Enhancement of immunostimulatory activity by dual substitution of C8-substituted guanine ribonucleosides: correlation with increased cytokine secretion.

Abstract

Guanine ribonucleosides with single substitutions at the C8 position (monosubstituted) or with dual substitutions at the C8 and N7 positions (disubstituted) up-regulate a spectrum of immunologic responses, including cytolytic responses to tumor cells. The current studies were undertaken to determine the effects of dual substitution on a number of nucleoside-inducible immunological parameters. To do so, two monosubstituted analogues, 8-bromoguanosine and 8-mercaptoguanosine, were directly compared with two disubstituted analogues, 7-methyl-8-oxoguanosine and 7-allyl-8-oxoguanosine (loxoribine). All of the compounds enhance natural killer (NK) activity, lymphocyte proliferation, and antibody production in dose-dependent fashion. However, the potency and maximal activity of the disubstituted analogues are considerably greater than those of the monosubstituted analogues. Spleen cells stimulated for 48 h with the disubstituted compounds produce immunoreactive interleukin (IL) 1 alpha, IL-6, tumor necrosis factor-alpha (TNF alpha), and interferon-gamma (IFN gamma). Monosubstituted analogues induce lower quantities of IL-6, TNF alpha, and IFN gamma and fail to induce detectable levels of IL-1 alpha. Total IFN activity, assessed by viral inhibition assay, is also lower for the monosubstituted analogues. Augmentation of antibody secretion by B cells is diminished for neither mono- nor disubstituted compounds upon incubation with anti-cytokine antibodies. In contrast, anti-IFN alpha beta markedly reduces the effects of monosubstituted analogues on NK activity but has less marked effects on NK induction by the disubstituted compounds. A similar pattern of differences is seen for lymphocyte proliferation. Thus, although the analogues induce synthesis of several cytokines, to date only IFN alpha beta appears directly involved in enhancement of NK activity and lymphocyte proliferation. The present data do not, however, exclude the existence of an autocrine stimulatory mechanism not susceptible to inhibition by anti-cytokine antibodies.