Sputum matrix metalloproteases: comparison between chronic obstructive pulmonary disease and asthma

Respiratory Medicine (25) 99, 73 71 Sputum matrix metalloproteases: comparison between chronic obstructive pulmonary disease and asthma S.V. Culpitt, D.F. Rogers, S.L. Traves, P.J. Barnes, L.E. Donnelly Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College London, Dovehouse Street, London SW3 6LY, UK Received 11 August 24 KEYWORDS Elastase; MMP; Neutrophil; Tissue inhibitors of metalloprotease; TIMP Summary Asthma and chronic obstructive pulmonary disease () are different conditions with contrasting airway inflammation and parenchymal disease patterns. A number of matrix metalloproteases (MMPs) are implicated in the pathophysiology of and asthma. Different profiles of airway MMPs may, therefore, be expected in asthma and. The present study compared MMP profiles in the airways of nonsmokers, non-symptomatic cigarette smokers, and patients with or asthma (n ¼ 15 subjects per group). Induced sputum was assessed for MMP-1, -2, -3, -8 and - 9, and tissue inhibitor of metalloproteases (TIMP)-1 by ELISA. Gelatinase activity was determined by zymography. Sputum from patients contained increased levels of MMP-1, -8 and -9 compared with the other groups (2 7-fold, depending upon group). MMP-9 activity was elevated in sputum by 3 12-fold above the other groups. Sputum from patients had 3-fold higher levels of TIMP-1 than samples from asthmatics or controls, but was not different to smokers. FEV 1 correlated negatively with MMP-1, -8, -9, MMP-9 activity and TIMP-1, whereas percent neutrophils in sputum correlated positively with MMP-1, -8, -9, TIMP-1 and MMP-9 activity. The MMP profile in differs to that in asthma and cigarette smokers. This may contribute to, or be a marker of, different pathophysiologies of asthma and. & 24 Elsevier Ltd. All rights reserved. Introduction Corresponding author. Tel.: +44 2 7352 8121x361; fax: +44 2 7351 8126. E-mail address: l.donnelly@imperial.ac.uk (L.E. Donnelly). Airway inflammation is a feature common to asthma and chronic obstructive pulmonary disease (). 1,2 However, the profile of the inflammation differs between the two conditions. 3 is a 954-6111/$ - see front matter & 24 Elsevier Ltd. All rights reserved. doi:1.116/j.rmed.24.1.22

74 debilitating respiratory disease characterised by progressive and largely irreversible airflow limitation. 4 Cigarette smoking is the major risk factor for development of, and smoking cessation is currently the only intervention that slows disease progression. 5,6 comprises three disease components, namely chronic bronchitis (mucous hypersecretion), chronic bronchiolitis (also known as small airways disease) and emphysema (alveolar destruction). 7 The contribution of each component to disease status and clinical presentation varies from one patient to another. Endogenous protease activity is implicated in the pathophysiology of each of these three disease components. 8,9 In contrast to, asthma is defined as a clinical entity characterised by variable airflow obstruction and increased airway responsiveness that is reversible, either spontaneously or with treatment. 1 Proteases are also implicated in part in the pathophysiology of asthma. 11,12 In particular, matrix metalloproteinases (MMPs), a large family of proteolytic enzymes that degrade the components of extracellular matrix, 13,14 are implicated in the pulmonary remodelling processes that underlie both asthma and. 15,16 A number of studies have examined the expression of individual MMPs in either asthma or and compared it with healthy control subjects. For example, there is increased expression of the collagenases MMP-1 and -8 in asthmatic airways compared with non-asthmatic controls 17,18 and in the airways of patients with emphysema compared with healthy subjects. 19,2 Similarly, the gelatinases MMP-2 and -9 are upregulated in asthmatic airways compared with healthy controls 21,22 and in the airways of patients with emphysema compared with healthy subjects. 23 In contrast, MMP-3 (stromelysin-1) is not up-regulated in asthma compared with healthy volunteers, 24 with no reports of its expression in the lung in. MMP activity is regulated by endogenous inhibitors termed tissue inhibitors of metalloproteinase (TIMPs), which are also elevated in asthma 25 and. 26 However, to the knowledge of the authors, there has been no direct comparison of airway MMP and TIMP expression between patients with asthma and patients with. The aim of the present study was to compare and contrast the concentrations of MMP-1, -2, -3, -8 and 9, together with TIMP-1, in induced sputum from patients with asthma and patients with. Nonsmoking subjects and non-symptomatic cigarette smokers were included as relevant controls. In addition, the enzymatic activity of MMP-9 was assessed using gelatin zymography. Moreover, glucocorticosteroids down-regulate MMP expression, and upregulate TIMP expression, in asthma. 27 Consequently, we recruited mild asthmatics who were not currently taking glucocorticosteroids, which might confuse interpretation of the data. In addition, they were matched for pharmacotherapy with the patients (bronchodilator therapy alone). Methods Subjects S.V. Culpitt et al. Four groups of subjects were recruited: 15 patients with (cigarette smokers), diagnosed according to current standard criteria, 4 15 patients with asthma (non-smokers), diagnosed according to current standard criteria, including a personal history of asthma, 1 15 current smokers without airways obstruction (FEV 1 48% predicted) and 15 non-smoking control subjects without lung disease (Table 1). Smokers and patients had a smoking history of 42 pack years. Six of the Table 1 Clinical characteristics of control subjects, asthmatic patients, cigarette smokers and patients *. Parameter Controls Asthmatics Non- smokers smokers Age (yr) 3.571.4 34.671.6 47.672.6 # 6.772.6 Sex (F:M) 9:6 9:6 1:5 3:12 FEV 1 % predicted 14.971.7 93.571.1** 1.971.2 45.273.1 # +++ FVC % predicted 14.271.7 12.571.1 1.7.9 93.171.6 ### + FEV 1 :FVC ratio.787.1.747.1.747.1.437.4 #++ Pack years 28.872.5 41.971. ++ *Po.5, **Po.1, Po.1 compared with equivalent value for controls. # Po.5, ### Po.1 compared with equivalent value for asthmatics. + Po.5, ++ Po.1, +++ Po.1 compared with equivalent value for smokers. FEV 1 ¼ forced expiratory volume in 1 s; FVC ¼ forced vital capacity. * Values are mean7sem for 15 subjects in each group. ### ++

Sputum MMPs in and asthma 75 patients could be classified as stage II, eight patients as stage III and one patient as stage IV. 4 None of the current smokers could be classified as stage. The asthmatic patients had bronchial hyperreactivity as evidenced by a PC 2 methacholine of o4 mg/ml. Otherwise, the asthmatic patients were considered to be mild and clinically stable, with no change in treatment for at least 8 weeks prior to study and no more than four short courses of oral corticosteroids (o2 weeks) during the preceding year. Asthmatic subjects maintained their current therapy (i.e. b 2 -agonists alone, with no inhaled or oral glucocorticosteroids for at least 8 weeks prior to the study), as did the patients (i.e. b 2 -agonists and/or anticholinergics only). Neither the asthmatic nor the patients had suffered an exacerbation for at least 8 weeks prior to the study. Smokers were unmedicated. The study was approved by the Ethics Committee of the Royal Brompton and Harefield NHS Trust, and subjects gave informed written consent. Sputum induction Sputum was induced via inhalation of hypertonic saline as previously described, and was processed for differential counts of inflammatory cells. 28 MMP measurements The concentrations of total MMP and TIMP-1 in sputum supernatant were determined using paired antibody quantitative ELISAs and appropriate blanks (Amersham Life Sciences, Little Chalfont, UK). The assay detected both active and latent forms of MMPs. The presence of dithiothreitol (DTT) (.5% w/v) in the sputum samples reduced the sensitivity of the assays. However, this was controlled for by inclusion of DTT in the standards for each ELISA. The lower limit of detection of the assays was 1.8 ng/ml for MMP-1,.4 ng/ml for MMP- 2, 2.4 ng/ml for MMP-3, 32 pg/ml for MMP-8,.6 ng/ml for MMP-9 and 1.3 ng/ml for TIMP-1. Zymography Sputum supernatant was incubated with b-galactosidase (3 i.u. per 1 ml) for 2 h with shaking at 37 1C to partially digest mucopolysaccharides in order to improve resolution of the sputum samples on the zymographic gel. Sputum protein content was measured using the BioRad protein assay reagent according to the manufacturer s instructions (BioRad Ltd., Hemel Hempstead, UK). Two micrograms of sputum protein was diluted with Tris-Glycine sodium dodecyl sulphate (SDS) sample running buffer (.25 M Tris-HCl containing glycerol 2% v/v, 4% w/v SDS and.5% w/v bromophenol blue), and zymography performed as described previously. 29 It should be noted that MMP-2 activity was not detected in any of the samples (see Results). Consequently, gelatinase activity in these samples was due to MMP-9. Standards of.1 mg human recombinant active MMP-9 (Oncogene Research Products, Cambridge, MA, USA) were resolved on each gel to provide a reference for gelatinase activity (MMP-2 and MMP-9) in the samples. Gels were analysed using the Gelworks (UVP Ltd., Cambridge, UK) software system which calculated a measurement of gelatinase activity according to band thickness and density. MMP-9 activity in the samples was expressed as a percentage of the MMP-9 standard for each gel. Statistical analysis Comparisons between subject groups were made using the Kruskal Wallis test followed by Dunn s multiple comparison test, or a Student s t-test where applicable. The significance of the relationship between two variables was assessed using Spearman s rank correlation coefficient. The null hypothesis was rejected at Po:5: Results Clinical characteristics of subjects The current smokers and patients with were older than both the asthmatic patients and the nonsmoking controls (Table 1). The patients with were older than the current smokers and had smoked more cigarettes. None of the current smokers had an FEV 1 :FVC ratio of o.7, indicating that they were not stage 1 subjects according to GOLD criteria. 4 We cannot anticipate whether or not these subjects would in later life develop and, therefore, have specifically not referred to them herein as healthy smokers. Inflammatory cell counts There were no significant differences in the total number of inflammatory cells recovered in sputum from any of the groups (Table 2). Patients with had a significantly reduced percentage macrophage count and an increased percentage neutrophil count compared with any of the other groups (Table 2). Eosinophils were not detected in

76 S.V. Culpitt et al. Table 2 Induced sputum total cell counts and differential cell counts *. Parameter Controls Asthmatics Non- smokers smokers Patient number 15 (9F) 15 (9F) 15 (1F) 15 (3F) Total Inflammatory cells ( 1 6 /ml) 2.37.5 2.17.4 1.97.3 5.271.5 Macrophages (%) 58.371.5 59.172.6 48.171.7 21.571.9 ### + Macrophages ( 1 6 /ml) 1.57.4 1.27.2.97.2.87.2 Neutrophils (%) 4.871.5 36.971.9 51.571.7 ## 77.771.9 ### + Neutrophils ( 1 6 /ml).97.2.87.2.97.1 4.371.3* ## *Po.5, Po.1 compared with equivalent value for controls. ## Po.1, ### Po.1 compared with the equivalent value for asthmatics. + Po.5, compared with the equivalent value for smokers. * Data are mean7sem for 15 subjects in each group. sputum of normal controls, smokers or patients, whereas patients with asthma had a mean % eosinophil count of 2.7% (s.e.m..8%). MMP concentrations MMP-1 was significantly increased in the sputum of patients with, with increases in median values of almost 4-fold above controls, 3-fold above asthmatics and 2-fold above smokers (Fig. 1, panel A). In contrast, there were no significant differences in concentrations of either MMP-2 or -3 between the four subject groups (Fig. 1, panels B and C). MMP-8, however, was increased in the sputum of patients with, with increases in median values of 4-fold above controls, 5-fold above asthmatics and 3-fold above smokers (Fig. 1, panel D). Similarly, MMP-9 was increased in the sputum of patients with, with increases in median values of 8.5-fold above controls, 6.5-fold above asthmatics and 4-fold above smokers (Fig. 1, panel E). In addition, there was an increase of 2- fold in the sputum of smokers above that of controls or asthmatics. TIMP-1 was increased in the sputum of patients with above that in controls and asthmatics (3-fold increase for both subject groups), but with no significant change compared with smokers (Fig. 1, panel F). In addition, TIMP-1 was elevated in the sputum of smokers compared with that of controls or asthmatics (3-fold increase for both). MMP activity Active MMP-9 (86 kda band) was significantly elevated in the sputum of patients with (Fig. 2, panel A), with increases in median values of 12-fold above controls, 5-fold above asthmatics and 3-fold above smokers (Fig. 2, panel B). In addition, MMP-9 activity was increased in the sputum of smokers compared with controls by 4.5-fold. There was no increase in activity in the sputum from the asthmatics (Fig. 2, panel B). MMP- 2 activity was not detected in any of the sputum samples (data not shown). The high molecular weight zones of lysis on the zymography gels corresponded to latent MMP-9. b-galactosidase did not lyse the gels (data not shown). Relationship between MMP and TIMP-1 with lung function and sputum inflammatory cells MMP-8 and -9 concentrations and activity correlated negatively with FEV 1 (Table 3; Fig. 3, panels A and D). Similarly, MMP-1 and TIMP-1 concentrations, but not MMP-2 or MMP-3, also correlated negatively with FEV 1. MMP-8 concentrations and MMP-9 concentrations and activity correlated positively with both the percentage (Table 3; Fig. 3, panels B and E) and number of neutrophils (Table 3). Similarly, MMP-1 and TIMP-1 concentrations, but not MMP-2 or MMP-3, also correlated positively with the percentage and number of neutrophils. MMP-8 concentrations and MMP-9 concentrations and activity correlated negatively with the percentage of macrophages (Table 3; Fig. 3, panels C and F). Similarly MMP-1 and TIMP-1 concentrations, but not MMP-2 or MMP-3, correlated negatively with the percentage of macrophages. No correlations were observed with macrophage numbers. There was also no correlation between MMP-8 levels and FEV 1 or neutrophil count, or between MMP-9 activity and FEV 1 or neutrophil count for any of the individual groups. Similarly, there was no correlation between MMP-9 activity, % neutrophils or % macrophages and age for any of the subject groups. There was also no correlation between age and MMP-8 concentrations for normal controls and patients. However,

Sputum MMPs in and asthma 77 75 * 4 [MMP-1] ng/ml 5 25 [MMP-2] ng/ml 3 2 1 (A) (B) [MMP-3] ng/ml 3 2 1 [MMP-8] ng/ml 9 8 3 2 1 * (C) (D) 2 * 2 [MMP-9] ng/ml 15 1 5 ** ** [TIMP-1] ng/ml 15 1 5 ** ** (E) (F) Figure 1 Concentrations of MMPs and tissue inhibitor of MMP (TIMP)-1 in induced sputum from control subjects, asthmatics, cigarette smokers and patients with. Panel A ¼ MMP-1, panel B ¼ MMP-2, panel C ¼ MMP-3, panel D ¼ MMP-8, panel E ¼ MMP-9 and panel F ¼ TIMP-1. Po:5; Po:1; Po:1: MMP-8 correlated negatively with age in the asthmatic group (r ¼ :57; Po:5) and positively with age in the smokers (r ¼ :58; Po:5). Discussion In the present study, MMP-1, -8, and -9, were increased in sputum from patients with compared with patients with asthma. To our knowledge, this is the first direct demonstration of differences in pulmonary MMP expression between asthma and. The levels of these MMPs in sputum were also increased compared with non-smokers and current cigarette smokers without. This is consistent with observations that these MMPs are elevated in the lungs of patients with. 2,23,3 The increase in concentration of MMP-9 in sputum from patients with may be related to smoking status, since MMP-9 levels were also elevated in the sputum from smokers, or may be due to age differences within our groups. However, it is noteworthy that there was no significant correlation between, for example, smoking history (pack years) and MMP-9 activity (r ¼ :33). Similarly, there was an overall lack of positive correlation between age and the inflammatory markers measured herein, except for MMP-8 in the smokers. Herein, there was no change in MMP-2 or MMP-3 in the sputum of patients. This differs from the observation of increased MMP- 2 levels in lung tissue from patients with emphysema. 31 The reason(s) underlying this discrepancy may relate to sampling differences. The present study used induced sputum whereas the cited study

78 (A) (B) C A A A S S MMP-9 activity (% of standard) 3 * 2 1 C C MMP-9 86 kda Figure 2 MMP-9 activity in induced sputum from control subjects, asthmatics, cigarette smokers and patients with. Panel A shows a representative gelatin zymogram of MMP-9 activity in individual subjects from the different groups: C ¼ controls, A ¼ asthmatic patients, S ¼ smokers and patients. MMP-9 is increased in the smokers and patients with. Panel B ¼ group data. Po:5; Po:1: S.V. Culpitt et al. examined lung tissue. Alternatively, the discrepancy could be related to different study populations since it is reported that MMP-2 activity is detected in induced sputum in only 25% of a cohort of patients. 32 In the present study, TIMP-1 was elevated in patients compared with the other groups. This is consistent with the observation that sputum TIMP-1 levels were enhanced in compared with healthy subjects. 26 Whilst elevated levels of TIMP-1 might be expected to nullify the increased expression of MMP-1 shown herein, TIMP-1 can bind to and inhibit all MMPs. 33 Therefore, the increase in TIMP-1 expression observed in may not be sufficient to counteract the effects of the numerous MMPs that are elevated in, leading to a protease antiprotease imbalance and an enhanced proteolytic environment. In the present study, levels of MMPs or TIMP-1 were not elevated in our asthmatic patients. This is in contrast to certain other studies. 17,18,21,25,32,34,35 The reason(s) for these discrepancies may reflect asthma severity, with our patients being milder than in the studies cited above. Consequently, it is possible that the lungs of our asthmatic patients exhibited less remodelling than those with more severe disease. Part of the remodelling process might be due to the activity of MMPs, which would be less marked in these milder patients. Consistent with this suggestion is the observation that MMP-9 levels are greater in BAL fluid and induced sputum of patients with severe asthma compared with patients with mild or moderate asthma. 34,36 The increase in MMP-9 may reflect an increase in neutrophils that are found in the induced sputum of patients with severe asthma and during exacerbations. 37,38 In the present study, lung function was inversely related to MMP-1, -8,-9 and TIMP-1 levels. This is consistent with a number of other diverse observations. 18,26,36,39 The negative correlation between MMP-1 and FEV 1 in the current study has not been reported previously, although polymorphisms in the MMP-1 and -12 genes, but not the MMP-9 gene, are associated with a rapid decline in lung function in cigarette smokers. 4 This relationship may be due to increased inflammatory cell infiltrate in these conditions, and consistent with the positive correlations observed between neutrophils and MMP-9 in asthmatic or sputum. 26,39,41 The differences in MMP expression between asthmatics and patients with seen herein may reflect chronicity of disease. Our asthmatic patients were relatively mild, whereas our patients were moderate to severe (Table 1). Table 3 Relationship between MMPs and TIMP-1 with lung function and sputum inflammatory cells *. FEV 1 Neutrophils ( 1 6 /ml) Neutrophils (%) Macrophages ( 1 6 /ml) Macrophages (%) MMP-1.42.43.61.6.6 MMP-2.8.12.19.17.15 MMP-3.16.3.3.2.6 MMP-8.51.3*.63.23.57 MMP-9.52.42.76.19.74 MMP-9 activity.66.45.72.18.73 TIMP-1.5.45.78.15.74 * Data are Spearman s rank correlation coefficients, *Po.5, Po.1.

Sputum MMPs in and asthma 79 [MMP-8] ng/ml 1 8 2 1 9 9 8 8 r=-.51 3 r=.63 3 r=-.57 [MMP-8] ng/ml 2 1 [MMP-8] ng/ml 2 1 2 4 6 8 1 12 25 5 75 1 (A) FEV 1 % predicted (B) % Neutrophils (C) 25 5 75 % Macrophages 3 3 3 MMP-9 activity (% control) 2 1 MMP-9 activity (% control) r=-.66 2 r=.72 2 r=-.73 1 MMP-9 activity (% control) 1 2 4 6 8 1 12 25 5 75 1 (D) FEV 1 % predicted (E) % Neutrophils (F) 25 5 75 % Macrophages Figure 3 Relationship between MMP-8 concentrations and MMP-9 activity with lung function and sputum inflammatory cells. Panels A C ¼ MMP-8 vs. FEV 1, % neutrophils and % macrophages respectively; panels D F ¼ MMP-9 vs. FEV 1,% neutrophils and % macrophages, respectively. J ¼ controls, K ¼ asthmatics, & ¼ smokers, ¼ patients. r ¼ Spearman s rank correlation coefficient. However, the two groups were matched for pharmacotherapy (bronchodilator treatment alone). This is relevant because inhaled glucocorticosteroids down-regulate expression of MMP-9 in asthmatics. 27 The lack of differences in any of the MMPs between the normal controls and the asthmatic patients possibly reflects the relative mildness of disease in the asthmatic group. The increased concentrations of MMP-1, -8 and -9 in the patients compared with the asthmatics may reflect the destructive nature of. Although airway remodelling is a feature of asthma, it is not the tissue destruction characteristic of emphysema. Therefore, the enhanced levels of MMP expression, together with MMP-9 activity, reflect the enhanced pulmonary proteolytic load in. In summary, the present study demonstrates increased concentrations of MMP-1, -8, -9 activity and TIMP-1 in induced sputum from patients compared with patients with mild asthma, cigarette smokers or control subjects. Cigarette smokers have increased levels of MMP-9 and TIMP-1 compared with asthmatics and controls, and greater MMP-9 activity than controls. MMP-8 concentrations and MMP-9 activity correlate negatively with FEV 1, and are positively correlated with the percentage and number of neutrophils. In conclusion, the MMP profile of patients differs to that seen in mild asthmatics, and may contribute to, or be a marker of, the different pathophysiologies of asthma and. References 1. Barnes PJ. The role of inflammation and anti-inflammatory medication in asthma. Respir Med 22;96(Suppl. A):S9 15. 2. Rennard SI. Inflammation and repair processes in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999;16:S12 6. 3. Sutherland ER, Martin RJ. Airway inflammation in chronic obstructive pulmonary disease: comparisons with asthma. J Allergy Clin Immunol 23;112:819 27. 4. National Heart Lung and Blood Institute World Health Organisation. Global initiative for chronic obstructive lung disease, 21;271. 5. Scanlon PD, Connett JE, Waller LA, Altose MD, Bailey WC, Buist AS. Smoking cessation and lung function in mild-tomoderate chronic obstructive pulmonary disease. The Lung Health Study. Am J Respir Crit Care Med 2;161:381 9. 6. Culpitt SV, Rogers DF. Evaluation of current pharmacotherapy of chronic obstructive pulmonary disease. Expert Opin Pharmacother 2;1:17 2.

71 S.V. Culpitt et al. 7. Barnes PJ. New concepts in chronic obstructive pulmonary disease. Annu Rev Med 23;54:113 29. 8. Nadel JA. Role of neutrophil elastase in hypersecretion during exacerbations, and proposed therapies. Chest 2;117:386S 9S. 9. Shapiro SD. Proteinases in chronic obstructive pulmonary disease. Biochem Soc Trans 22;3:98 12. 1. National Institutes of Health. Global initiative for asthma: pocket guide for asthma management and prevention, 22. 11. Nadel JA, Takeyama K, Agusti C. Role of neutrophil elastase in hypersecretion in asthma. Eur Respir J 1999;13:19 6. 12. Chiappara G, Gagliardo R, Siena A, Bonsignore MR, Bousquet J, Bonsignore G, Vignola AM. Airway remodelling in the pathogenesis of asthma. Curr Opin Allergy Clin Immunol 21;1:85 93. 13. Ohbayashi H. Matrix metalloproteinases in lung diseases. Curr Protein Pept Sci 22;3:49 21. 14. Donnelly LE, Rogers DF. Antiproteases and retinoids for treatment of. Expert Opin Ther Patents 23;13: 1345 72. 15. Kelly EA, Jarjour NN. Role of matrix metalloproteinases in asthma. Curr Opin Pulm Med 23;9:28 33. 16. Parks WC, Shapiro SD. Matrix metalloproteinases in lung biology. Respir Res 21;2:1 9. 17. Rajah R, Nachajon RV, Collins MH, Hakonarson H, Grunstein MM, Cohen P. Elevated levels of the IGF-binding protein protease MMP-1 in asthmatic airway smooth muscle. Am J Respir Cell Mol Biol 1999;2:199 28. 18. Prikk K, Maisi P, Pirila E, Reintam MA, Salo T, Sorsa T, Sepper R. Airway obstruction correlates with collagenase-2 (MMP-8) expression and activation in bronchial asthma. Lab Invest 22;82:1535 45. 19. Imai K, Dalal SS, Chen ES, Downey R, Schulman LL, Ginsburg M, D Armiento J. Human collagenase (matrix metalloproteinase-1) expression in the lungs of patients with emphysema. Am J Respir Crit Care Med 21;163:786 91. 2. Betsuyaku T, Nishimura M, Takeyabu K, Tanino M, Venge P, Xu S, Kawakami Y. Neutrophil granule proteins in bronchoalveolar lavage fluid from subjects with subclinical emphysema. Am J Respir Crit Care Med 1999;159:1985 91. 21. Maisi P, Prikk K, Sepper R, Pirila E, Salo T, Hietanen J, Sorsa T. Soluble membrane-type 1 matrix metalloproteinase (MT1- MMP) and gelatinase A (MMP-2) in induced sputum and bronchoalveolar lavage fluid of human bronchial asthma and bronchiectasis. APMIS 22;11:771 82. 22. Suzuki R, Kato T, Miyazaki Y, Iwata M, Noda Y, Takagi K, Nakashima N, Torii K. Matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in sputum from patients with bronchial asthma. J Asthma 21;38:477 84. 23. Finlay GA, Russell KJ, McMahon KJ, D Arcy EM, Masterson JB, FitzGerald MX, O Connor CM. Elevated levels of matrix metalloproteinases in bronchoalveolar lavage fluid of emphysematous patients. Thorax 1997;52:52 6. 24. Dahlen B, Shute J, Howarth P. Immunohistochemical localisation of the matrix metalloproteinases MMP-3 and MMP-9 within the airways in asthma. Thorax 1999;54:59 6. 25. Tanaka H, Miyazaki N, Oashi K, Tanaka S, Ohmichi M, Abe S. Sputum matrix metalloproteinase-9: tissue inhibitor of metalloproteinase-1 ratio in acute asthma. J Allergy Clin Immunol 2;15:9 5. 26. Beeh KM, Beier J, Kornmann O, Buhl R. Sputum matrix metalloproteinase-9, tissue inhibitor of metalloprotinease- 1, and their molar ratio in patients with chronic obstructive pulmonary disease, idiopathic pulmonary fibrosis and healthy subjects. Respir Med 23;97:634 9. 27. Hoshino M, Takahashi M, Takai Y, Sim J. Inhaled corticosteroids decrease subepithelial collagen deposition by modulation of the balance between matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in asthma. J Allergy Clin Immunol 1999;14:356 63. 28. Culpitt SV, De Matos C, Russell RE, Donnelly LE, Rogers DF, Barnes PJ. Effect of theophylline on induced sputum inflammatory indices and neutrophil chemotaxis in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 22;165:1371 6. 29. Russell RE, Culpitt SV, DeMatos C, Donnelly L, Smith M, Wiggins J, Barnes PJ. Release and activity of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by alveolar macrophages from patients with chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 22;26:62 9. 3. Imai K, Dalal SS, Chen ES, Downey R, Schulman LL, Ginsburg M, D Armiento J. Human collagenase (matrix metalloproteinase-1) expression in the lungs of patients with emphysema. Am J Respir Crit Care Med 21;163:786 91. 31. Ohnishi K, Takagi M, Kurokawa Y, Satomi S, Konttinen YT. Matrix metalloproteinase-mediated extracellular matrix protein degradation in human pulmonary emphysema. Lab Invest 1998;78:177 87. 32. Cataldo D, Munaut C, Noel A, Frankenne F, Bartsch P, Foidart JM, Louis R. MMP-2- and MMP-9-linked gelatinolytic activity in the sputum from patients with asthma and chronic obstructive pulmonary disease. Int Arch Allergy Immunol 2;123:259 67. 33. Brew K, Dinakarpandian D, Nagase H. Tissue inhibitors of metalloproteinases: evolution, structure and function. Biochim Biophys Acta 2;1477:267 83. 34. Mattos W, Lim S, Russell R, Jatakanon A, Chung KF, Barnes PJ. Matrix metalloproteinase-9 expression in asthma: effect of asthma severity, allergen challenge, and inhaled corticosteroids. Chest 22;122:1543 52. 35. Vignola AM, Riccobono L, Mirabella A, Profita M, Chanez P, Bellia V, Mautino G, D accardi P, Bousquet J, Bonsignore G. Sputum metalloproteinase-9/tissue inhibitor of metalloproteinase-1 ratio correlates with airflow obstruction in asthma and chronic bronchitis. Am J Respir Crit Care Med 1998;158:1945 5. 36. Wenzel SE, Balzar S, Cundall M, Chu HW. Subepithelial basement membrane immunoreactivity for matrix metalloproteinase 9: association with asthma severity, neutrophilic inflammation, and wound repair. J Allergy Clin Immunol 23;111:1345 52. 37. Jatakanon A, Uasuf C, Maziak W, Lim S, Chung KF, Barnes PJ. Neutrophilic inflammation in severe persistent asthma. Am J Respir Crit Care Med 1999;16:1532 9. 38. Fahy JV, Kim KW, Liu J, Boushey HA. Prominent neutrophilic inflammation in sputum from subjects with asthma exacerbation. J Allergy Clin Immunol 1995;95:843 52. 39. Cataldo DD, Bettiol J, Noel A, Bartsch P, Foidart JM, Louis R. Matrix metalloproteinase-9, but not tissue inhibitor of matrix metalloproteinase-1, increases in the sputum from allergic asthmatic patients after allergen challenge. Chest 22;122:1553 9. 4. Joos L, He JQ, Shepherdson MB, Connett JE, Anthonisen NR, Pare PD, Sandford AJ. The role of matrix metalloproteinase polymorphisms in the rate of decline in lung function. Hum Mol Genet 22;11:569 76. 41. Kelly EA, Busse WW, Jarjour NN. Increased matrix metalloproteinase-9 in the airway after allergen challenge. Am J Respir Crit Care Med 2;162:1157 61.