NOypiel.pdf

Nitric Oxide 10 (2004) 179–193

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Review

Nitric oxide function in the skin

M.-M. Cals-Grierson¤ and A.D. Ormerod

L’ Oréal Recherche, Clichy, FranceDepartment of Dermatology, Aberdeen Royal InWrmary, Scotland, UK

Received 7 October 2003; received in revised form 19 April 2004Available online 28 May 2004

Abstract

Endogenously produced nitric oxide (NO) has a remarkably diverse range of biological functions, including a role in neurotrans-mission, smooth muscle relaxation, and the response to immunogens. Over the last 10 years, it has become clear that this extraordi-nary molecular messenger also plays a vital role in the skin, orchestrating normal regulatory processes and underlying some of thepathophysiological ones. We thought it pertinent to review the current literature concerning the possible function of NO in normalskin, its clinical and pathological signiWcance, and the potential for therapeutic advances. The keratinocytes, which make up the bulkof the epidermis, constitutively express the neuronal isoform of NO synthase (NOS1), whereas the Wbroblasts in the dermis and othercell types in the skin express the endothelial isoform (NOS3). Under certain conditions, virtually all skin cells appear to be capable ofexpressing the inducible NOS isoform (NOS2). The expression of NOS2 is also strongly implicated in psoriasis and other inXamma-tory skin conditions. Constitutive, low level NO production in the skin seems to play a role in the maintenance of barrier functionand in determining blood Xow rate in the microvasculature. Higher levels of NOS activity, stimulated by ultraviolet (UV) light orskin wounding, initiate other more complex reactions that require the orchestration of various cell types in a variety of spatially andtemporally coordinated sets of responses. The NO liberated following UV irradiation plays a signiWcant role in initiating melanogen-esis, erythema, and immunosuppression. New evidence suggests that it may also be involved in protecting the keratinocytes againstUV-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, UVirradiation and skin wounding, the activation of constitutive NOS proceeds and overlaps with the expression of NOS2. Thus, at amacro-level, at least three diVerent rates of NO production can occur in the skin, which seem to play an important part in organizingthe skin’s unique adaptability and function. 2004 Elsevier Inc. All rights reserved.

Introduction

Evidence of nitric oxide (NO)1 synthesis by humanskin cells was Wrst reported just over 10 years ago [1].Since that time, and from a proposed role in non-speciWchost defense, it is now clear that NO plays a key role inorchestrating the skin’s response to external stimuli such

1089-8603/$ - see front matter 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.niox.2004.04.005

¤ Corresponding author.E-mail address: [email protected] (M.-M. Cals-Grierson).1 Abbreviations used: ADMA, asymmetric dimethylarginine; cGMP, gua

tide; EGF, epidermal growth factor; GSNO, S-nitrosoglutathione; IFN-�, cyte growth factor; L-NAME, N-nitro-L-arginine-methyl-ester; L-NMMAfactor-�B; NO, nitric oxide; NOS, nitric oxide synthase; PKG, protein kinareverse transcriptase-polymerase chain reaction; SNAP, S-nitroso-N-acetylfactor-�; UV, ultraviolet; VEGF, vascular endothelial growth factor.

as heat, ultraviolet (UV) light, response to infection, andwound healing, as well as possibly underlying certainpathological conditions.

The importance of NO-mediated signaling in the skinhas been reviewed by several authors [2–6] although pre-viously the emphasis has often been placed on patho-logic conditions. Here, we review the current literature of

nosine cyclic 3�–5�-monophosphate; CGRP, calcitonin gene-related pep-interferon-�; IL, interleukin; IP3, inositol trisphosphate; KGF, keratino-, N-monomethyl-L-arginine; LPS, lipopolysaccharide; NF-�B, nuclearse G; PGE2, prostaglandin E2; ROS, reactive oxygen species; RT-PCR,penicillamine; TGF, transforming growth factor; TNF�, tumor necrosis

mail to : [email protected]

180 M.-M. Cals-Grierson, A.D. Ormerod / Nitric Oxide 10 (2004) 179–193

NO in the skin and focus on NO-based signaling in nor-mal skin and contrast this with the pathological condi-tions.

Biochemistry of NO

In simple terms, NO is synthesized by the intracellularenzyme, NO synthase (NOS), in a two-step oxidation ofL-arginine, that produces equal parts of citrulline andNO [7–9]. Although, in the literature, it is almost takenfor granted that NOS produces the free radical, NO«, thisis still under debate, since other nitrogen oxide speciescould result from this catalytic process (see [10]).

The three main NOS isoforms currently identiWed are,NOS1, originally isolated from neuronal tissue (alsoknown as nNOS), NOS2 (or iNOS), an inducible iso-form, and NOS3 (or eNOS), predominant in the endo-thelium [10–12]. (An isoform named mtNOS has alsorecently been isolated in mitochondria [13].) They allexist as homodimers, with molecular weights between130 and 160 kDa, and all require the cofactors, Xavindinucleotide, Xavin mononucleotide, tetrahydrobiop-terin, and reduced nicotinamide adenine dinucleotidephosphate. They also require bound calmodulin, butwhereas NOS1 and NOS3 require Ca2+-calmodulin, theNOS2 appears to have lost this calcium dependence.

In addition to requiring these cofactors, the activity ofthe NOS isozymes is regulated by associated proteinsand by their localization inside cells [14,15]. For exam-ple, NOS3 in endothelial cells is regulated in part by itsdistribution between the caveolae (specialized plasmamembrane structures) and intracellular pools, in a pro-cess that involves palmitoylation and the recently char-acterized proteins, nosip and nostrin [16,17]. Furthermore, its activity is also controlled by dynamic associa-tions with regulatory proteins in the caveolae includingcaveolin-1, hsp90, and transmitter receptors, as well asby phosphorylation [18,19].

NOS1 and NOS2 are active as cytoplasmic enzymesand yet could also function to be membrane associatedproteins [15]. NOS1 possesses a PDZ-binding motif withwhich it can interact with a number of other proteins. Inparticular, via the PDZ motif, NOS1 is reported to makestimulatory association with the 5HT2b receptor and aninhibitory association with the calcium ATPase[10,20,21].

The role of endogenous NOS inhibitors in basic skinphysiology is still to be established. The arginine metab-olite, asymmetric dimethylarginine (ADMA), has beenshown to be an important competitive NOS inhibitor incardiovascular physiology [22] and it may also play arole in the skin. Another point of regulation is the supplyof the substrate, arginine, to NOS. Indeed, the competi-tion for arginine by other cellular pathways and thepresence of endogenous NOS inhibitors have beeninvoked to explain the “arginine paradox”; situations

where L-arginine supplementation stimulates NO syn-thesis, despite apparently saturating extracellular argi-nine concentrations [23,24].

NO is highly diVusible and highly reactive. Instead ofactivating downstream pathways via traditional recep-tor-mediated events, it modulates the activity of a num-ber of diverse molecules. Wink and Mitchell [25]classiWed these NO reactions as being either direct (onthe biological mediator) or indirect (involving reactivenitrogen and oxygen species). The direct downstreampathways consist mainly of interactions between NOand heme-containing proteins, the most important beingguanylate cyclase [11,26]. Activation of this enzyme, byNO, induces the production guanosine cyclic 3�–5�-monophosphate (cGMP) which in turn activates proteinkinase G (PKG). This downstream pathway is particu-larly important in mediating the eVects of the low levelsof NO production, which seem to occur with constitutiveNOS activation. The indirect downstream pathways,which become more important under high local concen-trations of NO, involve the formation of nitrogen oxidespecies such as N2O3, HNO (nitroxyl), and ONOO- (per-oxynitrite) [27]. These molecules, in turn, modify thiol-containing proteins, either by nitrosation (N2O3) oroxidation (HNO and ONOO-). The selection of whichindirect downstream pathway is chosen seems to depend,to some extent, on the redox potential of the cell [28–30].

The existence of biological NO donors in the cyto-plasm has been suggested for some time [31,32] but, theidentity and function of these remain unclear. Some ofthe early work of Furchgott and colleagues showed that‘stored’ NO in vascular tissue could be mobilized by UVirradiation to induce NO-dependent smooth musclerelaxation (see [33]). In the lung, a putative endogenousNO donor can induce the S-nitrosylation of 5-HT2receptors [34]. The evidence points to such stores asbeing perhaps S-nitrosoglutathione (GSNO) or a“GSNO-like compound,” although other nitrogen com-pounds may also be involved [33–35]. The existence of aputative NO store in the skin has previously been sug-gested [36,37], but its identity has not been elucidated.Since GSNO is readily formed from N2O3 and glutathi-one [9], it seems likely that GSNO may also represent anNO ‘store’ in skin cells that express NOS.

Control of expression of NOS

The constitutive NOS isoforms are expressed in cer-tain cell types as part of the mature phenotype, althoughit is now clear that the level of expression of theseenzymes can be modulated by circulating hormones. Forexample, the changing estrogen concentrations as foundduring the menstrual cycle and pregnancy can modulatethe expression of NOS1 [38,39], and the expression andactivity of NOS3 [40–42]. The eVect of estrogen on NOSexpression in skin cells is likely to underlie a variety of

M.-M. Cals-Grierson, A.D. Ormerod / Nitric Oxide 10 (2004) 179–193 181

estrogen-based skin reactions such as Xushing andhyperpigmentation [43,44]. UV-B irradiation (290–320 nm) has been reported to upregulate NOS1 mRNAin keratinocytes [45,46], whereas shear stress upregulatesNOS3 [47].

NOS2, on the other hand, is not usually found in nor-mal resting cells, but induced in response to inXamma-tory cytokines or a combination of cytokines andbacterial polysaccharides. Frequently, a synergismbetween diVerent external stimuli is observed, which ismediated by nuclear factor-�B (NF-�B) and the Jak-Statpathways [48]. Thus, NOS2 expression can be induced incultured keratinocytes with combinations of interferon-�(IFN-�)/tumor necrosis factor-� (TNF�), lipopolysac-charide (LPS)/IFN-�, IFN-�/interleukin-8 (IL-8), orTNF�/interleukin-1� (IL-1�) [1,49,50]. It is possible thatthe cocktail of cytokines to which cells are exposed maydetermine the rate and magnitude of NO production[51].

NOS localization in the skin

The skin, the largest organ of the body, is com-posed of three main layers, the epidermis, the dermis,and a sub-dermal layer, or hypodermis. The major celltypes that comprise these layers, including keratino-cytes, Wbroblasts, melanocytes, and endothelial cells,express NOS and appear capable of releasing NO (seeFig. 1).

Keratinocytes

Keratinocytes account for about 90–95% of the cellsin the epidermis [52]. They are formed continuously inthe proliferative, basal layer and undergo a tightly regu-lated process of diVerentiation as they progress upwards

and become part of the corniWed epithelium. This matu-ration process consists essentially of keratinization, butinvolves a number of morphological and metabolicevents as the cells diVerentiate and lose their nuclei.Although a few early reports suggested that keratino-cytes expressed NOS3 [53,54], this was put in doubt fol-lowing further experimentation that suggested perhapspost-translational processing of the NOS proteinallowed a cross-reaction with certain anti-NOS3 anti-bodies [55]. Most of the evidence now indicates thatkeratinocytes constitutively express NOS1. This hasbeen demonstrated using the reverse transcriptase-poly-merase chain reaction (RT-PCR) in unstimulatedhuman and animal keratinocytes and in transformedkeratinocyte cell lines [50,55,56] (Cals-Grierson unpub-lished results). Furthermore, Jackson et al. [55] found noinduction of NOS3 message following the exposure ofkeratinocytes to activating treatments, such as UV-Birradiation or IFN�. One recent study [57] however hasreported NOS3 mRNA expression in human primarykeratinocytes and in the human keratinocyte cell line,HaCaT, but with a very low level of protein transcrip-tion. Keratinocytes are also known to express NOS2message and protein following the exposure to inXam-matory cytokines [50,58] (Cals-Grierson unpublishedresults). It is notable that NOS2 expression is found in anumber of inXammatory skin conditions including pso-riasis, atopic dermatitis, and irritant and allergic contactdermatitis [59–62].

Fibroblasts

The Wbroblasts are the most abundant cell type in thedermis, where their role is to produce the Wbrousextracellular matrix that gives the skin its mechanicalresistance. 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.


express NOS3 [63]. This expression appears to be lowlevel as compared to porcine endocardial cells or humanumbilical endothelial cells and NOS immunocytochem-istry is heterogeneous, with the reaction intensity rang-ing from weak to strong [63,64]. The protein appears tobe localized to the cytoplasm with more intense stainingsometimes observed in the perinuclear area [63].Although Wbroblasts are known to possess calveolae,there is no evidence, as yet, to suggest that NOS3 is pref-erentially localized in these structures.

The expression of NOS2 in Wbroblasts following cyto-kine stimulation has also been shown [63]. Here too, theexpression of NOS2 immunoreactivity is markedly het-erogeneous, with only a minority of cells exhibiting apositive reaction [63,65]. This has been suggested toreXect a diVerence in the maturity of cells [65].

Endothelial cells

Projecting into the dermis from the underlying subcu-taneous layer are the papillary loops of the microvascu-lature [66], whose walls are composed of an endotheliumand a smooth muscle sheath. The endothelial cells ofnormal human skin express NOS3, as shown by Westernblot and immunocytochemistry [60,67]. These cells canalso be induced to express NOS2 mRNA by exposingthem to TNF�, or more potently, a combination ofTNF� and IFN�. Following exposure to these stimuli,NOS2 message is detectable within 6 h and NOS proteinincreases linearly over 72 h [68]. The involvement ofcytokines is not however obligatory, since UV-A (320–400 nm) can induce NOS2 expression in the “absence” ofcytokines [69]. The expression of NOS2 in a constitutivefashion has been observed (immunocytochemically) inthe dermal endothelial cells of patients with atopic orallergic contact dermatitis [60], indicating that, undercertain circumstances, long-term NOS2 expression mayoccur.

Melanocytes

The melanocytes are the pigment-producing, den-dritic cells of the skin, located in the suprabasal layer ofthe epidermis. They respond to UV irradiation bysynthesizing the pigment, melanin, which is stored inspecialized structures called melanosomes. These organ-elles are then transferred from the melanocytes to thesurrounding keratinocytes, producing coloration of theskin. Normal human melanocytes have been shown toexpress NOS3 mRNA [55], although immunoreactive-NOS1 was reported in one study [70]. NOS1 also seemsto be expressed by some malignant melanomas anddysplastic nevi (irregularly shaped and colored moles)[71]. Normal human melanocytes will express NOS2following incubation with LPS, TNF�, and IFN�[72,73].

Adipocytes

Adipocytes are the fat storage cells of the hypodermis.In rodents, these cells express NOS3 and can be acti-vated to express NOS2 [74]. In this cell type, the endoge-nously produced NO appears to be involved in theregulation of lipolysis, since NOS inhibition signiWcantlyreduces lipolysis, via a mechanism of action that involvescytoplasmic oxidation [75].

The development of adipose tissue is determined to alarge extent by circulating sex steroid hormones and inpathophysiological conditions, high levels of these ste-roids can lead to the over-development of adipose tissue.It is interesting to speculate that, considering the inXu-ence of estrogen on NOS, in certain cases of obesityinvolving high estrogen levels, the NO-mediated signal-ing in the adipocyte may be downregulated.

Other skin cell types

The Langerhans cells are antigen-presenting cells(originating in the bone marrow) that take up a positionin the suprabasal layer of the epidermis. It is doubtfulthat these cells express constitutive NOS, although thereis one report of immuno-positive NOS2 staining in Lan-gerhans cells in human neonatal foreskin, in situ [76]. Onthe other hand, no NOS2 expression was observed inunstimulated mouse Langerhans cells, nor in cells acti-vated by LPS or cytokines [77], nor in human psoriaticskin [78].

The outer root sheath in murine skin has beenreported to be immunoreactive for NOS1 [79], whereasthe cells of the arrector pili muscle, apocrine secretorygland, eccrine coiled duct, and a part of the eccrine secre-tory gland are all reported to exhibit NOS3 immunore-activity [80,54]. Dermal papilla cells derived from humanhair follicles have been shown to express NOS3 messageand protein [81]. The sebaceous glands are reported to belacking in NOS immunoreactivity [54], although the less-speciWc diaphorase staining has shown positive results insome species [82].

The role of NO in physiologic responses in the skin

Vasodilatation

The constitutive release of NO by the endothelial cellsof the microvasculature plays an important role in set-ting resting blood Xow rate. Using laser-Doppler Xow-metry to measure blood Xow, Lawrence and Brain [83]reported that the intradermal injection of the NOSinhibitor, L-NAME, signiWcantly reduced Xow rates inrat skin. In 1994, CoVman [84] reported a similar resultfollowing the perfusion of the NOS inhibitor, L-NMMA,into the brachial artery of volunteers and subsequent


experimental modiWcations have continued to supportthese results [85,86].

The vasoconstricting eVect of NOS inhibition isenhanced in locally warmed skin [85], whereas when thewhole body is warmed, intradermal injections of NOSinhibitors have relatively little eVect. This is due to a neu-rally mediated vasodilatation reXex, involving the releaseof calcitonin gene-related peptide (CGRP) as well as NO[85,87–89]. Although CGRP has intrinsic vasodilatatoryactivity, NO release is required for a complete vasodila-tor response [86,89]. Thus, NOS responds rapidly tolocal changes in temperature and neurogenic signalswith an increase in NO production that has a directeVect on the vasculature.

In unstimulated endothelial cells, at normal tempera-tures, it is likely that the primary activator of NOS isblood Xow shear stress [90], although circulating cyto-kines, steroids, and peptide hormones will also play apart [91]. In the response to heat, it is possible that thevanilloid receptor family of cationic channels, whichallow the entry of Ca2+ into the cell, may be important incontrolling the activity of constitutive NOS. Members ofthis heat-sensitive family of channels are expressed byneurons and keratinocytes [92–94].

In conclusion, the constitutive release of NO in theskin is involved in setting the rate of resting blood Xow,via a cGMP-dependent relaxation of the vascularsmooth muscle.

The response to UV irradiation

Irradiation of the skin by UV light induces a varietyof biologically active molecules [95,96]. Some of theseare created photochemically, by the modiWcation of vari-ous proteins, carbohydrates, and lipids, whereas othersare induced or released in consequence. Amongst theimportant diVusible transmitters released by UV-acti-vated keratinocytes are interleukin (IL)-1 [97], IL-6 [98],IL-8 [99], IL-10 [100], TNF-� [101], transforming growthfactor (TGF)-� [102], TGF-�1 [103], prostaglandin E2(PGE2) [104], endothelin-1 [105], and the pro-opiomela-nocortin peptides [106]. UV irradiation also directly acti-vates certain transmembrane receptors, such as theepidermal growth factor (EGF) receptor [107] and thekeratinocyte growth factor (KGF) receptor [108], whichgo on to activate downstream pathways and initiate theproduction of peroxide and reactive oxygen species(ROS).

In 1992, Deliconstantinos and colleagues [109]showed that the irradiation of cultured endothelial cellsby UV-B led to the dose-dependent increase in NO andcGMP. They went on to show that a similar responsecould be evoked in cultured keratinocytes [36] with sig-niWcant elevations in NO and [3H]citrulline; changes thatoccurred within 10 min of the UV irradiation. It thusbecame apparent that the endogenous generation of NO

plays a signiWcant role in the response to UV. Exactlyhow NO is involved within this stimulatory “soup” oftransmitter molecules, to elicit the various physiologicalresponses to UV, is still the subject of a great deal ofresearch projects.

UV-induced erythemaIt is well established that the exposure of skin to UV

irradiation causes erythema. The intensity of the ery-thema is proportional to exposure dose [110] and theonset of erythema occurs after a characteristic delay ofabout 8–24 h [110–112]. In 1993, Warren and colleagues[113] showed that an intradermal injection of L-NAMEprevented this erythema. Even when the injection wasmade 30 min before the predicted onset of erythema(18 h after irradiation in their model), the NOS inhibitorabolished the increase in blood Xow. A comparableresult was found in human volunteers, where the intra-dermal injection of NMMA attenuated the delayedblood Xow increase [114] and intradermal administra-tion of L-NAME produces a long-lasting (24 h) palloraround the UV-B irradiated injection site [80]. Rhodeset al. [115] also found that intradermal injections ofL-NAME were eVective in blocking the erythemicresponse, but also noted that NO acted in concert withPGE2, since the L-NAME-induced block was lost athigh doses of UV-B, when a stronger stimulation ofPGE2 occurs.

From work of Deliconstantinos and co-workers[36,109,116] and others, it has been proposed that UVirradiation augments both the activity and expression ofNOS1 and NOS3 [117,118]. In addition, UV irradiationwas also found to induce the expression (and thereforeactivation) of NOS2. In normal skin, NOS2 is measur-able at about 6 h after UV exposure, with a peak ofexpression at around 24 h and a return to resting levels ofexpression after 72 h [46]. Although UV-A irradiationappears to be suYcient to induce NOS2 expression [69],local cytokine release is likely to contribute signiWcantly[95,119]. The experimental inhibition of constitutive NOSduring the exposure to UV has been found to inhibit theinduction of NOS2 [120]. Therefore, in the early stages ofUV exposure there would appear to be a positive feed-back eVect of NO on NOS2 induction. The rise in intra-cellular calcium that occurs upon UV stimulation [121]appears to be the principal activator of the constitutiveNOS [36]. This calcium Xux is also essential for ROS pro-duction via the UV-activated EGF receptor [122].

While the delay for UV-induced erythema appears tocoincide with the expression of NOS2 [146], this maycoincidental. It is not clear from the work with NOS2-knockout mice if UV-induced erythema has the samedynamic as in wild-type mice.

Thus, the cutaneous production of NO appeared tobe a central component of the delayed-onset erythema.The source of this NO, whether it derives primarily from


constitutive or from inducible NOS, remains howeverunclear.

UV-induced melanogenesisThe evidence of a central role for NO in the induction

of melanogenesis has been apparent since the mid-1990swhen Roméro-Graillet et al. [56,123] found that UV irra-diated cultures of human melanocytes produced NO andthat the presence of NO was suYcient to induce melano-genesis. Their experiments showed that the eVect of UVon their cultures could be mimicked by exogenous NOor an analog of cGMP, and that melanogenesis wasblocked by inhibitors of cGMP-dependent kinase. Laterwork demonstrated that UV irradiated keratinocytesreleased suYcient NO to induce melanogenesis in kerati-nocyte/melanocyte co-cultures [123]. These results sug-gested therefore that NO could act as both an autocrineand paracrine regulator of melanogenesis. In vivo exper-iments on guinea pigs have shown that the topical appli-cation of L-NAME inhibits UV-induced melanogenesis,reducing melanin content and the number of histochem-ically positive melanocytes [124].

In addition to these eVects on melanogenesis, NO hasbeen found to enhance the dendritic branching of mela-nocytes [123] and facilitate the melatonin-induced aggre-gation of melanosomes [125]. Recent research suggeststhat NO may also be involved in setting the eumelanin/pheomelanin ratio in melanocytes, since the ratio ofthese two melanins increases if melanocytes are exposedto NO or histamine [126] and diVerences have also beennoted when melanocytes are cultured in close contactwith keratinocytes, as opposed to pure melanocyte cul-tures [127].

The induction of erythema and melanogenesis by UVirradiation linearly correlated [128], suggesting that simi-lar regulatory pathways are involved. There wouldappear to be a rapid activation of constitutive NOS, fol-lowed by an elevation of NO and cGMP (within 30 s)and other cytokines. At later time points, it is likely thatNOS2 takes on a more important role in maintainingNO production at an elevated level.

The downstream targets of NO in melanocytesremain to be fully elucidated. It is likely however that,via guanylate cyclase and PKG activation, NO plays acentral role in the activation of tyrosinase [123]. NO mayalso be involved in the induction of the tyrosinasemRNA message, which is induced within 2 h of an appli-cation in vitro of an NO donor [129]. Other NO-acti-vated pathways, however, might also be important. Inthe presence of oxygen, NO reacts with the melanin-related metabolites 5,6-dihydroxyindole and its 2-car-boxylic acid (DHICA) resulting in the deposition ofmelanin-like pigments [130]. Evidence from other cellsystems suggests that NO can reduce inositol trisphos-phate (IP3) synthesis [131], and reducing Ca2+ release viamodulation of the IP3 receptor [132,133].

UV-induced immunosuppression

In UV-induced immunosuppression, the acuteexposure of skin to UV irradiation induces a transientsuppression of both contact hypersensitivity anddelayed-type hypersensitivity and a temporally limiteddevelopment of transferable antigen-speciWc suppressorcells. In addition, the Langerhans cells, which areresponsible for antigen presentation, disappear from theepidermis [134]. Strongly implicated in this response isthe release of CGRP from peripheral neurons and cyto-kine stimulation, particularly TNF� and IL-10 [135–137]. However, some evidence suggests that NO might beinvolved in passing a ‘migration signal’ to the Langer-hans cells that stimulates them to migrate [138,139].

The elevated NO synthesis following NOS2 inductionhas a complex eVect on the immune system and in mod-els of skin transplantation, this is often associated withgraft rejection and speciWc NOS2 inhibitors have pro-longed graft survival [140]. It appears, in this situation,that the inhibition of NO enhances the release of Th2cytokines (IL-10 and IL-4) and reduces the release ofTh1 cytokines (IL-2 and INF�), thereby favoring toler-ance [140]. In NOS2-knockout (¡/¡) mice however,rejection occurs in a similar fashion to wild-type graftrecipients [141,142].

InXammation

A large body of work indicates that elevated levels ofNO are pro-inXammatory and many similarities existbetween UV-induced erythema and inXammation. Theexposure of skin to UV irradiation or to chemical irri-tants results in higher levels of NO synthesis and theproduction of pro-inXammatory cytokines [113,143]. Inexperiments involving human volunteers, the applicationof an NO-releasing emulsion to the skin has been shownto evoke local inXammation and other inXammatoryevents, including the loss of Langerhans cells and theinduction of apoptosis in keratinocytes [138]. If, on theother hand, the production of NO in the skin is blocked,then the inXammatory response is lessened. In guinea pigskin, the inXammatory response (edema formation) toan intradermal injection of bradykinin, histamine orplatelet-activation factor, is attenuated by the co-injec-tion of the NOS inhibitor, L-NAME [143], and in NOS2¡/¡ mice, experimentally induced inXammation, usingthe LPS-evoked plasma extravasation model, is reducedas compared to wild-type mice [144].

Normally, inXammation is self-regulating. This is duepartly to NO-induced nitrosylation of NF-�B, whichprevents the transcription factor from binding to theNOS2 promoter [145], but other points of feedback mayalso be involved. A lot of recent research has focused onthe role of ROS and particularly superoxide, which incombination with NO, forms peroxynitrite. This highly


reactive molecule can cause the nitrosation of tyrosine[138], DNA strand breakage, and activation of thepoly(ADP–ribose) polymerase pathway of necrotic celldeath [146]. (It should be pointed out, however, that theaction of myeloperoxidase may be responsible for sig-niWcant tyrosine nitration in inXamed tissue [147].)

When the self-regulation of inXammation breaksdown, severe and sometimes life-threatening dermatosescan occur. The Stevens–Johnson syndrome, an inXam-matory skin condition with toxic epidermal necrolysis, isnotable for its persistent upregulation of NOS2 mRNA[148].

Paradoxically, UV irradiation of inXammatory skinconditions can suppress NOS2 expression [149]. Inexperiments on keratinocytes and macrophages, UV-Birradiation interfered with NF-kB and Stat-1 DNA-binding activity to produce a transient (12 h) inhibitionof IFN�-stimulated NOS2 induction. In keratinocytes,there was a more marked activity of UV-B on phosphor-ylation of MAP kinases, suggesting that this pathwaymay also modulate UV-induced responses.

Apoptosis

The cellular response to intracellular NO concentra-tion increases seems to depend to a signiWcant extent onthe redox potential of the cell, which is itself inXuencedby the resting levels of NO. In human neuroblastomacells, for example, NO induces thioredoxin expression,via a PKG-dependent pathway, which helps to protectthe 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 beendemonstrated by experiments on NOS2 ¡/¡ or NOS3¡/¡ mice, which show signiWcantly higher numbers ofapoptotic skin cells following UV irradiation, than inwild-type mice [153]. The mechanism of this protectiveaction is still uncertain. An induction of Bcl-2 expressionand inhibition of caspase activation have been suggestedby some studies [154], but this fails to explain the rapid-ity of the response. Also evoked is the inhibition of ROS-mediated lipid peroxidation [155] and indeed, lipidperoxidation is a good in vivo measure of UV-inducedoxygen free radical production (a reaction suppressed bythe NO donor, sodium nitroprusside, and enhanced byL-NAME (a NOS inhibitor)) [120]. But, the involvementof a cGMP-mediated pathway (as reported for neuro-blastoma cells) cannot be excluded [153].

The keratinocytes of patients with lupus erythemato-sus have increased susceptibility to apoptosis and theelimination of these apoptotic cells appears impaired,leading to the formation of anti-nuclear antibodies.Kuhn et al. [46] have found that the induction of NOS2by UV irradiation in these patients is delayed as com-pared to normal individuals. Instead of NOS2 message

peaking at 24 h and subsiding on day 3, the lupuspatients exhibited a peak signal on day 3, which per-sisted for 25 days. It is paradoxical therefore that persis-tent NOS2 expression in keratinocytes and endothelialcells is a consistent feature of this disease [156].

Wound healing

NO signaling appears to play a vital role in woundhealing. The positive eVects of arginine supplementationand NO donors, coupled to the negative eVects of NOSinhibitors or the deletion of the NOS2 or NOS3 genes,have provided unassailable evidence of a key role forNO. This has been reviewed recently by Schwentker etal. in this journal [157] and by others [158,159] and willnot be discussed at length. However, a brief examinationof the signaling pathways that seem to be implicated inwound healing does seem to be relevant for an overallappreciation of the eVects of NO in the skin.

The expression of NOS2 is stimulated in wound tis-sue, particularly in the basal keratinocytes adjacent tothe wound [58]. The peak expression occurs after 4–6days [160,161] and it stays elevated for an extendedperiod (at least 3 weeks in the mouse) [160]. This timecourse of NOS2 expression is therefore somewhatretarded and extended as compared with UV-inducedNOS2 activity. It also submaximal, since production canbe further stimulated with combinations of LPS andcytokines [160].

One of the key functions of NO in wound healingseems to be its permissive inXuence on keratinocyte andWbroblast proliferation, which helps promote wound re-epithelialization [162,163]. One study has noted that lowconcentrations (0.01–0.25 mM) of NO stimulate cell divi-sion, whereas high concentrations (10.5 mM) are cyto-static [164]. Interestingly, it seems to be the inXuence ofthe superoxide anion in the cell that determines the mito-genic capacity of the NO signal [165]. Together, theyform peroxynitrite which dose-dependently inhibits pro-liferation. Removal of this anion reduces the inhibitoryeVect.

The likely importance of NO-modulated cytokine sig-naling in the wound healing process has been noted byothers [157] and of particular interest seem to be theNO-induced activation of TGF-b1 and enhancement ofIL-1 and IL-8 production. NO is also known to stimu-late epithelial cells to produce and release chemokines[159] and other growth mediators such as vascular endo-thelial growth factor (VEGF). Key players are also EGFand KGF [166,167]. KGF is released by activated Wbro-blasts (IL-1 activated in particular) and stimulates theproliferation and migration of keratinocytes. In addi-tion, high levels of VEGF are found at the hyperprolifer-ative epithelium of the wound, which appears to beimportant for keratinocyte proliferation and angiogene-sis [168].


As well as having a role in promoting cell prolifera-tion, NO also inXuences the production of collagen byWbroblasts. The inhibition of NOS by competitive inhib-itors has been shown to reduce collagen synthesis [160]and in NOS3-knockout mice, the tensile strength ofregenerating tissue was signiWcantly reduced as com-pared to the wild-type. Primary dermal Wbroblastsobtained from NOS2-knockout mice have been shownto synthesize less collagen compared to wild-type cells,but their production can be augmented by the additionof NO donors [169]. Concordant with this positive eVectof NO, topical estrogen, perhaps acting via a stimulatoryeVect on NOS activity, has been found to accelerate theprocess of wound healing in the aged [170].

Barrier function

The integrity of the corniWed envelope of the epithe-lium confers a barrier against water loss, abrasion, andchemical insult. Intrinsic to the corniWcation process arethe activity of transglutaminase and the induction andcross-linking of the terminal diVerentiation proteins,involucrin and loricrin. NO appears to inhibit this pro-cess via the modifying the thiol groups on transgluta-minase and, via the inhibition of the AP-1 transcriptionfactor, involucrin and loricrin synthesis [171]. We haverecently demonstrated that the topical application of L-NAME attenuates the impairment of barrier functionfollowing an application of the irritant, sodium laurylsulfate, but we were unable to demonstrate an enhancedimpairment (transepidermal water loss) by applicationof an NO donor (Exogenous Dermatology, in press).

An impaired barrier function is typical in dermatitis[172] and other inXammatory dermatoses [173,174] andit is notable that high levels of NOS2 expression arefound in these reactive plaques [50]. Some evidence sug-gests that the resulting elevated production of NO andperoxynitrite formation lead to the activation ofpoly(ADP–ribose) polymerase; an event which seems toinhibit the diVerentiation of keratinocytes and contrib-ute to impaired barrier function [147]. Peroxynitrite for-mation is also implicated in the aggravation of apoptosisfound in other inXammatory conditions, for instance inthe intestine [175]. Interestingly, in vitiligo, a depigment-ing disease of the skin characterized by the early death ofepidermal melanocytes, the loss of melanocytes may becaused by their over-sensitivity to UV-induced oxidativestress [176].

Antimicrobial eVects

The idea that NO may serve as non-speciWc hostdefense has been aired since the early 1990s [1,177] and itis now known that it exerts antimicrobial eVects ondiverse micro-organisms including fungi, yeast, bacteria,viruses, and protozoa [77,178–180]. Thus, as the front

line against the invasion by pathogens, the constitutiveand steady production of NO on the skin’s surface islikely to play an important role.

In 1996, it was shown that NO is produced on the sur-face of human skin by the action of commensal bacteria:the bacteria convert nitrate from sweat to nitrite, and theacidic environment of the skin’s surface releases the NOfrom the nitrite [179]. Earlier work had shown that thereplication of clinically relevant viruses, such as the poxand herpes viruses, was reduced (by about a 1000-fold)by physiological concentrations of NO [178] and it waslater shown that NOS2 ¡/¡ mice were more susceptibleto herpes infection and exhibited a greater frequency ofreactivation than wild-type mice [181]. In patients withpsoriasis, the constitutive induction of NOS2 and ele-vated NO synthesis may be responsible for the relativelyhigh protection against infection seen in this disease[182].

Although infectious pathogens such as Mycobacte-rium leprae (leprosy), Mycobacterium tuberculosis, andherpes zoster virus stimulate NO production (via NOS2)and this seems to limit their progression [183,184], theycan also produce severe inXammation, which, in certaincases, results in epidermal necrolysis and damage toperipheral nerve terminals. Sometimes, therefore, theinduction of NOS2 appears to be uncontrolled andresults in excessive inXammation with serious conse-quences. In an experimental model of pneumonia causedby intranasal herpes simplex, the administration of theNOS inhibitor, L-NMMA, improved survival and pul-monary compliance [185]. The results suggested that theimprovement was mediated by the attenuation of theinXammatory response.

Important targets for the antiviral eVect of NOinclude the inhibition of reverse transcriptase and zincWnger domains necessary for DNA-binding and tran-scription, viral ribonucleotide reductase, and viral enve-lope [186,187].

Pathologic conditions

Skin cancer

The role of NO-mediated signaling in the progressionof skin cancer remains uncertain. Although some evi-dence suggests that elevated NOS expression plays anactive role in progression, the situation is complex andnot all data are in concordance.

In an examination of pigment cell lesions, Ahmed andVan Den Oord [71] found NOS1 expression in benignnevi (moles) and, more frequently, in the basal compo-nent of dysplastic nevi and in primary melanomas dur-ing radial growth. The authors suggested that theenhanced release of NO in these situations may beresponsible for the increased number of blood vessels


observed in the papillary dermis of these lesions. Whenthe same group analyzed NOS2 expression [188], theyobserved some reactivity in nevi and primary melano-mas, but none in metastases. In contrast, Tschugguelet al. [189] did Wnd NOS2 expression in subcutaneousmetastases of melanoma, although they noted that itsexpression was inversely related to the tumor’smetastatic potential. Whereas, Kagoura et al. [190] onWnding that NOS2 immunoreactivity was relativelyweak in well-diVerentiated adenocarcinomas and strong(although heterogeneous) in poorly diVerentiated ones,suggested that NOS2 expression may reXect the degreeof proliferation of the tumor. Thus, a progression inNOS2 immunoreactivity was observed ranging fromBowen’s disease (a pre-cancerous epidermal lesion, 75%positive), through squamous cell carcinomas (75% posi-tive, but more intense), to metastatic carcinomas (80%positive) [190,191]. Interestingly, Fecker et al. [192] werehowever unable to induce NOS2 mRNA in melanomacell lines using combinations of TNF�, INF�, and LPS,whereas it could be induced in cultures of normal mela-nocytes.

Evidence for an angiogenic role for elevated NO pro-duction in experimentally induced tumors has been dem-onstrated. Jenkins et al. [193] found that NOS2transfected human adenocarcinoma cells formed fastergrowing and more vascularized tumors in nude micethan non-transfected, wild-type adenocarcinoma cells.

Clearly the role of NO in skin cancer is complex andthe pathological signiWcance of these Wndings remainsuncertain.

InXammatory skin disease

The expression of NOS2 in inXammatory lesions ofthe skin is expected and well established [194]. In lupuserythematosus, NOS2 expression is present throughoutthe epidermis [114]. High levels of NOS2 are also foundin patients with Sjogren’s syndrome (an autoimmunedisorder in which immune cells attack and destroy theglands that produce tears and saliva) who experiencephoto-aggravated erythemic reactions [195]. What is lessclear is the role of NO in persistent cutaneous inXamma-tion and why there is no self-regulatory feedback.

As in other inXammatory lesions where superoxide ispresent, NO forms peroxynitrite, which nitrosylates thiolgroups [196], leading to DNA strand breakage and acti-vation of the poly(ADP–ribose) polymerase pathway ofnecrotic cell death, as shown in a model of contacthypersensitivity in the mouse [146]. In psoriasis, theover-expression of NOS2 is also associated with anincreased, perhaps compensatory, arginase 1 activity,which may reduce the available substrate for NO pro-duction, has led to a hypothesis that NO productionmay be inadequate in these situations [24]. Supplementa-tion of NO with GSNO has recently been shown to

reduce the expression of inXammatory markers and inWl-tration of T cell [197]. Direct measurements of NO pro-duction in psoriatic lesions however have not found anyevidence of competitive NOS2 inhibition [194].

Conclusions

From this review of NO signaling in the skin, it can beappreciated that this messenger is strongly implicated ina number of diverse responses. This raises the question ofwhy there is not more evidence of signaling cross-talkwhen NO stimulates inappropriate targets. For example,hyperpigmentation following wounding or skin irrita-tion, or angiogenesis following UV irradiation can occur,but they are relatively rare. A more common examplemight be the pigmentation plaques of melasma that canappear following sun exposure and elevated plasmaestrogen. It is likely that the answer lies the repertoire ofcytokines and other intercellular messengers that areinduced and their spatial and temporal distributions.

Thus, is NO merely a facilitatory messenger? A useful,but an otherwise non-essential, component of the signal-ing pathways? It would certainly appear from the geneknockout experiments that the skin is remarkably robustin terms of physiological responses. However, thisapparent redundancy of multiple NOS isoforms is alsofound in diverse animal phyla and classes. In mammals,it would appear that there are at least two NOSexpressed in normal skin, plus another for “crisis man-agement” allowing for substantial compensation andoverlap of function.

The therapeutic potential of delivering NO to the skinis being explored by a number of laboratories, using, forexample, glyceryl trinitrate and S-nitroso-N-acetylpeni-cillamine (SNAP) for anal Wssures or cutaneous leish-maniasis [198–200], topical diazeniumdiolates [201] orthe co-application of a mild acid and nitrite, which canliberate NO in a controlled fashion [202,203]. This lattermethod has been shown to have therapeutic eYcacy indermatophyte fungal tinea infections, viral infectionsmolluscum contagiosum, viral warts, and in vitro showsactivity against bacterial pathogens, including the acnebacillus and Staphylococcus aureus [202–205]. This tech-nology has been investigated in treating the vasospasmof Raynaud’s phenomenon [206]. A sympatex membranecan be incorporated for more selective NO deliverywhich was eVective in killing Escherichia coli and S.aureus [207]. A topical polymer-based NONOate NOdonor improved wound closure in rats [208] and a poly-vinyl alcohol hydrogel NO donor has been tested inwound healing in diabetic animals, which resulted inmore rapid initial healing and enhanced extracellularmatrix production [209]. Lastly, nitroglycerine adminis-tration has been shown to reduce phorbol ester-inducedcarcinogenesis in a mouse model, with a 32% inhibition


of tumorigenesis [210], suggesting that, far from beingmutagens, topical NO donors may protect against skincancer and protect cells from the harmful eVects of ultra-violet irradiation.

It is expected that with better understanding of theprecise role of NO in the skin and with advancements inthe controlled topical delivery of NO, new treatments forsome dermatological conditions may be possible as wellas perhaps, one day, a control of true tanning of the skinwithout the need for exposure to UV light.

Acknowledgments

The authors thank Dr J. P. Grierson for helpful dis-cussion and editorial assistance.

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