Direct Signal Transduction Via Functional Interferon- Receptors In
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Leukemia (2002) 16, 1135–1142 2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu Direct signal transduction via functional interferon-␣ receptors in CD34+ hematopoietic stem cells J Giron-Michel3, D Weill1, G Bailly1, S Legras2, PC Nardeux1, B Azzarone3, MG Tovey1 and P Eid1 1Laboratoire d’Oncologie Virale, UPR 9045, CNRS, Villejuif, France; 2Laboratoire d’He´matologie, Hoˆpital Paul Brousse, Villejuif, France; 3Inserm U506, Hoˆpital Paul Brousse, Villejuif, France + Affinity purified, freshly isolated CD34 progenitors were host defense against virus infection and neoplastic disease.4–6 shown to express low levels of type I interferon (IFN) receptors ␣ ± ± Recombinant IFN- has found wide application in clinical (740 60 binding sites/cell, Kd 0.7 0.04 nM) determined by Scatchard’s analysis using a radiolabelled, neutralizing, mono- medicine for the treatment of chronic viral hepatitis, solid clonal antibody directed against the IFNAR1 chain of the tumors, and a variety of hematological malignancies including human type I IFN receptor. Treatment of freshly isolated (day hairy cell leukemia and chronic myelogenous leukemia + 0), highly purified (Ͼ95% pure) CD34 cells with recombinant (CML).7–9 The mechanism(s) of the anti-tumor effects of IFN- ␣ IFN- resulted in rapid tyrosine phosphorylation and activation ␣ remain poorly understood, although it is well established of STAT1, Tyk2 and JAK1 as shown by Western immunoblot- ␣ ting. Similarly, IFN treatment was shown by confocal that IFN- exerts both direct anti-proliferative and pro-apop- microscopy to result in rapid nuclear localization of the tran- totic effects, and indirect immune-mediated effects on tumor scription factors IRF1 and STAT2, demonstrating the presence cells 10.10,11 In the case of hematological malignancies such + of functional IFN receptors on freshly isolated (day 0) CD34 as CML, IFN-␣ has been reported to inhibit the proliferation cells. The number of specific type I IFN receptor binding sites of both normal and leukemic progenitor cells.12–18 It remains expressed on hematopoietic progenitor cells increased to ␣ some 1440 ± 40 per cell after 11 days of cultivation of CD34+ unclear, however, whether IFN- exerts a direct effect on the cells in vitro suggesting that receptor expression increases proliferation of hematopoietic stem cells, and their immediate with cell differentiation. IFN-mediated signal transduction and progeny committed progenitor cells, or an indirect effect on the inhibitory effect of IFN-␣ on 7 or 14 days CFU-GM and BFU- stromal cells in the bone marrow, and the synthesis of growth E colony formation was abrogated in the presence of the ␣ factors and other cytokines required for stem cell prolifer- anti-IFNAR1 mAb, indicating that IFN- acts directly on the pro- 19–22 liferation of human hematopoietic progenitor cells via receptor ation. activated signal transduction without excluding the induction Interferons exert their characteristic biological effects by of other cytokines or growth factors by residual accessory binding to high-affinity receptors on the cell surface resulting cells. in the rapid tyrosine phosphorylation and activation of signal Leukemia (2002) 16, 1135–1142. DOI: 10.1038/sj/leu/2402492 transducers and activators of transcription (STATs) which regu- Keywords: interferon receptors; CD34+ cells; anti-IFNAR1 late the transcription of interferon-responsive genes. Thus, binding of IFN-␣ to its cell surface receptor activates the receptor-associated Janus tyrosine kinases Tyk2 and JAK123,24 Introduction leading to phosphorylation of the two transmembrane poly- + peptides, IFNAR1 and IFNAR2, which constitute the type I IFN Lymphohematopoietic CD34 stem cells and progenitors are receptor. Both chains of the receptor are members of the cyto- able to proliferate and differentiate into multiple lymphoid lin- kine receptor super family and are both required for high- eages, and understanding the mechanisms which control this process remains one of the central questions of modern day affinity ligand binding and the establishment of biological biology. Biochemical studies on primitive primary hemopo- activity. Tyk2 and JAK1 then phosphorylate STAT1 and ietic cells have been hindered by the difficulty in obtaining STAT2, leading to the formation of a heterodimer which is sufficient quantities of highly purified CD34+ cells. Further- translocated to the nucleus where it associates with a p48 more, analysis of mRNAexpression levels using techniques DNAbinding protein, a member of the IRF family of transcrip- 25–27 such as the polymerase chain reaction (PCR) does not provide tion factors (IRF9), leading to the activation of the tran- information on protein functionality. Thus, we have adopted scription of those genes which contain an IFN-sensitive an alternative approach based on the use of direct binding response element (ISRE) within their promoter region. It is the studies (Scatchard analysis), which provides information on activation of a specific set of IFN-responsive genes within a the level of translation of a receptor product and the level of given cell type which gives rise to the establishment of the its expression at the cell surface, coupled with an analysis of characteristic biological activities of the interferons. receptor signaling leading to the transcriptional activation of We show herein, using Scatchard analysis and a highly spe- effector genes. cific radiolabeled monoclonal antibody directed against the The proliferation and differentiation of hematopoietic stem IFNAR1 chain of the type I IFN receptor, which inhibits bind- cells is regulated by a complex network of cytokines and ing to both receptor chains and which neutralizes the biologi- growth factors,1–3 including the type I interferons (IFN-␣, IFN- cal activity of all the type I interferons,28,29 that freshly isolated , IFN-, and IFN-) which are multifunctional cytokines that highly purified CD34+ cells (Ͼ95%) from human umbilical play an important role in the innate immune response and in cord blood, possess low levels of functional IFN receptors, the number of which increase during differentiation of myeloid progenitors in culture. These results suggest that IFN-␣ can Correspondence: P Eid, Laboratoire d’Oncologie Virale, CNRS-UPR exert direct effects on the proliferation of early myeloid 9045, 7, rue Guy Moquet-94801 Villejuif, France; Fax: 33-1 49 58 progenitors. 34 44 Received 10 September 2001; accepted 29 January 2002 Type I interferon receptors in CD34+ cells J Giron-Michel et al 1136 Materials and methods against purified human leukocyte IFN-␣ has been described previously.31 The preparation used in this study had a neu- Isolation of human CD34+ stem cells by affinity tralizing titer of 2 × 105 against 8 IU of recombinant IFN-␣2a. chromatography or by magnetic cell sorting Normal sheep serum was used as a control. (MiniMacs) Human umbilical cord blood (CB) was collected from full- Colony-forming cell assay term newborns following informed consent of the mother at the Hospital St Vincent de Paul (Paris, France). Cord blood CD34+ cells were seeded in StemGem medium mononuclear cells (CB-MNC) were isolated by density (1.077 (hatzfeldȰvjf.cnrs.fr; CNRS, Villejuif, France). The medium g/ml) gradient centrifugation (2000 rpm, 20 min) using Ficoll– was composed of 0.9% methylcellulose supplemented with Hypaque (Seromed, Berlin, Germany). After centrifugation 30% fetal calf serum (Dominique Deutsher, Brumath, France), CB-MNC present at the interface were harvested and washed 10 mg/ml transferrin, 1 mg/ml detoxified serum albumin + twice with phosphate-buffered saline (PBS). CD34 cells were (Sigma Chemical, St Louis, MO, USA), 2 mM glutamine (Life + − separated using the Ceprate LC CD34 kit (CellPro, Bothell, Technologies, Cergy-Pontoise, France), 5 × 10 5 M 2-mercap- WA, USA) to yield CD34+ cells with a purity ranging from 65 toethanol (Merck, Darmstadt, Germany), 1.7 units/ml of to 85%. For magnetic cell sorting, the procedure was perfor- recombinant human IL3, 10 units/ml of recombinant human med according to the manufacturer’s instructions. Blocking IL-6, 3 ng/ml of recombinant human SCF (all obtained from reagent and the antibody QBEND/10 from the direct CD34+ R&D Systems), and 18.3 units/ml of recombinant human GM- progenitor cell isolation kit (MACS; Miltenyi Biotec, Paris, CSF (Genetic Institute, Cambridge, MA, USA), 2 units/ml of France) were added simultaneously to the cell suspension, recombinant human EPO (Boehringer, Mannheim, Germany), mixed well and incubated for 30 min at 4°C on a rotating Approximately 400 enriched CD34+ cells were re-suspended wheel. After incubation, cells were washed with PBE in 0.4 ml Iscove’s modified Dulbecco’s medium (IMDM) sup- (phosphate-buffered saline (PBS), 0.5% bovine serum albumin plemented with 10% FCS and mixed with 2 ml of StemGem (BSA), 2 mM EDTA). The cells were re-suspended in 1 ml PBE methylcellulose and 1 ml of this mixture was plated in 35- and applied to the pre-filled column which was placed in a mm Petri dishes. Cultures were incubated for 21 days in 5% magnetic field. Cells were centrifuged (1500 r.p.m., 10 min, CO2 in air and 100% humidity. Each culture was plated in 4°C) and re-suspended with PB buffer (PBS, 0.5% BSA). Only triplicate. The number of colonies containing more than 50 CD34+ progenitor cells were specifically retained by the col- cells was evaluated on days 7 and 14 with an inverted micro- umn matrix and subsequently eluted by removing the column scope. CFU-GM and BFU-E colonies were determined after from the magnetic field and flushing with PB. Using this iso- 14 days of incubation. lation procedure, the purity of CD34+ cells was routinely greater than 95%. The purity of CD34+ cells was determined by incubating cells for 15 minutes at 4°C with PerCP-labeled Liquid suspension culture of day 11 progenitor cells anti-CD34+ antibody (HPCA-2, clone 8G12, Becton Dickin- son, San Jose, CA, USA). Background staining was determined Purified CD34+ cells were seeded at a concentration of 103/ml using an irrelevant control PE-IgG1 (Coultronics, Margency, in StemGem liquid medium and incubated at 37°C with 5% France), or Per-IgG1 (Becton Dickinson) for MACS.