Resistance of differentiated human airway epithelium to infection by rhinovirus

N. Lopez-Souza,1 G. Dolganov,2 R. Dubin,1 L. A. Sachs,3 L. Sassina,3 H. Sporer,1 S. Yagi,4 D. Schnurr,4 H. A. Boushey,2 and J. H. Widdicombe1,2,3

1Children's Hospital Oakland Research Institute, Oakland 94609; 2Cardiovascular Research Institute, University of California San Francisco 94143; 3Department of Human Physiology, University of California-Davis, Davis 95616-8664; and 4California Department of Health Services, Richmond, California 94804

Submitted 2 September 2003 ; accepted in final form 9 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Virtually all in vitro studies of the effects of rhinovirus on human airway epithelium have used cells grown under conditions known to produce low levels of differentiation. The relevance of the results to native epithelium is questionable. Here we grew primary cultures of human tracheal or nasal epithelium under three conditions. One condition produced pseudostratified, mucociliary cells virtually indistinguishable from native epithelium. The other two conditions produced undifferentiated squamous cells lacking cilia. Cells were infected for 6 h with rhinovirus-16. After a 24-h incubation period, we determined levels of viral RNA in the cells, numbers of infectious viral particles released in the mucosal medium, expression of a variety of epithelial cytokines and other proteins, release of IL-6 and IL-8, and transepithelial electrical resistance and voltage. After infection, levels of viral RNA in the poorly differentiated cells were 30 or 130 times those in the differentiated. Furthermore, expression of mRNA for inflammatory cytokines, release of infectious particles, and release of IL-6 and IL-8 were closely correlated with the degree of viral infection. Thus well-differentiated cells are much more resistant to viral infection and its functional consequences than are poorly differentiated cells from the same source.

ion transport; porous-bottomed inserts; short-circuit current; transepithelial electrical resistance


RHINOVIRUS (RV) causes severalfold increases in the output of a variety inflammatory cytokines from human airway epithelial cell lines (29, 41) and from primary cultures of human airway epithelium (18, 23, 31, 39). However, the cell lines used (A549 and BEAS-2B) generally do not form tight junctions (17, 25), and the primary cultures were grown on solid supports, an approach known to produce highly undifferentiated cells (36). We questioned whether results obtained on such cultures were really representative of viral infection of native epithelium. Accordingly, we have grown cells from the same tracheas and nasal scrapings under several different culture conditions to achieve a range of differentiation from a squamous to a full-blown pseudostratified, mucociliary phenotype. We then compared the susceptibility of these various cultures to viral infection.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tracheal epithelial cells were obtained by protease digestion postmortem, as described previously (38). They were suspended in a 1:1 mixture of DMEM and Ham's F-12 medium (DMEM-F-12) containing 5% FBS and seeded at 5 x 105 cells/cm2 on 1.13-cm2 Transwell polycarbonate inserts (no. 3401; Costar, Corning, NY) or glass coverslips (12 mm diameter) in the bottom of 24-well plates. All growth surfaces were coated with human placental collagen, as described previously (3). The day after plating, the DMEM-F-12 over the coverslips and over half the filters was replaced with "Gray's medium" (8), a medium whose composition (as used by us) is fully described elsewhere (22). The other half of the inserts received DMEM-F-12 supplemented with 2% Controlled Process Serum Replacement no. 1 (Sigma, St. Louis, MO), "CPSR medium." Cells in inserts were grown with an air-liquid interface (i.e., medium was only added to the outside of the insert, the cell's basolateral surface). Cells on coverslips were immersed in 0.6 ml of medium to an average depth of 3 mm. Gray's medium induces a pseudostratified mucociliary phenotype in cells on inserts, whereas cells grown in CPSR medium are squamous and undifferentiated (22). Immersion feeding on glass coverslips also produces squamous poorly differentiated cultures (36).

Nasal epithelial cell cultures were obtained as described (14). In brief, scrapings were obtained from healthy volunteers after local vasoconstriction and anesthesia. After each nostril was rinsed four times with saline solution (5 ml per rinse per nostril), the medial-inferior surface of the inferior turbinate was gently scraped three to four times in each nostril using a 3-mm metallic ear curette (Storz, Culver City, CA). The cells were pooled in 600 µl of DMEM-F-12 and used after one passage as described (14).

To measure transepithelial electrical resistance (Rte) and transepithelial potential difference (Vte) we added 500 µl of PBS to the cell's mucosal surface and then used a "chop-stick" voltmeter (Millicell ERS; Millipore Products, Bedford, MA). The measurements took ~1 min, after which the PBS was immediately removed. Cells on coverslips were 100% confluent (as revealed by an inverted microscope) and at least in some cases formed tight junctions, as indicated by the formation of "domes" (13).

Experiments were performed 2 wk after plating (~10 days after attaining confluence). At this point, RV-16 passage 3 (originally obtained from Elliot Dick of the University of Wisconsin Madison Medical School) at a tissue culture infectious dose (TCID50) of 106/ml was added to the mucosal surface of cells on inserts (100 µl) or coverslips (200 µl). Control cells received the same volume of medium (PBS). After 6 h (or 1 h where specified in the text), the viral suspension (or PBS) was removed, the mucosal surface was washed three times with 500 µl of PBS, and the final rinse was retained. Cells were then allowed to recover for 24 h with an air-liquid interface (if they were grown on porous-bottomed inserts). The mucosal surface (of cells on inserts) was then flushed with 500 µl of PBS, and the flushings were retained. The medium over the cells on coverslips was also removed and kept. Rte and Vte were then determined, and the basolateral medium (0.7 ml) was retained. The filters or coverslips, with attached cells, were cut or broken in half. One half was placed in RLT buffer (RNeasy lysis buffer; Qiagen, Valencia, CA). The other half was fixed in buffered formaldehyde (10 min) and stored in 70% ethanol.

To determine viral titer, samples were serially diluted in half-log units, and 100 µl of each dilution were inoculated in duplicate wells of confluent human fetal diploid lung cells in 96-well plates. Cytopathic effects were determined on the 1st, 2nd, and every other day thereafter up to the 14th day. The titer was calculated based from the reciprocal of highest dilution showing cytopathic effects using the Reed-Munsch formula (12).

Interleukin (IL)-8 and IL-6 were determined by ELISA with kits from R & D Systems (Minneapolis, MN).

Levels of RNA for RV-16 and mRNA for a variety of mammalian proteins were determined in the cell lysates by a recently described (5) two-step method of gene transcript profiling in which multiplex RT-PCR is combined with individual gene quantification via real-time PCR on generated cDNA product. This method was specifically designed for quantification of multiple low-abundance transcripts using as little as 2.5 fg total RNA/gene. Total RNA was isolated from epithelial cells using the RNeasy mini kit (Qiagen), including DNase I treatment with RNase-free RQ1 DNase (Promega, Madison, WI). Gene-specific primers for multiplex RT-PCR and TaqMan were designed using "Primer Express" software (Perkin-Elmer, Foster City, CA) based on sequencing data from NCBI databases and were purchased from Biosearch Technologies (Novato, CA). Both sets of the primers were nested, and RT-PCR products were within the 250-bp range (http://asthmagenomics.ucsf.edu). Reverse transcription was performed using PowerScript RT under conditions described by the manufacturer (Clontech, Palo Alto, CA). In all reactions, cDNA was synthesized in 20 µl using 5–10 ng of total RNA with 200 nM random hexamers. All the reactions contained 1 units of Superase RNase inhibitor (Ambion, Austin, TX). Optimization of multiplex hot-start PCR was done as described, using KlenTaq DNA polymerase (cDNA Advantage Mix from Clontech). Each PCR reaction (50 µl) contained 1–10 µl of cDNA from the RT step and up to 19 gene-specific primer sets, at 100 nM each. Before amplification, the reaction was heated (94°C, 2 min) to inactivate the anti-Taq antibody followed by 0–25 cycles with 94°C for 30 s, 55°C for 30 s, and 70°C for 45 s. Typically, 2.5–50 fg of total RNA were used in 10 µl of Universal Master Mix (Perkin-Elmer). All forward and reverse Taq-Man primers were optimized, and transcript quantifications were run in duplicates with RT cDNA controls on an ABI Prizm 7900 Sequence Detection System (PE Applied Biosystems, Foster City, CA). Raw data from ABI Prizm7900 were processed in Excel (Microsoft, Redmond, WA) spreadsheets using software that automated proper baseline selection and cycle threshold calculation for each of the genes on a 384-well plate, as described previously (5). We measured mRNA for IL-1{beta}, IL-6, IL-8, tumor necrosis factor (TNF)-{alpha}, regulated on activation normal T-cell expressed and secreted (RANTES), eotaxin-3, intercellular adhesion molecule (ICAM)-1, cystic fibrosis transmembrane conductance regulator (CFTR), chloride channel numbers 1 and 3 (CLC1 and CLC3, respectively), S100 calcium-binding protein A4 (S100A4), interferon-inducible double-stranded RNA-dependent protein kinase (PRKR), and transcript variant-{alpha} of B cell CLL/lymphoma 2 nuclear gene (BCL2A). mRNA levels were expressed as relative gene copy numbers (RGCN) that had been normalized to the most stable housekeeping genes across the specimens. At least six different housekeeping genes at a time were measured for every specimen. GeNorm software (33) was used to identify the two most stable specimens across the samples, and the geometric means of these two was used for normalization.

Unless specified, all steps in the fluorescence in situ hybridization (FISH) protocol were performed at room temperature. Cell sheets were digested for 10 min at 37°C in proteinase K (Sigma P-6556; 1 µg/ml, 0.1 M Tris, 50 mM EDTA, pH 7.6), followed by washes in 0.2% glycine in PBS (1 min) to block remaining proteinase and PBS (5 min). Tissues were then postfixed for 10 min in 4% paraformaldehyde in 0.1 M Na/K phosphate buffer (pH 7.4), followed by a 5-min PBS wash, a 2-min exposure to 0.1 M triethanolamine (TEA) at pH 8.0, acetylation for 10 min (0.1 M TEA, 0.25% acetic anhydride), and a PBS wash (5 min). This was followed by hybridization at 37°C overnight in a mixture of 1x hybridization solution (Sigma H-7140), 30% deionized formamide (molecular biology grade), 250 µg/ml tRNA (Sigma R-8759), 10% dextran sulfate, and 2 µg/ml each 3'-labeled digoxigenin oligonucleotide probes PB4 and PB5 (Lofstrand Laboratories, Gaithersburg, MD), as used by others (1). Next, cell sheets were washed in 30% deionized formamide in 2x sodium citrate/chloride buffer (SSC) for 10 min and two times in 2x SSC (10 min) and exposed for 5 min to blocking buffer (3% BSA, 4x SSC, 0.1% Tween 20). This was followed by a set of steps at 37°C in blocking buffer as follows: anti-digoxigenin-fluorescein-labeled antibody (1–207–741, Roche Diagnostics, Indianapolis, IN; 1:100 dilution; 1 h), a wash (60 min), biotinylated anti-fluorescein antibody (BA-0601, Vector Laboratories, Burlingame, CA; 1:100 dilution; 30 min), fluorescein-labeled avidin (A-3101, Vector Laboratories; 1:100 dilution; 60 min), and 2 washes (5 min each). Cells were then washed three times for 5 min each with PBS, followed by staining of nuclei with propidium iodide (0.5 µg/ml; 2 min) and a PBS wash (5 min). They were then placed on slides with 35 µl of "Fluorogard" anti-fade reagent (Bio-Rad, Richmond, CA) and covered with a coverslip.

For immunocytochemistry of centrin, fixed cell sheets were exposed to prechilled methanol (-20°C) for 10 min. They were then washed three times (5 min each) in PBS and blocked for 30 min at 37°C in 3% BSA and 0.1% Tween 20 in 4x SSC. This was followed by exposure (60 min, 37°C) to anti-human centrin antibody (a gift from Dr. J. L. Salisbury, Mayo Clinic, Rochester, MN). Cells were then rinsed in PBS (3 x 5 min), reblocked (5 min), and exposed to secondary antibody (goat anti-mouse-FITC, 1:100; Jackson Immunoresearch Laboratories, West Grove, PA) for 60 min. After being washed with PBS (3 x 5 min), the cells were exposed to propidium iodide (0.5 µg/ml) and mounted under a coverslip in anti-fade reagent. Specimens for both FISH and immunocytochemistry were viewed with a confocal microscope (model L510; Zeiss).

Differences between means were compared by ANOVA or t-test, with P < 0.05 being considered statistically significant.


    RESULTS
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 METHODS
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 REFERENCES
 
Tracheal epithelium. Figure 1 shows Z-projections of X-Y stacks obtained on the confocal microscope. The cells had been stained for cilia with anti-centrin antibody, and their nuclei were stained with propidium iodide. Figure 1A shows that cells grown on coverslips consisted of a single layer of highly squamous cells that lacked cilia and were ~5 µm in height. In CPSR medium (Fig. 1B), two layers of cells were present, but the cells were still also highly squamous (~10 µm in height) and also lacked cilia. By contrast, cells on inserts grown in Gray's medium were pseudostratified, 40 µm in height, and contained abundant cilia. These results are in excellent agreement with those of our earlier studies (22, 36).



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Fig. 1. Appearance of cells under different culture conditions. A: coverslip. B: insert in Controlled Process Serum Replacement no. 1 (CPSR) medium. C: insert in Gray's medium. Cells have been stained with propidium iodide (red) to reveal nuclei and anti-centrin antibody (yellow) to show cilia. Scale bar = 50 µm.

 

The cells in Gray's medium had Rte that was ~15 times greater than in CPSR medium (1,186 ± 105 vs. 77 ± 10 {Omega}·cm2, n = 10). Transepithelial voltage was 24.5 ± 0.5 mV in Gray's and 0.5 ± 0.04 mV in CPSR medium. These values correspond to equivalent short-circuit currents (Ieq = Vte/Rte) of 22 ± 2 µA/cm2 in Gray's medium and 6 ± 1 µA/cm2 in CPSR medium.

Exposure to mucosal virus led to declines in Rte (-28 ± 15% for cells grown in Gray's; -15 ± 5% for cells grown in CPSR medium). However, exposure to mucosal PBS led to equivalent declines (-49 ± 8% for Gray's; -14 ± 12% for CPSR medium). In Gray's medium, Ieq was reduced equally by exposure to virus (-67 ± 6%) or to PBS (-54 ± 4%). In CPSR medium, the changes in Ieq produced by virus or by PBS were variable, reflecting the low (and comparatively unreliable) estimates of Vte; but the changes with virus were not significantly different from zero, and not significantly different from those with PBS alone.

As described in METHODS, after a 6-h exposure to virus (or PBS alone), the mucosal surface was rinsed three times with PBS, and the final rinse was retained. After a 24-h postexposure period, the mucosal surface was again rinsed with PBS. Control cells showed no virus (TCID50 < 100.5) in the final rinse at the end of the 6-h exposure period nor in the rinse at the end of the 24-h postexposure period. In cells exposed to virus, no particles were present in the final rinse at the end of the exposure period. Thus cells started the postexposure period free of virus in the mucosal medium, and any viruses obtained from the mucosal surface of the cells at the end of the postexposure (incubation) period of necessity would have been generated by the cells themselves and not merely have been left over from the original inoculum. Two of four cell sheets grown in Gray's medium and exposed to virus had no detectable levels of virus on their mucosal surfaces at the end of the postexposure period, whereas two had TCID50 values of 10. Thus the average TCID50 for all cultures grown in Gray's medium was 4 ± 2 (n = 5). By contrast, for cells grown in CPSR medium, the TCID50 of virus in the mucosal rinse was 48,600 ± 21,400 (n = 5; range = 103.5 to 105). The mucosal medium of cells grown on coverslips had TCID50 of 3,095 ± 1,791 (n = 5; range = 102.5 to 104).

Low levels of viral mRNA were detectable in cell lysates from control (i.e., unexposed) cells from all three culture conditions (Table 1). It is possible that this represents cross-contamination during tissue processing. But, after viral exposure, viral RNA levels in the cell lysates increased from 19- to 900-fold (Table 1) so that the levels of expression became CPSR medium > coverslip >> Gray's medium in the relative ratios 130:26:1. Across the three culture conditions, the levels of viral RNA in the cells correlated with the viral titers in the mucosal rinse (Fig. 2).


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Table 1. Viral RNA levels in tracheal cells cultured under various conditions

 


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Fig. 2. Dependence of virus released on cytoplasmic viral RNA. From left to right and from bottom to top, the points are for cells on inserts in Gray's medium, for cells on coverslips, and for cells on inserts in CPSR medium. The line is the best least-squared power fit of y = 1.4x1.95. Data are means ± SE, n = 4 or 5 experiments. RGCN, relative gene copy numbers; TCID50, half-maximal tissue culture infectious dose.

 

By FISH, cells positive for RV-16 RNA were detectable in cells under all culture conditions (Fig. 3A). At higher magnification, two staining patterns were distinguishable. In some cells, the nucleus was entirely surrounded by viral particles and had largely disintegrated (Fig. 3B). In other cells, viral particles were found in the perinuclear region and had started to distort the nucleus from its usual spherical shape (Fig. 3C). Infected cells were invariably in the layer immediately adjacent to the mucosal surface (i.e., they were cells with an apical membrane). Once able to detect RV-16 RNA by FISH reliably, we addressed the issue of whether the increased viral production in CPSR medium compared with Gray's medium was the result of more infected cells or more virus produced per infected cell. When focused on the layer of nuclei immediately under the apical membrane, we found that, in Gray's medium, 1.4% of cells exposed to virus were positive for RV-16, as opposed to 0.0% of control, unexposed cells (Table 2), values that were statistically different from one another ({chi}2-test). By contrast, in CPSR medium, 9.6% of cells were infected (Table 2) significantly more ({chi}2-tests) than control cells in CPSR medium (0.5%) or cells exposed to virus in Gray's medium. For the various culture conditions, the baseline levels of ICAM-1 mRNA were in the same sequence as the degree of viral infection. Thus there were 8.7 ± 0.6 x 106 RGCN of ICAM-1 mRNA in cells in Gray's medium, 19.8 ± 4.4 x 106 RGCN in cells on coverslips, and 34.2 ± 3.1 x 106 RGCN in cells grown in CPSR medium.



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Fig. 3. Fluorescence in situ hybridization for rhinovirus RNA. A: low-magnification micrograph showing a pair of positive cells on an insert in Gray's medium. B: high-power magnification of the cell on bottom in A. In this cell, viral particles surround the nucleus, which has largely disintegrated. C: high-power view of infected cell in CPSR medium, in which a pool of perinuclear viral particles can be seen to distort the nucleus. Scale bars are 10 µm (A) or 2 µm (B and C).

 

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Table 2. Cells positive forRV16 RNA as determined by fluorescence in situ hybridization

 

In cells not exposed to virus, levels of mRNA for RANTES were similar for all three culture conditions. However, mRNAs for IL-6, IL-8, and TNF-{alpha} were significantly higher in undifferentiated than differentiated cells (Fig. 4). Levels of PRKR mRNA were in the order coverslip > Gray's > CPSR (ANOVA). The changes in mRNA of all these proteins paralleled the increases in cytosolic RV-16 RNA (Fig. 4). Thus, in Gray's medium, only the mRNAs for IL-6 and PRKR were significantly changed by viral infection (Table 3 and Fig 4). However, mRNA for IL-6, PRKR, RANTES, IL-8, and TNF-{alpha} were all significantly increased by virus in cells grown in CPSR medium, and these changes were all significantly greater than those seen in Gray's medium. Cells grown on coverslips showed changes in mRNAs intermediate between those seen in cells grown on inserts in either Gray's or CPSR medium (Fig. 4). For RV-16, RANTES, IL-6, and TNF-{alpha}, the virally induced changes in mRNA were significantly greater for cells in CPSR medium on inserts than in cells on coverslips.



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Fig. 4. Changes in mRNA induced by viral infection of tracheal cells. Y-axes show mRNA in RGCN with a variety of different scales. C, control cells unexposed to virus. V, cells exposed to virus. {square}, Gray's medium; {circ}, coverslips; {bullet}, CPSR medium. aVirally induced change significantly greater than 0. bChange greater than that seen in Gray's. cChange greater than that in cells on coverslips. Values are means (n = 4 or 5). SEs are shown when they are larger than the symbols used. Baseline mRNA levels (RGCN) in cells on coverslips were 51 [rhinovirus (RV)-16], 1,247 regulated on activation normal T-cell expressed and secreted (RANTES), 14,452 [interleukin (IL)-6], 271,362 (IL-8), 322,373 [tumor necrosis factor (TNF)-{alpha}], and 11,062,000 [interferon-inducible double-stranded RNA-dependent protein kinase (PRKR)].

 

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Table 3. Relative changes in mRNA levels

 

To determine more precisely the correlation between levels of viral infection and increased expression of specific mRNAs, for each tissue exposed to virus the increase in viral RNA was taken as the difference between the viral RNA in that tissue and the mean of the baseline value for viral RNA. Increases in mRNA for specific epithelial proteins was calculated in the same way, and the best least-squared linear regressions of the increase in specific epithelial mRNA on the increase in viral RNA were determined. The regressions were statistically significant for IL-6 (R = 0.954), RANTES (R = 0.921), TNF-{alpha} (R = 0.872), IL-8 (R = 0.836), and PRKR (R = 0.675); the regression for RANTES is shown in Fig. 5.



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Fig. 5. Dependence of virally induced change in RANTES mRNA on level of viral RNA in tracheal cells. Symbols represent individual tissues. See text for details.

 

Significant declines in mRNA for S100A4, CLC3, BCL2A, CLC1, and CFTR were induced by viral infection in cells on coverslips or on inserts in CPSR medium (Table 3). However, these mRNAs were not changed by viral infection of cells in Gray's medium. mRNAs for ICAM-1, eotaxin-3, and IL-1{beta} were not altered by viral infection of any of the three types of cells (Table 3).

In all experiments on cells grown on inserts in CPSR medium, viral infection increased output of IL-6 (Fig. 6), and in about one-half of the experiments release of IL-8 was also increased (Fig. 6). By contrast, in five experiments with cells on inserts in Gray's medium, RV infection usually failed to increase release of IL-6 and never altered release of IL-8. To better compare the responses of cells grown on inserts in the two different media, Table 4 shows results from experiments in which cells from the same trachea were grown on inserts in Gray's medium or CPSR medium, as well as on coverslips. In these experiments, viral infection had little effect on the output of IL-8 under any culture conditions (Table 4). However, viral infection increased IL-6 output across both the apical and basolateral membranes of cells on inserts in CPSR medium by from 2.5- to 4-fold (Table 4). Similarly, IL-6 output from cells on coverslips was increased ~2.5-fold by viral infection (Table 4). The absolute levels of IL-6 released from cells exposed to virus (in ng·cm-2·day-1 across apical and basolateral membranes combined) were in the order CPSR medium (2,540 ± 162) > coverslip (41 ± 5) > Gray's medium (20 ± 3).



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Fig. 6. Output of IL-6 (A) and IL-8 (B) across the basolateral membranes of tracheal cultures. {bullet}, Infected with virus; {circ}, control cells. Each point represents an individual cell sheet from the same culture.

 

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Table 4. Output of IL-8 and IL-6 from tracheal cells

 

Nasal epithelium. The effects of media on histology were similar for nasal and tracheal cells. Thus nasal cells grown in CPSR medium were ~5 µm in height and unciliated, whereas those in Gray's medium were ~50 µm in height with mucociliary phenotype (data not shown). Cells in Gray's medium had Rte of 504 ± 70 {Omega}·cm2 and Vte of 15 ± 3 mV. Changes in Rte and Ieq induced by viral exposure (-33 ± 14 and 20 ± 23%, respectively) were similar to those produced by medium alone (-28 ± 14 and 102 ± 7%). By contrast, cells in CPSR medium had Rte that was not significantly different from zero (7 ± 6 {Omega}·cm2) and had undetectable Vte.

In Gray's medium, viral infection increased RV-16 RNA significantly from 951 ± 348 to 152,000 ± 50,000 RGCN. By contrast, in CPSR medium, the increase was almost 100-fold greater, from 6,360 ± 1,570 to 12,300,000 ± 1,300,000 RGCN. It is notable that, for cells grown in CPSR medium, the increase in viral mRNA for the nasal cells (12,300 x 103 RGCN) was much greater than for tracheal cells (232 x 103 RGCN).

Although the mRNA for IL-8, IL-6, RANTES, TNF-{alpha}, IL-1{beta}, and PRKR all increased significantly after viral infection in CPSR medium, these mRNAs showed no change in cells grown in Gray's medium (Fig. 7). In agreement with results on tracheal cells, viral infection of cells in CPSR medium caused significant decreases in mRNA for S100A4 (-37%), CLC3 (-37%), and CFTR (-57%). Again, these mRNAs did not change significantly after viral infection in Gray's medium. mRNAs for eotaxin and CLC1 were not changed significantly by viral infection in either medium.



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Fig. 7. Virally induced changes in mRNA in nasal cells. {circ}, RV-16; {blacksquare}, IL-8; {blacktriangleup}, PRKR; {triangledown}, RANTES; {blacktriangledown}, IL-1{beta}; {blacktriangleup}, IL-6; {triangleup}, TNF-{alpha}. Values are means (n = 4 or 5). SEs are shown when they are larger than the symbols used. Virus induced significant changes in all 7 RNAs in CPSR medium. Only RV-16 RNA was altered significantly in Gray's medium.

 

Interestingly, the baseline levels of mRNA for ICAM-1 were similar for Gray's (11,400,000 ± 1,900,000 RGCN) and CPSR medium (7,500,000 ± 800,000 RGCN), although the increases in RV-16 RNA on viral infection differed almost 100-fold between the two conditions (152 ± 50 x 103 RGCN in Gray's and 12,300 ± 1,300 x 103 RGCN in CPSR medium). Although viral infection had no effect on ICAM-1 mRNA in Gray's medium (+12%), it was significantly increased (+139%) in CPSR medium.

The effects of viral infection on output of IL-6 and IL-8 from nasal cells are shown in Table 5. Viral infection had no effect on release of IL-8 and IL-6 from cells in Gray's medium but significantly increased basolateral and apical release of IL-6 and basolateral release of IL-8 from cells in CPSR medium.


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Table 5. Output of IL-8 and IL-6 from nasal cells

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
There have been several reports that cell lines of human airway epithelium, when infected with RV, increase their output of inflammatory cytokines. Thus 1 day after exposure to RV-14, BEAS-2B cells showed increased release of IL-8, IL-6, and granulocyte-macrophage colony stimulating factor (GMCSF) (29). A549 cells also show increased output of IL-6 in response to RV-14 (41). Similar results have been obtained on primary cultures of human tracheal epithelium. Terajima et al. (31), for instance, showed RV increased release of IL-6, IL-8, TNF-{alpha}, and IL-1{beta} from primary cultures of human tracheal epithelium, and the same group obtained similar results with primary cultures of human tracheal gland cells (39). Secretion of RANTES and GM-CSF from primary cultures of human tracheal epithelium is also increased by rhinoviral infection (23).

Several groups (37, 38, 40) have developed conditions that produce airway epithelial cultures consisting of polarized cells with excellent pseudostratified mucociliary histology. However, little attention has been paid to the phenotype of the primary cell cultures used in most studies of rhinoviral infection. Cells have invariably been grown on solid supports, a form of culture that produces cells with much lower levels of differentiation than does growth on porous-bottomed inserts (36). In other cases (23), the cells had been passaged up to five times, again a procedure that is known to reduce cell quality. In fact, we have found that primary cultures of airway surface epithelium (Widdicombe and Sachs, unpublished observation) or glands (7) cease to form tight junctions and polarize after four or five passages. We wondered, therefore, how applicable many of the in vitro studies of rhinoviral infection were to infections of pseudostratified mucociliary epithelium in vivo.

In an attempt to address this issue, we grew cells under three different conditions, two that produced dedifferentiated (i.e., squamous unciliated) cells and one that produced differentiated cells of pseudostratified mucociliary appearance. We then compared how they responded to infection with RV. We found that the undifferentiated cultures showed much higher levels of viral infection (measured in terms of both viral RNA in the cells and numbers of infectious particles released) than did differentiated cells. Furthermore, whereas the undifferentiated cells showed increased expression (and release) of a variety of inflammatory cytokines in response to viral infection, the well-differentiated cells did not respond.

Viral infection had no effects on measures of tissue barrier function (Rte) or metabolism (Ieq), nor were any signs of cytotoxicity seen under the inverted microscope for any of the culture conditions. Thus viability was apparently unaffected by viral infection. This is in agreement with results on confluent cultures using several rhinoviral strains (2, 18, 31), although RV-49 is cytotoxic (23).

Across the three culture conditions, there was good correlation between viral RNA produced in the cells and the numbers of infectious particles released in the mucosal medium (Fig. 2). The dependence of viral titer on cytoplasmic viral RNA was exponential, suggesting that, over the range of infection seen here, the efficiency of viral production increased with increasing levels of viral RNA. By FISH, we found that only cells in the upper layer of the epithelium were infected, and, in agreement with other results on primary cultures of human airway epithelium (15), these constituted only a small fraction of the total (1% in Gray's medium and 10% in CPSR medium). However, interpretation of this difference is complicated by the marked differences in histology between the two types of cell culture. Thus average apical membrane surface area of squamous dedifferentiated cells in general is approximately four times greater than for well-differentiated cultures (22, 36). Therefore, in terms of infected cells per square centimeter of epithelium, the difference between cells grown in CPSR medium vs. Gray's medium is reduced to ~3:1. However, the levels of infectious particles produced per unit area were 10,000-fold greater in CPSR medium than Gray's. Thus the main reason for the difference in viral titer between differentiated and dedifferentiated cultures is that dedifferentiated cells produce more virus particles per cell than do differentiated.

In tracheal cells, the baseline levels of ICAM-1 mRNA, the increase in RV-16 RNA after infection, and the numbers of infectious particles released were all in the order CPSR medium > coverslip > Gray's medium. Furthermore, for all individual cell sheets, the best least-squares linear regression for the dependence of RV-16 RNA on ICAM-1 mRNA was significant (R = 0.73, n = 15; for this analysis, we used the ICAM-1 mRNA levels in the infected cells, since this was not changed significantly by viral infection). This suggests that the levels of ICAM-1, which is the receptor for RV (27), are a primary determinant of the degree of viral infection. However, when results from nasal and tracheal cells were combined, the dependence of the mean increase of RV-16 RNA on the corresponding mean baseline level of ICAM-1 was no longer significant. This was because the nasal cells grown in CPSR medium gave a disproportionately large increase in RV-16 RNA for their baseline levels of ICAM-1 mRNA. Thus the numbers of virus bound may be significantly influenced by factors other than ICAM-1 levels. Specifically, we note that the nasal cells grown in CPSR medium were the only ones that had no functional tight junctions (Rte = 0) and suggest that this is in some way linked to their higher levels of infection with virus.

In well-differentiated cells, viral infection had little effect on the levels of expression of RANTES, IL-6, IL-8, or TNF-{alpha}. However, mRNA for all these inflammatory cytokines was increased markedly when undifferentiated cells grown in CPSR medium were infected. Across culture conditions, virally induced increases in mRNA for all these cytokines showed significant linear regressions on the levels of viral RNA in the infected cells. This presumably represents a defensive response on the part of the epithelium, for all these cytokines are capable of mounting an immune response to the infecting virus (4, 21, 28, 32). Changes in mRNA for IL-6 and RANTES correlated most closely with the levels of viral mRNA. However, whereas RANTES mRNA was little affected by culture conditions, in tracheal cells baseline levels of IL-6 mRNA varied over a 40-fold range. Thus RANTES mRNA (or perhaps secreted RANTES) may make the best surrogate marker for viral infection, a conclusion supported by the results of others (11, 19, 23).

Output of IL-8 was affected little by viral infection. Only cells with the highest viral loads (i.e., nasal cells in CPSR medium) showed an appreciable increase (~2-fold) in IL-8 release. By contrast, IL-6 release was increased from most of the different types of cell culture used here, with the largest increase (~4-fold) being from tracheal cells in CPSR medium. These virally induced changes in IL-6 and IL-8 output from cells in CPSR medium are similar to those reported by others (11, 31), who have also found that IL-6 output increases more in response to rhinoviral infection than does IL-8 output (31, 39). When, for all five combinations of culture conditions and cells, the virally induced changes in IL-6 release (in ng·cm-2·day-1) were plotted against the corresponding changes in IL-6 mRNA, the best least-squared linear regression was highly significant (R = 0.95).

The increases in PRKR on infection with RV were to be expected given the importance of this kinase in mediating the antiviral effects of interferons (9). Our finding that eotaxin mRNA levels were unaltered by rhinoviral infection is in apparent disagreement with a recent report (19). However, the latter study used BEAS-2B cells, and measured eotaxin-2, not eotaxin-3. Also, the effects were small. Thus, even with exposures to virus of 24 or 48 h, eotaxin-2 mRNA levels were increased not at all or approximately twofold, respectively.

In undifferentiated cells, the levels of mRNA for three different chloride channels (CFTR, CLC1, and CLC2) decreased (by 30–70%) on infection with RV. Given the small number of infected cells, these decreases seem likely to have occurred in uninfected cells, perhaps caused by the actions of cytokines released from the infected cells. Despite these changes in chloride channel mRNA, neither Vte nor Ieq across the cultures was changed by viral infection, a predictable result given that, under baseline conditions, active chloride secretion is of minor importance in the generation of Vte (38).

Several groups have reported that infection of airway epithelium with RVs upregulates ICAM-1 and its mRNA (2, 9, 24, 31, 39). In particular, one group found upregulation of ICAM-1 in primary cultures of human bronchial epithelium after 8 h of continuous exposure to virus (with no postexposure period; see Ref. 20). We were surprised therefore that our tracheal cells showed no change in ICAM-1 mRNA after 6 h of viral exposure and a 24-h postexposure period. We note, however, that our tracheal cells had formed tight junctions, as evidenced by finite transepithelial resistance of cells on inserts and the formation of domes by cells grown on glass coverslips. In the one set of cells that failed to form tight junctions and had zero Rte (i.e., nasal cells grown in CPSR medium), we found that viral exposure caused a significant 2.5-fold increase in ICAM-1 mRNA, a change that is similar to that reported by others. In our experience, attainment of confluency is not always associated with development of Rte. Thus we speculate that, although the primary cultures of human airway epithelium used by others were confluent, they may not have formed tight junctions, and this explains the difference between their and our results with ICAM-1.

In fact, our results show that the degree of viral infection and the extent of its sequelae are inversely related to Rte, which in airway epithelial cells reflects mainly the tightness of the tight junctions (35). Thus, for cells on inserts, tracheal cells in Gray's medium had Rte of 1,200 {Omega}·cm2 and 2,000 RGCN of viral RNA after infection; for nasal cells in Gray's medium, 500 {Omega}·cm2 and 150,000 RGCN; for tracheal cells in CPSR medium, 80 {Omega}·cm2 and 230,000 RGCN; for nasal cells in CPSR, 0 {Omega}·cm2 and 12,300,000 RGCN. Why should the opening of tight junctions and breakdown of barrier function result in increased infection by viruses? Perhaps loss of tight junctions could allow uptake of virus across the basolateral membrane; ICAM-1 is found on both basolateral and apical membranes of airway epithelial cells (30). In this regard it is worth noting that adenovirus, one of the viruses that causes colds (6), enters airway epithelial cells much more efficiently across the basolateral than the apical membrane (34). Alternatively, the breakdown in polarity resulting from loss of tight junctions would have global effects on protein trafficking and expression that could by unknown mechanisms enhance viral binding or viral production.

In conclusion, we show that rhinoviral infection and its sequelae are much more pronounced in undifferentiated than differentiated cultures of human airway epithelium, and circumstantial evidence suggests that this difference is accounted for by a breakdown of the tight junctions in the dedifferentiated cells. Whatever the exact mechanisms for the differences between undifferentiated and differentiated cells, it is important to ask why the differentiated cells are better protected from the effects of RV. Furthermore, if our results apply to native epithelium in vivo, then the ability of RV to cause colds and trigger asthma attacks may be markedly enhanced by prior damage to the airway epithelium. This conclusion is supported by the findings that smokers have increased mucosal airway permeability (10) and are subject to more frequent and worse colds than nonsmokers (16). They are also at higher risk of asthma attack (26).


    ACKNOWLEDGMENTS
 
We thank Craig Reading for technical assistance. Dr. Pedro Avila helped with the acquisition of nasal brushings.

GRANTS

This work was supported by National Institute of Allergy and Infectious Diseases Grant AI-50496 and by a grant from the Conselho Nacional de Desenvolvimento Científico e Tecnológico of Brazil to N. Lopez-Souza.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. H. Widdicombe, Dept. of Human Physiology, Univ. of California-Davis, Davis CA 95616-8664 (E-mail: jhwiddicombe{at}ucdavis.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.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bardin PG, Johnston SL, Sanderson G, Robinson BS, Pickett MA, Fraenkel DJ, and Holgate ST. Detection of rhinovirus infection of the nasal mucosa by oligonucleotide in situ hybridization. Am J Respir Cell Mol Biol 10: 207-213, 1994.[Abstract]
  2. Bianco A, Sethi SK, Allen JT, Knight RA, and Spiteri MA. Th2 cytokines exert a dominant influence on epithelial cell expression of the major group human rhinovirus receptor, ICAM-1. Eur Respir J 12: 619-626, 1998.[Abstract/Free Full Text]
  3. Coleman DL, Tuet IK, and Widdicombe JH. Electrical properties of dog tracheal epithelial cells grown in monolayer culture. Am J Physiol Cell Physiol 246: C355-C359, 1984.[Abstract]
  4. Conti P and DiGioacchino M. MCP-1 and RANTES are mediators of acute and chronic inflammation. Allergy Asthma Proc 22: 133-137, 2001.[ISI][Medline]
  5. Dolganov GM, Woodruff PG, Novikov AA, Zhang Y, Ferrando RE, Szubin R, and Fahy JV. A novel method of gene transcript profiling in airway biopsy homogenates reveals increased expression of a Na+-K+-Cl-cotransporter (NKCC1) in asthmatic subjects. Genome Res 11: 1473-1483, 2001.[Abstract/Free Full Text]
  6. Engel JP. Viral respiratory infections. Semin Respir Infect 10: 3-13, 1995.[Medline]
  7. Finkbeiner WE and Widdicombe JH. Serial propagation of cells from human tracheobronchial glands. In Vitro Cell Dev Biol Anim 30A: 817-818, 1994.
  8. Gray TE, Guzman K, Davis CW, Abdullah LH, and Nettesheim P. Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells. Am J Respir Cell Mol Biol 14: 104-112, 1996.[Abstract]
  9. Grunberg K, Sharon RF, Sont JK, In't Veen JC, Van Schadewijk WA, De Klerk EP, Dick CR, Van Krieken JH, and Sterk PJ. Rhinovirus-induced airway inflammation in asthma: effect of treatment with inhaled corticosteroids before and during experimental infection. Am J Respir Crit Care Med 164: 1816-1822., 2001.[Abstract/Free Full Text]
  10. Kennedy SM, Elwood RK, Wiggs BJR, and Pare PD. Increased airway mucosal permeability of smokers. Am Rev Respir Dis 129: 143-148, 1984.[ISI][Medline]
  11. Konno S, Grindle KA, Lee W-M, Schroth MK, Mosser AG, Brockman-Schneider RA, Busse WW, and Gern JE. Interferon-{gamma} enhances rhinovirus-induced RANTES secretion by airway epithelial cells. Am J Respir Cell Mol Biol 26: 594-601, 2002.[Abstract/Free Full Text]
  12. Lennette DA. General principles for laboratory diagnosis of viral rickettsial, chlamydial infections. In: Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, edited by Lennette EH, Lennette DA, and Lennette ET. Washington, DC: APHA, 1995, p. 3-26.
  13. Lever JE. Regulation of dome formation in differentiated epithelial cell cultures. J Supramol Struct 12: 259-272, 1979.[ISI][Medline]
  14. Lopez-Souza N, Avila PC, and Widdicombe JH. Polarized cultures of human airway epithelium from nasal scrapings and bronchial brushings. In Vitro Cell Dev Biol Anim. In press.
  15. Mosser AG, Brockman-Schneider RA, Amineva S, Burchell L, Sedgwick JB, Busse WW, and Gern JE. Similar frequency of rhinovirusinfectible cells in upper and lower airway epithelium. J Infect Dis 185: 734-743, 2002.[CrossRef][ISI][Medline]
  16. Murin S, Bilello KS, and Matthay R. Other smoking-affected pulmonary diseases. Clin Chest Med 21: 121-137, 2000.[ISI][Medline]
  17. Noah TL, Yankaskas JR, Carson JL, Gambling TM, Cazares LH, McKinnon KP, and Devlin RB. Tight junctions and mucin mRNA in BEAS-2B cells. In Vitro Cell Dev Biol Anim 31: 738-740, 1995.
  18. Papadopoulos NG, Bates PJ, Bardin PG, Papi A, Leir SH, Fraenkel DJ, Meyer J, Lackie PM, Sanderson G, Holgate ST, and Johnston SL. Rhinoviruses infect the lower airways. J Infect Dis 181: 1875-1884, 2000.[CrossRef][ISI][Medline]
  19. Papadopoulos NG, Papi A, Meyer J, Stanciu LA, Salvi S, Holgate ST, and Johnston SL. Rhinovirus infection up-regulates eotaxin and eotaxin-2 expression in bronchial epithelial cells. Clin Exp Allergy 31: 1060-1066, 2001.[CrossRef][ISI][Medline]
  20. Papi A and Johnston SL. Rhinovirus infection induces expression of its own receptor intercellular adhesion molecule 1 (ICAM-1) via increased NF-kappaB-mediated transcription. J Biol Chem 274: 9707-9720, 1999.[Abstract/Free Full Text]
  21. Rincon M, Anguita J, Nakamura T, Fikrig E, and Flavell RA. Interleukin (IL)-6 directs the differentiation of IL-4-producing CD4+ T cells. J Exp Med 185: 461-469, 1997.[Abstract/Free Full Text]
  22. Sachs LA, Finkbeiner WE, and Widdicombe JH. Effects of media on differentiation of cultured human tracheal epithelium. In Vitro Cell Dev Biol Anim 39A: 56-62, 2003.
  23. Schroth MK, Grimm E, Frindt P, Galagan DM, Konno SI, Love R, and Gern JE. Rhinovirus replication causes RANTES production in primary bronchial epithelial cells. Am J Respir Cell Mol Biol 20: 1220-1228, 1999.[Abstract/Free Full Text]
  24. Sethi SK, Bianco A, Allen JT, Knight RA, and Spiteri MA. Interferongamma (IFN-gamma) down-regulates the rhinovirus-induced expression of intercellular adhesion molecule-1 (ICAM-1) on human airway epithelial cells. Clin Exp Immunol 110: 362-369, 1997.[CrossRef][ISI][Medline]
  25. Shen BQ, Finkbeiner WE, Wine JJ, Mrsny RJ, and Widdicombe JH. Calu-3: a human airway epithelial cell line that shows cAMP-dependent Cl- secretion. Am J Physiol Lung Cell Mol Physiol 266: L493-L501, 1994.[Abstract/Free Full Text]
  26. Siroux V, Pin I, Oryszczyn MP, Le Moual N and Kauffmann F. Relationships of active smoking to asthma and asthma severity in the EGEA study. Epidemiological study on the genetics and environment of asthma. Eur Respir J 15: 470-477, 2000.[Abstract/Free Full Text]
  27. Staunton DE, Merluzzi VJ, Rothlein R, Barton R, Marlin SD, and Springer TA. A cell adhesion molecule, ICAM-1, is the major surface receptor for rhinoviruses. Cell 56: 849-853, 1989.[ISI][Medline]
  28. Strieter RM. Interleukin-8: a very important chemokine of the human airway epithelium. Am J Physiol Lung Cell Mol Physiol 283: L688-L689, 2002.[Free Full Text]
  29. Subauste MC, Jacoby DB, Richards SM, and Proud D. Infection of a human respiratory epithelial cell line with rhinovirus. Induction of cytokine release and modulation of susceptibility to infection by cytokine exposure. J Clin Invest 96: 549-557, 1995.[ISI][Medline]
  30. Taguchi M, Sampath D, Koga T, Castro M, Look DC, Nakajima S, and Holtzman MJ. Patterns for RANTES secretion and intercellular adhesion molecule 1 expression mediate transepithelial T cell traffic based on analyses in vitro and in vivo. J Exp Med 187: 1927-1940, 1998.[Abstract/Free Full Text]
  31. Terajima M, Yamaya M, Sekizawa K, Okinaga S, Suzuki T, Yamada N, Nakayama K, Ohrui T, Oshima T, Numazaki Y, and Sasaki H. Rhinovirus infection of primary cultures of human tracheal epithelium: role of ICAM-1 and IL-1beta. Am J Physiol Lung Cell Mol Physiol 273: L749-L759, 1997.[Abstract/Free Full Text]
  32. Thomas PS. Tumour necrosis factor-alpha: the role of this multifunctional cytokine in asthma. Immunol Cell Biol 79: 132-140, 2001.[CrossRef][ISI][Medline]
  33. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, and Speleman F Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes (Abstract). Genome Biol 3: 34, 2002.
  34. Walters RW, Grunst T, Bergelson JM, Finberg RW, Welsh MJ, and Zabner J. Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia. J Biol Chem 274: 10219-10226, 1999.[Abstract/Free Full Text]
  35. Welsh MJ, Smith PL, and Frizzell RA. Chloride secretion by canine tracheal epithelium. III. Membrane resistances and electromotive forces. J Membr Biol 71: 209-218, 1983.[ISI][Medline]
  36. Widdicombe JH, Sachs LA, and Finkbeiner WE. Effects of growth surface on differentiation of cultures of human tracheal epithelium. In Vitro Cell Dev Biol Anim. 39A: 51-55, 2003.
  37. Wu R, Nolan E, and Turner C. Expression of tracheal differentiated functions in serum-free hormone-supplemented medium. J Cell Physiol 125: 167-181, 1985.[ISI][Medline]
  38. Yamaya M, Finkbeiner WE, Chun SY, and Widdicombe JH. Differentiated structure and function of cultures from human tracheal epithelium. Am J Physiol Lung Cell Mol Physiol 262: L713-L724, 1992.[Abstract/Free Full Text]
  39. Yamaya M, Sekizawa K, Suzuki T, Yamada N, Furukawa M, Ishizuka S, Nakayama K, Terajima M, Numazaki Y, and Sasaki H. Infection of human respiratory submucosal glands with rhinovirus: effects on cytokine and ICAM-1 production. Am J Physiol Lung Cell Mol Physiol 277: L362-L371, 1999.[Abstract/Free Full Text]
  40. Yankaskas JR, Cotton CU, Knowles MR, Gatzy JT, and Boucher RC. Culture of human nasal epithelial cells on collagen matrix supports. A comparison of bioelectric properties of normal and cystic fibrosis epithelia. Am Rev Respir Dis 132: 1281-1287, 1985.[ISI][Medline]
  41. Zhu Z, Tang W, Ray A, Wu Y, Einarsson O, Landry ML, Gwaltney Jr J, and Elias JA. Rhinovirus stimulation of interleukin-6 in vivo and in vitro. Evidence for nuclear factor kappa B-dependent transcriptional activation. J Clin Invest 97: 421-430, 1996.[Abstract/Free Full Text]