The Microscopic Structure of the Epidermis and Its Derivatives

Joel J. Elias

University of California at San Francisco School of Medicine, San Francisco, California

A general review of the microscopic structure of the epidermis and those epidermal derivatives that are distributed widely over the skin and, therefore, may be of interest in considerations of mechanisms of percutaneous absorption, will be presented here. Both light and electron microscopic information will be discussed in order to give an integrated brief summary of the basic morphological picture.

The epithelial component of the skin, the epidermis, is classified histologically as a stratified squamous keratinizing epithelium. It is thickest on the palms and soles (Fig. 1) and thinner elsewhere on the body (Fig. 2). It lies on the connective tissue component of the skin, the dermis, in which are located the blood vessels and lymphatic vessels. Capillary loops in the dermis come to lie in close apposition to the underside of the epidermis. The epidermis, in common with other epithelia, is avascular. The living cells of the epidermis receive their nutrients by diffusion of substances from the underlying dermal capillaries through the basement membrane and then into the epithelium. Metabolic products of the cells enter the circulation by diffusion in the opposite direction.

As in the case of other epithelia, the epidermis lies on a basement membrane (basal lamina). This extracellular membrane, interposed between the basal cells of the epidermis and the connective tissue of the dermis, serves the important function of attaching the two tissues to each other. The point of contact of the epidermis with this structure is the basal cell membrane of the basal cells. Along this surface the basal cells show many hemidesmosomes, which increase the adherence of the basal cells (and therefore of the entire epidermis) to the basement membrane (and therefore to the dermis). In some locations, such as the renal glomerulus, the basal lamina has been shown to also play a role as a diffusion barrier to certain molecules.

The plane of contact between the epidermis and dermis is not straight but is an undulating surface, more so in some locations than others. Upward projections of connective tissue, the dermal papillae, alternate with complementary downgrowths of the epider-

This chapter is reproduced with permission from Bronaugh RL, Maibach HI, eds. Percutaneous Absorption: Mechanisms—Methodology—Drug Delivery. 2nd ed. New York: Marcel Dekker, Inc., 1989.

Figure 1 Thick epidermis from sole. The spiral channel through the extremely thick stratum corneum (sc) carries the secretion of a sweat gland to the surface. The stratum granulosum (sg) stands out clearly because its cells are filled with keratohyalin granules that stain intensely with hematoxylin. Hematoxylin and eosin. X100.

mis. This serves to increase the surface area of contact between the two and presumably, therefore, the attachment.

Within the epidermis are found four different cell types with different functions and embryologic origins: keratinocytes, melanocytes, Langerhans cells, and Merkel cells. These will be considered in turn.

The keratinocytes are derived from the embryonic surface ectoderm and differentiate into the stratified epithelium. Dead cells are constantly sloughed from the upper surface of the epidermis and are replaced by new cells being generated from the deep layers. It is generally considered that the basal layer is the major source of cell renewal in the epidermis. Lavker and Sun (1982) distinguish two types of basal cells, a stem cell type and a type that helps anchor the epidermis to the dermis, and an actively dividing su-prabasal cell population. The basal cells have desmosomes connecting them to surrounding cells and, as mentioned earlier, hemidesmosomes along the basal lamina surface. They have tonofilaments coursing through the cytoplasm and coming into close apposition to the desmosomes. These protein filaments are of the intermediate filament class and are made up principally of keratin. Basal cells have the usual cell organelles and free ribo-somes, the site of synthesis of intracytoplasmic proteins.

As a result of the proliferation of cells from the deeper layers the cells move upward through the epidermis toward the surface. As they do, they undergo differentiative changes

Figure 2 Thin epidermis. The strata spinosum, granulosum, and corneum are considerably thinner than in Figure 1. Hematoxylin and eosin. X200.

which allowed microscopists to define various layers. The cells from the basal layer enter the stratum spinosum, a layer whose thickness varies according to the total thickness of the epidermis. The layer derives its name from the fact that, with light microscopic methods, the surface of the cell is studded with many spiny projections. These meet similar projections from adjacent cells and the structure was called an intercellular bridge by early light microscopists (Fig. 3). Electron microscopy showed that the so-called ''intercellular bridges'' were really desmosomes, and the light microscopic appearance is an indication of how tightly the cells are held to each other at these points. The number of tonofilaments increases in the spinous cells (prickle cells) and they aggregate into coarse bundles—the tonofibrils—which were recognizable to light microscopists using special stains.

Electron microscopy reveals the formation within the spinous cells of a specific secretory granule. These small, membrane-bound granules form from the Golgi apparatus and are the membrane-coating granules (MCG; lamellar bodies; Odland bodies). They contain lipids of varying types which have become increasingly characterized chemically (Grayson and Elias, 1982; Wertz and Downing, 1982).

As the cells of the stratum spinosum migrate into the next layer there appear in their cytoplasm large numbers of granules that stain intensely with hematoxylin. These are the keratohyalin granules and their presence characterizes the stratum granulosum. Electron microscopy shows that the granules are not membrane bound but are free in the cytoplasm. Histidine-rich proteins (Murozuka et al., 1979; Lynley and Dale, 1983) have been identified in the granules. The tonofilaments come to lie in close relationship to the keratohyalin granules. The membrane-coating granules are mainly in the upper part of the granular cell.

When observed by either light or electron microscopy there is an abrupt transformation of the granular cell to the cornified cell with a loss of cell organelles. In thick epidermis, the first cornified cells stain more intensely with eosin and this layer has been called the stratum lucidum. The interior of the cornified cell consists of the keratin filaments, which appear pale in the usual electron microscopic preparations, and interposed between them a dark osmiophilic material. The interfilamentous matrix material has been shown to have derivations from the keratohyalin granule and is thought to serve the function of aggregation of the keratin filaments in the cornified cell (Murozuka et al., 1979; Lynley and Dale, 1983).

Figure 3 High power view of upper part of stratum spinosum and lower part of stratum granulosum. Note the many ''intercellular bridges'' (desmosomes) running between the cells, giving them a spiny appearance. When the cells move up into the stratum granulosum, kerato-hyalin granules (k) appear in their cytoplasm. Hematoxylin and eosin. xl000.

In the uppermost cells of the granular layer the membrane-coating granules move toward the cell surface, their membrane fuses with the cell membrane and their lipid contents are discharged into the intercellular space. Thus, the intercellular space in the cornified layer is filled with lipid material which is generally thought to be the principal water permeability barrier of the epidermis (Grayson and Elias, 1982; Wertz and Downing, 1982). The stratum corneum has been compared to a brick wall, with the bricks representing the cornified cells, surrounded completely by mortar, representing the MCG material (Elias, 1984).

The cornified cell is further strengthened by the addition of protein to the inner surface of the cell membrane. Two proteins that have been identified in this process are involucrin (Banks-Schlegel and Green, 1981; Simon and Green, 1984) and keratolinin (Zettergren et al., 1984). A transglutaminase cross-linking of the soluble proteins results in their fusion to the inner cell membrane to form the tough outer cell envelope of the cornified cell. Desmosomes between the cells persist in the cornified layer.

It can be seen that formation of an outer structure (stratum corneum) which can resist abrasion from the outside world and serve as a water barrier for a land-dwelling animal has proven incompatible with the properties of living cells. The living epidermal cells, therefore, die by an extremely specialized differentiative process that results in their non-living remains having the properties that made life on land a successful venture for vertebrates.

Distributed among the keratinocytes of the basal layer are cells of a different embry-ologic origin and function, the melanocytes. In the embryo, cells of the neural crest migrate from their site of origin to the various parts of the skin and take up a position in the basal layer of the epidermis. They differentiate into melanocytes and extend long cytoplasmic processes between the keratinocytes in the deep layers of the epidermis. Because they contain the enzyme tyrosinase they are able to convert tyrosine to dihydroxyphenylalanine (dopa) and the latter to dopaquinone with the subsequent formation of the pigmented polymer melanin. The tyrosinase is synthesized in the rough endoplasmic reticulum and transferred to the Golgi body. From the latter organelle, vesicles with an internal periodic structure are formed which contain the tyrosinase. These are the melanosomes, the melanin-synthesizing apparatus of the cell. Melanin is formed within the melanosome, and as it accumulates the internal structure of the melanosome becomes obscured. Seen with the light microscope the pigmented melanosome appears as the small brown melanin granule. The melanin granules are then transferred from the melanocyte's cytoplasmic extensions to the keratinocytes, and become especially prominent in the basal keratino-cyte's cytoplasm. In this position their ability to absorb ultraviolet radiation has a maximal effect in protecting the proliferating basal cell's DNA from the mutagenic effects of this radiation. Within the keratinocyte varying numbers of melanosomes are often contained within a single membrane-bound vesicle. The classic method of demonstrating melano-cytes is the dopa test. Sections of skin are placed in a solution of dopa and only the melanocytes turn a dark brown color (Fig. 4).

Within the epidermis is another population of cells which were first demonstrated by Langerhans in 1868. By placing skin in a solution of gold chloride he showed that a number of cells in the epidermis, particularly in the stratum spinosum, turned black. The cytoplasmic extensions of the cell give them a dendritic appearance. For many decades the nature of this cell type was unknown, including whether it was a living, dead, or dying cell. Electron microscopy showed that it was a viable cell in appearance, lacked desmosomes, and possessed a very unusual cytoplasmic structure—the Birbeck granule.

Figure 4 A thick section of the epidermis was made with the plane of section running parallel to the surface of the skin and including the deep layers of the epidermis. Dopa reaction shows whole melanocytes on surface view, illustrating their branching, dendritic nature. X340.

With the development of methods for identifying cell membrane receptors and markers in immune system cells it was shown that Langerhans cells originate in the bone marrow. They are now thought to be derived from circulating blood monocytes, with which they share common marker characteristics. The monocytes migrate into the epidermis and differentiate into Langerhans cells. Considerable evidence shows that these dendritic cells capture cutaneous antigens and present them to lymphocytes in the initiation of an immune response. Their population in the epidermis is apparently constantly replenished by the bloodborne monocytes.

Finally, a fourth cell type, the Merkel cell, can be found in the epidermis. These appear to be epithelial cells and are found in the basal layer. A characteristic feature is the presence of many small, dense granules in their cytoplasm. Sensory nerve endings form expanded terminations in close apposition to the surface of Merkel cells.

Hair follicles begin their formation as a downgrowth of cells from the surface epidermis into the underlying connective tissue. The growth extends into the deep dermis and subcutaneous tissue and forms in the deepest part of the structure a mass of proliferative cells—the hair matrix. The cells of the outermost part of the hair follicle, the external root sheath, are continuous with the surface epidermis. The deepest part of the hair follicle is indented by a connective tissue structure, the hair papilla, which brings blood vessels close to the actively dividing hair matrix cells (Fig. 5). As the cells in the matrix divide the new cells are pushed upward toward the surface. Those moving up the center of the hair follicle will differentiate into the hair itself. The structure of the hair, from the center to the outer surface, consists of the medulla (when present), the cortex and the cuticle of the hair. The cortex forms the major part of the hair. These cells accumulate keratin to a very high degree. They do not die abruptly as in the case of the surface epidermis. Instead, the nucleus of the cell gradually becomes denser and more pyknotic and eventually disappears. Keratohyalin granules are not seen with the light microscope. Cells moving up from the matrix in the region between the hair and the external root sheath form the internal root sheath. Here, the cells adjacent to the hair form the cuticle of the internal root sheath. Next is Huxley's layer and, adjacent to the external root sheath, Henle's layer. These cells accumulate conspicuous trichohyalin granules in their cytoplasm in the deeper part of the internal root sheath. The cells of the internal root sheath disintegrate higher up in the hair follicle and disappear at about the level of the sebaceous gland. Thereafter, the hair is found in the central space of the hair follicle without a surrounding internal root sheath.

Figure 5 The connective tissue hair papilla (p) indents into the base of the hair follicle. The follicle cells in the hair matrix region (m) show many mitotic figures. Iron hematoxylin and aniline blue. x!50.

When viewed with the light microscope the hair follicle is surrounded by an exceedingly thick basement membrane called the glassy membrane. Scattered among the kera-tinocytes in the hair matrix are melanocytes which transfer pigment to the forming hair cells and give the hair color. Hair growth is cyclic, with each follicle having alternating periods of growth and rest.

About a third of the way down the hair follicle from the surface epidermis, the sebaceous glands connect to the hair follicle. The sebaceous alveoli consist of a rounded, solid mass of epithelial cells surrounded by a basement membrane. The outer cells proliferate and the newly formed cells are pushed into the interior of the sebaceous alveolus. As they move in this direction they accumulate a complex of lipids and lipidlike substances. As the lipids fill the cell it begins to die and the nucleus becomes more and more pyknotic. The cells eventually disintegrate, releasing their oily contents by way of a short duct into the space of the hair follicle (Fig. 6). This is the classic example of holocrine secretion where the entire gland cell becomes the secretion. In some scattered locations (e.g., nipple) sebaceous glands can be found independent of the hair follicle. In other areas their size relative to the hair follicle is very large (Fig. 7). Because the lipids are extracted in the usual histologic preparations the cells typically appear very pale.

The major type of sweat gland in the human, the eccrine sweat gland, is distributed over practically all parts of the body. It produces a watery secretion which is conveyed to the surface of the skin where its evaporation plays an important thermoregulatory role. The eccrine glands arise as tubular downgrowths from the surface epidermis independent of hair follicles. The tubule extends deep into the dermis or the subcutaneous tissue level where it becomes coiled. The eccrine gland, therefore, is a simple coiled tubular gland.

Figure 6 Upper part of hair follicle. The hair (h) is shown emerging from the follicle (the lower part of the hair passed out of the plane of section). The sebaceous gland is shown emptying its secretion by way of the duct (d) into the space of the follicle. Iron hematoxylin and aniline blue. X50.

Figure 8 Section through a sweat gland. The pale structures are part of the secretory coiled tubule, the dark ones are part of the duct. Hematoxylin and eosin. x250.

The coiled segment at the blind-ending terminus represents the secretory portion of the gland. This leads to the duct portion of the gland which is also coiled. The duct then ascends toward the surface. When it reaches the underside of the epidermis a spiralling channel through it conveys the secretion to the skin surface (Fig. 1). It is not understood how this channel remains patent in an epidermis whose keratinocytes are constantly proliferating and migrating.

When viewed with the light microscope the two parts of the gland can be easily distinguished from each other (Fig. 8). Compared to the duct, the secretory portion is wider, has a larger lumen, its epithelial lining cells appear pale and many myoepithelial cells are present. The latter are contractile cells that are part of the epithelium, lying within the basement membrane. Their contraction is thought to forcefully expel the secretion toward the skin surface. With the electron microscope, two types of epithelial lining cells are seen in the secretory portion. The so-called dark cells have an extensive contact with the lumen of the tubule and have secretory granules containing glycoprotein substances. The clear cells are distinguished by abundant glycogen in their cytoplasm. Continuous with the tubule lumen are many intercellular canaliculi between the clear cells. It is thought that the clear cells secrete a more or less isotonic solution via these channels into the lumen. The duct portion is lined by two layers of epithelial cells and lacks myoepithelial cells. It is thought that electrolytes are absorbed from the lumen here, making the sweat hypotonic by the time it reaches the surface of the skin.

ACKNOWLEDGMENTS

I would like to express my appreciation to Ms. Linda Prentice and Ms. Simona Ikeda for the photomicrographic work.

REFERENCES

1. S Banks-Schlegel, H Green. Involucrin synthesis and tissue assembly by keratinocytes in natural and cultured human epithelia. J Cell Biol 90:732-737, 1981.

2. PM Elias. Stratum corneum lipids in health and disease. In: Progress in Diseases of the Skin, Vol. 2, R. Fleischmajer, ed. Grune and Stratton, San Diego, 1984, pp. 1-19.

3. S Grayson, PM Elias. Isolation and lipid biochemical characterization of stratum corneum membrane complexes: implications for the cutaneous permeability barrier. J Invest Dermatol 78: 128-135, 1982.

4. RM Lavker, T Sun. Heterogeneity in epidermal basal keratinocytes: morphological and functional correlations. Science 215:1239-1241, 1982.

5. AM Lynley, BA Dale. The characterization of human epidermal filaggrin: a histidine-rich, keratin filament-aggregating protein. Biochim Biophys Acta 744:28-35, 1983.

6. T Murozuka, K Fukuyama, WL Epstein. Immunochemical comparison of histidine-rich protein in keratohyalin granules and cornified cells. Biochim Biophys Acta 579:334-345, 1979.

7. M Simon, H Green. Participation of membrane-associated proteins in the formation of the cross-linked envelope of the keratinocyte. Cell 36:827-834, 1984.

8. PW Wertz, DT Downing. Glycolipids in mammalian epidermis: structure and function in the water barrier. Science 217:1261-1262, 1982.

9. JG Zettergren, LL Peterson, KD Wuepper. Keratolinin: the soluble substrate of epidermal transglutaminase from human and bovine tissue. Proc Natl Acad Sci USA 81:238-242, 1984.

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