ABSTRACT
Several types of psoriasiform dermatitis are associated with increased IL-36 cytokine activity in the skin. A rare, but severe, psoriasis-like disorder, generalized pustular psoriasis (GPP), is linked to loss-of-function mutations in the gene encoding IL-36RA, an important negative regulator of IL-36 signaling. To understand the effects of IL-36 dysregulation in a mouse model, we studied skin inflammation induced by intradermal injections of preactivated IL-36α. We found the immune cells infiltrating IL-36α-injected mouse skin to be of dramatically different composition than those infiltrating imiquimod-treated skin. The IL-36α-induced leukocyte population comprised nearly equal numbers of CD4+ αß T cells, neutrophils, and inflammatory dendritic cells, whereas the imiquimod-induced population comprised γδ T cells and neutrophils. Ligands for chemokine receptors CCR6 and CXCR2 are increased in both GPP and IL-36α-treated skin, which led us to test an optimized small-molecule antagonist (CCX624) targeting CCR6 and CXCR2 in the IL-36α model. CCX624 significantly reduced the T cell, neutrophil, and inflammatory dendritic cell infiltrates and was more effective than saturating levels of an anti-IL-17RA mAb at reducing inflammatory symptoms. These findings put CCR6 and CXCR2 forward as novel targets for a mechanistically distinct therapeutic approach for inflammatory skin diseases involving dysregulated IL-36 signaling, such as GPP.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Interleukin-1/toxicity , Psoriasis/immunology , Receptors, CCR6/antagonists & inhibitors , Receptors, Interleukin-8B/antagonists & inhibitors , Animals , Interleukin-1/immunology , Mice , Mice, Inbred BALB C , Psoriasis/chemically induced , Skin/drug effects , Skin/immunologyABSTRACT
Human and mouse neonates exhibit limited vaccine responses characterized by predominant Th2 and limited Th1 responses. Because IL-36 exerts a synergic adjuvant effect with IL-12, enhancing Th1 polarization in adult (AD) mice, we administered IL-36ß to neonatal (1-wk old) and AD control mice at the time of immunization with tetanus toxoid adsorbed to aluminum hydroxide (TT/Alum). Unexpectedly, the combination of IL-36ß with TT/Alum, which was well tolerated in AD mice, proved toxic and even lethal in neonates. This neonatal toxicity was associated with high Il36r mRNA expression in neonatal liver, resulting in increased cytokine production. Liver Il36r mRNA expression decreased with the termination of fetal liver hematopoiesis, and this decrease correlated with a complete protection from TT/Alum/IL-36ß-induced mortality. The combination of IL-36ß and TT/Alum induced the rapid production of TNF-α and IFN-γ by liver myeloid and lymphoid cells, respectively. These responses were less marked when IL-36ß was used alone, with no adverse effect. The toxicity of IL-36ß + TT/Alum was abrogated by the administration of a neutralizing anti-TNF-α Ab, confirming causality. In conclusion, liver myeloid cells in neonatal mice are an important source of proinflammatory cytokines that may lead to TNF-α-mediated toxicity and even lethality.
Subject(s)
Interleukin-1/toxicity , Liver/immunology , Myeloid Cells/immunology , Tumor Necrosis Factor-alpha/biosynthesis , Animals , Animals, Newborn , Cytokines/biosynthesis , Mice , Mice, Inbred C57BL , Tetanus Toxoid/toxicityABSTRACT
OBJECTIVE: Angiogenesis is tightly controlled by growth factors and cytokines in pathophysiological settings. Interleukin 37 (IL-37) is a newly identified cytokine of the IL-1 family, some members of which are important in inflammation and angiogenesis. However, the function of IL-37 in angiogenesis remains unknown. We aimed to explore the regulatory role of IL-37 in pathological and physiological angiogenesis. APPROACH AND RESULTS: We found that IL-37 was expressed and secreted in endothelial cells and upregulated under hypoxic conditions. IL-37 enhanced endothelial cell proliferation, capillary formation, migration, and vessel sprouting from aortic rings with potency comparable with that of vascular endothelial growth factor. IL-37 activates survival signals including extracellular signal-regulated kinase 1/2 and AKT in endothelial cells. IL-37 promoted vessel growth in implanted Matrigel plug in vivo in a dose-dependent manner with potency comparable with that of basic fibroblast growth factor. In the mouse model of retinal vascular development, neonatal mice administrated with IL-37 displayed increased neovascularization. We demonstrated further that IL-37 promoted pathological angiogenesis in the mouse model of oxygen-induced retinopathy. CONCLUSIONS: Our findings suggest that IL-37 is a novel and potent proangiogenic cytokine with essential role in pathophy siological settings.
Subject(s)
Angiogenesis Inducing Agents/pharmacology , Human Umbilical Vein Endothelial Cells/drug effects , Interleukin-1/pharmacology , Neovascularization, Physiologic/drug effects , Retinal Neovascularization/chemically induced , Retinopathy of Prematurity/chemically induced , Animals , Animals, Newborn , Cell Hypoxia , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Disease Models, Animal , Dose-Response Relationship, Drug , Extracellular Signal-Regulated MAP Kinases/metabolism , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Interleukin-1/metabolism , Interleukin-1/toxicity , Mice, Inbred C57BL , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , Retinal Neovascularization/metabolism , Retinal Neovascularization/pathology , Retinopathy of Prematurity/metabolism , Retinopathy of Prematurity/pathology , Time Factors , TransfectionABSTRACT
Treatment of cultures with toll-like receptor (TLR) ligands or cytokines has become a popular approach to investigate astrocyte neuroinflammatory responses and to simulate the neural environment in various CNS disorders. However, despite much effort, the mechanism of astrocyte activation such as their responses to the TLR ligands and IL-1 remain highly debated. We compared highly pure primary mouse and human astrocyte cultures in their ability to produce proinflammatory mediators (termed "A1") and immunoregulatory mediators (termed "A2") in response to LPS, poly IC, and IL-1 stimulation. In human astrocytes, IL-1 induced both A1 and A2 responses, poly IC induced mostly A2, and LPS induced neither. In mouse astrocytes, LPS induced mostly an A1-predominant response, poly IC induced both A1 and A2, and IL-1 neither. In addition, mouse astrocytes produce abundant IL-1 protein, whereas human astrocytes did not, despite robust IL-1 mRNA expression. Of the TLR4 receptor complex proteins, human astrocytes expressed TLR4 and MD2 but not CD14, whereas mouse astrocytes expressed all three. Mouse astrocyte CD14 (cell-associated and soluble) was potently upregulated by LPS. Silencing TLR4 or CD14 by siRNA suppressed LPS responses in mouse astrocytes. In vivo, astrocytes in LPS-injected mouse brains also expressed CD14. Our results show striking differences between human and mouse astrocytes in the use of TLR/IL-1R and subsequent downstream signaling and immune activation. IL-1 translational block in human astrocytes may be a built-in mechanism to prevent autocrine and paracrine cell activation and neuroinflammation. These results have important implications for translational research of human CNS diseases.
Subject(s)
Astrocytes/drug effects , Astrocytes/physiology , Interleukin-1/toxicity , Lipopolysaccharide Receptors/physiology , Lipopolysaccharides/toxicity , Animals , Animals, Newborn , Cells, Cultured , Fetus , Humans , Mice , Mice, Inbred C57BL , Species SpecificityABSTRACT
The proinflammatory cytokine interleukin-1 (IL-1) is a key mediator of inflammation in cerebral ischemia, but its precise mechanisms of action remain elusive. Temperature is critical to outcome in brain injury and given the importance of IL-1 in pyrogenesis this has clear mechanistic implications. IL-1 exacerbates ischemia independently of core (rectal) temperature. However, it is temperature in the ischemic brain that influences outcome and rectal temperature is likely to be a poor surrogate marker. This study tested the hypothesis that IL-1 exacerbates cerebral ischemia by increasing ischemic brain temperature. Wistar rats undergoing transient middle cerebral artery occlusion received either 4 microg/kg IL-1 (n=9) or vehicle (n=10) intraperitoneally. NMR-generated maps of brain temperature, tissue perfusion, and the trace of the diffusion tensor were collected during occlusion, early reperfusion, and at 24 hr. IL-1 significantly increased ischemic damage at 24 hr by 35% but rectal temperature did not vary significantly between groups. However, ischemic brain was 1.7 degrees C cooler on reperfusion in IL-1-treated animals (vs. vehicle) and a corresponding reduction in cerebral blood flow was identified in the ischemic striatum. Contrary to the stated hypothesis, IL-1 reduced ischemic brain temperature during reperfusion and this may be due to a reduction in tissue perfusion.
Subject(s)
Brain Ischemia/pathology , Interleukin-1/toxicity , Magnetic Resonance Spectroscopy/methods , Analysis of Variance , Animals , Body Temperature/drug effects , Linear Models , Phantoms, Imaging , Rats , Rats, WistarABSTRACT
Acute brain injury is associated with induction of hepatic chemokine expression, which is an essential element in the subsequent recruitment of leukocytes to the damaged brain. To further understand the significance of the hepatic inflammatory response, we focused on nuclear factor (NF)-kappa B, a pivotal regulator of inflammation. Nondestructive real-time whole-body imaging was undertaken in the 3XNF-kappa B-luciferase mouse to monitor NF-kappa B activation. Acute brain injury induced by intracerebral injection of interleukin-1 provoked rapid activation of hepatic and CNS NF-kappa B, with only minimal changes in other organs. Elevated NF-kappa B in the brain was limited to the site of the lesion, whereas hepatic NF-kappa B was widespread. The function of NF-kappa B in this model was determined by monitoring leukocyte recruitment to the liver and brain of nf kappa b1 mice, which lack the anti-inflammatory p50:p50 NF-kappa B homodimer. Brain injury in the nf kappa b1 mice was associated with increased neutrophil recruitment to the liver and brain compared with wild-type mice, thereby confirming a regulatory role for the NF-kappa B system. To determine the role of hepatic NF-kappa B, it was selectively inhibited by intravenous adenoviral-mediated delivery of an I kappa B alpha super-repressor. This treatment significantly reduced the numbers of neutrophils recruited to the brain. In conclusion, acute brain injury is associated with rapid and robust activation of hepatic NF-kappa B, which is required for efficient mobilization of circulating leukocytes to the brain.
Subject(s)
Brain Injuries/immunology , Chemotaxis, Leukocyte/immunology , Liver/metabolism , NF-kappa B/metabolism , Neutrophil Infiltration/immunology , Neutrophils/immunology , Animals , Brain/immunology , Brain/metabolism , Brain/physiopathology , Brain Injuries/metabolism , Down-Regulation/immunology , Encephalitis/immunology , Encephalitis/metabolism , Encephalitis/physiopathology , Genetic Vectors , HeLa Cells , Humans , I-kappa B Proteins/antagonists & inhibitors , I-kappa B Proteins/genetics , Interleukin-1/toxicity , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-KappaB Inhibitor alpha , NF-kappa B p50 Subunit/metabolism , Reaction Time/immunology , Repressor Proteins/pharmacology , Time Factors , Up-Regulation/immunologyABSTRACT
OBJECTIVE: To determine the effects of sodium hyaluronate (HA) in combination with methylprednisolone acetate (MPA) on interleukin-1 (IL-1)-induced inflammation in equine articular cartilage pellets. SAMPLE POPULATION: Chondrocytes collected from 7 horses euthanatized for problems unrelated to the musculoskeletal system. PROCEDURES: Chondrocyte pellets were treated with medium (negative control); medium containing IL-1 (positive control); or medium containing IL-1 with MPA only (0.05 or 0.5 mg/mL), HA only (0.2 or 2 mg/mL), or MPA (0.05 or 0.5 mg/mL) and HA (0.2 or 2 mg/mL) in combination. Proteoglycan (PG) synthesis was determined by incorporation of sulfur 35-labeled sodium sulfate into PGs. Glycosaminoglycan (GAG) content of the media and the pellets and total pellet DNA content were determined. RESULTS: Methylprednisolone acetate at 0.5 mg/mL caused an increase in PG synthesis, whereas HA had no effect alone. The combination of MPA, both 0.05 mg/mL and 0.5 mg/mL, with HA at 2 mg/mL increased PG synthesis, compared with IL-1-treated control. All treatment groups containing the high concentration of MPA (0.5 mg/mL) and the high concentration of HA (2.0 mg/mL) had pellets with increased GAG content. The addition of HA caused an increase in total GAG content in the media, regardless of MPA treatment. Cyclooxygenase-2 mRNA and aggrecan mRNA expression was significantly reduced with MPA treatment. Total pellet DNA content was unchanged by any treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that MPA in combination with HA has beneficial effects on PG metabolism of IL-1-treated equine chondrocytes.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Chondrocytes/drug effects , Horses/metabolism , Hyaluronic Acid/pharmacology , Methylprednisolone/analogs & derivatives , Proteoglycans/metabolism , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/pharmacology , Animals , Anti-Inflammatory Agents/administration & dosage , Cells, Cultured , Chondrocytes/cytology , Chondrocytes/metabolism , Hyaluronic Acid/administration & dosage , Inflammation/drug therapy , Interleukin-1/toxicity , Methylprednisolone/administration & dosage , Methylprednisolone/pharmacology , Methylprednisolone Acetate , Proteoglycans/analysis , Proteoglycans/drug effects , Sulfur Radioisotopes/analysisABSTRACT
BACKGROUND AND PURPOSE: The acute vascular inflammatory dysfunction associated with endotoxaemia may reflect an imbalance between matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs), induced by the endotoxin. This possibility was tested in rat aortic tissue. EXPERIMENTAL APPROACHES: Tone induced by phenylephrine in aortic rings was measured after exposure in vitro to ambient lipopolysaccharide (LPS) or the proinflammatory cytokine interleukin-1beta (IL-1beta) for 6h, with or without MMP inhibitors (doxycycline or GM6001). Gelatinase and MMP activities, TIMP proteins and contractility were measured in aortae taken from rats 6h after receiving LPS in vivo. KEY RESULTS: Inhibition of MMP prevented the loss of phenylephrine-induced tone in aortic rings after LPS or IL-1beta. IL-1beta also increased release of MMP-2 activity from aortic tissue. In aortae exposed in vivo to LPS, net gelatinase, MMP-9 activities and TIMP-1 protein levels were increased, whereas TIMP-4 was reduced. These aortae were hypocontractile to both phenylephrine and KCl. Hypocontractility was partially reversed by doxycycline ex vivo. CONCLUSIONS AND IMPLICATIONS: MMP inhibitors ameliorate vascular hyporeactivity induced by either LPS or IL-1beta in vitro. LPS in vivo alters the balance between MMPs and TIMPs, contributing to vascular dysfunction which is partially reversed by MMP inhibitors. Vascular MMPs are activated as a result of LPS or IL-1beta-induced stress and contribute to the hyporeactivity of blood vessels to vasoconstrictors.
Subject(s)
Endotoxemia/enzymology , Endotoxins/toxicity , Interleukin-1/toxicity , Matrix Metalloproteinases/metabolism , Vascular Diseases/chemically induced , Animals , Aorta, Thoracic , Blotting, Western , Collagenases/metabolism , Endotoxins/blood , Gelatinases/metabolism , In Vitro Techniques , Male , Muscle Contraction/drug effects , Muscle Proteins/metabolism , Muscle Tonus/drug effects , Muscle, Smooth, Vascular/drug effects , Nitrates/blood , Nitrites/blood , Phenylephrine/pharmacology , Rats , Rats, Sprague-Dawley , Tissue Inhibitor of Metalloproteinase-1/metabolism , Tissue Inhibitor of Metalloproteinases/metabolism , Vascular Diseases/enzymology , Vasoconstrictor Agents/pharmacology , Tissue Inhibitor of Metalloproteinase-4ABSTRACT
Interleukin (IL)-1 expression is induced rapidly in response to diverse CNS insults and is a key mediator of experimentally induced neuronal injury. However, the mechanisms of IL-1-induced neurotoxicity are unknown. The aim of the present study was to examine the toxic effects of IL-1 on rat cortical cell cultures. Treatment with IL-1beta did not affect the viability of pure cortical neurones. However, IL-1 treatment of cocultures of neurones with glia or purified astrocytes induced caspase activation resulting in neuronal death. Neuronal cell death induced by IL-1 was prevented by pre-treatment with the IL-1 receptor antagonist, the broad spectrum caspase inhibitor Boc-Asp-(OMe)-CH(2)F or the antioxidant alpha-tocopherol. The NMDA receptor antagonist dizolcipine (MK-801) attenuated cell death induced by low doses of IL-1beta but the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) had no effect. Inhibition of inducible nitric oxide synthase with N(omega)-nitro-l-arginine methyl ester had no effect on neuronal cell death induced by IL-1beta. Thus, IL-1 activates the IL-1 type 1 receptor in astrocytes to induce caspase-dependent neuronal death, which is dependent on the release of free radicals and may contribute to neuronal cell death in CNS diseases.
Subject(s)
Caspases/metabolism , Free Radicals/metabolism , Interleukin-1/toxicity , Neuroglia/physiology , Neurons/drug effects , Analysis of Variance , Animals , Cell Death/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Coculture Techniques/methods , Dose-Response Relationship, Drug , Drug Interactions , Embryo, Mammalian , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Agonists/pharmacology , Immunohistochemistry/methods , N-Methylaspartate/pharmacology , Nerve Tissue Proteins/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
Jeongshintang (JST) is a Korean herbal prescription, which has been successfully used for cerebral diseases. However, the anti-inflammatory effect of JST on Alzheimer's disease (AD) is still not fully understood. In this study, we investigated the effects of JST in attenuating the inflammatory response induced by interleukin (IL)-1beta plus beta-amyloid [1-42] fragment (A beta) in the human astrocyte cell line, U373MG. The production of IL-6, IL-8, and prostaglandin (PG)E2 was significantly increased by IL-1beta plus A beta (1-42) in a time-dependent manner (P < 0.05). JST significantly inhibited the IL-1beta plus A beta (1-42)-induced IL-6, IL-8, and PGE2 production at 24 h (P < 0.05). Maximal inhibition rate of IL-6, IL-8, and PGE2 production by JST was about 54.40%, 56.01%, and 44.06% respectively. JST (0.01-1 mg/ml) also attenuated the expression of cyclooxygenase (COX)-2 and activation of p38 MAPK induced by IL-1beta and A beta (1-42). These results demonstrated that JST has an anti-inflammatory effect, which might explain its beneficial effect in the treatment of various neurodegenerative diseases such as AD.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Neuroprotective Agents/pharmacology , Plant Extracts/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Amyloid beta-Peptides/toxicity , Astrocytoma , Cell Line, Tumor , Cyclooxygenase 2 , Dinoprostone/antagonists & inhibitors , Enzyme Activation/drug effects , Humans , Interleukin-1/toxicity , Interleukin-6/antagonists & inhibitors , Interleukin-8/antagonists & inhibitors , Membrane Proteins/antagonists & inhibitors , Peptide Fragments/toxicity , Protein Kinase Inhibitors/pharmacologyABSTRACT
BACKGROUND: Increased interleukin-1beta (IL-1) in the brain and periphery has been associated with neurodegenerative and psychiatric disorders. However, results from different IL-1 sources, administrating routes, doses and treatment duration were inconsistent and confused. The neuroendocrine-immune mechanism by which IL-1-induced behavioral changes occur is still unclear. METHODS: In the present study, the acute and sub-chronic effects of rat recombinant IL-1, following either intraperitoneal (ip) or intracerebroventricular (icv) injection, were studied on the behavior, corticosterone secretion, peripheral inflammatory responses and brain monoamines. RESULTS: In the open field apparatus, IL-1 (ip) increased locomotor activity but decreased the activity following icv administration. IL-1 had a greater anxiogenic effect in the elevated plus maze after icv than after ip administration. In the Morris water maze spatial memory was only impaired following sub-chronic and icv administration. Both acute and sub-chronic IL-1 increased the serum corticosterone concentration and decreased the release of the anti-inflammatory cytokine IL-10 from whole blood cultures. However, centrally administered IL-1 increased, while peripherally administered decreased, the release of PGE2 from blood cultures. After sub-chronic administration, the noradrenaline concentration was decreased in several limbic regions, while the turnovers of serotonin and dopamine were increased. DISCUSSION: These results suggest that 1) IL-1 effects depended on the dose, route and duration of administration, and 2) IL-1 enhances the responsiveness of rats to stressful environmental stimuli. In addition, the sub-chronic administration of IL-1 induces behavioral, neurotransmitter, hormonal and immune changes that may be causally implicated in the mechanism of some of psychiatric disorders such as depression.
Subject(s)
Behavior, Animal/drug effects , Brain/drug effects , Corticosterone/blood , Dinoprostone/blood , Interleukin-10/blood , Interleukin-1/toxicity , Neurotransmitter Agents/blood , Animals , Arousal/drug effects , Depression/blood , Depression/chemically induced , Dopamine/blood , Dose-Response Relationship, Drug , Drug Administration Schedule , Fear/drug effects , Injections, Intraperitoneal , Injections, Intraventricular , Interleukin-1/administration & dosage , Limbic System/drug effects , Male , Motor Activity/drug effects , Norepinephrine/blood , Rats , Rats, Wistar , Serotonin/bloodABSTRACT
Cartilage damage in osteoarthritis (OA) is considered an imbalance between catabolic and anabolic factors, favoring the catabolic side. We assessed whether adenoviral overexpression of transforming growth factor-beta (TGFbeta) enhanced cartilage repair and whether TGFbeta-induced fibrosis was blocked by local expression of the intracellular TGFbeta inhibitor Smad7. We inflicted cartilage damage by injection of interleukin-1 (IL-1) into murine knee joints. After 2 days, we injected an adenovirus encoding TGFbeta. On day 4, we measured proteoglycan (PG) synthesis and content. To examine whether we could block TGFbeta-induced fibrosis and stimulate cartilage repair simultaneously, we injected Ad-TGFbeta and Ad-Smad7. This was performed both after IL-1-induced damage and in a model of primary OA. In addition to PG in cartilage, synovial fibrosis was measured by determining the synovial width and the number of procollagen I-expressing cells. Adenoviral overexpression of TGFbeta restored the IL-1-induced reduction in PG content and increased PG synthesis. TGFbeta-induced an elevation in PG content in cartilage of the OA model. TGFbeta-induced synovial fibrosis was strongly diminished by simultaneous synovial overexpression of Smad7 in the synovial lining. Of great interest, overexpression of Smad7 did not reduce the repair-stimulating effect of TGFbeta on cartilage. Adenoviral overexpression of TGFbeta stimulated repair of IL-1- and OA-damaged cartilage. TGFbeta-induced synovial fibrosis was blocked by locally inhibiting TGFbeta signaling in the synovial lining by simultaneously transfecting it with an adenovirus overexpressing Smad7.
Subject(s)
Cartilage/pathology , Fibrosis/prevention & control , Smad7 Protein/physiology , Transforming Growth Factor beta/pharmacology , Wound Healing/drug effects , Animals , Cartilage/drug effects , Disease Models, Animal , Genetic Vectors , Interleukin-1/toxicity , Knee Joint/drug effects , Knee Joint/pathology , Mice , Mice, Inbred C57BL , Osteoarthritis/pathology , Osteoarthritis/prevention & control , Recombinant Proteins/pharmacology , Transfection , Transforming Growth Factor beta/geneticsABSTRACT
Pro-apoptotic cytokines are toxic to the pancreatic beta-cells and have been associated with the pathogenesis of Type 1 diabetes (T1D). Proteome analysis of IL-1beta exposed isolated rat islets identified galectin-3 (gal-3) as the most up-regulated protein. Here analysis of human and rat islets and insulinoma cells confirmed IL-1beta regulated gal-3 expression of several gal-3 isoforms and a complex in vivo expression profile during diabetes development in rats. Over-expression of gal-3 protected beta-cells against IL-1beta toxicity, with a complete blockage of JNK phosphorylation, essential for IL-1-mediated apoptosis. Mutation scanning of regulatory and coding regions of the gal-3 gene (LGALS3) identified six polymorphisms. A haplotype comprising three cSNPs showed significantly increased transmission to unaffected offspring in 257 T1D families and replicated in an independent set of 170 T1D families. In summary, combined proteome-transcriptome-genome and functional analyses identify gal-3 as a candidate gene/protein in T1D susceptibility that may prove valuable in future intervention/prevention strategies.
Subject(s)
Cytokines/toxicity , Diabetes Mellitus, Type 1/genetics , Diabetes Mellitus, Type 1/immunology , Galectin 3/physiology , Gene Expression Regulation , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/immunology , Animals , Apoptosis/genetics , Apoptosis/immunology , Diabetes Mellitus, Type 1/metabolism , Galectin 3/genetics , Gene Expression Regulation/drug effects , Genomics , Haplotypes , Humans , Insulin-Secreting Cells/chemistry , Interleukin-1/toxicity , Mutation , Phosphotransferases/metabolism , Polymorphism, Single Nucleotide , Proteomics , RNA, Messenger/analysis , RNA, Messenger/metabolism , Rats , Rats, Inbred StrainsABSTRACT
Nitric oxide (NO) has been reported to play an important role as an effector molecule in cytokine signal transduction in cardiomyocytes. A treatment of neonatal rat ventricular cardiomyocytes with interleukin-1 beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) induces apoptosis via an NO-dependent pathway. However, cardiomyocytes were more resistant to NO-dependent cell death in the presence of catalase, while producing inducible nitric oxide synthase. This paper reports that catalase stimulates the NF-kappaB-binding affinity. However, the NO synthase activity is abolished by the addition of catalase, suggesting that H(2)O(2) is involved in NO synthesis in a posttranslation state. The catalase-induced inhibition of NO was partially but significantly reversed by H(4)B, an important cofactor of NO synthesis. Treatment of myocytes with IL-1beta, TNF-alpha, and IFN-gamma induced a significant increase in the formation of peroxynitrite, and a pretreatment with catalase was found to quench the production of peroxynitrite. This paper shows that the catalase activity was significantly down-regulated by H(4)B in a concentration-dependent manner. The treatment of H(4)B induced reactive oxygen species (ROS) release in cardiac cell system. These results suggest that catalase interferes with NO and peroxynitrite production as well as with the related apoptosis of cardiomyocytes. This study also shows that the catalase-induced inhibition of NO release may be reversed by H(4)B by the release of ROS.
Subject(s)
Apoptosis/drug effects , Catalase/pharmacology , Cytokines/toxicity , Myocytes, Cardiac/drug effects , Nitric Oxide/antagonists & inhibitors , Animals , Biopterins/analogs & derivatives , Biopterins/pharmacology , Catalase/antagonists & inhibitors , Heart Ventricles/cytology , Interferon-Stimulated Gene Factor 3/toxicity , Interleukin-1/toxicity , Myocytes, Cardiac/cytology , NF-kappa B/metabolism , Nitric Oxide/biosynthesis , Peroxynitrous Acid/metabolism , Rats , Rats, Wistar , Reactive Oxygen Species/metabolism , Tumor Necrosis Factor-alpha/toxicityABSTRACT
The cytokine interleukin-1 (IL-1) is an established and important mediator of diverse forms of neuronal injury in experimental animals. However, its mechanisms of action remain largely unknown. We have reported previously that IL-1 markedly enhances excitotoxic injury induced in the rat by striatal administration of the excitotoxin alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), leading to widespread neuronal loss throughout the ipsilateral cortex. Here we tested the hypothesis that IL-1 causes this injury through induction and/or enhancement of seizure activity in the rat. Consistently with this hypothesis, intrastriatal injection of AMPA or AMPA with IL-1 in the rat brain increased c-Fos expression in regions similar to those in which c-Fos has been reported previously in response to seizures. A significant increase in cortical neuronal activity (number of c-Fos positive cells) was observed in response to AMPA with IL-1 compared with AMPA (8 hr after injection). Increased seizure duration [3,522 +/- 660 sec (SEM) vs. 1,415 +/- 301 sec; P < 0.001] and cell death volume (140 +/- 20 mm3 vs. 52 +/- 6 mm3; P < 0.001) were seen in response to coinfusion of AMPA with IL-1 vs. AMPA alone. In addition, the anticonvulsant diazepam (intraperitoneal) significantly reduced cell death (P < 0.001) and seizure duration (P < 0.001) induced by AMPA with IL-1, and a significant correlation was found between seizure duration and cell death volume. These findings support our hypothesis that IL-1 enhances excitotoxic injury by enhancement of seizures, which may be of relevance to IL-1 actions in other forms of neuronal injury, including cerebral ischemia.
Subject(s)
Corpus Striatum/drug effects , Interleukin-1/toxicity , Nerve Degeneration/etiology , Seizures/complications , Analysis of Variance , Animals , Anticonvulsants/therapeutic use , Cell Count/methods , Cell Death/drug effects , Corpus Striatum/pathology , Corpus Striatum/physiopathology , Diazepam/administration & dosage , Diazepam/therapeutic use , Disease Models, Animal , Drug Interactions , Electroencephalography/methods , Immunohistochemistry/methods , Male , Nerve Degeneration/drug therapy , Proto-Oncogene Proteins c-fos/metabolism , Rats , Rats, Sprague-Dawley , Seizures/drug therapy , Time Factors , alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid/toxicityABSTRACT
BACKGROUND: Whereas a primary role of interleukin-1beta (IL-1beta) in local bone remodelling and articular inflammation has been well established, the effect of prolonged systemic administration of this cytokine on total skeletal Ca, somatic growth and joint tissue has not yet been investigated. METHODS: Five groups of 14 rats each, aged 7-8 weeks, had miniosmotic pumps (Alzet 200 microl) implanted and primed to release 100, 200, 500, 1,000 and 2,000 ng/kg/24 h of human recombinant IL-1beta (rIL-1beta) daily for 14 days. On days 0 and 14 total skeletal mineral content (BMC) was assessed by means of X-ray absorptiometry and vertebral and tibial growth was measured by computer-assisted radiometry. On the same days, blood was drawn and analyzed for rat growth hormone (rGH), insulin-like growth factor (IGF-1), and osteocalcin. Also 24-hour urine was collected for d-pyridinoline (dpd) determinations. Hind- and forepaw diameter as a parameter of joint inflammation was assessed using a micrometric calliper. Subsequently the animals were sacrificed and one tibia dissected for measurement of trabecular volume by computerized histomorphometry. RESULTS: BMC decreased in a dose-dependent manner reaching significance at 1,000 and 2,000 ng/kg (p < 0.03 and 0.04) in close correlation with tibial trabecular volumes (r = 0.84; p < 0.02). Normal vertebral and tibial growth was recorded at all dosages. There was no evidence of joint involvement. Blood rGH and IGF-1 remained normal as did osteocalcin, the latter reflecting lack of osteoblast activation. In contrast dpd increased in a dose-dependent manner indicating enhanced bone matrix turnover. CONCLUSION: It is concluded that graded infusions of supraphysiological doses of rIL-1beta capable of inducing osteopenia did not affect skeletal growth in the absence of articular reaction. This is in contrast with the experience recorded in experimental arthritis in which growth retardation, in addition to osteopenia, may be caused by factors other than circulating IL-1beta.
Subject(s)
Bone Development/drug effects , Bone Diseases, Metabolic/chemically induced , Interleukin-1/toxicity , Joints/drug effects , Animals , Biomarkers , Bone Density/drug effects , Bone and Bones/drug effects , Bone and Bones/metabolism , Calcium/metabolism , Cartilage, Articular/drug effects , Corticosterone/blood , Dose-Response Relationship, Drug , Female , Foot/growth & development , Interleukin-1/administration & dosage , Rats , Rats, Wistar , Recombinant Proteins/pharmacology , Weight Gain/drug effectsABSTRACT
Transcription factor hypoxia-inducible factor (HIF)-1 protein accumulates and activates the transcription of genes that are of fundamental importance for oxygen homeostasis - including genes involved in energy metabolism, angiogenesis, vasomotor control, apoptosis, proliferation, and matrix production - under hypoxic conditions. We speculated that HIF-1alpha may have an important role in chondrocyte viability as a cell survival factor during the progression of osteoarthritis (OA). The expression of HIF-1alpha mRNA in human OA cartilage samples was analyzed by real-time PCR. We analyzed whether or not the catabolic factors IL-1beta and H2O2 induce the expression of HIF-1alpha in OA chondrocytes under normoxic and hypoxic conditions (O2 <6%). We investigated the levels of energy generation, cartilage matrix production, and apoptosis induction in HIF-1alpha-deficient chondrocytes under normoxic and hypoxic conditions. In articular cartilages from human OA patients, the expression of HIF-1alpha mRNA was higher in the degenerated regions than in the intact regions. Both IL-1beta and H2O2 accelerated mRNA and protein levels of HIF-1alpha in cultured chondrocytes. Inhibitors for phosphatidylinositol 3-kinase and p38 kinase caused a significant decrease in catabolic-factor-induced HIF-1alpha expression. HIF-1alpha-deficient chondrocytes did not maintain energy generation and cartilage matrix production under both normoxic and hypoxic conditions. Also, HIF-1alpha-deficient chondrocytes showed an acceleration of catabolic stress-induced apoptosis in vitro. Our findings in human OA cartilage show that HIF-1alpha expression in OA cartilage is associated with the progression of articular cartilage degeneration. Catabolic-stresses, IL-1beta, and oxidative stress induce the expression of HIF-1alpha in chondrocytes. Our results suggest an important role of stress-induced HIF-1alpha in the maintenance of chondrocyte viability in OA articular cartilage.
Subject(s)
Cartilage, Articular/metabolism , Chondrocytes/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis , Osteoarthritis, Knee/etiology , Osteoarthritis, Knee/metabolism , Oxidative Stress/physiology , Aged , Cartilage, Articular/cytology , Cartilage, Articular/drug effects , Cells, Cultured , Chondrocytes/drug effects , Enzyme Inhibitors/pharmacology , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Humans , Hydrogen Peroxide/toxicity , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Interleukin-1/toxicity , Male , Middle Aged , Osteoarthritis, Knee/genetics , Oxidative Stress/drug effectsABSTRACT
Proinflammatory cytokines are mediators of inflammatory and neuropathic pain. Here, we investigated pain-related behavior in rats after intraneural injection of different doses of rat recombinant interleukin-1beta (rrIL-1beta) and tumor necrosis factor-alpha (rrTNF) into the sciatic nerve. Doses ranged between 0.25 and 2500pg/ml for rrIL-1beta and 0.25-250pg/ml for rrTNF. Thermal hyperalgesia as measured according to the Hargreaves method was most prominent with 2.5pg/ml of rrIL-1beta or rrTNF. Mechanical allodynia as assessed using von Frey hairs was seen consistently with 2.5pg/ml of rrIL-1beta and 0.25-2.5pg/ml of rrTNF. Higher and lower doses had no significant effect on pain-related behavior. Morphometric analysis of semithin sections of the sciatic nerve 10 days after the injections revealed no significant fiber loss. The fiber size distribution was not significantly altered by any of the treatments. Particularly with injections of rrIL-1beta, an increase of epineurial macrophages was observed at all doses. The immunohistochemical expression of cellular markers of neuronal damage (activating transcription factor 3) or activation (phosphorylated p38 mitogen-activated kinase, NF-kappa B p65) in dorsal root ganglia (DRG) tended to increase with both cytokine injections. However, this did not reflect the extent of behavioral changes. In summary, we found a bell-shaped dose-response curve for the algesic effects of rrIL-1beta and rrTNF, peaking at doses equivalent to those of endogenous cytokines released locally after nerve injury. The absence of corresponding morphological changes in nerves supports the concept of a functional effect of the cytokines at these doses.
Subject(s)
Interleukin-1/toxicity , Pain Threshold/drug effects , Sciatic Neuropathy/chemically induced , Tumor Necrosis Factor-alpha/toxicity , Activating Transcription Factor 3 , Animals , Axons/metabolism , Axons/pathology , Behavior, Animal , Cell Size/drug effects , Dose-Response Relationship, Drug , Ectodysplasins , Female , Ganglia, Spinal/drug effects , Ganglia, Spinal/pathology , Immunohistochemistry/methods , Injections/methods , Membrane Proteins/metabolism , Mitogen-Activated Protein Kinase 13/metabolism , Myelin Sheath/metabolism , Myelin Sheath/pathology , Neurons/metabolism , Neurons/pathology , Pain Measurement/methods , Physical Stimulation/methods , Protein Serine-Threonine Kinases/metabolism , Rats , Rats, Sprague-Dawley , Reaction Time/drug effects , Sciatic Neuropathy/physiopathology , Transcription Factors/metabolism , NF-kappaB-Inducing KinaseABSTRACT
AIMS/HYPOTHESIS: Free radicals generated in mitochondria play a crucial role in the toxic effects of cytokines upon insulin-producing cells. This study therefore investigated the role of manganese superoxide dismutase (MnSOD) in cytokine-mediated toxicity in insulin-producing cells. METHODS: MnSOD was either stably overexpressed (MnSODsense) or stably suppressed (MnSODantisense) in insulin-producing RINm5F cells. Cell viability was quantified after incubation with different chemical reactive oxygen species (ROS) generators and with cytokines (IL-1beta alone or a mixture of IL-1beta, TNF-alpha and IFN-gamma). Additionally, cell proliferation and endogenous MnSOD protein expression were determined after exposure to cytokines. RESULTS: After incubation with hydrogen peroxide (H(2)O(2)) or hypoxanthine/xanthine oxidase no significant differences were observed in viability between control and MnSODsense or MnSODantisense clones. MnSOD overexpression reduced the viability of MnSODsense cells after exposure to the intracellular ROS generator menadione compared with control and MnSODantisense cells. MnSODsense cells also showed the highest susceptibility to cytokine toxicity with more than 75% loss of viability and a significant reduction of the proliferation rate after 72 h of incubation with a cytokine mixture. In comparison with control cells (67% viability loss), the reduction of viability in MnSODantisense cells was lower (50%), indicating a sensitising role of MnSOD in the progression of cytokine toxicity. The cell proliferation rate decreased in parallel to the reduction of cell viability. The MnSOD expression level after exposure to cytokines was also significantly lower in MnSODantisense cells than in control or MnSODsense cells. CONCLUSIONS/INTERPRETATION: The increase of the mitochondrial imbalance between the superoxide- and the H(2)O(2)-inactivating enzyme activities corresponds with a greater susceptibility to cytokines. Thus optimal antioxidative strategies to protect insulin-producing cells against cytokine toxicity may comprise a combined overexpression of H(2)O(2)-inactivating enzymes or suppression of MnSOD activity.
Subject(s)
Cytokines/toxicity , Insulin/biosynthesis , Mitochondria/enzymology , Reactive Oxygen Species/toxicity , Superoxide Dismutase/physiology , Blotting, Western , Cell Line , Cell Proliferation/drug effects , Cell Survival/drug effects , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Humans , Hydrogen Peroxide/pharmacology , Interferon-gamma/toxicity , Interleukin-1/toxicity , Oxidants/pharmacology , RNA, Antisense/biosynthesis , Superoxide Dismutase/biosynthesis , Superoxide Dismutase/genetics , Tetrazolium Salts , Thiazoles , Tumor Necrosis Factor-alpha/toxicity , Vitamin K 3/pharmacologyABSTRACT
The aim of the present study was to evaluate the in vitro chondroprotective effects of the lyophilised methanolic extract from flowering buds of Capparis Spinosa L (LECS). This plant, common to the Mediterranean basin, has been used by the traditional medicine for its diuretic and antihypertensive effects and also in certain pathological conditions related to uncontrolled lipid peroxidation. The extract contains many constituents, in particular some flavonoids (kaempferol and quercetin derivatives) and hydrocinammic acids with several known biological effects such as the anti-inflammatory and the antioxidant ones. In this study, we assayed the effect of LECS on human chondrocytes cultures stimulated by proinflammatory cytokine interleukin-1beta (IL-1beta) and we determined the production of key molecules released during chronic inflammatory events (nitric oxide, glycosaminoglycans, prostaglandins and reactive oxygen species). We observed that LECS was able to counteract the harmful effects induced by IL-1beta. This protection appeared to be greater than that elicited by indomethacin, which is usually employed in joint diseases. Since LECS possess a chondroprotective effect, it might be used in the management of cartilage damage during the inflammatory processes.