Lung-Homing of Endothelial Progenitor Cells and Airway Vascularization Is Only Partially Dependant on Eosinophils in a House Dust Mite-Exposed Mouse Model of Allergic Asthma

<div><p>Background</p><p>Asthmatic responses involve a systemic component where activation of the bone marrow leads to mobilization and lung-homing of progenitor cells. This traffic may be driven by stromal cell derived factor-1 (SDF-1), a potent progenitor chemoattractant. We have previously shown that airway angiogenesis, an early remodeling event, can be inhibited by preventing the migration of endothelial progenitor cells (EPC) to the lungs. Given intranasally, AMD3100, a CXCR4 antagonist that inhibits SDF-1 mediated effects, attenuated allergen-induced lung-homing of EPC, vascularization of pulmonary tissue, airway eosinophilia and development of airway hyperresponsiveness. Since SDF-1 is also an eosinophil chemoattractant, we investigated, using a transgenic eosinophil deficient mouse strain (PHIL) whether EPC lung accumulation and lung vascularization in allergic airway responses is dependent on eosinophilic inflammation.</p><p>Methods</p><p>Wild-type (WT) BALB/c and eosinophil deficient (PHIL) mice were sensitized to house dust mite (HDM) using a chronic exposure protocol and treated with AMD3100 to modulate SDF-1 stimulated progenitor traffic. Following HDM challenge, lung-extracted EPCs were enumerated along with airway inflammation, microvessel density (MVD) and airway methacholine responsiveness (AHR).</p><p>Results</p><p>Following Ag sensitization, both WT and PHIL mice exhibited HDM-induced increase in airway inflammation, EPC lung-accumulation, lung angiogenesis and AHR. Treatment with AMD3100 significantly attenuated outcome measures in both groups of mice. Significantly lower levels of EPC and a trend for lower vascularization were detected in PHIL versus WT mice.</p><p>Conclusions</p><p>This study shows that while allergen-induced lung-homing of endothelial progenitor cells, increased tissue vascularization and development lung dysfunction can occur in the absence of eosinophils, the presence of these cells worsens the pathology of the allergic response.</p></div

Airway responsiveness as measured by resistance to intranasal methacholine.

<p>Airway resistance curves to methacholine for (A) WT and (B) PHIL mice; (C) Maximal airway resistance in response to 25 mg/mL methacholine <i>i.n.</i> (n = 10 per group). Data are presented as the maximal resistance between 3.1–25 mg/mL minus the baseline value (0 mg/ml) for each mouse. In WT and PHIL mice, there was a significant increase in methacholine airway responsiveness in HDM versus SAL in WT and PHIL mice. This was significantly attenuated by treatment with AMD3100. Data are expressed as mean ± SEM. *p<0.05 compared with SAL; #p<0.05 compared with HDM.</p

Airway eosinophilia assessed by hematoxylin and eosin stain.

<p>In WT mice, house dust mite (HDM) exposure significantly increased eosinophils compared with saline (SAL). This was significantly attenuated by treatment with AMD3100. PHIL mice had negligible numbers of eosinophils. *p<0.05 compared with saline; #p<0.05 compared with HDM. Data are expressed as mean ± SEM. (n = 10 mice per group).</p

Airway inflammation as measured by bronchial alveolar lavage (BAL).

<p>Data are presented as mean ± SEM. TCC: total cell count. Cells were counted in bronchoalveolar lavage samples collected from WT and PHIL mice (n = 10 per group) sensitized by chronic exposure protocol with concurrent treatment with AMD3100. Measurements were made at 24 h after saline (SAL), house dust mite (HDM), or HDM+AMD3100 challenge. *p<0.05 compared to SAL. Data are expressed as mean ± SEM.</p><p>Airway inflammation as measured by bronchial alveolar lavage (BAL).</p

Lung Tissue Associate Levels of Pro-Angiogenic Cytokines and Chemokines.

<p>Tissue Associated levels of Vascular endothelial derived growth factor (VEGF), TSLP and IL-33 were determined in supernatants collected from digested lung tissue (n = 10 mice per group) by ELISA in samples collected 24 h post-challenge. In WT and PHIL mice, there is a significant increase in levels of VEGF and TSLP that were not attenuated by treatment with AMD3100 in both groups of mice. Compared to SAL, there was no allergen-induced production of IL-33 in either WT or PHIL mice. *: p<0.05 compared with saline (SAL); Data are presented as mean ± SEM.</p><p>Lung Tissue Associate Levels of Pro-Angiogenic Cytokines and Chemokines.</p

Chronic sensitisation and drug treatment protocol.

<p>Wild type (WT) and eosinophil deficient (PHIL +/+) BALB/c mice were sensitized to house dust mite (HDM; 15 µg/µl) or saline (SAL) <i>i.n.</i> on days 1–5 and 8–12 and challenged <i>i.n.</i> with HDM or SAL 3 days per week for the following 6 weeks. During the challenge phase, treatment groups were administered AMD3100 (15 mg·kg⁻¹) or vehicle (<i>i.n.</i>) 4 hours prior to HDM or SAL challenge. Outcome measures were made 24 h after the final HDM or SAL challenge (day 57).</p

Flow cytometric enumeration of lung-extracted primitive progenitor cells.

<p>(A) On a dot plot of linear side scatter vs Sca-1 FITC plot, a region R1 was drawn to determine Sca-1+ cells lymphomononuclear cells. From this region events were gated on a dot plot of Sca-1+/C-kit+ cells as shown in Figure panel A. Compared to isotype controls, primitive progenitors were identified as identified as Sca-1+c-kit+ cells. (B) Lung extracted cells were harvested 24 h after final i.n. challenge with saline (SAL), house dust mite (HDM) or HDM+AMD3100 (n = 10 per group). In WT and PHIL mice, a significant increase in Sca-1+c-kit+ cells was detected in HDM compared to SAL which was attenuated following treatment with AMD3100. There were significantly lower levels of primitive progenitors in PHIL compared to WT mice. *p<0.05 compared with SAL; #p<0.05 compared with HDM; $p<0.05 compared with WT HDM. Data expressed as mean ± SEM.</p

Microvessel density (MVD) assessed by immunostaining for von Willebrand factor in Lung tissue sections.

<p>(A) Images from 24 hrs post-final allergen challenge at 40X magnification. Enumerated vessels were 10 µm in diameter or less (arrows). Scale bars = 50 µm (n = 10 per group) (B) MVD levels increased significantly in HDM compared with SAL groups in both WT and PHIL mice which was attenuated by AMD3100. Between group comparisons showed a trend for lower MVD levels in PHIL versus WT mice (p = 0.058). *p<0.05 compared with SAL; #p<0.05 compared with HDM. Data are expressed as mean ± SEM.</p