Critical involvement of atypical chemokine receptor CXCR7 in allergic airway inflammation

Abstract

Trafficking and recruitment of immune cells to the site of inflammation with spatial and temporal synchronization is crucial for the development of allergic airway inflammation. Particularly, chemokines are known to be key players in these processes. Previous studies revealed that the CXCL12/CXCR4 axis plays an important role in regulating allergic airway inflammation. However, the role of CXCR7, a recently discovered second receptor for CXCL12, in regulating airway inflammation has not been explored. Initially, CXCR7 was considered as a decoy receptor; however, numerous subsequent studies revealed that engagement of CXCR7 triggered its own signalling or modulated CXCR4-mediated signalling. In the present study, we detected the expression of CXCR7 in airway epithelial cells. Use of a lentiviral delivery system to knock down the expression of CXCR7 in the lung of sensitized mice abrogated the cardinal features of asthma, indicating that CXCR7 plays a role in regulating allergic airway inflammation. The activation of mitogen-activated protein kinase and Akt signalling in response to CXCL12 in the mouse epithelial cell line MLE-12 was reduced when CXCR7 expression was knocked down. However, either knockdown or overexpression of CXCR7 in MLE-12 did not affect CXCL12-mediated calcium influx, indicating that CXCR7 does not modulate CXCR4-mediated signalling, and that it functions as a signalling receptor rather than a decoy receptor. Finally, we found that the expression of chemokine CCL2 is regulated by CXCR7/CXCL12-mediated signalling through β-arrestin in airway epithelial cells. Hence, regulating the expression of CCL2 in airway epithelial cells may be one mechanism by which CXCR7 participates in regulating allergic airway inflammation.

Keywords: CXCR7; airway epithelial cells; allergic airway inflammation.

Figure 1

Brief scheme of animal sensitization, treatment and challenge. Abbreviations: i.p., intraperitoneal; i.t., intratracheal; i.n., intranasal; OVA, ovalbumin; Alum, Imject Alum (containing 40 mg/mL aluminum hydroxide and 40 mg/mL magnesium hydroxide)(Pierce, Rockford, IL, USA). [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 2

Expression of CXCR7 in the lung. (a) The expression level of CXCR7 in the lung was analysed by Western blot analysis. The protein extract was prepared from the lungs of three naive and three ovalbumin (OVA) ‐sensitized and challenged inflamed BALB/c mice. Band intensity was quantified and analysed with image J software after normalizing the expression levels of protein to β‐actin. Numbers represent the mean ± SEM of relative expression level to naive‐1. *P < 0·001 compared with naive. Statistical significance was determined using Student's t‐test. (b) Frozen mouse lung sections derived from inflamed BALB/c mice were evaluated for the expression of CXCR7 (green) and CXCR4 (red) using immunofluorescence staining.

Figure 3

Reduced airway inflammation following CXCR7 knockdown (a) Bronchoalveolar lavage fluid (BALF) total cell number (left) and differential cell counts (right) following CXCR7 knockdown virus delivery and three ovalbumin (OVA) challenges were assessed by Pappenheim stain. Numbers represent the mean ± SEM of cell number (n ≥ 4). *P < 0·05; ***P < 0·001 compared with Lenti‐Control. Statistical significance was determined using one‐way anova. Data shown are representative of three independent experiments. (b) Representative lung sections stained with haematoxylin and eosin (H&E; ×200) or periodic acid‐Schiff (PAS; ×200). Arrows indicate areas of airway infiltrate (H&E) or presence of mucous substance (PAS). Numbers for H&E staining represent the mean ± SEM of infiltrating inflammatory cells in subepithelial and subendothelial area (mm²) in lung section (n = 3). Numbers for PAS staining represent the mean ± SEM of H‐Score (n = 3). **P < 0·01; ***P < 0·001 compared with Lenti‐Control. Statistical significance was determined by one‐way anova. NC: negative control, mice sensitized and challenged with PBS. PC: positive control, mice sensitized and challenged with OVA. Lenti‐Control: lentivirus carrying control vector was intratracheally administered to sensitized mice 2 weeks before challenge. Lenti‐CXCR7 shRNA: lentivirus carrying CXCR7 short hairpin RNA was intratracheally administered to sensitized mice 2 weeks before challenge.

Figure 4

Serum ovalbumin (OVA) ‐specific IgE and cytokine production was reduced in CXCR7 knockdown mice. (a) OVA‐specific IgE in the serum was measured by ELISA. Numbers represent the mean ± SEM of optical density (OD; n = 4). ***P < 0·001 compared with Lenti‐Control. Statistical significance was determined by one‐way anova. (b) Cytokine production by mediastinal lymph node (LN) cells isolated from mice following re‐stimulation with antigen‐presenting cells and OVA were analysed by ELISA. In each experiment, mediastinal LN cells in the same group were pooled for culturing. Data shown are representative of three independent experiments. Data for an additional two additional experiments are shown in the Supplementary material (Fig. S2). NC: negative control, mice sensitized and challenged with PBS. PC: positive control, mice sensitized and challenged with OVA. Lenti‐Control: lentivirus carrying control vector was intratracheally administered to sensitized mice 2 weeks before challenge. Lenti‐CXCR7 shRNA: lentivirus carrying CXCR7 short hairpin RNA was intratracheally administered to sensitized mice 2 weeks before challenge.

Figure 5

Reduced airway hyper‐responsiveness following CXCR7 knockdown. Airway resistance was measured 48 hr after the final challenge by invasive body plethysmography. Data are expressed as the mean ± SEM of pulmonary resistance (R_L) (n = 3). **P < 0.01; ***P < 0·001. Statistical significance was determined by one‐way anova. NC: negative control, mice sensitized and challenged with PBS. PC: positive control, mice sensitized and challenged with ovalbumin (OVA). Lenti‐Control: lentivirus carrying control vector was intratracheally administered to sensitized mice 2 weeks before challenge. Lenti‐CXCR7 shRNA: lentivirus carrying CXCR7 short hairpin RNA was intratracheally administered to sensitized mice 2 weeks before challenge.

Figure 6

CXCR7 participates in CXCL12‐mediated signalling in lung epithelial cells. (a) Protein extract was isolated from MLE‐12 cells, their derived control (bearing control vector, control), and CXCR7 knockdown cell line (bearing CXCR7 shRNA expression construct, CXCR7 short hairpin RNA) following stimulation with 100 ng/ml CXCL12 for the indicated time. Western blotting was performed using the indicated antibodies. Quantitative data are shown in the Supplementary material (Fig. S2). (b, c) Indicated cell lines were loaded with 10 nm/ml Fluo3 and stimulated with 250 ng/ml CXCL12. The changes in relative fluorescence intensity due to calcium influx were assessed. Data are expressed as the mean ± SEM of fluorescence change. Statistical significance was determined by one‐way anova. [Colour figure can be viewed at http://wileyonlinelibrary.com]

Figure 7

Production of CCL2 by airway epithelial cells was regulated by engagement of CXCR7. (a) The Mouse Cytokine Array Panel A purchased from R&D Systems was used to analyse the levels of cytokines in the bronchoalveolar fluid collected from indicated mice. Indicated cytokine is CCL2. (b) Concentrations of CCL2 in cell culture supernatant collected from MLE‐12 cell line stimulated with 100 ng/ml CXCL12 with or without 10 μm AMD3100 for 15 hr were determined by ELISA using a Duo Set ELISA kit (R&D Systems). (c) Concentrations of CCL2 in the cell culture supernatant collected from MLE‐12 cells, their derived control (bearing control vector, p), and CXCR7 overexpression cell line (bearing CXCR7 cDNA expression construct) stimulated with 100 ng/ml CXCL12 for 15 hr were analysed by ELISA. (d) Concentrations of CCL2 in the cell culture supernatant collected from MLE‐12, their derived control (bearing control vector), and β‐arrestin 2 knockdown cell line (bearing β‐arrestin 2 short hairpin RNA expression construct) following stimulation with 100 ng/ml CXCL12 for 15 hr were analysed by ELISA. Data were expressed as the mean ± SEM of protein concentration. ***P < 0·001. Statistical significance was determined by one‐way anova. [Colour figure can be viewed at http://wileyonlinelibrary.com]