Effect of Interleukin 10 on the Hematopoietic Progenitor Cells from Patients with Aplastic Anemia YOSHINOBU ASANO, SHOICHIRO SHIBATA, SHINJI KOBAYASHI, SEIICHI OKAMURA, YOSHIYUKI NIHO First Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan Key Words. IL-10 Aplastic anemia Interferon-γ Hematopoietic progenitor cells ABSTRACT The overproduction of cytokines with inhibitory effects on hematopoiesis is considered to play a role in the pathogenesis of aplastic anemia. While interleukin 10 (IL-10) is a cytokine production inhibitory factor, the possibility of immunosuppressive therapy using IL-10 for aplastic anemia has not been explored. In this study, therefore, we examined the effect of IL-10 on progenitor cells obtained from seven patients with severe aplastic anemia. Our study indicated that IL-10 dramatically enhanced the erythroid colony formation in a dosedependent manner in two of the seven cases examined. When we examined the concentration of cytokines in the culture supernatants of unstimulated bone marrow cells, the spontaneous production of interferon-γ (IFN-γ) was observed in one of these two cases, and this production was completely inhibited by addition of IL-10. These findings suggested that IL-10 enhanced the erythroid colony formation by inhibiting the pathological production of IFN-γ in this case. This study provides an experimental support for the clinical application of IL-10 in some patients with aplastic anemia. Stem Cells 1999;17:147-151 INTRODUCTION It has been reported that the overproduction of cytokines with inhibitory effects on hematopoiesis, including interferonγ (IFN-γ) and tumor necrosis factor-α (TNF-α), may play a role in the pathogenesis of aplastic anemia [1-3]. The effectiveness of cyclosporin A (CyA) therapy in treating aplastic anemia supports this hypothesis, because the main pharmacological effect of CyA is the inhibition of cytokine production by T lymphocytes. Recently, an improved response rate in patients with severe aplastic anemia was reported when CyA was given in combination with antilymphocyte globulin and G-CSF [4]. However, development of other immunosuppressive therapies for aplastic anemia is also needed, because the administration of CyA is contraindicated in some cases because of its side effects, especially renal toxicity. Interleukin 10 (IL-10) was originally identified by virtue of its ability to inhibit cytokine production in T helper 1 clones [5]. Recently, we reported that IL-10 could inhibit the cytokine production of leukemic cells, while it did not influence normal bone marrow colony formation [6]. These findings led us to explore the possibility of immunosuppressive therapy using IL-10 for aplastic anemia. In the present study, we examined the effect of IL-10 on the growth of hematopoietic progenitor cells from seven patients with severe aplastic anemia and found that IL-10 could enhance the erythroid colony formation in two cases. This is the first report concerning the effect of IL-10 on aplastic anemia. MATERIALS AND METHODS Patients Seven patients with severe idiopathic aplastic anemia were studied. Their profiles are shown in Table 1. Cases 1, 3, 4, 5, and 7 had not received any therapy at the time of sampling. In the remaining cases, prednisolone and CyA had been administered, but the bone marrow failure rate was not improved in spite of these immunosuppressive medications. Preparation of Cells Heparinized bone marrow samples were taken from the aplastic anemia patients listed above and three normal Correspondence: Dr. Yoshinobu Asano, First Department of Internal Medicine, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. Accepted for publication January 28, 1999. AlphaMed Press 1066-5099/99/$5.00/0 STEM CELLS 1999;17:147-151
148 Effect of IL-10 on Aplastic Anemia Table 1. Patient characteristics Peripheral blood count a Patient Age/sex Hb PLT WBC Clinical status at time n of sampling 1 72/M 10.9 47 2.5 Newly diagnosed 2 22/F 7.3 48 3.5 No response to PSL, CyA b 3 19/F 11.9 33 2.1 Newly diagnosed 4 32/F 6.7 28 2.7 Newly diagnosed 5 45/M 9.8 49 1.6 Newly diagnosed 6 33/F 7.4 58 2.9 No response to PSL, CyA 7 74/F 9.5 2 2.6 Newly diagnosed a Hemoglobin (Hb) (g/dl); Platelets (PLT) ( 10 9 /l); White blood cells (WBC) ( 10 9 /l). b PSL = prednisolone; CyA = cyclosporin A. volunteers after obtaining informed consent. Mononuclear cells were collected by density gradient centrifugation and used for the following assays. Cytokines Human recombinant IL-10 and GM-CSF were provided by Schering-Plough K.K. (Osaka, Japan), and human recombinant G-CSF and erythropoietin (Epo) were furnished by Kirin Brewery Co., Ltd. (Tokyo, Japan). Hematopoietic Colony Formation Assay The effect of IL-10 on hematopoietic progenitor cells was assessed using the colony formation technique in methylcellulose culture, as previously described [6, 7]. Briefly, 1 10 5 normal or 2 10 5 aplastic anemia bone marrow mononuclear cells/ml were cultured in Iscove s modified Dulbecco s medium (GIBCO; Grand Island, NY) containing 0.88% methylcellulose and 10% fetal calf serum ([FCS]; Flow Laboratories; McLean, Virginia) in a 35-mm tissue-culture dish (Lux 5221-R, Naperville, IL). All cultures were supplemented with 10 ng/ml of G-CSF, 10 ng/ml of GM-CSF, and 1 U/ml of Epo. The cultures were incubated in various concentrations of IL-10 in a moist atmosphere of 5% CO 2 in air at 37 C. After 14 days, colony-forming units-erythroid (CFU- E) and colony-forming units-granulocyte/macrophage (CFU- GM) were counted using an inverted microscope. The accuracy with which colonies could be identified by direct visualization was confirmed by random staining of cytospinprepared colonies with May-Giemsa stain. Measurement of TNF-α and IFN-γ Production by Bone Marrow Mononuclear Cells Bone marrow mononuclear cells were cultured at 10 6 cells/ml in various concentrations of IL-10 in RPMI 1640 medium (GIBCO) containing 10% FCS for 24 h. The levels of TNF-α and IFN-γ in cell-free culture supernatants were determined using double-sandwich enzyme-linked immunosorbent assay (ELISA) kits, purchased from R&D Systems, Minneapolis, MN. ELISA assays were performed in accordance with the guidelines of the manufacturer. Statistical Analysis Data are expressed as mean values ± standard deviation (SD). The Student s t-test was used to compare differences. RESULTS Effect of IL-10 on the Normal Hematopoietic Progenitor Cells First, we examined the effect of IL-10 on the growth of the hematopoietic progenitor cells obtained from three normal volunteers. Formation of colonies of CFU-GM and CFU-E was substantially stimulated by G-CSF, GM-CSF, and Epo in all cases examined. When various concentrations of IL-10 were added, the CFU-E formation was slightly enhanced in two cases (Fig. 1A). However, these phenomena were observed only sporadically, and dose-dependency was not observed. The CFU-GM formation was not influenced by IL-10 in any of the cases examined (Fig. 1B). Effect of IL-10 on the Aplastic Anemia Hematopoietic Progenitor Cells Next, we examined the effect of IL-10 on the growth of the hematopoietic progenitor cells obtained from seven patients with severe aplastic anemia. Compared with the normal bone marrow, the number of colonies stimulated by G-CSF, GM-CSF, and Epo was generally reduced but was sufficient for estimating the enhancing effect of IL-10. When various concentrations of IL-10 were added, the CFU-E formation was enhanced dose-dependently in two of the seven cases examined (Case 1 and Case 4), as shown in Figure 2A. In Case 1, the number of colonies of CFU-E formed in the presence of 10 ng/ml IL-10 was about twice the number formed in the absence of IL-10. In contrast, the CFU-GM formation was slightly enhanced only sporadically in two cases (Fig. 2B). Effect of IL-10 on the Production of TNF-α and IFN-γ from Bone Marrow Cells To clarify the mechanism involved in the enhancement of CFU-E formation by IL-10, we determined the concentration of TNF-α and IFN-γ in the supernatants of bone marrow cells (Table 2). Spontaneous production of TNF-α was observed in all of the normal volunteers and patients with aplastic anemia except for one case (Case 6). When various concentrations of
Asano, Shibata, Kobayashi et al. 149 Figure 1. Effect of IL-10 on CFU-E (A) and CFU-GM (B) formation stimulated by G-CSF (10 ng/ml), GM-CSF (10 ng/ml), and Epo (1 U/ml) in three normal volunteers. All data are shown as mean values ± SD from triplicate plates. *p < 0.05 compared with the number of colonies formed in the absence of IL-10. IL-10 were added, the production of TNF-α was inhibited in a dose-dependent manner. On the other hand, spontaneous production of IFN-γ was observed in only one case (Case 1), in which IL-10 dramatically enhanced CFU-E formation. When serial concentrations of IL-10 were added, the production of IFN-γ began to decline at the level of 0.01 ng/ml of IL-10 and was completely inhibited at the level of 1 ng/ml of IL-10. DISCUSSION Previous studies showed the overproduction of IFN-γ and TNF-α in patients with aplastic anemia and related these cytokines to bone marrow failure [1-3]. Zoumbos et al. reported that addition of antibodies to IFN-γ enhanced in vitro colony growth of bone marrow from patients with aplastic anemia [1]. Furthermore, Fas antigen expression was reported to be increased on CD34 + cells from patients Figure 2. Effect of IL-10 on CFU-E (A) and CFU-GM (B) formation stimulated by G-CSF (10 ng/ml), GM-CSF (10 ng/ml), and Epo (1 U/ml) in seven patients with severe aplastic anemia. All data are shown as mean values ± SD from triplicate plates. *p < 0.05, **p < 0.02 and ***p < 0.01 compared with the number of colonies formed in the absence of IL-10. with aplastic anemia [8], and we recently found that IFN-γ or TNF-α could induce the functional expression of Fas antigen on CD34 + cells [9]. These findings suggest that the inhibition of production or action of these cytokines may be useful for improving bone marrow failure. CyA therapy results in hematopoietic recovery in some patients with aplastic anemia, and detection of IFN-γ gene expression in unstimulated bone marrow cells is reported to be helpful in predicting a good response to CyA therapy [10]. Therefore, CyA is presumed to function by inhibiting the pathological production of IFN-γ. IL-10 is also known to inhibit the production of cytokines, and its clinical application is expected to be relatively harmless [11]. Nevertheless, the possibility of immunosuppressive therapy using IL-10 for aplastic anemia has not been explored. In this study, therefore, we examined the effect of IL-10 on progenitor
150 Effect of IL-10 on Aplastic Anemia Table 2. Effect of IL-10 on the production of cytokines from bone marrow cells Production of TNF-α IL-10 (ng/ml) cells obtained from seven patients with severe aplastic anemia. Our study indicated that IL-10 significantly enhanced the CFU-E formation in a dose-dependent manner in two of the seven cases (Case 1 and Case 4). On the other hand, the enhancement by IL-10 of CFU-GM formation was quite weak, and dose-dependency was not observed. When we examined the concentrations of TNF-α and IFN-γ in the culture supernatants of unstimulated bone marrow cells, the production of IFN-γ was observed in only one case (Case 1), in which IL-10 enhanced CFU-E formation. As shown in Table 2, IL-10 inhibited this production in a dose-dependent manner. These findings suggest that IL-10 enhanced CFU-E formation by inhibiting the overproduction of IFN-γ in this case. Although IL-10 could also inhibit the production of TNF-α, this phenomenon may not directly contribute to the enhancement by IL-10 of CFU-E formation, because TNF-α was also produced in other cases (Cases 2, 3, 5, 7), in which IL-10 did not enhance the CFU-E formation. The mechanism involved in the enhancement by IL-10 of Production of IFN-γ IL-10 (ng/ml) Samples Control 0.01 0.1 1 Control 0.01 0.1 1 Normal volunteers Case A 60 a 37 ND b ND ND ND ND ND Case B 40 41 27 ND ND ND ND ND Case C 68 53 42 ND ND ND ND ND Aplastic anemia Case 1 60 59 31 ND 82 52 21 ND Case 2 85 44 ND ND ND ND ND ND Case 3 68 31 ND ND ND ND ND ND Case 4 65 59 35 ND ND ND ND ND Case 5 45 46 30 ND ND ND ND ND Case 6 ND ND ND ND ND ND ND ND Case 7 72 54 33 ND ND ND ND ND a pg/ml. Mean values of triplicate cultures, with SD always <10%. b ND = not detected (below sensitivity of ELISA). CFU-E formation in Case 4 is unknown, but several possibilities can be considered. For example, the bone marrow progenitors may be so sensitive to TNF-α that the inhibition of its production by IL-10 is critical in this case. Another possibility is that IL-10 may inhibit the production of other cytokines with inhibitory effects on hematopoiesis. In order to clarify the functional mechanism of the effects of IL-10 on CFU-E formation, further studies are necessary. Recently, Chernoff et al. reported the results of a phase I study of IL-10 in healthy volunteers [11]. According to their report, serum levels of IL- 10 exceeded 10 ng/ml at 3 h after the injection of 25 mg/kg of IL-10, without any severe side effects. Our studies indicate that CFU-E formation was enhanced twofold in the presence of 10 ng/ml of IL-10. Since this enhancement by IL-10 was observed in only two of the seven cases examined, IL-10 is not a universally effective stimulator for aplastic anemia bone marrow. However, it may be possible to utilize IL-10 as a new supportive therapeutic tool for some patients with aplastic anemia. ACKNOWLEDGMENTS We are grateful to Schering-Plough K.K. for providing human recombinant IL-10 and GM-CSF, and to Kirin Brewery Co., Ltd. for providing human recombinant G-CSF and Epo. This work was partially supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (No. 10181219, 10153249), the Foundation for the Advancement of Clinical Medicine, and Kaibara Morikazu Medical Science Promotion Foundation. REFERENCES 1 Zoumbos NC, Gascon P, Djeu JY et al. Interferon is a mediator of hematopoietic suppression in aplastic anemia in vitro and possibly in vivo. Proc Natl Acad Sci USA 1985;82:188-190. 2 Hinterberger W, Adolf G, Aichinger G et al. Further evidence for lymphokine overproduction in severe aplastic anemia. Blood 1988;72:266-272. 3 Schultz JC, Shahidi NT. Detection of tumor necrosis factor-α in bone marrow plasma and peripheral blood plasma from patients with aplastic anemia. Am J Hematol 1994;45:32-38. 4 Bacigalupo A, Broccia G, Corda G et al. Antilymphocyte globulin, cyclosporin, and granulocyte colony-stimulating factor in patients with acquired severe aplastic anemia
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