Inhibitory effects of interleukin 12 on retroviral gene transduction into CD34 cord blood myeloid progenitors mediated by induction of tumor necrosis factor-alpha.
Interleukin 12 (IL-12), a heterodimeric cytokine with potent biologic activity, was evaluated for effects on retroviral-mediated gene transduction into human myeloid progenitor cells in vitro. Cord blood CD34 cells were prestimulated with Steel factor (SLF), IL-3, GM-CSF, and erythropoietin (Epo) in the presence and absence of 5-80 ng/ml IL-12 for 40 hr in suspension culture prior to gene transduction using viral supernatant collected from a packaging cell line containing the pLNL6 vector encoding Neo sequences. After gene transduction, cells were assayed for colony formation stimulated by Epo, GM-CSF, IL-3, and SLF, and gene transduction efficiency was determined by the percentage of G418 resistant (R) colonies and confirmed by PCR analysis. IL-12 dose-dependently inhibited retroviral-mediated gene transduction into human cord blood CD34 granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitors. These suppressive effects could be neutralized by incubation of IL-12 with polyclonal antihuman IL-12. IL-12 had no inhibitory effects directly on colony formation. To understand the possible mechanisms for this suppression, ELISA assays were used to detect the release of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, which could potentially have been induced by IL-12 from CD34 cells. TNF-alpha protein release was significantly increased in CD34 cells incubated with IL-12. No detectable levels of IFN-gamma were noted. Anti-TNF-alpha, but not anti-IFN-gamma, blocked the inhibitory effects of IL-12 on gene transduction. Moreover, TNF-alpha, but not IFN-gamma, suppressed gene transfer to the same degree as IL-12. No change of amphotropic receptor mRNA expression was noted by Northern blot analysis in cells treated with or without IL-12. The results suggest that the suppressive effects of IL-12 on retroviral gene transduction are, at least in part, mediated by IL-12 induction of the release of TNF-alpha.
Xiao
M
M
Department of Medicine (Hematology/Oncology), Indiana University School of Medicine, Indianapolis, USA.
Li
Z H
ZH
McMahel
J
J
Broxmeyer
H E
HE
Lu
L
L
D015703:Antigens, CD / Q000097:blood*; D018952:Antigens, CD34 / Q000097:blood*; D001483:Base Sequence; D017931:DNA Primers; D005312:Fetal Blood; D017930:Genes, Reporter; D005822:Genetic Vectors; D006133:Growth Substances / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000276:immunology; D006801:Humans; D007231:Infant, Newborn; D018664:Interleukin-12 / Q000494:pharmacology*; D019868:Kanamycin Kinase; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis; D016133:Polymerase Chain Reaction; D012190:Retroviridae; D014162:Transfection / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors / Q000494:pharmacology*
D015703:Antigens, CD; D018952:Antigens, CD34; D017931:DNA Primers; D006133:Growth Substances; D014409:Tumor Necrosis Factor-alpha; D018664:Interleukin-12; D017853:Phosphotransferases (Alcohol Group Acceptor); D019868:Kanamycin Kinase