Department of Pathology, University of British Columbia, Vancouver, British Columbia, Canada V6T 2B5
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ABSTRACT |
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Tumor necrosis
factor (TNF)- is released from alveolar macrophages after
phagocytosis of mineral fibers. To determine whether TNF-
affects
the binding of fibers to epithelial cells, we exposed rat tracheal
explants to TNF-
or to culture medium alone, followed by a
suspension of amosite asbestos or fiberglass (MMVF10). Loosely adherent
fibers were removed from the surface with a standardized washing
technique, and the number of bound fibers was determined by scanning
electron microscopy. Increasing doses of TNF-
produced increases in
fiber binding. This effect was abolished by an anti-TNF-
antibody,
the proteasome inhibitor MG-132, and the nuclear factor (NF)-
B
inhibitor pyrrolidine dithiocarbamate. Gel shift and Western blot
analyses confirmed that TNF-
activated NF-
B and depleted I
B in
this system and that these effects were prevented by MG-132 and
pyrrolidine dithiocarbamate. These observations indicate that TNF-
increases epithelial fiber binding by a NF-
B-dependent mechanism.
They also suggest that mineral particles may cause pathological lesions
via an autocrine-like process in which the response evoked by
particles, for example, macrophage TNF-
production, acts to enhance
subsequent interactions of particles with tissue.
tumor necrosis factor-; particle adhesion; nuclear factor-
B; MG-132; pyrrolidine dithiocarbamate
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INTRODUCTION |
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ADHESION (binding) of mineral particles to the epithelial cell surface is the first step in a variety of mineral dust-induced pathological reactions. Some of these reactions are probably mediated directly on the cell surface. For example, Zanella et al. (27) reported that surface interactions of crocidolite asbestos with the epidermal growth factor receptor on rat pleural mesothelial cells induced autophosphorylation of the receptor, with subsequent upregulation of c-fos expression and apoptosis. Tsuda et al. (25) found that the adhesion of glass or crocidolite fibers to the surface of A549 cells (a model of type II cells) and the subsequent stretching of the cells caused them to elaborate enhanced amounts of the neutrophil chemoattractant interleukin (IL)-8.
Binding of fibers to the cell surface is also believed to be a
prerequisite for fiber uptake, and fiber uptake leads to a wide variety
of pathological processes including intracellular oxidative damage to
lipids, proteins, and DNA; nuclear factor (NF)-B activation, with
subsequent induction of proinflammatory cytokines; and, depending on
the nature of the particle, induction of fibrogenic mediators either
within the epithelial cell or after passage of the particles through
the cells to the underlying interstitial tissues (4, 7, 9, 10,
15).
Although the adhesion and uptake of mineral particles by pulmonary epithelial cells are processes seen with any particle that contacts the cell surface (7), the factors that govern adhesion are poorly defined. For positively charged particles such as chrysotile asbestos, carbonyl iron spheres, or aluminum spheres, binding to negatively charged sialic acid residues plays a role because binding can be abolished by pretreatment with neuraminidase (3, 13). There is some evidence to suggest that coating of the negatively charged asbestos fibers amosite and crocidolite with fibronectin or vitronectin and subsequent adhesion to cell surface integrins is important in increasing binding, although blocking the integrins does not completely abolish binding (2, 5, 6, 25). Heparin and polyinosinic acid but not polyanionic chondroitin sulfate have been found to decrease the binding of a variety of compact particles to A549 cells (23), suggesting that a macrophage scavenger-type receptor might be involved, and Palecanda et al. (21) have recently reported that TiO2, Fe2O3, and latex beads bind to hamster alveolar macrophages via a receptor that is analogous to the human macrophage scavenger receptor MARCO. Whether this receptor system functions on lung epithelial cells has not yet been determined. Churg et al. (8) observed that cigarette smoke, a source of reactive oxygen species (ROS), increased amosite asbestos binding to tracheal epithelial cells; this effect could be completely abolished by treatment of the fibers with deferoxamine and decreased with mannitol, suggesting that surface iron on the fibers played an important role, probably by generating the hydroxyl radical from ROS in the smoke.
Inhaled mineral particles also evoke a complex set of reactions in the
alveolar spaces and airway lumens. All particles induce an alveolar
macrophage influx, and phagocytosis of inhaled particles by macrophages
is often postulated as an initial and central event in
particle-associated pathology (19). One of the results of phagocytosis of most types of mineral particles by macrophages is the
generation of tumor necrosis factor (TNF)-, a proinflammatory cytokine that turns on cell signaling pathways and induces synthesis of
other cytokines as well as of surface adhesion molecules (11, 19,
26). Production of TNF-
is also seen as a later event in
epithelial cells in whole animal models of asbestos inhalation (18). In this study, we used amosite asbestos and a
fibrous glass, MMVF10, as model particles to ask whether the TNF-
response can potentiate the reactions of fibers with epithelial cells
by increasing surface binding.
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MATERIALS AND METHODS |
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Explant Preparation
Tracheal explants were prepared from 250-g Sprague-Dawley rats as previously described (8) and divided into treatment groups of 4 explants each. The following treatment groups were created.Dust only. The explants were submerged in Dulbecco's minimal Eagle's medium (DMEM) without serum for 3 h (to keep total submersion time for all groups constant) at 37°C, placed on agarose-DMEM plates in an air-CO2 incubator at 37°C for 2 h (8), and then placed in a suspension of 500 µg/cm2 of International Union Against Cancer (UICC) amosite asbestos (kindly supplied by Dr. J. C. Wagner, Medical Research Council Pneumoconiosis Unit, Cardiff, UK) or a glass fiber, MMVF10 (Thermal Insulation Manufacturer's Association, Stanford, CT), for 1 h. The explants were subsequently washed to remove loosely adherent fibers and prepared for scanning electron microscopy (SEM) as described in Determination of Fiber Binding.
TNF- plus dust and antibodies to TNF-
plus dust.
The explants were submerged in DMEM alone for 1 h and then in DMEM
containing variable amounts of recombinant human TNF-
(specific
activity >2 × 107 U/mg; Life Technologies,
Rockville, MD) for 2 h, followed by air-CO2 culture
for 2 h, dust exposure as in Dust only for 1 h, and washing and preparation for SEM. To show specificity, additional experiments were performed in which TNF-
at 20 ng/ml was first mixed
with excess goat polyclonal antibody to human TNF-
(Santa Cruz
Biotechnology) for 1 h, and the explants were then carried forward
as above. As a control, nonimmune serum was used instead of
anti-TNF-
.
Proteasome inhibitor MG-132 plus TNF- plus dust.
The explants were submerged in 0.5 µM MG-132 (Peptide Institute,
Osaka, Japan) in 0.1% DMSO-DMEM for 1 h, followed by TNF-
(20 ng/ml) for 2 h, air-CO2 culture for 2 h, and
finally dust exposure for 1 h. To test the effects of MG-132 on
baseline fiber adhesion, additional explants were exposed to MG-132 and
dust but not to TNF-
.
NF-B inhibitor pyrrolidine dithiocarbamate.
The explants were submerged in 200 µM pyrrolidine dithiocarbamate
(PDTC; Sigma) in culture medium for 2 h, and then the same protocol as in Proteasome inhibitor MG-132 plus TNF-
plus
dust was followed but with the addition of PDTC to the medium.
Non-dust-exposed explants for IB Western blot and NF-
B gel
shift assay.
Three additional treatment groups of explants were created; initial
studies showed that six explants per group were required to obtain
reliable signals. One group was exposed to DMEM alone, another to DMEM
followed by 20 ng/ml of TNF-
, and a third to 0.5 µM MG-132 for
1 h and then to 20 ng/ml of TNF-
. The explants were snap-frozen
and assayed as described in Western Blots for I
B. The
same protocol was followed, with additional explants exposed to 200 µM PDTC.
Determination of Fiber Binding
Fiber binding was determined as previously described (8). Exposure of the explants to the dusts resulted in initial coating of the epithelial surface with a layer of fibers, most of which were very loosely adherent. To remove fibers not bound to the surface, each explant was very slowly dipped once in four different containers of fresh culture medium. Churg et al. (8) have previously shown that this approach removes the vast majority of fibers and leads to quite reproducible levels of residual fibers bound to the epithelial surface; we also found that four washes were adequate, with little change in the number of adherent fibers if more than four washes were used. The explants were then dried under vacuum and examined by SEM. SEM photographs of randomly selected fields were taken at ×1,000 and printed. The proportion of the surface occupied by fibers (areal fraction of fibers) was determined with a 42-point transparent overlay by counting the points that fell on fibers versus the points that fell on tissue (8). Differences in the number of adherent fibers among treatment groups were determined by analysis of variance.Western Blots for IB
NF-B Gel Shift Assay
Cytotoxicity Assay
Groups of five explants were treated with culture medium only (control), amosite asbestos, TNF- ![]() |
RESULTS |
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Figure 1 shows the effects of
increasing concentrations of TNF- on amosite asbestos binding. Fiber
adhesion significantly and progressively increased above the control
levels with doses of 5 and 10 ng/ml of TNF-
; at doses of 20 ng/ml,
no further increase in binding was seen, perhaps suggesting that the
possible expression or exposure of the actual binding agent (see
DISCUSSION) can only be increased to a limited amount.
Figure 2 shows the effects of pretreating
TNF-
with polyclonal anti-TNF-
antibodies. Antibodies to TNF-
abolished the increase in fiber concentration. Nonimmune serum did not
prevent the TNF-
effect (data not shown). These observations support
the idea that increased adhesion is specifically driven by TNF-
.
Figure 3 shows that MG-132 abolishes the
TNF-
-mediated increases in amosite binding, implying that NF-
B
activation is playing a role. Figure 4
shows the dose data and similar effects of MG-132 on MMVF10 binding.
TNF-
increased the binding of this synthetic mineral fiber to the
explants, although the dose response was somewhat different from that
for amosite because greater TNF-
doses were needed to show an
effect. MG-132 again prevented the TNF-
effect. Figure
5 shows that the NF-
B inhibitor PDTC
also prevented increases in fiber binding after TNF-
exposure, again suggesting that this process is driven through NF-
B activation. To
prove that TNF-
does activate NF-
B in this explant system and
that the inhibitors prevented activation, gel shift assays for NF-
B
and Western blots for I
B levels were run after exposure of the
explants to TNF-
or TNF-
plus MG-132 (Figs.
6 and
7) or TNF-
or TNF-
plus
PDTC (Figs. 8 and
9). In both instances, the inhibitors
prevented the increase in TNF-
-induced NF-
B nuclear translocation, as shown in the gel shifts, and ameliorated I
B degradation, confirming that inhibition of fiber binding correlated with inhibition of NF-
B activation.
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DISCUSSION |
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In this study, we looked at the effects of exogenous TNF- on
fiber binding to tracheal epithelial cells. A word about our technical
approach is in order. We are defining a "bound" particle as one
that is resistant to removal by a simple washing technique. Review of
the literature shows that there is no generally utilized method for
measuring fiber binding. A wide variety of approaches including
examination of cultured cells by confocal scanning microscopy to
attempt to separate surface from internalized particles
(2); a fairly simple SEM technique similar to ours
(3, 13); fractionation of cells with radiolabeled
membranes and subsequent measurement of radioactivity adherent to
centrifuged fibers (5, 6); and measurement of changes in
light scattering by flow cytometry (23) have all been
reported. Not only are these methods quite different and the results
difficult to compare, but with any of these methods, harsh enough
treatment will remove all fibers from the cells. Thus the definition of
a bound fiber is in many respects arbitrary. Nonetheless, the method of
Churg et al. (8) is simple and reproducible and shows
consistent effects with various treatments, suggesting that it provides
useful data on binding. An additional advantage of using tracheal
explants for evaluating binding is that fiber uptake is very slow, with
very few fibers seen in the epithelial cells even at 24 h
(9); thus we do not need to be concerned that the surface
assay has missed bound fibers that have already entered the cells.
Our present data indicate that TNF- increases fiber binding to the
surface of tracheal epithelial cells and that this process appears to
be mediated by TNF-
-induced activation of NF-
B. NF-
B consists
of a cytoplasmic complex of two proteins (typically p50 and p65) and an
inhibitory protein, I
B-
or I
B-
. TNF-
and other
inflammatory or oxidant mediators can activate NF-
B by causing
ubiquitination and subsequent degradation of I
B, allowing the
p65/p50 complex to translocate into the nucleus and bind to recognition
sequences in the promoters of a wide variety of genes important in
acute inflammatory responses (1, 16). MG-132 is a
proteasome inhibitor that prevents degradation of I
B and thus
inhibits the TNF-
-driven pathway of NF-
B activation
(16). Our gel shift and Western blot data confirm that
TNF-
does cause I
B degradation and NF-
B activation and that
MG-132 prevents NF-
B activation and I
B degradation in our explant
system. The fact that a second NF-
B inhibitor, PDTC, also prevents
the effects of TNF-
further adds to the idea that increased adhesion
is driven through a NF-
B-dependent mechanism. It is, of course,
possible that increases in adhesion and NF-
B activation are two
TNF-
-driven but unrelated effects, but this seems to be a much less
likely explanation for our observations.
What is not as yet clear is the mechanism by which TNF- or NF-
B
increases surface binding. TNF-
increases the production of a
variety of substances that are expressed on the epithelial cell surface
or exported into the lumen. In bronchial epithelial cells, TNF-
, via
NF-
B, is known to induce the adhesion molecule intercellular
adhesion molecule-1 in bronchial epithelial cells (22),
and TNF-
also induces mucus secretion (12) in these cells. Other candidate binding substances include fibronectin, which is
produced in increased amounts in A549 cells after coal dust plus
TNF-
exposure (17), and MARCO-type scavenger receptors, which probably exist on pulmonary epithelial cells (21,
23), although this has yet to be confirmed. This list is not
comprehensive and other possible candidate molecules, particularly cell
surface integrins such as
v
5
(2) or even the TNF-
-responsive
1- and
2-integrins that bind neutrophils and eosinophils to
bronchial epithelial cells (14), certainly exist. This
list is, of course, speculative, but it is of interest that amosite
asbestos and MMVF10 show a different dose response to TNF-
, and it
is possible that the binding agents differ for these two fibers.
Our observations also suggest the novel possibility that the interaction of dusts and macrophages or epithelial cells is not the one-way street that is typically assumed in most hypothetical schemes of events after dust inhalation; i.e., what is usually proposed is that dusts interact with both macrophages and tissues to elicit a variety of cytokine and oxidant mediators that are then assumed to be sufficient in and of themselves to establish subsequent biochemical and molecular responses and eventual pathological changes such as interstitial fibrosis, with the dust particle that evoked the response becoming (conceptually) almost irrelevant to the process. (19).
The notion that particle-evoked responses can influence subsequent
interactions of particles and pulmonary epithelial cells has been
little explored. What we observe here suggests that a dust-evoked
cytokine, TNF-, acts to increase dust binding to epithelial cells
and thus presumably to increase both surface interactions, as described
in the introduction, and particle uptake, with, eventually, increased
activation of cell signaling pathways and increased mediator
production. With asbestos and glass fibers, one of those increased
intracellular mediators is TNF-
(18, 20, 26); thus not
only does TNF-
affect downstream interactions of particles with the
epithelia, but by doing so, it potentially upregulates epithelial
TNF-
production in a type of autocrine-like feedback loop.
It is interesting in this context that Stringer and Kobzik
(24) showed that TNF- primes A549 cells for IL-8
release after subsequent particle contact. Stringer and Kobzik intended
their experiments to mimic the effects of air pollutant particles
in individuals with preexisting inflammatory disease. However, the Stringer and Kobzik data could be viewed in much the same way as ours,
in that TNF-
increases release of IL-8 after particle adhesion; IL-8
attracts neutrophils that release ROS, and, as Churg (7)
has discussed in detail elsewhere, ROS cause respiratory epithelia to
increase the uptake of most mineral particles and thus again to
increase mediator (including TNF-
) production.
This study has only looked at the effects of TNF- on fiber binding,
but the same phenomena might occur with compact particles including air
pollutant particles. We propose that mineral particles may cause
pathological lesions not only by an initial reaction with macrophages
and epithelial cells that results in mediator production but also via a
feedback loop in which the dust-evoked mediator increases subsequent
interactions of the dust with epithelial cells and thus magnifies the response.
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ACKNOWLEDGEMENTS |
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This study was supported by Grant MA8051 from the Medical Research Council of Canada.
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. Churg, Dept. of Pathology, Univ. of British Columbia, 2211 Wesbrook Mall, Vancouver, BC, Canada V6T 2B5 (E-mail: achurg{at}interchange.ubc.ca).
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.
Received 7 January 2000; accepted in final form 14 May 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Blackwell, TS,
and
Christman JW.
The role of nuclear factor-B in cytokine gene regulation.
Am J Respir Cell Mol Biol
17:
3-9,
1998
2.
Boylan, AM,
Sanan DA,
Sheppard D,
and
Broaddus VC.
Vitronectin enhances internalization of crocidolite asbestos by rabbit pleural mesothelial cells via the integrin v
5.
J Clin Invest
96:
1987-2001,
1995[ISI][Medline].
3.
Brody, AR,
George G,
and
Hill LH.
Interactions of chrysotile and crocidolite asbestos with red blood cell membranes.
Lab Invest
49:
468-475,
1983[ISI][Medline].
4.
Brody, AR,
Liu J,
Brass D,
and
Corti M.
Analyzing the genes and peptide growth factors expressed in lung cells in vivo consequent to asbestos exposure and in vitro.
Environ Health Perspect
105, Suppl 5:
1165-1171,
1997[ISI][Medline].
5.
Brown, RC,
Sara EA,
Hoskins JA,
and
Evans CE.
Factors affecting the interaction of asbestos fibres with mammalian cells: a study using cells in suspension.
Ann Occup Hyg
35:
25-34,
1991[ISI][Medline].
6.
Brown, RC,
Sara EA,
Hoskins JA,
and
Houghton CE.
Interaction between mineral fibres and cell surface receptors studied with amosite and surface derivatized amosite asbestos.
Ann Occup Hyg
38, Suppl 1:
587-593,
1994.
7.
Churg, A.
The uptake of mineral particles by pulmonary epithelial cells.
Am J Respir Crit Care Med
154:
1124-1140,
1996[ISI][Medline].
8.
Churg, A,
Sun J-P,
and
Zay K.
Cigarette smoke increases amosite asbestos fiber binding to the surface of tracheal epithelial cells via a surface iron-dependent mechanism.
Am J Physiol Lung Cell Mol Physiol
275:
L502-L508,
1998
9.
Dai, J,
Gilks B,
Price K,
and
Churg A.
Mineral dusts directly induce epithelial and interstitial fibrogenic mediators and matrix components in the airway wall.
Am J Respir Crit Care Med
158:
1907-1913,
1998
10.
Driscoll, KE,
Carter JM,
Howard BW,
Hassenbein D,
Janssen YMW,
and
Mossman BT.
Crocidolite activates NF-B and MIP-2 gene expression in rat alveolar epithelial cells. Role of mitochondrial-derived oxidants.
Environ Health Perspect
106, Suppl 5:
1171-1174,
1998[ISI][Medline].
11.
Driscoll, KE,
Hassenbein DG,
Carter JM,
Kunkel SL,
Quinlan TR,
and
Mossman BT.
TNF and increased chemokine expression in rat lung after particle exposure.
Toxicol Lett
82-83:
483-498,
1995.
12.
Fischer, BM,
Rochelle LG,
Voynow JA,
Akley NJ,
and
Adler KB.
Tumor necrosis factor stimulates mucin secretion and cyclic GMP production by guinea pig tracheal epithelial cells in vitro.
Am J Respir Cell Mol Biol
20:
413-422,
1999
13.
Gallagher, JE,
George G,
and
Brody AR.
Sialic acid mediates the initial binding of positively charged inorganic particles to alveolar macrophage membranes.
Am Rev Respir Dis
135:
1345-1352,
1987[ISI][Medline].
14.
Jagels, MA,
Daffern PJ,
Zuraw BL,
and
Hugli TE.
Mechanisms and regulation of polymorphonuclear leukocyte and eosinophil adherence to human airway epithelial cells.
Am J Respir Cell Mol Biol
21:
418-427,
1999
15.
Janssen-Heininger, YMW,
Macara I,
and
Mossman BT.
Cooperativity between oxidants and tumor necrosis factor in the activation of nuclear factor (NF-B).
Am J Respir Cell Mol Biol
20:
942-952,
1999
16.
Lee, DH,
and
Goldberg AL.
Proteasome inhibitors.
Trends Cell Biol
8:
397-403,
1999[ISI].
17.
Lee, YC,
and
Rannels DE.
Regulation of extracellular matrix synthesis by TNF- and TGF-
1 in type II cells exposed to coal dust.
Am J Physiol Lung Cell Mol Physiol
275:
L637-L644,
1998
18.
Liu, JY,
Brass DM,
Hoyle GW,
and
Brody AR.
TNF- receptor knockout mice are protected from the fibroproliferative effects of inhaled asbestos fibers.
Am J Pathol
153:
1839-1847,
1998
19.
Mossman, BT,
and
Churg A.
Mechanisms in the pathogenesis of asbestosis and silicosis.
Am J Respir Crit Care Med
157:
1666-1680,
1998
20.
Murata-Kamiya, N,
Tsutsui T,
Fujino A,
Kasa H,
and
Kaji H.
Determination of carcinogenic potential of mineral fibers by 8-hydroxydeoxyguanosine as a marker of oxidative DNA damage in mammalian cells.
Int Arch Occup Environ Health
70:
321-326,
1997[ISI][Medline].
21.
Palecanda, A,
Paulauskis J,
Al-Mutairi E,
Imrich A,
Qin G,
Suzuki H,
Kodama T,
Tryggvason K,
Koziel H,
and
Kobzik L.
Role of the scavenger receptor MARCO in alveolar macrophage binding of unopsonized environmental particles.
J Exp Med
189:
1497-1506,
1999
22.
Roebuck, KA.
Oxidant stress regulation of IL-8 and ICAM-1 gene expression: differential activation and binding of the transcription factors AP-1 and NF-B.
Int J Mol Med
4:
223-230,
1999[ISI][Medline].
23.
Stringer, B,
Imrich A,
and
Kobzik L.
Lung epithelial cell (A549) interaction with unopsonized environmental particulates.
Exp Lung Res
22:
495-508,
1996[ISI][Medline].
24.
Stringer, B,
and
Kobzik L.
Environmental particulate-mediated cytokine production in lung epithelial cells (A549): role of preexisting inflammation and oxidant stress.
J Toxicol Environ Health
55:
31-44,
1998[ISI].
25.
Tsuda, A,
Stringer BK,
Mijailovich SM,
Roger RA,
Hamada K,
and
Gray MI.
Alveolar cell stretching in the presence of fibrous particles induces interleukin-8 responses.
Am J Respir Cell Mol Biol
21:
455-462,
1999
26.
Ye, J,
Shi X,
Jones W,
Rojanasakul Y,
Cheng N,
Schwegler-Berry D,
Baron P,
Deye GJ,
Li C,
and
Castranova V.
Critical role of glass fiber length in TNF- production and transcription factor activation in macrophage.
Am J Physiol Lung Cell Mol Physiol
276:
L426-L434,
1999
27.
Zanella, CL,
Timblin CR,
Cummins A,
Jung M,
Goldberg J,
Raabe R,
Tritton TR,
and
Mossman BT.
Asbestos-induced phosphorylation of epidermal growth factor receptor is linked to c-fos and apoptosis.
Am J Physiol Lung Cell Mol Physiol
277:
L684-L693,
1999