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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