Tumor necrosis factor inhibitor: purification, NH2−terminal amino acidsequence and evidence for anti‐inflammatory and immunomodulatory activities

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Abstract
The urine of some febrile patients has been shown to contain a tumor necrosis factor-alpha-inhibiting activity (TNF-α INH) when tested in a cytotoxicity assay using the TNF-susceptible cell line L-929. The inhibitor was purified to homogeneity using a simple three-step procedure which included a TNF-α affinity column, cation exchange and reverse-phase chromatography. TheNH2−terminal amino acid sequence of the inhibitor showed no sequence similarity with proteins in the data bases used. Using gel filtration, it was shown that TNF-α and the inhibitor form a stable complex which eluted with a molecular weight of about 75000. This value corresponds to the sum of the inhibitor (∼︁ 30000) andTNF-α (∼︁45000–50000) molecular weight. The TNF-α INH blocked prostaglandin E2 production by dermal fibroblasts in a dose-dependent manner, providing evidence for antiinflammatory activity. TNF-α INH also blocked class I antigen expression in a dose-dependent manner as measured using the human Colo 205 tumor cell line. Furthermore, TNF-α INH affected TNF-α synergism with IFN-γ-induced HLA-DR antigen expression but had no effect on IFN-γ activity. The data presented demonstrate that TNF-γ bioactivity can be regulated at the protein level.