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.

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Erythropoietin (EPO) is an important component in the treatment of cancer patients for improvement of cancer related anemia. EPO treatment for cancer related anemia is usually combined with chemotherapy. Cyclophosphamide (CP) is a known cytotoxic alkylating agent widely used in cancer chemotherapy. While at high doses it functions as an immunosuppressive agent, the anti-neoplastic activities of CP at low doses are attributed to enhancement of cellular and humoral immunity e.g. (Berd et al., Cancer Res; 1984). We have previously shown that EPO displays anti-neoplastic activities (Mittelman, 2001, 2004) and that EPO treatment is associated with enhancement of both the humoral and cellular immune responses (Prutchi-Sagiv 2006, Katz 2007). Here we focused on a murine model of DNP-KLH-injection, used to assess the humoral response in mice. Recently we demonstrated that administration of high doses of EPO (180U×3) to DNP-KLH-injected C57BL mice resulted in an increase in anti-DNP immunoglobulin G1 (IgG1) production. The present study was designed to examine the effect of combining low dose CP (12.5mg/kg ×2) used to achieve an anti-neoplastic activity, with a lower dose of recombinant human EPO (rHuEPO; 90U×3) on the humoral immune response of the DNP-KLH-injected mice, thus simulating the conditions of patient care. Hence, we compared anti-DNP Ig serum levels in DNP-KLH-injected C57BL mice that were treated with either EPO or CP alone, or the combination of CP and EPO (CP-EPO). CP treatment alone resulted in increased levels of serum anti-DNP IgG1 (O.D.(CP) = 0.38±0.06 vs O.D.(Non treated) = 0.18±0.064). In contrast, EPO treatment alone enhanced serum levels of IgG2 (O.D.(EPO) = 0.47±0.09 vs O.D.(Non treated) = 0.18±0.069). CP or EPO alone did not affect the total levels of anti-DNP total Ig (O.D.(EPO) = 0.37±0.07 vs O.D.(Non treated) = 0.28±0.04). Yet, the combined CP-EPO treatment resulted in increased levels of anti-DNP total Ig (O.D.(EPO+CP) = 0.48±0.05 vs O.D.(EPO or CP) = 0.37±0.04), maintaining the higher levels of IgG1 (O.D.(EPO+CP) = 0.38±0.06) and IgG2 (O.D.(EPO+CP) = 0.49±0.1). In summary, the combined CP-EPO treatment additively improved immunoglobulin production, compared to treatment with CP or EPO alone. We thus demonstrate that in context of chemotherapy treatment as usually administered in the clinic, EPO can enhance humoral immunity alongside its erythropoietic activities. Our findings emphasize the role of EPO as an immunomodulator, particularly when given as treatment in a combined therapeutic panel