American Journal of Hematology 40:245-251 (1992)
Effect of Interleukin-l, Tumor Necrosis Factor-a, and Intetferon-a on the Blast Cells of Acute Myeloblastic Leukemia Anna Carter, llana Silvian-Draxler, and llana Tatarsky Department of Hematology, The Bruce Rappaport Faculty of Medicine and Rappapport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology (A.C., IS.-D., I.T.); Rambam Medical Center (A.C., I.T.), Haifa, Israel
In this study, we further establishedthe role of interleukin-la (IL-la), interleukin-1p, tumor necrosis factor-a (TNF-a), and interferon-a (IFN-a) as regulators of proliferation of acute myeloid leukemia (AML) cells. AML cells from 8 of 15 patients incorporated high levels of 3H-thymidine ('H-TdR) in the absence of exogenous growth factors. The spontaneous DNA synthesis could be abrogated with monospecific antibodies directed toward IL-1a, IL-1p, or TNF-a, as well as with antigranulocyte-macrophage colony-stimulating factor (GM-CSF). Human recombinant GM-CSF reversed the inhibitory action of each of these antibodies and reinduced DNA synthesis in AML cells. Thus, in these cases, constitutively produced IL-1 or TNF-a had stimulated the synthesis of GM-CSF, which resulted in GMCSF-dependent proliferation of AML blasts. Exogenous IL-1 up-regulatedthe endogenous production of GM-CSF, suggesting a positive regulation of autocrine growth factor production. We also present evidence that TNF-a may exert both stimulative as well as inhibitory effects on DNA synthesis in AML cells. The enhancing effect of TNF-a was mediated through the induciion of GM-CSF production, as stimulation of DNA synthesis in AML blasts could be abrogated with anti-GM-CSF antibody. A concentration-dependent inhibitory effect of TNF-w on 3H-TdR incorporation into AML blasts was observed only when these cells were grown in the absence of GM-CSF. Finally, we show that human recombinant IFN-a is a potent inhibitor of AML cell proliferation in vitro. 4 1992 Wiley-Liss,
Inc.
Key words: leukemia, interleukin-1, tumor necrosis factor-a, interferon-a, proliferation, differentiation
INTRODUCTION Recently, it has been shown that the blast cells of acute myeloblastic leukemia (AML) proliferate in culture in the presence of exogenous growth factors such as interleukin-3 (IL-3), granulocyte macrophage (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) [ 1-81. The growth regulatory cytokines such as interleukin- I (IL- 1 ) and tumor necrosis factor-a (TNF-a) are also capable of influencing the growth of AML cells in vitro. IL- 1 stimulates the proliferation of AML blasts through the induction of GM-CSF production [9,10]. TNF-a is a potent modulator of AML cell growth [ 1 I ] . It enhances the stimulative responses of AML blasts to GM-CSF [ 121 and effectively suppresses the clonogenic growth of leukemia cells and leukemia-derived cell lines [ 131. Recent data indicate that some AML blasts constitutively secrete IL- 1 , GM-CSF, and TNF-a and use these
0 1992 Wiley-Liss, Inc.
factors as autocrine growth stimulators [14-16]. In the present study, we further investigated how IL-I and TNF-a stimulate the proliferation of AML cells in vitro. We also studied the effects of human recombinant (rh) interferon-& (IFN-a) on the growth of AML blasts in culture. In this report, we show that a substantial proportion of primary human AML cells spontaneously produce IL-la, IL-Ip, and TNF-a and use these activities to stimulate their own growth through the induction of endogenous GM-CSF production. We also provide evidence that exogenous IL- l as well as TNF-a are capable
Received for publication May 8. 1991; accepted November 18, 1991. Address reprint requests to Dr. Anna Carter, Department of Heniatology, The Bruce Rappaport Faculty of Medicine. Technion-Israel Institute of Technology, P.O.B. 9649. 31096 Haifa, Israel.
246
Carter et al.
TABLE 1. AML Cells ~
Case No. 1
2 3 4 5 6 7 8 9 10
II 12 13 14 15
Age/Sex
20 F 56 M 32 M 45 F 64 F 44 F 17 M 31 M 12 M 42 F 51 M 48 M 66 F 45 M 64 M
FAB classificationa
Source of cellsb
MI MI MI MI MI M2 M2 M2 M3 M4 M4 M4 MS M5
BM PB BM BM PB BM BM BM BM BM BM PB BM BM PB
M5
~~
% blasts
99 100
99 97 95 98 93 99 92 97 93 98 100 93 98
"French- American-British classification. bBM, bone marrow, PB, peripheral blood.
of inducing GM-CSF synthesis in some AML cells, suggesting a positive regulation of autocrine growth factor production. Furthermore, co-stimulation with GM-CSF and IL- 1 or TNF-a may show strong synergism in eliciting leukemia cell growth. Finally, we demonstrate that TNF-a is a potent inhibitor of AML cell proliferation. This effect may indicate the applicability of this cytokine against human leukemia cell growth.
M 1 , M2, and M3 blast samples contained < 1 % monocytes, as determined by morphology, cytochernistry, and immunofluorescence analysis using OKT3 (Ortho, Raritan, NJ) and MY4 MoABs, respectively. M4 and M5 leukemic blasts were morphologically free of matureappearing monocytes, as was judged by morphological inspection of Wright-stained cytospin preparations. The viability of cells as assessed with the trypan blue day exclusion always exceeded 95%. For experiments, highly purified, fresh leukemic blasts were resuspended at 0.5 X 10' cells/mL in culture medium consisting of Iscove's modified Dulbecco's medium (IMDM, Gibco, Grand Island, NY) supplemented M with L-glutamine (2 mmol/L), 2% FCS, and mercaptoethanol (Merck, Darmstadt, Germany). Proliferative responses of blasts were assayed by the tritiated thymidine ('H-TdR) incorporation assay. Differentiation of cultured cells was evaluated by morphological criteria. Cytokines and Neutralizing Monospecific Antibodies
Human recombinant IL- I a and p were generous gifts from Immunex Corporation (Seattle, WA). Each of these two cytokines has a specific activity of 3 X lo6 U/mg protein and is 99% pure. Human recombinant TNF-a was a generous gift from the Santory Institute for Biomedical Research (Osaka, Japan). This factor has a specific activity of 2 X lo6 U/mg and is 99% pure. Human recombinant GM-CSF (specific activity 9.3 X 19' U/mg, purity >95%) was generously supplied by the Genetics Institute MATERIALS AND METHODS (Cambridge, MA). Human recombinant IFN-2a was Patients and Separation of AML Cells kindly provided by Hoffmann La Roche (Basel, SwitzerPeripheral blood or bone marrow aspirate samples land). Monospecific antibodies. Polyclonal sheep anti-GMwere obtained at diagnosis, with informed consent, from 15 AML patients diagnosed according to the French- CSF was also a gift from the Genetics Institute. PolyAmerican-British (FAB) Committee [ 171 and immu- clonal rabbit anti-IL-la, anti-IL-lp, and anti-TNF-a nophenotyped. All samples contained more than 90% were purchased from Genzyme Corp. (Boston, MA). Cell cultures. DNA synthesis was measured by 'Hblasts (Table I). Blood or marrow samples were diluted in TdR uptake in freshly isolated target leukemia cells. The Hank's balanced salt solution (HBSS, Gibco Laboratoassay was performed in triplicate in flat-bottomed 96ries, Grand Island, NY), the cell suspension was sepawell culture plates (NUNC), using a total of 1 X 10' rated over Ficoll-Hypaque (
Effect of Interleukin-l, Tumor Necrosis Factor-a, and Intetferon-a on the Blast Cells of Acute Myeloblastic Leukemia Anna Carter, llana Silvian-Draxler, and llana Tatarsky Department of Hematology, The Bruce Rappaport Faculty of Medicine and Rappapport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology (A.C., IS.-D., I.T.); Rambam Medical Center (A.C., I.T.), Haifa, Israel
In this study, we further establishedthe role of interleukin-la (IL-la), interleukin-1p, tumor necrosis factor-a (TNF-a), and interferon-a (IFN-a) as regulators of proliferation of acute myeloid leukemia (AML) cells. AML cells from 8 of 15 patients incorporated high levels of 3H-thymidine ('H-TdR) in the absence of exogenous growth factors. The spontaneous DNA synthesis could be abrogated with monospecific antibodies directed toward IL-1a, IL-1p, or TNF-a, as well as with antigranulocyte-macrophage colony-stimulating factor (GM-CSF). Human recombinant GM-CSF reversed the inhibitory action of each of these antibodies and reinduced DNA synthesis in AML cells. Thus, in these cases, constitutively produced IL-1 or TNF-a had stimulated the synthesis of GM-CSF, which resulted in GMCSF-dependent proliferation of AML blasts. Exogenous IL-1 up-regulatedthe endogenous production of GM-CSF, suggesting a positive regulation of autocrine growth factor production. We also present evidence that TNF-a may exert both stimulative as well as inhibitory effects on DNA synthesis in AML cells. The enhancing effect of TNF-a was mediated through the induciion of GM-CSF production, as stimulation of DNA synthesis in AML blasts could be abrogated with anti-GM-CSF antibody. A concentration-dependent inhibitory effect of TNF-w on 3H-TdR incorporation into AML blasts was observed only when these cells were grown in the absence of GM-CSF. Finally, we show that human recombinant IFN-a is a potent inhibitor of AML cell proliferation in vitro. 4 1992 Wiley-Liss,
Inc.
Key words: leukemia, interleukin-1, tumor necrosis factor-a, interferon-a, proliferation, differentiation
INTRODUCTION Recently, it has been shown that the blast cells of acute myeloblastic leukemia (AML) proliferate in culture in the presence of exogenous growth factors such as interleukin-3 (IL-3), granulocyte macrophage (GM-CSF), and granulocyte colony-stimulating factor (G-CSF) [ 1-81. The growth regulatory cytokines such as interleukin- I (IL- 1 ) and tumor necrosis factor-a (TNF-a) are also capable of influencing the growth of AML cells in vitro. IL- 1 stimulates the proliferation of AML blasts through the induction of GM-CSF production [9,10]. TNF-a is a potent modulator of AML cell growth [ 1 I ] . It enhances the stimulative responses of AML blasts to GM-CSF [ 121 and effectively suppresses the clonogenic growth of leukemia cells and leukemia-derived cell lines [ 131. Recent data indicate that some AML blasts constitutively secrete IL- 1 , GM-CSF, and TNF-a and use these
0 1992 Wiley-Liss, Inc.
factors as autocrine growth stimulators [14-16]. In the present study, we further investigated how IL-I and TNF-a stimulate the proliferation of AML cells in vitro. We also studied the effects of human recombinant (rh) interferon-& (IFN-a) on the growth of AML blasts in culture. In this report, we show that a substantial proportion of primary human AML cells spontaneously produce IL-la, IL-Ip, and TNF-a and use these activities to stimulate their own growth through the induction of endogenous GM-CSF production. We also provide evidence that exogenous IL- l as well as TNF-a are capable
Received for publication May 8. 1991; accepted November 18, 1991. Address reprint requests to Dr. Anna Carter, Department of Heniatology, The Bruce Rappaport Faculty of Medicine. Technion-Israel Institute of Technology, P.O.B. 9649. 31096 Haifa, Israel.
246
Carter et al.
TABLE 1. AML Cells ~
Case No. 1
2 3 4 5 6 7 8 9 10
II 12 13 14 15
Age/Sex
20 F 56 M 32 M 45 F 64 F 44 F 17 M 31 M 12 M 42 F 51 M 48 M 66 F 45 M 64 M
FAB classificationa
Source of cellsb
MI MI MI MI MI M2 M2 M2 M3 M4 M4 M4 MS M5
BM PB BM BM PB BM BM BM BM BM BM PB BM BM PB
M5
~~
% blasts
99 100
99 97 95 98 93 99 92 97 93 98 100 93 98
"French- American-British classification. bBM, bone marrow, PB, peripheral blood.
of inducing GM-CSF synthesis in some AML cells, suggesting a positive regulation of autocrine growth factor production. Furthermore, co-stimulation with GM-CSF and IL- 1 or TNF-a may show strong synergism in eliciting leukemia cell growth. Finally, we demonstrate that TNF-a is a potent inhibitor of AML cell proliferation. This effect may indicate the applicability of this cytokine against human leukemia cell growth.
M 1 , M2, and M3 blast samples contained < 1 % monocytes, as determined by morphology, cytochernistry, and immunofluorescence analysis using OKT3 (Ortho, Raritan, NJ) and MY4 MoABs, respectively. M4 and M5 leukemic blasts were morphologically free of matureappearing monocytes, as was judged by morphological inspection of Wright-stained cytospin preparations. The viability of cells as assessed with the trypan blue day exclusion always exceeded 95%. For experiments, highly purified, fresh leukemic blasts were resuspended at 0.5 X 10' cells/mL in culture medium consisting of Iscove's modified Dulbecco's medium (IMDM, Gibco, Grand Island, NY) supplemented M with L-glutamine (2 mmol/L), 2% FCS, and mercaptoethanol (Merck, Darmstadt, Germany). Proliferative responses of blasts were assayed by the tritiated thymidine ('H-TdR) incorporation assay. Differentiation of cultured cells was evaluated by morphological criteria. Cytokines and Neutralizing Monospecific Antibodies
Human recombinant IL- I a and p were generous gifts from Immunex Corporation (Seattle, WA). Each of these two cytokines has a specific activity of 3 X lo6 U/mg protein and is 99% pure. Human recombinant TNF-a was a generous gift from the Santory Institute for Biomedical Research (Osaka, Japan). This factor has a specific activity of 2 X lo6 U/mg and is 99% pure. Human recombinant GM-CSF (specific activity 9.3 X 19' U/mg, purity >95%) was generously supplied by the Genetics Institute MATERIALS AND METHODS (Cambridge, MA). Human recombinant IFN-2a was Patients and Separation of AML Cells kindly provided by Hoffmann La Roche (Basel, SwitzerPeripheral blood or bone marrow aspirate samples land). Monospecific antibodies. Polyclonal sheep anti-GMwere obtained at diagnosis, with informed consent, from 15 AML patients diagnosed according to the French- CSF was also a gift from the Genetics Institute. PolyAmerican-British (FAB) Committee [ 171 and immu- clonal rabbit anti-IL-la, anti-IL-lp, and anti-TNF-a nophenotyped. All samples contained more than 90% were purchased from Genzyme Corp. (Boston, MA). Cell cultures. DNA synthesis was measured by 'Hblasts (Table I). Blood or marrow samples were diluted in TdR uptake in freshly isolated target leukemia cells. The Hank's balanced salt solution (HBSS, Gibco Laboratoassay was performed in triplicate in flat-bottomed 96ries, Grand Island, NY), the cell suspension was sepawell culture plates (NUNC), using a total of 1 X 10' rated over Ficoll-Hypaque (
