CXCR2 mediates the recruitment of endothelial progenitor cells during allergic airways remodeling

Abstract

Airway remodeling is a central feature of asthma and includes the formation of new peribronchial blood vessels, which is termed angiogenesis. In a number of disease models, bone marrow-derived endothelial progenitor cells (EPCs) have been shown to contribute to the angiogenic response. In this study we set out to determine whether EPCs were recruited into the lungs in a model of allergic airways disease and to identify the factors regulating EPC trafficking in this model. We observed a significant increase in the number of peribronchial blood vessels at day 24, during the acute inflammatory phase of the model. This angiogenic response was associated with an increase in the quantity of EPCs recoverable from the lung. These EPCs formed colonies after 21 days in culture and were shown to express CD31, von Willebrand factor, and vascular endothelial growth factor (VEGF) receptor 2, but were negative for CD45 and CD14. The influx in EPCs was associated with a significant increase in the proangiogenic factors VEGF-A and the CXCR2 ligands, CXCL1 and CXCL2. However, we show directly that, while the CXCL1 and CXCL2 chemokines can recruit EPCs into the lungs of allergen-sensitized mice, VEGF-A was ineffective in this respect. Further, the blockade of CXCR2 significantly reduced EPC numbers in the lungs after allergen exposure and led to a decrease in the numbers of peribronchial blood vessels after allergen challenge with no effect on inflammation. The data presented here provide in vivo evidence that CXCR2 is critical for both EPC recruitment and the angiogenic response in this model of allergic inflammation of the airways.

Figure 1

Acute and prolonged ovalbumin (OVA) challenge leads to peribronchial angiogenesis. Representative photomicrographs of immunostaining for von Willebrand factor of lung sections from alum controls (A) and OVA-challenged mice up to days 24 (B) or 55 (C). Magnification ×400. (D) Bars represent mean ± SEM of the number of blood vessels per square millimeter from alum- and OVA-treated mice at indicated time points. Data derived from 4–6 mice.

Figure 2

Acute allergen challenge leads to an increase of EPCs in the lungs. (A) Bars represent mean ± SEM of the numbers of EPCs at 21 days of culture, as described in Materials and Methods. * represents p < .05 compared to alum controls. Photomicrograph represents lung EPC colonies in bright-field; magnification ×50 (inset: ×400). (B) Representative photomicrographs from EPCs immunostained for CD31-Alexa 488/DAPI; vWF-Alexa 564/DAPI, and VEGFR2/Alexa 564/DAPI. (C) Representative photomicrographs from EPC colonies positive for Ac-LDL uptake and GS-lectin. (D) Representative photomicrographs from hematopoietic colonies (left and middle) or EPCs (right) immunostained for CD45- and CD14-positive monocytes. Data derived from 8–12 mice. Abbreviations: Ac-LDL, acetylated low-density lipoprotein; DAPI, 4′,6-diamidino-2-phenylindole; EPC, endothelial progenitor cell; GS-lectin, Griffonia simplicifolia isolectin; vWF, von Willebrand factor.

Figure 3

Acute allergen challenge leads to increased levels of CXCR2 ligands and VEGF-A in lung homogenates. CXCL1, CXCL2, and VEGF-A levels in lung tissue homogenates were measured in samples from alum controls or samples from mice challenged with OVA at the indicated time points. Data are expressed as individual values and median. * represents p < .05 compared to alum controls. Data derived from 6 mice. Abbreviations: OVA, ovalbumin; VEGF, vascular endothelial growth factor.

Figure 4

CXCL1 in association with CXCL2 is able to induce EPC recruitment to the lungs of sensitized mice. Mice were sensitized with alum or OVA (intraperitoneal) on days 0 and 12. On day 18 mice were challenged with CXCL1 in association with CXCL2, VEGF-A, or PBS intranasally. (A) Data represent mean ± SEM of neutrophils × 10⁵ per mL bronchoalveolar lavage fluid. (B) Data represent mean ± SEM of the numbers of EPCs per lung. n = 4–6 mice. * represents p < .05 compared to PBS-instilled mice. Abbreviations: EPC, endothelial progenitor cell; PBS, phosphate-buffered saline; OVA, ovalbumin; VEGF, vascular endothelial growth factor.

Figure 5

Administration of anti-CXCR2 decreased EPC recruitment to the lungs induced by allergen challenge. Mice were sensitized with alum or OVA (intraperitoneal) on days 0 and 12, and challenged with OVA aerosol between days 18 and 23. Selected mice were treated with anti-CXCR2 antibody or isotype controls on days 18 and 21. (A) EPC quantities in the lungs were analyzed 24 hours after the last allergen challenge. Animals were left to rest for another 6 days and EPCs from lung (B) and blood (C) were analyzed at day 30. Bars represent mean ± SEM of the numbers of EPCs. n = 4–9 mice. * represents p < .05 compared to alum controls. ** represents p < .05 compared to OVA-isotype–treated mice. Abbreviations: EPC, endothelial progenitor cell; OVA, ovalbumin.

Figure 6

Effect of CXCR2 blockade on peribronchial angiogenesis after allergen challenge. (A) Representative photomicrographs of immunostaining for von Willebrand factor of lung sections from alum- and OVA-challenged mice treated with isotype control or anti-CXCR2. (B) Bars represent mean ± SEM of vessels/mm² immunostained for von Willebrand factor. (C, D) Total number of cells recovered from lung digests and bronchoalveolar lavage fluid, respectively. (E, F) Numbers of eosinophils (SiglecF-positive cells) and Th2 cells (CD3+CD4+T1ST2+ cells) in lung digests. (G) Bars represent mean ± SEM SDF-1α levels measured in lung tissue homogenates. * represents p < .05 compared to alum controls. ** represents p < .05 compared to OVA-isotype–treated mice. Data derived from 4-6 mice. Abbreviations: EPC, endothelial progenitor cell; OVA, ovalbumin; SDF-1α, stromal cell–derived factor-1α.