Br. J. exp. Path. (1974) 55, 275. ULTRASTRUCTURAL CHANGES IN LUNG FIBROBLAST CULTURES EXPOSED TO CHRYSOTILE ASBESTOS R. J. RICHARDS,* P. M. HEXT,* G. BLUNDELL, W. J. HENDERSON AND B. E. VOLCANIt From the Department of Biochemistry,* University College, Cardiff, Wales, U.K. and the Tenovus Institute, University Hospital of Wales, Heath Park, Cardiff, U.K. Received for publication February 15, 1974 Summary.-Early changes in the ultrastructure of lung fibroblasts exposed to chrysotile asbestos in vitro are described. The dust exposed cells rapidly undergo a " maturation process " reflected by highly dilated endoplasmic cisternae, irregularity in nuclear outline and composition and the appearance of membrane microvilli which are all characteristics of normal cells in the stationary phase. It is suggested the surviving cells undergoing this" matura - tion process " may account in part for the higher synthetic activity, leading to increased collagen levels, in chrysotile exposed cultures. RECENT biochemical and morphological studies (Richards, Wusteman and Dodgson, 1971; Richards and Morris, 1973; Richards and Jacoby, 1973) have suggested that considerable alterations occur in lung fibroblast cells upon direct exposure to chrysotile asbestos in vitro. Biochemical data (Richards and Morris, 1973) have indicated that cells exposed to chrysotile have a higher cell mat collagen, increased protein levels and an altered glycosaminoglyean (unsulphated/ sulphated) ratio released into the culture medium. It has been suggested that these in vitro effects are either similar or equivalent to a fibrogenic response produced bv chrysotile in vivo. Morphological studies have shown that chrysotile asbestos is cytotoxic to lung fibroblasts (Richards and Jacoby, 1973) and to other cell types (Allison, 1970; Miller and Harington, 1972; Beck, Holt and Nasrallah, 1971; Sakabe, Koshi and Hayashi, 1970; Suzuki, Churg and Ono, 1972). The dust causes considerable cell lysis in fibroblast cultures coincidental with nuclear, nucleolar and cytoplasmic alterations, after which the cell population recovers and the majority of cells appear perfectly normal (Richards and Jacoby, 1973). Light microscopical studies, however, provide insufficient data, in that clear visualization of the dust particles is impossible and detailed nuclear and cytoplasmic alterations caused by chrysotile cannot be seen. Thus, the present electron microscopical study was designed to study these aberrations in more detail. An attempt was made to determine the means of fibre attachment to the plasma membrane, whether these asbestos fibres enter the cells, if entry occurred then the size and distribution of the dust, and finally to assess any alterations in fine strtucture that resulted from dust attachment or ingestion. The overall aim was to correlate these results with the previous biochemical and morphological data in the search for mechanisms of dust fibrosis. t Dr Volcani's present address is the University of California, La Jolla, California 92037, U.S.A.
2,76 RICHARDS, HEXT, BLUNDELL, HENDERSON AND VOLCANI MATERIALS AND METHODS Cell cultures. Rabbit lung fibroblasts were cultured in an identical manner to that described previously (Richards et al., 1971). Subcultures were set up by seeding approximately 12 x 105 cells (the number being constant for any one experiment) on to glass coverslips (35 x 10 mm) in Leighton tubes. The culture medium, 1 ml of 20% foetal bovine seruin in WVaymouth's medium containing additional ascorbic acid (final concentration 50 Itg/ml) wxas changed twice weekly. A single dose (50 tg) of chrysotile A (UICC sample) was added in 0 1 ml of medium 199 to test cultures 24 houirs after subculturing. Control cultlures were given 0 1 ml of medium 199. At various stages of growth, control and test cultures were removed and processed in an identical manner for electron microscopy. Fixation, embedding and staining of cultures. After removal of the culture medium, the inonolayers on glass coverslips were washed in freshly prepared sodium cacodylate/sucrose buffer ph 7 2 for 10 min, then prefixed in 10% glutaraldehyde in sodium cacodylate/sucrose buffer ph 7 2 for 1 hour on ice. The coverslips were left in buffer wash 16-20 hours at low (4 ) temperature for convenience of handling. They were then post fixed in 10% osmium tetroxide in cacodylate/sucrose buffer for 1 hour on ice. Following an initial wash in buffer, the cultures were dehydrated in a series of graded alcohols. Propylene oxide and Araldite were then added. To facilitate encapsulation, the Araldite plus tridimethylaminomethyl phenyl (DMP 30) was precured partially at 60 to obtain a more viscous consistency. A thin layer of this precured Araldite was poured over the monolayer culture onto the cover slip and allowed to become more viscous. The remainder was used to fill the capsules. The viscous nature of the precuired Araldite enabled the capsules to be inverted on to the top of the Araldite coated monolayer cultures, without loss of the Araldite from the capsules before hardening. They were then placed in the 60 oven to cure for 24 hours. After curing, the capsules were separated and mounted in the microtome chuck. The glass was removed with a scalpel blade to expose the embedded monolayer. With this procedure the cells were presented horizontally to the knife. Both glass and diamond knives were tried, the latter giving the best results, especially with the cultures containing asbestos particles. The sections were stained with uranyl acetate and lead citrate. To increase ultrastructural contrast some samples were pre-stained with 1% aqueous uranyl acetate after post-fixation. RESULTS Untreated normal cultures Logarithmic phase. A normal cell during the early stages of logarithmic growth is shown (Fig. 1). The oval or rounded nucleus has little chromatin around the edges and contains several well defined nucleoli, some of which show multicomplex patterns. Large vacuoles are prominent in the cytoplasm, the majority being round although in the early stages of logarithmic growth many are elongated. These are attributed to be lipid containing vacuoles in the lining cell but appear as empty vacuoles in the electron micrographs due to the extraction of the lipid material during the fixation process. Usually these vacuoles are found associated in groups towards the end of the cells. The cytoplasm contains a large number of ribosomes, some of which are attached to the endoplasmic reticulum but the majority are in the form of free polysomes. Mitochondria of varying size and shape are present, the majority in a perinuclear position. Single membrane bodies, presumably secondary lysosomes, are often found to contain whorls of membrane-like material in the early stages of culture (Fig. 2). The cell surface appears as a very indistinct amorphous coat in the majority of cells, no true membrane being discernible (Fig. 3). In close association with this outer coat, microfilaments can be seen running parallel to the surface (Fig. 3). Similar filaments also run throughout the cytoplasm. Stationary phase. During the stationary phase one of the main functions of the fibroblast is to synthesize collagen fibres and these have been laid down in the intercellular matrix (Fig. 4). Prominent microvilli also extend from the cell
LUNG FIBROBLAST CULTURES 277 surface into this matrix and may become budded off (Fig. 4). The majority of cells have a distinct plasma membrane with very few patches of the amorphous coat remaining. During the stationary phase a few cells have the normal rounded nuclear appearance observed during logarithmic growth but the majority of cells have irregular nuclear outlines. These irregular nuclei (see Fig. 4) have few discernible nucleoli and appear to have more chromatin around the edge of the nuclear membrane. Lipid vacuoles are still present in large numbers, constituting in some cells approximately 60% of the cell volume. Whilst the elongated lipid vacuoles are no longer present, granular bodies of varying sizes are now found associated with the groups of these vacuoles (Fig. 4, 5). Early stationary phase fibroblasts (Day 9, in culture) apparently show little alteration in the ribosomal and endoplasmic reticulum content compared with cells in logarithmic growth. However, one major difference is that the endoplasmic cisternae have become highly dilated in stationary phase cells (Fig. 4, 5). There is an increase in the number of densely stained bodies in stationary phase cells (Fig. 5) and a well developed golgi is present in many of the cells. Mitochondria are considerably longer than those found in cells in logarithmic growth and are often found in large numbers towards the ends of the cell (Fig. 6). (ultures exposed to chrysotile asbestos Logarithmic phase. Within 41 hours of exposure to chrysotile asbestos the fibroblasts have taken up a considerable number of dust fibres (Fig. 7). Serial sectioning of the cells has clearly shown that many of the asbestos fibres are intracellular. At sites where fibres appear to be entering a cell there is invariably a dense area surrounding the point of contact (Fig. 7). Nuclear aberration occurs in many cells within 41 hours of exposure to dust and after 24 hours nuclei are often grossly distorted, together with large abnormal nucleoli (Fig. 8). Change in the endoplasmic cisternae also appears to be very rapid upon exposure to chrysotile. Fibroblasts exposed to chrysotile for just 41 hours have a highly dilated endoplasmic cisternae throughout much of the cell, in an identical manner to that noted during the stationary phase in normal cells. This effect, however, is not apparent after 24 hours of dust exposure. Mitochondria and dense bodies in dust exposed cells are similar in number and shape to normal cells. In many instances where dust fibres were found within the cell a distinct membrane could be seen surrounding the fibres (Fig. 9, 10), suggesting that intracellular asbestos fibres were membrane bound and not in intimate contact with the cytoplasm or cytoplasmic organelles. The surface layer of cells exposed to dust for 41 hours has regions of the amorphous coat together with its associated microfilaments. After 24 hours of exposure the surface has a large number of microvilli extending into the intracellular matrix (Fig. 9, 10). Microvilli were rarely seen in normal cells in the equivalent time period. Stationary phase. A high proportion of cells exposed to chrysotile asbestos for 8 days contain little dust and have the appearance of normal cells at varying stages of development. However, there are still many cells which contain dust fibres in very appreciable amounts (Fig. I1). The nuclei in the cells containing a high
278 RICHARDS, HEXT, BLUNDELL, HENDERSON AND VOLCANI proportion of dust have highly irregular outlines and some cells are binucleate. Mitochondria in these dust-containing cells appear more numerous and prominent and are less elongated than those found in the equivalent unexposed cell (Fig. 11). The golgi is extremely well developed, covering a large area in the centre of the cell (Fig. 11). In the initial stages of the stationary phase (up to Day 9 in culture) collagen fibres are rarely observed in the intercellular matrix, in direct contrast to that found in unexposed cells. However, after 13 days exposure to dust the cells have begun to produce collagen (Fig. 12). Electron microscope microanalyser (EMMA) studies have shown that the particles within the cells have the correct magnesium/silicon ratio, as would be expected of chrysotile asbestos fibres. DISCUSSION The structure of the normal rabbit lung fibroblasts used in the present study compares favourably with that found by previous investigators (Goldberg and Green, 1964; Comings and Okada, 1970) using other fibroblast cell lines. The change noted by Comings and Okada (1970) as human fibroblasts passed from logarithmic to the stationary phase of growth, i.e. dilation of the endoplasmic cisternae, increase in autophagic vacuoles and loss of polyribosome configurations EXPLANATION OF PLATES FIG. 1. Normal fibroblast in logarithmic growth (24 hours after subculture). N, nucleus; Nu, nucleolus; Lv, lipid vacuoles. x 10,000. FIG. 2.-Normal fibroblast in logarithmic growth (48 hours in culture) showing membrane-like whorls in vacuoles (V) and the golgi (G). x 23,000. FIG. 3. Normal fibroblast in logarithmic growth (48 hours in culture). Microfilaments (Mf) are present running in bundles parallel to the cell surface. The latter appears as an amorphous coat (C) as opposed to a distinct membrane. Poly ribosomes (arrowed) and mitchondria (M) are present throughout most of the cytoplasm. x 15,000. FIG. 4.-Normal fibroblast during the stationary phase of growth (9 days in culture) Collagenlike fibre (Cf) and microvilli (Mv) can be seen in the intracellular matrix. The nuclei (N) have become irregular in outline and the endoplasmic cisternae (arrowed) is highly dilated. A granular body (Gb) is found in association with lipid vacuoles. x 3750. FIG. 5. Normal cell in the stationary phase of growth showing a large granular body (Gb) and many dense bodies (Db). In addition, there are extensive areas of dilated endoplasmic cisternae (arrowed); Mv, microvillus. x 4500. FIG. 6. Normal fibroblast after 9 days in culture showing elongated mitochondria (arrowed) which are generally present towards the cell periphery. x 4500. FIG. 7. Lung fibroblast after 4j hours exposure to chryostile asbestos (2 days in culture). Dust fibres (Af) can be on the edge of the cell and within the cytoplasm. The arrow indicates the point of contact between the dust fibre and the cell. M, mitochondrion; C, amorphous coat; Mf, microfilaments. x 15,000. FIG. 8. A highly irregular nucleus (N) observed after 24 hours exposure to chrysotile asbestos. Nu, nucleolus. x 4 500. FIG. 9.-Fibroblast after 24 hours exposure to chrysotile asbestos showing the different orientations of dust at the cell surface. Microvilli (Mv), some of which contain asbestos are present at the cell surface. x 7000. FIG. 10.-Higher magnification of the microvillus (Mv) shown boxed in Fig. 9. The asbestos fibres (Af) are apparently membrane bound (arrowed). x 40,000. FIG. 1 1.-Perinuclear region of a fibroblast exposed to asbestos for 8 days (9 days in culture). Large numbers of densely stained mitochondria (M) and extensive golgi (G) together with asbestos fibres fill this area. A second type of granular body (Gb2) is also present. x 11,500. FIG. 12.-Fibroblast 13 days after exposure to asbestos. A small amount of asbestos dust (arrowed) is found within the cytoplasm probably as a result of secondary uptake. There is extensive development of collagen-like fibres (Cf) in the intercellular matrix. x 7000.
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LUNG FIBROBLAST CULTURES 279 were all found to occur in the lung fibroblasts. However, no increase was noted in the number of cytoplasmic microfilaments (see Perdue, 1973) in the lung cells in stationary phase, as found with the human fibroblasts. The present ultrastructural study has clearly shown, particularly by serial sectioning, that chrysotile fibres of a considerable size range (up to 15 /am) enter the fibroblast cells and are found within all areas of the cytoplasm (often in vacuoles) but never within the nucleus. The dust fibres (proved to be chrysotile asbestos by the EMMA studies) are phagocytozed very rapidly (no comparison has yet beeni madle with alveolar macrophages) and there is apparently no specific orientation of the dust fibres to the cell surface. It has been noticed that an electron dense area exists at the point of contact between the dust fibre and the cell membraiie which is perhaps analogous to previous observations on the early contact between fibroblast cells (Heaysman and Pegrum, 1973). XVhilst it is accepted that cells exhibit a greater phagocytic potential in vitro than in vivo, the important fact that fibroblasts may be capable of taking up asbestos fibres in vivo, and the subsequent effects this may have in the fibrotic reaction, would seem to warrant further investigation. One of the first ultrastructural changes induced by chrysotile (within 41 hours exposure) is that the endoplasmic cisternae become highly dilated, suggesting increased cellular synthetic or secretory activity. However, after 24 hours of dust exposure this effect is no longer noticeable, suggesting either a reversion of the endoplasmic cisternae to its normal appearance or the death of the cells displaying this feature after 4- hours exposure. During the first 24 hours of dust exposure the nuclei of chrysotile exposed cells are found to transform from the normal log phase oval or round shape to irregular, distorted shapes containing ill-defined nucleoli. The general appearance of these transformed nuclei is similar to that of normal cells in the stationary growth phase. Another major difference between normal and chrysotile treated fibroblasts after 24 hours of dust exposure is the presence of considerable numbers of microvilli in the latter cells. The most obvious conclusion is that the presence of the microvilli is associated with the phagocytic activity of the cells but microvilli are apparent in many cells only after the dust has been taken up. The presence of microvilli is a characteristic feature of normal cells in the stationary phase of growth. Thus, the irregular nuclear shape, the presence of highly dilated endoplasmic cisternae and the occurrence of microvilli, i.e. all characteristics of normal fibroblasts in the stationary phase of growth (Day 8 + in culture) are found to occur in cells exposed to chrysotile after just 24 hours (Day 2 in culture). This suggests that after the initial exposure to chrysotile the fibroblasts undergo a very rapid " maturation process ", taking on the structure and hence presumably function of a cell in the stationary phase. Hence, it could be speculated that these cells which suirvived the dust exposure would be active synthetically (i.e. in glycosaminoglycan, protein and collagen production) and not actively committed to cell division. Previous histological findings (Richards and Jacoby, 1973) have shown that mitoses are rare in the early stages of culture in chrysotile exposed cells. Both light and electron microscope studies have shown the presence of binucleate cells in these cultures, suggesting abnormalities in cell division. After an initial decimation of the fibroblast population by the chrysotile dust,
280 RICHARDS, HEXT, BLUNDELL, HENDERSON AND VOLCANI the surviving cells show a remarkable recovery (Richards and Jacoby, 1973). Electron micrographs at this stage (Day 9 in culture) show a completely mixed cell population a major proportion of which are dust-free and exhibit the structure of normal cells in logarithmic growth. Some cells have a small number of dust particles in the cytoplasm (secondary uptake?) whilst others are almost totally packed with chrysotile fibres and such cells always have a high complement of mitochondria. At this stage in culture the production of collagen fibres by the dust-exposed fibroblasts is rare (presumably because most of the cells are still in logarithmic growth) whereas in 9-day old normal cultures collagen fibres are clearly evident. Previous biochemical estimations confirm these findings (Richards and Morris, 1973). Shortly after this time period and up to Day 24, however, the chrysotile exposed fibroblasts produce significantly higher amounts of collagen than the normal untreated cultures (Richards and Morris, 1973). Whether the stimulus for this effect originates from the surviving dust laden cells or from the relatively dust-free replacement (altered?) population is unknown. Further detailed electron microscope studies of cells at varying stages of stationary growth combined with detailed biochemical studies of collagen turnover are required in order to further elucidate the changes in fibroblasts during the production of " fibrosis " in vitro by chrysotile asbestos. We are grateful to Mr W. Edwards for excellent assistance with cell cultures. One of us (P.M.H.) wishes to thank the Medical Research Council for financial support. REFERENCES ALLISON, A. C. (1970) Effects of Silica and Asbestos on Cells in Culture. In Inhaled Particles, III, Vol. 1. Proc. Int. Symnp., London, U.K. September 14-23, 1970. Surrey: Unwin Brothers Ltd. BECK, E. G., HOLT, P. F. & NASRALLAH, E. T (1971) Effects of Chrysotile and Acidtreated Chrysotile on Macrophage Cultures. Br. J. industr. Med., 28, 179. COMINGS, D. E. & OKADO, T. A. (1970) Electron Microscopy of Human Fibroblasts in Tissue Culture during Logarithmic and Confluent States of Growth. Expl Cell Res., 61, 295. GOLDBERG, B. & GREEN, H. (1964) An Analysis of Collagen Secretion by Established Mouse Fibroblast Lines. J. cell. Biol., 22, 227. HARINGON, J. S., MILLER, K. & MACNAB, E. (1971) Hemolysis by Asbestos. Environ. Rees., 4, 95. HEAYSMAN, J. E. M. & PEGRUM, S. M. (1973) Early Contacts between Fibroblasts. Expl cell Res., 78, 71. MILLER, K. & HARINGTON, J. S. (1972) Some Biochemical Effects of Asbestos on Macrophages. Br. J. exp. Path., 53, 397. PERDUE, J. F. (1973) The Distribution, Ultrastructure and Chemistry of Microfilaments in Cultured Chick embryo Fibroblasts. J. cell Biol., 58, 265. RICHARDS, R. J. & JACOBY, F. (1974) Lung Fibroblasts and Dust. In Symposium of Tissue Culture in Medical Research. M.R.C. Llando ugh Hospital, Penarth. Glamorgan, U.K. April 11-13, 1973. London: Heinemann. In the press. RICHARDS, R. J. & MORRIS, T. G. (1973) Collagen and Mucopolys accharide Production in Growing Lung Fibroblasts Exposed to Chrysotile Asbestos. Life Sci., 12, 441. RICHARDS, R. J., WUSTEMAN, F. S. & DODGSON, K. S. (1971) The Direct Effects of Dust on Lung Fibroblasts grown in vitro. Life Sci., 10, 1149.
LUNG FIBROBLAST CULTURES 281 SAKABE, H., KOSHI, K. & HAYASHI, H. (1970) On the Cell Toxicity of Mineral Dusts. In Inhaled Particles, III, Vol. 1. Proc. Int. Symp., London, U.K. September 14-23, 1970. Surrey: Unwin Brothers Ltd. SUZUKI, Y., CHURG, J. & ONO, T. (1962) Phagocytic Activity of the Alveolar Epithelial Cells in Pulmonary Asbestosis. Am. J. Path., 69, 373.