Degenerative and regenerative changes in epidemrmal organ culture: A morphological study wity reference to membrane-coating granules

Summary Membrane-coating granules (MCG) are poorly understood lamellate organelles unique to keratinized epithelia. This study provides data on a skin model for future in vitro investigations of MCG. Porcine ear epidermal, organ cultures were used under standard cell culture conditions. This system was selected because it is easily established and, following a degenerative period in which MCG are lost, regenerates to form a highly differentiated epidermis. The epidermis appeared healthy during the first 2 din vitro and contained MCG but lost keratohyalin granules (KHG). Overt degenerative changes were evident in the upper epidermis on Day 3, and MCG were now bloated. By Day 4 only one to three layers of viable undifferentiated cells remained. In the overlying necrotic epidermis MCG were rare, presumably due to the bursting of bloated MCG. Epidermal regeneration began around Day 5 and by Day 7 there, were 8 to 13 layers, including a, rudimentary parakeratotic stratum corneum (up to 4 layers). The stratum granulosum (two to three layers) now containe immature KHG and poorly lamellate MCG, but only amorphous material extracellularly. By Day 11 there were three to four layers of granular cells as in vivo, and an orthokeratotic stratum corneum (two to four layers). Improved cornification coincided with an increased number of mature KHG and cross-banded MCG, and lamellate MCG contents extracellularly. This model of epidermal regeneration will factiliate studies into the role played by MCG in keratinization because the epithelium initially lacked MCG but later expressed all the major morphologic features of epidermis. Furthermore the mechanisms by which MCG translaction and extrusion are effected may be probed, by the inclusion of such agents as antimicrotubular drugs and calcium ionophores. This work was supported by a grant from Unilever Research, Port Sunlight, England.     

An electron microscopic study of the human epidermal keratinocyte

Summary 1. The epidermis of the flexor surface of the upper arm of human subjects was studied with the electron microscope. 2. The cytoplasm of the keratinocytes in the basal layer contained many tonofilaments, ribosomes and other cell organelles. The tonofilaments were arranged singly or in loose bundles and many were attached to the inner membrane of the desmosomes. Along the basal border of the cells pinocytotic vesicles could be seen at different stages of development. 3. The keratinocytes in the stratum spinosum differed from those in the basal layer in two main ways: (a) The tonofilaments were grouped together into large compact bundles known as tonofibrils and it was possible to determine a definite beading or cross banding along the length of some of the filaments. (b) The cells were assuming a flattened shape. 4. The keratinocytes in the stratum granulosum possessed large numbers of irregularly shaped keratohyaline granules. The granules were strongly osmiophilic and were always situated on a meshwork of tonofibrils. The keratohyaline granules had no internal structure. The nuclei and mitochondria showed evidence of degeneration. 5. The keratinocytes in the stratum corneum were long and flattened. The cell walls showed increased electron density and were considerably thickened. The cytoplasm was filled with closely packed fibres separated by a small amount of lucent matrix. The fibres were grouped together in bundles running in different directions within the flattened squames. The fibres had along their entire length alternating areas of high and low electron density. The keratohyalin granules had disappeared and nothing remained of the nuclei or the organelles. In the deepest cells of this region the fibres were sometimes loosely packed leaving large irregular open spaces. This area corresponded to the stratum lucidum. In the most superficial layers of the stratum corneum the fibres appeared to be breaking down so that little remained within the keratinocyte except large lucent spaces. The desmosomes showed distinct structural changes. 6. An attempt was made to correlate the structural changes in the different epidermal layers with the process of keratinization. The possible part that keratohyalin may play in the process of thickening of the cell walls was discussed. The relationship between the desmosome and its dynamic environment was considered.I wish to express my sincere thanks to Dr. David Hilding of the Department of Otolaryngology for the use of an R.C.A. electron microscope and other facilities in his laboratory. This research was supported by the United States Public Health Service and American Cancer Society grants. USPHS CA 04679-07, NB 03995.     

Ultrastructural studies of the transitional zone in the nasopharyngeal epithelium, with special reference to the keratinizing process in the mouse

In the nasopharynx of the SMA mouse, the 'intermediate epithelium' occupies the transitional zone between the ciliated columnar and the stratified squamous epithelia. The intermediate epithelium showed gradations ranging from ciliated stratified low-columnar through stratified cuboidal to stratified squamous type. It is suggested that the intermediate epithelium shows the various stages of the epithelium transforming from the ciliated columnar to the stratified squamous epithelium, and that the basal cells of the ciliated columnar epithelium serve as the germinal layer for the transformation. The intermediate epithelium containing a few keratohyalin granules and many membrane-coating granules represented earlier stages of keratinization. The width of the microprojections in the stratified squamous epithelium was about doubled compared to that in the intermediate epithelium. It is suggested that the difference in width is caused by cell membrane distortion associated with keratinization and is regarded as an important marker of the start of keratinization.     

Electron microscope studies of the human epidermis; the cell boundaries and topography of the stratum malpighii

The thin skin of the left upper quadrant of the human abdomen has been studied by electron microscopy. Tissue removed with a high speed rotary punch was fixed in osmium tetroxide or potassium permanganate. The latter fixative in our preparations is superior to osmium for the demonstration of epidermal cell membranes and certain other membranous structures of the epidermis. The cytoplasmic membranes of basal cells and cells of the stratum granulosum have been found to be relatively straight, while those of most spinous cells are sharply scalloped. The deep cells of the stratum spinosum in the rete ridge area show cell membranes and cytoplasmic structure intermediate between true basal cells and most cells of the stratum spinosum. The extracellular material of the desmosome has been found to consist of alternate dark and light laminae similar to those described by Odland (13) and Horstmann and Knoop (7).     

Fine structure of the filiform papilla of beagle dogs

Light and electron microscopic examination of the dorsal lingual epithelium of beagle dogs (Canis domesticus) revealed three different regions: that anterior to the filiform papillae, that posterior to the papillae, and an interpapillary region. Whereas the basal and suprabasal cells are similar throughout, differences characterize the intermediate and surface layers. Keratohyalin granules are common in the intermediate layers in the anterior and interpapillary regions, tonofibrils are prominent in the posterior region, and no keratohyalin granules occur. The surface layer of the interpapillary region is not keratinized, that of the anterior region shows soft keratinization, and that of the posterior region shows hard keratinization. The perimeter of keratohyalin granules is composed of ribosomes 10-20 nm in diameter.     

Electron Microscope Studies of the Human Epidermis : The Cell Boundaries and Topography of the Stratum Malpighii

The thin skin of the left upper quadrant of the human abdomen has been studied by electron microscopy. Tissue removed with a high speed rotary punch was fixed in osmium tetroxide or potassium permanganate. The latter fixative in our preparations is superior to osmium for the demonstration of epidermal cell membranes and certain other membranous structures of the epidermis. The cytoplasmic membranes of basal cells and cells of the stratum granulosum have been found to be relatively straight, while those of most spinous cells are sharply scalloped. The deep cells of the stratum spinosum in the rete ridge area show cell membranes and cytoplasmic structure intermediate between true basal cells and most cells of the stratum spinosum. The extracellular material of the desmosome has been found to consist of alternate dark and light laminae similar to those described by Odland (13) and Horstmann and Knoop (7).     

Age-related changes in the ultrastructural organization of the human gingival epithelium

By means of transmissive and scanning electron microscopy 103 gingival bioptates in practically healthy persons at the age of 18-80 years have been studied. At ageing essential changes take place in all structural elements of the epithelium. The basal membrane is intermittent and loose. In cytoplasm of the cells of the basal layer epithelium the amount of microfilaments increases essentially, and as a result it becomes electron opaque. Tonofibrillar fasciculi of the spinous layer cells are fragmented, their contours are indistinct. In cytoplasm of the granular layer cells amount of keratohyalin granules increases, their size becomes large and their typical form is lost. In cytoplasm of the basal, spinous and granular layer cells the amount of organells decreases. Mitochondria acquire the appearance of electron translucent cavities with discomplexic, and sometimes, destroyed cristae. Rather great changes occur in intercellular interrelations. In all the layers some intercellular spaces are widen, in the spaces formed isolated desmosomes and other debries of cellular structures are formed. Sharp changes of microrelief of the granular layer epitheliocytes are observed. The ultrastructural rearrangements of epitheliocytes, revealed in the human gingiva, demonstrate certain disturbances in keratinization processes, in mechanical firmness, as well as in barrier function of the epithelial layer.     

A quantitative ultrastructural analysis of changes in hamster cheek-pouch epithelium treated with vitamin A

Summary The ultrastructural changes induced by the topical application of retinol acetate on hamster cheek pouch epithelium were evaluated using stereological analysis. Electron micrographs were prepared of the basal and superficial regions of the nucleated cell layer of the epithelium obtained from 3 treated and 3 control animals and examined at two levels of magnification. A total of 528 micrographs were analyzed using a coherent double lattice test system. Although the mean thickness of the nucleated cell layer did not change significantly after 10 days of treatment with retinol acetate the formation of keratinized squames was completely inhibited. This was paralleled by significant changes in the volume density of a number of organelles in both the basal and superficial strata. Rough endoplasmic reticulum increased significantly whereas filaments, which maintained a constant diameter of approximately 9 nm, keratohyalin granules and membrane-coating granules decreased in both strata. Desmosomes also showed a significant decrease in numerical area density in the treated tissues. In contrast, no changes were observed in the volume density of the Golgi apparatus, free ribosomes or mitochondria in the treated epithelium. It is concluded that this treatment provides an epithelium lacking all features of keratinization and may be a useful model for examining metabolic activities specifically associated with keratinization.     

Absolute volume of differentiating cells in the epithelium of the human hard palate

Summary In previous studies the differentiation of the epithelium in the human hard palate has been described stereologically using parameters expressed per unit tissue volume. Since single epithelial cells represent the true biological units of this tissue, it became necessary to estimate the absolute size of such cells in order to transform density data into absolute data. Therefore, in the present study, a stereological method (originally developed for myocyte volume determination) was tested in terms of its applicability to stratified epithelia; the absolute size of differentiating epithelial cells was determined in the epithelium of the human hard palate. The results suggest that (1) rather precise determination of epithelial cell size is possible by using the modified myocyte volume determination, and (2) the average cell volumes are 926 ± 148, 4,111 ± 1,619, 4,394 ± 551 m³ for the stratum basale, the upper stratum spinosum and the stratum granulosum, respectively. The results are discussed with respect to methodology and to differentiation phenomena in the epithelium of the human hard palate.     

Development of chicken epidermis cultured with embryo extract

The epidermis from 11-day-old chick embryo shank skin was cultured with 11-day-old chick embryo extract. The growth and the differentiation of the epidermis in culture were studied histologically, electron microscopically and with polyacrylamide gel electrophoresis of keratin proteins. The epidermis cultured with the chick embryo extract proliferated and stratum structures developed simultaneously with the increase in epidermal cell layers. Finally, a keratinized layer was observed after 10 days in culture. Electron microscopic observations revealed that tonofilaments were produced after 2 days in culture and increased thereafter with culture time, becoming condensed with desmosomes. Keratohyaline granules were observed in 7-day cultures. These keratinization characteristics occurring during culture showed the general characteristics of the alpha stratum observed in the skin of in ovo embryos during the early stages of development. However, the development of peridermal and subperidermal granules was poor and the stratum granulosum, which develops at the late stages between the stratum intermedium and the stratum corneum, was not observed. Polyacrylamide gel electrophoresis of S-carboxymethylated keratin proteins showed that the keratin protein band patterns of the culture differed from those of in ovo skin epidermis.     

Biosynthetic pathways of filaggrin and loricrin--two major proteins expressed by terminally differentiated epidermal keratinocytes

We have used immunoelectron microscopy to map the biosynthetic pathways of loricrin and filaggrin in epidermal keratinocytes at successive stages of differentiation in newborn mouse skin. The filaggrin epitope is first detected in large, irregularly shaped, keratohyalin granules (F-granules) in the stratum granulosum, and then distributed throughout the cytoplasms of the innermost layers of stratum corneum cells. We conclude that the poly-protein filaggrin precursor is first accumulated in F-granules, from which it is subsequently released and processed into filaggrin, and becomes associated with the densely packed bundles of keratin filaments inside stratum corneum cells. Its diminished visibility in the outer layers correlates with the known degradation of filaggrin to free amino acids. Loricrin is first detected in small round keratohyalin granules (L-granules), and subsequently at the periphery of cells throughout the stratum corneum. Labeling of purified keratinocyte envelopes establishes that this loricrin epitope is exposed only at their inner (cytoplasmic) surface. Thus loricrin is initially accumulated in L-granules, to be released at a specifically programmed stage of keratinocyte maturation, and incorporated into the covalently cross-linked lining of the cell envelope. Since loricrin is rich in cysteine, L-granules account for the sulfur-rich keratohyalin granules described earlier. Proposals are made to rationalize why, subsequent to synthesis, filaggrin precursor and loricrin should be segregated both from each other and from the rest of the cytoplasm.     

Proliferation of human embryonic and fetal epidermal cells in organ culture

The morphology of human embryonic and fetal skin growth in organ culture at the air-medium interface was examined, and the labeling indices of the epidermal cells in such cultures were determined. The two-layered epidermis of embryonic specimens increased to five or six cell layers after 21 days in culture, and the periderm in such cultures changed from a flat cell type to one with many blebs. The organelles in the epidermal cells remained unchanged. Fetal epidermis, however, differentiated when grown in this organ culture system from three layers (basal, intermediate, and periderm) to an adult-type epidermis with basal, spinous, granular, and cornified cell layers. Keratohyalin granules, lamellar granules, and bundles of keratin filaments, organelles associated with epidermal cell differentiation, were observed in the suprabasal cells of such cultures. The periderm in these fetal cultures formed blebs early but was sloughed with the stratum corneum in older cultures. The rate of differentiation of the fetal epidermis in organ culture was related to the initial age of the specimen cultured, with the older specimens differentiating at a faster rate than the younger specimens. Labeling indices (LIs) of embryonic and fetal epidermis and periderm were determined. The LI for embryonic basal cells was 8.5% and for periderm was 8%. The fetal LIs were 7% for basal cells, 1% for intermediate cells, and 3% for periderm. The ability to maintain viable pieces of skin in organ culture affords a model for studying normal and abnormal human epidermal differentiation from fetal biopsies and for investigating proliferative diseases.     

Immunocytochemical and autoradiographic studies on the process of keratinization in avian epidermis suggests absence of keratohyalin

The process of keratinization in apteric avian epidermis and in scutate scales of some avian species has been studied by autoradiography for histidine and immunohistochemistry for keratins and other epidermal proteins. Acidic or basic alpha-keratins are present in basal, spinosus, and transitional layers, but are not seen in the corneous layer. Keratinization-specific alpha-keratins (AE2-positive) are observed in the corneous layer of apteric epidermis but not in that of scutate scales, which contain mainly beta-keratin. Alpha-keratin bundles accumulate along the plasma membrane of transitional cells of apteric epidermis. In contrast to the situation in scutate scales, in the transitional layer and in the lowermost part of the corneous layer of apteric epidermis, filaggrin-like, loricrin-like, and transglutaminase immunoreactivities are present. The lack of isopeptide bond immunoreactivity suggests that undetectable isopeptide bonds are present in avian keratinocytes. Using immunogold ultrastructural immunocytochemistry a low but localized loricrin-like and, less, filaggrin-like labeling is seen over round-oval granules or vesicles among keratin bundles of upper spinosus and transitional keratinocytes of apteric epidermis. Filaggrin-and loricrin-labeling are absent in alpha-keratin bundles localized along the plasma membrane and in the corneous layer, formerly considered keratohyalin. Using ultrastructural autoradiography for tritiated histidine, occasional trace grains are seen among these alpha-keratin bundles. A different mechanism of redistribution of matrix and corneous cell envelope proteins probably operates in avian keratinocytes as compared to that of mammals. Keratin bundles are compacted around the lipid-core of apteric epidermis keratinocytes, which do not form complex chemico/mechanical-resistant corneous cell envelopes as in mammalian keratinocytes. These observations suggest that low amounts of matrix proteins are present among keratin bundles of avian keratinocytes and that keratohyalin granules are absent.     

Lectins as Markers of Human Epidermal Cell Differentiation

The expression of sugar residues on human epidermal cells was investigated by means of lectin binding, as a way of determining membrane structural changes occurring during the differentiation of the epidermis. Fourteen lectins of different sugar specificity were conjugated with fluorescein isothiocyanate (FITC-lectins) and tested in fluorescence microscopy on frozen sections of normal human epidermis. In parallel, FITC-lectins were tested on psoriatic-involved epidermis to visualize differences in the expression of sugar residues that might occur during abnormal epidermal differentiation. No labelling could be obtained with lectins from Bandeira simplicifolia I, Dolichos biflorus, Limulus polyhemus, Tetragonolobus purpureas, Ulex europeus I , and Triticum vulgaris (group 1 lectins). A "pemphigus-like" intercellular labelling of the whole epidermis, except the stratum corneum, was obtained with lectins from Canavalia ensiformis, Maclura pomifera, Phaseolus vulgaris , and Ricinus communis I (group 2 lectins). A selective intercellular labelling of the stratum spinosum and the stratum granulosum was seen in normal epidermis with lectins from Arachis hypogaea, Glycine max, Helix pomatia , and Sophora japonica (group 3 lectins). In psoriatic epidermis, not only the basal cell layer, but also cells from the adjacent lower stratum spinosum were found to be negative, using FITC-lectins of group 3. These data indicate that the expression of lectin binding sites in normal epidermis differs according to the maturation of the cell from the basal cell to the more mature keratinocyte in the stratum granulosum. They suggest that lectins may be used as markers of epidermal cells in various stages of normal and abnormal differentiation.     

Changes in the microstructure of the human epidermis following local vacuum exfoliation]

Structural rearrangements of the human epidermis have been studied after its local vacuum exfoliation. Blisters have been formed in 48 men-volunteers by means of negative pressure up to 0.7 kg/cm2 and during the following 72 h structure of the exfoliated epidermis has been investigated. Immediately after the blister formation the epidermal basal layer is traumatized, a part of its cells die in some time after the lesion. In the center of most of the cells of the spinous layer there is a large vacuole which presses back the nucleus. However, the whole epidermis is not ruined, and during 24 h actively regenerates. The remaining viable cells into the blister lumen. By the end of the first 24 h span they practically cover from below the whole surface of the exfoliated epidermis. In the cells of the spinous layer amount and size of vacuoles decrease, the nuclei return to the central position. In 48 h in the spinous layer keratohyalin granules are revealed, moreover, in the cells, arranging on the border with the basal layer. By 72 h within the epidermis of the number of necrotic areas sharply increases. All the arrangements in the epidermal structure occur at the absence of mitotic division of cells.     

Differentiation of human scalp hair follicle keratinocytes in culture

The morphology of human scalp hair follicle keratinocytes, cultured on the bovine eye lens capsule, is studied by light and electron microscopy. The hair follicle keratinocytes in the stratified cultures are characterized by the presence of numerous tonofilaments, desmosomes and lysosomes and by the presence of glycogen accumulations. The cells in the upper layers develop a cornified envelope. Moreover, an incomplete basal lamina is found between the capsule and the basal cells. However, some features of epidermal keratinocytes in vivo, such as keratohyalin granules and stratum corneum formation, are absent. Analysis of the polypeptides by sodium dodecylsulfate polyacrylamide gel electrophoresis also reveals differences between the cultured hair follicle cells and epidermis, whilst the patterns of cultured cells and hair follicle sheaths are similar. The morphological and protein biosynthetic aspects of terminal differentiation of the keratinocytes in vitro are correlated. These results are discussed in the light of the findings with cultured epidermal keratinocytes, reported in the literature.     

The binding of epidermolytic toxin from Staphylococcus aureus to mouse epidermal tissue

Summary Fluorescein-labelled epidermolytic toxin (FTC-toxin) ofStaphylococcus aureus and ferritin—toxin conjugate have been prepared and purified. FTC-toxin bound selectively to cryostat and resin-impregnated sections of neonatal mouse skin. Binding was localized at the keratohyalin granules and in the stratum corneum. In an epidermal cell (granular, spinous and basal) preparation, only keratohyalin granules of the granular cells bound FTC-toxin. Ferritin—toxin conjugate bound to skin sections at the same two sites as FTC-toxin and was competitive with the binding of free toxin. Keratohyalin granules in unstained sections had a novel patched appearance under the electron microscope, and the ferritin—toxin conjugate bound preferentially to the electron-lucent areas. In the stratum corneum it was shown by quantitative estimation that the target density decreased as the surface of the tissue was approached.     

Electron microscopical demonstration of thiols and disulphides in the porcine epidermis

Summary This study describes the electron microscopical distribution of free thiols and disulphides in the epidermis of the domestic pig and the wild boar, as compared to light microscopical histochemistry. With the silver methenamine method, silver labelling of thiols was clearly achieved on the keratohyalin and cytofilament accumulations in the cells of the living epidermis and the plasma membrane of granular cells. To a certain extent, the envelope and cytoplasm of young corneocytes reacted equally intensively. Disulphides were very abundant in the filaments, keratohyalin granules, and cell envelope of granular cells, and, particularly, in the envelope (marginal band) of corneal cells; the latter structure being distinctly delineated from the background. As a specific feature, the viable epidermis of the wild boar stained strongly for disulphides. The results obtained are discussed in view of actual concepts of epidermal keratinization and corneal cell function.     

1,25-Dihydroxyvitamin D3 stimulates specifically the last steps of epidermal differentiation of cultured human keratinocytes

Human keratinocytes grown on deepidermized dermis (DED) are able to reconstruct a morphologically normal stratified and keratinized epidermis. This culture system is suitable for studying in vitro the effects of various hormones and factors on epidermal differentiation, and the goal of the present work was to study the effect of vitamin D. We found that the hormonal form of vitamin D3, 1,25-dihydroxyvitamin D3, produced very specific alterations in epidermal architecture in a dose-dependent manner, consisting of significant reduction of the nucleated layers of the epithelium, but not of the stratum corneum, which was instead slightly thickened. The study of stage-specific differentiation markers showed that the two extreme layers of epidermis, i.e. the basal layer and the stratum corneum, were unaffected by the hormone, but that the reduction involved specifically the intermediate differentiation compartment, i.e. the spinous and granular layers. It was shown that the reduction of the intermediate compartment provoked by 1,25-dihydroxyvitamin D3 is not due to a block in the proliferation of basal cells or to inhibition of their differentiation into suprabasal cells, but to stimulation of the terminal differentiation of suprabasal cells into corneocytes.     

Epidermolytic toxin binds to components in the epidermis of a resistant species

Ferritin-labeled epidermolytic toxin selectively bound to resin-impregnated sections of toxin-resistant rat skin. Binding was confined to the keratohyalin granules of the stratum granulosum and to the cytoplasm of cells in the stratum corneum. Single granules, and equivalent regions in composite granules, failed to bind the conjugate. Preferential binding of the ferritin-toxin probe created anastomosed arrays in the large granules. The scoring density within corneal cells varied with the position of the cell in the stratum corneum. It reached a maximum in the fourth cell from the granular-corneal junction, then decreased to a negligible value in the outermost corneal layers. Three groups of toxin-binding polypeptides were identified in epidermal extracts. These include profilaggrin, a group of three histone H1 polypeptides and histone H3. Filaggrin did not react with toxin on Western blots. The findings demonstrate that sensitivity to epidermolytic toxin is not solely dependent on the presence of target material. It is suggested that species-specific differences in the morphology of keratohyalin granules and in metabolism of profilaggrin may be important factors in intoxication.