Yonago Acta medica 1997;40:43–52

Cadmium Induces Interleukin-8 in Alveolar Epithelial Type II Cells (A549)
Katsuhiko Fukuda, Yutaka Hitsuda and Tadashi Igishi
Third Department of Internal Medicine, Faculty of Medicine, Tottori University, Yonago
683, Japan
Cadmium (Cd) is a contaminant in cigarette smoke and may play an important role in
the pathogenesis of smoking-related emphysema. It is reported that repeated Cd
exposure causes emphysema after neutrophil infiltration in the rat lung. Interleukin-8(IL-8) is a major neutrophil chemotactic factor commonly involved in a variety of
pulmonary disorders including emphysema. The aim of this study is to elucidate theeffect of Cd on IL-8 production of alveolar epithelial type II cells, using the A549 cellline. We used 1–100 µM of cadmium chloride (CdCl2) to stimulate the cells for a 48 h
period. IL-8 production (2957 ± 137 pg/mL) without any cytotoxic effect was observedin the cell supernatants after 24 h exposure to 50 µM CdCl 2. The Cd-removed
supernatant caused neutrophil chemotaxis and was inhibited by the anti-IL-8 antibody.
Cd-induced IL-8 was inhibited by EDTA, and the inhibition was blocked by copper (II)β or tumor necrosis factor-α
α antibodies did
chloride. The addition of anti-interleukin-1β
not diminish IL-8 release induced by Cd. These results suggest that Cd increases the
production of IL-8 without any cytotoxic effect in alveolar epithelial cells, which may
be an important factor in the developmental process of cigarette smoking-related
emphysema.
key words: A549; alveolar epithelial type II cell; cadmium chloride; emphysema; interleukin-8

correlated with the Cd content of the lung tissue(Hirst et al., 1973).
It has been elucidated that neutrophil orneutrophil elastase plays a principal role in theestablishment of emphysema in smokers.
Recently, it is reported that an increase in theneutrophil influx in smokers was regulated byinterleukin-8 (IL-8), a potent neutrophil
chemotactic cytokine (Waltz and Kunkel,
1989). Although IL-8 is produced by various
types of cells in the lung (Bagglioni et al.,1989), the alveolar epithelial type II cell (type IIcell) is known to be one of the major sources ofthis cytokine.
In this study, we investigated the effects of
Cd on IL-8 production in type II cells.

Cadmium (Cd) is not only an air pollutant, but
is also one component of cigarette smoke. Cd
inhalation can cause mainly 2 types of lungdamage depending on its concentration. Acutechemical pneumonitis and bronchitis after ahigh concentration of Cd inhalation are believed to be the result of direct toxic effect of Cd on
the lung tissue (Nermery, 1990). On the other
hand, a low concentration of Cd inhalation cancause lung emphysema in rats. Since then, Cd
within tobacco smoke has been thought to be acausative factor of cigarette-related emphysema
in humans. Human lungs with emphysema had
a significantly higher Cd content than thosefrom a nonemphysematous age-matched control group and the degree of emphysema was

Abbreviations: BALF, bronchoalveolar lavage fluids; D-MEM/F-12, Dulbecco’s modified Eagle medium:
nutrient mixture F-12; FBS, fetal bovine serum; IL-8, interleukin-8; MTS, 3-(4,5-dimethylthiazol-2-yi)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; PBS, phosphate-buffered saline;
PMS, phenazine methosulfate

43

K. Fukuda et al.

Fig. 1. A confluent monolayer of A549 cells from passage level 82nd was cultured in D-MEM/F-12 media
with 10% FBS. The cells were stained with a tannic acid and polyclome stain. The dark cytoplasmic granules
are the lamellar bodies. × 400.

µg/mL amphotericin B (Wako Pure Chemical
Industries Ltd., Osaka, Japan). Cultures wereincubated in a humidified 95% air and 5% CO2
atmosphere at 37˚C for 16 h to achieve confluence. A549 monolayers were washed withphosphate-buffered saline (PBS), replaced withserum free media (D-MEM/F-12) and incubated for 2 h. Cadmium chloride (CdCl 2;
Wako) and other stimulants were suspended in
fresh complete media for additional times (8,16, 24 and 48 h). For blocking studies, A549
cultures were incubated with 100 neutralizing
U/mL of monoclonal antibodies to humaninterleukin-1β (IL-1β) or tumor necrosis factorα (TNF-α; Genzyme, Cambridge, MA) 30 min
after the addition of either Cd, TNF-α (10 U/mL) or IL-1β (10 U/mL). After each time ofincubation with stimulants, the supernatants
were filter sterilized, and stored at –30˚C until
the IL-8 enzyme-linked immunosorbent assay(ELISA). The cells were lysed with 0.05%
triton X-100 in PBS or freeze-thawed 4 times
by alcohol/dry ice and a 37˚C water bathaccording to Gaffar and colleagues (1986) and
Rosenthal and co-workers (1994). The cell

Materials and Methods

Cell preparation
The A549 cells (American Type Culture
Collection, Rockville, MD) we used are fromthe epithelial type II cell line derived from apatient with alveolar cell carcinoma (Leiber etal., 1976). The A549 cell line retains thefeatures of type II cells, such as a cuboidal
appearance, cytoplasmic multilamellar bodies
and the synthesis of surfactants. We usedconfluent cultures of the A549 cell line at the82nd to 86th passages (Fig. 1) in the present
study. After trypsinization, the cells wereplated at a concentration of 3 × 105 cells/well to
24-well microtiter plates (Nunc, Roskilde,
Denmark) in 1 mL of Dulbecco’s modified
Eagle medium: nutrient mixture F-12, 1:1 (DMEM/F-12) media containing 365 mg/L Lglutamine (Life Technologies, Inc., Grand
Island, NY), 10% heat-inactivated fetal bovine
serum (FBS; Life Technologies), 100 units/mL
penicillin, 100 µg/mL streptomycin and 0.25
44

Cd induces IL-8 in A549

lysates were centrifuged and suspended in fresh
media to quantify intracellular IL-8. CdCl 2,
copper (II) chloride (CuCl 2; Wako), EDTA,
lipopolysaccharide (LPS; W E. coli 055: B5;
Difco Laboratories, Detroit, MI) and otherreagents were dissolved in distilled water andsterilized with a MILLEX-GS 0.22 µm filter(Millipore Ltd., Tokyo, Japan) before use.

bated for 2 h with an enzyme-linked polyclonal
antibody specific for IL-8. Following a wash toremove any unbound antibody-enzyme reagent,peroxidase substrate was added and incubatedfor 20 min. Then the reaction was terminatedwith sulphuric acid and the intensity of the color
was measured at 450 nm in the plate leader(MPR-A4i).

Viability and cytotoxic assay

Chemotactic assay

Cell viability was evaluated with the tetrazolium compound, MTS [3-(4,5-dimethylthiazol-2-yi)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium, inner salt]
assay (MTS assay) and trypan blue exclusion todefine the cytotoxic threshold dose. The culture
media were removed at 8, 16, 24 and 48 h after
incubation of the cells with Cd (1, 5, 10, 50 and
100 µM), 20 µL of the MTS/phenazine methosulfate (PMS) solution (Promega Corporation,
Woods Hollow Road, WI) were added to theculture wells and agitated cell monolayers in
0.2 mL of D-MEM/F-12 media. After 1 h, a
100 µL sample of each extraction was placed in
duplicate in a 96-well plate and the amount offormazan production was assayed by measuring
the density at 492 nm using a plate leader(MPR-A4i; Tosoh, Tokyo, Japan).
A549 cell monolayers with 50 µM Cdtreatment or without treatment were detachedusing trypsin, centrifuged and resuspended in
fresh media with tripan blue and inspectedunder the haemocytometer. Viable cells wereexpressed as a percentage of the values obtained in the initial incubation.

Chemotactic experiments were done using amodified Boyden chamber method (Cates et al.,1978). EDTA at 10–4 M was added to the Cdcontaining samples after 24 h exposure, andfiltrated using Ultrafree-C3 (molecular cut-off
3000; Millipore) to remove the Cd-EDTA
complex. Human neutrophils, prepared fromnormal human peripheral blood, were separated
and adjusted by Hanks’ Balanced Salt Solution(Life Technologies) at a concentration of 2 ×
106 cells/mL. The isolated neutrophils were atleast 97% viable by trypan blue exclusion and
95% pure. Cd-removed supernatants (218 µL)
were added to the bottom of Blind-Wellchemotaxis chambers (Neuro Probe, Inc., Cabin
John, MD). The positive control was 10–7 M Nformyl-methionyl-leucyl-phenylalanine (Wako)
and the negative was D-MEM/F-12 alone. The
neutrophil suspensions (200 µL) were added tothe top chambers after separating from thebottom chambers by 3-µm polycarbonate filters(PVP-free; Portics Corp., Livermore, CA). For
neutralizing studies, the test samples wereincubated with a 1:100 dilution of monoclonalanti-human IL-8 antibody (Genzyme) at 37˚C
for 1 h before adding to the chamber. The
chambers were incubated in humidified 95% air
and 5% CO2 at 37˚C for 1 h. The filters were
removed, fixed in methanol and stained by
May-Giemsa. Neutrophils that migrated to thebottom chambers were counted by using a × 400
magnification in 10 fields. Chemotactic activity was shown as a percentage of the positive
control as previously described by Standifordand colleagues (1990).

IL-8 assay
The concentration of IL-8 was measured using
a specific ELISA kit (Amersham Internationalplc, Bucks, United Kingdom). This assay employs the quantitative immunometric, “sandwich” enzyme immunoassay technique. Briefly, 100 µL of samples and standards, both induplicate, were added with assay diluent to a
microtiter plate precoated with a monoclonal
antibody specific to IL-8. After washing away
any unbound sample proteins, these were incu45

K. Fukuda et al.

Absorbance 492 nm

1.4
1.2

A

∗

∗

∗

1.0

∗

0.8

∗

0.6

∗

0.4
0.2

0

8

16

24

48 (h)

200

Viable cells (% total)

Control
Cd 1 µM
Cd 5 µM
Cd 10 µM
Cd 50 µM
Cd 100 µM

180

B

160
140

Fig. 2. Effect of Cd on A549 cell viability.
A : MTS assay for the indicatedconcentrations of Cd and time intervals over48 h. Each value represented the mean from
6 replicate wells; *P < 0.01. B: The numberof viable cells was monitored by trypan blue
exclusion for the indicated time intervals.
The open and closed circles respectively
indicate untreated control cells and 50 µM
Cd-exposed viable cells. The values are
expressed as a percentage of viable cells
counted in initial incubations. The data areexpressed as mean ± SD from 6 independent
experiments; *P < 0.05.

120

∗

100

Control
Cd 50 µM

80
60

0

8

16

24

48 (h)h (P < 0.01), began to decrease at 16 h, andlowered significantly as compared with
controls at 24 and 48 h (P < 0.01).
Figure 2B shows the result of the trypan
blue exclusion for the indicated time intervals.
Up to 24 h, there were no significant differences
between 50 µM Cd-treated cells and the controls. At 48 h, the number of viable cells with
Cd treatment was significantly smaller than thatof the controls (P < 0.05).

Statistical analysis
Data are expressed as mean ± SD. The KruskalWallis and Mann-Whitney U tests were used
for statistical comparisons of results between
different groups, and were considered significant when P values were < 0.05.

Results

IL-8 contents in supernatants and celllysates

MTS assay and trypan blue exclusion

A549 cells cultured in the presence of Cd at aconcentration of 1, 5, 10 and 50 µM induced
steep time- and dose-dependent IL-8 release. In
every time and dose, Cd-exposed cell cultures
showed significantly (P < 0.01) high IL-8 levels
compared with non-exposed controls. At 24 hpostexposure, 50 µM Cd-induced IL-8 releases
reached a plateau (2957 ± 137 pg/mL), resulting
in a 15-fold increase when compared with untreated controls (190±11pg/mL)(Fig. 3A).

The result of the MTS assay is shown in Fig. 2A.
Up to 24 h, the absorbance of 1, 5, 10 and 50µM Cd-exposed cells did not decrease compared with non-treated control cells (controls).
In 50 µM Cd-exposed cells, MTS activities weresignificantly higher than that of the controls at
8, 16 and 24 h (P < 0.01, respectively). In 100µM exposure of Cd, the absorbance wassignificantly higher than that of the controls at 846

IL-8 in supernatant (pg/mL)

Cd induces IL-8 in A549

3500
3000

A

2500

Control
Cd 1 µM
Cd 5 µM
Cd 10 µM
Cd 50 µM
Cd 100 µM

2000

∗

1500
1000
500

∗
∗
∗
∗
∗

∗

∗∗ ∗

∗

∗

∗

∗

∗

∗∗

∗

Fig. 3. A: The concentration of IL-8 in the
supernatants of A549 cells stimulated withvaried concentrations of Cd for 8, 16, 24 and48 h. The data are expressed as mean ± SD
from 6 independent experiments; *P < 0.01.
B: The concentration of IL-8 in lysates ofA549 cells. The data are expressed as mean ±SD from 6 independent experiments; *P <0.05, **P < 0.01.

08

IL-8 in lysate (pg/mL)

∗

∗

16

24

∗

90
80

B

48

(h)

∗ ∗∗

∗∗

70

Control, triton X
Control, freeze-thaw
Cd, triton X
Cd, freeze-thaw

60
50
40
30
20
10
0

0

8

16

24

48 (h)

Intracellular IL-8 concentrations were
quantified after the cells were lysed with 0.05%
triton X-100 or repeated freeze-thawing. At 8 h
and 16 h, 50 µM Cd-exposed cell lysates fromthe freeze-thaw treatment showed significantly(8 h; P < 0.01, 16 h; P < 0.05) high IL-8 levels
compared with the controls. At 24 h, the IL-8
contents in the Cd-exposed cell lysates weresignificantly greater than those of the nonexposed controls (triton X-100 treated; P <0.05, freeze-thaw; P < 0.01) (Fig. 3B). But
these IL-8 levels were much lower than those of
supernatants derived from Cd-exposed cell
cultures.

IL-8 (pg/mL)

7000
6000
5000
4000
3000
2000
1000
0

Control

LPS

Cd

TNF-α

IL-1β

100 µg/mL

50 M

10 U/mL

10 U/mL

Fig. 4. The concentration of IL-8 in the supernatantsof A549 cells stimulated by pretoxic doses of LPS,
Cd, TNF-α and IL-1β for 24 h. The data areexpressed as mean ± SD from 6 independent experiments.

IL-8 release induced by different stimulants
5032 ± 113 pg/mL and 5809 ± 121 pg/mL,
respectively. All of them were significantly
higher than that of controls (P < 0.01). Cdinduced IL-8 production was about 2.5 times
potent compared with LPS.

As shown in Fig. 4, peak levels of IL-8 release
were observed after 24 h exposure. The IL-8
levels induced by non-cytotoxic doses of LPS,
Cd, TNF-α- and IL-1β-treated cell cultures
were 1169 ± 159 pg/mL, 2957 ± 137 pg/mL,
47

K. Fukuda et al.

Neutrophil migration(% of positive control)60∗

Chemotactic activity of cell culture supernatants

50

As shown in Fig. 5, culture supernatants from
Cd-treated A549 cell suspensions induced
significant chemotactic responses comparedwith the controls (P < 0.01). This activity was
inhibited by 52% in the presence of the anti-IL8 antibody.

40
30
20
10
0

Cd

Cd

Con
trol

+ an

Effect of EDTA and CuCl2 on IL-8 releaseinduced by Cd

ti-IL
-8

EDTA at 100 µM inhibited 50 µM Cd-induced
IL-8 production to about 86%, but 100 µg/mL
LPS-induced IL-8 production was not inhibitedby 1–100 µM EDTA (Fig. 6A). The inhibition
was blocked by the presence of CuCl2 at 100µM. The cultured cells exposed to CuCl2 solely
produced a significantly large amount of IL-8
(1270 ± 129 pg/mL) compared with the controls(P < 0.01). These IL-8 levels were significantly
lower than those produced by the cells whichwere solely exposed to 50 µM Cd (Fig. 6B).

Fig. 5. Chemotactic activity of the supernatants of
A549 cells. The activity was represented as neutrophil migration and was expressed as a percentage ofthe positive control (10–7 M formylmethionyleucylphenylalanine) from 6 independent experiments; *P< 0.05.

A
IL-8 (pg/mL)

3500
3000

Effects of anti-TNF- α and anti-IL-1 β
antibodies

2500
2000
1500

Anti-TNF-α or anti-IL-1β monoclonal antibodies failed to block IL-8 release from Cdtreated and non-treated cells. The addition ofanti-TNF-α or anti-IL-1β monoclonal antibodies completely blocked the IL-8 release induced by exogenous TNF-α or IL-1β, respectively (Fig. 7).

1000
500
0

EDTA (µM)–

IL-8 (pg/mL)

3500

–

1

10

100

B

3000

Discussion

2500
2000

Emphysema in humans takes several different
forms: centrilobular, panlobular, paracicatricaland localized types (Foster et al., 1993). The
varying morphologic features of these forms ofemphysema suggest that they differ in patho-

1500
1000
500
0

Cd
50 µM –EDTA 100 µM –Cu(µM) –

+ – +
– + +
– – –

+
+

+
+

+ –
+ –

1

10 100 100

Fig. 6. A: The effect of EDTA on Cd- and LPS-induced IL-8 in A549 cells. Cells were incubated with either
50 µM Cd (stripe columns) and 100 µg/mL LPS (solid columns), respectively. The data are expressed asmean ± SD from 6 independent experiments. B: The effect of EDTA and CuCl2 on Cd-induced IL-8 in A549
cells. The data are expressed as mean ± SD from 6 independent experiments.

48

Cd induces IL-8 in A549

7000

IL-8 (pg/mL)

6000
5000
4000
3000
2000
1000
0

IL-1β

IL-1 β+ ant
i-IL1βti-TNNF-α
anti-T
L-1β

anti-I

n
α+ a
TNF-α
α

TNF-

Cd +

Cd

Cd +

rol

α
TNFAntiIL-1β

Anti-

Cont

Fig. 7. The effect of anti-TNF-α and antiIL-1β antibodies. Cd (50 µM), TNF-α (10U/mL) and IL-1β (10 U/mL) were added 30
min before the antibodies (100 U/mL), andIL-8 in the cell cultures was measured after
24 h incubation. Values represent mean ±SD of 6 cultures obtained from representative experiments.

Fαgenesis. Centrilobular emphysema associatedwith cigarette smoking is the most common
form and represents air space enlargement of
respiratory bronchioles. Cd is a contaminant ofcigarette smoke and it has been suggested that it
plays an important role in cigarette smoking
emphysema, because repeated exposure to subtoxic concentrations of Cd can induce destructive peribronchial lesions in rat lungs resembling human centrilobular emphysema (Snider
et al., 1973, 1988). However, the mechanism of
this has not been elucidated even until the present time.
Neutrophils are accepted now as the predominant effector cells for emphysema incigarette smokers. Neutrophils increase in
number in bronchoalveolar lavage fluids (BALF)
and lung tissues in smokers (Hunninghake et al.,
1979; Hunninghake and Crystal, 1983) andsmoking increases the activity of neutrophil
elastase in BALF (Fera et al., 1986). Snider andcolleagues (1973, 1988) also observed that pulmonary neutrophilia occurs immediately after
Cd exposure in rats.
IL-8, a potent chemotactic cytokine, was recently reported to increase in the airway secretion of patients with emphysema (Yamamoto etal., 1996), and it has been suggested to enhance
the recruitment of neutrophils in the lung
(Bagglioni et al., 1989). Although the alveolar
macrophages are major producers of IL-8
(Cooper et al., 1973), alveolar type II cells also

produce IL-8 within the alveoli (Standiford etal., 1991). In emphysema, type II cells are important as a source of IL-8, because hyperplasiaof these cells has been observed in the alveolar
walls of human emphysematous lesions (Nagai
and Konno, 1993) and proliferation of type II
cells also occurred after repeated Cd inhalationin the rat lung (Hayes et al., 1976).
Since type II cells were difficult to obtain inpure culture, we investigated the effect of Cd on
the IL-8 production using A549, type II cellline. Cells of the A549 cell line, at low and highpassage levels, have properties of typical alveolar epithelial type II cells (Leiber et al., 1976).
However, between the 20th and 27th sublines ofA549, a different Cd-cytotoxic response wasobserved (Kang et al., 1990). In our preliminary studies, there was no difference in Cd
cytotoxicity among the 82nd to 86th passagesof the A549 sublines which we used, and cytotoxic threshold dose of these sublines werehigher than those of younger sublines.
In the present study, dose- and time-dependent
IL-8 production was found in 1, 5 and 10 µM of
Cd until 48 h postexposure, and in 50 µM Cd up to
24 h without any significant decrease in cell
viability (Figs. 2 and 3A). There was no difference in the level of IL-8 induced by 50 µM Cd
at 24 and 48 h. This may be due to presecreted
IL-8 remaining in the supernatant without
decay. The marked reduction of the IL-8 levelat 100 µM Cd was probably due to the severe49

K. Fukuda et al.

cytotoxic effect of Cd, because MTS activity
was reduced after 16 h postexposure. These
results indicate that 50 µM Cd is optimal for inducing IL-8 secretion without cytotoxic effect.
Up to 24 h, 50 µM Cd-treated cells showedsignificantly high MTS activity and marked IL8 production (Figs. 2A and 3A), although Cdexposed viable cells did not increase in number,determined by trypan blue exclusion (Fig. 2B).
MTS activity represents mitochondrial metabolization of living cells. In alveolar macrophages, Cd was reported to be bound strongly tomitochondrial dehydrogenases and behave as
an inhibitor of electron transport (Mustafa andCross, 1971). On the other hand, numerous
mitochondria and their hypertrophy have been
observed in type II cells from Cd-treated rats
(Hart et al., 1990; Murthy and Holovack, 1991).
The high MTS activity of cells exposed to 50µM Cd in the present study may be explainedby the phenomenon of hormesis, the elevationof cellular metabolic responses at subtoxic concentrations of cell toxicants (Swisher, 1989).
Accelerated mitochondrial functions may
contribute to high MTS activity in our study.
To evaluate the total production of IL-8,
intracellular IL-8 contents were also examinedby triton X and freeze-thaw treatments (Fig.
3B). The levels of IL-8 obtained with thesetreatments was negligible compared with thoseof supernatants of Cd-exposed cells. Accordingly, the amount of extracellular IL-8 secretion
was thought to be nearly equal to total IL-8 production.
Chemotactic activity for human neutrophils
was examined to determine whether IL-8 induced by Cd in the present study was biologically
active. It is reported that Cd itself inhibits chemotactic activity (Ward et al., 1986). Since, the 50µM Cd-containing medium also allowed few
neutrophil to migrate in our preliminary studies,
ultrafiltration was performed to remove Cd
before the assay. These supernatants induced
potent neutrophil chemotactic activity, and the
addition of anti-IL-8 inhibited this activity significantly (Fig. 5). These results indicate that Cdinduced IL-8 is a major neutrophil chemotactic
factor in the present study. Therefore, neutro-

phil infiltration in the lung following Cd exposure may be mainly due to the action of IL-8.
For a further demonstration that endotoxin
contaminants are not responsible, but Cd itself
induced IL-8 production, we examined the effects of a chelating agent for divalent cations,
EDTA. Cd was a stronger stimulant for IL-8
production than LPS in A549 cells. Cd-induced
IL-8 production was inhibited by EDTA, andthis was blocked by the addition of CuCl2. Cu 2+
binding affinity for EDTA is stronger than thatof Cd2+. Uncoupling Cu 2+ may displace Cd2+
and the effect of Cd2+ liberated from EDTA
during IL-8 production was revived when the
EDTA binding capacity was saturated by Cu2+ .
These results suggest that the induction of IL-8
could be ascribed mainly to this uncoupling
cation, Cd2+.
TNF-α and IL-1β were reported to be mainly generated by alveolar macrophages, andwere potent stimuli for the induction of IL-8 bytype II cells (Kunkel et al., 1991). IL-8 production by A549 cells and bronchial epithelial cells
has been shown to be induced by TNF-α andIL-1β (Standiford et al., 1991; Cromwell et al.,1992). Moreover, pulmonary tumors produce avariety of growth factors and cytokines that
may act in both autocrine and paracrine fashion(Wang et al., 1996). To determine whether A549,
a characterized human lung adenocarcinomacell line, produced TNF-α or IL-1β in response
to Cd, neutralizing experiments of TNF-α andIL-1β were conducted using the monoclonal
antibodies. As shown in Fig. 7, co-incubationof Cd with these monoclonal antibodies did notdiminish the level of IL-8 concentration,
indicating that A549 produced little TNF-α orIL-1β in response to Cd.
In summary, the present study demonstrates
that Cd increased the production of biologically
active IL-8 by A549, alveolar epithelial type II
cells, suggesting that Cd plays an important rolein the development of cigarette smoking emphysema.

50

Cd induces IL-8 in A549

V, Crystal R. Inflammatory and immune process
in the human lung in health and disease. Evaluation by bronchoalveolar lavage. Am J Pathol
1979;97:149–206.
12 Hunninghake G, Crystal RC. Cigarette smoking
and lung destruction. Accumulation of neutrophils in the lungs of cigarette smokers. Am Rev
Resir Dis 1983;128:833–838.
13 Kang YJ, Nuutero ST, Clapper JA, Jenkins P,
Enger MD. Cellular cadmium responses in subpopulations T20 and T27 of human lung carcinoma A549 cells. Toxicology 1990;61:195–203.
14 Kunkel SL, Stanford T, Kasahara K, Strieter RM.
Interleukin-8 (IL-8): the major neutrophil chemotactic factor in the lung. Exp Lung Res 1991;17:
17–23.
15 Leiber M, Smith B, Szakal A, Nelson-Rees W,
Todaro G. A continuos tumor cell line from human lung carcinoma with properties of type II
alveolar epithelial cells. Int J Cancer 1976;17:
62–71.
16 Murthy RC, Holovack MJ. Ultrastructuralchange in rat lung exposed to combination of Cd,
zinc, copper, and nickel. J Submicrosc Cytol
Pathol 1991;23:289–293.
17 Mustafa MG, Cross CE. Pulmonary alveolar
macrophage. Oxidative metabolism of isolated
cells and mitochondria and effect of Cd ion onelectron- and energy-transfer reactions. Biochemistry 1971;10:4176–7185.
18 Nagai A, Konno K. Alveolar type II cells incentrilobular emphysema in lesions of humanlung: an ultrastructual study. Nippon Kyoubu
Shikkan Gakkai Zassi 1993;31:421–425 (in Japanese).
19 Nermery B. Metal toxicity and the respiratory
tract. Eur Respir J 1990;3:202–219.
20 Rosenthal GJ, Germolec DR, Blanka ME,
Corsini E, Simeonova P, Pollock P, et al. Asbestos stimulate IL-8 production from human lungepithelial cells. J Immunol 1994;153:3237–3244.
21 Snider GL, Hayes JA, Korthy AL, Lewis GP.
Centrilobular emphysema experimentally induced by Cd chloride aerosol. Am Rev Respir Dis
1973;108:40–48.
22 Snider GL, Lucey EC, Faris B, Yangja JL. Cadmium chloride-induced air-space enlargementwith interstitial pulmonary fibrosis is not associate with destruction of lung elastin. Am Rev
Respir Dis 1988;137:918–923.
23 Standiford TJ, Kunkel SL, Basha MA, Chensue
SW, Lynch JP, Toews GB, et al. Interleukin-8
gene expression by pulmonary epithelial cell line:a model for cytokine networks in the lung. J Clin
Invest 1990;86:1945–1953.
24 Standiford TJ, Kunkel SL, Phan SH, Rollins BJ,
Strieter RM. Alveolar macrophage-derived
cytokines induce monocyte chemoattractant
protein-1 expression from human pulmonary type

Acknowledgments: We express our appreciation toProf. Takao Sasaki, Third Dept. of Internal Medicine, Prof. Hideaki Nakayama, Dept. of Hygiene andProf. Eisaku Ohama, Div. of Neuropathology, Inst.
of Neurological Sciences, Faculty of Medicine,
Tottori University, for their kind advice. We wish to
thank Dr. Toshiyuki Tanaka, Dept. of General
Education, Tottori University College of Medical
Care Technology for his invaluable help and advice.

References
1 Bagglioni M, Walz A, Kunkel SL. Neutrophil
activating peptide-1/interleukin-8, a novel cytokine that activates neutrophils. J Clin Invest
1989;84:1045–1049.
2 Cates KL, Ray CE, Quie PG. Modified Boydenchamber method of measuring polymorphonuclear leukocyte chemotaxis. New York: Raven
Press, 1978:67–71.
3 Cooper WC, Tabershaw IR, Nelson KW. Laboratory studies of workers in lead smelting andrefining. In: Purdom PW, ed. Environmental
health aspects of lead. Luxembourg: CID, 1973:
517–530.
4 Cromwell O, Hamid Q, Corrigan CJ, Barkans J,
Meng Q, Collins PD, et al. Expression and generation of interleukin-8, IL-6 and granulocytemacrophage colony-stimulating factor by bronchial epithelial cells and enhancement of IL-1β
and tumor necrosis factor-α. Immunology 1992;
77:330–337.
5 Fera T, Abboud RT, Richter A, Johal SS. Acuteeffect of smoking on elastase-like esterase
activity and immunologic neutrophil elastaselevel in bronchoalveolar lavage fluid. Am Rev
Respir Dis 1986;133:568–573.
6 Foster WL Jr, Gimenez EI, Roubidoux MA,
Sherrier RH, Roggi VL, et al. The emphysematous: radiologic-pathologic correlations. Radiographics 1993;13:311–328.
7 Gaffar SA, Surh CD, Glassy MC. Variations inthe secretion of monoclonal antibodies byhuman-human hybridomas. Hybridoma 1986;
5:93–105.
8 Hart BA, Voss GW, Shtos MA, Doherty J.
Cross-tolerance to hyperoxia following Cd aerosol pretreatment. Toxicol Appl Phamacol 1990;
103:255–270.
9 Hayes JA, Snider GL, Palmer KC. The evolutionof biochemical damage in the rat lung after acute
Cd exposure. Am Rev Respir Dis 1976;113:121–
130.
10 Hirst RN, Perry HM, Crux MG, Pierce JA.
Elevated Cd concentration in emphysematous
lungs. Am Rev Respir Dis 1973;108:30–39.
11 Hunninghake G, Gadek J, Kawanami O, Ferrans51

K. Fukuda et al.

II-like epithelial cells. J Biol Chem 1991;266:
9912–9918
25 Swisher DA, Cormier M, Jihnson J, Ledger PW.
A cytotoxicity assay using nomal human keratinocytes: characterization and applications.
Mod Dermatol 1989;4:131–137.
26 Yamamoto C, Yoneda T, Yosikawa M, Fu A,
Tokuyama T, Okamoto Y. Interleukin-8 in sputum from patients with pulmonary emphysema.
Nippon Kyoubu Shikkan Gakkai Zassi 1996;34:
174–179 (in Japanese).
27 Walz BMA, Kunkel SL. Neutrophile-activating

peptide-1/interleukin-8, a novel cytokine that
activates neutrophils. J Clin Invest 1989;84:
1045–1049.
28 Ward PA, Goldschmidt P, Greene ND. Suppressive effect of metal salts on leukocyte andfibroblastic function. J Reticul Soc 1975;18;313–
318.
29 Wang J, Huang M, Lee P. Interleukin-8 inhibits
non-small cell lung cancer proliferation: apossible role for regulation of tumor growth byautocrine and paracrine pathway. J Interferon
Cytokine Res 1996;16:53–60.

(Received December 20, 1996, Accepted January 13, 1997)

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