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Marie-Luise Humpert et al.
DOI: 10.1002/eji.201343907
Eur. J. Immunol. 2014. 44: 694–705
CXCR7 influences the migration of B cells during maturation Marie-Luise Humpert1,2 , Dora Pinto1 , David Jarrossay1 and Marcus Thelen1 1 2
Institute for Research in Biomedicine, Bellinzona, Switzerland Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland
The atypical chemokine receptor CXCR7 binds the chemokines CXCL12 and CXCL11. The receptor is widely expressed and was shown to tune CXCR12-induced responses of CXCR4. Here, the function of CXCR7 was examined at late stages of human B-cell maturation, when B cells differentiate into Ab-secreting plasmablasts. We identified two populations of CXCR7+ cells in tonsillar lymphocytes, one being presumably memory B cells or early plasmablasts (FSClow CD19+ CD38mid ) and the other being plasmablasts or early plasma cells (FSChigh CD19+ CD38+ ). CXCR7 is expressed on CD19+ CD27+ memory B cells, on CD19+ CD38+ CD138− and intracellular immunoglobulin high plasmablasts, but not on CD19+ CD138+ icIghigh plasma cells. The differential expression pattern suggests a potential contribution of the scavenger receptor in final B-cell maturation. On in vitro differentiating B cells, we found a marked inverse correlation between CXCR7 and CXCR5 cell surface levels, whereas expression of CXCR4 remained almost constant. Migration assays performed with tonsillar mononuclear cells or in vitro differentiated cells revealed that inhibition of CXCR7 markedly increases chemotaxis toward CXCL12, especially at late stages of B-cell maturation. Chemotaxis was attenuated in the presence of CXCR4 antagonists, confirming that migration is CXCR4 mediated. Our findings unequivocally demonstrate a novel role for CXCR7 in regulating the migration of plasmablasts during B-cell maturation.
Keywords: Atypical chemokine receptor
r
B cells
r
CXCR4 r CXCR5 r CXCR7
Additional supporting information may be found in the online version of this article at the publisher’s web-site
Introduction After birth, B lymphopoiesis occurs in the BM, where cells progress through pro-B cell and pre-B cell stages. By expression of a functional BCR, B cells acquire antigen specificity and enter the periphery as transitional immature B cells, eventually maturing into follicular or marginal zone B cells [1]. Follicular B cells circulate
Correspondence: Dr. Marcus Thelen e-mail:
[email protected] C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
between lymphoid organs and form GCs within secondary lymphoid organs upon activation. The GC splits up into two histological distinct compartments, the dark zone, where B cells are tightly packed and proliferate, and the light zone. Within the GC, CXCL12 is preferentially expressed in the dark zone, whereas CXCL13, which is also expressed in the follicle, is mainly found in the light zone. B cells may modulate the surface expression of CXCR5 and move between the zones of the GC attracted by CXCL12 and CXCL13 [2]. Activated B cells can grow and differentiate into plasmablasts in extrafollicular foci or form GCs within follicles [3, 4]. Within the GC, activated B cells can differentiate into memory or plasma
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cells [5]. Cells expressing a BCR with high avidity for an antigen differentiate preferentially along the plasma cell pathway and this process might be regulated by expression of factors such as the B-lymphocyte maturation-induced-protein-1, BCL-6, and X-box binding protein-1 [6–8] or cytokines such as IL-6 or APRIL [3]. Others showed that Bcl-2, the cytokine IL-21, and SWAP70, which functions in activation, homing, and class switching of B cells, and is strongly upregulated in GC B cells but absent in plasma cells, are involved in commitment of GC B cells into memory B or plasma cells [9–11]. Human memory B cells are characterized by the expression of CD19, CD20, and CD27. Reactivated memory B cells differentiate into Ab-secreting plasmablasts and, in general, home to the BM or into the mucosa where they become long-lived plasma cells, depending on their chemokine receptor expression [9,12]. B cells alter the expression of chemokine receptors when they exit the GC and initiate migration to the BM, mucosa, or sites of inflammation [12,13]. Both, CCR7 and CXCR5, generally expressed by follicular B cells under homeostatic conditions, are downregulated, whereas CXCR4 expression is stable in order to enable plasma cell homing to the splenic red pulp, LN medullary cords, and BM [14, 15]. Precursors of plasma cells, generally termed plasmablasts, display high levels of surface CD27, CD38, and have a high intracellular immunoglobulin (icIg) content, whereas surface expression of CD20 is downregulated and the proteoglycan CD138 is barely expressed [16]. The rare peripheral blood plasma cells are characterized by high expression of CD27 and CD38, Ig secretion, and an increasing presence of CD138 [17]. Isolated plasma cells die within 3 days by apoptosis, but using BM-derived stromal cells as feeder cells remarkably improves plasma cell survival [18–20]. In agreement with this in vitro observation, it was shown that in vivo plasma cells, which express VLA-4 and CXCR4, in the BM colocalize with stromal cells expressing VCAM-1 and CXCL12 and may require additional survival factors [17, 20, 21]. It was thought that CXCR4 and its ligand CXCL12 represent a unique couple [22], but this view changed when it was shown that CXCL12 can bind and partially signal through CXCR7 [23–25]. Expression of CXCR7 mRNA in leukocytes is amply reported [24, 26]. In contrast, expression of CXCR7 on leukocytes was controversial [27, 28]. However, functional CXCR7 protein expression on lymphocytes was demonstrated and recently confirmed by different molecular and proteomic approaches [23, 26, 28, 29]. Although CXCR4 is a typical chemokine receptor activating cellular processes such as chemotaxis, proliferation, apoptosis, survival, and differentiation [30,31], CXCR7 is an atypical chemokine receptor, which binds CXCL12 and CXCL11 [23, 32]. Atypical chemokine receptors are unable to activate conventional signaling pathways, but by acting as scavengers they are important regulators of chemokine functions [24]. CXCR7, which binds CXCL12 with about tenfold higher affinity than CXCR4, can control the availability of CXCL12 and therefore shape functional chemokine gradients for the CXCR4 [23, 32–34]. In line with this, it was shown that scavenging activity of CXCR7 expressed on stroma cells is essential for the directional migration of primordial germ cells in zebrafish [35]. Furthermore, overexpression studies sug C 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Cellular immune response
gest that CXCR7 may form heterodimers with CXCR4 affecting the signaling properties of CXCR4 [36–38]. Here, we report two populations of CXCR7+ cells on tonsillar lymphocytes, one being presumably memory B cells or early plasmablasts (FSClow CD19+ CD38mid ) and the other plasmablasts or early plasma cells (FSChigh CD19+ CD38high ). An in vitro B-cell maturation assay reveals an inverse correlation between CXCR7 and CXCR5 cell surface levels, whereas expression of CXCR4 remains almost constant. Moreover, inhibition of CXCR7 markedly increases chemotaxis toward CXCL12 at late stages of B-cell maturation. The results suggest that the scavenger activity of CXCR7 reduces the level of CXCL12 in the medium, thereby limiting the migratory capacity of CXCR4. Taken together the findings suggest an important role of CXCR7 in regulating the migration of plasmablasts and plasma cells.
Results Expression of CXCR7 on human peripheral blood and tonsillar B and T cells We demonstrated that approximately 9% of PBLs express detectable levels of CXCR7. When the population was gated on CD19+ cells, which represent a minor fraction of total lymphocytes (99% purity, B cells were isolated from PBMCs using human CD19 magnetic MicroBeads according to the manufacturer’s instructions (MACS, Miltenyi Biotec) and subsequently stained with CD19, CD27, and CD38 to identify memory B cells (CD19+ , CD27+ , and CD38low ). For the in vitro culture assay, memory B cells were stimulated with 2.5 μg/mL CpG 2006 (5 GGGGGACGATCGTCGGGGGG-3 ) for 12 h, washed with B-cell medium, and plated on hTERT+ MSCs (20 000 cells/well) in a 96-well plate. Cells were harvested after indicated days, washed and stained with appropriate Abs to analyze the maturation by FACS analysis. Tonsils were obtained from routine tonsillectomies performed at the “Ospedale San Giovanni”, Bellinzona. After mincing, tonsils were treated with 1 mg/mL collagenase D and 1mg/mL DNase (Roche Diagnostics) for 70 min at 37◦ C. TMCs were prepared by Ficoll-Paque density centrifugation as previR (Corning) ously described [28]. For FACS analysis and Transwell migration assays, B cells were isolated through negative selection by using the StemSepTM Human B Cell Enrichment Kit according to the manufacturer’s instructions (StemCell technologies). CXCR7+ cells were sorted from TMCs by staining with the anti-CXCR7 mAb 9C4 and an appropriate secondary goat anti-mouse IgG1 Ab and subsequent sorted using magnetic MicroBeads according to the manufacturer’s instructions (MACS, Miltenyi Biotec).
Acknowledgements: This study was supported by funds obtained from the Jost Reinhold Foundation, the Gottfried und Julia Bangerter-Rhyner-Stiftung, Basel, the Helmut Horten Foundation, Novartis Foundation for Medical-Biological Research, and the Fondazione Ticinese per la Ricerca sul Cancro (M-L.H. and M.T.). Conflict of interest: The authors declare no financial or commercial conflict of interest.
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Abbreviations: FSC: forward scatter · icIg: intracellular immunoglobulin · S1P2 : sphingosine 1-phosphate receptor type 2 · TMC: tonsillar mononuclear cell
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Received: 17/7/2013 Revised: 31/10/2013 Accepted: 15/11/2013 Accepted article online: 20/11/2013
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