Actin-Containing Cells in Human Pulmonary Fibrosis KENNETH B. ADLER, PhD, JOHN E. CRAIGHEAD, MD, N. V. VALLYATHAN, PhD and JOHN N. EVANS, PhD Lung tissue from five patients with varying degrees of diffuse pulmonary fibrosis was examined for the pres- ence of nonmuscle cells containing the contractile pro- tein actin. Histochemical studies using light- and elec- tron-microscopic techniques suggested that the fibrotic tissue, in areas lacking smooth muscle, had within it numerous cells with cytoplasmic microfilaments com- posed of actin. Similar cells rarely were observed in NONMUSCLE CELLS with cytoplasmic filaments normally are found in the interstitium of the mamma- lian lung. These cells have been variously termed myo- fibroblasts,' contractile interstitial cells,' capillary pericytes,2,3 and intermediate cells.2 There is reason to believe these cells possess contractile properties, since the filaments are organized into bundles, and physio- logic studies have demonstrated contractility of nor- mal lung tissue in vitro. 1,4,5 Work by Gabbiani et al6 documented proliferation of myofibroblasts in fibrous tissue, and Majno et a17 showed that cell-rich granulation tissue in an open wound possesses contractile properties. On the basis of these observations, we hypothesized that lung tis- sue in pulmonary fibrosis contains an increased num- ber of nonmuscle cells with the potential for contrac- tility (in addition to the more obvious smooth muscle, which is present in variable amounts). The morpho- logic studies recorded here were undertaken in an ef- fort to establish the validity of this hypothesis, using lung tissue from patients with diffuse pulmonary fi- brosis. Physiologic studies in this laboratory8 have demonstrated increased contractility of lung from ani- mals with experimentally induced fibrosis and human patients with the naturally occurring disease. Materials and Methods Lung Tissue Patients with diffuse fibrotic pulmonary disease are evaluated systematically by an investigative team of From the Departments of Pathology and Physiology-Biophysics University of Vermont, College of Medicine, Burlington, Vermont control lung tissue. The orientation of these cells within the parenchyma, as well as the arrangement and den- sity of the filaments in the cell, suggests a contractile function. Cells with contractile properties might play a role in the altered dynamics of the lung in diffuse fibrotic pulmonary disease. (Am J Pathol 1981, 102:427-437) the Vermont Pulmonary Special Center of Research (SCOR), and biopsy specimens of lung tissue are ob- tained as appropriate to establish diagnosis. Samples of lung routinely are examined by conventional mor- phologic techniques and selectively by transmission electron microscopy. In addition, specimens are frozen at - 70 C for future study. One of us (J.E.C.) selected five cases for detailed study. Their histologic features showed them to be representative of a spectrum of fibrotic lesions. The specimens were chosen specifically, however, because the lung tissue did not exhibit interstitial smooth mus- cle cells in sections stained with Beibrich scarlet-acid fuchsin as a component of the Masson trichrome tech- nique. Relevant clinical and physiologic data on the patients are recorded in Table 1. Fresh lung tissue from three age-matched male pa- tients undergoing either lobectomy or pneumonec- tomy for bronchogenic carcinoma was obtained. These specimens revealed only minimal amounts of focal interstitial fibrosis, and thus served as control Supported in part by NIH Grants HL-14212 (SCOR) and HL-21539 and by a grant from the American Lung Associa- tion. Dr. Adler is a Postdoctoral Fellow of the American Lung Association. Dr. Vallyathan's current address is ALFORD-NIOSH, 944 Chestnut Ridge Road, Morgantown, WV 26505. Accepted for publication September 24, 1980. Address reprint requests to Kenneth B. Adler, PhD, De- partment of Pathology, University of Vermont, College of Medicine, Burlington, VT 05405. 0002-9440/81/0313-0427$01.05 i) American Association of Pathologists 427
Transcript
Actin-Containing Cells in Human Pulmonary Fibrosis
KENNETH B. ADLER, PhD,JOHN E. CRAIGHEAD, MD,
N. V. VALLYATHAN, PhD andJOHN N. EVANS, PhD
Lung tissue from five patients with varying degrees ofdiffuse pulmonary fibrosis was examined for the pres-ence of nonmuscle cells containing the contractile pro-tein actin. Histochemical studies using light- and elec-tron-microscopic techniques suggested that the fibrotictissue, in areas lacking smooth muscle, had within itnumerous cells with cytoplasmic microfilaments com-posed of actin. Similar cells rarely were observed in
NONMUSCLE CELLS with cytoplasmic filamentsnormally are found in the interstitium of the mamma-lian lung. These cells have been variously termed myo-fibroblasts,' contractile interstitial cells,' capillarypericytes,2,3 and intermediate cells.2 There is reason tobelieve these cells possess contractile properties, sincethe filaments are organized into bundles, and physio-logic studies have demonstrated contractility of nor-mal lung tissue in vitro. 1,4,5Work by Gabbiani et al6 documented proliferation
of myofibroblasts in fibrous tissue, and Majno et a17showed that cell-rich granulation tissue in an openwound possesses contractile properties. On the basisof these observations, we hypothesized that lung tis-sue in pulmonary fibrosis contains an increased num-ber of nonmuscle cells with the potential for contrac-tility (in addition to the more obvious smooth muscle,which is present in variable amounts). The morpho-logic studies recorded here were undertaken in an ef-fort to establish the validity of this hypothesis, usinglung tissue from patients with diffuse pulmonary fi-brosis. Physiologic studies in this laboratory8 havedemonstrated increased contractility of lung from ani-mals with experimentally induced fibrosis and humanpatients with the naturally occurring disease.
Materials and Methods
Lung Tissue
Patients with diffuse fibrotic pulmonary disease areevaluated systematically by an investigative team of
From the Departments of Pathology and Physiology-BiophysicsUniversity of Vermont, College of Medicine, Burlington, Vermont
control lung tissue. The orientation ofthese cells withinthe parenchyma, as well as the arrangement and den-sity of the filaments in the cell, suggests a contractilefunction. Cells with contractile properties might play arole in the altered dynamics of the lung in diffusefibrotic pulmonary disease. (Am J Pathol 1981,102:427-437)
the Vermont Pulmonary Special Center of Research(SCOR), and biopsy specimens of lung tissue are ob-tained as appropriate to establish diagnosis. Samplesof lung routinely are examined by conventional mor-phologic techniques and selectively by transmissionelectron microscopy. In addition, specimens arefrozen at - 70 C for future study.One of us (J.E.C.) selected five cases for detailed
study. Their histologic features showed them to berepresentative of a spectrum of fibrotic lesions. Thespecimens were chosen specifically, however, becausethe lung tissue did not exhibit interstitial smooth mus-cle cells in sections stained with Beibrich scarlet-acidfuchsin as a component of the Masson trichrome tech-nique. Relevant clinical and physiologic data on thepatients are recorded in Table 1.
Fresh lung tissue from three age-matched male pa-tients undergoing either lobectomy or pneumonec-tomy for bronchogenic carcinoma was obtained.These specimens revealed only minimal amounts offocal interstitial fibrosis, and thus served as control
Supported in part by NIH Grants HL-14212 (SCOR) andHL-21539 and by a grant from the American Lung Associa-tion.
Dr. Adler is a Postdoctoral Fellow of the American LungAssociation.
Dr. Vallyathan's current address is ALFORD-NIOSH,944 Chestnut Ridge Road, Morgantown, WV 26505.
Accepted for publication September 24, 1980.Address reprint requests to Kenneth B. Adler, PhD, De-
partment of Pathology, University of Vermont, College ofMedicine, Burlington, VT 05405.
0002-9440/81/0313-0427$01.05 i) American Association of Pathologists
427
428 ADLER ET AL
tissue. They were excised from areas distant from thetumor; histologic evaluation confirmed the absence ofthe neoplasm in the samples (Figure 1).
Histologic and Histochemical Procedures
A piece of lung tissue, approximately 5 sq mm, was
fixed in 1O0o phosphate-buffered formalin and em-
bedded in Paraplast after dehydration in ethanol andxylene. Serial 4-M-thick sections were prepared. Thefirst section was stained by the Masson trichrometechnique to identify muscle and connective tissue. Anadjacent section was processed for the localization ofthe contractile protein actin with the use of the indirectimmunofluorescence procedure of Wang and Gold-berg.9 This technique capitalizes on the binding of cel-lular actin by the enzyme deoxyribonuclease I.'0 Inbrief, tissue sections on glass slides were deparaffin-ized with xylene and immersed in a graded series of di-lute solutions of ethanol. After rinsing in water, thetissue sections were incubated in a solution of bovinepancreatic DNase I and then in a dilute solution ofrabbit antiserum to DNase I. Finally, they were
treated with fluorescein-labeled goat antirabbit globu-lin. The DNase I-anti-DNase I technique has been re-
ported to stain actin specifically.9"10 In addition, we
have carried out standard control procedures to dem-onstrate this specific staining (see Table 2).
Pieces of lung tissue that had been fixed at the timeof surgery in 4% glutaraldehyde (0.1 M Na cacodylatebuffer, pH 7.2) were postfixed in 07e OS04 for 2hours. After dehydration in ethanol and propyleneoxide, the tissues were embedded in Epon. Ultrathin
sections were prepared and mounted on copper grids.The sections were stained with a saturated solution ofuranyl acetate in 50% ethanol for 10 minutes and bybasic lead citrate for 5 minutes before examinationwith a Philips 201 transmission electron microscope(TEM).Samples of lung tissue that had been frozen at the
time of surgery were studied with the use of heavymeromyosin (HMM) for the identification of actin fil-aments in cells. I HMM binds specifically to actin fila-ments at a reproducible periodicity of approximately350 A, giving the filaments a characteristic "arrow-head" configuration. HMM was separated from rab-bit skeletal muscle myosin by trypsin digestion, as de-scribed by Margossian and Lowey.'2 Pieces of lungtissue, approximately 2 sq mm, were incubated in de-creasing concentrations of buffered (pH 7.0) glycerolin an isotonic salt solution for 72 hours at 4 C. The tis-sues then were immersed in a 2-4 mg/ml solution ofHMM in 0.5 M KCl for 18 hours at 4 C. I The stainingprocedure was controlled by the incubation of tissuesfrom the same lungs in 0.5 M KCl that contained no
HMM. Subsequently, tissues were rinsed in 0.5 MKCl, fixed in cold 4%7o glutaraldehyde, postfixed,stained, and examined by TEM as described above.
Results
Lung tissue from the 5 members of the study group
exhibited varying degrees of diffuse fibrosis (Figure 1
B-F). Muscle cells identifiable by Beibrich scarlet-acidfuchsin (Masson's trichrome stain) were not present inthe parenchyma, except for those elements associated
Table 1-Clinical and Functional Data on Patients With Pulmonary Fibrosis
Abbreviations: VC = vital capacity; % pred = percent of predicted value; FEV = forced expiratory volume in 1 second; TLC = total lungcapacity; DL = pulmonary diffusing capacity.0 = no fibrosis; + + + + = severe, extensive fibrosis.
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I ..
I -
Figure 1-Representative sections of parenchyma from control (A) and 5 fibrotic patients (B-F), stained by Masson's trichrome. Thickened in-terstitial areas with increased collagen (arrowheads) and cellularity were characteristic of all 5 fibrotic lungs. Interstitial muscle (other thanthat associated with airways or vessels) was not observed In any of these tissues. (x 350)
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Table 2-Tests for Specificity of DNase I-Anti-DNase ITechnique for Staining Actin
Fluorescence in fibroblastTreatment (hamster) monolayer
2. No treatment (autofluores- No fibers seen; weak, spottycence) perinuclear fluorescence
3. lmmunoabsorption- No fibers seenincubation of DNase-l withactin prior to treatment
4. DNase-l:Normal rabbit No fibers seenserum:F*-GAR globulin
5. No DNase l:X-DNase l: No fibers seen; weak peri-F*-GAR globulin nuclear fluorescence
6. F*-GAR globulin only No fibers seen; weak cellularfluorescence
7. DNase l:X-DNase l:GAR No fibers seenglobulin:F*-GAR globulin
8. Incubation with Cytochala- "Asterisks," starlike frag-sin Bt 0.1 mg/ml for 1 hour; ments, around nucleus;then DNase-l:X-DNase-l: No fibers seenF*-GAR globulin
* X-DNase = antiserum raised in rabbits against bovine pancre-atic DNase I.
t F*-GAR globulin = fluorescein-labeled goat antirabbit globulin.t Cytochalasin B is a drug that specifically disrupts actin filaments.
with vessels and airways. The lungs of the 3 controlpatients showed minimal degrees of focal interstitialfibrosis but otherwise were unremarkable.Examination of the lung tissue from the 5 patients
by the method of Wang and Goldberg9 revealed actin-containing cells scattered throughout the fibrotic pa-renchyma (Figure 2 B-F). In contrast, actin rarely wasobserved in the interstitial cells of lung tissue fromcontrol subjects (Figure 2A). The fibrotic lungs ap-peared to be composed of a greater number of cells ofthis type than the control lungs.
Ultrastructural study of the lesions in the 5 lungs re-vealed numerous cells with cytoplasmic microfila-ments 40-80 A in diameter (Figures 3-5). A rigid mor-phologic characterization of these cells was not possi-ble, because they exhibited a diversity of fine struc-tural features and were distorted by the associated ac-cumulations of collagen and elastin. The cytoplasm ofthe entire cell was filled with bundles of filaments or-ganized into parallel arrays. Often the filaments ap-peared to attach to the cytoplasmic face of the plasma-lemma. These features suggest a possible contractilefunction. Many of these interstitial cells possessed cy-toplasmic extensions located adjacent to capillariesand epithelia. Their anatomic position in the intersti-
tium was similar to the contractile interstitial cells(CIC) described by Kapanci et al' (Figure 6A).
Filaments in the cytoplasm of cells populating thefibrotic parenchyma of the 5 lungs showed "arrow-heads" when glycerol-treated tissue was incubated in asolution of HMM (Figure 7). This observation sup-ported the conclusion, based on the histochemicaltechnique of Wang and Goldberg,9 that the filamentsin these cells contained actin. Since glycerol treatmentdistorted the fine-structural morphology of the cells,especially the plasmalemma and intracellular mem-branes, it was not possible to define their fine struc-tural features after using this technique.
Discussion
Although diffuse pulmonary fibrosis has been thesubject of numerous morphologic studies, l 3-l6 cells inthe interstitial fibrous tissue remain poorly character-ized. The results of our study indicate that the fibroticparenchyma contains large numbers of cells havingfilaments of actin in their cytoplasm. These cells aresmall, thin, and have numerous cytoplasmic exten-sions. Since their features cannot be defined by light-microscopic examination, histochemical and TEMtechniques are required to identify them. The fibroticlung appears to contain a greater number of these cellsthan nonfibrotic pulmonary tissue.The fine structural features of the cells we describe
strongly suggest that they have a contractile function.They could be related to the CICI and the capillarypericyte2,3 (Figure 6) because their topographicalposition in the interstitium is similar, and they possesscytoplasmic processes that appear to abut the base-ment membranes of nearby epithelium. However,they differ in several ways. The entire cytoplasm is fill-ed with filaments, imparting an electron-dense ap-pearance to the cell. In contrast, both CIC and capil-lary pericytes contain discrete bundles of microfila-ments restricted to cytoplasmic processes. In addition,the cells are more prominent in the interstitium thaneither the CIC or capillary pericyte. Although the cellsalso possess certain fine-structural features of smoothmuscle (eg, dense bodies, pinocytotic vesicles, base-ment membrane components), their small size, round,eccentric nuclei and abundant rough endoplasmicreticulum indicate different cytologic properties. Inaddition, the cells lack a complete basement mem-brane and are not stained by Beibrich scarlet-acidfuchsin using the Masson trichrome technique. At thistime, the origin of these contractile cells is uncertain.The presence of certain characteristics of CIC,
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PULMONARY FIBROSIS 431
Figure 2-Representative sections of parenchyma from control (A) and 5 fibrotic patients (B-F), stained by the DNase l-anti-DNase tech-nique9 and examined in the fluorescent microscope. The lungs of all five patients with fibrosis contain many cells with actin. In comparison,control tissue shows a few, discrete areas containing actin. (x 600)
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capillary pericytes, and smooth muscle suggests thatthe cells originate from CIC or capillary pericytes andmay be in the process of becoming smooth musclecells. However, their derivation from undifferentiatedmesenchymal elements in the fibrotic tissue also ispossible.
It is of interest that proliferation of contractile cellsis characteristic of several lung disorders. Hyperplasiaof smooth muscle in interstitial scars is a pathologicprocess that often accompanies fibrotic disease.'7 Thesmooth muscle observed in such lesions is readily iden-tified histochemically and ultrastructurally (Figure 7).
Figure 3-Interstitum of lung of a patient with pulmonary fibrosis (No. 4). Cells containing cytoplasmic filaments (F) with dense bodies (*)abound, as do fibrils of collagen (COL). A = air.space. Inset-Processes from these cells, filled with filaments, abut the basement membrane(BM) of the pulmonary epithelium (EP) at arrows. (Uranyl acetate and lead citrate, x 8700; Inset, x 18,500)
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......... ..... --
A
Vol. 102 * No. 3 PULMONARY FIBROSIS 433
S.':v;=' ~~~~~~~~~~~~~~~~~~" -Z-^
.,-,
1.O.pm..> . l.Zm. j.Q.g ;-. .-.; A-@
'~~~~~~~~~~~- ,, 4_
Figure 4-Cells with cytoplasmic filaments (F) in interstitium of the lung of a patient with pulmonary fibrosis (No. 2). A-Cells with promi-nent accumulations of filaments are interdigitated with collagen (COL). B-The cells contain filaments with dense bodies (*) and possibleattachment plaques to the inner face of the plasmalemma (arrowheads), basement membrane components (BM), and rough endoplasmic retic-ulum in electron-lucent areas. (Uranyl acetate and lead citrate, A, x 12,700; B, x 32,500)
Kq.
434 ADLER ET AL
Lymphangiomyomatosis, a rare, idiopathic condi-tion, also is characterized by the luxuriant prolifera-tion of microfilament-containing cells in the intersti-tium of the lung.'8 The pathogenetic basis for thisunique lesion remains to be defined, although somehave suggested that either the pericyte or CIC is thecell of origin. 19 Relatedly, Meyrick and Reid have sug-gested that hypoxia induces proliferation of filament-
containing "intermediate cells" in pulmonary ves-sels.20Our study shows that interstitial areas of lungs
which are characterized histologically as "fibrotic"contain nonmuscular cells with an apparent contrac-tile function. These cells probably are members of a"family" of contractile cells that exhibit morphologicfeatures ranging from smooth muscle to fibroblast.2'
bt ~ ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ rtl' i .-
00 , , (; '4'L O.,
Figure 5-A cell with filaments that occupy large areas of cytoplasm in interstitium of lung from a patient with pulmonary fibrosis (No. 5). Fila-ments are decorated with HMM in characteristic "arrowhead" configuration (inset), confirming that the filaments are composed of actin. Largeareas of collagen (COL) are observed. The severe alterations in cellular structure are due to incubation in glycerol solutions (see text). (Uranylacetate and lead citrate, x 46,300; inset, x 135,600)
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PULMONARY FIBROSIS 435
Figure 6-Lung tissue from a "control" patient without pulmonary fibrosis. Interstitial areas contain cell types with cytoplasmic filaments thatdiffer from the cells described above in fibrotic lungs. A-Contractile interstitial cell (CIC) spans the alveolar septum between two airspaces (A). The filaments (F) appear to concentrate in cytoplasmic processes that contact the basement membranes of the pulmonary epithe-lium (EP) and capillary endothelium (EN). B-Process of capillary pericyte (PER) abuts the basement membrane of capillary endothelium(EN). Pinocytotic vesicles (PV) are seen on the adluminal side, and bundles of filaments (F) are observed on the luminal side. Both these celltypes obviously differ from the filament-filled cells observed in the fibrotic interstitium (Figures 3-5). CT = connective tissue; RBC = redblood cell. (Uranyl acetate and lead citrate, A, x 23,000; B, x 20,500)
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436 ADLER ET AL
.LOpm.7B
Figure 7-Lung tissue from a patient with hyperplasia of smooth muscle accompanying fibrosis (not one of the 5 fibrotic patients described in,the text). A-In contrast to the lung tissue described in this report, the parenchyma of this lung is filled with bundles of smooth muscle,identifiable by trichrome staining (arrow). Masson's trichrome, (x250) B-Ultrastructurally these smooth muscle cells appear spindle-shaped, long, and narrow, with a centrally located, ovoid nucleus and thick bundles of cytoplasmic myofilaments with dense bodies. They aremuch larger than the cells we observed in the fibrotic interstitium, and complex cytoplasmic processes are absent from these highly electron-dense cells. Pinocytotic vesicles (PV) are small and highly ordered. These features are not compatible with the filament-containing cells as il-lustrated in Figures 3 and 4. (Uranyl acetate and lead citrate, x 7200)
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Vol. 102 * No. 3 PULMONARY FIBROSIS 437
Interstitial cells having the capacity to generate forcecould influence the mechanical properties of the fi-brotic lung. Reduced compliance, usually attributedto increased connective tissue, could be attributed, atleast in part, to an active contractile component. Stud-ies in our laboratory support this hypothesis. Ourwork indicates that strips of parenchyma from bothhumans with pulmonary fibrosis and animals with ex-perimentally induced disease have contractile proper-ties which exceed normal tissue.8,22 Further studies areneeded to clarify the origin of these contractile cellsand their precise role in influencing the pathophysiol-ogy of the fibrotic lung.
References
1. Kapanci Y, Assimacopoulos A, Irle C, Zwahlen A, Gab-biani G: "Contractile interstitial cells" in pulmonary al-veolar septa: A possible regulator of ventilation/perfu-sion ratio? J Cell Biol 1974, 60:375-392
5. Adler KB, Vallyathan NV, Craighead JE, Evans JN:Morphological and physiological characterization ofcontractile properties of rabbit peripheral lung (Abstr).Am Rev Respir Dis 1979, 119:285
6. Gabbiani G, Hirschel BJ, Ryan GB, Statkov PR, MajnoG: Granulation tissue as a contractile organ: A study ofstructure and function. J Exp Med 1972, 135:719-734
7. Majno G, Gabbiani G, Hirschel BJ, Ryan GB, StatkovPR. Contraction of granulation tissue in vitro: Similar-ity to smooth muscle. Science 1971, 173:548-550
8. Adler KB, Kelley J, Evans JN: Morphological and phar-macomechanical evidence of increased contractility inhuman and rat fibrotic lungs (Abstr). Am Rev RespirDis 1980, 121:309
9. Wang E, Goldberg AR: Binding of deoxyribonuclease Ito actin: A new way to visualize microfilament bundlesin nonmuscle cells. J Histochem Cytochem 1978, 26:745-749
10. Lazarides E, Lindberg V: Actin is the naturally occur-ring inhibitor of deoxyribonuclease I. Proc Natl AcadSci (USA) 1974, 71:4742-4746
11. Ishikawa H, Bischoff R, Holtzer H: Formation of ar-rowhead complexes with heavy meromyosin in a varietyof cell types. J Cell Biol 1969, 43:312-328
12. Margossian SS, Lowey S: Substructure of the myosinmolecule: III. Preparation of single-headed derivativesof myosin. J Mol Biol 1973, 74:301-311
13. Crystal RG, Fulmer JD, Roberts WC, Moss ML, LineBR, Reynolds HY: Idiopathic pulmonary fibrosis: Clin-ical, histologic, radiographic, physiologic, scinti-graphic, cytologic, and biochemical aspects. Ann InternMed 1976, 85:769-788
14. Scadding JG, Hinson KFW: Diffuse fibrosing alveolitis(diffuse interstitial fibrosis of the lungs). Correlations ofhistology at biopsy with prognosis. Thorax 1967, 22:291-304
15. Livingstone JL, Lewis JG, Reid L, Jefferson KE: Dif-fuse interstitial pulmonary fibrosis. J Clin Invest 1979,63:665-676
16. Kapanci Y, Mo Costobella P, Gabbiani G: Location andfunction of contractile interstitial cells in the lungs.Lung Cells in Disease. Edited byA Bouhuys. New York,Elsevier/North Holland Biomedical Press, 1976, pp 69-84
17. Davies D, Macfarlane A, Darke CS, Dodge OG: Muscu-lar hyperplasia ("cirrhosis") of the lung and bronchial di-latations as features of chronic diffuse fibrosing alveo-litis. Thorax 1966, 21:272-289
18. Corrin B, Liebow AA, Friedman PJ: Pulmonary lymph-angiomyomatosis: A review. Am J Pathol 1975, 79:347-382
19. Kane PB, Lane BP, Cordice JWV, Greenberg GM: Ul-trastructure of the proliferating cells in pulmonarylymphangiomyomatosis. Arch Pathol Lab Med 1978,102:618-622
20. Meyrick B, Reid L: Hypoxia and incorporation of3H-thymidine by cells of the rat pulmonary arteries andalveolar wall. Am J Pathol 1979, 96:51-70
21. Majno G: The story of the myofibroblasts. Am J SurgPathol 1979, 3:535-542
