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ABSTRACT |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In this study, we evaluated the effects of respiratory syncytial virus
(RSV) infection on nitric oxide (NO) production in human airway
epithelial cells. In addition, we evaluated whether T-helper type 1 (Th1)- and Th2-type cytokines modulate the release of NO in response to RSV infection. To do this, we infected monolayers of
A549 cells with RSV and determined nitrite levels in the supernatant fluids. We also measured nitrite levels in human small-airway epithelial cells (SAEC) in primary culture and in the bronchoalveolar lavage fluid (BALF) obtained from Balb/c mice after RSV infection. To further support our observations in these analyses, we
performed immunocytochemistry and Western blot analysis for inducible nitric oxide synthase (iNOS) in A549 cells. To evaluate the
regulation of NO production in response to RSV, we performed
experiments in the absence and presence of the Th1 and Th2 type
cytokines: interferon (IFN)-
, interleukin (IL)-4, and IL-13. In addition, we assessed the inhibitory effect of dexamethasone on iNOS
in RSV infected A549 cells. Results were expressed in terms of
nmol/mg protein and shown as percents of control values (mean ± SE). RSV increased the release of nitrites in A549 cells, SAEC, and
BALF. The increase in nitrite levels was supported by immunocytochemistry and Western blot analysis for iNOS protein in A549
cells, indicating activation of iNOS in response to RSV infection.
IFN- and IL-13 did not affect the RSV-induced increase in NO production. By contrast, IL-4 and dexamethasone suppressed the release of NO in response to RSV infection. These observations show
that RSV infection leads to activation of iNOS within the airway
epithelium and that IL-4 and dexamethasone inhibit the production of NO in response to RSV infection.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Respiratory syncytial virus (RSV) is an important respiratory pathogen in infants and young children (1, 2). Although various genetic and environmental factors may determine the outcome of RSV infection on airway function, an association between RSV in early life and long-term pulmonary abnormalities has been repeatedly suggested (3). In this respect, recent work has indicated that RSV bronchiolitis in infancy is an important risk factor for the development of asthma, airway dysfunction, and sensitization to allergens (4, 6). Despite the potential importance of RSV in the pathogenesis of lung inflammation, only recently have animal models been used for the study of basic mechanisms of airway dysfunction. These studies have shown that RSV is capable of producing significant changes in the autonomic regulation of airway smooth muscle (ASM) (7), as well as airway hyperresponsiveness (AHR) and enhanced sensitization to aeroallergens (10).
Several cells and cell products are involved in the pathogenesis of virus-induced airway inflammation (15). The importance of the airway epithelium in the host response to respiratory pathogens has been increasingly accepted, owing to its newly recognized metabolic activities (16). Epithelial cells have been proposed to be involved in airway injury and remodeling by releasing products capable of modulating the function of inflammatory and immune cells (15, 16). In this respect, recent studies have described important changes in the metabolic function of epithelial cells in response to RSV infection (17).
Epithelial-derived nitric oxide (NO) plays a prominent role in cell signaling within the respiratory tract and has been implicated in the pathogenesis of a number of airway diseases (25). A variety of lung cells may produce NO from L-arginine through action of the enzyme nitric oxide synthase (NOS) (28). Constitutive isoforms of NOS appear to be involved in the regulation of endothelial and nerve cell functions (25, 27). The inducible isoform of NOS (iNOS), highly expressed in the airway epithelium after exposure to proinflammatory cytokines and oxidants, has been implicated in the pathogenesis of airway inflammation (25, 29). Additionally, recent studies have shown that exhaled NO is increased in wheezy infants, thus suggesting that changes in NO production could be induced by viral respiratory illnesses in early life (33, 34). Moreover, patients with asthma have a marked increase in exhaled NO, as well as showing an increased immunolabeling for iNOS in epithelial biopsies when compared with control subjects (35).
In this study, we evaluated whether RSV infection produces changes in NO production in human airway epithelial
cells (A549 and small-airway epithelial cells [SAEC]) and in a
well-established murine model of RSV infection. In addition,
we determined whether Th1- and Th2-type cytokines modulate the release of NO in response to RSV infection. We found
that RSV infection enhances the release of NO and leads to
activation of the iNOS in airway epithelium. Furthermore, the
airway microenvironment appears to influence the production
of NO in that interleukin (IL)-4, but not IL-13 or interferon
(IFN)-, appears to inhibit the release of NO in response to
RSV infection.
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The human lung carcinoma cell line (A549) and human RSV (strain A2) used in our studies were purchased from American Type Culture Collection (Rockville, MD). A human SAEC line in primary culture was obtained from Clonetics Corporation (San Diego, CA). Female BALB/c mice (8 wk of age), obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN), were used in our in vivo investigations. All procedures used in these investigations were approved by the Animal Care and Use Committee of the University of Texas Health Science Center at Houston and conformed to National Institutes of Health guidelines. The mouse monoclonal antibodies to macrophage iNOS were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) and Transduction Laboratories (Lexington, KY). The reagents for gel electrophoresis were purchased from Bio-Rad Laboratories, Life Science Group (Richmond, CA). The enhanced chemiluminescence (ECL) detecting system and films were obtained from Amersham Life Science (Arlington Heights, IL). All media and test reagents were obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise indicated.
Culture of Airway Epithelial Cells
Airway epithelial cells from the human lung carcinoma cell line A549 were used for our studies. These cells have been shown to retain their type II alveolar epithelial cell features (17), and provide a useful model for the study of RSV replication (20, 21). For experiments, cells were seeded in six-well plates and incubated at 37° C in 5% CO2. Cells were grown in Ham's F12 culture medium (Kaighn's modification) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), penicillin (5 U/ml), and streptomycin (5 µg/ml). When reaching 80% to 90% confluence, cells were washed twice with phosphate-buffered saline (PBS) and incubated in serum-free and phenol red-free F12 medium (Gibco BRL, Grand Island, NY) for 24 h before infection and/or treatment with various agents. In selected experiments, human SAEC were also studied. These cells were grown in SAEC culture medium (Clonetics Corp.) supplemented with bovine pituitary extract (30 µg/ml), hydrocortisone (0.5 µg/ml), human recombinant epidermal growth factor (0.5 ng/ml), epinephrine (0.5 µg/ml), transferrin (10 µg/ml), insulin (5 µg/ml), retinoic acid (0.1 ng/ml), triiodothyronine (6.5 ng/ml), gentamycin (50 µg/ml), amphotericin-B (50 ng/ml), and fatty acid-free bovine serum albumin (BSA) (0.5 mg/ml). The cells were incubated at 37° C in ventilated tissue culture flasks with 5% CO2. When confluent, the cells were seeded in 12-well culture plates at a density of 0.2 × 105 cells/well.
RSV Infection
Human RSV of strain A2 was used in all experiments. The virus stock
of RSV was diluted in culture medium to a defined multiplicity of infection (MOI) of 0.001, 0.01, 0.1, and 1. The last of these MOIs is defined as the number of plaque-forming units (pfu)/number of A549
cells. Monolayers of A549 cells were exposed to RSV for 2 h and then
washed and incubated. Cells and supernatant fluids were removed at
various times after RSV infection, and were stored at 
80° C until
needed for assays. SAEC cells were infected with human RSV at an
MOI of 0.1 in a similar manner. Control samples were prepared from
uninfected cultures that were processed in the same manner as the infected cultures. RSV infection was confirmed by plaque-forming assay in Hep-2 monolayers as previously described (7, 8). Cell viability
was determined by trypan blue exclusion. To corroborate our in vitro
observations, we measured nitrite levels in a murine model of RSV infection (39). After sedation with ketamine and xylazine, mice were infected with 107 pfu of virus. This experimental protocol has been previously shown to produce viral replication within the respiratory tract of mice and other mammalian models (39). RSV infection was confirmed by determining virus titers in lung tissues as previously described (7, 8). Control animals were given uninfected culture medium or nonreplicative RSV in a manner similar to the infected mice.
Nitrite Determination
Nitrite concentrations were determined in the supernatant fluid of
A549 cells and SAEC, and in the bronchoalveolar lavage fluid
(BALF) of mice through a modification of the Griess reaction. After
1 ml of supernatant fluid or BALF was collected from each well, an
equal volume of a mixture (1:1) of 1% sulfanilamide and 0.1% naphthylethylenediamine in 2% (vol/vol) phosphoric acid was added to
each sample. After incubation at room temperature, the optical density of the reaction product was measured at a wavelength of 540 nm
and the nitrite concentration was calculated with sodium nitrite as a
standard. To determine the content of total cellular protein, cells were
washed twice with PBS and collected for bicinchoninic acid (BCA)
protein assay (Pierce Inc., Rockford, IL). To evaluate the effects of
Th1- and Th2-type cytokines on nitrite production, we performed experiments with A549 cells in the absence and presence of IL-4 (5 to 50 ng/ml), IL-13 (1 to 50 ng/ml), and IFN- (1 to 50 ng/ml). For comparison, cells were incubated with cytomix, containing IL-1
(10 ng/ml),
tumor necrosis factor-
(10 ng/ml), and IFN- (10 ng/ml) as a positive
control (30). In addition, dexamethasone (DEX; 10
6 M) was used in
selected experiments because of its known inhibitory effect on iNOS
(30). Results were expressed in terms of nmol/mg protein and shown
as percents of control (mean ± SE).
Immunocytochemistry
Immunolabeling of iNOS was performed in A549 cells and SAEC grown on tissue culture slides. Control and RSV-infected cells were processed as described earlier. In addition, in selected experiments, cells were incubated with nonreplicative RSV, using either ultraviolet-(UV)-irradiated virus or treatment with Ribavirin (100 µM). Cells were fixed with 1% paraformaldehyde, washed in buffered sucrose solution, and permeabilized in Triton X-100. iNOS protein was detected with a Vectastain Universal Elite Avidin-Biotin-Complex (ABC) Kit (Vector Laboratories, Burlingame, CA), using mouse monoclonal antimacrophage iNOS antibody.
Western Blot Analysis
A549 cells were homogenized in 0.05 M Tris-HCl buffer, pH 7.4, containing 1 µM pepstatin A, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM ethylenediamine tetracetic acid, and 1% NP-40. The samples were centrifuged at high speed for 10 min. The supernatant was collected, and its iNOS protein content was enhanced with 2',5'-Sepharose 4B (Amersham, Pharmacia Biotech, Piscataway, NJ). The protein concentration in the samples was determined with a BCA protein assay kit, using BSA as the standard. Equal amounts of protein (10 to
20 µg) were diluted in 0.05 M Tris-HCl buffer, pH 7.4, containing 2%
sodium dodecylsulfate (SDS), 0.01% bromophenol blue, 0.7% -mercaptoethanol, and 10% glycerol. Gel electrophoresis was done on
7.5% SDS-polyacrylamide gels. Immunoblot analysis was done with a
mouse monoclonal antibody to macrophage iNOS. The blotted protein was detected with an ECL system and recorded on high-performance chemiluminescence film.
Statistical Analysis
All data were normalized for protein content and expressed as mean ± SE. Student's paired and unpaired t tests were used as appropriate, to evaluate differences (40). A value of p < 0.05 was considered significant.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Unstimulated A549 cells have been previously shown to produce measurable amounts of nitrite (30), and represent a useful model for the study of RSV replication (20, 21). To evaluate whether RSV infection produces changes in nitrite production, we exposed A549 to RSV at various MOIs for 48 h and confirmed infection with a plaque-forming assay in Hep-2 monolayers (7, 8). As previously shown by Merolla and colleagues (20), the peak of viral replication was obtained at 48 h after infection (data not shown). RSV-infected cells produced enhanced levels of nitrite as compared with control cells, by 140.3 ± 17.7% (n = 4), 151.2 ± 12.6% (n = 3), 236.4 ± 15.6% (n = 7), 340.0 ± 52.0% (n = 8) of the corresponding control values for MOIs of 0.001, 0.01, 0.1, and 1, respectively (Figure 1). At an MOI of 0.1, RSV infection increased nitrite production significantly at 24 h and 48 h after infection, by 183.3 ± 13.9% (n = 7) and 236.4 ± 15.6% (n = 8) of the corresponding control values, respectively (Figure 2). The increase in nitrite production was abolished by using UV-irradiated RSV or treatment with Ribavirin, suggesting that viral replication is necessary for the increased production of NO by infected epithelial cells. To further support our observations, we measured nitrite levels in human SAEC in primary culture. At a MOI of 0.1, nitrite levels were increased at 48 h after RSV infection as compared with those for control cells, by 227.0 ± 19.4% (n = 4) of the control value (Figure 3). Furthermore, we evaluated nitrite concentrations in the BALF obtained from control and RSV-infected Balb/c mice. As shown in Figure 4, RSV produced an increase in nitrite levels at 4, 6, and 10 d after infection as compared with those of control animals by 145.2 ± 12.7% (n = 6), 221.0 ± 18.3 (n = 5), and 289.1 ± 24.3 (n = 7) of the corresponding control values, respectively.
|
0.01 as compared with uninfected control
cells. *Values were significantly greater (p < 0.05) than those obtained
with control cells. Results were corrected for protein content, expressed in
nmol/mg protein, and shown as percents of control values (mean ± SE).
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Immunocytochemistry for iNOS with murine monoclonal antibodies is shown in Figure 5. Unstimulated A549 cells showed no significant staining for iNOS (Figure 5A). By contrast, a distinct immunoreactivity was detected at 24 h and 48 h after infection. Figure 5B shows a representative field of epithelial cells after RSV infection, with cytosolic immunostaining for iNOS in the perinuclear region. The majority of cells within each group displayed normal morphology. A549 cells treated with nonreplicative RSV (UV-irradiated or Ribavirin-treated virus) were similar to control uninfected cells and did not display immunoreactivity for iNOS (Figure 5C). Similarly, RSV infection induced positive staining for iNOS in SAEC in primary culture (Figure 6A), whereas uninfected control cells showed no significant staining (Figure 6B). Western blot analysis, performed with A549 cells at 48 h after infection, confirmed an RSV-induced increase in iNOS protein expression (Figure 7). In selected studies, we did not detect either eNOS or nNOS proteins in control or RSV-infected A549 cells (data not shown).
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To evaluate the effects of exogenous substances on the
basal production of nitrite, we incubated A549 cells with
agents capable of affecting NO release. As shown in Figure 8,
cytomix increased NO release by 179.3 ± 6.2% (n = 4) over the
control value. By contrast, IL-4, IL-13, IFN-, and DEX did
not affect basal nitrite production, giving values that were 91.9 ± 7.6% (n = 4), 87.2 ± 6.0% (n = 4), 109.4 ± 15.4% (n = 4), and
84.0 ± 6.6% (n = 4) of the corresponding controls, respectively.
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To evaluate whether Th1- and Th2-type cytokines modulate the nitrite production that occurs in response to RSV infection, we infected A549 cells in the presence of IL-4, IL-13,
and IFN-. We also assessed the inhibitory effect of DEX on
NO production. After 48 h, RSV increased nitrite production
to 222.4 ± 6.6% (n = 5) of the control value (Figure 9). Treatment with IL-4 suppressed the increase in NO produced in
response to RSV, giving values of 166.8% (n = 2), 139.0%
(n = 2) and 104.2 ± 3.9% (n = 4) of the corresponding controls, at 5, 25, and 50 ng/ml, respectively. IL-13 (1, 10, and
50 ng/ml) and IFN- (1, 10, and 50 ng/ml) did not affect the
RSV-induced increase in nitrite production (Figures 10 and
11). DEX significantly suppressed the increase in NO produced in response to RSV infection, to 73.3 ± 4.2% (n = 7) of
the control value.
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DISCUSSION |
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METHODS
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DISCUSSION
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The ability of RSV to cause airway disease in humans has been recognized for approximately four decades (41), and an association between RSV infection early in life and long-term pulmonary abnormalities has been repeatedly suggested (3). Despite the potential importance of this association in the pathogenesis of airway inflammation, the mechanisms leading to RSV-induced airway dysfunction are poorly understood. As a result, the development of therapeutic agents and of an effective vaccine for such infection remains a major challenge (42).
Clinical and laboratory studies have suggested a number of mechanisms by which RSV may alter airway functions (15). Our laboratory has recently investigated the effects of RSV on neural control of the airways in vitro, using well established models of RSV infection. These studies showed that RSV produces acute and long-lasting effects on autonomic regulation of the airways, leading to an imbalance between neurally mediated contractile and relaxant responses of the ASM (7- 9). More recently, AHR and inflammation associated with enhanced sensitization to aeroallergens have also been described, in other animal models in vivo (10). Taken together, these observations support the findings in human studies and suggest that RSV infection may lead to changes in airway and immune functions.
Although several types of cells are likely to be involved in virus-induced airway injury, the airway epithelium is a primary target of respiratory viruses and may play an important role in the host response to RSV (15, 16). In this respect, recent work has shown important changes in the metabolic function of airway epithelial cells in response to RSV infection, including changes in the expression of adhesion molecules, synthesis and release of proinflammatory cytokines and chemokines, and activation of transcriptional factors (17). This suggests that epithelial cells initiate the inflammatory response to RSV by translating viral signals within the airways and thereby capturing and activating immune cells.
NO plays an important role in cell signaling within the respiratory tract and has been implicated in the pathogenesis of a number of inflammatory airway diseases including asthma (25). Lung cells may produce NO from L-arginine through the action of constitutive NOS or via activation of iNOS by proinflammatory cytokines and oxidants (28). Of significant clinical interest are the recent observations that patients with asthma have a marked increase in exhaled NO and a greater staining for iNOS in epithelial biopsies when compared with control subjects (35). In addition, exhaled NO has been shown to be increased in "wheezy" infants, thus suggesting that changes in NO production could be induced by viral respiratory illnesses early in life (33, 34).
In the present study, we demonstrated that RSV enhances
the capacity of A549 human airway epithelial cells
a well-
established cell line for studies of RSV infection as well as NO
production (20, 43)to release NO. This was shown by detecting increased levels of nitrite in the supernatant fluids of
A549 cells after RSV infection. Furthermore, enhanced production of nitrite levels was detected in the supernatant fluid
of SAEC in primary culture after RSV infection. Increased nitrite levels were also confirmed in a well established model of
RSV infection in vivo. The increase in nitrite was supported
by the increase in iNOS protein observed through immunocytochemistry and Western blot analysis. The effect of RSV on
iNOS expression in epithelial cells appears to depend on viral
replication, since nonreplicative RSV failed to increase either
NO release or iNOS protein immunolabeling in our cell culture system. These findings support and extend recent work
on the effect of RSV infection on iNOS expression in human
respiratory epithelial cells (44). Although RSV alone did not
produce changes in either nitrite levels or iNOS protein, an increase in iNOS messenger RNA (mRNA) expression, that
was further enhanced by exposure to exogenous cytokines,
was described after RSV replication (44).
The functional consequences of RSV-induced production of NO are uncertain, since NO may have effects on various target cells within the lung. Furthermore, the direct and indirect effects of NO-derived intermediate metabolites are not apparent from our studies. NO is a potent vasodilator and may contribute to the increased blood flow and vascular permeability seen during viral infections (25, 27). NO is also the mediator of the nonadrenergic, noncholinergic inhibitory response in human airways (45). Therefore, a bronchodilator response produced by enhanced levels of NO could be beneficial in conditions associated with loss of airway control. Of interest is that a deficiency in NO production has been shown to be responsible for virus-induced AHR in vivo in a guinea pig model of parainfluenza virus infection (46). However, high levels of NO appear to have cytotoxic effects on airway epithelial cells (47), suggesting that enhanced production of NO may be partly responsible for the shedding of epithelial cells seen during RSV infection. Furthermore, NO has been shown to be a chemotactic factor for a variety of inflammatory cells including eosinophils (48). In light of these observations, changes in NOS activation after RSV infection are likely to result in alterations of cellular functions within the respiratory tract.
There is increasing evidence that NO is involved in nonspecific defense mechanisms in the lung, either directly or through interactions with macrophages and T cells (25, 27, 49). In this
respect, NO has been shown to inhibit the proliferation of
Th1-type cells and the production of their metabolic products, IFN- and IL-2 (49). By contrast, NO does not appear to directly affect Th2-type cytokines (25, 27, 49). In the present
study, we found that the Th1/Th2 balance may affect the ability of epithelial cells to produce NO in response to RSV infection. Although Th1-type cytokines do not alter the enhanced
release of nitrites in our system, IL-4, but not IL-13, reduces
the production of NO in response to RSV infection. Although
the degree of inhibition of iNOS expression by IL-4 is not evident from our studies, previous work with transformed and
primary human lung epithelial cells has shown an inhibitory effect of IL-4 at the level of transcription after exposure to cytomix (50). Similarly, DEX, a potent inhibitor of iNOS
expression (30), blocked the release of NO induced by RSV in
our study. The clinical significance of the latter finding is uncertain, since NO is involved in regulatory, protective, and
detrimental functions. Furthermore, corticosteroids affect the
function of several other immune cells and cell products within
the lung in vivo. As a result, the suppression of NO production
and/or the use of corticosteroids could have variable effects on
target cells. Nevertheless, in clinical studies, the use of corticosteroids has not produced significant changes in the outcome of RSV-induced bronchiolitis (51).
The importance of NO and other reactive oxygen species in the pathogenesis of viral diseases involving the respiratory tract has recently been confirmed by in vivo studies. In murine models of viral pneumonia, iNOS activity and mRNA expression were significantly increased within lung tissue by viral replication (52). Of interest was that inhibition of NO production improved the histologic changes and the survival of the mice without affecting the propagation of viruses within the respiratory tract (52). Therefore, pulmonary injury can be attenuated by administration of NO inhibitors regardless of viral propagation in the lung. Since our present work was not designed to link changes in NO production mechanistically to viral replication, detailed studies with cell culture systems and animal models should define the role of RSV-induced NO in airway inflammation.
In summary, we have shown that RSV enhances the production of NO by airway epithelial cells both in vitro and in vivo. This effect appears to depend on viral replication since nonreplicative RSV failed to increase NO and iNOS protein in our cell culture system. Th2-type cytokines (IL-4) appear to be involved in the regulation of NO production in response to RSV infection. Our findings support previous work in inflammatory lung diseases and could provide mechanistic information about the activation and modulation of iNOS in response to an important human respiratory pathogen. In light of these observations, NO may represent an important signaling molecule in initiation of the inflammatory response to viruses by modulating the recruitment and activation of immune cells. It is therefore possible that complex interactions between T cells and iNOS, produced by genetic and/or environmental factors, could contribute to the final airway response to RSV. Future studies are needed to determine the level of production of NO and its metabolites in response to RSV infection.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Giuseppe N. Colasurdo, M.D., Department of Pediatrics, UT-Houston Medical School, 6431 Fannin, MSB 3.146A, Houston, TX 77030. E-mail: Giuseppe.N.Colasurdo{at}uth.tmc.edu
(Received in original form December 14, 1999 and in revised form July 28, 2000).
Acknowledgments: The authors thank Drs. Okan Elidemir, Michael Blackburn, Andrew P. Morris, Carl White, and Jamey Miller for their comments in the preparation of this manuscript.
Supported by Grant HL-03196 from the National Institutes of Health.
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References |
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DISCUSSION
REFERENCES
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