1 Department of Pharmaceutical Sciences, University of Connecticut; Storrs, Connecticut 06269; and Departments of 2 Pathology and 3 Medicine, University of Vermont, Burlington, Vermont 05405
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ABSTRACT |
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Occupational exposure to
crystalline silica is associated with the development of pulmonary
inflammation and silicosis, yet how silica initiates pulmonary fibrosis
and which cell types are involved are unclear. In studies here, we
hypothesized that silica particles interact initially with pulmonary
epithelial cells and alveolar macrophages (AMs) to cause
transcriptional activation of nuclear factor (NF)-B-regulated genes
encoding inflammatory cytokines. Exposure of NF-
B luciferase
reporter mice intratracheally to silica or lipopolysaccharide (LPS),
but not the nonfibrogenic particle titanium dioxide (TiO2),
increased immunoreactivity of luciferase protein in bronchiolar
epithelial cells and AMs. Ribonuclease protection assays revealed
significant (P
0.05) increases in mRNA levels of
inducible nitric oxide synthase, tumor necrosis factor-
, macrophage
inflammatory protein-2, macrophage chemotactic protein-1 (MCP-1),
interferon-
, interleukin (IL)-6, and IL-12 in lung homogenates of
reporter mice after exposures to silica or LPS. Immunoreactivity of
MCP-1 in these animals was localized to AMs and epithelial cells. These
data are the first to show activation of NF-
B in situ by fibrogenic
particles in pulmonary epithelial cells and AMs. Increased expression
of NF-
B-related inflammatory cytokines by these cell types, which
first encounter silica after inhalation, may be critical to the
initiation of silica-associated lung diseases, thus providing a
rationale for focusing on NF-
B in preventive and therapeutic strategies.
silica; transgenic mice; nuclear factor-B; silicosis; inflammation
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INTRODUCTION |
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CRYSTALLINE SILICA IS
ASSOCIATED in the workplace with the development of silicosis
(1) and recently has been categorized as a human
carcinogen in lung (21). Although the mechanisms of
pulmonary fibrosis by silica are under investigation in a number of
laboratories (reviewed in Ref. 28), the critical events
involved in initiation of pulmonary inflammation and lung disease are
obscure. In human and rodent models, exposure to silica particles
elicits a significant and sustained inflammatory response characterized by the influx of inflammatory cells (8, 16) and increased expression of inflammatory cytokines such as tumor necrosis factor (TNF)- (19, 29), interleukin (IL)-1 (35),
inducible nitric oxide synthase (iNOS) (4, 33), and
interferon (IFN)-
(12, 13). Regulation of these
cytokines may be dependent upon the activation of the transcription
factor, nuclear factor (NF)-
B, because the promoter regions of many
of these genes are known to contain binding sites for this
transcription factor (5).
NF-B is a ubiquitous transcription factor that can be activated by
cytokines, reactive oxygen species (ROS), growth factors, bacteria and
viruses, ultraviolet irradiation, and inorganic particles (reviewed in
Ref. 25). The regulation of NF-
B and its degradation are topics of contemporary interest, as many inducible genes that encode cytokines, chemokines, adhesion molecules, growth factors, enzymes, and transcription factors contain binding sites for NF-
B within their promoter or enhancer regions (34). Moreover,
modulation of the NF-
B pathway may be critical to treatment of a
number of respiratory and other inflammatory diseases in which
NF-
B-related genes are upregulated (2).
We and others have shown in a number of in vitro models that the
fibrogenic minerals silica and asbestos cause NF-B activation (9, 10, 22). Moreover, increases in immunoreactivity of p65, a transcriptionally active subunit of NF-
B, have been
demonstrated in bronchiolar epithelium and fibrotic lesions of rats
after inhalation of asbestos (23). Bronchoalveolar lavage
(BAL) cells from rats exposed to silica by intratracheal instillation
also showed NF-
B activation as determined by electromobility shift
assay (32). However, whether silica causes
transcriptional activation of NF-
B-dependent gene expression in the
lung after exposures in vivo is unclear. We addressed this question
using intratracheal instillation of
-quartz silica into NF-
B
luciferase reporter mice (27) backcrossed into C57BL/6
mice, a strain susceptible to the development of fibrotic lung disease
by asbestos or silica. This protocol gives rise to pulmonary
inflammation and silicosis (8). In addition, we used
ribonuclease protection assays (RPA) to determine whether mRNA levels
of genes encoding specific inflammatory cytokines were increased in the
lung after silica exposures and immunocytochemistry to determine the
cell types involved in NF-
B transactivation and cytokine expression.
The specificity of these responses was determined using a known
inflammatory agent, lipopolysaccharide (LPS), and a nonpathogenic
control particle, TiO2 (15, 24).
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METHODS |
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NF-B luciferase reporter mice.
The NF-
B luciferase reporter mice were made using the
pBIIX-luciferase construct with two copies of the
B sequence from the Ig
intronic enhancer and were characterized as described previously (27). Thus increases in luciferase
protein expression demonstrate functional NF-
B activation of the
luciferase reporter construct, a tandem repeat of the canonical NF-
B
response element found in the Ig
enhancer. The transcript was
expressed in all cell types (27). Mice were backcrossed
4× onto a C57BL/6 background, a strain exhibiting inflammation and
fibrosis in response to silica or asbestos (28). Animals
were housed at the University of Vermont under controlled conditions of
temperature, humidity, and light and provided food and water ad
libitum. Animal facilities are American Association for Accreditation
of Laboratory Animal Care approved and operated under the supervision
of the Institutional Animal Care and Use Committee of the University of Vermont.
Exposures to particles.
Silica particles (-quartz; Min-U-Sil 5; 0.6 µm, mean equivalent
spherical diameter; Pennsylvania Glass and Sand, Pittsburgh, PA) were
administered to mice as previously described (8). Briefly,
mice were anesthetized with pentobarbital sodium (40 mg/kg ip), and
their tracheas were exposed by dissection. Silica particles were
instilled intratracheally at 1 mg/0.05 ml sterile Ca2+-Mg2+-free phosphate-buffered saline
(CMF-PBS)/mouse. LPS (026:86; Sigma, St. Louis, MO) was instilled at 2 µg/mouse (100 µg/kg) in 0.05 ml sterile CMF-PBS. TiO2
(fine, 0.25-µm mean equivalent spherical diameter; obtained from Dr.
Gunter Oberdörster, University of Rochester, Rochester, NY), a
nonpathogenic control particle, was instilled at 1 mg/mouse in 0.05 ml
of sterile CMF-PBS. Vehicle control animals received a single
intratracheal injection of 0.05 ml sterile CMF-PBS. Mortality in all
groups was <5%.
BAL.
Animals were killed by an overdose of pentobarbital sodium at 4, 24, or
72 h after exposures to particles, LPS, or PBS (sham vehicle
control). Chest cavities were opened, and lungs were cannulated via the
trachea with polyethylene tubing. Lungs were then lavaged in situ six
times with CMF-PBS at a volume of 1 ml for each lavage. All lavage
(BAL) fluid was centrifuged at 250 g for 10 min at 4°C,
and the cell pellets were resuspended in 1% bovine serum albumin (BSA)
in CMF-PBS. A portion of these cells was then stained with 1% crystal
violet and counted under light microscopy for total cell number.
Another portion of the cells was diluted in 1% BSA/CMF-PBS and
centrifuged onto slides in a Shandon cytocentrifuge (Shandon Southern
Products, Cheshire, England). The cells were either stained with
LeukoStat Stain Kit (Fisher Scientific, Pittsburgh, PA) and identified
by nuclear morphology or fixed in 2% paraformaldehyde (PFA)/CMF-PBS
and stored at 20°C. The remaining cells were evaluated for
luciferase enzymatic activity.
Detection of NF-B activation by immunostaining for luciferase
protein.
After lung lavage, two left lobes of the lung were inflated at a
constant pressure of 25 cmH2O and preserved in 4%
PFA/CMF-PBS. All lungs were embedded in paraffin and sectioned. Lung
sections (3 µm thickness) and BAL cells were evaluated for the
presence of luciferase protein by immunostaining. Lung sections were
deparaffinized in xylene for 5 min 3× and rehydrated through graded
ethanols. Both lung sections and fixed BAL cells were equilibrated in
CMF-PBS. The sections and cells were boiled in 0.1 M citrate buffer (pH 6.0) for antigen retrieval and then incubated in 3%
H2O2 to dampen endogenous peroxidase activity.
BAL cells were then permeabilized with 1% SDS. Both lung sections and
BAL cells were incubated overnight at 4°C in 2% normal goat serum
(Jackson ImmunoResearch Labs, Westgrove, PA) to reduce nonspecific
protein binding. The following day, the sections and cells were
incubated with rabbit anti-luciferase antibody (0.5 µg/ml) (Cortex
Biochem, San Leandro, CA) for 1 h at room temperature (RT). After
three washes in CMF-PBS, immunoreactivity was detected using the
anti-rabbit IgG Vectastain ABC Elite kit (Vector Laboratories,
Burlingame, CA) and diaminobenzidine (DAB) as a chromogen according to
the manufacturer's protocols. After color development, lung
sections and BAL cells were counterstained with hematoxylin. Lung
sections were then dehydrated, cleared, and mounted on slides in
VectaMount (Vector Laboratories), and BAL cells were mounted on slides
in glycerol gelatin (Sigma) before examination by light microscopy.
Negative controls consisted of lung sections incubated with secondary
antibody alone or rabbit IgG type-matched monoclonal antibody (Zymed
Laboratories, San Francisco, CA) in place of primary antibody.
RPA for inflammatory chemokines and cytokines.
Total RNA was isolated from frozen lavaged lung tissues (right lobe) as
described previously (31), quantitated by absorbance at
260 nm, and analyzed using an RPA system with a multiprobe template set
for iNOS, IL-12, TNF-, IL-6, macrophage inflammatory protein
(MIP)-2, TGF-
, macrophage chemotactic protein (MCP)-1, and IFN-
according to manufacturer's instructions (Riboquant; PharMingen, San
Diego, CA). RNA duplexes were isolated by extraction/precipitation, dissolved in 5 µl of gel loading buffer, and electrophoresed in standard 5% acrylamide/urea sequencing gels. After gels were dried, autoradiograms were developed and quantitated using a Bio-Rad phosphorimager (Bio-Rad, Hercules, CA). Results were normalized to
expression of the housekeeping gene L32.
Immunohistochemistry for MCP-1 protein.
To determine whether elevated mRNA levels of genes encoding
inflammatory chemokines and cytokines in lung homogenates reflected increases in protein and the cell types expressing these proteins, immunocytochemistry using an antibody to an NF-B-regulated
inflammatory chemokine, MCP-1, was performed on lung sections. Lung
sections were deparaffinized in xylene for 5 min 3×, rehydrated
through graded ethanols, and equilibrated in CMF-PBS. The sections were boiled in 0.1 M citrate buffer, pH 6.0, for antigen retrieval and then
incubated in 3% H2O2 to dampen endogenous
peroxidase activity. Lung sections were incubated overnight at 4°C in
2% normal rabbit serum (Jackson ImmunoResearch Labs) to reduce
nonspecific protein binding. The following day, the sections and cells
were incubated with goat anti-MCP-1 antibody (2 µg/ml) (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at RT. After three washes in CMF-PBS, immunoreactivity was detected using the anti-goat IgG
Vectastain ABC Elite kit (Vector Laboratories) and DAB as a chromogen
according to the manufacturer's protocols. After color development,
lung sections and BAL cells were counterstained with hematoxylin. Lung
sections were then dehydrated, cleared, and mounted in VectaMount
(Vector Laboratories) before examination by light microscopy. Negative
controls consisted of lung sections incubated with secondary antibody
alone or rabbit IgG type-matched monoclonal antibody (Zymed
Laboratories) in place of primary antibody.
Statistical analysis. Data are expressed as means ± SE. Differences between groups were evaluated by analysis of variance using the Student-Newman-Keuls procedure to correct for multiple comparisons. A P value of less than or equal to 0.05 was considered significant.
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RESULTS |
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Instillation of silica or LPS causes protracted inflammation in
mouse lungs.
Instillation of silica particles (1 mg/C57BL/6 luciferase reporter
mice) initiated a prominent inflammatory response as evidenced by
increased numbers of polymorphonuclear leukocytes (PMNs)
(P 0.05) as early as 4 h postinstillation (Fig.
1, A and B). This cellular inflammatory response was still apparent at 72 h when increases (P
0.05) in total cell numbers also were
observed. Exposure of mice to LPS (2 µg) also elicited a marked
inflammatory response comprising increased total cell numbers and
elevations in the proportions of PMNs and alveolar macrophages (AMs) at
all time points. Instillation of the nonfibrogenic particle
TiO2 did not elicit significant increases in total cell
numbers or percentages of PMNs.
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Luciferase protein is increased in AMs and bronchiolar epithelial
cells in luciferase reporter mice exposed to silica or LPS.
NF-B reporter mice were also assessed for luciferase activity as an
indication of transcriptional activation of NF-
B gene expression
(27). Homogenized whole lung tissue after exposures to
agents did not demonstrate significantly increased enzymatic activity
above that seen in PBS-instilled mice (data not shown), presumably
because epithelial cells and AMs are only a small fraction of lung
tissue. However, examination of lung tissue and BAL cells by
immunohistochemistry revealed focally increased luciferase immunoreactivity in both AMs and epithelial cells of silica- and LPS-instilled mice (Fig. 2). Compared
with PBS controls (Fig. 2A), exposure of mice to LPS (Fig.
2B), an agent activating NF-
B in lung (6),
or to silica for 24 or 72 h (Fig. 2, C and
D) elicited increases in luciferase protein in both AMs
(arrow) and in bronchiolar epithelial cells. At 72 h
postinstillation, less intense staining was detected in lung sections
from mice exposed to LPS (data not shown). Increases in staining were
not observed in alveolar epithelial cells from asbestos or LPS-exposed
mice. No staining was seen in lung tissue from mice exposed to
TiO2 despite its accumulation in lung (Fig. 2E,
arrow) or in silica-exposed lungs stained with secondary antibody alone
(Fig. 2F). Moreover, no staining was observed in lung
tissues from wild-type C57BL/6 mice.
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Instillation of silica or LPS causes an increase in mRNA levels of
NF-B-related inflammatory cytokines.
Possible consequences of NF-
B activation after exposure to silica or
LPS were investigated by examining lung homogenates for increased mRNA
levels of a number of NF-
B-associated or -regulated inflammatory
cytokines in C57BL/6 wild-type mice. Figure
3 is a representative autoradiograph from
an RPA on lung homogenates of mice injected with PBS, LPS, or silica.
Figure 4 shows quantitation by
phosphorimaging of steady-state mRNA levels of these cytokines in lung
homogenates of mice at 4 and 24 h after instillation of agents.
Compared with sham (PBS) mice, instillation of silica elicited a
significant (P
0.05) increase in mRNA of several inflammatory cytokines (iNOS, MIP-2, MCP-1, IFN-
, IL-12, and IL-6)
as early as 4 h postinstillation (Fig. 4A). Significant increases in mRNA levels of TNF-
, iNOS, MIP-2, MCP-1, and IFN-
also occurred at 24 h (Fig. 4B), whereas only mRNA
levels of IFN-
were increased at 72 h postinstillation of
silica (data not shown). LPS caused elevations in mRNA levels of all
cytokines at 4 h, which persisted, with the exception of TGF-
1,
for 24 h. Increases in mRNA levels of cytokines were not observed
with instillation of TiO2 (data not shown) and were
identical to saline-instilled controls.
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MCP-1 protein is increased in AMs and epithelial cells in mice
exposed to silica.
To document whether increased mRNA levels of an NF-B-regulated gene,
MCP-1 (38), reflected increased protein and the cell types
involved, lung sections from all groups were evaluated for MCP-1
protein using immunohistochemistry. As shown in Fig.
5, sham mice instilled with PBS
demonstrated some constitutive expression of MCP-1 in bronchiolar
epithelial cells (Fig. 5A). Whereas LPS increased MCP-1
immunoreactivity primarily in AMs (Fig. 5D), silica elicited
marked increases in immunoreactivity of MCP-1 in AMs and bronchiolar
epithelium (Fig. 5, E and F). No staining was found in lung tissue from mice exposed to secondary antibody alone (Fig. 5B).
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DISCUSSION |
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Silicosis is associated with occupational exposures to crystalline
silica in stone cutting, quarrying, and mining (1, 21). Increases in pulmonary inflammation may be related to the development of fibrotic lung disease, although the critical molecular events involved in initiation of inflammation are unclear. Work here demonstrates for the first time that fibrogenic silica particles in
vivo activate the transcription factor NF-B in AMs and bronchiolar epithelial cells of the lung. The epithelial cell specificity of
transactivation is supported by our previous work showing increased localization of p65 protein in bronchiolar epithelium of mice exposed
to asbestos by inhalation (23). NF-
B transactivation may participate in the regulation of key inflammatory mediators and the
development of inflammation and fibrosis, features of these model
systems. Although trends were observed, significant increases in
luciferase enzymatic activity were seen in neither whole lung
homogenates nor BAL cell pellets of silica-exposed mice. However,
because increased immunoreactivity of luciferase protein occurred only
in a small population of cells in the lung, activity may have been
diluted beyond the limits of detection in homogenates from an entire
lung lobe or in BAL pellets containing many neutrophils.
Activation of NF-B in epithelial cells or AMs may result in the
transcriptional initiation of a diverse set of genes important in
perpetuating or attenuating immune and inflammatory responses (reviewed
in Ref. 25). Thus steady-state mRNA levels of several inflammatory cytokines and chemokines with NF-
B sites in their promoter regions were measured in lung homogenates of silica-, LPS-, or
TiO2-exposed mice. Significant increases in mRNA levels of
TNF-
, iNOS, MIP-2, MCP-1, IFN-
, IL-12, and IL-6 were detected after instillation of silica or LPS, a known inflammatory agent and
positive control in our studies.
The elaboration of these cytokines by minerals inducing inflammation
and fibrosis may be complex and interrelated (reviewed in Ref.
28). For example, intratracheal administration of silica primes AM to release TNF- after in vitro exposures to LPS
(15). Intratracheal instillation of silica also increases
TNF-
mRNA levels in lung tissue (30) and release of
TNF-
protein from BAL cells (19, 29). After inhalation
of silica, persistent overexpression of TNF-
mRNA (measured by in
situ hybridization) occurs in alveolar epithelial cells and aggregate
lesions in the lungs as well as in mononuclear cells in BAL
(12). The critical relevance of TNF-
to fibrosis is
demonstrated by experiments in which administration of human
recombinant soluble TNF receptor prevents silicosis and
bleomycin-induced fibrosis in mice (30).
IFN- synergistically enhances TNF-
-induced NF-
B
transactivation by a mechanism involving I
B degradation
(11). In studies here, elevated and protracted mRNA levels
of IFN-
were observed in lung for as long as 72 h after
exposure to silica. Inhalation of silica also elicits overproduction of
IFN-
by lymphocytes in mice developing silicosis (12,
13). Moreover, in rats inhaling silica, increased IFN-
and
IL-12 mRNA levels occur primarily in thoracic lymph nodes
(18).
Silica also increased mRNA levels of the potent chemokine MIP-2, a
factor upregulated by TNF- and synthesized by bronchiolar and
alveolar type II epithelial cells in vitro (14). Increased levels of MIP-2 mRNA are also detected in the lungs and BAL cells of
rats after intratracheal instillation of silica (14, 39).
The fact that the epithelial cell may be a major source of cytokines and chemokines after exposures to silica in vivo is supported by our data and several recent reports. In human bronchial epithelial cells (BEAS-2B) cocultured with inorganic particles, increased IL-6 mRNA, a cytokine elevated in BAL fluids from pneumoconiosis patients (37), occurs (17). In addition, increased MCP-1 mRNA levels are seen in a mouse type II epithelial cell line (MLE) after exposure in vitro to silica (3).
We document here increased immunoreactivity of MCP-1 in both AMs and
epithelial cells of silica-exposed mice at sites corresponding to
transactivation of NF-B-dependent gene expression (Figs. 3, 6).
MCP-1, an NF-
B-regulated gene, is produced by fibroblasts, macrophages, and epithelial cells in vitro and is a potent chemotactic factor for circulating monocytes (26). The possibility
that this chemokine participates in the pathogenesis of
particle-induced lung injury is supported by studies showing increased
levels of soluble MCP-1 in BAL cells and its increased immunoreactivity in alveolar type II epithelial cells in lung sections from patients with coal worker's pneumoconiosis (7). MCP-1 has also
been implicated in fibrosis via its regulation of profibrotic cytokine generation and matrix deposition. For example, T helper (Th) 2 type
fibroblasts from murine fibrotic pulmonary granulomas generate twofold
more MCP-1 than Th1 type fibroblasts from nonfibrotic lungs
(20).
In summary, our data are the first to show transactivation of
NF-B-dependent gene expression in vivo by fibrogenic particles in
bronchiolar epithelial cells and AMs. These changes were observed after
instillation of LPS, but not in response to TiO2, a
nonfibrogenic particle (24), and were accompanied by
increased mRNA levels of NF-
B-related cytokines and chemokines in
lung tissue. A key NF-
B-regulated cytokine, MCP-1, was localized in
epithelial cells and AMs corresponding to sites of NF-
B
transactivation. Further studies using transgenic mice in which NF-
B
is inhibited in bronchiolar epithelial cells (30a) will be
necessary to determine a cause-effect relationship. Increased
expression of NF-
B-related inflammatory cytokines by epithelial
cells and macrophages may initiate critical molecular events leading to
the development of fibrotic lung disease, suggesting modulation of this
transcription factor in approaches to prevent and treat pulmonary fibrosis.
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ACKNOWLEDGEMENTS |
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We acknowledge the valuable technical assistance of Ingrid Berlanger and Andrew Cummins and the secretarial assistance of Laurie Sabens. Dr. Douglas Taatjes provided expertise on cell imaging approaches and photomicroscopy.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants RO1 HL-39469 and RO1 ES/HL-09213.
Address for reprint requests and other correspondence: B. T. Mossman, Dept. of Pathology, Univ. of Vermont College of Medicine, Burlington, VT 05405 (E-mail: bmossman{at}zoo.uvm.edu).
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.
10.1152/ajplung.00327.2001
Received 14 August 2001; accepted in final form 9 November 2001.
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