IL-4 induces ICAM-1 expression in human bronchial epithelial
cells and potentiates TNF-
Ilja
Striz1,
Tadashi
Mio1,
Yuichi
Adachi1,
Peggy
Heires1,
Richard A.
Robbins2,
John R.
Spurzem2,
Mary J.
Illig1,
Debra J.
Romberger2, and
Stephen I.
Rennard1
1 Pulmonary and Critical Care
Medicine Section, Department of Internal Medicine, University of
Nebraska Medical Center, Omaha 68198-5300; and
2 Veterans Affairs Hospital,
Omaha, Nebraska 68105
 |
ABSTRACT |
Interleukin (IL)-4 is thought to contribute to the
Th2 type of immune response and hence the development of allergic
reactions such as asthma. In asthmatic patients, the airway epithelium
expresses increased amounts of the cell surface adhesion molecule
intercellular adhesion molecule (ICAM)-1 (CD54). One cytokine capable
of inducing ICAM-1 in airway epithelial cells, tumor necrosis
factor-
(TNF-
), is present in asthma. This study evaluated if
IL-4 either alone or together with TNF-
costimulation might modulate
CD54 expression by human bronchial epithelial cells (HBECs). CD54
positivity increased in response to IL-4 (16 ± 2% positive vs. 3 ± 1%, P < 0.01); greater induction of CD54 resulted from TNF-
(45 ± 2%,
P < 0.001). Costimulation with
TNF-
plus IL-4 further augmented expression (56 ± 1%,
P < 0.05). Immunoperoxidase results
were confirmed by flow cytometry. RT-PCR revealed no increase in ICAM-1
mRNA expression under control conditions or after stimulation with IL-4
alone. TNF-
increased IL-4 mRNA, and IL-4 potentiated this.
Functionally, IL-4 augmented the adhesion of THP-1 monocyte/macrophage
cells to monolayers of HBECs both alone and in the presence of TNF-
.
We conclude that 1) IL-4 augments
epithelial cell ICAM-1 expression,
2) IL-4 potentiates the adhesion of
THP-1 monocyte/macrophage cells to epithelial cells, and
3) modulation of epithelial cell
ICAM-1 expression by IL-4 may play a role in the immunopathology of
bronchial asthma.
interleukin-4; interleukin-10; interleukin-13; intercellular
adhesion molecule-1; tumor necrosis factor-
 |
INTRODUCTION |
THE EXPRESSION OF intercellular adhesion molecule-1
(ICAM-1, CD54) on the membrane of bronchial epithelial cells is thought to be involved in leukocyte trafficking and activation during bronchial
inflammation (7). Previous studies have shown upregulation of ICAM-1
molecules on the bronchial epithelium of asthmatics in comparison with
that in normal subjects (6, 46). Because blockade of ICAM-1 can
attenuate asthmatic responses (47), the increased ICAM-1 in asthma
appears to have an important role in asthma pathogenesis. Inflammatory
cytokine production by alveolar macrophages (29), T lymphocytes (2), or
mast cells (9) may be one mechanism by which the increased expression
of ICAM-1 in asthma may occur. In vitro, several cytokines, including
tumor necrosis factor (TNF)-
, which is reported to be increased in asthma (10, 19), have been demonstrated to upregulate ICAM-1 expression
by the airway epithelium (1).
In asthma, Th2 lymphocytes are prominent in the airways and are thought
to drive the immunopathology of the disease. In this regard,
interleukin (IL)-4 hyperproduction is a prominent feature in the
cytokine profile of atopic asthmatic subjects (37). This multifunctional cytokine can drive the development of Th2 cells (39)
and inhibit Th1-type effector functions (34). It can also function as a
switch factor for IgG1 and IgE production by B cells (35). Regarding
ICAM-1 expression, a stimulatory effect of IL-4 has been found in mast
cells (43) and fibroblasts (36), but minimal effects have been reported
on endothelial cells (44).
The purpose of the present study was to evaluate the hypothesis that
IL-4 could modulate ICAM-1 expression of human bronchial epithelial
cells (HBECs) either directly or by modulating the responsiveness of
the cells to TNF-
. In addition, the activity of IL-4 was compared
with the related Th2-derived cytokines IL-10 and IL-13.
 |
METHODS |
Preparation of HBECs. HBECs were
obtained from a donor undergoing bronchoscopy for evaluation of a lung
mass with a modification of the method of Kelsen et al. (24). Informed
consent was obtained in agreement with a protocol approved by the
Institutional Review Board for the Protection of Human Subjects at the
University of Nebraska Medical Center. After premedication with
meperidine and atropine and local anesthesia with lidocaine, the
flexible fiber-optic bronchoscope (Olympus model T or equivalent) was
introduced into a proximal main stem or lobar bronchus. The brushing
was performed in an area of normal-appearing mucosa under direct visual
guidance. The brush was then retracted, and dissociated cells were
recovered by vortexing the brush in ice-cold MEM for several seconds.
The harvested cell suspension was filtered through a 100-µm Nitex filter (Tetko, Elmsford, NY) to remove mucus and then was treated for
10 min with 50 µg/ml DNase to eliminate clumping. Finally, the cell
pellet was resuspended in culture medium, cell number was determined
using a hemocytometer, and cell viability was assessed by trypan blue
exclusion. For most of the experiments, cells from a single donor were
used. Cells were also prepared from a second individual with a
modification of the method of Lee et al. (26) using tissue harvested at
rapid autopsy (25).
Cell culture techniques. Cells were
cultured under serum-free conditions as described in detail previously
(5) using a 1:1 mixture of RPMI 1640 medium (GIBCO BRL, Grand Island,
NY) and LHC-9 (prepared from LHC basal; Biofluids, Rockville, MD). Bovine pituitary extract for supplementation of LHC-9 was prepared as
described (8). Cells were plated on Vitrogen 100 (Collagen, Palo Alto,
CA)-coated tissue culture dishes at 37°C in a humidified atmosphere
of 5% CO2, and culture media were
changed every 1-3 days as indicated. Second- or third-passage
cells were harvested by brief trypsinization (0.25% trypsin-EDTA;
GIBCO BRL), washed two times in Hanks' balanced salt solution (HBSS),
and stored in liquid nitrogen until used. Frozen cells were gently
thawed at 37°C, resuspended in LHC-9-RPMI 1640 medium, and directly
plated in LHC9-RPMI 1640 medium as described above. Cells were then
grown as needed, taking care to passage cells before they reached dense confluency. Cells from passages 4-7 were used for
experiments after the viability (>97%) was checked; purity of
cultures was routinely assessed with anti-human cytokeratin antibody
(MAK-6; Triton, Alameda, CA) staining, which is highly specific for
cells of epithelial origin. At the time of initial
isolation, >95 of cells stained positively for cytokines.
This technique of cell preparation permits the routine reevaluation of
identical cells. Because there is some variability among strains of
cells prepared from different individuals, the majority of experiments
performed in the current study were done using a single strain of cells
isolated from a normal-appearing airway of an individual with carcinoma
of the lung. For confirmation purposes, selected experiments were
performed with additional cell strains as indicated.
Cytokine stimulation. Cells were
allowed to grow to near confluence after which the culture medium
LHC9-RPMI 1640 was removed and changed to growth factor-depleted
LHC9-RPMI 1640 medium (LHCD-RPMI) to exclude the effect of supplements.
Cytokines were dissolved in LHCD-RPMI. Cytokines used included IL-4
(Genzyme, Cambridge, MA), IL-10 and IL-13 (both PeproTech, Rocky Hill,
NJ), and TNF-
(R&D Systems, Minneapolis, MN). After a 24-h exposure
to cytokines, cells were harvested by a method that had proved not to
affect immunoperoxidase staining for ICAM-1 expression. Briefly, cells were washed one time with LHCD-RPMI and briefly exposed to 0.25% trypsin-EDTA (GIBCO BRL). After cell detachment, which could be observed visually, the cells were harvested, and trypsin was inhibited by a 0.02% soybean trypsin inhibitor (GIBCO BRL). Cells were then washed in LHCD-RPMI and used for immunophenotypic analysis.
Immunoperoxidase staining of ICAM-1.
Immunocytochemical analysis for the determination of the percentage of
CD54- positive cells was done with an immunoperoxidase slide assay as
described in detail (12) with minor modification. Briefly, 10 µl of
cell suspensions (2 × 105
cells/ml) were transferred to reaction areas of adhesive slides (Bio-Rad), and after attachment, cells were fixed with 0.05%
glutaraldehyde. Cells were then washed with 8 g of NaCl, 0.4 g of KCl,
and 1 M HEPES, pH 7.4, in 1,000 ml of distilled water (NKH buffer) and were preincubated with gelatin-blocking medium to prevent Ig binding to
the glass surface. The staining procedure included the following steps:
1) 5-min incubation with anti-CD54
monoclonal antibody 84H10 (Amac) diluted 1:50 in gelatin-blocking
medium; 2) 5-min incubation with
rabbit anti-mouse Ig (Dako); 3)
5-min incubation with swine anti-rabbit Ig (Dako);
4) 5-min incubation with
peroxidase-anti-peroxidase complex from rabbit (Dako; all antibodies
were diluted 1:30 in NKH buffer; and
5) incubation with
aminoethylcarbazole (Sigma) for 10 min. To evaluate the reaction, the
slides were viewed under a light microscope, and a positive reaction
was denoted by the presence of red, granular staining of the cell
membrane. As an isotype-identical control, monoclonal antibody IOM2
(Amac) was used; the antibody showed a negative reaction with HBECs and
a strongly positive reaction with peripheral blood monocytes.
Quantitative indirect immunofluorescence
analysis. For a quantitative evaluation of ICAM-1
density on the membrane of stimulated HBECs, flow cytometry was used as
previously described (14). HBECs obtained by gentle trypsinization were
washed in PBS containing 0.02 mM sodium azide and 1% BSA and were
incubated with a monoclonal antibody for human ICAM-1 diluted 1:50 in
PBS for 30 min on ice. After two washes, the cells were incubated with
FITC-labeled goat anti-mouse antibody (Becton Dickinson), 1:50 in PBS,
for 30 min on ice. After two additional washes, cells were resuspended
in formaldehyde-containing fixative medium and were stored at 4°C in the dark until flow cytometry analysis by fluorescence-activated cell sorter (Becton Dickinson, Sunnyvale, CA).
mRNA analysis. Total cellular RNA was
extracted from adherent HBECs cultured in six-well culture plates
(Falcon, Lincoln Park, NJ) using a modification of the method of
Chomczynski and Sacchi (11). After the cells were washed with HBSS,
they were solubilized in 4 M guanidinium thiocyanate, 25 mM sodium
citrate, 0.5 sarcosyl, and 0.1 M 2-mercaptoethanol. The resulting cell
lysate was acidified with sodium acetate, pH 4.0, and extracted with
phenol and chloroform-isopentyl alcohol. After precipitation with
isopropanol, the resulting pellet was washed with ethanol, dissolved in
0.5% SDS, and stored at
70°C until use.
RT-PCR of ICAM-1 mRNA. Total cellular
RNA (1 µg) was denatured at 95°C for 5 min and incubated at
42°C for 60 min in 20 µl of mixture consisting of 50 mM KCl, 10 mM Tris · HCl (pH 8.3), 2.5 mM
MgCl2, 1 U/µl RNasin (RNA-GUARD;
Pharmacia, Piscataway, NJ), 100 pM random hexamer (Pharmacia), 1 mM
each dATP, dCTP, dGTP, and dTTP (Perkin-Elmer, Norwalk, CT), and 200 units Superscript RT enzyme (GIBCO BRL). For each reaction mixture, 2 µl of the RT product were added to PCR buffer (1.5 mM
MgCl2, 50 mM KCl, 10 mM
Tris · HCl, pH 8.3, and 0.01% gelatin; Perkin-Elmer)
along with 1 mM each primer, 1 mM each dATP, dCTP, dGTP, and dTTP
(Perkin-Elmer), and 2.5 units AmpliTaq polymerase
(Perkin-Elmer). Oligonucleotide primers used were the human ICAM-1 PCR
primer pair purchased from R&D Systems. The reaction mixture was
overlaid with paraffin oil, and the DNA sequence was amplified in a
thermal cycler (MJ Research, Watertown, MA). Thirty cycles were
performed with initial denaturation at 94°C for 4 min, followed by
cycles of denaturing at 94°C for 45 s, annealing at 55°C for 45 s, and extension at 72°C for 45 s, with a final extension at
72°C for 10 min. The amplification products were separated
electrophoretically on a 1.5% 3:1 NuSieve agarose gel and were
visualized with ethidium bromide staining.
For comparison, PCR was also performed with primers for
glyceraldehyde-3-phosphate dehydrogenase (Stratagene, La Jolla, CA).
Macrophage attachment assay. After
reaching confluency, HBECs cultured in 96-well tissue culture plates
(Falcon) were exposed to cytokines for 24 h. Cells were then washed and
cocultured for 15 min with 0.3 × 106 THP-1 monocyte/macrophage
cells (American Type Culture Collection, Manassas, VA) per well. These
cells had been cultured in RPMI 1640 medium supplemented with 10% FCS
and 5 × 10
5 M
2-mercaptoethanol. Before attachment assay, THP-1 cells were labeled
with the fluorescent dye
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF;
Molecular Probes, Eugene, OR; see Ref. 13). After the incubation, the
top of the plate was hermetically covered, the plate was gently
inverted, and nonadherent cells were sedimented for 5 min. All wells
were then gently washed with MEM and HBSS (phenol red free). Attached
cells were solubilized with 0.3% Triton in distilled water. The cell
lysate was then transferred to a 96-well Microfluor plate (Dynatech,
Chantilly, VT) and evaluated by an automatic microfluorometer
Fluoroscan (Perkin-Elmer) at 485/535-nm excitation/emission wavelengths.
Statistics. Nonparametric statistics,
the Kruskal-Wallis and Mann-Whitney tests, were used to compare groups
and paired data, respectively. Spearman's rank correlations were used
for the statistical analysis of differences in the membrane expression
of CD54 on cytokine-stimulated cells and for the changes in the
attachment rate of THP-1 cells to HBEC monolayers.
P values below 0.05 were considered significant.
 |
RESULTS |
Modulation of HBEC ICAM-1 expression by
IL-4. IL-4 increased the percentage of HBECs that
expressed ICAM-1 in a concentration-dependent manner (Fig.
1). At the maximum dose tested, ~40% of
HBECs expressed ICAM-1, a percentage similar to that induced by 5 ng/ml
TNF-
. When IL-4 was added in the presence of 5 ng/ml TNF-
, it
further augmented HBEC ICAM-1 expression (Fig. 1). For a comparison, we also evaluated IL-4 together with single concentrations of other Th2-related cytokines, IL-13 and IL-10, to induce ICAM-1 both alone and
in the presence of TNF-
(Table 1).
Although there is more variability between experiments than within an
experiment, results are consistent (Table 1). IL-13 has been shown to
stimulate ICAM-1 expression by HBECs similarly to IL-4; however, it
does not potentiate the effect of TNF-
in these cells.

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Fig. 1.
Concentration-dependent effect of interleukin (IL)-4 on intercellular
adhesion molecule (ICAM)-1 expression on human bronchial epithelial
cells (HBECs). HBECs were cultured in 6-well tissue culture plates
until reaching confluency and then were stimulated with increasing
concentrations of IL-4 (0.1-20 ng/ml) in the presence or absence
of tumor necrosis factor- (TNF- ; 5 ng/ml). After 24 h, cells were
recovered by a gentle trypsinization and were stained for ICAM-1
expression by the immunoperoxidase slide assay. IL-4 increased CD54
expression in a concentration-dependent manner, reaching a peak at 5 ng/ml. The effect of IL-4 in combination with TNF- on the induction
of CD54 showed a similar concentration dependence. LHCD, growth
factor-depleted LHC9.
|
|
To confirm the results obtained by visual scoring, flow cytometric
analysis was performed and yielded results similar to those obtained by
direct counting (Fig. 2). Compared with
unstimulated HBECs (Fig. 2A), cells
cultured in the presence of IL-4 (Fig. 2B) and TNF-
(Fig.
2C) demonstrated increased staining.
The combination of IL-4 and TNF-
further augmented staining (Fig.
2D). The quantitative data are
expressed in Fig. 3; both the mean
fluorescence intensity and the median value (in parentheses) increased
from 49 (21) for unstimulated cells to 93 (34) for IL-4-, to 297 (195)
for TNF-
-, and to 489 (308) for IL-4- and TNF-
-stimulated cells.

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Fig. 2.
Flow cytometric analysis of CD54 expression on HBECs after cytokine
stimulation. HBECs were cultured for 24 h in the presence of IL-4 or
TNF- or costimulated with IL-4 plus TNF- (all cytokines in 10 ng/ml concentration). Cells were then detached by trypsinization and
stained for CD54 using an indirect immunofluorescence assay with the
monoclonal antibody 84H10 and FITC-labeled rabbit anti-mouse second
antibody. Samples were then evaluated by fluorescence-activated cell
sorter (FACS) scan calibrated for counting of epithelial cells. Flow
cytometry showed low basal expression of CD54
(A) and a slight increase in
fluorescence intensity in samples stimulated with IL-4
(B). TNF- stimulation resulted in
significant enhancement of CD54 staining
(C), and the highest level of
immunofluorescence was reached by HBECs stimulated with both IL-4 and
TNF- (D).
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Fig. 3.
Quantitative immunofluorescence analysis of CD54 expression by HBECs
stimulated by cytokines. Confluent HBECs were exposed to IL-4, TNF- ,
and a combination of TNF- plus IL-4 for 24 h. Cells were
then gently trypsinized, washed, and stained for CD54 by indirect
immunofluorescence using the monoclonal antibody 84H10 to ICAM-1
(CD54). Staining of HBECs was determined by FACScan. Stimulation with
IL-4 showed only a moderate increase in the mean fluorescence
and median values for CD54 immunostaining, whereas TNF- induced a
more marked expression of CD54. Costimulation with TNF- plus IL-4
resulted in augmented expression of CD54 on the surface of HBECs. Data
represent a single representative experiment designed to confirm
results obtained by the immunoperoxidase assay and show a similar
pattern of CD54 induction.
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ICAM-1 mRNA expression in IL-4- and
TNF-
-stimulated HBECs. Using the more
sensitive method of RT-PCR, constitutive levels of ICAM-1 mRNA were
demonstrable, and no increase was observed after IL-4 exposure (Fig.
4). TNF-
exposure induced readily
detectable ICAM-1 mRNA, and IL-4 augmented this.

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Fig. 4.
ICAM-1 mRNA expression by RT-PCR in HBECs after exposure to TNF- and
IL-4 separately and in combination. HBECs were cultured in the absence
or presence of IL-4 (10 ng/ml), TNF- (10 ng/ml), or both cytokines
for 8 h. mRNA was extracted, and RT-PCR was performed as described in
METHODS. Lane 1, negative
control (PCR done without cDNA template); lane
2, unstimulated HBECs; lane
3, IL-4-stimulated cells; lane
4, TNF- -stimulated cells; lane
5, IL-4- and TNF- -stimulated cells;
lane 6, kb size markers (nos. on
right). PCR for glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was done separately because of proximity of bands; however, the
same RT product and PCR cycling utilized for ICAM-1 PCR were used.
|
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Attachment of THP-1 monocytes/macrophages to
stimulated HBECs. To assess if the increased ICAM-1
expression was associated with altered function of the HBECs, binding
of the monocyte/macrophage cell line THP-1 was measured. Binding of
BCECF-labeled THP-1 cells to HBECs (Fig. 5)
increased significantly when the epithelial monolayer was preincubated
with IL-4 (146 ± 1% of control,
P = 0.03) or TNF-
(152 ± 1%,
P = 0.01). The combination of both
cytokines further increased attachment of THP-1 cells (202 ± 10%
of control, P = 0.012). The absolute
values of fluorescence intensity are presented in Fig. 5. The
anti-ICAM-1 antibody 84H10 partially inhibited THP-1 binding under all
conditions (Fig. 6).

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Fig. 5.
Attachment of THP-1 cells (monocytes/macrophages) to stimulated HBECs.
HBECs were cultured in 96-well tissue culture plates until reaching
confluency after which they were stimulated with IL-4 (10 ng/ml),
TNF- (10 ng/ml), or both cytokines for 24 h. Cells were then washed
and incubated for 15 min together with THP-1 cells previously labeled
with the fluorescent dye
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF).
HBEC monolayers were then rinsed to remove unattached cells, the
attached cells were solubilized with 3% Triton, and the fluorescence
intensity of the resulting lysate reflecting number of attached THP-1
cells was evaluated by microfluorometer. IL-4 and TNF- stimulated
THP-1 binding to HBECs, and the combination of both cytokines further
augmented attachment.
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Fig. 6.
Inhibition of THP-1 attachment to stimulated HBECs by anti-ICAM-1
antibody. HBECs were stimulated by IL-4 (10 ng/ml), TNF- (10 ng/ml),
and both cytokines for 24 h in a 96-well tissue culture plate. Some
cells from each condition were preincubated for 10 min with anti-ICAM-1
monoclonal antibody 84H10 (5 µg/ml), whereas others remained in
serum-free medium LHCD-RPMI 1640. Monolayers of HBECs were then
cocultured for 15 min with THP-1 monocytes/macrophages prelabeled by
BCECF fluorescence dye. THP-1 attachment to HBECs was evaluated by
measurement of the mean fluorescence of the cell lysate of washed
monolayers using microfluorometer. The rate of inhibition of THP-1
attachment by anti-ICAM-1 antibody is expressed as a percentage
decrease of mean fluorescence measured in each condition. Blocking by
anti-ICAM-1 antibody resulted in a partial inhibition of THP-1 binding
to HBECs (~20-30% of total attachment) in all conditions
studied.
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Effects of other Th2 cytokines. At a
concentration of 10 ng/ml, at which the effect of IL-4 was maximal,
IL-13 stimulated ICAM-1 expression slightly and IL-10 was without
effect (Table 1). Neither IL-10 nor IL-13, however, altered the
TNF-
-induced expression of ICAM-1. Thus the effects of IL-4 differed
markedly from those of IL-10 and IL-13.
 |
DISCUSSION |
In addition to airway hyperreactivity, asthma is characterized by the
presence of activated immune cells in the airway epithelium (4, 21).
Lymphocytes producing cytokines IL-4, IL-5, and IL-13, called Th2
according to a previously described murine nomenclature, are thought to
play a particularly important role (2). These cells are prominent in
the asthmatic airway epithelium, and cytokines released by these cells
can drive both eosinophil accumulation and B-cell production of IgE.
The epithelial cells that line the airways are also involved in the
immune response, both producing cytokines (38) and expressing increased
amounts of cell surface adhesion receptors for inflammatory cells (18,
23, 41, 46, 47). The present study suggests that Th2 cells, through the production of IL-4, may also drive this aspect of the immune response in asthma.
Increased expression of the cell surface adhesion receptor ICAM-1 has
been demonstrated by immunohistochemistry in asthma (18, 46). This
increased expression is accompanied by increased mRNA expression as
demonstrated by in situ hybridization. Several mediators have been
reported to increase ICAM-1 expression in in vitro cultures of human
airway epithelial cells, including histamine (45) and the Th1- and
inflammatory-derived cell cytokines TNF-
, IL-1
, and
interferon-
(30). Although histamine may play a role in asthma, Th1
cells are generally felt to be lacking in asthma and to have the
potential to suppress an asthmatic Th2-type response. The present study
demonstrates that Th2 cytokines can also upregulate epithelial cell
ICAM-1. Moreover, IL-4 and TNF-
, a cytokine released by Th2 as well
as Th1 cells, can interact cooperatively to further increase ICAM-1
expression. Activation of Th2 lymphocytes in the airway epithelium,
therefore, may drive not only the eosinophil and the B-cell response
but may also contribute to the epithelial changes characteristic of
asthma. We have derived our data using cells from nonatopic and
nonasthmatic subjects; nevertheless, further studies with cells from
airway brushings of patients with bronchial asthma would be of
potential interest. Such studies may reveal either an acquired or a
genetically based difference in cytokine induction of ICAM-1 by
epithelial cells.
Under control conditions, no detectable ICAM-1 mRNA was detected in the
present study, presumably because levels were below the limit of
detectability. This result agrees with both the in vitro studies of
Look et al. (30), who were also unable to detect ICAM-1 mRNA, and the
in situ hybridization studies of normal airway epithelium of Vignola et
al. (46), who detected no ICAM-1 mRNA. Despite the inability to detect
mRNA, faintly detectable surface expression of ICAM-1 was observed in a
small percentage of control cells. Although IL-4 increased surface
expression, mRNA was not increased. TNF-
, in contrast, markedly
increased ICAM-1 mRNA. IL-4 together with TNF-
potentiated the
increased ICAM-1 mRNA expression in parallel with its effect on surface
molecule expression.
The IL-4 and IL-13 genes share a common intron-exon structure on
chromosome 5q, and the degree of homology between IL-4 and IL-13
protein sequences is ~30% (48). IL-13 shares many of the known
activities of IL-4 on monocytes. Both cytokines induce morphological transformation of monocytes into cells of dendritic appearance (15),
downregulate CD14 and Fc
receptors, and upregulate the
expression of class II major histocompatibility (MHC) antigens (16).
Both cytokines inhibit proinflammatory cytokine release (20, 32),
nitric oxide production (28), and killing of intracellular parasites
(40). IL-4 and IL-13 also have similar activities on B cells, inducing
their proliferation, differentiation, and Ig switching to the synthesis
of IgE (3). However, IL-13 fails to activate T cells in contrast to the
growth-promoting effect of IL-4 (48). Our data suggest another unique
activity of IL-4 compared with that of IL-13, since IL-4 augments
TNF-
-induced expression of ICAM-1 by HBECs, but IL-13 does not. It
remains possible, of course, that IL-13 may exert such effects but may be much less potent in this regard than IL-4.
IL-10 is another Th2-derived cytokine with some effects that resemble
IL-4. IL-10 is a potent immunosuppressant of macrophage function,
although it augments both the proliferation and differentiation of B
cells (33). Whereas there are several IL-4-like effects of IL-10, this
cytokine has been found to inhibit the upregulation of class II MHC
molecules on monocytes induced by IL-4 (17). Also, the effects of IL-10
on NK cells differ from those of IL-4 in that IL-4 inhibits their
activity, whereas IL-10 enhances their interferon production and
lymphokine-activated killer activity (22). We found a very mild
inhibitory effect of IL-10 on ICAM-1 induction by HBECs in contrast to
a clear stimulatory effect of IL-4.
Adhesion of human leukocytes to the airway epithelium is mediated by
both ICAM-1-dependent and ICAM-1-independent mechanisms (42).
Upregulation of ICAM-1 surface expression was accompanied by increased
binding of THP-1 cells, suggesting that increased expression altered
the functional activity of the HBECs. However, anti-ICAM-1 antibody
caused only partial inhibition of THP-1 monocytes/macrophages attachment to HBECs in both unstimulated and cytokine-induced cells.
Our incomplete inhibition of THP-1 binding with anti-ICAM-1 antibody is
in agreement with similar blocking experiments of others (42) and
suggests multiligand-mediated interactions between leukocytes and
epithelial cells. Thus we cannot exclude that, in addition to
IL-4-mediated increase of ICAM-1 expression, this cytokine also induces
increased expression of other adhesion receptors on the epithelial cell surface.
Our results in which IL-4 and TNF-
interacted to increase THP-1
binding are also consistent with the animal model studies of Leung et
al. (27). In these studies, IL-4 and TNF-
interacted to increase
both cell surface adhesion receptor expression and differentiation of a
myeloid cell line. Although the functional significance of epithelial
cell ICAM-1 expression in asthma is unknown, some evidence suggests
that it may play a pathogenetic role and therefore may be a therapeutic
target. Specifically, ICAM-1 blockade by specific antibodies has been
shown to attenuate the symptoms of experimental bronchial
hypersensitivity induced in primates (47). A possible link between
cytokines derived from Th2 cells and the induction of adhesion
molecules on the airway epithelium suggests an alternative strategy to
affect epithelial cell ICAM-1 expression therapeutically. In this
regard, anti-IL-4 therapy using soluble IL-4 receptors has been
recently tested in animal models of inflammation (31).
In summary, IL-4 stimulates HBEC expression of ICAM-1 (CD54). IL-4 also
potentiates the induction of ICAM-1 induced by TNF-
. Of the
IL-4-related cytokines, IL-13 also induced ICAM-1 expression, whereas
IL-10 lacked this activity, and neither potentiated TNF-
-induced ICAM-1 expression. The ability of Th2-derived cytokines to induce the
expression of ICAM-1 on airway epithelial cells can lead to increased
adhesion of inflammatory cells. This pathway may contribute to the
pathogenesis of bronchial asthma, a condition in which both Th2-derived
cytokines and TNF-
are present in high concentrations.
 |
ACKNOWLEDGEMENTS |
We are grateful to K. Nelson, A. Heires, R. Ertl, and L. Tate for
technical assistance and to L. Richards for editing the manuscript. We
also thank the Department of Anesthesiology of the University of
Nebraska Medical Center for providing the microfluorometer Fluoroscan
for the evaluation of attachment assays.
 |
FOOTNOTES |
This work was supported by Fogarty International Research Fellowship
F05 TW04895-01.
Address for reprint requests and other correspondence: S. I. Rennard,
Univ. of Nebraska Medical Center, 985300 Nebraska Medical Center,
Omaha, NE 68198-5300 (E-mail: srennard{at}unmc.edu).
Received 6 February 1997; accepted in final form 5 March 1999.
 |
REFERENCES |
1.
Adler, K. B.,
B. M. Fischer,
D. T. Wright,
L. A. Cohn,
and
S. Becker.
Interactions between respiratory epithelial cells and cytokines: relationships to lung inflammation.
Ann. NY Acad. Sci.
725:
128-145,
1994[Abstract].
2.
Agostini, C.,
M. Chilosi,
R. Zambello,
L. Trentin,
and
G. Semenzato.
Pulmonary immune cells in health and disease: lymphocytes.
Eur. Respir. J.
6:
1378-1401,
1993[Abstract].
3.
Aversa, G.,
J. Punnonen,
B. G. Cocks,
R. de Waal Malefyt,
F. Vega,
S. M. Zurawski,
G. Zurawski,
and
J. E. de Vries.
An interleukin 4 (IL-4) mutant protein inhibits both IL-4 or IL-13-induced human immunoglobulin G4 (IgG4) and IgE synthesis and B cell proliferation: support for a common and IL-13 receptors.
J. Exp. Med.
178:
2213-2218,
1993[Abstract].
4.
Barnes, P. J.
Cytokines as mediators of chronic asthma.
Am. J. Respir. Crit. Care Med.
150:
S42-S49,
1994[Medline].
5.
Beckmann, J. D.,
H. Takizawa,
D. Romberger,
M. Illig,
L. Claassen,
K. Rickard,
and
S. I. Rennard.
Serum-free culture of fractionated bovine bronchial epithelial cells.
In Vitro Cell. Dev. Biol.
28A:
39-46,
1992.
6.
Bentley, A. W.,
S. R. Durham,
D. S. Robinson,
G. Menz,
C. S. Storz,
O. Cromwell,
A. B. Kay,
and
A. J. Wardlaw.
Expression of endothelial and leukocyte adhesion molecules intercellular adhesion molecule-1, E-selection, and vascular cell adhesion molecule-1 in the bronchial mucosa in steady-state and allergen-induced asthma.
J. Allergy Clin. Immunol.
92:
857-868,
1993[Medline].
7.
Bloemen, P. G.,
M. C. Van den Tweel,
P. A. Henricks,
F. Engels,
M. J. Van de Velde,
F. J. Blomjous,
and
F. P. Nijkamp.
Stimulation of both human bronchial epithelium and neutrophils is needed for maximal interactive adhesion.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L80-L87,
1996[Abstract/Free Full Text].
8.
Boyce, S. T.,
and
R. G. Ham.
Cultivation, frozen storage and clonal growth of normal human epidermal keratinocytes in serum-free media.
J. Tissue Cult. Methods
9:
83-93,
1985.
9.
Bradding, P.,
J. A. Roberts,
K. M. Britten,
S. Montefort,
R. Djukanovic,
R. Mueller,
C. H. Heusser,
P. H. Howarth,
and
S. T. Holgate.
Interleukin-4, -5, -6 and tumor necrosis factor-alpha in normal and asthmatic airways: evidence for the human mast cell as a source of these cytokines.
Am. J. Respir. Cell Mol. Biol.
10:
471-480,
1994[Abstract].
10.
Broide, D. H.,
M. Lotz,
A. J. Cuomo,
D. A. Coburn,
E. C. Federman,
and
S. I. Wasserman.
Cytokines in symptomatic asthma airways.
J. Allergy Clin. Immunol.
89:
958-967,
1992[Medline].
11.
Chomczynski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidine thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
12.
Costabel, U.,
K. J. Bross,
and
H. Matthys.
A new method for the demonstration of surface antigens on bronchoalveolar lavage cells.
Bull. Eur. Physiopathol. Respir.
21:
381-387,
1985[Medline].
13.
De Clerck, L. S.,
C. H. Bridts,
A. M. Mertens,
M. M. Moens,
and
W. J. Stevens.
Use of fluorescent dyes in the determination of adherence of human leukocytes to endothelial cells and the effect of fluorochromes on cellular function.
J. Immunol. Methods
172:
115-124,
1994[Medline].
14.
De Rose, V.,
R. A. Robbins,
R. M. Snider,
J. R. Spurzem,
G. M. Thiele,
S. I. Rennard,
and
I. Rubinstein.
Substance P increases neutrophil adhesion to bronchial epithelial cells.
J. Immunol.
152:
1339-1346,
1994[Abstract/Free Full Text].
15.
De Waal Malefyt, R.,
C. G. Figdor,
and
J. E. de Vries.
Effects of interleukin 4 on monocyte functions: comparison to IL-13.
Res. Immunol.
144:
629-633,
1994.
16.
De Waal Malefyt, R.,
C. G. Figdor,
R. Huijbens,
S. Mohan-Peterson,
B. Bennett,
J. Culpepper,
W. Dang,
G. Zurawski,
and
J. E. de Vries.
Effects of IL-13 on phenotype, cytokine production and cytotoxic function of human monocytes.
J. Immunol.
151:
6370-6381,
1993[Abstract/Free Full Text].
17.
De Waal Malefyt, R.,
H. Yssel,
M.-G. Roncarolo,
H. Spits,
and
J. E. de Vries.
Interleukin-10.
Curr. Opin. Immunol.
4:
314-320,
1992[Medline].
18.
Gosset, P.,
I. Tillie-Leblond,
A. Janin,
C. H. Marquette,
M. C. Copin,
B. Wallaert,
and
A. B. Tonnel.
Increased expression of ELAM-1, ICAM-1 and VCAM-1 on bronchial biopsies from allergic asthmatic patients.
Ann. NY Acad. Sci.
725:
163-172,
1994[Medline].
19.
Gosset, P.,
A. Tsicopoulos,
B. Wallaert,
C. Vannimenus,
M. Joseph,
A.-B. Tonnel,
and
A. Capron.
Increased secretion of tumor necrosis factor alpha and interleukin-6 by alveolar macrophages consecutive to the development of the late asthmatic reaction.
J. Allergy Clin. Immunol.
88:
561-571,
1991[Medline].
20.
Hart, P. H.,
G. F. Vitti,
D. R. Burgess,
G. A. Whitty,
D. S. Piccoli,
and
J. A. Hamilton.
Potential anti-inflammatory effects of interleukin-4: suppression of human monocyte tumor necrosis factor alpha, interleukin-1 and prostaglandin E2.
Proc. Natl. Acad. Sci. USA
86:
3803-3807,
1989[Abstract].
21.
Howarth, P. H.,
P. Bradding,
D. Qunit,
A. E. Redington,
and
S. T. Holgate.
Cytokines and airway inflammation.
Ann. NY Acad. Sci.
725:
69-82,
1994[Medline].
22.
Hsu, D. H.,
K. W. Moore,
and
H. Spits.
Differential effects of interleukin-4 and -10 on interleukin-2 induced interferon-gamma synthesis and lymphokine-activated killer activity.
Int. Immunol.
4:
563-569,
1992[Abstract].
23.
Kauffmann, F.,
D. Drouet,
J. Lelouch,
and
D. Brille.
Twelve years spirometric changes among Paris area workers.
Int. J. Epidemiol.
8:
201-212,
1979[Abstract].
24.
Kelsen, S. G.,
I. A. Mardini,
S. Zhou,
J. L. Benovic,
and
N. C. Higgins.
A technique to harvest viable tracheobronchial epithelial cells from living human donors.
Am. J. Respir. Cell Mol. Biol.
7:
66-72,
1992[Medline].
25.
Lechner, J. F.,
A. Haugen,
H. Autrup,
I. A. McClendon,
B. F. Trump,
and
C. C. Harris.
Clonal growth of epithelial cells from normal adult human bronchus.
Cancer Res.
41:
2294-2304,
1981[Abstract].
26.
Lee, T.,
R. Wu,
A. R. Brody,
J. C. Barrett,
and
P. Nettesheim.
Growth and differentiation of hamster tracheal epithelial cells in culture.
Exp. Lung Res.
6:
27-45,
1984[Medline].
27.
Leung, K. N.,
N. K. Mak,
M. C. Fung,
and
A. J. Hapel.
Synergistic effect of IL-4 and TNF-
in the induction of monocytic differentiation of a mouse myeloid leukaemic cell line (WEHI-3B JCS).
Immunology
81:
65-72,
1994[Medline].
28.
Liew, F. Y.,
Y. Li,
A. Severen,
S. Millot,
J. Schmidt,
M. Salter,
and
S. A. Moncada.
A possible novel pathway of regulation of murine T helper type-2 (Th2) cells of a Th1 cell activity via the modulation of the induction of nitric oxide synthase on macrophages.
Eur. J. Immunol.
21:
2489-2494,
1991[Medline].
29.
Lohman-Matthes, M.-L.,
C. Steinmuller,
and
G. Franke-Ullmann.
Pulmonary macrophages.
Eur. Respir. J.
7:
1678-1689,
1994[Abstract/Free Full Text].
30.
Look, D. C.,
S. R. Rapp,
B. T. Keller,
and
M. J. Holtzman.
Selective induction of intercellular adhesion molecule-1 by interferon-
in human airway epithelial cells.
Am. J. Physiol.
263 (Lung Cell. Mol. Physiol. 7):
L79-L87,
1992[Abstract/Free Full Text].
31.
Maliszewski, C. R.,
T. A. Sato,
B. Davison,
C. A. Jacobs,
F. D. Finkelman,
and
W. C. Fanslow.
In vivo biological effects of recombinant soluble interleukin-4 receptor.
Proc. Soc. Exp. Biol. Med.
206:
233-237,
1994[Abstract].
32.
Minty, A.,
P. Chalon,
J.-M. Derocq,
X. Demont,
J.-C. Guillemont,
M. Kaghad,
C. Labit,
P. Leplatois,
P. Liauzun,
B. Miloux,
C. Minty,
P. Casellas,
G. Loison,
J. Lupker,
D. Shire,
P. Ferrara,
and
D. Caput.
Interleukin-13 is a new human lymphokine regulating inflammatory and immune responses.
Nature
362:
248-250,
1993[Medline].
33.
Moore, K. W.,
A. O'Gara,
R. de Waal Malefyt,
P. Vieira,
and
T. R. Mosmann.
Interleukin-10.
Annu. Rev. Immunol.
11:
165-190,
1993[Medline].
34.
O'Garra, A.,
S. E. Macatonia,
C.-S. Hsieh,
and
K. M. Murphy.
Regulatory role of IL-4 and other cytokines in T helper cell development in an a
TCR transgenic mouse system.
Res. Immunol.
144:
620-625,
1994.
35.
Paul, W. E.
The role of IL-4 in the regulation of
cell development, growth and differentiation.
In: Structure and Function, edited by H. Spits. Boca Raton, FL: CRC, 1994, p. 58-73.
36.
Piela-Smith, T. H.,
G. Broketa,
A. Hand,
and
J. P. Korn.
Regulation of ICAM-1 expression and function in human dermal fibroblasts by IL-4.
J. Immunol.
148:
1375-1381,
1992[Abstract/Free Full Text].
37.
Ricci, M.
IL-4: a key cytokine in atopy.
Clin. Exp. Allergy
24:
801-812,
1994[Medline].
38.
Robbins, R. A.,
and
S. I. Rennard.
Biology of airway epithelial cells.
In: The Lung: Scientific Foundations (2nd ed.), edited by R. G. Crystal,
J. B. West,
E. R. Weibel,
and P. J. Barnes. Philadelphia: Lippincott-Raven, 1997, p. 445-457.
39.
Swain, S. L.
IL-4 dictates T-cell differentiation.
Res. Immunol.
144:
616-620,
1994.
40.
Thompson, A. W.,
and
M. T. Lotze.
Interleukins 13, 14 and 15.
In: The Cytokine Handbook, edited by A. W. Thompson. San Diego, CA: Academic, 1994, p. 257-263.
41.
Tosi, M. F.,
A. Hamedani,
J. Brosovich,
and
S. E. Alpert.
ICAM-1-independent, CD18-dependent adhesion between neutrophils and human airway epithelial cells exposed in vitro to ozone.
J. Immunol.
152:
1935-1942,
1994[Abstract/Free Full Text].
42.
Tosi, M. F.,
J. M. Stark,
C. W. Smith,
A. Hamedani,
D. C. Gruenert,
and
M. D. Infeld.
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,
1992[Medline].
43.
Valent, P.,
D. Bevec,
D. Maurer,
J. Besener,
F. DiPadova,
J. H. Butterfield,
W. Speiser,
O. Majdic,
K. Lechner,
and
P. Bettelheim.
Interleukin-4 promotes expression of mast cell ICAM-1 antigen.
Proc. Natl. Acad. Sci. USA
88:
3339-3342,
1991[Abstract].
44.
Verdegaal, E. M. E.,
H. Beekhuizen,
I. Blokland,
and
R. van Furth.
Increased adhesion of human monocytes to IL-4 stimulated human venous endothelial cells via CD11/CD18, and very late antigen-4 (VLA-4)/vascular cell adhesion molecule-1 (VCAM-1)-dependent mechanisms.
Clin. Exp. Immunol.
93:
292-298,
1993[Medline].
45.
Vignola, A. M.,
A. M. Campbell,
P. Chanez,
P. Lacoste,
F. B. Michel,
P. Godard,
and
J. Bousquet.
Activation by histamine of bronchial epithelial cells from nonasthmatic subjects.
Am. J. Respir. Cell Mol. Biol.
9:
411-417,
1993[Medline].
46.
Vignola, A. M.,
P. Chanez,
A. M. Campbell,
A. M. Pinel,
J. Bousquet,
and
F.-B. Michel.
Quantification and localization of HLA-DR and intercellular adhesion molecule-1 (ICAM-1) molecules on bronchial epithelial cells of asthmatics using confocal microscopy.
Clin. Exp. Immunol.
96:
104-109,
1994[Medline].
47.
Wegner, C. D.,
R. H. Gundel,
P. Reilly,
N. Haynes,
L. G. Letts,
and
R. Rothlein.
Intercellular adhesion molecule-1 (ICAM-2) in the pathogenesis of asthma.
Science
247:
456-459,
1990[Medline].
48.
Zurawski, G.,
and
J. E. de Vries.
Interleukin 13, an interleukin 4-like cytokine that acts on monocytes and B cells, but not on T cells.
Immunol. Today
15:
19-26,
1994[Medline].
Am J Physiol Lung Cell Mol Physiol 277(1):L58-L64
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