Delayed-Amelanotic (DAM or Smyth) Chicken: Dysfunction In Vivo and In Vitro*

Raymond E. Boissy, Ph.D., G isela M oellmann, Ph.D., Audrey T. Trainer, B.S., J. Robert Smyth, Jr. , Ph.D., and A aron B. Lerner, M.D. , Ph.D. Depart ment of Dernntology. Yak University Sc hool of M edi cine, New Hoven. Connecti cut; ond Department of Vctcrin ory and Aninul Science. University of Massachusetts UHS), Amherst, Massachuse tts, U.S.A.

C hickens of the autoimmune delaycd-amclanotic (DAM of dopa-oxidase and acid-phosphatase activities in eDAM o r Smyth) lin e develop postnata l feather amelanosis and feather m elanocytes suggest that melanogenesis and au­ severe visual defects, both of w hi ch arc pres umed to be tophagocytosis of melanosom es occur in tandem and that due to a dysfunction of mclanocytes and :1 subsequent au­ the rates of both arc hi gher in these cells than in m elano­ toimmune res ponse that eliminJtes pig ment cells. In this cytes of no rmall y pigmented control chi ckens. Assays for report we elucidate further th e mclanocytic defect. We tyrosin ase activity in feather follicles indicate a hyperme­ present a m o rpho logic analys is of the mildly affected erratic lanization in eDA M feathers and in the pigmented fea thers (eDAM) g roup of Sm yth chi cken w hose pa rtial depig­ of young Smyth chicks prior to the onset of depigmen­ mentation and lack of visua l impairment resemble human tation. Finall y, we report on the establishment of pure, vitili go m o re so than do th e complete ;11 n ebnosis and blind­ proli ferative cultures of neural crest-derived m elanocytes ness in the classicJI Smyth line. Histologica ll y, the se­ from contro l and Smyth chicken embryos. T he degener­ q u ential events lead in g to amelanosis in the young Sm yth ati ve events in Sm yth chicken melanocyte cultures mimic chicken occur simultaneously in the feJthers of adult eDAM in part those of the cell s in vivo and are therefore indica tive Smyth chickens, and the infiltrati on o f th e feather pulp of a geneti c defect thJt is independent of the immune sys­ with mononuclea r leukocytes correl ates w ith the extent of tem. J lin; est Dcnnatol 86:1 49-156, 1986 local pigmentary abnormali ty. Cytochemical locali zations

ost of the m elanin in mamm;1ls and birds is lo­ the keratinizing epithelium and a rc shed with the feathers. At the ca ted in the integument and eyes, w here it a b­ o nset of feather regeneration after a m o lt o r afi:er plucking, new sorbs UV and visible radiation. Pi g mcntatio n of melanocytcs appear w hich arc presumed to be recruited fro m a avian fe

0022-202X/86/$03.50 Copy ri ght © 1986 by T he Society for In vestigative Dermatology. In c.

149 150 IIO ISSY ET AI. T il E .JUU I ~NA I OF IN VESTICATIVE IJEHM ATOLOGY

a. ..J :::> 300 . .. a. I ~ (/) 2 ~ 0 w 1- >- u 0 200 :::> u"'l ...J a: z 0z 10 0 0 :::; u. 0 5 0 t 0: w o ·-·- '- __, - _j_ 0 20 40 60 BO 100 PERCENT AFFECTED BARBS Figure 1. Photomicrograph of 2-!.1.111 cross-sectional segment of regen­ crating f(:.trher front ~ n adult d )AM bird. The barb ridge·s display scvcr.tl Figure 2. Corrcbtion o f M N L in fi ltration with the pcrcemagc of ab­ mdanocytic stages of degeneration: ( I ) normal, dendritic mc bnocytc; ( 2) nornJall y pigmented barb ridge·s pn cross-scctillnal area of rq.o;l'nera ti ng aekndritic clump; and (3) absence of mclanocytes. Ill~ = ln rh ridge. feather (n = 23) t.Jk cn li-om 5 adult eDAMs. (lntiltr:Jti o n is expressed a I' = pulp. 1!.11· = 22 fLIIl. the number of brgc MNLs residing in a 0.4-111 JTJ2 a rea immediatel y sur­ ro undin l:\ the ccntr:tl axial artery o f the pulp shaft.) The corrdarion coef­ fi cient is 0.72 and positive at rhe 0.1% le vel. If the 2 va lu es at the bottom right arc ignored, this coefficient increases ru O.i!9. suggcstinr-; chat leu­ the t\:~ th e r pulp. T he basic melanocyte dd\:n. however, is in­ dependent or the immune syste m :ts evidenced by the behavior kocytic infiltration and pigment loss arc correlated cvc 1 ~ ;norc positively. The linear rcgre·ss io n line shown rcpn.:sents the lartn conditio n . · of isolated embryoni c ce ll s in v itro. MATEI\IALS AND METHODS Birds Adult Smyth birds used lo r the morpho logic and cyto­ and then processed fo r electron microscopy. For the localization chemica l studies were o f the erratic DAM (eDA M) g ro up JIIJ . oracici ph osphataSL' :tctivity, split feathe rs were fixed w ith quarter­ These birds were partiall y ann:Lmotic and h:td no demo nstrable strength Karnovsky's fixative (room temperature, 4 h , pH 7.2), visual impairment. The embryos used lo r cell culture, and the rinsed in buHi.:r, and cross-sectioned on a freezin g microtome chi cks used for the tyrosinase assays, were fro m a randoml y bred (40 f.Llll ). ThL· sL·ct io ns were collected in cold 50 mM acetate buffer 1 Smyth population JIIJ . Control chickens were or the Brown (c ') (pl-1 5.0) and incubated in Gomori/rhl o ride m edium (2 h , 35°C) 1 JI 6 J and Light Brown Leghorn (c ) Jl ojlines. Each control line c onsistin~ or" Gomori's medium JI 7 J as nwdified by de j o n g et is homozygous for the respective all ele at the major plumage colo r aiJ IHJ, with {3-g lycerophosph:tte (sodium salt) as ~ ub st rate. Con­ 1 locus. The Smyth line was develo ped from the c ' line in w hi ch trol sectio ns w ere in cubated w itho ut substrate. All sections were anH:lanosis occurs sporadicall y. T he c 1 line is not prone to de­ rinsed in buffer and processed for electron microscopy. velopin g amelanosis. Tyrosinase Assay Tyrosin:t sc activity in extracts o f regener­ Morphologic and Cytochen'lical Techniques l{ q!;enerating ating fi.::tthers was m easured in duplica te by the method of Pom­ fe athers were plucked alter 2 weeks or g rowth, s plit lo ng itudi­ cr:llltzj i'J j as m odifi ed by 1-lal:tban et alj 21> /. From each bird (set' nall y, ami processed for li ght or electro n microscopy as published T able I) on alternate breast tracts, 12 feathers were plucked each in detail elsewhere JI 2 J. week to induce l'i.:at lt er rq.;e ne ration . Beginning at day of h:u ch, ltcgencratinp; feathers were ~ l so prep:tred rDr ultrastructural this procedure provicJec\ :\ Week ly supply or 2-wcek-oJd rerTett­ loca Ii zati o n or dopa-oX idase (tyrosinase) and :tcid-phosph:nase ac­ crati n ~ feathers. beginning at 2 weeks of age. T he testing ~vas ti vities. Aldehyde-fi :-.ed split leathers were in c u b~te d at 37°C, for discontinued at II Wl'eks, o r ea rli er if a m elanosis developed. For J It , w ith O. I'X, dopa in sodium cacod yl:ttL' butTer (0.2 M , pl-1 7.2) each weekly assay, rhe 12 relevant t(::nhers were plu cked and tht•

Figure 3. Ekctmn micrographs of ab­ no nHal Jlld :uJOSO ill l.: S . ''· Section of a llJL"­ I:Jn ocyte front a pigmcmcd rL·g~ n cr:lling li:.nhc r of" an adu lt e n AM bird. This cell 0)11t :ti 11 S llLlll Y .lhllo rrn :l l llH.' I.l!lU SO ill ~ S with pi g mented ~x t c n s i o n s (11 1'1'<'11' .<) , small pi g nH'nln.l ves icles (t1 1Tcl/ JJ /u ·n ds), ;tnd ek·c­ tn>n-lu ccnt ch:nnhcrs (a ~ frri ~ ks); also pre­ llll'l ,Jnusn lncs (ope''' tl tTOII'). ~nd ~1 vicll-dl'­ vdopnl C:nl g i app

contai ning 1% N onidet P-40 (NP-40; Sigma C hemica l Co., Ar­ lin gton Heights, Illinois). Tissucs fro m each bird were pooled and soni ca ted, and the lysates were divided into 3 equal aliquots and stored frozen ( - 20°C) until assayed. Cell Culture Pure, proli fe rative cultures of from neural crests of Sm yth and control chi cken embryos were estab­ li shed and processed fo r elcctron microscopy by methods pub­ lished in detail elsewhere 121).

RESULTS Histology of Regenerating eDAM Feathers Fea thers that had been all owed to regencrate for 2 weeks after plucking had both pi gmented and amelanotic arcas. As seen by li ght micros­ co py, mela nocytcs were :~ b se nt from the amclanoti c areas. In the pigmented areas, all stages of melanocyti c abno rmality, fr om sli ghtl y crooked dendrites to com pletely adcndritic cell s [1 2), were present and di stributed rando m ly (F ig I). Abnormal mclanocytcs wen; abundant regardless of the age of the bird or the duration of the erratic amelanosis. The 8 birds cxamined ranged in age fr om 3 months to 2 yea rs, and the interval between first and la st bi opsies in the sa me bird was as long as 8 months. Large mononuclea r lcukocytes (MNL) wcre fr equently ob­ sc rved in the center of th e pulp, es peciall y surrounding the ax ial artery. We therefore correlated the number of MNL w ith the percentage of barb ridges containing mo rphologicall y abnormal mdanocytcs (F ig 2). Although both parameters differed widely in the 23 cross-secti onal sa mplcs of fea ther cxamined from 5 ad ult el AM chi ckens, there was signifi ca nt positive correlati on among them , as shown by a high correlation coefficient (0 . 72) and by linear regression. At the far end o f the scalc this correlation began to coll apse.: . We relate this coll apse to the o bservati on that in thc Sm yth chi cken thc infi ltration subsides at the time when fcather amclanosis bcco mes extensive 11 2]. Ultrastructure and Cytoche1nistry Me!J noso mes in nor­ mall y dendritic melanocytes of the pigmented regions were ex­ Figure 4. Seri al sections t hr oug h - ~ set of abno rmal mdanosomcs !1; a tremely irregular (Fi g 3) in comparison with controls (Fig 3a i11sCt). melanocyte fro m a regenerating leather of an adult eDAM b1rd. I he They had jagged pro trusions, rounded blcbs, and narrow pi g­ composite fi gure suggests that the dccrro n- lu cent regio ns ~rc irrcgubrly shaped chambers limited by fenestrated, parfl~ll y mda1n zed Cistern ae that mcntcd extensions that generall y bo rdcred on electron-lucent re­ may enclose one o r several rn cbnosomcs. N 11111 bcrs i11 ea ch pn 11 cl dcs1 gnatc gions (F ig 3a) . Small pigmented vesicles were present in the pe­ individual granules secti oned seriall y. Bnr = 0. 1(, J..Lnl. riphcral zones of some of the mclanosomcs. Premclanosomes often contain ed nonpigmented vesicles of similar size. The pi g­ mentcd extcnsions, which arc characteristi c of Sm yth chicken basal tips (0.5 em) were cut off ~ nd split longitudinally . The feath er mdanocytcs [1 2 1. were morc numero us and more cxten­ epithelia l b yers w ere freed fr om the central pulp and_peripheral sive in the erratic birds (Fig 3 1> ). They were cqual ly apparent in keratin shea th and placed Into 3 ml of phosphate-bufh:red sa l me stained and unstained sections, suggesting that thei r ele ctron den-

Figure 5. Do pa r.:a cti on in mcbnocytcs from reg.:neratin g fea thers of adult birds. a, Melanocyte fro m an e• control chi cken with few dopa-positi ve cyropb smic ves­ icl es o r tubules (n rro111h cnds), :1!1 rcstrict.:d to the area of the Golgi apparatus. No te larg.: number of mebnized mcbnoson1 cs. /3nr = 0.5 p.m . b, Md:mocytc fro m eDAM chicken demo nstrating numerous and widel y d istributed d; pa-posiri ve cyto­ pbsm ic com ponents and comparativel y few melanized mdanosomcs. Bar 0. 5 J..LIII. 152 UDISSY ET AL TH E JOUHNAL OF INVESTIG ATIVE DEHMATOLOGY

sity was due to osmium-reactive melanin polymer. As shown in serial sections, the pigmented extensions create fenestrated, often con voluted and pleomorphic chambers around the m clanosomes (Fig 4). Prcmclanosomes were generall y normal but a few pos­ sessed fo lded membrane protrusions w ith dimensions conforming to the pig men ted extensions of melanized mclanosom es. C lose apposition of melanosom es and coalescence of the pig­ mented extensions occurred in the m ore advanced pathologic states, i.e. , in m elanocytes w ith irregular dendrites. Within ad­ cndritic melanocytcs most melanosomes were clustered. T he clus­ ters were of two types, containing: (1) melanosomes loosely packed with degenerative debris and enclosed by a li m iting m embrane; or (2) tightly packed melanosom es with electro n-dense material j o ining them. Occasionall y all mclanosomes within a given sec­ tion were sequestered in one huge, m embrane-bounded com­ partment w hich also contained much m elanin debris. T hese de­ generative changes resemble those described fo r t he dcvclopmcm of complete am elanosis in the Smyth (DAM) chicken [1 2]. M clanocytes of regenerating feathers of e+ control chickens contained dopa-positive mclanosomes, tubules, and vesicles in the Golgi area (Fig Sa). Reaction in the Golg i apparatus w as restricted to the trans-most cisterna. T he remainder of the p eri­ ka ryon and the dendrites were devoid of reaction product. In contrast, do pa-positive cytoplasmic components in dendritic feather melanocytes from eDAM Smyth chickens were more numero us and d istributed over a wider area (Fig Sb). Reactive vesicles and tubules were present not only in the immediate Golgi area but all around the nucleus and within the dendrites. ln some cell s the Figure 6. Pathologic changes affectin g th e mclanosomes of mcl:mocytes electron-lucent chambers surro unding mclanosomcs were fill ed from regenerating fea thers of adu lt eDAM birds. Selective compartmen­ ta li zation of mcl anoso mes. ( /) Type-! compartment: membrane-boun ded, with floccular deposits of reaction product. In melanocytes with loose a~g r eg atc of mcbnosomes md mebnin debris. (2) Type-2 com­ clustered or compartmentalized melanosomes the dopa-positive partment: co mpact convoluted aggrega te of mclanosomcs joined by c:lcc­ vesicles and tubules were drasticall y decreased in number and in tron-dense material. Bar = 0. 7 p.m. ln .w. Hi stochemi ca l loca li za tion of som e cases absent. acid-p hosphata se activit y in melanocy te from similar source. (3) Typc-2 Acid-phosphatase activity, on the other hand, correlated pos­ co rnpartn1e1H with extensive lead depos ition. (4) Type- ! compartment itively with the extent of mclanosomc compartmentalization. In with minimal lead depositi on: armwlll•ad points to pres umed primary ly­ the least pathologic m clanocytcs, the reaction product was re­ sosome. Bar = 0.4 p.m. stricted to a few small vesicles ncar the Golgi apparatus. O n occasion, lead deposits were seen within the Golgi region an d in primary lysosomcs in those mclanocytcs in which melanosomal aggregation h ad begun. When mclanosome compartments w ere

Table I. Relative Tyrosinase Activities in Regenerating Feathers From Developing Smyth and Control C hickens Age at Biopsy (weeks) Age at O nset of hi ·k Sex 2 3 4 5 6 7 9 10 II Amclanosis (weeks)

1 c ' (n = 4) IUO" 100 100 100 100 100 100 100 100 100 F 187" 328 262 140 23 1 86 106 17Y 237 - ' 1 M 2 16 ' 308 293 262 28 1 242 159 324 250 312 c ' (n = 2) F 98 84 72 73 62 61 73 66 52 c' (n = 2) M IR I:l 122 123 107 11 5 111 87 86 71 cD 22 F (,y 62 5 cD IY38 F 126 95 117 7 cD 1 F 121 34 102 135 7 cD 1933 M 1% 102 151 128 23 7 cD 13 F 80 153 140 188 202 59 23 9 cD 14 M 75 202 107 95 178 104 77 63 34 11 cD 1Y19 M 62 98 11 5 95 133 147 174 55 48 74 12 cD I'120 M 70 171 155 113 145 166 148 91 78 14 eD 1934 F 186 158 100 124 206 109 163 11 3 84 11 eD 2 F 156 130 45 185 249 199 232 182 87 24 n O 1921 F 120 108 75 11 5 104 157 66 75 124 nD 1936 M 11 6 80 96 161 68 132 131 78 59 nD 1939 M 88 YS 11 4 142 103 159 241 11 9 110

11 Key: r :111d l' t = con trol lines. c D = Sm yth lin e chickens th:u went on to develo p co mplete :unclanosis. d ) = Sm yth line chi ckens th at de velo ped erratic amdanosis. nD = Sm yth line chi ckens that did not develop amcl:mosis over 8 months of o bservation.

"Except for ", ;t il va lues given arc pcrccnlagcs w ilh respect to sex- and age-matched t.'h contro ls. '' Avera ges of actua l values recorded for ,J> females and ma les as cpm 3H/ mg protcin/20 min. (Dashes indi cate that no mcasur c- mcms wert made. VOL. 86. NO. 2 FEBRUARY 1986 MELANOCYTE DYSFUNCTION IN A VI AN VITILIGO 153

Retohve cumulative overooe fore, regenerating feathers representing the same age periods and of tyrosinou OCIIIIII)' Of 7 Wllkl sa me sex were assayed si multaneously. The average of dupli c:tte measurem ents was expressed as the percentage of the averages 100% fo r the r" con trols (Table 1). With each assay, regenerating feathers fr o m completely am elano ti c Smyth chi ckens and from pure albin o chi ckens [221 were used as negati ve con trols. T he relative values 101 % ' for tyrosinase activity in the Sm yth chickens varied over a w ide range. Sixty-three percen t were above e" control levels, with a

121% large percentage being twice as hi g h. These high values were distributed randomly throug ho ut the developmental sequence, withou t an o bvious pattern that might suggest a grad ual in crease !54% or decrease in tyrosinase activity. A ll Smyth chicks, even those that never develo ped feather amclanosis, exhibited at least one period of elevated tyrosinase JCtiv ity d uring the first I 0 weeks 113% after hatch. T hirty-seven percent of the tyrosinase values were below the corresponding c" control values. The m aj ority of these Figure 7. Graphi c summary of the rel ative: tyrosinase activities tabulated low values was obtained within the 2- 3 weeks prior to the de­ individuall y in Table I. The width of each strip at ea ch weekly time point velopment of feather a melanosis. represents the average of the percenta ge: of tyrosina se activities in Table The relative tyrosinase activities per week for the 3 groups o f 1 for a given group of birds. Like row I in Table I, strip I represents the Smyth line chickens and the 2 con trol lines were averaged and 1 reference 100% tyrosina se activities of e' birds aga inst whi ch all other summari zed graphicall y in Fig 7. Each of the 3 Smyth line gro ups activities have been co mpared. The percentage va lu es given on th e right sho wed peri ods of elevated ty rosinase acti vity, and the group that were obtain ed by averagin g the percentage week ly tyrosinase activities eventuall y became com pletely am elanotic (cO), had a coll ective for each group horizontall y through week 7. They arc a relati ve meas ure " burst" of tyrosinase activity at 7 weeks of age, 2 weeks before of rhe cumulati ve tyrosinase acti vities for each group of birds up to 2 weeks prior to the average time of onset of a melanosis in cD birds, whi ch the average o nset of am elanosis. Up to that time, the relati ve occurred at 9 weeks (arrow). ': cf respecti ve dopa reactions in electron cumulative average of tyrosinase activity in the gro ups of Smyth micrographs of Fig 6. For letter designati on of birds, see Table I. line chickens was higher than that of the control lines, including the e~> line from which the Smyth chicken had been developed. T he sm ooth sequence of relative va lues o btained for tyrosinase prevalent, prim ary lysosomcs were abundant thro ug hout the cy­ activities of the e+ con tro l feathers affi rmed the reli ability of the toplasm and m any were loc:ttcd :tdj:tcen t to the fo rming com­ experimental design and the validity of comparing all va lues as partmen ts. The large compartments of melanosomes conta111ed percentages o f average c~> va lues . T herefore, the temporal va ri a­ extensive lead deposits (Fig 6 i11 se t). T he type- I clusters of mcl­ ti ons in relative tyrosinase ati v ities within the di fferent groups of anosomcs (Fig 6) often contained a m in imal am ount oflead w hich Sm y th chi cks refl ect real flu ctuati ons and indica te a sporad ic or. appeared to increase with the am o unt of extramelanosom al mel­ in cDAMs, fina l h yperactivity. anin debris present (Fig 6 i11 ser). By comparison, the ti g hrl y packed Neural Crest-Derived Melanocytes in Culture Pure, pro­ type-2 clusters contained much lead deposit. liferative cul tures of pigmented m elanocytes from embryos of the Tyrosinase Activity in Regenerating Feathers of Growing c', e'' , and Sm yth line genotypes have been maintained for up to Chicks Weekly le vels of ty rosinase activity were assayed 111 13 weeks. Initiall y, the cultures of contro l m elanocytes and those regenerating feathers of chicks beginning at 2 weeks of age. The of Sm yth chicken melanocytes were m orpho logica ll y identic:t l 13 Smyth line chicks were categorized according to the plumage (F ig 8a,b). 13y 4-6 weeks a small percentage of Sm yth chi cken pigmentati o n at 24 weeks. Preliminary measurements lud d if­ melanocytes had developed large, pig m ented cytoplasmic vacu­ fered sig nificantly among assays and between the sexes. T here- o les (Fig Sb i11 set). By 12 weeks, cultures of c+ m elanocytes re-

•.. Figure 8. Ph ase-contrast photomicro­ graphs of pure cu ltures of proliferative ncur:~l crest-derived chi cken mebnocytcs. Cell densities shown arc representative of respective culture dis hes. a, Pigmented mcl anocytes fro m c+ embryos. den ton­ strating brge ce ll bodies with 2- 6 den­ drites which branch occasionall y. Cells maint:~in c d 50 days through 4 subculttt res . Bar = 10 IJ.m . b, Pi gmented ml'ianocytcs ~ - .. from Smyth lin e embryos. Cell s mai n­ tain ed 50 days through 4 sub cul tures. Oc­ " ' cas ionall y a large, pi gmented vesicle (nr· rowlu·nd) ca n be see n (i 11 set). Bnr = I U IJ.m: i>~ sc t bar = 6 ~J.m . c. e+; mela nocytes main­ tained for 90 days through 3 subcultures and demonstrating norm al morphology. Bnr = 10 IJ.lll . d. Smyth line melanocy tes m :~intained fo r 90 days through 3 subcul ­ .. tures, demonstrating a change to larger, less de ndritic ce ll s (n rrilll') and an in crea se ~f in the number and size of pi gmented vac­ uoles (nrro u,licnd). Nore red uced cell den­ sit y. Bnr = 10 IJ.lll . d ~ · T H E JO URNAL OF IN VESTIGATIVE DERMATOLOGY 154 BOISS Y ET AL

many m elanized m elanosomes (Fig 9a) which, surprising ly, dif­ fered significa ntl y from the m elanin g ranules synthesized in vivo by feather mclanocytcs (cf Fi g 3 i11 set). G ranules in vitro were m ostly spherica l, contained an irregular arrangement of melan­ o filaments, and had Aoccular deposits of melanin. Melanogenesis was active in the cultured cell s as indica ted b y the extensi ve Golgi apparatus and the numero us premelanosomes (Fi g 9a i11 set) . Small membrane-bounded compartments containing m elanin g ranules were observed in 39% (n = 12) of c; and 33%, (n = 2) of e1' m clanocytes. T he ultrathin profiles contained no m o re than 3 g ranules, and there were no m ore than 3 compartments per section. The fine structure of cultured Sm yth m elanocytcs differed dr:~stica ll y from that o f the control cells. O nly 10% (n = 4) of ultrathin sections were free of compartm ents. T he other 90% (n = 35) contained compartments, all of which were considerably larger than those in the contro l cell s. A m ~~ o rit y of the affected Sm yth mclanocytes (83%) contained compartments with as m any as 12 g ranules per sectional view (F ig 9b) ; the rem aining 17% contained even larger accumulations. O n occasion we observed huge compartments w hich , in addition to melanin g ranules, con­ tained degenerative material, probably of melanosomal o rigin, and resembled the large autophagosomcs in the mclanocytes of regenerating feathers of eDA M birds (Fig 9r) . D ISCUSSION T he findi ngs reported here clarify fu rther the events leading to a m elanosis of feathers in chi ckens of the Smyth line, including errati ca ll y depig mcnting, eDA M birds. A basic defect of the Smyth line is aberrant melani za tion in feather mclanocytcs. Two li nes of evidence support this conclusio n: (1) synthesis of abnormal m elanosom es associated w ith an in c r c:~se in dop:~ -po s itivc cyto­ plasmic components; :~ nd (2) sporadic in creases and generall y higher le vels of tyrosinase activity within prea mclano ti c feathers. The m o rpho logic manifestati ons and the high level of histochemical dopa activity precede the onset of acid-phosphatase activity and the appearance of pulp immunocytes. Cytopl as mic components containing dopa reaction product after incubation with the m el­ anin precursor arc presumed to be sites of tyrosinase. These com­ ponents are numerous and distributed m ore widely in eDAM mclanocytcs than in the e+ contro ls. In fa ct, tyrosinase assays in these 2 lines reAcct this distribution. We must also consider the mo rphologica ll y abno rmal m ela­ nosom cs. It is possible that the pig m ented m embranous exten­ sions arc a consequence o f a high rate of transport of tyrosinaS<' to mclanosom es. In mammalian mclanocytcs and melano ma cells. tyrosinase is presumed to be transported from the Golg i apparatus to premelanosomes via coated vesicles 123,24 1, i.e., via a discon­ tinuous system . In heav il y melani zin g avian and amphibian me­ bnocytcs, tyrosinase is tho ught to be transported, in addition, through tubules or cisternae r25 ,26 J. The numero us pig m ented extensions may be a manifestation of such a continuous transporr system. Overa cti ve melanizatio n ca n have detrimental conse­ Figure 9. Electron mi crographs of pi gmented mdanocytcs from pure quences for mclanocytes. M elanin precursors, phenols and h y­ cul tures of pro li fcrJtivc nc urJl crcs[-dt:ri vcd c hicken nH.: bnocytcs, an ain­ droquinoncs, are toxic to ce ll s [27 J, and it has been shown that tain ed for 70 days. n, Melanocyte from e' embryo demonstratin g ab un ­ they ca n destroy pigment cell s [28-301 and fibroblasts [28 ]. da nce of mcla noso mcs immediately outside the Go lgi region and ex­ Regardin g the selecti ve autopha gocytosis of mclanosom es in tendin g in to dendrite (arrow). Notice the fl occular depos iti on of melanin feather mel anocytes, it is li kely that the extensive auto phagic within th e granules (arrow l u · a r~ and the lac k of a wel l-orga ni zed mJtri x acti vity is detrimental to the melanocyte and leads to cell ular (a sterisk) in prcmd :mosomes (i11 ser). /Jao· = 0. 7 j.L111 ; i11 scr har = 0.25 j.Lnl. necrosis. Rupture of necrotic m clanocytes would cause the release /J , Melanocyte from Smyth embryo dem onstrat in g compartmentaliza ti on of heav il y pi gmented mcl anoso mcs into small va cuoles throughout the of m elanocytic antigens which could, in turn, stimulate an im­ entire cdl bod y. /Jar = 0.7 j.Lll1 . c, Melanocyte from same culture as in mune response against m elanocytcs. At this point another m ajor (b). dem onstratin g large membrane-bounded co mpartment with numer­ physiologic defect of the Sm yth line com es to the fo re: a h yper­ ous mdanosomcs and (kbris (A). Bar = 0. 7 j.Lill. active immune response 114, IS J. We have previously reported that mclanocytes are rem oved fro m the choroid of Smyth chick­ ens by MNL 11 21. Macrophages and lym phocytes appear in the taincd their original mo rphology (Fig 8c). At that time the Sm yth cho roidal connective tissue and sinusoidal system w hile auto­ chicken melanocytes had become large and less dendritic and the phagocytosis of m clanosom es occurs in som e of the choroidal number and size of pi gmented vacuoles had in creased (l: ig 8d). mclanocytes. This proximity suggests that m ebnocytes in t h ~ Ten-week- old cultures were examined with the electron mi­ choroid of Smyth line chi ckens can be targets in the immum· croscope. Melanocytcs of both control lin es (c ' and e") contained response. VOL. 86, N O. 2 FE URUAHY 19H(, M ELANOC YTE DYSFUN TION IN AVIAN V ITILIGO 155

Probably, even presumptive mclanocytcs arc targ ets. In feather 10. Millikan LE, H ook JUt Manning PJ: lmmunobiologyofmclano m a. tissu e of eDAM birds immunocytes appe:1red exclusively in the Yak J 13i o l Mcd 46:63 1- 645, 1973 ' central pu lp. T h e infiltrates did n ot reach the diffe n.: ntiated me­ II. Sm ythJRJr. Boissy RE, Fite KV: The DAM chic ken: a modd for lanocytcs located w ithin the epithelium . Neverthe less, the cxtel1t spontaneous postnata l cutaneo us and ocular amclanosis. J Hercd of pulp inAammation correlated positively w ith the extent of 72: 151-1 56, 198 1 melanocyte pathology and the number of a melano tic barbs. A 12. 13oissy RE, Sm yth jl( Jr, Fire K V: Progress ive cytologica l changes derm a l reserve pool of unpig mented ml'iano blasrs is presumed to d urmg the development of dela yed feather :nnclanosis and asso­ exis t o utside the feather fo llicle I2 J. At the o n set of feather re­ ciated choro id al defects in the DAM chicken lin e-a vitiligo model. generation, some of these cell s would mig rate into the feather Amj Pach a l 111 :197-202, 1983 ' b laste ma, prob:~bl y mov ing a lo n g b lood vesse ls 13 1]. O n ce inside 13. Sm yth Jl~ Jr, 13o issy I~ E. Fitc KV, Albert DM : J~ e tinal d ys trophy the feathers , the mig ra to ry melan ocytes enter the epidermal colbr assoCiated w ah a postnatal amcbnosis in the chi ckcn. In vest [1 ], synthesize melanosomes fo r trans fer to the kcratinocytes 132 /. Ophthalmo l Vis Sci 20:799-803. 19R I a nd become incorporated in to the emerging feather [33 /. The 14. Lamont SJ , Sm yth jl{ Jr: E rti.·cr of bursectomy o n develo pment o f melanocytes mig rating thro u g h loose connective tissues wou ld a spontaneous postnata l a melanosis. C lin lmmunol lmmunopatho l be vulnerable to attack b y s timulated immunocytcs. 2 1:407-411 . 19H I Human patients w ith vitili go. for which the Smyth line c hic ke n 15. Lamont SJ. 13o issy. RE, Smyth Jl~ Jr: Hun10 ral immunl' response is being inves tigated as a model, a lso have been implicated in and expressw n o ( spo ntaneous postnatal :11ndanosis in DAM line having an unbalanced immune syste m 17] because a sig nifica nt ch1 ckens. lmmuno l Commun 11 :12 1- 127, 19H2 per centage of patients have elevated levels of a ntithyroid, anti­ 16. Brumbaugh JA, H olbndcr WF: A further stud y o f the E pattern s mooth-muscle, antiparietal-ccll, or antinuclear antibodies in their locus 111 the fowl. Iowa Sr:n e J Sci 41) :5 1-64. )')(,5 serum [34,35]. Serum antibodies against c ultured n o rmal m elan­ 17. Gom ori G: Microscopi c Histochemistry. Univ of C hi cago Press, ocytes in patients w ith vitiligo have been fo und recently by two C h1 cago, I 952. pp I H9- 19-l g roups of investigators 136,37]. Also, in certain patients, ly m­ IR de Jong ASH, 1-I:Jk 1]. VanDuiji 1', Dacms WT: A new dynamic phocytic infiltrates have been o bser ved a t the pigmented bo rde rs m odl'i system fo r the study o f ca prurc reactions fo r ditTu sa bk o f e nlarg ing white areas [381 and within the n o rma ll y pi g mented compounds in cyrochemistry. II. Effe ct o f the compositio n of the e pidermis [39J. Melanocyte d ysfun ction has also bee n desc ribed mcubatio n mecli um o n the trapping of phosphHc' ions in acid fo r human vitiligo patie nts [34] and in the Belg ian Tcrvurcn d og phosph:1t:1 sc cy tochemistry. 1-lisrochem J II : 145- 16 1. 1979 I 'J. 1 [8 ]. At the level of li g ht mic roscopy, enlarg ed, hig hl y d o pa­ Pom erantz S J-1 : L-Tyrosinc-3.5-· 1-1 assay fo r tyrosin ase development positive mcla nocy tcs arc often seen at the bo rde r of advancing Ill skm of newbo rn hamster. Sciencc 164:838-839, 1969 lesions [34!, and in some cases the depig mcnting borders a rc 20. H a~ab : m R, N ordlund J , Francke U. Mocllmann G. Eisenstadt JM : hyperpigmcnted in comparison with the surrounding n o rmall y Supermebnoti c hybrids derived fro m m ouse· mcbno mas and no r­ mal m ouse cell s. Somati c Cell Genet 6:29-44. I 9SO pigmented e piderm is 134]. Occasional border m c lanocytes l13 vc 2 1. sequestered their mclanosomcs into a single large vacuole (our 13 o issy . RE. Ha laban R: Es rablishrncnt o f pro liferative, pure cultures own o bservatio ns) . o f p1 g mcntcd chi cken mclanocytes fro m neural rubes. J In ves t Derm:n o l R-1: 151>- 16 1, I 9R5 Finally , we h ave demons trated that neural c res t-derived m c­ 22. la n ocytes from embryos of Smyth line c hickens likewise develo p 13 o issy R E. 1-l alaban H. Moellmann GE, Lern er AJ3 : Pure cul tures of mclanocytcs from neural rubes o f tyrosinase positive and ty­ a utophagosorncs w hen isolated in cell culture, w h ereas melano­ rosmase negati ve albmo chi ckens: an initial chara cteriza ti on. J Cell cytes from con tro l birds continue to thri ve. T his finding is com­ B1ol 99:386a, 1984 patible with our h y pothesis d e rived fro m the study ofm c lanocytcs 23. Maul GG: Golgi-melanosom c relationship in human melano m a in in situ, th at a basic gen e tic defect of the Smyth line is expressed VItro. J U ltr:lsrrucr l{es 26: 163- 176. 1969 by the m e lanoc yte population . C ultured m c lan ocytcs fro m the S myth line and contro l chickens will becom e an impo rtant tool 24. Mishima Y. lmo kawa G, Ogura H : Functi onal and three-dimen­ SIOnal differentiation of sm ooth membrane structures in m cbn­ for fu rthe r s tudy of this d e fec t. ogenesis, Pi g ment C ell 4: Part I of l'rocccdings of the lOth lnter­ n;nional Pig me11t C ell Conferen ce. Cambridge, Massachusetts, 1977. Ed1ted by SN Klaus. S Karger, Basel / N ew Yo rk , 1979 pp REFERE N CES 277-290 ' 25. I. Lu cas AM. Stettcnheim 1'1~ : The g rowth and colo r of feathers, Avian Eppig JJ Jr. Dtn11 ont JN: Cytochemica l locali za ti on of tyrosinase Anatomy-lnregumenr (Part II ). Agricultural Res Service. USDA. act1 v1ry m p1g m cnted epithelial cel ls o f Rm111 pipic11 .< :md Xc11np 11 s 1972, pp 341 - 4 19 lncl'ts larvae. J U ln·astrucr Hes 39:397-41 0. 1972 26. 2. FoulksJG: An ana lysis o f the source o fmclano pho res in regenerating Maul GG. 13rumbaugh JA: O n the possible fun cti o n o f coated ves­ feathers. l'hysiol Zoo I I 6:351-380, I 943 ICles Ill melan ogenes is o f th e re generating fowl feather. J C ell Bioi 4!>:4 1- 48, 197 1 ' 3. Mayer TC: Enh:m ccm cnt of mclanocytc dcvclopmcnt fro m pi ebald 27. neural crest by a f.1 vorablc ti%ue environnlcllt. J)ev 13iol 56:255-21i2. Hochstein l'. Cohen G: The cyto toxicity of melanin precursors. Ann 1977 NY Aca d Sci 100:876-886, 1963 4. Jim bow K, Szabo G, Fitzpatrick TB: Ultrastructure i11 vesti garion o f 28. Halaban R. Lerner AB: Tyrosin ase and inhibition o f pro liferation of aurophagocytosis of m ebnosomes a11d programmed ce ll death of mdano ma cells and fibroblasts. Exp Cell l ~e s 108: I 19-125, 1977 melanocytes in White Lcgho rn feather: a stud y of mo rphogeneti c 29. Pa w elek J, . Korner A, Bergstrom A. Bologni a J : New regubrions events leading to h ypo melanosis . Dev Bioi 36:8-23. 1974 of melanm b10synthcs1s and the :lll todes trucri on of melano m a cel ls. 5. Witkop CJ Jr: Albinism . Adv Hum Genet 2:6 1- 142. 197 1 Nature 286:6 17-619, 1980 6. Bleehen SS: Hisro logy o f vitili go. Pi g ment C ell 5: Part II of thc 30 Wi ck MM. 13 ye rs L. Frei E Ill : Selecti ve toxi city o f L-d o pa fo r Proceedin gs o f the lOth ln tcrnational Pi gm ent C dl Conference. m ebno ma ce ll s. Science 197:468-469. 1977 Cambridge, Massachusetts, 1977. Edited by SN Klaus. S Karger, 31. Rawles M E: The mig rati on of mclano bbsrs after hatching inro pi g­ Basel/New York, 1979, pp 54-6 1. mem-frec skm grafts ot the common fow l. l'hysio l Zool 17: 167-183 7. Lerner AB: On the etiology of vitiligo and g ra y hair. Am J Med 1944 ' 5 1:141 - 147. 1971 32. Ruprecht I< W: Pi g mcntierung dcr Duncn fedc r von Gnll11 s do 111 csrims 8. Mahaffey MB, Yarbroug h KM, Munnell JF: Focal loss of pi gm ent L. ; Li chtund cl ektro ncnmikroskopischc Untersuchungen zur Mc­ in the Belg ian Tervuren dog. JAm Vet Med Assoc 173:390-396, lanosomcnubertragung. Z Z dlfo rsch 11 2:396-4 13. 197 1 1978 33. Bowers RR. Chun DW: T yrosinase and acid phosphatase pHhways 9. Lerner AB, Nordlund JJ : Vitiligo: loss of pi g ment in skin and hair. Ill rcgcnerat111 g feather mebnocyres of the fowl. Pi g ment C ell JpnJ Dermarol 5:1-8, 1978 I 9H I. Proceedings of the lith lntcrnJtio nal Pi g ment' Cell Con- 156 lJO I SS Y ET AL THE JOUHNAL OF INVESTIGATIVE DEHMATOLOGY

ferencc, Scndai, Japan, 1980. Edited by M Seiji. Univ of T okyo 37. Mocllm ann GE, Kra ss P, Halaban R. Kuklinska E, Lerner AB: On Press, Tokyo, 1980, pp 285-293 the subject of se rum antibodies to mclanocytes in vitiligo (abstr). 34. N o rdlund JJ, Lerner AB: Viti li go-It is important? Arch Dermarol J Invest Dermatol 84:333-334. 1985 I 18:5-8, 1982 38. 13hawan J, Bh utani LK: Possible lymphocyte-mediated melanocyte 35. Woolf.so n H. Finn O A, MacKie RM, McQueen A, Mac Sween and keratinocytc damage in vitili go. Yak J Bioi Mcd 53:448, 1980 RNM: Serum anti-tumo ur antibodies and auto-antibodies in vi­ 39. Mocllmann G, Klcin-Angcrcr S, Scoll ay DA. N o rdlund JJ, Lerner tiligo. Br J Dcrmato l 92:395-400, 1975 AB: Extracellular granular material and degeneration of kcratin­ 36. Naughton G K, Eisin ger M, Bystryn J-C: Antibodi es to no rmal hu­ ocytes in the no rmall y pi gmented epidermis of patients w ith vi­ Imn mebnocytcs in vitiligo. J Exp M ~ d 158:246-25 1, 1983 tiligo. J In vest Dcrmatol 79:321-330, 1982