Differential Responsiveness of Langerhans Cell Subsets of Varying Phenotypic States in Normal Human Epidermis

Akihiko Shibaki, "'t Laurent Meunier, * ~ Chisei Ra,§ Shil"tii Shirnada,~ Akira Ohkawara:t and Kevin D . Cooper* II *lmmunodermatology Unit, Department of Dermatology, University of Mkhigan Medical School, Ann Arbor, Michigan , U.S .A.; tDepartment of Dermatology, Hokkaido University School of Medicine, Sapporo, J apan; *Dcpartment of Dermatology, St. Charles Hospital , University of Montpellier, France; §Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan; ~Department of Demlatology, Tokyo University Branch Hospi~a l , Tokyo, Japan; and IiAnn Arbor Veterans Administration Medical Center, Ann Arbor, Michigan, U.S.A.

Epidennal Langerhans cell heterogeneity is poorly understood with regard to phenotypic characteris­tics, such as the expression of human leukocyte anti­gen (HLA)-DR, integrin, and Fc receptor molecules, as well as functional characteristics, such as the ability to process and present antigens or produce cytokines during various phases of immigration and maturation. Technical limitations of Langer hans cell number have limited functional assays on putative Langerhans cell subsets in i" vivo epidermis. There­fore, we used flow cytometry for simultaneous phe­notypic and functional assessment at the single-cell level within the Langerhans cell population. Freshly isolated human epidermal cell suspensions were stained with a battery of monoclonal antibodies, including anti-HLA-DR, -CD la, -CDlc, -CDllc, -FcI'RII, and -FcERI. Two distinct Langerhans cell subsets were identified by their different levels of HLA-DR expression. The DRHi subset expressed higher amounts of CDl1c and exhibited greater cy­toplasmic complexity and higher baseline calcium than the DRLo subset (p ::; 0.03 for each). Some subjects also expressed high levels of FCERI in the

Antigen-specific cutan eous immune responses are ini­tiated through the activation of epi.dermal and der­mal antigen-presenting cells (APCs) , whkh then induce T-cell proliferation and Iymphokine produc­tion . T he dendritic APC lineage population of nor­

mal epidermis, Langerhans cells [l ,2], are criti cal for the induction of primary immune responses agail1st contact allergens [3.4], al­lografts [5], and chronic i.nflammatory skin diseases such as atopi c dermatitis [6J . Langerhans cells acquire a unique phenotypic and functional state upon loca lization within epidermis [6,7] and un-

Manuscript received March 29. 1994; revised Septel11ber 8, 1994; ac­cepted for publjca tion September 14, 1994.

Reprint requests to: Dr. Akihiko Sitibaki, North 15, West 7, Kita-ku, Sapporo, Japan.

Abbreviations: APC, antigen-presenting cell ; MCF, mean chalmel fluo­rescence.

DRHi, CDllcHi subset. To determine whether these

phenotypic subsets may exhibit differential signal­transduction functional properties, Langerhans cells were partially enriched over Ficoll-Hypaque and their cytosolic calcium mobilization after the addi­tion of ionomycin was analyzed using the calcium indicator, indo-1, in conjunction with quantitative analysis of HLA-DR expression. By this real-time flow cytometric analysis, a new subpopulation was revealed within the DR Lo Langerhans cell subset. This subset increased its cytosolic calcium concen­tration much more than the other two subsets (change in indo-l blue:violet emission ratio of 37.33 ± 2.34 in the Hi Flux DRLo subset versus 13.23 ± 0.29 in the Lo Flux DRLo subset, and versus 7.6 ± 2.99 in the Lo Flux DR Hi subset). These data indicate that functional, as well as phenotypic, subsets of Langer­hans cells exist within nonnal human epidermis. Their responses to physiologic stimuli may relate to maturational stage or the level of ilt vivo activation. Key words: HLA-DR atttige"slcalci",,,lsigllal traltsdltctiolll 19B. ] 1ltvest Dertnatol 104:42-46, 1995

dergo further modulation after stimulation with antigen, which includes emigration, enlargement, and phenotypic changes such as increased class n major hjstocompatibility complex (MHC) expres­sion [8 -1 0] . Similarly to cultured Langerhans cells, ill "ill{) trigger­ing of Langer hans cells also leads to a loss of the capacity to process native protein antigens and the acquisition of more potent antigen­presenting capacity for restiJlg T cells [2,11 ,12]. Langerhans cell modulation is thought to be mediated at least in part by activated keratinocyte production of granulocyte macrophage colony-stimu­lating factor [13] and tumor necrosis factor-a [14,15], or interleu­kin-1/3 produced by the Langerhans cell population itself [16]. However, the key mechanisms causing Langerhans cell modulation remain to be resolved. .

Cytosoljc calcium mobilization and production of dillcylglycerol induced by phosphatidyl i.nositol turnover act synergistically to activate protein kinase C (PKC) , a major signal transduction

0022-202XI951$09.50 • SSDIO022-202X(94)00260-E • Copyright © 1995 by The Society for Investigative Dermatology, Inc.

42

VOL. 104. NO. I J ANUARY 1995

pathway that links external sign als to in tracc llular cvcnts. Lan gcr­hans cell s contain high amounts of PKC,B L 17 ,18] and app ear to have unde rgon c m o dulatio n through th is pathway in les ional p soriasis, as ev iden ced b y red uccd Lange rhans cell PKC,B [1 8] . T h e latte r finding pe rhap s accounts for t he lac k of th e activity of Langerh ans cells in a lloan t igen presentation b y les io n al epidermal cell s [1 9] . Furth er ill lIillo evide n cc that calc ium and phospho Lipid­depe nde nt PKC is impo rtant for Langerhans cell function is pro­vided by th e finding that ph ospha t idyl se rine enh ances contact sensitization in m ice [20] . T h c stu dy of La n gerhan s cell rcceptors and the fo llowing sign al transduc;tion cvcnts has b ecn h ampc red by the limi tcd numbe rs of Langerhans cell s availab lc fo r study and by the difl:i c ul ty of preparing tota lly pure samplcs of these cells. Howeve r, Langerh an s cell s can bc iso lated rcprodu cibly e lectron­ica lly to extrem e ly high purity by Row cytomctry [21] , and sign al

transdu ctio n involving cytosolic calcium m obilization also can bc visua lized in rea l time by this m eth od [22] . Mul t iparameter Row cytometry indeed a ll owed identification of distinct Lan gerhans cell subsets in some sa mples of hum an e pide rmis o n the b as is of both surface m arke rs and cytosolic calcium m obiliz atio n.

MATEIUALS AND METHODS

Antibodics Monoclonal antibody (MoAb) AEI"l..24 was used to detect express ion of the a subunit of hUlllan high-altinity IgE FceR..! rcceptor (FcElUa). · Othcr antibodies were purchased 1:;'0111 the fo llowing sources : phycoerythrin (PE)-conjugated MoAb to CO l a (T6-RD1) and PE-conju­gated mouSc IgG l . Coul te r Immu nology (Hialeah. FL) : PE and biotin­conjugatcd MoAb to human leukocyte antigcn (HLA)-DR, MoAb to CO li c (anti-Lcu-M5), and PE-conjugatcd mo usc IgG2a, Bccton Dickinson (Mounta in Vicw, CAl; MoAb to CO l a (OKT6) . O rtho Diagnostic Systems (Raritan, NJ) ; MoA b to FcyR.I1 (IV.3), Medarcx. Inc. (Annandale, NJ); M oAb to C O l c, Amac (Westbrook, ME); PE and flu o resccin isothiocyanate (F1TC)-conjugated goat anti-mo use IgG2b, Calrag Laboratorics Inc. (South San Francisco, CAl; FITC-conjugated goat anti- mo use IgG l , Boehringe r (Indianapo lis. IN) ; streptav idin conjugated with allophycocy:min , Bio lllcda Corpora tion (Foster City, CAl. Isotypc contro l mo usc antibodies were o bta ined fi'om Sigma Chemical Co. (St. Lo ui s. MO) and Phanningen (San Diego, CAl·

EpidcrmaJ CclI Suspcnsions Ski n was obta ined from no rmal adu lt voluntcers by keratome biopsy after info rmcd consent, followed by over­night incuba tion with Dispasc (50 U/m l) (Co ll aborativc Research. Wa ltham , MA) , as described [7]. Epidermal sheets were separated fi,om dermis, tr)'psinized , teased in to a cell suspension , and fi ltered thro ugh a nylon mesh to achicve a singlc-ce ll suspcnsion. Disaggrega ted cell s wcre resuspcnded with app ropriate media for thc fo llowing procedures.

Quantification of Surfacc Antigcn Expression T he expression of surface antigens was IncaslIrcd by direct and indirect inlillUllofi uo [csccnce st:lining with the appropriate MoAbs, fo llowed by quantitative ana lysis by fl ow cytomctry. essentiall y as desc libed previously [8]. For all analytic experiments, epidermal cell suspensions wcre incubated with heat-aggre­ga ted (65 °C X 20 min) puriflcd human IgG (Sigma) to block nonspecific binding of the MoAbs to the IgG Fc receptors. For double-marker fluoresccnce, PE-conjugated anti-HLA-DR was combined with appropriate primary antibodies and thcn incubatcd with FIT goat anti- mo use IgG l or IgG2b. For triplc- marker flu orescence. biotin-conjugated anti-HLA-D R was combined with two additional primary antibodies of difFerent isotypes, fo llowed by visua lization with streptavidin allo phycocyanin. PE goat anti­mo usC IgG2b. and FITC goat anti-mOllse IgG l. Norma l human serum (10%) and no rmal goat serum (1 0%) were added at cach step to block nonspecific binding.

CclJ Preparation for Cytosolic CaJcimn Measurcmcnt To o bt:Jin sufti cient flow rates of La nge rh :lll s cells fo r experimcnts measuring cytosolic ca lcium. epidcrmal cell suspensions wcre partially enriched in Langerhans cell s by Fico ll Aotation [21.23] . Epidermal cell suspcnsions were placed on the same vo lume of a Ficol l-Hypaque grad ient (Pharmacia. UppsaIa. Sweden) and ccntrifuged fo r 30 min at room tcmperature. Ccll s located at

• Kuromitsu S, Katoh H, Ogino T. I-Iirose T. Yasuda S. Furuichi K. Okumura K, Ra C: Establi shment of monoclonal antibodies specific fo r human FCE.lUa chain (abstr). ProCJJl II Sor IIIIIIIIIIW/ 22:541, , 992. Suzuki K. H irose T. Jin Z, Yagita H. Okulllura K, Ra C : Ana lysis of the exprcssion of hum:m FCER.I a exodomain contai ning chimera molecules (abstr). ProCJplI Soc 111111111110 / 23:625, '1993.

I1 .. ESPONS IVENESS O F LANGEIU-IANS CELL SUnSETS IN EPID EKM IS 43

thc interface were collected. resuspended at 1 X 10" cells/ml in phosphate­bufFered sa line with 1% fctal bovinc serum contairilng 1 J.LM indo-1 AM (Molccular Probes, Eugenc, O R). and incubatcd in thc dark for 30 min at 30°C . T his temperaturc was used to avoid possible dye compartmentaliza­tion. Cell s were washed twicc in phosphate-bufFered saline with 1 % fetal bovine serum and thcn sta ined with PE-coltiugated anti-HLA-DR for 30 min at room temperature. After washing, the cells were resuspended in the sa me buR'er and kept at room tempcrature until fl ow cytometric analysis.

Cytosolic Calciulll Measurcmcnt Langerhans cell cytosolic calcium measurements were performed with an Epics Elite flow cytomcter. equipped with an air-cooled argon lascr for PE cxcitation at 488 rlm and an air-cooled ultraviolct laser for excitation of indo-l at 350 nm. Samples wcrc kcpt at 37°C using a circulating watcr bath during data acquisition. Changc of cytosolic calcium concentration was measured by calculating the ratio of the fl uorcsccnce intensities at two difFcrent cmission wavelengths, wlilch are at 405 nm (violet color) and 485 nm (bluc color). Indo-l shi fts the emission spectrum upon binding calcium (via increased calcium conccntration), and thus the emiss ion pcak at 405 n111 incrcases rcciprocally with a dccreasc in cmission at 485 nm . As a result, thc ratio of blue to violet fluoresccnce decreases as the calcium concentration increascs . Cells with inadcquate loading of dye wcrc cxcluded fro lll ana lysis by plotting b lue versus violet flu orescence intensity. Langerhans cell s in the suspensions were selected by visualizing thcir I-ILA-DK expression (PE flu orescence), and the cytosolic calc ium of this selected population was measured for 30 seconds before and 210 seconds after thc addition of 10 J.LM calcium ionophore, ionomycin (Ca lbiochelll . La J olla , CAl. Changcs in calcium concen tration after stimulation were quantified eithcr as the raw blue:violet fluorcscencc intensity ratio or as the change (dclta) in bluc:violet ratio relativc to basel ine. All signa ls were stored in Listmode. down-loaded onto optical disks (M itsubishi), and analyzed using the Elitc software version 3 program (Coul ter) for DOS-based computers.

Statistical Analysis T he mcan chan ncl fluorescence (MCF) intensity of Langerhans ccll markers and the blue:vio let ratio of indo-l fluorescence were co llcctcd in each individual subject, pooled for thc group mcan and standard error of thc mean (SEM) , an d compared using Student t test for paired data.

RESULTS

Identification of Two Langerhans Cell Subsets in Human Epidermis That Express Distinct Amounts of HLA-DR Upon the cxa mi n ation of Lan gerhan s cell HLA-DR e"-"Pression in conjunction with their cytoplasmic complexity (90 0 light scatter), a mino r but rcproducible Lan gerh ans cell subset could be identi.fied re lative to the isotype contro l stainin g (Fig 1). HLA-DR expres­sio n of this minor Lan gerhans celJ subset (DRHi). which represents 16.3 ::'::: 1.58% of Langer han s cells , was quite high (MCF ::'::: SEM = 351 ::'::: 67.3) rela tive to the m ajor Lan gerh an s cell subset (DRI..O)

(MCF ::'::: SEM = 66.2 ::'::: 16.3) (Table I) . Tn additio n . these two Lan gerh an s cell subsets d emonstrated distinct degrees of cytoplas­mic complcxity; the DRHi subset contain ed m ore membrane-water interfaces th an did th e DR Lo subset (p = 0.030) (Table I). Because

:l ----

~ ~ '" u u ",<= <= e,," a:" "'u

=j o~ -;~ ' .. <~

11>0 -,0

"" J::@ 0:;::

• Ew ., r. .~.

W ~ J , ~ r

~

"".,.....,..,.",r . ~ . ... " .. It ... u"

Cytoplasmic C~mplexity CytoplasmiC Complexity

Figurc 1. Langcrhans cclIs arc divisiblc into two distinct subscts b y thcir HLA-DR exprcssion and cytoplasmic cOlllplcxity. Cytoplas­mic complexity (log 90° light scatter) of fres hly isolatcd epidermal cells is plotted vc rsus PE fluorescence intensity. LIft: isotypc control staining with PE-conjugatcd mouse IgG2a. R igh,: epidermal cells stained \vith PE­conjugated anti- HLA-DR. A small Langcrhans cell subset intensely cx­pressing HLA-DR (D K"';) is obscrvcd above the m'\ior HLA-DR + popu­lation (DRLu

).

44 SHIBAKJ ET AL

Table I. Phenotypic and Optical Characteristics of Resting Langerhans Cell Subsets"

Percent of DR +

DR intensity/' Cytoplasmic complexity' Baseline indo-1 e

DIl~H

16.3 :!: 1.58 351 :!: 67.3

9.05 :!: 2.31 36 .4 :!: 3.08

II Data represent average 2:: SEM; 11 = 1. 2. /, MCF ofHLA-DR sta ining. , Log 90° light scatter.

DRLo

83.7 :!: 1.58 66.2 :!: 16.3 5.81 :!: 1.19'/ 47.0 :!: 1.59'/

d p = 0.03 (cycoplosmic complexity) and p = 0.01 (baseline indo-I). t' B:lsclinc ralio of bluc:violet fluorescence after indo-1 loading.

cell size (forward light scatter) did not differ between the two subse ts (not shown), the high DR expression likely represents increased DR density, and the increased cytoplasmic complexity of the DR

,-,i subset likely indicates increased surface membrane

folding and/or cytoplasmic complexity [23J. Electron mi croscopy of purified subsets was not performed, but may be useful to characterize t1us finding further .

Fc Receptor, {32 Integrin, and COl Expression on the DRLo

and Hi Subsets Surface expression of FcylUI (n = 6), CDllc (n = 6), CDla (n = 5), Fc€lU (n = 3), and CDlc (n = 3) was quantified in each Langerhans cell subset in conjunction with HLA-DR quantification by double- or triple-color flow cytometry. In each of six subjects tested, the DRHi Langerhan s cell subset expressed higher amounts of CDllc (ax{32 integrin) than did the DR L o subset (p = 0:026) (Table II). In some individuals (two of six subjects), the DRHi subset expressed almost 10 times more CDllc than did the DR L o subset (Fig 2a). Thus, in such subjects, the DRHi , CDllcHi subset could be visualized on a dot plot as a compJetely separate cluster above and to the right of the main Langerhans cell population (Fig 2a). The increased CDl1c expres­sion on the DR'-

Ii Langerhans cells was not a finding common to all Langerhans cell surface markers because no such difference in surface staining was observed for CDla and FcyRII (Table II). However, more than two thirds of th e subj ects demonstrated a more subtle difference in CDllc (Fig 2c) . A similar distribution of subjects (one third) exhibited very high FCERI expression on the DRHi Langerhans cell subset (Fig 2b). CDlc, a marker for dendritic APC lineage in humans (7J, also demonstrated a trend toward higher expression in the DRHi subset, but this was not statistically significant (p = 0.11) .

Langerhans Cell Subset Definition by the Ability to Mobi­lize Cytosolic Calcium To determille whether these two Langerhans cell subsets differ in their flillctiona l attributes, we measured their capacity to mobilize cytosoli c calcium along with simultaneous HLA-DR quantification by multiparameter flow cy­tometry. Langerhans cells were selected on the basis of their HLA-DR expression and visualized as PE fluorescence illtensity (Fig 3a). The DRHi subset was identified in this subject as a DR intensity of more than 600 fluorescence units (Fig 3a) . After gating on the DR + Langerhans cells (Fig 3") and acquiring the baseline indo-l fluorescence ratio ("pre") ionomycin was added to the sample and sampling flow resumed within 5 seconds (/,/"eak). As seen in Fig 3c, the DRHi Langerhans cell subset had a slightly lower baseline indo-l fluorescence ratio compared with the DRLo subset

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

(p = 0.01) (Table I). This difference in the baseline indo-l fluorescence ratio was seen in seven of eight different experiments (Table I). WitlUn 30 seconds of exposure to ionomycin, essentially all Langerhans cells exlubited a marked calcium increase (Fig 3"). However, two distinct patterns of calcium flux among Langerhans cells were observed. One Langerhans cell subset exhibited a brisk but moderate degree of calcium flux, reaching a blue:violet fluo­rescence ratio of approximately 30, with some recovery thereafter (Fig 3"). The other subset exhibited an extreme degree of calcium mobilization, reaching a blue:violet fluorescence ratio of 12, with­out evidence of recovery. These distinct subsets were seen in six of e ight different experiments (Fig 4) .

To detennine whether the two Langerhans cell subsets defined by calcium mobilization were related to the phenotypically defined subsets, the indo-l blue:violet ratio before (Fig 3b, "pre") and after (Fig 3", "post") ionophore treatment was plotted against DR intensity. Before ionomycul exposure, the two Langerhans cell subsets of varying DR ultensity could be distinguished clearly (Fig 3c, pre). By comparing the Langerhans cell blue:violet ratio to DR intensity after ionomycin exposure (Fig 3d, post), it is evident tha't the L3ngerhans cells that exlubited a marked degree of calcium flux represented mainly the DR Lo subset. However, only somewhat less than half of the DRLO subset demonstrated this extreme calcium­flux phenotype. In contrast to the brisk illcrease of cytosolic calcium by the DR Lo subsets, the majority of DRHi Langerhans cells responded very weakly, with nUnunal calcium flux ulduced by the addition ofionomycin (Fig 3d, DR Hi) relative to their pretreat­ment state (Fig 3c, DRHi).

Thus, calcium mobilization capacity, combUled with DR expn;s­sion, identifies three Langerhans cell subsets in normal human epidermis. Pooled data on these Langerhans cell ' subsets from multiple subjects are summarized in Table III. After stimulation with ionomycul, the Hi Flux DR Lo subset showed the lowest cytoplasmic complexity as well as the lowest DR ultensity and the lughest calcium mobilization capacity (delta blue:violet ratio). The La Flux DR Lo and the Lo Flux DRHi subsets had mid- and high cytoplasmic complexity, respectively (Table III).

DISCUSSION

This study indicates the presence of several Langerhans cell subsets that have not only phenotypic differences, but also distinct func­tional and optica l properties. By multicolor surface staining, we could identify two distinct Langerhans cell subsets. These two subsets have differential expression of the class II MHC molecule, HLA-DR, and the {32 integrin, CDllc. Tlus difl:erence of surface marker expression was not due to nonspecific bUlding of MoAb, because the expression of other surf.,ce markers such as FcyRII and CDla was not different in these two subsets. A difference in cell size as the bas is for elevated DR and CDlic fluorescen ce per cell also was excluded because there was no difference Ul the forward light scatter between these subsets. Ovenught treatment with dispase at 4°C may have modulatory effects on Langerhans cells, but the conclusion regarding differentially responsive subsets of Langer hans cells would still hold because each subset received the same treatment with dispase. The mea ning of distulct surface marker expression and functional responsiveness in Langerhans cell subsets is not yet clear, but may be related to particul ar states of differen­tiation or focal recent ill. lIillo activa tion .

Langerhans cells clearly can undergo maturational changes after external stimulation. Upon contact allergeri exposure in. 11;110 a.nd ill

Table II. Expression of CDllc, COl Molecules, and Fc Receptors by Resting Langerhans Cell Subsets"

CD11 c CD1a CD1c FclU FclUI

DR~1i 29.8 :!: 8.55 197 :!: 53.9 32.9 :!: 10.2 2.06 :!: 0.382 34.3 :!: 9.88 DRLo 12.7 :!: 4.69 182 :!: 24.2 16.3 :!: 3.72 1.69 :!: 0.507 30.2 :!: 5.79 P value 0.026 0.87 0.11 0.47 0.94

" Da ta represe nt average MC F ± SEM .

VOL. 104, NO. 1 JANUARY 1995

a b

CD11c (PE fluorescence) FceRI (ATC fluorescence)

c d . '

n ,.,r--, - ..,.....,-,.,..-,-rn"'''- r-T""TT 1 n u. u ..

CD11c (PE fluorescence) FceRI (FITC fluorescence)

Figure 2. Range of patterns of DRHI Langerhans cell subset ex­pression of CDllc and FceRI. Results are derived from two di1ferent individuals (subject 1 = n,b; subject 2 = c,d). nand c: electronically selected HLA-DR + epidermal cells (visualized as APC fluorescence) are co-stained with PE mouse IgG2b (colllo"I'plo(.<) or with PE anti-CDllb (dot plots). DR>'; Langerhans cells exhibi t moderate (c) to large (n) rightward shifts in PE fluorescence (relative to the isotype control contour plot), and are labeled DR>';, CDll CHi, whereas DR Lo Langerhans cells with less of a rightward shift in PE flu orescence are labeled DR La , CDllcLa

. band d: HLA-DR + epidemlal ceils are costained with mouse IgGl (COIllO" I' plots) or anti-FcER! (dol plots). As with CDllc, some subjects exhibited a very large rightward shift in FcElU flu orescence intensity by their DR H, subset (b), whereas in other subjects the delta in FcElU MCF was similar in both the DRHi and DR La Langerhans cell subsets (d).

/Jitra, Langerhans cells change their class II MHC antigen expression [8-10]. After short-teml culture, Langerhans cells alter their phe­notypic properties upon removal from epidermis and/or in response to cytokin e exposure and become more similar to lymphoid dendritic ce lJs [2,24,25] . For example, human Langerhans cells increase their class II MHC antigen expression, and their expression of CD1a and FcrRII is decreased [24,25]. Thus, the DRHi , CDllcHi Langerh ans cell subset may represent a population that has exp el;enced recent ;1/. /J;/JO activation and commenced matura­tional changes, but that has not emigrated from the epidermis. T lus con cept is supported further by the abili ty of chemoattractants to up-regulate CD11.c expression in m onocytes [26]. Because {32 in tegrin s are connected to the cytoskeleton and fWlction as adhe­sion molecules [27], variations in CD 1.1 c may regulate Langerhans cell morphology, function, or intraepiderm al position. On the other h and , the DRHi subset recently m ay have immigrated to the epidernus [1,28].

T here are severa l reports suggestin g the presence of Langerhans cell subsets in normal skin by flow cytometry [2,21,29,30]. Fur­thermore, two Langerhans cell populations, determined by distinct levels of class II MHC antigen expression , em erged 24 h after epicutaneous application of the hapten trinitrochloroben zen e to naive mice [9]. T h ese reports are consistent with o ur results in humans demonstrating both constitutive phenotypic and fWlCtion­ally e licited Langerhans cell subsets. In addition , we observed lug h expression of FceRI by th e DRHi , CD11cHi Langerhans cell subset in som e individuals. FceRI itself can trigger calcium flux upon external crosslinking in other cell types [31,32] and also can function to capture antigens for antigen presentation [33].

RESPONS IVENESS OF LANGERHANS CELL SUBSETS IN EPIDERMIS 45

B I:

a:Ql cli: 'Ql

4('" ...J o x:~

W e:..

a

Cytoplasmic Complexity

Gated on pre c

HLA-DR (PE fluorescence)

-~ Ql ... :20 >'0 0-= -~

Gated on DR+

~ 8 : -I: mQl _t.l 0" o I!! ;:0 «I~ a:;:

TIme (sec)

Gated on post

b

d

HLA-DR (PE fluorescence)

Figure 3. Cytosolic calcium mobilization in HLA-DR+ epidermal cells after stimulation with ionomycin. HLA-DR + epidemlal cells were selected electronically (a); DR + epidermal cells were stimulated with ionomycin at 30 seconds (break), and the ratio of the two different emission wavelengths of indo-l was analyzed up to 4 min (b) . Cells before and 90 seconds after ionomycin stimulation were selected (pre and post in b, respectively) and plotted by HLA-DR expression on the :.,' ax:is and indo-l ratio on the}' axis (c,d). DR Hi and DR La subsets were identified in DR + epidennal cells before ionomycil1 stimulation (c). After stimulation, the DR La subset was divided further into Hi Flux and Lo FhD': subsets (d).

Elevations in b aseline cystolic calcium have been reported after surface receptor stimulation in other immunologic cell types [34]. Dilferential b aseline indo-1 fluorescence ratios between DRHi and DR Lo suggest tI,at ti,e DR Hi Langerh ans cell subset may represent Langerhans cells that have elevated their baseline cytosolic CalCiunl after recent endogenous stimulation by the surrowlding nUlieu, either as a result of h avin g recently traversed into tile epidermis or as a result of local intraepidennal perturbations. Differen tial indo-l fluorescence intensity as a result of n onw1iform loading, dye bleaching, or cell tluckness is avoided by using ratio analysis of the two enuss iol1 wavelengths of indo-1.

We noted distinct calcium flux intensity levels in Langerhans cell subsets after addition of calcium ionophore . Again, we suspect that the DR Hi subset may have been activated recently ill 11;110, to account for its refractoriness to further calciu1111110bilization. These

l- SO w~

..J'7 00 n=6 -'0 >c: 40 0::' I-W wU 30 ::I z ..Jw mU u.C/) 20 O::g 00 -::I 10 I-...J 4(u. a:

0

Responder

Figure 4. Grouped data demonstrating the reproducibility of the differential response by Langerhans cells after ionomycin stimula­tion. Low-responder and high-responder Langerhans cell subsets were observed in six of eight subjects.

46 SHIJ3AIG ET A L

Table III. Relation of Langerhans Cell Subsets, Identified by Elicited CalciUln Flux, to Class II MHC

Expression and Cytoplasmic Complexity

D R Cytoplasmic R atio" In tcnsity Complexity

Hi Flux OR Lo 37.33 ± 2.34 1.00 2.53 ± 0.42 Lo Flux DRlo 13.23 ± 0.29 1.1 9 ± 0 .03 5 .45 ± 2.64 Lo Flux D R "; 7.60 ± 2 .99 6.54 :t 0.71 15.38 ± 8 .1 8

" Ratio: the ratio ofbluc:violet fluorescence at the time of gate "pre" m_inus the ratio of blue:vio lc t Auorescence at the time of ga te "post."

diiferentially respon sive Lan gerha ns cell subset s also exhibite d

distin ct cytoplasmic complexity, p oss ibly b eca u se the subsets o f

murine cla ss II M HC + e p id e rma l cells v ary ill d e n sity and c an

ind u ce sen sitization or to le ran ce to antigen s (35]. We rule d out

diffe re nti al compartm e nta liza ti o n of illdo -1 by assessing the p a t tem

of h o m ogen eou s cytopl as m ic fl uo rescen ce b y fluorescen ce micros­copy (data n ot sh o wn) .

In summary, u sing multicolor flow cytom e try and simultaneou s

ana lysis of su rface m a rke r exprcssio n ill conjunc ti o n w ith c al cium

mobilizatio n , we iden tified phenotypica lly and fun c ti o n al1 y di stinc t

Langerh an s cell s ubsets ill huma n e pidermis. P h e notypic ally, La n gerh an s ceBs w e re divide d into DRI-!; , C Dll c l-!; and DI~ Lo ,

CDllclo s ubse ts, whic h showe d di stinc t baseline c alcium levels

a nd cytoplasmic com p lexi ty. Hig h expression o f FCEIU a nd CD l c

also was a ssociated w it h the DRI-I;, C Dll c H; subse t ill some

su bj ects. Langerha n s .ce ll s ubsets wer e c h a racte ri zed fu rth er as Hi Flux DR LO, Lo F lux D R Lo, and Lo F lux DRI-I; subsets b y tne ir

d istin ct resp onses to calcium io n o phore. T h ese d a ta indica te t h e

p resen ce of multiple Lan gerh a n s cell subse ts w i thin human e pide r­

mis, w hic h m ay rep resent La n gerh a n s ceBs in di ffe re nt stages of

activa ti o n , m aturation , a nd respo n siven ess to externa l m o dulatio n .

T his lVork lVas SII)JJlorted ill pm1 by NIAMS gralll RD-l A R 41642-01A '1 (/lid U"i/wer COIporatio" a"d M"ltip"rpose A ,tl"ilis Cell /ergro,,! N IH ]>60 AR20557.

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