Transgenic smooth muscle expression of the human CysLT1 receptor induces enhanced responsiveness of murine airways to leukotriene D4

Guochang Yang,1 Angela Haczku,2 Hang Chen,2 Viviane Martin,1 Helen Galczenski,3 Yaniv Tomer,2 Christopher R. Van Beisen,2 Jilly F. Evans,3 Reynold A. Panettieri,2 and Colin D. Funk1

1Center for Experimental Therapeutics, Department of Pharmacology, 2Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104; and 3Department of Pharmacology, Merck Research Laboratories, West Point, Pennsylvania 19486

Submitted 27 October 2003 ; accepted in final form 15 December 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cysteinyl leukotrienes (CysLTs) exert potent proinflammatory actions and contribute to many of the symptoms of asthma. Using a model of allergic sensitization and airway challenge with Aspergillus fumigatus (Af), we have found that Th2-type inflammation and airway hyperresponsiveness (AHR) to methacholine (MCh) were associated with increased LTD4 responsiveness in mice. To explore the importance of increased CysLT signaling in airway smooth muscle function, we generated transgenic mice that overexpress the human CysLT1 receptor (hCysLT1R) via the {alpha}-actin promoter. These receptors were expressed abundantly and induced intracellular calcium mobilization in airway smooth muscle cells from transgenic mice. Force generation in tracheal ring preparations ex vivo and airway reactivity in vivo in response to LTD4 were greatly amplified in hCysLT1R-overexpressing mice, indicating that the enhanced signaling induces coordinated functional changes of the intact airway smooth muscle. The increase of AHR imposed by overexpression of the hCysLT1R was greater in transgenic BALB/c mice than in transgenic B6 x SJL mice. In addition, sensitization- and challenge-induced increases in airway responsiveness were significantly greater in transgenic mice than that of nontransgenic mice compared with their respective nonsensitized controls. The amplified AHR in sensitized transgenic mice was not due to an enhanced airway inflammation and was not associated with similar enhancement in MCh responsiveness. These results indicate that a selective hCysLT1R-induced contractile mechanism synergizes with allergic AHR. We speculate that hCysLT1R signaling contributes to a hypercontractile state of the airway smooth muscle.

asthma; allergic airway inflammation


ASTHMA, A SYNDROME CHARACTERIZED by recurrent respiratory symptoms, such as cough, wheezing, and chest tightness, is associated with airway hyperresponsiveness (AHR), inflammation, and remodeling (10, 29). Many different inflammatory cells and mediators are involved in asthma (3, 23), among which are the cysteinyl leukotrienes (CysLTs), comprising leukotriene (LT) C4, LTD4, and LTE4, derived from eosinophils and mast cells. These lipid mediators are chiefly implicated in bronchoconstriction and chronic airway inflammation in asthma. CysLTs promote airway edema, bronchial smooth muscle contraction and proliferation, enhanced mucus secretion, and eosinophil recruitment (8, 9, 22, 30). Of the two known and characterized CysLT receptor subtypes, the human CysLT1 receptor (hCysLT1R) is responsible for the preponderance of CysLT airway actions and is the site of action of three antileukotriene drugs that have been shown to be clinically efficacious in chronic asthma, namely montelukast (Singulair), zafirlukast (Accolate), and pranlukast (Onon) (9, 14, 30, 39, 41).

Mouse models are commonly used to study mechanisms of allergic airway inflammation due, in part, to the vast resources of induced mutant strains that can implicate specific gene products in pathogenesis (28, 31). Previously, transgenic mice overexpressing the LTB4 receptor (B-LT1) in leukocytes have been generated to investigate LTB4-enhanced responses in inflammation (5). Leukotrienes are involved in the eosinophil recruitment, mucus secretion, and nonspecific bronchoconstrictor responses in mice in addition to more chronic remodeling changes (11, 21, 22, 26). However, mice are refractory to direct leukotriene challenge in terms of bronchoconstrictor responses compared with guinea pigs (1, 35), and this limits their usefulness with respect to human asthma. To overcome this difficulty we have overexpressed the human CysLT1 receptor in murine airway smooth muscle with a view to enhance leukotriene responses and to explore coupling and signaling of this human G protein-coupled receptor (GPCR) with endogenous signaling components.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Transgenic mice. Smooth muscle-specific expression of the hCysLT1R in mice was achieved with the mouse smooth muscle {alpha}-actin promoter (13). A 1.1-kb fragment encoding the hCysLT1R, generated by PCR from genomic DNA, was subcloned into the plasmid SMP8-pBLCAT3 (13, 42) downstream of a 3.6-kb mouse vascular smooth muscle {alpha}-actin promoter fragment with exon 1, first intron, and part of exon 2, termed SMP8 (gift from Arthur Strauch, Ohio State University). The SV40 early polyadenylation signal sequence was subcloned 3' to the hCysLT1R coding region. Orientation of each fragment was confirmed by sequence analysis and restriction enzyme digestion. A 4.8-kb fragment was excised from the plasmid by AatII and BspEI digestion, purified, and microinjected into male pronuclei of fertilized zygotes from superovulated B6 x SJL F1 mice for subsequent implantation into pseudopregnant recipients at the University of Pennsylvania Transgenic & Chimeric Mouse Facility. Founder mice expressing the transgene were mated with nontransgenic B6 x SJL F1 mice. Hemizygous transgene-positive progeny were identified by Southern blot analysis of genomic DNA derived from tail clips. Hemizygous B6 x SJL mice from subsequent generations between the ages of 10 and 14 wk from two to three separate founder lines were used for all studies. One line of mice was backcrossed seven times to the BALB/c genetic background and tested for LTD4 responses (see RESULTS). Experiments using animals have been approved by the animal care and use committee at the University of Pennsylvania.

RT-PCR. Total RNA was prepared from various mouse tissues using TRIzol reagent (Life Technologies). Total RNA (2 µg) was converted to cDNA by reverse transcription (RT, GIBCO protocol) in a total volume of 20 µl. PCR was performed in a final volume of 50 µl containing 2 µl of RT reaction product. Samples were placed in a thermal cycler (Perkin-Elmer, Norwalk, CT) for 30 cycles consisting of 2 min of denaturation at 94°C, 45 s of annealing at 58°C, and 1 min of extension at 72°C, followed by a final 7-min extension at 72°C. hCysLT1R (557 bp) was amplified with receptor gene-specific primers, downstream primer, 5'-AAACTATACTTTACATATTTCTTCTCC-3' oligonucleotide (+1,017 to +991; +1 corresponds to the A nucleotide of the AUG start codon), and upstream primer, 5'-CCAGTTCTCCATTTCTAATGGC-3' oligonucleotide (+461 to 482), respectively. Samples were subjected to parallel amplification of the constitutively expressed, mouse {beta}-actin gene using the following primers: 5'-GTGACGAGGCCCAGAGCAAGAG-3' as sense and 5'-AGGGGCCGGACTCATCGTACTC-3' as antisense. A 10-µl aliquot was electrophoresed in 0.7% agarose and stained with ethidium bromide. No PCR products were obtained when reverse transcriptase was omitted.

Ribonuclease protection assays. The hCysLT1R lung expression was analyzed by ribonuclease protection assay using an RPA III kit (Ambion). A [{alpha}-32P]UTP (800 Ci/mmol, Perkin-Elmer Life Sciences)-labeled antisense RNA probe corresponding to the 5'-end 335 bp of hCysLT1R open reading frame plus a 64-bp adapter sequence was synthesized by in vitro transcription with SP6 RNA polymerase (Ambion) in the presence of ribonuclease inhibitor at 37°C for 1 h. DNase I (Ambion) was added after transcription, and the mixture was incubated at 37°C for 30 min to remove template. The 399-bp labeled probe was gel purified. Total RNA (10 µg) from mouse lungs was mixed with 7x104 cpm probe and coprecipitated with NH4OAc and ethanol. The precipitated pellets were resuspended in hybridization buffer and incubated at 42°C overnight. A mixture of RNase A/RNase T1 diluted in digestion buffer was added to hybridization tubes and incubated for 30 min at 37°C to yield a 335-bp protected fragment. RNase was inactivated, and samples were precipitated and resuspended in gel loading buffer II, denatured, and electrophoresed in a 5% denaturing polyacrylamide urea gel. Radiolabeled probes were detected by exposure to X-ray film. Yeast tRNA served as a negative control.

Fluorescence in situ hybridization. To localize transgene expression within the lung, we performed in situ hybridization on lung sections (12). Briefly, frozen mouse lungs were embedded in optimal cutting temperature solution, sectioned at 6 µm, thaw-mounted onto Superfrost Plus slides, and fixed for 30 min in 4% paraformaldehyde. Oligonucleotide antisense and sense CysLT1R mRNA probes were prepared and biotin-labeled (synthesized by University of Pennsylvania Nucleic Acid Facility): 5'-AGATACTGTCAGATTTCCTGTTTCATCCAT-3' and 5'-ATGATTTTTAGTTTGATTGTCTTGTGGGGG-3', and sense probe 5'-ATGGATGAAACAGGAAATCTGACAGTATCT-3' and 5'-CCCCCACAAGACAATCAAACTAAAAATCAT-3'.

We detected bound probe with tyramide signal amplification-direct red fluorescence using the in situ hybridization tyramide amplification kit (NEN Life Sciences, Boston, MA) according to the manufacturer's instructions. Mixed oligonucleotide probes were used that limit detection to that of the transgene only.

Airway smooth muscle cell cultures. Tracheal smooth muscle cells (TSMCs) were cultured from explants of excised tracheae exactly as described (37). The explants were incubated at 37°C in a humidified environment of 95% air-5% CO2 and passaged at a 1:4 ratio when confluence was reached. The authenticity of the smooth muscle cell cultures was determined by immunohistochemistry with {alpha}-smooth muscle actin staining with at least 90% of the cells positive. The radioligand binding and Ca2+ assays were performed on confluent cells at matched passage numbers.

Radioligand binding studies. Cultured TSMC membranes were prepared as described previously for transfected cells (24). Briefly, detached smooth muscle cells from primary cultures were harvested from passages 5–6. The cells were homogenized in cold buffer A (10 mM HEPES, 2 mM EDTA, and 0.37 mg/ml protease inhibitor mixture, pH 7.4) with a Dounce B homogenizer, followed by nitrogen cavitation at 1,100–1,300 psi for 15 min. The cell homogenate was centrifuged at 10,000 g, and the supernatant was recentrifuged at 100,000 g for 30 min. The pellet was homogenized with a Dounce A homogenizer and suspended in buffer A. For determination of expression, radioligand binding was carried out with 0.5 nM [3H]LTD4 in buffer containing 10 mM HEPES and 20 mM CaCl2, pH 7.4, in the presence of 20 mM L-penicillamine at room temperature for 1 h. Binding was stopped with cold wash buffer (10 mM HEPES and 0.01% bovine serum albumin, pH 8.0), and the bound radioactivity was separated by filtration and washing over Whatman GF/B filters. Radioactivity was quantitated by liquid scintillation counting. Specific binding was determined by subtracting nonspecific binding in the presence of 1 µM LTD4 from total binding.

Measurement of agonist-induced intracellular calcium mobilization. Cultured TSMCs were plated onto poly-D-lysine-treated black-coated microplates (Biocoat) at 5 x 104 cells/well. Twenty-four hours later the cells were loaded with Fluo-4 calcium indicator dye (Molecular Probes) in the presence of 2.5 mM probenecid. After washing them three times with Hanks' balanced salt solution containing 20 mM HEPES and 2.5 mM probenecid, we treated the cells with various concentrations of LTC4 or LTD4 (Cayman Chemical); maximum fluorescence indicating the changes in intracellular calcium concentrations was measured in a Molecular Devices Fluorometric Imaging Plate Reader. Data were analyzed by nonlinear regression with PRISM software (GraphPad, San Diego, CA).

Ex vivo smooth muscle studies. Contractility experiments were carried out with mouse tracheal strips using techniques as described (38). Briefly, tracheae were excised and dissected free of surrounding tissues, cut into rings of ~3–4 mm in length, placed in 10-ml water-jacketed organ baths containing Krebs-Henseleit solution, and connected via silk suture to Grass FT03C force-displacement transducers; changes in ring tension were measured. Mechanical responses were recorded isometrically using the MP100WS/Acknowledge data acquisition system (BIOPAC Systems, Goleta, CA). All initial tensions were set to ~0.5 g and maintained for 1 h, after which a steady-state tension level was attained. The composition of the Krebs-Henseleit solution, which was gassed with 95% O2 and 5% CO2 and maintained at 37°C, was (in mM): 113.0 NaCl, 4.8 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 25.0 NaHCO3, and 5.5 glucose. After the equilibration period and before construction of LTD4 concentration-response curves, tissues were exposed to 10 µM carbachol to test for tissue viability. After plateau of this contraction, tissues were washed several times over 30–60 min until the tension returned to baseline level. The preparations were then left for at least 30 min before the start of the experiment. LTD4 concentration-response curves (2 nM–2 µM) were generated. At the end of the experiment, tissues were exposed again to 10 µM carbachol, and agonist-induced responses in each tissue were expressed as a percentage of this reference contraction ("% postcarbachol maximal"). Concentration-response relations were fitted to a logistic equation.

Allergenic sensitization and challenge with Aspergillus fumigatus. Mice were sensitized by intraperitoneal injections with 20 µg of Aspergillus fumigatus (Af; Bayer Pharmaceuticals, Elkhart, IN) together with 20 mg of alum (Imject Alum; Pierce, Rockford, IL) in 100-µl volume on days 0 and 14, followed by intranasal challenge on days 25, 26, and 27 with 25 µl of Af extract in PBS (12.5 µg in 21% glycerol/PBS). Intranasal treatment was carried out essentially as described previously (27). Briefly, sensitized and control mice were anesthetized by isoflurane inhalation, and 25 µl of Af extract or vehicle were applied to the left nare, respectively. The studies were performed 24 h after the third intranasal treatment. Naïve mice that received intranasal glycerol treatment alone showed no difference from nonsensitized, nonexposed normal mice in any of the study parameters that we investigated, including lung histology, bronchoalveolar lavage (BAL) cellular content, immunoglobulin and cytokine profile, and airway responses to acetylcholine (not shown).

In vivo airway physiology. Airway responsiveness to methacholine (MCh) was measured noninvasively in conscious, unrestrained mice with a whole body plethysmograph (Buxco Electronics, Troy, NY) (16, 20) and the dimensionless parameter enhanced pause (Penh). Penh reflects changes in the wave form of the box pressure signal from both inspiration and expiration and combines it with the timing comparison of early and late expiration (pause). Penh = (Te/Tr - 1) x PEF/PIF, where Te is expiratory time (the time from the end of inspiration to the start of the next inspiration), Tr is relaxation time [the time of pressure decay to 36% of the total expiratory pressure signal (area under the box pressure signal in expiration)], PEF is peak expiratory flow (ml/s), and PIF is peak inspiratory flow (ml/s). Increases in Penh are related to an increase in pulmonary resistance (20). MCh dose responses were generated after baseline readings were taken and saline inhalation was performed. Increasing concentrations of MCh, between 6.25 and 50 mg/ml, were aerosolized for 1 min each, and responses were measured and averaged over a 4-min period. The degree of bronchoconstriction was expressed as the percentage change in Penh relative to the baseline.

Invasive airway function measurements of lung resistance (RL) and dynamic compliance (Cdyn) were carried out as previously described (17). Briefly, mice were anesthetized, cannulated, and ventilated (140 breaths/min, 0.2-ml tidal volume) following administration of pancuronium bromide (1.0 mg/kg). Transduced alveolar pressure and airflow rate (Validyne DP45 and DP103) were used to calculate RL and Cdyn by a computer (Buxco Electronics). Baseline RL values were established, and after administration of saline LTD4 was given intravenously at concentrations ranging from 8 to 128 µg/kg in five increments.

BAL analysis for total and differential cell count and cytokine content. Lungs were lavaged with sterile saline (1 ml, three times), and total and differential cell counts were performed as described previously (1618). To remove cells the BAL samples were centrifuged at 400 g for 10 min at 4°C. Cytokine levels in cell free supernatants of the BAL and serum IgE levels were determined by ELISA using antibodies, recombinant cytokines, and standard IgE from PharMingen (San Diego, CA) or Immuno-Biological Labs (Gunma, Japan) for KC (mouse homolog of human GRO/MGSA).

Lung tissue histology. After lavage, lungs were inflated with 0.5 ml of paraformaldehyde (4% with sodium cacodylate 0.1 M, pH 7.3) and were fixed in the same solution for histological analysis. Paraffin sections prepared from the lungs of naïve and sensitized mice were stained with hematoxylin and eosin for evaluation of airway inflammation or with Congo red (Sigma Diagnostics) specifically for eosinophils (44) and with periodic acid-Schiff (PAS) for mucus secretion (15).

Data analysis. Data were expressed as means ± SE. Time courses and dose responses were compared by ANOVA. To test differences between individual groups, we performed Student's t-test assuming equal variances. Correlations were investigated by regression analysis. A P value of <0.05 was considered as significant. Data were analyzed with the Sigmastat standard statistical package (Jandel Scientific).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
LTD4 responsiveness is significantly increased in mice sensitized and challenged with Af. The protocol for sensitization (Fig. 1A) was selected on the basis of the known parameters of the human allergic response and our previous experience with acute models of locally elicited inflammation in mice (1619). Because leukocyte trafficking characterizes airway inflammation in asthma, the numbers of inflammatory cells recovered from the BAL fluid were assessed following sensitization and challenge with Af (Fig. 1, B and C). Af challenge induced a predominantly perivascular and peribronchial inflammatory infiltrate. Mucus secretion and goblet cell hyperplasia in the sensitized and challenged mice are shown by positive PAS staining (Fig. 1B, bottom right). Naïve mice were free of cellular infiltration and mucus (Fig. 1, B left panels and C). A significant influx of eosinophils (P < 0.01) and, to a lesser extent, neutrophils and lymphocytes (P < 0.05) trafficked into the airways of mice sensitized and challenged with Af (Fig. 1C).



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Fig. 1. Sensitization and challenge with Aspergillus fumigatus (Af) induce airway hyperresponsiveness to leukotriene (LT) D4. A: protocol for allergic sensitization and challenge. Mice were sensitized and boosted on days 0 and 14 with Af extract and alum as described in MATERIALS AND METHODS. Groups received intranasal Af challenges on days 25, 26, and 27, and mice were killed 24 h after the last Af challenge. B: representative paraffin-embedded, Congo red-stained (top) and periodic acid-Schiff (PAS)-stained (bottom) lung sections prepared from the left lung showing airways of naïve mice (left) and mice sensitized and challenged with Af (right). Sections were evaluated under light microscopy (n = 5). C: absolute numbers of bronchoalveolar lavage (BAL) leukocytes were derived from counts in modified Wright's-stained (Kwik-Diff) cytospin preparations and the total cell number in each sample (n = 17–36). MP, macrophages; EP, eosinophils; NP, neutrophils; LC, lymphocytes. Cytokine levels in BAL fluid (n = 12–14, D) IgE levels in serum (n = 3–6, E) were determined by ELISA. RANTES, regulated on activation normal T-expressed and presumably secreted. Baseline enhanced pause (Penh, F) and time of expiration measurements (G) were carried out in conscious mice 24 h after their last allergen challenge (n = 14–22). Methacholine (MCh, H) and LTD4 responsiveness (I) were measured using increasing doses of inhaled MCh (n = 17–22) or LTD4 (n = 6), respectively. Provocative concentration (PC) values were calculated using the dose-response curve and represent the PC that achieves 50, 100, or 200% change above the baseline values (PC50, PC100, PC200, respectively). Data are expressed as means ± SE for all panels. *P < 0.05; **P < 0.01 Af vs. naïve mice (N).

 

The underlying mediators of the allergic airway inflammation in the cell-free supernatants of the BAL fluid were investigated by assessment of the levels of TNF-{alpha}, IFN-{gamma}, IL-13, and chemokines such as KC (cytokine-induced neutrophil chemoattractant, a murine homolog of human IL-8) and regulated on activation normal T-expressed and presumably secreted (RANTES) (Fig. 1D). Sensitization and challenge with Af increased levels of IL-13, KC, and RANTES. In contrast, IFN-{gamma} and TNF-{alpha} were significantly decreased in the BAL fluid following sensitization and challenge, indicating a predominance of a T helper (Th) 2 cytokine profile. (Fig. 1D). Serum IgE levels were significantly increased upon sensitization and challenge with the allergen (144 ± 54 ng/ml naïve, n = 6 vs. 8,879 ± 2,018 ng/ml sensitized, n = 3) (Fig. 1E). Animals sensitized with Af antigen but challenged with vehicle (glycerol) showed no significant alterations in their inflammatory cell profile in the lung (not shown) or the BAL fluid (Fig. 1C, hatched bars) when compared with naïve, nonsensitized mice (Fig. 1, B left panels and C open bars) and did not develop changes in their cytokine levels (data not shown).

The functional consequences of allergic airway inflammation elicited by sensitization and exposure to Af were investigated by lung function measurements performed both at baseline and following challenges with various concentrations of inhaled MCh. Baseline Penh measurements showed highly significant increases (P < 0.01) after challenge of sensitized mice (n = 22) when compared with naïve, nonsensitized controls (n = 14, Fig. 1F). In addition to the Penh values, an indicator of small airway obstruction, the Te of these mice was also monitored. There were significant increases (P < 0.01) after challenge of sensitized mice when compared with naïve, nonsensitized controls (Fig. 1G). These results corroborated the presence of airway obstruction in the sensitized and challenged animals. We assessed airway responsiveness to nonspecific stimuli (MCh and LTD4; Fig. 1, H and I, respectively) by calculating the provocative concentrations (PC) that were necessary to achieve 50, 100, or 200% increase above the baseline Penh value (PC50, PC100, and PC200, respectively). Sensitized and challenged mice exhibited airway hyperresponsiveness to both inhaled MCh and LTD4 when compared with nonsensitized controls (P < 0.05 for PC100 and P < 0.01 for PC200). Thus whereas normal mice show relatively low responsiveness of airways to leukotrienes, mice that were sensitized and challenged with Af demonstrate markedly amplified Penh values to inhaled LTD4.

Identification and characterization of expression levels in transgenic mice. To characterize the mechanisms of action of CysLTs on airway smooth muscle function, we generated hCysLT1R transgenic mice. Eight hCysLT1R founder mice were identified from a total of 45 mice screened. Subsequent matings with nontransgenic mice showed that five of eight founders were mosaic, and three of these died prematurely. Three founder mice lines were established (denoted as lines 1, 4, and 31), and the transgene was inherited in ~50% of the offspring with equal distribution between both sexes. Hemizygous mice from lines 1 and 31 (and in some cases line 4) were further characterized with respect to transgene copy number, mRNA expression, receptor density, functional signaling, and histological analysis. Southern blot analysis of tail-clip genomic DNA along with titrated known quantities of transgenic construct revealed that the transgene copy number was about four to five copies for lines 1 and 31 and ~10 copies for line 4 (Fig. 2A). Mice from each line appeared overtly normal and healthy. To confirm that the hCysLT1R transgene was expressed in smooth muscle-rich tissues of positive mice, total RNA was prepared from a variety of tissues of transgenic animals and subjected to semiquantitative RT-PCR. As shown in Fig. 2B, a 557-bp fragment corresponding to human CysLT1R mRNA was present in the smooth muscle-rich tissues of stomach, bladder, intestine, uterus, and lung but not detectable in heart and skeletal muscle. In a specific test designed to discriminate against any native mouse CysLT1R expression, ribonuclease protection assay with RNA from total lung using an antisense RNA probe corresponding to the 5'-end 335 bp of hCysLT1R open reading frame (45 mismatches and 6-bp deletion compared with mouse sequence in corresponding region) indicated a protected hCysLT1R band was present in lungs from transgenic mice but not from nontransgenic littermates (Fig. 2C). Quantitation of band density showed there was no significant difference in transgene expression between lines 1 and 31.



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Fig. 2. Generation of human cysteinyl leukotriene-1 receptor (hCysLT1R) mice. A: assessment of transgene copy number. Genomic DNA samples (10 µg) from the 3 founder lines designated TG1, TG4, and TG31 were electrophoresed adjacent to titrated amounts of transgene plasmid and hybridized with a [32P]hCysLT1R-labeled probe. B: total RNA was prepared from the indicated tissues and subjected to RT-PCR analysis with hCysLT1R-specific and mouse {beta}-actin primers for the 557- and 939-bp products, respectively. C: ribonuclease protection assay detects hCysLT1R expression in lung of transgenic (TG) lines of mice TG1 and TG31 but not nontransgenic littermates (NTG). Mouse {beta}-actin served as control.

 

Fluorescence in situ hybridization was performed to verify that expression of the transgene was directed to airway and/or vascular smooth muscle of lungs. Using species-specific oligonucleotide antisense probe labeling with biotin, we selectively detected hCysLT1R mRNA in peribronchiolar regions and along small blood vessels (Fig. 3) in the transgenic mice but not in nontransgenic littermates. These data plainly indicate that expression of the hCysLT1R transcript was limited to airway smooth muscle and vascular smooth muscle in lungs.



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Fig. 3. Fluorescence in situ hybridization detection of hCysLT1R expression in smooth muscle of TG mouse airways (A) and vasculature (B) but not in NTG mouse airway (C). Hybridization with anti-sense (A–C) and sense control (D) probes was carried out with tyramide signal amplification-direct red fluorescence using tyramide amplification as described in MATERIALS AND METHODS. Nuclei were stained blue with 4',6'-diamidino-2-phenylindole.

 

Functional characterization of CysLT1R in TSMCs. hCysLT1R expression was quantitated next by radioligand binding assays with [3H]LTD4. Because the fluorescence in situ hybridization experiments indicated that most hCysLT1Rs localized to smooth muscle cells, a minor cell type of the whole lung parenchyma, we switched to [3H]LTD4 binding in smooth muscle cells cultured from tracheal explants of which >90% of these cultured cells had the morphologic characteristics of smooth muscle cells and stained positive for smooth muscle {alpha}-actin. Saturation radioligand binding indicated that CysLT1R was notably overexpressed in TSMCs from transgenic mice (Fig. 4A). hCysLT1R density in transgenic lines 31 and 1 was 37 and 26 times greater than that of cells from nontransgenic mice (3,492 and 2,505 vs. 94 dpm/mg protein, respectively, n = 3, P < 0.001) with 1 µM unlabeled LTD4 used to define nonspecific binding.



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Fig. 4. hCysLT1R overexpression on tracheal smooth muscle cells from transgenic lines leads to enhanced Ca2+ signaling upon leukotriene addition. A: specific radioligand binding of [3H]LTD4 (0.5 nM) to membranes prepared from NTG and TG lines 31 and 1 subtracted for nonspecific binding in the presence of cold LTD4 (1 µM); n = 3. B–E: dose-response curves (B, C) and time course (D, E) for changes in fluorescence signal upon LTD4 (B, D) and LTC4 (C, E) addition to Fluo-4-loaded tracheal smooth muscle cells. D and E are representative tracings (n = 3).

 

We investigated whether overexpression of hCysLT1R was associated with an augmentation of the biological responsiveness to LTD4 and LTC4. Receptor function was evaluated by fluorescence assay in Fluo-4-loaded TSMC upon agonist activation. TSMCs from nontransgenic mice showed a small response to LTD4 and LTC4, consistent with their basal expression of mouse CysLT1R (Fig. 4, B–E). TSMCs from lines 1 and 31 responded to LTD4 and LTC4 with marked, dose-dependent elevations of intracellular calcium (Fig. 4, B–E). The enhanced responsiveness of TSMCs from transgenic mice to LTD4 and LTC4 was associated with an increased maximal response. The increase was rapid in onset, reaching maximum levels within 10 s, and decayed back to baseline levels in 60–90 s (Fig. 4, D and E). LTD4 was more potent than LTC4, which is consistent with the known pharmacological properties of this receptor (33). TSMCs of transgenic and nontransgenic mice responded weakly to LTB4, with no differences between groups (data not shown).

Functional responses to LTD4 in tracheal rings and in vivo. To assess whether the amplified signaling observed in transgenic TSMC results in regulation of coordinated smooth muscle function of the airway, we first measured responses to LTD4 ex vivo using tracheal ring preparations. The constriction response to carbachol was equivalent between rings derived from each group, indicating no inherent contractile differences to muscarinic agonist challenge (Fig. 5A). The force generation in response to LTD4 was found to be markedly enhanced in tracheal rings of transgenic mice (Fig. 5B, n = 6, P < 0.05), whereas the EC50 values were within the same range between transgenic and nontransgenic rings (15–25 nM).



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Fig. 5. Overexpression of hCysLT1R enhances force generation in tracheal rings upon LTD4 administration and increases airway resistance to intravenous LTD4 in vivo. A: tracheal ring preparations from TG and NTG mice were tested for force generation in response to carbachol (10 µM) from lines 31 and 1. Tension responsiveness (mg tension/mg weight) was expressed as relative change from the mean response (230.0 ± 18.1) of NTG line 31. Results represent means ± SE of n = 6 preparations in each group. B: increasing concentrations of LTD4 were added to the tracheal ring preparation as described and the g tension responsiveness was expressed as relative change from the mean response of the NTG line 31 to 10 µM carbachol. Results represent means ± SE of n = 6 preparations in each group. C: anesthetized, intubated mice from line 31 were investigated for their lung resistance (RL) in response to increasing concentrations of intravenously administered LTD4 as described. Baseline RL values in the NTG and TG groups were 1.484 ± 0.053 and 1.462 ± 0.014 cmH2O·ml-1·s-1, respectively (n = 6 in each group).

 

Because transgenic mice overexpressing hCysLT1R in smooth muscle have enhanced signaling in isolated cells and tracheal rings, we sought to determine whether this resulted in altered physiological function of the airways in vivo. Experiments were carried out in intubated, anesthetized mice in response to intravenous LTD4 administration. Enhanced airway resistance (RL) to LTD4 was observed at all tested doses in the transgenic mice vs. nontransgenic littermates (Fig. 5C; ANOVA, P < 0.001, n = 6 in each group, at each dose) with no signs of anaphylaxis.

LTD4 responsiveness is significantly increased in BALB/c mice overexpressing hCysLT1R. C57BL/6 and SJL strains of mice are relatively hyporesponsive to nonspecific airway stimuli and resistant to development of allergic AHR compared with the hyperresponsive BALB/c mouse strain (32, 43). To investigate the effects of hCysLT1R overexpression on LTD4 responsiveness in these different strains, one line of the C57BL/6 x SJL F1 mice was backcrossed to the BALB/c genetic background for seven generations and tested for responses to inhaled LTD4. Results indicate that the LTD4 PC50 of the C57BL/6 x SJL mice was 8.8 ± 0.8 µM, whereas in the BALB/c mice it was 4.4 ± 0.4 µM (P < 0.05). Transgenic BALB/c mice had significantly increased LTD4 responsiveness compared with transgenic C57BL/6 x SJL mice (Fig. 6, A and B; relative increase 61 ± 8% vs. 37 ± 5% compared with nontransgenic strains at highest concentration tested; P < 0.05, n = 6 in each group).



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Fig. 6. LTD4 responsiveness is enhanced in TG hCysLT1R (A) BALB/c genetic background vs. C57BL/6 x SJL (B) mice. LTD4 responsiveness was measured using increasing concentrations of inhaled LTD4 as described in MATERIALS AND METHODS. The first points (0 on the x-axis) represent the response given to saline inhalation. Results are expressed as % increase over the baseline Penh values (means ± SE). {circ}, NTG mice (n = 6); {bullet}, TG mice (n = 6). P < 0.001 TG vs. NTG dose-response curves (ANOVA).

 

LTD4, but not MCh, responsiveness is augmented following allergic sensitization and challenge of mice overexpressing hCysLT1R. To investigate the effects of allergic sensitization and challenge on smooth muscle responsiveness to LTD4 in vivo, we assessed Penh following inhalational administration of various concentrations of LTD4 or MCh. Compared with nonsensitized controls there were significantly increased LTD4 responses in both the transgenic and the nontransgenic strains following sensitization and exposure to Af (P < 0.001 naïve nontransgenic vs. sensitized nontransgenic and P < 0.0001 naïve transgenic vs. sensitized transgenic; n = 6 in each group at each dose) (Fig. 7B). ANOVA between the LTD4 dose responses of nontransgenic and transgenic mice sensitized and challenged with Af, unlike the measurements at baseline or in response to MCh challenge (Fig. 7A), revealed a highly significant difference (P < 0.001). In addition, the relative changes in LTD4 responsiveness between the naïve and sensitized groups were also significantly greater in the hCysLT1R transgenic mice than in the nontransgenic mice (P < 0.05, ANOVA), indicating that allergic airway sensitization potentiated responsiveness to LTD4 in the transgenic mice.



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Fig. 7. Allergic airway inflammation significantly enhances LTD4 responsiveness following inhalational administration. MCh (A) and LTD4 (B) responsiveness were measured using increasing doses of inhaled LTD4 as described in MATERIALS AND METHODS using TG line 31. The first points (0 on the x-axis) represent the response given to saline inhalation. Results are expressed as % increase over the baseline Penh values (means ± SE). {square}, NTG/N, n = 6; {blacksquare}, NTG/Af-sensitized and challenged mice (Af, n = 6); {circ}, TG/N (n = 6); {bullet}, TG/Af (n = 6). A "delta value" was calculated for each concentration as the difference of the average % change between naïve and sensitized mice, both in the NTG and in the TG animals. P < 0.001 and P < 0.0001 Af vs. N for LTD4 dose-response curves in the NTG and the TG strains, respectively; P < 0.001 TG vs. NTG in the Af-sensitized groups (ANOVA).

 

To investigate whether the amplified LTD4 responsiveness was associated with increased airway inflammation in the hCysLT1R transgenic mice, we compared the parameters of a Th2-type inflammatory response with nontransgenic mice. In the 10- to 12-wk-old animals, no significant differences were seen in the extent or composition of the inflammatory cell influx (lymphocytes, neutrophils, eosinophils, and macrophages), mucus secretion, and BAL cytokine levels (IL-13, KC, and RANTES) between the nontransgenic and the transgenic mice sensitized and exposed to Af. These results indicate that overexpression of the CysLT1R on smooth muscles does not result in enhancement of the acute inflammatory response given to allergenic challenge of sensitized mice.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In a model of allergic airway inflammation due to a major airborne fungus responsible for disorders including allergic bronchopulmonary aspergillosis and IgE-mediated asthma, we have demonstrated that transgenic overexpression of hCysLT1R on smooth muscle cells significantly augments allergen-induced airway hyperresponsiveness to LTD4. This implies that increased expression of hCysLT1R on smooth muscles, a pathological situation that could mimic some aspects of human asthmatic airway conditions (40), may significantly contribute to the altered airway physiology during the asthmatic response. In addition, we demonstrate that the increase in airway reactivity was not based on an enhanced inflammatory response, as the transgenic and nontransgenic mice mounted comparable cellular and humoral inflammatory changes. Furthermore, the enhanced AHR to LTD4 was not a result of potentiation of smooth muscle function, since responsiveness to MCh and carbachol was similar between the transgenic and nontransgenic strains. Thus our results demonstrate a unique mechanism in which CysLT1R signaling synergizes with smooth muscle hyperresponsiveness. Because the {alpha}-actin promoter driving the hCysLT1R expression was smooth muscle specific, these data suggest that airway inflammation may serve as a "rheostat" affecting bronchomotor tone that can enhance myogenic responses to contractile agonists. This type of leukotriene response has not been described in a relevant airway model, especially in mice that are normally hyporesponsive to leukotrienes (35).

Endogenous mouse CysLT1 receptors are expressed in two alternatively spliced forms, a long form (13 extra amino acids at the NH2 terminus relative to the human receptor) and a short form analogous to the human receptor (34, 36). The results of our study indicate that hCysLT1Rs can couple to endogenous mouse signaling pathways (most likely Gq/phospholipase C) in airway smooth muscle to enhance contractility in tracheal rings and allergic airways. The relative importance of the NH2-terminal extension in smooth muscle function in mice remains to be discovered.

These hCysLT1R transgenic mice will be useful for studies seeking to examine leukotriene actions within smooth muscle of airways. Previously, leukotriene-deficient mice (4) or mice treated with a CysLT1R antagonist (montelukast) were shown to have defective recruitment of eosinophils into airways after ovalbumin-induced allergic airway inflammation, reduced mucus secretion, and variable bronchoconstrictor responses to MCh airway challenge (11, 21, 22, 26), suggesting that endogenous leukotrienes participate in many airway inflammatory responses. Eum et al. (11) have also shown that instillation of LTD4 elicited enhanced cellular response when given before, but not after, allergen challenge, suggesting that CysLTs may play a significant role in the evolution of airway inflammatory responses. Recent evidence suggests that airway smooth muscle may serve an immunomodulatory/proinflammatory role by secreting cytokines and chemokines such as eotaxin (2, 6). Here we reveal that hCysLT1R-mediated signals in smooth muscle cells do not play a significant role in eliciting proinflammatory alterations in airways during the acute allergic response and cannot explain the marked hyperresponsiveness of the animals to LTD4.

Endogenous hCysLT1R is expressed in myeloid cells (12) in addition to airway and vascular smooth muscle. In our studies, expression of the hCysLT1R gene was detected in airway smooth muscle, vessels of the lung, and in bladder, uterus, and stomach but not in macrophages. The interplay of CysLT1 actions on macrophages and smooth muscle may be essential to recapitulate the complete phenotype of proinflammatory leukotriene actions in airways. Transgenic overexpression of the {beta}-adrenergic receptor (37) or insulin-like growth factor binding protein-4 (42) using the same {alpha}-actin promoter results in similar expression profiles. Interestingly, the {beta}-adrenergic receptor-overexpressing mice demonstrated many opposing effects to those observed with hCysLT1R mice, including enhanced relaxation of tracheal rings in response to receptor-specific agonist and diminished bronchial hyperreactivity. McGraw et al. (37) found evidence for spontaneous activation of this GPCR as there were significantly elevated levels of cAMP at baseline in their TSMC cultures. Overexpression of GPCR can induce ligand-independent constitutive activation that is consistent with some multistate models of GPCR, whereby some receptors attain an active conformation and influence downstream coupling (25, 37). Although it is relatively easy to detect this constitutive activation in a GPCR system coupled to adenylyl cyclase, it is not as clear with the Fluo-4-loaded cell assay for enhanced Ca2+-signaling/Gq coupling used here. So, we cannot say with certainty that there is an increased pool of constitutively active hCysLT1R in the transgenic mice.

Future studies with hCysLT1R-overexpressing mice will address the role of this receptor in other smooth muscle-rich tissues such as the bladder and in chronic airway inflammation models (7, 22) for effects on airway remodeling and bronchial hyperresponsiveness.

In conclusion, novel transgenic hCysLT1R-overexpressing mice have been generated and show enhanced leukotriene-dependent responses in vivo and in vitro. These mice represent a useful preclinical testing model for allergic airway inflammation studies.


    ACKNOWLEDGMENTS
 
The authors express gratitude to Dr. Arthur Strauch for supplying the mouse smooth muscle actin promoter plasmid.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-58464 (C. D. Funk), HL-67663, and HL-55301 (R. A. Panettieri). A. Haczku is a recipient of the Parker B. Francis Fellowship.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. D. Funk, Center for Experimental Therapeutics, Rm. 814BRBII/III, Univ. of Pennsylvania, 421 Curie Blvd., Philadelphia, PA 19104-6160 (E-mail: colin{at}spirit.gcrc.upenn.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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