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* From the Department of Pharmaceutical Sciences (Dr. Hubbard), University of Connecticut, Storrs, CT; and the Departments of Pathology (Drs. Timblin and Mossman) and Medicine (Dr. Rincon), University of Vermont, Burlington, VT.
Correspondence to: Brooke T. Mossman, PhD, Department of Pathology, A-145 Medical Alumni Building, University of Vermont College of Medicine, Burlington, VT 05405; e-mail: bmossman{at}zoo.uvm.edu
Exposure to
silica or asbestos elicits a striking
inflammatory response that is characterized by the influx of
inflammatory cells, increased expression of inflammatory cytokines,
cell injury, and compensatory cell proliferation.1
The
regulation of these events may be dependent on the activation of
transcription factors such as nuclear factor-
B (NF-B) and/or
activator protein-1 (AP-1) since the promoter regions of many genes
involved in inflammation, proliferation, and/or apoptosis are known to
contain binding sites for these transcription factors.2
3
Previous work from this laboratory has demonstrated increased AP-1
binding to DNA and AP-1-dependent gene transcription in mesothelial
cells and tracheal epithelial cells after exposure to asbestos in
vitro.4
5
We and others6
7
also have
shown that asbestos causes NF-B activation in these cell types
in vitro and in an inhalation model of asbestosis. However,
whether silica causes transcriptional activation of NF-B and
AP-1-dependent gene expression in the lung after exposures in
vivo is unclear.
Materials and Methods
We addressed this question using intratracheal instillation of
-quartz silica (Minusil at 1 or 2 mg in phosphate-buffered
saline solution) into NF-[]B)8
(1 mg per mouse) and
AP-19
(2 mg per mouse) luciferase reporter mice
back-crossed into the C57/BL6 strain. Control animals received
phosphate-buffered saline solution alone or fine TiO2
(obtained from Dr. Gunter Oberdorster, University of Rochester), as a
nonfibrogenic particle. At time points from 4 to 72 h, mice
underwent BAL for total and differential cell counts and luciferase
activity. In addition, whole-lung homogenates were evaluated for
luciferase activity and messenger RNA levels of fos/jun
family members (AP-1 mice) and cytokine genes (NF-B) using a
ribonuclease protection assay (RPA) according to the manufacturer’s
instructions (Pharmingen; San Diego, CA). Lung sections were also
evaluated for luciferase protein as detected by
immunocytochemistry.3
In additional experiments, AP-1
luciferase reporter mice were exposed by inhalation to silica (70
mg/m3) or crocidolite asbestos (National
Institute for Environmental Health Sciences reference sample; 8.5
mg/m3) for 72 h.
Results and Conclusions
The instillation of silica particles into either NF-[]B or
AP-1 luciferase reporter mice elicited significant increases in
inflammatory cells in BAL fluid and elevations in proportions of
neutrophils, occurring as early as 4 h after instillation and
remaining elevated for 72 h. Instillation of the positive
controls, lipopolysaccharide (2 µg per mouse) (NF-B mice), or
phorbol myristate acetate (PMA) (2 µg per mouse) (AP-1 mice) also
elicited significant cellular inflammation at these times.
The analysis of lung homogenates or BAL cells for luciferase activity
did not reveal significant increases by silica. However, when
luciferase expression was examined by immunohistochemistry, both
strains of mice demonstrated increased luciferase protein in both
bronchiolar epithelial cells and alveolar macrophages (AMs) in response
to silica. Peak luciferase immunoreactivity occurred at 24 h after
instillation. This response appeared to be specific for fibrogenic
particles, since exposure to TiO2 (1 mg/mL) did
not elicit increases in luciferase immunoreactivity in NF-B reporter
mice, and we have shown previously3
that luciferase
protein is increased in the alveolar epithelium of AP-1 mice inhaling
asbestos. In studies herein, the instillation of the positive controls,
lipopolysaccharide (NF-B mice), or PMA (AP-1 mice) also elicited
marked luciferase expression in AMs and epithelial cells.
To determine whether increased luciferase immunoreactivity preceded alterations in steady-state messenger RNA levels of fos/jun family members that comprise the AP-1 transcription factor, RPA was performed at 72 h on the lungs of AP-1 mice exposed to silica by intratracheal instillation (Fig 1 , top, A) or inhalation (Fig 1 , bottom, B). Interestingly, the instillation or inhalation of silica particles elicited a significant decrease (p < 0.05 by analysis of variance) in messenger RNA levels of c-jun, whereas levels of other family members (ie, junB, junD, c-fos, fra-1, fra-2, or fosB) were unchanged in comparison to sham control mice. Mice exposed to PMA (instillation) also showed a decrease in c-jun messenger RNA. Further studies are needed to determine whether decreases in c-jun messenger RNA reflect compensatory decreases in c-jun protein in epithelial cells.
|
]B activation,
messenger RNA levels of several cytokines the promoter regions of which
are known to contain binding sites for NF-B were measured by RPA at
4, 24, and 72 h. The instillation of silica particles elicited a
significant increase in steady-state messenger RNA levels of several
inflammatory cytokines (iNOS, MIP-2, MCP-1, interferon
[ IFN]-
, interleukin [ IL]-1, IL-2, and IL-6) as early as
4 h after instillation. Significant increases in specific
messenger RNA species also were seen at 24 h (TNF-, iNOS,
MIP-2, MCP-1, IFN-) and 72 h (IFN-) after instillation.
Increases in MCP-1 messenger RNA levels by silica were very
striking and were accompanied by increases in immunoreactivity of
MCP-1 protein in AMs and alveolar type II epithelial cells (manuscript
in preparation). Our results suggest that silica initiates inflammation and epithelial cell alterations through the activation of multiple transcription factors in vivo, which in turn may govern critical events (ie, cell injury, proliferation, and increased expression of inflammatory cytokines) in fibrogenesis.
Footnotes
Abbreviations:
AM = alveolar macrophage; AP-1 = activator protein-1; IFN = interferon;
IL = interleukin; iNOS = inducible nitric oxide synthase;
MCP = monocyte chemotactic protein; NF-B = nuclear factor-B;
PMA = phorbol myristate acetate; RPA = ribonuclease protection
assay;
Supported by National Institutes of Health grants HL39469 and ES/HL09213.
References
B in cytokine gene regulation. Am J Respir Cell Mol Biol 17,3-9B (NF-B) DNA binding activity and NF-B dependent gene expression in tracheal epithelial cells. Proc Natl Acad Sci U S A 92,8458-8462]B DNA binding activity in rat lung epithelial and pleural mesothelial cells. Am J Pathol 151,389-401[Abstract]
]B activity and enhancement of apoptosis by the neuropeptide calcitonin gene-related peptide. J Biol Chem 257,15114-15121[CrossRef]
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