In vitro investigations of hematopoiesis have been performed almost exclusively at ambient (21%) oxygen tensions. In vivo tissue and marrow oxygen tensions, however, have been estimated at 23-40 mmHg (2-5% O2). Studies from our laboratory indicate physiologic oxygen tensions augment in vitro granulopoiesis and erythropoiesis and that T cells and monocytes (Mo) can respond to variations in O2 tensions by collaborating to produce burst-promoting activity (BPA). Augmentation in the production or release of BPA from Mo and T cells could represent an additional mechanism mediating the response of erythropoiesis to alterations in tissue and marrow O2 tensions. Studies indicate low oxygen tensions affect the release of growth factors from Mo and cause increased cloning efficiency of human CFU-GM associated with altered progenitor sensitivity to purified granulopoietins. We hypothesize that the study of hematopoietic progenitors at low O2 tensions may more closely simulate physiologic growth of marrow stem cells in vivo, that variable O2 tensions exert a regulatory role on in vitro hematopoiesis and that release of hematopoietic growth factors from Mo and T cells occurs as a response to variation in O2 tensions. In many studies of patients with COPD and hypoxemia, increase in RBC mass is less than expected and is not explained by impaired Ep release or decreases in BM iron reutilization; our initial studies indicate marrow BFU-E but not CFU-GM from these patients fail to show augmentation at 5% O2. We hypothesize that impaired erythroid augmentation under (sub-)physiologic O2 tensions may contribute to the pathogenesis of anemia in patients with COPD, hypoxemia or inflammation. In this proposal, we plan to investigate in detail the effects of physiologic O2 tensions on erythropoiesis. Specifically, we plan to 1) address the question of how varying physiologic O2 tensions affect intrinsic properties of enriched marrow BFU-E and CFU-E (colony size and differentiation, cell cycle status and progenitor sensitivity to purified erythropoietic growth modulators). We will extend our prior work to obtain increasingly purified populations of erythroid progenitors using sequential immunoabsorption and multi-parameter cytofluorography. We will 2) study effects of physiologic O2 tensions on the ability of purified T cells, Mo, fibroblasts and endothelial cells to regulate growth of marrow erythroid bursts. These experiments will incorporate two separate assays for BPA and a serum-substituted culture system. We will specifically examine how varying physiologic O2 tensions affect the transcription and release of defined erythropoietic growth factors from purified accessory cell populations. We will 3) investigate the mechanism of impaired erythroid augmentation under physiologic O2 tensions in selected patients with chronic pulmonary disease, hypoxemia or anemia. We will directly assess production of specific erythropoietic growth modulators form purified patient T cells and Mo and will study possible mechanisms for impaired collaboration between patient Mo, and T cells in the generation of BPA.
