Mechanisms of caspase-1 activation by P2X7receptor-mediated K+release

Abstract

The mechanisms underlying caspase-1 activation and IL-1β processing during inflammatory activation of monocytes and macrophages are not well defined. Here, we describe an in vitro proteolytic processing assay that allows for comparison of caspase-1 regulatory components in a cell-free system separately from the confounding issue of IL-1β secretion. Analysis of in vitro IL-1β and caspase-1 processing in lysates from unstimulated Bac1 murine macrophages indicated a slow rate of basal caspase-1 activation and proteolytic maturation of IL-1β. In contrast, brief (5 min) treatment of intact macrophages with extracellular ATP (as an activator of the P2X7receptor) or nigericin before cell lysis markedly accelerated the in vitro processing of caspase-1 and IL-1β. This acceleration of in vitro processing was strictly dependent on loss of intracellular K+from the intact cells. The induction of in vitro caspase-1 activation by lysis per se or by K+loss before lysis was sensitive to pretreatment of intact macrophages with the tyrphostin AG-126 or bromoenol lactone, an inhibitor of Ca2+-independent phospholipase A2. Caspase-1 activation and IL-1β processing in lysates from unstimulated macrophages were also accelerated by addition of recombinant ASC, a previously identified adapter protein that directly associates with caspase-1. These data indicate that increased K+efflux via P2X7nucleotide receptor stimulation activates AG-126- and bromoenol lactone-sensitive signaling pathways in murine macrophages that result in stably maintained signals for caspase-1 regulation in cell-free assays.