Acne Vulgaris – Comedogenesis: The Role of Inflammation, Androgens, Sebum Lipid and Cytokines
Despite extensive research over the past century, the pathogenesis of acne vulgaris remains unclear. In particular, the sequences of events that are involved in comedogenesis have yet to be elucidated. Comedogenesis is one of the four primary etiological factors of acne; the others are increased sebum production and secretion, follicular duct colonization with Propionibacterium acnes (P. acnes), and the inflammatory response. These four factors should not be seen as distinct phases, but rather as closely interrelated mechanisms that ultimately lead to the clinical disease acne vulgaris.
That comedones are essential lesions in acne was first suggested in 1960 (1). It is widely accepted that their pathogenesis is multifactorial, attributable to follicular hyperkeratinization, increased sebum production, increased androgen activity, alteration of sebum lipid quality, dysregulation of cutaneous steroido-genesis, neuropeptide production, inflammation, and follicular hypercolonization by the P. acnes bacteria (2,3).
This post focuses on the current etiologic aspects of comedogenesis, the factors controlling comedo formation and the various comedo subtypes.
COMEDONES: AN OVERVIEW
Acne vulgaris begins with the formation of microcomedones, subclinical lesions characterized by follicular epithelial hyperproliferation. These lesions can fur-ther evolve into inflammatory lesions or noninflammatory comedones, of which there are two types, open and closed. Clinically invisible, microcomedones are lesions present in regions of apparently healthy skin in a patient with acne (4).
They can only be observed via surface biopsy, a technique that employs cya-noacrylate glue to remove the follicular contents from the skin surface. These can then be microscopically examined (5). Histologic examination of micro-comedones reveals dilated pilosebaceous ducts containing excessive keratino-cytes and modified sebum, with a prominent granular layer in the ductal epithelium (4).
Under normal circumstances, keratinocytes are loosely arranged in normal pilosebaceous follicles. The flow of sebum transports them to the surface of the skin upon desquamation, maintaining a balance between new and desquamated keratinocytes. However, in comedones, this balance is disturbed, leading to keratinocyte accumulation in the pilosebaceous duct.
Hyperproliferation of basal keratinocytes, which line the wall of the infrainfundibulum, plays a central role in this accumulation (6,7). As more keratinous material accumulates, the follicular wall continues to distend and become thinner. Concurrently, sebaceous glands begin to atrophy and are replaced by undifferentiated epithelial cells. The thin walled, fully developed comedone contains very few, if any, sebaceous cells. The open comedo contains keratinous material arranged in a concentric lamellar fashion. The closed com-edone contains less compact keratinous material and has a narrow follicular orifice.
The distal section of the pilosebaceous follicle, which is located above the junction of the sebaceous canal, is called the infundibulum. The infundibulum is composed of two parts: the deep infrainfundibulum (lower four-fifths) and the distal acroinfundibulum (upper fifth). The keratinocytes of the infrainfundibulum differentiate in a different manner than the epidermal keratinocytes and often have a subtle granular layer. Electronmicroscopically, the pattern of hyper-keratinization demonstrates retention hyperkeratosis with increased number and size of keratohyaline granules, accumulation of lipid droplets, and folding of the retained squames on themselves as a result of pressure effects (8). At the ultrastructural level, the follicular keratinocytes present in comedones display increased numbers of desmosomes and tonofilaments (9).
The mechanism of follicular hyperkeratinization is still unclear, and it is thought that several factors are responsible. These include changes in sebum lipid composition, abnormal responses to androgens, local cytokine production, and later, the presence and effects of P. acnes, each of which will be described below.
Keratinocyte hyperproliferation and retention have been demonstrated by labeling comedones with 3H-thymidine, a marker for cell proliferation (6). Immunohistochemical confirmation has been provided using a monoclonal antibody to Ki-67, another cellular marker for proliferation, which labels increased numbers of ductal keratinocytes (10). Further evidence of this ductal hyperproliferation was the increased expression of keratin K6, K16, and K17, the keratin markers of hyperproliferation in the wall of microcomedones and com-edones (11).
In addition, clinically normal follicles in acne-prone skin have also been shown to overexpress Ki-67 and K16, which suggests that topical therapy should be applied not only to lesions but also to the surrounding healthy-looking skin as well (12). Further studies have provided evidence that the expression of keratin markers is upregulated by inflammatory cytokines, such as interleukin-1 (IL-1) and IFN-oc, and growth factors, including TGF-oc and EGF. This provides evi-dence for the involvement of inflammatory responses in the very early stage of acne lesion development (13 15).
Comedogenesis might also be due to reduced desquamation caused by increased cohesion between ductal keratinocytes, or a combination of kerati-nocyte hyperproliferation and reduced desquamation (6). Hydrolytic enzymes, produced by lamellar granules, are required for desquamation. When decreased lamellar granules were discovered in the follicle wall of comedones, it was assumed that lower levels of hydrolytic enzymes were available for desquama-tion (8). P. acnes biofilm production is believed to be another cause of increased cohesiveness of keratinocytes (16).
In summary, when more keratinocytes are produced or less keratinocytes are separated, they accumulate in the pilosebaceous duct, creating a bottleneck phenomenon and resulting in the formation of a nonvisible lesion, the micro-comedone.
THE ROLE OF INFLAMMATION IN COMEDOGENESIS
Inflammation had generally been considered a secondary event in acne patho-genesis until it was demonstrated that inflammatory events in fact occur in the earliest stage of acne lesion development (17). Inflammatory markers can be detected even before hyperproliferation and abnormal differentiation of kerati-nocytes. The role of IL-1oc in cutaneous inflammation and keratinocyte prolif-eration has since been closely studied. IL-1oc is present in the perifollicular epidermis of uninvolved skin in acne patients before hyperproliferation or abnormal differentiation of the follicular epithelium takes place (17). IL-1oc has been reported to induce in vitro and in vivo hyperkeratinization in the follicular infundibulum (18). IL-1oc is comedogenic in pilosebaceous units (PSUs) that have been isolated in vitro (18,19). Addition of IL-1oc to an isolated piloseba-ceous infundibulum in vitro results in comedo-like hypercornification. An IL-1oc antagonist can inhibit this reaction (19). When released into the dermis, IL-1oc initiates an inflammatory response. Further, enough IL-1a has been shown to be present in a comedo for this to occur (20). The observed inflammatory response around uninvolved follicles consists of an infiltrate of CD4+ lymphocytes and macrophages (17).
It has been hypothesized that the cytokines produced in the follicle might be responsible for activating local endothelial cells, causing the upregulation of inflammatory vascular markers in the vasculature around the pilosebaceous follicles of the uninvolved skin (17). Further, it was suggested that the entire process is initiated by an increase in IL-1a activity. However, a matter of con-troversy exists about which factors might be responsible for the increased IL-1a expression and release (17).
THE ROLE OF SEBUM LIPID COMPOSITION ABNORMALITIES IN COMEDOGENESIS
Sebum, produced by sebaceous glands, is a complex mixture of triglycerides, wax esters, squalene, and, to a lesser extent, cholesterol and phospholipids. Abnormalities in its content are considered among the main factors implicated in acne pathogenesis, playing a role in both comedogenesis and the development of inflammatory reactions that lead to clinical acne lesions. Sebum production and secretion is a necessary condition for acne vulgaris, although hypersecretion is not sufficient to initiate lesion development. However, sebum in acne patients is both quantitatively and qualitatively different from that of the skin of normal individuals. The accumulation of comedogenic sebum components also plays a role.
In the late 1960s, studies with a rabbit ear model showed that sebaceous lipid abnormalities could all trigger hypercornification. These abnormalities include increased fatty acid, squalene, and squalene oxide, and decreased linoleic acid (21 23). Although it is unclear whether animal models accurately reflect the human sebaceous follicles, substances that are comedogenic in this model are capable of inducing comedones in the human model (5).
In the 1970s, comedogenesis was thought to be triggered by the presence of excessive follicular free fatty acids (24), the production of which is metab-olized by bacterial lipases, predominantly from P. acnes and Staphylococcus epidermidis (25). Although this idea was later discounted, both topical and oral antibiotics have been shown to reduce comedones (26). Whether this reduction is due to the antibacterial effects of the antibiotics or the direct inhibition of lipase production is unclear. Regardless, it has since been discovered that the sebum lipid abnormalities that play a role in comedogenesis are more complex than a simple increase in amount.
One such abnormality is lipid polarity. Skin surface lipids in acne patients, as well as lipids in open and closed comedones, have an increased polar lipid content as compared with the skin surface lipids obtained from controls. These polar lipids appear to be derived mainly from the oxidation of squalene, a sebum-specific lipid, to squalene peroxide. Excessive accumulation of these peroxides may promote inflammatory changes in comedones, and their accumulation in comedones may lead to an increase in IL-1a expression via NF-xB and exacerbate comedogenesis by triggering follicular keratinization (27).
A link between a low sebum level of linoleic acid, an essential fatty acid, and comedogenesis was suggested in 1986 (23). Diminished linoleic acid content in intrafollicular sphingolipids may be involved in follicular hyperkeratosis, a crucial event in comedogenesis. Hyperkeratosis could possibly lead to a diminished epidermal barrier function, increased transepidermal water loss, and a scaly dermatosis. This can later predispose the follicular wall to increased permeability to proinflammatory substances (28). Further evidence of the importance of linoleic acid is provided by the fact that topically applied linoleic acid reduces the size of microcomedones (29). The concentration of linoleic acid in the sebum of acne patients increases after treatment with either oral iso-tretinoin or cyproterone acetate (30), although studies have found that this effect relates to the higher sebum secretion rate in acne patients rather than a direct effect on linoleic acid. It has been suggested that the linoleic acid level in human sebum depends on both the quantity of linoleic acid present in each sebaceous cell at the start of its differentiation and the extent to which this initial linoleic acid content is diluted by the sebum synthesized by each sebaceous cell (22). There is an inverse relationship between linoleic acid levels in sebum and the sebum secretion rate as endogenous lipid synthesis may dilute this essential fatty acid (23).
More recent studies have focused on the saturation pattern of fatty acids in acne. These have revealed differences in the ratio of saturated to unsaturated fatty acids in the skin surface triglycerides between acne patients and controls (31). In particular, an increased ratio of unsaturated to saturated fatty acids seems to be associated with increased sebogenesis.
THE ROLE OF ANDROGENS IN COMEDOGENESIS
Androgens influence various cutaneous functions, including sebaceous gland growth and differentiation, hair growth, epidermal barrier function, and wound healing. They play a central role in the stimulation of sebum production and keratinocyte proliferation (32,33).
Prohormone androgens, primarily produced in the gonads and adrenal glands, are converted in the skin to the more potent testosterone and dihy-drotestosterone (DHT). Type 1 5oc-reductase, which is located within both the infrainfundibulum part of the duct and the sebaceous gland, catalyzes the cutaneous conversion of testosterone to the more potent DHT (34). The andro-gens then initiate changes in the sebaceous glands and pilosebaceous canals, both of which express androgen receptors. Androgens affect sebocytes and infun-dibular keratinocytes in a complex manner, having an effect on differentiation and proliferation of sebocytes, lipogenesis, and comedogenesis.
The role of androgens in the pathogenesis of acne has long been recog-nized. The principal link between androgens and acne is sebum, as the sebaceous glands are highly androgen sensitive. A significant proportion of patients with acne have systemic androgen abnormalities. Women experiencing excessive androgen states, as in polycystic ovary syndrome, suffer from acne (35). Skin in acne patients displays a higher androgen receptor density and higher 5oc-reductase activity than nonacne skin (34,36). Systemic or topical administration of androgens (testosterone and DHT) or anabolic steroids increases the size and function of sebaceous glands (32). The close timing between the onset of microcomedonal acne during the prepubertal period and the adrenarchal rise in serum levels of DHEAS has been well documented (37,38). On the other hand, men who have been castrated before puberty, or individuals without functional androgen receptors, such as patients with androgen-insensitivity syndrome, neither produce sebum nor develop acne (39). Diet can also influence come-dogenesis (40). In particular, dairy milk contains testosterone precursors (41) that, after conversion to DHT via testosterone, stimulate the PSU. The comedogenicity of milk is thought to be due to both testosterone precursors and 5oc-reduced molecules (42,43). The consumption of milk might also exert a comedogenic effect via the IGF-1 pathway via the stimulation of androgen synthesis (44).
The role of androgens in follicular hyperkeratinization, and thus the pos-sibility that local androgens might directly contribute to comedo formation, has been investigated in an in vitro study of cultured keratinocytes from the epi-dermis and the follicular infrainfundibulum. This study demonstrated a higher activity of type 1 5oc-reductase in the infrainfundibular region, the region which is affected by hypercornification, as compared to epidermal keratinocytes, from subjects both with and without acne. Patients with acne have a slightly higher level of this enzyme compared to patients without acne, indicating increased capacity for producing androgens (33). The role of androgens in comedo for-mation, and whether the increase in enzyme level is a consequence of acne or its cause, remains to be determined. However, it is known that androgenic stimu-lation leads to excessive ductal and infundibular hyperkeratinization. This effect is potentiated by synergistic growth factors, neuropeptides and IL-1oc, and hyperproliferation and hypercornification of the follicular wall could be blocked by the addition of IL-1 receptor antagonist (19).
Regarding the hormonal response, increased DHT may act on infundibular keratinocytes leading to abnormal hyperkeratinization (45). It remains to be determined whether higher activity of the type 1 5a-reductase detected in the follicular infrainfundibulum is associated with the abnormal differentiation of keratinocytes (45).
Further evidence for the comedogenic effects of androgens is the obser-vation that anti-androgens reduce comedones. Oral contraceptives containing cyproterone acetate, such as Diane® and Dianette®, have a direct effect on comedogenesis (30).
THE ROLE OF CYTOKINES IN COMEDOGENESIS
There is increasing evidence that immune phenomena underlie both comedo-genesis and the formation of inflammatory acne lesions (12,19,20,46,47). Cytokines, some of the most potent and diverse of the body’s inflammatory mediators, have become the focus of study as possible comedogenic substances. Of particular interest is IL-1a, which has been found to be present in the inflammatory cytokine content of comedones (48). IL-1a seems to be produced by ductal keratinocytes in comedones (49) and, as previously mentioned, plays a role in the androgenic stimulation in comedogenesis. Indeed, elevated levels have been detected in most open comedones of acne vulgaris (20).
Direct evidence for involvement of IL-1a in comedogenesis has been demonstrated. The addition of IL-1a to an in vitro medium of normal PSUs leads to hyperproliferation and abnormal differentiation in isolated pilosebaceous follicles. Thus, IL-1a leads to comedonal features in the absence of other mediators. The addition of an IL-1a receptor antagonist to experimental acne systems inhibits the growth of comedones, confirming that this is indeed an IL-1a-specific response (12,19,47).
Because IL-1a influences hypercornification of the infundibulum in vitro, it might be responsible for creating the keratinous mass as well as the inflam-matory response by inducing the production of vascular endothelial growth factor in dermal papilla cells and follicular keratinocytes of the PSU (50). Indeed, comedones have been found to contain enough IL-1a activity to initiate inflammation when released into the dermis. Further, it has been postulated that this cytokine causes the scaling seen in many inflammatory skin diseases (12,18,19,46,47). Aldana et al. reported increased IL-1a immunoreactivity in the follicle wall of PSUs of uninvolved skin and comedones (48,51). Recent evidence suggests that inflammatory events not only occur post hyper-proliferation but are also involved in the early stages of acne lesion initiation, before the development of microcomedonal features (17). However, it is cur-rently not clear what factors are responsible for the increased expression and release of IL-1a.
THE ROLE OF PROPIONIBACTERIUM ACNES
P. acnes is a gram-positive bacterium whose close association with acne vulgaris has long been recognized (52). Initially thought to be the primary causative factor of acne lesions, P. acnes is now regarded as one of several interconnected factors in the pathophysiology of acne vulgaris. The role that P. acnes plays in comedogenesis has not yet been entirely elucidated. However, studies have shown that P. acnes is frequently present in high concentrations in microcomedones (53); and that it plays a part in the induction of cytokines, integrins, and inflammation; and that the biofilm produced by the bacteria might have a role in follicular hyperkeratinization. P. acnes is not only important in the development of inflammatory acne lesions but also in the for-mation of the microcomedo. However, the presence of microorganisms is not a strict prerequisite for comedo formation (54). Further, P. acnes belongs to the resident cutaneous flora. It is possible, however, that P. acnes may play a role in comedogenesis by secreting lipase that hydrolyzes the triglycerides of sebum into free fatty acids and glycerol. Free fatty acids are likely to be comedogenic.
Cytokines are present in normal sebaceous glands (55), but under pathological conditions, the amount of cytokines released increases significantly. P. acnes, by acting on TLR-2, has been shown to be able to induce cytokines in the pilosebaceous unit (56,57). IL-6 and IL-8 are thus produced by follicular keratinocytes and IL-8 and IL-12 by macrophages. Barely detectable in the sebaceous glands of healthy skin, IL-6 concentrations are slightly higher in the uninvolved skin of acne patients and significantly higher in the acne-involved skin of the same patients (58). IL-1a has been implicated in the hypercornification of the infundibulum, which plays a central role in comedo-genesis (19).
The importance of the bacteria in the induction and maintenance of the inflammatory phase of acne is well recognized. In contrast to what was initially thought, it is now generally accepted that inflammation is not only the result of comedone rupture but is also involved in initiating comedogenesis. This is achieved via the elaboration of IL-1a, whose expression and production is induced by P. acnes. An innate immune response to P. acnes also induces inflammation.
Integrin and Filaggrin
In vitro studies have confirmed that P. acnes is able to modulate the differen-tiation and proliferation of keratinocytes, by inducing the expression of integrin and filaggrin (filament-aggregating protein) derived from P. acnes (59,60), and thus playing a role in the formation of comedones. Biopsies of acne lesions that the keratinocyte differentiation alterations are associated with altered integrin expression (17). Integrins, which are cell adhesion proteins, play an important role in the modulation of keratinocyte proliferation and differentiation. In 1998, it was suggested that integrins might play a role in the initial events of acne lesion formation, as abnormal oc2 and a3 integrin expression was observed around comedones and uninvolved follicles of acne patients, whereas the basal membrane was still intact (61). This disorder of integrin expression appears to coincide with the inflammatory events, suggesting that it precedes hyper-proliferation, although whether it is responsible for it remains to be seen. Abnormal infundibular keratinization has been associated with a disorder in terminal differentiation of infundibular keratinocytes, which is related to increased filaggrin expression (62). Acneic skin has been shown to carry large amounts of filaggrin in the intermediate layers of the sebaceous duct and infundibulum, suggesting a premature terminal differentiation process in these particular areas of the PSU. Additionally, electron microscopy images have shown an increase in the number of keratohyaline granules in acne skin (62).
P. acnes biofilms appear to play a role in comedogenesis (16). A biofilm is an aggregation of microorganisms encased within a polysaccharide lining secreted by the microorganisms upon adherence to a surface. This lining allows for intermicrobial coherence as well as adherence to the surface. It has been recently discovered that P. acnes resides within a biofilm in the follicles, allowing the bacteria to adhere to the interior follicular surface. A P. acnes biofilm that penetrates into the sebum may act like an adhesive, leading to the increased sebum stickiness and thus corneocyte cohesiveness seen in the keratin plug in microcomedones. The recent decoding of the genome of P. acnes further sup-ports the existence of a P. acnes biofilm (63,64). Further, the presence of a biofilm greatly increases bacterial resistance to antibiotic therapy.
THE DIFFERENT COMEDONAL SUBTYPES
Comedones can be categorized into several different subtypes, based on several factors including morphology, size, and chronology of appearance. In most patients, the various subtypes of comedones coexist. However, it is occasionally important to differentiate the different types to prescribe the appropriate treatment.
Microcomedones represent the first subclinical acne lesions. These precursor lesions, which are clinically invisible, and only evident histologically, can be found in uninvolved skin of acne-prone individuals (4). Despite their subclinical nature, they require special therapeutic attention, because they represent the initial lesions that can eventually change into both noninflammatory and inflammatory acne lesions. Further, the number of microcomedones increases in correlation with worsening of acne severity (7).
As previously discussed, the factors that induce microcomedones include aberrant proliferation and differentiation of the follicular epithelium, excessive sebum production, and inflammatory events. Microcomedones can be sampled and studied by using cyanoacrylate follicular biopsies to sample material from the upper portion of the follicular duct (29).
Open and Closed Comedones
Open and closed comedones are the primary characteristics of noninflammatory acne. They are easily recognizable, follicle-based lesions that develop from microcomedones. Comedones may be open, as with blackheads, or closed, as with whiteheads, depending on the size of the follicular opening (65). Closed comedones are typically small, white-, or skin-colored papules and are usually found in higher numbers than open comedones. These lesions frequently go unrecognized. Bright light and stretching of the skin are sometimes necessary for visualization (66). Open comedones are then recognizable as papules with a dark central plug. Melanin deposition and lipid oxidation give the material in the follicle the typical black color (66).
Sand Paper Comedones
Sand paper comedones are confluent closed comedones frequently found on the forehead, which give the skin a rough, “sandpaper-like” feel (67). These dreaded comedones can be very difficult to treat, being frequently resistant to topical retinoid therapy and antibiotic treatment. Treatment with oral isotretinoin is often necessary for resistant cases.
As with closed comedones, stretching of the skin may be necessary to see these lesions. Despite their large size, up to 1 cm in diameter, they are located deeper in the skin, away from the surface. They are probably best treated with cautery under local anesthesia.
Macrocomedones refer to closed or more commonly open comedones that are larger than 1 mm. The treatment of these cosmetically unflattering lesions may be very challenging. They are known for their potentially poor response to, or to slow down the effect of, oral isotretinoin (68). Macrocomedones can produce devastating acne flares, particularly if patients are inappropriately prescribed oral isotretinoin (69). Different treatment options have been proposed such as cautery therapy, specific extraction techniques, and photodynamic therapy (70,71).
Corticosteroids, androgens, and anabolic steroids are known potential triggers for comedonal drug-induced acne (67).
Chloracne is caused by exposure to halogenated aromatic compounds via cuta-neous, pulmonary, or gastrointestinal exposure. This type of acne is primarily characterized by numerous persistent comedones that are frequently abnormally large. These large comedones can become confluent and form plaques. The lesion location is also typical: lesions in pre- and postauricular lesions and in the axillae or male genital region are well recognized. Systemic effects may persist years after exposure to the chloracne-producing agent.
Nevus comedonicus, first described by Kofmann in 1895, is a rare devel-opmental abnormality of the PSU that presents as plaque of confluent come-dones. The nevus comedonicus syndrome is characterized by a combination of nevus comedonicus with ipsilateral ocular, skeletal, or central nervous system abnormalities (72). Its treatment is challenging and numerous therapies have been reported, including surgical excision, dermabrasion, manual extraction, topical retinoids, oral isotretinoin, and laser treatments (73).
Acne conglobata is an uncommon, often therapy-resistant nodulocystic condition usually seen in males. This disorder typically begins in adulthood and is characterized by papules, pustules, nodules, abscesses, draining sinus tracts, and characteristically grouped polypored comedones involving the posterior neck and trunk (74).
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