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.

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE) is a novel cysteine protease that cleaves the 31-kD inactive cytoplasmic IL-1 beta precursor into active extracellular 17-kD IL-1 beta. 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 p10 subunits. Generation of active ICE is affected by the removal of an 11-kD NH2-terminal precursor domain (p11) 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-1 beta 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 p11 epitopes were not detected on the surfaces of these monocytes. Likewise, labeling of fixed ultrathin cryosections of monocytes 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-1 beta were both localized upon the cell membrane after stimulation of IL-1 beta secretion. Lipopolysaccaride-primed monocytes that synthesized, but did not secrete IL-1 beta, exhibited only cytoplasmic staining. The data suggests that mature IL-1 beta is generated via cleavage of the 31-kD inactive cytoplasmic IL-1 beta precursor by ICE after association with the plasma membrane during secretion.

IL-1 beta-converting enzyme is present in monocytic cells as an inactive 45-kDa precursor.

The major form of IL-1 beta-converting enzyme (ICE) identified in THP.1 monocytic cells and human monocytes is the 45-kDa precursor protein (p45), which is found in the cytoplasm. Cytoplasmic extracts of these cells show no pIL-1 beta cleavage activity, indicating that the p45 has no detectable catalytic activity. pIL-1 beta cleavage activity can only be observed after incubation in vitro when p45 breaks down to the active p20 form of the enzyme. LPS stimulation of human monocytes or THP.1 monocytic cells results in no change in the amount of p45 or its activity and no detectable appearance of p20 ICE. Immunoprecipitation of [35S]Met-labeled LPS-stimulated monocyte extracts revealed only p45 with no other co-precipitating protein. The inability to identify active ICE in stimulated monocytic cells was probably a reflection of the very low levels of active ICE present.

Dissociation of IL-1 beta synthesis and secretion in human blood monocytes stimulated with bacterial cell wall products.

Human blood monocytes synthesize but do not secrete IL-1 beta in response to low doses of bacterial cell-wall products. With this observation, we have developed a two-step staged release assay that separates IL-1 beta synthesis from secretion. This assay can be used to identify secretagogues or inhibitors of IL-1 beta secretion as well as the biochemical events leading to IL-1 beta release. Human blood monocytes are first treated with low (< 50 pg/ml) doses of LPS, which causes the synthesis of intracellular proIL-1 beta. Release of intracellular IL-1 beta can be induced by further treatment with 100 ng/ml of LPS or 1 x 10(6) CFU/ml of heat-killed Staphylococcus aureus. The amount and the efficiency of IL-1 beta secretion in the staged release assay was comparable with that of a standard method of treating blood monocytes with a single dose of 100 ng/ml LPS. Ongoing protein synthesis was not required for IL-1 beta secretion because mature IL-1 beta release occurred in the presence of the protein synthesis inhibitor cycloheximide. We have compared the effects of four different inhibitors of cytokine synthesis on IL-1 beta production in the standard and staged release assays. We find that dexamethasone or IL-10, when added together with 100 ng/ml LPS, inhibits IL-1 beta production with IC50 levels of 0.2 microM and 2.0 ng/ml, respectively. The IC50 levels increase greater than 50-fold when tested against monocytes pretreated with 50 pg/ml of LPS. These data suggest that dexamethasone and IL-10 have little effect on IL-1 beta secretion. Conversely, the IL-1 beta converting enzyme inhibitor, Ac-Tyr-Val-Ala-Asp-CHO (L-709,049) and the anti-inflammatory agent [5-(4-pyridyl)6(4-fluorophenyl)-2,3-dihydroimidazo(2,1-b)thiazole] (SK&F 86002) inhibited IL-1 beta release in both the standard and staged release assays with IC50 of 1 microM. The data suggest that L-709,049 and SK&F 86002 interfere with steps involved in IL-1 beta secretion, as opposed to synthesis. The results also document a novel method for delineating separate events in the pathway required for IL-1 beta biosynthesis and further distinguish two classes of compounds capable of modulating IL-1 beta secretion.

A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes.

Interleukin-1 beta (IL-1 beta)-converting enzyme cleaves the IL-1 beta precursor to mature IL-1 beta, an important mediator of inflammation. The identification of the enzyme as a unique cysteine protease and the design of potent peptide aldehyde inhibitors are described. Purification and cloning of the complementary DNA indicates that IL-1 beta-converting enzyme is composed of two nonidentical subunits that are derived from a single proenzyme, possibly by autoproteolysis. Selective inhibition of the enzyme in human blood monocytes blocks production of mature IL-1 beta, indicating that it is a potential therapeutic target.

IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31-kDa IL-1 alpha.

IL-1 converting enzyme (ICE) specifically cleaves the human IL-1 beta precursor at two sequence-related sites: Asp27-Gly28 (site 1) and Asp116-Ala117 (site 2). Cleavage at Asp116-Ala117 results in the generation of mature, biologically active IL-1 beta. ICE is unusual in that preferred cleavage at Asp-X bonds (where X is a small hydrophobic residue), has not been described for any other eukaryotic protease. To further examine the substrate specificity of ICE, proteins that contain Asp-X linkages including transferrin, actin, complement factor 9, the murine IL-1 beta precursor, and human and murine IL-1 alpha precursors, were assayed for cleavage by 500-fold purified ICE. The human and murine IL-1 beta precursors were the only proteins cleaved by ICE, demonstrating that ICE is an IL-1 beta convertase. Analysis of human IL-1 beta precursor mutants containing amino acid substitutions or deletions within each processing site demonstrated that omission or replacement of Asp at site 1 or site 2 prevented cleavage by ICE. To quantitatively assess the substrate requirements of ICE, a peptide-based cleavage assay was established using a 14-mer spanning site 2. Cleavage between Asp [P1] and Ala [P1']2 was demonstrated. Replacement of Asp with Ala, Glu, or Asn resulted in a greater than 100-fold reduction in cleavage activity. The rank order in position P1' was Gly greater than Ala much greater than Leu greater than Lys greater than Glu. Substitutions at P2'-P4' and P6' had relatively little effect on cleavage activity. These results show that ICE is a highly specific IL-1 beta convertase with absolute requirements for Asp in P1 and a small hydrophobic amino acid in P1'.

Probing the role of interleukin-1 beta convertase in interleukin-1 beta secretion.

Interleukin-1 beta must be processed from its precursor form of 31.5 kDa to its mature form of 17 kDa in order to elaborate its wide array of bioactivities. The recent identification of a monocyte-specific endoprotease, termed interleukin-1 beta-converting enzyme (ICE), capable of generating authentic, bioactive 17 kDa IL-1 beta suggests that this protease may serve a specific role in the processing and subsequent secretion of IL-1 beta. To test this hypothesis, we describe initial attempts to establish a monocytic cell-based system to test if mutant preIL-1 beta molecules which are poor substrates for ICE in vitro will be processed and secreted by monocytic cells.

Identification of a monocyte specific pre-interleukin 1 beta convertase activity.

Interleukin 1 (IL-1) is a lymphokine secreted by monocytes in response to a variety of inflammatory stimuli. IL-1 beta, the predominant form of IL-1 produced by human monocytes, is synthesized as an inactive precursor of 31 kDa and is cleaved at Asp116-Ala117 to yield a 17.5-kDa extracellular form. The exact cellular site of cleavage and mechanism of secretion is at present unknown. We have prepared cell-free postnuclear extracts from freshly isolated human monocytes as well as THP.1 cells, a human monocyte-like cell line, and various blood lymphocytes and fibroblast cell lines. Using pre-IL-1 beta synthesized by in vitro transcription and translation, we have shown that only extracts derived from human monocytes and THP.1 cells were capable of cleaving precursor IL-1 beta to authentic mature IL-1 beta. Subcellular fractionation of the extracts suggested that the processing activity is found in the cytosol of monocytes or monocyte-like cell lines. The cleavage product of this protease is identical to authentic IL-1 beta as shown by mobility on SDS/PAGE and amino acid sequence analysis of the [3H]leucine-labeled product. The cleavage product is also capable of binding to the IL-1 receptor found on fibroblast membranes. Finally, mutation of Asp116----Ala116 rendered the IL-1 beta precursor resistant to cleavage by the processing activity. We conclude that a protease activity found only in monocytes will specifically process IL-1 beta to an active form.