Toxicology, 60 (1990) 41--52 Elsevier Scientific Publishers Ireland Ltd.
Physiologic and toxicologic responses of alveolar type II cells Jacob N. Finkelstein Department o f Pediatrics and Environmental Health Sciences Center, University o f Rochester School o f Medicine and Dentistry, Rochester, N Y (U.S.A.)
(Received September 7th, 1989; accepted September 12th, 1989)
Summary This report summarizes recent data regarding the direct responses of the type I1 alveolar epithelial cell to agents that are known to produce lung injury. These responses are not limited to cytotoxicity or cell death, but include alterations in the known differentiated functions of this cell type. Among the functions assessed and shown to be altered by toxic agents are: (l) synthesis and secretion of pulmonary surfactant; and (2) proliferation and renewal of the alveolar type I cell population. Agents such as ionizing radiation, CdCl2 and hyperoxia are shown to directly alter pulmonary surfactant phospholipid synthesis and secretion by type II cells in a manner consistent with their known effect at the whole animal level. Changes in protein synthesis are also observed. In addition, information is presented which suggests that pulmonary epithelial proliferation and repair is a complex process mediated, in part, by complex cell-cell interaction in the pulmonary parenchyma. In particular, the alveolar macrophage may play a significant role through its ability to synthesize and secrete potent growth factors that influence type II cell growth.
Key words: Type II cell; Radiation; Hyperoxia; Growth factors; Pulmonary surfactant; Secretion
My goal in this report is to briefly discuss the type II cell a n d its responses in lung i n j u r y . It has b e e n o u r w o r k i n g hypothesis that the type II cell plays a n i m p o r t a n t role in m o d u l a t i n g the response to b o t h acute a n d chronic l u n g i n j u r y a n d that a better u n d e r s t a n d i n g o f its f u n c t i o n s a n d o f its repertoire o f responses would enable us to: (1) develop a m e a n s o f accurately assessing lung i n j u r y ; (2) m o d i f y responses to reduce sensitivity o f the lung to toxic agents. Figure 1 shows a d i a g r a m a t i c r e p r e s e n t a t i o n o f a n alveolus, highlighting the 2 epithelial cells that m a k e u p the alveolar surface; the type I cell a n d the type II cell. The type I cell is a s q u a m o u s epithelial cell that acts to line the alveolar surface a n d provides a
Address all correspondence to: Jacob N. Finkelstein, Ph.D., Department of Pediatrics; Box 777,
University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642, U.S.A. 0300-483x/90/$03.50 © 1990Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
41
ALVEOLAR E P I T H E L I A L CELLS
Tyoe I c e l l s : -
squamous, form most of the l i n i n g of the gas d i f f u s i o n region
- endstage
TyDe I |
cell
ce11~;
-
cuboidal
-
synthesize and secrete s u r f a c t a n t
- low basal level of division - p r o g e n i t o r of
the Type
I cell
Fig. 1. The pulmonary alveolar region. Modified from [18].
barrier for gas diffusion. Typically the type I cells make up 9 0 - - 9 5 % of the surface area of the deep lung, while only counting for 4 - - 5 % of the cells [1,2]. In contrast, the type II cell or granular pneumocyte is a cuboidal epithelial cell. It makes up about 5°/o of the surface area, while accounting for 10--15% of the cells [1,2]. The fact that the type II cell represents such a low percentage of the total lung cell population makes detailed biochemical and cell biologic investigations of its functions difficult. Although metabolically quite active, it has been difficult to specifically assess its type II cell function without the development of a highly purified cell population. Studies on the isolation and purification of type II cells have been ongoing for the last 10--12 years; but it is really only in the last 5 or 6 years that clear details have emerged about type II cell properties and specific functions in the lung. Those are: (1) the synthesis and secretion of pulmonary surfactant; and (2) its ability to function as a stem cell, renewing the epithelium in the event of type I cell death. If these are its principle functions, then the relevant toxicologic question is how would these functions be influenced by known lung toxicants? In other words, in the cases of lung injury how would synthesis and secretion of surfactant or the ability of the type II cell to proliferate and renew the epithelium be influenced? What I will try to do is provide a brief overview of some data from my laboratory relevant to characterizing the properties of an in vitro type II cell model that we have been developing, and then try to place that data into the context of a number of known lung injury models to show how the type II cell and its functions could be affected. Over the past 10 years my laboratory has developed a method for isolation
42
and purification o f type II cells f r o m adult rabbit lung which makes use o f the instillation o f a defined mixture o f proteases and density gradient centrifugation to give a p o p u l a t i o n o f type II cells which is 90--95°7o pure [3--6]. Cells isolated by this technique are identified as type II cells based on the presence o f lamellar bodies, microvilli and a b u n d a n t endoplasmic reticulum as well as phospholipid composition [3]. In addition, we have used specific biochemical measures o f metabolic activity to demonstrate that the cell p o p u l a t i o n that we isolate is type II cell like and also maintains functions that one would expect type II cells to have in vivo. These include assays for enzymes o f phospholipid biosynthesis, as well as measurements o f choline and fatty acid i n c o r p o r a t i o n into various specific phospholipid species [4--6]. In order to p e r f o r m some o f the studies relative to surfactant secretion and growth, we have also developed methods for culturing type II cells in a m e d i u m consisting o f D M E / F 1 2 with 10o70 fetal bovine serum on a type I collagen matrix. We have observed, that over a 7-day period the cells begin to flatten and lose their lamellar bodies, and as it has been demonstrated by m a n y other laboratories, no longer maintain type II cell like properties. However, during the first 48 h in culture, we have shown that m a n y o f the properties o f the type II cell are maintained. One such property, which is shown in Fig. 2 is the ability o f the type II cell to respond to specific agonists by secreting previously labelled surfactant phospholipids. This figure shows the relative response o f rabbit type II ceils to the beta adrenergic agonist, terbutaline, a p h o r b a l ester analog, T P A , and a purinergic agonist, A T P . As you can see, the cells respond quite vigorously to the 3 agonists c o m p a r e d to the unstimulated basal control. The most potent agonist in our system has been A T P and that is consistent with data
4-
z 0
I---- 3Ld Cr C) LLI CO 2ILl > I,I [12
BASAL
TERB
TPA
ATP
(n = 9)
(n = 8)
(n = g)
(n = 7)
Fig. 2. Surfactant secretion by rabbit type II cells in vitro. Type II cells from adult rabbits are cultured for 18--20 h in DME/FI2 media containing 10e/0 FBS plus 2/aCi/ml [3H]cholinechloride. After extensive washing to remove unincorporated label cells are incubated for 1 h in media without serum followed by a second hour in media containing the specific agonist listed. Relative secretion is the ratio of the label released by the cells during the 1 h stimulation composed to that rdeased during the previous hour. Unstimulated controls (both hours) represent basal secretion. Concentrations of agonists used are: terbutaline 10/aM, TPA 100 ng/ml and ATP 10 ~M.
43
1400 1200 n rl v I.l.l X¢
1000
/A•O•...•O
800
NO
n
600. +
DRUG
400. 200. OI 0
I
I
I
I
5
10 TIME (aIN)
15
20
Fig. 3. Effect of secretory agonists on 22Na uptake. Freshly isolated type II cells suspended in Hank's balanced salt solution (107/ml) were preincubated for 10 min with 300/aM ouabain after which 2/aCi/ ml 2JNa+ was added. At the intervals noted aliquots of cells were removed and quenched with cold BSS with 200 ~M amiloride. Cells were then spun through an oil cushion to remove adherent radioactivity and counted. Correction for trapped media was made using [~4C]sucrose. Each point represents at least 4 replicates repeated 3 - - 4 times. O; No Drug; TPA; /x; Terbutaline.
seen by other laboratories and in other species. One of the questions that we began to ask is what is the nature of the signal that the type II cell transmits across its cell membrane to stimulate the process of secretion. After a detailed examination of some of the ionic influences [7], we determined that one of the earliest signals observed in the type II cell in response to a specific agonist, is an increase in sodium flux [8]. Data in Fig. 3 show an increase in net sodium uptake by type II cells immediately after the addition of either T P A or terbutaline. We then examined the mechanism of this increase in sodium flux and aspects of its control. One pathway by which cells regulate their intracellular sodium is through the activation of a Na ÷, H ÷ exchange. The activation of this ion channel necessitates that for sodium to enter the cell hydrogen ions must be pumped out. As a consequence, one would predict that there should be an intracellular p H shift in response to a secretory signal that would stimulate this kind of exchange. We TABLE I EFFECT OF S E C R E T A G O G U E S ON TYPE I1 CELL I N T R A C E L L U L A R pH Treatment
ApH
T P A (100 nM) Terbutaline (10/aM) Phorbol didecanoate (100 nM) DMSO (Control)
0.11 0.10 0.01 0.01
_ _+ _+ _+
0.02 0.02 0.01 0.01
Type 1I cells were incubated at 37°C in buffer (1 × 108/ml) containing 0.1 m M dimethyl oxazolidine2-4-dione (DMO) plus 1.0 /aCi/ml ['4C]DMD. After 5 min, cells were aliquoted and drug or solvent carrier added. After an additional 5 rain, cells were spun through an oil cushion to remove adherent label and counted. Analysis of pH used a modification of the Henderson-Hasselbach equation.
44
thus measured the change in intracellular p H , using both fluorescent probes and radiolabelled markers, in response to the addition of various secretory signals to the type II cell, and have shown (Table I) that as cells are stimulated to secrete, their intercellular p H increases. That is consistent with a loss of H ÷ ions in exchange for the entry of Na ÷. With such a response identified, we sought to examine some specific toxic agent and determine whether its affects on surfactant secretion may be mediated through changes in this sodium proton exchange. A large number of agents are known to alter surfactant; either its synthesis or secretion. We first chose to use ionizing radiation, since we have had some long standing interest in the effect of ionizing radiation on the lung and a well Characterized model was available. Some of our early observations of this model have shown, using electron microscopy, that type II cells lose their lamellar bodies immediately following a single dose of ionizing radiation [9]. As early as 1 h after radiation, the number of lamellar bodies per cell dropped dramatically, and that this decrease was maintained through about 4 weeks after irradiation. We first sought to develop a biochemical assay to corrolate this morphologic observation. Surfactant phospholipid content of the bronchoalveolar lavage was measured after single doses of irradiation. We determined that over time there was a dramatic increase in lavage surfactant content that persisted over the first 28 days, but occurred as early as 1 h after radiation, in agreement with our morphologic observations [10,11]. We then investigated whether this apparent increase in secretion was a direct type II cell effect, or indirectly mediated through the effects of radiation on another cell type. In order to answer this question, we used our type II cell model to examine the direct effects of radiation on the type II cell. Type II cells in culture responded to ionizing radiation in the manner identical to that seen in vivo [12]. That is, the radiation dose response curve for secretion in vitro was virtually superimposable on the dose response curve for in vivo surfactant release. When Na ÷ uptake was measured in type II cells under these same conditions, a dramatic increase was observed with the identical doseresponse relationship as seen for secretion. This suggests that the acute effect of ionizing radiation on these cells is somehow mediated by the activation of the same ion channel as is seen with physiologic stimuli. In addition to the studies on secretion, we have also looked at the biosynthetic response of the type II cell to a single dose of radiation. We have found that cells isolated f r o m in vivo treated animals exhibit changes in phospholipid synthesis rates as measured by precursor incorporation. More importantly, the nature of the phospholipid synthesized by type II cells reflects a decrease in the amount of disaturated phosphatidylcholine and phosphatidylglycerol synthesis. This suggests that these cells, although still capable of phospholipid synthesis, are no longer making the specific phospholipid components of surfactant, but mainly making phospholipids for maintenance of cell membrane. In addition to the phospholipid effects, it is now known that surfactant has specific protein components. In recent studies we examined whether any of the proteins that are part of surfactant could be useful in detecting damage caused by ionizing radiation. Using a panel of monoclonal antibodies against surfactant components we determined that one of those antibodies detected a surfactant
45
associated protein that we have been able to detect in circulation after a single dose of radiation [13]. In fact, the release of this protein into plasma follows a strict dose response curve and correlates very well with the onset of radiation pneumonitis [13]. We are hopeful that this might be a very useful indicator of lung injury in other circumstances and are currently pursuing this actively. Recently, we have also looked at the effects on surfactant secretion of other agents known to induce lung injury. We found that when 10/aM cadmium chloride, which has been shown to alter surfactant levels in vivo, is added to cultures of type II cells, secretion, either basal or stimulated by adrenergic agonists or TPA, is inhibited by 50070. Addition of 1 /aM cadmium caused less of an effect. In fact, there is some indication that a slight stimulation of basal secretion occurred. We are continuing to do studies on both the inhibition and the possible enhancement of the secretory response and trying to characterize the mechanism of the effect of cadmium. We have also examined the consequences of in vivo hyperoxia on the in vitro response of type II cells to secretion. We have observed that after a 64 h exposure to 100070 oxygen, followed by recovery in room air, that there appears to be a decrease in secretory responsiveness of the type II cells in the immediate postexposure period (Fig. 4). This seems to be consistant with the in vivo data [14] which showed that during a similar time period, the amount of lavageable surfactant present in the lung decreased. One of the problems that I eluded to earlier about type II cells in vitro is that they lose their differentiated properties over time in culture. For example, it has been very difficult to measure synthesis of any of the surfactant apoproteins in type II cells after isolation, although abundant synthesis occurs in vivo. We attempted to characterize protein synthesis after cell isolation, to look at some of
z
oeo L,.J
50
25
•
III
O_
50
-1
I
I
I
I
I
I
I
0
1
2
,5
4
5
6
DAYS RECOVERYAFTER 0 2 EXPOSURE Fig. 4. Effect of in vivo hyperoxia on in vitro secretory response to a /J-adrenergic agonist. Type II cells were isolated from rabbits exposed to hyperoxia for 64 h followed by removal to room air for the time indicated. After overnight culture in media containing /~Ci/ml choline, choline secretory response to 10/aM terbutaline was measured. Results represent at least 3 replicates repeated in 3 separate preparations of cells.
46
the abnormal states that might be generated after toxic insult in vivo or perhaps in vitro. These studies showed that during the 7-day period in culture there are drastic changes in protein synthesis patterns and one can see loss a n d / o r gain of particular proteins and appearance of new ones using 2D gel electrophoresis after [35Slmethionine o r [3H]leucine labelling. Interestingly, we recently showed [15], that during the first 2--18 h after isolation, the type II cells are characterized, by what can best be described, as a stress response. Our results suggest a true heatshock type response in type II cells immediately after isolation. Protein synthesis is drastically inhibited for the first 10--14 h after isolation except for a select number o f proteins (i.e. 10 or 12) [15]. Characterization of the proteins by 2D gel electrophoresis was consistent with known heat shock proteins, particularly the 70 kD and the 90 kD heat shock protein families [15]. We have examined the time course of their appearance after isolation and have compared this directly to the effect of heat shock in type II cells and showed that there are many similarities (see detailed discussion in [15]). By 24 h, protein synthesis appeared to be normal and we thus thought it would be possible to look at the effects of in vitro stresses on protein synthesis in type II cells. Because of our interest in hyperoxic effects both those generated by oxygen as well as the oxidant radicals that are produced by ionizing radiation, we thought to look in vitro at effects of oxidants, or those things that deplete antioxidants, on protein synthesis. We have compared in vitro the effects of hydrogen peroxide, 95% O2, and glutathione depletion by diethylmaleate. In each of these cases we saw protein synthesis responses very similar to heat shock (i.e. induction of 70 and 90 kD proteins). Although, in order to see a stress response with in vitro hyperoxia it was necessary to deplete glutathione with pretreatment of diethylmaleate. In addition to the standard heat shock response, we also saw induction o f a new protein at 32 kD. Preliminary data suggest this may be related to changes in antioxidant enzymes. This was observed (Figs. 5a and 5b) regardless of whether the response was directly related to hyperoxia like hydrogen peroxide, or heat shock or glutathione depletion. We are continuing to explore this effect further. But the results suggest that in response to many pulmonary toxicants, the type II cell does alter its repertoire of protein synthesis which may aid in developing some degree of resistance or tolerance to those agents. As I discussed earlier, a second fundamental property of the type II cell is the ability to proliferate and replace the alveolar epithelium under the circumstances where lung injury has caused the loss of type I cells and the denudation of the basement membrane. We have recently begun to assess whether our in vitro type II cell culture model would allow us to study proliferation in vitro. We have shown [16] that type II cells, even after isolation and the fact that they are beginning to lose their differentiated properties, are capable of at least 1 or 2 rounds of cell division in culture. We have confirmed this proliferation both by looking at the cultures over time and showing that the number of cells increases, and also by autoradiography and measurement of [3H]thymidine incorporation. In unstimulated type II cell cultures, we found a peak in cell proliferation between 24 and 48 h post-isolation. We then determined if we could cause this proliferation to be stimulated beyond that basal rate that we observed just through the
47
A 2 0 0 -"
10069-
46-
30-
14-
A
48
B
C
D
E
F
B 200--
1006g--
6--
30--
14-A
B
C
D
E
Fig. 5. Effects of in vitro stress on proteins synthesized by type 1I cells. Twenty-four hour cultures of type II cells were treated as described, pulsed with [3H]leucine for 1 h and solubilized. Proteins were separated by SDS-polyacrylamide gel electrophoresis on 10%0 gels and subjected to fluorography. An equal number of counts was applied to each lane. (A) A: Control; B: 100 laM diethylmaleate (DEM), 24 h; C: 950/0 O:, 24 h; D: 95% 02 and 100 taM DEM, 24 h; E: 95% 02 48 h; F: 45°C, 30 rain, 2 h recovery. (B) A" Control; B: 0.4 mM H202 4 h recovery; C: 0.4 mM H20: 8 h recovery; D: 0.4 mM H202 16 h recovery; E: 0.4 mM HzO2 24 h recovery.
49
,. z D
200
.J.,~ 0::0
8o F--Z Z--
"'
50
w" w n
EGF
PDCF
TGF# IGF-I TNFa
IL-I A M C M
Fig. 6. Response of cultured type II cells to purified growth factors. Twenty-four hour type II cell cultures were treated with the growth factor indicated for a subsequent 24-h period in the presence of [3H]thymidine. After extensive washing counts incorporated into DNA were measured and reported as percent change compared to basal proliferation. EGF 100 ng/ml; PDGF 2 half-max units; TGF/3 5 ng/ml IGF-1 100 ng/ml TNFa 1600 units/ml; IL-1 20 units/ml. AMCM, Activated macrophage conditioned medium 1:2 dilution
presence of serum in the cultures. Our first studies examined the role of specific growth factors in the regulation of type II cell proliferation and in Fig. 6 we show that type II cells respond quite vigorously to the addition of epidermal growth factor with a 30--50% increase in [3H]thymidine incorporation. In addition, we also looked at a number of other purified growth factors to determine what affect they might have on a type II cell. In general, these growth factors which include interleukin I, platelet derived growth factor, tumor necrosis factor, and insulin-like growth factor I, had no real affect on the type II cell as there was no change in thymidine incorporation or in cell number. In addition to these purified growth factors, we found that conditioned media from particle stimulated macrophages also contains some mitogen for type II cells. It was known from previous in vivo hyperoxia studies [14] that 2--3 days after exposure to 64 h of 100% oxygen, these appeared to be a peak of type II cell proliferation and replacement of type I cells. What we wondered was whether or not this effect was controlled by some particular growth factor and whether this might be related to the dramatic influx of inflammatory cells that occurs in the hyperoxic animals at the same time. We took free cells recovered by bronchoalveolar lavage from animals sacrificed at varying times after in vivo hyperoxia, placed them in culture for 24 h, took this conditioned media and added it to type II cell cultures. We found [16,17] an approximate peak in production of a proliferative activity between 2 and 3 days after exposure. This coincided quite closely with our observed in vivo effects. In order to further characterize this growth factor, we utilized the particle stimulated macrophage model and began to examine in more detail the effects of the particle, in this case, opsonized zymosan, on the production of the type II cell growth factor. We have characterized this further and found optimal release of the type II cell growth factor at 1 mg/ml zymosan. We
50
TABLE II THE RELATIVE ABILITY OF VARIOUS PARTICLES TO STIMULATE MACROPHAGEDERIVED TYPE II CELL GROWTH FACTOR PRODUCTION AND RELEASE Panicle
Opsonized"
Unopsonizeda
Zymosan (1.0 mg/ml) TiO2 (anatase) (0.6 mg/ml) Amorphous silica (0.1 mg/ml) Carbon (0.4 mg/ml) Polyvinyl chloride beads (PVC) (0.6 mg/ ml)
198.9 170.2 166.4 157.4 160.7
150.7 152.5 130.6 129.2 137.2
_ 6.1 _ 4.3 _ 2.2 ___ 4.8 _ 3.5
± 4.2 ± 1.9 _+ 2.6 ± 5.1 _+ 1.6
aData is expressed as percent of control [3H]thymidine incorporation. Macrophages (107/ml) were cultured in the presence of the particle shown with or without prior opsonization of the particles with serum. After 24 h the media was removed, filtered to remove particles and debris and tested for its ability to stimulate [3H]thymidine uptake by cultured type II cells as described in Fig. 6.
have also shown that release o f the factor continues for at least 72 h after initiation o f the m a c r o p h a g e cultures with peak p r o d u c t i o n between 24 and 48 h. The growth factor exhibited sensitivity to heat and trypsin suggesting that it is a protein. Preliminary gel filtration and ultrafiltration experiments indicate its molecular weight to be between 25 and 32 kD. We have also f o u n d that this protein will bind weakly to heparin Sepharose eluting at 0.4 M sodium chloride, a concentration m u c h lower than that observed acidic or basic fibroblast growth factor. This indicates that the heparin Sepharose binding was m o r e related to an ion exchange type binding rather than a specific interaction with heparin. We are continuing to characterize and purify the g r o w t h factor still further, with a view to produce antibodies to it and begin to better characterize its interaction with the type II cell and factors that control its p r o d u c t i o n . In addition, z y m o s a n is not the only particle that caused the p r o d u c t i o n o f this growth factor. As s h o w n in Table II, other particles, including titanium dioxide could also stimulate m a c r o p h a g e release o f this growth factor. This could be very interesting in light o f the effects o f high concentrations o f nuisance dusts on the generation o f fibrosis and other lung injuries. W h a t these results suggest is that one needs to look again at a model o f the cell interactions in lung injury and discuss not only direct effects o f lung texicants on the type II cell, but the effects o f those agents on cells such as the alveolar m a c r o p h a g e . Then we can consider the interactions between a stimulated m a c r o p h a g e and an injured II cell or perhaps a lung fibroblast. This clearly points out that in this tissue, just like in m a n y others, the response to any particular toxic insult is a consequence o f multicellular interactions and cellular c o m m u n i c a t i o n . In s u m m a r y , I would like to conclude by saying that our studies have indicated that by looking at specific biochemical properties o f the type II cell, we can begin to get a m o r e detailed picture o f h o w the lung could respond to toxic agents and that an in vitro model o f type II cell cultures could be very useful in beginning to understand the m e c h a n i s m by which agents affect lung and cause lung injury. In addition, investigations o f m o d u l a t i o n o f lung injury could begin
51
t o b e a p p r o a c h e d u s i n g a t y p e II cell m o d e l as l o n g as o n e t a k e s c a r e f u l c o n s i d e r ation o f the possible cellular i n t e r a c t i o n s t h a t c o u l d exist in the lung.
Acknowledgements The author gratefully acknowledges the contributions of current and former students, Richard Gallo, Mary Brandes, Susan Robey-Bond and Carol Stapanowich. Excellent technical assistance was provided by Christina Kramer. Also a c k n o w l e d g e d are the c o l l a b o r a t i o n s o f D o n a l d S h a p i r o , Philip R u b i n and D a v i d P e n n e y . T h e w o r k r e p o r t e d h a s b e e n s u p p o r t e d in p a r t b y U S P H S G r a n t s CA27791, HL32476, HL37388, HL36543 and NIEHS Training Grant ES07026.
References 1 2 3 4 5 6 7 8 9 10 11
12
13
14 15 16 17 18
52
R. Breeze and M. Terk, Cellular structure, function and organization in the lower respiratory tract. Environ. Health Perspect., 55 (1984) 3. P. Gehr and J.D. Crapo, Morphometric analysis of the gas exchange region of the lung, in D.E. Gardner, J.D. Crapo and E.J. Massaro (Eds.), Raven Press, NY 1988, p. 1. J.N. Finkelstein and R.D. Mavis, Biochemical evidence for internal proteolytic damage during isolation of type I1 alveolar epithelial cells. Lung, 156 (1979) 243. J.N. Finkelstein and D.L. Shapiro, Isolation of type It alveolar epithelial cells using low protease. Lung, 160 (1982) 85. J.N. Finkelstein, W.M. Maniscalco and D.L. Shapiro, Properties of freshly isolated type II alveolar epithelial cells. Biochim. Biophys. Acta, 762 (1983) 398. W.M Maniscalco, J.N. Finkelstein and A.B. Parkhurst, De novo fatty acid synthesis by freshly isolated alveolar type II epithelial cells. Biochim. Biophys. Acta, 751 (1983)462. R.L. Gallo, J.N. Finkelstein and R.H. Notter, Characterization of the membrane potential of alveolar type II cells by the use of ionic probes. Biochim. Biophys. Acta, 771 (1984) 217. R.L. Gallo, J.N. Finkelstein, R.H. Notter and D.L. Shapiro, Alterations in the membrane potentials accompanying exposure to secretagogues. Fed. Proc., 43 (1984) 1846. D. Penney, D. Siemann, P. Rubin, D. Shapiro, J. Finkelstein and R. Cooper, Early and late effects of irradiation of the mouse lung: A review. Scan. Microsc., 1 (1982) 413. P. Rubin, D.L. Shapiro, J.N. Finkelstein and D.P. Penney, The early release of surfactant following lung irradiation of alveolar type 11 cells. Int. J. Radiat. Oncol. Biol. Phys., 6 (1980) 75. P. Rubin, J.N. Finkelstein, D.W. Siemann, D.L. Shapiro, P. Van Houtte and D.P. Penney, Predictive biochemical assays for late radiation effects. Int. J. Radiat. Oncol. Biol. Phys., 12 (1986) 469. D.L. Shapiro, J.N. Finkelstein, D.P. Penney, D. Siemann and P. Rubin, Radiation induced secretion of surfactant from cell cultures of type 11 pneumocytes: An in vitro model of in vivo radiation toxicity. Int. J. Rad. Oncol. Biol. Phys., 10 (1984) 375. P. Rubin, S. McDonald, P. Maasilta, J.N. Finkelstein, D.L. Shapiro, D. Penney and P. Gregory, Serum markers for prediction of pulmonary radiation syndromes. Part I. Surfactant Apoprotein. Int. J. Radiat. Oncol. Biol. Phys., 1989. In press. B.A. Holm, J.N. Finkelstein, S. Matalon and R.H. Notter, Changes in Type II pneumocytes during ARDS injury and recovery from severe hyperoxia. J. Appl. Physiol., 1988. In press. M.E. Brandes and J.N. Finkelstein, Induction of the stress response by isolation of rabbit type II pneumocytes. Exp. Lung Res., 1988. In press. M.E. Brandes and J.N. Finkelstein, Production of growth modulators for type 1I epithelial ceils by activated alveolar macrophages. FASEB J., 2 (1988) A1010. M.E. Brandes and J.N. Finkelstein, Responses of the type II pneumocyte and alveolar macrophage to hyperoxia in vivo and in vitro. Am. Rev. Res. Dis., 137 (1988) 79. A.W. Ham, Histology, 7th edn., J.B. Lippincott Co., Philadelphia, PA, 1974, p. 731.
Physiologic and toxicologic responses of alveolar type II cells Jacob N. Finkelstein Department o f Pediatrics and Environmental Health Sciences Center, University o f Rochester School o f Medicine and Dentistry, Rochester, N Y (U.S.A.)
(Received September 7th, 1989; accepted September 12th, 1989)
Summary This report summarizes recent data regarding the direct responses of the type I1 alveolar epithelial cell to agents that are known to produce lung injury. These responses are not limited to cytotoxicity or cell death, but include alterations in the known differentiated functions of this cell type. Among the functions assessed and shown to be altered by toxic agents are: (l) synthesis and secretion of pulmonary surfactant; and (2) proliferation and renewal of the alveolar type I cell population. Agents such as ionizing radiation, CdCl2 and hyperoxia are shown to directly alter pulmonary surfactant phospholipid synthesis and secretion by type II cells in a manner consistent with their known effect at the whole animal level. Changes in protein synthesis are also observed. In addition, information is presented which suggests that pulmonary epithelial proliferation and repair is a complex process mediated, in part, by complex cell-cell interaction in the pulmonary parenchyma. In particular, the alveolar macrophage may play a significant role through its ability to synthesize and secrete potent growth factors that influence type II cell growth.
Key words: Type II cell; Radiation; Hyperoxia; Growth factors; Pulmonary surfactant; Secretion
My goal in this report is to briefly discuss the type II cell a n d its responses in lung i n j u r y . It has b e e n o u r w o r k i n g hypothesis that the type II cell plays a n i m p o r t a n t role in m o d u l a t i n g the response to b o t h acute a n d chronic l u n g i n j u r y a n d that a better u n d e r s t a n d i n g o f its f u n c t i o n s a n d o f its repertoire o f responses would enable us to: (1) develop a m e a n s o f accurately assessing lung i n j u r y ; (2) m o d i f y responses to reduce sensitivity o f the lung to toxic agents. Figure 1 shows a d i a g r a m a t i c r e p r e s e n t a t i o n o f a n alveolus, highlighting the 2 epithelial cells that m a k e u p the alveolar surface; the type I cell a n d the type II cell. The type I cell is a s q u a m o u s epithelial cell that acts to line the alveolar surface a n d provides a
Address all correspondence to: Jacob N. Finkelstein, Ph.D., Department of Pediatrics; Box 777,
University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642, U.S.A. 0300-483x/90/$03.50 © 1990Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
41
ALVEOLAR E P I T H E L I A L CELLS
Tyoe I c e l l s : -
squamous, form most of the l i n i n g of the gas d i f f u s i o n region
- endstage
TyDe I |
cell
ce11~;
-
cuboidal
-
synthesize and secrete s u r f a c t a n t
- low basal level of division - p r o g e n i t o r of
the Type
I cell
Fig. 1. The pulmonary alveolar region. Modified from [18].
barrier for gas diffusion. Typically the type I cells make up 9 0 - - 9 5 % of the surface area of the deep lung, while only counting for 4 - - 5 % of the cells [1,2]. In contrast, the type II cell or granular pneumocyte is a cuboidal epithelial cell. It makes up about 5°/o of the surface area, while accounting for 10--15% of the cells [1,2]. The fact that the type II cell represents such a low percentage of the total lung cell population makes detailed biochemical and cell biologic investigations of its functions difficult. Although metabolically quite active, it has been difficult to specifically assess its type II cell function without the development of a highly purified cell population. Studies on the isolation and purification of type II cells have been ongoing for the last 10--12 years; but it is really only in the last 5 or 6 years that clear details have emerged about type II cell properties and specific functions in the lung. Those are: (1) the synthesis and secretion of pulmonary surfactant; and (2) its ability to function as a stem cell, renewing the epithelium in the event of type I cell death. If these are its principle functions, then the relevant toxicologic question is how would these functions be influenced by known lung toxicants? In other words, in the cases of lung injury how would synthesis and secretion of surfactant or the ability of the type II cell to proliferate and renew the epithelium be influenced? What I will try to do is provide a brief overview of some data from my laboratory relevant to characterizing the properties of an in vitro type II cell model that we have been developing, and then try to place that data into the context of a number of known lung injury models to show how the type II cell and its functions could be affected. Over the past 10 years my laboratory has developed a method for isolation
42
and purification o f type II cells f r o m adult rabbit lung which makes use o f the instillation o f a defined mixture o f proteases and density gradient centrifugation to give a p o p u l a t i o n o f type II cells which is 90--95°7o pure [3--6]. Cells isolated by this technique are identified as type II cells based on the presence o f lamellar bodies, microvilli and a b u n d a n t endoplasmic reticulum as well as phospholipid composition [3]. In addition, we have used specific biochemical measures o f metabolic activity to demonstrate that the cell p o p u l a t i o n that we isolate is type II cell like and also maintains functions that one would expect type II cells to have in vivo. These include assays for enzymes o f phospholipid biosynthesis, as well as measurements o f choline and fatty acid i n c o r p o r a t i o n into various specific phospholipid species [4--6]. In order to p e r f o r m some o f the studies relative to surfactant secretion and growth, we have also developed methods for culturing type II cells in a m e d i u m consisting o f D M E / F 1 2 with 10o70 fetal bovine serum on a type I collagen matrix. We have observed, that over a 7-day period the cells begin to flatten and lose their lamellar bodies, and as it has been demonstrated by m a n y other laboratories, no longer maintain type II cell like properties. However, during the first 48 h in culture, we have shown that m a n y o f the properties o f the type II cell are maintained. One such property, which is shown in Fig. 2 is the ability o f the type II cell to respond to specific agonists by secreting previously labelled surfactant phospholipids. This figure shows the relative response o f rabbit type II ceils to the beta adrenergic agonist, terbutaline, a p h o r b a l ester analog, T P A , and a purinergic agonist, A T P . As you can see, the cells respond quite vigorously to the 3 agonists c o m p a r e d to the unstimulated basal control. The most potent agonist in our system has been A T P and that is consistent with data
4-
z 0
I---- 3Ld Cr C) LLI CO 2ILl > I,I [12
BASAL
TERB
TPA
ATP
(n = 9)
(n = 8)
(n = g)
(n = 7)
Fig. 2. Surfactant secretion by rabbit type II cells in vitro. Type II cells from adult rabbits are cultured for 18--20 h in DME/FI2 media containing 10e/0 FBS plus 2/aCi/ml [3H]cholinechloride. After extensive washing to remove unincorporated label cells are incubated for 1 h in media without serum followed by a second hour in media containing the specific agonist listed. Relative secretion is the ratio of the label released by the cells during the 1 h stimulation composed to that rdeased during the previous hour. Unstimulated controls (both hours) represent basal secretion. Concentrations of agonists used are: terbutaline 10/aM, TPA 100 ng/ml and ATP 10 ~M.
43
1400 1200 n rl v I.l.l X¢
1000
/A•O•...•O
800
NO
n
600. +
DRUG
400. 200. OI 0
I
I
I
I
5
10 TIME (aIN)
15
20
Fig. 3. Effect of secretory agonists on 22Na uptake. Freshly isolated type II cells suspended in Hank's balanced salt solution (107/ml) were preincubated for 10 min with 300/aM ouabain after which 2/aCi/ ml 2JNa+ was added. At the intervals noted aliquots of cells were removed and quenched with cold BSS with 200 ~M amiloride. Cells were then spun through an oil cushion to remove adherent radioactivity and counted. Correction for trapped media was made using [~4C]sucrose. Each point represents at least 4 replicates repeated 3 - - 4 times. O; No Drug; TPA; /x; Terbutaline.
seen by other laboratories and in other species. One of the questions that we began to ask is what is the nature of the signal that the type II cell transmits across its cell membrane to stimulate the process of secretion. After a detailed examination of some of the ionic influences [7], we determined that one of the earliest signals observed in the type II cell in response to a specific agonist, is an increase in sodium flux [8]. Data in Fig. 3 show an increase in net sodium uptake by type II cells immediately after the addition of either T P A or terbutaline. We then examined the mechanism of this increase in sodium flux and aspects of its control. One pathway by which cells regulate their intracellular sodium is through the activation of a Na ÷, H ÷ exchange. The activation of this ion channel necessitates that for sodium to enter the cell hydrogen ions must be pumped out. As a consequence, one would predict that there should be an intracellular p H shift in response to a secretory signal that would stimulate this kind of exchange. We TABLE I EFFECT OF S E C R E T A G O G U E S ON TYPE I1 CELL I N T R A C E L L U L A R pH Treatment
ApH
T P A (100 nM) Terbutaline (10/aM) Phorbol didecanoate (100 nM) DMSO (Control)
0.11 0.10 0.01 0.01
_ _+ _+ _+
0.02 0.02 0.01 0.01
Type 1I cells were incubated at 37°C in buffer (1 × 108/ml) containing 0.1 m M dimethyl oxazolidine2-4-dione (DMO) plus 1.0 /aCi/ml ['4C]DMD. After 5 min, cells were aliquoted and drug or solvent carrier added. After an additional 5 rain, cells were spun through an oil cushion to remove adherent label and counted. Analysis of pH used a modification of the Henderson-Hasselbach equation.
44
thus measured the change in intracellular p H , using both fluorescent probes and radiolabelled markers, in response to the addition of various secretory signals to the type II cell, and have shown (Table I) that as cells are stimulated to secrete, their intercellular p H increases. That is consistent with a loss of H ÷ ions in exchange for the entry of Na ÷. With such a response identified, we sought to examine some specific toxic agent and determine whether its affects on surfactant secretion may be mediated through changes in this sodium proton exchange. A large number of agents are known to alter surfactant; either its synthesis or secretion. We first chose to use ionizing radiation, since we have had some long standing interest in the effect of ionizing radiation on the lung and a well Characterized model was available. Some of our early observations of this model have shown, using electron microscopy, that type II cells lose their lamellar bodies immediately following a single dose of ionizing radiation [9]. As early as 1 h after radiation, the number of lamellar bodies per cell dropped dramatically, and that this decrease was maintained through about 4 weeks after irradiation. We first sought to develop a biochemical assay to corrolate this morphologic observation. Surfactant phospholipid content of the bronchoalveolar lavage was measured after single doses of irradiation. We determined that over time there was a dramatic increase in lavage surfactant content that persisted over the first 28 days, but occurred as early as 1 h after radiation, in agreement with our morphologic observations [10,11]. We then investigated whether this apparent increase in secretion was a direct type II cell effect, or indirectly mediated through the effects of radiation on another cell type. In order to answer this question, we used our type II cell model to examine the direct effects of radiation on the type II cell. Type II cells in culture responded to ionizing radiation in the manner identical to that seen in vivo [12]. That is, the radiation dose response curve for secretion in vitro was virtually superimposable on the dose response curve for in vivo surfactant release. When Na ÷ uptake was measured in type II cells under these same conditions, a dramatic increase was observed with the identical doseresponse relationship as seen for secretion. This suggests that the acute effect of ionizing radiation on these cells is somehow mediated by the activation of the same ion channel as is seen with physiologic stimuli. In addition to the studies on secretion, we have also looked at the biosynthetic response of the type II cell to a single dose of radiation. We have found that cells isolated f r o m in vivo treated animals exhibit changes in phospholipid synthesis rates as measured by precursor incorporation. More importantly, the nature of the phospholipid synthesized by type II cells reflects a decrease in the amount of disaturated phosphatidylcholine and phosphatidylglycerol synthesis. This suggests that these cells, although still capable of phospholipid synthesis, are no longer making the specific phospholipid components of surfactant, but mainly making phospholipids for maintenance of cell membrane. In addition to the phospholipid effects, it is now known that surfactant has specific protein components. In recent studies we examined whether any of the proteins that are part of surfactant could be useful in detecting damage caused by ionizing radiation. Using a panel of monoclonal antibodies against surfactant components we determined that one of those antibodies detected a surfactant
45
associated protein that we have been able to detect in circulation after a single dose of radiation [13]. In fact, the release of this protein into plasma follows a strict dose response curve and correlates very well with the onset of radiation pneumonitis [13]. We are hopeful that this might be a very useful indicator of lung injury in other circumstances and are currently pursuing this actively. Recently, we have also looked at the effects on surfactant secretion of other agents known to induce lung injury. We found that when 10/aM cadmium chloride, which has been shown to alter surfactant levels in vivo, is added to cultures of type II cells, secretion, either basal or stimulated by adrenergic agonists or TPA, is inhibited by 50070. Addition of 1 /aM cadmium caused less of an effect. In fact, there is some indication that a slight stimulation of basal secretion occurred. We are continuing to do studies on both the inhibition and the possible enhancement of the secretory response and trying to characterize the mechanism of the effect of cadmium. We have also examined the consequences of in vivo hyperoxia on the in vitro response of type II cells to secretion. We have observed that after a 64 h exposure to 100070 oxygen, followed by recovery in room air, that there appears to be a decrease in secretory responsiveness of the type II cells in the immediate postexposure period (Fig. 4). This seems to be consistant with the in vivo data [14] which showed that during a similar time period, the amount of lavageable surfactant present in the lung decreased. One of the problems that I eluded to earlier about type II cells in vitro is that they lose their differentiated properties over time in culture. For example, it has been very difficult to measure synthesis of any of the surfactant apoproteins in type II cells after isolation, although abundant synthesis occurs in vivo. We attempted to characterize protein synthesis after cell isolation, to look at some of
z
oeo L,.J
50
25
•
III
O_
50
-1
I
I
I
I
I
I
I
0
1
2
,5
4
5
6
DAYS RECOVERYAFTER 0 2 EXPOSURE Fig. 4. Effect of in vivo hyperoxia on in vitro secretory response to a /J-adrenergic agonist. Type II cells were isolated from rabbits exposed to hyperoxia for 64 h followed by removal to room air for the time indicated. After overnight culture in media containing /~Ci/ml choline, choline secretory response to 10/aM terbutaline was measured. Results represent at least 3 replicates repeated in 3 separate preparations of cells.
46
the abnormal states that might be generated after toxic insult in vivo or perhaps in vitro. These studies showed that during the 7-day period in culture there are drastic changes in protein synthesis patterns and one can see loss a n d / o r gain of particular proteins and appearance of new ones using 2D gel electrophoresis after [35Slmethionine o r [3H]leucine labelling. Interestingly, we recently showed [15], that during the first 2--18 h after isolation, the type II cells are characterized, by what can best be described, as a stress response. Our results suggest a true heatshock type response in type II cells immediately after isolation. Protein synthesis is drastically inhibited for the first 10--14 h after isolation except for a select number o f proteins (i.e. 10 or 12) [15]. Characterization of the proteins by 2D gel electrophoresis was consistent with known heat shock proteins, particularly the 70 kD and the 90 kD heat shock protein families [15]. We have examined the time course of their appearance after isolation and have compared this directly to the effect of heat shock in type II cells and showed that there are many similarities (see detailed discussion in [15]). By 24 h, protein synthesis appeared to be normal and we thus thought it would be possible to look at the effects of in vitro stresses on protein synthesis in type II cells. Because of our interest in hyperoxic effects both those generated by oxygen as well as the oxidant radicals that are produced by ionizing radiation, we thought to look in vitro at effects of oxidants, or those things that deplete antioxidants, on protein synthesis. We have compared in vitro the effects of hydrogen peroxide, 95% O2, and glutathione depletion by diethylmaleate. In each of these cases we saw protein synthesis responses very similar to heat shock (i.e. induction of 70 and 90 kD proteins). Although, in order to see a stress response with in vitro hyperoxia it was necessary to deplete glutathione with pretreatment of diethylmaleate. In addition to the standard heat shock response, we also saw induction o f a new protein at 32 kD. Preliminary data suggest this may be related to changes in antioxidant enzymes. This was observed (Figs. 5a and 5b) regardless of whether the response was directly related to hyperoxia like hydrogen peroxide, or heat shock or glutathione depletion. We are continuing to explore this effect further. But the results suggest that in response to many pulmonary toxicants, the type II cell does alter its repertoire of protein synthesis which may aid in developing some degree of resistance or tolerance to those agents. As I discussed earlier, a second fundamental property of the type II cell is the ability to proliferate and replace the alveolar epithelium under the circumstances where lung injury has caused the loss of type I cells and the denudation of the basement membrane. We have recently begun to assess whether our in vitro type II cell culture model would allow us to study proliferation in vitro. We have shown [16] that type II cells, even after isolation and the fact that they are beginning to lose their differentiated properties, are capable of at least 1 or 2 rounds of cell division in culture. We have confirmed this proliferation both by looking at the cultures over time and showing that the number of cells increases, and also by autoradiography and measurement of [3H]thymidine incorporation. In unstimulated type II cell cultures, we found a peak in cell proliferation between 24 and 48 h post-isolation. We then determined if we could cause this proliferation to be stimulated beyond that basal rate that we observed just through the
47
A 2 0 0 -"
10069-
46-
30-
14-
A
48
B
C
D
E
F
B 200--
1006g--
6--
30--
14-A
B
C
D
E
Fig. 5. Effects of in vitro stress on proteins synthesized by type 1I cells. Twenty-four hour cultures of type II cells were treated as described, pulsed with [3H]leucine for 1 h and solubilized. Proteins were separated by SDS-polyacrylamide gel electrophoresis on 10%0 gels and subjected to fluorography. An equal number of counts was applied to each lane. (A) A: Control; B: 100 laM diethylmaleate (DEM), 24 h; C: 950/0 O:, 24 h; D: 95% 02 and 100 taM DEM, 24 h; E: 95% 02 48 h; F: 45°C, 30 rain, 2 h recovery. (B) A" Control; B: 0.4 mM H202 4 h recovery; C: 0.4 mM H20: 8 h recovery; D: 0.4 mM H202 16 h recovery; E: 0.4 mM HzO2 24 h recovery.
49
,. z D
200
.J.,~ 0::0
8o F--Z Z--
"'
50
w" w n
EGF
PDCF
TGF# IGF-I TNFa
IL-I A M C M
Fig. 6. Response of cultured type II cells to purified growth factors. Twenty-four hour type II cell cultures were treated with the growth factor indicated for a subsequent 24-h period in the presence of [3H]thymidine. After extensive washing counts incorporated into DNA were measured and reported as percent change compared to basal proliferation. EGF 100 ng/ml; PDGF 2 half-max units; TGF/3 5 ng/ml IGF-1 100 ng/ml TNFa 1600 units/ml; IL-1 20 units/ml. AMCM, Activated macrophage conditioned medium 1:2 dilution
presence of serum in the cultures. Our first studies examined the role of specific growth factors in the regulation of type II cell proliferation and in Fig. 6 we show that type II cells respond quite vigorously to the addition of epidermal growth factor with a 30--50% increase in [3H]thymidine incorporation. In addition, we also looked at a number of other purified growth factors to determine what affect they might have on a type II cell. In general, these growth factors which include interleukin I, platelet derived growth factor, tumor necrosis factor, and insulin-like growth factor I, had no real affect on the type II cell as there was no change in thymidine incorporation or in cell number. In addition to these purified growth factors, we found that conditioned media from particle stimulated macrophages also contains some mitogen for type II cells. It was known from previous in vivo hyperoxia studies [14] that 2--3 days after exposure to 64 h of 100% oxygen, these appeared to be a peak of type II cell proliferation and replacement of type I cells. What we wondered was whether or not this effect was controlled by some particular growth factor and whether this might be related to the dramatic influx of inflammatory cells that occurs in the hyperoxic animals at the same time. We took free cells recovered by bronchoalveolar lavage from animals sacrificed at varying times after in vivo hyperoxia, placed them in culture for 24 h, took this conditioned media and added it to type II cell cultures. We found [16,17] an approximate peak in production of a proliferative activity between 2 and 3 days after exposure. This coincided quite closely with our observed in vivo effects. In order to further characterize this growth factor, we utilized the particle stimulated macrophage model and began to examine in more detail the effects of the particle, in this case, opsonized zymosan, on the production of the type II cell growth factor. We have characterized this further and found optimal release of the type II cell growth factor at 1 mg/ml zymosan. We
50
TABLE II THE RELATIVE ABILITY OF VARIOUS PARTICLES TO STIMULATE MACROPHAGEDERIVED TYPE II CELL GROWTH FACTOR PRODUCTION AND RELEASE Panicle
Opsonized"
Unopsonizeda
Zymosan (1.0 mg/ml) TiO2 (anatase) (0.6 mg/ml) Amorphous silica (0.1 mg/ml) Carbon (0.4 mg/ml) Polyvinyl chloride beads (PVC) (0.6 mg/ ml)
198.9 170.2 166.4 157.4 160.7
150.7 152.5 130.6 129.2 137.2
_ 6.1 _ 4.3 _ 2.2 ___ 4.8 _ 3.5
± 4.2 ± 1.9 _+ 2.6 ± 5.1 _+ 1.6
aData is expressed as percent of control [3H]thymidine incorporation. Macrophages (107/ml) were cultured in the presence of the particle shown with or without prior opsonization of the particles with serum. After 24 h the media was removed, filtered to remove particles and debris and tested for its ability to stimulate [3H]thymidine uptake by cultured type II cells as described in Fig. 6.
have also shown that release o f the factor continues for at least 72 h after initiation o f the m a c r o p h a g e cultures with peak p r o d u c t i o n between 24 and 48 h. The growth factor exhibited sensitivity to heat and trypsin suggesting that it is a protein. Preliminary gel filtration and ultrafiltration experiments indicate its molecular weight to be between 25 and 32 kD. We have also f o u n d that this protein will bind weakly to heparin Sepharose eluting at 0.4 M sodium chloride, a concentration m u c h lower than that observed acidic or basic fibroblast growth factor. This indicates that the heparin Sepharose binding was m o r e related to an ion exchange type binding rather than a specific interaction with heparin. We are continuing to characterize and purify the g r o w t h factor still further, with a view to produce antibodies to it and begin to better characterize its interaction with the type II cell and factors that control its p r o d u c t i o n . In addition, z y m o s a n is not the only particle that caused the p r o d u c t i o n o f this growth factor. As s h o w n in Table II, other particles, including titanium dioxide could also stimulate m a c r o p h a g e release o f this growth factor. This could be very interesting in light o f the effects o f high concentrations o f nuisance dusts on the generation o f fibrosis and other lung injuries. W h a t these results suggest is that one needs to look again at a model o f the cell interactions in lung injury and discuss not only direct effects o f lung texicants on the type II cell, but the effects o f those agents on cells such as the alveolar m a c r o p h a g e . Then we can consider the interactions between a stimulated m a c r o p h a g e and an injured II cell or perhaps a lung fibroblast. This clearly points out that in this tissue, just like in m a n y others, the response to any particular toxic insult is a consequence o f multicellular interactions and cellular c o m m u n i c a t i o n . In s u m m a r y , I would like to conclude by saying that our studies have indicated that by looking at specific biochemical properties o f the type II cell, we can begin to get a m o r e detailed picture o f h o w the lung could respond to toxic agents and that an in vitro model o f type II cell cultures could be very useful in beginning to understand the m e c h a n i s m by which agents affect lung and cause lung injury. In addition, investigations o f m o d u l a t i o n o f lung injury could begin
51
t o b e a p p r o a c h e d u s i n g a t y p e II cell m o d e l as l o n g as o n e t a k e s c a r e f u l c o n s i d e r ation o f the possible cellular i n t e r a c t i o n s t h a t c o u l d exist in the lung.
Acknowledgements The author gratefully acknowledges the contributions of current and former students, Richard Gallo, Mary Brandes, Susan Robey-Bond and Carol Stapanowich. Excellent technical assistance was provided by Christina Kramer. Also a c k n o w l e d g e d are the c o l l a b o r a t i o n s o f D o n a l d S h a p i r o , Philip R u b i n and D a v i d P e n n e y . T h e w o r k r e p o r t e d h a s b e e n s u p p o r t e d in p a r t b y U S P H S G r a n t s CA27791, HL32476, HL37388, HL36543 and NIEHS Training Grant ES07026.
References 1 2 3 4 5 6 7 8 9 10 11
12
13
14 15 16 17 18
52
R. Breeze and M. Terk, Cellular structure, function and organization in the lower respiratory tract. Environ. Health Perspect., 55 (1984) 3. P. Gehr and J.D. Crapo, Morphometric analysis of the gas exchange region of the lung, in D.E. Gardner, J.D. Crapo and E.J. Massaro (Eds.), Raven Press, NY 1988, p. 1. J.N. Finkelstein and R.D. Mavis, Biochemical evidence for internal proteolytic damage during isolation of type I1 alveolar epithelial cells. Lung, 156 (1979) 243. J.N. Finkelstein and D.L. Shapiro, Isolation of type It alveolar epithelial cells using low protease. Lung, 160 (1982) 85. J.N. Finkelstein, W.M. Maniscalco and D.L. Shapiro, Properties of freshly isolated type II alveolar epithelial cells. Biochim. Biophys. Acta, 762 (1983) 398. W.M Maniscalco, J.N. Finkelstein and A.B. Parkhurst, De novo fatty acid synthesis by freshly isolated alveolar type II epithelial cells. Biochim. Biophys. Acta, 751 (1983)462. R.L. Gallo, J.N. Finkelstein and R.H. Notter, Characterization of the membrane potential of alveolar type II cells by the use of ionic probes. Biochim. Biophys. Acta, 771 (1984) 217. R.L. Gallo, J.N. Finkelstein, R.H. Notter and D.L. Shapiro, Alterations in the membrane potentials accompanying exposure to secretagogues. Fed. Proc., 43 (1984) 1846. D. Penney, D. Siemann, P. Rubin, D. Shapiro, J. Finkelstein and R. Cooper, Early and late effects of irradiation of the mouse lung: A review. Scan. Microsc., 1 (1982) 413. P. Rubin, D.L. Shapiro, J.N. Finkelstein and D.P. Penney, The early release of surfactant following lung irradiation of alveolar type 11 cells. Int. J. Radiat. Oncol. Biol. Phys., 6 (1980) 75. P. Rubin, J.N. Finkelstein, D.W. Siemann, D.L. Shapiro, P. Van Houtte and D.P. Penney, Predictive biochemical assays for late radiation effects. Int. J. Radiat. Oncol. Biol. Phys., 12 (1986) 469. D.L. Shapiro, J.N. Finkelstein, D.P. Penney, D. Siemann and P. Rubin, Radiation induced secretion of surfactant from cell cultures of type 11 pneumocytes: An in vitro model of in vivo radiation toxicity. Int. J. Rad. Oncol. Biol. Phys., 10 (1984) 375. P. Rubin, S. McDonald, P. Maasilta, J.N. Finkelstein, D.L. Shapiro, D. Penney and P. Gregory, Serum markers for prediction of pulmonary radiation syndromes. Part I. Surfactant Apoprotein. Int. J. Radiat. Oncol. Biol. Phys., 1989. In press. B.A. Holm, J.N. Finkelstein, S. Matalon and R.H. Notter, Changes in Type II pneumocytes during ARDS injury and recovery from severe hyperoxia. J. Appl. Physiol., 1988. In press. M.E. Brandes and J.N. Finkelstein, Induction of the stress response by isolation of rabbit type II pneumocytes. Exp. Lung Res., 1988. In press. M.E. Brandes and J.N. Finkelstein, Production of growth modulators for type 1I epithelial ceils by activated alveolar macrophages. FASEB J., 2 (1988) A1010. M.E. Brandes and J.N. Finkelstein, Responses of the type II pneumocyte and alveolar macrophage to hyperoxia in vivo and in vitro. Am. Rev. Res. Dis., 137 (1988) 79. A.W. Ham, Histology, 7th edn., J.B. Lippincott Co., Philadelphia, PA, 1974, p. 731.
