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Persistence of Acholeplasma laidlawii in an established cell line RL 19
Exp. Path. 24, 207-211 (1983) Friedrich Schiller University Jena, Institute of Pathology (Head: Prof. Dr. sc. med. Dr. h. c. F. BOLCK)
Persistence of Acholeplasma laidlawii in an established cell line RL 19 1. Host-parasite morphologyl)
By G.
NEUPERT
and TH.
STERBA
With 2 figures Received January 12, 1983) Address for correspondence: Dr. rer. nat. G. NEUPERT, Institut fiir Pathologie, Friedrich-SchillerUniversitat, Ziegelmiihlenweg 1, DDR - 6900 Jena
Key w 0 r d s: mycoplasma; Acholeplasma laidlawii; liver cell line ; transmission electron microscopy; scanning electron microscopy; ethylenediamine tetraacetate (EDTA)
Summary The cell-parasite relationship of epitheloid cells from the established liver cell line RL 19 persistently infected with Acholeplasma laidlawii was studied by transmission electron microscopy and scanning electron microscopy. The predominantly filamentous, branched A. laidlawii were arranged horizontally on the cell surface between microvilli at the cell margin. The cell adhesion, spreading, growth pattern and cell surface shape were not altered in relation to uninfected cultures of the same cell line.
Introduction The persistent infection of mycoplasmas in continuous cell lines is well-known and a widespread problem. Several membrane-associated mycoplasmas that cause no overt cytopathogenic effect on the host cells, however are of interest for the function of the host cell membrane. For example it has been shown that mycoplasma-infected cell lines are more susceptible to the nonspecific cytolytic action of lymphocytes than are mycoplasma-free cultures (ALDRIDGE et al. 1977; BROOKS et al. 1979). In this study we investigated the surface properties of the host cell-mycoplasma relationship in a defined cell culture system. The attachment of A.laidlawii to host cells has been studied morphologically. The purpose of the present work was to find possible local alterations at the host cell membrane.
Materials and Methods The cell line RL 19 maintained in our laboratory was established by use of liver from neonatal Wistar rats in 1973, details have been described by KARSTEN et al. (1975). The cells were cultured in Eagle'S Minimal Essential Medium (MEM) with 10 % heat-inactivated calf serum and gentamycin. Cells were routinely passaged every 7 days with 0.25 % trypsin and 0.02 % EDTA in phosphate buffered saline (PBS). A part of cell cultures were continuously treated with tylosine solution (Gibco). . The cultures were checked routinely for mycoplasma contamination after the method of CHEN (1977) using the DNA stain bisbenzimidazol, compound 33258 (Hoechst) and observed in the fluorescence microscope. 1) Dedicated to Prof. Dr. se. med. Dr. h. c. F. BOLCK on occasion of his 65th birthday.
207
Cultured cells and cell supernatants were examined by direct culture in PPL01) broth or agar supplemented with different additions in order to determine the mycoplasmas. The coverslips with cells were rinsed in filtered PBS, then fixed in 2.5 % glutaraldehyde in PBS for 30 min at room temperature and afterwards washed twice in PBS. The cells were dehydrated through increasing concentrations of acetone and then transferred into dried 100% acetone. The specimens were critical point dried with liquid CO 2 in a critical point dryer (Balzers Union), sputtercoated with gold and observed in a SEM (JSM 35, Jeol) at an angle of 30°. For transmission electron microscopy (TEM) the monolayers were fixed with 2.5 % glutaraldehyde in 0.1 M Na-cacodylate buffer, pH 7.2 for 30 min, followed by treatment with 1 % buffered osmium tetroxide for 30 min. After dehydration in ethanol the cells were in situ embedded in Mikropal and separated from the glass by means of hot water (80 DC). Ultrathin sections were contrasted with uranyl acetate and lead citrate before being examined in a Tesla 500 electron microscope.
Results The epitheloid cells of the RL 19 line grew in cord-like patterns and by confluence in closely adherent pavement-like monolayer (fig. 1 a). The cell surface is covered with short thick microvilli varying from eell to cell especially at the close contact zones to neighbouring cells.
Fig. 1. a) Sub confluent monolayer of RL 19 cells in living state, NOMARSKI interference contrast. x 200. b) Mycoplasma-infected RL 19 cells stained with bisbenzimidazol, fluorescence mieroscopy. x 200. Besides DNA-stained cell nuclei mycoplasmas appear as brightly fluorescent particles localized exclusively at the cell periphery. c) Thin section through mycoplasmas (M) and microvilli of RL 19 cells in monolayer culture. x 1,GOO. The darkly contrasted vesicles are mycoplasmas. 1) pleuropneumonia-like organism
208
Fig. 2. SEM micrographs of the surface of RL 19 cells persistently infected with A. laidlawii. a) Single epitheloid cell from a confluent monolayer with individual rod-shaped A. laidlawii at the cell ma,rgin (arrows). x 2,000. b) Highly magnified area of the cell surface with individual mycoplasmas. x 10,000. c) and d) Cluster of mycoplasmas attached to epitheloid cells a.long the cell-cell contact wne. c) 15,000, d) 10,000. e) Numerous A. laidl((wii of variable morphology at the cell margin. x 5,200.
209
After the 52nd subculture on the surface of the cells mycoplasmas were demonstrated with TEM and SEM as well as by DNA staining (fig. 1 b, c, 2) and were serologically classified as A. laidlawii. This spontaneous contamination has developed a 4 years persisting infection. 30 min after cell seeding single rod-shaped mycoplasmas lie horizontally upon the smooth lamellipodium of spread cells. The filamentous A. laidlawii are swollen at their ends and show a pronounced tendency to form branching structures (fig. 2b). When epitheloid cells take up contact with neighbouring cells the mycoplasmas are aligned mostly parallel to the cell margin and are lying between the microvilli and intercellular gaps of the adjoining cell membranes. In confluent and superconfluent cultures the filamentous, branched A. laidlawii are predominantly arranged in clusters along the cell borders (fig. 2c, d). The clusters were associated with the surface of epitheloid cells, though, as analysed in thin sections, the mycoplasmas were always separated from the host cell membrane by a minute gaps. Several mycoplasmas, especially in clusters are erected vertically and from the ends spherical bodies tied off (fig. 2c, d). The pleomorphism of A. laidlawii ranges from long filamentous rods to short coccoid chains. In quiescent cultures (without subculturing) the liver cells form a continuous network of collagen fibers upon the monolayer. The mycoplasmas then lie between the collagen fibers. In electron microscopic sections the sizes of microvilli and mycoplasmas are about equal, but the mycoplasmas have a high intracellular density that distinguishes them from the cytoplasma of the host cells. A. laidlawii evidenced no morphologically visible cytopathogenic effect on the host cells. The intimate association between adhering A. laidlawii and the host cells provides no situation in which the host cell membrane was attached or locally damaged. The cell adhesion on the substrate, spreading, growth pattern and cell surface shape were not altered as compared to tylosine treated uncontamined cultures of the same cell line. The scanning electron micrographs indicate that the host cell surfaces are neither alterated nor damaged by attachment of mycoplasmas. This suggests that no toxic metabolites are excreted by the mycoplasmas.
Discussion BeRides others mycoplasmas A. laidlawii is considered to be the most common mycoplasma strain found in contaminated cell cultures of various species. In this study it has been shown that the interaction of A. laidlawii with a defined type of host cells can lead to persistent infection. The possibility to spread the mycoplasmas from subculture to subculture is very manifold despite of preventive measures (MCGARRITY 1976). Reports about the effects of non-pathogenic mycoplasmas on host cell morphology in culture are scarce (BROWN et al. 1974). ZUCKER-FRANKLIN et al. (1966) reported on development of surface irregularities on host cells and BOATMAN et al. (1976) found a 26 % increase in host cell size. Despite of observation for many years we found no morphological alterations on the surface of persistently infected cells under different culture conditions. It is remarkable that the A. laidlawii did not cover the whole cell surface, but the cell-cell contact zone only.
Acknowledgements The mycoplasmas were determined by Dr. H. PFUTZNER, Institute of Animal Bacteriological Infectious Diseases of the Academy of Agricultural Sciences J ena. We thank Mrs. I.-M. HERRMANN, Department of Electron Microscopy, Friedrich Schiller University Jena, Medical School, for her expert technical assistance and Mr. H. MULLER, GDR Academy of Sciences, Optics and Spectroscopy, Jena, for the operation of the SEM (JSM 35, JEOL).
Literature ALDRIDGE, K. E., B. S. COLE and J. R. WARD, Mycoplasma-dependent activation of normallymphocytes: induction of a lymphocyte-mediated cytotoxicity for allogeneic and syngeneic mouse target cells. Infect. Immun. 18, 377-392 (1977).
210
BOATMAN, K, F. CARTWRIGHT and G. KENNY, Morphology, morphometry and electron microscopy of HeLa cells infected with bovine mycoplasma. Cell Tiss. Res. 170, 1-16 (1976). BROOKS, C. G., R C. REES and R H. LEACH, High nonspecific reactivity of normal lymphocytes against mycoplasma infected target cells in cytotoxicity assays. Eur. J. Immunol. 9, 159-171 (1979). BROWN, S., M. TEPLITZ and J.-P. REVEL, Interaction of mycoplasmas with cell cultures as visualized by electron microscopy. Proe. Nat. Acad. Sci. DSA 71, 464-468 (1974). CHEN, T. R, In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell. Res. 104, 255-262 (1977). KARSTEN, D., G. NEUPERT and R TUUST, Characteristics of an established epithelial cell line (RL 19) from rat liver. Exp. Path. 12, 88-99 (1976). MCGARRITY, G. J., Spread and control of mycoplasmal infection of cell cultures. In vitro 12, 643-648 (1976). ZUCKER-FRANKLIN, D., M. DAVIDSON and 1. THOMAS, The interaction of mycoplasmas with mammalian cells. 1. HeLa ccUs, neutrophils, and eosinophils. J. expo Med. 124, 521-531 (1966).
211
Persistence of Acholeplasma laidlawii in an established cell line RL 19 1. Host-parasite morphologyl)
By G.
NEUPERT
and TH.
STERBA
With 2 figures Received January 12, 1983) Address for correspondence: Dr. rer. nat. G. NEUPERT, Institut fiir Pathologie, Friedrich-SchillerUniversitat, Ziegelmiihlenweg 1, DDR - 6900 Jena
Key w 0 r d s: mycoplasma; Acholeplasma laidlawii; liver cell line ; transmission electron microscopy; scanning electron microscopy; ethylenediamine tetraacetate (EDTA)
Summary The cell-parasite relationship of epitheloid cells from the established liver cell line RL 19 persistently infected with Acholeplasma laidlawii was studied by transmission electron microscopy and scanning electron microscopy. The predominantly filamentous, branched A. laidlawii were arranged horizontally on the cell surface between microvilli at the cell margin. The cell adhesion, spreading, growth pattern and cell surface shape were not altered in relation to uninfected cultures of the same cell line.
Introduction The persistent infection of mycoplasmas in continuous cell lines is well-known and a widespread problem. Several membrane-associated mycoplasmas that cause no overt cytopathogenic effect on the host cells, however are of interest for the function of the host cell membrane. For example it has been shown that mycoplasma-infected cell lines are more susceptible to the nonspecific cytolytic action of lymphocytes than are mycoplasma-free cultures (ALDRIDGE et al. 1977; BROOKS et al. 1979). In this study we investigated the surface properties of the host cell-mycoplasma relationship in a defined cell culture system. The attachment of A.laidlawii to host cells has been studied morphologically. The purpose of the present work was to find possible local alterations at the host cell membrane.
Materials and Methods The cell line RL 19 maintained in our laboratory was established by use of liver from neonatal Wistar rats in 1973, details have been described by KARSTEN et al. (1975). The cells were cultured in Eagle'S Minimal Essential Medium (MEM) with 10 % heat-inactivated calf serum and gentamycin. Cells were routinely passaged every 7 days with 0.25 % trypsin and 0.02 % EDTA in phosphate buffered saline (PBS). A part of cell cultures were continuously treated with tylosine solution (Gibco). . The cultures were checked routinely for mycoplasma contamination after the method of CHEN (1977) using the DNA stain bisbenzimidazol, compound 33258 (Hoechst) and observed in the fluorescence microscope. 1) Dedicated to Prof. Dr. se. med. Dr. h. c. F. BOLCK on occasion of his 65th birthday.
207
Cultured cells and cell supernatants were examined by direct culture in PPL01) broth or agar supplemented with different additions in order to determine the mycoplasmas. The coverslips with cells were rinsed in filtered PBS, then fixed in 2.5 % glutaraldehyde in PBS for 30 min at room temperature and afterwards washed twice in PBS. The cells were dehydrated through increasing concentrations of acetone and then transferred into dried 100% acetone. The specimens were critical point dried with liquid CO 2 in a critical point dryer (Balzers Union), sputtercoated with gold and observed in a SEM (JSM 35, Jeol) at an angle of 30°. For transmission electron microscopy (TEM) the monolayers were fixed with 2.5 % glutaraldehyde in 0.1 M Na-cacodylate buffer, pH 7.2 for 30 min, followed by treatment with 1 % buffered osmium tetroxide for 30 min. After dehydration in ethanol the cells were in situ embedded in Mikropal and separated from the glass by means of hot water (80 DC). Ultrathin sections were contrasted with uranyl acetate and lead citrate before being examined in a Tesla 500 electron microscope.
Results The epitheloid cells of the RL 19 line grew in cord-like patterns and by confluence in closely adherent pavement-like monolayer (fig. 1 a). The cell surface is covered with short thick microvilli varying from eell to cell especially at the close contact zones to neighbouring cells.
Fig. 1. a) Sub confluent monolayer of RL 19 cells in living state, NOMARSKI interference contrast. x 200. b) Mycoplasma-infected RL 19 cells stained with bisbenzimidazol, fluorescence mieroscopy. x 200. Besides DNA-stained cell nuclei mycoplasmas appear as brightly fluorescent particles localized exclusively at the cell periphery. c) Thin section through mycoplasmas (M) and microvilli of RL 19 cells in monolayer culture. x 1,GOO. The darkly contrasted vesicles are mycoplasmas. 1) pleuropneumonia-like organism
208
Fig. 2. SEM micrographs of the surface of RL 19 cells persistently infected with A. laidlawii. a) Single epitheloid cell from a confluent monolayer with individual rod-shaped A. laidlawii at the cell ma,rgin (arrows). x 2,000. b) Highly magnified area of the cell surface with individual mycoplasmas. x 10,000. c) and d) Cluster of mycoplasmas attached to epitheloid cells a.long the cell-cell contact wne. c) 15,000, d) 10,000. e) Numerous A. laidl((wii of variable morphology at the cell margin. x 5,200.
209
After the 52nd subculture on the surface of the cells mycoplasmas were demonstrated with TEM and SEM as well as by DNA staining (fig. 1 b, c, 2) and were serologically classified as A. laidlawii. This spontaneous contamination has developed a 4 years persisting infection. 30 min after cell seeding single rod-shaped mycoplasmas lie horizontally upon the smooth lamellipodium of spread cells. The filamentous A. laidlawii are swollen at their ends and show a pronounced tendency to form branching structures (fig. 2b). When epitheloid cells take up contact with neighbouring cells the mycoplasmas are aligned mostly parallel to the cell margin and are lying between the microvilli and intercellular gaps of the adjoining cell membranes. In confluent and superconfluent cultures the filamentous, branched A. laidlawii are predominantly arranged in clusters along the cell borders (fig. 2c, d). The clusters were associated with the surface of epitheloid cells, though, as analysed in thin sections, the mycoplasmas were always separated from the host cell membrane by a minute gaps. Several mycoplasmas, especially in clusters are erected vertically and from the ends spherical bodies tied off (fig. 2c, d). The pleomorphism of A. laidlawii ranges from long filamentous rods to short coccoid chains. In quiescent cultures (without subculturing) the liver cells form a continuous network of collagen fibers upon the monolayer. The mycoplasmas then lie between the collagen fibers. In electron microscopic sections the sizes of microvilli and mycoplasmas are about equal, but the mycoplasmas have a high intracellular density that distinguishes them from the cytoplasma of the host cells. A. laidlawii evidenced no morphologically visible cytopathogenic effect on the host cells. The intimate association between adhering A. laidlawii and the host cells provides no situation in which the host cell membrane was attached or locally damaged. The cell adhesion on the substrate, spreading, growth pattern and cell surface shape were not altered as compared to tylosine treated uncontamined cultures of the same cell line. The scanning electron micrographs indicate that the host cell surfaces are neither alterated nor damaged by attachment of mycoplasmas. This suggests that no toxic metabolites are excreted by the mycoplasmas.
Discussion BeRides others mycoplasmas A. laidlawii is considered to be the most common mycoplasma strain found in contaminated cell cultures of various species. In this study it has been shown that the interaction of A. laidlawii with a defined type of host cells can lead to persistent infection. The possibility to spread the mycoplasmas from subculture to subculture is very manifold despite of preventive measures (MCGARRITY 1976). Reports about the effects of non-pathogenic mycoplasmas on host cell morphology in culture are scarce (BROWN et al. 1974). ZUCKER-FRANKLIN et al. (1966) reported on development of surface irregularities on host cells and BOATMAN et al. (1976) found a 26 % increase in host cell size. Despite of observation for many years we found no morphological alterations on the surface of persistently infected cells under different culture conditions. It is remarkable that the A. laidlawii did not cover the whole cell surface, but the cell-cell contact zone only.
Acknowledgements The mycoplasmas were determined by Dr. H. PFUTZNER, Institute of Animal Bacteriological Infectious Diseases of the Academy of Agricultural Sciences J ena. We thank Mrs. I.-M. HERRMANN, Department of Electron Microscopy, Friedrich Schiller University Jena, Medical School, for her expert technical assistance and Mr. H. MULLER, GDR Academy of Sciences, Optics and Spectroscopy, Jena, for the operation of the SEM (JSM 35, JEOL).
Literature ALDRIDGE, K. E., B. S. COLE and J. R. WARD, Mycoplasma-dependent activation of normallymphocytes: induction of a lymphocyte-mediated cytotoxicity for allogeneic and syngeneic mouse target cells. Infect. Immun. 18, 377-392 (1977).
210
BOATMAN, K, F. CARTWRIGHT and G. KENNY, Morphology, morphometry and electron microscopy of HeLa cells infected with bovine mycoplasma. Cell Tiss. Res. 170, 1-16 (1976). BROOKS, C. G., R C. REES and R H. LEACH, High nonspecific reactivity of normal lymphocytes against mycoplasma infected target cells in cytotoxicity assays. Eur. J. Immunol. 9, 159-171 (1979). BROWN, S., M. TEPLITZ and J.-P. REVEL, Interaction of mycoplasmas with cell cultures as visualized by electron microscopy. Proe. Nat. Acad. Sci. DSA 71, 464-468 (1974). CHEN, T. R, In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell. Res. 104, 255-262 (1977). KARSTEN, D., G. NEUPERT and R TUUST, Characteristics of an established epithelial cell line (RL 19) from rat liver. Exp. Path. 12, 88-99 (1976). MCGARRITY, G. J., Spread and control of mycoplasmal infection of cell cultures. In vitro 12, 643-648 (1976). ZUCKER-FRANKLIN, D., M. DAVIDSON and 1. THOMAS, The interaction of mycoplasmas with mammalian cells. 1. HeLa ccUs, neutrophils, and eosinophils. J. expo Med. 124, 521-531 (1966).
211