Post-translational processing of murine IL-1: evidence that ATP-induced release of IL-1 alpha and IL-1 beta occurs via a similar mechanism.

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-1 beta 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-1 beta initiates within 7.5 min of ATP addition; 17-kDa mature IL-1 beta is observed first intracellularly and subsequently extracellularly. In contrast, ATP-treated cells do not contain 17-kDa IL-1 alpha. Macrophages exposed to ATP continuously or only for a 15-min pulse release IL-1 alpha, IL-1 beta, and lactate dehydrogenase (LDH). Proteolytic maturation of IL-1 beta exceeds that of IL-1 alpha 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-1 beta and prevent release of pro-IL-1 alpha/beta 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-1 beta-converting enzyme inhibitor blocks proteolysis of IL-1 beta, but it does not prevent release of pro-IL-1 alpha, pro-IL-1 beta, or LDH. These results indicate that ATP stimulates externalization of both IL-1 alpha and IL-1 beta. 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.