Activation of caspase-1 dependent interleukins in developmental brain trauma

Focal mechanical cortical trauma triggers diffuse apoptotic neurodegeneration in the developing rat brain which is associated with invasion of brain tissue with inflammatory mediators. We hypothesized that caspase-1 and the two caspase-1-processed cytokines, interleukin (IL)-1beta and IL-18, are involved in trauma-induced neuronal cell death in the developing brain. 7-day-old Wistar rats or C57/BL6 mice were subjected to head trauma using a weight drop device. Animals were sacrificed at defined time points following trauma and brains were processed for histology and molecular analyses. Neuronal cell death in the immature brain peaked at 12-24 h and was accompanied by a marked increase of mRNA and protein levels for caspase-1, IL-1beta and IL-18 within 2 to 12 h following the injury. Caspase-1 levels were elevated for 72 h, whereas IL-1beta decreased earlier at 48 h. IL-18 remained high over a period of 3 days and decreased to normal levels by day 7 after the injury. Intraperitoneal injection of recombinant human IL-18-binding protein (IL-18BP), a specific inhibitor of IL-18, attenuated traumatic brain injury. Mice deficient in IL-18 (IL-18-/-) were protected against trauma-induced brain damage. These findings indicate that IL-18 is involved in trauma-induced neuronal cell death in the immature rodent brain and might serve as a potential therapeutic target.     

Hypoxic induction of caspase-11/caspase-1/interleukin-1beta in brain microglia

Caspase-11 is an inducible protease that plays an important role in both inflammation and apoptosis. Inflammatory stimuli induce and activate caspase-11, which is required for the activation of caspase-1 or interleukin-1beta (IL-1beta) converting enzyme (ICE). Caspase-1 in turn mediates the maturation of proinflammatory cytokines such as IL-1beta, which is one of the crucial mediators of neurodegeneration in the central nervous system. Here, we report that hypoxic exposure of cultured brain microglia (BV-2 mouse microglia cells and rat primary microglial cultures) induces expression and activation of caspase-11, which is accompanied by activation of caspase-1 and secretion of mature IL-1beta and IL-18. Hypoxic induction of caspase-11 was observed in both mRNA and protein levels, and was mediated through p38 mitogen-activated protein kinase pathway. Transient global ischemia in rats also induced caspase-11 expression and IL-1beta production in hippocampus supporting our in vitro findings. Caspase-11-expressing cells in hippocampus were morphologically identified as microglia. Taken together, our results indicate that hypoxia induces a sequential event-caspase-11 induction, caspase-1 activation, and IL-1beta release-in brain microglia, and point out the importance of initial caspase-11 induction in hypoxia-induced inflammatory activation of microglia.     

Blockade of caspase-1 increases neurogenesis in the aged hippocampus

Adult hippocampal neurogenesis dramatically decreases with increasing age, and it has been proposed that this decline contributes to age-related memory deficits. Central inflammation contributes significantly to the decrease in neurogenesis associated with ageing. Interleukin-1beta is a proinflammatory cytokine initially synthesized as an inactive precursor that is cleaved by caspase-1 to generate the biologically active mature form. Whether IL-1beta affects neurogenesis in the aged hippocampus is unknown. Here we analysed cells positive for 5-bromo-2-deoxyuridine (BrdU; 50 mg/kg) in animals in which cleavage of IL-1beta was inhibited by the caspase-1 inhibitor Ac-YVAD-CMK (10 pmol). Aged (22 months) and young (4 months) rats received Ac-YVAD-CMK for 28 days intracerebroventricularly through a brain infusion cannula connected to an osmotic minipump. Starting on day 14, animals received a daily injection of BrdU for five consecutive days. Unbiased stereology analyses performed 10 days after the last injection of BrdU revealed that the total number of newborn cells generated over a 5-day period was higher in young rats than in aged rats. In addition, there was a 53% increase in the number of BrdU-labelled cells of the aged Ac-YVAD-CMK-treated rats compared to aged controls. Immunofluorescence studies were performed to identify the cellular phenotype of BrdU-labelled cells. The increase in BrdU-positive cells was not due to a change in the proportion of cells expressing neuronal or glial phenotypes in the subgranular zone. These findings demonstrate that the intracerebroventricular administration of Ac-YVAD-CMK reversed the decrease in hippocampal neurogenesis associated with ageing.     

Improvement of memory for context by inhibition of caspase-1 in aged rats

Impaired learning and memory is a common pathologic feature associated with numerous neurologic disorders. There is strong evidence that central inflammation contributes significantly to the progression of several neurodegenerative diseases as well as to the ageing process. For example, in aged rats an increase in interleukin-1beta (IL-1beta) is implicated in the decline of synaptic plasticity in the hippocampus and impaired performance on cognitive tasks such as contextual fear conditioning. IL-1beta is a proinflammatory cytokine initially synthesized in an inactive precursor form that is cleaved by caspase-1 to generate the biologically mature form. In the present study, cleavage of IL-1beta was chronically inhibited using a specific caspase-1 inhibitor (Ac-YVAD-CMK; 10 pmol) in both aged (22 month) and young (4 month) rats. Both groups received Ac-YVAD-CMK for 28 days intracerebroventricularly through a brain infusion cannula connected to an osmotic minipump. On day 20 the animals were trained in contextual fear conditioning, and memory for context was tested on day 22. Chronic infusion of a specific caspase-1 inhibitor in aged rats ameliorated age-related increases in hippocampal IL-1beta and improved memory for context.     

Inactivation of caspase-1 in rodent brain: a novel anticonvulsive strategy

PURPOSE: Cytokines and related inflammatory mediators are rapidly synthesized in the brain during seizures. We previously found that intracerebral administration of interleukin-1 (IL-1)-beta has proconvulsant effects, whereas its endogenous receptor antagonist (IL-1Ra) mediates potent anticonvulsant actions in various models of limbic seizures. In this study, we investigated whether seizures can be effectively inhibited by blocking the brain production of IL-1beta, by using selective inhibitors of interleukin-converting enzyme (ICE/caspase-1) or through caspase-1 gene deletion. METHODS: Caspase-1 was selectively blocked by using pralnacasan or VX-765. IL-1beta release was induced in mouse organotypic hippocampal slice cultures by proinflammatory stimuli [lipopolysaccharide (LPS) + adenosine triphosphate (ATP)] and measured with enzyme-linked immunosorbent assay (ELISA). IL-1beta production during seizures was measured in the rat hippocampus by Western blot. Seizures were induced in freely moving mice and rats by intrahippocampal injection of kainic acid and recorded by EEG analysis. RESULTS: Caspase-1 inhibition reduced the release of IL-1beta in organotypic slices exposed to LPS+ATP. Administration of pralnacasan (intracerebroventricular, 50 microg) or VX-765 (intraperitoneal, 25-200 mg/kg) to rats blocked seizure-induced production of IL-1beta in the hippocampus, and resulted in a twofold delay in seizure onset and 50% reduction in seizure duration. Mice with caspase-1 gene deletion showed a 70% reduction in seizures and an approximate fourfold delay in their onset. CONCLUSIONS: Inhibition of caspase-1 represents an effective and novel anticonvulsive strategy, which acts by selectively reducing the brain availability of IL-1beta.     

Expression of inducible nitric oxide synthase, interleukin-1 and caspase-1 in HIV-1 encephalitis

Inflammatory cytokines and enzymes such as IL-1 and inducible nitric oxide synthase (iNOS) may play an important role in the pathogenesis of AIDS dementia, a condition associated with infection of the CNS cells by the HIV-1. In this report, we investigated the expression of iNOS, IL-1, and caspase-1 (interleukin-1 converting enzyme) in HIV-1 encephalitis (HIVE) by immunocytochemistry and analyzed their expression with respect to HIV-1 infection and glial activation. In HIVE, all three molecules were expressed at high levels in areas of HIV-1 infection (microglial nodules with HIV-1 p24 immunoreactivity) and in areas of diffuse white matter gliosis. Expression was cell-type specific, with IL-1 and caspase-1 being expressed in macrophages and microglia, and iNOS in activated astrocytes. Multinucleated giant cells, a hallmark of virally infected cells, showed intense staining for both IL-1 and caspase-1, suggesting induction of these molecules by HIV-1. Double immunocytochemistry demonstrated a regional co-localization of astrocyte iNOS and microglial IL-1 and caspase-1. These results support the notion that autocrine and paracrine interactions between HIV-1 infected macrophages and microglia, activated microglia, and astrocytes lead to expression of proinflammatory and neurotoxic molecules. iNOS and caspase-1 may provide additional therapeutic targets for HIVE.     

Neuronal expression of caspase-1 immunoreactivity in the rat central nervous system

Caspase-1/interleukin-1beta (IL-1beta)-converting enzyme (ICE) cleaves IL-1beta and IL-18 precursor proteins to the active forms of these proinflammatory cytokines. Since both cytokines are constitutively expressed in the brain, we investigated whether this is also the case for caspase-1. Using an antibody raised against the p10-subunit of the active enzyme, constitutive expression of caspase-1 immunoreactivity was found in nerve cells in the arcuate nucleus and in nerve fibres throughout the brain. Co-localisation with alpha-melanocyte stimulating hormone was demonstrated. The distribution pattern of caspase-1 immunoreactive structures is consistent with a role to produce mature IL-1beta in regions where IL-1beta mediates fever and sleep.     

The age-related increase in IL-1 type I receptor in rat hippocampus is coupled with an increase in caspase-3 activation

Evidence from several studies indicates that expression of interleukin-1beta (IL-1beta) and IL-1 type I receptor is particularly high in hippocampus, and it has recently been shown that the concentration of IL-1beta is increased in the hippocampus of the aged rat. Here we report that this increase is coupled with an increase in expression of IL-1 type I receptor and increased activity of IL-1 receptor-associated kinase. The evidence presented indicates that the age-related increase in activity of the mitogen-activated protein kinases, Jun N-terminal kinase (JNK) and p38, was accompanied by enhanced caspase-3 activity. Analysis of colocalization of activated caspase-3 with activated p38 (p-p38) suggested that p-p38 was necessary for activation of caspase-3; while in vitro analysis indicated that the IL-1beta-induced increase in caspase-3 activity was abrogated by the p38 inhibitor, SB203580. The IL-1beta-induced increase in caspase-3 activity in vitro was also abrogated by vasoactive intestinal peptide, which is a JNK inhibitor; however, colocalization of activated JNK (p-JNK) and activated caspase-3 did not clearly identify JNK as an upstream activator of caspase-3. We propose that these changes are indicative of cell death in aged hippocampus and suggest that they contribute to the age-related decrease in long-term potentiation in perforant path granule cell synapses.     

Excitotoxic brain damage in the rat induces interleukin-1beta protein in microglia and astrocytes: correlation with the progression of cell death

Interleukin-1 beta (IL-1beta) has been proposed as a mediator of several forms of brain damage, including that induced by excitotoxins. In vitro studies suggest that glial cells are the effector cells of IL-1beta-mediated neurodegeneration. We have investigated the expression of IL-1beta protein by glial cells in vivo in response to NMDA receptor-mediated excitotoxicity in the rat parietal cortex and striatum. Expression of IL-1beta by glial cells was investigated using immunocytochemistry 30 min to 7 days after infusion of the NMDA agonist cis-2,4-methanoglutamate (MGlu; 10 nmol) into the cortex. Early expression (1-4 h) of IL-1beta by microglia was directly related to lesion development. Later expression by microglia (up to 24 h), and by astrocytes (2-7 days), was widespread compared to the area involved in excitotoxic cell death and co-localised with areas of reactive gliosis. Infusion of MGlu into the striatum induced a similar temporal pattern of IL-1beta expression by microglia and astrocytes. However, IL-1beta-expressing glial cells were localised strictly to the area of striatal cell death. Infusion of PBS or a subtoxic dose of MGlu into the cortex or striatum induced only limited neuronal death and negligible glial IL-1beta expression. These studies reveal that IL-1beta is expressed specifically by microglia during the early response to excitotoxicity in the adult rat cortex and striatum. However, the widespread and delayed IL-1beta expression by astrocytes suggests diverse roles for IL-1beta in response to excitotoxicity.     

Transient ischemia-induced changes of interleukin-2 and its receptor beta immunoreactivity and levels in the gerbil hippocampal CA1 region

Interlukin-2 (IL-2) is an important cytokine in the brain: IL-2 and its receptors are involved with inflammatory processes. Chronological changes in IL-2 level in serum, and IL-2 and its receptor (IL-2 receptor beta, IL-2Rbeta) immunoreactivities and levels were examined in the hippocampal CA1 region after transient forebrain ischemia in gerbils. IL-2 level in serum significantly decreased 12 h after ischemia/reperfusion. IL-2 immunoreactivity was detected in the somata of pyramidal cells in sham-operated group. At 15 min after ischemia, IL-2 immunoreactivity was shown in non-pyramidal cells as well as pyramidal cells. One day after ischemia, IL-2 immunoreactivity was lowest, and IL-2 immunoreactivity is shown in non-pyramidal cells from 2 days after ischemia. Four days after ischemia, IL-2 immunoreactivity was shown in dying pyramidal cells. IL-2Rbeta immunoreactivity in the sham-operated and 15 min-3 min post-ischemic groups is detected in the cell membrane of pyramidal cells. From 3 h after ischemia, IL-2Rbeta immunoreactivity is found in cytoplasm and nuclei, but not in cell membrane. IL-2Rbeta immunoreactivity decreases from 6 h after ischemia and is shown mainly in non-pyramidal cells from 3 days after ischemia. The data of Western blot analyses for IL-2 and IL-2Rbeta was similar to the immunohistochemical data. IL-2 infusion into cerebrospinal fluid did not protect hippocampal neurons from ischemic damage. These results suggest that IL-2 and IL-2Rbeta show malfunction from 3 h after ischemia, and exogenous IL-2 does not protect ischemic neuronal damage.     

Increased expression of mRNA encoding interleukin-1beta and caspase-1, and the secreted isoform of interleukin-1 receptor antagonist in the rat brain following systemic kainic acid administration

Kainic acid, an analogue of glutamate, injected systemically to rats evokes seizures that are accompanied by nerve cell damage primarily in the limbic system. In the present study, we have analyzed the temporal profile of the expression of the cytokines interleukin-1beta (IL-1beta) and IL-1 receptor antagonist (IL-1ra), and the related IL-1beta-converting enzyme (ICE/caspase-1), in different regions of the rat brain in response to peripheral kainic acid administration (10 mg/kg, i.p.). In situ hybridization histochemistry experiments revealed that IL-1beta mRNA-expressing cells, morphologically identified as microglial cells, were mainly localized to regions showing pronounced neuronal degeneration; hippocampus, thalamus, amygdala, and certain cortical regions. The strongest expression of IL-1beta mRNA was observed after 12 hr in these regions. A weak induction of the IL-1beta mRNA expression was observed already at 2 hr. Similar results were obtained by RT-PCR analysis, showing a significantly increased expression of IL-1beta mRNA in the hippocampus and amygdala after 12 hr. In addition, RT-PCR analysis revealed that IL-1ra mRNA, and specifically mRNA encoding the secreted isoform of IL-1ra (sIL-1ra), was strongly induced in the hippocampus and amygdala at 12 and 24 hr post-injection. RT-PCR analysis of mRNA encoding caspase-1 showed a significantly increased expression in the amygdala after 12 hr. In conclusion, in response to systemic kainic acid injection IL-1beta mRNA is rapidly induced and followed by induction of IL-1ra mRNA and caspase-1 mRNA, supporting a role of the IL-1 system in the inflammatory response during excitotoxic damage.     

Zaprinast, an inhibitor of cGMP-selective phosphodiesterases, enhances the secretion of TNF-alpha and IL-1beta and the expression of iNOS and MHC class II molecules in rat microglial cells

Proinflammatory cytokines produced by activated glial cells may in turn augment the immune/inflammatory reactions of glial cells through autocrine and paracrine routes. The NO/cGMP signaling represents one of the reactions of activated glial cells. We investigated whether the production of proinflammatory cytokines by glial cells is affected by NO-dependent downstream cGMP signaling. In primary cultures of mixed astrocytes and microglial cells, zaprinast (0.1 mM), an inhibitor of cGMP-selective phosphodiesterases, enhanced the basal and LPS (1.0 microg/ml)-induced secretion of TNF-alpha and IL-1beta. Zaprinast also enhanced NO production induced by LPS or IFN-gamma (100 U/ml), and in microglial cell cultures, but not in astrocyte cultures, zaprinast enhanced the basal and the IFN-gamma-induced production of the cytokines, TNF-alpha and IL-1beta, and of NO. This upregulation by zaprinast was partially inhibited by KT5823 (1.0 microM), an inhibitor of protein kinase G. The LPS-induced production of TNF-alpha, IL-1beta, and NO was inhibited by ODQ (50 microM), an inhibitor of soluble guanylyl cyclase, and by KT5823. Immunohistochemical analysis of mixed glial cell cultures showed that LPS/IFN-gamma-induced iNOS expression and the enhanced expression of iNOS by zaprinast were restricted to microglial cells. Zaprinast enhanced the IFN-gamma (200 U/ml)-induced expression of MHC Class II molecules in astrocytes and microglial cells in mixed cultures, but did not enhance this IFN-gamma-induced expression in pure astrocytes, which lacked paracrine TNF-alpha from microglial cells. Summarizing, zaprinast, which is associated with cGMP/protein kinase G signaling, may augment central immune/inflammatory reactions, possibly via the increased production of TNF-alpha and IL-1beta by activated microglial cells.     

Increased expression of caspase-1 and interleukin-18 in peripheral blood mononuclear cells in patients with multiple sclerosis

Multiple sclerosis (MS) is supposedly a T-cell mediated autoimmune disorder of the central nervous system. Cytokines and other molecules involved in the regulation of apoptosis are thought to be of importance for the pathogenesis of MS. In this study, the mRNA levels of interleukin 18 (IL-18), IL-1beta and their processing enzyme caspase-1 were quantified by a competitive RT-PCR method in unstimulated peripheral blood mononuclear cells (PBMCs) in MS patients never treated with disease modifying drugs. Western blot was used to support the expression pattern at the protein level. We found that the expression of caspase-1 and IL-18 was significantly increased in MS patients compared with healthy controls. Analysis of clinical subgroups revealed that caspase-1 was increased in all subgroups, whereas IL-18 was upregulated in chronic progression (P=0.001) and relapsing MS patients in remission (P=0.002) but not significantly during relapses (P=0.12). mRNA levels of IL-1beta were not significantly altered in MS except for a possible decrease in chronic progression (P=0.03). An increased IL-18 expression, potentially augmented at the mature protein level, may indicate a pathway worth considering in future therapeutic strategies in MS.     

Interleukin-1 beta, but not IL-1 alpha, mediates nerve growth factor secretion from rat astrocytes via type I IL-1 receptor.

In astrocytes, nerve growth factor (NGF) synthesis and secretion is stimulated by the cytokine interleukin-1 beta (IL-1 beta). In the present study, the role of IL-1 receptor binding sites in the regulation of NGF release was evaluated by determining the pharmacological properties of astroglially localized IL-1 receptors, and, by comparing the effects of both the agonists (IL-1 alpha and IL-1 beta) and the antagonist (IL-1ra)-members of the IL-1 family on NGF secretion from rat neonatal cortical astrocytes in primary culture. Using receptor-binding studies, binding of [(125)I] IL-1 beta to cultured astrocytes was saturable and of high affinity. Mean values for the K(D) and B(max) were calculated to be 60.7+/-7.4 pM and 2.5+/-0.1 fmol mg(-1) protein, respectively. The binding was rapid and readily reversible. IL-1 receptor agonists IL-1 alpha (K(i) of 341.1 pM) and IL-1 beta (K(i) 59.9 pM), as well as the antagonist IL-1ra (K(i) 257.6 pM), displaced specific [(125)I] IL-1 beta binding from cultured astrocytes in a monophasic manner. Anti-IL-1RI antibody completely blocked specific [(125)I] IL-1 beta binding while anti-IL-1RII antibody had no inhibitory effect. Exposure of cultured astrocytes to IL-1 alpha and IL-1 beta revealed the functional difference between the agonists in influencing NGF release. In contrast to IL-1 beta (10 U/ml), which caused a 3-fold increase in NGF secretion compared to control cells, IL-1 alpha by itself had no stimulatory action on NGF release. The simultaneous application of IL-1 alpha and IL-1 beta elicited no additive response. IL-1ra had no effect on basal NGF release but dose-dependently inhibited the stimulatory response induced by IL-1 beta. We concluded that IL-1 beta-induced NGF secretion from cultured rat cortical astrocytes is mediated by functional type I IL-1 receptors, whereas IL-1 alpha and IL-1ra, in spite of their affinity for IL-1RI, have no effect on NGF secretion from these cells. Type II IL-1R is not present on rat neonatal cortical astrocytes.     

Inhibition of caspase-1-like activity by Ac-Tyr-Val-Ala-Asp-chloromethyl ketone induces long-lasting neuroprotection in cerebral ischemia through apoptosis reduction and decrease of proinflammatory cytokines.

Broad spectrum caspase inhibitors have been found to reduce neurodegeneration caused by cerebral ischemia. We studied whether blockade of group I caspases, mainly caspase-1, using the inhibitor Ac-YVAD.cmk reduced infarct volume and produced prolonged neuroprotection. Ac-YVAD.cmk (300 ng/rat) was injected intracerebroventricularly 10 min after permanent middle cerebral artery occlusion in the rat. Drug treatment induced a significant reduction of infarct volume not only 24 hr after ischemia (total damage, percentage of hemisphere volume: control, 41.1 +/- 2.3%; treated, 26.5 +/- 2.1%; p < 0.05) but also 6 d later (total damage: control, 30.6 +/- 2.2%; treated, 23.0 +/- 2.2%; p < 0.05). Ac-YVAD. cmk treatment resulted in a reduction not only of caspase-1 (control, 100 +/- 20.3%; treated, 3.4 +/- 10.4%; p < 0.01) but also of caspase-3 (control, 100 +/- 30.3%; treated, 13.2 +/- 9.5%; p < 0.05) activity at 24 hr and led to a parallel decrease of apoptosis as measured by nucleosome quantitation (control, 100 +/- 11.8%; treated, 47 +/- 5.9%; p < 0.05). Six days after treatment no differences in these parameters could be detected between control and treated animals. Likewise, brain levels of the proinflammatory cytokines IL-1beta and TNF-alpha were reduced at 24 hr (39.5 +/- 23.7 and 51.9 +/- 10.3% of control, respectively) but not at 6 d. Other cytokines, IL-10, MCP-1, MIP-2, and the gaseous mediator nitric oxide, were not modified by the treatment. These findings indicate that blockade of caspase-1-like activity induces a long-lasting neuroprotective effect that, in our experimental conditions, takes place in the early stages of damage progression. Finally, this effect is achieved by interfering with both apoptotic and inflammatory mechanisms.     

Peripheral levels of caspase-1 mRNA correlate with disease activity in patients with multiple sclerosis; a preliminary study

The cysteine protease caspase-1 plays a crucial part in the inflammatory process due to its ability to proteolitically activate proinflammatory cytokine precursors, such as interleukin (IL)-1beta and IL-18. Multiple sclerosis is a chronic inflammatory demyelinating disease of the CNS in which the pathogenic process is mainly orchestrated by proinflammatory cytokines. The role of caspase-1 in multiple sclerosis was evaluated by measuring its mRNA levels in peripheral blood mononuclear cells (PBMCs) from seven patients with relapsing-remitting multiple sclerosis every 15 days over a 1 year period. The recorded levels were compared with clinical and MRI evidence of disease activity. Brain MRI was performed monthly in each patient. Caspase-1 mRNA levels were significantly increased in PBMCs from patients with multiple sclerosis compared with healthy controls (p<0.001). In patients with multiple sclerosis, a twofold to threefold increase of caspase-1 mRNA mean level was found in the week preceding an acute attack (p<0. 05). The magnitude of caspase-1 mRNA increase correlated with the number of new (p=0.01) but not persisting gadolinium enhancing brain MRI lesions. In conclusion, caspase-1 might be involved in the immune mediated process underlying CNS inflammation and might represent a suitable peripheral immunological marker of disease activity in multiple sclerosis.     

Caspase-1-processed interleukins in hyperoxia-induced cell death in the developing brain

Infants born prematurely may develop neurocognitive deficits without an obvious cause. Oxygen, which is widely used in neonatal medicine, constitutes one possible contributing neurotoxic factor, because it can trigger neuronal apoptosis in the developing brain of rodents. We hypothesized that two caspase-1-processed cytokines, interleukin (IL)-1beta and IL-18, are involved in oxygen-induced neuronal cell death. Six-day-old Wistar rats or C57/BL6 mice were exposed to 80% oxygen for various time periods (2, 6, 12, 24, and 48 hours). Neuronal cell death in the brain, as assessed by Fluoro-Jade B and silver staining, peaked at 12 to 24 hours and was preceded by a marked increase in mRNA and protein levels of caspase 1, IL-1beta, IL-18, and IL-18 receptor alpha (IL-18Ralpha). Intraperitoneal injection of recombinant human IL-18-binding protein, a specific inhibitor of IL-18, attenuated hyperoxic brain injury. Mice deficient in IL-1 receptor-associated kinase 4 (IRAK-4), which is pivotal for both IL-1beta and IL-18 signal transduction, were protected against oxygen-mediated neurotoxicity. These findings causally link IL-1beta and IL-18 to hyperoxia-induced cell death in the immature brain. These cytokines might serve as useful targets for therapeutic approaches aimed at preserving neuronal function in the immature brain, which is exquisitely sensitive to a variety of iatrogenic measures including oxygen.