Background. Tumor necrosis factor-α (TNF-α) and thrombopoietin (TPO) have been shown to sustain differentiation and proliferation of CD34+ cells toward dendritic cells (DCs) in the presence of multi-acting cytokines. We hypothesized that co-stimulation of TPO and TNF-α generate megakaryocytic progenitors and DCs together from human CD34+ cells and that interaction of these cells may provide a physiological and/or a pathological role of DCs in megakaryopoiesis.
Materials and Methods. Highly purified human CD34+ cells were cultured with TPO, with or without TNF-α, in plasma-depleted medium and induced to undergo megakaryocytic differentiation. We enumerated megakaryocytic progenitor cells using the specific markers CD41, CD42b, and CD61, and DCs using CD4, CD11c, CD80, CD83, CD86, and CD123. The character and roles of co-developing non-megakaryocytic cells in the presence of TNF-α were analyzed by fluorescence-activated cell sorter, enzyme immunohistochemistry, confocal microscopy, and autologous mixed lymphocyte reaction. Cytokine production was assessed using a cytometric bead array system.
Results. When CD34+ cells were cultured for 7 days in the presence of TPO, the generated cells predominantly expressed CD41 (95±2%), CD42b (54±12%), and CD61 (96±2%), while rarely expressing CD11c (1.6±1.3%), CD80 (0.1±0.1%), CD83 (0.8±0.6%), or CD86 (3.3±1.9%). The addition of TNF-α significantly decreased the number of cells expressing CD41 (3.0±0.6%), CD42b (3.3±1.0%), or CD61 (3.2±0.9%), but did not affect the number of total cells. In the presence of TNF-α, the generated cells expressed major histocompatibility complex (MHC) class I (100%) plus MHC class II (100%). A substantial number of cells became positive for CD11c (37±1%), and even co-stimulatory molecules such as CD80 (2.4±1.9%), CD83 (8±4%), and CD86 (18±7%). Immature CD11c+ DCs were physically associated with apoptotic and CD61+ cells and capable of endocytosing CD61+ cells. Most of the CD11c+ cells co-expressed the c-mpl TPO receptor, CD4, and CD123 and about one half of CD11c+ cells co-expressed CD86. The DCs generated by TNF-α and TPO, but not those by TNF-α alone, facilitated autologous T cell proliferation in some extent, although cytokine production from activated T cells were low. We also confirmed engulfment of CD61+ cells and their fragment by CD11c+ cells in bone marrow cells from patients with hemophagocytic syndromes.
Conclusions. This is the first report showing that in the presence of TNF-α, the non-megakaryocytic cells with typical feature of DCs are co-generated from human CD34+ cells during megakaryocytic differentiation by TPO. The CD4+ CD11c+ CD123+ DCs physically associates with and phagocytose developing or dying immature megakaryocytic cells. Similar phenomenon showing engulfment of CD61+ fragment by CD11c+ cells was also observed in bone marrow cells from patients with hemophagocytic syndrome. Therefore, it may be conceivable that DCs with phagocytic activity during the development in bone marrow may play a crucial role in the maintenance of tolerance for self-substances derived from hematopoietic progenitor cells.