w w w . r e u m a t o l o g i a . c o m . b r
REVISTA
BRASILEIRA
DE
REUMATOLOGIA
Original
article
MHC
class
I
antigens,
CD4
and
CD8
expressions
in
polymyositis
and
dermatomyositis
Carla
Renata
Grac¸a
∗,
João
Aris
Kouyoumdjian
FaculdadedeMedicinadeSãoJosédoRioPreto,RioPreto,SP,Brazila
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received3June2014 Accepted6October2014 Availableonline5January2015
Keywords:
Musclepathology
Muscleimmunohistochemistry Musclebiopsy
Majorhistocompatibilitycomplex classIantigens(MHC-I)
Inflammatorymyopathies
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Objective:Toanalyzethefrequenciesoftheexpressionofmajorhistocompatibilitycomplex classI(MHC-I)antigens,andCD4andCD8cellsinskeletalmuscleinpolymyositis(PM)and
dermatomyositis(DM).
Methods:Thiswasaretrospectivestudyof34PMcases,8DMcases,and29controlpatients withnon-inflammatorymyopathies.
Results:MHC-Iantigenswereexpressedinthesarcolemmaand/orsarcoplasmin79.4%ofPM cases,62.5%ofDMcases,and27.6%ofcontrols(CD4expressionwasobservedin76.5%,75%, and13.8%,respectively).TherewasahighsuspicionofPM/DM(mainlyPM)inparticipants inwhomMHC-IantigensandCD4wereco-expressed.In14.3%ofPM/DMcases,weobserved MHC-Iantigensexpressionalone,withoutinflammatorycells.
Conclusion: MHC-IantigensexpressionandCD4positivitymightaddtostrongdiagnostic suspicionofPM/DM.Nocellularinfiltrationwasobservedinapproximately14.3%ofsuch cases.
©2014ElsevierEditoraLtda.Allrightsreserved.
Expressão
de
antígenos
MHC
classe
I
e
de
células
CD4
e
CD8
na
polimiosite
e
dermatomiosite
Palavras-chave:
Patologiamuscular
Imuno-histoquímicamuscular Biópsiamuscular
Antígenosdecomplexoprincipalde histocompatibilidadeclasseI (MHC-I)
Miopatiasinflamatórias
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e
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o
Objetivo:Analisarasfrequênciasdeexpressãodosantígenosdecomplexoprincipalde histo-compatibilidadeclasseI(MHC-I)ecélulasCD4eCD8nomúsculoesqueléticonapolimiosite
(PM)edermatomiosite(DM).
Métodos:Estudoretrospectivode34casosdePM,8casosdeDMe29controlescommiopatias nãoinflamatórias.
Resultados: OsantígenosMHC-Iexpressaram-senosarcolemae/ousarcoplasmaem79,4%
doscasosdePM,62,5%doscasosdeDMe27,6%doscontroles(aexpressãodeCD4foi
∗ Correspondingauthor.
E-mail:cgraca@hotmail.com(C.R.Grac¸a).
http://dx.doi.org/10.1016/j.rbre.2014.10.005
groupofautoimmune diseases that are characterized clin-ically by weakness and inflammation in skeletal muscle. Accurate diagnosis is critical, as IM are potentially treat-able myopathies. Based on clinical and histopathological characteristics, three main subgroups of IM were defined: polymyositis(PM),dermatomyositis(DM),andinclusionbody myositis(IBM).Thediagnosisofthesediseasesisbasedon clinicalexaminationandlaboratorytests, particularly crea-tinekinase,electromyogram,andmusclepathologyfindings obtainedafterbiopsy.1–3Thehallmarksforcorrectdiagnosis
onmuscle biopsyare muscle fibernecrosis(usually in iso-lated spots)and the presenceof inflammatorycells inthe perimysialandendomysialregions,andalsooftenin perivas-cularregions.Atypicalfinding islymphocyticnon-necrotic fiberinvasion,which issoon replacedbymacrophagesand T-cellsafteritbecomesnecrotic.4Perifascicularatrophyis
spe-cifictoand ahallmark featureofDM,as rimmed-vacuoles areforIBM.4Itiswellrecognizedthattheabsenceof
inflam-matoryinfiltratesdoesnotexcludeanIM.Inthesecases,the presenceofmajorhistocompatibilitycomplexclassI(MHC-I) antigensinsarcolemmaand/orsarcoplasmmightcontribute todiagnostic suspicionofIM, althoughit isnotspecific to IM.
Innormalmusclefibers,MHC-Iantigensareonlydetected on blood vessels and can be easily seen on endomysial capillaries.Incontrast,inIMMHC-Iantigensexpressionwere observedonthesarcolemmaandalsointernally(sarcoplasm) inseveralfibers.5,6MHC-Iantigensinductionandexpression
occursearly, frequentlybeforethe inflammatoryinfiltrates, andcontinuesthroughouttheevolutionofthischronic dis-ease, even with the use of immunosuppression and after apparentclinicalremission.1,7–10
Regenerating and/or immature muscle fibers exhibited consistent MHC I antigens sarcolemmal expression irre-spective of disease;11 because of that, it is important to
distinguishthisnormalfinding fromtheabnormallabeling on mature fibers by using a marker for immaturity, such as neonatalmyosin.4 Theexpression ofMHC-I in the
sar-colemmaand/orthe sarcoplasmofmaturemusclefibers is abnormaland representsauseful toolforthe diagnosis of IM, particularly in the absence of inflammatoryinfiltrates, musclefibernecrosis,rimmedvacuoles,orperifascicular atro-phy.
The present study was figured out to emphasize the importance of routinely making the MHC I antigens together with subtypes of T cells expression on muscle
Material
and
methods
Patients
Seventy-one patients assisted at the Hospital de Base, Faculdade de Medicina de São José do Rio Preto (FAMERP) were studied, from June 2005 to June 2013. They were referred for muscle biopsy from several medical specialties, particularly neurology and rheuma-tology.
Twopatientgroupswereconstitutedforevaluation.Group 1 consisted of 42 patients with a consistent clinical pic-ture and muscle pathology characteristic ofPM or DM, as follows: muscle necrosis, endomysial and/or perivascular inflammatory infiltrate (Fig. 1A and B), invasion of non-necrotic musclefibers,and/orperifascicularatrophy.Group 2 consisted of29 patients referred formuscle biopsy with suspicionofamyopathyother thanIM,butwithnormalor non-specificandnon-inflammatorymusclepathology abnor-malities.
Musclebiopsy
All muscle biopsies from Deltoideus were performed by a physicianspecializedinneuromusculardisorders,usingan opentechniqueunderlocalanesthesia.Eachmusclesample wasforwarded totheLaboratoryofNeuromuscular Investi-gation in a fresh state with no fixatives or additives, and was immediately frozen in liquid nitrogen and stored at
−176◦C until processing. Frozen muscle specimens were
cut into sections of 5m thickness using a cryostat at a
temperature of −30◦C, and slices were mounted on glass
Fig.1–Deltoideusmusclebiopsyfromapatientwithinflammatorymyopathy.(A)Musclefibernecrosisandendomysial inflammatoryinfiltrate(hematoxylinandeosin).(B)Increasedlysosomalactivity(acidphosphatase).
Statistics
AChi-squaredtestwasusedforthecomparisonoftwo propor-tions,expressedasapercentage.p-values<0.05weredefined asstatisticallysignificant.
Ethics
The study was approved by the ethics committee of the FAMERP.
Results
Group1encompassedof42 patients(PMorDM):28female (66.7%) and 14 male (33.3%), mean age 44.7±19.9 years (range6–80years).Thirty-fourofthese42cases(80.1%)were PMpatients [22 female (64.7%) and 12 male (35.3%); mean age 49.3±17.7 years (range 8–80 years)]. The remaining 8 cases(19.9%)were DMpatients[6female(75%)and 2male (25%);meanage25.3±17.4years(range6–51years)].Group2 consistedof29patients:17female(58.6%)and12male(41.4%), meanage34.2±21.9years(range1–71years).
Thefrequency of MHC-I antigens expressionin muscle fibersareshownindetailintable.Fig.2A–Cdepictsits expres-sion in the sarcolemma or sarcoplasm, as well as lack of expression.MHC-Iantigensexpressionineithersarcoplasm orsarcolemmawereobservedin79.4%ofPMpatients,62.5% ofDM patients, and 27.6% ofcontrols. NoMHC-I antigens expressionwereobservedineitherthesarcolemmaorthe sar-coplasmin20.6%ofPMpatients,37.5%ofDMpatients,and 72.4%ofcontrols.
Thefrequenciesofantibodypositivity forCD4and CD8 aredetailedinTable1.Fig.3A–CdepictsCD4/CD8expression mainlyintheendomysium.ThemajorityofPMcases(76.5%) testedpositiveforCD4expression,while23.5%testednegative forbothCD4andCD8.Similarly,75%ofDMcasestested pos-itiveforCD4,and25%testednegativeforbothCD4andCD8. EitherCD4orCD8expressionwasobservedin24.1%of con-trolsamples;however,expressionofbothCD4andCD8was observedin0%ofcontrolsamples,and75.9%testednegative forbothCD4andCD8.
MHC-Iantigensexpressionineitherthesarcoplasmorthe sarcolemma were observedtogether with CD4positivityin 88.2%ofPM,50%ofDM,and3.5%ofcontrolsamples.In com-parison,MHC-Iantigensexpressionineitherthesarcoplasm orthesarcolemmawereobservedtogetherwithCD8positivity in35.3%ofPM,12.5%ofDM,and0%ofcontrolsamples.
In5PMcases(14.7%)andin1DMcase(12.5%),MHC-I anti-genswereexpressedeitherinsarcolemmaorsarcoplasmin theabsenceofbothCD4andCD8.
Discussion
ThepresentstudydemonstratedthattheexpressionofMHC-I antigensineitherthesarcolemmaorthesarcoplasmoccurred moreofteninpatientswithPM/DMthanincontrols,although onlythedifferencebetweencontrolsandPMpatientswas sta-tisticallysignificant.Overall,MHC-Iantigenswereexpressed in79.4%ofPM/DMpatients.Overall,thesensitivityofthetest fordiagnosingIMwas78%,similartoours(79.4%).12When
weconsideredonlysarcolemmalMHC-Iantigensexpression, there was no significant difference between controls and eitherPMorDMpatients.Whenweconsideredonly sarcoplas-micMHC-Iantigensexpression,thedifferencewassignificant only betweencontrols versusPM patients. MHC-I antigens were notexpressedinbothsarcolemmaandsarcoplasmin mostcontrols,whichdifferedsignificantlyonlyfromPMcases. AccordingKarpatietal.,11inPMthemajorityofmusclefibers
exhibitedstrongsarcolemmalMHC-Iantigensexpressionand inDM,musclefiberssituatedperifascicularlyordistributedin randomclustersrevealedstrongexpression.
Overall,76.2%ofPM/DMpatientstestedpositiveforCD4 and/or CD8.Thepositivityofinflammatorycells(CD4) was higherincaseswithPM/DMversuscontrols,withasignificant differenceforboth.CD8positivitywaslesspronouncedbut stillsignificantforPMcases,althoughnotforDMcases.The rateatwhichcontrolstestednegativeforCD4andCD8was sig-nificantwhencomparedwithbothPMandDM.Noneonthe controlswerepositiveforbothCD4andCD8andrepresented astrikingfinding.
themostcommonfindinginPMversuscontrols,andthiswas themostusefulfindingforPMdiagnosis.Thisfindingwasless usefulforDMdiagnosis,althoughitwasstillausefulfindingin thatcontext.Itisalsoworthwhiletoemphasizethatwedidnot observeanyassociationbetweenMHC-Iantigensexpression (eitherinsarcolemmaorsarcoplasm)andCD8expressionin controlsamples.Thisfindinglikelyrepresentsaveryuseful tooltoruleoutPM/DM.
Weobservednoinflammatoryinfiltratein5PMand1DM cases,althoughMHC-Iantigenswereexpressedeitherinthe sarcolemmaorinthesarcoplasm.Thisfindingshowedthat approximately15%ofPM/DMcasescouldnotbediagnosed basedonlyoninflammatoryinfiltrate,corroboratingthe state-mentfromDalakas1thattheexpressionofMHC-Iantigensare
ausefulmarkertoconfirmthediagnosisofIMevenwhenthere isnoevidenceofinflammatorycellsinthemusclebiopsy.
According to muscle biopsy results from van der Pas etal.,13 theexpressionofMHC-Iantigenswereobservedin
67% patients withDM and in 61% ofpatients withPM. In DMcases, the immunohistochemicalanalysisrevealed sig-nificantlyhigherMHC-I antigensexpressioninthejuvenile form(96.4%)thanintheadultform(50%).12MHC-Iantigens
expressionwere observedin11% ofbiopsiesfrom patients withmusculardystrophyandin4%ofbiopsiesfrompatients withamiscellaneous neuromusculardisorder,totaling 15% (lessthanourobservedexpressionof27.6%).
Most muscle fibers invadedbyCD4 and/or CD8express MHC-I antigenson the surface.7 However,as noted above,
sometimes the MHC-I antigens expression were observed withoutinvasionbymononuclearcells.Nybergetal.14hasalso
emphasizedtheimportanceofMHC-Iantigensexpressionin inactivechronicPMorDMwithpersistentmuscleweakness intheabsenceofbothinflammatoryinfiltratesandsignsof inflammationon magneticresonance imaging.In addition, the expressionofMHC-I antigensarenotmodifiedbyprior treatmentwithimmunosuppressivedrugs,althoughvander Pas etal.13 reportedadecrease ofsensitivityforthe MHC-I
antigenstestafter4weeksofimmunosuppressivetherapy. It should be emphasized that MHC-I antigens can also be expressed in muscular dystrophies (mainly dysferlin deficiency, which clinically presents as limb-girdle dystro-phy and distal myopathy).15 In these cases, an increased
inflammatoryresponsewasobservedtogetherwithanactive dystrophic pattern.The cellularinfiltratessuggest that the
Table1–ExpressionofMHC-Iantigens(sarcolemmaandsarcoplasm),CD4andCD8inmusclebiopsiesfrompatients
withpolymyositis(PM),dermatomyositis(DM)andcontrols(C).
PM DM Controls p p
N 34 8 29 PM/C DM/C
Age 49.3±19.9 25.3±17.4 34.2±21.9
Male 35.3%(12) 25%(2) 41.4%(12)
Female 64.7%(22) 75%(6) 58.6%(17)
MHC-I(+)sarcolemma 47.1%(25) 62.5%(5) 24.1%(7) 0.1037 0.1035
MHC-I(+)sarcoplasma 73.5%(16) 50.0%(4) 13.8%(4) <0.0001 0.0860
MHC-I(+)both 41.2%(14) 50.0%(4) 10.3%(3) 0.0135 0.0424
MHC-I(+)(either) 79.4%(27) 62.5%(5) 27.6%(8) 0.0001 0.1579
MHC-I(−)both 20.6%(7) 37.5%(3) 72.4%(21) 0.0001 0.1579
CD8(+) 38.2%(13) 12.5%(1) 10.3%(3) 0.0247 0.6410
CD4(+) 76.5%(26) 75.0%(6) 13.8%(4) <0.0001 0.0027
CD4andCD8(+) 38.2%(13) 12.5%(1) None(0) 0.0006 0.4846
CD4and/orCD8(+) 76.5%(26) 75.0%(6) 24.1%(7) 0.0001 0.0243
CD4andCD8(−) 23.5%(8) 25.0%(2) 75.9%(22) 0.0001 0.0243
MHC-I(either)andCD4(+) 88.2%(30) 50.0%(4) 3.5%(1) <0.0001 0.0048
MHC-I(either)andCD8(+) 35.3%(12) 12.5%(1) None(0) 0.0012 0.4846
Fig.3–CD4andCD8expressions:(A)CD4-positive;(B)CD4-negative;(C)CD8-positive;(D)CD8-negative.
inflammatoryreactionissecondarytonecrosis.MHC-I anti-gens were overexpressed mainly in association with fiber phagocytosisandregeneration.15,16Indysferlinopathies,CD8
lymphocytesarerareandTlymphocytesinvademusclefibers onlyoccasionally,whilebotharecommonfindingsinPM.17
Dysferlinopathyshouldbeconsideredinthedifferential diag-nosisofIMthatareunresponsivetosteroids.
Conclusion
The presence of MHC-I antigens and subtypes of T cells expressioncouldbeusefultohelpthecliniciandifferentiatein thosecaseswheredifferentialbetweeninflammatoryversus othernon-inflammatorymyopathiescases,sometimes diffi-culttodistinguishonclinicalgrounds.MHC-I antigenswas moreoftenexpressedinPM;morecellswerepositiveforCD4 inPMandDM;MHC-Iantigenswereexpressedwithout inflam-matorycellsin14.6%ofPM/DMcases;thereisahighsuspicion ofPM/DM(mainlyPM)whenMHC-Iantigenswereexpressed incombinationwithCD4positivity;andthereisahigh proba-bilitytoruleoutbothPMandDMintheabsenceofbothMHC-I antigensandCD4expression.
Funding
BAP,BolsadeAuxílioàPesquisa,FaculdadedeMedicinadeSão JosédoRioPreto,SãoPaulo,Brazil.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
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