Sebum is an oily liquid containing triglycerides, free fatty acids, wax esters, squa- lene, and a little cholesterol produced by the gland. It is modified by bacteria that hydrolyze triglycerides to produce free fatty acids, thus a sample of skin surface lipids has a different composition compared to sebum produced by the gland (Table 2).
The delay between sebum synthesis, as measured by the incorporation of injected 14-C acetate into forehead skin of four healthy male subjects, and the sub- sequent excretion of radiolabeled sebum was determined to be eight days (56). This was similar to the five days reported for the delay between the onset of fasting and initial change in composition of skin surface lipids reported by Pochi et al. in 1970 (57). However, the overall process from sebocyte cell division to cell rupture is longer, about 14 days (58). In the beard and scalp follicles, it is believed that the hairs act as a wick to facilitate the passage of sebum to the surface of the skin. In follicles, which possess a vellus hair, the sebum may pool and form a follicular reservoir (59). Thus, the rate of sebum excretion onto the skin surface is a function of the rate of sebocyte proliferation, lipid synthesis, cell lysis, and the rate of flow through the follicular reservoir.
Sebum secretion is increased around puberty under the influence of andro- gens, concomitant with sebaceous gland enlargement. In human males, sebum secretion continues until the age of 80, but in females it drops significantly in the decade after menopause (60). In elderly individuals, sebaceous glands may undergo hyperplasia, but this does not seem to result in an increase in sebum output (61).
Function of Sebum
In animals, sebum may provide odor: it contains pheromones and may also con- dition the hair. However, in humans, the function of sebum is unclear (5) and it has been speculated that the sebaceous glands are vestigial (20). There is now an increasing body of evidence indicating that the sebaceous gland and the components of sebum play a crucial role in the homeostasis of skin (6). Sebum transports vitamin E, a lipophilic antioxidant, to the skin surface where it may play a key role in protecting skin surface lipids from peroxidation (62). Gly- cerol, a major component of sebaceous triglycerides, may play a role in maintaining epidermal barrier function, since asebic mice lacking sebum and, therefore, glycerol have a poorly functioning epidermal barrier (63). Other proposed functions include antibacterial, lubrication, and/or to provide precursor substrates for epidermal metabolism and synthesis of lipids and/or vitamin D (5). In equine follicles, the sebaceous gland and sebum are required to regulate growth of the hair sheath (64) and an intact sebaceous gland in sheep and human terminal follicles was shown to be required for the inner root sheath breakdown (65,66). Whether this holds true for human sebaceous and vellus follicles, remains to be determined. Alo- pecia is present in asebic mice with hypoplastic sebaceous glands, that may also indicate a role of the sebaceous gland in hair follicle growth (67).
It seems unlikely that lack of sebum is a causative factor in the production of dry skin. The distribution of sebaceous glands and amount of sebum produced does not correlate with dry skin. Dry skin in the elderly is, apparently, not related to sebum output (68), nor does a low sebum output in prepubertal children lead to dry skin (69). In addition, subjective self-assessment of skin type as dry, normal, or oily does not always correlate with the amount of sebum as measured using a sebumeter (70).
Human sebum is quite distinct in composition and biological complexity compared to that of other animals, and also compared to epidermal lipids synthesized by keratinocytes. It is unique in containing high levels of squalene and characteristic free fatty acids, for which the rate-limiting enzymes of the biosynthetic pathways are 3-hydroxy-3-methylglutaryl (HMG) CoA reductase and acetyl CoA carboxy- lase, respectively. Both enzymes are inactivated by phosphorylation through a cAMP-activated protein kinase. Comparison of the kinetic parameters for these enzymes with those previously described in the literature found in other organs, indicates that the sebaceous gland enzymes have similar affinities for substrates and similar responses to allosteric effectors such as citrate. However, there are key points of differences and these will be discussed in turn.
Human sebum contains a high percentage of squalene, in contrast to the epidermal lipids, which contain a higher proportion of cholesterol. In the sebaceous glands, therefore, the cholesterol biosynthetic pathway appears to be partly arrested in the steps after the production of squalene. This may be due to low activity of squa- lene epoxidase, or other enzyme of cholesterol biosynthesis, or the availability of substrates. For example, a ready supply of acetate appears to direct lipogenesis toward squalene biosynthesis, while glucose, glutamine, and isoleucine preferen- tially result in more triacylglyceride production in vitro (71). Limiting levels of
NADPH had previously been suggested, but this is not believed to be responsible for the low levels of cholesterol produced. Since squalene is unique to sebum, it is often used as a marker to differentiate sebaceous lipids from epidermal lipids.
The activity of HMG CoA reductase in sebocytes regulates the amount of squalene produced. This can be reduced by 200 mg/mL of low-density lipoproteins (LDL) in isolated glands (72), similar to LDL downregulation of cholesterol pro- duction in fibroblasts, which may work via HMG CoA reductase suppression. Cholesterol itself is a weak repressor of HMG CoA reductase, with the oxygenated cholesterol derivatives mediating suppression of the enzyme. Thus, low concen- trations of 25-hydroxycholesterol (2 mg/mL) did not have an effect on squalene syn- thesis in sebocytes. However, synthesis was reduced by 65% by a 10-fold higher concentration of 25-hydroxycholesterol (73). Mevalonate, the end product of the reaction catalyzed by HMG CoA reductase, can downregulate the reaction and a structurally related compound, mevinolin (lovastatin), inhibits the synthesis of squalene and cholesterol in isolated sebaceous glands (74).
Other compounds that inhibit cholesterol synthesis such as oral aluminum nicotinate and clofibrate had no effect on sebum production (75). Eicosa-5:8:11:14- tetraynoic acid, given orally, suppressed squalene synthesis by 13% to 64% between subjects (76), but its site of action has never been determined.
Free Fatty Acids
Sebum contains numerous fatty acids and many are quite unique in structure, including a wide variety of straight, branched, saturated, and unsaturated fatty acids (77). In human sebum, about 27% of fatty acids chains are saturated, with the greatest proportion being unsaturated (68%). The vast majority of these are monounsaturated (64%) and about 4% are diunsaturated, with the remainder being composed of fatty acid chains longer than 22 carbons (78). Monounsaturated fatty acids of sebum have a double bond usually inserted at position D9. However, in human sebum there is an unusual placing of the double bond at D6 to produce sapienic acid. Sapienic acid is a very abundant and important monounsaturated fatty acid with 16-carbons and a cis double bond located at the sixth carbon from the carboxyl terminal. This fatty acid has been implicated in acne and is produced by an enzyme unique to sebaceous glands, the D6 desaturase, which has recently been isolated from human skin, and its expression is demonstrated in human sebac- eous glands (79). This is the first time that a sebaceous gland-specific functional marker has been demonstrated.
In mice, a similar enzyme has been located in sebaceous glands producing unsaturation between carbons 9 and 10, in other words, a D9 desaturase. Loss of expression of this enzyme in mice results in profound effects with hypoplastic sebaceous glands and an asebic phenotype (80). Although the reason for the pre- sence of these unique fatty acids is not understood, it is speculated that they may impart fluidity on the sebum allowing it to reach the skin surface. An area of active interest is, whether subtle changes in these lipids occur causing blockage in the duct leading to skin diseases such as acne (81) or not. The ratio of D6:D9 fatty acids depends on the rate of sebum secretion—in prepubertal children, and in sebum from the vernix caeosa, as well as elderly individuals the D9 fatty acid pre- dominates. The D6 form is found most commonly in sebum from adults and increases concomittantly with sebum excretion rate. This is thought to be due to a higher level of the D6 desaturase enzyme located in differentiating sebocytes,
compared to the enzyme responsible for producing the D9 unsaturated fatty acids, which predominates in undifferentiated sebocytes (82). Thus, as sebum secretion increases, the D6 fatty acid increases and dilutes the D9 fraction. Since high levels of sebum production are implicated in acne, the increased content of sapienate may be relevant to the pathogenesis of this disease.
Diunsaturated fatty acids in sebum have double bonds at positions D5 and D8 or at positions D7 and D10. The major form of dienoic acid was identified as
18:2 D5:8 and named sebaleic acid. This is presumably synthesized by action of the D6 desaturase on palmitic acid (16:0) to produce 16:1 D6, which then undergoes further desaturation at the 5,6 position following chain elongation to 18 carbons (83). Sebaleic acid is thought to be a major component of sebaceous membrane phospholipids and this may explain the increased levels associated with increased rates of sebum excretion and acne. Another important dienoic acid of sebum is linoleic acid (18:2 D9,12), and the levels are inversely related to sebum excretion, with lower levels being found in subjects with high sebum excretion rates (84). As the cells enlarge during differentiation, they may need additional diunsaturated fatty acids for membrane synthesis and presumably synthesize sebaleic acid as a subsititute for linoleic acid, thus explaining the change in ratio of these fatty acids accompanying high sebum excretion (81). There is evidence that linoleic acid from sebum is incorporated into epidermal acylceramides, but when levels of sebum are low it is replaced by sapienic acid, and this could impair barrier function.
There is evidence to suggest that the types of fatty acids synthesized by indi- viduals is controlled by genotype (85) and is unchanged by fluctuations in diet or metabolism. Site to site variations in free fatty acids in skin surface lipids have also been reported (86). It is important to note that the types of fatty acids syn- thesized seem to vary significantly during the life of an individual: there are several reported examples of this. In addition to those mentioned earlier, a similar effect is seen with some branched chain fatty acids (82). Although human fatty acids are predominantly straight chain, branched chain components were detected with gas chromatography, and saturated fatty acids of human sebum were found to contain methyl branches on one or more of the even numbered carbon atoms throughout the chain (87). The terminally isobranched 15 and 17 carbon species, often comprising wax esters, occur in higher proportions in the sebum of young children, when sebum secretion is low, and in lower quantities
in adult sebum (88).
Another enzyme involved in fatty acid synthesis is acetyl CoA carboxylase and this controls the rate-limiting step of this biosynthetic pathway. It exists in differ- ent isoforms, but it is not known whether any of these forms predominates in sebo- cytes. These cells incorporate fatty acids into triglycerides and wax esters. Palmitate, palmitoleate, stearate, and oleate seem to be the main fatty acids used for further esterification in the gland (89). Of all the fatty acids investigated, linoleic acid was unique, since it was preferentially broken down to 2-carbon units to fuel further fatty acid synthesis, such as palmitic and oleic acids, and squalene synthesis.
The processes of lipid biosynthesis and terminal differentiation in human sebocytes are quite well understood, but the pathways that control holocrine secretion are still unknown. Immortalized humans (SZ95) sebocytes were found to exhibit DNA fragmentation after a six-hours culture followed by increased lactate dehydrogenase release after 24 hours, indicating cell damage, indicating at least in culture this cell line that lipid release is accompanied by apoptosis (90).
Regulation of Pilosebaceous Unit Activity
There are many controlling influences on the pilosebaceous unit (Fig. 3). The devel- opment of model systems for the sebaceous gland and duct has made it easier to study these pathways in vitro.
Perhaps the most profound and well-known effect of hormones on the pilosebac- eous unit is the one caused by androgens, more specifically in causing sebaceous gland enlargement, sebocyte proliferation, and lipid metabolism (91,92). It is well established that the increase in lipid production and enlargement of the sebaceous glands at puberty is attributable to an increase in circulating androgens from the testes in males, the ovaries in females, and the adrenal glands of both sexes. Androgen-insensitive subjects do not produce sebum (93). Furthermore, the local application of testosterone to skin resulted in a 15-fold increase in sebum excretion rate, although admittedly this was a small study in which 2% testosterone cream was applied to the foreheads of prepubertal boys. In fact, three subjects failed to respond, but this study does demonstrate that skin can respond to local androgens (94).
Androgen sensitivity of pilosebaceous units has been confirmed by the posi- tive detection of androgen receptors in pilosebaceous units by immunohistochem- istry (Fig. 4) (95 – 97) and by their isolation from sebaceous gland cells (98). However, it has not been possible to demonstrate differences in receptor levels that explain androgen dependent dermatoses, such as acne or the different rates of sebum production in different individuals. There is a strong correlation between sebum excretion rate and acne grade (99,100), but it has not been possible
FIGURE 3 A schematic representation of potential pathways in sebocytes that can regulate lipogenesis. Abbreviations: CAMP, cyclic adenosine monophosphate; CRH, corticotrophic releasing hormone; EGF, epidermal growth factor; FGF, fibroblast growth factor; GH, growth hormone; IGF, insulin-like growth factors; KGF, keratinocyte growth factor; MSH, melanocyte stimulating hormone; PPARs, peroxisome proliferator-activated receptors; TGF, transforming growth factor.
FIGURE 4 Immunohistochemical labeling of a cross-section through the infundibulum of a sebaceous follicle stained with a monoclonal antibody for androgen receptors. Positively stained nuclei are dark gray.
to relate the rate of sebum production to differences in androgen receptor levels in different acne patients.
Androgen receptor antagonists, such as oral cyproterone acetate and spirono- lactone compete with androgens for the androgen receptor-binding site (101), and while both compounds effectively decrease sebum production, they also have feminizing side-effects. Since their effect is not specific for skin, their use is restricted to women. Antiandrogens also decrease follicular impactions (see later) (102), but this may be secondary to a reduction in sebum flow or a change in sebac- eous lipid composition.
An important feature of the effect of androgens on pilosebaceous unit metab- olism is the metabolic conversion of the androgens themselves, and much work has been done in the last decade to define the pathways of androgen metabolism. It is now clear that pilosebaceous units possess all the steroid metabolizing enzymes needed to convert dehydroepiandrosterone to the most potent androgen, dihydro- testosterone (DHT), including 3-b-hydroxsteroid dehydrogenase (103 – 105), and
5-a-reductase (5-a-R) (106). These latter two enzymes exist in several isoforms: type 2 isozyme of 17 b-hydroxy steroid dehydrogenase ( b-HSD) is predominant in sebaceous glands and type 1 5a-R is highest in sebaceous glands (107,108), iso- lated and cultured infundibular keratinocytes, and in epidermis (109). This would explain why 5a-R II inhibitors such as finasteride, do not produce a signifi- cant reduction in sebum output (93).
Furthermore, sebocytes have the biosynthetic capacity to produce their own androgens from cholesterol through the cytochrome P450 side-chain cleavage system (P450scc) (110). Along with its cofactors, adrenodoxin, adrenodoxin reductase, and the transcription factor, steroidogenic factor 1, P450scc converts cholesterol to pregnenolone, which is also the precursor for estrogen synthesis. Positive staining using antibodies to these proteins was demonstrated in hair follicles in human facial skin, and biochemical activity was demonstrated in vitro confirming that sebac- eous glands are locally steroidogenic with the capacity to produce the highly potent DHT from cholesterol. The conclusion from this body of work is that the skin has its own capacity to metabolize androgens suggesting that the skin exercises local control over the ultimate effects of circulating androgens on the target tissue.
Genes for lipid biosynthesis are regulated in sebaceous glands by androgen- dependent transcription (111). Topical application of 0.005% DHT to hamster ear activated the steroid regulating element (SRE) binding proteins in sebaceous glands, with an upregulation of the mRNA expression for enzymes involved in lipid production, including HMG CoA reductase and synthase, glycerol
3-phosphate acyltransferase, and stearoyl CoA desaturase. SREs are present in the promoter regions of the genes for these enzymes (112).
In contrast to the stimulatory effect of androgens on the sebaceous glands, estrogens and compounds that have estrogenic activity, such as phenol red (113), reduce lipo- genesis in vitro. In vivo, the effect of estrogens is contradictory, although at pharma- cological doses estrogens are sebosuppressive in rat preputial gland (114) and in humans also producing feminizing side effects (115). This action of estrogens is indirect and occurs by inhibiting the adrenals and gonads via the pituitary, thereby reducing the production of androgens. During hormonal treatments, such as hormone replacement therapy (HRT), the effect on skin surface lipids depends on the predominant hormone given. Skin surface lipids are increased during combined HRT, possibly reflecting stimulatory effects of the progestogen component on sebaceous gland activity, while estrogen alone has a sebum- suppressive action (116).
Local effects of estrogens have been demonstrated (115). Thus, ethinyl estra- diol is capable of reducing sebum secretion when applied to the forehead. In addition, sebum secretion was reduced at sites on the forehead distal to the site of application. In agreement with a local effect, both a and b receptor subtypes for estrogens are detected in sebaceous glands of human terminal follicles in both basal and partially differentiated sebocytes (117). There was also widespread expression of estrogen receptor b in the terminal hair follicle, where it was localized to the nuclei of the outer root sheath, epithelial matrix, dermal papilla cells, and in the cells of the bulge (118).
Retinoids exert dramatic, dose-dependent effects on sebocytes in vitro and in vivo. The effect of retinoids is mediated through nuclear receptors [retinoic acid receptor (RAR), retinoid X receptor (RXR)] expressed in sebocytes (119), and in vitro, sebocytes respond differently to retinoids depending upon whether RAR or RXR agonists are given. Activation of RAR by all-trans-retinoic acid (all-trans-RA) mediated the antiproliferative and antidifferentiation effects, while RXR agonists caused differentiation and only weak proliferation in rat preputial cells (120).
Vitamin A was essential for the proliferation, lipogenesis, and differentiation of immortalized human sebocytes in vitro (121), but these cells showed different responses to other retinoids and the effects were dose dependent. In delipidized serum (i.e., in the absence of vitamin A), isotretinoin [13-cis-retinoic acid (13-cis-
RA)] and acitretin at concentrations 10
M enhanced sebocyte proliferation and
lipid synthesis, whereas at concentrations .1027M – sebocyte proliferation was inhi- bited (121). In contrast, with cells cultured in media containing vitamin A, further addition of either isotretinoin or to a lesser extent, all-trans-RA at concentrations ranging from 1028M to 1025M inhibited cell proliferation and lipid synthesis, and altered keratin expression (122). For 13-cis-RA to affect sebocyte proliferation,
differentiation, and lipogenesis, it must apparently be first isomerized to all-trans-
RA (123), so it is perhaps not surprising that these retinoids show similar effects.
Other culture systems support the inhibitory role for retinoids in sebaceous glands. Retinoids significantly inhibited proliferation in the rank order
13-cis-RA . all-trans-RA acitretin in immortalized sebocytes (124) and in sebac- eous gland organ culture, 13-cis-RA caused a significant decrease in lipogenesis over seven days (113,125), and reduced thymidine uptake in pilosebaceous duct organ culture (126).
Clinically, 13-cis-RA is given orally for severe acne at doses of 1 mg/kg/day for four months and produces a rapid reduction in sebum excretion, for example, down to 20% of its former level within four weeks (127). This is due to sebaceous gland atrophy as demonstrated by histological methods (128) but the mechanism involved is unknown. 13-cis-RA and the derivatives, 3,4-didehydroretinoic acid, and 3,4-didehydroretinol are potent competitive inhibitors of the oxidative
3 a-HSD, potentially downregulating the production of potent androgens, DHT, and androstandione, in vitro (129). 13-cis-RA does not however have any effect on 5-a-R responsible for the production of DHT (107). There is also a strong increase in cellular retinoic acid binding protein II expression in the sebaceous gland, and to some extent, in the infundibular structures compared with the level of expression in the epidermis in biopsies obtained from patients treated with 13-cis-RA. Strongest staining was always found in layers of suprabasal sebocytes lacking lipid droplets, and staining was absent in the basal layers (130).
The effect of topical vitamin D on human sebocytes and sebum secretion has not been documented, although receptors for vitamin D have been demonstrated in the nuclei of basal sebocytes (131,132). In a culture system of hamster auricular sebocytes, 1-a,25-dihydroxyvitamin D3, decreased the triglyceride level by 34.3%, but augmented the accumulation of wax esters by 30%, with no difference in the level of cholesterol produced (133).
Peroxisome Proliferator-Activated Receptors
Peroxisome proliferator-activated receptors (PPARs) are a subfamily of so-called orphan receptors belonging to the nonsteroid receptor family of nuclear hormone receptors. Several major types of PPARs have been identified. Activation of PPARs regulates many of the lipid metabolic genes contained in peroxisomes, mito- chondria, and microsomes, with the net effect of stimulating lipogenesis. Adipo- cytes, for example, are induced to differentiate and accumulate lipid through PPARg2. Therefore, there is interest in PPARs functioning as regulators of lipogen- esis in sebocytes (134,135). Certainly in rat preputial sebocytes, PPARg is selectively expressed and has been implicated in the regulation of sebocyte differentiation (136). Fatty acids are PPAR ligands and act as regulators of lipogenesis (137). For example, linoleic acid incubation for 48 hours in immortalized sebocytes induced abundant lipid synthesis. Furthermore, the effect of androgens appears to be addi- tive with the PPAR pathways: androgens appear to upregulate downstream PPAR expression, which in turn influences terminal differentiation of sebocytes (137,138). PPARs can also form heterodimers with RXRs and there is some evidence that there is cooperation between RXR agonists and PPAR agonists in sebocyte growth and development (139). This is a complex area and the role of these receptors in lipid
production in sebocytes is just beginning to be understood with the help of model systems.
Thyroid hormones exert an effect on sebum secretion since thyroidectomy decreases the rate of sebum secretion in rats and administration of thyroxine reverses this effect (140). Increases in sebum secretion rate were observed when thyroxine was given to hypothyroid patients (141), and immunohistochem- ical localization of thyroid hormone nuclear receptors has been demonstrated in human scalp follicles in the nuclei of the outer root sheath cells, dermal papilla cells, sheath cells, and sebaceous gland cells (142). Other than this, little attention has been paid to the effect of thyroid hormones on sebum secretion.
Melanocortins are small peptides, including a-melanocyte stimulating hormone (aMSH) and adrenocorticotrophic hormone (ACTH), known to regulate immune functions, lipid metabolism, and pigmentation pathways. These small peptides are derived from a larger precursor, proopiomelanocortin (POMC), a 241 amino acid peptide secreted by the pituitary, which is cleaved to liberate the melanocor- tins. Evidence has existed since the 1970s demonstrating that the sebaceous gland is influenced by pituitary secretions. For example, removal of the posterior pituitary in rats significantly reduced lipid secretion from rat preputial glands, addition of aMSH stimulated a dose-dependent increase in sebum secretion in rats (143), and patients with hypopituitarism show reduced sebum secretion (141). The expression of POMC-derived peptides in hair follicles in mice varies during the hair cycle, with high levels of b-endorphin expressed during anagen and catagen (144).
The biological effects of melanocortins are mediated through specific trans- membrane receptors, known as melanocortin receptors, which are coupled to G-proteins to modulate intracellular levels of cAMP. To date, five receptor subtypes (MCR1 – 5) have been cloned (145) and, of these, MCR-1 was detected in human sebaceous glands using immunostaining (146), and in an immortalized human sebocyte cell line (147). MCR-5 expression was demonstrated in microdissected human sebaceous glands (148), but could not be detected using immunostaining of sections. Specific transcripts for MCR-1, but not for MCR-5, were present in an immortalized sebocyte cell line and these sebocytes responded to aMSH by modu- lating interleukin-8 (IL-8) secretion (147). In rat preputial cells, MCR-5 was detected perhaps explaining the earlier observations of the effect of melanocortin on these cells (143); and in transgenic mice, targeted disruption of MCR-5 gave marked reduction of sebum secretion (149). Other investigators showed that sebocytes derived from normal human facial skin were stimulated to differentiate and produce prominent lipid droplets when POMC-derived peptides, aMSH, and ACTH were added, and this correlated with an increase in expression of MCR-5 (150). Together, these data support the regulation of human sebum production by melanocortins and may offer an explanation for the link between stress and acne, with high circulating levels of ACTH acting directly on the sebaceous gland to mediate sebum production (148).
Growth Factors and Neuropeptides
EGF, TGF-a, basic FGF, and keratinocyte growth factor (KGF) all inhibit lipogenesis and with the exception of KGF are mitogenic in cultured hamster sebocytes (133,151). In organ culture of human sebaceous glands, EGF produced a dose- dependent inhibition of lipid synthesis and inhibited DNA synthesis (152), and removal of EGF from the medium caused an increase in the rate of lipogenesis without affecting cell turnover (113). In vivo, the presence of EGF inhibited the sebaceous gland differentiation in sheep (153), and in hamster ears, it stimulated the sebaceous glands to proliferate (154). Receptors for EGF are found in sebaceous glands (155). Based on this evidence, Kealey and colleagues proposed that EGF may be, in part, responsible for sebaceous gland atrophy observed during acne lesion formation (7,152). EGF can also induce changes in sebum composition. Ridden (156), reported that there was a fall in the amount of squalene and a corresponding rise in cholesterol, although this was not corroborated by other researchers (157).
Sebocyte proliferation is stimulated by insulin, thyroid-stimulating hormone, and hydrocortisone (158). In rat preputial cells, growth hormone (GH) increased sebocyte differentiation and was more potent than either insulin-like growth factors (IGFs) or insulin. Furthermore, GH enhanced the effect of DHT on sebocyte differentiation perhaps complementing the effect of androgens in increasing sebum during puberty. IGFs, in contrast, had a greater effect on increasing DNA synthesis compared to GH, and had no effect on the response of the cell to DHT (159), although a role for IGF in causing the observed increase in sebum production at puberty has not been ruled out. This suggests that antagonists of IGF may offer a way of decreasing sebum synthesis.
Corticotrophic-releasing hormone (CRH), a key regulator in the central nervous system, has been implicated in sebum production (160). CRH and its recep- tor are expressed in cultured sebocytes, where the hormone appears to upregulate expression of 3 b-HSD—a key enzyme in the androgen biosynthetic pathways. Therefore, CRH could indirectly influence lipid synthesis by modifying the local production of androgens.
Incubation of sebaceous glands in organ culture with neuropeptides (e.g., calcitonin gene-related peptide, substance P, vasoactive intestinal polypeptide, or neuropeptide Y), or NGF up to a final concentration of 1027M showed that only substance P had any effect on sebaceous gland ultrastructure as observed in the electron microscope, and this effect was dose dependent (161). Skin samples cultured with substance P showed sebaceous glands with an increased gland area (in sections) compared with control samples. The sebocytes were engorged with numerous lipid droplets.
Expression of IL-1a and b was demonstrated in sebaceous glands by immunohis- tochemistry (162), and mRNA for IL-1a was detected in cultured sebocytes (163). No change in staining pattern was observed when sebum was extracted from the samples, indicating that IL-1 is not associated with sebum. The function of IL-1 in normal sebaceous glands is unclear, although in vitro IL-1a and tumor necrosis factor a, inhibited sebaceous lipogenesis in sebaceous gland organ culture and induced de-differentiation of human sebocytes into a keratinocyte-like phenotype (152). In organ culture, IL-1a at 1 ng/mL promoted ductal cornification (164) and
upregulated mRNA and protein synthesis of vascular endothelial growth factor (VEGF) (165). Thus in follicular diseases, liberation of IL-1 into the dermis may contribute toward inflammation and increased levels of VEGF by follicular kerati- nocytes can stimulate angiogenesis. In acne, high levels of IL-1a bioactivity existed in comedonal contents (166).
SECTION THREE: DISEASES
Disorders Affecting Pilosebaceous Unit Biology
Various disorders affect pilosebaceous units, although these diseases are rarely life threatening. Three types of skin cysts exist: epidermoid cysts result from squamous metaplasia of a damaged sebaceous gland, while trichilemmal cysts and steatocys- toma are both genetically determined structural aberrations of the pilosebaceous duct. Accumulation of material in the follicular lumen results in distension of the follicle, leading to the formation of noninflamed lesions that are typical of acne, which is the most common follicular disease. In terminal follicles, plugging of the pilosebaceous duct may occur and result in keratosis pilaris. Inflammation around follicles is often seen at the skin surface, for example in folliculitis or acne. In folliculitis, there is extensive colonization of the follicular lumen by microflora.
Acne is the most common follicular disorder and accounts for the majority of der- matological visits by patients between the ages of 15 to 45 years, with treatment costs likely to exceed $1 billion in the United States (167). Problems associated with follicles often occur on the face and thus can cause great psychological pro- blems for the sufferer. Four main factors contribute to acne: increased sebum pro- duction, ductal hypercornification, increased bacterial activity in the duct, and inflammation. The changes that occur in the pilosebaceous biology will be reviewed briefly.
Changes in the Pilosebaceous Unit in Acne
There is no doubt that sebum plays an important etiological role in acne (99,168 –
170). Patients with acne secrete more sebum than unaffected individuals and sebo- suppressive treatments alleviate acne: the greater the inhibition the more profound the clinical response. In addition to elevated sebum excretion, it is well-established that acne is associated histologically and clinically with hypercornification of the duct epithelium (7,38,171). Ductal hypercornification results initially in the for- mation of microcomedones and eventually comedones, a process termed comedo- genesis. On the basis of a detailed histological study Kligman (7) concluded that comedogenesis began in the infrainfundibulum and was closely followed by hyper- keratosis of the sebaceous duct. This is now generally accepted to a reasonable description of what is likely to happen.
Essentially, microcomedones are those follicles containing “impactions,” “follicular casts,” or “sebaceous filaments” of corneocytes, bacteria, sebum, and vellus hairs within the follicular lumen. There are no clinical signs of these changes occurring, although they are more common in patients with acne (7,172). Microcomedones may develop either into noninflamed, or inflamed lesions, or even resolve (173). The contents of the normal infundibulum from nonacne
FIGURE 5 Environmental scanning electron micrograph of a microcomedone removed from a pilosebaceous duct. Keratinocytes are seen covering multiple hairs.
control patients were easy to extrude and contained numerous empty spaces in the cells which appeared to have lost their contents and had no clear cytoskeleton. In contrast, the contents of follicles from acne patients were hard to extrude and the cells were firm and compact (7).
An extensive ultrastructural study on the cornified material in follicular impactions was carried out by Al Baghdadi (174). He showed that corneocytes are arranged into three layers in the follicular impaction: a loosely attached outer layer; a densely packed middle layer; and a further loosely attached layer in the middle of the cast. Lipid droplets occur inside the cornified cells (38,175) and as a result the corneocytes become balloon-like in appearance. Abnormal lamellar inclusions of lipid have been described as a marker for abnormal keratinization (176). Scanning electron microscopy of comedonal contents revealed a complex system of numerous internal channels or “canaliculi” composed of concentric lamellae of corneocytes enclosing bacteria and sebum (177) (Fig. 5).
The overall composition of follicular casts was reported to be water (20 – 60%), lipids (20%), proteins (15%), and microorganisms (178). Bladon et al. (179) analyzed the protein content of comedones and found both epidermal keratins and degraded keratins to be present in the comedones.
Biochemical Changes in the Infundibulum During Comedogenesis
The process of comedogenesis is thought to be due to hyperproliferation of ductal keratinocytes, inadequate separation of the ductal corneocytes, or a combination of both factors, resulting in microcomedones. Hyperproliferation of basal keratino- cytes in acne has been demonstrated (180,181) and correlates with keratin 16 expression, a marker for hyperproliferation suprabasally (182). Aldana et al. (183) proposed a cycling of normal follicles through different levels of expression of Ki-67 (a proliferation marker) and keratin 16 and that these represent different stages of development of the microcomedone. The point at which both Ki-67 and keratin 16 are coexpressed by the follicle is when the follicle is susceptible to comedogenic changes (184).
A more extensive investigation of adhesion in follicles is required. In epider- mis, intercellular adhesion is mediated by lipids, cellular adhesion molecules, and desmosomes. At the present time, the relative importance of follicular intercellular lipids in normal and acne follicles is unclear due to the experimental difficulties encountered obtaining sufficient material for analysis. Hypercornification from the retention of desmosomes was described to be a contributory factor to the formation of acne lesions. However, no differences were detected between staining for these epitopes in the wall of normal and comedonal follicles (29).
Although it is clear that we now know a great deal about the pilosebaceous unit at the structural, biochemical, and physiological levels, there are still considerable gaps in our understanding. Much of the early work was descriptive biology of the structure obtained using immunohistochemical studies. Over the last decade, several in vitro models systems (isolated organ culture, cultured sebocytes) have made it easier to study the cellular processes in the follicle, including sebum pro- duction and the effect of inhibitors and stimulators. There is also now a good deal of information about the chemistry and biosynthesis of sebum components, and we also have a much better understanding of the cell biology of the pilosebac- eous duct. Even so, we are still unable to precisely define the cause of one of the most common skin diseases, namely acne, which affects a very large proportion of the population globally.
The author wishes to acknowledge and thank Professor W Cunliffe for kindly allowing the use of Figure 1B, and the journal “Retinoids and Lipid Soluble Vitamins in Clinical Practice” for kindly allowing the use of Figures 1A, 2, and 3.