title: The kinetics of gene expression and maturation of IL-1 alpha after induction with the surface coat of Trypanosoma brucei rhodesiense or lipopolysaccharide.
abstract: The purpose of this study was threefold: to determine if the variant surface coat glycoprotein (VSG) of Trypanosoma brucei rhodesiense induces IL-1 alpha; to study the kinetics of IL-1 alpha transcription, maturation and secretion; and to compare VSG to LPS in its ability to induce IL-1 alpha. VSG was added to cultures of the P388D1 murine macrophage cell line. RNA was dotted onto nitrocellulose and hybridized with a murine IL-1 alpha cDNA probe. Maximal production of IL-1 alpha mRNA occurred in a dose- and time-dependent manner, peaking at 25 micrograms/ml VSG, within 2 h. Induction of IL-1 alpha was not due to contaminants because 1) absorption of VSG with a mAb abrogated IL-1 alpha mRNA synthesis, 2) the addition of polymyxin B did not affect mRNA levels, and 3) cellular IL-1 alpha was detectable in VSG-treated splenocytes from endotoxin nonresponder C3H/HeJ mice. Murine splenic macrophages also had enhanced levels of IL-1 alpha mRNA after administration of VSG in vivo or during an acute infection. Antiserum generated against the synthetic peptide SGDDSKYPV (amino acids 177-185 from the murine IL-1 alpha sequence) was used to measure the levels of the 33-, 22-, and 14-kDa proteins in cell lysates and medium of VSG-stimulated P388D1 cells. The 22-kDa protein was the predominant cellular form until secretion started. Secretion of the 14-kDa form began abruptly 6 to 8 h after the addition of VSG. By 12 h, the 33-kDa precursor was the major cytoplasmic form. In comparative analyses, LPS-stimulated P388D1 cells produced more transcript, generated peak levels of 22-kDa protein 3 h earlier, and began to secrete the 14-kDa molecule 5 h earlier. The rate of IL-1 alpha accumulation in the medium was linear between 6 and 24 h after LPS treatment, but began to drop by 8 h in VSG-treated cells. Functional (comitogenic) IL-1 activity was also detected in media from VSG-treated splenic macrophages and P388D1 cells. Activity peaked at 50 micrograms/ml and was lost if 0.2% IL-1 antisera were added to the cultures.
authors.affiliation: Department of Biological Sciences, University of Notre Dame, IN 46556.
authors.forename: S
authors.initials: S
authors.lastname: Mathias
authors.forename: R
authors.initials: R
authors.lastname: Perez
authors.forename: P
authors.initials: P
authors.lastname: Diffley
mesh_terms: D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D007375:Interleukin-1 / Q000032:analysis / Q000235:genetics*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D014158:Transcription, Genetic / Q000187:drug effects; D014346:Trypanosoma brucei brucei / Q000276:immunology*; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000494:pharmacology*
chemical_list: D007375:Interleukin-1; D008070:Lipopolysaccharides; D014643:Variant Surface Glycoproteins, Trypanosoma