The Immunostimulatory Compound 7-Allyl-8-Oxoguanosine (Loxoribine) Induces a Distinct Subset of Murine Cytokines

C8- and N7,C8-substituted guanine ribonucleosides comprise a class of molecules with potent immunostimulatory activity for a variety of humoral and cellular immune responses. Although it has been suggested that the immunostimulatory activity may be partially mediated by cytokine production, to date there has been no systematic evaluation of the spectrum of cytokines elicited by these nucleosides. In this study, we examine the cytokines produced by murine spleen cells in response to the di-substituted guanosine analog loxoribine (7-allyl-8-oxoguanosine). First, the levels of cytokine mRNA in spleens from vehicle- or loxoribine-treated mice were compared using PCR analysis with a panel of cytokine-specific primers. Enhancement of IL-1α, TNF-α, TNF-β, IL-6, IFN-α, and IFN-γ mRNA was seen in the spleens of loxoribine-treated mice. IL-12 mRNA responses were more complex, with an increase in the p40 chain and a decrease in the p35 chain. In contrast, no increase was seen for mRNA levels of IL-2, IL-3, IL-4, IL-5, IL-7, or GM-CSF. ELISA assays on the supernatants of loxoribine-treated spleen cells demonstrated that IL-1α, IL-6, TNF-α, and IFN-γ were all produced in a dose-dependent fashion with TNF-α produced first, followed by IL-6 and IFN-γ, and last by IL-1α. IFN-αβ activity rose as quickly as TNF-α, leveling off at 8 to 12 hr, and was supplanted by a later-occurring surge of IFN-γ production. IL-1a, IL-6, TNF-α, and IFN-γ, were also detected in the sera of mice injected with loxoribine. When antibodies against the relevant cytokines were tested, only anti-IFN-αβ inhibited NK activity or lymphocyte proliferation and, in both cases, activity was partially restored by the addition of exogenous IFN-αβ. Taken together, these data indicate that loxoribine induces the production of a selective cohort of cytokines all of which have been shown to have immunostimulatory activity. However, only IFN-αβ appears to play a role in the enhancement of NK activity and lymphocyte proliferation.