Yonago Acta medica 1999;42:11–20

An Inhibitory Effect of Dexamethasone on A549 Cell Adhesion to
Neutrophils: Possible Involvement of Nuclear Factor-Kappa B
Hirokazu Tokuyasu, Katsuyuki Tomita and Yutaka Hitsuda
Third Department of Internal Medicine, Faculty of Medicine, Tottori University, Yonago 6830826, Japan
In order to clarify the effects of dexamethasone on epithelial cells at the transcription
factor level, protein synthesis level and functional level, we examined neutrophil adhesion to A549 cells (an immortalized human type II alveolar epithelial cell line), expression of intercellular adhesion molecule-1 (ICAM-1), and translocation of nuclear
factor-kappa B (NF-κB) into nuclei. The intranuclear localization of fluorescently
stained NF-κB and an adhesion assay between neutrophils and A549 cells was analyzed
quantitatively using laser scanning cytometry. ICAM-1 expression on A549 cells wasmeasured by flow cytometric analysis. Stimulation of A549 cells with tumor necrosis
factor (TNF)-α caused a time- and dose-dependent increase in their adhesiveness forneutrophils. ICAM-1 expression on A549 cells after stimulation with TNF-α for 12 h
was significantly up-regulated, compared with unstimulated cells (P < 0.05). Intranuclear NF-κB was induced 10 min after stimulation with TNF-α and was increased ina TNF-α-dose-dependent manner. Dexamethasone suppressed TNF-α-induced NF-κB
translocation in A549 cells by approximately 60%, ICAM-1 expression on A549 cells
by approximately 40%, and A549 adhesiveness for neutrophils completely. Therefore,
we suggest that the inhibition of A549 adhesiveness for neutrophils by dexamethasone
may be due in part to the suppression of NF-κB entry into nuclei and ICAM-1 expression in A549 cells.
Key words: alveolar epithelial cells; adhesion assay; ICAM-1; laser scanning cytometry;
neutrophils; NF-κB

neutrophils to alveolar epithelial cells may be
an important event of the inflammatory process.
Glucocorticoids are widely used as immunosuppressive and anti-inflammatory agents.
The synthetic glucocorticoid dexamethasone
suppressed nuclear factor-kappa B (NF-κB)
activity of tumor necrosis factor (TNF)-αactivated A549 cells (Adcock et al., 1996). NFκB is a ubiquitous transcription factor involvedin the regulation of a variety of genes encoding
pro-inflammatory mediators, especially intercellular adhesion molecule-1 (ICAM-1). It wasreported that dexamethasone suppressed the

Injury to alveolar epithelial cells is a prominent
feature in a variety of lung diseases. In adultrespiratory distress syndrome, destruction ofalveolar epithelial cells and increased numberof neutrophils within the alveoli have been noted to be frequent and early findings (Nash et al.,
1974; Bachofen and Weibel, 1977). Stark andcolleagues (1996a, 1996b) reported that A549
cells infected by respiratory virus increased
levels of adhesion of neutrophils. Simon andcolleagues (1986) have demonstrated thatneutrophils can adhere to rat pulmonary type II
cells and mediate cytotoxicity. The adhesion ofAbbreviations: AM, acetoxymethylester; BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle
medium; FITC, fluorescein isothiocyanate; GM-CSF, granulocyte-macrophage colony-stimulating factor;
HBSS, Hanks’ balanced salt solution; ICAM-1, intercellular adhesion molecule-1; LSC, laser scanning cytomety; NF-κB, nuclear factor-kappa B; PBS, phosphate buffered saline; PI, propidium iodide; PLP, periodate
lysine paraformaldehyde; TNF, tumor necrosis factor

11

H. Tokuyasu et al.

Jose, CA) in Dulbecco’s modified Eagle
medium/nutrient mixture F-12 (DMEM/F12;
Gibco BRL, Grand Island, NY) supplemented
with 10% heat-inactivated fetal bovine serum(Cansera, Rexdale, ON, Canada), 100 units/mL
penicillin, and 100 mg/mL streptomycin (BioWhittaker, Walkersville, MD) in an incubator
containing 5% CO2 at 37°C. At confluency, thecells were detached with trypsin-EDTA(Kurabo Co., Ltd., Osaka, Japan) and subcultured on 22 × 24 mm2 coverglasses (Matsunami
Glass Ind., Ltd., Osaka) in 35 mm diameter
dishes (Falcon 1008; Becton Dickinson) andthen incubated to allow the formation of semiconfluent monolayers. The culture medium
was replaced with serum free medium (DMEM/
F12) and incubated over night, which resulted
in confluent monolayers.

expression of ICAM-1 on TNF-α-activatedA549 cells (Rothlein et al., 1988) and the neutrophil-to-bronchial epithelial cell line adhesion(Kawasaki et al., 1998). However, in respiratory epithelial cells the effect of dexamethasone
on transcription factor level, protein level, andfunction is still unclear.
Traditionally, adhesion assays to detect cellto-cell interaction have been measured by thecounting of 51[Cr] labeled cells. Recently, thefluorogenic dye calcein acetomethylester (AM)has provided a fast and highly sensitive method
for analyzing labeled cells which adhere to cellmonolayers (Clerck et al., 1994; van Kessel etal., 1994; Martens et al., 1995). Calcein AM isa non-fluorescent compound, and once loaded
into cells, is cleaved by endogenous esterases to
produce the highly fluorescent and well-retained
dye calcein. In this method, the number oflabeled cells has been assessed by a fluorometer
after cell lysis. It is difficult to assess cell-tocell adhesion fairly when unlabeled cells frommonolayers such as A549 cells are undergoingproliferative and rapid growth. To count the
number of monolayer cells may be an important
method for assessing adhesion interaction
objectively. So we quantitatively analyzed andvisualized the adhesion between calceinstained neutrophils and DNA-stained epithelial
cells using laser scanning cytometry (LSC).
The aim of the present study was to assay
for adhesion between neutrophils and A549
cells by LSC. Furthermore, we examined theeffects of dexamethasone on neutrophil-toA549 cell adhesion, ICAM-1 expression, and
NF-κB translocation in A549 cells.

Neutrophil isolation
Neutrophils were obtained from heparinizedvenous blood of healthy volunteers. Thirty
minutes after sedimentation of erythrocytes in 6
% dextran (Nycomed As, Oslo, Norway)-saline,
the supernatant layer containing leukocytes wascentrifuged in Ficoll-Paque (Pharmacia, Uppsala,Sweden) for 30 min at 400 × g at room temperature. The supernatant containing lymphocytes
was discarded, and contaminating erythrocytes
within the pellet were lysed by hypoosmosiswith hypotonic saline (0.2%) for 15 s. Hypertonic saline (1.6%) was added immediately tomaintain isotonicity. This procedure was repeated twice. After washing in phosphatebuffered saline (PBS) without Ca2+ and Mg2+
(Handai Bacterial Research, Osaka) and centrifugation, the pellet was resuspended in 1 mL
Hanks’ balanced salt solution (HBSS; Gibco
BRL) without Ca2+ and Mg2+. Viability was >95% by trypan blue exclusion and > 95% of thecells were neutrophils by May-Giemsa staining.

Materials and Methods

Human respiratory epithelial cell cultures
A549 (American Type Culture Collection,
Rockville, MD), the epithelial type II cell line
derived from a patient with alveolar cell
carcinoma (Lieber et al., 1976) was selected asa source of respiratory epithelial cell for thesestudies. A549 cells were cultured in culture
dishes (Falcon 3002; Becton Dickinson, San

Calcein AM labeling of neutrophils
Stock solutions were prepared by adding 100
mL of fresh dimethyl sulfoxide (Wako Life
Science Co., Ltd., Osaka) to 1 mg of calcein
AM (Molecular Probes, Eugene, OR). Aliquots
12Effect of dexamethasone

macrophage colony-stimulating factor (GMCSF) was added to 2.5 × 106 of calcein-labeledneutrophils supplemented with 1 mL of HBSS
with Ca2+ and Mg2+. After incubation for 30
min at 37°C in 5% CO2, the plates were washed
six times with 1 mL of HBSS without Ca2+ andMg2+ to remove nonadherent neutrophils. We
confirmed the adhesion of neutrophils to A549
cells under microscopy. A coverglass in eachdish was mounted cell-side down on each slideand sealed with nail polish. The slide was placed on the microscope stage.

of the stock solution were stored at –20°C.
Before the assay, the calcein AM was dissolved
and added to the neutrophil suspension at a final
concentration of 1 µM for 30 min at 37°C and
5% CO2. Incubation time was set taking into
account the possible increase in the release ofcalcein after this optimal period (van Kessel etal., 1994). Labeled cells were washed twice in
HBSS without Ca2+ and Mg2+ and resuspended
in HBSS with Ca2+ and Mg2+ at 2.5 × 106 cells
per mL before the cell-to-cell adhesion assay.

Fixation and preparations

NF-κB staining of A549 cells

A549 cells were stimulated with or without
TNF-α (Genzyme Corporation, Cambridge,MA) for 12 h for adhesion assay and detectionof ICAM-1, or 30 min for detection of NF-κB.
Cells in the dexamethasone group were pretreated with dexamethasone (Sigma ChemicalCo., St. Louis, MO) for 30 min at various concentrations before stimulation with TNF-α.
After washing twice in PBS, the cells were fixed at room temperature in freshly preparedperiodate lysine paraformaldehyde (PLP) fixative that consisted of 37 mmol/L phosphatebuffer, pH 7.4, containing 10 mmol/L sodium
m-per-iodate (Wako), 75 mmol/L lysine(Wako) and 2% paraformaldehyde (Wako).
This procedure has been shown to preserve theantigenicity of many protein and carbohydrate
determinants (McLean and Nakane, 1974).
After removing the supernatant, the monolayers
were washed twice with PBS. Then they were
washed with 100 mM glycine, and 0.1% bovine
serum albumin (BSA; Sigma) in HBSS without
Ca2+ and Mg2+ (blocking solution). The dishes
were stored at 4°C with blocking solution. Before use in the adhesion assays they were washed with PBS, and then 1 mg/mL ribonuclease A(Sigma) and 100 mg/mL propidium iodide (PI)
(Sigma) were added to each dish for 30 min at
4°C. After removing the supernatant, themonolayers were washed twice with PBS.

A549 cells in the dexamethasone group werepretreated with dexamethasone at various concentrations 30 min before TNF-α stimulation.
After washing twice in PBS, the cells wereprefixed in 2% formalin (Wako) in PBS, pH
7.4, for 10 min at room temperature. After
thorough washing in PBS, the cells were fixedwith methanol-acetone at –20°C for 10 min.
Nonspecific antibody binding sites were thenblocked with 10% normal goat serum (Chemicon,
Temecula, CA) in PBS with 1% BSA. Coverglasses were incubated with anti-Rel A rabbit
polyclonal antibody (SC-109; Santa Cruz Biotechnology, Santa Cruz, CA,) at a final dilutionof 1:100 for 1 h at room temperature. Afterwashing thoroughly 3 times with PBS, cells
were incubated for 30 min at room temperature
with 5 mg/mL goat anti-rabbit IgG fluorescein
isothiocyanate (FITC)-conjugated antibody(Santa Cruz). After each incubation with
antibodies, cells were extensively washed with
PBS. Nuclei were stained for 10 min with 50
mg/mL PI and 200 mg/mL ribonuclease A andwashed again. As a negative control, rabbit IgG(Santa Cruz) was used in place of the firstantibody at the same dilution.

Detection of adhesion and of intranuclear
NF-κB by LSC
For adhesion assay and measuring intranuclear
NF-κB staining, LSC (LSC101, Olympus Co.,Tokyo, Japan) was used as previously described(Newton and Millette, 1992; Clatch et al., 1996).

Adhesion assay of neutrophils to A549
monolayers
For the adhesion assay, 10 ng/mL granulocyte13

H. Tokuyasu et al.

Cell number of A549 cells

Dual-channel imaging was performed with 2
photomultipliers, using a band-pass filter specifically designed to detect FITC fluorescenceand a long-pass (590 nm) filter for detecting PI
fluorescence. Images were generated using
integral computational and display software.
To assess the mean fluorescence intensity of
NF-κB translocated into nuclei, each nucleus
was identified by the areas counter stained with
PI. For the adhesion assays, staining with FITC
for neutrophils and PI for A549 cells was used
for cell counting. Firstly, we confirmed theaccuracy of the instrument. When cells of thesame sample and the same area were counted
repeatedly by LSC, the liability to error, calculating the mean value of the squares of theseerrors, was 0.4% (n = 10). Secondly, we chose
1 mm2 as the scan area in this experiment. Thisis because the cell number counted by LSC
closely correlated to the scan area (r = 0.998,
P < 0.0001) (Fig. 1A) and the scan time would
be too long if the area were over this size.
Thirdly, we checked the correlation between
the A549 cell number and the neutrophil number in adhesion to each other since A549 cells
were immortalized and were rapid-growth cells.
The result showed that the number of A549
cells was positively correlated with the adherent
neutrophils (Fig. 1B). Then the adhesion assay
was performed. After counting the number ofA549 cells, the number of adherent neutrophils
was examined in the same scan area. Each sample was counted in triplicate. The rate of theadhesion of neutrophils to A549 cells was calculated as follows:

A

80000

60000

Adherent rate (%) =40000

the total numberof A549 cells

× 100

r = 0.998
P < 0.0001

20000

0
0

20

40

60

80

100

120 (mm2)

Scan area
Cell number of neutrophils

the total number ofadherent neutrophils

200

A549 cells were pretreated with dexamethasone
30 min before 50 ng/mL TNF-α was added. At
the end of the 12 h incubation period, the cells
were washed twice with PBS. After trypsinization, the cells were centrifuged for 5 min at 400
× g, and at 4°C. The supernatant was discarded
and the pellet was suspended with PBS. This
procedure was repeated twice. Then the cells
were fixed in PLP fixative at room temperaturefor 10 min. After thorough washing in PBS, thecells were resuspended in 300 mL PBS containing 0.1% sodium azide (Wako) and 1%
BSA (staining buffer). Then, they were incubated with FITC-conjugated mouse antihuman-ICAM-1 monoclonal antibody (B-C14,
IgG1; Serotec, Oxford, United Kingdom) at a
final dilution of 1:100 for 1 h in the dark at 4°C.
Next they were washed twice in staining buffer
and resuspended in staining buffer. Flow cytometry was performed with 1.0 × 106 cells per
sample by FACSCalibur (Becton Dickinson,

B

150

100

r = 0.996
P < 0.0001

0
0

600

700

800

900

Detection of ICAM-1 on A549 cells by flow
cytometry

1000

Cell number of A549 cells
Fig. 1. Relationship between the cell number ofA549 cells and scan area (A), or between the cellnumber of A549 cells and that of neutrophils (B).
For the relationship between A549 cells number andscan area, the number of PI-stained cells wasmeasured at different sites on the same sample,
which was confluent on the glass, by LSC. For the
relationship between A549 cell number andneutrophil number, we analyzed a 1 mm2 scan area
after adhesion of GM-CSF-stimulated and calceinstained neutrophils to 50 ng/mL TNF-α-stimulated
and PI-stained A549 cells.

14

Effect of dexamethasone

Mountain View, CA). Fluorescence measured
in the fluorescence-1 channel was quantified asmean fluorescence intensity units.

Results

Statistical analysis

The adhesiveness of A549 cells forneutrophils

Statistical analysis was performed with the Stat
View 4.0 statistics package (Abacus Concepts,
Berkeley, CA). Data are expressed as mean ±SD. The nonparametric Mann-Whitney U test
was used for statistical analysis of the unpaired
data. In addition, the relationship between scan
area and the number of A549 cells, and that between the A549 cell number and the neutrophil
number were analyzed by Spearman’s rank sum
test.

Fluorescent microscopy
Figure 2 is a representative micrograph ofneutrophil adhesion to a confluent monolayer ofA549 cells, which was pretreated without (Fig.
2A) or with TNF-α (Fig. 2D). Neutrophil adhesion increased significantly on exposure to
TNF-α. In addition, LSC was available to visualize and separate A549 cells (Figs. 2B and E)
and neutrophils (Figs. 2C and F) by calcein- andPI-staining, respectively.

A

D

B

E

C

F
20 µm

Fig. 2. Neutrophil adhesion to an underlying confluent monolayer of A549 cells after no treatment (A, B andC) and after pretreatment with TNF-α (50 ng/mL) for 12 h (D, E and F). These were shown by microscopic
image (A and D), red fluorescence image (B and E) for PI stained A549 cells, and fluorescence image (C andF) for calcein-stained neutrophils from the same sample.

15

H. Tokuyasu et al.

(%)
25

A
*

Adherent rate

20

15

*

10

5

0
(–)

25

6

12

(%)

B

(h)

100

(ng/mL)*20

Adherent rate

24

Incubation time

15

10

5

0
0

10

50

Concentration of TNF-α
Fig. 3. A549 adhesiveness for neutrophils after
stimulation with TNF-α (50 ng/mL) for different
periods (A) and that with different concentrations of
TNF-α (B) for 12 h. Results are expressed as mean ±SD; n = 6. *P < 0.05, compared with no stimulation.

Effect of dexamethasone on the adhesiveness of A549 cells for neutrophils
Pretreatment with dexamethasone at a low
dose (10–8 M) could inhibit the TNF-α-induced
neutrophil adhesion to A549 cells (P < 0.05). As
shown in Fig. 4, 10–7 M dexamethasone completely inhibited neutrophil adhesion to A549
cells.

(%)
20*
Adherent rate

16

ICAM-1 expression on A549 cells

12

8

†

4

†

Concentration-related dexamethasone suppression of TNF-α-induced ICAM-1 expression on
A549 cells is shown in Fig. 5. TNF-α stimulation caused a significant increase in ICAM-1
expression compared with the unstimulatedgroup (mean fluorescence intensity; 13.3 ± 5.6
versus 103.4 ± 15.8, P < 0.05). At a dose of 10–7
M, dexamethasone suppressed TNF-α-induced
ICAM-1 up-regulation by approximately 40%.
Using 10–6 M of dexamethasone did not increase
the suppression, indicating that dexamethasone
only partially blocks the TNF-α-induced ICAM-1
up-regulation.

†

0

[18]

Pretreatment (–)

TNF-αEffect of TNF-α on the adhesiveness of A549
cells for neutrophils
Stimulation of A549 cells with TNF-α caused a time-dependent increase in their adhesiveness for GM-CSF stimulated neutrophils (Fig.
3A). The adherent rate began to increase after
stimulating A549 cells with TNF-α for 6 h compared with the control group (2.1 ± 1.6% versus
5.4 ± 5.2%) and reached its peak at 12 h (10.6 ±
9.2% ). When stimulation lasted for 24 h, nofurther increase was observed (6.8 ± 3.5%).
The effect of different doses of TNF-α on A549
adhesiveness for neutrophils is shown in Fig.
3B. TNF-α pretreatment increased the adherent
rate in a dose-dependent manner within 50 ng/
mL. Although there was a trend for the adherent rate to decline with high doses of TNF-α(100 ng/mL) compared with 50 ng/mL, thisdifference did not reach statistical significance.

(–)

[18]

[6]

[6]

[6]

(–)

Dex
10–8 MDex
10–7 MDex
10–6 M

Pretreatment for 30 min
TNF-α 50 ng/mL for 12 hFig. 4. Suppression of A549 adhesiveness forneutrophils by dexamethasone (Dex). A549 cells ineach group except for the controls were treated with
TNF-α (50 ng/mL) for 12 h. Cells in the Dex groupwere incubated with various concentrations of Dex
30 min before treatment with TNF-α. Results are
expressed as mean ± SD. [ ] = number of samples.
*P < 0.05, compared with no stimulation. †p < 0.05,
compared with TNF-α stimulation.

16

Effect of dexamethasone

Table 1. Time response of translocation of
p65 into nuclei

Mean fluorescence intensity

140

*

120

Stimulation time
(min)

100

†

†

80
60
40
20
0

Pretreatment (–)

TNF-α

(–)

Dex
10–7 M
(–)

(–)

Dex
10–8 M

Dex
Dex
10–7 M 10–6 M

Pretreatment for 30 min

Fluorescenceintensity017.6 ± 4.8
1051.6 ± 7.2*
30
51.9 ±10.3*
60
44.1 ± 3.2*
A549 cells were stimulated with 50 ng/mL TNF-α
for various times.
Data are expressed as mean ± SD of 8 individual
experiments.
* P < 0.01, compared with no stimulation.

TNF-α 50 ng/mL for 12 hner. Intranuclear p65 staining was induced 10 min
after stimulation with TNF-α, without a decline instaining intensity over the subsequent 1 h.

Fig. 5. Suppression of TNF-α-induced ICAM-1
expression on A549 cells by dexamethasone (Dex).
Cells were incubated with various concentrations of
Dex 30 min prior to addition of 50 ng/mL TNF-α.
ICAM-1 levels were measured by flow cytometry
analysis. Results are expressed as mean ± SD; n = 5.
*P < 0.05, compared with no stimulation. †P < 0.05,
compared with TNF-α stimulation.

Intranuclear NF-κB of A549 cells
NF-κB was located in the cytosol of unstimulated A549 cells and exposure to TNF-α caused
NF-κB to translocate into the nuclei in a time(Table 1) and dose- (Table 2) dependent man-

Effect of dexamethasone on NF-κB translocation into nuclei
Pretreatment with dexamethasone suppressed translocation of NF-κB into nuclei in the presence of TNF-α stimulation. Quantitative image
analysis using LSC revealed that the green
fluorescence level in A549 cell nuclei from theunstimulated cells and the cells treated by dexamethasone alone was significantly less than thecells treated by TNF-α (P < 0.01). Dexamethasone suppressed the TNF-α-induced translocation of NF-κB into nuclei by approximately
60% (Table 3).

Table 2. Dose response of translocation of
p65 into nuclei by TNF-α

Table 3. Inhibition of translocation of p65
into nuclei by dexamethasone

NF-κB translocation into nuclei of A549
cells

TNF-α
(ng/mL)

Fluorescenceintensity

Dexamethasone(M)019.6 ± 6.1
0.1
26.2 ± 1.9†
1.0
32.2 ± 3.8*
1034.4 ± 3.8*
50
53.0 ± 8.9*
A549 cells were stimulated with various concentrations of TNF-α for 30 min.
Data are expressed as mean ± SD of 10 individual
experiments.
* P < 0.01, compared with no stimulation.
†
P < 0.05, compared with no stimulation.

TNF-α(ng/mL)

Fluorescenceintensity(–)(–)
17.8 ± 3.3(–)
17.4 ± 3.2
10–7
(–)
50
41.8 ± 4.1†
50
34.0 ± 2.2*
10–850
28.0 ± 2.5*
10–7
10–6
50
28.9 ± 1.2*
A549 cells were pretreated with various concentrations of dexamethasone for 30 min, and thenwere stimulated with 50 ng/mL TNF-α for 30 min.
Data are expressed as mean ± SD of 8 individual
experiments.
* P < 0.01, compared with TNF-α stimulation.
† P < 0.01, compared with no stimulation.

17

H. Tokuyasu et al.

suppression of NF-κB translocation and theaction of transcription factors other than NFκB.
The present study showed that TNF-α increases the adhesiveness of alveolar epithelial
cells for neutrophils. The cell-to-cell adhesiveinteractions are mediated through adhesion
molecules. Tosi and coworkers (1992) described that enhanced neutrophil adhesion to TNFα-treated airway epithelial cells was dependenton neutrophil CD18 integrins and epithelial cell
expression of ICAM-1. Our results are coincident with Burke-Gaffney and Hellewell (1996)
who demonstrated that TNF-α caused ICAM-1
up-regulation in A549 epithelial cells. As foradhesion assays, we found that A549 cells
adhesiveness for neutrophils after stimulation
with TNF-α increased significantly. This is different from a previous report (Thornhill et al.,1993) which showed that stimulation of A549
cells with TNF-α for 4 h could not increase
neutrophil adhesion. As to this discrepancy,
two reasons could be considered. First, stimulation for 4 h may be too short to up-regulateadhesion molecules. Atsuta and colleagues(1997) reported that ICAM-1 messenger RNA
expression was induced in the bronchial epithelial cell line after TNF-α treatment for 2 h.
Secondly, the neutrophils used in our study
were stimulated with GM-CSF. Previous reports indicated that GM-CSF-treated neutrophils increased the expression of adhesion molecules such as CD11b/CD18 (Yong and Linch,
1992; Novella et al., 1997). In addition, our results showed that the suppressive role of dexamethasone exhibited at the functional level andprotein level is different. This indicates thepossibility that ICAM-1-mediated adhesion ofneutrophils to epithelial cells requires theseadhesion molecules to be up-regulated to someextent.
The increase in adhesiveness of airway epithelial cells for neutrophils following stimulation by cytokines is considered to be an important mechanism whereby neutrophils injury
and exfoliate epithelial cells at sites of inflammation (Tosi et al., 1992). Simon and associates (1986) have demonstrated that neutrophils
can adhere to alveolar epithelial cells and medi-

Discussion
We assessed the adhesion of neutrophils to
A549 cells using the quantitative and visual
method, LSC. The results have demonstrated
that pretreatment of A549 cells with dexamethasone suppressed simultaneously their adhesiveness for neutrophils, ICAM-1 expression
and nuclear translocation of NF-κB in the presence of TNF-α.
NF-κB controls genes encoding proinflammatory cytokines, chemokines and adhesion molecules, and is therefore an important
regulatory factor in immune and inflammatory
responses. NF-κB is capable of being transported into nuclei and interacting with specific
NF-κB-like binding sites in the promoters of
responsive genes (Siebenlist et al., 1994). Previous work and our present results have demonstrated that the proinflammatory cytokines,
TNF-α and interleukin-1β cause NF-κB activation in A549 epithelial cells (Newton et al., 1996).
In this study, we only assessed the translocationof NF-κB into nuclei and did not directly analyze DNA-NF-κB binding with electrophoretic
mobility shift assay. We found that nuclear
translocation of NF-κB by TNF-α occurred
within the first 30 min of stimulation, anddecayed rapidly and then declined more slowly.
These results are in agreement with a previous
study using electrophoretic mobility shift assay(Newton et al., 1996). Dexamethasone has
been reported to reduce ICAM-1 messenger
RNA levels and its expression in bronchial
epithelial cells (van de Stolpe et al., 1993). We
demonstrated that dexamethasone suppressed
the TNF-α-induced translocation of NF-κB and
ICAM-1 expression to a similar extent in A549
cells. These results suggest one action of dexamethasone at the transcriptional level. There
are multiple binding sites for transcription factors, such as NF-κB, activating protein 1 and
CCAAT/enhancer-binding protein β in the upstream regions of ICAM-1 genes, which also
encode important targets for glucocorticoid
repression (Adcock and Newton, 1998). So, wesuggest that dexamethasone partially suppresses ICAM-1 expression on A549 cells by partial18Effect of dexamethasone

for neutrophils by dexamethasone might be due
in part to the suppression of NF-κB entry into
nuclei and the subsequent suppression of the
ICAM-1 expression.

ate cytotoxicty. Glucocorticoids are widelyused as immunosuppressive and anti-inflammatory agents. Repression of NF-κB-dependent
gene expression is one of the key characteristics
by which glucocorticoids exert their effects. It
was previously reported that in mild asthmatic
subjects treatment with corticosteroids suppressed the activation of NF-κB in the bronchial
mucosa obtained by bronchial biopsy (Demoly etal., 1995). The mechanisms of anti-inflammatory
effects of dexamethasone are not well-known.
We agree with the reports of Shimoyama andcolleagues (1997) who demonstrated that the
suppressive effect of dexamethasone on NF-κB
translocation into nuclei in corneal endothelium
was dissociated from that of the endothelium
adhesiveness for neutrophils. In our results,
10–7 M dexamethasone was able to completely
inhibit adhesiveness for neutrophils whereas
the suppression of NF-κB was approximately
60%. We suggested that this dissociation of
suppressive effects might be attributed to thedissociation between protein level and functional level. Further study is necessary to solve
the suppression mechanisms of the dexamethasone from transcription factor level to functional level.
For quantitative detection of the adherent
rate, we used non-radiolabeled fluorochromes
to stain cells and visualize them by LSC. It waspossible for LSC to discriminate each different
kind of cells by staining the cells one after
another, as calcein-stained neutrophils and PIstained A549 cells. LSC is a useful tool for performing immunophenotypic analysis of cells ona slide rather than in a fluid stream (Clatch etal., 1996). We confirmed that LSC could quantitatively count the number of cells and might
be an optimal instrument for adhesion assays
when monolayers are proliferative cells, such as
cancer cell lines, because the number of adherent neutrophils was dependent on the number of cells in the A549 monolayer.
In conclusion, we measured adherent rates
of calcein-stained neutrophils to PI-stained
A549 cells using the LSC quantitative method.
It might also be a useful method for proliferative monolayer cells. We also demonstrated
that the inhibition of A549 cells adhesivenessAcknowledgments: The authors wish to thank Prof.
Takao Sasaki, Third Dept. of Internal Medicine,
Faculty of Medicine, Tottori Univ., for his kind advice
and valuable suggestions during the course of the study.
We would also like to thank Prof. Shigetsugu Ohgi,
Second Dept. of Surgery, Faculty of Medicine, Tottori
Univ. and Prof. Mitsuo Oshimura, Dept. of Molecular
and Cell Genetics, School of Life Science, Faculty of
Medicine, Tottori Univ., for reading this manuscript
and offering helpful suggestions.

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(Received November 30, Accepted December 9, 1998)

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