the topical glucocorticoids and the skin–mechanisms of action:...

Topical glucocorticoids and theskin–mechanisms of action: anupdate

A. Ahluwalia

Centre for Clinical Pharmacology, University CollegeLondon, The Cruciform Project, The Rayne Institute,5 University Street, London WC1E 6JJ, UK

Tel: +44(0)171 209 6614Fax: +44(0)171 813 2846Email: [email protected]

THE topical glucocorticoids (GCs) represent the tr eat-m ent of choice for m any types of inflam m atoryde rm atose s . Despite th e ex ten sive use of this c lass ofdrugs as firs t line th erapy th e m echanism of the iraction is uncer tain . It is c lear that the m ultiplicity ofactions of the topical GCs is an im portant facet ofthe ir scope in th e tr eatm ent of derm al disorders . Theaim of th is update is to review past and curren ttheorie s r egarding how th ese agen ts m igh t work.Curren t understanding of th e m olecular m echanism sof GC action has advanced s ign ifican tly ove r th e pastdecade w ith the realisation th at m ultiple system s areresponsible for transduction of GC effects at a m olec-ular leve l. Th e tw o prim ary m odes of action are v iain te raction directly w ith DNA or indirectly th roughm odulation of specific trans crip tion factors : the end-poin t in both cases be ing m odulation of specificprotein synth esis . Both of th es e m echan ism s w ill bediscus sed. In particular th is r evie w w ill concen trateon the poss ibility that a GC-inducible prote in , term edlipocortin 1, m ay have a s ign ifican t role to play in thean ti-inflam m atory actions of the se drugs . Addition-ally it has becom e apparen t that several in flam m atoryenzym es induced in in flam m ation are s ites of in hibi-tory action of th e GCs, and the poss ibility that th isoccurs in th e skin w ill be dis cussed paying par ticularatten tion to th e in ducible phospholipase A2 , nitr icox ide synth ase and cycloox ygenase system s .

Key w ords : hipocortin 1, annexin 1, dexamethasone,inflammation, nitric oxide synthase, cyclooxygenase, phos-pholipase A2

Introduction

Topical glucocorticoid (GC) treatment of inflamma-tory disease was first recognized as a viable mode oftherapy by the ophthalmologists. These specialistswere quick to realize the potential of the GCs ininflammatory ocular conditions following Hench’sdescription in 1949 of his, Noble Prize winning,studies demonstrating the anti-inflammatory effects ofsteroid treatment in rheumatoid arthritis.1 The derma-tologists followed suit confirming the efficacy ofthese agents in skin disease. These rather humblebeginnings initiated the ensuing revolutionary chan-ges in the treatment of inflammatory skin conditionscommencing with the first, albeit unsuccessful, trialswith topical cortisone in 19502 and 1951.3 Howeversoon after, in 1952, Sulzberger and Witten publishedthe first well-controlled trial demonstrating the clear-cut benefits of topical hydrocortisone in patients w ithinflammatory dermatological conditions.4 The 1960swere the golden era for the GCs with the introductionof the first synthetic congeners (e.g. prednisolone)into the clinic, followed soon after by the fluorinated

derivatives (such as dexamethasone) possessing muchincreased potency. The unmatched efficacy of theseagents when treating skin disease has in part resultedin the neglect afforded to understanding the exactmechanisms of action of the topical GCs. Theexistence of a facile assay system for estimation ofpotency, termed the vasoconstrictor assay (describedlater), aided and abetted this situation. More recently,however, understanding of the mechanisms of steroidaction, in systems other than the skin, has advancedsignificantly and several groups are now pursuing thisnewly gained knowledge with respect to the skin.This article reviews the past and present theories ofthe mechanism of action of the glucocorticoids withspecific reference to their use in the treatment of skindisease.

Mechanism of ActionThe GCs have a multiplicity of actions; anti-inflamma-tory, immunomodulatory, vasoconstrictor, gluconeo-genic, anti-mitotic to name a few (see Table 1). It isbelieved that several of these actions contribute to the

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Invited Review

Mediators of Inflammation, 7, 183–193 (1998)

therapeutic efficacy of these drugs in the treatment ofskin disease. Indeed it is often this multi-prongedattack that endows the GCs with the considerablygreater therapeutic potency above other modes oftreatment. On the downside however it is thisplethora of disparate effects that may preclude theiruse, since to date it has not been possible to separatethe required actions of the GCs from those that causesignificant unwanted side-effects. Due to the diversityof conditions that the GCs may be used to treat it isimportant to appreciate that any particular action of aGC may be beneficial in the treatment of one diseaseyet responsible for a confounding side effect inanother. For instance, the anti-mitotic nature of GCs isthe property upon which their use in psoriasis isbased,5 yet in the treatment of other inflammatorydermatoses results in skin atrophy and may often bethe reason for cessation of topical GC treatment.

Topical GCs are prescribed for the treatment ofmany inflammatory conditions and today representthe drug type of choice for the treatment of mostdermal inflammatory disease (Table 2). This is not tosay that the topical GCs are a panacea for all skindisease, but perhaps may be regarded as the mosteffective single class of drugs providing such broadspectrum therapeutic benefit. The anti-inflammatoryaction of the topical GCs has variously been attributedto several different mechanisms, and it is now clearthat it is a combination of the different properties ofthe GCs that act in concert to provide the therapeuticadvantages of this class of drugs in the treatment ofinflammatory skin disease. Additionally both the anti-mitotic and vasoconstrictor nature of the GCs areproperties which, most likely, contribute to resolu-tion of certain inflammatory skin disease. The anti-

mitotic effect is believed to confer the beneficialeffects of topical GCs in the treatment of psoriasis, adisease characterized by a high cell turnover rate (forreview see Van De Kerkhof and Van Erp6). Thevascular effect i.e. vasoconstriction, also termed‘blanching’ when applied to the skin surface, hasbeen suggested to contribute to the anti-inflammatoryeffects of these drugs by virtue of the decreased bloodflow to the inflamed site, however the mechanism forthis effect is still unclear (for review see Walter andWilliams7). Additionally it is this blanching responsewhich forms the basis of the standard assay for theassessment of the potency of topical GCs.8,9 Theimmunosuppressive nature of these agents also con-fer benefit in treatment of dermal diseases and indeedexperimentally it is clear that steroids block delayedhypersensitivity responses in the skin.

Molecular mechanism of action

Topica l GCs interact with GC receptorThe specific biological actions of the topical steroidsare brought about by interaction with the GCreceptor. In the 1960s and 1970s several studiesprovided circumstantial evidence supporting theconcept of steroid binding sites in skin by demonstra-tion, for example, of [14C]-cortisol retention in thestratum corneum10 and identification of solubleproteins with high affinity for certain GCs in rat skinhomogenates.11 It was in 1971 that definitive evi-dence for the ex istence of these receptors in mouseskin was obtained,12 followed by confirmation ofthese receptors in humans, firstly in cultured humankeratinocytes and langerhan cells and then humanepidermis.13 –15

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184 Mediators of Inflammation · Vol 7 · 1998

Table 1. Mechanisms of anti-inflammatory action of thetopical glucocorticoids

Action Effect

Inhibition ofphospholipase A2 activity

Decreased production oflipid mediators(prostaglandins,leukotrienes, platelet-activating factor)

Inhibition ofcyclooygenase induction

Decreased prostaglandinproduction

Inhibition of nitric oxidesynthase induction

Decreased nitric oxideproduction

Inhibition of cytokineproduction

Suppression of cell-mediated inflammation

Inhibition of mast cellactivity and reduction ofmast cell number

Decreased levels of mastcell inflammatorymediators (histamine,5-HT)

Vasoconstriction Decreased local bloodflow

Table 2. Inflammatory dermal diseases treated with topicalglucocorticoids

Level of treatment Disease

Treatment of choice EczemaContact dermatitisAtopic dermatitis

Lichen planusLichen simplex chronicus

Chronic dermatitisNeurodermatitis

Insect and arthropod bitesBurns and sunburnsKeloids

Useful alternative oradjunctive therapy

PsoriasisSeborrhoeic dermatitisChronic lupuserythematosusAlopecia areataAcne

Isolated examples ofsuccessful treatment

Bullous pemphigoidCutaneous mastocytosisVitiligo

The subcellular localization of the GC receptor is amatter of debate. Early immunocytochemical studiesconcluded that the GR in the unoccupied stateremains primarily in the cytoplasm and it is only uponoccupation with a GC, to form a GC-receptorcomplex (GC-R), that it translocates to thenucleus.16,17 This pattern of activity is quite differentfrom all of the other members of the steroid receptorfamily i.e. oestrogen, progesterone and androgenreceptors, however more recently it has been demon-strated that the mineralocorticoid receptor functionsin a similar manner.18 In contrast some studies suggestthat the receptor is solely nuclear,19,20 and the authorsin these cases attribute this apparent difference inlocalization to immunocytochemical procedure.Recent studies agree with cytoplasmic alocalization ofthe receptor but in association with the microtubulenetwork (for review see Akner et a l.21). This associa-tion with cytoskeletal filaments may explain how theGC on binding to the receptor is transported to thenucleus. In contrast some studies show no associationof the cytosolic receptor with microtubular fila-ments.22 The prevailing belief at the present time ishowever that the receptor is cytoplasmic and trans-locates to the nucleus once bound to GC.

Specific GC functionality is conferred by binding tothe receptor and this has been demonstrated for manyof those effects which are anti-inflammatory. Forexample both oestradiol and testosterone, full GCreceptor antagonists, were shown to block theblanching activity of clobetasol-propionate in thehuman forearm.23 This was supported by later studieswith the GC receptor antagonist, RU38486, whereprior systemic treatment of human volunteers with theantagonist completely blocked the blanching responseto topically applied clobetasol-propionate.24

GC-R complex activity(i) Interactions with DNA. Classically the mechanismof action of the GCs was thought to be brought aboutby changes in gene expression. It is now clear,however, that there are two modes of action of theactivated GC receptor; those via interactions directlywith DNA and those through protein–protein inter-actions w ith transcription factors. The end-point ineach case is the same i.e. the alteration of thesynthesis of key proteins.

Once the GC crosses the cell membrane it isthought that it binds with cytoplasmic receptor (R) toform a GC-R complex. The unliganded receptor is aheterotetrameric complex comprised of heat-shockproteins (hsp), hsp70 and hsp90, and chaperoneimmunophilins (for reviews see Pralt25 and Smith andToft26). The sequelae following GC-R complex bind-ing with DNA have been characterized and it isthought that interaction with genomic DNA accountsfor most GC-induced effects (for a recent review onGC-receptor binding mechanisms see Brann et a l.27).

Briefly GC-receptor binding causes a conformationalchange of the receptor with consequent shedding ofthe DNA-binding domain capping protein, hsp90.28,29

Exposure of the DNA-binding site allows binding ofthe GC-R complex to the GC response element (GRE).This interaction stimulates alterations in transcription,either positively or negatively, and thereby translationof proteins. One particular anti-inflammatory proteinthat is induced in this way is lipocortin 1 (LC1), alsoknown as annexin 1, a member of the annexinfamily.30 This protein has attracted considerable inter-est as an important mediator of the anti-inflammatoryaction of the GCs and this review will highlight theactivity of this protein in particular.

(ii) Pro tein –protein interactions . Whilst a con-siderable component of GC-R complex activity hasbeen attributed to interaction with GREs, generepression may also be modulated indirectly byprotein–protein interactions between the activatedGR and other transcription factors thereby alteringprotein synthesis without binding to GREs. Of partic-ular interest is the inhibitory effect of GCs on twoessential regulatory transcription factors for severalgenes involved in the inflammatory process; AP-1 andNFk B. These factors regulate the expression ofinflammatory cytokines and adhesion molecules andindeed inhibition of the activity of these transcriptionfactors has profound effects on inflammatory andimmune responses.31,32

Whilst it seems clear that the GC-R complex bindsdirectly with AP-1, thereby blocking AP-1-dependentgene expression,33 the mode of interference withNFk B is less clear. NFk B exists constitutively in thecytosol as a heterodimer (p50 and p65 subunits), andis maintained in an inactive form by association withIk B.34 Exposure to inflammatory stimuli results inrapid degradation of Ik B allowing free NFk B totranslocate to the nucleus where it binds w ithpromoter elements on certain inflammatory genes.Originally it was thought that the GC-R complex bindsdirectly with NFk B, however in 1995 it was shown, ina cultured monocytic T-cell hybridoma cell line, thatthe activated receptor may decrease free NFk B levelsby upregulating Ik B transcription.35,36 Increased Ik B,in turn, would sequester NFk B thereby depressing thelevels of free NFk B available for binding to targetgenes. This theory however has not been uniformlyaccepted with studies, in primary endothelial cellcultures, showing that GC-induced repression ofNFk B is not related to elevations of Ik B but rather todirect binding of the activated GC receptor toNFk B.37 The complication of the latter mechanism isthat once GC-R complexes have been formed thecomplex is rapidly translocated to the nucleus whilstthe protein–protein interaction, proposed betweenNFk B and the GC-R complex presumably must becytoplasmic. Studies in the adenocarcinoma A549 cellline suggest that in the absence of an inflammatory

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Mediators of Inflammation · Vol 7 · 1998 185

stimulus this classical translocation does take place,however in the presence of an inflammatory cyto-kine, such as tumour necrosis factor alpha (TNFa ),GC-R complexes remain within the cytoplasm wherethey are then available for interaction with transcrip-tion factors.38 How this retention in the cytoplasm isbrought about is unclear but the authors indicate thatsuch a mechanism would allow cytoplasmic protein–protein interaction.

Without doubt the modulation of NFk B by the GCsrepresents an important potential mechanism of anti-inflammatory action. Interpretation of studies demon-strating GC-induced suppression of NFk B levels arecomplicated by the fact that, in the large part, theconcentrations of GC used for these studies have beenhigh (0.1–1 m M). Whether these concentrations invivo relate to those achieved during treatment39 isuncertain. Additionally other studies show that thenon-steroidal anti-inflammatory drugs (NSAIDs) alsohave the capacity to effectively suppress NFk Blevels.40 Since there is an enormous disparity betweenthe effects of the GCs and the effects of the NSAIDs thissuggests that mechanisms other than protein–proteininteractions are also important in mediating the effectsof the GCs at a molecular level.

Inhibition of pro-inflammatory enzyme activity

The inflammatory response in the skin involvesseveral soluble mediators. The prostanoids and nitricoxide (NO) represent two important groups ofinflammatory mediator41 that have attracted consider-able attention as sites of anti-inflammatory action ofthe GCs. The pathways involved in the production ofthese two types of mediator are known to be prone toinhibition by the GCs and it is now clear thatdecreased production of either of these class ofmediators has a significant impact on many aspects ofan inflammatory response.41 With respect to theprostanoids the GCs suppress the induction ofphospholipase A2 (PLA2) and cyclooxygenase-2 (COX-2), both enzymes involved in the phospholipid-arachidonate cascade (see Fig. 1). Importantly inhibi-tion of PLA2 activity w ill not only suppress prostanoidlevels but will also attenuate production of theleukotrienes and platelet-activating factor; otherimportant mediators of inflammation. Similarly it isthe synthesis of NO that is targeted by the GCs i.e.inhibition of NO synthase induction, an enzymeimperative for NO synthesis during inflammation.41

Overproduction of either prostanoids or NO isbrought about by the induction of certain inducibleenzymes that belong to the class of early responsegenes. Interestingly it seems that at a molecular levelit is the early response genes which are particularlysensitive to GC action. These mechanisms of GCaction will be discussed below with particular refer-ence to their action in skin.

Phospho lipa se A2 (PLA2 )The first significant studies attempting to determinethe site of antiinflammatory action of the GCspinpointed the enzyme PLA2 as the most probablesite. PLA2 activity results in conversion of membranephospholipids into arachidonic acid and lysoPAF.Arachidonic acid is the substrate for both COX-1 andCOX-2 enzymes, the activities of which producevarious prostanoids some of which possess inflamma-tory activity. It was in 1974 that the first reportdemonstrating that hydrocortisone reduced the out-put of prostaglandin (PG) E-like substances, in exer-cised dogs, was published.42 Soon after this initialobservation it became clear that this decreasedprostanoid production was a consequence of sup-pressed arachidonic acid release, implicating PLA2 asthe site of GC action rather than direct inhibition ofCOX activity.43,44 This appears to be true for topicalGCs in the skin since abnormally high levels ofarachidonic acid, in psoriatic plaques, are reduced bytopical GC treatment.45,46 Furthermore PLA2 activityin psoriatic plaque samples is suppressed in thosepatients previously treated with topical GC.47 It haseven been proposed that measurement of PLA2

activity in psoriatics could efficiently form the basis ofa model for the assessment of topical GC potency. Inlight of this evidence it was suggested that a

A. Ahluwalia


FIG. 1. The lipid mediator cascade.

reduction, by the GCs, of the elevated levels of PLA2

was primarily responsible for the benefits of thistreatment in disease such as psoriasis and eczema.48

Whilst it is clear that GC treatment attenuates theabnormally high levels of arachidonate products inpsoriatic skin it remains unclear as to what eventcomes first; decreases in the levels of these sub-stances and consequently plaque resolution or plaqueclearance followed by normalization of prostanoidlevels.

The mechanism of GC-induced inhibition of PLA2

was first unravelled by the pharmacologists usingclassical bioassay techniques, in this case the guinea-pig perfused isolated lung preparation. The guinea-piglung can be stimulated to release prostanoids byperfusion with either the substrate, arachidonic acid,or by other substances such as histamine or bradyki-nin. Using these techniques a series of elegantexperiments demonstrated that the GC, dexametha-sone, inhibited prostanoid release induced by theprostanoid-releasing substances but not by addition ofthe substrate, and most importantly that this processrequired de novo protein synthesis.49,50 Soon afterthese seminal experiments the protein responsible forthis effect was identified by several independentgroups simultaneously. In 1984 these groups cametogether and proposed a common nomenclature forthe protein, lipocortin 1 (LC1).51 Since this timeseveral different groups have demonstrated the anti-inflammatory potential of this protein in pathologicalsituations. Additionally the role of this protein as aphysiological regulator was proposed in 1992 byVishwanath et a l.52 This group showed that the levelsof both LC1 mRNA and protein were significantlydecreased in adrenalectomized rats. From these find-ings this group proposed that LC1 may be animportant defence mechanism and decreases in thelevels of this protein may predispose to enhancedinflammatory or immunological processes. In muchthe same way as the GCs the mechanism of action ofLC1 was believed initially to be solely via inhibition ofPLA2 , however recent advances suggest that thisinducible protein possesses multiple sites of action ininflammation which are both cellular and non-cellular(for review see Perretti53).

LC1 was thought to inhibit conversion of mem-brane phospholipids to arachidonic acid by a directaction on PLA2 and indeed there are recent studieswith porcine splenic PLA2 which concur with thisproposition.54 More recently however this theory hascome under considerable scrutiny. In vitro studiesusing Escherichia co li cells or porcine pancreaticPLA2 show clearly that LC1 inhibits PLA2 activity,55

however this effect was not due to direct bindingwith the enzyme but rather by binding to thephospholipid substrate. This results in depletion ofsubstrate availability and hence apparent decreasedPLA2 activity. Studies measuring PLA2 activity in full

thickness human skin homogenates agree with thesefindings showing that LC1-induced inhibition of PLA2

activity is a function of substrate concentration andmoreover, that LC1 competes with PLA2 for phospho-lipid substrate.56 In contrast recent studies suggestthat phosphorylation of PLA2 precedes and procuresenzyme activation and further that this phosphoryla-tion is sensitive to dexamethasone in a LC1-depend-ent manner.57 Finally the GCs may alter PLA2 expres-sion itself by an effect at the level of the gene whichpossesses a GRE (e.g. Schalkwijk et a l.58). Whateverthe mechanism of the induced decrease in conversionof membrane phospholipids into arachidonic acid theend-point remains the same i.e. resultant decrease inarachidonate products. Up till now there have beenno direct studies either using epidermal cells inculture or in vivo to demonstrate directly that LC1, orany other lipocortins, alter the levels of arachidonateproducts in the skin and are likewise associated witha resolution of the disease.

(i) Lipo co rtin 1 in skinIn 1989 Fava and co-workers were the first to identifybasally expressed LC1 in rat, porcine and humanskin.59 The exact cellular location of this proteinremains somewhat unclear w ith contradictory resultsfrom several different groups. Using antibodies gen-erated by their group Fava showed, immunohis-tochemically, that LC1 could be found in discreteareas. The protein was situated predominantly in basalkeratinocytes of the basement membrane. Staining forLC1 was also present in the epithelium of sweatglands in pig and human skin. Additionally, in humanskin, there was an intense staining in the epithelialcells of hair follicles. However other studies haveidentified a different cellular localization for LC1 inthe skin. For instance Kitajima’s study in 1991localized basal LC1 expression to the suprabasal layerof human skin, predominantly keratinocyte.60 It hasbeen suggested that these differences between stud-ies may reflect binding patterns of antibodies gen-erated against different epitopes of the LC1 moleculeand suggest that LC1 may adopt different intercellularconformational states.

The intracellular location of LC1 is also con-troversial. Indeed it is now believed that there aremultiple pools of LC1 with only certain compart-ments conferring the biological activity of this pro-tein. Figure 2 shows a schematic of the different poolsof LC1 which have been identified. In the skin LC1was found within the cytoplasm of epidermal cells invitro ,60 an observation supported more recently bystudies in human skin biopsies, where LC1 was foundin the cytoplasm of cells of the upper and middleepidermal layers.61 Of interest is the demonstrationthat in disease the intracellular localization of LC1appears to change. In lesional psoriatic skin LC1 isfound only in the cell membrane with no staining in



the cytoplasm, demonstrating an apparent transloca-tion of the protein.60,62 It is possible that themovement of LC1 to the cell membrane is an activeprotective response of the cell in disease and, assuggested by the authors, may occur to allow thebinding of LC1 to phospholipids thereby decreasinginflammatory prostanoid production. However themovement of LC1 was also associated with increasesin the EGF receptor/tyrosine kinase, for which LC1 isa substrate, and therefore it could be that the netavailability of LC1 is in fact reduced. All of the studiesmentioned so far have concentrated on the expres-sion of basal LC1 in skin and its possible alteration inskin disease. We however addressed the concept thatLC1 may be a mediator of the anti-inflammatoryeffects of topical GC therapy.

(ii) LCI expres s ion in skin is GC-sens itive . Studiesin rat have clearly demonstrated that topical GCtreatment of the skin results in alterations in LC1expression. Although measurement of the total LC1content in skin homogenates showed no differencesbetween GC-treated and vehicle-treated skin, exam-ination of the pericellular compartment of LC1 usingWestern blotting, demonstrated that a pericellularpool of LC1 increases following topical GC treatment(betamethasone-17-valerate 0.018 mg/cm2 in ace-tone).63 Figure 2 shows schematic representation ofthe pools of LC1 thought to exist in the cell. Recentlyit has been suggested that the pericellular compart-ment of LC1 provides the biological activity of thisprotein. This proposal is supported by the fact thatinduced LC1 expression, in vivo , may be neutralizedwith antibody generated against this protein.64,65

Indeed pericellular LC1 expression, induced in ratskin, increased to a maximum at approximately 3 h

following topical treatment returning to basal levelsby 18 h. This alteration of LC1 levels was alsotemporally associated with anti-inflammatory activitywith approximately 50% inhibition of oedema pro-duced in response to a range of putative mediators ofinflammation.66 This change in LC1 levels was aspecific effect of the GC since pretreatment ofanimals with the selective GC receptor antagonist,RU38486, prevented these differences. The situationin man is somewhat similar with an increase in LC1expression in the pericellular compartment over asimilar time course (A. Ahluwalia and R. J. Flower,unpublished observations). Unlike these studies oth-ers have found no increase in total LC1 levelsfollowing GC treatment of epidermal cells in vitro .67

This group showed that 16 h following treatment ofcells with dexamethasone there was a significantdecrease in the total expression of LC1 in both thecytosol and membrane of these cells.67 Howeverthese studies looked neither at shorter time pointsfollowing treatment nor at the different compart-ments independently. If there had been an increase inthe expression of the protein pericellularly it may bethat this increase is achieved via the translocation ofLC1 from either the intracellular or membranouscompartments without necessarily a change in totalLC1 content. This of course in turn would result in adecrease in the LC1 levels in the latter compartmentswhich may explain the decreases in the protein seenin the epidermal cells at the 16 h timepoint. In A549adenocarcinoma cells pericellular LC1 expressionincreased following dexamethasone treatment, reach-ing a maximum after 2 h.64 Recently this same grouphave shown, by measuring incorporation of labelledamino acid, that following GC treatment new protein

A. Ahluwalia


FIG. 2. Cellular distribution of distinct compartments of lipocortin 1.

synthesized by the cells was specifically directed tothe cell surface in contrast with incorporation intothe intracellular compartment in the absence ofGC.68

In a separate series of investigations the involve-ment of induced LC1 in procuring the effects oftopical GC treatment in vivo have also been investi-gated. Our group have shown that the same topicalGC regimen, as used in the above studies, resulted inan inhibition of the oedema produced in response toneurogenic stimulation; electrical stimulation of thesaphenous nerve. Furthermore prior LC1 immuno-neutralization of rats, by systemic treatment w ithantibody, partially inhibited the anti-oedema effects ofthe GC.69 In another model of skin inflammationusing intradermal administration of various putativemediators of inflammation, including platelet activat-ing factor and 5-hydroxytryptamine, the inhibitoryeffects of topical GC treatment was also reversed byprior immunoneutralization with LC1 antibody.66

These results together suggest that at least some ofthe anti-inflammatory effects of the topical GCs in theskin are brought about by the GC-inducible proteinLC1.

The beneficial effects of the topical GCs in skindisease are not however restricted solely to their anti-inflammatory properties. The anti-mitotic nature ofthese agents has been proposed to provide benefits inpsoriasis where the cell turnover rate of the skin isconsiderably elevated above basal. Although there arenow topical formulations believed to have little effecton cell turnover but retaining significant anti-inflam-matory activity such as fluticasone.70 Studies innormal skin showed that topical GC treatmentresulted in a significant decrease in epidermal mitosesin comparison with that in vehicle controls71 andsimilar findings have been shown in psoriatic skin.72

Dexamethasone has profound anti-proliferativeeffects on the A549 cell line and moreover theseeffects are associated with increases in LC1 andblocked by pretreatment of the cells with specificneutralizing antiserum to LC1.64 Studies using thehairless mouse comparing the mitotic index and3H-thymidine uptake in normal and GC-treated skinsuggest that the effect of the GC may be a non-specificcell cycle effect or at the G1 interphase period.73 Ona more speculative note it would be interesting todetermine whether the anti-mitotic effects of thetopical GCs are brought about by LC1. This may thenidentify novel therapeutic opportunities for the treat-ment of psoriasis and importantly possibly identifythe mechanism of skin thinning, a significant side-effect of topical GC treatment.

Cyclooxygenase (COX)Over the past decade there has been a growing bodyof evidence demonstrating that the GCs alter prosta-noid synthesis by modulating the pathway at several

levels. Studies in various cells types in vitro , includinghuman dermal fibroblasts, have shown that the GCdexamethasone inhibits cytokine-stimulated COX syn-thesis.74 Whilst early studies demonstrated clearly thatGCs had no effect on normal eicosanoid synthesis ineither mice or man, other studies show that insituations where expression of COX has been upregu-lated, such as following treatment with pro-inflamma-tory substances including cytokines andlipopolysaccharide, prostanoid synthesis becomessensitive to GC treatment.75 These differences whilstinitially puzzling have been explained with thediscovery that there are two isoforms of this enzyme;the constitutively expressed enzyme found in mostcell types called COX-1, and an inducible form foundin inflammatory cell types called COX-2.76 The COX-1and COX-2 genes have now been cloned and showapproximately 60% homology.77–79 It appears thatwhilst the induction of COX-2 is sensitive to GCtreatment the expression of COX-1 is not (seeHerschmann79 for review). Furthermore GC-induceddecreases in COX-2 expression is due to a decrease inthe stability of the mRNA rather than an effect ontranscription per se.80,81

In vitro it is clear that it is the up-regulatedprostanoid synthesis stimulated by COX-2 that is GC-sensitive. In vivo animal studies, however haveuncovered the fact that endogenous GCs are impor-tant regulators of basal prostanoid production sincethe lethal response to LPS in animals is more severe inadrenalectomized animals.82,83 Additionally adrena-lectomy of mice results in substantial increases inCOX expression and prostanoid production in perito-neal macrophages, that is associated with anincreased lethality.84 Later studies showed that thisenhanced COX expression was of the COX-2 iso-form85 suggesting that constitutive expression ofCOX-2 in murine peritoneal macrophages is tonicallyregulated by endogenous glucocorticoid.

Not all studies are consistent with the hypothesisthat GCs downregulate COX activity and in somecases it seems that GC treatment has no effect orconversely elevates prostanoid production (forreview see Duval and Freyss-Beguin86). In murinebone-marrow-derived mast cells dexamethsone selec-tively induces COX-1 expression with consequentelevation of PG formation.87 Whilst this potentiatoryeffect may at first appear contradictory on closerinspection this elevation may be advantageous to theorganism since in certain situations PGE2 , at least, isanti-inflammatory. In rat and murine skin in vivo ,PGE2 inhibits oedema formation induced by inflam-matory stimuli including platelet-activating factor andzymosan activated serum.88 The authors demon-strated that this inhibitory effect of the PGs ismediated by the prostanoid EP3 receptor. Support forthese findings in man show that misoprostol, theselective EP3 receptor agonist, inhibits antigen-



induced cutaneous late response in atopic patients.89

Inhibition of elevated prostanoid production andenhancement of, or even no effect upon, basalprostanoid production would provide an idealapproach for anti-inflammatory treatment w ith block-ade of the damaging over-production of PGs andpreservation of basal, potentially anti-inflammatory,PG production.

Nitric oxide s ynthase (NOS)Nitric oxide (NO) plays an important role in severalsystems modelling physiological and pathologicalprocesses in the skin including; vasodilatation, inflam-mation, immunomodulation and oxidative damage tocells and tissues (for review see Lyons90). As such NOrepresents another important possible site of actionof the GCs. NO is synthesized by a family of enzymes:the nitric oxide synthases of which there are threeisoforms–the constitutive endothelial eNOS and neu-ronal nNOS (also collectively known as cNOS) and theinducible iNOS. GCs inhibit the induction of iNOSwhilst leaving cNOS activity untouched. Studies in theearly 1990s demonstrated that the GCs inhibited NOproduction only in situations where NO productionwas elevated by inflammatory substances such as LPSor cytokines, much in the same way as the COXsystem. The similarity between the two enzymefamilies extends further in that cNOS like COX-1, andin contrast to iNOS or COX-2, is resistant to modula-tion by GC treatment.90

NOS is expressed in skin and this has beendemonstrated in animal91 and human skin.92 Indeedintradermal application of an NO synthase inhibitorcauses decreases in local skin blood flow in animals93

and man.94 Thus it appears that NO is involved inbasal dermal blood flow. Recent evidence showssignificant increases in NO production in severaldermal pathologies and it has been postulated thatthis elevated NO contributes to the aetiology ofcertain skin diseases. Studies using the NOS inhibitorssuggest a role for NO in erythema and oedemaformation in psoriasis and atopic dermatitis.94,95

Additionally iNOS is present in lesional psoriaticskin96,97 and specifically in involved sites in patientswith atopic dermatitis and allergic contact dermati-tis.96 It is clear that enhanced NO production, due toiNOS activity, may have a significant role to play ininflammatory skin conditions.

Thus it may be possible that resolution of inflamma-tory skin conditions with the GCs may be due, at leastin part, to the inhibition of iNOS induction in the skin.There have been no studies, as of yet, which haveclearly demonstrated that following topical GC treat-ment there is a consequent reduction of iNOS activityin sites subjected to an inflammatory stimulus such asLPS or carrageenin or indeed in human skinbiopsies.

Modulation of mast cell activity

An alternative mechanism for the anti-inflammatoryaction of GCs has been proposed to be the modula-tion of mast cell numbers and activity. Degranulationof mast cells with consequent release of histamineplays a major role in hypersensitivity responses aswell as contributing to other types of inflammatoryskin diseased states. Mast cells possess high affinityIgE receptors and in atopic individuals exposure toantigen results in degranulation since the antigenbinds to the IgE attached to the receptor. The mastcell contains several pro-inflammatory mediators anddegranulation will consequently release these pro-inflammatory substances, such as histamine and PGs,into the local environment.98 Therefore inhibition ofthis mast cell component should have significant anti-inflammatory effects. Topical GC treatment causes adecrease in mast cell numbers in the skin99 and thisdecrease in numbers is responsible for the decrease inhistamine content of the treated skin. Despite initialstudies with human skin in vitro showing that GCtreatment, for only 2 h, attenuates histamine release inresponse to allergen challenge100 early studies in theairways, in vivo , suggested that the GCs had no effecton histamine release in an allergic reaction. Howeverit has become clear that duration of treatment w ithGC is an important determinant of the efficacy ofthese agents in the skin.100 Indeed a 1–2 weektreatment with topical GC causes complete block ofthe allergen-induced late response and attenuates theimmediate wheal and flare response in the skin of thehuman forearm.101,102 Recent studies also show GC-induced reduction of IgE receptor-mediated expres-sion of pro-inflammatory cytokine in murine bonemarrow derived mast cells.103

Inhibition of cytokine activity

It is clear that the expression and activity of severalimportant inflammatory cytokines may be suppressedby prior treatment w ith glucocorticoids. Suppressionof cytokines expression, in general, appears not to bedue to interaction of the GC-R complex with GREs,since most cytokine genes do not possess a GREelement. The only clear exception to this is the IL-6gene104,105 although there is some evidence for thepresence of a GRE on the IL-8 gene of a humanfibrosarcoma cell line106 and more recently a negativeregulatory region containing a negative GRE has beenidentified in the human IL-1b gene.107 Reportssuggest that the transcription of several cytokines islowered by the GCs following inactivation of specifictranscription factors, such as AP-1 and NFk B, asdescribed earlier. This mode of action has beensuggested to be responsible for the attenuated pro-duction of IL-6, IL-8 and IL-2.105,108,109 Finally thethird mechanism for GC-induced inhibition of cyto-

A. Ahluwalia


kine expression is through the enhancement ofmRNA instability, i.e. a shortened half-life, for certaincytokines (for e.g. see Lee et a l.110). As mentioned theGCs also inhibit the activity of many cytokines andthere is a vast literature documenting these actionsand this review will not discuss these further. Ofparticular interest however have been the demonstra-tions that the GC-sensitive actions of the cytokines inmodels of cellular migration, specifically IL-1b and IL-8, are mediated partially by LC1.111 Additionally inpsoriatic skin both IL-1 and IL-6 are significantlyelevated112,113 and both of these studies postulatethat these cytokines may be implicated with thepathology of the disease.

SummaryThe topical GCs still remain one of the most effectiveand popular forms of treatment of various inflamma-tory skin disease states. These agents are clearly acomplex class of drugs that modulate the activity ofseveral pivotal processes and mediators of inflamma-tion. Modulation of the levels and/or compartmentali-zation of the GC-inducible, anti-inflammatory, anti-proliferative protein, LC1, following topical GCtreatment provides solid evidence supporting thesuggestion that this protein plays an important role inthe action of topical GCs. Exploration of the possibil-ities of more specific treatment of skin disease withLC1 related products may provide novel more efficientmodalities for treatment of inflammatory skin disease.

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ACKNOWLEDGEMENTS. I would like to thank the British Heart Foundationfor their support and Professor R. J. Flower and Dr Mauro Perretti for theirhelpful discussions. I would also like to thank Dr Adrian Hobbs for his kindhelp in preparation of this manuscript.



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