Vol. 21, No. 1

INFECTION AND IMMUNITY, July 1978, p. 333-336 0019-9567/78/0021-0333$02.00 Copyright © 1978 American Society for Microbiology

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Ciliated Respiratory Epithelial Monolayers: New Model for Mycoplasma pneumoniae Infection MICHAEL G. GABRIDGE,* HOLLY GUNDERSON, SUSAN L. SCHAEFFER, AND Y. DEE BARDEN-STAHL Department of Microbiology and School of Basic Medical Sciences, University of Illinois, Urbana, Illinois 61801 Received for publication 28 February 1978

Hamster respiratory epithelial cells were cultured in a monolayer format, and 20% of the cells were ciliated. Mycoplasma pneumoniae attached to the epithelial cells in a neuraminidase-specific fashion and induced ciliostasis and cytonecrosis.

Organ, or explant, cultures of mammalian ciliated respiratory epithelium serve as valuable in vitro models for the study of mycoplasmal pneumonia infections (2, 10). However, as with most explant systems, these multicellular complexes cannot be used to obtain detailed information on the individual cell associations involved in a host-parasite interaction. This type of high resolution data is best provided by monolayer cell cultures. To date, monolayer cultures of differentiated respiratory cells (7, 11, 13) have not been widely used because of difficulties encountered in the preparation and maintenance of these fragile and normally nonreplicating cells. We have developed a combination enzyme-outgrowth technique to produce ciliated respiratory epithelial monolayers and have employed these cultures as target tissue for Mycoplasma pneumoniae. One to six hamster tracheal ring explants (8) were placed in vials containing 1 ml of 0.01% collagenase (Grand Island Biological Co. [GIBCO], Grand Island, N.Y.) in Waymouth's MAB 87/3 basal medium (GIBCO) and were gently rotated (1 rpm) for 90 min at 370C. Explants were then placed on cover slips in 34-mm petri dishes containing 0.8 ml of Waymouth's medium supplemented with 10% fetal bovine serum and 200 U of penicillin G (final pH, 7.4) per ml. Incubation was at 360C in 5% C02 (in air, water saturated). Epithelial outgrowth, in monolayer form, began on day 1 to 2 and progressed for up to 4 days or more (typical areas were then about one to two times the diameter of the explants). Clusters of three explants per cover slip formed monolayers averaging 3,244 cells after 4 days of growth, at which time the parent explant was typically removed. Based on a microscopic examination of over 18,000 individual cells, at least 18% of the cells in the monolayer contained intact, beating cilia. Scanning electron micros-

copy (Fig. 1) revealed that tufts of cilia, apparently representing the top surface from the former position of the pseudostratified cells in the trachea, were clearly discernible. Ciliary motion continued in the monolayer sheets for several days after the removal of the explants, and the latter could be recultured (without further enzyme treatment) to provide additional "crops" of cells for up to six additional passages, though with decreasing effectiveness. Each passage would yield fewer cells with a perceptably lower fraction of ciliated cells. Cilia in these new "crops" would continue to beat actively for 3 to 7 days. The uptake of tritiated mycoplasmas by monolayers was significant and was primarily neuraminidase sensitive (Table 1). This was analogous to results previously noted (9) with tracheal explant cultures, as was the diminution of M. pneumoniae attachment after brief pretreatment of the monolayer with glutaraldehyde or unlabeled M. pneumoniae cells (strain PI 1428, less than 15 passages since isolated from a patient, was used throughout this study). These binding experiments were corroborated with visual observations of M. pneumoniae cells attached to the epithelial monolayer. Monolayers which had been maintained for 3 days after infection were fixed and stained with the DNAspecific Hoechst 33258 compound (1). With UV illumination, uninfected monolayers had brightly fluorescent, yellow-green nuclei prominent against a clear black background. Infected cultures, in spite of extensive rinsing, showed both the bright nuclei and numerous fluorescent particles (short filaments) over the cell (Fig. 2). The fluorescent particles were the shape, size, and arrangement expected for M. pneumoniae cells. Recovery experiments (Table 2) indicated that monolayers could bind approximately 10% of the available inoculum. M. pneumoniae was 333

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FIG. 1. Scanning electron micrograph (original magnification, x 2,000) of monolayer developed from hamster tracheal epithelium. Note cilia and cell boundaries. TABLE 1. Effect of various pretreatments on the attachment of radioactive (3H amino acids) M. pneumoniae (9) to ciliated monolayers after 2h at36oCa Treatment

Saline (control) Neuraminidase (5

Mycoplasma attach% Control ment (cpm/cover

slip) 8,995 (+ 596) 2,131 (± 219)

100 24

U/mi) 77 M. pneumoniae 6,891 (± 592) (ca. 106 CFU/ ml)b 16 Glutaraldehyde 1,453 (± 295) (3% x 45 min) Infected with CFU for 2 h at 36WC, rinsed three times in phosphate-buffered saline, and blotted. Mean data (± standard error of the mean) from four replicate cover slip cultures, each with outgrowth resulting from a 4-day incubation period. b Unlabeled ("cold") organisms. a

recovered after treating rinsed cover slips with 0.8 ml of neuraminidase (5 U/ml) for 45 min at 370C. Cover slips were washed and agitated in saline with a Vortex mixer, and the neuraminidase and washes were pooled and assayed for

viable mycoplasmas with standard plating techniques (9). After 72 h, the mean cell associated mycoplasma titer was still in excess of 3 x 106 colony-forming units (CFU) per cover slip. When M. pneumoniae cells were added to Waymouth's medium in the absence of epithelial cells, titers rapidly fell approximately 100-fold in 72 h. The significantly improved mycoplasma survival in the monolayer cultures suggests that the epithelial cells are actually contributing to the mycoplasma multiplication and/or survival. The cytopathic effect which accompanied infection was pronounced. When monolayers had been developed for a 4-day period and then infected with M. pneumoniae, the cell sheets would appear normal for 24 h, but would then show significant decreases in the intactness of the sheet, the vigor of ciliary beating, and the absolute number of ciliated cells. This apparently is the first report of the infection of tracheal epithelial cells in cell culture with M. pneumoniae, though infection and/or association has been noted in various cell cultures (3, 6, 12, 14). Our proposed model has definite advantages for the study of this and other respiratory, host-parasite interactions be-

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FIG. 2. Micrograph (original magnification, x 1,000) of prominent nuclei and mycoplasmas in ciliated monolayer cultures stained with Hoechst 33258 stain and illuminated by incident UV light. Monolayer was infected 72 h previously with M. pneumoniae.

TABLE 2. Number of viable cells (CFU) ofM. pneumoniae recoverable from ciliated monolayer cultures Time (h Postn- No. of replicates Viable mycoplasmas fection)b 0 24 48 72

eliae

(CFU/culture) 5 1.3 x 107 9 1.3 x 107 11 8.5 x 10' 5 3.4 x 106 a Mean data from three separate experiments. Inoculum was ca. 1.5 x 10' CFU per ml; each cover slip contained the epithelial outgrowth from three hamster tracheal rings cultivated in Waymouth's MAB 87/3 medium for about 6 days. b Infected and recovered as described in footnote a of Table 1.

cause: (i) the target tissue is equivalent to that which is naturally infected; (ii) events occurring on the cellular level can be easily observed and quantitated; (iii) the artifactual "cut" surfaces (i.e., non-epithelial lined) in tracheal explants

are not present to physically entrap organisms in a nonspecific fashion; and (iv) the actively beating cilia provide a convenient and sensitive parameter with which to monitor epithelial cell activity. Given the fact that ciliated respiratory epithelial tissue is being used with increasing frequency in studies of pathogenic microbiology (2, 9, 10), cystic fibrosis detection (4), and environmental toxicology (5), this unique ciliated monolayer system should have widespread application. ACKNOWLEDGMENTS This project was supported by grant Al 12559 from the National Institutes of Health, a Cystic Fibrosis Foundation Research Grant, and a University of Illinois Biomedical Sciences Support Grant. Electron microscopy was done through the courtesy of R. MacLeod and the University of Illinois Center for Electron Microscopy. LITERATURE CITED 1. Chen, T. R. 1977. In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258

stain. Exp. Cell Res. 104:255-262.

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2. Cherry, J. D., and D. Taylor-Robinson. 1973. Mycoplasma pathogenicity studies in organ cultures. Ann. N.Y. Acad. Sci. 225:290-303. 3. Clyde, W. A. 1961. Demonstration of Eaton's agent in tissue culture. Proc. Soc. Exp. Biol. Med. 107:715-718. 4. Conover, J. H., et al. 1973. Studies on ciliary dyskinesia factor in cystic fibrosis. I. Bioassay and heterozygote detection in serum. Pediatr. Res. 7:220-223. 5. Donnelly, G. M., H. E. McKean, C. S. Heird, and J. Green. 1974. Ciliostasis as a bioassay. Arch. Environ. Health 28:350-355. 6. Fernald, G. W. 1972. In vitro response of human lymphocytes to Mycoplasma pneumoniae. Infect. Immun. 5:552-558. 7. Frazier, M. E., J. G. Hadley, T. K. Andrews, and H. Drucker. 1975. Use of thermolysin for the dissociation of lung tissue into cellular components. Lab. Invest. 33:231-238. 8. Gabridge, M. G. 1976. Hamster trachea organ cultures, p. 75-80. In V. J. Evans, V. P. Perry, and M. M. Vincent (ed.), Tissue culture association manual, vol. 1. Tissue Culture Association, Rockville, Md. 9. Gabridge, M. G., Y. D. Barden-Stahl, R. B. Polisky,

and J. A. Engelhardt. 1977. Differences in the attachment of Mycoplasma pneumoniae cells and membranes to tracheal epithelium. Infect. Immun. 16:766-772. 10. Gabridge, M. G., and Y. D. Barden-Stahl. 1978. Role of adenine in the pathogenesis of Mycoplasma pneumoniae infections of tracheal epithelium. Med. Microbiol. Immunol., in press. 11. Indo, K., and R. B. Wilson. 1976. Cell interaction between epithelial and mesenchymal components in primary sheets of fetal rat lung cells, and the effects of 3methylcholanthrene treatment. J. Natl. Cancer Inst.

57:1333-1340. 12. Larin, N. M., N. V. Saxby, and D. Buggey. 1969. Quantitative aspects of Mycoplasma pneumoniae-cell relationships in cultures of lung diploid fibroblasts. J. Hyg. 67:375-385. 13. Nevo, A. C., Z. Weisman, and J. Sade. 1975. Cell proliferation and cell differentiation in tissue cultures of adult muco-ciliary epithelia. Differentiation 3:79-90. 14. Powell, D. A., and W. A. Clyde. 1975. Opsonin-reversible resistance of Mycoplasma pneumoniae to in vitro phagocytosis by alveolar macrophages. Infect. Immun. 11:540-550.

Ciliated respiratory epithelial monolayers: new model for Mycoplasma pneumoniae infection.

Vol. 21, No. 1 INFECTION AND IMMUNITY, July 1978, p. 333-336 0019-9567/78/0021-0333$02.00 Copyright © 1978 American Society for Microbiology Printed...
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