The Type 1 Alveolar Lining Cells of the Mammalian Lung II. In Vitro Identification Via the Cell Surface and Ultrastructure of Isolated Cells From Adult Rabbit Lung Robert M. Rosenbaum, PhD, and Paul Picciano, PhD

Using a newly described dissociation and isolation technique, Type 1 alveolar lining cells were obtained from adult rabbit lung within a heterogeneous population. Identification of many lung cell types in this mixed population was by a) comparison of isolated cells with in situ lung cells in lung sections using identical parallel staining, b) stepwise ultrastructural examination of cells during all stages of lung dissociation so that intercellular associations were monitored throughout, and c) Type 1 cell surface changes following collagenase treatment. This phenomenon was studied with both electron and light microscopy, the latter employing tetrachrome staining of basophilic blebs as well as characteristic staining of nucleus and cytoplasm. Following their isolation, most Type 1 cells lost their surface blebs and assumed a "relaxed" state. In this condition, Type 1 cells were exposed to cytochalasin D (CD) for various times and at several concentrations. Surface knobs, having all the characteristics of zeiotic knobs produced in a number of cultured cell lines by exposure to CD, were produced in isolated Type 1 epithelial cells within 45 minutes. The reaction to CD was temperature-dependent, proceeding mmally at 37 C with inhibition at lower temperatures and was inhibited by antimetabolites such as dinitrophenol and 2-deoxyglucose in the presence of CD. As with established cell lines, formation of zeiotic knobs at the isolated Type 1 cell surface appeared closely related to microfilamentous nets located beneath the plasmalemma. The density of this net appeared to vary as isolated Type 1 cells underwent expansion and contraction in response to CD. Zeiotic knobs were formed as the result of herniation of endoplasm through the cell cortex. The significance of such a labile cortical zone is considered in relation to the deformation changes Type 1 cells undergo during inflation-deflation of alveoli and the folding-unfolding of alveolar lining cells as a result of lung volume changes. (Am J Pathol 90:123-144, 1978)

THE SQUAMOUS OR TYPE 1 EPITHELIAL CELL, one of the four cell types found in the lung that may be considered unique to that organ,' plays a major integumentory role in maintaining the air-blood barrier at the alveolar level.2 Several characteristics distinguish this cell type, including a high degree of topographic differentiation,3'4 great susceptibility to alveolar injury,5'6 and the possibility of a transcellular pinocytic transport mechanism.7 From the Department of Patholog-. Albert Einstein College of Medicine. Bronx. New- York Supported by Program Project HL-1613- and Contract HR5-2952 from the National Heart. Lung and Blood Institute Dr Picciano is a recipient of Young Investigator Grant HL-21396 from the National Heart. Lung and Blood Institute. Accepted for publication September 8. 1977 Address reprint requests to Dr Robert M. Rosenbaum. Department of Pathology. Albert Einstein College of \Medicine. 1300 Mlorris Park Avenue. Bronx. NY 10461 123

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We have described the dissociation and separation of rabbit lung into cell fractions, including enriched fractions of Type 1 alveolar epithelial cells.8 Initiallv, these cells were recognized by observations at each stage of separation using the electron microscope and an analine dye method tinctoriallv selective for a number of epithelial cell types from lung.8 UTltimately, it was deemed desirable to devise a means of recognizing freshly isolated Type 1 cells rapidly within an in vitro heterogeneous lung cell population. Due to the extensive outer perimeter of these cells,4 coupled wvith their fragility and apparent elasticity, isolated Txpe 1 cells may assume a range of irregular shapes and sizes, thereby making recognition difficult in the case of primary isolation. However, a common property of freshly isolated Type 1 cells from lung appears to be transient disruption of the cell surface, especially following secondary treatments with collagenase 8 employed in the separation procedure. The present study deals with the basis of such cell surface disruptions during isolation and the use of experimental modifications of the Type 1 epithelial cell surface suitable both for recognition and for exploration of its ultrastructure. Materials and Methods Male New Zealand rabbits weighing 0.5 to 1.3 kg were used for these experiments. Animals w-ere heparinized 8 and killed with pentobarbital sodium (Nembutal) (100 mg 'kg). The lungs w-ere prepared. lavaged. and dissociated as described by Picciano and Rosenbaum." Cell Enrichmnent Fractions enriched in T%-pe 1 cells were obtained using unit gravity separation on a 3.0 to 6.0%c Ficoll gradient prepared mvith Eagle's minimal essential medium (MEM). 0.01%5 EDTA. and 0.1% bovine serum albumin (BSA).' Light and Elctron Mcroscopy For light microscopy. 0.2-ml samples of Type 1 cell preparations w-ere spun onto slides

using a Shandon cy tocentrifuge. Pellets w-ere fixed in Bouin-Hollande fixative and stained with a modification of Herlant's tetrachrome stain.9 For electron microscopy. fixation wvith cold (4 C) 2.3%c glutaraldehyde prepared in 0.2 NM sodium cacodvlate buffer (pH 7.2) for 2 to .3 hours took place after Type 1 cell suspensions had been placed in BEENI capsules (size 00. conical tip) and centrifuged (500 x g. 10 min) using an EFFA adaptor for the capsules. Preparations were postfixed in 2.3% OSO4 prepared in 0.2 NM sodium cacodvlate (pH 7.2) for an additional 2 hours. The pellet mvas dehydrated. cleared. and embedded in Araldite-Epon. Sections were examined with a Siemens Elmiskop 102. To obtain sections across the flattened aspect of Type 1 cells, cells were spun onto coverslips using the cytocentrifuge. This preparation was fixed in glutaraldehyde as described above. postfixed in OSO4., dehy-drated. cleared, and everted with the cells down over a BEENM capsule completelx filled with embedding medium. The entire preparation was frozen in liquid nitrogen and the coverslip was snapped off. transferring the cells from the glass to the uppermost region of the embedding medium. Following polymerization, cells were sectioned across their flattened aspect.

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Examination of Ling Cells and Exposure to Active Agents Living cells suspended in NM EM 0. 1 % BSA were examined with negative phase contrast microscopy using a Reichert inverted microscope equipped w-ith a Polaroid film camera. Cytochalasin D (Sigma. St. Louis. Mo.) (CD) was used with or without 0.025% dimethvlsulfoxide (DM SO) prepared in MEM 0.1% BSA using preparations similar to those described by Miranda et al.10 A stock solution was prepared at a concentration of 100 9g ml and diluted to obtain a range of final concentrations from 0.1 to 1.5 lig ml. For some observations, dyes such as methylene blue and toluidine blue were added in dilute

concentrations for better visualization of cells. Metabolic inhibitors were 2,4-dinitrophenol (DNP). KCN. iodacetamide (IAA), 2deoxyglucose (DOG), antimycin A (AMA), and \-ethvlmaleimide (EMI). These were prepared in MENM BSA at the concentrations listed in Table 1 and used in experiments in which cells were exposed to CD (0.36 ;g/ml for 30 to 60 minutes) in glucose-free MEM 'BSA with or without DMSO. Inhibitor was then added to final concentrations as seen in Table 1 and incubation continued for an additional 45 minutes. Cells bathed in and washed free of CD were trapped by means of a glass wool matrix which prevented translocation during washing. Only cells held b%- the matrix wvere used for surface area studies as described below. Quantitation of Cell Planar Area For determination of planar area, cells were placed in an unruled hemocvtometer chamber and thus were in "suspension." They were photographed with Polaroid film against a Zeiss stage and vernier ocular micrometer. thus standardizing the area of the hemocxvtometer chamber. Four to 6 cells were counted for each area determination at desired time points. The film was enlarged X 5 to provide an image of the cell large enough to permit accurate planimetry of an optical midline section with a Salmoiraghi compensating polar planimeter directlv in square millimeters (error = 0.2%).

Results

Presumptive T-pe 1 alveolar lining cells in newly isolated dispersed heterogeneous lung cell populations invariably showed the light microscope pattern depicted in Figure 1. This consisted of Type 1 cell surfaces Table 1-Effects of Inhibitors of Energy Metabolism on CD-induced Zeiosis of Isolated Type 1 Alveolar Epithelium Inhibitor

Controls untreated Dinitrophenol KCN lodacetamide 2-Deoxyglucose AntimycinA N-Ethylmaleimide

Concentration (molar)

Percentinhibition of zeiosis

2.5 x 1 0-l

96 32 25 98 96 75

2.5X10-' 10-2 5.0 x 10-2 5.0 x 104 2.5 x 10-2

Zeiosis was produced with 0.36 yg/ml CD exposure for 45 minutes, followed by an additional 45-minute exposure to inhibitor in the continued presence of CD. Controls proceeded in CD through the total 90-minute exposure. Percent inhibition was calculated by comparing inhibitor treated cells with controls treated with CD alone.

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containing varying numbers of surface protuberances (Figure 2). With the modification of Herlant's tetrachrome stain we employed, these sites stained an intense deep blue while the remainder of the cytoplasm appeared blue-green and the nucleus red-purple. Most cells at this time showed occasional small foci of peripheral basophilia (Figure 2) associated with basophilic stalks originating from the cell periphery. Reaction to Enzm and Ctocalasin D (Ught Mc

When presumptive Type 1 epithelial cells were allowed to stand following isolation (120 minutes +) the majority no longer showed surface protuberances as described above. The typical appearance at this time was that of a cell with irregularly shaped cytoplasmic margins, with the nucleus pushed to one side (Figure 3A). To test effects of the collagenase (Sigma, Type II) and trypsin (Difco, 1: 250) used in dispersing cells from whole lung, presumptive Type 1 cells in heterogeneous cell preparations, maintained in MEM/BSA at 37 C, were exposed at 1 hour following isolation to these enzymes at various concentrations and times as outlined in Table 2. Both collagenase and trypsin at concentrations suitable for cell release from intact lungs were unable to produce protuberances at the surface of presumptive Type 1 cells (Table 2). Isolated cells, with no detectable signs of surface activity at the level of light microscopy were exposed to each enzyme for up to 60 minutes, removed, washed by centrifugation (500 X g, 10 min), placed in MEM/BSA and observed for an additional 90 minutes (Table 2) for appearance of knobs at their surface. No surface knobs were seen at anv time as the result of such enzyme treatment. The appearance of protuberances on the surface of cells has often been described as "blebbing."'1"2 Godman et al,14 in studying this phenomenon in established cultured cell lines, referred to the process as "zeiosis." Since freshly isolated presumptive Type 1 cells were seen to undergo such zeiosis during isolation and to distinguish such knobs from hydropic vacuoles or blebs which can bulge at the cell surface in some pathologic situations, it was our intention to see whether CD, known to produce zeiosis, could also alter the cell surface in a similar manner in isolated Type 1 cells. In contrast to the negative results obtained with collagenase and trypsin, cells treated with CD at various concentrations and times (Table 2) showed intense surface changes during the first 15 minutes following removal from CD into MEM/BSA only. The knobs gradually disappeared when CD-treated cells remained in MEM/BSA over the next 3 hours. Ninetv minutes after removal from CD, most cells had lost their surface

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Table 2-Tests for Collagenase, Trypsin, and Cytochalasin D as Active Agents Active agent (all exposures at 37 C)

Collagenase

Percent isolated type 1 cells* showing surface Exposure knobs at times following transfer to MEM/BSA

Concentration 0.01%

0.1%

Trypsin

0.01%

0.1%

Cytochalasin D

0.1 jug/ml

0.36 gg/ml

0.9,g/ml

time

(min)

5 min

15 min

30 min

60 min

15 30 60 15 30 60

3 2 8 3 6 7

0 0 0 0 0 3

0 0 0 0 0 0

0 0 0 0 0 0

-

15 30 60 15 30 60

0

0 0 0 0 0 0

0 0 0 0 0 0

-

3 0 0 3

0 0 0 0 0 0

15 30 15 30 15 30

86 92 90 98 95 97

90 95 97 96 94 96

62 77 79 68 89 87

41 52 53 57 61 58

3 1 2 8 9 7

1

90 min -

-

-

Minimum count = 100 cells.

knobs (Table 2). Some, however, showed a few isolated knobs at their surface. Effects of CD on individual Tvpe 1 cells are shown in Figure 3, representing typical stages of development of the zeiotic alterations in presumptive Tvpe 1 cells. Following exposure to CD for 15 minutes, cells were returned to MEM/BSA for observation. Within 10 minutes, individual cells began to show surface changes following treatment with CD (Figure 3B). A surface reaction, marked bv the appearance of numerous stalks with terminal knobs, was evident 45 minutes following initiation of treatment (Figure 3C) and remained 30 minutes after removal from CD. Recoven from CD was first noticeable bv 90 minutes. Type 1 cells appeared reduced in area and had lost most surface blebs within 120 minutes (Figure 3E). Since, in a heterogeneous population of dispersed lung cells, no other cell tvpe showed this response, the presence of zeiotic knobs was noted as unique to Type 1 cells. Cl Changs Duing ft Zeiotic Cyce

Since the earliest cell changes occurring after application of CD appeared to be expansion and contraction of affected cells,1'1'l we measured

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the planar area of living cells exposed to CD during an entire zeiotic cvele. Planimetric determinations of the area of an optical section occupied by a Type 1 epithelial cell exposed to CD for 15 minutes are presented in Textfigure 1. Of particular interest were periods of expansion and contraction vielding significant changes in relation to prior area. For example, 25 minutes following initial exposure to CD and representing 10 minutes removal from CD, cells underwent a 40% increase over prior area. As shown in Text-figure 2, in which an entire exposure cycle is graphed, this series of expansions and contractions finally resulted in a return to essentiallv the same area occupied at the start of the experiment. Effects of I rbi of Meblism

A characteristic of CD-induced zeiosis in cultured cells was the inhibitorv effect of low temperatures on the aggregation and dispersal of

T(3min)

Area I configuration Expansion (+) Contraction (-I

Percent of

prior cell area

F~~~~~~~~~~~~~~~~~~~~~~

r°0 uJ

*

LL a:

OCD c 0

0c 00

3

(+)

10

(H

Er x uJ Lu

25

:

(+)

45

(+)

12

60

(-)

21

120

(-)

240

26

TEXTr-FIGURE 1-Tracings of the surface area of a (typical) rabbit Type 1 epithelial cell from enlarged phase contrast photomicrographs. (See Figure 3.) The cell was exposed to 0.36 jg ml CD for approximately 15 minutes starting at zero time. After 15 minutes in CD MIENi'BSA, the cell was placed in MENM 'BSA for recoverv. Planimetric measurements of the area occupied by the cell, trapped in glass wool matrix, were made at regular intervals, and the average percent change over the presvious area s as recorded.

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January 1978

TExr-FicGun 2-Changes in avercell area marked by the plasmalemma in newly released rabbit Type 1 epithelial cells exposed to 0.36 g /ml CD. Note the contractions and expansions, the latter peaking after removal of the cells from CD. There is a rapid decline in av erage surface area, leading to restoration of normal area to the equivalent seen at the start of the experiment. Each point represents an average of four cells.

age

0

x

E 4

rime (min;

surface knobs.14 Low temperature inhibition of CD-induced knobs dispersed on Type 1 cell surfaces was studied as shown in Table 3. For these experiments, the length of exposure time to CD was varied, since we desired to observe effects of temperature on production and maintenance of zeiosis rather than on recoverv. In the presence of CD at 37 C, increase in the exposure time produced a greater number of cells with dispersed knobs. When treated cells were removed from media containing CD and further incubated at 37 C, recovery of these cells from zeiotic effects took place, as described earlier. At room temperature (23 C), reduction in the number of cells maintaining dispersed zeiotic knobs was noted despite Table 3-Effects of Temperature on Dispersion of CD-induced Zeiotic Knobs at the Surface of Isolated Rabbit Type 1 cells

Temperature (C) 37

Time in lower temperature (min)

Total time in CD (min)

Percent cells with dispersed knobs

-

10 30 60 90 30 60 90 30 60 90 30 60 90

73 82 91 97 85 67

-

23 14 4

15 45 75 15 45 75 15 45 75

64

63 37 8 63 9 3

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continuous exposure to CD (Table 3). At 14 C and 4 C, the ability to undergo zeiosis initially was markedly reduced (Table 3). These results suggested that, at low temperatures, induction of events leading to production of zeiosis was inhibited and that, even with continuous exposure to CD, dispersion of zeiotic knobs already present could not be adequately maintained. The possibility that CD induction of these phenomena required energy was suggested by the above temperature experiments. The likelihood that production of zeiosis might be energy-linked was suggested by studies employing inhibitors of energy metabolism (Table 1). In this series of experiments, Type 1 cells in suspension with other rabbit lung cells were exposed to CD for 45 minutes followed by addition of a given inhibitor to the preparations. The most marked inhibition of zeiosis was brought about with DOG (5.0 X 102 M), AMA (5.0 X 10' M), and DNP (2.5 X 10- M). The sulfydryl inhibitors gave variable results since IAA produced only a 25% inhibition of zeiosis while EMI caused a 75% inhibition. KCN also produced a minimal inhibition. Comparison Beten CD4nduced Sufc Phenme arod Sufce Chanes hdued Drig Typ 1 Cd

Ultrastructural evidence that those cells we recognized as Type 1 alveolar lining epithelium were indeed the squamous alveolar lining cell of lung includes a) the observation of ultrastructural features and alveolar lining cell interrelationships made throughout the entire period of cell isolation and b) electron microscopic observations on newly isolated Type 1 epithelial cells prior to and after induction of surface phenomena following exposure to CD. Following final collagenase treatment during the isolation procedure,8 cells recognized as Type 1 pneumocytes appeared as in Figure 4A. Large numbers of fully formed zeiotic knobs erect on single or branched stalks arising from endoplasmic extensions of the cell were evident. Initially, these stalks were most numerous along the alveolar surface of the cell. Provided that sections were such as to provide several knobs sectioned through their centers, dense osmiophilic material in the cortical region of the cell was observed (Figure 4B). The stalks of the knobs were also partially filled with this dense material, while the knobs themselves contained less dense endoplasm, usually containing free ribosomes or, occasionally, with endoplasmic organelles such as endoplasmic reticulum (ER) fragments of mitochondria. Several such zeiotic protrusions are shown in Figure 5. The lower region of the stalks demonstrated a compact osmiophilic mass appearing virtually identical to the microfilamentous felt

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described by others in established cultured cell lines and in lymphocvtes."'5"l This material extended along the cortex of the cell proper to give the osmiophilic band evident at the base of the zeiotic protuberances (Figure 4B). Most typically, cortical microfilamentous felt extended into the endoplasmic region of the Type 1 cell for a depth of 1.5 to 3.0 A (Figure 6). While further details of its ultrastructure are not yet fullv understood, such felt appeared composed of branched or ravelled fibrils 4 to 8 nm in diameter. Under the conditions we studied, this cortical felt was at its greatest density when underlying zeiotic knobs, as described by others.14 The knob was formed of plasmalemma but contained no subplasmalemmal microfilaments. It was generally filled with ribosomal units (Figure 5), although other endoplasmic components such as fragments of ER, lipid, and dense bodies were encountered (Figure 4B). With the light microscope, the knobs appeared phase dense (Figures 3C and 3D) and stained intensely with cationic dyes such as methylene blue and toluidine blue under conditions selective for binding with RNA. In the cortex beneath the plasma membrane, numerous small rough-coated vesicles were found (Figures 4A, 4B, and 5). By 100 to 120 minutes following the initiation of trypsin perfusion, isolated Type 1 alveolar lining cells showed dispersal of zeiotic knobs about their surfaces (Figure 7). Many of these knobs contained ribosomal aggregates. Beyond this time, although these cells maintained an expanded surface area, the gradual loss of knobs was evident. The sequence of cell changes as they appeared throughout the release cycle and the times at which these occurred are summarized in Text-figure 3. The surface response of Type 1 cells exposed to CD compared with those recently released from lung by action of collagenase and trypsin made it of interest to compare cells exposed to these treatments. A Type 1 cell from a preparation enriched by separation of cell types at unit gravity is shown in Figure 6. Enriched type 1 cell fractions 8 were exposed to 0.36 jg/ml CD for 45 minutes, spun onto glass coverslips with the cvtocentrifuge, and ultimately placed in embedding medium so as to allow sectioning through the entire flattened aspect of the cell. Such a section was essentially equivalent to an optical section of the living Type 1 cell seen in Figure 3C. The entire surface of the cell in Figure 6 was covered with single or branched zeiotic knobs arising from a cortex packed with what appeared to be masses of subplasmalemmel microfilaments. This material extended into the stalks of zeiotic knobs and frequently filled the knob itself. At several points, the knobs showed contents similar to endoplasm. The subcortical region was filled with numerous small vesicles, while the endoplasm contained larger vesicles. We could not ascertain

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Air WM

hbn,

I

cm Fl AF_9IE

71 _t. 1mUdZid

AMaw

z

mw.,

6 L!Zlonl Ar

C i-oodDellpa

of Zi.tk Knolsat hobw Surh.a, RNA IRb)ommi AWtdn I_

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TExr-riuRE 3-Schematic sequence of Type 1 epithelial cell conformational changes throughout a single zeiotic cycle during dissociation from lung. rtmes indicated represent the following completed periods of digestion: 30 minutes = trypsin perfusion; 60 minutes = collagenase digestion; 80-120 minutes = final trypsinization. For details of methodology see article by Picciano and Rosenbaum.'

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whether the latter had formed from fusion of the smaller cortical vesicles. A cell newly isolated following final trypsinization (110 to 120 minutes) is shown in Figure 7. It had not been exposed to CD. The number of zeiotic knobs at the cell surface was not as large as with exposure to CD (compare Figures 6 and 7), but cortical felt was present in the peripheral regions; the terminal knobs contained endoplasm in which ribosomes were generally evident. Small vesicles appeared in the cortical and subcortical endoplasmic regions although not in the numbers seen following treatment with CD. Ultashucture of the Relxed solaed Type 1 Cd

Following final tryptic digestion of lung, isolated Type 1 epithelial cells achieved a relaxed state (+ 120 minutes). This was characterized by the absence of zeiosis and a complex, irregular, extended cellular circumference formed by the plasmalemma. The endoplasm contained dilated ER filled with a fine granular material (Figure 8). In addition, numerous membrane-bound vacuoles were present; small, coated vesicles could be detected in the cortical region (Figure 8). Occasionally, a remnant of a zeiotic knob was seen, but there were no signs of the cortical microfilamentous region identified with large numbers of knobs.

Discssin This study, describing surface alterations in Type 1 alveolar lining epithelium which occur during dissociation from lung tissue, provides a firm basis for recognition of this isolated cell type at the light and electron microscopic level. With the study of Picciano and Rosenbaum,8 this study provides the first reported specific isolation of this major cell component of the air-blood barrier of the mammalian lung. In addition, this paper describes a degree of subplasmalemmal differentiation not previously reported in this cell type. Verification of the identity of the Type 1 pneumocyte in vitro comes from several lines of evidence. First, a comparison of selective staining properties of Type 1 alveolar lining cells in situ in paraffin-embedded lung with those of dissociated Type 1 cells in heterogeneous or enriched light microscopic preparations was possible with use of a tetrachrome aniline dye method.8 The staining properties of embedded cells appeared identical to those of the isolated Type 1 cells. Secondly, our stepwise ultrastructural examination at all stages of the enzymatic dissociation process, including that of gradient enrichment, provided evidence that surface blebs are unique to Type 1 cells whether these are partially separated or fully

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isolated from lung." Since no other cell type in heterogeneous or enriched cell populations demonstrated this phenomenon, we regard the appearance of surface blebs under the conditions described in these studies as a firm basis for recognition of the Type 1 cell in vitro. The original studies of Costero and Pomerat 17 introduced the term "zeiosis" to describe the protrusion of cytoplasmic knobs at the cell surface. The essential feature of this process, as emphasized by Godman et al,14 involves herniation of endoplasm through the cell cortex. This distinguishes zeiosis from cortical edema or hydropic vacuolation which occurs at the cell surface or beneath it and is generally due to failure of membrane-associated ion pump mechanisms following cellular injury. Zeiosis has been described in physiologic,13'1718 pathologic,19 20 and experimental situations 19 and has been particularly identified with epithelium.13'21 In lung cells, zeiosis was brought about during cell isolation procedures as well as by exposure to CD. We believe, therefore, that Type 1 cells possess an intrinsic capacity to undergo endoplasmic herniation in response to these stimuli. Miranda et al 10,15 and Godman et al 14 describe generalized cell contraction which causes increased intracellular pressure as the basis of CDinduced zeiosis. The zeiotic protrusions we described in freshly isolated Type 1 cells after exposure to collagenase 8 suggested that rapid release of these cells resulted in increased intracellular pressure. As the intracellular pressure became stabilized in isolated cells, the knobs receded. The presence of previously undescribed cortical microfilaments could serve to stabilize the Type 1 cell in such instances. The basophilic nature of zeiotic protuberances appears to find its basis in the generalized distribution of ribosomes throughout the Type 1 cell. As confirmed by ultrastructural examinations of newly isolated Type 1 cells, free ribosomes are present in large numbers in the cytoplasm." The aggregation of the free ribosomes in the zeiotic knobs, a characteristic of zeiosis, appears to account for the basophilia exhibited by these protuberances. In addition, a paucity of mitochondria and organized endoplasmic reticulum is characteristic of this cell type, although isolated rabbit Type 1 cells, such as we described, invariably showed enlarged cisternae with a granular content. The presence of a rich microfilamentous zone beneath the surface of the Type 1 alveolar lining cells may be related to the highly complex topography associated with this cell,'4 as well as to the large surface area an individual Type 1 cell in situ may cover.4 The role of microfilaments in accommodating the deformational changes that these alveolar lining cells may encounter during changes in alveolar volume has not been deter-

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mined. Although during breathing such changes may not be great,2' 23 the histologic pattern during atelectasis u"2 indicates significant capacity to alter alveolar volume on the part of the lung. Radford 1* listed alveolar lining cells as possible factors accounting for the static mechanical properties of the lung. Plastic properties, which might be expected to originate from cell deformations, are of particular interest. Such properties may be necessary to deal with rapid changes in intracellular pressure, a natural environmental condition for these cells. Experimental fluid-filled adult lungs show hysteresis, suggesting that plastoelastic behavior may be of little consequence. Fetal lungs, whose alveoli are lined with cuboidal epithelium and filled with fluid, show marked static hysteresis of their volume-pressure curves,2',' indicating a degree of plastic behavior on the part of the tissue itself. Glazier et al " and Kuno and Staub 29 have shown that alveoli change shape very little and may even remain constant over the range of 25 to 100% of the lung volume. At low lung volumes, the data suggest that alveoli tend to fold up with no further decrease in surface area.2'20 Gil and Weibel,l employing carefully controlled fixation, '32 noted sizeable numbers of collapsed alveoli at all lung volumes, although these increased in numbers at lower inflation-deflation volumes. Alveoli in various stages of inflation thus seem to be natural part of the alveolar recruitment process for establishing lung volume changes related to pressure-volume hysteresis. With atelectasis, an extreme example is provided in which thickened interalveolar septums often contain rows of capillaries arranged spatially according to the folding or "pleating" of the alveolar 22 The Type 1 cell is especially involved in such severe deformasurface.= tive changes by virtue of the large surface it covers. At some locations, these cells may serve to smooth out depressions or irregularities where the alveolus itself is unable to retain its spherical shape.31 At other locations, where pleating of the collapsed alveolar surface is evident, Type 1 cells may become so deformed as to form a cleft in the air-cell interface.4'22,31 Kapanci et al 31 described contractile interstitial cells, located as pillars crossing the alveolar septums, which resemble fibroblasts but contain microfilament bundles of 40 to 80A diameter and stain with anti-actin and antimyosin antibodies. The location of these cells makes them likely candidates for bringing about active folding of the alveolus surface and expansion and contraction of the alveolar space. Although regarded as functioning primarily as an air-tissue barrier with little specialized intrinsic equipment, data presented in this study and by others cited above may indicate other previously unrecognized functions of the Type 1 pneumocyte. Substantiation of this possibility could be achieved by use of well-preserved isolated Type 1 cells.

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ROSENBAUM AND PICCIANO

American Joumal of Pathology

Refereces 1. Sorokin S: The cells of the lungs. Morphology of experimental respiratory carcinogenesis. Edited by P Nettesheim, MF Hahn Jr, JW Deatherage. U. S. Atomic Energy Commission, Washington, DC, 1970, pp 3-44 2. Naimark A: Clinical implications on research in lung disease. Lung Cells in Disease. Edited by A Bouhuys. Amsterdam, Elsevier North Holland Biomedical Press, 1976, pp 315328 3. Weibel ER: The mysterv of "non-nucleated plates" in the alveolar epithelium of the lung explained. Acta Anat (Basel) 78:425-443, 1971 4. Weibel ER, Gehr P, Haies D, Gil J, Bachofen M: The cell population of the normal lung.2 pp 3-18 5. Bachofen M, Weibel ER: Basic pattem of tissue repair in human lungs following unspecific injury. Chest 65:14S-19S, 1974 6. Yamamoto E, Wittner M, Rosenbaum RM: Resistance and susceptibility to oxygen toxicity by cell types of the gas-blood barrier of the rat lung. Am J Pathol 59:409-435, 1970 7. Dominguez EAM, Liebow AA, Bensch KG: Studies on the pulmonary air-tissue barrier. I. Absorption of albumin by the alveolar wall. Lab Invest 16:905-911, 1967 8. Picciano P, Rosenbaum RM: The Tvpe 1 alveolar lining cell of the mammalian lung. I. Isolation and enrichment from dissociated adult rabbit lung. Am J Pathol 90:99-122, 1978 9. Kraicer J, Herlant M, Duclos P: Changes in adenohypophyseal cytology and nucleic acid content in the rat 32 days after bilateral adrenalectomy and the chronic injection of cortisol. Can J Physiol Pharmacol 45:947-956, 1967 10. Miranda AF, Godman GC, Deitch AD, Tanenbaum SW: Action of cvtochalasin D on cells of established lines. 1. Early events. J Cell Biol 61:481-500, 1974 11. Porter K, Prescott K, Frye J: Changes in surface morphology of Chinese hamster ovarv cells during the cell cycle. J Cell Biol 57:815-836, 1973 12. Trinkaus J: Modes of cell locomotion in vivo. Locomotion of the Tissue Cells. Ciba Foundation Symposium 14 (new series), 1973. Edited bv R Porter, DW Fitzsimmons. Amsterdam, Elsevier Scientific Publishing Co., 1973, pp 233-244 13. Harris A: Cell surface movements related to cell locomotion.U pp 3-26 14. Godman GC, Miranda AF, Deitch AD, Tanenbaum SW: Action of cvtochalasin D on cells of established lines. III. Zeiosis and movements at the cell surface. J Cell Biol 64:644-667, 1975 15. Miranda AF, Godman GC, Tanenbaum SW: Action of cvtochalasin D on cells of established lines. II. Cortex and microfilaments. J Cell Biol 62:406-423, 1974 16. Spooner BS, Yamada KM, Wessels, NK: Microfilaments and cell locomotion. J Cell Biol 49:595-613, 1971 17. Costero I, Pomerat CM: Cultivation of neurons from the adult human cerebral and cerebellar cortex. Am J Anat 89:405468, 1951 18. Haemmerli G, Striuli P, Lindenmann R: Mikrokinematographische und electron mikroscopische Beobauchtungen an Zelloberflichen und Zellcontakten der menschlichen Carcinom-Zellkulturlinie HEp2. Virchows Arch [Cell Pathol] 8:143-161, 1971 19. Rose GG: Zeiosis. 1. Ejection of cell nuclei into zeiotic blebs. J Roy Micr Soc 86:87-102, 1966 20. Bessis M: Studies on cell agony and death. Cellular Injury. Edited by A Rouch, J Knight. Boston, Little, Brown & Co., 1964 21. Puck TT, Waldren CA, Hsie A: Membrane dynamics in the action of dibutN-rvl adenosine 3':5' cyclic monophosphate and testosterone on mammalian cells. Proc Natl Acad Sci USA 69:1943-1947, 1972

Vol. 90, No. 1 January 1978

ALVEOLAR LINING CELL IDENTIFICATION

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22. Gil J, Weibel ER: Morphological study of pressure-volume hysteresis in rat lungs fixed bv vascular perfusion. Respir Phvsiol 15:190-213, 1972 23. Forrest JB: The effect of changes in lung volume on the size and shape of alveoli. J Phvsiol (Lond) 210:533-547, 1970 24. Krahl VE: The expansion of pulmonary alveoli in the newborn mouse. Anat Rec 115:448, 1953 25. Krahl VE: Factors influencing the histologic appearance of the lung. Anat Rec 124:321, 1956 26. Radford EP Jr: Static mechanical properties of mammalian lungs. Handbook of Physiology, Section 3, Vol 1. Respiration. American Physiological Society. Edited by WO Fenn, H Rahn. 1964, Bethesda, Md., Williams & Wilkins Co., pp 429-449 27. Agostoni E, Taglietti A, Agostoni AF, Setnikar I: Mechanical aspects of the first breath. J Appl Physiol 13:344-348, 1958 28. Glazier JB, Hughes JMB, Maloney JE, West JB: Vertical gradient of alveolar size in lungs of dogs frozen intact. J Appl Phvsiol 23:694-705, 1967 29. Kuno K, Staub NC: Acute mechanical effects of lung volume changes on artificial microholes in alveolar walls. J Appl Physiol 24:83-92, 1968 30. Klingele TG, Staub NC: Alveolar shape changes with volume in isolated, air-filled lobes of cat lung. J Appl Phvsiol 28:411414, 1970 31. Kapanci Y, Costabella P, Gabbiani G: Location and function of contractile interstitial cells of the lungs.2 pp 69-84 32. Gil J: Preservation of tissues for electron microscopy under phvsiological criteria. Techniques of Biochemical and Biophysical Cvtologv, Vol 3. Edited by D Glick, RNM Rosenbaum. New York, Wiley Interscience, 1977, pp 19-44

The authors wish to gratefully acknowledge the expert assistance of Ms. Yvonne Kress in electron microscopy.

Fiu 1-Cytocentrifuge preparation of a freshly isolated heterogeneous lung cell suspension from rabbit stained by a modification of Herlant's tetrachrome method after fixation in Bouin-Hollande fixative. Whole Type 1 alveolar lining cells all show some degree of cytoplasmic blebbing. Asterisks mark several typical Type 1 cells. (x 400)

Figure 2-Light micrograph of whole rabbit lung cells obtained from a cytocentrifuge coverslip preparation fixed in Bouin-Hollande fixative soon after isolation and stained with modified Herlants Tetrachrome stain. Two Type 1 alveolar lining cells (asterisks) show extreme stages of zeiosis following isolation. The upper cell is damaged and shows severe blebbing; the lower cell, representative of most Type 1 cells at this time, is well preserved and still shows well dispersed areas of basophilia. The other two cells in this figure may be categorized as "airway" cells and show no sign of blebbing. (x 800)

Fge 3-A freshly isolated, rabbit Type 1 epithelial cell maintained in MEM/0.1% BSA to which 0.36 uig/ml cytochalasin D (CD) had been added at 0 time for 15 minutes, following which the cell was returned to MEM/BSA. A-0 time. B-10 minutes. C-45 minutes. D-60 minutes. E-120 minutes. Note the appearance of cytoplasmic blebs beginning with 10 minutes exposure to CD and peaking at 45 minutes. Blebs frequently show dense areas near their tip. The nucleus exhibits conformation changes throughout the exposure time. After 120 minutes exposure to CD, Type 1 cells may undergo fragmentation, which may be starting in E. (x 800)

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Fgure 4A-Electron micrograph of a rabbit Type 1 alveolar lining cell following final collagenase digestion (60 minutes). Note the intense blebbing of the alveolar border of the cell. as = alveolar space. B.-Detail of a cytoplasmic extension

of the cell shown in A. Note intensety osmiophilic zone marking the original external border of the cell (arrow). Numerous, closely packed knobs or blebs extend from this zone. The central region of these knobs is hollow (asterisk) and contains endoplasm, some organelles, or free ribosomes. (See Figure 5.) Numerous vesicles (cv) are present in the cortical region of the cell. (A, x 18,000; B, x 24,000)

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Figure 5-Electron micrograph of the surface knobs seen in a freshly isolated rabbit Type 1 alveolar

lining cell. The cortex, from which the knobs originate, consists of densely compacted masses of microfilaments. The knobs show a pattern indicating their protrusion from the cortical felt at a point marked by arrows. The upper portions of each knob (asterisks) contain endoplasm rich in ribosomes and poor in cortical microfilaments. The outer limit of these knobs is marked by the external plasma membrane of the cell. Several rough coated vesicles (cv) appear dispersed in the cortical region. (x

60,000)

Fgu 6-Electron micrograph of an isolated rabbit Type 1 alveolar lining cell following

gradient enrichment. Coils were exposed to CD (0.36 gg/ml) for 45 minutes. Numerous zeiotic knobs have formed above a compact layer of cortical felt (arrows) and have become dispersed about the surface of the entire cell. Some knobs show endoplasm containing a mixture of microfilamentous felt as well as ribosomes. There are numerous subcortical vacuoles. (x

26,000)

Fgu 7-A Type 1 rabbit alveolar lining cell following trypsin perfusion and collagenase treatment of lung with final release by trypsin digestion in vitro at approximately 1 10 minutes from the start of the trypsin perfusion. As with cytochalasin D treatment for 45 minutes, there is dispersion of zeiotic knobs over the entire cell perimeter. Note accumulation of microfilamentous felt in the cortical region beneath the knobs and extending up into the stalk of each knob (arrows). (x 26,000)

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Figue 8-A rabbit Type 1 alveolar lining cell following final tryptic digestion of lung tissue (120 minutes +). The cell is in a "relaxed" state showing extensions of the cytoplasmic circumference and elaborate infoldings of the outer limiting membrane. Cisternae appear filled with a granular material and are edged by ribosomes (small arrows). Coated vesicles (cv) are present in the cortical region and clusters of ribosomes (r) appear in the endoplasmic regions. A remnant of a zeiotic knob is present (large arrow). (x 40,000)

The type 1 alveolar lining cells of the mammalian lung. II. In vitro identification via the cell surface and ultrastructure of isolated cells from adult rabbit lung.

The Type 1 Alveolar Lining Cells of the Mammalian Lung II. In Vitro Identification Via the Cell Surface and Ultrastructure of Isolated Cells From Adul...
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