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2007년 8월 박사학위논문

The The

The The distribution distribution distribution of distribution of of of dendritic dendritic dendritic dendritic cells cells cells cells in

in in

in healthy healthy healthy and healthy and and and inflamed inflamed inflamed inflamed human human human human gingival

gingival gingival

gingival tissue tissue tissue tissue

조 선 대 학 교 대 학 원

치 의 학 과

송 광 은

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The The The

The distribution distribution distribution of distribution of of of dendritic dendritic dendritic dendritic cells cells cells cells in in

in in healthy healthy healthy and healthy and and and inflamed inflamed inflamed inflamed human human human human gingival

gingival gingival

gingival tissue tissue tissue tissue

임상적으로 건강한 치은조직과 염증성 치은조직에서 수지상 세포의 분포에 관한 면역조직화학적 연구

2007년 8월 24일

조 선 대 학 교 대 학 원

치 의 학 과

송 광 은

(3)

The The The

The distribution distribution distribution of distribution of of of dendritic dendritic dendritic dendritic cells cells cells cells in in

in in healthy healthy healthy and healthy and and and inflamed inflamed inflamed inflamed human human human human gingival

gingival gingival

gingival tissue tissue tissue tissue

지도교수 김 병 옥

이 논문을 치의학 박사학위신청 논문으로 제출함.

2007년 4월

조 선 대 학 교 대 학 원

치 의 학 과

송 광 은

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송광은의 박사학위논문을 인준함

위원장 전남대학교 교 수 김 영 준 인 위 원 조선대학교 교 수 윤 정 훈 인 위 원 조선대학교 교 수 안 상 건 인 위 원 조선대학교 교 수 장 현 선 인 위 원 조선대학교 교 수 김 병 옥 인

2007년 6월

조 선 대 학 교 대 학 원

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Abstract

I.Introduction···1

II.MaterialsandMethods···2

III.Results···3

IV.Discussion···6

References ···8

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Table.1.ThehistologicalfindingsoftheLangerhanscellsinnormalgingivaand inflammatorytissue.···4

Table2.ThedistributionpercentageoftheDendriticcellsinnormalgingiva.···4

Table3.ThedistributionpercentageoftheDendriticcells

ininflammatorygingiva.···4

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Fig 1.CD4+,CD1a+,and CD83+ cellswasweakly shown in normalgingiva.a. CD4+

cells,b.CD1a+cells,c.CD83+cells, Immunohistochemicalstaining×200.···5

Fig 2.CD1a+,CD4+,andCD83+ cellsarestrongly observedininflammatory gingiva.a.

CD4+cells,b.CD1a+cells,c.CD83+cells,Immunohistochemicalstaining×200.···5

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국 국

국문문문초초초록록록

임상상적적으으로로 건건강강한한 치치은은과과 염염증증성성 치치은은에에서서 수수지지상상 세세포포의 분포포에에 관관한한 면면역역조조직직화화학학적적 연연구

SohngKwang-Eun,D.D.S.

Advisor:Prof.Kim Byung-Ock,Ph.D.

DepartmentofDentistry,

GraduateSchoolofChosunUniversity

수지상세포는 치태관련 치주염에 대한 숙주반응에서 면역학적 역할을 하는 것으로 간 주되고 있는 바,이 연구는 임상적으로 건강한 치은과 염증성 치은조직에서 랑거한스세 포의 분포를 면역조직화학적으로 확인하기 위해서 시행되었다.

치은조직은 14명의 환자에서 치주치료 (임상치관확장술,치은절제술,치은판막술,발치 등)도중에 침윤마취하에서 채취되었다.채취된 조직은 10% 중성포르말린이 함유된 용 액에 즉시 고정되었으며,통상적인 방법으로 처리되었고 파라핀내에 포매되었다.파라핀 플록은 6㎛ 두께의 박편들로 만들어졌으며,조직학적으로 관찰하기 위하여 이 박편들 은 hematoxylin-eosin으로 염색되었다.치은조직내의 염증 정도에 따라 수지상세포들의 분포를 관찰하기 위하여,T-cell,미성숙한 수지상세포 그리고 성숙한 수지상세포에 각 각 특이한 항체 (CD4,CD1a,CD83)를 이용하여 박편을 면역조직화학적으로 염색하였 다.

이 연구결과,정상조직에 비하여 염증조직에서 수지상세포가 더 많이 발현되는 경향을 보였으나,염증성 치은조직에서의 수지상세포는 염증의 형태와 심도에 따라 CD4+

T-cell,CD1a+ immatureDCs,CD83+ matureDCs는 다르게 나타났다.따라서 염증성 치은조직 뿐만아니라 임플란트 주위조직에서 DCs의 역할과 형태에 대한 보다 포괄적인 연구가 필요하리라 생각되었다.

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Dendritic cells (DCs) are very heterogeneous group of antigen-presenting cells.

Mostmucosalsurfaceshaveatleasttwo subsetsofDCs.Langerhanscells(LCs) are dendritic,nonkeratinocytes which reside in the suprabasallayers ofepithelial cellsin theskin,oral,nasal,esophageal,pulmonary,virginal,and rectalmucosa.1,2) Turville et al.3) reported DCs subpopulation in the gingiva closely resembles a subpopulation ofdermalDCs in the skin.Similarimmature DCs populations have beenreportedinthelaminapropriaofothermucosaltissuesliketherectum,uterus, and cervix and in lymphoid tissues such as lymph nodes,tonsils,and spleen.4,5) Cirrincione etal.6)reported thatthe contactoccurs between the mature DCs and lymphocytesininflamedgingiva.

In gingiva,LCs are found in oralepithelium ofnormalgingiva and in smaller amountsinthesulcularepithelium,buttheyareprobablyabsentfrom thejunctional epithelium ofnormalgingiva7).They belong tothemononuclearphagocytessystem as modified monocytes derived from the bone marrow,contain elongated granules and are considered macrophages with possible antigenic properties,and play an important role in the immune reaction as antigen-presenting cells for lymphocytes8,9).They areimmature,9͠ 10㎛ diameterin blood.Theirkey receptors in interactions with antigens are IAM-1, LFA-1, MHC class Ⅱ, and CD 1.

Importantfunctionsininflammationaretheprocessingandpresentationofantigen10). They ingestantigen locally and transporttheantigen to thelymph nodesthrough theafferentlymphatics.8,11,12)

ItisimpossibletodistinguishLCs from otherdendriticcellssuchasmelanocytes byroutinehematoxylinandeosinstainedmaterial.Theyhaveanorganelle(Birbeck granule)whichappearstobeuniquetothem.Theywerelabeledwithgoldchloride andobservedthroughaprimitivelightmicroscope.13,14)

Immunohistologicalstudies on human gingivalLCs have been carried outusing antibodiesagainstHLA-DR,orLangerin,orCD1amoleculethesedays.15-17)

CD1a is a markerofimmature dendritic cells and LCs in the epidermis18,19),and CD83isamarkerofmaturedendriticcells20).

Jotwanietal.21)showed thatin human chronicperiodontitis,CD1a+ immatureLCs predominantly infiltrate the gingival epithelium, whereas CD83+ mature mDCs specificallyinfiltratetheCD4+lymphoid-richlaminapropria.

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Jotwani and Cutler1) reported that the lamina propria in chronic periodontitis containsCD83+maturedendriticcells(mDCs)andCD4+T-cells.Theyshow that the gingiva contains DDCs in the lamina propria,which increase in number during chronic periodontitis. DDCs, LCs and B-cells co express CD83 in chronic periodontitisandcontributetothemDCspool.

Recently,Bodineau et al.22) demonstrated that the tissue inhibitors of matrix metralloproteinase-positiveLCsweremainly observed in theupperepitheliallayers, the otherhand,marix metralloproteinase 9-positive LCs were observed especially during periodontitis and ine the basalepitheliallayer or crossing the basement membrane.

An immunologic role in the host response to plaque-associated periodontitis is postulated forLCs.CD1a+ LCs appearto be principalleukocytes involved in the responseoftheoralmucosalepithelium to infection23).ThespecificroleofLCsis unclearatpresent.Thesedays,researchabouttheroleofLC inthepathogenesisof chronic periodontitis is in progress.The purpose of this in situ study was to immunohistochemically confirm the distribution of LCs in epithelium and lamina propriaofpatientswithplaque-associatedperiodontitis.

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1)Selectionofpatients

Twenty sitesfrom 14patients,with amean ageof47years(range11to68)wereevaluated.All the patients were medically healthy.Informed consent was obtained form all subjects before commencementofthestudy.

Gingivaltissue specimens from 14 subjects were excised under infiltrative anesthesia by sharp dissection during the periodontaltherapy (clinicalcrown lengthening,gingivectomy,flap operation, extraction,etc.).

2)Methods

The specimen was immediately fixed in a solution containing 10% neutralformalin.They were processedroutinely andwereembeddedinparaffin,Theparaffinblocksweresectionedin 6㎛ thin slices and the sections were stained with hematoxylin-eosin forgeneralhistologicalobservations.

The sections were evaluated under a light microscope for detection of normal epithelium, inflammatoryepithelium (gingivitisandchronicperiodontitis).

For the immunohistochemistry,monoclonalmouse anti-human antibodies were purchased from DakoCytomation Inc.(Glostrup,Denmark).The primary Abs used were CD4,CD83,and CD1a.

Sections were stained by the biotin-streptavidin-peroxidase method (Vectastain ABC-AP kit, Burlingame,CA,USA.).The paraffin sections were pre-incubated with 1.5 % blocking normal serum dilutedin1X PBS for20min,andincubatedforovernightwith primary antibody (anti-CD4, CD83,and CD1a) diluted (1:200) in the diluted normalserum.After washing in 1X PBS,the sections were incubated for 30 min at room temperature with diluted biotinylated universal sencondary antibody.Thesectionswerethen rinsedbriefly with 1X PBS,reacted with Vectastain ABC-AP Reagent for 30 min. After color development with 0.05% DAB (diaminobenzidine tetrahydrochloride, 나라이름),thesectionswerewashed and counterstained with hematoxylin.The number of positively stained cells in the normalepithelium and inflammatory epithelium were comparedwitheachother.

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Wehaveevaluated immatureCD1a+ LCsand matureCD83+ DCsby using immunohistochemistry withintheepithelium andlaminapropriaofthegingivainhealthyandinflammatorygingivaltissues (Table1-2).

Microscopicanalysisofhealthy epithelium revealed thepresenceoflargenumbersofCD1a+ LCs, butonlyafew CD83+matureDCs.

Transitionfrom healthtochronicinflammatorytissuewasmarkedbyincreaseinnumbersofCD4+

T cellsandDCs(Fig.1-3),bothofwhichwerepredominantlypresentinlaminapropria.

In inflammatory gingivaltissues,therewasa slightincreasein thenumbersofCD1a+ - labeled cellsin theepithelium.However,thetransition from health to chronicperiodontitiswasassociated with a increase in numbers of LCs in the epithelium,and was associated with a increase in numbersofmatureCD83+DCsinthelaminapropria.

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Table1.ThehistologicalfindingsoftheLangerhanscellsinnormalgingivaand inflammatorytissue.

No Age Sex Dx Primary Antibody

CD4 CD83 CD1a

1 68 F normal gingiva ― ― +

2 25 F normal gingiva + ― ―

3 25 F inflammatory gingiva ++ ― ―

4 51 F inflammatory gingiva ― + ―

5 66 F inflammatory gingiva + + ―

6 46 F inflammatory gingiva + ― ++

7 57 F normal gingiva + ― ―

8 50 F inflammatory connective

tissue + ― ―

9 50 F inflammatory gingiva ++ ― +

10 50 F inflammatory gingiva ― + +

11 11 F inflammatory gingiva ― ― +

12 55 F normal gingiva ― + +

13 51 F inflammatory gingiva + + +

14 62 F inflammatory gingiva + + ―

15 62 F inflammatory gingiva + ― +

16 52 F normal gingiva ― + +

17 39 F inflammatory gingiva + ― ―

Table2.ThedistributionpercentageoftheDendriticcellsinnormalgingiva.

Primary Antibody Total

cases

CD4 CD83 CD1a

Cases(%) 2 (40%) 2(40%) 3(60%) 5

Table3.ThedistributionpercentageoftheDendriticcellsininflammatorygingiva.

Primary Antibody Total

cases

CD4 CD83 CD1a

Cases(%) 9 (75%) 5(42%) 6(50%) 12

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a. b.

c.

Fig 1.CD4+,CD1a+,and CD83+ cells was weakly shown in normalgingiva.a. CD4+ cells,b.

CD1a+cells,c.CD83+cells, Immunohistochemicalstaining×200.

a. b.

c.

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Fig2.CD1a+,CD4+,andCD83+cellsarestronglyobservedininflammatorygingiva.a. CD4+cells, b.CD1a+cells,c.CD83+cells,Immunohistochemicalstaining×200.

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Figure

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References

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