NOypiel PDF

Nitric Oxide 10 (2004) 179193
www.elsevier.com/locate/yniox
Review
Nitric oxide function in the skin

M.-M. Cals-Grierson and A.D. Ormerod
L Oral Recherche, Clichy, France
Department of Dermatology, Aberdeen Royal InWrmary, Scotland, UK
Received 7 October 2003; received in revised form 19 April 2004
Available online 28 May 2004
Abstract
Endogenously produced nitric oxide (NO) has a remarkably diverse range of biological functions, including a role in neurotransmission, smooth muscle relaxation, and the response to immunogens. Over the last 10 years, it has become clear that this extraordinary molecular messenger also plays a vital role in the skin, orchestrating normal regulatory processes and underlying some of the
pathophysiological ones. We thought it pertinent to review the current literature concerning the possible function of NO in normal
skin, its clinical and pathological signiWcance, and the potential for therapeutic advances. The keratinocytes, which make up the bulk
of the epidermis, constitutively express the neuronal isoform of NO synthase (NOS1), whereas the Wbroblasts in the dermis and other
cell types in the skin express the endothelial isoform (NOS3). Under certain conditions, virtually all skin cells appear to be capable of
expressing the inducible NOS isoform (NOS2). The expression of NOS2 is also strongly implicated in psoriasis and other inXammatory skin conditions. Constitutive, low level NO production in the skin seems to play a role in the maintenance of barrier function
and in determining blood Xow rate in the microvasculature. Higher levels of NOS activity, stimulated by ultraviolet (UV) light or
skin wounding, initiate other more complex reactions that require the orchestration of various cell types in a variety of spatially and
temporally coordinated sets of responses. The NO liberated following UV irradiation plays a signiWcant role in initiating melanogenesis, erythema, and immunosuppression. New evidence suggests that it may also be involved in protecting the keratinocytes against
UV-induced apoptosis. The enhanced NOS activity in skin wounding (reviewed recently in this journal [Nitric oxide 7 (2002) 1])
appears to be important in guiding the inWltrating white blood cells and initiating the inXammation. In response to both insults, UV
irradiation and skin wounding, the activation of constitutive NOS proceeds and overlaps with the expression of NOS2. Thus, at a
macro-level, at least three diVerent rates of NO production can occur in the skin, which seem to play an important part in organizing
the skins unique adaptability and function.
2004 Elsevier Inc. All rights reserved.
Introduction
Evidence of nitric oxide (NO)1 synthesis by human
skin cells was Wrst reported just over 10 years ago [1].
Since that time, and from a proposed role in non-speciWc
host defense, it is now clear that NO plays a key role in
orchestrating the skins response to external stimuli such
as heat, ultraviolet (UV) light, response to infection, and

wound healing, as well as possibly underlying certain
pathological conditions.
The importance of NO-mediated signaling in the skin
has been reviewed by several authors [26] although previously the emphasis has often been placed on pathologic conditions. Here, we review the current literature of
Corresponding author.
E-mail address: mmcals-grierson@rd.loreal.com (M.-M. Cals-Grierson).
1
Abbreviations used: ADMA, asymmetric dimethylarginine; cGMP, guanosine cyclic 35-monophosphate; CGRP, calcitonin gene-related peptide; EGF, epidermal growth factor; GSNO, S-nitrosoglutathione; IFN-, interferon-; IL, interleukin; IP3, inositol trisphosphate; KGF, keratinocyte growth factor; L-NAME, N-nitro-L-arginine-methyl-ester; L-NMMA, N-monomethyl-L-arginine; LPS, lipopolysaccharide; NF-B, nuclear
factor-B; NO, nitric oxide; NOS, nitric oxide synthase; PKG, protein kinase G; PGE2, prostaglandin E2; ROS, reactive oxygen species; RT-PCR,
reverse transcriptase-polymerase chain reaction; SNAP, S-nitroso-N-acetylpenicillamine; TGF, transforming growth factor; TNF, tumor necrosis
factor-; UV, ultraviolet; VEGF, vascular endothelial growth factor.
1089-8603/$ - see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.niox.2004.04.005
180
M.-M. Cals-Grierson, A.D. Ormerod / Nitric Oxide 10 (2004) 179193
NO in the skin and focus on NO-based signaling in normal skin and contrast this with the pathological conditions.
Biochemistry of NO
In simple terms, NO is synthesized by the intracellular
enzyme, NO synthase (NOS), in a two-step oxidation of
L-arginine, that produces equal parts of citrulline and
NO [79]. Although, in the literature, it is almost taken
for granted that NOS produces the free radical, NO, this
is still under debate, since other nitrogen oxide species
could result from this catalytic process (see [10]).
The three main NOS isoforms currently identiWed are,
NOS1, originally isolated from neuronal tissue (also
known as nNOS), NOS2 (or iNOS), an inducible isoform, and NOS3 (or eNOS), predominant in the endothelium [1012]. (An isoform named mtNOS has also
recently been isolated in mitochondria [13].) They all
exist as homodimers, with molecular weights between
130 and 160 kDa, and all require the cofactors, Xavin
dinucleotide, Xavin mononucleotide, tetrahydrobiopterin, and reduced nicotinamide adenine dinucleotide
phosphate. They also require bound calmodulin, but
whereas NOS1 and NOS3 require Ca2+-calmodulin, the
NOS2 appears to have lost this calcium dependence.
In addition to requiring these cofactors, the activity of
the NOS isozymes is regulated by associated proteins
and by their localization inside cells [14,15]. For example, NOS3 in endothelial cells is regulated in part by its
distribution between the caveolae (specialized plasma
membrane structures) and intracellular pools, in a process that involves palmitoylation and the recently characterized proteins, nosip and nostrin [16,17]. Further
more, its activity is also controlled by dynamic associations with regulatory proteins in the caveolae including
caveolin-1, hsp90, and transmitter receptors, as well as
by phosphorylation [18,19].
NOS1 and NOS2 are active as cytoplasmic enzymes
and yet could also function to be membrane associated
proteins [15]. NOS1 possesses a PDZ-binding motif with
which it can interact with a number of other proteins. In
particular, via the PDZ motif, NOS1 is reported to make
stimulatory association with the 5HT2b receptor and an
inhibitory association with the calcium ATPase
[10,20,21].
The role of endogenous NOS inhibitors in basic skin
physiology is still to be established. The arginine metabolite, asymmetric dimethylarginine (ADMA), has been
shown to be an important competitive NOS inhibitor in
cardiovascular physiology [22] and it may also play a
role in the skin. Another point of regulation is the supply
of the substrate, arginine, to NOS. Indeed, the competition for arginine by other cellular pathways and the
presence of endogenous NOS inhibitors have been
invoked to explain the arginine paradox; situations
where L-arginine supplementation stimulates NO synthesis, despite apparently saturating extracellular arginine concentrations [23,24].
NO is highly diVusible and highly reactive. Instead of
activating downstream pathways via traditional receptor-mediated events, it modulates the activity of a number of diverse molecules. Wink and Mitchell [25]
classiWed these NO reactions as being either direct (on
the biological mediator) or indirect (involving reactive
nitrogen and oxygen species). The direct downstream
pathways consist mainly of interactions between NO
and heme-containing proteins, the most important being
guanylate cyclase [11,26]. Activation of this enzyme, by
NO, induces the production guanosine cyclic 35monophosphate (cGMP) which in turn activates protein
kinase G (PKG). This downstream pathway is particularly important in mediating the eVects of the low levels
of NO production, which seem to occur with constitutive
NOS activation. The indirect downstream pathways,
which become more important under high local concentrations of NO, involve the formation of nitrogen oxide
species such as N2O3, HNO (nitroxyl), and ONOO- (peroxynitrite) [27]. These molecules, in turn, modify thiolcontaining proteins, either by nitrosation (N2O3) or
oxidation (HNO and ONOO-). The selection of which
indirect downstream pathway is chosen seems to depend,
to some extent, on the redox potential of the cell [2830].
The existence of biological NO donors in the cytoplasm has been suggested for some time [31,32] but, the
identity and function of these remain unclear. Some of
the early work of Furchgott and colleagues showed that
stored NO in vascular tissue could be mobilized by UV
irradiation to induce NO-dependent smooth muscle
relaxation (see [33]). In the lung, a putative endogenous
NO donor can induce the S-nitrosylation of 5-HT2
receptors [34]. The evidence points to such stores as
being perhaps S-nitrosoglutathione (GSNO) or a
GSNO-like compound, although other nitrogen compounds may also be involved [3335]. The existence of a
putative NO store in the skin has previously been suggested [36,37], but its identity has not been elucidated.
Since GSNO is readily formed from N2O3 and glutathione [9], it seems likely that GSNO may also represent an
NO store in skin cells that express NOS.
Control of expression of NOS
The constitutive NOS isoforms are expressed in certain cell types as part of the mature phenotype, although
it is now clear that the level of expression of these
enzymes can be modulated by circulating hormones. For
example, the changing estrogen concentrations as found
during the menstrual cycle and pregnancy can modulate
the expression of NOS1 [38,39], and the expression and
activity of NOS3 [4042]. The eVect of estrogen on NOS
expression in skin cells is likely to underlie a variety of
estrogen-based skin reactions such as Xushing and

hyperpigmentation [43,44]. UV-B irradiation (290
320 nm) has been reported to upregulate NOS1 mRNA
in keratinocytes [45,46], whereas shear stress upregulates
NOS3 [47].
NOS2, on the other hand, is not usually found in normal resting cells, but induced in response to inXammatory cytokines or a combination of cytokines and
bacterial polysaccharides. Frequently, a synergism
between diVerent external stimuli is observed, which is
mediated by nuclear factor-B (NF-B) and the Jak-Stat
pathways [48]. Thus, NOS2 expression can be induced in
cultured keratinocytes with combinations of interferon-
(IFN-)/tumor necrosis factor- (TNF), lipopolysaccharide (LPS)/IFN-, IFN-/interleukin-8 (IL-8), or
TNF/interleukin-1 (IL-1) [1,49,50]. It is possible that
the cocktail of cytokines to which cells are exposed may
determine the rate and magnitude of NO production
[51].
NOS localization in the skin

The skin, the largest organ of the body, is composed of three main layers, the epidermis, the dermis,
and a sub-dermal layer, or hypodermis. The major cell
types that comprise these layers, including keratinocytes, Wbroblasts, melanocytes, and endothelial cells,
express NOS and appear capable of releasing NO (see
Fig. 1).
181
and become part of the corniWed epithelium. This maturation process consists essentially of keratinization, but
involves a number of morphological and metabolic
events as the cells diVerentiate and lose their nuclei.
Although a few early reports suggested that keratinocytes expressed NOS3 [53,54], this was put in doubt following further experimentation that suggested perhaps
post-translational processing of the NOS protein
allowed a cross-reaction with certain anti-NOS3 antibodies [55]. Most of the evidence now indicates that
keratinocytes constitutively express NOS1. This has
been demonstrated using the reverse transcriptase-polymerase chain reaction (RT-PCR) in unstimulated
human and animal keratinocytes and in transformed
keratinocyte cell lines [50,55,56] (Cals-Grierson unpublished results). Furthermore, Jackson et al. [55] found no
induction of NOS3 message following the exposure of
keratinocytes to activating treatments, such as UV-B
irradiation or IFN. One recent study [57] however has
reported NOS3 mRNA expression in human primary
keratinocytes and in the human keratinocyte cell line,
HaCaT, but with a very low level of protein transcription. Keratinocytes are also known to express NOS2
message and protein following the exposure to inXammatory cytokines [50,58] (Cals-Grierson unpublished
results). It is notable that NOS2 expression is found in a
number of inXammatory skin conditions including psoriasis, atopic dermatitis, and irritant and allergic contact
dermatitis [5962].
Fibroblasts
Keratinocytes
Keratinocytes account for about 9095% of the cells
in the epidermis [52]. They are formed continuously in
the proliferative, basal layer and undergo a tightly regulated process of diVerentiation as they progress upwards
The Wbroblasts are the most abundant cell type in the

dermis, where their role is to produce the Wbrous
extracellular matrix that gives the skin its mechanical
resistance. Using RT-PCR and immunocytochemistry,
human skin-derived Wbroblasts have been shown to
Fig. 1. A simpliWed diagram of the upper layers of the skin showing the major cell types and their respective expressions of the NOS isoforms.
182
express NOS3 [63]. This expression appears to be low

level as compared to porcine endocardial cells or human
umbilical endothelial cells and NOS immunocytochemistry is heterogeneous, with the reaction intensity ranging from weak to strong [63,64]. The protein appears to
be localized to the cytoplasm with more intense staining
sometimes observed in the perinuclear area [63].
Although Wbroblasts are known to possess calveolae,
there is no evidence, as yet, to suggest that NOS3 is preferentially localized in these structures.
The expression of NOS2 in Wbroblasts following cytokine stimulation has also been shown [63]. Here too, the
expression of NOS2 immunoreactivity is markedly heterogeneous, with only a minority of cells exhibiting a
positive reaction [63,65]. This has been suggested to
reXect a diVerence in the maturity of cells [65].
Adipocytes
Adipocytes are the fat storage cells of the hypodermis.
In rodents, these cells express NOS3 and can be activated to express NOS2 [74]. In this cell type, the endogenously produced NO appears to be involved in the
regulation of lipolysis, since NOS inhibition signiWcantly
reduces lipolysis, via a mechanism of action that involves
cytoplasmic oxidation [75].
The development of adipose tissue is determined to a
large extent by circulating sex steroid hormones and in
pathophysiological conditions, high levels of these steroids can lead to the over-development of adipose tissue.
It is interesting to speculate that, considering the inXuence of estrogen on NOS, in certain cases of obesity
involving high estrogen levels, the NO-mediated signaling in the adipocyte may be downregulated.
Endothelial cells
Other skin cell types
Projecting into the dermis from the underlying subcutaneous layer are the papillary loops of the microvasculature [66], whose walls are composed of an endothelium
and a smooth muscle sheath. The endothelial cells of
normal human skin express NOS3, as shown by Western
blot and immunocytochemistry [60,67]. These cells can
also be induced to express NOS2 mRNA by exposing
them to TNF, or more potently, a combination of
TNF and IFN. Following exposure to these stimuli,
NOS2 message is detectable within 6 h and NOS protein
increases linearly over 72 h [68]. The involvement of
cytokines is not however obligatory, since UV-A (320
400 nm) can induce NOS2 expression in the absence of
cytokines [69]. The expression of NOS2 in a constitutive
fashion has been observed (immunocytochemically) in
the dermal endothelial cells of patients with atopic or
allergic contact dermatitis [60], indicating that, under
certain circumstances, long-term NOS2 expression may
occur.
Melanocytes
The melanocytes are the pigment-producing, dendritic cells of the skin, located in the suprabasal layer of
the epidermis. They respond to UV irradiation by
synthesizing the pigment, melanin, which is stored in
specialized structures called melanosomes. These organelles are then transferred from the melanocytes to the
surrounding keratinocytes, producing coloration of the
skin. Normal human melanocytes have been shown to
express NOS3 mRNA [55], although immunoreactiveNOS1 was reported in one study [70]. NOS1 also seems
to be expressed by some malignant melanomas and
dysplastic nevi (irregularly shaped and colored moles)
[71]. Normal human melanocytes will express NOS2
following incubation with LPS, TNF, and IFN
[72,73].
The Langerhans cells are antigen-presenting cells

(originating in the bone marrow) that take up a position
in the suprabasal layer of the epidermis. It is doubtful
that these cells express constitutive NOS, although there
is one report of immuno-positive NOS2 staining in Langerhans cells in human neonatal foreskin, in situ [76]. On
the other hand, no NOS2 expression was observed in
unstimulated mouse Langerhans cells, nor in cells activated by LPS or cytokines [77], nor in human psoriatic
skin [78].
The outer root sheath in murine skin has been
reported to be immunoreactive for NOS1 [79], whereas
the cells of the arrector pili muscle, apocrine secretory
gland, eccrine coiled duct, and a part of the eccrine secretory gland are all reported to exhibit NOS3 immunoreactivity [80,54]. Dermal papilla cells derived from human
hair follicles have been shown to express NOS3 message
and protein [81]. The sebaceous glands are reported to be
lacking in NOS immunoreactivity [54], although the lessspeciWc diaphorase staining has shown positive results in
some species [82].
The role of NO in physiologic responses in the skin

Vasodilatation
The constitutive release of NO by the endothelial cells
of the microvasculature plays an important role in setting resting blood Xow rate. Using laser-Doppler Xowmetry to measure blood Xow, Lawrence and Brain [83]
reported that the intradermal injection of the NOS
inhibitor, L-NAME, signiWcantly reduced Xow rates in
rat skin. In 1994, CoVman [84] reported a similar result
following the perfusion of the NOS inhibitor, L-NMMA,
into the brachial artery of volunteers and subsequent
experimental modiWcations have continued to support

these results [85,86].
The vasoconstricting eVect of NOS inhibition is
enhanced in locally warmed skin [85], whereas when the
whole body is warmed, intradermal injections of NOS
inhibitors have relatively little eVect. This is due to a neurally mediated vasodilatation reXex, involving the release
of calcitonin gene-related peptide (CGRP) as well as NO
[85,8789]. Although CGRP has intrinsic vasodilatatory
activity, NO release is required for a complete vasodilator response [86,89]. Thus, NOS responds rapidly to
local changes in temperature and neurogenic signals
with an increase in NO production that has a direct
eVect on the vasculature.
In unstimulated endothelial cells, at normal temperatures, it is likely that the primary activator of NOS is
blood Xow shear stress [90], although circulating cytokines, steroids, and peptide hormones will also play a
part [91]. In the response to heat, it is possible that the
vanilloid receptor family of cationic channels, which
allow the entry of Ca2+ into the cell, may be important in
controlling the activity of constitutive NOS. Members of
this heat-sensitive family of channels are expressed by
neurons and keratinocytes [9294].
In conclusion, the constitutive release of NO in the
skin is involved in setting the rate of resting blood Xow,
via a cGMP-dependent relaxation of the vascular
smooth muscle.
The response to UV irradiation
Irradiation of the skin by UV light induces a variety
of biologically active molecules [95,96]. Some of these
are created photochemically, by the modiWcation of various proteins, carbohydrates, and lipids, whereas others
are induced or released in consequence. Amongst the
important diVusible transmitters released by UV-activated keratinocytes are interleukin (IL)-1 [97], IL-6 [98],
IL-8 [99], IL-10 [100], TNF- [101], transforming growth
factor (TGF)- [102], TGF-1 [103], prostaglandin E2
(PGE2) [104], endothelin-1 [105], and the pro-opiomelanocortin peptides [106]. UV irradiation also directly activates certain transmembrane receptors, such as the
epidermal growth factor (EGF) receptor [107] and the
keratinocyte growth factor (KGF) receptor [108], which
go on to activate downstream pathways and initiate the
production of peroxide and reactive oxygen species
(ROS).
In 1992, Deliconstantinos and colleagues [109]
showed that the irradiation of cultured endothelial cells
by UV-B led to the dose-dependent increase in NO and
cGMP. They went on to show that a similar response
could be evoked in cultured keratinocytes [36] with signiWcant elevations in NO and [3H]citrulline; changes that
occurred within 10 min of the UV irradiation. It thus
became apparent that the endogenous generation of NO
183
plays a signiWcant role in the response to UV. Exactly

how NO is involved within this stimulatory soup of
transmitter molecules, to elicit the various physiological
responses to UV, is still the subject of a great deal of
research projects.
UV-induced erythema
It is well established that the exposure of skin to UV
irradiation causes erythema. The intensity of the erythema is proportional to exposure dose [110] and the
onset of erythema occurs after a characteristic delay of
about 824 h [110112]. In 1993, Warren and colleagues
[113] showed that an intradermal injection of L-NAME
prevented this erythema. Even when the injection was
made 30 min before the predicted onset of erythema
(18 h after irradiation in their model), the NOS inhibitor
abolished the increase in blood Xow. A comparable
result was found in human volunteers, where the intradermal injection of NMMA attenuated the delayed
blood Xow increase [114] and intradermal administration of L-NAME produces a long-lasting (24 h) pallor
around the UV-B irradiated injection site [80]. Rhodes
et al. [115] also found that intradermal injections of
L-NAME were eVective in blocking the erythemic
response, but also noted that NO acted in concert with
PGE2, since the L-NAME-induced block was lost at
high doses of UV-B, when a stronger stimulation of
PGE2 occurs.
From work of Deliconstantinos and co-workers
[36,109,116] and others, it has been proposed that UV
irradiation augments both the activity and expression of
NOS1 and NOS3 [117,118]. In addition, UV irradiation
was also found to induce the expression (and therefore
activation) of NOS2. In normal skin, NOS2 is measurable at about 6 h after UV exposure, with a peak of
expression at around 24 h and a return to resting levels of
expression after 72 h [46]. Although UV-A irradiation
appears to be suYcient to induce NOS2 expression [69],
local cytokine release is likely to contribute signiWcantly
[95,119]. The experimental inhibition of constitutive NOS
during the exposure to UV has been found to inhibit the
induction of NOS2 [120]. Therefore, in the early stages of
UV exposure there would appear to be a positive feedback eVect of NO on NOS2 induction. The rise in intracellular calcium that occurs upon UV stimulation [121]
appears to be the principal activator of the constitutive
NOS [36]. This calcium Xux is also essential for ROS production via the UV-activated EGF receptor [122].
While the delay for UV-induced erythema appears to
coincide with the expression of NOS2 [146], this may
coincidental. It is not clear from the work with NOS2knockout mice if UV-induced erythema has the same
dynamic as in wild-type mice.
Thus, the cutaneous production of NO appeared to
be a central component of the delayed-onset erythema.
The source of this NO, whether it derives primarily from
184
constitutive or from inducible NOS, remains however

unclear.
UV-induced melanogenesis
The evidence of a central role for NO in the induction
of melanogenesis has been apparent since the mid-1990s
when Romro-Graillet et al. [56,123] found that UV irradiated cultures of human melanocytes produced NO and
that the presence of NO was suYcient to induce melanogenesis. Their experiments showed that the eVect of UV
on their cultures could be mimicked by exogenous NO
or an analog of cGMP, and that melanogenesis was
blocked by inhibitors of cGMP-dependent kinase. Later
work demonstrated that UV irradiated keratinocytes
released suYcient NO to induce melanogenesis in keratinocyte/melanocyte co-cultures [123]. These results suggested therefore that NO could act as both an autocrine
and paracrine regulator of melanogenesis. In vivo experiments on guinea pigs have shown that the topical application of L-NAME inhibits UV-induced melanogenesis,
reducing melanin content and the number of histochemically positive melanocytes [124].
In addition to these eVects on melanogenesis, NO has
been found to enhance the dendritic branching of melanocytes [123] and facilitate the melatonin-induced aggregation of melanosomes [125]. Recent research suggests
that NO may also be involved in setting the eumelanin/
pheomelanin ratio in melanocytes, since the ratio of
these two melanins increases if melanocytes are exposed
to NO or histamine [126] and diVerences have also been
noted when melanocytes are cultured in close contact
with keratinocytes, as opposed to pure melanocyte cultures [127].
The induction of erythema and melanogenesis by UV
irradiation linearly correlated [128], suggesting that similar regulatory pathways are involved. There would
appear to be a rapid activation of constitutive NOS, followed by an elevation of NO and cGMP (within 30 s)
and other cytokines. At later time points, it is likely that
NOS2 takes on a more important role in maintaining
NO production at an elevated level.
The downstream targets of NO in melanocytes
remain to be fully elucidated. It is likely however that,
via guanylate cyclase and PKG activation, NO plays a
central role in the activation of tyrosinase [123]. NO may
also be involved in the induction of the tyrosinase
mRNA message, which is induced within 2 h of an application in vitro of an NO donor [129]. Other NO-activated pathways, however, might also be important. In
the presence of oxygen, NO reacts with the melaninrelated metabolites 5,6-dihydroxyindole and its 2-carboxylic acid (DHICA) resulting in the deposition of
melanin-like pigments [130]. Evidence from other cell
systems suggests that NO can reduce inositol trisphosphate (IP3) synthesis [131], and reducing Ca2+ release via
modulation of the IP3 receptor [132,133].
UV-induced immunosuppression
In UV-induced immunosuppression, the acute
exposure of skin to UV irradiation induces a transient
suppression of both contact hypersensitivity and
delayed-type hypersensitivity and a temporally limited
development of transferable antigen-speciWc suppressor
cells. In addition, the Langerhans cells, which are
responsible for antigen presentation, disappear from the
epidermis [134]. Strongly implicated in this response is
the release of CGRP from peripheral neurons and cytokine stimulation, particularly TNF and IL-10 [135
137]. However, some evidence suggests that NO might be
involved in passing a migration signal to the Langerhans cells that stimulates them to migrate [138,139].
The elevated NO synthesis following NOS2 induction
has a complex eVect on the immune system and in models of skin transplantation, this is often associated with
graft rejection and speciWc NOS2 inhibitors have prolonged graft survival [140]. It appears, in this situation,
that the inhibition of NO enhances the release of Th2
cytokines (IL-10 and IL-4) and reduces the release of
Th1 cytokines (IL-2 and INF), thereby favoring tolerance [140]. In NOS2-knockout (/) mice however,
rejection occurs in a similar fashion to wild-type graft
recipients [141,142].
InXammation
A large body of work indicates that elevated levels of
NO are pro-inXammatory and many similarities exist
between UV-induced erythema and inXammation. The
exposure of skin to UV irradiation or to chemical irritants results in higher levels of NO synthesis and the
production of pro-inXammatory cytokines [113,143]. In
experiments involving human volunteers, the application
of an NO-releasing emulsion to the skin has been shown
to evoke local inXammation and other inXammatory
events, including the loss of Langerhans cells and the
induction of apoptosis in keratinocytes [138]. If, on the
other hand, the production of NO in the skin is blocked,
then the inXammatory response is lessened. In guinea pig
skin, the inXammatory response (edema formation) to
an intradermal injection of bradykinin, histamine or
platelet-activation factor, is attenuated by the co-injection of the NOS inhibitor, L-NAME [143], and in NOS2
/ mice, experimentally induced inXammation, using
the LPS-evoked plasma extravasation model, is reduced
as compared to wild-type mice [144].
Normally, inXammation is self-regulating. This is due
partly to NO-induced nitrosylation of NF-B, which
prevents the transcription factor from binding to the
NOS2 promoter [145], but other points of feedback may
also be involved. A lot of recent research has focused on
the role of ROS and particularly superoxide, which in
combination with NO, forms peroxynitrite. This highly
reactive molecule can cause the nitrosation of tyrosine

[138], DNA strand breakage, and activation of the
poly(ADPribose) polymerase pathway of necrotic cell
death [146]. (It should be pointed out, however, that the
action of myeloperoxidase may be responsible for signiWcant tyrosine nitration in inXamed tissue [147].)
When the self-regulation of inXammation breaks
down, severe and sometimes life-threatening dermatoses
can occur. The StevensJohnson syndrome, an inXammatory skin condition with toxic epidermal necrolysis, is
notable for its persistent upregulation of NOS2 mRNA
[148].
Paradoxically, UV irradiation of inXammatory skin
conditions can suppress NOS2 expression [149]. In
experiments on keratinocytes and macrophages, UV-B
irradiation interfered with NF-kB and Stat-1 DNAbinding activity to produce a transient (12 h) inhibition
of IFN-stimulated NOS2 induction. In keratinocytes,
there was a more marked activity of UV-B on phosphorylation of MAP kinases, suggesting that this pathway
may also modulate UV-induced responses.
Apoptosis
The cellular response to intracellular NO concentration increases seems to depend to a signiWcant extent on
the redox potential of the cell, which is itself inXuenced
by the resting levels of NO. In human neuroblastoma
cells, for example, NO induces thioredoxin expression,
via a PKG-dependent pathway, which helps to protect
the cells from oxidative stress and apoptosis [150]. But,
higher levels of NO can promote apoptosis [28,151],
earning it the epithet the Janus-faced molecule [152].
The protective eVects of NO in the skin have been
demonstrated by experiments on NOS2 / or NOS3
/ mice, which show signiWcantly higher numbers of
apoptotic skin cells following UV irradiation, than in
wild-type mice [153]. The mechanism of this protective
action is still uncertain. An induction of Bcl-2 expression
and inhibition of caspase activation have been suggested
by some studies [154], but this fails to explain the rapidity of the response. Also evoked is the inhibition of ROSmediated lipid peroxidation [155] and indeed, lipid
peroxidation is a good in vivo measure of UV-induced
oxygen free radical production (a reaction suppressed by
the NO donor, sodium nitroprusside, and enhanced by
L-NAME (a NOS inhibitor)) [120]. But, the involvement
of a cGMP-mediated pathway (as reported for neuroblastoma cells) cannot be excluded [153].
The keratinocytes of patients with lupus erythematosus have increased susceptibility to apoptosis and the
elimination of these apoptotic cells appears impaired,
leading to the formation of anti-nuclear antibodies.
Kuhn et al. [46] have found that the induction of NOS2
by UV irradiation in these patients is delayed as compared to normal individuals. Instead of NOS2 message
185
peaking at 24 h and subsiding on day 3, the lupus

patients exhibited a peak signal on day 3, which persisted for 25 days. It is paradoxical therefore that persistent NOS2 expression in keratinocytes and endothelial
cells is a consistent feature of this disease [156].
Wound healing
NO signaling appears to play a vital role in wound
healing. The positive eVects of arginine supplementation
and NO donors, coupled to the negative eVects of NOS
inhibitors or the deletion of the NOS2 or NOS3 genes,
have provided unassailable evidence of a key role for
NO. This has been reviewed recently by Schwentker et
al. in this journal [157] and by others [158,159] and will
not be discussed at length. However, a brief examination
of the signaling pathways that seem to be implicated in
wound healing does seem to be relevant for an overall
appreciation of the eVects of NO in the skin.
The expression of NOS2 is stimulated in wound tissue, particularly in the basal keratinocytes adjacent to
the wound [58]. The peak expression occurs after 46
days [160,161] and it stays elevated for an extended
period (at least 3 weeks in the mouse) [160]. This time
course of NOS2 expression is therefore somewhat
retarded and extended as compared with UV-induced
NOS2 activity. It also submaximal, since production can
be further stimulated with combinations of LPS and
cytokines [160].
One of the key functions of NO in wound healing
seems to be its permissive inXuence on keratinocyte and
Wbroblast proliferation, which helps promote wound reepithelialization [162,163]. One study has noted that low
concentrations (0.010.25 mM) of NO stimulate cell division, whereas high concentrations (10.5 mM) are cytostatic [164]. Interestingly, it seems to be the inXuence of
the superoxide anion in the cell that determines the mitogenic capacity of the NO signal [165]. Together, they
form peroxynitrite which dose-dependently inhibits proliferation. Removal of this anion reduces the inhibitory
eVect.
The likely importance of NO-modulated cytokine signaling in the wound healing process has been noted by
others [157] and of particular interest seem to be the
NO-induced activation of TGF-b1 and enhancement of
IL-1 and IL-8 production. NO is also known to stimulate epithelial cells to produce and release chemokines
[159] and other growth mediators such as vascular endothelial growth factor (VEGF). Key players are also EGF
and KGF [166,167]. KGF is released by activated Wbroblasts (IL-1 activated in particular) and stimulates the
proliferation and migration of keratinocytes. In addition, high levels of VEGF are found at the hyperproliferative epithelium of the wound, which appears to be
important for keratinocyte proliferation and angiogenesis [168].
186
As well as having a role in promoting cell proliferation, NO also inXuences the production of collagen by
Wbroblasts. The inhibition of NOS by competitive inhibitors has been shown to reduce collagen synthesis [160]
and in NOS3-knockout mice, the tensile strength of
regenerating tissue was signiWcantly reduced as compared to the wild-type. Primary dermal Wbroblasts
obtained from NOS2-knockout mice have been shown
to synthesize less collagen compared to wild-type cells,
but their production can be augmented by the addition
of NO donors [169]. Concordant with this positive eVect
of NO, topical estrogen, perhaps acting via a stimulatory
eVect on NOS activity, has been found to accelerate the
process of wound healing in the aged [170].
Barrier function
The integrity of the corniWed envelope of the epithelium confers a barrier against water loss, abrasion, and
chemical insult. Intrinsic to the corniWcation process are
the activity of transglutaminase and the induction and
cross-linking of the terminal diVerentiation proteins,
involucrin and loricrin. NO appears to inhibit this process via the modifying the thiol groups on transglutaminase and, via the inhibition of the AP-1 transcription
factor, involucrin and loricrin synthesis [171]. We have
recently demonstrated that the topical application of LNAME attenuates the impairment of barrier function
following an application of the irritant, sodium lauryl
sulfate, but we were unable to demonstrate an enhanced
impairment (transepidermal water loss) by application
of an NO donor (Exogenous Dermatology, in press).
An impaired barrier function is typical in dermatitis
[172] and other inXammatory dermatoses [173,174] and
it is notable that high levels of NOS2 expression are
found in these reactive plaques [50]. Some evidence suggests that the resulting elevated production of NO and
peroxynitrite formation lead to the activation of
poly(ADPribose) polymerase; an event which seems to
inhibit the diVerentiation of keratinocytes and contribute to impaired barrier function [147]. Peroxynitrite formation is also implicated in the aggravation of apoptosis
found in other inXammatory conditions, for instance in
the intestine [175]. Interestingly, in vitiligo, a depigmenting disease of the skin characterized by the early death of
epidermal melanocytes, the loss of melanocytes may be
caused by their over-sensitivity to UV-induced oxidative
stress [176].
Antimicrobial eVects
The idea that NO may serve as non-speciWc host
defense has been aired since the early 1990s [1,177] and it
is now known that it exerts antimicrobial eVects on
diverse micro-organisms including fungi, yeast, bacteria,
viruses, and protozoa [77,178180]. Thus, as the front
line against the invasion by pathogens, the constitutive

and steady production of NO on the skins surface is
likely to play an important role.
In 1996, it was shown that NO is produced on the surface of human skin by the action of commensal bacteria:
the bacteria convert nitrate from sweat to nitrite, and the
acidic environment of the skins surface releases the NO
from the nitrite [179]. Earlier work had shown that the
replication of clinically relevant viruses, such as the pox
and herpes viruses, was reduced (by about a 1000-fold)
by physiological concentrations of NO [178] and it was
later shown that NOS2 / mice were more susceptible
to herpes infection and exhibited a greater frequency of
reactivation than wild-type mice [181]. In patients with
psoriasis, the constitutive induction of NOS2 and elevated NO synthesis may be responsible for the relatively
high protection against infection seen in this disease
[182].
Although infectious pathogens such as Mycobacterium leprae (leprosy), Mycobacterium tuberculosis, and
herpes zoster virus stimulate NO production (via NOS2)
and this seems to limit their progression [183,184], they
can also produce severe inXammation, which, in certain
cases, results in epidermal necrolysis and damage to
peripheral nerve terminals. Sometimes, therefore, the
induction of NOS2 appears to be uncontrolled and
results in excessive inXammation with serious consequences. In an experimental model of pneumonia caused
by intranasal herpes simplex, the administration of the
NOS inhibitor, L-NMMA, improved survival and pulmonary compliance [185]. The results suggested that the
improvement was mediated by the attenuation of the
inXammatory response.
Important targets for the antiviral eVect of NO
include the inhibition of reverse transcriptase and zinc
Wnger domains necessary for DNA-binding and transcription, viral ribonucleotide reductase, and viral envelope [186,187].
Pathologic conditions
Skin cancer
The role of NO-mediated signaling in the progression
of skin cancer remains uncertain. Although some evidence suggests that elevated NOS expression plays an
active role in progression, the situation is complex and
not all data are in concordance.
In an examination of pigment cell lesions, Ahmed and
Van Den Oord [71] found NOS1 expression in benign
nevi (moles) and, more frequently, in the basal component of dysplastic nevi and in primary melanomas during radial growth. The authors suggested that the
enhanced release of NO in these situations may be
responsible for the increased number of blood vessels
observed in the papillary dermis of these lesions. When

the same group analyzed NOS2 expression [188], they
observed some reactivity in nevi and primary melanomas, but none in metastases. In contrast, Tschugguel
et al. [189] did Wnd NOS2 expression in subcutaneous
metastases of melanoma, although they noted that its
expression was inversely related to the tumors
metastatic potential. Whereas, Kagoura et al. [190] on
Wnding that NOS2 immunoreactivity was relatively
weak in well-diVerentiated adenocarcinomas and strong
(although heterogeneous) in poorly diVerentiated ones,
suggested that NOS2 expression may reXect the degree
of proliferation of the tumor. Thus, a progression in
NOS2 immunoreactivity was observed ranging from
Bowens disease (a pre-cancerous epidermal lesion, 75%
positive), through squamous cell carcinomas (75% positive, but more intense), to metastatic carcinomas (80%
positive) [190,191]. Interestingly, Fecker et al. [192] were
however unable to induce NOS2 mRNA in melanoma
cell lines using combinations of TNF, INF, and LPS,
whereas it could be induced in cultures of normal melanocytes.
Evidence for an angiogenic role for elevated NO production in experimentally induced tumors has been demonstrated. Jenkins et al. [193] found that NOS2
transfected human adenocarcinoma cells formed faster
growing and more vascularized tumors in nude mice
than non-transfected, wild-type adenocarcinoma cells.
Clearly the role of NO in skin cancer is complex and
the pathological signiWcance of these Wndings remains
uncertain.
InXammatory skin disease
The expression of NOS2 in inXammatory lesions of
the skin is expected and well established [194]. In lupus
erythematosus, NOS2 expression is present throughout
the epidermis [114]. High levels of NOS2 are also found
in patients with Sjogrens syndrome (an autoimmune
disorder in which immune cells attack and destroy the
glands that produce tears and saliva) who experience
photo-aggravated erythemic reactions [195]. What is less
clear is the role of NO in persistent cutaneous inXammation and why there is no self-regulatory feedback.
As in other inXammatory lesions where superoxide is
present, NO forms peroxynitrite, which nitrosylates thiol
groups [196], leading to DNA strand breakage and activation of the poly(ADPribose) polymerase pathway of
necrotic cell death, as shown in a model of contact
hypersensitivity in the mouse [146]. In psoriasis, the
over-expression of NOS2 is also associated with an
increased, perhaps compensatory, arginase 1 activity,
which may reduce the available substrate for NO production, has led to a hypothesis that NO production
may be inadequate in these situations [24]. Supplementation of NO with GSNO has recently been shown to
187
reduce the expression of inXammatory markers and inWltration of T cell [197]. Direct measurements of NO production in psoriatic lesions however have not found any
evidence of competitive NOS2 inhibition [194].
Conclusions
From this review of NO signaling in the skin, it can be
appreciated that this messenger is strongly implicated in
a number of diverse responses. This raises the question of
why there is not more evidence of signaling cross-talk
when NO stimulates inappropriate targets. For example,
hyperpigmentation following wounding or skin irritation, or angiogenesis following UV irradiation can occur,
but they are relatively rare. A more common example
might be the pigmentation plaques of melasma that can
appear following sun exposure and elevated plasma
estrogen. It is likely that the answer lies the repertoire of
cytokines and other intercellular messengers that are
induced and their spatial and temporal distributions.
Thus, is NO merely a facilitatory messenger? A useful,
but an otherwise non-essential, component of the signaling pathways? It would certainly appear from the gene
knockout experiments that the skin is remarkably robust
in terms of physiological responses. However, this
apparent redundancy of multiple NOS isoforms is also
found in diverse animal phyla and classes. In mammals,
it would appear that there are at least two NOS
expressed in normal skin, plus another for crisis management allowing for substantial compensation and
overlap of function.
The therapeutic potential of delivering NO to the skin
is being explored by a number of laboratories, using, for
example, glyceryl trinitrate and S-nitroso-N-acetylpenicillamine (SNAP) for anal Wssures or cutaneous leishmaniasis [198200], topical diazeniumdiolates [201] or
the co-application of a mild acid and nitrite, which can
liberate NO in a controlled fashion [202,203]. This latter
method has been shown to have therapeutic eYcacy in
dermatophyte fungal tinea infections, viral infections
molluscum contagiosum, viral warts, and in vitro shows
activity against bacterial pathogens, including the acne
bacillus and Staphylococcus aureus [202205]. This technology has been investigated in treating the vasospasm
of Raynauds phenomenon [206]. A sympatex membrane
can be incorporated for more selective NO delivery
which was eVective in killing Escherichia coli and S.
aureus [207]. A topical polymer-based NONOate NO
donor improved wound closure in rats [208] and a polyvinyl alcohol hydrogel NO donor has been tested in
wound healing in diabetic animals, which resulted in
more rapid initial healing and enhanced extracellular
matrix production [209]. Lastly, nitroglycerine administration has been shown to reduce phorbol ester-induced
carcinogenesis in a mouse model, with a 32% inhibition
188
of tumorigenesis [210], suggesting that, far from being

mutagens, topical NO donors may protect against skin
cancer and protect cells from the harmful eVects of ultraviolet irradiation.
It is expected that with better understanding of the
precise role of NO in the skin and with advancements in
the controlled topical delivery of NO, new treatments for
some dermatological conditions may be possible as well
as perhaps, one day, a control of true tanning of the skin
without the need for exposure to UV light.
Acknowledgments
The authors thank Dr J. P. Grierson for helpful discussion and editorial assistance.
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Nitric Oxide 10 (2004) 179193

Nitric oxide function in the skin

as heat, ultraviolet (UV) light, response to infection, and

M.-M. Cals-Grierson, A.D. Ormerod / Nitric Oxide 10 (2004) 179193

M.-M. Cals-Grierson, A.D. Ormerod / Nitric Oxide 10 (2004) 179193

estrogen-based skin reactions such as Xushing and

NOS localization in the skin

The Wbroblasts are the most abundant cell type in the

M.-M. Cals-Grierson, A.D. Ormerod / Nitric Oxide 10 (2004) 179193

express NOS3 [63]. This expression appears to be low

The Langerhans cells are antigen-presenting cells

The role of NO in physiologic responses in the skin

M.-M. Cals-Grierson, A.D. Ormerod / Nitric Oxide 10 (2004) 179193

experimental modiWcations have continued to support

plays a signiWcant role in the response to UV. Exactly

M.-M. Cals-Grierson, A.D. Ormerod / Nitric Oxide 10 (2004) 179193

constitutive or from inducible NOS, remains however

M.-M. Cals-Grierson, A.D. Ormerod / Nitric Oxide 10 (2004) 179193

reactive molecule can cause the nitrosation of tyrosine

peaking at 24 h and subsiding on day 3, the lupus

M.-M. Cals-Grierson, A.D. Ormerod / Nitric Oxide 10 (2004) 179193

line against the invasion by pathogens, the constitutive

M.-M. Cals-Grierson, A.D. Ormerod / Nitric Oxide 10 (2004) 179193

observed in the papillary dermis of these lesions. When

M.-M. Cals-Grierson, A.D. Ormerod / Nitric Oxide 10 (2004) 179193

of tumorigenesis [210], suggesting that, far from being

[16] J. Dedio, P. Konig, P. Wohlfart, C. Schroeder, W. Kummer, W.

M.-M. Cals-Grierson, A.D. Ormerod / Nitric Oxide 10 (2004) 179193

[51] A.T. Jacobs, L.J. Ignarro, Lipopolysaccharide-induced expression

M.-M. Cals-Grierson, A.D. Ormerod / Nitric Oxide 10 (2004) 179193

M.-M. Cals-Grierson, A.D. Ormerod / Nitric Oxide 10 (2004) 179193

[127] C. Duval, N.P. Smit, A.M. Kolb, M. Regnier, S. Pavel, R.

M.-M. Cals-Grierson, A.D. Ormerod / Nitric Oxide 10 (2004) 179193

M.-M. Cals-Grierson, A.D. Ormerod / Nitric Oxide 10 (2004) 179193

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