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

Abstract

Interleukin 1 (IL-1) is a lymphokine secreted by monocytes in response to a variety of inflammatory stimuli. IL-1fB the predominant form of IL-1 produced by human monocytes, is synthesized as an inactive precursor of 31 kDa and is cleaved at Asp"'6-Ala"17 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) provided the first substantive evidence that, in a mouse monocyte cell line, IL-1 was synthesized as a cell-associated precursor that could be chased into an extracellular 17-kDa form. Subsequently, reports emerged which suggested that a 31-kDa form of IL-1i3 was associated with human monocytes (2-5) and that this material was cleaved to release the mature form (2, 4, 5). These studies were corroborated by cDNA sequence data from a number of species which indicated that IL-1 mRNA encodes a larger protein than that identified as mature secreted IL-1 (6-10). As precursor IL-183 (pre-IL-1,j)is unable to bind to IL-1 receptors and is biologically inactive (11), some form of proteolytic processing is apparently required to release active IL-1p8. While the kinetics of IL-1 synthesis and secretion has been analyzed in some detail, little has been uncovered about the mechanism by which IL-1 is synthesized, processed, and secreted. Analysis ofthe predicted amino acid sequence from pre-IL-1,3 cDNA has not revealed the presence of a unique hydrophobic signal sequence domain, common to most secreted proteins (6)(7)(8)(9)(10)12). The N-terminal amino acid of mature monocyte IL-1p from humans has been sequenced by a number of investigators as Ala"17 (6, 13), suggesting that a cleavage site exists between Asp'6 and Ala"7. While the first 116 residues may be considered a signal sequence of sorts, it is not recognized as such by otherwise competent endoplasmic reticulum membranes (G.L., unpublished observation). Young et al. (14) showed that mature pre-IL-1P was not secreted from hamster fibroblasts that were stably transformed with pre-IL-1,i cDNA. Instead, large amounts of the precursor accumulated in the cytoplasm of the cell (14). Lomedico et al. (12) The processing of IL-1f3 has recently been investigated by using purified recombinant precursor as a substrate (5, 17).Hazuda et al. (5) showed that pre-IL-1f3, when added to intact human blood monocytes, was not cleaved or processed in any fashion, arguing against an extracellular site of processing. In another report, a potential pre-IL-1,8 cleavage activity was identified in a pelletable compartment of KG-1 cells, a neutrophil-like cell line. This enzymatic activity was able to generate IL-1 activity of similar size to authentic IL-1 from a partially purified pre-IL-1f3 substrate (17). However, the products were not sequenced and the site ofcleavage was not identified.In this report, we describe an in vitro processing system in which mature 17.

Inflammatory caspases and inflammasomes: master switches of inflammation

“…30,31 A technical breakthrough in the identification of the mechanisms of proIL1b maturation was the discovery an in vitro assay to monitor proIL-1b maturation. 2 By this assay, mature IL-1b can be generated by incubation of proIL-1b with partially fractionated extracts from human monocytes or the monocyte-like cell line THP-1. This assay was used to biochemically purify and sequence the IL-1b converting enzyme (ICE) or caspase-1.…”

Caspases as the Key Effectors of Inflammatory Responses Against Bacterial Infection

“…In 1989, caspase-1 was first identified as ''monocyte specific pre-interleukin 1b convertase'' called as ICE, in cell lysates of human monocytes (Black et al 1989b;Kostura et al 1989). The cDNA encoding ICE (caspase-1) was cloned in 1992 (Cerretti et al 1992;Thornberry et al 1992).…”

“…The first caspase identified in mammalian cells was the interleukin (IL)-1b-converting enzyme (ICE, now known as caspase-1). ICE cleaves pro-IL-1b to yield active cytokine; however, it does so in the absence of apoptosis induction (Black et al 1989a;Kostura et al 1989). Based on multiple reports revealing the functional roles and activation mechanisms of caspases, caspases are now divided into three functional groups: apoptosis induction (caspase-2, -3, -6, -7, -8, -9, and -10) (Guo et al 2002;Kischkel et al 2001;Parrish et al 2013), inflammatory responses (caspase-1, -4, -5, and -12) (Bian et al 2011;Black et al 1989a;Kostura et al 1989;Lamkanfi et al 2004;Sollberger et al 2012), and differentiation (caspase-14) (Denecker et al 2008) (Fig.…”

Selective induction of apoptosis in Hep 3B cells by topoisomerase I inhibitors: evidence for a protease-dependent pathway that does not activate cysteine protease P32.

“…Standard curves were generated with AMC and AFC (40). Cytosolic extracts from the THP-1 human monocytic leukemia cell line and from etoposidetreated HL-60 cells were used as positive controls for ICE-like and CPP32-like protease activity, respectively (41)(42)(43).…”