Ultrastructural and cytochemical comparison of cultured normal and cystic fibrosis fibroblasts

EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

33, l&t-121

(1980)

Ultrastructural and Cytochemical Comparison of Cultured Normal and Cystic Fibrosis Fibroblastsl S. S. SPICER, P. A. DI SANT’AGNESE, Research

Pathology,

Received

R. A. VINCENT,

Medical University of South Charleston, South Carolina November

Carolina, 29403

28, 1979, and in revised

form

JR., AND M. ULANE 171 Ashley

May

Avenue,

14, 1980

Fibroblasts were cultured from skin biopsies of normal donors and cystic fibrosis (CF) patients, and compared morphologically and cytochemically by light and electron microscopy. Normal and CF fibroblasts revealed by light microscopy a comparable number of cytoplasmic bodies which contained acidic complex carbohydrate and acid phosphatase. These increased in prevalence with the number of days in culture, the time in culture between the last passage and assay, and the percentage of noncycling cells. The fibroblasts did not exhibit extensive or intense metachromasia. Normal and CF cells displayed similar fine structural morphologic features, including a comparable population of dense bodies. These contained acid phosphatase and became labeled by ferritin pinocytosed from the medium. The dense bodies were thus identified as secondary lysosomes engaged, at least in part, in the degradation of endocytosed material. The CF and normal cells exhibited no difference by light microscopy in reactivity to stains for neutral, sialic acid-rich, and sulfated complex carbohydrate. By electron microscopy, no difference in the content of acid complex carbohydrate was demonstrable with dialyzed iron. The amount and distribution of antimonateprecipitable cations was observed ultrastructurally and found to be similar in CF and normal cells. By morphologic or cytochemical criteria, it was not possible to distinguish CF from normal cells or to provide a basis for the extensive cellular metachromasia observed by some investigators in CF cells examined by light microscopy.

INTRODUCTION Fibroblasts grown in culture from patients with cystic fibrosis (CF) have been investigated extensively for a morphological (Danes and Beam, 1969; Taysi et al., 1969; Milunsky and Littlefield, 1969; Bartman et al., 1970; Kraus et al., 1971; Report, 1972) or biochemical (Matalon and Dorfman, 1968; Houck and Sharma, 1970; Wiesmann and Neufeld, 1970; Kraus et al., 1971; Benke et al., 1972; Klagsbrun and Farrell, 1973; Rennet-t et al., 1973; Fletcher and Lin, 1973; Hodes et al., 1974; Quissell and Pitot, 1974) aberration of possible diagnostic or pathognomonic significance. CF fibroblasts have been found, for example, to exhibit a characteristic cytoplasmic metachromasia, although this property has not been an invariable finding (Danes and Beam, 1969; Taysi et al., 1969; Milunsky and Littlefield, 1969; Bartman et al., 1970; Kraus et al., 1971). An increase in cytoplasmic dense bodies demonstrable by electron microscopy has been reported for CF tibroblasts (Bartman et a/., 1970). The latter structures, of presumed autophagic nature, conceivably could account for cytoplasmic metachromasia, inasmuch as lysosome-like bodies from which autophagosomes are thought to be derived often contain carbohydrate (Horn and Spicer, 1964) which is requisite for metachromatic staining (Schubert and Hamerman, 1956). In the present investigation, normal and CF fibroblasts at variable doubling levels and intervals following the last passage, were examined by light microscopic cytochemical meth’ This research was supported by NIH Grants AM-10956 and HR-2929 and a Cystic Fibrosis grant from United Health and Medical Research Foundation of South Carolina, Inc. 104 0014-4800/80/040104-18$0200/O Copyright @ 1980 by Academic Press, Inc. AU rights of reproduction in any form reserved.

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10s

ods for visualizing the acidic complex carbohydrate and acid phosphatase which characterize lysosomal bodies. In addition, several similarly matched normal and CF fibroblast strains were compared ultrastructurally for their morphologic features, content of acidic complex carbohydrate and acid phosphatase, and capacity to endocytose the tracer macromolecule, ferritin. Because disturbances in transport of electrolytes possibly underlie the pathophysiology of CF (di Sant’Agnese et al., 1953), the cultured fibroblasts were examined also with the Komnick method (Komnick, 1962) for the ultrastructural localization of antimonateprecipitable cation. MATERIALS

AND METHODS

Dermal biopsies were obtained with informed consent from patients whose clinical course and sweat tests established the diagnosis of CF (Tables T-III). Biopsies were obtained similarly from age-matched donors who exhibited normal sweat electrolyte levels and no clinical features of CF. The biopsies were cultured in a CO, incubator by conventional procedures (Greene, 1973) utilizing Eagle’s minimum essential medium supplemented with 10% fetal calf serum, 2 mM glutamine and 50 pg/ml neomycin sulfate or Dulbecco’s modified Eagle medium supplemented with 8% fetal calf serum and 1% antibiotic-antimycotic 100x mixture (GIBCO). Cells which grew out of the biopsy fragments were subcultured at intervals and examined by light and electron microscopy after a varying number of passages and at various times in culture after the last passage or change of medium. Cells were grown on coverslips in 35mm culture dishes for light microscopy and in 75cm2 culture flasks for electron microscopy. As an assessment of their relative capacity to carry on endocytic activity, normal and CF fibroblasts were exposed to medium containing ferritin for 24 to 48 hr. On three occasions age-matched normal and CF cultures were processed together for light microscopic study at a comparable number of passages and days following the last passage, utilizing different cell strains for each of the three experiments (see Table I). The coverslips cultures were fixed for 30 min in a 10% formalin solution buffered with 2% calcium acetate, a 6.25% glutaraldehyde solution buffered with 0.1 M cacodylate, pH 7.4, or Camoy fluid, prior to rinsing in TABLE I Staining for Carbohydrate in Cytoplasmic Bodies of Cultured Fibroblasts Reactivityb Donor”

Azure A, pH 4.5

Alcian blue (pH 2.5)periodic acid-Schiff

High iron diamine alcian blue

’ Normal and cystic fibrosis (CF) fibroblasts were cultured concurrently and examined together. This was repeated twice with different cultures each time. b The range of values denotes the abundance of stained granules in the least to the most reactive cells of the cultures. + denotes small numbers of weakly stained bodies. c Blue orthochromatic staining and purple metachromatic staining demonstrative of acidic complex carbohydrate was observed in both normal and CF cells. d Purple-stained bodies were observed in both normal and CF cells demonstrative of acidic complex carbohydrate. ’ Blue- to black-stained bodies demonstrative of acidic complex carbohydrate with carboxyl (blue staining) and sulfate (black staining) groups were observed in both normal and CF cells.

20 18 13 Fetal

33

11 10 13 10

Donor

Normal SaJO GM-37 GM-179 IMR-90

Carrier GM-1708

Cystic fibrosis GM-1957 GM- 1959 GM-1958 GM-1959

3 3 6 6

13

21 6 18 9

Passage level

4 4 9 8

18

28 8 23 19

Doubling level

74 NA’ 19 27

10

44 11 74 23

Noncyclinga cells (%I

673 681 197 197

212

694 77 691 553

Total time at 37°C (days)

NFd NFd 22 22

20

NFd 20 661 290

Time at 3PC since thawed Ways)

345 168 22 22

20

107 20 341 237

NA’ NA’ 0 to f 0 to 2

0

++to+++ 0 to 2 + to +++ 2to++

Azure A, pH 4.5

++ to +++ +to+++ *to++ *to++

0 to 2

++ to +++ 0 to k +++ to ++++ +to++

Alcian blue (pH 2.5)periodic acid- Schiff

Abundance of cytoplasmic bodiesb

for Various Periods in Culture

Time since last passage (days)

TABLE II Cytoplasmic Bodies in Fibroblasts Maintained

a The proportion of cells without labeled nuclei after exposure to tritiated thymidine for 3 days. b Lysosome-like structures stained blue to purple with azure A, and purple with al&n blue-periodic acid-Schiff. The values given reflect the range of prevalence of the stained bodies in different cells of a coverslip culture. 2 denotes a small number of weakly stained bodies. c Culture established here from a female donor. d Not frozen. e Not assayed.

Donor age Wirs)

Prevalence of Carbohydrate-Containing

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TABLE III Fibroblast Cultures Examined Ultrastructurally

Donor

Passage level

Time from last passage to fixation (days)

Normal DaRo DuWa JaPe DeRy BeWa RoTe

10 3 14 8 2 3

7 7 12 and 12 and 16 and 12 and

Cystic fibrosis RoMi ChAn RoCo JaPi PaWi PeMi

I5 2 3 4 4” 6

7 9 9 9 16 and 17 3

Time from last medium change to fixation (days)

13 13 17 13 4 5 5 5 9 2

LIAfter storage in liquid nitrogen.

FIG. 1. Small granules with staining demonstrative of acidic mucosubstance were found in the cytoplasm of cultured fibroblasts. Cells from CF patients and controls appeared comparably reactive. Coverslip stained with azure A at pH 4. x 800. FIG. 2. Cultured tibroblasts contain numerous small cytoplasmic bodies which apparently correspond with the dense bodies identified as heterophagic bodies at the ultrastructural level. These structures appeared comparable in cells cultured from normal donors and CF patients. Epoxy thick section stained with alkaline toluidine blue. x 1000.

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distilled water and staining with cytochemical methods. Coverslips were stained with azure A at pH 4.5 to localize carboxyl- and sulfate-containing mucosubstances and with a high iron diamine-alcian blue sequence which differentiated black-stained glycoconjugates containing sulfate esters from blue-stained mucosubstances possessing carboxyl but not sulfate groups (Spicer ef al., 1967). An alcian blue (pH 2.5)-periodic acid-Schiff (PAS) (Mowry and Winkler, 1965) sequence was also employed to distinguish blue- to purple-stained complex carbohydrate possessing acid groups from magenta-colored neutral mucosubstances. A few coverslips of CF and normal cells were allowed to dry in air after staining, prior to rinsing in xylol and mounting. These specimens appeared optimally stained. Coverslips fixed 3 min with formalin solution were also incubated 60 min at 37°C in a pH 5.0 medium for localizing acid phosphatase (Barka and Anderson, 1962). In addition, four normal, one carrier, and three CF tibroblast cultures were examined on coverslips at varying intervals with the alcian blue-PAS method (see Table II). These same cultures were examined for PAS reactivity after exposure to medium containing tritiated thymidine (0.1 pCi/ml, 20 Ci/mmol) for 3 days, formalin furation, and autoradiography (NTB3 emulsion, l-week exposure). With this procedure, it was possible to distinguish cycling cells which had incorporated tritiated thymidine during replicative DNA synthesis by the presence of autoradiographic silver grains over cell nuclei (Vincent and Huang, 1976). The staining of dense bodies was compared for the cycling and noncycling cells of each culture. The proportion of noncycling (unlabeled) cells in each culture was also determined from these preparations. The cultures were additionally examined for cytoplasmic vacuolation after staining with 0.5% toluidine blue in a 1% sodium borate solution. The cultures were not examined cytogenetically. Electron microscopic examination was carried out on cells in the 2nd to 15th passage and at 2 to 9 days following the last passage (Table III). Initial processing for electron microscopy entailed release of the cells from the culture flask by exposure to 0.25% trypsin for 2 to 3 min. Medium containing serum was then added to diminish protease activity and to allow some recovery from the effects of trypsinization. After a rinse in PBS, the suspended cells were fixed for 30 to 60 min by the addition of an equal volume of 6% glutaraldehyde in 0.1 M cacodylate, FIG. 3. A portion of a fibroblast cultured from a cystic fibrosis (CF) patient shows several stacks of Golgi lamellae (G) with closely associated rough- and smooth-coated vesicles and small vacuoles. Irregularly dilated cistemae of granular reticulum contain moderately dense (arrows) and less dense amorphous secretory product. The denser material often lies in the proximity of Golgi cistemae. Pleomorphic dense bodies (D) with heterogeneous content populate the cytoplasm. x 15,900. Figures 3 through 15, 21 and 22 illustrate uranyl acetate-lead citrate-stained thin sections of specimens fixed sequentially with glutaraldehyde and osmium tetroxide and processed for ultrastructural morphologic examination. The remaining electron microscopic figures illustrate cytochemical preparations. FIG. 4. A cistema of granular reticulum in a tibroblast grown from another CF patient contains dense material that appears immiscible with surrounding less dense luminal content. Such dense foci occurred comparably in normal and CF cells. ~22,500. FIG. 5. Small vesicles suggestive of endocytic or exocytic activity border a region under the plasmalemma of a tibroblast from a CF patient. x 12,800. FIG. 6. A Golgi zone from a CF tibroblast consists of closely stacked cistemae and numerous small vesicles lying between the convex (forming) face and the neighboring cistemae of granular reticulum above. Small vesicles and mitochondria border the presumed maturing face of the complex below. These features resemble those observed in cells from normal subjects. ~23,900. FIG. 7. This centriole and its bordering microtubules occupying an area near the Golgi complex of another CF tibroblast resemble such structures seen in normal cells. ~20,600.

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loo 60

CYSTIC

FIBROSIS

SoJo

&mid

CYSTIC

Fl9ROSlS

GM-1706 brrer)

60

60 40 20 i

L 100 SO

100 ;II ‘E L GM-37

80 60

60 YI 40 fj 20 E ; 100 60 60

NORMAL 100 SOJO 80

VI 40 i GM-179

E

;

GM-1958

5 20 I$ 100 B 60

l&+oF

Staring

of Granules

m w

GM-1956

GM-179

,

I idyI

,- , ,

0 0.5 1.0 1.5 20 2.5 30

0 0.5 IO 15 292.530 Stammg of Granules

CHART 1. The staining of cytoplasmic granules in 100 cells of normal and cystic fibrosis cultures with the alcian blue (pH 2.5)-periodic acid-Schiff procedure. Cells were categorized from 0 to 3.0 for the abundance of granules reactive to the stain, with higher values indicative of more abundant stained granules. The prevalence of cells with abundant stained cytoplasmic granules correleted with the time they had been in culture since the last passage (Table II). In this and the next two charts, culture GM-1959 was assayed at passage 6 and the other cultures were assayed at the passage level given in Table II. CHART 2. The abundance of cytoplasmic granules reactive to the periodic acid-Schiff method in 100 cycling (solid bars) and 100 noncycling (open bars) cells. Cells were exposed to tritiated thymidine for 3 days, fixed, autoradiographed, stained, and examined for dense bodies as described in Chart 1. Those cells which were capable of replicative DNA synthesis during the 3-day exposure to tritiated thymidine incorporated radiolabel into their nuclei and were identified in autoradiographs by the presence ofnumerous black silver grains over their nuclei. The abundance of stained granules was then assessed in the cycling (labeled) and noncycling (unlabeled) cells. Cycling and noncycling cells with few stained granules could be found in most cultures. But in cultures characterized by marked variability among cells in the abundance of stained granules, the noncycling cells appeared to contain the most abundant granules.

pII 7.4. Alternatively, cells were fixed by flooding with 3% glutaraldehyde and subsequently scraped free with a rubber policeman. Fixed cells were shipped in phosphate-buffered sucrose and on receipt were processed for morphologic and cytochemical examination. A CF and a comparable normal fibroblast strain established and grown in culture flasks in Charleston were also processed for electron microscopy. An aliquot of several experimental and control specimens was pelleted, sectioned in the cryostat, and stained with the dialyzed iron (DI) reagent for visualizing acid mucosubstances ultrastructurally or processed for the demonstration of acid phosphatase (Wetzel et al., 1966). A portion of ail specimens reserved for morphologic examination was fored for 60 min and, together with the cryostat sections, was rinsed and postfixed 1 hr in 2% osmium tetroxide prior to routine dehydration through graded alcohols and embedment in Epon. To localize antimonate reactive cations, an ahquot of some of the CF and normal cultures was fixed with a pyroantimonate-osmium tetroxide solution alone (Komnick, 1962),

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05 10 1.5202530 0510 15202530 Extent of Vacuolotm

CHART 3. The extent of cytoplasmic vacuolation revealed after staining with 0.5% toluidine blue. Most cultures did not exhibit significant vacuolation. One hundred cells were examined in each culture.

and was then rinsed briefly, dehydrated, and embedded in Epon. Ultrathin sections of cytochemical preparations were examined without heavy metal staining in an AEI 6B or Hitachi HS-8 electron microscope. Thin sections of morphologic preparations were examined after staining with a many1 acetate-lead citrate sequence . RESULTS Light Microscopy

Fibroblasts grown on coverslips from biopsies of CF and normal subjects disclosed the usual spindle contour and a central nucleus. The abundance of small bodies that stained with methods for complex carbohydrate appeared greatest in cells ftxed with Camoy fluid, somewhat less in those fixed with buffered formalin and still less in glutaraldehyde-fixed cells. With either of these fixatives, the fibroblasts displayed cytoplasmic granules which varied widely in prevalence. Some cells contained few or no granules; others displayed a moderate number of numerous reactive granules that bordered the poles of the nucleus and ranged from the limit of light microscopic resolution to approximately 2 grn in size. By cytochemical methods, the cytoplasmic bodies in the cultured fibroblasts revealed content of complex carbohydrate. The granules stained a metachromatic purple with azure A and, thus, disclosed presence of acid mucosubstance (Fig. 1). With the high iron diamine-alcian blue sequence, most granules stained black, demonstrative of sulfate glycoconjugate, but a few granules in occasional cells stained blue, demonstrative of carboxyl-rich, sulfate-free mucosubstance. The majority of the cytoplasmic granules stained with both alcian blue and the PAS method, appearing purple with the combined sequence of these techniques. The range of staining for carboxyl-rich or sulfated complex carbohydrate, or for periodate-reactive glycoconjugates did not differ significantly between normal and CF cultures (Table I). Cultured fibroblast lines from normal and CF donors ap-

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peared comparable also in disclosing numerous cells with few‘ to moderately abundant granules that stained for acid phosphatase. Cells in toluidine-blue stained epoxy sections of specimens processed for electron microscopy as a rule exhibited a rounded contour and an irregular nucleus surrounded by abundant cytoplasm containing numerous dark-stained cytoplasmic bodies (Fig. 2). Cells in epoxy thick sections of CF cultures did not differ discernibly from those of normal cultures. Differences in cytoplasmic granules reactive cytochemically for complex carbohydrate were evident in cultures with different histories in vitro, although each were cultured under similar conditions and assayed at the same time. The abundance of granules increased with the number of days in culture, the time in culture since the last passage and the percentage of noncycling cells (Table II). The prevalence of cells with few, moderately numerous, or abundant reactive granules appeared comparable in the CF and normal cultures (Tables I and II). No differences between the CF and normal cells were detected with respect to granule morphology. Further characterization of the different cultures listed in Table II for alcian blue-PAS-stained cytoplasmic bodies is given in Chart 1. Cultures with significant proportions of noncycling cells exhibited considerable cellular variability in content of stained granules, whereas cultures with few noncycling cells exhibited homogeneous cell populations with few or no reactive granules. Granule reactivity to PAS was considerably greater in the noncycling than cycling cells of the former cultures (Chart 2). Thus, in cultures with abundant cytoplasmic granules, reactivity appeared to be attributable to the presence of noncycling cells. The cultures were essentially unremarkable with respect to cytoplasmic vacuolation (Chart 3). Electron

Microscopy

Cells processed for this study disclosed the usual cytologic structures as described previously (Bartman et al., 1970). The cell profiles generally enclosed a variable portion of an irregular nucleus which often was deeply indented by cytoplasm and contained moderately dispersed heterochromatin. Nucleolar profiles, when present, commonly appeared near the margin of the nucleus, in contact with heterochromatin. Some nucleoli showed a nucleolonemal arrangement of the granular and dense components with a central pars amorpha, but others appeared more solid. FIG. 8. This profile of a tibroblast from a normal culture reveals numerous crowded vacuoles with lucent content and occasionally an eccentric dense focus. Some vacuoles, reminiscent of storage vacuoles in fibroblasts of patients with mucopolysaccharidoses, could theoretically account for metachromasia in the cultured cells, but were observed in relatively few cells in comparable numbers in CF and normal fibroblasts. Dense bodies lie between vacuoles and elsewhere in the hyaloplasm. Hayloplasm composed largely of a microtilamentous component separates cistemae of granular reticulum and mitochondria. x6500. FIG. 9. In a tibroblast from another normal subject, a lucent vacuole borders a dense body containing heterogeneous material. x 35,600. FIG. 10. In a cell from a normal fibroblast culture, broad bands of microtilaments intervene between mitochondria, dilated cistemae of granular reticulum and dense bodies. The latter vary widely in structure and content. x 13,600. FIG. 11. Dense bodies in a cell from a CF fibroblast culture vary in content but generally resemble those in cultured normal cells (cf. Fig. 10). These dense bodies apparently correspond with the small bodies stained at the light microscopy level with cytochemical methods for visualizing complex carbohydrate. x 30,OflO.

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A moderate number of microvilli or irregular processes protruded randomly from the surface of the fibroblasts. Empty-appearing vesicles, presumably of pinocytic nature, bordered some regions of the plasmalemma (Fig. 5). The cells enclosed moderately numerous, irregular, dilated cistemae of rough endoplasmic reticulum in which occasional accumulations of denser material lay separated from less dense substance distending the cistemal lumen (Figs. 3,4). These denser foci appeared more centrally located near Golgi elements in some cell profiles (Fig. 3). The Golgi complex commonly neighbored the nucleus and in a favorable plane of section exhibited parallel, collapsed cistemae closely bordered by smooth and rough-coated vesicles (Figs. 3,6) and, in some cell profiles, by a centriole with associated microtubules (Fig. 7). A variable number of widely scattered dense bodies infiltrated the tibroblast cytoplasm (Figs, 3, 8, 11). These pleomorphic structures enclosed in varying proportions lucent foci and areas with moderately dense granular or membranous material. Some cell profiles also enclosed few to numerous round vacuolar structures which were generally crowded together near the nucleus and, when abundant, filled much of the cytoplasm at one pole of the cell (Fig. 8). These vacuoles were limited by a unit membrane and were filled by a lucent, flocculent content, often including a focus of dense material in the periphery (Fig. 9). A few to very abundant microfilaments usually infiltrated the hyaloplasm. In extreme cases these filaments largely filled the cell to the exclusion of the usual structures (Fig. 10). Free polysomes populated the hyaloplasm in abundance in some cell profiles (Fig. 15). Lipid droplets occasionally occurred free in the cytoplasm (Fig. 12) and, FIG. 12. Lucent lipid droplets in a fibroblast from a CF patient have a dense periphery with myelinlike material. Lipid droplets occurred infrequently in most cell stains. x 12,800. FIG. 13. Glycogen particles surround a lipid droplet in a fibroblast from another CF patient. x 16,900. FIG. 14. Glycogen pools fill extensive areas in the periphery of a fibroblast from a CF patient. A membrane and myelin t&tires lie in the glycogen area. Glycogen varied widely in normal and CF cells and, although apparently somewhat more abundant in cells from two of the CF patients, would be difficult to assess quantitatively by electron microscopy. x 15,000. FIG. 15. Hyaloplasm in this cell from a normal culture contains numerous free polysomes. Collections of ribosomes were infrequent in normal and CF cells. A somewhat dilated cistema of granular reticulum is continuous with the nuclear envelope and encloses a projection of cytoplasm into its lumen. x 11,700. FIG. 16. Precipitates demonstrating antimonate-reactive cations rim irregular lipid droplets and occupy a dense body in a cell from a CF patient. Lighter deposits line the inner face of the plasmalemma. Antimonate precipitates appeared similar in normal and CF cells. Antimonate-osmium tetroxide fixation. Unstained thin section. x 11,300. FIG. 17. Like Fig. 16, showing the precipitates in the rim of lipid droplets in another cell. ~20,000. FIG. 18. Structures corresponding with the cytoplasmic dense bodies evident morphologically (cf. Figs. 8- 11) contain heavy deposits indicative of acid phosphatase in this tibroblast from a CF culture. Golgi elements below the nucleus stain strongly. Acid phosphatase preparation unstained thin section. x 8800. FIG. 19. The surface of the plasmalemma of a glutaraldehyde-fixed normal cell shows a layer of acidic complex carbohydrate. Cytoplasmic dense bodies did not clearly evidence content of acid mucosubstance in glutaraldehyde-fixed cells, although their intrinsic density, probably attributable to lipid, could have obscured staining. Dialyzed iron (DI)-stained cryostat section. Unstained thin section. ~20,000. FIG. 20. A normal fibroblast fixed with buffered formalin discloses staining of the outer surface of the plasmalemma (upper left) as well as light reactivity in cytoplasmic bodies (arrows), possibly corresponding with the dense bodies of morphologic preparations. DI-stained cryostat section. Unstained thin section. x 12,500.

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in some instances, were intimately associated with abundant glycogen (Fig. 13). Glycogen particles in some cells appeared scattered in the hyaloplasm and, in others, aggregated into large confluent pools which commonly occupied the cell periphery, often filling blunt, pseudopod-like processes (Fig. 14). Glycogen aggregates varied widely in different cells within and between specimens, and were not characteristically present or absent in profiles of CF or normal donors. The largest peripheral glycogen pools with enclosed myelin figures occurred in fibroblasts from a CF patient. Otherwise, fibroblasts cultured from CF patients afforded no evidence for a change in abundance, distribution, or morphology of their cytoplasmic structures, including cytoplasmic dense bodies and vacuole-like structures. As at the light microscopic level, the cytoplasmic dense bodies of presumed lysosomal nature in cells at high population doubling level markedly exceeded in number those at low doubling levels but age-matched CF and normal cultures did not differ notably from one another at either stage. Cells fixed with the antimonate-osmium tetroxide solution revealed antimonate deposits lining the plasmalemma (Fig. 16) and rimming the rather irregular lipid droplets (Figs. 16, 17). The dense bodies also contained antimonate precipitates. Reaction product, indicative of acid phosphatase, filled structures that corresponded with the dense bodies in morphologic preparations (Fig. 18), further identifying these bodies as lysosomal organelles. Golgi cistemae showed reactivity (Fig. 18) and nucleolar foci that appeared to correspond with the pars amorpha also exhibited acid phosphatase reaction product. Control sections incubated in the absence of glycerophosphate substrate lacked these precipitates. Fibroblasts stained with dialyzed iron for the ultrastructural localization of acidic complex carbohydrate disclosed a thin layer of densification on the outer surface of the plasmalemma (Figs. 19, 20). In glutaraldehyde-fixed specimens, density was not clearly imparted to dense bodies by this method; but in formalin-fixed cells, the material in dense bodies appeared to be stained by dialyzed iron. Fibroblasts from CF and normal subjects revealed no distinct difference in cytochemical reactivity for antimonate reaction cation, acid phosphatase, or acidic complex carbohydrate. Cells exposed to ferritin for 24 hr exhibited cytoplasmic dense bodies with sparse to abundant incorporated particles (Figs. 21, 22). The ferritin was evident in small vesicles as well as dense bodies and in the later occupied all or a part of the structure. The prevalence of ferritin-loaded bodies appeared roughly comparable in normal and CF cells (Table IV). Fibroblasts incubated 2 hr with the tracer disclosed on the average only one or two ferritin-labeled phagosomes per cell profile. It was not possible to relate the number of fen-kin-labeled vesicles and dense bodies to the total number of these structures present in each profile. This is because the lack of stain in the sections (necessary to distinguish the presence of tracer) made it difficult to detect all small vesicles and dense bodies, especially those without tracer. However, the similarity in the number of small vesicles and dense bodies observed in stained profiles of CF and normal cells suggests the percentage of the total number of small vesicles and dense bodies which incorporated tracer is probably also similar in the CF and normal cells. DISCUSSION The variable but similar staining for complex carbohydrates in fibroblasts from normal and CF donors raises a question as to the basis for the reported extensive metachromasia in CF tibroblasts (Danes and Beam, 1969; Beam and Danes,

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FIG. 21. A cell from a CF fibroblast culture encloses dense bodies tilled with ferritin particles. Uptake of ferritin from the culture medium into the cytoplasmic dense bodies identified these structures as at least partially heterophagic in origin. Ferritin particles vary from absent to very abundant in different dense bodies, occupying only a part of some bodies. Routine processing for morphologic examination after 24-hr exposure to ferritin-loaded culture medium. Unstained thin section. x7500. FIG. 22. Another fibroblast from the culture shown in Fig. 21. x 11,600.

1970). Metachromasia in fibroblasts could most reasonably be attributed to complex carbohydrates, which possess carboxyl and sulfate groups that bind basic dyes in required proximity. Acid mucosubstances account for most of the known histologic metachromasia (Schubert and Hamerman, 1956), aside from the less distinct B-metachromasia attributable to ribonucleic acid (Flax and Himes, 1952), and the pathological metachromasia attributable to sulfatides in metachromatic leukodystrophy of the central nervous system (Moser, 1960). The metachromasia previously observed in CF tibroblasts could not likely be accounted for by ribonucleic acid or pathologic lipid accumulation, but conceivably is attributable to production of complex carbohydrate under conditions that differed in previous studies from those prevailing in this investigation. Under culture methods employed in this study, however, it was not possible to demonstrate the presence of increased amounts of complex carbohydrate or other potentially metachromatic substances in fibroblasts from CF patients. The ultrastructural examination concurs with that at the light microscope level TABLE IV Number of Labeled Cytoplasmic Dense Bodies after 24 hr exposure to Ferritin

Culture

Number of cell profiles examined

Normal Cystic fibrosis

22 33

Fe&in-containing bodies 175 265

Ferritin-containing bodies per cell profile 7.2 8.0

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in failing to visualize in CF flbroblasts an increase of organelles that could account for metachromasia as do cytoplasmic granules in mast cells or leukocytes and mucous droplets in mucigenic epithelium (Schubert and Hamerman, 1956; Horn and Spicer, 1964; Spicer et al., 1967). The vacuolar structures encountered in occasional control and test cells provide a possible fine structural basis for metachromasia at the light microscopic level as they resemble ultrastructurally the vacuoles found in fibroblasts of patients with Hurler’s disease (DeCloux and Friederici, 1969; Bartman and Blanc, 1970) and shown by electron microscopy to contain acidic complex carbohydrates (Spicer et al., 1978). However, cells with these vacuoles occurred so infrequently in the specimens examined that it was not possible to correlate ultrastmctural vacuolation with the presence of metachromasia at the light microscope level. Investigation of factors influencing prevalence of these vacuoles, such as the number of days after last subculturing and an alkaline shit? in the pH of the medium, did not yield conclusive evidence for an effect. The basis for the occurrence and the nature of the vacuoles remains undetermined. Vacuoles sufficiently large to be detected by light microscopy were observed in the CF and normal cells, but they were not very prevalent or more abundant in the CF cells (Chart 3). Cytoplasmic dense bodies, on the other hand, appeared to correspond in abundance, size and distribution with granules evidencing complex carbohydrate by light microscopic cytochemistry. These bodies, therefore, constitute a potential basis for metachromasia. The structure and abundance of dense bodies varied comparably between different cells and different specimens for fibroblast cultures derived from normal donors and from CF patients. A relation was evident between abundance of dense bodies and the number of days between the last change of medium and fixation. The bodies also appeared increased with the number of population doubling levels for a given cell strain. These bodies appear, from their heterogeneous content and pleomorphic structure, to be secondary lysosomes concerned with autophagy or heterophagy, rather than secretory organelles. Their increase with time in culture suggests accumulation of undigestible material in the process of turnover of cell components during autophagic activity. However, the dense bodies are shown here by their ferritin uptake to function also in heterophagy and to be concerned, at least in part, with degrading endocytosed matter. The prevalence of autophagic, residual, and multivesicular bodies has been shown to be increased in old (passage 40 and over) diploid human cultures (Lipetz and Cristofalo, 1972). The proportion of noncycling cells also appears increased in senescent cultures (Cristofalo, 1972; Cristofalo and Sharf, 1973; Vincent and Haung, 1976). Our results (Chart 2) suggest that the accumulation of lysosomal bodies occurs to a greater extent in the noncycling cells of the cultures, and that the ,increased number of lysosomal bodies in cells of senescent cultures may be attributable in part to the increased proportion of noncycling cells in the senescent cultures. Although definitive fine structural differences between normal and CF tibroblasts were not demonstrated, the present morphologic examination cannot be considered to eliminate biochemical differences. Despite the lack of fine structural and cytochemical evidence for an abnormality of cultured fibroblasts from CF patients, these cultured cells may conceivably express the defective gene at a molecular level without undergoing changes detected by methods utilized here.

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Such a possibility derives support from the absence of apparent pathognomonic ultrastructural change in sweat glands of CF patients (Munger et al., 1961), despite their abnormal electrolyte transport capacity (di Sant’Agnese et al., 1953). On the other hand, small quantitative or qualitative fine structural differences could exert a clinical effect over a prolonged period in the CF patient, and exist as yet undetected in the cultured fibroblasts. More precise morphometric determinations perhaps could elucidate such a potential difference. The CF fibroblasts, for example, often evidenced abundant glycogen, some of which appeared sequestered in vacuoles. However, some normal cells also disclosed conspicuous glycogen pools in the peripheral cytoplasm. Because of variability between and within specimens, uncertainty as to the degree of preservation of glycogen in the cytoplasm with glutaraldehyde furation, and difficulty in quantitating glycogen morphometrically , the question of increased glycogen in cultured CF fibroblasts indicated biochemically (Pallavicini ei al., 1970) could not be established ultrastructurally. Fibroblasts grown in culture displayed certain striking cytologic features in both CF and normal cell strains. In some cells, for example, abundant microfdaments comprised a major cell constituent. These filaments resembled in general morphology those filling adrenocorticotrophic cells involved in the Crook’s hyaline pathologic change (Porcile and Racadot, 1966), suggesting cell involution. Narrower filaments that consist of actin-like proteins on the basis of their size, and that may function in cell motility, were also observed in fibroblasts, but were relatively infrequent. The preponderant intermediate sized filaments observed here seem not to serve a contractile capacity in fibroblasts. Additionally, the content of cistemae of the granular reticulum appeared heterogeneous in some normal and CF fibroblasts. Aggregation of luminal content in rough endoplasmic reticulum possibly reflects a stage in passage of secretory material from their cistemae, as these coalescent foci occasionally were observed in regions of the reticulum proximal to Golgi lamellae. The nature of this presumed secretory material in the moderately abundant profiles of granular reticulum remains uncertain, but presumably the material is not collagen since cultured fibroblasts secrete little collagen. A proteoglycan seems a more likely explanation as fibroblasts are known to secrete glycosaminoglycans into the culture medium (Matalon and Dorfman, 1968; Welch and Roberts, 1975). ACKNOWLEDGMENTS The skilled technical assistance of Ms. N. T. Brissie and B. J. Hall and the secretarial and editorial assistance of Ms. Dot Smith and Fran Cameron are gratefully acknowledged. We also thank Mr. J. F. Rogers for making available culture SaJo.

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TAYSI, K, KISTENMACHER, M. L., PUNNETT, H. H., et al. (1969) Limitations of metachromasia as a diagnostic aid in pediatrics. N. Engl. J. Med. 281, 1108- 1111. VINCENT, R. A., JR., and HUANG, P. C. (1976) The proportion of cells labeled with tritiated thymidine as a function of population doubling level in cultures of fetal, adult, mutant, and tumor origin. Exp. Cell. Res. 102, 31-42. WELCH, D. W., and ROBERTS, R. M. (1975). Complex saccharide metabolism in cystic fibrosis tibroblasts. Pediatr. Res. 9, 698-702. WETZEL, M. G., WETZEL, B. K., and SPICER, S. S. (1966) Ultrastructural localization of acid mucosubstances in the mouse colon with iron-containing stains. J. Cell. Biol. 30(2), 299-315. WIESMANN, U., and NEUFELD, E. F. (1970) Metabolism of sulfated mucopolysaccharide in cultured tibroblasts from cystic fibrosis patients. J. Pediarr. 77, 685-690.