Lung Inflammation and Atopy Muscarinic Receptor Expression

The Journal of Immunology

Maternal Exposure to Secondhand Cigarette Smoke Primes the Lung for Induction of Phosphodiesterase-4D5 Isozyme and Exacerbated Th2 Responses: Rolipram Attenuates the Airway Hyperreactivity and Muscarinic Receptor Expression but Not Lung Inﬂammation and Atopy1

Shashi P. Singh,* Neerad C. Mishra,* Jules Rir-sima-ah,* Mathew Campen,* Viswanath Kurup,† Seddigheh Razani-Boroujerdi,* and Mohan L. Sopori2*

Airway hyperreactivity (AHR), lung inflammation, and atopy are clinical signs of allergic asthma. Gestational exposure to cigarette smoke (CS) markedly increases the risk for childhood allergic asthma. Muscarinic receptors regulate airway smooth muscle tone, and asthmatics exhibit increased AHR to muscarinic agonists. We have previously reported that in a murine model of bronchopulmonary aspergillosis, maternal exposure to mainstream CS increases AHR after acute intratracheal administration of Aspergillus fumigatus extract. However, the mechanism by which gestational CS induces allergic asthma is unclear. We now show for the first time that, compared with controls, mice exposed prenatally to secondhand CS exhibit increased lung inflammation (predominant infiltration by eosinophils and polymorphs), atopy, and airway resistance, and produce proinflammatory cytokines (IL-4, IL-5, IL-6, and IL-13, but not IL-2 or IFN-␥). These changes, which occur only after an allergen (A. fumigatus extract) treatment, are correlated with marked up-regulated lung expression of M1, M2, and M3 muscarinic receptors and phosphodiesterase (PDE)4D5 isozyme. Interestingly, the PDE4-selective inhibitor rolipram attenuates the increase in AHR, muscarinic receptors, and PDE4D5, but fails to down-regulate lung inflammation, Th2 cytokines, or serum IgE levels. Thus, the fetus is extraordinarily sensitive to CS, inducing allergic asthma after postnatal exposure to allergens. Although the increased AHR might reflect increased PDE4D5 and muscarinic receptor expression, the mechanisms underlying atopy and lung inflammation are unrelated to the PDE4 activity. Thus, PDE4 inhibitors might ease AHR, but are unlikely to attenuate lung inflammation and atopy associated with childhood allergic asthma. The Journal of Immunology, 2009, 183: 2115–2121.

he adverse health effects of cigarette smoke (CS)3 are well sponses differently (12, 13). For example, some allergic diseases recognized, and smoking is associated with increased risk and ulcerative colitis are less common in adult smokers than non- T for lung cancer and respiratory infections (1). Increasing smokers (1, 14–18), whereas ex-smokers are more likely to develop evidence suggests that chronic exposure to environmental or sec- asthma than current smokers (19, 20). Moreover, in animal models, ondhand CS (SS) also causes significant health effects (2–4). chronic exposure of adult animals to mainstream CS or nicotine sup- Moreover, strong epidemiological evidence indicates parental presses innate and adaptive immune responses (1, 21–24), and even smoking, particularly maternal smoking during pregnancy, in- SS moderates some parameters of allergic asthma in mice (25). In creases the risk of allergic asthma in children (4–10). Yet in the Brown Norway rats, chronic exposure to nicotine attenuates the rag- United States alone, nearly 12% of prospective mothers continue weed/house dust mite-induced lung inflammation and atopy (13). to smoke during pregnancy (11). Interestingly, prenatal and post- Thus, in adult humans and animals, chronic CS/nicotine exposure natal exposure to CS may affect immune and inflammatory re- may attenuate some parameters of allergic inflammation in the lung. In contrast, in utero exposure to mainstream CS exacerbates allergic

*Respiratory Immunology Division, Lovelace Respiratory Research Institute, Albu- and inflammatory responses in the offspring (26), and it is likely that querque, NM 87108; and †Veterans Affairs Medical Center and Medical College of the mechanisms by which CS modulates the allergic responses in Wisconsin, Milwaukee, WI 53295 utero and during adult life do not totally overlap. Received for publication March 13, 2009. Accepted for publication May 26, 2009. The mechanism(s) by which gestational exposure to CS affects The costs of publication of this article were defrayed in part by the payment of page the lung function in children is not clearly understood. In an es- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tablished murine model of bronchopulmonary aspergillosis, in 1 This work was supported in part by grants from Flight Attendant Medical Re- which Aspergillus fumigatus extract (Af) induces allergic asthma search Institute and the National Institutes of Health (R01 DAO17003 and R01 (26, 27), we have shown that exposure of mothers to mainstream DAO4208-17). CS throughout the gestational period increases airway hyperreac- 2 Address correspondence and reprint requests to Dr. Mohan L. Sopori, Lovelace tivity (AHR) after an acute exposure to the allergen (Af) and the Respiratory Research Institute, 2425 Ridgecrest Drive SE, Albuquerque, NM 87108. E-mail address: [email protected] increased AHR is related to elevated expression of phosphodies- 3 Abbreviations used in this paper: CS, cigarette smoke; Af, Apergillus fumigatus terase (PDE)4 in the lungs of the progeny (26). However, unlike extract; AHR, airway hyperreactivity; BAL, bronchoalveolar lavage; BALF, BAL chronic Af sensitization (27), single exposure to the allergen did fluid; EOS, eosinophil; FA, filtered air; i.t., intratracheal; mAChR, muscarinic acetylcholine receptor; MCh, methacholine; PDE, phosphodiesterase; qPCR, quantitative not induce significant lung inflammation and atopy (26). There- RT-PCR; RL, airway resistance; RP, rolipram; SS, secondhand CS. fore, in addition to the mechanism of allergen-induced increase in www.jimmunol.org/cgi/doi/10.4049/jimmunol.0900826 2116 SS EXPOSURE IN UTERO PROMOTES ALLERGIC ASTHMA

AHR, the effects of gestational CS exposure on lung inflammation Bronchoalveolar lavage (BAL) and cell collection and atopy are unknown. In this communication, we show that fe- Mice were anesthetized and euthanized by exsanguination 24 h after the tuses are highly sensitive to CS, and maternal exposure to even SS last allergen (Af) challenge. Before excision of the lungs, the trachea was strongly exacerbates the allergen-induced AHR through up-regu- surgically exposed and cannulated. The left lung lobe was tied off with a lated expression of M1, M2, and M3 muscarinic receptors and the silk thread suture, and the right lobe was lavaged with 1 ml of PBS (2ϫ) PDE4 isozyme PDE4D5 in the lung. In addition, SS markedly and pooled for each animal. BAL cells were collected by centrifugation and resuspended in PBS. Approximately 5 ϫ 104 cells from each sample were intensifies lung inflammation, Th2 cytokine production, and atopy cytospun on duplicate slides and stained with Diff Quik (Baxter Health- induced through allergic sensitization. Although the PDE4-selec- care) to score eosinophils (EOS), macrophages, neutrophils, and lympho- tive inhibitor rolipram (RP) decreased muscarinic receptor expres- cytes using standard hemocytologic criteria. At least 200 cells from each sion and AHR, it essentially failed to affect the allergen-induced slide were counted to obtain the differential cell count. atopy and lung inflammation. Analysis of cytokines/chemokines in the BAL fluid (BALF) BALF was analyzed for cytokines and chemokines by the commercially Materials and Methods available mouse cytokine Ten-Plex ELISA kit (BioSource International; Animals Invitrogen) and used according to the manufacturer’s directions. Cytokine concentrations were determined using Bioplex manager software with Pathogen-free BALB/c mice were obtained from the Frederick Cancer four-parameter data analysis. The sensitivity of the assay was Ͻ10 pg/ml. Research Facility. Animals were housed in shoe box-type plastic cages with hardwood chip bedding and conditioned to whole-body exposure Quantitative RT-PCR (qPCR) analysis in exposure chambers (H1000; Hazleton Systems) for 2 wk before exposure to SS. The chamber temperature was maintained at 26 Ϯ 2°C, Total RNA was extracted from the lung samples using TRI reagent (Mo- and lights were set to a 12-h on/off cycle. Food and water were provided lecular Research Center) and quantified following the manufacturer’s in- ad libitum. All animal protocols used in this study were approved by structions. The lung expression of cytokines (IL-4, IL-5, IL-6, and IL-13), Lovelace Respiratory Research Institute’s Institutional Animal Care muscarinic receptors (M1, M2, and M3), 18S RNA, and GAPDH was and Use Committee. determined by qPCR using the One-Step RT-PCR Master Mix and specific labeled primer/probe set (Applied Biosystems). The relative expression of Abs and reagents each mRNA was calculated, as previously described (13). Ab to PDE4 (ab14628) was obtained from Abcam. All the other reagents, Total serum IgE unless otherwise stated, were bought from Sigma-Aldrich. Blood from the mice was collected on the day of sacrifice, centrifuged in a serum separator, and stored at Ϫ80°C until analysis. Total serum IgE CS generation and exposure titers were determined by the ELISA method, as previously described (30). Mice were exposed to whole-body SS (the smoke released from the burn- Western blot analysis ing end of a cigarette) or filtered air (FA) for 6 h/day, 7 days/wk, as described (26). Briefly, a smoking machine (type 1300; AMESA Electron- Lung tissues were homogenized in radioimmunoprecipitation assay buffer ics) generated two 70-cm3 puffs/min from a research cigarette (type 2R1; (20 mM Tris, 150 mM NaCl, 20 mM ␤-glyceryl-phosphate, 1% Triton-X,

Tobacco Health Research Institute), and the smoke was captured from the 10 mM NaF, 5 mM EDTA, 1 mM Na3VO4, protease inhibitors, 1 mM lit end of the cigarette with a plastic manifold placed above it. Mice were PMSF, and 1 ␮g/ml each of aprotinin, antipain, and leupeptin) at 4°C. placed in whole-body chambers and exposed to either FA or SS (total Protein content of the extracts was determined by the bicinchoninic acid particulate matter: 1.52 Ϯ 0.41 mg/m3). This level of SS exposure simu- protein assay kit (Pierce), according to the manufacturer’s directions. The lates the conditions to which fetuses are likely to be exposed through pa- samples were electrophoresed on 10% SDS-PAGE and transferred onto ternal smoking, is slightly lower than the average SS concentration of 2 nitrocellulose membrane (Bio-Rad). Membranes were blocked using 5% mg/m3 found in most smoking bars, and is ϳ70-fold less than the amount nonfat dry milk in TBST for1hatroom temperature and probed with of CS inhaled by an average two-pack/day smoker (26, 28). Adult (3- to primary Ab to PDE4 (rabbit polyclonal ab14628; Abcam) overnight at 4°C. 4-mo-old) male and female mice were separately acclimatized either to SS The blots were washed three times with TBST, incubated for1hatroom or FA for 2 wk and then paired for mating under the same exposure con- temperature with HRP-conjugated anti-rabbit serum (Santa Cruz Biotech- ditions. After ascertaining pregnancy by vaginal smear, pregnant mice nology) at room temperature, and developed by ECL (Amersham Bio- were separated, housed singly in plastic cages, and continued to receive SS sciences) on an x-ray film. or FA until the pups were born. The mothers and pups continued to be exposed to FA or SS until the pups were weaned at 3 wk of age. Statistical analysis All data were analyzed using GraphPad Prism software 3.0 using Student’s Allergen (Af) sensitization and challenge t test or by two-way ANOVA. Results are presented as the means Ϯ SD of A lyophilized culture filtrate preparation (29, 30) of Af was used as the the combined experiments. The differences with p value of Յ0.05 were allergic sensitization, as described previously (27). Briefly, mice were sen- considered statistically significant. sitized intratracheally (i.t.) with Af (50 ␮g/0.1 ml sterile endotoxin-free saline or saline alone) and subsequently challenged i.t. with 100 ␮g/0.1 ml Results Af extract three times at 5-day intervals. Where indicated, RP (250 ␮g/kg) PDE4D5 expression is up-regulated in the lungs of mice was injected i.p. into animals at the time of Af sensitization. Control ani- exposed to maternal SS mals received sterile saline alone. We have previously shown that the activity of PDE4 is higher in Pulmonary function test animals exposed in utero to mainstream CS (26). However, the identity of the PDE4 isozyme(s) affected by gestational exposure At 48 h after the last Af/saline challenge, airway resistance (RL) was measured by the Flexivent system (SCIREQ). Briefly, mice were anesthetized to CS was not known. Also, it was not clear whether the increased by i.p. injection of avertin (250 mg/kg). A small incision was made in the enzymatic activity reflected increased PDE4 protein and whether trachea through which a 20-gauge needle hub was inserted; a saline-filled the increase was independent of allergen sensitization. Although catheter was placed in the thoracic esophagus via the mouth to obtain transthoracic pressure. The mouse was placed on the Flexivent apparatus there are nine isoforms of PDE4, including alternative spliced and ventilated through the tracheal cannula. Then the paralytic doxacurium products (31), under these conditions the Western blot analysis of (0.5 mg/kg) was administered i.p. Heart rate and electrocardiogram were the lung homogenates showed the presence of only three immu- monitored (via a Grass Instruments Recorder with Tachograph). AHR was noreactive forms of PDE4 isozymes in the mouse lung that corre- assessed using increasing doses of aerosolized methacholine (MCh). RL and elastance were measured by manipulation of ventilatory patterns and spond to 105 kDa (PDE4D5), 95 kDa (PDE4D3), and 79 kDa measurement of upstream and downstream pressures. The peak responses (PDE4A/B) (Fig. 1). Results indicate that prenatal exposure to SS at each MCh concentration were used for data analysis. or Af sensitization of control animals alone does not significantly The Journal of Immunology 2117

FIGURE 1. Maternal exposure to SS increases allergen-induced lung expression of PDE4D5. Lung homogenate (75 ␮g) was fractionated using SDS-PAGE, transferred onto nitrocellulose membrane, and probed with anti-PDE4 Ab. The ﬁgure represents the response of three animals from each group: FA, FA/Af, SS, SS/Af, and SS/Af treated with RP. alter their content in the lung. However, after Af sensitization, the lungs from SS-exposed animals (SS/Af) exhibit increased PDE4 proteins, particularly the PDE4D5 isoform of the enzyme. Treat- ment of SS/Af animals with the PDE4-selective inhibitor RP completely blocked the rise in PDE4 content, including that of the PDE4D5 isoform. These data suggest that prenatal exposure to CS per se does not elevate PDE4, but primes the lung to make more PDE4, particularly the PDE4D5 isoform, after an allergic challenge. Moreover, essentially all the increase in PDE4D5 is blocked by RP treatment.

Increased RL in Af-sensitized mice exposed to maternal CS Children exposed to tobacco smoke either through maternal smoking or exposure of the mother to SS are at higher risk of developing asthma (32). We have demonstrated that prenatal exposure to mainstream CS increases AHR detected by Baxco plethysmogra- FIGURE 2. Maternal SS exposure strongly increases MCh-induced and phy (26). However, the validity of the Baxco system to measure RP-insensitive RL in Af-treated animals. Indicated groups of animals were bronchoconstriction has been strongly questioned (33). Therefore, examined for RL by the Flexivent system, as described in Materials and Methods. A, Peak values for R are plotted against each MCh concentra- to determine whether maternal exposure to CS affected AHR, we L tion, and the values represent mean Ϯ SD of five to seven mice/group in used the Flexivent system to measure R in response to MCh in L one experiment. The difference in the p value between SS plus Af group animals exposed prenatally to SS. Results presented in Fig. 2A with other groups was Յ0.001. Inset, Because the response of SS/Af to (inset) show that maternal exposure to SS slightly, but significantly, MCh was much higher than other animals, the y-axis scale was expanded increased AHR at the baseline. Af sensitization of FA-exposed ani- to visualize differences in the response between other groups (n ϭ 5–7/ mals significantly increased the AHR response; however, compared group). B, Doses of MCh needed to elicit 50% (ED50) of the peak response with animals exposed to FA or FA plus Af, the increase in the RL in in SS-exposed animals before and after Af and Af plus RP treatment. Bars .Represents p Յ 0.001 ,ءءء .response to MCh of prenatally SS-exposed animals sensitized with Af are mean Ϯ SD was extremely dramatic (Fig. 2A). Moreover, as seen by changes in the ED50 for MCh, treatment of animals with the PDE4-selective and EOS were over 3-fold higher in SS/Af mice than in the FA/Af inhibitor RP significantly attenuated the MCh-induced RL in Af-sensitized SS-treated animals (Fig. 2B). Thus, gestational exposure to SS mice (35 Ϯ 12, and 31 Ϯ 11 vs 9 Ϯ 5 and 12 Ϯ 3, respectively). primes the lung for increased allergen-induced AHR, and the increase Interestingly, RP did not significantly alter the total number or is at least partly related to the increased lung PDE expression after the distribution of leukocyte subtypes in the SS/Af BAL. These results allergic sensitization. suggest that maternal exposure to SS primes the lung for increased inflammatory responses, and the increase is not significantly re- Maternal SS exposure increases lung leukocytic infiltration after lated to changes in the lung PDE4 activity. allergic sensitization Allergic asthma is associated with elevated numbers of EOS and neutrophils in the lung (34, 35). To determine whether maternal exposure to SS promoted leukocytic infiltration in the lung before and after an allergen challenge, prenatally FA- and SS-exposed animals were challenged through i.t. instillation with Af extracts. BAL cells were collected and stained for differential cell count. The total number of BAL cells in FA- and SS-exposed animals without Af sensitization was similar (6–8 ϫ 104); however, after Af challenge, the number of cells in FA/Af rose to 44 Ϯ 7 ϫ 104, and the number was further increased to 96 Ϯ 14 ϫ 104 in SS/Af animals. Moreover, as shown in Fig. 3, before Af sensitization, FIGURE 3. Changes in leukocyte subtypes in the BAL cells after ma- macrophages represented the predominant cell population in FA- ternal SS exposure, Af treatment, and Af plus RP treatment. Differential and SS-exposed animals; following Af sensitization, however, the cell count was accessed by staining cytospin slides using standard hemo- composition of BAL cells changed. The percentage of EOS and cytologic criteria (n ϭ 4–6/group). Two hundred cells were counted per polymorphs relative to macrophages increased significantly, and slide (MACS, macrophage; PMN, neutrophils; LYMPH, lymphocytes). the increase was markedly higher in Af-sensitized animals exposed Bars represents mean Ϯ SD. Differences in the p values between SS and SS maternally to SS. Thus, in the 200 cells counted, neutrophils plus Af and SS plus Af plus RP are Յ0.01. 2118 SS EXPOSURE IN UTERO PROMOTES ALLERGIC ASTHMA

FIGURE 4. Maternal exposure to SS strongly up-regulates cytokine expression in the lung after Af sensitization that is insensitive to RP. Cytokine levels Indicates a signiﬁcant ,ء .in BALF (A) and expression in the lung (B) were determined by ELISA (n ϭ 4–6/group) and qPCR (n ϭ 3/group), respectively .p Յ 0.001 ,ءءء p Յ 0.01; and ,ءء ;change at p Յ 0.05

Maternal exposure to SS increases the propensity of Th2 asthma (38). To investigate whether increased AHR in animals cytokine production in the lung maternally exposed to SS and sensitized with Af was related to the To ascertain whether the increased lung inflammation in mater- effects of SS exposure on the mAChR expression in the lung, we nally SS-exposed animals is associated with increased production determined the expression of M1, M2, and M3 mAChRs in the of proinflammatory cytokines, we determined the expression of lung by qPCR. These three mAChR subtypes have been detected various proinflammatory cytokines by ELISA in the BALF and by in murine and human airways (39). It is clear from the results qPCR of the lung mRNA in animals exposed prenatally to FA or presented in Fig. 6 that compared with Af-sensitized FA animals, SS before and after sensitization with Af. Compared with FA/Af, the mRNA expression of M1, M2, and M3 receptors is markedly ϳ levels of the Th2 cytokines (IL-4, IL-5, IL-6, and IL-13) were up-regulated in Af-sensitized, SS-exposed animals by 43-, 13-, significantly higher in the BALF from SS/Af animals (Fig. 4A). and 6-fold, respectively. Moreover, RP treatment completely Similarly, the lung mRNA expression for IL-4, IL-5, IL-6, and blocked the increase in expression of these mAChR subtypes, in- IL-13 was significantly higher in the SS/Af than the FA/Af group dicating a crucial role for PDE4 in the maternal smoke-induced (Fig. 4B). There was no detectable level of IFN-␥ or IL-2 in any elevated expression of mAChRs. BALF sample, and the lung mRNA level of IFN-␥ was low and unaltered by any treatment (data not shown). Moreover, RP had no Discussion significant effect on the expression of Th2 cytokines. Thus, it is In utero exposure to parental smoking is associated with an in- likely that the maternal exposure to SS induces epigenetic changes creased risk for development of childhood atopy and asthma (40), that primarily increase the propensity of the lung to up-regulate the indicating that direct maternal smoking as well as exposure of expression of Th2 cytokines after Af sensitization, and the SS- mothers to SS may be significant factors in eliciting childhood induced changes in Th2 cytokine expression are not significantly asthma. To study the role of maternal SS exposure in childhood related to the increase in PDE4 activity. allergic asthma, we used the murine model of allergic bronchopulmonary aspergillosis, in which Aspergillus proteins act as al- Maternal exposure to SS increases serum IgE levels lergens and increase the total and Aspergillus-reactive IgE in adult animals (27, 37). Allergic bronchopulmonary aspergillosis is also Atopy is the hallmark of allergic asthma, and the Th2 cytokines seen in humans, and IgE from these patients reacts with Aspergil- IL-4/IL-13 are critical in the production of IgE (36). Moreover, Af lus proteins (29). In the Af-sensitized animals, the allergic re- sensitization stimulates IgE production in the murine model of sponse is further aggravated after the administration of rIL-4, in- aspergillosis (37). To determine the effect of maternal exposure to dicating a significant role of this cytokine in the allergic response SS on IgE, total IgE was assayed in the serum from the FA- and maternal SS-exposed mice before and after Af sensitization. Sen- sitization with Af significantly increased the serum IgE concentration of FA and SS animals; however, compared with FA-exposed mice, the serum level of IgE was nearly 3-fold higher in the SS-exposed animals after Af sensitization. Moreover, RP treatment failed to significantly alter the serum concentration of IgE (Fig. 5). These results suggest that maternal SS exposure increases serum IgE in response to allergic sensitization, but the increased IgE levels are not linked to changes in the lung PDE4 activity.

Maternal exposure to SS up-regulates the muscarinic acetylcholine receptor (mAChR) expression in the lung FIGURE 5. Maternal exposure to SS exacerbates atopy following Af sensitization that is insensitive to RP. Total serum IgE was determined by Lungs have an abundant expression of mAChRs. These receptors a commercially developed kit, as described in Materials and Methods (n ϭ .p Յ 0.01 ,ءء .(modulate airway smooth muscle contraction and affect AHR and 4–6/group The Journal of Immunology 2119

selective inhibitor RP blocked the rise in PDE4D5 expression and the increased AHR observed in Af-sensitized SS animals, suggest- ing that the two responses are interrelated. In recent years, the pharmaceutical industry has actively fol- lowed the development of subtype-specific PDE4 inhibitors for the treatment of asthma, and several compounds such as roflumilast, filaminast (43), and cilomilast are in clinical trials (44). Results with novel PDE4 inhibitors BAY 19-8004 (45) and roflumilast (46) have shown only marginal clinical benefits in asthmatics and chronic obstructive pulmonary disease patients. Similarly, despite the initial optimism, the clinical trials of many other PDE4 inhibitors have been discontinued primarily due to the low therapeutic ratio that severely limits the dose that can be administered to humans (43). Our data clearly show that despite ameliorating AHR and blocking increased expression of PDE4D5, RP has no significant effect on lung inflammatory parameters, including leukocytic infiltration and Th2 cytokine/chemokine expression. These data are similar to those obtained in the PDE4DϪ/Ϫ mice, in which the disruption of the PDE4D gene attenuated AHR without affecting the inflammatory response in the lung (47). However, some PDE4 inhibitors have decreased lung inflammation in the murine OVA model (48). Although it is conceivable that some PDE4 inhibitors may have beneficial effects on lung inflammation (48, 49), at present it is not clear whether the anti-inflammatory properties of these inhibitors are related to PDE4 activity. Indeed, the anti- inflammatory property of theophylline is manifested at drug levels

FIGURE 6. Maternal exposure to SS up-regulates the expression of M1, well below the Ki for PDE inhibition and might relate to its his- M2, and M3 muscarinic receptors in the lung. The lung expression of tone-modulating activity (50, 51). In general, the problem is the muscarinic receptors was determined by qPCR, as detailed in Materials complexity of PDE4, because it includes 4 gene families and 20 ϭ and Methods (n 3/group). A, M1; B, M2; and C, M3. Bars represents splice variants (52), and the expression of these isozymes in mul- mean Ϯ SD of triplicate values. tiple cell types (44, 53). Given the critical role of cAMP as the second messenger in numerous biological functions, a global PDE4 inhibitor is likely to have undesirable side effects. The iden- (29). In addition, BAL cells from Af-sensitized animals produce tification of PDE4D5 in these studies as the RP-sensitive PDE4D increased levels of proinflammatory cytokines after in vitro stim- isozyme suggests the possibility of targeting this isoform for ther- ulation (41). Thus, this model simulates typical characteristics of apeutic interventions to reduce AHR. With anti-inflammatory (par- allergic asthma, including increased AHR, inflammation, and atopy. We have used this model to demonstrate that maternal ex- ticularly Th2-specific) drugs, it could be useful in controlling al- posure to mainstream CS increases AHR that is causally related to lergic asthma in children. An agent without PDE4D5 inhibition increased PDE4 activity (26). However, the experiment repre- may lack sufficient therapeutic efficacy to control AHR in children sented a single exposure to Af, which did not induce significant exposed to maternal CS. lung inflammation or atopy (26). Because of these concerns, in the Although the precise mechanism by which PDE4D5 controls AHR is not clear, cAMP, the substrate for PDE4, is a bronchodi- current studies we measured RL by the Flexivent system and after Af sensitization conditions that produced significant increases in lator (55), and decreased levels of cAMP in the lung (26) are likely lung inflammation and IgE levels. to increase bronchoconstriction. However, the major effect of SS It is clear from our results that, similar to mainstream CS ex- exposure on AHR is attained only after Af sensitization, which is posure (26), compared with controls, gestational exposure to SS associated with increased lung expression of muscarinic receptors M1, M2, and M3. Five muscarinic receptor subtypes (M1–M5) slightly increases the baseline RL, and the difference becomes highly exaggerated when the animals are sensitized with Af. These have been identified (56); however, the lung mainly expresses the results suggest that the potential for higher AHR is established M1, M2, and M3 subtypes (39, 56). Although M1, M2, and M3 during in utero SS exposure, but requires allergic sensitization for receptors participate in bronchoconstriction, M3 accounts for most the full expression. Therefore, it is likely that CS exposure during of the bronchoconstriction in the normal lung (39). Our data show fetal development produced conditions that lead to an increased that compared with controls, the expression of all three muscarinic propensity for AHR. Moreover, the fact that the concentration of receptors is strongly up-regulated in SS-exposed lungs. Therefore, SS to which the animals were exposed during gestation (1.52 Ϯ it is likely that the increased muscarinic receptor expression ac- 0.41 mg/m3 total particulate matter) is nearly 70-fold lower than counts for the increased sensitivity of these animals to MCh-in- the level of exposure seen in a two-pack/day human smoker (42) duced RL. RP attenuates AHR and the increased muscarinic re- or the mice exposed to mainstream CS in our earlier studies (26) ceptor expression in SS-exposed lungs, indicating a causal suggests that the fetus is extraordinarily sensitive to CS. Af sen- relationship between the PDE4D5 activity and muscarinic receptor sitization of prenatally SS-exposed animals also led to increased expression in allergic asthma. However, the mechanistic basis of expression of the 105-kDa isoform of PDE4-PDE4D5, whereas the this relationship is not clear at present. Although RP essentially expression of PDE4D3 (95 kDa) was not significantly affected by blocked the allergen-induced rise in PDE4D5 expression and mus- the allergen and SS exposure. Other variants of the PDE4D isoen- carinic receptor expression, it did not completely eliminate the zyme were not detectable under our assay conditions. The PDE4- allergen-induced AHR. It is possible that other mediators, such as 2120 SS EXPOSURE IN UTERO PROMOTES ALLERGIC ASTHMA mast cell-derived serotonin and histamine that cause bronchial hy- 3. Bradley, J. P., L. B. Bacharier, J. Bonfiglio, K. B. Schechtman, R. Strunk, perresponsiveness through receptors distinct from muscarinic re- G. Storch, and M. Castro. 2005. Severity of respiratory syncytial virus bronchi- olitis is affected by cigarette smoke exposure and atopy. Pediatrics 115: e7–e14. ceptors (57), are not affected by PDE inhibitors. 4. Environmental Protection Agency. 1992. Respiratory Health Effects of Passive In addition to AHR and inflammation, the clinical signature Smoking: Lung Cancer and Other Disorders. US EPA Office Research and De- velopment, Washington, D.C. of allergic asthma is atopy (25), and the serum IgE levels are 5. Cliver, S. P., R. L. Goldenberg, G. R. Cutter, H. J. Hoffman, R. O. Davis, and significantly higher in Af-sensitized SS animals than in FA- K. G. Nelson. 1995. The effect of cigarette smoking on neonatal anthropometric exposed animals. However, as with lung inflammation, RP measurements. Obstet. Gynecol. 85: 625–630. 6. Wen, S. W., R. L. Goldenberg, G. R. Cutter, H. J. Hoffman, S. P. Cliver, treatment did not decrease the allergen-induced IgE response, R. O. Davis, and M. B. DuBard. 1990. Smoking, maternal age, fetal growth, and indicating that atopy in these animals is essentially independent gestational age at delivery. Am. J. Obstet. Gynecol. 162: 53–58. of PDE4 activity. The inability of RP to control atopy is un- 7. Taylor, B., and J. Wadsworth. 1987. Maternal smoking during pregnancy and lower respiratory tract illness in early life. Arch. Dis. Child. 62: 786–791. derstandable, because the RP treatment failed to significantly 8. Sekhon, H. S., B. J. Proskocil, J. A. Clark, and E. R. Spindel. 2004. Prenatal suppress the production of IL-4/IL-13 and IL-6 in SS-exposed, nicotine exposure increases connective tissue expression in foetal monkey pul- Af-sensitized animals. These cytokines are critical in IgE iso- monary vessels. Eur. Respir. J. 23: 906–915. 9. Tager, I. B., J. P. Hanrahan, T. D. Tosteson, R. G. Castile, R. W. Brown, type switching and B cell proliferation/differentiation, respec- S. T. Weiss, and F. E. Speizer. 1993. Lung function, pre- and post-natal smoke tively (36, 58). Moreover, the development of marked lung exposure, and wheezing in the first year of life. Am. Rev. Respir. Dis. 147: 811–817. eosinophilia persisted in RP-treated, SS-exposed animals, sug- 10. Cunningham, J., D. W. Dockery, and F. E. Speizer. 1994. Maternal smoking gesting a lack of effect on IL-5 and eotaxin, the main EOS during pregnancy as a predictor of lung function in children. Am. J. Epidemiol. proliferation/differentiation cytokine (59), and the potent eosin- 139: 1139–1152. 11. Mathews, T. J. 2001. Smoking during pregnancy in the 1990s. Natl. Vital Stat. ophilic chemoattractant (60), respectively. Thus, it is likely that Rep. 49: 1–14. the inability of RP to control the inflammation and atopy in our 12. Singh, S. P., S. Razani-Boroujerdi, J. C. Penã-Philippides, R. J. Langley, allergic asthma model is its failure to control the allergen-in- N. C. Mishra, and M. L. Sopori. 2006. Early postnatal exposure to cigarette smoke impairs the antigen-specific T-cell responses in the spleen. Toxicol. Lett. duced Th2 cytokine/chemokine production. 167: 231–237. Mechanistically, the connection between maternal CS exposure 13. Mishra, N. C., J. Rir-Sima-ah, R. J. Langley, S. P. Singh, J. C. Penã-Philippides, during gestation and the increased susceptibility to allergic asthma T. Koga, S. Razani-Boroujerdi, J. Hutt, M. Campen, K. C. Kim, et al. 2008. Nicotine primarily suppresses lung Th2 but not goblet cell and muscle cell re- remains somewhat undefined. Although these animals were ex- sponses to allergens. J. Immunol. 180: 7655–7663. posed to CS both prenatally and perinatally to simulate the poten- 14. Warren, C. P. 1977. Extrinsic allergic alveolitis: a disease commoner in non- smokers. Thorax 32: 567–569. tial maternal exposure, we have observed that neonatal exposure to 15. Calkins, B. M. 1989. A meta-analysis of the role of smoking in inflammatory CS does not affect AHR (26) and, in fact, neonatal or adult expo- bowel disease. Dig. Dis. Sci. 34: 1841–1854. sure to CS or nicotine causes immunosuppression (1, 12, 13). In- 16. Baron, J. A. 1996. Beneficial effects of nicotine and cigarette smoking: the real, the possible, and the spurious. Br. Med. Bull. 52: 58–73. terestingly, the major difference in AHR between FA- and SS- 17. Netscher, D. T., and J. Clamon. 1994. Smoking: adverse effects on outcome for exposed animals is manifested after exposure to Af, indicating that plastic surgical patients. Plast. Surg. Nurs. 14: 205–210. gestational CS creates the conditions that allow allergens to induce 18. Eskenazi, B., and M. L. Warner. 1997. Epidemiology of endometriosis. Obstet. Gynecol. Clin. North Am. 24: 235–258. excessive AHR and lung inflammation. This is likely through epi- 19. Baldacci, S., P. Modena, L. Carrozi, M. Pedreschi, M. Vellutini, P. Biavati, genetic changes associated with gestational exposure to CS that M. Simoni, T. Sapigni, G. Viegi, P. Paoletti, and C. Giuntini. 1996. Skin prick encourages higher expression of Th2 cytokine/chemokine genes, test reactivity to common aeroallergens in relation to total IgE, respiratory symptoms, and smoking in a general population sample of northern Italy. Allergy 51: PDE4D5, and muscarinic receptors. Because changes in methyl- 149–156. ation alter gene transcription (50, 51), we examined the methyl- 20. Troisi, R. J., F. E. Speizer, B. Rosner, D. Trichopoulos, and W. C. Willett. 1995. Cigarette smoking and incidence of chronic bronchitis and asthma in women. ation status of PDE4 and M1 muscarinic receptor genes in FA- and Chest 108: 1557–1561. SS-exposed animals. Our preliminary results indicate that PDE4 21. Kalra, R., S. P. Singh, J. C. Penã-Philippides, R. J. Langley, S. Razani-Boroujerdi, essentially lacked methyl islands and the M1 gene is methylated to and M. L. Sopori. 2004. Immunosuppressive and anti-inflammatory effects of nico- ϳ tine administered by patch in an animal model. Clin. Diagn. Lab. Immunol. 11: only 5% (our unpublished observations). Therefore, it is unlikely 563–568. that methylation of these genes contributes to potential changes in 22. Razani-Boroujerdi, S., S. P. Singh, C. Knall, F. F. Hahn, J. C. Penã-Philippides, the gene expression during allergic asthma in animals exposed R. Kalra, R. J. Langley, and M. L. Sopori. 2004. Chronic nicotine inhibits inflammation and promotes influenza infection. Cell. Immunol. 230: 1–9. prenatally to CS. It is possible that changes in the methylation 23. Wang, H., M. Yu, M. Ochani, C. A. Amella, M. Tanovic, S. Susarla, J. H. Li, status of other genes such as cytokines and chemokines or mech- H. Wang, H. Yang, L. Ulloa, et al. 2003. Nicotinic acetylcholine receptor ␣7 anisms other than gene methylation such as covalent modifications subunit is an essential regulator of inflammation. Nature 421: 384–388. 24. Saeed, R. W., S. Varma, T. Peng-Nemeroff, B. Sherry, D. Balakhaneh, J. Huston, of the histone tails, nucleosome occupancy and turnover, and high- K. J. Tracey, Y. Al-Abed, and C. N. Metz. 2005. Cholinergic stimulation blocks er-order chromatin folding (61) are involved in the epigenetic endothelial cell activation and leukocyte recruitment during inflammation. J. Exp. Med. 201: 1113–1123. modulation by gestational CS. 25. Thomson, N. C., R. Chaudhari, and E. Livingston. 2004. Asthma and cigarette smoking. Eur. Respir. J. 24: 822–833. 26. Singh, S. P., E. G. Barrett, R. Kalra, S. Razani-Boroujerdi, R. J. Langley, Acknowledgments V. Kurup, Y. Tesfaigzi, and M. L. Sopori. 2003. Prenatal cigarette smoke de- We express our deep appreciation to Dr. Ted Barrett for his assistance in creases lung cAMP and increases airway hyperresponsiveness. Am. J. Respir. Crit. Care Med. 168: 342–347. developing animal protocols for these studies, Steve Randock for help with 27. Kurup, V. P., S. Mauze, H. Choi, B. W. Seymour, and R. L. Coffman. 1992. A graphics, and Paula Bradley for editorial help. murine model of allergic bronchopulmonary aspergillosis with elevated eosinophils and IgE. J. Immunol. 148: 3783–3788. 28. Barrett, E. G., J. Wilder, T. H. March, T. Espindola, and D. E. Bice. 2002. Disclosures Cigarette smoke-induced airway hyperresponsiveness is not dependent on ele- The authors have no financial conflict of interest. vated immunoglobulin and eosinophilic inflammation in a mouse model of allergic airway disease. Am. J. Respir. Crit. Care Med. 165: 1410–1418. 29. Kurup, V. P. 2005. Aspergillus antigens: which are important? Med. Mycol. 43(Suppl. 1): S189–S196. References 30. Brummund, W., A. Resnick, J. N. Fink, and V. P. Kurup. 1987. Aspergillus 1. Sopori, M. 2002. Effects of cigarette smoke on the immune system. Nat. Rev. fumigatus-specific antibodies in allergic bronchopulmonary aspergillosis and as- Immunol. 2: 372–377. pergilloma: evidence for a polyclonal antibody response. J. Clin. Microbiol. 2. DiFranza, J. F., A. Aligne, and M. Weitzman. 2004. Prenatal and postnatal en- 25: 5–9. vironmental tobacco smoke exposure and children’s health. Pediatrics 113: 31. Levallet, G., J. Levallet, H. Bouraima-Lelong, and P. J. Bonnamy. 2007. Expres- 1007–1015. sion of cAMP-phosphodiesterase PDE4D isoforms and age-related changes in The Journal of Immunology 2121

follicle-stimulating hormone-stimulated activities in immature rat sertoli cells. 47. Hansen, G., S. Jin, D. T. Umetsu, and M. Conti. 2000. Absence of muscarinic Biol. Reprod. 76: 794–803. cholinergic airway responses in mice deficient in the cyclic nucleotide phospho- 32. Weitzman, M., S. Gortmaker, and A. Sobol. 1990. Racial, social, and environ- diesterase PDE4D. Proc. Natl. Acad. Sci. USA 97: 6751–6756. mental risks for childhood asthma. Am. J. Dis. Child. 144: 1189–1194. 48. Deng, Y. M., Q. M. Xie, H. F. Tang, J. G. Sun, J. F. Deng, J. Q. Chen, and 33. Lundblad, L. K., C. G. Irvin, A. Adler, and J. H. Bates. 2002. A reevaluation of S. Y. Yang. 2006. Effects of ciclamilast, a new PDE 4 PDE4 inhibitor, on airway the validity of unrestrained plethysmography in mice. J. Appl. Physiol. 93: hyperresponsiveness, PDE4D expression and airway inflammation in a murine 1198–1207. model of asthma. Eur. J. Pharmacol. 547: 125–135. 34. Hauk, P. J., M. Krawiec, J. Murphy, J. Boguniewicz, A. Schiltz, E. Goleva, 49. Wollin, L., D. S. Bundschuh, A. Wohlsen, D. Marx, and R. Beume. 2006. Inhi- A. H. Liu, and D. Y. Leung. 2008. Neutrophilic airway inflammation and asso- bition of airway hyperresponsiveness and pulmonary inflammation by roflumilast ciation with bacterial lipopolysaccharide in children with asthma and wheezing. and other PDE4 inhibitors. Pulm. Pharmacol. Ther. 19: 343–352. Pediatr. Pulmonol. 43: 916–923. 35. Jones, C. A., and P. G. Holt. 2000. Immunopathology of allergy and asthma in 50. Barnes, P. J., I. M. Adcock, and K. Ito. 2005. Histone acetylation and deacety- childhood. Am. J. Respir. Crit. Care Med. 162: S36–S39. lation: importance in inflammatory lung diseases. Eur. Respir. J. 25: 552–563. 36. Poulsen, L. K., and L. Hummelshoj. 2007. Triggers of IgE class switching and 51. Adcock, I. M., K. Ito, and P. J. Barnes. 2005. Histone deacetylation: an important allergy development. Ann. Med. 39: 440–456. mechanism in inflammatory lung diseases. COPD 2: 445–455. 37. Kurup, V. P., and G. Grunig. 2002. Animal models of allergic bronchopulmonary 52. Lynch, M. J., G. S. Baillie, and M. D. Houslay. 2007. cAMP-specific phospho- aspergillosis. Mycopathologia 153: 165–177. diesterase-4D5 (PDE4D5) provides a paradigm for understanding the unique non- 38. Gosens, R., J. Zaagsma, H. Meurs, and A. J. Halayko. 2006. Muscarinic receptor redundant roles that PDE4 isoforms play in shaping compartmentalized cAMP signaling in the pathophysiology of asthma and COPD. Respir. Res. 7: 73–88. cell signalling. Biochem. Soc. Trans. 35: 938–941. 39. Struckmann, N., S. Schwering, S. Wiegand, A. Gschnell, M. Yamada, 53. Houslay, M. D., P. Schafer, and K. Y. Zhang. 2005. Keynote review: phospho- W. Kummer, J. Wess, and R. V. Haberberger. 2003. Role of muscarinic receptor diesterase-4 as a therapeutic target. Drug Discov. Today 10: 1503–1519. subtypes in the constriction of peripheral airways: studies on receptor-deficient 54. Mehats, C., S.-L. C. Jin, J. Wahlstrom, E. Law, D. L. Umetsu, and M. Conti. mice. Mol. Pharmacol. 64: 1444–1451. 2003. PDE4D plays a critical role in the control of airway smooth muscle con- 40. Floreani, A. A., and S. I. Rennard. 1999. The role of cigarette smoke in the traction. FASEB J. 17: 1831–1841. pathogenesis of asthma and as a trigger for acute symptoms. Curr. Opin. Pulm. Med. 5: 38–46. 55. Caulfield, M. P., and N. J. M. Birdsall. 1998. International Union of Pharmacol- 41. Seymour, B. W., J. L. Peake, K. E. Pinkerton, V. P. Kurup, and L. J. Gershwin. ogy. XVII. Classification of muscaranic acetylcholine receptors. Pharmacol. Rev. 2005. Second-hand smoke increases nitric oxide and alters the IgE response in a 50: 279–290. murine model of allergic aspergillosis. Clin. Dev. Immunol. 12: 113–124. 56. Hislop, A. A., J. C. Mak, J. A. Reader, P. J. Barnes, and S. G. Haworth. 1998. 42. Finch, G. L., K. J. Nikula, B. T. Chen, E. B. Barr, I. Y. Chang, and C. H. Hobbs. Muscaranic receptor subtype in the porcine lung during postnatal development. 1995. Effect of chronic cigarette smoke exposure on lung clearance of tracer Eur. J. Pharmacol. 359: 211–221. particles inhaled by rats. Fundam. Appl. Toxicol. 24: 76–85. 57. Nagai, T., D. J. Kim, R. J. Delay, and S. D. Roper. 1996. Neuromodulation of 43. Giembycz, M. A. 2008. Can the anti-inflammatory potential of PDE4 inhibitors transduction and signal processing in the end organs of taste. Chem. Senses 21: be realized: guarded optimism or wishful thinking? Br. J. Pharmacol. 155: 353–365. 288–290. 58. Kishimoto, T. 2006. Interleukin-6: discovery of a pleiotropic cytokine. Arthritis 44. Spina, D. 2008. PDE4 inhibitors: current status. Br. J. Pharmacol. 155: 308–315. Res. Ther. 8(Suppl. 2): S2. 45. Grootendorst, D. C., S. A. Gauw, N. Benschop, P. J. Sterk, P. S. Hiemstra, and K. F. Rabe. 2003. Efficacy of the novel phosphodiesterase-4 inhibitor BAY 19- 59. Foster, P. S., H. F. Rosenberg, K. L. Asquith, and R. K. Kumar. 2008. Targeting Curr. Mol. Med. 8004 on lung function and airway inflammation in asthma and chronic obstructive eosinophils in asthma. 8: 585–590. pulmonary disease (COPD). Pulm. Pharmacol. Ther. 16: 341–347. 60. Conroy, D. M., and T. J. Williams. 2001. Eotaxin and the attraction of eosinophils 46. Field, S. K. 2008. Roflumilast: an oral, once-daily selective PDE-4 inhibitor for to the asthmatic lung. Respir. Res. 2: 150–156. the management of COPD and asthma. Expert Opin. Investig. Drugs 17: 61. Van der Maarel, S. M. 2008. Epigenetic mechanisms in health and disease. Ann. 811–818. Rheum. Dis. 67(Suppl. 3): iii97–100.