Murine Cytomegalovirus Infection Alters Th1/Th2 Expression, Decreases Airway Eosinophilia, and Enhances Mucus Production in Allergic Airway This information is current as of September 24, 2021. Carol A. Wu, Lynn Puddington, Herbert E. Whiteley, Carmen A. Yiamouyiannis, Craig M. Schramm, Fusaini Mohammadu and Roger S. Thrall J Immunol 2001; 167:2798-2807; ;

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References This article cites 55 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/167/5/2798.full#ref-list-1 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Murine Cytomegalovirus Infection Alters Th1/Th2 Cytokine Expression, Decreases Airway Eosinophilia, and Enhances Mucus Production in Allergic Airway Disease1

Carol A. Wu,2* Lynn Puddington,* Herbert E. Whiteley,† Carmen A. Yiamouyiannis,‡ Craig M. Schramm,§ Fusaini Mohammadu,* and Roger S. Thrall*

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 chal- lenged and MCMV/challenged mice, the cellular profile differed dramatically. After 10 days of OVA-aerosol challenge, comprised 64% of the total leukocyte population in BAL fluid from challenged mice compared with 11% in MCMV/challenged Downloaded from mice. 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␣␤ 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-␥ and IL-10 mRNAs, as well as decreased levels of IL-4, IL-5, and IL-13 mRNAs. Elevated levels of IFN-␥ and reduced levels of IL-5 were also demonstrated in BAL fluid from MCMV/challenged mice. Histological evaluation of lung sections revealed extensive http://www.jimmunol.org/ 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. The Journal of Immunology, 2001, 167: 2798–2807.

pidemiological evidence supports a close relationship be- Although not widely included in epidemiological studies on tween respiratory viral infections and exacerbation of , human CMV (HCMV)3 has been associated with asthma asthma, with viral upper respiratory infections coinciding exacerbations in adults (7). HCMV, a ␤ herpesvirus, is recognized E by guest on September 24, 2021 with asthma attacks in 80–85% of school-age children and 44% of as an opportunistic pulmonary pathogen and is a major cause of adults (1, 2). Studies of individuals with asthma demonstrate a pneumonia in immunosuppressed and lung trans- clear correlation between the presence of and an increase in plantation recipients (reviewed in Ref. 8). In lung samples, alve- asthma symptom scores, an increased need for medication, and a olar epithelial cells represent the majority of infected cells and are decrease in pulmonary function (reviewed in Ref. 3). Respiratory fully permissive for HCMV replication (9–11). Although less fre- syncytial virus and parainfluenza virus are common in quent, HCMV infection of the bronchial has also been young children, whereas rhinovirus is a frequent etiological agent reported (9, 12). HCMV is ubiquitous in nature with 40–100% of in older children and adults (reviewed in Refs. 3–5). However, any the adult population becoming infected (13). Primary infection is virus capable of eliciting an acute respiratory infection has the generally unremarkable, but chronic infection with intermittent vi- potential to exacerbate asthma (6). The mechanisms by which vi- ral shedding and the establishment of latency occurs even in im- ruses induce the onset of an asthma attack or increase its severity munocompetent individuals (reviewed in Ref. 14). Reactivation of are not fully understood, but are likely to involve multiple path- latent HCMV in vitro can be triggered by IL-4 or IFN-␥ (15) ways that include alterations in airway inflammation through the that are elevated in the blood and bronchoalveolar la- activation of T lymphocytes and the release of inflammatory cell vage (BAL) fluid of individuals with asthma (16, 17). mediators. 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 CD4ϩ and CD8ϩ T response, which is nec- Departments of *Medicine and §Pediatrics, University of Connecticut School of Med- icine, Farmington, CT 06030; †Department of Pathobiology, University of Connect- essary to mediate viral clearance from the and pe- icut, Storrs, CT 06269; and ‡Department of Science and Mathematics, Capital Com- ripheral organs, respectively (19–21). These two subsets of T lym- munity College, Hartford, CT 06105 phocytes have been shown to play a role in the progression of Received for publication November 8, 2000. Accepted for publication July 3, 2001. allergic airway inflammation and airway hyperresponsiveness (22, The costs of publication of this article were defrayed in part by the payment of page 23). In addition, MCMV infection induces a strong Th1 response, charges. This article must therefore be hereby marked advertisement in accordance characterized by the production of IFN-␥, which helps regulate with 18 U.S.C. Section 1734 solely to indicate this fact. acute, chronic, and latent viral infection (21, 24–28). A therapeutic 1 Funding for these studies was provided by grants from the American Lung Asso- ciation (to C.A.W.) and National Institutes of Health Grant AI-43573 (to R.S.T.). 2 Address correspondence and reprint requests to Dr. Carol A. Wu, Division of In- 3 Abbreviations used in this paper: HCMV, human CMV; MCMV, murine CMV; fectious , University of Connecticut Health Center, 263 Farmington Avenue, BAL, bronchoalveolar lavage; PAS, diastase-periodic acid-Schiff; Penh, enhanced Farmington, CT 06030-3212. E-mail address: [email protected] pause; FSC, forward scatter; SSC, side scatter.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 2799 role for IFN-␥ has been suggested in various murine models of for 30 min at 4°C. After staining, the cells were washed twice with the asthma (29–31). Therefore, MCMV may potentially alter the pro- above PBS solution, and relative fluorescence intensities were determined gression of airway inflammation through the proliferation of virus- by flow cytometric analysis using a FACSCalibur (BD Biosciences, San Jose, CA). specific T lymphocytes or changes in cytokine expression. In this report, we examined the influence of concomitant MCMV infec- Lung histology and quantitative image analysis tion on the development of allergic airway disease in the OVA- At the time of sacrifice, unmanipulated, noninflated lung tissue was re- induced murine model. moved from animals, fixed in a 10% buffered formalin solution, and em- bedded in paraffin. Tissue sections were stained with H&E for general Materials and Methods morphology and diastase-periodic acid-Schiff (PAS) for the detection of Animals mucins by the Department of Pathobiology at the University of Connect- icut (Storrs, CT). Quantitative analysis of PAS-stained lung sections from Male and female C57BL/6J mice, purchased from The Jackson Laboratory MCMV/sensitized, challenged, and MCMV/challenged mice was per- (Bar Harbor, ME), were housed at the University of Connecticut Health formed as follows. Black and white digital images of lung sections were Center. All testing and animal manipulations were preapproved by the captured using a Carl Zeiss (Thornwood, NY) Axiovert 135 inverted mi- Animal Care Committee at the University of Connecticut Health Center croscope, a Photometrics EEV37 PXL CCD camera (Roper Scientific, and followed the guidelines established by the U.S. Animal Welfare Act. Trenton, NJ), and EasyPXL software (F. R. Morgan, University of Con- necticut Health Center). The resulting images were assembled and ana- Study protocol lyzed using Adobe PhotoShop 5.0.2 and Scion Image Beta 3b. The outer Sensitization and OVA-aerosol challenge of C57BL/6J mice has been pre- boundary of each airway was defined and the total area determined by pixel viously described for our model of OVA-induced allergic airway disease count. Using the black and white pixel function, the unstained (unobstruct- (32). Briefly, challenged mice (representing allergic airway disease) were ed) area of the airway lumen (white pixels) was calculated and expressed Downloaded from sensitized with three weekly i.p. injections of 25 ␮g OVA, grade V (Sigma, as a percentage of the total area. A distinction between small (less than half St. Louis, MO) suspended in alum. One week later the animals were placed of the visual field) and medium (greater than half of the visual field) air- ϫ in a nose only exposure chamber and challenged with a 1% OVA aerosol ways was made at a magnitude of 10. Analysis was performed with the generated by a Lovelace nebulizer (In-Tox Products, Albuquerque, NM). assistance of the Center for Biomedical Imaging Technology at the Uni- The estimated daily inhaled OVA dose was 80 ␮g/mouse. This procedure versity of Connecticut Health Center (Farmington, CT). 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 Determination of pulmonary function http://www.jimmunol.org/ were sacrificed, and analysis of BAL fluid, lung tissue, and blood samples Pulmonary function in challenged, MCMV/challenged, sensitized, and was performed. An outline of this protocol is presented in Fig. 1A. Sensi- MCMV/sensitized mice was assessed in awake, unrestrained mice by tized mice were included in these studies as a control and received three whole-body barometric plethysmography (33). Briefly, mice were placed weekly i.p. injections of OVA/alum but were not exposed to OVA-aerosol in the main chamber of a whole-body plethysmograph (Buxco Electronics, challenge. Sharon, CT) and exposed for 2 min to aerosolized saline or increasing For the studies with MCMV, MCMV/challenged mice were sensitized concentrations of methacholine from 3 to 100 mg/ml. with three weekly injections of OVA/alum and infected intranasally with variables including tidal volume, respiratory frequency, inspiratory/expi- MCMV (detailed below) 7 days before the start of OVA-aerosol challenge. ratory times, and changes in box pressure were recorded before and during Two control groups were included in these studies: 1) MCMV/sensitized aerosolization and for 4 min after each exposure. The maximal enhanced mice, which were sensitized with three weekly i.p. injections of OVA/alum

pause (Penh) value response to methacholine was recorded at each dose. To by guest on September 24, 2021 and infected with MCMV, but were not exposed to OVA-aerosol chal- assess airway sensitivity, the interpolated concentration of methacholine lenge; and 2) MCMV alone mice, which were infected with MCMV, but needed to increase the Penh value to2U(aϳ5-fold increase over baseline) were not sensitized with OVA/alum or exposed to OVA-aerosol challenge. was calculated. As plateau responses were not obtained, a conventional Virus propagation and infection half-maximal methacholine concentration could not be calculated, and the Penh-2 value was selected as the portion of the dose-response curve where MCMV strain K181, purchased from American Type Culture Collection greatest changes in sensitivity would be manifested. (Manassas, VA), was commercially screened for other pathogens and scored negative. The virus was routinely propagated in mouse embryo RNase protection assays cells, maintained in DMEM containing 10% FCS (Gemini Bio-Products, Calabasa, CA), 100 U/ml , and 50 ␮g/ml streptomycin. Total RNA was isolated from ϳ100 mg of fresh lung tissue after homog- Animals were anesthetized with 0.2 ml of a 1/10 mixture of ketamine enization in 1 ml of Ultraspec RNA solution (Biotecx Laboratories, Hous- (90 mg/ml) and xylazine (10 mg/ml) and inoculated intranasally with 1.5 ϫ ton, TX). 32P-labeled riboprobes were generated using an in vitro tran- 104 PFU of MCMV. Infected animals were housed in isolation apart from scription kit (BD PharMingen) and the mouse cytokine multiprobe uninfected animals and showed no signs of illness (weight loss, changes in template set, mCK-1 (BD PharMingen), according to the manufacturer’s appearance and apparent behavior, etc.). At the end of each experiment, a specifications. These antisense probes were hybridized with total RNA, portion of the lung was processed to determine viral load for infected and then treated with a mixture containing RNase A ϩ T1 from a RNase pro- uninfected animals using a standard plaque assay. All lung tissue from tection kit (BD PharMingen). The resulting hybrids were resolved on a 6% uninfected animals was negative for MCMV. polyacrylamide-urea gel and analyzed by autoradiography.

BAL fluid analysis Measurement of cytokines Twenty-four hours after the final OVA-aerosol challenge, the lungs from BAL fluid was recovered from challenged and MCMV/challenged mice each animal were lavaged in situ with five 1-ml aliquots of sterile saline after 3 or 10 days of OVA-aerosol challenge, along with BAL fluid from (33). Total leukocyte counts were scored using a hemocytometer, and vi- sensitized, MCMV/sensitized, and MCMV alone mice sacrificed on the ability was determined by trypan blue dye exclusion. Leukocyte subsets same days. BAL fluid was concentrated 10-fold using an Amicon (Beverly, (eosinophils, , or lymphocytes) were enumerated in BAL fluid MA) Centriplus YM-10 filtration device and examined by ELISA for the using cytocentrifuged preparations stained with May-Gru¨nwald/Giemsa. presence of IL-5, IL-10, and IFN-␥ (Pierce Endogen, Rockford, IL) and Further characterization of the lymphocyte population of leukocytes was IL-13 (R&D Systems, Minneapolis, MN). The limits of detection for IL-5, performed by fluorescence flow cytometry using mAbs against the follow- IL-10, IFN-␥, and IL-13 were 5, 12, 10, and 1.5 pg/ml, respectively. In ing Ags: CD45 (clone 30-F11), TCR␤ (H57.597), CD3⑀ (500A2), or CD8 addition, blood was obtained from challenged, MCMV/challenged, and (53-6.7) (all purchased from BD PharMingen, San Diego, CA) or CD4 control mice by cardiac puncture before sacrifice, to measure circulating (GK1.5) (purchased from BD Collaborative Technologies, Bedford, MA). IL-5 by ELISA. These Abs were conjugated with biotin, PE, FITC, or allophycocyanine. Biotin-conjugated Abs were detected with streptavidin-Cy5 (Jackson Im- Statistical analysis munoResearch Laboratories, West Grove, PA) or PE-Cy7 (Caltag Labo- ratories, San Francisco, CA). For fluorescence flow cytometry, BAL cells Groups were compared by Student’s unpaired t test. ANOVA was used to Ͻ were washed in PBS containing 0.2% BSA and 0.1% NaN3. Aliquots of compare Penh measurements. Values of p equal to or 0.05 were consid- 104–105 cells were incubated with 100 ␮l of the appropriately diluted Abs ered significant. All data are expressed as the mean Ϯ SE. 2800 MCMV INFECTION ALTERS ALLERGIC AIRWAY DISEASE

Results 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, demon- strated elevated levels of macrophages, eosinophils, and lympho- cytes in their BAL fluid (33). The goal of this study was to deter- mine the influence of MCMV, an opportunistic respiratory pathogen, in this model. Our strategy is outlined in Fig. 1A. Mice received three weekly injections of OVA/alum and were infected intranasally with MCMV before the start of OVA-aerosol chal- lenge. 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 ϫ 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. 1B,

peak viral load was observed on day 7, with virus titers rapidly Downloaded from 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 http://www.jimmunol.org/ OVA-aerosol-challenged (MCMV/challenged) mice was com- pared with uninfected OVA-aerosol-challenged (challenged) mice after 3 days of OVA-aerosol exposure. It has been established in FIGURE 1. Rationale for the model of OVA-induced allergic airway this model that increases in BAL fluid cells are noticeable on day disease. A, Treatments in the model including three weekly i.p. injections 3, but have not reached maximum levels in challenged animals of OVA/alum, with the last injection given 1 wk before the start of OVA- (32), allowing exacerbations caused by viral infection to be scored. aerosol challenge. In some groups, mice were infected intranasally with As shown in Table I, total leukocytes increased from 2.5 ϫ 104 in MCMV 1 wk before OVA-aerosol challenge (Ⅺ). Samples were collected naive animals to 14 ϫ 104 in challenged mice ( p Ͻ 0.05), repre- at various times after OVA-aerosol challenge, as indicated in the text. B,To senting a marked augmentation of and determine the time of peak viral load in the lung, naive C57BL/6J mice by guest on September 24, 2021 ϫ 4 populations. Airway inflammation, as determined by the total were infected intranasally with 1.5 10 PFU of MCMV strain K181. number of leukocytes recovered from BAL fluid, was also present Four, 7, 10, 14, 21, and 42 days after infection, animals were sacrificed, lung tissue was removed, and viral titers were determined by standard in MCMV/challenged animals (18.7 ϫ 104 cells; p Ͻ 0.02); how- plaque assay (n ϭ 4). The results are expressed as PFU per gram of lung ever, the profile of cells differed. The BAL fluid from MCMV/ tissue at various times after infection. Interestingly, OVA-aerosol chal- challenged mice contained fewer eosinophils and an enhanced pro- lenge did not affect the time course of viral clearance from the lung in portion of lymphocytes. No significant changes in the total number MCMV/challenged mice. 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). MCMV infection vs concomitant MCMV infection with allergic To aid in the interpretation of our data, two additional control airway inflammation. As shown in Table I, a 2.5-fold increase in groups were included in these studies. MCMV/sensitized control the total number of leukocytes in BAL fluid was observed in mice received three weekly i.p. injections of OVA/alum and were MCMV/sensitized mice when compared with sensitized mice; infected intranasally with MCMV, but did not receive OVA-aero- however, this increase was not statistically significant ( p ϭ 0.07). sol challenge. This control group helped us discern the effects of MCMV/sensitized mice also displayed an increase in the number

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

Total Leukocytes Macrophages Eosinophils Lymphocytes ϫ 104 ϫ 104 ϫ 104 ϫ 104

Naive 2.5 Ϯ 0.5 2.5 Ϯ 0.5 0 0 Sensitized 4.9 Ϯ 0.9 4.9 Ϯ 0.9 0 0 MCMV/sensitized 12.0 Ϯ 2.4 6.9 Ϯ 1.8 0.4 Ϯ 0.3 4.8 Ϯ 1.6 Challenged 14.0 Ϯ 4.1* 7.6 Ϯ 1.4 5.2 Ϯ 2.5 1.0 Ϯ 0.3 MCMV/challenged 18.7 Ϯ 3.8* 8.9 Ϯ 1.1 0.4 Ϯ 0.1 9.3 Ϯ 2.7†

a Mice were sensitized with three weekly injections of OVA/alum, then uninfected (challenged) and infected (MCMV/ challenged) animals were exposed to aerosolized OVA for 3 days. Twenty-four hours after the last OVA-aerosol challenge, mice were sacrificed, and differential analysis was performed on BAL fluid cells stained with May-Gru¨nwald/Giemsa. Control animals included naive mice (no treatment), sensitized mice (i.p. injections of OVA/alum, no aerosol challenge), and MCMV/sensitized p Ͻ 0.05 when compared to naive. †, p Ͻ ,ء .(i.p. injections of OVA/alum and infection with MCMV, but no aerosol challenge) 0.02 when compared to challenged. n ϭ 5. The Journal of Immunology 2801 of lymphocytes present in BAL fluid, but again this difference was lower at all time points examined ( p Ͻ 0.006). Thus, MCMV not statistically significant when compared with sensitized mice infection appears to suppress eosinophilia in this model. ( p ϭ 0.06). Our final control group of mice, referred to as MCMV A comparable percentage of macrophages was found in the alone, was infected intranasally with MCMV, but was not sensi- BAL fluid of both groups after 3 or 7 days of OVA-aerosol chal- tized with OVA/alum or challenged with OVA aerosol. Although lenge (Fig. 2), but the percentage increased in MCMV/challenged the total number of leukocytes recovered in BAL fluid from mice after 10 days ( p Ͻ 0.01). In addition, the percentage of lym- MCMV alone mice was reduced 50% when compared with phocytes in MCMV/challenged mice was significantly higher MCMV/sensitized mice, the cellular profiles were similar. Macro- when compared with challenged mice, 45 vs 7% after 3 days ( p Ͻ phages, lymphocytes, and eosinophils comprised 61, 38, and 1% of 0.001), and 30 vs 11% after 10 days ( p Ͻ 0.02). the leukocyte population, respectively, in MCMV alone mice com- pared with 59, 40, and 1% in MCMV/sensitized mice. MCMV infection augments the number of CD8ϩ T lymphocytes The decrease in the number of eosinophils found in BAL fluid in BAL fluid from MCMV/challenged mice may be attributed to a delay in eo- ϩ We have previously demonstrated a significant increase in CD4 sinophil infiltration to the lungs or a suppression in eosinophil TCR␣␤ lymphocytes in association with airway inflammation and recruitment normally observed in challenged mice. To distinguish eosinophilia in challenged mice (32). To examine the effect of between these possibilities, BAL fluid analysis was performed af- MCMV infection on the recruitment of T lymphocytes to the lung, ter 3, 7, and 10 days of OVA-aerosol challenge, comparing chal- flow cytometric analysis was performed on total BAL fluid leuko- lenged and MCMV/challenged mice. The total number of leuko- ϩ

cytes (all CD45 cells) collected from challenged and MCMV/ Downloaded from cytes recovered from BAL fluid peaked on day 7 with 44 ϫ 104 challenged mice (Fig. 3A). The forward scatter (FSC) vs side scat- and 50 ϫ 104 BAL cells present in challenged and MCMV/chal- ter (SSC) properties for leukocyte subsets are well established and lenged mice, respectively. As shown in Fig. 2, the percentage of the lymphocyte populations are circled, representing 9 and 46% for eosinophils present in challenged animals increased from 33% on challenged and MCMV/challenged mice after 7 days of OVA- day 3 to 64% on day 10 ( p Ͻ 0.008), with peak eosinophilia aerosol challenge. These findings are in good agreement with the occurring between days 7 and 10. The percentage of eosinophils in differential analysis of BAL fluid presented in Fig. 2. The major the BAL fluid from MCMV/challenged animals was significantly http://www.jimmunol.org/ leukocyte population in BAL fluid from challenged mice has FSC vs SSC properties typical of eosinophils, whereas the other prom- inent leukocyte population in MCMV/challenged mice has FSC vs SSC properties typical of macrophages. In addition, a marked shift in the ratio of CD4ϩ to CD8ϩ TCR␣␤ lymphocytes infiltrating the lung was observed (Fig. 3B). CD8ϩ TCR␣␤ lymphocytes com- prised 65 and 70% of the population after 3 and 7 days of OVA- aerosol exposure in MCMV/challenged mice. In challenged mice, ϩ

CD8 TCR␣␤ lymphocytes decreased from 46 to 15%, whereas by guest on September 24, 2021 CD4ϩ TCR␣␤ 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. 3C. After 3 days of OVA-aerosol challenge, the number of CD4ϩ and CD8ϩ TCR␣␤ lymphocytes in MCMV/challenged mice was 22.6 ϫ 103 and 54.5 ϫ 103 with a ratio of 0.4. In challenged animals, the number of CD4ϩ and CD8ϩ TCR␣␤ lymphocytes was 5.9 ϫ 103 and 3.6 ϫ 103 or a ratio of 1.6. A decrease in the ratio of CD4ϩ to CD8ϩ TCR␣␤ lymphocytes was also observed in MCMV/challenged mice after 7 days of OVA-aerosol challenge. This enhanced recruitment of lymphocytes to the lung and dra- matic augmentation of CD8ϩ TCR␣␤ lymphocytes in MCMV/ challenged mice is indicative of a host cell-mediated immune re- sponse against the virus.

Increased mucus secretion occurs during MCMV infection in OVA-aerosol-challenged mice Histological evaluations by H&E and PAS staining were per- formed on formalin-fixed, paraffin-embedded sections of unin- flated, nonmanipulated lungs from challenged and MCMV/chal- FIGURE 2. Suppression of BAL fluid eosinophilia in MCMV/chal- lenged mice. The lungs of challenged mice demonstrated a dense lenged mice. BAL fluid was collected from challenged and MCMV/chal- peribronchial inflammation consisting of lymphoplasmacytic cells lenged mice after 3, 7, and 10 days of OVA-aerosol exposure. Nucleated and eosinophils (Fig. 4C). There were also areas of perivascular cells were counted, and the BAL fluid cell differential was obtained from inflammation and slight peribronchial epithelial and smooth mus- cytocentrifuged preparations stained with May-Gru¨nwald/Giemsa. Data are cle hypertrophy. MCMV/challenged mice had more intense bron- expressed as the relative percentage of lymphocytes, macrophages, and eosinophils present at each time point (n ϭ 5). The total numbers of leu- chial epithelial cell hypertrophy/hyperplasia (D). No evidence of kocytes recovered from BAL fluid after 3, 7, and 10 days of OVA-aerosol histologic damage was found in naive (data not shown), sensitized challenge were 14 ϫ 104,44ϫ 104, and 27 ϫ 104 for challenged mice, and (A), or MCMV/sensitized (B) mice. 19 ϫ 104,50ϫ 104, and 17 ϫ 104 for MCMV/challenged mice, Few PAS positive staining cells were observed in sensitized respectively. mice (Fig. 4A), MCMV/sensitized mice (B), or naive animals (data 2802 MCMV INFECTION ALTERS ALLERGIC AIRWAY DISEASE Downloaded from

FIGURE 3. MCMV infection altered the ratio of CD4ϩ to CD8ϩ TCR␣␤ 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. 2. B, CD45ϩ http://www.jimmunol.org/ lymphocytes from BAL fluid of challenged and MCMV/challenged mice were positively gated for TCR␣␤ 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 ␣␤ TCR (TCR␣␤), and the number of CD4ϩ and CD8ϩ TCR␣␤ lymphocytes were determined for challenged and MCMV/challenged after 3 days of OVA-aerosol exposure (n ϭ 5).

not shown). Histological examination of lung sections from chal- days of OVA-aerosol exposure, mucus occlusions were not found by guest on September 24, 2021 lenged mice after 7 days of OVA-aerosol exposure revealed an in either group (data not shown). increase in PAS staining in bronchoepithelial cells (C), but the To calculate the level of airway obstruction caused by mucus cells exhibited a normal morphology. In contrast, MCMV/chal- plugging, digital images of small and medium airways from chal- lenged mice exhibited intense positive PAS staining, and the cells lenged, MCMV/challenged, and MCMV/sensitized mice were appeared elongated and more abundant, indicative of epithelial cell captured for quantitative image analysis (detailed in Materials and hypertrophy/hyperplasia (D). This dramatic mucus plugging, fre- Methods). After 7 days of OVA-aerosol challenge, blockage of quently seen in the airways of MCMV/challenged mice after 7 MCMV/challenged airways was significant in both small ( p Ͻ days of OVA-aerosol challenge, was not observed in challenged 0.02) and medium ( p Ͻ 0.001) airways (Fig. 5). Occlusion of animals. Furthermore, although positive PAS staining was noted in airways from challenged mice was not statistically significant the lungs of both challenged and MCMV/challenged mice after 3 when compared with MCMV/sensitized control mice.

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. The Journal of Immunology 2803

FIGURE 5. Quantitative image analysis of mucus obstruction in air- ways of MCMV/challenged mice. Lung tissue from MCMV/sensitized, challenged, and MCMV/challenged mice were examined for mucus pro-

duction by PAS staining as shown in Fig. 4. Black and white digital images Downloaded from of these lung sections were analyzed to determine the percentage of un- obstructed (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 ϫ10 (n ϭ 3–9). When compared with MCMV/ indicates a p value ءء indicates a p value Ͻ0.02, and ء ,sensitized mice

Ͻ0.0001. http://www.jimmunol.org/

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

To investigate whether MCMV infection altered pulmonary func- FIGURE 6. Development of airway hyperresponsiveness in challenged, tion in mice with allergic airway disease, Penh values were com- but not MCMV/challenged mice. A, Baseline responses to increasing doses pared between challenged and MCMV/challenged mice. We and of aerosolized methacholine (0–100 mg/ml) were assessed by whole-body others have demonstrated that maximal cholinergic hyperreactivity plethysmography for challenged and MCMV/challenged mice 1 day before by guest on September 24, 2021 occurs before the development of peak airway inflammation (i.e., initiation of OVA-aerosol challenge. No differences attributed to MCMV after 3–7 days of OVA-aerosol challenge in this model; Ref. 32). infection alone were noted. B, Airway hyperresponsiveness to methacho- Accordingly, serial changes in airway responsiveness were mea- line was measured in challenged and MCMV/challenged mice 12 h after sured in conscious, unrestrained mice after 3 and 6 days of OVA- the 3rd and 6th days of OVA-aerosol challenge by whole-body plethys- mography. The concentration of methacholine required to increase Penh aerosol challenge using whole-body plethysmography. At base- p Ͻ 0.05 ,ء .(values to2U(ϳ5-fold above baseline) was calculated (n ϭ 5 line, Penh responses to increasing doses of methacholine did not when compared with baseline values for challenged mice. differ between challenged and MCMV/challenged mice (Fig. 6A; p Ͼ 0.05). Methacholine dose-response relationships were as- sessed again 12 h after the third and sixth OVA-aerosol challenges tion was examined in both groups. Measurements of total IgE were and were compared in individual mice with their baseline re- calculated for challenged and MCMV/challenged mice after 3, 7, sponses. Challenged mice developed increased responsiveness to and 10 days of OVA-aerosol exposure. Increased serum IgE levels methacholine after 3 days of OVA-aerosol challenge, as demon- were observed for both challenged (range 1.67–3.13 ␮g/ml) and strated by a significant leftward shift in their dose-response rela- MCMV/challenged (range 0.68–2.84 ␮g/ml) mice in comparison tionships ( p Ͻ 0.05) and a 2- to 3-fold decrease in the methacho- to naive mice (0.02 ␮g/ml), but no differences were noted between line concentration eliciting a Penh of 2 U (Fig. 6B). This challenged and MCMV/challenged mice. Thus, MCMV infection heightened airway responsiveness persisted after the sixth day of does not appear to influence IgE production in this model of al- OVA-aerosol challenge. In contrast, MCMV/challenged mice did lergic airway disease. not demonstrate airway hyperresponsiveness after OVA-aerosol challenge. Their methacholine dose-response relationships and MCMV influences the balance of Th1/Th2 mRNA synthesis in Penh-2 values were statistically unchanged from baseline measure- the lung ments ( p ϭ 0.6 after 3 days; p ϭ 0.3 after 6 days). In addition, the Cytokines are important mediators in airway inflammation and can change in Penh-2 values after OVA-aerosol exposure was signif- regulate excessive production of mucus, as well as the recruitment icantly different between challenged and MCMV/challenged mice of eosinophils to the lung. To determine whether MCMV infection ( p Ͻ 0.03). alters the local cytokine environment in the lung, RNase protection assays were performed. Total RNA, isolated from lungs of chal- Elevated levels of total serum IgE after OVA-aerosol challenge lenged and MCMV/challenged mice, was hybridized with ribo- Ag-induced IgE synthesis has been associated with airway hyper- probes specific for Th1 and Th2 cytokines. The results obtained responsiveness in murine models of allergic airway disease (32, from challenged mice are shown in Fig. 7A. IL-5 and IL-13 34–37). As increased hyperresponsiveness was observed in chal- mRNAs were observed after 3 days of OVA-aerosol challenge lenged mice, but not MCMV/challenged mice, serum IgE produc- (lanes 5 and 6) and persisted throughout the time course (lanes 2804 MCMV INFECTION ALTERS ALLERGIC AIRWAY DISEASE

served in challenged (7.8 Ϯ 4.4 pg/ml) and MCMV/challenged (6.7 Ϯ 4.9 pg/ml) mice after 3 days of OVA-aerosol challenge. These levels slowly declined to 2.8 Ϯ 1.6 and 3.4 Ϯ 0.6 pg/ml Downloaded from http://www.jimmunol.org/

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 ϩ 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 chal- by guest on September 24, 2021 lenged mice. B, Results from MCMV/sensitized and MCMV/challenged mice. A nonspecific radioactive mark partially coincides with the IFN-␥ fragment in B, lane 4.

7Ð10). 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 1Ð4). 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 load- ing of RNA. The cytokine mRNA profiles from the lungs of MCMV/chal- lenged mice are shown in Fig. 7B. Expression of IL-10 and IFN-␥ mRNAs was observed after 3 days of OVA-aerosol exposure (lanes 3 and 4); however, the level of these cytokines diminished with time (lanes 5Ð7). IL-5 and IL-13 mRNAs were first detected after 7 days of OVA-aerosol challenge (lanes 5 and 6), and no FIGURE 8. Th1/Th2 cytokine expression is altered in BAL fluid from evidence of IL-4 mRNA was found at any time point examined. MCMV/challenged mice. BAL fluid was recovered from challenged and The synthesis of IL-10 and IFN-␥ was also detected in MCMV/ MCMV/challenged mice after 3 or 10 days of OVA-aerosol challenge (n ϭ sensitized mice, albeit at lower levels (lanes 1 and 2), whereas 6 for each group). The samples were concentrated 10-fold using Amicon expression of IL-4, IL-5, and IL-13 mRNAs were not found in this Centriplus YM-10 filtration units and assayed for IL-5 (A), IL-13 (B), and control group. Similar to the results shown in Fig. 7A, consistent IFN-␥ (C) by ELISA. Controls included concentrated BAL fluid from sen- expression of IL-15 was observed throughout the time course in sitized and MCMV/sensitized mice collected at the same time points (n ϭ MCMV/challenged and MCMV/sensitized mice. 6 for each group). Cytokine levels in sensitized mice were not significantly above baseline for any of these cytokines. Elevated levels of IFN-␥ were The induction of both local and systemic IL-5 has been reported observed in MCMV/sensitized control mice at the 3-day time point, but not during airway inflammation; however, a recent study indicates that at 10 days. No increase in IL-5 or IL-13 was observed in MCMV/sensitized circulating, not local, IL-5 may be required for the development of mice at either time point. Similarly, elevated levels of IFN-␥, but no in- pulmonary eosinophilia (38). Therefore, serum from challenged crease in IL-5 or IL-13, was noted at the 3-day time point for MCMV alone p Ͻ 0.05 when compared with sensitized controls. ϩ, p Ͻ 0.05 ,ء .and MCMV/challenged mice was examined for the presence of mice circulating IL-5 by ELISA. An increase in serum IL-5 was ob- when comparing MCMV/challenged with challenged mice. The Journal of Immunology 2805 after 14 days of OVA-aerosol exposure in challenged and MCMV/ found in any of our control groups. These findings are in good challenged mice, respectively. Serum IL-5 in sensitized controls agreement with previous reports indicating that elevated Th2 cy- was 2.7 Ϯ 0.2 pg/ml. These findings suggest that an increase in tokines play a pivotal role in the development and pathogenesis of circulating IL-5 was present in both challenged and MCMV/ allergic airway disease and asthma (reviewed in Refs. 39 and 40). challenged mice. In contrast, decreased expression of IL-4, IL-5, and IL-13 mRNAs, Finally, the levels of IL-5, IL-10, IL-13, and IFN-␥ were mea- as well as increased IFN-␥ mRNA production, was observed in sured by ELISA in concentrated BAL fluid from challenged and MCMV/challenged mice when compared with challenged mice. MCMV/challenged mice. A significant increase in IL-5 was de- Decreased expression of IL-5 and IL-13 and increased expression tected in challenged mice after 3 days of OVA-aerosol challenge of IFN-␥ were also demonstrated in BAL fluid recovered from when compared with sensitized controls (Fig. 8A; p Ͻ 0.03). After MCMV/challenged mice. Polarization toward a Th1 response has 10 days of OVA-aerosol challenge, the level of IL-5 in challenged been documented for other viral, bacterial, and protozoan infec- mice returned to baseline. In contrast, no increase in IL-5 was tions (reviewed in Ref. 41), although the idea that a Th1 response observed in MCMV/challenged mice after 3 or 10 days of OVA- can counterbalance Th2-induced airway inflammation remains aerosol challenge. This 20-fold difference in BAL fluid IL-5 levels controversial (42). between challenged and MCMV/challenged mice after 3 days of The decrease in BAL fluid eosinophilia observed in MCMV/ OVA-aerosol exposure was significant ( p Ͻ 0.01). An increase in challenged mice is most likely due to an inability of these animals IL-13 was found in BAL fluid from challenged mice when com- to generate an IL-5 response. IL-5, a cytokine necessary for the pared with sensitized controls (Fig. 8B; p Ͻ 0.02). Although IL-13 regulation of eosinophil growth, differentiation, activation, and was detected in BAL fluid from MCMV/challenged mice after 3 survival, plays a critical role in the recruitment of eosinophils to Downloaded from days of OVA-aerosol challenge, the level of IL-13 was not signif- the lung (43–45). In our studies, decreased levels of IL-5 mRNA icantly elevated when compared with controls ( p ϭ 0.20). Again, in lung tissue and decreased IL-5 levels in BAL fluid correlated the level of IL-13 in BAL fluid returned to baseline in both groups with reduced eosinophilia in MCMV/challenged mice. This reduc- after 10 days of OVA-aerosol challenge. Expression of IL-5 and tion in eosinophilia was observed at all time points examined, in- IL-13 was not observed in concentrated BAL fluid recovered from cluding times when no mucus plugging was found (i.e., after 3 sensitized, MCMV/sensitized, or MCMV alone control mice. In days of OVA-aerosol challenge). Therefore, decreased BAL fluid http://www.jimmunol.org/ addition, IL-10 was not detected in challenged or MCMV/chal- eosinophilia most likely cannot be attributed to technical difficul- lenged mice after 3 or 10 days of OVA-aerosol challenge and was ties involving BAL cell recovery in the presence of increased mu- not present in BAL fluid from sensitized or MCMV/sensitized cus production. No differences in serum IL-5 were detected be- control mice. tween challenged and MCMV/challenged mice after 3, 7, or 10 Increased production of IFN-␥ was observed in concentrated days of OVA-aerosol challenge. BAL fluid from challenged mice after 3 days of OVA-aerosol ex- Enhanced mucus production and epithelial cell hypertrophy/hy- posure when compared with sensitized controls (Fig. 8C; p Ͻ perplasia were observed in MCMV/challenged mice. Such changes 0.05). IFN-␥ synthesis was further elevated in MCMV/challenged in lung histology have typically been associated with a Th2 phe- mice after 3 days of OVA-aerosol challenge when compared with notype and IL-13 gene expression (46–48). Surprisingly, the lev- by guest on September 24, 2021 challenged animals ( p Ͻ 0.04). In both groups, IFN-␥ levels de- els of IL-13 in BAL fluid from MCMV/challenged mice were not creased to baseline after 10 days of OVA-aerosol exposure. As significantly elevated above controls (sensitized, MCMV/sensi- expected, elevated levels of IFN-␥ were found in BAL fluid re- tized, or MCMV alone mice) or background noise. In contrast, covered from MCMV/sensitized (132 pg/ml) mice when compared elevated levels of IL-13 were measured in BAL fluid from chal- with sensitized control mice (10 pg/ml) that were sacrificed at the lenged mice, which do not exhibit mucus plugging or epithelial same time as mice exposed to 3 days of OVA-aerosol challenge. cell hypertrophy/hyperplasia. Although IL-13 mRNA was ob- This significant increase in IFN-␥ levels ( p Ͻ 0.02) most likely served in the lungs of MCMV/challenged mice, expression was reflects the antiviral response of the host to MCMV infection. In- detected at only one time point (after 7 days of OVA-aerosol chal- deed, elevated levels of IFN-␥ were also observed in BAL fluid lenge), whereas IL-13 expression was noted at all time points in from MCMV alone controls (327 pg/ml). These values were not challenged mice. These findings suggest that other factors, in ad- statistically different from MCMV/sensitized mice ( p ϭ 0.07). dition to IL-13, are critical for mucus hypersecretion. A recent Thus, both MCMV infection and allergic airway disease appear to study indicates that IL-10 may be a key contributor to mucus hy- contribute to IFN-␥ production in MCMV/challenged mice. persecretion in allergic airway disease, as IL-10 knockout mice display diminished goblet cell development and mucus secretion Discussion (49). Gelfand and colleagues (50) have demonstrated that the ad- In this study, we examined the influence of MCMV, an opportu- ministration of IL-10 to either OVA-sensitized/challenged IL-10 nistic respiratory pathogen, on a murine model of OVA-induced knockout mice or OVA-sensitized/challenged wild-type mice allergic airway inflammation. As compared with challenged mice, heightened mucin production and goblet cell hyperplasia. In our MCMV/challenged mice exhibited 1) a decrease in Th2 cytokines studies, IL-10 was not detected in BAL fluid from either chal- present in lung tissue and BAL fluid, 2) a decrease in BAL fluid lenged or MCMV/challenged mice; however, increased levels of eosinophilia, 3) histological evidence of enhanced mucus plugging IL-10 mRNA were found in lung tissue from both groups. Inter- and bronchial epithelial cell hypertrophy/hyperplasia, and 4) an estingly, the kinetics of IL-10 gene expression differed with tran- increase in lymphocytes recovered from BAL fluid, which was sient expression of IL-10 mRNA appearing earlier in MCMV/chal- associated with a decrease in the ratio of CD4ϩ to CD8ϩ TCR␣␤ lenged mice (after 3 days of OVA-aerosol challenge) and lymphocytes. decreasing rapidly thereafter. In contrast, IL-10 mRNA synthesis Elevated levels of IL-4, IL-5, IL-10, and IL-13 mRNAs were was not observed until 7 days of OVA-aerosol exposure in chal- demonstrated in the lungs of challenged mice. In addition, in- lenged mice, and although mucin synthesis was increased in these creased levels of IL-5 and IL-13 protein were found in BAL fluid mice, mucus plugging was not observed. As the effects of IL-10 on from challenged mice. This Th2 phenotype corresponded with in- allergic airway inflammation are likely to be influenced by inter- creased eosinophilia and airway hyperresponsiveness, and was not actions with other cytokines, such differences in kinetic expression 2806 MCMV INFECTION ALTERS ALLERGIC AIRWAY DISEASE may be important. IL-10 mRNA synthesis was also noted in con- Acknowledgments trol MCMV/sensitized mice, and these mice do not develop aller- We gratefully acknowledge Dr. John Shanley for the gift of MCMV and his gic airway disease or exhibit mucus plugging and epithelial cell assistance during viral infection. We thank Drs. Michelle Cloutier and Leo hypertrophy/hyperplasia. Thus, mucus plugging and epithelial cell LeFrancois for critical review of the manuscript; our colleagues in the hypertrophy/hyperplasia observed in MCMV/challenged mice Pulmonary Research Consortium for their encouragement and helpful dis- cannot be directly attributed to IL-10 production, and other, yet cussions; and Linda Guernsey, Grace Nicksa, and Caroline Benkovich for their excellent technical assistance. 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 References 1. Johnston, S. L., P. K. Pattemore, G. Sanderson, S. Smith, F. Lampe, L. Josephs, in challenged mice using barometric whole-body plethysmogra- P. Symington, S. O’Tolle, S. H. Myint, D. A. J. Tyrrell, and S. T. Holgate. 1995. phy. Penh values obtained after 3 and 6 days of OVA-aerosol Community study of role of viral infections in exacerbations of asthma in 9–11 challenge demonstrated an increase in sensitivity to methacholine, year old children. Br. Med. J. 310:1225. 2. Nicholson, K. G., J. Kent, and D. C. Ireland. 1993. Respiratory viruses and indicated by a leftward shift in the dose-response curve, and an exacerbations of asthma in adults. Br. Med. J. 307:982. increase in reactivity, indicated by a decrease in the methacholine 3. Pattemore, P. K., S. L. Johnston, and P. G. Bardin. 1992. Viruses as precipitants concentration necessary to elicit a Penh of 2 U. In contrast, of asthma symptoms. I. Epidemiology. Clin. Exp. 22:325. 4. Busse, W. W., and J. E. Gern. 1997. Updates on cells and cytokines: viruses in MCMV/challenged mice did not display increased airway hyper- asthma. J. Allergy Clin. Immunol. 100:147. responsiveness. Enhanced Penh values have been shown to corre- 5. Folkerts, G., W. W. Busse, F. P. Nijkamp, R. Sorkness, and J. E. Gern. 1998. Virus-induced airway hyperresponsiveness and asthma. Am. J. Respir. Crit. Care late with increased pulmonary resistance, increased IgE produc- Med. 157:1708. Downloaded from tion, and increased pulmonary eosinophilia (51). Furthermore, 6. Monto, A. S. 1995. Epidemiology of respiratory viruses in persons with and studies have shown that both IL-5 and eosinophils are essential for without asthma and COPD. Am. J. Respir. Crit. Care Med. 151:1653. 7. Atmar, R. L., E. Guy, K. K. Guntupalli, J. L. Zimmerman, V. D. Bandi, B. D.B- the development of airway responsiveness during the late, but not axter, and S. B. Greenberg. 1998. Respiratory tract viral infections in inner-city early, asthmatic response in the mouse (52). In our model, in- asthmatic adults. Arch. Intern. Med. 158:2453. 8. Salomon, N., and D. C. Perlman. 1999. Cytomegalovirus pneumonia. Semin. creased Penh values in challenged mice were associated with in- Respir. Infect. 14:353.

creased eosinophilia and elevated levels of IL-5 in BAL fluid and 9. Singer, C., A. Grefte, B. Plachter, A. S. H. Gouw, T. H. The, and G. Jahn. 1995. http://www.jimmunol.org/ lung tissue, whereas the absence of airway hyperresponsiveness in Fibroblasts, epithelial cells, endothelial cells and smooth muscle cells are major targets of human cytomegalovirus infection in lung and gastrointestinal tissues. MCMV/challenged mice paralleled a reduction in airway eosino- J. Gen. Virol. 76:741. philia and undetectable levels of IL-5. Still, it was surprising that 10. Ng Bautista, C. L., and D. D. Sedmak. 1995. Cytomegalovirus infection is as- sociated with absence of alveolar epithelial cell HLA class II antigen expression. the extensive mucus plugging and epithelial cell hypertrophy/hy- J. Infect. Dis. 171:39. perplasia observed in MCMV/challenged mice did not lead to 11. Aukrust, P., I. N. Farstand, S. S. Froland, and E. Holter. 1992. Cytomegalovirus changes in pulmonary function as determined by whole-body (CMV) pneumonitis in AIDS patients: the result of intensive CMV replication? Eur. Respir. J. 5:362. plethysmography. Mucus plugging was most prevalent in small 12. Vasudevan, V. P., D. A. Mascarenhas, P. Klapper, and S. Lomvardias. 1990. airways, and changes in pulmonary function associated with in- Cytomegalovirus necrotizing bronchiolitis with HIV infection. Chest 97:483. 13. Ho, M. 1990. Epidemiology of cytomegalovirus infection. Rev. Infect. Dis. 12: by guest on September 24, 2021 creased obstruction of small airways may not be detectable by this 701. approach. 14. Britt, W. J., and C. A. Alford. 1996. Cytomegalovirus. In Fields Virology. NK cells (53) and CD8ϩ T lymphocytes (54) represent the ini- B. N. Fields, D. M. Knipe, and P. M. Howley, eds. Lippincott-Raven Publishers, Philadelphia, PA, p. 2493. tial response of the host to acute infection with MCMV. Activation 15. Hahn, G., R. Jones, and E. S. Mocarski. 1998. Cytomegalovirus remains latent in of NK cells peaks between 3 and 5 days after infection and is a common precursor of dendritic and myeloid cells. Proc. Natl. Acad. Sci. USA characterized by the induction of IFN-␥ (25). A second burst of 95:3937. ϩ 16. Walker, C., E. Bode, L. Boer, T. T. Hansel, K. Blaser, and J. J. Virchow. 1992. IFN-␥ synthesis correlates with the proliferation of CD8 T lym- Allergic and nonallergic asthmatics have distinct patterns of T-cell activation and phocytes 7–10 days after infection (55). These antiviral responses cytokine production in peripheral blood and bronchoalveolar lavage. Am. Rev. ϩ Respir. Dis. 146:109. are likely to account for the elevated levels of CD8 T lympho- 17. Krug, N., J. Madden, A. E. Redington, P. Lackie, R. Djukanovic, U. Schauer, cytes and, in part, for the increase in IFN-␥ observed in MCMV/ S. T. Holgate, A. J. Frew, and P. H. Howarth. 1996. T-cell cytokine profile challenged mice. Indeed, rapid viral clearance from the lungs of evaluated at the single cell level in BAL and blood in allergic asthma. Am. J. Respir. Cell Mol. Biol. 14:319. MCMV/challenged mice was observed in our studies. However, 18. Hudson, J. B. 1979. The murine cytomegalovirus as a model for the study of viral IFN-␥ was also detected in BAL fluid from challenged mice, albeit pathogenesis and persistent infections. Arch. Virol. 62:1. 19. Jonjic, S., W. Mutter, F. Weiland, M. J. Reddehase, and U. H. Koszinowski. to a lesser extent than MCMV/challenged mice, suggesting that 1989. Site-restricted persistent cytomegalovirus infection after selective long- allergic airway inflammation also contributes to IFN-␥ production. term depletion of CD4ϩ T lymphocytes. J. Exp. Med. 169:1199. 20. Jonjic, S., I. Pavic, P. Lucin, D. Rukavina, and U. H. Koszinowski. 1990. Effi- Expression of both Th1 and Th2 cytokines in BAL fluid from ϩ cacious control of cytomegalovirus infection after long-term depletion of CD8 OVA Ag-challenged mice has been reported by others (56). T lymphocytes. J. Virol. 64:5457. In summary, our results demonstrate that MCMV infection in 21. Lucin, P., I. Pavic, B. Polic, S. Jonjic, and U. H. Koszinowski. 1992. ␥ interferon- dependent clearance of cytomegalovirus infection in salivary glands. J. Virol. this model of allergic airway inflammation can modulate the dis- 66:1977. ease process in multiple ways. A reduction in Th2 cytokines, par- 22. Hogan, S. P., A. Koskinen, K. I. Mattaei, I. G. Young, and P. S. Foster. 1998. ϩ ticularly IL-5, was associated with a decrease in BAL fluid eosin- -5 producing CD4 T cells play a pivotal role in aeroallergen-induced eosinophilia, bronchial hyperreactivity, and lung damage in mice. Am. J. Respir. ophilia in MCMV/challenged mice, which is suggestive of Crit. Care Med. 157:210. decreased lung injury. In contrast, MCMV/challenged mice also 23. Hamelmann, E., A. Oshiba, J. Paluh, K. Bradley, J. Loader, T. A. Potter, G. L. Larsen, and E. W. Gelfand. 1996. Requirement for CD8ϩ T cells in the exhibited enhanced mucus plugging and epithelial cell hypertro- development of airway hyperresponsiveness in a murine model of airway sensi- phy/hyperplasia, which is usually indicative of exacerbation of al- tization. J. Exp. Med. 183:1719. lergic airway inflammation. Together, these findings highlight the 24. Heise, M. T., and H. W. Virgin. 1995. The independent role of IFN-␥ and TNF-␣ in macrophage activation during murine cytomegalovirus and herpes sim- complex nature of allergic airway disease, especially with respect plex virus infection. J. Virol. 69:904. to concomitant upper respiratory viral infections. Furthermore, 25. Orange, J. S., B. Wang, C. Terhorst, and C. A. Biron. 1995. Requirement for natural killer cell-produced interferon ␥ in defense against murine cytomegalo- they suggest that eosinophilia can occur independently of excess virus infection and enhancement of this defense pathway by interleukin 12 ad- mucus production and epithelial cell hypertrophy/hyperplasia. ministration. J. Exp. Med. 182:1045. The Journal of Immunology 2807

26. Hengel, H., P. Lucin, S. Jonjic, T. Ruppert, and U. H. Koszinowski. 1994. Res- 43. Lopez, A. F., C. J. Sanderson, J. R. Gamble, H. D. Campbell, I. G. Young, and toration of cytomegalovirus antigen presentation by ␥ interferon combats viral M. A. Vadas. 1988. Recombinant human interleukin 5 is a selective activator of escape. J. Virol. 68:289. human eosinophil function. J. Exp. Med. 167:219. 27. Presti, R. M., J. L. Pollock, A. J. DalCanto, A. K. O’Guin, and H. W. Virgin. 44. Yamaguchi, Y., Y. Hayashi, Y. Sugama, Y. Miura, T. Kasahara, S. Kitamura, 1998. Interferon ␥ regulates acute and latent murine cytomegalovirus infection M. Torisu, S. Mita, A. Tominaga, K. Takatsu, and T. Suda. 1988. Highly purified and chronic disease of the great vessels. J. Exp. Med. 188:577. murine interleukin-5, IL-5, stimulates eosinophil function and prolongs in vitro 28. Lucin, P., S. Jonjic, M. Messerle, B. Polic, H. Hengel, and U. H. Koszinowski. survival. J. Exp. Med. 167:1737. 1994. Late phase inhibition of murine cytomegalovirus replication by synergistic 45. Campbell, H. D., W. Q. Tucker, Y. Hort, M. E. Martinson, G. Mayo, action of interferon-␥ and tumour necrosis factor. J. Gen. Virol. 75:101. E. J. Clutterbuck, C. J. Sanderson, and I. G. Young. 1987. Molecular cloning, 29. Lack, G., H. Renz, J. Saloga, K. Bradley, J. Loader, D. Y. M. Leung, and nucleotide sequence, and expression of the gene encoding human eosinophil dif- E. W. Gelfand. 1994. Nebulized but not parenteral IFN-␥ decreases IgE produc- ferentiation factor interleukin 5. Proc. Natl. Acad. Sci. USA 84:6629. tion and normalized airway function in a murine model of allergen sensitization. 46. Zhu, A., R. J. Homer, A. Wang, Q. Chen, G. P. Geba, J. Wang, Y. Zhang, and J. Immunol. 152:2546. J. A. Elias. 1999. Pulmonary expression of interleukin-13 causes inflammation, 30. Li, X.-M., R. K. Chopra, T.-Y. Chou, B. H. Schofield, M. Wills-Karp, and S.-K. mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and Huang. 1996. Mucosal IFN-␥ gene transfer inhibits pulmonary allergic responses eotaxin production. J. Clin. Invest. 103:779. in mice. J. Immunol. 157:3216. 47. Wills-Karp, M., J. Luyimbazi, X. Xu, B. Schofield, T. Y. Neben, C. L. Karp, and 31. Kung, T. T., D. M. Stelts, J. A. Zurcher, H. Jones, S. P. Umland, R. W. Egan, D. D. Donaldson. 1998. Interleukin-13: central mediator of allergic asthma. Sci- ␥ W. Kreutner, and R. W. Chapman. 1995. Interferon- and antibodies to inter- ence 282:2258. leukin-5 and interleukin-4 inhibit the pulmonary eosinophilia in allergic mice. 48. Grunig, G., M. Warnock, A. E. Wakil, R. Venkaya, F. Brombacher, Inflamm. Res. 44:S185. D. M. Rennick, D. Sheppard, M. Mohrs, D. D. Donaldson, R. M. Locksley, and 32. Yiamouyiannis, C. A., C. M. Schramm, L. Puddington, P. Stengel, D. B. Corry. 1998. Requirement for IL-13 independently of IL-4 in experimental H. E. Whiteley, and R. S. Thrall. 1999. Shifts in lung lymphocyte profiles cor- asthma. Science 282:2261. relate with the sequential development of acute allergic and chronic tolerant 49. Yang, X., S. Wang, Y. Fan, and X. Han. 2000. IL-10 deficiency prevents IL-5 stages in a murine asthma model. Am. J. Pathol. 154:1911. overproduction and eosinophilic inflammation in a murine model of asthma-like 33. Jacky, J. P. 1978. A plethysmograph for long-term measurements of ventilation reaction. Eur. J. Immunol. 30:382. Downloaded from in unrestrained animals. J. Appl. Physiol. 45:644. 50. Makela, M. J., A. Kanehiro, L. Borish, A. Dakhama, J. Loader, A. Joetham, 34. Renz, H., H. R. Smith, J. E. Henson, B. S. Ray, C. G. Irvin, and E. W. Gelfand. Z. Xing, M. Jordana, G. L. Larsen, and E. W. Gelfand. 2000. IL-10 is necessary 1992. Aerosolized antigen exposure without adjuvant causes increased IgE pro- for the expression of airway hyperresponsiveness but not pulmonary inflamma- duction and increased airway responsiveness in the mouse. J. Allergy Clin. Im- tion after allergic sensitization. Proc. Natl. Acad. Sci. USA 97:6007. munol. 89:1127. 35. Lack, G., A. Oshiba, K. L.Bradley, J. E. Loader, D. Amran, G. L. Larsen, and 51. Hamelmann, E., J. Schwarze, K. Takeda, A. Oshiba, G. L. Larsen, C. G. Irvin, E. W. Gelfand. 1995. Transfer of immediate hypersensitivity and airway hyper- and E. W. Gelfand. 1997. Non-invasive measurement of airway responsiveness in responsiveness by IgE-positive B cells. Am. J. Respir. Crit. Care Med. 152:1765. allergic mice using barometric plethysmography. Am. J. Respir. Crit. Care Med. 156:766. 36. Eum, S. Y., S. Haile, J. Lefort, M. Huerre, and B. B. Vargaftig. 1995. Eosinophil http://www.jimmunol.org/ recruitment into the respiratory epithelium following antigen challenge in hyper- 52. Cieslewica, G., A. Tomkinson, A. Adler, C. Duez, J. Schwarze, K. Takeda, IgE mice is accompanied by interleukin 5-dependent bronchial hyperresponsive- K. A. Larson, J. J. Lee, C. G. Irvin, and E. W. Gelfand. 1999. The late, but not ness. Proc. Natl. Acad. Sci. USA 92:12290. early, asthmatic response is dependent on IL-5 and correlates with eosinophil 37. Coyle, A. J., K. Wagner, C. Bertrand, S. Tsuyuki, J. Brews, and C. Heusser. 1996. infiltration. J. Clin. Invest. 140:301. Central role of immunoglobulin (Ig) E in the induction of lung eosinophil infil- 53. Bancroft, G. J., G. R. Shellam, and J. E. Chalmer. 1981. Genetic influences on the tration and T helper 2 cell cytokine production: inhibition by a non-anaphylac- augmentation of natural killer cells during murine cytomegalovirus infection: togenic anti-IgE antibody. J. Exp. Med. 183:1303. correlation with patterns of resistance. J. Immunol. 126:988. 38. Wang, J., K. Palmer, J. Lotvall, S. Milan X.-F. Lei, K. I. Matthaei, J. Gauldie, 54. Reddehase, M. J., W. Mutter, K. Munch, H. J. Buhring, and U. H. Koszinowski. ϩ M. D. Inman, M. Jordana, and Z. Xing. 1998. Circulating, but not local lung, IL-5 1987. CD8 positive T lymphocytes specific for murine cytomegalovirus imme- is required for the development of antigen-induced airways eosinophilia. J. Clin. diate-early antigens mediate protective immunity. J. Virol. 61:3102. Invest. 102:1132. 55. Orange, J. S., and C. A. Biron. 1996. An absolute and restricted requirement for

39. Chung, K. F., and P. J. Barnes. 1999. Cytokines in asthma. Thorax 54:825. IL-12 in natural killer cell IFN-␥ production and antiviral defence: studies of by guest on September 24, 2021 40. Wills-Karp, M. 1999. Immunologic basis of antigen-induced airway hyperre- natural killer and T cell responses in contrasting viral infections. J. Immunol. sponsiveness. Annu. Rev. Immunol. 17:255. 156:1138. 41. Mosmann, T. R., and S. Sad. 1996. The expanding universe of T-cell subsets: 56. Kuperman, D., B. Schofield, M. Wills-Karp, and M. J. Grusby. 1998. Signal Th1, Th2 and more. Immunol. Today 17:138. transducer and activator of transcription factor 6 (Stat6)-deficient mice are pro- 42. Umetsu, D. T., and R. H. DeKruyff. 1999. Interleukin-10: the missing link in tected from antigen-induced airway hyperresponsiveness and mucus production. asthma regulation? Am. J. Respir. Cell Mol. Biol. 21:562. J. Exp. Med. 187:939.