Lilach Lifshitz

Macrophages as novel target cells for erythropoietin

Background Our 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 Methods The 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. Results Erythropoietin 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. Conclusions Our 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.

Erythropoietin enhances immune responses in mice

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 anti‐neoplastic 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.

Erythropoietin treatment leads to reduced blood glucose levels and body mass: Insights from murine models

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(-/-)), and ob/ob mice susceptible to diabetes and obesity. The data presented herein demonstrate EPO-mediated decrease in blood glucose levels in all mice models tested. Moreover, in the ob/ob mice, we observed EPO-mediated 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.

Non-erythroid activities of erythropoietin: Functional effects on murine dendritic cells

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.

Non-erythroid effects of erythropoietin: Are neutrophils a target?

We have previously shown that erythropoietin (EPO) potentiates the immune response. Analysis of various possible cellular mediators was performed on EPO-injected mice and transgenic mice overexpressing human EPO (tg6). Here we present our studies on neutrophils, peritoneal (casein induced), and from the peripheral blood, spleen and bone marrow. Neutrophil counts were elevated in peripheral blood and spleens of the tg6 mice, yet, no other EPO-associated effects were detected in the count and function of the different neutrophil populations. Hence, neutrophils are probably not mediators of the EPO immunological effects, although their counts may be affected by extreme EPO levels.

Increased eosinophilic responses in splenectomized patients

BACKGROUND The spleen is a key organ within the immune system. Its removal is known to bring about adverse effects such as an increased susceptibility to overwhelming infection. Few reports have suggested that the spleen may play a role in controlling eosinophilic responses, mostly based on animal models. OBJECTIVES To examine whether the human spleen impacts eosinophil numbers in the blood. METHODS We have retrospectively analyzed eosinophil counts and medical records of 29 patients who had undergone splenectomy between 2000 and 2010. Statistical comparison was performed between post-splenectomy blood counts and both pre-splenectomy and control values. Data regarding the clinical settings around hypereosinophilia events were obtained from patient charts. RESULTS An increased rate of eosinophilia was observed after splenectomy as compared with normal individuals. Furthermore, a considerable proportion of patients who had undergone splenectomies (8/29) presented peak eosinophil numbers exceeding 1,000/mm(3), reaching a maximum of 3,070/mm(3). These values were mostly encountered perioperatively or during episodes of acute infection. CONCLUSIONS Our data indicate that impaired control of eosinophilic responses is a long-term post-splenectomy effect and is evident in the context of acute stress. We suggest that the spleen plays a significant role in controlling eosinophil levels and that these cells may mediate some of the harmful consequences observed after removal of the spleen.

Erythropoietin receptor is detectable on peripheral blood lymphocytes and its expression increases in activated T lymphocytes (reply)

We have read with interest the letter of Lisowska et al. , submitted to Haematologica in response to our manuscript “Macrophages as novel target cells for erythropoietin” by Lifshitz et al. , published in Haematologica in June 2010. In their commentary, Lisowska et al. claim that: (1) we have

Erythropoietin and cyclophosphamide combination treatment additively enhances antibody production in mice.

termine the possible effects of such combination treatments on the production of anti-DNP antibodies. Cyclophosphamide (CP) is a known cytotoxic alkylating agent widely used in cancer chemotherapy. While it functions as an immunosuppressive agent at high doses, the anti-neoplastic activities of CP at low doses are attributed to enhancement of cellular and humoral immunity. Augmentation of the immune response following lowdose CP treatment was observed in both murine models and humans injected with various tumor antigens [9] . These effects were attributed to the ability of low-dose CP to specifically inhibit the activity and proliferation of suppressive Th CD25+ cells [10] . Here, we chose to focus on the humoral immunomodulatory effects of lower doses of EPO combined with lowdose CP, thus simulating the routine clinical conditions, yet under experimentally controlled conditions, in the absence of a malignant disease. The present study was designed to compare serum levels of anti-DNP immunoglobulin in DNP-KLH-injected C57BL/6 mice that were treated with either low doses of EPO or with CP alone, or with the combination of both Erythropoietin (EPO), a key therapy for several types of anemia, e.g. cancer-related anemia [1] , is also known to display non-erythroid effects, such as heart failure improvement and neuroprotection [2] . It should be noted that there are ongoing safety concerns regarding EPO treatment [3] . Although EPO receptor (EPO-R) expression has been reported in various solid tumors, its functionality is still controversial [4] . Among the non-erythroid effects of EPO, accumulating data show that EPO can affect the immune system, including both cellular and humoral responses. We have previously shown that EPO displays anti-neoplastic activity in patients with multiple myeloma [5] . This EPO activity was also manifested in murine multiple myeloma models [6] and was related to stimulatory effects on both humoral and cellular immune responses [7, 8] . In addition, we demonstrated that administration of high doses of EPO [180 U recombinant human EPO (rHuEPO) thrice/week] to dinitrophenyl-keyhole limpet hemocyanin (DNP-KLH)-injected BALB/c mice resulted in an increase in anti-DNP IgG1 production [8] . Thus far, our reports have focused on EPO as the only treatment. However, in the context of clinical treatment for cancer-related anemia, EPO is usually administered along with chemotherapy. In view of the fact that chemotherapy agents may act on the immune system, we decided to deReceived: August 18, 2009 Accepted after revision: October 12, 2009 Published online: December 23, 2009