Role of p38 mitogen-activated protein kinase isoforms in murine skin inflammation induced by 12-O-tetradecanoylphorbol 13-acetate

p38 mitogen-activated protein kinase plays a pivotal role in skin inflammation. The purpose of this study was to investigate the role of the various p38 isoforms. p38 beta/delta-knockout-C57BL/6 mice were generated, studied in a 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced skin inflammation model and compared with wild-type mice. The inflammatory response was determined by ear thickness, myeloperoxidase activity and histology. mRNA and protein expression of interleukin (IL)-1 beta and IL-6 was determined by quantitative real-time reverse transcription PCR and enzyme-linked immunoassay. In both groups application of TPA resulted in a significant increase in inflammation, and pretreatment with the p38 alpha/beta inhibitor, SB202190 resulted in a significant inhibition. A significantly slower onset but prolonged duration of the response was seen in p38 beta/delta knockout mice. This was paralleled by a significant, but transient, lower IL-1 beta and IL-6 protein expression in p38 beta/delta knockout mice. Although the p38 alpha isoform is important, our data also demonstrate an important role of the p38 beta and/or delta isoforms in the regulation of TPA-induced skin inflammation.</p

Modulation of TGF-beta signaling by proinflammatory cytokines in articular chondrocytes.

OBJECTIVE: The normal structure and function of articular cartilage are the result of a precisely balanced interaction between anabolic and catabolic processes. The transforming growth factor-beta (TGF-beta) family of growth factors generally exerts an anabolic or repair response; in contrast, proinflammatory cytokines such as interleukin 1 beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) exert a strong catabolic effect. Recent evidence has shown that IL-1beta, and TNF-alpha, and the TGF-beta signaling pathways share an antagonistic relationship. The aim of this study was to determine whether the modulation of the response of articular chondrocytes to TGF-beta by IL-1beta or TNF-alpha signaling pathways occurs through regulation of activity and availability of mothers against DPP (Drosophila) human homologue (Smad) proteins. METHODS: Human articular chondrocytes isolated from knee joints from patients with osteoarthritis (OA) or normal bovine chondrocytes were cultured in suspension in poly-(2-hydroxyethyl methacrylate)-coated dishes with either 10% fetal bovine serum media or serum-deprived media 6h before treatment with IL-1beta alone, TNF-alpha alone or IL-1beta followed by TGF-beta. Nuclear extracts were examined by electrophoretic mobility-shift assays (EMSA) for nuclear factor-kappa B (NF-kappaB) and Smad3/4 deoxyribonucleic acid (DNA) binding. Nuclear extracts were also subjected to the TranSignal Protein/DNA array (Panomics, Redwood City, CA) enabling the simultaneous semiquantitative assessment of DNA-binding activity of 54 different transcription factors. Nuclear phospho-Smad2/3 and total Smad7 protein expression in whole cell lysates were studied by Western blot. Cytoplasmic Smad7, type II collagen alpha 1 (COL2A1), aggrecan and SRY-related high mobility group-Box gene 9 (SOX-9) mRNA expression were measured by real-time polymerase chain reaction (PCR). RESULTS: The DNA-binding activity of Smad3/4 in the TranSignal Protein/DNA array was downregulated by TNF-alpha (46%) or IL-1beta treatment (42%). EMSA analysis showed a consistent reduction in Smad3/4 DNA-binding activity in human articular chondrocytes treated with IL-1beta or TNF-alpha. TGF-beta-induced Smad3/4 DNA-binding activity and Smad2/3 phosphorylation were also reduced following pretreatment with IL-1beta in human OA and bovine chondrocytes. Real-time PCR and Western blot analysis showed that IL-1beta partially reversed the TGF-beta stimulation of Smad7 mRNA and protein levels in TGF-beta-treated human OA cells. In contrast, TGF-beta-stimulated COL2A1, aggrecan, and SOX-9 mRNA levels were abrogated by IL-1beta. CONCLUSIONS: IL-1beta or TNF-alpha exerted a suppressive effect on Smad3/4 DNA-binding activity in human articular chondrocytes, as well as on TGF-beta-induced stimulation of Smad3/4 DNA-binding activity and Smad2/3 phosphorylation in human OA and bovine articular chondrocytes. IL-1beta partially reversed the increase in TGF-beta-stimulated Smad7 mRNA or protein levels suggesting that Smad7 may not be involved in the suppression of TGF-beta signaling induced by IL-1beta or TNF-alpha in articular chondrocytes. The balance between the IL-1beta or TNF-alpha and the TGF-beta signaling pathways is crucial for maintenance of articular cartilage homeostasis and its disruption likely plays a substantial role in the pathogenesis of OA

Nlrp6 promotes recovery after peripheral nerve injury independently of inflammasomes

Background: NOD-like receptors (Nlrs) are key regulators of immune responses during infection and autoimmunity. A subset of Nlrs assembles inflammasomes, molecular platforms that are activated in response to endogenous danger and microbial ligands and that control release of interleukin (IL)-1 beta and IL-18. However, their role in response to injury in the nervous system is less understood. Methods: In this study, we investigated the expression profile of major inflammasome components in the peripheral nervous system (PNS) and explored the physiological role of different Nlrs upon acute nerve injury in mice. Results: While in basal conditions, predominantly members of NOD-like receptor B (Nlrb) subfamily (NLR family, apoptosis inhibitory proteins (NAIPs)) and Nlrc subfamily (ICE-protease activating factor (IPAF)/NOD) are detected in the sciatic nerve, injury causes a shift towards expression of the Nlrp family. Sterile nerve injury also leads to an increase in expression of the Nlrb subfamily, while bacteria trigger expression of the Nlrc subfamily. Interestingly, loss of Nlrp6 led to strongly impaired nerve function upon nerve crush. Loss of the inflammasome adaptor apoptosis-associated speck-like protein containing a CARD (ASC) and effector caspase-1 and caspase-11 did not affect sciatic nerve function, suggesting that Nlrp6 contributed to recovery after peripheral nerve injury independently of inflammasomes. In line with this, we did not detect release of mature IL-1 beta upon acute nerve injury despite potent induction of pro-IL-1 beta and inflammasome components Nlrp3 and Nlrp1. However, Nlrp6 deficiency was associated with increased pro-inflammatory extracellular regulated MAP kinase (ERK) signaling, suggesting that hyperinflammation in the absence of Nlrp6 exacerbated peripheral nerve injury. Conclusions: Together, our observations suggest that Nlrp6 contributes to recovery from peripheral nerve injury by dampening inflammatory responses independently of IL-1 beta and inflammasomes

Regulation of human lung fibroblast alpha 1(I) procollagen gene expression by tumor necrosis factor alpha, interleukin-1 beta, and prostaglandin E2.

We investigated the participation of prostaglandin (PG) E2 in the regulation of the alpha 1(I) procollagen gene expression by tumor necrosis factor alpha (TNF alpha), and interleukin-1 beta (IL-1 beta) in normal adult human lung fibroblasts. TNF alpha (100 units/ml) and IL-1 beta (100 units/ml) stimulated the production of PGE2 and caused a dose-dependent inhibition of up to 54 and 66%, respectively, of the production of type I procollagen. Preincubation of cultures with indomethacin partially reversed the inhibition of procollagen production induced by the cytokines. Cytokine-stimulated endogenous fibroblast PG accounted for 35 and 68% of the inhibition induced by TNF alpha and IL-1 beta, respectively. Steady-state mRNA levels for alpha 1(I) procollagen paralleled the changes in collagen production. The transcription rate of the alpha 1(I) procollagen gene was reduced by 58% by TNF alpha and by 43% by IL-1 beta. Cytokine-stimulated endogenous PG production accounted for half of these effects. These results indicate that TNF alpha and IL-1 beta inhibit the expression of the alpha 1(I) procollagen gene in human lung fibroblasts at the transcriptional level by a PGE2-independent effect as well as through the effect of endogenous fibroblast PGE2 released under the stimulus of the cytokines

Mediators and Cytokines in Persistent Allergic Rhinitis and Nonallergic Rhinitis with Eosinophilia Syndrome

Background: Patients with nonallergic rhinitis with eosinophilia syndrome (NARES) show typical symptoms of persistent allergic rhinitis (PAR). The aim of the present study was to compare nasal cytokine patterns between NARES and PAR. Methods: Nasal secretions of 31 patients suffering from NARES, 20 patients with PAR to house dust mite and 21 healthy controls were collected using the cotton wool method and analyzed for interleukin (IL)-1 beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 beta (MIP-1 beta) by Bio-Plex Cytokine Assay as well as eosinophil cationic protein (ECP) and tryptase by UniCAP-FEIA. Results: NARES and PAR presented elevated levels of tryptase, while ECP was markedly increased solely in NARES compared to both the controls and PAR. Elevated levels of IL-1 beta, IL-17, IFN-gamma, TNF-alpha and MCP-1 were found in NARES compared to the controls as well as PAR. MIP-1 beta was elevated in NARES and PAR, while IL-4, IL-6 and G-CSF showed increased levels in NARES, and IL-5 was elevated in PAR only. Conclusions: In patients with NARES and PAR, eosinophils and mast cells appear to be the pivotal cells of inflammation, reflected by high levels of tryptase and ECP as well as IL-5 and GM-CSF as factors for eosinophil migration and survival. The elevated levels of proinflammatory cytokines in NARES may indicate the chronic, self-perpetuating process of inflammation in NARES which seems to be more pronounced than in PAR. IL-17 might be a factor for neutrophilic infiltration or be responsible for remodeling processes in NARES. Copyright (C) 2012 S. Karger AG, Base

Mechanisms of dysregulation of low-density lipoprotein receptor expression in vascular smooth muscle cells by inflammatory cytokines

Objective - Although inflammation is a recognized feature of atherosclerosis, the impact of inflammation on cellular cholesterol homeostasis is unclear. This study focuses on the molecular mechanisms by which inflammatory cytokines disrupt low-density lipoprotein (LDL) receptor regulation.Methods and Results - IL-1 beta enhanced transformation of vascular smooth muscle cells into foam cells by increasing uptake of unmodified LDL via LDL receptors and by enhancing cholesterol esterification as demonstrated by Oil Red O staining and direct assay of intracellular cholesterol concentrations. In the absence of IL-1 beta, a high concentration of LDL decreased LDL receptor promoter activity, mRNA synthesis and protein expression. However, IL-1 beta enhanced LDL receptor expression, overriding the suppression usually induced by a high concentration of LDL and inappropriately increasing LDL uptake. Exposure to IL-1 beta also caused overexpression of the sterol regulatory element binding protein ( SREBP) cleavage-activating protein ( SCAP), and enhanced its translocation from the endoplasmic reticulum to the Golgi, where it is known to cleave SREBP, thereby enhancing LDL receptor gene expression.Conclusions - These observations demonstrate that IL-1 beta disrupts cholesterol-mediated LDL receptor feedback regulation, permitting intracellular accumulation of unmodified LDL and causing foam cell formation. The implication of these findings is that inflammatory cytokines may contribute to intracellular LDL accumulation without previous modification of the lipoprotein

Heme Oxygenase-1 Expression Affects Murine Abdominal Aortic Aneurysm Progression.

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease

Effects of interleukin-1 beta on thyrotropin secretion and thyroid hormone uptake in cultured rat anterior pituitary cells

The effects of interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha) on basal and TRH-induced TSH release, and the effects of IL-1 beta on the uptake of [125I]T3 and [125I]T4 and on nuclear binding of [125I]T3 were examined. Furthermore, the release of other anterior pituitary hormones in the presence of IL-1 beta was measured. Anterior pituitary cells from male Wistar rats were cultured for 3 days in medium containing 10% FCS. Incubation were performed at 37 C in medium with 0.5% BSA for measurement of [125I]T3 uptake and with 0.1% BSA for measurement of [125I]T4 uptake. Exposure to IL-1 beta (1 pM-1 nM) or TNF alpha (100 pM) for 2-4 h resulted in a significant decline in TSH release, which was almost 50% (P < 0.05) for 1 nM IL-1 beta and 24% (P < 0.05) for 100 pM TNF alpha. Measurement of other anterior pituitary hormones (FSH, LH, PRL, and ACTH) in the same incubation medium showed that IL-1 beta did not alter their release. When the effects of IL-1 beta (1 pM-1 nM) and TNF alpha (100 pM) on TRH-induced TSH release were measured in short term experiments, the inhibitory effects had disappeared. The addition of 1-100 nM octreotide, a somatostatin analog, resulted in a decrease in TRH-induced TSH release up to 33% of the control value (P < 0.05). Exposure to dexamethasone (1 nM to 1 microM) affected basal and TRH-induced TSH release similar to the effect of IL-1 beta. The 15-min uptake of [125I]T3 and [125I]T4, expressed as femtomoles per pM free hormone, was not affected by the presence of IL-1 beta (1-100 pM). When IL-1 beta (100 pM) was present during 3 days of culture, TSH release was reduced to 88 +/- 2% of the control value (P < 0.05). This effect was not associated with an altered [125I]T3 uptake (15 min to 4 h) or with any change in nuclear T3 binding. We conclude that 1) IL-1 beta decreases TSH release by a direct action on the pituitary; 2) this effect is not due to elevated thyroid hormone uptake or increase T3 nuclear occupancy; 3) IL-1 beta does not affect TRH-induced TSH release or the release of other anterior pituitary hormones; and 4) TNF alpha affects basal and TRH-induced TSH release in the same way as IL-1 beta

TGFβ (transforming growth factor-β) blockade induces a human-like disease in a nondissecting mouse model of abdominal aortic aneurysm

Objective-Current experimental models of abdominal aortic aneurysm (AAA) do not accurately reproduce the major features of human AAA. We hypothesized that blockade of TGF beta (transforming growth factor-beta) activity-a guardian of vascular integrity and immune homeostasis-would impair vascular healing in models of nondissecting AAA and would lead to sustained aneurysmal growth until rupture. Approach and Results-Here, we test this hypothesis in the elastase-induced AAA model in mice. We analyze AAA development and progression using ultrasound in vivo, synchrotron-based ultrahigh resolution imaging ex vivo, and a combination of biological, histological, and flow cytometry-based cellular and molecular approaches in vitro. Systemic blockade of TGF beta using a monoclonal antibody induces a transition from a self-contained aortic dilatation to a model of sustained aneurysmal growth, associated with the formation of an intraluminal thrombus. AAA growth is associated with wall disruption but no medial dissection and culminates in fatal transmural aortic wall rupture. TGF beta blockade enhances leukocyte infiltration both in the aortic wall and the intraluminal thrombus and aggravates extracellular matrix degradation. Early blockade of IL-1 beta or monocyte-dependent responses substantially limits AAA severity. However, blockade of IL-1 beta after disease initiation has no effect on AAA progression to rupture. Conclusions-Endogenous TGF beta activity is required for the healing of AAA. TGF beta blockade may be harnessed to generate new models of AAA with better relevance to the human disease. We expect that the new models will improve our understanding of the pathophysiology of AAA and will be useful in the identification of new therapeutic targets