Expression of transferrin receptors during erythroid maturation

Copyright @ 1983 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/83/040361-06$02.00/0

ExDerimental Cell Research 144 (1983) 361-366

Expression

of Transferrin

Receptors

during

Erythroid

Maturation

MICHAEL A. HORTON Hemopoiesis Research Group, Department of Hematology, St Bartholomew’s Hospital Medical College, London ECIA 7BE, UK

SUMMARY A monoclonal antibody, F111/2Dl, produced after immunisation of C3HMe mice with the human erythroleukemia cell line, K562, is described. It detects cell surface determinants of similar distribution to those characterised by the OKT-9 monoclonal antibody, which has been shown to identify the transferrin receptor. The F111/2Dl antibody, as welI as OKT-9, has been used to investigate the distribution of transfertin receptors during erythroid maturation in normal bone marrow and peripheral blood, and on the K562 cell line during erythroid differentiation, induced in vitro.

Various cell membrane glycoproteins are expressed in a characteristic pattern as part of the sequence of changes that occur during the maturation of early, but morphologically recognisable, elements of the erythroid cell lineage to the mature red blood cell [l]. The distribution of receptors for the iron transport protein on erythroid stem cells has been investigated using antibodies specific for antigenic determinants on the transferrin receptor [2] and much is known about the biochemistry of receptor-mediated iron uptake by reticulocytes [3]. However, the distribution of transferrin receptors on the morphologically recognisable erythroid elements in bone marrow has not been studied in detail. The derivation of a hybridoma producing antibodies with similar characteristics as OKT-9, which is known to react with the non-iron binding portion of the transferrin receptor [4], has allowed us to investigate its distribution during erythroid maturation in normal bone marrow and peripheral blood and on the K562 erythroleukemia cell line, during induced erythroid differentiation.

MATERIALS AND METHODS Derivation of monoclonal antibody, Fllll2DI C3HEIe mice were immunised intraperitoneally with 10~10~ KS62 (clone 3) cells, and boosted intravenously, to yield a source of immune spleen cells. Three days after the last immunisation, spleen cells were taken and fused with NS-1 myeloma cells using standard techniques [S]. Supematants from cultures containing growing hybrids were screened by binding assays (FITC or ‘*%labelled anti-MIg(FAb)z) using K562, erythrocytes or bone marrow as targets. Of several clones producing antibodies with similar properties, the twice cloned fusion product, F111/2Dl was selected for study as it preferentially bound to K562, failed to react with erythrocytes and labelled a subpopulation of bone marrow cells. Its properties are described herein, as part of a study of the distribution of transfertin receptors on hemopoietic cells.

Other antisera Monoclonal antibody to the transfertin receptor, OKT-9 (Ortho Pharmaceutical Corp., USA) and a conventional antiserum to transferrin (goat anti-human transferrin; Technicon Corp., USA) were used. 24-831817

362 Horton

Exp Cell Res 144 (1983)

Cell fines The K562 (clone 3) cells were a gift from Dr L. C. Andersson, University of Helsinki, Finland. It was grown in suspension culture in RPM1 1640 medium supplemented with 10% fetal calf serum (FCS) and maintained in logarithmic phase growth by 1 in 10 dilution in fresh medium, thrice weekly.

Induction of differentiation Inductions were performed by exposure of 105/ml logarithmically growing K562 cells to 0.5 mM bovine hemin, 1 mM sodium butyrate, lo-’ M 4a-phorbol or lo-’ M 12-O-tetradecanoylphorbol-13acetate (TPA) (Sigma Chemical Co., UK) for 3 days, and compared with control cultures in which inducers were omitted 161.Hemin and butyrate have been shown to stimulate [7, 81 and TPA inhibit [9, 101erythroid differentiation in K562 cells.

Bone marrow preparation and assay for transferrin receptors Bone marrow samples were obtained from hematologically normal subjects and fractionated by centrifugation over ‘Histopaque’ (Sigma Chemical Co., UK). The distribution of transferrin receptors on bone marrow erythroid cells was analysed by indirect immunofluorescence 161or rosetting with protein A containing S. uureus Cowan I bacteria [l 11as previously described, and compared with the distribution of the erythroid specific membrane component, glycophorin A, defined by monoclonal antibodies [123.

Transferrin receptors on reticulocytes The binding of F111/2Dl, OKT-9 and antibodies to transferrin to peripheral blood cells during the reticulocyte response, induced on treatment of a patient with severe megaloblastic anemia, was analysed by indirect immunofluorescence.

RESULTS AND DISCUSSION Production and properties of FlI112Dl

monoclonal antibody

Fusions with spleen cells immune to KS62 surface molecules resulted in several hybrids producing antibodies with similar properties of binding to KS62 cells and a fraction of bone marrow cells, but not mature erythrocytes; antibodies with such reactivity would potentially be useful in characterising early events in erythropoiesis, as K562 probably represents an erythroid precursor cell, transTable 1. Expression of Fllll2Dl determinant on uninduced K.562cells and after 3 days in the presence of various inducing agents Inducing agent”, f

Ceil line

None

DMSO (1%)

K562 (Clone 3)

+++b

+++

Sodium butyrate (1 nw

Hemin/ BSA (0.05 mM)

o-20%

+++d

+c

4 a-Phorbol TPA (lo-’ M) (lo-’ M) +++

-e

0 See Materials and Methods for induction conditions. b Approx. 90% cells strongly positive, less than 10% giving weak fluorescence. Induction with DMSO, hemin/BSA and 4 a-phorbol gave staining equivalent to controls. c Reduced cell growth with only minimal increase in benzidine staining for hemoglobin. d Control fluorescence with F111/2Dl, but marked increase in hemoglobin synthesis. c No detectable binding of F111/2Dl antibody. f Identical results as with F111/2Dl obtained using OKT-9 antibody.

Transferrin

Exp Cell Res 144 (1983)

receptors on erythroid cells

formed at a relatively early differentiation stage [6]. The F111/2Dl clone was selected for further study. The distribution of the determinant detected by the F111/2Dl antibody was wide-spread and demonstrable on all cultured hemopoietic cell lines tested (data not shown); this included all sublines of the K562 erythroleukemia cell line, which was used as immunogen in the production of the hybridoma secreting F111/2Dl. However, we could not demonstrate binding to mature human red blood cells or peripheral blood leukocytes; in contrast, the antibody detected a subpopulation of bone marrow cells, recognisable as maturing erythroid precursors. The distribution of the antigen seen by Fl lU2Dl was thus identical to that bound by OKT-9 [4] and other monoclonal antibodies [13-151, which have been shown to react with the transfer-tin receptor [4, 151.Further evidence for considering that F111/2Dl detects determinants on the transferrin receptor comes from binding experiments. In these the binding of 1251-labelled F111/2Dl aflinitypurified antibodies to K562 was inhibited by either pre- or coincubation with unlabelled transfer&; preliminary studies had excluded the possibility that this monoclonal antibody reacted with transferrin itself. The epitope recognised differed from that seen by OKT-9 as the two antibodies did not cross-block each other; further F111/2Dl binding was not inhibited by the 4F2 antibody [14], detecting another proliferation-related determinant. Although definitive biochemical analyses of the F111/2Dl antigen have not been performed, our assumption that it recognises the transferrin receptor are borne out by the identical reactivity seen with both OKT-9 and antisera detecting receptor-bound transferr-in in the results reported herein. Reactivity of Fllll2DI with KS62 cells Binding of F111/2Dl ,antibody to uninduced and induced K562 cells was studied by indirect immunofluorescence (table 1). The majority of control K562 cells bound F111/2Dl. Exposure to hemin, which is known to induce hemoglobinisation in this cell line [7], did not alter transferrin receptor expression; DMSO, a differentiation inducer of murine erythroleukemia [17] cells, but not K562 [6, 81, was without effect, whereas sodium butyrate, which resulted in a reduction in cell Table 2. Distribution of Fllll2Dl bound transferrin

and glycophorin

and OKT-9 transferrin receptor determinants, A on bone marrow erythroid precursors

Cell type”

F111/2Dl

OKT9

Tlf

Glycophorin A

Proerythroblast Basophilic normoblast Polychromatic nonnoblast Orthochromatic normoblast Reticulocyte Mature erythrocyte

-l+b +++ +++ +++ (+I’ -

-/+ +++ +++ +++ (+I -

-I+ +++ +++ +++ (+)

-I+ +++ +++ +++ +++ +++

a No labelling of myeloid, monocytic or lymphoid cells was observed. b Some proerythroblasts are negative. c Only some reticulocytes bind F111/2Dl, OKT-9 and antibodies to transferrin. See fig. 2.

363

364 Horton

Exp Cell Res 144 (1983)

Fig. 1. Binding of antibody to transferrin receptor (F111/2DI), to normal bone marrow cells, as detected by rosetting with protein A containing staphylococci. a, Pronormoblast; b, normoblast; c, reticulocyte; d, mature erythrocyte; e, granulocytic cells;f, lymphocyte.

growth but not differentiation, reduced Flll/ZDl binding. TPA, but not its inactive phorbol ester analogue, 4-a-phorbol, blocked hemoglobin synthesis [lo], reduced cell growth and also inhibited F111/2Dl expression. Thus, the expression of transfer& receptors in K562, as detected by F111/2Dl (and OKT-9, data not shown), is not related to the degree of erythroid differentiation of KS62 cells, but more to its proliferative status. Expression of transferrin receptors on normal bone marrow erythroid cells Preliminary screening of bone marrow by immunofluorescence suggested that F111/2Dl bound to recognisable nucleated erythroblasts and some unidentifiable cells. To study the distribution of transferrin receptors on the different hemopoietic lineages, and especially during the morphologically recognisable stages of erythroid differentiation, the binding of F111/2Dl was visualised using a protein A S. aureus rosetting technique [ 111;this allows the cell type binding antibody to be identified following staining of cell preparations. F111/2Dl was found only to bind

Transferrin

Exp Cell Res 144 (1983) Vitamin

B, 2 200pgm

i0

50

40

I-

40

30

I-

30

2 g I? E z a E

20

receptors on erythroid

I s ::

3 .g

I-

20

d

Ip 10

10

/!

0 -101

N 3

5

........T........_... 4 7

9

11

13

0

Fig. 2. Binding of 0, F111/2Dl antibody; *, antibodies to transferrin and transferrin receptor (OKT-9) to peripheral blood erythrocytes, during the reticulocyte response (0, total; *, heavily granulated reticulocytes) following treatment of a severe case of megaloblastic anaemia.

15

DAYS

to erythroid cells (fig. 1, table 2) with increasing reactivity from the earliest proerythroblast to orthochromic normoblasts; only a fraction of reticulocytes expressed immunologically detectable transferrin receptors and no binding was seen to mature erythrocytes. An identical pattern of reactivity was seen using OKT-9 and anti-transfer-tin antisera. Glycophorin A was found only on erythroid cells and in a similar distribution to the transferrin receptor, except that it was also detectable on all reticulocytes and red cells (table 2); the distibution of glycophorin A is thus in agreement with that previously reported, using conventionally raised antisera [ 111. Reticulocyte heterogeneity transferrin receptor

demonstrated

with antibodies

to the

The bone marrow studies showed that the transferrin receptor, as detected using immunological techniques, was not expressed on all reticulocytes. The binding of F111/2Dl to peripheral blood reticulocytes was investigated in a patient with severe megaloblastic anemia during the response following treatment (fig. 2). Cells were detected in the peripheral blood which bound F111/2Dl, as well as OKT-9 and anti-transferrin antisera. At the onset of hematological recovery, a cohort of reticulocytes appeared which strongly expressed transferrin receptors; these accounted for approx. 20% of the total reticulocyte population. The positive cells rapidly waned at a time when the total number of reticulocytes was unaltered, and corresponded to a subpopulation of the heavily granulated reticulocytes which are released first from the bone marrow. From this study it was clear that the distribution of transferrin receptors in bone marrow, as detected by monoclonal antibodies and S. aureus rosetting, is restricted to cells of the erythroid cell lineage; this result essentially agrees with the findings of Lebman et al. [I51 using a different antibody and cell sorting, although binding to proerythroblasts was less prominent in our study. Transferrin receptors appear at the earliest morphologically identifiable stages of erythropoiesis and are strongly expressed throughout the different maturational stages, up to and including a subpopulation of reticulocytes. This distribution corre-

cells

365

366 Horton

Exe Cell Res 144 (1983)

sponds to the period of maximal iron uptake by bone marrow erythroid cells [ 181, as mediated by transferrin and transferrin receptors. The demonstration that only some reticulocytes express transfer& receptors suggests that either there may be some degree of heterogeneity in their distribution or that the sensitivity of the immunological techniques used was too low to detect low receptor numbers on later, more mature reticulocytes. In contrast, the expression of transferrin receptors in the K562 cell line seems to be related more to the proliferative state of the cell and not to the degree of induced erythroid differentiation. Further, our data confirms that the synthesis of erythrocyte membrane and other red cell specific proteins follows a programmed pattern in normal bone marrow and that the coordination of these changes can be disrupted in malignancy. The above findings, taken with the known distribution of transferrin receptors on a wide range non-erythroid cell types [4, 13-161, including morphologically unidentifiable erythroid progenitors [2], highlight the possibility that the transfertin receptor may have two functions [19]. The expression of the transferrin receptor in various proliferating [4, 13-16, 191, and even non-proliferative cell types [19], suggests that it may have some unique, and as yet undefined, relationship to the regulation of the balance between cell division and differentiation, distinct from its function as a mediator of iron transport during eryfhropoiesis. Studies using a wide range of monoclonal antibodies to different antigenic determinants on the transferrin receptor may help in the elucidation of this question. M. A. H. is a Wellcome Trust Senior Research Fellow in Clinical Science.

REFERENCES 1. Fukda, M, Fukuda, M N, Papayannopoulu, T & Hakomori, S-I, Proc natl acad sci US 77 (1980) 3474. 2. Seif, C A, Robinson, J B, Lam, G, Greaves, M F & Hardisty, R M, Brit j haemato149 (1981) 132.

3. Jandl, J H & Katz, J H, J clin invest 42 (1%3) 314. 4. Sutherland, R, Delia, D, Schneider, C, Newman, R, Kemshead, J T & Greaves, M, Proc natl acad sci US 78 (1981) 4515. 5. Kohler, G & Milstein, C, Nature 256 (1975) 495. 6. Horton, M A, Cedar, S H & Edwards, P A W, Stand j haemato127 (1981) 231. 7. Rutherford, T R, Clegg, J B & Weatherall, D J, Nature 280 (1979) 164. 8. Andersson, L C, Jokinen, M & Gahmberg, C G, Nature 278 (1979) 364. 9. Fukuda, M, Cancer res 41 (1981) 4621. 10. Horton, M A, Cedar, S H, Maryanka, D, Mills, S C & Turberville, C, Proc 3rd conf. on hemoglobin switching (ed A W Nienhuis & G Stamatoyannopoulos). Alan R. Liss, New York. In press. 11. Gahmberg, C G, Jokinen, M & Andersson, L C, Blood 52 (1978) 379. 12. Edwards, P A W, Biochem sot trans 8 (1980) 334. 13. Omary, M B, Trowbridge, I S & Minowada, J, Nature 286 (1980) 888. 14. Haynes, B E, Immunol rev 57 (1981) 127. 15. Lebman, D, Trucco, M, Bottero, L, Lange, B, Pessano, S & Roverra, G, Blood 59 (1982) 671. 16. Trowbridge, I S & Omary, M B, Proc natl acad sci US 78 (1981) 3039. 17. Friend, C, Scher, W, Holland, J G & Sato, T, Proc natl acad sci US 68 (1971) 378. 18. Glass, J, Lavidor, L M & Robinson, S H, J cell biol 65 (1975) 298. 19. Faulk, W P & Galbraith, G M P, Proc roy sot B 204 (1979) 83. Received August 24, 1982 Revised version received December 6, 1982 Printed

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