ム湾滋αなゲ rん ど沢力β "初αFたDな どαsgs‐ 1990;49:50ど 告506 ProductiOn Of prostaglandin E2 histamine by c10ned rheumatoid ェ nduced by synovial cells M Sasano,M Goto,K NishiOka Abstract (PBS).Thcse synovial tissues were treatcd with PBS+0'250/。 trypsin(Nakarai chcmicals L〔 d) K y o t o , J a p a n ) f o r 4 0 m Ci )n au ft tc cs r a wt h i3 c7 h° thc cells in thc supernatants、 vcrc collec[cd. Thc cclls Mrcrc琳 ′ ashed thrcc tilncs in PBS,suspcndcd in Nutricnt Mixture「 12(HAМ ― F12;Gibco LabOratOries, Grand lsiand, Ncw York)USA) supplemcntcd with loO/。 fctal bOvine scrum(FBS; GibcO),5× lo 5М 2-mcrcaptocthanol(Nakarai), 100U/ml penicillin,and loo卜 tg/ml streptomycin PFOduCtiOn of prostagiandin E2,With Or withOut (GibcO), and cultured at 37°C in a hLlェ1lidifled atII10spherc Of 950/O air and 5% carbOn diOxidc。 FibrOblast likc and macrOphagc like cclls cach comprised 40_50°/。and dcndritic cclls-50/O of the Articular synOviurn is considcrcd to bc the mostto[al ccll pOpulation frOm thc Original synovial ilnportant site of chronェ c inaalnlmatlon in rheuI1la_ specirncns,SubcultLlred ASC were establishcd by toid arth:itiS(RA).Daycr夕 どα′havc shOwn that treating the prinlary adhcrent synovial ccll culture ctllturcd adhcrent sy1loviAl cells(ASC)diSpersed with O・o50/。trypsin fOr fivc nlinurcs at 37° C)tilcn rcplating at a reduced cell dcnsity, 縄 駆 艦 晶 !群 謙 盛 科難 3S tO i浮 the destructiol1 0f 緊 cartilage and tO chを 血ges in immune fLInCtiois,2 CLONING OF SYNOVIAL CELLS . and its synthcsis incrcases in responsc to variousSynOvial cell ciOning was lerformcd On nine inflaIIIInatory stiinuli〕 including interleLlkin-1.3 patients by lilflliting dilutiOn,正 )ispersed cclls werc Rcccnt histOchelnical studics have shO、 vn that bOth suspellded in thc cLllturc llicdiurn and distributed nlast cclls and macrophages accumurate at sitcs Ofin each wcl1 0f MicrOtcst platcs(Falcon 3072)at a cartilage crOsiOnt and synOvium5 6 in rheumatOid density of O・ 6 cells/、 vell. Thcy、vcre incubatcd at iOints,as wcll as a largc amount Of histaminc in 37°C in a hulllidiflcd atrl10sphcre of 950/O air and some rheumatOid synOviai nuids.7 Histanlinc can 59るcarbOil diOxidc, The day aftcr thc c10ning of also stimulate ASC tO prOdLlce prostagiandin E2・ thc synovial cells cach、 rell、 げas carefully cxaIIlined Thcse studics did nOt prOvidc any informatiOn, ヽ′ ith a IIlicroscope to asccrtain that Only a singic ho、vever, On which types Of cclls arc responsiblc cell cxistcd.IFtwO Or more cells、 vcre Observed the for the prodЧ ctiOn Or prostagiandin E2 aS Synovial、 vell、 vas excluded from thc study.After 30 days Of cells cOrisisted Of hcterogeneous ccll pOpulatiOns. priIIlary culture cach adhcrent cioned ccll vas 、 To OvcrcOme this、ve ciOned these synovitti cells.In vO、veeks by trypsin trcatrllcnt。 passaged once in t、 otlr previoLIs papcr9 ciOned adherent synOvial cells compriscd thrce maiOr populatiOns, classitted morpho10gically as fibrOblast like,dendritic,and PROSTAGLANDIN E2 PRODUCTIoN OF nlacrOphage likc cells. In this sttldy we tried tO SUBCULTURED,AND CLONED ASC investigate prostaglandin E2 prOductiOn induced by histarninc by rheulllatOid synovial cells,usi1lg thrce diffcrcnt typcs Of ciOlled cells, 熱F盟 縄縛1器 号 ぢ 甲潔館 Institute of Rlleunlat01ogy, Tokyo WOmcn's Medical Collcge, TOkyo 舟i Sasano 卜l G o t O K NishiOka 帥n 齢樹 群 謂詣 E)ivisiOn of Rhcumatic CorrcsI)OndCncc tO: lう rルl Goto, 舟I e t h O d s PREPARAT10N OF SYNOVIAL CELLS SynOvial tissue spctヽ lillerls、 rcrc obtailled from thc knCe iOints Of nine patients(Cight wOmen, onc naan; age 52・ 7 (10・1), rallgc 40_73 years)、 vith -70°C until prOstagiandin E2 aSSay,Aftcr the c i a s s i c O r d c f l n i t c R A ( d L l r a t i o n O f d i s 3c a s supernatants e ll・ had becn c01lectcd thc cclls wcrc 2))rangc t、vO t0 26 ycars, crythrocytc sedi一 (9・ 器 紆嶺 開貞 路晶縄ギ 鮮 運 mcntatiOn rate:58・ 5(29・ 3),rantte108 2(算 mm/arst houlヽ )at the timc Of synOvcctomy.The spccimells t 胡 u 縄y , 濫i曲 文 謡t岳 3器3i督 格ettce縄 絆 卒 品 さ nよ せ獣 齢 s t c r o i d aゴ 母 l anti_inaarnll.atory agellts;scven patients u臨 ) 、verc also rccciving slllall dOscs Of cOrticOsterOids 輸1論luメ 春 :繊句 、 vere flbroblast likc cclls. C 0 1 l c g c〕 ,。 】 x l16,NS BLD. 163)Japan (<5 mg/day).Atter rci110Va1 0f the adiposc tissucs AcccPtCd thr ptlblicati011 the specimens、 vcre Ftllinced intO snaall pieccs,thcn 6 Scptcnlhcr 1989 、 vashcd thrcc tilncs in phOsphate buffcrcd salilae 505 ttFarご ガrs ′ 'ど α ,1冴 fれ ′ θ ,7″r71ttPFlα θ ど2ら ご どメ crfθ "げ prθ sraF′ Prθ り"θ vcrc obscrvcd duction by thesc subculturcd ASC、 throughout the sccond to fourth passages,Produc― tion of prostagiandin E2 by ASC ill rcsponsc to histaHlinc is shown in thc flgurc.Prostaglandin E2 C瑞 n∬ ゴ 鼎 錨 投 s ゃ辞 曜 端 : 翼S 溢 簿 according to a paired′test. Although the prosta一 7P ig2 αg′ α,,ど す Prθsι `Cτ どうy ″f″ どを ,/θどtrcriθ 71,srα ル2す PIC'メα″九2r伊!r α′ご 冴な. r力 β 夕初a r ο す どッ, 2υ θす 'Carcs どαご 72ク θ力7ど ',7ど i,2ど 2 prθsrα grα7rど ん 2tr ,「 ,!ガ タ ,2g2イ θ Cθ 湾 ご で ″ rα rテ θ ,す υ'α ′ r夕 ブ ルrttr w"θ cガr″ o/α r r attrt7兄 cttrs/rθ ,れ 乃″ 警控 部輪t(枕 gianditt E2 COnCentrations varicd considerably among individual cuiturc salmplcs) thc mcan 岳 3000 prostagialldin E2 COnCentration relcascd froIIl thc ASC was highcst at a histaminc conccntratiOn of 再′ e thus uscd histanlinc at this 5× 10 6 m01/1. ｀ concentration in the subsequent cxperinlcnts.The addition of histalline incrcascd prostaglandin E2 relcase by all flvc adhercnt synovial cell cuitures tested)and there、vas no significant change in the numbcr ofviable cells throughout thc cxperinlcnts, ,1ど α′Fす ′ !tPsそ りfrん rrθ,.zθ '22 `ど ,ち α′ c力β う`7rs r2pr2sF,2,ど =5).(リ ム 初β α,2岱どルリ(,こ 2 Cr2α ∫ タ Sを肥"ごα77rか ・ 宅 所r/2 0<θ θ5ルοttpar″ F,1り α,,″ も ご ″rrθ ′ r力 θ冴れど sど θ (働す srα α,2ど F ,2ど PCど 2=prθ 2. g′ 抵 20CXl 0 1x10-8 5X10 7 5X10 6 5X10 5 Histamine concentration(mol/1) PROSTACLANDIN E2 1N THE CULTURE SUPERNATANT FROヽ なCLONED ASC Thc cuiture supernatants froHl thc 14 cloncs of rhcumatoid synovial cclls in thc prcsence or verc assaycd for prostaglandirl absencc ofhistamillc、 vs a relativel),small numbcr of E2・The table sho、 dcndritic cclls(abOut 200 cclls/well)and macro― Om phagc like cells(abOut 1000 Cells/wcll)fI・ vere passaged with trypsin trcat― blast like cells、 ment and replated at a dcnsity of about 100,500, or 1000 cclls/Lrell,rcspcctively,in 96-well culture 鮒 鮒 養絆 品 t盤 品 i揮域 堤 n器 ゴ軽 iよ 41-121l pg/rlll, macrophage likc cells: 51-4393 broblast pg/ml).Sinilarly,but to a lcsscr cxtcnt)丘 like cclis(abOllt 3000 cells/well)sCCreted prosta― giandin E2 SpOntaneously(84-634 pg/11■ 1).COn_ platcs.Thc cuitures became conflucnt after thrcc to six days,and thc approxilnate numbCr of the vcll for dendritic cclls, 1000 cclls was 主200 cells/、 鑑 :電 1鉛 撚 軽 r鑑 旨 鍔 藻 :錨 汀cll for macrophagc like cells, and 3000 cclls/、 rcll for flbroblast like celis)respectively. macrophage like cclls, and flbroblast like cells. cells/、 Dendritic and macrophage likc cells produccd After ihc medium 、 vas rcmoved aliquots of 2abbut cight to 10 tirncs lnore prostaglandin E2 than verc merca Dethanol frec mediunl to bc testcd ヽ flbroblast like cclls on a ccll to ccll basis,and the 10 6Aな added ili the prcsence or abscncc of 5× v ccn flbroblast like and macro一 histalllinc. Thc supcrnatantsヽ vCre C01lectcd after diffcrenccs bet、 -70°C phage likc or dcndritic celis 、verc signiflcant 24 hou,s of additional culture and kept a〔 until prostagiandin E2 aSSay. Prostaどlandin E2 in tlle cuiture mcdia was incrcased thc production ofprOstagiandin E2 by al mcasured by radioimmunoassay kit(Ncw tす pcs ofadherentsynovialcell ciones,Prostaglandin England Nuclcar,Boston,Massachusetts,USA) E2prOdLlction in dendritic and fibroblast like cells aftcr extraction and silicic acid colurlan chromato― vicc vas signiflcantly c1lhanced by about t、as IIluch vere 、 graphy)as describcd previ()usly.10 Rcsults 、 1). compared with spontancous relcasc whcn they cxpressed as pg of prostaglandin E2/1nl(SE沖 were stimulatcd with histaminc(flbrOblast like cclls=p<0・01)and dcndritic cells=p<0・ 001 by paircd どtest). Although thc incrcase of prosta― Results giandin E2 in maCrophage likc cells 、 vas not PROSTAGLANDIN E,IN THE CULTURE 1!ぜ 品蓋途 ∬ t鮮 ittW☆ 塩噛進艦盤品 signiflcant becausc of the largc variability among SUPERNATANT「 ROルl THE SUBCULTURED ASC ｀ salnplcs, thc mean conccntration of increased ■hc ASC spontaneously rcleased considcrablc prostagiandin E2 Stimtllated by histarnine in amounts of prostagiandin E2 intO the culturc vas about four tilnes as gRcat aftcr thc second to fourth passagcs. macrophagc like cells、 mediulll evcl■ 「hc ability of No signiflcallt changes in prostagiandin E2 prO― as that for spontaneous relcasc. ア r cメ′ ttfα s jど母″2θ ,7ι r施2を ,'2α rθ ど αが九crι 冴″Cガθ'2 bjlダθ″τ れ ど2ク 'つ α″どす Srag′ ど クrθ rれゴ材c夕 ザ 仰p ど ′ ど 72Fど ご α Crθ ″ r ,2泌 ,b夕 x,771arθ Apprθ な/ pそF F r ザ C冴 〕 ,り(Sど) rθ α αれ ど,″ tど2(pゴ″ ゴ sと ど′ ″S ,こ ど θ Spっ″rα ルカ!,と rrfβ ど 九″Srて で1,'ど acrophagc likc 舟【 1000 1241(387) 4199(2095) (0840) 449 (103) 449) (0・ 749(278) ( 0 0 5 0 ) Dcndritic Fibroblast like 200 3000 裾圏 'ど プ rて で,,rt7r? (格 科￨] 2∫4 2 ざ f ,, ,t ど ご and iibroblast like cells V a l u c s a r c t h c m c a n ( S)EoМ f 1 4scparatc cxpcrimcnts,cach cionc in a sct of dCndritic)n3acrophagc likc、 ア c in the mcditHn ithout thc additioll of histalllir、 14 diflercnt cioncs with and 、 o送 研 劇 Ю 劇 ぷ ф錬 劇 河 s h d C h a t c c t 田 論 h 晩 ∞К卸 闘 ふ 皿 11乱 ltta群 漁胡 agiandin E2 COnCentratio,1)/(SPOni induccd pros〔 ぷ 臣 灘盟 3盛 路嶺 506 pttgi甘 占 ぽ tttCics盟 諾 憲 !魯 辞 瑞 注 格 器 豊 ギ 「 Sα5α "θ,Cθ rθ ,ハ お力わたα macrOphagc like cclls f fol10wcd by Sbroblast l cclls ProdLICCd the mOst prostaglandill E2 in rcspollsc to histarlinc. Discuss1011 温 能 etti鮪 縮 総 ettf繰 ;群 1塩 ア novial fluid suggcst that 錨就淵摘 鳥 持 鷲 点 盤:盤 電 :繁 ettt° 盤縛盟i盟 患 F督 熱斜鮮sを 紺 掛 shOwn tO stimulate prostagiandin E2 prOductiOn in cells vere 、 activatcd tO incrcase thc amounts Of 盟 tittei縦 開 productiOn Of prostagian i韓 縄 轄 艦 、 vhich typc Of cclls were rcspOnsiblc fOr the indLIced prOdtictiOn of prostaglandin E2,hO｀ Vever) 離総品 鮒撚鎧1鑑 辮 like,Illacrophage like,and dendritic)9 and tried tO idcntify thOse rcsponsible fOr thc spontancous Or induced production of prOstagiandin E2・ Thc rcsults prcsentcd here suggcst that ciOned ASC)as well as unseparatcd ASC,can bc stirnll― lated by histalnine tO producc prostaglandill E2 cven af〔er iOng tcrm culturc. Each Of the thrce typcs Of synOvial cell ciOncs from flve differellt patients 、vith RA produccd prOstaglandin E2 spontaneously and was stimulated tO prOducc additiOnal prostagiandin E2 by histaFI■ ine.None Of the f0110、ving ilndices affected the histarnine mcdiated prOductiOn of prostaglandin E2 by ASC: diseasc activity; drugs; duratiOn Of disease; and 4B韻 paticnt's agc. The nunbcr of cxperilllcnts,hO、 hん cvcr, was quitc lilnitcd. Thc ability of cclls tO respond tO histall■ inc On a cclltO cell basis wasin thc fol10wing orderi lrllacrophage likc cclls,>dendritic CCllS>flbrOblast like cclls.舟 IacrOphage like cclls produccd the lnost prostagiandin E2 in reSpOnse〔 O histarnine, Thc histalnlne cOnccntratlons uscd and thc llle〔 hod Of stimulatiOn in the present experirllents vcre silnilar tO thOse rcportcd tO enhance thc ヽ 鮮 ざ, す 撫 嵐 s 間 盟 野亀 r t t l 撤 選 8 鰹 to thc anaphyia〔 oxins C3a)C4a,and C5a dcrivcd frOm the intcractions Mた th thc cOinplement and ょ rnmunc cOmplexes, mast cclls present in the 坑 盤認 将 総艦 群縄 狩 十 eS the prOductiOn Of prOstagiandin E2 by ASC is nOt cicar,bllt it has bccll reported that productiOn of prOstaglandin E2 lnduced by histaFlline ls mediated vla the histarll】 nc Hl rcccptor.8 14 MOrcOvcr, carly in thc culturc ASC rclcased 野 脊 翻 i13登 ''i群 総 晟【E載 i濫 鑑 鎗 12-、 vcll platc,but the prOstagiandin E2 COnCCIltra― tion in culture declined rapidly 亭 ′ ith tilne.18 rtttttЖ !統 岳 器撤品号 る 絆沈材 済獣 社.輯よ
© Copyright 2024 ExpyDoc
