Figure 1

CXCL12 induces internalization of CXCR4 and surface translocation of CXCR7 conferring differential receptor availability on monocyte surface. (a) (i) Western blot analysis for the expression of CXCR4 (immunoreactive band at 43 KD) and CXCR7 (at 35, 50 KD) in untreated resting monocytes. Data are representative of three independent western blot analysis performed with monocytes isolated from three independent healthy donors. (ii) Representative immunofluorescence confocal microscopic images showing relative intracellular localization of CXCR4 (Alexa Fluor 647-red) and CXCR7 (Alexa Fluor 488-green) in untreated and CXCL12-treated (1 μg/ml) monocytes. Internalized CXCR4 and externalized CXCR7 among CXCL12-treated monocytes are indicated by white arrows. Bar=5 μm. Images are representative of three independent experiments performed with three healthy donors. Relevant control stainings are shown in Supplementary Figure 1. (iii) Flow-cytometric dot plot (left) showing percentage of CXCR4⁺ and CXCR7⁺ monocytes under resting conditions (94 and 61%, respectively) and following CXCL12 (1 μg/ml) treatment, causing downregulation of CXCR4⁺ monocytes owing to receptor internalization and an increase in CXCR7⁺ monocytes due to enhanced surface exposure. Flow-cytometry histograms showing the relative surface expression of CXCR4 and CXCR7 in the presence (red) and absence (gray) of CXCL12. Corresponding bar diagram shows a significant (**P<0.01) increase in MFI for CXCR7-PE in CXCL12-treated monocytes and a concomitant (**P<0.01) decrease in MFI for CXCR4-PE. Data represent mean±S.E.M. of five independent experiments. (b) (i) Bar diagram showing the relative number of monocytes migrated in response to CXCL12 (1 μg/ml), MIF (200 ng/ml), and MCP-1 (50 ng/ml). *P<0.05 as compared with control. Data are mean±S.E.M. of four independent chemotaxis analyses with monocytes from three donors. (ii) Flow-cytometric histograms showing the relative surface expression of CXCR4 and CXCR7 on the surface of migrated monocytes in response to CXCL12, MIF and MCP-1. Gray fills indicate control or basal level of expression and red lines indicate monocytes migrated toward CXCL12, MIF and MCP-1. (iii) Bar diagram showing surface expression of CXCR4 and enhanced surface expression of CXCR7 among monocytes following migration in response to CXCL12 and MIF but not MCP-1. Data represent mean±S.E.M. of three independent chemotaxis analyses with monocytes from four donors. (iv) Flow-cytometric density plot showing the relative surface expression of CXCR4 and CXCR7 on migrating monocytes showing relative surface expression of CXCR4–CXCR7 in response to CXCL12 or MIF as compared with control. (c) (i) Bar diagram of flow-cytometric data showing the number of platelets as detected in the peritoneal fluid collected from mice following induction of peritonitis by thioglycolate as compared with control injection of NaCl. Data represent mean±S.E.M. derived from seven mice in each group. *P<0.05 as compared with NaCl-treated group. Right, Flow-cytometric dot plot of FSC-SSC showing infiltrated platelets in the peritoneal lavage as indicated within the square and in conjugation or aggregate formed with other cells. (ii) Immunofluorescence confocal microscopic images showing the presence of Mac-3⁺ monocytes (in red) and F4/80 labelled macrophages (in red) in combination with CD42b⁺ platelets (in green). Bar=5 μm. (iii) Bar diagram of flow-cytometric data showing the relative percentage of monocyte platelets (CD14⁺CD42b⁺) and macrophage platelet (F4/80⁺CD42b⁺) aggregates detected in peripheral blood and peritoneal lavage following thioglycollate-induced peritonitis in mice. ***P<0.001 as compared with monocyte platelet aggregates detected in peripheral blood. Data represent mean±S.E.M. derived from seven mice in each group. Immunofluorescence confocal microscopic images showing the expression of CXCL12 (iv in green), CXCR4 (v in green), and CXCR7 (vi in green) among Mac-3+ (in red) peripheral blood monocytes and infiltrated peritoneal monocytes following induction of peritonitis in mice. Bar=5 μm