|
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
ABSTRACT |
|---|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
To investigate whether rhinovirus infection impairs epithelial barrier functions, human rhinovirus 14 (HRV-14) was infected to primary cultures of human tracheal epithelial cells and experiments were
performed on Day 2 after HRV-14 infection. Hydrogen peroxide (H2O2; 3 × 10
4 M) increased electrical conductance (G) across the epithelial cell sheet measured with Ussing's chamber methods. Exposure of the epithelial cells to HRV-14 had no effect on H2O2-induced increases in G and [3H]mannitol
flux through the cultured epithelium in the control condition, but it markedly potentiated H2O2-
induced increases in both parameters in IL-1
(100 U/ml) pretreated condition. However, pretreatment with TNF-
(100 U/ml) was without effect. IL-1 enhanced the intercellular adhesion molecule-1
(ICAM-1) expression assessed by immunohistochemical analysis and susceptibility of epithelial cells to
HRV-14 infection. An antibody to ICAM-1 inhibited HRV-14 infection of epithelial cells and abolished
H2O2-induced increases in G and [3H]mannitol flux in IL-1-pretreated epithelial cells with HRV-14 infection. These results suggest that rhinovirus infection may reduce barrier functions in the airway epithelium in association with upregulation of ICAM-1 expression.
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Several investigators have reported that rhinoviruses are the most common infectious exacerbants in inflammatory airway diseases such as bronchial asthma (1), chronic bronchitis (1, 3), and sinusitis (6, 7). Despite the high prevalence of rhinovirus infections, the pathogenesis of the subsequent disease process is incompletely understood. Respiratory tract epithelium is the primary target for respiratory viruses, and several viruses cause airway epithelial cell injury. Influenza A infection induces diffuse inflammation of bronchi, trachea, and larynx with desquamation of ciliated epithelial cells down to the basal layer (8). Likewise, ciliary defects in the nasal mucosa of children were observed with influenza types A and B, parainfluenza types 1, 2, and 3, adenovirus, respiratory syncytial virus, and herpes simplex virus infections (9). A study with human nasal cultured epithelium demonstrated a marked cytopathic effect followed by destruction of the cell monolayer with both influenza type A and adenovirus infections (10). However, no cytopathic effect on epithelial cells was observed with rhinovirus infection, despite active viral replication being demonstrated (10). Lack of changes in the morphology or integrity after rhinovirus infection was also reported in the transformed cell line derived from human bronchial epithelial cells (BEAS-2B cells) (11).
Intercellular adhesion molecule-1 (ICAM-1), the receptor
for the major group of rhinoviruses (12, 13), is known to be expressed by epithelial cells and upregulated by several cytokines such as tumor necrosis factor (TNF)-, interferon
(IFN)-
, and interleukin (IL)-1 (14). Subauste and coworkers (11) reported that the susceptibility of epithelial cells
to infection by human rhinovirus 14 (HRV-14) can be increased by upregulation of ICAM-1 in the BEAS-2B cells,
and epithelial cells infected with HRV-14 produce IL-6, IL-8,
and granulocyte macrophage-colony stimulating factor (GM-
CSF) (11). These proinflammatory cytokines may attract monocytes, neutrophils, and eosinophils to the inflammatory sites where oxygen radicals may be released from inflammatory
cells (17). Oxygen radicals cause airway epithelial injury,
leading the epithelium to hyperpermeable state (20, 21).
To test the hypothesis that rhinovirus infection impairs functional integrity of the airway epithelium, we investigated whether HRV-14 infection exaggerates oxygen radical-induced hyperpermeability in the cultured human tracheal epithelium. We also examined whether upregulation of ICAM-1 modifies the susceptibility of the epithelial cells to HRV-14 infection and viral infection-induced effects on the epithelial cells.
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Human Tracheal Epithelial Cell Culture
Tracheas were obtained from 49 patients (mean age 67 yr; range, 41 to 83 yr) without overt pulmonary disease at autopsy between 3 and 10 h after death under a protocol permitted by the Tohoku University Ethics Committee. Culture methods have been described elsewhere in detail (21, 22). In brief, tracheas were rinsed in ice-cold and sterile phosphate-buffered saline (PBS) to remove mucus and debris, opened longitudinally along the anterior surface, and mounted in a stretched position in a dissection tray. The surface epithelium was scored into longitudinal strips and pulled off the submucosa. The tissue strips were rinsed five times in PBS containing 5 mM dithiothreitol and then twice in PBS alone. The tissue strips were placed into conical tubes (Coster, Cambridge, MA) containing protease (Sigma type XIV, 0.4 mg/ml; Sigma Chemical, St. Louis, MO) dissolved in PBS. They were put overnight in the refrigerator at 4° C. The enzyme was then competitively inhibited by the addition of fetal calf serum (FCS) to a final concentration of 2.5%, and small sheets of cells were dislodged from the epithelial strips by vigorous agitation. The denuded strips were removed, and the sheets of cells remaining were dispersed by repeated aspiration in a 10-ml pipette.
Cells were pelleted (200 g, 10 min) and suspended in a mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12 containing 5% FCS (50/50, vol/vol). Cell counts and estimates of viability were made using a hemocytometer and trypan blue, and cells were plated at 106 viable cells/cm2 onto Millicell CM inserts (0.45-µm pore size and 0.6-cm2 area; Millipore Products Division, Bedford, MA). This medium was replaced by serum-free DMEM/Ham's F-12 containing 2% Ultroser G serum substitute (IBF Biotechnics, Savage, MD) on the first day after plating. Cells were grown with an air interface (i.e., no medium added to the mucosal surface). Cell culture media were obtained from GIBCO/BRL Life Technologies (Palo Alto, CA) and were supplemented with penicillin (105 U/L), streptomycin (100 mg/L), gentamicin (50 mg/L), and amphotericin B (2.5 mg/L). Millicell inserts were coated with vitrogen gels. To make vitrogen gels, 10-fold minimum essential medium, 0.1 N NaOH, and vitrogen solution (Collagen, Palo Alto, CA) were mixed at 4° C (10/10/80, vol/vol/ vol). After mixing, 0.15 ml/cm2 of this solution was added to the Millicell inserts. Vitrogen gels were formed by incubation at 37° C for 1 h and were used within 2 h of manufacture.
To determine whether cultured cells can form tight junctions, we
performed parallel cultures of human tracheal epithelial cells on Millicell CM inserts to measure electrical resistance and short-circuit current using Ussing chamber methods described subsequently. When
cells cultured under these conditions become differentiated and form
tight junctions without contamination of fibroblasts, they have values
of > 40 
· cm2 for resistance and > 10 µA/cm2 for short-circuit current (22). Therefore, cultured human tracheal epithelial cells were
judged as cells able to form tight junctions and were used for the following experiments when cells on Millicell CM inserts had high resistance (> 40 · cm2) and high short-circuit current (> 10 µA/cm2).
Human Embryonic Fibroblast Cell Culture
Human embryonic fibroblast cells were cultured in a Roux type bottle (Iwaki Glass Co., Chiba, Japan) sealed with a rubber plug in minimum essential medium containing 10% FCS and were supplemented with penicillin (5 × 104 U/L) and streptomycin (50 mg/L) (23). Confluency was achieved at 7 d, at which time cells were collected by trypsinization (0.05% trypsin and 0.02% EDTA). Cells (1.5 × 105 cells/ml) suspended in minimum essential medium containing 10% FCS were then plated in glass tubes (15 × 105 mm; Iwaki Glass) sealed with rubber plugs, and cultured at 37° C.
Viral Stocks
HRV-14 was prepared in our laboratory from patients with common
cold as described previously (16, 23). Stocks of HRV-14 were generated by infecting human embryonic fibroblast cells cultured in glass
tubes in 1 ml of the minimum essential medium supplemented with
2% -globulin free calf serum (GGFCS; GIBCO/BRL Life Technologies), penicillin (5 × 104 U/L), and streptomycin (50 mg/L) at 33° C. Cultures were grown in glass tubes in 1 ml of minimum essential medium supplemented with 2% GGFCS for several days until cytopathic
effects were obvious, after which the cultures were frozen at 
80° C,
thawed, and sonicated. The virus-containing fluid so obtained was frozen in aliquots at 80° C. The content of viral stock solutions was determined using the human embryonic fibroblast cell assay described
below.
Detection and Titration of Viruses
HRV-14 was detected by exposing confluent human embryonic fibroblast cells in glass tubes to serial 10-fold dilutions of virus-containing medium in minimum essential medium supplemented with 2% GGFCS. The glass tubes were then incubated at 33° C for 7 d and the cytopathic effects of viruses on human embryonic fibroblast cells were observed using an inverted microscope (MIT-2; Olympus, Tokyo, Japan) as reported previously (23). The amount of specimen required to infect 50% of human embryonic fibroblast cells (TCID50) was determined.
Viral Infection of Human Tracheal Epithelial Cells
Medium in the serosal side was removed from confluent monolayers
of human tracheal epithelial cells on Day 7 and replaced with 0.4 ml of
DMEM/Ham's F-12 medium containing 2% Ultroser G serum substitute. HRV-14 was added to the mucosal surface of the cultured epithelium at a concentration of 103 TCID50/ml. After 60-min incubation
at 33° C, the viral solution of the mucosal surface was removed and
cells were washed twice with 0.5 ml of fresh medium. Cells were then
fed with DMEM/Ham's F-12 medium containing 2% Ultroser G serum substitute with penicillin (10 5 U/L), streptomycin (100 mg/L),
gentamicin (50 mg/ml), and amphotericin B (2.5 mg/L) and cultured
at 33° C. Culture medium in the serosal side was removed at 1 hr, 2, 4, and 6 d after infection and stored at 80° C for the determination of
viral content. Viral content in the culture medium is expressed as
TCID50 units/ml.
Ussing Chamber Study
For studies in Ussing chambers, Millicell inserts with their attached cells without edge damage were mounted in a modified Ussing chamber (21, 22). Experiments were performed on Day 2 after HRV-14 infection of sham infection (control) in a Krebs-Henseleit solution with the following composition (mM): 118 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 NaH2PO4, 25.5 NaHCO3, and 5.6 glucose. The solution was maintained at 37° C and aerated continuously by bubbling with a mixture of 95% O2-5% CO2 (pH 7.4). Transepithelial resistance (R) and electrical conductance (G) were determined from the current produced by the fixed transepithelial potential difference (PD) (pulse width, 200 ms; intensity, 0.5 mV; frequency, 0.05 Hz).
Experimental Protocol
To examine whether hydrogen peroxide (H2O2; from 104 M to 3 × 103 M) alters viability of cultured human tracheal epithelial cells, cells were incubated with each concentration of H2O2 for 60 min. The
estimates of cell viability were done using trypan blue and by measuring the amount of lactate dehydrogenase (LDH) in the medium as
previously reported (24). The maximal concentration of H2O2 that did
not alter cell viability, as assessed by the exclusion of trypan blue and
the amount of LDH in the medium, was obtained at 3 × 104 M (Table 1). In preliminary studies, we also found that H2O2 at the concentration of 3 × 104 M reproducibly increased G in IL-1 (100 U/ml)
pretreated cells with HRV-14 infection. Therefore, we used this concentration of H2O2 in the following experiments.
|
To examine the effects of H2O2 on G in the cultured tracheal epithelium, we added H2O2 (3 × 104 M) into the Krebs-Henseleit solution after the baseline value was obtained. H2O2 was added to both
sides of the Ussing chamber.
Because HRV-14 uses ICAM-1 as its cellular receptor, we examined the effects of cytokines that are known to increase the expression of ICAM-1 on airway epithelial cells (14, 15) on the susceptibility of
the cells to HRV-14 infection and H2O2-induced increases in G. Confluent monolayers of human tracheal epithelial cells on Day 7 were
incubated, therefore, for 16 h with either medium alone (control),
TNF- (100 U/ml; Genzyme, Cambridge, MA), or IL-1 (100 U/ml;
Ohtsuka, Tokushima, Japan) at 37° C. This concentration of IL-1
was shown earlier to be optimal for inducing ICAM-1 expression of
primary cultures of human tracheal epithelial cells (16). The concentration of TNF- was adjusted to that of IL-1. After the removal of
cytokines, cells were exposed to HRV-14 at a concentration of 10 3
TCID50/ml for 60 min.
We also tested the effects of the anti-ICAM-1 antibody on the susceptibility of the cells to HRV-14 infection and H2O2-induced increases in G. After treatment with IL-1 (100 U/ml) as described previously, the mucosal side of cells was incubated for 1 h with medium
alone, with medium containing a mouse monoclonal anti-human antibody to ICAM-1 (100 µg/ml; 84H10; Immunotech, Marseille, France), or with medium containing a class-matched monoclonal antibody to
human leukocyte antigen (HLA) class 1 (100 µg/ml; Immunotech). 84H10 recognizes the ICAM-1 functional domain (25) and an antibody to HLA class 1 binds to the epithelium without blocking rhinovirus interactions with the cell. After excess antibodies were washed off,
cells were exposed to HRV-14 (103 TCID50/ml) for either 15 or 60 min
before rinsing and adding fresh DMEM-Ham's F-12 containing 2%
Ultroser G serum substitute supplemented with 105 U/L penicillin, 100 mg/L streptomycin, 50 mg/L gentamicin, and 2.5 mg/L amphotericin
B. The viral content of this medium was then assessed at various times
after infection. To confirm that increases in G induced by H2O2 in IL-1-pretreated cells with HRV-14 infection were due to the effects of
HRV-14 infection and not a contaminant present in the viral stock,
the ability of ultraviolet (UV)-inactivated virus to induce increases in
G was also examined. UV inactivation was performed as described
previously (26).
Mannitol Flux Studies
Measurement of permeability across epithelial cell sheets was performed by methods described by Cooper and coworkers (27) with
D-[3H]mannitol, producing a behavior similar to albumin (28). Epithelial cells cultured on Millicell inserts were treated with five different kinds of regimen: no cytokine treatment with either HRV-14 (n = 7)
sham infection (n = 7), IL-1 (100 U/ml) pretreatment with either
HRV-14 (n = 7) or sham infection (n = 7), or TNF- (100 U/ml) pretreatment with HRV-14 infection (n = 7). We also examined the effects of UV-inactivated virus on mannitol flux in IL-1 (100 U/ml)
pretreated cells. Experiments were performed on Day 2 after either
HRV-14 infection (103 TCID50/ml) or sham infection. Cells, put on 24-well plates (FALCON; Becton Dickinson, Lincoln Park, NJ), were
rinsed with PBS and culture medium was added to both sides of the
cell sheets. Culture medium contained 2 mM nonradioactive mannitol
to minimize the D-[3H]mannitol (0.05 µM)-induced changes in osmolarity. To match the fluid level, we added 0.2 ml and 0.7 ml of medium
to the mucosal and serosal sides, respectively. D-[3H]mannitol (1 µCi/
ml, 0.05 µM; Daiichi Kagaku Yakuhin, Tokyo, Japan) was added to
either the mucosal or serosal side of the tissue (hot side). Cell sheets
were then reincubated at 37° C in a 5% CO2 incubator for 60 min.
Whole volumes of the medium (0.2 or 0.7 ml) were taken for liquid
scintillation counting from either the serosal or mucosal side of the
cell sheets (cold side) every 60 min, and the same volume of fresh cold
medium was replaced to the cold side of the cell sheets. For the first 60 min, the medium in both sides (hot and cold sides) did not contain
H2O2. After the first sampling at 60 min, the hot side medium containing D-[3H]mannitol was taken and replaced by fresh radioactive medium containing D-[3H]mannitol supplemented with H2O2 (3 × 104 M).
In the cold side, fresh medium with added H2O2 was replaced after the
first sampling period at 60 min.
We also tested the effects of the anti-ICAM-1 antibody on H2O2-induced increases in mannitol flux. After treatment with IL-1 (100 U/ml), the mucosal side of cells was incubated for 1 h with medium
containing 84H10 (100 µg/ml) or with medium containing a class-matched monoclonal antibody to HLA class 1 (100 µg/ml). After
washing off excess antibodies, cells were exposed to HRV-14 (10 3
TCID50/ml) for 60 min.
To examine whether an increased viral load is a mechanism responsible for increases in G and mannitol flux in IL-1-pretreated cells with HRV-14 infection (103 TCID50/ml), cells were exposed to
HRV-14 (104 TCID50/ml) for 60 min and studies of the Ussing chamber and mannitol flux were performed on Day 2 after HRV-14 infection.
Immunohistochemical Analysis
Immunohistochemical analysis for ICAM-1 expression in human tracheal epithelial cells was done as described previously (29). Human
tracheal epithelial cells were cultured on Vitrogen gels on Millicell inserts (0.45 µm pore size and 0.6 cm2 area) (16, 30). Cell sheets were
fixed in periodate-lysine-paraformaldehyde at 4° C for 2 h. After
washing in sucrose (10%, 15%, 20%/PBS), they were embedded in
optimal cutting temperature (OCT) compound (Miles Laboratories,
Naperville, IL) in liquid nitrogen and stored at 70° C until use. The
staining of cryostat sections (6 µm) was performed with the alkaline
phosphatase-anti-alkaline phosphatase (APAAP) method (31). The
first antibody was a mouse monoclonal antibody to human ICAM-1
(84H10; Immunotech), and the second antibody was a rabbit antibody
to mouse immunoglobulins (Dako Japan Co., Ltd., Tokyo, Japan).
The final staining was performed using APAAP complex and fast red
(Dako Japan Co., Ltd.), and counterstained with hematoxylin. For
negative control, cells were treated with mouse immunoglobulin instead of the monoclonal antibody to ICAM-1.
Statistical Analysis
Values are reported as means ± SE. For statistical analysis, we used a two-way repeated measure analysis of variance. A value of p < 0.05 was considered significant; n refers to the number of donors from which cultured epithelial cells were used.
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
HRV-14 Infection of Human Tracheal Epithelial Cells
Exposing confluent human tracheal epithelial cell monolayers
to HRV-14 (103 TCID50/ml) consistently led to infection. Collection of culture medium at differing times after viral exposure revealed no detectable virus at 1 h after infection. However, the viral titers of culture media collected during the first
2 d, 2 to 4 d, and 4 to 6 d after infection each contained significant levels of virus in the control condition (Figure 1). Pretreatment of cells with IL-1 (100 U/ml) further increased viral titer levels compared with control, whereas that with TNF-
(100 U/ml) was without effect (Figure 1). Human tracheal cell
viability, as assessed by the exclusion of trypan blue, was consistently > 97% in HRV-14-infected culture. Cell counts 24 h after infection were not significantly different from those in
noninfected cells (1.0 ± 0.1 × 106 in noninfected cells versus
1.0 ± 0.2 × 106 in infected cells, p > 0.50, n = 8).
|
Ussing Chamber Experiments
Figure 2 shows the time course of H2O2-induced effects on G
in the cultured tracheal epithelium. The baseline values of G were 3.0 ± 0.3 mS/cm2 in control cells with sham infection,
3.2 ± 0.3 mS/cm2 in control cells with HRV-14 infection, 3.0 ± 0.3 mS/cm2 in TNF--pretreated cells with sham infection, 2.7 ± 0.3 mS/cm2 in TNF--pretreated cells with HRV-14 infection,
3.1 ± 0.3 mS/cm2 in IL-1-pretreated cells with sham infection, and 3.3 ± 0.3 mS/cm2 in IL-1-pretreated cells with
HRV-14 infection, and there were no significant differences
among them (p > 0.20, n = 8). The baseline values of G were
stable with time for 60 min in the absence of H2O2. In the
presence of H2O2, increases in G did not differ significantly between sham and HRV-14 infections in control (p > 0.20, n = 8) and TNF--pretreated cells (p > 0.20, n = 8) throughout the experiments. However, HRV-14 infection significantly
enhanced increases in G induced by H2O2 in IL-1-pretreated
cells at intervals of 30 to 60 min after the addition of H2O2
(Figure 2).
|
Effects of an Antibody to ICAM-1 on HRV-14 Infection and G
Incubation of cells with a mouse monoclonal antibody to
ICAM-1 (84H10; 100 µg/ml) completely blocked HRV-14 infection, as assessed by the absence of detectable viral titers in the
supernatants recovered during the first 2 d after 15 min of
HRV-14 exposure (1.2 ± 0.1 log TCID50 units in control and
0 ± 0 log TCID50 units in 84H10). Likewise, viral titers collected during the first 2 d after 60 min of HRV-14 exposure were
significantly decreased by 84H10 (0.5 ± 0.2 log TCID50 units,
p < 0.01, n = 8) from control values (2.0 ± 0.1 log TCID50
units, n = 8). However, a class-matched monoclonal antibody
to HLA class 1 failed to alter viral titers in the supernatants collected during the first 2 d after 15 min of viral exposure (1.3 ± 0.2 log TCID50 units, p > 0.50, n = 8) and 60 min of viral exposure (2.2 ± 0.3 log TCID50 units, p > 0.20, n = 8). Likewise,
treatment of cells with 84H10 completely inhibited increases
in G induced by H2O2 in IL-1-pretreated cells on Day 2 after
HRV-14 infection. However, a class-matched monoclonal antibody to HLA class 1 did not alter H2O2-induced increases in G in IL-1-pretreated cells with HRV-14 infection (Figure 3). H2O2 (3 × 104 M; 60 min) caused increases in G in IL-1-pretreated cells with UV-inactivated HRV-14 infection (3.5 ± 0.4 mS/cm2) to the degree similar to those in cells with sham infection (3.6 ± 0.6 mS/cm2) (p > 0.50, n = 8).
|
Mannitol Flux Studies
Increases in transepithelial [3H]mannitol flux from mucosa to
serosa (Figure 4A) and from serosa to mucosa (Figure 4B) induced by H2O2 did not differ significantly among cells with
HRV-14 or sham infection alone, TNF--pretreated cells with
HRV-14 infection, and IL-1-pretreated cells with sham infection (p > 0.10) (Figure 4). Likewise, H2O2 caused increases in
mannitol flux from mucosa to serosa (151 ± 20 cpm/30 min)
and from serosa to mucosa (161 ± 23 cpm/30 min) in IL-1-pretreated cells with UV-inactivated HRV-14 infection to the
degree similar to those in cells with sham infection (p > 0.50, n = 7). However, H2O2 significantly increased mannitol flux
from mucosa to serosa and from serosa to mucosa in IL-1-pretreated cells with HRV-14 infection compared with cells
with sham infection (Figure 4). An antibody to ICAM-1 inhibited increases in mannitol flux from mucosa to serosa (144 ± 19 cpm/30 min) and from serosa to mucosa (155 ± 20 cpm/30
min) in IL-1-pretreated cells with HRV-14 infection to the
degree similar to those in cells with sham infection (p > 0.50, n = 7). In contrast, a class-matched monoclonal antibody to
HLA class 1 failed to alter H2O2-induced increases in mannitol flux in IL-1-pretreated cells with HRV-14 infection
(686 ± 41 cpm/30 min from mucosa to serosa and 672 ± 35 cpm/30 min from serosa to mucosa, p > 0.20, n = 7) compared
with IL-1-pretreated cells with HRV-14 infection (Figure 4).
|
When cells were exposed to HRV-14 at a concentration of
104 TCID50/ml, H2O2 alone increased G and increases in G
were significantly different at 30 min (4.6 ± 0.5 mS/cm2, p < 0.05, n = 7), at 45 min (6.9 ± 0.6 mS/cm2, p < 0.01, n = 7), and
at 60 min (9.8 ± 0.9 mS/cm2, p < 0.01, n = 7) after the addition
from the baseline G (3.0 ± 0.3 mS/cm2, n = 7). Likewise, H2O2
induced increases in mannitol flux from mucosa to serosa
(641 ± 52 cpm/30 min, p < 0.01, n = 7) and from serosa to mucosa (622 ± 49 cpm/30 min, p < 0.01, n = 7) compared with
the baseline values (71 ± 17 cpm/30 min from mucosa to serosa and 65 ± 14 cpm/30 min from serosa to mucosa, n = 7) in
cells with HRV-14 infection (104 TCID50/ml). Increases in G
at intervals of 30 to 60 min and mannitol flux from mucosa to
serosa and serosa to mucosa after the addition of H2O2 in cells
with HRV-14 infection (104 TCID50/ml) did not differ significantly from those in IL-1-pretreated cells with HRV-14 infection (103 TCID50/ml) (p > 0.20).
Immunohistochemical Analysis
Treatment with IL-1 (100 U/ml) increased ICAM-1 expression in the cultured human tracheal epithelial cells (Figure
5C). Increased ICAM-1 expression was observed in both mucosal and serosal sides. In contrast, TNF- (100 U/ml) failed
to alter the ICAM-1 expression (Figure 5D) compared with
control (Figure 5B). No significant signal was detected in the
negative control cells (Figure 5A).
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
The rhinovirus infection to human airway tissues has been reported previously in nasal polyp explants (32) and monolayer cultures of nasal epithelial cells (10), but these cultures contain other cell types such as fibroblasts. Subauste and colleagues (11) showed that HRV-14 can infect to BEAS-2B cells, which are transfected with an adenovirus 12-SV 40 hybrid virus. In this experiment, we report rhinovirus infection of primary cultures of the human tracheal epithelial cells that do not contain other cell types.
It is known that cytokines can be generated by numerous
cell types within the airways in response to a variety of stimuli and conditions. It has been shown that exposure of epithelial cells to cytokines such as IL-1 and TNF- can upregulate the expression of ICAM-1 on these cells (15). In the primary cultures of human tracheal epithelia, HRV-14 infection stimulated epithelial cell IL-1 production and IL-1 increased the
expression of ICAM-1 especially on the mucosal surface of
the epithelial cells (16). The present study showed that IL-1
increases susceptibility to infection with HRV-14, and that increased susceptibility to infection is blocked by a monoclonal
antibody to ICAM-1, consistent with its ability to increase the
expression of ICAM-1. However inhibition became less consistent at longer incubation time (e.g., 1 h), presumably because of the high affinity of the virus for its receptor and of the
requirement for very few viral particles to enter the cell to induce infection (11). In contrast, TNF- did not alter the expression of ICAM-1 in the present study. The reason for the
inability of TNF- to induce upregulation of ICAM-1 is uncertain. The ability of the cultured human tracheal epithelial cells
responding to TNF- may be low as reported previously (16).
In inflammatory airway diseases, epithelial cells have been reported to express a greater amount of adhesion molecules including ICAM-1 (1, 11, 14, 15, 33), suggesting that inflamed airways are more susceptible to rhinovirus infection compared with normal airways. In fact, human rhinoviruses can precipitate asthmatic attacks in susceptible children and these children also experience more rhinovirus colds than their nonasthmatic counterparts (1).
A consistent observation is that rhinovirus infection is not cytotoxic for human tracheal epithelial cells, in that cell viability was > 97% after infection. This is consistent with the observed lack of epithelial damage in cultured fragments of human trachea (34) and nasal polyps (32), and with the observation of no cytopathic effects on the epithelial cells in vitro (10).
The baseline values of G and mannitol flux were similar between infected and noninfected epithelial cells irrespective of IL-1 pretreatment. Furthermore, HRV-14 infection alone
had no effect on H2O2-induced increases in G. These results
are still consistent with the morphological observations described earlier. However, H2O2-induced increases in G and
mannitol flux were markedly potentiated by HRV-14 infection in epithelial cells pretreated with IL-1. Increases in G
and mannitol flux were inhibited by blockade of infection with
an antibody to ICAM-1 or by inactivation of the virus with
UV light, indicating that these phenomena were due to viral
infection and not to a contaminant present in the viral stocks.
The reason why IL-1 enhances hyperpermeability induced by oxygen radicals is uncertain. The cell membrane is a
critical site of free radical reactions for several reasons (35).
Extracellularly generated free radicals must cross the cell
membrane before reacting with other cell components. The
unsaturated fatty acids present in membranes and transmembrane proteins containing oxidizable amino acids are susceptible to free radical damage. However, biochemical changes in
the cell membrane may not explain the virus-induced exaggeration of oxygen radical-induced hyperpermeability in the epithelial cells because virus-induced effects appear to be dependent on the pretreatment with IL-1. IL-1 caused an
increased expression of ICAM-1 on epithelial cells on both
mucosal and serosal sides and increases in susceptibility to
HRV-14 infection. In contrast, TNF- failed to alter H2O2-
induced increases in G as well as both the susceptibility to
HRV-14 infection and ICAM-1 expression. Furthermore, exposure of the epithelial cells to a tenfold higher concentration
of HRV-14 mimicked the effects of IL-1 pretreatment on increases in G and mannitol flux. Therefore, increases in susceptibility to rhinoviruses and subsequent viral replication in the
cells may exaggerate oxygen radical-induced hyperpermeability in the epithelial cells pretreated with IL-1.
Infection of the epithelial cells with rhinoviruses produces cytokines (11, 16) which draw inflammatory cells into the airways, resulting in the generation of H2O2. These events lead to tissue injury and a hyperpermeability, which may lead to a greater penetration of allergen to cells involved in the allergic reaction. Because the size of most allergen particles is too large to reach to the lower airways, and because more allergen particles deposit in the trachea and large bronchi than in the distal airways (36, 37), hyperpermeability of the tracheal epithelium observed in the present study may be important for the penetration of allergen and therefore relevant to the pathogenesis in asthma.
However, it should be noted here that the in vitro model
system used in the present study may not simply reflect the
barrier function of the tracheal epithelium in vivo. This model
bypassed the need for inflammatory cell infiltration, and H2O2
contributing to tissue injury was experimentally provided. Furthermore, the concentration of H2O2 employed in the present
study (3 × 104 M) might exceed the physiological range, although the precise concentration of H2O2 in the tissue released from inflammatory cells remains unknown. Given previous demonstrations that micromolar concentrations of H2O2
can be detected in normal human serum (38), we have employed the lowest range of H2O2 concentrations that reproducibly injure epithelial cells in this model system.
In conclusion, the present study suggests that rhinovirus infection may reduce barrier functions in the airway epithelium in association with upregulation of ICAM-1 expression.
| |
Footnotes |
|---|
Correspondence and requests for reprints should be addressed to Hidetada Sasaki, M.D., Professor and Chairman, Department of Geriatric Medicine, Tohoku University School of Medicine, Aoba-ku, Seiryo-machi 1-1 Sendai 980, Japan.
(Received in original form July 29, 1996 and in revised form February 4, 1998).
| |
References |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1. Stanway, G. 1994. Rhinoviruses. In R. G. Webster and A. Granoff, editors. Encyclopedia of Virology, Vol. III. B. Academic Press, London. 1253-1259.
2. Minor, T. E., E. C. Dick, J. W. Baker, J. J. Ouellette, M. Cohen, and C. E. Reed. 1976. Rhinovirus and influenza type A infections as precipitants of asthma. Am. Rev. Respir. Dis. 113: 149-153 [Medline].
3. Lambert, H. P., and H. Stern. 1972. Infective factors in exacerbations of bronchitis and asthma. B.M.J. 3: 323-327 .
4. Gwaltney, J. M.. 1995. Rhinovirus infection of the normal human airway. Am. J. Respir. Crit. Care Med. 152(Suppl.): S36-S39 .
5.
Johnston, S. L.,
P. K. Pattemore,
G. Sanderson,
S. Smith,
F. Lampe,
L. Josephs,
P. Symington,
S. O'Toole,
S. H. Myint,
D. A. J. Tyrrell, and
S. T. Holgate.
1995.
Community study of role of viral infections in
exacerbations of asthma in 9-11 year old children.
B.M.J.
310:
1225-1229
6. Turner, B. W., W. S. Cail, J. O. Hendley, F. G. Hayden, W. J. Doyle, J. V. Sorrentino, and J. M. Gwaltney. 1992. Physiologic abnormalities of the paranasal sinuses during experimental rhinovirus colds. J. Allergy Clin. Immunol. 90: 474-468 [Medline].
7.
Gwaltney, J. M.,
C. D. Phillips,
R. D. Miller, and
D. K. Riker.
1994.
Computed tomographic study of the common cold.
N. Engl. J. Med.
330:
25-30
8. Hers, J. F.. 1996. Disturbance of ciliated epithelium due to influenza virus. Am. Rev. Respir. Dis. 93: 162-171 .
9. Carson, J. L., A. M. Collier, S. Shin, and -chin, and M. S. Hu. 1985. Acquired ciliary defects in nasal epithelium of children with acute viral upper respiratory infections. N. Engl. J. Med. 31: 463-468 .
10. Winther, B., J. M. Gwaltney, and J. O. Hendley. 1990. Respiratory virus infection of monolayer cultures of human nasal epithelial cells. Am. Rev. Respir. Dis. 141: 839-845 [Medline].
11. Subauste, M. C., D. B. Jacoby, S. M. Richards, and D. Proud. 1995. Infection of a human respiratory epithelial cell line with rhinovirus: induction of cytokine release and modulation of susceptibility to infections by cytokine exposure. J. Clin. Invest. 96: 549-557 .
12. Greve, J. M., G. Davis, A. M. Meyer, C. P. Forte, S. C. Yost, C. W. Marlor, M. E. Kamarck, and A. McClelland. 1989. The major human rhinovirus receptor is ICAM-1. Cell 56: 839-847 [Medline].
13. Staunton, D. E., V. J. Merluzzi, R. Rothlein, R. Barton, S. D. Marlin, and T. A. Springer. 1989. A cell adhesion molecule ICAM-1, is the major surface receptor for rhinoviruses. Cell 56: 849-853 [Medline].
14.
Wegner, C. D.,
R. H. Gundel,
P. Reilly,
N. Haynes,
L. G. Letts, and
R. Rothlein.
1990.
Intercellular adhesion molecule-1 (ICAM-1) in the
pathogenesis of asthma.
Science
247:
456-459
15. Tosi, M. F., J. M. Stark, C. W. Smith, A. Hamedani, D. C. Gruenert, and M. D. Infeld. 1992. Induction of ICAM-1 expression on human airway epithelial cells by inflammatory cytokines: effects on neutrophil-epithelial cell adhesion. Am. J. Respir. Cell Mol. Biol. 7: 214-221 .
16.
Terajima, M.,
M. Yamaya,
K. Sekizawa,
S. Okinaga,
T. Suzuki,
N. Yamada,
K. Nakayama,
T. Ohrui,
T. Oshima,
Y. Numazaki, and
H. Sasaki.
1997.
Rhinovirus infection of primary cultures of human tracheal epithelium: role of ICAM-1 and IL-1.
Am. J. Physiol.
273:
L749-L759
17. Farber, J. L., M. E. Kyle, and J. B. Coleman. 1990. Biology of disease: mechanisms of cell injury by activated oxygen species. Lab. Invest. 62: 670-679 [Medline].
18. Sanders, S. P., J. L. Zweier, S. J. Harrison, M. A. Trush, S. J. Rembish, and M. C. Liu. 1995. Spontaneous oxygen radical production at sites of antigen challenge in allergic subjects. Am. J. Respir. Crit. Care Med. 151: 1725-1733 [Abstract].
19. Johnston, S. L.. 1995. Natural and experimental rhinovirus infections of the lower respiratory tract. Am. J. Respir. Crit. Care Med. 152(Suppl.): S46-S52 .
20. Welsh, M. J., D. M. Shasby, and R. M. Husted. 1985. Oxidants increase paracellular permeability in cultured epithelial cell line. J. Clin. Invest. 76: 1155-1168 .
21.
Yamaya, M.,
K. Sekizawa,
T. Masuda,
M. Morikawa,
T. Sawai, and
H. Sasaki.
1995.
Oxidants affect permeability and repair of the cultured
human tracheal epithelium.
Am. J. Physiol.
268:
L284-L293
22.
Yamaya, M.,
W. E. Finkbeiner,
S. Y. Chun, and
J. H. Widdicombe.
1992.
Differentiated structure and function of cultures from human tracheal
epithelium.
Am. J. Physiol.
262:
L713-L724
23. Numazaki, Y., T. Oshima, A. Ohmi, A. Tanaka, Y. Oizumi, S. Komatsu, T. Takagi, M. Karahashi, and N. Ishida. 1987. A microplate method for isolation of viruses from infants and children with acute respiratory infections. Microbiol. Immunol. 31: 1085-1095 [Medline].
24. Amador, E., L. E. Dorfman, and W. E. C. Wacker. 1963. Serum lactic dehydrogenase activity: an analytical assessment of current assays. Clin. Chem. 9: 391-399 [Abstract].
25. Makgoba, M. W., M. E. Sanders, G. E. Ginther-Luce, M. L. Dustin, T. A. Springer, E. A. Clark, P. Mannoni, and S. Shaw. 1988. ICAM-1 a ligand for LFA-1-dependent adhesion of B, T and myeloid cells. Nature 331: 86-88 [Medline].
26. Hughes, J. H., M. Mitchell, and V. V. Hamparian. 1979. Rhinoviruses: kinetics of ultraviolet inactivation and effects of UV and heat on immunogenicity. Arch. Virol. 61: 313-319 [Medline].
27.
Cooper, J. A.,
P. J. Del Vecchio,
F. L. Minner,
K. E. Burhop,
W. M. Selig,
J. G. N. Garcia, and
A. B. Malik.
1987.
Measurement of albumin
permeability across endothelial monolayers in vitro.
J. Appl. Physiol.
62:
1076-1083
28.
Kondo, M.,
W. E. Finkbeiner, and
J. H. Widdicombe.
1992.
Changes in
permeability of dog tracheal epithelium in response to hydrostatic
pressure.
Am. J. Physiol.
262:
L176-L182
29. Ohkawara, Y., K. Yamauchi, N. Maruyama, H. Hoshi, I. Ohno, M. Honma, Y. Tanno, G. Tamura, K. Shirato, and H. Ohtani. 1995. In situ expression of the cell adhesion molecules in bronchial tissues from asthmatics with air flow limitation: in vivo evidence of VCAM-1/ VLA-4 interaction in selective eosinophil infiltration. Am. J. Respir. Cell Mol. Biol. 12: 4-12 [Abstract].
30.
Gruenert, D. C.,
W. E. Finkbeiner, and
J. H. Widdicombe.
1995.
Culture
and transformation of human airway epithelial cells.
Am. J. Physiol.
268:
L347-L360
31. Cordell, J. L., B. Falini, and W. N. Erber. 1984. Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J. Histochem. Cytochem. 32: 219-229 [Abstract].
32. Hamory, B. H., J. O. Hendley, and J. M. Gwaltney. 1977. Rhinovirus growth in nasal polyp organ culture. Proc. Soc. Exp. Biol. Med. 155: 577-582 [Medline].
33. Pilewski, J. M., and S. M. Albelda. 1995. Cell adhesion molecules in asthma: homing, activation, and airway remodeling. Am. J. Respir. Cell Mol. Biol. 12: 1-3 [Medline].
34. Hoorn, B., and D. A. J. Tyrrell. 1966. Effects of some viruses on ciliated cells. Am. Rev. Respir. Dis. 93: 156-161 .
35. Freeman, B. A., and J. D. Crapo. 1982. Biology of disease: free radicals and tissue injury. Lab. Invest. 47: 412-426 [Medline].
36. Busse, W. W., C. E. Reed, and J. H. Hoehne. 1972. Where is the allergic reaction in ragweed asthma? II. Demonstration of ragweed antigen in airborne particles smaller than pollen. J. Allergy Clin. Immunol. 50: 289-293 [Medline].
37. Michel, F. B., J. P. Marty, L. Quet, and P. Cour. 1977. Penetration of inhaled pollen into the respiratory tract. Am. Rev. Respir. Dis. 115: 609-616 [Medline].
38. Varma, S. D., and P. S. Devamanoharan. 1991. Hydrogen peroxide in human blood. Free Rad. Res. Comm. 14: 125-131 [Medline].
This article has been cited by other articles:
 |
|
 |
|
  T. Suzuki, M. Yamaya, M. Kamanaka, Y. X. Jia, K. Nakayama, M. Hosoda, N. Yamada, H. Nishimura, K. Sekizawa, and H. Sasaki Type 2 rhinovirus infection of cultured human tracheal epithelial cells: role of LDL receptor Am J Physiol Lung Cell Mol Physiol, March 1, 2001; 280(3): L409 - L420. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Yamaya, K. Sekizawa, T. Suzuki, N. Yamada, M. Furukawa, S. Ishizuka, K. Nakayama, M. Terajima, Y. Numazaki, and H. Sasaki Infection of human respiratory submucosal glands with rhinovirus: effects on cytokine and ICAM-1 production Am J Physiol Lung Cell Mol Physiol, August 1, 1999; 277(2): L362 - L371. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Proc. Am. Thorac. Soc. | Am. J. Respir. Cell Mol. Biol. |