Key words: Asthma, remodeling, MMP-9, TIMP-1, ISS. 1 The potential role of MMP-9 in matrix remodeling

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1 Remodeling associated expression of matrix metalloproteinase 9 but not tissue inhibitor of metalloproteinase 1 in airway epithelium: Modulation by immunostimulatory DNA Jae Youn Cho, MD, PhD, a Marina Miller, MD, PhD, a Kirsti McElwain, BS, a Shauna McElwain, BS, a Jung Yeon Shim, MD, a,b Eyal Raz, MD, a and David H. Broide, MBChB a San Diego, Calif, and Seoul, Korea 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. (J Allergy Clin Immunol 2006;117: ) From a the Department of Medicine, University of California San Diego, and b the Sungkyunkwan University School of Medicine, Seoul. Dr Broide is supported by National Institutes of Health grant AI Received for publication April 4, 2005; revised December 1, 2005; accepted for publication December 5, Reprint requests: David Broide, MBChB, University of California San Diego, Basic Science Building, Room 5090, 9500 Gilman Drive, La Jolla, CA dbroide@ucsd.edu /$32.00 Ó 2006 American Academy of Allergy, Asthma and Immunology doi: /j.jaci Key words: Asthma, remodeling, MMP-9, TIMP-1, ISS Matrix metalloproteinases (MMPs) are a family of extracellular proteases that are responsible for the degradation of the extracellular matrix during tissue remodeling. 1 The potential role of MMP-9 in matrix remodeling in asthma is suggested from studies in human beings with asthma demonstrating elevated levels of MMP-9 in bronchoalveolar lavage (BAL) and sputum Studies in mouse models of asthma have also demonstrated an important role for MMP-9 in allergen-induced acute airway inflammatory responses but have not yet investigated the role of MMP-9 in airway remodeling. MMPs are synthesized as secreted or transmembrane proenzymes and are processed to an active form by the removal of an amino terminal propeptide which, when not cleaved, blocks access to the catalytic site. 1 Tissue inhibitor of metalloproteinases (TIMPs) are specific inhibitors of MMPs that bind noncovalently in a 1:1 molar ratio with activated MMPs. 1 TIMPs are produced by a wide variety of cells and are induced by several cytokines and growth factors. In contrast with previous studies that have examined the role of MMP-9 in mediating the acute inflammatory response to ovalbumin challenge in mouse models of asthma, in this study we have investigated the airway pattern of expression of MMP-9 and its inhibitor TIMP-1 during episodes of airway remodeling induced in a mouse model by repetitive allergen challenge. Because MMPs have the potential to play an important role in the degradation of the extracellular matrix during tissue remodeling, we considered it important to determine whether MMP-9, an MMP that is highly expressed in sputum and BAL in patients with asthma, 2-11 is expressed in the airway during periods of airway remodeling, and whether TIMP-1, an endogenous inhibitor of MMP-9, is expressed in the same anatomical distribution in the remodeled airway. The mouse model of airway remodeling we have used is associated with allergen-induced structural changes in the airway including peribronchial fibrosis with deposition of collagen III and collagen V, and increased thickness of the peribronchial smooth muscle layer The T H 2 cytokine dependence of the allergen induced airway remodeling is suggested from studies in IL-5 deficient mice that have significantly reduced levels of airway remodeling. 17 Studies in human beings with asthma have also

2 J ALLERGY CLIN IMMUNOL VOLUME 117, NUMBER 3 Cho et al 619 Abbreviations used BAL: Bronchoalveolar lavage CpG: Cytosine phosphorothioate guanosine ISS: Immunostimulatory sequences of DNA MMP: Matrix metalloproteinase TIMP: Tissue inhibitor of metalloproteinase TLR-9: Toll receptor 9 demonstrated an important role for IL-5 in airway remodeling. 19 In addition to examining the expression of MMP-9 and TIMP-1 in the remodeled airways of mice challenged with allergen, we have also investigated whether immunostimulatory sequences (ISSs) of DNA containing a cytosine phosphorothioate guanosine (CpG) DNA motif modulate allergen induced expression of MMP-9 and TIMP-1 in the remodeled airway. Previous studies have demonstrated that ISS is effective in inhibiting acute allergen induced eosinophilic airway inflammation, T H 2 cytokine expression, mucus expression, and airway hyperreactivity In addition, when ISS is administered before allergen challenge in mice, it can prevent features of airway remodeling including the development of peribronchial fibrosis. 16 More recent studies have also demonstrated that ISS can reverse established airway remodeling in mice when ISS is first administered after the development of allergen induced airway remodeling. 18 Currently there is no information regarding the effect of ISS on allergen-induced expression of MMP-9, which has been implicated in tissue remodeling. We have therefore investigated whether ISS modulates expression of MMP-9 and its inhibitor TIMP-1 in the remodeled airways of allergenchallenged mice. METHODS Induction of allergen-induced airway remodeling The methods we have used to administer ovalbumin allergen to induce airway remodeling in mice have previously been described In brief, BALB/c mice (16 mice/group; Jackson Laboratory, Bar Harbor, Me) were used when they reached 8 to 10 weeks of age. Mice were immunized subcutaneously on days 0, 7, 14, and 21 with 25 mg ovalbumin (grade V; Sigma-Aldrich, St Louis, Mo) adsorbed to 1 mg alum (Sigma-Aldrich) in 200 ml normal saline. Intranasal ovalbumin challenges (20 ng/50 ml in PBS) were administered on days 27, 29, and 31 under isoflurane (Vedco, Inc, St Joseph, Mo) anesthesia. Intranasal ovalbumin challenges were then repeated twice a week for 1 to 3 months. Age-matched and sex-matched control mice were sensitized but not challenged with ovalbumin during the 3-month study. Mice were killed 24 hours after the final ovalbumin challenge, and BAL fluid and lungs were analyzed. All animal experimental protocols were approved by the University of California, San Diego, Animal Subjects Committee. Therapeutic intervention with ISS Different groups of mice (16 mice/group) were administered intraperitoneal endotoxin-free (<1 ng/mg DNA) phosphorothioate ISSoligodeoxynucleotide (ODN) (5 9 -TGACTGTGAACGTTCGAGAT GA-3 9 ; Trilink, San Diego, Calif; 100 mg in 100 ml sterile, endotoxin-free PBS), mutated ODN (M-ODN) (5 9 -TGACTGTGAAGG TTGGAGATGA-3 9 ), which lacks the CpG motif present in ISS, or diluent control starting 1 day before the first intranasal ovalbumin challenge on day 27, and then continuing every other week 1 day before intranasal challenges for 3 months. Measurement of MMP-9 Gelatin zymography. Gelatin zymogaphy 12 was performed to detect MMP-9 in BAL fluid in the different groups of mice. Samples of BAL fluid were added to loading buffer and separated in 10% SDSpolyacrylamide gels that contained 0.1% gelatin. SDS was removed by two 30-minute washes with 2.5% Triton-X 100 before incubation of gels for 24 hours at 37 C in developing buffer (BioRad, Hercules, Calif). Gelatin gels were stained with 0.25% Coomassie Blue and the optical density of the MMP-9 band determined by Unscan-it software (Silk Scientific, Orem, Utah). Results are expressed as MMP-9 zymography units (arbitrary units/ml) 13 on the basis of the computer analysis. A MMP-9 standard (Chemicon, Temecula, Calif) was used as a positive control. ELISA. The activity of MMP-9 in lung was assayed by ELISA (sensitivity 5 ng/ml) according to the manufacturer s instructions (Chemicon). Homogenized lung supernatants were prepared by using methods previously described in this laboratory for measurement of cytokines in lung. 16,18 Immunohistochemical detection of MMP-9 in lung sections. A primary mab directed against MMP-9 (Chemicon) was used for immunohistochemical detection of MMP-9 in the lung sections using the immunoperoxidase method as previously described in this laboratory Total peribronchial MMP-9 immunoreactivity was assessed and includes all immunoreactivity for MMP-9 extending from the lumenal aspect of the epithelium to the adventitia (ie, epithelium, submucosa, adventitia). The immunostained lung sections were also analyzed to determine the anatomical distribution of expression of MMP-9 in the airway (eg, epithelial versus peribronchial inflammatory cells). The total area of MMP-9 immunostaining (peribronchial MMP-9 immunoreactivity) as well as MMP-9 immunostaining in epithelium or in peribronchial inflammatory cells in each paraffin embedded lung was outlined and quantified by using a light microscope attached to an image analysis system (Image-Pro Plus; Media Cybernetics, Silver Springs, Md). The unit area of computer program images were calibrated and standardized with a slide micrometer. Results are expressed as the area of immunostaining per micrometer length of basement membrane of bronchioles 150 to 200 mm of internal diameter. Between 3 and 5 bronchioles were counted in each slide. For all experiments that require morphometric analysis of lung tissues, the slides are coded and analyzed by technicians blind to the study group. Each slide is also analyzed in the same predetermined sequence to minimize observer bias. All slides to be analyzed in a particular experiment are stained in the same batch under identical staining conditions and analyzed under the same light microscope conditions (magnification, gain, illumination). Immunohistochemical detection of Toll receptor 9 in airway epithelial cells in lung sections. A primary mab directed against Toll receptor 9 (TLR-9; Chemicon) was used for immunohistochemical detection of TLR-9 in the lung sections using the immunoperoxidase method as previously described in this laboratory Effect of ISS on macrophage MMP-9 expression in vitro. To determine whether ISS directly modulates levels of macrophage expression of MMP-9, bone marrow derived macrophages were incubated with LPS (10 mg/ml) in triplicate for 24 hours in the presence or absence of ISS or M-ODN (10 mg/ml) as previously described in this laboratory. 18 Supernatants were collected and assayed for MMP-9 by using the zymography assay as described.

3 620 Cho et al J ALLERGY CLIN IMMUNOL MARCH 2006 FIG 1. Peribronchial distribution of expression of MMP-9 and TIMP-1: modulation by ISS. Minimal levels of either MMP-9 (A) or TIMP-1 (D) immunostaining are noted in nonovalbumin-challenged mice. Repetitive ovalbumin (OVA) challenge induced significantly increased levels of expression of MMP-9 (B) and TIMP-1 (E) that were significantly inhibited by ISS (C and F). Repetitive OVA challenge induced MMP-9 (B), but not TIMP-1 (E), expression in epithelial cells. Measurement of TIMP-1 TIMP-1 ELISA. The concentrations of TIMP-1 in homogenized lung supernatants was assayed by ELISA (sensitivity 37 pg/ml) according to the manufacturer s instructions (R&D Systems Inc, Minneapolis, Minn). Immunohistochemical detection of TIMP-1 in lung sections. Immunohistochemical detection of TIMP-1 in lung sections used a primary mab directed against TIMP-1 (Chemicon), and similar immunohistochemistry and image analysis methods as described for quantitation of epithelial versus peribronchial inflammatory cell expression of MMP-9. Quantitation of peribronchial fibrosis Peribronchial trichrome staining. The area of peribronchial trichrome staining in paraffin embedded lung was outlined and quantified by using a light microscope attached to an image analysis system as previously described Results are expressed as the area of trichrome staining per micrometer length of basement membrane of bronchioles 150 to 200 mm of internal diameter. At least 10 bronchioles were counted in each slide. Lung collagen assay. The amount of lung collagen was measured as previously described in this laboratory with a collagen assay kit that uses a dye reagent that selectively binds to the [Gly-X-Y]n tripeptide sequence of mammalian collagens (Biocolor, Newtownabbey, United Kingdom). In all experiments, a collagen standard was used to calibrate the assay. BAL eosinophil levels Bronchoalveolar lavage eosinophil counts were performed as previously described Statistical analysis Results in the different groups of mice were compared by ANOVA using the nonparametric Kruskal-Wallis test followed by posttesting using the Dunn multiple comparison of means. All results are presented as means 6 SEMs. A statistical software package (Graph Pad Prism, San Diego, Calif) was used for the analysis. P values of <.05 were considered statistically significant. RESULTS Immunohistochemical detection of epithelial cell vs peribronchial cells expressing MMP-9 and TIMP-1 in the remodeled airway: Effect of ISS Minimal peribronchial expression of either MMP-9 (Fig 1, A)orTIMP-1(Fig 1, D) was noted in nonovalbuminchallenged mice by immunohistochemistry. In contrast, both MMP-9 (Fig 1, B) and TIMP-1 (Fig 1, E) were significantly expressed after repetitive ovalbumin challenge for 3 months in the remodeled airway. However, the peribronchial distribution of MMP-9 expression differed from that of TIMP-1 expression in the remodeled airway in that MMP-9 was expressed in airway epithelium (Fig 1, B) whereas TIMP-1 was not expressed in airway epithelium (Fig 1, E). Both MMP-9 (Fig 1, B) and TIMP-1 (Fig 1, E) were expressed in peribronchial inflammatory cells and the peribronchial extracellular matrix. Quantitative analysis of MMP-9 expression by immunohistochemistry and image analysis demonstrated that ovalbumin challenge for 1 or 3 months induced significant increased numbers of MMP-9 1 peribronchial inflammatory cells (ovalbumin vs no ovalbumin; P 5.001; Fig 2, A) as well as significant increased levels of MMP-9 immunostaining in airway epithelial cells (ovalbumin vs no ovalbumin; P 5.001; Fig 2, B). ISS significantly reduced the numbers of MMP-9 1 peribronchial inflammatory cells

4 J ALLERGY CLIN IMMUNOL VOLUME 117, NUMBER 3 Cho et al 621 FIG 2. Expression of MMP-9: modulation by ISS. Levels of MMP-9 were assessed by counting the number of MMP-9 1 peribronchial cells (A), epithelial MMP-9 immunostaining (B), MMP-9 as assessed by ELISA (C), zymography (D and E), and peribronchial MMP-9 immunostaining (F). D, Lane 1 is MMP-9 standard, lane 2 is no ovalbumin (OVA), lane 3 is OVA, and lane 4 is OVA 1 ISS. (ovalbumin vs ovalbumin 1 ISS; P 5.001; Fig 1, B and C; Fig 2, A), as well as levels of MMP-9 immunostaining in airway epithelial cells (ovalbumin vs ovalbumin 1 ISS; P 5.001; Fig 1, B and C; Fig 2, B) when administered for 1 or 3 months. Quantitative analysis of TIMP-1 expression by immunohistochemistry and image analysis demonstrated that ovalbumin challenge for 1 or 3 months increased the numbers of TIMP-1 1 peribronchial inflammatory cells (ovalbumin vs no ovalbumin; P 5.001; Fig 1, D and E; Fig 3, A) but did not induce significant TIMP-1 compared with MMP-9 immunostaining in airway epithelial cells (Fig 1, B and E; Fig 2, B; Fig 3, B). ISS significantly reduced the number of TIMP-1 1 peribronchial inflammatory cells (ovalbumin vs ovalbumin 1 ISS; P 5.001; Fig 1, E and F; Fig 3, A). Quantitation of MMP-9 in lung by ELISA, zymography, and peribronchial immunostaining: Effect of ISS Significantly increased lung levels of MMP-9 were detected after ovalbumin challenge for 1 or 3 months using 3 different methods to quantitate MMP-9, including MMP-9 ELISA ( vs ng MMP-9/mg lung protein; ovalbumin 3 months vs no ovalbumin 3 months; P 5.001; Fig 2, C), MMP-9 zymography ( vs MMP-9 zymography units; ovalbumin 3 months vs no ovalbumin 3 months; P 5.001; Fig 2, D and E), and peribronchial MMP-9 immunostaining and image analysis quantitation ( vs mm 2 /mm circumference of bronchiole; ovalbumin 3 months vs no ovalbumin 3 months; P 5.001; Fig 2, F). Level of inhibition of MMP-9 in remodeled airway induced by ISS Levels of inhibition of MMP-9 induced by ISS were in general greater after 3 months of ISS administration compared with 1 month of ISS administration (Fig 2). Five different quantitative measurements of MMP-9 (MMP-9 1 immunostaining of epithelium; MMP-9 1 peribronchial cells; peribronchial MMP-9 immunostaining which includes extracellular matrix deposition of MMP- 9; ELISA; zymography) were used to assess the effect of ISS on levels of MMP-9 in the remodeled airway. Administration of ISS for 3 months significantly reduced the number of peribronchial MMP-9 1 cells (P 5.001; Fig 2, A), epithelial MMP-9 immunostaining (P 5.001; Fig 2, B), and levels of peribronchial MMP-9 immunostaining (P 5.001; Fig 2, F), and also had a statistically significant effect on reducing levels of MMP-9 as assessed by ELISA (P 5.05; Fig 2, C) and by MMP-9 zymography (Fig 2, E; P 5.01; ISS 1 ovalbumin vs ovalbumin). Administration of ISS for 1 month significantly reduced the number of peribronchial MMP-9 1 cells (P 5.001; Fig 2, A) and epithelial MMP-9 immunostaining (P 5.001; Fig 2, B) as well as levels of lung MMP-9 as assessed by ELISA (P 5.003; Fig 2, C; ISS 1 ovalbumin vs ovalbumin), and modestly reduced levels of BAL MMP-9 (P 5.05; Fig 2, E), but did not significantly reduce peribronchial MMP-9 immunostaining (Fig 2, F).

5 622 Cho et al J ALLERGY CLIN IMMUNOL MARCH 2006 FIG 3. Expression of TIMP-1 and collagen: modulation by ISS. The number of TIMP-1 1 peribronchial cells (A), epithelial TIMP-1 immunostaining (B), lung TIMP-1 measured by ELISA (C), peribronchial TIMP-1 stained area (D), levels of lung collagen (E), and peribronchial trichrome staining (F) were assessed at 1 and 3 months in mice challenged with ovalbumin (OVA) and treated with ISS. Repetitive ovalbumin challenge induces expression of TIMP-1 Increased numbers of TIMP-1 1 peribronchial cells (P 5.001; Fig 3, A) and TIMP-1 in the peribronchial extracellular matrix (Fig 1, E) were detected by immunohistochemistry after ovalbumin challenge. In contrast, ovalbumin challenge induced minimal TIMP-1 immunostaining in airway epithelial cells (Fig 1, E;Fig 3, B). Levels of lung TIMP-1 as measured by ELISA were also significantly increased in mice exposed to repetitive ovalbumin challenge ( vs ng TIMP-1/mg lung protein; ovalbumin 3 months vs no ovalbumin 3 months; P 5.001; Fig 3, C). The area of peribronchial TIMP-1 immunostaining in mice repetitively challenged with ovalbumin was also significantly greater than in control nonovalbumin-challenged mice ( vs mm 2 /mm circumference of bronchiole; P 5.001; Fig 3, D). Effect of ISS on TIMP-1 expression in vivo Immunostimulatory sequences of DNA administered for either 1 or 3 months significantly reduced levels of lung TIMP-1 as assessed by significant reductions in the number of peribronchial TIMP-1 1 cells (P 5.001; Fig 3, A) and level of peribronchial TIMP-1 immunostaining (P 5.001; Fig 3, D) in mice exposed to repetitive ovalbumin challenge. ISS administered for 3 months, but not for 1 month, reduced levels of lung TIMP-1 as assessed by ELISA (P 5.01; Fig 3, C). Effect of ISS on peribronchial collagen deposition Levels of total lung collagen (P 5.001; Fig 3, E) and the area of peribronchial trichrome staining (P 5.001; Fig 3, F) were significantly greater in mice that were repetitively challenged with ovalbumin for 1 or 3 months compared with control nonovalbumin-challenged mice. Administration of ISS to mice repetitively challenged with ovalbumin significantly reduced levels of lung collagen (P 5.05; Fig 3, E) at both 1 and 3 months. ISS reduced the area of peribronchial trichrome (P 5.001) staining at 3 months, but not at 1 month (Fig 3, F). Comparison of MMP-9, TIMP-1, and collagen expression after 1 month or 3 months of allergen challenge Overall, these studies show that MMP-9 (Fig 2), TIMP- 1(Fig 3), and lung collagen (Fig 3, E and F) are significantly induced after ovalbumin challenge for 1 month at approximately similar levels as noted after 3 months of ovalbumin challenge. The effect of ISS on reducing levels of MMP-9, TIMP-1, and collagen is not significantly different at 1 versus 3 months, although there appears to be a trend for greater ISS inhibitory effects on reducing levels of MMP-9 (Fig 2, E and F), TIMP-1 (Fig 3, C), and trichrome staining (Fig 3, F) at 3 months compared with 1 month. In contrast with the inhibitory effect of ISS on MMP-9, TIMP-1, and fibrosis, administration of an M-ODN to mice for as long as 3 months had no significant

6 J ALLERGY CLIN IMMUNOL VOLUME 117, NUMBER 3 Cho et al 623 FIG 4. Expression of TLR-9 by airway epithelial cells. Lung sections of nonovalbumin-challenged mice were stained with a control species and isotype matched antibody (A), and an anti TLR-9 Ab (B). After ovalbumin (OVA) challenge, epithelial cells in lung sections stained with an anti TLR-9 Ab continued to express TLR-9, as did peribronchial mononuclear cells (C). effect on reducing any of the parameters of ovalbumin induced MMP-9, TIMP-1, collagen, or trichrome staining (data not shown). Expression of TLR-9 the receptor for ISS by airway epithelial cells Lung sections of nonovalbumin-challenged mice stained with an anti TLR-9 Ab demonstrated significant staining of airway epithelial cells (Fig 4, B). A control species and isotype matched antibody did not stain airway epithelial cells in nonovalbumin-challenged lung sections (Fig 4, A). After ovalbumin challenge epithelial cells continued to express TLR-9, as did peribronchial mononuclear cells (Fig 4, C). Effect of ISS on macrophage MMP-9 expression in vitro Bone marrow derived macrophages incubated with LPS expressed significantly higher levels of MMP-9 in the supernatant compared with cells cultured in media alone ( vs MMP-9 zymography units; LPS vs no LPS; P 5.01). Incubation of bone marrow derived macrophages with ISS significantly reduced levels of MMP-9 expression in the supernatant ( vs MMP-9 zymography units; ISS 1 LPS vs LPS; P 5.05). Effect of ISS on BAL eosinophilia The absolute number of BAL eosinophils in mice sensitized to ovalbumin and repetitively challenged with ovalbumin was significantly greater than in control nonovalbumin-challenged mice ( vs BAL eosinophils; ovalbumin 3 months vs no ovalbumin 3 months; P ). Administration of ISS before initiation of repetitive ovalbumin challenges significantly reduced the absolute number of BAL eosinophils ( vs BAL eosinophils; ISS 1 ovalbumin 3 months vs ovalbumin 3 months; P 5.001). DISCUSSION In this study, we have demonstrated that allergen induced airway remodeling is associated with significantly increased levels of expression of MMP-9 and its inhibitor TIMP-1. Interestingly, immunostaining of lungs derived from mice with remodeled airways demonstrated a distinctly different pattern of MMP-9 expression compared with TIMP-1 expression in the remodeled airway, with MMP-9, but not TIMP-1, expressed in airway epithelium. Thus, the localized absence of expression of TIMP-1, an endogenous MMP-9 inhibitor, in the airway epithelium of mice with remodeled airways could contribute to the continued function of MMP-9 in the airway epithelium. In addition, we have demonstrated that ISS can inhibit expression of MMP-9 in airway epithelium in vivo, a tissue site where TIMP-1 is not significantly expressed in the remodeled airway. We have also demonstrated that airway epithelial cells in mouse lung express TLR-9, the receptor for ISS, raising the possibility that ISS could inhibit MMP-9 by acting directly on airway epithelial cells and/or acting indirectly on epithelial cells through effects on innate immune cells expressing TLR-9. To determine whether ISS could be acting on either innate immune cells (ie, TLR-9 expressing macrophages) or airway epithelial cells to inhibit MMP-9 expression, we performed in vitro studies with macrophages and epithelial cells to determine whether ISS inhibited MMP-9 expression. These studies demonstrated that ISS inhibited macrophage expression of MMP-9, suggesting that ISS effects on macrophages could contribute to reduced MMP-9 expression in the remodeled airway. Although we demonstrated that TLR-9 was expressed by a mouse airway epithelial cell line MLE-12 (data not shown), we were unable to induce MMP-9 expression in this cell line, making assessment of the ability of ISS to modulate airway epithelial MMP- 9 expression in this cell line not feasible (data not shown). Interestingly, the human airway epithelial transformed cell line BEAS 2B and primary human bronchial epithelial cells express TLR-9 as assessed by RT-PCR. 23 However,

7 624 Cho et al J ALLERGY CLIN IMMUNOL MARCH 2006 incubation of these epithelial cells with CpG did not induce GM-CSF or macrophage inflammatory protein 3-a release. 23 Thus, both mouse and human airway epithelial cells express TLR-9, but its function in epithelial cells in terms of modulating allergen induced MMP-9 or other mediator generation is currently unknown. To determine whether MMP-9 plays a role in the pathogenesis of airway remodeling, we have recently performed studies of airway remodeling in MMP-9 deficient mice (Lim et al, unpublished data, January 2006). In these studies, we demonstrated that ovalbumin-challenged MMP-9 deficient mice have reduced levels of peribronchial fibrosis compared with ovalbumin-challenged wildtype mice, suggesting that MMP-9 does play a role in promoting airway remodeling. However, because airway remodeling is reduced but not ablated in MMP-9 deficient mice, MMPs other than MMP-9 and/or alternate mediators that regulate collagen synthesis and breakdown are also likely to contribute to airway remodeling. We have also demonstrated that repetitive ovalbumin challenge not only induces expression of MMP-9 but also induces expression of an inhibitor of MMP-9, TIMP-1. At the transcriptional level, MMP-9 and TIMP-1 may be coregulated, such that activators of MMP-9 and TIMP-1 transcription (eg, LPS) will affect both genes in the same direction. 24 The effect of ISS on MMP-9 and TIMP-1 transcription is similar to that of corticosteroids, which are also able to inhibit both MMP-9 and TIMP-1 expression by macrophages. 25 The net effect of induction of MMP-9 as well as its inhibitor TIMP-1 on MMP-9 function will be dependent on the ratios of MMP-9 and TIMP-1 expressed, the localization of MMP-9 and TIMP-1 in the airway, and MMP-9 regulatory pathways other than TIMPs that play key roles in localizing the pericellular proteolytic activity of MMPs. 25 One of the assays that we have used to measure MMP-9 in BAL fluid is a functional assay assessing cleavage of the MMP-9 substrate gelatin. This functional MMP-9 zymography assay demonstrated significantly increased levels of MMP-9 bioactivity in BAL fluid after ovalbumin challenge, suggesting that although TIMP-1 had also been induced by repetitive ovalbumin challenge, it did not prevent repetitive ovalbumin challenge from inducing significant MMP-9 bioactivity in BAL. In summary, in this study we have demonstrated that allergen induced airway remodeling is associated with expression of MMP-9 in the remodeled airway in airway epithelium, as well as in peribronchial cells and the extracellular matrix. Administration of ISS to mice exposed to repetitive ovalbumin challenge significantly reduces levels of MMP-9 expression and peribronchial fibrosis. In vitro studies demonstrate that ISS can directly inhibit macrophage expression of MMP-9, and further studies are needed to determine whether ISS directly inhibits MMP-9 expression by airway epithelial cells that we have identified to express TLR-9 in lung sections. 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