The Type 1 Alveolar Lining Cells of the Mammalian Lung

The Type 1 Alveolar Lining Cells of the MammalianLung

II. In Vitro Identification Via the Cell Surface andUltrastructure 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 liningcells were obtained from adult rabbit lung within a heterogeneous population. Identifi-cation of many lung cell types in this mixed population was by a) comparison of isolatedcells with in situ lung cells in lung sections using identical parallel staining, b) stepwiseultrastructural examination of cells during all stages of lung dissociation so that inter-cellular associations were monitored throughout, and c) Type 1 cell surface changesfollowing collagenase treatment. This phenomenon was studied with both electron andlight microscopy, the latter employing tetrachrome staining of basophilic blebs as wellas characteristic staining of nucleus and cytoplasm. Following their isolation, mostType 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 severalconcentrations. Surface knobs, having all the characteristics of zeiotic knobs producedin a number of cultured cell lines by exposure to CD, were produced in isolated Type 1epithelial cells within 45 minutes. The reaction to CD was temperature-dependent,proceeding mmally at 37 C with inhibition at lower temperatures and was inhibitedby antimetabolites such as dinitrophenol and 2-deoxyglucose in the presence of CD. Aswith established cell lines, formation of zeiotic knobs at the isolated Type 1 cell surfaceappeared closely related to microfilamentous nets located beneath the plasmalemma.The density of this net appeared to vary as isolated Type 1 cells underwent expansionand contraction in response to CD. Zeiotic knobs were formed as the result of hernia-tion of endoplasm through the cell cortex. The significance of such a labile cortical zoneis considered in relation to the deformation changes Type 1 cells undergo duringinflation-deflation of alveoli and the folding-unfolding of alveolar lining cells as aresult of lung volume changes. (Am J Pathol 90:123-144, 1978)

THE SQUAMOUS OR TYPE 1 EPITHELIAL CELL, one of the four celltypes found in the lung that may be considered unique to that organ,'plays a major integumentory role in maintaining the air-blood barrier atthe alveolar level.2 Several characteristics distinguish this cell type, in-cluding a high degree of topographic differentiation,3'4 great susceptibilityto alveolar injury,5'6 and the possibility of a transcellular pinocytic trans-port mechanism.7

From the Department of Patholog-. Albert Einstein College of Medicine. Bronx. New- YorkSupported 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 theNational Heart. Lung and Blood Institute.

Accepted for publication September 8. 1977Address reprint requests to Dr Robert M. Rosenbaum. Department of Pathology. Albert Einstein

College of \Medicine. 1300 Mlorris Park Avenue. Bronx. NY 10461123

124 ROSENBAUM AND PICCIANO American Journalof Pathology

We have described the dissociation and separation of rabbit lung intocell fractions, including enriched fractions of Type 1 alveolar epithelialcells.8 Initiallv, these cells were recognized by observations at each stageof separation using the electron microscope and an analine dye methodtinctoriallv selective for a number of epithelial cell types from lung.8UTltimately, it was deemed desirable to devise a means of recognizingfreshly isolated Type 1 cells rapidly within an in vitro heterogeneous lungcell population. Due to the extensive outer perimeter of these cells,4coupled wvith their fragility and apparent elasticity, isolated Txpe 1 cellsmay assume a range of irregular shapes and sizes, thereby making recog-nition difficult in the case of primary isolation. However, a commonproperty of freshly isolated Type 1 cells from lung appears to be transientdisruption of the cell surface, especially following secondary treatmentswith 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 1epithelial cell surface suitable both for recognition and for exploration ofits 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) (100mg 'kg). The lungs w-ere prepared. lavaged. and dissociated as described by Picciano andRosenbaum."

Cell EnrichmnentFractions 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%5EDTA. and 0.1% bovine serum albumin (BSA).'

Light and Elctron McroscopyFor light microscopy. 0.2-ml samples of Type 1 cell preparations w-ere spun onto slides

using a Shandon cytocentrifuge. Pellets w-ere fixed in Bouin-Hollande fixative and stainedwith a modification of Herlant's tetrachrome stain.9

For electron microscopy. fixation wvith cold (4 C) 2.3%c glutaraldehyde prepared in 0.2 NMsodium cacodvlate buffer (pH 7.2) for 2 to .3 hours took place after Type 1 cell suspensionshad 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% OSO4prepared in 0.2 NM sodium cacodvlate (pH 7.2) for an additional 2 hours. The pellet mvasdehydrated. cleared. and embedded in Araldite-Epon. Sections were examined with aSiemens Elmiskop 102. To obtain sections across the flattened aspect of Type 1 cells, cellswere spun onto coverslips using the cytocentrifuge. This preparation was fixed in glutaral-dehyde as described above. postfixed in OSO4., dehy-drated. cleared, and everted with thecells down over a BEENM capsule completelx filled with embedding medium. The entirepreparation was frozen in liquid nitrogen and the coverslip was snapped off. transferringthe cells from the glass to the uppermost region of the embedding medium. Followingpolymerization, cells were sectioned across their flattened aspect.

Vol. 90, No. 1 ALVEOLAR LINING CELL IDENTIFICATION 125January 1978

Examination of Ling Cells and Exposure to Active AgentsLiving cells suspended in NMEM 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% dimeth-

vlsulfoxide (DM SO) prepared in MEM 0.1% BSA using preparations similar to thosedescribed by Miranda et al.10 A stock solution was prepared at a concentration of 1009g ml and diluted to obtain a range of final concentrations from 0.1 to 1.5 lig ml. Forsome observations, dyes such as methylene blue and toluidine blue were added in diluteconcentrations for better visualization of cells.

Metabolic inhibitors were 2,4-dinitrophenol (DNP). KCN. iodacetamide (IAA), 2-deoxyglucose (DOG), antimycin A (AMA), and \-ethvlmaleimide (EMI). These wereprepared in MENM BSA at the concentrations listed in Table 1 and used in experiments inwhich cells were exposed to CD (0.36 ;g/ml for 30 to 60 minutes) in glucose-freeMEM 'BSA with or without DMSO. Inhibitor was then added to final concentrations asseen in Table 1 and incubation continued for an additional 45 minutes. Cells bathed in andwashed free of CD were trapped by means of a glass wool matrix which preventedtranslocation during washing. Only cells held b%- the matrix wvere used for surface areastudies as described below.

Quantitation of Cell Planar AreaFor determination of planar area, cells were placed in an unruled hemocvtometer

chamber and thus were in "suspension." They were photographed with Polaroid filmagainst a Zeiss stage and vernier ocular micrometer. thus standardizing the area of thehemocxvtometer chamber. Four to 6 cells were counted for each area determination atdesired time points. The film was enlarged X 5 to provide an image of the cell largeenough to permit accurate planimetry of an optical midline section with a Salmoiraghicompensating polar planimeter directlv in square millimeters (error = 0.2%).

ResultsPresumptive T-pe 1 alveolar lining cells in newly isolated dispersed

heterogeneous lung cell populations invariably showed the light micro-scope 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 IsolatedType 1 Alveolar Epithelium

Inhibitor Concentration (molar) Percentinhibition of zeiosis

Controls untreatedDinitrophenol 2.5 x 1 0-l 96KCN 2.5X10-' 32lodacetamide 10-2 252-Deoxyglucose 5.0 x 10-2 98AntimycinA 5.0 x 104 96N-Ethylmaleimide 2.5 x 10-2 75

Zeiosis was produced with 0.36 yg/ml CD exposure for 45 minutes, followed by anadditional 45-minute exposure to inhibitor in the continued presence of CD. Controlsproceeded in CD through the total 90-minute exposure.

Percent inhibition was calculated by comparing inhibitor treated cells with controlstreated with CD alone.

126 ROSENBAUM AND PICCIANO American Joumalof Patholgy

containing varying numbers of surface protuberances (Figure 2). With themodification of Herlant's tetrachrome stain we employed, these sitesstained an intense deep blue while the remainder of the cytoplasm ap-peared blue-green and the nucleus red-purple. Most cells at this timeshowed occasional small foci of peripheral basophilia (Figure 2) associatedwith 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 follow-ing isolation (120 minutes +) the majority no longer showed surfaceprotuberances as described above. The typical appearance at this time wasthat of a cell with irregularly shaped cytoplasmic margins, with thenucleus 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 cellsin heterogeneous cell preparations, maintained in MEM/BSA at 37 C,were exposed at 1 hour following isolation to these enzymes at variousconcentrations and times as outlined in Table 2. Both collagenase andtrypsin at concentrations suitable for cell release from intact lungs wereunable to produce protuberances at the surface of presumptive Type 1cells (Table 2). Isolated cells, with no detectable signs of surface activity atthe level of light microscopy were exposed to each enzyme for up to 60minutes, removed, washed by centrifugation (500 X g, 10 min), placed inMEM/BSA and observed for an additional 90 minutes (Table 2) forappearance of knobs at their surface. No surface knobs were seen at anvtime 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 phenome-non in established cultured cell lines, referred to the process as "zeiosis."Since freshly isolated presumptive Type 1 cells were seen to undergo suchzeiosis during isolation and to distinguish such knobs from hydropicvacuoles or blebs which can bulge at the cell surface in some pathologicsituations, it was our intention to see whether CD, known to producezeiosis, could also alter the cell surface in a similar manner in isolatedType 1 cells.

In contrast to the negative results obtained with collagenase and tryp-sin, cells treated with CD at various concentrations and times (Table 2)showed intense surface changes during the first 15 minutes followingremoval from CD into MEM/BSA only. The knobs gradually disappearedwhen CD-treated cells remained in MEM/BSA over the next 3 hours.Ninetv minutes after removal from CD, most cells had lost their surface

Vol. 90, No. 1January 1978

ALVEOLAR LINING CELL IDENTIFICATION 127

Table 2-Tests for Collagenase, Trypsin, and Cytochalasin D as Active Agents

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

(all exposures Concen- timeat 37 C) tration (min) 5 min 15 min 30 min 60 min 90 min

Collagenase 0.01% 15 3 0 0 0 -

30 2 0 0 0 -60 8 0 0 0 -

0.1% 15 3 0 0 0 -30 6 0 0 0 -60 7 3 0 0 -

Trypsin 0.01% 15 0 0 0 0 -

30 1 0 0 0 -60 3 0 0 0 -

0.1% 15 0 0 0 0 -30 0 0 0 0 -60 3 0 0 0 -

Cytochalasin D 0.1 jug/ml 15 86 90 62 41 330 92 95 77 52 1

0.36 gg/ml 15 90 97 79 53 230 98 96 68 57 8

0.9,g/ml 15 95 94 89 61 930 97 96 87 58 7

Minimum count = 100 cells.

knobs (Table 2). Some, however,surface.

showed a few isolated knobs at their

Effects of CD on individual Tvpe 1 cells are shown in Figure 3, repre-senting typical stages of development of the zeiotic alterations in pre-sumptive Tvpe 1 cells. Following exposure to CD for 15 minutes, cellswere returned to MEM/BSA for observation. Within 10 minutes, individ-ual cells began to show surface changes following treatment with CD(Figure 3B). A surface reaction, marked bv the appearance of numerousstalks with terminal knobs, was evident 45 minutes following initiation oftreatment (Figure 3C) and remained 30 minutes after removal from CD.Recoven from CD was first noticeable bv 90 minutes. Type 1 cellsappeared reduced in area and had lost most surface blebs within 120minutes (Figure 3E). Since, in a heterogeneous population of dispersedlung cells, no other cell tvpe showed this response, the presence of zeioticknobs was noted as unique to Type 1 cells.

Cl Changs Duing ft Zeiotic Cyce

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

128 ROSENBAUM AND PICCIANO American Journalof Pathology

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 aType 1 epithelial cell exposed to CD for 15 minutes are presented in Text-figure 1. Of particular interest were periods of expansion and contractionvielding significant changes in relation to prior area. For example, 25minutes following initial exposure to CD and representing 10 minutesremoval from CD, cells underwent a 40% increase over prior area. Asshown in Text-figure 2, in which an entire exposure cycle is graphed, thisseries of expansions and contractions finally resulted in a return to essen-tiallv 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 inhib-itorv effect of low temperatures on the aggregation and dispersal of

T(3min) I configuration

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25 :

45

60

120

240

AreaExpansion (+)Contraction (-IF~~~~~~~~~~~~~~~~~~~~~~

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TEXTr-FIGURE 1-Tracings of thesurface area of a (typical) rab-bit Type 1 epithelial cell from en-larged phase contrast photo-micrographs. (See Figure 3.) Thecell was exposed to 0.36 jg mlCD for approximately 15 minutesstarting at zero time. After 15minutes in CD MIENi'BSA, thecell was placed in MENM 'BSA forrecoverv. Planimetric measure-ments of the area occupied by thecell, trapped in glass wool matrix,

12 were made at regular intervals,and the average percent changeover the presvious area s as re-corded.

21

26

Vol. 90, No. 1January 1978

TExr-FicGun 2-Changes in aver-

age cell area marked by the plasma-lemma in newly released rabbitType 1 epithelial cells exposedto 0.36 g /ml CD. Note the con-

tractions and expansions, the latterpeaking after removal of the cellsfrom CD. There is a rapid declinein average surface area, leadingto restoration of normal area to theequivalent seen at the start of theexperiment. Each point representsan average of four cells.

ALVEOLAR LINING CELL IDENTIFICATION

0

x

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4

rime (min;

surface knobs.14 Low temperature inhibition of CD-induced knobs dis-persed on Type 1 cell surfaces was studied as shown in Table 3. For theseexperiments, the length of exposure time to CD was varied, since we

desired to observe effects of temperature on production and maintenanceof zeiosis rather than on recoverv. In the presence of CD at 37 C, increasein the exposure time produced a greater number of cells with dispersedknobs. When treated cells were removed from media containing CD andfurther incubated at 37 C, recovery of these cells from zeiotic effects tookplace, as described earlier. At room temperature (23 C), reduction in thenumber of cells maintaining dispersed zeiotic knobs was noted despite

Table 3-Effects of Temperature on Dispersion of CD-induced Zeiotic Knobs at the Surfaceof Isolated Rabbit Type 1 cells

Time in lower Total time in Percent cells withTemperature (C) temperature (min) CD (min) dispersed knobs

37 - 10 73- 30 82- 60 91- 90 97

23 15 30 8545 60 6775 90 64

14 15 30 6345 60 3775 90 8

4 15 30 6345 60 975 90 3

129

130 ROSENBAUM AND PICCIANO American Joumalof Pathology

continuous exposure to CD (Table 3). At 14 C and 4 C, the ability toundergo zeiosis initially was markedly reduced (Table 3). These resultssuggested that, at low temperatures, induction of events leading to pro-duction of zeiosis was inhibited and that, even with continuous exposureto CD, dispersion of zeiotic knobs already present could not be adequatelymaintained.The possibility that CD induction of these phenomena required energy

was suggested by the above temperature experiments. The likelihood thatproduction of zeiosis might be energy-linked was suggested by studiesemploying inhibitors of energy metabolism (Table 1). In this series ofexperiments, Type 1 cells in suspension with other rabbit lung cells wereexposed to CD for 45 minutes followed by addition of a given inhibitor tothe preparations. The most marked inhibition of zeiosis was broughtabout with DOG (5.0 X 102 M), AMA (5.0 X 10' M), and DNP (2.5 X10- M). The sulfydryl inhibitors gave variable results since IAA producedonly a 25% inhibition of zeiosis while EMI caused a 75% inhibition. KCNalso 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 alveo-lar lining epithelium were indeed the squamous alveolar lining cell oflung includes a) the observation of ultrastructural features and alveolarlining cell interrelationships made throughout the entire period of cellisolation and b) electron microscopic observations on newly isolated Type1 epithelial cells prior to and after induction of surface phenomenafollowing exposure to CD.

Following final collagenase treatment during the isolation procedure,8cells recognized as Type 1 pneumocytes appeared as in Figure 4A. Largenumbers of fully formed zeiotic knobs erect on single or branched stalksarising 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 sectionedthrough their centers, dense osmiophilic material in the cortical region ofthe cell was observed (Figure 4B). The stalks of the knobs were alsopartially filled with this dense material, while the knobs themselves con-tained less dense endoplasm, usually containing free ribosomes or, occa-sionally, with endoplasmic organelles such as endoplasmic reticulum (ER)fragments of mitochondria. Several such zeiotic protrusions are shown inFigure 5. The lower region of the stalks demonstrated a compact osmio-philic mass appearing virtually identical to the microfilamentous felt


described by others in established cultured cell lines and in lympho-cvtes."'5"l This material extended along the cortex of the cell proper togive the osmiophilic band evident at the base of the zeiotic protuberances(Figure 4B). Most typically, cortical microfilamentous felt extended intothe 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 fullvunderstood, such felt appeared composed of branched or ravelled fibrils 4to 8 nm in diameter. Under the conditions we studied, this cortical feltwas at its greatest density when underlying zeiotic knobs, as described byothers.14 The knob was formed of plasmalemma but contained no sub-plasmalemmal microfilaments. It was generally filled with ribosomal units(Figure 5), although other endoplasmic components such as fragments ofER, lipid, and dense bodies were encountered (Figure 4B). With the lightmicroscope, the knobs appeared phase dense (Figures 3C and 3D) andstained intensely with cationic dyes such as methylene blue and toluidineblue under conditions selective for binding with RNA. In the cortexbeneath the plasma membrane, numerous small rough-coated vesicleswere 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 knobsabout their surfaces (Figure 7). Many of these knobs contained ribosomalaggregates. Beyond this time, although these cells maintained an ex-panded surface area, the gradual loss of knobs was evident. The sequenceof cell changes as they appeared throughout the release cycle and thetimes 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 trypsinmade it of interest to compare cells exposed to these treatments. A Type 1cell from a preparation enriched by separation of cell types at unit gravityis shown in Figure 6. Enriched type 1 cell fractions 8 were exposed to 0.36jg/ml CD for 45 minutes, spun onto glass coverslips with the cvto-centrifuge, and ultimately placed in embedding medium so as to allowsectioning through the entire flattened aspect of the cell. Such a sectionwas essentially equivalent to an optical section of the living Type 1 cellseen in Figure 3C. The entire surface of the cell in Figure 6 was coveredwith single or branched zeiotic knobs arising from a cortex packed withwhat appeared to be masses of subplasmalemmel microfilaments. Thismaterial extended into the stalks of zeiotic knobs and frequently filled theknob itself. At several points, the knobs showed contents similar to endo-plasm. The subcortical region was filled with numerous small vesicles,while the endoplasm contained larger vesicles. We could not ascertain

132 ROSENBAUM AND PICCIANO American Joumalof Paology

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TExr-riuRE 3-Schematic sequence of Type 1 epithelial cell conformational changes throughout asingle zeiotic cycle during dissociation from lung. rtmes indicated represent the following completedperiods of digestion: 30 minutes = trypsin perfusion; 60 minutes = collagenase digestion; 80-120minutes = 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 ofzeiotic knobs at the cell surface was not as large as with exposure toCD (compare Figures 6 and 7), but cortical felt was present in theperipheral regions; the terminal knobs contained endoplasm in whichribosomes were generally evident. Small vesicles appeared in the corticaland subcortical endoplasmic regions although not in the numbers seenfollowing treatment with CD.

Ultashucture of the Relxed solaed Type 1 Cd

Following final tryptic digestion of lung, isolated Type 1 epithelial cellsachieved a relaxed state (+ 120 minutes). This was characterized by theabsence of zeiosis and a complex, irregular, extended cellular circum-ference formed by the plasmalemma. The endoplasm contained dilatedER filled with a fine granular material (Figure 8). In addition, numerousmembrane-bound vacuoles were present; small, coated vesicles could bedetected in the cortical region (Figure 8). Occasionally, a remnant of azeiotic knob was seen, but there were no signs of the cortical micro-filamentous region identified with large numbers of knobs.

DiscssinThis study, describing surface alterations in Type 1 alveolar lining

epithelium which occur during dissociation from lung tissue, provides afirm basis for recognition of this isolated cell type at the light and electronmicroscopic level. With the study of Picciano and Rosenbaum,8 this studyprovides the first reported specific isolation of this major cell component ofthe air-blood barrier of the mammalian lung. In addition, this paperdescribes a degree of subplasmalemmal differentiation not previouslyreported in this cell type.

Verification of the identity of the Type 1 pneumocyte in vitro comesfrom several lines of evidence. First, a comparison of selective stainingproperties of Type 1 alveolar lining cells in situ in paraffin-embedded lungwith those of dissociated Type 1 cells in heterogeneous or enriched lightmicroscopic preparations was possible with use of a tetrachrome anilinedye method.8 The staining properties of embedded cells appeared identicalto those of the isolated Type 1 cells. Secondly, our stepwise ultrastructuralexamination at all stages of the enzymatic dissociation process, includingthat of gradient enrichment, provided evidence that surface blebs areunique to Type 1 cells whether these are partially separated or fully


isolated from lung." Since no other cell type in heterogeneous or enrichedcell populations demonstrated this phenomenon, we regard the appear-ance of surface blebs under the conditions described in these studies as afirm 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 cellsurface. The essential feature of this process, as emphasized by Godman etal,14 involves herniation of endoplasm through the cell cortex. This distin-guishes zeiosis from cortical edema or hydropic vacuolation which occursat the cell surface or beneath it and is generally due to failure of mem-brane-associated ion pump mechanisms following cellular injury. Zeiosishas been described in physiologic,13'1718 pathologic,19 20 and experimentalsituations 19 and has been particularly identified with epithelium.13'21 Inlung cells, zeiosis was brought about during cell isolation procedures aswell as by exposure to CD. We believe, therefore, that Type 1 cellspossess an intrinsic capacity to undergo endoplasmic herniation in re-sponse to these stimuli.

Miranda et al 10,15 and Godman et al 14 describe generalized cell con-traction which causes increased intracellular pressure as the basis of CD-induced zeiosis. The zeiotic protrusions we described in freshly isolatedType 1 cells after exposure to collagenase 8 suggested that rapid release ofthese cells resulted in increased intracellular pressure. As the intracellularpressure became stabilized in isolated cells, the knobs receded. Thepresence of previously undescribed cortical microfilaments could serve tostabilize 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 1cells, free ribosomes are present in large numbers in the cytoplasm." Theaggregation of the free ribosomes in the zeiotic knobs, a characteristic ofzeiosis, appears to account for the basophilia exhibited by these protuber-ances. In addition, a paucity of mitochondria and organized endoplasmicreticulum is characteristic of this cell type, although isolated rabbit Type 1cells, such as we described, invariably showed enlarged cisternae with agranular 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 topogra-phy associated with this cell,'4 as well as to the large surface area anindividual Type 1 cell in situ may cover.4 The role of microfilaments inaccommodating the deformational changes that these alveolar lining cellsmay encounter during changes in alveolar volume has not been deter-


mined. Although during breathing such changes may not be great,2'23 thehistologic pattern during atelectasis u"2 indicates significant capacity toalter alveolar volume on the part of the lung. Radford 1* listed alveolarlining cells as possible factors accounting for the static mechanical proper-ties of the lung. Plastic properties, which might be expected to originatefrom cell deformations, are of particular interest. Such properties may benecessary to deal with rapid changes in intracellular pressure, a naturalenvironmental condition for these cells. Experimental fluid-filled adultlungs show hysteresis, suggesting that plastoelastic behavior may be oflittle consequence. Fetal lungs, whose alveoli are lined with cuboidalepithelium and filled with fluid, show marked static hysteresis of theirvolume-pressure curves,2',' indicating a degree of plastic behavior on thepart of the tissue itself. Glazier et al " and Kuno and Staub 29 have shownthat alveoli change shape very little and may even remain constant overthe range of 25 to 100% of the lung volume. At low lung volumes, the datasuggest that alveoli tend to fold up with no further decrease in surfacearea.2'20 Gil and Weibel,l employing carefully controlled fixation, '32noted sizeable numbers of collapsed alveoli at all lung volumes, althoughthese increased in numbers at lower inflation-deflation volumes. Alveoliin various stages of inflation thus seem to be natural part of the alveolarrecruitment process for establishing lung volume changes related to pres-sure-volume hysteresis. With atelectasis, an extreme example is providedin which thickened interalveolar septums often contain rows of capillariesarranged spatially according to the folding or "pleating" of the alveolarsurface.=22 The Type 1 cell is especially involved in such severe deforma-tive changes by virtue of the large surface it covers. At some locations,these cells may serve to smooth out depressions or irregularities where thealveolus itself is unable to retain its spherical shape.31 At other locations,where pleating of the collapsed alveolar surface is evident, Type 1 cellsmay 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 pillarscrossing the alveolar septums, which resemble fibroblasts but containmicrofilament bundles of 40 to 80A diameter and stain with anti-actin andantimyosin antibodies. The location of these cells makes them likelycandidates for bringing about active folding of the alveolus surface andexpansion and contraction of the alveolar space.

Although regarded as functioning primarily as an air-tissue barrier withlittle specialized intrinsic equipment, data presented in this study and byothers cited above may indicate other previously unrecognized functionsof the Type 1 pneumocyte. Substantiation of this possibility could beachieved by use of well-preserved isolated Type 1 cells.


Refereces

1. Sorokin S: The cells of the lungs. Morphology of experimental respiratory carcino-genesis. Edited by P Nettesheim, MF Hahn Jr, JW Deatherage. U. S. Atomic EnergyCommission, Washington, DC, 1970, pp 3-44

2. Naimark A: Clinical implications on research in lung disease. Lung Cells in Dis-ease. 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 ofthe 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 normallung.2 pp 3-18

5. Bachofen M, Weibel ER: Basic pattem of tissue repair in human lungs followingunspecific injury. Chest 65:14S-19S, 1974

6. Yamamoto E, Wittner M, Rosenbaum RM: Resistance and susceptibility to oxygentoxicity by cell types of the gas-blood barrier of the rat lung. Am J Pathol59:409-435, 1970

7. Dominguez EAM, Liebow AA, Bensch KG: Studies on the pulmonary air-tissuebarrier. 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 mammalianlung. I. Isolation and enrichment from dissociated adult rabbit lung. Am J Pathol90:99-122, 1978

9. Kraicer J, Herlant M, Duclos P: Changes in adenohypophyseal cytology andnucleic acid content in the rat 32 days after bilateral adrenalectomy and the chronicinjection of cortisol. Can J Physiol Pharmacol 45:947-956, 1967

10. Miranda AF, Godman GC, Deitch AD, Tanenbaum SW: Action of cvtochalasin Don 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 hamsterovarv 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. CibaFoundation 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 3-2614. 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 Biol64:644-667, 1975

15. Miranda AF, Godman GC, Tanenbaum SW: Action of cvtochalasin D on cells ofestablished lines. II. Cortex and microfilaments. J Cell Biol 62:406-423, 1974

16. Spooner BS, Yamada KM, Wessels, NK: Microfilaments and cell locomotion. J CellBiol 49:595-613, 1971

17. Costero I, Pomerat CM: Cultivation of neurons from the adult human cerebral andcerebellar cortex. Am J Anat 89:405468, 1951

18. Haemmerli G, Striuli P, Lindenmann R: Mikrokinematographische und electronmikroscopische Beobauchtungen an Zelloberflichen und Zellcontakten der men-schlichen 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 Soc86:87-102, 1966

20. Bessis M: Studies on cell agony and death. Cellular Injury. Edited by A Rouch, JKnight. Boston, Little, Brown & Co., 1964

21. Puck TT, Waldren CA, Hsie A: Membrane dynamics in the action of dibutN-rvladenosine 3':5' cyclic monophosphate and testosterone on mammalian cells. ProcNatl Acad Sci USA 69:1943-1947, 1972


22. Gil J, Weibel ER: Morphological study of pressure-volume hysteresis in rat lungsfixed 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. JPhvsiol (Lond) 210:533-547, 1970

24. Krahl VE: The expansion of pulmonary alveoli in the newborn mouse. Anat Rec115:448, 1953

25. Krahl VE: Factors influencing the histologic appearance of the lung. Anat Rec124:321, 1956

26. Radford EP Jr: Static mechanical properties of mammalian lungs. Handbook ofPhysiology, Section 3, Vol 1. Respiration. American Physiological Society. Edited byWO 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 firstbreath. J Appl Physiol 13:344-348, 1958

28. Glazier JB, Hughes JMB, Maloney JE, West JB: Vertical gradient of alveolar sizein 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 artificialmicroholes in alveolar walls. J Appl Physiol 24:83-92, 1968

30. Klingele TG, Staub NC: Alveolar shape changes with volume in isolated, air-filledlobes of cat lung. J Appl Phvsiol 28:411414, 1970

31. Kapanci Y, Costabella P, Gabbiani G: Location and function of contractile inter-stitial 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, RNMRosenbaum. New York, Wiley Interscience, 1977, pp 19-44

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

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

Figure 2-Light micrograph of whole rabbit lung cells obtained from a cytocentrifuge coverslippreparation fixed in Bouin-Hollande fixative soon after isolation and stained with modifiedHerlants Tetrachrome stain. Two Type 1 alveolar lining cells (asterisks) show extreme stagesof zeiosis following isolation. The upper cell is damaged and shows severe blebbing; the lowercell, representative of most Type 1 cells at this time, is well preserved and still shows welldispersed 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 towhich 0.36 uig/ml cytochalasin D (CD) had been added at 0 time for 15 minutes, followingwhich the cell was returned to MEM/BSA. A-0 time. B-10 minutes. C-45 min-utes. D-60 minutes. E-120 minutes. Note the appearance of cytoplasmic blebsbeginning with 10 minutes exposure to CD and peaking at 45 minutes. Blebs frequently showdense areas near their tip. The nucleus exhibits conformation changes throughout the ex-posure 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 extensionof the cell shown in A. Note intensety osmiophilic zone marking the original external border of the cell (arrow). Numer-ous, closely packed knobs or blebs extend from this zone. The central region of these knobs is hollow (asterisk) andcontains endoplasm, some organelles, or free ribosomes. (See Figure 5.) Numerous vesicles (cv) are present in thecortical 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 alveolarlining cell. The cortex, from which the knobs originate, consists of densely compacted masses ofmicrofilaments. The knobs show a pattern indicating their protrusion from the cortical felt at a pointmarked by arrows. The upper portions of each knob (asterisks) contain endoplasm rich in ribosomesand poor in cortical microfilaments. The outer limit of these knobs is marked by the external plasmamembrane of the cell. Several rough coated vesicles (cv) appear dispersed in the cortical region. (x60,000)

Fgu 6-Electron micrograph of an isolated rabbit Type 1 alveolar lining cell followinggradient enrichment. Coils were exposed to CD (0.36 gg/ml) for 45 minutes. Numerous zeioticknobs have formed above a compact layer of cortical felt (arrows) and have become dis-persed about the surface of the entire cell. Some knobs show endoplasm containing a mixtureof microfilamentous felt as well as ribosomes. There are numerous subcortical vacuoles. (x26,000)

Fgu 7-A Type 1 rabbit alveolar lining cell following trypsin perfusion and collagenasetreatment of lung with final release by trypsin digestion in vitro at approximately 1 10 minutesfrom the start of the trypsin perfusion. As with cytochalasin D treatment for 45 minutes, there isdispersion of zeiotic knobs over the entire cell perimeter. Note accumulation of micro-filamentous felt in the cortical region beneath the knobs and extending up into the stalk ofeach 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 ina "relaxed" state showing extensions of the cytoplasmic circumference and elaborate infoldings of the outer limitingmembrane. Cisternae appear filled with a granular material and are edged by ribosomes (small arrows). Coatedvesicles (cv) are present in the cortical region and clusters of ribosomes (r) appear in the endoplasmic regions. Aremnant of a zeiotic knob is present (large arrow). (x 40,000)

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The Type 1 Alveolar Lining Cells of the Mammalian Lung