IFN-c, IL-21, and IL-10 Co-Expression in Evolving Autoimmune Vitiligo Lesions of Smyth Line Chickens

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1 ORIGINAL ARTICLE IFN-c, IL-21, nd IL-10 Co-Expression in Evolving Autoimmune Vitiligo Lesions of Smyth Line Chickens Fengying Shi 1 nd Gisel F. Erf 1 The Smyth line (SL) of chicken is n excellent niml model for humn utoimmune vitiligo. In SL vitiligo (SLV), postntl loss of melnocytes in fethers ppers to e due to cell-medited immunity. In this study, leukocyte infiltrtion nd ssocited expression (RNA) of immune function-relted cytokines in growing fethers were investigted throughout SLV development nd progression. Both leukocyte infiltrtion nd cytokine expression levels strted to increse ner visile SLV onset (erly SLV), reched pek levels during ctive SLV, nd decresed to ner pre-vitiligo levels fter complete loss of melnocytes. Specificlly, significnt increses were noticed in reltive proportions of T cells, B cells, nd mjor histocomptiility complex (MHC) II-expressing cells during ctive SLV. Levels of T-cell infiltrtion were higher thn those of B cells, with more CD8 þ thn CD4 þ cells throughout SLV. Elevted leukocyte infiltrtion in erly nd ctive SLV ws ccompnied y incresed levels of cytokine expression, especilly in IFN-g, IL-10, nd IL-21. Low expression of IL-4 nd IL-17 did not suggest importnt roles of Th2 nd Th17 cells in SLV pthogenesis. Tken together, SLV ppers to e Th1-polrized utoimmune disese, wherey IFN-g expression is strongly ssocited with prllel increses in IL-10 nd IL-21, prticulrly during erly nd ctive stges of SLV. Journl of Investigtive Dermtology (2012) 132, ; doi: /jid ; pulished online 24 Novemer 2011 INTRODUCTION The mutnt Smyth line (SL) of chicken is n excellent niml model for the study of utoimmune vitiligo (Wick et l., 2006) due to mny phenotypic nd etiopthologicl similrities etween SL nd humn vitiligo, nd the multifctoril nture, high incidence, nd spontneous onset of SL vitiligo (SLV; Smyth, 1989; Erf, 2010). In chickens, melnocytes, the trget cells in vitiligo, re locted in growing fethers (Figure 1; Smyth, 1989). Growing fethers cn e esily removed nd cn regenerte, nd hence the developing utoimmune lesion cn e monitored throughout SLV in the sme individul. Moreover, the living portion of the growing fether (fether tip; Figure 1) provides sufficient tissue smple for vrious post-collection nlyses. Similr to humn utoimmune vitiligo, oth humorl nd cellulr immunity hve een implicted in SLV, with more prominent role ttriuted to cellulr immunity in melnocyte loss sed on phenotypicl nlysis of infiltrting leukocytes (Erf et l., 1995; Shrest et l., 1997; Wng nd Erf, 2004) nd 1 Deprtment of Poultry Science, University of Arknss Division of Agriculture, Fyetteville, Arknss, USA Correspondence: Gisel F. Erf, Deprtment of Poultry Science, University of Arknss Division of Agriculture, Fyetteville, Arknss 72701, USA. E-mil: gferf@urk.edu Arevitions: inos, inducile nitric oxide synthse; MHC II, mjor histocomptiility complex II; SL, Smyth line; SLV, Smyth line vitiligo; Th, T helper Received 22 April 2011; revised 14 Septemer 2011; ccepted 5 Octoer 2011; pulished online 24 Novemer 2011 oservtion of IFN-g expression in fether smples collected from SL chickens with ctive SLV (Wng, 2001; Shi et l., 2009). Moreover, the presence of melnocytes-specific cellmedited immunity ws demonstrted in vivo sed on the delyed wttle-swelling response to injection of syngeneic melnocyte lystes in chickens with SLV (Wng nd Erf, 2003). Although phenotypic nlyses of infiltrting cells in crosssections of ctive lesions nd in fether pulp cell suspensions support more prominent role of cell-medited immunity in melnocyte loss in SLV (Erf et l., 1995; Shrest et l., 1997; Wng nd Erf, 2004), immune functionl ctivities ssocited with SLV development hve not een exmined. The ojective of this study ws to monitor cytokine gene expression nd determine leukocyte infiltrtion profiles in fether tips (Figure 1) collected over the course of SLV development from SLV chickens. Specificlly, using two-step quntittive reverse trnscriptse PCR, gene expression profiles were estlished for inducile nitric oxide synthse (inos) nd cytokines of innte immunity (IL-1, IL-6, IL-8, IL-10, IL-12, IL-12, nd IL-15), signture cytokines of T helper (Th)1, Th2, nd Th17 cells (IFN-g, IL-4 nd IL-17F, respectively), nd IL-21, pleiotropic cytokine implicted in orgn-specific utoimmune diseses. To relte gene expression with leukocyte infiltrtion, the presence of mcrophges nd T- nd B-lymphocyte susets were lso exmined in longitudinl sections prepred from fether tips. Knowledge gined from this study will shed more light on the pthogenic mechnisms involved in the utoimmune loss of melnocytes in SLV. 642 Journl of Investigtive Dermtology (2012), Volume 132 & 2012 The Society for Investigtive Dermtology

2 Approximte loction of cp Fether tip Percentge of stined cells (% of re) NV EV AV CV Vitiligo stte MHC II-expressing cell T cell B cell Mcrophge Sheth Br ridge Figure 2. Infiltrtion profiles of leukocytes in fether tips collected t different sttes of SLV. Mjor histocomptiility complex (MHC) II-expressing cells, T cells, B cells, nd mcrophges were identified y indirect immunoperoxidse stining using I, TCR gd nd TCR, Bu-1, nd KUL01 mouse nti-chicken mas, respectively. Stined sections were exmined t 40 originl mgnifiction, nd the mount of stined cells ws expressed s the percentge of the whole fether section re nlyzed (% of re). NV: fether tips from three SL chickens tht never developed SLV; EV, AV, nd CV: fether tips were collected from seven SLV chickens just efore, during, nd fter SLV development, respectively. Ech r represents the men±se. (, ) For ech cell type, mens without common letters re different (Pp0.05). Pulp Cp Figure 1. Morphology of 2- to 3-week-old growing fethers from SL chickens. () From left to right: normlly pigmented, prtilly depigmented, nd completely depigmented growing fethers from SL chickens tht developed SLV. Growing fethers cn e collected from SLV chickens over the whole course of SLV. The living prt of growing fethers (newest growth to the epiderml cp) is referred to s fether tip. () Microstructure of the newest growth of fether tip with norml pigmenttion; lyers shown from the outside to inside re sheth, r ridge, nd pulp. (c) A cp formed y the epiderml lyer enclosing the pulp. Longitudinl sections were stined with hemtoxylin nd eosin stin nd exmined t 40 (, c) originl mgnifiction under right-field microscope. Br ¼ 1 mm. RESULTS Leukocyte infiltrtion in growing fethers collected t different sttes of SLV For ech cell type, the reltive mount of stining ws expressed s the percentge of the longitudinl sections nlyzed (% of re). Leukocyte infiltrtion dt from ech individul were grouped with respect to vitiligo stte: NV (fether tips from SL chickens tht never developed vitiligo), EV, AV, nd CV (fether tips from SLV chickens just efore visile SLV onset, during ctive, nd fter complete depigmenttion, respectively). The infiltrting leukocytes consisted primrily of T nd B lymphocytes, which reched pek levels during AV (Pp0.05). Although proportiontely fewer mcrophges thn T nd B lymphocytes were present in SLV fethers, mcrophge levels were lredy elevted in EV nd remined elevted in AV (Pp0.05; Figure 2). The reltive proportions of mjor histocomptiility complex (MHC) II-expressing cells were higher thn those of other leukocytes, independent of SLV stte. Except for MHC II-expressing cells, the proportions of T cells, B cells, nd mcrophges returned to ner NV levels when melnocyte loss ws complete (CV, Figure 2). Both types of TCR-defined T cells ( nd gd TCR) followed the sme infiltrtion trend (Tle 1). However, wheres the reltive proportions of T cells were gretly elevted (Pp0.05) in AV compred with NV smples, those of gd T cells did not differ etween NV, EV, AV, nd CV smples. CD4 þ nd CD8 þ cells reched pek levels in AV smples compred with NV, EV, nd CV smples (Pp0.05, Tle 1). At ll SLV sttes, there were more CD8-expressing cells thn CD4-expressing cells, with CD4:CD8 rtios eing 643

3 Tle 1. Reltive proportions of leukocytes (% of re) 1 in fether tips 2 from SL chickens with nd without SLV Vitiligo stte 3 NV EV AV CV T cells ( TCR+) 0.70±0.17,4 3.95±1.57, 4.92± ±0.67, T cells (gd TCR+) 0.47± ± ± ±0.35 CD4+ lymphocytes 0.61± ± ± ±0.51 CD8+ lymphocytes 1.17± ± ± ±0.60 CD4+:CD ± ±0.08, 0.87± ±0.13 B cells (Bu-1+) 0.25± ± ± ±0.46 IgM+ cells 0.31± ± ± ±0.48 T:B ± ±1.10, 1.52± ±0.69, 1 Leukocyte types were determined y indirect immunoperoxidse stining. Primry unleled ntiodies were mouse-nti-chicken mas to TCR (cocktil of 1 nd 2 TCR), gd TCR, CD4, CD8, Bu-1 (B-cell mrker) nd IgM. Binding of primry ntiodies ws detected y iotinylted horse-nti-mouse IgG (H+L) with vidin-iotin peroxidse complex regents nd using 3,3-diminoenzidine s the sustrte. Imge nlysis ws crried out t 40 originl mgnifiction under rightfield microscope. The mount of stined cells in fether tip sections ws expressed s the percentge of the whole re nlyzed (% of re). 2 For ech smple collection, three fether tips were lid prllel in the sme orienttion in n luminum cup contining Tissue-Tek OCT freezing medium nd snp frozen with liquid nitrogen. Longitudinl sections (6 mm) were cut t 19 1C, fixed in cetone, nd sujected to indirect immunohistochemicl stining. 3 Vitiligo stte, NV, EV, AV, or CV mens fether tips were from SL chickens tht never developed SLV (n=3), from vitiliginous SL chickens (n=7) just (o2 weeks) efore, during, or fter SLV development. 4 Dt re shown s men±se., Dt in the sme row without common letters re different t Pp T cells include oth nd gd TCR+ T cells. lowest in NV smples, intermedite in EV nd CV smples, nd highest (Pp0.05) in AV smples (Tle 1). On the sis of the proportions of IgM þ cells t the vrious sttes of SLV, the mjority of infiltrting B cells (Bu-1 þ ) ppered to e IgM þ cells (Tle 1). Clculted T- to B-cell rtios (TCR þ cells vs. Bu-1 þ cells) were lower (Pp0.05) in AV smples (1.52±0.21) thn in NV smples (5.16±2.66) ecuse of reltively greter increse in B-cell thn in T-cell infiltrtion in AV smples compred with other sttes of SLV (Tle 1). Exmintion of hemtoxylin nd eosin-stined formldehyde-fixed fether sections confirmed the mononucler nture of the leukocyte infiltrte nd the notle sence of grnulocytes, including heterophils (vin equivlent to neutrophils), t ll vitiligo sttes (dt not shown). In ddition, nturl killer cells do not pper to mke up significnt portion of pulp-infiltrting lymphocytes in SLV sed on flow cytometric nlysis for CD8 þ lymphocytes without surfce CD3 (ritrry definition of vin nturl killer cells; (Goel et l., 1994)) in pulp cell suspensions from ctive utoimmune lesions (dt not shown). Profiles of cytokine expression throughout SLV development Owing to limited vilility of chicken cytokine-specific ntiodies, cytokine expression ws exmined t the trnscriptome level using quntittive reverse trnscriptse PCR. Reltive expression ws clculted y the DDC t method (Wong nd Medrno, 2005) nd ws expressed s fold chnge with respect to fether tips collected throughout the course of the study from NV SL chickens. As ech vitiliginous chicken developed SLV t different weeks of ge, to demonstrte the generl trend of cytokine expression in SLV development nd progression, dt for ech ird were ligned with respect to time of visile SLV onset (set s 0). In fether tips collected from SLV chickens severl weeks (42) efore SLV onset (BV), expression levels for ll trgets were similr to those of NV smples, with the exception of IL-8, IL-10, nd IL-21, which exhiited X3 fold higher levels thn NV smples (Figure 3). In fether tips collected from SLV chickens closer to (p2 weeks efore; EV) SLV onset, prllel increses in the reltive expression of IL-8 nd IL-10 could e oserved, with IL-10 reching higher thn BV levels in smples collected 1 week ( 1) efore SLV onset (Tle 2 nd Figure 3). With SLV onset, expression of IL-8, IL-10, IL-21, nd IFN-g incresed compred with BV smples, with ll four cytokines simultneously reching pek levels nd remining elevted until 3 weeks post SLV onset (AV; Po0.05; Tle 2 nd Figure 3). This ctive phse of SLV ws ccompnied with significnt, lthough much lower, increses in IL-1, IL-6, IL-12, IL-15, nd inos (Tle 2 nd Figure 3). A notle exception to this expression pttern ws IL-4, which did not increse ove BV levels nd exhiited the highest reltive expression t much lter time points thn other trgets (Tle 2). With the progression of SLV, levels of most trgets returned to BV levels, especilly in white growing fethers collected from irds tht exhiited complete depigmenttion for t lest 1 week (CV; Figure 3). Throughout the course of SLV development, from initition, progression to complete pigmenttion loss, the ctul reltive expression vlue of chemokine IL-8 nd cytokines IL-10, IL-21, nd IFN-g, nevertheless, differed gretly etween irds (Tle 2). However, the comintion of these cytokines, especilly IL-10, IL-21, nd IFN-g, emerged s the signture cytokine profile during the erly stge nd ctive progression of SLV in ll vitiliginous chickens. Expression of IFN-g t the protein level ws confirmed in pilot study y indirect immunohistochemistry using mouse nti-chicken IFN-g ntiodies developed y Dr Lillehoj (Animl nd Nturl Resources Institute, USDA-ARS, Beltsville, MD; dt not shown). Reltive expression levels for IL-12 nd IL-17F could not e determined sed on the formule of the DDC t method, ecuse no C t vlue ws detected in the clirtor smple. The reltively low expression of IL-12 nd IL-17F in SLV cn, however, e inferred sed on clcultion of the DC t vlue (C t of trget C t of endogenous 28S control), which is reltive mesure of trget gene expression within smple. DISCUSSION The SL chicken is n excellent niml model for humn utoimmune vitiligo s suggested y the mny phenotypic nd etiologicl similrities etween the two cses, which re summrized in Tle 3. Although SLV is multifctoril 644 Journl of Investigtive Dermtology (2012), Volume 132

4 Fold chnge in gene expression Fold chnge in gene expression BV CV Time in weeks with respect to vitiligo (0) IL-21 IL-10 IFN-γ IL-1β IL-6 IL-8 IL-12β IL-15 inos 0 BV CV Time in weeks with respect to vitiligo onset (0) Figure 3. Time course of the reltive expression of cytokines nd inducile nitric oxide synthse (inos) in growing fethers collected from seven SLV chickens throughout vitiligo development. Reltive expression ws clculted y the DDC t method, using cdna pool mde from growing fether of three SL chickens tht never developed vitiligo s the clirtor nd chicken 28S s the endogenous control gene. The X-xis represents the time (weeks) of fether tip collection with respect to vitiligo onset (0); BV nd CV represent dt from fethers collected from the seven SL chickens 42 weeks efore vitiligo nd 41 week fter complete pigmenttion loss, respectively; weeks 2 nd 1 represent erly vitiligo (EV) nd weeks 0 4 correspond to ctive vitiligo (AV). () Expression pttern of IL-21, IL-10, nd IFN-g. () Expression pttern of IL-1, IL-6, IL-8, IL-12, IL-15 nd inos. Also see Tle 2. disese in nture, the immune system ws shown to hve criticl role in SLV expression in susceptile chickens. Humorl immunity ws initilly implied y immunosuppressive effects of ursectomy (excision of genertive orgn for B cells), which resulted in decresed incidence nd severity of SLV in SL chickens (Lmont nd Smyth, 1981; Boissy et l., 1984). Findings from the current study lso Tle 2. Men of highest reltive expression 1, men time of highest reltive expression, nd times of significnt elevtion (Pp0.05) of cytokines nd inos in growing fethers collected efore nd throughout the development of vitiligo in SL chickens Trget gene Highest expression men 2 Time (weeks) 3 Times elevted 4 IFN-g 31.82± ±0.43 0, 1, 2, 3 IL ± ±0.53 1, 0, 1, 2, 3 IL ± ±0.46 0, 1, 2, 3 IL ± ±0.43 0, 1, 3 IL ± ±0.78 None IL ± ±0.53 0, 1, 2, 3 IL ± ± IL ± ±0.77 0, 2 IL ± ± inos 7.61± ±0.55 1, 2 1 Reltive expression ws expressed s fold chnges with respect to expression levels in fether smples from three non-vitiliginous SL chickens (clirtor smple). 2 Men±SEM of the seven SL chickens highest expression level of ech trget gene during SLV development. 3 Men time±sem (n=7) in weeks (with respect to SLV onset; week 0) when highest expression occurred. 4 Time in weeks with respect to SLV onset (week 0) when trgets were significntly (Pp0.05) elevted compred with pigmented growing fethers collected from the seven SLV chickens more thn 2 weeks efore SLV onset (BV smples). Also see Figure 3. support potentil role of B cells, s their infiltrtion into the trget tissue ws ssocited with SLV development (Figure 2 nd Tle 1). The reson for B-cell infiltrtion nd the role of B cells in the ctive SLV lesion is not cler from this study. B cells will follow similr infiltrtion signls s T cells nd cn prticipte in cellulr immunity through ntigen uptke nd presenttion, s well s ntiody secretion. Considering the lte nd low expression of IL-4 (Tle 2), cytokine supportive of humorl immunity nd isotype switching, together with the predominnt IgM þ phenotype of infiltrting B cells (Tle 1), it seems tht IL-4 is not responsile for B-cell ctivities in this utoimmune response. Evidence for B-cell prticiption in vitiligo is lso provided y the presence of melnocyte-specific utontiodies in ser of vitiliginous SL chickens nd vitiligo ptients (Hrning et l., 1991; Austin et l., 1992). Furthermore, destructive effect of vitiligo ntiodies on humn melnocytes vi complementmedited nd ntiody-dependent cellulr cytotoxicity ws demonstrted in vitro nd in vivo (Cui et l., 1993; Gilhr et l., 1995). From the current study, it is resonle to speculte tht utontiodies nd fether-infiltrting B cells re more importnt in mplifying thn inititing melnocyte loss in SLV. Phenotypic chrcteristics of infiltrting leukocytes nd cytokine profiles in the current study confirm more prominent involvement of cell-medited, specificlly Th1- medited, thn humorl immune ctivity in melnocyte loss 645

5 Tle 3. Comprison etween vitiligo in SL chickens (SLV) nd in humns SLV Humn vitiligo References Min trget tissue Growing fethers Skin Phenotype Symptom Depigmenttion in growing fethers Depigmenttion in the skin Etiology Genetics Immunology Associted utoimmune disorders Onset Autoimmune thyroiditis, lopeci-like fethering defect Erly puerty to young dults (6 14 weeks of ge) Autoimmune thyroiditis nd other disorders of immune origin Erly puerty to young dults (10 30 yers of ge) Erf, 2010; Spritz, 2010 Smyth, 1989; Erf, 2010 Severity Errtic to complete Errtic, occsionlly complete Smyth, 1989; Erf, 2010 Vitiligo susceptiility genes Multifctoril (genetic, immunologicl, metolic, nd environmentl fctors) Multigenic, identifiction in progress Multifctoril (genetic, immunologicl, metolic, nd environmentl fctors) Multigenic, cndidte genes identified Erf, 2010; Boissy nd Nordlund, 2011 Wick et l., 2006; Spritz, 2010 Intrinsic melnocyte defects Yes Yes Boissy et l., 1986, 1991 Trget tissue infiltrting leukocytes Melnocyte-specific utontiodies Cytokines 1 nd inos in the ffected tissue Mcrophges, CD4, CD8, nd B cells Mcrophges, CD4 nd CD8 cells Erf et l., 1995; Le Poole et l., 1996 Yes Yes Hrning et l., 1991; Austin et l., 1992 mifn-g mifn-g Wng, 2001; Grimes et l., 2004 mil-10 mil-10 Grimes et l., 2004 mil-6 mil-6 Moretti et l., 2002, 2009 IL-4 no chnge IL-4 no chnge Grimes et l., 2004 IL-17 low levels of expression mil-17 Bssiouny nd Shker, 2011 mil-21, mil-1, mil-8, mil-12, mil-15, nd minos Not determined Metolism Oxidtive stress Yes Yes Schllreuter et l., 1999; Erf et l., 2005 Environmentl fctor Virl ssocition Positive ssocition with live HVT vccintion t htch Possile ssocition with HCV nd HIV Arevitions: HCV, heptitis C virus; HVT, herpesvirus of turkey; inos, inducile nitric oxide synthse. 1 The expression profile of cytokines nd inos in the growing fether ws determined in the current study. Erf et l., 2001; Tsuoi et l., 2006; Seyedlinghi et l., 2009 s previously suggested (Wng, 2001; Wng nd Erf, 2003; Shi et l., 2009). Compred with the reltive proportions of B (Bu-1 þ ) cells, those of T cells, including nd gd TCR þ cells, were numericlly higher t ll vitiligo sttes (Tle 1 nd Figure 2). Moreover, lthough oth nd gd T cells infiltrted, the infiltrtion of T cells ws proportiontely greter during ctive SLV (Tle 1), n oservtion consistent with previous reports (Erf et l., 1995; Shrest et l., 1997). Moreover, in line with previous reports on SLV (Erf et l., 1995; Wng nd Erf, 2004) nd in vitiligo ptients (Le Poole et l., 1996; Le Gl et l., 2001), the current study showed the predominnce of CD8 þ compred with CD4 þ cells t ll vitiligo sttes (Tle 1). Predominnce of cytotoxic lymphocytes in ctive SLV lesions, their close juxtposition, nd ggregtion round poptotic melnocytes (Wng nd Erf, 2004), together with mcrophge infiltrtion t erly nd ctive stges of SLV, further supports Th1-medited, melnocyte-specific cellulr response in SLV. Mcrophges, which re importnt immune system ctivtors nd effector cells of cell-medited immunity, were lso present in perilesionl skin from vitiligo ptients (Le Poole et l., 1996). IFN-g, which is the signture cytokine of Th1-type cellmedited immunity nd ws mrkedly incresed in smples collected t nd fter SLV onset (Tle 2 nd Figure 3), my hve centrl role in the SLV pthomechnism. IFN-g my function in severl wys during SLV expression nd progression. IFN-g is importnt in mcrophge ctivtion, phgocytic ctivity, cytokine nd chemokine production, MHC I nd II expression, nd NO production due to trnsctivtion of inos (As et l., 2010). In the current study, incresed expression of IL-1, IL-6, IL-8, IL-12, nd inos indictes mcrophge ctivtion, which, ecuse of the reltively low levels of mcrophge infiltrtion, does not pper to e mjor mechnism y which IFN-g exerts its effects in SLV (Tle 2, Figures 2 nd 3). IFN-g ws shown to increse utophgosome formtion in humn CD4 þ T cells (Son et l., 2010) nd mcrophges (Shi nd Kehrl, 2010). Autophgosomes were reported in melnocytes from SL 646 Journl of Investigtive Dermtology (2012), Volume 132

6 chickens tht developed vitiligo (Boissy et l., 1983, 1986), which my hve een promoted y elevted levels of IFN-g in the environment of the melnocyte. In ddition, IFN-g is le to stimulte the expression of MHC I nd MHC II molecules on vrious types of cells. In the current study, the heightened nd sustined proportions of MHC II-expressing cells in EV, AV, nd CV smples compred with NV smples (Figure 2) re strong indiction of n immunologiclly ctive site, with IFN-g supporting ntigen presenttion nd mcrophge, B-cell, nd T-cell ctivtion. Finlly, IFN-g hs een shown to hve inhiitory effects on Th17-cell development from CD4 þ precursor cells (Kelchtermns et l., 2008), which my explin the reltively low IL-17F expression nd the sence of heterophils (vin equivlent to neutrophils) in fethers from vitiliginous SL chickens in the current study. On the sis of the ove discussion, IFN-g ppers to hve n essentil role in SLV pthogenesis. As IFN-g expression ws not preceded y IL-12 nd IL-15 (two cytokines cple of IFN-g induction) production, the question regrding the mechnisms driving IFN-g expression needs to e ddressed. Review of recent literture reveled importnt positive interreltionships etween IFN-g nd IL-10 nd IL-21, the other cytokines with mrked expression in EV nd AV smples (Tle 2 nd Figure 3). IL-10 is generlly known s cytokine with nti-inflmmtory nd immunosuppressive ctivities (As et l., 2010). IL-10 hs, however, recently een shown in humns nd mice to hve immunostimultory effects y incresing IFN-g production nd ctivtion of nturl killer cells nd cytotoxic T lymphocytes (Shit et l., 1998; Luw et l., 2000; Tilg et l., 2002). A pre-stimulted immune system nd the presence of high levels of IL-10 re two importnt fctors tht fvor immunostimultory effects of IL-10. Both these preconditions re met in SLV. Mrked elevtion of IL-10 expression ws oserved in the current study, together with heightened inflmmtory immune ctivities, such s mononucler cell infiltrtion, expression of IL-8 chemokine, IFN-g, nd MHC II expression in EV nd AV fether tips. The source of immune ctivtion my e the routine vccintion t htching with live herpesvirus of turkey ginst lymphomcusing Mrek s disese virus. Herpesvirus of turkey trnsloctes to nd infects the fether follicle epithelium (Cho, 1975), nd hs een identified s relile environmentl trigger of SLV expression in susceptile SL chickens (Erf et l., 2001). Recent cytokine gene expression nlysis t the trnscriptome level y Adul-Creem et l. (2008) showed tht herpesvirus of turkey dministrtion in chickens resulted in concomitnt increse in IFN-g nd IL-10 expression in fethers reching pek levels t 7 dys nd returning to levels of control y 10 dys post vccintion. IL-21, cytokine with pleiotropic functions, is the trget of intensive investigtions prtilly ecuse of its ility to induce differentition nd expnsion of Th17 cells in chronic inflmmtory diseses (Nuriev et l., 2007). Low expression of IL-17F nd the sence of heterophils in SLV lesions do not justify pthologicl role of IL-21 through Th17 cells in SLV. IL-21 ws lso shown to e required for effector function nd sustinility of CD8 þ T cells, including cytotoxic ctivities nd IFN-g production, in chronic virl infection (Elsesser et l., 2009; Frohlich et l., 2009; Yi et l., 2009); inhiition of Foxp3 þ Treg cell development, which indirectly enhnced ctivity of CD8 þ T cells (Li nd Yee, 2008); nd induction of IL-10 expression in ctivted CD4 nd CD8 T cells in mouse model of systemic lupus erythemtosus (Spolski et l., 2009). Therefore, overexpression of IL-21 my hve n underestimted role in the pthogenesis of SLV directly through its positive effects on cytotoxic T-cell function nd IFN-g expression, nd indirectly through its support of immunostimultory effects of IL-10. Tken together, SLV is n utoimmune disese in which melnocyte loss is ssocited with mononucler leukocyte infiltrtion consisting predominntly of T cells (with more CD8 þ thn CD4 þ cells), followed y B cells nd mcrophges. Cytokine expression in the trget tissue in the sme individuls throughout SLV development reveled concomitnt increses in the reltive expression of IFN-g, IL-10, nd IL-21 efore nd during ctive melnocytes loss. The mechnisms underlying this cytokine signture nd the interreltionship of immune cells nd cytokines in melnocyte loss need to e further exmined to gin insight into the etiology nd pthology of the spontneous expression of vitiligo in susceptile SL chickens. MATERIALS AND METHODS Experimentl design Animls. Twenty SL chicks, homozygous for the B101 MHC hplotype, were rndomly selected from 20 fmilies t the time of htching, vccinted with live herpesvirus of turkey (Fort Dodge Animl Helth, Fort Dodge, IA), nd ssigned to one floor pen t the Arknss Experiment Sttion Poultry Frm in Fyetteville, Arknss. A stndrd rering protocol ws followed, nd feed nd wter were provided d liitum. Animl use ws pproved y the University of Arknss Institutionl Animl Cre nd Use Committee. Fether collection Smples (six fether tips per ird; Figure 1) were collected weekly from chickens strting t 5 nd ending t 18 weeks of ge. Hlf of the fether tips were snp-frozen in Tissue-Tek OCT freezing medium (Skur Finetek, Torrnce, CA) nd the other hlf were plced in RNAlter (Qigen Vlenci, CA). All smples were stored t 80 1C until use. SLV ws scored t ech fether collection following previous criteri (Wng nd Erf, 2003). Bsiclly, SLV ws given score of 1 to 5 if the newest growth of 0%, p20, 420 nd p60, 460 nd p99, nd 100% of the growing fethers demonstrted visile depigmenttion. Upon completion of smple collection, fether tips from seven SL chickens tht developed SLV nd reched score of 5 during the 18-week period (SLV chickens) nd three SL chickens tht never developed SLV (NV chickens) were used in this study. Indirect immunoperoxidse stining of growing fethers Longitudinl sections (6 mm thick) were cut t 19 1C in cryostt (CM3050-S, Leic Microsystems, Bnnockurn, IL), processed nd immunochemiclly stined s descried in Erf et l. (1995). Primry ntiodies were mouse mas specific for chicken TCR gd (ctcr gd), ctcr1, ctcr2, ccd4, ccd8, cbu-1 (B cells), cigm, ckul

7 (mcrophges), nd cmhc II molecules (Southern Biotechnology Assocites, Birminghm, AL). Imge nlysis Imge nlysis ws crried out under right-field microscope ( 40 mgnifiction), nd ImgePro Plus softwre ws used (Medi Cyernetics, Silver Spring, MD). The mount of positively stined cells ws expressed s the percentge of the whole tissue section (epidermis nd pulp) nlyzed (% re). All evlutions were mde y the sme person. RNA isoltion, quntifiction, nd cdna synthesis For ech chicken nd time point, the three fether tips preserved in RNAlter were homogenized y Tissue Teror (BioSpec Products, Brtlesville, OK, Model: ) in lysis uffer provided in the Qigen RNesy Mini kit (Qigen, Vlenci, CA). Totl RNA ws isolted from homogentes with on-column DNA digestion (Qigen). RNA ws eluted in 30 ml RNse-, DNse-free wter nd stored t 80 1C until use. RNA integrity nd concentrtion were determined s previously descried (Hml et l., 2010). RNA (175 ng per smple) ws trnscried to cdna in 30 ml rection volume using High-Cpcity cdna reverse trnscription kit ccording to the mnufcturer s protocol (Applied Biosystems, Foster City, CA). The resulting cdna ws liquotted nd stored t 80 1C until nlysis. Reltive expression of cytokines nd inos Primers nd proes used in this study re ville upon request. Reltime PCR ws performed ccording to Hml et l. (2010). The clirtor smple ws pool of cdna prepred from fether tips of the three SL chickens without vitiligo (NV). The reltive gene expression ws determined y the ddc t method (Wong nd Medrno, 2005). Sttisticl nlysis To determine the effect of SLV stte, one-wy nlysis of vrince (SYSTAT softwre, Chicgo, IL) ws conducted using the generl liner model procedure, followed y Fisher s LSD multiple mens comprison. For the time course of gene expression, EV, AV, nd CV smples were compred with BV smples collected from the sme SLV chickens. 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