Unité de recherche pulmonaire, Faculté de médecine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4
Submitted 26 November 2002 ; accepted in final form 19 May 2003
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ABSTRACT |
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CC chemokines; inflammation; asthma; regulated on activation, normal T-cell expressed, and presumably secreted; monocyte chemotactic protein
Recently, zinc has been identified as an important factor in the airway epithelium (38). Zinc exists in two distinct pools, the first being tightly bound and an integral part of >300 enzymes, of transcription factors and of structural proteins. The second pool is more labile and dynamic and is rapidly exchangeable (38). Its intracellular levels are dictated by a dynamic process involving three main mechanisms: buffered by zinc-binding proteins such as metallothionein (6, 25), transported by plasma membrane zinc transporters such as ZnT-1 (27), and sequestered by vesicular membranes through ZnT-2 and ZnT-3 (25, 26). It has been suggested that zinc can be a significant factor in the biology of the respiratory epithelium (5, 38). As is extensively reviewed by Shankar and Prasad (31), zinc is very important for specific cells of the immune system. For example, zinc is required for T lymphocyte proliferation in response to interleukin (IL)-1, phytohemagglutinin, concanavalin A, or IL-2. Moreover, it has been shown that zinc alters the expression and function of lymphocyte surface molecules governing cell-cell interactions (21) and enhances the transcription and expression of ICAM-1 on the surface of lymphoid cells but not on fibroblasts (19).
Because zinc is important for immune function and has been shown to be important in the airway epithelium, we proposed that the pharmacological modulation of zinc can influence epithelial cytokine production. The aim of this study was therefore to investigate the effect of zinc modulation, using zinc chelators, on the expression and secretion of C-C chemokines from several lung cell lines.
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METHODS |
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Experimental protocols. The following procedures and times were
respected throughout the study. All cell lines were seeded and grown to
90% confluence (unless otherwise specified) in their respective media as
described in Cell culture. Cells were washed three times with
phosphate-buffered saline (PBS), and media were replaced with incomplete media
(without FBS). A 24-h equilibration period was respected. Media were
discarded, fresh incomplete medium was added, and cells were pretreated with
either 2,3-dimercapto-1-propanesulfonic acid (DMPS, 18 h) or
N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine
(TPEN, 1 h; Sigma, St. Louis, MO). Cells were then washed, incomplete medium
was added, and cells were stimulated with TNF-
(10 ng/ml; Peprotech,
Rocky Hill, NJ) for 4 h. After this 4-h stimulation period, the cells
underwent the procedures described below.
Zinquin fluorescence. A549 cells were seeded and grown to
70-80% confluence on sterile glass coverslips in six-well plates and
washed as described above. Cells were then pretreated with 1.5 mM DMPS (18 h)
or 20 µM TPEN (1 h), washed three times with PBS, and 25 µM Zinquin
[TRC, Toronto, ON, Canada; excitation/emission (ex/em): 368 nm/490 nm] in PBS
was added for 30 min at 37° C. Coverslips were then mounted with
Vectashield mounting medium (Vector Laboratories, Burlingame, CA), and cells
were analyzed under an Axioskop 2 fluorescence microscope (Carl Zeiss,
Thornwood, NY). Photomicrographs were captured with a SPOT-3 color digital
camera (Diagnostic Instruments, St. Sterling Heights, MI). The images were
processed with SPOT software. Zinquin fluorescence was captured with the
4,6-diamidino-2-phenylindole (DAPI)/Hoechst/AMCA filter (ex 360/40 nm, em
460/50 nm), an exposure time of 4 s, and a signal gain of 2. Nonspecific
cellular fluorescence at 535 nm, as an indicator of the presence of cells, was
captured with the FITC/BODIPI/FLUO-3/DiO filter (ex 480/30 nm, em 535/40 nm)
with an exposure time of 2 s and a signal gain of 1. A single dilution of
Zinquin was used in each set of experiments to ensure that all coverslips
received the same concentration of dye, and all experimental conditions were
rigorously followed between coverslips and sets of experiments. The results
shown are representative of the results obtained in each separate set of
experiments.
RNA extraction and Northern blot analysis. After having been
exposed to experimental conditions, cells were harvested with the TRIzol
reagent. Total RNA was extracted with a one-step guanidium-phenol chloroform
extraction procedure according to the TRIzol manufacturer's protocol
(GIBCO-BRL). We carried out RNA quantification by reading the optical density
of the sample at 260 nm with a Beckman spectrophotometer, and RNA quality was
monitored by simultaneous optical density reading at 280 nm. Northern blot
analysis was performed by the electrophoresis of 20-µg samples of total
cell RNA in a 1% agarose-6% formaldehyde MOPS-buffered gel. The RNA was then
transferred onto a Hybond-N+ membrane (Amersham Pharmacia Biotech,
Buckinghamshire, UK) for analysis. Membranes were prehybridized for 4 h at
68°C in a solution containing 600 mM NaCl, 120 mM Tris, 8 mM EDTA, 10%
dextran sulfate, 0.2% SDS, 0.1% Na4P2O7, and
625 µg/ml heparin at pH 7.4. Hybridization was carried out overnight at
68° C in the same solution containing the radioactively labeled probe.
Membranes were hybridized sequentially with the eotaxin, IL-8, or
glyceraldehyde phosphate dehydrogenase housekeeping gene (GAPDH, as control
for RNA quantity and integrity) probes labeled with the Multiprime DNA
labeling system using [-32P]dCTP (Amersham Life Science,
Oakville, ON, Canada). The eotaxin probe was obtained from PCR amplification
of a 222-bp fragment of eotaxin cDNA, the IL-8 probe was an EcoRI
fragment spanning 500 bp of the coding region of IL-8, and the GAPDH probe was
a 1-kb PstI cDNA fragment (ATCC). The membranes were then washed once
at room temperature (RT) for 20 min in 2x SSC, three times at 68°C
for 20 min in 0.1% SDS/0.1% SSC, and rinsed at RT in 0.1% SSC. The membranes
were then exposed to Kodak XAR5 film (Eastman Kodak, Rochester, NY) with an
intensifying screen at -80°C. Autoradiographs were quantified with a UMAX
PowerLook II scanner and the NIH Image 1.62 software (National Institutes of
Health, Bethesda, MD). Results shown are ratios of chemokine-GAPDH
(housekeeping gene) densitometric units.
Eotaxin ELISA. Eotaxin protein levels in cell culture supernatants
were measured with commercially available ELISA kits (Biosource International,
Montreal, PQ, Canada). The limit of detection of this assay was 2 pg/ml for
eotaxin. In brief, eotaxin protein levels were measured in cell culture
supernatants from cells having been cultured in 24-well plates, treated
according to experimental protocols, and following a 4-h stimulation with
TNF- (10 ng/ml). After the stimulation period, cells were washed three
times with PBS, and fresh medium was added; 48 h later, supernatants were
collected and stored for analysis.
GATA Western blot. The presence of GATA transcription factor
immunoreactivity was assessed by Western blot analysis in whole cell extracts
from A549 cells. A549 cells were cultured and stimulated with TNF- (10
ng/ml) for 4 h. Whole cell extracts were prepared in a lysis buffer containing
50 mM Tris, 10 mM EDTA, 5 mM EGTA, 10 mM NaF, 10 mM NaPP, 0.5 mM
Na2VO4, 1 tablet Mini Complete (protease inhibitor
mix)/10 ml, 1 mM PMSF, and 1% Nonidet P-40. Cells were incubated in the lysis
buffer for 5 min and sonicated. Homogenates were centrifuged 10 min at 10,000
g (4°C), and the supernatant fractions were collected. Protein
content was determined by the Bio-Rad assay (Bio-Rad Laboratories, Hercules,
CA). Standard Western blot analyses were used to determine protein expression.
Fifty micrograms of proteins were separated by SDS-polyacrylamide gel
electrophoresis (10% resolving gel) and transferred to polyvinylidene
difluoride membranes. Membranes were blocked for 2 h at room temperature with
5% nonfat milk in Tris-buffered saline with 0.1% Tween 20. GATA
immunoreactivity was identified with the M-20 GATA-1 antibody (Santa Cruz
Biotechnology, Santa Cruz, CA). Membranes were incubated with the GATA-1
primary antibody (1:1,000) overnight at 4°C, washed, and incubated for 1 h
with a horseradish peroxidase-labeled anti-goat IgG. (1:8,000; Sigma).
Immunoreactive bands were revealed with the enhanced chemiluminescence
detection system (ECL+; Amersham Canada). Cellular extracts obtained form
megakaryocytic DAMI cells were used as positive controls in these experiments
(22).
NF-B and GATA-1 EMSA. The ability of zinc
chelators to modulate NF-
B and GATA-1 binding was assessed by EMSA.
Cells were cultured and treated as described above. After stimulation, cells
were scraped with a rubber policeman in PBS at 4° C and centrifuged, and
nuclear extracts were prepared at 4°C in lysis buffer (60 mM KCl, 10 mM
HEPES, 1 mM EDTA, 1 mM DTT, and 0.5% Nonidet P-40, pH 8.0) containing 1
tablet/10 ml Mini Complete (protease inhibitor mix), and 1 mM Pefabloc SC (all
reagents from Boehringer Mannheim, Laval, QC, Canada). After centrifugation
cell nuclei were resuspended in 250 mM Tris · HCl, 60 mM KCl, 1 mM DTT,
1 mM Pefabloc SC, containing 1 tablet/10 ml Mini Complete, pH 7.8, lysed by
freezing-thawing, and centrifuged at 13,000 g for 10 min at 4°C.
The supernatant containing nuclear extracts was supplemented with 20%
glycerol, and total protein content was determined by the Bio-Rad assay
(Bio-Rad Laboratories). Fractionated nuclear extracts (3 µg total protein)
were added at RT for 30 min to 10 mM Tris · HCl, 50 mM NaCl, 1 mM DTT,
1 mM EDTA, 5% glycerol containing 0.25 µg poly(dI-dC), and 7.1 fmol
32P-end-labeled double-stranded oligonucleotide with a
high-affinity binding matrix as follows (binding site is underlined):
NF-
B: 5'-(AGTTGAGGGGACTTTCCCAGGC)-3',
3'-(TCAACTCCCCTGAAAGGGTCCG)-5'; GATA-1:
5'-(GTGCGACCAGATATGTCACCACCACATCACTTTTAG)-3',
3'-(CACGCTGGTCTATACAGTGGTGGTGTAGTGAAAATC)-5'. DNA-binding
reactions were analyzed on 5% nondenaturing polyacrylamide gels (37.5:1
acrylamide/bis-acrylamide) for NF-
B and on 4% polyacrylamide gels (60:1
acrylamide/bisacrylamide) for GATA-1. Dried gels were exposed to Kodak XAR5
film (Eastman Kodak). Supershift experiments were performed with the N-6
anti-GATA-1 antibody (Santa Cruz Biotechnology). Binding buffer, poly(dI-dC)
(0.25 µg), nuclear extract, and antibody were mixed and incubated for 30
min at 4°C. The 32P-labeled GATA-1 probe was then added and
incubated of 15 min at RT. Samples were then immediately loaded onto gels for
analysis.
RNase protection assay. Total RNA was isolated from A549 cells by
the TRIzol method as described in RNA extraction and Northern blot
analysis and used in the standard PharMingen (San Diego, CA) RNase
protection protocol as follows. The multiprobe template set hCK5, containing
DNA templates for lymphotactin; regulated on activation, normal T-cell
expressed, and presumably secreted (RANTES); IFN--inducible protein;
macrophage inflammatory protein (MIP)-1
; MIP-1
; MCP-1; IL-8;
I-309; L32; and GAPDH (PharMingen) was used to synthesize
[
-32P]UTP (NEN Life Science Products, Boston, MA)-labeled
probes in the presence of a GACU pool using a T7 RNA polymerase. Probes were
hybridized overnight with 5 µg of target RNA, followed by RNase digestion
and proteinase K treatment. Samples were chloroform-extracted,
ethanol-precipitated in the presence of ammonium acetate, and loaded on an
acrylamideurea sequencing gel in 0.53 M Tris-borate-EDTA buffer. After
electrophoresis at 50 W for 1-2 h, the gel was adsorbed to filter paper and
dried under vacuum. The dried gel blot was exposed to a phosphor screen for
phosphor imagery analysis using ImageQuant software (Molecular Dynamics,
Sunnyvale, CA). The intensity of each band was analyzed and normalized to the
values of the housekeeping gene GAPDH, and normalized values were used to
quantify expression.
Statistical analysis. All data are presented as means ± SE. To assess statistical significance between treatments, we analyzed data using Student's t-test. A P value of <0.05 was considered as statistically significant.
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RESULTS |
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Effects of DMPS and TPEN on eotaxin mRNA expression in A549 and BEAS-2B
cell lines. To explore the effect of zinc chelation on eotaxin mRNA
induced by TNF-, we preincubated A549 cells with either DMPS for 18 h
(18-24 h being optimal preincubation times as determined by time-course
experiments; data not shown) or TPEN for 1 h and stimulated these cells with
TNF-
. A549 cells stimulated for 4 h with TNF-
showed marked
eotaxin mRNA expression. This increase in eotaxin mRNA induced by TNF-
was also a dose- and time-dependent phenomenon (data not shown). In a few
cases, a basal amount of eotaxin mRNA was detected in these cells. In each
experiment the effects of medium alone and of TNF-
(positive control)
were examined. In A549 cells both DMPS and TPEN significantly and dose
dependently inhibited TNF-
induced eotaxin mRNA expression as
determined by Northern blot analysis (Fig.
2A). DMPS was most effective at 2.0 mM, inhibiting
TNF-
-induced eotaxin mRNA expression by 84.5% ± 0.8%; however, a
statistically significant reduction of eotaxin mRNA was observed at all other
concentrations (0.5-1.5 mM). TPEN was most effective at the 25 µM
concentration, achieving 57.5 ± 2.1% inhibition. Treatment of cells
with the TPEN diluent DMSO, tested at an amount equivalent to that contained
in the TPEN preparation, altered neither basal nor TNF-
-induced eotaxin
mRNA expression. Similar results were observed in the bronchial epithelial
cell line BEAS-2B stimulated and pretreated in the same manner. In BEAS-2B
cells, DMPS dose dependently inhibited TNF-
-induced eotaxin mRNA
expression, achieving maximal inhibition at the 2.0 mM concentration (81.3
± 1.1% inhibition) (Fig.
2B). However, in this cell line, TPEN was more effective
in inhibiting eotaxin mRNA expression than in A549 cells. TPEN achieved
maximal inhibition (77.5 ± 7.1%) of TNF-
-induced eotaxin mRNA
expression at the 25 µM concentration. DMPS and TPEN treatments were tested
for cytotoxicity with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) assay (10) under
the same experimental conditions as described in the Cell culture and
Experimental protocols sections. No cytotoxicity was observed for any
of the compounds compared with medium alone (data not shown).
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Effects of DMPS and TPEN on IL-8 mRNA expression. To ascertain the
specificity of the zinc chelation treatments, we tested the ability of DMPS
and TPEN to inhibit the mRNA expression of a structurally and functionally
unrelated chemokine, IL-8. In this set of experiments, A549 cells were treated
in exactly the same conditions as for the eotaxin experiments.
TNF--stimulated A549 cells showed a marked increase in IL-8 mRNA
expression. However, in contrast to eotaxin, TNF-
-induced IL-8 mRNA
expression was not inhibited by zinc chelation treatments
(Fig. 2C). In this
case, a slight but nonsignificant increase in IL-8 mRNA was observed at the
1.5 mM DPMS and 25 µM TPEN concentrations.
Effect of exogenous zinc on the inhibitory effect of DMPS. We
investigated whether TNF--induced eotaxin mRNA expression could be
reversed by the addition of varying concentrations of zinc. We added exogenous
zinc acetate at varying ratios to DMPS (1:1 being one molecule of DMPS to one
zinc ion) 20 min before adding it to cell media. At the 1:1 ratio, cell death
was observed due to an excess of zinc. At the 3:1 ratio, enough DMPS was free
to chelate a small amount of intracellular labile zinc and therefore retained
10% of its inhibitory effect. In contrast, at the 2:1 ratio (2 mM DMPS/1
mM zinc acetate), exogenous zinc completely abolished the inhibitory effect of
DMPS on TNF-
-induced eotaxin mRNA expression. In this set of
experiments, 2.0 mM DMPS alone achieved
60% inhibition of
TNF-
-induced eotaxin mRNA expression
(Fig. 3).
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Effects of DMPS and TPEN on eotaxin protein production. In
preliminary experiments we tested eotaxin release from the A549 and BEAS-2B
cell lines, but only very small amounts of eotaxin protein (5-10 pg/ml, also
the limit of detection of the ELISA kit) could be detected under our
experimental conditions. This was also observed by Fujizawa et al.
(7) when cells were stimulated
with TNF- alone. In this set of experiments, both DMPS and TPEN reduced
eotaxin production; however, the concentrations obtained were at the limit of
detection of the ELISA kits and were therefore deemed inadequate for the
proper evaluation of the effects of zinc chelators on eotaxin production. To
remain in the same experimental conditions and to avoid the use of
supplemental or different stimuli such as LPS, IL-1
, or IL-4, we chose
the fibroblastic cell line HFL-1 to investigate the effect of DMPS and TPEN on
eotaxin at the protein level, because of the large amount of eotaxin that
these cells can produce and for their importance in producing eotaxin under
inflammatory conditions
(35).
Effects of DMPS and TPEN on eotaxin mRNA in the fibroblastic cell line
HFL-1. In a first series of experiments we tested the effects of DMPS and
TPEN at the eotaxin mRNA level in HFL-1 cells to verify that the effects were
similar to those demonstrated with the epithelial (A549 and BEAS-2B) cell
lines. HFL-1 cells were treated similarly to the A549 and BEAS-2B cells as
described in Effects of DMPS and TPEN on eotaxin mRNA expression in A549
and BEAS-2B cell lines. The effects of both chelators were similar to
those observed with the two previous cell lines. DMPS dose dependently
inhibited the TNF--induced eotaxin mRNA expression with maximal
inhibition (69.5 ± 5.2%) at the 2.0 mM concentration
(Fig. 4A). TPEN also
significantly inhibited TNF-
-induced eotaxin mRNA expression. However,
in this case, although dose dependent, up to 41 ± 0.2% inhibition was
still observed at the lowest (1 µM) TPEN dose used
(Fig. 4A). At the TPEN
dose of 25 µM, a 73.9 ± 6.6% inhibition was observed.
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Effects of DMPS and TPEN on eotaxin at the protein level. The
effects of DMPS and TPEN on eotaxin protein secretion, as assessed by ELISA in
the supernatants of HFL-1 cells, were similar. The data in
Fig. 4B demonstrate
that a significant amount of eotaxin was produced by HFL-1 cells (2,750 pg/ml)
in a 48-h period following stimulation with TNF-. HFL-1 cells also
produced a constitutive quantity of eotaxin (480 pg/ml) without stimulation.
DMPS dose dependently inhibited TNF-
-induced eotaxin protein secretion
from HFL-1 cells (Fig.
4B). Maximal inhibition (38 ± 4.6%) was observed
at the highest DMPS dose used (2.0 mM). The lowest (0.5 mM) DMPS dose used had
little or no effect on eotaxin protein secretion. Similarly, TPEN dose
dependently inhibited TNF-
-induced eotaxin protein secretion from HFL-1
cells (Fig. 4B).
Maximal inhibition (45 ± 6.1%) was achieved by the 25 µM TPEN
concentration. However, at the lowest concentration of TPEN used (1 µM),
29.7 ± 6.3% inhibition was still observed.
GATA immunoreactivity. We assessed GATA immunoreactivity in
cellular extracts obtained from A549 cells. In this set of experiments, A549
cells were cultured and stimulated with TNF- (10 ng/ml) for 4 h, and
cellular extracts were prepared. Figure
5 shows that GATA immunoreactivity using the GATA-1 antibody was
observed in cell lysates from A549 cells in the form of a 47-kDa protein
(lanes 1 through 3). This was compared with a positive
control obtained from the lysates of megakaryocytic DAMI cells (lane
4). With 50 µg of total proteins, A549 cells exhibited GATA-1
immunoreactivity to a lesser degree than the megakaryocytic DAMI cells.
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GATA-1 and NF-B EMSA. In the EMSA experiments,
TNF-
clearly induced NF-
B activation
(Fig. 6A, arrow).
However, neither DMPS (2.0 mM) nor TPEN (25 µM) treatments seemed to
inhibit the TNF-
-induced NF-
B activation.
Figure 6B (arrow)
shows GATA-1 binding. GATA-1 had constitutive binding in A549 cells as is
shown by binding of the probe in the nuclear extracts from cells treated with
medium alone. TNF-
stimulation increased the binding of the GATA-1
probe (lane 2). Neither DMPS nor TPEN alone affected the basal GATA-1
binding (lanes 3 and 5). However, both DMPS and TPEN reduced
the TNF-
-induced GATA-1 activation (lanes 4 and 6).
Furthermore, supershift experiments (Fig.
6C) using an anti-GATA-1 antibody confirmed that GATA-1
was indeed involved in DNA binding.
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Effects of DMPS on RANTES, MCP-1, and IL-8 mRNAs. To further study
the specificity of the effects of DMPS on TNF--induced mRNA inhibition,
we carried out RNase protection assays on total RNA from A549 cells treated
with medium alone, stimulated for 4 h with TNF-
, or pretreated with
DMPS (1.5 mM for 18 h) and stimulated with TNF-
. mRNAs for RANTES and
MCP-1 (CC chemokine family members) and IL-8 (CXC chemokine family member) and
GAPDH were examined. Figure 7
shows that cells treated with medium alone express little or no mRNA for the
three chemokines studied (lane 2). In contrast, lane 3 shows
a significant increase in mRNA for all three chemokines studied following
TNF-
stimulation of the cells compared with the medium control. IL-8
and MCP-1 had the greatest increase in mRNA, whereas RANTES had about half the
level of expression of the other two chemokines in our experimental
conditions. Lane 4 shows that pretreatment of cells with DMPS
signifi-cantly decreased TNF-
-induced mRNA for the C-C chemokine family
members RANTES and MCP-1 (75 and 80%, respectively). Conversely, as previously
shown by Northern blot analysis, the CXC chemokine IL-8 induction was
unaffected by DMPS pretreatment. In lanes 2-4 GAPDH was affected
neither by TNF-
stimulation nor by DMPS pretreatment and shows equal
loading.
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DISCUSSION |
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In this study, we clearly show that eotaxin mRNA expression is influenced
by intracellular zinc concentrations. In fact, our results show that the
widely recognized zinc chelator TPEN
(3) and the clinically used
heavy metal chelator DMPS (37)
inhibit the mRNA expression of the proinflammatory chemokine eotaxin in three
different human lung cell lines (BEAS-2B, a bronchial epithelial cell line;
A549, an alveolar epithelial cell line; and HFL-1, a fibroblastic cell line).
As is clearly demonstrated by Northern blot analysis experiments, the cell
lines used in this study all respond very well to TNF- stimulation,
leading to increased eotaxin mRNA expression. These results show that the cell
lines used are valid models for the study of TNF-
-induced chemokine
mRNA expression. This is supported by the findings of Lilly et al.
(17), where A549 cells were
used to determine the time course of TNF-
-induced eotaxin mRNA
expression. Furthermore, in other in vitro studies involving eotaxin, BEAS-2B
cells (7,
32) and HFL-1 cells
(30) were used as models to
study the effects of various proinflammatory cytokines on mRNA expression.
Our results show that in all three cell lines studied DMPS and TPEN acted
dose dependently to inhibit eotaxin mRNA expression induced by TNF-.
Moreover, an inhibition of
80% was achieved at the highest concentrations
of DMPS and TPEN used. In vitro studies have shown that exogenously
administered zinc could influence the expression of molecules such as ICAM-1
(19), but none have
demonstrated the effects of pharmacological alterations of the intracellular
concentrations of zinc on proinflammatory cytokine mRNA expression. The
relevance of our results can be seen by the many studies showing an increased
production of eosinophil-active chemokines at the sites of allergic
inflammatory reactions, such as those found in asthma
(36). Our results clearly
indicate that the inhibitory effect of the chelators was not a disruptive
effect on cellular function, since the mRNA of the housekeeping gene GAPDH was
not affected by the treatment. The GAPDH mRNA was used in all Northern blot
analysis experiments as a control of equal loading of total RNA on the gels
and as a control of maintained cellular function. However, to further
determine the specificity of action of DMPS, we compared the effects of the
compound on TNF-
-stimulated eotaxin mRNA expression with those found
for the neutrophil-active cytokine IL-8. Even at the highest concentration of
DMPS or TPEN used, the TNF-
-stimulated IL-8 mRNA expression was not
altered. These results suggest that at the concentrations used, zinc chelators
do not disrupt the housekeeping functions of the cell but specifically alter
the expression of certain inducible chemokines (in this case eotaxin), while
not affecting the expression of other cytokines such as IL-8. The nontoxic
effects of the zinc chelators are supported by the cytotoxicity experiments
using the MTT assay in which the chelators were tested at the maximal
concentrations in the same experimental conditions as described for Northern
blot analysis and compared with medium alone. The MTT assay demonstrated that
all the treatments used in this study were nontoxic to the cells.
The proposed mechanism by which DMPS could chelate divalent cations such as
zinc is through its two free thiol groups. We therefore tested the
reversibility of the inhibitory effect of DMPS on TNF--induced eotaxin
mRNA expression by the addition of exogenous zinc. When A549 cells were
incubated with a combination of exogenous zinc and DMPS treatment, the
inhibitory effect of DMPS was lost. At a molar ratio of 3:1 (DMPS/zinc
acetate), the inhibitory action of DMPS was partially repressed, whereas at a
molar ratio of 2:1, the inhibitory effect of DMPS was completely abolished.
This suggests that a two-molecule complex of the chelator is needed to
efficiently bind one zinc ion. This is in accordance with the fact that
dimercaprol (2,3-dimercaptopropanol, a molecular cousin of DMPS) also
functions at the 2:1 ratio to heavy metal
(8). Together these results
show that DMPS and TPEN through zinc chelation specifically inhibit
TNF-
-induced eotaxin mRNA expression. It should, however, be noted that
DMPS can also bind other physiologically important metals such as copper and
to a much lesser extent iron, magnesium, and manganese.
A recent study of zinc in the airway epithelium (38) showed an important role for this cation within the lung. The use of the sulfoamidoquinoline-based UV-excitable zinc fluoroprobe Zinquin enabled the authors to show the presence of labile zinc lining the apical and luminal sides of the entire length of the conducting airways. Using the Zinquin probe, Tang et al. (34) demonstrated that the potent zinc chelator TPEN could compete with the labile pool of zinc, which is further evidence that Zinquin interacts with the physiologically relevant levels of labile zinc. Our results show that DMPS also interacts with this physiologically important pool of labile zinc, since there was a significant difference between the fluorescence of Zinquin-loaded untreated A549 cells and that of DMPS-treated cells. In this series of experiments DMPS clearly diminished Zinquin fluorescence in fluoroprobe-loaded cells compared with untreated fluoroprobe-loaded cells.
At the protein level, our results show that the production of
TNF--induced eotaxin protein was dose dependently inhibited by
pretreatment of cells with DMPS or TPEN. An inhibition of up to 50% of eotaxin
release was observed at the highest chelator doses used. These results are in
accordance with those found at the mRNA level, where both chelators
significantly reduced the amount of eotaxin mRNA after TNF-
stimulation.
Given the efficiency by which zinc chelators inhibited TNF--induced
eotaxin mRNA expression, we investigated the effects of the clinically used
DMPS chelator on the mRNA expression of other C-C chemokines (RANTES and
MCP-1) and cytokines (IL-8). Our results show that TNF-
stimulation
induced the expression of RANTES, MCP-1, and IL-8. These results are in
accordance with other studies
(1,
16,
29). Interestingly, we
demonstrate that the zinc chelator DMPS selectively inhibited the
TNF-
-induced mRNA expression of the C-C type chemokines RANTES and
MCP-1, while leaving unaffected the TNF-
-induced CXC-type chemokine
IL-8. These results confirm our previous Northern blot analysis results.
Together the results demonstrated herein lead to the interesting hypothesis
that zinc chelators could selectively inhibit the C-C chemokine family without
influencing other chemokine families such as the CXC type. However, this
hypothesis warrants further investigation since it is derived from in vitro
studies where the CXC chemokine family was represented only by IL-8.
Many cis-regulatory elements have been identified in the promoter
regions of eotaxin, RANTES, and MCP-1
(4,
11,
20,
23,
39). The most commonly
identified sites relate to the transcription factors NF-B, activator
protein (AP)-1, and the signal transducer and activator of transcription
family. These transcription factors were regarded as having little or no
dependence on zinc ions. However, recent studies by Mackenzie et al.
(18), Ho and Ames
(12), and Oteiza et al.
(24) show that the activities
of transcription factors NF-
B and AP-1 could be modulated by reduced
zinc concentrations in the culture medium in various cell types. Conversely,
the zinc-finger family of transcription factors requires the binding of one or
more zinc ions to ensure proper folding and DNA binding. From the analysis of
the eotaxin, RANTES, MCP-1, and IL-8 promoter regions using the TRANSFAC
database (41), many potential
binding sites for the zinc-finger transcription factor GATA-1 were identified
in the C-C chemokine promoters, whereas none were found for the IL-8 promoter,
suggesting that this factor could be involved in the mechanism of action of
the zinc chelators DMPS and TPEN. In fact, we observed that the zinc chelators
had no effect on the TNF-
-mediated activation of NF-
B, which
appeared to be zinc insensitive under our experimental conditions in A549
cells. This indeed supports the results showing that DMPS or TPEN treatments
did not affect TNF-
-induced IL-8 mRNA expression, where NF-
B is
the most important transcription factor for IL-8. In the studies mentioned
above involving NF-
B and AP-1 and their dependency on zinc, NF-
B
activity was reduced by exposure to low zinc concentrations in the culture
medium in IMR-32, C6, and 3T3 cells. However, in 3T3 cells, AP-1 activity was
increased by low-culture-medium zinc, and, on the other hand, AP-1 activity
was decreased by low-culture-medium zinc in rat glioma C6 cells. This suggests
that various cell types may have different responses to reduced zinc that may
be dependent on exposure time and/or threshold levels. Conversely, DMPS and
TPEN both reduced the binding of the zinc-dependent transcription factor
GATA-1 in TNF-
-stimulated cells. These results suggest that GATA-1
could be an important regulatory factor in the transcription complex of these
chemokines, possibly participating in the enhancement of mRNA transcription.
This, however, does not eliminate the involvement of other zinc-dependent
factors, nor does it exclude effects of the chelators on other parts of signal
transduction. Site-directed mutagenesis in the promoters of these chemokines
would shed some light on the relative importance of GATA transcription factors
for the transcription of these chemokines; however, this was outside the scope
of this study.
In summary, this study shows that intracellular zinc clearly plays a key
role in regulating TNF--induced C-C chemokine mRNA expression and
protein release in respiratory epithelial cells and in fibroblasts. Zinc
chelators act efficiently in depleting cellular labile zinc, therefore
reducing its availability for zinc-dependent transcription factors. The
inhibitory effect of zinc chelators on inflammatory cytokine-induced mRNA
expression seems specific to the C-C chemokine family, whereas it leaves the
CXC family unaffected. Furthermore, zinc chelation influenced the binding of
the zinc-finger transcription factor GATA-1. To our knowledge this is the
first study to clearly demonstrate the importance of the intracellular labile
zinc content for the expression of specific proinflammatory chemokines from
human lung cells.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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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. Section 1734 solely to indicate this fact.
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REFERENCES |
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