leupeptins and Inflammation

Information regarding cellular anti-inflammatory and immunomodulatory attributes of leupeptin with respect to modulation of perturbed macrophage function and lymphocytes has not yet been delineated, particularly in the context of ROS-cytokines-autophagy-inflammatory signalling cascades. Therefore, the present study identified the attributes and mechanisms of leupeptin, from actinomycetes, in relation to excessive oxidative stress mediated disrupted immune homeostasis and inflammatory mechanism in activated macrophages and lymphocytes. Results revealed that leupeptin treatment showed noticeable inhibition in the production of NO, ROS, mitochondrial membrane potential and phagocytosis activity in LPS-stimulated macrophages. These findings were accompanied by reduction in TNF-α, IL-1β, IL-6, IFN-γ/IL-10 ratio, endopeptidases, oxidative effectors (Cox-2, IL-5, IL-15, IL-17, COX-2), iNOS with concomitant increase in Arg 1, Msr 1 and Mrc - 1exprssion in leupeptin treatment. Additionally, compared to LPS-challenged cells, marked alleviation in MDC, lysotracker staining, beclin-1, LC3B expression, and enhanced p62 levels in leupeptin exposed cells indicate the reversal of impaired autophagy flux. Subsequently, oxi-inflammatory signalling analysis demonstrated p-PTEN, p-NF-κB, p-PI3K, p-Akt, p-p38, and ERK1/2 upregulation decisively thwarted by leupeptin administration. In silico analysis further implied its target selectivity to these cascades. Furthermore, decreased proliferation index and Th1, Th2/IL-10 cytokines ratio in mitogen-challenged splenic lymphocytes confers its role in mitigating unwarranted inflammation mediated by disrupted regulation of adaptive immune cells. Together, these findings signify the attributes of leupeptin as an alternative anti-inflammatory strategy and affirm it as a promising natural entity to modulate immune-mediated response during inflammatory disorder.

Topics: Anti-Inflammatory Agents; Autophagy; Cyclooxygenase 2; Cytokines; Homeostasis; Humans; Inflammation; Interleukin-10; Leupeptins; Lipopolysaccharides; Lymphocytes; Macrophages; NF-kappa B; Oxidation-Reduction; Reactive Oxygen Species

Mesenchymal stem cells have an important potential in the treatment of age-related diseases. In the last years, small extracellular vesicles derived from these stem cells have been proposed as cell-free therapies. Cellular senescence and proinflammatory activation are involved in the loss of therapeutic capacity and in the phenomenon called inflamm-aging. The regulators of these two biological processes in mesenchymal stem cells are not well-known. In this study, we found that p65 is activated during cellular senescence and inflammatory activation in human umbilical cord-derived mesenchymal stem cell. To demonstrate the central role of p65 in these two processes, we used small-molecular inhibitors of p65, such as JSH-23, MG-132 and curcumin. We found that the inhibition of p65 prevents the cellular senescence phenotype in human umbilical cord-derived mesenchymal stem cells. Besides, p65 inhibition produced the inactivation of proinflammatory molecules as components of a senescence-associated secretory phenotype (SASP) (interleukin-6 and interleukin-8 (IL-6 and IL-8)). Additionally, we found that the inhibition of p65 prevents the transmission of paracrine senescence between mesenchymal stem cells and the proinflammatory message through small extracellular vesicles. Our work highlights the important role of p65 and its inhibition to restore the loss of functionality of small extracellular vesicles from senescent mesenchymal stem cells and their inflamm-aging signature.

Topics: Adolescent; Adult; Cell Proliferation; Cells, Cultured; Cellular Senescence; Curcumin; DNA Damage; Female; Humans; Inflammation; Leupeptins; Mesenchymal Stem Cells; Nanoparticles; Paracrine Communication; Phenotype; Phenylenediamines; Transcription Factor RelA; Umbilical Cord

Neuroinflammation is a hallmark in intracerebral hemorrhage (ICH) that induces secondary brain injury, leading to neuronal cell death. ER stress-triggered apoptosis and proteostasis disruption caused neuroinflammation to play an important role in various neurological disorders. The consequences of ER stress and proteostasis disruption have rarely been studied during the course of ICH development.. ICH was induced by collagenase VII-S intrastriatal infusion. Animals were sacrificed at 0, 3, 6, 24, and 72 h post-ICH. Rats were determined for body weight changes, hematoma volume, and neurological deficits. Brain tissues were harvested for molecular signaling analysis either for ELISA, immunoblotting, immunoprecipitation, RT-qPCR, protein aggregation, or for histological examination. A non-selective proteasome inhibitor, MG132, was administered into the right striatum three hours prior to ICH induction.. ICH-induced acute proteasome over-activation caused the early degradation of the endoplasmic reticulum (ER) chaperone GRP78 and IκB protein. These exacerbations were accompanied by the elevation of pro-apoptotic CCAAT-enhancer-binding protein homologous protein (CHOP) and pro-inflammatory cytokines expression via nuclear factor-kappa B (NF-κB) signal activation. Pre-treatment with proteasome inhibitor MG132 significantly ameliorated the ICH-induced ER stress/proteostasis disruption, pro-inflammatory cytokines, neuronal cells apoptosis, and neurological deficits.. ICH induced rapid proteasome over-activation, leading to an exaggeration of the ER stress/proteostasis disruption, and neuroinflammation might be a critical event in acute ICH pathology.

Topics: Animals; Apoptosis; Cerebral Hemorrhage; Cytokines; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Stress; Inflammation; Leupeptins; Male; Neuroimmunomodulation; NF-kappa B; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Signal Transduction

Diabetic nephropathy (DN), the leading cause of end-stage renal disease (ESRD). To date, mounting evidence has shown that inflammation may contribute to the pathogenesis of DN. Recent reports have shown that proteasome inhibitors display cytoprotection by reducing the phosphorylation of Akt, a serine/threonine kinase, plays a critical role in cellular survival and metabolism and can crosstalk with inflammation. Therefore, we hypothesized that MG132, specific proteasome inhibitor, could provide renoprotection by suppressing Akt-mediated inflammation in DN. In vivo, male Sprague-Dawley rats were divided into normal control group (NC), diabetic nephropathy group (DN), DN model plus MG132 treatment group (MG132), and DN model plus deguelin treatment group (Deguelin)(deguelin, a specific inhibitor of Akt). In vitro, a human glomerular mesangial cell lines (HMCs) was exposed to 5.5 mmol/L glucose (CON), 30 mmol/L glucose (HG), 30 mmol/L glucose with 0.5 umol/L MG132 (MG132) and 30 mmol/L glucose with 5 umol/L deguelin (Deguelin). Compared with NC, DN showed a significant increase in the urinary protein excretion rate and inflammatory cytokines, as well as p-Akt. Compared with CON, HMCs co-cultured with HG was notably proliferated, which is in accord with α-smooth muscle actin (α-SMA) expression. These alterations were inhibited by administration of MG132 or deguelin. In conclusion, MG132 significantly inhibits the development of DN by regulating Akt phosphorylation-mediated inflammatory activation.

Topics: Animals; Cell Line; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Glomerular Mesangium; Glucose; Humans; Inflammation; Inflammation Mediators; Kidney; Leupeptins; Male; Mesangial Cells; Proteasome Inhibitors; Protective Agents; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Rotenone; Signal Transduction

Ziziphus spina-christi (L.) Desf. (Christ's Thorn Jujube) is a wild tree today found in Jordan, Israel, Egypt, and some parts of Africa, which was already in use as a medicinal plant in Ancient Egypt. In ancient Egyptian prescriptions, it was used in remedies against swellings, pain, and heat, and thus should have anti-inflammatory effects. Nowadays, Z. spina-christi, is used in Egypt (by Bedouins, and Nubians), the Arabian Peninsula, Jordan, Iraq, and Morocco against a wide range of illnesses, most of them associated with inflammation. Pharmacological research undertaken to date suggests that it possesses anti-inflammatory, hypoglycemic, hypotensive and anti-microbial effects. The transcription factor NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) is critical in inflammation, proliferation and involved in various types of cancer. Identification of new anti-inflammatory compounds might be an effective strategy to target inflammatory disorders and cancer. Therefore, extracts from Z. spina-christi are investigated in terms of their anti-inflammatory effects. Our intention is to evaluate the effects of Z. spina-christi described in ancient Egyptian papyri, and to show whether the effects can be proven with modern pharmacological methods. Furthermore, we determine the active ingredients in crude extracts for their inhibitory activity toward NF-κB pathway.. To determine the active ingredients of Z. spina-christi, we fractionated the extracts for bioassays and identified the active compounds. Epigallocatechin, gallocatechin, spinosin, 6''' feruloylspinosin and 6''' sinapoylspinosin and crude extracts of seed, leaf, root or stem were analyzed for their effect on NF-κB DNA binding by electromobility shift assay (EMSA) and nuclear translocation of NF-κB-p65 by Western blot analysis. The binding mode of the compounds to NF-κB pathway proteins was compared with the known inhibitor, MG-132, by in silico molecular docking calculations. Log10IC50 values of gallocatechin and epigallocatechin as two main compounds of the plant were correlated to the microarray-based mRNA expression of 79 inflammation-related genes in cell lines of the National Cancer Institute (NCI, USA) as determined. The expression of 17 genes significantly correlated to the log10IC50 values for gallocatechin or epigallocatechin.. Nuclear p65 protein level decreased upon treatment with each extract and compound. Root and seed extracts inhibited NF-κB-DNA binding as shown by EMSA. The compounds showed comparable binding energies and similar docking poses as MG-132 on the target proteins.. Z. spina-christi might possess anti-inflammatory activity as assumed by ancient Egyptian prescriptions. Five compounds contributed to this bioactivity, i.e. epigallocatechin, gallocatechin, spinosin, 6''' feruloylspinosin and 6''' sinapoylspinosin as shown in vitro and in silico.

Topics: Anti-Inflammatory Agents; Cell Line, Tumor; Egypt, Ancient; Herbal Medicine; History, Ancient; Humans; Inflammation; Leupeptins; Molecular Docking Simulation; Plant Extracts; Plant Roots; Plant Stems; Plants, Medicinal; Seeds; Transcription Factor RelA; Ziziphus

While inflammation with aberrant activation of NF-κB pathway is a hallmark of cystic fibrosis (CF), the molecular mechanisms underlying the link between CFTR defect and activation of NF-κB-mediated pro-inflammatory response remain elusive. Here, we investigated the link between CFTR defect and NF-κB activation in ΔF508cftr-/- mouse intestine and human intestinal epithelial cell lines. Our results show that the NF-κB/COX-2/PGE2 pathway is activated whereas the β-catenin pathway is suppressed in CF mouse intestine and CFTR-knockdown cells. Activation of β-catenin pathway by GSK3 inhibitors suppresses CFTR mutation/knockdown-induced NF-κB/COX-2/PGE2 pathway in ΔF508 mouse intestine and CFTR-knockdown cells. In contrast, suppression of β-catenin signaling induces the nuclear translocation of NF-κB. In addition, CFTR co-localizes and interacts with β-catenin while CFTR mutation disrupts the interaction between NF-κB and β-catenin in mouse intestine. Treatment with proteasome inhibitor MG132 completely reverses the reduced expression of β-catenin in Caco-2 cells. Collectively, these results indicate that CFTR stabilizes β-catenin and prevents its degradation, defect of which results in the activation of NF-κB-mediated inflammatory cascade. The present study has demonstrated a previously unsuspected interaction between CFTR and β-catenin that regulates NF-κB nuclear translocation in mouse intestine. Therefore, our study provides novel insights into the physiological function of CFTR and pathogenesis of CF-related diseases in addition to the NF-κB-mediated intestinal inflammation seen in CF.

Topics: Active Transport, Cell Nucleus; Animals; beta Catenin; Caco-2 Cells; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Glycogen Synthase Kinase 3; Humans; Inflammation; Intestine, Small; Leupeptins; Mice; Mice, Inbred CFTR; Mutation; NF-kappa B p50 Subunit; Signal Transduction

The detailed knowledge about the contribution of immunoproteasome to the neuroinflammation in ischemic stroke is still not available. The immunoreactivity of low molecular mass peptide 2 (LMP2) and low molecular mass peptide 7 (LMP7) was evident in the ipsilateral ischemic cerebral cortex and striatum following transient middle cerebral artery occlusion (MCAO). Both LMP2 and LMP7 increased as early as 4 h after the MCAO, further increased at 24 h, peaked at 72 h and decreased 7 days later. LMP2 and LMP7 were mainly present in astrocytes and microglia/macrophage cells, respectively. LMP2 knockdown by shRNA (short hairpin RNA) markedly reduced the levels of LMP2 and LMP7 protein and caused 75.5 and 78.6% decrease in the caspase-like (C-L) and chymotrypsin-like (CT-L) activities, respectively. Compared with cont-shRNA group (39.7%, infarction volumes/total ipsilateral hemisphere), the infarction volumes were reduced to 22.5% in LMP2-shRNA group. Additionally, LMP2 knockdown significantly reduced activated astrocytes and microglia, the expression nuclear factor kappa B (NF-κB) p65, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) and caused less accumulation of ischemia-induced protein ubiquitination compared with MG132. These findings demonstrate that inhibition of LMP2 significantly attenuates inflammatory reaction and offers neuroprotection against focal cerebral ischemia in rats, suggesting that selective immunoproteasome inhibitors may be a promising strategy for stroke treatment.

Topics: Animals; Astrocytes; Cerebral Infarction; Cysteine Endopeptidases; Disease Models, Animal; Gene Knockdown Techniques; Infarction, Middle Cerebral Artery; Inflammation; Interleukin-1beta; Leupeptins; Male; Microglia; NF-kappa B; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Subunits; Protein Transport; Rats, Sprague-Dawley; RNA, Small Interfering; Stroke; Tumor Necrosis Factor-alpha; Ubiquitinated Proteins

Nuclear factor-kappa B (NF-κB) is a ubiquitously expressed protein complex regulating the transcription of genes involved in inflammation and pain. Increased NF-κB activity in immune and nervous system cells is linked to several chronic pain conditions in humans as well as inflammation and nerve injury-evoked pain in animals. A recent in vitro study further demonstrates that increased NF-κB activity in astrocytes decreases transcription of catechol-o-methyltransferase (COMT), an enzyme that inactivates catecholamines that cause pain. The purpose of the present study was to examine the relationship between systemic and astrocytic NF-κB activity, pain, and COMT expression in an animal model of inflammation. Results demonstrated that administration of the inflammatory stimulant complete Freund's adjuvant (CFA) led to increased pain and decreased COMT protein expression in an NF-κB-dependent manner. Specifically, we found that rats and mice receiving intraplantar CFA exhibited increased behavioral responses to mechanical and thermal heat stimuli. CFA-evoked pain was blocked in rats receiving a pre-emptive systemic dose of the NF-κB inhibitor MG132 and exacerbated in IKKca mice with constitutive NF-κB activity in astrocytes. Furthermore, we observed NF-κB-linked reductions in COMT expression in midbrain at 6h and 1d following CFA in rats and at 1h and 1d in forebrain and midbrain following CFA in IKKca mice. Collectively, these results demonstrate that systemic and astrocytic NF-κB activity drive inflammatory pain and regulate the expression of COMT in forebrain and midbrain structures.

Topics: Animals; Astrocytes; Brain; Catechol O-Methyltransferase; Disease Models, Animal; Freund's Adjuvant; Hot Temperature; Hyperalgesia; Inflammation; Leupeptins; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Pain; Pain Measurement; Physical Stimulation; Rats; Rats, Sprague-Dawley

Recent studies have linked necrotic cell death and proteolysis of inflammatory proteins to the adaptive immune response mediated by the lysosome-destabilizing adjuvants, alum and Leu-Leu-OMe (LLOMe). However, the mechanism by which lysosome-destabilizing agents trigger necrosis and proteolysis of inflammatory proteins is poorly understood. The proteasome is a cellular complex that has been shown to regulate both necrotic cell death and proteolysis of inflammatory proteins. We found that the peptide aldehyde proteasome inhibitors, MG115 and MG132, block lysosome rupture, degradation of inflammatory proteins and necrotic cell death mediated by the lysosome-destabilizing peptide LLOMe. However, non-aldehyde proteasome inhibitors failed to prevent LLOMe-induced cell death suggesting that aldehyde proteasome inhibitors triggered a pleotropic effect. We have previously shown that cathepsin C controls lysosome rupture, necrotic cell death and the adaptive immune response mediated by LLOMe. Using recombinant cathepsin C, we found that aldehyde proteasome inhibitors directly block cathepsin C, which presumably prevents LLOMe toxicity. The cathepsin B inhibitor CA-074-Me also blocks lysosome rupture and necrotic cell death mediated by a wide range of necrosis inducers, including LLOMe. Using cathepsin-deficient cells and recombinant cathepsins, we demonstrate that the cathepsins B and C are not required for the CA-074-Me block of necrotic cell death. Taken together, our findings demonstrate that lysosome-destabilizing adjuvants trigger an early proteolytic cascade, involving cathepsin C and a CA-074-Me-dependent protease. Identification of these early events leading to lysosome rupture will be crucial in our understanding of processes controlling necrotic cell death and immune responses mediated by lysosome-destabilizing adjuvants.

Topics: Adjuvants, Pharmaceutic; Aldehydes; Animals; Antigens, Bacterial; Bacterial Toxins; Cathepsin B; Cathepsin C; Dipeptides; Inflammation; Leupeptins; Lysosomes; Mice, Inbred BALB C; Mice, Inbred C57BL; Necrosis; Peptides; Proteasome Inhibitors; Proteolysis

The forkhead family transcription factor FOXP3 is critical for the differentiation and function of CD4(+) CD25(+) regulatory T cells (Treg). How FOXP3 protein level is negatively regulated under the inflammatory microenvironment is largely unknown. Here we report that the combination of transforming growth factor-beta (TGF-β) and IL-6 treatment (IL-6/TGF-β) can synergistically downregulate FOXP3 at the posttranslational level by promoting FOXP3 protein degradation. In our FOXP3 overexpression model, we found that IL-6/TGF-β treatment upregulated IL-6R expression but did not affect the stability of FOXP3 mRNA. Moreover, we found that the proteasome inhibitor MG132 could inhibit IL-6/TGF-β-mediated downregulation of FOXP3 protein, which reveals a potential pathway for modulating Treg activity by preventing FOXP3 degradation during inflammation.

Topics: Cellular Microenvironment; Down-Regulation; Drug Synergism; Forkhead Transcription Factors; Humans; Inflammation; Interleukin-6; Jurkat Cells; Leupeptins; Proteolysis; Receptors, Interleukin-6; RNA, Messenger; Signal Transduction; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

The ubiquitin-proteasome pathway (UPP) has been indicated to contribute to dysfunction of endothelial cells (ECs). Nevertheless, the relationship between UPP and vascular complications of uraemia remains unknown. We aimed to determine whether the UPP is activated in vascular ECs when cultured with uraemic serum, and to examine the role of the UPP on dysfunction of ECs in uraemia. Rabbit aortic endothelial cells (RAECs) were cultured with normal serum or different concentrations of uraemic serum. The expression of the ubiquitin-activating enzyme (E1), an indicator of the UPP, was detected by real-time RT-PCR and Western blot; proteasome activity was determined by fluorescence spectrophotometry; and nuclear factor-κB (NF-κB) activity and expression, as well as tumour necrosis factor-α (TNF-α) expression, were also detected. We found that the expression of E1 and the activities of three kinds of proteasomes were increased significantly in RAECs after incubation with uraemic serum. Proliferation of RAECs was increased significantly by incubation with 3-15% uraemic serum but decreased markedly when incubated with uraemic serum above 15% (increased apoptosis). Incubation of RAECs with uraemic serum induced increased NF-B DNA-binding activity and nuclear translocation of NF-κB, decreased nitric oxide production and increased expression of TNF-α, which is the final effector of inflammatory activation of cells. All of these responses in RAECs were suppressed by the specific proteasome inhibitor, MG132. The inhibition of inflammatory responses by MG132 was further supported by a parallel experiment with pyrrolidine dithiocarbamate, a specific inhibitor of κNF-B. These findings suggest that the UPP was activated in RAECs by administration of uraemic serum, and played a pivotal role in the dysfunction of vascular ECs, such as inflammatory activation.

Topics: Animals; Apoptosis; Cell Cycle; Cell Nucleus; Cell Proliferation; Cells, Cultured; DNA-Binding Proteins; Endothelial Cells; Inflammation; Leupeptins; NF-kappa B; Nitric Oxide; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Protein Transport; Pyrrolidines; Rabbits; Signal Transduction; Thiocarbamates; Tumor Necrosis Factor-alpha; Ubiquitin; Ubiquitin-Activating Enzymes; Uremia

Tumor necrosis factor (TNF)-α, a homotrimeric, pleiotropic cytokine, is secreted in response to inflammatory stimuli in diseases such as rheumatoid arthritis and inflammatory bowel disease. TNF-α mediates both apoptosis and inflammation, stimulating an inflammatory cascade through the non-canonical pathway of NF-κB activation, leading to increased nuclear RelB and p52. In contrast, the common food additive carrageenan (CGN) stimulates inflammation through both the canonical and non-canonical pathways of NF-κB activation and utilizes the adaptor molecule BCL10 (B-cell leukemia/lymphoma 10). In a series of experiments, colonic epithelial cells and mouse embryonic fibroblasts were treated with TNF-α and carrageenan in order to simulate the possible effects of exposure to dietary CGN in the setting of a TNF-α-mediated inflammatory disease process. A marked increase in secretion of IL-8 occurred, attributable to synergistic effects on phosphorylated NF-κB-inducing kinase (NIK) in the non-canonical pathway. TNF-α induced the ubiquitination of TRAF2 (TNF receptor-associated factor 2), which interacts with NIK, and CGN induced phosphorylation of BCL10, leading to increased NIK phosphorylation. These results suggest that TNF-α and CGN in combination act to increase NIK phosphorylation, thereby increasing activation of the non-canonical pathway of NF-κB activation. In contrast, the apoptotic effects of TNF-α, including activation of caspase-8 and PARP-1 (poly(ADP-ribose) polymerase 1) fragmentation, were markedly reduced in the presence of CGN, and CGN caused reduced expression of Fas. These findings demonstrate that exposure to CGN drives TNF-α-stimulated cells toward inflammation rather than toward apoptotic cell death and suggest that CGN exposure may compromise the effectiveness of anti-TNF-α therapy.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; B-Cell CLL-Lymphoma 10 Protein; Carrageenan; Fibroblasts; Food Additives; Humans; I-kappa B Proteins; Inflammation; Interleukin-8; Leupeptins; Mice; NF-KappaB Inhibitor alpha; NF-kappaB-Inducing Kinase; Protein Serine-Threonine Kinases; Tumor Necrosis Factor Receptor-Associated Peptides and Proteins; Tumor Necrosis Factor-alpha

Macrophage-triggered chronic inflammation and smooth muscle cell-initiated vascular remodeling are two major pathophysiologic events during atherogenesis. Major histocompatibility class II (MHC II) transactivator (CIITA) is a key mediator of these processes through transcriptional regulation of interferon gamma (IFN-gamma) induced MHC II activation and type I collagen repression. Transcriptional activity of CIITA is regulated by multiple post-translational modifications. Here we report that CIITA and histone deacetylase 2 (HDAC2) interact in smooth muscle cells and macrophages as assayed by co-immunoprecipitations. HDAC2 deacetylates CIITA whereas both the HDAC inhibitor trichostatin A (TSA) and over-expression of HDAC2 interfering RNA increase CIITA acetylation. HDAC2 down-regulates CIITA recruitment to target promoters as evidenced by chromatin immunoprecipitation assays, and suppresses MHC II activation and collagen repression mediated by CIITA in luciferase reporter assays. Quantitative PCR reveals that TSA enhances MHC II activation and collagen repression by IFN-gamma. Wild type but not enzyme-deficient HDAC2 promotes the degradation of CIITA protein, whereas TSA and the proteasome inhibitor MG132 restore CIITA activity by stabilizing CIITA protein and increasing its association with target promoters. Furthermore, TSA treatment enhances the association of CIITA with the transcription factor RFX5, which ameliorates the down-regulation of CIITA recruitment to target promoters by HDAC2. In conclusion, our data suggest that HDAC2 antagonizes CIITA activity by committing CIITA to protein degradation and decreasing the interaction of CIITA with RFX5 in a deacetylation-dependent manner. Therefore, modulating CIITA activity by targeting HDAC2 may provide potential anti-atherogenic strategies.

Topics: Acetylation; Animals; Atherosclerosis; Cell Line; Collagen Type I; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Histocompatibility Antigens Class II; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Inflammation; Interferon-gamma; Leupeptins; Macrophages; Mice; Myocytes, Smooth Muscle; Nuclear Proteins; Promoter Regions, Genetic; Protein Processing, Post-Translational; Regulatory Factor X Transcription Factors; Repressor Proteins; Trans-Activators; Transcription Factors; Transcription, Genetic

Topics: Acetylation; Animals; Atherosclerosis; Cell Line; Collagen Type I; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Histocompatibility Antigens Class II; Histone Deacetylase 2; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Inflammation; Interferon-gamma; Leupeptins; Macrophages; Mice; Myocytes, Smooth Muscle; Nuclear Proteins; Promoter Regions, Genetic; Protein Processing, Post-Translational; Regulatory Factor X Transcription Factors; Repressor Proteins; Trans-Activators; Transcription Factors; Transcription, Genetic

Insulin-resistant states are commonly associated with chronic inflammation and hepatic overproduction of apolipoprotein B100 (apoB100), leading to hypertriglyceridemia and a metabolic dyslipidemic profile. Molecular mechanisms linking hepatic inflammatory cascades and the pathways of apoB100-lipoprotein production are, however, unknown. In the present study, we employed a diet-induced, insulin-resistant hamster model, as well as cell culture studies, to investigate the potential link between activation of hepatic inflammatory nuclear factor-kappaB (NF-kappaB) signaling cascade and the synthesis and secretion of apoB100-containing lipoproteins. Using an established insulin-resistant animal model, the fructose-fed hamster, we found that feeding fructose (previously shown to induce hepatic inflammation) for as little as 4 days reduced hepatic IkappaB (inhibitor of NF-kappaB) level, indicating activation of the inflammatory NF-kappaB cascade. Importantly, IKK (IkappaB kinase) inhibition was found to suppress apoB100 overproduction in fructose-fed hamster hepatocytes. As IKK, the upstream activator of NF-kappaB has been shown to inhibit insulin signaling, and insulin is a major regulator of apoB100, we modulated IKK activity in primary hamster hepatocytes and HepG2 cells and assessed the effects on hepatic apoB100 biosynthesis. Inhibition of the IKK-NF-kappaB pathway by BMS345541 and activation of the pathway by adenoviral-mediated IKK overexpression decreased and increased newly synthesized apoB100 levels, respectively. Pulse-chase and metabolic labeling experiments revealed that IKK activation regulates apoB100 levels at the levels of apoB100 biosynthesis and protein stability. Inhibition of the IKK-NF-kappaB pathway significantly enhanced proteasomal degradation of hepatic apoB100, while direct IKK activation led to reduced degradation and increased apoB100 mRNA translation. Together, our results reveal important links between modulation of the inflammatory IKK-NF-kappaB signaling cascade and hepatic synthesis and secretion of apoB100-containing lipoproteins. Hepatic inflammation may be an important underlying factor in hepatic apoB100 overproduction observed in insulin resistance.

Topics: Administration, Oral; Animals; Apolipoprotein B-100; Cell Line, Tumor; Cell-Free System; Cricetinae; Cysteine Proteinase Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fructose; Gene Expression; Hepatocytes; Humans; I-kappa B Kinase; I-kappa B Proteins; Imidazoles; Inflammation; Leupeptins; Lipid Metabolism; Lipoproteins; Lipoproteins, VLDL; Liver; Male; Mesocricetus; NF-kappa B; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Biosynthesis; Protein Kinase Inhibitors; Quinoxalines; Transfection; Tumor Necrosis Factor-alpha

The ability of human neutrophils to express a variety of genes encoding inflammatory mediators is well documented, and mounting evidence suggests that neutrophil-derived cytokines and chemokines contribute to the recruitment of discrete leukocyte populations at inflammatory sites. Despite this, our understanding of the signaling intermediates governing the generation of inflammatory cytokines by neutrophils remains fragmentary. Here, we report that inhibitors of the p38 MAPK and MEK pathways substantially diminish the release of (and in the case of p38 inhibitors, the gene expression of) several inflammatory cytokines in neutrophils stimulated with LPS or TNF. In addition, various NF-kappaB inhibitors were found to profoundly impede the inducible gene expression and release of inflammatory cytokines in these cells. The MAPK inhibitors did not affect NF-kappaB activation; instead, the transcriptional effects of the p38 MAPK inhibitor appear to involve transcriptional factor IID. Conversely, the NF-kappaB inhibitors failed to affect the activation of MAPKs. Finally, the MAPK inhibitors were found to prevent the activation a key component of the translational machinery, S6 ribosomal protein, in keeping with their post-transcriptional impact on cytokine generation. To our knowledge, this constitutes the first demonstration that in neutrophils, the inducible expression of proinflammatory cytokines by physiological stimuli largely reflects the ability of the latter to activate NF-kappaB and selected MAPK pathways. Our data also raise the possibility that NF-kappaB or MAPK inhibitors could be useful in the treatment of inflammatory disorders in which neutrophils predominate.

Topics: Boronic Acids; Cell Differentiation; Cytokines; Humans; Inflammation; Leupeptins; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Neutrophils; NF-kappa B; Nitriles; Proline; Protein Kinase Inhibitors; Structure-Activity Relationship; Sulfones; Thiocarbamates

Interleukin-25 (IL-25) is a novel T-helper-2 (Th2) cytokine of the IL-17 family that plays a key role in allergic inflammation. Recent studies reported that over-expression of IL-25 in mouse induces eosinophilia. We investigated the effect of IL-25 on the expression of several adhesion molecules on human eosinophils and the underlying intracellular mechanisms.. Viability of eosinophils was measured by annexin V-fluorescein isothiocyanate (FITC) assay. Gene expression and surface expression of intercellular adhesion molecule (ICAM)-1 (CD54), ICAM-3 (CD50), L-selectin (CD62L), leukocyte function-associated antigen (LFA-1) (CD11a/CD18) and very late antigen-4 (VLA-4, CD49d/CD29) on eosinophils were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry, respectively. Adhesion of eosinophils to fibronectin was assessed using the fibronectin-coated insert system.. Viability of eosinophils was significantly enhanced by IL-25 from 41% to 76% dose-dependently. IL-25 could significantly upregulate the surface expression of ICAM-1, but suppress those of ICAM-3 and L-selectin on eosinophils in a dose-dependent manner. Adhesion of eosinophils to fibronectin was also significantly enhanced by IL-25. Besides, pre-incubation with p38 mitogen-activated protein kinases (MAPK) inhibitor SB203580, C-Jun NH2-terminal protein kinases (JNK) inhibitor SP600125 and proteosome inhibitor MG-132 could significantly restrain the effects of IL-25 on surface expression of L-selectin, ICAM-1 and ICAM-3, respectively, and also on the adhesion of eosinophils onto fibronectin (all P < 0.05).. Our findings suggest an essential role of IL-25 in enhancing survival and regulating surface expression of ICAM-1, ICAM-3 and L-selectin on human eosinophils through the activation of p38 MAPK, JNK and nuclear factor (NF)-kappaB pathways, thereby shedding light on the molecular mechanisms of IL-25-induced eosinophilia in allergic inflammation.

Topics: Apoptosis; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Eosinophilia; Eosinophils; Gene Expression Regulation; Humans; Hypersensitivity; Imidazoles; Inflammation; Interleukin-17; Interleukins; JNK Mitogen-Activated Protein Kinases; Leupeptins; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Pyridines; RNA, Messenger

Excessive activation of calpains has been implicated in the pathophysiology of inflammation, trauma, and ischemia reperfusion injury. Here, we investigated the effects of calpain inhibition on myocardial dysfunction and inflammation induced by endotoxin in rats. Rats were treated i.v. with endotoxin (10 mg/kg) or endotoxin plus calpain inhibitors and were then prepared after 4 h for myocardial contractility assessment, detection of endothelium leukocyte interactions, and plasma TNF-alpha, nitrite/nitrate, and endocan levels. Compared with vehicle-treated rats, hearts from endotoxin-treated rats had reduced systolic performance that was partially prevented by calpain inhibitors, i.e., acetyl-leucyl-leucyl-arginal (leupeptin), carbobenzoxy-valyl-phenylalanial (calpain inhibitor III), and N-acetyl-leucinyl-leucinyl-norleucinal (ALLN). Leupeptin and calpain inhibitor III reduced plasma TNF-alpha levels in endotoxin-treated rats. ALLN reduced plasma TNF-alpha and nitrite/nitrate levels in endotoxin-treated rats. Endotoxin treatment increased mesenteric venule leukocyte rolling (10 +/- 3 leukocytes/min vs. 44 +/- 10 leukocytes/min; P < 0.01) and adhesion (2 +/- 2 leukocytes/min vs. 15 +/- 3 leukocytes/min; P < 0.01), which was reduced by calpain inhibitors. Attenuation of leukocyte endothelium interactions observed in calpain inhibitor-treated rats with sepsis was associated with increases in plasma anti-adhesion molecule endocan. In conclusion, calpain inhibitors improved endotoxin-induced cardiac dysfunction, which may be attributed to the modulation of endothelium leukocyte interactions in the inflamed vasculature.

Topics: Animals; Cardiomyopathies; Endotoxins; Glycoproteins; Heart; In Vitro Techniques; Inflammation; Injections, Intravenous; Leukocyte Rolling; Leupeptins; Male; Myocardial Contraction; Myocardium; Nitrates; Nitrites; Rats; Rats, Sprague-Dawley; Shock, Septic; Tumor Necrosis Factor-alpha

The ubiquitin-proteasome system is the major intracellular protein degradation pathway in eucaryotic cells. It regulates central mediators of proliferation, inflammation, and apoptosis that are fundamental pathomechanisms in the development of vascular restenosis.. Effects of proteasome inhibition on neointima formation were studied in a balloon injury model in the rat carotid artery. Local application of the proteasome inhibitor MG132 (1 mmol/L) resulted in significant inhibition of intimal hyperplasia, that is, by 74% (P=0.008). This effect was accompanied by decreased proliferation, reduced infiltration of macrophages, and prolonged apoptosis, as determined by immunohistochemical and TUNEL analyses. Functional effects of proteasome inhibition on proliferation, activation of nuclear factor kappa B, and apoptosis were further characterized in rat primary vascular smooth muscle cells. MG132 dose-dependently inhibited vascular smooth muscle cell proliferation with 50% inhibition at 10 micromol/L. TNFalpha-induced degradation of IkappaBalpha and beta was blocked, and activation of nuclear factor kappa B was suppressed in a concentration-dependent manner in bandshift assays. Moreover, proteasome inhibition (1 to 50 micromol/L MG132) induced apoptotic cell death up to 80%, as confirmed by DNA/Histone-ELISA and TUNEL-FACS analysis. Specificity of proteasome inhibition was shown by accumulation of multiubiquitinylated proteins and accumulation of specific proteasomal substrates.. These proof-of-principle experiments demonstrate that inhibition of the ubiquitin-proteasome system effectively reduces neointima formation in vivo, which corresponds to strong antiproliferative, anti-inflammatory, and proapoptotic effects in vitro and in vivo. Our data suggest the ubiquitin-proteasome system as a new target in the prevention of vascular restenosis.

Topics: Angioplasty, Balloon; Animals; Apoptosis; Carotid Stenosis; Cell Division; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Graft Occlusion, Vascular; Inflammation; Leupeptins; Multienzyme Complexes; Muscle, Smooth, Vascular; NF-kappa B; Proteasome Endopeptidase Complex; Rats; Rats, Wistar; Substrate Specificity; Ubiquitin

The role that the nuclear factor (NF)-kappa B plays in regulating the biosynthesis of interleukin (IL)-1 beta, an inflammatory cytokine, has been investigated in vitro. Irreversible inhibition of the proteasome complex by carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132; 1-50 microM) had no inhibitory effect on lipopolysaccharide (LPS)-mediated IL-1 beta biosynthesis. Furthermore, selective inhibition of NF-kappa B by the action of caffeic acid phenylethyl ester (CAPE; 1-100 microM) and sulfasalazine (SSA; 0.1-10 mM), a potent and irreversible inhibitor of NF-kappa B, partially attenuated but did not abolish LPS-dependent IL-1 beta secretion. Incorporation of a selectively permeant inhibitor of NF-kappa B, SN-50 (1-20 microM), a peptide which contains the nuclear localization sequence (NLS) for the p50 NF-kappa B subunit and the amino-terminal sequence of Kaposi fibroblast growth factor to promote cell permeability, attenuated in a dose-dependent manner LPS-mediated release of IL-1 beta. It is concluded that the NF-kapp B pathway is partially implicated and its blockade attenuates but does not abrogate LPS-dependent IL-1 beta biosynthesis in alveolar epithelial cells.

Topics: Animals; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Fetus; Inflammation; Interleukin-1; Leupeptins; Lipopolysaccharides; Multienzyme Complexes; NF-kappa B; Proteasome Endopeptidase Complex; Pulmonary Alveoli; Rats; Respiratory Mucosa; Sulfasalazine

The activation of nuclear factor-kappaB (NF-kappaB) is required for the induction of many of the adhesion molecules and chemokines involved in the inflammatory leukocyte recruitment to the kidney. Here we studied the effects of NF-kappaB inhibition on the machinery crucial for monocyte infiltration of the glomerulus during inflammation. In mesangial cells (MC), the protease inhibitors MG-132 and N-alpha-tosyl-L-lysine chloromethyl ketone or adenoviral overexpression of IkappaB-alpha prevented the complete IkappaB-alpha degradation following tumor necrosis factor-alpha (TNF-alpha) stimulation. This resulted in a marked inhibition of TNF-alpha-induced expression of mRNA and protein for the immunoglobulin molecules intracellular adhesion molecule-1 and vascular cell adhesion molecule-1 and the chemokines growth-related oncogene-alpha, monocyte chemoattractant protein-1, interleukin-8, or fractalkine in MC. Finally, the inhibition of IkappaB-alpha degradation or IkappaB-alpha overexpression suppressed the chemokine-induced transendothelial monocyte chemotaxis toward MC and the chemokine-triggered firm adhesion of monocytic cells to MC. The inhibition of NF-kappaB by pharmacological intervention or gene transfer may present a multimodal approach to control the machinery propagating inflammatory recruitment of monocytes during glomerular disease.

Topics: Adenoviridae; Cell Movement; Cells, Cultured; Chemokines; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Flow Cytometry; Glomerular Mesangium; Humans; I-kappa B Proteins; Immunohistochemistry; Inflammation; Intercellular Adhesion Molecule-1; Leupeptins; Monocytes; NF-kappa B; NF-KappaB Inhibitor alpha; Serine Proteinase Inhibitors; Tosyllysine Chloromethyl Ketone; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Inflammation involves the sequential activation of signaling pathways leading to the production of both pro- and anti-inflammatory mediators. Although much attention has focused on pro-inflammatory pathways that initiate inflammation, relatively little is known about the mechanisms that switch off inflammation and resolve the inflammatory response. The transcription factor NF-kappaB is thought to have a central role in the induction of pro-inflammatory gene expression and has attracted interest as a new target for the treatment of inflammatory disease. We show here that NF-kappaB activation in leukocytes recruited during the onset of inflammation is associated with pro-inflammatory gene expression, whereas such activation during the resolution of inflammation is associated with the expression of anti-inflammatory genes and the induction of apoptosis. Inhibition of NF-kappaB during the resolution of inflammation protracts the inflammatory response and prevents apoptosis. This suggests that NF-kappaB has an anti-inflammatory role in vivo involving the regulation of inflammatory resolution.

Topics: Animals; Anti-Infective Agents; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; bcl-2-Associated X Protein; Carrageenan; Cysteine Endopeptidases; Female; Granuloma; Inflammation; Leukocytes; Leupeptins; Male; Mice; Multienzyme Complexes; NF-kappa B; Nitriles; Pleurisy; Proteasome Endopeptidase Complex; Protein Binding; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrrolidines; Rats; Sulfones; Thiocarbamates; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Suppressor Protein p53

Sulfur mustard provokes an acute inflammatory response in skin. To determine if keratinocytes regulate this response and whether three potential vesicant antagonists can counteract adverse changes, specimens of EpiDerm (MatTek Corp., Ashland, MA), a human skin model of differentiating keratinocytes, were exposed 2 h to humidified air with or without 2-chloroethyl ethyl sulfide (CEES, 1.72-1.73 mg/L/min) with or without 10 mM niacinamide, a poly (ADP-ribose) polymerase (PARP) inhibitor, 25 microM CGS9343B (calmodulin antagonist), or 8.4 mM leupeptin (cysteine protease inhibitor). After a 22-h incubation, levels of interleukin-1 alpha (IL-1alpha), its receptor antagonist (IL-1Ra), soluble type II receptor (sIL-1RII) and prostaglandin-E(2) (PGE(2)) were determined. Methylthiazole tetrazolium (MTT) viability tests and histological observations were also conducted. PGE(2) levels were abundant but unaffected by CEES regardless of antagonist presence. Total amounts (media plus lysate) of IL-1alpha, IL-1Ra, and sIL-1RII were reduced with CEES irrespective of antagonist. CEES promoted the release of IL-1Ra. Exposure of EpiDerm to CEES in the presence of the vesicant antagonists did not improve viability or counteract histological damage. We conclude CEES depresses total IL-1alpha and related cytokines, does not affect PGE(2) release, and adverse changes associated with CEES-exposed EpiDerm are not ameliorated by these particular antagonists. Dramatically increased (5- to 10-fold) release of IL-1Ra may provide a useful marker for cytotoxicity. The high level of IL-1Ra and increased release with injury suggest a primary function in down-regulating IL-1 inflammatory responses in skin.

Topics: Benzimidazoles; Biomarkers; Calmodulin; Cells, Cultured; Cysteine Proteinase Inhibitors; Dinoprostone; Drug Synergism; Enzyme Inhibitors; Humans; Inflammation; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Irritants; Keratinocytes; Leupeptins; Mustard Gas; Niacinamide; Poly(ADP-ribose) Polymerase Inhibitors; Receptors, Interleukin-1; Receptors, Interleukin-1 Type II; Sialoglycoproteins

The effect of proteinase inhibitors such as TLCK, TPCK and leupeptin on vascular permeability was investigated in the carrageenin-air-pouch inflammation in rats. When each inhibitor was injected into the air-pouch immediately after carrageenin injection, TLCK, TPCK and leupeptin caused a rapid and significant increase in vascular permeability. The TLCK- and TPCK-induced increase declined gradually, whereas leupeptin inhibited the vascular permeability after the temporary increase. When the inhibitors were injected 5 h after carrageenin injection, TLCK and TPCK increased the vascular permeability, whereas leupeptin was without effect. Cyproheptadine, an anti-histamine and antiserotonin drug, inhibited the leupeptin-induced temporary increase, but failed to inhibit the TLCK- and TPCK-induced increase in vascular permeability. These results suggest that the leupeptin-induced increase in vascular permeability was mediated by histamine and serotonin, while TLCK and TPCK may increase vascular permeability as a result of a direct action on endothelial cells.

Topics: Animals; Capillary Permeability; Carrageenan; Inflammation; Kinetics; Leupeptins; Protease Inhibitors; Rats; Rats, Inbred Strains; Time Factors; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone

Proteinase inhibitors were evaluated for their anti-inflammatory actions on carrageenin-induced inflammation in rats. The development of granulation tissue and the exudate were markedly suppressed by a single injection of L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) into the carrageenin-air-pouch immediately after carrageenin injection, whereas a single injection of TPCK at 12 or 24 hr after carrageenin injection was less effective or slightly effective respectively. These results suggest that proteinase inhibitors exert their anti-inflammatory actions by interfering with the initial inflammatory reactions after carrageenin injection. When the wet weight of granulation tissue and the weight of exudate were measured on day 4 after the simultaneous injection of carrageenin and inhibitors, a single injection of serine- and thiol-proteinase inhibitors including TPCK, leupeptin, antipain, chymostatin and cystamine suppressed the development of granulation tissue, though EDTA and o-phenanthroline, metallo-proteinase inhibitors, were also effective at a high dose. Exudate was reduced by treatment with TPCK in a dose-dependent manner, while EDTA and o-phenanthroline were effective only at a high dose. On the other hand, the migration of polymorphonuclear leukocytes into the carrageenin-air-pouch (the inflammatory lesion) was markedly suppressed by TPCK and leupeptin, while a high dose of cystamine and o-phenanthroline was slightly effective, and antipain, chymostatin, pepstatin, elastatinal, EDTA, trans-1-aminomethylcyclohexane 4-carboxylic acid and aprotinin were without effect.

Topics: Animals; Carrageenan; Cell Movement; Cystamine; Inflammation; Leupeptins; Male; Neutrophils; Phenanthrolines; Protease Inhibitors; Rats; Rats, Inbred Strains; Tosylphenylalanyl Chloromethyl Ketone