Abstracts of the Langerhans Cells Fourth International Workshop

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Abstracts of the Langerhans Cells Fourth International \^orkshop August 24-26, 1995, The Netherlands

Local Organizing Committee Bert Ian VcrincLT (chairnian) A. Miokc !Vl(.>niniaas (secretary) Jan 1). lio.s C]ajla M . r . Bniyn7A'el-Kot>inLMi Martin 1. KapsenliLMg I'lmnias A. Lii^cr

International Advisory Board |an 1), Bos (chairman) Marcel li.M. I ennissen (,secretary) Colelte Deziitter-Damhuyant Stefan CIrahbe Steplien I. Kat? Cerald Scluiler CJeoru; Stingl j . Wayne Streilein

Financial Underwriters Ada Medica Cyananiid Beneltix (Netherlands) Emopenn liinninioderniatology Society nlsevier Science (ialdernia Netherlands CJlaxo

janssen I'liarinaceutics KNAW Koche Netherlands Sando/ Netlierlanils Sandoz S\\ it/erlantl Schering Netherlands Yainanonchi Hiirope 'llic foiirtli intoniatioiKil vvorUshi^p o n l.angerhiuis fells \v;is an ;ibstriu-tcd nu'cuni; ori;ani/ed U. iiu-lndr prcsenUuions iVoin iiiviled truest spe;ikers as well as oral and poster presentations. T h e pa^es ihat follow i.iclnde die abstracts tor review and citalion. W e are grale(\il for the entluisi;,stic- support ofall participants, our .supporters, and especially our lour s|H'cial guests. Ralph Sleiinnan, David |. Harrison, llninias liiebei. and 1 rits Kcmiiig.

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• C:opyri,u:lit < ' ' 1995 hy T h e Society for Investij^ative Dermatology. Inc.

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1 C O N T A C T ALLERGENS INDUCE MIGRATION OF LANGERHANS CELLS IN H U M A N SKIN CULTURES: IL-lfi IS CRITICAL MEDIATOR. £H_M. Pistoor'''. A. Rambukkana'"'. M. Kroezen-. M.L. Kapsenberg'. I.D. Bo.s' and p i c T D a s " ' Dcpartmenls of 'Dermatology, 'Cell Biology and Histology and 'Pathology, ' J ^ e m i c Medical Center, University of Amsterdam, The Netherlands. C o n t a c t allergens sensitize the immune system by activation of epidermal Langerhans ^ells (LC), whereas irritants do not. In order to develop an in vitro assay for the .Justification of contact allergenic compounds, we analyzed the effects of contact lillergens and irritants on LC in human skin cultures. Skin biopsies were treated with contact allergens (DNFB, NiSO,, oxazolon, diphencyprone, K,Cr,O,, DNCB) or irritants (SDS, croton oil, nonanoic acid, propylene glycol, benzalkonium chloride). JtnmunohistDchemical staining of cryostate seetions of 24 hour contact allergen-treated biopsies showed a dramatic reduction of the number of LC in epidermis and an accumulation of remaining LC at the epidermal-dermal junction. In contrast, irritants arid solvents did not show this effect. Morphometrical analysis of cryostat sections and flowcy t o metrical analysis of epidermal cells supported these findings, suggesting that human skin cultures provide a promising model to identify potential contact allergens. Investigating the mechanisms of contact allergen-induced LC migration, immunohistochemical staining for cytokines showed that lL-18 protein is pnsduced rapidly after application of contact allergens. Treatment of human skin cultures with rhIL-lfi, but not with r h T N F - a , rhIL-la or rhGM-CSF, led to LC migration as seen in the biopsies after treatment with contact allergens. Moreover, contact allergen-induced LC migration could partly be blocked by the treatment of skin cultures with anti-IL-lfS polycional antibodies, but not with antibodies directed against IL-la, TNF-a and GM-CSF. These results suggest that IL-lfi plays a critical role in the induction of LC migration.

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MHC CLASS I+/CLASS 11" DENDRITIC CELLS ARE ABLE TO INDUCE DELAYED-TYPE HYPERSENSITIVITY RESPONSES. A. Kolesaric, G. Stingl, A. Elbe. DIAID, Dept. of Demiatology, Univ. of Viemia Medical School, VIRCC, Austiia. We have generated a CD45+/MHC class l+(H-2k)/MHC class II-/CD80+ dendritic cell line (80/1) from murine skin which is able to stimulate haptenspecific proliferative responses of naive CD8+ bnt not of CD4+ lymph node T cells in vitro. To investigate whether this dendritic cell line is also able to prime naive T cells in vivo, we nsed the contact hypersensitivity model, wbicb is generally considered to be mediated by CD4+ T cells. For tbis prospect, 80/1 cells, and for control puiposes, other MHC class I+(H-2k)/MHC class 11" cell types were derivatized in vitio with trinitrophenyl (TNP) and injected (lO*"' cells/mouse) subcntaneonsly into female C3H tnice. As a system control, mice were sensitized epicntaneously with trinitrochlorobenzene (TNCB) on tbe sbaved abdomen. Sensitized and nonsensitized mice were ear challenged 5 days later witli TNCB. Suiprisingly, TNP-derivatized 80/1 cells were able to induce significant (p<0.05) contact hypersensitivity responses 24 bonrs after challenge. In contiast, injection of TNP-derivatized L929 or RI.l cells and nonhapten-derivatized cells failed to induce any significant ear swelling response dm ing tbe obsewation petiod of 24-72 bours after challenge. Our fmding tbat MMC class I+/MHC class 11" dendritic cells are capable of initiating a primary delayed type bypersensitivity response leads us to conclude that CD8+ T cells play a key role in these reactions.

ALTERATION OF CYTOKINE EXPRESSION BY CALCITONIN GENBRELATED PEPTIDE (CGRP). I. Hosoi. H. Torii. F. Fox. '/.. Yaii. A. H. pj^nk and R. D. Granstein. MGM/Harvard Cutaneotis Biology Research Center, Massachusetts General Hospital and Harvard Medical Scbool, Boston, MA, USA, Department ol' Dermatology, University of Pennsylvania School of Medicine, Philadelpbia, PA, USA C G R P inbibits autigen presentation by Langerbans cells (LC) and macrophages (Mcfi). To examine whether CGRP elTccts migbt be mediated, in part, by modulation of cytokine expression, we examined tbe ability ol CGRP to alter IL-I P, IL-12, and IL-10 expression by LC and peritoneal exudate cells (I>EC). Expostire of tbioglycollatc-elicited PBC to 100 nM rat CGRP-1 + I |.lg/nil of LPS for 3 b testilted in inhibition of mRNA expression for IL- 1 P, and tbe p40 and p35 subunits of IL-12, (by RT-PCR) compared to PEC treated witb LPS alone. Culture of PEC for 24 h in LPS + CGRP resulted in significantly less IL-I|i production dian seen with cells incubated in L P S alone wbile IL-10 production induced by LPS was augmented by coculture witb CGRP (by ELISA). CGRP also potentiated the LPS-induced increase in IL-10 mRNA. LC were eniicbed to 85% (iom BALB/c epidermal cells using imnuinoniagnetic tecbniques. Cnltnre in CGRP + LPS inbibited induction of mRNA for the p40 subunit of IL-12 compared to culture in LPS alone (by RT-PCR). mRNA for IL-10 could not be detected in LC under these conditions. These results stiggest tbat some of the effects t)f CGRP on LC a n d M(|) function may be mediated by effects on cytokines.

EPIDERMAL LANGERHANS CELLS AND DERMAL ANTIGEN PRESENTING CELLS ARE DIFFERENTIALLY INHIBITED BY TRANSFORMING GROWTH FACTOR BETA-1. Toshiaki Nakamura. J. Wavne Streilein. Schepens Eye Research Institute and Department of Deraiatology, Han'ard Medical School, Boston, MA. Transforming growth factor-beta 1 (TGF-(il), a naturally occurring cytokine with potent immunosuppressive properties, has been reported to be produced by many different types of cells, including activated T cells, activated macrophages, and keratinocytes. We have previously reported that pretreatment with TGF-{il in vitro inhibits ability of cultured, but not fre.sh, Langerhans cells to activate alloreactive T cells, suggesting that not all cutaneous antigen presenting cells (APC) are equally susceptible to the deleterious effects of this cytokine. The present study was designed to determine whether intraeutaiieously injected TGF-fif can interfere with tfie induction of contact hypersensitivity to dinitrofluorobenzene (DNFB). TGF-pl (fOng/200;;!) was injected intradermally into abdominal skin of BALB/c mice. 2 hr later, the injected site was painted epieutaneously with either "conventional" (lS5/jg) or "optimal" (L5/^g) sensitizing doses of DNFB (Kurimoto ct ul, J. of Invest. Dennatol. 101:132-136, 1993). Eive days later, the ears were challenged with diluteDNFB. Intense CH was obsersed in mice that received an optimal sensitizing dose of DNFB on TGF-tif treated skin, whereas only weak CH was detected in mice that reeei\ed a conventional sensitizing dose of hapten on TGF-f\l treated skin. Sinee conventional sensitizing doses of DNFB arc toxie to LC, CH induction at this dose of hapten appeares to rely primarily on extra-epiderma! (dermal?) antigen presenting cells. We speculate that TGF-pf can have a deleterious effect on CH induction in circumstances if the relevant APCs are located in the dennis, rather than the epidermis.

CELLS CIRCULATING IN HUMAN SKIN LYMPH CONTRIBUTE TO THE INCREASED IL-10 PRODUCTION IN THE ELICITATION OF ALLERGIC CONTACT DERMATITIS. Qt) U. Brand. N. Yawalkar. Th. HiinzikRr. L R. Braathen. Dermatologieal Clinic, University of Berne, Switzerland Recent reports suggested an immunomodulatoiy role for IL-tO in contact hypersensitivity. In the present study, IL-10 and IFN-T protein levels were measured in normal skin lymph, in lymph derived trom irritant and allergic (induction and elicitation phase) contact dermatitis as well a s in skin blister fluid obtained trom a strong elicitation reaction. Increased IL-10 and IFN•I levels were detected during the elicitation phase ot allergic contact dermatitis, in a primary allergic skin reaction as well as In the blister tluld trom the strong elicitation reaction. In elicitation reactions, the mean daily lymph levels ot the IL-10 output increased trom baseline valties around 1 pg/h up to 129 pg/h, reaching maximum two days atter the peak ol the skin reaction. The mean daily levels ot the IFN-7 output, on the other hand exhibited a time-course inversely correlated with IL-10, with baseline values around 10 pg/h and a maximum ol 58 pg/h. tjsing a reverse transcriptase-polymerase chain reaction, the expression ot IL-10 and IFN-7 in eells trom the lymph as well as trom the blister fluid was examined. Signals tor IFN-7 were not found, specific transcripts lor IL-10 were detected in all samples examined. The IL10 mRNA signal, however, was markedly stronger in lymph and epidermal blister cells obtained during the elicitation reactions as compared to that in cells from normal skin and trom t h e induction phase of allergic contact dermatitis. In conclusion, in human skin lymph derived from irritant and allergic contact dermafilis, IL-10 is selectively increased in the elicitation phase of allergic contact dermatitis, indicating a role for IL-10 in limiting and down-regulating this type of T-cell mediated skin reaction. Besides keratinocytes cells circulating in the lymph may also contribute to IL-10 production. Thereby, IL-10 probably acts by inhibition ot cytokines and / or of the function ot antigen-presenting cells, a n d might exert its anti-intlammatocy activities not only in the skin, but also in the skin associated lymphoid tissue.

SUBTRACTIVE CLONING OF GENES EXPRESSED PREFERENTIALLY BY DENDRITIC CELLS, K. Arii-/.umi, I'.R. Bergstrcs.ser, A. Taka.shimj. UT Southwestern Medical Ctr. Dallas, TX, USA Dendritic cells (DC) ,irc a specific subset of leukocytes that exhibits potent antigen presenting capacity. We sought to identify Irenes that are expressed preferentially by DC, by employing a subtractive cl^NA cloning strategy. A XZaplI cDNA library was prepared from XS.S2 cells, a murine epidermal-derived DC line that resembles resident epidermal Langerhans cells in many important respects. After conversion into a phagemid library, it was hybridized with mRNA isolated from the murine macrophage line, J774; 200-fold enrichment was achieved by this subtraction. 12,000 clones were analyzed by colony Itybridization with total cDNA probes from XS52 or J774 cells. 250 clones selected in this procedtire were then examined by slot blotting. 14S clones were selected and finally analyzed by Northern blotting. 49 clones were confirmed for their preferential expression by XS52 cells. Sequencing these clones revealed f8 clones that were identical to IL-t/5, CfO, cathepsin C, spermidine acelyl transferasc, or YPT-1 (a ras-related protein). On the other hand, .^1 clones (6 different genes) showed no significant lic)niologies to known molectiles. 1E4, one of the DC-specific novel genes, was expressed prelerentially 111 spleen and thymus among 12 tested tissues and exclusively by the XS52 cells among 10 cell lines. IVA mRNA was also detectable in Ia* epidermal cells (i.e., Langerhans cells) isolated from BALB/c mice. We conclude that DC are distinguishable from other leukocytes by the selective expression of specific genes. Characterization of these genes will lead to a better understanding of tiic identity and immunological properties of DC.

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8 ESTABLISHMENT OF A CELL LINE WITH FEATURES OF EARLY DENDRITIC CELL PRECURSORS FROM MOUSE FETAL SKIN, G. Girolomonji, M B . Lutz^, S. Pastorei. C.U. ABmann^, A. Cavanji, P. Ricciardi-CastaqnoliZ. ^Lab. of Immunology, IDI, IRCCS, Rome; 2CNR Center of Cytopharmacobgy, Milan. During ontogeny, the skin is populated by MHC class II" dendritic cell (DC) precursors that then mature in efficient antigen-presenting cells. To better characterize skin DC progenitors, we generated a myeloid cell line (FSDC) from mouse fetal skin by infecting cell suspensions with a retroviral vector carrying an env'^^'^-myc^^^ fusion gene. These cells displayed a dendritic morphology and their proliferation in serum-free medium was promoted by GM-CSF, but not M-CSF. FSDC expressed strong membrane ATPase activity and a phenotype consistent with a myeloid precursor (CD45+, H-2<'+, I-A^*, CD54+, CD11b+, B7.2+, B7.1-, CD11C+, 2.4G2+, F4/80+, CD44+, 2F8+, Sca1+, Sca-2+, ER-MP 12", NLDC-145-, J U d " , B220-, T h y . r , CD3-). FSDC stimulated allogeneic T cells poorly, but markedly increased this function after treatment with GM-CSF, GM-CSF and iL-4 or IFN-v, but not with SCF, IL-1 a or TNFa. IFN-Y was required for in vitro presentation of haptens to primed T cells. However, even after cytokine activation, FSDC were much less potent than adult epidermal Langerhans cells. In contrast to A20 B cells or peritoneal macrophages, FSDC derivatized with haptens and injected either i.v. or s.c. could induce contact sensitivity responses in naive syngeneic mice. Results indicate that mouse fetal skin harbors myeloid precursors possessing a surface macrophage/immature DC-iike phenotype and priming capacity in vivo. These cells need further differentiation and activation signals (e.g., cytokines) to express their antigen presenting potential in vitro.

I'lil'TlDH-LOADING Ol- MHC CLAS,S II MOLHCULliS IN THK ENDOCYTIC PATHWAY 01- H U M A N DI-NDRITIC CF.LLS C A N O C C U R AI-ThR RI-INTI-RNAI IZATION Ol' HLA-DR-INVARIANT CHAIN COMPLEXES IROM THE Ci;i.L SURl-ACE. C. Saudniis (1). H. de la ,Salk- (7.1. D. Spelin.-r m. A. Buhhol |4)_W^ H Friclmaii HI. .I.-P. Caymiavc (5). .1. Sahiiiiero ( U . and P . Hanau (2). (1) UMR 144, hislilul Curie, Paris; IN,S1';RM (2) CJF 94-03 and (5) U.31I, E.T.S., Slrasbnurg; (.il INSliRM U.74, Insliliil de Virologie, Slrasbnurg; and (4) Service d'Onco-llenialolopie. Hnpilal dc Hiiuleplerre, Sliashourg, F'rance. .. Invariant chains (li, CD74) are expressed al the surface ol" nn)ni)cyle-derived dendrilic cells (I3C). Combining nielabolie pulse-chase labeling with biolinylaliim ol surtace prolcins, we could ilenionstrale Ihat HEA-DR molecules ass.icialccl with li (uP-Ii complexes) appear at lhe cell surface of DC within .10 to 45 ntin al'ler Ihctr synthesis. Quantitative analysis .showed that 20-2.'i % of the newly synthesized u|i-li eoniplexc-s do actually reach lhe cell surface of the DC. The.se complexes, conlaining both p31 and p4l Ibrms of li, are rapidely reinleriialized and associated Ii chains are efficiently degiaded^ Under the eleclron ntiero.scope, DC incubated at 4°C with gold-labeled anti-CD74 F(ab ) fracments show "old particles within elalhrin-eualed pils. Once internalized, eonloeal mic-roscopy shows that wilhiti .10 min Ii coloeali/.e with the MHC elass II cotiipartnieiil. electron n'lieroscopy confirms ihat li enter the ctidocytic pathway and reach the MHC class II comparltnenl. l-inally, combining metabolic pulse-chase labeling and biotmylation of siu-raec proleius with iuiniunoprecipilaliou wilh the iiiAb L243, whieh rceogni/.es DRa|l complexes devoid of intael Ii, we show Ihat the a(i-Ii eomplexes reaehing the eell surlace of DC are first reiiuernalized, then converted to E243 reactive a|l dimers and linally become SDS-slable. i.e., peplide-loaded. These findings suggest that, together witli inlernali/.ed antigens, a(l-li complexes can iralTie Honi the DC surface through the enlire endocytic pathway permitting capture of distinct delerminants made available utider differing conditions of pH and proleolytic aelivity. In eonelusion ; lwo dislitiet MHC eluss II tralTickiiig pathways can lead to the generation o( functional class ll-peptide complexes in DC.

10 AI'PEARANCK OF A NOVKL EPIDERMAL FcERI-^/CDla-^ DENDRITIC CELL POPULATION IN LESIONAL SKIN OE ATOPIC ECZEMA, A, Wollenhert!*, ,S.P.Wen*, J. Hiibctstock*. S. Kral't*, D. Hanau" and T. Hiebet*. *Depl. of Detniatology, LLidwig-Maximiliaits-Univer.sity, Munich, Gcniiutiy; "Etablissctnent dc Tritnsftision ,Sungtline, Strasbourg, Fratice. I iuman epidermal dendritic cells are subjected to regulatory .signals derived frotil Ihcir celltilar mictocnvironmcitt which, beside altering their phenotype and function, ai.so leads to the recruilctncnt of oUicr cell types in the iitnanitiiutory skin silc. Epideitnal cells were isolated front variou.s iitflatntnatory skin diseases, e,g. atopic eczema (AE), allergic contact eczema (ACE), psoriasis vulgaris (PV) and normal skin (NS) anti lhe expression of FceRI, FCYRII, CDIb, CD23, IlLA-DR and CD36 was detertiiined on CDta-tcpidential dendritic cells. Thereby, FceRI was significantly upregulated in AB as compared to NS or ACli. iHirthertnote, FceRI-exptcssion correlated .significantly to the serum igE-level, stiggesting a link between tlie regulatory meehanisms involved in IgEsynthesis and Fct.RI-cxpression on LC. FcyfUI was moderately expressed in all skin satiiples examined, btit upregulated in PV. Cn3fi was present in all inflammatory skin samples, but absent in NS. CD23 was negative in NS, but present in AE and PV. In fact these variatiotis were most due to the picsence of two distinct cell popttlations; a first, CD|ahright/CDlb"SE/rcERltl'"VI'CYRII'''"VHI.A-DRbrigl>l/ CD.Vibi'gl" and a second, quantitatively nioie important population with CDla'''"VCD I f/'i'iVFct-Rlbiigl'V Fc7RIl''''"/HLA-DR'"iglil/CD3()"''gl". As shown by double htfieling in imtiiitnoelectronmicroscopy, this second poptilation was tlltrastiuctuially devoid of Birbeck gtanulcs, unrcactive with the Birbeck granule .specific-LAG iiiAb and was not found in NS satiiplcs. In conclusion, we provide evidence for the dc turn) itiiigration in iilflamtnatory skin sites of a CDla+ dendritic cell population which is distinct Ironi classical LC and specifically expresses high levels of FccRl in AE. •

PERIPHERAL BLOOD DENDRITIC CELLS EXPRESS FUNCTIONAL FCERI, D. Maurer. C. Ebner, B. Reininger, E. Fiebiger, D, Kraft, J,-P. Kinet, iL_StjngL DIAID, Dept. of Dermatology, Gen. and Exp, Pathol,, Univ. of Vienna Med, Sch., Vienna, Austria and Mol. All. and Immunol, Sect., NIAID, NIH, Rockville, iVID, U.S.A. The unique accessory functions of dendritic celis (DC) in the generation and modulation of antigen-specific immune responses opens the attractive possibility to use these ceils for immunotherapy of cancer, infectious diseases and allergy. In recent efforts, we could identify a numerically minor cell population present in the peripheral blood that homogeneously expresses the high affinity IgE receptor (FceRI) but lacks expression of lineage-defining anfigens, i.e., CD3/TcR, CD14, CD19, CD34, and CD56. Detailed immunophenofyping confirmed fhat these cells are indeed a (sub-) population of peripheral blood DC (HLA-DRhigh, HLA-DQhigh, CD4high, high hih , hih : l , l , l , l , The expression of the cutaneous lymphocyte-associated antigen CLA, as defined by the mAb HECA-452, strongly suggests that this DC population has skin homing properties and, therefore, may constitute a precursor pool for FccRI-expressing skin DC. After having established the exact immunophenotype of this DC population, we were enabled to isolate FCERIexpressing DC from peripheral blood by fluorescence-activated cell sorting and, consequently, to investigate their immunostimulatory properties. These experiments revealed that FceRl-positive DC have the capacity to eiicit both primary (allogeneic MLR) and secondary (IgE-mediated, FccRI-dependent allergen presentation) T cell responses.

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ROLE OF SERUM FACrOR(S) IN RBOULATING INTRA EPIDERMAL LANGBRMANS CELL FUNCTION. Y. Xie, J.W. Streilein Schepens Bye Research Institute & Depiutnienl of Dermatology, Harvard Medical Schcxil, Boston, MA 02114 L,angerhans cells (LC) freshly obtained Itom epidermis display a unititie pattern of fLinctional properties, including eflieicnt prcx;essing ol exogenous antigens, and aetivation of primed and alloreactive, but not naive or autoreactive, T eells. When exposed to GMCSF in vitio, fresh LC rapidly adopt a distinctly different lunclional program that enables them to activate unprimcd a.s well as autoreaetive T eells. Since the GM-CSF gene in keratiniKytes is "open" in resting skin, we have wondered why intraepidermal LC do not spontaneously acquite the "cultured" phenotype. We have obtitined evidenee that monse serum contains a lactor(s) tfial is relevant to this paradox. Fresh LC culttited in scrum of normal mice, but not of other s|3ecies, failed to up-regul;Ke expression of class fl MHC, B7-1 and 137-2, or to acquire the capacity to activate syngcncie, naive T eells. This inhibitory effect of mouse serum was not re\ersed by adding exogenous GM-CSF or IL-1 to the culture tnalium. The inhibition was tiot merely non-specilie toxicity, since mouse serum did not suppress the eapacity of fresh LC to activate primed or alloieiictive T eells, nor did it interfere with the unicjue functional properties of already cultured LC. The inhibitoiy activity in mouse serum eorresp KK)kD, is heat stable (KXP C x 30 min), and is resistant to trypsin digestion. We speculate that Ijingerhans cells maintain their "fresh" functional program in normal epidertiiis because a systemic (serum) factor protects them from the function-altering effects of GM CSF.

ULTRASTRUC'lURAL ANO PHENOTYPIC CRlinRIA MAY DISTINGUISH LANGERHANS CilLL AND DllNDRinC CI-I.L PRHCURSOR SUDSIHS GliNl-RATED ITiOM HUMAN CD34+ HLMATOPOIIinC PROOILNirORS, C, Dey.mtcr-D.iinlnivaiil. C, Oiux. M,-J. Slaaiict, C, Jaaiucl. C Massncricr. ,1, Baiiclicrca\i. D. Sclimill. INSHI^M Dcmialology Resaircli U34f>, Schering-Plough. Lab. for Iintniiiiologicil Research, I.yon, France. We have NIIOWII in humans, the cooperalioii of GM-CSF and 'INl'a in lhe/;i vilro generation of (iendrilic cell subsets from C'D34+ henialopoictic progenitor cells. Two developing subsets ;irc di.slingui.shed by sixicifie phenolyix; (CDla+/CD14-, CDla-/CDI4+) and cliaraclcrislic nltnisinicturc More Ihaii 90% of the CDla+ cells (at day 5) expressed one or more of tlic following iiltnistnictiiral lealiires : ' 'nuiltivesicular Ixxlies, -Mense btxlies displaying niyelin-like uniliinieltar inlenial slnietures iuid clcctron-lncenl iKxIies from sliort-lo-long to eiirved rods imd ^ViuItilamelUir internal inclusions. D)' electron mieroseopy we followal the evolution of the different states of differentiation on the basis of: cell sha)>c, ullrastnicture and CDla, CD14 iinligen levels (quantitative indirect inmiuiiogold lalx;liiip with 5nni-j;ol(i gnuiules (gg)) from day 8 to 19 (2 experiments). From d:iy 8 to 19, tlio tli,sap|>ejireance of CD14 correlated to an increjise of the % of CDla+ cells (20-36% to 35-57%) ;uid to a dccrciuse of the ^'e of CD]a+ eells with sjKcific ultrastructural features suggested the existence of 2 distinct CDla+ cell subsets. In the C"Dla+/ullraKtructural iii;irkers+ cell subset : 'Hhe density of CDla inerejised from dtiy 8 (319 328 gg/ KH);(in) to rcacli :m O])ti]num at day 14 (352 447 gg/ IOO;^ni) Uicn decrcjiscd lo day 19 (115186 gg/;viii). -^'immature forms of Birbeck granules (By) were noted in tlie first 2 weeks of culture whiU" the highest frc(|ucney of typicjil 13g ivjis reiiched at day 14-16 (25 38% ofcells) to jirogessively decreased until day 19 {(i%). In the C:Dla+/ullrastruetur:il markers- : l)the density of CDla kept on decreasing from day 8 to 19 (339-253 gg/ lOO^m to 143-149 gg/KXl/^ni), -)no Bg-relatcd stnictures or chiiracteristic ultrastructure was noted. Tlie existence of these 2 distinel dendritic eell s\ibsets was eonfinned by rcciilturing separately CDIii+/CD14- and CDla-/CDI4+ precursor subsets iilter FACS sorting (2 e\|)edmcnts). At day 13, the Cn)la+/CD14- precursors leading to the Bg+ (30 62%)/ultrastruciuriil markers+ (83 92%)/CDla+ eell subset are unetiuivociilly UmgerhiUis cell precursors whereas the CDU/CDI4+ precursors leading to Bg-/ultrastnictural markers/CD I a+ cell subset need further exploration to lie related to precursors of known dendritie cells sucb as ixrripheral dendritie eells or dennal dendritic cells.

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KERAriNOCYrF.-Di:RIVI-;D I1.-15 I'NHANCI-S .ACCliSSORY FUNCTIONS ()|F.PIDIIRMAL LC AND BLOOD Dl-NDR! IIC Cl-l.l.S. II. .loinilcit. U. Kiihn. C. vtnller. M. Wolli. S. Lolimann. .1. Saloua. P. Becker. .1. Kiiori, A.H. Ivnk. Clinical Res. Unit. Oept. of Dermatol.. Univ. of Mainz, Mainz. I'RG. T h e novel cytokine IL-15 has recently been identified as a kcratiiioeyte (KC) produet in our elinieal reseach group. As l.angerhans cells (LC) and dendritie cells (DC) ure known to express II.-2R that is also used for sigtialling by 11.-15. elTeets of IL-15 on tlie imtmniostiniukitory capacity ol" these eells were sttidied. Therefore human epidermal cells (EC) were eultuted in the presence olTI.-l5. anti-IL-15. or controls Ibr 241l and enriched for I,C by density gradient, [^e.sulting Iraetions (containing -10% I.C) were used as APC in allogeneie proliferation assays Ibr naive T cells. Addition of 2t)ng/nil oi' IL-I 5 resulted in a 4-rold enhancement of LC-APC I'unetion meastired by tliymidine incorporation. In contrast, addition of an anti-Il.-15-antisertmi redtieed the allostirmilatoiy capacity ol'LC to almo.st baekgrotuid levels. When added to eultun,>s of blood-derived DC grown with GM-CSI- and IL-'I. 11.-15 (20ng/ml from day 5 of cultt.ire) induced ehange.s in morphology and ftinetion of the cells. Phenotypleally. DC grown in the presence of OM-CSI- and IL-15 were similar to eells grown in GM-CSl' and lL-4 expressing high amounts ofMHC clas.s 11. CD80. CD86. CD40. CD54. CDla. with no CD14. CD3. CD2. or CD19 being detectable, l-tmetionally. DC grown with ILI 5 were 4-8-tinics more potent in indticing T eell proliferation in an allogetieie MLR for naive T eells. Turthermore addition ol'an anti-lL-15-antisertim to DC cultures (CiMC S F + IL-4) redttced the allostimulatory capacity ofthe cells hy half. In aggregate otir data support the notion that I1.-15 is an importani maturation litctor of human LC and D C . enhancing the immunostimulatory capaeity of these cells.

INTERLEUKIN-12 IS PRODUCED BY DENDRITIC CELLS INCLUDING LANGERHANS CELLS AND MEDIATES TH1 DEVELOPMENT. Franz Koch, C. Heufler, U. Stanzl, A. Enkl.N/l. WysockaS. N^J5omanL and_G. Schu[ec. Departments of Dermatology, University of Innsbruck, Austria and University of Mainz^, Germany, and The Wistar Institute^, Philadelphia, PA. lnterleukin-12 is the most critical cytokine for driving a T cell response towards a Thl type. Dendritic cells (DCs) are highly specialized accessory cells for the stimulation of resting T cells in vitro and in vivo. We investigated at the levels of gene expression, protein production and bioactivity whether DCs were a source of IL-12 and would thus influence the Thl/Th2 balance. We found that murine DCs stirnulaled substantial IFNy production in established Thl cells. When used as stimulators they induced Th1 development from naive T cells by an IL-12-dependent mechanism. We detected mRNA for both IL12 chains (p40 > p35). mRNA as well as IL-12 protein production was increased by treating DC with fixed staphylococoi. Supernatants from pure DC cultures including Langerhans cells induced IFNy secretion in Th1 clones indicating that the IL-12 protein was produced in a bioactive form. Finally, in situ hybridization showed that T cell contact markedly upregulated IL-12 expression in DCs. In conclusion, we have found that IL-12 is produced by murine and human DCs notably w/f/iou/the need for stimuli such as bacteria, LPS or exogenously added cytokines (IL-12) or anti-cytokines (anti-IL-4). This capacity is enhanced upon contact with T cells. This renders DCs capable of skewing the immune response towards T h l . Therefore, IL-12 constitutes a hitherto unrecognized, soluble key molecule that is critical for the immunoslimulatory function of DCs.

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T CELL-DEPENDENT TERMINAL MATURATION OF DENDRITIC CELLS. A. Takashima. T. Kitaiima, M. Ogoshi. K. Ariiy.umi, P.R. Bcrptrcsser. Department of Dermatology, UT Southwestern Medical Ctr, Dallas, TX, USA Dendritic cells (DC) are known to deliver activation signals to T cells during antigen presentation. Here we report that responding T cells deliver signals back to D C , thereby altering their phenotypic and functional properties. XS.')2 cells, a murine cpidernial-deri .nd DC line that resembles resident epidermal Langerlians cells in many importani respects, e.\hibited a series of profound changes upon interaction with HDK-1 T cells, a KLH-specific Till clone. Finst, XS52 cells secreted rel.itively large amounts of biologically active (17.5 kD) forms of IL-1/3. T h i s event was associated with a .striking upregulation of IL-1/3 mRNA expression and with increased mRNA expression and enzymatic activity for the IL-1/3 converting enzyme (ICE). IL-1/3 secretion required coupling of surface la molecules and B7-related molecules with relevant ligands on T cells. T cell-dependent lL-1/3 secretion was bloeked by the peptide inhibitor of ICE, Ac-WAD-CHO. Second, XS52 cells, whicli ordinarily prolifer.ite vigorously to CSF-1, lost tbis mitotic potential. With respect to mechanisms, interferon-7 (IFN7), which is secreted by HDK-1 T cells upon activation by XS52 cells, abrogated surface expression of CSF-1 receptors. Finally, XS.'i2 cells, which ordin,irily express low levels of B7-2, acquired elevated expression of this co-stimulatory molecule. "I'lits change was also mediated by IFN7 secreted by the responding T cells. All of these cbangcs occurred rapidly (within 6-24 hr) and required both T cells and antigen. Because each of these changes has also been observed as Langcrbans cells mature in culture, we propose tbat they represent T cell-dependent terminal maturation of DC, a process that is required for effective antigen presentation.

.STIMULATION OF T CKLL POPULATIONS HY I50V1NI-: DON'ORI'l'lC CI-M.l.S C.J. Howard. P. Sopp. I, Briiwnlie. K.R. Parsons. P..I. Chaplin. I.-S. Kwori!.' ;uul R.A. Collins. Institute (oc Ariinuil Health. Com[iti)ii. Near Newluny. Berksllire. RG20 7NN UK. Afferent lyaipli veiled cells (ALVC) collected from cattle hy lymphatic caniuilation after .surgical renio\'al ofthe prescapiilai- lyin[ili imde weie iderititleti hy tlieir expiession of tlle 210 kDa liloleciile BoWCt) and large size, and sorted tor fuiictiiuial investigations. Isolated cells had not heen cultured or otherwise manipulated ami their propertie.s can he considered to retleet those of cells in viva. ALVC stimulated proliferative response.s in sorted allogeneic C D 4 ' and CDS' T cells hut not WCI ' -,;i"i 'fCR' T cells. This was related to Ci:)28 expre.ssion hy the T cells. CD2S niRNA was evident in Ihe .MHC class I and class II restricted al0 'VCR T cell populations hy RT-PCR hut not in llie y/S T C R ' 'V cell poiHiIatioii. .\n analysis, hy R'l'-PCR. ol'the CMokines produced hy sorted ALVC revealed transcripts encoding IL-Uv. ll.-lf'i. IL-IO. T N F a . GMCSF. ll'Nvi. ll-N/3 htit not IL-:. lL-4, IL-6. lL-7 or IFN7. Two distinct suhpopulatinnsof ALVC were evideul that were CDl la ' or CDl hr. The latter populalion was 10-100 times more effective than die former al stimulaling alloproliferalive responses iu siuled C D 4 ' and C D S ' T cells. Bolh ALVC suhpopulatious expressed MHC ela.ss I and class II molecules al a couiparaMe high level but a numher of other molecules were idenlil'ied widi uiAhs thai were restricted to either the CDl l a ' or CDl la' suhset. Biudiug of human C'rLA-4 fusion protein was similar for holh ALVC suhset.s indicaliug that differences iu stimulalory capaeity were nol due to CDSO (B7.I) or CDSd (B7.2) expression and are likely to involve other, as yet unidentirieil. suifa- ' moleeules.

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CHARACTERIZATION OF ACCHSSORY CELL FUNCTION IN XS52 DENDRITIC CELL LINE. G. Sciiuhniaehers. M. Moliamadzadch, P.R. Bergstres.ser, A. Takashittia. UT Soutltwestern Medical Ctr. Dallas, TX, USA We have established recently a scrie.s of dendritie ceil (DC) lines from tlie epidermis of tlewborn BALU/e tnice. These litics, tertlied X,S series, resemble resident epidertnal Langerhans eells (LC) hy: a) surface phenotype, b) antigen presenting eapaeity. and c) cytokine and cytokitle receptor mRNA profiles. Mere we report tliat tlie XS52 line possesses tltc capacity to provide eostimulatory (or aeeessory eell) sigtials to naive T cells. 7-irradiated XS52 eells atigtnented sttbstantially (> 10fold) proliferative responses of CD4' T cells (isolated frotn BALB/e spleen) to suboptimal doses of soluble anti-CD3 niAh or Con A. As few as 5 x 10^ XS52 cells were suffieient to promote the growth of 1 x 10'' respotiding T cells, indieatitig their relatively high efficiency. By eontrast, other skin-derived eell lities (e.g.. Pam 212 keratinocyte and NS47 stronial cell lines) of BALI3/e origin, even with higher numbers, failed to augtiient T eell proliferatiotl, validatitlg tlic cell type-speeifieity. Although anti-la mAb hlocked eompleteiy XS52-depetKlent activation of tlie KLHdependent Thl clone llDK-1. the same mAb had no effect on the accessory cell function of XS52 cells. On the oUier hand. CTLA4-lg or itiAb against B7-1 or B7-2. each of which ititerfercd witIt the activation of IIDK-I T eells. also blocked accessory eell function. Fitially, FACS puritication studies revealed that XS52 eells provide eostimulatory signals not only to CD4' T cells, hut also to CDS' T cells. These results indicate that tlie XS52 line resembles epidertnal LC by tlleir aeeessory cell function, and support the recent concept that B7-relatcd tnoleeules expressed oti DC play an essential role in tlie activation of diffetent T eell subsets.

DENDRITIC CELL (DC) - EXTRACELLULAR MATRIX (ECM) INTERACTION: CULTURE OF BONE MARROW-DERIVED DENDRITIC CELLS ON COLLAGEN-COATED PLATES STIMULATES ALLOANTIGEN PRESENTATION A N D CYTOKINE PRODUCTION. K _ J M i i i k j ; _ J l A . Ltti;ct\ T. .Sehwarz .-md .S. Gi,-tbbi-. Lttdwig Boltzmann Inst. for Cell Biology ^irid Itiimunobiology of the Skin, Dept. of Dermatology, University of Miinster, O-rmany. Altei-antigenic stimulation, Langerhans eells niigrale through conneetivc tisstie on their way towaid.s lytriphatie tissues, which is accompanied by altered function and morphology, e.g. uptegulation of lL-l and IL-6 prodtiction, la-expie.ssion and allostitiuilatoi-y eapaeity. To iiive.stigate a possible tole of lsCM proteins in this proee.ss, DC were generated by eultttte of tnotise hone manow eells in the presence of 100 U/ml GM-CSi- I'tir fi days, Ig-.sedimentation and additional tivernight ettlttue, tysulting in >7O'7r ptire DC. These cells were then plated on collagen (COD- or libroneetin (FN)-eoated dishes for ?• days in tneditim without exogenotis cylokines. and assayed for eytokine prodtietion and expression of MHC elass' II and eostitmilatory tnoleeules. DC viability i\;niaincd >'-X)7ii dtiring enlltuc and was tinaffeeted by COL or FN. DC etilture on COL resulted in a 4-fold inetea.si: of their stimulatory eapaeity in allogeneic mixed lymphoeyte reaetiotis. compaa^d to eontmls grown on FN-coatcd plates or on tegular plastie dishes. Moreover. IL-I and lL-6 bioaetivity in the supernatants was ineiea.sed signiUcantly after cultui-e of DC on COLeoated plates, compared to DC etiltttred on FN or plasiie. Produetion of TNFa remained constant, lti addition, DC grown on CC)L-eoated dishes tlisplayed stistained expression of heat-stable antigen (USA), whereas expression of I-.V atul vai-iotis eostimulatory surface moleeitles was tinaffeeted. These data indicate that BCM ptotcins diffcretitially titodil'y DC lunclion in vitro, suggesting a possible itivoKeniciit ol ECM protcitis in regttlation of DC. funetion in sitti.

II IE lOUKNM. OF INVESTK^AI'IVE I)EH.MATOLO(;V

862

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IL-I. BUT NOTTNF-
METABOLIC CAPABILITIES OF LANGERHANS CELLS. C.A, Elmels, C. Anderson, H. Mukhtar. Dept of Dermatology, Case Western Reserve University, Cleveland, Ohio, USA. Although Langerhans cells {LC) are known to be potent initialors of cell mediated immune responses to highly reactive chemicals, the extent to which they patticipate in cell-mediated immunily to compounds that require metabolic activation is unknown. The purpose of this investigation was to assess the enzymalic capacity of LC, employing the polyaromatic hydrocarbons (PAHs) benzo(a)pyrcne (BP) and dimethylbenz(a)anlhraeene (DMBA) as protolypic agents. These eompounds are cutaneous carcinogens that are metabolized by the cylochrome P450 dependent enzytne system. Incubation of llie XS52 LC-Iike dendritic cell line with [^H] BP resulted in the production of significant quantities of diols, quinones and phenols. Metabolites weie not detected when [-^H] BP was placed in cultures without cells, indicatitig Ihat oxidation ofthe compound was not responsible for the effect. Incubation of XS52 cells wilh GM-CSF increased metabolism to the diol metabolites (i.e. carcinogenic metabolites) whereas there was no change in metabolism lo the quinonos and phenols. When applied topically to the skin of mice, both DMBA and BP produced a significant contact hypersensitivity response. Depletion of LC ffom DMBA conjugated epidermal cell suspensions abrogated their ability to initiate contact sensitivity to that agent, Evidenee ihat conversion of the parent compound lo a reactive nielabolile was necessaiy for development of contact hypersensilivity to PAHs included the fact that the response to DMBA only occurred in sttains of mice thai eould metabolize PAHs and that inhibitors of PAH metabolism reduced DMBA contact hypersensitivity. The implications of these experiments are that LC are able to tnetabolize foreign compounds and that, at least foi" some contact allergens, the melabolie status of LC is a key determinant of individual susceptibility lo allei'gie contact dennatitis.

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PMENOTYPIC AND FUNCTIONAL CHARACTERIZATION OF TUMOR ASSOCIATED DENDRITIC CELLS. F.O. Nestle. F. Raboud. B.J. Nickoloff. G. Burg. Department of Dermatology, Univeisity ol Zurich Medical School, Zurich, Switzerland and Department of Pathology University of Michigan, Ann Arbor, MI, Immune surveillance of skin cancer itivolves the stimulation of effector T cells by lumor-tierivcd anligen-presentitig cells (APCs). An effective APC must not only display processed antigen in the cotitext of MHC moleeules but also express co-stimulatory molecules lliat bind and activate T eells. One of the most comtnon skin Itimors is basal cell carcinoma (BCC). To investigate the expiession ofthe eo-slimulalory molecules CD80 (B7-1) and CDK6 (B7-2) on BCCs (n=l()), serial eryostat sections were stained with appropriate antibodies and B7 positive APCs were counted a.s % of HLA-DR positive celis with dendritic morphology. While only 1-2 % of intratumoral and 5-10 % of perituuioral APCs expressed CD80 or CD86, iti inflammatory control biopsies (allergic contact dermatitis and atopic dermatitis) 40-70% of APCs expressed CD80 and CD86. We nexl isolated dendritic cells from shave biopsies of BCCs (TUDC). TUDCs were non-adherent to plastic, displayed a typical dendritic morphology and expressed high levels of MllC-class II molectiles on Eheir surface. When those cells were compared to DCs from normal skin (NNDCs) for presentation of superatUigens (staphyllococcal enlertoxin A and B) lo T cells, TUDCs were consistently weaker (4050%) stimulators of T eell proliferation than NNDCs. 3-day culture in GM-CSF containing uicdia reconstituted lhe immunostimulatory capacity of TUDCs. Furthermore t;x|>iession of CD80 and CD 86 was assessed by flow eytometry. in a represenlalive experiuient 16% of TUDCs expressed CD80 (eompaied to 48% of NNDCs) and 18% of TUDCs CDHfi (compared lo 5K% of NNDCs). Thus in one ofthe tnost comuion skin eaiicers proniineni collections of APCs are deficient in importani co-slimuiatory molecules in-vivo and in-vitro, as well as weak stimulalors of T cell proliferation invitro. The ability of tumor eells lo suppress APC eo-stimulalory aetivity may be one mechanism by wliich uiulignuiU cells evade tho hosl immune system.

LANGERHANS CELLS ACCUMULATE IN SKIN TUMOURS DUE TO REDUCED LEVELS OF BIOACTIVE TUMOUR NECROSIS FACTOR-a, BUT ARE ABLE TO INDUCE ANTI-TUMOUR IMMUNITY. GajX-M; Halliday. Diana M. Rubel. Kylie S. Yuen and Lois L. ravanaglk Department of Dermatology, tiniversity of Sydney at Royal Prince Alfred Hospital, Sydney, Australia. It has previously been shown that Langerhans cells (LC) accumulate in skin tumours due to production of factor(s) by tumour cells (Halliday et al.. Immunology, 77:13, 1992). To determine factors which lead to this accumulation, bioactive TNFo, GM-CSF, and IL-3 were quantitated in a range of tumour lines with differing abilities to accumulate LC. There was no detectable IL-3, and GM-CSF did not correlate with LC. Those tumours which accumulated LC contained significantly lower levels of bioactive TNFa than tumours which did not accumulate LC. Skin grafts or epidermal cell suspensions from above one of the tumours were able to induce protective anti-tumour immunity in naive mice. The tumours with high LC densities were all progressors; those which did not accumulate LC were regressors. Hence we propose that the low level of TNFa produced by some tumour lines is insufficient for LC migration from the tumour, thus inhibiting the induction of antitumour immunity. If given the opportunity to migrate to local lymph nodes, these LC are able to provide protective immunity.

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LANGERHANS CELL FUNCTION DURING CARCINOGEN INDUCED IMMUNOSUPPRESSION. ILK. Muller, S.I. Rage. A.R. Chilcott. A.D. Grzegorczck. G.W. Dandie and G.M. Woods. Department of Pathology, University of Tasmania, Hobart, Australia, 7000. The complete chemical carcinogen DMBA depletes the skin of Langerhans cells (LC) and application of antigen through LC depleted skin induces antigen specific immunosuppression via mechanisms which require definition. This study investigated the function of the residual LC by (i) applying the fluorescent antigen FITC through DMBA treated murine skin and isolating the FITC+ cells from the draining lymph nodes and (ii) analysing the function of the LC collected in the afferent lymph draining DMBA treated sheijp skin. After DMBA treatment, fewer FITC+ cells reached the lymph nodes; these cells failed to induce antigen specific proliferation of naive syngeneic T cells and had a reduced ability to cluster with T cells due to a preferential reduction in cluster formation with CD4+ T cells. On further examination it was found that these cells could still bind effectively to CD8+ T cells without inducing proliferation. Analysis of culture supernatants from cocultures of LC migrating from DMBA treated sheep skin and antigen specific T cells revealed a significant reduction in IL-lfi production. Thus DMBA induced immunosuppression is due to a number of events - reduced ability of LC to convey antigen to the draining lymph nodes, reduced binding with CD4+ T cells and a failure to produce IL-IB.

INHIBITION OF LANGERHANS CELL MIGRATION BY TUMOUR DERIVED FACTOR(S) IS RESPONSIBLE FOR THEIR ACCUMULATION IN THE EPIDERMIS. Andrew D. Lucas. Gary M. Hallidav. Department of Medicine (Dermatology), University of Sydney at RPAH, Sydney, Australia. Skin tumour derived factor(s) (TDF) cause an accumulation ot Langerhans cells (LC) when applied to normal murine epidermis {Halliday,ef a/. Immunology 77:13, 1992). The aim of this study was to determine the mechanism by which TDF increases LC. Parental skin grafted onto F1 hybrids recipients demonstrated that the accumulated LC were of parental not FI phenotype and thus derived from the skin, not bone marrow precursors. This indicated that the LC did not accumulate diie to chemoattraction into the skin but must have arisen from in situ mitosis or inhibition of migration out of skin. Incorporation of the thymidine analogue bromo-deoxyuridine (BrdU) did not show any evidence for TDF-induced LC mitosis. LC migration from the epidermis, induced by fluorescein isothiocyanate (FITC), was lower in TDF treated skin than controls. In controls FITC decreased the number of epidermal LC but did not have this effect in TDF treated mice, in support of this, the draining auricular lymph nodes of control FITC-treated mice contained larger numbers of LC than the lymph nodes of FITC/TDF-treated mice. We hypothesise that skin tumours produce factor(s) which inhibit LC rnigration to iocal lymph nodes and that this leads to the LC accumulation in tumours, which has been observed in many studies.

veil.

Aii.si ii..'\(ris

.s. NO. (.

25 DNA DAMAGE IN CUTANEOUS ANTIGEN PRESENTING CELLS IS CAUSALLY RELATED TO LOCAL SUPPRESSION OF CONTACT ALLERGY IN UV-IRRADIATBD MICE. A.A. Vink. E.M. Slrickliind. C D . fiiican;!. I.. Rozal. n . B . Yarosli2. and M.L. Krinkc Depanment of Imimiiiology, UT M.D. Anderson Cancer Center, Houston TX, 'TNO Nutrition and Food Researeh. Rijswijk, The Netherlands, and 2Applied Genetics, Inc., Ereepotl, NY. DNA datiiage in ttie form of cyclobutane pyritiiidine liitiiers (CPD) is an itnportant molecular event in suppression of the contact hypersensitivity (CHS) response by U V B . Whether keratinocytes, which produce mediators that can tnodify the activity of A P C , or cutaneous APC themselves ate the relevatit targets for DNA damage is not known, ln these studies we test the hypothesis that DNA damage to etitaneous APC is responsible for their impaired ability to induee CHS in UVB-irradiated miee. C311 miee w e r e exposed to .•) kj/m^ UVB from FS40 stuilatnps and sensitized epicutaneously, 3 day.s later on ihe exposed skin witii FITC. APC were collected frotn the drainitig lymph nodes (DLN) 24 hr later and enriched hy metrizatiiide centrifugation. Sotiie of the Fn'C+ APC eotitained DNA damage detectable witli a monoclonal antibody against C P D and itiitiiunoperoxidase staining. "This cell poptilatioti was deficient in its ability to induce CHS when injected into the'footpads of normal C3H mice. Tiiese cells were incubated in vitro with liposomes containing a photolyase (photosomes), which, upon activation with visible light, rapidly tepairs CPD. This treatment reduced the number of CPD+ cells in the preparation and completely restored their ability to induce Cl IS to F I T C in recipient mice. Photosome or light treattiienl alone or light given before photosotne ttealnient did not restote activity. Thus, repair of CPD in vitro restored the antigen-presenting activity of the cutaneous APC from UV-irradiated tnice, suggesting that DNA datnage to etitaneous APC are responsible for this immtinostippressive effect of UV.

863

26 LANGERHANS CELL CYT()SKF.LETAL DER ANG1-:M1-NT .'\ND THE DFiLETERlOUS EFFECTS OF I'VB R A D l . \ r l O N ON CONTACT HYPERSENSIVITY. S.Bacci..l.\V.Stivilein. Schcpi-ns Eye Reseatch Institute and Department of Dermatology, HarvartI Medical School, Rostiiii MA. 02114. Acute low dose UVB radiation of tiiotiSL' .skin impairs the induction of eontaet hypersen.sivity (CH) to loeally appiietl dinitrtintiorobeir/etie (DNFB). and aitets the morphologic appearance of resident Latigerhati.s cells (LCV In iespon.se to UVB. Ia+ eells of the epidermis lo.w their dendrites and a.sstime a swollen, rotitidi'd shape. Similar effects on CH and LC arc achieved when skin is trcateil with subitiOatiimatory tloses o\' rceombituuit tiitiritiL- tumour tieciosis t'actoi-alpha (TNFa) atid c/.v-urocanic acid (UCA). In an effort to detertiiine whether the morphologic changes observed among UVB-expo.sed LC lesull from produclive re-anangetnent or from palhologic disrt^ition of the eyto.skeleton. tnoti.se skin was treated with vinhlastine. an anti-tnitolie alkaloid that disrupts tiic cytoskeleton by causing mierotubule disassemhly. When DNFB was applied to \'inblasIine-lreatL"d skin. Cl 1 indtictioti was grossly impaired, atid epidermal la bearing cells displayed a rounded shape with tew. if any. dendrites. Moreover, itnmunoliistoeliemieal stain for Ihc intetniediate filanieiit. vitnentin. revealed that vinblastine treated LC no longer displayed polymeti/ed vimentin. iiidieating that the eyto.skeleton had been disrupted, rather thau leananged. LC frotn UVB-e.x'posed. or TNFa or UCA-treated skin also failed to express eytopla.sniie .staitiing lor \'itnentin. Cyclophostatnide. an ami mitotic agetit that tkies nm alter the eyto.skeleton. neither impaited t i l induclion nor altered LC morphology or vimentin staining. We eotiehide that theie is a link between eytoskeletai integtity ol LC and their capacity to futiction as antigen pre.scnting cells during CH induction, and that cytoskeletai derangetnent of LC is an impottant nieeliatiisni by wliieli UVB i"adiation (peiliaps via UCA atul TNFa) ititeifeies with etitaneous immutiit\'.

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DEFINING THE ROLB(S) OF LANGERHANS CELLS IN THE IMMUNE ABNORMALITIES EVOKED BY ULTRAVIOLET B RADIATION OF SKIN. .I.W. Streilein. I. Kutitnoto. R. Dai,Sehe|x;ns Eye Researeh Instittite and Dep;lt-ttnent of Demiatology, Har\atd Medical Sehtxil, Boston, MA, USA. Acute, low dose ultraviolet B radiation (UVR - ACtO J/ni - x 4 days) is toxic to epidemial L;ingerlians cells (LC), elicit.s dertnal inllaninutCion, itnpairs eontaet hyper.sensitiN ity (Cl 1) itiduetioti in UVB-stiseeptible tniee exposed to a eonventional dose of dinittofluotobcnzene (DNFB), and induees hapten-speeifie toleratiee when DNFB is applied to the irtxidiatcd surfaee. By diffetcntially exatnitiitig the iti wm antigen presenting properties of hapteti-dcrivati/.ed epidetinal and dennal eells han ested from UVR-exposed skin, we have detertilined (a) that phagix-ytie dertnal eells. bttt not cpidemiiil eells, ftotn UVB-resistunt mice can itiduce CH, and (b) that non-phagoeytie dermal eells frotn hotli UVB-S atid UVB-R tniee ean tnduee loler.mee. Thus, exeept by their absetice, LC appear HO/ to participte in the imtnune reaetivities obset^ed after the typical acule, low dose UVR teginien. When UVR was limited lo a single ex|X)stite ( 4 « ) J/tn=) whieh pertutbs. bul is not lethal to, LC, CH induction was impaired wi/y in UVB-S mice expo.sed lo an oplimal sensitizing dose of DNFB, and no lolemnce W;LS obsetved in these miee. Finally, haplen-dcnvatized epidemial eells prep;ued ftotn UVB exposed .skin of UVB-R. but not UVB-S, miee indtieed CH when injeetcd into naive, .syngeneic tccipicnts. These obset\alions itidicale (a) that a single low dose UVR e.sposute selectively acts pritnarily within the epidertnis, (b) that at this dose LC of UVB-R tniee tetain their ability to prcn ide APC lutietion leaditig to CH induetion. atid (c) thai UVR-exposed LC of UVB-S miee do not genet-ate tolerance. Sitice higher doses of UVR \ irtually elitninate LC from llie skin, CH induetion in UVB-R tniee and loleratice in UVB-S mice excised to higher UVR doses nuisl be dictated b.v ettlatieous cells nllter tttnti LC.

IIVB-Uiufiatioii Pcrttirb.s the 1 tinctional I'.xprcssion of IJ7-1 and B7-2 Coslimiilatoiy MoUcuIcs on Ihimaii i.an^eiiians f ells (LC) .I.M. Weiss. A. Rcnkl. RAV. DcnCckL I:. SchopfatKi S.V, Simon Dopt. Dcniiatol.. iM'ciburg, (icrniaii} ln jiro\ious .studies, we lm\'e sluiwn UX'H-radiation to con\ert IX' IVoin iimmogenic lo lolerogenic APC hy inlerferiiig \\itli niembrane-boiind cosliniulatory signals. Reeenlly. B7-I. B7-2 costimulatory nioleeulcs (tin Ai*C) weic demonstrated to deli\'er inijiottanl eoslinuilaU)ry signals ihiough inlcraetion with tlieir eonntcrteceptors Cn2S atul Cri.A-4 (on \ cell.s). We therefore qiieslioned whether I'VB would affect the functional exjire.ssion oi' B7-1 or B7-2 on LC. Vo addres.s this question. B7-I-, B7-2-e\pression was studied on htmian l.C h>' 3-color nowe\'tonietr\' using CDla, B7-1 or B7-2 and the vital dye 7-AAl) as niarkers. Little, if any. B7-'l or B7-2 was detected on freshly isolated LC. However, following 4S h of culture hoth B7-1 and B7-2 were markedly upregulated on LC. To test whether these molecules were functional, primary, mixed leukocyte reactions (MI R) were performed in the presence or ahsenee of hloekitig niAh to B7-1 or B7-2. Only anti-B7-2 , hut not antiB7-1 inhihited the MLR. indicating that ihe allostimulatory capaeity of humati LC tlepends primarily on the functional expression o{ B7-2. \ A'B-ratliation dosedependently (50-2'0() h'wv) inhihited the eulture-induced upregulation of B7-1 and B72 on LC, without ititerferitig with their viahility. rurthcrniore. LC exposed to the same lluences of L'VB (UVB-LC) failed completely to sLimulate an MLR. I'inally. we wished to determine whether the UVB-induced suppression of B7-1 and B7-2 was related to the inahility o'i UVB-LC to stimulate an MLR. In this ease, exogenous triggering o{ the B7-counler-reeeplors CI')2S or CLLA-4 on the '!" cells may pre\'ent their proliferatix'e Linresponsi\'eness. Indeed, addition of sohihle anti-CD28 mAh to the eocullures of ii\'B-LC and allogeneic 'f cells pai'tiallv resloretl pi-oliferation. We conchulo. that the suppressi\e effects of low-dose I !VB-radiation on ihe .MH' function of LC are. at least in part, due to an itiliihition of functional B7-i- and B7-2expression.

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EFFECTS OF ULTRAVIOLET LIGHT ON OVINE LANGERHANS CELLS Geoffrey W Dandle. Gavin J. clvdRsdale. Fiona J Radcliff.Irene Jacobs and H. Konrad Huller. Department o f Pathology, University of Tasmania, Hobart, Australia. The effects of UVB light on sheep Langerhans cells (LC) was studied by cannulating the afferent lymphatic vessel draining a defined area of skin and collecting lymph for investigation. The parameters studied include: the pattern of LC migration, LC morphology and the antigen presenting capacity of these cells. These changes in LC were correlated with cutaneous damage caused by eguivalent doses of UV light. UVB light triggered LC migration in a dose dependent manner with significant increases in the number of LC leaving irradiated skin within hours of exposure and again 3 - 4 days later. Electron microscopic examination of LC migrating from skin exposed to low dose (3,600 J/H') UVB between 48 and 72 hours post irradiation showed a loss of dendritic processes. In parallel studies, migrating LC were found to be incapable of normal antigen presentation after exposure to this dose of UV light. This loss of function occured between 48 - 72 hours post irradiation and remained for at least 14 days. It is concluded that loss of dendritic processes may be an important factor in the altered function of these eells after UV exposure.

HAFIliN TRE.VrMnNT SriMliI..ATl-S .\ 95 kDa T">PH IV COLLAGliNASli SECRETION FROM MLIRINE EPIDERMAL LANGERHANS CELLS. Yasunobtt Kobayaslii. Skin Cate R&D Division. R&D Headquatlers. SUNSTAR INC.'Takatsuki. .lapati. ltl otder to know how cpidcttnal l^mgethatis cells (LC) |)eticltalc through Ihc basetiietil tticnilitatte, we cxatnitied if sonie of tnattix nielalloptotcitiases (M.MP) ate itivolved in Ihe tnigration properly of LC. I'sitig gelatiti-substratc cn/.ymogratn, we fottttd that LC enriched epidertnal cells ohiaitied ftotn the ears of 3% TNCB-paitited tnice secreted a 95 kDa gelatitiaso. Highly etirichtnetit of LC by the tiiagtietic cell sotter via \-i\ atitigctis tevealed thai the gclatittase itidticed by epictttatteott.s TNCH treatment was LC-derived one. Western blot analysis using a specific ni.\b .showed that this gclatitiase was a 95 kDa type IV collagctiase. the so called tnttrittc MMP-9. Other haplens, DNCB and DNEI3 tteattnetit also indnced a 95 kDa type IV eollagenase secretion of LC-enriched epidertnal cells. By conttast. little eollagenase itidttetion was obsetved by SI.S painting. These testtlls suggest Ihat a 95 kDa type IV collagettasc indttced by epietttaneons hapten application tnay be involved in the inigtatioti capacity of LC. This tnay allow LC to pettelrate throngh the basetiicnt tnetnbratte in the early event of the itiductioti phase iti hapteti-indtieed eontaet hy[x?r sensitivity.

864

J()UU.NA1 Ol- INVESriOAllVi; DHKMATOLCKIY

ABSIKACrS

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ROLE OF BI INTBGRINS IN THE IN VITRO MIGRATION CAPACITY OF HUMAN EPIDERMAL LANGERHANS CELLS. Y. Kobavashi ill. M-J. Slaqucl. C. Dc'/Liitcr-Dambuvant. D. Schmilt. INSERM U346, aflilic CNRS, Dcrnialology Research Unit, E. Herriot i-lo.sp., Lyon, France.'')Sunslur Inc, Takalsuki, Osaka, Japan. Epidermal Liingeihan.s cells (LC) lunelion its mobile messenger cells which aclually carry antigens Irom (he skin to the lymph ntxies. However, lhe driving and regulatory mechanisms lor LC motility remain to be investigated. During the journey to the regional lymph nodes, LC encounter and make contact with various extracellular matrices (HCM). The major receptors by whieh cells attach to the ECM molecules appear to be integrins and particularly Bl integrins. We studied the elTect ol' some extracellular matrix proteins on the migration capacity of epidermal dendritic LC /// vilro., and in\'cstigated the role of BI integrins in this prt.x;ess, using two migration assays; the chemo-invasion assay (matrigel assay) and the phagokinetic track assay. Although freshly isolated human epidermal I^mgcrhans cells exhibited a very low migratory capacity, they showed a marked increase in cell migration alter in vilro contact with liaptcns. Type I and type IV collagens, and libronectin promoted the migration ot these hapteni/cd-Langerhans cells. Using as inhibitory probes monospecilic antibodies that rccogni/.e integrin subunits, we found that bl(x:king the BI subunit inhibited hapten-induccd latngerhans cells invasion through matrigel and also inhibited their random migration on type I and type IV collagens, and on libronectin. The addition of anti-(x() stibtinit anlib(xly inhibited the invasion of Langerhans cells through matrix-txuind himinin (matrigel) hy 45^7<.. Bhxjking the a5 subunit significantly inhibited migration on fibronectin but not on collagen matrices. Our dala indicate that the extracellular matrix plays se\'eral imix>rtanl roles in epidermal l..xingerhans cells migration from the epidermis to lhe dciniis, and this is regulated by some of the |H integiins including at least
DENDRITIC CELLS OF SKIN AND ORAL MUCOSA, MIGRATORY CAPACITIES AND THEIR ROLE IN ORAL TOLERANCE G. Kraal. F..J.G. van Wilsem. D. Wolvers and R.J.S. Schcper. Department of Cell Biology and Pathology, Vrije Universileit, Amsterdam, The Netherlands. The oral mucosa is an important silc lo induce immunological tolerance to protein antigens. Because of the importance of tolerance indtiction as a possible way to modulate allergie reitclivity we wished to study the mechanisms involved in effieient toleranee induction via the oral mucosa. Dendritie Langerhans' cells in both skin and oral epithelium are the first eells to encounter antigen. By applying fluoresceinated hapten on skin or oral mucosa of the mouse we eould demonstrate similar migratory aetivilies of the Langerhans from both sites to the draining lymi)h nodes. To see if any functional differences between the langerhans cells deriving from the two different sites would exist we sttidied the antigen presentation capacity of the two cell types. However, no differences in antigen presenting capacity were seen after stimulation of the dendritie eells via skin or oral mucosa. Transfer in vivo of dendritic cells from either site did not restilt in loleranee induction but instead both cell types were able to induce delayed type hypersensitivity reactions. Analyzing the eytokines produeed in the draining lymph node a skewing towards Th2 was seen after oral antigen application. The results indicate ihal dendritic eells do not directly influenee the direction of the generated immune responses, but that local factors in the microenvironment of lhe draining lymph node are of crucial importance for the induction of oral tolerance.

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LF|-iiC:'r Ol- tlVU I R R A D I A T I O N O N THli MIGRATORY PROPERTIES AND FUNCTIONAL CAPACITIES 01- HUMAN LANGERHANS CELLS C D . RichlL-rs. E. Reits. A.M. van Pelt. M J . Hniikstra*. J.S, du Pont* aiul E.W.A. Kitnipcrdiik. Departmeni of Cell Biology and Imiminiilogy, Faculty of Medicine. Vrije Universileit, Amsterdam and *lJuriis Research Institute, Beverwijk, The Netherlands. Il has been shown that UVB irradiation causes immunosuppression both in mice and human. After ex[io.sure of .skin to UVB, the induction of contact hypersensitivity i.s impaired. UVB irradiation inliuences the Langerhans cells (LC) in the epidermis hut the exact mechanism hy which the tolerance is achievetl in vivo is not yet fully understood. Recently, we have .shown that after culture of human split skin, CDla positive dendritic cells (DC) migrated "spontaneously" into the medium. Part the C D t a + eells were epidermal Langerhans cells as shown by the presence of Birheck granules, other cells expressed CDlh, a marker of dermal DC. In this siudy, we investigated the intluence of UVB irradiation on the migratory aiitl liiiictioiial capacities of skin DC. The skin was divided in two pans of e(|ual size. One part was Tir.st inadiated with 15 mJ/cnr UVB liglit, using a Philips lamp (40W/I2), the other was direeily placed in the ineJium. After 24h i)f euliiire, the non-adliereni niigraied cells were harvested and coimted. The inimber ofcells that had inlgraled out ofthe irradiated skin was 35-60% lower. The percentage of cells expressing CDlh was higher in the population that migrated out of the UVB treated skin. Furthermore, the capacity of tliese cells to stimulate ailogeneic T cells of this population was lower in comparison with tlie eells migratetl from untreated skin. This could be due to the lower vlaliility of the cells (5-10%) and the lower expression of LI-A-3 after UVB treatment. Expression of MHC cia.ss II was not different. These data indicate tliat after UVB irradiation. LC are impaired in their migratory capacities. Prohably, the used dose did not iiiHuencc migration of dermal DC hut alters their allostimutatiiig function.

l U J M A N D E N D R I T I C C E L L S S L C R E T E , B I N D A N D I N I E R N A L I Z H lHH S O L U B L E , 1 ' R A N S M E M B R A N E DOMAIN-DBLliTHD FcYRIIa2. M. Fallcr (I.LX Monciiit (2). 11. dc la Salle (11. A, Bolibol (3). W.-H. Fridman (2). .1 -P Cazcnavjj (4). .L-L. 'IVillaud (2). and D, Ilanau (1).INSERM (1) C.IF 'M-()3 and (4) Li.311. H.T.S., Strasbourg; (2) INSERM U.255, Institut Curie. Paris: and (3) Service d'Oncoilcnuiiologie. Hopilal tic Ilaulepierre. StrasboLU'g, Franco. Monocyte-derived dendritic cells (DC) express two forms of I-cyRIF: a ineinbiaiicassociated form. FcyRIIal. detected by the anti-I'cyRII niAh IV.3, and a soluble sccreteti form. Fc7Rlla2. Existence o f t h e latter form was demonstrated both at the mRNA and at the protein level. Indeed. RT-PCR cxperimcnis performed on DC mKNA icvcaletl the presence of (he transcript encoding the transinembrane doniain-deiL'ted Fc7Rlla2. Moreover. DC supernatants contain l-"e7Rila2 as shown (i) by an BLISA. using the mAb IV.3 as capture antibody and polycional antibodies directcti against the intracytoplasmic tail ol" FcYRIla2 as revealing antibodies and (ii) by Western blot analysis. The latter experiment - performed wilh polycional antibodies directed against the entire intracytoplasmic tail or against a peptide locateel near the C-tei"ininal part of llic intracellular region of Fc7RIIa2 - demonstrated that both a complete fonn of Fc7RIIa2 and a C-tei'minal truncated form were presenl in DC supernatants. Intciestinglyaddition of TNF-(x (10 ng/ml) to the ctilture medium rctluccs (i) expression of botli FcyRllal and I-C7R!la2 iiiRNA, (ii) expiession of the membrane-associated I-VyRIIal and (iii) the level of produclion of soluble f*C7RII by DC. Finally, using gokl-Iahclcti icconibinanl r-C7Rlla2 we obsei'vcd that DC bind l'V7Rlla2 which is then intcrnali/.eti by rcccptor-mediatctl enducytosis. The nattirc of the receplor which binds l'V7Rlhi2 remains unknown just as the significance of this internalization [cmains unknou'ii.

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PURIFICATION OF HUMAN PERIPHERAL BLOOD DENDRITIC CELLS BY COUNTERFLOW CENTRIFUGAL ELUTRIATION AND HIGH GRADIENT MAGNETIC CELL SORTING. M. Schmilt-Eqenolf. D. Maurer. G. Stinql. DIAID, Dept. of Dermatology, Univ. of Vienna Medical School, Vienna, Austria. Human peripheral blood dendritic cells (DC) are phenotypically defined as lineage marker-negative and HLA-DR-positive cells. Previously, DC have been enriched from human peripheral blood by means of positive selection for HLA-DR-expressing cells and depletion of T cells, monocytes, NK cells, and B cells employing antibodies against CD3, CD11b, CD16, and CD19, respectively. We used counterflow centrifugal elutriation to preenrich DC by depleting lymphocytes, residual erythrocytes, dead cells and cell debris from Ficoll-separated PBMC. Thereafter, cells were subjected to high gradient magnetic cell sorting (MACS) to remove still contaminating CD3-, CDUb-, CD56-, and CD19- bearing cells. The resulting cell population homogeneously expressed high levels of HLA-DR but was lineage markernegative as determined by FACS analysis. Interestingly, the reactivity of mAb 15-1, directed against the a-chain of FccRI, identified two subpopulations within this highly enriched DC population. Comparative phenotyping of these two subsets revealed no differences in the expression of DC-related cell surface structures. Based on these results, we postulate that the expression of FctRi may not only enable DC to specifically bind IgE but, furthermore, may define two functionally distinct DC populations.

IN VITRO INFECTION OF CD34.f PRECURSORS OF DC/LC THROUGH THEIR DIFFERENTIATION. A,S, diarhonpiff'jajgrricr^. C, Jacauct'. C. Mii5.sncri.Tl. M.M. Ficrs', E. Mallei^. C. Dcyiillcr-Dimiliiivanll. D. Schmill'. 'lNSBRM U346, •tNRS/bioMSricLX, Lyon, France.

Langerhans cells (LC) can be generated in vitro by culturing CD34 + heniatopoietie progenitors with GM-CSF + TNFa. In seropositive patients, bone marrow CD34-I- cells were rarely found infected or not by HlV-1. Nevertheless they were shown sensitive to infected in vitro by HIV-t. In this study, we tested Ihe sensitivity to HIV-I infection of in vitro generated LC along their differentiation and we investigated the effect of such an infection on the in vitro differentiation. Phenotypic controls were performed by FACS analysis atday6 with the presence of CDl-f cell population and by transmission electron microscopy at day 13 for ultrastructural differentiation with the presence of Birbeck granule (Bg). CD34+ cells were purified from eord blood PBL by a magnetic separation. Cell suspensions were infected with either a T-lytnphotroplc, syncytium-inducing (SI) isolate (HxB2) or macrophage-tropic, non-syncytium-inducing (NSI) isolate (Ba-L). Cell infection was controled for the presence of the provirat DNA from cellular DNA by means of PCR amplification in gag genes. Viral particle release was measured by p24 antigen production in the culture supernatant. A significant high level of p24 production was noted on day 16 of post-infection only when infection was carried with Ba-L isolate on cells generated after 6 days in culture with GM/CSF + TNFa. No infection of CD34-^ progenitor cells was obtained either with Ba-L isolate or HxB2. The sensitivity of LC/DC precursors to NSI isolate (Ba-L) seems to coincide with the early stage of differentiation (CDta antigen appearance). The infection did not alter the differentiation of in vitro generated LC which present their specific ultrastructural marker of epidermal environment, Bg at day 13 of culture as compiu'cd to control culture. The prclitninary results enlight the sensitivity of HIV infection of a differentiated population of DC/LC generated in vitro from Cp34-f progenitors. Next investigation will be to clarify the precise relationship of the infected cells to the dendritic cell family (LC peripheral DC, dermal DC ...).

vol..

11)5. NO.

AiiSi'RACrs

i)i:c:r;MHi;n.

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HETEROGENEITY OF DENDRITIC CELLS: IDENTIFICATION OF TWO DISTINCT SUBSETS. M,0, Canning, .J.M.W, vnn Haarst, H.J. do Wit, H.A, Draxhacio. Department of Immunology, Erasmus University, Botterdnm, t h e Netherlands. Previously, we establislied that cells willi a morphology and function of dendritic cells (DC) could be maturated from human peripheral blood: cells with a dendritic morphology, MHC class II positivity, woak-toabscnt acid phosphataso reactivity nnd strong capability to stimulate naivG T cell proliferation werD obtained f r o m monocytos after exposure to the thyroid Hormone tri iodothyronino (T31(Mooii cl al, J Endocrinol 1994: 1 AO: 503-512). Subjecting these cells to a chemotaxis assay using tho chemoattractant n-formyl methionylleucyl-phenylalanine (fMLP) in the Boyclen Chamber, followed by Magnetic Cell Separation (MACS) employing moriocional antibodies to CD3, CD19. CD56 and CD141LeijM31 resulted in 7 4 ± 8 % of The cells displaying a dendritic morphology and phenotype. Further MACS purification of these cells using iho monoclonal antibody M y l ICDl-l) resulted in distinct My4- and My4-(• subsets. The My4- cells, 71 ±10 % of which exhibited a dendritic morphology, were positive (or HLA-DR, L25 and RFDl. These cells were aiso positive for and produced the calcium-bindino protein S-100, whereas tfie My4 + colls, 92 ± 8 % of which displayed a dendritic morpholooy wero negalivo in this respect, My4- cells were excellent stimulators of T cell proliferation (median PI-=18B 5 at stimulator-to-T cell r.ilio 1:10, P<0.05), in stark contrast lo My4+ cells which were extremely poor in stimulating T cells to proliferate (median Pl-10,25 at slimulator-to-T cell ratio 1:10, P<0.05). The phagocytosing capabilities of the two subsets also differed: My4 + cells were better at phagocytosis (median = 32 5 %phagocytosis at 50 min.) than My4- cells (median = 14,5 % pfiagocytosis at 10 min., P
MONOCYTES ARE THE PRECURSORS OF F C E R I / C D I A POSITIVE DENDRITIC CELLS OBTAINED BY CULTURE OF ADULT PERIPHERAL BLOOD MONONUCLEAR CELLS.

M. Kriimcr. T. Bieber. Department of Dermatology, Ludwig-Maximilians University of Munich, Germany. Beside tlie production of CDla+ Langoihans cells (LC) /// strich) scnsu, i.e. eontaining Birbeck granules, using CD34+ precursors from cord blood cultured in tlie presence of TNF-a and GM-CSF (Caux et al. Nature 1992), it has been reported that IL-4 and GM-CSF induce the phenotypie and functional differentiation of monocytes into CDla+ dendritic cells (DC)(SaIusto ct al. J,Exp.Med. 1994). On the other hand, the culture of plastic adherent mononuclear cells with IL-4 and GM-CSF has been proposed as a simple and convenient technique for the generation of CDla+ LC from yet to be defined progenitors from adult blood (Romani et al, J.Exp.Med, 1994). We used the latter protocole to study in details the emergence of CDla+/FceRl+ DC /// vitro. Kinetic experiments revealed a lapid (within 24 h) surface expression of FccRl on CD 14+ monocytes cultured in the presence of IL-4 and GM-CSF. These cells then progressively became dendritic and express CDla+ while CD 14 expression decreased and completely disappeared by day 6-7, At that time point, almost all previously CDI4+ cells had become CDla+ DC. Maximal FccRl expression was noted by day 45 and then progressively decreased. The addition of IL-9 lead to a modest but significant increase in the FeeRI expression. We conclude that FccRlf/CDIa+ DC obtained by culture of adult mononuclear cells with IL-4 and GM-CSF are derived from CD14+ monocytes and that II.,-9 modulates the FceRl expression on these cells. Whether these DC will give rise to LC /// ,slriclo sc/isu remains to be determined.

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EXPRESSION OF THE IL-2 RECEPTOR T . - C H A I N BY MURINE EPIDERMAL CELL SUBPOPULATIONS. M. Mohamady-adeh. K. Ariir.umi. D. Kdclb.-ium. P.R. Bcr|;stre.ssLM-. A. Takashima. Department of Dermatology, UT Southwestern Medical Center, Dallas, TX, USA The IL-2 receptor (R) complex is composed of three components, a, (3 and 7,, each of which is required for high affinity binding of IL-2. The 7,-chain, which is also an essential component for IL-4R, IL-7R and IL-15R, plays n critical ii-nmunological role, as evidenced by the fact that its mutation causes X-linked severe combined immunodeficiency. Here we exatnined the expression of the 7,chain by murine epidermal cells, i.e., Langerhans cells (LC), dendritic epidermal T cells (DETC), and keratinocytcs (KC). 7,-chain mRNA was detectable by RT-PCR in unfractionated epidermal cells isolated from BALB/c mice, as well as in each subpopulation: 1-A* cells (LC), Thy-T cells (DETC), and I-A/Thy-l" cells (KC). Likewise, 7^-chain mRNA was detected in the XS52 epidermal-derived dendritic cell (DC) line, 7-17 DETC line, and Pam 212 KC line. Thus, each epidermal subpopulation lias the potential to expre.ss this receptor. FACS analysis revealed significant 7,-chain surface expression on 7-17 DETC. Importantly, expression was regulated by the state of cell activation, including upregulation by Con A. Anti-7,chain iiiAb blocked the proliferative responsiveness of this line to IL-2, lL-7, or IL1 5, indicating that the 7,-chain on DETC surface is functionally active. Only marginal, if any, stirface 7,-chain expression was detected on unstimulated XS52 DC o r Pam 212 KC, suggesting a requirement for cell activ.uion. We propose th.u exogenous stimuli may modulate cutaneous immunity, not only by altering the local production of cytokines, but also by regulating the expression of the 7,-chain by epidermal cells.

MURINE EPIDERMAL LANGERHANS CELLS DO NOT EXPRESS INDUCIBLE NITRIC OXIDE SYNTHASE. H. Moll. C. Boprian. C RlntikResearch Center for Inleelious Diseases, University of Wiirzburg and Institute of Clinical Microbiology and Immunology, University of Erlangen-Niirnberg, Germany. In Lm/iHM/»Vj-infected macrophages, the formation of nitrogen oxides is critical for intracellular killing of the parasites. Their production is mediated by the inducible nitric oxide synthase (iNOS). We have recently shown that, in addition to macrophiiges, epidermal Langerhans eells can phagoeytose L. tnajor. It was therefore of interest to analyze whether Langerhans cells display the same leishmanicidal effector mechanism. For this purpose, pure Langerhans cells were collected, using single-cell picking, and were infected with L. major and/or activated with different cytokines in the presence or absenee of LPS in vitro. Subsequently, the cells were analyzed for expression of iNOS mRNA using RT-PCR. In contrast to macrophages, Langerhans cells did not express iNOS mRNA. On the other hand, significant levels of iNOS mRNA could be detected in unseleeted epidertnal cells, the majority of which consists of keratinocytes. These results indicate that in the L. /H^/'or-infected skin, activated macrophages and keratinocytes, but not Langerhans eells have the ability to express iNOS activity. The implications of this finding for the interaction of Langerhans cells with intracellular pathogens will be discussed.

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EXPRESSION OF CUTANEOUS LYMPHOCYTE-ASSOCIATED ANTIGEN ON HUMAN LANGERHANS CELLS. N.Yasaka, K.Nakamura, M.FtJrue, K.Tamaki. Department of Dermatology, Yamanashi Medical University, University of Tokyo, Japan. Cutaneous lymphocyte-associated antigen (CLA) defined by monoclonal antibody (MoAb) HECA-452 has been shown to be preferentially expressed on cutaneous T cells. The CLA expression has been regarded as a homing molecule of T cells to t h e skin in various inflammatory cutaneous disorders. We investigated the significance of CLA expression on Langerhans cells (LC) and found that, in normal skin, some epidermal LC express LC express CLA, and that most dermal CDla positive cells express CLA. When normal skin was organ cultured, the percentage of CLA positive cells in LC and dermal CDla positive cells decreased appreciably. In diseased skin, epidermal LC increased in number and most LC expressed CLA. Thus, this study suggests that the CLA expression on LC may play as a homing molecule of LC to the skin.

QUANTITATIVE AND 3-DIN4ENSIONAL ANALYSIS OF I^MAN LANGERHANS" CELLS IN EPIDERMAL SHEETS AND VERTICAL SKIN SECTIONS. A. Emilson, A. Sclicynius. Department of Clinical Immunology, Karolinska Hospital, Stockholm, Sweden. Confocal laser scanning microscopy was used to analyze and compare Langerlians' cells (LCs) in nomiai skin of 6 subjects. Acetone-fixed cpidennal sheets and 25-fim thick vertical skin sections were incubated witli fluorosceinisotliiocyanate-conjugated mouse monoclonal antibodies directed against HLADR. An individual llireshold setting algoritlim compensating for the differences in background fluorescence was applied to identify specific fluorescence. No statistically significant difference was found in the relative volume of epidermal HLA-DR reactivity between epidermal sheets (14 i 5%) and vertical skin sections (13±6%)orinthenumberofdendritesperHLA-DR+LCs(7.8i3.l and 5.9 ± 3.1, n = 58, respectively). However, statistically significant higher background noise was found in vertical sections tlian in epidermal sheets, lliree-dimensional (3-D) reconstructions of HLA-DR+ LCs revealed a concentration of HLA-DR to one or a few intracellular vesicles in 42 of 58 analyzed LCs in epidermal sheets and ill 18 of 58 analyzed LCs in vertical sections. Direct contact between dendrites from different LCs was not found. The results indicate that both skin forms arc suitable for quantitative studies. Due to less background intensity and larger tissue volume, detailed 3-D analysis of LCs is preferably performed on epidermal sheets tlian on vertical sections.

866

AIISIRACIS

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QUANTITATIVIC AND QUALITATIVIi CliANGES IN LANGERHANS CELLS IN IRRITANT DERMATITIS USING CONFOCAL MICROSCOPY. H . Slinhldnllnh. D.S. CiiriiiinL'liaiii'-. !•:. McVIILu-. R. Forsev*. c Snncls*. R.D. AkIrlclL'e. S.E.M. llowk-' & ,I,A.A. Ihinli-r. Dcpartiiienls of Dei-maloloHy and •Palliolofiy, Uiiiverslly of Edinburgh. ScoUand. U.K. Conftical la.sc-r seaniiinfj niicrixsropy (CLSM) is a powerful Lool for oblairiiii;^ morphological and (|uanLilallvc liiforiiiation IVoni thick tissue; s c d i o n s s u c h a s c-oinplele suction blister roofs. We used CLSM to investigate changes in CDIa+ve LanKc-i'lians cells (LC) in irritant reactions induced in \7 volunteers and 22 patients wiUl cliroiiic irritant deriliatitis. Nonanoic acid (80%) was applied under occlusion to the forearms for 1, 6 or 24h. Suction blisters were raised and the roofs snap-frozeii. Blister roofs were stained with CD hi antibody oveniinht, incubated with FITClalx^lled shee|) anti-mouse !^G anlibody and optically sectioned by CLSM.

BIRBIXK GRANUL[:S AND CORI-.D TUBIIL[-S IN CIRCUI.A TING LANGFiRllANS CFI.I.S Ol- M I C I ' Miya Kobnyaslii". Satoni DoJ-'. Takeshi Hoshinoli Department ol" Anatomy'' and Pediatrics^', Nagoya Univeisily School ol~Medicine, Nagoya 4(>6, Japan. l.angcrlians cells (l.f') are known to exist in ihe epidennis. dermis and superlicial lymph nodes In addition lo Birbeck granules, we have reported that I..C have another characteristic organeila, a coied tubule. I.X can be classified into ? cell types; liirbeck granule (Bg)-eoiuaining LC, coied tubule (('t)-containing IX~ as well as Bg and Ctcontaining IX' They all belong to the same IX' fainily Ct-LC occurred frequently in the derniis and ils draining lymph node, but none in llie epidermis ol'the same animal.In the deiniis ol'untieated mice, Ct-LC occurred se|)arately trom Bg-LC. LC with Bg :ind Ct occurred only afler the subculaneous injeclion o f saline, thus (biniing active lymph (low 'fhis also caused the increase of"Ct-LC in the paracortical aiea ofdraining lymph nodes.

Low power exaiiilriatioii showed an inci'ease in both % fluoi'esceiiee and ahsolnle nninbers of CDIa+ve LC Ih post irritation (21.3/ low power fic-ld) compared to normal eoiitrols ( 1 7 , 2 / l p n . At 6h this was still increiised iii volunteers (22.8/lpO bnl had started to fall iu patients (15.4/lpn. However, hy 24h there was a sifinifieant decrease in Ihe niimbei' of LC in both [jati(;nis and volunteers - 8. t (p
In contact sensilization with I''ITC, LC in the dermis contained many cored tubules in their cyloplasm lig-LC charaeleri/.e the LC that travel through the epidermis and other keratinizing epithelia. While, Ct-LC may not travel through the kei'atinizing epithelia. The distribution o f Ct-LC may not be restricted to the skin and its draining system, btit may be more wide spread in the interstitial connective tissue, fhe molecular dynamics ofthe exiraeellular [natrix components in ihe interstitial connective tissue eould be crucial lo facilitate migration route o f I.X\ Circulation o f LC family in ihe connective tissue has some clue to solve the role o f LC in allergic response

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INTERLEtJKIN-12 GENE EXPRESSION IN HUMAN SKIN DERIVED CD1a+ DENDRITIC LYMPH CELLS. N. Yawalkar. C.U. Rrand I R Rraathfin Dermatological Clinic. University ot Berne, Inselspitai, 3010 Berne, Switzerland Dendritic cells represent a widely distributed system of antigen presenling cells specialised to initiate primary immune response. Althotigh dendrilic cells purilied predominantly Irom the skin have been extensively investigated, cytokine gene expression ol CD1a+ dendritic cells from human skin lymph has yel lo be examined. Recent reporis point to a role for Interleukin-12 (IL-12), which is predominantly secreted by peripheral blood antigen presenting cells, in regulating T and NK cell function, macrophage activation and initiation of Th1 type celi responses. To investigate if human skin derived CD1a+ dendritic iymph cells are a source of IL-12, we cannulated microsurgically a skin draining lymph vessel on the lower legs of 5 healttiy volunteers. Altogether, 10 ditterent samples, each consisting of 1 x 10^ lymph cells, were investigated. In 4 out of fhe 10 samples, CD1a+ dendritic lymph cells were isolated and purified by positive selocfion using mouse anfi-CD1a monoclonal antibodies and sheep anti-mouse antibody-coated Dynabeads. Messenger RNA levels were estimated by a nested reverse franscriplase polymerase chain reaction (nRTPCR) method. Total RNA was extracted from fhe cells, reverse transcribed to cDNA and ampiified using specific primers for Ihe target gene. Amplified products were sized by electrophoresis and visualised by ethidium bromide. Expression of IL-12 p40 and p35 mRNA was defected in all samples, both whole lymph samples as well as the highly enriched CDla+ dendritic cell population. Funhermore, the identity of the PCR-amplified DNA-product was confirmed by sequence analysis. Our findings demonstrate that human skin derived CD1a+ dendritic lymph cells produce IL-12 mRNA and may therefore be an important source of IL-12. Thus one might speculate that these CD1a+ dendrilic cells, through their IL-12 producing capacity, might signiticantly intluence the balance of Th1 versus Th2 reacfions ultimately occurring.

MURiNE LANGERHANS CELLS DO NOT PRODUCE BIOACTIVE IL-12. J. Schv^nn, S. Froscb. A.B. Reske-Kunz. Clinical Research Unit, Department of Dermatology, Johannes Gutenberg-University, D55101 Mainz, Germany. Tho Thl done Klli5 specitic tor GAT efficiently downregulates IL-2 receptors following previous contact with the antigen and retums to a resting state. These T cells respond to presentation of GAT by splenic APC in the context of I-A,,''AB' with IL-2 production, IL-2 receptor upregulalion and proliferation. Recently we demonstrated that antigen-specific proliferation of Kill5 T cells depended on a B7-CD28-interaction and the presence of IL-12 as costimulatory signals. We showed that IL-12 enhanced upregulation of high-affinify iL-2 receptors and IL-2-dependent proliferation ot KIII5 T cells. In the present study, we used this model as a highly sensitive assay tor bioactive IL-12. Because both treshly isolated Langerfians cells (LC) and short-lerm cultured LC (cLC) are able lo provide aii knovm membrane-bound costimulatory signals tor activation of CD4+ T clone cells, especially B7-1 and B7-2, induction ot Kill5 T cell proliteration should solely depend of the capacity of LC to generate bioactive IL-12. KIII5 T clone ceils were stimuiatod in fhe presence or absence ot GAT presented by irradiated (20 gray), syngeneic APC. The APC tested were spleen cells (SC), LC and cLC (both >93% pure after magnetic bead enrichment), SC presented GAT to Kill5 T cells and induced proliferation of these T cells. Addition ot neutralizing anti-iL-12 anlibody abrogated proliferation, GAT presenting LC and cLC did not induce proliteration ot KIII5 T cells. Addition of IL-12 resulted in a dose-dependent proliferative response. These data indicate that both freshly prepared LC and short-term cultured cLC do not produce bioactive IL-12 and theretore do not gualify as accessory ceiis for the induction of proliferation ot resting Thl cells

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ROI.i: Ol- CDla, B7-2 AND ICAM-l LYMIMKKVIM ALLOGr.NF.IC IN IliRAC MA. Vidnl'. I. Caoisi'. D. Sclitnifi^. Uiiiversidiid Austtal de C'liile, Valdivia. I leriiot, Lyon, l-rancc.

IN iltlMAN I.ANGIlRlfANS Cr.LL-T I ION, M. Conclia'. .1. I'cKtiet-Navairo^. llnstifttto dc Ilistologia y I'alologia, Chile, ^INSliRM U346, ilopifal. Kd.

To activate I' cells, l.aiigerhatis cells (LC) expose itniintnogctiic peptide/MllC chi.s.s II fnigrnents iitid deliver co-stiniulatoiy signals. We liave investigated the adhesion and activating molectiles e\ptesscd by cell cltistets fontied hctweeti higlily etitiched huinan epidettnal I.C co-cniftned with diffetent atnotinf.s of allogeiieic I' cells. Kinetic experiments siiowed that both the period of celhilar iiiteiaetion and I.C/lyinphocyfc ratio legnhitcd chister loimation. hi contrast to IVcshly isolated I.C and ?• days cnlttiied I.C with GM-CSF, I,C-T cell cUisteis expressed an increased iinniunoieactivity to CDla, ICAM-l and B7 bnt not I.I'A3. The I X - T cell chtsteiing vvas efficiently hlocked by anti-C'Dla, -137-2, and - I C A M - l , while anti-li7-l and -CIMO showed a niodciafe effect. At innuinoclcctroti microscopy, liiibeck grannie containing-LC revealed CDla, B71, B7-2, ( D4(), atld ICAM-l expression oti disctete plastna tnetnbtanc zones thai were in clo.se contact with I cells. Intereelhilar :? nni bridges formed between I.C and r cells were itntntinoteactive to I C A M - l . Otir testilts .sttggesi thai I) CDla, B7-2 and ICAM-l are |)otent tnoleetiles that drive the LC self peptide presentation to I eells; 2) their expression is inereascd dnring the allogeneic ititeiaction; and 3) ICAM-l tnoleetiles fonn cotiiplex jttnelioti .strttcttites in Ihe transient tight association of I.C fo alloreaelive T cells. -

EXPRES.SION AND FtJNCTION OF n 7 - l (CD8t)) AND D7-2 (CDR(,) ON HUMAN ISPfDfiRMAL LANGERHAN.S CELL.S. F.M. Ratti.s. J. Pcgucl-Navarro. M..I. .Stacliict, C—Dcztittcr-Daiiilnivaiit. * ! ' . Ciiiirtcllciiiont. * G . Ri;il/iiii:il<, n . .Schmitt, INSERM tl34r), f'av, R, Hi'ipital E, Hcrriot. (,9.147 Lyon. France. •Centre dc rcchcrchcs PCD. Saiiil-Jean dc Brayc, I-rancc. In addition (o T cell receptor triggering, activatiim of T cells requires costinuilatory signals thai have been shown lo he mainly initialed through r D 2 8 . At Iea.st two di,slin(;t ligands ior CD28 have heen de.scrihed at the surface of various antigen presenting cells (APC): [ i 7 - l (CDSO) and U7-2 (CD8()). In this study, we analyzed the expression and function ol' both B7-I and 1)7-2 on liuinan Langerhans eells ( L C ) , llie anligeii presenting cell Irom epidermis. Humau LC ireshly isolated Irom ihe epideruiis (TLC) expres.sed sigiiitieanl level of li7-2, whieii was increased upon a sliorl in vitrii culture. By contrast, B7-I was undeleelable on TLC but appears al the cell surface atter 1 lo 3-day in vitro culture. The role of B7-I and B7-2 on hnman LC has heen analyzed hy adding specitic iiiAhs at the beginning ot ini.xcd skin cell lymphocyte reactions. Ami B7-2. bnl not anli B 7 - i . niAhs produced near complete iuhibition of aliogeucic as weii as recall anligen induced T eell prolii'eratiou vlicu ILC were used as A l ' C . Similar resnlts have been obtained nsing .1-day cultured LC, allllougli tliese cells express B7-I at Ilieir suri'ace. Collectively, llie.se results demonstrate that fJ7-2 is expressed early at the LC snrlace and that it plays a crucial role in human LC costiinnlatory function with little, it auy, dependence on B7-1 expression.

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Ati.STRACrS

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N O R M A L HUMAN LANGKRMANS CELLS EXI'RIuSS THE VITAMIN D R E C El'TOR, AND THEIR CO-S IIMULArORV A( T l V n ^ WAS M O D I LATEl) UY IHE VH AMIN 1)3 ANALOGUE CALCH'OTRIOL. T N o t t n a n Datir. B. Mollcr'. II. Snlvsii-n' atid K.K.timhallc.' 'Dcpt. of pci-matology. NJcpt. of Clitiical Iriinuinology. Uiiivor.sity oi Aatluis. Detitnark. A possible mecliatiistii of local f cell activatioti is thtougii atitigcti prcscntitig cells in the skin. In phaniiaeological concentrations l,25-dthydto\y\itatiitn D, the biolouically active fottii of vitaiiiin D and calcipottiol the synthetic analogue with similar receptor binding, ha.s beeti shown lo ha\e potent in vitto itiiniunoinhibilotA' propetlies on activated (CD45R0') T cells and on cytokine ptodttetion (lL-1 a. IL6 a n d TNI-a) iti lunnan blood tiionocytes. Ihe putpose ofthe piesetit stttdy was to investigate tlie in vitto effect of 1.25- diliydtoxy\'itatiiiti and calctpotrtol on LC to induce autologus T cell ptolifetation. LC enriched cptdetinal cell suspensions (75 ±22% LC) wete prepated ftotii tiortiial httniati skin fVotu patietits tttidergoing p l a s t i c suigety. LC were co-culluied with autologus T cells in the presence or absence of 1.2'5-dihydto\y\'itatiiin D,. calcipott iol. atid atitigeti (1>1'D or Catidida). 1 25-dlhydto.\y\'itatiiiti atid calcipotriol induced a sigtiiflcaiit itlhibitioti of atuigeti speciftc accessory eell futictioti. l'vttiliennore. the vitamin D receptor was deteeted b y Westetii atialysis in the isolated LC. Tlow c\ totiietT>' did tiot. as expected, show iiny siiiuificant eliatige in IILA-DR exptessioti. this finding was lutther suppotted by init"iiunoliistoeheiiiical studies. In sutiitnaiA. out data show tliat ealcipotriol was a b l e to dectcase antigen specific T eell ptoliferation. independetit on the number o f rctiiaininu. keratinoeytes. This itidieatcs that 1.25-dthydto\yvttatiitn D and calcipottiol had direct effect on LC atid sttggests that humati LCs are tatgets ot vitamin D, atid calcipottiol. 1 herefore. vitatiiin O, analogues tiia>' be betieltetal tti s k i n disoidets in wliich activated LC ate presutued to pla\' a tole. •

A POSITIVE. ATOPY PATCH TESl' REACTION REQUIRES THE PRESENCE OE AEEERGEN-SPECH'IC T CEELS AND IGE-BEARING EANGERHANS CELLS IN CLINICALLY NON-INVOLVED ATOPIC DERMATITIS SKIN. li..C._v;m_Rei.isc.n.X..ycr&Lui.s^:r. :nK-p_cn,_l::XlJ.-nug!^ckkWiM£diu.L.JJ<^.KJet\&^^^ Iii.liaii_/VCL_yan_Lep_ci:cnz_Vau Oijk. R.A. dc Wci^cr.* G,C. Miiddc.** and C.A.F.M. Rruijn/ccl-Kooincn. Dcparliiicnls of Dcrmatology/AMergology and *Paliiolog\'. Univursily Hospital. Utrcclil. Tlic Netherlands and **Saiidoz Research Institute, Vienna. Austria. We aimed to elucidate the role ol" ailcrgen-specific T cells and cpidcrnia! Langerhans cells (LC) in the niechanism underlying the triggering of atopic dcrniatis (AD) lesions b\' skin-penetrating allergen. First, poKelonal T cell lines were generated from clinieally [ioninvolved skin of AD patients with a positive atopy paieh test (APT) reaetion (APT* AD palients) to house dust mite. Throe conlro! groups were included, tiamcly non-atopic subjects, alopie suhjccts without AD and AD patients with a negative AP T reaction (APTAD patients). Dpt specificity was detected in eliiiically non-involved skin ot'all Ai*"ri AD jxitienls, but not in these subjects only. Nevertheless, the presence of allergen-speciHc T cells was crucial Ibr the generation of an APT reaction. Second, by imniuiiohistolog>\ clinically non-involved skin ot'all subjects was screened for the presence of epidermal LC that expressed the high aTfinity reeept' non-in\'olved skin, initiates the allergic skin inllammation towards skinpcnetratiiig allergens in AD.

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A ROLE FOR IL-10 IN HAPTEN-SPECIFIC TOLERANCE INDUCED BY A C U T E LOW DOSE ULTRAVIOLET B RADIATION OF SKIN. H. Niizeki. J. vy. streilein. Schepens Eye Research Institute and Department of Dermatology, Harvard Medical School, Boston, MA, USA. lnterleukin-10 (IL-10) is an immunosuppressive cytokine that can be secreted by keratinocytes foilowing exposure to ultravioiet-B radiation (UVR). Depending upon dose and timing, UVR can create iocai and/or systemic defects in cutaneous immunity. We wished to determine whether I L - 1 0 could mimic the local effects of UVR on either contact hypersensitivity ( C H ) induction and tolerance. A non-infiammatory dose of mouse recombinant IL-10 (200 ng) was injected intradermaliy into shaved abdominal skin of C3H/HeN mice. When epidermal sheets were prepared within 30 minutes from injected skin, fluorescence microscopy revealed normai numbers of la-bearing dendritic ceiis with typical Langerhans ceil configuration. Moreover, when DNFB (185 |.g) was painted on IL-10injected skin, CH was induced which was equai in intensity to that induced through skin that received PBS injection (positive control). Thus, at this d o s e , IL-10 appears to have no deleterious effects on cutaneous antigen presenting celis. Subsequently, mice that first encountered DNFB via lL-10treated dorsal skin 14 days later. When the ears of these mice were chalienged 5 days later, CH was significantly reduced (60+4 mm) compared to positive controls (100±7 mm) that first encountered DNFB via PBStreated skin. These findings are consistent with the view that UVRdependent IL-10 may be important in hapten-specifie tolerance induction, but that IL-10 may not be contribute to failed CH induction when hapten is painted on skin immediately after acute UVR.

LANGERHANS CELLS UPON TREATMENTS AFI-ECTING CONTACT FlYPER•SENSFFIVITY: IMMUNOHLSTOCHEMLSTRY AND ELECTRON MlCROSCOin. S. Bacci. F. Prii'nano. P. Rotnagnoli anci .LW. Sti'eilcin. The Schepens Eye Rescaivh institute, Hai^varci Medical Sdiool. Boston, Massachusetts, and Department of Human Anmoiuy and Histology. University of I-lorcncc. Italy. Ultraviolet-B (UVB) radiation alters the function of Langerhans cells (LC) and leads to partial inhibition of contact hypersensitivity (Cli). The action of UVB in LC seems to be mediated, in secjuence. by c/.v-urocanic acii.1 (('/.v-UCA) and tumor iieci'osis factor (TND-a. Comparable inhibition of CM can be achieved b>' using high doses ol" allergen ( =20 times the usual ones) for the sensitizing pulse. To better define the effeeis ol' these agents on LC. we bave treated miee with UVB. L-LV-UCA. TNF-a. and DNi-B (10 or 1X9 |.tg) and have analyzed the epidermis by immunohistochemistry and electron mieroscopy. Ail treatments, except DNFB at 10 ug/ml, gave the following results: by fluofcscence mieroscopy. there was a retiuction of la positive epidermal cells: the fesidtial la positive cells appeared round; and the expression of vimentin became low or absent. Hy electron microscop>\ dendritic cells wei'c demonstrated in niuiibers (lelative to those o( basal keratinocytes) similar to controls; however. the\' were poor in eytoplasmic vesicles, vacuoles and intermediate filaments. The section profiles oi' dendrites were tlecreaseti in number. poiEiting to a reduction in their numbci" aiid/oi' length. The results suggest that; (!) LC are moiiified in shape, expression and loealizatjon of la antigens, organization of the cytoplasm ami presmnabiy membrane tui-nover, by the treatments ajiplietl here: and (2) niodifieatioiis of the cytoskeleton can play a role in the response o( LC to treatment and, in more general wa\\ in the reuuiation of their ilifl'erentiation anti function.

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|L-(S PRODUCTION BY DRAINING LYMPH NODH CKLL.S IN UVB RESISTANT A N D UVB St.lSCI-:PTIBLE STRAINS OI- MICB. MB. l.;ir)nin. R..I. Dcarmuil' M. Mortal. 1. Kimhcr'. Dcparlmctit ot Medical Microbiology. Utii\crsity ol Minbttrgh .Vicdical Sclux)l, Edinburgh. IZcncca, Central To\iciiloiJ\ l,aboratory, Aldcrley Park. Chcshiic T h e induetion pha.se ol skin sensili/ation in tiiiee ts characten/ed by the .stitnulation ol prolilcratiNe responses in lymph nodes draining the site ol exposure. Proliterati\e activity has been shown prc\iiHlsty to be ass(K;iated with the production by draining lymph node cells (LNC) ol itUerleukin-6 (IL-6); the main source ol whtch appears lo be dendritie cells. As lL-6 i.s known to be an importani cosCimuUuor in T lymphocyte activation, we have compared the prixiuetion by LNC ot this cytokine in two strains ot mice. BALB/e and C3H-HeN. which are eon.sidercd. respectively, lo display resistance or suseepttbilily to ultraviolet B (UVB)-nicdiated immunosuppre.ssion. Mice were treated on the dorsuni of both ears with l7' << oxa/jlone (O.x) in 4:1 acetone;olivc oil (AOO). or with AOO atone. Three days later draining (auricular) lymph nodes were excised and a .single cell .suspension ol LNC prepared. LNC were cultured lor 24 or 4ii houns. The concenlralion ol IL-h (pg/ml) in the supcrnatant.s w;us measuted by enzyme-linked immunosorbcnl as.say. Exposure ol either strain ol mice to vehicle alone failed to stimulate the prixiuction ot detectable levels (I5() pg/ml) ol lL-6. Scnsiti/iition ol BALB/c strain mice with Ox pro\oked a vigorous proliterative response by LNC (mca.sured by ineorptiration ot radiolabelted thymidine) and high levels ol IL-6. In eontrast. although LNC prepared trom sensitized C3H-HeN miee exhibited cotnparablc prolilerativc activity, the ptxxiuction ot lL-6 was substantially less. These data reveal marked strain dilTerences in the stimulation ol IL-6 resp
LANGERHANS' CELL HISTIOCYTOSIS IS A MODEL OF PROLIEERATION DIFEERENTIATION AND ACTIVATION OE LANGERHANS' CELLS. J.F. Emilc. N. Brousse Dep;utement of Pathology, Neeker-Enfaius Malades hospital, Paris, France The initiation of the immune response relies on Langerhans' cells (LC), which process the antigens and present them to the T lymphocytes. Many reeent studies demonstrated that Granulocytc/Macrophage-Colotiy Stitmilatitig-Factor (GM-CSF) is necessary for the proliferation, differentiation and activation of LC in vitro. However, few data are available coneerning the eontrol of human LC irt vivo, Langerhans' cell histiocytosis is a clonal proliferation of LC. Using iit sittt itiimutiohistochemistry on frozen LCH tissue samples, we demon.'itrated that LCH eells eontain GM-CSF. Cultivated LC in presence of GM-CSF, but not freshly extracted LC, express activation niiu'kers and the GM-CSE reeeptor. We have shown tliat lymph node interdigitating dendritic cells but not intra-epidemial LC aLso express activation markers and the GM-CSF receptor. Furthermore, with in .S/JH immunohistochemistry, we also shown that LCII cells express activation markers snch as LFA-1, CD4, CD24, CD68 and B7-1 and the GM-CSF rece|)tor. Therefore, LCH could be a valuable model to study the cytokines involved in the proliferation, differentiation and activation of LC, and the interaction of LC with T cells.

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ADSTRACTS

INVI;,STK;ATIV17. IMIRMAIOLOGY

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T H E PRESENCE OP CYfOKINriS IN LANGHRflANS' CELL MISTIOCYTOSIS AND NORMAL LANGERl-lANS' CELI^ Ian H. de Graal. Ricnk Y.T. TammJnga.JViikc Datn-Meiring, WHetn A Kamps ami ^ l m Timcns. Department of Pathology ;md tlie Ciiildrcn's Cancer Center, University Hospital Groningen, Tiic Netherlands. I^ingerhans' eell histiocytosis (LCH) is characterized by an accunuilation and proliFeration of cells witJi a l-angcrhans' cell {LC) phenotype, indicaled by liie expression on the LCH cells of CDla, S-100, and the presence ofBirbeck' granules. It has been postulated that LCH is caused by an immunologic dysregulation, causing activated LCs lo migrate to and to accumulate al difierent sites. Central features of normal immunologic regulation involve the production of cytokines and expression of adhesion molecules. In tliis study we investigiiied the presence ol a numher of cytokines in LCH lesions {n=15) and in LCs in normal skin and dermatopatliic reaction. We found LCH cells :uid LCs in normal skin to stain for IL1(1, IL-l|i, GM-CSF, TGlMt, TGF-|1, TNP-a and IPN-y, in contrast to normal activated LCs in dermatopatliic reaction that were negative for these cytokines. We previously showed that LCH cells in part resemble activated LCs, but have hampered expression of adliesion iTiolecules. The presence oi these cytokines in LCH cells resembles tJiat of normal LCs, and may indicale that a "down-regulatory" signal may be lacking in LCLI auisjng partial or aberrant activation resuhing in an accumulation and/or proliferation of LCs.

BONE MARROW-DERIVED DENDRITIC CELLS INDUCE PROTECTIVE TUMOR IMMUNITY AGAINST A MURINE SQUAMOUS CELL CARCINOMA, KLN205. K. Mahnko. *P. Ricciardi-Castagnoli. T A. L i i p c r ^ Schwar/, and S. Grabhc. Ludwig Boltzmatin Insl. for Cell Biology and Immunobiologv of the Skin, Dept. of Dermatology, University ol' Miirister, Gemiany. *DepaitnieiU ot Pharmacology, University of Milan, Italy. Murine epidermal cell.s have been shown to present tumor associated anligens (TAA) for the generation of protective antiliimor immunily in vivo. These elTecls arc inediali-'d hy IA+ dendritie cpidcnnal Langerhans cells (LC). Accordingly, this sludy quesiioncd, whether treatment of mice with bone-manow derived dendritic cells (BmDC) or lhe splenic dendrite cell line CB-I is capable lo induce tumor immunity against tlie murine KLN carcinoma. BmDC were generated from bone manow of DBA/2N mice by culUiiv in lhe presence of 100 U/ml GM-CSF for 6 days, Ig scdimenlation and additional overnight cultui-e, resulting in >7() % pure DC. BmDC as well as the CB-1 eells were pulsed with free/e thaw-lysates from KLN cells (1x1 O^/ml) as source of TAA and naive DBA/2N mice were immunized twice al weekly inleivals with 3 x 10^ cells, followed by s.c. injection of 2 x 10-'^ KLN cells. Tlie tumor growlh was suppressed signiRcanily only in BmDC treated mice, when compared lo control- or CB>i inhaled mice {mciu\ tumor volume 35 days after challenge: BmDC: 50 mni^; control: 400 mm^; CB-I: 480 mm^). Moreover, most of lhe the BmDC treated mice were free of tumors, compared lo control mice. In contrast to previous data using LC, BmDC only indticed proleclivc tumor immunity when injected s.c. al the sile of subsequent tumor challenge, bul not al a distanl skin site. Therefore il is suggestive that dendrilic cells from different tissue origins may have differential eapacity lo induce protective tumor immunity.

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GI-NERATION OF DENTRITIC CELLS FROM PERIPHERAL BLOOD FOR INDUCTION OF CTL AGAINST HUMAN MELANOMA CELLS. S. Alijagic. P. Moller. M. Artuc. K Jurgovsky. T, Dorbic. B.M, Czamctzki. D, Schadendorf. Virchow Klinik, Depart, of Dermatology, Humboldt Univcrsilat zu Burlin. Gcnnany, Various mclanosomal prolcins have recently been identified which arc recognized in conjunction with selected HLA elass I molecules by specific CTLs in melanoma patients opening new avenues for tumor vaccination strategics. Dendritic antigen presenting cells are considered to be the most effective stimulators of T cell responses. The use of dendritic cells has been proposed to generate therapeutic responses to tumor antigens in cancer patients. Mere we describe the generation of dendritie cells (DC) from leukoeytc-enriehed buffy coats (40 ml) using GM-CSF (800 U/ml) and IL-4 (500 U/ml) yielding on average 2x10^ DCs after 8 days. Flow eytomctrie analysis revealed a loss of CDI4 and CD34 witli high expression of CDla. CDlla,b,c. CD44. CD 45RO, CD54, HLA-elqss i and class II and intermediate levels of CD23, CD26, and CD80. Function was validated in experiments showing that eultured DCs could stimulate proliferation of allogcnic T cells. Furthermore, antigen presenting capacity was tested by DC loaded with tetanus toxoid leading to a stimulation of T cell proliferation up to 2()-fold at low DC: T eell ratios (L 50 and 1; 100). In order to become independent of already identified antigenic peptides and HLA class I molecules restricting the general application of such vaccination approach, we tested whether transfection of tumor antigens into DCs might overcome those limitations. DCs were transfected with two reporter genes (CAT, D-gai) using various tcehniques (liposomcs, receptor-mediated transfer, ballistie transfer, microinjections). Transfection efficacies were difFieult to determine because of the fast intracellular protein degradation and processing. However, CAT as well as tyrosinase mRNA were detectable after transfeetion by RT-PCR suggesting protein synthesis. Whether DCs transfected with the tyrosinase gene are capable to induce a CTL response in naive T cells is currently under investigation.

LANCiIiRIlANSCI-:LLS ANDTlIi-; I'ATl KXIBNI'ISIS O i ' C U T A N i i O U S T t'l^LL L Y M P I i O M A S (CTCL): A Rl>i-VA1,IIATION. Cl. Rowden. P, C't^p. S, D c a n J ^ Walsh. Department of I'tilliology. Dalhousie (Iiiiversity. I lalifax, N.S., Canada. As part of an hypothesis proposed in 1976 linking LCs with the cpidermotropism typical of C"l"C'Ls(Bril. .1. Dcrmatol. ')5:665), injury and death tiflhc inciting IX's was suggested. Subsequent \'erillcation of this proposal has been tiil'llcull to achie\x" using elcelron microscopic melhotls. Application of in silu hybridi/ation tcchniLiucs lo label cleaved DNA, allowed us lo reinvestigated a scries o f C T C I . cases in an allempl to dociimcnl lhe presence ol apoptosis in the response of LCs to tbe upitiertiidlropiL' 1 lympliocytes. Anlibodies reactive lo C D l a antigen in paraffin sections, together witii SlOO were used to itlcntify and double .stain LCs in addition to the TUNI'I, staining technique oi'nick end kibclling. Apoplosis was iiolcd in lhe expected ciiidcrmal sites sucb as in the granular hiycr. but there was no significant involvement of LCs wilh this form of cell deatb. Some of the inllltraling T lynipbocylcs, bowe\'cr. showed pt>silivc staining and evidence of apopUisis. The suggested palbogcncsis of CTC'Ls in\'ol\'ing cbronic antigen persistence, does nol appear Ui iiivoKe ticslruclion of LCs in the epidermis altbougb iheir fale after migration tn the lymph nodes remains unsolved.

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IMMIJNOMODULATION OK LANGERHANS CELLS DURING IIERI'KS SIMPLEX VIRUS REACTIVATION. ShiyanthijyianLcKaim!Bli,Tm, Tcrry_I_Hill. NejIA wn!ianis^.University of Bristol, Dept of Patliology and Microbiology, Bristol, BS8 lTD, UK Langerhans cells (LC) have a crucial role in detemiining immune responses to herpes simplex vims (HSV) In a nuirine model, certain chemicals (DMSO, xylene and retinoic acid) can reactivate latent HSV in the sensory ganglia. Of these, only xylene and retinoic acid cause symptomatic viral infection in the skin. Tlierefore, understanding tlie effects of these chemicals on the skin will provide an important insight into why only certain individuals get symptomatic herpetie recurrences We have demonstrated that tlie in vilrn phenotypic maturation of LC, as assessed by tlie upregulation of la, ICAM-1, CD45 and Mac-I antigens, was not affected by the in vivo administration of DMSO, xylene and retinoic acid, DMSO, however, induced the appearance of 10-20 times more la on LC in vivo Such changes did not occur after treatment with retinoic acid or xylene. We have also investigated tlie effect of each ofthe chemicals on LC migration to tlie lymph nodes following topical exposure to the contact sensitiser FITC DMSO had no effect on LC migration whereas, xylene and retinoie aeid acted in a synergistic manner witli FITC to increase tlie loss of LC from the epidermis In addition, tlie infiltration of leukocytes and the production of cytokines in tlie skin after topical application of tlie chemicals was investigated. At the time when virus would be arriving in tlie epidermis, numbers of PMN are substantially reduced after treatment with all of tlie chemicals. Treatment witli xylene and retinoic acid, but not DMSO, also caused the almost complete loss of T-cells from tlie dermis. Tlierefore, whereas those chemicals which are associated with the causation of symptomatic skin disease stimulate LC migration from the epidennis and cause the loss of T-cells and PMN from the dermis, DMSO appears to enhance the immune competency of tlie skin by promoting tlie maturation of LC (increasing la and decreasing F4/80 expression).

T H E CELLS 01- O R A L MUCOSA IMMUNI- SYSTP.M UPON HIV INI-ECTION. N. Piinpinclli. G. Ficai'i":i. P. Roni:ignnli. II Dernialology Clinic, Institute of OtiontognalhO'Sloniatology anti Department of Human Analomy and Histology, University of l-'lorenee, Italy. 'I'hc pi'cscncc and hill dillcrcntiaiidn of immune cells arc prci"cc]iiisiic,s to mount efficient local imniuiie responses in ihc skin and oral mucosa. Those i-esponscs arc defective upon HIV infection, but tbe basis of ibc defect is not complcicly understood. To address tbis issue, wo liavo invcstigatotl by inimunohistochomistry ami election microscopy on the pioscncc. disiribiitioii and fcatuies of iiiimuiio colls in tbo oral mucosa of 10 bealthy HIV-ucgativo and 40 HlV-positivo subjects with clinically healthy mucosa (7 cases) oi' with diseases strongly associated with illV infeetion (hairy leukoplakia, 22 cases, periodoiital tliseaso, 7 cases, ei'ytliematous and pseudomembranous candidiasis. 6 and 4 cases respeclively). The results show (i) that tbe mueosal dendritic cells of IllV-infectet! subjects, even of clinically healthy ones, arc defective in the oxpression of IlLA-DR antigons and (ii) that a local, coll-metliated immune response takes place in the inflammatory diseases of the muoosa during HIV intcction. but is abnormal under several respects. This re.sponse does iiol bonefii'or the recruitment of Cf34+ T-cells, even in cases with normal circulaling CD4-f- celi counts; the Langoi'haiis cells whicb take pai't in it exhibit altered ultiasti-ucturo and reduced expression of functionally meaningful molecules (CDl la, CD34); the infilirating CD8+ lymphocytes are associated wilh C D l a + Langorlians cells, instead o r C D 3 6 + tleudritic maerophagcs. Those abnormalities can explain [he sovere clinical course of theso disorders in HlV-infectotl patients and, since they are progi'ossively moi'e markoti from crythcmatous eandicliasis lo periodonta! disease, pseudomembranous candidiasis and hairy loukoplakia, can explain ihe different prognostic significanco of these tlisoasos.

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VARIATIONS IN THE DISTRIBUTION OF BI INTEGRINS ON HUMAN EPIDERMAL LANGERHANS CELLS DURING SHORT-TERM CULTURE. M-.l. ,Staauct, alY. Kobavashi . C. Jaccniet, C. Dc/ullcr-DainbiivanI, D. Schmitl. INSERM U346, alfilid CNRS, Dermatology Research Unit, E. Herriot Hosp., L\(in, France. a)Sunstar Inc, Takatsuki, Osaka, Japan. Though much of the functional activity ol LC dciKiids on their mobility, the mechanisms underlying the migration ol LC remain poorly understoixl. HI integrins, known to mediate cell-eell and eell-e.\tracel!ular maln.\ interactions, appear relevant to L C migration. In order to determine whether the e.xpression of BI integiins change when LC migrate IVom the epidermis to lymph nixies, »c compared the expression of BI integrin.s of freshly isolated epidermal LC (ll^C) and cultuted LC (cLC) which ate considered as the equivalent of LC that have tnigrated to lymph nodes. LC w ere maintained for 24 h and 72 h at 37°C in RPMI-10% FCS. In some e.\|x;riments, LC were incubated for 24 h in RPMI-10% FCS in presence of l(X)ng/tiil rhGM-CSF or lOOUI/ml rhTNFa. The expiession of BI integrins was appreciated by imnuiiiogold stainings and FACS analysis. I'LC express clearly detectable levels of n4BI, (t5Bl and (16131 and low but detectable levels of alHl, «2BI and a3BI. We observed that n l , cx2, a3 and a6 integrin stibunits wete rapidly dow n legulated and no more expressed after 72 h of culture, and that only it4 and a slight exptession of u5 integiins wete maintained on cLC. Interesttngly, alter 3 days of culture, while the level of expression of
TNFa INDUCES MIGRATION OF LANGERHANS CELLS IN MURINE SKIN ORGAN CULTURES. M_onica Zanella, F. Koch, U. Orlner, M. Heine, G, Schuler, and N. Rj^iTianL Depl. of Dermatology, University of Innsbruck, Austria, Migration and maturation of dendritic ceils (DCs) are regulated by cytokines. Experiments such as the intradermal injection of TNFa, that caused a rapid depletion of Langerhans cells (LC) from the skin, underscore the relevance of this cytokine. We therefore decided to examine the effects of TNFa on the migration of LC in a different system. In a murine ear-skin organ culture model we evaluated the number of DC that emigrated into the culture medium. TNFa was added to these cultures at different concentrations. We obseived a dose-dependent effeci of TNFa: Low doses of TNFa (50-100 U/ml) enhanced the emigration of LC (167% of emigration without TNFa). High doses of TNFa (>1000 U/ml) suppressed the emigration, probably due to toxicity. Alternatively, intraperitoneal injection of a neutralizing anti-TNFa mAb (rat lgG2a) before the onset of the organ culture caused a significant decrease cf LC emigration indicating the presence of endogenous TNFa. Approximately 60% less DC emigrated under these conditions as compared to the injection of an isotype-matched control mAb. The numbers of LC remaining in the epidermis and detectable by immunohistochemistry corresponded to the numbers of emigrants in a reciprocal fashion. We conclude that TNFa is crucially involved in the mechanism of DC migration. Whether it acts directly on LC cr indirectly by the induction of a cytokine cascade (e.g. IL-1 B) remains to be determined.

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PATHWAY OF LANGERHANS CELL MIGRATION IN HUMAN SKIN ORGAN CtJLTURES. MichaeJ Ljjka_s, .H, Stossel, N...S.epp, G^^chuteL and_M- RojuanL Department of Dermatology, University of Innsbruck, Innsbruck, Austria, In murine skin organ cultures migrating dendritic cells (DCs) have been shown to accumulate in the dermis in a distinct, string-like pattern ("cords"). Electron microscopy proved that these "cords" represented lymphatic vessels. We wondered vi/hether human cutaneous DCs would follow the same route of emigration. To this end, organ cultures of human skin were set up. Epidermal sheets and tangential thick sections of whole skin were studied. Over three days of culture the density of Langerhans cells (i.e, MHC class 11+ cells) in epidermal sheets dropped to 30-50% of the starting values. On sections immunostained with mAb "LAG", specific for Birbeck granules, we consistently observed accumulations of LAG+ cells. They were arranged in a non-random, string-like fashion. Double-labeling experiments showed that these cells were strongly MHC class Ih- and thus qualified as Langerhans cells. They were not contained in blood vessels (identified with mAb PAL-E). Ultrastructurally these cells exhibited all features of mature DCs, They were located in wide lymphatic vessels that were separated from the surrounding dermal tissue by a thin layer of endothelium, typically devoid of a basement membrane. We conclude, that human cutaneous DCs leave the skin via lymphatic vessels both in vivo and in the in vitro culture system described here. This model may therefore prove useful for the investigation of mechanisms of DC migration in human skin.

TIIEEXI'RKSSION OK VL,\-) AND VLA-6 BY LANGICUHANS CELLS:T11E1U POTENTIAL ROLIC IN KEGULAI ING LANGEKIIANS CELL MIGUAIION Abigail A. Price, Ian Kimber^ and Ann Agcr, Division of Cellular Imnninology, National Institute tor Medical Research. London, NW7 lAA, U.K, and 'Zciicca Central Toxicology Laboratory, Aldcrlcy Park, Cheshire, SK10 4TJ, U.K, We have investigated the roles of adhesion receptors on the Langerhans Cell (LC) surface for components of the extracellular inalrix in regulating the initial stages of LC migration, t'ollowing topical apjilication o!" a chemical allergen. Cell surface expression ofthe Hbroncctin receptor, \'LA-4, and the laniinin receptor, VLA-6, has been compared wilh that of clenthitic cells (DC) from draining lyiniih nodes in Ba!b/c miee. LC were isolated trom ear skin oi uiiticatetl mice tising trypsin digestion and enriehed tor on a l.ymphoprep gradient. DC were enriched from atiricular lymph nodes 18 hours following topical a|)plication of \% oxazolone on a Metrizamide gradient, LC and DC were slaincd tor MIIC Class II and the expression of »., and a,, integrin subunits on Class II positive cells determined by tlow cytometric analysis. LC, in epidermal sheets and following i.solalion, expressed low levels of a'4 integrins in eomparison with DC, In contrast, a siibptipulatioii of isolaietl LC (10%) cxprcsscti high levels of u,, integrins ( as high as on keratinocytes), whereas DC did not express detectable levels of a,, integrins. The skill explain model is currently being used to test the role of changes in the expression of VLA-4 and VLA-6 oit tX' ;n regulating their migration out of the skin.