TNF-inhibitors Inhibit Hemophagocytosis Of Codeveloping Hematopoietic Progenitors By Dendritic Cells In Culture

TNF-inhibitors inhibit hemophagocytosis ofcodeveloping hematopoietic progenitors bydendritic cells in cultureObjectiveHemophagocytic Syndrome (HPS) is a rare disorder characterized by theincreasing of mococytes and phagocytic cells. The recent study demonstrated thatdendritic cells (DCs) may be the phagocytic cells of Hemophagocytic Syndrome. Thetumor necrosis factor-α(TNF-α) has been reported to be overproducted in HPSpatients. In vivo and in vitro erythropoiesis is inhibited by TNF-α. Codevelopment ofdendritic cells along with erythroid differentiation from human CD34⁺ cells is inducedby TNF-αand the dendritic cells showed activity of phagocytosis.Methods一,ReagentsBovine serum albumin(BSA), Iscove's Modified Dulbecco's Medium (IMDM)and Propidium iodide(PI) were purchased from Sigma. Fetal calf serum (FCS) waspurchased from Hyclone. Penicillin and streptomycin were purchased from FlowLaboratories Inc. Insulin was from Calbiochems. Insulin (porcine sodium, activity 28.9U/mg) was obtained from Wako Pure Chemical Industries. Interleukin-3 (IL-3) andstem cell factor (SCF) were kind gifts of the Kirin Brewery Co. Ltd., and erythropoietin(EPO) from Chugai Pharmaceutical Co. Vitamin B12 was purchased from Eisai andfolic acid was from Takeda. Etanercept was from Takeda and Infliximab was fromTanabe. Fluorescein isothiocyanate (FITC)-labeled GPA was purchased from Becton Dickinson. The FITC-labeled CD11c was purchased from Dako. The PE-labeledCD11c was from Beckman Coulter. The PE-labeled CD123, the PE-labeled CD4 andthe FITC-labeled CD4 were purchased from BD Pharmingen.二,Purification of human CD34+ cellsGranulocye colony stimulating factor mobilized human peripheral blood CD34⁺cells were purified with CliniMACS from healthy volunteers, who had signed informedconsent forms approved by the Akita University School of Medicine Committee for theProtection of Human Subjects, and stored in liquid nitrogen until use.三,Liquid suspension culture of purified CD34+ cellsThe thawed CD34⁺ cells were suspended in IMDM containing 20% FCS and 100U/ml DNAse and then were washed twice with IMDM containing 10% FCS and0.3%BSA. Next, the cells were seeded at 2×10⁴ to 4×10⁴ cells/ml in IMDM containing10% heat-inactivated pooled human AB serum, 1% BSA, 10μg/ml insulin, 0.5μg/mlvitamin B12, 15μg/ml folic acid, 12.5μMβ-mercaptoethanol, 50 U/ml penicillin, and50 U/ml streptomycin in the presence of 10ng/ml IL-3, 10ng/ml SCF and 2 IU/ml EPOat 37℃in a 5% CO2 incubator. After incubation for the indicated days, the cells werecollected.å››,cytospin slideCells were spun onto slides using Cytospin, dyed with May-Giemsa after airdrying, and then observed under microscope.五,Flow cytometryThe cells collected from the cultures were washed twice, using centrifugation andpipette aspiration, with phosphate-buffered saline (PBS) containing 3% FCS, and0.05% NaN3 (staining medium) and stained with FITC- and PE-labeled mAbs, andthen were analyzed using a FACS Calibur^TM (Becton Dickinson).å…­,Statistical analysisDifferences were determined using spss12.0. Results一,1μg/ml Etanercept and 2000μg/ml Infliximab almostinhibited 100ng/ml TNF-αcompletelyHuman CD34⁺ cells with a purity of 98% were plated at a concentration of 4×10⁴/ml with 100ng/ml TNF-α, 0-10μg/ml Etanercept or 0-2000μg/ml Infliximab.The purified CD34⁺ cells cultured for 7 days in liquid phase with IL3, SCF and EPOdifferentiated into erythroid progenitor cells. In the presence of TNF-α, the proportionof GPA⁺ cells markedly decreased, whereas DC increased. Etanercept and Infliximabalmost inhibited 100ng/ml TNF-αcompletely at the concentration of 1μg/ml and2000μg/ml, which is the same as the negative control.二,1μg/ml Etanercept and 2000μg/ml Infliximab inhibited10ng/ml TNF-α三,Etanercept and Infliximab's effects on TNF-αdecreasedwith the timeThe time course of Etanercept and Infliximab showed that Etanercept andInfliximab's effects on TNF-αdecreased with the time. It was the best whenEtanercept and Infliximab were added in at the same time of TNF-α.DisscussionCytokine research now shows clearly that HPS results from uncontrolled,dysregulated cellular immunereactivity that can be initiated by a number of underlyingdiseases. HPS is a culmination of a cascade of overproduction of cytokines includingIFN-γ, sIL-2R, TNF-α, IL-2, IL-6, IL-12, and IL18. Although it is not entirely clearwhich cytokines are pivotal in HPS, TNF-αmay play a key role. So neutralization ofTNF-αin the early phase of inflammatory may be effective in the treatment of HPS.Henzan.et had reported a case describing successful treatment of HPS with Infliximab. To date, anti-TNF-αtherapy with Etanercept has been reported in twopatients for diseases involving histiocytic-lineage cells: macrophage activationsyndrome(MAS) and Langerhans' cell histiocytosis.ConclusionNeutralization of TNF-αin the early phase of inflammatory may be effective inthe treatment of HPS.