title: Immunochemical and biochemical characterization of purified canine interferon-gamma. Production of a monoclonal antibody, affinity purification, and its effect on mixed lymphocyte culture and mixed lymphocyte kidney culture reactions.
abstract: We have advanced the hypothesis that the primary autolymphoproliferative response of dog T cells in mixed lymphocyte kidney cultures (MLKC) results from their recognition of tissue-specific (kidney-associated) antigen(s) presented in conjunction with class II MHC antigens. Lymphocyte culture-derived supernatants had been found previously to upregulate class II antigen expression on kidney cells and enhance T cell activation. In the present study we have isolated and characterized dog IFN-gamma, a class II-inducing substance that is secreted in the culture supernatant of activated T lymphocytes. Dog IFN-gamma was induced with A-23187 and PMA and purified stepwise using controlled-pore glass, Mono Q anion exchange chromatography, and Superose 6-gel filtration on FPLC. The purification resulted in two molecules of 42 Kd and 31 Kd molecular weights. An IgG1 monoclonal antibody was engendered to these molecules. With this mAb reagent, in immunochemical experiments, we have developed a sensitive ELISA and a method for purifying dog IFN-gamma by affinity chromatography. Species specificity studies indicated that purified dog IFN-gamma reacted with a polyclonal rabbit antihuman IFN-gamma, but not with a mAb to human IFN-gamma. However, the antidog IFN-gamma mAb that was generated also reacted with recombinant human IFN-gamma. In in vitro biological studies, the purified IFN-gamma (two mol. wt. species) upregulated the expression of canine class II MHC molecules on dog tubular epithelial cells and the dog kidney epithelial cell line (MDCK). The antidog IFN-gamma mAb blocked T cell proliferative response to kidney cell and, by inference, the interaction between endogenously released IFN-gamma in vitro with its cell surface receptor, thus inhibiting the induced upregulation of class II. Interestingly, although antidog IFN-gamma markedly blocked the MLKC (10 micrograms mAb/well), there was no effect on the allogeneic MLC. This observation indicates that the cytokine IFN-gamma may be a uniquely key substance amplifying the immune response of T cells to tissue-associated antigens on surrogate antigen-presenting cells that require induced upregulation of class II MHC antigen expression (MLKC), in contrast to reactions in which these antigens are already constitutively expressed on the antigen-presenting cells (mixed lymphocyte culture).
authors.affiliation: Department of Surgery, University of Miami School of Medicine, Florida.
authors.forename: L
authors.initials: L
authors.lastname: Fuller
authors.forename: J
authors.initials: J
authors.lastname: Fernandez
authors.forename: S
authors.initials: S
authors.lastname: Zheng
authors.forename: M
authors.initials: M
authors.lastname: Carreno
authors.forename: V
authors.initials: V
authors.lastname: Esquenazi
authors.forename: W C
authors.initials: WC
authors.lastname: Yang
authors.forename: J
authors.initials: J
authors.lastname: Miller
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology; D002846:Chromatography, Affinity; D004285:Dogs; D005260:Female; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis; D007371:Interferon-gamma / Q000276:immunology / Q000302:isolation & purification*; D007668:Kidney / Q000276:immunology*; D007959:Lymphocyte Culture Test, Mixed; D008214:Lymphocytes / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C
chemical_list: D000911:Antibodies, Monoclonal; D000949:Histocompatibility Antigens Class II; D007371:Interferon-gamma