tosylphenylalanyl-chloromethyl-ketone and Inflammation

This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein-specific protease of interest and results can be measured in both real time and as endpoint fluorescence assays on a flow cytometer. Endpoint assays are easily adapted to microplate format for flow cytometry high-throughput analysis and inhibitor screening.

Topics: Animals; Biotinylation; Flow Cytometry; Fluorescence Resonance Energy Transfer; Green Fluorescent Proteins; High-Throughput Screening Assays; Humans; Inflammation; Kinetics; Microspheres; Peptide Hydrolases; Peptides; Reproducibility of Results; Temperature

This study is focused on the links between the major products of inflammation and cell damage induced by the administration of lipopolysaccharide (LPS) from Salmonella typhimurium in embryonal cardiomyocytes. LPS treatment for 72 hours induced transcription factor NF-kappaB activation, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expression, nitric oxide (NO) and tumor necrosis factor (TNF)-alpha release. Moreover, LPS administration induced a significant cell loss, reversed by the NO-synthases inhibitor, suggesting a relationship between cell damage and iNOS-dependent NO overproduction. Cell death was reversed by the specific NF-kappaB inhibitor, TPCK, whereas COX-2 specific inhibitor determined an increase of cell damage in terms of apoptosis, as observed by YO-PRO immunostaining, DNA laddering analysis and caspase-3 activation. Overall these findings evidenced a selective role for NF-kappaB in mediating NO-induced cell damage and a protective action by COX-2 in LPS-treated embryonal cardiomyocytes. The reflection of these experiments on human cardiac pathology will be discussed.

Topics: Animals; Apoptosis; Caspase 3; Cells, Cultured; Chick Embryo; Cyclooxygenase 2; Enzyme Inhibitors; Heart; In Vitro Techniques; Inflammation; Lipopolysaccharides; Myocytes, Cardiac; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Signal Transduction; Tosylphenylalanyl Chloromethyl Ketone; Tumor Necrosis Factor-alpha

Recent evidence suggests that the mechanism of manganese (Mn) neurotoxicity involves activation of microglia and/or astrocytes; as a consequence, neurons adjacent to the activated microglia may be injured. Mn modulation of proinflammatory cytokine expression by microglia has not been investigated. Therefore, the objectives of this research were to (1) assess whether Mn induces proinflammatory cytokine expression and/or modulates lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines and (2) investigate possible mechanisms for such an induction. N9 microglia were exposed in vitro to increasing concentrations (50-1000 microM) of Mn in the presence or absence of LPS (10, 100, or 500 ng/ml). After various incubation times (up to 48 h), media levels of several cytokines and nitric oxide (NO) were determined, as was the expression of the inducible form of NO synthase (iNOS). Lactate dehydrogenase (LDH) release into the medium and the cellular uptake of Neutral Red were used as general measures for cytotoxicity. In the absence of LPS, Mn moderately increased interleukin-6 and tumor necrosis factor alpha (TNF-a) production only at higher Mn concentrations, which were cytotoxic. At all LPS doses, however, proinflammatory cytokine production was dose-dependently increased by Mn. Similarly, LPS-induced NO production and iNOS expression were substantially enhanced by Mn. Pharmacological manipulations indicated that nuclear factor kappa B (NFkappaB) activation is critical for the observed enhancement of cytokine and NO production. Within the context of inflammation, increased production of proinflammatory cytokines and NO by Mn could be an important part of the mechanism by which Mn exerts its neurotoxicity.

Topics: Acetylcysteine; Animals; Buthionine Sulfoximine; Cells, Cultured; Chromans; Cytokines; Enzyme Inhibitors; Inflammation; L-Lactate Dehydrogenase; Lipopolysaccharides; Manganese Poisoning; Mice; Microglia; NF-kappa B; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Pentoxifylline; Phosphodiesterase Inhibitors; Serine Proteinase Inhibitors; Tosylphenylalanyl Chloromethyl Ketone

We reported previously that tumor cells isolated from metastases of the in vitro transformed squamous cell carcinoma line Pam 212 exhibit an elevation in constitutive production of proinflmmatory cytokines interleukin (IL)-1alpha, IL-6, granulocyte-macrophage colony-stimulating factor, and KC (the murine homologue of chemokine Gro-alpha). The basis for constitutive expression of these cytokines after tumor progression in vivo is unknown. Regulation of the expression of these proinflammatory cytokines involves transcription factor nuclear factor kappaB (NF-kappaB), which can be activated by cytokines such as tumor necrosis factor (TNF)-alpha. In this study, we compared the constitutive and TNF-alpha-induced expression of proinflammatory cytokines in parental Pam 212 and metastatic LY-2 and LY-8 cell lines and determined the relationship of cytokine expression to activation of NF-kappaB. We found that the metastatic cell lines exhibited an increase in constitutive and TNF-alpha-inducible expression of proinflammatory cytokines when compared with parental Pam 212 cells. The increased cytokine expression was associated with an increase in constitutive and TNF-alpha-inducible activation of NF-kappaB as demonstrated by electrophoretic mobility shift assay and luciferase-reporter gene assay. Constitutive nuclear localization of NF-kappaB p65 was observed in LY-2 and LY-8 cells in culture and in tumor specimens but rarely in Pam 212 cells, consistent with the constitutive activation of NF-kappaB in tumor cels after selection in vivo. Induction of NF-kappaB by TNF-alpha was inhibited by the addition of protease inhibitors calpain inhibitor I and N-tosyl-phechloromethyl ketone and antioxidant 1-pyrrolidinecarbodithioic acid, whereas constitutive activation of NF-kappaB and cytokine KC mRNA expression was inhibited by N-tosyl-phechloromethyl ketone alone. Overexpression of a human Ikappa(B)alpha dominant suppresser in Pam 212 cells inhibited TNF-alpha-induced NF-kappaB binding activity and KC expression. These data indicate that activation of NF-kappaB contributes to increased expression of proinflammatory cytokines during metastatic tumor progression of squamous cell carcinoma, and that distinct mechanisms may be involved in the regulation of constitutive and TNF-alpha-induced activation of NF-kappaB in squamous cell carcinoma.

Topics: Animals; Carcinoma, Squamous Cell; Cell Nucleus; Cytokines; Cytoplasm; DNA-Binding Proteins; Gene Expression Regulation, Neoplastic; Genes, Reporter; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; I-kappa B Proteins; Inflammation; Interleukin-1; Interleukin-6; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Proteins; Neoplasm Transplantation; NF-kappa B; NF-KappaB Inhibitor alpha; Promoter Regions, Genetic; Protease Inhibitors; Pyrrolidines; Recombinant Fusion Proteins; Selection, Genetic; Thiocarbamates; Tosylphenylalanyl Chloromethyl Ketone; Transcription Factor RelA; Transplantation, Heterologous; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

In this report, we demonstrate that NF-kappa B, a ubiquitous transcription factor, plays an essential role in silica-induced inflammatory mediator production in the mouse macrophage cell line RAW 264.7. Compared to the effect of lipopolysaccharide (LPS), silica mediated a stronger activation of NF-kappa B p50/p50 homodimer at early phase of poststimulation. Furthermore, activation of NF-kappa B by silica and LPS appears to be mediated by different signal transduction pathways. Both silica and LPS increased mRNA expression in these cells for cyclooxygenase II, inducible nitric oxide synthase, tumor necrosis factor-alpha and interleukin-1 alpha. This expression was attenuated along with the inhibition of NF-kappa B activation.

Topics: Animals; Arginine; Base Sequence; Cell Line; Consensus Sequence; DNA; Enzyme Induction; Gene Expression; Inflammation; Interleukin-1; Isoenzymes; Lipopolysaccharides; Macrophages, Alveolar; Mice; Molecular Sequence Data; NF-kappa B; Nitric Oxide Synthase; Oligodeoxyribonucleotides; omega-N-Methylarginine; Prostaglandin-Endoperoxide Synthases; Protease Inhibitors; RNA, Messenger; Silicon Dioxide; Tosylphenylalanyl Chloromethyl Ketone; Transcription, Genetic; Tumor Necrosis Factor-alpha; Tyrosine

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

Phorbol myristate acetate (PMA), a potent inflammatory agent with tumor-promoting activity, was examined for its effect on the growth of Mycobacterium lepraemurium (MLM) in the left hind footpad of mice. When the animals were infected with 10(4) MLM and received multiple injections of 3 micrograms of PMA in the infection site weekly during the first 2 months and biweekly thereafter, the growth of the bacilli was markedly enhanced. PMA injection in the infection site resulted in severe footpad swelling accompanied by inflammatory signs such as redness, edema, induration, and sometimes ulcer. Acetic acid, as potent an inflammatory and hyperplastic agent as PMA but without any appreciable tumor-promoting action, did not stimulate MLM growth when it was injected biweekly in the site of infection with MLM at a dose of 30 mumol per injection. When mice were infected with 10(8) MLM, proper elimination of bacilli from the infection site was observed during the first 3 months. In this case, multiple injections of PMA in the infection site resulted in the enhancement of the elimination of MLM by host defense mechanisms, although PMA caused as severe inflammation as that observed when MLM infection was produced with a small inoculum (10(4) MLM). In both cases, dexamethasone was synergistic with, but indomethacin and L-l-tosylamide-2-phenyl-ethylchloromethyl ketone were antagonistic to, the effect of PMA.

Topics: Acetates; Acetic Acid; Animals; Dexamethasone; Female; Hypersensitivity, Delayed; Indomethacin; Inflammation; Mice; Mycobacterium Infections; Mycobacterium lepraemurium; 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