Interleukin-12 (IL-12) and IL-18 synergistically induce the fungicidal activity of murine peritoneal exudate cells against Cryptococcus neoformans through production of gamma interferon by natural killer cells

We examined the ability of interleukin-12 (IL-12) and IL-18 to induce the production of gamma interferon (IFN-γ) and nitric oxide (NO) by murine peritoneal exudate cells (PEC) and to stimulate the growth-inhibitory activity of these cells against Cryptococcus neoformans. PEC produced IFN-γ and NO when stimulated with a combination of IL-12 and IL-18 but little or no IFN-γ or NO when either cytokine was used alone. PEC anticryptococcal activity was mediated by IFN-γ and NO production, since it was completely inhibited by a neutralizing anti-IFN-γ monoclonal antibody (MAb) and N(G)-monomethyl-L-arginine, a competitive inhibitor of NO synthesis, respectively. To identify the IFN-γ- producing cells among PEC stimulated with IL-12 and IL-18, we depleted NK cells, γδ T cells, or CD4⁺ T cells by treating PEC with specific Abs and complement. NK cell depletion strongly suppressed IFN-γ production and almost completely inhibited NO production and anticryptococcal activity, while depletion of other cells had no such influence. Alternatively, purified NK cells by two cycles of glass adherence and magnetic separation with anti- CD3, -CD4, -CD8, and -B220 MAbs produced a greater amount of IFN- γ by stimulation with IL-12 and IL-18 than unseparated non-glass- adherent PEC. Our results demonstrated that IL-12 and IL-18 synergistically induced NO-dependent anticryptococcal activity of PEC by stimulating NK cells to produce IFN-γ.