(c) Wild-type c57BL/6 mouse tissue sections were stained with biotin-conjugated 11G8 by the sensitive IHC method

(c) Wild-type c57BL/6 mouse tissue sections were stained with biotin-conjugated 11G8 by the sensitive IHC method. CXCL12-mediated cell migration for therapeutic purposes. results in Arry-520 (Filanesib) increased CXCL12 levels in the bloodstream.19,26 Wang experiments and were formulated in 10% captisol for experiments (subcutaneous dosing). CXCR7?/? mice were generated at ChemoCentryx and have been described previously.37 All animal procedures were approved by the ChemoCentryx Institutional Animal Care and Use Committee. CXCL12 uptake assay CXCL12 (final concentration 1 nm) was added to a flask of human umbilical cord endothelial cells (HUVEC passage 3; Lonza, Inc., Allendale, NJ), after which CCX771 or CCX704 (final concentration 1 m) was added to the cells. At multiple time-points, an aliquot of the medium was removed from the flask and frozen. After all of the aliquots were Arry-520 (Filanesib) collected, they were thawed and analysed for CXCL12 levels using a CXCL12 ELISA kit (R&D Systems). In vivo -and = 3) and 1674 102 pg/ml CXCL12 in CCX771-treated mice (= 9). Hence, genetic deletion or pharmacological inhibition of CXCR7 resulted in fourfold to fivefold increases in plasma CXCL12 levels. Similar effects with several selective CXCR7 inhibitors, including CCX771, have been seen in mice, rats and dogs (data not shown). Hence, CXCR7 regulates plasma CXCL12 levels. Open in a separate window Figure 1 Regulation of CXCL12 levels by CXCR7 and 005 versus TB; one-way analysis of variance (anova) with Tukey’s post tests), indicating that HUVECs express CXCR7. The experiment was repeated twice. (b) Amount of CXCL12 in the Arry-520 (Filanesib) culture medium of HUVECs at multiple time-points after addition of CXCL12 and either CCX771, CCX704 or a vehicle control. Only CCX771 blocked the removal of CXCL12 by the HUVEC cells (* 005 versus CCX704; two way anova with repeated measures and Tukey’s post tests). The experiment was repeated twice. (c) CXCL12 levels in plasma of CXCR7+/+, CXCR7+/? and CXCR7?/? mice (10 age-matched mice each, comprising five males and five females). The mice were littermates, born from CXCR7+/? parents and sampled at a variety of ages (12C35 weeks). CXCL12 levels were elevated in CXCR7?/? mice (* 005 versus CXCR7+/+ mice; two-way anova with Tukey’s post tests). (d) CXCL12 levels in plasma of 10-week-old female C3H/HeJ mice treated with 30 mg/kg of CCX771 (nine mice) or vehicle control (three mice), sampled 1-hr post-dosing. CCX771 increased plasma CXCL12 levels (* 005 versus vehicle; Student’s 005 versus vehicle; one-way analysis of variance with Tukey’s post GFAP tests) into the air pouch. The number of cells in air pouches lacking CXCL12 indicates non-CXCL12-mediated cell recruitment. = 7 (no CXCL12), = 10 (CXCL12 + vehicle) and = 9 (CXCL12 + CCX771) mice. The experiment was repeated three times. Error bars indicate SEM. Evaluation of CXCR7 expression in mice Competitive [125I]CXCL12 binding assayTo investigate the mechanism by which CXCR7 regulates plasma CXCL12 levels, we sought to determine where CXCR7 protein is expressed. Towards this end, we first analysed a subset of organs in healthy mice using a competitive [125I]CXCL12 binding assay. In this assay, CXCR7 is identified by the ability of CXCR7-specific compounds (such as CCX771) and the other CXCR7 chemokine ligand CXCL11, but not the CXCR4-specific antagonist AMD3100, to inhibit the binding of [125I]CXCL12 to cells or cell membranes. In one of our previous studies, we performed the competitive [125I]CXCL12 binding assay on cells isolated from mouse heart, liver and lung by mechanical dispersion and observed no CXCR7-specific [125I]CXCL12 binding to these cells.12 To look for CXCR7 protein inside these cells, as well as cells (e.g. endothelial cells) that may have been excluded using the dispersion method, we homogenized whole organs from multiple strains of mice (C57BL/6, BALB/c, C3H/Hej and 129S) as well as CXCR7?/? mice. Homogenates of wild-type mouse heart, kidney, lung and spleen, but not liver, displayed the CXCR7 profile in the competitive [125I]CXCL12 binding assay (Fig. 3). In contrast, Arry-520 (Filanesib) homogenates of the CXCR7?/? mouse organs did not display the CXCR7 binding profile, confirming the specificity of the assay (Fig. 3). Hence, CXCR7 protein is indeed expressed in these organs.