Interleukin-l f3 and Granulocyte-Macrophage Colony-Sthnulating Factor Mediate Langerhans Cell Maturation Differently

Hiroaki Ozawa , Satoshi Nakagawa, Hachiro Tagami, and Setsuya Aiba Departmcnt o f Dermatology. T ohok u University School of Meoicine. Scnd"i. Japan

It has been reported that the ill vivo maturation of Langerhans cells after hapten painting is mediated by IL-II3 while Langerhans celllnaturation after ill IIU,'O culture is mediated by granulocyte-macrophage col­ony- stimulating factor (GM-CSF). To clarify the rea­son for this discrepancy, we exanlined the expression ofIa antigen and several co-stitnulatory molecules on Langerhans cells that were activated by ill .fit,'o cul­ture, by hapten painting, or by an intradermal injec­tion of several cytokines. Both cultured Langerhans cells and those activated by hapten painting in­creased the expression of la antigen and all the co-stimulatory molecules (i.e., intercellular adhesion molecule-I [ICAM-I], B7-I, B7-2, and CD40). In contrast, an intradermal injection of interleukin-113 (IL-If3) or tumor necrosis factor-a (TNF-a) increased the expression of Ia antigen, ICAM-I, B7-2, and CD40, but not that ofB7-1. These data indicate that

Recentl y it ha s been demonstrated , by several authors [1. 2,1 4,23,24,29 J that Langerhan s cells can be m aturated both ill IIi"O and ill "ifro, Maturated Lange rhan s ce ll s in crease their expression of major histocompatibility co mplex (M HC) class II antigcn

(Ag) and show en hanced antigcn prescnting fun ction [4.21 ,30]. W itmer- Pack cf 111 [32J and )-ieuAer ef III [1 3] have demonstrated that the maturati on of Langerhans cell s that occu rs after ill IIifm

short-term culture is mediated by granulocytc-macrophagc colon y­sti mulating factor (GM-CSF) and that inte rl cukin-1 (rL- l) en­hances the e ffcct of GM- C SF. On the other hand, Enk cf 111 [10] havc fo und that the increase in Langcrhans cell-derivcd IL-1 f3 mRNA signal strength is the first even t in the process of ill I, ill" maturation of Langerhans cells aftcr hapten painting . In addition, they showed that the cutaneou s inj ec tion of IL-1 f3 res ul ted in increased exp ress io n of M.H C class II Ag .. Ild en hanced antigen presenting fun ction by Langerhans ccll s suggesti ng that ill lIillo

ma turation o f La ngerhans ce ll s th;lt occurs after hapten painting is mediated by I L-I f3 18]. T herefore, if Langerhans cell maturation induced by the cutan eous injection of IL-'l f3 is the sa me ph en om-

Manu script rcceivcd Fcbruary II , 1995; rev ised October 26, 1995; acceptcd for publication October 311 . 1995.

Reprint requcsts to: I r. SctSLlya Aiba. Departll1e nt of DerIllatology, Toh oku Un iversity School of Mcdicinc . 1- 1, Sciryo-machi, Aoba-k u . Sendai. 980-77 , J apan.

Abbrev iation: TNCU. 2.4 .G-trinitroch lorobcnzenc.

IL-113 or TNF-a is not sufficient to induce B7- I expression on Langerhans cells ill .';110. Subsequently we examined the effect of anti-cytokine antibodies (Abs) on the expression of those molecules on cul­tured Langerhans cells. While none of the Abs to IL-II3, TNF-a, or GM-CSF changed the upregulation ofla antigen, ICAM-I, or CD40 on cultured Langer­hans cells, anti-GM-CSF Ab suppressed that of B7-I and B7-2. Taken together, our present results suggest that IL-113 is required for the upregulation of la, ICAM-I, B7-2, and CD40, while GM-CSF is required for the upregulation of B7-I and B7-2, although it still remains unclear why the injected GM-CSF could not augluent B7-I expression on Langerhau.s cells i71 IIillo and why anti-IL-113 Ab did not suppress the upregulation of la, ICAM-I, or CD40 on cultured Langerhans cells. Key words: B71tllllJ.ol' IIec"'osis facto' .... al CD40Icl'tok;IIe. ] IIlIIest Demratol106:441-445, 1996

en on as that occurring on Langerhans cells that mature after hapten painting, there remains a discrepancy in the reported beh avio r of cytolcines that mediate the m aturation of L1ngerhans cells between ;11 pilll) and ill ";(1"0.

It is also we l1 known that accessory molecul es are rcquired for antigen-presenting cells to stimulate T cell s 17,15,22,28,3 1]. T hcre­fore, in this study examining the phenotypic changes in Langerhans cells maturated ill I/ifro aud ill II illO, we havc analyzed the contribu­tion of diffe rent cytolcincs in the maturatio n process of Langerhans cell s unde r ill l,iv(I and ill IIifm situ ations.

MATERJALS AN I METHODS

M.iee Specifi c pathogen-fi'ee I" malc OAU3/c AJclmice. 8-10 wk of age. were obtaincd fro lll the Institute of Expcl;m ental Animals. Tohoku Uni­ve rsity School of Mcdicine,

Antibodies We uscd thc followi ng m onoclonal antiboo ies (MoAbs) for in1ll'11Inosta ining : anti-intcrcc llula r ad hes ion molecule-I (ICAM- I) (KA T­I). anti-CD-IO (HM-40-3) (gifts fi'ol11 Dr. Yagita and Dr. Kato. Dcpartment of Iml11unology. Julltcndo University. Tokyo . Japan). aI10-U7-1 (07 I B01). <I nti-07-2 (GL- I) (PharMingell. Sail Diego. C Al, ano fluorcscein isothio­cyanate (FITC)-conjugated anti-I-E (d. k haplotypcs) (l'hmMingen) MoAbs. Phycoe r),tltrin-conjugated ami-rilt IgG (TAGO, UlII'lingill1lc . CAl o r bioon ylatcd anti-hamster I ~G (Vcctor Labonn:ories. 13l11'linga m e. CAl followed by phycocrythrin -conjugated avidi n (Uecton-Dickinsoll. Sa il J ose. CAl wcre used as second-stcp rl'agcnts , Isotype-lI1atched ral control Ab (lgG 2• and IgG21,). halllste r IgG and FITC-colljuga ted rat control IgG oh Ab (PharMingcn) ,vere llsed <1 S negative contro ls. In o rd er to cxanlinc the clrccts of anti-cytokinc Abs 011 th e upreguiation of adhesion molecul es or

0022-202X/ 96 /S10,50 • Copyright Ii) 1996 by T hc Society for Ill vestigative l)e rInato lob')' . Inc.

441

442 OZAWA ET il l

MH C class JI Ag by ill " itro cul tured Langerh"n s ce ll s, we used goat anti-murine GM-CSF, anti-murine tUl110r necrosis f.,cto r-CI' (TNF-a). and anti-1I1urine IL-1 {3 Ab frol11 R&D System (M inneapo li s. MN). Purifi ed Goat Ig was purchased frolll ZY MED Laboratories. Inc. (San Francisco. CAl.

Chcmicals and Cytokincs 2.4 .6-trinitrochlo robcnzcnc (TNCB) was purchased from Tokyo Kase i Laborato ries (Tokyo, Japan) and was IHcpared in acetone/oli ve o il (1 :4) at 3% fi nal concentration. R.ecombi nan t murin e GM-CSF, recombinant murine TNF-CI' . and recombinant murine IL-1 (3 wcre all obtained frol11 R &D Systcm. T hese cytokincs were suspen ded in phosphate-buffered salinc (PHS) containing 0.2'!!') bovine serum albumin (BSA).

Chcmical Trcatmcnt and Cytolcinc Injcction After anesthetized with )I-hydroxybutyri c acid (Sigma Chemical Co .. St. Lo uis. MO) , BALB/c mice we re injected illtrade rmall y in each ear with 50 o r 250 Ilg of recombinant murine rG M-CSF, 50 ng of TNF-u, o r 50 ng of lL-1 {3. Another gro up of mice werc injected with PBS con ta ining 0.2% BSA as a negative control. As a positive control, other mice wcre pain ted on t h c ca rs with 3% TNCI3 in acctone/oLlve oil (1 :4) . In most experiments. epjdermal ce ll suspens ions wcre prepared 16 h after the treatmcnts.

Preparation of Epidermal Cell Suspcnsion Epidermal cell suspen­sions werc prepa red by treating the split skjn sheets of cars of nontrca ted o r chcrni caJl y treated mice, or those injected with va rio us cytokjnes with 1000 U/ 1111 Dispase (Sanko Pure Chemical Inc., Tokyo, J apan) in RPMf-1640 sup plemented w ith 1 % fetal bovine se rum fo r 2 h as described previo usly r3.19] . In order to ob tain a Langerhans cell- enrich ed population, these cpidcrmal cell suspensions were applied onto Lympho lite M density g radi­en ts (Ccdarlane Laborato ri es Limited. Canada ) and centr ifuged at 1400 rpm fo r 10 111in nt roonl tel1lpCnlturc. T he in terface cell s were recovered. which contained 5-10'10 I-E-positive cell s (Lange rhans) in addition to keratino­cytes.

Langerhans Cell Culture R .. PMI 1640 medium supplemented with 10'X. fe tal bov ille serum , 2 mM glutamine (G ibco Laboratories . Chagrin Fa ll s, OH). penicillin. streptom ycin and fungizone antibiotic so lu tio n (Gibco) . non-essentia l amino acid so lution (G ibeo) . sodium pyruva te solution (Gibeo). 10 111M N-2-hydroxyethylpiperazine-N '-2-e thancsulfon.ic acid buffer solution (G ibco), 5 X 10- ' M 2-ME. and 1 p,g/ml indomethac in (Sigma) (complete medium) was used for the culture . Enriched Langerhans cell suspcns ions ( 1.5 X 10" / ml) were cultured in the complete m e dium using 24-well plate (Falcon) in the presence of goat Ig or MoAb to GM-CSF, TNF- CI' , o r I L- l {3. T he surface expression of various adhesion molecules oil Langerhans ccll s was exam ined by flo w cy tom etry 72 h aftc r culture.

F low CytOJnetry For fl ow cytometry, cell suspensions were first incu­bated with various MoAbs to mouse adhesion molecules o r isotype-matched rat or hamster control Abs fo r 30 min on icc, washed with PllS supp le­m ented with 1% feta l bovine serum and 0.02% NaN , (washing buller) , and rhen incubated with ph ycoerythrin-conjuga ted anti-rar- Ig (TAGO). Or biotinylated an ti-hamste r IgG followcd by phycoerythrin-conjugate d av i­d in. After being w"shed with washing buffer, ce lls were seriall y treated with 50 /Lg/ml of rat Ig for 10 min and with FITe -I E o r FITC-rat control Ab for 30 min on icc. Afte r being washed again, they werc ana lyzed On a FACSc:l n using Lysis " software (Becton Dickjnson) . LlI1gerhans cell s as identi fi ed by intense reacti vity with FITC-I:lbeled anti-I -E MoAb were gated for the analysis of MI-I C class " Ag or costimulatory molecule express ion. and 1110 re t han 3.000 events were acq uired for each ana lysis. Mean flu orescence intensity (MFI) was dctcrmined for e<l ch surface mo leculc. "nd the cll:ccts of cytokines were eva luated by percent<l ge M PI as c<llculatcd usin g the to llowing form ul a 15,6,171:

% MFI

( MPI of Langerh"ns cell streated with cytokj11l!(s) . T NCB. or Ab )

= MFI of Langerhans cell., treated with 0.2% I3 SA/PBS o r medium OJ11y

X lOO'X,

RESULTS

Both Langerhans Cells after TNCB Painting and Those Cultured ill Vitro Could Upregulate the Expression of MHC Class II Ag, ICAM-l, B7-1 , B7-2, and CD40 We have reported that ilt lIillo maturated Langerhan s cell s in crease their ex press ion of MH C class II Ag togethe r with e nhan ced antigen presentin g func tion like Lan gerhans cells c ul ture d ill I'ilro [1]. Recen tly, culture d Langerhans cell s h ave been sh own to c nhan ce

a: UI m ::E ::J Z -' -' w u

MHC CLASS II AG

THE JOUR.NAL O F INV EST IGATI VE DERMAT O LOGY

ICAM· 1 B7·1 B7·2 CD40

FLUORESCENCE INTENSITY

F igure 1. Both Langerhans cells after TNCB painting and those cultured ill (Jitra lIpregulate the expression of MHC class nAg, ICAM-1, B7-1, B7-2, and CD40. Langerhans cell s maturated ill "illll br TNCB painting (n- e) and cultured Langerhans ce ll s (F-") were cxamined for the expression of MHC class " Ag (nJ), IC AM-1 (II,S), 137- 1 (t,") . J3D-2 (d,i). and CD40 (ed) after gating I-E positive ce lls on FITC lIerSlI' side sca tter contour plo ts. T he profiles of Langerhans cell s maturated by TNCB painting and cultured Langerhans cd Is were indica ted by open histograms. and those representing fi'e shl y iso lated Langcrhans ce lls arc indica ted by shaded ones.

their co- stimulatory molecules (e.g. , ICA M-l , B7-1) [21 ,30] . T h erefore, in this study we examined w hethe r Langerhans ceUs maturated ilt lI il lO afte r hapte n painting also upregulate those costimu lato ry m o lec ul es. Figure 1 presents representative da ta fi'om e ig h t different exp erim e n ts. N o ntreated fi'eshly iso la ted Lang­e rhans cell s ex pressed MHC c lass II Ag, ICAM-I , 137- 2, and CD40, but did n ot e xpress B7-1. On the other hand , b oth cultured Langerhans cell s and t hose treated w ith TNCB painting ill Ii i 1'0

sh owed upregulation of MHC cla ss ]( Ag, ICA M-l, B7-1, B7-2. and CD40 expression , alth o ug h the magnitude of th e e xpression of these molecules o n Langerhans cell s m aturated b y TNCB painting was mu ch less than that on culture d Langerhans cell s.

The Intradermal Injection ofCytokines Could Not Upregu­late B7-1 011 Langerhans Cells ill Vivo I L-1 f3 and G M-C SF are re po rte d to b e in volved in Lan gerhan s cellm3turatio n ill 1';1'0 and ill

(lilro, resp ec tive ly [8, 13,32] . In addition , acco rding to E nk cI nl [8] . the cutaneou s injection of I L-l f3 induce th e phenotypic as well as functio n al maturatio n in Lan gerlwns cells ;11 (lhlo similar to that in th e se ceUs after h apten painting. We exam ine d thc express ion of MHC class II Ag, ICAM-l , B7-1 , B7- 2, and CD40 o n Langerhans cell s 16 h after intrad ermal injecti o n ofGM-CSF, T NF-a . o r lL- l{3, and compared their expressio n with those on Langerhans cell s after TNCB pain ting (Fig 2) . Lange rhans cell s from the mice injected w ith IL-1 f3 o r T NF-a enhan ced the e xpressio n of MHC class II Ag. !CAM-l. B7-2, and CD40 compared with those injected w ith 0.2% bovine serum a lbumin in PBS as contro l, w hil e no significant cha n ges w e re recogniza bl e o n th e ir expressio n ofB7-1. Intradermal inj ection of G M-CSF in two diffe re nt doses, e ithe r 50 or 250 n gl ear, cou ld n ot affect tl1e express io n on Langerhans ce ll s of a\l the molec ul es we studie d. Furthermore, we perfo rmed a kjnetic study in which we prepared epide rm al ce ll su spe n sio n s at th e va rio us time inte rval s (i.e., 1,4,8, 16, and 48 h) after G M-CSF, IL-1 f3 injection , or TNCB treatment (Fig 3). We found that th e m ost re markable phenotypic changes o n Langerhans ceUs w e re observ ed at 16 h afte r the trea tm ents and that the ph en otype o f Lange rhan s ce ll s after G M-CSF injec tion was not diffe rent from that after PBS injec tion at each t ime point. R ecently, Kampgen et nl l1 6] have re po rted that IL-l induces G M-CSF receptor expressio n o n Langerhans ceLI s. T herefo re, it is con ceiva bl e th at the si mple inj ec tion of GM-CSF cannot affect th e ph en otypes of Langerhans ce ll s because of insuf­fici ent numbers of expressed G M-CS F recepto rs o n fresh ly iso lated

VOL. 106 , NO.3 MARC H 1996

300r-~(.71~M~H~C~C~LA7S~S~I~I--'

3°OUillbI ICAM-'

200

100

o 1 2 3 .. 5

300~ (el B7-'

200

100

o 1 2 3 .. 5

• PBS

III GM-CSF • TNFa

~ Il-1P o TNCB PAINTING

300r-~(d~I=B7~_2~~-----'

200

100 ~ I I o I 2 3

300r-~(.-IC-D-4-0--------~

EXPERIMENT NUMBER

Figure 2, The intradermal injection of cytokines cannot upregu­late B7-1 on Langerhalls cells ill I';VO, Langerhall s cell s treated by the intradermal injection of G M-CSF, T N F-a, Il- l13, or by T N CB painting were examined for the expressio n of MHC class II Ag (a), ICAM-l (b), B7- 1 (c), B7-2 (d), and CD40 (e) after gating I-E-pos it ive cell s on FITC versus side scatter contour plots. M FI was determined for each surface molecule, and the effects of cytokines were eva luated by percentage MI' I as calculated using the fo llowing formula:

% MFl

__ (MFI of Langerhans cells treated w ith cytokine(s) o r TNCB) X 100'Y.,

MFI of Lange rh ans cells trented with 0.2% BSA/ PBS

Langerhalls cells. To exclude such a poss ibility, we injected at first IL-l,8, and at diffe rent time intervals (0, 4, 8, 16 h) thereafter we examined the effect of GM-CSF; however, these combined injec­tions did not alter the effects of injectin g lL-1,8 alone (data not shown).

The Upregulation ofB7-1 Expression on Cultured Langer­hans Cells Was Downregulated by Anti-GM-CSF Ab and Anti-TNF-a Ab From ill 11;'10 study, we could not specifY the cytokine(s) that mediate B7-1 upregulation . Thus, we examin ed the effects of Abs to several cytokin es on the upregul ation ofB7-1 , MHC class II Ag, and other accessory molecules on Langerhans cells cultured for 72 h (Fig 4). When we added 20 /-Lg/ml of anti-GM-CSF or anti-TNF-a Ab to the cul ture of epidermal cells, the expression ofB7-1 on Langerhans cells was inhibited , whereas the addition o f 20 /-Lg/ml of anti-IL-1,8 Ab could not suppress its expression a t all. In addition, only anti-G M-CSF Ab exerted a partial inhibito ,-y effect on the upregulation of B7-2 expressio n. Non e of the Abs to GM-CSF, TNF- a, or IL-l,8 changed the expression of MHC class II Ag, lCAM-l, o r CD40.

DISCUSSION

In tius study, we have demonstrated that Langerhans cells m atu­rated ill lIillo by hapten painting also upregulate the co-stimulato ry molecules as well as MHC class II Ag. In other words, Langerhans cells that mature ill v ivo seem to b e almost identical with those that mature ill lIill'O, although the latter express the co- stimulatory molecules much more than the former.

We did demonstrate that the phenotype of Langer hans cells after intradermal injection ofIL-1,B was diffe rent from that of Langer hans cells after hapten painting. Langerhans ce LIs o btained after intra­dermal injection of IL-l,8 did not upregulate B7-1, whereas ;11 11 ;110

LA NGER.HANS CELL MATUR.ATION IN "/VO AND IN VITRO 443

> 300 l-e;; Z W I-~ W U z W U (f) W CC o ;:, -' u.. z c:t: w :!:

200

-'-0-- B7-' (GM-CSF) -0- B7- ' (IL-' Pl ....£l- B7- ' TNCB

---0-- ICAM-' (GM-CSF)

-D- ICAM- ' (IL-' Pl -0- ICAM- ' (TNCB)

----IJ.--- MHC class \I (GM-CSF) ---b- MHC class \I (IL-' Pl ~ MHC class \I (TNCB)

~ 100 ¥--=~ __ .-__ ~ __ .-__ ~ __ -r __ ~ __ -. __ ~ __ ~ o 4 8 16 48

TIME AFTER TREATMENT (HOURS)

Figure 3_ The kineti c studies of the expression of MHC class II Ag and co-stimula tory molecules after injection of various cytokines_ Prc pnri n g cpidc nna.l cell slI spensio 11 were started at va riou s cinlcs (c.g .. ]. 4 , 8, 16, o r 48 h) afte r GM-CSF (---). l L-I13 (-) injecti o n. or TNCB (_) trcatnlCl1t. Treated Langerhans cells were cxanlincd for the expression of MH C class II Ag (6 ) . ICAM- 'I (0 ), and B7- 1 (0 ) after gating l-E-positive cell s o n PITC lIerslls side scatter co ntour plots. T he exprcssion ofB7-1 o n La ngerhans ce lls was not different fi'om that of 0.2% BSA/ PBS injection as a negative con tro l at all time po int aftcr GM-CSF injection. indicating that intradem,al injection of GM-CSF had not m ade La nge rh"ns cells left fro m the epidermis at 16 h aftcr injection. In .. ddition. the upregulatio n of M H C class II Ag and ICA M-l after IL- t 13 injection o r TNCB trea tmcnt had started at 4 h and beca rn e Illost marked '16 h after treatlllen ts respectively.

hapten treatm en t definite ly increased 137-1. This observed differ­en ce indicates that the si mpl e inj ec tion of IL-l (3 could not be a substitute for the m ed iato rs in vo lved in L:lJ1gerh ans cell maturation after h apten painting and th at other signals arc required for the full simulation of Langerhans cells. T herefore, we next examined whether TNF-a or GM- CSF, which were suggested to affect Langerhans cell fun ction ill 'Iil ro 1' 13, 18,32), a uld upreglllate the expressio n of B7-1.. Unexpectedly, we could not recogni ze the upregulatio n ofB7-1 expression either by the in tradermal injection of 50 o r 250 ng/ea r of GM-CSF, o r by th at of T NF-a.

O n the other hand , anti-GM-CSF Ab and , altho ugh less effi­ciently , anti-TNF-a Ab could suppress the llpregulation of137-1. on cultured Lan gerhans cells, sugges ting that G M-CSF an d T N F-a mediate the in crease in expressioll of 137-1 on Langerhans cell s ill

vitro . These ill "i llO and ill II il l'O data suggest that Langerhans ceUs require both IL-l,8 and GM-CSF for their fu ll maturation; the former is responsible for the llpregllla tio n of MHC class II Ag, ICAM-l, 137-2, and CD40, w hik the latter is fo r that of 137-1 .

When we assume, however, that GM- CSf upregllJate th e ex­press ion ofB7-1 , w hj le LL-l ,8 increase the e xpression of MHC class II Ag, (CAM-I , 137- 2, and CD40, at least two qu es tions ari se. The first is w hy we could not recognize the upregulatio n ofB7-1 afte r GM-CS,F il'jection. We can think of three possible explanations. T he first o ne is that the amount ofGM-CSF we inj ected is too small to induce the phe notypic changes of La nger hails ce lls. To excl ude this possibility, w e measured GM-CSF concen tration of the culture superna ta nt of epidermal cells , in w hich B7-1 upreguJation on Langerhans cells was induced . T he avera ge GM- CSF con centration in repeated experimen ts ranged fi-om 400 to 500 pg/ ml (data not shown), w hereas we injected 1 o r 5 /-Lg / ml, far exceeding amounts ofGM-CSF in OUl' experiments. T he second possible explanation is that GM-CSF actuall y cann ot reach o r bind th e receptors on epiderm al Lan gerhans cells. It is well known that g lycosaminogly­ca ns bind to GM-CSF [11 ,25]. The glycosa rnin oglycan that is abundantly presen t in the dermis as well as at the de rm o-epidermal junction may bind to GM-CSF to inhibit its further difli.lsioll to

4 4 4 OZAWA ET AI.

>-t:: f/)

z W t-~ 150

W 0 Z 100 W 0 f/) W 50 a: 0 :::I ...J LL Z 41: W :; ~

(b)ICAM· 1

• hamster 19

IJ anti-GM-CSF

• anti-TNF a

~ anli-IL-113

EXPERIMENT NUMBER

Figure 4. B7-1 e" pression on cultured Langer-hans cells is dOWJJ­regula ted b y anti-GM-CSF Ab and anti-TNF-a Ab. C ultured Lang­erhall s cells trea ted by anti-GM-CS F A b, anti-T N F-a Ab or 311 0-I L- 1 J3 Ab were examined fo r the expression of MH C class II Ag (11) , ICA M - l ( II ), and B7-·\ (c), B 7-2 (d) and C D 40 (r) after ga ting \-E posit ive c ells On FITC " C/"S IIS

side .,catter conto ur plo ts. MFI W<lS determined for each surface m o lecul e. and the e/fects of cytokines were cv,dllated by percentage MF I as calculated using the fo llowing fo rmula:

( MFI o fLallge rhallS cell s trc<lted wi th A b )

";., M PI = X 100% M FI of Lall gc rhallS cell s treated wieh m edium only

epidermal La ngerhans cells . T h e third p ossibili ty is that SOlUe facto r(s) may exist in the dermis whi ch inhibi ts the ;11 11;110 m atur;l­ti on ofLaligerhan s cells indu ced by G M-C SF injection. Streil e in ef

ai' re po rted th at the conve rsio n of fi'eshly isolated to elrl tured Langerhans cells ill vit ro fails to occur if normal mo u se serum is presen t in the culturc fluid . We also think that the presen ce o f such a f,lcto r m ay explain wh y intradermal injectio n of GM-CSF fails to induce the ph eno typi c changes .

R ecently, G rabbe ei a/ [1 2 J dc mon stra ted d efici e nt antigen­presen ting cirpacity of Langerhans cell s isolated fTo m athyn1ie (1/1/ 11/1/) mice. In addition, they fo und that prein cubatio n o f th ese Lange rhans cell s with G M-CSF reconstituted allo antigen-presel1t­ing fun ctio n. W e speculated th at if G M-CSF wa s resp o nsible fo r 137-1 upregul ati on on Langel'hans cells, 137-1 upregulatio n on Lan ger/w lls cells afccr T N CB pain ting should b e d ecreased in athymic (1111 / 111') mice . W e fo und that La n gerhans cell s fi'om athymi c ("II / II") mi ce showed signifi cantl y decreased upregularion o f 137-1 afte r TNC 13 painting when compare d wi th those fi 'om c uth ymic controls (/1,, /+ or + / + ), wh ere as th e upregulation of MHC class Il Ag, IC AM-I , ill1d C D40 exprcss ion in LangeThan s cell s wa s not significantly diffe rent amon g t hese anima ls (dara not shown) . T hese data al so suggest tha t the incr'cased ex press io n o f MH C class II Ag. l C AM-1 , and C D 40 on Langerha.n s cells afte r hapten paintin g is media ted b y a stimulation pathway diffe rent fro rn that required fo r the upregulat.ion o f1l 7-1 and tha t Langerhans cells fro m athymic mi ce are defec ti ve o nly in the la tte r pathway .

T he second questio n is w hy ant i-IL-1 (3 Ab co uld n o t s uppress th e upl'egula tion of MH C class II Ag, IC AM-l . D7-2, 0 )· C D 40 on cultured Langerhans cells. Witmer-Pack ('/ ((I [3 2] re ported tha t the additi on of l1--1 alo ne to the highly purified L1l1 gerhan s cell culture

I Strcilcin JW , X ic Y: A scrum ractor(s) that governs fl.lnc ti o nal propcr­ti c.~ of epidermal Lang crhans cell s. ) 1Il11es/ D erllla/o/ 104:567, 1995 (abstr.)

T H E J OU R NAL 0 1' IN VESTIGATIVE DErlMAT LOGY

co uld n o t enhan ce th eir an tigen-presenting func tio n . H euRer CI ll/

[1 3] de monstrated that none o f anti-cytoki ne Abs coul d suppress t h e llpreglllati on of MH C class II Ag tha t occurs on cultu red L ange rh ans cells. O m' presellt observation is consistent w ith these d ata. but it is \lo t proper to judge that lL-1f3 is no t: in volved in the upregula tion of MH C class II Ag, ICA M-I . 137-2, o r C D40 in c u.lttll'ed Langerhans cells only ba sed on these findings. Enk d II / [9] h a ve fo und that increased IL-l{3 mRNA signa l is acc lImuloted in Langerhans ce lls as earl y as '/ 5 min ;rfce r exposure to contact a lle rgens. T h erefore , it is speculolted that the production of lL- lf3 a lso starts in Langerh ans cells at an extrem ely earl y stage in culture . It took about 3 h fo r us to prepare Langerhans ce ll- enriched popula tio n and to sta rt their culture with anti-IL-l {3 Ab. T hus, "'~ c annot exclude the possibility that durin g this timc in terval. La ngerhans cells aI'e already stimula ted to increase MHC class II Ag, IC AM.-1 , 11 7- 2. and C D 40 express ion .

As fo r the I'egulation of D7- 2 express io ll, we found that anri­G M-CS F Ab slightly bu t clearly inhibi ted its augmentatio n in o ur ill Il itm culture study of Lan gerhans cells . larsen C( II/ [2()] recently d e m onstra ted that Langcrh ans cells frol11 cultures containing anti­G M-C SF expressed decreased level s of CTLA-4-co unter receptor a nd tha t GM-CS F added in th e culture ca n upl'egulate the expres­sion of both D7-1. and B7-2 0 \1 splenic dendriric ceJJ s. T hese data s ugges t tb at GM-CS F regula te bo th 137-1 and B7-2 expression of d endritic cells including Langerhans celJs.

O ur present experiments showed that the intradermal injection o f T NF-a increased the expression o fMH C class 11 Ag, rCA M-i . B7-2, and C D 40 . and that ;lnti-T N F-a Ab suppressed the upregu­la tio n of B7-1 on c u.l tured Langerhans cells. R ecently, Sallusto el II/ [26J have also dem o nstrated that T NF-a increased the ex press ion o f MHC class IT Ag, rC AM-l, and B7-l o n human dendritic cells that w ere obtained by the cu lture o f periphera l blood t1l ononu clear cells w ith GM-C SF and lL-4 . T NF-a also playa rol e in L111gerhans ce ll maturatio n w hich is distinc t from that of IL-l f3 and G M-CSF.

Human Langerhans cells, which a re po tent an tigen-prcsenri11g cells in the skin. have been shown to express C D40 [23 ]. It has nor yet been rep orted , however , w h ether murine Lan gerhans cells express C D40 o r w hether they upregul ate it wh en they becorne n1ature ill "i"" or ill IIi/l'o. In this ex pe riment, we de m o nstrated rhar nlurin e tj'eshl y isola ted Langerh ans cells al so ex press C D 40 mole­c l1l e and they upregulate C D40 during [II "ilro culture o r after h a pten painting . Th e express ion of C D 40 on Langerhans cells may play a c rucia l role in the ir antigen presentation.

Finally, becau se we did not exam ine th e produ ctio n o f other c)' tokines after the injec tion of IL-l (3 , T NF- a, or G M-CSF, we could n o t comple tely exclude th e p ossibiljty th,lt the cytokines induced by IL-l {3 injection m ;lY aflect the pl1 enotypic changcs on La ngerhan s cells ill IIi llo .

T bis sllIdy /lias slIp/", rled by Crall/s·il/ -A id ji". Scierrli/ir n Nl'flld, 06 -15 -1JIJ "ml 0 7670929 Ji·o", (he M;lIisII1' ~r Ed"f", ;"" ,)"!,"", " li d ('Y 1(, 1' Nn /w llI/"i FII/ II ,dll(ioll.

R E FER EN CES

I . Aiha S. Katz Sl : Phc ll or),pil.: :'Illd fun cli onal ch ar.H:tcristics of in vivn-acti";Hl!d

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protein Hill-igcn s is MHC-ocpcJldc ll t. J //11/111/1101 I -4 6:247<}-2487. 199 f 3. . Aiha S . M"~lt ko T . Hl.1sok" Wll M. Hashimoto Y: Monocloual :l llrihodks ro

various m orpho logic skin compo ne ll ts of hllllla ll skin. J (" II('S' Dt'nulIr,,1 g 1 :498-502. 1983

4 . Aiba S. N;tk;1g;1 wn S. Ozawil H . Miynkc K. V<1git;t H . T<1gam; H : Up-rc.gul-:ltion o f cr4 integ-rin 0 11 ac ti vated L:lIlgcrllil lls cells: i111 :l lys is o( ad Jl c .~io l1 Itlo lccuics on LlIIgcrilans cells relatin g 10 their m igratio ll fro lll skin to dra ill il1 g: IYlllph !lodes.

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histncol1lp il tibl1i ty (om p lcx dilSS I .md (( mo lecule c...-xprcssio lt 0 11 cpidenllal Lallgcrh.lIls cells by sonle of the cytokillCS rch.!4\sc.d by kcr;,tillocyrcs and T cells. /i"r) /"''''''/1 0/ 24:2889-2895. 1994

(, . C lwlI g: C H . FuniC M. Ta lllaki 1(: B7- t cxprcssio ll o f L 1l1 gcdl:iIIS ("'cJb is lip- regulated by p ro infl:IlTI I11;lto ry cytokil1 cs. alld is down-rcg-tlJ ~l l'cd hy imer­

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