Cell Biology of the Pilosebaceous Unit Helen Knaggs, Department of Research and Development, Nu Skin Enterprises, Provo, Utah, U.S.A.
This chapter reviews the structure and function of the pilosebaceous unit and the controlling influences on the pilosebaceous unit and sebum secretion. The chapter is divided into three sections. Section I gives an account of the structure and function of the normal pilosebaceous unit; Section II describes the biochemistry and regulation of pilosebaceous unit biology; and finally, Section III deals briefly with the biochemical changes occurring in the pilosebaceous duct in acne.
Structure of the Pilosebaceous Unit
In humans, pilosebaceous units or pilosebaceous follicles are found on all skin sur- faces, apart from the palms of the hands and soles of the feet. Essentially, they are invaginations of the epidermis into the dermis. Each comprises a duct, which ends in the dermal papilla, a hair fiber (or pilus) produced by the dermal papilla, a sebac- eous gland and its associated sebaceous duct. The duct supports and protects the hair fiber and also drains sebum produced by the sebaceous gland and carries it to the skin surface. In addition, in split thickness wounds, the cells of the ductal epi- thelium are a source of proliferating keratinocytes, which migrate to re-epithelialize the wound (1). A specialized population of epithelial cells called stem cells, located in the bulge region situated below the sebaceous gland, are believed to be crucial for this (2). These cells are pluripotent and can also differentiate in some circumstances to produce ductal keratinocytes and sebocytes (3,4). Both the hair and sebum are products of pilosebaceous follicles, emerging onto the skin surface. Sebum is a holocrine secretion from the sebaceous gland cells or sebocytes, which means that the cells are destroyed when sebum is released. The function of sebum in humans is unclear, but as will be discussed later it may play a role in several skin functions (5,6).
According to Kligman (7), three types of pilosebaceous units may be distin- guished histologically based on the relative proportions of duct, gland, and hair in each: the terminal follicle, the vellus follicle, and the sebaceous follicle. Terminal follicles produce long hairs and are found, for example, on the scalp (Fig. 1). These pilosebaceous ducts are long, relative to those of other follicles, penetrating deep into the dermis, and the associated sebaceous glands are relatively small. The hair acts as a wick facilitating the passage of sebum and possibly also desquamating ductal cells to the surface of the skin. In contrast, vellus and sebaceous follicles have small, vestigial hairs and relatively large sebaceous glands. Sebaceous follicles are distinguished from vellus follicles by their large sebaceous gland and large follicu- lar orifices (pores), which are visible clinically to the naked eye. However, based on
FIGURE 1 (A) Basic structure of a terminal pilosebaceous unit in anagen (growth phase). The hair penetrates deep into the scalp and the associated sebaceous gland is relatively small. (B) Structures of the different types of pilosebaceous units. The hair, sebaceous gland, and duct vary in size relative to each other.
microdissection of skin, studies (8,9) appear to show that there is a spectrum of different sized follicles as compared to the three types described by Kligman.
Neonatal pilosebaceous units begin as clusters of basal epidermal cells (pre- germs) that bud down into the dermis during the third and fourth months follow- ing conception (10). Budding is believed to be controlled by inductive messages, transmitted from the mesenchyme to the pregerms (11) and proper hair follicle development is dependent on a series of several inductive factors travelling between epithelial and mesenchymal follicle progenitors (12). Regulation of Wnt, b-catenin, and hedgehog signaling pathways directs proper follicle morphogenesis, along with many growth factors including epidermal growth factors (EGFs), trans- forming growth factors (TGFs), bone morphogenic proteins, fibroblast growth factors (FGFs), and neurotrophins (13). Specialized mesenchymal cells accumulate
beneath and around the pregerms and direct the elongation of the cells to form oblique hair pegs in the dermis. By approximately the fifth month, the pilosebaceous units are clearly visible and the keratinization of the duct begins. At this stage there are no follicular orifices. As cells in the center of what is to be the duct senesce, a central canal forms allowing the development of the hair fiber. The growth of the hair upwards removes the cellular debris from the canal and produces the follicular pore. It is believed that no new pilosebaceous units are formed after birth (14). However, recent work indicates that the epidermis can still maintain its capacity to produce follicles, if it is combined with embryonic trichogenic dermis by transplantation (15).
Sebaceous glands form from the outgrowths of the outer root sheaths of hair follicles and are clearly visible by the 15th week of fetal life (16). The factors sur- rounding the development of sebaceous glands are complex, although there are data supporting a critical role for the hedgehog-signaling pathway. Thus, inhibition of hedgehog signaling blocked the development of sebocytes, while activation led to a striking increase in size and number of sebaceous glands in transgenic mice (17,18). In the embryo, sebum secretion contributes to the vernix caseosa and the amniotic fluid. During the neonatal period, sebaceous gland function is regulated by both fetal steroid synthesis and maternal androgens. At parturition, the sebac- eous glands are well developed, largely due to the influence of the maternal andro- gens. However, during the first six months of life they become small and atrophic, remaining so until puberty, when they mature and become active under the influence of pubertal androgens.
As mentioned earlier, all skin areas excluding the glabrous skin, that is, the palmar (palms) and plantar (soles), possess pilosebaceous units. Although the number of secreting follicles, and consequently sebum output, varies greatly between individ- uals, the distribution and shape of follicles tend to follow the same pattern over the human body (19). The highest density of sebaceous and vellus follicles is found on the face, especially on the forehead, where there may be as many as 900 glands/ cm2 in some areas (20), but the number varies according to the study: Blume et al. (21) determined a density of 423 follicles/cm2, Pagnoni et al. (22) found 455 fol- licles/cm2 on the lateral forehead and up to 1220 follicles/cm2 in the nose area, and most recently, Otberg et al. (23) determined a number of 292 follicles/cm2 on the forehead. Nevertheless, there is agreement that sebum output is maximal on the forehead, nose, and chin, the so-called “t-zone” and decreases toward the outer edges of the face (22). Elsewhere on the body, there may be fewer than 100 pilosebaceous units/cm2 (23). The face contains the largest sebaceous glands, but despite this the ducts serving these glands are smallest on the face, particularly, on the forehead and have a significantly smaller pore compared to those found on the back. It has been calculated that the smaller duct creates a five times greater resistance to sebum output on the forehead compared to the back (24). This greater resistance exerted by the small duct on sebum flow, may, in part, explain the prevalence of pilosebaceous diseases on the face, involving high sebum secretion, such as acne.
Other types of sebaceous glands are found in humans distributed over epithelial surfaces and open directly onto the surfaces upon which they secrete. These so-called free sebaceous glands are particularly prevalent in transitional areas between skin and
mucosal membranes for example, anogenital region, lips, and meibomian glands in the eyelids. Sebaceous glands are also located in oral mucosa (called Fordyce spots), the digestive and respiratory tract, and the areoles of the nipples.
Structure of the Pilosebaceous Duct
The pilosebaceous duct is lined by a stratified, squamous epithelium consisting of keratinocytes. The duct lumen is frequently colonized by bacteria that rely on the keratinocytes and sebum as a source of nutrients.
Several distinct anatomical parts of the duct can be recognized. The opening of the duct onto the surface of the skin is the orifice or ostium. The sebaceous follicles on the back frequently group together and emerge through one orifice (25), while those on the face and chest show no such “grouping” (26). Grouped follicles may be more prone to bacterial colonization, which may, in turn, have important impli- cations for follicular diseases. The size of the duct orifice can decrease by as much as
50% in conditions of high humidity due to hydration of keratin in the pilosebaceous duct (27). During this time, sebum excretion is significantly reduced, but then increases above its original level when the duct reverts to its original size.
The region of duct from the orifice to the point of insertion of the sebaceous duct is called the infundibulum. The hair shaft lies unprotected in the infundibu- lum, while in the lower duct (i.e., below the insertion of the sebaceous gland) it is surrounded by the inner and outer root sheaths (Fig. 1).
In terminal follicles, the whole of the infundibular lining closely resembles the interfollicular epidermis, the cells of which undergo the same basic pattern of term- inal differentiation, as do those of the epidermis. However, in sebaceous and vellus follicles only the upper fifth resembles the contiguous epidermis. This region is termed the acroinfundibulum. The lower four-fifths of the duct, down to the inser- tion of the sebaceous duct, the infrainfundibulum, display distinct structural and functional differences when compared with the epidermis. The granular layer is thin and inconspicuous and the keratinocytes often contain glycogen granules in contrast to those of the acroinfundibulum. The cornified layer that develops from this is thin, being composed of only two to three layers, and the corneocytes have been described as fragile (28), as opposed to the interfollicular epidermis, where the stratum corneum is well defined. Based upon histological observations, there appears to be little adherence between the ductal cells or corneocytes (28), and as a consequence it is thought that they slough off and disintegrate to form a loose disorganized mass within the lumen which is normally eliminated along with sebum and shed vellus hairs (7). Desmosomes are present between the cells of the follicular epithelium and there is no difference in their expression in acroinfun- dibulum versus infrainfundibulum (29). The ductal stratum corneum is observed to be relatively thick near the orifice, but it thins as it extends down the follicular canal until it is almost absent at the junction with the sebaceous duct. At these sites, it is thought that the stratum corneum may present an incomplete barrier, allowing the entry of microorganisms and drugs.
The sebaceous duct is lined by a thin epithelium consisting mainly of a basal and granular layer. These cells contain small lipid droplets, whose origin is unclear, but they are thought to be derived from the secretion of the sebaceous glands (7), keratin filaments, and sparse keratohyalin granules. The horny cells here are fragile and are sloughed off in the stream of sebum (7).
FIGURE 2 All follicles produce a hair from the dermal papilla, and hair growth is cyclical, comprising anagen (growth), catagen (breakdown), and telogen (resting) phases successively.
All follicles produce a hair from the dermal papilla and the hair growth is cyclical, comprising of anagen (growth), catagen (breakdown), and telogen (resting) phases, successively (Fig. 2). The amount of hair growth in any area of the body, therefore, depends on several factors, including the duration of anagen, the proportion of time spent in anagen, and the growth rate of the hair (Table 1). The length of a hair shaft is directly proportional to the time the follicle grows (anagen). At the end of an anagen, the follicle stops growing and regresses, transit- ing in terminal follicles from a deep lower follicle portion in the reticular dermis to a shallow structure in the papillary dermis. Both FGF5 and EGF are believed to play a role in the anagen – catagen switch; transgenic mice lacking FGF5 have an extended anagen phase and have an angora phenotype (30), while topical addition of EGF to sheep can cause spontaneous induction of catagen (31). In the catagen phase, hair growth is arrested by a sudden shrinkage of the lower follicle and papilla. The dermal papilla is released from the hair bulb, accompanied with degradation of the lower follicle followed by apoptosis (32). In order to achieve this, the lower portion from which the hair has been growing begins to break down aided by a perifollicular mast cell infiltrate that may function to remove some of the excess components and end products of apoptosis. During this process, the lower follicle is destroyed and the dermis is remodeled appropriately. In telogen, the follicle is quiescent and no hair growth occurs and finally the hair is shed. The Wnt/b catenin pathway plays an important role in telogen – anagen transition and reactiva- tion of follicle growth during postnatal hair cycling hair follicle (33). More is known about hair follicle cycling in terminal follicles and the influence of steroid hormones
TABLE 1 Characteristics of the Hair Cycle in Vellus and Terminal Follicles
and receptor expression, compared to sebaceous and vellus follicles. Terminal hair follicle cycling is well reviewed by Stenn and Paus (34).
The Cells of the Pilosebaceous Duct
The duct is lined with keratinocytes, which undergo the same basic pattern of term- inal differentiation as do those of the epidermis. This process of terminal differen- tiation of keratinocytes has been well characterized for the epidermis, but is less well understood for the pilosebaceous duct, largely due to the small amounts of material available for experimentation. Most of the information comes from immuno- histochemical studies. Our present understanding of keratinocyte biochemistry within the infundibulum of terminal, vellus, and sebaceous follicles is outlined briefly in the next paragraph.
In the epidermis, cytokeratins form the major structural proteins of keratino- cytes, forming the cytoskeleton. Reports on the expression of keratins in the ducts of terminal follicles vary according to the antisera used (35 – 37). Moll et al. (36) reported that the expression of keratins in the infundibulum of terminal follicles resembled that of the interfollicular epidermis. However, other researchers made very specific antibodies to the highly variable C-terminal region of keratins and reported finding the hyperproliferative keratin 16 suprabasally in the infundibu- lum, indicating that follicular epithelium may naturally have a higher cell turnover compared to interfollicular epithelium.
Keratins 1 and 10, normally found suprabasally in epidermis, are expressed throughout the infundibulum of sebaceous and vellus follicles, except for the area around the point of insertion of the sebaceous duct, where expression of K16 and K17 was noted, suprabasally (9). It is surprising that no gross differences were reported between the acroinfundibulum and the infrainfundibulum despite the fact that the latter is reported to produce only a thin stratum granulosum and stratum corneum in the sebaceous follicle (38). In comparison with terminal follicles, sebaceous follicles were found to have less extensive expression of keratins
16 and 19 in the outer root sheath of the lower duct (39,40), suggesting that the follicular epithelia of lower ducts in terminal follicles are more hyperproliferative than in other follicles. The keratin pairs 1 and 10, 5 and 14, and 16 and 17 were expressed in the sebaceous duct cells.
Histological observations made by Plewig and Kligman (28) led them to describe the cornified envelopes formed in the infrainfundibulum as fragile, since they disintegrated more readily than did those of the acroinfundibulum. These purely histological observations have never been followed up by isolating and examining the shapes of cornified envelopes from ducts. In fact, ductal corni- fied envelopes were shown to be antigenically identical throughout the infundibu- lum, but different from interfollicular cornified envelopes (41). Rupniak et al. (42) also showed that psoriatic keratinocytes express epitopes similar to those expressed by the cornified envelopes of keratinocytes in the follicular duct.
Immunohistochemical staining for transglutaminase demonstrated that this enzyme is expressed throughout the follicular epithelium of sebaceous follicles (H. Knaggs, unpublished observations). A hair follicle specific transglutaminase, which is structurally distinct from the other types of transglutaminase, exists in the inner root sheath and in the medulla of the terminal hair follicles (43,44). Transglutaminase catalyzes the crosslinking of various precursor proteins via
g-glutamyl-1-lysine bonds in a calcium-dependent acyl transfer reaction. Proteins that can be incorporated into the cornified envelope include desmosomal com- ponents, compounds obtained following organelle destruction, and involucrin. Staining for involucrin in the duct is restricted to the upper spinous and granular layers at the cell boundaries (45). Other transglutaminase substrates such as loricrin, keratolinin, pancornulins (46), and sciellin (47) may be more important than involucrin in cornified envelope formation, although the location of these pro- teins within the sebaceous follicle has not been reported. In terminal follicles of rats, loricrin is found predominantly in the upper duct (48).
The lumen of the duct provides a suitable environment for bacterial coloniza- tion, but recently it has been demonstrated that the keratinocytes of follicles are a source of antimicrobial peptides called defensins (49).
Structure of the Pilosebaceous Gland
All sebaceous glands are similar in structure. They consist of either a single lobule (acinus) or a collection of acini. The glands are separated from the dermis by a connective tissue capsule, consisting of fine collagen fibers, fibroblasts, and a capillary plexus. The ultrastructure of human sebaceous cells does not vary significantly from one skin site to another nor does the ultrastructure of sebocytes of prepubertal children differ significantly from that of adults, implying that increased levels of androgens at puberty do not induce gross ultrastructural changes (50).
In human skin, the cells of sebaceous glands, called sebocytes, which are modified keratinocytes, can be divided into three major cell types determined by structure: undifferentiated or dividing, differentiated, and mature. The undifferen- tiated cells are attached to the basement membrane by hemidesmosomes. These cells tend to be cuboidal and are characterized by the possession of large nuclei, numerous mitochondria, tonofilaments, small golgi bodies, a high free ribosome, and glycogen granule content. No sebum is apparent in these cells. A second pool of dividing cells has been described to occur near the insertion of the sebac- eous duct. These cells have a higher labeling index with tritiated thymidine (19224%) compared to the germinative cells at the periphery of the gland (8210%), implying that these cells have a higher turnover rate. Langerhans cells, which participate in the immune function of skin, are also found among the undif- ferentiated layer.
The sebocytes differentiate centripetally, that is, toward the center of the lobule. They take on a rounded appearance and the volume of cytoplasm decreases as the cells become filled with lipid containing vacuoles. The cells develop an exten- sive golgi apparatus, smooth endoplasmic reticulum, numerous mitochondria, free ribosomes, and glycogen. As differentiation progresses, lysosomes become appar- ent, which are thought to originate from the golgi apparatus. These are enriched with acid phosphatase activity. The mature cells in the center of the gland, near the insertion of the sebaceous duct, are approximately 100 to 150 times larger in volume than the basal cells. At this point, cytoplasmic organelles and nuclei degen- erate and the mature cells disintegrate to produce the oily liquid sebum, a so-called holocrine secretion.
In sebaceous glands, the release of sebum from the mature cells into the sebac- eous duct is thought to be a consequence of physical displacement of mature cells by new cells from the basal layer. Acid esterases and phosphatases have been
demonstrated histochemically in the central portion of sebaceous glands (51), where they may be involved in holocrine secretion (52).
It was generally accepted that sebaceous glands were not innervated (53) until a very specific silver staining procedure was used on sections of sebaceous glands. This showed that nerve fibers do, in fact, penetrate the connective tissue capsule of the gland and enter between the lobules, reaching the inner part of the sebaceous lobule (54). These fibers may play a role in the holocrine secretion of sebum or they may secrete neuropeptides into sebum before the sebum exits from the gland. Certainly in acne, there is evidence of increased innervation of the sebaceous gland with increased expression of nerve growth factor (NGF) (55).