The First Department of Internal Medicine, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan
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ABSTRACT |
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Reactive oxygen intermediates (ROIs) play an
important role in the initiation and progression of lung diseases. In
this study, we investigated whether ROIs were involved in the induction
of interleukin (IL)-6 in human bronchial epithelial cells. We exposed normal human bronchial epithelial cells as well as a human bronchial epithelial cell line, HS-24, to ROIs. We measured the amount of IL-6 in
the culture supernatants using ELISA and the IL-6 mRNA levels using
RT-PCR. Superoxide anions (O2), but
not hydrogen peroxide
(H2O2),
increased IL-6 production. To examine whether it is a cell
type-specific mechanism of airway epithelial cells, the experiments
were also performed in human lung fibroblasts, WI-38-40. In WI-38-40
cells, neither O
2 nor
H2O2
increased IL-6 production. In contrast, tumor necrosis factor (TNF)-
(200 U/ml) induced IL-6 at the protein and mRNA levels in both airway
epithelial cells and lung fibroblasts. This cytokine-induced IL-6
production was significantly suppressed by several antioxidants,
including dimethyl sulfoxide (DMSO), in airway epithelial cells. In
WI-38-40 cells, DMSO was not able to suppress IL-6 production induced
by TNF-
. Pretreatment with DMSO recovered the TNF-
-induced
depletion of intracellular reduced glutathione in HS-24 cells. These
findings indicate that oxidant stress specifically induces IL-6
production in human bronchial epithelial cells and that in these cells
ROIs may be involved in IL-6 production after stimulation with
cytokines such as TNF-
. Presumably, ROIs participate in the local
immune response in lung diseases via IL-6 release from bronchial
epithelial cells.
airway epithelial cells; interleukin-6; reactive oxygen intermediates; reduced glutathione; superoxide anions
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INTRODUCTION |
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NUMEROUS STUDIES (15, 30) have revealed that reactive oxygen intermediates (ROIs) play a crucial role in the initiation and progression of a wide range of diseases and in the regulation of a number of important biological processes. The lung is a major target organ for oxidant injury because ROIs are generated by inflammatory cells and by chemotherapeutic agents that concentrate in the lung. Furthermore, various forms of lung diseases require O2 therapy, which adds to the oxidant burden on the lung (15, 16, 30). In the lung, ROIs can induce a variety of lesions in the respiratory tract as well as in the pulmonary vasculature and parenchyma (36, 37).
It has been reported that bronchial epithelial cells release soluble
mediators on exposure to ROIs (2, 11). Adler et al. (2) have
demonstrated that ROIs stimulate release of high-molecular-weight glycoconjugates from rodent respiratory epithelial cells in vitro. DeForge et al. (11) have reported that ROIs induce the production of
interleukin (IL)-8 in A549 human type II pulmonary epithelial cells.
There is also growing evidence that ROIs can be released by many types
of cells in response to a variety of stimuli, such as tumor necrosis
factor (TNF)- and lipopolysaccharide (3, 14, 23, 24), and that ROIs
can serve as intracellular signals for gene activation involving
specific transcription factors such as nuclear factor (NF)-
B (31,
32).
IL-6 is a cytokine produced by a variety of cells including human
bronchial epithelial cells (8, 35). IL-6 has multiple biological
activities, which include proliferation of hemopoietic stem cells,
potentiation of T-cell proliferative responses, and induction of
hepatic acute-phase protein synthesis (4). Several experimental studies
(8, 35) have demonstrated that the production of IL-6 in airway
epithelial cells is modulated by various inflammatory stimuli such as
TNF-. Recently, Simeonova et al. (33) have reported that asbestos
induces IL-6 in human bronchial epithelial cells via oxidative stress.
Despite these studies, there has been no detailed study on IL-6 release
in response to ROIs in human airway epithelial cells, including normal
human bronchial epithelial (NHBE) cells.
In the present investigation, using NHBE cells as well as a human
bronchial epithelial cell line, HS-24, we studied the effects of ROIs
on IL-6 release. We also examined whether antioxidants might modulate
IL-6 production in these cells stimulated with cytokines such as
TNF-. Finally, we tested whether ROIs were cell type-specific
stimuli for IL-6 production in airway epithelial cells.
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MATERIALS AND METHODS |
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Reagents. Xanthine (X), xanthine
oxidase (XO), ferricytochrome c,
superoxide dismutase (SOD), and 1,3-dimethyl-2-thiourea (DMTU) were
purchased from Sigma (Tokyo, Japan).
4'-Hydroxy-3'-methoxyacetophenone was purchased from
Aldrich (Tokyo, Japan). Dimethyl sulfoxide (DMSO) was purchased from
Wako Pure Chemical Industries (Osaka, Japan). Hydrogen peroxide
(H2O2)
was purchased from Santoku Chemical Industries (Tokyo, Japan). TNF-
was a gift from Dainippon Pharmaceutical (Osaka, Japan). IL-1
was a
gift from Otsuka Pharmaceutical (Tokyo, Japan).
Cell culture. Cryopreserved primary NHBE cells were purchased from Clonetics (San Diego, CA) and grown in 100-mm tissue culture dishes in bronchial epithelial cell growth medium supplied by Clonetics. The cultures were incubated at 37°C in a humidified, 95% air-5% CO2 atmosphere. After trypsinization, the cells were subcultured in 24-well culture plates (Corning, New York, NY) at a seeding density of 0.5 × 105 cells/well. When the cells were 50-60% confluent, the medium was changed to bronchial epithelial cell growth medium without hydrocortisone, and the cells were allowed to grow for an additional 24 h. NHBE cells were used within the first five passages.
HS-24 cells, a tumor cell line with properties of human bronchial epithelial cells, were generously provided by Dr. Thomas Muley (Thoraxklinikum, Heidelberg, Germany) (6, 28, 29). This cell line has been used as a model of human airway epithelial cells in the literature (1, 22, 25). WI-38-40 cells, human lung fibroblasts, were obtained from Health Science Research Resources Bank (Osaka, Japan). Stock cultures of cells were grown in a humidified atmosphere containing 95% air-5% CO2 and split once weekly by trypsinization. HS-24 cells were maintained in RPMI 1640 medium, and WI-38-40 cells were maintained in MEM (Nissui Pharmaceutical, Tokyo, Japan). Both media were supplemented with 100 U/ml of penicillin, 100 µg/ml of streptomycin, 2 mM L-glutamine, 25 mM HEPES, and 10% heat-inactivated FCS (GIBCO BRL, Grand Island, NY). The phenotype and stimulated IL-6 production of HS-24 cells remained stable between passages. WI-38-40 cells were used within the first six passages.
Measurement of superoxide anions. The
production of superoxide anions (O2)
by X plus XO was measured as the reduction in ferricytochrome
c with the method described by Pick
and Mizel (26). X (0.7 mM) and various concentrations of XO (0-20
mU/ml) were incubated with 100 µl of reaction solution containing 160 µM ferricytochrome c in Hanks'
balanced salt solution without phenol red at pH 7.4. Then the amount of
ferricytochrome c reduced by
O
2 was measured as the change in absorbance at 550 nm with an iEMS Reader MF microplate reader (Labsystems). Blank wells containing medium and ferricytochrome c were used as controls. The amount of
O
2 production per well was calculated
as reported by Pick and Mizel with the following formula: nanomoles of
O
2 per well = (absorbance at 550 nm × 100)/6.3.
Measurement of IL-6. IL-6 was measured with an ELISA kit (Quantikine human IL-6 ELISA kit, R&D Systems, Minneapolis, MN).
RNA isolation. Total cellular RNA was extracted from the cells with the acid guanidinium thiocyanate-phenol-chloroform extraction method with ISOGEN (Nippon Gene, Tokyo, Japan) (7).
RT-PCR. Two micrograms of total RNA were reverse transcribed to cDNA after annealing with 100 pmol of oligo(dT)12-18 primer (Pharmacia Biotechnology, Uppsala, Sweden) in the presence of 100 U of Moloney murine leukemia virus RT (GIBCO BRL, Life Technologies, Gaithersburg, MD), 10 U of RNasin (Promega, Madison, WI), 1 mM dithiothreitol, 25 pmol of random primer (Takara Shuzo, Kyoto, Japan), and 10 pmol of each deoxynucleotide (Takara Shuzo) in a total volume of 10 µl for 1 h at 37°C. Two microliters of resultant cDNA preparation were used directly for each amplification reaction. PCR was done in a 50-µl reaction mixture containing 20 pmol of each primer (see below), 20 pmol of each deoxynucleotide, and 1.25 U of Taq DNA polymerase (Takara Shuzo). Amplification of a specific PCR product was carried out separately in a different tube. The primers used were IL-6 sense primer, 5'-GGCTGAAAAAGATGGATGCT-3'; IL-6 antisense primer, 5'-CCTGCTTCACCACCTTCTG-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense primer, 5'-CAAAAGGGTCATCATCTCTG-3'; and GAPDH antisense primer, 5'-CCTGCTTCACCACCTTCTTG-3'. These primer sets yield PCR products of 303 and 446 bp for IL-6 and GAPDH, respectively. Reaction mixtures were incubated in a Perkin-Elmer Cetus DNA thermal cycler for 25-30 cycles (denaturation, 1 min at 94°C; annealing, 2 min at 60°C; and extension, 1 min at 72°C). Aliquots of the PCR products were subjected to agarose gel electrophoresis in Tris-borate-EDTA buffer and visualized by ethidium bromide staining.
Lactate dehydrogenase cytotoxicity
assay. Lactate dehydrogenase (LDH) release from the
cells was determined colorimetrically with an LDH assay kit
(LDH-Cytotoxic Test Wako, Wako Pure Chemical Industries, Osaka, Japan)
(10, 18). Maximum releasable LDH was assessed by incubating cells with
0.2% Tween 20, and background release of LDH was evaluated by
incubating cells in medium alone. At the end of the incubation period,
LDH release in the supernatant was quantitated. The percentage of
cytotoxicity was calculated as follows:
[(A B)/(C
B)] × 100, where
A is LDH (in optical density) released into the medium of the test sample,
B is LDH released from control cells
(i.e., background release), and C is
LDH released from cells treated with 0.2% Tween 20.
Colorimetric assay for reduced glutathione. Intracellular reduced glutathione (GSH) content was determined colorimetrically with a commercially available kit (BIOXYTECH GSH-400, OXIS International, Portland, OR) (5).
Statistical analysis. We repeated each type of experiment at least three times and confirmed that similar data were obtained. The results, obtained in triplicate, are presented as means ± SD, and comparisons were made with one-way ANOVA with Fisher's post hoc test. A P value of <0.05 was considered significant. Data were analyzed on a Macintosh PowerBook 180c computer (Apple Computer, Cupertino, CA) with StatView-J 4.5 (Abacus Concepts, Berkeley, CA).
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RESULTS |
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O2 enhances
IL-6 production in bronchial epithelial cells. The
initial experiments were performed to determine whether ROIs were
capable of inducing IL-6 production in human bronchial epithelial
cells. For these experiments, NHBE and HS-24 cells were incubated in
the presence of an O
2-generating
system consisting of X (0.7 mM) and increasing amounts of XO. The
amount of O
2 generated by this
reaction was dependent on the concentration of XO, and the release of
O
2 was ~10
nmol · ml
1 · min
1
at 20 mU/ml of XO (Fig. 1). Alternatively,
these cells were exposed to
H2O2.
XO induced a dose-dependent release of IL-6 in NHBE and HS-24 cells
(Fig. 2, A
and C). An XO enzyme activity of 5.0 mU/ml led to a twofold increase in IL-6 secretion in NHBE cells. And incubation of HS-24 cells with XO (20 mU/ml) induced a 1.8-fold increase in IL-6 production. Percent cytotoxicity measured by an LDH
cytotoxicity assay was 2.8 ± 1.0% in XO (5.0 mU/ml)-exposed NHBE
cells and 1.2 ± 1.0% in XO (20 mU/ml)-exposed HS-24 cells. Higher
concentrations of XO were not tested because they may cause cell damage
as manifested by morphological changes of the monolayer or cell
detachment (data not shown) and by the LDH cytotoxicity assay. In
contrast, neither cell increased IL-6 production on exposure to
H2O2
(Fig. 2, B and
D). Incubation of HS-24 cells in the
presence of the
H2O2-generating
enzyme glucose oxidase did not modulate the release of IL-6 protein
(data not shown). Next, the time-dependent secretion of IL-6 protein
was determined in HS-24 cells after stimulation with XO (20 mU/ml). As
shown in Fig.
3A,
stimulation with XO resulted in a significant increase in the levels of
IL-6 at 12 and 24 h compared with those with 0.7 mM X alone
(P < 0.0001). Consistent with these
protein data, stimulation of HS-24 cells with XO caused the maximum
elevation in IL-6 mRNA levels at 6 h, whereas XO did not modulate
control GAPDH transcript levels (Fig.
3B). To confirm that the effect of
XO on the release of IL-6 was due to its
O
2 synthesis, SOD and catalase,
O
2 and
H2O2
scavengers, respectively, were added to HS-24 cells before stimulation
with XO (20 mU/ml). SOD (250 U/ml) significantly suppressed the IL-6 production and IL-6 mRNA expression induced by XO, whereas catalase (500 U/ml) did not modulate the IL-6 response to XO (Fig.
4). These experiments indicate that
O
2, but not H2O2,
induced IL-6 production in human bronchial epithelial cells.
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Antioxidants suppress IL-6 production in bronchial
epithelial cells. TNF- was dose dependently capable
of inducing IL-6 release from NHBE and HS-24 cells (Fig.
5, A and
C). To determine whether ROIs were
involved in IL-6 production in bronchial epithelial cells
stimulated with TNF-
, NHBE and HS-24 cells were incubated for 24 h with TNF-
(200 U/ml) in the presence of various
concentrations of an antioxidant, DMSO. DMSO, a known hydroxyl radical
(· OH) scavenger, has been found to have beneficial effects
in a variety of disease models and can easily penetrate through the
cell membranes (38). Measurement of IL-6 levels in the supernatants
demonstrated that TNF-
(200 U/ml) caused a markedly enhanced
production of IL-6, and increasing DMSO concentrations resulted in an
essentially linear dose-dependent suppression of TNF-
-induced IL-6
production (Fig. 5, B and
D). DMSO was also able to suppress
the IL-1
-induced increase in IL-6 release from HS-24 cells (data not
shown). A time-course kinetic study has shown that DMSO (1%)
significantly suppressed TNF-
-induced IL-6 release from HS-24 cells
after a 24-h incubation (P < 0.0001;
data not shown). In accordance with these protein data, TNF-
(200 U/ml) significantly increased the levels of IL-6 mRNA in HS-24 cells
and DMSO (1.0%) markedly reduced those induced with the cytokine (Fig.
5E). In contrast, TNF-
and DMSO did not change the levels of GAPDH mRNA. These results agree
with the previous reports (3, 14, 23, 24) that TNF-
exerts some of
its effects by stimulating production of ROIs in many types of cells.
To further confirm that ROIs are involved in IL-6 production in airway
epithelial cells, other antioxidants were studied in NHBE and HS-24
cells. DMTU (1 and 10 mM) and
4'-hydroxy-3'-methoxyacetophenone (100 µg/ml)
significantly inhibited TNF-
-induced IL-6 production (Fig.
6).
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DMSO recovers TNF--induced depletion
of the intracellular GSH. Treatment with TNF-
significantly decreased the intracellular GSH content of HS-24 cells.
Pretreatment of this culture with DMSO recovered the TNF-
-induced
decrease in GSH (Table 1).
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ROIs are not involved in the production of IL-6 in
lung fibroblasts. To determine whether the involvement
of ROIs in IL-6 production is specific for bronchial epithelial cells,
we tested the ability of X plus XO and
H2O2
to stimulate IL-6 production in human lung fibroblast cells, WI-38-40.
We used lower concentrations of ROIs on WI-38-40 cells because an LDH
cytotoxicity assay indicated that WI-38-40 cells were much more
sensitive to the cytotoxic effects of ROIs than airway epithelial cells
(data not shown). Neither XO nor
H2O2
increased the production of IL-6 in WI-38-40 cells (Table
2). Next, we stimulated WI-38-40 cells with
200 U/ml of TNF- in the presence and absence of DMSO and measured the amount of IL-6 in the supernatants at a 24-h time point. TNF-
significantly increased the production of IL-6 in WI-38-40 cells. However, DMSO was not able to inhibit the TNF-
-induced IL-6
enhancement in both protein and mRNA levels (Fig.
7). These results suggest that ROIs may not
be involved in the production of IL-6 in human lung fibroblasts.
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DISCUSSION |
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IL-6 is a multifunctional pleiotropic cytokine involved in the
modulation of the immune response and inflammation (4). Recent evidence
(8, 12, 13, 35) revealed that IL-6 is produced by a variety of cell
types in the lung, including alveolar macrophages, lung fibroblasts,
endothelial cells, and airway epithelial cells under appropriate
stimulation. Increased levels of IL-6 have been detected in the serum
and bronchoalveolar lavage fluid in various pathological conditions
such as bronchial asthma and idiopathic pulmonary fibrosis (19, 40). In
the present study, we investigated the effects of ROIs on the
production of IL-6 in human bronchial epithelial cells. XO-derived
ROIs, but not H2O2,
induced the production of IL-6. Antioxidants significantly suppressed
IL-6 release from airway epithelial cells stimulated with TNF-.
These findings relevant to ROIs were not observed in human lung fibroblasts.
Our results indicate that XO-derived ROIs induce a dose-dependent
increase in IL-6 production in human airway epithelial cells. However,
it is difficult to identify the specific ROI responsible for the
induction of IL-6 because the various species are highly reactive and
often reactive with each other. For instance,
O2 formed by the action of XO on X is
rapidly dismutated to
H2O2, which, in turn, is converted in the presence of
Fe2+ to (· OH).
O
2 also can contribute to the
formation of · OH by converting
Fe3+ to
Fe2+. We found that the addition
of an O
2 scavenger, SOD, resulted in
the inhibition of the IL-6 response to X plus XO stimulation,
suggesting that O
2 may be, at least in
part, involved in IL-6 production in human bronchial epithelial cells.
Unexpectedly,
H2O2
per se did not seem to be implicated in IL-6 induction. This was also
indicated by the result that
H2O2
enzymatically generated by the reaction of glucose and glucose oxidase
did not enhance IL-6 production (data not shown). Another possibility
is that extracellularly generated XO-derived ROIs may penetrate through
the cell membranes and change into other kinds of ROIs intracellularly,
and it is these intracellularly produced ROIs that induce IL-6 release.
The extent of IL-6 release induced by X plus XO in airway epithelial
cells was significant but small compared with that seen in
TNF--treated cells. It should be considered that ROIs produced by X
plus XO may be short-lived and can quickly decompose in medium (9).
Presumably, the actual concentrations of ROIs that induced the
production of IL-6 in cell cultures were much lower than the theoretical ones.
Consistent with our findings, it has been observed that ROIs are capable of inducing the production of soluble mediators in a variety of cells. Vischer et al. (39) have demonstrated that ROIs induce von Willebrand factor release from human vascular endothelial cells. It has also been reported that ROIs are implicated in high-molecular-weight glycoconjugate secretion from rodent tracheal epithelial cells and in IL-1 decoy receptor release from human myelomonocytic cells (2, 27).
Our results show that antioxidants including DMSO can suppress the
induction of IL-6 in response to TNF- in human bronchial epithelial
cells. Generation of ROIs on exposure to a variety of inflammatory
stimuli has been reported for many types of cells including bronchial
epithelial cells (3, 14, 21, 23, 24). In human endothelial cells,
TNF-
and interferon-
stimulate O
2 release (23). Lopez et al. (21)
have reported that various stimuli, including platelet-activating
factor and neutrophil elastase, stimulate bovine bronchial epithelial cells to produce and release
H2O2.
Our results suggest that intracellular ROIs produced by TNF-
stimulation may be the signals as second messengers to produce IL-6
from bronchial epithelial cells. Our data that TNF-
induced the
depletion of intracellular GSH in HS-24 cells and that DMSO recovered
this decreased GSH further support this notion. Transcriptional
regulatory factors such as NF-
B can be rapidly activated by ROIs
(31, 32) and bind to their consensus enhancer sequences in a number of
cytokine genes including IL-6 (20). Antioxidants such as DMSO and DMTU
have been reported to decrease NF-
B binding activity (31, 34). The
findings of our study would support the view that NF-
B activates the
IL-6 promoter through oxidants generated by the effects of TNF-
.
The effects of ROIs on IL-6 production appeared to be cell-type specific. Although XO-derived ROIs significantly induced IL-6 production in bronchial epithelial cells, the same oxygen-free radicals had no IL-6 stimulatory effect on WI-38-40 cells. DMSO that is potent in inhibiting IL-6 production in human bronchial epithelial cells did not reduce the production of the same cytokine in WI-38-40 cells. The mechanisms stimulating IL-6 production are diverse (4), and probably lung fibroblasts may use another set of transcription factors to induce IL-6 gene expression than that used in bronchial epithelial cells.
In summary, the present study demonstrates that ROIs are involved in the production of IL-6 in human bronchial epithelial cells. Taken together with the relationship between ROIs and other cytokines (11), bronchial epithelial cells are actively involved in inflammatory lung diseases on exposure to oxidant burden.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Maruyama, The First Dept. of Internal Medicine, Faculty of Medicine, Toyama Medical and Pharmaceutical Univ., 2630 Sugitani, Toyama 930-0194, Japan (E-mail: mmaruyam-tym{at}umin.ac.jp).
Received 3 August 1998; accepted in final form 4 February 1999.
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