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The pleiotropic cytokine TNF has been implicated in the regulation of many immune and inflammatory responses in vivo, and in addition exerts a wide range of effects on target cells in vitro. However, although two cell surface receptors for TNF have been identified, and their cDNAs cloned, the amino acid residues necessary for the biological activity of TNF have not been characterized. We have therefore constructed derivatives of TNF termed 'muteins', in which the first 3 to 7 amino acids of native TNF-alpha have been replaced, using synthetic cDNA expressed in E. coli. In the present study we compare the effects of native TNF-alpha and muteins III, IV, V and VI in several different in vitro systems and in one in vivo model. We observed binding to the p75 TNF receptor on Jijoye Burkitt lymphoma cells with native TNF-alpha and mutein III alone, whereas the p55 TNF receptor on the human epithelioid carcinoma cell line HeLa bound TNF-alpha, mutein III and mutein V. Muteins IV and VI failed to recognize either TNF receptor. WEHI 164 fibrosarcoma cells were killed by muteins III, V and VI. Human umbilical vein endothelial cells responded to native TNF-alpha and to muteins III, IV and V, but not to mutein VI, by increasing the surface expression of ICAM-1 antigen and secretion of the cytokines GM-CSF and IL-6. All four compounds were pro-inflammatory in a mouse in vivo model. The results presented in this report confirm that N-terminal amino acids are critical for both receptor binding and biological activity of TNF-alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Type

Journal article

Journal

Cytokine

Publication Date

03/1993

Volume

5

Pages

125 - 132

Keywords

Amino Acid Sequence, Animals, Binding Sites, Cell Adhesion Molecules, Cells, Cultured, Endothelium, Vascular, Granulocyte-Macrophage Colony-Stimulating Factor, Humans, Inflammation, Intercellular Adhesion Molecule-1, Interleukin-6, Male, Mice, Mice, Inbred BALB C, Molecular Sequence Data, Receptors, Cell Surface, Receptors, Tumor Necrosis Factor, Tumor Cells, Cultured, Tumor Necrosis Factor-alpha