Ensing, Geert Jacob

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Dissertations of the University of Groningen

   University of GroningenThe radioactive labeling of monocytes.Ensing, Geert JacobIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1985Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Ensing, G. J. (1985). The radioactive labeling of monocytes. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 04-06-20223hapter 9: SUMMARY, SAMENVATTING.Ír l i th the aim of studylng a posslble relacionshlp between circulat ing monocytesand Sternberg-Reed cel-1s lnvesÈigatlons were sËarted on the speclf ic label ingof  monocy tes .In thls thesls the l l terature on the pert lnent data has been revlewed and aseries of experlments on the monocyte labe1lng procedure has been descrlbed.I n  Chap te r  I  t he  ob jec t i ve  o f  Ehese  l nvesE lga t l ons ,  t o  r e l a te  t he  o r l g i n  o fthe Sternberg-Reed ce11 ln HodgkÍnrs d lsease to c l rcuLat ing roonocytes and Ëhusto the mononuclear phagocyt ic  system, rdas stated.A review of the relevant l l terature on the characterlst ics and origln ofSternberg-Reed ce11s was presenÈed ln Chapter 2. I t  was concluded that noclear answer to the question, fron whlch celL type the Sternberg-Reed cel l  1soriglnating, can be glven by coroparing Èhe characEerlst ics of the Sternberg-Reed ce11 wlth other cel l  types because of three reasons:l .  The d i f fe ren t  techn lques  used by  the  var lous  lnves t iga tors .2: The snaLl number of patlents examlned.3. The malignant nature of Ehe Sternberg-Reed cel l .Therefore another approach, conslst lng of the adnlnlstrat lon of labeled ce11sthat wÍ11 mature lnto Sternberg-Reed cel l-s was suggested and the requlrernentsfor thls approach were given.In Chapter 3, the propert les and functlons of monocytes ln the hunan bodylogeEher wlth the aval lable lnfornatlon on monocyte klnetics were reviewed.The nonocyte 1s actlve nalnly agalnst lnvadlng rnlcroorganÍsrns by engulf lngthen dlrect ly or after belng st inulated co do so by lymphocytes.Next the probl-erns encountered by neasuring accurately the funcÈlon ofnonocytes, were discussed. These problems focus on the fact that oonocyÈes donot possess functlons that are unÍque when compared wlth the other nhiÈe bloodcel1s. Therefore methods to separate monocyÈes are necessary. AtÈentlon wasdrawn to the most pronising of these nethods: counterf low centrtfugatione luÈr ia t lon  ( "e lu t r la t lon" ) .Flnal ly nonocyte functlon under paÈhologlc conditLons, wíth special attentlonto l{odgklnrs dlsease, rÍas revleríed short ly. The general conclusion was thatin nost dlseases the lntrÍn8lc nonocyte functlon ís nornal but that 1n sornel ns tances  ( ln fecË ions ,  Hodgk ln rs  d isease)  the  nonocy tes  are  ac t lva ted  by  thecond l t lons  caused by  the  d lsease.In Chapter 4 the principles of ce11 1abellng rd-l th radloacÈive conpounds wered l s c u s s e d .These princlples can be separated inuo t\ro parts:1. Total separatlon of the part icular cel l  populaclon to be labeled andsubsequent  labe l lng  w l th  a  non-spec l f l c  rad lopharmaceut lca l .2 .  spec i f l c  ce l l  1abe1 lng  ln  a  n íxÈure  o f  ce lL  types  based on  a  we l l  de f lnedaff lntty of uhe cel1 under study for the radiopharmaceutLcal used.Next the radionucl ides Ëhat can be used for cel l  label ing purposes wered lscussed w i th  spec ia l  a t ten t lon  fo ,  11 l ln  and iEs  che la tes .The meEhods that nay be used ln order to achieve cel1 label lng by Lhe secondprlnclple mentloned above were summarized and the reports concernÍng Í lonocytelabel lng were reviewed. I t  was concluded Ehat, at present, no rel lable rnethoder ( lsLs  fo r  t .he  spec l f i c  labe l lng  o f  rnonocyEes.F lna l l y  the  pr inc ip les  o f  rad lodos Í rne t ry  were  d lscussed shor t l y .  Th is  sec t Íonwas focussed on the radlat lon dose Lhe labeled cel1s recelve because of thein t race l lu la r  loca1 lzed rad  loac t iv l t y .  ïhe  rad  la t lon  burden 1s  h tgh  incomparison !o amounts of radiat lon known to affect cel1 viabl l i ty. The rnethodused to calculate the dose frorn lnErace1lu1ar1-y radioactivl ty nay give over-estimated values. I t  rdas suggested that lnvestlgatlons comparlng theblologlcal effects of external- ard lnternal lrradlat lon of cel ls w111 have tobe performed. The consequences of the radlat ion dose were dlscussed and i t  wasconcluded thaE, due to the relatÍve lnsenslt ivl ty of the Ínonocyte, probably nornajor loss ln ce11 functlon wl11 occur. Radiat lon-induced nonocyte-relalednallgnancies ríere consldered funprobabl-e.In Chapter 5 a newly developed nethod for label lng nonocytes specíf ical ly byphagocytosls of l l l rrr-F"-col1oid \r l thout apparent loss of cel ls was descrlbecri n  d e t a l l .Th is  method cons is ts  o f  severa l  s teps :1. Blood leukocytes are depleted fron erythrocytes by acceleratedsedlÍnentatlon of the erythrocytes.2. A nononuclear cel l  suspenslon 1s prepared by centr l fugation on a two-layerhunan serum albunln (HSA) gradtent.3. After washlng Ehe ce1ls, label ing ls accornpltshed by lncubating the cel lswl th  l l l I n -Fe -co l l o i d .  The  rnonocy tes  phagocy tosed  t he  co l l o i d  pa rE i c l es .4.  The non-phagocytosed col lo id is  solubl l lzed by ACD and the labeledmonocytes are resuspended ln autologous plasma.The  ce11  suspens lon  ob ta l ned  a f t e r  cen t r i f uga t i on  on  HSA was  co roposed  o fnonocytes and lynphocytes.  The recovery of  monocytes was compleËe ln normalsand  i n  pa t l enËs  w i t h  new ly  d i agnosed  Hodgk in r s  d i sease .Autoradlography of  the labeled mononuclear cel ls  showed that :1.  MonocyLes are labeled to a s lgni f icant ly  h igher level  Èhan lyrnphocytes andgranulocytes,  resul t ing in a d i f ferent la l  label lng of  about 90 % in nornalsand  i n  pa t l en t s  w i t h  new ly  d l agnosed  Hodgk in ' s  d i sease .2 .  Mos t  o f  t he  au to rad iog raph l c  g ra l ns  we re  l oca l l zed  i n  t he  cy top lasn  o f  t heÍnonocy tes .The víabi1 l ty  and ster i l i ty  of  the labeled mononuclear cel1s proved to begood .I t  was concluded that  monocyËes can be labeled specl f ical ly  by phagocytosls of111 l r r -F " - "o11o ld ,  w l t h  good  v i ab l l l t y  a fÈe rwa rds .In Chapter 6 some in-v i t ro funct ions of  labeled monocytes were compared wi ththose  o f  non - l abe led  monocy tes .No s lgni f icanE dl f ferences were found in phagocytoslng capaci ty (neasured ascheml luminescence as wel l  as by nlcroscope) uslng opsonlzed zymosan part ic les.When che ( labeled) monocytes were cul tured for  up to 7 days no ef fect  oflabe1lng on nonocyte/macrophage recovery couLd be found. The amounl  ofcel lbound radloacÈlv i ty  decreased rapid ly:  af tet  7 days of  cul ture about 20%of the ln l t la l  amoun! was cel lbound. No re-ut l l lsat lon of  the Eracer could bede tec ted .At  least .  Ëwo factors are re levant  for  th ls d imlnishing ce1l-boundrad loac t l v l t y :1 .  D l v í s i on  o f  t he  monocy tes  du r l ng  cu l t u re ,  r esu l t l ng  l n  a  r d l l u t i on r  o f  t heanoun t  o f  r ad íoac t l v l t y  pe r  ce1 l .2.  A "secret lon" process thaÈ could be part ly  tnhtbt ted by disodiuncrornoglycate af ter  sevaral  days of  cul ture.I t  was concluded f roro these exper inents Ehat because of  the rapid loss ofce l l bound  rad ioac t l v lÈy  nonocyÈes  l abe led  *1 th  l l l I n -Fe -co1 lo l d  canno t  be  usedfor  long-term klnet lc  studíes ln-v ivo.Therefore,  the synthesls and label ing of  another conpound, a cel lobiose-- 1 0 4 -tyranine adduct, that ! ías orpected to show a rnuch slower loss fron the ceIl ,was perforned (Chapter 7). IE is known that cel" loblose cannot be metabollzedby  ce l l s ,  thus ,  once in  the  lysosomes,  1 t  l s  ra ther  s tab le  there .  In  o rder  tolabel cel loblose with 125ï an adduct rí1th Eyranine was narle and purlf Íed.SubsequenLly the tyrarnlne part of this adduct was rad io-1od Ínated. Thesynthesis ard label lng procedure proved to be rather easy.Severa l  a t tempts  to  labe l  monocyÈes w i th  th ls  adduc t  were  descr Íbed:1. ïhe use of the "plggy-back" systen wlth non-radloactive In-Fe-col l-oid asthe phagocytosed part lcle. A nlnor anounË of 125t becane celLbound wlth a lowdifferential label lng of the nonocytes. I t  was concluded that thls ls probablycauseil  by the rel-at ively low phagocytoslng capaclty of monocytes under thecond i t lons  used.2, The use of 125r-1.b"1"d aggregated lnrounoglobulln. Efforrs to 1abelaggregated Ímmunoglobulln wlth the 125t-tyramlne-cel loblose adduct fal led toy íe1d a  spec l f l c  ac t l v l t y  su f f l c ien t ly  h lgh  to  per fo rn  au torad lography .  Wi thaggregated lmrnunoglobulln, labeled with 125I (by the chloramÍne-T method) to ahlgher speclf ic act ivi ty so that autoradlography could be perforned, a veryl-ow dlf ferentlal nonocyte label lng (Less than 30%) was found.3. The use of l lposornes labeled wÍth a l ipld-phase rnarker (14c-cholesteryloleate). A Low uptake of radloactivl ty by the nononuclear ce11s was measured,an uptake that rras too low to warrant further studÍes. I t  sras concluded thaLthls l-ow uptake was probably caused by the low phagocyEoslng capaclty of thenonocytes under the condít lons used.The concluslon of thls chapter was that wlLh Ehe experimenEs conducted Ít  waslnpossible to Iabel monocytes spectf icaLly wlth the 125t-tyramlne-cel1obloseadduc t.F lna11y ,  ln  Chapter  8  th ree  poss ib le  d i rec t lons  fo r  fu tu re  inves t lga t Íons  1nnonocyte label lng were mentloned:1. Inproved ce1l separatlon nethods ln order to produce pure monocytepopulat ions without loss of any specif lc subpopulatÍon.2. The use of cel l  label ing agents that a11ow 1abelíng 1n pLasroa ni l ieu.3. The use of labeled monoclonal antibodÍes agalnst monocyte nerobraneanElgens .

The radioactive labeling of monocytes.

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The radioactive labeling of monocytes.

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