ISOLATION OF AN INHIBITOR OF TUMOR NECROSIS FACTOR-ALPHA-MEDIATED CYTOTOXICITY LIBERATED FROM CHEMOTAXIN-STIMULATED EQUINE WHITE BLOOD-CELLPOPULATIONS

Objectives of this investigation were to extract and isolate protein f ractions inhibitory to the cytotoxic properties of tumor necrosis fact or-alpha (TNF-alpha). In this context, mixed populations of WBC were h arvested from equine blood and were stimulated with a combination of a synthetic chemotactic peptide and a calcium ionophore. Several method s were subsequently applied for the initial preparation of cell-free c rude protein extracts, including fractional precipitation with gradien t concentrations of ammonium sulfate and preparative-scale isoelectric focusing. In addition, protein fractions were harvested from extracts of concentrated equine urine. Protein extracts of urinary origin were further separated by gel-filtration column chromatography. Identifica tion of protein fractions possessing properties inhibitory to the cyto toxic characteristics of TNF-alpha was facilitated by a tissue culture -based technique for the biological assay of TNF-alpha-mediated cytoto xicity. Purified protein extracts possessed a marked ability to inhibi t or neutralize the cytotoxic properties of TNF-alpha, on the basis of survival of murine fibrosarcoma cell populations, compared with appro priate negative and positive reference controls. Relative purity of in hibitors and estimation of approximate molecular weight were establish ed by conventional reducing and nonreducing sodium dodecyl sulfate-pol yacrylamide gel electrophoresis analysis. Equine inhibitory protein fr actions from mixed WBC populations, purified in the manner described, had molecular weights of 70,000 to 80,000 and 28,000. An analogous pro tein fraction of 28 kDa also was isolated from equine concentrated uri ne. Estimated isoelectric point of TNF-alpha inhibitor protein fractio ns was between pH of 5.5 and 6.1. These physical characteristics of eq uine TNF-alpha inhibitor protein fractions were similar to those descr ibed for a membrane-associated TNF-alpha receptor protein shed from ch emotaxin- and calcium-ionophor-stimulated human WBC populations.