title: Purification and biologic characterization of a specific tumor necrosis factor alpha inhibitor.
abstract: The purpose of the present investigation was to purify a urine-derived tumor necrosis factor alpha inhibitor (TNF alpha INH) and to characterize its mechanism of action. For the purification procedure, urine was concentrated and TNF alpha INH purified by ion-exchange chromatographies, gel filtration, TNF alpha affinity column, and reverse-phase chromatography. The TNF alpha INH migrates with an apparent Mr of approximately 33,000 when estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis run under both reducing and nonreducing conditions. Elution of TNF alpha INH activity from the gel yields also a approximately 33,000-Da inhibitory fraction. Besides inhibiting TNF alpha-induced cytotoxicity in L929 cells in the presence of actinomycin D, the TNF alpha INH impeded in a dose-dependent manner prostaglandin E2 production and expression of cell-associated interleukin-1 by human dermal fibroblasts. Therefore, TNF alpha INH is active on both actinomycin D-treated and untreated cells. In contrast to TNF alpha, TNF beta-induced cytotoxicity was only slightly affected by the inhibitor. This specificity was confirmed by the fact that it affected neither interleukin-1 alpha nor interleukin-1 beta biologic activities. The mechanism of action of TNF alpha INH involves blocking of 125I-TNF alpha binding to the promonocytic cell line U937. Moreover, preincubation of 125I-TNF alpha with TNF alpha INH increased binding inhibition, suggesting an interaction between TNF alpha and the inhibitor.
authors.affiliation: Division of Immunology and Allergy, Hans Wilsdorf Laboratory, Hôpital Cantonal Universitaire, Geneva, Switzerland.
authors.forename: P
authors.initials: P
authors.lastname: Seckinger
authors.forename: S
authors.initials: S
authors.lastname: Isaaz
authors.forename: J M
authors.initials: JM
authors.lastname: Dayer
mesh_terms: D002460:Cell Line; D002470:Cell Survival; D002853:Chromatography, Liquid; D003609:Dactinomycin / Q000494:pharmacology; D015232:Dinoprostone / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts; D006801:Humans; D006868:Hydrolysis; D053590:Interleukin 1 Receptor Antagonist Protein; D011506:Proteins / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor; D012795:Sialoglycoproteins; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D011506:Proteins; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D012795:Sialoglycoproteins; D014409:Tumor Necrosis Factor-alpha; D003609:Dactinomycin; D015232:Dinoprostone