Departments of 1 Environmental Medicine and 2 Pediatrics, University of Rochester School of Medicine, Rochester, New York 14642
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ABSTRACT |
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We
have shown previously that epithelial cells may contribute to the
inflammatory response in the lung after exposure to crystalline silica
through the production of and response to specific chemokines and
cytokines. However, the exact cellular and molecular responses of
epithelial cells to silica exposure remain unclear. We hypothesize that
non-oxidant-mediated silica-cell interactions lead to the upregulation
of tumor necrosis factor- (TNF-
), whereby TNF-
-induced generation of reactive oxygen species (ROS) leads to the activation of
the monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-2 genes. Using a murine alveolar type II cell line, murine lung epithelial (MLE)-15, we measured the early changes in
TNF-
, MCP-1, and MIP-2 mRNA species after exposure of the cells to
18 µg/cm2 silica (cristobalite)
in combination with various antioxidants. Total mRNA was isolated and
assayed using an RNase protection assay after 6 h of particle exposure.
We found that extracellular GSH could completely attenuate the
cristobalite-induced expression of MCP-1 and MIP-2 mRNAs, whereas
TNF-
mRNA levels were unaltered. We also found using the
oxidant-sensitive dye
6-carboxy-2',7'-dichlorodihydrofluorescein diacetate
di(acetoxymethyl ester) that treatment of MLE-15 cells with
cristobalite and TNF-
(1 ng/ml) resulted in ROS production. This ROS
production could be inhibited with extracellular GSH treatment, and in
the case of cristobalite-induced ROS, inhibition was also achieved with
an anti-TNF-
antibody. The results support the hypothesis that
TNF-
mediates cristobalite-induced MCP-1 and MIP-2 expression
through the generation of ROS.
airway epithelium; reactive oxygen species; monocyte chemotactic
protein-1; macrophage inflammatory protein-2; tumor necrosis factor-
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INTRODUCTION |
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INHALATION OF CRYSTALLINE silica particles can result
in the development of an inflammatory and fibrotic disease in the lung (10). There is growing evidence that the lung epithelium plays a
significant role in silica-induced inflammation by mediating inflammatory cell recruitment through the release of specific chemokines. Previously, using an established cell line and primary rat
type II cells in an in vitro silica exposure model, Driscoll and
co-workers (5) observed that type II cells respond directly to silica
(-quartz) by increasing expression of macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant. More
importantly, the same study also showed the localization of MIP-2 mRNA
expression in type II cells after in vivo silica exposure (5). We have
shown recently that MIP-2, monocyte chemotactic protein (MCP)-1, and
RANTES are expressed in a murine alveolar type II cell line after
exposure to silica (cristobalite; see Ref. 2). The results of our
previous study also indicate that silica-induced expression of type II
cell tumor necrosis factor-
(TNF-
) plays a critical role in the
upregulation of the MCP-1 and MIP-2 genes (2). The question of how
silica-type II cell interactions lead to the expression of TNF-
and
how TNF-
subsequently mediates MCP-1 and MIP-2 gene expression
remains to be elucidated.
There are two potential mechanisms by which silica-cell interactions
may lead to cellular activation; they include
1) cell activation via a distinct
activating receptor(s) involved in binding silica particles and/or
2) cell activation by aspects of the
ingested particles chemistry, e.g., surface characteristics. In support of the second hypothesis, increasing evidence supports the role for
particle-associated reactive oxygen species (ROS) as mediators of
pulmonary inflammation and damage after silica exposure (31). ROS can
form either on the surface of silica, especially after its fracture
(8), or through the generation of a respiratory burst caused by the
phagocytosis of silica (30). The addition of the antioxidant
N-acetyl-L-cysteine (NAC)
has been shown to decrease residual oil fly ash (ROFA) and
-quartz-mediated interleukin (IL)-8 production by ~50% in normal
and TNF-
-primed A549 (epithelial type II cell line) cells (29).
Additionally, treatment with the free radical trapper,
N-t-butyl-
-phenylnitrone,
decreases silica-induced expression of TNF-
mRNA in alveolar
macrophages and attenuates overall lung injury (9). Both of these
studies have speculated that silica-associated components (e.g.,
transition metals/surface free radicals) mediate oxidant stress and
subsequent cellular activation.
We hypothesize, however, that non-oxidant-mediated silica-cell
interactions lead to the upregulation of TNF-, whereby
TNF-
-induced generation of ROS leads to the activation of the MCP-1
and MIP-2 genes in type II cells. To test our hypothesis, we exposed a
murine lung epithelial (MLE)-15 cell line to cristobalite in
combination with various antioxidants (DMSO, extracellular GSH, and
NAC) and then measured the changes in TNF-
, MCP-1, and MIP-2 mRNA
levels. In addition, we measured the TNF-
-induced expression of
TNF-
, MCP-1, and MIP-2 mRNA in MLE-15 cells. We also
measured the cristobalite- and TNF-
-induced generation of ROS within
individual cells using an oxidant-sensitive fluorescent dye and
examined whether GSH or anti-TNF-
antibody (cristobalite only)
treatment altered this ROS production.
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MATERIALS AND METHODS |
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Particles and reagents. Cristobalite, a form of crystalline silica (gift from Dr. D. Hemenway, University of Vermont, Burlington, VT), was size selected using a cascade cyclone sampler (series 280 Cyclade; Sierra Instruments, Carmel Valley, CA) with an ~1.2-µm particle diameter cutoff. The average particle diameter was measured using a scanning electron microscope equipped with an imaging software package (50 particles were measured and the average was calculated). Because silica particles are not perfect spheres, the average diameter was determined by measuring the longest and shortest diameter of each particle, then averaging the measurements. Mean particle diameter was equal to 0.64 ± 0.05 µm, and particle size ranged from 0.08 to 1.5 µm. Silica was baked at 180°C for 16 h to inactivate any possible endotoxin contamination. Silica particle suspensions were sonicated for 30 s before addition to cell culture exposure experiments. Throughout the studies presented in this paper, we utilized a particle dose of 18 µg/cm2 = 100 µg/ml. This particle dose has been shown to be slightly cytotoxic after 6 h of exposure in MLE-15 cells as measured by lactate dehydrogenase (LDH) release (2).
NAC was purchased from ICN Biomedicals (Aurora, OH), DMSO was from
American Type Culture Collection (Manassas, VA), and
DL-buthionine-[S,R]-sulfoximine (BSO) was from Schweizerhall (South Plainfield, NJ). Extracellular GSH
was purchased from Sigma (St. Louis, MO). Recombinant mouse interferon- (IFN-
), TNF-
, and rabbit anti-mouse TNF-
polyclonal neutralizing antibody were purchased from Genzyme
(Cambridge, MA).
Cell culture. MLE-15 cells are a cell line immortalized from lung tumors of transgenic mice containing the simian virus 40 large T antigen under the transcriptional control of the regulatory sequences derived from the human surfactant protein (SP)-C promoter region (18, 34). The MLE-15 cell line maintains many of the morphological characteristics and gene expression patterns consistent with that seen in nonciliated bronchiolar and alveolar type II epithelial cells. More specifically, MLE-15 cells maintain a typical polygonal epithelial cell morphology and retain multilamellar inclusion bodies (18, 34). In addition, the MLE-15 cell line maintains the ability to express SP-A, SP-B, and SP-C (18, 34). Cells were maintained in 5 ml of DMEM-F-12 medium with 2% FBS and 10 µg/ml gentamicin. For stimulation experiments, cells were plated (2 × 106 cells) in 60-mm cell culture dishes (DMEM-F-12-1% FBS) coated with type I collagen and were allowed to grow for 48 h (~5 × 106 cells). After the growth period, cells were washed with Hanks' balanced salt solution (calcium chloride, magnesium chloride, magnesium sulfate, and phenol red free), and the medium was replaced with 5 ml of fresh DMEM-F-12-0% FBS. Cristobalite or the agent of interest was added immediately after medium replacement.
For inhibitor studies, extracellular GSH (50 mM), DMSO (1%), or NAC (30 mM) was added immediately after medium replacement and 0.5 h before the addition of cristobalite. In contrast, BSO (0.4 mM) was added 24 h before medium replacement. Fresh BSO (0.4 mM) was then added immediately after medium replacement and 0.5 h before the addition of cristobalite. NAC and extracellular GSH were prepared in DMEM-F-12, and the pH was adjusted to 7.4. It should also be noted that three different exposure periods (1, 3, or 6 h) were utilized throughout these experiments. As previously described (2), maximal cristobalite-induced gene activation (MCP-1 and MIP-2 mRNAs) in MLE-15 cells occurs at ~6-9 h of exposure. Thus it is assumed that cristobalite-induced oxidant stress (if it is involved in gene activation) will develop before changes in mRNA levels are seen. Thus, in the present study, although we measured changes in mRNA levels after 6 h of particle exposure, we measured particle-induced ROS production after 1 and 3 h of particle exposure.
RNase protection assay. Total RNA was
isolated from MLE-15 cells using TRIZOL Reagent (GIBCO BRL, Grand
Island, NY). RNase protection analysis was carried out using a
previously described protocol (15). Briefly, a riboprobe template
encoding for MIP-2, MCP-1, TNF-, L32 (mouse ribosomal protein L32),
and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased in
kit form (Riboquant Kit; PharMingen, San Diego, CA). For synthesis of
radiolabeled probe, the final reaction mixture (10 µl) contained 70 µCi of [
-32P]UTP
(3,000 Ci/mmol; New England Nuclear, Cambridge, MA), 10 µmol of UTP,
500 µmol each of GTP, ATP, and CTP, 10 µmol of dithiothreitol, 1× transcription buffer, 12 units of RNasin, 8 units of T7
polymerase (all from Promega, Madison, WI), and an equimolar pool of
linearized template (60 ng total). After 1 h at 37°C, the mixture
was treated with DNase buffer (90 µl) and RQ1-DNase (2 units;
Promega) for 30 min at 37°C. Probes were purified by extractions
with phenol-chloroform and precipitation with ethanol.
Dried probes were dissolved [3 × 105
counts · min
1
(cpm) · µl
1]
in hybridization buffer (80% formamide, 0.4 M NaCl, 1 mM EDTA, and 40 mM PIPES, pH 6.6) and were added (2 µl; 3 × 105 cpm/µl) to tubes containing
sample RNA (10 µg) dissolved in 8 µl of hybridization buffer.
Samples were heated at 80°C for 3 min and incubated at 56°C for
16 h. The single-strand RNA was then digested by addition
(100 µl) of a solution of RNase A (0.2 µg/ml; Sigma) and RNase T1
(600 U/ml; GIBCO BRL) in 10 mM Tris, 300 mM NaCl, and 5 mM EDTA, pH
7.5. After incubation (30 min, 37°C), samples were treated with 18 µl of a mixture of proteinase K (1 mg/ml; GIBCO BRL), SDS (5%), and
yeast tRNA (200 µg/ml). RNA duplexes were isolated by
phenol-chloroform extraction and ethanol precipitation,
dissolved in 5 µl of gel loading buffer (65% formamide, 5.5 mM EDTA,
and dyes), and electrophoresed in standard 5% acrylamide-8 M urea
sequencing gels. Dried gels were placed on X-AR film (Kodak, Rochester,
NY) with intensifying screens and were developed at
80°C for
18 h. For certain data displays, dried gels were developed on
phosphorimager screens, and gel band intensity was analyzed using a
phosphorimager (Molecular Dynamics, Sunnyvale, CA). For
quantitation of gel band intensity, all values were normalized using
constitutively expressed GAPDH mRNA levels.
Detection of ROS generation with 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate di(acetoxymethyl ester) staining. MLE-15 cells were grown as previously described except for one important change that was necessary for allowing viewing of the cells under a fluorescent microscope. The MLE-15 cells were grown on collagen-covered micro cover glasses (24 × 40 mm; VWR Scientific) inserted in the bottom of 60-mm culture dishes. Immediately after exposure to cristobalite for 1 or 3 h, MLE-15 cells were washed one time with DMEM (no phenol red) and stained for 20 min at 37°C with 3 µM 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate di(acetoxymethyl ester) (C-DCDHF-DA; Molecular Probes, Eugene, OR) in prewarmed DMEM (no phenol red) as described (11). After incubation with dye, cells were washed two times with DMEM (no phenol red). Each sample was independently stained with fresh dye solution so that samples were exposed to the same amount of dye for the same time. The micro cover glasses containing the MLE-15 cells were removed from their respective culture dishes and then adhered separately to individual glass microscope slides. To maintain cellular viability while viewing under the microscope, several drops of DMEM (no phenol red) were added to the micro cover glasses, and then another micro cover glass was placed on top. MLE-15 cells were viewed with fluorescence microscopy and photographed. Images were obtained on an Olympus AX70 Microscope (Olympus America, Lake Success, NY) using Image Pro Plus software (Media Cybernetics, Silver Spring, MD). Samples were epi-illuminated by a 100-watt mercury lamp and were viewed with fluorescein filters (B2E cube). Fields were viewed at ×40 magnification and numerical aperture of 0.85 and were acquired with a charge-coupled device color video camera (DXC-9000; Sony) under computer control with 1/60-s integration time. Illumination caused increased fluorescence because of oxidation of the dye; thus each field was exposed to light for exactly the same time. The average relative fluorescence intensity for 50 cells (cells that were clumped on top of each other or too close together to separate their individual fluorescence were not measured) in three separate experiments was determined as previously described (25) using Image Pro Plus software (Media Cybernetics). Briefly, relative fluorescence intensities for each condition were determined, combined, and partitioned into four brightness classes (1-4). Class 1 represents the lowest fluorescence intensity, and class 4 represents the highest fluorescence intensity.
Statistics. Results from the phosphorimager analysis and ROS quantitation are reported as means ± SE. Statistical comparisons were made using a one-way ANOVA with Tukey-Kramer multiple comparison test, with significance defined as P < 0.05.
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RESULTS |
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Cristobalite and TNF- induce ROS generation in
MLE-15 cells. We used the oxidant-sensitive dye
C-DCDHF-DA to detect ROS production in cristobalite- and
TNF-
-stimulated MLE-15 cells. After dye uptake, intracellular
esterases hydrolyze the ester bonds, releasing the intact
nonfluorescent substrate. This reduced substrate is oxidized by ROS to
the fluorescent species carboxydichlorofluorescein, which is retained
by living cells. MLE-15 cells were stimulated with either cristobalite
or TNF-
for 1 or 3 h and then loaded for 20 min with 3 µM
C-DCDHF-DA. Cells were examined for fluorescence intensity under a
fluorescence microscope. Figure
1 shows the fluorescence
images of MLE-15 cells after C-DCDHF-DA staining. Control cells
exhibited a low intensity of fluorescence (Fig. 1,
A, B,
A1, and
B1). In contrast, both
cristobalite- and TNF-
-stimulated cells showed an increased level of
fluorescence (Fig. 1, C-F and C1-F1). TNF-
-induced
fluorescence appeared to peak after 1 h and then decrease, whereas
cristobalite did not induce fluorescence until 3 h of exposure.
Preincubation of the cells for 0.5 h with 50 mM extracellular GSH
reduced the cristobalite- and TNF-
-induced fluorescence to near
control levels (Fig. 1, G,
H, G1
and H1). Also, treatment with an
anti-TNF-
antibody reduced the cristobalite-induced fluorescence to
control levels (Fig. 1, I and
I1). Treatment with extracellular
GSH or anti-TNF-
alone led to fluorescence levels similar to those
of untreated controls (data not shown).
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Cristobalite-induced ROS generation mediates chemokine
mRNA expression. Using various antioxidants and
modifiers of cellular oxidant status in the presence or absence of
cristobalite, we examined whether cytokine and chemokine expression in
MLE-15 cells could be altered. Treatment with the hydroxyl scavenger
DMSO, extracellular GSH, or NAC decreased cristobalite-induced MIP-2 and MCP-1 mRNA levels by 81 and 49%, 99 and 97%, and 70 and 53%, respectively (Fig. 2,
A-C). Treatment with BSO, which
inhibits -glutamylcysteine synthetase and subsequently reduces
intracellular GSH levels, also led to a reduction in
cristobalite-induced MIP-2 (42%) and MCP-1 (42%) mRNA levels.
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GSH-mediated regulation of cristobalite-,
TNF--, and IFN-
-induced cytokine and
chemokine mRNA expression. As shown in Figs. 1 and 2,
extracellular GSH can inhibit cristobalite- and TNF-
-induced ROS
production and attenuate cristobalite-induced MCP-1 and MIP-2 mRNA
production in MLE-15 cells. To further support our hypothesis that
cristobalite-induced chemokine expression is mediated by TNF-
-induced ROS, we examined MCP-1, MIP-2, and TNF-
mRNA levels in MLE-15 cells after treatment with cristobalite or TNF-
in combination with extracellular GSH. We found that both cristobalite and
TNF-
could elevate TNF-
mRNA levels; however, on coincubation with extracellular GSH, only TNF-
-induced TNF-
mRNA was
completely inhibited (Fig. 3,
A and
B). In contrast, MCP-1 and MIP-2
mRNA levels were completely attenuated after treatment with
cristobalite or TNF-
in combination with extracellular GSH (Fig.
4,
A-C). Interestingly, even when BSO
was used to deplete intracellular GSH levels, the addition of
extracellular GSH greatly reduced cristobalite-induced TNF-
mRNA
levels (Fig. 3, A and
B). Also, cristobalite-induced MCP-1
and MIP-2 mRNA levels were completely attenuated after BSO and
extracellular GSH treatment (Fig. 4, A-C).
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We have shown previously (3) that IFN- can synergistically elevate
cristobalite-induced MCP-1 mRNA levels but has no effect on TNF-
or
MIP-2 mRNA levels in MLE-15 cells. In the present study, we found that
treatment with extracellular GSH had only a slight effect on
cristobalite-IFN-
-induced MCP-1 mRNA levels (Fig. 4,
A-C). Interestingly, the
addition of IFN-
in combination with cristobalite and extracellular
GSH appears to inhibit the expected expression of TNF-
mRNA in
MLE-15 cells (Fig. 3, A and B).
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DISCUSSION |
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We have shown previously, using an anti-TNF- antibody, that
cristobalite-induced activation of the MCP-1 and MIP-2 genes in the
MLE-15 cells is mediated by TNF-
(2). This finding is further
supported by the results of another study that found that passive
immunization of mice against TNF-
markedly attenuates the increases
in lung MIP-2 mRNA seen in response to
-quartz (4). Thus, in the
present study, we set out to better characterize the mechanisms through
which cristobalite-type II cell interactions lead to increased
chemokine expression. We hypothesized that non-oxidant-mediated cristobalite-cell interactions lead to the upregulation of TNF-
, whereby TNF-
-induced generation of ROS leads to the activation of
the MCP-1 and MIP-2 genes in type II cells. In support of this hypothesis, we have found that the cristobalite-induced generation of
ROS in MLE-15 cells can be attenuated by treating the cells with an
anti-TNF-
antibody. In addition, pretreatment with extracellular GSH
inhibits cristobalite- and TNF-
-induced production of ROS. At the
level of gene expression, we found that treatment with various
antioxidants inhibits cristobalite- and TNF-
-induced expression of
MCP-1 and MIP-2 mRNAs in MLE-15 cells. More importantly, we show that
antioxidant treatment does not inhibit the direct cristobalite-induced
expression of TNF-
mRNA. Although the level of cristobalite-induced
TNF-
mRNA appears to be lower after antioxidant treatment, we
believe this is due to the inhibition of secondarily produced TNF-
autocrine interactions and not the inhibition of the initial
cristobalite-cell interactions. We have also shown that TNF-
-induced
TNF-
mRNA can be completely inhibited with extracellular GSH pretreatment.
Our results compliment earlier findings by Stringer and Kobzik (29)
that show that antioxidant treatment can attenuate the -quartz-induced production of IL-8 in A549 cells. However, our proposed mechanism of silica-induced oxidant stress and cellular activation differs. The same investigators have shown that silica and
other environmental particulates (e.g., ROFA,
TiO2, and iron oxide) can interact
with type II cells and alveolar macrophages via scavenger-type
receptors (22, 28). Thus the investigators hypothesize that, because
the scavenger receptors mediate uptake of both inert (e.g.,
TiO2) and proinflammatory
(
-quartz) particles without evidence of receptor-mediated cell
activation, then particle-associated components (e.g., transition
metals or surface free radicals) are likely to mediate intracellular
oxidant stress and proinflammatory activation (28). Interestingly, the
same study shows that inhibitors of the scavenger receptors,
polyinosinic acid and heparin, only inhibited
-quartz binding to
A549 cells by ~35 and ~45%, respectively (28). The authors concede
in their discussion that multiple receptor types are likely to
contribute to particle binding and uptake by epithelial cells. Our
results in conjunction with these previous studies (22, 28) support the
idea that non-oxidant-mediated cristobalite-cell interactions (e.g.,
receptor binding) may lead to the upregulation of TNF-
, whereby
TNF-
-induced generation of ROS leads to the activation of MCP-1 and
MIP-2 genes in type II cells. It should be noted that other
non-receptor-mediated particle-cell interactions could also potentially
mediate the observed cellular activation. However, to our knowledge,
there is no evidence in the literature that describes the simple
physical act of non-receptor-mediated endocytosis causing cellular
activation. Future studies need to be conducted to identify what other
receptor- or non-receptor-mediated particle-cell events lead to
cellular activation.
In contrast, if the alternative hypotheses that silica-associated free
radicals and/or a silica-induced respiratory burst are responsible for
initiating the expression of MCP-1, MIP-2, and TNF-, we should have
observed the following results instead: 1) treatment with an anti-TNF-
antibody does not alter cristobalite-induced ROS or MCP-1 and MIP-2
mRNA levels and 2) antioxidant
treatment not only inhibits cristobalite-induced MCP-1 and MIP-2 mRNA
expression but also inhibits TNF-
mRNA. However, we are not
suggesting that this alternative hypothesis is never involved in
silica-induced activation of type II cells. The fact that we used
"aged" cristobalite, which contains very few surface free
radicals in comparison with freshly fractured silica (8), could explain
why we did not observe a direct silica- and ROS-mediated activation of
the MLE-15 cells.
The idea that TNF- leads to oxidant production and subsequent gene
activation is not new. Evidence exists showing that antioxidants inhibit TNF-
-mediated stimulation of IL-8, MCP-1, and collagenase expression in cultured human synovial cells (26). Also, a previous study utilizing human endothelial cells indicated that TNF-
induces nuclear factor-
B activation and the resultant E-selectin gene expression by a pathway that involves formation of ROS and that E-selectin expression can be inhibited by the antioxidant NAC (25). In
addition, TNF-
-induced expression of cyclooxygenase-2 in rat
mesangial cells can be inhibited with antioxidants and inhibitors of
NADPH oxidase (6). However, the exact mechanism whereby TNF-
triggers oxidant production in type II cells is not known.
The effects of ROS within the lung are counterbalanced by a complex system of enzymatic and nonenzymatic antioxidants located both intracellularly and extracellularly (14). Little is known about the antioxidant status of the lung after exposure to silica and the involvement of individual antioxidants in lung defense against silica-induced cell injury. Previous studies in rats have shown that, after inhalation of silica, mRNA levels for the antioxidant enzymes catalase and manganese-containing superoxide dismutase (Mn SOD) are elevated in lung homogenates (19, 20). More specifically, another study found that silica-induced increases in Mn SOD gene expression are localized to type II epithelial cells and alveolar macrophages (16).
GSH plays a major role in the antioxidant system by acting as a
substrate for GSH peroxidase. GSH peroxidase utilizes GSH as a
reductant to reduce toxic peroxides (21). Depletion of GSH potentiates
cellular injury due to oxidant stress (24), and stimulation of
processes that support maintenance of GSH protects against injury (1,
3). In the present study, we utilized extracellular GSH and NAC to
examine the role of ROS in cristobalite-induced cytokine and chemokine
expression. Previous investigators have shown that extracellular GSH
can elevate intracellular GSH levels and protect alveolar macrophages
(7), intestinal epithelial cells (23), and kidney cells (12) from
oxidative injury. NAC is a thiol compound that can act as a cysteine
source for the repletion of intracellular GSH and act as a direct
scavenger of ROS (1, 3). Our results showed that extracellular GSH and NAC (cristobalite gene activation) can inhibit cristobalite- and TNF--induced oxidant stress and gene activation (e.g., MIP-2 and
MCP-1) in MLE-15 cells. These results suggest that GSH levels may play
an important role in mediating the cristobalite or TNF-
-induced response. Future experiments should be performed to examine the exact
role GSH levels play in mediating the cristobalite- and TNF-
-induced
response in type II cells.
One might speculate that a decrease in intracellular GSH would lead to an increase in cristobalite-induced ROS and subsequently an increase in MCP-1 and MIP-2 mRNA levels. Interestingly, we found that, even though BSO treatment in combination with cristobalite led to a significant decrease in total GSH levels (unpublished results), cristobalite-induced MCP-1 and MIP-2 mRNA levels actually decreased. A simple explanation may be that after BSO treatment the cells become unable to respond efficiently to oxidant stress, and the addition of cristobalite, which is already cytotoxic to the MLE-15 cells (2), just leads to an increase in cell death (24). Thus fewer cells are able to respond to cristobalite treatment by elevating cytokine and chemokine levels. Treatment with t-butylhydroquinone (TBHQ), a monofunctional phase II enzyme inducer that produces ROS in combination with BSO, has been shown to elevate LDH levels above TBHQ treatment alone in the rat lung epithelial L2 cell line (24).
We have also found that the addition of extracellular GSH to BSO- and
cristobalite-treated MLE-15 cells can still completely attenuate the
cristobalite-induced expression of MCP-1 and MIP-2. Although several
authors, mostly from the same research group, have described uptake of
intact GSH by type II cells (13), we found that MLE-15 cells cannot
(unpublished results). Others have also found it difficult to
demonstrate any uptake of intact GSH in type II cells (32). The
depletion of GSH in type II cells has been shown to elevate
-glutamyltransferase activity (33), which would
subsequently elevate intracellular cysteine levels, via the breakdown
of extracellular GSH, in an effort to replete intracellular GSH levels.
Even though the synthesis of new GSH is blocked in our experiment, an
elevation in cysteine, which has antioxidant activity itself (17, 27),
could explain why the cristobalite-induced chemokine response is still
attenuated in the presence of extracellular GSH and BSO.
Previously, we have shown that IFN- synergistically elevates
cristobalite-induced MCP-1 mRNA levels without influencing MIP-2 mRNA
levels (2). In the present study, we have found that extracellular GSH
in combination with IFN-
-cristobalite treatment inhibited MIP-2 mRNA
expression, whereas MCP-1 mRNA levels were only minimally altered. This
lends further support to our hypothesis that IFN-
can enhance the
cristobalite-induced expression of MCP-1 through a pathway independent
of TNF-
and the generation of ROS. Interestingly, IFN-
either
alone or in combination with cristobalite has no effect on TNF-
mRNA
expression. However, IFN-
-cristobalite treatment in the presence of
extracellular GSH leads to the inhibition of TNF-
mRNA levels. The
cellular and molecular mechanisms contributing to this observation are unknown.
In summary, the present study shows that non-oxidant-mediated
cristobalite-cell interactions lead to the upregulation of TNF-, whereby TNF-
-induced generation of ROS leads to the activation of
the MCP-1 and MIP-2 genes. In addition, in the presence of IFN-
,
other non-TNF-
/oxidant-mediated pathways appear to be activated
after cristobalite exposure. These results support the idea that
silica-cell interactions can lead to cellular activation through
multiple pathways (oxidant/nonoxidant mediated).
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institute of Environmental Health Sciences Grants ES-04872, ES-01247, and ES-07026 and Center for Indoor Air Research Grants CA-27791 and CA-11051.
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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: J. N. Finkelstein, Box 777, Dept. of Pediatrics, Univ. of Rochester School of Medicine, Rochester, NY 14642 (E-mail: finj{at}ehsct7.envmed.rochester.edu).
Received 2 December 1998; accepted in final form 22 February 1999.
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