title: Interferon-alpha (IFN-alpha) inhibits granulocyte-macrophage colony-stimulating factor (GM-CSF) expression at the post-transcriptional level in murine bone marrow stromal cells.
abstract: Recently it has been shown that IFN-alpha inhibits expression of GM-CSF in adherent cells of human long-term bone marrow cultures (LTBMC) stimulated with interleukin-1 (IL-1), tumour necrosis factor-alpha (TNF-alpha) or endotoxin. The murine bone marrow stromal cell line +/+(-1).LDA11 was used to further define regulatory mechanisms of IFN-alpha inhibition on GM-CSF expression. This cell line originated from a murine Dexter type culture and exhibits a preadipocytic phenotype. As in human LTBMC, we could demonstrate a inhibitory effect of IFN-alpha co-incubation on GM-CSF activity in serum-free supernatants of +/+(-1).LDA11 stromal cell cultures stimulated with IL-1 or TNF-alpha or the combination of IL-1 plus TNF-alpha. IFN-alpha inhibitory effect on GM-CSF expression was shown to be dose dependent with minimal response at 10 U/ml and maximal inhibition at a dose of 500 U/ml. Northern blot analysis confirmed these data at the mRNA level. Reprobing of Northern blots for interleukin-6 (IL-6) mRNA showed increased expression after IFN-alpha incubation, demonstrating specific and differential regulatory effects of IFN-alpha on cytokine production in bone marrow stromal cells. Inhibition of GM-CSF mRNA by IFN-alpha was time dependent, starting at about 90-120 min post-treatment. Cycloheximide (CHX) incubation abolished the inhibitory effect of IFN-alpha on GM-CSF expression, suggesting the requirement of a labile protein. Reporter gene studies were used in order to evaluate the effect of IFN-alpha incubation on GM-CSF mRNA transcription in stromal cells. For this purpose, GM-CSF promoter fragments were subcloned into a luciferase expression vector. Neither constitutive nor TNF-alpha stimulated GM-CSF transcription was inhibited by IFN-alpha coincubation. On the other hand, actinomycin-D chase experiments revealed a reduced GM-CSF mRNA stability after IFN-alpha incubation. The induction of a RNAase, possibly a 2-5A-dependent RNAase, by IFN-alpha may be a possible cause for the increased GM-CSF mRNA decay. These results show a regulatory role for IFN-alpha in the bone marrow microenvironment possibly involved in the myelosuppressive effect of IFN-alpha therapy or viral infections.
authors.affiliation: III. Department of Medicine, Johannes Gutenber-Universität, Mainz, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Göllner
authors.forename: M J
authors.initials: MJ
authors.lastname: Aman
authors.forename: H P
authors.initials: HP
authors.lastname: Steffens
authors.forename: C
authors.initials: C
authors.lastname: Huber
authors.forename: C
authors.initials: C
authors.lastname: Peschel
authors.forename: H G
authors.initials: HG
authors.lastname: Derigs
mesh_terms: D000818:Animals; D001854:Bone Marrow Cells; D002460:Cell Line; D004306:Dose-Response Relationship, Immunologic; D015536:Down-Regulation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics; D007370:Interferon Type I / Q000494:pharmacology*; D007375:Interleukin-1 / Q000037:antagonists & inhibitors; D051379:Mice; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors; D017154:Stromal Cells; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors
chemical_list: D007370:Interferon Type I; D007375:Interleukin-1; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D016178:Granulocyte-Macrophage Colony-Stimulating Factor