Colocalization of the IL-12 receptor and FcγRIIIa to natural killer cell lipid rafts leads to activation of ERK and enhanced production of interferon-γ

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Abstract
Natural killer (NK) cells express an activating receptor for the Fc portion of IgG (FcγRIIIa) that mediates interferon (IFN)–γ production in response to antibody (Ab)–coated targets. We have previously demonstrated that NK cells activated with interleukin-12 (IL-12) in the presence of immobilized IgG secrete 10-fold or more higher levels of IFN-γ as compared with stimulation with either agent alone. We examined the intracellular signaling pathways responsible for this synergistic IFN-γ production. NK cells costimulated via the FcR and the IL-12 receptor (IL-12R) exhibited enhanced levels of activated STAT4 and Syk as compared with NK cells stimulated through either receptor alone. Extracellular signal–regulated kinase (ERK) was also synergistically activated under these conditions. Studies with specific chemical inhibitors revealed that the activation of ERK was dependent on the activation of PI3-K, whose activation was dependent on Syk, and that sequential activation of these molecules was required for NK cell IFN-γ production in response to FcR and IL-12 stimulation. Retroviral transfection of ERK1 into primary human NK cells substantially increased IFN-γ production in response to immobilized IgG and IL-12, while transfection of human NK cells with a dominant-negative ERK1 abrogated IFN-γ production. Confocal microscopy and cellular fractionation experiments revealed that FcγRIIIa and the IL-12R colocalized to areas of lipid raft microdomains in response to costimulation with IgG and IL-12. Chemical disruption of lipid rafts inhibited ERK signaling in response to costimulation and significantly inhibited IFN-γ production. These data suggest that dual recruitment of FcγRIIIa and the IL-12R to lipid raft microdomains allows for enhanced activation of downstream signaling events that lead to IFN-γ production.

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