Respiratory syncytial virus upregulates expression of the substance P receptor in rat lungs

Giovanni Piedimonte1,2,3, Maria M. Rodriguez1,4, Katherine A. King1, Stafford McLean5, and Xiaobo Jiang1

Departments of 1 Pediatrics, 2 Medicine, 3 Pharmacology, and 4 Pathology, University of Miami School of Medicine, Miami, Florida 33136; and 5 Central Research Division, Pfizer, Inc., Groton, Connecticut 06340


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Respiratory syncytial virus (RSV) is a major respiratory pathogen in infants. The first goal of this study was to determine whether the infection following endotracheal inoculation of RSV in Fischer 344 rats results in increased inflammatory responses to substance P (SP) either released by capsaicin from sensory nerves or injected into the circulation. Five days after inoculation, the extravasation of Evans blue-labeled albumin after capsaicin or SP was significantly greater in RSV-infected airways than in pathogen-free controls. The peptide-degrading activity of the regulatory enzyme neutral endopeptidase was unaffected by RSV. However, SP(NK1) receptor mRNA levels increased fivefold in RSV-infected lungs, and the density of SP binding sites in the bronchial mucosa increased threefold. These data suggest that RSV makes the airways abnormally susceptible to the proinflammatory effects of SP by upregulating SP(NK1) receptor gene expression, thereby increasing the density of these receptors on target cells. This effect may contribute to the inflammatory reaction to the virus and could be a target for the therapy of RSV disease and its sequelae.

airway inflammation; asthma; bronchiolitis; neurokinin receptors; sensory nerves


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

RESPIRATORY SYNCYTIAL virus (RSV) infection presents a large public health burden worldwide (14). One-half of all infants become infected with RSV in the 1st yr of life, and by 3 yr of age, essentially all children have been infected with RSV at least once (13). RSV is estimated to cause up to 90% of all childhood bronchiolitis and up to 40% of all pediatric pneumonias (14), resulting in 90,000 hospitalizations and 4,500 deaths annually in the United States (15). Because immunity after the first infection is not complete, RSV is a common cause of respiratory infections also in older children and adults (14). Furthermore, there is growing concern that early RSV infection is an important risk factor for the development of chronic asthma, particularly in children with predisposing genetic factors (7). The mechanisms by which this virus causes airway inflammation and hyperreactivity are unclear.

Previous studies have shown that other common respiratory viruses, such as parainfluenza viruses, can potentiate neurogenically mediated inflammatory responses in the infected airways (33, 36). Neurogenic inflammation is caused by the local release of substance P (SP) and other peptide neurotransmitters with proinflammatory properties from unmyelinated sensory nerves forming a dense network within and beneath the epithelial lining of the respiratory mucosa (31). It is believed that virus-induced potentiation of neurogenic inflammation is caused by the loss of the peptide-degrading activity of neutral endopeptidase (NEP), a membrane-bound enzyme localized primarily in the airway epithelium, which can be damaged during viral infections (35).

In the present study, we investigated whether RSV lower respiratory tract infection in adult rats results in an increased inflammatory response to SP either released from its natural source (C-type sensory nerve fibers) or injected directly into the circulation. We also explored the mechanism of this action by studying the effect of RSV on key regulatory elements of the neurogenic inflammatory pathway, i.e., NEP enzymatic activity and expression of the high-affinity SP receptor [also known as neurokinin type 1 (NK1) receptor] (39).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Adult (12 wk of age) male pathogen-free Fischer 344 (F-344) rats were obtained from Charles River Breeding Laboratories (Raleigh, NC). To prevent microbial contamination, groups of two or three rats each 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). We used separate rooms for housing infected and pathogen-free rats; both were serviced by specifically trained husbandry technicians. All manipulations were conducted inside a class 100 laminar flow hood. 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 chemicals and heat. Experimental procedures followed in this study were approved by the Division of Veterinary Resources of the University of Miami School of Medicine.

Preparation of RSV suspensions. HEp-2 cells (American Type Culture Collection, Manassas, VA) 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 unit of human RSV long strain (American Type Culture Collection), and the infection was allowed to proceed at 37°C in a 5% CO2 atmosphere until >75% of cells exhibited a cytopathic effect. Cell debris was removed by centrifugation at 9,500 rpm for 20 min in a refrigerated (4°C) centrifuge. Aliquots of the virus stock were snap frozen in liquid nitrogen and stored at -70°C. Before inoculation, the virus stock was titrated and diluted as needed for a final titer of 5 × 104 50% tissue culture infective dose (TCID50) in 0.1 ml. Supernatants and cell lysates from virus-free flasks of HEp-2 cells in MEM were harvested, centrifuged, and divided into aliquots following the same protocol to obtain the virus-free medium used as a negative control.

Virus inoculation. We elected to inoculate endotracheally rather than intranasally to avoid the possibility of having a subpopulation of our rats with a localized upper respiratory infection without extension to the lower airways. Rats were anesthetized with pentobarbital sodium (50 mg/kg ip). The vocal cords were visualized with a rodent laryngoscope, and the trachea was carefully intubated with a 16-gauge cannula (37). An 18-gauge inner cannula connected to a tuberculin syringe was passed through the endotracheal cannula to deposit the inoculum over the airway mucosa between the cricoid cartilage and the first tracheal ring. The rats used in this study were inoculated with 100 µl of RSV suspensions containing 5 × 104 TCID50. Control rats were dosed with 100 µl of virus-free medium.

Virus detection. The trachea, main stem bronchi, and lungs were fixed in 10% buffered formalin, embedded in paraffin, and cut in 3-µm-thick sections. Staining with hematoxylin and eosin was performed for histopathological analysis. Immunoperoxidase staining for RSV detection was performed on the Formalin-fixed sections after the paraffin was melted in an oven at 37°C overnight. The slides were deparaffinized in a xylene bath and dehydrated in decreasing concentrations of ethanol. Endogenous peroxidase activity was blocked by incubation in 6% hydrogen peroxide for 5 min. The sections were incubated for 30 min in a humidity chamber with a 1:400 dilution of a pool of mouse 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 shown to maximize the sensitivity of RSV detection (40). The localization of anti-RSV antibodies was delineated by the streptavidin-biotin peroxidase complex method with use of an immunostaining kit (DAKO, Carpinteria, CA) and developed with the 3,3'-diaminobenzidine tetrahydrochloride chromogen. Finally, the slides were counterstained with hematoxylin for 15 s. With this technique, cells expressing viral antigens are stained with a dark-brown precipitate lining the cell membrane and cytoplasm. All slides were coded and interpreted by a pathologist who did not know whether the section corresponded to an RSV- or a medium-inoculated animal.

Albumin extravasation. Rats were anesthetized with pentobarbital sodium. Evans blue dye (30 mg/kg iv over 5 s) was injected to measure the extravasation of albumin from airway blood vessels (42). Immediately after the injection of the tracer, separate groups of RSV-infected and pathogen-free rats received an intravenous injection of vehicle (1 ml/kg), capsaicin (75 µg/kg over 2 min), or SP (1 µg/kg over 20 s).

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 PBS (Sigma, St. Louis, MO) with a syringe pump set at the rate of 50 ml/min. 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, weighed, and incubated in 1 ml of formamide (Sigma) 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 a wavelength of 620 nm. The quantity of Evans blue dye extravasated in the airway tissues, expressed in nanograms per milligram of wet weight, was interpolated from a standard curve of Evans blue concentrations (0.5-10 µg/ml).

Peptidase activity. The extrapulmonary airways and the lungs were dissected after perfusion through the heart with 100 ml of sterile PBS. The tissues were weighed, frozen in dry ice, and then homogenized in a buffer containing 50 mM Tris, 10 µM phenylmethylsulfonyl fluoride, and 10 nM pepstatin A (pH 7.5). After centrifugation at 1,000 rpm for 4 min in a refrigerated (4°C) centrifuge, the supernatants were transferred to new vials and stored at -70°C. NEP activity was measured by a fluorometric assay with the substrate glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine (Glu-MNA; Enzyme Systems Products, Dublin, CA) as described previously (43). All samples were incubated in duplicate with 40 µM Glu-MNA and 1 µg of aminopeptidase M in a final volume of 250 µl of buffer (50 mM Tris · HCl, pH 7.5) for 30 min at 37°C. One-half of the samples were preincubated with the specific NEP inhibitor phosphoramidon (10-6 M) for 10 min before addition of the substrate. The generation of MNA was measured fluorometrically (340-nm excitation wavelength, 425-nm emission wavelength). Enzymatic activity was interpolated from a standard curve of MNA concentrations and normalized to protein concentration.

RT-PCR. SP(NK1) receptor mRNA levels in lung tissues were measured by semiquantitative RT-PCR on the basis of previously published work (21). Total cellular RNA was extracted from lung tissue homogenates 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 20-µl final volume of reaction buffer containing 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 5 mM MgCl2, dNTP at 1 mM each, 1 U/µl RNase inhibitor, and 2.5 µM oligo(dT)16 primer. Moloney murine leukemia virus reverse transcriptase (2.5 U/µl; Perkin-Elmer, Foster City, CA) was added to each tube, and the reaction was allowed to proceed for 10 min at room temperature, then for 30 min at 42°C. Five-microliter aliquots of the synthesized cDNA (corresponding to 100 ng of RNA) were added to 45 µl of PCR mixture containing 5 µl of 10× PCR buffer, 2 µl of deoxynucleotides (0.4 mM each), 1 µl of 3'- and 5'-specific primers (0.2 µM each), and 0.25 µl of AmpliTaq Gold DNA polymerase (5 U/µl; Perkin-Elmer).

PCR amplification of the rat SP(NK1) receptor was performed using the following primer sequences: sense 5'-CATCAACCCAGATCTCTACC-3', targeting bases 1371-1391, and antisense 5'-GCTGGAGCTTTCTGTCATGGA-3', targeting bases 1735-1755. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified simultaneously as an internal standard with the following primer sequences: sense 5'-TGAAGGTCGGTGTCAACGGATTTGGC-3', targeting bases 35-60, and antisense 5'-CATGTAGGCCATGAGGTCCACCAC-3', targeting bases 994-1017. As a positive control, we used the full-length rat SP(NK1) receptor cDNA (46). Amplification was initiated with 10 min of denaturation at 94°C for 1 cycle followed by 35 cycles at 94°C for 45 s, 55°C for 45 s, and 72°C for 1 min with a thermal cycler (GeneAmp PCR System 9600, Perkin-Elmer). After the last cycle of amplification, the samples were incubated for 10 min at 72°C. RNA concentrations and PCR cycler 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 2% agarose gel at 45 V for 120 min. The cDNA bands were visualized by ultraviolet illumination after the gels were stained with 0.5 mg/ml ethidium bromide dissolved in Tris-borate-EDTA buffer (89 mM Tris, 89 mM boric acid, 2.5 mM EDTA, pH 8.2). The gels were photographed, and the films were scanned and analyzed with a computerized densitometer.

Autoradiography. Lungs removed after vascular perfusion with PBS were frozen in isopentane and stored at -70°C. Tissues were embedded, cut at 20 µm thickness, thaw mounted onto gelatin-coated slides, and stored at -70°C until used. Slides were brought to room temperature by preincubation for 15 min at 22°C in medium containing 50 mM Tris · HCl, 5 mM MnCl2, 0.02% BSA, 20 µg/ml bacitracin, 4 µg/ml leupeptin, and 2 µg/ml chymostatin (pH 7.3). The slides were then incubated for 60 min at 22°C in the same medium supplemented with 125I-labeled Bolton-Hunter-coupled SP (29, 38). Nonspecific binding was determined by incubation of adjacent tissue sections under the same conditions in the presence of 1 µM unlabeled SP. After incubation, the slides were washed twice for 5 min in ice-cold buffer, rinsed in distilled water to remove buffer salts, and then dried under a stream of air. All sections were placed in light-tight X-ray cassettes, apposed to tritium-sensitive ultrafilm for 4 days, and developed using standard photographic procedures. The ultrafilms and the corresponding hematoxylin and eosin-stained sections were scanned and digitally superimposed to analyze the localization of SP binding sites. The density of SP binding sites in the intrapulmonary airways was assessed as OD in each section obtained from RSV-infected (n = 12) and pathogen-free lungs (n = 8) by use of a constant magnification (×4). OD measurements were corrected for nonspecific binding and expressed as percent increase over background.

Experimental protocols. Preliminary experiments were performed to assess whether RSV antigens could be detected in the respiratory tract of adult F-344 rats after endotracheal inoculation. Two groups of rats inoculated with RSV or virus-free medium were killed 5 days after inoculation, and the airways and lungs were processed for immunoperoxidase identification of RSV antigens and histopathological analysis. The 5-day time point was chosen on the basis of previous work with viral respiratory infections in rats (33, 36, 37).

To determine whether the endotracheal administration of RSV resulted in increased neurogenic inflammatory responses, 10 rats were inoculated with 5 × 104 TCID50 of RSV. A control group of 12 rats was dosed with virus-free medium. Five days after inoculation, capsaicin was injected into one-half of the RSV-infected rats and one-half of the pathogen-free controls to stimulate sensory nerves in the airway mucosa (16). The other RSV-infected and pathogen-free rats received an injection of vehicle. To determine whether the potentiating mechanism of RSV on neurogenic inflammation was operating at a presynaptic or a postsynaptic level, a group of RSV-infected rats and a group of pathogen-free controls (n = 6 rats each) received an intravenous injection of SP 5 days after inoculation.

To determine whether the inflammatory reaction associated with RSV infection can be prevented by blocking the SP(NK1) receptor, two groups of rats inoculated 5 days earlier with RSV were pretreated with an injection of CP-122721 (10 mg/kg sc, n = 6) 60 min before the injection of capsaicin. This selective antagonist binds noncompetitively the SP(NK1) receptor, with nanomolar affinity producing long-lasting blockade (28), and thus generating a pharmacological SP(NK1) receptor "knockout." Controls were injected with 0.9% NaCl (1 ml/kg sc, n = 4).

To determine whether the potentiation of SP-induced inflammation in RSV-infected rats was mediated by downregulation of NEP, we measured in duplicate the activity of this enzyme in RSV-infected rats and pathogen-free controls 5 days after inoculation (n = 5/group). One-half of the samples were preincubated with the specific inhibitor phosphoramidon (17) to separate true NEP activity from the activity of other NEP-like peptidases.

To determine whether RSV infection was altering SP(NK1) receptor gene expression, we compared the levels of mRNA extracted from the right lung of RSV-infected and pathogen-free rats killed for the vascular permeability studies 5 days after inoculation (n = 5-6/group). To confirm that changes in gene expression translated into different receptor density, SP binding sites were visualized by autoradiography and quantified by densitometry in lung sections from RSV-infected and pathogen-free rats.

Drugs. All drugs used for the in vivo experiments were delivered in a volume of 1 ml/kg body wt. Evans blue dye was dissolved in 0.9% NaCl. Capsaicin (8-methyl-N-vanillyl-6-nonenamide; Sigma) was dissolved in a vehicle with a final concentration of 0.75% ethanol, 0.375% Tween 80, and 0.85% NaCl in aqueous solution. SP (Sigma) was dissolved in distilled water to obtain the stock solution and then diluted with 0.9% NaCl to final concentration. CP-122721 [(+)-(2S,3S)-3-(2-methoxy-5-trifluoromethoxybenzyl)amino-2-phenylpiperidine; Pfizer Central Research Division] was dissolved in 0.9% NaCl immediately before administration.

Statistical analysis. Values are means ± SE. The effects of RSV on mean values of Evans blue extravasation and NEP enzymatic activity were analyzed by two-factor ANOVA (47). Multiple comparisons between means were performed with Fisher's protected least significant difference test (44). Data obtained by densitometry analysis of RT-PCR and autoradiography products were compared by unpaired Student's t-test. Statistical analysis was performed using the software SuperANOVA (Abacus Concepts, Berkeley, CA). Differences with P < 0.05 were considered significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

RSV detection. Immunoperoxidase staining with specific monoclonal antibodies performed on lung sections from adult F-344 rats killed 5 days after the endotracheal inoculation of RSV revealed the presence of RSV antigens on the membranes and in the cytoplasm of bronchiolar epithelial cells (Fig. 1). No virus was detected in the epithelium of more proximal airways, including the trachea and large bronchi. The preliminary experiments designed to assess the efficiency of endotracheal inoculation confirmed the presence of viral antigens in the bronchiolar epithelium of all rats inoculated with RSV and in none of the rats receiving virus-free medium. The histopathological changes caused by RSV in the airways of F-344 rats were quite variable, ranging from mild epithelial damage to focal bronchopneumonia with predominantly mononuclear cell infiltration (Fig. 2).


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Fig. 1.   Lung section from a Fischer 344 (F-344) rat killed 5 days after intratracheal inoculation of respiratory syncytial virus (RSV). Immunoperoxidase staining was performed using a pool of mouse monoclonal antibodies composed of 4 clones specific for matrix-2 protein, phosphoprotein, fusion protein, and nuclear protein of human RSV. Dark-brown reaction reveals presence of viral antigens on membranes and in cytoplasm of bronchiolar epithelial cells. There was no detectable virus in lungs of control rats inoculated with virus-free medium. Internal scale, 25 µm.



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Fig. 2.   Photomicrograph of hematoxylin and eosin-stained section obtained from lung of RSV-infected F-344 rat killed 5 days after inoculation. Wall of a bronchiole (B) and surrounding pulmonary parenchyma are infiltrated by numerous mononuclear leukocytes, consistent with focal acute bronchopneumonia. Internal scale, 25 µm.

Vascular permeability. In rats injected with vehicle 5 days after inoculation (Fig. 3), RSV infection caused no significant increase in the extravasation of Evans blue-labeled albumin in the respiratory tract. The difference between RSV-infected and pathogen-free rats was not significant in the extrapulmonary airways (P = 0.23) and the intrapulmonary airways (P = 0.49).


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Fig. 3.   Potentiation of airway neurogenic inflammation 5 days after endotracheal administration of RSV or virus-free medium measured in extrapulmonary and intrapulmonary airways of F-344 rats. In rats injected with vehicle, RSV by itself caused only minimal increase in vascular permeability compared with pathogen-free controls. Increase in vascular permeability elicited in extrapulmonary airways by capsaicin and by exogenous substance P (SP) was significantly larger in RSV-inoculated than in pathogen-free rats. In intrapulmonary airways, vascular permeability increased only after injection of SP in RSV-inoculated rats. *** Significantly different from pathogen-free controls: P < 0.001.

After pharmacological stimulation of sensory nerves with capsaicin, Evans blue extravasation in the extrapulmonary airways (Fig. 3) of RSV-infected rats increased significantly and was approximately twice that measured in pathogen-free controls injected with the same dose of capsaicin (P = 0.0001). In contrast, capsaicin had no effect on vascular permeability in the intrapulmonary airways (Fig. 3) of RSV-infected rats (P = 0.61) or pathogen-free controls (P = 0.64).

Mimicking the effect of capsaicin, the intravascular injection of exogenous SP increased Evans blue extravasation in the extrapulmonary airways (Fig. 3) of RSV-infected rats, which was significantly different from pathogen-free controls (P = 0.0001). In contrast to capsaicin, SP also increased significantly Evans blue extravasation in the intrapulmonary airways (Fig. 3) of RSV-infected rats (P = 0.0002).

Selective antagonism of the SP(NK1) receptor with CP-122721 potently inhibited the extravasation of Evans blue-labeled albumin caused by capsaicin in the extrapulmonary airways of RSV-infected rats (38.3 ± 2.8 vs. 119.4 ± 14.7 ng/mg, P = 0.0002). After treatment with this inhibitor, neurogenic albumin extravasation in RSV-infected rats was similar to that in pathogen-free controls (P = 0.32).

NEP activity. NEP activity was measured to verify whether the exaggerated neurogenic inflammation found in the respiratory tract of RSV-infected rats is due to reduced enzymatic degradation of SP as shown previously with other respiratory viruses (influenza and parainfluenza). In this model, most of the Glu-MNA cleaving activity in the extrapulmonary airways (Fig. 4) was blocked by phosphoramidon (90 ± 2% in pathogen-free controls and 86 ± 3% in RSV-infected rats) and was, therefore, attributable to NEP. No significant difference in the activity of this enzyme was found between RSV-infected and pathogen-free rats in the extrapulmonary airways (115.8 ± 12.1 vs. 112.6 ± 10.0 pmol MNA · h-1 · µg protein-1; P = 0.84).


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Fig. 4.   Neutral endopeptidase (NEP)-like enzymatic activity in extrapulmonary and intrapulmonary airways of F-344 rats 5 days after endotracheal administration of RSV or virus-free medium. Activity was assessed by measuring fluorometrically generation of free methoxy-2-naphthylamine (MNA) from substrate glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine. In each bar, bottom segment represents component inhibited by specific NEP inhibitor phosphoramidon; top segment represents noninhibitable component. No significant difference in enzymatic activity was found between RSV-infected and pathogen-free rats.

In the lungs (Fig. 4), phosphoramidon inhibited approximately one-half of the total Glu-MNA cleaving activity (56 ± 2% in pathogen-free controls and 53 ± 1% in RSV-infected rats). There was a trend for increased cleaving activity in RSV-infected rats, but the difference in phosphoramidon-inhibitable activity (277.7 ± 41.1 vs. 220.4 ± 12.9 pmol MNA · h-1 · µg protein-1; P = 0.22) and noninhibitable activity (244.6 ± 37.3 vs. 176.0 ± 11.5 pmol MNA · h-1 · µg protein-1; P = 0.12) was insignificant.

SP(NK1) receptor. Semiquantitative RT-PCR analysis of the SP(NK1) receptor mRNA from the lung tissues consistently revealed a much stronger signal in RSV-infected rats than in pathogen-free controls. After normalization of the densitometry measurements to the internal standard (the housekeeping gene GAPDH; Fig. 5, top), the level of SP(NK1) receptor mRNA was 4.6-fold higher in the lungs of RSV-infected rats 5 days after inoculation (Fig. 5, bottom) than in pathogen-free controls (P = 0.0001; see Fig. 7, top).


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Fig. 5.   Amplification of SP(NK1) receptor mRNA from lung tissues of F-344 rats 5 days after endotracheal inoculation of RSV or virus-free medium. Total RNA was reverse transcribed to cDNA, amplified by PCR with use of primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and for SP(NK1) receptor, and analyzed by electrophoresis on an ethidium bromide-stained agarose gel. +, Amplification product of full-length rat SP(NK1) receptor cDNA, used as a positive control. Each band was obtained from lungs of a different animal (6 pathogen-free and 5 RSV-infected rats).

Analysis of the localization of SP binding sites in sections from RSV-infected and pathogen-free lungs revealed specific binding overlying the bronchial mucosa and the wall of the adjacent arterial and venous pulmonary vessels (Fig. 6). No binding was observed over the airway smooth muscle and alveolar tissue. Consistent with the observed change in gene expression, the density of binding sites assessed as OD was threefold higher in the airways of RSV-infected lungs than in pathogen-free controls (P = 0.0002; Fig. 7, bottom).



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Fig. 6.   Autoradiographic mapping of SP binding sites in lungs of RSV-infected (A-D) and pathogen-free (E-H) rats 5 days after inoculation. Left to right: schematic diagrams (A and E), hematoxylin and eosin-stained preparations (B and F), autoradiographic images (C and G), and autoradiography digitally superimposed to hematoxylin and eosin (D and H). Nonspecific binding for RSV-infected (I-L) and pathogen-free (M-P) rats was determined by incubation of adjacent tissue sections under same conditions in presence of 1 µM unlabeled SP. Specific SP binding was detected over bronchial mucosa and wall of adjacent pulmonary vessels and was consistently and markedly increased in infected airways. No significant binding was detected over airway smooth muscle and alveolar tissue. Internal scale, 0.5 mm.



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Fig. 7.   RSV-induced upregulation of SP receptor at gene and protein level. Densitometry analysis of RT-PCR bands normalized to internal control GAPDH (top) revealed ~5-fold increase in SP(NK1) receptor mRNA from lungs of RSV-infected rats compared with pathogen-free controls. Accordingly, density of SP binding sites (bottom) increased 3-fold in bronchial mucosa of RSV-infected lungs. *** Significantly different from pathogen-free controls: P < 0.001.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study shows for the first time that lower respiratory tract infection with RSV in adult F-344 rats causes a potentiation of neurogenically mediated inflammatory reactions as manifested by the exaggerated increase in microvascular permeability in response to endogenous and exogenous SP observed 5 days after inoculation of the virus. Selective antagonism of the SP(NK1) receptor with CP-122721 (28) abolished the effect of RSV on airway neurogenic inflammation.

The potentiation of capsaicin-induced neurogenic inflammation was significant in the extrapulmonary, but not in the intrapulmonary, airways of RSV-infected rats. This observation suggests that the density of capsaicin-sensitive C-type nerves is highest in the proximal airways and progressively decreases in more distal airways, confirming previous observations (26). However, our study shows that intrapulmonary airways exposed to RSV respond to exogenous SP with a large increase in vascular permeability, suggesting that RSV-infected distal airways become hyperresponsive to intravascular SP, which may participate in the inflammatory and immune responses against this virus. Thus our findings suggest that SP may exert at least part of its biological effects distant from the release sites and are consistent with other studies that demonstrated a significant mismatch in the localization of SP-containing nerves and SP receptors in the rat brain (23) and lungs (18). It is also possible that SP is locally released in the distal airways by cellular sources other than nerve fibers.

The presence of RSV in the lower respiratory tract 5 days after endotracheal inoculation was confirmed by immunohistochemical localization of specific viral antigens in the bronchiolar epithelium. However, viral antigens could not be detected in the proximal airways of RSV-inoculated rats where we found a strong potentiation of neurogenically mediated inflammation. This observation suggests that the influence of RSV on the regulatory elements of the neurogenic inflammatory pathway may require the release of intermediate factor(s) from the infected bronchiolar epithelium or inflammatory cell infiltrate. This factor may be one of the proinflammatory cytokines released from RSV-infected epithelial cells in vitro, such as the granulocyte-macrophage colony-stimulating factor, interleukin-6 (30), RANTES (41), or interleukin-11 (12).

In this study, RSV infection of adult F-344 rats was obtained using a relatively small inoculum (5 × 104 TCID50) and caused variable histopathological changes, ranging from mild epithelial damage to focal bronchopneumonia with predominantly mononuclear cell infiltration. Preliminary experiments suggest that a possible factor for the limited tissue damage observed is that adult rats may be less susceptible than newborn or weanling rats to the pathogenetic effects of RSV infection as reported previously with parainfluenza virus (8). Another important characteristic of this F-344 rat model is that no evidence of viral antigens can be found in the lungs 30 days after inoculation of RSV (G. Piedimonte, unpublished observation). Thus the time course of RSV infection in adult F-344 rats resembles the typical course in humans, in contrast to other animal models (e.g., guinea pigs) that are unable to clear this virus even after several months (10). Therefore, our model may be useful to study the long-term physiological abnormalities after a mild and transient lower respiratory tract infection with RSV.

Effect of RSV on NEP enzymatic activity. Our data indicate that 5 days after the endotracheal inoculation of RSV, when neurogenic inflammation is markedly potentiated by this virus, NEP-like activity in the respiratory tract is not affected significantly. Thus the mechanism of RSV-induced potentiation, in contrast to other respiratory viruses (35), cannot be explained on the basis of reduced enzymatic inactivation of SP after release from sensory nerves. Our data also show that the enzymatic activity in the extrapulmonary airways is almost entirely inhibited by the specific NEP inhibitor phosphoramidon, whereas in the intrapulmonary airways, there is a larger contribution from other peptidases insensitive to phosphoramidon inhibition. We found a trend of increased enzymatic activity in the intrapulmonary airways of RSV-inoculated rats, but this trend was far from significant.

Previous studies in rats with murine parainfluenza type I (Sendai) virus revealed that viral respiratory infections strongly potentiate the increase in permeability of tracheal blood vessels and the adherence of neutrophils to the vascular endothelium produced by neurogenic inflammation (33, 36). This effect was observed 5-6 days after inoculation of Sendai virus when the pathological changes are maximal. The primary mechanism responsible for the virus-induced potentiation appeared to be the loss of epithelial cells rich in NEP, a peptide-degrading enzyme that modulates the biological activity of SP and other tachykinins (35). This enzymatic activity is concentrated in the basal layer of the respiratory epithelium and is markedly reduced in the airways of rodents infected with influenza or parainfluenza viruses (3, 11, 19). However, this mechanism alone cannot explain the increased response to intravenous SP or the potentiation produced by other pathogens (e.g., Mycoplasma pulmonis) in the absence of significant epithelial damage (27). More recent studies have shown that airway neurogenic inflammation can be potentiated by nonreplicating recombinant adenovirus in rats with intact respiratory epithelium (37) and with a normal level of NEP-like activity (34). Similarly, the data shown in the present study suggest that the potentiation of neurogenic inflammation associated with the presence of RSV in the respiratory tract involves a different postsynaptic mechanism(s) independent from the activity of peptide-degrading enzymes.

Effect of RSV on SP(NK1) receptor mRNA. SP binds with high affinity to the NK1 tachykinin receptor subtype (39). In our study, semiquantitative RT-PCR analysis revealed that the expression of the gene encoding this receptor is strongly upregulated in the lung tissues of rats inoculated 5 days earlier with RSV. At the same time, the density of binding sites for SP visualized by autoradiography is markedly increased in the bronchial mucosa of RSV-infected lungs. The distribution of binding sites visualized in our preparations is remarkably consistent with previous immunohistochemical studies in rat lungs with use of anti-NK1 antibodies (18).

Part of the increase in SP(NK1) receptor mRNA may be due to the influx of inflammatory cells bearing the receptor. However, our autoradiography preparations clearly show SP binding overlying the airway epithelium and the vascular walls as represented in Fig. 6. These preparations also consistently show a dramatic difference in epithelial and endothelial binding between pathogen-free and infected airways, suggesting overexpression of SP binding sites in the structural elements of RSV-infected airways.

Our genetic, pharmacological, and physiological data show for the first time that the potentiating effect of RSV on neurogenic inflammation in the respiratory tract of rats is mediated by overexpression of SP(NK1) receptors in infected tissues via increased mRNA levels, which could derive from changes in transcription rate or message stability. Our present data offer no information concerning other aspects of the neurogenic inflammatory pathway, such as receptor affinity and recycling or intracellular signal transduction after ligand-receptor binding, which may also be affected during viral infections. In addition, the autoradiographic technique used in this study was specifically aimed at the quantitative analysis of receptor expression and lacks the resolution necessary to identify individual substructures within the respiratory mucosa, particularly the endothelial cells of postcapillary venules that are responsible for inflammatory plasma extravasation. Immunohistochemical studies with anti-NK1 antibody have shown that this receptor is expressed at very low density on the surface of postcapillary venular endothelium in the tracheal mucosa of pathogen-free F-344 rats (5).

Previously published data (22) suggested a reduction in SP receptor number and agonist affinity in the trachea of guinea pigs during the 1st wk after inoculation of parainfluenza virus type 3. Several technical aspects of that study, including animal species and infecting virus, are different from our model. However, it should be noted that the data published by Kudlacz and co-workers (22) propose a surprising lack of correlation between the physiological responses to SP, which were enhanced in infected animals, and the expression of SP receptors, which were apparently downregulated. In our study, we found concordance between physiological responses to SP, SP(NK1) receptor mRNA levels, and ligand-receptor binding, all being markedly and consistently upregulated in RSV-infected rats.

The sensory nerves of the upper (2) and lower (24) respiratory tract of several species, including humans, contain SP-immunoreactive fibers in the airway epithelium and smooth muscle and around blood vessels and submucosal glands. In addition to the effect on vascular permeability, SP binding to its high-affinity receptor (NK1) stimulates leukocyte adhesion to the vascular endothelium (20, 25), endothelium-dependent vasodilation (32), gland secretion (1, 4), and mucociliary clearance (45). These proinflammatory properties of SP in the respiratory tract suggest that the SP-NK1 receptor interaction may play an important role in the pathogenesis of airway inflammation.

The importance of the SP(NK1) receptor in the organization of pulmonary inflammatory reactions has been highlighted recently by the observation that gene-targeted deletion of this receptor in mice prevents the acute inflammatory responses associated with immune complex-mediated lung injury, including plasma extravasation and neutrophil influx (6). Furthermore, selective pharmacological inhibition of the SP(NK1) receptor in mice markedly reduces leukocyte influx in the lungs after antigen challenge (21). These recent studies expand an already large body of evidence indicating that SP and other neuropeptides play an important modulatory role upstream from other inflammatory pathways in the respiratory tract (9). In addition, together with our present finding of the upregulation of the SP(NK1) receptor in RSV-infected lungs, these studies suggest that SP may play a role in the inflammatory processes affecting the distal airways and the lung parenchyma, whereas most previous studies had focused on the large airways.

Conclusions. This study shows that RSV renders the proximal and distal airways of adult rats abnormally susceptible to the proinflammatory effect of the neuropeptide SP. However, RSV does not affect the enzymatic activity of NEP as previously reported for other viruses, and, therefore, its effect cannot be explained with decreased catabolism of SP after being released from nerve fibers. Rather, the increased levels of SP(NK1) receptor mRNA and the increased number of SP binding sites in the lungs of RSV-infected rats suggest that the potentiating effect of RSV is linked to upregulation of this receptor due to increased gene expression. The potentiation of neurogenic-mediated inflammation in response to RSV infection may contribute to the pulmonary inflammatory reaction to this virus observed in humans. If so, pharmacological modulation of this inflammatory pathway via receptor antagonists may minimize the acute response to RSV and perhaps influence the development of airway hyperresponsiveness observed in a large proportion of children with a history of RSV bronchiolitis.


    ACKNOWLEDGEMENTS

We thank Dr. James Krause for providing the full-length SP(NK1) receptor cDNA and Dr. John A. Lowe III for providing the SP(NK1) receptor antagonist CP-122721. We also thank Dr. Adam Wanner for intellectual contribution.


    FOOTNOTES

This research project was supported in part by the 1997 Career Investigator Award of the American Lung Association of Florida and by a research grant from MedImmune, Inc., to G. Piedimonte.

Some of the findings reported in this paper were presented at the 1997 North American Cystic Fibrosis Conference and at the 1998 American Thoracic Society/American Lung Association Conference.

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: G. Piedimonte, Pediatric Pulmonology Div., University of Miami School of Medicine, 1601 NW 12th Ave., Miami, FL 33136 (E-mail: gpiedimo{at}med.miami.edu).

Received 8 February 1999; accepted in final form 10 June 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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