Investigating the factors that regulate the rate of intracellular IL-1β degradation (IRM12P.652)

Abstract

Abstract Interleukin (IL)-1β is a key player in the initiation and orchestration of inflammation. The secretion of IL-1β requires 2 independent processes; an initial signal to induce the upregulation of the inactive precursor (pro-IL-1β) and, a second signal to drive cleavage and subsequent secretion. In previous investigations using murine dendritic cells, we have demonstrated that in lipopolysaccharide-stimulated cells, pro-IL-1β is polyubiquitinated and degraded by the proteasome, resulting in a rapid reduction in the amount of intracellular IL-1β available for secretion. The current study has employed an immortalized bone marrow derived murine macrophage cell line that stably expresses fluorescent IL-1β to track the rate of degradation. Here, proteasome inhibition using MG132 led to an increase in cell fluorescence compared with baseline expression, as measured by flow cytometry. This was coupled with a corresponding increase in IL-1β protein levels, as measured by ELISA, and an increase in polyubiquitinated IL-1β, as detected by Western blot. These data indicate that polyubiquitination and subsequent proteasomal degradation of IL-1β are also features of macrophages and independent of cell activation. Furthermore, fluorescence is a reliable readout for measuring degradation in this cell line and thus, represents a useful tool to measure factors that regulate the rate of IL-1β degradation and potentially, the potency of the inflammatory response.