Journal o f Immunological Methods, 31 (1979) 193--195
193
© Elsevier/North-Holland Biopaedical Press Short c o m m u n i c a t i o n R A D I O T O X I C I T Y OF 111INDIUM
G. KRAAL and A.A. GELDOF Department o f Histology, Free University, P.O. Box 7161, 1007 MC Amsterdam, The Netherlands
(Received 1 March 1979, accepted 6 August 1979)
Bone marrow cells were labelled with various concentrations of 11lindium.oxine and their capacity to form colonies (CFU-s) in an adoptive transfer was investigated. Labelling with more than 0.1 pCi/ml/lO 7 cells impaired colony formation. It is concluded that the 11lindium_oxine complex is detrimental to cell proliferation.
INTRODUCTION The advantages of the lipid-soluble 111Indium-oxine com pl ex for labelling blood c o m p o n e n t s such as l y m p h o c y t e s , p o l y m o r p h s and platelets have been r e p o r t e d (McAfee and Thakur, 1976; Segal et al., 1976; Thakur, 1978). The short half-life (68 h) and the possibility of external det ect i on of em i t t ed radiation have made it a suitable radioisotope for clinical studies. After the c o m p l e x has entered the cell the l~lIndium dissociates from the oxine and binds firmly to c o m p o u n d s in the cytoplasm and possibly to DNA (Thakur et al., 1977a). The spontaneous release of 111Indium is very low and the label released is n o t re-utilized (Frost et al., 1978). Although migration and localization patterns of 11~Indium-oxine-labelled cells are specific (Thakur et al., 1977b; Frost et al., 1978) effects of the labelling on cell f unc t i on by radiation damage have been r e p o r t e d (Rannie et al., 1977; Segal et al., 1978}. Haemo p o ietic stem cells are very sensitive to radiation damage after entering the cell cycle (Duplan and Feinendegen, 1970). In order to study the effect o f l'~Indium on proliferation h a e m o p o i e t i c stem cells were incubated with various concent r a t i ons of ' ~ I n d i u m - o x i n e and the ability to form colonies (CFU-s) in an adoptive transfer was investigated. MATERIALS AND METHODS Animals
CBA/J mice, 6--8 weeks old were obtained from Zentralinstitut fiir Versuchstierzucht, Hannover, F.R.G. T h e y were given acidified water (pH 3) and commercial mouse f o o d ad l i b i t u m .
194
Radioactive labelling and CFU-s determination 111Indium (INS 1, A m e r s h a m R a d i o c h e m i c a l s , Great Britain) was chelated with o x i n e a c c o r d i n g to the m e t h o d o f T h a k u r et al. ( 1 9 7 7 b ) . B o n e m a r r o w cells were o b t a i n e d b y flushing the f e m o r a l cavities with H a n k ' s Balanced Salt S o l u t i o n (HBSS). A f t e r washing, the cells were incub a t e d for 20 min at r o o m t e m p e r a t u r e at a c o n c e n t r a t i o n o f 107/ml with d i f f e r e n t c o n c e n t r a t i o n s o f 111Indium as indicated. C o n t r o l i n c u b a t i o n was p e r f o r m e d with o u t d a t e d (cold) ~11Indium-oxine (30 days old). The cells were w a s h e d twice with HBSS and 2 X l 0 s cells were injected i n t r a v e n o u s l y into lethally irradiated recipients. Details o f the irradiation p r o c e d u r e have been described elsewhere (Kraal and B o d e n 1978). A f t e r 8 d a y s the spleens were r e m o v e d and the colonies were c o u n t e d after f i x a t i o n o f the spleens in B o u i n ' s s o l u t i o n f o r 24 h. The a m o u n t o f r a d i o a c t i v i t y injected with 2 X 10 s b o n e m a r r o w cells was d e t e r m i n e d in a dual c h a n n e l g a m m a scintillation c o u n t e r (LKB). RESULTS AND DISCUSSION A f t e r i n c u b a t i o n o f b o n e m a r r o w cells with various c o n c e n t r a t i o n s o f 111Indium-oxine 2 X 10 s cells were injected into irradiated recipients. Table 1 represents the n u m b e r o f colonies c o u n t e d after 8 d a y s and the r a d i o a c t i v i t y injected with 2 X l 0 s cells. T h e c a p a c i t y t o f o r m colonies was m a r k e d l y impaired a f t e r i n c u b a t i o n o f the b o n e m a r r o w cells with m o r e t h a n 0.1 pCi/ml. This i m p a i r m e n t was caused b y r a d i a t i o n d a m a g e o f the 111Indiu m because i n c u b a t i o n o f the cells with cold 111Indium at a c o n c e n t r a t i o n e q u i v a l e n t t o 10 p C i / m l gave n o r m a l n u m b e r s o f CFU-s. These results are in a c c o r d a n c e with t h e findings o f Segal et al. (1978). These a u t h o r s describe r e d u c e d tritiated t h y m i d i n e i n c o r p o r a t i o n in cells labelled with 40 pCi 111Indium b u t n o t in cells labelled with cold 111 I n d i u m . This e f f e c t c o u l d be f o u n d o n l y after 27 h and h a d n o influence on r o s e t t e - f o r m i n g capacity. TABLE 1 THE NUMBER OF CFU-s IS EXPRESSED AS THE MEAN (-+ S.E.) OF 5 SPLEENS. INCUBATION IN HBSS AND WITH OUTDATED INDIUM-OXINE WAS PERFORMED AS CONTROL The radioactivity associated with 2 x l0 s bone marrow cells after labelling was expressed as cpm. 1111ndium-oxine (/~Ci/ml)
CFU-s/2 x 10 s bone marrow cells
Radioactivity/2 x 10 s bone marrow cells (cpm)
control 0.1 pCi 1.0 pCi 10.0 pCi 10.0 pCi cold
20.20 23.67 11.50 2.75 20.75
30 69 673 20,509 19
-+ 2.17 + 5.51 -+ 2.08 + 2.36 -+ 2.99
195 T h e r e l a t i o n s h i p b e t w e e n u p t a k e o f label a n d t h e various c o n c e n t r a t i o n s is n o t linear (Table 1) a n d i n d i c a t e s i n e f f i c i e n t labelling at l o w e r c o n c e n t r a tions. This, t o g e t h e r w i t h t h e small p e r c e n t a g e o f CFU-s p r e s e n t in t h e s u s p e n s i o n s (20.20-+ 2 . 1 7 / 2 X l 0 s cells), m a k e s it likely t h a t t h e CFU-s d e t e c t e d at t h e various c o n c e n t r a t i o n s were n o t labelled. T h e r e f o r e we a s s u m e t h a t labelling w i t h 111Indium-oxine at c o n c e n t r a t i o n s n e e d e d to achieve u n i f o r m labelling a n d g o o d cell d e t e c t i o n is d e t r i m e n t a l to cell p r o liferation. A f t e r labelling cell surfaces are n o t a f f e c t e d a n d loss o f f u n c t i o n a l a c t i v i t y is o n l y m a n i f e s t e d a f t e r s o m e t i m e (Segal et al., 1978). This indicates t h a t the t ~ l I n d i u m - o x i n e c o m p l e x is suitable f o r s h o r t t i m e m i g r a t i o n studies b u t in e x p e r i m e n t s in w h i c h m i t o t i c a c t i v i t y o f t h e labelled cells o c c u r s e.g. in g r a f t vs h o s t r e a c t i o n s , its d e t r i m e n t a l e f f e c t s m u s t be seriously c o n s i d e r e d . ACKNOWLEDGEMENTS We are i n d e b t e d to Prof. Dr. H.L. L a n g e v o o r t a n d Dr. N. Van R o o i j e n f o r t h e i r h e l p f u l advice a n d criticism d u r i n g t h e p r e p a r a t i o n o f t h e m a n u s c r i p t , a n d to Mr. A.J. C o o p s f o r facilities in t h e ' R a d i o - N u c l i d e n C e n t r u m ' a n d f o r p r e p a r i n g t h e 11lindium_oxine" REFERENCES Duplan, J.F. and L.E. Feinendegen, 1970, Proc. Soc. Exp. Biol. Med. 134,319. Frost, P., J. Smith and H. Frost, 1978, Proc. Soc. Exp. Biol. Med. 157, 61. Kraal, G. and D. Boden, 1978, Cell Immunol. 39, 219. McAfee, J.G. and M.L. Thakur, 1976, J. Nucl. Med. 17,480. Rannie, G.H., M.L. Thakur and W.L. Ford, 1977, Clin. Exp. Immunol. 29,509. Segal, A.W., R.N. Arnat, M.L. Thakur and J.P. Lavender, 1976, Lancet ii, 1056. Segal, A.W., P. Deteix, R. Garcia, P. Tooth, G.D. Zanelli and A.C. Allison, 1978, J. Nucl. Med. 19, 1238. Thakur, M.L., 1978, Int. J. Appl. Rad. Isotopes 28,183. Thakur, M.L., A.W. Segal, L. Louis, M.J. Welch, J. Hopkins and T.J. Peters, 1977a, J. Nucl. Med. 18, 1022. Thakur, M.L., R.E. Coleman and M.J. Welch, 1977b, J. Lab. Clin. Med. 89,217.
193
© Elsevier/North-Holland Biopaedical Press Short c o m m u n i c a t i o n R A D I O T O X I C I T Y OF 111INDIUM
G. KRAAL and A.A. GELDOF Department o f Histology, Free University, P.O. Box 7161, 1007 MC Amsterdam, The Netherlands
(Received 1 March 1979, accepted 6 August 1979)
Bone marrow cells were labelled with various concentrations of 11lindium.oxine and their capacity to form colonies (CFU-s) in an adoptive transfer was investigated. Labelling with more than 0.1 pCi/ml/lO 7 cells impaired colony formation. It is concluded that the 11lindium_oxine complex is detrimental to cell proliferation.
INTRODUCTION The advantages of the lipid-soluble 111Indium-oxine com pl ex for labelling blood c o m p o n e n t s such as l y m p h o c y t e s , p o l y m o r p h s and platelets have been r e p o r t e d (McAfee and Thakur, 1976; Segal et al., 1976; Thakur, 1978). The short half-life (68 h) and the possibility of external det ect i on of em i t t ed radiation have made it a suitable radioisotope for clinical studies. After the c o m p l e x has entered the cell the l~lIndium dissociates from the oxine and binds firmly to c o m p o u n d s in the cytoplasm and possibly to DNA (Thakur et al., 1977a). The spontaneous release of 111Indium is very low and the label released is n o t re-utilized (Frost et al., 1978). Although migration and localization patterns of 11~Indium-oxine-labelled cells are specific (Thakur et al., 1977b; Frost et al., 1978) effects of the labelling on cell f unc t i on by radiation damage have been r e p o r t e d (Rannie et al., 1977; Segal et al., 1978}. Haemo p o ietic stem cells are very sensitive to radiation damage after entering the cell cycle (Duplan and Feinendegen, 1970). In order to study the effect o f l'~Indium on proliferation h a e m o p o i e t i c stem cells were incubated with various concent r a t i ons of ' ~ I n d i u m - o x i n e and the ability to form colonies (CFU-s) in an adoptive transfer was investigated. MATERIALS AND METHODS Animals
CBA/J mice, 6--8 weeks old were obtained from Zentralinstitut fiir Versuchstierzucht, Hannover, F.R.G. T h e y were given acidified water (pH 3) and commercial mouse f o o d ad l i b i t u m .
194
Radioactive labelling and CFU-s determination 111Indium (INS 1, A m e r s h a m R a d i o c h e m i c a l s , Great Britain) was chelated with o x i n e a c c o r d i n g to the m e t h o d o f T h a k u r et al. ( 1 9 7 7 b ) . B o n e m a r r o w cells were o b t a i n e d b y flushing the f e m o r a l cavities with H a n k ' s Balanced Salt S o l u t i o n (HBSS). A f t e r washing, the cells were incub a t e d for 20 min at r o o m t e m p e r a t u r e at a c o n c e n t r a t i o n o f 107/ml with d i f f e r e n t c o n c e n t r a t i o n s o f 111Indium as indicated. C o n t r o l i n c u b a t i o n was p e r f o r m e d with o u t d a t e d (cold) ~11Indium-oxine (30 days old). The cells were w a s h e d twice with HBSS and 2 X l 0 s cells were injected i n t r a v e n o u s l y into lethally irradiated recipients. Details o f the irradiation p r o c e d u r e have been described elsewhere (Kraal and B o d e n 1978). A f t e r 8 d a y s the spleens were r e m o v e d and the colonies were c o u n t e d after f i x a t i o n o f the spleens in B o u i n ' s s o l u t i o n f o r 24 h. The a m o u n t o f r a d i o a c t i v i t y injected with 2 X 10 s b o n e m a r r o w cells was d e t e r m i n e d in a dual c h a n n e l g a m m a scintillation c o u n t e r (LKB). RESULTS AND DISCUSSION A f t e r i n c u b a t i o n o f b o n e m a r r o w cells with various c o n c e n t r a t i o n s o f 111Indium-oxine 2 X 10 s cells were injected into irradiated recipients. Table 1 represents the n u m b e r o f colonies c o u n t e d after 8 d a y s and the r a d i o a c t i v i t y injected with 2 X l 0 s cells. T h e c a p a c i t y t o f o r m colonies was m a r k e d l y impaired a f t e r i n c u b a t i o n o f the b o n e m a r r o w cells with m o r e t h a n 0.1 pCi/ml. This i m p a i r m e n t was caused b y r a d i a t i o n d a m a g e o f the 111Indiu m because i n c u b a t i o n o f the cells with cold 111Indium at a c o n c e n t r a t i o n e q u i v a l e n t t o 10 p C i / m l gave n o r m a l n u m b e r s o f CFU-s. These results are in a c c o r d a n c e with t h e findings o f Segal et al. (1978). These a u t h o r s describe r e d u c e d tritiated t h y m i d i n e i n c o r p o r a t i o n in cells labelled with 40 pCi 111Indium b u t n o t in cells labelled with cold 111 I n d i u m . This e f f e c t c o u l d be f o u n d o n l y after 27 h and h a d n o influence on r o s e t t e - f o r m i n g capacity. TABLE 1 THE NUMBER OF CFU-s IS EXPRESSED AS THE MEAN (-+ S.E.) OF 5 SPLEENS. INCUBATION IN HBSS AND WITH OUTDATED INDIUM-OXINE WAS PERFORMED AS CONTROL The radioactivity associated with 2 x l0 s bone marrow cells after labelling was expressed as cpm. 1111ndium-oxine (/~Ci/ml)
CFU-s/2 x 10 s bone marrow cells
Radioactivity/2 x 10 s bone marrow cells (cpm)
control 0.1 pCi 1.0 pCi 10.0 pCi 10.0 pCi cold
20.20 23.67 11.50 2.75 20.75
30 69 673 20,509 19
-+ 2.17 + 5.51 -+ 2.08 + 2.36 -+ 2.99
195 T h e r e l a t i o n s h i p b e t w e e n u p t a k e o f label a n d t h e various c o n c e n t r a t i o n s is n o t linear (Table 1) a n d i n d i c a t e s i n e f f i c i e n t labelling at l o w e r c o n c e n t r a tions. This, t o g e t h e r w i t h t h e small p e r c e n t a g e o f CFU-s p r e s e n t in t h e s u s p e n s i o n s (20.20-+ 2 . 1 7 / 2 X l 0 s cells), m a k e s it likely t h a t t h e CFU-s d e t e c t e d at t h e various c o n c e n t r a t i o n s were n o t labelled. T h e r e f o r e we a s s u m e t h a t labelling w i t h 111Indium-oxine at c o n c e n t r a t i o n s n e e d e d to achieve u n i f o r m labelling a n d g o o d cell d e t e c t i o n is d e t r i m e n t a l to cell p r o liferation. A f t e r labelling cell surfaces are n o t a f f e c t e d a n d loss o f f u n c t i o n a l a c t i v i t y is o n l y m a n i f e s t e d a f t e r s o m e t i m e (Segal et al., 1978). This indicates t h a t the t ~ l I n d i u m - o x i n e c o m p l e x is suitable f o r s h o r t t i m e m i g r a t i o n studies b u t in e x p e r i m e n t s in w h i c h m i t o t i c a c t i v i t y o f t h e labelled cells o c c u r s e.g. in g r a f t vs h o s t r e a c t i o n s , its d e t r i m e n t a l e f f e c t s m u s t be seriously c o n s i d e r e d . ACKNOWLEDGEMENTS We are i n d e b t e d to Prof. Dr. H.L. L a n g e v o o r t a n d Dr. N. Van R o o i j e n f o r t h e i r h e l p f u l advice a n d criticism d u r i n g t h e p r e p a r a t i o n o f t h e m a n u s c r i p t , a n d to Mr. A.J. C o o p s f o r facilities in t h e ' R a d i o - N u c l i d e n C e n t r u m ' a n d f o r p r e p a r i n g t h e 11lindium_oxine" REFERENCES Duplan, J.F. and L.E. Feinendegen, 1970, Proc. Soc. Exp. Biol. Med. 134,319. Frost, P., J. Smith and H. Frost, 1978, Proc. Soc. Exp. Biol. Med. 157, 61. Kraal, G. and D. Boden, 1978, Cell Immunol. 39, 219. McAfee, J.G. and M.L. Thakur, 1976, J. Nucl. Med. 17,480. Rannie, G.H., M.L. Thakur and W.L. Ford, 1977, Clin. Exp. Immunol. 29,509. Segal, A.W., R.N. Arnat, M.L. Thakur and J.P. Lavender, 1976, Lancet ii, 1056. Segal, A.W., P. Deteix, R. Garcia, P. Tooth, G.D. Zanelli and A.C. Allison, 1978, J. Nucl. Med. 19, 1238. Thakur, M.L., 1978, Int. J. Appl. Rad. Isotopes 28,183. Thakur, M.L., A.W. Segal, L. Louis, M.J. Welch, J. Hopkins and T.J. Peters, 1977a, J. Nucl. Med. 18, 1022. Thakur, M.L., R.E. Coleman and M.J. Welch, 1977b, J. Lab. Clin. Med. 89,217.
