Stimulation of human gamma interferon production by diterpene esters

Abstract

The diterpene ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and several structurally related compounds were tested for their ability to stimulate interferon (IFN) production in primary cultures of human leukocytes. In cultures of Ficoll-Hypaque-purified mononuclear cells, TPA treatment alone induced only low levels of IFN, but TPA pretreatment of cells caused significant enhancement of IFN yields produced with phytohemagglutinin or several other T cell mitogens. In cultures of unprocessed cells derived from plateletpheresis residues or buffy coats, TPA treatment alone induced high levels of IFN and costimulation with TPA and phytohemagglutinin produced some further enhancement of IFN production. Phorbol 12,13-dibutyrate was comparable to TPA in its ability to enhance phytohemagglutinin-induced IFN production. Several other phorbol ester analogs were also active, but maximal stimulation occurred only at higher drug concentrations. Mezerein, a structurally related diterpene ester, was at least as active as TPA in stimulating IFN production in either Ficoll-Hypaque-purified or unprocessed cells. IFN produced after stimulation with TPA or mezerein, singly or in combination with phytohemagglutinin, had several properties characteristic of IFN-gamma, e.g., it was largely inactivated by dialysis at pH 2, or after exposure to sodium dodecyl sulfate, whereas it was not neutralized by antibody to IFN-alpha and IFN-beta. The stimulatory effect of diterpene esters has proved helpful in producing IFN-gamma for physicochemical analysis and other studies.

Interrelationships of human interferon-gamma with lymphotoxin and monocyte cytotoxin.

“…Combined treatment with TPA and PHA produced a synergistic enhancement in the production of both lymphokine activities which was particularly marked in total cells derived from the plateletpheresis residue. It was noted earlier that cultures from buffy coats generally produce lower yields of IFN-gamma than cells from plateletpheresis residues (32). Nonadherent cells from bully coats were more responsive to all inducing treatments; nonadherent cells from plateletpheresis residues were more responsive to PHA and about equally responsive to the other inducing treatments as total cells.…”

“…Preparation of Conditioned Medium Containing IFN-gamma and Lymphotoxin. The procedure for obtaining high yields of IFN-gamma by combined stimulation of human PBL with the tumor-promoting agent, TPA, and the T cell mitogen, PHA, has been described (32). Briefly, plateletpheresis residues were washed once to remove plasma and seeded at a density of 6 x 106 white blood cells/ml in serum-free RPMI 1640 medium supplemented with gentamycin (50 #g/ml), Hepes (6 raM), Tricine (3 mM), and 20 ng/ml TPA.…”

“…Assay for IFN Activity. The antiviral activity of IFN-gamma was assayed by inhibition of the cytopathic effect of encephalomyocarditis virus in human FS-4 fibroblasts in 96well microtiter tissue culture plates as described elsewhere (32). An internal laboratory standard of human IFN-gamma was included in each assay and titers of IFN-gamma were expressed in laboratory units.…”

Evidence for a deficiency of interferon production in patients with chronic hepatitis B virus infection acquired in adult life

“…y-Interferon Production: Peripheral blood mononuclear cells (2 x lofi) in RPMI 1640 medium with 5% fetal calf serum were incubated with 5 ng per ml 12-O-tetradecanoyl phorbol 13 acetate (Sigma Chemical Co., St. Louis, Mo.) (13) and 5 mg per ml phytohemagglutinin (Wellcome Research Laboratories, TuckahOe, NY) for 48 hr as above.…”

Cytolytic activity of interferon‐gamma and its synergism with 5‐fluorouracil

“…Recombinant human IFN-gamma (rIFN-amma) with a specific activity of approximately al., 1982) was provided by Genentech (South San Francisco, CA). The antiviral activity of IFN-gamma was assayed by inhibition of the cytopathic effect of encephalomyocarditis virus in human FS-4 fibroblasts in 96-well plates (Have11 and VilEek, 1972;Yip et al, 1981). A laboratory standard for human IFN-gamma was included in the assays; all IFN-gamma titers reported correspond to the value of the NIH human IFNgamma standard Gg 23-901-530.…”