Macrophages, but Not Langerhans Cell-like Cells of Dendritic Lineage, Express the CD36 Molecule in Norntal Huntan Derntis: Relevance to Downregulatory Cutaneous Intntune Responses?
Antonietta Lonati,* Nlieke A. Mommaas;l" Giorgio Pasolini,* Antonio Lavazza,i: Geoffrey Rowden,§ and Guiseppe De Panfilis* ' Department of Dermatology, Sped"li C ivi li , J3rescia , Italy; t Department of Dermatology, University Hospital, Leiden, T he Netherlands; :1:Zooprophylacti c Institute, Brescia , Ita ly; and §Dalhousie University, Halifax, Nova Scotia, Canada
The CD36 molecule has been shown to be associated including fibroblasts, lymphocytes, and mast cells, as with subsets of peripheral blood monocyte/macro well as demlal fibers, were not decorated with gold; phages and, in cells isolated from either ultraviolet dermal Langerhans cell-like dendritic cells (LC/DC), (UV)-irradiated or diseased skin, to induce down though they did show gold labeling in some intracy regulatory immune responses. Although macro toplasmic organelles, did not show any gold particles phages are certainly present within normal human along their plaslna Inelnbranes. Therefore, although dermis, whether they normally express CD36 is still a macrophages in normal human dermis exhibit vari matter of debate. In this study, we investigated der ability with regard to their localization and shape, mal CD36-expressing macrophages ill sitll using the they regularly and constitutively expressed CD36. gold immunoelectron microscopic technique on tis CD36 molecules may be considered a useful marker sue ultracryosections. This is a very sensitive and for macrophages in normal human dermis and may specific method, and its results clearly reflect the in furthermore confer on macrophages, or a subpopu vivo immunophenotypic constitutive situation. Mac lation thereof, intriguing functional properties (e.g., rophages in normal human dermis were variously downregulatory capacity vel'SIIS upregulatory capacity shaped from round to dendritic and were localized subserved by LC/DC) within the cutaneous immune either immediately beneath the epidermis, in peri system. Key words: dermal celis/0Kl\15 IIIolloclOllal allti vascular areas, or in intervascular zones. Macro bodY/II ltraclyomiCl'otomyli""'lJI1Ioelectl'otll icl'oscopy. ] Illvest phages showed consistent gold-positive staining on Del'matol 106:96-101, 1996 their cell surface. In contrast, other dermal cells,
urrent concepts of skin immunity suggest that the is still the subject of debate [6]. Ultraviolet (UV)-ilTadiated [7,8] end result of an interaction with hapten or antigen at and lesional [9,10] skins have been shown to harbor CD36-positi e chis environmental interface is determined by the APC, which when isolated are able to induce ill JJilm downregula balance between upregulatory influences, mediated tory immune responses. Similarly, a population of circulating via epidermal Langerhans cells and, possibly, via CDJ6-positive monocytcs is known to be capable of such activity rCelated dermal antigen-presenting cell s (APC) [1), and downregu [11 ). latory responses fr om a balancing immunomodulatory APC (2) . In contrast to results concerning CDJ6-positive downrcgulatory H o wever, while downregulatory APC have been characterized in APC in UV-irradiated/lesional human skin, parallel data have not murine skin [3-5], the absence of similar cells in normal human skin been obtained in normal human skin. In fact, as far as normal human epidermis is concerned, Langerhans ceUs do not constitu tively express the CD36 molecule (reviewed in [12]). Furthermore, Manuscript received February I I, 1995; revised July 17, 1995: accepted the hypothesized presence of a CDJ6-positive APC in normal for publication August 3, 1995. human epidermis is still a matter of debate [12-14]. The possible Parts of tltis work were presented at: (1 ) the 6th Illlmunodermatology expression of CDJ6 by dermal APC, including dermal macro Symposium, June 24-26, 1994, Munster, Germany (abstract published in phages [15] and dermal Langerhans cell-like cell s of dendritic Exp Ocrlll"loJ 3:134, 1994 l abst. J); and (2) the 22nd Annual Meeting of the lineage (LC/DC) [16,17) is still typified by conflicting results Society for C utancous Ultrastructure Rcsearch (where it was given by [15-20). Four contrasting lines of evidence have emerged so far Gio rgio Pa solini and awarded as the best oral presentation), June 23-24, concerning dermal APC as stained by OKM5 monoclonal antibody 1995, Heidelberg, Germany (abstract in press in) 1IIIIcs t 0,.,.11111101, 1995). R ep"int reqllests to: Dr. Gi useppe De Panfil is , Department of D enl1iltol (moAb) (which reacts with the CDJ6 molecule): (i) dermal perivas og)', Sped"li C ivili, 1-25125 Brescia, Italy. cular dendritic DR-positive macrophages arc CD36 negative [18]; Abhreviations: APe . ;lI1tj g cl1-prc <~e ntjn g ccll (s); LC/DC, LallgerhallS (ii) dermal "dendrocytes", showing features common to mono cell- li ke dendritic ccll (s). cyte/J11acrophages, are mostly DR positive and CD36 positive [1 9];
0022-202X/96/S10.50 • Copyri ght © <1996 by The Society for Investigative Dermatology, Inc.
96 VOL. 106. N O. I J ANUARY 19% DEItMAL MACROI'H AGES EXPR£SS C D36 97
(iii) d ermal macroph ages are m ostly CD36 positive, but are difficult (iii) prein cubation with the un co l~ju ~ated goat anti-mouse IgG antibody to differentiate fi'om endothelial cells [15]; and (iv) o nly 10-1 5°;;, of before the gold-conjugated antibody: and (iv) replacement of the gold DR-positive cell s are CD36 p ositive in human dermal cell suspen conjugated antibody by the unconjugated particles. si ons [20]. Statistical Analysis For the four types of negative controls. 300 macro T his curre nt con fus ion regarding the classification of dermal phages on control ultracryosections were scru tin.ized. and the score of gold CD36-expressing APC is mainly due to the relatively low sen sitiv labeling for the controls was compared with the above-mentioned number ity of the marker analyses and to thc lack of precise identification at of gold particles coun ted on the surfa e of OKM5-reactive macrophages, the single cell level of LC/DC and macrophages in routine usin g a oll c-\va y nl1ai ysis of the va rian ce r27]. immunocytochcmical sections or in fluorescen ce-activated cell sorter analyses [21-23]. It is n o t possible to ascertain the shape and R ESULTS localization of d erm al APC, and their possible CD36 expression, w hen cells aTe enzymaticall y isolated fi-om normal skin . in this Various Dermal Cells Exhibit the CD36 Molecule in Normal study, we therefore uti.1ized an ill sitll immunoelectron microscopy Human Skin Differently VaJ;ous types of dermal cells within (IEM) system b ased on immunocolloidal gold lab eling of tissu e thc human n01"m al de rmis were recognized by their establish ed ultracryosections [24,25]. T his technique demonstrates a very high ultrastructural characters [28]. We n oted LC/DC, mast cells, sensitivity for the CD36 immullo labe ling, thereb y revealing even lymphocytes, fibroblasts, and macrophages. Whereas mastcells, minimal antigenic levels. Additionall y, dermal LC/DC and macro lymphocytes, and fibroblasts were easily rccognized by their dis phages were simultaneously demonstrated at the single cell level by tinctive ultrastructural morphology, the definite differcntiation their ultrastructural characters. Thus, it was possible to precisely between macrophages and Birbeck grannIe-negative cells of the assess the CD36 expression by d em1al cell s, thereby reflecting the LC/DC scri es was sometimes di fficul t. Indeed, we used the foll ow i ll II ;VO situation in normal human skin. ing morphological crite ria to d iffe rentiate macroph ages from LCI DC: LC/DC have a folded, or even indented, nucleus, mitoch on MATER.lALS AND METHODS dria, some endo/lysosomal structures, moderately well- developed Normal Human Skin Three healthy volunteers participated in the study Golg i apparatus, a rough cndoplasmic reticulum [29,30], and a after informed conse nt. Punch biopsies were obtain ed fi'om the trunk. spind le to round-oval outer shape, with numerous short vill ous Immunoreagents and Reagents T he moAb reacti ve with CD36 mol projections exhibited by the plasma m embrane l31]; macrophages ecul e (OKMS) was provided by Prof. G. Rowden (Hali fax, Nova Scotia, sometim es showed enlongatcd cytoplasmic proccsses (see below) Canada). The 10 nm-sized coll oidal gold-conjugated goat anti-mouse IgG'1 that cxtended w ithin the interstitial m atrix and contained some antibody, used for immunogold labeling, was purcha sed frO Il1 Bio Cell phagolysosom es [32], lipid bodies, and a lysosom e-rich cytoplasm (Cardiff, UK) . As controls, a purified mouse unreactive IgG '1 frac tion and a [7,33]. W hile m ast ceUs, lymphocytes, and fibroblasts did n ot show purified unlabeled goat anti-mouse IgG antibody were obtained from gold granules b oth on th e plasma m embrane and w ithin th e Chemi con [nt.ernational (EI Segundo, CAl. Unconjugated colloidal gold cytoplasm, LC/DC sh owed som e gold particles within som e intra particles (10 nm sized) were purchased rrom Orrho Pharmaceutica l Co. (Raritan, NJ). cytoplasmic organ ell es, but no staining on the cell surface (Fig 1). O n the oth er hand, macrophages showed gold particles on their U1tracryonticrotomy Biopsies were immediately immersed in para plasma m embrane (Figs 2, 3). fo nnaldchyde 2% in 0.1 M phosphate buffer, pH 7.4. T he specimcns were cut in small blocks, which were kept in the sa me fixative for 1 h at roO I11 Macrophages in Nonnal Human Dermis Express CD36 on temperature and at least 18 h at 4°C. T hey were cryoprotected by the Cell Surface Sin ce macrophages were the only dermal cells immersion in 2.3 M sucrose in phosphate-buffered sa lin e (PBS) for 30 min, showing cell surface CD36 expression, we particularly focu sed on mounted O il copper specimen holders, and frozen by rapid plunging in liquid nitrogen. Specimen holders were transferred to the ultracryomic these cell s. Macropbages o bsel'ved ill siill were variously shaped. rotome R eichert-Jung Ultracut E with cryoattachement FC 40. After the Some m acrophages displayed multiple thin, elongated cytoplasmic qual.ity of specimens was assessed by semithin sections (0.5-1 J..l1n), ultrathin processes (Fig 2) extending in the dermal matrix. thus showing sections (50 nm) were obtained. The ul tracryosections were picked up with apparently dendritic morphology. On thc other h and, other mac a platinum loop fi ll ed with 2.3 M sucrose in PB S. and the drops were placed roph ages were less dendritic, and apparently quite ro und. Dermal on carbon-coated colloidon grids 0 11 gelatin plates (2% gelatin in PBS with m acrophagcs were located (i) b eneath the dermal-epidermal junc 0.01% sodium azid e) at 4°C. tion (Fig 2); (ii) in the proximity of dermal vessels (Fig 3); and (iii) III Sitllltnml1nociectron Microscopy After Roatin g for 10 min at room f.'1 r fi'om both epidermis and vessels in the intervascular zones. temperature in previously melted gelatin plates, grids carrying ultra cryosec tions were subj ected to a bloclcing pretreatment in cluding PB S-l% bovinl' The Gold Staining of Macrophages Is Significant when serum albumin, 0.2% sodium azide, alld 10% decomplemented human A.B Semiquantitatively Evaluated We have focused our observa serum for 30 min, then washed on drops of incubation buffer (0.15% glycin . tion o n 300 dermal macroph ages located in the upper dermis and in 0. 1% gelatin, and 0.1 % bovine serum albumin in PBS) , and fina ll y incubated perivascular areas. All the scrutin.ized cells were gold labeled on wi th the above-mentioned moAb at concentration of 5- '10 ,""g/ ml fo r 1 h at room temperature. After the first incubation, the grids were washed in their surface (Figs 2b,3/) , although with val; able labeling intensity. PBS/Glycin (three times for 5 min) and incubated with goat anti-mouse T he semiquantitative estimation of the gold IEM label.in g, carried antibody diluted 1140 in 1 % bovine se rum albumin in PBS for 1 h. After out by counting the number of gold particles bound to the plasma subsequent was ltin gs in PBS and disti ll ed water, the sections were sra ined m embrane of macrophages per cell section (midplane), revealed with uranyl acetate, embedded in methylcellulosa, and viewed at a Philips 69.85 ::+: 53.47 (mean ::+: SO) gold granules per macrophage section CM10 electron microscope. (Table I), thus clearly d emonstrating [34] the OKM5 reactivity of Sctniql1antitative Analysis of the Gold Labeling Three hundred macl·ophages. macrophages within the tissue ultracryosections were scrutinizcd cell by ceU under the electron mi croscope. T he number of gold particles per section of Positive Controls T h e con trols fo r th e m oAb were positive, as labeled macrophage surface was scored and the mean (::': SD) calculated. th e majority of peripheral b lood m ononuclear cells showing the Controls for the Monoclonal Antibody Positive controls for the u ltrastructural characteristics of monocytes were labeled, in agree OKMS moAb were performed on peripheral blood mononuclear cell s, m ent w ith previously p ublish ed data [11 ,35,36]. usi ng a similar gold immunoelectron microscopy protocol, with slight modifications appropriate for the peripheral bl ood mononuclear cells Negative Controls All the contro ls for the method specificity investigations, as already described (26). gave con sistently n egative results. T h e macrophage staining in th e Controls for the Methods Specificity Four negative controls were different contro l section s was always lower t.han the above m en performed: (i) repl acement of the OKM5 rnoAb by the IgGl mouse tioned results by a statistically significant margin (p < 0.005 fo r all unreactive fi'action; (ii) replacement of the OKMS moAb by PBS /Glycin ; th c four negative controls) (Table I). 98 LONATI I1T I lL T H E JOURNAL OF I N V ESTIGATIVE DER.MATOLOGY
d
Figure 1. Dermal Langerhans cell-like dentritic cells (LC/DC) show CD36 staining w ithin some organelles. /11 s;11I IEM labeling of a portion of sllperfi cial no rmal hllman dermis. a) At tillS magllificatio n the gold labeling is no t vi sible. R ather, a LC/OC is recogni zable b), its lI ltrastrllctural characters. Sc ale Iwr. 2.5 J.Lm . b) High magnifIcation of an area of Fig la. Even at this m agni fica ti on. gold granllies are no t visible. MlIltiple elo ngated, thin cytoplasmic processes of the LC/OC arc clearly o bserved. T he arrowheads indicate the dermal-epidermal jllncti on . T he boxed areas arc seen enlarged in Figs lc and l d. Sen le bar, 1 J.Lm . c) I,-Ugh magllificati o n of one of the boxed areas of Fig lb. No gold granules arc present along the plasma m embrane of th e LC/OC. By contras t. o nc cytoplasmic o rganell e contains some gold particles. Sen le bar. 0.2 p.m . d) High magnificatio n of o ne of the boxed areas of Fig 11,. Some intra cytoplasmic organelles of different electro n densi ty "re seell, showing gold labeling. Sen le bar, 0.2 P.1ll. VOL. 106 . NO. I JANUARY 1996 DEltMAL MACIlOPHAGES EXPIlESS CD36 99
Figure 2. Dermal macrophages (Mph) express CD36 on the cell surface. E" sil" IEM labeling of a portion of superficial normal human dennis. a) Two dermal cell s arc visible. located not far l"om the dermal-epidermal junction (arr"" 'heads). O ne of th em is a CD36-lIegative fibroblast (F), the other one is a CD36-expressing macrophage (Mph). At this magnification, however, the gold granul es present on the cell surface of the macrophage arc not visible. T he boxed area is seen enlarged in Fig 2 1, . Sra le bnr, 2 .5 /L111. b) High magnification of the boxed area of Fig 2a. Gold particles (amllll.') are well visible at this magnification alo ng the ph,sm,., mem brtlll e of the macrophage, while 11 0 gold particles arc present on the cell surface of the facing cell. Sca le lin 1', 0.5 fLm .
DlSCUSSION analysis of cell s suspended fr0111 normal human dermis demon strated that rhe sm all dermal cell subset closely related to LC/DC We have shown that, am o ng vari o us types of cell s harbored in APC lineage was clearly CD36 negative [20]. LC/DC are known to normal human dermis recognized by their peculiar ultrastructural express CD36 in som e pathological conditions, however, as i.n features [7,28-33], macrophages are the only ones expressin g the atopic dermatitis [37,38] and mycosis fungoides [39]. D uring the CD36 m olecul e on their cell surface. O ther cell types, including Langerhans celJ repopulation phase in the period of immwlologic fibroblasts, mast- cell s, lymphocytes, and LC/DC, do not show reconstituti9n after allogenic bone marrow transplantation [40], CD36- specific gold labeling along their plas ma m embranes. T he CD36 is also expressed as such cell s. C learl y, there are ditFerences present results permit som e clari fication of the current controver sies regarding the CD36 expression by dermal APC in normal between no rmal and pathological situations. It m ay be thar Lan human dermis. APC of LC/DC lineage, as identified by their gerh ans cell precursors transiently express CD36 en route to the ultrastructural characters [29 - 33], did not show any gold labeling epidermis, and epidermaJ Langerh ans cen s may differentiate into along their plasmn m embrane, while APC showing the ultrastruc CD36-positive dermal cell s on migratin g to the dermis (reviewed in ture of macrophages [7,28 -33] clearly showed gold particles on the [1 2]). We favor the suggestion that cells of th e skin exhibit some celJ surface. At least two recent studies seem to corroborate these phenotypic plasticity, which is stro ngly i.nRuenced by local tissue findin gs. All CD36-positive dermal ceUs were clearly negati ve for microenvironment [20]. In this sch eme, LC/DC m ay be induced to rhe LC/DC m arker CDl a [1 2], and the expression of CD36 011 express certain surface determinants, including CD36 molecul e, CD1-negative, DR-positive sll spended dermal cell s was consistent depending on their ill lIilf{) culture conditions [12]. In this respect, it with a macrophage derivation, w hereas isolated dermal LC/DC is not surprising that abo ut 10%, of dendritic cell s isolated and were CD36 negative [20]. cultured from normal human dermis acquire a weak CD36 staining The present in vestigatio n clearly dem onstrates that LC/DC do [16], w hereas unstimulated LC/DC investigated ill silll in the nor constitu tively express CD36 in normal human dermis ill lIillo. In context of the normal human dermal enviro nment, as in the present agreement w ith our ill sitll results, triple- color Row cytometric investigation , do not express CD36. 100 LONATI ET AL THE JO URNAL OF INVESTIGATIVE DER.MATOLOGY
Table I. Sentiquantitative Evaluation of the CD36 Expression by Macrophages in Norntal Huntan Dermis"
Control C036-Exprcssing Macrophages Macrophages
0.13 ± O.4 b 69.85 ± 53.47' p < 0.005'
" Number o f 1 0 fll11 -sized gold p:lrriclcs alo ng the plasma membraue per secrion of CD3G-cxprcsslng Inacrophagcs and control (obtained by o mission or primary anti body) macro phagcs in normal skin (i ll silll gold IEM labelin g of ul tr;lcryoscctions) . I, Mean ± SD.
t' O ne-way analysis of the variance (11 = 300).
equipped to express the CD36 molecule on the plasma membrane as weJl if appropriately stimulated. It is noteworthy that cytokines such as interferon-y seem to be capable of inducing and upregu- lating the CD36 expression on target cell s, such as keratinocytes [41] and endothelial cell s [42]. Although this study is limited to a morphological investigation, our results demonstrate that the heterogeneous dermal macro phagic population of normal human skin constitutively expresses CD36 molecules, which may confer to this popul ation intriguing functional properties in the context of the cutaneous environment. It is noteworthy that CD36-positive macrophages in the skin are also, at least mostly, CDllb negative [43,44], thus being pheno typically identical to the peripheral blood subset of CD36-positive, CD11b-negative monocytes [11]. Such cells are able to induce downregulatory immune responses, as indicated by their capacity to stimulate autologous T cells in the absence of antigen [11] . In addition, even within the cuta neous environment, CD36-positive APC were demonstrated to have suppressive capabilities, in UV irradiated and diseased skin, as above mentioned [7-10]. Further more, macrophages, isolated 6.. om UV-irradiated skin, synthesized and released interl eukin-l0 (IL-l0), which is known to block th e ability ofLC/DC to activate T helper 1 cells while spari.ng T help er 2 function, thereby inducing immunologic tolerance. I T he APC type (e.g., macrophage vcrSIlS LC/DC) may thus inAuence the cytokines produced from T cell s and determine the final balance of an immune response (e.g., downregulatory verslls upregulatory) [45]. We hypothesize that dermal macrophages may fun ction as above, since they can release IL-10 to trigger " downregulatory" dermal immune responses [46]. The opposite function may involve dermal LC/DC, as w ell as epidermal Langerhans cells 1 and spleen/ bone marrow dendritic ceLIs / which release IL-12. T lus cytokin e is a potent inducer of interferon-y production, cytotoxic T lympho cytes, and T helper 1 upregulatory dermal immune responses [47] . Unlike dermal macrophages, dermal LC/DC show an exclusive ability to potently present antigen to T lymphocytes [20] and normal skin-derived dermal human dendritic cells mediated a T-cell response of T helper 1 pattern, with high levels of IL-2 and interferon-y but not IL-4 or IL-l0 [16]. T he upregulatory aUo stimulation induced by normal human dermal cell s suspensions was mainly sub served by CD1a-positive LC/DC, and not by CD1 a F ig ure 3. Dermal macrophages express CD36 on th e cell surface. negative macrophages [48]. III silll IEM labeling of a peri vascular area of normal human dermis. II) A macrophage is vis ible not f., r fro m a dermal vessel (JI). T he macrophage (I'tl11lI, ) expresses C036, although gold granules on the cell m embrane arc Tile allillors IIIallk )1. Mlllder (D"})a rlll, ell r ~r Der/l/al.olngy, Leidell , Tl,e Ndll er no t visible at thi s magnifi cation. T he boxed arca is shown enl arged in Fig lallds), G. BnI/{/lIi (isrilllio Zo{)projila(/ico, Brescia, Ir aly), alld M. Marcelli 31, . S(ale bar, 0.5 11.111. II) High magnificatio n of the boxed arca ill Fig 3a. (De1'1II1/1/elll oj Denllolology, Brescia , Iialy) Jo r III eir e;re""ml ,"(IIl1ienl ass islal/ce. Gold particles arc visible at th is magni fication 0 11 the plasm a m embrane of the macrophage. w hile the (,Icing dermal fibers (C) are not labeled. Swlc uar, 0.25 /1-nl.
I Cooper KD el al : Opposing cytokine production by no rmal Langerhal1s cells (IL-·12) and UV -induced macrophages (lL-l0). J [,/IIesl Denll alol Our new observation that dermal LC/DC, although not showing 103:433, 1994 (abst.) CD36-specifi c gold labeling on the cell surface, do show staining 2 Hcufler C I-I el al: Dendritic cell s arc source of interIcukin-12. J IlI lIcsl of intracytoplas mic organ ell es suggests that these cell s may be Denllalo/l 03:418. 1994 (abst.) VOL. 106. NO. I JANUAR.Y 1996 DElZN!AL MAcrtOPHAGE ' EXPRESS CD36 101
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