76

Biochimica et Biophysica Acta, 584 (1979) 76--83 © Elsevier/North-Holland Biomedical Press

BBA 28844

TRANSFERRIN RECEPTORS DURING RABBIT RETICULOCYTE MATURATION

FRANCESCO M. VAN BOCKXMEER and EVAN H. MORGAN

Department of Physiology, University of Western Australia, Nedlands, Western Australia, 6009 (Australia) (Received September 19th, 1978)

Key words: Transferrin receptor; Maturation; Iron assimilation; (Rabbit, Reticulocyte)

Summary Experiments were performed to examine the fate of transferrin receptors in reticulocytes as these cells mature in vivo to erythrocytes. Reticulocytosis, synchronized by administration of actinomycin D, was induced in adult rabbits. Simultaneous measurements were made of haematological parameters and the interaction between transferrin and reticulocytes while the cells matured in vivo to erythrocytes. As the reticulocytes matured there was a parallel decline in their ability to take up transferrin and transferrin iron. At the same time, there was a proportionate decrease in the density of receptors for transferrin on the reticulocyte surface. The affinity of the receptors for transferrin remained unaltered during the maturation process. It was concluded that the inability of erythrocytes to take up transferrin or its iron is due primarily to the loss of transferrin receptors from the maturing reticulocyte surface.

Introduction

Immature erythroid cells, up to and including reticulocytes, acquire iron from the circulating plasma protein transferrin and utilize it for haemoglobin synthesis [ 1--3]. Four stages are recognized in this process; namely, the binding of transferrin to specific receptors on the cell surface [4--7]; the endocytotic internalization of the transferrin-receptor complex [8,9,10]; the removal of iron from transferrin and its incorporation into haem, and finally the release of transferrin from the cell, enabling it to function in further cycles of plasma-tocell iron exchange [1,3]. The iron requirements of cells at different stages of erythroid cell differentiation are variable, reflecting on their rates of haemoglobin synthesis and

77 haemoglobin content. Maximum transferrin and iron uptake capacities have been observed in early and intermediate normoblasts (Kailis, S.G. and Morgan, E.H., unpublished) [11,12]. These capacities are maintained in late normoblasts and early reticulocytes but disappear with reticulocyte maturation into erythrocytes (Kailis, S.G. and Morgan, E.H., unpublished). It has frequently been proposed that the inability of mature erythrocytes to take up transferrin or its iron is due to a loss, during reticulocyte maturation, of transferrin receptors from the cell membrane [6,11,13]. No direct evidence to support this hypothesis has been presented. The work reported here examines the fate of transferrin receptors in reticulocytes obtained from anaemic rabbits, as the cells mature in vivo to erythrocytes. The rabbits were treated with actinomycin D [12] (Kailis, S.G. and Morgan, E.H., unpublished) in order to synchronize reticulocyte production. The experiments were designed to give information on the following parameters: the binding affinity of transferrin for receptors during maturation; the cell membrane receptor density; the rate of transferrin uptake by the cells following initial receptor binding, which is very likely an indicator of the transferrin endocytotic capacity of the cells [5,10]; and the rate of iron accumulation by the cells from internalized transferrin. Materials and Methods Actinomycin D was obtained from Merck, Sharpe and Dome, West Point, PA, U.S.A.; the radioisotopes S9Fe (FeC13, spec. act. 10--30 pCi/g Fe) and 12sI (as NaI, carrier free) from the Radiochemical Centre, Amersham, U.K.; and the non-ionic detergent, Teric 12A9 (poly(oxyethylene) ( n = 9) dodecyl alcohol, referred to as 'Teric' in the following text) was a gift from I.C.I. Ltd., Perth, Western Australia. Rabbit transferrin, isolated from pooled iron saturated serum [14] was labelled with 12sI using the iodine monochloride method of McFarlane and labelled with S9Fe as previously described [15]. Recticulocyte counts, haematocrits, protein concentration and radioactivity were measured as described in Ref. 5. Reticulocytosis was induced in adult rabbits by six daily injections of phenylhydrazine [16]. Cells collected by bleeding from a marginal ear vein into heparlnized tubes were washed three times with 0.15 M NaC1 at 4"C and 'buffy coat' cells were removed by aspiration during washing cycles. On the day following the last phenylhydrazine dose, actinomycin D dissolved in sterile 0.15 M NaC1 was injected into the rabbits in a dose of 100 pg/kg body weight [12]. Blood was collected 0, 3, 5, 6, 7, 10 and 12 days after actinomycin D treatment. For uptake studies, cell suspensions were incubated with doubly labelled iron-saturated transferrin as described in an earlier work [16]. Transferrin and iron uptake were measured as pg/ml reticulocytes/h. For the determination of reticulocyte receptor activities, ghost preparations were extracted with Teric to solubilize the receptors [ 5,7]. A chromatographic assay procedure, based on the Hummel-Dreyer principle [7,17], was used to measure the receptor-transferrin binding activity in Teric extracts. Plots of the amount of transferrin bound/receptor aliquot versus transferrin concentration were analysed, using a computerized non-linear regression method, to provide estimates for both the affinity of transferrin for the receptor and the density of

78

receptors on cell membranes after actinomycin D treatment [7]. Mean cell volumes, for use in this analysis, were obtained from red cell volume distributions using a Coulter Counter and from haematocrit values, red cell counts and reticulocyte percentage in the cell suspensions [ 7].

Results

Characteristics of reticulocytes following antinomycin D treatment• During phenylhydrazine treatment the rabbits were rendered severely anaemic and reticulocytotic. Thus, a marked reduction was observed in whole blood haematocrit (halved) haemoglobin concentration (to 40%) and the number of red cells per dm 3 (to 40%) relative to normal values obtained prior to treatment. Mean cell volumes and reticulocyte percentages rose, respectively, from approximately 65 fl to 105 fl and 2% to 70%. The results presented in this work were those obtained from one animal. Quantitatively similar results were obtained with another animal treated in an identical manner and with seven rabbits treated with actinomycin D followmg a haemorrhagm induced anaemias (Kailis, S.G. and Morgan, E.H., personal communication). Fig. 1 shows that the reticolocyte percentage dropped sharply from 70% at day 0 to a minimum of 5% at day 5 and increased again over the next 5 days to reach a m a x i m u m of 73 seven days after actinomycin D administration. These data also indicate that the

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79 anaemic condition persisted for the twelve day sampling period. The cells collected at the various time intervals were stained with new methylene blue and examined by phase contrast light microscopy (×1000 magnification) and photographed on a calibrated graticule. On the basis of the size of the reticulocytes and on their stained reticulum content, individual cells were classified as early, intermediate and late reticulocytes. All cell suspensions examined contained various proportions of reticulocytes at different stages of maturity. However, cells obtained on days 0, 5 and 6 were mainly early reticulocytes, day 7 intermediate and those on days 3, 10 and 12 predominantly late reticulocytes. Incubation of reticulocytes with transferrin. The cells obtained at different time intervals were incubated with SgFe and 12SI-labelled transferrin. If it is presumed that the previous classification of reticulocytes into early, intermediate and late reflects their ability to take up transferrin and its iron then the results on Fig. 2 are in reasonable agreement with this classification. Maximum rates of iron uptake were observed on days 0, 5 and 6, intermediate rates on days 3 and 7 and lowered rates on days 10 and 12. Morphologically, day 0 and 3 reticulocytes were clearly predominantly early and late reticulocytes, respectively, although on a functional basis they appear to be intermediate in nature. The initial rate of transferrin uptake by the reticulocytes and the amount of trans-

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Fig. 2. Iron u p t a k e ability b y i n t a c t r e t i c u l o c y t e s f r o m transferrin at various t i m e s • f t e r a c t i n i m y c i n D t r e • t m e n t . S a m p l e s o f r e t i c u l o c y t e w e r e i n c u b a t e d w i t h 12 S I-, s 9 F e . l a b e n e d transfen~in at 2 m g / m l and t h e rate o f iron u p t • k e b y t h e cells, e x p r e s s e d as ~ m o l / m l r e t i c u l o e y t e s p e r h w a s d e t e r m i n e d . Cells • p p e a r i n g o n day 6 w e r e classified as early; t h o s e on day 7 and 1 0 as i n t e r m e d i • t e , and o n d a y 1 2 as late reticulocytes.

80 TABLE I BINDING CONSTANTS OF TRANSFERRIN BINDING TO RECEPTORS R a b b i t b l o o d cells s a m p l e d a t v a r i o u s t i m e s f o l l o w i n g a c t i n o m y c i n D d o s a g e w e r e t r e a t e d to y i e l d r e c e p t o r c o n t a i n i n g e x t r a c t s as d e s c r i b e d in Materials a n d M e t h o d s . T h e s e e x t r a c t s w e r e a s s a y e d for t~ansferrin b i n d i n g a c t i v i t y as d e s c r i b e d in Fig. 3. T h e b i n d i n g c o n s t a n t s w e r e d e r i v e d f r o m t h e s e d a t a as d e s c r i b e d in Materials a n d M e t h o d s a n d are e x p r e s s e d in t h e f o r m o f m e a n -+ s t a n d a r d e r r o r o f t h e m e a n . B m a x : t h e a s s y m p t o t i c a m o u n t o f t r a n s f e r r i n b o u n d / r e c e p t o r a l i q u o t a t s a t u r a t i n g t r a n s f e r r i n c o n c e n t r a t i o n s . Ka: the equilibrium constant of association of transferrin for the receptor. Time after actinomycin D administration (days)

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Fig. 3. Plots o f t h e a m o u n t o f t r a n s f e r r i n b o u n d / r e c e p t o r a l i q u o t versus t r a n s f e r r i n c o n c e n t r a t i o n . B l o o d was r e m o v e d from the circulation of rabbits at various times after actinomycin D t r e a t m e n t . The receptorc o n t a i n i n g e x t r a c t s p r e p a r e d f r o m t h e s e cells w e r e a s s a y e d f o r t z a n s f e r r i n b i n d i n g a c t i v i t y in t h e p r e s e n c e o f d i f f e r e n t c o n c e n t r a t i o n s of 12 $ I-labelled t r a n s f e r r i n (specific a c t i v i t y 19 c p m / n g t r a n s f e r r i n ) . B i n d i n g a c t i v i t y , e x p r e s s e d as # g t r a n s f e r r i n b o u n d / r e c e p t o r a l i q u o t , w a s m e a s u r e d o n d a y O, • ~', d a y 3, • i ; d a y 6, • A; d a y 9, o o, a n d d a y 10, o o.

81

ferrin taken up after 40 min incubation gave results similar to those obtained for the rates of iron uptake by these cells. Receptor activities during reticulocyte maturation. Extracts, containing solubilised transferrin receptors, were prepared from cells collected on days 0, 3, 5, 6, 7, 10 and 12 after actinomycin D treatment. The transferrin binding activity of these extracts was measured in the presence o f different transferrin concentrations under conditions where equilibrium was maintained between transfertin and the receptor [7]. The plots of the amount of transferrin bound/receptor aliquot versus the free transferrin concentration are shown in Fig. 3. The data was analysed using an iterative Gauss-Raphson least-squares method [7] to yield estimates for the equilibrium constant of association (Ka) of transferrin for the receptor and the maximum receptor-transferrin binding capacity (Bmax) at saturation levels of transferrin concentration (Table I). The data points and curves fitted to them, when plotted according to the method of Scatchard, gave linear relationships between transferrin bound over transferrin concentration versus the amount of transferrin bound. Thus, as reported elsewhere [ 7 ], binding of transferrin to its receptor occurs in a specific, reversible and saturable manner and only one class of receptor is involved. The low reticulocyte percentage suspensions obtained on days 5 and 12 could not be analysed in this manner as the receptor activities were too low for reliable K a estimates to be performed on them. The K a values (Table I) obtained from cells

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82 at different stages of maturation were not significantly different and this indicate no dependence on reticulocyte maturity. Number of receptors per reticulocyte during maturation. From estimates of mean reticulocyte volumes and the Bmax values obtained during the analysis of the data in Fig. 3, the average n u m b e r of transferrin receptors per reticulocyte at various stages of maturation was determined (Fig. 4). This figure shows that the transferrin-receptor density per pm 2 of cell surface area correlates well with the observed rates of iron uptake by intact cells (Fig. 2). Again, it is evident that cells morphologically classified as early and late, on days 0 and 3, respectively, have intermediate values for receptor densities as well as rates of iron or transferrin uptake. The cells obtained on day 6 can be classified as early reticulocytes on the bases o f their morphology, ability to take up iron and transferrin, and on their receptor densities. On a similar basis, it is also evident that over 50% of these cells had matured to intermediate reticulocytes within 24 h (by day 7). Similarly, cells obtained over the next 72 h (days 10 and 12) had matured to late reticulocytes. Discussion

Actinomycin D interferes with the normal proliferative and differentiation processes occurring in erythropoietic stem cells by a selective inhibition of DNA-dependent RNA synthesis, thereby interrupting red cell progenitor production. Upon cessation of its action, renewed red cell production is synchronized [12,18--21]. In anaemic rabbits, a large number of reticulocytes appeared in the circulation about 6 days after actinomycin D treatment (Fig. 1). Morphologically and functionally, the majority of the cells were synchronized at the same developmental stage, that is, very early reticulocytes. These cells exhibited near maximal values for mean cell volume, stained reticulum content, transferrin endocytotic capacity, their ability to take up iron from transferrin, and membrane transferrin-receptor density. As the reticuloccytes matured in vivo, the values for these morphological and functional indices declines rapidly in a parallel manner (Figs. 1, 2 and 4). The striking feature of these results was the close correlation between the loss of membrane receptors for transferrin and the loss by reticulocytes of their ability to take up transferrin or its iron. In the interaction between transferrin and reticulocytes the rate of iron assimilation by the cells is 'paced' or limited by the intracellular availability of transferrin [7,10]. The rate of transferrin internalization seems to be governed by the transferrin endocytotic capacity of the cells and the degree of occupation of membrane receptors by transferrin [1,7,8,10]. It may be concluded therefore, that the rates of transferrin and iron uptake by reticulocytes is paced by the transferrin-membrane receptor interaction. Observed reductions, during reticulocyte maturation, of the transferrin uptake capacity could be due to both a reduction in the affinity of transferrin for the receptor and the loss of receptors from the cell membrane. The results presented here indicate that the affinity of transferrin for its receptor is unaltered during reticulocyte maturation (Table I). There was, however, a marked loss of receptors as the cells matured. Thus it m a y be concluded that the prim-

83 ary event leading to the inability of immature erythrocytes to take up transferrin or its iron, is the complete loss o f transferrin receptors from the maturing reticulocyte surface. At the same time as the receptors are lost various other morphological and metabolic cellular attributes are also lost [13,22--24]. How these factors are interrelated has yet to be determined. Acknowledgements This investigation was supported by grants from the Australian Research Grants Committee and the University of Western Australia. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Jandl, J.H. and Katz, J. (1963) J. Clin. Invest. 42, 314--336 Morgan, E.H. and Laurell, C-B. (1963) Brit. J. Haematol. 9, 471--483 Morgan, E.H. (1964) Brit. J. Haematol. 10, 442--452 Van Bockxmeer, F.M., Hemmaplardh, D. and Morgan, E.H. (1975) in Proteins of Iron Storage and Transport in Biochemistxy and Medicine (Crichton, R.R., ed,), North-Holland, Amsterdam Van Bockxmeer, F.M. and Morgan, E.H. (1977) Biochim. Biophys. Acta 468, 437--450 Leibman, A. and Aisen, P. (1977) Biochemistry 16, 1268--1272 Van Bockxmeer, F.M., Yates, G.K. and Morgan, E.H. (1978) Eux. J. Biochem., in press Morgan, E.H. and Appleton, T.C. (1969) Nature 223, 1371--1372 Sullivan, A.L., Grasso, J.A. and Weintraub, L. (1976) Blood 47, 133--143 Hemmapla~dh, D. and Morgan, E.H, (1977) Brit. J. Haematol. 36, 85--96 Lajtha, L.G. and Suit, H.D. (1955) Brit. J. Haematol. 1, 55--61 Hershko, Ch., Schwartz, R. and Izak, R. (1969) Brit. J. Haematol. 17, 569--579 Heywood, J.D., Ganzoni, A. and Hillman, R.S. (1972) Brit. J. Haematol. 23, 351--361 Baker, E., Shaw, D.C. and Morgan, E.H. (1968) Biochemistry 7, 1371--1378 Hemmaplardh, D. and Morgan, E.H. (1974) Biochim. Biophys. Acta 373, 84--99 Baker, E. and Morgan, E.H. (1969) Biochemistry S, 1133--1141 Hummel, J.P. and Dreyer, W.J. (1972) Biochim. Biophys. Acta 6 3 , 5 3 2 - - 5 3 4 Izak, G., Karsai, A., Eylon, L. and Hershko, Ch. (1971) J. Lab. Clin. Med. 77,916--922 Izak, G., Karsai, A., Eylon, L. and Hershko, Ch. (1971) J. Lab. Clin. Med. 77,923--930 Izak, G. and Karsai, A. (1972) Blood 39, 814--925 Zuckerman, K.S., Sullivan, R. and Quesenberry, P.J. (1978) Blood 51,957---969 Lodish, H.F., Small, B. and Chang, H. (1975) Dev. Biol. 47, 59--67 Gasko, O. and Danon, D. (1974) Brit. J. Haematol. 28, 463---470 Hulea, S.A. and Amstein, H.R.V. (1977) Biochim. Biophys. Acta 476, 131--148