Burst-Promoting Activity in Anemia and hlycythemia

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1 International Journal of Cell Cloning 4: (1986) Burst-Promoting Activity in Anemia and hlycythemia Hiromi Fukamachi", Akio Urabe", Tsunehiro Saito", Fumimuro Takaku", Mamoru Kubota "The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Tokyo 113 Japan; bapplied Bioscience Laboratory, Kirin Brewery Co., Lid., Maebashi, Gunma 371 Japan Key Words. Burst-promoting activity * Erythropoietin - Anemia * Polycythemia Abstract. Burst-promoting activity (BPA) in the sera of patients with various types of anemia and polycythemia was compared with that of normal subjects by an in vitro method using mouse bone marrow cells. The control culture contained normal human AB serum instead of sample materials. Results were expressed as a percentage of burst numbers in control cultures. Serum erythropoietin (Epo) levels were determined by a radioimmunoassay. Serum BPA in patients with aplastic anemia ( %, mean f SD) was significantly higher than that in normal subjects (112.1 f 29.1%, Wilcoxon's rank sum test, P < 0.05). However, serum BPA in patients with uremic anemia (122.2 f 26.5%), polycythemia Vera (101.9 f 19.5%) and stress polycythemia ( fr 25.6%) was not significantly different from normal subjects. There was a correlation between serum BPA and Epo titers in patients with aplastic anemia and paroxysmal nocturnal hemoglobinuria (r = 0.81, t test, P < 0.001). Introduction In both mouse and human models, transit of the erythroid pathway in culture requires two distinct growth factors: erythroid burst-promoting activity (BPA) at early stages and erythropoietin (Epo) at later stages [l, 21. Human Epo has been purified [3] and serum Epo levels in various diseases have been determined by a radioimmunoassay [4, 5, 61. Recently BPA, derived from a human T lymphoblast cell line, also has been purified to homogeneity [7]. The presence of high levels of BPA was found in the sera of patients with aplastic anemia [8]. However, there have been only a few reports on human serum BPA as yet [9, 10, 201. In this study we report a comparison of BPA in the sera of Correspondence: Hiromi Fukamachi, The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Tokyo 113 Japan. Received April 18, 1985; accepted October 31, Press, Inc.

2 BPA in Anemia and Polycythemia 75 patients with various types of anemia and polycythemia by an in vitro method using mouse bone marrow cells. Materials and Methods Adherent-Phagocytic Cell Removal Bone marrow cells of BDF, female mice (Shizuoka Laboratory Animal Center, Shizuoka, Japan) capable of adherence to, or phagocytosis of, carbonyl iron particles were removed by applying a magnetic field after incubation at 37.C for 30 min in alpha medium (Flow Laboratories, McLean, VA) containing 10% fetal calf serum (FCS; Flow) and 4 mg/ml carbonyl iron (Sigma Chemical Co., St. Louis, MO). Cells remaining in suspension after application of the magnetic field were used for assay of BPA. Cell Culture Mouse bone marrow cells (4 X 10s/ml) depleted of phagocytic cells were suspended in alpha medium containing 20% FCS, 1% bovine serum albumin (BSA; Armour Pharmaceutical, Chicago, IL), 1 X 10-4M 2-mercaptoethanol (Sigma) and 10% test material in a final volume of 2.5 ml in plastic capped 17 X 100 mm tubes (Becton Dickinson Co., Oxnard, CA), and were incubated for 24 h at 37 C in humidified 5% C02 in air. After incubation, the cells were washed twice with alpha medium and resuspended in alpha medium containing 0.8% methylcellulose (Dow Chemical Co., Midland, MI), 20% FCS, 1% BSA, 1 X 10-4M 2-mercaptoethanol and 1 U/ml Epo (Connaught Laboratories, Willowdale, Ontario, Canada). The cell suspensions were transferred into 35 mm Petri dishes (Becton Dickinson). Each dish contained 1 ml of the cell suspension. For each sample, 4 Petri dishes were set up and incubated for 7 days at 37 C in humidified 5% C02 in air. Scoring of Cultures and Expression of Activities Erythroid bursts were enumerated directly in methylcellulose cultures using an inverted microscope (X 40). Red- or pink-colored aggregates appearing as isolated groups of several colonies or as single large colonies composed of 200 or more erythroblasts were scored as erythroid bursts. The control culture contained normal human AB serum (GIBCO Laboratories, Grand Island, NY) instead of sample materials. The mean f standard deviation of burst-forming efficiency in 16 experiments containing 10% human AB serum was bursts/4 X lo5 cells seeded. Results were expressed as a percentage of burst numbers of control cultures. For statistical analysis, Student s t test and Wilcoxon s rank sum test were used. Preparation of Spleen Conditioned Medium (PWM-SCM) BDF, mouse spleen cells were incubated for 7 days at a concentration of 1 X lo6 cells per ml in HBlOl medium (Hana Media, Inc., Berkeley, CA) containing 1% pokeweed mitogen (GIBCO). After incubation, the medium was centrifuged for 10 min at 400 X g. The supernatant was then harvested and filtered through Millipore filters (0.45 pm; Millipore Corporation, Bedford, MA).

3 FukamachiIUrabelSaitolTakakulKubota 76 Serum Samples Serum samples were obtained from 16 normal individuals, 6 patients with polycythefia Vera, 8 patients with stress polycythemia, 2 patients with paroxysmal nocturnal hemoglobinuria (PNH), 17 patients with aplastic anemia and 19 patients with uremic anemia. After inactivation at 56 C for 30 min, the serum was sterilized by Millipore filtration (0.45 am) and stored at -20 C until use. Radioimmunoassay of Epo Serum Epo was determined by a radioimmunoassay, as described by Sherwood and Goldwasser [ll]. Epo was purified by the method of Miyake et al [3] and labeled by the iodogen method [12]. All serum samples and Epo standards were assayed in duplicate at three or more concentrations of the sample. Results The effect of varying the concentration of PWM-SCM on the erythroid burst growth is shown in Figure 1. A dose-dependent increase of the erythroid burst numbers was observed by the preincubation with PWM-SCM. The activity of PNH patients sera was also investigated (Fig. 2). Preincubation with PNH patients sera resulted in a dose-dependent increase in erythroid burst numbers. Figure 3 shows the effects of BPA in the sera of patients with various types of anemia and polycythemia. Serum BPA of 17 aplastic anemia patients (155.4 f 56.7%, mean f SD) was significantly higher than that of the 16 normal volunteers (112.1 f 29.l%, P < 0.05, Wilcoxon s rank sum test). However, some aplastic anemia patients showed normal or rather decreased BPA. Serum BPA in 19 cases of uremic anemia, 6 cases of polycythemia Vera and 6 cases of stress polycythemia was k 26.5%, f 19.5% and f 25.6%, respectively. The levels were not significantly different from those of normal subjects. Serum BPA of 2 patients with PNH was 155.8% and 156.3%, respectively. Both were higher than normal values. Serum BPA in patients with both aplastic anemia and PNH was higher than that in normal subjects. Figure 4 shows the relationship between serum Epo titers and BPA in these patients. Both parameters correlated significantly (r = 0.81, P < 0.001, I test). There were no correlations, however, between serum Epo titers and BPA in the cases other than aplastic anemia and PNH. Discussion When mouse bone marrow cells were cultured with 10% human serum without

4 BPA in Anemia and Polycythemia 77 Fig. 1. Dose-response effect of pokeweed mitogen-stimulated spleen conditioned medium on the production of erythroid bursts by 4 x lo5 BDF, mouse bone marrow cells. Fig. 2. Effect of varying the concentration of the serum of paroxysmal nocturnal hemoglobinuria patients on erythroid burst formation by 4 x 10s mouse bone marrow cells.

5 Fukamachi/Urabe/Saito/Takaku/Kubota 78 Fig. 3. Comparison of burst-promoting in the sera of patients with various types of anemia and polycythemia. The bars indicate mean BPA. Mean BPA in patients with aplastic anemia is significantly elevated (P c 0.05). one day of preincubation, only a few erythroid bursts were observed. Human serum may contain toxic factor(s) for mouse bone marrow cells. Our preliminary test suggested that mouse bone marrow cells were more sensitive to BPA than human peripheral blood mononuclear cells were. In this study, we used a two-stage method for the assay of BPA in human serum utilizing mouse nonadherent nonphagocytic bone marrow cells. The most widely used source of BPA is lectin-stimulated lymphocytes. FWM-SCM and phytohemagglutinin-stimulated human leukocyte conditioned medium (PHA-EM) also increase the growth of erythroid bursts in cultures of mouse or human bone marrow cells [13, 141. In this study, a dose-dependent increase of the erythroid burst numbers was shown by the preincubation with

6 BPA in Anemia and Polycythemia 79 Fig. 4. Relationship between serum erythropoietin (Epo) and burst-promoting activity (BPA) in patients with aplastic anemia and paroxysmal nocturnal hemoglobinuria. Epo and BPA are highly significantly correlated (r = 0.81, P < 0.001). PWM-SCM (Fig. 1). Increase of erythroid burst numbers also was shown by the preincubation with PHA-EM (data not shown). Immunoreactive Epo was not detected in the PWM-SCM and PHA-EM we used, suggesting that the erythroid bursts formed in this culture method are stimulated by BPA in the sample materials. When we examined the relationship between the erythroid burst numbers and PNH patients sera, which seemed to contain high levels of BPA, a dose-dependent increase of erythroid burst numbers by the preincubation with PNH patients sera was observed (Fig. 2). These results suggest that the numbers of erythroid bursts obtained in this method represent the amount of BPA present in the human test sera.

7 Fukamac hilurabe1 SaitolTakakulKubota 80 Serum BPA of aplastic anemia patients was significantly higher than that of normal subjects (P < 0.05). This result is similar to the data reported by Nissen et al. [8] who have found high BPA in sera of patients with aplastic anemia by an in vitro method using human bone marrow cells, although some aplastic anemia patients in our study showed normal or rather decreased BPA (Fig. 3). Serum BPA of two patients with PNH was higher than that of normal subjects. BPA in the sera of uremic anemia patients was slightly higher than that of normal subjects, but not significantly different. Recently, evidence has accumulated that both T lymphocytes and monocytes produce BPA. Golde et al. found that the human T lymphoblast cell line, Mo, produces BPA, and they have purified it to homogeneity [l]. Another human T lymphocyte cell line, ATCC. CCL199 [15]; the murine macrophage cell line, WEHI-3 [16]; and two human monocyte cell lines, GCT [17] and U-937 [18], also have been reported to produce BPA. Epo is produced mainly in the kidney, and serum Epo levels of patients with uremic anemia are known to be low [4]. Serum BPA of uremic anemia patients was not so high as that of aplastic anemia. However, there was a correlation between serum BPA and Epo titers in patients with aplastic anemia and PNH (r = 0.81, P < 0.001). The existence of a fraction of BPA without Epo activity in the sera of patients with aplastic anemia [19] supports our results that there are high levels of BPA in the sera of aplastic anemia patients, although possibly Epo in the Serum samples might have augmented the burst numbers. BPA may reflect the anemic state of human subjects as serum Epo titers do. Our method described here is convenient and will favor clinical screening of a broad spectrum of diseases. References Golde DW, Westbrook CA, Lusis AJ. Erythroid-potentiating activity. In: Guroff G, ed. Growth and maturation factors. Vol. 2. New York: John Wiley & Sons, 1984: Porter PN, Ogawa M. Erythroid burst-promoting activity (BPA). In: Dunn CDR, ed. Current concepts in erythropoiesis. New York: John Wiley & Sons, 1983: Miyake T, Kung CK-H, Goldwasser E. Purification of human erythropoietin. J Biol Chem 1977;252: Zaroulis CG, Hoffman BJ, Kourides IA. Serum concentrations of erythropoietin measured by radioimmunoassay in hematologic disorders and chronic renal failure. Am J Hernatol 1981;11: Garcia JF, Ebbe SN, Hollander L, Cutting HO, Miller ME, Cronkite EP. Radioimmunoassay for erythropoietin: circulating levels in normal and polycythemic human beings. J Lab Clin Med 1982:99:

8 BPA in Anemia and Polycythemia Rege AB, Brookins J, Fisher JW.A radioimmunoassay for erythropoietin: serum levels in normal human subjects and patients with hemopoietic disorders. J Lab Clin Med 1982;lOO: Westbrook CA, Gasson JC, Gerber SE, Selsted ME, Golde DW. Purification and characterization of human T lymphocyte-derived erythroid-potentiating activity. J Biol Chem 1984;259: Nissen C, Iscove NN, Speck B. High burst-promoting activity (BPA) in serum of patients with acquired aplastic anemia. In: Baum SJ, Lendney GD, eds. Experimental hematology today. New York: Springer-Verlag, 1979: Kigasawa H, Nishihira H. High human erythroid burst-forming activity in sera from childhood aplastic anemia in semisolid agar. Br J Haematol 1980;46: Terasawa T, It0 T, Matsuda M, Azuma T, Kasai S. High erythroid burst-promoting activity in sera from umbilical cord blood. Jap J Physiol 1984;34: Sherwood JB, Goldwasser E. A radioimmunoassay for erythropoietin. Blood 1979;54: Garcia JF, Sherwood J, Goldwasser E. Radioimmunoassay of erythropoietin. Blood Cells 1979;5: Humphries RK, Eaves AC, Eaves CJ. Characterization of a primitive erythropoietic progenitor found in mouse marrow before and after several weeks in culture. Blood 1979 ; Meytes D, Ma A, Ortega JA, Shore NA, Dukes PP. Human erythroid burst-promoting activity produced by phytohemagglutinin-stimulated, radioresistant peripheral blood mononuclear cells. Blood 1979;54: Hamburger AW. Enhancement of human erythroid progenitor cell growth by media conditioned by a human T lymphocyte line. Blood 1980;56: Iscove NN, Schreier M. Involvement of T cells and macrophages in generation of burst-promoting activity (BPA). Abstract. Exp Hematol l979;7(suppl 6):4. Abboud CN, DiPersio JF, Brennan JK, Lichtman MA. Erythropoietic enhancing activity derived from a human cell line: similarity to colony-stimulating activity. Abstract. Clin Res 1980;28:303a. Ascensao JL, Key NE, Earenfight-Engler T, Koren HS, Zanjani ES. Production of erythroid potentiating factor@) by a human monocyte cell line. Blood 1981; Okamoto T, Kanamaru A, Hara H, Nagai K. Burst-promoting activity (BPA) without erythropietin (Ep) activity in sera of aplastic anemia patients fractionated by chromatofocusing column. Exp Hematol 1982;lO: Dukes PP, Ma A, Clemons GK, Meytes D. Measurement of human erythroid burstpromoting activity by a specific cell culture assay. Exp Hematol 1985;l3:59-66.