Interleukin-1beta Converting Enzyme Research Articles

Cloning of cDNA for canine interleukin-18 and canine interleukin-1beta converting enzyme and expression of canine interleukin-18.

Cloning of canine interleukin-18 (IL-18) and canine interleukin-1beta converting enzyme (ICE) cDNA was carried out by using murine IL-18 cDNA and human ICE cDNA, respectively, as probes. Sequence homology to known sequences of human, mouse, or rat genes was noted at nucleotide and amino acid levels. Canine IL-18 mRNA was expressed in various canine organs, whereas canine ICE mRNA was expressed in only a few, particularly in the brain and testis. Cloned canine IL-18 cDNA was expressed in Escherichia coli. The resulting protein promoted induction of canine interferon-y (IFN-y) from stimulated canine lymphocytes. Canine IL-18 and canine IL-12 produced canine IFN-gamma synergistically. Canine IL-18 suppressed the growth of tumor cells transplanted in SCID mice. Cloned canine IL-18 should prove useful as an anticancer agent.

Nucleotide sequence of equine caspase-1 cDNA.

Caspases are a family of cysteine proteases which have important roles in activation of cytokines and in apoptosis. Caspase-1, or interleukin-1 beta converting enzyme (ICE), promotes maturation of interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18) by proteolytic cleavage of precursor forms to generate biologically active peptides. We report the cloning and sequencing of equine caspase-1 cDNA. Equine caspase-1 is 405 amino acids in length and has 72% and 63% identity to human and mouse caspase-1, respectively, at the amino acid level. Sites of proteolytic cleavage and catalytic activity as identified in human caspase-1, are conserved.

Molecular abnormalities of the brain in Down syndrome: relevance to Alzheimer's neurodegeneration.

Down syndrome is caused by over-expression of genes located within a segment of chromosome 21, termed the Down locus. Down syndrome is associated with developmental abnormalities of the central nervous system that result in mental retardation and age-dependent Alzheimer-type neurodegeneration. Some of the neurodegenerative lesions, including A beta amyloid deposition, apoptotic cell death, and aberrant dendritic arborization, are in part due to constitutively increased expression of genes that encode the amyloid precursor protein, superoxide dismutase I, and S100-beta, and located within the Down locus. However, neurodegeneration in Down syndrome is also associated with aberrant expression of genes that are not linked to the Down locus, including the growth associated protein, GAP-43, nitric oxide synthase 3, neuronal thread protein, and pro-apoptosis genes such as p53, Bax, and interleukin-1 beta-converting enzyme. Increased expression of these non-Down locus genes correlates with proliferation of dystrophic neurites and apoptotic cell death, two important correlates of cognitive impairment in Alzheimer's disease. This article reviews the functional importance of abnormal gene expression in relation to Alzheimer-type neurodegeneration in brains of individuals with Down syndrome.

Streptococcal pyrogenic exotoxin B induces apoptosis and reduces phagocytic activity in U937 cells.

Treatment of U937 human monocyte-like cells with Streptococcus pyogenes led to an induction of apoptosis in these cells. A comparison between the wild-type strain and its isogenic protease-negative mutant indicated that the production of streptococcal pyrogenic exotoxin B (SPE B), a cysteine protease, caused a greater extent of apoptosis in U937 cells. Further study using purified SPE B showed that this protease alone could induce U937 cells to undergo apoptosis, which was characterized by morphologic changes, DNA fragmentation laddering on the gel, and an increase in the percentages of hypodiploid cells. The protease activity of SPE B was required for apoptosis to proceed, since treatment with cysteine protease inhibitor E64 or heat inactivation abrogated this death-inducing effect. The SPE B-induced apoptosis pathway was interleukin-1beta converting enzyme (ICE) family protease dependent. Further experiments showed that the phagocytic activity of U937 cells was reduced by SPE B. Treatment with E64 and heat inactivation both abrogated this phagocytosis-inhibitory effect. Taken together, the present data show that SPE B not only possesses the ability to induce apoptosis in monocytic cells but also helps bacteria to resist phagocytosis by host cells.

Structure and chromosome localization of the human CASP8 gene.

The human CASP8 gene, whose product is also known as caspase 8 and FLICE, encodes an interleukin-1beta converting enzyme (ICE)-related cysteine protease that is activated by the engagement of several different death receptors. Caspase 8 is immediately recruited to the Fas receptor once it oligomerizes, and its protease activity is crucial for the apoptotic response generated by the resulting death-inducing signaling complex (DISC). We report here that the CASP8 gene contains at least 11 exons spanning approximately 30kb on human chromosome band 2q33-34. This region of human chromosome 2 was previously reported as the location of the CASP10 gene, whose product is closely related to caspase 8. Chromosome 2 band q33-34 is also involved in tumorigenesis, with loss of heterogeneity (LOH) being reported in a number of tumors. We also report EcoRI and HindIII polymorphisms that may prove to be useful in disease analysis. Both caspases 8 and 10 contain long pro-domains with duplicated death effector domains (DEDs), as well as their corresponding cysteine protease catalytic domains. Thus, it appears that CASP8 and CASP10 have evolved by tandem gene duplication, much like the CASP1, CASP4 and CASP5 gene cluster on human chromosome 11q22.2-22.3.

Up-regulation of ICH-1L protein by thromboxane A2 antagonists enhances cisplatin-induced apoptosis in non-small-cell lung-cancer cell lines.

We evaluated the effect of thromboxane A2 (TXA2) blockade on cisplatin-induced apoptosis in non-small-cell lung cancer (NSCLC) cell lines. Cisplatin induced apoptosis in PC/9 and PC-9/CDDP in a dose-dependent manner. Treatment with specific TXA2 antagonist, calcium 5(Z)-1R,2S,3S,4S-7-[3-phenylsulfonylaminobicyclo[2,2,1]hept-2-yl]- 5-heptonoate hydrate (S-1452) and 5(Z-6-[(1R,2R,3R,4S)-3-(N-4-bromobenzenesulfonyl aminomethyl) bicyclo[2,2,1]heptane-2-yl]-hex-5-enoic acid (ONO-NT-126), enhanced the cisplatin-induced apoptosis in each cell line. Acetyl-L-aspartyl-glutamyl-valyl-aspart-1-aldehyde (Ac-DEVD-CHO) inhibited cisplatin-induced apoptosis and enhancement of the apoptosis by TXA2 blockade, but acetyl-L-tyrosyl-valyl-alanyl-aspart-1-aldehyde (Ac-YVAD-CHO) had no effect on the apoptosis. There was no difference in the interleukin-1beta-converting enzyme (ICE) protease protein expression in either cell line. Cysteine protease p32(CPP32) protein expression was lower in PC-9/CDDP but was not changed by S-1452, cisplatin, or cotreatment with cisplatin and S-1452. Ice and Ced-3 homolog (ICH-1L) expression was significantly lower in PC-9/CDDP and was up-regulated by S-1452 or ONO-NT-126. These data suggest that ICH-1L might play a critical role in cisplatin-induced apoptosis and that TXA2 blockade up-regulates ICH-1L protein expression. Overexpression of ICH-1L and treatment with cisplatin might result in an increase in apoptosis in NSCLC cell lines.

Caspase 1-independent IL-1beta release and inflammation induced by the apoptosis inducer Fas ligand.

Fas ligand is a well-characterized apoptosis inducer. Here we demonstrate that Fas ligand induces the processing and secretion of interleukin-1beta (IL-1beta) in peritoneal exudate cells. This IL-1beta secretion is independent of IL-1beta converting enzyme (caspase 1), yet it is inhibited by caspase inhibitors, indicating that a caspase(s) in addition to IL-1beta converting enzyme can process IL-1beta. Inoculation of tumor cells expressing Fas ligand into wild-type mice induces a massive neutrophil infiltration that is, in contrast, suppressed in IL-1alpha/beta knockout mice. These results demonstrate a newly discovered role for Fas ligand in inflammation, and challenge the dogma that apoptosis does not induce inflammation.

Redox regulation of the caspases during apoptosis.

Apoptosis is now widely recognized as being a distinct process of importance both in normal physiology and pathology. In the current paradigm for apoptotic cell death, the activity of a family of proteases, caspases, related to interleukin-1 beta-converting enzyme (ICE) orchestrates the multiple downstream events, such as cell shrinkage, membrane blebbing, glutathione (GSH) efflux, and chromatin degradation that constitute apoptosis. Recent studies suggest that mitochondria could be the principle sensor and that the release of mitochondrial factors, such as cytochrome c, is the critical event governing the fate of the cell.--One of the most reproducible inducers of apoptosis is mild oxidative stress, although it is unclear how an oxidative stimulus can activate the caspase cascade. Oxidative modification of proteins and lipids has also been observed in cells undergoing apoptosis in response to nonoxidative stimuli, suggesting that intracellular oxidation may be a general feature of the effector phase of apoptosis. The caspases themselves are cysteine-dependent enzymes and, as such, appear to be redox sensitive. Indeed, our recent work on hydrogen peroxide-mediated apoptosis suggests that prolonged or excessive oxidative stress can actually prevent caspase activation. A physiological example of this is the NADPH oxidase-derived oxidants generated by stimulated neutrophils that prevent caspase activation in these cells. Pursuant to these findings, stimulated neutrophils appear to use a specialized caspase-independent pathway to initiate phosphatidylserine (PS) exposure and subsequent phagocytic clearance. The possible implications of these dual roles for reactive oxygen species in apoptosis, that is, induction and inhibition of caspases, are discussed in the present review.

Apoptosis of undifferentiated progenitors and granule cell precursors in the postnatal human cerebellar cortex correlates with expression of BCL‐2, ICE, and CPP32 proteins

Naturally occurring apoptotic cells have been demonstrated in the postnatal cerebellum of rodents (Wood et al. [1993] Neuron 11:621-632; Krueger et al. [1995] J. Neurosci. 15:3366-3374). The nature of these cells differs among species: they are considered to be granule cells in mouse and astrocytes in rat. We labeled proliferating and apoptotic cells in the postnatal human cerebellar cortex by using antibodies against the Ki-67/proliferating cell nuclear antigen and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling method for fragmented DNA. We also immunocytochemically detected some proteins encoded by genes modulating apoptosis and specific markers of neuronal/glial differentiation. Proliferating cells were observed from birth to 4 months, representing 31-35% of cells within the external granular layer (EGL). Apoptotic cells were detected during the first 3 months and corresponded to 5-7% of EGL cells. Much lower percentages were calculated in other cortical layers and white matter. The balance between proliferation and apoptosis was quantitatively favorable to the latter during the first postnatal week. Expression of BCL-2, CPP32, and interleukin-1beta-converting enzyme (ICE) proteins was spatially and developmentally regulated in parallel with apoptosis. Apoptotic cells were often CPP32/ICE immunoreactive but negative for BCL-2. Some apoptotic cells were positive for vimentin and, less frequently, for alpha-internexin or type-III beta tubulin, but never expressed the glial fibrillary acidic protein. This study demonstrates that apoptosis is a significant phenomenon in early postnatal development of human cerebellar cortex and shares some of the regulatory mechanisms described in other vertebrates.

Lipopolysaccharide-induced fever is dissociated from apoptotic cell death in the rat brain

Immune system activation induces increase in expression level and enzymatic activity of interleukin-1 beta converting enzyme (ICE) in rat brain. As ICE has been implicated in apoptotic cell death, a possible link may exist between immune system activation by bacterial endotoxic lipopolysaccharide (LPS) and apoptosis in rat brain. The aim of this study was to investigate possible effect of acute (5.5 h) or chronic (5 days) intraperitoneal (i.p.) administration and central injection of LPS on brain apoptotic cell death. Body temperature was continuously monitored for fever, a hallmark of immune activation. Detection of apoptotic cell death was carried out by using in situ labelling of DNA fragmentation in various brain structures. Despite the chronic or the acute pyrogenic effects of LPS, no evidence for apoptotic cell death was observed in any of the brain areas analysed, including hippocampus, hypothalamus, area postrema, subfornical organ, organum vasculosum of the lamina terminalis and nucleus tractus solitaris. Other well-known sites of apoptotic cell death, including brain of ischemic rat, mammary gland of post-lactating rat and rat intestine as well as Dnase-treated rat brain slices, were used as positive controls. These results suggest that ICE activation during fever development is dissociated from cell death by apoptosis in rat brain. Unlike peripheral targets of immunocompetent cytokines, a protective system, yet to be defined, may be present in the central nervous system and block the deleterious effects of infectious agents and cytokines.

Interleukin-1 beta, interleukin-18, and the interleukin-1 beta converting enzyme.

When injected intravenously into humans and animals, interleukin-1 beta (IL-1 beta) is perhaps the most potent of the endogenous pyrogens. However, IL-1 beta is initially synthesized as a relatively inactive precursor molecule (proIL-1 beta) which lacks a signal peptide and hence remains inside the cell. To be active as a fever-producing molecule, proIL-1 beta must first be processed to an active mature molecule and secreted. Although several enzymes associated with inflammatory tissues are capable of processing proIL-1 beta into an active molecule in the extracellular compartment, the IL-1 beta converting enzyme (ICE, also called caspase-1) cuts intracellular proIL-1 beta after the aspartic acid residue in position 116, resulting in a highly active mature IL-1 beta that is secreted into the extracellular space. IL-18 is also initially synthesized as an inactive precursor molecule (proIL-18) lacking a signal peptide. IL-18 is a member of the IL-1 family, and like IL-1 beta, proIL-18 is cleaved by ICE to yield an active molecule. However, unlike IL-1 beta, IL-18 is not an endogenous pyrogen following intraperitoneal injection into mice. Nevertheless, IL-18 may contribute to inflammation and fever because IL-18 is a potent inducer of tumor necrosis factor, chemokines, and interferon-gamma production.

Partial Purification and Characterization of Two Distinct Types of Caspases from Human Epidermis

Recent observations demonstrated that interleukin-1beta converting enzyme family proteases, now referred to as caspase family, play central roles in apoptosis, or programmed cell death. In this study, we tried to isolate and characterize epidermal caspases. By DEAE-Sephacel anion-exchange chromatography, human cornified cell extract showed two caspase-like fractions (F-I and F-II) with different substrate specificities. These were further purified by Sephacryl S-200, Mono Q ion exchange and Superose 6 gel chromatography. F-I showed a molecular weight of 30 kDa and specifically hydrolyzed acetyl-Asp-Glu-Val-Asp-methylcoumarinamide, a fluorogenic substrate for caspase-3 (CPP32) with a Km value of 13.8 microM. F-I generated a characteristic 85 kDa fragment from poly(ADP-ribose) polymerase. Inhibitor susceptibility of F-I was very similar to that of caspase-3, further confirming the caspase-3-like properties of F-I. In contrast, the molecular weight of F-II was estimated to be 110 kDa, which was much higher than the other caspases. F-II equally hydrolyzed acetyl-Asp-Glu-Val-Asp-methylcoumarinamide, and acetyl-Tyr-Val-Ala-Asp-methylcoumarinamide, caspase-1 (interleukin-1beta converting enzyme)-specific substrate, and was inhibited by acetyl-Tyr-Val-Ala-Asp-aldehyde and acetyl-Tyr-Val-Ala-Asp-aldehyde. Affinity labeling using biotinylated YVAD-cmk demonstrated several positive bands ranging from 25 to 35 kDa, supporting the hypothesis that F-II is a complex of multiple caspases. Reverse transcriptase-polymerase chain reaction analysis demonstrated that among known caspases tested, caspase-1, -2, -3, -4, and -7 were expressed in cultured human keratinocytes. These results suggest that multiple caspases are synthesized in human keratinocytes and are involved in terminal differentiation.

Role of caspases in N-methyl-D-aspartate-induced apoptosis in cerebrocortical neurons.

Overactivation of glutamate receptors mediates neuronal death in several acute and chronic neurodegenerative diseases. The intracellular processes underlying this form of death, however, remain poorly understood. Depending on the severity of insult, N-methyl-D-aspartate (NMDA) receptor activation induces either apoptosis or necrosis. Cysteine proteases related to interleukin-1beta-converting enzyme (ICE), recently termed caspases, appear necessary for neuronal apoptosis in vivo and in vitro. To determine whether caspases play a role in NMDA-induced apoptosis, we used two functionally distinct approaches to decrease substrate cleavage by caspases. One is a novel peptide (V-ICEinh) that contains the caspase catalytic site and acts as a pseudoenzyme that binds caspase substrates and prevents their cleavage. The other is a pseudosubstrate peptide (Z-VAD x fmk) that inhibits caspase activity. Pretreatment with either V-ICEinh or Z-VAD-fmk protects cerebrocortical neurons from NMDA-induced apoptosis, suggesting a role for caspases in NMDA-induced apoptosis. To explore the signaling pathways involved, we looked at the effects of NMDA receptor activation on Ca2+ influx, production of reactive oxygen species (ROS), mitochondrial membrane potential, and lipid peroxidation. Neither NMDA-induced Ca2+ influx nor the initial collapse of mitochondrial membrane potential could be prevented by pretreatment with V-ICEinh or Z-VAD x fmk. In contrast, ROS formation and lipid peroxidation were completely blocked by both V-ICEinh and Z-VAD x fmk. Taken together, our results suggest that Ca2+ influx and mitochondrial depolarization occur upstream from caspase activation, whereas ROS formation and lipid peroxidation may be downstream events in the cascade leading to cortical neuronal apoptosis.

Antisense telomerase treatment: induction of two distinct pathways, apoptosis and differentiation.

Telomerase, the enzyme that elongates telomeric DNA (TTAGGG)n, may be involved in cellular immortality and oncogenesis. To investigate the effect of inhibition of telomerase on tumor cells, we transfected the antisense vector against the human telomerase RNA into human malignant glioma cells exhibiting telomerase activity. After 30 doublings, some subpopulations of transfectants expressed a high level of interleukin-1beta-converting enzyme (ICE) protein and underwent apoptosis. In contrast, other subpopulations also showed enhanced ICE protein but escaped from apoptotic crisis and continued to grow, although their DNA synthesis, invasive ability, and tumorigenicity in nude mice were significantly reduced. Surviving cells demonstrated increased expression of glial fibrillary acidic protein and decreased motility, consistent with a more differentiated state. These cells also contained enhanced expression of the cyclin-dependent kinase inhibitors (CDKIs) p21 and p27. Treatment of surviving nonapoptotic cells with antisense oligonucleotides against p27, but not p21, induced apoptotic cell death, suggesting that p27 may have protected differentiating glioma cells from apoptosis. These data show that treatment with antisense telomerase inhibits telomerase activity and subsequently induces either apoptosis or differentiation. Regulation of these two distinct pathways may be dependent on the expression of ICE or CDKIs.

Differential Regulation of Discrete Apoptotic Pathways by Ras

The products of the ras genes are known to regulate cell proliferation and differentiation; recently, they have been found to play a role in apoptosis. The expression of oncogenic p21(ras) in a number of cell types, including Jurkat (a human T lymphoblastoid cell line) and murine fibroblasts, makes the cells susceptible to apoptosis following suppression of protein kinase C (PKC) activity (PKC/Ras-mediated apoptosis). Engagement of Fas antigen, a potent effector of apoptosis, activates cellular p21(ras), which may be required for completion of the cell death program. To further investigate the role of p21(ras) in the regulation of apoptosis, the cellular mechanisms employed in these two apoptotic processes in which Ras activity is involved (PKC/Ras-related and Fas-triggered apoptosis), was explored. Increasing p21(ras) activity by expressing v-ras or by treatment with an antisense oligonucleotide to the GTPase-activating protein was found to accelerate the Fas-mediated apoptotic process in Jurkat and mouse LF cells. PKC/Ras-related apoptosis was associated with, and required, cell cycle progression, accompanied by the expression of the G1/S cyclins. In contrast, Fas engagement, although inducing a vigorous and PKC-independent activation of endogenous p21(ras), did not alter cell cycle progression, nor did it require such progression for apoptosis. Both the protein synthesis inhibitor cycloheximide and cyclin E antisense oligonucleotides partially abolished PKC/Ras-mediated apoptosis but had only a moderate effect on Fas-induced apoptosis. In contrast, the CED-3/interleukin-1beta-converting enzyme (ICE) protease inhibitor Z-VADfmk efficiently suppressed Fas-induced apoptosis and only marginally inhibited PKC/Ras-mediated apoptosis. Induction of both pathways resulted in activation of the Jun NH2-terminal kinase/JUN signaling system. These results suggest that different cell death programs, such as PKC/Ras-mediated and Fas-mediated apoptosis, may be interconnected via p21(ras) and perhaps Jun NH2-terminal kinase/JUN. In response to various death stimuli, p21(ras) may act as a common intermediate regulator in the transduction of apoptotic signals.

The regulation of reactive oxygen species production during programmed cell death.

Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. The role of ROS in cell death caused by oxidative glutamate toxicity was studied in an immortalized mouse hippocampal cell line (HT22). The causal relationship between ROS production and glutathione (GSH) levels, gene expression, caspase activity, and cytosolic Ca2+ concentration was examined. An initial 5-10-fold increase in ROS after glutamate addition is temporally correlated with GSH depletion. This early increase is followed by an explosive burst of ROS production to 200-400-fold above control values. The source of this burst is the mitochondrial electron transport chain, while only 5-10% of the maximum ROS production is caused by GSH depletion. Macromolecular synthesis inhibitors as well as Ac-YVAD-cmk, an interleukin 1beta-converting enzyme protease inhibitor, block the late burst of ROS production and protect HT22 cells from glutamate toxicity when added early in the death program. Inhibition of intracellular Ca2+ cycling and the influx of extracellular Ca2+ also blocks maximum ROS production and protects the cells. The conclusion is that GSH depletion is not sufficient to cause the maximal mitochondrial ROS production, and that there is an early requirement for protease activation, changes in gene expression, and a late requirement for Ca2+ mobilization.

Immunohistochemical study of Fas, Fas ligand and interleukin-1 beta converting enzyme expression in human prostatic cancer.

To determine the cellular expression of Fas, Fas ligand and interleukin-1 beta converting enzyme (ICE) in prostatic cancer. Specimens of prostate were obtained from 21 patients (mean age 66 years, SD 5) undergoing radical prostatectomy for prostatic cancer. Nine of the 21 patients had received endocrine therapy before surgery. Specimens were also obtained from 10 patients with benign prostatic hypertrophy (BPH) and during autopsy from 10 patients who had died from hormone-unresponsive prostate cancer. Paraffin-embedded sections were cut from the specimens and stained immunohistochemically to detect Fas, Fas ligand and ICE. Fas was expressed in all 21 of the cancer specimens while Fas ligand was detected in none and ICE was expressed in 11. The difference between the expression of Fas and ICE was significant (P < 0.001). ICE was expressed in nine of 12 patients who were untreated before surgery and in two of nine treated with endocrine therapy (P < 0.05). Fas expression was detected in the specimens from all 10 patients with BPH and in all 10 autopsy specimens; the expression, tended to be more marked than that in specimens from total prostatectomy (P < 0.1). The expression of ICE was weaker than that of Fas in the total prostatectomy specimens, suggesting a possible interruption of the apoptotic signalling pathway. Prostates with BPH and hormone-unresponsive cancer therefore showed no appreciable change in Fas expression when compared with total prostatectomy samples.

Induction of apoptosis in thecal/interstitial cells: action of transforming growth factor (TGF) alpha plus TGF beta on bcl-2 and interleukin-1 beta-converting enzyme.

Follicular atresia is characterized by the initial rapid loss of granulosa cells by apoptosis, followed by the loss of thecal cells at a slower rate. We have previously shown that treatment of subconfluent cultures of thecal/interstitial cells (T/I) with transforming growth factor (TGF) alpha plus TGF beta caused chromatin condensation and internucleosomal fragmentation characteristic of apoptosis, whereas in the presence of either TGF alpha or TGF beta alone the cells remained healthy. In this study we have examined the effect of TGF alpha and TGF beta alone and in combination on the levels of mRNA encoding bcl-2 and interleukin-1 beta-converting enzyme (ICE) in T/I cells using a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay. Bcl-2, a cell survival gene, has been implicated in regulating the balance between cell proliferation and cell death in physiological processes. ICE, the homolog of the C. elegans cell death gene, ced-3, is also involved in apoptotic signal transduction. The levels of mRNA encoding specific PCR products for bcl-2 (430 bp) and ICE (453 bp) were amplified from T/I cell cDNA. Untreated T/I cells and TGF alpha- or TGF beta-treated cells contained comparable levels of bcl-2 mRNA. Treatment of T/I cells with TGF alpha plus TGF beta significantly decreased the levels of bcl-2 mRNA expression. TGF alpha plus TGF beta caused a significant decrease in bcl-2 mRNA levels within 3 h of treatment of T/I cells, followed by a progressive decline to 10% of control levels after 24 h of treatment. In contrast, in control T/I cells, the levels of ICE mRNA were low. TGF alpha plus TGF beta caused a progressive increase in ICE mRNA, reaching levels 2- and 3-fold higher than control cells after 5 and 7 h respectively. DNA analysis showed that DNA fragmentation, indicative of apoptosis, occurred after 10 h of treatment with TGF alpha plus TGF beta. These studies demonstrated that treatment of T/I cells with TGF alpha plus TGF beta influenced gene expression of bcl-2 and ICE prior to the time at which DNA fragmentation was observed. We propose that the gene products of bcl-2 and ICE are involved in the apoptotic signal transduction pathway induced by TGF alpha plus TGF beta in T/I cells.

Peptidyl beta-homo-aspartals: specific inhibitors of interleukin-1 beta converting enzyme and its homologues (caspases).

Inhibition of interleukin-1 beta converting enzyme (ICE), apopain, papain, thrombin and trypsin with substrate like peptidyl L- and D-alpha-aldehydes and their L-beta-homo-aldehyde analogues was investigated. The L-beta-homo-aspartals appear to be specific inhibitors for ICE and its homologues; the other enzymes were not inhibited with such L-beta-homo aldehydes. Papain shows tolerance for D-residues at P1 depending on their chiral stability.

The interleukin-1 system: receptors, ligands, and ICE in the brain and their involvement in the fever response.

The ligands and the receptors of the interleukin 1 (IL-1) system constitute a highly inducible set of proteins whose expression in infection and inflammation is of key importance in the host defense. The IL-1 system participates in the stimulation of the immune system, the neuroendocrine system, and the neuroimmune system. The major soluble and secreted agonist of the system, IL-1 beta, has been studied by mutational and transgenic approaches. Furthermore, involvement of the signal-transducing type I IL-1 receptor (IL-1RI), in fever and other responses, has been studied by null mutation technique. We describe the inducible expression of the two agonists, IL-1 alpha (31 kDa and 17 kDa) and IL-1 beta (17 kDa) and of the IL-1 receptor subtypes IL-1RI and IL-1RII in the brain and in the adrenals (as well as in the pituitary cell line AtT20). We also describe an additional member of the IL-1 family: the IL-1 receptor antagonist (IL-1ra), an endogenous antagonist to IL-1 alpha and IL-1 beta. Furthermore, the IL-1 beta-converting enzyme (ICE) and its differential regulation and expression in brain and adrenals is also discussed. Fever is a systemic response to intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) injection of IL-1 alpha or IL-1 beta. IL-1 beta-induced fever can be blocked by IL-1ra pretreatment. The fever response seems to be mediated via the IL-1RI as inferred from studies with receptor subtype-specific mutants of IL-1 beta and from studies in IL-1RI knock-out (IL-1RI KO) mice. IL-1 beta knock-out mice showed a hyperresponsive fever to both IL-1 agonists, IL-1 alpha and IL-1 beta, as well as to LPS.