Tumour-necrosis factor from the rabbit. I. Mode of action, specificity and physicochemical properties

Abstract

Summary.-Sera from rabbits injected with BCG and then with endotoxin contain a factor (tumour-necrosis factor TNF) which, even at high dilutions, is cytotoxic in vitro for mouse L cells and some other cell lines. Using a 51Cr-release assay, cytotoxicity was detected as early as 7-8 h after addition of TNF serum to L cells and cell death was evident microscopically by 24 h. TNF was cytotoxic at 37°C but not at 21°C or 4°C, and acted on both dividing and non-dividing cells. The antimetabolites sodium azide and dinitrophenol partially protected L cells from TNF, suggesting that actively metabolizing cells are the most sensitive. Treatment of L cells with trypsin did not delay cytotoxicity nor was cytotoxicity inhibited in the presence of various saccharide derivatives of cell-surface glycoproteins. Rabbit TNF was remarkably stable with a mol. wt. of 40-50,000. It was eluted with the more acidic serum proteins on ion-exchange chromatography, but precipitated in 50o-saturated ammonium sulphate. Sensitivity to TNF could not be correlated with tumourigenicity of several animal and human lines tested nor with the production of C-type viruses.

Tumour-necrosis factor from the rabbit. III. Relationship to interferons

“…Only some cell lines are affected by TNF, and there is no correlation with the production of C-type or other viruses, tumorigenicity, growth rate or mycoplasma contamination (Matthews & Watkins, 1978, and unpublished observations).…”

“…Further evidence that the TNF and interferon in TNF serum are separate entities is that TNF purified sequentially by ammonium sulphate precipitation, ionexchange chromatography and gel filtration (Matthews & Watkins, 1978) had an interferon titre of < 160 but a TNF titre of 5120.…”

“…Rabbit TNF inhibited the growth of a human melanoma cell line, and also had effects on certain mouse and rabbit cell lines, whereas the anti-cellular effects of interferons are reported to be species-specific. TUMOUR-NECROSIS FACTOR (TNF) is a substance which causes necrosis of some transplantable tumours (Carswell et al, 1975) and is cytotoxic or growth-inhibitory in vitro to a number of cell lines (Carswell et al, 1975;Matthews & Watkins, 1978). TNF is released into the blood after endotoxin injection in animals which have been pretreated with agents such as Bacillus Calmette-Guerin (BCG) or Corynebacterium parvum, which induce macrophage hyperplasia.…”

Differences in tumour necrosis factor productive ability among rodents

“…(Algire et al, 1952). In contrast to LPS, TNF displays a direct cytotoxic activity for cultured tumour cells of both mouse and human origin Matthews & Watkins, 1978;Old, 1976). The hypothesis that LPS causes release of a tumouricidal factor, TNF, from activated macrophages has been confirmed (Matthews, 1978;Mannel et al, 1980;Satomi et al, 1981).…”

“…TNF exhibits a direct cytotoxicity against murine or human cancer cells in vitro but has no cytotoxicity against normal or embryonal cells Matthews & Watkins, 1978;Old, 1976). The in vitro cytotoxic activity is of particular interest because the observed properties of tumour cell sensitivity and species independence are characteristic of the in vitro toxic activity of activated macrophages (Oettgen et al, 1980;Piessens et al, 1957).…”