Endogenous Relaxin Regulates Collagen Deposition in an Animal Model of Allergic Airway Disease
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《内分泌学杂志》
Howard Florey Institute of Experimental Physiology and Medicine (I.M., G.W.T., C.S.S.), University of Melbourne, Parkville, Victoria 3010, Australia
Murdoch Children’s Research Institute (N.R.S., S.G.R., M.L.K.T.), Royal Childrens Hospital, Parkville, Victoria 3052, Australia
Abstract
We examined the relationship among relaxin (a peptide hormone that stimulates collagen degradation), airway fibrosis, other changes of airway remodeling, and airway hyperresponsiveness (AHR) in an animal model of allergic airway disease. Eight- to 10-wk-old relaxin gene-knockout (RLX–/–) and wild-type (RLX+/+) mice were sensitized with ovalbumin (OVA) or saline ip at d 0 and 14 and challenged three times per week for 6 wk with nebulized 2.5% OVA or saline. Saline-treated control RLX+/+ and RLX–/– mice had equivalent collagen expression and baseline airway responses. OVA-treated RLX–/– mice developed airway inflammation equivalent to that in OVA-treated RLX+/+ mice. However, OVA-treated RLX–/– mice had markedly increased lung collagen deposition as compared with OVA-treated RLX+/+ and saline-treated mice (all P < 0.05). Collagen was predominantly deposited in the subepithelial basement membrane region and submucosal regions in both OVA-treated RLX+/+ and RLX–/– mice. The increased collagen measured in OVA-treated RLX–/– mice was associated with reduced matrix metalloproteinase (MMP)-9 (P < 0.02) expression and failure to up-regulate matrix metalloproteinase-2 expression, compared with levels in OVA-treated RLX+/+ mice. Goblet cell numbers were equivalent in OVA-treated RLX–/– and RLX+/+ mice and increased, compared with saline-treated animals. Both OVA-treated RLX+/+ and RLX–/– mice developed similar degrees of AHR after OVA treatment. These findings demonstrate a critical role for relaxin in the inhibition of lung collagen deposition during an allergic inflammatory response. Increased deposition of collagen per se did not influence airway epithelial structure or AHR.
Introduction
STRUCTURAL CHANGES IN the airway wall (airway remodeling) are characteristic features of allergic airway disease (AAD) that can occur early in the course of disease (1), contribute to airway hyperresponsiveness (AHR) (2), and lead to irreversible airway obstruction (1, 2). Airway remodeling is characterized by increased deposition of collagen (fibrosis) in the subepithelial basement membrane region and in the submucosal layers (3), smooth muscle hypertrophy and hyperplasia, fibroblast hyperplasia, epithelial metaplasia, and goblet cell proliferation (1). Collagen deposition in the subepithelial basement membrane region is a hallmark of airway remodeling in AAD (4); however, the cellular and molecular events that lead to the remodeling process are poorly understood. In particular, the factors regulating collagen deposition associated with airway remodeling in AAD have not been clearly defined, and the relationship between collagen deposition and other structural changes in the epithelium remain unclear. Furthermore, although corticosteroid antiinflammatory treatment can reduce symptoms, improve lung function, and modify bronchial hyperresponsiveness, these agents are not effective in reversing established airway collagen deposition or other structural changes of remodeling (5, 6). Consequently, there are currently no treatments that effectively reverse established collagen deposition or other structural changes in AAD.
Relaxin is a small dimeric peptide hormone secreted by the ovary into the blood in highest amounts during pregnancy and generally associated with female reproduction (7). However, the most consistent biological effect of relaxin is its ability to stimulate the breakdown of collagen. Relaxin stimulates collagen breakdown in preparation for successful parturition (7). Relaxin treatment has been demonstrated to inhibit fibrosis in a bleomycin-induced model of lung fibrosis (8), by inhibiting TGF-induced matrix protein production (8) and increasing the expression of the collagen-degrading enzymes, matrix metalloproteinases (MMPs) (8, 9). Furthermore, relaxin administration has been reported to prevent the development of collagen deposition associated with airway inflammation in ovalbumin (OVA)-sensitized guinea pigs (10) and OVA-sensitized challenged BALB/c mice (11), highlighting the antifibrotic potential of exogenous relaxin therapy. However, the role that endogenous relaxin plays in airway fibrosis and remodeling is yet to be determined.
Recently we generated a relaxin gene knockout (RLX–/–) mouse to further our understanding of the physiological actions of relaxin (12), which lacks the major stored and circulating form of relaxin. RLX–/– mice developed an age-related progression of fibrosis in several tissues (13, 14, 15), including the airway and lung that was first detectable at 6–9 months of age (15). Furthermore, treatment of RLX–/– mice with recombinant human relaxin-2 relaxin successfully reversed established fibrosis in the airways and lung (15). These findings suggested an important role for endogenous relaxin in regulating collagen deposition within the lung and further confirmed the potential role for exogenous relaxin in the treatment of fibrosis associated with airway and lung disorders. Based on these combined findings, we hypothesize that endogenous relaxin protects the airways and lung from fibrosis in response to AAD.
The 9-wk murine model of AAD mimics a majority of the changes observed in human asthma including: the recruitment of eosinophils, goblet cell hyperplasia, and/or metaplasia and a distinctive pattern of subepithelial fibrosis (16, 17, 18, 19), although no changes in smooth muscle thickening are detected. Whereas this model reflects several changes seen in human AAD, it is strain dependent (16, 17, 18, 19, 20). In the present study, we examined the role of endogenous relaxin in the regulation of collagen deposition and airway remodeling using this model of AAD on RLX–/– mice. The relationship among airway fibrosis, other structural changes of remodeling, and AHR was also examined. The RLX–/– mouse is an ideal model to study the specific contributions of endogenous relaxin to airway fibrosis in AAD.
Materials and Methods
Animals
Relaxin knockout mice were generated as described previously (12). All female relaxin wild-type (RLX+/+) and RLX–/– mice used in this study were generated from relaxin heterozygous (C57BLk6J/129SV) parents (12) and were age-matched littermates. Eight- to 10-wk-old RLX+/+ and RLX–/– mice with similar baseline collagen levels (15) were used in all studies. The animals were housed in a controlled environment and maintained as detailed before (15). These experiments were approved by the Howard Florey Institute and the Royal Children’s Hospital Animal Experimental Ethics Committees, which adhere to the Australian Code of Practice for the care and use of laboratory animals for scientific purposes.
OVA-induced allergic airway disease model
An OVA-induced model of AAD was used as previously described (21). Eight- to 10-wk-old RLX+/+ and RLX–/– mice were sensitized with an ip injection of 10 μg of grade V OVA (Sigma Chemical, St. Louis, MO) and 1 mg of aluminum potassium sulfate (Sigma) in 0.5 ml saline on d 0 and 14. Mice were then challenged 3 d/wk for 6 wk (from d 21 to 63) with an aerosol of 2.5% (wt/vol) OVA in saline solution for 30 min (21). Vehicle-treated mice received 0.5 ml saline alone ip on d 0 and 14 and were challenged with saline aerosols.
Analysis of AHR
Methacholine (MCh)-induced airway reactivity was assessed 24 h after the final aerosol challenge by invasive plethysmography (Buxco Electronics, Troy, NY). Briefly, mice were anesthetized by ip injection of ketamine (200 μg/g) and xylazine (10 μg/g), tracheostomized, and the jugular vein cannulated. Mice were ventilated with a small animal respirator (Harvard Apparatus, Holliston, MA) delivering 0.01 ml/g body weight at a rate of 120 strokes/min in a mouse plethysmograph chamber. Increasing MCh doses were delivered iv and airway resistance and compliance measured (Biosystem XA version 2.7.9; Buxco Electronics) for 2 min after each dose. Results are expressed as the maximal resistance after each dose of MCh minus baseline (PBS alone) resistance.
Determination of OVA-specific antibody titers by ELISA
Serum was obtained 24 h after the final OVA or saline challenge and stored at –20 C. Levels of OVA-specific IgE were determined by ELISA, as described previously (21).
Bronchoalveolar lavage (BAL)
After measurement of airway reactivity, BAL was carried out, as detailed previously (21). Total viable cell counts were determined in a hemocytometer using trypan blue exclusion. Differential counts of eosinophils, neutrophils, lymphocytes, and monocytes/macrophages were determined on cytospin smears of BAL samples (4 x 105 cells) from individual mice stained with DiffQuick (Life Technologies, Auckland, New Zealand) after counting 300 cells. Results are expressed as total cell number x 104.
Tissue collection
Lung tissues were weighed (total lung weight) and then separated into individual lobes for various analyses including hydroxyproline analysis, histological analysis, and MMP zymography.
Lung histopathology
The right lung lobe and trachea were fixed in 10% neutral buffered formalin for 18–24 h and routinely processed. Serial 5-μm sections taken every 100 μm were stained with hematoxylin and eosin for assessment of peribronchial inflammation, with Masson trichrome for assessment of collagen and Alcian blue-periodic acid Schiff (AB-PAS) for assessment of goblet cells.
Morphometric analysis of structural changes
Images of lung tissue sections were captured using a Digital camera (Q Imaging, Burnaby, British Columbia, Canada). A minimum of five bronchi measuring 150–350 μm luminal diameter were analyzed per mouse for the parameters described below using Image Pro-Discovery software (Media Cybernetics, Silver Spring, MD), which was calibrated with a reference micrometer slide. The thickness of the bronchial epithelial layer was measured by tracing around the basement membrane and the luminal surface of epithelial cells and calculating the area between these lines, using a digitizer (Aiptek, Irvine, CA). Total collagen thickness was similarly measured by tracing around the outer extent of the total collagen layer in the submucosal region and around the basement membrane and the area between these lines calculated. Total areas were calculated by subtracting the inner area from the outer area. These areas were expressed per length (micrometers) of basement membrane to account for variation in bronchial diameters. Goblet cells were counted in AB-PAS stained sections and expressed as number of cells per 100 μm of basement membrane.
Hydroxyproline analysis of lung collagen
A portion of each lung sample from saline and OVA-treated RLX+/+ and RLX–/– mice were treated as described previously to determine their hydroxyproline contents (22). Hydroxyproline values were then converted to collagen content by multiplying by a factor of 6.94 (based on hydroxyproline representing approximately 14.4% of the amino acid composition of collagen, in most mammalian tissues) (23) and further expressed as a proportion of the tissue dry weight (collagen concentration).
Zymography of MMP expression
Total MMPs were extracted from a similar portion of each lung tissue from saline and OVA-treated RLX+/+ and RLX–/– mice and analyzed by gelatin zymography as described previously (24). The resulting bands on the zymograph were analyzed by densitometry using a GS 710 densitometer (Bio-Rad Laboratories, Richmond, CA) and Quantity-One software (Bio-Rad). The mean ± SE density of each MMP was graphed and expressed as the relative ratio of the values in the saline-treated RLX+/+ group, which was expressed as 1.
Statistical analysis
The results were analyzed using a one-way ANOVA; with Newman-Keuls tests for multiple comparisons between groups. Lung functional studies were analyzed with a two-way ANOVA, with Bonferroni posttest. In this paper P < 0.05 is described as statistically significant. Morphometry was expressed as median with 95% confidence interval and analyzed using the Mann-Whitney test.
Results
RLX–/– mice have normal sensitization and airway inflammation responses to OVA
To confirm that both RLX+/+ and RLX–/– mice were adequately sensitized to OVA, serum levels of OVA-specific IgE were measured in mice treated with either OVA or saline (controls). Serum levels of OVA-specific IgE in OVA-treated RLX–/– mice were equivalent to those of OVA-treated RLX+/+ mice, and both were significantly (P < 0.001) higher than levels in saline-treated controls (Fig. 1). As expected saline-treated mice did not develop any IgE response to OVA.
Both RLX+/+ and RLX–/– mice demonstrated increased total and differential BAL cell counts after OVA sensitization-treatment. There were no significant differences in total cell counts between OVA-treated RLX+/+ and RLX–/– mice. There was a marked increase in the total number of BAL eosinophils in both the RLX+/+ and RLX–/– OVA-treated mice (P < 0.001), compared with saline-treated mice (Table 1). The increased numbers of eosinophils confirmed that the chronic model of AAD (17) could be induced in RLX+/+ and RLX–/– mice. Increases in BAL neutrophils (P < 0.001), monocytes/macrophages (P < 0.001), and lymphocytes (P < 0.05) were also observed in OVA-treated mice, compared with that measured from saline-treated animals (Table 1). These combined data demonstrated that RLX+/+ and RLX–/– mice were able to mount IgE and airway inflammation responses after OVA sensitization.
Histologic analysis of lung tissues from RLX +/+ and RLX –/– mice
Lung tissue sections from saline or OVA-treated RLX+/+ and RLX–/– mice were stained with Masson trichrome to determine the relative amount and distribution of collagen deposition within the airway/lung of RLX–/– and RLX+/+ mice. Saline-treated RLX+/+ (Fig. 2A) and RLX–/– (Fig. 2B) mice had minimal collagen staining in the airway and lung. OVA-treated RLX+/+ mice demonstrated a modest increase in collagen staining in the airways (Fig. 2C), when compared with saline-treated RLX+/+ controls. A further marked increase in collagen deposition was observed in OVA-treated RLX–/– mice (Fig. 2D), compared with that observed in OVA-treated RLX+/+ mice. The increase in collagen was observed predominantly in the subepithelial basement membrane (BM) regions of the airway wall and in the submucosal regions surrounding airway structures and the larger blood vessels, with minimal deposition in the lung parenchyma (Fig. 2D).
Morphometric analysis of the total area of collagen deposition in lung tissue sections confirmed these changes. There was significantly increased collagen in the airways of OVA-treated RLX+/+ mice, compared with that in saline-treated RLX+/+ mice (P < 0.02; Fig. 2E). A further marked increase in total area of collagen was observed in OVA-treated RLX–/– mice, compared with OVA-treated RLX+/+ mice (P < 0.001; Fig. 2E).
Hydroxyproline analysis of total lung collagen, reflected what was observed by histological and morphometric examination (Fig. 2F). The lungs of saline-treated RLX+/+ and RLX–/– mice had similar levels of collagen (approximately 850 μg/lung). OVA-treated RLX+/+ mice had a 15% increase in lung collagen content, which was not significantly different (P = 0.16) from that measured from saline-treated animals. However, OVA-treated RLX–/– mice had a 26% (P < 0.05; Fig. 2F) increase in total lung collagen content, compared with lung tissues obtained from the saline-treated groups. Taken together, these findings indicate that OVA sensitization and treatment of mice was associated with increased airway fibrosis that was exaggerated in the absence of relaxin.
MMP expression and activity in saline or OVA-treated RLX+/+ and RLX–/– mice
Gelatin zymography of lung tissue from saline and OVA-treated RLX+/+ and RLX–/– mice was used to demonstrate changes in the expression and activity of MMP-9 (gelatinase-B) and MMP-2 (gelatinase-A). Densitometric analysis of the zymographs demonstrated no significant differences in either MMP-9 (Fig. 3A) or MMP-2 (Fig. 3B) expression in the lungs of saline-treated RLX+/+ mice and saline-treated RLX–/– mice. OVA treatment of RLX+/+ mice had no obvious effects on MMP-9 expression (Fig. 3A) but did induce a significant increase (60%, P < 0.05) in MMP-2 expression, compared with that measured in the lung of saline-treated mice (Fig. 3B). Conversely, in OVA-treated RLX–/– mice, a significant decrease in MMP-9 expression (P < 0.02) was observed, compared with levels measured in OVA-treated RLX+/+ mice (Fig. 3A), whereas no changes in MMP-2 expression were observed, compared with that measured in the lungs of saline-treated animals (Fig. 3B). As a result, the levels of both matrix-degrading proteinases, MMP-9 and MMP-2 (both P < 0.02), were significantly lower in OVA-treated RLX–/– mice, compared with levels detected in OVA-treated RLX+/+ mice.
Functional consequences of relaxin deficiency
To determine whether the observed differences in airway collagen deposition were associated with changes in lung function, AHR was determined by invasive plethysmography in RLX+/+ and RLX–/– mice. Both saline-treated RLX+/+ and RLX–/– mice exhibited standard responses to MCh that were not significantly different (Fig. 4). Both groups of OVA-treated mice developed increased reactivity to MCh, with an increased gradient in the dose-response curve as compared with values obtained from respective saline-treated control mice (P < 0.001) (Fig. 4). AHR in OVA-treated RLX–/– mice was equivalent to that of OVA-treated RLX+/+ mice. Therefore, OVA treatment was associated with increased airway reactivity in both RLX+/+ and RLX–/– mice that was independent of the influence of relaxin.
Other airway remodeling changes: airway epithelial thickness and goblet cell hyperplasia
Lung tissue sections from RLX+/+ and RLX–/– mice were also examined for other structural changes associated with airway remodeling. Morphometric examination of lung tissue sections from saline- and OVA-treated RLX+/+ and RLX–/– mice revealed that there were no significant alterations in epithelial thickness in any of the groups studied (Fig. 5A). There was, however, a significant increase in goblet cell numbers in both the OVA-treated RLX+/+ and OVA-treated RLX–/– mice (P < 0.001), compared with the saline-treated control animals (Fig. 5B). There was no significant difference in goblet cell numbers between OVA-treated RLX+/+ and RLX–/– mice. These findings indicated that relaxin deficiency was associated with increased airway fibrosis but did not influence these other structural changes of airway remodeling in this animal model of AAD.
Discussion
In this study, we have demonstrated a number of key findings. First, long-term OVA treatment of mice was associated with increased collagen deposition and airway fibrosis, which was exaggerated in the absence of relaxin, i.e. OVA-treated RLX–/– mice demonstrated significantly higher airway collagen deposition, compared with OVA-treated RLX+/+ mice. These findings confirm an important role for endogenous relaxin in the regulation of lung collagen turnover in response to AAD. The distribution of collagen in OVA-treated RLX–/– mice was predominantly in the epithelial BM regions of the airway wall and in the submucosal regions surrounding the airways consistent with the consequences of airway remodeling in asthma (3). Second, the increased collagen detected in OVA-treated RLX–/– mice (compared with levels detected in OVA-treated RLX+/+ mice) was mediated in part by a failure for up-regulation of MMP-2 and a significant reduction in MMP-9 expression. Third, the increased collagen detected in OVA-treated mice did not affect AHR or other structural changes within the airway. These combined findings confirm that relaxin down-regulates collagen expression in adverse AAD.
The data from both the saline and OVA-treated RLX+/+ and RLX–/– mice indicate that the long-term model of allergic airway disease was established, as described previously (16, 17, 18, 19). OVA-specific IgE was increased in OVA-treated mice but not in saline controls. Furthermore, inflammatory cells were moderately increased in OVA-treated but not saline-treated mice with a significant increase in numbers of eosinophils. This influx of eosinophils during AAD is critical because activated eosinophils are a source of TGF, which is a potent profibrotic agent. TGF induces fibroblasts to proliferate and differentiate into myofibroblasts, which are the major source of collagen in active fibrotic sites (26). Furthermore, many of the changes that represent airway remodeling in asthma were observed. The most striking of these was the area of collagen deposition in the subepithelial basement membrane regions of the airway wall in the submucosa surrounding airway structures but not in the lung parenchyma. Goblet cell proliferation was also noted but smooth muscle thickness was unaltered in this model as previously described by Kumar et al. (16). Therefore, this model can be considered to be reflective of human asthma.
To investigate the mechanisms by which relaxin may regulate collagen deposition in the airway/lung, MMP-2 and MMP-9 expression was examined in the saline and OVA-treated RLX+/+ and RLX–/– mice. Collagen deposition is controlled at several levels, first at the level of collagen gene expression, and in particular, by the balance between the MMPs that degrade fibrillar matrix proteins and the tissue inhibitors of MMPs that inhibit the activity of MMPs. The gelatinases MMP-9 and MMP-2 primarily degrade basement membrane components such as type IV collagen, fibronectin, and elastin but have also been shown to degrade type I collagen (27).
In this study a decrease in MMP-9 was demonstrated in the lungs of OVA-treated RLX–/– mice (compared with values in OVA-treated RLX+/+ mice), and a failure of up-regulation of MMP-2 expression was also identified. This suggests that a relative deficiency of these MMPs in OVA-treated RLX–/– mice was one means by which excessive collagen accumulation (fibrosis) was accelerated during AAD, in the absence of relaxin. These findings are consistent with previous studies, demonstrating that exogenous relaxin administration up-regulates either or both MMP-2 and MMP-9 in other reproductive (28) and nonreproductive (29) organs, with the differential regulation of these enzymes most likely the result of species specificity. Also consistent with this, decreased MMP-9 has been reported in chronic asthma (30) and correlated with increased deposition of collagen and fibronectin. Paradoxically, however, increased MMP-9 and an elevated MMP-9 to tissue inhibitor of MMP-1 ratio has been demonstrated in acute asthma (31, 32, 33). Therefore, MMP-9 may play a dual role in the course of asthma, reflecting its differential regulation in the early vs. later stages of disease. Because MMP-9 can degrade matrix proteins of the BM, increased MMP-9 in acute stages of asthma may contribute to the reduced integrity of airway epithelium and the aberrant epithelial repair process, whereas reduced MMP-9 in the later stages of asthma may lead to increased deposition of collagen and fibronectin resulting in BM thickening and airway fibrosis. Furthermore, MMPs are required to allow migration and regression of inflammatory cells (34); MMP-2 is found to be preferentially secreted by fibroblasts and lung epithelial cells, whereas MMP-9 is preferentially secreted by inflammatory cells including macrophages, lymphocytes, neutrophils, and eosinophils (26, 35).
Mucous cell metaplasia and hyperplasia are key features of both acute and chronic asthma with the consequent oversecretion of mucus contributing to the pathology of the disease. There is growing evidence that epithelial and connective tissue changes in asthma are interrelated via the epithelial mesenchymal trophic unit (36). Relaxin is known to influence both epithelial and connective tissues. For example, the vaginal and prostatic epithelium of relaxin-1 null mice is altered (13, 37), and relaxin stimulates bronchial epithelial cell protein kinase activation, migration, and ciliary beating in vitro (38) and induces the proliferation of human amniotic epithelium (25). In our study, the increased fibrosis associated with the absence of relaxin did not alter goblet cell numbers or epithelial thickness. There are two possible explanations for this; one possibility is that relaxin and fibrosis per se do not drive other structural changes of remodeling. Alternatively, longer periods (>6 wk) of aberrant matrix proteins are required to induce further changes in the epithelium.
AHR is a central feature of asthma and correlates closely with ongoing symptoms. Persistent AHR is a consistent finding in chronic symptomatic asthma; however, the specific contributions by individual components of airway remodeling to AHR are not known. Whereas it has been suggested that airway fibrosis may protect against AHR, in our study, it was found that AHR was not influenced by airway fibrosis per se.
We have previously shown that recombinant human relaxin treatment of 9- and 12-month-old RLX–/– mice resulted in reversal of collagen accumulation, which in turn restored lung structure and function, particularly when treatment was applied in the early stages of fibrosis (15). Relaxin also decreased the development of collagen deposition associated with airway inflammation in previously reported OVA sensitized models of AAD (10, 11). In those previous studies (11), collagen accumulation was elevated after 2 wk but was only significantly increased after 4–8 wk of OVA aerosols, which is consistent with what was seen in this model of AAD. Our present findings demonstrate for the first time an important role of endogenous relaxin in the regulation of airway fibrosis and remodeling in AAD and highlight the potential use of relaxin to reverse and/or prevent airway fibrosis in asthma. Our findings indicate that relaxin plays a critical protective role in the inhibition of collagen deposition associated with a long-term allergic inflammatory response in the airway and suggest that AAD may be associated with a relative deficiency of relaxin expression during the allergic inflammatory response. As such, relaxin may represent an effective therapeutic agent for the prevention or reversal of airway fibrosis in AAD. Relaxin may regulate the extracellular matrix either directly or indirectly via its effects on matrix proteins such as collagen and fibronectin (8, 9, 13, 14, 15). Additionally, relaxin did not appear to affect other aspects of AAD including AHR and epithelial architecture.
Acknowledgments
The authors thank Chongxin Zhao and Jacquetta Wainwright for their technical assistance.
Footnotes
This work was supported in part by a Howard Florey Institute block grant from the National Health and Medical Research Council of Australia and a Murdoch Children’s Research Institute project grant (to M.L.K.T., N.R.S., and C.S.S.). I.M. is the recipient of a Howard Florey Institute Postgraduate Scholarship.
A portion of this work was presented in the conference proceedings of the 4th International Conference on Relaxin and Related Peptides, Jackson Hole, Wyoming, September 2004.
All listed authors have nothing to declare.
First Published Online October 27, 2005
Abbreviations: AAD, Allergic airway disease; AB-PAS, Alcian blue-periodic acid Schiff; AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage; BM, basement membrane; MCh, methacholine; MMP, matrix metalloproteinase; OVA, ovalbumin; RLX+/+, relaxin wild type; RLX–/–, relaxin gene knockout.
Accepted for publication October 18, 2005.
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Murdoch Children’s Research Institute (N.R.S., S.G.R., M.L.K.T.), Royal Childrens Hospital, Parkville, Victoria 3052, Australia
Abstract
We examined the relationship among relaxin (a peptide hormone that stimulates collagen degradation), airway fibrosis, other changes of airway remodeling, and airway hyperresponsiveness (AHR) in an animal model of allergic airway disease. Eight- to 10-wk-old relaxin gene-knockout (RLX–/–) and wild-type (RLX+/+) mice were sensitized with ovalbumin (OVA) or saline ip at d 0 and 14 and challenged three times per week for 6 wk with nebulized 2.5% OVA or saline. Saline-treated control RLX+/+ and RLX–/– mice had equivalent collagen expression and baseline airway responses. OVA-treated RLX–/– mice developed airway inflammation equivalent to that in OVA-treated RLX+/+ mice. However, OVA-treated RLX–/– mice had markedly increased lung collagen deposition as compared with OVA-treated RLX+/+ and saline-treated mice (all P < 0.05). Collagen was predominantly deposited in the subepithelial basement membrane region and submucosal regions in both OVA-treated RLX+/+ and RLX–/– mice. The increased collagen measured in OVA-treated RLX–/– mice was associated with reduced matrix metalloproteinase (MMP)-9 (P < 0.02) expression and failure to up-regulate matrix metalloproteinase-2 expression, compared with levels in OVA-treated RLX+/+ mice. Goblet cell numbers were equivalent in OVA-treated RLX–/– and RLX+/+ mice and increased, compared with saline-treated animals. Both OVA-treated RLX+/+ and RLX–/– mice developed similar degrees of AHR after OVA treatment. These findings demonstrate a critical role for relaxin in the inhibition of lung collagen deposition during an allergic inflammatory response. Increased deposition of collagen per se did not influence airway epithelial structure or AHR.
Introduction
STRUCTURAL CHANGES IN the airway wall (airway remodeling) are characteristic features of allergic airway disease (AAD) that can occur early in the course of disease (1), contribute to airway hyperresponsiveness (AHR) (2), and lead to irreversible airway obstruction (1, 2). Airway remodeling is characterized by increased deposition of collagen (fibrosis) in the subepithelial basement membrane region and in the submucosal layers (3), smooth muscle hypertrophy and hyperplasia, fibroblast hyperplasia, epithelial metaplasia, and goblet cell proliferation (1). Collagen deposition in the subepithelial basement membrane region is a hallmark of airway remodeling in AAD (4); however, the cellular and molecular events that lead to the remodeling process are poorly understood. In particular, the factors regulating collagen deposition associated with airway remodeling in AAD have not been clearly defined, and the relationship between collagen deposition and other structural changes in the epithelium remain unclear. Furthermore, although corticosteroid antiinflammatory treatment can reduce symptoms, improve lung function, and modify bronchial hyperresponsiveness, these agents are not effective in reversing established airway collagen deposition or other structural changes of remodeling (5, 6). Consequently, there are currently no treatments that effectively reverse established collagen deposition or other structural changes in AAD.
Relaxin is a small dimeric peptide hormone secreted by the ovary into the blood in highest amounts during pregnancy and generally associated with female reproduction (7). However, the most consistent biological effect of relaxin is its ability to stimulate the breakdown of collagen. Relaxin stimulates collagen breakdown in preparation for successful parturition (7). Relaxin treatment has been demonstrated to inhibit fibrosis in a bleomycin-induced model of lung fibrosis (8), by inhibiting TGF-induced matrix protein production (8) and increasing the expression of the collagen-degrading enzymes, matrix metalloproteinases (MMPs) (8, 9). Furthermore, relaxin administration has been reported to prevent the development of collagen deposition associated with airway inflammation in ovalbumin (OVA)-sensitized guinea pigs (10) and OVA-sensitized challenged BALB/c mice (11), highlighting the antifibrotic potential of exogenous relaxin therapy. However, the role that endogenous relaxin plays in airway fibrosis and remodeling is yet to be determined.
Recently we generated a relaxin gene knockout (RLX–/–) mouse to further our understanding of the physiological actions of relaxin (12), which lacks the major stored and circulating form of relaxin. RLX–/– mice developed an age-related progression of fibrosis in several tissues (13, 14, 15), including the airway and lung that was first detectable at 6–9 months of age (15). Furthermore, treatment of RLX–/– mice with recombinant human relaxin-2 relaxin successfully reversed established fibrosis in the airways and lung (15). These findings suggested an important role for endogenous relaxin in regulating collagen deposition within the lung and further confirmed the potential role for exogenous relaxin in the treatment of fibrosis associated with airway and lung disorders. Based on these combined findings, we hypothesize that endogenous relaxin protects the airways and lung from fibrosis in response to AAD.
The 9-wk murine model of AAD mimics a majority of the changes observed in human asthma including: the recruitment of eosinophils, goblet cell hyperplasia, and/or metaplasia and a distinctive pattern of subepithelial fibrosis (16, 17, 18, 19), although no changes in smooth muscle thickening are detected. Whereas this model reflects several changes seen in human AAD, it is strain dependent (16, 17, 18, 19, 20). In the present study, we examined the role of endogenous relaxin in the regulation of collagen deposition and airway remodeling using this model of AAD on RLX–/– mice. The relationship among airway fibrosis, other structural changes of remodeling, and AHR was also examined. The RLX–/– mouse is an ideal model to study the specific contributions of endogenous relaxin to airway fibrosis in AAD.
Materials and Methods
Animals
Relaxin knockout mice were generated as described previously (12). All female relaxin wild-type (RLX+/+) and RLX–/– mice used in this study were generated from relaxin heterozygous (C57BLk6J/129SV) parents (12) and were age-matched littermates. Eight- to 10-wk-old RLX+/+ and RLX–/– mice with similar baseline collagen levels (15) were used in all studies. The animals were housed in a controlled environment and maintained as detailed before (15). These experiments were approved by the Howard Florey Institute and the Royal Children’s Hospital Animal Experimental Ethics Committees, which adhere to the Australian Code of Practice for the care and use of laboratory animals for scientific purposes.
OVA-induced allergic airway disease model
An OVA-induced model of AAD was used as previously described (21). Eight- to 10-wk-old RLX+/+ and RLX–/– mice were sensitized with an ip injection of 10 μg of grade V OVA (Sigma Chemical, St. Louis, MO) and 1 mg of aluminum potassium sulfate (Sigma) in 0.5 ml saline on d 0 and 14. Mice were then challenged 3 d/wk for 6 wk (from d 21 to 63) with an aerosol of 2.5% (wt/vol) OVA in saline solution for 30 min (21). Vehicle-treated mice received 0.5 ml saline alone ip on d 0 and 14 and were challenged with saline aerosols.
Analysis of AHR
Methacholine (MCh)-induced airway reactivity was assessed 24 h after the final aerosol challenge by invasive plethysmography (Buxco Electronics, Troy, NY). Briefly, mice were anesthetized by ip injection of ketamine (200 μg/g) and xylazine (10 μg/g), tracheostomized, and the jugular vein cannulated. Mice were ventilated with a small animal respirator (Harvard Apparatus, Holliston, MA) delivering 0.01 ml/g body weight at a rate of 120 strokes/min in a mouse plethysmograph chamber. Increasing MCh doses were delivered iv and airway resistance and compliance measured (Biosystem XA version 2.7.9; Buxco Electronics) for 2 min after each dose. Results are expressed as the maximal resistance after each dose of MCh minus baseline (PBS alone) resistance.
Determination of OVA-specific antibody titers by ELISA
Serum was obtained 24 h after the final OVA or saline challenge and stored at –20 C. Levels of OVA-specific IgE were determined by ELISA, as described previously (21).
Bronchoalveolar lavage (BAL)
After measurement of airway reactivity, BAL was carried out, as detailed previously (21). Total viable cell counts were determined in a hemocytometer using trypan blue exclusion. Differential counts of eosinophils, neutrophils, lymphocytes, and monocytes/macrophages were determined on cytospin smears of BAL samples (4 x 105 cells) from individual mice stained with DiffQuick (Life Technologies, Auckland, New Zealand) after counting 300 cells. Results are expressed as total cell number x 104.
Tissue collection
Lung tissues were weighed (total lung weight) and then separated into individual lobes for various analyses including hydroxyproline analysis, histological analysis, and MMP zymography.
Lung histopathology
The right lung lobe and trachea were fixed in 10% neutral buffered formalin for 18–24 h and routinely processed. Serial 5-μm sections taken every 100 μm were stained with hematoxylin and eosin for assessment of peribronchial inflammation, with Masson trichrome for assessment of collagen and Alcian blue-periodic acid Schiff (AB-PAS) for assessment of goblet cells.
Morphometric analysis of structural changes
Images of lung tissue sections were captured using a Digital camera (Q Imaging, Burnaby, British Columbia, Canada). A minimum of five bronchi measuring 150–350 μm luminal diameter were analyzed per mouse for the parameters described below using Image Pro-Discovery software (Media Cybernetics, Silver Spring, MD), which was calibrated with a reference micrometer slide. The thickness of the bronchial epithelial layer was measured by tracing around the basement membrane and the luminal surface of epithelial cells and calculating the area between these lines, using a digitizer (Aiptek, Irvine, CA). Total collagen thickness was similarly measured by tracing around the outer extent of the total collagen layer in the submucosal region and around the basement membrane and the area between these lines calculated. Total areas were calculated by subtracting the inner area from the outer area. These areas were expressed per length (micrometers) of basement membrane to account for variation in bronchial diameters. Goblet cells were counted in AB-PAS stained sections and expressed as number of cells per 100 μm of basement membrane.
Hydroxyproline analysis of lung collagen
A portion of each lung sample from saline and OVA-treated RLX+/+ and RLX–/– mice were treated as described previously to determine their hydroxyproline contents (22). Hydroxyproline values were then converted to collagen content by multiplying by a factor of 6.94 (based on hydroxyproline representing approximately 14.4% of the amino acid composition of collagen, in most mammalian tissues) (23) and further expressed as a proportion of the tissue dry weight (collagen concentration).
Zymography of MMP expression
Total MMPs were extracted from a similar portion of each lung tissue from saline and OVA-treated RLX+/+ and RLX–/– mice and analyzed by gelatin zymography as described previously (24). The resulting bands on the zymograph were analyzed by densitometry using a GS 710 densitometer (Bio-Rad Laboratories, Richmond, CA) and Quantity-One software (Bio-Rad). The mean ± SE density of each MMP was graphed and expressed as the relative ratio of the values in the saline-treated RLX+/+ group, which was expressed as 1.
Statistical analysis
The results were analyzed using a one-way ANOVA; with Newman-Keuls tests for multiple comparisons between groups. Lung functional studies were analyzed with a two-way ANOVA, with Bonferroni posttest. In this paper P < 0.05 is described as statistically significant. Morphometry was expressed as median with 95% confidence interval and analyzed using the Mann-Whitney test.
Results
RLX–/– mice have normal sensitization and airway inflammation responses to OVA
To confirm that both RLX+/+ and RLX–/– mice were adequately sensitized to OVA, serum levels of OVA-specific IgE were measured in mice treated with either OVA or saline (controls). Serum levels of OVA-specific IgE in OVA-treated RLX–/– mice were equivalent to those of OVA-treated RLX+/+ mice, and both were significantly (P < 0.001) higher than levels in saline-treated controls (Fig. 1). As expected saline-treated mice did not develop any IgE response to OVA.
Both RLX+/+ and RLX–/– mice demonstrated increased total and differential BAL cell counts after OVA sensitization-treatment. There were no significant differences in total cell counts between OVA-treated RLX+/+ and RLX–/– mice. There was a marked increase in the total number of BAL eosinophils in both the RLX+/+ and RLX–/– OVA-treated mice (P < 0.001), compared with saline-treated mice (Table 1). The increased numbers of eosinophils confirmed that the chronic model of AAD (17) could be induced in RLX+/+ and RLX–/– mice. Increases in BAL neutrophils (P < 0.001), monocytes/macrophages (P < 0.001), and lymphocytes (P < 0.05) were also observed in OVA-treated mice, compared with that measured from saline-treated animals (Table 1). These combined data demonstrated that RLX+/+ and RLX–/– mice were able to mount IgE and airway inflammation responses after OVA sensitization.
Histologic analysis of lung tissues from RLX +/+ and RLX –/– mice
Lung tissue sections from saline or OVA-treated RLX+/+ and RLX–/– mice were stained with Masson trichrome to determine the relative amount and distribution of collagen deposition within the airway/lung of RLX–/– and RLX+/+ mice. Saline-treated RLX+/+ (Fig. 2A) and RLX–/– (Fig. 2B) mice had minimal collagen staining in the airway and lung. OVA-treated RLX+/+ mice demonstrated a modest increase in collagen staining in the airways (Fig. 2C), when compared with saline-treated RLX+/+ controls. A further marked increase in collagen deposition was observed in OVA-treated RLX–/– mice (Fig. 2D), compared with that observed in OVA-treated RLX+/+ mice. The increase in collagen was observed predominantly in the subepithelial basement membrane (BM) regions of the airway wall and in the submucosal regions surrounding airway structures and the larger blood vessels, with minimal deposition in the lung parenchyma (Fig. 2D).
Morphometric analysis of the total area of collagen deposition in lung tissue sections confirmed these changes. There was significantly increased collagen in the airways of OVA-treated RLX+/+ mice, compared with that in saline-treated RLX+/+ mice (P < 0.02; Fig. 2E). A further marked increase in total area of collagen was observed in OVA-treated RLX–/– mice, compared with OVA-treated RLX+/+ mice (P < 0.001; Fig. 2E).
Hydroxyproline analysis of total lung collagen, reflected what was observed by histological and morphometric examination (Fig. 2F). The lungs of saline-treated RLX+/+ and RLX–/– mice had similar levels of collagen (approximately 850 μg/lung). OVA-treated RLX+/+ mice had a 15% increase in lung collagen content, which was not significantly different (P = 0.16) from that measured from saline-treated animals. However, OVA-treated RLX–/– mice had a 26% (P < 0.05; Fig. 2F) increase in total lung collagen content, compared with lung tissues obtained from the saline-treated groups. Taken together, these findings indicate that OVA sensitization and treatment of mice was associated with increased airway fibrosis that was exaggerated in the absence of relaxin.
MMP expression and activity in saline or OVA-treated RLX+/+ and RLX–/– mice
Gelatin zymography of lung tissue from saline and OVA-treated RLX+/+ and RLX–/– mice was used to demonstrate changes in the expression and activity of MMP-9 (gelatinase-B) and MMP-2 (gelatinase-A). Densitometric analysis of the zymographs demonstrated no significant differences in either MMP-9 (Fig. 3A) or MMP-2 (Fig. 3B) expression in the lungs of saline-treated RLX+/+ mice and saline-treated RLX–/– mice. OVA treatment of RLX+/+ mice had no obvious effects on MMP-9 expression (Fig. 3A) but did induce a significant increase (60%, P < 0.05) in MMP-2 expression, compared with that measured in the lung of saline-treated mice (Fig. 3B). Conversely, in OVA-treated RLX–/– mice, a significant decrease in MMP-9 expression (P < 0.02) was observed, compared with levels measured in OVA-treated RLX+/+ mice (Fig. 3A), whereas no changes in MMP-2 expression were observed, compared with that measured in the lungs of saline-treated animals (Fig. 3B). As a result, the levels of both matrix-degrading proteinases, MMP-9 and MMP-2 (both P < 0.02), were significantly lower in OVA-treated RLX–/– mice, compared with levels detected in OVA-treated RLX+/+ mice.
Functional consequences of relaxin deficiency
To determine whether the observed differences in airway collagen deposition were associated with changes in lung function, AHR was determined by invasive plethysmography in RLX+/+ and RLX–/– mice. Both saline-treated RLX+/+ and RLX–/– mice exhibited standard responses to MCh that were not significantly different (Fig. 4). Both groups of OVA-treated mice developed increased reactivity to MCh, with an increased gradient in the dose-response curve as compared with values obtained from respective saline-treated control mice (P < 0.001) (Fig. 4). AHR in OVA-treated RLX–/– mice was equivalent to that of OVA-treated RLX+/+ mice. Therefore, OVA treatment was associated with increased airway reactivity in both RLX+/+ and RLX–/– mice that was independent of the influence of relaxin.
Other airway remodeling changes: airway epithelial thickness and goblet cell hyperplasia
Lung tissue sections from RLX+/+ and RLX–/– mice were also examined for other structural changes associated with airway remodeling. Morphometric examination of lung tissue sections from saline- and OVA-treated RLX+/+ and RLX–/– mice revealed that there were no significant alterations in epithelial thickness in any of the groups studied (Fig. 5A). There was, however, a significant increase in goblet cell numbers in both the OVA-treated RLX+/+ and OVA-treated RLX–/– mice (P < 0.001), compared with the saline-treated control animals (Fig. 5B). There was no significant difference in goblet cell numbers between OVA-treated RLX+/+ and RLX–/– mice. These findings indicated that relaxin deficiency was associated with increased airway fibrosis but did not influence these other structural changes of airway remodeling in this animal model of AAD.
Discussion
In this study, we have demonstrated a number of key findings. First, long-term OVA treatment of mice was associated with increased collagen deposition and airway fibrosis, which was exaggerated in the absence of relaxin, i.e. OVA-treated RLX–/– mice demonstrated significantly higher airway collagen deposition, compared with OVA-treated RLX+/+ mice. These findings confirm an important role for endogenous relaxin in the regulation of lung collagen turnover in response to AAD. The distribution of collagen in OVA-treated RLX–/– mice was predominantly in the epithelial BM regions of the airway wall and in the submucosal regions surrounding the airways consistent with the consequences of airway remodeling in asthma (3). Second, the increased collagen detected in OVA-treated RLX–/– mice (compared with levels detected in OVA-treated RLX+/+ mice) was mediated in part by a failure for up-regulation of MMP-2 and a significant reduction in MMP-9 expression. Third, the increased collagen detected in OVA-treated mice did not affect AHR or other structural changes within the airway. These combined findings confirm that relaxin down-regulates collagen expression in adverse AAD.
The data from both the saline and OVA-treated RLX+/+ and RLX–/– mice indicate that the long-term model of allergic airway disease was established, as described previously (16, 17, 18, 19). OVA-specific IgE was increased in OVA-treated mice but not in saline controls. Furthermore, inflammatory cells were moderately increased in OVA-treated but not saline-treated mice with a significant increase in numbers of eosinophils. This influx of eosinophils during AAD is critical because activated eosinophils are a source of TGF, which is a potent profibrotic agent. TGF induces fibroblasts to proliferate and differentiate into myofibroblasts, which are the major source of collagen in active fibrotic sites (26). Furthermore, many of the changes that represent airway remodeling in asthma were observed. The most striking of these was the area of collagen deposition in the subepithelial basement membrane regions of the airway wall in the submucosa surrounding airway structures but not in the lung parenchyma. Goblet cell proliferation was also noted but smooth muscle thickness was unaltered in this model as previously described by Kumar et al. (16). Therefore, this model can be considered to be reflective of human asthma.
To investigate the mechanisms by which relaxin may regulate collagen deposition in the airway/lung, MMP-2 and MMP-9 expression was examined in the saline and OVA-treated RLX+/+ and RLX–/– mice. Collagen deposition is controlled at several levels, first at the level of collagen gene expression, and in particular, by the balance between the MMPs that degrade fibrillar matrix proteins and the tissue inhibitors of MMPs that inhibit the activity of MMPs. The gelatinases MMP-9 and MMP-2 primarily degrade basement membrane components such as type IV collagen, fibronectin, and elastin but have also been shown to degrade type I collagen (27).
In this study a decrease in MMP-9 was demonstrated in the lungs of OVA-treated RLX–/– mice (compared with values in OVA-treated RLX+/+ mice), and a failure of up-regulation of MMP-2 expression was also identified. This suggests that a relative deficiency of these MMPs in OVA-treated RLX–/– mice was one means by which excessive collagen accumulation (fibrosis) was accelerated during AAD, in the absence of relaxin. These findings are consistent with previous studies, demonstrating that exogenous relaxin administration up-regulates either or both MMP-2 and MMP-9 in other reproductive (28) and nonreproductive (29) organs, with the differential regulation of these enzymes most likely the result of species specificity. Also consistent with this, decreased MMP-9 has been reported in chronic asthma (30) and correlated with increased deposition of collagen and fibronectin. Paradoxically, however, increased MMP-9 and an elevated MMP-9 to tissue inhibitor of MMP-1 ratio has been demonstrated in acute asthma (31, 32, 33). Therefore, MMP-9 may play a dual role in the course of asthma, reflecting its differential regulation in the early vs. later stages of disease. Because MMP-9 can degrade matrix proteins of the BM, increased MMP-9 in acute stages of asthma may contribute to the reduced integrity of airway epithelium and the aberrant epithelial repair process, whereas reduced MMP-9 in the later stages of asthma may lead to increased deposition of collagen and fibronectin resulting in BM thickening and airway fibrosis. Furthermore, MMPs are required to allow migration and regression of inflammatory cells (34); MMP-2 is found to be preferentially secreted by fibroblasts and lung epithelial cells, whereas MMP-9 is preferentially secreted by inflammatory cells including macrophages, lymphocytes, neutrophils, and eosinophils (26, 35).
Mucous cell metaplasia and hyperplasia are key features of both acute and chronic asthma with the consequent oversecretion of mucus contributing to the pathology of the disease. There is growing evidence that epithelial and connective tissue changes in asthma are interrelated via the epithelial mesenchymal trophic unit (36). Relaxin is known to influence both epithelial and connective tissues. For example, the vaginal and prostatic epithelium of relaxin-1 null mice is altered (13, 37), and relaxin stimulates bronchial epithelial cell protein kinase activation, migration, and ciliary beating in vitro (38) and induces the proliferation of human amniotic epithelium (25). In our study, the increased fibrosis associated with the absence of relaxin did not alter goblet cell numbers or epithelial thickness. There are two possible explanations for this; one possibility is that relaxin and fibrosis per se do not drive other structural changes of remodeling. Alternatively, longer periods (>6 wk) of aberrant matrix proteins are required to induce further changes in the epithelium.
AHR is a central feature of asthma and correlates closely with ongoing symptoms. Persistent AHR is a consistent finding in chronic symptomatic asthma; however, the specific contributions by individual components of airway remodeling to AHR are not known. Whereas it has been suggested that airway fibrosis may protect against AHR, in our study, it was found that AHR was not influenced by airway fibrosis per se.
We have previously shown that recombinant human relaxin treatment of 9- and 12-month-old RLX–/– mice resulted in reversal of collagen accumulation, which in turn restored lung structure and function, particularly when treatment was applied in the early stages of fibrosis (15). Relaxin also decreased the development of collagen deposition associated with airway inflammation in previously reported OVA sensitized models of AAD (10, 11). In those previous studies (11), collagen accumulation was elevated after 2 wk but was only significantly increased after 4–8 wk of OVA aerosols, which is consistent with what was seen in this model of AAD. Our present findings demonstrate for the first time an important role of endogenous relaxin in the regulation of airway fibrosis and remodeling in AAD and highlight the potential use of relaxin to reverse and/or prevent airway fibrosis in asthma. Our findings indicate that relaxin plays a critical protective role in the inhibition of collagen deposition associated with a long-term allergic inflammatory response in the airway and suggest that AAD may be associated with a relative deficiency of relaxin expression during the allergic inflammatory response. As such, relaxin may represent an effective therapeutic agent for the prevention or reversal of airway fibrosis in AAD. Relaxin may regulate the extracellular matrix either directly or indirectly via its effects on matrix proteins such as collagen and fibronectin (8, 9, 13, 14, 15). Additionally, relaxin did not appear to affect other aspects of AAD including AHR and epithelial architecture.
Acknowledgments
The authors thank Chongxin Zhao and Jacquetta Wainwright for their technical assistance.
Footnotes
This work was supported in part by a Howard Florey Institute block grant from the National Health and Medical Research Council of Australia and a Murdoch Children’s Research Institute project grant (to M.L.K.T., N.R.S., and C.S.S.). I.M. is the recipient of a Howard Florey Institute Postgraduate Scholarship.
A portion of this work was presented in the conference proceedings of the 4th International Conference on Relaxin and Related Peptides, Jackson Hole, Wyoming, September 2004.
All listed authors have nothing to declare.
First Published Online October 27, 2005
Abbreviations: AAD, Allergic airway disease; AB-PAS, Alcian blue-periodic acid Schiff; AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage; BM, basement membrane; MCh, methacholine; MMP, matrix metalloproteinase; OVA, ovalbumin; RLX+/+, relaxin wild type; RLX–/–, relaxin gene knockout.
Accepted for publication October 18, 2005.
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