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Permeability of the epidermis and the phagocytic activity of keratinocytes
1971 by Academic Press, Inc.
176
J. ULTRASTRUCTURE RESEARCH
36, 176-190 (1971)
Permeability of the Epidermis and the Phagocytic Activity of Keratinocytes Ultrastructural
Studies with Thorotrast
as a M a r k e r 1
KLAUS WOLFF AND HERBERT HONIGSMANN
Department of Dermatology (I.), University of Vienna, A-1090 Vienna, Austria Received September 15, 1970 This paper details investigations on the permeability of the intercellular spaces of guinea pig epidermis and the phagocytic capacity of keratinocytes as studied with a long-term tracer (Thorotrast) at the ultrastructural level. Immediately after injection into the skin, the tracer penetrates the basal lamina and permeates the intercellular compartment of the epidermis. A barrier to further spread is located in the upper stratum granulosum. Thorotrast fails to penetrate the desmosomes, and this contrasts the findings obtained with low molecular weight proteins. Five minutes after the injection the tracer is found in large vacuoles within keratinocytes which probably represent the "paranuclear cisternae" described previously and are believed to be continuous with the intercellular space. Osmotic pressure leads to a rupture of some of these vacuoles in the stratum granulosum and to cytolysis of some cells. Active uptake of Thorotrast by keratinocytes commences approximately 1 hour after the injection and continues as long as a tracer depot is present within the dermis. The internalization of the marker is accomplished by means of single membrane-limited phagosomes which transfer Thorotrast into the interior of the cells. Since Thorotrast is indigestible, it is stored in the vacuolar system of keratinocytes and is eliminated from the epidermis during the keratinization process. The results of the present study underscore the excellent permeability of the intercellular spaces for small particles; the readiness of keratinocytes to phagocytose even indigestible material, and the fact that substances released in the dermis diffuse into the epidermis by a continuous flow as long as a sufficient supply is maintained by the dermal depot. The most important biological function of epidermal cells is the synthesis of the structural proteins which constitute the basic c o m p o n e n t of keratin. Consequently, most investigations on the epidermis have been preoccupied with this synthetizing capacity of its cells. However, keratinocytes also possess resorptive potentials x This work was supported in part by Fonds zur FSrderung der wissenschaftlichen Forschung, Vienna, and Schering, AG, Berlin.
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FIG. 1. Five minutes after injection the tracer is present in the dermis, extending as far as the basal lamina (BL). Thorotrast has entered the intercellular spaces of the epidermis (arrows) and is seen in a paranuclear vacuole (V). Two pinocytotic vesicles at the basal cell membrane contain a few Thorotrast particles (thin arrows) N, nucleus; M, melanosome, x 25 600. Inset: An enlargement of one of the pinocytotic infoldings containing tracer material, x 91 200. 1 2 - 711839 J. Ultrastructure Research
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which are based on mechanisms of heterophagy (11, 12, 23, 24). Recent studies have shown that tracer proteins injected into the dermis rapidly spread into the epithelium, where they permeate the intercellular spaces (18, 19). The keratinocytes avidly ingest these proteins and degrade them within their lysosomal system (23, 24). This experimental approach provides a model for studies on mechanisms that may be operative in the uptake of endogenous substances by epidermal cells. In order to determine whether the phagocytic properties of keratinocytes are of a more general or a discriminatory nature, it was now considered desirable to extend these studies by employing physically and chemically different markers. The present paper is based on investigations with Thorotrast, a colloidal thorium dioxide stabilized with dextrin. This substance can be easily identified at the ultrastructural level, it does not damage the cells (14, 15) and it differs from the tracers employed so far both with respect to particle size and chemistry. The inorganic moiety cannot be decomposed by the cells and thus offers the opportunity to study the process by which such substances are eliminated from the epidermis.
MATERIALS AND METHODS Albino and white-spotted guinea pigs were used in these experiments. Aliquots of 0.I ml Thorotrast [a colloidal solution of thorium dioxide stabilized with dextrin; particle size in the order of 50-125 •, with most in the range of 70-90 A (according to the manufacturer); obtained from Fellows Testagar, Inc., Detroit, lot 15 098] were injected intracutaneously into multiple sites of dorsal skin of the ears. Biopsies were performed after 5 minutes, 1, 3, 12, 24, and 48 hours, and 3, 4, 5, 7, 9, and 14 days after injection. The tissue was minced with a razor blade and fixed immediately in 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) at 4°C for 2-6 hours. After several rinses and overnight storage in cacodylate buffer, the tissue was posffixed in 1 To osmium tetroxide in barbital acetate buffer (pH 7.3) and stained en bloc in 0.5% uranyl acetate in barbital acetate buffer. Some specimens were osmicated without glutaraldehyde fixation. After rapid dehydration the tissue was embedded in Epon 812. Ultrathin sections were cut on a Reichert OM U2 ultramicrotome, mounted on Formvar-coated copper grids, and stained with uranyl acetate and lead citrate. The specimens were examined with a Zeiss EM 9S electron microscope. Controls AIiquots of 0.l ml of a saturated solution of dextrin and of saline, respectively, were injected into control animals. The experimental conditions and the processing of the tissue were as described for the main experiment.
FI6. 2. Higher magnification of the dermal-epidermal junction shown in Fig. 1. Most of the Thorotrast is on the dermal side of the basal lamina (BL), and only a few particles are seen beyond this structure (arrow). The intercellular space (ICS) of the epidermis shows normal dimensions, but in one area it is expanded by aggregates of tracer particles. The pinocytotic infoldings (PV) of the basal cell membrane are devoid of Thorotrast. C, collagen. × 91 200.
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RESULTS 1. Five minutes after the injection the tracer was diffusely distributed between the collagen fibers and ceils of the dermis. A definite stop occurred at the epidermal basal lamina but small amounts of Thorotrast were observed beyond this barrier (Figs. 1 and 2) and in some areas the marker was seen to have entered the intercellular spaces of the epidermis (Figs. 1 and 2). Most micropinocytotic infoldings of the cytomembranes of basal cells were devoid of the tracer, but, occasionally, a pinocytotic vesicle contained a few dense particles (Fig. 1). The intercellular spaces of the epidermis appeared normal, but in some specimens they were dilated and the surfaces of the keratinocytes formed villuslike cytoplasmic projections. Desmosomes were present in normal numbers and showed no structural alterations. Variable amounts of Thorotrast were present in the intercellular spaces of the entire viable epidermis, usually appearing in focal aggregates (Figs. 2 and 3). There was a decreasing gradient of intercellular tracer toward the upper epidermal layers. The intercellular spaces of the upper stratum granulosum which contain the lamellae derived from Odland bodies (21) were regularly devoid of Thorotrast. Although the tracer was frequently seen to have accumulated at one end of a desmosome, it was never observed within the desmosomes proper (Fig. 3). Even at this early stage of the process the tracer was observed intracellularly. Thorotrast was regularly found in large vacuoles of the keratinocytes where it showed a tendency to clump and to accumulate on the inner surface of the delimiting membrane (Fig. 4a). Most of the vacuoles contained only sparse amounts of the tracer (Fig. 4a), but some were packed with the dense particles (Fig. 1). They were present in a considerable number of keratinocytes and often acquired such monstrous dimensions that they distorted the nucleus and displaced it to the cell periphery (Fig. 4a). Subserial sections revealed that the vacuoles were truly intracellular and did not represent dilatations of the intercellular spaces bulging into the cells. In some keratinocytes of the granular layer these vacuoles had ruptured spilling the tracer into the cytoplasm (Fig. 4 b) where it exhibited a tendency to coat keratohyalin granules (Fig. 4b). These cells contained Thorotrast diffusely throughout their cytoplasm and showed signs of cytolysis. One hour after the injection, an increased number of granular and spinous cells contained ruptured vacuoles and a diffuse dispersion of Thorotrast. One to 48 hours after the injection dermal macrophages and fibroblasts were observed to exhibit phagocytic activity and to take up Thorotrast from the dermal depot. There was a decrease of the amount of intercellular tracer within the epidermis and a reduction of the number of cells containing giant vacuoles or exhibiting an intracytoplasmic dispersion of the tracer. After 48 hours no such cells were present
FIG. 3. (a) Dilated intercellular spaces of the epidermis containing sparse amounts of tracer (arrows). Villuse-like projections (V) are seen on the surfaces of keratinocytes; the desmosomes (D) contain no Thorotrast particles. Five minutes after injection. × 91 200. (b) Convoluted intercellular space of the epidermis, exhibiting small focal aggregates of Thorotrast (arrows). The intercellular space is not widened, pinocytotic vesicles (PV) and a desmosome (D) are devoid of tracer. Three hours after injection, x 91 200. Inset: intercellular accumulation of Thorotrast at one end of a desmosome. One hour after injection, x 54 000.
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in the viable epidermis, but the lower layers of the stratum corneum contained a population of cells which lacked a typical keratin pattern and held Thorotrast within the confines of their membranes. At this time small deposits of Thorotrast were also seen within the intercellular cement of the horny layer. 2. Active uptake of the tracer by keratinocytes was first observed 1 h o u r after injection and was pronounced in the 3 hour specimens. Micropinocytotic infoldings of the plasma membranes rarely contained tracer, but the peripheral cytoplasm of keratinocytes regularly exhibited a considerable number of phagosomes which were packed with dense particles (Fig. 5). These vacuolar structures were limited by a trilaminar membrane; they were usually round or oval and measured up to 0.5/~ in diameter, occasionally attaining even larger dimensions (Figs. 5 and 6). In the initial phases of the ingestion process phagosomes were confined to the basal and lower spinous layers, but after 48 hours they were numerous in all strata of the epithelium. Most frequently they were seen in a paranuclear position or in the vicinity of the Golgi region (Fig. 5 c, d). Membrane-limited melanosome complexes also contained Thorotrast particles (Fig. 6 b). This interesting association of ingested endogenous and exogenous materials within a single phagocytic vacuole is the subject of a separate investigation. 3. Three to 14 days after the injection, variable amounts of the tracer were still present in the dermis; however, there was a considerable shift to intracellular localization as now most of the Thorotrast was observed within macrophages. No tracer was detected in the intercellular compartment of the epidermis, but, apparently, the keratinocytes did not cease to engulf Thorotrast as phagosomes charged with electron dense particles continued to appear within their cytoplasm. As the interval after the injection increased there was a reduction of the number of phagosomes and of the amount of their electron dense contents. There was a definite relationship between the magnitude of the phagosome population within the epidermis and the type of distribution of Thorotrast within the dermis. Epidermal phagosomes were encountered only as long as extracellular Thorotrast was present in the connective tissue (in some instances this was the case as late as 14 days after the injection). Biopsy specimens in which all detectable tracer in the dermis had been engulfed by macrophages exhibited no phagosomes within the epithelium. From the third day on Thorotrast was also observed within fully keratinized Fie. 4. (a) Giant vacuole (V)in a spinous cell 1 hour after injection of Thorotrast. The tracer forms small clumps and tends to attach to the vacuolar membrane (arrows). The nucleus (N) is indented and displaced to one side of the cell. M, mitochondria; T, tonofilaments, x 18 0C0. (b) Ruptured giant vacuole (V) exhibiting a collapsing delimiting membrane which is coated by Thorotrast particles (arrows). Thorotrast is also seen to be diffusely distributed within the cytoplasm (arrows). Twenty four hours after injection. N, nucleus; T, tonofilaments. × 18 000. Inset: Keratohyalin (KI4) coated by Thorotrast (arrows). x 54 000.
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cells of the stratum corneum (Fig. 6c). The tracer was aggregated into well defined intracellular foci which exhibited similar shapes and sizes and a similar distribution as the phagosomes seen in the viable keratinocytes. Occasionally, small amounts of the marker were diffusely dispersed between the keratin fibrils and within the intercellular space. Intraepidermal Langerhans cells also exhibited phagocytic activity, but they engulfed much less tracer than the keratinocytes. Thorotrast was confined to occasional phagosomes within their cytoplasm, but at no time was it observed within the specific Langerhans cell granules. Melanocytes were always devoid of Thorotrast. 4. Controls. Specimens of skin injected with dextrin exhibited morphologic changes which were identical with those of the main experiment. In the early phases after dextrin injection the keratinocytes exhibited giant vacuoles which lacked electron dense contents. These giant vacuoles were regularly absent from the controls injected with saline. DISCUSSION The results of the present report bear on problems of intraepidermal permeability, on the induction of phagocytosis by keratinocytes and on the elimination of foreign material from the epidermis. For reasons of clarity these different aspects are discussed separately.
The permeability of the intercellular space of the epidermis Considerable amounts of the tracer reached the epidermis despite the fact that the bulk of Thorotrast had been stopped at the basal lamina. This indicates that, although the lamina proper may not be permeable to particles of this size (70-90 A), fenestrations or gaps must exist within this structure. The rapid spread of the tracer within the epidermis reemphasizes the excellent permeability of the intercellular spaces demonstrated in earlier experiments (18, 19, 22). However, the desmosomes were not permeated by Thorotrast, and this contrasts with the findings obtained with smaller tracer molecules, such as horseradish peroxidase (18) or the lanthanum complex (22), which readily diffuse through the attachment devices. The distance between the outer leaflet of the cell membrane and the intercellular contact layer (13) [M-line (17)] of the desmosome measures less than 100 A and, since the diameter of the individual Thorotrast particles approaches this order of magnitude, their size may account for their inability to enter these structures. In addition, the restriction of the desmosomal permeability to Thorotrast may have been partly due to the glycocalyx present in these structures (22) and to an effective enlargement of the tracer particles by hydration. However, as desmosomes represent only focal areas of intercellular contact (5), they did not interfere with the overall spread of the marker within the extracellular compartment.
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Fia. 5. (a) Single membrane-limited phagosomes (P) in the peripheral cytoplasm of a keratinocyte. In contrast to the intercellular space (ICS) which contains only sparse amounts of Thorotrast (arrows), the phagosomes are densely packed with tracer particles. One hour after injection, x 25 600. (b, c and d). Different examples of phagosomal structures (P), some of which have acquired considerable dimensions. Note the dense packing of Thorotrast within these phagosomes. M, mitochondria; N, nucleus; G, Golgi apparatus, x 25 600.
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The intercellular space of the epidermis is sealed from the external milieu by a barrier which prevents the loss of extracellular fluid through the surface of the skin. Actually, this barrier is formed by the entire stratum corneum and portions of the transitional layer but with regard to the outward permeability of the intercellular spaces it probably starts at different levels for different compounds, depending on the chemistry and physical properties of these substances. As regards the small molecules of horseradish peroxidase [(radius ~ 25-30 • (7)] the barrier has been shown to start at that particular level of the epidermis where the lamellar structures of Odland bodies (21) [membrane coating granules (9), keratinosomes (20)] appear within the intercellular space (18, 19). It has been proposed that these lamellae seal the intercellular compartment and, in addition, alter its contents enzymatically (18). As for Thorotrast, the present investigation has failed to pinpoint the exact site of the barrier since the intercellular spaces were not filled continuously by the tracer. However, in the early stages of the experiment the marker was neither found between the intercellular lamellae nor beyond this zone and this, by inference, locates the lower limits of the barrier to the same level as for peroxidase (18). The fact that, 24 hours after the injection, the tracer was observed between the lamellae does not reflect a penetration phenomenon since these lamellae are continuously discharged into the intercellular spaces (9, 21), which at this time may have already contained Thorotrast. This also explains the presence of tracer in the intercellular cement of the stratum corneum as the marker probably accompanied the cells as they moved upward into the horny layer. The nature of the giant vacuoles awaits clarification. Since the vacuoles were observed immediately after the injection and appeared much earlier than the phagosomes, they should not be ascribed to endocytic uptake mechanisms but rather to preexisting cavities which had filled up from the intercellular space. Recently, evidence has been presented that paranuclear cisternae exist in keratinocytes which are continuous with the intercellular compartment and are permeated by horseradish peroxidase immediately after the tracer has entered the epidermis (18). It does not appear unreasonable to assume that the giant vacuoles observed in our experiment correspond to such dilated cisternae. The Thorotrast solution employed in this investigation was hypertonic and, consequently, osmotic forces may have been responsible for the expansion of these cavities and their subsequent rupture. This conclusion is supported by the fact that (hypertonic) dextrin also led to the formation FIG. 6. (a) Higher magnification of a phagosome containing Thorotrast. The arrows denote the trilaminar delimiting membrane, x 91 200. (b) Melanosome complex of a keratinocyte which contains Thorotrast within its delimiting membrane. The arrows mark some of the melanosomes. 24 hours after injection, x 18 000. (c) Thorotrast within a horny cell. Five days after injection. ME, melanosomes, x 43 500.
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of giant vacuoles whereas the injection of saline did not. However, it remains to be proved whether this interpretation is correct and whether, in the lysed cells, the affinity of Thorotrast to keratohyalin was due to a selective physical chemical effect or was nonspecific.
2. Phagocytosis of Thorotrast by keratinocytes The results of our study show that keratinocytes can be induced to engulf and store substances which cannot be utilized metabolically. The internalization of the tracer was accomplished by means of single membrane-limited phagosomes which were probably formed by infoldings of the plasma membrane. This process was a very rapid and efficient one, as can be inferred from a comparison of the relatively scant amounts of tracer within the intercellular spaces and the relatively large number of phagosomes within the cells. The readiness of keratinocytes to engulf the marker may also explain the observation that, in later phases of the process, phagosomes continued to appear within the cells although no tracer was detected in the intercellular spaces. Apparently, the Thorotrast particles were removed by the epidermal cells immediately after they had reached the intercellular compartment. The studies on protein uptake by keratinocytes have shown that acid phosphatase is incorporated into phagosomes which are thereby equipped enzymatically to degrade the phagocytosed material (23). The dynamics of the lysosomal system of the epidermis (24) conform to the general outlines for lysosomes as proposed by de Duve and Wattiaux (4), and it may be assumed that a similar transformation of phagosomes into (phago)lysosomes had occurred in the present experiment. The visualization of this process is, unfortunately, subject to technical limitations since it is impossible to distinguish Thorotrast particles from the reaction product of acid phosphatase. Thorotrast was stored in the vacuolar apparatus of keratinocytes until the cells underwent keratinization. In this respect the results of the present investigation differ decisively from those obtained with the tracer protein (23). While it may be assumed that the stabilizer dextrin was decomposed within the lysosomal system of the keratinocytes the inorganic moiety of Thorotrast certainly was not, and this afforded the opportunity to follow the further fate of the ingested material until it was eliminated from the epidermis. As keratinocytes remove an indigestible and nonusable material from the intercellular spaces they perform a scavenger function. A recent theory (16) has ascribed such a function to Langerhans cells, which, on morphological grounds, have been considered to represent epidermal macrophages (6). This contention has been questioned on the basis of experimental evidence which has shown that the phagocytic potentials of Langerhans cells are far less than those of keratinocytes (25). The
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results of the present study offer additional evidence against this epidermal "macrophage theory." Thorotrast was regularly observed within melanosome complexes of keratinocytes. Since the cytoplasm of melanocytes was always devoid of tracer particles, only two alternatives remain to explain this surprising phenomenon: Thorotrast was either incorporated into melanosome complexes by fusion of the complexes with phagosomes or the melanosomes and the tracer had been taken up simultaneously from the intercellular spaces. The second alternative would, however, necessitate a revision of the present concept on pigment transfer mechanisms (1, 8, 10). This problem is presently investigated in our laboratory. 3. The elimination of the tracer from the epidermis The foreign material was removed from the epidermis by two separate mechanisms. One consisted of the shedding of those cells which, after rupture of their giant vacuoles, had exhibited a diffuse dispersion of tracer within their cytoplasm and had lysed. The second mode of Thorotrast elimination was brought about by the keratinization of cells which had engulfed the tracer actively and had stored this material within their cytoplasm. This type of elimination was observed as early as 3 days after injection, and this time interval is well below the transit time of the guinea pig epidermis (2, 3). This may be explained by the fact that the tracer was not only engulfed by the basal cells but also by keratinocytes which were already moving upward to the horny layer. In addition, the injection of the tracer into the dermis may have represented a sufficient stimulus for a transient increase of the turnover rate of epidermal cells. The length of the period during which the tracer was taken up and eliminated by keratinocytes depended on the availability of an extracellular depot in the dermis. This indicates that a constant flow of Thorotrast into the epithelium was maintained and permits an important conclusion: substances injected into the dermis reach the epidermal compartment not only by force of the hydrodynamic pressure of the injection but diffuse upward as long as the dermal supply lasts. This model demonstrates that endogenous compounds released into the dermal compartment may reach the epithelium by a similarly continuous flow. We thank Mrs. Lotte Polasek and Mrs. Susan Csegezi for their excellent technical assistance. REFERENCES 1. BIRBECK,M. S. C., MERCER, E. H. and BARNICOT,N. A., Exp. Cell Res. 10, 505 (1956). 2. CHRISTOPHERS,E., Arch. Klin. Exp. Dermatol. 236, 161 (1970). 3. CHRISTOPHERS,E. and BRAUN-FALCO, O., Arch. Klin. Exp. Dermatol. 231, 85 (1967).
190 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
WOLFF AND HONIGSMANN DE DUVE, C. and WATTIAUX,R., Ann. Rev. Physiol. 28, 435 (1966). FARQUHAR,M. G. and PALADE, G. E., J. CellBioL 17, 375 (1963). HASmMOTO,K. and TARNOWSKI,W. M., Arch. Dermatol. 97, 450 (1968). KARNOVSKY,M. J., J. CellBiol. 35, 213 (1967). KLAUS, S. N., Arch. Dermatol, 100, 765 (1969). MATOLTS7,A. G. and PARA~KAL, P. F., J. Cell Biol. 24, 297 (1965). MOTTAZ,J. H. and ZELICKSON,A. S., J. Invest. Dermatol. 49, 605 (1967). -ibid. 54, 272 (1970). NORDQUIST,R. E., OLSON, R. L. and EVERETT, M. A., Arch. Dermatol. 94, 482 (1966). ODLAND, G. F., J. Biophys. Biochem. CytoL 4, 529 (1958). PADAWER,J., J. Cell Biol. 40, 747 (1969). -ibid. 43, 100a (1969). PRUNIERAS,M., J. Invest. Dermatol. 52, 1 (1969). RUPEC, M., Arch. Klin. Exp. Dermatol. 224, 32 (1966). SCHREINER,E. and WOLFF, K., Arch. Klin. Exp. Dermatol. 235, 78 (1969). -Acta Histochem., in press. WILGRAM,G. F., KIDD, R. L., KRAWCZYK,W. S. and COLE, P. L., Arch. Dermatol. 101, 505 (1970). WOLFF, K. and HOLUBAR,K., Arch. Klin. Exp. Dermatol. 231, 1 (1968). WOLFF, K. and SCHREINER,E., J. Invest. Dermatol. 51, 418 (1968). -Arch. Klin. Exp. Dermatol. 235, 203 (1969). -Arch. Dermatol. 101, 276 (1970). -J. Invest. Dermatol 54, 37 (1970).
176
J. ULTRASTRUCTURE RESEARCH
36, 176-190 (1971)
Permeability of the Epidermis and the Phagocytic Activity of Keratinocytes Ultrastructural
Studies with Thorotrast
as a M a r k e r 1
KLAUS WOLFF AND HERBERT HONIGSMANN
Department of Dermatology (I.), University of Vienna, A-1090 Vienna, Austria Received September 15, 1970 This paper details investigations on the permeability of the intercellular spaces of guinea pig epidermis and the phagocytic capacity of keratinocytes as studied with a long-term tracer (Thorotrast) at the ultrastructural level. Immediately after injection into the skin, the tracer penetrates the basal lamina and permeates the intercellular compartment of the epidermis. A barrier to further spread is located in the upper stratum granulosum. Thorotrast fails to penetrate the desmosomes, and this contrasts the findings obtained with low molecular weight proteins. Five minutes after the injection the tracer is found in large vacuoles within keratinocytes which probably represent the "paranuclear cisternae" described previously and are believed to be continuous with the intercellular space. Osmotic pressure leads to a rupture of some of these vacuoles in the stratum granulosum and to cytolysis of some cells. Active uptake of Thorotrast by keratinocytes commences approximately 1 hour after the injection and continues as long as a tracer depot is present within the dermis. The internalization of the marker is accomplished by means of single membrane-limited phagosomes which transfer Thorotrast into the interior of the cells. Since Thorotrast is indigestible, it is stored in the vacuolar system of keratinocytes and is eliminated from the epidermis during the keratinization process. The results of the present study underscore the excellent permeability of the intercellular spaces for small particles; the readiness of keratinocytes to phagocytose even indigestible material, and the fact that substances released in the dermis diffuse into the epidermis by a continuous flow as long as a sufficient supply is maintained by the dermal depot. The most important biological function of epidermal cells is the synthesis of the structural proteins which constitute the basic c o m p o n e n t of keratin. Consequently, most investigations on the epidermis have been preoccupied with this synthetizing capacity of its cells. However, keratinocytes also possess resorptive potentials x This work was supported in part by Fonds zur FSrderung der wissenschaftlichen Forschung, Vienna, and Schering, AG, Berlin.
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FIG. 1. Five minutes after injection the tracer is present in the dermis, extending as far as the basal lamina (BL). Thorotrast has entered the intercellular spaces of the epidermis (arrows) and is seen in a paranuclear vacuole (V). Two pinocytotic vesicles at the basal cell membrane contain a few Thorotrast particles (thin arrows) N, nucleus; M, melanosome, x 25 600. Inset: An enlargement of one of the pinocytotic infoldings containing tracer material, x 91 200. 1 2 - 711839 J. Ultrastructure Research
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which are based on mechanisms of heterophagy (11, 12, 23, 24). Recent studies have shown that tracer proteins injected into the dermis rapidly spread into the epithelium, where they permeate the intercellular spaces (18, 19). The keratinocytes avidly ingest these proteins and degrade them within their lysosomal system (23, 24). This experimental approach provides a model for studies on mechanisms that may be operative in the uptake of endogenous substances by epidermal cells. In order to determine whether the phagocytic properties of keratinocytes are of a more general or a discriminatory nature, it was now considered desirable to extend these studies by employing physically and chemically different markers. The present paper is based on investigations with Thorotrast, a colloidal thorium dioxide stabilized with dextrin. This substance can be easily identified at the ultrastructural level, it does not damage the cells (14, 15) and it differs from the tracers employed so far both with respect to particle size and chemistry. The inorganic moiety cannot be decomposed by the cells and thus offers the opportunity to study the process by which such substances are eliminated from the epidermis.
MATERIALS AND METHODS Albino and white-spotted guinea pigs were used in these experiments. Aliquots of 0.I ml Thorotrast [a colloidal solution of thorium dioxide stabilized with dextrin; particle size in the order of 50-125 •, with most in the range of 70-90 A (according to the manufacturer); obtained from Fellows Testagar, Inc., Detroit, lot 15 098] were injected intracutaneously into multiple sites of dorsal skin of the ears. Biopsies were performed after 5 minutes, 1, 3, 12, 24, and 48 hours, and 3, 4, 5, 7, 9, and 14 days after injection. The tissue was minced with a razor blade and fixed immediately in 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) at 4°C for 2-6 hours. After several rinses and overnight storage in cacodylate buffer, the tissue was posffixed in 1 To osmium tetroxide in barbital acetate buffer (pH 7.3) and stained en bloc in 0.5% uranyl acetate in barbital acetate buffer. Some specimens were osmicated without glutaraldehyde fixation. After rapid dehydration the tissue was embedded in Epon 812. Ultrathin sections were cut on a Reichert OM U2 ultramicrotome, mounted on Formvar-coated copper grids, and stained with uranyl acetate and lead citrate. The specimens were examined with a Zeiss EM 9S electron microscope. Controls AIiquots of 0.l ml of a saturated solution of dextrin and of saline, respectively, were injected into control animals. The experimental conditions and the processing of the tissue were as described for the main experiment.
FI6. 2. Higher magnification of the dermal-epidermal junction shown in Fig. 1. Most of the Thorotrast is on the dermal side of the basal lamina (BL), and only a few particles are seen beyond this structure (arrow). The intercellular space (ICS) of the epidermis shows normal dimensions, but in one area it is expanded by aggregates of tracer particles. The pinocytotic infoldings (PV) of the basal cell membrane are devoid of Thorotrast. C, collagen. × 91 200.
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RESULTS 1. Five minutes after the injection the tracer was diffusely distributed between the collagen fibers and ceils of the dermis. A definite stop occurred at the epidermal basal lamina but small amounts of Thorotrast were observed beyond this barrier (Figs. 1 and 2) and in some areas the marker was seen to have entered the intercellular spaces of the epidermis (Figs. 1 and 2). Most micropinocytotic infoldings of the cytomembranes of basal cells were devoid of the tracer, but, occasionally, a pinocytotic vesicle contained a few dense particles (Fig. 1). The intercellular spaces of the epidermis appeared normal, but in some specimens they were dilated and the surfaces of the keratinocytes formed villuslike cytoplasmic projections. Desmosomes were present in normal numbers and showed no structural alterations. Variable amounts of Thorotrast were present in the intercellular spaces of the entire viable epidermis, usually appearing in focal aggregates (Figs. 2 and 3). There was a decreasing gradient of intercellular tracer toward the upper epidermal layers. The intercellular spaces of the upper stratum granulosum which contain the lamellae derived from Odland bodies (21) were regularly devoid of Thorotrast. Although the tracer was frequently seen to have accumulated at one end of a desmosome, it was never observed within the desmosomes proper (Fig. 3). Even at this early stage of the process the tracer was observed intracellularly. Thorotrast was regularly found in large vacuoles of the keratinocytes where it showed a tendency to clump and to accumulate on the inner surface of the delimiting membrane (Fig. 4a). Most of the vacuoles contained only sparse amounts of the tracer (Fig. 4a), but some were packed with the dense particles (Fig. 1). They were present in a considerable number of keratinocytes and often acquired such monstrous dimensions that they distorted the nucleus and displaced it to the cell periphery (Fig. 4a). Subserial sections revealed that the vacuoles were truly intracellular and did not represent dilatations of the intercellular spaces bulging into the cells. In some keratinocytes of the granular layer these vacuoles had ruptured spilling the tracer into the cytoplasm (Fig. 4 b) where it exhibited a tendency to coat keratohyalin granules (Fig. 4b). These cells contained Thorotrast diffusely throughout their cytoplasm and showed signs of cytolysis. One hour after the injection, an increased number of granular and spinous cells contained ruptured vacuoles and a diffuse dispersion of Thorotrast. One to 48 hours after the injection dermal macrophages and fibroblasts were observed to exhibit phagocytic activity and to take up Thorotrast from the dermal depot. There was a decrease of the amount of intercellular tracer within the epidermis and a reduction of the number of cells containing giant vacuoles or exhibiting an intracytoplasmic dispersion of the tracer. After 48 hours no such cells were present
FIG. 3. (a) Dilated intercellular spaces of the epidermis containing sparse amounts of tracer (arrows). Villuse-like projections (V) are seen on the surfaces of keratinocytes; the desmosomes (D) contain no Thorotrast particles. Five minutes after injection. × 91 200. (b) Convoluted intercellular space of the epidermis, exhibiting small focal aggregates of Thorotrast (arrows). The intercellular space is not widened, pinocytotic vesicles (PV) and a desmosome (D) are devoid of tracer. Three hours after injection, x 91 200. Inset: intercellular accumulation of Thorotrast at one end of a desmosome. One hour after injection, x 54 000.
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in the viable epidermis, but the lower layers of the stratum corneum contained a population of cells which lacked a typical keratin pattern and held Thorotrast within the confines of their membranes. At this time small deposits of Thorotrast were also seen within the intercellular cement of the horny layer. 2. Active uptake of the tracer by keratinocytes was first observed 1 h o u r after injection and was pronounced in the 3 hour specimens. Micropinocytotic infoldings of the plasma membranes rarely contained tracer, but the peripheral cytoplasm of keratinocytes regularly exhibited a considerable number of phagosomes which were packed with dense particles (Fig. 5). These vacuolar structures were limited by a trilaminar membrane; they were usually round or oval and measured up to 0.5/~ in diameter, occasionally attaining even larger dimensions (Figs. 5 and 6). In the initial phases of the ingestion process phagosomes were confined to the basal and lower spinous layers, but after 48 hours they were numerous in all strata of the epithelium. Most frequently they were seen in a paranuclear position or in the vicinity of the Golgi region (Fig. 5 c, d). Membrane-limited melanosome complexes also contained Thorotrast particles (Fig. 6 b). This interesting association of ingested endogenous and exogenous materials within a single phagocytic vacuole is the subject of a separate investigation. 3. Three to 14 days after the injection, variable amounts of the tracer were still present in the dermis; however, there was a considerable shift to intracellular localization as now most of the Thorotrast was observed within macrophages. No tracer was detected in the intercellular compartment of the epidermis, but, apparently, the keratinocytes did not cease to engulf Thorotrast as phagosomes charged with electron dense particles continued to appear within their cytoplasm. As the interval after the injection increased there was a reduction of the number of phagosomes and of the amount of their electron dense contents. There was a definite relationship between the magnitude of the phagosome population within the epidermis and the type of distribution of Thorotrast within the dermis. Epidermal phagosomes were encountered only as long as extracellular Thorotrast was present in the connective tissue (in some instances this was the case as late as 14 days after the injection). Biopsy specimens in which all detectable tracer in the dermis had been engulfed by macrophages exhibited no phagosomes within the epithelium. From the third day on Thorotrast was also observed within fully keratinized Fie. 4. (a) Giant vacuole (V)in a spinous cell 1 hour after injection of Thorotrast. The tracer forms small clumps and tends to attach to the vacuolar membrane (arrows). The nucleus (N) is indented and displaced to one side of the cell. M, mitochondria; T, tonofilaments, x 18 0C0. (b) Ruptured giant vacuole (V) exhibiting a collapsing delimiting membrane which is coated by Thorotrast particles (arrows). Thorotrast is also seen to be diffusely distributed within the cytoplasm (arrows). Twenty four hours after injection. N, nucleus; T, tonofilaments. × 18 000. Inset: Keratohyalin (KI4) coated by Thorotrast (arrows). x 54 000.
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cells of the stratum corneum (Fig. 6c). The tracer was aggregated into well defined intracellular foci which exhibited similar shapes and sizes and a similar distribution as the phagosomes seen in the viable keratinocytes. Occasionally, small amounts of the marker were diffusely dispersed between the keratin fibrils and within the intercellular space. Intraepidermal Langerhans cells also exhibited phagocytic activity, but they engulfed much less tracer than the keratinocytes. Thorotrast was confined to occasional phagosomes within their cytoplasm, but at no time was it observed within the specific Langerhans cell granules. Melanocytes were always devoid of Thorotrast. 4. Controls. Specimens of skin injected with dextrin exhibited morphologic changes which were identical with those of the main experiment. In the early phases after dextrin injection the keratinocytes exhibited giant vacuoles which lacked electron dense contents. These giant vacuoles were regularly absent from the controls injected with saline. DISCUSSION The results of the present report bear on problems of intraepidermal permeability, on the induction of phagocytosis by keratinocytes and on the elimination of foreign material from the epidermis. For reasons of clarity these different aspects are discussed separately.
The permeability of the intercellular space of the epidermis Considerable amounts of the tracer reached the epidermis despite the fact that the bulk of Thorotrast had been stopped at the basal lamina. This indicates that, although the lamina proper may not be permeable to particles of this size (70-90 A), fenestrations or gaps must exist within this structure. The rapid spread of the tracer within the epidermis reemphasizes the excellent permeability of the intercellular spaces demonstrated in earlier experiments (18, 19, 22). However, the desmosomes were not permeated by Thorotrast, and this contrasts with the findings obtained with smaller tracer molecules, such as horseradish peroxidase (18) or the lanthanum complex (22), which readily diffuse through the attachment devices. The distance between the outer leaflet of the cell membrane and the intercellular contact layer (13) [M-line (17)] of the desmosome measures less than 100 A and, since the diameter of the individual Thorotrast particles approaches this order of magnitude, their size may account for their inability to enter these structures. In addition, the restriction of the desmosomal permeability to Thorotrast may have been partly due to the glycocalyx present in these structures (22) and to an effective enlargement of the tracer particles by hydration. However, as desmosomes represent only focal areas of intercellular contact (5), they did not interfere with the overall spread of the marker within the extracellular compartment.
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Fia. 5. (a) Single membrane-limited phagosomes (P) in the peripheral cytoplasm of a keratinocyte. In contrast to the intercellular space (ICS) which contains only sparse amounts of Thorotrast (arrows), the phagosomes are densely packed with tracer particles. One hour after injection, x 25 600. (b, c and d). Different examples of phagosomal structures (P), some of which have acquired considerable dimensions. Note the dense packing of Thorotrast within these phagosomes. M, mitochondria; N, nucleus; G, Golgi apparatus, x 25 600.
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The intercellular space of the epidermis is sealed from the external milieu by a barrier which prevents the loss of extracellular fluid through the surface of the skin. Actually, this barrier is formed by the entire stratum corneum and portions of the transitional layer but with regard to the outward permeability of the intercellular spaces it probably starts at different levels for different compounds, depending on the chemistry and physical properties of these substances. As regards the small molecules of horseradish peroxidase [(radius ~ 25-30 • (7)] the barrier has been shown to start at that particular level of the epidermis where the lamellar structures of Odland bodies (21) [membrane coating granules (9), keratinosomes (20)] appear within the intercellular space (18, 19). It has been proposed that these lamellae seal the intercellular compartment and, in addition, alter its contents enzymatically (18). As for Thorotrast, the present investigation has failed to pinpoint the exact site of the barrier since the intercellular spaces were not filled continuously by the tracer. However, in the early stages of the experiment the marker was neither found between the intercellular lamellae nor beyond this zone and this, by inference, locates the lower limits of the barrier to the same level as for peroxidase (18). The fact that, 24 hours after the injection, the tracer was observed between the lamellae does not reflect a penetration phenomenon since these lamellae are continuously discharged into the intercellular spaces (9, 21), which at this time may have already contained Thorotrast. This also explains the presence of tracer in the intercellular cement of the stratum corneum as the marker probably accompanied the cells as they moved upward into the horny layer. The nature of the giant vacuoles awaits clarification. Since the vacuoles were observed immediately after the injection and appeared much earlier than the phagosomes, they should not be ascribed to endocytic uptake mechanisms but rather to preexisting cavities which had filled up from the intercellular space. Recently, evidence has been presented that paranuclear cisternae exist in keratinocytes which are continuous with the intercellular compartment and are permeated by horseradish peroxidase immediately after the tracer has entered the epidermis (18). It does not appear unreasonable to assume that the giant vacuoles observed in our experiment correspond to such dilated cisternae. The Thorotrast solution employed in this investigation was hypertonic and, consequently, osmotic forces may have been responsible for the expansion of these cavities and their subsequent rupture. This conclusion is supported by the fact that (hypertonic) dextrin also led to the formation FIG. 6. (a) Higher magnification of a phagosome containing Thorotrast. The arrows denote the trilaminar delimiting membrane, x 91 200. (b) Melanosome complex of a keratinocyte which contains Thorotrast within its delimiting membrane. The arrows mark some of the melanosomes. 24 hours after injection, x 18 000. (c) Thorotrast within a horny cell. Five days after injection. ME, melanosomes, x 43 500.
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of giant vacuoles whereas the injection of saline did not. However, it remains to be proved whether this interpretation is correct and whether, in the lysed cells, the affinity of Thorotrast to keratohyalin was due to a selective physical chemical effect or was nonspecific.
2. Phagocytosis of Thorotrast by keratinocytes The results of our study show that keratinocytes can be induced to engulf and store substances which cannot be utilized metabolically. The internalization of the tracer was accomplished by means of single membrane-limited phagosomes which were probably formed by infoldings of the plasma membrane. This process was a very rapid and efficient one, as can be inferred from a comparison of the relatively scant amounts of tracer within the intercellular spaces and the relatively large number of phagosomes within the cells. The readiness of keratinocytes to engulf the marker may also explain the observation that, in later phases of the process, phagosomes continued to appear within the cells although no tracer was detected in the intercellular spaces. Apparently, the Thorotrast particles were removed by the epidermal cells immediately after they had reached the intercellular compartment. The studies on protein uptake by keratinocytes have shown that acid phosphatase is incorporated into phagosomes which are thereby equipped enzymatically to degrade the phagocytosed material (23). The dynamics of the lysosomal system of the epidermis (24) conform to the general outlines for lysosomes as proposed by de Duve and Wattiaux (4), and it may be assumed that a similar transformation of phagosomes into (phago)lysosomes had occurred in the present experiment. The visualization of this process is, unfortunately, subject to technical limitations since it is impossible to distinguish Thorotrast particles from the reaction product of acid phosphatase. Thorotrast was stored in the vacuolar apparatus of keratinocytes until the cells underwent keratinization. In this respect the results of the present investigation differ decisively from those obtained with the tracer protein (23). While it may be assumed that the stabilizer dextrin was decomposed within the lysosomal system of the keratinocytes the inorganic moiety of Thorotrast certainly was not, and this afforded the opportunity to follow the further fate of the ingested material until it was eliminated from the epidermis. As keratinocytes remove an indigestible and nonusable material from the intercellular spaces they perform a scavenger function. A recent theory (16) has ascribed such a function to Langerhans cells, which, on morphological grounds, have been considered to represent epidermal macrophages (6). This contention has been questioned on the basis of experimental evidence which has shown that the phagocytic potentials of Langerhans cells are far less than those of keratinocytes (25). The
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results of the present study offer additional evidence against this epidermal "macrophage theory." Thorotrast was regularly observed within melanosome complexes of keratinocytes. Since the cytoplasm of melanocytes was always devoid of tracer particles, only two alternatives remain to explain this surprising phenomenon: Thorotrast was either incorporated into melanosome complexes by fusion of the complexes with phagosomes or the melanosomes and the tracer had been taken up simultaneously from the intercellular spaces. The second alternative would, however, necessitate a revision of the present concept on pigment transfer mechanisms (1, 8, 10). This problem is presently investigated in our laboratory. 3. The elimination of the tracer from the epidermis The foreign material was removed from the epidermis by two separate mechanisms. One consisted of the shedding of those cells which, after rupture of their giant vacuoles, had exhibited a diffuse dispersion of tracer within their cytoplasm and had lysed. The second mode of Thorotrast elimination was brought about by the keratinization of cells which had engulfed the tracer actively and had stored this material within their cytoplasm. This type of elimination was observed as early as 3 days after injection, and this time interval is well below the transit time of the guinea pig epidermis (2, 3). This may be explained by the fact that the tracer was not only engulfed by the basal cells but also by keratinocytes which were already moving upward to the horny layer. In addition, the injection of the tracer into the dermis may have represented a sufficient stimulus for a transient increase of the turnover rate of epidermal cells. The length of the period during which the tracer was taken up and eliminated by keratinocytes depended on the availability of an extracellular depot in the dermis. This indicates that a constant flow of Thorotrast into the epithelium was maintained and permits an important conclusion: substances injected into the dermis reach the epidermal compartment not only by force of the hydrodynamic pressure of the injection but diffuse upward as long as the dermal supply lasts. This model demonstrates that endogenous compounds released into the dermal compartment may reach the epithelium by a similarly continuous flow. We thank Mrs. Lotte Polasek and Mrs. Susan Csegezi for their excellent technical assistance. REFERENCES 1. BIRBECK,M. S. C., MERCER, E. H. and BARNICOT,N. A., Exp. Cell Res. 10, 505 (1956). 2. CHRISTOPHERS,E., Arch. Klin. Exp. Dermatol. 236, 161 (1970). 3. CHRISTOPHERS,E. and BRAUN-FALCO, O., Arch. Klin. Exp. Dermatol. 231, 85 (1967).
190 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
WOLFF AND HONIGSMANN DE DUVE, C. and WATTIAUX,R., Ann. Rev. Physiol. 28, 435 (1966). FARQUHAR,M. G. and PALADE, G. E., J. CellBioL 17, 375 (1963). HASmMOTO,K. and TARNOWSKI,W. M., Arch. Dermatol. 97, 450 (1968). KARNOVSKY,M. J., J. CellBiol. 35, 213 (1967). KLAUS, S. N., Arch. Dermatol, 100, 765 (1969). MATOLTS7,A. G. and PARA~KAL, P. F., J. Cell Biol. 24, 297 (1965). MOTTAZ,J. H. and ZELICKSON,A. S., J. Invest. Dermatol. 49, 605 (1967). -ibid. 54, 272 (1970). NORDQUIST,R. E., OLSON, R. L. and EVERETT, M. A., Arch. Dermatol. 94, 482 (1966). ODLAND, G. F., J. Biophys. Biochem. CytoL 4, 529 (1958). PADAWER,J., J. Cell Biol. 40, 747 (1969). -ibid. 43, 100a (1969). PRUNIERAS,M., J. Invest. Dermatol. 52, 1 (1969). RUPEC, M., Arch. Klin. Exp. Dermatol. 224, 32 (1966). SCHREINER,E. and WOLFF, K., Arch. Klin. Exp. Dermatol. 235, 78 (1969). -Acta Histochem., in press. WILGRAM,G. F., KIDD, R. L., KRAWCZYK,W. S. and COLE, P. L., Arch. Dermatol. 101, 505 (1970). WOLFF, K. and HOLUBAR,K., Arch. Klin. Exp. Dermatol. 231, 1 (1968). WOLFF, K. and SCHREINER,E., J. Invest. Dermatol. 51, 418 (1968). -Arch. Klin. Exp. Dermatol. 235, 203 (1969). -Arch. Dermatol. 101, 276 (1970). -J. Invest. Dermatol 54, 37 (1970).