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

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 LF 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.

Sendai virus induces high levels of tumor necrosis factor mRNA in human peripheral blood leukocytes

“…Cytotoxins produced by the immurne system are a heterogeneous group of proteins, the nature of which depends upon the variety of inducing agent and target cell involved (16)(17)(18)33). As the cytotoxin TNF was originally named for a protein produced in erndotoxin-treated mice (1), most studies of TNF have focused on induction by endotoxin, although certain mitogens and phorbol myristate acetate have also been shown to induce TNF (34).…”

“…That report, however, may offer evidence for the existence of a cytotoxin closely-related to the cloned TNF, since the majority of the cytotoxic activity in that study eluted on a gel filtration column with a relative molecular mass of approximately 14,000 daltons. Cloned TNF has a monomer size of 17,000 daltons and usually behaves as a 40,000 to 45,000 dalton multimer on gel filtration columns (2,5,33).…”

Tumor necrosis factor: specific binding and internalization in sensitive and resistant cells.

“…TNF is functionally defined as a protein producing hemorrhagic necrosis of some tumors in experimental animals and cytolytic or cytostatic effects in tumor cells in culture. Since the major cellular source of TNF is the macrophage (1)(2)(3)(4)(5)(6), TNF is thought to be a mediator of the cytotoxic activity of macrophages against tumor cells (7). Recently, human TNF was purified to homogeneity (8) and cDNA for human TNF was cloned, sequenced, and expressed in Escherichia coli (9)(10)(11).…”

Evaluation of recombinant human tumor necrosis factor by scheduled intratumoral administration in mice bearing transplantable tumors.

“…Tumor necrosis factor (TNF), a cytokine released by activated monocytes and/or macrophages, shows in vitro cytotoxicity to a wide variety of human and animal malignant cells including cell lines such as mouse L cells, P388, HL-60, ML-1, HeLa, and Raji and fresh cells of cancer patients in primary culture (Carswell et al 1975; Ha et al 1985; Haranaka et al 1985; Kull et al 1985; Watanabe et al 1985; Yoshie et al 1986) and causes hemorrhagic necrosis followed by tumor regression in transplanted tumors in mice (Matthews 1981(Matthews , 1983; Fisch and Gifford 1983; Kelker et al 1985). Human TNF has been purified and the cDNA was cloned and expressed in Escherichia coli in several laboratories (Pennica et al 1984; Marmenout et al 1985; Shirai et al 1985).…”