ANATOMY

12 May

Cell  Biology  of the  Pilosebaceous Unit Helen Knaggs, Department of Research and Development, Nu Skin Enterprises, Provo, Utah, U.S.A.

INTRODUCTION

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

ANATOMY

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.

Distribution

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).

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