Yonago Acta medica 1999;42:21–29

Alveolar Epithelial Cell Line, A549 Cells Inhibit Neutrophil Apoptosis
Yasushi Kometani, Katsuyuki Tomita and Yutaka Hitsuda
Third Department of Internal Medicine, Faculty of Medicine, Tottori University, Yonago 6830826, Japan
Though cellular localization at the site of an inflammatory challenge is the critical firststep in a neutrophil response, little has been known about the effects of alveolar epithelial cells on the dynamics of neutrophils. To investigate these effects, we used humanpurified neutrophils incubated with monolayers of human alveolar epithelial cells (A549),
quiescent or preactivated with recombinant human tumor necrosis factor (TNF)-α for 24
h. Laser scanning cytometry (LSC) was employed to detect nuclear morphological changes
and quantitate DNA strand breaks in neutrophils. The experiments revealed that A549
cells inhibited neutrophil apoptosis, and that this inhibitory effect was enhanced whenthe A549 cells were preactivated with TNF-α for 24 h. These results suggest that alveolar
epithelial cells may be potentially able to contribute to promote inflammatory
mechanisms by delaying neutrophil apoptosis.
Key words: apoptosis; A549; laser scanning cytometry; terminal deoxynucleotidyl transferase-mediateddUTP nick end-labeling method

neutrophil nuclear morphological changes andDNA strand breaks incubated with monolayersof A549 cells, quiescent or preactivated withrecombinant human tumor necrosis factor(TNF)-α for 24 h. TNF-α is an important cytokine, active in the inflammation in the lung,
whose sources are mainly macrophages. Because alveolar macrophages are in the front lineof pulmonary defense and a rich source ofcytokines, they are uniquely capable of stimulating the alveolar epithelium by secretion of
TNF-α. The experiment was performed bylaser scanning cytometry (LSC), which combines the advantages of both flow and image
cytometry (Kamentsky and Kamentsky, 1991).

In many lung diseases, neutrophils contribute to
the pathogenesis of these diseases (Diaz et al.,
1989; Sibille and Reynolds, 1990). An important factor determining whether inflammation
resolves or progresses to a chronic inflammatory state is the process by which recruited neutrophils and their potentially noxious contents
are removed from inflamed tissues (Haslett etal., 1989). Normally, these terminally differentiated phagocytes undergo spontaneous apoptosis and then lead to macrophage ingestion(Cox et al., 1995). This may represent a neutrophil removal mechanism that is important bothin the control of inflammatory tissue injury andfor the normal resolution process of inflammation. Even though neutrophil recruitment
from the bloodstream to respiratory tissue is theinitial step of alveolar inflammation, the effectsof alveolar epithelial cells on the dynamics ofneutrophils has not been previously studied. So
the hypothesis has been proposed that alveolar
epithelial cells might modulate neutrophil
apoptosis. To test this hypothesis, we studied

Subjects and Methods

Monolayer preparation of A549 cells
The human A549 alveolar epithelial cell line
(ATCC, Rockville, MD) shows similar features

Abbreviations: D-MEM/F-12, Dulbecco’s modified Eagle medium/nutrient mixture F-12; FBS, fetal bovine
serum; FITC, fluorescein isothiocyanate; ICAM-1, intercellular adhesion molecule-1; LSC, laser scanning cytometry; PBS, phosphate-buffered saline; PI, propidium iodide; TNF, tumor necrosis factor; TUNEL, terminal
deoxynucleotidyl transferase-mediated dUTP nick end-labeling21

Y. Kometani et al.

as type II alveolar epithelial cells. The cells
were grown as monolayers in 35 mm tissue
culture dishes incubated in 100% humidity and
5% CO2 at 37˚C. Dulbecco’s modified Eagle
medium/nutrient mixture F-12 (D-MEM/F-12)
media containing 365 mg/L L-glutamine (LifeTechnologies, Inc., Grand Island, NY), supplemented with 10% heat-inactivated fetal bovine
serum (FBS; Life Technologies), 100 units/mL
penicillin, and 100 µg/mL streptomycin (Wako
Pure Chemical Industries Ltd., Osaka, Japan)
was used as the growth media. The cells thatform the monolayers were harvested withtrypsin (0.25%) and EDTA (0.1%) in Mg2+- andCa 2+ -free phosphate buffered saline (PBS)(Research Institute for Medical Disease, Osaka)
centrifuged at low speed (250 × g, 5 min), resuspended in fresh medium and plated at a concentration of 2 × 105 cells/dish. The cells weregrown to confluence on tissue culture dishes for
3 to 4 days. Monolayer integrity was ensured
by phase contrast microscopy.

Incubation of neutrophils with A549 cells
in a preactivated state with TNF-α
After A549 cells were pretreated with 100 ng/
mL TNF-α (Genzyme, Cambridge, MA) for 24 hand then washed carefully twice with culture medium, neutrophils were plated 1 mL onto tissue
culture dish coated with confluent monolayersof activated A549 cells, and were allowed to be
incubated for an indicated length of time.
Neutrophils incubated in medium alone served
as the control.

Slide staining and LSC scanning protocol
After incubation at 6 and 24 h, the supernatant
which contained the neutrophils were collected
and fixed with 4% formalin (3 × 106 cells/mL)
for 10 min, then pipetted 50 µL onto glass microscope slides, and thereafter allowed to air dry.
In order to detect morphological changes andDNA strand breaks, the samples were processed using the TdT kit (Apop Tag Direct;
ONCOR, Inc., Gaithersburg, MD) employing
the terminal deoxynucleotidyl transferasemediated dUTP nick end-labeling (TUNEL)
method, according to the manufacturers instructions. TUNEL is a method of choice for rapid
identification and quantification of DNA
fragmentation (Gavrieli et al., 1992). DNA strand
breaks were directly labeled with fluorescein
isothiocyanate (FITC) conjugated deoxynucleotides in a single step reaction. The cells werecounterstained with the DNA intercalating fluorochrome propidium iodide (PI; 10 µg/mL,
Sigma Chemical Co., St. Louis, MO) solution
containing 0.1% RNase (Sigma). After staining, coverslips were immediately placed on theslides and sealed with nail polish.

Isolation of neutrophils
Neutrophils were isolated from the peripheral
blood of healthy volunteers, as previously described by Ferrante and Thong (1980), employing Ficoll-Hypaque gradient centrifugation(Pharmacia, Uppsala, Sweden). Only neutrophil preparations with a purity greater than 95%(as determined by microscopic examination ofsmears of the cell preparation) and viability
greater than 95% (as assessed by using trypan
blue exclusion) were used for these studies.
Neutrophils were finally resuspended in DMEM/F-12 at a concentration 2.5 × 106 cells/mL.

Incubation of neutrophils with A549 cells
in a quiescent state

LSC measurement

After A549 cells were washed carefully twicewith culture medium, neutrophils were plated 1
mL onto a tissue culture dish coated with confluent monolayers of A549 cells, and wereallowed to be incubated for an indicated lengthof time.

We used the LSC of Kamentsky and Kamentsky(1991), build up by Olympus Optics Co. (LSC
101; Tokyo, Japan). The cells were illuminatedwith a 488 nm wavelength light from an argon
ion laser and their emission was separated by a
570 nm dichroic mirror. Green FITC (530 ± 20nm) and red PI (> 610 nm) fluorescences were22

A549 cells inhibit neutrophil apoptosisaPI fluorescence peak

b

b
a
c

c
PI fluorescence value
Fig. 1. A 2-parameter (PI fluorescence value versus PI fluorescence peak) cytogram of suspension
neutrophils at 24 h. The cells producing the data points in each of the 3 regions labelled a to c wereautomatically relocated, and a gallery of digital photographs for each of the regions were created.

measured by separate photomultipliers. In mostcases, a number greater than 5,000 cells were
examined for approximately 10 min. Overlapping nuclei were automatically excluded fromthe counting by special statistical filters. Threeparameters were used for expressing nuclear
features. Among the features caluculated were
the peak, corresponding to the highest pixel
value within the segmenting contour, the area,
corresponding to the total number of pixels
within the segmenting contour, and the value,
which is the sum of all pixel values within thesegmenting contour. Data were expressed using
2-parameter dots of possible permutations ofthese 3 parameters.

which is above and left of region a, was created
to cover the cluster of cells that appeared mainly at 6 h. Region c, which is lower to region a,was created to cover the cluster of cells thatappeared mainly at 24 h. The cells in each indicated region were automatically counted andexpressed as a percentage of the total number.
In the experiment using 2 fluorochromes, the
FITC positive cells were separated by a rectangular gating region (Fig. 3A) in a cytogram
showing PI fluorescence value and FITC
fluorescence value. These gated FITC positive
cells were automatically relocated in a cytogram showing PI fluorescence value versus PI
fluorescence peak (Fig. 3B). TUNEL positive
cells were counted and also expressed as apercentage of the total cell number.

Gating region in the cytograms
We defined 3 gating regions in a cytogram of PI
fluorescence value versus PI fluorescence peak(Fig. 1). The cluster of cells that were not foundat the beginning of the present experimentappeared at incubation time (Fig. 2). Region acontains all the neutrophils at 0 h. Region b,

Inspection of nucleus
In every experiment, the cells producing thedata points were automatically relocated andobserved by microscope or digital photos.

23

A

PI fluorescence peak

Y. Kometani et al.

PI

B

PI fluorescence peakPI fluorescence value

PI fluorescence peak

C

cen

fluo

res

cen

ce

ce

pea

pea

6h

PI

PI fluorescence value

res

0h

PI

PI fluorescence value

fluo

fluo

24 h

res

cen

ce

pea

k

k

k

P

P

o
I flu

o
I flu

P

res

res

o
I flu

ce

c

res

v
nce

e
enc

c

alu

val

e
enc

e

ue

val

ue

Fig. 2. A PI fluorescence value versus PI fluorescence peak cytogram and 2-parameter histogram ofsuspension neutrophils were made at each indicated time point. A: newly purified neutrophils, B: after 6 h
incubation, C: after 24 h incubation. The cluster of cells that were not found in the control cytogram (A)appeared at incubation time (B and C). 3D histograms show a smooth peak at 0 h, and then another smaller
peak appearing through 6 and 24 h.

Statistics

between different groups, and P values less than
0.05 were considered to be statistically significant. These statistical analyses were performed
with the Stat View 4.11 statistics software(Abacus Concepts, Berkeley, CA).

All the data were presented as mean ± SD. The
Kruskal-Wallis and Mann-Whitney U tests
were used for statistical comparisons of results

24

A549 cells inhibit neutrophil apoptosis

A
FITC fluorescence value

C

10 µm

PI fluorescence value

B
PI fluorescence peakbaFig. 3A. A cytogram of PI fluorescence value versus FITC
fluorescence value represents suspension neutrophils at 24 husing the TUNEL method. The rectangular region contains
neutrophils that incorporated FITC labeled deoxynucleotide
in the reaction catalyzed by TdT.
Fig. 3B. TUNEL positive cells in rectangular region in Fig.
3A were relocated in a cytogram of PI fluorescence value
versus PI fluorescence peak.

cFig. 3C. Direct inspection showed small nuclei with hypercondensation of chromatin labeled with fluorochromeconjugated deoxynucleotide.

PI fluorescence value

each indicated region was not significantly
different compared with that of suspension
neutrophil cultures. After 24 h, the percentageof region a was higher (P = 0.01), while thepercentage of both region b and c were lower (P= 0.02 and P = 0.01, respectively) than those ofsuspension neutrophils (Fig. 4). Representative
cytograms are shown in Fig. 5A.

Results

Inspection of neutrophil nuclei in eachindicated region
In every experiment, the cells contained inregion a exhibited lobulated nuclei. The cells inregion b had bright and small nuclei comparedwith the cells in region a. The cells in region cwere all in various sizes of nuclei with the least
dense chromatin of any of the cell groups(Fig. 1).

The differential percentage of neutrophils
incubated with A549 cells in a preactivated state with TNF-α
After 6 h, the percentage of region a was higher(P = 0.01), while the percentage of region c was
lower (P = 0.01) than those of suspension neutrophils. After 24 h, the percentage of region a
was higher (P = 0.01) and the percentage ofboth region b and c were lower (P = 0.01, both)

The differential percentage of neutrophils incubated with A549 cells in a quiescent state
After 6 h of incubation time, the percentage of25

Y. Kometani et al.

*†

100

A

than those of suspension neutrophils. Furthermore, the percentage of region a at 6 h was
higher and the percentage of region c at both 6
and 24 h were lower (P = 0.01 and P = 0.03 for
6 h and 24 h, respectively) than those of neutrophils incubated with quiescent A549 cells (Fig.
4). Representative cytograms are shown in Fig.
5B.

* *

Region c

Neutrophil number (% of total number)
Region b
Region a

80

60

40

20

0

6h

24 h

TUNEL positive cells

20

B

FITC positive cells showing small nuclei withhypercondensation of chromatin labeled with
FITC-conjugated deoxynucleotides (Fig. 3C)
were almost all located in region b, some extending to region c (Fig. 3B). At 6 h, these
FITC positive cell percentages were not significantly different, but at 24 h, the percentage washigher in suspension neutrophils than in neutrophils in the other 2 groups (Fig. 4D).

15

10

*

*

5

0

6h

24 h

40

C
Discussion

30

Apoptosis or programmed cell death is a mechanism of cellular death believed to play a pivotal role in a wide variety of physiological conditions (Thompson, 1995), and its deregulation
has been proposed to contribute to the pathogenesis of many diseases (Newell et al., 1990).
The morphological event characterizing apoptosis is nuclear pyknosis associated with chromatin condensation and the biological “hallmark” is internucleosomal chromatin fragmentation (Wyllie et al., 1980). The apoptosis process does not elicit an inflammatory response,
nor does it damage adjacent cells (Fesus et al.,1991) because apoptotic neutrophils are rapidly
ingested by alveolar macrophages (Cox et al.,1995).
In our experiment, TUNEL positive cells
were almost all located in region b, some extending to region c. Taking into account that
TUNEL is a method of choice for rapid identification and quantification of DNA fragmentation, the cells in region b which were strongly
TUNEL positive with small condensed nuclei
were thought to be apoptotic cells when the nuclear chromatin had begun to condense. Be-

20

*

10

*†

*†

TUNEL positive cell number(% of total number)

0

6h
6

24 h

D

5
4

* *

3
2
1
0

6h

24 h

Control
Neutrophils incubated with quiescent A549 cells
Neutrophils incubated with preactivated A549 cells

Fig. 4. Differential percentages of neutrophils ineach indicated region (a, b and c) and percentages of
TUNEL positive cells. The data are expressed asmean ± SD from 8 independent experiments. *P <0.05 compared with control neutrophils. †P < 0.05
compared with neutrophils incubated with quiescentA549 cells.

26

PI fluorescence peakA

PI fluorescence peakA549 cells inhibit neutrophil apoptosis

PI fluorescence value

PI fluorescence value

6h

24 h

Fig. 5. Representative cytograms of neutrophils incubated with quiescent A549
cells at 6 and 24 h (A), andneutrophils incubated withpreactivated A549 cells at 6
and 24 h (B).

PI fluorescence peakB

PI fluorescence peak

Neutrophils incubated with quiescent A549 cells

PI fluorescence value

PI fluorescence value

6h

24 h
Neutrophils incubated with preactivated A549 cells
present the static measurement of a dynamic
process and reflect the equilibrium between thedevelopment of apoptosis and phagocytosis of
apoptotic neutrophils by macrophages. Theoretically, inhibition of neutrophil apoptosis
could result in increased numbers of viable
neutrophils. This may prolong the neutrophil
alveolitis and contribute to acute lung injury.
But from the stand point of host defense, thisshould be desirable because the active life ofneutrophils may be prolonged in order to fightinvading microbes and to allow time for recruited monocytes, which are unable to take up apoptotic neutrophils, to differentiate into phagocytically competent macrophages.
When the lung is in an inflammatory state,neutrophils can survive longer than those in aphysiological state, thus possibly preparing
against external noxious factor which can cause
TNF-α secretion from alveolar macrophages.

sides, the cells in region c were supposed to be
apoptotic bodies which were all in various sizesof nuclei with the least dense chromatin of anyof the cell groups. In examining the images, it
became clear that the cells progressed frompurified control cells in region a to apoptotic
cells in region b, and then to possible apoptotic
bodies in region c.
Comparison of neutrophils incubated withquiescent alveolar epithelial cells to those ofsuspension neutrophil cultures demonstrated
that quiescent alveolar epithelial cells delayed
neutrophil apoptosis, and this inhibitory effectagainst neutrophil apoptosis was enhanced
when A549 cells had been treated with TNF-αfor 24 h prior to their use for neutrophil plating.
These data suggest that alveolar type II cells
may be potentially able to contribute in promoting inflammation by delaying neutrophil apoptosis. Estimates of neutrophil apoptosis re27

Y. Kometani et al.

Taken together, we supposed that alveolar epithelial cells are not only serving simply as apassive barrier but also play an active role inself defense by prolonging neutrophil survival
and progress inflammatory response for thehost in both physiological and pathologicalstates.
As previously reported, some cytokines
affect the rate of neutrophil apoptosis (Brach etal., 1992; Lee et al., 1993). Indeed, TNF-α treatment of alveolar epithelial cells could affect therate of neutrophil apoptosis by causing the production of interleukin-6 which can delay
neutrophil apoptosis (Biffl et al., 1996); ourexperimental conditions, in which the epithelial
cells were washed twice to remove TNF-α andconditioned medium containing cytokines before plating of neutrophils, make this possibility
unlikely.
On the other hand, Ginis and Faller (1997)
reported that upregulation of adhesion molecules on the surface of the vascular endothelium
delayed neutrophil apoptosis. Due to their low
baseline expression of adhesion molecules(Rothlein et al., 1988), A549 cells were selected
as a source of alveolar epithelial cells for thepresent study. But TNF-α stimulation specifically upregulates intercellular adhesion
molecule-1 (ICAM-1) expression on A549 cells(Arnold and König, 1996). Signals derived
from these adhesion molecules on alveolar epithelial cells might affect neutrophil apoptosis.
More detailed research should be undertaken to
determine the cause of this effect.
One advantage of LSC is the possibility ofcell staining on slides which eliminates cell loss
which inevitably occurs due to repeated centrifugations during sample preparation for flow
cytometry. Another advantage stems from thepossibility of localization of particular cells ona slide for visual microscopic inspection, after
the initial measurement and automatic relocation (Kamentsky and Kamentsky, 1991).
Because the specimen is spatially fixed on a
glass microscope slide, individual specimens orcells can be analysed repeatedly if desired. The
instrument, thus, combines advantages of bothflow and image cytometry. Li and coworkers(1996) used LSC for the detection of DNA

strand breaks labeled with fluorochrome inapoptotic cells and proposed its advantages.
The methods for the detection of apoptosis areusually laborious and time consuming. In addition, it can be difficult to get quantitative information on the number of apoptotic cells. However, LSC can identify features of cells fromfluorescent measurements and provide us with
rapid quantitative information in a convenient
way because its preparation requirements areminimal.
In summary, the experiments revealed thatincubation of neutrophils with A549 cells inhibited neutrophil apoptosis, and that this inhibitory effect was enhanced when the A549 cells
were preactivated with TNF-α for 24 h. A
greater understanding of alveolar epithelial celland neutrophil interaction would provide new
insights into the pathophysiology of lung diseases, which in turn may promote the development of novel approaches to their treatment.

Ackowledgments: We express our appreciation to Prof.
Takao Sasaki, Third Dept. of Internal Medicine, Prof.
Nobuaki Kaibara, First Dept. of Surgery, and Prof.
Tadashi Terada, Second Dept. of Pathology, Faculty of
Medicine, Tottori University, for their kind advice.

References
1 Arnold R, König W. ICAM-1 expression andlow-molecular-weight G-protein activation ofhuman bronchial epithelial cells(A549) infectedwith RSV. J Leukoc Biol 1996;60:766–771.
2 Biffl WL, Moore EE, Moore FA, Barnet CC Jr,
Carl VS, Peterson VM. Interleukin-6 delays
neutrophil apoptosis. Arch Surg 1996;131:24–30.
3 Brach MA, de Vos S, Gruss HJ, Hermann F. Prolongation of survival of human polymorphonuclear neutrophils by granulocyte-macrophagecolony-stimulating factor is caused by inhibitionof programmed cell death. Blood 1992;80:2920–
2924.
4 Cox G, Crossly J, Xing Z. Macrophage engulfment of apoptotic neutrophils contribute to theresolution of acute pulmonary inflammation in
vivo. Am J Respir Cell Mol Biol 1995;12:232–
237.
5 Diaz P, Gonzales MC, Galleguillos FR, Ancic P,
Cromwell O, Shepherd D, et al. Leukocytes andmediators in bronchoalveolar lavage during

28

A549 cells inhibit neutrophil apoptosis

Biol 1993;54:283–288.
14 Li X, Melamed MR, Darzynkiewicz Z. Detectionof apoptosis and DNA replication by differential
labeling of DNA strand beaks with fluorochromes of different color. Exp Cell Res 1996;
222:28–37.
15 Newell MK, Haughn LJ, Maroun CR, Julius MH.
Death of mature T-cell receptor antigen. Nature
1990;347:286–289.
16 Rothlein R, Czajkowski M, O’Neill MM, Marlin
SD, Mainolfi E, et al. Induction of intercellularadhesion molecule-1 on primary and continuous
cell lines by pro-inflammatory cytokines. J Immunol 1988;141:1665–1669.
17 Sibille Y, Reynolds HY. Macrophages andpolymorphonuclear neutrophils in lung defense
and injury. Am Rev Respir Dis 1990;141:471–
501.
18 Thompson C. Apoptosis in the pathogenesis andtreatment of disease. Science 1995;267:1456–
1462.
19 Tosi MF, Stark JM, Smith CW, Hamedani A,
Gruenert DC, Infeld MD. Induction of ICAM-1
expression on human airway epithelial cells by
inflammatory cytokines: effects on neutrophilepithelial cell adhesion. Am J Respir Cell Mol
Biol 1992;7:214–221.
20 Wyllie AH, Kerr JFR, Currie AR. Cell death: thesignificance of apoptosis. Int Rev Cytol 1980;
68:51–306.

allergen-induced late-phase asthmatic reactions.
Am Rev Respir Dis 1989;139:1383–1389.
6 Ferrante A, Thong YH. Optimal conditions for
simultaneous purification of mononuclear andpolymorphonuclear leukocytes from humanblood by the Hypaque-Ficoll method. J Immunol
Methods 1980;36:109–117.
7 Fesus L, Davies PJA, Piacentini M. Apoptosis:
molecular mechanisms in programmed cell death.
Eur J Cell Biol 1991;56:170–177.
8 Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell death in situ via
specific labeling of nuclear DNA fragmentation.
J Cell Biol 1992;119:493–501.
9 Ginis I, Faller DV. Protection from apoptosis in
human neutrophils is determined by the surfaceof adhesion. Am J Physiol 1997;272(1 Pt 1):
C295–C309.
10 Golstein P, Ojcius DM, Young JD. Cell deathmechanisms and immune system. Immunol Rev
1991;121:29–65.
11 Haslett C, Savill JS, Meagher LC. The
neutrophil. Curr Opin Immunol 1989;2:10–18.
12 Kamentsky LA, Kamentsky LD. Microscopebased multiparameter laser scanning cytometeryielding data comparable to flow cytometry data.
Cytometry 1991;12:381–387.
13 Lee A, Whyte MK, Haslett C. Inhibition of apoptosis and prolongation of neutrophil functional
longevity by inflammatory mediators. J Leukoc(Received December 2, Accepted December 8, 1998)

29