Cutaneous Acute Graft-Versus-Host Disease to Minor Histocompatibility Antigens in a Murine Model: Histologic Analysis and Correlation to Clinical Disease
Cutaneous Acute Graft-Versus-Host Disease to Minor Histocompatibility Antigens in a Murine Model: Histologic Analysis and Correlation to Clinical Disease
James Ferrara, M.D., Francisco]. Guillen, M.D.,* Barry Slcckman, B.S., Stcvcn J. Burakoff, M.D., and George F. Murphy, M.D. Departments ol Pediatrics and Pathology, 1-brvard Medical School. Divisions of Ped iatric Oncology. Dan ,I-Farbcr Ca ncer Institute, and Dermatopathology. Brigham and Women's Hospital. Boston, Massac hu se tts, U.S. A.
Graft-vers us-h ost disease (GVHD) can occur in bone m ar vacuolization, cxocytosis, and sa tellitosis of mononuclea r row-transplant recipients even when donor and host arc cell s in the epide rmis. Dyskeratosis was observed o nl y in identically match ed at the m aj o r his tocompatibility com animals receiving T cell s, and proved to be rhe mos t reli:-rblc plex. GVHD in this context prest~m ab l y arises because of histologic pa ra m eter of disease w ith the number of dys differences in mmor histocompatibiltty antigens. Munnc keratotic cell s per linea r millimeter of e pidermis correlating GVHD to minor his tocompatibility antigens has been stud both with severity of clinical disease and w ith the number ied in an effort to determine whether skin is a target of the of transplanted T cell s. Ultras truc tural examination re immune response in this model system . T cell-depleted vealed exocytosis of mononuclear cell s into the epidermis 7 marrow cell s (10 ) from BlO.BR (H-2k) mice were sup where they were frequently apposed to degen erating an d plemented with varying numbe rs of nylo n wool-enriched n ecrotic keratinocytes. These data indicate that the skin is sple nic B10.BR T cells and transplanted mtraven o usly mto an info rmative target o rgan for study of experimental GVHD irradiated (11 00 R) CBA (H-2k) mice. Sequential biopsies to minor histocompatibility antigen s. J fnflcsr Ocnna/(J/ of ear skin were obtained at weekly intervals over a 7-week 86:371-375, '1986 period. Histopathologic evaluation revealed basal cell layer
raft-versus-host disease (GVHD) is a commonly en marrow inoculum [I]. Althoug h transplant recipiems develo p countered, frequently devasta ting complication of pronounced signs ofGVJ-1]), in cl ud in g copi ous diarrhea and weight bone marrow transplantation (BMT). GVHD is loss, these findin gs arc not entirely specifi c to GVHD and ca n thought to result fr om antigenic differences between occur in immunocompromised and infected hosrs. Furthermo re. host and donor cell s, although the precise nature of cutaneo us alterations ha ve not been documented previously in Gthe effector cells and their molecular targets is unknown. The rhis model despite the fact thar it has been srudi L· d in g reat derail majority of human BMT occurs between donors and recipients [1]. This absence of skin disease not onl y Ius raised questions who are identical at the m aJOr histocompatibdity complex (MHC), about the clinica l applica bility of this model, but ha s led ro rhe yet significa nt numbers of these patients develop GVHD. GVHD impression that GV HD of the skin OIIi y rarely occurs in th e mou c in this context is presumed to result fr om differences 111 mmor [2 J, lin1iring the usefulness of murine mo,icls fo r studies of rh e histocompatibility antigens between donor and host. pathogenesis of sk in GV HD ro mino r hisrocomparibiliry ami We have studied a murine model of GV HD to minor histo gens. T hi s study presents hi stologic evid ence of alterations in th e compatibility antigen differences that is clinica ll y relevant to hu skin of animals w irh clinica l signs of GV HD th at bea r a striking man disease. T he strain combin ations employed (BlO.BR _,. CBA) sim ibrity to the lesions described in human disease. We conclu de have been shown to produce lethal GVHD with clinical severity that in this model, as in other murim: models, acute skin G VHD proportio nal to the number ofT lymphocytes added to the donor directed against mino r hi stocompatibility antigens is present and is li kely to be demonstrable in the majority of model systems despite minimal macroscopic evid ence of disease. Manuscript received July Hi, 1985; accepted for publication November 7, 1985. MATEIUALS AND METHODS Supported by a 13iomcdical Research Support Grant, National In stitutes of Hea lth, a gift lrolll the Dyson Foundation, and by United States Public Marrow Harvesting and Treatment with Monoclonal An Health Service gratH 1 R01 CA40358-0 1 awarded by the National Cancer tibody and Complement B 10. B R (H-2') mi ce were sacri ficed Institute, Department ol Hea lth and Human Services. by cervical dislocation. Fe murs and 1ibias were removed sur *V isiting Fu lbright Scholar, Department of Pathology, University of gica ll y and Ru shed with Eagle's minimal essential medium ro Ali ca nte, Alicante, Spai n. which had been added 3'Yo prcscrcencd feta l ca lf serum , 2 mM Reprint req uests to: James Ferrara, M.D. , Division of Pedi atric On 1..-g lut ::~ min c, penicillin (100 J.Lg/ml). and streptom ycin (10() J.Lg/ml). cology. Dana-Fa rber Ca nce r ln stitut<', Boston, Ma ssachu se tts 02 11 5. A suspension of 50 X 10" harvested bo ne marrow ce ll s was in Abbreviations: BMT: bone marrow transplantation cubated for 40 min at 4°C w irh I ml ofanri-Thy \.2 mo noclo nal GVHD: graft-vers us-host disease antibody (New England Nuclear, Boston, Massachuse tts) ar a MHC: major histocompatibility complex dilution ofJO- -' in LIS medium (M .A. 13ioproducts, Walkersville. MST: median survival time Maryland). The cel ls were then pelktcd. rhc supcrn.1ra nt dis-
0022-202X/86/S03.50 Copyright © 1986 by The Society for In vestiga ti ve IJ L' nnarology. Inc.
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environment, effector cell s were harvested, washed, counted, and assayed fo r specific cytolytic activity in a 5 1Cr release assay. This assay was performed as described previously [2] w ith some m od 1 ifi ca tions. BrieRy, 5 X 10 ' target cell s (P815:H-2d) were labeled with 100 J.L C i Nal 1Cr04 (N ew Eng land Nuclear) fo r 90 min at 37°C in complete culture medium. Labeled target cell s were washed 80 3 times and 104 cell s were mixed w ith varying numbers of effector ce ll s in 0.2 ml of complete medium in 96-well V-bottomed plates (Linbro). Supernatant was harvested to determine radioactivity released. The percentage of chro mium released was calculated as Untreated Marrow percent specifi c release: 5 1Cr released by immune cell s - 5 1Cr released by no rmal cells/maximum 5 1C r released (with 1% de oxycholate) - 5 1C r released by normal cell s. Tissue and Histopathologic Examination Serial biopsies o f car skin were obtained at the time of transplantation, sequentially every 4-8 days (average every 7 days) for 7 weeks, and at the time of dea th. Biopsies were fu ll thickness, measured 8 X 10 Treated Marrow+0 . 1~T Cella 20 mm, and were performed in alternatin g sides. Care was taken to sa mple skin not adjacent to previous hea ling surgica l wounds. T iss ue was immediately immersed in 10% neutral buffered for Treated Marrow mali n, fix ed for at least 24 h, and processed for routine paraffm histology. Sections 6 J.L m thick and cut 40 J.LI11 apart were evaluated 6 24 96 for the fol lowin g parameters: maturation disa rray, basal cell layer vacuoli za tion, number of dyskeratotic epidermal cells, number E:T Ratio of lymphocytes in dermis and epidermis, satellitosis of lympho Figure 1. Eliminati on ofT cell s from dono r marrow by anti-Thy 1. 2 cy tes about dys keratotic cell s, li chen planus-like changes, and and co mplement trea tment. Untre;Jtcd (• ) and trea ted (e ) C57BL/6 sclcrodcrmoid changes. Dyskeratotic cell s were enumerated per (H-2h) marrow were placed in a 5-da y mi xed lymphocyte cultu re with linear mil li meter of epidermis with the aid of an ocular g rid mi 151)() H irradiated 13ALB/c (H-2d) stimulator splcnocytcs. N ylon wool cro meter. Total epidermal length evaluated per biopsy specimen pa ss a!-;ecl ll(> T ce ll s (S X IO') (0. 1'}\, of to ta l culture) were
0. 1 M sodium cacodylate buffer (pH 7.4) fo r 2 h. Postfi xati on that the severity of these signs reAects the tempo of posttranspl ant was achieved with 2% osmium tetroxide for 2 h; aft er 2 15-min mo rtality. rinses with 0. I M sodium cacod ylate buffer, the ti ssue was de Histologic Graft-Versus-Host Disease Ea r skin appea red h y drated in g raded ethanol solutions and embedded in an E pon sli ghtl y ro ughened in animals with cl inical G VHD. This w as no t Araldite mixture. One micro m eter-thick secti ons were cut on a a constant fmding, however, and did not reliably correlate w ith Porter Blum MT-2 ultramicrotom e and stain ed with Gi emsa re othn clini ca l signs of disease. Histologic an alysis of sequential agent for li ght microscopy. Ultrathin secti ons subsequmtly cut ca r bi o psics revealed focal progressing to diffus e basa l cell layer for electro n microscopy w ere stained w ith uranyl acetate and hyperplasia (in crease of the basa l cell la yer to m ore than one ce ll lead citrate and examined with a JEO L J EM 100S electron mi in thickness). progressive basa l cell vacuo li za tion, epidermal and cr oscope. folli cular dys keratosis (cellular degenerati on and death defin ed by nuclea r pyknosis o r fragmentati on and cytoplasmic hypcrcosi RES ULTS nophilia). epi dermal maturation disa rray [disorderl y change in Effectiveness of Marrow Treatment M arrow depleti on ofT axis fro m vertica l (bas al cell ) to hori zontal (s tratum corneum cell s was tested in a 5 -d ::~y C TL 51 C r release assay. As shown in cell )]. and scant but increasing infiltrates of superficial dermal Fig 1, untreated marrow exhibited a vigorous all ogeneic res ponse mononuclear cell s during the first 7 weeks of the di sease (F igs 3. but treated m arrow show ed no killing above ba ckg round. even 4). T hese changes were m ost striking during the second to thit·d w h en cultured in the presence of conc::1na valin A supernatant. weeks post-BMT and in animals that had received 1 X I 07 spleni c Additio n of 0.1 'Yo T cell s to the treated m arrow at the initi ::l ti on T cell s in the m arrow in oculum (Fi g 4). In some bio psies, pe of the 5-day culture produced small but specifi c lys is at hi gh ripheral ;Jggrega ti on of intrae pidermal m ononuclea r ce ll s about effector to target rati os. Thus antibod y and complement trea t dyskerato ti c keratinocytes (satelli tosis) was obscrvcd . m e nt o f the m arrow was able to eliminate all cytotoxic T lym T he mos t easil y quantifiable hi stopatho logic alteratio n was ep p h ocytes and their precursors at le as t to the 0. I 'Yo level. iderm al and folli cul ar dyskeratosis. The number of dys keratoti c ce ll s per linear millimeter of epid ermis correla ted w ith the pres Clinical Graft-Versus-Host Disease Fi g 2A shows the sur ence of clini ca l CVHD in all animals studied (Fig 5). T he number v ival curves fo r transpl ant recipients. T he group receivin g a T of dyskeratotic ce ll s peaked during the second to thi rd weeks cell-depl eted m arrow in oculum supplemented by I X T ce ll s l(f post-13M T in animals w ith clinica l G VHD. Control animals re h a d a m edian surviv ::1 l time (MST ) of 20 days, w hereas the g roup ceivin g marrow but no T ce ll s occasionall y showed small numbers receivi ng 1 X 10'' T cell s had a M ST of g re::1 ter than 70 da ys . of similar ce ll s d uring the fi rsr to second week post-BMT, pos T h e g ro up reccivin g T cell-depleted marrow alonc had IOU % sibly related to irradiati on. surv iva l at 100 days . Animals in each group were weighed twice weekl y fo r the first m onth posttransplant and then wcekl y (F ig Electron Microscopy Sequential analys is reveale d gradual ac 28). C linica l m anifes tatio ns of acute G V 1-1 I) in clu ded in acti vit y. cumulati on o f mononucle ar ce ll s in the dermis during the fi rst 2 r u fA ed fur, hunched posture, ri nd copio us di arrhea; these signs weeks post- 13M T. Focall y, these ce ll s fo rmed small aggregates correlated with w ei g ht loss. A comparison o f Fi g 2A and B shows wi thin the epidermis and hair fo lli cles w here they were frequcmly
Figure 3. Histopatho logy of cuta neous acute G VHD. Seria l ca r biopsies o f C JJ A mice transplanted with I O'' T ce ll-depicted n llJ.I1 R 1 m arrow ce ll s and 10 ' B'IO .I11 ~ T cell s. I f = Da y 0 (m agni fica ti on = x 40) tf/0, d18, d25, tf29 = da ys I 0, 18. 25, ;md 29 postt ra nsplant. res pecti vely ( X 100). 374 FE IUlARA ET AL T il E JOU ilNAL OF I NVESTil;ATIVE D EHMATO L OGY
r '· . ~
Figure 4. H is t op~t h o l ogy o l· 1- J.Lm sections (l(f T cells; d ~y 14) . Nu merous mononu clea r ce ll s, some in appositi on tO dys keratotic basa l ke ratin ocytcs. arc present in th e lower hall of th e ep id er mis ( X 250).
apposed to d egene rating and necro ti c basa l kcratinocytes (Fig 6). Even tually this p rocess became w idespn :ad, resulting in d isrup ti o n ofinterccllularjuncti o ns and correlating w ith the appea rance of basa l cell layer vacuo li zatio n b y li g ht microscop y. T he infiltrating m o no nuclear cell s wen: of 2 types. T he f1rst was a larger histi ocyte-like cL· ll (20-40 J.Lnl in diam eter). that contained a ro und to ovoid nucleus and p llJgocytoscd m aterial associated w ith cyto pl asmic lysosom es. T his m aterial was o ft en Figure 6. T ransmiss ion ele ctron microscopy. A portio n of a necrotic knatinocytc (*) is associated with ~ nl ononuclc:lr cells ((/() within the composed of aggregates of tonofi lamcnts derived fr o m necro ti c epidermis (E). /) = dermis: lwr = 1.0 J.Lill. kcratinocytcs. The second was a smalkr ly mphocyte-li ke cell (10 -20 J.Lm ) con taining an info ld ed nucleus and scant com plc mcm s of cyto pl asmic o rganelles. !3oth cell s were o bserved in the dermis and epidermis, a ltho ug h degene ratio n and necrosis o f ad microscopic alte rati o n of anHe G VHD in the s kin ha ve been jacem cell s was observed only in the epiLkrmis and hair fol li cles. previously d ocumented in this clinicall y rclcvallt and well-ch ar Langcrhans cell s wne 11ot obsnved in biopsiL· s studied by elccrro n acteri zed murin e m odel altho ug h skin C VHD was hte r described 111 icroscopy. in simibr syste llls 13-S J. Macroscopic skin changes (includin <> DISCUSSION alopecia) were obser ved in different m odels of GV HD w m i n o~ histocompatibility antigens b y both Ibppaporr et all 3 1 and H am This study dcK liJn <.: Jll S the occurrence of hi sto logic a lterati o ns il tO n et al 141. C harl ey ct :II 16 1 and H a milto n and Pa rkma n J71 chara cteri sti c of JClltc cutaneous G VHD in a murine m odel of ha ve each published corrcla ti o ns of m acroscopic an d microscopic C VHJ) to mino r histocolllpatibili ry antigens. Neithn g ross no r analysis of ski n G VHD in H-2 identical marrow-transp l::uH re cipients. No alo pecia was eviden t durin g clinical G VHD in the p resent m o del an d macroscopic sk in changes werL· minima l, yet histologic am lysis o f sequential car biopsies shows not o nl y that ca r skin is a reli able and accessible source of ti ssue. but that the 4 .0 chan ges o facllr L' skin G VHD arc indeed in evidence in this m odel system. Thus skin GV HD to mino r histocompatibility antigen s e3.o is probably universal in murine syste m s w hen looked for vv ith appropri ate histo logic techniques. { The specifi cit y of cutaneous histop:tth o logic alterations in acute Ill 2 .0 CVHI) has been ques ti o ned JSI. We have o bsn ved histologic =Q) chan ges simibr ro mild acute G VHD in human and murine skin u 1.0 presumably as a result ofprctr:m splant conditio ning regim e n s [9]. In this m ot~ el , however, the cxtcnr of epidnmal d a mage (num ber of d ys keratotic cell s) was an impo rtant discriminating tcaturc between cutaneous changes associated w ith clinical GV I:-ID and those o bserved in contro l an imals. T he f1nding of large number Days Post Transplant of d yske ratoti c epiderma l cell s correlated w ith the presen ce of m o n o nuclear cell infil trates in rhe supe rfi cial dermis and epidermis Figure 5. D ys kcraroric cells as :1 11 index of cutaneous :~ c ute CVI-ID. (Fi gs 3, 4), and dyskeratosis has recen tly been shown to correlate Lcilla ll y ir ra diated ( 11 ()1 1 I ~) C UA mi ce (n = 3 pe r group) we re tr :~ n s pla iJ te d with T cell-depleted H I (1.13H with th e addition of noT cel ls (0T ), w ith severity of di sease in an in vitro p redictive test for GV H D Ill'' T cell s ( Ill'' T). :1nd 107 T cell s ( 11 17 T ). Se rial car bi opsies we re to mino r histocompatibility antig ens 11 0 1. C cllubr necrosis h a examined by light Jlli croscopy and th e nul\lber of dyskeratotic ce ll s per also proved to be a sig ni fica n t index o f inunune-mediated ti ssue lin c:1r millimeter o f epi derm is (cc ll ~,/ nllll ) wnc counted. Total len gth of rL:jec ti o n i11 o rgans o the r than skin. such as in the setting o f cardiac epidermis exa mined was 4(1 ± 10 mm /binpsy. transplantatio n 1111 . VOL. sr,. NO. 4 AI' IUL 1')8(, ACUTE C l iVI) AND IIISTOCOMI'ATII.l iLI TY ANTIGENS 375
U ltrastructurall y, we observed frequent apposition of mono 3. lbppapo rt H , Klulil A, 1-l:!lle-l'annenko 0 , Pritchard L, Dantchev nuclear cell s to degeneratin g o r d yin g epidermal and fo lli cular D , Mathe G: 1-li sto pat.h o logic sequence o f evcnrs in adu lt mice k e ratinocytes. This resembled "satellite cel l d ys keratosis," a phe undergoin g lethal g raft-versus-host reaction developed across nomenon well documented in experimental acute GV I-:ID 11 2,1 31. H-2 and/o r non- H-2 histocompatibility barriers. Am J Pathol Keratinocytes sho wing similar alterations were not observed in %: 12 1- 142, 1979 d e pendent of appostion to n1 ononuclca r cell s, suggesting that 4. H amilton BL, Bevan MJ, Parkman R: Anti-r<: ci pient cytotoxic T direct contact between these ce ll s (as opposed to re lease of a lymphocyte p recurso rs arc present in the spl eens o f mic<: w ith acute g raft versus host d isease due to mino r hi stocompatibility soluble Jy mphocytotoxin ) m ay m ediate target cell kil lin g. We did ;111ti gens. J lmmuno l 12fi:621-625, 198 1 not observe parti cipation of Lan gcrhans ce ll s in these cellular 5. C lam an HN, Jaffee BD, 1-lu ffJ C: Sck roderma-like changes in mouse interactio ns, a find ing consistent w ith previous studies in our chronic g raft-vs-host disease (absrr). J Invest Dermatol 80:32 , laboratory o f human GVHD 191. 19R3 This stud y dem onstrates that sequential bi opsy of murine car 6. C harl ey M il, .l:l o ugert .JL, Hamilto n 13L, Gi lliam J N , Somhcimcr skin is an effective wa y of m oni torin g the histologic evolution of H I): Murine g raft-versus-host sk in disease: a chro no logie and acute GVHD to mino r histocompatibility antigens w here no m ac qu:111titativc analys is of two histologic patterns. J In vest Dermato l roscopic disea se is rc3_di ly id enti fiab le. T hi s murine system all ows Rl :4 12-417, 1983 for the producn on o t acute G VHD With system1 c and cutaneous 7 . H amil ton BL, Parkman R: Acute and chro nic graft-ve rsus-host dis manifestations th at arc proportional to the number of dono r splenic ca se induct•d by mino r histocompatibil ity a n ~i ge ns in mice. T rans T cel ls added to the T ccll-dcplctcd m arrow in oculum . Mo rtality plam ati o n 36: 150-155, 19H3 and weig ht loss arc not specifi c fo r GV HD and the findin g o f 8. Sale GE, Lerner KG, Barker EA, Shulman HM , T ho mas E D: T he histopatho logic parametc_rs ? f di sease activity in the skin enhances skin bi o psy in the d ia gnosis of acute g raft-v<: rsus-host disease in the experimenta l utlhty ot th1 s and other murme models of G VHD. m an. AmJ l'atho l S9:()2 f-636, 1')77 Dctermjnati o n o f the effects of depletion of subsets of splenic T 9. Murph y GF. M cro t Y, T o ng AFK, Smith !3. Mihm M Jr: Deple cells added to the dono r in oculum and of immunotherapeutic ti o n and repopulation o f <:pid ermal dendritic c<: ll s after a ll ogeneic s trategies to abrogate acute GV HD to minor hi stocompatibility bone marrow tr:m sp lantati o n in humans. J In vest Dermatol antigens is now possible usin g skin as an a ccessible target organ_ 84:2 10-214, 1985 of study. Final ly, because th1 s m odel nl ll ll l CS most settings o t 10. Vog<: lsang G V. H ess AD, lk rkm:Jn AW, T utschka I'J , Farmn E R. human .BMT (MHC - matched dono r and recipi ent), it provides Converse PJ , Sa ntos FW: An i11 uirw pr<:d icti vc test fo r graft versus host discas<: in patients with geno typic l-ILA-identica l ho ne m ar a n experimental setting_ relevan t to the pathophys io logy and Jm row tra nspbnts. N Eng! J Med 3 13:6-1 5-(,5 1, 1985 m uno hisropatho logy o t cutaneous G VH D 111 humans. II . llilling ham ME: D iagnosis o f cardiac r<:jccti o n by endo m yocard ial bi o psy. Hea rt T ransplant I :25-30, 1982 R EFERENCES 12. WoodrutrjM. Hansen .J S. Good RS, Santos GW. Sla vin HE: T he 1. Ko rngold R, Spre 11 tj: LetllJ! CVHJ ) across mino r hi stocompatibility pa tho logy of the g raft-wrsus- host r<:actio n (G VHI1) in adu lts re barrie rs. N ature of the dt<.·ctor cc:JJ · and ro le o t" 1-1-2 complex. ceiving bo n<: marrow transpbnrs. Transplant Proc S:675-684, 197() Jmmuno l J ~cv 7 1:5-2'), 1983 13. WoodruffJM. l3utcher W I, 1-l cll cnstcin LF: Ea rl y secondary disease 2. Burakoft" SJ, Germain HN, Dorf M E. IJenacLTra f 13 : Inh ibition o f in the rhesus mo nkey. II . E lectro n microscopy of changes in mu cell-m<:dia tcd cyto lysis o f trinitrophen yl-dcrivcd ta rget cell s b y cous mem bran<:s and external epithelia as demonstrated in the H-2 complex. Proc Nat! Acad Sci U S A 73:625-(>29. 1')7(, tongue and lip. Lab In vest 27:85-98, 1972