Post-Translational Processing of Murine IL-1: Evidence that ATP-Induced Release of IL-1α and IL-1β Occurs via a Similar Mechanism

Abstract

In response to LPS, peritoneal macrophages produce IL-1, but, for the most part, newly synthesized cytokine molecules remain cell associated. Externalization and proteolytic processing of pro-IL-1β can be initiated by extracellular ATP. In this study, kinetics and inhibitor sensitivity of the stimulus-coupled mechanism were investigated with [35S]methionine-labeled macrophages. Optimal ATP concentrations required to promote cytokine post-translational processing suggest the involvement of a P2Z type of receptor. Proteolysis of pro-IL-1β initiates within 7.5 min of ATP addition; 17-kDa mature IL-1β is observed first intracellularly and subsequently extracellularly. In contrast, ATP-treated cells do not contain 17-kDa IL-1α. Macrophages exposed to ATP continuously or only for a 15-min pulse release IL-1α, IL-1β, and lactate dehydrogenase (LDH). Proteolytic maturation of IL-1β exceeds that of IL-1α in both formats, but pulsed cells process the externalized cytokines more efficiently. Ethacrynic acid and DIDS (4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid) block ATP-induced proteolysis of pro-IL-1β and prevent release of pro-IL-1α/β and LDH; they do not inhibit ATP-induced K+ (86Rb+) efflux. Ethacrynic acid inhibits release of both forms of IL-1 with a similar concentration dependence; within the arrested cells, procytokines accumulate in a Triton-insoluble fraction. An IL-1β-converting enzyme inhibitor blocks proteolysis of IL-1β, but it does not prevent release of pro-IL-1α, pro-IL-1β, or LDH. These results indicate that ATP stimulates externalization of both IL-1α and IL-1β. The ATP-induced cytokine release mechanism is accompanied by cell death and requires activity of an anion transport inhibitor-sensitive component, but this pathway operates independently of cytokine proteolytic processing.