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Actin-Containing Microfilaments of Pulmonary Epithelial Cells Provide a Mechanism for Translocating Asbestos to the Interstitium
Actin-Containing Mlcrofilaments of Pulmonary Epithelial Cells Provide a Mechanism for Translocatlng Asbestos to the Interstitium
la
AS
Arnold R. Brody, Ph.D.;* Lila H. Hill, B.S.;• WilliamS . Stirewalt, Ph.D.;t and Kenneth B. Adler, Ph.D. t
chrysotile asbestos fibers impact upon epithelial I nhaled cells which line the alveolar region of the lung.' Many of these fibers are taken up by alveolar epithelial cells.' During the 1-month period after a 1-hr exposure to chrysotile asbestos, numerous fibers are translocated to basement membranes as well as to interstitial cells and connective tissue.u The interstitial compartment at the alveolar level is the primary region affected during the pathogenesis of asbestosis. 3 We propose that actin-containing micro6laments of the alveolar epithelium provide a mechanism by which inhaled asbestos fibers are translocated to the pulmonary interstitium. The evidence to support this hypothesis has been achieved through ultrastructural studies of asbestosfilament associations in vivo and by quantitative biochemical studies of asbestos-epithelial interactions in vitro. MATERIALS AND METHODS
White rats were exposed to an aerosol of chrysotile asbestos (-15 mv/m3 , respirable mass} fur only 1 hour. Groups of animals were sacrificed immediately after exposure and at 24 and 48 hrs, and 1 month post-exposure. Animals were anesthet4ed with pentobarbital sodium (Nembutol) and the trachea was clamped. The lungs were expanded and fixed fur electron microscopy by perfusion of Karnovsky's fixative through the right ventricle and pulmonary artery fur 10 minutes. The lungs with the trachea still clamped then were removed in toto from the chest cavity and immersed in fresh fixative. DetaiJs of asbestos exposure, lung perfusion and dissection have been published. 1 To stabilize intracellular microfilaments, fresh lung tissue was dissected into 3 mm 3 blocks and immersed in 25% glycerol:75% KCI. The tissue then was placed in a 0.5% KCl solution containing dialyzed heavy-meromyosin fur 18 hrs.• Blocks were then fixed and embedded fur conventional electronic microscopy. For in vitro studies, asbestos fibers were added to organ cultures of tracheal explants and to cultures of cells from an established tracheal epithelial cell line. • Conventional electron microscopic studies were carried out on these cultures. In addition, biochemical quantitation of polymerized and non-polymerized actin proteins were carried out befOre apd after treating the cultured epithelial cells with asbestos. The amount of actin was determined by measurement of DNase I inhibition according to recently described methods. •
REsur.:rs
AND DISCUSSION
Inhaled asbestos fibers have been found within the cytoplasm of type I alveolar epithelial cells immediately after a 1-hr exposure and through the 48-hr post-exposure period (Fig 1). The asbestos fibers generally were not membranebound, but they had clear associations with intracellular •Laboratory of Pulmonary Function and Toxicology, National Institute of Environmental Health Sciences, Research 1iiangle Park NC. ' tDepartment of Pathology, University ofVermont College of Medicine, Burlington. Reprint re~Btl: Dr. Brody, Box 12233, Research Triangle lbrk, North Carolina 27709
FIGURE Ia. Type 1 alveolar epithelium (Ep) contains chrysotile asbestos fibrils (arrows) which were inhaled 24 hr earlier. The air spare (AS), basement membrane (BM), and underlying connective tissue (C) are indicated. A suggestion of microfilament accumulation is seen (arrowheads). b) An alveolar epithelial cell (Ep) contains asbestos fibrils (arrow) . An array of microfilaments (arrowheads) appears to radiate from the fibrils.
filaments which measured approximately 50-70 Ain diameter (Fig 1). Heavy meromyosin (HMM) treated tissues showed clear associations between the asbestos fibers and microfilaments (Fig 2). Since glycerinated tissue generally was swollen and disrupted, only well-stabilized complexes of microfilaments were retained during the fixation and embedding structures fur electron microscopy. Inasmuch as HMM is reported to stabilize polymerized microfilaments, 7 commonly we were able to find web-like complexes around intraepithelial asbestos fibers (Fig 2). Ultrastructural studies of cultured tracheal explants and epithelial cells demonstrated clear associations of filaments with asbestos fibers (Fig 3). If the intracellular microfilaments which have been observed are composed of contractile actin proteins, we should be able to measure increased levels of intracellular polymerized actin after treatment of the cells with asbestos in vitro. Data shown in Table 1 suggests that this is the case since the levels of filamentous actin were substantially increased over the nontreated controls. This CHEST I 83 I 5 I May. 1983 I Supplement
118
2 3 4
5
6
7 8
9
10
alveolar epithelium and pulmonary macrophages. Am Rev Respir Dis 1981; 123:670-79 Brody AR, Hill LH . Interstitial accumulation of inhaled chrysotile asbestos fibers and consequent formation of microcalcifications. Am J Pathol1982; 109:107-14 WagnerJC, BerryG, SkidmoreJW, TimbrellV. Theelfectsofthe inhalation of asbestos in rats. Br J Cancer 1974; 29:252-69 Adler KB, Craighead JE, Vallyathan NV, Evans JN. Actincontaining cells in human pulmonary fibrosis . Am J Pathol1981; 102:427-37 Mossman Bl: Ezerman EB, Adler KB, Craighead JE. Isolation and spontaneous transformation of cloned lines of hamster tracheal epithelial cells. Cancer Res 1980; 40:4403-09 Fox JE, Docktor ME, Phillips DR. An improved method for determining the actin filament content of non-muscle cells by the DNase I inhibition assay. Anal Biochem 1981; 117:170-77 'liotter JA. The organization of actin in spreading macrophages. Exp Cell Res 1981; 132:235-48 Adler KB, Brody AR, Craighead JE. Studies on the mechanism of mucin secretion by cells of the porcine tracheal epithelium. Proc Soc Exp Bioi and Med 1981; 166:37-44 Hartwig JH, Davies WA, Stossal TP. Evidence for contractile protein translocation in macrophage spreading, phagocytosis and phagolysosome fOrmation. J Cell Biol1977; 75:956-67 Barak LS, Nothnagel EA, DeMarco EF, WB WW. Differential staining of actin in metaphase spindles with phallacidin and floure~nt DNase: Is actin involved in chromosomal movement? Proc Nat Acad Sci 1981; 78:3034-38
Granulocytes Alter Endothelial Cell Cytoskeletons* Potential Contribution To Regulation Of Vascular Permeability D. Michael Shasby, M.D., F.C.C.P.; Sandra S. Shasby, M.D.; }amu M. Sullivan, M.D. ; Michael]. Peach, M.D.
FIGURE 2. Lung tissue treated with heavy meromyosin appears lucent and poorly &xed, but actin-containing microJilaments are stabilized in the process. This alveolar epithelial cell (Ep) exhibits a clear complex of microfilaments (arTOWheads) associated with asbestos fibrils (arTOW.t). FIGURE 3. Asbestos fibers (arTOW.t) in epithelial cells (Ep) of tracheal organ cultures are associated with complexes of micro6laments (arTOWheads) .
experiment was repeated twice with nearly identical results . Soluble actin monomers are rapidly polymerized into filamentous structures during cell movement, phagocytosis and intracellular transport. Actin-containing microfilaments are known to transport organelles such as mucin granules, 8 lysosomes, • and chromosomes. 10 Accordingly, we propose that similar contractile microfilaments provide the motive fOrce to translocate asbestos fibers through the epithelium to the lung interstitium where they interact with fibroblasts and macrophages, 2 cell types which are likely to play a major role in the pathogenesis of asbestosis . REFERENCES
1 Brody AR, Hill LH, Adkins B, O'Connor RW. Chrysotile asbestos inhalation in rats: deposition pattern and reaction of
18ble 1-~e cfFilamentoua (~)Actin Hamater Traclteal Epithelial CeU.
in
Untreated
1tLg Chrysotile Asbestos
2tLg Asbestos
12%
25%
21%
128
·nigh
permeability lung edema is characterized by increased permeability of the lung microvasculature to serum proteins. This increased permeability can occur in the absence of gross widespread endothelial destruction, and sections from lungs with experimental high permeability edema often show gaps between apparently undamaged endothelial cells during periods of leakage, and absence of the gaps when leakage has stopped a few hours later. 1 Ultrastructural studies of systemic endothelium have identified similar gaps in postcapillary venules exposed to histamine,1 and the recent recognition that endothelial histamine receptors are concentrated in the postcapillary venules helps to explain the fOcality of the histamine effect. 3 Until recently described, a rapidly reversible increase in lung microvascular permeability caused by intravascular activation of complement and both gaps between apparently healthy endothelial cells and fOcal endothelial injury appeared to contribute to the increased permeability. • Savion et al5 recently commented on the potential role of endothelial cell cytoskeletons in maintaining the closely apposed and flattened morphology that is necessary for the endothelium to resist sheer stress and pressure. Several studies have suggested that cytoskeletons of epithelial cells •From the Departments of Medicine and Pharmacology, University of Virginia Scnool of Medicine, Charlottesville. Supported in part by BRSG 5 S07 RR05431-19. Lung DetenM, Injury and Repair
la
AS
Arnold R. Brody, Ph.D.;* Lila H. Hill, B.S.;• WilliamS . Stirewalt, Ph.D.;t and Kenneth B. Adler, Ph.D. t
chrysotile asbestos fibers impact upon epithelial I nhaled cells which line the alveolar region of the lung.' Many of these fibers are taken up by alveolar epithelial cells.' During the 1-month period after a 1-hr exposure to chrysotile asbestos, numerous fibers are translocated to basement membranes as well as to interstitial cells and connective tissue.u The interstitial compartment at the alveolar level is the primary region affected during the pathogenesis of asbestosis. 3 We propose that actin-containing micro6laments of the alveolar epithelium provide a mechanism by which inhaled asbestos fibers are translocated to the pulmonary interstitium. The evidence to support this hypothesis has been achieved through ultrastructural studies of asbestosfilament associations in vivo and by quantitative biochemical studies of asbestos-epithelial interactions in vitro. MATERIALS AND METHODS
White rats were exposed to an aerosol of chrysotile asbestos (-15 mv/m3 , respirable mass} fur only 1 hour. Groups of animals were sacrificed immediately after exposure and at 24 and 48 hrs, and 1 month post-exposure. Animals were anesthet4ed with pentobarbital sodium (Nembutol) and the trachea was clamped. The lungs were expanded and fixed fur electron microscopy by perfusion of Karnovsky's fixative through the right ventricle and pulmonary artery fur 10 minutes. The lungs with the trachea still clamped then were removed in toto from the chest cavity and immersed in fresh fixative. DetaiJs of asbestos exposure, lung perfusion and dissection have been published. 1 To stabilize intracellular microfilaments, fresh lung tissue was dissected into 3 mm 3 blocks and immersed in 25% glycerol:75% KCI. The tissue then was placed in a 0.5% KCl solution containing dialyzed heavy-meromyosin fur 18 hrs.• Blocks were then fixed and embedded fur conventional electronic microscopy. For in vitro studies, asbestos fibers were added to organ cultures of tracheal explants and to cultures of cells from an established tracheal epithelial cell line. • Conventional electron microscopic studies were carried out on these cultures. In addition, biochemical quantitation of polymerized and non-polymerized actin proteins were carried out befOre apd after treating the cultured epithelial cells with asbestos. The amount of actin was determined by measurement of DNase I inhibition according to recently described methods. •
REsur.:rs
AND DISCUSSION
Inhaled asbestos fibers have been found within the cytoplasm of type I alveolar epithelial cells immediately after a 1-hr exposure and through the 48-hr post-exposure period (Fig 1). The asbestos fibers generally were not membranebound, but they had clear associations with intracellular •Laboratory of Pulmonary Function and Toxicology, National Institute of Environmental Health Sciences, Research 1iiangle Park NC. ' tDepartment of Pathology, University ofVermont College of Medicine, Burlington. Reprint re~Btl: Dr. Brody, Box 12233, Research Triangle lbrk, North Carolina 27709
FIGURE Ia. Type 1 alveolar epithelium (Ep) contains chrysotile asbestos fibrils (arrows) which were inhaled 24 hr earlier. The air spare (AS), basement membrane (BM), and underlying connective tissue (C) are indicated. A suggestion of microfilament accumulation is seen (arrowheads). b) An alveolar epithelial cell (Ep) contains asbestos fibrils (arrow) . An array of microfilaments (arrowheads) appears to radiate from the fibrils.
filaments which measured approximately 50-70 Ain diameter (Fig 1). Heavy meromyosin (HMM) treated tissues showed clear associations between the asbestos fibers and microfilaments (Fig 2). Since glycerinated tissue generally was swollen and disrupted, only well-stabilized complexes of microfilaments were retained during the fixation and embedding structures fur electron microscopy. Inasmuch as HMM is reported to stabilize polymerized microfilaments, 7 commonly we were able to find web-like complexes around intraepithelial asbestos fibers (Fig 2). Ultrastructural studies of cultured tracheal explants and epithelial cells demonstrated clear associations of filaments with asbestos fibers (Fig 3). If the intracellular microfilaments which have been observed are composed of contractile actin proteins, we should be able to measure increased levels of intracellular polymerized actin after treatment of the cells with asbestos in vitro. Data shown in Table 1 suggests that this is the case since the levels of filamentous actin were substantially increased over the nontreated controls. This CHEST I 83 I 5 I May. 1983 I Supplement
118
2 3 4
5
6
7 8
9
10
alveolar epithelium and pulmonary macrophages. Am Rev Respir Dis 1981; 123:670-79 Brody AR, Hill LH . Interstitial accumulation of inhaled chrysotile asbestos fibers and consequent formation of microcalcifications. Am J Pathol1982; 109:107-14 WagnerJC, BerryG, SkidmoreJW, TimbrellV. Theelfectsofthe inhalation of asbestos in rats. Br J Cancer 1974; 29:252-69 Adler KB, Craighead JE, Vallyathan NV, Evans JN. Actincontaining cells in human pulmonary fibrosis . Am J Pathol1981; 102:427-37 Mossman Bl: Ezerman EB, Adler KB, Craighead JE. Isolation and spontaneous transformation of cloned lines of hamster tracheal epithelial cells. Cancer Res 1980; 40:4403-09 Fox JE, Docktor ME, Phillips DR. An improved method for determining the actin filament content of non-muscle cells by the DNase I inhibition assay. Anal Biochem 1981; 117:170-77 'liotter JA. The organization of actin in spreading macrophages. Exp Cell Res 1981; 132:235-48 Adler KB, Brody AR, Craighead JE. Studies on the mechanism of mucin secretion by cells of the porcine tracheal epithelium. Proc Soc Exp Bioi and Med 1981; 166:37-44 Hartwig JH, Davies WA, Stossal TP. Evidence for contractile protein translocation in macrophage spreading, phagocytosis and phagolysosome fOrmation. J Cell Biol1977; 75:956-67 Barak LS, Nothnagel EA, DeMarco EF, WB WW. Differential staining of actin in metaphase spindles with phallacidin and floure~nt DNase: Is actin involved in chromosomal movement? Proc Nat Acad Sci 1981; 78:3034-38
Granulocytes Alter Endothelial Cell Cytoskeletons* Potential Contribution To Regulation Of Vascular Permeability D. Michael Shasby, M.D., F.C.C.P.; Sandra S. Shasby, M.D.; }amu M. Sullivan, M.D. ; Michael]. Peach, M.D.
FIGURE 2. Lung tissue treated with heavy meromyosin appears lucent and poorly &xed, but actin-containing microJilaments are stabilized in the process. This alveolar epithelial cell (Ep) exhibits a clear complex of microfilaments (arTOWheads) associated with asbestos fibrils (arTOW.t). FIGURE 3. Asbestos fibers (arTOW.t) in epithelial cells (Ep) of tracheal organ cultures are associated with complexes of micro6laments (arTOWheads) .
experiment was repeated twice with nearly identical results . Soluble actin monomers are rapidly polymerized into filamentous structures during cell movement, phagocytosis and intracellular transport. Actin-containing microfilaments are known to transport organelles such as mucin granules, 8 lysosomes, • and chromosomes. 10 Accordingly, we propose that similar contractile microfilaments provide the motive fOrce to translocate asbestos fibers through the epithelium to the lung interstitium where they interact with fibroblasts and macrophages, 2 cell types which are likely to play a major role in the pathogenesis of asbestosis . REFERENCES
1 Brody AR, Hill LH, Adkins B, O'Connor RW. Chrysotile asbestos inhalation in rats: deposition pattern and reaction of
18ble 1-~e cfFilamentoua (~)Actin Hamater Traclteal Epithelial CeU.
in
Untreated
1tLg Chrysotile Asbestos
2tLg Asbestos
12%
25%
21%
128
·nigh
permeability lung edema is characterized by increased permeability of the lung microvasculature to serum proteins. This increased permeability can occur in the absence of gross widespread endothelial destruction, and sections from lungs with experimental high permeability edema often show gaps between apparently undamaged endothelial cells during periods of leakage, and absence of the gaps when leakage has stopped a few hours later. 1 Ultrastructural studies of systemic endothelium have identified similar gaps in postcapillary venules exposed to histamine,1 and the recent recognition that endothelial histamine receptors are concentrated in the postcapillary venules helps to explain the fOcality of the histamine effect. 3 Until recently described, a rapidly reversible increase in lung microvascular permeability caused by intravascular activation of complement and both gaps between apparently healthy endothelial cells and fOcal endothelial injury appeared to contribute to the increased permeability. • Savion et al5 recently commented on the potential role of endothelial cell cytoskeletons in maintaining the closely apposed and flattened morphology that is necessary for the endothelium to resist sheer stress and pressure. Several studies have suggested that cytoskeletons of epithelial cells •From the Departments of Medicine and Pharmacology, University of Virginia Scnool of Medicine, Charlottesville. Supported in part by BRSG 5 S07 RR05431-19. Lung DetenM, Injury and Repair