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1. Introduction
2. Skin as a
neuroimmunoendocrine organ
3. Neuropeptides in skin
inflammation
4. Neuropeptides: focus on skin
cells function
5. Neuropeptides in diabetic
wound healing
6. Expert opinion
Review
Role of neuropeptides in skin
inflammation and its involvement
in diabetic wound healing
Lucı
´lia da Silva, Euge
´nia Carvalho & Maria Teresa Cruz
†
†
Universidade de Coimbra, Coimbra, Portugal
Importance of the field: In 2010, the world prevalence of diabetes is 6.4%,
affecting 285 million adults. Diabetic patients are at risk of developing neu-
ropathy and delayed wound healing that can culminate in incurable diabetic
foot ulcerations (DFUs) or even foot amputation.
Areas covered in this review: The contrast between cellular and molecular
events of wound healing and diabetic wound healing processes is character-
ized. Neuropeptides released from the autonomous nervous system and skin
cells reveal a major role in the immunity of wound healing. Therefore, the
signaling pathways that induce pro/anti-inflammatory cytokines expression
and its involvement in diabetic wound healing are discussed. The involvement
of neuropeptides in the activation, growth, migration and maturation of
skin cells, like keratinocytes, Langerhans cells, macrophages and mast cells,
are described.
What the reader will gain: This review attempts to address the role of neuro-
peptides in skin inflammation, focusing on signal transduction, inflammatory
mediators and pro/anti-inflammatory function, occurring in each cell type, as
well as, its connection with diabetic wound healing.
Take home message: Understanding the role of neuropeptides in the
skin, their application on skin wounds could be a potential therapy for skin
pathologies, like the problematic and prevalent DFUs.
Keywords: diabetic foot ulcers, inflammation, neuropeptides, signaling pathways, skin cells,
wound healing
Expert Opin. Biol. Ther. (2010) 10(10):1427-1439
1. Introduction
Wound healing is a dynamic process that involves the complexity of the immune
system, including a variety of immune cells and inflammatory mediators. Wound
healing can be divided into four overlapping phases: homeostasis/coagulation,
inflammation, migration-proliferation and remodeling. Homeostasis lasts 2 -- 3h,
the fibrin plug is formed and aggregated platelets release pro-inflammatory media-
tors such as cytokines and growth factors. Cytokines recruit neutrophils and mono-
cytes to the wound site, activating the inflammatory phase of wound healing, lasting
from hours to days. The proliferation phase occurs in different cell types such as
epithelial cells, endothelial cells and fibroblasts. After cytokine stimulation, these
cells migrate to the wound site -- migration phase. Extracellular matrix (ECM) is
deposited and begins the angiogenesis and re-epithelialisation, contraction and
closure of the wound. The remodeling phase (which can last several weeks) is
characterized by ECM adjustment and formation of scar tissue. Wound healing
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involves the activation of different cells, namely keratinocytes
(KCs), fibroblasts, endothelial cells, Langerhans cells, macro-
phages (MFs), mast cells (MCs) and platelets, inflammatory
mediators, such as growth factors and cytokines, and it also
requires complex biological and molecular events
that induce cell migration, cell proliferation and ECM
deposition [1].
Currently, diabetes affects over 285 million adults. As a
consequence, diabetic people may develop coronary heart dis-
ease, chronic kidney disease, diabetic ketoacidosis, diabetic
retinopathy and obesity. Additionally, diabetic people can
develop wound healing difficulties, persisting in three main
body areas: the eye, skin and bones.
Diabetic keratopathy is a consequence of defective healing
in corneal epithelium injuries, which is important as a barrier
against ocular infection in the eyes. Patients with keratopathy
have abnormalities in corneal re-epithelialization associated
with persistent epithelial defects, infectious corneal ulcers,
decreased corneal sensitivity, increased epithelial fragility, sec-
ondary scaring, punctuate keratopathy, edema and loss of
vision [2].
Diabetic foot ulceration (DFU) is a painless and harmful
disease that also affects diabetic people and is characterized
by delayed wound healing, the consequence of various causes.
Sensory denervation -- neuropathy -- is an emerging risk of
the development of DFUs. In addition to neuropathy, these
patients have impaired angiogenic response and decreased
blood supply to the site of injury [1,3]. Delayed wound healing
can also be explained by cytokine deregulation, inhibiting
the last phase of wound healing: migration-proliferation and
remodeling [4]. Accordingly, TNF-a, IL-1, IL-6 and CCL5
are released from immune cells and adipocytes, in type 2
diabetic patients [5]. Nevertheless, other inflamma-
tory mediators, like growth factors, specifically basic fibroblast
growth factor (bFGF), platelet-derived growth factor (PDGF)
and VEGF are diminished in expression or their activities are
impaired. Diabetic wounded skin also presents impaired
expression of ECM proteins and misbalance between the
accumulation of ECM components and their remodeling by
MMPs [6].
Focusing on skin cells from DFUs, like KCs and
fibroblasts, these cells have deregulated migration and proli-
feration [7]. In hyperglycemia and inflammatory conditions,
leukocytes (mainly neutrophils) also have defective migra-
tion to the wound [1,4]. Neutrophils have deficient chemo-
tactic, phagocytic and microbicidal activities, augmenting
the susceptibility and severity of infection in diabetes [8].
Macrophages undergo morphological transformations and
decrease the release of cytokines, like TNF, IL-1 and
VEGF [9]. Lack of upregulation of chemokines, cytokines
and growth factors in KCs and dermal endothelial cells, in
the margin of chronic diabetic foot ulcers, associated with
the reduced influx of immune cells, may lead to poor
formation of granulation tissue and chronicity of ulcer
epithelialization [10].
Considering all these deficiencies in the skin of diabetic
people, an extra disturbance, such as a physical stress, can be
a potential cause of foot ulcers or even bone healing problems,
like the Charcot neuroarthropathy of the foot (characterized
by weakened bones that can fracture) [1], that may lead to a
final foot amputation.
2. Skin as a neuroimmunoendocrine organ
Neuropeptides circulate between the brain and peripheral
organs, through the CNS, autonomous nervous system and
sensory nervous system. The sensory nervous system includes
sensory receptors that sense internal or external, chemical or
physical alterations, namely chemoreceptors, mechanorecep-
tors, noniceptors, photoreceptors and thermoreceptors, as
well as afferent sensory neurons that transmit the perceived
information. Sensory neurons have the cell body located in
the dorsal root ganglia of the spinal nerve, in the spinal
cord, or in the brain and can have different conducting
fibers, namely A fibers (a,band d) and C fibers. A fibers
are myelinated and highly conducting, C fibers are unmye-
lianted and slow-conducting. The autonomous nervous sys-
tem includes parasympathetic ganglia (close to the target
organ), efferent parasympathetic motor neurons, sympathetic
ganglia (located in the spinal cord) and efferent sympathetic
motor neurons [11]. To understand the relevance and function
of these nerves, the pungent agent from chilli peppers --
capsaicin -- that results in the depletion of sensory neuropep-
tides and permanent degeneration of small-diameter C-fibers
by binding to the transient receptor potential vanilloid
1 (TRPV1), as well as, it’s antagonist -- capsazepine -- have
been used in research [12].
Article highlights.
.Wound healing is a dynamic process that involves the
activation, migration and proliferation of cells, the
production of growth factors, chemokines and cytokines
and extracellular matrix adjustment.
.Skin injury stimulus may activate sensory neurons to
induce the release of neuropeptides in the site of injury.
.Substance P, calcitonin-gene-related peptide and
vasointestinal polypeptide are able to induce a
pro-inflammatory response in keratinocytes, as well as,
keratinocyte proliferation.
.Actual knowledge of neuropeptides function in
Langerhans cells only reveals an anti-inflammatory
condition.
.Vasointestinal polypeptide diminishes TNF-arelease and
superoxide anion production in macrophages. Other
neuropeptides are also implicated in IL-10 release and
in the increase of LPS-induced cytokines production.
.Higher levels of neurotensin are found in diabetic
wound healing although the role of neurotensin is
not clear.
This box summarizes key points contained in the article.
Neuropeptides in skin inflammation and diabetic wound healing
1428 Expert Opin. Biol. Ther. (2010) 10(10)
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Endogenous factors, such as pH, cytokines, kinins, hista-
mine and proteases and exogenous agents, like allergens,
bacteria and UV radiation [13] activate sensory nerve endings.
After injury, mast cells release histamine which activates
sensory neurons via H1 receptors, inducing orthodromic
stimulation to spinal cord and antidromic stimulation to sur-
rounding skin cells, triggering the release of neuropeptides [14].
Orthodromic stimulation is characterized by the transmission
of stimuli through unmyelinated C or myelinated A primary
afferent nerve fibers, via dorsal root ganglia to the spinal
cord and then to specific areas of the CNS, resulting in the
perception of pain, burning or other symptoms. Conse-
quently, it results in the release of neuropeptides in the site
of the skin stimuli by efferent autonomous motor neurons.
Antidromic stimulation is characterized by axon reflex in the
neighboring afferent nerve fibers with a final release of neuro-
peptides. The analgesic morphine can activate opioid recep-
tors located on primary afferent sensory nerve terminals,
reduce the excitability of these neurons and suppress the anti-
dromic release of pro-inflammatory neuropeptides, like sub-
stance P (SP) and neurokinin (NKA). Consequently,
providing this analgesic to ulcer patients delays wound heal-
ing [15]. Finally, neuropeptides released from cutaneous nerves
act on target cells via paracrine, juxtacrine and endoncrine
pathways, similar to cytokines.
In the last three decades, neuropeptides received further
attention from the scientific community. These molecules
are significant in wound healing, not only in the adult stage,
but also in fetal skin wound healing. In a study with fetal
rats during skin development, nerves and neuropeptides
were upregulated and contributed to the survival and regener-
ation of peripheral neurons and modulated an ideal wound
healing process -- a scarless wound healing [16]. However,
higher levels of nerves and neuropeptides with reduced neutral
endopeptidase (NEP) levels were shown in human and
porcine hypertrophic scars with exuberant inflammation [17].
These results reveal the importance of neuropeptides in
wound healing and so, their role in skin inflammation and
in skin cells is discussed later.
3. Neuropeptides in skin inflammation
Neuropeptides have appeared as key regulators of inflamma-
tion and inflammatory processes per se, giving new insights
in the understanding of the skin’s immune system. Neuropep-
tides may be released in the skin by the autonomous nervous
system and also by skin cells, such as keratinocytes, endothe-
lial cells, fibroblasts, epidermal dendritic cells and Langerhans
cells [18,19]. Neuropeptides may induce the activation of
immune cells (neutrophils, macrophages, antigen-presenting
dendritic cells and lymphocytes). As a result, T-lymphocytes
may release specific anti or pro-inflammatory mediators (cyto-
kines, chemokines and growth factors), B cells can produce
antibodies and Langerhans cells may modulate antigen
presentation [11]. Accordingly, inflammatory mediators are
able to start and end skin inflammation and determine impor-
tant immune functions, such as proliferation/differentiation/
apoptosis of skin cells, vasodilation/vasoconstriction, vessel
permeability, angiogensesis, T cell chemotaxis to the site
of injury, mast cell degranulation and histamine release, pro/
anti-inflammatory cytokine release and improvement of
wound healing. Neuropeptides may regulate cytokine release
and the inflammatory response, as well as their own function,
by inducing its degradation with cell-associated neuropeptide-
degrading peptidases, like NEP, and by controlling nerve
growth [20].
Neuropeptides predominantly interact with GPCR. These
receptors exist in different cell types in the skin, including
keratinocytes, microvascular endothelial cells, merkel cells,
fibroblasts and all immune cells of the skin and activate
different signaling pathways and mediators, namely cAMP,
the phosphoinositol/calcium, the diacylglicerol/PKC, NF-kB,
PI3K/Akt and MAPK pathways.
4. Neuropeptides: focus on skin cells function
The following section delineates the involvement of different
neuropeptides, namely SP, vasointestinal polypeptide (VIP),
calcitonin gene-related peptide (CGRP), pituitary adenylate
cyclase activating polypeptide (PACAP), neuropeptide Y
(NPY), corticotropin releasing hormone (CRH), pro-
opiomelanocortin peptides (POMC peptides), melanocyte-
stimulating hormone (MSH), secretoneurin (SN), urocortin
and neurotensin (NT), in the activation, growth, migration
and maturation of specific skin cells, such as KCs, Langerhans
cells, macrophages and mast cells. In addition, this review
aims to distinguish the signaling pathways that induce the
expression of pro/anti-inflammatory cytokines.
4.1 Immune responses on keratinocytes (KCs)
Keratinocytes are an abundant and important cell type in the
epidermis [21]. KCs occur in the innermost layer (basal layer)
of the epidermis and after cell division, some of them remain
in the basal layer as stem cells, whereas others migrate into the
upper epidermal layers and differentiate. As long as KCs dif-
ferentiate, they synthesize the major structural components
of the stratum corneum, like different structural and catalytic
proteins, namely involucrin, keratins, filaggrin and trans-
glutaminase. Additionally, the stratum corneum becomes
composed of disulfide-linked keratin filaments surrounded
by a cross-linked envelope of protein and all these structures
are held together by desmosomal junctions and lipids released
from lamellar bodies. This surface layer of the skin (stratum
corneum) consists of death KCs. Indeed, KCs go through
biochemical and morphological changes, having a primary
role in the stratum corneum structure.
KCs are involved in immune skin diseases, such as psoria-
sis, itching, contact hypersensitivity and skin responses to
UV radiation and some studies have been done to determine
their role in these diseases. In fact, neuropeptides may be
da Silva, Carvalho & Cruz
Expert Opin. Biol. Ther. (2010) 10(10) 1429
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behind the role of KCs in proliferation, migration, differenti-
ation and immunity, as their receptors are expressed in these
cells and they are known to regulate immune function.
4.1.1 Proliferation, migration and differentiation of
keratinocytes
Some neuropeptides are known to induce KC proliferation,
such as CGRP, VIP and a-MSH; to inhibit KC proliferation
inducing its differentiation, such as CRH; and to support
KC migration, like SP and VIP. The role of SP has been
controversial. Studies in mice infer that SP mediates stress-
induced hair-growth-inhibitory effects, including perifollic-
ular neurogenic inflammation, hair follicle keratinocyte
apoptosis, inhibition of hair follicle epithelium prolifera-
tion and premature hair follicle regression (catagen induc-
tion) [22]. In contrast, Zhou et al. affirm that elevated levels
of SP are essential to induce the progression of anagen (the
growing phase) hair cycling through murine keratinocyte
growth promotion [23]. In acanthosis, a skin disease charac-
terized by diffuse epidermal hyperplasia, ultraviolet irradia-
tions induce murine KC proliferation, a result of an
increase in CGRP synthesis [24]. UV light also increases
a-MSH receptor (MC-1R) activity in human immortalized
KCs, inducing the expression of Pro-opiomelanocortin
(POMC) genes and human epidermal KC proliferation,
when stimulated by a-MSH [25]. However, Thioredoxin,
an antioxidant enzyme, is released from UVB-irradiated
human KCs and suppresses the production and secretion
of a-MSH, adrenocortiotrophic hormone (ACTH) and the
expression of POMC [26].
KCs proliferation is often associated with the effect of neuro-
peptides. However, some neuropeptides induce differentiation,
like CRH, which activates adenylate cyclase, induces Ca
2+
influx and PKC activation, altering the NF-kB signaling path-
way, in mouse epidermal KCs [27]. CRH increases activator
protein 1 (AP-1) binding activity, which triggers the expression
of cytokeratin 1 and involucrin and inhibits cytokeratin 14,
increasing cell granularity and cell size, and activating KC dif-
ferentiation. This differentiation process also occurs in HaCaT
keratinocytes, turning the epidermis more protected [28].In
contrast, in human keratinocytes, CGRP and VIP, but not
NPY, rapidly stimulate the synthesis of DNA and KC prolifer-
ation in a dose-dependent way and through adenylyl cyclase
activation, since cAMP levels increase [29].
VIP induces human KC migration to the matrix, in a
dose-dependent way. Loss of the C-terminus of VIP, with
the N-terminal region remaining intact, abrogates both migra-
tion and colonization of the matrix [30]. Membrane-bound
peptidases are able to control neuropeptide signaling and
modulate immune responses. Some aminopeptidases, amino-
peptidase N and NEP, are involved in the inhibition of neu-
ropeptide signaling and consequently, in the inhibition of
KC function. In KCs, aminopeptidase N inhibits human
KC growth [31] and NEP, a cell surface metallopeptidase
that degrades SP, regulates SP functions. An example of this
response can be obtained from unwounded skin from diabetic
mice that shows increased NEP activity, inhibiting wound
closure kinetics [32].
4.1.2 a-MSH and CRH induce anti-inflammatory
responses in KCs
a-MSH is important in the reduction of infection and
inflammatory processes, as it downregulates adhesion mole-
cules (such as integrins: b1 and heat shock surface protein
70), the expression of pro-inflammatory cytokines (in human
KCs cell lines activated by Staphylococcus aureus)[33] and indu-
ces KCs to produce the anti-inflammatory cytokine IL-10 [34].
a-MSH also stimulates IL-10 expression in cultured human
keratinocyte cells [35].
CRH downregulates IL-18 expression, a member of the
IL-1 family, which is a key mediator of peripheral inflammation
and host defense responses, in human HaCaT keratinocytes, by
the activation of the p38/MAPK pathway [36].
4.1.3 SP, CGRP, VIP and ACTH induce
pro-inflammatory responses in KCs
SP augments the expression of IL-1ain murine PAM212
KCs, when binding to NK-2R [37], and the production of
IFN-d, IL-1band IL-8 in human epidermal KCs [38]. Not
only SP, but also CGRP and VIP, are able to induce the
IL-1aand IL-8 upregulation and influence the expression of
proNGF/NGF and their secretion from cultured human
KCs [39]. CGRP also stimulates both constitutive NOS acti-
vity and NO generation, via nitrosothiol, mediating a
NO pro-inflammatory response. NO has a pivotal role in
cutaneous immune function, cellular differentiation, cytokine
expression and apoptosis [40].
ACTH has a regulatory effect on IL-18 expression in
HaCat KCs. ACTH binding to MC-1R and MC-2R induces
the expression and secretion of IL-18, through p38/MAPK
and ERK pathways [41].
4.2 Immune responses on dendritic cells (DCs)
Dendritic cells received this name because of the long
membrane extensions they possess, that are similar to den-
drites of nerve cells. There are many types of dendritic cells:
Langerhans cells (LCs), interstitial dendritic cells, myeloid
cells and lymphoid dendritic cells; even though, the main
function of mature dendritic cells remains unchanged - the
presentation of the antigen to T
H
cells. In inflammation,
mature and immature forms of LCs and interstitial dendritic
cells capture the antigen, by phagocytosis or endocytosis,
and then migrate into draining lymph nodes, functioning as
antigen-presenting cells (APCs) which, in turn, initiate T
H
cell responses. DCs give important information to T and B
cells about inflammation, such as the nature of the pathologic
insult and the type of tissue in which the inflammation
occurred [42]. Taking these essential functions of DCs into
consideration, it is easy to comprehend that impairment of
these cells leads to immune diseases.
Neuropeptides in skin inflammation and diabetic wound healing
1430 Expert Opin. Biol. Ther. (2010) 10(10)
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The role of neuropeptides in the regulation of DCs is
well documented, essentially the density of LCs, DC
migration and maturation, in the antigen-presenting function
and in the inhibition/upregulation of the immune system.
Later in this paper, we examine DCs immune regulation by
different neuropeptides.
4.2.1 SP, CGRP, VIP, Secretin, SN and stressor
neuropeptides induce migration and maturation
of LCs
Some neuropeptides play a chemotactic role in the migration
and maturation of blood-derived DCs. CGRP, VIP, Secretin
and SN induce immature DC migration, while SP stimulates
its migration only slightly [43].
SP receptors (NK-1) are present in normal murine and
human dendritic cells and can elicit transcription factor acti-
vation in DCs [44]. Mathers et al. used the gene gun to deliver
transgenic (tg) antigen to the skin of C57BL/6 mice and the
selective NK-1R agonist. The administration of NK-1R ago-
nist induced NF-kB-mediated migration of activated LCs to
skin-draining lymph nodes, increasing the expression of tg
Ag in the epidermis and stimulating LCs to induce both
T
H
1andT
C
1 cellular responses and antibody mediated
responses [45].
A well-established murine sound stress model (frequency of
300 Hz and an intensity of 75 dB, during 24h, at intervals of
15 sec) induces DC maturation and migration. Accordingly,
some activation-related molecules are upregulated, such as
MHC class II and CD11c, inducing cellular activation and trig-
gering neutrophil respiratory burst and langerin expression.
Langerin is an intradermal transmembrane cell surface antigen
receptor that binds to the mannose cell wall of pathogens,
inducing its internalization into LCs Birbeck granules and pro-
viding access to a nonclassical antigen-processing pathway. Not
only maturation is stimulated, but also migration, as can be
observed by the upregulation of the adhesion molecule intracel-
lular adhesion molecule 1 (ICAM-1), in murine skin [46]. Since
sound stress may enhance stressor neuropeptides, like CGRP,
SP, NT, CRH and a-MSH, these can be responsible for DC
maturation and migration to blood vessels.
4.2.2 CGRP, VIP, PACAP and a-MSH mediate
anti-inflammatory and T
H
2 responses in LCs
Tori et al. determined the anti-inflammatory properties
of CGRP, in Langerhans cells [47]. Indeed, CGRP inhibits
antigen-presenting function and enhances anti-inflammatory
cytokines like IL-10. Since CGRP acts in DC cells, mature
and immature DCs express type 1 CGRP receptors that
regulate the interaction of LCs with T cells. Indeed, CGRP
downregulates CD86 (a cell surface ligand that provides
co-stimulatory signals essential for T cell activation and
survival), HLA-DR (involved in the antigen presenting cell
function to T cells) and T cell proliferation, in human den-
dritic cells [48]. In a recent study in murine dendritic cells,
the authors verified that CGRP also inhibits TLR-stimulated
production of inflammatory mediators, mediated by the
cAMP/PKA pathway, leading to a rapid upregulation of the
transcriptional repressor, the inducible cAMP early repressor
(ICER). Additionally, CGRP also attenuated serum TNF-a
levels [49].
a-MSH also induced the anti-inflammatory function on
DCs, inducing IL-10 release. In addition, PACAP and VIP
suppress Langerhans cell antigen presentation and modulate
cytokine production. In a LPS-induced inflammatory study,
NF-kB is activated in both a LC-like cell line (XS52) and
an epidermal LC. Treatment of cells with these neuropeptides
suppresses the phosphorylation of p-IKKband consequently
inhibits the normal activation of the NF-kB pathway [50].
Indeed, T
H
1 response is inhibited, while T
H
2 immune
response is enhanced [51].
4.3 Immune responses on macrophages (MFs)
Macrophages are immunologically known for their phago-
cytic capacity and destruction of pathogens. These cells can
be dispersed throughout the body or be resident in parti-
cular tissues, becoming fixed macrophages; whereas others
remain motile and are called free, or wandering macro-
phages. Free macrophages travel by amoeboid movement
throughout tissues.
Macrophages may become activated (and do phagocytosis)
by a variety of stimuli, namely antigens of bacteria, and
become even more activated by cytokines secreted by activated
T
H
cells (like IFN-d), by mediators of the inflammatory
response and by components of the bacterial cell wall. Acti-
vated macrophages are potent immune cells, as they have
increased ability to kill ingested microbes, competence to pro-
duce antimicrobial and cytotoxic substances to destroy phago-
cytosed microorganisms and finally, the capability to activate
T cells, through the secretion of inflammatory mediators and
the expression of higher levels of class II MHC molecules,
essential to the antigen-presenting function.
In the skin, macrophages essentially occur when a
microbe reaches and enters the skin and can be harmful in
immune skin diseases. The regulatory role of neuropeptides
in macrophages is dicussed in the following sections.
4.3.1 VIP, PACAP, STT and POMC peptides (ACTH
and a/g-MSH) mediate anti-inflammatory responses
in MFs
VIP and PACAP downregulate the expression of TNF-ain
LPS-stimulated peritoneal macrophages and RAW264.7 cells.
These neuropeptides change the composition of the CRE-
binding complex in the TNF-apromoter (characteristic of
unstimulated cells), decreasing AP-1 binding. TNF-adown-
regulation occurs through the vasoactive intestinal peptide/
pituitary adenylate cyclase activating peptide receptor 1
(VPAC1) receptor and the cAMP/PKA pathway, where the
MAPK kinase kinase 1 (MEKK1)/MAPK kinase 4 (MEK4)/
JNK pathway is inhibited and the phosphorylated c-Jun and
the stimulation of JunB reduced [52].
da Silva, Carvalho & Cruz
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Melanocortin peptides modulate the release of cytokines
and the expression of adhesion molecules, as they induce
the anti-inflammatory cytokine IL-10, in RAW264.7 cells.
Furthermore, melanocortin peptides, ACTH(1 -- 39) or
melanotan II (MTII), do not alter ERK1/2 and JNK phos-
phorylation, but activate p38 phosphorylation and the accu-
mulation of intracellular cAMP, leading to a final activation
of the PKA pathway [53] which is involved in IL-10 increase.
In the same lines, Lipton et al. also showed that a-MSH reduces
gene expression and the production of pro-inflammatory pepti-
des, as well as modulating NO production, through the NF-kB
pathway [54].
g-MSH binding to melanocortin receptor (MR)-1 receptor
induces an anti-inflammatory response in macrophages,
downregulating the production of the pro-inflammatory cyto-
kines IL-2, IL-1, TNF-aand IFN-d, downregulating the
APCs costimulatory molecules CD86 and CD40, and stimu-
lating the production of the anti-inflammatory cytokine
IL-10 [55].
In RAW264.7 macrophages, Somatostatin increases the
anti-inflammatory glucocorticoid response by upregulating
glucocorticoid receptor (GR) expression and signaling, by
limiting the calpain-specific cleavage of GR-associated
Hsp90. Then, GR transactivates a minimal promoter contain-
ing two glucocorticoid response elements, interfering with the
activation of the NF-kB pathway [56].
4.3.2 SP, corticotropin-releasing factor (CRF) and
urocortin activate pro-inflammatory signaling
pathways in MFs
SP binding to NK-1R activates two independent but conver-
gent pro-inflammatory signaling pathways. In murine mac-
rophages, it induces the activation of PKCa,PKCdand e,
as well as, the downstream ERK1/2 and NF-kBcascade,
which drives the transcription of inducible chemokines.
The PI3K/Akt pathway is also activated by SP and is impor-
tant at later stages of the SP-induced cellular inflammatory
response [57,58].
CRF and its related peptides urocortins (Ucn) 1 and 2 aug-
ment the production of LPS-induced pro-inflammatory cyto-
kines. Experiments performed with the murine macrophage
cell line RAW264.7 and primary murine peritoneal macro-
phages demonstrated that cells treated with CRF, Ucn1 or
Ucn2, increase TLR4 mRNA levels and enhance the inflam-
matory response. This effect is mediated by CRF2 receptor,
via activation of the transcription factors PU.1 (important in
hematopoiesis) and AP-1 [59].
4.3.3 NPY and VIP/PACAP regulate oxidative burst in
MFs
Macrophage function is regulated by NPY receptors. NPY and
peptide tyrosine tyrosine (PYY), through Y1 and Y2 receptors,
increase oxidative burst in phorbol myristate acetate (PMA)-
stimulated rat peritoneal macrophages, due to the activation
of PKC. Though, NPY binding to Y2 receptors suppresses
zymosan-stimulated cells and, when binding to Y5 receptors,
mediates the suppression of the oxidative burst [60].
In human macrophages, VIP binding to VIP/PACAP
receptors decreases superoxide anion production, which is
associated with a decrease in cAMP levels. Deficiencies in
cAMP cascade, during the terminal differentiation of macro-
phages, alter VIP/PACAP receptor subtypes expression and
the functional activity of the stimulatory and inhibitory
Gs/Gi subunits of adenylate cyclase [61]. In the same lines,
Delgado et al. showed that PACAP-38 binding to type I
PACAP receptor, through PKC activation, stimulates phago-
cytosis and production of superoxide anion, in peritoneal
macrophages [62]. Indeed, type I PACAP receptor is involved
in the stimulation of the macrophages’ activity, while VIP
receptors are involved in the inhibition of the function
of macrophages.
4.4 Immune responses on mast cells (MCs)
Mast-cell precursors are formed in the bone marrow during
hematopoiesis and released into the blood as undifferentiated
cells; they do not differentiate until they leave the blood
and enter the tissues. MCs can be found in a wide variety of
tissues, including the skin. These cells have large numbers
of cytoplasmic granules that contain histamine and other
pharmacologically active substances that are important for the
inflammatory response and in the development of allergies [42].
MCs express a high-affinity receptor for the Fc region of
the antibody IgE (FceRI). When crossing with IgE, mast
cells become coated with IgE and activated. Consequently,
activated MCs trigger a G-protein-associated intracellular
pathway, the phospholipase C (PLC)/inositol pathway. As a
consequence a rapid release of histamine, inflammatory
mediators and eicosanoids, specifically prostaglandin D2
and leukotriene C4 occurs [63].
In contrast to some human MCs that are associated with
mucosal surfaces such as lung, adenoids, tonsils and intestine,
skin MCs can be stimulated in a concentration-related man-
ner to release histamine. Accordingly, various molecules
were determined to induce this release, like the neuropeptides
SP, CRF, Ucn, VIP, somatostatin, a-MSH and NPY.
Human skin MCs are distinct from the other human mast
cells so far studied in their ability to respond to basic non-
immunological stimuli [64] and this may reflect a specialized
function of these cells.
Subsequent to MC activation, these cells release a variety of
vasoactive and pro-inflammatory mediators. Neuropeptides
may control a variety of homeostatic functions, such as
blood flow, histamine-related angiogenesis [65] and fibroblast
proliferation. Such processes help to remodel tissues during
wound healing and atopic dermatitis, as increased numbers
of MCs have been noticed around inflamed rat skin and areas
of developing fibrosis [42]. Therefore, the role of some neuro-
peptides in mast cells activation and in the release of different
inflammatory mediators into the skin is presented in the
following sections.
Neuropeptides in skin inflammation and diabetic wound healing
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4.4.1 Neuropeptides activate MCs and the
pro-inflammatory response
Studies performed with different classes of MCs, namely
rat peritoneal MCs, serosal MCs and bone-marrow-derived
MCs, have shown that SP induces the activation and
consequent degranulation of mast cells, by Ca
2+
increase,
via the band dsubunits of G(i2) and G(i3) GTP-binding
protein, and via G proteins that couple to PLC (Gp), with
further PKC and ERK-- MAPK pathway activation [66].
SP is involved in the expression and release of cytokines, as
it induces the expression and secretion of TNF-a,ina
dose-dependent way, in a murine mast cell line (CFTL12).
This mast cell induction is quite selective, since SP does
not stimulate the production of other known cytokines:
IL-1, IL-3, IL-4, IL-6 or GM-CSF [67].Furthermore,
SP-induced production of TNF-aand histamine secretion
by peritoneal mast cells occurs via p38 and JNK MAPKs
pathways, being PI3K and upstream component of the
JNK pathway [68].
In human skin mast cells, Gibbs et al. found that not only
the pro-inflammatory cytokine TNF-awas released through
an IgE-dependent activation by SP, but also IL-8. The cells
were also incapable of secreting IL-4, IL-5 or IL-13, indicat-
ing again the selectivity for the type of cytokine released [69].
Additionally, Chien et al. observed in rats that SP induced
the release of reactive oxygen species (ROS) in the whole
blood by mast cell degranulation [70]. In human and rat
mast cells, SP induced the infiltration of eosinophil into the
skin, the expression of intercellular adhesion molecules and
leukocyte adhesion [71].
SP may also be involved in the enhancement of angio-
genesis and melanoma cell growth [65],asSPisexpressedin
cutaneous melanocytic lesions. This function involves mast
cells, as MC-derived histamine has potent vasoactive effects
(angiogenesis) and promotes tissue fibroplasia. In addition
to histamine, MCs contain many other angiogenic factors
and a variety of cytokines, growth factors and proteo-
lytic enzymes implicated in tissue remodeling and normal/
tumor-associated neoangiogenesis.
CGRP receptor and its associated proteins, namely calcito-
nin receptor-like (CRLR), receptor (G protein-coupled) activ-
ity modifying protein (RAMP)2 and RAMP3, are expressed
in rat peritoneal mast cells, whereas RAMP1 is not. Adreno-
medullin stimulates histamine release via its receptor
(CRLR/RAMP2 or 3), but CGRP, via the CGRP receptor
(CRLR/RAMP1), is not able to activate mast cells to release
histamine [72]. Also, CGRP is a potent and long-acting
vasodilator in human mast cells, being regulated by tryptase,
an enzyme that cleaves CGRP and finishes CGRP signaling,
controling blood flow [73].
PACAP induces the degranulation of rat peritoneal mast
cells, in a PACAP-receptor-independent manner. This process
probably occurs via a direct activation of the heterotrimeric
G-proteins of the Gi-type, leading to a sequential activation
of PLC [74].
The POMC system is the central coordinator of the sys-
temic endocrine responses to sustained stress. It has been
recently discovered that human mast cells also display regu-
lated production of POMC peptides and release a-MSH in
an IgE-dependent way. In fact, a-MSH also plays an immu-
nomodulatory role during the inflammatory and allergic reac-
tions of the skin, in a pro-inflammatory way by releasing
histamine [75].
CRH induces the release of VEGF from human mast cells,
via selective activation of the cAMP/PKA/p38 MAPK signal-
ing pathway, but not by extracellular signal regulated kinase
(ERK) or JNK pathways [76]. Indeed, after activation, human
mast cells express CRH receptors that mediate the release of
cytokines and other pro-inflammatory mediators [77].
Human mast cells synthesize and secrete both CRF and Ucn
in response to acute psychological stress, after FceRI crosslink-
ing. Urocortin has pro-inflammatory actions in mast cells,
leading to its degranulation and inducing skin vascular per-
meability. This Ucn effect is connected with stress-induced
disorders, such as atopic dermatitis or psoriasis [78].
4.4.2 Neuropeptides induce MCs mediated
immunosuppression
The work of Hart et al. in mice demonstrated that
cis-urocanic acid (UCA) and UVB induce the release of
neuropeptides from sensory c-fibers that trigger mast cell
degranulation and histamine/TNF-arelease. Indeed, hista-
mine, but not TNF-a, is responsible for the induction of
the immunosuppressive response, which is proportional to
the density of mast cells [79]. However, immunosuppression
mediated by UCA and UVB only occurs when neuropeptides
are released, demonstrating the importance of these neuro-
peptides in immunosuppression. As a result, T
H
2 immune
response is enhanced and T
H
1 response diminished.
5. Neuropeptides in diabetic wound healing
5.1 Denervation of diabetic wounded skin
Neuropathy is a possible cause of delayed diabetic wound
healing and this may be caused by altered metabolism of
glucose via the polyol pathway resulting in sorbitol accumula-
tion, ischemia, superoxide-induced free-radical formation and
impaired axonal transport [80]. Diabetic skin has motor, sen-
sory and autonomic fiber denervation: sensory neuropathy
limits the sensations of pain, pressure, temperature, and
others; autonomic denervation causes arteriovenous shunting,
leading to vasodilation in small arteries; motor neuropathy
causes weakness and wasting of small intrinsic muscles [81].
Chronic nerve compression in addition to neuropathy, may
lead to amputation. Recent approaches in clinical surgery --
neurosurgical prevention -- showed relief of pain in 88%
and restoration of sensation in 79% of patients, preventing
ulceration and amputation [82].
Diabetic neuropathy appears to be the cause of delayed
wound healing. Accordingly, the work of Gibran and
da Silva, Carvalho & Cruz
Expert Opin. Biol. Ther. (2010) 10(10) 1433
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colleagues demonstrated fewer nerves in the epidermis and
papillary dermis of skin from diabetic human subjects and
diabetic (db/db) murine skin [83]. Ever since, db/db murine
skin has been a model to study the role of nerves in cutaneous
injury, as these mice developed neuropathy and delayed
healing. Additionally, diminished release of neuropeptides
from nerves may be the cause of impaired wound healing,
as Engin et al. demonstrated delayed wound contraction
and loss of neuropeptide secretion from nerve endings in
denervated tissues [84].
Indeed, further studies were done to identify which
neuropeptides are decreased due to the denervation and
what function they had in the skin that was impaired.
However, some other neuropeptides may have higher release
and improved function. In the next section studies are pre-
sented that indicate the importance of neuropeptides in
wound healing.
5.2 Decreased SP, CGRP, NPY and POMC peptides in
diabetic wound healing
As mentioned, SP promotes a pro-inflammatory environ-
ment, inducing vasodilation, angiogenesis, leukocyte chemo-
taxis and adhesion to endothelial cells, which ensures the
extravasation, migration and subsequent accumulation of
leukocytes into the site of injury. Some mice studies show
that in normal wound healing, SP release induced by stress
augments the growing nerves and connection of SP to mast
cells, leading to a rise in apoptotic cells in the skin [85].Addi-
tionally, SP upregulates NO production, enhancing wound
closure kinetics and epithelialization [86],italsoincreases
the density of neutrophils, dendritic cells, endothelial cells
and macrophages, and triggers the production of TNF-a,
IL-1-b, IL-2 and IL-6 by T-lymphocytes [87]. In contrast,
in skin biopsies of diabetic wounds, SP levels are reduced
due to elevated levels of the degrading enzyme NEP [88]
and reduced SP-positive nerve fibres [89].Eventhough,
exogenous SP treatment improves wound healing kinetics
in animal models [90].
CGRP is known to induce neurogenic inflammation,
inducing a pro-inflammatory response in different cells,
including the formation of new vessels, important during
physiological and pathological wound healing [91]. In wound
healing, inflammation may induce CGRP release, and CGRP
is able to activate inflammatory pathways [92]. Diabetes
decreases the expression, release and action of CGRP, resulting
in reduced CGRP-mediated vasodilation [93].
In type 1 and type 2 diabetic patients, NPY levels are
increased in the hypothalamus, however NPY levels in the
skin are reduced [94]. NPY binding to Y2 receptors is
important in angiogenesis and, according to Ekstrand and
collegues, this binding induces the formation of microvessels
with distinct vascular tree-like structures. As a result, the dele-
tion of Y2 receptor in diabetic mice results in the blockage of
NPY-induced angiogenesis and delayed wound healing [95].
These consequences might explain a possible cause of delayed
wound healing in diabetic patients, reinforcing the impor-
tance of NPY in both the inflammatory and angiogenic phases
of wound healing.
In diabetic rats, POMC peptides, like ACTH and a-MSH,
were less expressed. However, Gohshi et al. demonstrated
contradictory results in short-term (1 week) diabetic rats,
detecting an increase of ACTH (POMC peptide) release, via
the cAMP pathway [96]. In long-term diabetic rats (8 weeks),
the results were already concordant, since ACTH release
decreases, which may be explained by an alteration in the
properties of the L-type Ca
2+
channel coupled with the CRF
receptor, or with the CRF receptor itself. Knowing the anti-
inflammatory role of POMC peptides, less is known about
how its decreased levels affect wound healing.
5.3 Increased CRF and NT levels in diabetic wound
healing
Type 2 diabetic patients have subclinical hypercortisolism.
This is a consequence of Hypothalamic-- pituitary-- adrenal
axis hyperactivation, CRF stimulation and pituitary adrenal
peptides release. Impaired stress response is responsible for
the decrease of pituitary and adrenal sensitivity, as well as
the glucocorticoid negative feedback sensitivity [97]. Accord-
ingly, chronic stress delays wound healing in humans and
rodents [98]. In a study with a rhesus monkey model of wound
healing, stress decreased IL-8 and CCL3 expression [99],as
IL-1band PDGF, in a stress model of mouse wound healing.
Thus, the authors infer that glucocorticoid release mediated
by stress is responsible for delayed wound healing.
Studies with pancreas and intestines from diabetic mice
have shown elevated levels of neurotensin (NT). In fact,
NT may be implicated in the pathogenesis of diabetic
wound healing [100]. However, studies concerning the
involvement of NT in diabetic wound healing have not
been performed yet.
6. Expert opinion
This review shows the importance of neuropeptides in skin
inflammation with special focus on diabetic wound healing,
an examined area, but definitely not explored in detail. The
role of diverse neuropeptides has been much studied in the
brain, but poorly studied in peripheral neurologic regulated
organs, like the skin.
From the reviewed studies, neuropeptides play a crucial
role in immune cells responses in the skin, and diminished
levels have been correlated with skin disorders, namely
diabetic wound healing. Recently, several reports have
highlighted the role of neuropeptides in the immune system.
As such, a more detailed study of the function of each
neuropeptide may help to determine which neuropeptide is
more important in the skin, both in physiological and patho-
logical conditions, and to what extent. Although some neuro-
peptides such as, SP, VIP and CGRP have been partially
studied in the skin, the roles of neurotensin and somatostatin
Neuropeptides in skin inflammation and diabetic wound healing
1434 Expert Opin. Biol. Ther. (2010) 10(10)
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at the skin level is mostly unknown and should be studied. It
would be important in future research to address the effects of
NT or somatostatin in the skin, with particular emphasis on
skin pathologies. Finally, up until now no neuropeptides
have been used as therapies for impaired skin wound healing
that occurs in diseases like diabetes, atopic dermatitis or
hypersensitivity reactions. These could be a promising therapy
for wound healing.
6.1 Conclusion
In conclusion, SP and CGRP are decreased in diabetic wound
healing, explaining the reduced angiogenesis, which in turn
diminishes the supply of inflammatory mediators to the site
of injury. However, the pro-inflammatory neuropeptides
CRH and NT are enhanced in diabetic wound healing, while
the anti-inflammatory NPY and POMC neuropeptides are
diminished as it is summarized in Table 1. Taken together,
although angiogenesis and inflammatory mediators become
decreased in diabetic wound healing, pro-inflammatory neu-
ropeptides are preferentially released in the skin, rather than
anti-inflammatory neuropeptides and, per se, shaping a
pro-inflammatory phase in the wounds. These conclusions
are in accordance to the American Diabetes Association
(ADA) definition of chronic wound. ADA defined a diabetic
wound as a wound which fails to ‘continuously progress
toward healing’. The pro-inflammatory response maintains
the healing process in the inflammatory phase, inhibiting
the progress to the other phases of migration-proliferation
and remodeling.
Ultimately, future research in wound healing should
address the role of these inflammatory modulators on skin
diseases, like diabetic wound healing.
Declaration of interest
This work was supported by the Portuguese Government’s
Fundac¸a
˜o para a Cie
ˆncia e a Tecnologia -- Project PPDC/
SAV-OSH/099762/2008 (MT Cruz) and PTDC/SAU-MII/
098567/2008 (E Carvalho) -- and by FSD/JDRF/Novo
Nordisk European Programme in Type 1 Diabetes Research
(E Carvalho).
Table 1. Balance between anti- and pro-inflammatory
neuropeptides in diabetic wound healing.
Neuropeptides Levels in diabetic
wound healing
CRF/CRH, NT "
SP, NPY, CGRP, POMC
peptides (ACTH and a-MSH)
#
Normal type: Anti-inflammatory neuropeptides; Bold type: Pro-inflammatory
neuropeptides.
da Silva, Carvalho & Cruz
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Bibliography
1. Singer A, Clark RA. Cutaneous wound
healing. N Engl J Med 1999;341:738-46
2. Cisarik-Fredenburg P. Discoveries in
research on diabetic keratopathy.
Optometry 2001;72:691-704
3. Galiano R. Topical vascular endothelial
growth factor accelerates diabetic wound
healing through increased angiogenesis
and by mobilizing and recruiting bone
marrow-- derived cells. Am J Pathol
2004;164:1935-47
4. Goren I, Muller E, Pfeilschifter J,
Frank S. Severely impaired insulin
signaling in chronic wounds of diabetic
ob/ob mice: a potential role of tumor
necrosis factor-alpha. Am J Pathol
2006;168:765-77
5. Bogdanski P. Influence of insulin therapy
on expression of chemokine receptor
CCR5 and selected inflammatory
markers in patients with type 2 diabetes
mellitus. Int J Clin Pharmacol Ther
2007;45:563-7
6. Lobmann R. Expression of matrix
metalloproteinases and their inhibitors in
the wounds of diabetic and non-diabetic
patients. Diabetologia 2002;45:1011-16
7. Loots M, Kenter SB, Au FL. Fibroblasts
derived from chronic diabetic ulcers
differ in their response to stimulation
with EGF, IGFI, bFGF and PDGF-AB
compared to controls. Eur J Cell Biol
2002;81:153-60
8. Stegenga M. Effect of acute
hyperglycaemia and/or hyperinsulinaemia
on proinflammatory gene expression,
cytokine production and neutrophil
function in humans. Diabet Med
2008;25:157-64
9. Zykova S, Jenssen TG, Berdal M, et al.
Altered cytokine and nitric oxide
secretion in vitro by macrophages from
diabetic type II-like db/db mice. Diabetes
2000;49:1451-58
10. Galkowska H, Wojewodzka U,
Olszewski WL. Chemokines, cytokines,
and growth factors in keratinocytes and
dermal endothelial cells in the margin
of chronic diabetic foot ulcers.
Wound Repair Regen 2006;14:558-65
11. Roosterman D, Goerge T,
Schneider SW, et al. Neuronal
control of skin function: the skin
as a neuroimmunoendocrine organ.
Physiol Rev 2006;86:1309-79
12. Bevan S, Hothi S, Hughes G, et al.
Capsazepine: a competitive antagonist
of the sensory neurone excitant capsaicin.
Br J Pharmacol 1992;107:544-52
13. Wollenberg A, Bieber T. Topical
immunomodulatory agents and their
targets in inflammatory skin diseases.
Transplant Proc 2001;33:2212-16
14. Maggi C. The pharmacology of the
efferent function of sensory nerves.
J Auton Pharmacol 1991;11:173-208
15. Rook J, Hasan W, McCarson KE.
Morphine-induced early delays in
wound closure: involvement of sensory
neuropeptides and modification of
neurokinin receptor expression.
Biochem Pharmacol 2009;77:1747-55
16. Antony A, Kong W, Lorenz HP.
Upregulation of neurodevelopmental
genes during scarless healing.
Ann Plast Surg 2010;64:247-50
17. Scott J, Muangman P, Gibran NS.
Making sense of hypertrophic scar:
a role of nerves. Wound Repair Regen
2007;15:S27-31
18. Leung M, Wong CC. Expressions of
putative neurotransmitters and neuronal
growth related genes in Merkel
cell-neurite complexes of the rats.
Life Sci 2000;66:1481-90
19. Wang H, Xing L, Li W, et al.
Production and secretion of calcitonin
gene-related peptide from human
lymphocytes. J Neuroimmunol
2002;130:155-62
20. Olerud J, Usui ML, Seckin D, et al.
Neutral endopeptidase expression and
distribution in human skin and wounds.
J Invest Dermatol 1999;112:873-81
21. Fitzpatrick T, Eisen AZ, Wolff K,
et al. Dermatology in General Medicine.
5th edition. McGraw-Hill Books,
New York; 1978;1:57-61
22. Peters E, Arck PC, Paus R. Hair growth
inhibition by psychoemotional stress:
a mouse model for neural mechanisms
in hair growth control. Exp Dermatol
2006;15:1-13
23. Zhou Z, Kawana S, Aoki E, et al.
Dynamic changes in nerve growth factor
and substance P in the murine hair cycle
induced by depilation. J Dermatol
2006;33:833-41
24. Seike M, Ikeda M, Morimoto A,
et al. Increased synthesis of calcitonin
gene-related peptide stimulates
keratinocyte proliferation in murine
UVB-irradiated skin. J Dermatol Sci
2002;28:135-43
25. Chakraborty A, Pawelek J. MSH
receptors in immortalized human
epidermal keratinocytes: a potential
mechanism for coordinate regulation
of the epidermal-melanin unit.
J Cell Physiol 1993;157:344-50
26. Funasaka Y, Chakraborty AK, Yodoi J,
Ichihashi M. The effect of thioredoxin
on the expression of
proopiomelanocortin-derived peptides,
the melanocortin 1 receptor and cell
survival of normal human keratinocytes.
J Invest Dermatol 2001;6:32-7
27. Lee Y, Dlugosz AA, McKay R,
et al. Definition by specific antisense
oligonucleotides of a role for protein
kinase Calpha in expression of
differentiation markers in normal and
neoplastic mouse epidermal keratinocytes.
Mol Carcinog 1997;18:44-53
28. Zbytek B, Pikula M, Slominski RM,
et al. Corticotropin-releasing hormone
triggers differentiation in HaCaT
keratinocytes. Br J Dermatol
2005;152:474-80
29. Takahashi K, Nakanishi S, Imamura S.
Direct effects of cutaneous neuropeptides
on adenylyl cyclase activity and
proliferation in a keratinocyte cell line:
stimulation of cyclic AMP formation
by CGRP and VIP/PHM, and inhibition
by NPY through G protein-coupled
receptors. J Invest Dermatol
1993;101:646-51
30. Wollina U, Huschenbeck J, Knoll B,
et al. Vasoactive intestinal peptide
supports induced migration of human
keratinocytes and their colonization
of an artificial polyurethane matrix.
Regul Pept 1997;70:29-36
31. Gabrilovac J, Cupic B, Breljak D, et al.
Expression of CD13/aminopeptidase N
and CD10/neutral endopeptidase on
cultured human keratinocytes.
Immunol Lett 2004;91:39-47
32. Spenny M, Muangman P, Sullivan SR,
et al. Neutral endopeptidase inhibition
in diabetic wound repair.
Wound Repair Regen 2002;10:295-301
33. Donnarumma G, Paoletti I,
Buommino E, et al. alpha-MSH reduces
the internalization of Staphylococcus
Neuropeptides in skin inflammation and diabetic wound healing
1436 Expert Opin. Biol. Ther. (2010) 10(10)
Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by Northwestern University on 03/24/15
For personal use only.
aureus and down-regulates HSP 70,
integrins and cytokine expression
in human keratinocyte cell lines.
Exp Dermatol 2004;13:748-54
34. Seiffert K, Granstein RD. Neuropeptides
and neuroendocrine hormones in
ultraviolet radiation-induced
immunosuppression. Methods
2002;28:97-103
35. Redondo P, Garcia-Foncillas J,
Okroujnov I, Bandres E. alpha-MSH
regulates interleukin-10 expression by
human keratinocytes. Arch Dermatol Res
1998;290:425-8
36. Park H, Kim HJ, Lee JH, et al.
Corticotropin-releasing hormone (CRH)
downregulates interleukin-18 expression
in human HaCaT keratinocytes by
activation of p38 mitogen-activated
protein kinase (MAPK) pathway.
J Invest Dermatol 2005;124:751-5
37. Song I, Bunnett NW, Olerud JE,
et al. Substance P induction of murine
keratinocyte PAM 212 interleukin
1 production is mediated by the
neurokinin 2 receptor (NK-2R).
Exp Dermatol 2000;9:42-52
38. Liu J, Zhao YZ, Peng C, et al. Effect of
cetirizine hydrochloride on the expression
of substance P receptor and cytokines
production in human epidermal
keratinocytes and dermal fibroblasts.
Yao Xue Xue Bao 2008;43:383-7
39. Dallos A, Kiss M, Polyanka H, et al.
Effects of the neuropeptides substance P,
calcitonin gene-related peptide, vasoactive
intestinal polypeptide and galanin on the
production of nerve growth factor and
inflammatory cytokines in cultured
human keratinocytes. Neuropeptides
2006;40:251-63
40. Yaping E, Golden SC, Shalita AR, et al.
Neuropeptide (calcitonin gene-related
peptide) induction of nitric oxide in
human keratinocytes in vitro.
J Invest Dermatol 2006;126:1994-2001
41. Park H, Kim HJ, Lee JY, et al.
Adrenocorticotropin hormone stimulates
interleukin-18 expression in human
HaCaT keratinocytes. J Invest Dermatol
2007;127:1210-16
42. Goldsby R, Kindt TJ, Osborne BA.
Kuby Immunology. 5th edition. W.H.
Freeman & Co, New York; 2003;4-43
43. Dunzendorfer S, Kaser A, Meierhofer C,
et al. Cutting edge: peripheral
neuropeptides attract immature and
arrest mature blood-derived dendritic
cells. J Immunol 2001;166:2167-72
44. Marriott I, Bost KL. Expression of
authentic substance P receptors in
murine and human dendritic cells.
J Neuroimmunol 2001;114:131-41
45. Mathers A, Tckacheva OA,
Janelsins BM, et al. In vivo signaling
through the neurokinin 1 receptor
favors transgene expression by
Langerhans cells and promotes the
generation of Th1- and Tc1-biased
immune responses. J Immunol
2007;178:7006-17
46. Joachim R, Handjiski B, Blois SM, et al.
Stress-induced neurogenic inflammation
in murine skin skews dendritic cells
towards maturation and migration:
key role of intercellular adhesion
molecule-1/leukocyte function-associated
antigen interactions. Am J Pathol
2008;173:1379-88
47. Tori H, Tamaki K, Granstein RD.
The effect of neuropeptides/hormones
on Langerhans cells. J Dermatol Sci
1998;20:21-8
48. Carucci J, Ignatius R, Wei Y, et al.
Calcitonin gene-related peptide decreases
expression of HLA-DR and CD86 by
human dendritic cells and dampens
dendritic cell-driven T cell-proliferative
responses via the type I calcitonin
gene-related peptide receptor. J Immunol
2000;164:3494-9
49. Harzenetter M, Novotny AR, Gais P,
et al. Negative regulation of TLR
responses by the neuropeptide CGRP is
mediated by the transcriptional repressor
ICER. J Immunol 2007;179:607-15
50. Ding W, Wagner JA, Granstein RD.
CGRP, PACAP, and VIP modulate
Langerhans cell function by inhibiting
NF-kappaB activation. J Invest Dermatol
2007;127(10):2357-67
51. Ding W, Stohl LL, Wagner JA,
Granstein RD. Calcitonin gene-related
peptide biases Langerhans cells toward
Th2-type immunity. J Immunol
2008;181:6020-26
52. Delgado M, Ganea D. Vasoactive
intestinal peptide and pituitary adenylate
cyclase-activating polypeptide inhibit
interleukin-12 transcription by regulating
nuclear factor kappaB and Ets activation.
J Biol Chem 1999;274:31930-40
53. Lam C, Perretti M, Getting SJ.
Melanocortin receptor signaling in
RAW264.7 macrophage cell line.
Peptides 2006;27:404-12
54. Lipton J, Zhao H, Ichiyama T, et al.
Mechanisms of antiinflammatory action
of alpha-MSH peptides. In vivo and
in vitro evidence. Ann NY Acad Sci
1999;20:173-82
55. Lotti T, Bianchi B, Ghersetich I, et al.
Can the brain inhibit inflammation
generated in the skin? The lesson of
alpha-melanocyte-stimulating hormone.
Int J Dermatol 2002;41:311-18
56. Bellocq A, Doublier S, Suberville S,
et al. Somatostatin increases
glucocorticoid binding and signaling
in macrophages by blocking the
calpain-specific cleavage of Hsp 90.
J Biol Chem 1999;274:36891-6
57. Sun J, Ramnath RD, Tamizhselvi R,
Bhatia M. Neurokinin A engages
neurokinin-1 receptor to induce
NF-kappaB-dependent gene expression
in murine macrophages: implications of
ERK1/2 and PI 3-kinase/Akt pathways.
Am J Physiol Cell Physiol
2008;295:C679-691
58. Sun J, Ramnath RD, Tamizhselvi R,
Bhatia M. Role of protein kinase C
and phosphoinositide 3-kinase-Akt in
substance P-induced proinflammatory
pathways in mouse macrophages.
FASEB J 2009;23:997-1010
59. Tsatsanis C, Androulidaki A, Alissafi T,
et al. Corticotropin-releasing factor and
the urocortins induce the expression of
TLR4 in macrophages via activation of
the transcription factors PU.1 and AP-1.
J Immunol 2006;176(3):1869-77
60. Dimitrijevic M, Vujic V,
Beck-Sickinger A, Von Horsten S.
Neuropeptide Y and its receptor subtypes
specifically modulate rat peritoneal
macrophage functions in vitro: counter
regulation through Y1 and Y2/
5 receptors. Regul Pept 2005;124:163-72
61. Chedeville A, Mirossay L, Chastre E,
et al. Interaction of VIP, PACAP and
related peptides in normal and leukemic
human monocytes and macrophages.
FEBS Lett 1993;319:171-6
62. Delgado M, Garrido E, de La Fuente M,
Gomariz RP. Pituitary adenylate
cyclase-activating polypeptide (PACAP-
38) stimulates rat peritoneal macrophage
functions. Peptides 1996;17(7):1097-105
63. Church M, el-Lati S, Caulfield JP.
Neuropeptide induced secretion from
da Silva, Carvalho & Cruz
Expert Opin. Biol. Ther. (2010) 10(10) 1437
Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by Northwestern University on 03/24/15
For personal use only.
human skin mast cells. Int Arch Allergy
Appl Immunol 1991;9:310-18
64. Lowman M, Benyon RC, Church MK.
Characterization of neuropeptide-induced
histamine release from human dispersed
skin mast cells. Br J Pharmacol
1988;95:121-30
65. Khare V, Albino AP, Reed JA. The
neuropeptide/mast cell secretagogue
substance P is expressed in cutaneous
melanocytic lesions. J Cutan Pathol
1998;25:2-10
66. Okabe T, Hide M, Hiragun T, et al.
Bone marrow derived mast cell acquire
responsiveness to substance P with Ca2+
signals and release of leukotriene B4 via
mitogen-activated protein kinase.
J Neuroimmunol 2006;181:1-12
67. Ansel JC, Song I, Quinlan KL, et al.
Interactions of the skin and nervous
system. J Invest Dermatol 1997;2:23-6
68. Azzolina A, Guarneri P, Lampiasi N.
Involvement of p38 and JNK MAPKs
pathways in Substance P-induced
production of TNF-alpha by peritoneal
mast cells. Cytokine 2002;18:72-80
69. Gibbs B, Wierecky J, Welker P, et al.
Human skin mast cells rapidly release
preformed and newly generated
TNF-alpha and IL-8 following
stimulation with anti-IgE and other
secretagogues. Exp Dermatol
2001;10:312-20
70. Chien C, Yu HJ, Lin TB, et al.
Substance P via NK1 receptor
facilitates hyperactive bladder
afferent signaling via action of ROS.
Am J Physiol Renal Physiol
2003;284:F840-851
71. Matsuda H, Kawakita K, Kiso Y,
et al. Substance P induces granulocyte
infiltration through degranulation of
mast cells. J Immunol 1989;142:927-31
72. Matsushima M, Kitaichi K,
Tatsumi Y, et al. The expression of
mRNA for calcitonin receptor-like
receptor/receptor-activity modifying
proteins in rat peritoneal mast cells.
Eur J Pharmacol 2003;464:111-14
73. Walls A, Brain SD, Desai A, et al.
Human mast cell tryptase attenuates
the vasodilator activity of calcitonin
gene-related peptide.
J Biochem Pharmacol 1992;43:1243-8
74. Seebeck J, Kruse ML,
Schmidt-Choudhury A, et al.
Pituitary adenylate cyclase activating
polypeptide induces multiple signaling
pathways in rat peritoneal mast cells.
Eur J Pharmacol 1998;352:343-50
75. Artuc M, Bohm M, Grutzkau A, et al.
Human mast cells in the neurohormonal
network: expression of POMC, detection
of precursor proteases, and evidence for
IgE-dependent secretion of alpha-MSH.
J Invest Dermatol 2006;126:1934-6
76. Cao J, Cetrulo CL, Theoharides TC.
Corticotropin-releasing hormone
induces vascular endothelial growth
factor release from human mast cells
via the cAMP/protein kinase A/p38
mitogen-activated protein kinase
pathway. Mol Pharmacol
2006;69:998-1006
77. Theoharides T, Donelan JM,
Papadopoulou N, et al. Mast cells
as targets of corticotropin-releasing
factor and related peptides.
Trends Pharmacol Sci 2004;25:563-8
78. Singh L, Boucher W, Pang X, et al.
Potent mast cell degranulation and
vascular permeability triggered by
urocortin through activation of
corticotropin-releasing hormone
receptors. J Pharmacol Exp Ther
1999;288:1349-56
79. Hart P, Townley SL,
Grimbaldeston MA, et al. Mast
cells, neuropeptides, histamine, and
prostaglandins in UV-induced systemic
immunosuppression. Methods
2002;28:79-89
80. Apfel SC. Neurotrophic factors
and diabetic peripheral neuropathy.
Eur Neurol 1999;41:27-34
81. Dinh T, Veves A. International Textbook
of Diabetes Mellitus. 3rd edition.
John Wiley & Sons, Chichester;
2004;1315-32
82. Dellon AL. Neurosurgical prevention
of ulceration and amputation by
decompression of lower etremity
peripheral nerves in diabetic neuropathy:
update 2006. Acta Neurochur Suppl
2007;100:149-51
83. Gibran N, Jang YC, Isik FF, et al.
Diminished neuropeptide levels
contribute to the impaired cutaneous
healing response associated with diabetes
mellitus. J Surg Res 2002;108:122-8
84. Engin C, Demirkan F, Ayhan S,
et al. Delayed effect of denervation
on wound contraction in rat skin.
Plast Reconstr Surg 1996;98:1063-7
85. Peters E, Kuhlmei A, Tobin DJ, et al.
Stress exposure modulates peptidergic
innervation and degranulates mast cells
in murine skin. Brain Behav Immun
2005;19:252-62
86. Muangman P, Tamura RN, Muffley LA,
et al. Substance P enhances wound
closure in nitric oxide synthase knockout
mice. J Surg Res 2009;153:201-9
87. Delgado M, Leceta J, Ganea D.
Vasoactive intestinal peptide and
pituitary adenylate cyclase-activating
polypeptide inhibit the production of
inflammatory mediators by activated
microglia. J Leukoc Biol 2003;73:155-64
88. Antezana M, Sullivan SR, Usui M,
et al. Neutral endopeptidase activity is
increased in the skin of subjects with
diabetic ulcers. J Invest Dermatol
2002;119:1400-4
89. Lindberger M, Schroder HD,
Schultzberg M, et al. Nerve fibre
studies in skin biopsies in peripheral
neuropathies. I. Immunohistochemical
analysis of neuropeptides in diabetes
mellitus. J Neurol Sci 1989;93:289-96
90. Gibran N, Jang YC, Isik FF, et al.
Diminished neuropeptide levels
contribute to the impaired cutaneous
healing response associated with diabetes
mellitus. J Surg Res 2002;108:122-8
91. Toda M, Suzuki T, Hosono K, et al.
Roles of calcitonin gene-related peptide
in facilitation of wound healing and
angiogenesis. Biomed Pharmacother
2008;62:352-9
92. Ambalavanar R, Dessem D, Moutanni A,
et al. Muscle inflammation induces a
rapid increase in calcitonin gene-related
peptide (CGRP) mRNA that temporally
relates to CGRP immunoreactivity and
nociceptive behavior. Neuroscience
2006;143:875-84
93. Sheykhzade M, Dalsgaard GT,
Johansen T, Nyborg NC. The effect of
long-term streptozotocin-induced diabetes
on contractile and relaxation responses of
coronary arteries: selective attenuation of
CGRP-induced relaxations.
Br J Pharmacol 2000;129:1212-18
94. Wallengren J, Badendick K, Sundler F,
et al. Innervation of the skin of the
forearm in diabetic patients: relation
to nerve function. Acta Derm Venereol
1995;7:37-42
95. Ekstrand A, Cao R, Bjorndahl M, et al.
Deletion of neuropeptide Y (NPY)
Neuropeptides in skin inflammation and diabetic wound healing
1438 Expert Opin. Biol. Ther. (2010) 10(10)
Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by Northwestern University on 03/24/15
For personal use only.
2 receptor in mice results in blockage of
NPY-induced angiogenesis and delayed
wound healing. Proc Natl Acad Sci USA
2003;100:6033-8
96. Gohshi A, Honda K, Tominaga K, et al.
Changes in adrenocorticotropic hormone
(ACTH) release from the cultured
anterior pituitary cells of
streptozotocin-induced diabetic rats.
Biol Pharm Bull 1998;21:795-9
97. Chan O, Inouye K, Vranic M,
Matthews SG. Hyperactivation of the
hypothalamo-pituitary-adrenocortical axis
in streptozotocin-diabetes is associated
with reduced stress responsiveness and
decreased pituitary and adrenal
sensitivity. Endocrinology
2002;143:1761-8
98. Detillion C, Craft TK, Glasper ER,
et al. Social facilitation of wound
healing. Psychoneuroendocrinology
2004;29:1004-11
99. Kalin N, Shelton SE, Engeland CG,
et al. Stress decreases, while central
nucleus amygdala lesions increase,
IL-8 and MIP-1alpha gene expression
during tissue healing in non-human
primates. Brain Behav Immun
2006;20:564-8
100. Fernstrom M, Mirski MA, Carraway RE,
Leeman SE. Immunoreactive neurotensin
levels in pancreas: elevation in diabetic
rats and mice. Metabolism
1981;30:853-5
Affiliation
Lucı
´lia da Silva
1,2
BSc,
Euge
´nia Carvalho
2
PhD &
Maria Teresa Cruz
†2,3
PhD
†
Author for correspondence
1
Faculdade de Cie
ˆncias e Tecnologia,
Universidade de Coimbra,
Coimbra, Portugal
2
Centro de Neurocie
ˆncias e Biologia Celular,
Universidade de Coimbra,
Coimbra, Portugal
3
Faculdade de Farma
´cia,
Universidade de Coimbra,
Po
´lo das Cie
ˆncias da Saude,
Azinhaga de Santa Comba,
3000-548 Coimbra, Portugal
Tel: +351 239 480203; Fax: +351 239 480217;
E-mail: trosete@ff.uc.pt
da Silva, Carvalho & Cruz
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