Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Rhinovirus (RV) is the pathogen most frequently associated with exacerbations of asthma in susceptible children and adults (1, 2). Although mechanisms by which viral respiratory infections provoke asthma are incompletely understood, it is likely that airway epithelial cells participate in triggering local inflammation through the secretion of inflammatory cytokines and/or mediators. Although few studies have been performed using nontransformed epithelial cells, studies with epithelial cell lines indicate that in vitro infection with RV induces secretion of granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-6, IL-8, and IL-11 (3). Moreover, several of these cytokines are increased in nasal secretions during RV infection (3, 4, 6), and can have profound effects on inflammatory cells that are known to potentiate asthma. For example, IL-8 is a potent chemotactic agent for neutrophils and activated eosinophils (7). GM-CSF is a potent activator of eosinophil survival and adhesion molecule expression, and is a cofactor for eosinophil superoxide production and degranulation (8). Targeted expression of IL-11 in the lungs of transgenic mice leads to airway inflammation and bronchial hyperresponsiveness (9). These data suggest that epithelial cells could contribute to both eosinophil recruitment and activation in the context of RV respiratory infection, thus contributing to associated lower airway inflammation.

One key question is whether RV infections associated with asthma exacerbations extend into the lower airway. There is evidence to indicate that RV infection may involve the lower airway under some conditions. For example, RVs have been detected in upper airway secretions from children and adults with clinical bronchitis and/or pneumonia, and also from lower airway samples obtained at autopsy from an infant with fatal pneumonitis (10). In addition, RV infections produce lower airway inflammation, as indicated by increased submucosal lymphocytes and epithelial eosinophils in bronchial biopsies (11). Finally, although RV has proven difficult to culture from lower airway secretions obtained via bronchoalveolar lavage (BAL), RV RNA has been detected in experimentally infected volunteers in BAL cells using polymerase chain reaction (PCR) (12) and in bronchial biopsy specimens by in situ hybridization (13).

Although RV replicates and causes cytokine secretion in epithelial cell lines and in primary cultures of epithelial cells from the upper airway (5, 14), effects of RV on cultures of airway epithelial cells from the lungs are incompletely understood. To further define the role of bronchial epithelial (BE) cells in RV infection and the pathogenesis of asthma exacerbations, we inoculated primary cultures of BE cells with RV, and measured effects on viral replication, cell viability, adhesion molecule expression, and the production of the cytokines regulated on activation, normal T cells expressed and secreted (RANTES), IL-8, and GM-CSF.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Cell Culture

Human BE cells for primary culture were obtained from surgical specimens of normal human bronchi or trachea (lung transplant donors), isolated by the method described by Wu and colleagues (15), with modifications. Briefly, airway specimens were rinsed with phosphate-buffered saline (PBS) and then placed in dissociation solution consisting of modified Mg²⁺- and Ca²⁺-free minimum essential medium supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml), pronase (0.14%; Boehringer Mannheim, Indianapolis, IN), and DNase (100 µg/ml; Sigma Chemical Co., St. Louis, MO) for 24 to 36 h. After incubation, fetal calf serum (FCS) (Sigma) was added to a final concentration of 10%, and epithelial cells were detached from the stroma by gentle agitation. The cells were collected by centrifugation, washed, and suspended in Dulbecco's modified Eagle medium F12 media (GIBCO BRL, Grand Island, NY) with penicillin, streptomycin, and 5% FCS. To remove fibroblasts, the cell suspension was incubated (37°C, 5% CO₂) in uncoated tissue culture dishes for 2 to 6 h. Nonadherent cells were seeded into type VI collagen-coated tissue culture plates and flasks. All primary cell cultures were grown in serum-free hormonally supplemented media, basal essential growth media (BEGM; Clonetics, San Diego, CA), and were passaged up to five times. Epithelial cell purity was estimated by staining the cells with anticytokeratin (DAKO, Carpinteria, CA) or isotype control and analyzing with flow cytometry: less than 1% of the cells were cytokeratin-negative. Cell viability, as assessed by exclusion of trypan blue dye, was consistently greater than 90%.

RV Suspensions

RV serotypes 16 and 49 were grown in HeLa cells and WI-38 cells as previously described (16). Stock suspensions of RV16 contained 10^8-8.5 50% tissue culture infective dose/ ml (TCID₅₀/ml), and RV49 10^7-7.25 TCID₅₀/ml. An RV16 suspension that had been purified over a sucrose gradient (17) was used in some experiments, and was generously provided by Dr. Wai Ming Lee (Institute of Molecular Virology, University of Wisconsin-Madison, Madison, WI).

RV Replication Assay

Monolayers of BE cells grown in 24-well plates were incubated at room temperature for 90 min with RV16 (10⁷ TCID₅₀/ml) or RV49 (10⁶ TCID₅₀/ml) while gently rotating. Different maximal doses of RV16 and RV49 were used on the basis of the concentration of the virus available (e.g., RV16 stock concentration was 10^8.5 TCID₅₀/ml and RV49 stock concentration was 10⁷ TCID₅₀/ml). After incubation, the monolayers were washed with PBS to remove unbound virus, and the media were replaced. At 2 and 24 h, samples were quick-frozen in a dry ice and methanol bath and thawed in rapid succession three times to lyse the cells and release intracellular virus. The suspensions were collected, centrifuged to remove cellular debris, and the supernates were collected and stored at 70°C pending analysis. Ultraviolet (UV)-irradiated RV suspensions were prepared as previously described (16), and the irradiated virus suspensions were found to be noninfectious after this procedure. Quantitative RV cultures were performed as previously described (16).

Cell Viability Assay

BE cell viability was measured using an assay based on the intracellular enzymatic conversion of the nonfluorescent cell-permeable calcein acetoxymethyl ester to fluorescent calcein, which occurs in live but not dead cells (18). BE cells were incubated (72 h, 37°C, 5% CO₂) with RV16 (10³ to 10⁷ TCID₅₀/ml), UV-irradiated RV16 (equivalent to 10⁶ TCID₅₀/ml), RV49 (10³ to 10⁶ TCID₅₀/ml), UV-irradiated RV49 (equivalent to 10⁶ TCID₅₀/ml), or media alone. After incubation, the wells were rinsed and fresh media were added. Calcein was diluted 1:1,000 according to the manufacturer's instructions (Molecular Probes, Inc., Eugene, OR) and incubated (45 min, 37°C, 5% CO₂) with the cells. The plate was read with a microfluorometer (Model 7625; Packard Instruments, Meriden, CT) at excitation/emission wavelengths of 485/ 530 nm. Preliminary experiments were conducted in which 0 to 10⁵ live uninfected cells, or cells killed by incubation (10 min) with 0.1% saponin, were placed into wells and then processed as described previously. There was a linear relationship between the number of live cells per well and fluorescence, whereas wells containing dead cells had minimal fluorescence (data not shown). Data from virus-infected samples are expressed as percent calcein conversion, calculated with the following formula: 100 × (fluorescence of infected sample/fluorescence of uninfected sample).

Whole-Cell Enzyme-Linked Immunosorbent Assays

Effects of RV, macrophage supernates, and cytokines on epithelial cell intercellular adhesion molecule (ICAM)-1 expression were measured with whole-cell enzyme-linked immunosorbent assay (ELISA), as previously described (19, 20). Briefly, cell monolayers were incubated (24 h, 37°C, 5% CO₂) with RV16 (10⁵ to 10⁷ TCID₅₀/ml) or RV49 (10⁴ to 10⁶ TCID₅₀/ml) in 96-well plates with BEGM. The wells were washed in PBS with 3% milk (wt/vol) and 5% FCS, and then incubated (2 h, 37°C, 5% CO₂) with either 100 ng/ml of monoclonal anti-ICAM-1 (RR1; generously provided by Dr. R. Rothlein, Boehringer Ingelheim, Ridgefield, CT) or an isotype control (mouse immunoglobulin [Ig] G₁; Sigma) in blocking solution. After washing, the cells were incubated (2 h, 37°C, 5% CO₂) with sheep antimouse antibody conjugated with horseradish peroxidase (Sigma). After another wash, 3,3',5,5'-tetramethybenzidine (Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) was applied and the plates were incubated at room temperature until the greatest screening fluorescence at 660 nm was 0.5 to 0.7 optical density (OD). The reaction was stopped with 0.18 M H₂SO₄ and the final OD was read at 450 nm.

The whole-cell ELISA to measure human leukocyte-associated antigen-DR (HLA-DR) was similar to that described previously, with the following modifications. After incubation with RV, the BE cells were fixed with 1% paraformaldehyde for 15 min. After washing, the plates were blocked with 10% FCS in PBS for 1 h at 37°C. In addition, the primary (monoclonal anti-HLA-DR 25 ng/ml; Becton-Dickinson, San Jose, CA) and secondary (sheep antimouse) antibodies were diluted in PBS with 10% FCS, and incubations were 90 min. Results from both the ICAM-1 and HLA-DR whole-cell ELISAs are expressed in terms of OD.

Induction and Detection of RANTES Secretion from BE Cells

BE cells were grown in 96-well tissue culture plates with BEGM to near confluence. Twenty-four hours before adding inhibitors, the media were changed to BEGM without hydrocortisone, epidermal growth factor, epinephrine, or bovine pituitary hormone, in an effort to remove factors that could affect cytokine production. Preliminary experiments demonstrated that RANTES production was decreased in experiments performed in complete media compared with experiments in media lacking the factors described previously (data not shown). Further, no deleterious effects to the cells were noted when established monolayers were cultured in the medium without these additives for 72 h. In some experiments, BE cell monolayers were preincubated (1 h, room temperature, gentle shaking) with 20 µg/ml of either anti-ICAM-1 (RR1) or an isotype control before inoculation with RV16. The monolayers were then incubated (48 h, 37°C, 5% CO₂) with either purified RV16, RV49, or medium alone. After incubation, the supernates were collected and frozen at 70°C: RANTES and IL-8 proteins were measured by commercially available ELISA kits (R&D Systems, Minneapolis, MN; and Biosource, Camarillo, CA), and GM-CSF was measured in a standard sandwich ELISA by Dr. E. A. Becky Kelly (University of Wisconsin-Madison) (21).

Detection of RANTES Messenger RNA with Semiquantitative Reverse Transcriptase-PCR

RANTES messenger RNA (mRNA) in BE cells was analyzed with semiquantitative reverse transcriptase (RT)- PCR. Total cellular RNA was extracted using a one-step phenol/chloroform extraction reagent (RNA-STAT-60; Cinna/Leedo Medical Laboratories, Houston, TX) according to the manufacturer's instructions. After RNA was precipitated with 50% isopropanol and 100 µg glycogen and the pellet was washed in 70% ethanol, RNA was resuspended in 50 µl double-distilled diethylpyrocarbonate-treated water. The RNA was reverse transcribed by incubating (at 37°C for 1 h) 8 µl of the RNA solution along with 200 U RT (Superscript II; GIBCO BRL), 4 µl 5× reaction buffer, 0.01 M dithiothreitol, 40 U recombinant RNAsin (GIBCO BRL), 0.5 µg random primers, and 0.5 mM deoxynucleotide triphosphates in a total volume of 20 µl. The mixture containing complementary DNA (cDNA) was then diluted 1:5 in water and 4 µl was transferred to a 650-µl thin-walled PCR tube along with 2 U Taq DNA polymerase (Promega, Madison, WI), 5 µl 10× PCR buffer, 25 mM MgCl₂, and 20 µM of each primer, in a total reaction volume of 50 µl. Primer pairs for RANTES (22) and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) (Clontech, San Diego, CA) were selected to span introns; no amplification of genomic sequences was observed. The PCR mixture was overlaid with oil, denatured by heating to 94°C for 5 min, and subjected to denaturation at 94°C for 45 s, annealing at 60°C for 45 s, and then extension at 72°C for 2 min. The number of PCR cycles (RANTES, 33 cycles; G3PDH, 18 cycles) was chosen to maintain a linear relationship between mRNA and the PCR products so that quantitative comparisons of PCR products could be obtained. In addition, a reference standard curve consisting of 4-fold dilutions of cDNA from a highly positive sample was included in each PCR run. Controls in each PCR run included samples containing reagents but no cDNA, and positive control samples containing RANTES or G3PDH cDNA template. The PCR products were electrophoresed onto a 2.0% agarose gel, and the identity of the PCR products was verified by Southern blotting using a commercial kit (ECL System; Amersham, Arlington Heights, IL).

Statistical Analysis

Analysis of data was performed using computer software (SigmaStat; Jandel Scientific, San Rafael, CA). Results from quantitative viral cultures and cytokine values were log transformed; these values approximated a normal distribution. Changes in viral titers were evaluated with the two-tailed t test. Calcein conversion data, expressed as % conversion relative to uninfected control samples, were analyzed with analysis of variance (ANOVA) with a post hoc analysis (Fisher PLSD) test. Cytokine levels were analyzed with two-way ANOVA: the analysis included day of experiment and treatment group as factors, and pairwise comparisons were made with the Tukey test. The correlation between the concentration of hydrocortisone and RANTES secretion was analyzed by Spearman's rank sum test. Statistical significance was defined as P  0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

RV Replication in BE Cells

To determine whether RV could replicate in lower airway cells, BE cell monolayers were incubated with an excess of RV16 (10⁷ TCID₅₀/ml) or RV49 (10⁶ TCID₅₀/ml), washed to remove unbound virus, and incubated at either 33°C (a temperature that has been reported as optimal for RV replication) or 37°C (which approximates more closely the temperature of terminal airways in vivo) (23). Replication, as indicated by an increase in RV titer 24 h after viral attachment, occurred at both temperatures (Figure 1). Titers of RV16 increased by 2.0 log units at 33°C (Figure 1a) and 1.6 log units at 37°C (Figure 1b). Similar increases in viral titers (2.5 log units at 33°C and 1.6 log units at 37°C) were observed in cells inoculated with RV49.

View larger version (28K): [in this window] [in a new window]   Figure 1.   Replication of RV16 and RV49 in BE cells at 33° and 37°C. BE cells were incubated (90 min, room temperature) with an excess of RV16 (107 TCID50/ml) or RV49 (106 TCID50/ml) and then washed to remove nonadherent virus. After fresh media were added, parallel sets of samples were either lysed to determine the amount of bound virus (2-h samples), or incubated (33° or 37°C, 5% CO2) and then lysed the following day (24-h samples). RV replication, indicated as a positive TCID50/ml, occurred with (a) RV16 at 33°C (TCID50/ml = 2.0 log units, n = 7); (b) RV16 at 37°C (TCID50/ml = 1.6 log units, n = 3); (c) RV49 at 33°C (TCID50/ml = 2.5 log units, n = 7); and (d) RV49 at 37°C (TCID50/ml = 1.6 log units, n = 3). Data are expressed as geometric means; *P < 0.01, **P < 0.001.

Effect of RV Infection on BE Cell Viability

Despite the presence of vigorous viral replication, the morphology of BE cells incubated for 1 to 5 d with RV16 did not differ from uninfected cells. In contrast, RV49 infection caused cytopathic effects such as increased granularity, cell rounding, and detachment as early as 24 h after inoculation (Figure 2).

View larger version (219K): [in this window] [in a new window]   View larger version (232K): [in this window] [in a new window]   View larger version (212K): [in this window] [in a new window]   Figure 2.   Cytopathic effect of RV on BE cells. BE cell monolayers were inoculated with (a) medium from uninfected HeLa cells, (b) RV16 (107 TCID50/ml), and (c) RV49 (106 TCID50/ml), and were photographed after 24 h. Cells infected with RV49 (c) showed cytopathic effects, including increased granularity, rounding, detachment, and shrinkage.

To quantify these observations, BE cell viability was measured 72 h after incubation with control medium or with increasing amounts of RV16 or RV49 (Figure 3). RV16, at concentrations as high as 10⁷ TCID₅₀/ml, did not significantly diminish BE cell viability (Figure 3a). In contrast, BE cells infected with RV49 at 10⁶ TCID₅₀/ml had significantly diminished viability compared with control cells (Figure 3b). Incubation with UV-irradiated (noninfectious) RV16 or RV49 did not significantly diminish BE cell viability (Figure 3).

View larger version (21K): [in this window] [in a new window]   Figure 3.   BE cell viability after RV16 or RV49 infection. Incubation with RV16 (a) did not reduce BE cell viability, as indicated by no significant change in calcein conversion (n = 3). In contrast, BE cells incubated with 106 TCID50/ml RV49 (b) had significantly decreased viability (n = 5). Treating the virus with UV irradiation significantly blocked this effect. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01.

Epithelial cells incubated with RV49 for 5 d tended to have progressively greater virus-induced cytopathic effects (rounding, granularity) and reduced viability (calcein conversion), and there were trends toward reduced viability at 10⁴ to 10⁵ TCID₅₀/ml RV49 (data not shown). However, cells incubated for 5 d in the absence of virus were also starting to show cytopathology and reduced viability.

One potential explanation for the deleterious effects of large doses of RV49 is that RV nonstructural proteins (e.g., 2A protease), which are present in viral preparations obtained from cell lysates, interfere with cell metabolism (24) and could cause cell toxicity. If this were the case, the toxic effect of the viral suspension would be independent of the binding of intact virions to the cells. To test this possibility, we incubated epithelial cells with RV49 in the presence or absence of low-density lipoprotein (LDL; Sigma) that inhibits binding of minor-group RV to epithelial cells (25). Although LDL itself produced some reduction in calcein conversion, LDL blocked most of the RV49-induced cytotoxicity (_RV, Table 1).

Effect of RV Infection on ICAM-1 and HLA-DR Expression

BE cells express the cellular proteins ICAM-1 and HLA-DR in response to a variety of inflammatory stimuli. To determine whether RV infection induces ICAM-1 and/or HLA-DR expression, BE cells were incubated with partially purified RV16 or RV49 for 24 to 72 h and receptor expression was measured by whole-cell ELISA. Primary BE cells expressed basal levels of ICAM-1 that were not increased 24 h after inoculation with either RV16 (mean values 0.61 versus 0.63 OD units, n = 4) or RV49 (mean values 0.83 versus 0.78 OD units, n = 4). In addition, HLA-DR was not detectable on BE cells after incubation with RV16, RV49, or medium alone for 48 h (data not shown). In contrast, vigorous HLA-DR (2.1 OD units, mean, n = 4) and ICAM-1 (2.6 OD units, mean, n = 4) expression was induced with interferon- (10 U/ml), which was used as a positive control.

RANTES Secretion by RV-Infected Epithelial Cells

To determine whether RV infection induced production of proinflammatory cytokines, BE cell monolayers were incubated with RV16 or RV49 for 48 h and culture supernates were analyzed for RANTES protein and mRNA. BE cells incubated with RV16 (10⁷ TCID₅₀/ml) or RV49 (10⁶ TCID₅₀/ml) secreted significantly more RANTES than did uninfected control cells (243, 398, and 10 pg/ml, respectively; Figure 4). In addition, when RV infectivity was neutralized with UV irradiation RANTES secretion was significantly (~ 80%) reduced for both RV16 and RV49 (50 and 85 pg/ml, respectively), although RANTES secretion by cells inoculated with UV-inactivated virus was still significantly greater than that of control uninfected cells (Figure 4).

View larger version (11K): [in this window] [in a new window]   Figure 4.   Effects of RV and UV-inactivated RV on BE cell RANTES secretion. BE cell monolayers were inoculated with untreated or UV-irradiated RV16 (107 TCID50/ml, n = 4) or RV49 (106 TCID50/ml, n = 6), and supernates collected 48 h later were analyzed for RANTES protein. RV16 and RV49 each induced significantly greater RANTES production than did uninfected control cells (*P < 0.05). BE cells incubated with UV-irradiated RV secreted small but significant amounts of RANTES (*P < 0.05), but at significantly lower levels compared with untreated RV (P < 0.05). Data are expressed as geometric means ± SEM.

Incubation with RV16 or RV49 also increased steady-state RANTES mRNA levels but had no noticeable effect on expression of G3PDH (Figure 5a). Induction of RANTES mRNA was blocked by treating RV16 or RV49 with UV light before inoculation; or, for RV16, by preincubating the cells with anti-ICAM-1 monoclonal antibody (mAb) (Figure 5b).

View larger version (36K): [in this window] [in a new window]   View larger version (35K): [in this window] [in a new window]   Figure 5.   Effect of RV on RANTES mRNA in BE cells. (a) Monolayers of BE cells were incubated with RV16 (107 TCID50/ ml) or RV49 (106 TCID50/ml) for 24 h, and then RANTES and G3PDH mRNA were analyzed by RT-PCR and Southern blotting. Both RV16 and RV49 increased RANTES mRNA compared with uninfected control cells, whereas G3DPH mRNA expression was stable. (b) Monolayers of BE cells were incubated (24 h, 37°C) with either RV16 (107 TCID50/ml, lane 1), UV-irradiated RV16 (lane 2), RV49 (106 TCID50/ml, lane 3), UV-irradiated RV49 (lane 4), anti-ICAM-1 mAb (10 µg/ml, lane 5), or RV 16 plus anti-ICAM mAb (lane 6 ). The anti-ICAM mAb was applied 1 h before virus inoculation. RANTES mRNA and G3PDH mRNA were determined from total cellular RNA with RT-PCR and Southern blotting.

To further characterize patterns of RV-induced cytokine secretion, BE cells were incubated with purified RV16 (10⁷ TCID₅₀/ml) in the presence or absence of anti-ICAM-1 mAb. Uninfected cells secreted low amounts of RANTES (16 pg/ml), and incubation with purified RV16 significantly increased secretion of RANTES (239 pg/ml). RV-induced RANTES was significantly blocked by anti-ICAM-1 mAb (32 pg/ml), but not by an isotype control antibody (140 pg/ ml; Figure 6). In addition, RV also significantly enhanced secretion of IL-8 (3,013 versus 1,175 pg/ml control uninfected cells; Figure 6B) and GM-CSF (179 versus 32 pg/ml uninfected control cells; Figure 6C). In contrast to RV induction of RANTES, coincubation with anti-ICAM-1 did not inhibit virus-induced IL-8 or GM-CSF. Incubation of BE cells with anti-ICAM-1 mAb alone did not induce cytokine secretion (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 6.   Effects of anti-ICAM-1 mAb on RV-induced cytokine secretion. BE cell monolayers were incubated with or without - ICAM-1 mAb or an isotype control antibody (IgG) for 24 h and then inoculated with either RV16 (107 TCID50/ml) or medium alone for an additional 48 h, and supernatant fluids were analyzed for cytokines. RV16 induced significant amounts of RANTES (A), and similar trends were noted for IL-8 (B) and GM-CSF (C ). Preincubation with -ICAM-1 mAb, but not the control antibody, blocked RV16- induced BE cell RANTES (A) secretion, but did not significantly reduce secretion of IL-8 (B) or GM-CSF (C ). Data are expressed as geometric means ± SEM, n = 6 for RANTES and GM-CSF, and n = 8 for IL-8. *P = 0.05 versus uninfected control cells ("medium"), P < 0.05 versus cells inoculated with RV16.

Effect of Hydrocortisone on RV-Induced RANTES Secretion

Corticosteroids have been shown to inhibit cytokine-induced RANTES secretion from human airway epithelial cells (26). To determine whether hydrocortisone could also inhibit RV-induced RANTES secretion in BE cells, cell monolayers were incubated for 24 h with 0 to 10⁶ M hydrocortisone, then RV16 (10⁷ TCID₅₀/ml) or an equivalent amount of control medium was added to the wells, and cell supernates were sampled 48 h later. Increasing concentrations of hydrocortisone significantly suppressed BE-cell RANTES secretion (Figure 7): RANTES secretion was 50% inhibited at approximately 10⁸ M. 

View larger version (12K): [in this window] [in a new window]   Figure 7.   Effect of hydrocortisone on RV-induced RANTES secretion. BE cells were incubated for 24 h with 0 to 106 M hydrocortisone, and were then inoculated with RV16 (107 TCID50/ml) for an additional 48 h. Hydrocortisone significantly suppressed RV16-induced RANTES secretion (rs = 1.0, P < 0.05, n = 3). Data are expressed as geometric means ± SEM.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

To gain an understanding of mechanisms of asthma exacerbations triggered by RV infections, we studied the interactions between two different RV serotypes and nontransformed bronchial epithelial cells in vitro. Both RV16 and RV49 replicated vigorously in primary human lower airway BE cells during in vitro culture. However, the two different RV serotypes had distinct effects on epithelial cell morphology and viability: RV49 lysed most of the cells by 24 to 48 h, whereas RV16 infection produced minimal cytopathic effects and did not significantly diminish cell viability. Moreover, RV replication in primary BE cells was associated with selective activation: RANTES mRNA and protein were increased, and similar effects were observed for IL-8 and GM-CSF secretion, but RV infection did not induce increased expression of ICAM-1 or HLA-DR. Finally, RV-induced RANTES secretion was suppressed by treatment with hydrocortisone.

Several controls were included in these experiments to establish that the effects observed were virus-related. For example, effects caused by RV16 in HeLa cell lysate were reproduced using purified RV16. In addition, treating the viral suspensions with UV irradiation (which presumably damages viral RNA to prevent replication) reduced RANTES secretion by 80%, indicating that viral replication is required to fully induce RANTES secretion. The fact that inhibition of RANTES secretion by UV irradiation was incomplete suggests that events early in the viral replication cycle (such as binding and uncoating) that are unlikely to be affected by UV irradiation could make small but measurable contributions to induction of RANTES secretion. Interestingly, a mAb (RR1) specific for the viral binding domain of ICAM-1 significantly inhibited RV16-induced secretion of RANTES, but not IL-8 or GM-CSF. This information suggests that there may be mechanisms for RV proteins to activate epithelial cells that are independent of viral binding and replication, but additional studies are needed to test this hypothesis.

Our experiments demonstrate the feasibility of studying immune responses to RV infection using cultured BE cells. Primary cultures of nontransformed BE cells provide an informative model to test factors that affect viral replication and epithelial cell cytokine production while minimizing the chances of obtaining nonphysiologic responses that can occur in tumor cell lines or immortalized cells. Although use of cultured BE monolayers allows for detailed examination of RV/epithelial cell interactions, a potential limitation of these studies is that interactions between BE cells, airway inflammatory cells, and matrix proteins that occur in airway tissues are lacking in this in vitro model of isolated epithelial cells.

Whether RV replicates in lower airway cells during RV-induced exacerbations of asthma has been an unresolved question. Our findings demonstrate that BE cells have the necessary surface receptors and intracellular machinery to serve as hosts for RV infection in vivo. Further, our findings complement epidemiologic data and clinical research studies which imply that RV alters lower airway physiology (2, 27, 28), and to accomplish these changes may infect lower airway cells.

RVs replicate with the greatest efficiency at 33°C, and it has been suggested that this characteristic could limit the ability of RV to grow in the warmer environment of the lower airway. In fact, temperatures in the lower airways of humans have been directly measured, and although the lung parenchyma is at core temperature, the first few generations of airways are significantly cooler (23). For example, the temperature of a subsegmental bronchus is approximately 35°C during quiet breathing of room air, and becomes lower with hyperventilation or with breathing of cooler inspired air (23). These data, together with our findings that significant RV replication occurs in lower airway cells even at 37°C, indicate that temperatures throughout the human airway are conducive to RV replication.

The mechanisms by which RV infection of bronchial epithelium increases airway responsiveness and airway inflammation are not established, but do not necessarily involve extensive destruction of airway epithelium. In general, biopsies of nasal mucosa during acute RV infections have shown little cytopathology (29), although some studies have detected increased epithelial detachment or sloughing (30, 31). Accordingly, our results indicate that growth of RV16 in BE cell monolayers does not cause appreciable cell damage. Because the only known mechanism for the release of picornaviruses is via cell lysis, these findings imply that there may be a small subpopulation of epithelial cells that support RV replication during natural infections. This hypothesis has been verified by studies using in situ hybridization (14, 32), although the identity of this subpopulation of susceptible cells is as yet unknown.

In contrast to the effects of RV16, incubation of BE cells with high concentrations of RV49 produced appreciable cell lysis in vitro. Because LDL, which blocks viral binding, also inhibited most of the virus-induced changes in cell viability, these data suggest that the binding of intact viruses, and not some other component of the viral suspension, was mainly responsible for the reduction in cell viability. The increased cell destruction may have been due to greater viral binding and a larger yield of RV49 from cultured BE cells, although RV16 and RV49 titers could not be compared directly because they were determined in different cell lines. Additional studies will be needed to determine whether increased destruction of BE cells in tissue culture is indicative of greater epithelial cell damage, and potentially greater virulence, during RV infections in vivo.

We did not find that RV infection caused significant changes in epithelial cell expression of ICAM-1. One potential pitfall in our experiments is that the antibody used to detect ICAM-1 binds to an epitope that overlaps with the binding site for major-group RVs, and so RV16 could have interfered with the detection assay. However, RV49, which does not bind to ICAM-1 and therefore would not block the detection antibody, also did not cause increased ICAM-1 expression. In addition, our experiments do not rule out the possibility that ICAM-1 was induced on a small subpopulation of epithelial cells, and we are conducting additional experiments to test this hypothesis.

Other studies have examined the effects of RV infection on epithelial cell ICAM-1 expression, with conflicting results. Subauste and associates (5) incubated BEAS-2B epithelial cells with RV14 for up to 24 h and found no significant changes in ICAM-1 expression in infected cells. In contrast, Sethi and coworkers (33) incubated H292 cells with RV14 for up to 14 d and found ICAM-1 expression was increased after 4 d of incubation with virus, with peak expression occurring between 6 and 14 d after inoculation. Similarly, Terajima and colleagues (34) found that two RV serotypes (RV2 and RV14) significantly increased ICAM-1 mRNA in nontransformed monolayers of human tracheal epithelial cells after a 5-d incubation. In addition, tracheal cells appeared to have increased immunostaining for ICAM-1 2 d after inoculation with RV14, although a quantitative analysis was not performed. Several differences in experimental design, including the types of airway epithelial cells, the viral preparations and/or serotypes, and the temperature and length of incubation with virus, could account for the lack of consensus regarding the effect of RV infection on ICAM-1 expression. Additional studies are needed to resolve this issue.

Secretion of RANTES and other cytokines by BE cells during RV infections could have important effects on airway physiology, and these effects may be especially important in the context of asthma. For example, RANTES is a chemoattractant for eosinophils and memory T cells, and there is evidence that in mild asthma, both eosinophils and T cells are recruited to the lower airway mucosa early during the course of viral respiratory infections (11, 35). Further, Calhoun and colleagues (28) reported that experimentally induced RV16 infection increased antigen-induced eosinophil recruitment to the lower airway. These observations suggest that chemotactic factors such as RANTES are produced by lower airway cells, and may contribute to recruitment and/or activation of eosinophils and T cells to the lower airway during viral respiratory infections. Inhibition of RV-induced RANTES secretion by hydrocortisone suggests that corticosteroid medications may be able to interrupt this pathway of virus-induced inflammation. In addition, corticosteroids, which are commonly added to epithelial cell culture media such as BEGM, should be left out of the medium when studying virus-induced immune responses of epithelial cells in vitro.

In summary, our data indicate that nontransformed BE cells have the necessary surface receptors and intracellular factors to support RV replication, and the process of viral replication also activates these cells to secrete RANTES and potentially other inflammatory cytokines. Our findings support the concept that RV infection can extend into the lung and infect cells of the lower airway, and secretion of cytokines by infected epithelial cells in the lower airway provides a possible mechanism for the recruitment to the lower airway of inflammatory cells such as the eosinophil. In asthma, this increased lower airway inflammation could contribute to increased airway responsiveness and the pathogenesis of respiratory symptoms.