Prenatal Cigarette Smoke Decreases Lung cAMP and ... - ATS Journals

Prenatal Cigarette Smoke Decreases Lung cAMP and Increases Airway Hyperresponsiveness Shashi P. Singh, Edward G. Barrett, Roma Kalra, Seddigheh Razani-Boroujerdi, Raymond J. Langley, Viswanath Kurup, Yohannes Tesfaigzi, and Mohan L. Sopori Respiratory Immunology and Asthma Program, Lovelace Respiratory Research Institute, Albuquerque, New Mexico; and VA Medical Center and Medical College of Wisconsin, Milwaukee, Wisconsin

Epidemiologic studies suggest that in utero exposure to tobacco smoke, primarily through maternal smoking, increases the risk for asthma in children; however, the mechanism of this phenomenon is not clear. Cyclic adenosine monophosphate relaxes airway smooth muscles in the lung and acts as an antiasthmatic. In this study, we examined the effects of in utero cigarette smoke exposure of Balb/c mice on airway responsiveness, as determined by Penh measurements. Animals exposed prenatally but not postnatally to cigarette smoke exhibited increased airway hyperresponsiveness after a single intratracheal injection of Aspergillus fumigatus extract. The increased airway hyperresponsiveness was not associated with increased leukocyte migration or mucous production in the lung but was causally related to decreased lung cyclic adenosine monophosphate levels, increased phosphodiesterase-4 enzymatic activity, and phosphodiesterase-4D (PDE4D) isoform-specific messenger ribonucleic acid expression in the lung. Exposure of adult mice to cigarette smoke did not significantly alter airway responsiveness, cyclic adenosine monophosphate levels, or the phosphodiesterase activity. These results suggest that prenatal exposure to cigarette smoke affects lung airway reactivity by modulating the lung cyclic adenosine monophosphate levels through changes in phosphodiesterase-4D activity, and these effects are independent of significant mucous production or leukocyte recruitment into the lung. Keywords: asthma; phosphodiesterase-4D; Aspergillus fumigatus; airway responsiveness

Cigarette smoking is a major cause of morbidity and mortality worldwide, affecting the health of an individual at any age (1). Epidemiologic studies suggest that children from families of cigarette smokers have a higher incidence of asthma than children from families that do not smoke (2–6). The increasing incidence of asthma among children globally has been postulated to result from such factors as upper respiratory viral infections (7, 8), decreased breastfeeding (9), increased airway atopy (10, 11), prenatal and/or postnatal exposure to cigarette smoke (12), or decreased pulmonary bacterial infections in early childhood that tend to encourage mostly T-helper type 2 immune responses (13). Both prenatal and perinatal exposure to cigarette smoke might affect a child’s lung function; the mother’s smoking during the pregnancy rather than her smoking status after the delivery is better correlated to the development of childhood asthma and wheezing

(Received in original form November 1, 2002; accepted in final form May 29, 2003) Supported by grants from the National Institutes of Health (RO3HD38222–01, DA04208–14, and DA04208–07) and the Lovelace Respiratory Research Institute. The Lovelace Respiratory Research Institute is fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. Correspondence and requests for reprints should be addressed to Mohan L. Sopori, Ph.D., Respiratory and Immunology Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive SE, Albuquerque, NM 87108. E-mail: [email protected] Am J Respir Crit Care Med Vol 168. pp 342–347, 2003 Originally Published in Press as DOI: 10.1164/rccm.200211-1262OC on June 5, 2003 Internet address: www.atsjournals.org

(2, 5, 14). Although the epidemiologic evidence clearly associates the in utero exposure to cigarette smoke with elevated risk for reduced lung function (15), childhood atopy, and asthma (reviewed in 16), the precise mechanism of this phenomenon remains largely unknown. In humans and animals, chronic exposure to mainstream cigarette smoke (MS) (smoke inhaled by cigarette smokers) suppresses the immune system (1, 17, 18). However, it is unlikely that an increased propensity of asthma among children exposed in utero to MS results from a suppressed immune system. Recent observations suggest a critical role for lung cAMP levels in airway responses (19, 20). cAMP relaxes the airway smooth muscles and may inhibit airway remodeling associated with chronic asthma (21). cAMP concentration is regulated by its production and degradation by phosphodiesterases (PDEs) (22), and increased PDE expression exacerbates atopy (23). PDE inhibitors attenuate many asthma symptoms (19). These observations underscore the importance of PDE in regulating cAMP levels in the lung and manifestation of asthma symptoms. Mammals express multiple isoforms of cAMP-specific PDEs that differ in their biochemical properties and tissue expression (24). The PDE4D isoform is primarily expressed in the lung, and PDE4D-deficient mice show weaker airway hyperresponsiveness to antigenic and cholinergic challenges (25). Therefore, PDE4D may be critical in the development of airway hyperreactivity. Here we show for the first time that prenatal exposure of mice to cigarette smoke increases airway hyperreactivity as measured by Penh. This hyperresponsiveness is associated with increased PDE4 activity in the lung. These results provide a possible mechanism for the adverse effects of prenatal exposure to cigarette smoke on lung function and some asthma parameters. METHODS Animals Pathogen-free Balb/c male and female mice were obtained from the Frederick Cancer Research Facility (Frederick, MD). The Lovelace Institutional Animal Care and Use Committee approved all animal protocols. Animals were housed in shoebox-type plastic cages with hardwood chip bedding and were conditioned to whole-body exposure chambers (H1000; Hazelton Systems, Inc., Aberdeen, MD) for 2 weeks before beginning the exposures. Chamber temperature was maintained at 26 ⫾ 2⬚C, and lights were set to a 12-hour on/off cycle. Food and water were provided ad libitum.

Cigarette Smoke Generation and Exposure Mice were exposed to whole-body MS, sidestream cigarette smoke (SS; the smoke released from the burning end of a cigarette), or filtered air (FA) for 6 hours/day, 7 days/week using methods as described (26). Briefly, two 70-cm3 puffs per minute from research cigarettes (Type 2R1; Tobacco Health Research Institute, Lexington, KY) were generated by a smoking machine (Type 1300; AMESA Electronics, Geneva, Switzerland). The desired smoke concentration within the exposure chambers was achieved by adjusting the flow rates of cigarette smoke and FA. The concentration of MS in the exposure chambers, expressed as the concentra-

Singh, Barrett, Kalra, et al.: Prenatal Cigarette Smoke Decreases Lung cAMP

tion of total particulate matter, was 103 ⫾ 1.0 mg/m3 (average ⫾ SEM). This is approximately equivalent to the daily cigarette smoke exposure of a human who smokes two packs (27). SS consisted of the smoke from the lit ends of the cigarettes that was diluted with FA to yield a total particulate matter concentration of 5 mg/m3 in the exposure chamber. This SS concentration approximates a child’s exposure to parental and environmental tobacco smoke (26). For prenatal exposure, adult (3–4 months old) male and female mice were acclimatized to either MS or FA exposures for 2 weeks and were then paired for mating. Pregnant mice were separated and moved to chambers for continuing daily exposure to either MS or FA until pups were born. Mothers and pups from each group were divided into two subgroups for postnatal exposure to either FA or SS. At 3 weeks after birth, pups from each group were weaned, and the postnatal exposures were continued for 7–12 additional weeks. Exposure groups are designated as MS/FA or MS/SS (prenatal MS followed postnatally with FA or SS) or FA/FA or FA/SS (prenatal FA followed postnatally by FA or SS).

Aspergillus Extract Administration Aspergillus fumigates extracts (Aspergillus extracts) were prepared as described previously (28) and represent a mixture of culture filtrate and mycelial extract from 2-week cultures grown in a synthetic broth. We chose an extract preparation from A. fumigatus primarily because it is associated with a number of human pulmonary diseases, including hypersensitivity pneumonitis, allergic asthma, and some atopic inflammatory responses (29, 30). The use of Aspergillus extract as opposed to live Aspergillus limits the complications associated with replicating organisms. To administer Aspergillus extract, mice (10–18 weeks old) were lightly anesthetized by intraperitoneal injection of 0.1 ml of 0.2% xylazine and 1% ketamine mixture and were inoculated intratracheally with a single injection of the extract (200 ␮g in 100 ␮L of sterile saline).

Airway Hyperresponsiveness Unless mentioned otherwise, in this study airway hyperresponsiveness is defined as an increase in Penh values. Penh was determined on 10- to 12-week-old animals at 48 hours after intratracheal inoculation with Aspergillus extract. Airway hyperresponsiveness was assessed by methacholine-induced airflow obstruction (Penh) in conscious mice placed in a whole-body plethysmograph (Buxco Electronics, Troy, New York). Penh ⫽ ([expiratory time/relaxation time] ⫺ 1) ⫻ (peak expiratory flow/peak inspiratory flow) where Penh ⫽ enhanced pause (dimensionless), peak expiratory flow ⫽ ml/second, and peak inspiratory flow ⫽ ml/second. Each mouse was placed in a chamber, and the chamber–pressure–time wave was measured continuously via a transducer connected to a computer dataacquisition system. Penh was measured by exposing mice for 1 minute to saline (0.9% NaCl) for baseline values, followed by incremental doses of aerosolized methacholine (6, 12, and 25 mg/ml). Animals were monitored for 10 minutes after each methacholine challenge (25, 26), and the values for the first 5 minutes were averaged for Penh calculations. There is some dispute as to whether Penh truly represents airway reactivity (31); however, several studies have shown an excellent correlation between increased Penh values and bronchoconstriction (25, 32, 33). A recent report (34) clearly documents that a single-chamber plethysmograph, as used in our study, accurately measures airway reactivity. Thus, although Penh measurements might have limitations in accurately depicting changes in lung physiology, it is a very useful indicator of changes in airway mechanics.

Isolation of Bronchoalveolar Lavage Cells Bronchoalveolar lavage (BAL) fluid and BAL cells were obtained as described (28). Briefly, lungs were lavaged by three serial instillations of 0.8 ml of phosphate-buffered saline (PBS) into the tracheal tube by inserting a catheter, and the samples were pooled for each animal. Cells from the BAL fluids were obtained by centrifugation (300 ⫻ g for 10 minutes at 4⬚ C), resuspended in 0.2 ml of PBS, and counted in a hemocytometer. BAL cell differentials were determined by placing 5 ⫻ 104 cells in 100 ␮L of PBS on a cytospin slide and were centrifuged

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(Cytospin 2; Shandon, Cheshire, UK). The slides were stained with Hema-Tek Stain Pak, a modified Wright-Giemsa stain (Fisher Diagnostics, Pittsburgh, PA). At least 200 cells were differentially counted by light microscopy and were assigned to various subsets by conventional morphologic criteria.

Assay for PDE4 Activity Lungs were homogenized in a buffer containing 50-mM Tris-HCl (pH 7.8), 150-mM NaCl, 10-mM MgCl2, 10-mM 2-mercaptoethanol, 1-mM ethyleneglycol-bis-(␤-aminoethyl ether)-N,N⬘-tetraacetic acid, 50-mM sodium fluoride, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.5% sodium dodecyl sulfate, 50-mM benzamidine, 0.5-␮g/ml leupeptine, 0.7-␮g/ml pepstatin, 4-␮g/ml aprotinin, 10-␮g/ml soybean trypsin inhibitor, and 2-mM phenylmethylsulfonyl fluoride in an all-glass Dounce homogenizer. The homogenate was centrifuged at 20,000 ⫻ g for 15 minutes, and supernatants were tested for PDE activity (35). Lung homogenates were first tested at various dilutions to determine the linearity of the PDE assay, and the enzymatic activity was found to be linear between 2.5–30 ␮g of the homogenate protein (data not shown). All subsequent assays were run at 15–20 ␮g of homogenate protein. The detection limit of the assay was 2.5 ␮g or less of homogenate protein. Total PDE and PDE4 (rolipram-sensitive) activities were determined essentially by the method described by Thompson and colleagues (36). Briefly, in a final volume of 200 ␮l, the reaction mixture contained 40-mM Tris-HCl (pH 8.0), 10-mM MgCl2, 1.25-mM 2-mercaptoethanol, 20-␮g bovine serum albumin, 1-␮M [3H]cAMP (approximately 0.1 ␮Ci), and 25 ␮L of the test supernatant. After the incubation at 34⬚C for 10 minutes, 200 ␮L of 40-mM Tris-HCl (pH 7.5) was added to the reaction mixture, and the tubes were placed in a boiling water bath for 2 minutes. The incubation tube was allowed to come to room temperature (approximately 30 minutes) and was treated with 50 ␮L of Ophiophagus hannah snake venom (1 mg/ml) to hydrolyze 5⬘-AMP to adenosine by the nucleotidase in the venom; 500 ␮L of a 1:3 slurry of Bio-Rad resin (AG1-X2, 200–400 mesh) in deionized water were added to each tube to remove the charged nucleotides. After centrifugation for 10 minutes, 250-␮L aliquots of the supernatant were counted in a liquid scintillation counter. To determine PDE4 activity, parallel assays were run in the presence of 10-␮M rolipram (Sigma, St. Louis, MO), and the rolipram-insensitive activity was subtracted from the total PDE activity (36). Protein concentration was determined by using bicinchoninic acid Protein Assay Reagent (Pierce, Rockford, IL), and the enzyme activity was expressed as pmol cAMP hydrolyzed min⫺1 mg⫺1 protein.

Determination of cAMP Levels Lungs were homogenized in cold 5% trichloroacetic acid in a glass– Teflon tissue homogenizer (30 strokes). The homogenates were centrifuged at 600 ⫻ g for 10 minutes, and the supernatants were extracted with water-saturated ether. The aqueous extracts were dried in a Speed Vacuum concentrator (Savant, Farmingdale, NY). The dried samples were reconstituted in the assay buffer (Sigma) and used in the cAMP assay. cAMP was assayed using an immunoassay kit (Sigma). The assay is highly sensitive and can detect cAMP in the femtomolar range.

Assay for PDE4D mRNA Expression PDE4D-specific mRNA was determined by reverse transcriptasepolymerase chain reaction. RNA purification and reverse transcriptasepolymerase chain reaction were performed using commercial reagents (Molecular Research Center, Inc., Cincinnati, OH). First-strand cDNA was generated from 5 ␮g of total RNA using oligo-dT to prime the reverse transcription according to the supplied protocol (Invitrogen Corp., Carlsbad, CA). The completed first-strand cDNA reaction product (2 ␮l) was directly amplified by polymerase chain reaction after the addition of 200 ng of specific primers and 1 U of Gold Taq DNA polymerase (Perkin Elmer, Norwalk, CT). PDE4D-specific primers for the polymerase chain reaction were as follows: forward, 5⬘-CGGAACTT GCCTTGATGTACAAC-3⬘, and reverse, 5⬘-AGAGGCGTTGTGCTT GTCACAC-3⬘. Commercially available primers (Clontech, Palo Alto, CA) were used to amplify glyceraldehyde phosphate dehydrogenase to serve as control. After the hot start at 94⬚C for 10 minutes, the polymerase chain reaction was run for 35 cycles (94⬚C, 30 seconds; 60⬚C, 30 seconds; and 72⬚C, 30 seconds) and a final extension at 72⬚C

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for 5 minutes in a Perkin Elmer, Model 2,400 polymerase chain reaction machine. Aliquots of polymerase chain reaction products were separated by electrophoresis on 1.5% agarose gels and visualized with ethidium bromide.

Statistical Analysis The statistical significance of Penh values in response to methacholine was determined by two-way analysis of variance using SAS software. Other results were analyzed by the unpaired t test using Excel software. A value of p ⭐ 0.05 was considered significant.

RESULTS Prenatal Exposure to Cigarette Smoke Increases Airway Hyperresponsiveness as Measured by Penh

To determine the effects of prenatal and/or postnatal exposure to cigarette smoke on airway reactivity, Balb/c mice were exposed prenatally to FA or MS. After birth, the pups from each group were subdivided into two groups and were exposed postnatally to FA or SS, leading to four groups of animals exposed to prenatal/ postnatal combinations of FA/FA, MS/FA, FA/SS, and MS/SS. At 10 weeks after postnatal exposure, animals were inoculated intratracheally with Aspergillus extracts, and Penh values were determined at the indicated concentrations of methacholine at the predetermined peak response time of 48 hours after the extract inoculation. Penh values of MS/FA and MS/SS mice were significantly higher than FA/FA and FA/SS mice, and postnatal SS exposure did not significantly affect Penh values in animals exposed prenatally to FA or MS (Figure 1). The airway hyperresponsiveness of all animals decreased at 72 hours after the Aspergillus extract administration. Nonetheless, differences between various groups were still discernible (data not shown). Thus, prenatal exposure to mainstream, but not to postnatal SS, increased Penh in response to Aspergillus inoculation in the lung. Because SS did not significantly affect Penh, subsequent studies used only FA/FA and MS/FA animals.

Effects of Prenatal MS on Body and Lung Weights

Prenatal exposure to cigarette smoke leads to decreased birth weights in humans and animals (37). Therefore, it is possible that the differences in Penh values between prenatal MS and FA exposure reflected differences in body or lung weights of these animals. However, at 3 weeks after birth, differences in body weights were insignificant between the animal groups (data not shown), and at 10 weeks, the average body weights between the MS/FA and FA/FA groups were comparable (i.e., 23.4 ⫾ 1.2 g, n ⫽ 15, and 24.0 ⫾ 0.9, n ⫽ 16, in the MS/FA and FA/ FA animals, respectively). Similarly, lung weights of these animals were also comparable (MS/FA 0.29 ⫾ 0.015 g, n ⫽ 4; FA/FA, 0.29 ⫾ 0.015 g, n ⫽ 4). Therefore, differences in body and lung weights were not major factors in the increased Penh observed in MS/FA animals. Effects of Prenatal Exposure to MS on Leukocyte Migration into the Lung

Multiple intranasal administrations of Aspergillus extracts induce inflammation and accumulation of lymphocytes, neutrophils, and eosinophils in the lung (30). To determine whether a single intratracheal inoculation of Aspergillus extract affected the leukocyte migration into the lungs of MS/FA and FA/FA mice, we stained BAL cells from the two groups of mice and enumerated BAL leukocyte populations microscopically. Aspergillus extract-treated mice had higher numbers of eosinophils than saline-treated mice; however, prenatal MS-exposed animals did not exhibit significant differences in the number or subpopulation distribution of leukocytes in the BAL (Figure 2). Moreover, the similar histopathology of the lung tissue from MS/FA and FA/FA animals did not indicate a significant presence of lymphocytes or intraepithelial stored mucosubstances (data not shown). Thus, the higher Penh in MS/FA animals after the Aspergillus extract administration did not result from increased mucous production or significant leukocyte migration into the lung. Effects of MS Exposure on Penh in Adult Mice

Because prenatal exposure to MS enhanced Penh, we evaluated whether exposure of adult (3–4 months old) mice to MS also affected the Penh in response to Aspergillus extract. A 10-week exposure of adult mice to MS did not significantly affect the Penh response to Aspergillus extract (data not shown). Thus, unlike the prenatal exposure, adult exposure to MS did not alter Penh in response to a single inoculation of Aspergillus extract. These results suggest that the increased propensity to develop airway hyperresponsiveness, as measured by Penh, resulted primarily from prenatal exposure to MS.

Figure 1. Airway hyperresponsiveness (Penh) in mice prenatally and postnatally exposed to cigarette smoke in response to a methacholine (MCh) challenge 48 hours after intratracheal inoculation of Aspergillus extract. FA ⫽ filtered air; MS ⫽ mainstream cigarette smoke; SS ⫽ sidestream cigarette smoke. Penh values are the means ⫾ SEM (n ⫽ 7–9 mice per group).

Figure 2. Cellular composition of bronchoalveolar lavage (BAL) fluids from MS- and FA-exposed mice intratracheally challenged with Aspergillus extract (Asp). EOS ⫽ eosinophils; LYM ⫽ lymphocytes; MAC ⫽ macrophage; NEU ⫽ neutrophils. Data are expressed as cells ⫾ SEM based on differentials of at least 200 cells (n ⫽ 8 mice per group).

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Prenatal Exposure to MS Stimulates PDE4D mRNA Expression

Figure 3. cAMP concentration in the lung homogenates from mice exposed (A ) prenatally or (B ) during adulthood to MS or FA. Extracts from lung homogenates were used for the cAMP immunoassay after the acetylation protocol. Data are the mean ⫾ SEM (n ⫽ 4 mice per group, *p ⬍ 0.0004).

Prenatal Exposure to MS Decreases Lung cAMP Levels

Lung cAMP levels are negatively related to airway hyperresponsiveness (20, 21). To determine whether the effects of prenatal exposure of MS on Penh reflected changes in the lung cAMP levels, we measured the cAMP concentration in the trichloroacetic acid-soluble lung extracts of 10-week-old MS/FA and FA/ FA mice at 48 hours after the Aspergillus extract administration. The lung cAMP levels of MS/FA mice were significantly lower than in FA/FA animals (Figure 3A). On the other hand, exposure of adult mice to MS for 10 weeks did not affect the lung cAMP levels (Figure 3B). Thus, prenatal but not postnatal exposure to MS inhibited the rise in lung cAMP levels after Aspergillus extract inoculation, augmenting the propensity for increased airway reactivity. Prenatal Exposure to MS Increases Lung PDE4 Activity

The cAMP-dependent PDE4 isoform, PDE4D, plays a pivotal role in regulating cAMP levels in the lung (13). To ascertain whether prenatal exposure to MS affected lung cAMP levels through changes in the PDE4 enzymatic activity, we determined the PDE4 activity in MS/FA and FA/FA lung homogenates at 48 hours after Aspergillus extract inoculation. Compared with FA/ FA, the rolipram-sensitive PDE4 activity in MS/FA lungs was significantly increased (approximately twofold; Figure 4A); however, adult mice exposed to MS for 10 weeks did not exhibit PDE4 activity that was higher than control animals (Figure 4B). These data suggest that the higher PDE4 activity in the lungs of prenatally MS-exposed animals decreased the lung cAMP levels.

Figure 4. Phosphodiesterase (PDE) 4 activity after exposure to MS or FA (A ) prenatally or (B ) during adulthood. Aliquots of lung homogenates were assayed for PDE activity in the absence (total PDE) and presence of 10-␮M rolipram (rolipram-insensitive PDE activity). Rolipram-insensitive activity was subtracted from the total PDE activity to determine PDE4 activity. Data are the mean ⫾ SEM (n ⫽ 5–7 mice per group; *p ⬍ 0.003).

PDE4D is a lung-specific isoform of cAMP-specific PDE4 that regulates the airway hyperreactivity to cholinergic stimulation (25). To ascertain whether the higher lung PDE4 activity in MS/ FA animals reflected increased transcription of the PDE4D gene, we isolated RNA from the lungs of MS/FA and FA/FA mice at 48 hours after Aspergillus extract administration and analyzed the RNA for PDE4D-specific mRNA expression by reverse transcriptase-polymerase chain reaction. MS/FA animals had significantly higher levels of PDE4D mRNA (approximately twofold) than FA/FA animals (Figure 5). Exposure to mainstream smoke did not affect the expression of the housekeeping gene glyceraldehyde phosphate dehydrogenase. These results suggest that prenatal exposure to MS enhanced the transcription of the PDE4D gene.

DISCUSSION Asthma is usually associated with lung inflammation, increased mucous production, and increased airway hyperresponsiveness (24); however, all asthma attacks are not necessarily associated with mucous production or lung inflammation. For example, in susceptible children, exercise typically induces airway hyperresponsiveness within a very short time, and a significant number of children showing exercise-induced asthma do not have a history of asthma (38, 39). The ␤2-adrenergic system through production of cAMP is very important in bronchodilation, and ␤-agonists attenuate exercise-induced asthma without significantly affecting airway inflammation (40–42). Airway epithelial cells elicit various cAMP-dependent processes that affect airway caliber, hyperplasia of smooth muscles, influx and oxidative burst of eosinophils and neutrophils, and airway hyperresponsiveness (43). PDE4, a cAMPspecific PDE, plays a critical role in lung asthmatic responses, and PDE4-selective inhibitors (e.g., rolipram, cilomilast, SCH 351,591) are beneficial in treating asthma (25, 44, 45). PDE4D, an isoform of PDE4, is expressed specifically in the lung, and PDE4D knockout mice exhibit reduced airway hyperresponsiveness to cholinergic stimulation (25). In utero exposure to MS increases the risk of asthma (2–6, 14–16), and in this study, we tested whether in utero exposure to MS augments airway hyperresponsiveness by modulating the lung cAMP levels. Methacholine-induced bronchoconstriction is often used to evaluate the therapeutic potential of antiasthma therapies in human volunteers (46). Mice exposed prenatally to MS and inoculated Aspergillus extract showed a significant rise in Penh values in response to methacholine. Thus, muscarinic cholinergic signaling is an important trigger in the manifestation of changes in airway hyperresponsiveness induced by in utero exposure to MS. Moreover, increases in Penh seen in prenatally smoke-exposed mice required stimulation with Aspergillus extract. This is in line

Figure 5. PDE4D mRNA expression in the lungs of mice exposed prenatally to MS and FA (representative data from five of seven mice per group tested for PDE4D mRNA expression). BP ⫽ base pair size marker; GAPDH ⫽ glyceraldehyde phosphate dehydrogenase, a housekeeping gene. (A ) Three of five control (FA/FA) animals showed undetectable levels of PDE4D mRNA; (B ) two FA/FA animals showed that some basal level of PDE4D expression was increased by cigarette smoke exposure.

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with observations that human asthma attacks are generally associated with pulmonary infections or exposure to environmental allergens (47, 48). An important inference from the study is that postnatal (Day 1–10 weeks of life) exposure to SS or a 10-week MS exposure during adulthood did not significantly affect Penh, indicating that under these conditions prenatal MS exposure is critical in creating a milieu for increased airway reactivity. Nonetheless, it is possible that inflammation and mucous production associated with repeated immunization with an allergen/antigen might cause elevated Penh at higher concentrations of methacholine in postnatally cigarette smoke–exposed animals (26). In human subjects, in utero exposure to MS has been strongly linked to a higher incidence of childhood asthma, whereas postnatal exposure to SS, to some degree, may predispose children to airway hyperresponsiveness (49). Prenatal exposure to cigarette smoke decreases the birth weights of offspring (38), and thus, it is possible that differences in airway hyperresponsiveness between MS/FA- and FA/FA-exposed animals reflected changes in their body or lung weights. However, we did not note any significant difference in body or wet-lung weights between the groups after 3 weeks of age. Repeated immunizations with Aspergillus extract produce atopic lung inflammation accompanied by mucous production and infiltration of monocytes, granulocytes, and lymphocytes (30). It is possible that the differences in Penh between prenatally smoke-exposed and control animals reflected differences in the recruitment of inflammatory cells into the lung. However, other than comparable increases in eosinophils, a single inoculation of Aspergillus extract did not produce a significant amount of mucous or leukocyte infiltration into the lung. Thus, changes in airway hyperresponsiveness between experimental groups did not result from differences in leukocytic infiltration into the lung in response to Aspergillus extract. Although these results do not exclude the possibility that prenatal smoking also exacerbates airway hyperresponsiveness associated with lung inflammation (30), those conditions were not tested in this study. Nonetheless, it is clear that the increased Penh reading in MS-exposed animals did not result from an increased recruitment of inflammatory leukocytes into the lung. This facet of airway hyperresponsiveness might be comparable to exercise-induced bronchoconstriction, which does not necessarily require a pulmonary inflammatory response (42). Moreover, in response to repeated antigenic challenges, PDE4D-deficient mice showed lung inflammation that was comparable to wild-type mice with a significant increase in airway hyperresponsiveness (25). Higher airway reactivity detected in mice exposed in utero to cigarette smoke is not the result of increased IgE antibody levels (data not shown), eosinophils, or mucus accumulation in the bronchus. Thus, changes in Penh are not always dependent on the inflammatory response, involving accumulation of leukocytes and mucoid substances in the lung. Because lung cAMP levels affect airway hyperresponsiveness, we explored the possibility that prenatal exposure to MS altered these levels. Indeed, at 48 hours after Aspergillus extract inoculation, lung cAMP levels were significantly lower in MS/FA than in FA/FA mice. Rolipram-sensitive PDE (PDE4) controls the cAMP levels in the lung; this enzymatic activity was approximately twofold higher in MS/FA than FA/FA animals. Thus, increased PDE4 activity in the MS/FA lungs may decrease cAMP and increase Penh in response to a cholinergic stimulation. The PDE4D isoform of PDE4 plays a critical role in airway hyperresponsiveness, and PDE4D-deficient animals exhibit poor airway hyperresponsiveness to cholinergic stimulation (25, 43). Therefore, we determined whether prenatal exposure to cigarette smoke altered the expression of PDE4D-specific mRNA in the lung after Aspergillus extract administration. PDE4D mRNA levels were

significantly higher in prenatally cigarette smoke exposed than in control animals. Thus, prenatal exposure to MS increases the potential of the lung to upregulate the transcription of the PDE4D gene. Although the mechanism by which prenatal exposure to MS modulates the transcription of the PDE4D gene is unclear, preliminary unpublished observations suggest that the ability of prenatally smoke-exposed animals to upregulate PDE4 activity is lost around 15–18 weeks of age. Given that the average lifespan of a Balb/c laboratory mouse is approximately 30 months, 15–18 weeks translates into the human equivalent of approximately 8–10 years. This is reminiscent of a childhood asthma condition that attenuates with age in many children (50). The PDE4 enzyme has been implicated in the relaxation of the human bronchus, and in vitro studies indicate that the selective PDE4 inhibitors, rolipram and denbufylline, relax human and guinea pig bronchus and trachea, respectively (51, 52). Because PDE4 is the major PDE isoform present in the human lung (25, 52), inhibitors of PDE4 activity might be clinically useful as bronchodilators. Cilomilast, a second-generation PDE4 inhibitor, has promise in attenuating asthma in clinical trials (53). Thus, isoenzyme-selective PDE inhibitors, particularly PDE4D inhibitors, might have therapeutic value in attenuating asthma symptoms. Acknowledgment : The authors thank Paula Bradley for editing the manuscript, Steve Randock for the graphics, and James Aden for the statistical evaluation of the data.

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