Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections in infants (1), resulting in more than 90,000 hospitalizations and 4,500 deaths annually in the United States. In addition, there is a growing concern that early RSV infection is an important risk factor for the development of recurrent wheezing and childhood asthma (2, 3). Because immunity after the first infection is not complete, RSV is a common cause of respiratory infections also in older children and adults, but the clinical manifestations usually remain confined to the upper airways.

The mechanisms of RSV-induced airway inflammation and hyperreactivity are still unclear. Previous studies have proposed that early RSV infection in young rodents may cause dysregulation of the nonadrenergic, noncholinergic (NANC) nervous system in the developing respiratory tract of young rodents, thus potentiating the bronchoconstrictive effect of tachykinin peptides like substance P (SP) against the bronchorelaxant effect of the vasoactive intestinal peptide (VIP) (4). More recently, we found that RSV infection in adult Fischer F-344 rats is associated with upregulation of the messenger RNA (mRNA) encoding the high-affinity SP receptor neurokinin (NK) 1, which translates into a large increase in SP binding sites expressed by the airway epithelium and vascular endothelium (5). This effect determines exaggerated inflammation after nerve stimulation (neurogenic inflammation) in the extrapulmonary airways, but no difference can be found in the intrapulmonary airways due to the low density of peptidergic fibers.

However, little information is available concerning neurogenic inflammation caused by RSV infection early in life. The effects of this virus in the lower respiratory tract during a critical period of the development of neural pathways and of the maturation of immune competence may trigger inflammatory responses qualitatively and/or quantitatively different from those found in adult airways. This information is important considering that the acute and chronic manifestations of RSV disease are strongly age-dependent.

Therefore, in the present study we developed a model of RSV infection in weanling F-344 rats and measured the changes in neurogenic-mediated inflammation during the acute viral infection. We also assessed the protective effect of a selective NK1 receptor antagonist (CP-122721) (6) and of a humanized monoclonal antibody specific for the viral fusion (F) glycoprotein (palivizumab) (7) against RSV-induced neurogenic inflammation. Finally, we compared the effect of RSV on the expression of key receptors mediating the inflammatory and immune effects of NANC neuropeptides, the tachykinin receptor subtypes NK1 and NK2 (8), and the VIP receptors subtypes VIPR1 and VIPR2 (9).

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animals

We used weanling rats, strain Fischer F-344, born in our barrier facility to pathogen-free, timed-pregnant dams from Charles River Breeding Laboratories (Raleigh, NC). To prevent microbial contamination, groups of two dams 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) (5, 10). We used separate rooms for housing infected and pathogen-free rats, both 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 both 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 and Inoculation of RSV

HEp-2 cells from the American Type Culture Collection (ATCC; Rockville, MD) were grown in Eagle's minimum essential medium (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 long strain (ATCC) and the infection was allowed to proceed at 37°C in 5% CO₂ atmosphere until more than 75% of the cells exhibited a cytopathic effect. Cell debris were removed by centrifugation at 9,500 rpm for 20 min in a refrigerated centrifuge (4°C). 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 TCID₅₀ (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.

Rats were inoculated intranasally at 18 ± 2 d of age under methoxyflurane anesthesia (0.2 ml/kg administered by open drop; Metofane; Mallinckrodt Veterinary, Mundelein, IL). A volume of 20 µl of RSV suspension containing 5 × 10⁴ TCID₅₀ in 0.1 ml was deposited in each nostril. Control weanling rats were dosed with 20 µl per nostril of virus-free medium.

Virus Detection and Histopathology

The right lung from each animal was fixed in 10% buffered formalin, embedded in paraffin, and cut in 3-µm-thick sections. Hematoxylin and eosin staining was performed for histopathologic 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 3% hydrogen peroxide for 5 min. A jar was filled with target retrieval diluted 1:10 (DAKO, Carpinteria, CA) and was heated at 90°C for 30 min in a steamer. The slides were placed in the jar for 20 min, allowed to cool for 30 min, and then left overnight in phosphate buffered saline (PBS; Sigma, St. Louis, MO) at room temperature.

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 (M) 2 protein, phosphoprotein (P), fusion protein (F), and nuclear protein (N) of human RSV (Vector Laboratories, Burlingame, CA). This technique has been shown to maximize the sensitivity of RSV detection (11). The localization of anti-RSV antibodies was delineated by the streptavidin-biotin peroxidase complex method using an immunostaining kit (DAKO) 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 brown precipitate lining the cell membrane and cytoplasm.

All slides were coded and were interpreted separately by two pathologists who did not know whether the section corresponded to an RSV-inoculated or to a medium-inoculated animal. Histopathologic changes were graded as minimal (occasional inflammatory cells), mild (five to 10 inflammatory cells in three or more adjacent alveoli), moderate (10 to 20 inflammatory cells per alveolus), or severe (> 20 inflammatory cells per alveolus).

Albumin Extravasation

Rats were anesthetized with sodium pentobarbital. Evans blue dye (30 mg/kg intravenously over 5 s) was injected to measure the extravasation of albumin from airway blood vessels (12). Immediately after the injection of the tracer, groups of RSV-infected and pathogen-free rats received a 2-min intravenous infusion of 75 µg/kg of capsaicin (8-methyl-N-vanillyl-6-nonenamide; Sigma) dissolved in a vehicle with a final concentration of 0.75% ethanol, 0.375% Tween 80, and 0.85% NaCl in aqueous solution. Control rats received an infusion of vehicle (1 ml/kg). All chemicals were delivered in a volume of 1 ml/kg of body weight.

Five minutes after the injection of the tracer, the chest was opened and a 22-gauge cannula was inserted into the ascending aorta through the left ventricle. After incision of the left atrium, the circulation was perfused with 40 ml of PBS over 1 min using a syringe pump. The extrapulmonary airways (from the first tracheal ring to the end of the mainstem 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 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 to 10 µg/ml).

Reverse Transcription/Polymerase Chain Reaction

The mRNA levels of neuropeptide receptors in lung tissues were measured by semiquantitative reverse transcription/polymerase chain reaction (RT-PCR) based on previously published work (5, 13). 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 complementary DNA (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 8.4), 50 mM KCl, 5 mM MgCl₂, 1 mM of each deoxynucleotide triphosphate, 1 U/µl RNAse inhibitor, and 2.5 µM oligo-d(T)₁₆ primer. Moloney murine leukemia virus reverse transcriptase (2.5 U/µl; GIBCO-BRL) was added to each tube and the reaction was allowed to proceed for 50 min at 42°C, then for 15 min at 72°C. Aliquots of 5 µl of the synthesized cDNA (corresponding to 100 ng RNA) were added to a 45-µl PCR mixture containing 5 µl 10× PCR buffer, 2 µl deoxynucleotides (0.4 mM each), 1 µl 3' and 5' specific primers (0.2 µM each), and 0.25 µl AmpliTaq Gold DNA polymerase (5 U/µl; Perkin-Elmer, Foster City, CA).

PCR amplification of the neuropeptide receptors was performed using the following primer sequences: NK1 sense, 5'-CATCAACCCAGATCTCTACC-3', antisense, 5'-GCTGGAGCTTTCTGTCATGGA-3'; NK2 sense, 5'-CATCACTGTGGACGAGGGGG-3', antisense, 5'-TGTCTTCCTCAGTTGGTGTC-3'; VIPR1 sense, 5'-GCCCGCAGC-ACGAGTGTGAGTACC-3', antisense, 5'-TTGCTGCTCATCCAGACTCGATGC-3'; VIPR2 sense, 5'-TCCCAGCAGGTGTTTCCTGGCCTAC-3', antisense, 5'-CGAGCCTCTTGTACTGTGACTGGTC-3'. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified simultaneously as an internal standard using the following primer sequences: sense, 5'-TGAAGGTCGGTGTCAACGGATTTGGC-3', antisense, 5'-CATGTAGGCCATGAGGTCCACCAC-3'. Amplification was initiated with 10 min denaturation at 94°C for one cycle 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). 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 2% agarose gel at 45 V for 120 min. The cDNA bands were visualized by ultraviolet illumination after staining the gels 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 the films were scanned and analyzed with a computerized densitometer.

Experimental Protocols

In each experiment, body weight was measured at the end of the 5-d incubation period and the right lung was removed after perfusion for RSV detection and histopathologic analysis.

To determine whether the intranasal administration of RSV resulted in increased neurogenic inflammatory responses, 15 rats were inoculated with RSV and 15 rats were dosed with virus-free medium. Five days after inoculation, capsaicin was injected into nine RSV-infected rats and eight pathogen-free rats to stimulate sensory nerves in the respiratory tract (14). The other six RSV-infected and seven pathogen-free rats received an injection of vehicle used to dissolve the capsaicin.

To determine whether the neurogenic inflammatory reaction associated with RSV infection can be prevented by blocking SP binding to its high-affinity receptor (NK1 receptor), two groups of weanling rats inoculated 5 d earlier with RSV were pretreated with a subcutaneous injection of CP-122721 ([+]-[2S-3S]-3-[2-methoxy-5-trifluoromethoxybenzyl]-amino-2-phenyl-piperidine; Pfizer Central Research Division, Groton, CT; 10 mg/kg, n = 5) 60 min before the injection of capsaicin. This selective antagonist binds noncompetitively the NK1 receptor with nanomolar affinity producing a long-lasting blockade (5, 6). Controls were injected subcutaneously with 0.9% NaCl (1 ml/kg; n = 5), which was used to dissolve CP-122721.

To assess whether a monoclonal antibody (mAb) specific for the viral F protein is protective against RSV-induced neurogenic inflammation in the intrapulmonary airways, a group of weanling rats was treated 24 h before the inoculation of RSV with a subcutaneous injection of palivizumab (MedImmune, Inc., Gaithersburg, MD; 15 mg/kg in 0.9% NaCl; n = 5 rats) or its vehicle (0.9% NaCl; 1 ml/kg; n = 5 rats). A control group of weanling rats (n = 5) was injected with vehicle 24 h before intranasal administration of virus-free medium. The dose of mAb used in this study corresponds to the dose currently used in routine clinical practice on the basis of previous studies conducted in animal models (15, 16).

To determine the levels of mRNAs encoding neuropeptide receptors, we extracted total RNA from lung homogenates of pathogen-free rats (n = 5) and of RSV-infected rats with or without mAb prophylaxis (n = 6 rats per group) and performed semiquantitative RT-PCR amplification using primers specific for the NK1, NK2, VIPR1, and VIPR2 receptors. The NK3 tachykinin receptor subtype was not investigated because it has not been detected in structural cells of the respiratory tract (17, 18) or in cells with inflammatory or immune functions (8).

Statistical Analysis

Data are expressed as the mean ± standard error of the mean. 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 (19). Multiple comparisons between means were performed with the Fisher Protected Least Significant Difference test (20). Statistical analysis was performed using the software StatView version 5.0 (Abacus Concepts, Berkeley, CA). Differences having a P value < 0.05 were considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

RSV Detection and Histopathology

Immunoperoxidase staining with specific mAbs performed on lung sections from weanling F-344 rats killed 5 d after the endotracheal inoculation of RSV at the age of 23 ± 2 d revealed the presence of RSV antigens on the membranes and in the cytoplasm of bronchiolar epithelial cells (Figure 1). Hematoxylin and eosin-stained microscopic sections from rats inoculated with RSV revealed interstitial and peribronchial cellular infiltrates made predominantly of monocytes and lymphocytes, which were graded from mild to moderate by our pathologists (Figure 2). RSV-infected rats were also characterized by the presence of hyperplastic bronchial-associated lymphoid tissue compared with pathogen-free control rats, confirming our previous report (21). The lungs of control rats dosed with virus-free medium had no evidence of viral antigens and the pathologic changes in rats were graded minimal to none.

View larger version (142K): [in this window] [in a new window]   Figure 1.   Lung sections from 23-day-old Fischer F-344 rats killed 5 d after the intranasal inoculation of virus-free medium (A) or 40 µl of a suspension containing 5 × 104 TCID50/0.1 ml of RSV (B). Immunoperoxidase staining was performed using a pool of mouse mAbs composed of four clones specific for the proteins M2, P, F, and N of human RSV. The brown reaction reveals the presence of viral antigens on the membranes and in the cytoplasm of bronchiolar epithelial cells from the RSV-inoculated rats. There was no detectable virus in the lungs of control rats inoculated with virus-free medium. Internal scale = 50 µm.

View larger version (71K): [in this window] [in a new window]   Figure 2.   Photomicrographs of hematoxylin and eosin-stained sections obtained from the lungs of 23-day-old F-344 rats killed 5 d after inoculation of virus-free medium (A) or RSV suspension (B). The peribronchial areas and the surrounding pulmonary interstitium of RSV-inoculated rats are infiltrated by numerous mononuclear leukocytes. No significant pathologic changes were noted in the lungs of medium-inoculated control rats. Internal scale = 200 µm.

Body Weight

The average final body weight measured at the end of the 5-d incubation period before the vascular permeability experiments was significantly lower in the RSV-inoculated weanling rats (30.3 ± 0.9 g, n = 25 rats) than in the medium-inoculated controls (36.2 ± 1.7 g, n = 20 rats; P = 0.03). Analysis of the effect of mAb prophylaxis shows that this antibody had no effect on final body weight, which was similar to nontreated RSV-infected rats (32.6 ± 1.0 versus 33.8 ± 0.4 g; P = 0.4) and significantly lower than pathogen-free control rats (45.4 ± 1.1 g; P = 0.0001).

Vascular Permeability

In the absence of sensory nerve stimulation, the extravasation of Evans blue-labeled albumin measured in the intrapulmonary and extrapulmonary airways of RSV-infected weanling rats was not significantly different from age-matched pathogen-free rats inoculated with medium obtained from virus-free cell cultures (Figure 3; P > 0.5). Stimulation of capsaicin-sensitive sensory nerves 5 d after inoculation caused a larger increase of Evans blue-labeled albumin extravasation in the intrapulmonary airways of RSV-infected rats compared with pathogen-free control rats (Figure 3, top; P = 0.01). In contrast, analysis of the extrapulmonary airways showed no difference in albumin extravasation between infected and pathogen-free rats (Figure 3, bottom; P = 0.3).

View larger version (33K): [in this window] [in a new window]   Figure 3.   Potentiation of airway neurogenic inflammation 5 d after the endotracheal administration of RSV or virus-free medium measured in the intrapulmonary airways (top panel) and extrapulmonary airways (bottom panel) of weanling F-344 rats. In rats injected with vehicle (left columns), RSV did not change vascular permeability compared with pathogen-free controls. However, the increase in vascular permeability elicited in the intrapulmonary airways by capsaicin (right columns) was significantly larger in RSV-infected rats (solid bars) than in pathogen-free controls (hatched bars). In the extrapulmonary airways, vascular permeability did not change significantly. *P < 0.05, significantly different from pathogen-free control rats.

Selective antagonism of the high-affinity SP receptor with CP-122721 completely abolished the extravasation of Evans blue-labeled albumin caused by capsaicin in the intrapulmonary airways of RSV-infected weanling rats (39.3 ± 7.4 versus 115.7 ± 7.1 ng/mg; P < 0.0001).

Also, RSV-induced potentiation of neurogenic inflammation in the intrapulmonary airways of weanling rats was reduced significantly by the prophylactic administration of mAb 24 h before inoculation of the virus (Figure 4; P = 0.02). Albumin extravasation in mAb-treated infected rats was larger than in pathogen-free control rats, although this difference did not reach statistical significance (P = 0.06).

View larger version (22K): [in this window] [in a new window]   Figure 4.   Protective effect of palivizumab (mAb) on the potentiation of neurogenic inflammation induced by RSV in the intrapulmonary airways of weanling F-344 rats. RSV was inoculated intranasally 24 h after the injection of mAb (hatched column) or its vehicle (solid column). Control rats (open column) were inoculated with virus-free medium and injected with vehicle. The extravasation of Evans blue-labeled albumin produced by capsaicin in RSV-infected weanling rats was reduced significantly by pretreatment with mAb. *P < 0.05, significantly different from RSV-infected rats treated with vehicle.

Receptors Expression

Semiquantitative RT-PCR analysis of lung tissues for mRNAs encoding neuropeptide receptors revealed significant qualitative and quantitative differences induced by RSV (Figure 5). After normalization of the densitometry measurements to the internal standard (encoding the housekeeping gene GAPDH), the level of NK1 receptor mRNA in the lungs of RSV-infected rats 5 d after inoculation was 3.7-fold higher than in pathogen-free control rats (Figure 6, top left; P = 0.002). NK1 mRNA levels from the lungs of RSV-infected rats pretreated with mAb were significantly lower than in nontreated infected rats (P = 0.003) and were similar to pathogen-free control rats (P = 0.7). VIPR1 mRNA also increased in RSV-infected rats (Figure 6, top right; P = 0.01), but the magnitude of this increase (+43%) was consistently much smaller compared with the NK1 receptor. The difference in VIPR1 receptor mRNA between RSV-infected rats pretreated with mAb and pathogen-free controls was smaller and not statistically significant (P = 0.3). The levels of mRNA encoding NK2 and VIPR2 receptors (Figure 6, bottom) were not affected by the virus (P > 0.4) or by mAb (P > 0.5).

View larger version (48K): [in this window] [in a new window]   Figure 5.   Amplification of neuropeptide receptor mRNA from the lung tissues of F-344 rats 5 d after intranasal inoculation of virus-free medium (lanes 2 to 6) or RSV without (lanes 7 to 12) or with (lanes 13 to 18) palivizumab (mAb) prophylaxis. Each band was obtained from the lungs of a different animal. Total RNA was reverse-transcribed to cDNA, amplified by PCR using primers specific for GAPDH (top panel) and for the NK1, NK2, VIPR1, and VIPR2 receptors, and analyzed by electrophoresis on an ethidium bromide-stained agarose gel. Lane 1 shows the ladder of molecular weight standards. The size of amplification products was 999 bp for GAPDH, 380 bp for NK1, 491 bp for NK2, 311 bp for VIPR1, and 271 bp for VIPR2.

View larger version (26K): [in this window] [in a new window]   Figure 6.   Modulation of neuropeptide receptor expression by RSV. Densitometry analysis of RT-PCR bands normalized to the internal control GAPDH revealed an approximately fourfold increase in NK1 receptor mRNA from the lungs of RSV-infected rats (solid bars) compared with pathogen-free controls (open bars), whereas the VIPR1 receptor mRNA increased by approximately 40%. mRNA levels for the NK2 and VIPR2 receptor subtypes were not affected by the virus. Palivizumab (mAb) prevented upregulation of the NK1 receptor in RSV-inoculated rats (hatched bars). *P < 0.05; **P < 0.01, significantly different from pathogen-free controls.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

This study shows that lower respiratory tract infection with RSV in weanling Fischer F-344 rats inoculated 18 ± 2 d after birth causes potentiation of neurogenic-mediated inflammatory reactions, as manifested by the exaggerated increase in microvascular permeability in response to stimulation of capsaicin-sensitive sensory nerves observed 5 d after inoculation of the virus. Selective antagonism of the high-affinity SP receptor (NK1 receptor) abolished the effect of RSV on airway neurogenic inflammation.

The potentiation of capsaicin-induced neurogenic inflammation was significant in the intrapulmonary, but not in the extrapulmonary, airways of RSV-infected weanling rats. Therefore, the response to capsaicin of the airway blood vessel in young rats is qualitatively different from RSV-infected adult rats that have extrapulmonary airways much more sensitive to capsaicin than are their intrapulmonary airways (5). The difference found between young and adult rats suggests age-dependent variability in the distribution of capsaicin-sensitive sensory nerves along the respiratory tract. Our previous studies in adult rats also showed that despite the relative paucity of sensory nerves (22), RSV-infected distal airways become hyperresponsive to intravascular SP due to the upregulation of NK1 receptors (5), which may represent a remnant of the neurogenic pathway lost during development. Thus, the mismatch in the localization of SP-containing nerves and SP receptors found in several organs of adult animals (e.g., brain [23] and lungs [24]) could be explained by the progressive change in the architecture of the peripheral nervous system during development. Furthermore, we speculate that the different clinical manifestations of RSV disease in infants (lower respiratory tract involvement with bronchiolitis) versus children and adults (signs and symptoms limited to the upper airways) may result at least in part from age-related differences in one or more inflammatory pathways and should not be determined by purely anatomic factors (e.g., airway caliber).

Our data also show that the prophylactic administration of a monoclonal immunoglobulin G (IgG) antibody against the surface F glycoprotein of RSV reduces the effect of this virus on neurogenic-mediated inflammation in the intrapulmonary airways of weanling rats. Passively acquired RSV-neutralizing antibodies do not protect against infection of the upper airways but rather oppose spreading of the virus to the lower respiratory tract (10, 25), suggesting that the presence of viral particles in the bronchiolar epithelium is important for the development of neurogenic inflammation in the lungs. Thus, we speculate that RSV infections limited to the upper airways have no pulmonary consequences caused by neural reflexes or other indirect mechanisms, which may be involved in the pathogenesis of bronchial obstruction in patients infected with other respiratory viruses (e.g., rhinoviruses) (26).

An incidental finding was the significant difference in body weight between pathogen-free and RSV-infected weanling rats, which was not prevented by mAb prophylaxis. This finding has been extremely consistent in a large number of experiments and we have also found that the difference in weight remains significant even when the animals are killed 30 d after inoculation (G. Piedimonte, unpublished observation), suggesting that RSV may have other nonrespiratory effects interfering with growth and that these effects may be long-lasting.

Effect of RSV on Receptor mRNA

This study reveals that the expression of the gene encoding the high-affinity SP receptor (NK1 receptor) is upregulated in the lungs of weanling rats inoculated 5 d earlier with RSV. Pretreatment with a mAb against the F protein of RSV prevented upregulation of the NK1 receptor. The increase in NK1 receptor transcripts measured in young rats was of the same magnitude measured in RSV-infected adult rats (12 to 14 wk old) versus age-matched, pathogen-free control rats. In contrast, RSV increased to a much lesser degree the expression of VIPR1 receptors and did not affect the expression of NK2 and VIPR2 receptor subtypes. These data suggest that RSV differentially modulates the expression of specific neuropeptide receptors, causing an imbalance between the proinflammatory (SP-dependent) and antiinflammatory (VIP-dependent) components of the NANC nervous system in the respiratory tract and favoring the development and maintenance of airway inflammation. In addition, because SP and VIP modulate several immune functions by exerting opposite influences, the peptidergic imbalance caused by RSV may link the neurogenic and immunoinflammatory mechanisms proposed by different authors to explain the pathophysiology of RSV disease and its sequelae (2).

Part of the increase in NK1 receptor mRNA may be due to the influx of inflammatory cells bearing the receptor. However, our previous study using autoradiography techniques revealed SP binding overlying the airway epithelium and the vascular walls, as well as a dramatic difference in epithelial and endothelial binding between pathogen-free and infected airways, suggesting overexpression of SP binding sites in structural elements of RSV-infected airways (5).

The sensory nerves of the upper and lower respiratory tract of several species, including humans, contain a rich supply of SP- and VIP-immunoreactive fibers in the airway epithelium and smooth muscle, and around blood vessels and submucosal glands (27). SP effects are generally proinflammatory and immunostimulatory (17, 28). In addition, SP is a potent mitogen for several cell types, including smooth muscle (29), fibroblasts (30), and endothelial cells (31), and therefore may play a role in airway remodeling. In contrast, VIP exerts a broad spectrum of antiinflammatory and immunoinhibitory activities in the lungs (9, 32), and by inhibiting airway smooth muscle proliferation (33), it may also protect against airway remodeling, possibly antagonizing the mitogenic effect of SP.

The biologic effects of SP and VIP are mediated by rhodopsin-like receptors with seven transmembrane domains coupled to G proteins. Each mammalian tachykinin (SP, NKA, and NKB) can act as a full agonist on all three neurokinin receptor subtypes if present at sufficiently high concentration (17). However, SP, NKA, and NKB display preferential affinity for NK1, NK2, and NK3 receptors, respectively (8). The NK1 receptor subtype mediates most of the inflammatory, immune, and mitogenic effects of SP. Although the NK2 receptor primarily modulates airway smooth muscle tone, it is also involved in neurogenic inflammation via the activation of alveolar macrophages (34). The low level of NK2 receptor mRNA found in RSV-infected weanling rats is consistent with the observation that this virus does not affect bronchial smooth muscle responsiveness to NKA in our model (G. Piedimonte, unpublished observation). VIP binds with 10-fold higher affinity to VIPR1 than to VIPR2, but the two receptor subtypes differ primarily for distribution and effector functions (9). In general, immune cells can express both receptors, and the regulation of relative concentrations may be critical in the modulation of migration and activation.

This study shows that RSV renders the intrapulmonary airways of weanling rats abnormally susceptible to the proinflammatory effects mediated by sensory nerves. In contrast, RSV-infected adult rats manifest potentiation of neurogenic inflammation exclusively in the extrapulmonary airways. The selectively increased levels of mRNA encoding the NK1 receptor in the lungs of RSV-infected weanling rats suggest that the effect of RSV is linked to upregulation of this receptor. In fact, selective antagonism of the NK1 receptor abolishes RSV-induced potentiation of airway neurogenic inflammation. The limited effect of RSV on the mRNAs encoding receptor subtypes specific for the antiinflammatory VIP suggests that this virus may cause dysregulation of the NANC system in the respiratory tract, favoring the proinflammatory influence of SP. We have also found that preventing lower respiratory tract infection with a mAb against the F protein of RSV inhibits NK1 receptor upregulation and the potentiation of neurogenic inflammation in the lungs. RSV modulation of neuropeptide activity at the receptor level may contribute to the pulmonary inflammatory response to RSV infection and may predispose infants to the development of persistent airway inflammation. Pharmacologic modulation of this pathway may limit the severity of acute RSV disease and may protect against subsequent respiratory sequelae, particularly childhood asthma.