First International Meeting onNeurobiology of the Skin

13–15 February 2004, Munster, Germany

Speaker abstracts

Neurocutaneous anatomy, developmentalbiology and physiology

Organization of the peripheral nervous system

C. Sternini

Departments of Medicine and Neurobiology, David Geffen School

of Medicine at UCLA, Los Angeles, CA, USA

The peripheral nervous system comprises the autonomic andsensory (afferent) nervous systems. Major advances in our under-standing of the autonomic and sensory transmission and functioninclude the recognition of the phenotypic expression of a varietyof transmitters and modulators that often coexist in individualneurons, the concept of co-transmission and chemical coding, theevidence for local effector functions of primary afferent nerves,and the discovery of plasticity of both the autonomic and thesensory nervous system during development, aging, diseasesstates, and inflammation. Co-transmission or plurichemicaltransmission, which indicates the release of more than onechemical messenger from the same neuron, enables autonomicand sensory neurons to exert a fine and highly regulated controlof various functions such as circulation and immune response.The concept of chemical coding, in which the combination oftransmitters/modulators is established, allows the identificationof functional classes of neurons with their projections and targets.In addition to transmitters and modulators, autonomic andsensory neurons express multiple receptors, including G-protein-coupled and ion-gated receptors, further supporting thecomplexity of autonomic and sensory transmission and function.Autonomic neurons regulate the internal environment and main-tain multiple homeostatic functions, and sensory neurons act asreceptive structures that activate their targets in response tostimulation but also exert effector functions including the controlof blood flow and vascular permeability, maintenance ofmineralized tissue, and regulation of gene expression.

Neurophysiology of pain

H. O. Handwerker

Department of Physiology and Experimental Pathophysiology,

University of Erlangen, Erlangen, Germany

The nociceptive system supports two sensory functions, pain anditch. Itch has often been regarded as a minor form of pain.Recently, it has been shown, however, that the pruritic system issupported by its own peripheral and central neuronal pathwayswhich are closely associated, although antagonistic in some

respect. Both the pruritic and the algesic system have their ownprimary nociceptive afferents. These nociceptive afferents areunique among sensory receptors in their capacity to becomesensitized following exposure to noxious stimuli. Consequencesof sensitization are increased spike discharges to stimulation anddecreased thresholds. These phenomena may be formally concep-tualized as leftward shift of the stimulus response function. Hyper-algesia was traditionally seen as the perceptual correlate ofsensitization of the algogenic system. The respective sensoryphenomena in the pruritic system have been called hyperknesis.Both hyperalgesia and hyperknesis encompass decrease in sensorythresholds and increased sensation (pain or itch) to suprathresholdstimuli, together with spontaneous pain or itch. However, at leastin the pain system, the simple conception of hyperalgesia as alinear corollary of sensitization of a uniform nociceptor populationis inadequate in the light of the diversity of hyperalgesias which aresubserved by various peripheral and central neuronal mechanisms,to different degrees. Likewise, different forms of hyperknesis existwhich are dependent either on peripheral or on central nervousmechanisms. The pathophysiology of different forms of hyperal-gesia and hyperknesis in dermatological diseases will be discussed.

Tachykinins can partly explain the link within the neuroendocrine-

immune-hematopoietic axis: novel role for mesenchymal stem cells

P. Rameshwar1,2, K. E. Corcoran2, S. J. Greco2, H. Kang2 and

H. Patel1

1New Jersey Medical School, Department of Medicine, and2Graduate School of Biomedical Sciences, University of

Medicine and Dentistry, Newark, NJ, USA

The tachykinins are amongmajor regulators of bonemarrow (BM)functions. The BM is resident to two stem cells: hematopoietic stemcells (HSCs) and mesenchymal stem cells (MSCs). The adult BMhas a finite number of HSCs that are required to replenish theimmune system throughout life. MSCs surround BM vasculature,while HSCs are located close to the endosteal regions. The mechan-isms by which the tachykinins regulate hematopoiesis requirefurther research. Innervated fibers in the BM form synapse-likestructures with MSCs. We propose that tachykinin-mediatedeffects on MSCs are relevant to hematopoiesis, because MSCs: 1)generate hematopoietic supporting stromal cells, 2) regulate themovement of cells in and out of the BM, and 3) regulate inflam-matory responses in the BM. This study focuses on the truncatedtachykinin receptor (NK-1-Tr). Its expression and regulation onMSCs mirrors the brain, but contrasts BM stroma. NK-1-Tr ispredominantly expressed on MSCs. Because of limited receptordesensitization, we propose that NK-1-Tr on MSCs could allowrapid responses to the tachykinins so as to maintain the vascular/barrier functions of the BM, regulate immune responses to infec-tious agents that could be threat toBMfailure, and respond to rapidloss of blood. Part of the mechanisms by which the tachykininsregulate MSCs functions involve novel interactions between

Experimental Dermatology 2004: 13: 567–591 Copyright # Blackwell Munksgaard 2004Blackwell Munksgaard . Printed in Denmark

EXPERIMENTAL DERMATOLOGYISSN 0906–6705

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IFN-g andMHC class II. The findings described in this study holdclues to the role of tachykinins on hematopoiesis and adult stem cellfunctions in the BM. This report adds to an understanding of thecrosstalk within the neural-immune-hematopoietic axis.

Pigmentation

b-Endorphin/b MSH – two neglected melanotropins?

K. U. Schallreuter1,2, J. D. Spencer

1, N. C. J. Gibbons

1,

E. M. J. Peters1, J. M. Carder

1and L. K. Marles

1

1Clinical and Experimental Dermatology University of Bradford,

and2Institute for Pigmentation, Disorders in association with

Ernst-Moritz-Arndt Universitat Greifswald/Germany and the

University of Bradford, Bradford, UK

POMC processing in human melanocytes has been widely docu-mented, and the a-MSH/MC1R/cAMP cascade has been impli-cated in the control of pigmentation. Only very recently, a roleof b-endorphin, one cleavage product of b-LPH, has been demon-strated to influence melanocyte growth, dendricity and melaninbiosynthesis via the m-opiate receptor. However, much earlier, itwas shown that b-MSH, the other cleavage product of b-LPH,controls melanogenesis and melanin transfer in amphibians. Todate, a specific receptor for b-MSH has not been identified.Earlier POMC processing has been found in melanosomes. There-fore, an MC1R-independent role of a-MSH was postulated anddemonstrated in control of 6-tetrahydrobiopterin (6BH4)-inhibited tyrosinase. Utilizing the depigmentation disordervitiligo, we were now able to follow the fate of epidermal POMCprocessing in the presence of mM levels of hydrogen peroxide(H2O2). In vitiligo epidermal PC2 and 7B2 protein expression isincreased, whereas a-MSH, b-MSH and b-endorphin are signifi-cantly decreased. Analysis of the peptide sequences revealed in allthree cases H2O2 oxidation targets such as methionine and tryp-tophan yielding significant structural alterations. Moreover, wehave identified a new function of b-MSH due to its capacity tobind the important cofactor 6BH4 as well as its isomer 7BH4.Hence, we propose for the first time that b-MSH can control boththe supply of L-tyrosine from L-phenylalanine via phenylalaninehydroxylase and L-Dopa synthesis via tyrosinase hydroxylase inmelanocytes and keratinocytes. Therefore, both melanogenesisand catecholamine synthesis could be regulated by this peptide.

a-MSH and cAMP signalling in normal human melanocytes

R. Busca

INSERM U385, University of Nice, Nice, France

Melanocytes are neural crest-derived skin cells specialized in thesynthesis of melanin pigments responsible, in human, for skinand hair colour. The pro-opiomelanocortin peptide, a-MSH is astrong melanogenic agent secreted by keratinocytes followingUV radiation. a-MSH through the binding to the MC1R andactivation of the cyclic AMP pathway plays a pivotal role inmelanocyte differentiation and in the regulation of skin pig-mentation. During the last few years, we have elucidated themolecular events linking the cAMP pathway to melanogenesisupregulation. This cascade involves the activation of proteinkinase A and CREB transcription factor, leading to the upregu-lation of the expression of microphthalmia-associated transcrip-tion factor (MITF). MITF binds and activates the melanogenicgene promoters thereby increasing their expression, whichresults in an increased melanin synthesis. Beyond this simplifiedscheme, other intracellular signalling pathways are regulatedby cAMP and participate to the regulation of melanocyte dif-ferentiation. Indeed, cAMP inhibits the phosphatidyl inositol3-kinase pathway, leading to the inhibition of AKT and to theactivation of GSK3b. This kinase phosphorylates MITF andallows its binding to the target sequence. Such pathways areinvolved in the upregulation of melanogenesis. a-MSH and

cAMP signalling also regulate melanocyte dendricity, and mela-nosome transport through the inhibition of the Rho GTPasecascade that function downstream the PI3 kinase. It should bealso mentioned that cAMP activates the ERK pathway througha melanocyte-specific pathway involving Ras and B-Raf.The activation of ERK and RSK1 leads to the phosphorylation ofMITF and target MITF to the proteasome degradation pathway.Interestingly, several proteins involved in melanocyte differentiationby a-MSH (MC1R, PI3K, B-Raf and MITF) have also beenimplicated in the development of melanoma, suggesting that thecAMP pathway could influence melanocyte transformation.

Regulation of MC1R signalling by G-protein-coupled receptor

kinases

J. C. Garcıa-Borron

Department of Biochemistry and Molecular Biology, School of

Medicine, University of Murcia, Murcia, Spain

The melanocortin 1 receptor (MC1R) is a key regulator ofmelanocyte proliferation and differentiation and a major deter-minant of human skin phototype and skin cancer risk. Althoughthe regulation of MC1R gene expression is fairly well under-stood, little is known about regulatory mechanisms acting at theprotein level. In particular, no information is available onhomologous desensitization of MC1R signalling. We studiedMC1R and Mc1r desensitization and found that: 1) MC1Rand Mc1r in melanoma cells undergo homologous desensitiza-tion, demonstrated by decreases in cAMP contents upon con-tinuous exposure to agonists, 2) desensitization is not dependenton PKA, PKC, calcium mobilization or MAPKs but is agonistdose dependent, suggesting a role of receptor occupancy, 3)melanoma cells express two members of the GRK family ofserine/threonine kinases, GRK2 and GRK6, 4. These kinasesare expressed in normal melanocytes, 5) in cotransfectionexperiments performed with HEK 293T cells, GRK2 stronglyimpairs agonist-dependent signalling by MC1R or Mc1r, 6)expression of a dominant negative GRK2 mutant in melanomacells increases their cAMP response to MC1R agonists, 7)cotransfection of HEK 293T cells with GRK6 and MC1R inhi-bits both basal and agonist-dependent signalling, and 8) cAMPproduction in agonist-stimulated melanoma cells is stronglyimpaired by enrichment with GRK6 following stable transfec-tion. Therefore, GRK2 and GRK6 are key regulators of MC1Rsignalling and may be important determinants of normal andpathological skin pigmentation.

Red hair, fair skin and melanoma – melanocortin 1 receptor

J. L. Rees1, K. Waterston1, L. Naysmith1, C. Oh1, A. Hennessy1,

Y. Bisset1, B. Diffey2, S. Ito3 and K. Wakamatsu3

1Department of Dermatology, The University of Edinburgh,

Edinburgh, 2Department of Medical Physics, The University of

Newcastle, Newcastle, UK, and3School of Health Sciences,

Fujita Health Authority, Toyoake, Japan

We have previously shown that the MC1R is a key determinantof pigmentary phenotype in man. A range of common anduncommon alleles show diminished function leading to a changein the relative amounts of eumelanin and pheomelanin. Asexpected, these particular allelic variants are associated withboth non-melanoma and melanoma skin cancer and other pig-mentary phenotypic characteristics such as freckling. We haverecently shown that even against very different genetic back-grounds, the MC1R variants show a phenotypic effect [J InvestDermatol 2003: 121 (1): 207]. We will present data to explainhow the human pigmentary phenotypes can be quantified moreappropriately, in terms of both hair melanins and cutaneousresponse to ultraviolet radiation (submitted and in press). Ourresults, we would argue, are relevant to those interested inmelanocortin signalling in skin and to studies of the genetics ofhuman skin colour and evolution of skin colour.

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568

The role of accessory proteins in melanocortin receptor signaling

G. Barsh, S. Candille, L. He, S. Aradhya and J. Kerns

Department of Genetics and Department of Pediatrics, Stanford

University School of Medicine, Stanford, CA, USA

Switching from eumelanin to pheomelanin synthesis during hairgrowth is accomplished by transient synthesis of agouti protein,an inverse agonist for the melanocortin 1 receptor (Mc1r). Theability of agouti to signal via the Mc1r requires two additionalgenes, Attractin (Atrn) and Mahogunin (Mgrn1), which encodea type I transmembrane protein and an E3 ubiquitin ligase,respectively. Atrn and Mgrn1 are genetically upstream of theMc1r, but transgenic and biochemical studies indicate that allthree genes act in a melanocyte-autonomous manner. To gainadditional insight into pigment-type switching, we have carriedout biochemical and cell biologic studies which suggest thatMgrnand Atrn are part of a conserved biochemical and genetic path-way that acts to regulate Mc1r-dependent signaling. We have alsoused an additional genetic model system based on coat color indogs, in which we find that dominant inheritance of black coatcolor is caused neither by Mc1r nor by agouti, but instead mapsto a region not previously implicated in pigmentation genetics.

Pilosebaceous biology

Cutaneous neuroimmunology – lessons from the hair follicle

R. Paus1, N. Ito1, T. Ito1, E. Peters2, E. Bodo3, S. Liotiri1,

T. Biro3 and P. Arck2

1Department of Dermatology, University Hospital Hamburg-

Eppendorf, University of Hamburg, Hamburg, 2Department of

Internal Medicine, Charite, Virchow Campus, Berlin, Germany,

and 3Department of Physiology, University of Debrecen,

Debrecen, Hungary

The hair follicle offers an exquisite model for the experimentalexploration of key issues of cutaneous neuroimmunology, forexample, how local, intracutaneous and systemic stress–responsesystems are integrated with the skin immune system and withepithelial–mesenchymal interactions (as they occur during hairfollicle growth and cycling). Previously, we had shown that skinmast cells, which operate as central switchboards of inflammationand tissue remodelling, also are important regulators of hairgrowth in mice and that endogenous, immunomodulatory mastcell secretagogues are potent hair growth modulators. This is trueboth for secretagogues that are generated by the hair follicleepithelium itself (e.g. ACTH) and for mast cell-activating neuro-peptides synthesized by the sensory hair follicle innervation (e.g.SP). Also, we had shown that the prototypic stress-associatedneuropeptide, SP, plays a crucial role in mediating the hairgrowth-inhibitory, mast cell-activating, inflammation- and cata-gen-promoting properties of chronic psychoemotional stress onmurine hair follicles. Now, we show that the immunomodulatoryand mast cell-activating neurotrophin, NGF, is also cruciallyinvolved in mediating the inhibitory effects of stress on murinehair growth. Furthermore, the central, stress-related neuro-hormone CRH, a recognized mast cell secretagogue which isexpressed by the hair follicle epithelium, also is a hair growthinhibitor and activates a fully functional peripheral equivalent ofthe hypothalamic-pituitary-adrenal axis within organ-culturedhuman scalp hair follicles, including the synthesis and secretionof cortisol as well as the induction of classical feedback loops. Wealso demonstrate that one of the melanocortins whose intra-follicular synthesis is stimulated by CRH (a-MSH) is a potentsuppressor of MHC class I expression in situ and is thus capableof restoring the collapsed immune privilege of human anagen hairbulbs, while SP upregulates the ectopic expression of MHC classI, thus endangering the hair follicle immune privilege. Finally, weshow that vanilloids long exploited as experimental tools forneuroimmunological research in the skin (capsaicin) can, infact, directly modulate human hair growth via the stimulation

of vanilloid receptors (VR1) expressed by the follicle epithelium,in addition to stimulating vanilloid expressed by skin mast cells.Therefore, the hair follicle offers an ideal, highly instructiveand clinically most relevant research model for dissecting hownervous system, central and peripheral (neuro-) endocrine signal-ling loops and the immune system interact in order to adapt skinfunctions to changing environmental conditions (e.g. in responseto external stressors, by alterating, e.g. keratinocyte proliferation/apoptosis, skin immune status, as well as defined cutaneousmetabolic and endocrine activities).

Expression and regulation of pro-opiomelanocortin-derived

peptides in human hair growth and pigmentation

D. J. Tobin

Department of Biomedical Sciences, University of Bradford,

Bradford, UK

Human skin provides for the local cleavage of pro-opiomelano-cortin (POMC) to yield multiple peptide products, e.g. a-MSH,ACTH and b-endorphin (b-end). a-MSH and ACTH are welldocumented to regulate human skin pigmentation. We haverecently shown that human epidermal melanocytes express afully functioning b-end/m-opiate receptor system, and that b-endhas potent melanogenic, mitogenic and dendritogenic effects incultured epidermal melanocytes. However, little is known abouttheir role in human hair follicle (HF) biology. We have charac-terized the expression and regulation of b-end, ACTH, a-MSHand their receptors in human haired scalp and in cultured HFmelanocytes, keratinocytes and fibroblasts by immunochemistry,detection of mRNA transcripts and by assessment of potentialmelanogenic, dendritogenic and mitogenic effects of these pep-tides in vitro. These studies show that the POMC peptide systemis expressed in HF cells as a function of their anatomic locationand differentiation status during the hair growth cycle. All threepeptides exhibit similar melanogenic, mitogenic and dendrito-genic effects in cultured HF melanocytes. Moreover, while thein situ expression profiles of ACTH and a-MSH are similar, theydiffer strikingly from b-end for all follicular cell populations. Onesuch example is the persistent expression of the former in the hairinductive follicular papilla (FP) throughout the entire hair cycle(but are expressed variably in HF keratinocytes and mela-nocytes). However, b-end expression is low to undetectable inthe FP during hair growth. In toto, these results indicate thatPOMC peptides are involved in regulating human HF growthand pigmentation and that alterations in POMC homeostasismay be contribute to pathology.

Neuroendocrine regulation of the sebaceous gland

Ch. C. Zouboulis

Department of Dermatology, Charite-University Medicine Berlin,

Campus Benjamin Franklin, Berlin, Germany

The sebaceous gland is the organ conferring upon the skinan endocrine function. It also seems to be involved in responsesto stress expressing receptors for neuropeptides such ascorticotropin-releasing hormone (CRH), a-melanocyte-stimulating hormone (a-MSH), b-endorphin, vasoactiveintestinal polypeptide, neuropeptide Y, and calcitonin gene-related peptide. CRH is the most proximal element of thehypothalamic-pituitary-adrenal (HPA) axis, and it acts ascentral coordinator for neuroendocrine and behavioralresponses to stress. The CRH/CRH-binding protein/CRHreceptor loop is expressed in human sebocytes in vivo andin vitro and is regulated and biologically active in culturedsebocytes. CRH may be an autocrine sebocyte factor that exertshomeostatic lipogenic activity by controlling the expression ofD5-3b-hydroxysteroid dehydrogenase, whereas testosterone andgrowth hormone induce CRH negative feedback. a-MSH andcalcitonin gene-related peptide are not produced by humansebocytes and downregulate the enhanced IL-8 synthesis in IL-1b-challenged cells, therefore exhibiting an anti-inflammatory

Abstracts

569

effect. b-endorphin is also a paracrine neuropeptide for humansebocytes which suppresses their proliferation induced byepidermal growth factor in Ca2þ-rich medium. Moreover, sub-stance P-immune reactive nerves are localized near the sebac-eous glands and can stimulate undifferentiated sebocytes ofthe outmost sebaceous gland layer to express neutral endo-peptidase, a substance P-inactivatimg molecule. In addition,substance P stimulates lipid synthesis in human sebocytes.In conclusion, sebaceous glands may be involved in ahypothalamus-pituitary-skin pathway conceptually similar tothat of the HPA axis. The current data implicate neuropeptidesto possibly play a strategic role in the regulation of sebaceousgland activity in close coordination with other hormonesmaking likely that they are involved in the development ofvarious skin diseases.

Tumour biology

Melanoma targeting by melanocortin peptides: current status and

future perspectives

A. N. Eberle1, S. Froidevaux

1, M. Calame-Christe

1, H. Tanner

1,

S. Knecht1, J. Schuhmacher2, R. Saffrich2 and M. Henze2

1Laboratory of Endocrinology, Department of Research,

University Hospital and University Children’s Hospital Basel,

Basel, Switzerland, and 2Department of Diagnostic and

Therapeutic Radiology, German Cancer Research Center,

Heidelberg, Germany

Melanoma tumour targeting was investigated with radiolabelleda-MSH peptides and with a-MSH derivatives attached to largecarriers such as liposomes. The main focus of this paper will becurrent targeting concepts of melanoma (a-melanoma tumourdiagnosis and internal radiotherapy) using a-MSH peptidesfor specific delivery of diagnostic and therapeutic radiometals.Several new a-MSH analogues (MSH1-n) were synthesized in ourlaboratory and conjugated to 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA), a universal metal chelator.The resulting DOTA-MSH1-n derivatives were found to retaingood binding capacity to the melanoma cell MC1 receptor(MC1R) in the low nanomolar range. In vivo tissue distributionof 5 mCi [111In]-labelled DOTA-MSH in female B6D2F1 micewith intracutaneous B16F1 melanoma tumours and with micro-metastases in the lung and liver demonstrated that the radio-ligands accumulated specifically in the tumour tissue, reachinga maximum, for example with DOTA-MSH4, of 9.43+ 1.06%I.D./g 4-h postinjection. Co-injection of an excess of a-MSH(50 mg) blocked the MC1Rs and hence reduced the 4-h tumouruptake by an average of 90%, which indicates that radioliganduptake by the melanoma tumours was a receptor-mediatedprocess. Blood clearance was very rapid and 4 h after injection,the blood-associated radioactivity was as little as 0.03+ 0.00%I.D./g. This was associated with a fast elimination of the radio-activity from all MC1R-negative tissues, except the kidneyswhich serve as main excretory organ. The ratios of radioactivityin melanoma tissue to that in non-target tissues 4 h after injec-tion were all above 10 and often greater than 100, except for thekidneys. The identification of radiopeptide structures yieldingreduced retention of radioactivity by the kidneys but neverthe-less excellent tumour uptake is currently the main goal of ourstudies. The specificity of targeting melanoma metastases usingradiolabelled MSH peptides was further analyzed by positron-emission tomography (PET) as well as with autoradiography oftumour tissue sections with surrounding healthy tissue after invivo injection of the radiopeptides into tumour-bearing animals:the radioactivity was concentrated exclusively in and localizeduniformly throughout the tumour tissue. Non-radioactiveapproaches to MC1R-mediated melanoma targeting includecytotoxic MSH–peptides, MSH–carrier conjugates and MSH–liposome constructs. A brief summary of the current state of the

different approaches including their advantages and disadvan-tages will be presented.

Sigma receptors as novel target structures for cancer

chemotherapeutics

W. D. Bowen

Laboratory of Medicinal Chemistry, NIDDK, NIH, Bethesda,

MD, USA

Sigma receptors bind several important classes of psychotropicdrugs. Two pharmacologically defined subtypes exist, termedsigma-1 and sigma-2. Sigma receptors are widely distributedacross tissues. Both subtypes are highly expressed in tumor celllines, including breast, lung, and prostate tumor cellines as wellas neuroblastomas, gliomas, and melanomas. Furthermore,sigma-2 receptors become more upregulated in rapidly dividingcells. Activation of sigma-2 receptors results in apoptotic celldeath. The signaling pathway involves changes in intracellularcalcium levels as well as increases in the cellular sphingolipidsceramide and sphingosylphosphorylcholine. The apoptoticeffect of sigma-2 agonists is not affected by the p53 status ofthe cells and is independent of caspase activity. Subtoxic dosesof sigma-2 receptor agonists markedly potentiate the cytotoxiceffect of doxorubicin and actinomycin-D in breast tumorcellines, indicating a chemosensitizing effect. The very highdensity of Sigma receptors in tumor cells indicates that theymay also be useful targets for development of non-invasivetumor imaging agents. Several radiolabeled sigma ligandsshow favorable biodistribution and tumor uptake, and mela-noma cell tumor xenografts have been successfully imaged innude mice. Taken together, these results suggest that sigma-2receptors are involved in the regulation of cell proliferation andsurvival and activate a novel pathway of programmed celldeath. It may be possible to exploit this pathway and the hightumor expression of sigma-2 receptors to develop agents for thetreatment and diagnosis of drug-resistant tumors.

The role of neurotrophins in brain metastasis of malignant

melanoma cells

G. L. Nicolson

Department of Molecular Pathology, The Institute for Molecular

Medicine, Huntington Beach, CA, USA

We have examined the role of neurotrophin receptors andneurotrophins in brain invasion and colonization of malignantmelanoma cells. Using mouse and human melanoma variant cellsublines that have the capacity to form brain tumor colonies innude mice, we studied the effects of neurotrophins and growthfactors on malignant properties. The high brain-colonizingmelanoma lines were characterized by high expression of thelow-affinity nerve growth factor (NGF) receptor p75NTR,which forms transmembrane complexes containing theneurotrophin and its receptors, but they expressed very lowamounts if any of the high-affinity neurotrophin receptortrkA. In the presence of brain endothelial cell-derived motilityfactors, NGF and other neurotrophins, such as neurotrophin-3(NT-3), stimulate release of basement membrane degradativeenzymes and significantly increase the invasion of endothelialcell extracellular matrix and Matigel-coated filters. Enzymessuch as the collagenase-degrading enzyme MMP-2 and heparansulfate-degrading enzyme heparanase are increased in theirsynthesis and release from the high brain-colonizing but not byother melanoma variant lines by NGF or NT-3. The increase inheparanase can be blocked by antisense resulting in decreasedinvasion and capacity to colonize brain. Neurotrophins alsostimulate the synthesis and release of autocrine growthfactors by brain-colonizing melanoma cells. Brain-colonizingmelanoma cells also respond to paracrine growth factors inthe brain, and one of the important paracrine growth factorsin brain metastasis has been identified as a transferrin.

Abstracts

570

Examination of the invasion front in brains colonized by thebrain-colonizing melanoma cells revealed high concentrations ofNGF and other neurotrophins (NT-3) at the interface betweenmelanoma cells and adjoining normal brain tissue (withextensive gliosis) that gradually diminished with distance fromthe invasion front. Using immunohistochemical techniques todetect neurotrophins, uninvolved brain tissue (adult animals)possessed very low or undetectable concentrations of theseneurotrophins. Trophic factors, autocrine factors, paracrinegrowth factors and other factors may determine whethermetastatic melanoma cells can successfully invade, colonizeand grow in the CNS.

Inflammation and infection

An evolutionary concept for changes of synovial tissue innervation

in patients with rheumatoid arthritis

R. H. Straub1and H. O. Besedovsky

2

1Department of Internal Medicine I, University Hospital

Regensburg, 93042 Regensburg, and2Institute of Normal and

Pathological Physiology, University of Marburg, 35033

Marburg, Germany

The pathogenesis of chronic disabling inflammatory diseases(CDIDs) is partly understood. The presently used conceptsfocus mainly on abnormalities of the immune system, but thisview is incomplete. The presented concept is a new frameworkfor the pathogenesis of CDIDs. It integrates evolutionarytheories with the classical immunological standpoint, which isfurther linked with a neuroendocrine immune view of erroneoushomeostatic adaptation of the other supersystems (nervous sys-tem, endocrine system and reproductive system): 1) In CDIDs,the loss of tolerance against self and harmless foreign antigensleads to continuous immune aggression which is dependent on amultifactorial genetically polymorph background (the initia-tion); 2) However, advantageous or disadvantageous adaptationto CDIDs were not evolutionary conserved, because CDIDsseverely impaired reproduction or appeared after the reproduc-tive phase and, thus, imply a strong negative selection pressure;3) Reactions of all supersystems are evolutionary conserved fortransient inflammatory reactions such as the elimination ofinfectious agents, wound healing, foreign body reaction andmany others; and 4) The sum of the false reactions of all super-systems, conserved for transient inflammation, provides thepathogenetic background for the chronification of CDIDs,because a continuous aggressive situation is created (the chron-ification). The human disease of rheumatoid arthritis is used asa prototypic CDID to illustrate the integrated view point. Thesynovial tissue innervation is in the focus of this concept.

Potential role of corticrotropin-releasing hormone in skin

physiology and pathology

A. Slominski

Department of Pathology, University of Tennessee HSC,

Memphis, TN, USA

Previously, we have demonstrated the expression of corticotro-pin-releasing hormone (CRH) and related urocortin peptides andtheir corresponding CRH receptors (CRH-Rs) in the skin andidentified new alternatively spliced CRH-R1 isoforms (FASEB J2001; 15: 1678–1693). These receptors are functional, bindingCRH and related urocortin peptides and activating intracellulartransduction pathways, to modify skin cell phenotype includingproliferation, differentiation and immune activity. Thus, CRH-Rs ligands can act as local growth factors and cytokines. Inconjunction with the cutaneous expression of POMC peptidesand their receptors as well as the expression of genes codingenzymes of corticosteroidogenic pathway, these observationsalso demonstrate integrated expression of the regulatory elements

of the hypothalamic-pituitary-adrenal (HPA) axis in mammalianskin (Physiol Rev 2000; 80: 979–1020; Endocrine Rev: 21: 457–487). This cutaneous CRH/POMC system appears to be highlyresponsive to the common stressor ultraviolet radiation (UVR),cutaneous pathology or physiologic changes such as those asso-ciated with the hair cycle. Therefore, we have proposed that thissystem would be structurally analogous to the central HPA axis,with CRH, urocortin and CRH receptors playing a central role inthe regulation of cutaneous reactions. Most recently, we founddifferential, spatiotemporal selectivity and species-restrictedexpression of CRH-Rs, suggesting that they may be evolutionarydriven to adapt to the species predominant determinant of skinfunction: human, solar radiation and thermal energy; mice, haircycle and production of chemical messengers.

The TRPV1: a possible role in GERD, asthma and migraine

P. Geppetti

Department of Critical Care Medicine and Surgery, Clinical

Pharmacology Unit, Medical School, University of Florence,

Viale Pieraccini 6, 50139 Florence, Italy

The transient receptor potential vanilloid type 1 (TRPV1) is anon-selective ion channel that belongs to the TRP family ofchannels that are activated by vanilloid molecules, includingcapsaicin. The cloned TRPV1 is a thermosensor, gated by tem-perature (43–52�C) and low pH. Additional putative endo-genous activators of the TRPV1 are the cannabinoid receptoragonist, anandamide, N-arachydonoil-dopamine and certainlipoxigenase metabolites of arachidonic acid, as 12-HPETEand LTB4. The TRPV1 is expressed selectively in a subpopu-lation of primary sensory neurons with C- and A-delta fibreswhich also express NGF receptors, the neuropeptides, substanceP (SP), neurokinin A and calcitonin gene-related peptide(CGRP). These neurons being activated by different sensory(mechanichal, thermal and chemical) modalities are defined aspolymodal nociceptors, and the peptides released from theirperipheral terminals cause neurogenic inflammation. TRPV1seems to be coupled with PLC and PIP2 hydrolysis results inchannel activation. Additional modes of TRPV1 sensitizationcomprise PKC- and PKA-dependent pathways. Activation ofeither G-protein-coupled receptors or tyrosine kinase receptorscauses TRPV1 sensitization. Of pathophysiological interest isthe finding that endogenous and exogenous molecules may alsocause TRPV1. Recently, we found that exposure to 0.3–3%ethanol causes a remarkable increase in Ca2þ mobilization incapsaicin-sensitive cultured DRG neurons of newborn rat, aneffect that was inhibited by the TRPV1 antagonist capsazepine.Wild-type human embryonic kidney (HEK) cells did notrespond to ethanol, but transfection with the human TRPV1conferred to these cells the ability to respond to ethanol (Ca2þ

mobilization) in a capsazepine-sensitive manner. Electrophysio-logical studies showed that ethanol dramatically potentiatedcurrents activated by anandamide and protons, and that ethanolreduced the threshold temperature for TRPV1 activation byabout 8�C. Thus, in the presence of ethanol, the physiologicaltemperature of 37�C is a sufficient stimulus to activate TRPV1on sensory neurons and activate their afferent and efferentfunctions. Ethanol can trigger attacks of asthma and migraineand aggravates the symptoms of GERD. Exposure to ethanolcaused a Ca2þ-dependent release of neuropeptides (SP/CGRP)from slices of rat esophagus and dura mater and guinea-pigairways and increased plasma extravasation in the rat oeso-phagus and dura mater and caused bronchoconstriction in vitroand in vivo in guinea pigs. All these responses were inhibitedselectively by capsazepine. Activation of sensory neurons andneurogenic inflammatory responses via TRPV1 stimulation maycontribute to the mechanism of attacks of migraine and to theworsening of GERD and asthma symptoms precipitated byalcohol ingestion.

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571

Role of protease-activated receptor-2 during cutaneous inflam-

mation and the immune response

M. Steinhoff

Department of Dermatology and Boltzmann Institute for

Immunobiology of the Skin, University of Muenster, Muenster,

Germany

Protease-activated receptors (PARs) constitute a new subfamilyof G-protein-coupled receptors with seven transmembranedomains which are activated by various serine proteases suchas thrombin, cathepsin G, trypsin or tryptase, and bacterialproteases or mite antigens, for example. PAR2 is a receptor formast cell tryptase or house dust mite allergens, which is releasedduring inflammation and allergic reactions. In the skin, PAR2 isdiversely expressed by keratinocytes, endothelial cells, andoccasionally sensory nerves of human skin in various diseasestates. Moreover, immunocompetent cells such as T cells andneutrophils express functional PAR2, thereby contributing toinflammation and host defense. Own data revealed that PAR2contributes to neurogenic inflammation by releasing neu-ropeptides from sensory nerves resulting in oedema, plasmaextravasation and infiltration of neutrophils. Thus, mast cellsmay communicate with sensory nerves in inflammatory tissuesby activating PAR2 via tryptase. Moreover, PAR2 agonistsupregulate the expression of certain cell-adhesion moleculesand cytokines such as interleukin-6 and interleukin-8 on dermalmicrovascular endothelial cells or regulate neutrophil migration,indicating that PAR2 plays an important role in leucocyte/endothelial interactions. These effects may be partly mediatedby NF-kB, an important transcription factor during inflamma-tion and immune response. PAR2 stimulation results in the acti-vation of NF-kB on microvascular endothelial cells andkeratinocytes, thereby regulating ICAM-1 expression. We alsodemonstrate evidence for a diverse expression of PAR2 in variousskin diseases and highlight the recent knowledge about theimportant role of PAR2 during inflammation and the immuneresponse. Together, PAR2-modulating agents may be new toolsfor the treatment of inflammatory and allergic diseases in theskin.

Trafficking of neurokinin receptors: regulation, mechanism and

function

N. Bunnett

Department of Surgery and Department of Physiology, University

of California, San Francisco, CA, USA

Cellular responses to agonists of G-protein-coupled receptors(GPCRs) depend in large part on the trafficking of receptorsbetween the plasma membrane and intracellular locations.Receptor activation usually triggers rapid endocytosis of recep-tors, which either recycle to the cell surface or are targeted fordegradation, depending on the receptor in question and thenature of the stimulation. Activation of neurokinin receptors(NKRs) induces membrane translocation of G-protein receptorkinases, which phosphorylate the receptors and b-arrestins,which interact with phosphorylated receptors. b-arrestins: 1)uncouple receptors from G-proteins to mediate desensitization;2) are adaptors for clathrin and AP-2 and mediate clathrin anddynamin-dependent endocytosis of receptors; and 3) interactwith components of the MAP kinase pathway such as src, andthereby determine the subcellular location and function of acti-vated MAP kinases. The fate of endocytosed NKRs depends onthe receptor and the nature of the stimulus. Transient stimula-tion with low concentrations of SP (1 nM, 10min) induces rapidrecycling of the NK1R from superficially located endosomes bya mechanism that is mediated by rab4a and rab11a. Higherconcentrations of SP (10 nM) induce rab5a-dependent traffick-ing of the NK1R to perinuclear sorting endosomes and agradual recycling to the plasma membrane. Continuous stimula-tion with high concentrations of SP (100 nM, 180min) inducesNK1R ubiquitination and trafficking for degradation. The fate

of endocytosed receptors also depends on their interaction withb-arrestins. The NK1R forms stable high-affinity interactionswith both b-arrestins 1 and 2 at the plasma membrane and inendosomes, whereas the NK3R interacts transiently only with b-arrestin 2 at the cell surface. The nature of these interactions isspecified by domains in the intracellular loop 3 and the carboxylterminus and determine the rate of recycling and resensitizationof these receptors.

Antimicrobial actions of a-MSH peptides – implications for

mucocutaneous host defense

A. Catania1, P. Grieco

2, C. Rossi

1, G. Colombo

1, S. Gatti

3and

J. M. Lipton4

1Department of Internal Medicine, Ospedale Maggiore di Milano

IRCCS, 20122 Milan, 2Department of Pharmaceutical Chemistry

and Toxicology, University of Naples, 80131 Naples,3Department of Surgery, Ospedale Maggiore di Milano IRCCS,

20122 Milan, Italy, and 4Zengen Inc., Woodland Hills CA 91367,

USA

Endogenous antimicrobial peptides are components of theinnate host defense system that prevents microbial penetrationbefore the time-consuming adaptive immunity starts. We haverecently demonstrated that a-melanocyte-stimulating hormone(a-MSH) has antimicrobial effects. The antimicrobial influencesof a-MSH are exerted through a unique mechanism, whichappears to be linked to the cAMP-inducing activity of the pep-tide. This mechanism mimics the influence of a-MSH in mam-malian cells in which the peptide binds to G-protein-linkedmelanocortin receptors, activates adenylyl cyclase, and increasescAMP. In an attempt to improve the antimicrobial activity ofa-MSH and to better understand the peptide structure–activityrelations, we designed and synthesized novel peptide analogs. Inthis structure–activity study, we discovered several compoundsthat have greater antimicrobial activity than a-MSH. The pep-tide [DNal-7, Phe-12]-a-MSH (6–13) was the most potent of theanalogs tested. This compound killed almost 100% of Candidacells and had substantial antimicrobial effects against Gram-posi-tive and Gram-negative bacteria. Enhanced antimicrobial activityof the Phe-12-substituted peptides was the most distinctive featurerelative to effects in mammalian cells. The results are veryencouraging in that they show the great potential of a-MSHpeptides as a truly novel class of antimicrobial compounds.

Immunology

Mast cells and their role in the neuro-immune-endocrine axis

J. Bienenstock

Department of Pathology and Molecular Medicine, McMaster

University, Hamilton, Ontario, Canada

It has become clear that the immune and nervous systems com-municate constantly to maintain homeostasis and a coordinatedand continuing adaptive response to an ever-changing environ-ment. Evidence from mast cell nerve communication, as anexample of this interaction, has been obtained in a variety oftissues and circumstances, most especially in the intestine andskin. Bidirectional communication has been shown in vivo,ex vivo, in vitro and in coculture experiments involving the twocell types. Examples will be given of these various situations andinvolve normal physiological situations and those involved inresponse to infection and inflammation as well as in response toultraviolet light. More recent examples of the importance ofmast cells in the regulation of central nervous activity includingthe secretion of hormones by the pituitary gland, and therebythe regulation of the HPA axis as well as involvement inbehavioural change will be addressed. Through its potentialcommunication with the nervous system, the mast cell can beregarded as a sentinel cell or receptor, especially located at

Abstracts

572

surfaces exposed to the environment, which specifically andnon-specifically react to molecules and substances, foreign tothe organism, so as to help orchestrate the complex andintegrated responses required to maintain homeostasis.

Effects of neuropeptides and hormones on Langerhans cells

R. D. Granstein

Department of Dermatology, Weill Medical College of Cornell

University, New York, NY, USA

Significant evidence suggests that the nervous and immune sys-tems have regulatory interactions within the skin. Langerhanscells (LCs) are dendritic antigen-presenting cells that residewithin the epidermis. By laser confocal scanning microscopy,LCs in human skin have been found to be frequently in ana-tomic association with epidermal nerves. Additionally, a minor-ity of LCs have been found by immunohistochemistry to havethe neuropeptide calcitonin gene-related peptide (CGRP) on ornear their cell surfaces. Functional studies have demonstratedthat CGRP, pituitary adenylate cyclase-activating polypeptide,and vasoactive intestinal peptide inhibit antigen presentation byLCs, at least in some assays. Epinephrine and norepinephrinealso have been shown to inhibit LC antigen presentation in vitro.Some of these agents appear to exert their effects throughregulation of the expression of cytokines and costimulatorymolecules. Furthermore, some of these agents inhibit theacquisition of contact hypersensitivity after intradermal admin-istration. As a whole, these findings suggest a regulatory locus ofinteraction between the immune system and the nervous systemwithin the skin.

Functional role of somatostatin receptors in neuroendocrine and

immune cells

P. M. van Hagen

Department of Internal Medicine/Clinical Immunology, Erasmus

Medical Centre, Rotterdam, The Netherlands

Somatostatin is a neuropeptide that is widely distributedthroughout the body. It was first identified as a growth hormonerelease-inhibiting factor synthesized in the hypothalamus.Outside the central nervous system (CNS), the peptide is presentin endocrine as well as non-endocrine tissues. Somatostatinfunctions as a peptide with a generally inhibitory action in theCNS and endocrine system. In the CNS, it can act as a neuro-transmitter, while in peripheral tissues, it regulates endocrineand exocrine secretion and acts as a modulator of motor activityin the gastrointestinal tract. Besides these actions, somatostatinhas also been shown to have antiproliferative effects in vitro.Somatostatin binds to five different subtype receptors (sst)which are differently expressed by various tissues. These recep-tors have been described also in the immune system of variousspecies including humans. It is therefore a long known fact thathuman immune cells and their progenitors can express somato-statin receptors. As a consequence, ssts were described inprimary and secondary human immune organs. Somatostatinproduced by immune cells may act as an autocrine or paracrineregulator within the local immune microenvironment in mice.The synthesis, however, of somatostatin has not been demon-strated in human immune cells. In a recent study, the expressionof another somatostatin-like peptide; cortistatin-17 (CST) wasfound in human lymphoid tissues, immune cells and lymphoidcell lines. On the basis of these observations, a role for CST as anendogenous ligand for sst in the human immune system, ratherthan SS itself was hypothesized. Somatostatin receptor expressioncan be detected in vivo somatostatin receptor scintigraphy afterinjection of 111In-labelled octreotide, a somatostatin analogue.This technique is used extensively for the localization of neuro-endocrine tumours and other malignancies that express high levelsof sst. Among the non-neuroendocrine tumours that can bevisualized by octreotide scintigraphy are malignant lymphomas,both T and B non-Hodgkin’s lymphomas and Hodgkin’s disease

lymphomas. In a number of infectious diseases (e.g. tuberculosis),autoimmune diseases (e.g. Graves’ ophthalmopathy) and otherimmune-mediated diseases (e.g. sarcoidosis and rheumatoidarthritis), the sites of inflammation can also be visualized. Basedon the receptor pattern in autoimmune diseases, controlledstudies are warranted to investigate the efficacy of somatostatinanalogues in the treatment of autoimmune diseases like rheuma-toid arthritis. In rheumatoid arthritis, not only immune cellscan be targeted by these analogues but also synoviocytes andsynovial blood vessels. Moreover, receptor expression duringtreatment in such diseases can be monitored by octreotidescintigraphy.

Vasoactive intestinal peptide and pituitary adenylate cyclase-

activating polypeptide as modulators of innate and adaptive

immunity

M. Delgado

Institute Parasitologia y Biomedicina, CSIC, Granada, Spain

Recent reports identified neural pathways, both hard-wiring andsoluble mediators, that control and adjust the peripheralimmune response. Immune organs are innervated by fibres richin neurotransmitters and neuropeptides, which are released ininflammatory conditions. Here, we focus on the immunomodu-latory role of two neuropeptides, vasoactive intestinal peptide(VIP) and pituitary adenylate cyclase-activating polypeptide(PACAP). VIP/PACAP are present and released from bothinnervation and immune cells, particularly Th2 cells, andimmune cells express receptors for VIP/PACAP. VIP/PACAPhave a general anti-inflammatory effect, both in innate and inadaptive immunity. In innate immunity, VIP/PACAP inhibitthe production of proinflammatory cytokines and chemokinesfrom macrophages, microglia and dendritic cells. In addition,VIP/PACAP reduce the expression of costimulatory moleculeson the antigen-presenting cells, and therefore reduce stimulationof antigen-specific CD4þ T cells. In terms of adaptive immunity,VIP/PACAP promote Th2-type responses and reduce the pro-inflammatory Th1-type responses. VIP is rapidly transforminginto something more than a mere neuropeptide. In evolvingscientifically from a neuropeptide to a novel agent for modifyingimmune function, and, possibly a cytokine-like molecule, VIPresearch has engaged many physiologists, molecular biologists,biochemists, endocrinologists and pharmacologist, and it is aparadigm to explore mutual interactions between neural andneuroendocrine links in health and disease. Recognition of thecentral functions that VIP plays in cellular processes is focusingour attention on this very important peptide as exciting newcandidates for therapeutic intervention and drug development.

Neuropeptides, a never-ending story? Termination of cutaneous

inflammation by proteolytic peptidases

T. E. Scholzen and T. A. Luger

Ludwig-Boltzmann Institute, Department of Dermatology,

University of Munster, Munster, Germany

Neuropeptide-specific peptidases such as neutral endopeptidase(NEP, CD10) and angiotensin-converting enzyme (ACE,CD143) effectively control the bioavailability of neuropeptidesreleased from sensory nerves, immune and skin cells duringneurogenic inflammation. Drug inhibition or genomic deletionof NEP or ACE results in a substance P (SP-) and bradykinin-dependent augmentation of murine allergic contact dermatitis(ACD) by affecting ACD sensitization and elicitation. The func-tional absence of NEP enhanced ACD inflammation by pro-moting bone marrow-derived dendritic cell (BmDC) maturationand function. In vitro haptenized BmDCs from NEP–/– miceneurokinin-1 receptor-dependently stimulated proliferation ofantigen-specific NEP–/– and NEPþ/þ T cells with higher efficacycompared to NEPþ/þ-mice BmDCs. Importantly, adoptivetransfer of in vitro haptenized DC from NEP–/– into wild-typemice significantly promoted ACD in comparison with transfer

Abstracts

573

of NEPþ/þDC. Likewise, hapten uptake into DC from regionallymph nodes during ACD sensitization is increased in NEP–/–

mice compared to normal mice. Moreover, in CD10- andCD143-expressing human dermal microvascular endothelialcells and keratinocytes, UV light and inflammatory mediatorsregulated mRNA and protein expression, as well as proteolyticactivity of these peptidases, which may be important for cellsurvival and the outcome of an inflammatory response. Like-wise, NEP and ACE are also involved in the proteolytic proces-sing of neuroendocrine hormones such as adrenocorticotropinand a-melanocyte-stimulating hormone. Thus, present dataindicate that ACE and NEP by proteolytic cleavage of peptidemediators have a significant role in controlling cutaneousinflammatory responses.

Neurotrophins in allergic disease of skin and lung: modulators of

immunological and neuronal plasticity

H. Renz, C. Hahn and W. A. Nockher

Department of Clinical Chemistry and Molecular Diagnostics,

Philipps University Marburg, Baldingerstr., D-35033 Marburg,

Germany

Allergies are chronic inflammatory disease of the skin, lung andgut. Atopic dermatitis represents the main manifestation of theskin, and bronchial asthma is the leading condition in the lowerrespiratory tract. Both conditions are due to an inappropriateimmune response to harmless environmental antigens (or aller-gens). There is growing evidence for a close interaction betweenthe immune and nervous system in the pathophysiology ofallergies. Recent evidence from our group indicates that neuro-trophin production including nerve growth factor (NGF) andbrain-derived neurotrophic factor (BDNF) are elevated in thoseorgans. Both, residential cells including airway epithelium andmigratory cells including macrophages, T cells and eosinophilsserve as important sources. The effects of increased neurotro-phin production are bidirectional. On the one hand, they controlsensory nerve fibres in terms of function, neuropeptide synthesisand survival. On the other side, neurotrophins also serve asimportant survival factors particularly for inflammatory cellssuch as eosinophils, T cells and macrophages. Utilizing themodel of segmental allergen provocation of mild to moderateasthmatic patients, it has been shown that neurotrophins pre-vent sufficiently apoptotic cell death of lung, but not bloodeosinophils. In addition, they augment the ongoing inflamma-tory reaction. The functional interaction between neurotrophinsand immune and nerve cells has been extensively studied in bothhuman and mouse models of experimental allergic asthma. Inthe latter system, the crucial role of the pan-neurotrophin recep-tor p75 has been investigated in p75 NTR–/– mice. Furthermore,NGF transgenic animals have been utilized to assess the con-tribution of NGF. The role of BDNF on differentiation andfunction of B-lymphocytes has been identified in BDNF–/– mice.In conclusion, our data support the concept that neurotrophinsmediate immunological and neuronal plasticity within theneuro-immune network of allergic disease in the lung and skin.

Interaction between the immune and neuroendocrine system –

focus on dendritic cells and skin

C. L. Butts and E. M. Sternberg

National Institute of Mental Health/NIH, Bethesda, MD, USA

Autoimmune diseases present clinically with many differences indisease pattern and are characterized by dysregulation of theimmune response to inflammation, pain, disease, and stiffness.Cells of the innate immune system, such as dendritic cells (DCs),have been shown to stimulate production of autoantibodies andself-recognized T cells that induce autoimmune disease. DCs areconsidered amongst the most important of the antigen-present-ing cells because of their highly efficient ability to present anti-genic peptides in the presence of MHC class II molecules thatactivates naıve lymphocytes. They also secrete cytokines and

express costimulatory cell-surface molecules that facilitate lym-phocyte activation. These cells are responsive to regulation bythe hypothalamic-pituitary-adrenal (HPA) axis, largely throughthe actions of the glucocorticoids. In addition to their long-recognized usefulness in pharmacotherapy of autoimmunediseases, glucocorticoids secreted the adrenal glands play animportant physiologic anti-inflammatory role in regulatinginnate immunity. Interruption of this negative feedback loop bygenetic, surgical, or pharmacological means leads to enhancedsusceptibility to autoimmune/inflammatory disease. Evidence forthe regulatory role of the HPA axis in autoimmune/inflammatorydisease and its implications for treatment will be discussed.

Vasoactive peptides, immune response andwound healing

Neurogenic modulation and vasoactive peptides in microvascular

biology

S. D. Brain, A. D. Grant, C. Tam, E. Pinter, A. Starr, J. Keeble

and N. Clark

Centre for Cardiovascular Biology and Medicine, King’s College,

Guy’s Campus, London SE1 1UL, UK

Acute neurogenic inflammation is observed after topical applicationof the TrpV1 agonist capsaicin to the mouse ear. Inflammatoryoedema is not detectable in the substance PNK1 receptor knockoutmouse, indicating the importance of this receptor in mediatingneurogenic oedema formation.However, neurogenic vasodilatationremains not only in the NK1 receptor knockout mouse but also inthe CGRP knockout mouse after sensory nerve stimulation. Inter-estingly, it is abolishedwhenboth theCGRPandsubstancePdilatorpathways are blocked. This has led us to suggest that there is afacilitatory interaction between endogenously released CGRP andsubstance P, such that when both are present there is redundancy interms of dilatormechanisms. This could be relevant to the apparentmaintenance of peripheral vasodilator tone, when either NK1 orCGRP receptor antagonists are given systemically for evaluation asnovel therapeutic agents. The relative activity of CGRP and thestructurally related non-neuropeptide adrenomedullin have beencompared. Adrenomedullin is less potent as a vasodilator, butfunctionally relevant concentrations can be induced in inflamedskin. Both peptides, as a consequence of their vasodilator activity,influence inflammatory processes (e.g. oedema and cell accumula-tion) acting through theCGRP receptor. Thus, adrenomedullin canact as a non-neuronally derived CGRP agonist in skin.

Neural regulation of endothelial cell-mediated inflammation

J. C. Ansel

Department of Dermatology, Northwestern University, Chicago,

IL, USA

There is much evidence that neurocutaneous interactions mayplay an important role in modulating a wide variety of biologi-cal processes in the skin including inflammation. Perhaps, thekey cell type for directing inflammatory processes in the skin isthe dermal microvascular endothelial cell (DMEC). All leuko-cyte trafficking during cutaneous inflammation is mediated byDMEC. This presentation will review various ways in which thecutaneous neurosensory system can modulate DMEC activities,and how this may lead to novel approaches in our understand-ing and treatment of inflammatory skin disease.

Neurogenic inflammation of the airways and skin

P. Baluk and D. M. McDonald

Cardiovascular Research Institute and Department of Anatomy,

University of California, San Francisco, CA, USA

According to its original definition, the main feature of neuro-genic inflammation is plasma leakage induced by the stimulation

Abstracts

574

of peripheral sensory nerves. The plasma leakage from post-capillary venules is accompanied by increased blood flow dueto dilatation of upstream arterioles, and by other phenom-ena, including leukocyte adhesion and migration. Neurogenicinflammation occurs in the airways, skin, and parts of theintestinal, urinary, and reproductive tract of man and animals,but varies markedly in its magnitude and extent. In skin,neurogenic inflammation is manifested as wheal and flare.Both phenomena are mediated by neuropeptides released fromunmyelinated sensory nerve fibers via stimulation of capsaicin-sensitive vanilloid receptors. Substance P is mainly responsiblefor the plasma leakage, acting via NK-1 receptors present ontarget blood vessels, whereas calcitonin gene-related peptide andsubstance P both induce vasodilatation. Sensory neuropeptidesalso trigger release of histamine from mast cells, which contri-butes substantially to plasma leakage in the skin, but less so inthe airways. The increase in vascular permeability is due to afocal, transient, and fully reversible formation of gaps locatedbetween endothelial cell junctions. Neurogenic inflammationcan be inhibited by preventing the stimulation of sensory nerves,by depleting them of their neuropeptide transmitters, bypresynaptic inhibition of transmitter release, or by blockingneuropeptide receptors. Anti-inflammatory drugs such asb-adrenergic agonists and steroids can reduce neurogenicinflammation by stabilizing endothelial cells.

Receptors for calcitonin gene-related peptide and adrenomedullin:

implications for skin cell biology

J. A. Fischer, L. M. Ittner and W. Born

Research Laboratory for Calcium Metabolism, University of

Zurich, Zurich, Switzerland

The specificity of a G-protein-coupled calcitonin receptor(CTR) and a CT receptor-like receptor (CLR) for calcitoningene-related peptide (CGRP), adrenomedullin (AM) and amylinis defined by the heterodimeric non-covalent association withthree receptor-activity-modifying proteins (RAMPs). Chemicalcross-linking of proteins at the cell surface and immunoprecipi-tation have identified [125I]CGRP/CLR/RAMP1, [125I]AM/CLR/RAMP2 and -3 as well as [125I]CGRP/CTR/RAMP1,[125I]amylin/CTR/RAMP1 and -RAMP3 complexes. CLR/RAMP1 defines a CGRP receptor. CLR/RAMP2 and -3 corres-pond to AM1 and AM2 receptor isotypes, respectively. TheAM1 receptor cross-reacts with CGRP at high and the AM2receptor at low concentrations. With the N-terminal deletion ofamino acids 14–20 of the mouse, CLR-selective inactivation ofAM over CGRP receptor function was obtained. As a result,functional interaction with AM was no longer possible. Over-expression of the CLR in transgenic mice together with theendogenous RAMP2 results in thinning of the hairs duringpostnatal development (L. M. Ittner et al. conference poster).In conclusion, the extreme N-terminus of the CLR and theextracellular N-terminal domains of RAMP1 and -2 containamino acid residues that provide AM- or CGRP-bindingselectivity of the CLR/RAMP complexes. Hair development isattenuated, resulting in the thinning of the hairs and eventuallyalopecia during postnatal development.

The role of neuropeptides and neuropeptide-degrading enzymes in

wound healing

John E. Olerud

Department Dermatology, University of Washington, Seattle,

WA, USA

Thirty to 40% of diabetic patients develop sensory neuropathy.Neuropathy is a major causal factor in diabetic ulcers. Only31% of neuropathic diabetic ulcers heal in 20 weeks. Patientswith neuropathy have a 15.5-fold excess risk of amputation.Diabetic patients with neuropathy particularly lose epidermaland papillary dermal sensory nerves which release neuropep-

tides such as substance P (SP). Neutral endopeptidase (NEP),the enzyme that degrades SP, is dramatically over expressed inpatients with diabetic neuropathy. SP has positive effect onwound healing. Treatment strategies related to the nervoussystem for prevention and treatment of diabetic ulcers currentlybeing studied include prevention of neuropathy with tight con-trol of blood glucose, application of neuropeptides, nervegrowth factors (NGF), and antagonists of NEP.

Relevance of leptin for cutaneous wound healing

I. Goren, J. Pfeilschifter and S. Frank

Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang

Goethe-Universitat, Frankfurt am Main, Germany

Recently, we demonstrated a direct role for leptin in skin repair.However, pair-fed experiments with caloric restricted ob/obmice clearly suggested that resolution of insulin resistance alsorepresents a pivotal process that contributes to an acceleratedhealing. Immunohistochemistry revealed wound keratinocytesto be the insulin-sensitive skin compartment, as the cellsstrongly expressed the insulin receptor (InsR). We now focusedto analyze the expression and activation of insulin-signalingcomponents in wound keratinocytes of healthy, diabetic, andleptin-treated mice. First, we observed a downregulation of theInsR expression in normal wounds, which is precisely co-regulated by a downregulation and phosphorylation-dependentinactivation of its negative regulator, the InsR-associated pro-tein tyrosine phosphatase-1B (PTP-1B). In line, a strong expres-sion of InsR during late repair was associated with an increasedpresence of a dephosphorylated and thus active PTP-1B and theInsR substrate (IRS)-1- and -2-signaling molecules, suggestingthat the InsR, its negative regulator PTP-1B, and both IRSwere functionally connected during healing. Impaired healingconditions were characterized by a loss of InsR, IRS-1, and -2expression but paralleled by increased amounts of phosphor-ylation-inactived PTP-1B. This finding suggested a regulatorymechanism to compensate for the loss of insulin-signalingcomponents and to allow a more efficient signaling from thereduced amount of remaining InsR and IRS-1/-2 duringimpaired healing. Interestingly, systemic treatment of ob/obmice with leptin resolved the dysregulated availability ofthe insulin-signaling components: InsR/IRS expression wasupregulated, and the amount of dephosphorylated and thusactive PTP-1B co-increased in wounds of treated animals.Finally, our findings could be confirmed by analysis of in vivoactivation of protein kinase B as one possible readout for insulinsignaling.

Apoptosis and oxidative stress

Nerve growth factor and its role in epidermal homeostasis

C. Pincelli

Institute of Dermatology, University of Modena and Reggio

Emilia, Modena, Italy

Nerve growth factor (NGF) belongs to the neurotrophin (NT)family which also includes brain-derived neurotrophic factor(BDNF), neurotrophin-3 (NT-3) and NT-4/5. Human keratino-cytes synthesize and release all NT and express both the low-(p75NTR) and the high-affinity receptor (trk). Keratinocytestem cells (KSCs) express higher levels of NGF as comparedwith young transit amplifying (TA) cells, while NGF levels arealmost undetectable in TA cells. While trk is expressed in allbasal keratinocytes in a homogeneous pattern, p75 is expressedonly in a subpopulation of basal keratinocytes. p75 is stronglyexpressed in young TA cells, whereas it is scarcely visible in TAcells and absent in KSCs. Moreover, Caþþ strongly enhances p75expression in subconfluent cells. NGF stimulates keratinocyte

Abstracts

575

proliferation and protects keratinocytes from spontaneous andUVB-induced apoptosis. These activities are performed throughtrk receptor, while the role of p75 is more controversial. p75belongs to the TNF receptor superfamily, and it shares withother members of this family a death domain which signalsapoptosis upon binding to the ligands. BDNF or NT-4 induceskeratinocyte apoptosis through p75, while the trk inhibitorK252 augments cell death in p75-overexpressing keratinocytes.BDNF also activates c-JUN kinase (JNK) in p75-transfectedkeratinocytes. Taken together, these data point to a dual role ofNT in epidermal homeostasis.

a-MSH as a modulator of apoptosis in cutaneous biology

M. Bohm, T. A. Luger, T. Scholzen, T. Schwarz and A. Schwarz

Department of Dermatology and Ludwig Boltzmann Institute for

Cell Biology and Immunobiology of the Skin, University of

Munster, Munster, Germany

The molecular pathways regulating ultraviolet (UV) radiation-induced apoptosis of melanocytes, a cell population cruciallyinvolved in the protection of epidermal keratinocytes againstthe harmful effects of UV light, are poorly characterized. Weshow that the a-melanocyte-stimulating hormone (a-MSH)blocks UVB-induced apoptosis of normal human melanocytesin vitro. The effect of a-MSH is not restricted to melanocytes butis also operative in cells that do not produce melanin, forexample in human epidermal keratinocytes and in dermal fibro-blasts. a-MSH not only delays but also protects melanocytesfrom UVB-induced cell death. The anti-apoptotic activity ofa-MSH is not mediated by a filtering effect or induction ofmelanin synthesis. a-MSH also does not induce changes in thecell cycle distribution or expression of Bcl2, Bclx, CD95 (Fas/APO-1) and FasL. In contrast, a-MSH markedly reduces theformation of cyclobutane pyrimidine dimers induced by UVBradiation. Human dermal fibroblasts carrying a defective XPAgene are not protected from UVB-induced apoptosis by a-MSH.These results highlight a novel biological activity of a-MSH aswell as novel regulatory pathways within the UV response ofskin cells targeted by this neuropeptide.

Apoptotic pathways targeted by a-MSH in human melanocytes

Z. Abdel-Malek and A. L. Kadekaro

Department of Dermatology, University of Cincinnati, Cincinnati,

OH, USA

It is well established that treatment of human epidermal mela-nocytes with a-melanocortin (a-MSH) stimulates eumelaninsynthesis and proliferation (1–3). Also, a-MSH enables humanmelanocytes to overcome the arrest in G1 and induces melano-genesis following UV exposure (4). Recently, we discovered anew role of a-MSH as a survival factor that inhibits the UV-induced apoptosis of human melanocytes. The survival effect ofa-MSH is mediated by the activation of the melanocortin 1receptor (MC1R) and is absent in melanocytes with the loss offunction MC1R. Also, the survival effect of a-MSH is indepen-dent of stimulation of melanogenesis, because it is evident intyrosinase-negative albino melanocytes. The anti-apoptoticeffect of a-MSH involves the activation of the IP3kinase path-way and is inhibited by LY 294002. Moreover, treatment of UV-irradiated melanocytes with a-MSH activates Akt/PKB, thesubstrate for IP3kinase. a-MSH suppresses the apoptotic effectof UV by inhibiting the reduction in Bcl2 levels, possibly as aconsequence of activating the transcription factor Mitf in mela-nocytes. a-MSH confers photoprotection not only by stimulat-ing eumelanin synthesis but also by enhancing DNA repair,particularly nucleotide excision repair. We have found that a-MSH enhances the repair of UV-induced cyclobutane pyrimi-dine dimers and pyrimidine (6-4) pyrimidone photoproducts,

thus maintaining the genomic stability and inhibiting mutagen-esis of melanocytes. Our findings offer an explanation for theincreased susceptibility of individuals with the loss of functionmutations in theMC1R gene to sun-induced skin cancer, includ-ing melanoma.Supported by NIH grant R01 ES009110 and by Skin

Research Grant from Johnson and Johnson Skin ResearchCenter.

References

1. Abdel-Malek A, Swope V B, Suzuki J et al. Proc Natl AcadSci USA 1995: 92: 1789–1793.

2. Suzuki J, Cohe R D, Im S et al. Endocrinology 1996: 137:1627–1633.

3. Hunt G, Kyne S, Ito S et al. J Invest Dermatol 1995: 104: 83–85.4. Im S, Moto O, Peng F et al. Cancer Res 1998: 58: 47054.

Antioxidant and anti-inflammatory activities of melanocortin

peptides

J. W. Haycock1, R. P. Hill

1, R. J. Elliott

1, M. J. Wagner

2and

S. MacNeil1,2

1Sheffield University, Department of Engineering Materials,

Sir Robert Hadfield Building, Mappin Street, and 2Sheffield

University, Division of Clinical Sciences, Northern General

Hospital, Sheffield, UK

a-Melanocyte-stimulating hormone (a-MSH) has previouslybeen identified as a potent anti-inflammatory agent in varioustissues including the skin. It operates by binding to the melano-cortin-1 receptor (MC-1R) which results in the elevation ofcyclic AMP. a-MSH opposes the action of several proinflam-matory cytokines including tumour necrosis factor-a (TNF-a).We have shown that a-MSH can inhibit TNF-a-stimulatedactivation of nuclear factor-kB (NF-kB) in human cultured mela-nocytes, melanoma cells, keratinocytes, fibroblasts, Schwanncells and olfactory ensheathing cells. It also inhibits TNF-a-stimulated upregulation of intercellular adhesion molecule-1(ICAM-1) in many of these cells and can inhibit peroxide-stimulated activation of glutathione peroxidase, suggesting anantioxidant role. a-MSH is also able to stimulate intracellularcalcium release in keratinocytes and fibroblasts (which do notreadily show detectible cyclic AMP elevation) but only in thepresence of PIA (an adenosine agonist). The carboxyl terminaltripeptides KPV/KP-D-V are reported to be the minimalsequences necessary to convey anti-inflammatory potential, butevidence on how they act is not fully known. Stable transfectionof Chinese hamster ovary cells with MC-1R suggests that theKPV peptides operate by this receptor, at least by elevatingintracellular calcium. Elevation of cyclic AMP by these tri-peptides has not been detected in any cell type studied; however,calcium elevation can inhibit TNF-a-stimulated NF-kB activity(as for cyclic AMP). In conclusion, the MSH peptides conveyanti-inflammatory and antioxidant activity in many cell types inskin and nerve, by counteracting proinflammatory cytokinesignalling. The KPV peptides appear to act functionally via theMC-1R and can also elevate intracellular calcium.

Melatonin as an antioxidant – a role in human aging?

R. J. Reiter

Department of Cellular and Structural Biology, The University of

Texas Health Science Center, San Antonio, TX, USA

N-Acetyl-5-methoxytryptamine, commonly known as mela-tonin, was isolated and structurally identified by a dermato-logist, Aaron B. Lerner, in 1958. His interest stemmed fromthe fact that melatonin has potent skin-lightening effects inamphibians and some other non-mammalian species. Inhumans, however, melatonin is not capable of lightening skinor in reducing pigmentation. While melatonin synthesis is the

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576

best known to occur in the pineal gland, it is also produced inseveral other organs including the skin. Melatonin is a power-ful, multifaceted direct free radical scavenger and indirectantioxidant. Numerous studies have shown that melatoninproduction wanes with increasing age and its loss has beenspeculated to be consequential in free radical-mediated cellu-lar and organ deterioration that occurs in the elderly. Add-itionally, a number of free radical-related diseases, e.g.Alzheimer’s disease, Parkinsonism, cataract formation, etc.,may in part be a consequence of the loss of endogenousmelatonin production. The administration of melatonin inanimal models of these diseases typically defers their progres-sion and limits their severity. Likewise, due to its antioxidantproperties and via other mechanisms, melatonin may reduceskin aging. Melatonin, when applied topically to the skin ofhumans, has obvious protective effects against ultraviolet(UV) light-induced erythema. Also, lipid peroxidation inhuman skin fibroblasts due to their exposure to UV-B isreduced when melatonin is present.

Clinical perspectives and futureimplications for neurobiology of the skin

Novel agonists for cold receptors on sensory neurons

E. T. Wei

School of Public Health, University of California, Berkeley, CA,

USA

An exciting research topic these past 2 years has been theidentification of transient receptor potential (TRP) channelprotein receptors on sensory neurons that are linked to thermo-sensation. Two compounds, menthol and icilin, were shown toactivate the putative cold receptor, TRP-M8 (CMR1, trp-p8),in vitro. Here, some of the pharmacological properties of icilin aredescribed and compared tomenthol, summarized in the table below.

MentholStimulates TRP-M8 receptor, does not require extracellularcalcium for activity, and does not stimulate the ANKTM1receptor.Mint odorIrritant and harsh taste at high concentrationsDuration of action less than15min after topical applicationAfter systemic administration, lethal at high concentrationsdue to anesthetic activity

IcilinStimulates TRP-M8 and ANKTM1 receptor and requiresextracellular calcium for activityNo odorNo irritant activity on skin or mucous membranesAction on inflamed skin and mucous membranes (e.g.eyelids, lips, nasal, and anorectal) can last 4–8 hNo anesthetic activity, and no lethality at doses of up to1.5 g/kg intraperitoneally, but after systemic administrationproduces ‘wet dog shakes’ which are rapid, alternating con-tractions, and relaxations of the supination and pronationmuscles about the spinal axis

Acetylcholine receptors in cutaneous biology – receptorology and

therapeutic implications

S. Grando

Department of Dermatology, University of California, Davis, CA,

USA

Free cytotransmitter acetylcholine (ACh) is present in physio-logically relevant concentrations in human skin, and ACh and

cholinergic drugs alter vital functions of keratinocytes (KCs).KCs respond to ACh via classical ACh receptor types that useCa2þ as a second messenger. The repertoire of cholinergic recep-tors changes with cell maturation, so that at each stage of theirdevelopment, KCs respond to ACh via different combinations ofnicotinic (nAChR) and muscarinic (mAChR) receptors. BasalKCs respond to ACh predominantly via a3b2(b4)+a5 nAChRsand the M3 mAChR; prickle KCs have more a5-containinga3 nAChRs and also express a9 nAChR as well as M4 andM5 mAChRs; granular KCs posses mainly a7 nAChR andM1 mAChR. We used three independent approaches toelucidate role of each receptor in KC biology: 1) Subtypeselective drugs, 2) Gene silencing with small interfering RNA orantisense oligonucleotides, and 3) Gene knockout in transgenicmice. Obtained results indicate that KC nAChRs and mAChRexhibit synergistic control of cell adhesion and crawling locomo-tion. KC cell-cell adhesion is regulated through a3 and a9nAChRs as well as M3 mAChR. KC chemokinesis is controlledby a3 and a7 nAChRs and M3 and M4 mAChRs. KC chemo-taxis toward nicotinic agonists is mediated by a7 nAChR. Thesefindings offer novel insights into the mechanisms of ACh-mediated modulation of epidermalization and may aid thedevelopment of novel methods to promote wound healing andinhibit tumor metastasis.

a-MSH and fragments: potential therapeutic agents in

inflammation

T. Brzoska1, T. Kucharzik2, C. Maaser2, T. E. Scholzen1,

M. Bohm1 and T. A. Luger1

1Department of Dermatology and Ludwig Boltzmann Institute for

Cell Biology and Immunobiology of the Skin, and2Department of

Medicine B, University of Munster, Munster, Germany

a-Melanocyte-stimulating hormone (a-MSH) exerts numerousimmunomodulatory and anti-inflammatory activities, which atleast partly are mediated through the melanocortin receptor-1(MC-1R), expressed on monocytes, dermal fibroblasts, dendriticcells (DCs), endothelial, and epithelial cells. Accordingly,a-MSH downregulates the production of proinflammatorycytokines and the expression of costimulatory molecules onantigen-presenting cells (APCs) via inhibiting the activation oftranscription factors such as NF-kB, while upregulating the pro-duction of suppressor factors such as IL-10. Besides a-MSH, itsC-terminal-tripeptide KPV and the IL-1b-derived tripeptide KPTare capable of modulating APC functions. Using a mouse modelof contact hypersensitivity (CHS), systemic and epicutaneousapplication of a-MSH, KPV, or KPT inhibited CHS inductionand induced hapten-specific tolerance. However, using MC-1R-deficient mice (MC-1Re/e), tolerance induction was found to beindependent of MC-1R expression. To further investigate themechanisms responsible for tolerance induction, adoptivetransfer experiments were performed. a-MSH-treated haptenizedDCs inhibited CHS and induced hapten-specific tolerance, viainduction of regulatory T lymphocytes (Treg). In contrast, using amurine model of intestinal inflammation [Dextransulfate (DSS)-induced colitis], the expression of a functional MC-1R wasfound to be crucial for a-MSH to exert its anti-inflammatoryactivity; in wt mice, weight loss was reduced and the survivalrate significantly was improved upon treatment with a-MSHor KPV. However, DSS colitis was significantly aggravated inMC1-Re/e mice, resulting in the death of all animals. Bonemarrow transplantation from wt mice did not alter the courseof inflammation, indicating that MC-1R expression on non-hematopoietic cells is crucial for host defense. These findingsfurther support the therapeutic potential of a-MSH-relatedpeptides for the treatment of inflammatory, autoimmune, andallergic diseases.

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577

Current developments in treatment of pruritus: neurophysiologic

basis and clinical efficacy

S. Stander

Department of Dermatology, University of Munster, Munster,

Germany

Non-histaminergic pruritus of any origin is difficult to treat andrequires evaluation of new therapeutic strategies which wereoffered by recent neurophysiologic findings. For example, thediscovery of opioid receptors on mast cells and nerve fibersenables the effective administration of opioid receptor anta-gonists. Up to now, 130 patients with pruritus of different originwere successfully treated with the oral opioidantagonist naltrex-one. A significant therapeutic response was achieved under50–150mg daily in 66% of patients. In prurigo nodularis, nal-trexone also contributed to healing of the skin lesions. Tachy-phylaxis was infrequent, and adverse drug effects, in particularnausea, were of short duration. Only recently, the vanilloidreceptor 1 (VR1) was demonstrated on nerve fibers and mastcells what explains the antipruritic efficacy of the topicalapplication of capsaicin. Upon continual therapy with thisVR1-ligand, neuropeptides are depleted and the nerve fiber isdesensitized. A total of 53 patients with pruritus of differentorigin (prurigo nodularis, psoriasis, eczema, aquagenic pruritus,PUVA itch, hydroxyethyl starch-induced itch, and lymphoma)were selected to receive capsaicin four to six times daily ingradually increasing concentrations (0.025–0.1%). After cessa-tion of the symptoms of neurogenic inflammation, all of thepatients experienced a complete elimination of pruritus within12 days. In addition, capsaicin largely contributed to healing ofthe skin lesions in prurigo nodularis.

Clinical trial design to study the effects of pharmacological

intervention on different models of skin pain and inflammation –

methodology and first results

M. Rother1, M. Schmelz

2, J. Lehmann

1and M. Grossmann

3

1Research and Development, IDEA AG, Munich,

2Department of

Anaesthesiology, Faculty of Clinical Medicine, and3Institut fur

Klinische Pharmakologie GmbH, Mannheim, Germany

Skin photodamage due to excessive ultraviolet (UV) light expo-sure is followed by a series of biochemical and immunologicevents that cause inflammation including the release of prosta-glandins, lipoxygenase products, cytokines (e.g. tumour necrosisfactor-a), adhesion molecule, reactive oxygen radicals and mastcell-derived mediators, such as histamine and tryptase. In var-ious inflammatory pain conditions, including UVB sunburn,nociceptors are sensitized to mechanical and thermal stimuli atthe site of inflammation. This phenomenon, which leads tohyperalgesia, can be used to investigate pharmacological effectsof different drugs and drug formulations in the model of UVB-induced skin inflammation. Two additional models are usefulfor evaluating the direct, prostaglandin-independent, modula-tory effects on neuronal excitability of nociceptors (chemosensi-tivity): the chemically induced pain (capsaicin test) and itch(histamine test). IDEA-070 is a novel carrier-based topicaldosage form of an analgesic drug that acts by inhibitingcyclooxygenase (COX-1 and COX-2) and lipoxygenase, therebyreducing prostaglandin- and leukotriene-mediated inflam-matory reactions. We used all three described models in theframework of a phase I clinical study to assess efficacy ofIDEA-070. The results of an interim analysis of the UVBmodel from the first 12 healthy volunteers indicate thatIDEA-070 as well as hydrocortisone-21-acetate (HC) reducesthe UVB-induced heat hyperalgesia if applied immediatelyafter UV exposure. In contrast, only IDEA-070 is effectivewhen used 6 h after exposure to UVB. In addition, IDEA-070,but not HC, suppressed erythema development if used immedi-ately after UVB exposure as well as 6 h after UVB irradiation.

Clinical potential of Melanotan1 (NDP-a-MSH) in skin

protection – current status and future perspective

S. M. Humphrey1, T. K. T. Oo2 and St. C. Barnetson2

1Epitan Ltd, Melbourne, and 2Department of Medicine (Derma-

tology), Melanoma and Skin Cancer Research Institute, Sydney

Cancer Centre, University of Sydney at Royal Prince Alfred

Hospital, Sydney, Australia

There is good epidemiological evidence that melanin in theskin protects against sunburn and skin cancer resulting fromexcessive ultraviolet radiation (UVR) exposure. Melanin issynthesized in a multistep biochemical pathway within specia-lized cell types in human skin called melanocytes situated inthe epidermis. UV light causes an increase in melanocyte-stimulating hormone (a-MSH) receptor activity on cutaneousmelanocytes (MC1-R) that results in an increase of eumelaninwithin the epidermal melanocytes. Melanotan is a syntheticanalogue of a-MSH with potent melanogenic activity in animalsand humans. We have recently completed a double-blind,randomized controlled study in 80 healthy Caucasian adultsubjects. A fixed dose of Melanotan (0.16mg/kg/day) orplacebo in a 3 : 1 ratio was given as three cycles of daily sub-cutaneous injections over 10 days each month for threeconsecutive months. Changes in skin pigmentation were quanti-fied by serial skin reflectance measurements and UVR injury, asdetermined by �3 MED exposure to broadband UVR, wasassessed by measurement of apoptosis (sunburn cells). Resultsdemonstrated that a highly significant increase in skin melaninwas attained in all subjects on active treatment compared withplacebo, and this was especially pronounced in subjects withlighter skin types (Fitzpatrick I and II; P< 0.0001). In theselatter subjects, significant sunburn protection was also deter-mined. The ability of Melanotan to stimulate melaninproduction without the associated UV-induced skin damagemay prove a useful adjunct to current photoprotective strategiesto stem the marked rise in skin cancer incidence. Currently, weare studying the therapeutic efficacy of a slow-release implant ofMelanotan in skin disorders such as polymorphic light eruptionand psoriasis.

Poster abstracts

Co-culture of neurones with Merkel cells and keratinocytes

Y. Chateau1, G. Dorange1, J. F. Clement1, N. Rougier2 and

L. Misery1

1Laboratory of Cutaneous Neurobiology, University of Western

Brittany, Brest, and 2Biopredic International, Rennes, France

In order to provide a model for in vitro studies of the interac-tions between skin and the nervous system, we have performeda co-culture of epidermal cells and neurons. We used a tri-compartmented box with separated domains. In the central partof this box, we put an epidermal suspension (4 millions cells/ml)obtained from biopsies of human skin. Around this centraldomain, we disposed sensorial neurons from the dorsal rootganglia of rats. The periphery was occupied by neurons fromthe dorsal horn of spinal cord. Sensorial neurons grew in lowdensity (500 cells), on a glial layer, in a medium conditioned byastrocytes. After 15 days of culture, cells were fixed and stainedwith monoclonal antibodies directed against PGP 9.5, keratins,or cytokeratin 20 (Merkel cells). We obtained a co-culture withthree identifiable territories, equivalents of epidermis, rootganglia, and spinal cord. Nervous fibers specifically grew fromthe sensorial neurons to epidermal cells or to the spinal cordequivalent. We observed synapse-like contacts between nerveendings and Merkel cells or keratinocytes. This model allows us

Abstracts

578

to reconstruct in vitro an equivalent of sensitive nerve fibers,connected for one part to a spinal cord equivalent and on theother part to an epidermis equivalent. Such a model could be usedto understand the origin and the function of Merkel cells in theepidermis and to study synapses in the skin.

NMDA receptors influence the intracellular calcium concen-

tration and the expression of differentiation markers in HaCaT

cells

M. Fischer1, D. Glanz2, T. Klapperstueck1, J. Wohlrab1,

E. Fiedler1 and W. C. Marsch1

1Department of Dermatology and Venerology, and 2Institute of

Physiological Chemistry, Martin-Luther-University Halle-

Wittenberg, Halle (Saale), Germany

Ionotropic glutamate receptors (ligand-gated, ion-channelproteins) of the N-methyl-D-aspartate (NMDA) receptor typecould enable a transmembranous calcium influx from theextracellular space. Though ionotropic glutamate receptors arepredominantly neuronal receptors, they are also expressed innon-neuronal tissues like keratinocytes. Therefore, investiga-tions were performed to study the function of NMDA receptorsin HaCaT cells. The intracellular calcium concentration ofHaCaT cells was studied under the influence of the selectivereceptor agonist NMDA and the selective NMDA antagonistMK-801. The proliferation of HaCaT cells was investigatedusing the crystal-violet method. Furthermore, the expression ofCytokeratin 10 and Filaggrin was examined in HaCaT cells afterblocking NMDA receptors with MK-801. Using NMDA, therewas a significant increase in the number of HaCaT cells showingelevated intracellular calcium concentration, at a dose between25mM and 1mM (up to 84.6% of cells). The NMDA-associatedcalcium influx could be significantly suppressed by prior appli-cation of MK-801. There was no influence of NMDA on theproliferation of HaCaT cells. There was also no cytotoxic effectof NMDA (up to 1mM). The expression of Cytokeratin 10 andFilaggrin could be suppressed by blocking NMDA receptorswith MK-801. The investigations show that glutamate receptorsof the NMDA-type play a role in the differentiation of HaCaTcells by regulating their intracellular calcium concentration.

Synergistic regulation of neuropeptide levels by internal and

external stimuli

J. Hosoi, K. Inoue, Y. Ashida and T. Tsuchiya

Life Science Research Center, Shiseido, Yokohama, Japan

The skin is the most peripheral organ confronting the externalenvironment. We found that the level of substance P is regulatedby both internal and external stimuli. Mock interview inducedthe acute stress in human assessed by the measurement of serumcortisol. The serum level of substance P increased within 1 hafter the mock interview. Interestingly, the increase wassuppressed by inhalation of 1,3-dimethoxy-5-methylbenzene.Similar regulation was observed in mice. Furthermore, restraintor the intravenous administration of substance P induced theactivation of cutaneous mast cells. Housing under the conditionof lower humidity (about 30%) for 24 h caused the increase inthe substance P level both in peripheral blood and in the skin.Restraint for 2 h during the housing under the condition oflower humidity increased the substance P level further. Theactivation of cutaneous mast cells under the dry condition wasreported. These data suggest that cutaneous neuropeptide levelis regulated by both psychological and environmental mech-anisms. The regulation may cause the downregulation of thethreshold of the induction of itch and inflammation.

Glutamine and glutamine synthetase immunoreactivities in

Schwann cells of the rat glabrous skin

K. E. Miller1, K. Jones1, A. E. Wood1 and R. M. Kriebel2

1Department of Anatomy and Cell Biology, Oklahoma State

University Center for Health Sciences, Tulsa, OK, and2Department of Biomedical Sciences, Philadelphia College of

Osteopathic Medicine, Philadelphia, PA, USA

We have shown recently that glial cells of the peripheral nervoussystem contain substrates and enzymes related to the glutaminecycle, e.g. glutamine, glutamine synthetase, and glutamatedehydrogenase (Miller et al., Brain Res 2002: 945: 202–11). Thepresent study used immunohistochemistry to determine the cellu-lar distribution of glutamine and glutamine synthetase in the ratglabrous skin. Normal adult Sprague–Dawley rats were used forthese studies. Glutamine and glutamine synthetase were localizedwith both immunoperoxidase and immunofluoresent techniques.Glutamine and glutamine synthetase immunoreactivities wereabundant in Schwann cells of large dermal nerve bundles.An enrichment of glutamine and glutamine synthetase immuno-reactivies occurred in terminal and near-terminal Schwann cells indermal papilla and at the dermal–epidermal interface. Wehypothesize that the peripheral glial glutamine cycle is importantfor supplying neuronal energy demands in dermal nerve fibers.Furthermore, the peripheral glial glutamine cycle in terminalSchwann cells most likely produces glutamine for sensory term-inal uptake. This glutamine would be converted to glutamate forrelease from sensory afferents. Glutamate released from sensoryafferents may be important for the development of hyperalgesiaand allodynia. Terminal Schwann cells, therefore, are potentialtargets for modulating sensory afferent sensitivity during acuteand chronic pain.Supported by NIH AR47410 (KEM).

New insights into the nerve end organ of human skin

C. M. Reinisch1, W. Weninger

1,2, C. Mayer

1, K. Paiha

3,

H. Lassmann4and E. Tschachler

1,5

1Department of Dermatology, Brain Research Institute University

of Vienna Medical School, Vienna, Austria,2The Center for Blood

Research, Department of Pathology, Harvard Medical School,

Boston, MA, USA, 3Institute of Molecular Pathology, 4Division

of Neuroimmunology, Brain Research Institute University of

Vienna Medical School, Vienna, Austria, and 5Centre de

Recherches et d’Investigations Epidermiques et Sensorielles

(CE.R.I.E.S.), Neuilly, France

Bearing the sensory nerve end organ, the skin establishes contactto our environment. So far, the analysis of the cutaneous ner-vous system was dependent on the use of tissue serial sections.Because such samples inherently allow visualization of only asmall part of the mainly horizontally oriented nervous system ofthe skin, we searched for possibilities enabling a more compre-hensive view. Here, we present a method based on the immu-nostaining of dermal sheet preparations for subsequent analysisby electron microscopy and light, or laser scanning microscopy.We used antibodies against PgP9.5 and NCAM/CD56, bothshowing a regular network of fibers covering the entire super-ficial dermis. The bulk of free ending nerve fibers ramifiedwithin 25 mm of the dermo–epidermal junction, whereas belowthat only larger nerve bundles were present. Along the course ofnerve fibers, we observed NCAM/CD56þ protrusions with dia-meters ranging from 5 to 15 mm. We further characterized theseprotrusions demonstrating the ultrastructural features of ter-minal non-myelinating Schwann cells ensheathing nerve fibres.Depending on the body region, we detected between 140 andover 300 individual terminal Schwann cells/mm2 skin surface.In a double staining for NCAM/CD56 and vWF, we analyzedthe topographical relationship of the nerve end organ to theblood vessels of the skin. In conclusion, this novel methodallows for the first time a complex three-dimensional depiction

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579

of the cutaneous nervous system over several cm2. Additionally,terminal Schwann cells can be studied in detail in situ for thefirst time. Furthermore, application of this method may providenew impetus in the investigation of the cutaneous nerve endorgan under physiological and pathological conditions.

RAB4a- and RAB11a-dependent recycling and resensitization of

the neurokinin 1 receptor

D. Roosterman, F. Schmidlin, M. Steinhoff and N. W. Bunnett

Department of Surgery and Department of Physiology, University

of California, San Francisco, CA, USA, and Department of

Dermatology, University of Munster, Munster, Germany

Neurokinin 1 receptor (NK1R) is an important mediator ofdepression, pain, and inflammation. Understanding the mole-cular mechanisms regulating NK1R trafficking is of importancebecause of the essential role of receptor transport in regulatingcell function and probably disease. We examined NK1R traf-ficking and resensitization with regard to the concentrationdependency of the ligand, substance P. NK1R trafficking wascharacterized using GFP-coupled b-arrestin, rab5, rab4, andrab11. NK1R resensitization was calculated by calcium mobili-zation assays. NK1R stimulated with 10 nM substance P (SP)rapidly internalized. NK1R and b-arrestin were sequestrated inthe perinuclear location, as demonstrated by confocal micro-scopy. NK1R did not reappear at the cell surface 30min afteragonist stimulation. Expression of a dominant negative rab5amutant impaired translocation from superficial to perinuclearlocations at the stage of the early endosome. Similarly,b-arrestin and rab5a induced rapid internalization of NK1Rstimulated with 1 nM SP. In contrast, 1 nM SP mediated trans-port of receptor and b-arrestin from the plasma membrane toonly superficial vesicles. NK1R rapidly recycled after rab4 andrab11 activation. Stimulation with 1 nM SP did not inducesequestration of NK1R and associated proteins but resulted inreceptor resensitization in less than 5min. Overexpression ofrab5a-induced NK1R transport to perinuclear compartments.Thus, while b-arrestin appears to be important for receptorendocytosis, the transport of NK1R seems to be regulated byrab5a. This study identifies a concentration-dependent traffick-ing and resensitization mechanism of NK1R, indicating a newfunction of rab5 as a control protein for directing NK1R intodifferent intracellular compartments and suggesting a role ofrab11 in GPCR recycling via early sorting endosomes.

Brain-derived neurotrophic factor, epidermal growth factor, or

A-Raf-induced growth of HaCaT keratinocytes requires extra-

cellular signal-regulated protein kinase

O. G. Rossler and G. Thiel

Department of Medical Biochemistry and Molecular Biology,

University of Saarland Medical Center, Homburg, Germany

The epidermal growth factor (EGF) receptor plays an importantrole in epithelial cells by controlling cell proliferation and survi-val. Keratinocytes also express another class of receptor tyrosinekinases, the neurotrophin receptors. To analyze the biologicalrole of the neurotrophin brain-derived neurotrophic factor(BDNF) in keratinocytes, we expressed the BDNF receptorTrkB in immortalized human HaCaT keratinocytes. Stimula-tion of HaCaT-TrkB cells with BDNF induced DNA synthesis,an indication of proliferating cells. An analysis of the signaltransduction cascade revealed that the activated TrkB receptoreffectively utilized components of the EGF receptor signalingpathway to control cell proliferation. Mitogenic signalinginduced by BDNF or EGF was completely abrogated by theMAP kinase kinase inhibitor PD98059, whereas the inhibitionof phosphatidylinositol-3 (PI3) kinase by wortmannin only delayedthe proliferative response. The importance of the extracellularsignal-regulated protein kinase (ERK) signaling pathway for

growth of HaCaT keratinocytes was further demonstrated withHaCaT cells engineered to express an inducible A-Raf-estrogenreceptor fusion protein (DA-Raf:ER). HaCaT cells expressingDA-Raf:ER proliferated following the activation of mutantA-Raf protein kinase. Proliferation was completely inhibited byPD98059. Proliferation of HaCaT cells induced by EGF, BDNF,or DA-Raf:ER was also accompanied by the biosynthesis ofthe transcription factors Egr-1 and c-Jun, suggesting that theseproteins may be part of the mitogenic signaling cascade.

Docking and fusion sites in human endothelial cells imaged and

measured by using atomic force microscopy

S. W. Schneider1, R. Ossig2, T. Gorge2, R. Matzke2, P. Rogge2,

A. Niemeyer2 and H. Oberleithner2

1Department of Dermatology, and

2Department of Physiology,

University of Muenster, Muenster, Germany

The vascular endothelium with its salient location at the interfacebetween blood and tissue plays a pivotal role in the process ofblood coagulation and inflammation. The transition into a pro-coagulatory and proinflammatory state upon stimulation (i.e.neuropeptides) is referred to as endothelial cell activation. Onefundamental characteristic of this activation is the induction ofvon Willebrand factor (vWF), IL-8, and P-selectin exocytosis.These molecules are stored in large (up to 3mm) cone-like vesiclescalledWeibel Palade bodies (WPBs). By using atomic force micro-scopy (AFM), we are able to visualize the apical surface topo-graphy of human endothelial cells with nanometer resolution. Inaddition, AFM allows to measure local cell stiffness with a spatialresolution of 100nm. In previous studies, we showed thatendothelial cells have a readily releasable pool ofWPBs. In restingcells, this intracellular docked vesicle pool can be imaged asplasma membrane protrusions with a height of 140+ 50nm(+SEM; n¼ 8) and a diameter of 275+ 85nm (+SEM; n¼ 8).Stiffness measurements revealed that humps are characterized bydecreased cell membrane stiffness of 30% compared to surround-ing cell membrane due to a reduced subapical actin network. Afterstimulation of the cells with hyperosmolaric solutions or hista-mine, these docked WPBs immediately fuse with the plasmamembrane forming large (diameter: approximately 500nm) exo-cytotic pores and release vWF into the supernatant (measured byELISA) and expose P-selectin. Immunostaining of vWF wasfound to be localized next to the exocytotic pores imaged byAFM. The data indicate that human endothelial cells have areadily releasable pool of WPBs that allows the instantaneousrelease of vWF, IL-8, and exposure of P-selectin. These distinctareas of exocytosis are characterized by cell membrane protru-sions and decreased cell membrane stiffness due to a reduced actincortical network.

Solar-simulated, ultraviolet radiation-induced, upregulation of the

melanocortin-1 receptor, pro-opiomelanocortin, and a-melanocyte-stimulating hormone in human epidermis in vivoM. Schiller

1*, T. Brzoska

1* M. Bohm

1, D. Metze

1, T. Scholzen

1,

A. Rougier2and T. A. Luger

1

1Department of Dermatology and Ludwig Boltzmann Institute

for Cell Biology and Immunobiology of the Skin, University of

Munster, Munster, Germany, and 2La Roche-Posay Pharma-

ceutical Laboratories, Asnieres, France

*These authors contributed equally to this work.

Ultraviolet (UV) light is one of the most crucial environmentalfactors with regard to its capacity to induce skin cancer, pre-mature aging of the skin, and immunosuppression. AlthoughUV directly affects the function of epidermal cells, many ofthese effects are mediated by the induction of cytokines, growthfactors, and neuropeptides such as a-melanocyte-stimulatinghormone (a-MSH). Recently, in addition to its well-knownpigmentation inducing activity, a strong anti-inflammatory

Abstracts

580

as well as an immunomodulatory potential of a-MSH has beenrecognized. The aim of this study was to determine whether UVirradiation affects the expression of both a-MSH and the mela-nocortin-1 receptor (MC-1R) in human epidermis in vivo. Thevolar aspects of the forearms were exposed to twice the minimalerythema dose of solar simulating radiation (SSR). Three, 6, and24 h after irradiation, the pro-opiomelanocortin (POMC) andinterleukin-10 (IL-10) mRNA levels in suction blister-inducedepidermal sheets were considerably upregulated as detected bysemiquantitative RT-PCR. Furthermore, a-MSH and IL-10protein levels in blister fluids were significantly increased 24 hafter UV irradiation, an effect which could be abolished by theapplication of the broadspectrum sunscreen AntheliosXL1

prior to UV (SSR) exposure. In addition, enhanced MC-1RmRNA and receptor protein expression upon SSR wasascertained by RT-PCR and immunohistochemistry of theepidermal sheets, respectively. POMC-derived neuropeptidessuch as a-MSH may therefore play an important role inmodulating UV-induced inflammation.

Agonist-independent, high constitutive activity of the human mela-

nocortin 1 receptor

J. Sanchez-Mas, I. Gerritsen, J. C. Garcıa-Borron and

C. Jimenez-Cervantes

Department of Biochemistry and Molecular Biology, School of

Medicine, University of Murcia, Murcia, Spain

The melanocortin hormones act on epidermal melanocytes toincrease eumelanogenesis, melanocyte dendricity and likely mela-nosome transfer to keratinocytes. These actions are mediated bythe melanocortin 1 receptor (MC1R), positively coupled toadenylyl cyclase. Gain-of-function Mc1r alleles are associatedwith dark, eumelanic skin. Conversely, loss-of-function variantsor overexpression of agouti, the natural antagonist, yield yellow,pheomelanic furs. In humans, loss-of-function MC1R variantsare associated with fair skin, poor tanning and increased skincancer risk. Therefore, MC1R is a key regulator of mammalianpigmentation. An induction of constitutive pigmentation inamelanotic mouse melanoma cells following the expression ofMC1R has been reported, suggesting that this receptor mightdisplay agonist-independent activity, although this aspect hasnot yet been comparatively studied for MC1R and Mc1r. Weshow that the expression of MC1R in heterologous systemsleads to high agonist-independent increases in intracellularcAMP. This basal signalling is a function of the quantity ofreceptor expressed, is considerably higher for MC1R than Mc1rand is also observed in human melanoma cells overexpressingMC1R. Moreover, MC1R basal signalling is abolished orreduced by point mutations impairing the response to agonists.Lastly, the expression of wild-type MC1R, but not of loss-of-function mutants potently stimulates forskolin activation ofadenylyl cyclase, a feature characteristic of constitutively activeG-coupled receptors. Therefore, we conclude that MC1Rdisplays a strong agonist-independent constitutive activity.

Neurogenic skin inflammation in stress-triggered inhibition of hair

growth in mice is promoted via nerve growth factor-dependent

pathways

P.C.Arck1,E.M.J.Peters1,B.Handjiski1,A.Kuhlmei1andR.Paus2

1Department of Internal Medicine, Charite, University Medicine

Berlin, Berlin, and 2Department of Dermatology, University Hospital

Eppendorf, Hamburg, Germany

Recently, we have pointed to the existence of a brain-hair follicleaxis (BFA), with neuropeptide substance P (SP) as one can-didate mediator, to which stress-triggered hair loss is imputable.Based on findings indicating that levels of nerve growth factor(NGF) increase upon exposure to stressful events, which isparticularly striking within the context of the BFA, becauseNGF is known to increase the release of SP, we then aimed at

dissecting the role of NGF in stress-triggered hair loss. Weobserved increased expression of NGF, analyzed by real timePCR and immunohistochemistry, in stress-exposed mice witha depilation-induced hair cycle. Expression of NGF receptorp75 was also upregulated with stress, and TrkA receptor wasmoderately downregulated. Upon neutralization of NGF byantibody injection, stress-triggered premature onset of catagen,which was accompanied by apoptosis and increased number/activation of perifollicular mast cells and macrophages, wassignificantly inhibited. Interestingly, subcutaneous injection ofrecombinant NGF to mimick stress effects resulted in anincreased percentage of SP-positive neurons in dorsal root gang-lia. Taken together, our data indicate that an interactive com-munication network between sensory nerves and immune cellsin the skin is promoted by stress-triggered release of NGF andresults in mast cell activation and migration of macrophages, therelease of proinflammatory neuropeptides, i.e. SP. Such disequi-librium, which may be referred to as neurogenic inflammation,constitutes the prerequisite of increased hair loss.

A ‘hot’ new twist to hair biology – involvement of vanilloid

receptor-1 signaling in human hair growth control

E. Bodo1,2, T. Bıro1, A. Telek1, G. Czifra1, Z. Griger1, I. B. Toth1,

J. Lazar1, A. Meschalchin3, T. Ito2, A. Bettermann2, P. Pertile3,

L. Kovacs1 and R. Paus2

1Department of Physiology, University of Debrecen, Debrecen,

Hungary, 2Department of Dermatology, University of Hamburg,

Hamburg, Germany, and 3Cutech Srl, Padova, Italy

The functional role of VR1, which we and others have recentlyidentified on several epithelial and mesenchymal human skin cellpopulations, was investigated in the human hair follicle (HF), asa prototypic epithelial–mesenchymal interaction system. VR1immunoreactivity was confined to distinct epithelial compart-ments of HFs in anagen and catagen, while dermal papillafibroblasts and HF melanocytes were VR1 negative. In organculture, VR1 activation by capsaicin resulted in a dose-dependentand VR1-specific inhibition of hair shaft elongation, suppressionof proliferation, promotion of apoptosis, and induction ofcatagen transformation, possibly due to upregulation of a potenthair growth inhibitor TGFb2. Cultured outer root sheath (ORS),as well as HaCaT, keratinocytes also expressed functional VR1,whose stimulation inhibited proliferation, induced apoptosis, andelevated intracellular calcium concentration. Finally, VR1 stimu-lation of cultured ORS keratinocytes upregulated the expressionof recognized endogenous hair growth inhibitors (IL-1b andTGFb2) and downregulated the expression of stimulators (HGF,IGF-1, and SCF), while key differentiation markers (CK17,CK14, filaggrin, and involucrin) remained unaffected. In conclu-sion, VR1 is a significant novel player in human hair growthcontrol underscoring that its physiological functions in humanskin far extend beyond sensory neuron-coupled nociception.

Expression of MC-R, POMC and POMC peptides and evidence

for a immunoregulatory role of a-MSH in human dermal papilla

cells

M. Bohm1, M. Eickelmann

1, Z. Li

1, A. Vogt

2, U. Blume-

Peytavi2, G. Barsh3 and T. A. Luger1

1Department of Dermatology and Ludwig Boltzmann Institute

for Cell Biology and Immunobiology of the Skin, University of

Munster, Munster, 2Department of Dermatology, Center of

Experimental and Applied Cutaneous Physiology, University

Medical Center Charite, Humboldt University of Berlin, Berlin,

Germany, and 3Department of Genetics, Stanford University

School of Medicine, Stanford, CA, USA

Pro-opiomelanocortin (POMC)-derived peptides are well-known regulators of pigmentation and proliferation of epider-mal and hair follicle-derived melanocytes. We demonstrated

Abstracts

581

that human dermal papilla cells (DPCs), a distinct myofibro-blastic cell population of the hair follicle, participate in thecutaneous POMC system. DPCs in vitro and in situ expressedthe melanocortin receptor-1 (MC-1R) as well as MC-4R asshown by RT-PCR, immunofluorescence and immunohisto-chemistry. Expression of POMC but not agouti signalling pro-tein, a natural MC-1R/MC-4R antagonist, was also detectablein DPC. Generation of POMC peptides by DPCs in vitro wasdemonstrated by immunofluorescence and ELISA studiesrevealing the expression of both adrenocorticotropin and b-endorphin. To investigate the functional relevance of MC-Rexpression in DPCs, we examined the effect of a-MSH oninterferon-g (IFN-g)-induced expression of intercellular adhe-sion molecule-1 (ICAM-1), an adhesion molecule upregulated ininflammatory disorders of the hair follicle such as alopeciaareata. a-MSH markedly suppressed the IFN-g-mediatedupregulation of ICAM-1 in DPCs as shown by real-time PCRstudies, while a-MSH alone did not have any effect. Our datasuggest that melanocortins such as a-MSH mediate paracrineand autocrine effect in the dermal papilla whose disruption maycontribute to inflammatory diseases of the hair follicle.

In vitro interactions between sensory nerves, epidermis, hair

follicles and capillaries in a tissue-engineered reconstructed skin

V.Gagnon1,M.Gingras1,L.Germain1,H.D.Durham2andF.Berthod1

1Laboratoire d’Organogenese experimentale, Universite Laval,

Hopital Saint-Sacrement, and 2Montreal Neurological Institute,

McGill University,Montreal,Quebec, Canada

Recent findings have established that cutaneous nerves modulateboth skin homeostasis and various skin diseases, by influencingcell growth and differentiation, inflammation and wound healing.In order to study the influence of epidermis, hair follicles andcapillaries on sensory neurons, and vice-versa, we developed atissue-engineered model of innervated endothelialized recon-structed skin (MIERS). Mouse dorsal root ganglia neuronswere seeded on a collagen sponge populated with human fibro-blasts and human endothelial cells. Keratinocytes or mice new-born immature hair follicle buds were then seeded on the oppositeside of the MIERS to study their influence on sensory nervesgrowth, and vice versa. A vigorous neurite elongation wasdetected inside the reconstructed dermis after 14 and 31 days ofneurons culture. The presence of endothelial cells induced a sig-nificant increase of the neurite elongation after 14 days of culture.The addition of human keratinocytes totally avoided the twofolddecrease in the amount of neurites observed between 14 and 31days in controls. We have successfully developed the MIERS thatallowed us to study the effects of epidermis and capillaries onnerve growth. This model will be a useful tool to study themodulation of sensory nerves on wound healing, angiogenesis,hair growth and neurogenic inflammation in the skin.

Defective hair formation in calcitonin-like receptor transgenic

mice

L. M. Ittner1, K. Husmann

1, R. Muff

1, J. Gotz

2, S. Bonneick

3,

U. Suter3, W. Born

1and J. A. Fischer

1

1Research Laboratory for Calcium Metabolism,

2Division of

Psychiatry Research, University of Zurich, and 3Department of

Cell Biology, ETH Zurich, Zurich, Switzerland

The heterodimeric calcitonin-like receptor (CLR)/receptor-activity-modifying protein 2 (RAMP2) complex is an adreno-medullin (AM) receptor. Here, transgenic mice expressing aV5-CLR in hair follicles have been generated. Skin sections ofthe transgenic mice and of control littermates have been investi-gated. The hair diameter of the mice was measured during hairmorphogenesis and first hair cycle. Size and weight of V5-CLRtransgenic and control mice were indistinguishable. But afterday 12, the coat of the transgenic mice is waved. The number

of hairs with a small diameter was significantly higher in trans-genic mice as compared with control littermates (P< 0.001).There skin sections revealed immunoreactive V5-CLR in thebulb of cycling hair follicles. On in situ hybridization, RAMP2encoding mRNA colocalized with the V5-CLR protein. Auto-radiographic examination showed specific 125I-AM binding inthe same place. In conclusion, CLR/RAMP2-overexpressingmice reveal a defined phenotype with thinning of the hairsduring postnatal development.

CRH peptides modulate proliferation, melanogenesis and dendri-

city in human follicular melanocytes

S. Kauser1, A. Slominski

2, E. T. Wei

3and D. J. Tobi

1

1Department of Biomedical Sciences, University of Bradford,

Bradford, UK,2Department of Pathology, University of

Tennessee, Memphis, TN and3School of Public Health,

University of California, Berkeley, CA, USA

Corticotropin-releasing hormone (CRH) is the most proximalelement of the hypothalamic-pituitary-adrenal axis (HPA) andis the chief regulator of pituitary POMC gene expression andthe subsequent production and secretion of POMC peptides.Previously, our laboratories documented cutaneous expressionof CRH, urocortin and functional CRH receptors (CRH-Rs),suggesting their role in skin physiology and pathology. Humanskin predominately expressed CRH-R1 with CRH-R2 beingexpressed primarily in the adnexal structures. While CRH-Ractivity has been implicated in the regulation of epidermal cellfunction, a role for these receptors in human hair biology has notyet been demonstrated. This study was designed to investigate theeffects of modified CRH peptides (D-Glu20)-CRH, (D-Pro5)-CRH and (D-Pro4)-urocortin with respective selectivity forCRH-R1 and CRH-R2 on behaviour of cultured hair folliclemelanocytes (HFMs) derived from scalp of seven normal individ-uals. HFMs were stimulated with these peptides (10�7�10�10M)for 72h. (D-Glu20)-CRH (10�8M) and (D-Pro5)-CRH (10�9 and10�10M) markedly increased cell dendricity, melanogenesis andproliferation (P< 0.01) compared with unstimulated levels. While(D-Pro4)-urocortin failed to stimulate cell dendricity, this peptidedid stimulate melanogenesis (10�8M) (P< 0.01) and exhibited abiphasic proliferative response; stimulating pigment cell divisionat 10�7 and 10�8M (P< 0.01) but inhibiting proliferation at 10�9

and 10�10M (P< 0.01). Here, we demonstrate the existence offunctionally active CRH-Rs in cultured human scalp HFM andshow that signalling via these receptors modulates follicularmelanocyte dendricity, melanogenesis and proliferation. Thus,activation of CRH-Rs may have a pivotal role in the regulationof follicular melanocyte homeostasis.

CRH peptides modulate human hair follicle growth in vitro –

a preliminary study

S. Kauser1, A. Slominski

2, E. T. Wei

3and D. J. Tobin

1

1Department of Biomedical Sciences, University of Bradford,

Bradford, UK,2Department of Pathology, University of

Tennessee, Memphis, TN and3School of Public Health,

University of California, Berkeley, CA, USA

Mammalian skin may contain an equivalent of the hypo-thalamic-pituitary-adrenal axis (HPA), composed of locallyproduced corticotropin-releasing hormone (CRH) that, togetherwith signalling via CRH receptor 1 (CRH-R1) and CRH-R2,may regulate local homeostasis. Studies in murine skin havedemonstrated significant hair cycle-dependent fluctuations inthe expression of CRH and urocortin peptides and CRH-Rsgenes, suggesting a modulatory role for this signalling systemin hair growth/cycling. This study was designed to investigatethe effects of ligands showing increased selectivity for CRH-R1[(D-Glu20)-CRH (10�7 and 10�8M)] and CRH-R2 [(D-Pro5)-CRH (10�8 and 10�9M)] and (D-Pro4)-urocortin (10�7 and

Abstracts

582

10�8M) on human hair growth in ex vivo culture. (D-Pro5)-CRHcan also activate CRH-R1, while (D-Pro4)-urocortin is highlyselective for CRH-R2. Anagen hair follicles (HFs) were isolatedfrom human scalp and stimulated for 9 days, with 10 HFs testedper CRH peptide concentration. Preliminary findings indicatethat (D-Pro4)-urocortin (10�8M) stimulated a 79% mean hairfibre elongation compared to the initial HF length over the9-day study period. (D-Glu20)-CRH (10�8M) also stimulatedhair fibre elongation of 63% of initial length, while (D-Pro5)-CRH (10�9M), which inhibited hair fibre elongation, comparedto unstimulated controls. In agreement with our previousdetection of CRH-R1 and CRH-R2 in human HFs, the abovedata suggest the existence of a functionally active CRH peptide/receptor system in cultured human HFs and suggest that signal-ling via these receptors may participate in the regulation ofhuman hair growth/cycling in vivo.

Skin and hair follicle fibroblasts differentially express POMC

peptides, receptors and associated processing convertases during

the hair growth cycle and in vitro – implications for fibroblasttopographic differentiation in skin

S. Kauser1, R. O. Karoo

1, M. Bohm

2and D. J. Tobin

1

1Department of Biomedical Sciences, University of Bradford,

Bradford, UK, and 2Department of Dermatology, University of

Munster, Munster, Germany

Mesenchymal cells are involved in reciprocal mesenchymal–epithelial interactions during development and growth of skinand its appendages. Fibroblasts exhibit topographic differen-tiation and so constitute a highly diverse family of cells withdistinct and characteristic traits. This heterogeneity is also seenin the skin where hair growth inductive fibroblasts called folli-cular papilla (FP) fibroblasts are distinct from fibroblasts of theconnective tissue sheath (CTS), and both, in turn, are distinctfrom peripheral interfollicular dermal fibroblasts (DFs). POMCpeptides and their cognate receptors are expressed variably byseveral skin cells types, including fibroblasts. However, it is notclear how the POMC system is regulated in different skin fibro-blasts populations. We characterized the expression of thePOMC peptide family and their receptors and pro-hormoneconvertases (PCs) in human haired scalp during the hair growthcycle and in matched sets of DF, FP and CTS fibroblastscultured from normal adult male scalp. Expression ofPOMC peptides, PC 2 and m-OR in FP fibroblasts was highestduring anagen. By contrast, b-end, 7B2 and PC1 were broadlyundetectable during anagen but expression levels increased con-siderably during the apoptosis-driven catagen phase. Matchedsets of cultured FP and DF fibroblasts showed similar proteinexpression levels of a-MSH, ACTH, PC2, 7B2, m-OR andMC1-R. However, FP cells expressed higher levels of PC1 andb-end peptide and higher m-OR mRNA levels than DF cells.Thus, follicular and interfollicular fibroblasts represent hetero-geneous subpopulations that are likely to respond variably toPOMC peptides.

The murine hair follicle is a melatonin target

H. Kobayashi1,2, T. W. Dunlop

3, B. Tychsen

1, F. Conrad

1, T. Ito

1,

N. Ito1, S. Aiba

2, C. Carlberg

3and R. Paus

1

1Department of Dermatology, University of Hamburg, Hamburg,

Germany, 2Department of Dermatology, Tohoku University,

Serdai Japan, and 3Department of Biochemistry, University of

Kuopio, Kuopio Finland

The pineal hormone, melatonin exerts many functional effectson mammalian skin (e.g. melanogenesis inhibition, melanocytegrowth inhibition, and regulation of seasonal pelage hairgrowth). However, its cutaneous expression, regulation, andfunctional role are still obscure. The aim of this study was tocheck whether murine hair follicles are indeed direct, peripheralmelatonin targets which express melatonin membrane receptors

(MT1 and MT2) and orphan nuclear receptor a (RORa) whichinteract with melatonin. Immunohistochemistry revealed thatmurine hair follicle keratinocytes show both MT1-like immuno-reactivity (IR) and ROR-like IR, both of which changedsubstantially in a hair cycle-dependent manner. Both semiquan-titive RT-PCR for MT1 and MT2, and quantitive real-timePCR for MT1, MT2, and ROR on murine skin cDNA revealedthat all three genes are transcribed in normal mouse skin in haircycle-dependent manner. Functionally, melatonin significantlyinhibited the constitutional level of epidermal and hair folliclekeratinocyte apoptosis in short-term mouse skin organ culture.In conclusion, we here provide evidence that normal murine hairfollicles are prominent direct target for melatonin bioregulationwhich express MT1, MT2, and ROR, at least some of which arefunctionally active in situ. These receptors are regulated in a haircycle-dependent manner, suggesting a role of melatonin in haircycle control.

Induction of neuropeptides in skin innervating sensory neurons by

stress and nerve growth factor as a possible reason for hair growth

alteration

A. Kuhlmei1, Q. T. Dinh1, E. M. J. Peters1, J. Kandil1, R. Paus2

and P. C. Arck1

1Department of Internal Medicine, Charite, University Medicine

Berlin, Berlin, and 2Department of Dermatology, University

Hospital Eppendorf, Hamburg, Germany

Recently, we introduced a mouse model launching experimentalevidence for stress-induced hair growth inhibition (HGI), point-ing to the existence of a brain-hair follicle axis (BFA). Wesuggested that nerve growth factor (NGF), besides neuropeptidesubstance P (SP), is a candidate mediator along the BFA. Pub-lished data further indicate that stress-related neuropeptides,e.g. calcitonin gene-related peptide (CGRP) and SP may beinvolved in HGI. SP and CGRP are synthesized in dorsal rootganglia (DRG) and released after axonal transport in the skin.Thus, aim of the present study was to investigate the effect ofstress or subcutaneous injection of NGF, which mimics stressand regulates neuropeptide genes in sensory neurons, on theexpression of SP and CGRP in DRG. Anagen was induced inC57BL/6 mice by depilation and retrograde tracing wasemployed on day 9 post-depilation (PD). On day 14 PD, micewere either exposed to sound stress (n¼ 4) injected subcuta-neously with NGF (n¼ 4) or served as control (n¼ 4). On day16 PD, DRG (mean of 30/mouse) were harvested and SP andCGRP in skin-specific sensory neurons, as identified by thetracer dye, were labelled by immunohistochemistry andcounted. Stress exposure as well as NGF injection leads to asignificant induction of SP and CGRP in retrograde-labelledneurons. This allows us to conclude that sensitive dermal nervefibres are likely to originate from the presently identified neu-ropeptide-positive neurons. Peripheral activation of SP-expres-sing afferent nerve fibres via NGF-dependent pathways maycause neurogenic inflammation, eventually resulting in HGI.

Regulation of human hair follicle growth by neurokinin-1 receptor

ligands

S. Lachgar, S. Metenier, S. Quibeuf and M. Charveron

European Research Center on Skin, IRPF, Hotel-Dieu, Toulouse,

France

Recent reports revealed that neural mechanisms play a role inhair growth control. An induction of murine hair growth isobtained after treatment with substance P (SP). In this study,we examined the expression of a SP receptor: the neurokinin-1(NK1-R) during: 1) the human hair cycle, 2) on isolated hairfollicle, and 3) in cultured hair dermal papilla (HDPC) and theimpact of NK1-R ligands on the regulation of hair growth. Weused immunohistochemistry, Western blot and RT-PCR analy-sis to examine its expression. Anagen hair follicles showed astrong expression of NK1-R in dermal papilla and in the outer

Abstracts

583

root sheath. Weak expression of NK1-R was observed in hairfollicle at the catagen and telogen stages. Two isoforms of NK1-R protein corresponding to 44 kDa and 54 kDa were identifiedin cultured HDPC. A fragment of 640 bp corresponding toNK1-R was obtained by RT-PCR. The nested PCR showedthe expected band of NK1-R at 395 bp. HDPC treatment withSP increases the level ofNK1-R gene and protein. The increase isstronger with NK1-R-selective agonist [Sar9, Met (O2) 11]-SP.Ex vivo studies showed a significant dose-dependent stimulationof hair growth by SP and by NK1-R agonist. However, theNK1-R antagonist (L-732–138). showed a significant inhibitionof hair follicle growth. These results demonstrate the presence ofNK1-R in the growing human hair follicle. NK1-R is activatedby SP and NK1-R agonist. Ex vivo studies indicate that NK1-Rantagonist inhibited hair follicle growth.

Mouse reconstructed skin from hair buds developed innervated

hair follicles after grafting on athymic mice

D. Larouche, V. Gagnon, F. Berthod, A. Deschambeault and

L. Germain

LOEX/Hopital du Saint-Sacrement, Universite Laval, Quebec,

Canada

Recovery of sensation in grafted skin is a major concern inreconstructive surgery. Because the pilosebaceaous unit repre-sents an important tactile organ within the skin, we developed atissue-engineered reconstructed skin allowing the formation ofcomplete pilosebaceous units after grafting, and we evaluatednerve regeneration. This model is based on our self-assemblyapproach of tissue engineering. Sheets obtained after culturingmouse fibroblasts for 35 days with ascorbic acid, which allowscollagen synthesis, are superposed. Thereafter, hair buds takenfrom dermis of newborn mice after collagenase digestion orfreshly isolated newborn mice keratinocytes were seeded on thereconstructed dermis, cultured 10 days and grafted on athymicmice. One month after grafting, complete pilosebaceous unitswhere obtained in reconstructed skin containing initially hairbuds and were maintained up to 6 months. NF150 (neurofila-ments) immunostaining revealed systematical localization ofnerves around the pilosebaceous units 1 month after grafting.In contrast, nerves colonization was almost always absent whenhair buds were not included in the grafted reconstructed skin.However, after 6 months, the nerves were present in both skingrafted (with or without hairs). These results suggest that thepresence of hairs inside the reconstructed skin promotes nervesregeneration after grafting. Furthermore, the connection ofpilosebaceous units with nerves suggests that they may be func-tional for the detection of the tactile stimuli.

Detection of ACTH, MC-1R and MC-4R immunoreactivity in

human terminal and vellus hair follicles

K. Stieler1, A. Vogt1, Z. Li2, M. Bohm2 and U. Blume-Peytavi1

1Department of Dermatology, University Medicine Berlin, Charite

Campus Mitte, Berlin, and2Department of Dermatology,

University Munster, Munster, Germany

Pro-opiomelanocortin (POMC) peptides such as ACTH anda-MSH play an important role in the pigmentation of humanskin and hair. Both peptides bind to a family of transmembranereceptors known as the melanocortin receptors (MC-Rs). Toinvestigate the in situ expression of a-MSH, ACTH and MC-Rs, we performed immunohistochemical studies on humanterminal and vellus hair follicles. Immunoreactivity for ACTH,MC-1R and MC-4R was detected in both human terminal andvellus hair follicles, with a pronounced increase of stainingintensity in the suprabulbar and bulbar region of anagen hairfollicles. The MC-1R immunoreactivity in situ was in accord-ance with in vitro studies showing expression of MC-1R at theRNA level in human hair follicle keratinocytes. There was nodetectable difference in the staining pattern of strong, pigmentedterminal hair follicles as compared with fine, silky and non-

pigmented vellus hair follicles. These results indicate thatPOMC-derived peptides may play a regulatory role in thehuman hair follicle independent of the pigmentation process.The expression pattern of ACTH and its receptors MC-1R andMC-4R in the human hair follicle correlates with the immuneprivilege of the human hair follicle which is characterized by theabsence of immune cells in the bulbar region of anagen hairfollicles. Thus, ACTH may possibly be involved in the main-tenance of the peribulbar immune privilege in both, terminaland vellus hair follicles.

Substance P-induction of endothelial nitric oxide synthase

expression on human endothelial cells: modulation of this

neurogenic inflammation by an Avena Rhealba1oatmeal extract

M. F. Aries, C. Vaissiere and M. Charveron

Pierre Fabre Research Institute, CERPER, Cutaneous Cell

Biology Department, Toulouse, France

During cutaneous inflammatory diseases, the neuro-immuno-cutaneous system is impaired. In atopic dermatitis pathology,a perturbation of neuromediators release such as substanceP (SP), calcitonin gene-related peptide (CGRP) and bradykinin(BK) could be in part involved in the neurogenic inflammatorystate; both of these peptides identified in human skin are potentinducers of vasodilatation, may induce pruritus and couldmediate their effects via nitric oxide (NO). NO, considered asa major intra/intercellular messenger, is generated by NOsynthase (NOS) enzymes identified in several cell types in theskin. NO displaying vasodilator properties are constitutivelyreleased and can also be synthesized in response to inflamma-tory mediators such as SP neuropeptide. The aim of the presentstudy was first to determine whether SP, CGRP and BK wereable to stimulate NO release from human endothelial cells(HECs); the second objective was to induce neurogenic inflam-mation on HECs with SP (10–100 pM) and to evaluate theactivity of Avena Rhealba1 oatmeal – Roasting extract –(0.001–0.005%) on endothelial NOS (eNOS) mRNA expressionby RT-PCR. Avena Rhealba1 significantly inhibits endothelialcells substance P-induced expression of eNOS. Our resultsdemonstrate the regulator properties of Avena Rhealba1

with respect to neurogenic inflammatory response showingtherefore a real interest of Avena Rhealba1 in atopic dermatitisinflammatory pathology.

Melanocortin receptors in fibroblastic cell types of the skin –

in vitro and in vivo expression and functional relevanceM. Bohm1, S. Stander1, M. Eickelmann1, U. Blume-Peytavi2,

D. J. Tobin3 and T. A. Luger1

1Department of Dermatology and Ludwig Boltzmann Institute for

Cell Biology and Immunobiology of the Skin, University of

Munster, Munster, 2Department of Dermatology, Center of

Experimental and Applied Cutaneous Physiology, University

Medical Center Charite, Humboldt University of Berlin, Berlin,

Germany, and3Department of Biomedical Sciences, University of

Bradford, Bradford, UK

In contrast to the well-established role of melanocortins onmelanocyte function, the expression and relevance of melano-cortin receptors (MC-Rs) in skin fibroblasts is incompletelyunderstood. We recently showed that human dermal fibroblastsderived from foreskin express functional MC-1Rs which medi-ate an inhibitory action of a-melanocyte-stimulating hormone(a-MSH) on collagen metabolism (Bohm et al., J Biol Chem2004, in press). Here, we show by RT-PCR, immunofluores-cence, immunohistochemistry and immune electron microscopythat MC-1R expression is conserved in vitro and in vivo inadditional human fibroblastic cell types of the skin includingadult dermal fibroblasts, fibrosarcoma cells, connective tissuesheath fibroblasts and dermal papilla cells of the hair follicle.In vitro expression of MC-1R declines as a matter of cellular

Abstracts

584

senescence in dermal fibroblasts. Interestingly, a-MSH inhibitsthe inductive effect of interferon-g, an important proinflam-matory mediator in inflammatory and fibrotic skin diseases,on expression of adhesion molecules such as intercellularadhesion molecule-1 in human dermal fibroblasts. These datapoint towards an additional biological role of a-MSH as amodulator of inflammatory reactions in the connective tissuecompartment of the skin.

Sex differences in morphine modulation of cutaneous

inflammation: involvement of NK1 receptors

J. C. Elliott and D. T. Lysle

Biological Psychology Program, Department of Psychology,

University of North Carolina, Chapel Hill, NC, USA

Morphine produces an exacerbation of DNFB-induced cuta-neous inflammation when administered prior to antigen chal-lenge in a rat model of contact hypersensitivity (CHS). Recentevidence indicates that this exacerbation of inflammation issignificantly greater in females than that in males. This sexdifference in morphine modulation of CHS is due to the acti-vation of central m-opioid receptor pathways and appears to becorticosterone independent. Furthermore, the presence offemale gonadal hormones may account for observed the sexdifferences, because ovariectomy largely eliminates morphine’senhancement of CHS inflammation without affecting the basalCHS response. Given the involvement of substance P in thepathological exacerbation of CHS, we hypothesized that sex dif-ferences in NK1 receptor activation might account for morphine’sgreater effect on the expression of CHS in females. To that end,male and female CDF rats were treated with the selective NK1receptor antagonist SR140333 (1mg/kg, subcutaneously) 150minprior to and after DNFB challenge. SR140 333 treatment signifi-cantly reduced the magnitude of morphine’s enhancement of CHSin female, but not male rats without affecting the baseline CHSresponse in either sex. Preliminary data suggest that neuropeptidedepletion of the skin using capsaicin produces a similar pattern ofsex-dependent effects on morphine modulation of CHS. Takentogether, these experiments support the hypothesis that greateractivity of the peripheral NK1 receptor system in females mayin part account for sex differences in the magnitude of CHSfollowing morphine treatment.This project was supported by NIH grants DA15709 (D.T.L.)

and DA016836 (J.C.E). SR140 333 was generously provided bySanofi-Synthelabo.

Increased intraepidermal CGRP correlates with local immuno-

suppression after repeated broadband and narrowband UVB

F. J. Legat1,2,3, P. Wolf1, L. T. Jaiani4, R. Lang2, M. S. Wang3,

C. A. Armstrong4, J. C. Ansel4 and J. D. Glass3

1Department of Dermatology, Medical University Graz, Austria,2Department of Dermatology,

3Department of Neurology, Emory

University, Atlanta, GA, and4Department of Dermatology,

Northwestern University, Chicago, IL, USA

We investigated the effects of repeated broadband UVB (UVB-BB) and repeated narrowband UVB (UVB-NB) on intraepider-mal nerve fibers (ENF) immunoreactive (IR) for CGRP andon CHS to DNFB in hairless mice. Mice were exposed toequivalent subinflammatory UVB-BB (Kodacel-filtered FS20)or UVB-NB (TL01) 3� per week for 4 weeks. One, 3, or 7 daysafter the last UV exposure, the number of CGRP-IR ENF wasdetermined in exposed back skin. At the same time points, othermice were sensitized to DNFB on exposed back skin, challengedon abdominal skin 5 days later, and skin swelling was deter-mined at 24 h after challenge. UVB-BB and UVB-NB signifi-cantly increased the number of CGRP-IR ENF in exposed backskin and significantly suppressed the sensitization of animals toDNFB. For both treatments, these effects were maximal 1 day

after the last UV exposure. However, at 7 days after the lastUV exposure, in UVB-NB-irradiated animals, the number ofCGRP-IR ENF was still significantly increased and the sensiti-zation to DNFB significantly suppressed, while in UVB-BB-irradiated animals, these parameters were similar to that innon-irradiated controls. Thus, while repeated exposure toUVB-BB or UVB-NB or both induced increase of CGRP-IRENF as well as local immunosuppression, the effects afterrepeated UVB-NB were prolonged compared with that afterrepeated UVB-BB. This may contribute to the higher photo-therapeutic efficacy of UVB-NB vs. UVB-BB.

Regulation of galanin gene expression in human keratinocytes

K. Moritz1, E. Voglas

1, W. Sperl

1, J. W. Bauer

2and B. Kofler

1

1Department of Pediatrics, and

2Department of Dermatology,

General Hospital Salzburg, Salzburg, Austria

Recently, we have detected abundant expression of specificimmunoreactivity for galanin (GAL), a 29–30 amino acidneuropeptide, in the epidermis and in sweat glands in a non-neuronal distribution and to a lesser extent in nerve fibre bun-dles and smooth muscle of blood vessels. In order to uncover apossible function of GAL in human epidermis, we investigatedthe regulation of GAL mRNA expression in cultured primaryhuman keratinocytes (KCs). First, we determined whetherPMA, a protein kinase C activator which is known to be apotent upregulator of GAL gene expression in chromaffincells, has similar effects in KCs. Stimulation of human culturedprimary KC with PMA resulted in a sixfold increase of GALmRNA. Furthermore, we exposed KCs to a combination of theproinflammatory cytokines IL-1b and TNF-a which resulted ina twofold induction of GAL mRNA in comparison with asixfold induction of ICAM-1. Most interestingly, capsaicine,which usually leads to a secretion of neuropeptides from nervesendings, did not induce the secretion of GAL of KCs. In con-trast, constitutive GAL mRNA expression and peptide secretionwere reduced twofold upon treatment of KC with 10 mMcapsaicine for 20min and 24 h, respectively. To our knowledge,this is the first report on a direct regulatory effect by capsaicineon neuropeptide mRNA expression in non-neuronal cells.

Calcitonin gene-related peptide and adrenomedullin modulate

cytokine-induced microvascular endothelial cell cellular adhesion

molecule expression and NF-kB activationT. E. Scholzen1, M. Fastrich1, T. Brzoska1, C. A. Armstrong2,

J. C. Ansel2, R. Muff3, W. Born3, J. A. Fischer3 and T. A. Luger1

1Ludwig-Boltzmann Institute, Department of Dermatology,

University of Munster, Munster, Germany, 2Department of

Dermatology, Northwestern University of Chicago, Chicago, IL,

USA, and 3Department of Orthopedic Surgery and Medicine,

University of Zurich, Zurich, Switzerland

AM and the sensory neuropeptide CGRP are potent vasoactivemediators that activate high-affinity G-protein-coupled receptorsconsisting of receptor-activity modifying proteins (RAMPs) and aseven-transmembrane domain calcitonin receptor-like receptor(CRLR) with RAMP-1/CRLR as CGRP and RAMP2 or-3/CRLR as AM receptors. In this study, we have examined thepossibility that AM or CGRP modulate dermal microvascular ECadhesion molecule (ICAM-1 and VCAM-1) expression. PrimaryHDMEC or cells of the EC line HMEC-1 were transfected withcDNA expression vectors for an EGFP control, RAMP-1, RAMP-2andCRLRbyelectroporation,or leftuntransfected.StimulationofEC-overexpressing R1/CRLR or R2/CRLR with CGRP or AM(0.01–1000nM) resulted in a dose-dependent upregulation of intra-cellular cAMP. Importantly, when HDMEC transfected with R1/CRLR or R2/CRLRwere treated with TNFa in combination withCGRP or AM, these peptides interfered with the TNF-inducedexpression of ICAM-1 and VCAM-1 as well as the adhesion of

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585

lymphoblastoid cell lines to HDMEC monolayer in a biphasicmanner. Likewise, AM and CGRP modulated the activation ofnuclear factor kB (NF-kB) partly by inhibiting the TNFa-induceddegradation of cytosolic IkBa. Neither transfection with theorphan CRLR nor RAMPs alone was capable of mediating afull reduction of TNFa-induced ICAM-1 or VCAM-1 expression.In conclusion, CGRP and more pronounced AM are capable ofmodulating TNFa-induced EC CAM expression, which may be ofimportance for the regulation of leucocyte–endothelial cell inter-action during cutaneous neurogenic inflammation.This study was supported by the ‘‘Medizinische Forschungs-

gesellschaft Salzburg’’ and a grant of the Austrian Science Founda-tion (P14906).

Expression of vanilloid receptor subtype 1 (VR1/TRPV1) in the

skin – implications for neurogenic inflammation and nociceptive

sensations

S. Stander1,2, C. Moormann

2, M. Schumacher

3, M. Artuc

4,

T. A. Luger1,2, D. Metze

1and M. Steinhoff

1,2

1Department of Dermatology,

2Ludwig Boltzmann Institute for

Cell Biology and Immunobiology of the Skin, University of Munster,

Munster, Germany, 3Department of Anesthesia and Postoperative

Care, UCSF, San Francisco, CA, USA, and 4Department of

Dermatology, Charite, Humboldt-University of Berlin, Berlin,

Germany

The vanilloid receptor subtype 1 (VR1/TRPV1) is a non-selective cation channel that binds the vanilloid capsaicin andendogenous cannabinoids. In human skin, VR1 has recentlybeen shown to be expressed by keratinocytes in vitro and in vivo.To determine a precise localization of VR1 in other cutaneouscompartments in particular cutaneous nerve fibres, we investi-gated VR1 immunoreactivity as well as mRNA and proteinexpression in a series of normal and capsaicin-treated humanskin. VR1 immunoreactivity could be observed in cutaneoussensory nerve fibres, mast cells, epidermal keratinocytes, dermalblood vessels, the inner root sheet and infundibulum of hairfollicles, differentiated sebocytes, sweat gland ducts and thesecretory portion of eccrine sweat glands. Upon RT-PCR andWestern blot, the expression of VR1 was confirmed in primarymast cells and keratinocytes from human skin. During capsaicintherapy, VR1-receptor distribution was unchanged, while areduction of neuropeptides (substance P, calcitonin gene-relatedpeptide) was observed in nerve fibres. After cessation of capsai-cin therapy, neuropeptides re-accumulated in skin nerves. Inconclusion, VR1 is widely distributed in the skin, suggesting acentral role for this receptor, e.g. in nociception and inflammation.

The neuropeptide PACAP upregulates expression and release of

cytokines and cell adhesion molecules in human microvascular

endothelial cells via VPAC type 1 receptor

A. Steinhoff1, A. Grevelhorster

1, W. E. Schmidt

2, T. A. Luger

1

and M. Steinhoff1

1Department of Dermatology, University of Munster, Munster, and2Department of Medicine, University of Bochum, Bochum, Germany

Pituitary adenylate cyclase-activating peptide (PACAP) andvasoactive intestinal peptide (VIP) belong to the same super-family of neuropeptides which exert their effects by activatingG-protein-coupled receptors defined as PACAP. So far, threereceptor subtypes exist (PAC1R, VPACR-1 and VPACR-2).Because, PACAP appears to play a crucial role in cutaneousinflammation and vasoregulation, we examined the expressionand biological effects of this peptide in primary human dermalmicrovascular endothelial cells (HDMECs). We detected theexpression of PACAP and VPAC type 1 receptor at RNA andprotein level by RT-PCR and immunohistochemistry, indicatingan autocrine regulatory mechanism. cAMP assays revealedVPAC1R to be functional in these cells. RT-PCR showed upre-gulation of IL-8 in a time- and dose-dependent manner. ELISA

experiments confirmed release of IL-8 by HDMEC cells. Wealso investigated cell adhesion molecule expression after stimu-lation with PACAP. ICAM-1 mRNA was upregulated at 3 and6 h after treatment with PACAP, while VCAM was only upre-gulated maximally at 6 h after PACAP stimulation, indicatingthe regulation of cell adhesion molecule expression in humandermal endothelial cells via VPAC1R. Immunoreactivity forVPAC-1R was enhanced in microvascular endothelial cells ofpatients with atopic dermatitis and urticaria, indicating upregu-lation of this receptor in endothelial cells during cutaneousinflammation. In summary, VIP and PACAP may play animportant role in cutaneous neurogenic inflammation by acti-vating VPAC-1R on dermal microvascular endothelial cells.

Identification of novel genes regulated by a-melanocyte-stimulatinghormone in murine bone marrow-derived dendritic cells

T. Brzoska1, J. Ehrchen2, Z. Li1, T. A. Luger1 and M. Bohm1

1Department of Dermatology and Ludwig Boltzmann Institute for

Cell Biology and Immunobiology of the Skin, and 2Department of

Experimental Dermatology, University of Munster, Munster,

Germany

Many strains of evidence indicate that a-melanocyte-stimulatinghormone (a-MSH) elicits its immunomodulatory activity viabinding to melanocortin receptors (MC-Rs) expressed on mono-cytes and dendritic cells. In order to identify novel target genesregulated by a-MSH in these cells, we prepared bone marrow-derived dendritic cell precursors from BALB/c mice and treatedthem with GM-CSF and IL-4 for 6 days. The MC-R profile onthese immature dendritic cells was first determined by quantita-tive RT-PCR. Both transcripts for MC-1R and MC-5R weredetected in these cells. Cells were subsequently stimulated withdinitrobenzene sulfonic acid (DNBS), a-MSH or both sub-stances for 2 or 16 h. After RNA preparation, cDNA synthesisand in vitro transcripton hybridization of biotinylated cRNAsamples was performed on MG U74A Affymetrix gene chips.Data evaluation, cleansing, extraction and analysis of the morethan 12 000 cloned genes and expressed sequence tags wereperformed using the GENE DATA ANALYST vs. 1 Expres-sionist software. Filter criteria included a minimum thresholdof 100, normalization by the logarithmic mean and a qualitysetting of P< 0.04. Changes with a change factor of >2 wereregarded as significant. As expected, stimulation with DNBSresulted in induction or upregulation of genes encoding pro-inflammatory cytokines, growth factors, signal transductionintermediates and transcription factors. Treatment with a-MSHblocked the DNBS-driven upregulation of several known genessuch as IL-1 or CD86. On the other hand, a-MSHmodulated theexpression of several novel genes implicated in immunomodu-lation, e.g. IL-1b converting enzyme, IFN-g receptor, FK506-binding proteins or several neuropeptides and their receptors.These data indicate novel molecular targets by which a-MSHexerts its immunomodulatory activities in immunocompetentcells.

The Langerhans’ cell-like cell lines XS52 and XS106 express

mRNA for ciliary neurotrophic factor and neurotrophic factor 4/5

K. Seiffert1, J. A. Wagner2 and R. D. Granstein1

1Department of Dermatology, and 2Department of Neurology,

Weill Medical College of Cornell University, New York, NY,

USA

Neurotrophins are responsible for the survival and outgrowth ofnerves within the peripheral and central nervous systems. Thesefactors include brain-derived neurotrophic factor (BDNF),CNTF,NT 3, and NT4/5. We have previously shown that LCs lie in closeproximity to nerves and that several neuropeptides regulate LCfunction, implying that nerves send regulatory signals to LCs. Toevaluate the possibility that LC signal nerves by release of

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586

neurotrophins, we examined LC expression of neurotrophins byRT-PCR. To eliminate the possibility of contaminating keratino-cytes in highly enriched LC preparations, we utilized the LC-likecell lines XS52 (BALB/c derived) and XS106 (A/J derived) forinitial experiments. The RNA obtained was digested with DNaseto ensure complete absence of genomicDNA. Several independentRT-PCRs revealed expression of bands of the expected size forCTNF and NT4/5, but not for BDNF and NT3 in XS106 andXS52 cells. In contrast, the transformed keratinocyte cellline PAM212 expressed BDNF, as well as CTNF and NT4/5.Preliminary experiments with purified LC confirm the expressionof CTNF and NT4/5 and also show the expression of BDNF.However, we cannot be sure that BDNF expression is not due tokeratinocyte contamination. We conclude that LCs may regulatenerve cells by the release of neurotrophic factors.

Functional absence of neutral endopeptidase in mice promotes

hapten uptake, maturation and function of bone marrow-derived

dendritic cells

T. E. Scholzen1, M. Fastrich1, T. Brzoska1, C. A. Armstrong2,

J. C. Ansel2 and T. A. Luger1

1Ludwig-Boltzmann Institute, Department of Dermatology,

University of Munster, Munster, Germany, and 2Department of

Dermatology, Northwestern University, Chicago, IL, USA

The bioavailability of neuropeptides such as substance P (SP)released from sensory nerves or immune cells during skininflammation is effectively controlled by proteolytic peptidases.Acute inhibition or genomic deletion of neutral endopeptidase(NEP) results in a SP-dependent augmentation of murine aller-gic contact dermatitis (ACD) by affecting sensitization andelicitation phase. In this study, we address the hypothesis thatabsence of NEP may modulate ACD responses by affectingbone marrow-derived dendritic cell (BmDC) maturation andfunction. BmDCs were generated from NEP-deficient mice(C57BL/6J-NEP–/–) or wild-type controls (C57BL/6J). FACSanalysis revealed that d3, d6 and d7 NEP–/– BmDCs expressedsignificantly more DC cell-surface markers and costimulatorymolecules compared to NEPþ/þ mice BmDCs, in particularafter BmDC maturation with LPS. In MLR utilizing d8BmDCs pulsed 3 h in vitro with DNBS and T cells from in vivoDNFB-haptenized NEP–/– and NEPþ/þ mice, BmDCs fromNEP–/– animals promoted proliferation of T cells with higherefficacy compared to wild-type mice BmDCs. Likewise, T cellsfrom NEP–/– mice demonstrated a higher proliferative responseto Concavalin A stimulation or CD3/CD28 ligation comparedto NEPþ/þ mice. In addition, acute systemic NEP inhibition inNEPþ/þ mice prior to sensitization with fluorescein isothiocya-nate (FITC) after 24 h significantly augmented uptake of FITCin the CD11c-positive DC fraction from regional lymph nodesbut not from spleen compared to cells obtained from mice nottreated with the NEP inhibitor. These data indicate that func-tional absence of NEP may significantly control cutaneousACD inflammatory responses by promoting hapten uptake,DC maturation and T-cell stimulation.

Role of nerve growth factor in nerve cells/skin cells physio-

pathology

M. Coassin1, N. Costa2, B. Stampachiacchiere3, A. Lambiase1,

S. Bonini1 and L. Aloe3

1Ophthalmology, University ‘Campus Bio-Medico’, Rome,2Faculty of Pharmacy, University of ‘Magna Graecia’, Catanzaro,

and 3Institute of Neurobiology and Molecular Medicine, CNR,

Rome, Italy

Studies carried out in our laboratory showed that NGF has aprimary role in wound healing, induces healing in the skin of farmanimals and promotes healing of human pressure ulcers. Morerecently, it has been demonstrated that NGF has a role in eye

physiology and promotes healing of corneal ulcers in humans. Inthe present study, we evaluated the efficacy of topical NGFtreatment in a dog model of eye chronic disease to promoteepithelial healing, reduction of corneal scarring, increase sensoryinnervation and goblet cells density. English Bulldogs who hadundergone the surgical removal of the lachrymal gland developedchronic keratoconjunctivitis sicca. One eye of each dog wastreated twice daily with 100ml of NGF ointment for 1 month,while the fellow eye was used as control. Eyes were evaluated atbaseline and after 1 month of NGF. NGF treatment significantlyrecovered epithelial keratopathy, reduced corneal haze, improvedcorneal sensitivity and strongly increased the tear production aswell as the conjunctival goblet cell density. This study shows thattopical application of NGF improves ocular surface signs in dogswith dry eye. Overall, our findings suggest a crucial role of NGFin nerve cells/skin cells physiopathology.

Modelization of skin endothelium reactivity, modulation by neuro-

peptides

J. Franchi1, C. Crola2, C. Marteau1, M. Mitterrand2,

S. Schnebert1, C. Mahe1, P. Andre1 and C. Kieda2

1Laboratoires de Recherche et Developpement, LVMH Branche

Parfums et Cosmetiques, Saint Jean de Braye, and 2Centre de

BiophysiqueMoleculaire, UPRCNRS, Ch. Sadron, Orleans, France

The endothelial cells which constitute the vascular wall areorgano-specific and reflect the biological reaction taking placein the tissue underlying the endothelial layer. This is the key forreparation after local injury, as, for example, response to UVinduced production of neuromediators by neurosensory cellsand keratinocytes. Endothelial cells react to the injury, mainlyby the production of adhesion molecules to recruit competentcells which are selected to adhere extravasate and can operatelocal anti-inflammatory/immune reaction. We demonstrate thatthe skin model constituted here by the cooperation of keratino-cytes and skin-derived microvascular endothelial cells is specificto the dermal selective processes as compared with the reactionof other organ microvasculary endothelial cells. Consequently,our data confirm that in order to reproduce in vitro local bio-logical reactions it is indeed crucial to use the proper andrepresentative endothelial cells. This is especially illustrated inthis work by the conjugated effects produced by the UV of media-tors production by keratinocytes and the modulation of their sub-sequent effect on activation of endothelial cell adhesion capacity.

Processing of POMC peptides by dermal microvascular endo-

thelial cells: implication for EC biologic functions and skin

inflammation

T. E. Scholzen1, S. Koenig2, M. Fastrich1, M. Bohm1 and

T. A. Luger1

1Ludwig-Boltzmann Institute, Department of Dermatology, and2Integrative Functional Genomics, University of Munster,

Munster, Germany

Dermal microvascular endothelial cells (ECs) are both sourceand target of the pro-opiomelanocortin (POMC) peptidesACTH and a-melanocyte-stimulating hormone (a-MSH). Theavailability of neuropeptides as important modulators of innateand adaptive immune responses is controlled by neuropeptide-specific peptidases such as neutral endopeptidase (NEP) orangiotensin-converting enzyme (ACE). In this study, we havetested the possibility that NEP or ACE expressed by ECs mayinfluence the local bioavailability of POMC peptides. Incuba-tion of ACTH1�39 with cell membranes prepared from the highNEP-/low ACE-expressing microvascular EC line 1 (HMEC-1)or from low NEP-/high ACE-expressing primary human dermalECs (HDMECs) for 30–480min resulted in a decrease in ACTHimmunoreactivity (IR) over time in membrane supernatants thatcould be partially blocked with NEP inhibitors as detected byradioimmunoassay. In parallel, a-MSH IR increased peakingafter 60min. Fragments generated by incubation of HMEC-1 or

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587

HDMEC membranes with ACTH1�39, ACHT1�24 or a-MSH for1–120min were further analysed by mass spectroscopy. HMEC-1membranes generated peptide products which could be altered byinhibition of NEP, but not ACE. Likewise, HDMEC membranesfragmented ACTH similar to HMEC-1membranes in the presenceof NEP inhibitors. Some of the proteins can be assigned to regularproteolytic cleavage, while others seem to bemodified. Importantly,HMEC-1 and HDMEC membranes also slowly degradeda-MSH, suggesting that EC proteolytic peptidases locallycontrol ACTH/a-MSH bioavailability, which may be importantin controlling cutaneous inflammation.

Cytotoxicity of capsaicin on cultured human skin fibroblast

S. J. Kim1, Y.-H. Won

1and J.-K. Kim

2

1Department of Dermatology, and

2Department of Pharmacology,

Chonnam National University Medical School, Kwangju, Korea

Capsaicin has been shown the different biologic and toxic effectson the non-neuronal celIs and serveral transformed cells. Thepresent study aimed at evaluating the cytotoxic mechanism ofcapsacin on the cultured human skin fibroblast. Normal neo-natal human fibroblasts were used forMTT assay to measure thechanges of celI survival, while growth factors, receptor antago-nist, antioxidants and calcium modulators were pretreated orco-treated with capsaicin. Fibroblast survival was significantlystimulated with EGF (10 ng/ml), bFGF (10 ng/ml) and capsaze-pine (10 mM) but inhibited by cycloheximide (1mg/ml). When200 mM capsaicin is given to fibroblasts, chromatin condensa-tions were observed at 12 h, and cell survival rate was reduced to25–50% at 24 h. Vanilloid receptor antagonists, capsazepine andruthenium red did not prevent the toxic effect of capsaicin, and10 mM capsazepine rather paradoxically enhanced the cytotoxi-city. In contrast to bFGF (10 ng/ml), EGF (10, 100 ng/ml)enhanced the cytotoxicity of capsaicin. Neuropeptides, sub-stance P (1, 10 nM) and CGRP (1, 10 nM), and a structuralanalogue to capsaicin, tyrosine (0.3–1.2mM) did not affect thecytotoxicity. However, antioxidants trolox (100 mM) and ascor-bic acid (0.1, 0.3mM) reduced the capsaicin cytotoxicity. Ofcalcium-modulating agents, nifedifine, a Ca2þ channel blocker(10, 20mM) and cyclopiazonic acid, a Ca2þ-ATPase inhibitor inER (10 mM) did not influence the cytotoxicity, but BAPTA/AMas a chelater for cytoplasmic free calcium ion (10 mM) signifi-cantly decreased capsaicin cytotoxicity. Unlike cycloheximide, aprotein synthesis inhibitor, z-VAD-FMK, a non-specific caspaseinhibitor, prevented the capsaicin cytotoxicity. The DNA ladderand TUNEL-positive cells were observed from the capsaicin-treated fibroblasts and Western blot revealed caspase-3 activity.Thus, capsaicin-induced cytotoxicity on human skin fibroblasts ismore likely to suggest the mechanism of apoptotic pathwaywhere antioxidants may play a role to prevent it.

Evidence for epidermal acetylcholine accumulation after oxidative

stress by H2O2 and possible implications

S. Elwary1, N. Gibbons

1, H. Rokos

2, J. M. Wood

1and

K. U. Schallreuter1,2

1Clinical and Experimental Dermatology University of Bradford,

and2Institute for Pigmentation Disorders in association with

Ernst-Moritz-Arndt Universitat Greifswald/Germany and the

University of Bradford, Bradford, UK

Acetylcholine (Ach) has been shown to be synthesized de novoand degraded in the human skin (1), while the presence ofacetylcholinesterase (AchE), the degrading enzyme, was firstmentioned in 1989 (2). It is now accepted that H2O2-relatedoxidative stress is a major player in the development/acceler-ation of vitiligo (3). This oxidative stress affects the recycling ofthe essential cofactor (6R)-L-erythro 5,6,7,8 tetrahydrobiopterin(6BH4) via H2O2-mediated oxidation of Trp andMet residues inthe structure of pterin-4a carbinolamine dehydratase (PCD) and

dihydropteridine reductase (DHPR) (3). Only very recently,it was recognized that AchE is also affected and deactivated by10�3M H2O2 causing the accumulation of Ach in the epidermisof patients with vitiligo (4). One of the implications of oxidativestress in vitiligo includes pruritus, which was for a long timeattributed to the presence of epidermal H2O2 in the 10

�3M

range. In this context, a role for Ach has been suggested inassociation with pruritus, and therefore, we would like tosuggest that Ach accumulation caused by H2O2-mediateddeactivation of AchE may well initiate pruritus (5). The Achaccumulation can also explain the earlier documented decreasedsweating in patients with vitiligo (6).

References

1. Grando S A, Kist D A, Qi M et al. J Invest Dermatol 1993:101 (1): 32–6.

2. Iyengar B, Moore J, Wood J M et al. Acta Anat (Basel).1989: 136 (2): 139–41.

3. Schallreuter K U, Elwary S M, Gibbons N C et al. J InvestDermatol 2001: 116 (1): 167–74.

4. Schallreuter K U, Elwary S M, Gibbons N C et al. BBRC2004: 315: 502–8.

5. Johansson O andWang L. Neurobiology 1993: 1 (3): 201–206.6. Elwary S M, Headly K, Schallreuter K U et al. Br JDermatol 1997: 37(1): 81–5.

The neurotrophin network in human skin

A. Marconi1, M. Dumas2, C. Fila1, F. Truzzi1, P. Atzei1,

M. Pignatti1, F. Bonte2, A. Giannetti1 and C. Pincelli1

1Institute of Dermatology, University of Modena and Reggio

Emilia, Modena, Italy, and2LVMH Branche Parfums et

Cosmetiques, Saint Jean de Braye, France

The neurotrophin (NT) family includes nerve growth factor(NGF), brain-derived neurotrophic factor (BDNF), neurotro-phin-3 (NT-3) and NT-4/5. Keratinocytes synthesize and releaseall NTs. Keratinocytes express the low-affinity p75 receptorwhich binds all NTs, the high-affinity NGF receptor trkA andthe NT-3 receptor trkC. By contrast, keratinocytes express onlya truncated form of trkB, the BDNF high-affinity receptor.NT-3 stimulates keratinocyte proliferation. NTs other thanNGF fail to protect keratinocytes from UVB-induced apoptosis.While NGF decreases upon UVB irradiation, NT-3 and NT-4are upregulated. UVA dose dependently increases NT-3 levels.NT-3 and NGF stimulate each other release. Both fibroblastsand myofibroblasts synthesize and release all NTs, as well astheir trk receptors except for trkC. p75 is more expressed inmyofibroblast than in fibroblasts. NGF reduces secretion ofcollagen I in myofibroblasts. Myofibroblasts secrete more col-lagen I than fibroblasts. NGF reduces production of metallo-proteinase 2 (MMP2) in myofibroblasts. Melanocytes synthesizeand release all NTs. UVB irradiation upregulates the release ofNT-3, while it downregulates the release of NT-4. Melanocytesexpress p75, which is downregulated by UVB. Melanocytes alsoexpress trkA and the extracellular domain of trkB. Takentogether, these data indicate that a complex NT network existsin human skin with potential functions, partly to be determined.

Neurotrophins exert immunomodulatory functions on peripheral

blood eosinophils in atopic dermatitis

U. Raap1, N. Deneka1, C. Goltz1, M. Bruder1, H. Renz2, A. Kapp1

and B. Wedi1

1Department of Dermatology and Allergology, Hannover Medical

University, and 2Department of Clinical Chemistry and Molecular

Diagnostics, Philipps-University Marburg, Marburg, Germany

Recently, the functional role of neurotrophins on eosinophils inallergic asthma has been described. The aim of this study was to

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588

investigate the possible role of BDNF, NT-3, NT-4 and NGF onperipheral blood eosinophils in atopic dermatitis (AD). ADpatients were defined according to the criteria of Hanifin andRajika. Peripheral blood eosinophils were purified by CD16-negative selection (purity >98%) and stimulated with BDNF,NT-3, NT-4 (10, 50 and 100 ng/ml), or NGF (100, 500 and1000 ng/ml) for 24 up to 120 h. Apoptotic eosinophils wereinvestigated by determining their hypodiploid DNA peak andAnnexin V method, respectively. Chemotactic index wasassessed in a modified Boyden chamber assay and respiratoryburst by lucigenin-dependent chemiluminescence. Stimulationwith BDNF, NT-3 and NGF significantly inhibited the pro-grammed cell death of AD eosinophils (P< 0.05–0.01) at eachtime point (24 h up to 120 h in culture). Chemotactic index wassignificantly increased in AD eosinophils after stimulation withBDNF, NT-3 and NT-4 (P< 0.05–0.01). Respiratory burst ofAD eosinophils was not modified after stimulation with BDNF,NT-3, NT-4 or NGF. To summarize, BDNF, NT-3 and NGFsignificantly inhibited the programmed cell death of AD eosino-phils. However, BDNF, NT-3, NT-4 and NGF did not modu-late respiratory burst of AD peripheral blood eosinophils. Onthe other hand, BDNF, NT3 and NT-4 significantly inducedAD eosinophil chemotaxis. Taken together, neurotrophins havea functional role on peripheral blood eosinophils revealingneuro–immunological interactions in AD.Supported by HILF grant.

Neuronal sensitization for itch in patients with chronic pruritus

A. Ikoma1, H. Handwerker

2, Y. Miyachi

1and M. Schmelz

3

1Department of Dermatology, Kyoto University, Kyoto, Japan,2Department of Physiology 1, University of Erlangen, Erlangen,

and3Department of Anesthesiology, Mannheim, University of

Heidelberg, Heidelberg, Germany

Itch is one of the major symptoms of various skin diseases.Although specific neuronal pathways for itch were identifiedboth peripherally and centrally, they still fail to explain itchyskin observed in patients with chronic pruritus. In this study,sensitivity to itchy and painful stimuli in patients with atopicdermatitis was investigated. Histamine-prick evoked enormousitch in their lesional skin, while less itch in their non-lesionalskin than healthy subjects. Flare reaction was not significantlydifferent between their non-lesional and lesional skin, rathersmaller than healthy subjects. Mechanical (pin-pricks), electrical,heat and chemical (injection of pH3 solution) stimuli evokedintense itch in their lesional skin and partly also in their non-lesional skin, while only pain in healthy subjects. Itch was also,but not intensely, evoked in healthy subjects by injection of pH3solution after sufficient histamine stimuli. These results confirmthe presence of itchy skin with hyperkinesis (excessive itch by itchystimuli) and allokinesis (itch by non-itchy stimuli) in patients withatopic dermatitis, which is so intense that painful stimuli cannotsuppress but evoke itch, and suggest that neuronal sensitization isinvolved in their itch not only peripherally but also centrally.

A comparative study on the effects of naltrexone and loratadine on

uremic pruritus

E. Legroux-Crespel1, J. Cledes2 and L. Misery1

1Department of Dermatology, and 2Department of Nephrology,

University Hospital, Brest, France

Two recent studies have provided opposite results on the effi-cacy of naltrexone on uremic pruritus. We have performed athird study. We compared efficacy and tolerance of naltrexoneand loratadine on uremic pruritus. Among 296 hemodialysedpatients, 65 suffered from uremic pruritus. 52 patients partici-pated in the study. Patients were treated for 2 weeks withnaltrexone (50mg/day; 26 patients) or loratadine (10mg/day;26 patients), after a washout of 48 h. Pruritus intensity was

scored by a visual analog scale (VAS). Adverse events werecarefully searched. The two groups were statistically equivalent.There was no significant difference in the mean VAS scores aftertreatment, but naltrexone allowed a dramatic decrease of VASsores (D> 3/10) in seven patients. Adverse events (mainly nau-sea and sleep disturbances) were observed in 10 of 26 patients.We could notice that 22% of hemodialysed patients sufferedfrom uremic pruritus. Naltrexone was effective only in a subsetof patients. Adverse events were very frequent. The differencesof efficacy and tolerance between patients might be due tometabolism. Naltrexone might be considered as a second-linetreatment.

New chapter in skin biopsy: diagnostic tool for neurodegenerative

disorders

A. Palotas1,2, B. Penke2, L. Kemeny3, Z. Janka1 and J. Kalman1

1Department of Psychiatry, 2Department of Medical Chemistry,

and 3Department of Dermatology, University of Szeged, Szeged,

Hungary

The accumulation of the ubiquitous amyloid peptide in thebrain is a defining feature of Alzheimer’s disease. Consistentwith studies demonstrating alterations of various biochemicalprocesses of cells of peripheral tissues and the importance ofskin biopsy in the diagnosis of neurodegenerative disorders, weinvestigated whether differences in the basal intracellular freecalcium levels of lymphocytes and cultured cutaneous fibro-blasts derived from sporadic Alzheimer patients and from age-matched control individuals might be present. Calcium levelswere measured in Fura-2AM-loaded human fibroblasts by dual-wavelength spectrofluorimetry. Basal calcium levels appeared tobe higher in Alzheimer lymphocytes when compared withcontrol ones. Resting calcium concentration of Alzheimer fibro-blasts, however, has proved to be lower than that seen withcontrol cells. Exposure of cells to amyloid resulted in the eleva-tion in the Ca2þ level of both control cell types, however, that ofAlzheimer lymphocytes and fibroblasts did not differ consider-ably. Our test could prove useful in supporting the diagnosisof (sporadic) AD in patients suspected of suffering from thedisease. Also, this simple finding may serve as a springboardto monitoring Alzheimer pathology in the peripheral systems ofthe body.

Electrode design for skin electroporation with minimal nerve

stimulation

U. Pliquett

Physical and Biophysical Chemistry, University of Bielefeld,

Bielefeld, Germany

Electroporation is an efficient tool for transdermal delivery ofwater-soluble molecules sizing up to several kDa. The mainbarrier to these agents is the stratum corneum, a 15mm thinlayer of dead keratinized cells. Once this layer is charged(approximately 50V) by an outer electric field, structural rear-rangements of the lipids create aqueous pathways (electropora-tion). Due to the high electric field within the stratum corneum,(E¼ 50V/15 mm¼ 33 kV/cm) electrophoresis can drive chargedmolecules into the deeper skin layers. A major concern is thehigh electric field required, because nerve stimulation is incon-venient for the patient. Taking advantage of the fact that up to adepth of 50mm no nervous receptors appear, a confinement ofthe field within the upper 15 mm would circumvent sensation.Field confinement within the stratum corneum is arranged by aspecial electrode geometry, an array of 300 mm holes withina 0.5mm thick dielectric. The bottom, facing the stratumcorneum, is metalized with a gap to the holes. The size of thisgap (3–10 mm) is critical for the penetration depth of the electricfield between the metalized bottom and a distant electrode at theother side of the holes. A drug reservoir is implemented belowthe distant electrode and the dielectric.

Abstracts

589

IgE-independent atopic dermatitis is associated with a b-2adrenergic receptor gene polymorphism

A. M. Roguedas, M. P. Audrezet, V. Scotet, D. Dupre, C. Ferec

and L. Misery

Department of Dermatology and INSERM-EMI 0115 (Laboratory

of Molecular Genetics), University Hospital, Brest, France

IgE levels are not elevated in about 20% of patients with atopicdermatitis (AD). In this intrinsic AD (IAD), allergic mechan-isms are not very important and pathogeny could be mainlyneurogenic. b2-adrenergic receptors are localized on cellsinvolved in AD: Langerhans’ cells, keratinocytes and lympho-cytes. We wondered whether IAD could be associated with genepolymorphisms 16 and 27 of this receptor. We studied 98healthy subjects and 83 subjects suffering from DA (UKWPcriteria). IgE levels were normal in 12 of them and elevated in71 (EAD). After DNA extraction, the genotyping was done byPCR and Direct Sequencing of candidate gene. Statistical ana-lysis was performed with EPI-INFO 6.04 for w2 test. We found asignificant association of Gln27Glu polymorphism with IAD(P¼ 0.00071 and w2¼ 14.51). There was no difference betweenhealthy subjects and EAD patients. Adrenergic receptor ago-nists are known to attenuate the proliferative response of humanlymphocytes after activation, through the inhibition of inter-leukin-2 release. It is known that catecholamines inhibit theantigen-presenting capability of epidermal Langerhans’ cells.Long-term agonist-promoted downregulation of receptornumber is absent when glu is at position 27. We suggest thatthe suppression of inhibiting effects of catecholamines could beinvolved in IAD pathogeny. Dichotomic nature of AD (EADand IAD) is also associated with polymorphisms (SNP) of theinterleukin-4/interleukin-13 receptor gene and the differences ofcutaneous variables (transepidermal water loss, capacitance andpH). Altogether, these findings indicate that IAD patientsexhibit phenotypic and also genotypic features which differfrom those patients with EAD. Otherwise, the presence of thispolymorphism could provide an explication of rarity of hyper-tension with AD, because Glu27Gln has been identified as asusceptibility polymorphism for HTA.

Evidence for a proinflammatory role of proteinase-activated

receptor-2 during cutaneous inflammation in vivoM. Steinhoff1, S. Seeliger1, C. Derian2, R. Nawroth3,

C. Sunderkotter1, D. Metze1, D. Vestweber3, P. Andrade-Gordon2

and T. A. Luger1

1Department of Dermatology, University of Munster, Munster,

Germany, 2R. W. Johnson Pharmaceutical Res Institute, Spring

House, NJ, USA, and 3Department of Cell Biology, Center for

Molecular Biology of Inflammation (ZMBE), University of

Munster, Munster, Germany

Contact dermatitis (CD) is a frequent dermatological diseasewith a high socioeconomical impact characterized by acute tochronic inflammation of the skin, often leading to therapy-resistent eczema. Proteinase-activated receptor-2 (PAR-2), aG-protein-coupled receptor for certain serine proteases, is local-ized on keratinocytes, endothelial cells, and nerve fibers and hasbeen demonstrated to play a role during inflammation of severaltissues. However, the precise role of PAR-2 and the underlyingmechanism of PAR-2-induced regulation of inflammation arestill fragmentary. Therefore, we were interested in whether ornot PAR-2 is involved in cutaneous inflammation using a modelof experimentally induced allergic (ACD) and irritant (ICD)contact dermatitis. In wild-type (PAR-2þ/þ) mice, PAR-2 ago-nists induced an increased intradermal edema and enhancedplasma extravasation with a maximum between 3 and 24 h.These inflammatory responses were significantly diminished inPAR-2-deficient (PAR2–/–) mice and controls (vehicle). Morpho-logical analysis revealed a dramatic increase of spongiosis andintradermal edema along with enhanced infiltration of neu-

trophils and monocytes in PAR-2þ/þ mice as compared withPAR-2–/– mice. Interestingly, nitric oxide (NOS) inhibitors sig-nificantly diminished these effects, indicating a role of NO inPAR-2-induced inflammatory responses of the skin. Functionalstudies at the RNA and protein level further revealed PAR-2-induced upregulation of the cell adhesion molecules ICAM-1 andE-selectin by dermal microvascular endothelial cells duringinflammation, suggesting that PAR-2 directly regulates celladhesion molecule function during skin inflammation. PAR-2agonists also stimulated upregulation of mediators involved incutaneous inflammatory responses such as IL-6 and NO inmurine and human (dermal) endothelial cells. Together, theseresults strongly suggest a proinflammatory role of PAR-2 duringCD and probably other inflammatory dermatoses, especiallyduring the early phase characterized by edema, plasma extra-vasation, and recruitment of inflammatory cells to the site ofinflammation. Thus, PAR-2 antagonists may be therapeutictools for the treatment of inflammatory skin disorders such ascontact dermatitis and atopic eczema.

Stress modulates peptidergic innervation and alters the cutaneous

immune response: exacerbating pathomechanisms in atopic

dermatitis?

E. M. J. Peters, A. Kuhlmey,M. Knackstedt, R. Paus, B. F. Klapp

and P. C. Arck

Psychoneuroimmunology, Biomedical Research Center, Charite,

Humboldt University, Berlin, Germany

Stress is said to induce itchiness of the skin and exacerbateinflammatory skin diseases such as atopic dermatitis. In thiscontext, stress mediators such as the neuropeptide substance Pplay a role as immunmodulators and in a wider sense growthfactors. For example, we were recently able to show that stressor treatment of mice with substance P is associated with mastcell degranulation, increased cutaneous inflammation andincreased apoptosis in the hair follicle. However, local interac-tions between the nervous and immune systems, especially underperceived stress, have rarely been reported. Here, we show forthe first time, that 24 and 48 h after sonic stress exposure, thenumber of SP-immunoreactive nerve fibres in the back skin ofC57BL/6 mice with all there hair follicles in the resting phase ofthe hair cycle (telogen, low numbers of cutaneous nerve fibres)increased significantly over non-stressed mice with the strongestincrease after 24 h. Such substance P immunoreactive nervefibres contacted mast cells more frequently, which became sig-nificant after 48 h. At the same time, the percentage of degranu-lated mast cells increased significantly after 24 and 48 h with thestrongest increase after 48 h when apoptotic cells also becamesignificantly upregulated. The same stressor increased dermalinfiltration, e.g. by eosinophils in C57BL/6 mice with experi-mentally induced allergic dermatitis over mice that were eitherstressed or had allergic dermatitis as well as over untreatedcontrols. Increased infiltration was associated with increasedepidermal thickness in stressed mice with allergic dermatitisand with an increased number of VCAM-immunoreactiveblood vessels. At the same time, the percentage of degranulatedmast cells increased significantly, and the number of substanceP-immunoreactive peptidergic sensory nerve fibres decreased inthe acute allergic dermatitis lesions. By semiquantitative RT-PCR, allergic dermatitis increased cutaneous IL-4 and to a lesserdegree IFN-g production, but this was not affected by stress.Ultrastructural investigation showed unmyelinated peptidergicnerve fibres in a state of deterioration close to degranulatingmast cells and eosinophils in the skin of stressed mice withallergic dermatitis, suggesting a decreased number of substanceP-immunoreactive nerve fibres due to active release of SP. Thismay lead to an upregulation of endothelial adhesion moleculesand increased infiltration by immunocytes to the skin butat mRNA level does not alter the production of classical

Abstracts

590

atopy-related cytokines in skin. These data provide first evi-dence for stress-induced exacerbation of cutaneous allergic dis-eases such as atopic dermatitis by local interaction of theperipheral nervous system with substance P.

Proteinase-activated receptor-2 mediates itch: a novel pathway for

pruritus in human skin

M. Steinhoff1, U. Neisius

2,3, A. Ikoma

2, M. Fartasch

3, G. Heyer

3,

P. S. Skov4, T. A. Luger

1and M. Schmelz

2

1Department of Dermatology, University of Munster, Munster,2Department of Physiology and Experimental Pathophysiology,3Department of Dermatology, University of Erlangen, Erlangen,

Germany, and4The Reference Laboratory, University Copenhagen,

Copenhagen, Denmark

Proteinase-activated receptors are G-protein-coupled receptorswith seven-transmembrane domains activated by serine pro-teinases. PAR-2 is a receptor for mast cell tryptase, house dustmite allergens, bacterial antigens and trypsin, for example, indi-cating a role of PAR-2 during inflammation and immuneresponses. In the skin, PAR-2 is expressed by keratinocytes,endothelial cells, certain immune cells and nerves, suggesting abroad regulatory role of proteases in the skin. Recently, PAR-2has been demonstrated to be involved in neurogenic inflamma-tion. Therefore, we examined whether neuronal PAR-2 may beinvolved in pruritus of human skin. The endogenous PAR-2agonist tryptase was increased up to fourfold in atopic derma-titis (AD) patients. PAR-2 was markedly enhanced on primaryafferent nerve fibres in skin biopsies of AD patients. Intracuta-neous injection of endogenous PAR-2 agonists provokedenhanced and prolonged itch when applied intralesionally. Inter-estingly, itch upon mast cell degranulation prevailed despite localantihistamines in AD patients only. Thus, we identified enhancedPAR-2 signalling as a new link between inflammatory and sen-sory phenomena in AD patients. PAR-2 antagonists, thus, repre-sent a promising therapeutic target for the treatment of cutaneousneurogenic inflammation and pruritus.

Corticotropin-releasing hormone skin signalling is receptor

mediated and is predominant in the sebaceous glands

K. Orlowski, A. Schnitger, E. Glass and Ch. C. Zouboulis

Department of Dermatology, Charite-University Medicine Berlin,

Campus Benjamin Franklin, Berlin, Germany

There is increasing evidence that the sebaceous gland is involvedin responses to stress-expressing receptors for several neuropep-tides. Among them, corticotropin-releasing hormone (CRH), ahypothalamus-derived peptide was currently found to also beproduced in the skin. Urocortin, urotensin and sauvagine arerecently described members of the family of structurally relatedCRH-like peptides, with urocortin sharing a 45% homology toCRH. To better understand the effects of CRH and CRH-likepeptides on skin cells, the distribution of CRH, CRH receptorsI and II as well as CRH-binding protein in cultured skin cellswas determined. Moreover, the effects of CRH and CRH-likepeptides on proliferation and inflammatory signalling of CRH

receptors-expressing skin cells in vitro were investigated. Whilenative keratinocytes and SZ95 sebocytes expressed CRH, CRH-binding protein, and both CRH receptors, endothelial cells didnot express any of these molecules. There was a cell-specificeffect regarding cell proliferation. While CRH and urocortininhibit SZ95 sebocyte growth, we could not observe such effecton HaCaT keratinocytes. Urotensin and sauvagine were inactiveon both cell types. CRH and urocortin stimulated interleukin-6and interleukin-8 release from SZ95 sebocytes, but theyexhibited no effect on interleukin-1a and -b release. a-helicalCRH, a CRH antagonist, annulled the CRH effect, but not thatof urocortin, on SZ95 sebocyte proliferation and interleukinsynthesis. In conclusion, CRH, and to less extend urocortin, islikely to play a major role on the signalling of stress pathophy-siology in the skin. The sebaceous gland is more likely to be theCRH activity regulation centre in human skin.

b-endorphin modulates lipogenesis in human sebocytesM. Bohm

1, Z. Li

1, M. Ottaviani

2, M. Picardo

2, Ch. C. Zouboulis

3,

S. Stander1and T. A. Luger

1

1Department of Dermatology and Ludwig Boltzmann Institute

for Cell Biology and Immunobiology of the Skin, University of

Munster, Munster, Germany, 2Instituto Dermatologico San

Gallicano, Rome, Italy, and 3Department of Dermatology,

University Medical Center Benjamin Franklin, The Free

University of Berlin, Berlin, Germany

Previous research in our laboratories has demonstrated thatthe human sebaceous gland is a target organ for several stresshormones including a-melanocyte-stimulating hormone(a-MSH) and corticotropin-releasing hormone (CRH). Tofurther investigate the role of stress hormones in acne, we exam-ined the expression of opioid receptors (ORs) and the biologicalactions of b-endorphin (b-ED), a natural high-affinity ligand forthe m-OR (MOR) and low-affinity ligand for the D-OR (DOR),in human sebocytes. RT-PCR, Western immunoblotting andimmunofluorescence studies identified the MOR but not theDOR in the human sebocyte cell line SZ95. Expression of theMOR was also confirmed in the sebaceous gland in normalhuman adult skin and was detectable primarily in peripherallylocated sebocytes. SZ95 sebocytes did not express the b-EDprecursor pro-opiomelanocortin (POMC) as shown by RT-PCR analysis and Western immunoblotting and stimulation ofcells with prototypical POMC inducers such as tumour necrosisfactor-a, a-MSH, dbcAMP, phorbol ester and CRH failed toinduce POMC. On the functional level, b-ED significantly sup-pressed the growth of SZ95 sebocytes induced by epidermalgrowth factor in chemically defined calcium-rich medium. Onthe other hand, b-ED enhanced lipogenesis as shown by Nile redstaining and gas chromatography. Accordingly, b-ED dramati-cally increased the amount of C16 : 0, C16 : 1, C18 : 0, C18 : 1 andC18 : 2 fatty acids in an extent similar to linoleic acid used as apositive stimulus. Our data demonstrate that human sebocytesexpress the MOR and respond to b-ED with reduced in vitroproliferation and increased lipogenesis. These data establish afurther link between stress and acne, the latter in which excessivesebum production is an invariable feature.

Abstracts

591