Effect of a matrix metalloproteinase-12 inhibitor, S-1, on allergic airway disease phenotypes in mice
- First Online:
- Cite this article as:
- Yu, Y., Chiba, Y., Sakai, H. et al. Inflamm. Res. (2010) 59: 419. doi:10.1007/s00011-009-0153-0
- 77 Views
Matrix metalloproteinase-12 (MMP-12) has been reported to play an important role in chronic airway inflammatory diseases, but its detailed role in allergic airway disease is not well known. In this study, we investigated the expressions of MMP-12 and the effect of S-1, an MMP-12 inhibitor, in a mouse model of allergic airway inflammation.
Materials and methods
The expressions and activity of MMP-12 were measured by RT-PCR western blot and zymography, respectively. The locations in the airways of MMP-12 and elastin fiber were histologically studied. The mice were orally administered with S-1 during the period of antigen challenge. Bronchoalveolar lavage fluid (BALF) cells were counted, and the activity of MMP-12 in BALF was measured by zymography after the treatment with S-1.
The allergen challenge model resulted in increased eosinophil number in BALF and damage to elastin fiber. Upregulation of MMP-12 was also found in the airways of challenged mice. The increased eosinophil number in the BALF after antigen challenge was inhibited by S-1.
These findings suggest that MMP-12 may play an important role in the eosinophil infiltration of the allergic airway.
KeywordsMatrix metalloproteinase-12 (MMP-12)MMP-9S-1Airway inflammationAllergic airway disease
Matrix metalloproteinases (MMPs) are a family of proteinases with a zinc-dependent activity structure, which are activated at physiological pH and can degrade the extracellular matrix (ECM) and non-matrix substrates . Like other MMPs, MMP-12 is activated by the removal of its propeptide and hemopexin domains. When it is activated, MMP-12 can degrade ECM fibers such as elastin, which is one of the main components of pulmonary parenchyma . Overexpressions of MMP-12 were reportedly found in the airways of some chronic airway inflammatory diseases such as emphysema, COPD and allergic bronchial asthma [3–5], indicating that MMP-12 is involved in the pathogenesis of chronic airway inflammatory diseases .
Asthma is characterized by increased inflammatory cells in the airways and lungs and by upregulations of IgE and cytokines including interleukin-13 (IL-13). It has been reported that depletion of MMP-12 inhibited the inflammation of lung tissue in an allergen challenge model . The expression of MMP-12 is reported to be IL-13 dependent in the airways of mice with airway inflammation . These results indicate that MMP-12 is implicated in the pathogenesis of allergic airway disease. In our previous study, upregulation and increased activity of MMP-12 were found in the airways of rats with allergic inflammation . However, the detailed role of MMP-12 in the pathogenesis of allergic asthma is still unclear. On the other hand, matrix metalloproteinase-9 (MMP-9), which is also named gelatinase-B, is found to be increased in the bronchoalveolar lavage fluids (BALFs) of patients with asthma . The depletion of MMP-9 inhibited the inflammatory cells’ egression into the airway lumen in an experimental allergen challenge model . Furthermore, tissue inhibitor of metalloproteinase-1 (TIMP-1), the endogenous inhibitor of MMP-9, inhibited the inflammatory cells’ recruitment in the airways of a mouse allergen challenge model .
The present study investigated the changes in the expression of MMP-12 in the airways of mice with allergic airway disease. In addition, the effect of a selective inhibitor of MMP-12, S-1, on the antigen-induced allergic phenotypes was investigated to clarify the role of MMP-12 in the pathogenesis of allergic airway disease.
Materials and methods
Animals and antigen challenge
Male BALB/c mice (7-week old, specific pathogen-free) were used. All the experiments were approved by the Animal Care Committee at Hoshi University (Tokyo, Japan). Mice were sensitized by intraperitoneal injection of 8 μg ovalbumin (OA) (Sigma–Aldrich, St. Louis, MO) with 2 mg aluminum hydroxide (Pierce Biotechnology, Inc., Rockfold, IL, USA) on days 0 and 5. The sensitized mice were challenged with aerosolized OA-saline solution (5 mg/mL in saline) for 30 min on days 12, 16 and 20. A control group of mice received the same immunization schedule, but were exposed to saline aerosol instead of OA challenge.
The synthetic specific inhibitor of MMP-12, S-1, was kindly provided by Shionogi & Co., Ltd., Japan. S-1 is an N-sulfonylamino acid derivative synthesized by Shionogi & Co., Ltd. (Osaka, Japan), and its analog has also been previously reported . The in vitro assay system, using the method previously reported , revealed that S-1 has an ability to inhibit MMP-12 selectively: the 50% inhibitory concentration (IC50) to MMP-12 was 0.04 μM, while the IC50s to other MMPs, such as MMPs-2, 8, 9, 13 and 14, were more than 10 μM (unpublished observation of Shionogi & Co., Ltd.). It has also been reported that the N-sulfonylamino acid derivatives are orally active in mammals .
The S-1 or vehicle (ethanol:polyethylene glycol:water = 0.15:0.15:0.7; volume ratio) was orally administered (30 mg/kg) twice a day from the day before the first antigen challenge until the day of the last antigen challenge. The dosage and administration schedule of S-1 were decided based on a pharmacokinetic study in rats; the bioavailability was 30.3%, the maximum concentrations (Cmax) in plasma and lung tissue were 5.68 μg/mL and 1.63 μg/g tissue, respectively (observed at 4 h after the administration), and the area under the blood concentration time curve (AUC) was 85.5 μg × h/mL when S-1 (20 mg/kg) was administered orally.
Bronchoalveolar lavage (BAL)
Twenty-four hours after the last antigen challenge, mice were anesthetized with urethane (1.6 g/kg, i.p.). One microliter of phosphate buffered saline (PBS) was instilled into the lungs and lavaged three times. The BAL fluid (BALF) was collected and then centrifuged (1,000g, 10 min at room temperature). Then the supernatant was collected for the following experiments, and the pellet was resuspended with PBS after incubation with 10% neutral formalin for 10 min. Total cell counts were performed using a Thoma’s hemocytometer (Erma, Tokyo, Japan). Differentiated cell count was carried out under Diff-Quik staining (Sysmex, Kobe, Japan).
RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)
Lungs, with the main bronchi, were removed under anesthesia, quickly frozen with liquid nitrogen and crushed into powder by using Cryopress™ (CP-100W; Microtec, Co. Ltd., Chiba, Japan: 15 s × 3). Total RNA was isolated from each frozen sample with a one-step guanidium–phenol–chloroform extraction procedure using TRI Reagent™ (Sigma–Aldrich, St. Louis, MO). The mRNA levels of MMP-12 were examined by RT-PCR. Briefly, cDNAs were prepared from the total RNA (1.0 μg) by using RevertAid First Strand cDNA Synthesis Kit (Fermentas Inc., Hanover, MD) in a total volume of 50 μL reaction buffer containing 50 mM Tris–HCl, pH 8.3, 50 mM KCl, 4 mM MgCl2, 1 mM dNTP mixture, 1 U/μL RNase inhibitor, 10 ng/μL random 6 mers, and 200 U/μL M-MuL V reverse transcriptase. The reaction mixture was incubated for 10 min at 25°C followed by the reaction for 60 min at 42°C to synthesize the cDNAs. Reverse transcriptase was inactivated at 70°C for 5 min. Then the RT reaction mixture (1 μL) was subjected to PCR (0.1 μM forward and reverse primers, 0.025 U/L Taq DNA polymerase, 2 mM MgCl2, 0.2 mM dNTPs) in a final volume of 5 μL. The PCR primer sets used were: 5′-AGGTCAAGATGGATGAAGCGG-3′ (sense) and 5′-GAAGTAATGTTGGTGGCTGGACTC-3′ (antisense) for MMP-12, and 5′-CCATCACTGCCACTCAGAAGAC-3′ (sense) and 5′-TACTCCTTGGAGGCCATGTAGG-3′ (antisense) for GAPDH. The thermal cycle profile used was 1) denaturing for 15 s at 95°C, 2) annealing primers for 15 s at 55°C, 3) extending the primers for 60 s at 72°C. The PCR amplification was performed at 28 cycles according to the preliminary cycle dependence experiment. The PCR products were subjected to electrophoresis on 1.2% agarose gel and visualized by ethidium bromide staining.
Twenty-five microliters of BALF obtained from antigen challenged or sensitized animals was mixed with 3 × SDS sample buffer and heated at 100°C for 4 min. The mixtures were subjected to 10% SDS-PAGE. Proteins were then electrophoretically transferred for 2 h onto polyvinylidene difluoride (PVDF) membranes (Hybond-P, Amersham, UK) in transfer buffer (20% methanol containing 25 mM Tris and 192 mM glycine). After repeatedly washing with Tris buffer (20 mM Tris, 500 mM NaCl, pH 7.5) containing 0.1% Tween-20 (TTBS), the membranes were incubated with blocking buffer (5% skim milk in TTBS) for 3 h at room temperature as previously reported . Then the membranes were incubated with primary rabbit anti-MMP-12 antibody (1:1,000 dilution; Sigma–Aldrich, St. Louis, MO), which specifically recognizes the hinge region of MMP-12 and does not cross-react with other MMPs including MMPs-1, 2, 3 and 9, or primary mouse anti-MMP-9 antibody (1:1,000 dilution; AnaSpec, San Jose, CA, USA) in antibody buffer (2% bovine serum albumin in TTBS) for 12 h at room temperature. After washing with TTBS for 10 min six times, the membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5,000 dilution; Amersham, UK) or goat anti-mouse IgG (1:5,000 dilution; Amersham, UK) for 1.5 h at room temperature, and were washed with TTBS for 10 min six times. The blots were detected with an enhanced chemiluminescent method (ECL System; Amersham, UK) and quantitated by densitometry system (Atto Densitograph; Atto Co., Tokyo, Japan). Under these conditions, no detectable band was observed when incubation with the primary antibody was omitted (data not shown).
α-Casein and gelatin zymographies were carried out to measure the activities of MMP-12 and MMP-9, respectively. In brief, 25 μL of BALF obtained after the last antigen challenge was mixed with 2 × SDS sample buffer without heating and then subjected to 10% polyacrylamide gel containing α-casein (1 mg/mL; USB Corp., Cleveland, OH) or 7.5% polyacrylamide gel containing gelatin (1 mg/mL; Bio-Rad Laboratories, Hercules, CA). Electrophoresis was performed at 4°C, and the gels were then incubated for 30 min in fixation buffer (50% methanol, 10% acetic acid). Gels were then washed with 2.5% Triton X-100 for 15 min four times to remove SDS and equilibrated in incubation buffer (50 mM Tris–HCl, 5 mM CaCl2, 0.02% NaN3; pH 8.5 for MMP-12 or pH 7.6 for MMP-9) for 30 min at room temperature. Gels were then incubated in incubation buffer at 37°C overnight. After incubation, the gels were stained with 5% Coomassie brilliant blue (CBB) and destained with destaining buffer (30% methanol, 10% acetic acid). The gelatinolytic or caseinolytic bands were detected and quantitated by densitometry system (Atto Densitograph; Atto Co., Tokyo, Japan).
Lungs were fixed in 10% formaldehyde and embedded in Paraplast X-TRA™ paraffin (Fisher Healthcare, Houston, TX). Four micrometer sections were obtained from blocks and mounted on silane-coated glass slides, deparaffinized with xylene and graded ethanol. For the immunohistochemical examination, the sections were then incubated with rabbit anti-MMP-12 as the primary antibody by using a streptavidin–biotin immunoperoxidase method as described previously  with minor modifications. In brief, before immunostaining, rehydrated sections were pretreated by incubation in 0.5% Triton X-100 in PBS for 10 min for permeabilization, and were immersed in 0.3% hydrogen peroxide in 100% methanol for 30 min to remove endogenous peroxidase activity. The pretreated sections were washed with PBS and incubated in 5% skim milk in PBS for 1 h. The sections were then rinsed and incubated sequentially at room temperature in the following solutions: (a) primary antibody (diluted 1:500 in PBS containing 1% skim milk) overnight, (b) biotinylated goat anti-rabbit IgG (diluted 1:200 in PBS containing 1% skim milk) for 30 min, and (c) avidin-biotinylated peroxidase complexes (Vector Laboratory, Inc., Burlingame, CA) in PBS for 30 min. The bound peroxidase activity was visualized by incubation with 0.7 mg/mL 3,3′-diaminobenzidine-0.02% H2O2 in 60 mM Tris buffer (pH 7.5). Sections were rinsed in PBS after each step of the immunostaining procedure. Finally, the sections were counterstained with Hematoxylin QS (Vector Laboratories, Inc., Burlingame, CA), dehydrated, and mounted in permanent mounting medium. Under these conditions, the specimens without incubation with primary antibody showed no distinct staining. The total macrophages and MMP-12-positive macrophages in lungs were counted under 160× magnification by two different investigators. The average number of three random microscopic fields from a section was defined as N = 1.
Elastic Van Gieson (EVG) staining
Lung tissue sections were prepared as above. EVG staining was performed according to the manufacturer’s instructions (Muto Pure Chemicals Co., Ltd., Tokyo, Japan). Briefly, the sections were incubated with 70% ethanol-1% HCl for 1 min after deparaffinization. Then the sections were stained with Weigert’s Resorcin Fuchsin solution for 1 h. After washing with 70% ethanol-1% HCl, the sections were stained with ferric hematoxylin for 5 min. Then the sections were washed with 0.5% HCl and stained with Van Gieson solution for 1 min before dehydration. After preparing each lung specimen, the elastic fiber damage in the subepithelium was graded on a scale of 0–3+ by two blinded observers using the coded slides as previously reported . A 3+ grade implies seriously damaged elastin fiber layer, whereas 0 implies intact elastin fiber layer.
All the data were expressed as means with SEM. Statistical significance of difference was determined by unpaired Bonferroni/Dunn analysis. A value of p < 0.05 was considered significant.
Upregulation of MMP-12 in the airways after the antigen challenge
The activity of MMP-12 in BALF was determined by α-casein zymography. The 54 and 45 kD forms of MMP-12 were detected (Fig. 1c). The activities of MMP-12 were evaluated by the band density quantitated by the densitometry system mentioned in “Materials and methods”. The activity of MMP-12 was significantly increased after the antigen challenge (Fig. 1d).
The MMP-12 protein expressions in BALF were determined by western blot. The single and clear bands corresponding to the proenzyme (54 kD), intermediate form (45 kD) and 29 kD form of MMP-12 were detected (Fig. 1e). The levels of MMP-12 protein were evaluated by the band density quantified by the densitometry system mentioned in “Materials and methods”. No significant differences in the expressions of the proenzyme and intermediate form of MMP-12 were observed between the sensitized control and challenged groups, while the expression of 29 kD MMP-12 was significantly increased in the challenged group (Fig. 1f). The MMP-12 antibody used does not have cross reactivity with MMP-9 (Fig. 1g).
MMP-12 immunohistochemistry in lung tissues
Effect of S-1 on the increased number of eosinophils in BALF induced by the antigen challenge
Effects of S-1 on the MMP-9 expressions and MMP-12 activity after the antigen challenge
Histological change of the elastin fiber after antigen challenge
It has been previously reported that MMP-12 is increased in the airways of chronic respiratory diseases such as COPD, including emphysema [18, 19]. Increased MMP-12 mRNA expression was also found in patients with allergic bronchial asthma . However, the exact role of MMP-12 in these chronic airway diseases is still not clear. In the present study, the expression and activity of MMP-12 were studied in an allergen challenge model in mice. The effect of S-1, a specific MMP-12 inhibitor, was also investigated to determine the role of MMP-12 in the pathogenesis of allergic airway disease.
In the current allergic airway inflammation model , the expression and activity of MMP-12 were found to be increased at 24 h after the last antigen challenge. Human MMP-12 is composed of the 54 kD proenzyme, 45 kD intermediate form and 22 kD active form . The proenyzme, intermediate form and 29 kD form of MMP-12 were detected in BALF by western blot. The expression of the 29 kD form of MMP-12, which may be an intermediate form of the degraded MMP-12, was significantly increased after the antigen challenge. The 29 kD form of MMP-12 was also found to be increased in the BALFs of mice with allergic airway inflammation which were treated with granulocyte macrophage colony-stimulating factor (GM-CSF) before OA challenge . In the current study, the proenzyme and the 45 kD intermediate form, but not the active form, of MMP-12 were detected in α-casein zymograph. This observation in part might be due to the conditions used in zymography . The active form of MMP-12 was not detected by western blot analysis, either, in our study, likely because the antibody used recognizes only the hinge region, which does not exist in the activated form of MMP-12. The increased expression of MMP-12 in allergic airway disease is thought to be attributable to upregulated levels of cytokines such as IL-1β, TNF-α and IL-13 which are responsible for the pathological state of airways in asthma patients [23–25].
MMP-12 was detected mainly in bronchial epithelia and alveolar macrophages by the antibody used in the immunohistochemistry. It has been previously reported that MMP-12 is secreted by bronchial epithelial cells and mainly by macrophages . An increased number of MMP-12 positive macrophages was found in the present study after the antigen challenge, and may be responsible in part for the upregulation of MMP-12 protein level as detected by western blot.
It has been previously reported that depletion of MMP-12 can inhibit inflammatory cells’ infiltration of lung tissues in allergic mice . These observations indicate that MMP-12 may be responsible for the inflammatory cell egression in the pathogenesis of allergic airway disease. Although the mechanism of inflammatory cells’ egression into airway lumen is still unclear, it is thought to be regulated in part by inflammatory cell apoptosis, phagocytosis of alveolar macrophage and the integrity of the bronchial epithelium . MMPs, which degrade ECM, play an important role in inflammatory cells’ transmigration due to their proteolysis . MMP-12, also named metalloelastase, has been previously reported to play an important role in the recruitment and migration of inflammatory cells [28, 29]. It has also been previously reported that prednisolone can inhibit accumulation of eosinophil in BALF in the allergen challenge model that was used in the present studies . In the current study, the increased number of eosinophils in BALF after the antigen challenge was also inhibited by S-1 treatment, indicating that MMP-12 may also play an important role in eosinophil recruitment into airway lumen induced by antigen challenge.
Similarly, the increased MMP-9 level in the airways of asthma patients has been reported to be inhibited by glucocorticoids . In our study, the expressions of MMP-9 which increased after antigen challenge were also inhibited by treatment with S-1. The increase in inflammatory cells such as eosinophils, one of the sources of MMP-9, is also thought to be responsible for the increased expression of MMP-9 [31, 32]. In an IL-13-induced airway inflammation model, the expression of MMP-9 was found to be dependent on the expression of MMP-12 . In this study, the effect of a specific inhibitor of MMP-12, S-1, was investigated in mice with allergic airway inflammation. Interestingly, in vivo treatment with S-1 had no effect on in vitro MMP-12 activity measured by zymography (Fig. 4). This observation indicates that the mode of action of S-1 might be a reversible association with MMP-12, and that the protein-drug complex formed in vivo might be dissociated under the in vitro process of zymography, as previously reported . Indeed, N-sulfonylamino acid derivatives were reported to reversibly combine with target MMP(s) ( and unpublished observations of Shionogi & Co., Ltd.). One of the important findings of our current study is that the increased MMP-9 expressions after antigen challenge were suppressed by the in vivo treatment with S-1 (Fig. 4b, c). Because of its selectivity and properties in in vitro experiments as described above, it is likely that S-1 inhibited the activity of MMP-9 indirectly, which resulted from reducing its protein expression. It is thus possible that MMP-9 activity might be modulated by MMP-12 cooperatively in allergic airway inflammation. This is also supported by the result that increased MMP-9 activity in the airways was inhibited by the depletion of MMP-12 in another allergen challenge model .
Elastin represents about 2.5% (wt/wt) of the dry weight of lung and is widely distributed in lung tissue . MMP-12 is able to degrade different substrates, including elastin . In the current murine allergic disease model, the presence of thin linear elastin fiber indicating damage of elastin fiber  in the bronchial subepithelium of challenged animals was also reported as in the cases of patients with asthma and in another allergen challenge model [36, 37]. This histological change of elastin fiber was found in the subepithelium of challenged animals even after S-1 treatment. The increased histological score that indicated the damage of elastin fiber in challenged animals was partly reduced by treatment with S-1. Neutrophil elastase is known to be an elastase which is different from MMP-12 and activated in asthma patients . A possible reason for the nonsignificant effect of S-1 may be that the accumulation of neutrophils in BALF, which produce neutrophil elastase, was not inhibited by treatment with S-1 after antigen challenge (Fig. 3).
In conclusion, MMP-12, which has been previously reported to be involved in the pathogenesis of airway inflammatory diseases, is thought to be a potential target for the treatment of chronic airway inflammatory diseases such as allergic bronchial asthma. In the present study, we demonstrated the upregulation of MMP-12 in the airways of allergen challenged mice and suggest that MMP-12 may play an important role in the eosinophil egression into lung lumen and the upregulation of MMP-9 in the pathogenesis of allergic airway disease.