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Original Article
Autoimmune and non-autoimmune thyroid dise ase s have
diffe re nt patte rns of ce llular HLA class II e xpre ssion
Service of Endocrinology and Department of Pathologic Anatomy,
School of Medical Sciences - State University of Campinas, Campinas, Brazil
Denise Engelbrecht Zantut-Wittmann, Luís Henrique Barbosa Boechat, Glauce Aparecida Pinto, Miriam Aparecida da Silva Trevisan, José VassalloINTRODUCTION
C la ss II a ntig e ns o f the huma n ma jo r histo co mpatibility co mplex (MHC), the human leuko cyte antig ens (HLA) DP, DQ and DR, are central elements in the presentatio n o f exo geno us a ntig e ns to T C D4 + lymp ho c yte s a nd the regulatio n o f immune respo nse. Expressio n o f these glyco pro teins o n the cell surface is restricted to B lymp ho c yte s, ma c ro p ha g e s, o the r a ntig e n-presenting cells and the capillary endo thelium.1 So me autho rs described aberrant HLA class II expressio n antig ens in fo llicular thyro id cells in G raves’ disease (G D)2 and Hashimo to thyro iditis (HT)3. Cell cultures fro m no rmal thyro ids o r G D cases generally express HLA-DR in fo llicles near areas infiltrated by lympho cytes, when stimulated by
γ
-interfero n.2 -4 These cells pro bably present self-antig ens to the immune system and perpetuate the auto immune pro cess.5 HLA class II expressio n has also been o bserved in so me no rmal thyro ids, a to xic multino d ula r g o ite r (A M G ), thyro id adeno mas and carcino mas.6 ,7 As there has been no repo rt o n the to po graphical pattern o f HLA-DR expressio n in thyro cytes, the aim o f the present stud y w a s to d e ta il this a sp e c t in no rma l, auto immune and no n-auto immune thyro id tissue, using the immuno pero xidase technique.ABSTRACT
Contex t: Surface HLA-DR antigen is usually o nly expressed by antigen-presenting cells (APC). In auto immune thyro id disease, fo llicle cells functio n as APC, thus expressing HLA-DR. Ho wever, no n-auto immune thyro id diseases may also express surface class II antigens.
Objective: To evaluate the presence and pattern o f HLA class II expressio n in auto immune and no n-auto immune thyro id diso rders.
Design: Retro spective: histo patho lo gical and immuno histo chemical analysis.
Loca tion: Referral center, university ho spital.
Sa mple: Ten histo lo g ically no rmal thyro ids, 1 1 G raves’ disease, 7 Hashimo to ’s thyro iditis, 1 0 ato xic multino dular g o iter and 3 to xic adeno mas were analyzed by
immuno histo chemistry, using a mo no clo nal antibo dy anti-HLA-DR.
M ain M easurements: The presence of these antigens in thyroid follicular cells and their relation to inflammatory infiltrate was evaluated. The pattern of HLA-DR expression in thyroid follicular cells was analyzed: membrane, cytoplasmic or both.
Results: Altho ugh HLA-DR antigens were sparsely present in o ne o f the 8 no rmal thyro ids, in 6 o f the 9 ato xic multino dular go iter and in 2 o f the 3 to xic adeno mas a net po sitivity co uld be seen in large areas. In all 5 Hashimo to ’s thyro iditis and in 7 o f the 1 0 G raves’ disease cases. This expressio n o ccurred in fo llicle cells either in co ntact with inflammato ry cells o r no t. In no n-auto immune thyro id disease, HLA-DR po sitivity was essentially cyto plasmic, whereas in G raves’ disease and Hashimo to thyro iditis it was mainly in cell membranes.
Conclusions: It is suggested that the HLA class II expressio n o n the surface o f fo llicle cells co uld be related to auto -antigen presentatio n to the immune system by these cells, leading to inflammatio n.
Key-w ords: G raves’ disease. Auto immune thyro iditis. HLA-DR antigens. Immuno histo chemistry
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METHODS
Thyro id surgical specimens fro m 8 no rmal individuals, 1 0 with G D, 5 with HT, 9 with AMG and 3 with to xic adeno ma (TA) were fixed in 1 0 % fo rma lin, emb edded in pa ra ffin, sta ined with he ma to xylin a nd e o sin a nd se le c te d fo r immuno histo c he mic a l te c hniq ue s. Sp e c ific immuno staining was perfo rmed to identify HLA-DR using a mo dified streptavidin-bio tin-pero xidase te c hniq ue .8 Mo no c lo na l prima ry a nti-HLA-DR antibo dy (Dako ), was diluted at 1 :1 0 in 0 .1 % BSA. Detectio n o f the streptavidin-bio tin-pero xidase co mplex was achieved with diamino benzidine and slight co unterstaining with hemato xylin. At the end o f the reactio n, slides were mo unted with Entellan (Merck). Immuno stained sectio ns were sco red fo r the expressio n o f HLA-DR using the fo llo wing criteria: a) presence o f HLA-DR po sitive fo llicular cells in co ntact with inflammato ry infiltrate; b) presence of DR positive follicular cells in the absence o f inflammato ry infiltrate; c) presence o f DR po sitive inflammato ry infiltrate and d) presence o f DR po sitive inflammato ry infiltrate in the absence o f DR po sitive fo llicular cells.
The p a tte rn o f HLA -DR e xp re ssio n in fo llicular cells was classified as: 1 ) restricted to the cell membrane; 2 ) restricted to the cyto plasm and 3 ) in bo th, cell membrane and cyto plasm.
RESULTS
HLA-DR expressio n in o ne o f the 8 no rmal thyro id tissue cases, 6 o f the 9 AMG and 2 o f the
3 TA was restricted to small scattered gro ups o f fo llicular cells. Ho wever, in 7 o f the 1 0 G D cases and especially in all 5 HT cases, expressio n was mo re diffuse and extensive, generally o bserved in clo se relatio n to a lympho plasmacytic infiltrate. In additio n, in 6 G D and 4 HT cases, po sitive fo llicles were also fo und o utside these areas.
The p a tte rn o f HLA -DR e xp re ssio n in fo llicular cells in AMG (5 o f 6 ) and in TA (2 o f 2 ) co uld o nly be verified restricted to the cyto plasm. In o ne no rma l thyro id tha t presented a sma ll gro up o f DR po sitive fo llicular cells, the expressio n was in the membrane. In G D, HLA-DR was always expressed in the membrane (7 o f 7 ) and in 2 cases it was also o bserved in the cyto plasm. In 5 o f the 5 HT c a ses, the po sitive memb ra ne staining was mo re intense and was acco mpanied by cyto plasmic expressio n in 4 o f the 5 . (Fig ure 1 , Table 1 )
DISCUSSION
This study has sho wn that vario us benig n thyro id diseases may express HLA class II, in a g reement with o ther a utho rs.6 Ho wever, the p a tte rn o f e xp re ssio n w a s d iffe re nt w he n a uto immune a nd no n-a uto immune thyro id diseases were co mpared. O nly in auto immune p ro c e sse s w a s the re sig nific a nt, e xte nsive membrane expressio n o f HLA-DR whereas in no n-auto immune pro cesses it was g enerally limited to the cyto plasm.
The genes o f the human MHC situated in the HLA regio n are related to the immune respo nse
Ta ble 1 - Pa tterns of HLA-DR ex pression in thyrocytes of norma l tissue a nd of tissue from pa tients w ith
Gra ves’ disea se, Ha shimoto’s thyroiditis, a tox ic multinodula r goiter a nd tox ic a denoma
Diag no sis N umber HLA-DR HLA-DR expressio n type
o f cases expressio n Membrane Cyto plasmic Bo th
N o rmal 8 1 1 0 0
G raves’ disease 1 0 7 5 0 2
Hashimo to ’s thyro iditis 5 5 1 0 4 Multino dular g o iter 9 6 1 5 0
To xic adeno ma 3 2 0 2 0
Tota l 3 5 2 1 8 7 6
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to pro te in a ntig e ns. Thus, C D4 + o r C D8 + T lympho cytes o nly reco gnize peptides pro cessed by antigen-presenting cells, the macro phages, B lympho cytes, capillary endo thelium and dendritic cells, when asso ciated with pro ducts o f the MHC genes expressed o n the cell surface.9 In the 1 9 8 0 ’s the ina deq ua te expressio n o f HLA c la ss II in fo llic ula r c e lls o f G D a nd HT c a se s w a s demo nstrated, mo st frequently in areas clo se to the lympho plasmacytic infiltrate. This suggests that the se thyro c yte s c o uld p la y a ro le in the develo pment and perpetuatio n o f the auto immune disturbance.6 ,7 ,1 0 In ag reement with these data, in o ur study a significant number o f po sitive cases (1 2 o ut o f 2 1 ) exhibited areas o f fo llicular cells expressing HLA-DR in the presence o f inflammato ry cells. In 1 9 o f these 2 1 cases there was also HLA-DR e xp re ssio n in a re a s d ista nt fro m the inflammato ry infiltrate.
Sub se q ue nt re p o rts ha ve sho w n tha t fo llicular cells in AMG , TA and carcino mas also express HLA-DR, especially in relatio n to fo ci o f infla mma to ry infiltra te. Studies using prima ry cultures o f no rmal thyro id cells, G D and o ther thyro id d ise a se s, ha ve d e mo nstra te d tha t thyro cytes in auto immune diseases co uld functio n a s a ntig en-presenting c ells, whic h a ppa rently do es no t o ccur in no n-auto immune pro cesses.6 ,1 1 In o ther investig atio ns using cultures o f thyro id murine cells o r FRTL-5 , it was o bserved that these cells can express the class II MHC mo lecules, but were unable to present antig ens.1 2 ,1 3 These co nflicting results may reflect co ntaminatio n o f the thyro id cell cultures by dendritic cells, which are abundant in this tissue and are po werful antig en-presenting cells.1 4
O ur find ing s ind ic a te a fund a me nta l difference in the pattern o f HLA class II expressio n in the fo llicular cells. Thus, in no n-auto immune thyro id disea ses, suc h a s AMG a nd TA, this expressio n was almo st exclusively o bserved in cell c yto pla sm, a nd in G D a nd HT, HLA-DR wa s expressed o n the cell memb ra ne in a ll ca ses irre sp e c tive o f the p re se nc e o f c yto p la smic expressio n. The pattern o f HLA-DR expressio n o n the cell membrane in auto immune thyro id diseases c o nfirms tha t the se thyro c yte s pla y a ro le in
antigenic presentatio n. O n the o ther hand, in cells o f no n-a uto immune thyro id d ise a se s suc h expressio n may reflect a general activatio n o f the mechanisms of cell proliferation and such activation may no t result in ho rmo ne o verpro ductio n, since it was also o bserved in AMG .5 ,6 ,1 2 ,1 3 ,1 5
O ur re sults p ro vid e e vid e nc e tha t, independent o f the thyro id functio n, class II MHC p ro d uc ts a re e xp re sse d o n the surfa c e o f thyro cytes in G D and HT, and that there are clear-c ut d iffe re nclear-c e s in the p a tte rns o f HLA -DR expressio n in auto immune and no n-auto immune thyro id diseases.
Sao Paulo Med J/Rev Paul Med 1999; 117(4):161-4.
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Sao Paulo Med J/Rev Paul Med 1999; 117(4):161-4.
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Denise Engelbrecht Za ntut-W ittm a nn - Do uto ra em Medicina Interna - Disciplina de Endo crino lo g ia – FCM-UN ICAMP
Luís Henrique Ba rbosa Boecha t - Mestre em Clínica Médica
Gla uce Apa recida Pinto - Do uto ra em Ciências-Departamento de Anato mia Pato ló g ica - FCM-UN ICAMP
M iria m Apa recida da Silva Trevisa n - LivreDo cente -Departamento de Anato mia Pato ló g ica - FCM-UN ICAMP
José Va ssa llo - Do uto r em Medicina - Departamento de Anato mia Pato ló g ica - FCM-UN ICAMP
Sources of Funding: N o t declared
Conflict of interest: N o t declared
La st received: 1 8 January 1 9 9 9
Accepted: 8 February 1 9 9 9
Address for correspondence:
Denise Eng elbrecht Zantut-W ittmann
Disciplina de Endo crino lo g ia - Departamento de Clínica Médica - Faculdade de Ciências Médicas da Universidade Estadual de Campinas - PO Bo x 6 1 1 1
Campinas/ SP - Brasil - CEP 1 3 0 8 3 -9 7 0 E-mail: hwittmann@ TRT1 5 .g o v.br
RESUMO
Contex to: Normalmente, apenas células apresentado ras de antígeno s expressam o HLA-DR em sua superfície. Na do ença tiro idiana auto -imune, as células fo liculares adquirem o papel de apresentar antígeno s e assim expressam o HLA-DR. Entretanto , do enças tiro idianas não auto -imunes também po dem expressar antígeno s de classe II. Objetivo: Avaliar a presença e o padrão de expressão do HLA de classe II em tiró ides de pacientes co m do enças tiro idianas auto -imunes e não auto -imunes. Tipo de estudo: Retro spectivo histo pato ló gico e imuno histo químico . Loca l: Centro de referência, ho spital universitário . Amostra: 1 0 tiró ides histo lo gicamente no rmais, 1 1 co m do ença de G raves, 7 co m tiro idite de Hashimo to , 1 0 co m bó cio multino dular ató xico e 3 co m adeno ma tó xico fo ram analisadas através de imuno histo química usando um antico rpo mo no clo nal anti-HLA-DR. Va riá veis estuda da s: Avaliamo s a presença desses antígeno s na célula fo licular tiro idiana, sua relação co m o infiltrado inflamató rio e o padrão de expressão do HLA-DR nas células fo liculares, se presente na membrana, no cito plasma o u em ambo s. Resulta dos: A expressão do HLA-DR o co rreu fo calmente em uma das 8 tiró ides no rmais, 6 do s 9 caso s de bó cio multino dular ató xico e em 2 do s 3 adeno mas tó xico s. Entretanto , fo i nítida sua po sitividade em áreas extensas de células fo liculares tiro idianas no s 5 caso s de tiro idite de Hashimo to e, em meno r grau, em 7 do s 1 0 caso s de do ença de G raves, quer em asso ciação ao infiltrado inflamató rio , lo nge dele o u apenas nas células linfó ides. Nas do enças tiro idianas não auto -imunes, o padrão de expressão do HLA-DR fo i
