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The Journal of Immunology, 2001, 167: 2798-2807.
Copyright © 2001 by The American Association of Immunologists

Murine Cytomegalovirus Infection Alters Th1/Th2 Cytokine Expression, Decreases Airway Eosinophilia, and Enhances Mucus Production in Allergic Airway Disease1

Carol A. Wu2,*, Lynn Puddington*, Herbert E. Whiteley{ddagger}, Carmen A. Yiamouyiannis§, Craig M. Schramm{dagger}, Fusaini Mohammadu* and Roger S. Thrall*

Departments of * Medicine and {dagger} Pediatrics, University of Connecticut School of Medicine, Farmington, CT 06030; {ddagger} Department of Pathobiology, University of Connecticut, Storrs, CT 06269; and § Department of Science and Mathematics, Capital Community College, Hartford, CT 06105


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Concomitant infection of murine CMV (MCMV), an opportunistic respiratory pathogen, altered Th1/Th2 cytokine expression, decreased bronchoalveolar lavage (BAL) fluid eosinophilia, and increased mucus production in a murine model of OVA-induced allergic airway disease. Although no change in the total number of leukocytes infiltrating the lung was observed between challenged and MCMV/challenged mice, the cellular profile differed dramatically. After 10 days of OVA-aerosol challenge, eosinophils comprised 64% of the total leukocyte population in BAL fluid from challenged mice compared with 11% in MCMV/challenged mice. Lymphocytes increased from 11% in challenged mice to 30% in MCMV/challenged mice, and this increase corresponded with an increase in the ratio of CD8+ to CD4+TCR{alpha}{beta} lymphocytes. The decline in BAL fluid eosinophilia was associated with a change in local Th1/Th2 cytokine profiles. Enhanced levels of IL-4, IL-5, IL-10, and IL-13 were detected in lung tissue from challenged mice by RNase protection assays. In contrast, MCMV/challenged mice transiently expressed elevated levels of IFN-{gamma} and IL-10 mRNAs, as well as decreased levels of IL-4, IL-5, and IL-13 mRNAs. Elevated levels of IFN-{gamma} and reduced levels of IL-5 were also demonstrated in BAL fluid from MCMV/challenged mice. Histological evaluation of lung sections revealed extensive mucus plugging and epithelial cell hypertrophy/hyperplasia only in MCMV/challenged mice. Interestingly, the development of airway hyperresponsiveness was observed in challenged mice, not MCMV/challenged mice. Thus, MCMV infection can modulate allergic airway inflammation, and these findings suggest that enhanced mucus production may occur independently of BAL fluid eosinophilia.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Epidemiological evidence supports a close relationship between respiratory viral infections and exacerbation of asthma, with viral upper respiratory infections coinciding with asthma attacks in 80–85% of school-age children and 44% of adults (1, 2). Studies of individuals with asthma demonstrate a clear correlation between the presence of virus and an increase in asthma symptom scores, an increased need for medication, and a decrease in pulmonary function (reviewed in Ref. 3). Respiratory syncytial virus and parainfluenza virus are common viruses in young children, whereas rhinovirus is a frequent etiological agent in older children and adults (reviewed in Refs. 3, 4, 5). However, any virus capable of eliciting an acute respiratory infection has the potential to exacerbate asthma (6). The mechanisms by which viruses induce the onset of an asthma attack or increase its severity are not fully understood, but are likely to involve multiple pathways that include alterations in airway inflammation through the activation of T lymphocytes and the release of inflammatory cell mediators.

Although not widely included in epidemiological studies on asthma, human CMV (HCMV)3 has been associated with asthma exacerbations in adults (7). HCMV, a {beta} herpesvirus, is recognized as an opportunistic pulmonary pathogen and is a major cause of pneumonia in immunosuppressed bone marrow and lung transplantation recipients (reviewed in Ref. 8). In lung samples, alveolar epithelial cells represent the majority of infected cells and are fully permissive for HCMV replication (9, 10, 11). Although less frequent, HCMV infection of the bronchial epithelium has also been reported (9, 12). HCMV is ubiquitous in nature with 40–100% of the adult population becoming infected (13). Primary infection is generally unremarkable, but chronic infection with intermittent viral shedding and the establishment of latency occurs even in immunocompetent individuals (reviewed in Ref. 14). Reactivation of latent HCMV in vitro can be triggered by IL-4 or IFN-{gamma} (15) cytokines that are elevated in the blood and bronchoalveolar lavage (BAL) fluid of individuals with asthma (16, 17).

Murine CMV (MCMV) shares many biological properties and a similar disease spectrum with HCMV, making it a useful model for understanding HCMV pathogenesis (18). MCMV infection elicits a strong CD4+ and CD8+ T lymphocyte response, which is necessary to mediate viral clearance from the salivary gland and peripheral organs, respectively (19, 20, 21). These two subsets of T lymphocytes have been shown to play a role in the progression of allergic airway inflammation and airway hyperresponsiveness (22, 23). In addition, MCMV infection induces a strong Th1 response, characterized by the production of IFN-{gamma}, which helps regulate acute, chronic, and latent viral infection (21, 24, 25, 26, 27, 28). A therapeutic role for IFN-{gamma} has been suggested in various murine models of asthma (29, 30, 31). Therefore, MCMV may potentially alter the progression of airway inflammation through the proliferation of virus-specific T lymphocytes or changes in cytokine expression. In this report, we examined the influence of concomitant MCMV infection on the development of allergic airway disease in the OVA-induced murine model.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Male and female C57BL/6J mice, purchased from The Jackson Laboratory (Bar Harbor, ME), were housed at the University of Connecticut Health Center. All testing and animal manipulations were preapproved by the Animal Care Committee at the University of Connecticut Health Center and followed the guidelines established by the U.S. Animal Welfare Act.

Study protocol

Sensitization and OVA-aerosol challenge of C57BL/6J mice has been previously described for our model of OVA-induced allergic airway disease (32). Briefly, challenged mice (representing allergic airway disease) were sensitized with three weekly i.p. injections of 25 µg OVA, grade V (Sigma, St. Louis, MO) suspended in alum. One week later the animals were placed in a nose only exposure chamber and challenged with a 1% OVA aerosol generated by a Lovelace nebulizer (In-Tox Products, Albuquerque, NM). The estimated daily inhaled OVA dose was 80 µg/mouse. This procedure was repeated daily for 1 h/day for 3, 7, 10, or 14 days, as indicated in the text. Twenty-four hours after the last OVA-aerosol challenge, the animals were sacrificed, and analysis of BAL fluid, lung tissue, and blood samples was performed. An outline of this protocol is presented in Fig. 1GoA. Sensitized mice were included in these studies as a control and received three weekly i.p. injections of OVA/alum but were not exposed to OVA-aerosol challenge.



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  		FIGURE 1. Rationale for the model of OVA-induced allergic airway disease. A, Treatments in the model including three weekly i.p. injections of OVA/alum, with the last injection given 1 wk before the start of OVA-aerosol challenge. In some groups, mice were infected intranasally with MCMV 1 wk before OVA-aerosol challenge ({square}). Samples were collected at various times after OVA-aerosol challenge, as indicated in the text. B, To determine the time of peak viral load in the lung, naive C57BL/6J mice were infected intranasally with 1.5 x 104 PFU of MCMV strain K181. Four, 7, 10, 14, 21, and 42 days after infection, animals were sacrificed, lung tissue was removed, and viral titers were determined by standard plaque assay (n = 4). The results are expressed as PFU per gram of lung tissue at various times after infection. Interestingly, OVA-aerosol challenge did not affect the time course of viral clearance from the lung in MCMV/challenged mice.

 
For the studies with MCMV, MCMV/challenged mice were sensitized with three weekly injections of OVA/alum and infected intranasally with MCMV (detailed below) 7 days before the start of OVA-aerosol challenge. Two control groups were included in these studies: 1) MCMV/sensitized mice, which were sensitized with three weekly i.p. injections of OVA/alum and infected with MCMV, but were not exposed to OVA-aerosol challenge; and 2) MCMV alone mice, which were infected with MCMV, but were not sensitized with OVA/alum or exposed to OVA-aerosol challenge.

Virus propagation and infection

MCMV strain K181, purchased from American Type Culture Collection (Manassas, VA), was commercially screened for other pathogens and scored negative. The virus was routinely propagated in mouse embryo cells, maintained in DMEM containing 10% FCS (Gemini Bio-Products, Calabasa, CA), 100 U/ml penicillin, and 50 µg/ml streptomycin.

Animals were anesthetized with 0.2 ml of a 1/10 mixture of ketamine (90 mg/ml) and xylazine (10 mg/ml) and inoculated intranasally with 1.5 x 104 PFU of MCMV. Infected animals were housed in isolation apart from uninfected animals and showed no signs of illness (weight loss, changes in appearance and apparent behavior, etc.). At the end of each experiment, a portion of the lung was processed to determine viral load for infected and uninfected animals using a standard plaque assay. All lung tissue from uninfected animals was negative for MCMV.

BAL fluid analysis

Twenty-four hours after the final OVA-aerosol challenge, the lungs from each animal were lavaged in situ with five 1-ml aliquots of sterile saline (33). Total leukocyte counts were scored using a hemocytometer, and viability was determined by trypan blue dye exclusion. Leukocyte subsets (eosinophils, macrophages, or lymphocytes) were enumerated in BAL fluid using cytocentrifuged preparations stained with May-Grünwald/Giemsa.

Further characterization of the lymphocyte population of leukocytes was performed by fluorescence flow cytometry using mAbs against the following Ags: CD45 (clone 30-F11), TCR{beta} (H57.597), CD3{epsilon} (500A2), or CD8 (53-6.7) (all purchased from BD PharMingen, San Diego, CA) or CD4 (GK1.5) (purchased from BD Collaborative Technologies, Bedford, MA). These Abs were conjugated with biotin, PE, FITC, or allophycocyanine. Biotin-conjugated Abs were detected with streptavidin-Cy5 (Jackson ImmunoResearch Laboratories, West Grove, PA) or PE-Cy7 (Caltag Laboratories, San Francisco, CA). For fluorescence flow cytometry, BAL cells were washed in PBS containing 0.2% BSA and 0.1% NaN3. Aliquots of 104–105 cells were incubated with 100 µl of the appropriately diluted Abs for 30 min at 4°C. After staining, the cells were washed twice with the above PBS solution, and relative fluorescence intensities were determined by flow cytometric analysis using a FACSCalibur (BD Biosciences, San Jose, CA).

Lung histology and quantitative image analysis

At the time of sacrifice, unmanipulated, noninflated lung tissue was removed from animals, fixed in a 10% buffered formalin solution, and embedded in paraffin. Tissue sections were stained with H&E for general morphology and diastase-periodic acid-Schiff (PAS) for the detection of mucins by the Department of Pathobiology at the University of Connecticut (Storrs, CT). Quantitative analysis of PAS-stained lung sections from MCMV/sensitized, challenged, and MCMV/challenged mice was performed as follows. Black and white digital images of lung sections were captured using a Carl Zeiss (Thornwood, NY) Axiovert 135 inverted microscope, a Photometrics EEV37 PXL CCD camera (Roper Scientific, Trenton, NJ), and EasyPXL software (F. R. Morgan, University of Connecticut Health Center). The resulting images were assembled and analyzed using Adobe PhotoShop 5.0.2 and Scion Image Beta 3b. The outer boundary of each airway was defined and the total area determined by pixel count. Using the black and white pixel function, the unstained (unobstructed) area of the airway lumen (white pixels) was calculated and expressed as a percentage of the total area. A distinction between small (less than half of the visual field) and medium (greater than half of the visual field) airways was made at a magnitude of x10. Analysis was performed with the assistance of the Center for Biomedical Imaging Technology at the University of Connecticut Health Center (Farmington, CT).

Determination of pulmonary function

Pulmonary function in challenged, MCMV/challenged, sensitized, and MCMV/sensitized mice was assessed in awake, unrestrained mice by whole-body barometric plethysmography (33). Briefly, mice were placed in the main chamber of a whole-body plethysmograph (Buxco Electronics, Sharon, CT) and exposed for 2 min to aerosolized saline or increasing concentrations of methacholine from 3 to 100 mg/ml. Respiratory system variables including tidal volume, respiratory frequency, inspiratory/expiratory times, and changes in box pressure were recorded before and during aerosolization and for 4 min after each exposure. The maximal enhanced pause (Penh) value response to methacholine was recorded at each dose. To assess airway sensitivity, the interpolated concentration of methacholine needed to increase the Penh value to 2 U (a ~5-fold increase over baseline) was calculated. As plateau responses were not obtained, a conventional half-maximal methacholine concentration could not be calculated, and the Penh-2 value was selected as the portion of the dose-response curve where greatest changes in sensitivity would be manifested.

RNase protection assays

Total RNA was isolated from ~100 mg of fresh lung tissue after homogenization in 1 ml of Ultraspec RNA solution (Biotecx Laboratories, Houston, TX). 32P-labeled riboprobes were generated using an in vitro transcription kit (BD PharMingen) and the mouse cytokine multiprobe template set, mCK-1 (BD PharMingen), according to the manufacturer’s specifications. These antisense probes were hybridized with total RNA, then treated with a mixture containing RNase A + T1 from a RNase protection kit (BD PharMingen). The resulting hybrids were resolved on a 6% polyacrylamide-urea gel and analyzed by autoradiography.

Measurement of cytokines

BAL fluid was recovered from challenged and MCMV/challenged mice after 3 or 10 days of OVA-aerosol challenge, along with BAL fluid from sensitized, MCMV/sensitized, and MCMV alone mice sacrificed on the same days. BAL fluid was concentrated 10-fold using an Amicon (Beverly, MA) Centriplus YM-10 filtration device and examined by ELISA for the presence of IL-5, IL-10, and IFN-{gamma} (Pierce Endogen, Rockford, IL) and IL-13 (R&D Systems, Minneapolis, MN). The limits of detection for IL-5, IL-10, IFN-{gamma}, and IL-13 were 5, 12, 10, and 1.5 pg/ml, respectively. In addition, blood was obtained from challenged, MCMV/challenged, and control mice by cardiac puncture before sacrifice, to measure circulating IL-5 by ELISA.

Statistical analysis

Groups were compared by Student’s unpaired t test. ANOVA was used to compare Penh measurements. Values of p equal to or <0.05 were considered significant. All data are expressed as the mean ± SE.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Establishing a protocol for MCMV infection in the OVA-induced model of allergic airway disease

We have previously reported that mice, sensitized with injections of OVA/alum and exposed to OVA-aerosol challenge, demonstrated elevated levels of macrophages, eosinophils, and lymphocytes in their BAL fluid (33). The goal of this study was to determine the influence of MCMV, an opportunistic respiratory pathogen, in this model. Our strategy is outlined in Fig. 1GoA. Mice received three weekly injections of OVA/alum and were infected intranasally with MCMV before the start of OVA-aerosol challenge. In developing the protocol, we sought to maximize viral load in the lung at the initiation of OVA-aerosol challenge. Thus, a time course of viral infection in naive animals was performed. C57BL/6J mice were infected intranasally with 1.5 x 104 PFU of MCMV and at various times after infection, viral load in the lung was determined by a standard plaque assay. As shown in Fig. 1GoB, peak viral load was observed on day 7, with virus titers rapidly decreasing thereafter. Based on these findings, mice were infected with MCMV 7 days before the start of OVA-aerosol challenge.

MCMV infection reduces eosinophilia and enhances lymphocyte recruitment to the airway

The profile of leukocytes present in BAL fluid in MCMV-infected OVA-aerosol-challenged (MCMV/challenged) mice was compared with uninfected OVA-aerosol-challenged (challenged) mice after 3 days of OVA-aerosol exposure. It has been established in this model that increases in BAL fluid cells are noticeable on day 3, but have not reached maximum levels in challenged animals (32), allowing exacerbations caused by viral infection to be scored. As shown in Table IGo, total leukocytes increased from 2.5 x 104 in naive animals to 14 x 104 in challenged mice (p < 0.05), representing a marked augmentation of eosinophil and macrophage populations. Airway inflammation, as determined by the total number of leukocytes recovered from BAL fluid, was also present in MCMV/challenged animals (18.7 x 104 cells; p < 0.02); however, the profile of cells differed. The BAL fluid from MCMV/challenged mice contained fewer eosinophils and an enhanced proportion of lymphocytes. No significant changes in the total number of leukocytes were noted between the control groups of naive mice and sensitized mice (three i.p. injections with OVA/alum, but no OVA-aerosol challenge).


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  		Table I. BAL fluid analysis of leukocytes from challenged and MCMV/challenged mice1

 
To aid in the interpretation of our data, two additional control groups were included in these studies. MCMV/sensitized control mice received three weekly i.p. injections of OVA/alum and were infected intranasally with MCMV, but did not receive OVA-aerosol challenge. This control group helped us discern the effects of MCMV infection vs concomitant MCMV infection with allergic airway inflammation. As shown in Table IGo, a 2.5-fold increase in the total number of leukocytes in BAL fluid was observed in MCMV/sensitized mice when compared with sensitized mice; however, this increase was not statistically significant (p = 0.07). MCMV/sensitized mice also displayed an increase in the number of lymphocytes present in BAL fluid, but again this difference was not statistically significant when compared with sensitized mice (p = 0.06). Our final control group of mice, referred to as MCMV alone, was infected intranasally with MCMV, but was not sensitized with OVA/alum or challenged with OVA aerosol. Although the total number of leukocytes recovered in BAL fluid from MCMV alone mice was reduced 50% when compared with MCMV/sensitized mice, the cellular profiles were similar. Macrophages, lymphocytes, and eosinophils comprised 61, 38, and 1% of the leukocyte population, respectively, in MCMV alone mice compared with 59, 40, and 1% in MCMV/sensitized mice.

The decrease in the number of eosinophils found in BAL fluid from MCMV/challenged mice may be attributed to a delay in eosinophil infiltration to the lungs or a suppression in eosinophil recruitment normally observed in challenged mice. To distinguish between these possibilities, BAL fluid analysis was performed after 3, 7, and 10 days of OVA-aerosol challenge, comparing challenged and MCMV/challenged mice. The total number of leukocytes recovered from BAL fluid peaked on day 7 with 44 x 104 and 50 x 104 BAL cells present in challenged and MCMV/challenged mice, respectively. As shown in Fig. 2Go, the percentage of eosinophils present in challenged animals increased from 33% on day 3 to 64% on day 10 (p < 0.008), with peak eosinophilia occurring between days 7 and 10. The percentage of eosinophils in the BAL fluid from MCMV/challenged animals was significantly lower at all time points examined (p < 0.006). Thus, MCMV infection appears to suppress eosinophilia in this model.



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  		FIGURE 2. Suppression of BAL fluid eosinophilia in MCMV/challenged mice. BAL fluid was collected from challenged and MCMV/challenged mice after 3, 7, and 10 days of OVA-aerosol exposure. Nucleated cells were counted, and the BAL fluid cell differential was obtained from cytocentrifuged preparations stained with May-Grünwald/Giemsa. Data are expressed as the relative percentage of lymphocytes, macrophages, and eosinophils present at each time point (n = 5). The total numbers of leukocytes recovered from BAL fluid after 3, 7, and 10 days of OVA-aerosol challenge were 14 x 104, 44 x 104, and 27 x 104 for challenged mice, and 19 x 104, 50 x 104, and 17 x 104 for MCMV/challenged mice, respectively.

 
A comparable percentage of macrophages was found in the BAL fluid of both groups after 3 or 7 days of OVA-aerosol challenge (Fig. 2Go), but the percentage increased in MCMV/challenged mice after 10 days (p < 0.01). In addition, the percentage of lymphocytes in MCMV/challenged mice was significantly higher when compared with challenged mice, 45 vs 7% after 3 days (p < 0.001), and 30 vs 11% after 10 days (p < 0.02).

MCMV infection augments the number of CD8+ T lymphocytes in BAL fluid

We have previously demonstrated a significant increase in CD4+ TCR{alpha}{beta} lymphocytes in association with airway inflammation and eosinophilia in challenged mice (32). To examine the effect of MCMV infection on the recruitment of T lymphocytes to the lung, flow cytometric analysis was performed on total BAL fluid leukocytes (all CD45+ cells) collected from challenged and MCMV/challenged mice (Fig. 3GoA). The forward scatter (FSC) vs side scatter (SSC) properties for leukocyte subsets are well established and the lymphocyte populations are circled, representing 9 and 46% for challenged and MCMV/challenged mice after 7 days of OVA-aerosol challenge. These findings are in good agreement with the differential analysis of BAL fluid presented in Fig. 2Go. The major leukocyte population in BAL fluid from challenged mice has FSC vs SSC properties typical of eosinophils, whereas the other prominent leukocyte population in MCMV/challenged mice has FSC vs SSC properties typical of macrophages. In addition, a marked shift in the ratio of CD4+ to CD8+ TCR{alpha}{beta} lymphocytes infiltrating the lung was observed (Fig. 3GoB). CD8+ TCR{alpha}{beta} lymphocytes comprised 65 and 70% of the population after 3 and 7 days of OVA-aerosol exposure in MCMV/challenged mice. In challenged mice, CD8+ TCR{alpha}{beta} lymphocytes decreased from 46 to 15%, whereas CD4+ TCR{alpha}{beta} lymphocytes increased from 41 to 71% after 3 and 7 days of OVA-aerosol challenge, respectively. These alterations in the number and ratio of T lymphocytes are detailed in Fig. 3GoC. After 3 days of OVA-aerosol challenge, the number of CD4+ and CD8+ TCR{alpha}{beta} lymphocytes in MCMV/challenged mice was 22.6 x 103 and 54.5 x 103 with a ratio of 0.4. In challenged animals, the number of CD4+ and CD8+ TCR{alpha}{beta} lymphocytes was 5.9 x 103 and 3.6 x 103 or a ratio of 1.6. A decrease in the ratio of CD4+ to CD8+ TCR{alpha}{beta} lymphocytes was also observed in MCMV/challenged mice after 7 days of OVA-aerosol challenge. This enhanced recruitment of lymphocytes to the lung and dramatic augmentation of CD8+ TCR{alpha}{beta} lymphocytes in MCMV/challenged mice is indicative of a host cell-mediated immune response against the virus.



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  		FIGURE 3. MCMV infection altered the ratio of CD4+ to CD8+ TCR{alpha}{beta} lymphocytes in this model of allergic airway disease. A, Light scatter analysis of BAL fluid leukocytes from challenged and MCMV/challenged mice after 7 days of OVA-aerosol challenge is shown, with linear FSC vs linear SSC as parameters. The percentage represents the lymphocyte subset of total leukocytes expressing CD45. Note that the majority of BAL fluid leukocytes in challenged mice, but not MCMV/challenged mice, exhibited FSC vs SSC properties typical of eosinophils, in agreement with the results in Fig. 2Go. B, CD45+ lymphocytes from BAL fluid of challenged and MCMV/challenged mice were positively gated for TCR{alpha}{beta} and analyzed for the expression of CD4 and CD8 by fluorescence flow cytometry after 3 and 7 days of OVA-aerosol exposure. Data are presented on a 4-decade log scale. Further characterization of the T lymphocyte populations present in BAL fluid is shown in C. The total number of lymphocytes, the number of T lymphocytes bearing the {alpha}{beta} TCR (TCR{alpha}{beta}), and the number of CD4+ and CD8+ TCR{alpha}{beta} lymphocytes were determined for challenged and MCMV/challenged after 3 days of OVA-aerosol exposure (n = 5).

 
Increased mucus secretion occurs during MCMV infection in OVA-aerosol-challenged mice

Histological evaluations by H&E and PAS staining were performed on formalin-fixed, paraffin-embedded sections of uninflated, nonmanipulated lungs from challenged and MCMV/challenged mice. The lungs of challenged mice demonstrated a dense peribronchial inflammation consisting of lymphoplasmacytic cells and eosinophils (Fig. 4GoC). There were also areas of perivascular inflammation and slight peribronchial epithelial and smooth muscle hypertrophy. MCMV/challenged mice had more intense bronchial epithelial cell hypertrophy/hyperplasia (D). No evidence of histologic damage was found in naive (data not shown), sensitized (A), or MCMV/sensitized (B) mice.



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  		FIGURE 4. Mucus plugging was observed only in MCMV/challenged mice. Histological evaluation of lung tissue from challenged (C) and MCMV/challenged mice (D) after 7 days of OVA-aerosol challenge was performed with H&E and PAS stain. H&E- and PAS-stained lung sections from sensitized (A) and MCMV/sensitized (B) mice served as controls.

 
Few PAS positive staining cells were observed in sensitized mice (Fig. 4GoA), MCMV/sensitized mice (B), or naive animals (data not shown). Histological examination of lung sections from challenged mice after 7 days of OVA-aerosol exposure revealed an increase in PAS staining in bronchoepithelial cells (C), but the cells exhibited a normal morphology. In contrast, MCMV/challenged mice exhibited intense positive PAS staining, and the cells appeared elongated and more abundant, indicative of epithelial cell hypertrophy/hyperplasia (D). This dramatic mucus plugging, frequently seen in the airways of MCMV/challenged mice after 7 days of OVA-aerosol challenge, was not observed in challenged animals. Furthermore, although positive PAS staining was noted in the lungs of both challenged and MCMV/challenged mice after 3 days of OVA-aerosol exposure, mucus occlusions were not found in either group (data not shown).

To calculate the level of airway obstruction caused by mucus plugging, digital images of small and medium airways from challenged, MCMV/challenged, and MCMV/sensitized mice were captured for quantitative image analysis (detailed in Materials and Methods). After 7 days of OVA-aerosol challenge, blockage of MCMV/challenged airways was significant in both small (p < 0.02) and medium (p < 0.001) airways (Fig. 5Go). Occlusion of airways from challenged mice was not statistically significant when compared with MCMV/sensitized control mice.



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  		FIGURE 5. Quantitative image analysis of mucus obstruction in airways of MCMV/challenged mice. Lung tissue from MCMV/sensitized, challenged, and MCMV/challenged mice were examined for mucus production by PAS staining as shown in Fig. 4Go. Black and white digital images of these lung sections were analyzed to determine the percentage of unobstructed (unstained) area relative to the total area of the airway (detailed in Materials and Methods). A distinction between small (less than half of the visual field) and medium (greater than half of the visual field) airways was made at a magnitude of x10 (n = 3–9). When compared with MCMV/sensitized mice, * indicates a p value <0.02, and ** indicates a p value <0.0001.

 
Challenged mice, but not MCMV/challenged mice, developed airway hyperresponsiveness

To investigate whether MCMV infection altered pulmonary function in mice with allergic airway disease, Penh values were compared between challenged and MCMV/challenged mice. We and others have demonstrated that maximal cholinergic hyperreactivity occurs before the development of peak airway inflammation (i.e., after 3–7 days of OVA-aerosol challenge in this model; Ref. 32). Accordingly, serial changes in airway responsiveness were measured in conscious, unrestrained mice after 3 and 6 days of OVA-aerosol challenge using whole-body plethysmography. At baseline, Penh responses to increasing doses of methacholine did not differ between challenged and MCMV/challenged mice (Fig. 6GoA; p > 0.05). Methacholine dose-response relationships were assessed again 12 h after the third and sixth OVA-aerosol challenges and were compared in individual mice with their baseline responses. Challenged mice developed increased responsiveness to methacholine after 3 days of OVA-aerosol challenge, as demonstrated by a significant leftward shift in their dose-response relationships (p < 0.05) and a 2- to 3-fold decrease in the methacholine concentration eliciting a Penh of 2 U (Fig. 6GoB). This heightened airway responsiveness persisted after the sixth day of OVA-aerosol challenge. In contrast, MCMV/challenged mice did not demonstrate airway hyperresponsiveness after OVA-aerosol challenge. Their methacholine dose-response relationships and Penh-2 values were statistically unchanged from baseline measurements (p = 0.6 after 3 days; p = 0.3 after 6 days). In addition, the change in Penh-2 values after OVA-aerosol exposure was significantly different between challenged and MCMV/challenged mice (p < 0.03).



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  		FIGURE 6. Development of airway hyperresponsiveness in challenged, but not MCMV/challenged mice. A, Baseline responses to increasing doses of aerosolized methacholine (0–100 mg/ml) were assessed by whole-body plethysmography for challenged and MCMV/challenged mice 1 day before initiation of OVA-aerosol challenge. No differences attributed to MCMV infection alone were noted. B, Airway hyperresponsiveness to methacholine was measured in challenged and MCMV/challenged mice 12 h after the 3rd and 6th days of OVA-aerosol challenge by whole-body plethysmography. The concentration of methacholine required to increase Penh values to 2 U (~5-fold above baseline) was calculated (n = 5). *, p < 0.05 when compared with baseline values for challenged mice.

 
Elevated levels of total serum IgE after OVA-aerosol challenge

Ag-induced IgE synthesis has been associated with airway hyperresponsiveness in murine models of allergic airway disease (32, 34, 35, 36, 37). As increased hyperresponsiveness was observed in challenged mice, but not MCMV/challenged mice, serum IgE production was examined in both groups. Measurements of total IgE were calculated for challenged and MCMV/challenged mice after 3, 7, and 10 days of OVA-aerosol exposure. Increased serum IgE levels were observed for both challenged (range 1.67–3.13 µg/ml) and MCMV/challenged (range 0.68–2.84 µg/ml) mice in comparison to naive mice (0.02 µg/ml), but no differences were noted between challenged and MCMV/challenged mice. Thus, MCMV infection does not appear to influence IgE production in this model of allergic airway disease.

MCMV influences the balance of Th1/Th2 mRNA synthesis in the lung

Cytokines are important mediators in airway inflammation and can regulate excessive production of mucus, as well as the recruitment of eosinophils to the lung. To determine whether MCMV infection alters the local cytokine environment in the lung, RNase protection assays were performed. Total RNA, isolated from lungs of challenged and MCMV/challenged mice, was hybridized with riboprobes specific for Th1 and Th2 cytokines. The results obtained from challenged mice are shown in Fig. 7GoA. IL-5 and IL-13 mRNAs were observed after 3 days of OVA-aerosol challenge (lanes 5 and 6) and persisted throughout the time course (lanes 7–10). On day 7, the synthesis of IL-10 mRNA was noted (lanes 7 and 8), and IL-4 mRNA was detected on day 14 (lanes 9 and 10). Expression of IL-4, IL-5, IL-10, or IL-13 was not observed in naive or sensitized controls (lanes 1–4). Comparable levels of IL-15 mRNA were observed in naive, sensitized, and challenged mice, allowing IL-15 to serve as an internal control for equal loading of RNA.



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  		FIGURE 7. Reduced levels of Th2 cytokine mRNAs in lung tissue from MCMV/challenged mice. RNase protection assays were performed on total RNA isolated from lung tissue of challenged and MCMV/challenged mice after 3, 7, and 14 days of OVA-aerosol exposure. A series of Th1/Th2 riboprobes were generated and hybridized with total RNA. After treatment with a mixture of RNase A + T1, the resulting hybrids were resolved on a 0.6% polyacrylamide-urea gel. GAPDH was used as a housekeeping gene for comparative purposes. A, Results from naive, sensitized, and challenged mice. B, Results from MCMV/sensitized and MCMV/challenged mice. A nonspecific radioactive mark partially coincides with the IFN-{gamma} fragment in B, lane 4.

 
The cytokine mRNA profiles from the lungs of MCMV/challenged mice are shown in Fig. 7GoB. Expression of IL-10 and IFN-{gamma} mRNAs was observed after 3 days of OVA-aerosol exposure (lanes 3 and 4); however, the level of these cytokines diminished with time (lanes 5–7). IL-5 and IL-13 mRNAs were first detected after 7 days of OVA-aerosol challenge (lanes 5 and 6), and no evidence of IL-4 mRNA was found at any time point examined. The synthesis of IL-10 and IFN-{gamma} was also detected in MCMV/sensitized mice, albeit at lower levels (lanes 1 and 2), whereas expression of IL-4, IL-5, and IL-13 mRNAs were not found in this control group. Similar to the results shown in Fig. 7GoA, consistent expression of IL-15 was observed throughout the time course in MCMV/challenged and MCMV/sensitized mice.

The induction of both local and systemic IL-5 has been reported during airway inflammation; however, a recent study indicates that circulating, not local, IL-5 may be required for the development of pulmonary eosinophilia (38). Therefore, serum from challenged and MCMV/challenged mice was examined for the presence of circulating IL-5 by ELISA. An increase in serum IL-5 was observed in challenged (7.8 ± 4.4 pg/ml) and MCMV/challenged (6.7 ± 4.9 pg/ml) mice after 3 days of OVA-aerosol challenge. These levels slowly declined to 2.8 ± 1.6 and 3.4 ± 0.6 pg/ml after 14 days of OVA-aerosol exposure in challenged and MCMV/challenged mice, respectively. Serum IL-5 in sensitized controls was 2.7 ± 0.2 pg/ml. These findings suggest that an increase in circulating IL-5 was present in both challenged and MCMV/ challenged mice.

Finally, the levels of IL-5, IL-10, IL-13, and IFN-{gamma} were measured by ELISA in concentrated BAL fluid from challenged and MCMV/challenged mice. A significant increase in IL-5 was detected in challenged mice after 3 days of OVA-aerosol challenge when compared with sensitized controls (Fig. 8GoA; p < 0.03). After 10 days of OVA-aerosol challenge, the level of IL-5 in challenged mice returned to baseline. In contrast, no increase in IL-5 was observed in MCMV/challenged mice after 3 or 10 days of OVA-aerosol challenge. This 20-fold difference in BAL fluid IL-5 levels between challenged and MCMV/challenged mice after 3 days of OVA-aerosol exposure was significant (p < 0.01). An increase in IL-13 was found in BAL fluid from challenged mice when compared with sensitized controls (Fig. 8GoB; p < 0.02). Although IL-13 was detected in BAL fluid from MCMV/challenged mice after 3 days of OVA-aerosol challenge, the level of IL-13 was not significantly elevated when compared with controls (p = 0.20). Again, the level of IL-13 in BAL fluid returned to baseline in both groups after 10 days of OVA-aerosol challenge. Expression of IL-5 and IL-13 was not observed in concentrated BAL fluid recovered from sensitized, MCMV/sensitized, or MCMV alone control mice. In addition, IL-10 was not detected in challenged or MCMV/challenged mice after 3 or 10 days of OVA-aerosol challenge and was not present in BAL fluid from sensitized or MCMV/sensitized control mice.



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  		FIGURE 8. Th1/Th2 cytokine expression is altered in BAL fluid from MCMV/challenged mice. BAL fluid was recovered from challenged and MCMV/challenged mice after 3 or 10 days of OVA-aerosol challenge (n = 6 for each group). The samples were concentrated 10-fold using Amicon Centriplus YM-10 filtration units and assayed for IL-5 (A), IL-13 (B), and IFN-{gamma} (C) by ELISA. Controls included concentrated BAL fluid from sensitized and MCMV/sensitized mice collected at the same time points (n = 6 for each group). Cytokine levels in sensitized mice were not significantly above baseline for any of these cytokines. Elevated levels of IFN-{gamma} were observed in MCMV/sensitized control mice at the 3-day time point, but not at 10 days. No increase in IL-5 or IL-13 was observed in MCMV/sensitized mice at either time point. Similarly, elevated levels of IFN-{gamma}, but no increase in IL-5 or IL-13, was noted at the 3-day time point for MCMV alone mice. *, p < 0.05 when compared with sensitized controls. +, p < 0.05 when comparing MCMV/challenged with challenged mice.

 
Increased production of IFN-{gamma} was observed in concentrated BAL fluid from challenged mice after 3 days of OVA-aerosol exposure when compared with sensitized controls (Fig. 8GoC; p < 0.05). IFN-{gamma} synthesis was further elevated in MCMV/challenged mice after 3 days of OVA-aerosol challenge when compared with challenged animals (p < 0.04). In both groups, IFN-{gamma} levels decreased to baseline after 10 days of OVA-aerosol exposure. As expected, elevated levels of IFN-{gamma} were found in BAL fluid recovered from MCMV/sensitized (132 pg/ml) mice when compared with sensitized control mice (10 pg/ml) that were sacrificed at the same time as mice exposed to 3 days of OVA-aerosol challenge. This significant increase in IFN-{gamma} levels (p < 0.02) most likely reflects the antiviral response of the host to MCMV infection. Indeed, elevated levels of IFN-{gamma} were also observed in BAL fluid from MCMV alone controls (327 pg/ml). These values were not statistically different from MCMV/sensitized mice (p = 0.07). Thus, both MCMV infection and allergic airway disease appear to contribute to IFN-{gamma} production in MCMV/challenged mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we examined the influence of MCMV, an opportunistic respiratory pathogen, on a murine model of OVA-induced allergic airway inflammation. As compared with challenged mice, MCMV/challenged mice exhibited 1) a decrease in Th2 cytokines present in lung tissue and BAL fluid, 2) a decrease in BAL fluid eosinophilia, 3) histological evidence of enhanced mucus plugging and bronchial epithelial cell hypertrophy/hyperplasia, and 4) an increase in lymphocytes recovered from BAL fluid, which was associated with a decrease in the ratio of CD4+ to CD8+ TCR{alpha}{beta} lymphocytes.

Elevated levels of IL-4, IL-5, IL-10, and IL-13 mRNAs were demonstrated in the lungs of challenged mice. In addition, increased levels of IL-5 and IL-13 protein were found in BAL fluid from challenged mice. This Th2 phenotype corresponded with increased eosinophilia and airway hyperresponsiveness, and was not found in any of our control groups. These findings are in good agreement with previous reports indicating that elevated Th2 cytokines play a pivotal role in the development and pathogenesis of allergic airway disease and asthma (reviewed in Refs. 39 and 40). In contrast, decreased expression of IL-4, IL-5, and IL-13 mRNAs, as well as increased IFN-{gamma} mRNA production, was observed in MCMV/challenged mice when compared with challenged mice. Decreased expression of IL-5 and IL-13 and increased expression of IFN-{gamma} were also demonstrated in BAL fluid recovered from MCMV/challenged mice. Polarization toward a Th1 response has been documented for other viral, bacterial, and protozoan infections (reviewed in Ref. 41), although the idea that a Th1 response can counterbalance Th2-induced airway inflammation remains controversial (42).

The decrease in BAL fluid eosinophilia observed in MCMV/challenged mice is most likely due to an inability of these animals to generate an IL-5 response. IL-5, a cytokine necessary for the regulation of eosinophil growth, differentiation, activation, and survival, plays a critical role in the recruitment of eosinophils to the lung (43, 44, 45). In our studies, decreased levels of IL-5 mRNA in lung tissue and decreased IL-5 levels in BAL fluid correlated with reduced eosinophilia in MCMV/challenged mice. This reduction in eosinophilia was observed at all time points examined, including times when no mucus plugging was found (i.e., after 3 days of OVA-aerosol challenge). Therefore, decreased BAL fluid eosinophilia most likely cannot be attributed to technical difficulties involving BAL cell recovery in the presence of increased mucus production. No differences in serum IL-5 were detected between challenged and MCMV/challenged mice after 3, 7, or 10 days of OVA-aerosol challenge.

Enhanced mucus production and epithelial cell hypertrophy/hyperplasia were observed in MCMV/challenged mice. Such changes in lung histology have typically been associated with a Th2 phenotype and IL-13 gene expression (46, 47, 48). Surprisingly, the levels of IL-13 in BAL fluid from MCMV/challenged mice were not significantly elevated above controls (sensitized, MCMV/sensitized, or MCMV alone mice) or background noise. In contrast, elevated levels of IL-13 were measured in BAL fluid from challenged mice, which do not exhibit mucus plugging or epithelial cell hypertrophy/hyperplasia. Although IL-13 mRNA was observed in the lungs of MCMV/challenged mice, expression was detected at only one time point (after 7 days of OVA-aerosol challenge), whereas IL-13 expression was noted at all time points in challenged mice. These findings suggest that other factors, in addition to IL-13, are critical for mucus hypersecretion. A recent study indicates that IL-10 may be a key contributor to mucus hypersecretion in allergic airway disease, as IL-10 knockout mice display diminished goblet cell development and mucus secretion (49). Gelfand and colleagues (50) have demonstrated that the administration of IL-10 to either OVA-sensitized/challenged IL-10 knockout mice or OVA-sensitized/challenged wild-type mice heightened mucin production and goblet cell hyperplasia. In our studies, IL-10 was not detected in BAL fluid from either challenged or MCMV/challenged mice; however, increased levels of IL-10 mRNA were found in lung tissue from both groups. Interestingly, the kinetics of IL-10 gene expression differed with transient expression of IL-10 mRNA appearing earlier in MCMV/challenged mice (after 3 days of OVA-aerosol challenge) and decreasing rapidly thereafter. In contrast, IL-10 mRNA synthesis was not observed until 7 days of OVA-aerosol exposure in challenged mice, and although mucin synthesis was increased in these mice, mucus plugging was not observed. As the effects of IL-10 on allergic airway inflammation are likely to be influenced by interactions with other cytokines, such differences in kinetic expression may be important. IL-10 mRNA synthesis was also noted in control MCMV/sensitized mice, and these mice do not develop allergic airway disease or exhibit mucus plugging and epithelial cell hypertrophy/hyperplasia. Thus, mucus plugging and epithelial cell hypertrophy/hyperplasia observed in MCMV/challenged mice cannot be directly attributed to IL-10 production, and other, yet unidentified factors induced by allergic airway disease are likely to be involved. Studies addressing this issue are currently underway.

The development of airway hyperresponsiveness was observed in challenged mice using barometric whole-body plethysmography. Penh values obtained after 3 and 6 days of OVA-aerosol challenge demonstrated an increase in sensitivity to methacholine, indicated by a leftward shift in the dose-response curve, and an increase in reactivity, indicated by a decrease in the methacholine concentration necessary to elicit a Penh of 2 U. In contrast, MCMV/challenged mice did not display increased airway hyperresponsiveness. Enhanced Penh values have been shown to correlate with increased pulmonary resistance, increased IgE production, and increased pulmonary eosinophilia (51). Furthermore, studies have shown that both IL-5 and eosinophils are essential for the development of airway responsiveness during the late, but not early, asthmatic response in the mouse (52). In our model, increased Penh values in challenged mice were associated with increased eosinophilia and elevated levels of IL-5 in BAL fluid and lung tissue, whereas the absence of airway hyperresponsiveness in MCMV/challenged mice paralleled a reduction in airway eosinophilia and undetectable levels of IL-5. Still, it was surprising that the extensive mucus plugging and epithelial cell hypertrophy/hyperplasia observed in MCMV/challenged mice did not lead to changes in pulmonary function as determined by whole-body plethysmography. Mucus plugging was most prevalent in small airways, and changes in pulmonary function associated with increased obstruction of small airways may not be detectable by this approach.

NK cells (53) and CD8+ T lymphocytes (54) represent the initial response of the host to acute infection with MCMV. Activation of NK cells peaks between 3 and 5 days after infection and is characterized by the induction of IFN-{gamma} (25). A second burst of IFN-{gamma} synthesis correlates with the proliferation of CD8+ T lymphocytes 7–10 days after infection (55). These antiviral responses are likely to account for the elevated levels of CD8+ T lymphocytes and, in part, for the increase in IFN-{gamma} observed in MCMV/challenged mice. Indeed, rapid viral clearance from the lungs of MCMV/challenged mice was observed in our studies. However, IFN-{gamma} was also detected in BAL fluid from challenged mice, albeit to a lesser extent than MCMV/challenged mice, suggesting that allergic airway inflammation also contributes to IFN-{gamma} production. Expression of both Th1 and Th2 cytokines in BAL fluid from OVA Ag-challenged mice has been reported by others (56).

In summary, our results demonstrate that MCMV infection in this model of allergic airway inflammation can modulate the disease process in multiple ways. A reduction in Th2 cytokines, particularly IL-5, was associated with a decrease in BAL fluid eosinophilia in MCMV/challenged mice, which is suggestive of decreased lung injury. In contrast, MCMV/challenged mice also exhibited enhanced mucus plugging and epithelial cell hypertrophy/hyperplasia, which is usually indicative of exacerbation of allergic airway inflammation. Together, these findings highlight the complex nature of allergic airway disease, especially with respect to concomitant upper respiratory viral infections. Furthermore, they suggest that eosinophilia can occur independently of excess mucus production and epithelial cell hypertrophy/hyperplasia.


   Acknowledgments
 
We gratefully acknowledge Dr. John Shanley for the gift of MCMV and his assistance during viral infection. We thank Drs. Michelle Cloutier and Leo LeFrancois for critical review of the manuscript; our colleagues in the Pulmonary Research Consortium for their encouragement and helpful discussions; and Linda Guernsey, Grace Nicksa, and Caroline Benkovich for their excellent technical assistance.


   Footnotes
 
1 Funding for these studies was provided by grants from the American Lung Association (to C.A.W.) and National Institutes of Health Grant AI-43573 (to R.S.T.). Back

2 Address correspondence and reprint requests to Dr. Carol A. Wu, Division of Infectious Diseases, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3212. E-mail address: cawu{at}nso1.uchc.edu Back

3 Abbreviations used in this paper: HCMV, human CMV; MCMV, murine CMV; BAL, bronchoalveolar lavage; PAS, diastase-periodic acid-Schiff; Penh, enhanced pause; FSC, forward scatter; SSC, side scatter. Back

Received for publication November 8, 2000. Accepted for publication July 3, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Johnston, S. L., P. K. Pattemore, G. Sanderson, S. Smith, F. Lampe, L. Josephs, P. Symington, S. O’Tolle, S. H. Myint, D. A. J. Tyrrell, S. T. Holgate. 1995. Community study of role of viral infections in exacerbations of asthma in 9–11 year old children. Br. Med. J. 310:1225.[Abstract/Free Full Text]
  2. Nicholson, K. G., J. Kent, D. C. Ireland. 1993. Respiratory viruses and exacerbations of asthma in adults. Br. Med. J. 307:982.
  3. Pattemore, P. K., S. L. Johnston, P. G. Bardin. 1992. Viruses as precipitants of asthma symptoms. I. Epidemiology. Clin. Exp. Allergy 22:325.[Medline]
  4. Busse, W. W., J. E. Gern. 1997. Updates on cells and cytokines: viruses in asthma. J. Allergy Clin. Immunol. 100:147.[Medline]
  5. Folkerts, G., W. W. Busse, F. P. Nijkamp, R. Sorkness, J. E. Gern. 1998. Virus-induced airway hyperresponsiveness and asthma. Am. J. Respir. Crit. Care Med. 157:1708.[Free Full Text]
  6. Monto, A. S.. 1995. Epidemiology of respiratory viruses in persons with and without asthma and COPD. Am. J. Respir. Crit. Care Med. 151:1653.[Abstract]
  7. Atmar, R. L., E. Guy, K. K. Guntupalli, J. L. Zimmerman, V. D. Bandi, B. D. Baxter, S. B. Greenberg. 1998. Respiratory tract viral infections in inner-city asthmatic adults. Arch. Intern. Med. 158:2453.[Abstract/Free Full Text]
  8. Salomon, N., D. C. Perlman. 1999. Cytomegalovirus pneumonia. Semin. Respir. Infect. 14:353.[Medline]
  9. Singer, C., A. Grefte, B. Plachter, A. S. H. Gouw, T. H. The, G. Jahn. 1995. Fibroblasts, epithelial cells, endothelial cells and smooth muscle cells are major targets of human cytomegalovirus infection in lung and gastrointestinal tissues. J. Gen. Virol. 76:741.[Abstract/Free Full Text]
  10. Ng Bautista, C. L., D. D. Sedmak. 1995. Cytomegalovirus infection is associated with absence of alveolar epithelial cell HLA class II antigen expression. J. Infect. Dis. 171:39.[Medline]
  11. Aukrust, P., I. N. Farstand, S. S. Froland, E. Holter. 1992. Cytomegalovirus (CMV) pneumonitis in AIDS patients: the result of intensive CMV replication?. Eur. Respir. J. 5:362.[Abstract]
  12. Vasudevan, V. P., D. A. Mascarenhas, P. Klapper, S. Lomvardias. 1990. Cytomegalovirus necrotizing bronchiolitis with HIV infection. Chest 97:483.[Abstract/Free Full Text]
  13. Ho, M.. 1990. Epidemiology of cytomegalovirus infection. Rev. Infect. Dis. 12:701.
  14. Britt, W. J., C. A. Alford. 1996. Cytomegalovirus. B. N. Fields, and D. M. Knipe, and P. M. Howley, eds. Fields Virology 2493. Lippincott-Raven Publishers, Philadelphia, PA.
  15. Hahn, G., R. Jones, E. S. Mocarski. 1998. Cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells. Proc. Natl. Acad. Sci. USA 95:3937.[Abstract/Free Full Text]
  16. Walker, C., E. Bode, L. Boer, T. T. Hansel, K. Blaser, J. J. Virchow. 1992. Allergic and nonallergic asthmatics have distinct patterns of T-cell activation and cytokine production in peripheral blood and bronchoalveolar lavage. Am. Rev. Respir. Dis. 146:109.[Medline]
  17. Krug, N., J. Madden, A. E. Redington, P. Lackie, R. Djukanovic, U. Schauer, S. T. Holgate, A. J. Frew, P. H. Howarth. 1996. T-cell cytokine profile evaluated at the single cell level in BAL and blood in allergic asthma. Am. J. Respir. Cell Mol. Biol. 14:319.[Abstract]
  18. Hudson, J. B.. 1979. The murine cytomegalovirus as a model for the study of viral pathogenesis and persistent infections. Arch. Virol. 62:1.[Medline]
  19. Jonjic, S., W. Mutter, F. Weiland, M. J. Reddehase, U. H. Koszinowski. 1989. Site-restricted persistent cytomegalovirus infection after selective long-term depletion of CD4+ T lymphocytes. J. Exp. Med. 169:1199.[Abstract/Free Full Text]
  20. Jonjic, S., I. Pavic, P. Lucin, D. Rukavina, U. H. Koszinowski. 1990. Efficacious control of cytomegalovirus infection after long-term depletion of CD8+ T lymphocytes. J. Virol. 64:5457.[Abstract/Free Full Text]
  21. Lucin, P., I. Pavic, B. Polic, S. Jonjic, U. H. Koszinowski. 1992. {gamma} interferon-dependent clearance of cytomegalovirus infection in salivary glands. J. Virol. 66:1977.[Abstract/Free Full Text]
  22. Hogan, S. P., A. Koskinen, K. I. Mattaei, I. G. Young, P. S. Foster. 1998. Interleukin-5 producing CD4+ T cells play a pivotal role in aeroallergen-induced eosinophilia, bronchial hyperreactivity, and lung damage in mice. Am. J. Respir. Crit. Care Med. 157:210.[Abstract/Free Full Text]
  23. Hamelmann, E., A. Oshiba, J. Paluh, K. Bradley, J. Loader, T. A. Potter, G. L. Larsen, E. W. Gelfand. 1996. Requirement for CD8+ T cells in the development of airway hyperresponsiveness in a murine model of airway sensitization. J. Exp. Med. 183:1719.[Abstract/Free Full Text]
  24. Heise, M. T., H. W. Virgin. 1995. The T cell independent role of IFN-{gamma} and TNF-{alpha} in macrophage activation during murine cytomegalovirus and herpes simplex virus infection. J. Virol. 69:904.[Abstract]
  25. Orange, J. S., B. Wang, C. Terhorst, C. A. Biron. 1995. Requirement for natural killer cell-produced interferon {gamma} in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin 12 administration. J. Exp. Med. 182:1045.[Abstract/Free Full Text]
  26. Hengel, H., P. Lucin, S. Jonjic, T. Ruppert, U. H. Koszinowski. 1994. Restoration of cytomegalovirus antigen presentation by {gamma} interferon combats viral escape. J. Virol. 68:289.[Abstract/Free Full Text]
  27. Presti, R. M., J. L. Pollock, A. J. DalCanto, A. K. O’Guin, H. W. Virgin. 1998. Interferon {gamma} regulates acute and latent murine cytomegalovirus infection and chronic disease of the great vessels. J. Exp. Med. 188:577.[Abstract/Free Full Text]
  28. Lucin, P., S. Jonjic, M. Messerle, B. Polic, H. Hengel, U. H. Koszinowski. 1994. Late phase inhibition of murine cytomegalovirus replication by synergistic action of interferon-{gamma} and tumour necrosis factor. J. Gen. Virol. 75:101.[Abstract/Free Full Text]
  29. Lack, G., H. Renz, J. Saloga, K. Bradley, J. Loader, D. Y. M. Leung, E. W. Gelfand. 1994. Nebulized but not parenteral IFN-{gamma} decreases IgE production and normalized airway function in a murine model of allergen sensitization. J. Immunol. 152:2546.[Abstract]
  30. Li, X.-M., R. K. Chopra, T.-Y. Chou, B. H. Schofield, M. Wills-Karp, S.-K. Huang. 1996. Mucosal IFN-{gamma} gene transfer inhibits pulmonary allergic responses in mice. J. Immunol. 157:3216.[Abstract]
  31. Kung, T. T., D. M. Stelts, J. A. Zurcher, H. Jones, S. P. Umland, R. W. Egan, W. Kreutner, R. W. Chapman. 1995. Interferon-{gamma} and antibodies to interleukin-5 and interleukin-4 inhibit the pulmonary eosinophilia in allergic mice. Inflamm. Res. 44:S185.
  32. Yiamouyiannis, C. A., C. M. Schramm, L. Puddington, P. Stengel, H. E. Whiteley, R. S. Thrall. 1999. Shifts in lung lymphocyte profiles correlate with the sequential development of acute allergic and chronic tolerant stages in a murine asthma model. Am. J. Pathol. 154:1911.[Abstract/Free Full Text]
  33. Jacky, J. P.. 1978. A plethysmograph for long-term measurements of ventilation in unrestrained animals. J. Appl. Physiol. 45:644.[Abstract/Free Full Text]
  34. Renz, H., H. R. Smith, J. E. Henson, B. S. Ray, C. G. Irvin, E. W. Gelfand. 1992. Aerosolized antigen exposure without adjuvant causes increased IgE production and increased airway responsiveness in the mouse. J. Allergy Clin. Immunol. 89:1127.[Medline]
  35. Lack, G., A. Oshiba, K. L. Bradley, J. E. Loader, D. Amran, G. L. Larsen, E. W. Gelfand. 1995. Transfer of immediate hypersensitivity and airway hyperresponsiveness by IgE-positive B cells. Am. J. Respir. Crit. Care Med. 152:1765.[Abstract]
  36. Eum, S. Y., S. Haile, J. Lefort, M. Huerre, B. B. Vargaftig. 1995. Eosinophil recruitment into the respiratory epithelium following antigen challenge in hyper-IgE mice is accompanied by interleukin 5-dependent bronchial hyperresponsiveness. Proc. Natl. Acad. Sci. USA 92:12290.[Abstract/Free Full Text]
  37. Coyle, A. J., K. Wagner, C. Bertrand, S. Tsuyuki, J. Brews, C. Heusser. 1996. Central role of immunoglobulin (Ig) E in the induction of lung eosinophil infiltration and T helper 2 cell cytokine production: inhibition by a non-anaphylactogenic anti-IgE antibody. J. Exp. Med. 183:1303.[Abstract/Free Full Text]
  38. Wang, J., K. Palmer, J. Lotvall, S. Milan X.-F. Lei, K. I. Matthaei, J. Gauldie, M. D. Inman, M. Jordana, Z. Xing. 1998. Circulating, but not local lung, IL-5 is required for the development of antigen-induced airways eosinophilia. J. Clin. Invest. 102:1132.[Medline]
  39. Chung, K. F., P. J. Barnes. 1999. Cytokines in asthma. Thorax 54:825.[Free Full Text]
  40. Wills-Karp, M.. 1999. Immunologic basis of antigen-induced airway hyperresponsiveness. Annu. Rev. Immunol. 17:255.[Medline]
  41. Mosmann, T. R., S. Sad. 1996. The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol. Today 17:138.[Medline]
  42. Umetsu, D. T., R. H. DeKruyff. 1999. Interleukin-10: the missing link in asthma regulation?. Am. J. Respir. Cell Mol. Biol. 21:562.[Free Full Text]
  43. Lopez, A. F., C. J. Sanderson, J. R. Gamble, H. D. Campbell, I. G. Young, M. A. Vadas. 1988. Recombinant human interleukin 5 is a selective activator of human eosinophil function. J. Exp. Med. 167:219.[Abstract/Free Full Text]
  44. Yamaguchi, Y., Y. Hayashi, Y. Sugama, Y. Miura, T. Kasahara, S. Kitamura, M. Torisu, S. Mita, A. Tominaga, K. Takatsu, T. Suda. 1988. Highly purified murine interleukin-5, IL-5, stimulates eosinophil function and prolongs in vitro survival. J. Exp. Med. 167:1737.[Abstract/Free Full Text]
  45. Campbell, H. D., W. Q. Tucker, Y. Hort, M. E. Martinson, G. Mayo, E. J. Clutterbuck, C. J. Sanderson, I. G. Young. 1987. Molecular cloning, nucleotide sequence, and expression of the gene encoding human eosinophil differentiation factor interleukin 5. Proc. Natl. Acad. Sci. USA 84:6629.[Abstract/Free Full Text]
  46. Zhu, A., R. J. Homer, A. Wang, Q. Chen, G. P. Geba, J. Wang, Y. Zhang, J. A. Elias. 1999. Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J. Clin. Invest. 103:779.[Medline]
  47. Wills-Karp, M., J. Luyimbazi, X. Xu, B. Schofield, T. Y. Neben, C. L. Karp, D. D. Donaldson. 1998. Interleukin-13: central mediator of allergic asthma. Science 282:2258.[Abstract/Free Full Text]
  48. Grunig, G., M. Warnock, A. E. Wakil, R. Venkaya, F. Brombacher, D. M. Rennick, D. Sheppard, M. Mohrs, D. D. Donaldson, R. M. Locksley, D. B. Corry. 1998. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 282:2261.[Abstract/Free Full Text]
  49. Yang, X., S. Wang, Y. Fan, X. Han. 2000. IL-10 deficiency prevents IL-5 overproduction and eosinophilic inflammation in a murine model of asthma-like reaction. Eur. J. Immunol. 30:382.[Medline]
  50. Makela, M. J., A. Kanehiro, L. Borish, A. Dakhama, J. Loader, A. Joetham, Z. Xing, M. Jordana, G. L. Larsen, E. W. Gelfand. 2000. IL-10 is necessary for the expression of airway hyperresponsiveness but not pulmonary inflammation after allergic sensitization. Proc. Natl. Acad. Sci. USA 97:6007.[Abstract/Free Full Text]
  51. Hamelmann, E., J. Schwarze, K. Takeda, A. Oshiba, G. L. Larsen, C. G. Irvin, E. W. Gelfand. 1997. Non-invasive measurement of airway responsiveness in allergic mice using barometric plethysmography. Am. J. Respir. Crit. Care Med. 156:766.[Abstract/Free Full Text]
  52. Cieslewica, G., A. Tomkinson, A. Adler, C. Duez, J. Schwarze, K. Takeda, K. A. Larson, J. J. Lee, C. G. Irvin, E. W. Gelfand. 1999. The late, but not early, asthmatic response is dependent on IL-5 and correlates with eosinophil infiltration. J. Clin. Invest. 140:301.
  53. Bancroft, G. J., G. R. Shellam, J. E. Chalmer. 1981. Genetic influences on the augmentation of natural killer cells during murine cytomegalovirus infection: correlation with patterns of resistance. J. Immunol. 126:988.[Abstract]
  54. Reddehase, M. J., W. Mutter, K. Munch, H. J. Buhring, U. H. Koszinowski. 1987. CD8+ positive T lymphocytes specific for murine cytomegalovirus immediate-early antigens mediate protective immunity. J. Virol. 61:3102.[Abstract/Free Full Text]
  55. Orange, J. S., C. A. Biron. 1996. An absolute and restricted requirement for IL-12 in natural killer cell IFN-{gamma} production and antiviral defence: studies of natural killer and T cell responses in contrasting viral infections. J. Immunol. 156:1138.[Abstract]
  56. Kuperman, D., B. Schofield, M. Wills-Karp, M. J. Grusby. 1998. Signal transducer and activator of transcription factor 6 (Stat6)-deficient mice are protected from antigen-induced airway hyperresponsiveness and mucus production. J. Exp. Med. 187:939.[Abstract/Free Full Text]



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[Abstract] [Full Text] [PDF]

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