Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Primary cultures of adult rat hepatocytes (Fischer 344) were used as an in vitro metabolic activation system in immunotoxicological assays. Rat hepatocytes were isolated by a collagenase perfusion technique and cultured for 20 to 24 hr to allow the formation of a monolayer on collagen-coated plastic petri dishes. Spleen cells isolated from (C57BL/6 X C3H)F1 mice were cocultured with the hepatocytes along with the chemicals. Cyclophosphamide (CP) and Aflatoxin B1 (AFB1) were effectively activated in this coculture system and produced a dose-related suppression of the in vitro antibody responses to LPS, DNP-Ficoll, and SRBC in 3 hr. Neither CP (1 mM) nor AFB1 (10(-4) M) cultured with spleen cells alone produced any effects. Both CP and AFB1 also produced a dose-related suppression of the proliferative responses to LPS, Con A, and PHA. In contrast, up to 100 mM of N-nitrosodimethylamine (DMN) did not suppress any of these assays after a 3-hr incubation in the coculture system. These results indicate that a coculture system can be used to characterize the activity of immunosuppressive chemicals requiring metabolic activation.
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PMID:Immunosuppression induced by chemicals requiring metabolic activation in mixed cultures of rat hepatocytes and murine splenocytes. 308 86

Gold salts, auranofin (AF), aurothiomalate (ATM) and aurothioglucose (ATG) displayed immunosuppressive action in a series of in vitro assays which mimic the cell-cell interactions thought to occur in rheumatoid arthritis. The gold salts inhibited phytohaemagglutinin (PHA)-induced thymidine incorporation and gamma-IF production by peripheral blood mononuclear cells, as well as IL-2-induced proliferation of PHA-blasts. The separate addition of IL-2 and gamma-IF partly reversed the anti-proliferative effects of ATM and ATG; however, the addition of IL-1 had no effect. ATM and ATG inhibited PHA-stimulated IL-1 production by mononuclear cells but not spontaneous or LPS-induced IL-1 production by adherent monocytes. It was concluded that ATM and ATG inhibited lymphocyte function and lymphocyte-amplification of macrophage function. The anti-proliferative effects of AF were partly reversed by IL-2 but not by gamma-IF or IL-1. AF inhibited PHA-stimulated IL-1 production by mononuclear cells as well as spontaneous and LPS-induced production by adherent cells. It appeared that AF inhibited lymphocyte and macrophage function directly. AF also displayed potential anti-inflammatory activity in that it inhibited PGE2 and collagenase production by proteolytically dispersed rheumatoid synovial cells.
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PMID:Unique properties of auranofin as a potential anti-rheumatic drug. 309 56

Although some data suggest that macrophages in the reticuloendothelial system (RES) are important sources of thromboxane A2 (TxA2) and prostacyclin (PGI2) during endotoxic shock, we are unaware of data documenting the ability of hepatic macrophages (Kupffer cells) to release either TxA2 or PGI2 when exposed to lipopolysaccharide (endotoxin, LPS). In this study, Kupffer cells were examined for their ability to release prostaglandin E2 (PGE2), TxA2, and PGI2 following stimulation with 0, 1.0, 50.0, and 100.0 micrograms/ml of Escherichia coli LPS. Kupffer cells were obtained from rat livers by enzymatic digestion with 0.05% collagenase followed by enrichment of the macrophage population on the basis of differences in density and adherence among the various cell populations isolated. Based on several criteria (phagocytosis of opsonized sheep erythrocytes, positive staining for esterase and peroxidase, failure to replicate), 95% of adherent cells were Kupffer cells. After 4 days of incubation, cells were stimulated with various doses of LPS for 4 and 8 hr. Prostanoid concentrations in culture supernatants were determined by radioimmunoassay. Increasing doses of LPS significantly (P less than 0.001) increased the concentration of immunoreactive PGE2 (iPGE2) and iTxB2 (the stable metabolite of TxA2). The concentration of i6-keto-PFG1 alpha (stable metabolite of PGI2) increased following stimulation with 1.0 microgram/ml of LPS, but declined as the dose of LPS was increased. The results provide evidence that endotoxin-activated Kupffer cells, like other macrophage populations, release several metabolites of arachidonic acid. Kupffer cell-derived prostanoids, particularly TxA2, may be important mediators of some of the pathophysiologic manifestations of acute endotoxemia.
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PMID:Prostanoid production by lipopolysaccharide-stimulated Kupffer cells. 388 37

We have isolated and characterized the human cardiac mast cell (CMC) and compared this novel mast cell (MC type with MC obtained from uterus, skin, and lung. Heart tissue was obtained from 14 patients with cardiomyopathy (CMP, heart transplantation). CMC were isolated by enzymatic digestion using collagenase, pronase-E, hyaluronidase, and DNAse. Substantial amounts of CMC (0.5% to 1.5% of isolated cells) were found in the atrial appendages but not in ventricular digests or other sites of the heart (< 0.1%). In situ staining of atrial tissue revealed the presence of CMC in the myocardium (2.16 +/- 0.7 MC/mm2), endocardium (2.24 +/- 0.9 MC/mm2), and epicardium. As assessed by combined toluidine blue/immunofluorescence staining with monoclonal antibodies (MoAbs), isolated CMC expressed surface IgE, the receptor for stem cell factor (c-kit receptor/CD117), the p24 antigen (CD9), the Pgp-1 homing receptor (CD44), the pan leukocyte antigen (CD45), and the ICAM-1 antigen (CD54). CMC were not recognized by MoAbs to lymphocyte function associated antigen 2 (LFA-2; CD2), T-cell receptor (TcR; CD3), T4 antigen (CD4), LFA-1 alpha-chain (CD11a), C3biR alpha-chain (CD11b), CR4 alpha-chain (CD11c), LPS-R related Ag (CD14), 3-FAL/x-hapten (CD15), Fc gamma RIII (CD16), lactosylceramid (CDw17), the B-cell antigen CD19, or CR1 (CD35). In situ expression of leukocyte antigens on CMC was demonstrable by indirect immunoperoxidase staining technique and double-labeling immunohistochemistry. Almost all CMC (90%) reacted with MoAbs against tryptase and chymase and thus were MCTC. Cardiac mast cells were also stained by the heparin-binding dye Berberine sulfate and expressed measurable amounts of histamine (4.6 +/- 1.4 pg per cell). Cross linking of either IgE receptor or SCF receptor (c-kit) on CMC resulted in histamine secretion (non-specific release: < 6% of total histamine, alpha IgE induced: 12% to 52%; SCF-induced release: 9% to 18%), whereas neither substance P (a skin MC agonist) nor the basophil agonist FMLP showed an effect on CMC. Together, the CMC is an MCTC primarily located in the appendage of the atrium. This novel type of MC exhibits surface membrane antigen and functional properties similar to those of lung and uterus MC.
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PMID:The human cardiac mast cell: localization, isolation, phenotype, and functional characterization. 752 50

Hepatic macrophages and endothelial cells play an important role in the clearance of endotoxin from the portal circulation. These cells are activated by endotoxin to release reactive mediators including superoxide anion, hydrogen peroxide, and nitric oxide, which have been implicated in hepatic inflammation and tissue injury. In the present studies we analyzed mechanisms regulating the production of nitric oxide by hepatic macrophages and endothelial cells following in vivo exposure to endotoxin. Rats were injected intravenously with Escherichia coli lipopolysaccharide (LPS, 5 mg/kg). Cells were isolated from the animals 48 h later by in situ perfusion of the liver with collagenase and pronase followed by differential centrifugation and centrifugal elutriation. We found that macrophages and endothelial cells from both untreated and endotoxin-treated rats readily synthesized nitric oxide following in vitro stimulation with interferon-gamma (IFN-gamma) and LPS alone and in combination. This response was dependent on l-arginine and was blocked by two nitric oxide synthase inhibitors, NG-monomethyl-l-arginine and l-canavanine. Macrophages produced more nitric oxide in response to LPS or LPS plus IFN-gamma than endothelial cells. In addition, nitric oxide production by both cell types in response to LPS plus IFN-gamma was increased after treatment of rats with endotoxin. Macrophages appeared to be more sensitive than endothelial cells to the in vivo effects of this inflammatory stimulus. Northern and Western blot analysis demonstrated that nitric oxide production by macrophages and endothelial cells in response to LPS plus IFN-gamma was due to increased expression of an inducible form of nitric oxide synthase (iNOS) mRNA and protein. Using fluorescence image analysis, iNOS protein was found to be localized in the cytoplasm of the cells. Treatment of rats with endotoxin was associated with increased expression of iNOS protein in the macrophages. The phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also stimulated nitric oxide production by macrophages and endothelial cells from endotoxin-treated rats, although not as effectively as LPS and IFN-gamma. Macrophages were more responsive than endothelial cells to TPA. Furthermore, depletion of the cells of glutathione using buthionine sulfoximine had no major effect on nitric oxide production by macrophages but resulted in small but significant inhibition in endothelial cells. This suggests that this sulfhydryl-containing tripeptide does not regulate intracellular levels of reactive nitrogen intermediates in activated macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Distinct patterns of nitric oxide production in hepatic macrophages and endothelial cells following acute exposure of rats to endotoxin. 752 31

Inflammatory reactions induce the production of reactive oxygen species (ROS): the reverse sequence of these events is also true. Moreover, many components of these reactions interact with a synergistic effect. In this short comprehensive review we analyze some of these interactions which may have pathological effects. Inflammatory reactions are triggered off by exogenous or endogenous aggressions and are characterized by cellular and vascular events. The activated leucocytes leave the circulating blood and reach the site of the aggression where they release a large amount of ROS as well as the content of their granules. The granular content is made in a large part by molecules with killing and degradative activities such as myeloperoxidase, defensins, elastase, collagenase, cathepsins and lysozyme. The inflammatory reaction is beneficial for humans when its effects are limited to the pathogens. The insufficiency of a component of the inflammatory reaction such as the production of ROS which is seen, for example in chronic granulomatous disease, leads to severe and recurrent bacterial infections. In other situations inflammatory reactions are deleterious because they are directed against normal tissues instead or in addition to pathogens. In some cases the behaviour of the phagocytes is modified because they have been primed by inflammatory molecules such tumor necrosis factor, LPS, interleukins or interferons. Priming often leads to a decreased speed of locomotion of the leucocytes with an increased susceptibility to their stimuli. The combination of these effects leads to a premature release by the phagocytes of their killing and degradative factors. Production of ROS such as that seen during irradiation, drug metabolism, or ischemia followed by reperfusion for example, induces inflammatory reactions with a secondary amplification of ROS production. Acute ROS production can also lead to thrombosis, whereas chronic ROS production can induce a chronic inflammatory reaction of the endothelium with atherosclerosis as a possible consequence. Some examples are also given to show that ROS might control positively or negatively the activity of inflammatory molecules. The multiplicity of the cross reactions between ROS and inflammation allows to suggest that drugs that disconnect these two events might be therapeutically used.
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PMID:[Reactive oxygen species and inflammation]. 801 8

Injury results in altered hepatocyte protein synthesis including the production of acute-phase reactants. Evidence suggests that these hepatocyte products regulate macrophage function; however, their role in liver macrophage-mediated hepatocyte dysfunction following a second insult is poorly characterized. We hypothesize that IL-6-stimulated hepatocyte products alter liver macrophage responses to lipopolysaccharide, contributing to enhanced hepatocyte dysfunction. To test this hypothesis, hepatocytes, obtained by liver collagenase digestion, were treated with rIL-6 (murine, 300 units/ml) for 24 hr, and then liver macrophages, obtained by perfusion and pronase digestion, were added to establish cocultures. Cocultures were then stimulated with endotoxin (LPS, Escherichia coli O111:B4, 10 micrograms/ml) and hepatocyte dysfunction was assessed by determining secretory protein synthesis ([35S]methionine labeling, trichloracetic acid precipitation, and SDS-PAGE) and energy metabolism [mitochondrial respiration using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye]. Cultures of hepatocytes alone stimulated with IL-6, LPS, or sequential IL-6 followed by LPS demonstrate no difference in total secretory protein synthesis or mitochondrial respiration. In contrast, hepatocyte-liver macrophage cocultures demonstrate significantly reduced total secretory protein synthesis following sequential IL-6 followed by LPS ([35S]methionine cpm x 10(3): control, 33.8 +/- 8.5; LPS, 25.8 +/- 6.3; IL-6/LPS, 15.7 +/- 6.4; P < 0.05 vs control). This effect is specific to IL-6 since sequential TNF-alpha followed by LPS did not result in significant suppression of secretory protein synthesis. One-dimensional SDS-PAGE of labeled coculture secretory proteins demonstrates qualitative changes following sequential insult in vitro compared to controls.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Sequential insult enhances liver macrophage-signaled hepatocyte dysfunction. 804 Nov 36

Other investigators have shown that exogenously administered transforming growth factor-beta (TGF-beta) inhibits lymphocyte adherence to vascular endothelial cells (VEC). We examined the role of TGF-beta 1 as an autocrine mediator of lymphocyte adhesion to adult human VECs. VECs were harvested from eight saphenous or cadaveric iliac veins using 0.2% collagenase. Low-passage VECs in MCDB + 0.1% BSA were pretreated for 24 hr with monoclonal anti-TGF-beta 1 antibody (5 micrograms/ml), LPS (5 micrograms/ml), or IL-1 (10 U/ml). Adherence of fluorescently labeled lymphocytes to pretreated VECs was quantitated and results were expressed as relative adhesion compared to untreated control. Total mRNA from LPS- or IL-1-treated VECs was subjected to Northern analysis to determine relative TGF-beta 1 expression. Total TGF-beta 1 protein concentration in supernatants from LPS- or IL-1-treated VECs was determined by ELISA. Data (means +/- SEM) were analyzed by ANOVA with a Newman-Keuls posttest. Neutralizing endogenous TGF-beta 1 with anti-TGF-beta 1 antibody significantly increased adhesion of lymphocytes to VEC monolayers compared to control (125 +/- 3 vs 101 +/- 2%, P < 0.01, n = 8). The level of adhesion was equivalent to that seen with IL-1 stimulation (131 +/- 6%). Spearman correlation of lymphocyte adherence to IL-1- or LPS-treated VECs vs TGF-beta 1 mRNA expression or vs relative TGF-beta 1 protein concentration showed significant inverse relationships (r = -0.82, P < 0.001, and r = -0.87, P < 0.001, respectively). Endogenous TGF-beta 1's inhibitory effect on lymphocyte adhesion was blocked by a specific neutralizing antibody. VEC TGF-beta 1 mRNA expression and TGF-beta 1 production were inversely proportional to lymphocyte adhesion, suggesting down-regulation of TGF-beta 1 in response to proinflammatory cytokines. Together, these observations support the hypothesis that TGF-beta 1 has an autocrine inhibitory role in regulation of lymphocyte adhesion to VECs.
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PMID:Transforming growth factor-beta 1 serves as an autocrine inhibitor of human endothelial cell/lymphocyte adhesion. 853 71

Interstitial collagenase (MMP-1), a metalloproteinase produced by resident and inflammatory cells during connective tissue turnover, cleaves type I collagen fibrils. This catalytic event is rate limiting in remodeling of tissues rich in fibrillar collagen such as the skin and lungs. The regulation of collagenase expression is cell-type specific; bacterial LPS and zymosan, a yeast cell wall derivative, are potent inducers of collagenase expression in macrophages, but do not alter fibroblast collagenase expression. Since promoter elements controlling collagenase transcription in monocytic cells have not been previously defined, we sought to delineate responsive cis-acting elements of the collagenase promoter in transiently transfected human (U937) and murine (J774) monocytic cell lines. Deletion constructs containing as little as 72 bp of 5' -flanking sequence of the collagenase promoter were sufficient for LPS- or zymosan-mediated transcriptional induction, whereas phorbol inducibility exhibited an absolute requirement for upstream elements including the polyoma enhancer A-binding protein-3 site (-83 to -91) and TTCA sequence (-102 to -105) in both monocytic cells and fibroblasts. Mutagenesis of the activator protein-1 [AP-1] site at -72 abolished basal promoter activity and LPS/zymosan inducibility, while mutagenesis of an NF-kappaB-like site at -20 to -10 had no effect. Nuclear extracts from LPS- and zymosan-treated cells showed strong AP-1 activity by gel-shift analysis, and supershift analysis showed the AP-1 complexes contained specific members of both the jun and fos gene families. These data indicate that, in contrast to most LPS effects, AP-1, but not nuclear factor-kappaB, mediates LPS induction of collagenase transcription in macrophagelike cells. Furthermore, as compared to regulation by phorbol ester, collagenase induction in monocytic cells by cell wall derivatives of bacteria or yeast is largely independent of upstream promoter sequences.
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PMID:Monocytic cell type-specific transcriptional induction of collagenase. 862 73

We previously reported that both local and systemic factors relevant to the pathogenesis of periodontal disease can increase gingival collagenase activity in rats. Since the degradation of extracellular matrix is an essential feature of periodontal disease and this tissue breakdown requires multiple enzyme interactions, the current study was carried out to determine the effects of bacterial endotoxin (LPS) (a local factor) and diabetes (a systemic factor) on a panel of matrix-degrading enzymes (collagenase, gelatinase, elastase, and beta-glucuronidase) in the gingiva of rats. In addition, the effects of therapy with a semisynthetic tetracycline (minocycline) were investigated. Ten male, Sprague-Dawley rats were made diabetic by IV injection of streptozotocin. Four of the ten rats then received minocycline (10 mg/day) by oral gavage on a daily basis for 3 weeks. Nineteen nondiabetic rats served as controls and 9 of them received 10 microliters of E. coli LPS (10 mg/ml) by injection into the labial gingiva every other day during the last week of the study. The other 10 nondiabetic rats were sham injected with saline into the gingiva. At the end of the 3 week experimental period, gingival tissue and skin were dissected from each rat and extracted for enzyme analysis. Our results showed that diabetes markedly increased the four matrix-degrading enzyme activities in both gingiva and skin. In contrast, local LPS injection increased these enzyme activities in the gingiva alone. Systemic therapy with minocycline completely ameliorated these elevated enzyme levels in diabetic rats in both gingiva and skin. Minocycline added in vitro to the enzyme assay systems containing skin extract from diabetic rats also inhibited collagenase and gelatinase activities, but no inhibition was observed for elastase and beta-glucuronidase activities, indicating that the MMPs and other enzymes were inhibited by minocycline, during diabetes, by indirect and indirect mechanisms, respectively.
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PMID:Local and systemic factors in periodontal disease increase matrix-degrading enzyme activities in rat gingiva: effect of micocycline therapy. 882 70


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