In addition, CXCR7 expressed by MZ B cells and possibly additional cells, is a sink for CXCL12. of MZ B cells, and modified granulocyte homeostasis associated with increasing serum levels of CXCL12. CXCR7 therefore appears to function as a scavenger receptor for CXCL12 on MZ B cells. Rabbit Polyclonal to Cyclin H == Intro == Localization and retention of B cells in the marginal zone (MZ) depends on antigenic specificity,1,2interactions between integrins and their receptors,3and signaling from the lysophospholipid, S1P, through the S1P1receptor.4Although MZ B cells are considered nonrecirculatory, exposure to cognate Ag or bacterial products causes them to relocalize from Cipargamin your MZ to the splenic white pulp,4providing for efficient delivery of captured Ags to follicular dendritic cells.5 Although no direct evidence links chemokine involvement to MZ B-cell localization and retention, the MZs are strikingly reduced or absent in mice deficient in a series of factors that may act downstream of chemokine receptors.6Recent studies, however, showed that transcripts for the chemokine receptor, CXCR7, were expressed at much higher levels by mouse MZ than follicular (FO) B cells.79CXCR7 has variously been reported to function as the primary receptor and scavenger for CXCL12,8,10,11a modulator of CXCR4 activity, or to act directly on different tumor types.11,12This prompted us to determine whether CXCR7 and its ligands, CXCL11 and CXCL12,11,13might influence MZ B-cell biology by taking advantage of recently developed CXCR7-specific mAbs and small molecule inhibitors. We found that CXCR7 was indicated on MZ but not on FO B cells and that treatment with inhibitors disrupted the normal organization of the MZ, modified MZ B-cell figures, and improved serum levels of CXCL12, resulting in modified homeostasis of granulocytes. == Methods == == Circulation cytometry == Splenic single-cell suspensions were blocked having a Fc receptorblocking mAb, 2.4G2, and stained with fluorochrome-labeled Abs specific for B220, CD19, CD23, CD21, Gr-1, CD3, CD11b, L, 4, 1, and 47 (purchased from BD Biosciences or eBioscience). In some experiments, cells were stained with anti-CXCR7 mAb (11G8; provided by ChemoCentryx) followed by FITC-conjugated secondary Ab. An isotype control Ab was included as a negative control. The cells were analyzed by FACSCalibur or LSRII (BD Biosciences), and the data were analyzed by FlowJo Version 7.5.5 software (TreeStar). == Animals, treatment, serum CXCL12, and histologic analysis == FVB and B6 mice (2-6 weeks old) were purchased from your Jackson Laboratory. FVB mice have unusually large MZs,14whereas B6 mice are functionally deficient in CXCL11 because of a deletion in the coding sequence.15Msnow were injected subcutaneously with CXCR7 antagonists CCX754 (100 mg/kg twice daily) or CCX771 (30 mg/kg daily; provided by ChemoCentryx) for 2-7 days. The specificity, half-life, and effect of the inhibitors Cipargamin have been reported previously.11,16,17Control mice were injected with equivalent quantities Cipargamin of vehicles (10% Captisol). The spleens were analyzed by circulation cytometry and confocal microscopy, as explained previously, with the use of labeled mAb to IgM, metallophilic macrophages (MOMA-1), Cipargamin and marginal sinus epithelial cells (MAdCAM-1).18The slides were imaged at 20C with the use of 10 and 20 oil-immersion lenses on a Leica SP2 4-chanel confocal microscope. Images were subsequently processed using Adobe Photoshop. Serum levels of CXCL12 were quantified by ELISA (Mouse CXCL12/SDF-1; Quantikine ELISA kit; R&D Systems) and CCX754 levels by ChemoCentryx. All animal studies were performed under protocols of LIP-4 and LIP-6 authorized by National Institute of Allergy and Infectious Diseases Institutional Animal Care and Use Committee. == Results and conversation == == CXCR7 is definitely differentially indicated on subsets of B lineage cells == Although earlier studies indicated that CXCR7 transcripts and protein were indicated by a variety of lymphoid and nonlymphoid cell types in mice and humans,8,9,19recent analyses performed with the CXCR7-specific mAb 11G8 and additional Abs showed that CXCR7 protein is not indicated by human being peripheral blood T or B cells, natural killer cells, or monocytes or by mouse peripheral blood leukocytes.20Nonetheless, earlier reports of high levels of CXCR7 transcripts in mouse MZ B cells and their precursors8,9prompted us to reexamine this problem with the.