Immunomodulatory effects of sensory nerves during respiratory syncytial virus infection in rats

Alexander Auais,1 Becky Adkins,2 Galia Napchan,1 and Giovanni Piedimonte1,3,4

Departments of 1Pediatrics, 3Medicine, 4Molecular/Cellular Pharmacology, and 2Microbiology/Immunology, University of Miami School of Medicine, Miami, Florida 33136

Submitted 8 January 2003 ; accepted in final form 6 March 2003


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Respiratory syncytial virus (RSV) infection is associated with exaggerated neurogenic inflammation in the airways. This study sought to determine whether irritation of the mucosal sensory fibers affects the recruitment of lymphocytes and monocytes to RSV-infected airways. Pathogen-free rats were inoculated with RSV or with virus-free medium and were injected 5 days later with capsaicin to stimulate airway sensory nerves. Bronchoalveolar lavage was performed 1, 5, or 10 days after nerve stimulation, and samples were analyzed by differential cell count and flow cytometry. Without nerve stimulation, RSV caused a minimal increase in the number of lymphocytes and monocytes above pathogen-free control levels. After nerve stimulation, numerous lymphocytes, predominantly CD4+ T cells, and monocytes were recruited in the airways of infected rats, whereas no difference was found in pathogen-free controls. RSV induced overexpression of the neurokinin 1 (NK1) receptor for substance P on discrete lymphocyte subpopulations within the bronchial-associated lymphoid tissue (BALT), and treatment with a specific NK1 receptor antagonist abolished the recruitment of both lymphocytes and monocytes to infected airways. Our data suggest that airborne irritants stimulating mucosal sensory fibers during RSV infection exert important immunomodulatory effects by attracting to the infected airways selected lymphocyte subpopulations from the local BALT as well as monocytes.

airway inflammation; asthma; lymphocytes; monocytes; substance P


RESPIRATORY SYNCYTIAL VIRUS (RSV) is the most common respiratory pathogen in infancy, infecting nearly all children within the first two years of life (30) and resulting in >120,000 hospitalizations annually in the United States (29). Furthermore, there is growing evidence that RSV lower respiratory infection early in life is an important risk factor for the development of recurrent wheezing and asthma in later childhood (24). However, the mechanisms of airway inflammation and hyperreactivity during and after RSV infection are not fully understood.

Previous studies have proposed that early RSV infection causes dysregulation of the nonadrenergic, noncholinergic nervous system in the developing respiratory tract of young rodents, potentiating the bronchoconstrictive effect of tachykinin neuropeptides like substance P against the bronchorelaxant effect of the vasoactive intestinal peptide (3). We have recently found that RSV infection in rats is associated with upregulation of the mRNA encoding the high-affinity substance P receptor (neurokinin 1, NK1), which translates into a large increase of substance P binding sites expressed by the airway epithelium and vascular endothelium, causing strong potentiation of neurogenic-mediated inflammation (11, 26).

In addition to its bronchoconstrictive and proinflammatory effects, substance P is known to have important immunomodulatory properties and specifically regulates the functions of T and B lymphocytes, monocytes, and macrophages by affecting their migration, response to mitogens and allergens, and synthetic functions (5, 14). These effects of substance P are generally mediated via the NK1 receptor subtype, and several studies have confirmed expression of this receptor on immunocytes from rodents (2, 17) and humans (8, 12).

In the present study, we report for the first time overexpression of NK1 receptors in lymphocyte subpopulations localized in the bronchial-associated lymphoid tissue (BALT) during RSV infection. Extending these studies, we became interested in the functional significance of NK1 receptor-bearing immunocytes and hypothesized that T cell subpopulations within the BALT are "primed" by RSV to respond to activation of mucosal sensory fibers after the inhalation of airborne irritants. Therefore, we asked whether stimulation of sensory nerves alters the cellular components of bronchoalveolar lavage (BAL) fluid during infection in rats. Specifically, we examined both quantitatively and qualitatively lymphocytes and monocytes recovered from the BAL of acutely infected rats with and without sensorineural challenge with capsaicin (9). We also compared the lymphocyte subpopulations recovered from BAL with those extracted from lung tissues. Finally, we studied the effects of the selective NK1 receptor antagonist CP-122721 (18, 26) against this neurogenic-mediated flux of immunocytes in RSV-infected airways.


    METHODS
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 ABSTRACT
 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, rats were housed individually or in pairs in polycarbonate cages isolated by polyester filter covers, and 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, Hazelton, PA; see Ref. 26). We used separate rooms for housing infected and pathogen-free rats, both serviced by specially trained husbandry technicians. All manipulations were conducted in 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. The Division of Veterinary Resources of the University of Miami School of Medicine approved all experimental procedures followed in this study.

RSV preparation and inoculation. HEp-2 cells [American Type Culture Collection (ATCC), Rockville, MD] were grown in Eagle's MEM supplemented with 10% FBS (GIBCO-BRL, Grand Island, NY). 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 cytopathic effect (26). Cell debris was removed by centrifugation at 9,500 g 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 viral stock was titrated and diluted as needed for a final titer of 5 x 104 TCID50 (50% tissue culture infective dose) in 0.1 ml. Supernatants and cell lysates from virus-free flasks of HEp-2 cells in MEM were harvested, centrifuged, and separated into aliquots after the same protocol to obtain the virus-free medium used as a sham infection control.

Rats were inoculated endotracheally under pentobarbital sodium (50 mg/kg ip) anesthesia, as described previously (26). The vocal cords were visualized using a rodent laryngoscope, and the trachea was carefully intubated with a 16-gauge cannula. An 18-gauge inner cannula was passed through the endotracheal cannula to deposit the inoculum over the airway mucosa. The rats used in this study were inoculated with 100 µl RSV suspension containing 5 x 104 TCID50. Control rats received 100 µl virus-free medium.

Capsaicin administration. After inoculation (5 days) with RSV or virus-free medium, rats were reanesthetized with pentobarbital sodium (50 mg/kg ip), and the femoral vein was exposed. RSV-infected and pathogen-free rats received a 2-min intravenous infusion of 75 µg/kg capsaicin (8-methyl-N-vanillyl-6-nonenamide; Sigma, St. Louis, MO) dissolved in a vehicle having a final concentration of 0.75% ethanol, 0.375% Tween 80, and 0.85% NaCl in aqueous solution. Control rats were injected with the vehicle of capsaicin (1 ml/kg). Capsaicin has been used in several animal models to mimic exposure of the airways to airborne irritants (9), and ablation of capsaicin-sensitive nerves has been shown to inhibit the inflammatory response of the airway mucosa to tobacco smoke and mechanical and chemical irritants (13).

BAL. BAL was carried out in rats under pentobarbital sodium anesthesia by infusing 28 ml/kg PBS via a tracheal cannula. The fluid was instilled and withdrawn three times. An aliquot of the lavage fluid was separated for flow cytometry. Total cell count of the BAL was determined using a hemacytometer, and, after appropriate dilution to obtain 20,000–30,000 cells/ml, a microscope slide was prepared using a cytocentrifuge at 9,500 g for 5 min. The slides were fixed by air-drying and then stained with the Giemsa-Wright method. Cells were identified based on size and morphological features. Lymphocytes are typically smaller than the predominant monocyte/macrophage lineage cells and have a deeply staining indented nucleus and a thin rim of cytoplasm that stains clear blue with Wright reagent. We avoided any cell that had evidence of artifact or lysis. Differential cell count was performed on 200 cells, and the total number of lymphocytes was derived using the following formula: total cells in lavage fluid x (lymphocytes/200 cells). Monocyte counts were also derived from the same cytospin preparations using this method. All slides were coded and reviewed separately by two investigators in our laboratory who were blinded with regard to the experimental conditions associated with the cytospin preparations.

Once the BAL was completed, a catheter was inserted through the right ventricle, and the lungs were perfused with 4–6 ml PBS to remove any residual blood. Tissue suspensions obtained from excised lungs were prepared and analyzed by flow cytometry following the same procedures outlined for BAL samples.

Flow cytometry. All antibodies and staining reagents were purchased from Pharmingen (San Diego, CA). Combinations of monoclonal antibodies coupled to fluorescein, phycoerythrin, or biotin were used. Biotinylated monoclonal antibodies were developed using streptavidin cychrome. The antibody specificities used were biotinylated anti-rat CD3, phycoerythrin-labeled anti-rat CD4 and CD45RC, and FITC-labeled anti-rat CD8a and CD45. Isotype-matched monoclonal antibodies were used as negative controls and showed no reactivity.

Approximately 5 x 105 cells were incubated at 4°C for 20–30 min in 50 µl of staining medium (HBSS supplemented with 1% calf serum, 10 mM HEPES, and 4 mM sodium azide, pH 7.0) containing appropriate concentration of first-stage antibody. The cells were diluted with staining medium and centrifuged through an underlayer of calf serum. The cell pellet was resuspended in second-stage antibody and processed again following the steps described above. Samples were analyzed with a Becton-Dickinson FACScan flow cytometer and CellQuest software (San Jose, CA). Resting spleen cells were used to determine the lymphocyte gate using a combination of side scatter and forward scatter heights. We then determined the percentage of cells within the lymphocyte gate out of the total live cell population and obtained the number of CD3+/CD4+/CD8+ cells by multiplying the fraction of the respective cell population by the total number of lymphocytes in the sample.

Immunostaining. Immunoperoxidase staining for the all-T (CD3) and all-B (CD20) antigens and for the NK1 receptor was performed on formalin-fixed 3-µm-thick lung sections. The NK1 receptor was detected with a polyclonal antibody generated against a synthetic peptide corresponding to residues 273–287 of both rat and human receptors, and specificity was confirmed with blocking experiments using the peptide itself. Localization of the primary antibodies was delineated with the streptavidin-biotin peroxidase complex method using an immunostaining kit (DAKO, Carpinteria, CA) and developed with the 3,3'-diaminobenzidine tetrahydrochloride chromogen. With this technique, cells expressing target antigens are stained with a dark brown precipitate.

Autoradiography. Autoradiographic detection of substance P binding in rat lungs was performed using previously reported methodology (26). In brief, 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 on gelatin-coated slides, and stored at -70°C until use. 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, at 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 substance P (19, 27). The specificity of the binding was determined by incubation of adjacent tissue sections under the same conditions in the presence of 1 µM unlabeled substance P. After incubation, the slides were washed two times 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 substance P binding sites.

Experimental protocols. In preliminary studies, we noted marked hypertrophy of the BALT in lung sections from RSV-infected rats. We then characterized BALT lymphocytes by immunohistochemistry and used a specific antibody to detect expression of the NK1 receptor. In addition, substance P binding sites were visualized by autoradiography in lung sections from RSV-infected rats and pathogen-free controls (n = 5–6 rats/group).

To investigate the potential pathophysiological consequences of neuropeptide receptor overexpression on immunocytes, we studied the effect of sensory nerve stimulation on the recruitment of lymphocytes and monocytes. Pathogen-free F-344 rats were inoculated with either RSV suspension or virus-free medium, and 5 days later capsaicin was injected in some of the RSV-infected rats and some of the pathogen-free controls to stimulate sensory nerves in the airway mucosa; the other rats received an injection of the vehicle used to dissolve the capsaicin. Groups of rats underwent BAL at 1 day (n = 10 pathogen-free and 10 RSV-infected rats), 5 days (n = 9 pathogen-free and 9 RSV-infected rats), or 10 days (n = 12 pathogen-free and 11 RSV-infected rats) after capsaicin stimulation. Similarly, BAL was obtained at 1 day (n = 10 pathogen-free and 10 RSV-infected rats), 5 days (n = 14 pathogen-free and 13 RSV-infected rats), or 10 days (n = 6 pathogen-free and 6 RSV-infected rats) after injection of the vehicle of capsaicin. Cytospin preparations were analyzed to determine the total number of lymphocytes and monocytes recovered from BAL at the different time points. Subgroups of five to six rats from each treatment group were also used for flow cytometry to determine the relative contribution of CD4+ and CD8+ T lymphocyte subpopulations. A separate comparison was made between CD4+ and CD8+ cells recovered from the BAL and those extracted from the lung tissues of pathogen-free and RSV-infected rats 1 day after capsaicin stimulation (n = 6 rats/group).

To determine whether the effect of sensory nerves on the recruitment of immunocytes to the airways is mediated by substance P binding to its high-affinity NK1 receptor, groups of rats inoculated 5 days earlier with RSV were treated with a subcutaneous injection of the substance P antagonist CP-122721 [(+)-(2S-3S)-3-(2-methoxy-5-trifluoromethoxybenzyl)-amino-2-phenylpiperidine; 10 mg/kg; n = 9; Pfizer Central Research Division, Groton, CT], or its vehicle (0.9% NaCl; 1 ml/kg; n = 7 rats), 60 min before the injection of capsaicin and again 60 min before BAL, which was performed 1 day after capsaicin. This selective antagonist binds noncompetitively to the NK1 receptor with nanomolar affinity, producing long-lasting blockade (26) and thus generating a pharmacological NK1 receptor "knockout." BAL from these rats was analyzed for lymphocytes and monocyte counts and by flow cytometry for CD4+ and CD8+ subsets.

Statistical analysis. Data are expressed as means ± SE. Two-factor ANOVA was used to analyze differences between RSV-infected and pathogen-free rats in terms of cell counts and lymphocyte subpopulations found in BAL (37). Multiple comparisons between means were performed with the Fisher protected least significant difference test (34). 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.


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NK1 receptor expression. Histological analysis of lung sections revealed consistently marked hypertrophy of the BALT in RSV-infected airways compared with pathogen-free controls. Staining with a monoclonal antibody specific for the all-T antigen CD3 revealed that the BALT of RSV-infected rats was predominantly populated by T lymphocytes (Fig. 1), whereas no significant staining was obtained with an antibody specific for the all-B antigen CD20 (data not shown). Discrete T cell subpopulations within the BALT exhibited strong positivity to the antibody against the NK1 receptor, whereas minimal staining for this receptor was detected in pathogen-free controls (Fig. 1). Autoradiographic analysis of the localization of substance P binding in lung sections revealed a high density of binding sites overlying discrete areas of the BALT around RSV-infected airways (Fig. 2, top), whereas only sparse binding was observed over the BALT of pathogen-free controls (Fig. 2, bottom). Confirming our previous report (26), sections from RSV-infected lungs also revealed high-density substance P binding overlying the bronchial mucosa and the wall of the adjacent arterial and venous pulmonary vessels, whereas no binding was observed over the airway smooth muscle and alveolar tissue.



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Fig. 1. Lung sections from rats killed 5 days after the intratracheal inoculation of virus-free medium (top) or respiratory syncytial virus (RSV; bottom). Immunoperoxidase staining was performed using antibodies specific for the all-T antigen CD3 (left) and the high-affinity substance P receptor neurokinin 1 (NK1; right). The dark brown reaction reveals predominance of T cells in the hypertrophic bronchial-associated lymphoid tissue (BALT) of RSV-infected airways with strong overexpression of the NK1 receptor on discrete lymphocyte subpopulations. B, bronchiolar lumen. Internal scale = 40 µm.

 


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Fig. 2. Autoradiographic mapping of substance P binding sites in the BALT of lungs from 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 (H&E) preparations (B and F), autoradiographic images (C and G), and autoradiography digitally superimposed to H&E (D and H). Specific substance P binding was detected consistently over discrete areas of BALT in infected lungs, whereas only sparse binding was detected over the BALT in pathogen-free lungs. Internal scale = 0.5 mm.

 

Immunocyte counts. In the absence of nerve stimulation, differential cell counts from BAL showed a threefold increase in lymphocytes recovered from RSV-infected lungs compared with pathogen-free lungs (P = 0.04) 1 day after injection of the vehicle of capsaicin (Fig. 3). Recruitment of these cells during RSV infection was transient, being no longer significant by day 5 (P = 0.4) and returning to baseline by day 10 (P = 0.9). In contrast, the BAL of RSV-infected rats stimulated with capsaicin revealed a larger influx of lymphocytes at all time points analyzed. At day 1 postcapsaicin, RSV-infected rats had a 10-fold increase compared with pathogen-free rats (P < 0.0001), and this increase persisted at 5 (P = 0.002) and 10 (P = < 0.0001) days. Compared with unstimulated RSV-infected rats, lymphocyte counts were approximately two times as high in stimulated infected rats on day 1 postcapsaicin (P = 0.04), became fourfold higher by day 5 (P = 0.003), and were still significantly elevated at day 10, when unstimulated rats had already returned to baseline (P < 0.0001). In the absence of infection, capsaicin had no significant effect on lymphocyte recruitment to the airways at any time point analyzed (P > 0.2).



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Fig. 3. Neurogenic-mediated recruitment of lymphocytes in the airways of pathogen-free and RSV-infected rats. Stimulation with capsaicin during the infection attracted in the bronchoalveolar lavage (BAL) a larger number of lymphocytes compared with pathogen-free and infected, nonstimulated rats at all time points. *P < 0.05, **P < 0.01, and ***P < 0.001, significantly different from pathogen-free controls assigned to the same treatment group.

 

RSV-infected rats stimulated with capsaicin also had a larger number of monocytes in the BAL (Fig. 4). At day 1 postcapsaicin, RSV-infected rats had two times as many monocytes as pathogen-free controls (P = 0.04), and this increase persisted at 5 (P = 0.002) and 10 (P = 0.001) days. Compared with unstimulated RSV-infected rats, the monocytic response in stimulated rats peaked 5 days after nerve stimulation (P = 0.004) and was still eightfold higher 10 days after stimulation (P < 0.0001). In the absence of nerve stimulation, no significant difference was found between pathogen-free and RSV-infected rats at any time point (P > 0.1).



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Fig. 4. Neurogenic-mediated recruitment of monocytes in the airways of pathogen-free and RSV-infected rats. Stimulation with capsaicin during the infection attracted in the BAL a larger number of monocytes compared with pathogen-free and infected, nonstimulated rats at all time points. *P < 0.05 and **P < 0.01, significantly different from pathogen-free controls assigned to the same treatment group.

 

Lymphocyte subpopulations. In the absence of nerve stimulation, flow cytometry of BAL obtained 1 day after injection of the vehicle of capsaicin showed a significant increase in CD4+ cells (Fig. 5) from RSV-infected rats compared with pathogen-free controls (P = 0.006), which was still present at 5 days (P = 0.02) and resolved by 10 days (P = 0.5). In contrast, CD8+ cell counts (Fig. 6) were not significantly increased in unstimulated infected rats at any time point (P > 0.1).



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Fig. 5. Neurogenic-mediated recruitment of CD4+ cells in the airways of pathogen-free and RSV-infected rats. A larger number of CD4+ cells was found in the BAL from infected rats 1 day after stimulation with capsaicin compared with pathogen-free and infected, nonstimulated rats. *P < 0.05, **P < 0.01, and ***P < 0.001, significantly different from pathogen-free controls assigned to the same treatment group.

 


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Fig. 6. Neurogenic-mediated recruitment of CD8+ cells in the airways of pathogen-free and RSV-infected rats. A larger number of CD8+ cells was found in the BAL from infected rats 1 day after stimulation with capsaicin compared with pathogen-free and infected, nonstimulated rats. **P < 0.01 and ***P < 0.001, significantly different from pathogen-free controls assigned to the same treatment group.

 

Stimulation of sensory nerves in RSV-infected rats resulted in a fivefold increase in the number of CD4+ cells at 1 day postcapsaicin compared with unstimulated infected rats (P < 0.0001). After (5 days) the injection of capsaicin, there was no significant difference in CD4+ counts between stimulated and unstimulated infected rats (P = 0.5).

Flow cytometry also showed an approximate seven-fold increase in the number of CD8+ cells in the BAL of RSV-infected rats 1 day postcapsaicin compared with unstimulated infected rats (P < 0.0001), although total CD8+ counts were approximately one order of magnitude lower than CD4+ counts in all groups examined. Again, by day 5 postcapsaicin, there was no difference in the numbers of CD8+ cells between stimulated and unstimulated infected rats (P = 0.6).

A comparison of cell subpopulations recovered from BAL vs. lung tissue homogenates revealed that the accumulation of CD4+ (Fig. 7A) and CD8+ (Fig. 7B) cells in RSV-infected airways was accompanied by a reduction of the same cells in lung tissues and confirmed the large predominance of CD4+ cells flowing in the infected airways after nerve stimulation.



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Fig. 7. Comparison of the effects of sensory nerve stimulation on the lymphocyte subpopulations recovered from the BAL fluid (BALF) and those extracted from lung tissues. Both CD4+ (A) and CD8+ (B) cells were recruited in the airways and simultaneously decreased in the lung parenchyma, but the effect on CD4+ cells was proportionally much larger. **P < 0.01, significantly different from pathogen-free controls.

 

When negative isotype controls were analyzed for each sample, we found the degree of nonspecific cell binding to be nonsignificant at all time points.

NK1 receptor antagonism. Selective antagonism of the NK1 receptor with CP-122721 prevented the recruitment of immunocytes in the BAL of RSV-infected rats after capsaicin stimulation. Specifically, CP-122721 reduced capsaicin-induced recruitment of lymphocytes by 86% (P = 0.003; Fig. 8A) and monocytes by 71% (P < 0.0001; Fig. 8B). Flow cytometry showed a 74% reduction in CD4+ cells (P < 0.0001; Fig. 9A) and a 76% reduction in CD8+ cells (P < 0.0001; Fig. 9B) in RSV-infected rats pretreated with CP-122721 compared with infected rats injected with vehicle. The numbers of lymphocytes and monocytes recovered from the BAL of RSV-infected rats after blockade of the NK1 receptor were similar to the pathogen-free controls (P > 0.4).



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Fig. 8. Effect of selective NK1 receptor inhibition on neurogenic-mediated immunocyte influx in the airways of RSV-infected rats 1 day after capsaicin stimulation. Treatment with CP-122721 abolished the effect of sensory nerve stimulation on the recruitment of both lymphocytes (A) and monocytes (B) to the airways of RSV-infected rats. After NK1 receptor inhibition, RSV-infected rats were not different from pathogen-free controls. **P < 0.01 and ***P < 0.001, significantly different from nontreated RSV-infected rats.

 


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Fig. 9. Effect of selective NK1 receptor inhibition on neurogenic-mediated influx of CD4+ (A) and CD8+ (B) T lymphocytes in the airways of pathogen-free and RSV-infected rats 1 day after capsaicin stimulation. Treatment with CP-122721 abolished the effect of sensory nerve stimulation on the recruitment of both lymphocyte subpopulations to the airways of RSV-infected rats. After NK1 receptor inhibition, RSV-infected rats were not different from pathogen free controls. ***P < 0.001, significantly different from nontreated RSV-infected rats.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This is the first study to show that sensorineural stimulation during acute RSV infection leads to a markedly increased flux of lymphocytes and monocytes in the airways, with a ratio of ~20:1 monocytes-lymphocytes at all time points. This neurogenic-mediated recruitment of immunocytes is already measurable 1 day after nerve stimulation, peaks by day 5, and remains significant 10 days after a single stimulus, whereas, in the absence of sensorineural activation, immunocyte recruitment by the infection alone is much more limited and transient. Thus not only is the degree of the cellular response to the virus magnified by concurrent nerve irritation, the response is also sustained and persists after nonstimulated airways have returned to normal.

In our studies, RSV infection is obtained using a relatively small inoculum and causes in F-344 rats limited histopathological changes, including mild epithelial damage with predominantly mononuclear cell infiltration that resolve completely within a few weeks after inoculation, as described in previous work (11, 26). Thus our model reflects the immunoinflammatory response to mild and transient lower respiratory tract infections, which represent the largest majority of clinical RSV infections (6) and have been linked to higher risk for subsequent frequent or infrequent wheeze by recent epidemiological data (31).

Neurogenic-mediated recruitment of immunocytes in RSV-infected airways was abolished by selective pharmacological antagonism of the NK1 receptor, indicating that substance P binding to its high-affinity receptor is primarily responsible for this effect. The probable source of NK1 receptor-bearing lymphocytes is the BALT, where we identified clusters of cells staining deeply with an antibody specific for the NK1 receptor and binding radiolabeled SP avidly. This is also consistent with the simultaneous reduction of both CD4+ and CD8+ cells recovered from infected lung tissues. In addition, recruitment from the blood is unlikely because circulating lymphocytes, differently from those residing within the mucosal immune system, do not seem to express NK1 receptors (7). Thus our study confirms previous reports of substance P receptor upregulation by RSV (32) and provides evidence that the lymphocytes flowing in the airways originate from the mucosal immune system.

The influence of substance P on immunocyte recruitment during RSV infection appears to be not only quantitative but also qualitative, as suggested by the disproportionate number of CD4+ cells vs. CD8+ cells found in the BAL. This finding confirms the CD4+ phenotypic predominance in response to substance P previously reported in murine (33) and human (23) lymphocytes. However, the effect of sensory nerves on both CD4+ and CD8+ cells appears to resolve rapidly after stimulation with capsaicin; therefore, the late surge in lymphocyte counts measured 5 and 10 days later could not be accounted for in the flow cytometry data. There does not appear to be a cell population CD3+ but CD4-, CD8- that we could identify to account for the difference seen. We were also able to determine that the cells in question are not of B cell lineage using antibodies against B cell-specific markers (data not shown). It should be noted, however, that we set a very stringent lymphocyte gate on our samples based on resting spleen cells in an effort to overcome the background signal and obtain a true count of CD4+ and CD8+ cells, and the limited gate may have excluded activated lymphocytes from our flow cytometry analysis at days 5 and 10. Therefore, the differences noted between differential cell counts and flow cytometry data are probably related to the presence of activated lymphocytes and/or natural killer (NK) cells.

Previous studies using a murine model of RSV infection have found increased levels of substance P in the BAL (33) but have not explored the effects of irritant stimulation of C-type nerve fibers. Surprisingly, the experiments of substance P inhibition with anti-substance P F(ab)2 antibodies in the same model failed to show a significant effect on CD4+ cells, CD8+ cells, or macrophages, while altering the traffic of polymorphonuclear and NK cells. The differences between those studies and ours could derive from the species involved or by the use of different inhibitors of substance P (blocking antibodies vs. receptor antagonists) but may also derive from the different local levels of substance P and/or other immunomodulatory neuropeptides released upon irritation of C-type fibers.

Neuroimmune interactions. We have shown that RSV does not affect the enzymatic activity of neutral endopeptidase, as previously reported for other viruses (influenza, parainfluenza; see Ref. 25); therefore, its effects on neurogenic inflammation cannot be explained with decreased catabolism of substance P after being released from nerve fibers (26). Rather, the early inflammatory effects of substance P on the airway microvasculature in airways infected with RSV are potentiated because of upregulated expression of the NK1 receptor subtype (11, 26). Data from the present study suggest that the substance P-NK1 receptor interaction also plays an important immunomodulatory role by coordinating the recruitment to the infected airways of effector cells necessary for the ensuing specific and nonspecific immune response to the virus.

Specifically, our data show that RSV induces overexpression of the NK1 receptor on selected lymphocyte subpopulations within the BALT, thus priming these cells for the immunomodulatory effects of substance P. Release of this peptide from the dense supply of sensory afferents innervating the mucosal lymphoid aggregates (14) by airborne irritants inhaled during lower respiratory tract infections can initiate a cascade-like sequence by recruiting the CD4+ cells primed by the virus. In addition, because it has been shown that also nonneuronal cells such as lymphocytes and monocytes/macrophages express substance P and its receptors and release this peptide in response to capsaicin stimulation, they may amplify and propagate the chemotactic and modulatory functions of sensory nerves in an autocrine and/or paracrine fashion (8, 12). The subsequent activation of these cells with release of cytokines and chemokines is likely to be responsible for the second phase, characterized by the buildup in the airways of a large population of activated lymphocytes and monocytes. The neurogenic-mediated recruitment of monocytes is of special interest because these cells can promote airway inflammation and modulate immune responses via the production of cytokines like tumor necrosis factor-{alpha} (1, 22) and interleukins (IL)-1 (28), IL-6 (1), IL-8 (1), and IL-10 (21). In addition, RSV-infected monocytes may amplify and spread the infection by delivering a greater load of viral particles to the host tissue than the initial load infecting the monocyte itself (20).

Several studies have shown expression of receptors for substance P, as well as other neuropeptides, on human lymphocytes and monocytes/macrophages and upregulation of these receptors associated with chronic inflammatory conditions (14), supporting the involvement of substance P in human diseases involving immune dysregulation. For example, increased expression of substance P receptors and ectopic expression of these receptors in lymphoid tissues have been reported in patients with inflammatory bowel disease (15, 16). Also, lymphocytes and monocytes from patients with rheumatoid arthritis show increased proliferative responses (4) and altered expression of membrane markers (36) in response to substance P. It is possible that similar pathophysiological mechanisms contribute to the immunoinflammatory responses of the airways against viral infections and to the development of chronic sequelae like childhood asthma.

Neuroimmune remodeling. The interactions described in this study between sensory neuropeptides and immunocytes are only one example of the complex neuroimmune cross-talking that takes place during and after viral respiratory infections. In a recent study, we have shown that RSV lower respiratory tract infection is associated with a marked increase in the number of mast cells in the airway mucosa, which form clusters around nerve fibers and establish functional interactions through the formation of local neuron-mast cell feedback loops (35). Another recent study from our laboratory shows that RSV infection promotes a large increase in the expression of nerve growth factor (NGF) and neurotrophin receptors, which is responsible for the upregulation of the NK1 receptor expression and consequently for the exaggerated neurogenic inflammation in RSV-infected airways (10). Finally, we have found in our model that substance P concentration is markedly increased in lung tissues infected with RSV both during and after the infection and that the stimulation of these nerves is followed by the release of large amounts of substance P in the BAL even 30 days after inoculation of the virus, when the virus has already been cleared from the airways (G. Piedimonte, R. G. Hegele, A. Auais; unpublished observation). Because studies in several animal models have shown activation of sensory afferents in the airways by a variety of airborne irritants (13), all of these common triggers of airway inflammation are likely to produce effects similar to the experimental administration of capsaicin.

Combining our previous and current data, we speculate that RSV-induced release of NGF not only leads to long-term changes in the distribution and reactivity of sensory nerves across the respiratory tract but also primes different cellular effectors of immunity and allergy (e.g., lymphocytes, monocytes, mast cells), making them susceptible to the modulatory influence of neuropeptides like substance P. Remodeling of the submucosal neural network and the deriving cluster of neuroimmune interactions may link RSV infections occurring during critical developmental "windows" to the development of asthma during childhood. Based on this model, activation of the upregulated sensorineural pathways by airborne irritants would be responsible for the recurring bouts of airway inflammation and subsequent narrowing that continue long after the acute phase of the infection has cleared.

Our data show that stimulation of sensory afferents innervating the airway mucosa leads to a markedly increased lymphocytic and monocytic response in the BAL of RSV-infected rats, which is primarily mediated by upregulation of the high-affinity substance P receptor expression. The early influx of CD4+ and CD8+ cells is amplified by the subsequent buildup of activated lymphocytes, which is still significant 10 days after a single stimulation of the nerves. T lymphocytes are recruited from discrete subpopulations of cells overexpressing the substance P receptor that appear within the BALT during RSV infection. The dominant CD4+ population found in infected airways confirms that these cells are exquisitely sensitive to the chemotactic activity of substance P. Based on our observations, we propose that neuroimmune interactions play an important role in the pathophysiology of airway inflammation during, and possibly after, the course of RSV infection.


    ACKNOWLEDGMENTS
 
We thank Dr. James Krause for providing the antibody against the NK1 receptor, Dr. Stafford McLean for providing the NK1 receptor antagonist CP-122721 and helping with the autoradiography studies, and Drs. Rita Romaguera and Maria Rodriguez for helping with the histopathology. We also thank Drs. Xiaobo Jiang, Chengping Hu, Mian Xu, and Patricia Guevara for valuable technical assistance.

Some of the findings reported in this paper were presented at the 2001 American Thoracic Society Conference in San Francisco, CA, May 18–23, 2001, and at the 2002 American Thoracic Society Conference in Atlanta, GA, May 17–22, 2002.

This research was supported in part by National Heart, Lung, and Blood Institute Grant HL-61007 and an Investigator Award grant from the Allergy and Asthma Foundation of America to G. Piedimonte.


    FOOTNOTES
 

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.


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