Characterization of human tumor necrosis factor produced by peripheral blood monocytes and its separation from lymphotoxin

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Abstract
Cultures of human peripheral blood leukocytes (PBL) induced with phytohemagglutinin (PHA) and the phorbol ester 12‐0‐tetradecanoylphorbol 13‐acetate (TPA) produced two types of cytotoxic proteins, indistinguishable in the in vitro assay employing murine L 929 cells as targets. One of these proteins had the antigenic and physicochemical properties of lymphotoxin (LT). We have identified the other cytotoxin as tumor necrosis factor (TNF), mainly on the basis of antigenic cross‐reactivity demonstrated with antiserum to TNF, and also by its characteristic physicochemical properties and cell source. Unlike LT, PBL‐derived TNF did not bind to Concanavalin A‐Sepharose or to several other agglutinin‐Sepharose columns specific for carbohydrate moieties common in glycoproteins. The molecular weight of native TNF determined by gel filtration was approximately 40,000 while SDS‐PAGE revealed a single sharp peak of 16,500 ± 500. When cultures of monocytes and lymphocytes separated by elutriation were stimulated with PHA and/or TPA, monocytes were the major source of TNF. In contrast, only lymphocytes produced LT. A mixture of antisera to TNF and LT neutralized all cytotoxicity of crude human lymphokine preparations for L 929 cells, suggesting that TNF and LT are either the only, or the major, cytotoxic proteins present in such crude lymphokine preparations demonstrable in this assay.