Published ahead of print on February 2, 2006, doi:10.1164/rccm.200503-456OC
© 2006 American Thoracic Society doi: 10.1164/rccm.200503-456OC
Uteroglobin-related Protein 1 Expression Suppresses Allergic Airway Inflammation in MiceLaboratory of Metabolism, National Cancer Institute, National Institutes of Health; Science Applications International Corporation, Bethesda, Maryland; Department of Pharmacology, School of Pharmacy, Hoshi University, Tokyo; and Department of Pathology, Fukushima Medical University, Fukushima, Japan Correspondence and requests for reprints should be addressed to Shioko Kimura, Ph.D., Bldg. 37, Rm. 3112B, National Institutes of Health, Bethesda, MD 20892. E-mail: shioko{at}helix.nih.gov
Rationale: Uteroglobin-related protein (UGRP) 1, which is highly expressed in the epithelial cells of the airways, has been suggested to play a role in lung inflammation. Objectives: The aim of study was to understand the effect of overexpressed UGRP1 on lung inflammation in a mouse model of allergic airway inflammation. Methods: Ovalbumin-sensitized and -challenged mice, a model for allergic airway inflammation, were used in conjunction with recombinant adenovirus expressing UGRP1. Measurements and Main Results: We demonstrated that intranasal administration of adeno-UGRP1 successfully delivered UGRP1 to the epithelial cells of airways and markedly reduced the number of infiltrating inflammatory cells, particularly eosinophils, in lung tissue as well as the level of proinflammatory cytokines such as interleukin (IL)-4, IL-5, and IL-13 in bronchoalveolar lavage fluids. The healed phase of inflammation was clearly seen in the peripheral areas of adeno-UGRP1–treated mouse lungs. Conclusion: These results demonstrate that UGRP1 can suppress inflammation in the mouse model of allergic airway inflammation. Based on this result, we propose UGRP1 as a novel therapeutic candidate for treating lung inflammation such as is found in asthma.
Key Words: asthma • bronchoalveolar lavage • eosinophils • mouse model • ovalbumin Uteroglobin-related protein 1 (UGRP1), also called SCGB3A2, is a secretory protein of approximately 10 kD that is highly expressed in epithelial cells of the trachea, bronchus, and bronchioles (1). UGRP1 is a novel member of the uteroglobin/Clara cell secretory protein (UG/CCSP) gene superfamily, officially called secretoglobin (SCGB) (2). A prototypic UG/CCSP protein, SCGB1A1, exhibits several immunomodulatory and antiinflammatory effects in the lung (3, 4). Mouse and human UGRP1 share 81% amino acid sequence identity (1). By using fluorescent in situ hybridization, the human UGRP1 gene was assigned to chromosome 5q31-32 (5), one of the most extensively investigated chromosomal regions in the pathogenesis of asthma and an area that contains a cluster of genes encoding numerous Th2 cytokines such as interleukin (IL)-4, IL-5, IL-9, and IL-13 (6, 7). The UGRP1 mRNA level was reduced in the lungs of allergic inflammation model mice, which returned to normal after dexamethasone treatment (1). The reduced UGRP1 mRNA levels in the lungs were inversely correlated with increased levels of IL-5 and IL-9 in bronchoalveolar lavage (BAL) fluid obtained from allergic model mice (8, 9). Intranasal instillation of IL-5 or IL-9 to naive mice reduced UGRP1 mRNA levels in the lung, suggesting that IL-5 or IL-9 may directly or indirectly mediate UGRP1 gene expression in the lung (8, 9). Further, in a Japanese population of adult patients with asthma, a polymorphism (G/A) was identified at –112 bp of the human UGRP1 gene promoter that was associated with an increased risk of bronchial asthma (5). In addition, MARCO, a macrophage scavenger receptor with collagenous structure that is expressed in lung alveolar macrophages and is involved in pulmonary inflammation, was identified as a receptor for UGRP1 (10). Taken together, these findings suggest that UGRP1 may play a role in lung inflammation, and may be involved in the pathogenesis of asthma, although the functional roles of UGRP1 in airway physiology and pathophysiology are not known. In the present study, model mice for allergic airway inflammation were intranasally administered recombinant adenovirus that express UGRP1, and the effect of overexpressed UGRP1 on antigen-induced airway inflammation was investigated. The results provide direct evidence that up-regulation of UGRP1 in the airways can suppress allergic airway inflammation.
Animal Model and Adenoviral Infection Female BALB/c mice (6 wk old; 6–8 mice/group) were sensitized and challenged with ovalbumin (OVA) as previously described (8, 9). All experiments were performed according to the Using Animals in Intramural Research Guidelines (NIH Animal Research Advisory Committee, National Institutes of Health, Bethesda, MD) and approved by the Institutional Animal Care and Use Committee. Recombinant adenovirus expressing UGRP1 (Ad-UGRP1) and Ad-empty carrying only the vector were prepared by using the Adenovirus Expression Vector Kit (Takara Mirus Bio, Madison, WI). Purified adenovirus was intranasally instilled into the trachea of each animal under anesthesia of 2.5% tribromoethanol.
Quantitation of Cytokines, UGRP1, and OVA-specific IgE and IgG Levels
Recombinant His-tagged mouse UGRP1 protein was prepared as previously described (1). The final product of approximately 30 kD on sodium dodecyl sulfate polyacryamide gel consists of a His-tag protein (
Histologic Analysis
RNA Isolation and Real-Time Polymerase Chain Reaction
Statistical Analyses
UGRP1 Expression in Murine Model of Allergic Airway Inflammation When OVA-sensitized mice were challenged by aerosolized antigen, marked airway inflammation—that is, increased number of eosinophils and lymphocytes—and up-regulation of inflammatory cytokines such as IL-4, IL-5, and IL-13 in BAL fluids—was observed 24 h after the allergen inhalation as compared with OVA-sensitized control mice that had inhaled saline (Figures 1A and 1B) as reported (12). Histologic examination demonstrated severe lung inflammation in allergen-challenged mice as revealed by a marked increase in eosinophils (Figures 1D and 1F) but not in sensitized control mice that had inhaled saline (Figures 1C and 1E).
We previously reported that the level of UGRP1 expression is reduced in inflamed mouse lungs and is inversely correlated with increased levels of inflammatory cytokines in BAL fluid in allergic model mice (8, 9). To understand the possible mechanism of regulation of UGRP1 expression under inflammatory condition, we determined levels of UGRP1 mRNA in lung tissues and UGRP1 protein and cytokines, IL-4, IL-5, IL-10, and IL-13 in BAL fluids during the first 24 h after OVA challenge using RT-PCR and ELISA, respectively (Figure 2). IL-10 had previously been shown to increase UGRP1 expression when administered to mice (13). The levels of UGRP1 mRNA and all four cytokines showed a cyclic change during the first 24 h after antigen inhalation; the change of UGRP1 mRNA levels was in general inversely correlated to the changes of all four cytokine levels during the first 6 h, whereas both UGRP1 mRNA and cytokine levels increased after 12 h of challenge (Figure 2A vs. Figures 2C–2F). These data suggest that UGRP1 expression may be under complex regulation, and no single cytokine among those examined appears to be responsible for the changes of UGRP1 expression. Surprisingly, very high UGRP1 levels (within the order of µg/ml) were found in BAL fluids, even in naive mice. Further, despite the cyclical changes in UGRP1 mRNA levels, UGRP1 protein level in BAL fluids kept increasing during the entire 24 h, with the increase being minimal during 3 to 12 h (Figure 2B). As a result, 24 h after OVA challenge, the BAL fluid UGRP1 levels became approximately 50% higher than that found 1 h after OVA challenge or in sensitized controls 24 h after saline inhalation. Note that sensitization does not seem to affect UGRP1 expression. These results suggest that UGRP1 protein is likely to be secreted as soon as it is made. Further, a factor may be present that facilitates secretion of UGRP1 protein and maintains its stability in BAL fluids upon OVA challenge, resulting in the accumulation of UGRP1 in BAL fluids. Of further interest were the observations that OVA sensitization alone drastically increased IL-10 and IL-13 levels as compared with those of normal naive control mice (Figures 2E and 2F) and that IL-5 levels increased more than 20-fold within 1 h of OVA challenge (Figure 2D).
Exogenous Expression of UGRP1 in Lung Airways by Use of Recombinant Adenovirus Because the role of UGRP1 in lung inflammation was previously suggested, the effect of UGRP1 overexpression on airway inflammation was investigated. First, to deliver UGRP1 to the airways, a recombinant adenovirus that expresses mouse UGRP1 (Ad-UGRP1) was constructed. Adenovirus carrying an empty vector without inserted DNA (Ad-empty) was used as a control. The ability of Ad-UGRP1 to express UGRP1 was first examined by infecting SV40 transformed monkey kidney–derived COS-1 cells. Northern blot and immunocytochemical analyses revealed a marked UGRP1 mRNA and protein expression in cells 3 d after infection with 10 multiplicity of infection of Ad-UGRP1, whereas no UGRP1 mRNA or protein expression was observed in Ad-empty–infected COS-1 cells (data not shown). The recombinant adenovirus was used for delivery of UGRP1 to mouse airways. When Ad-UGRP1 (20 µl of 1 x 109 pfu/ml) was intranasally administered to naive mice (14), robust expression of UGRP1 mRNA was observed in lungs 3 d after infection, and the elevated expression continued for at least 2 d more as determined by northern blot analysis (Figures 3A and 3B). By immunohistochemistry performed 3 d after adenovirus infection, a dramatic increase in UGRP1 protein expression was demonstrated in lung epithelial cells of naive mice treated with Ad-UGRP1 as compared with naive mice infected with Ad-empty (data not shown). Similarly, when adenovirus infection was performed 3 d before saline challenge and their lungs were examined 24 h later, the sensitized, Ad-UGRP1–infected mice (Figures 3D and 3F) clearly showed increased expression of UGRP1 in their lung epithelial cells as compared with sensitized, Ad-empty–infected mice (Figures 3C and 3E). Thus, Ad-UGRP1 infection by intranasal administration can successfully overexpress UGRP1 in airway epithelial cells, the site where endogenous UGRP1 expression is found (1).
UGRP1 Levels in Lungs and BAL Fluids after 24 h of OVA Challenge To determine the effect of UGRP1 overexpression on allergen-induced airway inflammation, Ad-UGRP1 or Ad-empty was intranasally administered to OVA-challenged mice 3 d before the challenge and results were compared with those from mice that inhaled saline. First, the UGRP1 mRNA and protein levels in lungs and UGRP1 levels in BAL fluids were examined 24 h after OVA or saline challenge without adenovirus infection, using RT-PCR, quantification of positive UGRP1 immunostaining, and ELISA, respectively (Figures 4A–4C, respectively). For both lung UGRP1 mRNA and protein levels, the differences between sensitized control and OVA-challenged mice were not statistically significant although a trend of decreased expression in OVA-challenged mice was seen as previously reported (Figures 4A and 4B, lane 1 vs. 2) (8, 9). Contrary to this result, UGRP1 protein levels in BAL fluids were higher in OVA-challenged mice as compared with saline-administered sensitized control mice as seen in Figure 2 (Figure 4C, lane 2 vs. 1).
When mice were intranasally administered adenovirus, whether Ad-empty or Ad-UGRP1, levels of UGRP1 mRNA and protein in lung tissues and BAL fluids markedly increased as compared with sensitized control or OVA-challenged mice that did not receive adenovirus (Figures 4A–4C, lanes 3–6 vs. 1, 2). The tissue UGRP1 mRNA levels were further increased by the administration of Ad-UGRP1 as compared with Ad-empty (Figure 4A, lanes 5, 6 vs. 3, 4); however, differences were not observed between sensitized control and OVA-challenged mice (lanes 3 vs. 4; lanes 5 vs. 6). The differences in UGRP1 mRNA levels between Ad-empty– and Ad-UGRP1–infected, OVA-challenged mouse lungs were also clearly demonstrated by in situ hybridization (relative expression,1.0 vs. 1.25, respectively; Figures 4D and 4E). UGRP1 protein levels in lung tissues and BAL fluids did not exhibit statistically significant differences between Ad-empty– and Ad-UGRP1–treated (Figures 4B and 4C, lanes 3, 4 vs. 5, 6), or sensitized control and OVA-challenged group of mice (Figures 4B and 4C, lanes 3, 5 vs. 4, 6) although there was a trend that these levels were higher in the Ad-UGRP1–treated mice. Altogether, the results suggest that, in general, the levels of UGRP1 mRNA and protein in lung tissues and BAL fluids correlate with each other, further suggesting that UGRP1 may be secreted as soon as protein is produced and may accumulate stably in BAL fluids. The results also demonstrate that the use of adenovirus itself caused increase of UGRP1 levels, probably due to perturbation of the immune system caused by adenovirus (15, 16). Nonetheless, it can be said that the intranasal delivery of Ad-UGRP1 led to the overexpression of UGRP1 in the airways of mice.
Overexpression of UGRP1 Successfully Suppresses Lung Inflammation
Histologic examination of lung sections stained with hematoxylin and eosin also revealed a marked decrease in infiltrating inflammatory cells, especially eosinophils in Ad-UGRP1–infected, OVA-challenged mice (Figures 5D and 5F,) as compared with Ad-empty–infected, OVA-challenged animals (Figures 5C and 5E). Further, marked infiltration of inflammatory cells was observed in the peripheral areas of the Ad-empty–infected lungs (Figures 5G and 5H), whereas similar areas of the Ad-UGRP1–infected lungs exhibited a typical healed phase of inflammation, characterized by thickened alveolar wall and diminished alveolar cavities (Figure 5I). A few infiltrating mononuclear cells were present; however, no eosinophils were found. These results clearly demonstrate that recombinant adenovirus–induced overexpression of UGRP1 in the airways is able to suppress antigen-induced inflammation at least in a mouse model of allergic inflammation. Note that a majority of Ad-empty–treated, sensitized, control mouse lungs exhibited no inflammation with only occasional presence of inflammatory cells (data not shown).
UGRP1 was originally suggested to be involved in lung inflammation based on the following evidence: predominant expression in airways (1); amino acid sequence similarity to UG/CCSP (1), which exhibits antiinflammatory activity (4); localization of the human gene on chromosome 5q31-32 where an asthma susceptibility locus has been assigned (5–7); reduced expression of UGRP1 in inflamed mouse lungs that returned to normal after dexamethasone treatment (1); and a polymorphism found in the human UGRP1 gene promoter that is associated with an increased risk of asthma in a Japanese population (5), although the association of this polymorphism with asthma is currently controversial due to other reports describing no association (20, 21). Later, MARCO was identified as a possible UGRP1 receptor (10). The MARCO ligand, lipopolysaccharide, competes with UGRP1 for binding to MARCO and bacteria, and thus the MARCO–UGRP1 pair was suggested to be involved in inflammation and pathogen clearance in the lung. Recently, IL-10 was shown to induce UGRP1 gene expression in lung epithelial cells, suggesting that UGRP1 may be a potential modulator of IL-10 antiinflammatory activities (13). Despite all of these findings, direct evidence for an antiinflammatory role for UGRP1 has not been presented. In this study, we clearly demonstrated that overexpression of UGRP1 in mouse airways by the use of recombinant adenovirus can successfully suppress lung inflammation when examined in a mouse model of allergic inflammation. This is the first direct demonstration that UGRP1 is an antiinflammatory agent.
Treatment with adenovirus, whether Ad-empty or Ad-UGRP1, markedly induced UGRP1 mRNA and protein expression in lungs as well as protein expression in BAL fluid, suggesting that perturbation of the immune system by adenovirus caused an increase in UGRP1 gene expression. Administration of adenovirus is known to trigger a variety of innate immune responses in target cells, followed 5–7 d later by adaptive immunity (15, 16). The innate immune response includes activation of monocytes and macrophages, and up-regulation of inflammatory cytokines such as IL-6, IL-1 Time-course study of UGRP1 and cytokine levels during 24 h after OVA challenge demonstrated that lung UGRP1 mRNA levels exhibited cyclic changes in two phases; during the first 6 h, UGRP1 levels had an inverse correlation with those of IL-4, IL-5, IL-10, and IL-13, whereas after 12 h of challenge, levels of UGRP1 and all cytokines increased. These results do not seem to fit our previous observation in which administration of inflammatory cytokines to naive mice resulted in reduced levels of lung UGRP1 mRNA levels (8, 9), whereas IL-10 enhances UGRP1 expression (13). Because OVA challenge initiates very complicated immune responses, including release of a variety of cytokines and chemokines that interrelate with each other, administration of a single cytokine to mice may not explain the current observed phenomenon. Time-course studies on cytokines and inflammatory cells were previously performed days after sensitization and allergen-induced inflammation (22, 23), but not hours after challenge as reported in this study. The time-course results suggest that UGRP1 may be regulated by two sets of regulators; one of which takes over between 6 and 12 h after the challenge. Alternatively, there may be a yet unknown dominant regulator, or more than one, for controlling UGRP1 expression, and the apparent two-phase pattern of UGRP1 and cytokine regulation may be simply a reflection of the results of complex interplay between the expression of these cytokines. The unknown dominant regulator(s) could share the same signaling pathway as the one(s) that regulates the expression of cytokines/chemokines after adenovirus administration. Further, the fact that UGRP1 protein in BAL fluids increased after OVA challenge despite reduced expression of UGRP1 in lungs may suggest the presence of a factor that facilitates UGRP1 secretion and maintains stability of the protein in BAL fluids on OVA challenge. We previously reported that lung UGRP1 mRNA level was reduced when examined at 24 h after OVA challenge as compared with sensitized control lungs (8, 9). In the current study, we observed that the UGRP1 mRNA levels almost returned to the level of sensitized control mice 24 h after OVA challenge. This discrepancy may be due to mouse strain differences (Balb/c in the current study vs. 129Sv in the previous study). What regulates UGRP1 gene expression and accumulation in BAL fluids requires further investigation. A most impressive part of the UGRP1's antiinflammatory activity is the drastic suppression of eosinophilia observed in airways of Ad-UGRP1–treated and OVA-challenged mice in addition to the substantial reduction in the levels of proinflammatory cytokines, such as IL-4, IL-5, and IL-13 in the BAL fluids. Further, peripheral areas of the lungs of Ad-UGRP1–treated, OVA-challenged mice showed a striking histology of healed phase inflammation, which is in marked contrast to Ad-empty-treated, OVA-challenged mouse lungs that present active inflammation. This is rather surprising based on the relatively small overexpression of UGRP1 in Ad-UGRP1–treated, OVA-challenged lungs as compared with Ad-empty, OVA-challenged lungs. This small increase in UGRP1 expression, however, may be sufficient to suppress inflammation by directly or indirectly regulating the release of cytokines from cells involved in inflammation such as T cells and mast cells, resulting in an inhibition of eosinophilia and other allergic reactions, thus further leading to a cessation of inflammation. However, the threshold level of UGRP1 sufficient to suppress inflammation is not known. It is also surprising to learn that relatively high levels of UGRP1 protein are present in BAL fluids even in naive mice (within the order of µg/ml), suggesting that UGRP1 may be constantly expressed at high levels in lungs. The fact that UGRP1 appears to be relatively stable may also contribute to the high levels of accumulated UGRP1 protein in BAL fluids. This could be one of the reasons why no clear statistically significant increase was observed for UGRP1 protein levels after Ad-UGRP1 administration. Further studies are required to understand the mechanism for the antiinflammatory activity of UGRP1. In summary, we demonstrated for the first time that exogenously expressed UGRP1 in the airways suppresses allergic airway inflammation when mouse model of allergic inflammation is used. UGRP1 may provide a useful reagent that can be successfully used to suppress lung inflammation such as is found in allergic asthma and chronic obstructive pulmonary diseases.
The authors thank Frank Gonzalez for his critical review of the manuscript and Sayuri Hoshi for her analysis of histologic sections.
Supported by the Intramural Research Program of the National Institutes of Health, the National Cancer Institute, and the Center for Cancer Research.
* These authors contributed equally to this work. This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200503-456OC on February 2, 2006 Conflict of Interest Statement: None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form March 22, 2005; accepted in final form January 27, 2006
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TOP
ABSTRACT
METHODS
20 kD) and UGRP1 (
-actin. The PCR condition was 95°C, 15 min, 1 cycle, followed by 94°C, 15 s, 56°C, 20 s, and 72°C, 20 s, for 45 cycles for both primer pairs. 
, and IFN-
, and chemokines such as macrophage inflammatory protein (MIP)-2, INF-
B pathways are involved in the up-regulation of cytokines and chemokines (