Bioactive human recombinant tumor necrosis factor‐α: an unstable dimer?

Abstract

As determined by native polyacrylamide gel electrophoresis (PAGE) and gel chromatography the molecular mass of native tumor necrosis factor (TNF)-alpha was approximately 35 kDa. When incubated at low concentrations (less than 1 nM) 125I-labeled TNF-alpha and unlabeled TNF-alpha rapidly multimerized or dissociated into monomers and bioactivity decreased. Sodium dodecyl sulfate (SDS)-PAGE analysis of cross-linked 125I-labeled TNF-alpha demonstrated bands of multi- and trimeric TNF-alpha in addition to dominating bands of dimers and monomers. Tri-, di- and monomeric TNF-alpha were recovered from SDS-PAGE gels and allowed to renature. Of the original receptor-binding activity, 10%-15% was obtained with cross-linked TNF-alpha dimers, whereas none was recovered from preparations of trimeric TNF-alpha. Multimeric and monomeric TNF-alpha exhibited little or no binding activity, and cell-bound, cross-linked TNF-alpha which was dissociated from cellular binding sites was mainly dimeric. 125I-labeled TNF-alpha bound to lymphokine-activated killer (LAK) cells and binding kinetics were much similar (Kd approximately 100 pM) to those reported in other normal cell types. The number of receptors per LAK cell was approximately 4 x 10(3). Cross-linking of TNF-alpha to binding sites in U-937 and LAK cells yielded a receptor-ligand complex of about 80/90 kDa. At 37 degrees C, 125I-labeled TNF-alpha was rapidly internalized and degraded in L-929, U-937 and LAK cells. Degradation of ligand and recycling of receptors were blocked in the presence of methylamine. Methylamine significantly inhibited TNF-alpha-mediated cytolysis of L-929 cells and caused a quantitatively corresponding reduction in cellular TNF-alpha uptake, indicating that L-929 lysis was mediated by receptors.

Homogeneous Expansion of Human T-Regulatory Cells Via Tumor Necrosis Factor Receptor 2

“…While animal studies indicate that TNF induces proliferation of T regs , the evidence in humans, both in vivo and in vitro , is conflicting and the topic for review articles attempting to explain the contradictory literature3233. It has been speculated that the conflicting findings could be due in part to the manufacturing problems with TNF and its tendency in culture to aggregate into different complexes, perhaps with different signaling consequences34. Also TNF has two receptors, TNFR1 and TNFR2, each of which controls different signaling pathways.…”

Effect of Cytokines on Sperm Motility and Ionophore—Stimulated Acrosome Reaction

“…The reason might be that dimers andor trimers, which seem to be the bioactive form of TNF [24] and therefore the ones measured in the TNF bioassay used in this study, do not penetrate the dialysis bags. Nevertheless, a great number of other substances are produced from the mononuclear WBC, and therefore a possible effect on acrosome reactivity could not be excluded in advance.…”

“…Both TNF-a and IL-8 were stable during incubation for up to 24 h in the relevant dilution buffers used, while the activity of IFN-y was reduced to about 50% after 24 …”

Abdominal surgery reduces the ability of rat spleen cells to synthesize and secrete active tumour necrosis factor-alpha (TNF-α) by a multilevel regulation

“…Addition of LPS to these cells increased the expression of the 26-kD form of TNF-a and the 17-kD form of the protein in the cellular extracts by 2´15 6 0´3-fold (P < 0´002) and by 1´7 6 0´13-fold (P < 0´003), respectively. Another band of molecular weight of 33 kD, which probably represented the intracellular dimeric intermediate of TNF-a protein [21], also increased by 1´4 6 0´3-fold in the control splenocytes induced with LPS. This increase did not reach statistical signi®cance.…”