INTRODUCTION

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The rhinoviruses (RVs) are an important cause of common cold symptoms. Substantial evidence suggests that the host response in general, and that of proinflammatory cytokines in particular, may contribute to the symptoms associated with RV infection (1). The production of interleukin-8 (IL-8) and IL-6 in response to RV infection appears to be mediated by oxidative stress, with subsequent activation of nuclear factor-B (NF-B) (3, 5, 6). Recent data suggest that similar signaling pathways may be involved in the production of nitric oxide (NO) in cells (7). NO is produced in a wide variety of mammalian cells as a result of oxidation of L-arginine by NO synthases (NOS). Both constitutive, calcium-dependent NOS (cNOS) and inducible, calcium-independent NOS (iNOS) are present in human respiratory epithelium. These respiratory epithelial cells produce NO in vitro as a result of induction of iNOS in response to lipopolysaccharide (LPS) and various cytokines (10). The similarities in the signaling events for the cytokines and for NO, and the potential for NO production by respiratory epithelium, suggest that NO may be produced during viral upper respiratory infections.

NO has been associated with both enhancement and inhibition of elaboration of the proinflammatory cytokines (11). In addition to these effects, NO may play a role in viral infection by exerting an antiviral effect (15). In view of the activation of signaling pathways associated with NO production, and the potential effects of NO on virus replication and the production of proinflammatory cytokines, we investigated the role of NO in RV infection by investigating the effects of NO on RV replication and RV-induced IL-8 elaboration in two different respiratory cell lines and measuring the production of NO in nasal secretions during experimental human RV infection.

	METHODS

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Reagents

N-^GNitro-L-arginine (L-NNA), N-^Gnitro-L-arginine methyl ester (L-NAME) hydrochloride, S-nitroso-N-acetyl penicillamine (SNAP), and sodium nitroprusside (SNP) were purchased from Calbiochem, San Diego, CA. 3-(2-Hydroxy-2-nitro-1-prophylhydrazino)-1-propanamine (PAPA-NONOate) was purchased from Cayman Chemical Company (Ann Arbor, MI). All other chemicals used in the study were from Sigma Chemical Company (St. Louis, MO).

Cell Culture

All experiments were done with human respiratory cell lines maintained at 37° C in 5% CO₂. Human embryonic lung fibroblast cells (MRC-5; Biowhittaker, Walkersville, MD) were grown in Eagle's minimal essential medium (EMEM) supplemented with 10% fetal calf serum, 5 U/ml penicillin G sodium, and 5 µg/ml streptomycin. Cells were used for experiments at passages 21 through 25, within 2 d of the time the monolayers became confluent. Human bronchial epithelial cells (BEAS-2B; American Type Culture Collection [ATCC], Rockville, MD) were grown in bronchial epithelial growth medium (BEGM; Clonetics, Minneapolis, MN) supplemented with human recombinant epithelial growth factor (0.5 ng/ml), insulin (5 µg/ml), hydrocortisone (0.5 µg/ml), epinephrine (0.5 µg/ml), transferrin (10 µg/ml), gentamicin (50 µg/ml), and amphotericin B (50 ng/ml). All experiments with BEAS-2B cells were done with cells at passages 40 through 55, when the monolayers were 85 to 95% confluent.

Virus Preparation and Purification

RV type 39 (RV39) was grown in HeLa-I cells, an HeLa cell clone with increased surface expression of intercellular adhesion molecule-1 (ICAM-1) (provided by F. G. Hayden, University of Virginia Health Sciences Center, Charlottesville, VA). HeLa-I cells infected with RV39 were collected mechanically, lysed by freezing and thawing and clarified via centrifugation at 2,000 × g (Beckman GPR centrifuge; Beckman Instruments, Palo Alto, CA). The supernatants were then centrifuged at 125,000 × g at 4° C for 45 min with a Ti45 rotor (L8-70M centrifuge, Beckman Instruments). Partially purified virus was produced by modifying a published method (5). Briefly, after ultracentrifugation, the resulting viral pellet was resuspended in 200 µl phosphate-buffered saline (PBS) and overlaid onto a two-layer sucrose cushion containing 60% sucrose in PBS on the bottom layer and 30% sucrose in PBS on the top layer. After centrifugation at 110,000 × g (SW28 rotor) for 135 min at 4° C, the interface containing the virus was collected and resuspended in 50 ml of EMEM. The virus suspension was again centrifuged at 125,000 × g for 45 min at 4° C, and the resulting pellet was resuspended in EMEM with 1% bovine serum albumin, with aliquots of the suspension snap frozen in liquid nitrogen and stored at 70° C.

Viral Infection

BEAS-2B and MRC-5 cells were grown to confluence in 24-well tissue culture plates ( 10⁵ cells/well). Unless specified, the virus challenge was 100 × the median tissue-culture infective dose (TCID₅₀)/ cell for experiments in BEAS-2B cells and 10 × TCID₅₀/cell for experiments in MRC-5 cells. The cells were challenged with virus in a final volume of 1 ml/well and incubated at 33° C for 1 h to allow the absorption of virus. The cells were then washed three times with media and further incubated with the fresh media at 33° C. Supernatants were then collected at the specified time (6, 24, or 48 h) and stored at 80° C until analyzed for nitrite, IL-8 protein, or virus titers.

Assay for NO Synthesis

NO generation was determined by assaying culture supernatants for nitrite, a stable end product of NO (19). Briefly, four volumes of cell-free supernatants and 1 volume of Greiss reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2.5% H₃PO₄) were incubated at room temperature for 10 min. The reaction product was quantified at 550 nm against standards (4:1 [vol/vol] sodium nitrite standard:Greiss reagent) on an automated microplate reader (Anthos HTII; Anthos Labtec Instrument Co., Salzburg, Austria).

Measurement of IL-8 Protein

The IL-8 concentrations in cell culture supernatant specimens were determined with a commercially available enzyme-linked immunosorbent assay (ELISA) (Genzyme Diagnostics, Cambridge, MA). All assays were run in duplicate, using an automated spectrophotometric plate reader (Anthos HTII). Sample concentrations were determined from OD values, using a standard curve based on a linear regression.

Quantitation of Virus

Virus titrations were done in 96-well microtiter plates (Falcon Labware, Oxnard, CA). Serial 10-fold dilutions of each specimen were made, and 2 × 10 ⁴ MRC-5 cells were then added to each well. The plates were incubated at 33° C for 7 d and then examined for viral cytopathic effects (CPE). The virus titers were calculated according to standard methods (20).

Effect of NO Donors and iNOS Inhibitors

Studies were done with three different NO donors with different solubilities and different half-lives of NO release. Monolayers of BEAS-2B and MRC-5 cells in 24-well plates were preincubated with varying concentrations of SNAP (soluble in ethanol, half-life of NO release = ~ 2 h) or SNP (water soluble, stable NO donor) for 30 min at 37° C, and were then challenged with RV39. After incubation at 33° C for 1 h, the monolayers were washed three times with medium and the cells were further incubated with fresh medium or medium plus NO donors for 6 to 24 h at 33° C. PAPA-NONOate (soluble in 0.01 M NaOH, half-life of NO release = ~ 76 min at pH 7.4) was added only during and after the viral infection, with no preincubation. Two different NOS inhibitors, L-NNA, which is known to inhibit both constitutive and inducible NOS, and L-NAME, which primarily inhibits cNOS, were studied. Cell-culture monolayers of BEAS-2B and MRC-5 cells were incubated with each inhibitor for 2 h before the virus challenge and for 6 h after virus challenge, when supernatants were collected for assay of IL-8 concentrations. The effect of these procedures on cell viability was determined with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay.

MTT Assay for Cytotoxicity

The concentrations of the NO donors (SNAP, SNP, and PAPA-NONOate) and NOS inhibitors (L-NNA and L-NAME) that could be used without cytotoxicity were determined by assessing cell viability with the MTT cleavage assay (Sigma). MTT reduced to its purple formazan derivative (by active mitochondrial dehydrogenase) was quantified at 570 mm. There is a linear relationship between the formazan generated and the number of viable cells present (21).

NO in Nasal Secretions during Experimental RV Infections

NO production during RV colds was assessed by measuring nitrite (as described earlier) in the nasal lavage fluids of human volunteers with experimental RV infections. All specimens had been stored at 80° C and had been subjected to a single freeze-thaw cycle prior to being thawed for the nitrite assay. The stability of nitrite under these storage conditions was confirmed by comparison of nitrite levels in nasal secretions from RV-infected symptomatic subjects that had been stored for different lengths of time. Specimens stored for approximately 6 mo, 9 mo, and 1 yr had nitrite concentrations of 2.36 ± 0.61 µM, 2.17 ± 0.79 µM, and 2.18 ± 0.61 µM (mean ± SD), respectively. These values were within the standard deviation of the data shown. The specimens used for this assay were collected during a previously reported study that included sham-inoculated volunteers (4). Briefly, healthy adult volunteers were either challenged on study Day 0 with RV23 or sham challenged with balanced salt solution. Nasal lavage was done prior to virus challenge and again on each day for the next 5 d (study Days 1 through 5). Only specimens from subjects who had received the placebo were selected for assay of nitrite concentration. Specimens from RV-infected subjects were specifically limited to specimens from those subjects who had symptomatic infection (total symptom score > 6).

Statistical Analysis

When appropriate, the statistical significance of results was determined. All statistical testing was done with the nonparametric signed rank test. All data are reported as the mean ± SD of at least three replicate experiments.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Effect of NO on RV Replication

Cell culture monolayers of BEAS-2B and MRC-5 cells were challenged with RV39 in the presence of three different NO donors with different solubilities and half-lives of NO release. No effect of the NO donors on viral replication was detected. Geometric mean log₁₀ virus titers were 3.58 ± 0.12 TCID₅₀ in control cells, and ranged from 3.42 ± 0.24 to 3.75 ± 0.18 TCID₅₀ in cells treated with SNAP (100 or 500 µM), SNP (500 µM), or NONOate (500 or 1,000 µM).

Effect of RV Infection on NO Production

To determine whether RV39 infection results in production of NO, confluent monolayers of BEAS-2B and MRC-5 cells were grown in 24-well plates. These cells were challenged with RV39 at multiplicities of infection (MOIs) of 1, 10, and 100 TCID₅₀/cell. Cell-free supernatants were harvested after 24 or 48 h of incubation at 33° C in 5% CO₂ and were assayed for the presence of nitrite. The nitrite concentrations were similar in the supernatants from infected and control monolayers for both cell lines (Table 1). The concentrations of nitrite detected in supernatants from cells challenged with MOIs of 1 and 10 TCID₅₀/cell were similar to those from cells challenged with an MOI of 100 TCID₅₀/cell.

Effect of NO on RV-induced IL-8 Elaboration

Increased concentrations of NO had no effect on RV-induced IL-8 elaboration. Incubation of BEAS-2B (Figure 1A) or MRC-5 (Figure 1B) cells with SNAP at increasing concentrations resulted in increased concentrations of nitrite (suggesting normal release of NO) but had no effect on IL-8 elaboration in the first 6 h after RV challenge. Similar results were seen with varying concentrations of SNP (10 to 500 µM) and PAPA-NONOate (10 to 300 µM in BEAS-2B cells and 100 to 300 µM in MRC-5 cells). Even at the highest nontoxic concentration of these NO donors there was no detectable effect on RV-induced IL-8 elaboration (Figure 2). The IL-8 concentrations in cell culture supernatants collected 24 h after virus challenge in the presence of NO donors were equivalent to the concentrations in control cells challenged with virus but without exposure to NO donors (data not shown). The ability of NO donors to produce biologic effects in BEAS-2B cells was confirmed by demonstrating enhancement of tumor necrosis factor- (TNF-)-induced IL-8 elaboration by similar treatment. Medium from BEAS-2B cells incubated with TNF- (10 ng/ml) for 6 h contained 425 ± 25 pg/ml of IL-8, as compared with 65 ± 8 pg/ml in medium from control cells. Preincubation of the cells with SNAP at concentrations of 10, 100, and 500 µM resulted in IL-8 concentrations of 636 ± 52 pg/ml, 986 ± 55 pg/ml and 1650 ± 75 pg/ml, respectively.

View larger version (30K): [in this window] [in a new window]   Figure 1.   IL-8 and nitrite concentrations in cell culture supernatants collected from BEAS-2B (A) and MRC-5 (B) cells 6 h after challenge with RV39 in the presence of different concentrations of the NO donor SNAP.

View larger version (38K): [in this window] [in a new window]   Figure 2.   IL-8 concentration in cell culture supernatants collected 6 h after RV39 challenge of BEAS-2B and MRC-5 cells in the presence of either NO donors (SNP and NONOate) or inhibitors of iNOS (L-NNA and L-NAME).

Although RV39 infection did not cause any increase in NO production, detectable levels of nitrite were present in the uninfected control cells (Table 1), suggesting the constitutive presence of NO synthesis in these cells. We therefore investigated the role of NOS inhibitors on RV-induced IL-8 elaboration. Neither L-NNA nor L-NAME showed a significant effect on the concentration of IL-8 in supernatant media collected 6 h (Figure 2) or 24 h after RV challenge of either BEAS-2B or MRC-5 cells. The biologic activity of the NOS inhibitors in these cell lines was confirmed in experiments with TNF- and interferon- (IFN-). Media from BEAS-2B cells collected after 48 h incubation with TNF- (5 ng/ml) and IFN- (1 µg/ml) contained 7.16 ± 0.6 µM nitrite, as compared with 4.32 ± 0.58 µM nitrite in media from control cells. Media from BEAS-2B cells preincubated with L-NNA contained 4.76 ± 0.74 µM nitrite after stimulation with TNF- and IFN-. Similarly, media from MRC-5 cells contained 8.85 ± 0.52 µM and 3.83 ± 1.4 µM nitrite after stimulation for 48 h with TNF- and IFN- without or with pretreatment with L-NNA, respectively. Control cells without stimulation contained 3.98 ± 1.5 µM nitrite.

Nitrite and IL-8 in Human RV Infection

Nitrite concentrations in nasal secretions were not altered by RV infection. Nitrite concentrations on study Day 0 prior to virus challenge, study Day 2, on which peak symptoms occurred, and study Day 5, when symptoms were abating, were comparable and were no different from the nitrite concentrations in nasal secretions from sham-challenged volunteers (Figure 3). In contrast, as previously reported, IL-8 concentrations were significantly increased in the nasal secretions of symptomatic, infected subjects (4).

View larger version (19K): [in this window] [in a new window]   Figure 3.   Nitrite concentrations in nasal lavage fluid from asymptomatic sham-challenged or symptomatic RV-infected subjects before (Day 0), and 2 and 5 d after challenge. Median IL-8 concentrations are shown for the infected (solid line) and sham challenged (dotted line) subjects on study Days 0 to 4.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

NO, a signaling and effector molecule with diverse roles in both physiologic and pathophysiologic conditions, is produced in a wide variety of mammalian cells as a result of oxidation of L-arginine by NOS. Both cNOS and iNOS are present in human respiratory epithelium. These respiratory epithelial cells produce NO in response to LPS and various cytokines in vitro (10). Our data, however, suggest that NO does not play a role in either the response to or the pathogenesis of RV infections. We found no evidence that NO was produced in response to RV infection, and there was no effect of NO donors or inhibitors of NO production on either virus replication or virus- induced IL-8 elaboration.

Activation of NF-B appears to be an essential step in the signal transduction pathway that results in elaboration of IL-8 (22). Oxidative stress-induced signaling events appear to be involved in the activation of NF-B and the elaboration of IL-8 following RV challenge (6). Recent data suggest that oxidative stress with activation of NF-B is also involved in the signaling pathways that result in upregulation of iNOS and production of NO (7). Despite these similarities, the absence of NO production after RV challenge in vitro and in vivo suggests that the signal transduction pathways for IL-8 and iNOS, at least in response to this stimulus, are ultimately different. This conclusion is further supported by data showing that treatment of cells with mevastatin, an inhibitor of farnesylation of ras protein, has no effect on RV-stimulated IL-8 elaboration (P. Kaul, I. Singh, R. B. Turner, unpublished observations) despite a potent inhibitory effect of mevastatin on iNOS and cytokine elaboration after endotoxin stimulation of neural cells (8).

The production of NO in response to viral upper respiratory tract infection in human subjects has been examined in a limited number of studies. In three previous studies, NO was measured directly in expired air. NO concentrations were increased during upper respiratory infection in two studies that involved subjects with lower respiratory tract disease (23, 24). In each of these studies NO was measured in orally exhaled air. A subsequent study of subjects with disease limited to the upper respiratory tract found no change in NO concentrations in nasally expired air (25). In accord with previous observations, the concentrations of NO in nasal air were substantially higher than those in orally expired air in these studies. These studies, together with the results we report here, suggest that NO production may correlate better with the presence of lower respiratory tract disease than with the presence or absence of upper respiratory tract infection.

NO may play a role in viral infection by exerting an antiviral effect against some viruses. NO has been reported to inhibit the replication of both DNA and RNA viruses in vitro (15, 26). In contrast, NO has no effect on the replication of several other viruses (27, 28). The effect of NO on picornavirus replication is inconsistent. NO inhibits the replication of coxsackievirus B3 by inhibiting viral RNA synthesis both in vitro and in vivo (18, 29). Similarly, poliovirus and encephalomyocarditis virus are inhibited by NO (30, 31). In studies with BEAS-2B cells, RV replication was inhibited by NO at 24 h but was unaffected 48 h after challenge (32). In our studies, no effect on RV replication was detected 48 h after challenge of human embryonic lung fibroblast cells.

An alternate mechanism by which NO may play a role in the pathogenesis of viral infection is by modulating the inflammatory response. Murine pneumonitis caused by cytomegalovirus or by influenza virus appears to be mediated at least in part by NO or its byproducts (28, 33). Inhibition of NO production in these models was associated with amelioration of disease, although the exact mechanism of the NO-induced injury was not determined.

NO may modulate the inflammatory response by its effect on the elaboration of proinflammatory cytokines. NO has been shown to upregulate expression of the IL-8 gene under some conditions (13, 14). In contrast to this proinflammatory activity, under certain conditions NO may also inhibit cytokine elaboration. Treatment of alveolar macrophages with NO inhibits their elaboration of TNF-, IL-1, and macrophage inflammatory protein-1 in vitro (11). Similarly, treatment with NO inhalation significantly reduced the concentrations of IL-6 and IL-8 in bronchoalveolar lavage fluid from patients with adult respiratory distress syndrome (12).

Conflicting data have been reported about the effect of NO on proinflammatory cytokine elaboration in response to viral infection. Treatment of A549 cells, a pulmonary epithelial cell line, with inhibitors of iNOS had no effect on IL-8 elaboration in response to infection with respiratory syncytial virus (34). In contrast, a recent study found that elaboration of both IL-8 and IL-6 by BEAS-2B cells in response to infection with RV type 16 was significantly inhibited by pretreatment of the cells with NONOate (32). These data are in direct contrast to our findings of no effect of either NO donors or inhibitors on RV-induced elaboration of IL-8. Although different RV serotypes and challenge inocula were used in the two studies, there is no obvious explanation for the difference in the results of these studies. The RV16 used by Sanders and colleagues and the RV39 used in our studies are both in the major group of RVs that use ICAM-1 as a receptor. The role of NO in virus- induced IL-8 elaboration with the minor serotypes of RV has not been evaluated. Despite the conflicting data from in vitro studies, our study found no correlation between the concentration of NO (as assessed by nitrite concentration) and IL-8 in nasal lavage from human volunteers. Nitrite concentrations were similar in RV-infected and sham-challenged volunteers. In contrast, IL-8 levels were significantly increased in subjects with symptomatic infections. These results suggest that NO does not affect IL-8 elaboration in the upper respiratory tract during RV infections.