Melanocortin-5 receptor: a marker of human sebocyte differentiation

Melanocortin receptors (MC1R-MC5R) and their ligands (melanocyte-stimulating hormone (MSH) and adrenocorticotrophin hormone (ACTH)) have been shown to influence physiological functions of cells and organs, including exocrine glands. Since relatively little is known about MC5R expression and function in the human sebaceous gland, we examined expression of MC5R by immunohistochemistry and RT-PCR in human sebaceous cells in vivo and in vitro. In human skin, MC5R was detected only in differentiating, lipid-laden sebaceous cells but not in basal, undifferentiated sebaceous cells. Similarly, in cultured human sebocytes MC5R was only detectable at the onset of differentiation and in fully differentiated cells displaying prominent lipid granules. The lipid profile of the cultured and differentiated human sebocytes was shown to be human sebum-specific using (14)C-acetate labeling and high performance thin layer chromatography. Our studies suggest that MC5R is a marker of human sebocyte differentiation.     

Immunohistochemical markers of human sebaceous gland differentiation

Cryostat sections of human skin were stained with monoclonal antibodies to involucrin, a range of cytokeratins, epithelial membrane antigen (EMA), and an ovarian cystadenocarcinoma antibody (OM1) to identify combinations of antibodies that could be used to discriminate between basal and differentiated sebocytes and other cell types present in the pilosebaceous unit. Both the EMA and OM1 monoclonal antibodies specifically recognized differentiated sebocytes. No staining of basal sebocytes or other epidermal cell types was seen. Differentiated (but not basal) sebocytes were also stained by a cytokeratin 10 antibody (LH2). Conversely, the basal sebocytes were recognized by an antibody specific to basal keratinocytes (LH6). Cells of the sebaceous duct stained with both LH2 and LH6 and also with the anti-involucrin monoclonal antibody. Cytokeratin 4 has been detected in sebaceous glands by protein analysis but has not previously been detectable immunohistochemically. We show by immunofluorescence after limited proteolysis that cytokeratin 4 epitopes are distributed in all sebaceous gland cells, including the duct cells.     

Localization of sex steroid receptors in human skin

Sex steroid hormones are involved in regulation of skin development and functions as well as in some skin pathological events. To determine the sites of action of estrogens, androgens and progestins, studies have been performed during the recent years to accurately localize receptors for each steroid hormone in human skin. Androgen receptors (AR) have been localized in most keratinocytes in epidermis. In the dermis, AR was detected in about 10% of fibroblasts. In sebaceous glands, AR was observed in both basal cells and sebocytes. In hair follicles, AR expression was restricted to dermal papillar cells. In eccrine sweat glands, only few secretory cells were observed to express AR. Estrogen receptor (ER) alpha was poorly expressing, being restricted to sebocytes. In contrast, ERbeta was found to be highly expressed in the epidermis, sebaceous glands (basal cells and sebocytes) and eccrine sweat glands. In the hair follicle, ERbeta is widely expressed with strong nuclear staining in dermal papilla cells, inner sheath cells, matrix cells and outer sheath cells including the buldge region. Progesterone receptors (PR) staining was found in nuclei of some keratinocytes and in nuclei of basal cells and sebocytes in sebaceous glands. PR nuclear staining was also observed in dermal papilla cells of hair follicles and in eccrine sweat glands. This information on the differential localization of sex steroid receptors in human skin should be of great help for future investigation on the specific role of each steroid on skin and its appendages.     

Cidea Control of Lipid Storage and Secretion in Mouse and Human Sebaceous Glands

Sebaceous glands are skin appendages that secrete sebum onto hair follicles to lubricate the hair and maintain skin homeostasis. In this study, we demonstrated that Cidea is expressed at high levels in lipid-laden mature sebocytes and that Cidea deficiency led to dry hair and hair loss in aged mice. In addition, Cidea-deficient mice had markedly reduced levels of skin surface lipids, including triacylglycerides (TAGs) and wax diesters (WDEs), and these mice were defective in water repulsion and thermoregulation. Furthermore, we observed that Cidea-deficient sebocytes accumulated a large number of smaller-sized lipid droplets (LDs), whereas overexpression of Cidea in human SZ95 sebocytes resulted in increased lipid storage and the accumulation of large LDs. Importantly, Cidea was highly expressed in human sebaceous glands, and its expression levels were positively correlated with human sebum secretion. Our data revealed that Cidea is a crucial regulator of sebaceous gland lipid storage and sebum lipid secretion in mammals and humans.     

Normal fur development and sebum production depends on fatty acid 2-hydroxylase expression in sebaceous glands

2-Hydroxylated fatty acid (HFA)-containing sphingolipids are abundant in mammalian skin and are believed to play a role in the formation of the epidermal barrier. Fatty acid 2-hydroxylase (FA2H), required for the synthesis of 2-hydroxylated sphingolipids in various organs, is highly expressed in skin, and previous in vitro studies demonstrated its role in the synthesis of HFA sphingolipids in human keratinocytes. Unexpectedly, however, mice deficient in FA2H did not show significant changes in their epidermal HFA sphingolipids. Expression of FA2H in murine skin was restricted to the sebaceous glands, where it was required for synthesis of 2-hydroxylated glucosylceramide and a fraction of type II wax diesters. Absence of FA2H resulted in hyperproliferation of sebocytes and enlarged sebaceous glands during hair follicle morphogenesis and anagen (active growth phase) in adult mice. This was accompanied by a significant up-regulation of the epidermal growth factor receptor ligand epigen in sebocytes. Loss of FA2H significantly altered the composition and physicochemical properties of sebum, which often blocked the hair canal, apparently causing a delay in the hair fiber exit. Furthermore, mice lacking FA2H displayed a cycling alopecia with hair loss in telogen. These results underline the importance of the sebaceous glands and suggest a role of specific sebaceous gland or sebum lipids, synthesized by FA2H, in the hair follicle homeostasis.     

Identification and characterization of ABCB1-mediated and non-apoptotic sebum secretion in differentiated hamster sebocytes

Sebaceous glands secrete sebum onto the skin surface in a holocrine manner and as such a thin lipid layer is formed as a physiological barrier. In the present study, extracellular level of triacylglycerols (TG), a major sebum component, as well as intracellular TG accumulation was augmented in insulin-differentiated hamster sebocytes (DHS). The DHS exhibited phosphatidylserine exposure in an apoptosis-independent manner. In addition, intracellular ATP level and membrane-transporter activity using a substrate, Rhodamine 123, were highly detectable in the DHS rather than in the undifferentiated hamster sebocytes. A membrane-transporter activating reagent, 2'(3')-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP), enhanced transporter activity, extracellular TG level, and phosphatidylserine exposure in the DHS. Both transporter activity and TG secretion were suppressed by R-verapamil, a potent membrane-transporter inhibitor, in the BzATP-treated and untreated DHS. Furthermore, the gene expression and production of ATP-binding cassette subfamily B member 1 (ABCB1) were augmented in the DHS. ABCB1 was also detectable in sebaceous glands in the skin of hamsters. Moreover, the cell-differentiation- and BzATP-augmented transporter activity and TG secretion were dose-dependently inhibited by adding not only an ABCB1 antibody but also a selective inhibitor of ABCB1, PSC833. Thus, these results provide novel evidence that ABCB1 is involved in sebum secretion in the DHS, which is associated with non-apoptotic phosphatidylserine exposure and the increased level of intracellular ATP. These findings should accelerate the understanding of sebum secretion occurring in a holocrine-independent manner in sebaceous glands, and may contribute to the development of therapies for sebaceous gland disorders such as acne, seborrhea, and xerosis.     

Sexual hormones in human skin.

The skin locally synthesizes significant amounts of sexual hormones with intracrine or paracrine actions. The local level of each sexual steroid depends upon the expression of each of the androgen- and estrogen-synthesizing enzymes in each cell type, with sebaceous glands and sweat glands being the major contributors. Sebocytes express very little of the key enzyme, cytochrome P450c17, necessary for synthesis of the androgenic prohormones dehydroepiandrosterone and androstenedione, however, these prohormones can be converted by sebocytes and sweat glands, and probably also by dermal papilla cells, into more potent androgens like testosterone and dihydrotestosterone. Five major enzymes are involved in the activation and deactivation of androgens in skin. Androgens affect several functions of human skin, such as sebaceous gland growth and differentiation, hair growth, epidermal barrier homeostasis and wound healing. Their effects are mediated by binding to the nuclear androgen receptor. Changes of isoenzyme and/or androgen receptor levels may have important implications in the development of hyperandrogenism and the associated skin diseases such as acne, seborrhoea, hirsutism and androgenetic alopecia. On the other hand, estrogens have been implicated in skin aging, pigmentation, hair growth, sebum production and skin cancer. Estrogens exert their actions through intracellular receptors or via cell surface receptors, which activate specific second messenger signaling pathways. Recent studies suggest specific site-related distribution of ERalpha and ERbeta in human skin. In contrast, progestins play no role in the pathogenesis of skin disorders. However, they play a major role in the treatment of hirsutism and acne vulgaris, where they are prescribed as components of estrogen-progestin combination pills and as anti-androgens. These combinations enhance gonadotropin suppression of ovarian androgen production. Estrogen-progestin treatment can reduce the need for shaving by half and arrest progression of hirsutism of various etiologies, but do not necessarily reverse it. However, they reliably reduce acne. Cyproterone acetate and spironolactone are similarly effective as anti-androgens in reducing hirsutism, although there is wide variability in individual responses.     

Fenestrated capillaries in the ventral sebaceous gland of the Djungarian hamster, Phodopus sungorus

The ventral sebaceous gland of the Djungarian hamster is a macroscopically visible organ situated in the midventral area of the abdominal wall. It consists of densely packed acini arranged in lobules with common excretory ducts. The rich vascular network of the gland is characterized by fenestrated capillaries. Fenestrated endothelium has not yet been reported as a characteristic and regular finding within sebaceous glands. Results are discussed with regard to proliferation rate of sebocytes and the demand of fluid and nutrient supply.     

Characterization of the vitamin D endocrine system in human sebocytes in vitro

Sebocytes are sebum-producing cells that form the sebaceous glands. We investigated the role of sebocytes as target cells for vitamin D metabolites and the existence of an enzymatic machinery for the local synthesis and metabolism of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃, calcitriol], the biologically active vitamin D metabolite, in these cell types. Expression of vitamin D receptor (VDR), vitamin D-25-hydroxylase (25OHase), 25-hydroxyvitamin D-1α-hydroxylase (1αOHase), and 1,25-dihydroxyvitamin D-24-hydroxylase (24OHase) was detected in SZ95 sebocytes in vitro using real time quantitative polymerase chain reaction. Splice variants of 1αOHase were identified by nested touchdown polymerase chain reaction. We demonstrated that incubation of SZ95 sebocytes with 1,25(OH)₂D₃ resulted in a cell culture condition-, time-, and dose-dependent modulation of cell proliferation, cell cycle regulation, lipid content and interleukin-6/interleukin-8 secretion in vitro. RNA expression of VDR and 24OHase was upregulated along with vitamin D analogue treatment. Although several other splice variants of 1αOHase were detected, our findings indicate that the full length product represents the major 1αOHase gene product in SZ95 cells. In conclusion, SZ95 sebocytes express VDR and the enzymatic machinery to synthesize and metabolize biologically active vitamin D analogues. Sebocytes represent target cells for biologically active metabolites. Our findings indicate that the vitamin D endocrine system is of high importance for sebocyte function and physiology. We conclude that sebaceous glands represent potential targets for therapy with vitamin D analogues or for pharmacological modulation of 1,25(OH)₂D₃ synthesis/metabolism.     

Neuroendocrinology and neurobiology of sebaceous glands

The nervous system communicates with peripheral tissues through nerve fibres and the systemic release of hypothalamic and pituitary neurohormones. Communication between the nervous system and the largest human organ, skin, has traditionally received little attention. In particular, the neuro‐regulation of sebaceous glands (SGs), a major skin appendage, is rarely considered. Yet, it is clear that the SG is under stringent pituitary control, and forms a fascinating, clinically relevant peripheral target organ in which to study the neuroendocrine and neural regulation of epithelia. Sebum, the major secretory product of the SG, is composed of a complex mixture of lipids resulting from the holocrine secretion of specialised epithelial cells (sebocytes). It is indicative of a role of the neuroendocrine system in SG function that excess circulating levels of growth hormone, thyroxine or prolactin result in increased sebum production (seborrhoea). Conversely, growth hormone deficiency, hypothyroidism, and adrenal insufficiency result in reduced sebum production and dry skin. Furthermore, the androgen sensitivity of SGs appears to be under neuroendocrine control, as hypophysectomy (removal of the pituitary) renders SGs largely insensitive to stimulation by testosterone, which is crucial for maintaining SG homeostasis. However, several neurohormones, such as adrenocorticotropic hormone and α‐melanocyte‐stimulating hormone, can stimulate sebum production independently of either the testes or the adrenal glands, further underscoring the importance of neuroendocrine control in SG biology. Moreover, sebocytes synthesise several neurohormones and express their receptors, suggestive of the presence of neuro‐autocrine mechanisms of sebocyte modulation. Aside from the neuroendocrine system, it is conceivable that secretion of neuropeptides and neurotransmitters from cutaneous nerve endings may also act on sebocytes or their progenitors, given that the skin is richly innervated. However, to date, the neural controls of SG development and function remain poorly investigated and incompletely understood. Botulinum toxin‐mediated or facial paresis‐associated reduction of human sebum secretion suggests that cutaneous nerve‐derived substances modulate lipid and inflammatory cytokine synthesis by sebocytes, possibly implicating the nervous system in acne pathogenesis. Additionally, evidence suggests that cutaneous denervation in mice alters the expression of key regulators of SG homeostasis. In this review, we examine the current evidence regarding neuroendocrine and neurobiological regulation of human SG function in physiology and pathology. We further call attention to this line of research as an instructive model for probing and therapeutically manipulating the mechanistic links between the nervous system and mammalian skin.     

Two new monoclonal antibodies (LN-1, LN-2) reactive in B5 formalin-fixed, paraffin-embedded tissues with follicular center and mantle zone human B lymphocytes and derived tumors

Two monoclonal antibodies (LN-1, LN-2) reactive with B lymphocytes in B5 formalin-fixed, paraffin-embedded tissue sections have been produced by utilizing cell extracts from pokeweed mitogen-stimulated peripheral blood lymphocytes and diffuse histiocytic lymphoma SU-DHL-4 cells, respectively. Both monoclonal antibodies were initially identified by indirect immunofluorescence screening techniques on paraformaldehyde-acetone-fixed cell preparations. Specificity screens with 36 well-characterized human lymphoma and leukemia cell lines showed that both LN-1 and LN-2 stained cell lines of B cell lineage but were unreactive with those of T cell or, with one exception, myeloid derivation. Null cell acute lymphoblastic leukemia cell lines were found to be LN-2+ but LN-1-. The B cell specificity of these reagents was confirmed on 15 lymphoma and 17 leukemia biopsy specimens by using indirect immunofluorescence techniques. Immunoperoxidase staining of sections from B5-fixed, paraffin-embedded human lymphoid tissues showed that LN-1 bound to the cell membrane and cytoplasm of germinal center cells whereas LN-2 stained the nuclear membrane and cytoplasm of germinal center and mantle zone B lymphocytes as well as interfollicular histiocytes and thymic medullary dendritic cells. Both monoclonal antibodies failed to stain cortical thymocytes, lymph node T cells, and peripheral blood T and myeloid cells. Immunoperoxidase staining of 20 nonlymphoid human organs and tissues revealed that LN-1 reacted positively with red blood cell precursors of the bone marrow, ciliated epithelial cells of the bronchus, distal tubular cells of the kidney, and ductal cells from several organs including the breast and prostate. In contrast, LN-2 was unreactive with all human nonlymphoid organs and tissues including the bone marrow. Indirect immunofluorescence staining of a panel of 26 solid tumor cells lines showed that LN-1 was reactive with the majority of epithelium-derived cell lines, glioblastomas, and astrocytomas but was unreactive with neuroblastomas, small cell carcinoma of the lung, and sarcomas. LN-2 was unreactive with 25 of 26 of the solid tumor cell lines by these techniques. Immunobiochemical studies have shown that LN-1 recognizes a cell surface sialoantigen whereas LN-2 is directed against a 35,000 dalton nuclear membrane protein. Because of their high specificity for B cell tumors and their ability to stain B5-fixed, paraffin-embedded tissues, LN-1 and LN-2 are useful reagents for the diagnosis and classification of the human lymphomas and leukemias.     

Nicotinic acid suppresses sebaceous lipogenesis of human sebocytes via activating hydroxycarboxylic acid receptor 2 (HCA2)

Nicotinic acid (NA) activates hydroxycarboxylic acid receptor 2 (HCA₂), and it is widely used in treating dyslipidaemias. Since its side effects include skin dryness, whereas its deficiency can be accompanied by dyssebacia, characterized by sebaceous gland enlargement, we asked if HCA₂ is expressed on human sebocytes, and if NA influences sebocyte functions. By using human immortalized SZ95 sebocytes, we found that non‐cytotoxic (≤100 μmol/L; MTT‐assay) concentrations of NA had no effect on the homeostatic sebaceous lipogenesis (SLG; Nile Red), but normalized excessive, acne‐mimicking SLG induced by several lipogenic agents (arachidonic acid, anandamide, linoleic acid + testosterone; Nile Red; 48‐hr treatments). Moreover, it exerted significant anti‐proliferative actions (CyQUANT‐assay), and increased [Ca²⁺]_IC (Fluo‐4 AM‐based Ca²⁺‐measurement). Although NA did not prevent the lipopolysaccharide‐induced pro‐inflammatory response (up‐regulation [Q‐PCR] and release [ELISA] of several pro‐inflammatory cytokines) of the sebocytes, collectively, these data support the concept that NA may be effective in suppressing sebum production in vivo. While exploring the mechanism of the sebostatic actions, we found that sebocytes express HCA₂ (Q‐PCR, immunofluorescent labelling), siRNA‐mediated silencing of which prevented the NA‐induced Ca²⁺‐signal and the lipostatic action. Collectively, our data introduce NA, and HCA₂ activators in general, as novel, potent and most likely safe sebostatic agents, with possible anti‐acne potential.     

LN-6: a monoclonal antibody to vimentin expressed in non-hematopoietic mesenchymal cells and derived tumors and reactive in B5-fixed, paraffin-embedded tissues

We generated a monoclonal antibody (MAb), designated LN-6, directed against human vimentin, which retains its immunoreactivity in B5-fixed, paraffin-embedded tissues. Like other anti-vimentin MAb, LN-6 was found to be reactive with a wide spectrum of human sarcomas and normal cells of mesenchymal derivation. However, unlike other similar reagents, LN-6 was unreactive with normal and malignant human lymphoid cells and therefore displays a more restricted immunoreactivity. Because of its ability to stain routinely processed pathological tissues and its marked reactivity with human sarcomas, LN-6 is a unique reagent for the immunohistochemical diagnosis of human cancer.     

Fine needle aspiration cytology of a sebaceous lymphadenoma: a case report

BACKGROUND: Sebaceous lymphadenoma is a rare, benign neoplasm, histologically characterized by proliferating islands of epithelium with sebaceous glandular differentiation in a dense, lymphocytic background. The parotid gland is the most common site, and the patient usually presents with a well-circumscribed, enlarging and painless mass. Primary sebaceous lesions of the salivary glands are very rare entities and must be differentiated from more common, potentially malignant tumors. CASE: A 75-year-old male presented with a 6-month history of a mass in the tail of the parotid gland. The mass was not fixed or tender to palpation, was well delineated and measured 4 cm in greatest dimension. Fine needle aspiration (FNA) revealed a mixed population of large and small lymphocytes, including plasma cells and occasional tingible body macrophages. Scattered among the lymphocytes were 3-dimensional, cohesive aggregates of epithelial cells, many demonstrating the characteristic cytoplasmic vacuolization of sebocytes, surrounded by layers of basaloid cells. No mitoses or cellular pleomorphism was identified. These findings suggested a sebaceous lymphadenoma, confirmed on biopsy. CONCLUSION: Although sebaceous lymphadenoma is encountered infrequently, FNA findings can result in its accurate diagnosis.     

Immunological and histochemical analysis of regional variations of epidermal Langerhans cells in normal human skin

Summary Epidermal Langerhans' cells (LC) were enumerated in normal human skin from various anatomical sites using a monoclonal antibody (NA1/34) to human thymocyte antigen (HTA-1) and the standard ATPase reaction on frozen sections. The same population of cells was identified with each technique. LC densities were found to be significantly higher in hair bearing skin than in skin from the palm and sole. LC were also identified in hair follicles (where the numbers decreased from the superficial to the deep portions) and sebaceous glands but in no other adnexal structure. Normal numbers were encountered in patients who had received radiotherapy or systemic chemotherapy for malignant disease for periods of greater than two months before death. As LC are important antigen presenting cells, the variation in their density suggests that the immunological properties of normal skin may not be uniform throughout the body. This may be related to the varying anatomical distribution of some skin disorders with an immunological basis.     

Alterations of irisin concentrations in saliva and serum of obese and normal-weight subjects,before and after 45 min of a Turkish bath or running

The purpose of this study was to ascertain (1) whether human saliva contains irisin and whether its level correlates with serum irisin concentration, (2) whether salivary glands, eccrine glands and sebaceous glands in human skin produce irisin, (3) how the changes in saliva and serum irisin concentrations after the Turkish bath at 47 ± 3 °C compare with the changes caused by moderate exercise in obese and normal weight subjects. Seven obese male subjects and seven normal weight subjects were enrolled for Turkish bath. Seven obese male subjects and seven normal weight subjects were also enrolled for moderate outdoor exercise, and thirteen male normal weight subjects neither exercised nor showered at the Turkish bath. From each participant, 1.5 ml of saliva and 5 ml blood were collected simultaneously before and after the moderate exercise and Turkish bath. Salivary glands and eccrine and sebaceous glands in the skin were screened immunohistochemically for irisin while serum and saliva irisin were measured with an ELISA. Submandibular glands, eccrine glands and sebaceous glands in the human skin showed strong irisin immunoreactivity. Human saliva contained irisin and its level was significantly higher than the serum levels in both obese and normal weight subjects. However, irisin concentrations were more markedly increased in both saliva and serum samples from subjects who had showered at a Turkish bath than in obese subjects who had exercised or in normal weight subjects. Human submandibular glands, eccrine sweat glands and sebaceous glands synthesize irisin.     

Comparative responsiveness of supracaudal and other sebaceous glands in male and female guinea pigs to hormones

Different types of sebaceous glands in guinea pigs were chosen to study their comparative responsiveness to steroid hormones. Glands selected were (1) Sebaceous glands associated with rudimentary hair in the supracaudal gland; (2) Free sebaceous glands of the nipple; (3) Sebaceous glands associated with the hair. The results showed distinct differences in sensitivity among these different sebaceous glands and according to the sex of the experimental animal. The most responsive of the glands in males was the supracaudal gland; in the female the most responsive glands were the sebaceous glands of the nipple. Sebaceous glands associated with normal hair were relatively insensitive to changes in hormonal level. In all cases testosterone propionate was more potent in stimulating any of the sebaceous glands than progesterone. After gonadectomy, various sebaceous glands showed unequal states of depletion due to the different rates of differentiation and disintegration of their cells into sebum.     

The effect of hydrocortisone on DNA synthesis by sebaceous gland epithelial cells]

The autoradiography method demonstrated a single injection of hydrocortisone (10 mg per 100 g of body weight) to result in suppression of the cell transition from the G1 phase of the mitotic cycle to the S phase and in prolongation of the S phase of the epithelium of the acinar portion (sebocytes) and stratified epithelium of the excretory ducts of the sebaceous gland of rats. Later, after a decrease of the proliferative processes by hydrocortisone there occurred their increase which was higher than control. Hydrocortisone induced a more pronounced effect on the proliferative activity of the stratified epithelium of the excretory ducts than on the sebocytes of the acini.