-PE (clone 2 E 4), IgG1-FITC (clone IDC-1), IgG2-biotinylated (clone G 18-21), and IgG3-biotinylated (clone G 18-3). CD20-FITC, CD19-biotinylated, CD27-FITC, CD38-APC, CXCR3-PE, and CXCR4-PE were purchased from BD Pharmingen; streptavidin-peridinin chlorophyll protein (PerCP; BD Pharmingen) was used as a secondary reagent. Dead cells were excluded with DAPI dilactate (4',6-diamino-2-phenylindole, dilactate; 0.2 µg/mL; Molecular Probes, Eugene, OR). Cells were incubated with staining reagents in phosphate-buffered saline/bovine serum albumin (PBS/BSA, 0.5%) for 10 minutes on ice. Analysis was performed by flow cytometry (LSR I, Becton Dickinson). Cell cycles were measured by flow cytometry using 5- and 6-carboxyfluorescein diacetate–succinimidyl ester (CFDA-SE) as described (Molecular Probes).20 Chemotaxis assay A chemotaxis assay recently described14 was modified to analyze human cells. Chemotaxis assays were performed in 5-µm pore Transwell inserts (Costar, Corning, NY) coated with 50 µL human fibronectin (10 µg/mL; Sigma-Aldrich). Cells harvested from culture were washed and diluted in prewarmed (37°C) assay medium (RPMI 1640 supplemented with 0.5% BSA, low endotoxin; Sigma-Aldrich) at a concentration of 2.5 to 5 x 106 cells/mL. The lower Transwell chamber was filled with 600 µL assay medium supplemented with the chemokine CXCL9 (100 nM; R&D Systems) and 100 µL cells was added to upper chamber and incubated for 90 minutes at 37°C in a atmosphere of 5% CO2. Subsequently, IgG-secreting cell numbers before and after migration were quantified by solid-phase enzyme-linked immunospot (ELISPOT) assay. Migration toward medium alone was less than 2% (basal migration). ELISPOT IgA-, IgG-, or IgM-secreting cells were quantified by ELISPOT.21 Antibodies used for ELISPOT were goat anti–human isotype antibodies (Sigma-Aldrich; for coating used 5 µg/mL) and biotin labeled goat anti–human isotype antibodies (Sigma-Aldrich; for detection 1 µg/mL). Streptavidin-alkaline phosphatase (Roche, Mannheim, Germany) and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP; Sigma-Aldrich) were used for development as described.14
The expression of CXCR3 on memory B cells is correlated with coexpression of IgG1 In mice, the chemokine receptors CXCR3 and CXCR4 are important in the regulation of the localization of IgG-plasma cells formed within memory immune responses.14 In the present study, the expression of these chemokine receptors was determined on human memory B cells from peripheral blood of healthy donors and correlated to the expression of particular antibody isotypes by FACS. B cells were identified by the expression of CD19; naive and memory B cells were distinguished by the absence and presence of CD27 expression, respectively.22 About 6.4% ± 4.3% of naive B cells expressed CXCR3 and 87.6% ± 8.8% expressed CXCR4. Among the population of memory B cells, 32.1% ± 9.5% expressed CXCR3 and 68.1% ± 2.9% expressed CXCR4 (data not shown). No correlation was found between CXCR4 expression and the coexpression of the antibody isotypes IgG1, IgG2, IgG3, IgA, or IgM on individual cells (Figure 1). In contrast, CXCR3 expression was associated with the coexpression of IgG1. About 34.7% ± 3.9% of memory cells expressing IgG1, but only 10% ± 6.3% expressing IgG2 (P < .001), did coexpress CXCR3 (Figure 1). The differences in CXCR3 expression between memory B cells expressing IgG1 and IgG3 (P < .030) or IgM (P < .043) were also significant, but less pronounced.
Once induced in B cells, CXCR3 expression is part of the cellular memory To induce differentiation into plasma cells, B cells were stimulated by cytokines plus CD40 ligation or CpG as described in "Materials and methods." With both methods, following 8 days in culture, 10% to 40% of cells showed a CD20-/CD38++ plasma-cell phenotype (Figure 2A). As evaluated by ELISPOT, similar frequencies of cultured cells secreted antibodies (data not shown). In accordance with earlier results,23,24 under these stimulation conditions preferentially CD27+ memory B cells were activated to differentiate into plasma cells (data not shown).
To analyze possible changes in the expression of CXCR3 during their differentiation into plasma cells, before culture, CD19+ B cells were sorted into CXCR3- and CXCR3+ fractions by FACS. When cultures were started with CXCR3- or CXCR3+ B cells, between 13.7% ± 1.5% or 81.0% ± 4.8% of B cells and plasma cells, respectively, expressed this chemokine receptor at day 3 and day 8 (Figure 2B). This result shows that once induced in memory B cells, CXCR3 expression remains stable during their differentiation into plasma cells and is part of the individual cellular memory.
CXCR3 expression is induced by IFN-
The question how the expression of CXCR3 is induced in B cells was addressed next. Ligands for CXCR3 attract lymphocytes into inflamed tissues,25,26 favoring inflammatory cytokines like IFN-
To identify the factors regulating CXCR3 expression, CXCR3- B cells were activated by CD40 ligand together with IL-2 and IL-10 as described and cultures were supplemented with the candidate cytokines listed. When no additional cytokines were added, the frequency of CXCR3+ cells among the CD20+/CD38+/- B cells and CD20-/CD38++ plasma cells were 19.5% ± 3.8% at day 8 of culture. Addition of IL-1
In these experiments, as a source of CD40L, irradiated thymoma cells were used. To exclude the possibility that the expression of CXCR3 on B cells was not directly induced by IFN- , but by another factor expressed by the thymoma cells on stimulation with IFN-, in other experiments purified B cells were stimulated by CpG, in the absence of any other cell type. Again, addition of IFN- increased the frequency of CXCR3+ B cells and plasma cells up to 43.4% ± 2.% (Table 1).
Together, these experiments identified IFN-
Stimulation with IFN-
The expression of a chemokine receptor on a particular cell does not necessarily correlate with its capability to migrate toward the concentration gradients of the corresponding chemokines.30,31 Therefore, we tested whether the addition of IFN- CXCR4 is constitutively up-regulated during T-dependent and T-independent plasma-cell differentiation It had been shown in mice that plasmablasts formed in the course of a T-dependent immune response express CXCR4 and remain positive for this receptor during their differentiation into plasma cells.11,14 CXCR4-deficient plasma cells accumulate only in severely reduced numbers in the bone marrow.17 About 68.1% ± 2.9% of human peripheral blood CD27+ memory B cells express CXCR4 (Figure 1 and data not shown). Following stimulation, the frequency of CXCR4+ cells among the activated CD20-/CD38+/- B cells and CD20-/CD38++ plasma cells increased slightly to 90% and 95%, respectively (data not shown). To test whether this was due to up-regulation of this chemokine receptor or due to a selective recruitment of CXCR4+ B cells into the plasma-cell differentiation pathway, CXCR4- B cells were enriched to more than 98% purity. These cells, roughly 72% of them memory B cells, were activated by IL-2 and IL-10 and either CD40L, S aureus Cowan I (SAC), or CpG. These culture methods resemble T-dependent and T-independent B-cell stimulations, respectively. After 3 days, in all cultures, 50.1% ± 9.4% of B cells already expressed CXCR4. At day 8, 75.2% ± 4.8% of plasma cells expressed CXCR4 (Figure 5). About 87.6% ± 8.8% of isolated CD27- naive B cells expressed this chemokine receptor. Following stimulation under T-independent activation conditions by B-cell receptor cross-linking, these cells also maintained CXCR4 expression (data not shown). Together, these results indicate that differentiation of naive and memory B cells into plasma cells is generally accompanied by induction of the expression of this chemokine receptor. Up-regulation of the expression of CXCR3 and CXCR4 does not depend on cell-cycle progression Recent data revealed that the frequency of CXCR3+ T cells following activation increases with the number of cell divisions. In contrast, the frequency of CXCR4+ T cells decreases with cell-cycle progression.32,33
Here, we tested whether up-regulation of CXCR3 and CXCR4 on B cells is associated with the number of cell divisions. B cells negative for the respective chemokine receptor were stained with CFDA-SE and stimulated with CpG, IL-2, and IL-10. To induce the expression of CXCR3 on CXCR3- B cells, IFN- was added. At day 3 of the cultures, expression of CXCR3, CXCR4, and content of CFDA-SE was analyzed (Figure 6). A fraction of B cells that had not undergone a complete cell division had already up-regulated CXCR3 and CXCR4. The frequencies of B cells that had up-regulated these receptors did not significantly differ between those that had proliferated and those that had not. These results indicate that the expression of CXCR3 and CXCR4 on activated B cells does not depend on proliferation. This is in accordance with the observation that the induction of CXCR3 expression occurs early following activation.
CXCR3, CXCR4, and their cognate chemokines are important regulators for the homing of murine IgG-secreting plasma cells formed in memory immune responses.14 The presence of CXCR4 on human bone marrow plasma cells had been reported.34,35 The importance of CXCR4 for plasma-cell homing to the bone marrow had been clearly demonstrated in CXCR4-deficient fetal liver chimeras. In these mice, normal numbers of plasma cells are formed but compared to wild type, the numbers of plasma cells accumulating in the bone marrow are reduced to about 30%.17 Plasma-cell homing to bone marrow is a specific feature of T-dependent immune responses.36 These findings prompted us to question whether up-regulation of CXCR4 expression on plasma-cell precursors determines their capabilities to home to the bone marrow and whether induction of the expression of this receptor depends on T cell–derived signals. As shown in the present study, in peripheral blood, the great majority of all mature naive and memory B cells express CXCR4 and remain positive for this receptor following activation and differentiation into plasma cells. On activation of B cells by all stimuli tested, including T-independent signals, plasma-cell differentiation generally seems to be accompanied by the induction of CXCR4 expression. These results suggest that CXCR4 expression is not limited to plasma-cell precursors formed in T-dependent responses and that during plasma-cell differentiation, the decision for bone marrow homing is not made by the induction to express CXCR4. Recent results showed that all human bone marrow plasma cells also express CXCR6 and CCR10.34 It remains to be elucidated whether plasma-cell homing to the bone marrow is regulated by the modulation of CXCR4 function or by other chemokine receptors. Plasma cells can accumulate within chronically inflamed tissues due to in situ formation37 and due to efficient immigration from secondary lymphoid tissues.8 Ligands for CXCR3 likely attract plasma-cell precursors to sites of inflammation.14,38 There, the CXCR3 ligands CXCL9, CXCL10, and CXCL11 are expressed in high quantities, attracting activated CXCR3-bearing leukocytes.25 The production of CXCL9 in high endothelial venules surrounding the B-cell follicles18 may indicate that the expression of CXCR3 on activated B cells also modulates their recirculation through, or release from, the follicles, hence influencing their differentiation. This idea is supported by the observation that the expression of CXCR3 on B cells is induced early following activation, even before these cells had divided.
The data presented here identified IFN-
The observed results show that the induction of CXCR3 occurs within the first 3 days of plasma-cell differentiation, that is, before B cells acquire a plasma-cell phenotype, thus suggesting that up-regulation of CXCR3 expression occurs on activated B cells, but not during later differentiation stages on plasmablasts or plasma cells. In the course of an immune response in vivo, before day 3 of antigenic challenge, B cells are found in the follicular areas adjacent to T-helper cells.39 These T-effector cells are classified into Th1 and Th2 cells, originally defined by their capacity to secrete either IFN-
It could not be excluded that other factors than IFN- can induce CXCR3 expression on terminally differentiating B cells. However, the results presented in this study suggest that the following scenario is at least one mechanism leading to the accumulation of plasma cells within inflamed tissues. At that site, interferons, mainly IFN-, induce the expression of the CXCR3 ligands CXCL9, CXCL10, and CXCL11, allowing the attraction of CXCR3-bearing leucocytes.25 Antigen-presenting cells (APCs) immigrating from the site of inflammation induce the formation of IFN-–secreting Th1 cells in the draining lymph nodes. These T cells can stimulate activated B cells specific for the same antigen to express CXCR3, thus supporting their accumulation within the adjacent inflamed tissue.
It had been shown by Butcher and colleagues that peripheral antibody-secreting cells induced by vaccination via parenteral or mucosal routes differ in their homing potential resembled by the expression pattern of tissue specific adhesion molecules.47 The authors proposed that the site of original B-cell activation determines the preferential homing potentials of antibody-secreting cells to mucosal sites or the systemic compartment. The data presented here show that B cells stimulated with IFN-
We thank Toralf Kaiser and Katharina Raba for uncomplaining help by cell sorting. Prof L. Wieler, Katrin Moser, Miro Mostarac, and Velia Gerl are gratefully acknowledged for helpful discussions.
Submitted August 4, 2004; accepted January 14, 2005.
Prepublished online as Blood First Edition Paper, February 1, 2005; DOI 10.1182/blood-2004-08-2992.
Supported by the Deutsche Forschungsgemeinschaft Klinische Forschergruppe KFO 105 and by the European Community QLK2-CT-2001-01205 (Memovax).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: RudolfA. Manz, Deutsches Rheumaforschungszentrum, Department for Humoral Immunology, Schumannstrasse 21/22, D-10117 Berlin, Germany; e-mail: manz{at}drfz.de.
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Abstract
Introduction
