Differential Proteolytic Enzyme Activity in Eosinophilic and Neutrophilic Asthma
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美国呼吸和危急护理医学 2005年第9期
School of Medical Practice and Population Health
Medical Genetics, School of Biomedical Science, Hunter Medical Research Institute, The University of Newcastle, Callaghan
Department of Respiratory and Sleep Medicine
Immunology and Infectious Diseases, Hunter Medical Research Institute, John Hunter Hospital, New Lambton, Australia
ABSTRACT
Rationale: Asthma is characterized by both chronic inflammation and remodeling of the airways. Proteases are important mediators of inflammation, cytokine activation, and tissue remodeling. Objectives: This study investigated matrix metalloproteinase-9 (MMP-9) and neutrophil elastase (NE) enzyme activity in the sputum of subjects with different inflammatory phenotypes of asthma (eosinophilic, neutrophilic, and paucigranulocytic asthma) and in healthy control subjects. Methods and Measurements: Nonsmoking adults with asthma and healthy control sujects underwent hypertonic saline challenge and sputum induction. Selected sputum portions were dispersed with dithiothreitol and assayed for MMP-9 and NE enzyme activity. Main Results: Subjects with eosinophilic asthma had significantly more active MMP-9 (39 ng/ml) compared with those with neutrophilic asthma (10 ng/ml) and control subjects (2.5 ng/ml, p < 0.01). Although there were high levels of total MMP-9 in neutrophilic asthma (5,273 ng/ml), most (> 99%) was inactivated (and bound to tissue inhibitor of metalloproteinase-1). In neutrophilic asthma, more subjects had NE activity (39%) compared with both healthy control subjects (0%), subjects with eosinophilic asthma (6%), or subjects with paucigranulocytic asthma (0%, p < 0.05). There were strong and consistent positive correlations between interleukin-8, neutrophils, and proteolytic enzymes. MMP-9 was inversely correlated with NE (r = eC0.93). Conclusions: Proteolytic enzyme activity in asthma is dependent on the underlying inflammatory phenotype and is differentially regulated with MMP-9 activity a feature of eosinophilic inflammation, and active NE in neutrophilic inflammation.
Key Words: inflammation peptide hydrolases sputum
Proteolytic enzymes play an important role in tissue remodeling and repair in the airways. Levels of proteolytic enzymes including active neutrophil elastase (NE) (1) and matrix metalloproteinase-9 (MMP-9) (2) are increased in asthma, indicating an imbalance in the protease/antiprotease system. The role of proteolytic enzymes in specific inflammatory phenotypes of asthma is unknown. However, interleukin-8 (IL-8), a potent chemoattractant and activator of neutrophils, is increased in noneosinophilic asthma (3) and may be involved in the release of proteolytic enzymes into the airways. NE can induce production of IL-8 (4), and MMP-9 can increase IL-8 potency by augmenting aminoterminal processing of IL-8 (5).
MMP-9 (Gelatinase-B) is a member of a family of zinc-containing enzymes that degrade extracellular matrix and modulate cytokine activity and the activity of other proteases (6). MMP-9 has been identified in several inflammatory cells such as bronchial epithelial cells, neutrophils, mast cells, eosinophils, and macrophages (6, 7). MMP-9 is released as a proenzyme, which can then be activated, by a number of mechanisms including other MMPs (8), bacterial proteases (9), reactive oxygen species (10), and neutrophil proteins such as lipocalin (11). Tissue inhibitor of metalloproteinase (TIMP-1) is the major tissue inhibitor secreted in association with MMP-9 and binds with both the pro- and active forms of MMP-9, causing its inactivation. Levels of MMP-9 are increased in asthma compared with those seen in healthy control subjects (12), and also in severe asthma compared with mild asthma (13, 14).
NE is a serine protease, which can attack a number of proteins including lung elastin. The presence of free NE indicates a protease/antiprotease imbalance, which may play a role in the pathogenesis of chronic obstructive pulmonary disease and also in asthma. The key endogenous inhibitor of NE is 1-antitrypsin (1AT), and when inhibition is deficient or the enzyme load exceeds the inhibitor's capacity to inactivate, proteolytic lung degradation occurs (15). NE is increased in asthma, with levels inversely correlated to FEV1 predicted (1).
Secretory leukocyte protease inhibitor (SLPI) is a broad-spectrum inhibitor of mast cell and leukocyte serine proteases, and is produced by epithelial cells (16) and submucosal glands (17). Levels of SLPI are increased in subjects with pneumonia (18) and also in the peripheral airways of subjects with emphysema (19), but are decreased in chronic diseases such as diffuse panbronchiolitis (20). 1AT is a serine protease inhibitor produced by hepatocytes and is expressed by neutrophils (21), epithelial cells (22), and macrophages (23). Both 1AT and SLPI are involved in the counterbalance of NE activity. Airway levels of 1AT are increased in the sputum of subjects with asthma (1) and are related to the duration of disease and lung function (24).
The inflammatory process in asthma is heterogeneous (3), with noneosinophilic forms of asthma described, which are clinically indistinguishable from asthma with eosinophilic inflammation (3, 25eC27). The relevance of the IL-8/proteolytic enzyme mechanisms in different asthma phenotypes is not known. In this study we investigated the fluid phase levels of two important proteolytic enzymes, MMP-9 and NE, as well as the potent neutrophil chemoattractant IL-8 in subjects with different inflammatory phenotypes of asthma. We examined subjects with asthma who had elevated neutrophils (neutrophilic asthma), those with asthma who had elevated eosinophils (eosinophilic asthma), and those with asthma and normal cell counts (paucigranulocytic asthma), as well as healthy control subjects. We hypothesized that subjects with neutrophilic asthma would have increased proteolytic enzyme activation compared with other asthma phenotypes and healthy control subjects as indicated by increased levels of active NE and increased MMP-9.
Some of the results of this study have been previously reported in the form of abstracts (28, 29).
METHODS
Subjects
Stable symptomatic asthma (n = 93) was defined using ATS criteria (30) including a clinical diagnosis of asthma and airway hyperresponsiveness. Healthy subjects (n = 42) had no diagnosis of respiratory disease, an FEV1 greater than 80% predicted (31), and normal airway responsiveness. Current smokers were excluded.
Subjects gave written informed consent and underwent clinical assessment, spirometry, a combined hypertonic saline challenge, and sputum induction. The Hunter Area Health Service and University of Newcastle Research Ethics Committees approved this study.
The inflammatory phenotypes of the subjects with asthma were classified using induced sputum cell counts (Table 1). The upper limit of normal for sputum cell counts was taken as the 95th percentile of the normal population studied. Consequently, a sputum eosinophil percentage greater than or equal to 1% and a sputum neutrophil percentage greater than or equal to 61% were considered to be elevated. Subjects who had sputum neutrophils greater than or equal to 61% were classified as Neutrophilic Asthma (n = 26), subjects with sputum eosinophils greater than or equal to 1% were classified as Eosinophilic Asthma (n = 38), and the subjects with sputum neutrophils less than 61% and sputum eosinophils less than 1% were classified as Paucigranulocytic Asthma (n = 29).
Sputum induction.
Spirometry (Minato Autospiro AS-600; Minato Medical Science Co Ltd., Osaka, Japan) and combined bronchial provocation testing and sputum induction with hypertonic saline were performed as previously described (32).
Sputum analysis.
Sputum was selected from saliva and processed as described (32). The sample was dispersed with dithiothreitol, filtered, and assessments of total leucocyte count and viability were performed. The cell suspension was centrifuged and supernatant aspirated for storage at eC70°C. Cytospins were prepared, stained (May-Grewald-Giemsa), and a differential cell count obtained from 400 nonsquamous cells. The proportion of cells containing intracellular bacteria was determined (see online supplement).
Fluid phase mediator assessment.
The concentrations of MMP-9, TIMP-1, MMP-9 bound to TIMP-1, 1AT, SLPI, and IL-8 were determined by ELISA (R&D Systems, Minneapolis, MN). The validity of these mediator measurements in induced sputum processed using dithiothreitol is reported in the online supplement. Active NE was determined using a chromogenic substrate specific for human NE, n-methoxysuccinyl-l-alanyl-l-alanyl-prolyl-l-valyl-p-nitroanilide (Sigma, St. Louis, MO). Additional detail is provided in the online supplement.
Gelatin zymography.
Gelatinolytic activity in sputum supernatant was determined by zymography, and further detail can be found in the online supplement.
MMP-9 immunocytochemistry.
Intracellular MMP-9 was detected in sputum cytospins (APAAP technique) with an anti-human MMP-9 antibody (R&D Systems) as described previously (33) and in the online supplement.
Analysis.
Data were analyzed using Stata 7 (Stata Corporation, College Station, TX). Results are reported as median and interquartile range unless otherwise indicated. Analysis was performed using the two-sample Wilcoxon Rank Sum, Kruskal-Wallis test for more than two groups, and Fisher's exact test for categorical data. Associations between data were determined using the Spearman rank correlation. Results were reported as significant when p < 0.05.
RESULTS
Total MMP-9 levels (active and proeCMMP-9) in induced sputum from subjects with asthma varied depending on the inflammatory phenotype (Figure 1a). In neutrophilic asthma there were very high levels of total MMP-9 (5,723 ng/ml). However, active MMP-9 levels were low, representing only 0.4% of total MMP-9 (Figure 1b). In eosinophilic asthma there was significantly more active MMP-9 (39 ng/ml) than in neutrophilic asthma (10 ng/ml, p < 0.004), and 16% of total MMP-9 was active (Figure 1b). Total MMP-9 levels in eosinophilic asthma were similar to those in healthy control subjects, although active MMP-9 was significantly elevated (p < 0.004) above control values. In subjects with asthma and a normal cellular profile (paucigranulocytic asthma), the active MMP-9 results did not differ from healthy control subjects. However, total MMP-9 levels were elevated above control values (p < 0.004). Gelatinolytic activity was confirmed in the sputum of subjects with paucigranulocytic asthma and eosinophilic asthma (as represented by a band in the 88-kD region). Subjects with neutrophilic asthma and healthy control subjects had weaker bands in the 92-kD region, indicating the pro-form of the enzyme. A band in the region of 125 and 220 kD was apparent in subjects with eosinophilic and paucigranulocytic asthma, which may indicate the presence of high molecular weight forms and homodimeric MMP-9 (Figure 2) (34). MMP-9 was present in 7% of sputum cells, and was predominantly found in neutrophils and macrophages.
Active NE was detected in 39% of sputum samples from subjects with neutrophilic asthma, compared with 6% of subjects with eosinophilic asthma (p < 0.004, Figure 3). No samples were positive for active NE in the paucigranulocytic asthma group or in healthy control subjects. Concentrations of active elastase tended to be higher in subjects with neutrophilic asthma (107 e/ml) compared with those found in eosinophilic asthma (56 e/ml). However, these levels were not statistically significant (p = 0.3) due to the small numbers with active elastase. Levels of active MMP-9 showed a significant negative relationship with levels of active NE measured in sputum supernatant (Figure 4; r = eC0.9, p = 0.0001).
IL-8 levels were increased in subjects with asthma compared with healthy control subjects. Subjects with neutrophilic asthma had a median (IQR) of 15.4 ng/ml (8.3eC52.9) for IL-8, significantly more IL-8 than subjects with eosinophilic (3.1 [1.7eC9.9]) or paucigranulocytic (5.2 [2.1eC13.3]) asthma. Levels of fluid phase IL-8 were significantly correlated with total MMP-9 (r = 0.8, p < 0.0001) and sputum neutrophil percentage (r = 0.5, p < 0.0001). Sputum neutrophils also correlated with total MMP-9 (r = 0.7, p < 0.0001) levels among subjects with asthma. Sputum eosinophil levels showed no correlation with IL-8 or the proteolytic enzymes.
Subjects with neutrophilic asthma had more MMP-9 bound to TIMP-1 in the sputum supernatant compared with subjects with eosinophilic and paucigranulocytic asthma and with healthy control subjects (Figure 1c). TIMP-1 levels were significantly increased in subjects with neutrophilic (462 e/ml) asthma compared with subjects with paucigranulocytic asthma (40 e/ml) and healthy control subjects (11 e/ml), but were not statistically different from subjects with eosinophilic asthma (109 e/ml).
SLPI levels were significantly increased in subjects with asthma (17.6 [9.7eC28.9] e/ml) compared with healthy control subjects (12.1 [9.1eC29.3] e/ml; p = 0.02). However, subjects with neutrophilic asthma (20.1 [6.5eC28.1] e/ml) had SLPI levels similar to those of subjects with eosinophilic (15.1 [9.7eC25] e/ml) and paucigranulocytic asthma (22 [10.9eC34.5] e/ml).
1AT levels were significantly increased in subjects with neutrophilic asthma (49.3 [19.6eC61.4] e/ml) compared with subjects with eosinophilic (19.7 [9.9eC39.7] e/ml) and paucigranulocytic asthma (12.1 [9.1eC29.3] e/ml; p < 0.01). Subjects with asthma had significantly elevated levels of 1AT compared with healthy control subjects (20.6 e/ml vs. 3.8 e/ml, p < 0.0001).
DISCUSSION
This study showed that the activity levels of two important proteolytic enzymes, MMP-9 and NE, varied according to the inflammatory cell phenotype in the sputum of subjects with stable asthma (Figure 5). Active NE was a feature of asthma with neutrophilic inflammation. In contrast, eosinophilic asthma was characterized by active MMP-9 without free elastase, and there was an inverse relationship between these two important enzymes in asthma. These results highlight the heterogeneity of the inflammatory response in asthma and indicate differential regulation of protease activity in asthma. This may be due to ineffective antiprotease defenses. In neutrophilic asthma, total MMP-9 levels were very high; however, over 99% of this MMP-9 was inactive and there were high levels of MMP-9/TIMP-1 complex. In contrast, in eosinophilic asthma there were high levels of active MMP-9 and normal levels of MMP-9/TIMP complex. In neutrophilic asthma there were high levels of active NE, and although the inhibitors SLPI and 1AT were increased in asthma overall, only 1AT was preferentially increased in neutrophilic asthma.
MMP-9 is a proteolytic enzyme released from inflammatory cells and capable of degrading extracellular matrix. Neutrophils synthesize MMP-9 during the latter stages of differentiation, storing it in granules that are released upon neutrophil activation. A relationship between total MMP-9 and neutrophils was confirmed in this study and has been demonstrated previously in both asthma and chronic bronchitis (2).
Few studies have examined active MMP-9 levels in asthma. This study was the first to examine active MMP-9 in inflammatory phenotypes in asthma. In this study there was gelatinolytic activity present in sputum supernatant from subjects with eosinophilic and paucigranulocytic asthma, with low levels found in healthy control subjects. However, subjects with neutrophilic asthma showed no gelatinolytic activity in the 88-kD region. These results are consistent with the active MMP-9 results assessed using ELISA.
A deficiency of antiproteases may explain the differential enzyme activity observed. TIMP-1 is a potent inhibitor of MMP-9. There were increased levels of MMP-9 bound to TIMP-1 in subjects with neutrophilic asthma compared with eosinophilic asthma. This explains the low level of active MMP-9 enzyme present. TIMP-1 levels in neutrophilic asthma were 42 times above control values and sufficient to neutralize MMP-9 activity. TIMP-1 levels in eosinophilic asthma were only nine times higher than in healthy control subjects, and the antiprotease response to MMP-9 was therefore insufficient in eosinophilic asthma, where high levels of active MMP-9 were found. TIMP-1 levels in subjects with neutrophilic asthma (median 462 e/ml) were four times higher than those found in subjects with eosinophilic asthma (median 109 e/ml). This relative deficiency in TIMP-1 in subjects with eosinophilic asthma may explain the high proportion of active MMP-9 present in the sputum of subjects in this group.
In neutrophilic asthma, the antiprotease response to NE was also relatively deficient. Two antiproteases, 1AT and SLPI were examined in this study. Whereas 1AT levels were increased in neutrophilic asthma, SLPI levels were not increased leading to a relative excess of active NE. Although 1AT was increased in neutrophilic asthma, its functioning may be impaired, leading to free elastase activity. 1AT can be proteolytically inactivated to a form that is both a potent neutrophil chemoattractant and an activator of neutrophils, resulting in degranulation and superoxide generation (35). These mechanisms require further investigation in asthma.
The source of free NE in subjects with neutrophilic asthma is unclear. NE is released from activated neutrophils. When primed, neutrophils release less than 2% of NE into the extracellular space, but can transfer up to 12% to the cell membrane through granule bursts (36). We found very high cell viability, especially in the subjects with neutrophilic asthma, indicating the presence of high numbers of intact live cells. The role of neutrophil necrosis rather than apoptosis in asthma is unclear. In our study of virus-induced asthma we found high levels of lactate dehydrogenase (LDH) in sputum supernatant, indicating increased cell lysis (33). However, in a recent study of apoptosis and necrosis in bronchiectasis, similar levels of necrotic and apoptotic granulocytes were observed (37). Impairments of macrophage phagocytosis are thought to lead to secondary necrosis of neutrophils and therefore release of NE. Thus increased neutrophil turnover could be a source of free NE.
There was a strong inverse relationship between active MMP-9 and active NE, indicating that high levels of NE are present when levels of active MMP-9 are low. Conversely, NE levels were positively correlated with total MMP-9 levels, which suggests that NE may play a role in the inactivation of MMP-9 or prevention of its activation. Activated neutrophil products contribute to total MMP-9 levels through inactivation of TIMP-1. NE can inactivate TIMP-1 bound to proeCMMP-9 without destruction or activation of MMP-9 (38). This would result in an increase in the proeCMMP-9, as is observed in subjects with neutrophilic asthma. In addition, TIMP-1 can be inactivated by the oxidant HOCl released by neutrophil activation (39). The mechanisms behind this relationship are unclear and require further investigation.
These results demonstrate that there is proteolytic enzyme release in asthma. Subjects with neutrophilic asthma are characterized by the presence of active NE, whereas subjects with eosinophilic asthma were characterized by the presence of active MMP-9. The mechanisms for excess enzyme activity appeared to be an inadequate inhibitor response.
In healthy subjects there was a small amount of active MMP-9 present, consistent with the ongoing ECM turnover that occurs in tissues. Interestingly, no active NE was present in normal control subjects, despite neutrophils being present in the airway. This indicates either no neutrophil activation in healthy subjects, or effective antielastase mechanisms to control any elastase leak from airway neutrophils. Low levels of both SLPI and 1AT were detectable in the sputum supernatant from healthy control subjects.
IL-8 is a key chemokine leading to neutrophil infiltration in the lung, and there may be a positive feedback loop operating in neutrophilic asthma, leading to chronic inflammation. When cultured neutrophils are treated with IL-8, MMP-9 is released rapidly (40). Consistent with this, we found a close association between levels of IL-8 and MMP-9 in asthma. Recently it has been demonstrated that IL-8 is a substrate for activated MMP-9, and the resultant MMP-9eCmodified IL-8 molecule was 10- to 27-fold more potent in neutrophil activation (5). The correlations demonstrated in this study are consistent with a positive feedback cycle where IL-8 induces neutrophil influx and activation, which in turn releases MMP-9 that may cleave IL-8 to a more potent form. This may explain the intense neutrophil influx in these subjects. One of the more interesting and novel observations of this study was the correlation between neutrophils and inflammatory mediators. IL-8 levels were correlated with sputum neutrophils, and there were strong positive relationships among subjects with asthma between sputum IL-8 and total MMP-9, and between IL-8 and NE. The correlation observed between sputum neutrophils and IL-8 is not unexpected due the production of IL-8 by the respiratory epithelium and also by airway neutrophils themselves. The link between IL-8, NE, and MMP-9 is consistent with the positive feedback between these mediators, where NE can induce IL-8 production (4). These correlations support a proinflammatory mechanism linking IL-8 influx and neutrophil degranulation and consequent release of NE and MMP-9, thus supporting a chronic inflammatory and remodeling process.
Although a role for neutrophilic inflammation has been established in severe asthma (41) and exacerbations of asthma (42), neutrophils are not always considered important in the pathogenesis of stable asthma. Recently we (3) and others (25) have identified subjects with asthma in whom neutrophils are the dominant inflammatory cell in the sputum. These subjects form part of a larger group of subjects with noneosinophilic asthma. The mechanisms behind the symptoms and airway hyperresponsiveness in these subjects are unknown; however, neutrophils and their products may play an important role. It has been hypothesized that noneosinophilic asthma is driven by persistent innate immune activation (43). IL-8 is one of the proinflammatory cytokines released in response to innate immune receptor activation and signaling (44). The high IL-8 levels observed in neutrophilic asthma indicate potential activation of the innate immune system. Our data also reinforce the role of IL-8eCmediated neutrophil influx and proteolytic enzyme release as a relevant mechanism in noneosinophilic neutrophilic asthma. Future work will define the clinical consequences of these effects.
All studies were undertaken using induced sputum; however, changes in the airway lumen might be different from those occurring in the tissue. Induced sputum is thought to reflect events that occur in the large conducting airways. We believe there is a need for further examination of these mechanisms in airway tissue, as recent evidence suggests increased expression of MMP-9 in the sub-basement membrane of subjects with asthma (45). However, inflammatory processes occurring in the airway lumen are important and may influence subsequent changes in the tissue, and therefore study of these processes are both important and relevant.
In conclusion, there is differential expression of proteolytic enzyme activity in subjects with asthma. Neutrophilic asthma is accompanied by active NE, whereas active MMP-9 characterizes eosinophilic asthma. These effects seem to occur because antiprotease activity is inadequate. The results suggest an important role of proteolytic enzyme activity in asthma and highlight the potential for antiprotease therapy in asthma.
Acknowledgments
The authors thank Mrs. Ruth Roxby, Ms. Majella Maher, Ms. Naomi Timmins, Mrs. Glenda Walker, Ms. Rebecca Oldham, and Mrs. Kellie Fakes for their technical assistance.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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Medical Genetics, School of Biomedical Science, Hunter Medical Research Institute, The University of Newcastle, Callaghan
Department of Respiratory and Sleep Medicine
Immunology and Infectious Diseases, Hunter Medical Research Institute, John Hunter Hospital, New Lambton, Australia
ABSTRACT
Rationale: Asthma is characterized by both chronic inflammation and remodeling of the airways. Proteases are important mediators of inflammation, cytokine activation, and tissue remodeling. Objectives: This study investigated matrix metalloproteinase-9 (MMP-9) and neutrophil elastase (NE) enzyme activity in the sputum of subjects with different inflammatory phenotypes of asthma (eosinophilic, neutrophilic, and paucigranulocytic asthma) and in healthy control subjects. Methods and Measurements: Nonsmoking adults with asthma and healthy control sujects underwent hypertonic saline challenge and sputum induction. Selected sputum portions were dispersed with dithiothreitol and assayed for MMP-9 and NE enzyme activity. Main Results: Subjects with eosinophilic asthma had significantly more active MMP-9 (39 ng/ml) compared with those with neutrophilic asthma (10 ng/ml) and control subjects (2.5 ng/ml, p < 0.01). Although there were high levels of total MMP-9 in neutrophilic asthma (5,273 ng/ml), most (> 99%) was inactivated (and bound to tissue inhibitor of metalloproteinase-1). In neutrophilic asthma, more subjects had NE activity (39%) compared with both healthy control subjects (0%), subjects with eosinophilic asthma (6%), or subjects with paucigranulocytic asthma (0%, p < 0.05). There were strong and consistent positive correlations between interleukin-8, neutrophils, and proteolytic enzymes. MMP-9 was inversely correlated with NE (r = eC0.93). Conclusions: Proteolytic enzyme activity in asthma is dependent on the underlying inflammatory phenotype and is differentially regulated with MMP-9 activity a feature of eosinophilic inflammation, and active NE in neutrophilic inflammation.
Key Words: inflammation peptide hydrolases sputum
Proteolytic enzymes play an important role in tissue remodeling and repair in the airways. Levels of proteolytic enzymes including active neutrophil elastase (NE) (1) and matrix metalloproteinase-9 (MMP-9) (2) are increased in asthma, indicating an imbalance in the protease/antiprotease system. The role of proteolytic enzymes in specific inflammatory phenotypes of asthma is unknown. However, interleukin-8 (IL-8), a potent chemoattractant and activator of neutrophils, is increased in noneosinophilic asthma (3) and may be involved in the release of proteolytic enzymes into the airways. NE can induce production of IL-8 (4), and MMP-9 can increase IL-8 potency by augmenting aminoterminal processing of IL-8 (5).
MMP-9 (Gelatinase-B) is a member of a family of zinc-containing enzymes that degrade extracellular matrix and modulate cytokine activity and the activity of other proteases (6). MMP-9 has been identified in several inflammatory cells such as bronchial epithelial cells, neutrophils, mast cells, eosinophils, and macrophages (6, 7). MMP-9 is released as a proenzyme, which can then be activated, by a number of mechanisms including other MMPs (8), bacterial proteases (9), reactive oxygen species (10), and neutrophil proteins such as lipocalin (11). Tissue inhibitor of metalloproteinase (TIMP-1) is the major tissue inhibitor secreted in association with MMP-9 and binds with both the pro- and active forms of MMP-9, causing its inactivation. Levels of MMP-9 are increased in asthma compared with those seen in healthy control subjects (12), and also in severe asthma compared with mild asthma (13, 14).
NE is a serine protease, which can attack a number of proteins including lung elastin. The presence of free NE indicates a protease/antiprotease imbalance, which may play a role in the pathogenesis of chronic obstructive pulmonary disease and also in asthma. The key endogenous inhibitor of NE is 1-antitrypsin (1AT), and when inhibition is deficient or the enzyme load exceeds the inhibitor's capacity to inactivate, proteolytic lung degradation occurs (15). NE is increased in asthma, with levels inversely correlated to FEV1 predicted (1).
Secretory leukocyte protease inhibitor (SLPI) is a broad-spectrum inhibitor of mast cell and leukocyte serine proteases, and is produced by epithelial cells (16) and submucosal glands (17). Levels of SLPI are increased in subjects with pneumonia (18) and also in the peripheral airways of subjects with emphysema (19), but are decreased in chronic diseases such as diffuse panbronchiolitis (20). 1AT is a serine protease inhibitor produced by hepatocytes and is expressed by neutrophils (21), epithelial cells (22), and macrophages (23). Both 1AT and SLPI are involved in the counterbalance of NE activity. Airway levels of 1AT are increased in the sputum of subjects with asthma (1) and are related to the duration of disease and lung function (24).
The inflammatory process in asthma is heterogeneous (3), with noneosinophilic forms of asthma described, which are clinically indistinguishable from asthma with eosinophilic inflammation (3, 25eC27). The relevance of the IL-8/proteolytic enzyme mechanisms in different asthma phenotypes is not known. In this study we investigated the fluid phase levels of two important proteolytic enzymes, MMP-9 and NE, as well as the potent neutrophil chemoattractant IL-8 in subjects with different inflammatory phenotypes of asthma. We examined subjects with asthma who had elevated neutrophils (neutrophilic asthma), those with asthma who had elevated eosinophils (eosinophilic asthma), and those with asthma and normal cell counts (paucigranulocytic asthma), as well as healthy control subjects. We hypothesized that subjects with neutrophilic asthma would have increased proteolytic enzyme activation compared with other asthma phenotypes and healthy control subjects as indicated by increased levels of active NE and increased MMP-9.
Some of the results of this study have been previously reported in the form of abstracts (28, 29).
METHODS
Subjects
Stable symptomatic asthma (n = 93) was defined using ATS criteria (30) including a clinical diagnosis of asthma and airway hyperresponsiveness. Healthy subjects (n = 42) had no diagnosis of respiratory disease, an FEV1 greater than 80% predicted (31), and normal airway responsiveness. Current smokers were excluded.
Subjects gave written informed consent and underwent clinical assessment, spirometry, a combined hypertonic saline challenge, and sputum induction. The Hunter Area Health Service and University of Newcastle Research Ethics Committees approved this study.
The inflammatory phenotypes of the subjects with asthma were classified using induced sputum cell counts (Table 1). The upper limit of normal for sputum cell counts was taken as the 95th percentile of the normal population studied. Consequently, a sputum eosinophil percentage greater than or equal to 1% and a sputum neutrophil percentage greater than or equal to 61% were considered to be elevated. Subjects who had sputum neutrophils greater than or equal to 61% were classified as Neutrophilic Asthma (n = 26), subjects with sputum eosinophils greater than or equal to 1% were classified as Eosinophilic Asthma (n = 38), and the subjects with sputum neutrophils less than 61% and sputum eosinophils less than 1% were classified as Paucigranulocytic Asthma (n = 29).
Sputum induction.
Spirometry (Minato Autospiro AS-600; Minato Medical Science Co Ltd., Osaka, Japan) and combined bronchial provocation testing and sputum induction with hypertonic saline were performed as previously described (32).
Sputum analysis.
Sputum was selected from saliva and processed as described (32). The sample was dispersed with dithiothreitol, filtered, and assessments of total leucocyte count and viability were performed. The cell suspension was centrifuged and supernatant aspirated for storage at eC70°C. Cytospins were prepared, stained (May-Grewald-Giemsa), and a differential cell count obtained from 400 nonsquamous cells. The proportion of cells containing intracellular bacteria was determined (see online supplement).
Fluid phase mediator assessment.
The concentrations of MMP-9, TIMP-1, MMP-9 bound to TIMP-1, 1AT, SLPI, and IL-8 were determined by ELISA (R&D Systems, Minneapolis, MN). The validity of these mediator measurements in induced sputum processed using dithiothreitol is reported in the online supplement. Active NE was determined using a chromogenic substrate specific for human NE, n-methoxysuccinyl-l-alanyl-l-alanyl-prolyl-l-valyl-p-nitroanilide (Sigma, St. Louis, MO). Additional detail is provided in the online supplement.
Gelatin zymography.
Gelatinolytic activity in sputum supernatant was determined by zymography, and further detail can be found in the online supplement.
MMP-9 immunocytochemistry.
Intracellular MMP-9 was detected in sputum cytospins (APAAP technique) with an anti-human MMP-9 antibody (R&D Systems) as described previously (33) and in the online supplement.
Analysis.
Data were analyzed using Stata 7 (Stata Corporation, College Station, TX). Results are reported as median and interquartile range unless otherwise indicated. Analysis was performed using the two-sample Wilcoxon Rank Sum, Kruskal-Wallis test for more than two groups, and Fisher's exact test for categorical data. Associations between data were determined using the Spearman rank correlation. Results were reported as significant when p < 0.05.
RESULTS
Total MMP-9 levels (active and proeCMMP-9) in induced sputum from subjects with asthma varied depending on the inflammatory phenotype (Figure 1a). In neutrophilic asthma there were very high levels of total MMP-9 (5,723 ng/ml). However, active MMP-9 levels were low, representing only 0.4% of total MMP-9 (Figure 1b). In eosinophilic asthma there was significantly more active MMP-9 (39 ng/ml) than in neutrophilic asthma (10 ng/ml, p < 0.004), and 16% of total MMP-9 was active (Figure 1b). Total MMP-9 levels in eosinophilic asthma were similar to those in healthy control subjects, although active MMP-9 was significantly elevated (p < 0.004) above control values. In subjects with asthma and a normal cellular profile (paucigranulocytic asthma), the active MMP-9 results did not differ from healthy control subjects. However, total MMP-9 levels were elevated above control values (p < 0.004). Gelatinolytic activity was confirmed in the sputum of subjects with paucigranulocytic asthma and eosinophilic asthma (as represented by a band in the 88-kD region). Subjects with neutrophilic asthma and healthy control subjects had weaker bands in the 92-kD region, indicating the pro-form of the enzyme. A band in the region of 125 and 220 kD was apparent in subjects with eosinophilic and paucigranulocytic asthma, which may indicate the presence of high molecular weight forms and homodimeric MMP-9 (Figure 2) (34). MMP-9 was present in 7% of sputum cells, and was predominantly found in neutrophils and macrophages.
Active NE was detected in 39% of sputum samples from subjects with neutrophilic asthma, compared with 6% of subjects with eosinophilic asthma (p < 0.004, Figure 3). No samples were positive for active NE in the paucigranulocytic asthma group or in healthy control subjects. Concentrations of active elastase tended to be higher in subjects with neutrophilic asthma (107 e/ml) compared with those found in eosinophilic asthma (56 e/ml). However, these levels were not statistically significant (p = 0.3) due to the small numbers with active elastase. Levels of active MMP-9 showed a significant negative relationship with levels of active NE measured in sputum supernatant (Figure 4; r = eC0.9, p = 0.0001).
IL-8 levels were increased in subjects with asthma compared with healthy control subjects. Subjects with neutrophilic asthma had a median (IQR) of 15.4 ng/ml (8.3eC52.9) for IL-8, significantly more IL-8 than subjects with eosinophilic (3.1 [1.7eC9.9]) or paucigranulocytic (5.2 [2.1eC13.3]) asthma. Levels of fluid phase IL-8 were significantly correlated with total MMP-9 (r = 0.8, p < 0.0001) and sputum neutrophil percentage (r = 0.5, p < 0.0001). Sputum neutrophils also correlated with total MMP-9 (r = 0.7, p < 0.0001) levels among subjects with asthma. Sputum eosinophil levels showed no correlation with IL-8 or the proteolytic enzymes.
Subjects with neutrophilic asthma had more MMP-9 bound to TIMP-1 in the sputum supernatant compared with subjects with eosinophilic and paucigranulocytic asthma and with healthy control subjects (Figure 1c). TIMP-1 levels were significantly increased in subjects with neutrophilic (462 e/ml) asthma compared with subjects with paucigranulocytic asthma (40 e/ml) and healthy control subjects (11 e/ml), but were not statistically different from subjects with eosinophilic asthma (109 e/ml).
SLPI levels were significantly increased in subjects with asthma (17.6 [9.7eC28.9] e/ml) compared with healthy control subjects (12.1 [9.1eC29.3] e/ml; p = 0.02). However, subjects with neutrophilic asthma (20.1 [6.5eC28.1] e/ml) had SLPI levels similar to those of subjects with eosinophilic (15.1 [9.7eC25] e/ml) and paucigranulocytic asthma (22 [10.9eC34.5] e/ml).
1AT levels were significantly increased in subjects with neutrophilic asthma (49.3 [19.6eC61.4] e/ml) compared with subjects with eosinophilic (19.7 [9.9eC39.7] e/ml) and paucigranulocytic asthma (12.1 [9.1eC29.3] e/ml; p < 0.01). Subjects with asthma had significantly elevated levels of 1AT compared with healthy control subjects (20.6 e/ml vs. 3.8 e/ml, p < 0.0001).
DISCUSSION
This study showed that the activity levels of two important proteolytic enzymes, MMP-9 and NE, varied according to the inflammatory cell phenotype in the sputum of subjects with stable asthma (Figure 5). Active NE was a feature of asthma with neutrophilic inflammation. In contrast, eosinophilic asthma was characterized by active MMP-9 without free elastase, and there was an inverse relationship between these two important enzymes in asthma. These results highlight the heterogeneity of the inflammatory response in asthma and indicate differential regulation of protease activity in asthma. This may be due to ineffective antiprotease defenses. In neutrophilic asthma, total MMP-9 levels were very high; however, over 99% of this MMP-9 was inactive and there were high levels of MMP-9/TIMP-1 complex. In contrast, in eosinophilic asthma there were high levels of active MMP-9 and normal levels of MMP-9/TIMP complex. In neutrophilic asthma there were high levels of active NE, and although the inhibitors SLPI and 1AT were increased in asthma overall, only 1AT was preferentially increased in neutrophilic asthma.
MMP-9 is a proteolytic enzyme released from inflammatory cells and capable of degrading extracellular matrix. Neutrophils synthesize MMP-9 during the latter stages of differentiation, storing it in granules that are released upon neutrophil activation. A relationship between total MMP-9 and neutrophils was confirmed in this study and has been demonstrated previously in both asthma and chronic bronchitis (2).
Few studies have examined active MMP-9 levels in asthma. This study was the first to examine active MMP-9 in inflammatory phenotypes in asthma. In this study there was gelatinolytic activity present in sputum supernatant from subjects with eosinophilic and paucigranulocytic asthma, with low levels found in healthy control subjects. However, subjects with neutrophilic asthma showed no gelatinolytic activity in the 88-kD region. These results are consistent with the active MMP-9 results assessed using ELISA.
A deficiency of antiproteases may explain the differential enzyme activity observed. TIMP-1 is a potent inhibitor of MMP-9. There were increased levels of MMP-9 bound to TIMP-1 in subjects with neutrophilic asthma compared with eosinophilic asthma. This explains the low level of active MMP-9 enzyme present. TIMP-1 levels in neutrophilic asthma were 42 times above control values and sufficient to neutralize MMP-9 activity. TIMP-1 levels in eosinophilic asthma were only nine times higher than in healthy control subjects, and the antiprotease response to MMP-9 was therefore insufficient in eosinophilic asthma, where high levels of active MMP-9 were found. TIMP-1 levels in subjects with neutrophilic asthma (median 462 e/ml) were four times higher than those found in subjects with eosinophilic asthma (median 109 e/ml). This relative deficiency in TIMP-1 in subjects with eosinophilic asthma may explain the high proportion of active MMP-9 present in the sputum of subjects in this group.
In neutrophilic asthma, the antiprotease response to NE was also relatively deficient. Two antiproteases, 1AT and SLPI were examined in this study. Whereas 1AT levels were increased in neutrophilic asthma, SLPI levels were not increased leading to a relative excess of active NE. Although 1AT was increased in neutrophilic asthma, its functioning may be impaired, leading to free elastase activity. 1AT can be proteolytically inactivated to a form that is both a potent neutrophil chemoattractant and an activator of neutrophils, resulting in degranulation and superoxide generation (35). These mechanisms require further investigation in asthma.
The source of free NE in subjects with neutrophilic asthma is unclear. NE is released from activated neutrophils. When primed, neutrophils release less than 2% of NE into the extracellular space, but can transfer up to 12% to the cell membrane through granule bursts (36). We found very high cell viability, especially in the subjects with neutrophilic asthma, indicating the presence of high numbers of intact live cells. The role of neutrophil necrosis rather than apoptosis in asthma is unclear. In our study of virus-induced asthma we found high levels of lactate dehydrogenase (LDH) in sputum supernatant, indicating increased cell lysis (33). However, in a recent study of apoptosis and necrosis in bronchiectasis, similar levels of necrotic and apoptotic granulocytes were observed (37). Impairments of macrophage phagocytosis are thought to lead to secondary necrosis of neutrophils and therefore release of NE. Thus increased neutrophil turnover could be a source of free NE.
There was a strong inverse relationship between active MMP-9 and active NE, indicating that high levels of NE are present when levels of active MMP-9 are low. Conversely, NE levels were positively correlated with total MMP-9 levels, which suggests that NE may play a role in the inactivation of MMP-9 or prevention of its activation. Activated neutrophil products contribute to total MMP-9 levels through inactivation of TIMP-1. NE can inactivate TIMP-1 bound to proeCMMP-9 without destruction or activation of MMP-9 (38). This would result in an increase in the proeCMMP-9, as is observed in subjects with neutrophilic asthma. In addition, TIMP-1 can be inactivated by the oxidant HOCl released by neutrophil activation (39). The mechanisms behind this relationship are unclear and require further investigation.
These results demonstrate that there is proteolytic enzyme release in asthma. Subjects with neutrophilic asthma are characterized by the presence of active NE, whereas subjects with eosinophilic asthma were characterized by the presence of active MMP-9. The mechanisms for excess enzyme activity appeared to be an inadequate inhibitor response.
In healthy subjects there was a small amount of active MMP-9 present, consistent with the ongoing ECM turnover that occurs in tissues. Interestingly, no active NE was present in normal control subjects, despite neutrophils being present in the airway. This indicates either no neutrophil activation in healthy subjects, or effective antielastase mechanisms to control any elastase leak from airway neutrophils. Low levels of both SLPI and 1AT were detectable in the sputum supernatant from healthy control subjects.
IL-8 is a key chemokine leading to neutrophil infiltration in the lung, and there may be a positive feedback loop operating in neutrophilic asthma, leading to chronic inflammation. When cultured neutrophils are treated with IL-8, MMP-9 is released rapidly (40). Consistent with this, we found a close association between levels of IL-8 and MMP-9 in asthma. Recently it has been demonstrated that IL-8 is a substrate for activated MMP-9, and the resultant MMP-9eCmodified IL-8 molecule was 10- to 27-fold more potent in neutrophil activation (5). The correlations demonstrated in this study are consistent with a positive feedback cycle where IL-8 induces neutrophil influx and activation, which in turn releases MMP-9 that may cleave IL-8 to a more potent form. This may explain the intense neutrophil influx in these subjects. One of the more interesting and novel observations of this study was the correlation between neutrophils and inflammatory mediators. IL-8 levels were correlated with sputum neutrophils, and there were strong positive relationships among subjects with asthma between sputum IL-8 and total MMP-9, and between IL-8 and NE. The correlation observed between sputum neutrophils and IL-8 is not unexpected due the production of IL-8 by the respiratory epithelium and also by airway neutrophils themselves. The link between IL-8, NE, and MMP-9 is consistent with the positive feedback between these mediators, where NE can induce IL-8 production (4). These correlations support a proinflammatory mechanism linking IL-8 influx and neutrophil degranulation and consequent release of NE and MMP-9, thus supporting a chronic inflammatory and remodeling process.
Although a role for neutrophilic inflammation has been established in severe asthma (41) and exacerbations of asthma (42), neutrophils are not always considered important in the pathogenesis of stable asthma. Recently we (3) and others (25) have identified subjects with asthma in whom neutrophils are the dominant inflammatory cell in the sputum. These subjects form part of a larger group of subjects with noneosinophilic asthma. The mechanisms behind the symptoms and airway hyperresponsiveness in these subjects are unknown; however, neutrophils and their products may play an important role. It has been hypothesized that noneosinophilic asthma is driven by persistent innate immune activation (43). IL-8 is one of the proinflammatory cytokines released in response to innate immune receptor activation and signaling (44). The high IL-8 levels observed in neutrophilic asthma indicate potential activation of the innate immune system. Our data also reinforce the role of IL-8eCmediated neutrophil influx and proteolytic enzyme release as a relevant mechanism in noneosinophilic neutrophilic asthma. Future work will define the clinical consequences of these effects.
All studies were undertaken using induced sputum; however, changes in the airway lumen might be different from those occurring in the tissue. Induced sputum is thought to reflect events that occur in the large conducting airways. We believe there is a need for further examination of these mechanisms in airway tissue, as recent evidence suggests increased expression of MMP-9 in the sub-basement membrane of subjects with asthma (45). However, inflammatory processes occurring in the airway lumen are important and may influence subsequent changes in the tissue, and therefore study of these processes are both important and relevant.
In conclusion, there is differential expression of proteolytic enzyme activity in subjects with asthma. Neutrophilic asthma is accompanied by active NE, whereas active MMP-9 characterizes eosinophilic asthma. These effects seem to occur because antiprotease activity is inadequate. The results suggest an important role of proteolytic enzyme activity in asthma and highlight the potential for antiprotease therapy in asthma.
Acknowledgments
The authors thank Mrs. Ruth Roxby, Ms. Majella Maher, Ms. Naomi Timmins, Mrs. Glenda Walker, Ms. Rebecca Oldham, and Mrs. Kellie Fakes for their technical assistance.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
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