Characterisation of bovine transferrin receptor on normal activated and Theileria parva-transformed lymphocytes by a new monoclonal antibody

Veterinary immunology and Veterinary Immunology and Immunopathology 52 (19%) 65-76

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Characterisation of bovine transferrin receptor on normal activated and Theileria parua-transformed lymphocytes by a new monoclonal antibody Jan Naessens aT* , Dennis J. Grab bSc,Gerhard Fritsch d aILRI, P.O. Box 30709. Nairobi, Kenya b Department of Parasitology, Tulane Regional Primate Center, 18703 Three Rivers Road, Covington, LA 70433, USA ’ Depurhnent of Tropical Medicine, Tulane School of Public Health and Tropical Medicine, I501 Canal St.. New Orleans, LA 70117, iJSA ’ Children’s Cancer Research Institute. St. Anna Kinderspital, Kinderspitalgasse 6, 1090 Wien IX, Austria Accepted 18 September 1995

Abstract A murine IgM monoclonal antibody (mAb), IL-A77, has been generated that recognises the bovine transferrin receptor (TfR) and will be a useful tool to measure the activation state of bovine lymphocytes and macrophages. The antigen is detected on immature erythroid cells and proliferating lymphocytes. It is undetectable on resting lymphocytes, but appears within 24 h after stimulation with concanavalin A (ConA) or pokeweed mitogen (PWM). Immune precipitations of lysates of both labeled activated lymphocytes and bone marrow erythroid cells showed that, similar to human TfR, the bovine receptor is a disulfide-bonded dimer of two identical chains of M, 97000. A similar 97000 M, protein was eluted from a column containing immobilised bovine transferrin (Tf) using conditions known to elute the human TfR, and this protein was recognised by mAb IL-A77, proving that it detected bovine Tfii. Although the mAb inhibited binding of transferrin to its receptor, it did not block proliferation of Theileria purua-transformed or ConA-stimulated lymphocytes. When cells were metabolically labeled with 35S-methionine, a second 90000-M, TlR band was detected in Theileria parua-transformed cells, but not in stimulated lymphocytes. This form of the TfR was not expressed on the cell surface. It may be an

Abbreviations: ConA = Concanavalin A; mAb = Monoclonal antibody; PBMC = Peripheral mononuclear cells; PWM = Pokeweed mitogen; Tf = Transfenin; TfR = Transferrin receptor * Corresponding author. Tel.: 254-2-630743; fax: 254-2-631499; e-mail: [email protected]. 0165.2427/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDI 0165-2427(95)05537-l

blood

unusual precursor of the receptor. a parasite-modified receptor or it may be of parasite origin and necessary for transfer of iron into the intracellular parasite. Key~vords: Transferin

receptor;

Bovine; Monoclonal

antibody;

Thrileriu

purw

1. Introduction Surface activation markers have proven to be highly effective tools to determine the state of activation of leukocytes and are widely used for in vivo applications. Owing to a lack of such tools in ruminants, little is known about the function and the state of activation of leukocytes in disease situations. One early activation antigen which is expressed on virtually all activated and proliferating leukocytes, and therefore would make a useful pan-leukocyte activation marker, is the transferrin receptor. As both energy metabolism and DNA synthesis in a cell are catalysed by iron-containing enzymes, transferrin, the main iron-carrying serum component, is an essential growth factor. The receptor for the iron-transferrin complex is expressed on cells which need large amounts of iron, i.e. activated and proliferating cells, but also on immature cells from the erythroid lineage which need iron for heme formation (Iacopetta et al., 19831. After entering the cell by endocytosis and release of iron. both transferrin and the receptor are recycled Dautry-Varsat, 19861. Monoclonal antibodies (mAbs1 to human transferrin receptor (TfR) have been described (Omary et al., 1980; Trowbridge and Omary, 1981; Haynes et al., 1981) and were instrumental in the characterisation of its biochemical structure: a disulfide-bonded homodimer of M, 95 000 (Trowbridge and Omary. 198 1; Schneider et al.. 1982). At the Fourth International Workshop on human leucocyte differentiation antigens in Vienna, mAbs to TfR were assigned cluster designation CD71 (Schwarting and Stein, 1989). Since few leukemic cell lines from cattle have been established in culture, Theilevia parua-transformed lymphocytes and lectin-stimulated PBMC are used as sources of proliferating lymphocytes. Bovine lymphocytes infected in vitro by the protozoan parasite T. parva, easily establish continuously growing clones in vitro (Brown et al., 1973), although the transformed lymphocytes gradually lose their functional capacities (Naessens et al., 1985; Baldwin and Teale. 1987; Baldwin et al., 19881. The parasite exists as an intracellular schizont, multiplies in synchrony with the cell and its presence is essential to maintain the transformed state of the cell (Dobbelaere et al., 1988). We describe the generation of a mAb specific for bovine TfR and the characterisation of the receptor on erythroid and cultured lymphoid cells.

2. Materials and methods 2.1. Animals Cattle used as blood donors were healthy Bos Taurus and Bos indicus females castrated males. Balb/c and Fl(Balb/c X Swiss) mice were bred at ILRAD.

or

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2.2. Cell suspensions

Bone marrow cells were prepared from aspirations of bovine sternum as previously described (Fritsch et al., 1991; Muiya et al., 1993) and centrifuged on Ficoll-Paque (gravity 1.077; Pharmacia, Uppsala, Sweden). Peripheral blood mononuclear cells were prepared on Ficoll-Paque from venous blood mixed with Alsever’s solution (1: 1) as described previously (Naessens et al., 1985). 2.3. Preparation of mAb mAb IL-A77 (IgM) was obtained from a fusion of spleen cells from a mouse immunised with enriched B lymphocytes from a Trypanosoma congolense-infected cow. Afferent lymph cells from the infected animal were enriched for B cells by complement lysis using an IgM mAb against T cells (IL-A26; Baldwin et al., 1988) and contained about 30% activated B lymphocytes. A Balb/C mouse was three times injected intraperitoneally at 3 week intervals with 2 X lo6 enriched B cells in 0.5 ml phosphatebuffered saline (PBS). Four days after the last boost its spleen cells were fused with X63.Ag8.653 myeloma cells, as described previously (Naessens et al., 1985). Hybridomas were screened for binding to activated B cells from infected cattle or T. parua-infected bovine B cells (Baldwin et al., 1988). IgM-hybridoma IL-A77 was recloned before further analysis. Ascitic fluid was produced in Fl (Balb/c X Swiss) mice. 2.4. Cell cultures Transformed cell lines BL3 (Theilen et al., 1982) and BL20 (Morzaria et al., 1984; Olobo and Black, 1989) have been established from animals with bovine leukosis and were obtained from ATCC (Rockville, MD). T. parua-transformed bovine lymphocytes were prepared and grown in complete RPM1 1640 (Gibco, Paisley, UK) as described previously (Naessens et al., 1985). The alloreactive T cell lines have been described previously (Teale et al., 1985). To obtain lectin stimulated cells, peripheral blood mononuclear cells (PBMC) were resuspended in complete RPM1 1640 medium at 2 X 10’ per well in 96-well culture plates, and stimulated with 5 p,g ml-’ concanavalin A (ConA; Sigma Chemical Company, Poole, UK) or 2.5 p,g ml-’ pokeweed mitogen (PWM; Sigma). Cultures were maintained for prolonged periods by regularly stimulating with ConA and IL-2 containing supernatants, as previously decribed (Naessens et al., 1985). 2.5. Immunojluorescence and cell sorting About lo6 cells in 25 ~1 were added per well in a 96-well plate, and an equal volume of a lOOO-folddilution of ascitic fluid was added. Antibodies were left to react for 30 min at 4”C, and cells were then washed twice in PBS with 0.1% azide. Fluorescein isothiocyanate (FITC) labeled anti-mouse Ig (Sigma) was added (25 ~1 of a 40-fold dilution) and after 30 min cells were washed once in PBS. Cells were fixed and kept in 2% formalin at 4°C until analyzed by flow cytometry on a FACStar PLUS (Becton Dickinson, Sunnyvale, CA). No azide was used in the buffers before sorting.

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For inhibitions, 25 p_l ascitic fluid containing the competing mAb at a 20-fold dilution were added at the same time as the first mAb. Binding of the first antibody was revealed by an FITC-labeled Ig-class specific antibody (Southern Biotechnology Associates, Birmingham, AL, USA) (25 l.~l of a 1:40 dilution). 2.6. Immune precipitation

with mAb IL-A77

Two-day cultured ConA-stimulated PBMC or 5 X 10’ T. parua-infected cells were radio-iodinated by the lactoperoxidase method as described (Jones, 1980) or metabolically labeled with 35S-methionine (Naessens et al., 1985). The cells were grown overnight at 2 X lo6 ml- ’ in complete methionine-free RPM1 1640 medium (Gibco) containing 125 t&i [ 35S]-methionine ml- ’ . The cells were washed three times in PBS, lysed in 1 ml of a detergent buffer (10 mM Tris, 1% Nonidet P40, pH 7.5) containing protease inhibitors (1 mM phenylmethylsulfonyl-fluoride, 12.5 kg ml - ’ antipain, 40 pg ml-’ leupeptin and 2 p.g ml-’ cystatin) for 2 h on ice and centrifuged for 15 min at 10000 x g. Immobilized mAb was prepared by mixing 200 ~1 biotinylated anti-mouse IgM (Amersham International, Amersham, UK) with 100 pl of packed avidin-coated agarose beads (Sigma) overnight at 4°C. The beads were washed three times with cold PBS before 50 ~1 of ascitic fluid containing IgM antibody IL-A77 were added, and mixed for 2 h at room temperature. The beads were washed three times with cold PBS, and then added to the 1 ml radiolabeled cell lysate. The mixture was kept on ice and agitated every 5 min for a total of 2 h. The beads were washed seven times with 1 ml of washing buffer (50 mM Tris, 0.5% Nonidet-P40, 0.1% sodium azide, 0.15 M NaCl, 5 mM EDTA, 1 mg ml-’ ovalbumin, pH 7.5) and seven times with 1 ml of the same buffer without ovalbumin. During the final wash, the beads were transferred to a new tube and all supematant was carefully removed. The beads were then resuspended in 50 ~1 sample buffer (180 mM Tris, 6% sodium dodecyl sulfate (SDS), 22.5% glycerol, 0.06% phenol red) with or without 15% 2-mercaptoethanol for reducing or non-reducing conditions. Samples were eluted by heating at 100°C for 5 min before application on a 10% SDS-polyacrylamide gel. 2.7. Purification

of transferrin-binding

membrane proteins

Cells were labeled and lysed as described in the paragraph above. Bovine diFe-Tf (Sigma) was first repurified by high performance liquid chromatography (HPLC) on a TSK column before coupling to activated CH-Sepharose as recommended by the manufacturer (Pharmacia). The radiolabeled cell lysate (1 ml) was incubated with an equal volume of the DiFeTf-Sepharose slurry (1 ml containing 1 mg coupled Tf) and rotated for 1 h at room temperature. The slurry was then poured into an empty PD- 10 column (Pharmacia). The column was washed with 50 ml 25 mM HEPES, 100 mM NaCl, 1 mM EDTA, 0.1% (v/v) TX-100 detergent (Sigma), pH 7.4. The bound proteins were subsequently removed from the Tf-affinity column with 10 ml each of the following buffers: 100 mM Na-citrate, 0.1% TX-100, desferal (10 kg ml-‘), pH 5.0; 50 mM HEPES, 0.1% TX-100, desferal, pH 7.4; 50 mM HEPES, 0.1% TX-100,

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desferal, 1 M KCl, pH 7.4; and 100 mM glycine, 0.1% TX-100, pH 2.5. The wash and elution buffers also contained a cocktail of protease inhibitors (50 pg ml- ’ each of E64, leupeptin, antipain and 2 Fg cystatin ml-’ >. Fractions (1 ml> were collected from the column and immediately neutralized with 1 M Tris base. Aliquots (100 ~1) were counted in 10 ml Aquasol in a Beckman LS 6800 liquid scintillation counter. Peak fractions were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and direct autoradiography using B-Max film (Amersham). The [‘4C]-methylated M, rainbow markers used in SDS-PAGE were from Amersham (CFA 755).

3. Results 3.1. Cellular distribution MAb IL-A77 did not react with resting lymphocytes, monocytes or granulocytes from peripheral blood when tested in an immunofluorescent assay. Cells from thymus, lymph nodes, Peyer’s patches and spleen were also negative when stained for the antibody. MAb IL-A77 bound to in vitro cultured bovine cell lines: BL3, BL20, and all T. parua-transformed lymphocyte lines tested (20 out of 20). Staining of T. parua-infected B cell lines tended to be stronger than infected T cells (both a$-TCR+CD2+ and y,bTCR+CD2-). The line that expressed most antigen was a clone obtained from in vitro infected B cells that had been purified from ileal Peyer’s patch. Long-term cultured non-infected T cell clones, grown in the presence of IL2, were very weakly positive when stained with IL-A77. When resting cells were stimulated with ConA or PWM, they expressed the IL-A77 antigen within 24 h (Fig. I>. Expression increased until day 3, and then started to decline. By day 7 surface antigen was barely detectable, which is in agreement with its absence from long-term cultured T cell clones. A subpopulation of bone marrow cells stained strongly with the antibody (Fig. 2). Positive cells could be found in the fraction of small bone marrow cells, identified by a

Day

Log relative Fig. 1. Kinetics of IL-A77 pawn-infected B cell line.

antigen

expression

1

.

clay 2

fluorescence

on PBMC

after

ConA

stimulation

and on a Theileria

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Fig. 2. Dot plot of bovine bone marrow cells stained for IL-A77. Small cells, with low forward scatter, contain mostly normoblasts which are TfR-positive. Larger cells. containing cells of different lineages and in different stages, contain both TfR positive and negative cells.

low forward scatter, which contained immature, non-nucleated cells with a high forward scatter, which contained normoblasts myeloid lineage. 3.2. inhibition of Tf-Tfl

erythroid cells and in and some cells of the

binding

Binding of iodinated bovine transferrin to bone marrow cells was inhibited by purified IL-A77 antibodies (Fig. 3). Increasing amounts of IL-A77 inhibited the binding of radioactive transferrin to cells, while a control IgM antibody (P13) to a late bovine

-

MAb

+

Control MAC

+

Cold

‘. ‘. , 1.0

0.1

Concentration

IL-A77

transferrin

Medium

only

,

0.01

(mg/ml)

Fig. 3. Inhibition of binding of iodinated bovine Tf to bone marrow cells by purified IL-A77 and an irrelevant IgM annbody (PII+), and by cold Tf.

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kDa 200-

69-

14.3-

e

Fig. 4. SDS-PAGE of immunoprecipitated IL-A77 non-reducing (Lane 2) or reducing (Lane 3) conditions

antigen from 2 day ConA (hf, markers in Lane 1).

stimulated

cells

under

activation antigen (Nthaie and Naessens, 1993) did not inhibit. Cold Tf also inhibited the binding, but in a different manner (Fig. 3). 3.3. Afinity purijkation

with IL-A77

Immune precipitations from cell surface iodinated T. parua-infected cells revealed that the IL-A77 antigen, like human TfB, is a homodimer with a unit IV, of approximately 97 000. It migrated as a band of M, 97000 under reducing conditions in SDS-PAGE (Fig. 41, while a band of M, 190000 was observed in non-reducing conditions. 3.4. Afiniry purification

with

Tf

Lysates of metabolically labeled T. parua-transformed or ConA stimulated cells were passed over an iron-loaded transfer& column. The column was washed extensively with homogenization buffer at pH 7.4 to remove non-specifically bound proteins. Elution was performed in several steps (Fig. 5): first, the column was eluted with a pH 5.0 citrate buffer containing desferal to remove iron and produce apotransferrin (apo-Tf). A peak of radioactivity was eluted from the column. This was followed by a pH 7.4 HEPES/desferal buffer which did not elute any radioactivity (Fig. 5(B)). More protein was recovered after eluting with the same buffer containing 1 M KC1 (Fig. 5(C)). The remaining bound proteins were recovered with a low pH (2.5) glycine buffer (Fig. 5(D)). The proteins in each fraction were analyzed on SDS-PAGE (Fig. 6, Lanes l-4) and

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2OimO : .; L z

E 0”

1 OQOOO

0 0

20

10

40

30

Fraction

SO

Number

Fig. 5. Elution profiles of labeled cell surface proteins from ConA blast or T. parua-transformed lymphocytes bound to a bovine transferrin column with pH 5.0 (first peak); pH 7.4 (B); pH 7.4+ KC1 (Cl and pH2.5 (D) buffers.

compared with the iodinated TfR precipitated by IL-A77 from T. parua-infected cells (Fig. 6, Lane 5). The ConA-stimulated cells revealed a 97000 M, band in the fraction that was eluted with 1 M KC1 (Fig. 6, Lane 31, but not in the fractions eluted previously, nor in the fraction eluted with pH 2.5. This 97000 M, molecule migrated in the same position as the molecule precipitated by IL-A77 from surface-iodinated T. parua-infected cells. Furthermore, the radioactivity from this fraction could be bound by

1

2

3

4

5

6

7

Fig. 6. Comparison of molecules obtained by affinity purification with transferrin and mAb IL-A77: SDS-PAGE of 35S-methionine-labeled molecules from ConA-stimulated cells eluted from a bovine transferrin column with a pH 5 buffer (Lane 11, followed by a pH 7.4 buffer (Lane 21, followed by a pH 7.4+ KC1 buffer cells was (Lane 3) and followed by a pH 2.5 buffer (Lane 4). TfK from ‘251-surface-labeled T. purua-infected precipitated by IL-A77 (Lane 51. TfR was also precipitated with IL-A77 from 35S-methionine-labeled T. parua-infected cells (Lane 61 and 35S-methionine-labeled ConA-stimulated cells (Lane 7).

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69-

46Fig. 7. SDS-PAGE of ‘5S-methionine-labekd TfR molecules from T. parua-infected cells eluted from a bovine transferrin column with a pH 7.4 buffer (Lane 1) followed by a pH 7.4+ KC1 buffer (Lane 2) followed by a pH 2.5 buffer (Lane 3).

immobilized IL-A77, but not by immobilized control mAb P13, suggesting that the molecules purified by antibody and transferrin column are the same. Unexpectedly, the transferrin-binding protein eluted in the 1 M KC1 fraction from metabolically 35S-methionine-labeled T. parua-infected cells showed two bands with M, of 97000 and 90000 (Fig. 7). Both bands were also precipitated by IL-A77 (Fig. 6, Lane 6), and the higher molecular mass band migrates in the same position as the surface iodinated molecule precipitated from the same cells (Fig. 6, Lane 5) and as the metabolically labeled molecule from the proliferating lymphocytes (Fig. 6, Lane 7).

3.5. Inhibition of cell growth Growth of cells from a clone of T. parua-transformed bovine B lymphocytes was compared in the presence of 1 mg ml- ’ of IL-A77 or an irrelevant IgM antibody. Growth curves, determined by measuring the concentration of viable cells per day over a period of 8 days, were identical (not shown).

4. Discussion The tissue distribution of the antigen recognised by mAb IL-A77 conforms with human TfR. The bovine TW is expressed on immature erythroid cells and activated lymphoid cells, which are both cell types that require high amounts of iron. Expression of the receptor on resting lymphocytes can be induced within 24 h by activation with

lectins. Inhibition of transfenin binding by IL-A77 immunoglobulin gave further proof that the mAb reacted with TfR. Biochemically, the bovine receptor is analogous to the human receptor: a homodimer of a protein with an apparent M, of 97 000 in SDS-PAGE, covalently linked together by disulfide bonds. The receptor could also be purified from a detergent lysate by affinity chromatography on a Tf-Sepharose column. The receptor could be eluted in two steps: first by removing the iron from the immobilized Tf using desferal under mildly acidic conditions (pH 5.01, and secondly by eluting the receptor with a neutral buffer with a high salt concentration. This confirms that the bovine receptor, like the human TfR (Anderson et al., 1986), has affinity for Tf at neutral pH, and for apo-Tf at acidic pH. Analysis of the proteins in the different peaks by SDS-PAGE showed that under these conditions a 97000 MW protein was eluted from apo-Tf at neutral pH and high salt. This protein could be precipitated by mAb IL-A77, confirming that the same protein was affinity purified by transferrin and IL-A77. If the high salt concentration was omitted in the neutral buffer, little or no TfR was released from the immobilized Tf. A final wash of the column at pH 2.5 removed a number of surface proteins which might have non-specifically bound to the column. An interesting observation in our studies was the detection of an additional TfR molecule with a slightly lower M, (90000) in T.parua-infected cells. This lower band was identified by its affinity for transferrin and the monoclonal antibody. It was not observed in cell surface labeled T. parua-infected cells and is therefore not expressed on the cell membrane. Since it was also absent from proliferating ConA-stimulated lymphocytes, its existence seems to be related to the presence of the intracellular parasite. It could either be a receptor produced by the intracellular parasite or perhaps the bovine receptor modified by a parasite enzyme. This parasite-associated form may also be involved in transfer of iron from the cytoplasm to the intracellular parasite and could be vital for its survival. Another possibility is that the lower M, band represents a precursor molecule, which is present in a relatively high amount in the parasitized cells (and may not be targeted to the cell surface), but undetectable in the non-infected cells, owing to a slower rate of maturation of the protein or carbohydrate attachment in the parasitized cells. Although the antibody should interfere with the iron uptake of cultured bovine cells, it did not inhibit the growth of T. parua-transformed lymphocytes. Few mAbs to human TfR block cell growth. Nevertheless, a monoclonal to bovine transferrin receptor will be a useful tool for studying erythroid differentiation in cattle and for monitoring monocyte and lymphocyte activation (Keyna et al., 1991), particularly on B lymphocytes (Ashman, 1990).

Acknowledgements The authors would like to thank James G. Magondu and Peter F. Mucheru for assistance with flow cytometry and Dr. Janet Newson and Joseph Nthale for technical assistance. Jan Naessens was supported by the Belgian Administration of Development Cooperation. This is ILRAD Publication No. 1355.

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