Pulmonary emphysema is believed to result from an imbalance between proteolytic enzymes and their inhibitors. Multiple studies have examined the presence of various proteases within the bronchoalveolar lavage fluid from patients with chronic obstructive pulmonary disease (COPD). However, to date extensive examination of the lung parenchyma for the expression of destructive enzymes has not yet been determined. The following study examines the lung parenchyma of 23 patients with emphysema and 8 normal control samples for the expression of matrix matalloproteinase-1 (MMP-1), MMP-12, and MMP-9. We report here that interstitial collagenase (MMP-1) RNA, protein, and activity are present in the lung parenchyma of patients with emphysema and not in the lung of normal control subjects. In contrast, metalloelastase (MMP-12) expression is absent in these samples. Immunohistochemistry studies localized MMP-1 to the Type II pneumocyte in patients with emphysema and not normal control subjects or smokers without emphysema. This observation demonstrates that the lung is altered in emphysema such that the Type II pneumocyte secretes MMP-1 and suggests that MMP-1 may be an important enzyme involved in the destruction of the lung in the human disease. In addition, the induction of a proteolytic enzyme within the Type II pneumocyte suggests that the cells within the lung itself are capable of producing degradative enzymes in this disease process.
Chronic obstructive pulmonary disease (COPD), which includes emphysema and chronic bronchitis, is the fourth leading cause of death in the United States (1). Approximately 15 million Americans are affected by COPD, with smoking the major risk factor, accounting for more than 90% of cases seen worldwide (1). Despite the importance of the disease, there are no specific therapies available to limit or prevent the slow, progressive, destructive changes observed in COPD (2). Currently the major hypothesis for the pathogenesis of emphysema is the protease–antiprotease theory (3). This hypothesis states that an imbalance between the levels of degradative enzymes and their respective inhibitors damages the connective tissue matrix components of the lung. The major focus has concentrated on neutrophil elastase as the primary destructive protease, because of the experimental induction of emphysema through the intratracheal instillation of pancreatic and leukocyte elastase (4) and the identification of the early development of emphysema in α1-antitrypsin deficiency (5).
Support for the involvement of elastolytic enzymes in emphysema came from studies performed on a metalloelastase (matrix metalloproteinase-12, or MMP-12) knockout mouse (6). These mice are normal at baseline but when exposed to cigarette smoke at the human equivalent of 2,000 cigarettes per day, they do not develop emphysematous changes in the lung as do wild-type animals (6). Although these data suggest that the lack of the MMP-12 enzyme may reduce the susceptibility of the animals to emphysema, there is no direct evidence demonstrating that excess elastolytic activity of MMP-12 is critically involved in human emphysema formation. In addition, investigators examined human alveolar macrophages from patients with emphysema and normal volunteers and did not demonstrate increased levels of MMP-12 within the lavage fluid of the patients with emphysema (7).
Despite the reservations about the role of elastase or metalloelastase in emphysema, the evidence for other degradative enzymes and their substrates is even weaker. For example, when bacterial collagenase was injected into the lungs of rats, no emphysematous lesions developed (8). However, there are some correlative studies suggesting that collagenase, a matrix metalloproteinase that degrades the fibrillar collagens, may be involved in a number of lung diseases including respiratory disorders (9, 10). Furthermore, there are a number of observations suggesting that collagen is degraded or damaged in pulmonary emphysema. For example, antibodies to collagen have been found in the serum of patients with emphysema (11). In the inherited disorder Type VI Ehlers–Danlos syndrome, there is a decreased structural integrity of collagen fibrils and emphysematous changes were reported in these patients (12). It has also been shown that collagen is affected in the various emphysema animal models. In papain- induced emphysema, dissolution of collagen fibrils is seen shortly after instillation of the enzyme (13). Similarly, collagen is rapidly degraded in elastase-induced emphysema (14). Finally, short exposure to toxic amounts of oxygen in rats will lead to emphysematous changes and collagen degradation with no changes in elastin (15).
We targeted the human collagenase gene (MMP-1) to the lung and demonstrated that the animals developed emphysematous changes in their lung (16). The pathology seen in the MMP-1 transgenic mouse lungs is striking in its similarity to the morphological changes observed in human emphysema. A number of studies have reinforced the role of collagenase in emphysema. When Wright and Churg exposed guinea pigs to smoke, they were able to demonstrate that the collagen matrix was affected (17). Follow-up studies by Selman and coworkers (18) demonstrated that smoke exposure led to increased expression of interstitial collagenase and increased activity within the lung. Immunohistochemical studies demonstrate that the collagenase expression is present in the alveolar macrophages, alveolar lining cells and fibroblasts (18) of the guinea pigs after smoke exposure. Most recently, investigators have examined alveolar macrophages from 10 patients with emphysema and 10 normal volunteers and measured protease activity and mRNA levels (7). In this study, Finlay and coworkers (7) demonstrated that there were elevated levels of gelatinase B and interstitial collagenase mRNA in the macrophages of smokers. In contrast, the mRNA for metalloelastase was not detected. In addition, the investigators demonstrated an increase in collagenase activity in the macrophages from the patients with emphysema as compared with the normal subjects (7). The following study was undertaken to examine carefully the protease expression within the lung parenchyma itself, so as to better understand the role of these enzymes in the disease process.
Human lung samples were obtained from 1993 to 1997 at Columbia Presbyterian Medical Center (New York, NY; IRB X042 1) from a total of 37 patients. Lung samples were collected and placed in one of four catagories (emphysema; normal; α1-antitrypsin deficiency emphysema; and normal smokers) as follows:
Emphysema. The 23 patients with emphysema ranged in age from 35 to 71 yr, with a mean age of 59 ± 10.0 yr with 18 females (78%) and 5 males (22%). Five samples were obtained from recipient lungs during transplant; 16 samples were obtained during lung volume reduction procedures, and 2 samples were obtained from lungs harvested for transplantation but rejected because of emphysematous changes. All samples, except EM2 and EM18, were taken from patients who reportedly stopped smoking for at least 3 mo. The FEV1/FVC ratio in the patients ranged from 0.23 to 0.52, with the Dl CO (difussing capacity of lung for CO) ranging from 7 to 92% of predicted values.
Normal. Normal tissue was obtained from eight patients. The normal patients ranged in age from 19 to 55 yr with a mean of 32 ± 13.9 yr, with three females and five males. Four samples were obtained from donor lungs harvested for transplant but not used because of recipient complications, one was obtained from normal tissue after resection of a benign nodule, and three were obtained from accidental death victims.
α1-Antitrypsin deficiency emphysema. The three α1-antitrypsin deficiency patients consisted of two females and one male with an average age of 40 ± 1.2 yr. All were undergoing lung transplantation for end-stage emphysema.
Normal smokers. The final specimens were obtained from two patients who were long-term smokers but had normal lungs by morphometry. Both patients had undergone lung resection for removal of a neoplasm and tissue from the normal area was analyzed by immunohistochemistry in this study. The mRNA was not obtained from these specimens because the samples were first analyzed by pathology.
All the lung samples from the first three groups were prepared in the following manner. A 2 × 2 cm piece of lung was analyzed immediately after removal from the patient. When whole lungs were obtained from transplant recipients, several pieces from each lung were isolated and analyzed to determine reproducibility of the findings. Ninety percent of the first 20 tissue samples was used for RNA isolation (EM1– 15, EM17, EM18, EM21, N2, N4–8, A1, and A3) while the remaining portion was examined histologically. Subsequently, for the remaining eight samples (EM16, EM19, EM20, EM22, EM23, N1, N3, and A2) 5% was placed in 4% paraformaldehyde for histology, 25% was homogenized to prepare protein, and 70% was used to isolate total RNA. All the samples were examined in a blinded fashion for the presence of emphysema, fibrosis, and infection. All the patients with samples that had pathological evidence of inflammation indicative of ongoing infection or neoplastic changes were excluded from this study.
Total RNA was prepared from fresh tissue, using the guanidinium thiocyanate–cesium chloride method as previously described (16). The total RNA was finally resuspended in diethyl pyrocarbonate-treated water and the absorbance was read at A260 and A280. The quality of the RNA was checked on a 1.2% denaturing agarose gel to ensure the presence of the 28S and 18S ribosomal bands.
The primers were designed from exons 9 (5′-AGC ACA TGA CTT TCC TGG AAT TGG C-3′) and 10 (5′-ATT TTG TGT TAG AAG AGT TAT CC-3′) of the human collagenase 1 gene (MMP-1) (19). A polymerase chain reaction (PCR) product of 819 bp is expected from genomic DNA and a product of 619 bp is expected from RNA or cDNA. For the detection of the human metalloelastase mRNA (MMP-12), primers were designed from exons 9 (5′-ATG ATG AAA GGA GAC AGA TGA TGG-3′) and 10 (5′-ACA ACC AAA CCA GCT ATT GC-3′) (20). A genomic PCR product of 1.2 kb and a cDNA product of 200 bp are expected. As a positive control, mRNA was detected in cultured macrophages from the bronchoalveolar lavage fluid of a patient with acute rejection posttransplantation. For the detection of gelatinase B mRNA (MMP-9), primers were designed from exons 9 (5′-GGC AGG ACC GTC TCT ACT GGC GCG T-3′) and 10 (5′-CAG AAC AGA ATA CCA GTT TGT ATC-3′) (21). A cDNA product of 200 bp is expected. As a negative control, mRNA from mouse peritoneal macrophages was used.
The reverse transcription (RT) reaction was performed as previously described (22). Briefly, 1 μg of total RNA, primers (300 ng/μl each), 200 units of Superscript I (GIBCO-BRL, Gaithersburg, MD), and 0.4 mM deoxyribonucleic acid nucleotides from Pharmacia (Piscataway, NJ) in the presence of 1× first-strand buffer (GIBCO-BRL; 0.50 M Tris-HCl [pH 8.3], 0.075 M KCl, 3 mM MgCl2). The reaction was carried out at 37° C for 1 h. PCR amplification was performed with 2 μl of the cDNA, and 30 cycles under the following conditions: 1 min at 50° C, 1 min at 72° C, and 1 min at 94° C in a thermal DNA cycler machine (Hybaid, Franklin, MA). Ten microliters of the reaction product was then analyzed on a 1.0% agarose gel.
The Biotrak human MMP-1 enzyme-linked immunosorbent assay (ELISA) system (Amersham, Arlington Heights, IL) was used and the assay was performed according to the manufacturer protocol. Fresh human lung tissue was homogenized in TNC buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 10 mM CaCl2, 0.05% Brij 35, 0.02% NaN3), using a Polytron homogenizer. The homogenate was centrifuged to sediment any particulate matter. The samples were concentrated on Centricon-30 columns (Amicon, Danvers, MA) to an approximate volume of 1–3 ml. The total protein concentration was determined by the standard bicinchoninic acid (BCA) method (Pierce, Rockford, IL).
For subsequent protein assays, equal amounts of total protein were used. Before the collagen degradation assay, human lung homogenate in TNC buffer was treated with 1.5 mM p-aminophenylmercuric acetate (APMA) for 16 h at 37° C to activate latent MMP-1. Twenty micrograms of each sample was incubated with 14C-labeled Type I collagen (rat tail) in a total volume of 20 μl at 27° C for 16 h. The digestion products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 9% total acrylamide).
Briefly, a 600-bp human MMP-1 cDNA fragment from exons 9 and 10 was subcloned into pBluescript SK(+) (Stratagene, La Jolla, CA), linearized and used as a template to synthesize a single-strand antisense RNA probe. The RNA probe was labeled with digoxigenin according to the instructions of the manufacturer (Boehringer Mannheim, Mannheim, Germany). Tissue sections fixed in 4% paraformaldehyde were incubated in the hybridization buffer containing RNA probe at 5 ng/ml overnight at 42° C and washed under high-stringency conditions. Expression was visualized with a digoxigenin (Dig) detection kit (Boehringer Mannheim).
Tissue sections (3 μm) fixed in 10% neutral buffered formalin were subjected to both single labeling with an MMP-1-specific antibody and double-labeling procedures by sequential avidin–biotin–immunoalkaline phosphate and silver-enhanced immunogold (IGSS) reactions as previously described (23). When performing single-labeling experiments a specific MMP-1 monoclonal antibody (clone 41-E5; 1 μg/ml) was incubated with sections overnight followed by avidin–biotin complex (ABC) detection developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB; Sigma, St. Louis, MO). In the double-labeling experiments, labeling of human interstitial collagenase immunostaining was first performed with a specific monoclonal antibody (clone 41-E5; 0.02 μg/ml ), using the indirect IGSS procedure. Subsequently, rabbit IgG against human surfactant protein A (SP-A, 10 μg/ml; gift of F. McCormack, Cincinnatti, OH) was reacted and visualized by the immunoalkaline phosphate procedure. For negative controls, primary antibodies were replaced with either rabbit serum or nonimmune mouse IgG1 at matched protein concentrations. All immunohistochemical preparations were counterstained with hematoxylin.
Data were analyzed by the SAS (Statistical Analysis System, SAS, Cary, NC) computer program for Microsoft Windows version 6.12. The data listings, descriptive statistics, and frequency distributions were generated by SAS programming. The rates for collagenase protein expression were compared by the Fisher exact test.
The major etiological factor for emphysema in these patients was cigarette smoking. All the patients with emphysema demonstrated physiological parameters of functional lung deficits through changes in the FEV1/FVC ratio and Dl CO. Included in the study were three patients with α1-antitrypsin deficiency, a genetic disease leading to an increased incidence of emphysema, presumably because of the diminished activity of the inhibitor α1-antitrypsin (5).
RT-PCR analysis was performed for the presence of MMP-1, MMP-12, and gelatinase B (MMP-9). These molecules are secreted by macrophages present during lung injury and are involved in the degradation of the extracellular matrix of the lung (25). MMP-1 mRNA was detected in all 23 of the tissue samples from patients with emphysema by RT-PCR analysis, but in none of the lung samples of normal subjects (Figure 1). MMP-1 expression was present in one of three patients with α1-antitrypsin deficiency (Figure 1). In contrast to these findings, MMP-12 was found in only 1 of 23 samples from smoking-induced emphysema and in none of the α1-antitrypsin deficiency samples or normal control samples (Figure 2a). When the lung samples were analyzed for the presence of MMP-9, the enzyme was found in all analyzed emphysema samples (22 of 22) but was also present in the control samples (8 of 8) (Figure 2b). Quantitative RNase protection assays confirmed specific expression of MMP-1 mRNA in 22 of 23 samples but demonstrated no correlation between clinical severity of emphysema based on FEV1 and the mRNA levels (data not shown). In conclusion, when analyzed by RT-PCR and RNase protection assays, MMP-1 expression was detected in 22 of 23 (96%) of the patients with emphysema and in none of the normal subjects. This difference in rates was significant (p < 0.001).
Because emphysema is believed to result from an imbalance in proteases and their inhibitors, it was important to analyze the lung tissue for the presence of a major inhibitor of MMP-1, tissue inhibitor of metalloproteinase 1 (TIMP-1). Through Northern blot analysis, the TIMP-1 mRNA was found to be present in both normal and emphysema samples without a consistent correlation of levels with either group (data not shown). To correlate RNA expression with protein, an ELISA was performed on the tissue homogenate from the identical human lung samples assayed for RNA. MMP-1 protein was detected in the emphysema samples but not in the normal samples (Table 1). Collagenase activity was detectable only in the emphysema samples through a collagen degradation assay (data not shown).
|Human Sample||Protein (ng/ml )|
|N1 (normal)||< 1|
|N3 (normal)||< 1|
Although emphysema is believed to be due to an increase in elastase activity within the lung, a molecular analysis of the lung parenchyma from patients with emphysema has never been performed. Two major sources of elastolytic activity believed to be involved in emphysema development are MMP-12 in macrophages and neutrophil elastase (26). Neutrophil elastase mRNA is present only in bone marrow myelocytic precursor cells (27). As these cells differentiate and enter the circulation, neutrophil elastase transcripts disappear and the protein is packaged into secretory granules (27). Therefore, it was important to assay the lung tissue for neutrophil elastase activity. The samples in this study were analyzed for elastase activity using elastase substrate I from Calbiochem (La Jolla, CA) as per the manufacturer instructions and no significant activity was detected (data not shown). These results do not mean that elastin degradation by neutrophil elastase is not important in the development of emphysema, as this activity may be localized to closely adherant inflammatory cells during migration.
The enhanced MMP-1 expression in the emphysema samples in this study was not due to an inflammatory cell response because tissue specimens were obtained from clinically stable patients, and if MMP-1 expression were due to an increase in macrophages within the lung, MMP-12 expression would have been detected. However, because the latter was not found, it was important to determine the exact cell type expressing MMP-1. In situ hybridization and immunohistochemistry studies were undertaken to identify the cell type responsible for MMP-1 expression within the emphysematous lung. Surprisingly, MMP-1 mRNA was expressed in alveolar lining cells within the lungs of the patients with emphysema (Figure 3A). The normal lung alveolar lining cells did not react with the probe (Figure 3B). No significant reaction was obtained with the sense probe (data not shown). As an internal control, the resident macrophages in both the normal and emphysematous lungs stained positively.
The results were confirmed at the protein level through immunohistochemical staining (28). MMP-1 was immunolocalized to the alveolar lining cells in the emphysematous lung (Figure 3C). As in the in situ hybridization experiment the resident macrophages stained in both the normal and emphysematous lung (Figures 3C and 3D). To identify the exact cell type expressing MMP-1, double immunostaining was performed with an antibody against the surfactant A-1 protein (SP-A1) (28). The SP-A1-positive cells also reacted with the anti-MMP-1 antibody, indicating that the expression of MMP-1 was in the Type II pneumocytes in the lungs of the patients with emphysema (Figure 3C, inset). The Type II pneumocyte reacted with the anti-surfactant antibody but not with the anti-MMP-1 antibody in the lungs of the normal subjects (Figure 3D, inset).
To identify whether MMP-1 expression in the pneumocyte was specific to the emphysematous lung or could also be seen in a smoker's lung, two samples from patients who were smokers with normal lung tissue were examined for the presence of MMP-1 protein in the alveolar lining cells (Figure 4). Although occasional macrophage positive cells were seen to be stained in these samples (Figure 4C), the alveolar pneumocytes in these samples did not express MMP-1 protein (Figures 4A–4C).
The results presented in this study directly demonstrate MMP-1 expression in the Type II pneumocyte of patients with emphysema and provide a potential role for this enzyme in the pathogenesis of the disease. In this study, 90% of the patients with emphysema had stopped smoking at least 3 mo before the collection of tissue samples and yet demonstrated expression of MMP-1 within the lung parenchyma. Our findings demonstrate that there are long-term changes in protease expression in the lungs of patients with emphysema, which may play a role in the pathogenesis of disease after smoking cessation.
The human studies described are correlative in nature and cannot address causality. However, pertinent to this point are the transgenic mouse experiment (16), in which the animals developed emphysema directly as a result of increased MMP-1 in the lung. Thus, we know that constant expression of MMP-1 in the lung is detrimental to the lung structure. Furthermore, the levels of mRNA and protein seen in the human tissue are equivalent to or greater than that observed in the lungs of the mouse model (data not shown). Consequently, the levels of enzyme present in the human tissue should be sufficient to destroy the lung parenchyma.
The inflammatory mediators that initiate and sustain proteolytic injury to the lung remain undefined. It is apparent from multiple studies that inflammatory cells are recruited into the lung after smoke exposure (29). However, it is not clear whether this initial inflammatory reaction is harmful or whether the resultant upregulation of other proteolytic enzymes is the ultimate cause of the lung destruction. Hence, future studies will be required to identify and characterize the factors that can upregulate MMP-1 expression in the lungs of patients with emphysema.
In this study, the expression of a number of proteolytic enzymes that degrade the extracellular matrix were analyzed and no expression was found for elastase or MMP-12. As expected, MMP-9 expression was present in the normal lung samples (30) and also in the emphysematous lung samples. Absence of measurable elastase or MMP-12 does not exclude a pathogenic role for these enzymes, particularly early in the course of the disease (25). Studies have shown that smoking leads to an acute increase in neutrophil elastase-derived fibrinopeptides in the serum of patients (31). Most, but not all, patients in our study had stopped smoking before surgery. As we have examined tissue samples from patients with late-stage disease, other proteases may well be involved in the destruction of the parenchyma in earlier stages of the disease.
The high prevalence of females in this study contrasts with the higher rates of COPD in men shown in most series. The patients in our study may therefore represent a pathologically distinct subpopulation of patients with severe, early onset COPD as has been previously reported (32). Nevertheless the predominance in females does not necessarily invalidate the conclusions of our study as the results do not differ between males and females. Our normal control subjects are younger than the patients with emphysema; however, we do not believe that MMP-1 expression is related to age, as one of the normal samples came from a subject who was 63 yr of age and two of the patients with emphysema were under 45 yr of age. Emphysema normally affects patients in the fifth to sixth decade of life. As with all human studies it is difficult to obtain normal samples in this age range. Moreover, our data are consistent within all of the patients with emphysema and normal subjects studied regardless of age.
Previous studies have identified a variety of protease activities in the bronchoalveolar lavage fluid of smokers (7, 33). This lavage fluid is enriched with inflammatory cells during cigarette smoking and may not faithfully represent the milieu within the lung parenchyma (34). This is the first direct demonstration that the lung parenchyma is altered in emphysema such that a degradative enzyme is produced by the Type II pneumocyte. In humans the lung parenchyma is modified during the time period of smoking. We hypothesize that this modification could induce MMP-1 expression in the parenchyma of the lungs of patients predisposed to emphysema, and after patients have ceased smoking the pneumocyte may continue to express the enzyme within the lung. Therefore, even after they are no longer exposed to smoke, and the macrophage population diminishes, the pneumocytes are altered and constitutively express the enzyme such that chronic tissue destruction may continue. The lack of MMP-1 protein expression in the pneumocytes of the lung from smokers without emphysema strengthens the argument that MMP-1 is playing a role in the pathogenesis of the disease.
The present data suggest that MMP-1 is associated with the pathogenesis of human emphysema even after smoking has ceased. The combination of this study and the demonstration of lung destruction in transgenic animals through the overexpression of collagenase (MMP-1) (16) leads to the hypothesis that the inhibition of this enzyme may be a suitable target in the treatment of the disease. Controlled trials of metalloproteinase inhibitors in emphysema can further support this hypothesis and provide a novel therapeutic intervention in the treatment of this disease.
The authors thank Dr. Sudhir N. Dalal for the statistical analyses, and Drs. Kiran Chada, Alan Tall, Yale Enson, and Randolph Cole for critical reading of the manuscript. Antibodies against human MMP-1 were a kind gift from Dr. Yasunori Okada and Dr. Iwata. Antibodies against surfactant A protein were a kind gift of Dr. Francis McCormack.
|1.||Centers for Disease Control and Preevention. Mortality patterns—United States, JAMA 1993;270:2916-2917.|
|2.||Snider GLChronic obstructive pulmonary disease: risk factors, pathophysiology and pathogenesis. Annu Rev Med401989411429|
|3.||Tetley TDProteinase imbalance: its role in lung disease. Thorax481993560565|
|4.||Janoff A, Sloan B, Weinbaum G, Damiano V, Sandhaus RA, Elias J, Kimbel PExperimental emphysema induced with purified human neutrophil elastase: tissue localization of the instilled protease. Am Rev Respir Dis1151977461478|
|5.||Laurell CB, Erickson SThe electrophoretic α1-globin pattern of serum in α1-antitrypsin deficiency. Scand J Clin Lab Invest151963132140|
|6.||Hautamaki RD, Kobayashi DK, Senior R, Shapiro SDRequirement for macrophage elastase for cigarette smoke-induced emphysema in mice. Science277199720022004|
|7.||Finlay GA, O'Driscoll L, Russell KJ, D'Arcy EM, Masterson JB, Fitzgerald MX, O'Connor CMatrix metalloproteinase expression and production by alveolar macrophages in emphysema. Am J Respir Crit Care Med1561997240247|
|8.||Johanson WG, Pierce AKEffects of elastase, collagenase, and papain on structure and function of rat lungs in vitro. J Clin Invest511972288293|
|9.||Chrystal RG, Gadek JE, Ferrans VJ, Fulmer JD, Line BR, Hunninghake GWInterstitial lung disease: current concepts of pathogenesis staging and therapy. Am J Med701981542560|
|10.||Gadek J, Kelman J, Fells G, Weinberger S, Horwitz A, Reynolds H, Fulmer J, Crystal RCollagenase in the lower repiratory tract of patients with idiopathic pulmonary fibrosis. N Engl J Med3011979737742|
|11.||Micheali D, Fudenberg HHAntibodies to collagen in patients with emphysema. Clin Immunol Immunopathol31974187192|
|12.||Pinnell SR, Krane SM, Kenzora JE, Glimcher MJA heritable disorder of connective tissue, hydroxylysine-deficient collagen disease. N Engl J Med286197210131020|
|13.||Kilburn KH, Dowell AR, Pratt PCMorphological and biochemical assessment of papain-induced emphysema. Arch Intern Med1271971884890|
|14.||Yu SY, Keller NRSynthesis of lung collagen in hamsters with elastase-induced emphysema. Exp Mol Pathol2919783743|
|15.||Riley DJ, Kramer MJ, Kerr JS, Chae CU, Yu SY, Berg RADamage and repair of lung connective tissue in rats exposed to toxic levels of oxygen. Am Rev Respir Dis1351987441447|
|16.||D'Armiento J, Dalal SS, Berg RA, Chada KCollagenase expression in the lungs of transgenic mice causes pulmonary emphysema. Cell711992955961|
|17.||Wright JL, Churg ASmoke induced emphysema in guinea pigs is associated with morphometric evidence of collagen breakdown and repair. Am J Physiol2681995L17|
|18.||Selman M, Montano M, Ramos C, Vanda B, Becerril C, Delgado J, Sansores R, Pardo ATobacco smoke-induced lung emphysema in guinea pigs is associated with increased intersitial collagenase. Am J Physiol2711996L734L743|
|19.||Goldberg G, Wilhelm S, Kronberger A, Bauer EA, Grant GA, Eisen AZHuman fibroblast collagenase. J Biol Chem261198666006605|
|20.||Shapiro SD, Kobayashi DK, Ley TJCloning and characterization of a unique elastolytic metalloproteinase produced by human alveolar macrophages. J Biol Chem26819932382423829|
|21.||Collier IE, Wilhelm SM, Eisen AZH-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen. J Biol Chem263198865796587|
|22.||D'Armiento J, Dalal SS, Chada KTissue, temporal and inducible expression pattern of haptoglobin in mice. Gene19519971927|
|23.||Ross R, Masuda J, Raines EWLocalization of PDGF-B protein in macrophages in all phases of atherogenesis. Science248199010091012|
|24.||Zhang J, Fujimoto N, Iwata K, Sakai T, Okada Y, Hayakawa TAA one-step sandwich enzyme immunoassay for human matrix metalloproteinase-1 (interstitial collagenase) using monoclonal antibodies. Clin Chim Acta2191993114|
|25.||Shapiro SDThe pathogenesis of emphysema: the elastase:antielastase hypothesis 30 years later. Proc Assoc Am Physicians1071995346352|
|26.||Janoff AJElastases and emphysema. Current assessment of the protease-antiprotease hypothesis. Am Rev Respir Dis1321985417433|
|27.||Takahashi S, Barry AC, Factor SMCollagen degradation in ischaemic rat hearts. Biochem J2651990233241|
|28.||Katsuda S, Coltrera MD, Ross R, Gown AMHuman atherosclerosis: IV. Immunohistochemical analysis of cell activation and proliferation in lesions of young adults. Am J Pathol142199317871793|
|29.||Hogg JC. Structure and function of small airways. Chest 1980;77(Suppl): 279–282.|
|30.||Tokuraku M, Sato H, Murakami S, Okada Y, Watanabe Y, Seiki MActivation of the precursor of gelatinase A/72 kDa Type IV collagenase/ MMP-2 in lung carcinomas correlates with the expression of membrane-type matrix metalloproteinase (MT-MMP) and with lymph node metastasis. Int J Cancer641995355359|
|31.||Weitz JI, Crowley KA, Landman SL, Lipman BI, Yu JIncreased neutrophil elastase activity in cigarette smokers. Ann Intern Med1071987680682|
|32.||Silverman EK, Chapman HA, Drazen JM, Weiss ST, Rosner B, Campbell EJ, O'Donnell WJ, Reilly JJ, Ginns L, Mentzer S, Wain J, Speizer FEGenetic epidemiology of severe, early-onset chronic obstructive pulmonary disease: risk to relatives for airflow obstruction and chronic bronchitis. Am J Respir Crit Care Med157199817701778|
|33.||Tetley TD, Smith SF, Winning AJ, Foxall JM, Cooke NT, Burton GHThe acute effect of cigarette smoking on the neutrophil elastase inhibitory capacity of peripheral lung lavage from asymptomatic volunteers. Eur Respir J21989802810|
|34.||Merchant RK, Schwartz DA, Helmers RA, Dayton CS, Hunninghake GWBronchoalveolar lavage cellularity. Am Rev Respir Dis1461992488453|
Jeanine D'Armiento is a recipient of a Burroughs Wellcome Fund Career Award in the Biomedical Sciences. Kazushi Imai is a postdoctoral fellow from the Japan Society for the Promotion of Science for Research Abroad.
Present address for R.D.: Division of Thoracic Surgery, Memorial Sloan Kettering, New York, NY 10021.