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Howard Florey Institute of Experimental Physiology and Medicine (C.S.S., C.Z., G.W.T.) and Department of Biochemistry and Molecular Biology (C.S.S., G.W.T.), The University of Melbourne, Parkville, Victoria 3010, Australia; and Department of Allergy and Immunology (S.G.R., M.D.B., M.L.K.T.), Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, Victoria 3052, Australia
Address all correspondence and requests for reprints to: Associate Professor Mimi L. K. Tang, M.D., Ph.D., Department of Allergy and Immunology, Royal Children’s Hospital, Parkville, Victoria 3052, Australia. E-mail: mimi.tang{at}rch.org.au.
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Abstract |
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Introduction |
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More recently relaxin has been shown to have additional effects beyond its role in regulating collagen turnover. In particular, relaxin has been reported to regulate epithelial and mesenchymal remodeling processes (9) as part of its pleiotropic actions. Whereas relaxin-deficient mice aged 12 months developed increased fibrosis in the airways and lung, they were also noted to develop epithelial thickening and altered lung function (increased lung recoil) (4). These findings suggest that relaxin may regulate other aspects of airway/lung structure and function beyond its capacity to stimulate collagen metabolism and that these effects may be mediated via direct relaxin effects or via indirect effects of pathological fibrosis. However, the effects of relaxin on airway/lung structure and function in healthy and diseased states are yet to be fully understood.
Interestingly, the structural changes noted in relaxin-deficient mice aged 12 months are remarkably similar to those observed in asthma. Airway remodeling in asthma includes subepithelial basement membrane fibrosis, epithelial thickening, increased vascularity, increased smooth muscle hypertrophy and hyperplasia, goblet cell metaplasia, and neovascularization (10, 11). These structural changes contribute to loss of lung function and increased airway hyperresponsiveness (AHR). A predominance of Th2 lymphocytes is characteristic of allergic inflammation in asthma. Mast cells, eosinophils, and lymphocytes are recruited to the airway and Th2 lymphocytes release IL-4 and IL-13 that induce class switching to IgE. Airway remodeling may therefore be mediated by products of the Th2 pathway. However, the relationship between airway structural changes, airway inflammation, and AHR in asthma remains unclear, with the etiology of remodeling being not necessarily secondary to airway inflammation. Furthermore, mathematical modeling studies (12, 13) suggest that structural changes in the airway can contribute directly to AHR, although this has not been directly demonstrated.
In the present study, we examined the broader role of endogenous relaxin in the regulation of airway/lung structure and function. The relationship among airway structural changes, airway inflammation, and AHR were also examined, using an animal model of AAD.
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Materials and Methods |
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Ovalbumin-induced allergic airways disease model
An acute OVA-induced model of AAD was used as previously described (15, 16, 17). Rln+/+ and Rln–/– mice were sensitized with an ip injection of 10 µg grade V OVA (Sigma Chemical, St. Louis, MO) and 1 mg of aluminum potassium sulfate (alum; Sigma) in 0.5 ml saline on d 0. Mice were then challenged once daily on d 14–20 (30 min) with 1% (wt/vol) OVA in saline (16, 17). Aerosol challenge was carried out on groups of up to 16 mice in a whole-body inhalation exposure system attached to an ultrasonic nebulizer (NE-U07; Omron Corp., Tokyo, Japan) with an output of 1 ml/min and 1- to 5-µm particle size. Vehicle-treated mice received 0.5 ml of saline in 1 mg alum on d 0 and were challenged with saline alone on d 14–20.
Analysis of airway reactivity
On d 21, 24 h after the last OVA or saline challenge, unrestrained Rln+/+ (n = 6 saline treated, n = 8 OVA treated) and Rln–/– (n = 6 saline treated, n = 8 OVA treated) mice were assessed for methacholine (MCh)-induced airway reactivity, using noninvasive whole-body plethysmography (Buxco Electronics Inc., Troy, NY), as described previously (15, 16, 17, 18). Enhanced pause (Penh) was used as an indicator of airway resistance. Penh is a dimensionless value representing a function of the proportion of maximal expiratory to maximal inspiratory box pressure signals and the time of expiration. Mice were closely monitored and readings were taken during normal respiration.
MCh-induced airway reactivity was also analyzed by invasive plethysmography (Buxco Electronics) in Rln+/+ (n = 6 saline treated, n = 7 OVA treated) and Rln–/– (n = 6 saline treated, n = 6 OVA treated) mice. Mice were anesthetized by an ip injection of ketamine (200 µg/g) and xylazine (10 µg/g), tracheostomized, and the jugular vein cannulated. Increasing MCh doses were delivered iv and airway resistance and compliance measured for 2 min after each dose. Results were expressed as the maximal resistance after each dose of MCh minus baseline (PBS alone) resistance (5).
OVA-specific IgE ELISA
To validate the model of AAD and confirm that the mice were adequately sensitized to OVA, serum levels of OVA-specific IgE were determined by ELISA, as described previously (15, 16, 17).
Bronchoalveolar lavage (BAL)
To assess the influx of leukocytes into the airway lumen, BAL was performed on anesthetized mice on d 21, 24 h after the last OVA or saline challenge. Total viable cell counts were determined manually by trypan blue exclusion. Differential counts of eosinophils, neutrophils, lymphocytes, and monocytes were determined on cytospin smears of BAL samples (5 x 104 cells) stained with DiffQuick (Life Technologies, Auckland, New Zealand) after counting 300 cells.
Tissue collection
Lung tissues were isolated and weighed (total lung weight) and then separated into individual lobes for biochemical and histological analyses, as detailed below.
Hydroxyproline analysis of lung collagen
Lung tissues were treated as described previously (4, 19) to determine hydroxyproline content. Hydroxyproline values were converted to collagen content by multiplying by 6.94 (based on hydroxyproline representing 
14.4% of the amino acid composition of collagen in most mammalian tissues) (20).
Lung histology
A portion of lung tissue from OVA- and saline-treated RLX+/+ and RLX–/– mice (n = 6–10 per group) were treated as described previously (4). Three representative sections from each tissue were stained with hematoxylin and eosin (for morphological assessment), Masson trichrome (for assessment of fibrosis), and Alcian blue period acid Schiff (for identification of goblet cells). Morphometric analysis of airway structural changes including subepithelial collagen deposition, epithelial layer thickness, and goblet cell count was performed on bronchi with a diameter of 150–350 µm as described previously (5, 17, 21) using Image Pro-Discovery software (Media Cybernetics, Silver Spring, MD), which was calibrated with a reference micrometer slide. Twenty measurements were taken from at least five bronchi from each representative tissue section and the mean ± SE was determined.
Statistical analysis
Results were analyzed using a one-way ANOVA, using the Newman-Keuls test for multiple comparisons between groups. Airway reactivity was analyzed with a two-way ANOVA, with Bonferroni posttest. All data in this paper are presented as the mean ± SEM, with P < 0.05 described as statistically significant.
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Results |
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), whereas saline-treated Rln–/– mouse lung sections revealed markedly increased collagen deposition in the airways (Fig. 1B). The increased collagen staining in saline-treated Rln–/– mice was predominantly identified in the subepithelial basement membrane (BM) regions of the airway wall and in the adventitia of the airways, with minimal deposition in the lung parenchyma (Fig. 1B).
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). Collagen content in the lungs of saline-treated Rln–/– mice was significantly increased (by 33.7%, P < 0.05), compared with that measured in saline-treated Rln+/+ animals (Fig. 1E). Morphometric analysis of the total area of subepithelial collagen deposition in the BM of lung tissue sections demonstrated a 48.8% increase (P < 0.05) in the area of collagen deposition in saline-treated Rln–/– mice, compared with that measured in saline-treated Rln+/+ animals (Fig. 1F). These findings confirmed that Rln–/– mice aged 12 months have increased airway fibrosis.
Effects of relaxin deficiency on airway epithelial structure
Lung tissues from saline-treated Rln+/+ and Rln–/– mice were analyzed by histology/morphometry to measure changes in airway epithelial structure. Compared with saline-treated Rln+/+ mice, the epithelial layer of saline-treated Rln–/– mice demonstrated increased thickening (by 26%, P < 0.05) (Fig. 1, B and G). However, the absence of relaxin in Rln–/– mice did not result in any changes in goblet cell numbers (Fig. 1H).
Relaxin-deficient mice have increased AHR
To determine whether the structural changes in the airways of saline-treated Rln–/– mice were associated with physiological differences in lung function, methacholine-induced airway responses were assessed by noninvasive whole-body plethysmography (Fig. 2A) and direct (invasive) measurement of airway resistance (Fig. 2B). For noninvasive whole-body plethysmography, Penh was used as an indicator of airway resistance (Fig. 2A). Remarkably, saline-treated Rln–/– mice revealed a significant increase in airway resistance [by both Penh (Fig. 2A) and invasive plethysmography (Fig. 2B)], compared with that measured in Rln+/+ animals (both P < 0.05). These findings suggest that structural changes of airway fibrosis and/or epithelial thickening can influence lung function leading to increased AHR.
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As expected, OVA sensitized Rln+/+ mice developed increased serum levels of OVA-specific IgE (P < 0.05), compared with that measured in saline-treated control animals (Fig. 3A). Total BAL inflammatory cell counts were significantly elevated (P < 0.05) in OVA-treated Rln+/+ mice, compared with that measured in control animals (Fig. 3B). The airway epithelial layer of OVA-treated Rln+/+ mice was thickened (by 43%, P < 0.05) (Fig. 1, C and G), compared with saline-treated Rln+/+ mice. Goblet cell counts were also increased (P < 0.01) (Fig. 1H), compared with that measured in saline-treated Rln+/+ mice. The increased inflammation detected in OVA-treated Rln+/+ mice was not associated with increased fibrosis in these animals, as detected in Masson trichrome-stained lung sections (Fig. 1C), total lung collagen content (Fig. 1E), and morphometric analysis of subepithelial BM collagen (Fig. 1F), when compared with saline-treated control animals.
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) and invasive direct plethysmography (P < 0.05; Fig. 4B), compared with saline-treated Rln+/+ animals. For both noninvasive and invasive measures of airway resistance, AHR was found to be similar in OVA-sensitized/challenged Rln+/+ mice and nonsensitized/challenged Rln–/– mice. These findings demonstrate that increased AHR can be induced by inflammation (with goblet cell hyperplasia and epithelial thickening) or structural changes of airway fibrosis and/or epithelial thickness alone.
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Serum levels of OVA-specific IgE in OVA sensitized-challenged Rln–/– mice were equivalent to those of OVA sensitized Rln+/+ mice, and both were significantly higher (both P < 0.05) than levels in saline sensitized-challenged controls (Fig. 3A). Total BAL cell counts in OVA-treated Rln–/– mice were also equivalent to those of OVA sensitized Rln+/+ animals and significantly higher (P < 0.05) than BAL counts in both saline-treated Rln+/+ and Rln–/– mice (Fig. 3B). These findings demonstrated that Rln–/– mice mount normal IgE and airway inflammation responses after OVA sensitization and challenge that are equivalent to those of OVA-treated Rln+/+ mice.
OVA-treated Rln–/– mice demonstrated increased collagen deposition (fibrosis) in the subepithelial BM regions of the airway wall and surrounding the airway structures (Fig. 1D), compared with that measured in OVA-treated Rln+/+ (Fig. 1C) and saline-treated Rln+/+ mice (Fig. 1A). Total lung collagen content in OVA-treated Rln–/– animals was 43% (P < 0.05) higher than that measured in OVA-sensitized/challenged Rln+/+ mice and 61% (P < 0.01) higher than that in saline-treated Rln+/+ mice (Fig. 1E). More specifically, subepithelial collagen deposition in the BM of OVA-treated Rln–/– mice was increased, compared with that measured in OVA-treated Rln+/+ mice (by 21%; P < 0.05) and saline-treated control Rln+/+ mice (by 62.5%; P < 0.05). However, none of these measures in OVA-treated Rln–/– mice were significantly different from that in saline-treated Rln–/– animals (Fig. 1, E and F). Furthermore, epithelial thickness (by 24%, P < 0.05) (Fig. 1G) but not goblet cell counts (Fig. 1H) was significantly elevated in OVA-treated Rln–/– mice, compared with that measured in OVA-treated Rln+/+ animals. Thus, the absence of relaxin was associated with increased lung fibrosis and epithelial thickening, compared with wild-type control animals in both the healthy state and in an animal model of AAD.
Analysis of AHR in OVA-treated Rln–/– mice demonstrated significantly increased airway resistance, as measured by whole body plethysmography (P < 0.05; Fig. 4A) and invasive plethysmography (P < 0.05; Fig. 4B), compared with that measured in saline-treated Rln+/+ mice. However, the increased AHR measured in OVA-treated Rln–/– mice (inflammation and fibrosis) was not significantly different to that measured in saline-treated Rln–/– mice (fibrosis alone) and OVA-treated Rln+/+ mice (inflammation alone), suggesting that structural changes and airway inflammation do not have an additive effect on airway function.
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Discussion |
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The finding that epithelial thickening (in addition to increased airway/lung fibrosis) was increased in Rln–/– mice after only 7 d of OVA challenge points to the presence of important and close interactions between the extracellular matrix and epithelium and implicates a major role for relaxin in the regulation of airway epithelial structure and function in Rln–/– mice. This is consistent with a previous in vitro study (9) that demonstrated that relaxin stimulated the migration of bovine bronchial epithelial cells, resulting in the augmentation of airway repair and also improving clearance in the lung via a protein kinase A-dependent mechanism. In other studies, relaxin deficiency led to the inability of the epithelial lining in the vagina of pregnancy (22) and developing prostate (23) to proliferate, which was associated with these organs having increased collagen deposition, compared with that in wild-type mice (containing relaxin), confirming that relaxin was required for epithelial proliferation and growth in addition to its ability to regulate collagen turnover. Radiolabeled relaxin binds to the lung (24) and its major receptor, RXFP1, is expressed in this tissue (25). The cellular localization of RXFP1 in the lung has not been published, but we have preliminary immunohistochemical data showing RXFP1 to be present in bronchial epithelial cells in mice and humans (data not shown). RXFP1 has been shown to be expressed in epithelium and/or the stroma in other tissues in which it has been studied (25). Whether relaxin directly effects airway epithelial structure or indirectly influences it via regulation of collagen metabolism is still to be determined.
Interestingly, epithelial thickening was present in both saline-treated Rln–/– mice and OVA-treated Rln+/+ mice, and both groups of animals demonstrated equivalent increases in AHR. This raises the possibility that changes in epithelial structure in both mouse groups contributed to increased AHR. Thus, further studies to delineate the relative contributions of fibrosis and epithelial thickening to AHR are warranted. Of note, in our previously reported studies, young (8–10 wk old) Rln–/– mice had normal collagen expression and epithelial structure (5) and did not develop any changes in AHR above that observed for age-matched Rln+/+ animals. In this study, we demonstrated for the first time that relaxin deficiency was associated with increased AHR. Rln–/– mice aged 12 months had markedly increased AHR equivalent to that in wild-type mice with allergic airway inflammation. The increased AHR in Rln–/– mice aged 12 months may have resulted from increased fibrosis or increased epithelial thickening or both. Mathematical models support a contribution by both components of structural change (12, 13) because they predict that the increased wall thickness on the luminal side of the smooth muscle can lead to increased airway constriction. Studies in asthma have shown a strong correlation between subepithelial fibrosis and AHR, suggesting that fibrosis is likely to be important in this process. AHR is a central feature of asthma. The relative contributions of airway inflammation and airway remodeling to AHR have not been clearly defined. Moreover, the specific contributions by individual components of airway remodeling to AHR are not known. Our findings demonstrate that airway structural changes can result in AHR independent of airway inflammation. Wild-type mice sensitized challenged with OVA with airway inflammation and epithelial thickening (without any marked airway fibrosis), had a similar degree of AHR as that induced by increased collagen deposition (fibrosis) in the subepithelial BM region and epithelial thickening in Rln–/– mice.
One of the most striking findings of our study was that although Rln–/– mice sensitized challenged with OVA demonstrated significantly increased airway fibrosis and epithelial thickening over and above that measured in OVA-treated wild-type mice, AHR in these animals was not significantly different to that measured in saline-treated Rln–/– mice (associated with fibrosis alone) or OVA-treated Rln+/+ animals (associated with inflammation alone). Thus, although both airway structural changes and airway inflammation can individually lead to AHR, the combined presence of airway fibrosis and airway inflammation together did not have an additive effect on AHR. We hypothesize from these findings that the increased AHR induced by structural changes of fibrosis and epithelial thickening (in Rln–/– mice aged 12 months) reaches a certain threshold, and cannot be further elevated by additional increases in fibrosis (in OVA-treated Rln–/– mice) or induction of inflammation (in OVA treated Rln+/+ mice). Alternatively, it is possible that existing fibrosis may be protective against further deterioration in AHR that would usually occur with airway inflammation, i.e. airway remodeling may play a dual role in the development of AHR, with lesser degrees of airway fibrosis, leading to increased AHR, but more significant degrees of fibrosis being protective against further worsening of AHR. This latter possibility is supported by findings from the longitudinal MESCA study, which showed that children with abnormal lung function at 7–10 yr do not experience further deterioration in respiratory function over 3 subsequent decades despite ongoing airway inflammation (26).
Our present findings also confirm that relaxin plays a critical protective role in the inhibition of collagen deposition associated with an allergic inflammatory response in the airway and therefore may represent an effective therapeutic agent for the prevention and/or reversal of airway fibrosis in asthma (4, 6, 7, 8). We had previously shown that endogenous relaxin specifically regulates collagen turnover via induction of matrix metalloproteinase activity in young (8–10 wk old) mice subjected to a chronic (9 wk) model of AAD (5). In a separate study involving a mouse model of AAD (8), collagen deposition was significantly increased only after 4–8 wk of OVA aerosol challenge in wild-type animals. In the current study, collagen content and BM region thickening were significantly elevated in Rln–/– mice aged 12 months after only 7 d of OVA challenge, highlighting the important protective role that endogenous relaxin plays in the developing lung.
In conclusion, we have demonstrated an important novel role for relaxin in the regulation of epithelial structure and airway function (AHR), which may reflect a direct interaction of relaxin with the epithelium, or secondary effects of abnormal collagens interacting with the overlying epithelium. We have also demonstrated that relaxin deficiency can result in increased AHR, possibly secondary to structural changes in the airway.
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Footnotes |
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A portion of this work was presented at the 4th International Conference on Relaxin and Related Peptides, September 2004, Jackson Hole, WY.
Disclosure Summary: All authors have nothing to declare.
First Published Online June 21, 2007
1 C.S.S. and S.G.R. contributed equally to this work. 
Abbreviations: AAD, Allergic airways disease; AHR, airway hyperresponsiveness; BAL, bronchoalveolar lavage; BM, basement membrane; MCh, methacholine; OVA, ovalbumin; Penh, enhanced pause; Rln, relaxin; RXFP1, major receptor of relaxin.
Received May 1, 2007.
Accepted for publication June 11, 2007.
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