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Matrix metalloproteinases and their inhibitors in the nasal mucosa of patients with perennial allergic rhinitis
Matrix metalloproteinases and their inhibitors in the nasal mucosa of patients with perennial allergic rhinitis Azhar Shaida, FRCS,a Guy Kenyon, MD,a Jagdish Devalia, PhD,b Robert J. Davies, MD,a Thomas T. MacDonald, PhD, FRCPath,c and Sylvia L. F. Pender, PhDc London and Southampton, United Kingdom
Background: Allergic rhinitis and asthma show many similarities in their epithelial and inflammatory responses to allergens. However, one notable difference is that disruption and desquamation of the epithelium is a characteristic feature of asthma, whereas in perennial allergic rhinitis the epithelium is intact and thickened. One reason for this might be differing expression of matrix metalloproteinases (MMPs) or their inhibitors (TIMPs). There are few published data on the presence of MMPs or TIMPs in the nasal mucosa in rhinitis. Objective: The purpose of this study was to investigate MMP and TIMP mRNA and protein in nasal mucosa from subjects with perennial allergic rhinitis and from nonrhinitic control subjects. Methods: Biopsy specimens of nasal mucosa were taken from 10 well-characterized subjects with perennial allergic rhinitis and 10 nonrhinitic control subjects. MMP and TIMP mRNA was quantified through use of competitive RT-PCR, and protein was detected by means of Western blotting and ELISA. Results: TIMP-1 mRNA and TIMP-2 mRNA were present in nasal samples, but there was no significant difference between the 2 groups. Only small amounts of MMP-1, -2, -3, and -9 mRNA were detected in the same samples. The corresponding proteins were detected by means of Western blotting. TIMP-1 protein and TIMP-2 protein were quantified in tissue homogenates; there was no significant difference between the 2 groups. Conclusion: Our studies have demonstrated the presence of large amounts of TIMP-1 and TIMP-2 mRNA and protein in nasal mucosa. There is no upregulation of MMPs or changes in TIMP expression in the nasal mucosa of patients with allergic rhinitis. (J Allergy Clin Immunol 2001;108:791-6.) Key words: Rhinitic, nasal, matrix metalloproteinases, TIMPs, tissue inflammation, tissue remodeling
From the Departments of aOtolaryngology and bAcademic Respiratory Medicine, St Bartholomew’s and the Royal London School of Medicine and Dentistry, and cthe Division of Infection, Inflammation and Repair, University of Southampton, School of Medicine. Supported by the Special Trustees of the Royal London Hospitals NHS Trust, Department of Respiratory Medicine, The London Chest Hospital. Received for publication May 23, 2001; revised July 27, 2001; accepted for publication July 27, 2001. Reprint requests: Sylvia L. F. Pender, PhD, Tissue Remodeling and Repair Group, Division of Infection, Inflammation and Repair, Mailpoint 813, Level E, South Academic Block, Southampton General Hospital, Southampton SO16 6YD, United Kingdom. Copyright © 2001 by Mosby, Inc. 0091-6749/2001 $35.00 + 0 1/83/119024 doi:10.1067/mai.2001.119024
Abbreviations used ECM: Extracellular matrix MMP: Matrix metalloproteinase MMP-1: Interstitial collagenase MMP-2: Gelatinase A MMP-3: Stromelysin-1 MMP-9: Gelatinase B TIMP: Tissue inhibitor of metalloproteinase
Asthma and rhinitis are common clinical presentations of allergic airway disease. In recent years, the concept of the “unified airway” has gained increasing acceptance; the upper and lower airways show similar epithelial features and inflammatory reactions to irritants and allergens.1,2 The nose is lined principally by ciliated pseudostratified columnar epithelium, whereas the major bronchi are lined by a similar ciliated pseudostratified columnar epithelium that changes to simple cuboidal epithelium distally.3 The inflammatory responses to allergens are also similar in the nose and lung. There is infiltration by inflammatory cells (such as eosinophils and lymphocytes), mast cell degranulation, and local overexpression of cytokines (such as IL-2, IL-5, and GM-CSF).4 However, one striking difference noted between the inflammatory responses in the nose and lung concerns the integrity of the epithelium. A characteristic feature of asthma is disruption and desquamation of the epithelium,5 but in perennial rhinitis the epithelium is intact and thickened.6 This difference might reflect differences in the concentrations and types of inflammatory mediators at the 2 sites. Alternatively, there might be a difference in the inhibitors of inflammation at the 2 sites. For example, TGF-β is scarce in bronchial epithelial cells from individuals with asthma in comparison with controls, and it is thought that its absence might be implicated in epithelial disruption.7 Finally, there might be differences in matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinase (TIMPs) between the nose and the airways; this is particularly relevant inasmuch as these molecules are involved in tissue injury in a variety of other tissue types. The MMPs are a family of Ca2+-activated, Zn2+dependent endopeptidases that have the ability to degrade various components of the extracellular matrix 791
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(ECM) and basement membrane.8 They are implicated in normal physiologic tissue remodeling, inflammation, and tumor spread. Most of the enzymes are relatively substrate-specific, each being secreted as an inactive proenzyme and cleaved extracellularly to produce the active form. The extracellular activity of MMPs is regulated by TIMPs, which are produced by the same cell types that produce MMPs.9 Asthma is a disease of the airways involving ongoing inflammation and repair, with epithelial shedding and airway remodeling.10 Thickening of the basement membrane in bronchial biopsy specimens from patients with asthma was noted by Roche et al.11 Chetta et al12 confirmed basement membrane thickening in bronchial biopsy specimens from asthmatic subjects in comparison with control subjects, the degree of thickening correlating with the severity of the asthma. Chakir et al13 examined bronchial biopsy specimens from subjects with asthma, subjects with seasonal allergic rhinitis, and control subjects and found increased type I and III collagen and fibronectin in the basement membrane. This was greatest in asthmatic subjects and moderate in rhinitic subjects in comparison with control subjects. In addition, Chakir et al13 also demonstrated a network of myofibroblasts beneath the epithelium in bronchial biopsy specimens from rhinitic subjects as well as bronchial biopsy specimens from asthmatic subjects, suggesting that the subepithelial fibrosis is due to deposition of type I and III collagens and fibronectin and that similar processes might be occurring in asthma and rhinitis. Collagen deposition in the basement membrane in individuals with asthma might be associated with increased expression of gelatinase B,14 and Hoshino et al15 have recently reported that corticosteroid treatment of asthma can reduce subepithelial collagen deposition by downregulation of gelatinase B expression and upregulation of TIMP-1 expression. Mautino et al,16 however, found high levels of TIMP-1 in bronchoalveolar lavage fluid from untreated asthmatic subjects and suggested that high levels of TIMP-1 might be responsible for airway fibrosis. These studies suggest increased ECM turnover in asthma. MMPs and TIMPs are involved in collagen turnover in diffuse alveolar damage and idiopathic pulmonary fibrosis,17 and neutrophil collagenase activity correlates with severity of disease in bronchiectasis.18 Gelatinase B is overexpressed by eosinophils in asthmatic patients19 and has been implicated in migration of human bronchial epithelial cells.20 Interstitial collagenase can be induced by the asthma and rhinitis–associated proinflammatory eicosanoid leukotriene D4 in vitro.21 An imbalance between gelatinase B and TIMP-1 has been linked to airway remodeling and obstruction in asthma and bronchitis.22 We hypothesized that the relative preservation of epithelium seen in rhinitic inflammation in comparison with asthma might be due either to absence of MMPs in the nose or to the presence of large amounts of inhibitory TIMPs. Previous studies from our group have shown that interstitial collagenase (MMP-1), gelatinase A (MMP-2), stromelysin-1 (MMP-3), gelatinase B (MMP-
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9), TIMP-1, and TIMP-2 are associated with mucosal inflammation in the gut.23 Accordingly, the aims of this study were to determine whether these MMPs and TIMPs are present in nasal mucosa, whether there is any difference between nasal mucosa from subjects with perennial allergic rhinitis and that from nonallergic, nonrhinitic control subjects, and whether there is any imbalance between MMP and TIMP expression.
METHODS Subjects Ten well-characterized subjects with perennial allergic rhinitis were recruited. All subjects had symptoms of perennial allergic rhinitis at the time of biopsy. Nasal examination demonstrated signs consistent with rhinitic mucosal inflammation, such as pale swollen mucosa, red engorged mucosa, and excess mucus. All of these rhinitic subjects had symptoms of bilateral or alternating nasal obstruction and rhinorrhea for at least 1 hour a day throughout the year. Skin prick tests were performed in accordance with international standardized guidelines,24 and results in the rhinitic subjects were all positive to a perennial allergen, the house dust mite (Dermatophagoides pteronyssinus). Subjects were precluded from taking any anti-inflammatory medication that might interfere with the inflammatory response, such as steroid sprays or antihistamines, for 1 month before the study. In addition, subjects were not included in the study if they had smoked within the last 2 years or had other nasal problems such as polyps or nasal septal deviation. Any subject with a recent upper respiratory tract infection was deferred and recalled after 1 month. As controls, 10 nonrhinitic, nonallergic, nonsmoking subjects were recruited; they were subjected to the same exclusion criteria. For all subjects, the nasal mucosa was anesthetized through use of a 10% cocaine solution, and biopsies were taken approximately 1, 3, and 5 cm from the anterior end of the inferior turbinate. For each subject, some tissue was mounted in optimal cutting temperature medium and frozen in liquid nitrogen; the remaining tissue was snap-frozen in liquid nitrogen and stored at –70°C.
Western blot analysis Tissue samples were homogenized with lysis buffer (6 mol/L urea, 5 mmol/L CaCl2 in TBS; pH 7.6), and the protein concentration was measured through use of the Bio-Rad Protein Assay (BioRad Labs, Hemel Hempstead, United Kingdom). Similar amounts of protein were loaded into each lane, run on 10% SDS-PAGE under reducing conditions, and transferred to nitrocellulose (BioRad). The nitrocellulose was incubated with primary antibodies (polyclonal sheep antihuman MMP antibodies; 1:500 dilution; The Binding Site Ltd, Birmingham, United Kingdom) that recognized both the active and proenzyme forms of the MMPs. The secondary antibody used was rabbit antisheep IgG (1:2500 dilution) conjugated to horseradish peroxidase (Dako Ltd, Ely, Cambridgeshire, United Kingdom). An ECL Plus system (Amersham Pharmacia Biotech UK, Amersham, United Kingdom) was used to detect immunoreactivity. Sheep antimouse IgG (1/1000, Sigma Chemical, St Louis, Mo) was used as a negative control, and human fetal gut explant culture supernatants were used as a positive control.23 The intensity of the bands was quantified by image analysis.
ELISA Tissue was homogenized with lysis buffer. Concentrations of TIMP-1 and TIMP-2 were measured through use of commercially available Biotrak ELISA assay systems (Amersham Pharmacia Biotech, Amersham, United Kingdom) according to the manufacturer’s instructions. Sufficient protein for assay was available from
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8 rhinitic and 8 nonrhinitic subjects. Each assay was performed in duplicate. The optical density at 450 nm was read through use of a Titertek Multiscan Plus Elisa reader (EFLAB, Helsinki, Finland). The mean optical density of each duplicate standard assay was calculated and plotted against the concentration of the standard to construct a standard curve. From this, the mean optical reading for each subject could be used to determine the concentrations of TIMP-1 and TIMP-2 by interpolation.
Competitive RT-PCR The mRNAs for MMP-1, -2, -3, and -9 and TIMP-1 and -2 were detected through use of a quantitative competitive RT-PCR technique, as described previously.25 In brief, total RNA was extracted through use of a monophasic solution of phenol and guanidine isothiocyanate (TRIzol, Life Technologies, Paisley, United Kingdom) with chloroform and then precipitated through use of isopropanol. One microgram of total RNA was co-transcribed with serial dilutions of synthetic mRNA constructs encoding the primer sites for these metalloproteinases and their inhibitors (donated by Dr G. S. Schultz, University of Florida). Reverse transcription was performed by adding 0.5 µg Oligo d(T) (Amersham Pharmacia Biotech UK) and 200 units of reverse transcriptase (M-MLV reverse transcriptase, Life Technologies) per reaction. The resulting cDNA was used for PCR amplification with 25 pmol of 5′- and 3′- sequence-specific oligonucleotide primers at 58°C annealing for 35 cycles. The PCR products were electrophoresed in 1.5% agarose gels containing 0.1 µg/mL ethidium bromide. Band intensities were quantified by densitometry. Positive controls were mRNA from explants of human fetal gut in which the T cells had been activated with pokeweed mitogen and which were known to contain elevated levels of transcripts for MMPs.23 The lower limit of sensitivity was 1000 transcripts per microgram of total RNA.
FIG 1. Western blots for MMPs in rhinitic and control subjects. These results are representative of the results for all 10 rhinitic and all 10 nonrhinitic subjects. Fetal gut explant culture supernatants were used as positive controls.
Statistics The significance of the data was analyzed by means of a MannWhitney U test through use of the SPSS statistics program, version 10 (SPSS Inc, Chicago, Ill).
RESULTS MMP and TIMP protein production in perennial allergic rhinitic and nonrhinitic nasal mucosa Immunoreactive bands at the appropriate molecular weights for interstitial collagenase, gelatinase A, stromelysin-1, and gelatinase B were detected by means of Western blotting in 10 rhinitic and 10 nonrhinitic subjects. The latent forms of interstitial collagenase and stromelysin-1 in the rhinitic group were in general slightly higher than those of the nonrhinitic group, but lowmolecular-weight active forms of enzymes were not detected. However, there was no difference in gelatinase A or gelatinase B between these 2 groups (Fig 1). The concentrations of TIMP-1 and TIMP-2 protein in tissue homogenates were quantified by means of ELISA. TIMP-1 protein tended to be higher in rhinitic subjects (median, 54 pg/µg total protein; range, 13.29 to 115.9 pg/µg total protein) than in nonrhinitic subjects (median, 32 pg/µg total protein; range, 5.45 to 76.91 pg/µg total protein), but the difference was not statistically significant (P > .1; Mann Whitney U 2-tail test; Fig 2). TIMP2 protein was present in approximately equal amounts in
FIG 2. TIMP-1 and TIMP-2 protein, as determined by means of ELISA. TIMP-1 protein concentrations are higher in samples from rhinitic subjects (median, 54 pg/µg total protein) than in samples from nonrhinitic subjects (median, 32 pg/µg total protein), but this difference was not statistically significant (P > .1). There was no significant difference in TIMP-2 protein between rhinitic (median, 33 pg/µg total protein) and nonrhinitic subjects (median, 30 pg/µg total protein).
rhinitic subjects (median, 33 pg/µg total protein; range, 0 to 42.14 pg/µg total protein) and nonrhinitic subjects (median, 30 pg/µg total protein; range, 2.68 to 59.05 pg/µg total protein).
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TABLE I. Number of MMP and TIMP transcripts in nasal mucosa Median no. of transcripts per microgram of total RNA (range)
MMP-1 MMP-2 MMP-3 MMP-9 TIMP-1 TIMP-2
Rhinitic subjects
Nonrhinitic subjects
<1,000 (0-518) <1,000 (1-2,905) <1,000 (0-302) <1,000 (30-1,963) 107,194 (754-331,536) 51,837 (4,518-51,502)
<1,000 (0-862) <1,000 (158-1,137) <1,000 (0-0) <1,000 (35-2,173) 100,723 (6,937-433,949) 53,492 (5,943-301,228)
mRNA signal for TIMP-1 and TIMP-2 was detected in large amounts. The small numbers of MMP mRNA transcripts were not due to a problem with the RNA extracted from the biopsy specimens; large numbers of TIMP transcripts were obtained from the same source RNA. In addition, bands were detected in all samples through use of housing-keeping gene primers (GAPDH). Using stimulated fetal gut culture explant as a positive control produced high levels of MMP transcripts, which indicates that there was no failure of the RT-PCR technique.
MMP and TIMP mRNA expression in perennial allergic rhinitic and nonrhinitic nasal mucosa MMP transcripts were particularly low in most of the nasal samples. All of the median numbers were less than 1000 transcripts per microgram of total RNA; in particular, interstitial collagenase and stromelysin-1 mRNA transcripts were below this limit or nondetectable in all nasal samples (Table I). MMP-2 mRNA was detected consistently in most subjects, but the median number of transcripts was also less than 1000 per microgram of total RNA (range, 1 to 2905 in rhinitic samples and 158 to 1137 in nonrhinitic samples). Gelatinase B mRNA was also detected in most subjects; again the median number was less than 1000 transcripts per microgram of total RNA (range, 30 to 1963 in rhinitic subjects and 35 to 2173 in control subjects; Table I). In contrast, TIMP mRNA was significantly higher in the nasal mucosa. In the same samples, there was a median of 107,194 TIMP-1 transcripts in the rhinitic group and of 100,723 transcripts in the nonrhinitic group per microgram of total RNA. TIMP-2 mRNA expression was half that of TIMP-1 (51,837 transcripts in rhinitic and 53,492 transcripts in nonrhinitic nasal mucosa). There was no significant difference between the 2 subject groups for either TIMP (Table I).
DISCUSSION In this study, we have demonstrated the presence of large amounts of mRNA for TIMP-1 and TIMP-2 and shown that the corresponding protein is abundant in both rhinitic and nonrhinitic subjects. Although MMP proteins were detected by Western blotting, mRNAs for interstitial collagenase, gelatinase A, stromelysin-1, and gelatinase B were not detected in large quantities in the majority of samples. We can conclude, therefore, that
MMPs are not upregulated in the nasal mucosa in perennial allergic rhinitic subjects. It is paradoxical that MMPs are not elevated in rhinitis, given that the tissue contains inflammatory cells and that cytokines upregulate MMP production by fibroblasts.23 The pathophysiologic character of perennial allergic rhinitis has been reviewed in detail by other authors.26-28 The early phase of allergic rhinitis involves antigen cross-linking mast cell–bound IgE, resulting in mast cell degranulation and histamine release and producing the characteristic itching, sneezing, congestion, and rhinorrhea. The late-phase response is mediated by leukotrienes, prostaglandins, and cytokines such as IL-3, -4, -5, -6, -8, -9, -10, and -13, GM-CSF, and RANTES, released from TH2 lymphocytes, mast cells, and epithelial cells. Cytokines and other inflammatory mediators upregulate adhesion molecules such as vascular cell adhesion molecule 1 and E-selectin, which allow circulating eosinophils, basophils, and T cells to adhere to endothelial cells before diapedesis; chemoattractants, including eotaxin, IL-5, and RANTES, cause these cells to migrate to the area of inflammation. Further production and release of inflammatory mediators from these cells perpetuate the inflammatory response. Similar processes and inflammatory mediators and cells are seen in asthma. Secretion of MMPs by various cells is well documented. MMPs might be produced by activated fibroblasts.29,30 Interstitial collagenase is produced by most connective tissue cells, whereas neutrophil collagenase production is confined to neutrophil granules. The gelatinases degrade types IV, V, VII, and X collagens and elastin, and they are associated with macrophages and connective tissue cells.8 Activated macrophages can produce interstitial collagenase, gelatinases A and B, and stromelysin-1.31-33 Gelatinase B can also be produced by mast cells, which are an integral component of the inflammatory response.34 Activated T cells might produce gelatinase A and gelatinase B.35 However, some mediators might inhibit MMP activity— for example, IL-10, a cytokine produced by activated macrophages and TH2-type T cells, downregulates mucosal T-cell activation, MMP production, and loss of ECM in the gut.36 Regulation of MMP activity occurs at 3 levels: gene transcription, activation of the secreted proenzyme, and inhibition by specific and nonspecific inhibitors. Activation might be autocatalytic or by other MMPs, by membrane-type MMPs, or by tissue activators such as plasmin, kallikrein, and neutrophil elastase. Inhibition might be by nonspecific inhibitors such as α2-macroglobulin or specific inhibitors such as TIMPs, which inhibit the activated forms of MMPs by forming irreversible complexes. Thus there is evidence of MMP activity in the form of matrix turnover in asthma, and cells and mediators implicated in MMP secretion and inhibition are present in asthma and rhinitis. The available data on MMP expression in the airways implicate gelatinase A, gelatinase B, neutrophil collagenase, TIMP-1, and TIMP-2,15 and these proteins were therefore investigated in the present study.17-19,37-39
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There are few data available on MMP expression in the nose, though Lechapt-Zalcman et al40 recently demonstrated the presence of gelatinase A and gelatinase B in tissue from nasal polyps and nasal mucosa. This study has demonstrated the presence of MMP proteins in the nasal mucosa of rhinitic and nonrhinitic patients and accordingly shown that there is a potential for tissue damage and epithelial disruption. We did not find any significant difference in epithelial thickness between rhinitic and nonrhinitic groups (data not shown). The fact that in the rhinitic group the latent form of interstitial collagenase and stromelysin-1 were increased in Western blots suggests that more MMPs were being produced in the rhinitic condition. Perhaps this increase was insufficient to cause epithelial disruption. In addition, we found that there were large amounts of TIMP mRNA and protein present in the mucosa to inhibit MMP activity. The lack of any significant difference in TIMPs between rhinitic and nonrhinitic groups might be due to the presence of inflammation in the nasal mucosa in nonrhinitic subjects as a result of environmental irritants and toxins. In conclusion, this study demonstrates the presence of MMPs and hence the potential for tissue damage and epithelial shedding in the nasal mucosa. The findings of high levels of TIMP mRNA and protein support our hypothesis that TIMPs might be present in large amounts in the nasal mucosa and that they might suppress MMPmediated damage. This is not surprising, inasmuch as the role of the nose as gatekeeper to the remainder of the airways means that it is continually exposed to irritants and toxins; it would therefore be expected to have developed efficient inhibitory mechanisms. The mucosa of the lower airway might differ, and further work is required to compare nasal mucosa with bronchial mucosa. We thank Dr G. Schultz, University of Florida, for the gift of MMP template (National Eye Institute Grant EY-05587).
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34. Kanbe N, Tanaka A, Kanbe M, Itakura A, Kurosawa M, Matsuda H. Human mast cells produce matrix metalloproteinase 9. Eur J Immunol 1999;29:2645-9. 35. Leppert D, Waubant E, Galardy R, Bunnett NW, Hauser SL. T cell gelatinases mediate basement membrane transmigration in vitro. J Immunol 1995;154:4379-89. 36. Pender SL, Fell JM, Chamow SM, Ashkenazi A, MacDonald TT. A p55 TNF receptor immunoadhesin prevents T cell-mediated intestinal injury by inhibiting matrix metalloproteinase production. J Immunol 1998;160:4098-103. 37. Ferry G, Lonchampt M, Pennel L, de Nanteuil G, Canet E, Tucker GC.
Activation of MMP-9 by neutrophil elastase in an in vivo model of acute lung injury. FEBS Lett 1997;402:111-5. 38. Torii K, Iida K, Miyazaki Y, Saga S, Kondoh Y, Taniguchi H, et al. Higher concentrations of matrix metalloproteinases in bronchoalveolar lavage fluid of patients with adult respiratory distress syndrome. Am J Respir Crit Care Med 1997;155:43-6. 39. Sepper R, Konttinen YT, Sorsa T, Koski H. Gelatinolytic and type IV collagenolytic activity in bronchiectasis. Chest 1994;106:1129-33. 40. Lechapt-Zalcman E, Coste A, d’Ortho MP, Frisdal E, Harf A, Lafuma C, et al. Increased expression of matrix metalloproteinase-9 in nasal polyps. J Pathol 2001;193:233-41.
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Background: Allergic rhinitis and asthma show many similarities in their epithelial and inflammatory responses to allergens. However, one notable difference is that disruption and desquamation of the epithelium is a characteristic feature of asthma, whereas in perennial allergic rhinitis the epithelium is intact and thickened. One reason for this might be differing expression of matrix metalloproteinases (MMPs) or their inhibitors (TIMPs). There are few published data on the presence of MMPs or TIMPs in the nasal mucosa in rhinitis. Objective: The purpose of this study was to investigate MMP and TIMP mRNA and protein in nasal mucosa from subjects with perennial allergic rhinitis and from nonrhinitic control subjects. Methods: Biopsy specimens of nasal mucosa were taken from 10 well-characterized subjects with perennial allergic rhinitis and 10 nonrhinitic control subjects. MMP and TIMP mRNA was quantified through use of competitive RT-PCR, and protein was detected by means of Western blotting and ELISA. Results: TIMP-1 mRNA and TIMP-2 mRNA were present in nasal samples, but there was no significant difference between the 2 groups. Only small amounts of MMP-1, -2, -3, and -9 mRNA were detected in the same samples. The corresponding proteins were detected by means of Western blotting. TIMP-1 protein and TIMP-2 protein were quantified in tissue homogenates; there was no significant difference between the 2 groups. Conclusion: Our studies have demonstrated the presence of large amounts of TIMP-1 and TIMP-2 mRNA and protein in nasal mucosa. There is no upregulation of MMPs or changes in TIMP expression in the nasal mucosa of patients with allergic rhinitis. (J Allergy Clin Immunol 2001;108:791-6.) Key words: Rhinitic, nasal, matrix metalloproteinases, TIMPs, tissue inflammation, tissue remodeling
From the Departments of aOtolaryngology and bAcademic Respiratory Medicine, St Bartholomew’s and the Royal London School of Medicine and Dentistry, and cthe Division of Infection, Inflammation and Repair, University of Southampton, School of Medicine. Supported by the Special Trustees of the Royal London Hospitals NHS Trust, Department of Respiratory Medicine, The London Chest Hospital. Received for publication May 23, 2001; revised July 27, 2001; accepted for publication July 27, 2001. Reprint requests: Sylvia L. F. Pender, PhD, Tissue Remodeling and Repair Group, Division of Infection, Inflammation and Repair, Mailpoint 813, Level E, South Academic Block, Southampton General Hospital, Southampton SO16 6YD, United Kingdom. Copyright © 2001 by Mosby, Inc. 0091-6749/2001 $35.00 + 0 1/83/119024 doi:10.1067/mai.2001.119024
Abbreviations used ECM: Extracellular matrix MMP: Matrix metalloproteinase MMP-1: Interstitial collagenase MMP-2: Gelatinase A MMP-3: Stromelysin-1 MMP-9: Gelatinase B TIMP: Tissue inhibitor of metalloproteinase
Asthma and rhinitis are common clinical presentations of allergic airway disease. In recent years, the concept of the “unified airway” has gained increasing acceptance; the upper and lower airways show similar epithelial features and inflammatory reactions to irritants and allergens.1,2 The nose is lined principally by ciliated pseudostratified columnar epithelium, whereas the major bronchi are lined by a similar ciliated pseudostratified columnar epithelium that changes to simple cuboidal epithelium distally.3 The inflammatory responses to allergens are also similar in the nose and lung. There is infiltration by inflammatory cells (such as eosinophils and lymphocytes), mast cell degranulation, and local overexpression of cytokines (such as IL-2, IL-5, and GM-CSF).4 However, one striking difference noted between the inflammatory responses in the nose and lung concerns the integrity of the epithelium. A characteristic feature of asthma is disruption and desquamation of the epithelium,5 but in perennial rhinitis the epithelium is intact and thickened.6 This difference might reflect differences in the concentrations and types of inflammatory mediators at the 2 sites. Alternatively, there might be a difference in the inhibitors of inflammation at the 2 sites. For example, TGF-β is scarce in bronchial epithelial cells from individuals with asthma in comparison with controls, and it is thought that its absence might be implicated in epithelial disruption.7 Finally, there might be differences in matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinase (TIMPs) between the nose and the airways; this is particularly relevant inasmuch as these molecules are involved in tissue injury in a variety of other tissue types. The MMPs are a family of Ca2+-activated, Zn2+dependent endopeptidases that have the ability to degrade various components of the extracellular matrix 791
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(ECM) and basement membrane.8 They are implicated in normal physiologic tissue remodeling, inflammation, and tumor spread. Most of the enzymes are relatively substrate-specific, each being secreted as an inactive proenzyme and cleaved extracellularly to produce the active form. The extracellular activity of MMPs is regulated by TIMPs, which are produced by the same cell types that produce MMPs.9 Asthma is a disease of the airways involving ongoing inflammation and repair, with epithelial shedding and airway remodeling.10 Thickening of the basement membrane in bronchial biopsy specimens from patients with asthma was noted by Roche et al.11 Chetta et al12 confirmed basement membrane thickening in bronchial biopsy specimens from asthmatic subjects in comparison with control subjects, the degree of thickening correlating with the severity of the asthma. Chakir et al13 examined bronchial biopsy specimens from subjects with asthma, subjects with seasonal allergic rhinitis, and control subjects and found increased type I and III collagen and fibronectin in the basement membrane. This was greatest in asthmatic subjects and moderate in rhinitic subjects in comparison with control subjects. In addition, Chakir et al13 also demonstrated a network of myofibroblasts beneath the epithelium in bronchial biopsy specimens from rhinitic subjects as well as bronchial biopsy specimens from asthmatic subjects, suggesting that the subepithelial fibrosis is due to deposition of type I and III collagens and fibronectin and that similar processes might be occurring in asthma and rhinitis. Collagen deposition in the basement membrane in individuals with asthma might be associated with increased expression of gelatinase B,14 and Hoshino et al15 have recently reported that corticosteroid treatment of asthma can reduce subepithelial collagen deposition by downregulation of gelatinase B expression and upregulation of TIMP-1 expression. Mautino et al,16 however, found high levels of TIMP-1 in bronchoalveolar lavage fluid from untreated asthmatic subjects and suggested that high levels of TIMP-1 might be responsible for airway fibrosis. These studies suggest increased ECM turnover in asthma. MMPs and TIMPs are involved in collagen turnover in diffuse alveolar damage and idiopathic pulmonary fibrosis,17 and neutrophil collagenase activity correlates with severity of disease in bronchiectasis.18 Gelatinase B is overexpressed by eosinophils in asthmatic patients19 and has been implicated in migration of human bronchial epithelial cells.20 Interstitial collagenase can be induced by the asthma and rhinitis–associated proinflammatory eicosanoid leukotriene D4 in vitro.21 An imbalance between gelatinase B and TIMP-1 has been linked to airway remodeling and obstruction in asthma and bronchitis.22 We hypothesized that the relative preservation of epithelium seen in rhinitic inflammation in comparison with asthma might be due either to absence of MMPs in the nose or to the presence of large amounts of inhibitory TIMPs. Previous studies from our group have shown that interstitial collagenase (MMP-1), gelatinase A (MMP-2), stromelysin-1 (MMP-3), gelatinase B (MMP-
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9), TIMP-1, and TIMP-2 are associated with mucosal inflammation in the gut.23 Accordingly, the aims of this study were to determine whether these MMPs and TIMPs are present in nasal mucosa, whether there is any difference between nasal mucosa from subjects with perennial allergic rhinitis and that from nonallergic, nonrhinitic control subjects, and whether there is any imbalance between MMP and TIMP expression.
METHODS Subjects Ten well-characterized subjects with perennial allergic rhinitis were recruited. All subjects had symptoms of perennial allergic rhinitis at the time of biopsy. Nasal examination demonstrated signs consistent with rhinitic mucosal inflammation, such as pale swollen mucosa, red engorged mucosa, and excess mucus. All of these rhinitic subjects had symptoms of bilateral or alternating nasal obstruction and rhinorrhea for at least 1 hour a day throughout the year. Skin prick tests were performed in accordance with international standardized guidelines,24 and results in the rhinitic subjects were all positive to a perennial allergen, the house dust mite (Dermatophagoides pteronyssinus). Subjects were precluded from taking any anti-inflammatory medication that might interfere with the inflammatory response, such as steroid sprays or antihistamines, for 1 month before the study. In addition, subjects were not included in the study if they had smoked within the last 2 years or had other nasal problems such as polyps or nasal septal deviation. Any subject with a recent upper respiratory tract infection was deferred and recalled after 1 month. As controls, 10 nonrhinitic, nonallergic, nonsmoking subjects were recruited; they were subjected to the same exclusion criteria. For all subjects, the nasal mucosa was anesthetized through use of a 10% cocaine solution, and biopsies were taken approximately 1, 3, and 5 cm from the anterior end of the inferior turbinate. For each subject, some tissue was mounted in optimal cutting temperature medium and frozen in liquid nitrogen; the remaining tissue was snap-frozen in liquid nitrogen and stored at –70°C.
Western blot analysis Tissue samples were homogenized with lysis buffer (6 mol/L urea, 5 mmol/L CaCl2 in TBS; pH 7.6), and the protein concentration was measured through use of the Bio-Rad Protein Assay (BioRad Labs, Hemel Hempstead, United Kingdom). Similar amounts of protein were loaded into each lane, run on 10% SDS-PAGE under reducing conditions, and transferred to nitrocellulose (BioRad). The nitrocellulose was incubated with primary antibodies (polyclonal sheep antihuman MMP antibodies; 1:500 dilution; The Binding Site Ltd, Birmingham, United Kingdom) that recognized both the active and proenzyme forms of the MMPs. The secondary antibody used was rabbit antisheep IgG (1:2500 dilution) conjugated to horseradish peroxidase (Dako Ltd, Ely, Cambridgeshire, United Kingdom). An ECL Plus system (Amersham Pharmacia Biotech UK, Amersham, United Kingdom) was used to detect immunoreactivity. Sheep antimouse IgG (1/1000, Sigma Chemical, St Louis, Mo) was used as a negative control, and human fetal gut explant culture supernatants were used as a positive control.23 The intensity of the bands was quantified by image analysis.
ELISA Tissue was homogenized with lysis buffer. Concentrations of TIMP-1 and TIMP-2 were measured through use of commercially available Biotrak ELISA assay systems (Amersham Pharmacia Biotech, Amersham, United Kingdom) according to the manufacturer’s instructions. Sufficient protein for assay was available from
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8 rhinitic and 8 nonrhinitic subjects. Each assay was performed in duplicate. The optical density at 450 nm was read through use of a Titertek Multiscan Plus Elisa reader (EFLAB, Helsinki, Finland). The mean optical density of each duplicate standard assay was calculated and plotted against the concentration of the standard to construct a standard curve. From this, the mean optical reading for each subject could be used to determine the concentrations of TIMP-1 and TIMP-2 by interpolation.
Competitive RT-PCR The mRNAs for MMP-1, -2, -3, and -9 and TIMP-1 and -2 were detected through use of a quantitative competitive RT-PCR technique, as described previously.25 In brief, total RNA was extracted through use of a monophasic solution of phenol and guanidine isothiocyanate (TRIzol, Life Technologies, Paisley, United Kingdom) with chloroform and then precipitated through use of isopropanol. One microgram of total RNA was co-transcribed with serial dilutions of synthetic mRNA constructs encoding the primer sites for these metalloproteinases and their inhibitors (donated by Dr G. S. Schultz, University of Florida). Reverse transcription was performed by adding 0.5 µg Oligo d(T) (Amersham Pharmacia Biotech UK) and 200 units of reverse transcriptase (M-MLV reverse transcriptase, Life Technologies) per reaction. The resulting cDNA was used for PCR amplification with 25 pmol of 5′- and 3′- sequence-specific oligonucleotide primers at 58°C annealing for 35 cycles. The PCR products were electrophoresed in 1.5% agarose gels containing 0.1 µg/mL ethidium bromide. Band intensities were quantified by densitometry. Positive controls were mRNA from explants of human fetal gut in which the T cells had been activated with pokeweed mitogen and which were known to contain elevated levels of transcripts for MMPs.23 The lower limit of sensitivity was 1000 transcripts per microgram of total RNA.
FIG 1. Western blots for MMPs in rhinitic and control subjects. These results are representative of the results for all 10 rhinitic and all 10 nonrhinitic subjects. Fetal gut explant culture supernatants were used as positive controls.
Statistics The significance of the data was analyzed by means of a MannWhitney U test through use of the SPSS statistics program, version 10 (SPSS Inc, Chicago, Ill).
RESULTS MMP and TIMP protein production in perennial allergic rhinitic and nonrhinitic nasal mucosa Immunoreactive bands at the appropriate molecular weights for interstitial collagenase, gelatinase A, stromelysin-1, and gelatinase B were detected by means of Western blotting in 10 rhinitic and 10 nonrhinitic subjects. The latent forms of interstitial collagenase and stromelysin-1 in the rhinitic group were in general slightly higher than those of the nonrhinitic group, but lowmolecular-weight active forms of enzymes were not detected. However, there was no difference in gelatinase A or gelatinase B between these 2 groups (Fig 1). The concentrations of TIMP-1 and TIMP-2 protein in tissue homogenates were quantified by means of ELISA. TIMP-1 protein tended to be higher in rhinitic subjects (median, 54 pg/µg total protein; range, 13.29 to 115.9 pg/µg total protein) than in nonrhinitic subjects (median, 32 pg/µg total protein; range, 5.45 to 76.91 pg/µg total protein), but the difference was not statistically significant (P > .1; Mann Whitney U 2-tail test; Fig 2). TIMP2 protein was present in approximately equal amounts in
FIG 2. TIMP-1 and TIMP-2 protein, as determined by means of ELISA. TIMP-1 protein concentrations are higher in samples from rhinitic subjects (median, 54 pg/µg total protein) than in samples from nonrhinitic subjects (median, 32 pg/µg total protein), but this difference was not statistically significant (P > .1). There was no significant difference in TIMP-2 protein between rhinitic (median, 33 pg/µg total protein) and nonrhinitic subjects (median, 30 pg/µg total protein).
rhinitic subjects (median, 33 pg/µg total protein; range, 0 to 42.14 pg/µg total protein) and nonrhinitic subjects (median, 30 pg/µg total protein; range, 2.68 to 59.05 pg/µg total protein).
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TABLE I. Number of MMP and TIMP transcripts in nasal mucosa Median no. of transcripts per microgram of total RNA (range)
MMP-1 MMP-2 MMP-3 MMP-9 TIMP-1 TIMP-2
Rhinitic subjects
Nonrhinitic subjects
<1,000 (0-518) <1,000 (1-2,905) <1,000 (0-302) <1,000 (30-1,963) 107,194 (754-331,536) 51,837 (4,518-51,502)
<1,000 (0-862) <1,000 (158-1,137) <1,000 (0-0) <1,000 (35-2,173) 100,723 (6,937-433,949) 53,492 (5,943-301,228)
mRNA signal for TIMP-1 and TIMP-2 was detected in large amounts. The small numbers of MMP mRNA transcripts were not due to a problem with the RNA extracted from the biopsy specimens; large numbers of TIMP transcripts were obtained from the same source RNA. In addition, bands were detected in all samples through use of housing-keeping gene primers (GAPDH). Using stimulated fetal gut culture explant as a positive control produced high levels of MMP transcripts, which indicates that there was no failure of the RT-PCR technique.
MMP and TIMP mRNA expression in perennial allergic rhinitic and nonrhinitic nasal mucosa MMP transcripts were particularly low in most of the nasal samples. All of the median numbers were less than 1000 transcripts per microgram of total RNA; in particular, interstitial collagenase and stromelysin-1 mRNA transcripts were below this limit or nondetectable in all nasal samples (Table I). MMP-2 mRNA was detected consistently in most subjects, but the median number of transcripts was also less than 1000 per microgram of total RNA (range, 1 to 2905 in rhinitic samples and 158 to 1137 in nonrhinitic samples). Gelatinase B mRNA was also detected in most subjects; again the median number was less than 1000 transcripts per microgram of total RNA (range, 30 to 1963 in rhinitic subjects and 35 to 2173 in control subjects; Table I). In contrast, TIMP mRNA was significantly higher in the nasal mucosa. In the same samples, there was a median of 107,194 TIMP-1 transcripts in the rhinitic group and of 100,723 transcripts in the nonrhinitic group per microgram of total RNA. TIMP-2 mRNA expression was half that of TIMP-1 (51,837 transcripts in rhinitic and 53,492 transcripts in nonrhinitic nasal mucosa). There was no significant difference between the 2 subject groups for either TIMP (Table I).
DISCUSSION In this study, we have demonstrated the presence of large amounts of mRNA for TIMP-1 and TIMP-2 and shown that the corresponding protein is abundant in both rhinitic and nonrhinitic subjects. Although MMP proteins were detected by Western blotting, mRNAs for interstitial collagenase, gelatinase A, stromelysin-1, and gelatinase B were not detected in large quantities in the majority of samples. We can conclude, therefore, that
MMPs are not upregulated in the nasal mucosa in perennial allergic rhinitic subjects. It is paradoxical that MMPs are not elevated in rhinitis, given that the tissue contains inflammatory cells and that cytokines upregulate MMP production by fibroblasts.23 The pathophysiologic character of perennial allergic rhinitis has been reviewed in detail by other authors.26-28 The early phase of allergic rhinitis involves antigen cross-linking mast cell–bound IgE, resulting in mast cell degranulation and histamine release and producing the characteristic itching, sneezing, congestion, and rhinorrhea. The late-phase response is mediated by leukotrienes, prostaglandins, and cytokines such as IL-3, -4, -5, -6, -8, -9, -10, and -13, GM-CSF, and RANTES, released from TH2 lymphocytes, mast cells, and epithelial cells. Cytokines and other inflammatory mediators upregulate adhesion molecules such as vascular cell adhesion molecule 1 and E-selectin, which allow circulating eosinophils, basophils, and T cells to adhere to endothelial cells before diapedesis; chemoattractants, including eotaxin, IL-5, and RANTES, cause these cells to migrate to the area of inflammation. Further production and release of inflammatory mediators from these cells perpetuate the inflammatory response. Similar processes and inflammatory mediators and cells are seen in asthma. Secretion of MMPs by various cells is well documented. MMPs might be produced by activated fibroblasts.29,30 Interstitial collagenase is produced by most connective tissue cells, whereas neutrophil collagenase production is confined to neutrophil granules. The gelatinases degrade types IV, V, VII, and X collagens and elastin, and they are associated with macrophages and connective tissue cells.8 Activated macrophages can produce interstitial collagenase, gelatinases A and B, and stromelysin-1.31-33 Gelatinase B can also be produced by mast cells, which are an integral component of the inflammatory response.34 Activated T cells might produce gelatinase A and gelatinase B.35 However, some mediators might inhibit MMP activity— for example, IL-10, a cytokine produced by activated macrophages and TH2-type T cells, downregulates mucosal T-cell activation, MMP production, and loss of ECM in the gut.36 Regulation of MMP activity occurs at 3 levels: gene transcription, activation of the secreted proenzyme, and inhibition by specific and nonspecific inhibitors. Activation might be autocatalytic or by other MMPs, by membrane-type MMPs, or by tissue activators such as plasmin, kallikrein, and neutrophil elastase. Inhibition might be by nonspecific inhibitors such as α2-macroglobulin or specific inhibitors such as TIMPs, which inhibit the activated forms of MMPs by forming irreversible complexes. Thus there is evidence of MMP activity in the form of matrix turnover in asthma, and cells and mediators implicated in MMP secretion and inhibition are present in asthma and rhinitis. The available data on MMP expression in the airways implicate gelatinase A, gelatinase B, neutrophil collagenase, TIMP-1, and TIMP-2,15 and these proteins were therefore investigated in the present study.17-19,37-39
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There are few data available on MMP expression in the nose, though Lechapt-Zalcman et al40 recently demonstrated the presence of gelatinase A and gelatinase B in tissue from nasal polyps and nasal mucosa. This study has demonstrated the presence of MMP proteins in the nasal mucosa of rhinitic and nonrhinitic patients and accordingly shown that there is a potential for tissue damage and epithelial disruption. We did not find any significant difference in epithelial thickness between rhinitic and nonrhinitic groups (data not shown). The fact that in the rhinitic group the latent form of interstitial collagenase and stromelysin-1 were increased in Western blots suggests that more MMPs were being produced in the rhinitic condition. Perhaps this increase was insufficient to cause epithelial disruption. In addition, we found that there were large amounts of TIMP mRNA and protein present in the mucosa to inhibit MMP activity. The lack of any significant difference in TIMPs between rhinitic and nonrhinitic groups might be due to the presence of inflammation in the nasal mucosa in nonrhinitic subjects as a result of environmental irritants and toxins. In conclusion, this study demonstrates the presence of MMPs and hence the potential for tissue damage and epithelial shedding in the nasal mucosa. The findings of high levels of TIMP mRNA and protein support our hypothesis that TIMPs might be present in large amounts in the nasal mucosa and that they might suppress MMPmediated damage. This is not surprising, inasmuch as the role of the nose as gatekeeper to the remainder of the airways means that it is continually exposed to irritants and toxins; it would therefore be expected to have developed efficient inhibitory mechanisms. The mucosa of the lower airway might differ, and further work is required to compare nasal mucosa with bronchial mucosa. We thank Dr G. Schultz, University of Florida, for the gift of MMP template (National Eye Institute Grant EY-05587).
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34. Kanbe N, Tanaka A, Kanbe M, Itakura A, Kurosawa M, Matsuda H. Human mast cells produce matrix metalloproteinase 9. Eur J Immunol 1999;29:2645-9. 35. Leppert D, Waubant E, Galardy R, Bunnett NW, Hauser SL. T cell gelatinases mediate basement membrane transmigration in vitro. J Immunol 1995;154:4379-89. 36. Pender SL, Fell JM, Chamow SM, Ashkenazi A, MacDonald TT. A p55 TNF receptor immunoadhesin prevents T cell-mediated intestinal injury by inhibiting matrix metalloproteinase production. J Immunol 1998;160:4098-103. 37. Ferry G, Lonchampt M, Pennel L, de Nanteuil G, Canet E, Tucker GC.
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