© 2002 American Thoracic Society
Cigarette Smoke-induced Airway Hyperresponsiveness Is Not Dependent on Elevated Immunoglobulin and Eosinophilic Inflammation in a Mouse Model of Allergic Airway DiseaseRespiratory Immunology Program, Lovelace Respiratory Research Institute; and Department of Pathology, University of New Mexico, Albuquerque, New Mexico Correspondence and requests for reprints should be addressed to Edward G. Barrett, Ph.D, Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive SE, Albuquerque, NM 87108. E-mail: tbarrett{at}lrri.org
Epidemiologic studies suggest that children raised in homes of cigarette smokers have a higher incidence of asthma than children who are raised in homes of nonsmokers. We sought to develop an experimental model to understand the mechanisms involved. Female BALB/c mice were paired with male DO11.10 ovalbumin (OVA)-T cell receptor hemizygous (+/-) mice such that the offspring were either transgene positive (+/-) or negative (-/-). Mice were exposed to either air or mainstream cigarette smoke (100 mg/m3 total particulate matter, 6 hours/day, 7 days/week) during pregnancy. Immediately after birth, newborn mice were exposed for 4 weeks to either air or sidestream cigarette smoke (SS; 5 mg/m3 total particulate matter, 6 hours/day, 5 days/week) and then exposed for the following 6 weeks to either air, SS, OVA (5 mg/m3, 6 hours/day, 5 days/week) or a combination of OVA-SS. DO11.10 +/- offspring exposed to OVA had increased airway hyperresponsiveness (AHR) to methacholine challenge, total IgE, OVA-specific IgE and IgG1, lymphocytes, and neutrophils in bronchoalveolar lavage and perivascular and peribronchiolar inflammation. Exposure to SS alone caused a significant increase in AHR in both +/- and -/- mice. Transgene -/- mice did not exhibit AHR after OVA exposure unless it was delivered in combination with SS. When compared with OVA-only exposure, OVA-SS exposure decreased total IgE, OVA-specific IgE, and IgG1 amounts in +/- mice. These results indicate that exposure to SS after birth enhanced AHR in offspring that are both predisposed (+/-) and nonpredisposed (-/-) to develop an allergic response to OVA, but this AHR was not associated with elevated lung eosinophilia or OVA-specific Ig amounts.
Key Words: asthma • sensitization • environmental • pollution • tobacco
Exposure to tobacco smoke through either in utero or postnatal exposure profoundly affects the development of lung function and respiratory disease in children (1). Children born to smoking mothers are more likely to suffer from chronic bronchitis, wheezing, respiratory infections, and increased medical visits or hospital admissions for respiratory problems (2–7). In addition, in utero and postnatal tobacco smoke exposures may be risk factors for the development and exacerbation of childhood asthma (8, 9). Tobacco smoke exposure is associated with an increased prevalence (8) and severity of asthma (9, 10). Children of mothers smoking four cigarettes daily have a 14% increase in the prevalence of asthma, but children of mothers smoking 15 or more cigarettes daily have a 49% increased prevalence (11). The odds ratio on smoke exposure for the development of asthma increases further when associated with home dampness (odds ratio 1.3) or cat or dog exposure (odds ratio 8.0) (12). Controversy remains, however, about whether cigarette smoke exposure and the development of asthma in children are directly related. Several longitudinal studies examining infants up to 6 to 10 years of age found that exposure to cigarette smoke is not associated with the development of asthma (13, 14). In addition, atopic status to inhaled allergens, which is closely related to the development of childhood asthma, is not clearly associated with cigarette smoke exposure. When children are divided based on their atopic status, cigarette smoke exposure is only associated with wheeze and asthma in the nonatopic group (15, 16). Exposure to cigarette smoke increases sensitization to food allergens in the first few years of life (17) but is not associated with sensitization to inhaled allergens (17, 18). Evidence is also mixed as to whether exposure to cigarette smoke is associated with elevated IgE amounts in children (19, 20). If cigarette smoke exposure in utero or postnatally causes the development of allergic asthma in an individual who would otherwise not become asthmatic, then the same exposure should be associated with a change in the person's atopic status. Although some epidemiologic studies indicate that inhaled cigarette smoke is associated with the development of asthma in children, experimental data confirming or disputing these studies are lacking. Exposure of rats in utero to aged and diluted sidestream cigarette smoke (SS) induces airway hyperresponsiveness (AHR) in the offspring (21). SS has an adjuvant effect on eosinophils, allergen-specific antibodies, and Th2 cytokines (interleukin-4 and interleukin-10) in adult mice previously sensitized by injection with ovalbumin (OVA) and aluminum hydroxide (22). In this study, we sought to determine whether exposure to cigarette smoke in utero and/or postnatally leads to the development and/or exacerbation of allergic sensitization and/or allergic airway responses using a murine model of allergic airways disease.
Animals Female BALB/c mice (Frederick Cancer Research and Development Center, Frederick, MD) and male DO11.10 OVA-T cell receptor hemizygous (+/-) mice on a BALB/c background from a colony housed at the Lovelace Respiratory Research Institute were used in this study. DO11.10 mice bear the T-cell receptor for the OVA peptide 323–339 on an average of 40% of their T cells and are thus "genetically" susceptible to respond to OVA exposure without prior sensitization. Before exposure, animals were conditioned to the whole-body exposure chambers (H1000; Hazelton Systems, Inc., Aberdeen, MD) for 2 weeks. Mice were housed in shoebox-type plastic cages with hardwood chip bedding and were given food and water ad libitum inside the chambers. Chamber temperatures were maintained at 26 ± 2° C, and lights were on a 12-hour on/off cycle. After the conditioning period, female BALB/c mice (10–12 weeks old) were paired with male DO11.10 mice (10–14 weeks old) such that their offspring were either hemizygous (+/-) for the transgene or did not bear the transgene (-/-). Adult mice and offspring produced were exposed to cigarette smoke and/or OVA according to the schedule depicted in Figure 1 . Adult pregnant mice were exposed to mainstream cigarette smoke (MS) to simulate maternal exposure, whereas offspring were exposed to SS to simulate a child's exposure.
All animal procedures were performed in accordance with protocols approved by the Lovelace Respiratory Research Institute's Institutional Animal Care and Use Committee. The Lovelace Respiratory Research Institute is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care.
Exposures After exposure on gestational Day 19, pregnant mice from both MS- or Air-exposed groups were moved to exposure chambers for either Air or SS (6 hours/day, 5 days/week [Monday through Friday]) exposure. SS exposure conditions were generated by capturing the smoke from the lit end of the cigarette with a plastic manifold placed above the cigarettes, diluting with filtered air, and delivering to the exposure chambers. A target concentration of 5 mg TPM/m3 for SS was chosen to simulate what a child might be exposed to under the most extreme conditions (25, 26). The mass concentration of SS in the chambers was 5.24 ± 0.14 TPM/m3 (average ± SEM of 67 exposure days). Thus, offspring were born in either Air or SS. After exposure to Air or SS for 4 weeks, mice were exposed to Air, SS, OVA, or a combination of OVA and SS (OVA-SS) for an additional 6 weeks. Mothers remained with their offspring until they were 3 weeks old. OVA exposures were generated by aerosolizing (6 hours/day, 5 days/week) 1% heat-aggregated OVA (chicken egg, grade V; Sigma, St. Louis, MO), diluted with filtered air, and then delivered to the exposure chambers. The total mass concentration of OVA was determined by gravimetric analysis of filter samples taken every 2 hours during exposure. The mass concentration of OVA was 4.63 ± 0.14 mg/m3 (average ± SEM of 47 exposure days). Concentrations of OVA and SS for the combined OVA-SS exposure chamber were set by mimicking exposure settings (e.g., flow rates and diluting air) from OVA and SS-only chambers and measuring the total mass concentration. Target exposure concentration was 10 mg TPM/m3 (e.g., 5 mg/m3 OVA and 5 mg/m3 SS), with the actual being 9.24 ± 0.27 mg TPM/m3 (average ± SEM of 47 exposure days).
Airway Responsiveness
Pathologic Analysis After blood collection, some animals had their lungs instilled via the trachea with 10% buffered formalin and removed and fixed in the same solution. Animals used for histopathologic analysis were not subjected to airway response measurements or BAL. After paraffin embedding, sections for microscopic analysis were stained with hematoxylin, eosin, and alcian blue. Lung lesions for each animal, including alveolar septal infiltrates, perivascular infiltrates, and combined bronchus-associated lymphoid tissue hyperplasia, and peribronchiolar infiltrates were subjectively graded on a severity scale of minimal, mild, moderate, and marked (corresponding to numbers 1 [minimal] to 4 [marked]). Individual lesion scores were summed from each animal to create an overall histopathology score for each animal. A pathologist who was blinded to the exposure conditions evaluated all slides.
Analysis of OVA-Specific IgG1
Analysis of OVA-Specific IgE
Statistical Analysis
Airway Responsiveness The day after final exposure, mice were assessed for AHR by Mch challenge. Transgenic (Tg) +/- offspring exposed to inhaled OVA or SS after birth had a significant increase in AHR to Mch (Figure 2A) . In addition, the combination of OVA-SS exposure led to a significant increase in AHR above the Air-exposed offspring and offspring exposed to SS. In contrast, Tg -/- offspring exposed to OVA did not have an increase in AHR (Figure 2B). Differences in AHR between Tg -/- and Tg +/- offspring after OVA exposure were statistically significant (p < 0.05). As with the Tg +/- offspring, the combination of OVA-SS exposure in Tg -/- offspring led to increased AHR in comparison to Air-, OVA- and SS-exposed groups.
Exposing mice to MS in utero did not statistically alter AHR in comparison to mice exposed to Air in utero (Figures 2C and 2D). In contrast to the observed differences between mice exposed to Air in utero (e.g., Air:Air versus Air:OVA or Air:SS), the differences between MS groups (e.g., MS:Air versus MS:OVA or MS:SS) failed to achieve statistical significance. Only MS offspring exposed to OVA-SS after birth had a statistically significant increase in AHR when compared with the other MS groups. There was no statistical difference in AHR between Tg -/- and Tg +/- MS-exposed animals.
Pathologic Analyses
Tg +/- mice exposed to OVA or OVA-SS had higher inflammatory lesion scores than those of other mice (Table 2). Inflammation in these mice consisted of minimum-to-mild peribronchiolar and perivascular interstitial infiltrates of lymphocytes and macrophages mixed with occasional plasma cells and rare neutrophils or eosinophils (Figures 3 and 4) . Occasionally, mononuclear leukocytes around larger airways were organized into discrete nodules of bronchus associated lymphoid tissue (Figures 3C and 3E). In the parenchyma, macrophages and lymphocytes occasionally widened alveolar septa slightly (Figures 4C and 4D). A moderate number of neutrophils were present in alveolar septa (either interstitial or within the capillary bed), and a few neutrophils along with a few macrophages were present in scattered alveolar lumina. Mucous cell hyperplasia and hypertrophy were occasionally present in the larger airways of Tg +/- offspring exposed to OVA or OVA-SS (Figures 4A and 4B).
Analyses of Immunoglobulin Concentrations Amounts of total serum IgE were detected in all animals (Table 3). Total IgE amounts in Tg +/- OVA-exposed offspring were significantly higher than Air-exposed animals. Adding SS in combination with OVA (OVA-SS) in Tg +/- offspring significantly reduced total IgE amounts back to Air control amounts. Amounts of serum OVA-specific IgE and IgG1 were increased significantly in Tg +/- and -/- offspring exposed to OVA (Table 3). Although not statistically significant, there was a trend toward reduced OVA-specific IgE and IgG1 amounts when OVA was combined with SS (OVA-SS), especially in Tg +/- mice.
Epidemiologic and experimental evidence shows a strong association between exposure to cigarette smoke in utero and/or postnatally and decreased lung function (1). Exposure to cigarette smoke causes increased coughing (25, 26, 28), wheezing (2, 26), sputum production (26), AHR (29–31), and respiratory illnesses (4, 28, 32), as well as decreased forced expiratory volume (33). The evidence is particularly strong in newborns, infants, and children. In addition, SS can exacerbate existing asthmatic symptoms in humans and animals (22, 34). However, a direct association between cigarette smoke exposure and the induction of allergic asthma is absent. In this study, we developed a murine model in which genetically susceptible and nonsusceptible offspring were exposed to cigarette smoke during and/or after pregnancy in combination with an allergen. Our objective was to assess the effects of cigarette smoke exposure on the development and/or exacerbation of allergic sensitization and/or allergic airway disease. Genetically susceptible animals (Tg +/-) responded to chronic allergen (OVA) challenge with increased amounts of serum total IgE, serum OVA-specific IgE and IgG1, severity of histopathology lesions, numbers of lymphocytes and neutrophils in BAL fluid, and increased AHR. In contrast, nonsusceptible (Tg -/-) animals developed serum OVA-specific IgE and IgG1 antibodies and slightly increased BAL neutrophils (not statistically significant), but no other changes were observed in responses to chronic OVA challenge. Exposure to SS in both Tg +/- and Tg -/- animals resulted in an increase in AHR independent of any other major changes. Subsequently, when animals were exposed to OVA in combination with SS, there was an additional increase in AHR relative to SS or OVA exposure. This increase in AHR was not associated with increased antibody concentrations; in fact, the number of antibodies tended to be lower, especially in Tg +/- animals (statistically significant). In addition, there was no increase in lung inflammation (histopathology and BAL cell differentials) associated with the increased AHR. Exposing animals in utero to MS did not have a statistically significant effect on any endpoint measured. However, significant differences in AHR observed between Air:Air versus Air:SS or Air:OVA animals were absent between MS:Air versus MS:SS or MS:OVA-exposed animals. The reasons for this apparent loss of enhanced AHR with postnatal exposure to SS or OVA in MS-exposed animals are unknown. However, it appears that MS:Air-induced AHR may be higher than the Air:Air group, and thus, this slight change may have reduced the absolute and statistical difference between MS:Air and MS:SS and MS:OVA groups. Exposure of mice to cigarette smoke in utero (MS) and/or postnatally (SS) has not previously been reported. In utero exposure of rats to SS (1 mg/m3 TPM) with subsequent postnatal SS exposure increases Mch-induced AHR (21, 35). In contrast to our results, these studies did not show an increase in AHR following postnatal SS exposure alone. Differences in SS exposure concentration (1 versus 5 mg/m3 TPM) or species differences between the studies may account for the different results. However, there was agreement between studies showing that in utero smoke exposure alone did not increase AHR. Separating the independent effects of in utero and postnatal cigarette smoke in human studies is difficult because persons who smoke tend to do it both during and after pregnancy. There is clear evidence that infants from mothers who smoked during pregnancy have reduced lung function within 3 days of birth (36) and within the first 6 months of life (37). Guinea pigs exposed to MS in utero exhibit increased AHR up to 21 days after birth (38). The long period (10 weeks) between the end of in utero exposure and measurement of AHR may explain why we were unable to detect significant changes in our MS:Air-exposed mice. Although infants with reduced pulmonary function at birth still maintain reduced pulmonary function at 6 years, they no longer experience wheezing (39). The most direct evidence showing an independent effect of postnatal SS exposure comes from studies in China where few women smoke. Paternal smoking increased hospital admissions for respiratory illness in the first 18 months of life (40) and decreased pulmonary function in older children 8 to 16 years of age (41). In addition, 3 year olds exposed to SS by caregivers other than the parents had a threefold increase in wheezing and lower respiratory tract illness (42). Our results support the concept that postnatal SS exposure alone can lead to altered lung function. Other experimental studies have found that air pollution-related exposures can alter immune and lung function. Takano and colleagues found that diesel exhaust enhances lung inflammation and AHR but does not enhance antigen specific IgG or IgE (43). Several studies using different mouse models and exposure protocols have shown that exposure to diesel exhaust in combination with an allergen can induce allergen specific IgE production, lung inflammation, and enhanced AHR (44–46), thus suggesting that diesel exhaust may act as an immune adjuvant, in contrast to these findings. However, an important limitation of these diesel exposure studies is that significant results were only observed at diesel exposure amounts that would not be present in the ambient environment. The concentration of SS used in this study (5 mg/m3 TPM) is just outside the high end of the range (2 mg/m3 TPM) to which humans are exposed (47). However, infants and young children with smoking mothers may actually be exposed to much higher concentrations than those normally reported in homes, as they are often affected directly by the source. For example, higher urinary cotinine concentrations are found in children from smoking mothers than from smoking fathers (48). Urinary cotinine amounts are also higher in bottle-fed infants from smoking mothers than in adults exposed to SS (49). Based on the exposure protocol of 6 hours/day, 7 days/week, exposure of mice in this study to MS (100 mg/m3 TPM) is calculated to be approximately equivalent to a human smoking one to two packs of cigarettes per day, on the basis of TPM deposited in the lung per unit time per gram of lung tissue (24, 50). The mechanisms of cigarette smoke-induced AHR are not fully understood. In our studies, SS-induced AHR was not associated with an increase in lung inflammation. To our knowledge, this is the first study to examine chronic exposure to SS in newborn animals and to report AHR in conjunction with data on lung inflammation. However, we did not examine the proximal airways (e.g., trachea, main bronchi) and thus have no data on the relative extent that large airway inflammation may have contributed toward the development of AHR. Joad and colleagues (21, 35) have suggested that an increase in pulmonary neuroendocrine cells and neuroepithelial bodies may be associated with increased AHR after in utero-postnatal SS exposure in rats. However, the presence or absence of lung inflammation was not reported. Studies in adult guinea pigs show an association between MS-induced AHR and lung inflammation that can be abolished with specific neurokinin receptor antagonists, suggesting a role for tachykinins and a neural component (51). A cigarette smoke concentration that had no effect in nontreated guinea pigs' significantly elevated AHR in OVA-sensitized and OVA-challenged animals (52). Whether the increase in AHR was associated with a subsequent increase in lung inflammation was not determined. In addition, exposure to residual oil fly ash can increase AHR independent of increases in lung inflammation in OVA-sensitized and OVA-challenged mice, further supporting a discordance between AHR and lung inflammation in SS exposure (53). We have shown that chronic OVA exposure (6 weeks; 5 days/week) leads to increased amounts of serum total IgE, serum OVA-specific IgE and IgG1, severity of histopathology lesions, increased numbers of lymphocytes and neutrophils in BAL, and increased AHR in DO11.10 mice. Previously, we have shown that nonsensitized DO11.10 mice exposed once a week (1-hour nose only) to OVA for 3 weeks exhibit increased BAL neutrophils and lymphocytes (no eosinophils), mild peribronchiolar inflammation, and increased Mch-induced AHR (54). However, these mice did not produce a humoral response as measured by the lack of OVA-specific IgG1 and IgG2a or increased total IgE. In contrast, nonsensitized DO11.10 mice exposed to OVA (1 or 4 days, 20 minutes per day, whole body) do not develop increased Mch-induced AHR (55). Interestingly, these mice did exhibit a large increase in neutrophils and lymphocytes from BAL, minimal eosinophils from BAL, and no increase in serum total IgE. Differences in these studies suggest that the OVA exposure period (acute versus chronic) are critical in inducing AHR and OVA-specific antibodies in DO11.10 mice. A potential concern with the experimental design of this study was that the chronic nature of the OVA exposure while inducing an allergic type response in the Tg +/- mice might lead to tolerance in the Tg -/- mice. Development of tolerance could potentially mask any contribution that cigarette smoke exposure may have in the development of allergen sensitization. Previous studies indicated that repeated exposure (up to 10 days) of naive animals to aerosolized OVA leads to an inability to produce OVA-specific IgE antibodies to a subsequent immunogenic OVA challenge (tolerance), but the animals can produce normal secondary IgG responses to OVA challenge (56, 57). To our knowledge, only one other study has exposed naive mice to aerosolized OVA for up to 6 weeks. Temelkovski and colleagues found that naive mice exposed chronically to aerosolized OVA developed increased Mch-induced AHR, but serum OVA-specific IgE and BAL eosinophils were not detectable (58). In this study, OVA-exposed naive (Tg -/-) mice maintained elevated total and OVA-specific IgE amounts equivalent to those observed in the Tg +/- animals; however, an increase in AHR or BAL eosinophils was not observed. Disparity between the studies may reflect differences in OVA exposure amounts or the age of the animals at the start of exposure. Interestingly, when we began OVA exposure immediately after the mice were born, we could not detect any OVA-specific IgE or IgG1 in either Tg -/- or Tg +/- mice at the end of 6 weeks of exposure (unpublished results). The presence of significant amounts of serum OVA-specific IgE suggests that Tg -/- offspring developed some degree of sensitization to OVA but not complete tolerance. However, the lack of allergic inflammation (i.e., eosinophils and lymphocytes) in the lungs and the absence of increased Mch-induced AHR following OVA challenge suggest that the animals did not develop an allergic airway response. Exposing Tg -/- offspring to OVA in combination with SS did not enhance antibody concentrations or induce allergic inflammation in the lung. In fact, antibody amounts tended to remain the same or be lower after exposure to SS smoke. Children of smoking parents may have higher total serum IgE amounts (20), although other studies have reported no significant changes in concentrations of IgE (18, 19, 59). In general, chronic cigarette smoke exposure decreases IgG, IgA, and IgM antibody amounts (reviewed in 60). Interestingly, despite the lack of allergic inflammation and/or enhanced numbers of antibodies, SS in combination with OVA induced a significant increase in AHR above amounts seen with SS or OVA exposure alone in Tg -/- animals. The underlying mechanisms of such a response are not known but may involve a neurogenic component as described previously. In summary, chronic SS exposure alone altered lung function. SS exposure also enhanced existing symptoms of allergic airway disease (i.e., AHR). However, interpreting our results in terms of answering the following question is more problematic: "Does cigarette smoke exposure lead to the induction of asthma in an otherwise nonsusceptible individual?" Clearly, SS in combination with allergen did not induce an allergic airway response in nonsusceptible animals. Exposure to SS and allergen appeared to be more directly associated with independent alterations in AHR. If SS exposure is not associated with the development of asthma/allergic sensitization, what role does it play? Numerous studies show an association between SS exposure and the induction (8) or exacerbation (9, 10) of asthma. Cigarette smoke may act as a sensitivity trigger so that other potential triggers such as viral infections may have a greater impact in the induction of asthma. In addition, complex gene–cigarette smoke interactions may lead to an increased risk for the development of asthma in certain susceptible individuals. Polymorphisms of the ß2-adrenergic receptor that alone are not risk factor for asthma, but when present in an individual who smokes, significantly increase the risk for the development of asthma (61). Certainly cigarette smoke exposure is an avoidable risk factor for respiratory disease; however, the exact nature of its association with the induction of asthma is still not clear.
The authors acknowledge Fred Kleinschnitz for his expertise in operating the cigarette smoke and OVA generation systems. Supported by National Institutes of Health Grant 1RO1ES 08143–01A1 and Department of Energy Office of Biological and Environmental Research Cooperative Agreement DE-FC04–96AL76406. Received in original form June 11, 2001; accepted in final form November 15, 2001
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ABSTRACT
INTRODUCTION
p < 0.01 treatment versus Air:SS; n 
7 mice per group). (B) Tg -/- offspring, Air:Air, Air:OVA, Air:SS, or Air:OVA-SS (*p < 0.001 treatment versus Air:Air; **p < 0.0001 treatment versus Air:Air; 
