title: Soluble TNF-alpha receptors bind and neutralize over-expressed transmembrane TNF-alpha on macrophages, but do not inhibit its processing.
abstract: Tumor necrosis factor alpha (TNF-alpha) is initially synthesized as a type II integral membrane protein (transmembrane TNF-alpha) after macrophage activation. In this study we have investigated some aspects of the regulation of expression and biological activity of transmembrane TNF-alpha by both soluble TNF-alpha receptors (sTNF-alphaR) and inhibitors of TNF-alpha processing. We show, using the technique of receptor-mediated ligand precipitation (RMLP), that a dimeric construct of the type I sTNF-alphaR binds to transmembrane TNF-alpha, expressed on the mouse macrophage cell line RAW264.7, under cell culture conditions. This interaction between sTNF-alphaR and transmembrane TNF-alpha does not prevent processing and release of soluble TNF-alpha. A specific hydroxamic acid-based inhibitor of processing, BB1101 (British Biotech), was found to increase the total cellular levels of whole-cell, 26-kDa, precursor TNF-alpha by 2.2-fold. However, the inhibitor increased the levels of precursor TNF-alpha present solely on the cell surface (i.e., transmembrane TNF-alpha) by 5.1- to 7.5-fold. This increase in the levels of transmembrane TNF-alpha on the activated human monocytoid cell line mono mac 6 was associated with a similar (6.7-fold) increase in TNF-alpha-mediated cytotoxicity toward the human adenocarcinoma cell line Colo 205, which is sensitive only to the transmembrane form of TNF-alpha. Mono mac 6 cells, expressing transmembrane TNF-alpha, were found to be killing the Colo 205 target cells through apoptosis. This cytotoxicity could be neutralized by pre-incubating the mono mac 6 cells with either sTNF-alphaR or polyclonal anti-TNF-alpha serum.
authors.affiliation: Department of Pathology, University of Sydney, NSW, Australia.
authors.forename: A D
authors.initials: AD
authors.lastname: Watts
authors.forename: N H
authors.initials: NH
authors.lastname: Hunt
authors.forename: M C
authors.initials: MC
authors.lastname: Madigan
authors.forename: G
authors.initials: G
authors.lastname: Chaudhri
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000276:immunology; D001593:Benzyl Compounds; D002460:Cell Line; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic; D003907:Dexamethasone / Q000494:pharmacology; D019281:Dimerization; D004338:Drug Combinations; D006801:Humans; D008262:Macrophage Activation / Q000502:physiology; D008264:Macrophages / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D009363:Neoplasm Proteins / Q000378:metabolism* / Q000494:pharmacology; D010431:Pentoxifylline / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000276:immunology*; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism* / Q000502:physiology; D047889:Receptors, Tumor Necrosis Factor, Type II; D012995:Solubility; D013386:Succinates; D014407:Tumor Cells, Cultured; D053319:Tumor Necrosis Factor Decoy Receptors; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000378:metabolism*
chemical_list: C101468:BB 1101; D001593:Benzyl Compounds; D004338:Drug Combinations; D009363:Neoplasm Proteins; D018124:Receptors, Tumor Necrosis Factor; D047889:Receptors, Tumor Necrosis Factor, Type II; D013386:Succinates; D053319:Tumor Necrosis Factor Decoy Receptors; D014409:Tumor Necrosis Factor-alpha; D003907:Dexamethasone; D010431:Pentoxifylline