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Division of Pulmonary, Critical Care and Occupational Medicine, University of Iowa College of Medicine and Veterans Affairs Medical Center, Iowa City, IA 52242; and
University Medicine, University of Southampton, Southampton General Hospital, Southampton, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The biology of rhinovirus infections remains poorly understood. Rhinoviruses infect nasal epithelial cells (4) and cause a local inflammatory response associated with the production of kinins (5) and, in atopic subjects, histamine (6). The nature of the nasal mucosal cellular immune response to rhinoviral infection is poorly defined (7), although increased numbers of neutrophils, eosinophils, and lymphocytes have been demonstrated in nasal secretions in rhinovirus infections (8, 9). The cytokines implicated in the recruitment and activation of these inflammatory cells in rhinovirus infections are largely unknown. Increased levels of IL-1, -6, and -8 have been demonstrated in nasal washings taken from subjects with experimental rhinovirus infection (10, 11, 12), IL-11 has been detected in nasal aspirate samples from infants and young children with symptoms of upper respiratory tract infection, some of whom had wheeze (13), and we have also recently demonstrated increased levels of IL-8 and the neutrophil product myeloperoxidase in nasal aspirates from children with wild-type rhinovirus infections (14). IL-8 is a potent neutrophil chemoattractant and activator. An important role for IL 8 in controlling neutrophil-mediated inflammation in rhinovirus infection was suggested by the finding of close correlations between the nasal aspirate IL-8 and myeloperoxidase levels and between myeloperoxidase levels and upper respiratory symptom severity (14).
Much less is known about the lower airway response to rhinoviral infection in normal subjects; indeed, there has been considerable debate over whether rhinoviruses can infect the lower airway (15). Two recent studies have investigated the lower airway in normal subjects during experimental rhinovirus nasal infections. The first found evidence of rhinovirus replication in lower airway lavage cell populations (16), and the second found a marked CD3+, CD4+, and CD8+ lymphocyte and eosinophil infiltration in bronchial biopsies (17).
Eosinophil bronchial mucosal infiltration is a cardinal feature of asthma pathogenesis (18), and eosinophil infiltration has also been implicated in virus-induced asthma exacerbations. Experimental infection studies have demonstrated that in asthmatic or allergic rhinitic subjects, the eosinophil infiltrate provoked by rhinovirus infection alone or in association with concurrent allergen challenge is more prolonged than that in normal subjects (17, 19).
The precise mechanisms by which rhinovirus infections trigger exacerbations of asthma are not known. However, an important role for IL-8 is suggested by the recent demonstration of a correlation between IL-8 nasal lavage levels and increases in bronchial hyper-reactivity induced by rhinovirus experimental infections (12). Furthermore, IL-8 has been implicated in lymphocyte chemotaxis in vitro (20) and in eosinophil chemoattraction and activation in vivo and in vitro (21, 22).
Given the putative important role of IL-8 in virus-induced asthma exacerbations and the recent demonstration that rhinovirus infection stimulates the release of IL-8 from the transformed bronchial epithelial cell line BEAS 2B (23), we have evaluated the ability of rhinovirus to infect a pulmonary epithelial cell line (A549) and have investigated the mechanisms of release of IL-8. We found low grade productive, but noncytolytic, rhinovirus infection of the epithelium that caused a prolonged release of IL-8 protein and up-regulation of IL-8 mRNA. The induction of IL-8 was only partially dependent on viral replication.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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A549 cells, a transformed alveolar epithelial cell line (24), were obtained from American Type Culture Collection (ATCC, Rockville, MD). Cells were split twice weekly. Ohio HeLa cells were obtained from the Medical Research Council Common Cold Unit (Salisbury, U.K.) and were split weekly. Both cell lines were cultured at 37°C in 5% carbon dioxide in Eagle’s MEM. All tissue culture media (Cancer Center, University of Iowa, Iowa City, IA) contained 10% heat-inactivated FBS (HyClone, Logan, UT), 4 mM L-glutamine, and 80 µg/ml of gentamicin.
Viruses
Rhinovirus serotype 9 was obtained from the Medical Research Council Common Cold Unit, and its identity was confirmed by neutralization with specific antiserum (ATCC) (25). Rhinovirus type 9 was used for all studies described and was propagated in large quantities in Ohio HeLa cells. After development of a full cytopathic effect, the virus was harvested, clarified, aliquoted, and frozen at -70°C. The viral titer, determined as described below on a thawed vial of the stored frozen aliquots, was 2 x 106 tissue culture infective doses 50% (TCID50)4/ml. For all experiments, a new vial of virus was rapidly thawed at 37°C and was used immediately at a multiplicity of infection (MOI) of 1, except where indicated.
Virus titrations
Rhinovirus titers were determined in Ohio HeLa cell monolayers in 96-well microtiter plate assays; 125 µl of 5% MEM was added to each well, followed by 100 µl of HeLa cells in 5% MEM at 3 x 105/ml. The plates were cultured in 5% CO2 at 37°C. When confluent (24–48 h), 25 µl of sample or 10-fold dilutions thereof (eight replicates) were added to each well, and the plates were cultured for 5 days. The cytopathic effect (CPE) was determined by visual assessment and by assessment of the continuity of the monolayer after fixation in methanol and staining with 0.1% crystal violet. TCID50 values were computed as previously described (25).
Measurement of IL-8 protein
A549 cells were cultured at 2 x 106/ml in 2 ml of medium in 12-well plates. After 24 h (when 80% confluent), virus at an MOI of 1 or control medium was added, and incubation was continued for 6, 24, 48, 72, 96, and 120 h. Dose-response studies were conducted using 10-fold dilutions of virus stock and were harvested at 24 h. The effect of UV- and soluble ICAM-1 (sICAM)-inactivated virus was studied at 24 and 120 h. Supernatants were harvested and stored in aliquots at -70°C. IL-8 protein was measured by specific ELISA (R&D, Minneapolis, MN). The sensitivity of the assay was 4.7 pg/ml.
Measurement of IL-8 mRNA by Northern analysis
A549 cells were cultured for 18 h in 100-mm plates, and then virus was added at an MOI of 1. Cells were harvested at 1, 3, 6, 24, 48, and 96 h. Experiments were repeated between three and five times for time points up to 24 h and twice for the 48 and 96 h points. Whole cell RNA was extracted using RNAstat (Tel-Test B, Friendswood, TX) according to the manufacturer’s instructions. RNA was resuspended in 25 to 50 µl of water and quantitated by absorbance at 260 nm.
The RNA (10 µg in each lane) was fractionated on a 1.5% denaturing agarose gel containing 2.2 M formaldehyde (26). Escherichia coli 23S and 16S ribosomal RNA were run as m.w. markers. Equal RNA loading was confirmed by ethidium bromide staining of 28S and 18S ribosomal RNA.
A 350-bp IL-8 cDNA probe complementary to part of the IL-8 mRNA 5'
noncoding region was generated by an EcoRI digest of pGEM3Z,
provided by Joost J. Oppenheim (National Cancer Institute, Frederick,
MD). Probe (100 ng) was labeled using the BRL (Gaithersburg, MD) random
primer DNA labeling system, and 10 µl of [
-32P]CTP
(DuPont-New England Nuclear, Boston, MA) following the manufacturer’s
instructions. Unbound nucleotides were removed using a Sephadex G-50
column. The RNA was transferred to GeneScreen Plus (New England
Nuclear, Boston, MA) as recommended by the manufacturer and then UV
cross-linked to the nylon membrane. Membranes were prehybridized for
6 h in 50% formamide, 1 M NaCl, 10% dextran, 1% SDS, 0.05 M
Tris, and 1x Denhardt’s solution. Hybridization was conducted
overnight in fresh solution also containing 100 µg/ml salmon sperm
DNA (Sigma, St. Louis, MO) at 42°C with
[-32P]-labeled probe. Hybridized membranes were washed
twice in 1x SCC at room temperature for 5 min, twice in 1x SCC/0.1%
SDS at 65°C for 30 min, and once in 0.1x SCC at room temperature for
15 min. Autoradiography (XAR Kodak film, Sigma) was performed at
-70°C.
Rhinovirus inactivation
UV light inactivation of virus was accomplished by exposure to 1200 µJ/cm2 UV light for 30 min. Confirmation of inactivation was conducted by microtiter plate assays using the methods described above.
Inactivation of the virus was also investigated by determination of new viral protein synthesis by [35S]methionine incorporation and immunoprecipitation as follows. Live virus and UV-inactivated virus were inoculated onto 107 Ohio HeLa cells at an MOI of 1. After 30-min adsorption, 10 ml of 10% MEM was added, and cells were incubated in 5% CO2 at 37°C. After 6 h of incubation, the medium was replaced by methionine-free medium (Sigma). Thirty minutes later, [35S]methionine (DuPont-New England Nuclear) was added to a final concentration of 50 µCi/ml. Incubation was continued for 2 h, and the culture was then placed on ice. After being washed three times, cell pellets were collected and suspended in 1 ml of lysis buffer (1% Triton X-100 in PBS). Lysates were stored in 250-µl aliquots at -70°C for immunoprecipitation. Immunoprecipitation was performed on a 250-µl lysate aliquot, which was thawed, and an additional 750 µl of lysis buffer was added. Lysates were then sonicated and incubated at 4°C for 10 min. To reduce nonspecific binding, 50 µl of Staph protein A/Sepharose Fast Flow (Sigma) was added, and lysates were agitated at 4°C for 1 h, protein A/Sepharose was pelleted at 12,000 rpm in a microfuge for 1 min, and supernatant was removed. The protein A/Sepharose/polyclonal Ab complex (prepared by adding 5 µl of guinea pig polyclonal anti-rhinovirus type 9 Ab (ATCC) to 100 µl of Staph protein A-Sepharose and agitating at room temperature for 2 h) was then added to the supernatant, and agitation was continued at 4°C overnight. After overnight incubation, pellets were washed three times in PBS, and 50 µl of sample buffer (24% glycerol, 5% SDS, 12% ß-ME, 0.001 M bromophenol blue, and 1.5 M Tris-HCl pH 6.8) was added. The solution was heated at 95°C for 2 min and centrifuged, and the Staph protein A/Sepharose pellet was removed. Supernatant was loaded onto a 10% SDS-PAGE gel and electrophoresed at 30 mA. Gels were fixed in 40% methanol, 10% acetic acid, and 3% glycerol for 1 h, then in EnHance (DuPont-New England Nuclear) for 1 h, and washed for 30 min in water. The gel was dried, and autoradiography was performed.
Rhinovirus inactivation was also performed by precoating the virus with soluble receptor to occupy all the receptor binding sites on the virus capsids. Virus stock solutions were preincubated with recombinant sICAM-1 (donated by P. Esmon, Bayer Corp, Berkeley, CA) at a concentration of 1 mg/ml for 30 min at room temperature. Confirmation of inactivation was conducted by microtiter plate assays using the methods described above.
Rhinovirus infectivity in A549 cells
Rhinovirus titrations. A549 cells were cultured at 2 x 106/ml in 2 ml of medium on 12-well plates, and when 80% confluent, cells were inoculated with rhinovirus at an MOI of 1. After 30 min of incubation at room temperature to allow virus attachment, cells were washed three times, and fresh medium was added. Supernatants were harvested at 6, 24, 48, and 72 h and stored at -70°C. Rhinovirus titers were determined using microtiter plate assays as described previously (25), including rhinovirus type 9 neutralizing antiserum (ATCC) to confirm the identity of the virus particles produced as rhinovirus type 9. Cultures were observed and stained with crystal violet to detect virus-induced CPE.
Rhinovirus genomic and replicative strand RNA analysis by RT-PCR. A549 cells were cultured at 2 x 106/ml in 2 ml of medium on 12-well plates and, when 80% confluent, were inoculated with rhinovirus at an MOI of 1. After 30 min of incubation at room temperature to allow virus attachment, cells were washed three times, and fresh medium was added. Supernatants were then harvested immediately and at 6, 24, 48, 72, 96, and 120 h and stored at -70°C. Total RNA was extracted from 50-µl aliquots of supernatant using Tryzol (Life Technologies) according to the manufacturer’s instructions. Rhinovirus genomic (positive) and replicative (negative) strand RNAs were detected using RT-PCR and internal probe hybridization as previously described (27). For genomic strand RT-PCR, the primer OL27 was used for the RT step, and PCR was conducted as previously described (27), except that the probe hybridization was conducted using nonisotopic labeling with the Amersham ECL kit (Amersham, Aylesbury, U.K.) according to the manufacturer’s instructions. For the replicative strand RT-PCR, identical methods were used, except that the primer used for the RT step was OL26 (27).
Intracellular rhinovirus replication analysis by in situ hybridization. A549 cells were cultured in 100-mm plates and, when 80% confluent, were inoculated with rhinovirus at an MOI of 1. After 30 min of incubation at room temperature to allow virus attachment, cells were washed three times, and fresh medium was added. Cells were then harvested by gentle scraping immediately and at 6 and 24 h. Cells were washed in PBS and fixed in neutral buffered formalin (NBF) for 1 h at 4°C. Cells were then washed three times in PBS and resuspended in 0.2-ml clots of human plasma (4). Cell clots were fixed in NBF for 24 h and embedded in paraffin wax blocks. Five-micron sections were hybridized with 200 ng/ml of a mixture of four conserved rhinovirus-specific probes (28). The probe sequences used were: PB4, CAG GGG CCG GAG GAC TCA AGA TGA GCA CAC GCG GCT; PB5, TGC AGG CAG CCA CGC AGG CTA GAA CTC CGT CGC CG; PB6, ACA CGG ACA CCC AAA GTA GTT GGT CCC ATC CCG CAA; and PB7, ACA TCC TTA ACT GGG TCT GTG AAT TTA CTG GGG TCT.
Rhinovirus new protein synthesis. Rhinovirus new viral protein synthesis in A549 cells was assessed by [35S]methionine incorporation and immunoprecipitation as described above. Live virus was inoculated onto 107 A549 cells at an MOI of 1, and incubations with [35S]methionine in methionine-free medium were performed for 2 and 24 h.
Viability of rhinovirus-infected A549 cells. A549 cells were cultured in 100-mm plates and when 80% confluent were inoculated with rhinovirus at an MOI of 1. After 30 min of incubation at room temperature to allow virus attachment, cells were washed three times, and fresh medium was added. The medium was then removed from infected and control noninfected cells at various time points. Detached cells in the supernatant were counted, attached cells on the plates were trypsinized and then counted, and trypan blue exclusion was determined to assess viability. For viability, 300 cells were counted after incubation with trypan blue for 5 min, and a ratio of living to dead cells was derived.
Statistical analysis
Data are expressed the mean ± SEM. Testing for
significance in the time-course studies was performed using two-way
repeated measures analysis of variance, followed by Student’s paired
t test at each time point. Other comparisons were conducted
using Student’s paired t test. p 
0.05
was considered significant.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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To determine whether IL-8 protein was released from A549 cells in
response to rhinovirus in a dose-response manner, A549 pulmonary
epithelial cells were cultured to 80% confluence in 12-well tissue
culture plates and incubated with 10-fold dilutions of rhinovirus type
9 for 24 h. Supernatants were then harvested and assayed for IL-8.
As shown in Figure 1
A, the
pulmonary epithelial cell line released IL-8 in a dose-dependent manner
in response to rhinovirus. An MOI of 1 produced the greatest IL-8
release; studies were not conducted at higher MOIs to determine whether
this was maximal. All additional experiments were performed at this
dose of virus.
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B, IL-8 release was already elevated above the control
value at 6 h and continued to increase until 120 h. Cultures
were not continued beyond 120 h. IL-8 mRNA induction in response to rhinovirus
To determine whether the observed IL-8 protein release induced by
rhinovirus was accompanied by increased expression of IL-8 mRNA, we
investigated IL-8 mRNA induction by rhinovirus using Northern analysis.
A549 epithelial cells were cultured in 100-mm tissue culture plates.
Rhinovirus type 9 was added, and RNA was harvested at 1, 3, 6, 24, 48,
and 96 h. Northern blot analysis was performed to detect the
presence of IL-8 mRNA. The time course of IL-8 mRNA induction in
response to rhinovirus type 9 up to 24 h is shown in Figure 2. A consistent response to rhinovirus
type 9 was noted, with a detectable increase in mRNA at 1 h and a
peak between 3 and 24 h. Induction of IL-8 mRNA expression in
response to rhinovirus was still present at 48 h, although reduced
compared to that at earlier time points. No induction was detected at
96 h (data not shown).
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As the rhinovirus replicative cycle is thought to be 6 to 8 h (29), we wished to investigate whether the observed induction of IL-8 protein was a response to virus replication or to virus-receptor binding triggering intracellular signaling pathways. To investigate this, we used two methods of inactivation of rhinovirus: UV inactivation to abolish rhinovirus replication and precoating the virus with solubilized receptor (sICAM-1) to prevent virus receptor binding (30).
Confirmation of UV and sICAM inactivation of rhinovirus type 9 was conducted in microtiter plate titration assays as described above. In the titration assays, the titer of stock rhinovirus type 9 was 2 x 106 TCID50/ml, while that of inactivated virus by either method was zero, with no CPE observed even with undiluted inactivated virus stocks.
Inactivation of virus protein synthesis by UV light was also confirmed
by immunoprecipitation of 35S-labeled newly synthesized
viral proteins produced in Ohio HeLa cells. Untreated rhinovirus type 9
demonstrated active new virus protein synthesis between 6 and 8 h
after virus inoculation (Fig. 3,
lane 2), while there was a complete absence of new
viral protein synthesis with UV inactivated rhinovirus type 9 (Fig. 3, lane 3).
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To investigate whether inhibition of virus replication by UV light altered rhinovirus induction of IL-8 protein release and mRNA expression, A549 epithelial cells were incubated with UV-inactivated and live rhinovirus type 9 and with medium alone. Supernatants were harvested at 24 and 120 h and were analyzed for IL-8 release by ELISA. RNA was harvested at 24 h and was analyzed by Northern analysis.
IL-8 protein release and mRNA induction are shown in Figures 4 and 5. UV-inactivated rhinovirus type 9
and control medium resulted in significantly less IL-8 protein release
than live virus at both time points. However, UV-inactivated virus did
result in increased IL-8 release compared with that using medium alone,
with IL-8 levels being approximately half those of live rhinovirus type
9 (Fig. 4).
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). Again, the UV-inactivated virus
produced approximately half the signal of the live virus.
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), while live rhinovirus resulted
in significantly increased IL-8 release as previously observed.
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The results reported above suggested that at least part of the induction of IL-8 protein and mRNA may result from rhinovirus replication in A549 cells. However, the ability of rhinoviruses to replicate in lower respiratory epithelium is controversial. To investigate the capacity of rhinovirus type 9 to replicate in the pulmonary epithelial cell line A549, we conducted studies to investigate new viral protein production, release of viral RNA into cell supernatants, intracellular localization of viral RNA, and release of functional viral replicative units into the supernatant.
Production of new rhinovirus proteins
The production of newly synthesized viral proteins following
rhinovirus inoculation onto A549 cells was investigated by
immunoprecipitation of 35S-labeled newly synthesized
viral proteins. Immunoprecipitation after incubation of rhinovirus type
9 with Ohio HeLa cells in the presence of 35S-labeled
methionine for 2 h (between 6–8 h after infection; Fig. 3, lane 2) produced strong bands for several newly
synthesized viral proteins. Similar new protein synthesis was also
observed with 24-h incubation with Ohio HeLa cells (between 6–30 h
after infection) (31). In contrast, when rhinovirus type 9 was
incubated with A549 cells under exactly the same conditions there was
no detectable new viral protein synthesis after either the 2-h (Fig. 7, lane 2) or the 24-h
(Fig. 7, lane 3) incubations. We concluded that
immunoprecipitation was not sensitive enough to detect new viral
proteins under the conditions described, and therefore we investigated
viral protein production by Western analysis. Once again increased
rhinovirus protein synthesis was easily detected when rhinovirus type 9
was inoculated onto Ohio HeLa cells for 24 h, but no increase in
viral proteins was observed with rhinovirus type 9 inoculation onto
A549 cells for the same time period (data not shown).
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As the protein detection techniques of immunoprecipitation and Western analysis were unable to detect rhinovirus replication in A549 cells, we elected to use more sensitive methods and investigated the release of rhinovirus RNA into the supernatants of A549 cells with RT-PCR assays for the genomic and replicative strands of rhinovirus RNA.
A549 cells were inoculated with rhinovirus; after attachment, cells
were washed three times and fresh medium was added. Supernatants were
harvested at various time points, and genomic and replicative strand
RT-PCRs were conducted. There was no detectable replicative (-ve)
strand RNA immediately following addition of fresh medium after washing
(time zero, Fig. 8); there was then a
marked increase in replicative strand RNA at 6 h after
inoculation, followed by a gradual decline, until 96 h, after
which no replicative strand RNA was detected. The results for the
genomic (+ve) strand were similar; however, there was a weak signal
present at time zero, presumably from the inoculated virus, again there
was a marked increase in viral RNA at 6 h, followed by a gradual
decline to 120 h, when only a weak signal was still detectable
(Fig. 8).
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To ensure that the observed increases in viral RNA in the supernatants from A549 cells were not a result of either virus adsorption onto the cell surface followed by release of viral uncoated RNA after washing of the cells or of inefficient washing leading to persistence of viral RNA from the inoculum without active intracellular replication, we investigated intracellular rhinovirus replication using in situ hybridization.
A549 cells were cultured and inoculated with rhinovirus. Cells were
then harvested at 0, 6, and 24 h; resuspended in plasma clots;
fixed; and embedded in paraffin wax blocks. Sections were then examined
by in situ hybridization for the genomic (+ve) strand of rhinovirus
RNA. As shown in Figure 9, there was no
detectable signal in control noninfected cells or in cells harvested
immediately after inoculation and washing. At 6 and 24 h after
inoculation, increases in intracellular rhinoviral RNA expression were
clearly seen, demonstrating that new virus genomic RNA was being
produced inside the cells.
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Finally, we investigated the ability of A549 cells to
assemble functional viral replicative units and to release them into
the cell supernatant. Rhinovirus type 9 was inoculated onto A549 cells
and adsorbed for 30 min. Following washing to remove unattached virus,
fresh medium was added. Supernatants were harvested at 6, 24, 48, 72,
96, and 120 h. The production of functional viral replicative
units was assessed by microtiter plate titration assays of cell
supernatants as described above. Identification of the infectious units
as rhinovirus type 9 was confirmed by neutralization with rhinovirus
type 9-specific antiserum (ATCC). No infective virus was detectable in
supernatants from cells at 6 h after inoculation. Peak titers were
observed in A549 cell supernatants at 24 h after inoculation (Fig. 10); viral replication was just
detectable in the supernatants at 48 h postviral inoculation, but
not thereafter.
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To determine whether rhinovirus replication in A549 cells had any cytotoxic or cytopathologic effect, A549 cell cultures were infected with rhinovirus and observed for the development of CPE. A549 cell cultures were observed up to 120 h postviral inoculation, and at no stage was any rhinovirus-related CPE detected. Monolayers were also stained with crystal violet, and no disruption of the cell layer was observed at any time point.
In addition, the effects of viral infection on cell viability were
examined by trypan blue exclusion. Rhinovirus type 9 was inoculated
onto A549 cells and adsorbed for 30 min. Following washing to remove
unattached virus, fresh medium was added. Supernatants and attached
cells were harvested from infected and control noninfected cells at 24,
48, 72, 96, and 120 h. Detached cells in the supernatant were
100% trypan blue positive, and numbers were counted to assess shedding
of nonviable cells by the monolayer. At no time point was there any
difference between infected and noninfected supernatants (Table I). Attached cells on the plates were
trypsinized and counted, and trypan blue exclusion was determined to
assess viability. At no time point was there any difference in cell
viability between the infected and noninfected attached cells
(Table I).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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There are several previous reports demonstrating that epithelial cell lines release IL-6, IL-8, IL-11, and GM-CSF following respiratory syncytial virus infection (32, 33, 34, 35). Choi and Jacoby found increased IL-8 gene expression in human tracheal cells exposed to influenza virus (36). The role of rhinoviruses in promoting lower airway proinflammatory responses has received less attention until recent years, as rhinoviruses were until then thought to be principally limited to upper respiratory tract infections and the common cold. The recent data implicating rhinovirus infections in the majority of asthma exacerbations in both adults and children (1, 2, 3) has stimulated renewed interest in this virus type and its ability to infect the lower respiratory tract and provoke airway inflammation. In this regard, rhinovirus induction of cytokine protein and mRNA from the A549 pulmonary epithelial cell line has recently been demonstrated for IL-6 (10) and IL-11 (13); however, these studies did not examine the relationship between cytokine release and the ability of rhinoviruses to infect this cell line.
The majority of other respiratory viruses (adenovirus, influenza virus, respiratory syncytial virus, parainfluenza virus, and CMV) can infect the lower respiratory tract. However, evidence that this occurs with rhinoviruses is not conclusive. In vivo studies provide some support for this hypothesis, in that small particle aerosols of rhinovirus have been shown to produce tracheobronchitis (37). Cultures of sputum from children with wheezy bronchitis were more often positive for rhinovirus than were nasal swabs taken at the same time (38). In experimental infection studies, rhinoviruses have been cultured from bronchial lavage fluid (39), and more recently rhinovirus has been demonstrated by RT-PCR in cells recovered from bronchial lavage (16). However, in both these studies the possibility of contamination from the upper airway during the nasal introduction of the bronchoscope could not be excluded. Finally, rhinoviruses are associated with a variety of severe lower respiratory tract diseases, such as bronchiolitis and pneumonia (40, 41, 42), and have been isolated from lower respiratory postmortem specimens in a few case reports (15).
There is also some in vitro evidence to suggest that rhinoviruses are capable of replicating in lower respiratory epithelium. Subauste et al. studied the transformed bronchial epithelial cell line BEAS-2B and used virus titrations in supernatants and lysates of cell pellets, and RT-PCR on RNA extracted from the cell pellets to demonstrate an increase in the rhinovirus genomic (+ve) strand for the first 24 h after virus inoculation and increases in virus titrations up to 72 h postinoculation (23). Interestingly, the infection of BEAS 2B cells was noncytolytic, and there was no effect on cell viability.
In this study, we have demonstrated that rhinovirus type 9 is also
capable of initiating productive infection of the transformed pulmonary
epithelial cell line, A549. This was demonstrated by the finding of the
release of replicative (-ve) strand RNA into A549 cell supernatants,
the demonstration of intracellular replication by in situ
hybridization, and the release of functional viral replicative units
into the supernatant. However, the infection was low grade, with virus
being detectable only in neat supernatants in microtiter plate assays,
and the maximal titer observed at 24 h postinoculation was 64
TCID50/ml (Fig. 10). Similar to the BEAS 2B cells
(23), no CPE on the A549 cells was observed at any time point; there
was also no effect on cell viability (Table I). However, in contrast to
the BEAS 2B cells, in which viral replication appeared to continue up
to the final time point examined (72 h), infection of A549 cells was
terminated spontaneously around 24 h. Despite this, IL-8 protein
release was noted to increase up to 120 h after virus inoculation
(Fig. 1B), while IL-8 mRNA expression tended to
follow the pattern of infection more closely, peaking early between 3
and 24 h (Fig. 2) and returning to control levels by 96 h
(data not shown). This prolonged release of IL-8 may be a result of
changes in cellular function resulting in IL-8 protein release that
persist for some time even after the infection itself has resolved.
The observations that both UV-inactivated and live rhinovirus are able
to trigger IL-8 protein release and mRNA expression (although the
induction with UV-inactivated virus was about 50% that with live
virus; Figs. 4 and 5) suggest that rhinovirus binding to its cell
surface receptor, ICAM-1, may be responsible in part for the
increased IL-8 protein and mRNA. An alternative explanation would be
that the virus stocks contained other active compounds, such as other
cytokines. However, assays sensitive to <5 pg for likely candidates,
including IL-1 and TNF, were negative (data not shown). Furthermore,
the fact that induction of IL-8 protein release was completely
suppressed by preventing virus-receptor binding by precoating the virus
with sICAM suggests that the inoculum itself contained no other
stimulatory substances that were able to induced IL-8 protein release
or mRNA expression under these conditions. The possibility that
cross-linking of ICAM-1 by rhinovirus occurs is made likely by the fact
that each virus particle has 60 receptor binding sites (43). Further it
has been demonstrated that cross-linking of ICAM-1 by Ab leads to both
intracellular signaling (44) and cytokine release (45). We therefore
believe that the virus-receptor interaction is the most likely
explanation for the induction of IL-8 mRNA and protein release observed
with UV-inactivated virus, while the difference in IL-8 protein release
and mRNA induction observed between UV-inactivated and live virus is
probably due to viral replication within the pulmonary epithelial
cell line.
The observations of rhinovirus-induced IL-8 protein release and mRNA expression from a pulmonary epithelial cell line probably have relevance to human rhinovirus infections, as we have found increased levels of IL-8 in nasal aspirates during wild-type upper respiratory virus infections, the majority of which were due to rhinoviruses (14). We studied nasal secretions obtained during virus-induced exacerbations of asthma in school age children and found increased levels of neutrophil myeloperoxidase and IL-8 in the presence of rhinoviral infection. Interestingly, there were correlations between the levels of IL-8 and neutrophil myeloperoxidase and between myeloperoxidase and the severity of upper respiratory symptoms, suggesting that IL-8 may play an important role in neutrophil chemoattraction and activation in rhinovirus infections (14). These observations are supported by an experimental rhinovirus infection study in asthmatic subjects, in whom increased levels of nasal IL-8 were observed after rhinovirus infection (12). This study was also of particular interest, as the researchers found correlations between the levels of nasal IL-8 and the increases in bronchial hyper-reactivity induced by rhinovirus infection, again suggesting that IL-8 may play an important role in the pathogenesis of rhinovirus-induced asthma exacerbations. The findings of the present study and those reported by Subauste et al. (23) suggest that rhinovirus-induced release of IL-8 from lower airway epithelium may be an important source of this chemokine.
Eosinphil infiltration is an important feature of asthma (18), and there is increasing evidence that eosinophil recruitment and activation are also important in virus-induced asthma exacerbations. We have found increased levels of eosinophil major basic protein in nasal aspirates taken from children with rhinovirus-induced asthma exacerbations (L. M. Teran, M. C. Seminario, G. J. Gleich, and S. L. Johnston, unpublished observations). Calhoun et al. demonstrated increased allergen-induced bronchial lavage eosinophil numbers in atopic rhinitic subjects, compared with those in normal subjects, in the presence of rhinovirus experimental infections (19). We also investigated the lower airway cellular response to rhinovirus infection using experimentally induced rhinovirus infections in normal and asthmatic subjects. Both normal and asthmatic subjects had increased numbers of eosinophils in the bronchial mucosa at 4 days after rhinovirus infection. However, in the asthmatic subjects, the eosinophil infiltrate was still present when the subjects were rebiopsied 6 to 8 wk later, while in normal subjects, eosinophil numbers had returned to baseline levels (17). Given the possible role of IL-8 in eosinophil recruitment and activation (21, 22), and the fact that its role in this respect may be up-regulated in asthma (46), it is possible that this chemokine plays an important role in the recruitment and activation of both eosinophils and neutrophils in the context of rhinovirus-induced asthma exacerbations.
A prominent feature of both normal and asthmatic subjects in the experimental rhinovirus infection study was the increased numbers of CD3+, CD4+, and CD8+ lymphocytes in the bronchial mucosa at 4 days after rhinovirus infection (17). IL-8 is also a lymphocyte chemoattractant (47); it is therefore possible that rhinovirus-induced IL-8 secretion from pulmonary epithelium plays a role in the recruitment and activation of several cell types involved in the pathogenesis of virus-induced asthma. However, further definition of the role of this chemokine in virus-induced asthma exacerbations will come from clinical studies making assessments of the inter-relationships between IL-8 levels and the severity of clinical illness and of viral infection in normal and asthmatic subjects (48).
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2 Current address: University Medicine (810), Level D, Center Block, Southampton General Hospital, Tremona Rd., Southampton, United Kingdom SO16 6YD.
3 Address correspondence and reprint requests to Dr. Gary W. Hunninghake, C33-G General Hospital, University of Iowa Hospitals and Clinics, Iowa City, IA 52242.
4 Abbreviations used in this paper: TCID50, tissue culture 50% infective dose; MOI, multiplicity of infection; CPE, cytopathic effect; sICAM, soluble intercellular adhesion molecule; NBF, neutral buffered formalin.
Received for publication November 13, 1997. Accepted for publication February 17, 1998.
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B-dependent transcriptional activation. J. Clin. Invest. 97:421.[Medline]
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and interleukin-1. Eur. J. Immunol. 20:2591.[Medline]
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50%), but not completely, by UV inactivation of
rhinovirus replication.
