Leukotrienes mediate neurogenic inflammation in lungs of young rats infected with respiratory syncytial virus

Katrin Wedde-Beer, Chengping Hu, Maria M. Rodriguez, and Giovanni Piedimonte

Departments of Pediatrics, Medicine, Molecular/Cellular Pharmacology, and Pathology, University of Miami School of Medicine, Miami, Florida 33136


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Respiratory syncytial virus (RSV) infection potentiates neurogenic inflammation in rat airways. Because some vascular effects of sensory nerves are mediated by cysteinyl leukotrienes (cysLTs), we studied whether the receptor antagonist montelukast inhibits neurogenic plasma extravasation in RSV-infected rats. Pathogen-free rats were inoculated at 2 wk (weanlings) or 12 wk (adults) of age with RSV or virus-free medium and treated with montelukast or its vehicle starting 1 day before inoculation. Five days postinoculation, we measured the extravasation of Evans blue-labeled albumin in the respiratory tract after stimulation of sensory nerves with capsaicin. Montelukast had no effect in the extrapulmonary airways but abolished albumin extravasation in the intrapulmonary airways of RSV-infected rats, with a larger effect in weanlings than in adults. Increased concentrations of 5-lipoxygenase-encoding mRNA and cysLTs, as well as numerous mast cells, were detected in the lung tissues of RSV-infected weanling rats. These observations suggest that the release of neuropeptides from capsaicin-sensitive sensory nerves and nonneuronal cells in the lungs of RSV-infected young rats increases vascular permeability by promoting the release of leukotrienes from mast cells.

airway inflammation; asthma; bronchiolitis; mast cells; montelukast


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

RESPIRATORY SYNCYTIAL VIRUS (RSV) is the most common respiratory pathogen in infancy and presents a large public burden worldwide (25). Furthermore, there is mounting evidence that RSV infection in early childhood is an important risk factor for the development of asthma (12, 17). However, the pathogenetic mechanisms of RSV-induced airway inflammation and hyperreactivity remain largely unknown, and no effective therapeutic option is currently available to manage the acute and chronic clinical manifestations of this infection.

We have found that RSV lower respiratory tract infection potentiates sensory nerve-mediated (neurogenic) inflammation in rats via upregulation of the high-affinity receptor for the neuropeptide substance P (16). We have also shown that the pattern of neurogenic inflammatory responses during RSV infection is largely age dependent (9), affecting predominantly the distal (intrapulmonary) airways in weanlings and the proximal (extrapulmonary) airways in adults.

Clinical studies have correlated the severity of RSV respiratory disease in infants with the concentration of leukotriene C4 (LTC4) in their respiratory secretions (27, 28). In addition, in vitro studies have shown that RSV infection in bronchial epithelial cells induces expression of the 5-lipoxygenase (5-LO) gene, which plays a central role in the biosynthesis of leukotrienes, increasing transiently the concentration of leukotrienes in the supernatant (2). Because it has been proposed that some of the vascular effects of substance P involve the production/release of cysteinyl leukotrienes (cysLTs) (21), interactions between neuropeptides and leukotrienes may represent an important mechanism of airway inflammation in response to RSV infection.

We hypothesized that the exaggerated neurogenic inflammatory response in the airways of RSV-infected rats involves activation of the leukotriene pathway. If so, selective antagonism of the cysLT1 receptor, which mediates most of the proinflammatory effects of cysLTs (3), should reduce or abolish this effect. Thus the primary objective of this study was to determine whether inhibition of the cysLT1 receptor with montelukast (18) reduces the exaggerated increase in microvascular permeability evoked by the stimulation of sensory nerves with capsaicin in RSV-infected airways and whether this inhibitory effect is age dependent. Extending these studies, we analyzed virus-induced histopathological changes in lung sections to identify possible cellular sources of leukotriene synthesis, focusing on mast cells because of their demonstrated functional relationship with sensory nerves (1). Finally, we sought to determine whether 5-LO gene expression and cysLTs production increase in vivo in RSV-infected lungs and what the time course of these changes is during and after the infection.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. We used pathogen-free adult rats and weanling rats born to pathogen-free timed-pregnant leaders, strain Fischer 344 (F-344), from Charles River Breeding Laboratories (Raleigh, NC). Because previous studies have shown a profound effect of respiratory infections on neurogenic control and inflammatory responses in the respiratory tract (14, 15), all animals used in this study were maintained under strict barrier conditions from birth to death to prevent any microbial contamination. Groups of two rats or one pregnant leader were housed in polycarbonate cages isolated by polyester filter covers. These cages were placed on racks providing positive individual ventilation with class-100 air to each cage at the rate of approximately one cage change of air per minute (Maxi-Miser; Thoren Caging Systems, Hazleton, PA) (9, 13, 16). We used separate rooms for housing infected and pathogen-free rats, both serviced by specifically trained husbandry technicians. All manipulations were conducted inside class-100 laminar flow hoods. Bedding, water, and food were autoclaved before use and unpacked only under laminar flow. Cages and water bottles were run through a tunnel washer after every use and disinfected with both chemicals and heat. The Division of Veterinary Resources of the University of Miami School of Medicine approved all experimental procedures followed in this study.

Preparation and inoculation of RSV. RSV suspensions were prepared as described previously (9, 13, 16). In brief, HEp-2 cells from the American Type Culture Collection (ATCC; Rockville, MD) were grown in MEM (GIBCO-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (GIBCO-BRL). Confluent monolayers of HEp-2 cells were infected with 0.1 plaque-forming units of human RSV strain ALong (ATCC), and the infection was allowed to proceed at 37°C in 5% CO2 atmosphere until >75% of the cells exhibited a cytopathic effect. Cell debris was removed by centrifugation at 9,500 g for 20 min in a centrifuge refrigerated at 4°C. Aliquots of the virus stock were snap frozen in liquid nitrogen and stored at -80°C. Before inoculation, the virus stock was titrated and diluted as needed to obtain a final titer of 5 × 104 50% tissue culture infective dose in 0.1 ml. Supernatants and cell lysates from virus-free flasks of HEp-2 cells in Eagle's MEM were harvested, centrifuged, and aliquoted following the same protocol to obtain the virus-free medium used as a negative control. Weanling rats 2 wk of age and adult rats 12 wk of age were inoculated under pentobarbital sodium anesthesia with RSV suspension or virus-free medium as described previously (9, 13, 16).

Albumin extravasation. Five days after the inoculation, the rats were reanesthetized with pentobarbital sodium (50 mg/kg ip). Evans blue dye (30 mg/kg iv over 5 s) was injected into the femoral vein to measure the extravasation of albumin from airway blood vessels associated with neurogenic inflammation (23). Immediately after the injection of the tracer, the rats received a 2-min intravenous infusion of 75 µg/kg of capsaicin to stimulate sensory nerves in the respiratory tract (8). This dose of capsaicin was used because it is at the threshold of effectiveness in increasing airway vascular permeability in pathogen-free rats, in that it causes large plasma extravasation in infected rats (14, 15).

Five minutes after the injection of the tracer, the chest was opened, a cannula was inserted into the ascending aorta through the left ventricle, and the circulation was perfused for 2 min with phosphate-buffered saline (PBS) with a syringe pump set at the rate of 50 ml/min for adult rats and 25 ml/min for weanling rats (9, 16). The extrapulmonary airways (from the first tracheal ring to the end of the main stem bronchi) and the left lung were dissected and prepared for Evans blue extraction. The specimens, free of connective tissue and opened along the ventral midline, were blotted with bibulous paper, weighed, and incubated in 1 ml of formamide (Sigma, St. Louis, MO) at 50°C for 18 h to extract the extravasated Evans blue dye.

The extravasation of Evans blue-labeled albumin from the tracheobronchial microcirculation was quantified by measuring the optical density (OD) of the formamide extracts at 620 nm wavelength. The quantity of Evans blue dye extravasated in the airway tissues, expressed in nanogram per milligram of wet tissue weight, was interpolated from a standard curve of Evans blue concentrations (0.5-10 µg/ml).

RSV detection and histopathology. Immunoperoxidase staining for RSV detection and mast cell identification and hematoxylin/eosin staining for histopathological analysis were performed on formalin-fixed 3-µm-thick lung sections.

As described previously (9, 13, 16), sections for RSV detection were incubated with a pool of monoclonal antibodies composed of four clones specific for the matrix protein, phosphoprotein, fusion protein, and nuclear protein of human RSV (Vector Laboratories, Burlingame, CA). This technique has been show to maximize the sensitivity of RSV detection (20). Mast cells detection was performed on sections digested with trypsin for 5 min and incubated for 30 min with a mouse monoclonal antibody specific for mast cell tryptase (DAKO, Carpinteria, CA) (30). Localization of the primary antibodies was delineated with the streptavidin-biotin peroxidase complex method using an immunostaining kit (DAKO) and developed with the 3,3'-diaminobenzidine tetrahydrochloride chromogen. With this technique, cells expressing target antigens are stained with a dark brown precipitate lining the cell membrane and cytoplasm.

Mast cell counts were performed in lungs from five pathogen-free and six RSV-infected weanling rats. Twenty-five sections were obtained from each lung specimen. Using a magnification of ×40, we counted mast cells in a square region from each lung section centered on a bronchiolar airway and measuring 0.18 mm2 in area. Measurements were expressed as the number of mast cells per square millimeter of surface area. All slides were coded and were interpreted by two independent observers who did not know whether the section corresponded to a RSV-inoculated or to a medium-inoculated animal.

RT-PCR. 5-LO mRNA levels were measured in lung homogenates by semiquantitative RT-PCR. Total cellular RNA was extracted in 1 ml of Tri-Reagent solution (Molecular Research Center, Cincinnati, OH). For the synthesis of cDNA, 1 µg of RNA from each sample was resuspended in a 12-µl final volume of reaction buffer containing 0.5 µg/µl oligo(dT)12-18 primer, incubated at 70°C for 10 min, and then cooled on ice for 5 min. The reaction mixture was prepared by adding 2 µl of 10× PCR buffer, 2 µl of 25 mM MgCl2, 1 µl of 10 mM 2-deoxynucleotide 5'-triphosphate mix, and 2 µl of 0.1 M dithiothreitol to each sample, which was then incubated for 5 min at 42°C. After adding 1 µl (200 units) of SuperScript II reverse transcriptase (GIBCO-BRL) to each tube, we allowed the reaction to proceed for 50 min at 42°C and terminated it at 70°C for 15 min.

PCR amplification of 5-LO was performed using the following primer sequences: sense 5'-AGG CTG CAG TGA GAA GCATC-3', and antisense 5'-GCC AGT GGT TCT TGA CTC TC-3', designed to amplify a fragment corresponding to nucleotides 181-770 (24). The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified simultaneously as an internal standard with the following primer sequences: sense 5'-CTA CCC ACG GCA AGT TCA AT-3', targeting bases 176-195, and antisense 5'-CCT GTT GTT ATG GGG TCT GG-3', targeting bases 1,174-1,193 (4). Amplification was initiated with 10 min of denaturation at 94°C followed by 35 cycles at 94°C for 45 s, 55°C for 45 s, and 72°C for 1 min using a thermal cycler (GeneAmp PCR System 9600; Perkin-Elmer, Foster City, CA). After the last cycle of amplification, the samples were incubated for 10 min at 72°C.

RNA concentrations and PCR cycles were titrated to establish standard curves to document linearity and to permit semiquantitative analysis of signal strength. Amplified PCR products were separated by electrophoresis through a 1.5% agarose gel at 65 V for 120 min. The DNA bands were visualized by ultraviolet illumination after the gels were stained with 0.5 mg/ml ethidium bromide dissolved in Tris borate-ethylenediaminetetraacetic acid (EDTA) buffer (89 mM Tris, 89 mM boric acid, 2.5 mM EDTA, pH 8.2). The gels were photographed and analyzed by computerized densitometry.

CysLT enzyme immunoassay. CysLT concentration in lung tissues homogenates was measured using a commercial kit (Cayman Chemicals, Ann Arbor, MI). In brief, 100 mg of tissue from each lung sample were homogenized in 10 ml of PBS containing the lipoxygenase inhibitor nordihydroguaiaretic acid (6 µM) and 1 mM EDTA. After centrifugation at 1,500 g for 5 min, cysLTs were purified by adding 25 µl of resin to 1 ml of supernatant. The resin, separated by centrifugation at 5,000 g for 5 min, was resuspended in 0.5 ml of cold methanol and vortexed briefly to displace the adsorbed leukotrienes. This step was repeated twice, and the combined methanol washes were evaporated to dryness and immediately resuspended in buffer for analysis.

This enzyme-linked immunoassay is based on the competition between unlabeled cysLTs and a fixed quantity of cysLTs-acetylcholinesterase conjugate (cysLT tracer) for a limited number of binding sites on a cysLT-specific antiserum. The intensity of the color change generated by the acetylcholinesterase read at 405 nm wavelength is inversely proportional to the concentration of free cysLTs in the sample. CysLT standards and test samples were assayed in duplicate, and each sample was measured at two dilutions (50 µl of sample/well and 25 µl of sample + 25 µl of additional buffer/well). With this assay, cysLTs (specificity for LTC4 and LTD4 100%, specificity for LTE4 67%) can be identified with a lower detection limit of 13 pg/ml and cross-reactivity with leukotrienes A and B < 0.01%.

Drugs. All drugs used in the in vivo experiments were delivered in a volume of 1 ml/kg body wt. Evans blue was dissolved in 0.9% NaCl. Capsaicin (8-methyl-N-vanillyl-6-nonenamide; Sigma) was dissolved in a vehicle having a final concentration of 0.75% ethanol, 0.375% Tween 80, and 0.85% NaCl in aqueous solution. Montelukast sodium, {MK-0476, Singulair, Merck; [R-(E)]-1-[[[1-[3-[2-(7-chloro-2-quinolinyl)-ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]thio]methyl]cyclopropane acetic acid, monosodium salt} was dissolved in 0.9% NaCl to the final concentration of 10 mg/ml.

Experimental protocols. To determine the role of cysLTs in the mechanism of airway neurogenic inflammation, weanling and adult rats were treated once daily with an intraperitoneal injection of montelukast (10 mg/kg; n = 7-8 rats per group) or with its vehicle (0.9% NaCl; n = 6-8 rats per group), starting 1 day before the inoculation of RSV or virus-free medium. Each rat received a total of seven daily doses of montelukast or vehicle, and the last dose was given on day 5 postinoculation, 1 h before pharmacological stimulation of sensory nerves. Capsaicin-induced extravasation of Evans blue-labeled albumin from the airways' microvasculature was measured 5 days after inoculation. This interval between inoculation and measurement of vascular permeability was chosen on the basis of previous work with RSV infection in rats (9, 13, 16). Because of the larger inhibitory effect of montelukast found in the lungs of RSV-infected weanling rats, the ensuing experiments focused on our early-life infection model (9).

Sections from the lungs of RSV-infected and pathogen-free weanling rats killed 5 days after inoculation were stained for histopathological analysis and for identification of possible cellular sources of leukotriene synthesis. In particular, specific staining with antitryptase antibodies was performed for the detection and quantification of mast cells, which have been shown to have close spatial and functional relationships with sensory nerves fibers in many organ systems (1).

To determine whether the leukotriene synthetic pathway in the lungs is modified during and/or after RSV infection in vivo, groups of RSV-infected weanling rats paired to age-matched pathogen-free controls were killed at 3, 5, or 30 days after inoculation (n = 5 rats per group), and their lungs were analyzed for 5-LO mRNA and cysLT concentrations by semiquantitative RT-PCR and immunoassay, respectively.

Statistical analysis. Data are expressed as the mean ± SE. The effects of RSV on mean values of Evans blue extravasation and densitometry measurements of RT-PCR products were analyzed by two-factor analysis of variance (31). Multiple comparisons between means were performed with the Fisher's protected least significant differences test (29). Mast cell density was compared by unpaired Student's t-test. Statistical analysis was performed using the software StatView version 5.0.1 (SAS Institute, Cary, NC). Differences having a P value < 0.05 were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

Vascular permeability. In pathogen-free weanling rats stimulated with capsaicin (Fig. 1, left), there was no difference in intrapulmonary or extrapulmonary airway microvascular permeability after treatment with either montelukast (n = 5) or its vehicle (n = 6; P > 0.5). In RSV-infected weanling rats without montelukast treatment (n = 5), the increase in microvascular permeability evoked by sensory nerves stimulated with capsaicin 5 days after inoculation (Fig. 1, right) was much larger in the intrapulmonary airways than in the extrapulmonary airways (P = 0.009). Daily treatment with montelukast during RSV infection (n = 8) decreased vascular permeability significantly in the intrapulmonary airways (P < 0.0001) but not in the extrapulmonary airways (P = 0.08). Evans blue extravasation in the intrapulmonary airways of RSV-infected weanling rats treated with montelukast was approximately one-third of control values measured after administration of the vehicle and was not different from the intrapulmonary extravasation measured in age-matched pathogen-free rats (P = 0.8).


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Fig. 1.   Comparison of capsaicin-induced Evans blue extravasation in respiratory syncytial virus (RSV)-infected weanling rats (2 wk of age, bars on right) treated daily with montelukast (solid bars; n = 8) or its vehicle (hatched bars; n = 5). Age-matched pathogen-free controls (bars on left) were treated daily with montelukast (n = 5) or vehicle (n = 6) after being dosed with virus-free medium. Vascular permeability was measured 5 days after inoculation. Montelukast caused a large reduction of Evans blue extravasation in the intrapulmonary airways (B), but not in the extrapulmonary airways (A). *** P < 0.001, significantly different from vehicle-treated control rats.

In pathogen-free adult rats stimulated with capsaicin (Fig. 2, left), there was no difference in intrapulmonary or extrapulmonary airway microvascular permeability after treatment with either montelukast (n = 5) or its vehicle (n = 5; P > 0.05). The increase in microvascular permeability evoked by sensory nerves stimulated with capsaicin after 5 days of RSV infection (Fig. 2, right) was much larger in the extrapulmonary airways than in the intrapulmonary airways (P < 0.0001). Daily treatment with montelukast (n = 7) caused a small, nonsignificant decrease of capsaicin-induced Evans blue extravasation in the extrapulmonary airways compared with vehicle-treated rats (n = 6; P > 0.05). Despite the lower microvascular permeability of adult rat intrapulmonary airways stimulated with capsaicin, the inhibition of Evans blue extravasation caused by montelukast was statistically significant (P = 0.0008).


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Fig. 2.   Comparison of capsaicin-induced vascular permeability in RSV-infected adult rats (12 wk of age; bars on right) treated daily with montelukast (solid bars; n = 7) or its vehicle (hatched bars; n = 6). Age-matched pathogen-free controls (bars on left) were treated daily with montelukast (n = 5) or vehicle (n = 5) after being dosed with virus-free medium. Vascular permeability was measured 5 days after inoculation. Montelukast caused a small but significant reduction of Evans blue extravasation in the intrapulmonary airways (B), whereas the effect on the extrapulmonary airways (A) was not significant. *** P < 0.001 = significantly different from vehicle-treated control rats.

Histopathology. Immunoperoxidase staining with specific monoclonal antibodies performed on lung sections from weanling rats killed 5 days after the inoculation of RSV revealed the presence of RSV antigens on the membranes and in the cytoplasm of bronchiolar epithelial cells (Fig. 3). Viral antigens were also expressed within the hypertrophic bronchiolar-associated lymphoid tissue (BALT) and in some inflammatory cells within the lamina propria. No virus was detected in the airways of rats dosed with virus-free medium.


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Fig. 3.   Lung sections from weanling rats killed 5 days after the intranasal inoculation of virus-free medium (A) or RSV suspension (B). Immunoperoxidase staining with anti-RSV monoclonal antibodies reveals the presence of viral antigens on the membranes and in the cytoplasm of bronchiolar epithelial cells from the RSV-inoculated rats (open arrow). Viral antigens were also expressed within the hypertrophic bronchiolar-associated lymphoid tissue (BALT) and in some inflammatory cells within the lamina propria (solid arrows). There was no detectable virus in the lungs of control rats inoculated with virus-free medium. Internal scale = 50 µm.

The histopathological changes caused by RSV consisted of a predominantly mononuclear cellular infiltration in the interstitium and peribronchial areas, made predominantly of monocytes and lymphocytes with only sparse polymorphonuclear leukocytes (Fig. 4). Abundant lymphoid cells were also present in the bronchiolar lamina propria, and the BALT was consistently hypertrophic in infected airways. No eosinophils were detected in the inflammatory infiltrate. Numerous mast cells were identified in the lung sections from RSV-infected rats (Fig. 5), with an average sevenfold increase compared with the lungs of pathogen-free controls (114 ± 33 vs. 16 ± 6 mast cells/mm2; P = 0.03).


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Fig. 4.   Photomicrographs of hematoxylin and eosin-stained sections obtained from the lungs of weanling rats killed 5 days after intranasal inoculation of virus-free medium (A) or RSV suspension (B). The asterisks mark the lumen of bronchioles. In RSV-infected rats, peribronchial areas and the surrounding pulmonary interstitium were infiltrated by numerous mononuclear leukocytes, and abundant lymphoid cells were present in bronchiolar lamina propria. Solid arrows mark the hypertrophic BALT, and the open arrow marks mucous secretions with desquamated epithelium inside the bronchiolar lumen. No significant pathological changes were noted in the lungs of the pathogen-free control rats. Internal scale = 50 µm.



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Fig. 5.   Lung sections from weanling rats killed 5 days after the intranasal inoculation of virus-free medium (A) or RSV suspension (B). Mast cells were identified by immunohistochemistry using a monoclonal antibody specific for tryptase. An average 7-fold increase in mast cell density was found in the lung sections from RSV-infected rats compared with pathogen-free controls (right). Internal scale = 40 µm. * P < 0.05 = significantly different from pathogen-free rats.

Leukotriene synthesis. RT-PCR analysis of 5-LO mRNA levels in lung homogenates from weanling rats revealed a significant increase 5 days after the inoculation of RSV (Fig. 6) compared with age-matched pathogen-free control rats dosed with virus-free medium (0. 99 ± 0.06 vs. 0.71 ± 0.07 OD units; n = 5 rats per group; P = 0.02). Time-course analysis of densitometry measurements normalized to the internal standard GAPDH revealed that this increase was an early event, being maximal 3 days after inoculation (1. 68 ± 0.07 vs. 1.02 ± 0.09 OD units; n = 5 rats per group; P = 0.0004), but limited to the acute phase of the infection because no difference was found 30 days after inoculation between rats previously exposed to RSV and pathogen-free controls (0.97 ± 0.07 vs. 0.92 ± 0.05 OD units; n = 5 rats per group; P = 0.61). Enzyme-linked immunoassay revealed a transient increase of free cysLT concentration in the lung tissues of weanling rats 3 days after infection with RSV (4.69 ± 0.56 vs. 1.75 ± 0.29 pg/mg tissue; n = 5 rats per group; P = 0.002), with a rapid return to levels similar to pathogen-free controls by 5 days postinfection (2.93 ± 0.36 vs. 2.50 ± 0.27 pg/mg; n = 5 rats per group; P = 0.37). Figure 7 compares RSV-induced changes in 5-LO mRNA and cysLTs concentration in lung tissues expressed as a percentage of control values measured in age-matched pathogen-free rats.


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Fig. 6.   Amplification of 5-lipoxygenase (5-LO) mRNA from the lungs of weanling rats 5 days after intranasal inoculation with RSV (n = 5) or with virus-free medium (n = 5). RSV caused a significant increase in 5-LO expression compared with pathogen-free weanling rats. Each band was obtained from the lungs of a different animal. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M, ladder of molecular weight standards.



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Fig. 7.   Time course of the expression of 5-LO (A) mRNA and cysteinyl leukotriene (cysLT; B) concentration measured in the lungs of weanling rats after the inoculation of RSV. Data are expressed as %control values measured in age-matched rats dosed with virus-free medium (n = 5 pathogen-free rats and 5 RSV-infected rats/each time point). The acute phase of RSV infection was associated with increased expression of the 5-LO gene and transient production of cysLTs in the lungs. * P < 0.05 = significantly different from postinfection day 3.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study, which is the first using a leukotriene modifier in vivo in an animal model of RSV bronchiolitis, shows that the exaggerated neurogenic inflammation in the intrapulmonary airways of young rats infected with RSV involves the concomitant release of cysLTs and activation of the cysLT1 receptor, as manifested by the potent inhibitory effect of the receptor antagonist montelukast on capsaicin-induced plasma leakage. Montelukast also had a much smaller, but still significant, inhibitory effect in the intrapulmonary airways of RSV-infected adult rats, whereas no effect was found in the extrapulmonary airways of either young or adult rats. Our data also show that montelukast has no inhibitory activity in the absence of the viral infection, suggesting that RSV infection is associated with increased leukotriene synthesis in the respiratory tract due to changes in gene expression and/or recruitment of inflammatory cells into the airways.

Concerning the mechanisms modulating the leukotriene pathway during RSV infection, we found a significant increase in 5-LO expression and cysLT synthesis in the lungs of weanling rats during the acute phase of RSV infection. In the lungs, the enzymatic capacity to produce cysLTs (LTC4, LTD4, LTE4) from arachidonic acid derives primarily from mast cells and eosinophils (22). Histopathological analysis of infected lung tissues showed an expanded mast cell population, whereas no eosinophils were detected in the inflammatory infiltrate, and thus the former appears to be the most likely source of cysLTs. However, we cannot rule out other sources contributing 5-LO transcripts and cysLTs in infected lungs, such as monocytes/macrophages and/or other cell types that may acquire 5-LO enzymatic activity as a result of RSV infection, e.g., epithelial cells (2). Further studies involving immunostaining and in situ hybridization, as well as in vitro infection of specific cell types in culture, are necessary to answer this question conclusively.

Time-course analysis of infected lung tissues suggests that the effect of RSV on 5-LO gene expression is transient, being maximal at 3 days postinoculation, already reduced at 5 days, and resolved by 30 days. A similar profile was found measuring the concentration of cysLTs in the same tissues, with an almost complete return to pathogen-free levels by 5 days postinoculation, when the physiological endpoint (neurogenic inflammation) was measured. The time course of these RSV-induced changes in the leukotriene synthetic pathway in vivo parallels the results obtained in vitro using bronchial epithelial cell lines (2). Our data suggest that, following the early phase of the viral infection, leukotriene production/release rapidly returns to baseline levels but can be reactivated by stimulation of the numerous mast cells still present in the lung tissues, e.g., by substance P released upon stimulation of sensory nerve terminals. In addition, since it has been shown that also nonneuronal cells such as monocytes and macrophages express substance P and its receptors and release this peptide in response to capsaicin stimulation (7), it is possible that cells involved in the immune and inflammatory response to the infection contribute to this mechanism of mast cell activation.

Mast cell-nerve interactions. The inhibitory effect of montelukast on neurogenic-mediated inflammation in RSV bronchiolitis suggests that the activity of leukotriene-producing cells is modulated by sensory neurotransmitters via molecular interactions that become hyperactive during RSV infection. Whereas no spatial associations have been described so far between sensory nerves and eosinophils or macrophages, juxtapositions of substance P-containing sensory nerve fibers with mast cells have been reported in several organ systems (26), particularly the skin, central nervous system, and gastrointestinal tract, and there is strong evidence for functional mast cell-nerve interactions mediated by bidirectional exchange of chemical transmitters resulting in synergistically amplified inflammatory responses (1).

An example of the interactions between leukotrienes and neuropeptides in the respiratory tract is provided by the putative pathophysiological mechanism of hyperpnea-induced bronchospasm (6), which involves a sequence of events starting with the release of leukotrienes in response to mucosal damage (5), stimulation of sensory nerves with release of substance P, and final airway smooth muscle contraction (10).

In our previous work we have shown that RSV infection potentiates neurogenically mediated inflammation in the respiratory tract. Differently from parainfluenza and influenza viruses, RSV does not affect the peptide-degrading activity of neutral endopeptidase in the airways (16) but, rather, exerts its effects by upregulating gene expression of the high-affinity substance P [neurokinin 1 (NK1)] receptor (16). We have also shown age-dependent differences in the distribution of RSV-induced neurogenic inflammation (9), affecting predominantly the intrapulmonary airways of young rats and the extrapulmonary airways of adult rats. On the basis of the data presented in this study, we hypothesize that the increased susceptibility of the intrapulmonary airways of RSV-infected young rats to the inflammatory effects of sensory nerves is dependent, at least in part, on increased neurostimulation of mucosal mast cells with consequent release of cysLTs, which in turn can amplify the release of tachykinins from sensory nerves (11), forming a local neuron-mast cell feedback loop. RSV may exert this effect by inducing qualitative/quantitative changes in mast cell-nerve synapses, such as upregulation of NK1 receptor expression on mast cell membranes analogous to epithelial cells (16), vascular endothelial cells (16), and T lymphocytes (19) in RSV-infected airways. This hypothesis is also supported by our previous finding that selective NK1 receptor antagonism abolishes neurogenic plasma extravasation in the intrapulmonary airways of RSV-infected weanling rats (9).

Previous studies have suggested that cysLTs cause airway inflammation and smooth muscle spasm during RSV infection, accounting for the wheezing observed in bronchiolitis. Increased LTC4 levels were measured in the nasopharyngeal secretions of children during the acute phase of RSV infection, and their concentration was correlated with clinical severity, being higher in patients with lower respiratory tract involvement than in children with upper respiratory illness alone (27, 28). These clinical observations confirm the importance of cysLTs in the pathophysiology of RSV-induced airway inflammation in humans and, together with our present data, provide a strong rationale for clinical trials exploring the potential role of leukotriene modifiers in the therapy of RSV bronchiolitis.

In conclusion, this is the first study showing an anti-inflammatory effect of a leukotriene receptor antagonist in vivo in an animal model of RSV bronchiolitis. Montelukast potently inhibited neurogenically mediated inflammation in the intrapulmonary airways of infected weanling rats and, to a much lesser degree, in adult rats. No significant inhibitory effect was found in the extrapulmonary airways. Acute RSV infection was associated with increased expression of the 5-LO gene and transient cysLT production, probably deriving from the expanded mast cell population found in infected lungs. We speculate that leukotrienes may also be released via mast cell-nerve interactions that are amplified during RSV infections and, in turn, potentiate the inflammatory effects of neuropeptides like substance P, as illustrated in Fig. 8. If the same mechanisms are present in humans, leukotriene modifiers could be beneficial in the therapy of RSV bronchiolitis, and controller therapy of pediatric asthma with leukotriene modifiers may protect against virus-induced asthma exacerbations.


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Fig. 8.   Flow diagram illustrating the proposed model of leukotriene production/release via mast cell-nerve interactions that are amplified during RSV infections and, in turn, potentiate the inflammatory effects of neuropeptides like substance P. NK1, neurokinin 1.


    ACKNOWLEDGEMENTS

We thank Drs. Xiaobo Jiang and Mian Xu for technical assistance and Dr. Alexander Auais for helping with the illustrations. We also thank Zuleika Perez for helping with the mast cell counts.


    FOOTNOTES

This research was supported in part by National Heart, Lung, and Blood Institute Grant HL-61007 and by a research grant from Merck to G. Piedimonte. K. Wedde-Beer was supported by the Deutsche Forschungsgemeinschaft.

Some of the findings reported in this article were presented at the American Thoracic Society 2001 International Conference in San Francisco, CA.

Address for reprint requests and other correspondence: G. Piedimonte, Batchelor Children's Research Institute, Pediatric Pulmonology & Cystic Fibrosis Center, Univ. of Miami School of Medicine, 1580 NW 10th Ave. (D-820), Miami, FL 33136 (E-mail: gpiedimo{at}med.miami.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.

First published January 4, 2002;10.1152/ajplung.00323.2001

Received 13 August 2001; accepted in final form 14 December 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Lung Cell Mol Physiol 282(5):L1143-L1150
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