Characterization of a tumor necrosis factor alpha (TNF-alpha) inhibitor: evidence of immunological cross-reactivity with the TNF receptor.

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Abstract
Previous studies have shown that urine of febrile patients contains a tumor necrosis factor .alpha. inhibiting activity (TNF-.alpha. Inh) when tested in a cytotoxicity assay using the tumor necrosis factor .alpha.(TNF-.alpha.)-susceptible cell line L929. In the present study, we investigate the relationship between the TNF-.alpha. Inh and a potential soluble form of the receptor, as the former has been shown to block TNF-.alpha. activities by binding to the ligand. We demonstrate that human TNF-.alpha. is affected to a greater extent than is murine TNF-.alpha.. This species specificity of the inhibitor correlates with the binding studies of TNF receptor interactions already reported. We raised a polyclonal antibody to TNF-.alpha. Inh that neutralizes its activity and does not recognize TNF-.alpha.. Solubilized cross-linked 125I-labeled TNF-.alpha. receptor complex could be immunoprecipitated by using either anti-TNF-.alpha. or anti-TNF-.alpha. Inh antibody, suggesting immunological cross-reactivity between the receptor and the inhibitor. By using fluorescein isothiocyanate-coupled TNF-.alpha., it was possible to visualize by fluorescence-activated cell sorter analysis the TNF-.alpha. receptor on phytohemagglutinin/interleukin 2-activated T cells. A similar increase of immunofluorescence intensity of the activated T cells was observed by using anti-TNF-.alpha. Inh antibody revealed with a fluorescein isothiocyanate-coupled goat anti-rabbit IgG1 conjugate, suggesting that the TNF-.alpha. Inh is also expressed as a membrane protein. Taken together, our results suggest that the TNF-.alpha. Inh originally described might be a soluble form of the TNF receptor itself.