Neuropeptides and sebaceous glands

ARTICLE

Innervation in the skin

The skin is innervated by primary afferent sensory nerves, postganglionic cholinergic parasympathetic nerves, and postganglionic adrenergic and cholinergic sympathetic nerves. Sensory nerves are derived from the dorsal root ganglion and are present in all parts of the skin representing the initial somatic portion of the afferent sensory pathway. The cutaneous sensory nervous system comprises a network of fine C fibers within the skin that innervate multiple cell types and play an important role in inflammation [1]. The epidermis is also innervated by a three-dimensional network of unmyelinated nerve fibers with free branching endings that arise in the dermis. Sensory nerves not only function as an afferent system to conduct stimuli from the skin to the central nervous system, but they also act in an efferent neurosecretory fashion to stimulate target tissues through their terminals [2]. Various stimuli, such as noxious stimuli, may directly activate the peripheral endings of primary sensory neurons generating impulses that are conveyed centrally as well as, through antidromic axon-reflexes, peripherally. Upon release of neuropeptides (NPs) from sensory terminals, important visceromotor inflammation and trophic effects occur in the peripheral tissues. This release of proinflammatory NPs elicits a set of changes that are collectively referred to as neurogenic inflammation.

Neuropeptides

NPs are a heterogeneous group of several hundred biologically active peptides that are present in neurons of both the central and peripheral nervous systems and that are involved in the transmission of signals, not only between nerve cells, but also with the immune system where they appear to be critical mediators of different processes. They act as neuromodulators, neurotransmitters, neurohormones and hormones, and can manifest immunomodulatory activity, and contribute to the cross talk between the nervous and immune systems in the skin [3, 4]. Normal human skin expresses a variety of NPs that are either directly derived from sensory neurons or from skin cells such as keratinocytes, microvascular endothelial cells or fibroblasts. In addition, immune cells that either constitutively reside in the skin, such as mast cells (MCs), or that infiltrate the skin during inflammation have been reported to produce NPs [3]. Cutaneous nerve fibers can modulate inflammatory reactions through the local release of NPs, which are able to regulate both acute and chronic aspects of cutaneous inflammatory processes, such as vascular motility, cellular trafficking, activation and trophism [3-6]. Clinical evidence in support of a connection between NP secretion and the development of inflammation is found in various skin diseases, such as atopic dermatitis, psoriasis and alopecia areata, which are commonly exacerbated during pe-riods of emotional stress [4-6]. Indeed, stress has been shown to elicit the release of substance P (SP) [7], an NP belonging to the tachykinin family, which can induce neurogenic inflammation. SP is associated with multiple cellular responses, including vasodilation, increased blood flow, plasma extravasation, mast cell degranulation, the wheal and flare reaction via axon reflex, neutrophil and macrophage activation, modulation of the release of proinflammatory cytokines and chemokines, and the up-regulation of adhesion molecule expression required for trafficking of leukocytes [3].

NP-degrading enzymes

Tissue responsiveness to NPs depends on the presence of specific receptors and on the distribution of NP-degrading enzymes which play essential roles in the removal of NPs from the extracellular environment and which are thus important regulators in neurogenic inflammation. Recent studies indicate that neutral endopeptidase (NEP; EC 3.4.24.11; enkephalinase), a zinc metallo-protease, is a cell surface enzyme with the potential to degrade several NPs such as SP, and thereby terminate their biologic actions [4]. NEP in normal skin is localized in keratinocytes, vascular endothelial cells, fibroblasts, the outer root sheath of hair follicles and MCs [8, 9]. Administration of NEP inhibitors magnifies the proinflammatory effects of SP and other tachykinins in several tissues [10]. Thus, up-regulation of NEP is a potential mechanism for limiting the proinflammatory effects of NEP-degradable NPs, notably SP, by reducing the amounts of bioactive NPs.

Pathogenesis of acne

Acne vulgaris is a complex, chronic, and common skin disorder of pilosebaceous units which usually begins at the time of the sharp increase in androgen production that occurs during adolescence, which suggests that this disease is intimately controlled by androgenic hormones. The clinical manifestations of acne vulgaris range from non-inflammatory comedones to inflammatory papules, pustules and cysts. In most patients with acne, these types of lesions are usually intermingled to various extents. The multifactorial nature of acne has been elucidated in recent years [11]. Briefly, acne begins when the pilosebaceous ducts become plugged with keratinocytes to form comedones, sebum builds up and distends the follicles, and the anaerobe Propionibacterium acnes (P. acnes) proliferates in the sebum. If the comedo ruptures into the dermis, inflammation results and a pustule or papule forms. Many lines of clinical evidence suggest that components of the nervous system, such as psychological and neurogenic factors, can influence the course of acne. The disease has been reported to be initiated and/or exacerbated as a result of emotional or psychosocial stress, suggesting the involvement of neural-derived factors in the pathogenesis of acne.

Sebaceous glands and sebum production

Sebum is the by-product of the holocrine rupture of mature sebocytes. As mentioned above, sebaceous glands are responsive to androgenic hormones which stimulate excess sebum secretion [12]. The most important androgen is testosterone which is converted to dihydrotestosterone by the iso-enzyme 5alpha-reductase type 1 [13]. In acne patients, there are increased levels of 5alpha-reductase levels in the sebaceous glands where an increased number of androgen receptors are also found [13]. In a microcomedo, the sebum will be trapped behind a keratin plug. The follicle then becomes enlarged and contains a mixture of sebum and keratinous squamae, which eventually obliterates the normal architecture of the follicle and forms a thin-walled cystic lesion, known as a comedo. Sebaceous lipids consist of triglycerides, with significant quantities of wax esters and squalene and very small amounts of cholesterol and cholesterol esters. Lipases from the bacteria hydrolyze sebum triglycerides into free fatty acids that are both comedogenic and proinflammatory.

Effects of NPs on the morphology of sebaceous glands

Acne is a skin disorder of sebaceous follicles, which are equipped with large sebaceous glands and produce only fine vellus hairs. To examine whether cutaneous neurogenic factors affect the morphology of sebaceous glands, we used electron microscopy to observe alterations of sebaceous glands in skin organ culture treated with several kinds of NPs or with nerve growth factor (NGF). From the exterior aspect to the interior, normal sebaceous glands consist of the germinative, the undifferentiated and the differentiated sebaceous cell layers. Most sebaceous gland cells stimulated with SP contained numerous lipid droplets, which disintegrated to form an acellular sebum secretion, even in the peripheral area of the glands. These observations indicate that SP can accelerate lipid synthesis. There were numerous free ribosomes and mitochondria, followed by densely packed smooth-surfaced membranes of the endoplasmic reticulum, suggesting an active phase of lipid synthesis. Morphometric analysis using a computer-assisted image analysis system (NIH) revealed that of all agents tested, only SP induced significant increases in the area of sebaceous glands as well as in the size of individual sebaceous cells. Furthermore, SP significantly increases the number of sebum vacuoles in each differentiated sebaceous cell at the ultrastructural level. The number of sebum vacuoles induced by SP increased in a dose-dependent manner when various concentrations of SP were added to the culture medium [11]. These findings suggest that SP may stimulate the proliferation as well as the differentiation of sebaceous glands, and further, that it up-regulates lipid synthesis in sebaceous cells. The effects of immobilization-induced stress on plasma testosterone levels and lipogenesis were examined in sebaceous glands of Syrian hamsters, and immobilization-induced stress lowered testosterone secretion and testosterone levels in the skin, which resulted in decreased lipogenesis in the skin [14]. Although these data suggest that psychological or physiological stress can influence sebaceous gland function by inducing changes in the neuroendocrine system, they provide no appropriate explanation for the effects of stress-induced exacerbation of acne. Taking into account that stress can elicit SP release from peripheral nerves [7], it is tempting to speculate that SP might be involved to some extent in the stress-induced exacerbation of the disease.

Innervation of sebaceous glands and NGF

It is generally accepted that sebaceous glands are not innervated and that the peripheral nervous system has no effect on sebaceous gland biology. Indeed, nerve fibers, which are detected immunohistochemically using the general neuronal marker PGP 9.5, are rarely observed around sebaceous glands in normal facial skin. In contrast, facial skin from acne patients shows numerous fine nerve fibers, not only around but also within, sebaceous acini (Fig. 1). Ultrastructurally, myelinated nerve bundles are present around sebaceous glands, and numerous nerve terminals are observed in close apposition to sebaceous glands. Such increased numbers of nerve fibers, some of which even invade sebaceous acini, may result from increased secretion of NGF, the best-characterized member of the neurotrophin family, in sebaceous glands of acne-prone facial skin since NGF is essential for the survival, development, differentiation and function of peripheral sympathetic and sensory neurons, and acts as a neurotrophic molecule also stimulating the sprouting of nerve fibers in the skin [15]. Immunohistochemical analysis reveals that germinative cells, which are the outer most layer of the sebaceous acini, express high levels of NGF in acne patients (Fig. 2), although no immunoreactivity for NGF is observed in normal sebaceous glands. In addition, an increase in the expression of the high-affinity receptor for NGF (TrkA) is apparent on the germinative cells of sebaceous glands from acne patients compared with those in normal sebaceous glands.

The precise mechanism of the specific induction of NGF in sebaceous glands of acne patients is unclear. Although we treated organ cultures of human facial skin with SP, no expression of NGF was observed. It is possible that SP induces NGF expression in sebaceous glands via some proinflammatory cytokines since SP is considered to modulate cytokine synthesis [3]. In vitro, SP enhances the production and release of IL-1, IL-6, TNF-alpha and IFN-gamma by monocytes, of IL-1alpha, IL-1beta, IL-1 receptor antagonist, IL-8 and granulocyte/macrophage colony-stimulating factor by keratinocytes, of TNF-alpha by MCs, and of IL-2 synthesis and secretion by peripheral blood monocyte cells. Moreover, it is known that IL-4 production by murine T lymphocytes is stimulated by SP, and that IL-2 is able to increase IL-4 production by T cells. In vivo, SP increases the expression of mRNAs for IL-1beta, IL-3, IL-5, IL-6, TNF-alpha and IFN-gamma in all mucosal samples of allergic subjects [1, 3-5]. These data suggest that SP may directly or indirectly enhance cutaneous inflammatory reactions by inducing major proinflammatory cytokines. Interestingly enough, several cytokines, including IL-1, IL-6, TNF-alpha and IFN-gamma, are potent inducers of NGF synthesis in peripheral tissues and in the central nervous system. Taking into account the increased number of degranulating MCs in close apposition to sebaceous glands of acne patients, we examined the inducibility of NGF by MC-derived mediators and cytokines, including histamine, tryptase, chymase, leukotriene D4, prostaglandin E2, IL-4, IL-6, IL-8, TNF-alpha, IFN-gamma and platelet activating factor. As a result, IL-6 specifically induced the expression of NGF in normal facial skin in organ culture. Preincubation of explants with antibodies specific for the IL-6 receptor, followed by exposure to IL-6, abrogated the NGF induction. Immunoelectron microscopic study revealed the presence of IL-6 within specific granules of MCs around sebaceous glands in the skin from acne patients. The numbers of IL-6-positive MCs and IL-6-containing MC granules are significantly increased in acne patients compared with controls (Fig. 3). These findings suggest that MC-derived IL-6 has the potential to induce NGF in sebaceous cells, which may result in promoting innervation within and around sebaceous glands in acne.

NGF is also considered to be a primary candidate as a regulatory molecule in neuropeptidergic responses. Indeed, it has been shown that: 1) NGF is increased in nerves supplying inflamed skin, 2) injection of NGF in the skin reproduces the same neuronal peptidergic modification observed during experimental inflammation in rats, and 3) pre-treatment with anti-NGF serum prevents the NP induced changes at a neuronal level. It is therefore tempting to speculate that NGF plays an important role in spontaneous inflammatory dermatoses, such as atopic dermatitis, by modulating NP expression. There is increasing evidence that NGF, in addition to its actions within the nervous system, elicits a number of biologic effects on local and systemic cells of the immune-inflammatory compartment. In vivo, administration of NGF to neonatal rats increases the size and the number of MCs in several peripheral tissues, while in vitro, NGF induces mast cell degranulation and mediator release. NGF enhances survival, phagocytosis, and superoxide production of mature murine neutrophils, causes mediator release from basophils, stimulates T and B lymphocyte proliferation, and stimulates B-cell differentiation into immunoglobulin-secreting plasma cells [5, 15]. These data imply a possible relationship of NGF with the inflammatory process associated with acne.

SP-containing nerve fibers around sebaceous glands

Nerve fibers showing immunoreactivity for SP were rarely observed in skin specimens from facial skin of healthy volunteers devoid of acne lesions. On the other hand, facial skin specimens from acne patients showed a strong immunoreactivity for SP with many fine nerve fibers around the sebaceous glands. Some of them were located in close apposition to the sebaceous acini [16].

Expression of NEP in sebaceous glands

Immunohistochemical staining for NEP in normal facial skin was negative within sebaceous glands. In contrast, NEP was highly expressed in sebaceous glands in specimens from all acne patients examined in which immunoreactivity for NEP in sebaceous glands was restricted to the germinative cells. The NEP staining of germinative cells appeared to be cytoplasmic and cell membrane associated. There was a statistically significant difference in the percentage of NEP-positive sebaceous acini between acne patients and controls [16]. We next examined the effects of SP on NEP expression in sebaceous glands using organ-cultured skin. Although normal facial skin in organ culture supplemented with medium alone showed no staining for NEP in sebaceous cells, skin specimens stimulated with SP revealed prominent NEP staining in the germinative cells of the sebaceous acini, which was similar to the staining pattern of sebaceous glands in patients with acne. SP induced NEP expression in sebaceous glands in a dose-dependent manner [16]. Taking into account the lack of NEP expression in tissue not stimulated with SP, we suggest that sebaceous germinative cells begin to synthesize NEP following stimulation by SP.

The subcellular localization of NEP in sebaceous glands

To confirm the expression of NEP by immunohistochemistry and to examine the subcellular localization of NEP in sebaceous cells more precisely, we performed ultrastructural immunocytochemistry using an indirect immunoperoxidase technique. We report for the first time that NEP expression is restricted to the Golgi apparatus and the endoplasmic reticulum within sebaceous germinative cells (Fig. 4) [16], which indicates that NEP is synthesized via the protein synthesis pathway in the usual fashion. There was no labeling of nuclei, intracytoplasmic organelles or intercellular spaces. Immunoreactivity for NEP was not observed in any cytoplasmic structures within mature sebocytes. Although NEP is considered to be a cell surface enzyme, the transport and release processes of this protein to and from the plasma membrane in sebocytes are not evident at present.

Role of MCs in association with SP in acne inflammation

Increasing attention has been directed towards interactions between components of the nervous system and multiple target cells of the immune system. Communication between nerves and MCs is a prototypic demonstration of such neuroimmune interactions. Several studies have demonstrated that MCs are often found in close contact with nerves and that there may be functional interactions between MCs and the nervous system [17]. In addition, recent evidence suggests that SP is an important mediator in intimate nerve-MC cross talk [18]. When organ-cultured normal facial skin was exposed to SP or to an SP analogue which binds to the identical MC surface receptor but does not provoke significant histamine release, uniformly degranulated MCs adjacent to the sebaceous glands were observed exclusively in explants exposed only to SP at the electron microscopic level. Venules around sebaceous glands of specimens exposed to SP showed evidence of ELAM-1 induction after subsequent culture, but ELAM-1 was not induced by the SP analogue. Furthermore, preincubation of explants with the SP analogue or with the MC inhibitor, cromolyn sodium, abrogated the ability of SP to induce ELAM-1. These findings suggest that SP endogenously released by dermal nerve fibers may be important in the regulation of endothelial-leukocyte interaction via MCs. It has been demonstrated that the proinflammatory effect of ELAM-1 induced by MC degranulation products is inhibited by a blocking antiserum to TNF-alpha. Thus, SP contained within dermal nerve fibers may represent a crucial initial mediator of a cascade of cellular events involving MC degranulation and the release of proinflammatory cytokines such as TNF-alpha, with subsequent induction of adhesion molecules such as E-selectin on the adjacent venular endothelium [19]. This would then facilitate the local accumulation of blood leukocytes during the inflammatory response. We have recently used immunoelectron microscopy to demonstrate that SP is localized within specific granules of human skin MCs [20]. MC-derived SP may also affect the morphologic and immunologic alterations associated with sebaceous glands and may contribute to the development inflammatory events in acne.

Proliferation of MCs by SP

The finding that the number of MCs in the perisebaceous area is increased in the facial skin of acne patients compared with normal skin was unexpected. The importance of fibroblasts and a fibroblast-derived MC growth factor, stem cell factor (SCF), for MC growth has been clearly demonstrated using MC-deficient mutant mice. SP up-regulates soluble SCF expression by human fibroblasts (Fig. 5) in a dose-dependent manner (Fig. 6) in monolayer culture, as measured by an enzyme-linked immunosorbent assay. SCF mRNA was detected by reverse transcriptase-PCR in cultured human fibroblasts. A predicted 414 bp cDNA product was produced. When the PCR-bands were quantified and the results expressed as ratios of densitometric scores for SCF and GAPDH for each sample, the SCF message after treatment with 10² to 10⁴ ng/ml of SP was relatively more intense than was platelet-derived growth factor, a well-known SCF enhancer (data not shown). This finding suggests that SP can enhance MC proliferation by up-regulating SCF expression in fibroblasts.

Based on the sum of these data, we propose a possible mechanism for stress-induced exacerbation of acne from a neurogenic aspect, as presented in Fig. 7.

Article accepted on 21/3/02

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