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The role of iron and transferrin in lymphocyte transformation
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Immunology Today, vol. 4, No. 12, 1983
The role of iron and transferrin in lymphocyte transformation Jeremy H. Brock and Tryfonia Mainou-Fowler It has recently become apparent that many types of transformed cells possess membrane receptorsfor the plasma ironbinding protein transferrin. In this review Jeremy Brock and T~yfonia Mainou-Fowler discuss these observations in relation to transferrin's properties as an iron-transport protein, and the relative importance of iron and transferrin in lymphocyte transformation. Iron is essential to almost all known forms of life. In man about 70% of total body iron is present as haemoglobin, most of the remainder being stored as ferritin in the cells of the liver, spleen and bone marrow. However, small but significant quantities of iron are also required by other cells and tissues, including lymphocytes, for the synthesis of metabolically important compounds such as metalloenzymes and cytochromes. The expansion of clones of lymphocytes, an essential feature of immune responses, involves increased cellular metabolic activity and cell division, and hence de-novo synthesis of some ironcontaining compounds and an increased iron requirement. It is therefore possible that an inadequate delivery of iron might prevent lymphocyte transformation from proceeding optimally. Two lines of evidence support this contention, albeit indirectly. Firstly, several studies in h u m a n subjects and, more recently, in experimental animals have reported that iron deficiency can result in impaired cell-mediated immune responses ~. A second line of evidence suggesting a crucial role for both iron and transferrin in lymphocyte transformation comes from studies demonstrating that both are essential for transformation of lymphocytes in response to mitogens in serum-free medium2'3. The same is true for the successful propagation of many cell lines in serum-free media~. These findings, and their implications, will be considered in more detail below.
Iron deficiency and T-cell transformation A number of clinical studies have demonstrated that iron deficiency may cause reduced skin reactions to antigens such as Candida and PPD, and decrease the in-vitro response of peripheral blood lymphocytes to mitogenss-7. More recently, studies using mic& or rats 9have indicated that the priming oflymphocytes by antigen is not affected by iron deficiency, thus suggesting that it is either transformation or effector mechanisms which are impaired. We have recently investigated the role of iron on the' transformation step by studying the ability of lymph-node cells from normal or iron-deficient mice to transform in response to concanavalin A (Con A). In these experiments serum-free media were used, thus allowing the iron levels to be carefully controlled. It was found that whereas the iron concentration of the medium, which contained transferrin, had a considerable effect on lymphocyte transformation, there was no significant difference in the transformation of lymphocytes from University Department of Bacteriology and Immunology, Western Infirmary, Glasgow G11 6NT, UK.
normal or iron-deficient mice (Table I). Furthermore, transformation of lymphocytes from either group was lower in media supplemented with serum from irondeficient mice than when serum from normal mice was used, but addition of sufficient iron to bring the serum iron level of the deficient serum to that of the normal serum significantly improved its ability to promote transformation. These studies indicate that the lymphocytes of iron-deficient mice have a normal capacity to transform in response to mitogens but that the supply of transferrin-bound iron in the sera of deficient animals is, under these conditions at least, a limiting factor in allowing transformation and cell division to proceed at normal rates. These results emphasize the important role played by iron in lymphocyte transformation, and the way in which iron and transferrin interact with lymphocytes is discussed in the following sections. TABLE I. Response of lymphocytesfrom norma4and iron-deficient mice to concanavalinA. Culture medium:RPMI 1640 supplementedwith
Incorporationof 14C-thymidine (c.p.m.) into lymphocytes from Iron-replete mice
Apotransferrin, 50 #g ml i Transferrin, 20% iron-saturated, 50 #g ml- ~ 2.5% normal autologousserum 2.5 % iron-deficientautologous serum 2.5% iron-deficientautologous serum supplementedwith iron
Iron-deficient mice
1161 + 550
836 +- 164
3564 + 686 5569 ± 377
3590 -+318 5067 ± 777
3655 __.601
3751 -+601
4619± 621
4969 ± 846
Transferrin and iron uptake It is now well established that transferrin is the only physiological carrier of iron in normal serum. While nontransferrin-bound serum iron may be found in patients with severe iron overload whose serum transferrin is saturated 1°, earlier evidence that such a fraction also existed in normal serum" has now been refuted n. Transferrin is a glycoprotein of molecular weight about 80 000. It consists of a single polypeptide chain which is folded to give two largely independent and similar, but not identical, globular domains, each of which can bind one ferric ion. Binding of iron requires the synergistic binding of an anion, which under physiological conditions is either carbonate or bicarbonatet3. Since the affinity constant for the binding of iron to transferrin under physiological conditions is of the order of 102° 1 M- 1(Ref. © 1983, Elsevier Science Publishers B.V., Amsterdam 0167 4919/83/$01 IX~
348 14), it follows that cells must possess a special mechanism in order to acquire transferrin-bound iron. M a n y studies, initially with erythroid precursors but more recently with non-erythroid cells, have been carried out in an attempt to elucidate this mechanism. In the first step, transferrin interacts with a specific receptor on the cell membrane. Although initial studies with erythroid precursors yielded conflicting data on the nature of this receptoP 5, more recent studies using mainly transformed cell lines have resulted in the receptor being characterized as a glycoprotein of molecular weight about 190 000, consisting of two probably identical subunits 16-19. The receptor is phosphorylated2°and also carries palmitic acid residues 21'z2. The structure of the receptor has recently been reviewed by Newman et al. 23. The affinity of the receptor for transferrin varies according to whether or not the latter carries iron; for iron-saturated transferrin the affinity constant is of the order of 107-10~ 1 M- 1 (Refs 24-26). For iron-free (apo) transferrin, the affinity is about two orders of magnitude lower~5'27. It should be noted that some authors have published data not in accord with this last observation 28-3°, but in all these studies very small amounts of transferrin were used in the binding experiments and no account was taken of the possibility that apotransferrin could become saturated by traces of iron present in culture media and reagents. As will be discussed later, this is an important point which needs to be taken into account when assessing the individual functions of iron and transferrin in lymphocyte transformation. The transferrin receptors on human erythroid precursors and non-erythroid cells, and on the placenta (where they probably participate in iron transport to the fetus) possess similar structures and binding characteristics for transferrin and cross-react immunologically using conventional antisera 31'32.Certain monoclonal antibodies do however react preferentially with receptors from either erthyroid or non-erythroid cells33'34, although this may simply reflect differences in the accessibility of antigenic sites. It is unclear to what extent the transferrinreceptor interaction is species specific. Studies of iron uptake by reticulocytes have shown that within mammalian species transferrin will donate iron to cells of a heterologous species, albeit at a somewhat slower rate than the homologous protein 35'36, but chicken transferrin does not donate iron to rabbit reticulocytes37, or vice versa 3s. As discussed below, the question of species specificity may be important in determining the role of transferrin in lymphocyte transformation. The second step probably involves internalization of the transferrin-receptor complex by a process of receptormediated endocytosis39, although there is some evidence that in erythroid precursors iron removal from transferrin may occur at the membrane, without endocytosis4°'.1. Once internalized, subsequent steps are not entirely clear. It is possible that fusion of the endocytotic vacuole with a lysosome occurs. The resultant lowering of the p H in the phagolysosome would facilitate the third step of the iron-uptake process, namely release of iron from transferrin, since the complex is very susceptible to protonation. However, it is then necessary to explain how t,ransferrin escapes digestion, since it is well established that in the
Immunology Today, vol. 4, No. 12, 1983
final step transferrin is almost always released by the cell intact but without iron 29'~2a3. A clue may come from the observation that the transferrin-receptor complex is stabilized at p H 5 (Refs 17, 44). This would ensure that transferrin remained bound to its receptor in the phagolysosorne, which could protect it from proteolytic digestion. However, very recent data indicate that transferrin may instead pass through an acidic non-lysosomal compartment, thereby avoiding the possibility of degradation ~5. Recycling of the transferrin receptor is known to occur, and this would return the receptor-bound apotransferrin to the cell membrane, where it would be displaced by new iron-containing transferrin molecules. The iron is presumed to leave the endocytotic vesicle prior to reexpression of the receptor on the cell surface, though how this occurs has received scant attention. However, most if not all of the iron undoubtedly does remain in the cell, where it is thought to initially enter a labile iron poop 6, from which it may subsequently be used for synthesis of enzymes, cytochromes, etc., or incorporated into ferritin as storage iron. Alternative schemes for the interaction of transferrin with cells are shown in Fig. 1. The interaction of iron and t r a n s f e r r i n with lymphocytes In common with most other non-proliferating nonerythroid cells, resting lymphocytes possess few transferrin receptors; consequently an early event in transformation must be, if iron is needed, the synthesis and expression of these receptors. Larrick and Creswell ~ showed that when human peripheral blood lymphocytes respond to phytohaemagglutinin (PHA) or Con A their ability to bind transferrin markedly increases. Furthermore, both transferrin binding and iron uptake precede D N A synthesis43'40, and it is now d e a r that transforming lymphocytes acquire transferrin-bound iron by a mechanism similar to that described above, the transferrin being returned intact to the extracellular environment 43a9. The early requirement for iron may be due to the fact that ribonucleotide reductase, an enzyme involved in D N A synthesis, is iron dependent 5°. In the absence oftransferrin, lymphocytes transform poorly in response to mitogens in serum-free medium. This is true not only for T-cell mitogens such as Con A and P H A , but also for the response of mouse lymphocytes to lipopolysaccharide, a B-cell mitogen 3. Mouse T cells respond to Con A in serum-free medium equally well using mouse, human or bovine transferrins. However, other workers found that bovine transferrin failed to support transformation of human lymphocytes51, and, using immunofluorescence, that bovine transferrin does not bind to transferrin receptors of human lymphocytes52. These observations suggest that bovine transferrin cannot interact with the human transferrin receptor. While this may simply reflect a difference in the ability of mouse and h u m a n transferrin receptors to bind bovine transferrin, it is difficult, if transferrin and iron are indeed essential for lymphocyte transformation, to reconcile a failure of h u m a n lymphocytes to bind bovine transferrin with the undoubted growth-promoting properties of fetal calf serum. Failure to detect binding of bovine transferrin using immunofluorescence may simply reflect a relatively low binding affinity, as has
349
Immunology Today, voL 4, No. 12, 1983
b e e n s h o w n for the b i n d i n g of b o v i n e t r a n s f e r r i n to rat 53 a n d h a m s t e r 54cells. T h i s low affinity m a y b e c o m p e n s a t e d b y the relatively h i g h c o n c e n t r a t i o n of b o v i n e t r a n s f e r r i n present in cultures c o n t a i n i n g fetal calf serum: m o u s e l y m p h o c y t e s will r e s p o n d to C o n A in serum-free m e d i u m in the p r e s e n c e of as little as 0.5/~g m l - 1 of h u m a n transferrin 3, a n d optimally with 10-50/ag m l - 1. C u l t u r e s cont a i n i n g 10% fetal calf s e r u m will c o n t a i n a b o u t 500/~g m l - 1 of b o v i n e transferrin. To demonstrate that lymphocyte transformation requires the presence of b o t h iron a n d t r a n s f e r r i n it is necessary to show t h a t a p o t r a n s f e r r i n will not p r o m o t e t r a n s f o r m a t i o n . T h i s presents p r o b l e m s , since we h a v e f o u n d t h a t R P M I 1640 m e d i u m used for serum-free cultures, a l t h o u g h ostensibly iron-free, c o n t a i n s 6 - 1 0 ~g m l - l of i r o n 3. T h i s is sufficient to s a t u r a t e u p to a b o u t 7 gg m l 1 o f t r a n s f e r r i n , which, as m e n t i o n e d above, is m o r e t h a n e n o u g h to p r o m o t e l y m p h o c y t e t r a n s f o r m a t i o n . T o o v e r c o m e this p r o b l e m we h a v e investigated the effect of a d d i n g to the m e d i u m d e s f e r r i o x a m i n e - a m i c r o b i a l iron chelator w i t h a h i g h affinity for iron - p r i o r to a d d i n g a p o t r a n s f e r r i n , thus p r e v e n t i n g the latter f r o m b i n d i n g
the e n d o g e n o u s iron. A d d i t i o n of the chelator reduced t r a n s f o r m a t i o n in the p r e s e n c e of a p o t r a n s f e r r i n 3, thus s u p p o r t i n g the idea t h a t t r a n s f e r r i n is essential for l y m p h o c y t e t r a n s f o r m a t i o n ( a n d for the culture of cell lines) b e c a u s e it acts as a supplier of iron to the cells. A n u n u s u a l feature of the role played by t r a n s f e r r i n in the t r a n s f o r m a t i o n of m o u s e lymphocytes is the effect of iron saturation. It is k n o w n t h a t diferric t r a n s f e r r i n d o n a t e s iron to reticulocytes a b o u t seven times faster t h a n m o n o f e r r i c t r a n s f e r r i n ~5, so one would therefore expect fully s a t u r a t e d t r a n s f e r r i n to be the most effective form of the protein in p r o m o t i n g l y m p h o c y t e t r a n s f o r m a t i o n . H o w e v e r , optimal t r a n s f o r m a t i o n of m o u s e lymphocytes o c c u r r e d with t r a n s f e r r i n w h o s e iron s a t u r a t i o n was in the r a n g e of 3 0 - 7 0 % 3. At h i g h e r s a t u r a t i o n , t r a n s f o r m a t i o n was m a r k e d l y reduced. Several e x p l a n a t i o n s are possible. It h a s b e e n suggested t h a t t r a n s f e r r i n - b o u n d zinc is also essential for l y m p h o c y t e t r a n s f o r m a t i o n s6, a n d since t r a n s f e r r i n b i n d s iron with a h i g h e r affinity t h a n zinc, s a t u r a t i o n with the f o r m e r m e t a l would p r e v e n t b i n d i n g of the latter. T h e role of t r a n s f e r r i n as a zinc carrier has, however, b e e n q u e s t i o n e d 57. A n o t h e r possible explana!
(ii)
(i)
ATP ~
o ' ~ o,,~ _
~
Fo~*
(
•. . ~
%** ~o
Fig. 1. Proposed mechanisms for cellular uptake of transferrin-bound iron. (i) Scheme A. Mechanism involving endocytosis but without involvement oflysosomes, cf. Dautry-Varsat et al. 45 Step I : binding of monoferric or diferric transferrin to membrane receptors, causing aggregation. Step 2: endocytosis of transferrin-receptor complexes. Step 3: formation of endocytotic vacuole. Step 4: action of proton pump causes reduction in intravesicular pH and release of iron from transferrin, possibly helped by ATP which can act as an iron chelator, and stabilization of receptor-apostransferrin complex. Step 5: fusion of vacuole with the cell membrane and re-expression of receptors. Apotransferrin displaced by fresh iron-containing transferrin molecules.
Scheme B. Possible mechanism without endocytosis. Transferrin binds to the receptor as before, but iron is removed at the membrane by an unknown mechanism and taken up by a membrane ironbinding component cf. Glass et al. 4° from which iron passes to the cytoplasm. Apotransferrin is then displaced as above. (ii) Mechanisms involving lysosomes. Essentially as in (i) Scheme A, but some transfcrrin may also be taken up by fluid-phase endocytosis at step 2. At step 3, fusion with a lysosome occurs. Receptor-bound transferrin releases its iron due to lowered pH but is protected from degradation; unbound transferrin is degraded. At step 5, both degraded and undegraded transferrin are released. This mechanism may operate in fibroblasts47.
350 tion arises from the observation that lymphocytes, unlike many other types of cell, do not synthesize ferritin in response to increased iron levels, and hence do not increase their iron-storage capacity5s. At high transferrin saturation iron uptake may proceed too rapidly to allow adequate intracellular processing, with possible toxic effects and a consequent reduction in transformation. Although the need for transferrin for transformation of lymphocytes in response to mitogens seems to be well established, its role in mixed lymphocyte reactions ( M L R ) is less clear. Anderson et aL 5~ reported that M L R in serum-free medium required other factors in addition to transferrin, but Hsia et aL 59 reported that one-way M L R would proceed in media supplemented with only bovine serum albumin or, to a lesser extent, with gelatin. The presence of trace impurities, including perhaps transferrin, might be of importance in these studies. D o e s transferrin have a function in l y m p h o c y t e t r a n s f o r m a t i o n other t h a n to s u p p l y iron?
From the foregoing it would appear that transferrin is essential for lymphocyte transformation because of its activity as an iron donor. However, there is some evidence to suggest that this might be an oversimplification. Firstly, although low molecular weight iron chelates such as those formed with citrate or nitrilotriacetate cannot replace transferrin as a promotor of lymphocyte transformation, the cells can nevertheless take up iron supplied in this form - indeed, they do so much more rapidly than when iron is bound to transferrin 3'43. It is important to note that care was taken in these experiments to ensure that the chelators used were low molecular weight species, by using a 20-fold molar excess of citrate and a 4-fold excess of nitrilotriacetate in the complex solutions. Use of ferric chloride, or commercial ferric citrate in which the citrate to iron ratio is much lower, leads to hydrolysis and polymerization and non-specific binding of iron. The inhibition of E-rosette-formation by human lymphocytes exposed to iron in these forms 6°can probably be explained by non-specific binding of polymerized iron to the cell membrane. Low molecular weight chelates can, on the other hand, transport iron into the cell as shown by the fact that only 14-17 % of iron taken up by lymphocytes could be bound at 4°C by desferrioxamine, which would remove iron on the cell surface. If the cells can not only bind but also internalize iron supplied as low molecular weight chelates, it is not clear why such chelates cannot substitute for transferrin as carriers of the iron needed for in-vitro transformation. A second point of consideration is that lymphocytes possess storage iron (ferritin) which one might expect to be mobilized to meet increased needs during transformation if no exogenous iron is available, but seemingly this also cannot substitute for transferrinbound iron. Finally, compared to erythoid precursors, lymphocytes bind excessive amouiits of transferrin in relation to their iron uptake 61. Taken together, these observations suggest that the binding of the iron-transferrin complex to the membrane receptor might, in itself, be an essential step in the sequence of events leading to transformation, although it could also be argued thgt neither chelate- nor ferritin-derived iron enter the intracellular
Immunology Today, vol. 4, No. 12, 1983
Precursor IL-2 producer
Pre-effectorcell
I Antigen o r mitogen IL-1 Production of IL-2
Antigen or mitogen Expression of IL-2 receptors
Expression of transferrin receptors Monoferrie or diferrie transferrin Effector T-cell proliferation Fig. 2. A possible scheme for the involvement of transferrln in
T-cell transformation. (Transferrin may also be required at a comparable stage in B-celltransformation.) This scheme is supported by the work of Neckersand Cossman67. compartment necessary to permit the metabolic events leading to cell division. Nevertheless, several features of the transferrin-receptor interaction do resemble those of other systems, such as the interaction of antigen with membrane immunoglobulin in B cells which are known to act as triggers for events leading to transformation. The receptor, with its two subunits, is evidently bivalent, and indeed it is the major multi-subunit component of the membrane of transformed T-cell linesrL Transferrin also appears to act as a divalent ligand, since monoferric fragments obtained by proteolytic cleavage of the molecule to yield the isolated domains, each with its iron-binding site still intact, do not promote lymphocyte transformation 3. Binding of transferrin to the receptor on lymphocytes leads to patching or capping~s, a phenomenon also seen when antigen binds to surface immunoglobulin. The binding of the iron-transferrin complex (but not apotransferrin) to the membrane receptor might therefore constitute a signal which is necessary to allow cell division to proceed. The failure of apotransferrin to promote transformation could be due to its lower affinity for the receptor. However, one is here faced with the argument that these events may also be essential for iron uptake, and indeed the fact that monoferric transferrin fragments are very poor iron donors to reticulocytes64 tends to support this argument. It is thus difficult to distinguish experimentally between a signal arising from the binding of transferrin per se and one consequent upon the acquisition of transferrin-bound iron. We are currently investigating this problem using transferrin loaded with metals other than iron. Hamilton 6s has recently explored the role oftransferrin in the two separate events of blast transformation and cell division. Transferrin binding was clearly associated with the latter event; non-dividing blasts obtained from a 4-day culture of Con A-stimulated rat lymphocytes only expressed transferrin receptors after exposure to interleukin 2 (IL-2). It is thus possible that binding of IL-2 triggers expression of transferrin receptors, and that binding of transferrin to the receptor (and the resulting uptake of iron?) is then necessary before cell division occurs. The proposed sequence of events, which is modified from the scheme of Gillis 66, is shown in Fig. 2. Experimental evi-
351
Immunology Today, vol. 4, No. 12, 1983
dence for the role of IL-2 in the induction oftransferrin receptors has very recently been presented 67. In conclusion, there is little doubt that transferrin plays a crucial role in cell division, and in particular in lymphocyte transformation. The ubiquity of this protein, both in vivo and in media normally employed for in-vitro culture, has meant that its role has only become apparent with the recent development of serum-free culture media. Whether the role of transferrin in lymphocyte transformation is inextricably linked to its iron-donating properties remains an intriguing question for future investigation. References 1 Jacobs, A. (1977) in Recent Advances in Haematology 2 (Hoflbrand, A. V., Brain, M. C. and Hirsh, J., eds), p. 1, Churchill Livingstone, Edinburgh 2 Phillips, J. L. and Azari, P. (1975) Cell. Immunol. 15, 94 3 Brock, J. H. (1981) Immunology 43, 387 4 Barnes, D. and ~&ato, G. (1980) Anal. Biochem. 102, 255 5 Joynson, D. H. M.,Jacobs, A., Walker, D. M. and Dolby, A. E. (1972) Lancet ii, 1058 6 Chandra, R. K. (1975)J. Pediatr. 86, 899 7 Srikantia, S. G., Prasad, J. S., Bhaskaram, C. and Krishnamachari, K. A. V. R. (1976) Lancet i, 1307 8 Kuvibidila, S. G., Baliga, B. S. and Suskind, R. M. (1981)Am.J. Clin. Nutr. 34, 2635 9 Cummins, A. G., Duncombe, V. M., Bolin, T. D. et al. (1978) Gut 19, 823 10 Hershko, C., Graham, G., Bates, G. W. and Rachmilewitz, E. A. (1978) Br. , f Haematol. 40, 255 11 Sarkar, B. (1970) Can. J. Biochem. 48, 1339 12 Hahn, D. and Ganzoni, A. M. (1980) Biochim. Biophys. Acta 627, 250 13 Aisen, P. (1980) in Iron in Biochernist~y and Medicine H (Jacobs, A. and Worwood, M., eds), p. 87, Academic Press, London 14 Aisen, P., Leibman, A. and Zweier, J. (1978)J. BioL Chem. 253, 1930 15 Bezkorovainy, A. (1980) in The Biochemistry of Non-heme Iron, p. 127, Plenum Press, New York 16 Leibman, A. and Aisen, P. (1977) Biochemistry 16, 1268 17 Seligman, P. A., Schleicher, R. B. and Allen, R. H. (1979)J. Biol. Chem. 254, 9943 18 Coding, J. w . and Burns, G. F. (1981)J. lmmunol. 127, 1256 19 Sutherland, R., Delia, D., Schneider, C. et al. (1981)Proc. NatlAcad. Sci. USA 78, 4515 20 Schneider, C., Sutherland, R., Newman, R. and Greaves, M. (1982) J. Biol. Chem. 257, 8516 21 Omary, M. B. and Trowbridge, I. S. (1981),f Biol. Chem. 256, 4715 22 Omary, M. B. and Trowbridge, I. S. (1981)J. BioL Chem. 256, 12888 23 Newman, R., Schneider, C., Sutherland, R. et al. (1982) Trends Biochem. Sci. 7,397 24 Schulman, H. M., Wilczynska, A. and Ponka, P. (1981) Biochem. Biophys. Res. Commun. 100, 1523 25 Young, S. P. and Aisen, P. (1981)Hepatology 1, 114 26 Galbraith, R. M., Werner, P., Arnaud, P. and Galbraith, G. M. P. (1980)J. Clin. Invest. 66, 1135 27 Kornfeld, S. (1969) Biochim. Biophys. Acta 194, 25
28 Hamilton, T. A., Wada, H. G. and Sussman, H. H. (1979) Proc. Nat/ Aead. Sei. USA 76, 6406 29 Karin, M. and Mintz, B. (1981)J. BioL Chem. 256, 3245 30 Ward, J. H., Kushner, J. P. and Kaplan, J. (1982)J. Biol. Chem. 257, 10317 3! Enns, C. A. and Sussman, H. H. (1981)J. Biol. Chem. 256, 12620 32 Enns, C. A.., Shindelman, J. E., Tonik, S. E. and Sussman, H. H. (1981) Proc. NatlAcad. Sci. USA 78, 4222 33 Trowbridge, I. S., Lesley, J. and Schuhe, R. (1982)J. Cell. Physiol. 112, 403 34 Lebman, D., Trncco, M., Botero, L. et aL (1982) Blood 59, 671 35 Esparza, I. and Brock, J. H. (1980) Biochim. Biophys. Acta 622, 297 36 Van Bockxmeer, F. M. and Morgan, E. H. (1982) Comp. Biochem. Physiol 71A, 211 37 Zapolski, E. J. and Princiotto, J. V. (1976) Biochim. Biophys. Acta 421, 80 38 Keung, W.-M. and Azari, P. (1982)J. Biol. Chem. 257, 1184 39 Morgan, E. H. (1981)MoL Aspects Med. 4, 3 40 Glass, J., Nhfiez, M. T. and Robinson, S. H. (1980) Biochirn. Biophys. Acta 598, 293 41 Woodworth, R. C., Brown-Mason, A., Christensen, T. G. et al. (1982) Biochemistry 21, 4220 42 Jandl, J. H. and Katz, J. H. (1963)J. Clin. Invest. 42, 314 43 Brock, J. H. and Rankin, M. C. (1981) Immunology 43, 393 44 Wada, H. G., Hass, P. E. and Sussman, H. H. (1979)J. Biol Chem. 254, 12629 45 Dautry-Varsat, A., Ciechanover, A. and Lodish, H. F. (1983) Proc~ Natl Acad. Sci. USA 80, 2258 46 Jacobs, A. (1977) in Iron Metabolism (Ciba Foundation Symposium), p. 91, Elsevier, Amsterdam 47 Octave, J.-N., Schneider, Y.-J., Crichton, R. R. and Trouet, A (1981) Eur. J. Biochem. 115, 611 48 Larrick, J. W. and Cresswell, P. (1979)J. Supramol. Struct. 11, 579 49 Hamilton, T. A. (1983)J. Cell. Physiol. 114, 222 50 Hoflbrand, A. V., Ganeshaguru, K., Hooton, J. W. L. and Tattersall, M. H. N. (1976) Br. ~f Haematol. 33, 517 51 Anderson, W. L., Chase, C. G. and Tomasi, T. B. (1982) In Vitro 18, 766 52 Galbraith, G. M. P., Goust, J. M., Mercurio, S. M. and Galbraith, R. M. (1980) Clin. ImrnunoL Irnmunopathol. 16, 387 53 Verhoef, N. J., Kester, H. C. M., Noordeloos, P. J. and Leijsne, B. (1979) Int. J. Biochem. I0, 595 54 Messmer, T. O. (I973) Exp. Cell Res. 77, 404 55 Huebers, H. A., Csiba, E., Huebers, E. and Finch, C. A. (1983) Proc. Natl Acad. Sci. USA 80, 300 56 Phillips, J. L. (1978) Cell. lmrnunol. 35, 318 57 Chesters, J. K. and Will, M. (1981) Br. J. Nutr. 46, 111 58 Lema, M.J. and Sarcione, E. J. (1981) Comp. Biochem. Physiol. 69B, 287 59 Hsia, S., Wilkinson, R. S. and Amos, D. B. (1979)J. Immunol. Methods 27, 383 60 Nishiya, K., De Sousa, M., Tsoi, E. et al. (1980) Cell. Immunol. 53, 71 61 Bomford, A., Young, S. P., Noun-Aria, K. and Williams, R. (1983) Br. J. Haematol. 55, 93 62 Goding, J. W. and Harris, A. W. (1981)Proc. NatlAcad. Sci. USA 78, 4530 63 Galbraith, G. M. P. and Galbraith, R. M (1980) Clin. Exp. Imrnunol. 42, 285 64 Esparza, I. and Brock, J. H. (1980)Biochim. Biophys. Acta 624, 479 65 Hamilton, T. A. (1982)J. Cell. Physiol. 113, 40 66 Gillis, S. (1983)J. Clin. lmmunoL 3, 1 67 Neckers, L. M. and Cossman,J. (1983)Proc. NattAcad. Sci. USA 80, 3494
T h e v a l u e o f t h e l y m p h o c y t e as a p r o v i n g g r o u n d f o r t h e p r i n c i p l e s a n d t e c h niques of molecular biology has shifted the focus of modern immunology from the functional properties of mixed cell populations to the genes and surface molecules o f i n d i v i d u a l cells. M a n y i m m u n o l o g i s t s , t h e r e f o r e , a r e s e e k i n g b a s i c i n s t r u c t i o n i n m o l e c u l a r b i o l o g y a n d w i t h t h i s i n m i n d G i l m o u r H a r r i s h e r e r e v i e w s t w o recent books on the subject.
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a n d m e d i c i n e n o w t e n d to b e m u l t i a u t h o r e d - a n i n d i c a t i o n of the g r e a t exp a n s i o n of i n f o r m a t i o n in these fields w i t h the s u b s e q u e n t n e e d for specialization. I n c o n t r a s t this b o o k is b y a s i n g l e a u t h o r w h o h a s m a d e a b r a v e a t t e m p t to cover, in a s y s t e m a t i c m a n n e r , the a r e a of m o l e c u l a r biology.
T h e a u t h o r ' s i n t e n t was to p r o d u c e a m u l t i - p u r p o s e college t e x t b o o k a i m e d p a r t i c u l a r l y , o n e p r e s u m e s , at u n d e r g r a d u a t e s in A m e r i c a n u n i v e r s i t i e s . A w i d e field has b e e n c o v e r e d , r a n g i n g f r o m a n o u t l i n e of s o m e b a s i c bioc h e m i c a l m e t a b o l i c p a t h s a n d the desc r i p t i o n o f c e l l u l a r m a c r o m o l e c u l e s to the c o m p l e x i t i e s o f n u c l e i c acid s t r u c t u r e a n d f u n c t i o n s . I n the c o n t e n t s t h e r e are t h e r e f o r e topics c o m m o n l y a c c e p t e d as b e i n g in the c o n t e x t of m o l e c u l a r b i o l o g y p r o p e r , as well as c h a p t e r s d e s c r i b i n g the p h y s i o l o g y o f p r o k a r y o t i c cells a n d viruses. T h e r e s u l t of this w i d e c o v e r a g e is therefore a textbook which can only de~ s o m e w h a t s u p e r f i c i a l l y w i t h all the topics
Immunology Today, vol. 4, No. 12, 1983
The role of iron and transferrin in lymphocyte transformation Jeremy H. Brock and Tryfonia Mainou-Fowler It has recently become apparent that many types of transformed cells possess membrane receptorsfor the plasma ironbinding protein transferrin. In this review Jeremy Brock and T~yfonia Mainou-Fowler discuss these observations in relation to transferrin's properties as an iron-transport protein, and the relative importance of iron and transferrin in lymphocyte transformation. Iron is essential to almost all known forms of life. In man about 70% of total body iron is present as haemoglobin, most of the remainder being stored as ferritin in the cells of the liver, spleen and bone marrow. However, small but significant quantities of iron are also required by other cells and tissues, including lymphocytes, for the synthesis of metabolically important compounds such as metalloenzymes and cytochromes. The expansion of clones of lymphocytes, an essential feature of immune responses, involves increased cellular metabolic activity and cell division, and hence de-novo synthesis of some ironcontaining compounds and an increased iron requirement. It is therefore possible that an inadequate delivery of iron might prevent lymphocyte transformation from proceeding optimally. Two lines of evidence support this contention, albeit indirectly. Firstly, several studies in h u m a n subjects and, more recently, in experimental animals have reported that iron deficiency can result in impaired cell-mediated immune responses ~. A second line of evidence suggesting a crucial role for both iron and transferrin in lymphocyte transformation comes from studies demonstrating that both are essential for transformation of lymphocytes in response to mitogens in serum-free medium2'3. The same is true for the successful propagation of many cell lines in serum-free media~. These findings, and their implications, will be considered in more detail below.
Iron deficiency and T-cell transformation A number of clinical studies have demonstrated that iron deficiency may cause reduced skin reactions to antigens such as Candida and PPD, and decrease the in-vitro response of peripheral blood lymphocytes to mitogenss-7. More recently, studies using mic& or rats 9have indicated that the priming oflymphocytes by antigen is not affected by iron deficiency, thus suggesting that it is either transformation or effector mechanisms which are impaired. We have recently investigated the role of iron on the' transformation step by studying the ability of lymph-node cells from normal or iron-deficient mice to transform in response to concanavalin A (Con A). In these experiments serum-free media were used, thus allowing the iron levels to be carefully controlled. It was found that whereas the iron concentration of the medium, which contained transferrin, had a considerable effect on lymphocyte transformation, there was no significant difference in the transformation of lymphocytes from University Department of Bacteriology and Immunology, Western Infirmary, Glasgow G11 6NT, UK.
normal or iron-deficient mice (Table I). Furthermore, transformation of lymphocytes from either group was lower in media supplemented with serum from irondeficient mice than when serum from normal mice was used, but addition of sufficient iron to bring the serum iron level of the deficient serum to that of the normal serum significantly improved its ability to promote transformation. These studies indicate that the lymphocytes of iron-deficient mice have a normal capacity to transform in response to mitogens but that the supply of transferrin-bound iron in the sera of deficient animals is, under these conditions at least, a limiting factor in allowing transformation and cell division to proceed at normal rates. These results emphasize the important role played by iron in lymphocyte transformation, and the way in which iron and transferrin interact with lymphocytes is discussed in the following sections. TABLE I. Response of lymphocytesfrom norma4and iron-deficient mice to concanavalinA. Culture medium:RPMI 1640 supplementedwith
Incorporationof 14C-thymidine (c.p.m.) into lymphocytes from Iron-replete mice
Apotransferrin, 50 #g ml i Transferrin, 20% iron-saturated, 50 #g ml- ~ 2.5% normal autologousserum 2.5 % iron-deficientautologous serum 2.5% iron-deficientautologous serum supplementedwith iron
Iron-deficient mice
1161 + 550
836 +- 164
3564 + 686 5569 ± 377
3590 -+318 5067 ± 777
3655 __.601
3751 -+601
4619± 621
4969 ± 846
Transferrin and iron uptake It is now well established that transferrin is the only physiological carrier of iron in normal serum. While nontransferrin-bound serum iron may be found in patients with severe iron overload whose serum transferrin is saturated 1°, earlier evidence that such a fraction also existed in normal serum" has now been refuted n. Transferrin is a glycoprotein of molecular weight about 80 000. It consists of a single polypeptide chain which is folded to give two largely independent and similar, but not identical, globular domains, each of which can bind one ferric ion. Binding of iron requires the synergistic binding of an anion, which under physiological conditions is either carbonate or bicarbonatet3. Since the affinity constant for the binding of iron to transferrin under physiological conditions is of the order of 102° 1 M- 1(Ref. © 1983, Elsevier Science Publishers B.V., Amsterdam 0167 4919/83/$01 IX~
348 14), it follows that cells must possess a special mechanism in order to acquire transferrin-bound iron. M a n y studies, initially with erythroid precursors but more recently with non-erythroid cells, have been carried out in an attempt to elucidate this mechanism. In the first step, transferrin interacts with a specific receptor on the cell membrane. Although initial studies with erythroid precursors yielded conflicting data on the nature of this receptoP 5, more recent studies using mainly transformed cell lines have resulted in the receptor being characterized as a glycoprotein of molecular weight about 190 000, consisting of two probably identical subunits 16-19. The receptor is phosphorylated2°and also carries palmitic acid residues 21'z2. The structure of the receptor has recently been reviewed by Newman et al. 23. The affinity of the receptor for transferrin varies according to whether or not the latter carries iron; for iron-saturated transferrin the affinity constant is of the order of 107-10~ 1 M- 1 (Refs 24-26). For iron-free (apo) transferrin, the affinity is about two orders of magnitude lower~5'27. It should be noted that some authors have published data not in accord with this last observation 28-3°, but in all these studies very small amounts of transferrin were used in the binding experiments and no account was taken of the possibility that apotransferrin could become saturated by traces of iron present in culture media and reagents. As will be discussed later, this is an important point which needs to be taken into account when assessing the individual functions of iron and transferrin in lymphocyte transformation. The transferrin receptors on human erythroid precursors and non-erythroid cells, and on the placenta (where they probably participate in iron transport to the fetus) possess similar structures and binding characteristics for transferrin and cross-react immunologically using conventional antisera 31'32.Certain monoclonal antibodies do however react preferentially with receptors from either erthyroid or non-erythroid cells33'34, although this may simply reflect differences in the accessibility of antigenic sites. It is unclear to what extent the transferrinreceptor interaction is species specific. Studies of iron uptake by reticulocytes have shown that within mammalian species transferrin will donate iron to cells of a heterologous species, albeit at a somewhat slower rate than the homologous protein 35'36, but chicken transferrin does not donate iron to rabbit reticulocytes37, or vice versa 3s. As discussed below, the question of species specificity may be important in determining the role of transferrin in lymphocyte transformation. The second step probably involves internalization of the transferrin-receptor complex by a process of receptormediated endocytosis39, although there is some evidence that in erythroid precursors iron removal from transferrin may occur at the membrane, without endocytosis4°'.1. Once internalized, subsequent steps are not entirely clear. It is possible that fusion of the endocytotic vacuole with a lysosome occurs. The resultant lowering of the p H in the phagolysosome would facilitate the third step of the iron-uptake process, namely release of iron from transferrin, since the complex is very susceptible to protonation. However, it is then necessary to explain how t,ransferrin escapes digestion, since it is well established that in the
Immunology Today, vol. 4, No. 12, 1983
final step transferrin is almost always released by the cell intact but without iron 29'~2a3. A clue may come from the observation that the transferrin-receptor complex is stabilized at p H 5 (Refs 17, 44). This would ensure that transferrin remained bound to its receptor in the phagolysosorne, which could protect it from proteolytic digestion. However, very recent data indicate that transferrin may instead pass through an acidic non-lysosomal compartment, thereby avoiding the possibility of degradation ~5. Recycling of the transferrin receptor is known to occur, and this would return the receptor-bound apotransferrin to the cell membrane, where it would be displaced by new iron-containing transferrin molecules. The iron is presumed to leave the endocytotic vesicle prior to reexpression of the receptor on the cell surface, though how this occurs has received scant attention. However, most if not all of the iron undoubtedly does remain in the cell, where it is thought to initially enter a labile iron poop 6, from which it may subsequently be used for synthesis of enzymes, cytochromes, etc., or incorporated into ferritin as storage iron. Alternative schemes for the interaction of transferrin with cells are shown in Fig. 1. The interaction of iron and t r a n s f e r r i n with lymphocytes In common with most other non-proliferating nonerythroid cells, resting lymphocytes possess few transferrin receptors; consequently an early event in transformation must be, if iron is needed, the synthesis and expression of these receptors. Larrick and Creswell ~ showed that when human peripheral blood lymphocytes respond to phytohaemagglutinin (PHA) or Con A their ability to bind transferrin markedly increases. Furthermore, both transferrin binding and iron uptake precede D N A synthesis43'40, and it is now d e a r that transforming lymphocytes acquire transferrin-bound iron by a mechanism similar to that described above, the transferrin being returned intact to the extracellular environment 43a9. The early requirement for iron may be due to the fact that ribonucleotide reductase, an enzyme involved in D N A synthesis, is iron dependent 5°. In the absence oftransferrin, lymphocytes transform poorly in response to mitogens in serum-free medium. This is true not only for T-cell mitogens such as Con A and P H A , but also for the response of mouse lymphocytes to lipopolysaccharide, a B-cell mitogen 3. Mouse T cells respond to Con A in serum-free medium equally well using mouse, human or bovine transferrins. However, other workers found that bovine transferrin failed to support transformation of human lymphocytes51, and, using immunofluorescence, that bovine transferrin does not bind to transferrin receptors of human lymphocytes52. These observations suggest that bovine transferrin cannot interact with the human transferrin receptor. While this may simply reflect a difference in the ability of mouse and h u m a n transferrin receptors to bind bovine transferrin, it is difficult, if transferrin and iron are indeed essential for lymphocyte transformation, to reconcile a failure of h u m a n lymphocytes to bind bovine transferrin with the undoubted growth-promoting properties of fetal calf serum. Failure to detect binding of bovine transferrin using immunofluorescence may simply reflect a relatively low binding affinity, as has
349
Immunology Today, voL 4, No. 12, 1983
b e e n s h o w n for the b i n d i n g of b o v i n e t r a n s f e r r i n to rat 53 a n d h a m s t e r 54cells. T h i s low affinity m a y b e c o m p e n s a t e d b y the relatively h i g h c o n c e n t r a t i o n of b o v i n e t r a n s f e r r i n present in cultures c o n t a i n i n g fetal calf serum: m o u s e l y m p h o c y t e s will r e s p o n d to C o n A in serum-free m e d i u m in the p r e s e n c e of as little as 0.5/~g m l - 1 of h u m a n transferrin 3, a n d optimally with 10-50/ag m l - 1. C u l t u r e s cont a i n i n g 10% fetal calf s e r u m will c o n t a i n a b o u t 500/~g m l - 1 of b o v i n e transferrin. To demonstrate that lymphocyte transformation requires the presence of b o t h iron a n d t r a n s f e r r i n it is necessary to show t h a t a p o t r a n s f e r r i n will not p r o m o t e t r a n s f o r m a t i o n . T h i s presents p r o b l e m s , since we h a v e f o u n d t h a t R P M I 1640 m e d i u m used for serum-free cultures, a l t h o u g h ostensibly iron-free, c o n t a i n s 6 - 1 0 ~g m l - l of i r o n 3. T h i s is sufficient to s a t u r a t e u p to a b o u t 7 gg m l 1 o f t r a n s f e r r i n , which, as m e n t i o n e d above, is m o r e t h a n e n o u g h to p r o m o t e l y m p h o c y t e t r a n s f o r m a t i o n . T o o v e r c o m e this p r o b l e m we h a v e investigated the effect of a d d i n g to the m e d i u m d e s f e r r i o x a m i n e - a m i c r o b i a l iron chelator w i t h a h i g h affinity for iron - p r i o r to a d d i n g a p o t r a n s f e r r i n , thus p r e v e n t i n g the latter f r o m b i n d i n g
the e n d o g e n o u s iron. A d d i t i o n of the chelator reduced t r a n s f o r m a t i o n in the p r e s e n c e of a p o t r a n s f e r r i n 3, thus s u p p o r t i n g the idea t h a t t r a n s f e r r i n is essential for l y m p h o c y t e t r a n s f o r m a t i o n ( a n d for the culture of cell lines) b e c a u s e it acts as a supplier of iron to the cells. A n u n u s u a l feature of the role played by t r a n s f e r r i n in the t r a n s f o r m a t i o n of m o u s e lymphocytes is the effect of iron saturation. It is k n o w n t h a t diferric t r a n s f e r r i n d o n a t e s iron to reticulocytes a b o u t seven times faster t h a n m o n o f e r r i c t r a n s f e r r i n ~5, so one would therefore expect fully s a t u r a t e d t r a n s f e r r i n to be the most effective form of the protein in p r o m o t i n g l y m p h o c y t e t r a n s f o r m a t i o n . H o w e v e r , optimal t r a n s f o r m a t i o n of m o u s e lymphocytes o c c u r r e d with t r a n s f e r r i n w h o s e iron s a t u r a t i o n was in the r a n g e of 3 0 - 7 0 % 3. At h i g h e r s a t u r a t i o n , t r a n s f o r m a t i o n was m a r k e d l y reduced. Several e x p l a n a t i o n s are possible. It h a s b e e n suggested t h a t t r a n s f e r r i n - b o u n d zinc is also essential for l y m p h o c y t e t r a n s f o r m a t i o n s6, a n d since t r a n s f e r r i n b i n d s iron with a h i g h e r affinity t h a n zinc, s a t u r a t i o n with the f o r m e r m e t a l would p r e v e n t b i n d i n g of the latter. T h e role of t r a n s f e r r i n as a zinc carrier has, however, b e e n q u e s t i o n e d 57. A n o t h e r possible explana!
(ii)
(i)
ATP ~
o ' ~ o,,~ _
~
Fo~*
(
•. . ~
%** ~o
Fig. 1. Proposed mechanisms for cellular uptake of transferrin-bound iron. (i) Scheme A. Mechanism involving endocytosis but without involvement oflysosomes, cf. Dautry-Varsat et al. 45 Step I : binding of monoferric or diferric transferrin to membrane receptors, causing aggregation. Step 2: endocytosis of transferrin-receptor complexes. Step 3: formation of endocytotic vacuole. Step 4: action of proton pump causes reduction in intravesicular pH and release of iron from transferrin, possibly helped by ATP which can act as an iron chelator, and stabilization of receptor-apostransferrin complex. Step 5: fusion of vacuole with the cell membrane and re-expression of receptors. Apotransferrin displaced by fresh iron-containing transferrin molecules.
Scheme B. Possible mechanism without endocytosis. Transferrin binds to the receptor as before, but iron is removed at the membrane by an unknown mechanism and taken up by a membrane ironbinding component cf. Glass et al. 4° from which iron passes to the cytoplasm. Apotransferrin is then displaced as above. (ii) Mechanisms involving lysosomes. Essentially as in (i) Scheme A, but some transfcrrin may also be taken up by fluid-phase endocytosis at step 2. At step 3, fusion with a lysosome occurs. Receptor-bound transferrin releases its iron due to lowered pH but is protected from degradation; unbound transferrin is degraded. At step 5, both degraded and undegraded transferrin are released. This mechanism may operate in fibroblasts47.
350 tion arises from the observation that lymphocytes, unlike many other types of cell, do not synthesize ferritin in response to increased iron levels, and hence do not increase their iron-storage capacity5s. At high transferrin saturation iron uptake may proceed too rapidly to allow adequate intracellular processing, with possible toxic effects and a consequent reduction in transformation. Although the need for transferrin for transformation of lymphocytes in response to mitogens seems to be well established, its role in mixed lymphocyte reactions ( M L R ) is less clear. Anderson et aL 5~ reported that M L R in serum-free medium required other factors in addition to transferrin, but Hsia et aL 59 reported that one-way M L R would proceed in media supplemented with only bovine serum albumin or, to a lesser extent, with gelatin. The presence of trace impurities, including perhaps transferrin, might be of importance in these studies. D o e s transferrin have a function in l y m p h o c y t e t r a n s f o r m a t i o n other t h a n to s u p p l y iron?
From the foregoing it would appear that transferrin is essential for lymphocyte transformation because of its activity as an iron donor. However, there is some evidence to suggest that this might be an oversimplification. Firstly, although low molecular weight iron chelates such as those formed with citrate or nitrilotriacetate cannot replace transferrin as a promotor of lymphocyte transformation, the cells can nevertheless take up iron supplied in this form - indeed, they do so much more rapidly than when iron is bound to transferrin 3'43. It is important to note that care was taken in these experiments to ensure that the chelators used were low molecular weight species, by using a 20-fold molar excess of citrate and a 4-fold excess of nitrilotriacetate in the complex solutions. Use of ferric chloride, or commercial ferric citrate in which the citrate to iron ratio is much lower, leads to hydrolysis and polymerization and non-specific binding of iron. The inhibition of E-rosette-formation by human lymphocytes exposed to iron in these forms 6°can probably be explained by non-specific binding of polymerized iron to the cell membrane. Low molecular weight chelates can, on the other hand, transport iron into the cell as shown by the fact that only 14-17 % of iron taken up by lymphocytes could be bound at 4°C by desferrioxamine, which would remove iron on the cell surface. If the cells can not only bind but also internalize iron supplied as low molecular weight chelates, it is not clear why such chelates cannot substitute for transferrin as carriers of the iron needed for in-vitro transformation. A second point of consideration is that lymphocytes possess storage iron (ferritin) which one might expect to be mobilized to meet increased needs during transformation if no exogenous iron is available, but seemingly this also cannot substitute for transferrinbound iron. Finally, compared to erythoid precursors, lymphocytes bind excessive amouiits of transferrin in relation to their iron uptake 61. Taken together, these observations suggest that the binding of the iron-transferrin complex to the membrane receptor might, in itself, be an essential step in the sequence of events leading to transformation, although it could also be argued thgt neither chelate- nor ferritin-derived iron enter the intracellular
Immunology Today, vol. 4, No. 12, 1983
Precursor IL-2 producer
Pre-effectorcell
I Antigen o r mitogen IL-1 Production of IL-2
Antigen or mitogen Expression of IL-2 receptors
Expression of transferrin receptors Monoferrie or diferrie transferrin Effector T-cell proliferation Fig. 2. A possible scheme for the involvement of transferrln in
T-cell transformation. (Transferrin may also be required at a comparable stage in B-celltransformation.) This scheme is supported by the work of Neckersand Cossman67. compartment necessary to permit the metabolic events leading to cell division. Nevertheless, several features of the transferrin-receptor interaction do resemble those of other systems, such as the interaction of antigen with membrane immunoglobulin in B cells which are known to act as triggers for events leading to transformation. The receptor, with its two subunits, is evidently bivalent, and indeed it is the major multi-subunit component of the membrane of transformed T-cell linesrL Transferrin also appears to act as a divalent ligand, since monoferric fragments obtained by proteolytic cleavage of the molecule to yield the isolated domains, each with its iron-binding site still intact, do not promote lymphocyte transformation 3. Binding of transferrin to the receptor on lymphocytes leads to patching or capping~s, a phenomenon also seen when antigen binds to surface immunoglobulin. The binding of the iron-transferrin complex (but not apotransferrin) to the membrane receptor might therefore constitute a signal which is necessary to allow cell division to proceed. The failure of apotransferrin to promote transformation could be due to its lower affinity for the receptor. However, one is here faced with the argument that these events may also be essential for iron uptake, and indeed the fact that monoferric transferrin fragments are very poor iron donors to reticulocytes64 tends to support this argument. It is thus difficult to distinguish experimentally between a signal arising from the binding of transferrin per se and one consequent upon the acquisition of transferrin-bound iron. We are currently investigating this problem using transferrin loaded with metals other than iron. Hamilton 6s has recently explored the role oftransferrin in the two separate events of blast transformation and cell division. Transferrin binding was clearly associated with the latter event; non-dividing blasts obtained from a 4-day culture of Con A-stimulated rat lymphocytes only expressed transferrin receptors after exposure to interleukin 2 (IL-2). It is thus possible that binding of IL-2 triggers expression of transferrin receptors, and that binding of transferrin to the receptor (and the resulting uptake of iron?) is then necessary before cell division occurs. The proposed sequence of events, which is modified from the scheme of Gillis 66, is shown in Fig. 2. Experimental evi-
351
Immunology Today, vol. 4, No. 12, 1983
dence for the role of IL-2 in the induction oftransferrin receptors has very recently been presented 67. In conclusion, there is little doubt that transferrin plays a crucial role in cell division, and in particular in lymphocyte transformation. The ubiquity of this protein, both in vivo and in media normally employed for in-vitro culture, has meant that its role has only become apparent with the recent development of serum-free culture media. Whether the role of transferrin in lymphocyte transformation is inextricably linked to its iron-donating properties remains an intriguing question for future investigation. References 1 Jacobs, A. (1977) in Recent Advances in Haematology 2 (Hoflbrand, A. V., Brain, M. C. and Hirsh, J., eds), p. 1, Churchill Livingstone, Edinburgh 2 Phillips, J. L. and Azari, P. (1975) Cell. Immunol. 15, 94 3 Brock, J. H. (1981) Immunology 43, 387 4 Barnes, D. and ~&ato, G. (1980) Anal. Biochem. 102, 255 5 Joynson, D. H. M.,Jacobs, A., Walker, D. M. and Dolby, A. E. (1972) Lancet ii, 1058 6 Chandra, R. K. (1975)J. Pediatr. 86, 899 7 Srikantia, S. G., Prasad, J. S., Bhaskaram, C. and Krishnamachari, K. A. V. R. (1976) Lancet i, 1307 8 Kuvibidila, S. G., Baliga, B. S. and Suskind, R. M. (1981)Am.J. Clin. Nutr. 34, 2635 9 Cummins, A. G., Duncombe, V. M., Bolin, T. D. et al. (1978) Gut 19, 823 10 Hershko, C., Graham, G., Bates, G. W. and Rachmilewitz, E. A. (1978) Br. , f Haematol. 40, 255 11 Sarkar, B. (1970) Can. J. Biochem. 48, 1339 12 Hahn, D. and Ganzoni, A. M. (1980) Biochim. Biophys. Acta 627, 250 13 Aisen, P. (1980) in Iron in Biochernist~y and Medicine H (Jacobs, A. and Worwood, M., eds), p. 87, Academic Press, London 14 Aisen, P., Leibman, A. and Zweier, J. (1978)J. BioL Chem. 253, 1930 15 Bezkorovainy, A. (1980) in The Biochemistry of Non-heme Iron, p. 127, Plenum Press, New York 16 Leibman, A. and Aisen, P. (1977) Biochemistry 16, 1268 17 Seligman, P. A., Schleicher, R. B. and Allen, R. H. (1979)J. Biol. Chem. 254, 9943 18 Coding, J. w . and Burns, G. F. (1981)J. lmmunol. 127, 1256 19 Sutherland, R., Delia, D., Schneider, C. et al. (1981)Proc. NatlAcad. Sci. USA 78, 4515 20 Schneider, C., Sutherland, R., Newman, R. and Greaves, M. (1982) J. Biol. Chem. 257, 8516 21 Omary, M. B. and Trowbridge, I. S. (1981),f Biol. Chem. 256, 4715 22 Omary, M. B. and Trowbridge, I. S. (1981)J. BioL Chem. 256, 12888 23 Newman, R., Schneider, C., Sutherland, R. et al. (1982) Trends Biochem. Sci. 7,397 24 Schulman, H. M., Wilczynska, A. and Ponka, P. (1981) Biochem. Biophys. Res. Commun. 100, 1523 25 Young, S. P. and Aisen, P. (1981)Hepatology 1, 114 26 Galbraith, R. M., Werner, P., Arnaud, P. and Galbraith, G. M. P. (1980)J. Clin. Invest. 66, 1135 27 Kornfeld, S. (1969) Biochim. Biophys. Acta 194, 25
28 Hamilton, T. A., Wada, H. G. and Sussman, H. H. (1979) Proc. Nat/ Aead. Sei. USA 76, 6406 29 Karin, M. and Mintz, B. (1981)J. BioL Chem. 256, 3245 30 Ward, J. H., Kushner, J. P. and Kaplan, J. (1982)J. Biol. Chem. 257, 10317 3! Enns, C. A. and Sussman, H. H. (1981)J. Biol. Chem. 256, 12620 32 Enns, C. A.., Shindelman, J. E., Tonik, S. E. and Sussman, H. H. (1981) Proc. NatlAcad. Sci. USA 78, 4222 33 Trowbridge, I. S., Lesley, J. and Schuhe, R. (1982)J. Cell. Physiol. 112, 403 34 Lebman, D., Trncco, M., Botero, L. et aL (1982) Blood 59, 671 35 Esparza, I. and Brock, J. H. (1980) Biochim. Biophys. Acta 622, 297 36 Van Bockxmeer, F. M. and Morgan, E. H. (1982) Comp. Biochem. Physiol 71A, 211 37 Zapolski, E. J. and Princiotto, J. V. (1976) Biochim. Biophys. Acta 421, 80 38 Keung, W.-M. and Azari, P. (1982)J. Biol. Chem. 257, 1184 39 Morgan, E. H. (1981)MoL Aspects Med. 4, 3 40 Glass, J., Nhfiez, M. T. and Robinson, S. H. (1980) Biochirn. Biophys. Acta 598, 293 41 Woodworth, R. C., Brown-Mason, A., Christensen, T. G. et al. (1982) Biochemistry 21, 4220 42 Jandl, J. H. and Katz, J. H. (1963)J. Clin. Invest. 42, 314 43 Brock, J. H. and Rankin, M. C. (1981) Immunology 43, 393 44 Wada, H. G., Hass, P. E. and Sussman, H. H. (1979)J. Biol Chem. 254, 12629 45 Dautry-Varsat, A., Ciechanover, A. and Lodish, H. F. (1983) Proc~ Natl Acad. Sci. USA 80, 2258 46 Jacobs, A. (1977) in Iron Metabolism (Ciba Foundation Symposium), p. 91, Elsevier, Amsterdam 47 Octave, J.-N., Schneider, Y.-J., Crichton, R. R. and Trouet, A (1981) Eur. J. Biochem. 115, 611 48 Larrick, J. W. and Cresswell, P. (1979)J. Supramol. Struct. 11, 579 49 Hamilton, T. A. (1983)J. Cell. Physiol. 114, 222 50 Hoflbrand, A. V., Ganeshaguru, K., Hooton, J. W. L. and Tattersall, M. H. N. (1976) Br. ~f Haematol. 33, 517 51 Anderson, W. L., Chase, C. G. and Tomasi, T. B. (1982) In Vitro 18, 766 52 Galbraith, G. M. P., Goust, J. M., Mercurio, S. M. and Galbraith, R. M. (1980) Clin. ImrnunoL Irnmunopathol. 16, 387 53 Verhoef, N. J., Kester, H. C. M., Noordeloos, P. J. and Leijsne, B. (1979) Int. J. Biochem. I0, 595 54 Messmer, T. O. (I973) Exp. Cell Res. 77, 404 55 Huebers, H. A., Csiba, E., Huebers, E. and Finch, C. A. (1983) Proc. Natl Acad. Sci. USA 80, 300 56 Phillips, J. L. (1978) Cell. lmrnunol. 35, 318 57 Chesters, J. K. and Will, M. (1981) Br. J. Nutr. 46, 111 58 Lema, M.J. and Sarcione, E. J. (1981) Comp. Biochem. Physiol. 69B, 287 59 Hsia, S., Wilkinson, R. S. and Amos, D. B. (1979)J. Immunol. Methods 27, 383 60 Nishiya, K., De Sousa, M., Tsoi, E. et al. (1980) Cell. Immunol. 53, 71 61 Bomford, A., Young, S. P., Noun-Aria, K. and Williams, R. (1983) Br. J. Haematol. 55, 93 62 Goding, J. W. and Harris, A. W. (1981)Proc. NatlAcad. Sci. USA 78, 4530 63 Galbraith, G. M. P. and Galbraith, R. M (1980) Clin. Exp. Imrnunol. 42, 285 64 Esparza, I. and Brock, J. H. (1980)Biochim. Biophys. Acta 624, 479 65 Hamilton, T. A. (1982)J. Cell. Physiol. 113, 40 66 Gillis, S. (1983)J. Clin. lmmunoL 3, 1 67 Neckers, L. M. and Cossman,J. (1983)Proc. NattAcad. Sci. USA 80, 3494
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