Characterization of factor XIII containing-macrophages in lymph nodes with Hodgkin's disease

B . J. Cance (1987), 55, 421-426 © The Macmillan P ess L d., 1987~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~- Cha ac e iza ion o Fac o XIII con aining-mac ophages in lymph nodes wi h Hodgkin's disease R. Adany1, Z. Nemes2 & L. Muszbek' 'Depa men o Clinical Chemis y and 2Depa men o Pa hology, Uni e si y School o Medicine, Deb ecen, H-4012, Hunga y. Summa y A la ge numbe o cells con aing subuni a o blood coagula ion Fac o XIII (FXIII) was de ec ed by immunope oxidase s aining in lymph nodes wi h Hodgkin's disease. These ela i ely la ge, mul ipola , mononuclea cells we e o en ound in he immedia e icini y o malignan Hodgkin's cells. In ensi e cha ac e iza ion o hese cells ca ied ou by immuno luo escen and enzymecy ochemical echniques in double- and iple-labelling sys ems on he same sec ions clea ly demons a ed ha hey ep esen umou - associa ed mac ophages (TAMs). FXIII con aining-cells showed a-naph yl ace a e es e ase (ANAE) posi i i y, and we e labelled by monoclonal an i-Leu M3 an ibody, a monocy e/mac ophage ma ke , bu no a all o only e y weakly by an i-HLA-DR. Nei he alkaline phospha ase (ALP) no adenosine iphospha ase (ATPase) ac i i y could be de ec ed in hese cells and su p isingly, hey we e consis en ly nega i e o acid phospha ase (AcP) as well. The p esence o FXIII subuni a in umou -associa ed mac ophages sugges s ha his cell ype migh ha e an impo an ole in he s abiliza ion o ib in deposi s a ound umou cells. Fib in deposi ion is a a he equen inding in spon aneously-a ising as well as ansplan able human and animal umou s and has been implica ed in a ious aspec s o umou g ow h and me as asis (Rickles & Edwa ds, 1983; D o ak e al., 1983). Though mos o pe haps all malignan cells possess p ocoagulan ac i i ies (O'Mea a, 1958; Go don e al., 1975; 1979; Go don & C oss, 1981; Seme a o & Dona i, 1981) ha can ac i a e he coagula ion cascade, i is becoming inc easingly e iden ha umou -associa ed mac o- phages (TAMs) a e also in ol ed in in a- umo al ib in o ma ion (Edwa ds e al., 1981; E ans, 1982; Lo enze e al., 1983; Key, 1983). Mac ophages o , in iew o hei he e ogenei y (Hoppe e al., 1979; Poul e e al., 1983), ce ain o hei subse s could con ibu e o ex a ascula clo ing by wo mechanisms. (i) By exp essing issue ac o ac i i y hey can ini ia e he ex insic coagula ion pa hway (Rickles & Edwa ds, 1983; D o ak e al., 1983; Lo enze e al., 1983). (ii) They con ain a numbe o clo ing ac o s which, i sec e ed o eleased om damaged cells in o he in e s i ial space, p o ide all he componen s necessa y o ex insic h ombin o ma ion. The p oduc ion o i amin K-dependen clo ing ac o s (Fac o II, VII, IX, X) and Fac o V by mac ophages has been well es ablished (Os e ud e al., 1980; Lindahl e al., 1982; an Dam-Mie as e al., 1985; Chapman e al., 1985). Mos ecen ly he p esence o subuni a o Fac o XIII (FXIII), he enzyma ically-ac i e cons i uen o ib in s abilizing ac- o has also been demons a ed in human pe iphe al blood monocy es (Muszbek e al., 1985) and pe i oneal mac o- phages (Adany e al., 1985) in ou labo a o y. Independ- en ly, hese esul s ha e been con i med by Hen iksson e al. (1985). FXIII deli e ed by mac ophages in o he umou s oma migh ha e an impo an ole in he s abiliza ion o ib in o med ex a ascula ly and in i s ac i a ed o m as a ansglu aminase i migh exe o he biological unc ions as well. Thus, i was in e es ing o see i TAMs o ce ain subse s o hem con ain FXIII. The p esence o ib in deposi s in lymph nodes wi h Hodgkin's disease is a gene al obse a ion (Ha is e al., (1982; D o ak e al., 1983) and in hese lymph nodes mac ophage-like cells p e ail o e all o he cell ypes in a eas ich in ib illa in e cellula subs ance (S ille & Ka enkamp, 1978; Hansmann & Kaise ling, 1981). He e we show ha a dis inc cell popula ion in lymph nodes wi h Hodgkin's disease con ains subuni a o FXIII. The mac ophage na u e o hese cells is clea ly demons a ed and by using double- Co espondence: R. Adany. Recei ed 21 July 1986; and in e ised o m, 10 No embe 1986. and iple-labelling echniques he FXIII con aining subse o TAMs is ex ensi ely cha ac e ized. T iple labelling sys ems p o ide an excellen oppo uni y o ca y ou an exac cha ac e iza ion o a cell popula ion, because he di ec demons a ion o wo di e en an igens in combina ion wi h enzyme-cy ochemical eac ions in he same sec ion yields a p ecise ep esen a ion o he coincidence o h ee di e en cha ac e is ics. Ma e ials and me hods Lymph node biopsies we e ob ained om 12 pa ien s wi h Hodgkin's disease o nodula scle osing ype. Sec ions om non-neoplas ic, eac i e lymph nodes se ed as con ols. All specimens we e di ided in o wo pa s a he ime o su gical biopsy. One pa was ixed in 3.5% pa a o maldehyde ixa i e (4h, oom empe a u e) hen acuum embedded in pa a in and sec ioned in o 6pim slides, while he o he pa was snap- ozen and cu in a c yos a . Six m ozen sec ions we e ai -d ied, w apped in oil and s o ed a -20°C. Immunope oxidase s aining Fo maldehyde- ixed pa a in embedded sec ions we e de- waxed and ehyd a ed. Endogenous pe oxidase ac i i y was blocked by I % H202 in absolu e me hanol o 30 min a oom empe a u e. Sec ions we e diges ed wi h 0.1 % ypsin, in TRIS-bu e ed saline (pH 7.6) con aining 0.1 % calcium chlo ide a 37DC o 20min. Non-speci ic IgG binding was p e en ed by p eincuba ion wi h 20% no mal goa se um o 15 min. Sec ions we e co e ed o 2 h, a oom empe a u e wi h abbi an ise um agains FXIII subuni a (Beh ingwe ke AG, Ma bu g, Wes Ge many) dilu ed 1:25 wi h 20% no mal goa se um. The monospeci i y o his an ise um was e i ied by immunoblo ing on whole human plasma as well as on human pla ele and monocy e homogena e (Muszbek e al., 1985). An igen-an ibody eac ion was de ec ed by bio inyla ed an i- abbi IgG and a idin-bio inyla ed pe - oxidase complex (Vec as ain ABC ki ) (Vec o Labo a o ies, Bu lingame, CA). The speci ic pe oxidase ac i i y was isualized by 0.05% 3,3'-diaminobenzidine e ahyd o- chlo ide (DAB) (Sigma Co., S . Louis, MO), 0.01% H202 in 0.1 mol 1- 1 TRIS HCI bu e , pH 7.2. Coun e s aining was wi h Maye 's haema oxylin be o e dehyd a ion in g aded alcohol and moun ing wi h Canada balsam. On con ol slides no mal abbi se um a he same dilu ion was used ins ead o an i FXIII subuni a an ise um. PBS, pH 7.3, was used o an ibody dilu ion and in washing p ocedu es. B . J. Cance (1987), 55, 421-426 ,'-. The Macmillan P ess L d., 1987 422 R. ADANY e al. Immuno luo escen and enzyme-his ochemical echniques F ozen sec ions we e used o he double immunolabelling echniques combined wi h he cy ochemical localiza ion o se e al enzymes on he same slide (combined iple labelling sys ems). Immedia ely be o e immuno eac ions c yos a sec- ions we e unw apped and ixed in ace one a +4°C, o 10 min. A e washing in PBS, slides we e incuba ed wi h 1:200 dilu ion o an ise um agains FXIII subuni a o 2 h a oom empe a u e. As he seconda y an ise um, a 1:40 dilu ion o swine an i- abbi IgG luo esceina ed (Dakopa s a/s, Glos up, Denma k), was used (30 min incuba ion). In he nex s age his eac ion was combined ei he wi h he de ec ion o HLA-DR an igen o wi h he isualiza ion o Leu M3, a monocy e/mac ophage su ace ma ke . Sec ions we e incuba ed wi h 1:5 dilu ion o bio inyla ed mouse an i- human HLA-DR monoclonal an ibody (Bec on-Dickinson, Sunny ale, CA) o wi h he same dilu ion o mouse an i- human Leu M3 monoclonal an ibody conjuga ed wi h phyco- e y h in (Bec on-Dickinson, Sunny ale, CA) o 30 min, a oom empe a u e. The speci ic binding o bio inyla ed an i- HLA-DR an ibody was de ec ed by 1:40 dilu ion o s ep a idin-Texas ed (Ame sham, UK). In he case o nega i e con ols non-immune abbi se um and con ol mouse IgG om umou -bea ing BALB/c mice conjuga ed wi h phycoe y h in o FITC (Bec on Dickinson, Sunny ale, CA) we e subs i u ed o he i s an ibodies. Following double immuno luo escen s aining, sec ions we e moun ed in 50% glyce ol in PBS and examined unde an Op on ul a iole mic oscope equipped wi h an epi luo escence condenso con aining selec i e il e s o FITC and Texas ed/phycoe y h in. A e pho og aphs had been aken, co e -slips we e emo ed, sec ions we e washed ho oughly in dis illed wa e . As a hi d s ep, one o he ollowing enzymes was de ec ed: oc-nap hyl ace a e es e ase (ANAE) (Muelle e al., 1975), acid phospha ase (AcP) (Poul e e al., 1983), aden- osine iphospha ase (ATPase) (Poul e e al., 1983) and alkaline phospha ase (ALP) (Mason & Wools on, 1982). In he case o nega i e con ol slides in he enzymecy o- chemical eac ions ei he he subs a e was omi ed om he incuba ion medium, o be o e de elopmen o an enzyme eac ion he app op ia e enzyme ac i i y was blocked acco d- ing o he ecommenda ion o Poul e e al. (1983). Finally, sec ions we e emoun ed in 50% glyce ol in PBS and he ields o which pho og aphs had been p e iously aken we e iden i ied in no mal ligh mic oscope and epho og aphed. Using he abo e-men ioned immuno- and enzyme-his o- chemical eac ions all possible combina ions o iple label- ling we e pe o med on e e y biopsy specimen. Resul s Immunope oxidase s aining on pa a o maldehyde- ixed, pa a in embedded sec ions was used o e i y he p esence and de e mine he dis ibu ion o FXIII subuni a con aining cells in lymph nodes wi h Hodgkin's disease o nodula scle osing ype. As demons a ed in Figu es la, 4b, 5a and 6a p ac ically he whole a ea o lymph node was in il a ed by cells showing in ensi e s aining o FXIII. These cells we e o en ound in he immedia e icini y o malignan Hodgkin's cells sugges ing an in ima e ela ionship be ween he wo cell ypes (Figu e lb). FXIII con aining-cells in lymph nodes wi h Hodgkin's disease possess a mac ophage-like appea ance, hey a e ela - i ely la ge, mul ipola , mononuclea cells wi h nume ous acuoles in he cy oplasm. Thei de ailed cha ac e iza ion was pe o med by iple labelling (double immuno- luo escen + enzyme-cy ochemical) echniques. S aining o FXIII and he monocy e/mac ophage ma ke Leu M3 (Dimi iu-Bona e al., 1983) showed an iden ical dis ibu ion pa e n in each case (Figu e 3a, b). This ac oge he wi h hei de ini e ANAE posi i i y (Figu e 4b, c) clea ly iden- i ies FXIII con aining-cells as membe s o he mac ophage amily. These cells we e p edominan ly nega i e o HLA- DR hough occasionally e y weak posi i e s aining could also be de ec ed (Figu es 2, 4a, b). ALP (Figu e 5a, b) and ATPase ac i i ies (no shown) could no be de ec ed in FXIII con aining cells and su p isingly, hey we e consis - en ly nega i e o AcP (Figu e 6a, b), as well. The p esence o HLA-DR o AcP posi i e bu FXIII nega i e cells wi h mac ophage-like mo phological ea u es clea ly indica es ha FXIII is exp essed only in a ce ain subse o TAMs. In con as o lymph nodes wi h Hodgkin's disease, in eac i e lymph nodes, FXIII con aining cells we e localized almos exclusi ely in pe i ascula connec i e issue and in subcapsula o medulla y sinuses. Mac ophages in ollicles o eac i e lymph nodes iden i ied by s ong ANAE eac ion we e consis en ly nega i e o FXIII subuni a and only a ew posi i e cells could be de ec ed in he in e ollicula a eas (no shown). A de ailed cha ac e iza ion o FXIII con aining-cells in eac i e lymph nodes is published elsewhe e (Nemes e al., 1986). Discussion Plasma FXIII, like mos o he clo ing ac o s ci cula es as a zymogen. I is a e ame ic p o ein consis ing o wo ypes o subuni s (a2b2). The po en ial enzyma ic si e is loca ed on subuni a which can assume an ac i e con igu a ion only ollowing p o eoly ic clea age by h ombin and Ca2 + in- duced dissocia ion om he inhibi o y b subuni (see Muszbek & Laki, 1984 o e iew). I has been known o a long ime ha subuni a bu no b also exis s as an in acellula p o ein in pla ele s (Buluk, 1955), mega- ka yocy es (Kiesselbach & Wagne , 1972) and placen a (Bohn & Schwick, 1971). As men ioned ea lie he exis ence o subuni a in monocy es has also been e i ied (Muszbek e al., 1985) and hese cells e ain hei FXIII con en ollowing di e en ia ion in o pe i oneal mac ophages (Ad'any e al., 1985). Fu he mo e, i was shown ha FXIII con aining cells in he placen a a e also o mac ophage o igin (Adany & Muszbek, submi ed o publica ion). In his s udy a cell ype exp essing FXIII subuni a was de ec ed in lymph nodes wi h Hodgkin's disease. The mo pho- logical appea ance o FXIII con aining cells indica ed ha hey belong o TAMs, hus combina ions o immuno- and enzyme-his ochemical eac ions - gene ally used o iden i i- ca ion and/o pheno yping o mac ophages - we e applied o cha ac e ize hem. Thei mac ophage na u e was clea ly demons a ed by Leu M3 and ANAE posi i i y. A he same ime nega i e eac ions we e ound o HLA-DR, AcP, ALP and ATPase. The abo e esul s s ongly sugges ha his FXIII con aining-TAM cell ype is no iden ical wi h any o he mac ophage cell ypes cha ac e ized ea lie in no mal human lymph node. This conclusion is clea ly suppo ed by he ollowing da a: (i) FXIII con aining-mac ophages a e no iden ical wi h in e digi a ing e icula cells and den i ic cells o lymph node because he la e s a e s ongly posi i e o HLA-DR and ATPase (Janossy e al., 1980; S ein e al., 1980; Poul e e al., 1983). (ii) They di e om sinus his iocy es o no mal lymph node and om in lamma o y mac ophages, as well as by he absence o AcP ac i i y (Poul e e al., 1983). (iii) Being ALP nega i e hey may no be in e p e ed as ib oblas ic e iculum cells (Chilosi e al., 1981). On he o he hand, FXIII con aining-cells in lymph nodes wi h Hodgkin's disease a e no e en iden ical wi h o he FXIII con aining-cells o no mal lymph nodes which occu in he connec i e issue o capsule and in he sinus (Nemes e al., 1986). HLA-DR posi i i y o connec i e issue his iocy es as well as he in ensi e AcP eac ion o sinus mac ophages clea ly dis inguish he la e cell ypes om FXIII con aining-TAMs. FXIII IN TUMOUR ASSOCIATED MACROPHAGES 423 Figu e 1 Immunope oxidase s aining o FXIII subuni a combined wi h haema oxylin-ch omo ope coun e s aining on a sec ion o pa a o maldehyde- ixed, pa a in embedded lymph node wi h Hodgkin's disease, nodula scle osis ype. FXIII subuni a- con aining cells (in b own colou ) in aded p ac ically he whole a ea o lymph node (a) and we e equen ly ound in he immedia e icini y o malignan Hodgkin's cells (b). Ba = 25 gm (a) and 10pm (b). Figu e 2 Double immuno luo escen labelling o FXIII subuni a and HLA-DR on a c yos a sec ion o lymph node wi h Hodgkin's disease. In a double exposu e pho og aph g een FXIII con aining cells a e easily dis inguishable om Texas ed labelled HLA-DR posi i e ones. Ba =10 lOm. Figu e 3 In lymph node wi h Hodgkin's disease iden ical cells we e labelled wi h immuno luo escen s aining o FXIII subuni a (a) and o he monocy e/mac ophage su ace an igen Leu M3 (b). Ba = 10 pm. D 424 R. ADANY e al. Al C* . Figu e 4 T iple labelling o HLA-DR (a), FXIII subuni a (b) and ANAE (c) on he same a ea o lymph node wi h Hodgkin's disease. FXIII con aining cells a e ANAE posi i e (see a ows), bu HLA-DR nega i e. Ba = 25 im. Di e en ypes o mac ophages o igina e om a common bone ma ow p ecu so ( an Fu h, 1981) bu owing o an in ica e di e en ia ion p og am associa ed wi h he ex- p ession and disappea ance o a ious gene p oduc s (Dimi iu-Bona e al., 1983) hey show an ex eme pheno- ypic and unc ional di e si y. In conside a ion o his ac we belie e ha FXIII con aining-TAMs a e a subse o mac ophages di e en ia ed om blood monocy es o special unc ion(s) ela ed o malignan cell p oli e a ion. Indepen- den ly om FXIII con aining-mac ophages some AcP posi i e mac ophage-like cells we e also de ec ed. This ind- ing oge he wi h he p esence o HLA-DR posi i e, bu FXIII negn-i i e cclls in lymph nodes wi h Hodgkin's diseaise suppo he possibili y ha TAMs a e o be conside ed as an inhomogeneous cell popula ion. Ex a ascula ib in deposi ion occu s in he s oma and a hos - umou in e ace in mos o pe haps all malignan neoplasms (D o ak e al., 1983). The pa hological signi- icance and he o igin o ib in deposi s has no been su icien ly explo ed. Mos o he a ailable clinical and expe imen al da a, howe e , end o suppo * he iew ha he ac i a ion o clo ing sys em is bene icial o bo h umou p og ession and me as asis o ma ion. Mos ly on he basis o heo e ical conside a ions he ollowing hypo heses ha e been p oposed o he pa hogenic ole o ib in o med be ween and a ound umou cells: (1) i migh ha e a ba ie unc ion and in e e es wi h he hos 's immune esponse, (2) i could s imula e umou angiogenesis, (3) i may ha e a ole in he implan a ion o ci cula ing umou cells a me as a ic si es (D o ak e al., 1983). An impo an ole o FXIII - ib in s abilizing ac o - in any o he abo e mechanisms seems a he ob ious. By o ming ib inolysis- esis an ib in meshwo k and c osslinking ibn lla in e - cellula ma ix componen s, FXIII migh suppo he ba ie unc ion o migh e en be essen ial o i . The p ocess o angiogenesis and ib oplasia du ing umou g ow h and wound healing has been compa ed (D o ak e al., 1983) and i is well known ha wound healing is highly impai ed in FXIII de icien pa ien s (Ducke , 1972). A possible di ec e ec o FXIII on cell p oli e a ion, as obse ed in he case o ib oblas s (Beck e al., 1961), migh also ha e some implica ions o umou g ow h. A u he possibili y con- ce ns he ansglu aminase na u e o ac i a ed FXIII. As a ansglu aminase i can a ach hos p o eins co alen ly o he memb ane o umou cells and mask hei pu a i e 'non-sel ' cha ac e esul ing in inc eased immune esis ance. The la e idea is suppo ed by da a showing ha a achmen o ib inogen o he memb ane o YPC-1 plasmocy oma cells by issue ansglu aminase esul s in an inhibi ion o cell media ed cy o oxic esponse (Hunyadi e al., 1981). In umou s FXIII could ge in o he in e s i ial space om wo possible sou ces. I may leak om blood essels oge he wi h o he plasma p o eins due o enhanced mic o ascula pe meabili y o i may o igina e om he TAM subg oup desc ibed he e by ac i e sec e ion and/o elease ollowing cell des uc ion. Howe e , his ques ion has no been ad- d essed expe imen ally. Clea ly, u he in es iga ions a e o be ca ied ou o es he abo e hypo heses and es ablish he ole o FXIII as well as FXIII con aining-TAMs in he p og ession o malignan umou s. These s udies we e conduc ed in pa pu suan o a con ac wi h he Na ional Founda ion o Cance Resea ch, Be hesda, Ma yland, USA. We hank M R6be He endi o he expe echnical assis ance and M s Mi ia Kozma o he excellen sec e a ial wo k. FXIII IN TUMOUR ASSOCIATED MACROPHAGES 425 Figu e 5 Immuno luo escen s aining o FXIII subuni a (a) combined wi h he de ec ion o alkaline phospha ase (b) on he same sec ion o lymph node wi h Hodgkin's disease. As alkaline phospha ase ac i i y was e ealed wi h naph ol AS-MX plus Fas Red TR he end p oduc o he eac ion appea ed in ed colou no only in no mal ligh bu on he FITC channel o he luo escence mic oscope, as well. On he pho og aph i is supe imposed on FITC labelled immun eac ion o FXIII subuni a. FXIII con aining mac ophages a e nega i e o alkaline phospha ase. Ba = 25 pm. Figu e 6 In lymph node wi h Hodgkin's disease immuno luo escen s aining o FXIII subuni a (a) and acid phospha ase ac i i y de ec ion (b) isualize wo dis inc cell popula ions. Ba =25pm. 426 R. ADANY e al. Re e ences ADANY, R., BELKIN, A., VASILEVSKAYA, T. & MUSZBEK, L. (1985). Iden i ica ion o blood coagula ion ac o XIII in human pe i- oneal mac ophages. Eu . J. Cell Biol., 38, 171. BECK, E., DUCKERT, F. & ERNST, M. (1961). The in luence o ib in s abilizing ac o on he g ow h o ib oblas s in i o and wound healing. Th omb. Dia h. Haemo hag., 6, 485. BOHN, H. & SCHWICK, H.G. (1971). Isolie ung und Cha ak e- isie ung eines ib ins abilisie enden Fak o s aus menschlichen. Plazen en. A zneim. Fo sch., 21, 1432. BULUK, K. (1955). An unknown unc ion o blood pla ele s. Polski Tygod. Leka ., 10, 191. CHAPMAN, H.A., ALLEN, L.C. & STONE, O.L. (1985). Human al eola mac ophages syn hesize ac o VII in i o. J. Clin. In es ., 75, 2030. CHILOSI, M., PIZZOLO, G., MENESTRINA, F., IANNUCCI, A.M., BONETTI, F. & FIORE-DONATI, L. (1981). Enzyme his ochemis y on no mal and pa hologic pa a in-embedded lymphoid issues. Am. J. Clin. Pa hol., 76, 729. DIMITRIU-BONA, A., BURMESTER, G.R., WATERS, S.J. & WINCHESTER, R.J. (1983). Human mononuclea phagocy e di - e en ia ion an igens. I. Pa e ns o an igenic exp ession on he su ace o human monocy es and mac ophages de ined by mono- clonal an ibodies. J. Immunol., 130, 145. DUCKERT, F. (1972). Documen a ion o he plasma ac o XIII *de iciency in man. Ann. N.Y. Acad. Sci., 202, 190. DVORAK, H.F., SENGER, R.D. & DVORAK, A.M. (1983). Fib in as a componen o he umo s oma: o igins and biological sig- ni icance. Cance Me as . Re ., 2, 41. EDWARDS, R.L., RICKLES, E.R. & CRONLUND, M. (1981). Abno m- ali ies o blood coagula ion in pa ien s wi h cance : mononuclea cell issue ac o gene a ion. J. Lab. Clin. Med., 98, 917. EVANS, R. (1982). Mac ophages and neoplasms: new insigh s and hei implica ion in umo immunobiology. Cance Me as . Re ., 1, 227. GORDON, S.G. & CROSS, B.A. (1981). A ac o X-ac i a ing cys ein p o ease om malignan issue. J. Clin. In es ., 67, 1665. GORDON. S.G., FRANKS, J.J. & LEWIS, B.J. (1975). Cance p o- coagulan A: a ac o X ac i a ing p ocoagulan om malignan issue. Th ombos. Res., 61, 127. GORDON, S.G., FRANKS, J.J. & LEWIS, B.J. (1979). Compa ison o p ocoagulan ac i i ies in ex ac s o no mal and malignan human issue. J. Na !. Cance Ins ., 62, 773. HARRIS, N.L., DVORAK, A.M., SMITH, H. & DVORAK, H.F. (1982). Fib in deposi s in Hodgkin's disease. Am. J. Pa hol., 108, 119. HANSMANN, M.L. & KAISERLING, E. (1981). Elec on-mic oscopic aspec s o Hodgkin's disease. J. Cance Res. Clin. Oncol., 101, 135. HENRIKSSON, P., BECKER, S., LYNCH, G. & McDONAGH, J. (1985). Iden i ica ion o in acellula ac o XIII in human monocy es and mac ophages. J. Clin. In es ., 76, 528. HOPPER, K.E., WOOD, P.R. & NELSON, D.S. (1979). Mac ophage he e ogenei y. Vox. Sang., 36, 257. HUNYADI, J., SZEGEDI, G., SZABO, T., AHMED, A. & LAKI, K. (1981). Inc eased cy o oxic sensi i i y o YPC-1 umo cells om mice ea ed wi h ni osou eas. Cance Res., 41, 1677. JANOSSY, G., THOMAS, J.A., PIZZOLO, G. & 5 o he s. (1980). Immunohis ological diagnosis o lymphop oli e a i e diseases by selec ed combina ions o an ise a and monoclonal an ibodies. B . J. Cance , 42, 224. KEY, M. (1983). Mac ophages in cance me as ases and hei ele ance o me as a ic g ow h. Cance Me as . Re ., 2, 75. KIESSELBACH, T.H. & WAGNER, R.H. (1972). Demons a ion o ac o XIII in human megaka iocy es by a luo escen an ibody echnique. Ann. N.Y. Acad. Sci., 202, 318. LINDAHL, U., KOLSET, S.O., B0GWALD, J., 0STERUD, B. & SELJELID, R. (1982). S udies wi h a luminogenic subs a e on blood coagula ion ac o X/Xa p oduced by mouse pe i oneal mac ophages. Biochem. J., 206, 231. LORENZET, R., PERI, G., LOCATI, D. & 5 o he s. (1981). Gene a ion o p ocoagulan ac i i y by mononuclea phagocy es: a possible mechanism con ibu ing o blood clo ing ac i a ion wi hin malignan issues. Blood, 62, 271. MASON, D.Y. & WOOLSTON, R.E. (1982). Double immunoenzyma ic labelling. In Techniques in Immunocy ochemis y. Bullock, G.R. & Pe usz, P. (eds) Vol. 1, p. 135. Academic P ess: New Yo k. MUSZBEK, L., ADANY, R., SZEGEDI, G., POLGAR, J. & KAVAI, M. (1985). Fac o XIII o blood coagula ion in human monocy es. Th ombos. Res., 37, 401. MUSZBEK, L. & LAKI, K. (1984). In e ac ion o h ombin wi h p o eins o he han ib inogen ( h ombin suscep ible bonds). Ac i a ion o ac o XIII. In The Th ombin, Macho ich, R. (ed) p. 83. CRC P ess: Boca Ra on. MUELLER, J., BRUN DEL RE, G., BUERKI, H., KELLER, H.K., HESS, M.W. & COTTIER, H. (1975). Nonspeci ic acid es e ase ac i i y a c i e ion o di e en ia ion o T and B lymphocy es in mouse lymph nodes. Eu . J. Immunol., 5, 270. NEMES, Z., THOMAZY, V., ADANY, R. & MUSZBEK, L. (1986). Iden i ica ion o his iocy ic e iculum cells by he immunohis o- chemical demons a ion o ac o XIII (F-XIIIa) in human lymph nodes. J. Pa hol., 149, 121. O'MEARA, R.A.Q. (1958). Coagula i e p ope ies o cance s. I ish J. Med. Sci., 6, 474. 0STERUD, B., LINDAHL, U. & SELJELID, R. (1980). Mac ophages p oduce blood coagula ion ac o s. FEBS Le e s, 120, 41. POULTER, L.W., CHILOSI, M., SEYMOUR, G.J., HOBBS, S. & JANOSSY, G. (1983). Immuno luo escence memb ane s aining and cy ochemis y, applied in combina ion o analysing cell in e ac ions in si u. In Immunocy ochemis y. P ac ical applica- ions in pa hology and biology, Polak, J.M. & Van Noo den, S. (eds) p. 233. W igh PSG: B is ol. RICKLES, E.R. & EDWARDS, R.L. (1983). Ac i a ion o blood coagula ion in cance : T ousseau's synd ome e isi ed. Blood, 62, 14. SCHLAGER, S.I. & DRAY, S. (1975). Comple e umo eg ession wi h an ibody o ib in agmen E. J. Immunol., 115, 976. SEMERARO, N. & DONATI, M.B. (1981). Pa hways o blood clo ing ini ia ion by cance cells. In Malignancy and he hemos a ic sys em, Dona i, e al., (eds) p. 65. Ra en P ess: New Yo k. STEIN, H., BONK, A., TOLKSDORF, G., LENNERT, K., RODT, H. & GERDES, J. (1980). Immunohis ologic analysis o he o ganiz- a ion o no mal lymphoid issue and non-Hodgkin's lymphomas. J. His ochem. Cy ochem., 28, 746. STILLER, D. & KATENKAMP, D. (1978). In acellula subs ances in Hodgkin's lymphomas. Ul as uc u al in es iga ions. Vi chows A ch. A. Pa h. Ana . His ol., 380, 81. VAN DAM-MIERAS, M.C.E., MULLER, D.A., VAN DEIJK, W.A. & HEMKER, H.C. (1985). Clo ing ac o s sec e ed by monocy es and mac ophages: analy ical conside a ions. Th ombos. Res., 37, 9. VAN FURTH, R. (1981). Iden i ica ion o mononuclea phagocy es: o e iew and de ini ions. In Me hods Jo s udying mononuclea phagocy es, Adams, e al. (eds) p. 243. Academic P ess: New Yo k.