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Airway remodelling is a feature of asthma that contributes to loss of lung function. One of the central components of airway remodelling is subepithelial fibrosis. Interleukin (IL)-13 is a key T-helper 2 cytokine and is believed to be the central mediator of allergic asthma including remodelling, but the mechanism driving the latter has not been elucidated in human asthma. We hypothesised that IL-13 stimulates collagen type-1 production by the airway fibroblast in a matrix metalloproteinase (MMP)- and transforming growth factor (TGF)-β1-dependent manner in human asthma as compared to healthy controls. Fibroblasts were cultured from endobronchial biopsies in 14 subjects with mild asthma and 13 normal controls that underwent bronchoscopy. Airway fibroblasts were treated with various mediators including IL-13 and specific MMP-inhibitors. IL-13 significantly stimulated collagen type-1 production in asthma compared to normal controls. Inhibitors of MMP-2 significantly attenuated collagen production in asthma but had no effect in normal controls. IL-13 significantly increased total and active forms of TGF-β1, and this activation was blocked using an MMP-2 inhibitor. IL-13 activated endogenous MMP-2 in asthma patients as compared to normal controls. In an ex vivo model, IL-13 potentiates airway remodelling through a mechanism involving TGF-β1 and MMP-2. These effects provide insights into the mechanism involved in IL-13-directed airway remodelling in asthma.

Rationale: Obesity is associated with increased prevalence and severity of asthma. Adipose tissue macrophages can contribute to the systemic proinflammatory state associated with obesity. However, it remains unknown whether alveolar macrophages have a unique phenotype in overweight/obese patients with asthma. Objectives: We hypothesized that leptin levels would be increased in the bronchoalveolar lavage fluid from overweight/obese subjects and, furthermore, that leptin would alter the response of alveolar macrophages to bacterial LPS. Methods: Forty-two subjects with asthma and 46 healthy control subjects underwent research bronchoscopy. Bronchoalveolar lavage fluid from 66 was analyzed for the level of cellular inflammation, cytokines, and soluble leptin. Cultured primary macrophages from 22 subjects were exposed to LPS, leptin, or leptin plus LPS. Cytokines were measured in the supernatants. Measurements and Main Results: Leptin levels were increased in overweight/obese subjects, regardless of asthma status (P = 0.013), but were significantly higher in overweight/obese subjects with asthma. Observed levels of tumor necrosis factor-α were highest in overweight/obese subjects with asthma. Ex vivo studies of primary alveolar macrophages indicated that the response to LPS was most robust in alveolar macrophages from overweight/obese subjects with asthma and that preexposure to high-dose leptin enhanced the proinflammatory response. Leptin alone was sufficient to induce production of proinflammatory cytokines from macrophages derived from overweight/obese subjects with asthma. Conclusions: Ex vivo studies indicate that alveolar macrophages derived from overweight/obese subjects with asthma are uniquely sensitive to leptin. This macrophage phenotype, in the context of higher levels of soluble leptin, may contribute to the pathogenesis of airway disease associated with obesity.

Surfactant protein A (SP-A) regulates a variety of immune cell functions. We determined the ability of SP-A derived from normal and asthmatic subjects to modulate the inflammatory response elicited by Mycoplasma pneumoniae , a pathogen known to exacerbate asthma. Fourteen asthmatic and 10 normal control subjects underwent bronchoscopy with airway brushing and bronchoalveolar lavage (BAL). Total SP-A was extracted from BAL. The ratio of SP-A1 to total SP-A (SP-A1/SP-A) and the binding of total SP-A to M. pneumoniae membranes were determined. Airway epithelial cells from subjects were exposed to either normal or asthmatic SP-A before exposure to M. pneumoniae . IL-8 protein and MUC5AC mRNA were measured. Total BAL SP-A concentration did not differ between groups, but the percentage SP-A1 was significantly increased in BAL of asthmatic compared with normal subjects. SP-A1/SP-A significantly correlated with maximum binding of total SP-A to M. pneumoniae , but only in asthma. SP-A derived from asthmatic subjects did not significantly attenuate IL-8 and MUC5AC in the setting of M. pneumoniae infection compared with SP-A derived from normal subjects. We conclude that SP-A derived from asthmatic subjects does not abrogate inflammation effectively, and this dysfunction may be modulated by SP-A1/SP-A.

Elastin synthesis and degradation in the airway and lung parenchyma contribute to airway mechanics, including airway patency and elastic recoil. IL-13 mediates many features of asthma pathobiology, including airway remodeling, but the effects of IL-13 on elastin architecture in the airway wall are not known. We hypothesized that IL-13 modulates elastin expression in airway fibroblasts from subjects with allergic asthma. Twenty-five subjects with mild asthma (FEV1, 89 ± 3% predicted) and 30 normal control subjects (FEV1, 102 ± 2% predicted) underwent bronchoscopy with endobronchial biopsy. Elastic fibers were visualized in airway biopsy specimens using Weigert's resorcin-fuchsin elastic stain. Airway fibroblasts were exposed to IL-13; a pan-matrix metalloproteinase (MMP) inhibitor (GM6001); specific inhibitors to MMP-1, -2, -3, and -8; and combinations of IL-13 with MMP inhibitors in separate conditions in serum-free media for 48 hours. Elastin (ELN) expression as well as MMP secretion and activity were quantified. Results of this study show that elastic fiber staining of airway biopsy tissue was significantly associated with methacholine PC20 (i.e., the provocative concentration of methacholine resulting in a 20% fall in FEV1 levels) in patients with asthma. IL-13 significantly suppressed ELN expression in asthmatic airway fibroblasts as compared with normal control fibroblasts. The effect of IL-13 on ELN expression was significantly correlated with postbronchodilator FEV1/FVC in patients with asthma. MMP inhibition significantly stimulated ELN expression in patients with asthma as compared with normal control subjects. Specific inhibition of MMP-1 and MMP-2, but not MMP-3 or MMP-8, reversed the IL-13-induced suppression of ELN expression. In asthma, MMP-1 and MMP-2 mediate IL-13-induced suppression of ELN expression in airway fibroblasts.

To identify novel genes regulating the biologic response to lipopolysaccharide (LPS), we used a combination of quantitative trait locus (QTL) analysis and microarray-based gene expression studies of C57BL/6J x DBA/2J(BXD) F2 and recombinant inbred (RI) mice. A QTL affecting pulmonary TNF-alpha production was identified on chromosome 2, and a region affecting both polymorphonuclear leukocyte recruitment and TNF-alpha levels was identified on chromosome 11. Microarray analyses of unchallenged and LPS-challenged BXD RI strains identified approximately 500 genes whose expression was significantly changed by inhalation of LPS. Of these genes, 28 reside within the chromosomal regions identified by the QTL analyses, implicating these genes as high priority candidates for functional studies. Additional high priority candidate genes were identified based on their differential expression in mice having high and low responses to LPS. Functional studies of these genes are expected to reveal important molecular mechanisms regulating the magnitude of biologic responses to LPS.

Asthma is a chronic inflammatory disease in which airway epithelial cells are the first line of defense against exposure of the airway to infectious agents. Src homology protein (SHP)-1, a protein tyrosine phosphatase, is a negative regulator of signaling pathways that are critical to the development of asthma and host defense. We hypothesize that SHP-1 function is defective in asthma, contributing to the increased inflammatory response induced by Mycoplasma pneumoniae, a pathogen known to exacerbate asthma. M. pneumoniae significantly activated SHP-1 in airway epithelial cells collected from nonasthmatic subjects by bronchoscopy with airway brushing but not in cells from asthmatic subjects. In asthmatic airway epithelial cells, M. pneumoniae induced significant PI3K/Akt phosphorylation, NF-κB activation, and IL-8 production compared with nonasthmatic cells, which were reversed by SHP-1 overexpression. Conversely, SHP-1 knockdown significantly increased IL-8 production and PI3K/Akt and NF-κB activation in the setting of M. pneumoniae infection in nonasthmatic cells, but it did not exacerbate these three parameters already activated in asthmatic cells. Thus, SHP-1 plays a critical role in abrogating M. pneumoniae-induced IL-8 production in nonasthmatic airway epithelial cells through inhibition of PI3K/Akt and NF-κB activity, but it is defective in asthma, resulting in an enhanced inflammatory response to infection.

Invasive cell phenotypes have been demonstrated in malignant transformation, but not in other diseases, such as asthma. Cellular invasiveness is thought to be mediated by transforming growth factor (TGF)-β1 and matrix metalloproteinases (MMPs). IL-13 is a key T(H)2 cytokine that directs many features of airway remodeling through TGF-β1 and MMPs.We hypothesized that, in human asthma, IL-13 stimulates increased airway fibroblast invasiveness via TGF-β1 and MMPs in asthma compared with normal controls.Fibroblasts were cultured from endobronchial biopsies in 20 subjects with mild asthma (FEV(1): 90 ± 3.6% pred) and 17 normal control subjects (FEV(1): 102 ± 2.9% pred) who underwent bronchoscopy. Airway fibroblast invasiveness was investigated using Matrigel chambers. IL-13 or IL-13 with TGF-β1 neutralizing antibody or pan-MMP inhibitor (GM6001) was added to the lower chamber as a chemoattractant. Flow cytometry and immunohistochemistry were performed in a subset of subjects to evaluate IL-13 receptor levels.IL-13 significantly stimulated invasion in asthmatic airway fibroblasts, compared with normal control subjects. Inhibitors of both TGF-β1 and MMPs blocked IL-13-induced invasion in asthma, but had no effect in normal control subjects. At baseline, in airway tissue, IL-13 receptors were expressed in significantly higher levels in asthma, compared with normal control subjects. In airway fibroblasts, baseline IL-13Rα2 was reduced in asthma compared with normal control subjects.IL-13 potentiates airway fibroblast invasion through a mechanism involving TGF-β1 and MMPs. IL-13 receptor subunits are differentially expressed in asthma. These effects may result in IL-13-directed airway remodeling in asthma.

Background The characteristics of human asthma are chronic inflammation and airway remodeling. Hyaluronan, a major extracellular matrix component, accumulates during inflammatory lung diseases, including asthma. Hyaluronan fragments stimulate macrophages to produce inflammatory cytokines. We hypothesized that hyaluronan and its receptors would play a role in human asthma. Objective To investigate the role of hyaluronan and hyaluronan-binding proteins in human asthma. Methods Twenty-one subjects with asthma and 25 healthy control subjects underwent bronchoscopy with endobronchial biopsy and bronchoalveolar lavage. Fibroblasts were cultured, and hyaluronan and hyaluronan synthase expression was determined at baseline and after exposure to several mediators relevant to asthma pathobiology. The expression of hyaluronan-binding proteins CD44, TLR (Toll-like receptor)–2, and TLR4 on bronchoalveolar lavage macrophages was determined by flow cytometry. IL-8 production by macrophages in response to hyaluronan fragment stimulation was compared. Results Airway fibroblasts from patients with asthma produced significantly increased concentrations of lower-molecular-weight hyaluronan compared with those of normal fibroblasts. Hyaluronan synthase 2 mRNA was markedly increased in asthmatic fibroblasts. Asthmatic macrophages showed a decrease in cell surface CD44 expression and an increase in TLR2 and TLR4 expression. Macrophages from subjects with asthma showed an increase in responsiveness to low-molecular-weight hyaluronan stimulation, as demonstrated by increased IL-8 production. Conclusion Hyaluronan homeostasis is deranged in asthma, with increased production by fibroblasts and decreased CD44 expression on alveolar macrophages. Upregulation of TLR2 and TLR4 on macrophages with increased sensitivity to hyaluronan fragments suggests a novel proinflammatory mechanism by which persistence of hyaluronan fragments could contribute to chronic inflammation and airway remodeling in asthma.