Production and mechanism of secretion of interleukin-1b from the marine fish gilthead seabream

Abstract

Interleukin-1b (IL-1b) is a secretory cytokine lacking a signal peptide, and does not follow the classical endoplasmic reticulum to Golgi pathway of secretion. Its post-translational processing by IL-1b-converting enzyme (ICE) and subsequent release from activated macrophages requires ATP acting on P2X7 receptors. No information is available on the production and release of fish IL-1b, but the IL-1b gene sequences reported to date lack a conserved ICE recognition site. We show for the first time that lipopolysaccharide (LPS)/macrophage-activating factor (MAF)/bacterial DNA (VaDNA)-primed immune cells of fish accumulate intracellular IL-1b as a ~30 kDa polypeptide (proIL-1b). The combination of LPS and VaDNA was found to be synergistic, suggesting that each ligand is recognized by a different pattern recognition receptor (PRR). More importantly, addition of extracellular ATP does not promote IL-1b secretion by immune cells and fails to induce phosphatidylserine (PS) flip. In contrast, fish SAF-1 fibroblasts shed microvesicles containing a 22 kDa IL-1b form within 30 min of activation with ATP. Notably, the post-translational processing of IL-1b by SAF-1 cells is abrogated by a specific ICE inhibitor.