title: Interleukin 1 receptor antagonist (IL-1Ra) is an acute-phase protein.
abstract: Interleukin 1 receptor antagonist (IL-1Ra) levels are elevated in the blood of patients with a variety of infectious, immune, or traumatic conditions. To examine whether IL1Ra is produced by liver cells with characteristics resembling an acute-phase protein, human primary hepatocytes isolated from liver biopsies and HepG2 hepatoma cells were stimulated with IL-1beta, IL-6, and TNFalpha. IL-1Ra was present in the supernatants of both cells, with production significantly enhanced by IL-1beta, and by the combination of IL-1beta and IL-6. The term IL-1Ra refers to two different proteins encoded by the same gene, but generated by alternative splicing of two different first exons. One isoform is secreted (17-kD sIL-1Ra), and the other isoform remains in the cytoplasm (18-kD icIL-1Ra). By Western blot analysis, the supernatants of human hepatoma (HepG2) cells contained only sIL-1Ra, whereas the lysates contained a novel smaller molecular mass isoform of 16 kD. RT-PCR and ribonuclease protection assay with RNA from HepG2 cells showed that only sIL-1Ra mRNA was expressed, and confirmed the inducing effect of IL-1beta and IL-6. Transfection studies were performed using constructs containing the promoters of either sIL-1Ra or icIL-1Ra coupled to the luciferase reporter gene. The sIL-1Ra promoter was active in HepG2 cells stimulated by IL-1beta and/or IL-6, whereas the icIL-1Ra promoter was inactive. Mutation of binding sites for transcription factors NF-kappaB and/or C/EBP within the proximal sIL-1Ra promoter led to significant decreases in response to IL-1beta and IL-6 in comparison to the wild-type promoter. Electromobility gel shift assays confirmed the presence of NF-kappaB and C/EBP binding sites within the sIL-1Ra promoter, and indicated a significant increase in the binding activities of nuclear proteins from HepG2 cells treated with IL-1beta and IL-6. In summary, sIL-1Ra, but not icIL-1Ra, is produced by hepatocytes, and is regulated by proinflammatory cytokines as an acute-phase protein. In addition, NF-kappaB and C/EBP family members are likely to play important roles in the full expression of IL-1Ra by hepatocytes during inflammatory conditions.
authors.affiliation: Division of Rheumatology, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. gabay_c@defiance.uchsc.edu
authors.forename: C
authors.initials: C
authors.lastname: Gabay
authors.forename: M F
authors.initials: MF
authors.lastname: Smith
authors.forename: D
authors.initials: D
authors.lastname: Eidlen
authors.forename: W P
authors.initials: WP
authors.lastname: Arend
mesh_terms: D000209:Acute-Phase Proteins; D001665:Binding Sites; D006528:Carcinoma, Hepatocellular; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004586:Electrophoresis; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1 / Q000494:pharmacology; D015850:Interleukin-6 / Q000494:pharmacology; D008099:Liver / Q000378:metabolism*; D008113:Liver Neoplasms; D016328:NF-kappa B / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D000209:Acute-Phase Proteins; D004268:DNA-Binding Proteins; C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1; D015850:Interleukin-6; D016328:NF-kappa B; D012333:RNA, Messenger; D012795:Sialoglycoproteins; D014409:Tumor Necrosis Factor-alpha; D004247:DNA