Disruption of Glut1 in Hematopoietic Stem Cells Prevents Myelopoiesis and Enhanced Glucose Flux in Atheromatous Plaques of ApoE(-/-) Mice.

RATIONALE: Inflamed atherosclerotic plaques can be visualized by noninvasive positron emission and computed tomographic imaging with (18)F-fluorodeoxyglucose, a glucose analog, but the underlying mechanisms are poorly understood. OBJECTIVE: Here, we directly investigated the role of Glut1-mediated glucose uptake in apolipoprotein E-deficient (ApoE(-/-)) mouse model of atherosclerosis. METHODS AND RESULTS: We first showed that the enhanced glycolytic flux in atheromatous plaques of ApoE(-/-) mice was associated with the enhanced metabolic activity of hematopoietic stem and multipotential progenitor cells and higher Glut1 expression in these cells. Mechanistically, the regulation of Glut1 in ApoE(-/-) hematopoietic stem and multipotential progenitor cells was not because of alterations in hypoxia-inducible factor 1α signaling or the oxygenation status of the bone marrow but was the consequence of the activation of the common ß subunit of the granulocyte-macrophage colony-stimulating factor/interleukin-3 receptor driving glycolytic substrate utilization by mitochondria. By transplanting bone marrow from WT, Glut1(+/-), ApoE(-/-), and ApoE(-/-)Glut1(+/-) mice into hypercholesterolemic ApoE-deficient mice, we found that Glut1 deficiency reversed ApoE(-/-) hematopoietic stem and multipotential progenitor cell proliferation and expansion, which prevented the myelopoiesis and accelerated atherosclerosis of ApoE(-/-) mice transplanted with ApoE(-/-) bone marrow and resulted in reduced glucose uptake in the spleen and aortic arch of these mice. CONCLUSIONS: We identified that Glut1 connects the enhanced glucose uptake in atheromatous plaques of ApoE(-/-) mice with their myelopoiesis through regulation of hematopoietic stem and multipotential progenitor cell maintenance and myelomonocytic fate and suggests Glut1 as potential drug target for atherosclerosis.

Targeted diphtheria toxin to treat BPDCN.

In this issue of Blood, Frankel et al describe a novel treatment of blastic plasmacytoid dendritic cell neoplasm (BPDCN) using an engineered version of diphtheria toxin that is targeted to malignant cells via a fusion with interleukin (IL)3 (see panel A).

Activity of SL-401, a targeted therapy directed to interleukin-3 receptor, in blastic plasmacytoid dendritic cell neoplasm patients.

This is the first prospective study of treatment of patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN), an aggressive hematologic malignancy derived from plasmacytoid dendritic cells that typically involves the skin and rapidly progresses to a leukemia phase. Despite being initially responsive to intensive combination chemotherapy, most patients relapse and succumb to their disease. Because BPDCN blasts overexpress the interleukin-3 receptor (IL3R), the activity of SL-401, diptheria toxin (DT)388IL3 composed of the catalytic and translocation domains of DT fused to IL3, was evaluated in BPDCN patients in a phase 1-2 study. Eleven patients were treated with a single course of SL-401 at 12.5 µg/kg intravenously over 15 minutes daily for up to 5 doses; 3 patients who had initial responses to SL-401 received a second course in relapse. The most common adverse events including fever, chills, hypotension, edema, hypoalbuminemia, thrombocytopenia, and transaminasemia were transient. Seven of 9 evaluable (78%) BPDCN patients had major responses including 5 complete responses and 2 partial responses after a single course of SL-401. The median duration of responses was 5 months (range, 1-20+ months). Further studies of SL-401 in BPDCN including those involving multiple sequential courses, alternate schedules, and combinations with other therapeutics are warranted. This trial is registered at clinicaltrials.gov as #NCT00397579.

Autocrine TNF-α production supports CML stem and progenitor cell survival and enhances their proliferation.

Chronic myeloid leukemia (CML) stem cells are not dependent on BCR-ABL kinase for their survival, suggesting that kinase-independent mechanisms must contribute to their persistence. We observed that CML stem/progenitor cells (SPCs) produce tumor necrosis factor-α (TNF-α) in a kinase-independent fashion and at higher levels relative to their normal counterparts. We therefore investigated the role of TNF-α and found that it supports survival of CML SPCs by promoting nuclear factor κB/p65 pathway activity and expression of the interleukin 3 and granulocyte/macrophage-colony stimulating factor common ß-chain receptor. Furthermore, we demonstrate that in CML SPCs, inhibition of autocrine TNF-α signaling via a small-molecule TNF-α inhibitor induces apoptosis. Moreover TNF-α inhibition combined with nilotinib induces significantly more apoptosis relative to either treatment alone and a reduction in the absolute number of primitive quiescent CML stem cells. These results highlight a novel survival mechanism of CML SPCs and suggest a new putative therapeutic target for their eradication.

The inhibitor of interleukin-3 receptor protects against sepsis in a rat model of cecal ligation and puncture.

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. There are multiple cytokines involved in the process of sepsis. As an important upstream cytokine in inflammation, Interleukin-3 (IL-3) plays a crucial role during sepsis, however, its exact role is unclear. The purpose of this study is to discuss the role of IL-3 and its receptor in cecal ligation and puncture (CLP)-induced sepsis in a rat model. The Cluster of Differentiation 123 (CD123, IL-3 receptor alpha chain, IL-3Rac) antibody (anti-CD123) was used to directly target IL-3's receptor and alleviate the effect of IL-3 in the CLP + anti-CD123 group during the early stage of sepsis. CLP was performed in the CLP and CLP + anti-CD123 groups. The time points of observation included 12 h, 24 h, and 5d after the operation. The results showed that the rats in the CLP + anti-CD123 group had lower levels of lactate, serum tumor necrosis factor-α (TNF-α), Interleukin-1ß (IL-1ß), and Interleukin-6 (IL-6), and also exhibited a higher core temperature, mean arterial pressure (MAP), Oxygenation Index (PO2/FiO2), and end-tidal carbon dioxide (ETCO2) and serum Interleukin-10 (IL-10) levels after CLP than those in the CLP group. Additionally, administration of anti-CD123 led to a stable down-regulation of tyrosine phosphorylation of the IL-3 receptor, a decline in phosphorylation of the Janus kinase 2 (JAK2) protein, and the signal transduction and activation of transcription 5 (STAT5) proteins in lung tissues. Meanwhile, the study revealed that treatment of anti-CD123 can markedly attenuate histological damages in lung and kidney tissues, improve sublingual microcirculation, and prolong survival post sepsis. In conclusion, anti-CD123 reduces mortality and alleviates organ dysfunction by restraining the JAK2-STAT5 signaling pathway and reduces serum cytokines in the development of early sepsis in a rat model induced by CLP.

TGFbeta1 induces mast cell apoptosis.

Mast cells are potent effectors of the inflammatory response, playing an important role in atopy, bacterial immunity, and animal models of arthritis, multiple sclerosis, and heart disease. Hence controlling mast cell numbers and responsiveness is essential for preventing inflammatory disease. We demonstrate that the cytokine transforming growth factor (TGF) beta1 is a potent inducer of mast cell apoptosis, a finding that was consistent in cultured mouse bone marrow-derived mast cells, peritoneal mast cells, and human mast cells. Cell death appeared to be caused by TGF-mediated repression of interleukin-3 (IL-3) receptor expression and function, leading to mitochondrial damage and activation of an apoptotic cascade acting via p53 and caspases. Although IL-3 receptor expression was reduced within 1 day of TGFbeta1 stimulation, apoptosis required at least 3 days to occur. This delay in onset is postulated to allow protective mast cell effector functions, protecting the host from infection while preventing the establishment of chronic inflammation. Our data support the theory that TGFbeta1 is an inhibitor of mast cell survival. The widespread expression of TGFbeta1 offers this cytokine as an ideal candidate for control of mast cell homeostasis.

Multitargeted approach using antisense oligonucleotides for the treatment of asthma.

Asthma is characterized by inflammation and hyperresponsiveness related to the accumulation of inflammatory cells, particularly eosinophils, within the airways. We tested the hypothesis that a multitargeted approach is better than a single-targeted approach in a rat model of asthma. We simultaneously delivered oligonucleotides (ODNs) targeting the chemokine receptor CCR3 and the common beta chain subunit of the receptors for IL-3, IL-5, and GM-CSF at the time of ovalbumin challenge in sensitized Brown Norway rats. Fewer eosinophils were detected in bronchoalveolar lavage (BAL) of rats treated with both ODNs as compared to each ODN alone. Moreover, airway responsiveness to LTD(4) was significantly decreased at lower doses in the 2 ODN-treated groups compared to a single ODN. As ODN therapy has raised concerns of toxicity we therefore examined ODNs prepared with modified DNA bases, specifically 2'amino, 2'deoxyadenosine (DAP) in place of adenosine. In vivo, administration of individual DAP-ODN was efficacious in inhibiting airway hyperresponsiveness, whereas delivery of 2 DAP-ODNs (targeting CCR3 and common beta chain) reduced the influx not only of eosinophils but also lymphocytes and macrophages in the lungs of rats as compared to the unmodified ODNs. Blocking multiple inflammatory pathways simultaneously is more effective in preventing eosinophilia and airway hyperresponsiveness than inhibiting either pathway alone. The challenges associated with the development of a product containing two oligonucleotides in humans are discussed.

Receptor cross-linking on human plasmacytoid dendritic cells leads to the regulation of IFN-alpha production.

Plasmacytoid dendritic cells (PDC) are the natural type I IFN-producing cells that produce large amounts of IFN-alpha in response to viral stimulation. During attempts to isolate PDC from human PBMC, we observed that cross-linking a variety of cell surface receptors, including blood DC Ag (BDCA)-2, BDCA-4, CD4, or CD123 with Abs and immunobeads on PDC leads to inhibition of IFN-alpha production in response to HSV. To understand the mechanisms involved, a number of parameters were investigated. Cross-linking did not inhibit endocytosis of soluble Ag by PDC. Flow cytometry for annexin V and activated caspase-3 indicated that PDC are not undergoing apoptosis after receptor cross-linking. Cross-linking of CD123, but not the other receptors, caused the up-regulation of costimulatory molecules CD80 and CD86, as well as the down-regulation of CD62L, indicating PDC maturation. Thus, anti-CD123 Ab may be acting similar to the natural ligand, IL-3. Anti-phosphotyrosine Ab, as well as Ab to the IFN regulatory factor, IRF-7, was used in intracellular flow cytometry to elucidate the signaling pathways involved. Tyrosine phosphorylation occurred after cross-linking BDCA-2 and BDCA-4, but not CD4. Cross-linking did not affect IRF-7 levels in PDC, however, cross-linking BDCA-2, BDCA-4, and CD4, but not CD123, inhibited the ability of IRF-7 to translocate to the nucleus. Taken together, these results suggest that cross-linking BDCA-2, BDCA-4, and CD4 on PDC regulates IFN-alpha production at the level of IRF-7, while the decrease in IFN-alpha production after CD123 cross-linking is due to stimulation of the IL-3R and induction of PDC maturation.

Regulation of interleukin 3 receptor alpha chain (IL-3R alpha) on human monocytes by interleukin (IL)-4, IL-10, IL-13, and transforming growth factor beta (TGF-beta).

Human interleukin 3 receptor (IL-3R) is constitutively expressed on committed haematopoietic stem cells, where it mediates proliferation and differentiation. This receptor is also expressed by monocytes and may induce functional activation. Interleukin (IL)-4, IL-10, IL-13, and transforming growth factor beta (TGF-beta) have different effects on human monocytes. As IL-3R may be regulated by different cytokines, whether the above-mentioned cytokines were able to modulate the alpha chain of IL-3R (IL-3R alpha) on monocytes was examined. Effects on IL-3R alpha antigen (Ag) expression were analysed by direct immunofluorescence and flow cytometry. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect variations in IL-3R alpha mRNA expression. IL-10 and TGF-beta were found to downregulate IL-3R alpha Ag. In contrast, IL-4 and IL-13 both caused a dose- and time-dependent increase. A maximum effect was observed at 1 ng/ml of IL-4 for 18 h. Furthermore, in RT-PCR, IL-4 was found to slightly up-regulate IL-3R alpha mRNA expression. These observations suggest that IL-4 and IL-13 play a role in the regulation of IL-3R alpha expression and the effects of cytokines on human monocytes may be mediated, in part, through the regulation of IL-3R.

Structure of the activation domain of the GM-CSF/IL-3/IL-5 receptor common beta-chain bound to an antagonist.

Heterodimeric cytokine receptors generally consist of a major cytokine-binding subunit and a signaling subunit. The latter can transduce signals by more than 1 cytokine, as exemplified by the granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and IL-6 receptor systems. However, often the signaling subunits in isolation are unable to bind cytokines, a fact that has made it more difficult to obtain structural definition of their ligand-binding sites. This report details the crystal structure of the ligand-binding domain of the GM-CSF/IL-3/IL-5 receptor beta-chain (beta(c)) signaling subunit in complex with the Fab fragment of the antagonistic monoclonal antibody, BION-1. This is the first single antagonist of all 3 known eosinophil-producing cytokines, and it is therefore capable of regulating eosinophil-related diseases such as asthma. The structure reveals a fibronectin type III domain, and the antagonist-binding site involves major contributions from the loop between the B and C strands and overlaps the cytokine-binding site. Furthermore, tyrosine(421) (Tyr(421)), a key residue involved in receptor activation, lies in the neighboring loop between the F and G strands, although it is not immediately adjacent to the cytokine-binding residues in the B-C loop. Interestingly, functional experiments using receptors mutated across these loops demonstrate that they are cooperatively involved in full receptor activation. The experiments, however, reveal subtle differences between the B-C loop and Tyr(421), which is suggestive of distinct functional roles. The elucidation of the structure of the ligand-binding domain of beta(c) also suggests how different cytokines recognize a single receptor subunit, which may have implications for homologous receptor systems. (Blood. 2000;95:2491-2498)

Combination therapy for radiation-induced bone marrow aplasia in nonhuman primates using synthokine SC-55494 and recombinant human granulocyte colony-stimulating factor.

Combination cytokine therapy continues to be evaluated in an effort to stimulate multilineage hematopoietic reconstitution after bone marrow myelosuppression. This study evaluated the efficacy of combination therapy with the synthetic interleukin-3 receptor agonist, Synthokine-SC55494, and recombinant methionyl human granulocyte colony-stimulating factor (rhG-CSF) on platelet and neutrophil recovery in nonhuman primates exposed to total body 700 cGy 60Co gamma radiation. After irradiation on day (d) 0, cohorts of animals subcutaneously received single-agent protocols of either human serum albumin (HSA; every day [QD], 15 micrograms/kg/d, n = 10), Synthokine (twice daily [BID], 100, micrograms/kg/d, n = 15), rhG-CSF (QD, 10 micrograms/kg/d, n = 5), or a combination of Synthokine and rhG-CSF (BID, 100 and 10 micrograms/kg/d, respectively, n = 5) for 23 days beginning on d1. Complete blood counts were monitored for 60 days postirradiation and the durations of neutropenia (absolute neutrophil count < 500/microL) and thrombocytopenia (platelet count < 20,000/microL) were assessed. Animals were provided clinical support in the form of antibiotics, fresh irradiated whole blood, and fluids. All cytokine protocols significantly (P < .05) reduced the duration thrombocytopenia versus the HSA-treated animals. Only the combination protocol of Synthokine + rhG-CSF and rhG-CSF alone significantly shortened the period neutropenia (P < .05). The combined Synthokine/rhG-CSF protocol significantly improved platelet nadir versus Synthokine alone and HSA controls and neutrophil nadir versus rhG-CSF alone and HSA controls. All cytokine protocols decreased the time to recovery to preirradiation neutrophil and platelet values. The Synthokine/rhG-CSF protocol also reduced the transfusion requirements per treatment group to 0 among 5 animals as compared with 2 among 5 animals for Synthokine alone, 8 among 5 animals for rhG-CSF, and 17 among 10 animals for HSA. These data showed that the combination of Synthokine, SC-55494, and rhG-CSF further decreased the cytopenic periods and nadirs for both platelets and neutrophils relative to Synthokine and rhG-CSF monotherapy and suggest that this combination therapy would be effective against both neutropenia and thrombocytopenia consequent to drug- or radiation- induced myelosuppression.

Characterization of potential antagonists of human interleukin 5 demonstrates their cross-reactivity with receptors for interleukin 3 and granulocyte-macrophage colony-stimulating factor.

The ligand-binding alpha-chain of the human interleukin 5 (IL-5) receptor was expressed in its soluble form, lacking the transmembrane and cytoplasmic domains, from recombinant baculovirus. The soluble receptor was used in a scintillation proximity assay to identify two chemical compounds that inhibit binding of human IL-5 to the soluble receptor alpha chain with IC50 of 8 microM and 11 microM. These compounds also inhibited the interaction of human IL-5 with its membrane-bound receptor, composed of the ligand-binding alpha chain and signal-transducing beta chain, and prevented signaling through the receptor. Analysis by surface plasmon resonance and matrix-assisted laser-desorption/ionization mass spectrometry showed that the identified compounds bound irreversibly to the receptor at a 1:1 (mol/mol) ratio, suggesting a covalent interaction with the alpha chain of the human IL-5 receptor. Both compounds also inhibited the interaction of the receptors for interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF), which are involved in hematopoietic differentiation and activation of immune cells, thus eliminating them as potential therapeutic agents. The inhibition of the structurally closely related receptors for IL-5, IL-3 and GM-CSF by both compounds, while binding of interleukin-4 to its receptor was not affected, suggests that a similar reactive site exists in the ligand-binding domains of the receptors for IL-5, IL-3 and GM-CSF.

Monoclonal antibody 7G3 recognizes the N-terminal domain of the human interleukin-3 (IL-3) receptor alpha-chain and functions as a specific IL-3 receptor antagonist.

The human interleukin-3 receptor (IL-3R) is expressed on myeloid, lymphoid, and vascular endothelial cells, where it transduces IL-3-dependent signals leading to cell activation. Although IL-3R activation may play a role in hematopoiesis and immunity, its aberrant expression or excessive stimulation may contribute to pathologic conditions such as leukemia, lymphoma, and allergic reactions. We describe here the generation and characterization of a monoclonal antibody (MoAb), 7G3, which specifically binds to the IL-3R alpha-chain and completely abolishes its function. MoAb 7G3 immunoprecipitated and recognized in Western blots the IL-3R alpha-chain expressed by transfected cells and bound to primary cells expressing IL-3R alpha. MoAb 7G3 bound the IL-3R alpha-chain with a kd of 900 pmol/L and inhibited 125I-IL-3 binding to high- and low-affinity receptors in a dose-dependent manner. Conversely, IL-3 but not granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibited 125I-7G3 binding to high- and low-affinity IL-3Rs, indicating that MoAb 7G3 and IL-3 bind to common or adjacent sites. In keeping with the inhibition of IL-3 binding, MoAb 7G3 antagonized IL-3 biologic activities, namely stimulation of TF-1 cell proliferation, basophil histamine release, and IL-6 and IL-8 secretion from human endothelial cells. Two other anti-IL-3R alpha-chain MoAbs failed to inhibit IL-3 binding or function. Epitope mapping experiments using truncated IL-3R alpha-chain mutants and IL-3R alpha/GM-CSFR alpha chimeras revealed that 31 amino acids in the N-terminus of IL-3R alpha were required for MoAb 7G3 binding. MoAb 7G3 may be of clinical significance for antagonizing IL-3 in pathologic conditions such as some myeloid leukemias, follicular B-cell lymphoma, and allergy. Furthermore, these results implicate the N-terminal domain of IL-3R alpha in IL-3 binding. Since this domain is unique to the IL-3/GM-CSF/IL-5 receptor subfamily, it may represent a novel and common binding feature in these receptors.