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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Recent evidence has suggested 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 growth factors, chemokines, and cytokines. However, the exact cellular and molecular responses of epithelial cells to silica
exposure remains unclear. Using a murine alveolar type II cell line [murine lung epithelial (MLE)-15 cell line],
we measured the early changes in various cytokine and chemokine mRNA
species after exposure of the cells to 4-35
µg/cm2 of silica (cristobalite),
interferon (IFN)-, tumor necrosis factor (TNF)-
, and
lipopolysaccharide (LPS) alone or in combination. Total mRNA was
isolated and assayed with an RNase protection assay after 6 and 24 h of
exposure. Cristobalite exposure alone led to an increase in monocyte
chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-2,
and regulated on activation normal T cells expressed and secreted
(RANTES) mRNAs. Treatment with IFN-
alone increased MCP-1 mRNA
levels. Treatment with TNF-
or LPS alone led to an increase in MCP-1
and MIP-2 mRNA. The combination of cristobalite plus TNF-
led to an
additive increase in MCP-1 and MIP-2, whereas cristobalite plus IFN-
or LPS had a synergistic effect. We also found with a
TNF-
-neutralizing antibody that TNF-
plays a major role in
mediating the type II cell chemokine response to cristobalite exposure.
The results indicate that the cristobalite-induced chemokine response
in the lung epithelium is mediated in part by TNF-
and can be
enhanced by macrophage- and lymphocyte-derived inflammatory mediators
in an additive and synergistic fashion.
airway epithelium; interferon-; lipopolysaccharide; monocyte
chemotactic protein-1; macrophage inflammatory protein-2; regulated on
activation normal T cells expressed and secreted; tumor necrosis
factor-
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INTRODUCTION |
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INHALATION OF CRYSTALLINE SILICA can lead to the
development of pulmonary inflammation and fibrosis (16). Studies (15, 16, 22) examining the early inflammatory response in the lung after
silica exposure have typically focused on the contribution of
lymphocytes, macrophages, neutrophils, and their products. However, an
additional or alternate means by which silica may initiate an
inflammatory response is through direct interaction with the pulmonary
epithelium. Type II cells are uniquely situated at the interface
between the alveolar air space of the lung and the capillary
circulation, allowing them to respond to airborne stimuli and interact
with various cell types such as endothelial and mesenchymal cells,
alveolar macrophages, and other inflammatory cells (26). Type II cells
are known to play an important role in lung injury through the
synthesis and secretion of pulmonary surfactant and by acting as the
stem cell for the replacement of damaged type I epithelial cells (26).
Recently, using an established cell line and primary
rat type II cells in an in vitro silica exposure model, Driscoll and
co-workers (13) observed that type II cells respond directly to silica
(-quartz) by increasing expression of the chemokine mRNA species,
macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil
chemoattractant (CINC). More importantly, the same study also showed
the localization of MIP-2 mRNA expression in type II cells after in
vivo silica exposure. Thus it is likely that type II cells play a key
role in inflammatory cell recruitment through the release of
specific chemokines; however, the exact molecular and cellular
events leading to the type II cell response remain unclear.
The release of soluble inflammatory mediators such as tumor necrosis
factor (TNF)- and interferon (IFN)-
from silica-activated alveolar macrophages and lymphocytes will likely influence the nature
of the type II cell chemokine response to silica in vivo. Increased
expression of chemokines in the lung has been linked to the release of
the cytokines TNF-
and interleukin (IL)-1 from macrophages, which
act in an autocrine and/or paracrine fashion to stimulate the
release of chemokines from cells including macrophages, epithelial
cells, and fibroblasts (12, 13, 23, 30, 31). Exposure of the lung to
silica also leads to an influx of T lymphocytes, which are a source of
IFN-
production (21, 33). A study (24) that used cocultured primary
rat type II cells and alveolar macrophages observed that cell-cell
interactions modified the direct type II cell-particle response to coal
dust as measured by the synthesis and assembly of extracellular matrix
proteins. Various stimuli are known to cause type II cells to produce
inflammatory mediators such as IFN, MIP-2, CINC, IL-6, IL-8, and
monocyte chemotactic protein (MCP)-1 (8, 13, 12, 17, 23, 30, 31). Thus we hypothesize that the in vivo response of type II cells to silica exposure will depend on direct particle-cell interactions and on
paracrine stimulation by other silica-activated cells such as
macrophages and lymphocytes. In the context of the studies described
here, direct particle-cell interactions are defined as any
particle effect, such as formation of oxidants or binding to the cell, that involves only one cell type (i.e., type II cell).
In the present study, we utilized an in vitro model to examine the
mechanisms through which cristobalite exposure in the lung increases
the expression of specific cytokines and chemokines in type II cells.
We conducted studies examining both the direct particle-type II cell
interactions and the effects of cristobalite-induced macrophage- and
lymphocyte-derived inflammatory mediators on type II cell cytokine and
chemokine mRNA expression. We also examined whether these mediators or
other inflammatory stimuli such as LPS had a synergistic or additive
effect on epithelial chemokine expression when combined with silica
exposure. In addition, using an anti-TNF- antibody,
we investigated the role of TNF-
in particle-induced alterations in
type II cell chemokine expression. Our results support the hypothesis
that silica-induced alterations in chemokine expression in type II
cells are mediated by particle-cell interactions and paracrine
stimulation by other silica-activated cells involving, at least in
part, the production of TNF-
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MATERIALS AND METHODS |
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Particles and reagents. Cristobalite, a form of crystalline silica (a gift from Dr. D. Hemenway, University of Vermont, Burlington), was size selected with 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 with a scanning electron microscope equipped with an imaging software package where 50 particles were measured and the average was calculated. Mean particle size was 0.64 ± 0.05 µm, and particle size range was 0.08-1.5 µm. Silica was baked at 180°C for 16 h to inactivate any possible contaminating endotoxin. Silica particle suspensions were sonicated for 30 s before addition to cell culture exposure experiments. Throughout the studies presented in this paper, we utilized several particle doses described as follows: 4 µg/cm2 = 25 µg/ml, 9 µg/cm2 = 50 µg/ml, 18 µg/cm2 = 100 µg/ml, and 35 µg/cm2 = 200 µg/ml.
Lipopolysaccharide (LPS) from Escherichia
coli serotype 026:B6 was purchased from Sigma (St.
Louis, MO). Recombinant mouse IFN-, TNF-
, and rabbit anti-mouse
TNF-
polyclonal neutralizing antibody were purchased
from Genzyme (Cambridge, MA).
Cell culture. The murine lung epithelial (MLE)-15 cell line was 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 (19, 36). The MLE-15 cell line maintains many of the morphological characteristics and gene expression patterns consistent with those seen in nonciliated bronchiolar and alveolar type II epithelial cells. More specifically, the MLE-15 cell line maintains a typical polygonal epithelial cell morphology and retains multilamellar inclusion bodies (19, 36). In addition, the MLE-15 cell line maintains the ability to express SP-A, SP-B, and SP-C (19, 36). Cells were maintained in Dulbecco's modified Eagle's medium-Ham's F-12 medium (DMEM-F12) with 2% fetal bovine serum (FBS) and 10 µg/ml of gentamicin. For stimulation experiments, cells were plated (2 × 106 cells) in 60-mm cell culture dishes (DMEM-F12-1% FBS) coated with type I collagen and allowed to grow for 48 h (~5 × 106 cells). After the growth period, cells were washed with Hanks' balanced salt solution (without calcium chloride, magnesium chloride, magnesium sulfate, and phenol red) and the medium was replaced with fresh DMEM-F12-0% FBS. Silica and/or the agent of interest was added immediately after medium replacement.
Preparation of a "silica-macrophage"-conditioned medium was
carried out with the use of the mouse monocyte-macrophage cell line RAW
264.7 (RAW; American Type Culture Collection). RAW cells were
maintained in DMEM-F12 medium with 10% FBS and 10 µg/ml of gentamicin. For stimulation experiments, cells were plated (2 × 106 cells) in 60-mm cell culture
dishes (DMEM-F12-5% FBS) and allowed to grow for 48 h (~5 × 106 cells). Silica was added at 35 µg/cm2 for 6 h. After the
exposure period, the medium was removed and centrifuged at 300 g for 10 min to remove any particles
or cells. Conditioned medium was then added to MLE-15 cells for 3 h.
For TNF- antibody experiments, the antibody was added to the RAW cells 1 h before the end of the silica exposure period.
Cytotoxicity analysis. Cell death was evaluated by measuring lactate dehydrogenase (LDH) activity in the culture medium. Cells were cultured for either 6 or 24 h with varying doses of silica, and then the culture medium and cells were isolated. LDH activity was measured in the culture medium (Sigma LDH kit), and the cells were assayed for total protein with the micro bicinchoninic acid assay (Pierce, Rockford, IL).
RNase protection assay. Total RNA was
isolated from MLE-15 cells with TRIzol Reagent (GIBCO
BRL, Grand Island, NY). RNase protection analysis was carried out with
the use of a previously described protocol (18). Briefly, unlabeled
sense RNA encoding for eotaxin, IL-6, IFN-, IFN-
,
interferon-inducible protein (IP)-10, lymphotactin
(LTN), lymphotoxin (LT)-
, MIP-1
, MIP-1
, MIP-2, MCP-1, regulated on activation normal T cells expressed and
secreted (RANTES), T cell activated (TCA)-3, TNF-
, TNF-
, mouse
ribosomal protein L32 (L32), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased in kit form (Riboquant Kit, Pharmingen, San Diego, CA). For synthesis of radiolabeled antisense RNA, the final reaction mixture (10 µl) contained 70 µCi of
[
-32P]UTP (3,000 Ci/mmol; NEN, Cambridge, MA); 10 µmol of UTP; 500 µmol each of GTP,
ATP, and CTP; 10 µmol of dithiothreitol; 1× transcription
buffer; 12 U of RNasin; 8 U 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 RNase-free DNase (2 U;
Promega) for 30 min at 37°C, and the probes were purified by
extractions with phenol-chloroform and chloroform and precipitation
with ethanol. Dried probes were dissolved (3 × 105
counts · min
1 · µl
1)
in hybridization buffer (80% formamide, 0.4 M NaCl, 1 mM EDTA, and 40 mM PIPES, pH 6.6) and added (2 µl; 3 × 105
counts · min
1 · µl
1)
to tubes containing sample RNA (10 µg) dissolved in 8 µl of hybridization buffer. The samples were heated at 80°C for 3 min and
incubated at 56°C for 16 h. The single-stranded RNA
was then digested by addition of a solution (100 µl) 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 at 37°C), the 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). The RNA
duplexes were isolated by extraction and precipitation as above,
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 XAR 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 with a
phosphorimager (Molecular Dynamics, Sunnyvale, CA).
Statistics. Results from LDH and phosphorimager analyses are reported as means ± SE. Statistical comparisons were made with a one-way ANOVA with Tukey-Kramer multiple comparisons test, with significance defined as P < 0.05.
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RESULTS |
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Cristobalite-induced alveolar epithelial cell injury. Cristobalite caused a dose- and time-dependent increase in LDH release from MLE-15 cells (Fig. 1). Exposure of MLE-15 cells to 18 and 35 µg/cm2 of cristobalite for 6 h resulted in small but significant LDH releases. These LDH responses were 13 and 29%, respectively, of the total LDH release as determined by cell lysis of MLE-15 cells with sonication. LDH release from the MLE-15 cells was significantly increased for the 9 (12% total LDH), 18 (30% total LDH), and 35 µg/cm2 (52% total LDH) particle concentrations after exposure to cristobalite for 24 h.
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Effect of cristobalite exposure on epithelial cell
chemokine expression. To investigate whether chemokine
gene expression in murine alveolar type II epithelial cells could be
induced by direct interactions with cristobalite particles, we
performed in vitro exposure studies with MLE-15 cells. mRNA for the
chemokines eotaxin, IP-10, LTN, MCP-1, MIP-1, MIP-1
, MIP-2, and
TCA-3 was not detected in untreated cultures of MLE-15 cells (Fig.
2). When MLE-15 cells were exposed to 18 or
35 µg/cm2 of cristobalite for 6 h, MCP-1 and MIP-2 mRNA levels increased significantly. However, after
24 h of cristobalite exposure, both MCP-1 and MIP-2 mRNA levels were
reduced in the 18 µg/cm2
exposure group and absent in the 35 µg/cm2 exposure group.
Time-course experiments that used the 18 µg/cm2 dose showed even more
dramatically the rise and fall of MCP-1 and MIP-2 mRNA expression (Fig.
2). Unlike MCP-1 and MIP-2, RANTES mRNA expression did not rise until
after 9 h of cristobalite exposure and continued to increase after 24 h
of exposure (Fig. 2). mRNA for the chemokines eotaxin, IP-10, LTN,
MIP-1
, MIP-1
, and TCA-3 was not observed at any time point or
dose tested. Transcription of the GAPDH and L32 genes were constitutive
and were not altered by cristobalite exposure.
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Effects of alveolar macrophage- and lymphocyte-derived
cytokines on chemokine expression in epithelial cells.
The release of alveolar macrophage- and lymphocyte-derived inflammatory
mediators such as TNF- and IFN-
will likely influence the type II
cell response to silica in vivo. To investigate the influence of these mediators on type II cell expression of chemokines, we exposed MLE-15
cells to recombinant murine IFN-
and TNF-
either alone or in
combination with cristobalite. Treatment with TNF-
alone led to a
dose-dependent increase in MCP-1 and MIP-2 mRNAs (Fig. 3). The combination of TNF-
with
increasing concentrations of cristobalite led to a dose-dependent
increase in both MCP-1 and MIP-2 mRNAs (Fig 3). Unlike the
cristobalite-only exposures in which particle doses >18
µg/cm2 were required to initiate
a chemokine response, the combination of cristobalite and TNF-
led
to MCP-1 and MIP-2 mRNA responses at a particle dose of 9 µg/cm2. This response at a lower
particle concentration did not, however, correlate with an increase in
cellular cytotoxicity because LDH levels remained the same after the
combined treatment compared with cristobalite treatment alone (data not
shown). Quantitative measurements of changes in MIP-2 and MCP-1 mRNA
levels are shown in Fig. 4,
A and
B, respectively. For all treatment
plus cristobalite (9 µg/cm2)
groups, statistical comparisons were made with the treatment control
group (i.e., TNF-
, IFN-
, LPS) because
cristobalite (9 µg/cm2)
treatment alone does not induce chemokine expression. Unlike the
seemingly synergistic MIP-2 mRNA response seen with the combination of
9 µg/cm2 of cristobalite and
TNF-
, the combination of 18 µg/cm2 of cristobalite and
TNF-
was additive compared with the sum of cristobalite (18 µg/cm2) alone and TNF-
alone (Fig. 4A). A similar additive
response was seen for MCP-1 mRNA levels after the same combination of
18 µg/cm2 of cristobalite and
TNF-
(Fig. 4B).
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Unlike TNF-, treatment with IFN-
alone or in combination with
cristobalite only altered MCP-1 mRNA levels (Figs. 3 and 4). Also, both
9 µg/cm2 and 18 µg/cm2 particle doses in
combination with IFN-
led to a synergistic increase in MCP-1 mRNA
levels. As with the TNF-
-cristobalite exposures, the
IFN-
-cristobalite exposures also showed no increase in LDH levels
compared with cristobalite-treated cells (data not shown). Addition of
both IFN-
and TNF-
to MLE-15 cell cultures led to a synergistic
increase in MCP-1, MIP-2, and RANTES (Figs. 3 and 4).
Treatment with the inflammatory agent LPS led to an increase in MIP-2
and MCP-1 mRNA levels in the MLE-15 cells (Figs. 3 and 4). Although it
appears in Fig. 3 that TNF- (1 ng/ml) is the more potent inducer of
MCP-1 (vs. LPS), when the treatment groups were normalized to GAPDH
levels and quantitated with the phosphorimager, LPS was actually
slightly more effective. Treatment with LPS in combination with
cristobalite (9 and 18 µg/cm2)
led to a synergistic increase in MIP-2 and MCP-1 mRNA levels compared
with the sum of LPS alone and cristobalite alone (Fig. 4,
A and
B, respectively). Interestingly,
whereas treatment with LPS in combination with TNF-
led to a
synergistic increase in MIP-2 mRNA, the same treatment only led to an
additive increase in MCP-1 mRNA levels. Also, similarly to
TNF-
-IFN-
treatment, LPS-IFN-
treatment led to a
synergistic increase in MCP-1, MIP-2, and RANTES mRNA levels (Figs. 3
and 4).
None of the other chemokines (eotaxin, IP-10, LTN, MIP-1, MIP-1
,
or TCA-3) was detected after any treatment combination of TNF-
,
IFN-
, cristobalite, and LPS tested. Transcription of the GAPDH and L32 genes were constitutive and were not altered by
TNF-
/IFN-
/cristobalite/LPS exposure.
To further explore the silica-induced paracrine interactions between
epithelial cells and macrophages, we used a macrophage cell line to
generate a silica-macrophage-conditioned medium
(described in MATERIALS AND
METHODS). Treatment of the MLE-15 cells with the
conditioned medium led to an increase in MCP-1 and MIP-2 mRNA levels
(Fig. 5). Although virtually undetectable
here, MCP-1 mRNA levels can be detected more clearly if the RNase
protection blots are exposed to autoradiograph film for 2 days (results
not shown). The pattern of MCP-1 and MIP-2 expression was similar to
that seen after cristobalite or TNF- treatment of MLE-15 cells. We (unpublished data) and others (7) have shown that the RAW macrophage cells used to generate the silica-macrophage-conditioned medium produce
TNF-
protein after exposure to silica. To examine the role that
TNF-
played in regulating MCP-1 and MIP-2 mRNA expression in the
MLE-15 cells after treatment with the silica-macrophage-conditioned medium, we added a TNF-
-neutralizing antibody to the medium 1 h
before its addition to the MLE-15 cells. The results in Fig. 5 show
that treatment with the TNF-
antibody completely inhibited the
increase in MCP-1 mRNA and markedly attenuated the increase in MIP-2
mRNA.
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Contribution of epithelium-expressed cytokines in
regulating cristobalite-induced chemokine expression.
Previous studies suggested that type II cells have the ability to
produce the inflammatory mediators IFN (18) and IL-6 (8) after various
stimuli. To investigate whether the observed silica-induced alterations
in chemokine mRNA were the result of autocrine interactions with other
inflammatory mediators produced by the MLE-15 cells, we used the RNase
protection assay to measure mRNA levels for the following cytokines:
IL-6, IFN-, IFN-
, LT-
, TNF-
, TNF-
, transforming growth
factor (TGF)-
1, and TGF-
2 after various treatment conditions (Fig. 6). Results show that after treatment
with cristobalite, LPS, and TNF-
, there was an increase in TNF-
mRNA in the MLE-15 cells (Fig. 6). After correction for gel loading,
TGF-
1 was found to be constitutively expressed and was not altered
after any treatment (Fig. 7). An increase
in IFN-
and LT-
mRNA could be detected in all treatment groups
except the control and IFN-
groups if the RNase protection blots
were exposed to autoradiograph film for 2 days (results not shown).
Next, we treated the MLE-15 cells with cristobalite and/or an
anti-TNF-
antibody to assess whether TNF-
was involved in
regulating the cristobalite-induced alterations in MCP-1 and MIP-2
mRNAs. Antibody treatment completely eliminated the increase in MCP-1
and MIP-2 mRNAs (Fig. 5).
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DISCUSSION |
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The contribution of the type II cell to the pathogenesis of silicosis
remains largely unknown. However, increasing evidence implicates the
type II cell as a potential mediator of pulmonary recruitment and activation of inflammatory cells through the release of
a variety of chemokines (17, 24, 30, 31). Recently, Driscoll and
co-workers (13) reported that rat type II cells express mRNA for the
chemokines MIP-2 and CINC in response to direct interactions with
-quartz. The same authors also found that pretreatment with an
anti-MIP-2 antiserum before intratracheal instillation of
-quartz in
rats reduced by 60% the accumulation of neutrophils in bronchoalveolar
lavage fluid (BALF) (13). In the present study, we confirm and extend
these findings by using an in vitro murine type II cell silica exposure
model. We observed an increase in mRNA levels for the chemokines MCP-1, MIP-2, and RANTES in a time- and dose-dependent fashion after cristobalite exposure in MLE-15 cells. These chemokines can be divided
into two subgroups, designated as C-X-C or C-C, depending on their
structural, functional, and genomic characteristics (27). The C-X-C
chemokines, which include MIP-2, act principally on neutrophils,
stimulating their chemotaxis and activation (10, 27). The C-C
chemokines, which include MCP-1 and RANTES, are chemotactic for
monocytes and T lymphocytes (22, 27), although they also act on other
cell types such as basophils (22), eosinophils (20), and mast
cells (2).
Previously, MCP-1 has been detected in BALF, alveolar macrophages,
fibroblasts, and type II cells of patients with coal worker's pneumoconiosis (4) and in whole lung tissue of rats exposed to carbon
black (11). Also, MCP-1 gene expression and secretion by rat alveolar
macrophages after treatment with LPS, TNF-, and IL-1
has been
reported (5). However, until this study, the silica-induced expression
of MCP-1 and RANTES mRNAs in any lung cell type has not been reported.
Increases in RANTES gene expression have not been previously associated
with particle exposure in the lung. Other inflammatory lung disorders
such as respiratory syncytial virus infection have been shown to induce
RANTES in infected airway epithelial cells (3). Our
results along with those of others provide strong evidence for the
participation of type II epithelial cells in the recruitment of
monocytes, neutrophils, and T lymphocytes to the lung after silica exposure.
A critical question is whether the observed increases in cytokine and
chemokine message are independent of cristobalite-induced cell death. A
previous study (13) has shown that a rat epithelial cell line exposed
to 20 µg/cm2 of
-quartz can
elevate CINC and MIP-2 mRNA levels without a corresponding increase in
LDH levels. Our data suggest that at least at low doses of cristobalite
(9 µg/cm2) and in the presence
of TNF-
or IFN-
, a chemokine response can be generated, with no
increase in LDH levels. At higher doses of cristobalite (18 and 35 µg/cm2), interpretation of the
results in terms of a cell death-mediated component becomes
complicated. For example, we might speculate that the decrease in MCP-1
and MIP-2 mRNAs after 24 h is due to the high toxicity seen at this
time. However, at the same time MCP-1 and MIP-2 mRNA levels are
decreasing, RANTES levels are rising, thus contradicting the hypothesis
that increased toxicity is attenuating the chemokine response.
The direct response of type II cells to silica observed in vitro will
likely account for only part of the response observed in vivo where
paracrine stimulation by other silica-activated cell types is extremely
important. In the present study, we examined the effects of the
macrophage- and T lymphocyte-derived cytokines TNF- and IFN-
on
the cristobalite-induced chemokine response in the MLE-15 cells. The
observation that TNF-
treatment in the MLE-15 cells led to an
increase in MIP-2 and MCP-1 mRNA levels is consistent with previous
studies performed in human and rat epithelial type II-like cell lines
(13, 30). Previous studies that used the human lung carcinoma A549 (1)
and bronchial epithelial BEAS-2B (32) cell lines have also shown an
increase in RANTES mRNA and protein after at least 16 h of TNF-
or
IFN-
treatment. The fact that the duration of our TNF-
and
IFN-
exposures was only 6 h likely explains why we did not observe
an increase in RANTES mRNA expression. However, using the MLE-15 cells,
we did confirm a previous study (32) showing that the combination of TNF-
and IFN-
displays a marked synergism in inducing RANTES mRNA
expression in BEAS-2B cells. We also showed TNF-
-IFN-
and LPS-IFN-
synergistically elevated MCP-1 and MIP-2
mRNAs. These results suggest that the type II cell will respond to
multiple paracrine interactions with other surrounding cells, such as
alveolar macrophages and T lymphocytes, through the release of various chemokines.
When given in combination with cristobalite, TNF- or IFN-
lowers
the dose at which a particle-induced alteration in chemokine expression
is observed in MLE-15 cells. This suggests an in vivo situation where
silica-activated alveolar macrophages and T lymphocytes can modulate
the sensitivity and pattern of the epithelial chemokine response
through the release of TNF-
or IFN-
. Our
silica-macrophage-conditioned medium experiments further this
hypothesis by showing the ability of the conditioned medium to
stimulate a chemokine response in the MLE-15 cells. In addition,
TNF-
appears to be the primary cristobalite-induced
macrophage-derived mediator orchestrating this macrophage-induced
epithelial chemokine response, as evidenced by our TNF-
antibody
experiments in which the chemokine response induced by conditioned
medium is markedly attenuated after antibody treatment. Our observation
that bacterial endotoxin in combination with cristobalite can also
synergistically increase the MCP-1 and MIP-2 mRNA responses in MLE-15
cells suggests that other inflammatory stimuli can sensitize or
"prime" the epithelium, thereby enhancing the particle response.
This concept is in agreement with a previous study (9) that found
pretreatment of human alveolar macrophages with LPS followed by
exposure to silica dust led to an enhanced production of thromboxane
A2 and leukotriene
B4. These data suggest that the
induction of MCP-1 and MIP-2 gene expression in murine type II cells
after in vivo exposure to cristobalite will, at least in part, be
influenced by interactions with alveolar macrophages and other
inflammatory stimuli such as endotoxin.
We have found (unpublished results) that the in vitro
activation of MLE-15 chemokine expression could not be achieved with the less inflammatory dust titanium dioxide. This result suggests that
the chemokine response observed after cristobalite exposure is not the
result of a generic particle-cell interaction. Other investigators have
also found that the in vitro activation of epithelial chemokine
expression, although not unique to silica, is not the property of all
particles. For example, a previous study (13) found that the highly
inflammatory dust -quartz and crocidolite asbestos fibers led to an
increase in the epithelial expression of CINC and MIP-2, whereas the
less inflammatory dust titanium dioxide or man-made vitreous fibers
(MMVF)-10 did not increase chemokine expression. The same
study speculated that the differential effect of
-quartz,
crocidolite, titanium dioxide, and MMVF-10 glass fibers on CINC and
MIP-2 expression may be explained by the fact that both
-quartz and
crocidolite can give rise to reactive oxygen species.
The precise mechanism by which direct interactions between cristobalite
particles and lung epithelial cells lead to increased MCP-1 and MIP-2
gene expression remains uncertain. Silica can give rise to reactive
oxygen species as a result of its surface chemistry and/or by
stimulating the cellular generation of oxidants (6). In this respect,
reactive oxygen species have been shown to act as second messengers,
stimulating the translocation of the stress-responsive transcription
factor nuclear factor (NF)-B (29). The promoter regions for MCP-1
and MIP-2 contain the binding element for NF-
B (34, 35). A recent
study (14) demonstrated an increase in nuclear translocation of NF-
B
in rat lung epithelial cells exposed to
-quartz in vitro as well as
the ability of antioxidants to attenuate the
-quartz-induced NF-
B
translocation and MIP-2 gene expression in a rat lung epithelial cell
line. Cristobalite-induced MCP-1 gene activation may also involve
activator protein (AP)-1 binding inasmuch as previous studies (25, 27,
37) that used endothelial cells have shown both AP-1 and NF-
B
binding is required for maximal gene induction after IL-1 or
H2O2
exposure. Our study examined whether direct cristobalite-induced
alterations in MCP-1 and MIP-2 mRNAs were the result of autocrine
interactions with other inflammatory mediators produced by the MLE-15
cells. The results of our study suggest that cristobalite-induced
activation of epithelial TNF-
plays a critical role in the
activation of the MCP-1 and MIP-2 genes. Driscoll and co-workers (12)
have shown that passive immunization of mice against TNF-
markedly attenuates the increases in lung MIP-2 mRNA seen in response to
-quartz. The question of whether cristobalite-induced activation of
TNF-
or the subsequent TNF-
-induced activation of the MCP-1 and
MIP-2 genes is the result of oxidant stress was not examined in the
present study. However, evidence exists showing antioxidants inhibit
TNF-
-mediated stimulation of IL-8, MCP-1, and collagenase expression
in cultured human synovial cells (28).
Our results suggest that there are multiple mechanisms involved in
cristobalite-induced expression of the chemokines MCP-1, MIP-2, and
RANTES. For example, one potential mechanism is that chemokine
expression is mediated by the cristobalite-induced expression of
TNF-. This mechanism allows us to explain our conflicting results
showing a synergistic effect of TNF-
plus cristobalite treatment on
MCP-1 and MIP-2 mRNA levels at a 9 µg/cm2 particle dose and an
additive effect at an 18 µg/cm2
particle dose. We believe the observed synergism after the 9 µg/cm2 particle dose is in fact
an additive effect. The combination of cristobalite-induced TNF-
and
recombinant TNF-
increases the level of TNF above a threshold level,
subsequently leading to the increased expression of MCP-1 and MIP-2
mRNAs. This hypothesis is further supported by our observations showing
that the chemokine response is eliminated after addition of a TNF-
antibody. In contrast, our results showing that IFN-
in combination
with cristobalite leads to a synergistic increase in MCP-1 mRNA but has
no effect on cristobalite-induced MIP-2 mRNA levels suggest that an
alternate non-TNF-
-mediated pathway is stimulated. The argument
could be made, however, that the observed synergy between IFN-
and
cristobalite is just the sum of the effects mediated by IFN-
enhancing the previously described TNF-
-mediated particle-activated
pathway plus the effects of IFN-
treatment alone. However, if this
argument were true, we should have also seen an effect of IFN-
treatment on cristobalite-induced MIP-2 mRNA levels. Thus, in the
presence of IFN-
, cristobalite appears to be able to stimulate an
alternate non-TNF-
-mediated pathway, leading to the expression of
MCP-1.
In summary, the present study demonstrates that the early inflammatory
response in the lung after exposure to cristobalite, a form of
crystalline silica, is a dynamic process involving multiple cell types.
The type II cell plays a crucial role in this inflammatory response by
interacting directly with cristobalite particles or by interacting with
macrophage- and lymphocyte-derived mediators. In both cases, the
interactions lead to the generation of the chemokines MCP-1, MIP-2, and
RANTES from the type II cell. Our in vitro results also suggest that
cristobalite-induced TNF- from either the alveolar macrophage or the
type II cell is a key mediator of the type II cell chemokine response.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institute of Environmental Health Science Grants ES-04872, ES-01247, and ES-07026 and Center for Indoor Air Research Grant CA-27791.
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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: J. N. Finkelstein, Box 777, Dept. of Pediatrics, Univ. of Rochester School of Medicine, Rochester, NY 14642.
Received 17 June 1998; accepted in final form 24 August 1998.
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