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Viral respiratory infections commonly precipitate asthma exacerbation in humans, especially in children younger than 6 y of age(1). Furthermore, even in individuals without asthma, viral respiratory infections have been associated with abnormal physiology and airway hyperresponsiveness(2–6). Glucocorticoid therapy is commonly used for prevention and treatment of virus-induced exacerbations of asthma, but controversy persists regarding its efficacy(7, 8), the uncertainty having been renewed by negative findings in a recent controlled trial in children(9).

Airway inflammation is a prominent feature of asthma(10, 11), and of respiratory viral infections(12–14). Although it is likely that virus-induced airway inflammation contributes to airway dysfunction during acute viral illness, it is not known to what extent the airway dysfunction may be attenuated by suppressing the inflammatory response. We hypothesized that airway dysfunction during acute viral illness is caused by the inflammatory response, rather than direct viral effects on airway epithelium. Previously we reported that adult rats infected with Sendai virus exhibit airway obstruction and hyperresponsiveness analogous to virus-induced airway dysfunction in humans(15). This animal model allows sufficient control of variables to address the basic mechanisms underlying virus-induced airway dysfunction. The purpose of this study was to test whether suppression of inflammation, but not viral replication, with dexamethasone would attenuate virus-induced airway obstruction and hyperresponsiveness in rats.

METHODS

Preparation of animals. All methods and procedures were approved by the Animal Care and Use Committee of the University of Wisconsin. Forty-three adult male-specific pathogen-free Sprague-Dawley derived rats (CD strain, Charles River Breeding Laboratories, Raleigh, NC), weighing an average of 0.295 kg, were used for these experiments. All animals were allowed food and water ad libitum.

General experimental design. Rats were inoculated with parainfluenza type 1 (Sendai) virus or sham inoculated with sterile vehicle. Dexamethasone (0.5 mg/kg) or saline was injected s.c. daily, with the initial dose given 12 h after inoculation to avoid the potential interference with viral infection noted when steroid treatment was started before inoculation(16). Physiologic evaluations and BAL were performed on postinoculation d 4, 5, or 6, 24 h after the last dexamethasone or saline injection. Measurements included daily weights, lung mechanics (respiratory system resistance and dynamic compliance), responsiveness to i.v. methacholine, and BAL total and differential inflammatory cell counts. An additional group of six infected rats of comparable age was evaluated for viral titers and lung histology at 5 d postinoculation.

Viral inoculation. Rats were inoculated with parainfluenza type 1 (Sendai) virus strain P3193 by use of an aerosol exposure apparatus(Glas-Col, Terre Haute, IN) as described previously(15, 17–19). The apparatus introduced an aerosol of approximately 108 plaque-forming units of virus in 2 mL of chorioallantoic fluid into the animal chamber, and the rats breathed the virus-containing aerosol for 15 min. Control rats were inoculated with sterile chorioallantoic fluid in a similar manner. Animals were housed in microisolater cages after inoculation, and virus and control groups were studied at different times to ensure that control rats would have no viral exposure.

Instrumentation and measurement of pulmonary mechanics. Cannulae were placed in the trachea, femoral vein, and femoral artery of each rat after anesthesia was induced with urethane (1.2 g/kg intraperitoneally; Sigma Chemical Co., St. Louis, MO). Rats were placed in a constant-pressure rodent plethysmograph, pretreated with propranolol (2 mg/kg i.v.; Sigma Chemical Co.) to suppress adrenergic modulation of methacholine challenges, and then paralyzed with succinylcholine chloride (4 mg/kg i.v. initially and 2 mg/kg thereafter as needed; Sigma Chemical Co.). Rats were ventilated mechanically at 80 breaths/min with the tidal volume based on body weight and adjusted to maintain a normal arterial Pco2 of 4.5-6.0 kPa. Lungs were inflated to 2.9-kPa pressure each minute to prevent atelectasis, and accumulated secretions were removed from the airways by suctioning as necessary.

Resistance was calculated by the isovolume method at 50% tidal volume(model 6 Pulmonary Mechanics Analyzer, Buxco Electronics, Sharon, CT), using inflation pressure measured at the tracheal cannula, and flow and volume obtained from the plethysmograph transducer signal(15). Cdyn was determined from inflation pressure and tidal volume at points of zero flow. Pressure and flow signals were in phase to at least 7 Hz under test conditions. Adiabatic/isothermal transients were negligible in the range of flow rates encountered in these studies. Rrs was obtained from the computed resistance by subtracting the resistance of the tracheal cannula and its connector.

Methacholine challenge. Methacholine chloride (Sigma Chemical Co.) was injected as 1 mL/kg of increasing concentrations of the agent, followed by a flush of 0.3 mL of saline, both completed in 5 s. Responses to i.v. methacholine were recorded as the change in resistance after administration of the agent. Sensitivity to methacholine was determined as the log dose (nmol/kg) required to increase resistance by 20 Pa mL-1 s(PD20) by interpolation of the dose-response curve(15, 17).

Bronchoalveolar lavage and cell count. After physiologic measurements, rats were killed by exsanguination and air embolus. The thoracic cavity was then exposed with a midline incision along the sternum and the lungs were inflated to total lung capacity with Hanks' balanced salt solution without calcium and magnesium; the lavage was repeated for a total of five exchanges. Lavage fluid was centrifuged, and the cell pellet was resuspended in 1 mL of buffer. Total BAL cell counts were measured with a Hemo-W cell counter (Coulter Electronics, Hialeah, FL). Cytocentrifuge cell samples of lavage fluid were made with Cytospin (Shandon Lipshaw Inc., Pittsburgh, PA) and stained with Diff-Quik (Baxter Healthcare, Miami, FL). The differential cell count was determined from 200 stained cells.

Lung viral titers. Viral titers were measured in a separate group of six infected rats 5 d postinoculation. After exsanguination, the left mainstem bronchus was tied, and the left lung was removed, frozen in liquid N2, and stored at -80 °C. Plaque assays for infectious virus were performed on lung homogenates, using Madin-Darby bovine kidney cells(20). Right lungs were fixed by tracheal perfusion with 10% buffered formalin and were then evaluated for inflammatory changes. A semiquantitative inflammation score was assigned for each lung section in a blinded manner by a pathologist (W.L.C.), with scores ranging from 1 (mild bronchitis) to 3 (moderately severe, erosive, and suppurative bronchiolitis and interstitial pneumonia).

Data analysis. All statistical analyses were done using Systat version 5.03 software (Systat, Inc., Evanston, IL). Kruskal-Wallis one-way analysis of variance was used to detect differences among the four study groups; when significant, planned pairwise comparisons were conducted with the Mann-Whitney test. Data from postinoculation d 4, 5, and 6 were pooled for all variables except BAL lymphocytes, for which d 4 was excluded due to prior observations that virus-induced increases in lymphocytes do not occur before d 5. Mann-Whitney was also used to test for differences in viral titers. A p value ≤ 0.05 was considered to be significant.

RESULTS

Inflammatory response and viral titers. There was an increase in total BAL inflammatory cells recovered from the virus-inoculated saline-treated group compared with sham-inoculated controls, with a significant increase in the number of neutrophils and macrophages (Fig. 1). Total BAL lymphocyte counts were significantly elevated in the untreated, virus-inoculated rats on d 5 and 6 compared with untreated controls (Fig. 1). In contrast, rats treated with dexamethasone during viral infection had no significant influx of inflammatory cells into the airways, having BAL cell counts similar to those of the noninfected rats (Fig. 1).

Figure 1
figure 1

Leukocytes in BAL from Sendai virus infected and noninfected control rats treated with daily injections of dexamethasone or saline. Group medians are indicated by bars.

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Viral titers measured at postinoculation d 5 were 1-2 logs higher in the lungs of dexamethasone-treated rats than in lungs of untreated rats of the same inoculation batch (Fig. 2). Semiquantitative inflammation scores were lower in the dexamethasone-treated animals (Fig. 2), consistent with the BAL findings.

Figure 2
figure 2

Lung viral titers and semiquantitative inflammation scores of individual rats 5 d after inoculation with Sendai virus, and treated with daily injections of dexamethasone or saline.

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Pulmonary physiology. The virus-infected group had significant airway obstruction, as measured by differences in lung mechanics compared with sham-inoculated controls. Rats in the infected group had a reduction in Cdyn and an increase in Rrs (Fig. 3) compared with those of the noninfected control group. Dexamethasone treatment significantly attenuated virus associated deviations in Rrs, but it was less effective in preventing virus-induced changes in Cdyn (Fig. 3). Rrs and Cdyn were not significantly changed by dexamethasone treatment in noninfected rats (Fig. 3).

Figure 3
figure 3

Lung mechanics in rats with acute Sendai virus infection and in noninfected controls, treated with daily injections of dexamethasone or saline. Group medians are indicated by bars.

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Methacholine responsiveness. Significant airway hyperresponsiveness to i.v. methacholine was present in saline-treated rats of the virus-inoculated group compared with rats of the saline-treated sham-inoculated control group (Fig. 4). Dexamethasone treatment significantly attenuated, but did not abolish, development of virus-associated airway hyperresponsiveness to methacholine (Fig. 4). Methacholine responsiveness was not significantly altered by dexamethasone treatment in noninfected rats (Fig. 4).

Figure 4
figure 4

Responsiveness to i.v. methacholine in rats infected with Sendai virus and in noninfected controls, treated with daily injections of dexamethasone or saline. Group medians are indicated by bars.

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DISCUSSION

Respiratory viral illnesses in rats produce acute airway pathology and under some conditions also produce chronic airway abnormalities. In the 1st wk after inoculation with Sendai virus, previously healthy rats develop an acute bronchiolitis and pneumonitis characterized by neutrophilic inflammation, airway obstruction, airway hyperresponsiveness, and altered gas exchange; toward the end of the 1st wk the viral titers drop precipitously, coincident with lymphocytic infiltration and the appearance of neutralizing antibodies(15, 20–22). These manifestations of acute viral illness in the airways of previously healthy rats are consistent with those observed during acute respiratory viral illness in nonasthmatic humans(2–6) and are qualitatively similar for rats ranging in age from weanlings to adults, and among different strains of rats. Adult rats typically resolve all their virus-associated changes in airway morphology, histology, and physiology within 4-8 wk(15). However, after a single respiratory viral illness in the 1st mo of life, rats may develop chronic airway abnormalities that have similarities to human asthma, including chronic episodic airway obstruction with a reversible component(23), airway hyperresponsiveness(17, 24), chronic airway inflammation(21, 22, 24), and bronchiolar wall thickening with subepithelial fibrosis(19, 24). There appears to be a genetic predisposition to the development of these postviral asthma-like features, in that certain inbred strains, such as the Brown Norway, develop the postviral airway pathology readily, whereas another inbred strain, the Fisher 344, recovers from the infection without significant airway sequelae(22, 24). Treatment of rats with 3 d of high dose dexamethasone at a point several weeks after viral illness effectively suppresses the airway obstruction and inflammation, but the asthma-like syndrome returns within 3 wk of the last steroid dose(23, 25). The current study differs from these previous studies in that it addresses the effect of glucocorticoid treatment on the acute, rather than the chronic, airway manifestations of respiratory viral illness in rats.

The results of the current study support the hypothesis that airway dysfunction during acute viral respiratory infection is caused predominantly by corticosteroid-sensitive mechanisms, such as the inflammatory response. In the present study, dexamethasone treatment completely prevented the increased influx of inflammatory cells into the airways during acute viral illness, and markedly attenuated the virus-associated airway obstruction and hyperresponsiveness. In contrast, lung viral titers were increased in the dexamethasone-treated rats, suggesting that suppression of the inflammatory response, although effective for prevention of physiologic dysfunction of the airways, might also compromise viral clearance. The glucocorticoid dose used in this study was large, but analogous to doses commonly used to treat low birth weight human infants(26) and adults with severe acute exacerbations of asthma(27).

Acute respiratory viral infections have been associated with exacerbations of asthma, particularly in children, but also in adults(1, 28). Although the mechanisms of virus-induced airway dysfunction have not been defined precisely, viral infections cause an acute inflammatory response in the airways that could alter airway structure and function by a multitude of pathways(3, 29–31). Although glucocorticoids might be expected to inhibit the development of virus-induced airway dysfunction based on their antiinflammatory effects(32), the efficacy of steroids in virus-induced exacerbations of asthma remains controversial(7, 8). Contributing to the controversy are the variables of dose, duration, and timing of steroid therapy(7, 8), as well as the uncertainties of which exacerbations are caused by viral illness, even in the presence of viral-like symptoms(33). Our current study in rats allowed precise control of these variables and demonstrated unequivocal efficacy of glucocorticoid therapy in the prevention of airway dysfunction during acute respiratory viral infection. Although airway dysfunction due to acute viral illness in previously healthy airways cannot be equated with exacerbations of preexisting airway disease, it is likely that some of the pathophysiologic mechanisms responsible for the acute manifestations also contribute to virus-associated exacerbations of asthma.

In these experiments there was a potent uncoupling effect of dexamethasone on the transduction of the viral infection/replication signal to the inflammatory cell infiltration response. Although this study did not address the mechanism responsible for the uncoupling of virus-induced inflammation, the latter is likely due to glucocorticoid-mediated inhibition of the expression of key cytokines and adhesion molecules in the affected airway epithelium. In this regard, respiratory viral infection in mice has been reported to increase pulmonary expression of NF-κB a transcriptional factor that is involved in the activation of immunoregulatory genes in response to immune stimulators(34). Recent work has identified NF-κB as a primary target for the inhibitory effects of glucocorticoids(35, 36), which might explain not only why glucocorticoid therapy may be a potent inhibitor of virus-induced inflammation when administered during the early stages of viral infection, but also why glucocorticoid treatment may be much less effective when begun after the expression of proinflammatory cytokines and adhesion molecules has already occurred.

Although glucocorticoid treatment was efficacious in altering virus-associated airway dysfunction, a potential concern in the use of glucocorticoids during viral illness was the increase of pulmonary viral titers associated with steroid treatment; a number of previous observations are noteworthy in this regard. Children who developed respiratory syncytial virus-confirmed respiratory illness while on chronic systemic glucocorticoid therapy had larger peak viral titers and more prolonged viral shedding in nasal washes compared with those who had no steroid treatment and normal immune systems at the time of infection(37). However, there were no apparent differences in the clinical course of the viral illness, whether or not the children were on glucocorticoid therapy(37–39). In experimental rhinovirus infection, glucocorticoid treatment increased nasal viral titers significantly, but it had no significant effect on infection rate or duration of nasal viral shedding(40, 41). Other studies reporting effective glucocorticoid treatments for prevention of virus-induced asthma in children similarly had no evidence for an alteration of the length or severity of viral illness due to treatment, although no viral studies were done(42, 43). Local glucocorticoid treatment via nasal insufflation in Cotton rats infected with parainfluenza-3 caused a marked decrease in lung pathology, but also a log increase in viral titers(44), consistent with the findings in our current study. In contrast, experimental respiratory syncytial virus infection of gnotobiotic calves treated with dexamethasone resulted in not only increased peak viral titers and duration of shedding, but enhanced lung lesions in the treated animals(45). Whether steroid treatment during acute viral illness in rats affects the ultimate recovery from the infection remains a question to be addressed in future studies.

Finally, the ability to demonstrate such a marked attenuation of virus-induced physiologic and inflammatory airway changes in this animal model underscores the potential of the model for defining relevant pathways that could be altered similarly with agents having fewer side effects compared with glucocorticoids.