ISG15 is a ubiquitin-like protein that functions in innate immunity both as an intracellular protein modifier and as an extracellular signaling molecule that stimulates IFN-γ secretion. The extracellular function, important for resistance to mycobacterial disease, has remained biochemically uncharacterized. We have established an NK-92 cell-based assay for IFN-γ release, identified residues critical for ISG15 signaling, and identified the cell surface receptor as LFA-1 (CD11a/CD18; αLβ2 integrin). LFA-1 inhibition blocked IFN-γ secretion, splenocytes from CD11a^−/− mice did not respond to ISG15, and ISG15 bound directly to the αI domain of CD11a in vitro. ISG15 also enhanced secretion of IL-10, indicating a broader role for ISG15 in cytokine signaling. ISG15 engagement of LFA-1 led to the activation of SRC family kinases (SFKs) and SFK inhibition blocked cytokine secretion. These findings establish the molecular basis of the extracellular function of ISG15 and the initial outside-in signaling events that drive ISG15-dependent cytokine secretion.

ISG15是一种泛素样蛋白，具有先天免疫功能，既可以作为细胞内蛋白修饰剂，又可以作为刺激IFN-γ分泌的细胞外信号分子。对于抵抗分枝杆菌疾病重要的细胞外功能在生化上仍未表征。我们建立了基于NK-92细胞的IFN-γ释放测定，鉴定了对ISG15信号传导至关重要的残基，并将细胞表面受体鉴定为LFA-1（CD11a / CD18；αLβ2整联蛋白）。LFA-1抑制可阻断IFN-γ的分泌，CD11a ^-/-小鼠的脾细胞对ISG15无反应，并且ISG15在体外直接与CD11a的αI结构域结合。ISG15还增强了IL-10的分泌，表明ISG15在细胞因子信号传导中具有更广泛的作用。LSG-1的ISG15参与导致SRC家族激酶（SFK）的激活和SFK抑制阻止了细胞因子的分泌。这些发现建立了ISG15胞外功能的分子基础以及驱动ISG15依赖性细胞因子分泌的最初的由外而内的信号事件。