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A rat anti-recombinant mouse tumour necrosis factor-alpha (rmTNF-α) monoclonal IgM antibody (1F3F3) with high specific binding activity for rmTNF-α was generated. The 1F3F3 monoclonal antibody (mAb) neutralizes the cytotoxic activity in vitro of rmTN F-α on L929 cells and inhibits the binding of radiolabelled rmTNF-α to its putative receptor on L929 cells. The 1F3F3 mAb binds to monomeric, dimeric and trimeric rmTNF-α and does not bind to reduced rmTNF-α, indicating that the recognized epitope is sensitive to denaturation. Using the 1F3F3 mAb as a capturing antibody and a biotinylated anti-rTNF-α as a detecting antibody, we have developed a sandwich ELISA that can specifically detect biologically active mTNF-α with a detection limit of 10 pg mTNF-α/well. This assay correlates well with the classical L929 cristal violet assay for the detection of bioactive rmTNF- in biological fluids. The 1F3F3 mAb inhibits various in vitro biological activities of the rmTNF-α, such as the TNF-α-mediated tumouricidal activity of activated macrophages, the rmTNF-αdependent stimulation of neutrophil degranulation and the growth-promoting effect of rmTN F-α. In vivo the 1F3F3 mAb inhibits lipopolysaccharide (LPS)-induced endotoxic shock. In conclusion, the 1F3F3 mAb is a useful tool to probe rmTNF-α activity both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

The in vitro cytotoxic activity of two types of hepatic sinusoidal cells, i.e., natural killer (NK) cells and macrophages (Kupffer cells), was tested against a syngeneic rat colon adenocarcinoma cell line (DHD-K12). Purified hepatic NK cells (85% cells with large granular lymphocyte morphology) were spontaneously cytolytic, whereas Kupffer cells (90% pure) were not able to kill the DHD-K12 cells. This carcinoma cell line was found to be resistant to the action of mouse recombinant tumor necrosis factor which is considered as the major cytolytic molecule secreted by macrophages. However, colon carcinoma cells were readily lysed by soluble factors present in the culture supernatant of NK cells. It is postulated that hepatic NK cells, which are strategically located within the lumen of the sinusoids, may form a first line of defense to metastasizing colon carcinoma cells.