Interferon-gamma-inducing factor (IGIF, interleukin-18) is a recently described cytokine that shares structural features with the interleukin-1 (IL-1) family of proteins and functional properties with IL-12. Like IL-12, IGIF is a potent inducer of interferon (IFN)-gamma from T cells and natural killer cells. IGIF is synthesized as a biologically inactive precursor molecule (proIGIF). The cellular production of IL-1beta, a cytokine implicated in a variety of inflammatory diseases, requires cleavage of its precursor (proIL-1beta) at an Asp-X site by interleukin-1beta-converting enzyme (ICE, recently termed caspase-1). The Asp-X sequence at the putative processing site in proIGIF suggests that a protease such as caspase-1 might be involved in the maturation of IGIF. Here we demonstrate that caspase-1 processes proIGIF and proIL-1beta with equivalent efficiencies in vitro. A selective caspase-1 inhibitor blocks both lipopolysaccharide-induced IL-1beta and IFN-gamma production from human mononuclear cells. Furthermore, caspase-1-deficient mice are defective in lipopolysaccharide-induced IFN-gamma production. Our results thus implicate caspase-1 in the physiological production of IGIF and demonstrate that it plays a critical role in the regulation of multiple proinflammatory cytokines. Specific caspase-1 inhibitors would provide a new class of anti-inflammatory drugs with multipotent action.

Recent studies have shown that protein kinase C (PKC) δ is proteolytically activated at the onset of apoptosis induced by DNA-damaging agents, tumor necrosis factor, and anti-Fas antibody. However, the relationship of PKCδ cleavage to induction of apoptosis is unknown. The present studies demonstrate that full-length PKCδ is cleaved at DMQD330N to a catalytically active fragment by the cysteine protease CPP32. The results also demonstrate that overexpression of the catalytic kinase fragment in cells is associated with chromatin condensation, nuclear fragmentation, induction of sub-G1 phase DNA and lethality. By contrast, overexpression of full-length PKCδ or a kinase inactive PKCδ fragment had no detectable effect. The findings suggest that proteolytic activation of PKCδ by a CPP32-like protease contributes to phenotypic changes associated with apoptosis.

The baculovirus antiapoptotic protein p35 inhibited the proteolytic activity of human interleukin-1 beta converting enzyme (ICE) and three of its homologs in enzymatic assays. Coexpression of p35 prevented the autoproteolytic activation of ICE from its precursor form and blocked ICE-induced apoptosis. Inhibition of enzymatic activity correlated with the cleavage of p35 and the formation of a stable ICE-p35 complex. The ability of p35 to block apoptosis in different pathways and in distantly related organisms suggests a central and conserved role for ICE-like proteases in the induction of apoptosis.

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The MEK kinase TPL-2 (also known as Cot) is required for lipopolysaccharide (LPS) activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase cascade in macrophages and consequent upregulation of genes involved in innate immune responses. In resting cells, TPL-2 forms a stoichiometric complex with NF-B1 p105, which negatively regulates its MEK kinase activity. Here, it is shown that lipopolysaccharide (LPS) stimulation of primary macrophages causes the release of both long and short forms of TPL-2 from p105 and that TPL-2 MEK kinase activity is restricted to this p105-free pool. Activation of TPL-2, MEK, and ERK by LPS is also demonstrated to require proteasome-mediated proteolysis. p105 is known to be proteolysed by the proteasome following stimulus-induced phosphorylation of two serines in its PEST region by the IB kinase (IKK) complex. Expression of a p105 point mutant, which is not susceptible to signal-induced proteolysis, in RAW264.7 macrophages impairs LPS-induced release of TPL-2 from p105 and its subsequent activation of MEK. Furthermore, expression of wild-type but not mutant p105 reconstitutes LPS stimulation of MEK and ERK phosphorylation in primary NF-B1-deficient macrophages. Consistently, pharmacological blockade of IKK inhibits LPS-induced release of TPL-2 from p105 and TPL-2 activation. These data show that IKK-induced p105 proteolysis is essential for LPS activation of TPL-2, thus revealing a novel function of IKK in the regulation of the ERK MAP kinase cascade.

Interleukin-1 beta converting enzyme (ICE) is a cytoplasmic cysteine protease required for generating the bioactive form of the interleukin-1 beta cytokine from its inactive precursor. We report the identification of ICH-2, a novel human gene encoding a member of the ICE cysteine protease family, and characterization of its protein product. ICH-2 mRNA is widely expressed in human tissues in a pattern similar to, but distinct from, that of ICE. Overexpression of ICH-2 in insect cells induces apoptosis. Purified ICH-2 is functional as a protease in vitro. A comparison of the inhibitor profiles and substrate cleavage by ICH-2 and ICE shows that the enzymes share catalytic properties but may differ in substrate specificities, suggesting that the two enzymes have different functions in vivo.

The Bcl-2 family of proteins plays a critical role in controlling immune responses by regulating the expansion and contraction of activated lymphocyte clones by apoptosis. ABT-737, which was originally developed for oncology, is a potent inhibitor of Bcl-2, Bcl-xL, and Bcl-w protein function. There is evidence that Bcl-2–associated dysregulation of lymphocyte apoptosis may contribute to the pathogenesis of autoimmunity and lead to the development of autoimmune diseases. In this study, we report that ABT-737 treatment resulted in potent inhibition of lymphocyte proliferation as measured by in vitro mitogenic or ex vivo Ag-specific stimulation. More importantly, ABT-737 significantly reduced disease severity in tissue-specific and systemic animal models of autoimmunity. Bcl-2 family antagonism by ABT-737 was efficacious in treating animal models of arthritis and lupus. Our results suggest that treatment with a Bcl-2 family antagonist represents a novel and potentially attractive therapeutic approach for the clinical treatment of autoimmunity.

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