ABSTRACT
This study was aimed to investigate the influences of interferonalpha (IFN-alpha) on expressions of CCR7, interleukin10 (IL-10) and IL-12p70 in dendritic cells (DCs) from patients with chronic myeloid leukemia (CML). In addition to stem cell factor (SCF), granulocyte-macrophage colony stimulating factor (GM-CSF), tumor necrosis factor-alpha (TNF-alpha) and IL-4, IFN-alpha was added to the serum-free medium of DCs. After culture for 10-14 days, phenotypes and function of CML-DCs were evaluated respectively by flow cytometry and methyl thiazolyl tetrazolium (MTT) assay. Chromosome of DCs was analyzed by displaying G banding assay. The concentrations of IL-10 and IL-12P70 in supernatants were evaluated by enzyme-linked immunosorbent assay (ELISA). The results showed that the expressions of CD40, CD83, CD86 and CCR7 and the OD value in allogeneic mixed-lymphocyte reaction (MLR) in group with IFN-alpha (300 U/ml) were twice as high as those in group without IFN-alpha. The percentage of Ph1 positive cells and concentrations of IL-10 and IL-12 P70 were reduced in group with IFN-alpha. It is concluded that the defective phenotypes and functions of CML-DCs can be recruited partly by IFN-alpha. The mechanism may lie in the facts that expression of CCR7 and co-stimulatory molecules is promoted and the inhibitory effect of IL-10 on CML-DCs is relieved partly through the regulation of IFN-alpha.
Subject(s)
Humans , Cells, Cultured , Dendritic Cells , Cell Biology , Interferon-alpha , Pharmacology , Interleukin-10 , Genetics , Metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Genetics , Allergy and Immunology , Philadelphia Chromosome , Receptors, CCR7 , Genetics , MetabolismABSTRACT
The study was aimed to investigate the influence of interferon alpha (IFN-alpha) on the expressions of Fas and Fas ligand (FasL) in dendritic cells (DCs) from patients with chronic myeloid leukemia (CML). In addition to adding stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha) and interleukin 4 (IL-4), the IFN-alpha was added to the serum-free medium for DCs. After culturing for 10 - 14 days, cell phenotype and percentage of Ph(1) chromosome were detected by different methods. The expression of Fas or FasL on CML-DCs and cell cycle of DCs labeled with propidium iodine (PI) were measured by flow cytometry. The concentration of sFas in supernatants was analyzed by enzyme-linked immunosorbent assay (ELISA). The results indicated that the expression of co-stimulatory molecules were improved significantly while the percentages of Ph(1) positive cells decreased. The level of Fas on cells was up-regulated and the concentration of sFas decreased. However, the expression of FasL was negative. The ratio of apoptosis rose gradually while the concentration of IFN-alpha increased. It is concluded that IFN-alpha can accelerate the apoptosis of Ph(1) positive cells through Fas/FasL pathway, so the number of Ph(1) negative cells increases relatively.
Subject(s)
Adolescent , Adult , Child , Female , Humans , Male , Middle Aged , Young Adult , Apoptosis , Cells, Cultured , Culture Media , Pharmacology , Dendritic Cells , Cell Biology , Metabolism , Fas Ligand Protein , Genetics , Metabolism , Interferon-alpha , Pharmacology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Metabolism , Pathology , Philadelphia Chromosome , fas Receptor , Genetics , MetabolismABSTRACT
The study was aimed to investigate the extensive amplification and the cytotoxicity of dendritic cells (DC) derived from chronic myeloid leukemia cells. DC were cultured in two steps: firstly, extensive amplification in primary culture of CD34(+) or mononuclear cells isolated from CML patients' bone marrow and peripheral blood with rhFlt3-L and rhTPO for 7 days; secondly, inducing culture of DC with rhGM-CSF, rhTNF and rhIL-4 for 14 days. A system inducing DC directly were established for comparison. DC were identified by immunophenotype with flow cytometry, chromosome analysis by displaying G banding and electric microscopy analysis. The function of stimulating T cells proliferation and cytotoxicity of CML cells were confirmed through MTT assay. The results showed that after first extensive amplification in primary culture with rhFlt3-L and rhTPO for 7 days, CD34(+) cells had a total cell number with (77 +/- 5) fold expansion, and DC were (39 +/- 8)% of total cell respectively after induction culture of DC with rhGM-CSF, rhTNF and rhIL-4 for 14 days. Both the amplification of cell number and yield of DC were higher than the system without extensively culture (P < 0.01). Such DC could stimulate T cells to proliferate and kill leukemia cells finally. In conclusion, two-step culture method can obviously improve the cell number of DC required, that is better than inducing them directly. DC derived from CML cells induce the generation of anti-leukemia immunization.
Subject(s)
Adolescent , Adult , Aged , Child , Female , Humans , Male , Middle Aged , Antigens, CD34 , Allergy and Immunology , Cell Proliferation , Cells, Cultured , Cytotoxicity, Immunologic , Dendritic Cells , Allergy and Immunology , Pathology , Flow Cytometry , Immunophenotyping , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Allergy and Immunology , Pathology , T-Lymphocytes, Cytotoxic , Allergy and ImmunologyABSTRACT
To study the influence of IFN-alpha on function of CML-DC cultured in vitro and expression of chemokine and its chemokine receptor, bone marrow mononuclear cells from 13 CML patients were cultured in the fetal calf serum culture system supplemented with rhSCF, rhFlt-3L for expansion system, and adding rhGM-CSF, rhTNF-alpha, rhIL-4, with or without rhIFN-alpha to induce DCs. After incubation for two weeks, the phenotypes of CML-DC were analyzed by direct immunofluorescence and flow cytometry. The concentration of MIP-3beta expressed by CML-DC in the supernatant were analyzed by ELISA. The proliferative ability of T cells from healthy volunteers stimulated by CML-DCs were measured by MTT assay. The results showed that expression of CD86, CD83, CD40, MHC-I class molecules, CCR7, the concentration of MIP-3beta expressed by CML-DC, and the proliferative ability of T cells stimulated by CML-DCs in IFN-alpha group were all significantly higher than that in control group (P < 0.01). It is concluded that the immunophenotype of CML-DCs can be partially changed by IFN-alpha to accelerate the maturation of CML-DCs, enhance the capacity of CML-DCs, and stimulate allogeneic T lymphocyte proliferation.