Interleukin-1beta-converting enzyme/caspase-1 in human osteoarthritic tissues: localization and role in the maturation of interleukin-1beta and interleukin-18.
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OBJECTIVE: To study the expression and production of interleukin-1beta-converting enzyme (ICE) in human normal and osteoarthritic (OA) cartilage and synovium, quantitate the level of ICE in OA chondrocytes, and examine the relationship between the topographic distribution of ICE, interleukin-1beta (IL-1beta), and IL-18, as well as apoptosis of chondrocytes. METHODS: The expression and synthesis of ICE were investigated in human normal and OA cartilage and synovial membrane using in situ hybridization and immunohistochemical methods. The intracellular level of ICE in OA chondrocytes was also measured by enzyme-linked immunosorbent assay (ELISA). Furthermore, the topographic relationship between the presence of ICE and mature IL-1beta and IL-18 was examined by immunohistochemistry, and apoptotic chondrocytes by the TUNEL technique. RESULTS: ICE was expressed and synthesized in both human synovial membrane and cartilage, with a significantly greater number of cells staining positive in OA tissue than in normal tissue. ICE production was preferentially located in the superficial and upper intermediate layers of articular cartilage. With a specific ELISA, a level of 230.2+/-22.5 pg/5 x 10(5) cells (mean +/- SEM) of ICE was found in OA chondrocytes. In cartilage, IL-1beta and IL-18 stained positive at a topographic location similar to that of ICE. The production of mature IL-1beta in OA cartilage explants and chondrocytes was completely blocked by treatment with a specific ICE inhibitor, which also markedly diminished the number of IL-18-positive cells. The data show that there was no close relationship between the presence of ICE and the presence of apoptotic chondrocytes in OA cartilage. CONCLUSION: This study shows, for the first time, the presence of active ICE in human articular cartilage, with a markedly increased cellular level in OA tissue. The relationship between active IL-1beta and ICE suggests that ICE may promote OA progression by activating this proinflammatory cytokine. The role of IL-18 in pathologic cartilage is discussed. (+info)
Caspase-1 regulates the inflammatory process leading to autoimmune demyelination.
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T cell-mediated inflammation is considered to play a key role in the pathogenic mechanisms sustaining multiple sclerosis (MS). Caspase-1, formerly designated IL-1beta-converting enzyme, is crucially involved in immune-mediated inflammation because of its pivotal role in regulating the cellular export of IL-1beta and IL-18. We studied the role of caspase-1 in experimental autoimmune encephalomyelitis (EAE), the animal model for MS. Caspase-1 is transcriptionally induced during EAE, and its levels correlate with the clinical course and transcription rate of proinflammatory cytokines such as TNF-alpha, IL-1beta, IFN-gamma, and IL-6. A reduction of EAE incidence and severity is observed in caspase-1-deficient mice, depending on the immunogenicity and on the amount of the encephalitogenic myelin oligodendrocyte glycoprotein (MOG) peptide used. In caspase-1-deficient mice, reduced EAE incidence correlates with defective development of anti-MOG IFN-gamma-producing Th1 cells. Finally, pharmacological blockade of caspase-1 in Biozzi AB/H mice, immunized with spinal cord homogenate or MOG35-55 peptide, by the caspase-1-inhibitor Z-Val-Ala-dl -Asp-fluoromethylketone, significantly reduces EAE incidence in a preventive but not in a therapeutic protocol. These results indicate that caspase-1 plays an important role in the early stage of the immune-mediated inflammatory process leading to EAE, thus representing a possible therapeutic target in the acute phase of relapsing remitting MS. (+info)
B cell apoptosis triggered by antigen receptor ligation proceeds via a novel caspase-dependent pathway.
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In contrast to positive signaling leading to proliferation, the mechanisms involved in negative signaling culminating in apoptosis after B cell Ag receptor (BCR) ligation have received little study. We find that apoptosis induced by BCR cross-linking on EBV-negative mature and immature human B cell lines involves the following sequential, required events: a cyclosporin A-inhibitable, likely calcineurin-mediated step; and activation of caspase-2, -3, and -9. Caspase-2 is activated early and plays a major role in the apoptotic pathway, while caspase-9 is activated later in the apoptotic pathway and most likely functions to amplify the apoptotic signal. Caspase-8 and -1, which are activated by ligation of the CD95 and TNF-R1 death receptors, are not involved. Apoptosis induced by BCR ligation thus proceeds via a previously unreported intracellular signaling pathway. (+info)
Acidic environment causes apoptosis by increasing caspase activity.
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An exposure of HL-60 human promyelocytic leukaemia cells to acidic media with pH 6.2-6.6 caused an up-regulation of Bax protein expression within 2 h, which lasted for longer than 6 h. On the other hand, the apoptosis, as judged from PARP cleavage, DNA fragmentation and flow cytometric determination of cell population with sub-G1 DNA content, occurred after the cells were incubated in the acidic media for longer than 4 h. The PARP cleavage and DNA fragmentation in the cells exposed to an acidic environment could be effectively suppressed by inhibitors specific for ICE or CPP32, indicating that activation of these caspases is an essential step in acidic stress-induced apoptosis. It has been known that Bax is involved in the activation of caspases. Taken together, it appears that acidic stress first up-regulates Bax protein thereby activating caspases followed by PARP cleavage and DNA fragmentation. The observation that inhibition of either ICE or CPP32 could suppress acidic stress-induced apoptosis suggested that ICE activates pro-CPP32, which then cleaves PARP. Flow cytometric analysis indicated that acidic stress-induced apoptosis occurs mainly in G1 cells. The finding in the present study demonstrated that acidic intra-tumour environment may markedly perturb the tumour cell proliferation and tumour growth. (+info)
Role of caspases in hypoxia-induced necrosis of rat renal proximal tubules.
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The role of the caspases, a newly discovered group of cysteine proteases, was investigated in a model of hypoxia-induced necrotic injury of rat renal proximal tubules. An assay for caspases in freshly isolated rat proximal tubules was developed. There was a 40% increase in tubular caspase activity after 15 min of hypoxia in association with increased cell membrane damage as indicated by a threefold increase in lactate dehydrogenase release. The specific caspase inhibitor Z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-D-DCB) attenuated the increase in caspase activity during 15 min of hypoxia and markedly decreased lactate dehydrogenase release in a dose-dependent manner. In the proximal tubules, Z-D-DCB also inhibited the hypoxia-induced increase in calpain activity, another cysteine protease. In contrast, when Z-D-DCB was added to purified calpain in vitro, there was no inhibition of calpain activity. The calpain inhibitor (2)-3-(4-iodophenyl)-2-mercapto-2-propenoic acid (PD150606) also inhibited the hypoxia-induced increase in caspase activity in proximal tubules, but did not inhibit the activity of purified caspase 1 in vitro. In these experiments, caspase activity was detected with the fluorescence substrate Ac-Tyr-Val-Ala-Asp-7-amido-4-methyl coumarin (Ac-YVAD-AMC), which is preferentially cleaved by caspase 1. However, minimal caspase activity was detected with the fluorescence substrate Ac-Asp-Glu-Val-Asp-7-amido-4-methyl coumarin (Ac-DEVD-AMC), which is cleaved by caspases 2, 3, and 7. The present study in proximal tubules demonstrates that (1) caspase inhibition protects against necrotic injury by inhibition of hypoxia-induced caspase activity; and (2) caspase 1 may be the caspase involved. Thus, although the role of caspases in apoptotic cell death is well established, this study provides new evidence that caspases contribute to necrotic cell death as well. (+info)
IL-12-induced IFN-gamma is dependent on caspase-1 processing of the IL-18 precursor.
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IL-12 and IL-18 are IFN-gamma-inducing cytokines. In the present study, the role of endogenous IL-18 in the induction of IFN-gamma by IL-12 was investigated in mice. In the presence of a specific inhibitor of caspase-1 (also known as IL-1beta-converting enzyme, or ICE) IL-12-induced IFN-gamma from splenocytes was reduced by 85%. Using splenocytes from ICE-deficient mice, IL-12-induced IFN-gamma was reduced by 80%. However, the role of ICE was not through processing and release of IL-1beta. Neutralizing anti-IL-18 IgG reduced IL-12-induced IFN-gamma in splenocytes by 85%. Splenocytes cultured in vitro spontaneously released IL-18 into the extracellular compartment over time. Extracellular levels of IL-18 significantly correlated with IL-12-induced IFN-gamma and were reduced in cells obtained from ICE-deficient mice. In vivo, IL-12 administration increased circulating levels of IL-18 in wild-type mice but not in ICE-deficient mice. Both neutralization of IL-18 and ICE deficiency significantly reduced induction of circulating IFN-gamma in mice receiving IL-12. The IL-18 precursor was constitutively expressed in the livers and spleens of untreated mice. Furthermore, administration of IL-12 significantly increased liver-associated IL-18 levels. These data demonstrate that endogenous, ICE-cleaved IL-18 significantly contributes to induction of IFN-gamma by IL-12. (+info)
Inhibition of epidermal growth factor-induced interleukin-1beta-converting enzyme expression reduces proliferation in the pancreatic carcinoma cell line AsPC-1.
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It is suggested that interleukin-1beta-converting enzyme (ICE) and ICE-related proteases play an important role in programmed cell death (apoptosis). We investigated ICE expression in the human pancreatic carcinoma cell line AsPC-1 after stimulation with epidermal growth factor and found a time-dependent expression of active ICE induced by epidermal growth factor. Interestingly, ICE expression does not lead to apoptosis. Cell cycle analyses revealed that acetyl-Tyr-Val-Ala-Asp-chloromethylketone-specific and acetyl-Ala-Ala-Val-Ala-Leu-Leu-Pro-Ala-Val-Leu-Leu-Ala-Leu-Leu-Ala-Pro-T yr-Val-Ala-Asp-aldehyde-specific cell-permeable inhibitors of ICE significantly reduced the proliferation of AsPC-1 cells, which suggested a positive influence of ICE on the proliferation in human pancreatic carcinoma cells. (+info)
Overexpression of IL-1ra gene up-regulates interleukin-1beta converting enzyme (ICE) gene expression: possible mechanism underlying IL-1beta-resistance of cancer cells.
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We investigated the interaction of endogenous interleukin (IL)-1beta, IL-1ra, and interleukin-1beta converting enzyme (ICE) in four human urological cancer cell lines, KU-19-19, KU-1, KU-2 and KU-19-20. Northern blot analysis showed that IL-1beta gene was expressed in all cell lines. On the other hand, in KU-19-19 and KU-19-20, the gene expressions of both IL-1ra and ICE were suppressed. MTT assay revealed that IL-1beta (10 ng ml(-1)) promoted cell growth in KU-19-19 and KU-19-20, while it inhibited in KU-1 and KU-2. An ICE inhibitor, Acetyl-Tyr-Val-Ala-Asp-CHO (YVAD-CHO) blocked IL-1beta-induced growth inhibition in KU-1 and KU-2. Overexpression of the secretory type IL-1ra with adenovirus vector (AxlL-1ra) enhanced ICE gene expression, while exogenous IL-1ra (100 ng ml(-1)) did not enhance it. Furthermore, AxIL-1ra treatment promoted endogenous IL-1beta secretion and induced significant growth inhibition and apoptotic cell death on KU-19-19 and KU-19-20. Treatment with either IL-1ra (100 ng ml(-1)), IL-1beta antibody (100 microg ml(-1)), or YVAD-CHO blocked AxlL-1ra-induced cell death in KU-19-19 and KU-19-20. These results suggest that IL-1beta-sensitivity depends on the level of ICE gene expression, which is regulated by the level of endogenous slL-1ra expression. This is a first report on the intracellular function of slL-1ra and these findings may provide key insights into the mechanism underlying the viability of cancer cells. (+info)