Different distribution patterns of plasmacytoid dendritic cells in discoid lupus erythematosus and lichen planopilaris demonstrated by CD123 immunostaining,

Anais

Brasileiros

de

Dermatologia

www.anaisdedermatologia.org.br

INVESTIGATION

Different

distribution

patterns

of

plasmacytoid

dendritic

cells

in

discoid

lupus

erythematosus

and

lichen

planopilaris

demonstrated

by

CD123

immunostaining

夽,夽夽

Azadeh

Rakhshan

_,

_Parviz

_Toossi

_,

_Maliheh

_Amani

_,

_Sahar

_Dadkhahfar

_,

Arash

Bagheri

Hamidi

a_{DepartmentofPathology,Shohada-eTajrishHospital,ShahidBeheshtiUniversityofMedicalSciences,Tehran,Iran} b_{SkinResearchCenter,ShahidBeheshtiUniversityofMedicalSciences,Shohada-eTajrishHospital,Tehran,Iran}

Received14April2019;accepted10November2019 Availableonline20March2020

KEYWORDS Alopecia; Dendriticcells; Discoid; Lupuserythematosus Abstract

Background: Clinical and histological features may overlap between lichen planopilaris-associatedanddiscoidlupuserythematosus-associatedscarringalopecia.

Objectives: Theaimofthisstudywastodemonstratethecutaneousinfiltrationofplasmacytoid dendriticcellsandtocomparetheirdistributionpatternindiscoidlupuserythematosus and lichenplanopilaris.

Methods: Twenty-fourcasesofdiscoidlupuserythematosusand30casesoflichen planopila-riswereexaminedforimmunostainingoftheCD123marker.Thepercentageanddistribution patternofplasmacytoiddendriticcellsandthepresenceoftheplasmacytoiddendriticcells clusterswereevalutedinthesamples.

Results: Thenumberofplasmacytoiddendriticcellswashigherinthediscoidlupus erythema-tosusspecimens.Aggregationsof10cellsormore(largecluster)wereobservedinhalfofthe discoidlupuserythematosusspecimensandonly2lichenplanopilaris,with50%sensitivityand 93%specificityfordifferentiatingdiscoidlupuserythematosusfromlichenplanopilaris. Studylimitations: Incidenceandprevalenceofdiscoidlupuserythematosus-associatedscarring alopeciainthescalparelow,sothesamplessizeofourstudywassmall.

夽 _{Howtocitethisarticle:RakhshanA,ToossiP,AmaniM,DadkhahfarS,HamidiAB.Differentdistributionpatternsofplasmacytoiddendritic} cellsindiscoidlupuserythematosusandlichenplanopilarisdemonstratedbyCD123immunostaining.AnBrasDermatol.2020;95:307---13.

夽夽_{StudyconductedattheSkinResearchCenter,ShahidBeheshtiUniversityofMedicalSciences,Shohada-eTajrishHospital,Tehran,Iran.} ∗_{Correspondingauthor.}

E-mail:ml.amani@yahoo.com(M.Amani). https://doi.org/10.1016/j.abd.2019.11.005

0365-0596/©2020SociedadeBrasileiradeDermatologia.PublishedbyElsevierEspa˜na,S.L.U.ThisisanopenaccessarticleundertheCC BYlicense(http://creativecommons.org/licenses/by/4.0/).

Conclusions: Wesuggestthataplasmacytoiddendriticcellsclusterof10cellsormoreishighly specificfordistinguishingdiscoidlupuserythematosusfromlichenplanopilaris.Italsoappears thatCD123immunolabelingisvaluableinbothactiveandlatestagesofthedisease.

©2020SociedadeBrasileiradeDermatologia.PublishedbyElsevierEspa˜na,S.L.U.Thisisan openaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/).

Introduction

Scarringalopecia (cicatricial alopecia)refers to disorders that are characterized by the irreversible destruction of hairfolliclesandpermanentalopecia.1---4_{Scarringalopecia}

iscategorizedintoprimaryandsecondary.Primaryscarring alopecia(cicatricialalopecia)impliesdisordersthat direc-tlydamagehair follicles suchaslichenplanopilaris (LPP), discoidlupuserythematosus(DLE)anddissectingcellulitis. Secondaryscarringalopeciaiscausedbyunwanteddamage tohairfollicles asaresultofinflammatoryprocessessuch as tinea capitis, sarcoidosis, and radiation dermatites.2---4

LPP and DLE are the most common causes of scarring alopecia.5 _{Clinically,both LPP and DLE appear similar on}

the scalp making the diagnosis difficult. Several dermos-copicfindingssuchasfollicularplugs,speckled patternof blue-graydots and whiteareas have been reportedtobe characteristicofDLEandtargetpatternofblue-graydots, perifollicularscallingtobecharacteristicofLPP.6_However,

overlapofthefindingsmightmakethehistological evalua-tionnecessary.5 _{Sometimes,histologicalfeaturesmayalso}

overlapwith each other, especially in pauci-inflammatory latefibrosingstages5,7_{;consequently,establishingadefinite}

diagnosis may bechallenging anddifficult basedon clini-cal, dermoscopic and histological features alone.5 _These

concernsunderscore theneed for useful adjunct diagnos-tic techniques, including direct immunofluorescence5 _and

Immunohistochemistry(IHC).8,9

Plasmacytoid dendritic cells (PDCs) are antigen-presenting cells thatplay an importantrole inthe innate immuneresponse.Theyarenottypicallyfoundinthe nor-malskin,10_{butthepresenceofthesecellsininflammatory}

diseases, infections, and malignancies is notable.11 _For

example,thesecellshavebeenidentifiedintheskinlesions of patients with lupus erythematosus,12 _{lichen planus,}13

dermatomyositis,14_{Sjogrensyndrome,androsácea.}15

Seve-ralstudieshavedemonstratedthesignificantroleofPDCsin thepathogenesisoflupuserythematosusbyproducingtype 1interferon.ThisrolehasbeenrecognizedviaCD123 immu-nolabeling,asasurfacemarkerofPDCs.12,15,16_Thepresence

ofthesecells andtheirspecific distributionpatterninthe tissue may help to distinguish lupus erythematosus from otherinflammatorydisorderssuchasLPP.8,17,18

Giventhataproperdiagnosisofinflammatoryconditions thatleadtothedestructionofhairfolliclesismandatoryfor thepreventionofpermanentscarring,weaimedtoperform thecurrentstudytoevaluatethepresenceanddistribution patternofPDCsbyimmunostainingtheCD123markerin pati-entswithLPPandDLE,whicharethemostcommoncauses ofprimaryscarringalopecia.

Methods

Subjectsanddatacollection

Thisretrospectivestudywasbasedonavailabledataatthe PathologyDepartmentofShohada-eTajrishHospitalinthe period from January 2014 to April 2018. Only scalp lesi-onsthathadbothclinicalandhistopathologicalcriteriafor a diagnosis of scarring alopecia consistent with ‘‘discoid lupuserythematosus’’or‘‘lichenplanopilaris’’were inclu-ded. For all the patients, the histopathological diagnosis wasbasedonH&Estainedsectionsand3specialstainings, including PAS (Periodic Acid Schiff) for the evaluation of basement membranethickening, alcianblueat pH2.5for demonstrating thepresence and pattern of dermalmucin depositionandorceinstainingfortheevaluationofelastic fibers.The histopathologicalcriteriaforDLEwere compri-sedofinterfacedermatitisat theinterfollicular epidermis andthe perifollicularregion,denseperivascular and peri-adnexal lymphoid infiltration (both superficial and deep dermal),increaseddermalmucindeposition,andthickening ofthebasementmembrane.ThehistologicalcriteriaforLPP wereperifollicularinterfacedermatitisat thelevel ofthe infundibulumandtheisthmusandalackofinvolvementat theinferiorsegmentofthehair follicleandinterfollicular epidermis. Overall, 24 cases of DLE and 30 cases of LPP were included. Allthe caseswere categorizedinto2 sta-gesaccordingtotheamount of inflammationandfibrosis: theearly/activestage,characterizedbylymphocytic infil-trationandtheabsenceoffibrosis,andthelatestage,which showeddestroyedhairfolliclesandreplacementbyfibrous tractsandminimalorabsentlymphocyticinfiltration.19,20

Immunohistochemicalstaining

Formalin-fixed paraffin-embedded tissue sectionsfrom all the cases were immunostained for the CD123 marker, mouse anti-human CD123 (Interleukin-3 Receptor Alpha Chain) monoclonal antibody (Clone 7G3) (Master Diagnos-tica, Spain).Fromthe paraffinblocks,4␮m(micrometer) tissue sectionswereprepared oncharged slidesandthen incubated overnight at 37◦C. The sectionswere deparaf-finized, rehydrated, and exposed to epitope retrieval by heat (boiling tissue in EDTA buffer at pH 9 for 30min at 95◦C). The process was followed by rinsing in distilled waterfor3---5changesandcoolingatroomtemperaturefor 20min.Endogenousperoxidasewasblockedusinga peroxi-dasesolutionfor10minatroomtemperature.Theprimary antibody(whichwasreadytouse)wasincubatedfor30min, followed byEnVision for 30min at 37◦C and rinsing with

phosphate-buffered saline and the IHC wash buffer. Thereafter, the antibody was detected by DAB (3,3 -diaminobenzidine) for 2min and was, then, washed with distilledwater.Slidemountingwascarriedoutafter hema-toxylincounterstaining.

ScoringofCD123staining

Three parametersofPDCs were mainlyevaluatedon IHC-stainedsections(Table1):

(1) ThepercentageofPDCs(CD123+),calculatedby divi-ding the PDC count by the mononuclear cell count at 3 separate regions that had the most promi-nent infiltration in serial sections. Then, the PDC content was defined as score 1 (PDC<10%), score 2 (10%≤PDC<20%),andscore3(PDC≥20%)(18). (2) The predominant anatomic location of the PDC

infiltrate,includingtheperifollicular,perivascular, peri-eccrine,subcutaneous,intrafollicular,dermoepidermal junction,andintraepidermalregions.

(3) ThepresenceofthePDCcluster,definedasthe aggrega-tionofatleast5PDCs.Aggregationsof5---10PDCswere consideredassmallclustersandaggregationsof more than 10 PDCs were considered as large clusters.17,18

Additionally, theanatomiclocationof smallandlarge clusterswasdeterminedaccordingtothelocations des-cribedabove.

Statisticalanalysis

ThepercentageofPDCswascomparedbetweenthe2groups usingtheindependentsamplet-test.Thedistribution pat-ternsof PDCswerecomparedbetween DLEand LPPusing theFisherexacttest.Thepresenceofclusterswas compa-redbetweenthe2groupsusingthe2_{test.Ap-valueofless} than0.05wasconsideredstatisticallysignificant.

*Allproceduresperformedinthisstudyinvolvinghuman participantswereinaccordancewiththeethicalstandards ofourinstitutionalresearchcommitteeandwiththe1964 Helsinkideclarationanditslateramendmentsor compara-bleethicalstandards.

This study was approved by our institutional research committeewithno.‘‘IR.SBMU.SRC.REC.1395.43’’.

Results

Baselinecharacteristics

In total,54 IHC-stained slideswere examined. DLE speci-mensweretakenfrom24patients(7malesand17females) at amean ageof 45.33±12.47 years,andLPP specimens weretakenfrom30patients(12malesand18females)at a mean age of 49.50±13.52 years. There was no signifi-cant difference between the 2 groups regarding the age (p=0.245).Nine(37.5%)casesofDLEand15(50%)casesof LPPwereintheearly/activestageofdisease,and15(62.5%) casesofDLEand15(50%)casesofLPPwereinthelatestage ofdisease.Therewasnosignificantdifferencebetweenthe 2groupsregardingthestageofdisease(p=0.3580).

60 50 40 30 20 10 0 LPP Groups P ercentage of PDCs DLE

Figure1 Percentage ofplasmacytoiddendriticcellsin

dis-coidlupuserythematosus (DLE)andlichenplanopilaris (LPP). Middlepoint,median;Box,interquartilerange;Whisker,range (excludingoutliers).

Percentageandanatomicdistributionpatternof PDCs

The mean percentage of PDCs was 14.87±7.99 (range: 4%---34%) in the LPP cases and 25.45±10.78 (range: 10%---52%)intheDLEcases,whichshowedasignificant dif-ferencebetweenthe2groups(p<0.001)(Fig.1).IntheDLE cases,0/24(0%),6/24(25%), and18/24(75%)of the spe-cimenswere scored 1,2 and 3,respectively. For theLPP cases,9/30(30%),14/30(46.6%),and7/30 (23.3%)ofthe specimenswerescored1,2and3,correspondingly, which wasstatisticallysignificant(p<0.001)(Table1).

Theperifollicularregionwasthemostcommonlocation involved(allthecasesofDLEandLPPhadperifollicular infil-trations)(Fig.2).Perivascularinvolvementwasobservedin 29/30(96.7%)oftheLPPcasesand22/24(91.7%)oftheDLE cases,whichwasnotstatisticallysignificant(p=0.579).The infiltrationofthedermoepidermaljunctionwasdetectedin 15/24(62.5%)oftheDLEcasesand2/30(6.7%)oftheLPP cases,whichwasstatisticallysignificant(p<0.001). Subcu-taneousinvolvementwasidentifiedin11/24(45.8%)ofthe DLEcases and6/30 (20%) of theLPP cases;this differen-tiation wasstatistically significant (p=0.042). Perieccrine infiltrationwasobservedin 4/24(16.7%) ofthe DLEcases and0/30 oftheLPPcases,showingstatisticalsignificance (p=0.034). Interstitial infiltration was observed in 14/30 (46.7%)oftheLPPcasesand2/24(8.3%)oftheDLEcases, whichwasalsostatisticallysignificant(p=0.02). Intrafolli-cularinvolvementwasdetectedin3/24(12.5%)oftheDLE casesand0/30oftheLPPcases(Fig.2),whichwasnot sta-tisticallysignificant.IntraepidermalinfiltrationofPDCswas observedneitherintheDLEcasesnorintheLPPcases(Fig.3 showstheanatomicaldistributionpatternofPDCs).

PDCclusterformation

Fifteensmallclusters(aggregationof5---10cells)were iden-tified.Tenofthesesmallclusterswereobservedin10cases of LPP (33.33%) and 5 of them were observed in 4 cases ofDLE(16.7%). Thelocationoftheseclustersin bothLPP

Table1 Comparisonsofthepercentages,clusterformation,andanatomiclocationofPDCsbetweenDLEandLPP. DLE LPP p-Value MeanpercentageofPDCs 25.45±10.78 14.87±7.99 <0.001 PDCscore Score1(<10%) 0 9(30%) <0.001 Score2(10_≤PDC<20%) 6(25%) 14(46.6%) Score3(≥20%) 18(75%) 7(23.3%) Clusterformation Smallcluster 4(16.7%) 10(33.3%) 0.002 Largecluster 12(50%) 2(6.7%) AnatomiclocationofPDCs Epidermal 0 0 ---DEJ 15(62.5%) 2(6.7%) <0.001 Interstitial 2(8.3%) 14(46.7%) 0.020 Intrafollicular 3(12.5%) 0 0.082 Perifollicular 24(100%) 30(100%) ---Perivascular 22(91.7%) 29(96.7%) 0.579 Perieccrine 4(16.7%) 0 0.034 Subcutaneous 11(45.8%) 6(20%) 0.042

Smallcluster,aggregationsof5---10PDCs;largecluster,aggregationsof10PDCsormore;DLE,discoidlupuserythematosus;LPP,lichen planopilaris;PDCs,plasmacytoiddendriticcells;DEJ,dermoepidermalJunction.

Figure2 Representative discoidlupus erythematosus case:

CD123immunohistochemistrypresenceofPDCsatthe intrafol-licularandperifolliculararea(40_×).

andDLEwasperifollicularandperivascular.Twenty-sixlarge clusters(aggregationof 10cells ormore)were identified. Threeoftheselargeclustersweredetectedin2(6.7%)cases of LPP and 23 of them were identified in 12 (50%) cases ofDLE.Therewasasignificantdifferencebetweenthese2 groupsregardingclusterformation(p=0.002).Thepresence oflargeclustershad50%sensitivityand94%specificityfor thedifferentiationofDLEfromLPP(Fig.4AandB).

Theanatomicsitesofthelargeclusterswerealso iden-tified.The largeclustersintheDLEspecimenswereoften observedattheperifollicularregion(21/23oftheclusters). Onelarge cluster was observed at the dermal---epidermal junctionregion,andanotheronewasobservedatthe sub-cutis.IntheLPPspecimens,thelargeclusterswereobserved

attheperifollicular(1),perivascular(1),andinterstitial(1) regions.

Half of the LPP cases with small clusters were in the early/activestageofdiseaseandtheotherhalfwereinthe latestageofdisease,whichwasnotstatisticallysignificant (p=1.000).OfalltheDLEcaseswithsmallclusters,onecase hadactivediseaseand3caseswereatthelatestageof dise-ase,whichwasnotstatisticallysignificant (p=1.000).Two casesofLPPhadlargeclusters:1intheactivestageof dise-aseand theother onein thelatestage ofdisease, which wasnotstatisticallysignificant(p=1.000).Ofthe12cases of DLEwithlargeclusters,4cases hadactivediseaseand 8 cases wereat the late stageof disease, which wasnot statisticallysignificant(p=0.673).

Discussion

Lupuserythematosus isanautoimmunediseasethat invol-ves multiple organs. PDCs andtype 1interferonshave an importantroleinthepathogenesisoflupuserythematosus. StudieshavedemonstratedthatPDCsmigratefrombloodto thesiteofinflammationsuchastheskinandthekidneyin lupuserythematosus.16 _{Largenumbersandaggregationsof}

PDCshavebeendescribedinmanytypesofcutaneouslupus (e.g.acutelupusandDLE).PDCshavebeenshowntohave greaternumbersincutaneouslupuserythematosusthanin otherinflammatoryskin diseasessuchasdermatomyositis, rosacea,andlichenplanus.12,14,15,21_{Ourstudyshowedahigh}

percentageofPDCsinscarringalopeciaduetoDLEby com-parisonwithLPP(approximately26%vs.15%).

PDCs have been demonstrated in the mucosal and cutaneouslesionsoflichenplanus.22,23_{Thenumberofthese}

cellsvariesinthedifferentstudieshavingbeenconducted hitherto. In some studies, a low percentage of PDCs has been reportedinLPP(<10%)(8,17,18),whereasahigher

100 90 80 70 60 50 40 30 20 10 0

Perifollicular Perivascular Interstitium Subcutis

DLE LPP

P

e

rcentage of samples

Distribution of PDCs pattern

DEJ Perieccrine Intrafollicular

Figure3 Distributionpatternsofplasmacytoiddendriticcells(PDCs)inlichenplanopilaris(LPP)anddiscoidlupuserythematosus

(DLE).

A

B

Figure4 Representativelichenplanopilaris:presenceofPDCs(noclusterformation),20×(A)andrepresentativediscoidlupus

erythematosuscase:largecluster(aggregationof10cellsormore)ofplasmacytoiddendriticcells,40×(B).

percentage of PDCs in oral lichen planus (20%) has been shown.23 _{Inour study,thepercentage ofPDCs in LPPwas}

approximately15%, whichis higher thanthe figure in the studiesbyFeningandSeleimans.8,18

The predominant anatomicsite of PDC infiltration can alsovaryin theskin tissuesof both DLE andLPP.Sleiman et al. reported the presence of PDCs in deep dermis, perieccrine,anddermoepidermal junctionareasinalmost allDLEspecimens,whiletheinfiltrationofthesecellsatthe dermoepidermaljunctionwasobservedinonlyasmall num-berofLPPspecimensanddeepdermisandtheperieccrine regionwerenotinvolved inLPPspecimens.18 _Inthestudy

ofFeningetal.,perifollicularandperivascularinvolvement wasobservedinalmostallDLEspecimensandsmallnumbers ofLPPspecimens,aswellasperieccrineandperifollicular involvement,wereobservedinmanyDLEspecimensbutnot inLPPspecimens.Intheirstudy,theintrafollicular involve-mentofPDCswasobservedneitherinDLEnorinLPP.8_Inour

study,theperifollicularandperivascularregionsshowedthe highestdensityofPDCsinbothDLEandLPP.Vertical histo-pathologicalsectionsinourstudyenabledtheevaluationof epidermisandthedermoepidermaljunction,whichcannot beassessedwhenthesectionsarehorizontal.NeitherDLE

nor LPP showed the intraepidermal infiltration of PDCs. Theinvolvementofthedermal---epidermaljunctionandthe subcutaneous region was notable in our DLE specimens. We detected perieccrine and intrafollicular infiltration in asmallnumberoftheDLEcasesbutnotinanyoftheLPP cases(Fig.2).Interstitialinfiltrationwasobservedinmany ofthe LPPcasesand smallnumberof theDLE cases. Our studyisthefirststudyofitskindtoevaluatethe subcutane-ousregionforthepresenceofPDCsinDLEandLPP,andwe detectedthesecellsatthesubcutaneoustissueinbothDLE andLPP.ItshouldbenotedthatPDCsweremostlyobserved alongthedeeppartofhairfolliclesatthesubcutaneous tis-sueoftheLPPspecimens,whiletheyweremorescattered throughthesubcutisintheDLEspecimens(Fig.5).

In our study, we observed large clusters (aggregations of 10 cells or more) in half of the DLE specimens but in only 2 cases of LPP which had 50% sensitivity and 94% specificityfor differentiationofDLEfromLPP.Theselarge clusterswere often localized at the perifollicular region. Theodoreetal.reportedaggregationsof 20cells ormore in60%offaciallupusspecimens.15 _{Sleimanetal.detected}

aggregationsof10cellsormorein100%ofDLEcaseswith scarringalopecia.18 _{Other studies havedetected a few or}

A

B

Figure5 Presenceofplasmacytoiddendriticcellsscattered

throughthesubcutisinacaseofdiscoidlupuserythematosus, 40_×(A)andmostlyaroundafollicularstelaatthesubcutisina caseoflichenplanopilaris,40×(B).

noclustersofPDCsinothercausesofscarringalopeciasuch asLPP, frontal fibrosing alopecia, and central centrifugal cicatricial alopecia.8,17,18 _{Koliver et al. estimated that}

clusters of 5 cells or more had 77% sensitivity and 89% specificitytodistinguish DLEfrom LPP.17 _{In ourstudy,the}

smallclusters(aggregationof5---10cells)didnothaveany diagnosticvalueindifferentiatingLPPfromDLE.

Fening etal. excluded cases that did not have signifi-cantinflammationofmononuclearcellsandthenidentified aggregations of 10PDCs or moreand 20 PDCs or more in 94.1% and 82.4% of cases of DLE with scarring alopecia, respectively.8_{Accordingtoourstudy,clusterformationcan}

be seen in both active (prominent inflammation without fibrosis)andlatestages(prominentfibrosiswithminimalor withoutinflammation)ofdisease.

Conclusion

Basedonourstudy,itappears thatthedetectionof PDCs byimmunostainingtheCD123markerisanacceptable diag-nosticmethod for the diagnosis of DLE and the presence ofclustersof 10cells or morehashigh specificityfor the diagnosisofDLE-associatedscarringalopecia.Italso appe-arsthatCD123immunolabelingisvaluableinbothactiveand latestagesofdisease.

Financial

support

Nonedeclared.

Authors’

contributions

Azadeh Rakhshan: Elaboration and writing of the manus-cript; obtaining, analysis,and interpretation of the data; criticalreviewofthemanuscript.

ParvizToossi:Approvalofthefinalversionofthe manus-cript;conceptionandplanningofthestudy;criticalreview ofthemanuscript.

MalihehAmani: Statisticanalysis;conception and plan-ningofthestudy;elaborationandwritingofthemanuscript; obtaining,analysis,andinterpretationofthedata;effective participationinresearchorientation.

Sahar Dadkhahfar:Obtaining, analysis,and interpreta-tionofthedata;criticalreviewofthemanuscript.

ArashBagheriHamidi:Statisticanalysis;obtaining, analy-sis,andinterpretationofthedata.

Conflicts

of

interest

Nonedeclared.

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