Absence of alpha 4 but not beta 2 integrins restrains development of chronic allergic asthma using mouse genetic models

Abstract

Objective: Chronic asthma is characterized by ongoing recruitment of inflammatory cells and airway hyperresponsiveness leading to structural airway remodeling. Although alpha 4 beta 1 and beta2 integrins regulate leukocyte migration in inflammatory diseases and play decisive roles in acute asthma, their role has not been explored under the chronic asthma setting. To extend our earlier studies with alpha 4(Delta/Delta) and beta2(-/-) mice, which showed that both alpha 4 and beta2 integrins have nonredundant regulatory roles in acute ovalbumin (OVA)-induced asthma, we explored to what extent these molecular pathways control development of structural airway remodeling in chronic asthma.

Materials and methods: Control, alpha 4(Delta/Delta), and beta2(-/-) mouse groups, sensitized by intraperitoneal OVA as allergen, received intratracheal OVA periodically over days 8 to 55 to induce a chronic asthma phenotype. Post-OVA assessment of inflammation and pulmonary function (airway hyperresponsiveness), together with airway modeling measured by goblet cell metaplasia, collagen content of lung, and transforming growth factor beta1 expression in lung homogenates, were evaluated.

Results: In contrast to control and beta2(-/-) mice, alpha 4(Delta/Delta) mice failed to develop and maintain the composite chronic asthma phenotype evaluated as mentioned and subepithelial collagen content was comparable to baseline. These data indicate that beta2 integrins, although required for inflammatory migration in acute asthma, are dispensable for structural remodeling in chronic asthma.

Conclusion: alpha 4 integrins appear to have a regulatory role in directing transforming growth factor beta-induced collagen deposition and structural alterations in lung architecture likely through interactions of Th2 cells, eosinophils, or mast cells with endothelium, resident airway cells, and/or extracellular matrix.

Figure 1

(A) Diagram illustrating the experimental design for chronic asthma development. i.p. = intraperitoneal injection, i.t. = intratracheal instillation. (B) Lung function testing using whole-body plethysmography. Measurement of airway hyperresponsiveness at 24 hours after the last ovalbumin (OVA) challenge on day 54. The degree of bronchoconstriction to increasing doses of aerosolized methacholine was expressed as P_enh (percentage of air as control). *p < 0.01 compared to baseline values in saline-treated control mice, n = 8 mice per group. Control mice, α4-deficient mice, and β2-deficient mice were treated with aluminium sulfate (alum) and OVA. Measurements of airway resistance by invasive plethysmography [22] were found to be in full agreement with P_enh data. (C) Numbers of lymphoid and myeloid cells in blood (n/mL), bone marrow (n/femur), and spleen, and (D) total numbers of cells in bronchoalveolar lavage (BAL) fluid and lung parenchyma from the two genetic models and controls treated with alum or OVA. Data are from two independent experiments expressed as average ± standard error of the mean. n = 8 mice per group.

Figure 2

Total numbers of all leukocytes in bone marrow, peripheral blood, bronchoalveolar lavage fluid (BALf), and lung parenchyma in control, α4-deficient (α4Δ/Δ), and β2^−/− mice pre- and post-chronic ovalbumin (OVA) treatment. Total numbers (× 10E6) of cells were counted in a Beckman Coulter Counter and differentials (neutrophils, eosinophils, monocytes, etc.) were assessed by fluorescein-activated cell sorting (specific antigen expression) and morphology in smears. *p < 0.01 from control values post-OVA, n = 8 mice per group, C = Control mice.

Figure 3

Paraffin sections of lungs from ovalbumin-treated mice were stained with (A) Masson’s trichrome stain revealing the collagen depositions around the airways and (B) Martius scarlet blue for collagen and fibrin deposition in interstitial spaces of the parenchyma. Adjustments for brightness, contrast, and color balance using Adobe Photoshop were made in order to match in all six photographs.

Figure 4

(A) Concentration of cytokines (pg/mL) post-ovalbumin (OVA) (upper two panels) in bronchoalveolar lavage fluid (BALf) and plasma in control (white bars), α4^Δ/Δ (black bars), and β2^−/− (gray bars) mice (*p < 0.01 compared to post-OVA control), and (B) concentrations of OVA-specific immunoglobulin (Ig) E and IgG₁ (μg/mL) in plasma of OVA-treated mice in the three genotypes. Data (see Suppl. Table 2) are averaged from two independent experiments, all assays were run in triplicate, n = 8 mice per group, and *p < 0.01 compared to post-OVA control values. IFN-γ = interferon-γ; IL = interleukin; TNF-α = tumor necrosis factor-α.

Figure 5

(A) Soluble vascular cell adhesion molecule-1 (sVCAM-1) concentrations (ng/mL) in bronchoalveolar lavage fluid (BALf) and (B) in plasma measured by enzyme-linked immunosorbent assay (ELISA), (C) transforming growth factor-β1 (TGF-β1) levels (pg/mL) in BALf measured by ELISA, and (D) soluble collagen content (μg/mL) of lung measured by Sircol dye kit. Data are averaged from two independent experiments ± standard error of mean. All assays were run in triplicate, n = 8 mice/group, and *p < 0.01 compared to post-ovalbumin (OVA) control values.

Figure 6

Lung tissue sections, stained for transforming growth factor-β1 (TGF-β1) (anti-TGF-β1), 9EG7 (activated anti-β1 antibody), and vascular cell adhesion molecule-1 (VCAM-1) (anti-VCAM-1, MK/2) as described in Materials and Methods. (A) Images from Control (left) and α4^Δ/Δ lung (right) post-ovalbumin (OVA) treatment and challenge stained with anti-TGF-β1. (B) Images of β2^−/− lung (left) and α4^Δ/Δ lung (right) post-OVA stained with 9EG7. (C) Images of β2^−/− lung (left) and α4^Δ/Δ lung (right) post-OVA stained with anti-VCAM-1. There were no significant differences in labeling with 9EG7, but labeling for TGF-β and VCAM-1 was less intense in α4^Δ/Δ mice.