The interleukin-1 beta-converting enzyme (ICE) is localized on the external cell surface membranes and in the cytoplasmic ground substance of human monocytes by immuno-electron microscopy.

Abstract

SummaryInterleukin-1 [3 (IL-l[3)-converting enzyme (ICE) is a novel cysteine protease that cleaves the 31-kD inactive cytoplasmic IL-113 precursor into active extracellular 17-kD IL-113. The ICE gene product is a 45-kD proenzyme that requires proteolytic processing to activate ICE. Active ICE is a heterodimer consisting of equal amounts of p20 and p l0 subunits. Generation of active ICE is affected by the removal of an 11-kD NH2-terminal precursor domain (pl 1) and an internal 19-amino acid sequence that separates the 20-and 10-kD subunits. Immuno-electron microscopy was performed on human monocytes with immunoglobulins recognizing the active (p20) or precursor (p11) domains of ICE. Elutriated monocytes were stimulated with 50 pM lipopolysaccharide followed by heat-killed Staphylococcus aureus under conditions that induce maximal rates of IL-113 secretion. Ultrathin cryosections were cut from fixed frozen pellets of these monocytes and were immunogold labeled with either antibody. Active and precursor domain ICE epitopes were localized in the cytoplasmic ground substance, but they were not detected within the endoplasmic reticulum, the Golgi apparatus, and secretory granules of activated or inactive monocytes. Importantly, numerous ICE p20 epitopes were also observed on the extracellular surfaces of the cell membrane, and were concentrated on the microvilli. Very similar patterns of ICE localization were obtained with unstimulated blood monocytes. In contrast, ICE pl I epitopes were not detected on the surfaces of these rnonocytes. Likewise, labeling of fixed ultrathin cryosections ofmonocytes with a biotinylated irreversible ICE inhibitor [Ac-Tyr-Val-Lys(biotin)-Asp-(acyloxy)-methyl-ketone] showed that the compound localized on the outer cell surface as well, and to a lesser extent, within the cytoplasmic ground substance. Furthermore, antipeptide antibodies specific for either the mature or precursor domains of IL-113 were both localized upon the cell membrane after stimulation of IL-113 secretion. Lipopolysaccaride-primed monocytes that synthesized, but did not secrete IL-113, exhibited only cytoplasmic staining. The data suggests that mature IL-l[3 is generated via cleavage of the 31-kD inactive cytoplasmic IL-lJ3 precursor by ICE after association with the plasma membrane during secretion.

Caspase-1-dependent processing of pro-interleukin-1β is cytosolic and precedes cell death

“…Shedding of microvesicles from the plasma membrane has also been proposed as a mechanism of secretion (MacKenzie et al, 2001). These proposals superseded previous models in which non-specific release due to cell lysis (Hogquist et al, 1991) and passage through a plasma membrane pore (Singer et al, 1995) was considered. However, there is evidence in the literature that can support or explain all of these mechanisms and there is, therefore, still controversy over how IL-1␤ makes its cellular exit.…”

The neuroprotective agents chlomethiazole and SB203580 inhibit IL‐1β signalling but not its biosynthesis in rat cortical glial cells

“…Effects of CMZ or SB203580 on IL-1b release by cortical glial cells Cleavage of the IL-1b precursor protein is usually coupled to secretion (Singer et al 1995). In order to analyse the levels of IL-1b protein exocytosis we performed ELISA in the supplement medium after stimulating the cortical glial cells with LPS for 12-72 h. The increase of the IL-1b protein release was concomitant with the increase of the IL-1b protein precursor levels but showed lower inducibility.…”

Apoptosis-Associated Speck-Like Protein Containing a Caspase Recruitment Domain Is a Regulator of Procaspase-1 Activation

“…To prevent caspase-1-induced apoptosis when overexpressed, cysteine 285 of the catalytic domain of procaspase-1 was mutated to alanine (30). GFP-tagged procaspase-1 was distributed mainly to the nucleus and cell membrane of cells, as reported previously (31)(32)(33). In contrast, a control protein consisting of GFP fused to the PAAD domain of PAN10 was located diffusely throughout the cytoplasm of cells (Fig.…”