Vol. 25, No. 1

INFECTION AND IMMUNITY, July 1979, p. 446-454 0019-9567/79/07-0446/09$02.00/0

Interaction of Mycoplasma pneumoniae with Human Lung Fibroblasts: Characterization of the In Vitro Model MICHAEL G. GABRIDGE,'* DAVID TAYLOR-ROBINSON,2 HEATHER A. DAVIES,: AND ROBERT R. DOURMASHKIN3 School of Basic Medical Sciences, University of Illinois, Urbana, Illinois 61801,' and Division of Communicable Diseases2 and Electron Microscopy Section,3 Clinical Research Centre, Harrow, Middlesex HAl 3UJ, England

Received for publication 1 February 1979

The interaction of pathogenic Mycoplasma pneumoniae and host cells was studied in cell cultures of MRC-5 human lung fibroblasts. A comparison of results obtained with fibroblasts in a monolayer format and with hamster tracheal explant cultures indicated that the former can bind significantly larger numbers of mycoplasmas. In addition, the attachment was 96% specific, that is, mediated through a neuraminidase-sensitive receptor on the host cell. Uptake of mycoplasmas was directly related to the number of mycoplasma cells present in the inoculum, and attachment was virtually complete within a 30-min period at 370C. High doses of M. pneumoniae induced a marked cytopathic effect, whereas doses of -106 colony-forming units per ml produced grossly observable cell damage that was moderate and variable. Transmission electron microscopy studies indicated that attachment of M. pneumoniae to the surface of lung fibroblasts occurred with the specialized terminal structure or binding site oriented closest to the epithelial cell surface. The filamentous mycoplasma cells were spatially arranged in several configurations and were not limited to a vertical orientation. The advantages and disadvantages of human lung fibroblast monolayer cultures, in reference to other in vitro models, are discussed. A new mycoplasma agar medium (G-200 agar) with a defined tissue culture base and 10% horse serum is also described.

Various animal and in vitro models are available for the study of Mycoplasma pneumoniae infections. Hamster tracheal organ cultures have been quite popular and useful in such studies since the cilia provide a readily observable index of cell function (6, 13). In addition, various biochemical assays of cytopathology are available (8, 14, 15). Unfortunately, some difficulties can be encountered in the use of tracheal organ culture because of the complexity of cell types, mucus production, the small mass of tissue per explant, and a relatively high degree of nonspecific attachment. Monolayer cell cultures (5, 12), and particularly fibroblasts (2, 3, 22), are valuable models for mycoplasma host interactions. We have conducted an analysis of human lung fibroblast cell cultures as a potential in vitro model for M. pneumoniae infection. Our results indicate that such cells are sensitive to infection and rapidly adsorb mycoplasmas through a neuraminidase-sensitive mechanism. This cell culture model may prove to be especially useful in studies of the attachment process which constitutes the initial event in mycoplasma infections. 446

MATERIALS AND METHODS Mycoplasma. The source of virulent M. pneumoniae (strain PI 1428) and its cultivation in G-199 mycoplasma medium have been described (11, 16, 17). The medium used in this study was modified by the addition of 0.01 M HEPES (N-hydroxyethyl piperazine-N'-2-ethanesulfonic acid) and will be designated G199H. Organisms were used after they had been passaged 10 to 20 times in liquid medium subsequent to the original isolation. For labeling studies, mycoplasmas were cultivated for two passages in complete G-199 medium which contained 1 ,uCi of '4C-amino acids (protein hydrolysate, Amersham; 59 mCi/matom carbon) per mrl, followed by a final passage for 48 h at 5 ,tCi/ml. Mycoplasma organisms in the form of sheets adherent to the bottom surface of plastic tissue culture flasks were washed 4x with sterile phosphate-buffered saline (pH 7.4) before they were detached with glass beads (11). Fibroblast cultures. Basic characteristics of the MRC-5 human diploid fibroblast cell line have been described (19, 20). The cells, originally obtained from the lung tissue of a 14-week fetus, were maintained in BME which contained 10% fetal calf serum and 100 U of penicillin G per ml. Fibroblasts were cultivated on 13-mm glass cover slips in petri dishes and were incubated at 37°C in 5% CO2 in air.

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Quantitation. Cover slips with monolayers were incubated for 30 min at 370C with radioactive mycoplasmas and were then rinsed 3X in separate phosphate-buffered saline baths, with blotting at each step. Cell monolayers were dissolved by placing each cover slip into a scintillation vial which contained 0.3 ml of NCS tissue solubilizer (Amersham/Searle) and incubating overnight at 370C. Scintillation cocktail was then added, and the counts per minute per cover slip was determined with standard counting techniques. Explant cultures. Hamster tracheal explants were prepared and maintained in minimal essential medium with 10% fetal calf serum as described previously (7, 13). Groups of three explants were treated in the same manner as the cover slip cultures, i.e., they were incubated in 0.3 ml of G199H medium with mycoplasmas in disposable well plates for 30 min at 370C. Solubilization and counting techniques were likewise identical to those described for cell monolayers. Improved mycoplasma agar. To facilitate the counting of mycoplasma colonies (colony-forming units [CFU]), an improved mycoplasma solid medium (designated G-200 agar) was developed. The usual brain heart infusion base was replaced with defined tissue culture medium, and the horse serum content was reduced from the standard 20% to 10%. G-200 agar medium is clear, has little batch-to-batch variability, and promotes the rapid growth of mycoplasma colonies.

The base consisted of 60 ml of deionized water containing 1.3% Noble agar (Difco Laboratories, Detroit, Mich.) supplemented with a mixture of the following: medium 199 (10x), 4 ml; CMRL medium (10x), 4 ml; horse serum, 10 ml; fresh yeast extract, 10 ml; glutamine (1% aqueous), 0.5 ml; HEPES (1 M, aqueous), 1.0 ml; and LAG supplement solution, 12 ml. The LAG supplement solution contained 5 g of lactalbumin hydrolysate (tissue culture grade), 2.5 g of bovine serum albumin (fraction V), and 12.5 g of glucose per 250 ml of deionized water. The LAG solution was filter sterilized. All of the sterile supplementary components of the G-200 agar were added aseptically to the 60 ml of molten 1.3% agar. Plates (inoculated with 0.1 ml of mycoplasmas diluted in Difco PPLO broth) were incubated in sealed containers with air at 37°C. To assist in the visualization and enumeration of colonies, some plates that had been incubated for 7 to 10 days were overlaid with tetrazolium chloride or sheep erythrocytes. The former consisted of: 15 ml of molten 1% Noble agar, 7.5 ml of the G-200 complete supplement mixture, and 2.5 ml of 1% aqueous triphenyltetrazolium chloride. Two milliliters was gently poured over the colonies on G-200 agar in a 60-mm petri dish. Plates were given an additional 48 to 96 h of aerobic incubation before the bright red colonies were counted. The erythrocyte overlay contained 15 ml of molten 1% Noble agar, 10 ml of the G-200 complete supplement mixture, and 5 ml of washed sheep erythrocytes (50%o in Alsever solution). The overlay was used at 1.5 ml per plate. After 24 to 36 h of aerobic incubation at 37°C, hemolytic plaques surrounding each colony could be easily discerned and enumerated. Neuraminidase. Protease-free neuraminidase, ob-

447

tained from Behringwerke (West Germany), was diluted to 100 U/ml in calcium acetate buffer, pH 6.2 (1). Host cells were incubated in the neuraminidase solution for 60 min at 37°C prior to infection. Microscopy. For light microscopy, washed cell monolayers were fixed in formal-acetate-saline and stained with a standard Giemsa solution. For transmission electron microscopy, specimens were washed with phosphate-buffered saline and were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer which contained 1% tannic acid and 5% sucrose. After 2 h at room temperature, samples were washed 3x in buffer and then stored at 4°C. Cover slips and explants were then postfixed in 1% buffered osmium tetroxide at 4°C. The cell sheets were scraped from the cover slips, then embedded in 1.2% LGT agarose (Miles Laboratories Ltd., England) (18). All of the samples were dehydrated through alcohol and embedded in Spurr resin. Ultrathin sections were stained with uranyl acetate and lead citrate before being examined in a Philips EM 300 electron microscope. were

RESULTS

M. pneumoniae attachment. To assess the number of mycoplasma organisms that attached to MRC-5 lung fibroblasts, cover slip cultures were infected with 14C-labeled M. pneumoniae. After static incubation at 37°C for 30 min, the cover slips were rinsed and then assayed for uptake of radioactivity. Hamster tracheal explant cultures were processed similarly for comparison. The data (Table 1) show that the monolayer fibroblast cultures could adsorb significant numbers of mycoplasmas. Each cover slip accommodated more than 1Ox as many organisms (as expressed by uptake of radioactivity) as did groups of three explants. The vast majority of the counts associated with the fibroblasts represented specific attachment, i.e., they were receptor site mediated, in contrast to nonspecific trapping or label reutilization. This is shown by the results of the neuraminidase pretreatment of the cells. One half of each collection of specimens was preincubated for 60 min in 100 U of TABLE 1. Attachment of "4C-labeled M. pneumoniae to MRC-5 lung fibroblasts and to hamster tracheal explants with and without neuraminidase pretreatment Attachment

Target cells cells Target

(cpm)Y -Neur.

+Neur.

Neuraminidase

(% ~~~~~effect decrease)

96 306 8,217 Lung fibroblasts 59 325 788 Tracheal explants a Lung fibroblasts, cpm per 13-mm cover slip culture; tracheal explants, cpm per three explants. Mean data of five replicates from two experiments, with an inoculum of ca. 107 CFU/ml.

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neuramiriidase per ml; the other half was held

in calcium n acetate saline as a control. Neuraminidase pre treatment (Table 1) reduced the mycoplasma attachment to tracheal explants by only 59/I, so that approximately 40% of the attachmemnt was not mediated by the sialic acidcontainir ig receptor site. In contrast, nonspecific attachmemnt was reduced to 4% in lung fibroblast cultures. The ty,pical mycoplasma cell suspension used in these studies contained approximately 107 CFU/ml, equivalent to 1.7 x 105 cpm/ml. With the CFU,/cpm ratio of 59:1, the tracheal explants (assayed in groups of three) could bind approximately 800 cpm, or 5 x 104 mycoplasmas, whereas 4each cover slip of fibroblasts could bind 8,000 cprn, or 5 x 105 CFU of M. pneumoniae. Kinetiics of mycoplasma attachment. M. pneumonliae was taken up rapidly by the fibroblasts ev,en under static incubation conditions. Data fro:m a typical experiment are shown in Fig. 1. Liarge numbers of radioactive mycoplasmas weree associated with the cell monolayers within tiie first 10 min of incubation. The majority of 1the mycoplasmas were adsorbed within 20 min at 370C, with a slight increase noted over the next 40-min period. Cover slip cultures pretreated wvith neuraminidase took up significantly fewer orgranisms, and peak attachment occurred about thie same time as in untreated cultures. Little or no additional attachment to the neuraminidas;e-treated fibroblasts took place with continue(d incubation. Effect s of mycoplasma attachment and infectionr. Normal, uninfected fibroblasts displayed thie typical elongated, spindle-shape morphology associated with fibroblastic cells (Fig. 2). Cells reached confluency after 2 to 4 days 2,00

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when they were plated at approximately 105 cells/ml. After this time, multiplication and metabolism diminished significantly. When fibroblasts in monolayer format (nearly confluent) were incubated in the presence of M. pneumoniae (approximately 107 CFU/ml), cytotoxicity was detected (Fig. 2). After 48 h, monolayers that were infected with 106 to 107 CFU/ml had developed patches of lysed or detached cells. There was nearly complete destruction of monolayers when they were infected with 108 CFU/ ml. Effect of mycoplasma inoculum size. In an attempt to demonstrate the relationship between the number of infecting mycoplasmas and the ultimate uptake by fibroblasts, a series of cover slip cultures was established, and groups of three were infected with various dilutions of 14C-labeled M. pneumoniae. The representative data shown in Fig. 3 illustrate the direct relationship that was observed. The amount of radioactive label uptake was directly related to the quantity of mycoplasmas used for infection. When the numbers of cpm and CFU attached per cover slip were determined after a 30-min incubation period, approximately 7% of the mycoplasmas were taken up from the suspension. This was consistent at all high concentrations of mycoplasmas, up to and including 3 x 108 CFU/ ml (the highest concentration tested). These kinds of data from several experiments, along with the actual numbers (Coulter Counter) of the fibroblasts per cover slip, were used to determine the numbers of receptor sites per cell. It was found that at least 2,000 individual mycoplasmas attached to each fibroblast (maximum of approximately 2 X 107 CFU/104 fibroblasts per cover slip). Electron microscopy. Transmission electron microscopy was used to determine the spatial relationship between the mycoplasmas and the fibroblast cells, with special attention being devoted to the role of the M. pneumoniae binding site or attachment tip. The normal, uninfected fibroblast presented a smooth surface, with few projections and irregularities (Fig. 4A), in marked contrast to the ciliated cells of tracheal explant cultures. The latter (Fig. 4B) contained large numbers of cilia, interspersed with long, filamentous microvilli. There were also goblet cells and nonciliated secretary epithelial cells covered with short, thick microvilli. Observation of tracheal explants infected with M. pneumoniae showed that the organisms were

oriented with the binding site or attachment tip (i.e., the slightly constricted end of the filament in which there was an electron-dense core surrounded by an electron-transparent space) prox-

VOL. 25, 1979

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imal to the host cell (Fig. 5). In almost every coplasmas attached by way of the constricted instance, the mycoplasma filaments were ori- binding site (Fig. 6). Unlike attachment to cilented vertically and were positioned nearly up- iated explants, however, the infecting mycoplasright between the cilia and microvilli. This oc- mas had a variety of spatial orientations and curred even when the mycoplasmas were at- were rarely situated in a vertical, upright positached to the nonciliated cells within the tra- tion. They were normally lying at an acute angle cheal surface layer (Fig. 5B and C). of 450 or less, or were even parallel to the Infected human lung fibroblasts also had my- fibroblast cell surface, with the mycoplasma

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binding site being juxtaposed to the outer cell surface. Mycoplasmas were not found within fibroblasts. DISCUSSION M. pneumoniae, one of the causative agents of human atypical pneumonia, has been studied in embryonated eggs, cotton rats, hamsters, monkeys, and dogs. However, most of the recent studies on mechanisms of pathogenesis have utilized in vitro models. Cells in such systems are isolated from the complicating effects of animal age, physiological status, the endocrine system, and the normal microbial flora. In addition, environmental factors such as temperature, humidity, and nutrient supply can be carefully controlled and easily monitored in culture. Tracheal explant cultures from humans and hamsters have been particularly valuable in studies of M. pneumoniae since cytopathology can be conveniently gauged by using ciliostasis as an index of impaired cell function and metabolism (6). In addition, there are several biochemical/biophysical assays which can be used to objectively quantitate cell damage to tracheal epithelium (e.g., respiration [8], adenosine 5'triphosphate content [15], and dehydrogenase activity [14]). Unfortunately, tracheal explants have certain drawbacks for some studies, due to the complexity of cell types, the differential ciliation along the epithelial surface (9), a relatively small mass of tissue, a high degree of nonspecific mycoplasma attachment, and the mucociliary clearance mechanism. Many of these difficulties can be circumvented, however, through the use of

monolayer cell cultures. M. pneumoniae has been studied in several cell systems, ranging from monolayers of ciliated tracheal cells (12) to HeLa cells (27) and lung fibroblasts (22, 25). The latter model offers distinct advantages over the others: the cells originate from the natural organ target site for the infection, and large quantities of homogeneous, genetically characterized cells can be obtained for biochemical studies. Unfortunately, previous reports on M. pneumoniae infection of lung fibroblasts were limited primarily to establishing the numbers of mycoplasmas that could grow in or on cells (22, 25). We found it necessary to characterize accurately the infected MRC-5 diploid fibroblast cultures to evaluate their potential as a reliable model for the host-parasite interactive process. Our data indicate that large numbers of M. pneumoniae can attach to the surface of lung fibroblasts (Table 1). The number of mycoplasmas that attached to a 13-mm cover slip with a nearly confluent monolayer of cells was severalfold higher than that noted with hamster tracheal explants. This may have been due to an increase in available surface area. One cover slip has approximately fivefold more surface area than the three tracheal explants normally used in such studies. The absence of mucociliary clearance with the fibroblasts also probably contributed to the increased attachment to monolayers. Mycoplasma attachment that was specific or neuraminidase sensitive (and hence presumably mediated through the sialic acid-containing receptor site) was much greater in the fibroblast model. Specific attachment constituted 96% (Table 1) to 83% (Fig. 1) of the total mycoplasma uptake with fibroblasts, in contrast to the 50% normally seen with the explants. This is apparently related to the fact that the cut surfaces on a tracheal explant (generated by transversely slicing a thin ring of tissue from the tracheal cylinder) have large cavernous spaces in the subepithelial region. These spaces can physically trap mycoplasmas and even larger particles such as erythrocytes. These artificially formed surfaces, and even the outer surface of the trachea, can "bind" mycoplasmas nonspecifically and thereby introduce error in pathogen attachment studies. This artifact is eliminated with cover slip monolayers. Though there is potential for nonspecific attachment in that M. pneumoniae could attach to the glass surface itself, our results (Table 1) with neuraminidase show that the combination of confluency and short incubation periods minimizes this problem. The uptake of mycoplasmas by fibroblasts is faster than that noted with explants. Tracheal

MYCOPLASMA INFECTION OF LUNG FIBROBLASTS

VOL. 25, 1979

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