INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Keywords: IL-10; respiratory syncytial virus; allergen sensitization; lung function; eosinophilia

Although respiratory syncytial virus (RSV) bronchiolitis in early life is associated with later episodes of wheezing during childhood (1), the existing data are controversial concerning the role of RSV as a true risk factor in asthma development and allergic sensitization. The study by Sigurs and coworkers (2) found bronchiolitis during the first year of life to be a risk factor both for the development of asthma and sensitization to common allergens during the subsequent 2 yr. In the Tucson cohort study, however, the association of frequent wheezing and early RSV bronchiolitis was not associated with an increased risk of allergic sensitization (1). In another cohort study, positive serology for RSV infection correlated with aeroallergen sensitization during the first year of life but not at later times (3). It seems likely, therefore, that predisposing factors such as altered airway function and/or immunogenetic factors determine which children ultimately develop asthma.

RSV infection has been shown to induce expression of interleukin-10 (IL-10) in human macrophages (4) and in mouse pulmonary T cells (5). Interleukin-10 is an important regulatory cytokine that can mediate a number of biological activities (6). Data derived from both in vivo and in vitro studies suggest that the biological effects of IL-10 can vary depending on the surrounding cytokine and cellular milieu and timing of expression during the immune response. For example, in a murine allergic sensitization model, IL-10 suppressed or delayed development of pulmonary eosinophilia when administered at the time of antigen challenge: given 1 h after the challenge, the cytokine had no effect (7). In vitro, preincubation of resting T cell clones with IL-10 enhanced their capacity to produce cytokines after subsequent activation (8). However, when IL-10 was added during the activation step, inhibition of IL-2 synthesis was observed.

The role of IL-10 in asthma remains controversial. Some studies found IL-10 expression to be higher in subjects with asthma than in control subjects (9-11) whereas others found lower IL-10 levels (12-15). Diminished IL-10 production could result in Th2 cytokine skewing in allergic mice, as demonstrated in a model of bronchopulmonary aspergillosis (16). In a mouse model of allergic sensitization, we have recently shown that IL-10 may play an important role in the development of airway hyperresponsiveness (AHR). IL-10-deficient mice sensitized and challenged with ovalbumin (OVA) developed a robust pulmonary inflammatory response but not AHR (17). After reconstitution with IL-10 through adenovirus- mediated gene transfer, the mice developed AHR. van Scott and coworkers observed an increase in AHR despite a decrease in pulmonary inflammation when allergen-sensitized and challenged wild-type mice were administered recombinant IL-10 protein (18). In the present study, we further evaluated the role of IL-10 in allergic lung inflammation, analyzing the effects of RSV infection on airway function and inflammation in sensitized and challenged IL-10/ mice.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Animals

Homozygous IL-10-deficient mice (IL-10/) on a C57BL/6 background (C57BL/6-IL-10(tm1Cgn) (19) were originally obtained from Dr. Werner Müller, Cologne, Germany. These mice and littermate control mice were bred and housed in specific pathogen-free conditions and maintained on an OVA-free diet in the Biological Resources Center at National Jewish Medical and Research Center. Both female and male mice, 6-10 wk of age were used in the experiments. Control mice were matched with the deficient mice with regard to both age and sex in each experimental group.

Virus

Human respiratory syncytial virus (Long strain, type A) was obtained from American Type Culture Collection (ATCC; Manassas, VA) and was propagated in monolayers of HEp-2 cells (ATCC) grown in Eagle's minimum essential medium (Gibco, Grand Island, NY) supplemented with 2% fetal bovine serum. At maximum cytopathic effect, the cells were harvested and disrupted by sonication in the same culture medium. The suspension was clarified by centrifugation at 2,000 × g for 15 min at 18° C and the resulting supernatant was layered on top of a sucrose gradient (30% sucrose in 50 mM Tris buffered-normal saline solution containing 1 mM ethylenediaminetetraacetate [EDTA], pH 7.5) and further centrifuged at 100,000 × g for 2 h at 10° C. The pellet containing virus was resuspended in 10 mM phosphate-buffered saline (PBS) containing 15% sucrose and stored in aliquots at 70° C. Viral titer was determined by standard plaque assay combined with immunostaining for RSV. Uninfected HEp-2 cells were similarly processed to prepare a sham inoculum. Mice were inoculated under light anesthesia (Avertin 2.5%, 0.015 ml/g body weight) by intranasal administration of sham (or ultraviolet- irradiated RSV) or RSV inoculum (10⁶ plaque-forming units (pfu) in a total volume of 25 µl). Both the sham inoculum or UV-irradiated RSV were shown to be ineffective in inducing AHR or inflammatory changes.

For studying the effect of the RSV G protein on the eosinophilic response, two strains of RSV were used. One of these, the B1 strain expressed while the CP52 strain did not express the G and SH genes (20). These virus strains were propagated as described previously (21). Mice were infected with 10⁴ pfu of B1 or CP52 virus by intranasal inoculation as described above.

Sensitization, Airway Challenge, and Infection of Mice

Mice were sensitized by intraperitoneal injection of OVA (17) in alum or received PBS alone on Day 0 and Day 14. On Day 26 after initiation of the protocol, mice were infected under light anesthesia (Tribromo-ethanol 2.5%, 0.015 ml/g body weight) by intranasal inoculation of RSV (10⁶ PFU in 25 µl PBS). Controls were sham infected (UV-RSV or sham inoculum) in the same way. Mice were then challenged via the airways with OVA (1% in PBS) or PBS for 20 min on Days 28, 29, and 30. On Day 32, airway function was measured and specimens were collected for further analysis.

Determination of Airway Resistance and Dynamic Compliance

Airway resistance (RL) was determined before and after inhalation of aerosolized methacholine (MCh) in anesthetized, tracheostomized, and mechanically ventilated mice, as previously described (22). A four-way connector was attached to the tracheostomy tube with two ports connected to the inspiratory and expiratory sides of two ventilators. Aerosolized MCh was administered for 10 breaths at a rate of 60 breaths/min, tidal volume of 500 µl by the second ventilator. After each aerosol MCh challenge, the data were continuously collected for 1 to 5 min and maximum values of RL were taken to express changes in these functional parameters.

Monoclonal Antibody Treatments

Anti-mouse IL-5 monoclonal antibody (mAb), TRFK-5 (IgG_2b), was used in this study. One hundred micrograms of the stock mAb was diluted with PBS in a total volume of 100 µl, which was then given to sensitized mice as a single intravenous injection, 2 h before the first airway challenge. As a control, purified rat IgG_2b at the same dose and volume was administered.

Bronchoalveolar Lavage

After assessment of RL, lungs were lavaged (17). Cytospin slides were stained with Leukostat (Fisher Diagnostics, Pittsburgh, PA) and differentiated in a blinded fashion by counting at least 200 cells under light microscopy.

Measurement of Serum Immunoglobulins

Serum levels of total IgE, OVA-specific IgE, and IgG₁ were measured by ELISA as previously described (23).

Measurement of Cytokines in Bronchoalveolar Lavage Fluid

Interferon (IFN)-, IL-4, and IL-5 in the bronchoalveolar lavage fluid (BALF) supernatants were detected by enzyme immunoassay (EIA) as previously described (24). For IL-10, the OptEIA set was used according to the manufacturer's directions (PharMingen). For IL-13, a commercial kit was used (R&D Systems, Minneapolis, MN). Cytokine levels were determined by comparisons with the known standards. The limits of detection were 30 pg/ml for IL-10 and 10 pg/ml for the other cytokines.

Histologic and Immunohistochemistry Studies

After obtaining the BALF, lungs were inflated through the tracheal tube with 2 ml air and fixed in 10% formalin and blocks of lung tissue were prepared (17). Tissue sections, 5 µm thick, were affixed to microscope slides and deparaffinized. The slides were stained with hematoxylin and eosin (H&E), and periodic acid-Schiff (PAS) for identification of mucus-containing cells, and examined under light microscopy. For quantitating mucus staining, PAS-positive goblet cells in the airways were counted and the length of the basement membrane (BM) in each studied section was measured using NIH Image software (version 1.62). The results are given as mean number of PAS-positive goblet cells per millimeter of BM after evaluating several airways of three to five mice per group in a blinded fashion.

Cells containing major basic protein (MBP) in lung sections were identified by immunohistochemical staining as described using rabbit anti-mouse MBP (provided by Dr. J. Lee, Mayo Clinic Scottsdale, Scottsdale, AZ) (25). The slides were examined in a blinded fashion with a Nikon microscope equipped with a fluorescein filter system. Numbers of eosinophils in the perivascular, peribronchial, and peripheral tissues were evaluated using the IPLab2 software (Signal Analytics, Vienna, VA) for the Macintosh computer counting five sections per animal (three mice per group).

Statistical Analysis

The data were analyzed with the JMP statistical software package (SAS Institute Inc., Cary, NC). Analysis of variance was used to determine the levels of difference between all groups in measurements of RL. Comparisons for all pairs were performed by Tukey-Kramer honest significant difference (HSD) test. Significance levels were set at a p value of 0.05. Values for all measurements are expressed as mean ± SEM. Differences in cytokine levels between groups were analyzed by nonparametric ANOVA, the Kruskal-Wallis test. When significant differences between groups were observed, comparison for pairs was made by the Wilcoxon test with Bonferoni correction. Significance levels were set at a p value of 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Airway Hyperresponsiveness in Allergen-sensitized and Challenged Mice Infected with RSV

As shown in our previous study and confirmed here, IL-10- deficient mice that were sensitized and challenged with OVA (OS groups) (and in this case exposed to inactivated RSV, sham infection) did not develop significant changes in RL (Figure 1A) when compared with wild-type (WT) mice (Figure 1B). The airway response to inhaled MCh was also low in the IL-10/ mice infected with RSV (R) groups compared with the WT mice. Further, even following sensitization, RSV infection of sensitized (but not challenged) IL-10-deficient mice had only marginal effects (Figure 1A, ipR) without allergic-sensitization. However, when allergen-sensitized/challenged IL-10- deficient mice were infected with RSV, airway responsiveness was significantly enhanced (Figure 1A) (OR groups). An enhancement of AHR following RSV infection of sensitized and challenged mice was also seen (Figure 1B). Inactivated RSV induced only a marginal increase in AHR in the allergen sensitized and challenged mice, a response that was significantly lower than following live virus infection, indicating the importance for live virus infection in this response.

View larger version (0K): [in this window] [in a new window]   View larger version (0K): [in this window] [in a new window]   Figure 1.   Airway responsiveness to MCh in IL-10-deficient mice. Mice were sensitized and challenged with OVA and either infected with RSV (OR) or sham infected (inactivated RSV) (OS). Shown are IL-10/ mice after sham infection (sham) alone (S), RSV infection (R), ip sensitized but not challenged mice after RSV infection (ipR). Airway responsiveness was monitored by measuring lung resistance (RL) as described in METHODS. B shows lung resistance in WT mice; symbols as described for A. The results for each group are expressed as means ± SEM (n = 8-12 per group). *Significant differences (ANOVA and Tukey-Kramer, p < 0.05) are indicated (IL-10+/+/OR versus all other groups in A, IL-10+/+/OR versus IL-10+/+/OS in B). BL = baseline, SAL = saline. #Significant differences (p < 0.05) between WT/OS or WT/R and the control WT/S.

BAL Analysis of Cells

There were no significant differences in the cellular profile in BALF between naive IL-10/ and WT mice. After allergen sensitization and challenge, the percentage of eosinophils was significantly lower and neutrophil and macrophage percentages were higher in the IL-10/ mice than in the WT mice (Figure 2). RSV infection significantly increased the number of eosinophils in the IL-10/ mice, to the levels in WT mice. There were no detectable eosinophils in the BALF of nonsensitized and challenged mice.

View larger version (0K): [in this window] [in a new window]   Figure 2.   Cellular composition of BAL fluid. IL-10-deficient (IL-10/) and WT mice were sensitized/challenged and infected as described in METHODS. BAL fluid was obtained from the same groups described in the legend to Figure 1. The results for each group are expressed as means ± SEM. *Significant differences between the groups (for example, eosinophils between IL-10//OS and each of the three other groups) (ANOVA and Tukey-Kramer, p < 0.05).

Histopathology

The allergen sensitization and challenge protocol induced significant mononuclear and eosinophilic cell infiltration perivascularly and peribronchially in both the IL-10/ and WT mice, as we have shown earlier (17). In the H&E-stained sections, no obvious differences could be detected between sham- and RSV-infected animals after allergen challenge of sensitized mice (not shown). However, PAS staining of the sections clearly showed an increase in mucus production, that is, airway goblet cell hyperplasia, both in the IL-10/ and the WT mice after RSV infection (Figures 3 and 4).

View larger version (0K): [in this window] [in a new window]   Figure 3.   PAS-stained histological sections of murine lungs. Normal airways and vessels after sensitization with OVA and exposure to nebulized PBS in IL-10/ mice (A). Representative sections from an IL-10/ mouse after RSV infection (B), after OVA sensitization/challenge and sham-infection (C ), and OVA sensitization/challenge and RSV infection (D). Note the staining of single goblet cells within the respiratory epithelium in the RSV-infected OVA-sensitized/challenged mouse.

View larger version (0K): [in this window] [in a new window]   Figure 4.   Number of mucin-producing goblet cells is increased in allergen-sensitized/challenged mice infected with RSV. PAS-stained cells were counted in five sites on the airway epithelium (at least two separate airways) including three mice/group. In each studied site, length of the basement membrane (BM) was measured by image analyzing software and the results are given as mean number of goblet cells per millimeter of BM. *Significant difference between groups (p < 0.05).

Eosinophils in the pulmonary tissue were identified by immunofluorescence using an MBP-specific antibody (Figure 5). Eosinophil counts were significantly lower in the IL-10/ (Figure 6A) compared with the WT mice (Figure 6B). These decreases were seen when eosinophil numbers were quantitated in the perivascular, peribronchial, and peripheral airways. However, after RSV infection the numbers increased to levels observed in WT mice. These results paralleled those in the BALF (Figure 2).

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