Asthma can progress to subepithelial airway fibrosis, mediated in large part by transforming growth factor-␤ (TGF-␤). The scaffolding protein caveolin-1 (cav1) can inhibit the activity of TGF-␤, perhaps by forming membrane invaginations that enfold TGF-␤ receptors. The study goals were 1) to evaluate how allergen challenge affects lung expression of cav1 and the density of caveolae in vivo 2) to determine whether reduced cav1 expression is mediated by interleukin (IL)-4 and 3) to measure the effects of decreased expression of cav1 on TGF-␤ signaling. C57BL/6J, IL-4-deficient mice, and cav1-deficient mice, sensitized by intraperitoneal injections of phosphate-buffered saline or ovalbumin (OVA) at days 0 and 12, received intranasal phosphate-buffered saline or OVA challenges at days 24, 26, and 28. Additionally, another group of C57BL/6J mice received IL-4 by intratracheal instillation for 7 days. We confirmed that the OVA-allergen challenge increased eosinophilia and T-helper type 2-related cytokine levels (IL-4, IL-5, and IL-13) in bronchoalveolar lavage. Allergen challenge reduced lung cav1 mRNA abundance by 40%, cav1 protein by 30%, and the number of lung fibroblast caveolae by 50%. Administration of IL-4 in vivo also substantially decreased cav1 expression. In contrast, the allergen challenge did not decrease cav1 expression in IL-4-deficient mice. The reduced expression of cav1 was associated with activation of TGF-␤ signaling that was further enhanced in OVA-sensitized and challenged cav1-deficient mice. This study demonstrates a previously unknown modulation of TGF-␤ signaling by IL-4, via cav1, suggesting novel therapeutic targets for controlling the effects of TGF-␤ and thereby ameliorating pathological airway remodeling.Asthma is often associated with structural changes in the bronchioles, commonly referred to as airway remodeling (1). These alterations result from injury and repair processes regulated by several mediators, such as T-helper type 2 (Th2) 2 cytokines and transforming growth factor ␤ (TGF-␤). Cytokines modulate subepithelial fibrosis by regulating fibroblast proliferation and differentiation and by stimulating synthesis and stabilization of the proteins that constitute the extracellular matrix. In vitro, Th2-related cytokines regulate the secretion of connective tissue proteins, including collagens, fibronectin, and tenascin, all of which are involved in the thickening of the airway basement membrane (2-7). Interleukin-4 (IL-4) and IL-13 levels are notably increased in bronchoalveolar lavage (BAL) fluid of asthmatic patients (3,8). Inhibitors of IL-4 and IL-13 prevent the development of fibrosis (9, 10). IL-4-and IL-13-deficient mice develop less subepithelial fibrosis and goblet cell hyperplasia after allergen challenge, compared with their normal counterparts (11, 12), indicating the importance of IL-4 and IL-13 in driving airway remodeling.TGF-␤, one of the most potent regulators of inflammation and connective tissue synthesis, plays an integral role in the development of airway remodel...

Caveolin-1 (cav1) is a 22-kDa membrane protein essential to the formation of small invaginations in the plasma membrane, called caveolae. The cav1 gene is expressed primarily in adherent cells such as endothelial and smooth muscle cells and fibroblasts. Caveolae contain a variety of signaling receptors, and cav1 notably downregulates transforming growth factor (TGF)-β signal transduction. In pulmonary pathologies such as interstitial fibrosis or emphysema, altered mechanical properties of the lungs are often associated with abnormal ECM deposition. In this study, we examined the physiological functions and the deposition of ECM in cav1−/− mice at various ages (1–12 mo). Cav1−/− mice lack caveolae and by 3 mo of age have significant reduced lung compliance and increased elastance and airway resistance. Pulmonary extravasation of fluid, as part of the cav1−/− mouse phenotype, probably contributed to the alteration of compliance, which was compounded by a progressive increase in deposition of collagen fibrils in airways and parenchyma. We also found that the increased elastance was caused by abundant elastic fiber deposition primarily around airways in cav1−/− mice at least 3 mo old. These observed changes in the ECM composition probably also contribute to the increased airway resistance. The higher deposition of collagen and elastic fibers was associated with increased tropoelastin and col1α2 and col3α1 gene expression in lung tissues, which correlated tightly with increased TGF-β/Smad signal transduction. Our study illustrates that perturbation of cav1 function may contribute to several pulmonary pathologies as the result of the important role played by cav1, as part of the TGF-β signaling pathway, in the regulation of the pulmonary ECM.

A mouse model for allergic airway inflammation involving ovalbumin (OVA) sensitization and challenge has been developed that reproduces hallmark features of human asthma and has provided valuable insight into the mechanisms by which this disease occurs. Cellular infiltrate in lungs of mice used in this model have conventionally been evaluated using histological examination of tissue sections and light microscopic analysis of lung lavage samples. As an alternative or complementary approach for characterizing cellular infiltrate, we developed a multicolor fluorescence-activated cell sorter (FACS) method involving the simultaneous detection of seven different markers on lung cell suspensions: CD4, CD8, B220, CD11b, Gr-1, CD49b, and FcepsilonRI. Only some of these cell types increased in OVA-challenged mice compared to PBS controls, including the CD4(+), B220(+), CD11b(+), and FcepsilonRI(+) groups. We also examined subpopulations of cells for coexpression of these markers and dissected heterogeneous populations as further evaluation procedures to characterize the cellular infiltrate resulting from OVA challenge. Finally, we combined FACS with real-time PCR to analyze certain cell types in terms of mRNA levels for factors involved in asthma, including GATA-3 and IL-1beta. Overall, these FACS-based techniques provide a powerful approach for analyzing cellular profiles in lung tissue from mice used in the mouse model of asthma and may also prove valuable in evaluating cellular infiltrates for other models of inflammation and immune responses.

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