Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent studies have shown that infusion of the anticoagulant protein, activated protein C (APC), can ameliorate many of the systemic effects of endotoxemia in experimental animals, although the mechanisms in this action are unknown. We investigated the effects of APC on the responses of blood monocytes, alveolar macrophages, and cells of the monocyte line, THP-1, to stimulation in vitro by LPS, IFN-gamma, or PMA. Mononuclear phagocyte (MO) activation was associated with rapid production of TNF-alpha, down-regulation of the glycophosphatidylinositol-linked protein CD14 (the key MO receptor for complexes of LPS and LPS-binding protein responsible for intracellular signaling), and down-regulation of the related LPS-binding proteins CD11b and CD18. Addition of APC, but not the zymogen, PC, or active site-blocked APC, inhibited selected MO responses involving the CD14-dependent LPS-induced pathway of MO activation, or activation induced by IFN-gamma or PMA. Thus, APC inhibited the production of TNF-alpha and prevented down-regulation of membrane CD11b, CD14, and CD18, but had no effect on up-regulation of MHC class II, ICAM-1, or IL-2R, down-regulation of MO expression of another glycophosphatidylinositol-linked protein, CD59, or production of reactive oxygen intermediates. These data show that APC inhibits host cytokine production but maintains MO responses associated with adhesion, phagocytosis, and killing of Gram-negative bacteria, such that use of APC may be a logical and potent adjunctive therapy in select inflammatory diseases involving MO activation and damaging host cytokine overproduction.
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PMID:Selective inhibitory effects of the anticoagulant activated protein C on the responses of human mononuclear phagocytes to LPS, IFN-gamma, or phorbol ester. 752

Rationally attenuated strains of Salmonella expressing foreign proteins represent a potentially important vaccine delivery system. The characteristics of Ag presentation of influenza nucleoprotein expressed in an AroA- strain of Salmonella typhimurium (SL3262-pNP-2) have therefore been compared with those of soluble purified nucleoprotein (NP) and infectious influenza virus. This represents three distinct modes of internalization of the same protein into APC. Human monocytes and the monocytic leukemia cell line THP-1 infected with SL3261-pNP-2 were found to present several different epitopes from NP to human CD4+ class II-restricted T lymphocytes. Ag presentation to these T cell clones was enhanced by pretreatment of THP-1 cells with IFN-gamma but not TNF-alpha. Bacterial phagocytosis and Ag presentation of NP were increased after opsonization of Salmonella with immune serum. Macrophages infected with SL3261-pNP-2 were unable to present NP to class I-restricted T cells. In contrast, cells infected with live influenza virus, although recognized by NP-specific class I-restricted CTL, were inefficiently recognized by NP-specific class II-restricted T cells. Ag presentation to CD4+ T cell clones by monocytes of SL3261-pNP-2, purified recombinant NP, and live influenza virus, but not the synthetic peptide 206-229, was inhibited by chloroquine and the protease inhibitors pepstatin A and leupeptin, suggesting that the major route of processing in each case was via the exogenous pathway. T cell recognition of NP via all of these Ag delivery systems was also abrogated by cycloheximide and brefeldin A treatment, indicating a requirement for recently synthesized MHC class II molecules in presentation of whole NP after processing but not for the corresponding synthetic peptide.
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PMID:Comparison of antigen presentation of influenza A nucleoprotein expressed in attenuated AroA- Salmonella typhimurium with that of live virus. 768 Oct 81

Upon activation, mononuclear phagocytes (Mphi) play key roles in the development of septic shock and multiple host immune responses, but details of the regulation of Mphi activation are little understood. We recently showed that the physiologic anticoagulant molecule, activated protein C (APC), blocks responses of human blood Mphi, alveolar Mphi, or THP-1 cells induced by LPS, IFN-gamma, or PMA, including TNF-alpha production and down-regulation of several LPS binding-related proteins. We now report a possible mechanism of action through inhibition of the rapid intracellular calcium signaling that occurs at the onset of Mphi activation, and characterization of a specific Mphi receptor for APC. Flow cytometry studies using Fluo-3 showed that Mph activation by Fc-receptor cross-linking or rIFN-gamma caused a rapid increase in free intracellular calcium, a primary event in multiple signal transduction pathways, which was blocked by pretreatment with APC. Consistent with this, addition of APC inhibited PHA-induced T cell proliferation in a dose- and time-dependent manner. Peak suppression (> 70%) required addition of APC within the first hour of 72 hr cocultures of Mphi and lymphocytes, and proliferative responses were not restored by addition of IL-2 or TNF-alpha. Biochemical studies showed that 125I-labeled APC bound specifically to M phi in a time-dependent and saturable manner. Scatchard analysis indicated there were 180,690 binding sites for APC per cell, which were of high affinity (Kd value of 12.9 mM). Binding of 125I-APC was doubled by activation of Mphi with LPS, and bound APC was not displaced by the zymogen, protein C (PC), or by enzymatically inactive (diisopropyl fluorophosphate- or PPACK-treated) APC, indicating an absolute requirement for the active site of APC in its binding to Mphi. APC binding was blocked by a polyclonal Ab to human PC/APC, but not by protein S, factor Va or Xa, or a polyclonal antithrombomodulin antibody. When 125I-APC was crosslinked to its receptor, immunoprecipitated and analyzed by SDS-PAGE under reducing conditions, a covalent complex (110-115 kD) of 125I-APC (62 kD) and its receptor was seen. In addition, a Mphi membrane protein of 50-55 kD, as determined by SDS-PAGE, was affinity-purified using an APC-Affigel column, and confirmed by ligand binding. Taken together, our findings document the presence of a M phi surface receptor for APC, which appears distinct from a recently described endothelial receptor for PC and APC, and which may be involved in the inhibitory effects of APC on activation of human Mphi, including Mphi-dependent T cell proliferation.
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PMID:Binding of activated protein C to a specific receptor on human mononuclear phagocytes inhibits intracellular calcium signaling and monocyte-dependent proliferative responses. 854 85

The endothelial molecule thrombomodulin (TM) regulates hemostasis by binding thrombin and promoting conversion of protein C to activated protein C (aPC). Apart from its anticoagulant actions, aPC modulates mononuclear phagocyte (M phi) activation, including TNF-alpha production, indicating interrelationships of the coagulation and immune systems. While the endothelium is considered to be the prime regulator of aPC generation, TM recently has been identified M phi and neutrophils. This study analyzes TM membrane expression by human blood monocytes, alveolar macrophages, and U937 cells cultured in the presence of various stimuli. All except U937 cell expressed high levels of surface TM. Surprisingly, stimulation with LPS or TNF-alpha further up-regulated TM expression by M phi, whereas cultured endothelial cells (EC) showed decreased TM expression. However, noninflammatory stimuli induced qualitatively similar changes in M phi and EC; all-trans retinoic acid and prostaglandin E up-regulated surface TM, and PMA decreased TM expression. Changes in M phi TM expression were accompanied by alteration in functional activity. Thus, LPS increased the TM cofactor activity of THP-1 cells by 27 +/- 6.9% (p < 0.05), and PMA decreased their cofactor activity by 53.2 +/- 11.5% (p < 0.05).In addition, in vivo relevance was demonstrated by the presence of TM on intragraft inflammatory M phi during cardiac rejection, whereas adjacent EC lacked TM expression. These studies demonstrate that expression of TM on human M phi is regulated differently to EC with respect to inflammatory stimuli, suggesting the potential for extravascular M phi to promote local production of aPC.
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PMID:A physiologic anti-inflammatory pathway based on thrombomodulin expression and generation of activated protein C by human mononuclear phagocytes. 869 Sep 16

We have investigated the effects of oxidized low density lipoproteins (oxidized LDL) on the expression of TM by THP-1 monocytic cells. TM antigen levels and its cofactor activity for thrombin-dependent protein C activation were increased by oxidized LDL and accompanied by an increase in TM mRNA levels. Incubation of THP-1 cells with 300 microg/ml oxidized LDL for 24 h resulted in an 80% increase of cellular TM antigen levels. Native LDL and acetylated LDL did not affect the TM expression by these cells. The resultant aqueous phase after extraction of oxidized LDL by chloroform/methanol increased the TM antigen levels as well as oxidized LDL. Phorbol 12-myristate 13-acetate (PMA) also increased the TM antigen level 2.1 times the control and was accompanied by the adhesion of cells to plastic dishes and increasing macrophage cell surface antigen CD14 levels. In contrast, oxidized LDL did not induce differentiation to the macrophage. The present results indicate that oxidized LDL increases cellular TM antigen without cellular differentiation and that up-regulation of TM by oxidized LDL in monocytes may have some implication in atherosclerosis.
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PMID:Effect of oxidized low density lipoprotein on thrombomodulin expression by THP-1 cells. 936 89

Human mononuclear phagocytes (MO) express a functional form of thrombomodulin (TM), the anticoagulant molecule typically considered purely in the context of regulation of conversion of protein C (PC) to activated PC (aPC) by thrombin-bound TM at the endothelial cell surface. We have been interested in the anti-inflammatory actions of aPC, including its ability to suppress MO production of multiple pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1), leading us to consider whether MO surface expression of TM and resultant local aPC generation, might contribute to autoregulation of MO activation at sites of inflammation involving thrombin and fibrin formation. Since TNF-alpha and IL-1 are known to downregulate endothelial expression of TM, this study investigated the effects of TNF-alpha on production of TM by the monocytic leukemic cell line, THP-1. THP-1 cells display many monocyte-like properties, providing a convenient source for biochemical and molecular studies. Western blotting of lysates of THP-1 cells versus cultured human umbilical vein endothelial cells showed that after 24 h of stimulation, TNF-alpha decreased TM protein expression in endothelial but not THP-1 cells and comparable responses were noted by flow cytometry. Subsequent Northern blot analysis showed that at 24 h, TNF-alpha diminished TM steady state mRNA in endothelial but not THP-1 cells, although Northern analysis of the kinetics of TM steady state mRNA did show a rapid and transient modulation by TNF-alpha at 2 h of stimulation, which was confirmed by nuclear run-on analysis of the effect of TNF-alpha on TM gene transcription rates in THP-1 cells, analysis of protein expression by flow cytometry and Western blotting showed similar effects. In contrast to the divergent effects of TNF-alpha on THP-1 vs endothelial cells, agonists such as cyclic adenosine monophosphate (c-AMP) and phorbol ester (PMA) had comparable effects on THP-1 and endothelial cells, resulting in parallel increases or decreases in TM mRNA and protein expression, respectively. Hence, there is a 'split' in the nature of endothelial vs THP-1 cellular responses to TNF-alpha as compared to non-inflammatory stimuli, suggesting cell-specific differences in regulation of the TM promoter. We conclude that in contrast to its effects on TM expression by endothelial cells, exposure of THP-1 cells to TNF-alpha causes a rapid and transient decrease in TM mRNA production which is followed by sustained and high level expression, supporting the concept that MO expression of TM may contribute to regulation of MO activation and cytokine production at inflammatory sites.
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PMID:Differential effect of tumor necrosis factor-alpha on thrombomodulin gene expression by human monocytoid (THP-1) cell versus endothelial cells. 959 45

Expression of HLA and CD1b molecules was investigated in the THP-1 macrophage cell line within 2 weeks following phagocytosis of mycobacteria or Escherichia coli. During the first 2-3 days, cell surface expression of HLA class II and CD1b was drastically down-modulated, whereas HLA class I expression was up-modulated. In the following days both HLA class II and CD1b expression first returned to normal, then increased and finally returned to normal with kinetics similar to that observed for the steadily increased HLA class I. The initial down-modulation of HLA class II and CD1b cell surface antigens was absolutely dependent on phagocytosis of bacteria. Further studies indicated that initial HLA class II cell surface down-modulation (1) was not due to reduced transcription or biosynthesis of mature HLA class II heterodimers, (2) was only partially, if at all, rescued by treatment with IFN-gamma, although both mRNA and corresponding intracellular proteins increased up to sixfold with respect to untreated cells, and (3) resulted in failure of THP-1 cells to process and present mycobacterial antigens to HLA-DR-restricted antigen-specific T cell lines. The existence of a transient block of transport of mature HLA class II heterodimers to the cell surface in the first days after phagocytosis of bacteria may have negative and positive consequences: it decreases APC function early but it may increase it later by favoring optimal loading of bacterial antigens in cellular compartments at high concentration of antigen-presenting molecules.
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PMID:Distinct regulation of HLA class II and class I cell surface expression in the THP-1 macrophage cell line after bacterial phagocytosis. 1006 65

Activated protein C (APC) protects against sepsis in animal models and inhibits the lipopolysacharide (LPS)-induced elaboration of proinflammatory cytokines from monocytes. The molecular mechanism responsible for this property is unknown. We assessed the effect of APC on LPS-induced tumour necrosis factor alpha (TNF-alpha) production and on the activation of the central proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) in a THP-1 cell line. Cells were preincubated with varying concentrations of APC (200 microg/ml, 100 microg/ml and 20 microg/ml) before addition of LPS (100 ng/ml and 10 microg/ml). APC inhibited LPS-induced production of TNF-alpha both in the presence and absence of fetal calf serum (FCS), although the effect was less marked with 10% FCS. APC also inhibited LPS-induced activation of NF-kappaB, with APC (200 microg/ml) abolishing the effect of LPS (100 ng/ml). The ability of APC to inhibit LPS-induced translocation of NF-kappaB is likely to be a significant event given the critical role of the latter in the host inflammatory response.
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PMID:Activated protein C inhibits lipopolysaccharide-induced nuclear translocation of nuclear factor kappaB (NF-kappaB) and tumour necrosis factor alpha (TNF-alpha) production in the THP-1 monocytic cell line. 1093 Sep 89

The precise regulatory mechanisms of amplification and downregulation of the pro- and anti-inflammatory cytokines in the inflammatory response have not been fully delineated. Although activated protein C (APC) and its precursor protein C (PC) have recently been reported to be promising therapeutic agents in the management of meningococcal sepsis, direct evidence for the anti-inflammatory effect remains scarce. We report that APC inhibits in vitro the release of tumor necrosis factor (TNF) and macrophage migration inhibitory factor (MIF), two known cytokine mediators of bacterial septic shock, from lipopolysaccharide (LPS)-stimulated human monocytes. The THP-1 monocytic cell line, when stimulated with LPS and concomitant APC, exhibited a marked reduction in the release of TNF and MIF protein in a concentration-dependent manner compared to cells stimulated with LPS alone. This effect was observed only when incubations were performed in serum-free media, but not in the presence of 1-10% serum. Serum-mediated inhibition could only be overcome by increasing APC concentrations to far beyond physiological levels, suggesting the presence of endogenous serum-derived APC inhibitors. Inhibition of MIF release by APC was found to be independent of TNF, as stimulation of MIF release by LPS was unaltered in the presence of anti-TNF antibodies. Our data confirm that the suggested anti-inflammatory properties of APC are due to direct inhibition of the release of the pro-inflammatory monokine TNF, and imply that the anti-inflammatory action of APC is also mediated via inhibition of MIF release.
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PMID:Activated protein C inhibits tumor necrosis factor and macrophage migration inhibitory factor production in monocytes. 1102 25

The activation of the matrix metalloproteinase progelatinase A (MMP-2) has been of keen interest because an increase in MMP-2 activity has been implicated in disease states such as cancer and atherosclerosis. Activation of MMP-2 occurs on the surface of specific cell types in two steps. In the first step, primary cleavage of MMP-2 by a membrane-type matrix metalloproteinase generates an intermediate. A secondary cleavage and activation of the intermediate is thought to occur autocatalytically. Previous studies have shown that thrombin can also activate progelatinase A in the presence of endothelial cells. We show that this cell-dependent mechanism of MMP-2 activation also occurs with THP-1 cells and involves binding of thrombin to thrombomodulin present on the cell surface and generation of the anti-coagulant protein, activated protein C. We demonstrate that activated protein C is directly responsible for activation and cleavage of the gelatinase A intermediate. This work contributes new mechanistic insights into the activation of MMP-2 and provides a novel link between matrix metalloproteinase activation and anti-coagulation.
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PMID:Thrombin-thrombomodulin activation of protein C facilitates the activation of progelatinase A. 1129 49


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