TNF-α Inhibits Generation Of GPA+ Cells By CD34+ Cells

INTRODUCTIONThe anemia associated with chronic disease is one of the most common he-matologic disorders in clinical medicine. The important mediator of this syndrome is tumor necrosis factor -α ( TNF -α) . TNF -α, a multifunctional cytokine is mainly produced by activated macrophages and lymphocytes. In hem-αtopoiesis, TNF -α acts as both a positive and negative regulator of myeloid cell proliferation and differentiation. Effects of TNF - α can be mediated either directly or indirectly by inducing other cells to produce cytokines.TNF -α inhibits erythropoiesis in vivo and in vitro. Chronic exposure to TNF -α in vivo preferentially inhibits erythropoiesis in nude mice with an absolute monocytosis. In the cancer patients, there was a decrease in hemoglobin after 1 month of TNF treatment. Several in vitro studies extensively demonstrated the inhibitory effects of TNF -α on erythroid colony formation, but the mechanisms have remained unclear.We attempted to determine the extent to which erythroid progenitor cells are sensitive to TNF -α, and to relate this to the expression of TNFRs in the erythroid lineage. For this purpose, we used both an erythroid colony - forming assay as well as a method by which CFU - E and glycophorin A ( GPA; a specific erythroid marker) positive cells can be generated in liquid culture from purified human CD34 + cells in the presence of multiple cytokines, including stem cell factor (SCF) , interleukin -3 (IL-3) and erythropoietin (EPO). It lays a foundation to expound the etiology of ACD.MATERIALS AND METHODSReagentsBovine serum albumin (BSA) , Iscove's Modified Dulbeccos Medium (IM-DM) and propidium iodide ( PI) were purchased from Sigma. Fetal calf serum (FCS) , penicillin, and streptomycin were from Flow Laboratories Inc. Insulin (activity 26. 3 U/mg) was purchased from Calbiochems. Fluorescein isothiocya-nate (FITC) - labeled antibodies specific for CD34 were purchased from Becton Dickinson. Anti - GPA - PE was obtained from DAKO Japan Co. Anti - TNFR -1 ( CD120a) - PE and anti - TNFR - II ( CD120b) - PE were obtained from R&D systems and Caltag Laboratories. Recombinant human TNF -a was kindly donated by Mochida Pharmaceutical Co. Ltd. Recombinant human EPO was kindly donated by Chugai Pharmaceutical Co. Ltd. Recombinant human IL - 3 and SCF were kindly donated by Kirin Brewery Co. Ltd. Vitamin B12 and folic acid were obtained from Sankyo Pharmaceutical Co. Purification of human CD34+ cellsRecombinant human granulocyte colony - stimulating factor ( G - CSF) was administered to healthy volunteers. The mobilized peripheral blood (PB) CD34+ cells were isolated using immunomagnetic beads. The cells were then cryopreserved and stored in liquid nitrogen until use. Semisolid culture of progenitorsPurified human CD34 + cells and their progenies generated ex vivo were incubated in triplicate at a concentration of 500 cells/mL in flat - bottomed, 48 -well tissue culture plates, in 0.25 or 0.5 mL serum - containing fibrin clots with EPO at 2 U/mL, IL - 3 at 50 ng/mL and SCF at 50 ng/mL. After the indicated days of incubation at 37℃ in a 5% CO2/5% O2 incubator, the clots were fixed and stained with benzidine - hematoxylin. The aggregates that consisted of 8 to 49 hemoglobinized cells were defined as CFU - E. Liquid suspension culture of purified CD34+ cellsThe frozen PB CD34 + cells were thawed, suspended in IMDM containing 30% FCS and 100 U/mL DNase, then centrifuged at 400g for 5 minutes at 4℃. The cells were washed twice, then resuspended in IMDM containing 0.3% dei-onized BSA. Cells ranging from 0. 5 x 104 to 2. 0 x 104 cells/mL were suspended in a mixture containing 20% FCS, 10% heat - inactivated pooled human AB se-rum, 1% BSA, 10 mg/mL of insulin, vitamin B12 at 10 g/mL, and folic acid at 15 g/mL, with IL -3 at 100 U/mL, SCF at 100 ng/mL, and EPO at 4 U/ mL, in the presence of 5 x 10-5 mol/mL B - ME, penicillin at 50 U/mL and streptomycin at 50 U/mL, and IMDM. After incubation for the indicated days at 37 ℃ in a 5% CO2 and 95...