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Matrix metalloproteinases (MMPs) are a family of extracellular proteases that are responsible for the degradation of the extracellular matrix during tissue remodeling. We have used a mouse model of allergen-induced airway remodeling to determine whether MMP-9 plays a role in airway remodeling. MMP-9-deficient and wild-type (WT) mice were repetitively challenged intranasally with ovalbumin (OVA) antigen to develop features of airway remodeling including peribronchial fibrosis and increased thickness of the peribronchial smooth muscle layer. OVA-challenged MMP-9-deficient mice had less peribronchial fibrosis and total lung collagen compared with OVA-challenged WT mice. There was no reduction in mucus expression, smooth muscle thickness, or airway responsiveness in OVA-challenged MMP-9-deficient compared with OVA-challenged WT mice. OVA-challenged MMP-9-deficient mice had reduced levels of bronchoalveolar lavage (BAL) regulated on activation, normal T cell expressed, and secreted (RANTES), as well as reduced numbers of BAL and peribronchial eosinophils compared with OVA-challenged WT mice. There were no significant difference in levels of BAL eotaxin, thymus- and activation-regulated chemokine (TARC), or macrophage-derived chemokine (MDC) in OVA-challenged WT compared with MMP-9-deficient mice. Overall, this study demonstrates that MMP-9 may play a role in mediating selected aspects of allergen-induced airway remodeling (i.e., modest reduction in levels of peribronchial fibrosis) but does not play a significant role in mucus expression, smooth muscle thickness, or airway responsiveness.

Background Airway remodeling in asthma is associated with angiogenesis. Objective We have examined whether immunostimulatory sequences of DNA (ISSs) inhibit allergen-induced airway angiogenesis and expression of angiogenic cytokines in a mouse model of airway remodeling. Methods Mice sensitized to ovalbumin were challenged repetitively with ovalbumin for three months to develop airway remodeling and angiogenesis. Levels of angiogenesis were compared in ISS-treated and control mice. Results Mice challenged with ovalbumin developed significantly increased levels of peribronchial angiogenesis (increase in the number of CD31+ peribronchial small blood vessels) and an increase in the peribronchial vascular area as assessed by image analysis. Ovalbumin-induced peribronchial angiogenesis was associated with increased bronchoalveolar lavage levels of vascular endothelial growth factor (VEGF) and an increase in the number of peribronchial cells expressing VEGF. Treatment of mice with ISS before repetitive ovalbumin challenge significantly reduced the levels of peribronchial angiogenesis as well as the levels of bronchoalveolar lavage VEGF and the number of peribronchial cells expressing VEGF. ISS is unlikely to act directly on endothelial cells to inhibit angiogenesis because lung endothelial cells did not express Toll receptor 9, the receptor for ISS as assessed by RT-PCR. In vitro studies demonstrated that ISS inhibited macrophage expression of VEGF. Conclusion The ability of ISS to inhibit angiogenesis in vivo is likely to be mediated by several mechanisms, including ISS reducing the number of peribronchial inflammatory cells that express VEGF, ISS inhibiting expression of TH2 cytokines such as IL-13 that promote VEGF expression, and direct effects of ISS on macrophages to inhibit VEGF expression.

Background Matrix metalloproteinase 9 (MMP-9) and its tissue inhibitor of metalloproteinase 1 (TIMP-1) are hypothesized to play a role in the pathogenesis of airway remodeling in asthma. Objective We have used a mouse model of airway remodeling to determine the pattern of expression of MMP-9 and TIMP-1 in airway epithelium and peribronchial cells, and assess whether TIMP-1, an inhibitor of MMP-9, is expressed at the same sites in the airway. In addition, we have investigated whether immunostimulatory sequences (ISSs) of DNA modulate levels of expression of MMP-9, TIMP-1, and peribronchial fibrosis. Methods Levels of lung MMP-9 and TIMP-1 were assessed by zymography, ELISA, and immunohistochemistry. Results Repetitive ovalbumin challenge induced a significant increase in levels of MMP-9, TIMP-1, and peribronchial collagen deposition. The pattern of expression of MMP-9 and TIMP-1 in the remodeled airway was significantly different. MMP-9 but not TIMP-1 was expressed in airway epithelium, whereas both MMP-9 and TIMP-1 were expressed in peribronchial inflammatory cells. ISS significantly reduced expression of MMP-9 in airway epithelium (which immunostained positive for Toll receptor 9), as well as in peribronchial inflammatory cells. In vitro studies demonstrated that ISS inhibited bone marrow macrophage generation of MMP-9. Conclusion Allergen-induced peribronchial fibrosis is associated with expression of MMP-9 and TIMP-1 at different anatomical sites in the remodeled airway. The ability of ISS to inhibit the expression of MMP-9 in airway epithelium (a site where its inhibitor TIMP-1 is not induced by allergen challenge) may be important in determining whether ISS contributes to reductions in airway remodeling by reducing levels of MMP-9. Clinical implications Immunostimulatory sequences of DNA, which are being investigated as novel therapeutics in asthma, inhibit airway remodeling in mice as well as epithelial expression of MMP-9, an enzyme that degrades the extracellular matrix proteins surrounding the airway.

Few peribronchial mast cells are noted either in the lungs of naive mice or in the lungs of OVA-sensitized mice challenged acutely with OVA by inhalation. In this study, we demonstrate that OVA-sensitized mice exposed to repetitive OVA inhalation for 1–6 mo have a significant accumulation of peribronchial mast cells. This accumulation of peribronchial mast cells is associated with increased expression of the Th2 cell-derived mast cell growth factors, including IL-4 and IL-9, but not with the non-Th2 cell-derived mast cell growth factor, stem cell factor. Pretreating mice with immunostimulatory sequences (ISS) of DNA containing a CpG motif significantly inhibited the accumulation of peribronchial mast cells and the expression of IL-4 and IL-9. To determine whether mast cells express Toll-like receptor-9 (TLR-9; the receptor for ISS), TLR-9 expression by mouse bone marrow-derived mast cells (MBMMCs) was assessed by RT-PCR. MBMMCs strongly expressed TLR-9 and bound rhodamine-labeled ISS. However, incubation of MBMMCs with ISS in vitro neither inhibited MBMMC proliferation nor inhibited Ag/IgE-mediated MBMMC degranulation, but they did induce IL-6. Overall these studies demonstrate that mice exposed to repetitive OVA challenge, but not acute OVA challenge, have an accumulation of peribronchial mast cells and express increased levels of mast cell growth factors in the lung. Although mast cells express TLR-9, ISS does not directly inhibit mast cell proliferation in vitro, suggesting that ISS inhibits accumulation of peribronchial mast cells in vivo by indirect mechanism(s), which include inhibiting the lung expression of Th2 cell-derived mast cell growth factors.

In response to inflammation or injury, airway epithelial cells express inducible genes that may contribute to allergen-induced airway remodeling. To determine the contribution of epithelial cell NF-κB activation to the remodeling response, we generatedCC10-Cretg/IkkβΔ/Δmice in which NF-κB signaling through IκB kinase β (IKKβ) is selectively ablated in the airway epithelium by conditional Cre-recombinase expression from the Clara cell (CC10) promoter. Repetitive ovalbumin challenge of mice deficient in airway epithelial IKKβ prevented nuclear translocation of the RelA NF-κB subunit only in airway epithelial cells, resulting in significantly lower peribronchial fibrosis inCC10-Cretg/IkkβΔ/Δmice compared with littermate controls as assessed by peribronchial trichrome staining and total lung collagen content. Levels of airway mucus, airway eosinophils, and peribronchial CD4+cells in ovalbumin-challenged mice were also reduced significantly upon airway epithelialIkkβ ablation. The diminished inflammatory response was associated with reduced expression of NF-κB-regulated chemokines, including eotaxin-1 and thymus- and activation-regulated chemokine, which attract eosinophils and Th2 cells, respectively, into the airway. The number of peribronchial cells expressing TGF-β1, as well as TGF-β1 amounts in bronchoalveolar lavage, were also significantly reduced in mice deficient in airway epithelium IKKβ. Overall, these studies show an important role for NF-κB regulated genes in airway epithelium in allergen-induced airway remodeling, including peribronchial fibrosis and mucus production.