BackgroundOur original demonstration of immunomodulatory effects of erythropoietin in multiple myeloma led us to the search for the cells in the immune system that are direct targets for erythropoietin. The finding that lymphocytes do not express erythropoietin receptors led to the hypothesis that other cells act as direct targets and thus mediate the effects of erythropoietin. The finding that erythropoietin has effects on dendritic cells thus led to the question of whether macrophages act as target cells for erythropoietin. Design and MethodsThe effects of erythropoietin on macrophages were investigated both in-vivo and in-vitro. The in-vivo studies were performed on splenic macrophages and inflammatory peritoneal macrophages, comparing recombinant human erythropoietin-treated and untreated mice, as well as transgenic mice over-expressing human erythropoietin (tg6) and their control wild-type counterparts. The in-vitro effects of erythropoietin on macrophage surface markers and function were investigated in murine bone marrow-derived macrophages treated with recombinant human erythropoietin. ResultsErythropoietin was found to have effects on macrophages in both the in-vivo and in-vitro experiments. In-vivo treatment led to increased numbers of splenic macrophages, and of the splenic macrophages expressing CD11b, CD80 and major histocompatibility complex class II. The peritoneal inflammatory macrophages obtained from erythropoietin-treated mice displayed increased expression of F4/80, CD11b, CD80 and major histocompatibility complex class II, and augmented phagocytic activity. The macrophages derived in-vitro from bone marrow cells expressed erythropoietin receptor transcripts, and in-vitro stimulation with erythropoietin activated multiple signaling pathways, including signal transducer and activator of transcription (STAT)1 and 5, mitogen-activated protein kinase, phosphatidylinositol 3-kinase and nuclear factor kappa B. In-vitro erythropoietin treatment of these cells up-regulated their surface expression of CD11b, F4/80 and CD80, enhanced their phagocytic activity and nitric oxide secretion, and also led to augmented interleukin 12 secretion and decreased interleukin 10 secretion in response to lipopolysaccharide. ConclusionsOur results show that macrophages are direct targets of erythropoietin and that erythropoietin treatment enhances the pro-inflammatory activity and function of these cells. These findings point to a multifunctional role of erythropoietin and its potential clinical applications as an immunomodulating agent.Key words: erythropoietin receptor, pro-inflammation, macrophages, signal transducer and activator of transcription (STAT).Citation: Lifshitz L, Tabak G, Gassmann M, Mittelman M, and Neumann D. Macrophages as novel target cells for erythropoietin. Haematologica 2010;95(11):1823-1831. doi:10.3324/haematol.2010 This is an open-access paper. Macrophages as novel target cells for erythropoietin

Erythropoietin (EPO) regulates proliferation and differentiation of erythroid precursor cells into erythrocytes. The last decade has revealed non-renal sites of EPO production and extrahematopoietic expression of the EPO receptor, thus suggesting that EPO has pleiotropic functions. Here, we addressed the interplay between EPO/glucose metabolism/ body weight by employing a panel of relevant experimental murine models. The models focused on situations of increased EPO levels, including EPO-injected C57BL/6 and BALB/c mice, as well as transgenic mice (tg6) constitutively overexpressing human EPO, thus exposed to constantly high EPO serum levels. As experimental models for diabetes and obesity, we employed protein Tyr phosphatase 1B (PTP1B) knockout mice associated with resistance to diabetes (PTP1B K/K ), and ob/ob mice susceptible to diabetes and obesity. The data presented herein demonstrate EPOmediated decrease in blood glucose levels in all mice models tested. Moreover, in the ob/ob mice, we observed EPOmediated attenuation of body weight gain and reduction of hemoglobin A1c. Taken together, our data bear significant clinical implications of EPO treatment in the management of a wide range of metabolic diseases, thus adding an important novel therapeutic potential to this pleiotropic hormone.

Erythropoietin (EPO) is the main hormone that promotes proliferation and differentiation of erythroid progenitor cells via binding to its surface receptor (EPO-R). Recent studies suggest that this hormone may affect also other cell types, besides the red blood cell lineage. We have previously demonstrated that the immune system is a target of EPO; however, the direct target cells of EPO, as well as the molecular mechanisms underlying its role as an immunomodulator, are unknown. Here we present evidence for functional effects of EPO on dendritic cells (DCs), which are known to initiate the immune response. In-vivo experiments in EPO-injected mice and in transgenic mice over-expressing human EPO showed an increased splenic DC population with a higher cell surface expression of CD80 and CD86. Further analysis based on mouse models, showed that DCs derived in-vitro from bone marrow (BM-DCs) express EPO-R mRNA. In-vitro stimulation of these DCs with recombinant human EPO enhanced viability, upregulated CD80, CD86 and MHC class II and augmented the secretion of IL-12. Biochemical analysis of EPO mediated signaling in the BM-DCs showed activation of the AKT, MAPK and NF-kappaB pathways. EPO stimulation of the BM-DCs led to Tyr-phosphorylation of STAT3. The inability to detect EPO mediated activation of STAT5 in the BM-DCs, suggests that in DCs, STAT3 may play a more important role than STAT5 in EPO-R signaling. Taken together, our data support the premise that DCs are direct targets of EPO, thereby providing an insight to the immunomodulatory functions of EPO.

Erythropoietin (Epo) is the main erythropoietic hormone. Recombinant human Epo (rHuEpo) is thus used in clinical practice for the treatment of anemia. Accumulating data reveals that Epo exerts pleiotropic activities. We have previously shown an antineoplastic activity of Epo in murine multiple myeloma (MM) models, and in MM patients. Our findings that this anti-neoplastic effect operates via CD8 + T lymphocytes led us to hypothesize that Epo possesses a wider range of immunomodulatory functions. Here we demonstrate the effect of Epo on B lymphocyte responses, focusing on three experimental models: (i) tumor-bearing mice, (5T2 MM mouse); (ii) antigen-injected healthy mice; and (iii) antigen-injected transgenic mice (tg6), overexpressing human Epo. In the MM model, despite bone marrow dysfunction, Epo-treated mice retained higher levels of endogenous polyclonal immunoglobulins, compared to their untreated controls. In both Epo-treated wild type and tg6 mice, Epo effect was manifested in the higher levels of splenocyte proliferative response induced in vitro by lipopolysaccharide. Furthermore, these mice had increased in vivo production of anti-dinitrophenyl (DNP) antibodies following immunization with DNP-keyhole limpet hemocyanin. Epo-treated mice showed an enhanced immune response also to the clinically relevant hepatitis B surface antigen. These findings suggest a potential novel use of rHuEpo as an immunomodulator.See accompanying commentary: http://dx.doi. org/10.1002/eji.200737401 Introduction Erythropoietin (Epo), produced mainly in the adult kidney, is the major growth regulator of the erythroid cell lineage. Cloning of the Epo gene has led to the introduction of recombinant human Epo (rHuEpo) into clinical practice as a treatment for various anemias, including anemia related to chronic kidney disease and certain forms of cancer [1, 2]. Detection of the target receptor for Epo (EpoR) in cells other than erythroid progenitors, such as polymorphonuclear leukocytes, megakaryocytes, endothelial, myocardial and neural cells [3][4][5][6][7], suggests that Epo has other biological functions beyond erythropoiesis, and may have further potential therapeutic applications. These effects include improvement in congestive heart failure [8,9] and neuroprotection [10][11][12][13][14][15][16][17].* These authors contributed equally to this study. The idea that rHuEpo may have an important effect on the immune system, including both cellular and humoral type responses, derives from several lines of data, as recently reviewed [18]. For instance, clinical observations revealed that treatment with rHuEpo was associated with enhanced antibody production in hemodialysis patients, demonstrated for T cell-dependent antigens, such as tetanus toxoid and hepatitis B [19][20][21][22] as well as for the T cell-independent pneumococcal polysaccharide antigen [20]. Others have demonstrated enhancement of basal and mitogen-stimulated immunoglobulin production by cultured peripheral mononuclear cells (MNC) of dialysis patients treated ...

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4557 Erythropoietin (EPO) regulates proliferation and differentiation of erythroid precursor cells into erythrocytes. The last decade has revealed non-renal sites of EPO production and extrahematopoietic expression of the EPO-receptor (EPO-R), suggesting that EPO has pleiotropic functions. We have previously shown anti-cancer and immunomodulating effects of the hormone in humans and mice. Here we addressed the interplay among EPO, glucose metabolism, and body weight by employing a panel of relevant experimental murine models. The models focused on situations of increased EPO levels, including EPO-injected C57BL/6 and BALB/c mice, as well as transgenic mice (tg6) constitutively overexpressing human EPO and thus exposed to constantly high EPO serum levels. As experimental models for diabetes and obesity we employed protein Tyr phosphatase 1B (PTP1B) knockout mice associated with resistance to diabetes (PTP1B-/-), and ob/ob mice susceptible to diabetes and obesity. Our data demonstrated an EPO-associated decrease in blood glucose levels in all mice models tested. The tg6 mice, continuously exposed to EPO, were hypoglycemic even under non-fasting conditions. Blood glucose reduction in the experimental models tested was evident already one week following EPO administration in all models, including the PTP1B-/- and ob/ob mice. This conclusion gained further support by the glucose tolerence test (GTT). EPO overexpression or administration led to improved glucose clearance in all the tested murine models. Notably, in the ob/ob mice we observed EPO-associated attenuation of body weight gain and reduction of hemoglobin A1C. Preliminary clinical observations with several diabetic patients support these data and will be discussed further. Taken together our data bear significant clinical implications for EPO treatment in the management of a wide range of metabolic diseases and add an important novel therapeutic potential to this pleiotropic hormone. Disclosures: Mittelman: BioGAL- Start up (inactive): Equity Ownership, Patents & Royalties. Off Label Use: Non erythroid effects: immune, anti-cancer (all under investigation).

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