Respiration PILvsiology (1980) 41, 381-390 %3 Elsevier/North-Holland Biomedical Press
EFFECTS OF O X Y G E N O N P U L M O N A R Y M A C R O P H A G E S AND ALVEOLAR EPITHELIAL TYPE I1 CELLS IN CULTURE
J.E. S T U R R O C K , J . F . N U N N
and A . J . J O N E S
Division ofAnaesthesia, Clinical Research Centre, Warlord Road, Harrow, Middlesex HA l 3UJ, England
Abstract. Cultures of Chinese hamster lung fibroblasts and explants of 20-day-old rat lungs were exposed to 950~i oxygen with 51~, CO~ in vitro. The Chinese hamster cells had stopped dividing after 17 hours exposure and cell death occurred at a mean time of 67 hours (s.d. 15 hours). The rat lung cxplants showed macrophages moving over a monolayer of alveolar type II epithelial cells. Both cell types appeared to function normally for 24 hours but cell division in the type II cells was about 50'~;, of control between 12 and 24 hours of exposure and virtually ceased after 36 hours. Cell death commenced after 4 days and was complete in 9 days. Macrophages divided freely in the control cultures but only one division was seen during exposure to oxygen and that occurred during the first 24 hours. Motility was reduced by 50'!;i during the second day of exposure and stopped during the 3rd day. No live macrophages were seen after 4 days exposure. These culture systems appear very suitable for screening drugs for their protective effect against oxygen toxicity. Alveolar epithelium Cells in culture Hamster
Oxygen toxicity Pulmonary macrophages Rat
High oxygen concentrations may be required by patients who already have severe lung disease and it is then difficult to distinguish which pathological changes are due to oxygen toxicity and which to the original disease, Furthermore, the experimental study of pulmonary oxygen toxicity in healthy human volunteers is not practicable and a study of patients with irreversible brain damage has yielded equivocal results (Barber et al., 1970). Animal studies are beset by problems of species differences but Kistler et al. (1967) have demonstrated damage to the endothelial and alveolar epithelial Type I cells in the rat exposed to oxygen for 72 hours. Most strains of rats will not survive in oxygen for more than 72 hours, but monkeys (Macacus mulatta) may survive as long as 13 days in oxygen. However, in this species it was also found that type I alveolar cells were destroyed and partially replaced by a proliferation of type II cells (Kaplan e t al., 1969 ; Kapanci e t al., 1969). Accepted[or publication 18 March 1980 381
The difficulties of human and animal experimentation suggested it might be useful to examine the effects of hyperoxia on pulmonary tissue cultures. Not only might such a system be convenient for preliminary testing of protective substances, but new techniques, considered below, now permit the selective growth of different cell types including capillary endothelium, type II alveolar epithelium and pulmonary macrophages. We report studies of the effect of 95% oxygen on cultured explants of weanling rat lungs in a technique similar to that used by Nunn, Sharp and Kimball (1970) and also by Richters, Elliott and Sherwin (1978). This simple technique provides fibroblasts and type II alveolar epithelial cells in monolayer, while macrophages and some other cell types may also be present. We have compared the results with the effects of hyperoxia on Chinese hamster lung fibroblasts (V 79). This cell line, isolated by Ford and Yerganian, (1958) has the advantages of remarkable uniformity of appearance, doubling time and other parameters, whereas freshly isolated cells eventually lose the property of division at constant intervals, unless they become transformed. Nevertheless, after more than 20 years in culture, it is unlikely that this cell line still shows any special characteristics which are peculiar to pulmonary fibroblasts. Methods
CELLCULTURES Chinese hamster lung fibroblasts (V 79) were grown in monolayer in plastic culture bottles with optically flat sides, using Eagle's minimum essential medium with added antibiotics, glutamine and 10% fetal calf serum. This medium contains glucose at 1 g/1 (5.6 mmol/1) and sodium bicarbonate buffer, thus requiring exposure to 5% carbon dioxide in air. Lung explants were prepared by removing the lungs of 20-day-old weanling rats (Sprague Dawley strain) and chopping the lungs into fragments less than 1 mm~. These fragments were placed in the medium described above in the same type of culture bottle and incubated for 7 days, by which time growth extended as a monolayer for a distance of several millimetres around most of the tissue fragments.
PHOTOGRAPHY Cell culture bottles were mounted on the stage of an inverted Wild microscope with phase-contrast illumination and facilities for timelapse cinemicrography (16 mm). The microscope was enclosed in a box maintained at 37 °C (+ 1.0 °C). At first the assembly was exposed to ambient light during daylight hours but, since this appeared to inhibit the growth of the lung explants, we later enclosed the assembly in black paper. Transmitted light for the photography was confined to the period of the exposure (less than 1% of total time). One frame was exposed every two minutes.
EFFECTS OF OXYGEN ON PULMONARY CELLS IN CULTURE
EXPOSURE TO OXYGEN A continuous stream of gas (approximately 5 ml/h) was passed through a humidifier (also at 37°C) into the culture bottle and the effluent was ducted outside the hot-box assembly. As exposures continued for several days, there was danger of loss of water from the culture if the gas was not fully humidified at the temperature of the culture, or gain of water if the humidifier temperature should exceed that of the culture bottle. Temperatures of stage and humidifier were carefully monitored and the osmolality of the culture was measured at the beginning and end of the experiment. It was not practicable to hold the osmolality absolutely constant from the initial value of 274 m osmol/kg, but the changes (317 for control, 306 and 327 for O2-exposed) were insufficient to affect the viability of the culture (Pirt and Thackeray, 1964). Control studies were undertaken with pre-mixed cylinders of 5~o CO2 in air. Oxygen studies used 95% oxygen with 5% CO2 (B.O.C. medical gas). Films were analysed with a Spectro analysing projector and selected prints were made from the negative film. Cell motility was determined by measuring the movement of the centre of the nucleus every 20 min for periods of 5 hours.
Results CHINESE HAMSTER LUNG FIBROBLASTS These cells have indefinite capacity to survive in culture when exposed to air in light or in darkness. Inspection of 10 cultures exposed to 95% oxygen with 5~o carbon dioxide showed cell death within about 3 days. One culture was filmed and, during preliminary control exposure to 5~/ carbon dioxide in air, showed normal divisions and motility. After 14 hours a field containing 4 cells was selected and exposure to 9~"/oxygen was commenced. The four cells ceased normal movement and, one by ~/O one, rounded up as though for division although no division took place. The four cells finally underwent physical disruption and evident death at 47, 65, 77 and 80 hours after commencement of exposure to oxygen.
RAT LUNG EXPLANTS Outgrowths from the explant formed a monolayer of fusiform, flattened and intermediate cells, over which alveolar macrophages moved. The fusiform cells appeared to be typical fibroblasts and we concentrated our attention on the flattened cells (fig. la). It was not possible to make a positive identification of the cell type by the appearance under phase-contrast illumination, but a typical sample was examined by electronmicroscopy and all cells showed the lamellar bodies characteristic of
J.E. S T U R R O C K eta/.
Fig. 1. Still photographs from cine film No. 2 during exposure of lung explant to 95'~f, oxygen. (Magnification x250). (a) Commencement of exposure. Note dark macrophages (indicated by arrows} against background of alveolar type 11 epithelial cells, which are characterised by sharp nuclear membrane and dark nucleoli. (b) After 48 hours of exposure. Notc macrophages clustered round a dead cell
E F F E C T S O F O X Y G E N ON P U L M O N A R Y CELLS IN C U L T U R E
(indicated by arrow) after exhibiting chemoiaxis. (c)After 96 hours. There arc no macrophages and the type II cells have lost their normal appearance arid begun to show retraction. (d) After 216 hours. Widespread cell death and retraction, leaving bare areas with no cell cover.
J.E. S T U R R O C K et al.
40 20 0
5%C02 in air
2 3 4 Days of exposure
Fig. 2. Motility of macrophages (mean of 5, bars indicate +__1 SD).
alveolar type II epithelial cells. The macrophages were identified from the film on the grounds of their pattern of movement, which included chemotaxis towards and phagocytosis of dead cells. Two control preparations remained viable for at least 9 days during which filming was continued. Morphology and patterns of cell movement were unchanged. The second film was somewhat unusual in that it showed large numbers of macrophages and the rate of random movement of these cells was unchanged for at least 6 days (fig. 2). In this film there were several instances of chemotaxis and phagocytosis by the macrophages among which there was a total of 23 cell divisions uniformly spread throughout the 9 days. F o u r films were made during exposure to 95% oxygen. Each was preceded by 24 hours filming during exposure to 5~o CO2 in air as a control sequence. Results were similar and we refer below to two preparations kept in the dark between exposures. Film 1. Morphology, division and motility of type II cells were normal for the first 30 hours of exposure to 95% oxygen. Thereafter there were no further divisions (fig. 3) and the morphology of the type II cells began to change after 2 days exposure. The cells commenced to retract and lose their normal outline, leaving spaces with no cell cover. On the 6th day of exposure to 95~o oxygen the process of retraction accelerated and cell death was widespread. Few macrophages were seen in this film and no divisions of macrophages were observed. Their motility declined during the second day of exposure, ceasing completely on the third day. Most macrophages died during the 3rd and 4th days of exposure and none was seen after the 4th day. Film 2. Morphology and motility of both type II cells and macrophages were normal for the first day of exposure to 95~o oxygen. However, divisions of type II cells were already reduced by the end of the 1st day and only one further division was seen thereafter (fig. 3). The appearance of the type II cells was scarcely
EFFECTS OF OXYGEN ON PULMONARY
C E L L S IN C U L T U R E
Hours of exposure to oxygen
Fig. 3. D i v i s i o n s of a l v e o l a r type lI epithelial cells expressed as a p e r c e n t a g e of c o n t r o l (in the same field) as a function of d u r a t i o n of e x p o s u r e to 95% oxygen + 5% C O 2 .
changed after 48 hours exposure (fig. lb) but there was evident retraction after 96 hours exposure (fig. lc). The effect of gross retraction and cell death after 216 hours exposure is shown in fig. ld. Macrophage motility declined sharply during the 2nd day of exposure and ceased at the end of the 3rd day (fig. 2). However, an incident of chemotaxis and phagocytosis was observed after 48 hours of exposure (fig. l b). One single macrophage division was seen after 20 hours exposure to oxygen. All macrophages were dead by the 4th day (fig. lc). A parallel control culture grown in contact with 5% CO2 in air (but not filmed) grew normally throughout this period.
The cellular nature of pulmonary oxygen toxicity has been demonstrated by Kirstler et al. (1967), Kaplan et al. (1969) and Kapanci et al. (1969), who showed the sensitivity of endothelial and alveolar type I cells contrasting with the relative resistance of alveolar type II cells. There have, however, been few attempts to demonstrate these changes in vitro although this would have considerable advantages in the screening of the very large number of substances which are claimed to confer protection from the effects of high concentrations of oxygen. There are two reports of the effects of high oxygen concentrations on pulmonary organ cultures. Resnick, Brown and Vernier (1975) studied the effect of 48 hours exposure to high concentrations of oxygen on cultured whole fetal murine lungs. They reported inhibition of bronchial and bronchiolar development but did not comment on changes at the alveolar level. Guerrero, Rounds and Booher (1977) maintained thin slices of adult rat lung in modified Rose chambers for periods up to 4 weeks and observed that 95% oxygen/5% carbon dioxide was toxic although
J.E. STURROCK et
no details were given. Such techniques of organ culture present the major difficulty that there is a gradient of Po~ from the surface of the organ to its centre. It is not feasible to calculate the Po~ at any particular point in the organ culture and therefore histological changes cannot be directly related to Po~. It was for this reason that we chose to use a monolayer of cells bathed by medium which was in diffusion equilibrium with a selected gas mixture. The use of a cell model such as the Chinese hamster fibroblast has the advantages of a uniform culture of cells whose properties have been well characterised during the many years in which it has been grown. However, the cells have had ample opportunity for transformation and their behaviour cannot be directly related to that of any particular cell in living tissue. On the other hand, primary cells growing from an explant are recognizably similar to the parent cells in the tissue from which they have grown, with little opportunity for transformation. Nevertheless, there remains the difficult problem of identification of the cell types in a mixed culture. The pulmonary explant technique described has been widely used (Nunn, Sharp and Kimball, 1970; Richters, Elliott and Sherwin, 1978; Tyrell, Mika-Johnson and Chapple, 1979). From the central explant there spreads a monolayer of cells which may be classified into spindle shaped fibroblast-like cells, flattened polygonal cells and intermediate types, We studied exclusively areas of flattened polygonal cells which electronmicroscopy showed to contain lamellar bodies and which we consider to be alveolar epithelial type 11 cells. Two further cell types may be seen moving over the monolayer. Lymphocytes are visible during the first few days and may be identified by their characteristic pattern of movement on time-lapse cinemicrography (Nunn, Sharp and Kimball, 1970). However, they are no longer present 7 days after explanting and so were not seen in the present studies. The second cell type which moves over the monolayer is the macrophage, which will survive for at least 16 days. Divisions may be observed but we have never seen transformation into a fibroblast as reported by Davis (1967). The system we have described is particularly suitable for study of alveolar epithelial type II cells and macrophages. The technique is simple and highly repeatable. When exposed to 5!,'~,CO~ in air, our preparation was clearly viable tor at least 16 days during which morphology, motility and division of fibroblasts, type Ii epithelial cells and macrophages continued normally. Exposure to 9~":~,,,, oxygen causes rapid cessation of cell division and motility, with death of the macrophages at about the 4th day of exposure while the type II ceils survive to the 9th day. The chinese hamster cells are more sensitive to oxygen and we have already reported that exposure to 950.,i oxygen causes extensive chromosome damage in these cells within 24 hours of exposure and that colony forming ability is effectively zero after 48 hours exposure (Sturrock and Nunn, 1978). In recent years there has been much progress in the isolation of pure cell lines from the lung by means of enzymic dissociation and differential centrifugation. It is possible to obtain relatively pure clones of alveolar epithelial type II cells
EFFECTS OF O X Y G E N ON P U L M O N A R Y CELLS IN C U L T U R E
(Kikkawa and Yoneda, 1974; Tompa and Langenbach, 1979). Type 1 cells are of greater interest in relation to oxygen toxicity but are end cells and do not divide in culture. Guerrero, Rounds and Booher (1977) have described a technique for maintaining alveolar structure in organ culture and report the presence of type I cells in their organ cultures but, at present, there appears to be no method for cloning a monolayer of type I cells in vitro. Explants are more easily grown from young animals. However, neonatal rats are markedly resistant to oxygen toxicity, when compared with adult animals (Stevens and Autor, 1977)and show significant increases in superoxide dismutase activity (Yam et al., 1978). Travis et al. (1972) reported that respiratory failure did not develop in 6-h-old Wistar rats exposed to 100~i oxygen for 9 days, although there was some oedema and atelectasis on histological examination. Our explants were taken from 20-day-old rats which are less sensitive to oxygen than adult rats (Yam et al., 1978). Our cultures may therefore minimise the potential danger of oxygen. The effect of oxygen on the alveolar macrophage has received comparatively little attention. Our results suggest that these cells are very sensitive to oxygen although they may be replaced from the intra-vascular pool of macrophages, since the arterial Po, would not be significantly raised in a patient receiving oxygen for severe respiratory failure. We have shown that, although the macrophages no longer divide, chemotaxis and phagocytosis occur normally for as long as 2 days of exposure to oxygen. It has been shown that pulmonary macrophages are the only pulmonary cells to respond to hyperoxia by increased superoxide dismutase activity (Stevens and Autor, 1977). There is a considerable number of substances which have been claimed to mitigate the effects of oxygen toxicity (Smith, 1980). Clinical trials in man are not feasible and determination of efficacy in animals is extremely difficult. Preliminary screening by studying the effect on cell cultures seems to be a practical possibility. We are currently engaged in an assessment of the efficacy of steroids in preventing chromosome breaks in Chinese hamster cells exposed to 950,o oxygen. The lung explant method could be used for assessment of the effect on macrophages and alveolar type II epithelial cells. Boat et al. (1973) have described a cell model for studying the effect of oxygen on respiratory ciliary activity.
Acknowledgements We are indebted to Dr. R. Dourmashkin and Mr. D.B. Gunner for undertaking the electronmicroscopy on the type II epithelial cells.
J.E. S T U R R O C K et al.
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