Circulating human neutrophils from patients with severe inflammatory disorders such as erysipelas and sepsis are specifically desensitized to complement factor C5a stimulation but not to stimulation with other stimuli like N-formyl-methionyl-leucyl-phenylalanine (FMLP), interleukin-8 (IL-8), leukotriene B4 (LTB4), or platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). In this study, we raised the question whether factors released from polymorphonuclear leukocytes (PMNs) can specifically down-regulate C5a-dependent neutrophil functions. When neutrophils were preincubated with either neutrophil lysates or neutrophil degranulation supernatants, a complete inhibition of C5a-stimulated beta-glucuronidase release and chemotaxis could be observed, whereas FMLP-, IL-8-, LTB4- or PAF-dependent functions were not affected. Serine protease inhibitors like phenylmethylsulfonyl fluoride, antileukoprotease, or elafin abolished this effect. High-performance liquid chromatography of neutrophil degranulation supernatants revealed pronounced inhibition of C5a-dependent neutrophil functions in fractions exerting elastase or cathepsin G activity, but not in fractions exerting proteinase 3 activity. Using purified human leukocyte elastase (HLE), C5a responses like intracellular calcium influx, beta-glucuronidase release, and chemotaxis were also specifically inhibited. Our experiments show that the release of HLE or cathepsin G from neutrophils specifically down-regulates the responsiveness of neutrophils to C5a. Elastase and cathepsin G may therefore play an important role in the down-regulation of acute inflammation.
Ecotin is a dimeric periplasmic protein from Escherichia coli that has been shown to inhibit potently many trypsin-fold serine proteases of widely varying substrate specificity. To help elucidate the physiological function of ecotin, we examined the family of ecotin orthologues, which are present in a subset of Gram-negative bacteria. Phylogenetic analysis suggested that ecotin has an exogenous target, possibly neutrophil elastase. Recombinant protein was expressed and purified from E. coli, Yersinia pestis and Pseudomonas aeruginosa, all species that encounter the mammalian immune system, and also from the plant pathogen Pantoea citrea. Notably, the Pa. citrea variant inhibits neutrophil elastase 1000-fold less potently than the other orthologues. All four orthologues are dimeric proteins that potently inhibit (<10 pM) the pancreatic digestive proteases trypsin and chymotrypsin, while showing more variable inhibition (5 pM to 24 microM) of the blood proteases Factor Xa, thrombin and urokinase-type plasminogen activator. To test whether ecotin does, in fact, protect bacteria from neutrophil elastase, an ecotin-deficient strain was generated in E. coli. This strain is significantly more sensitive in cell-killing assays to human neutrophil elastase, which causes increased permeability of the outer membrane that persists even during renewed bacterial growth. Ecotin affects primarily the ability of E. coli to recover and grow following treatment with neutrophil elastase, rather than the actual rate of killing. This suggests that an important part of the antimicrobial mechanism of neutrophil elastase may be a periplasmic bacteriostatic effect of protease that has translocated across the damaged outer membrane.
Interacting selectively and non-covalently with a cytokine, any of a group of proteins that function to control the survival, growth and differentiation of tissues and cells, and which have autocrine and paracrine activity.
Evidence
1:
Inferred from Physical InteractionUniProtKB
There is evidence that neutrophil production is a balance between the proliferative action of granulocyte-colony-stimulating factor (G-CSF) and a negative feedback from mature neutrophils (the chalone). Two neutrophil serine proteases have been implicated in granulopoietic regulation: pro-proteinase 3 inhibits granulocyte macrophage-colony-forming unit (CFU-GM) growth, and elastase mutations cause cyclic and congenital neutropenia. We further studied the action of the neutrophil serine proteases (proteinase 3, elastase, azurocidin, and cathepsin G) on granulopoiesis in vitro. Elastase inhibited CFU-GM in methylcellulose culture. In serum-free suspension cultures of CD34+ cells, elastase completely abrogated the proliferation induced by G-CSF but not that of GM-CSF or stem cell factor (SCF). The blocking effect of elastase was prevented by inhibition of its enzymatic activity with phenylmethylsulfonyl fluoride (PMSF) or heat treatment. When exposed to enzymatically active elastase, G-CSF, but not GM-CSF or SCF, was rapidly cleaved and rendered inactive. These results support a role for neutrophil elastase in providing negative feedback to granulopoiesis by direct antagonism of G-CSF.
Components of kinin-forming systems operating at inflammatory sites are likely to interact with elastase that is released by recruited neutrophils and may, at least temporarily, constitute the major proteolytic activity present at these sites. The aim of this work was to determine the effect of kininogen degradation by human neutrophil elastase (HNE) on kinin generation by tissue and plasma kallikreins. We show that the digestion of both low molecular mass (LK) and high molecular mass (HK) forms of human kininogen by HNE renders them essentially unsusceptible to processing by human urinary kallikrein (tissue-type) and also significantly quenches the kinin release from HK by plasma kallikrein. Studies with synthetic model heptadecapeptide substrates, ISLMKRPPGFSPFRSSR and SLMKRPPGFSPFRSSRI, confirmed the inability of tissue kallikrein to process peptides at either termini of the internal kinin sequence, while plasma kallikrein was shown to release the kinin C-terminus relatively easily. The HNE-generated fragments of kininogens were separated by HPLC and the fractions containing internal kinin sequences were identified by a kinin-specific immunoenzymatic test after trypsin digestion. These fractions were analyzed by electrospray-ionization mass spectrometry. In this way, multiple peptides containing the kinin sequence flanked by only a few amino acid residues at each terminus were identified in elastase digests of both LK and HK. These results suggest that elastase may be involved in quenching the kinin-release cascade at the late stages of the inflammatory reaction.
J. Biol. Chem. 257, 9849-9854 (1982)[PubMed:6980881]
This study has examined the interaction between human leukocyte elastase and alpha 2-plasmin inhibitor, or C1 inactivator, inhibitors of proteases of the complement, kinin, coagulation, and fibrinolytic enzyme systems. Leukocyte elastase, in catalytic concentrations, progressively inactivates the plasmin inhibitory activity of both inhibitors. The C1s binding function of C1 inactivator is also destroyed by leukocyte elastase. The nature of the molecular events underlying the inactivation of these protease inhibitors was examined by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Loss of functional activity was accompanied by limited proteolytic cleavage of both inhibitors with the production of several characteristic derivative peptide chains. Leukocyte elastase cleaved alpha 2-plasmin inhibitor at two separate sites and generated lower molecular weight fragments similar to those produced by bovine beta-trypsin. C1 inactivator was hydrolyzed at three different regions on the molecule whereas beta-trypsin cleaved two regions in common with leukocyte elastase. These findings suggest that inactivation of alpha 2-plasmin inhibitor and C1 inactivator by leukocyte elastase released in the inflammatory reaction may potentiate pathological proteolysis. The limited digestion of these inhibitory proteins by leukocyte elastase may prove useful in studies of their primary structure.
Interacting selectively and non-covalently with heparin, any member of a group of glycosaminoglycans found mainly as an intracellular component of mast cells and which consist predominantly of alternating alpha-(1->4)-linked D-galactose and N-acetyl-D-glucosamine-6-sulfate residues.
According to the hitherto accepted view, neutrophils kill ingested microorganisms by subjecting them to high concentrations of highly toxic reactive oxygen species (ROS) and bringing about myeloperoxidase-catalysed halogenation. We show here that this simple scheme, which for many years has served as a satisfactory working hypothesis, is inadequate. We find that mice made deficient in neutrophil-granule proteases but normal in respect of superoxide production and iodinating capacity, are unable to resist staphylococcal and candidal infections. We also show that activation provokes the influx of an enormous concentration of ROS into the endocytic vacuole. The resulting accumulation of anionic charge is compensated for by a surge of K+ ions that cross the membrane in a pH-dependent manner. The consequent rise in ionic strength engenders the release of cationic granule proteins, including elastase and cathepsin G, from the anionic sulphated proteoglycan matrix. We show that it is the proteases, thus activated, that are primarily responsible for the destruction of the bacteria.
Catalysis of the hydrolysis of a peptide bond. A peptide bond is a covalent bond formed when the carbon atom from the carboxyl group of one amino acid shares electrons with the nitrogen atom from the amino group of a second amino acid.
According to the hitherto accepted view, neutrophils kill ingested microorganisms by subjecting them to high concentrations of highly toxic reactive oxygen species (ROS) and bringing about myeloperoxidase-catalysed halogenation. We show here that this simple scheme, which for many years has served as a satisfactory working hypothesis, is inadequate. We find that mice made deficient in neutrophil-granule proteases but normal in respect of superoxide production and iodinating capacity, are unable to resist staphylococcal and candidal infections. We also show that activation provokes the influx of an enormous concentration of ROS into the endocytic vacuole. The resulting accumulation of anionic charge is compensated for by a surge of K+ ions that cross the membrane in a pH-dependent manner. The consequent rise in ionic strength engenders the release of cationic granule proteins, including elastase and cathepsin G, from the anionic sulphated proteoglycan matrix. We show that it is the proteases, thus activated, that are primarily responsible for the destruction of the bacteria.
J. Biol. Chem. 257, 9849-9854 (1982)[PubMed:6980881]
This study has examined the interaction between human leukocyte elastase and alpha 2-plasmin inhibitor, or C1 inactivator, inhibitors of proteases of the complement, kinin, coagulation, and fibrinolytic enzyme systems. Leukocyte elastase, in catalytic concentrations, progressively inactivates the plasmin inhibitory activity of both inhibitors. The C1s binding function of C1 inactivator is also destroyed by leukocyte elastase. The nature of the molecular events underlying the inactivation of these protease inhibitors was examined by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Loss of functional activity was accompanied by limited proteolytic cleavage of both inhibitors with the production of several characteristic derivative peptide chains. Leukocyte elastase cleaved alpha 2-plasmin inhibitor at two separate sites and generated lower molecular weight fragments similar to those produced by bovine beta-trypsin. C1 inactivator was hydrolyzed at three different regions on the molecule whereas beta-trypsin cleaved two regions in common with leukocyte elastase. These findings suggest that inactivation of alpha 2-plasmin inhibitor and C1 inactivator by leukocyte elastase released in the inflammatory reaction may potentiate pathological proteolysis. The limited digestion of these inhibitory proteins by leukocyte elastase may prove useful in studies of their primary structure.
Interacting selectively and non-covalently with any protein or protein complex (a complex of two or more proteins that may include other nonprotein molecules).
Evidence
1:
Inferred from Physical InteractionIntAct
Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease associated with autoantibodies against the hemidesmosomal proteins BP180 and BP230. In the IgG passive transfer model of BP, blister formation is triggered by anti-BP180 IgG and depends on complement activation, mast cell degranulation, and neutrophil recruitment. Mice lacking neutrophil elastase (NE) do not develop experimental BP. Here, we demonstrated that NE degrades recombinant mouse BP180 within the immunodominant extracellular domain at amino acid positions 506 and 561, generating peptide p561 and peptide p506. Peptide p561 is chemotactic for neutrophils both in vitro and in vivo. Local injection of NE into B6 mice recruits neutrophils to the skin, and neutrophil infiltration is completely blocked by co-injection with the NE inhibitor α1-proteinase inhibitor. More importantly, NE directly cleaves BP180 in mouse and human skin, as well as the native human BP180 trimer molecule. These results demonstrate that (i) NE directly damages the extracellular matrix and (ii) NE degradation of mouse BP180 generates neutrophil chemotactic peptides that amplify disease severity at the early stage of the disease.
Evidence
2:
Inferred from Physical InteractionUniProtKB
The urokinase receptor (CD87) participates to the pericellular proteolytic potential of migrating cells and to the recruitment of leukocytes during inflammation. It consists of three structurally homologous domains, with the C-terminal domain D3 attached to cell membranes through a GPI anchor. CD87 is susceptible to an endoproteolytic processing removing the N-terminal domain D1 and generating truncated D2D3 membrane species, thus modulating CD87-associated functions. Full-length or truncated CD87 can be also released from cells via juxtamembrane cleavage by phospholipases and/or by yet unidentified proteinases. Using a recombinant CD87 and the CD87-positive monocytic U937 cell line and isolated blood monocytes, we show by protein immunoblotting and flow immunocytometry that the human neutrophil serine-proteinases elastase and cathepsin G cleave CD87 within the D1-D2 linker sequence, while in addition cathepsin G is highly efficient in cleaving the C terminus of D3. The combination of cathepsin G and elastase provided by degranulated neutrophils results in enzymatic cooperation leading to the release from monocytic cells of a truncated D2D3 species resembling that previously described in pathological body fluids. Using mass spectrometry analysis, the proteolytic fragmentation of synthetic peptides mapping the D1-D2 linker and D3 C-terminal domains identifies potential cleavage sites for each enzyme and suggests the existence of a mechanism regulating the CD87(D1-D2)-associated chemotactic activity. Finally, isolated or combined elastase and cathepsin G drastically reduce the capacity of cells to bind urokinase. Secretable leukocyte serine-proteinases are thus endowed with high potential for the regulation of CD87 expression and function on inflammatory cells.
Evidence
3:
Inferred from Physical InteractionUniProtKB
Human neutrophil elastase (HNE) has long been linked to the pathology of a variety of inflammatory diseases and therefore is a potential target for therapeutic intervention. At least two other serine proteases, proteinase 3 (Pr3) and cathepsin G, are stored within the same neutrophil primary granules as HNE and are released from the cell at the same time at inflammatory sites. HNE and Pr3 are structurally and functionally very similar, and no substrate is currently available that is preferentially cleaved by Pr3 rather than HNE. Discrimination between these two proteases is the first step in elucidating their relative contributions to the development and spread of inflammatory diseases. Therefore, we have prepared new fluorescent peptidyl substrates derived from natural target proteins of the serpin family. This was done because serpins are rapidly cleaved within their reactive site loop whether they act as protease substrates or inhibitors. The hydrolysis of peptide substrates reflects the specificity of the parent serpin including those from alpha-1-protease inhibitor and monocyte neutrophil elastase inhibitor, two potent inhibitors of elastase and Pr3. More specific substrates for these proteases were derived from the reactive site loop of plasminogen activator inhibitor 1, proteinase inhibitors 6 and 9, and from the related viral cytokine response modifier A (CrmA). This improved specificity was obtained by using a cysteinyl residue at P1 for Pr3 and an Ile residue for HNE and because of occupation of protease S' subsites. These substrates enabled us to quantify nanomolar concentrations of HNE and Pr3 that were free in solution or bound at the neutrophil surface. As membrane-bound proteases resist inhibition by endogenous inhibitors, measuring their activity at the surface of neutrophils may be a great help in understanding their role during inflammation.
Evidence
4:
Inferred from Physical InteractionUniProtKB
J. Leukoc. Biol. 72, 538-545 (2002)[PubMed:12223522]
CD40 is a crucial element in the process of fibroblast activation. We demonstrated that treatment of human gingival fibroblast (HGF) with human leukocyte elastase (HLE), a neutrophil serine protease, down-regulated the expression of CD40 and binding to the CD40 ligand (CD40L) using flow cytometry. The other neutrophil serine proteases, cathepsin G and proteinase 3, exhibited markedly less activity for CD40 reduction. The CD40 reduction by HLE was also observed in skin and lung fibroblasts, but not in monocytes, macrophages, and dendritic cells. The reduction resulted from direct proteolysis by HLE on the cell surface, because HLE reduced CD40 on fixed HGF and also on cell lysates and membranes. HLE treatment of HGF decreases interleukin (IL)-8 and macrophage chemoattractant protein-1 production by HGF when stimulated by CD40L, but not by IL-1alpha, suggesting that HLE inhibited a CD40-dependent cell activation. These results suggest that HLE possesses an anti-inflammatory effect for the HGF-mediated inflammatory process.
Catalysis of the hydrolysis of internal, alpha-peptide bonds in a polypeptide chain by a catalytic mechanism that involves a catalytic triad consisting of a serine nucleophile that is activated by a proton relay involving an acidic residue (e.g. aspartate or glutamate) and a basic residue (usually histidine).
Circulating human neutrophils from patients with severe inflammatory disorders such as erysipelas and sepsis are specifically desensitized to complement factor C5a stimulation but not to stimulation with other stimuli like N-formyl-methionyl-leucyl-phenylalanine (FMLP), interleukin-8 (IL-8), leukotriene B4 (LTB4), or platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). In this study, we raised the question whether factors released from polymorphonuclear leukocytes (PMNs) can specifically down-regulate C5a-dependent neutrophil functions. When neutrophils were preincubated with either neutrophil lysates or neutrophil degranulation supernatants, a complete inhibition of C5a-stimulated beta-glucuronidase release and chemotaxis could be observed, whereas FMLP-, IL-8-, LTB4- or PAF-dependent functions were not affected. Serine protease inhibitors like phenylmethylsulfonyl fluoride, antileukoprotease, or elafin abolished this effect. High-performance liquid chromatography of neutrophil degranulation supernatants revealed pronounced inhibition of C5a-dependent neutrophil functions in fractions exerting elastase or cathepsin G activity, but not in fractions exerting proteinase 3 activity. Using purified human leukocyte elastase (HLE), C5a responses like intracellular calcium influx, beta-glucuronidase release, and chemotaxis were also specifically inhibited. Our experiments show that the release of HLE or cathepsin G from neutrophils specifically down-regulates the responsiveness of neutrophils to C5a. Elastase and cathepsin G may therefore play an important role in the down-regulation of acute inflammation.
Any process that stops, prevents, or reduces the frequency, rate or extent of the chemical reactions and pathways resulting in the formation of chemokines.
J. Leukoc. Biol. 72, 538-545 (2002)[PubMed:12223522]
CD40 is a crucial element in the process of fibroblast activation. We demonstrated that treatment of human gingival fibroblast (HGF) with human leukocyte elastase (HLE), a neutrophil serine protease, down-regulated the expression of CD40 and binding to the CD40 ligand (CD40L) using flow cytometry. The other neutrophil serine proteases, cathepsin G and proteinase 3, exhibited markedly less activity for CD40 reduction. The CD40 reduction by HLE was also observed in skin and lung fibroblasts, but not in monocytes, macrophages, and dendritic cells. The reduction resulted from direct proteolysis by HLE on the cell surface, because HLE reduced CD40 on fixed HGF and also on cell lysates and membranes. HLE treatment of HGF decreases interleukin (IL)-8 and macrophage chemoattractant protein-1 production by HGF when stimulated by CD40L, but not by IL-1alpha, suggesting that HLE inhibited a CD40-dependent cell activation. These results suggest that HLE possesses an anti-inflammatory effect for the HGF-mediated inflammatory process.
Any process that stops, prevents, or reduces the frequency, rate or extent of the directed movement of a motile cell or organism in response to a specific chemical concentration gradient.
Circulating human neutrophils from patients with severe inflammatory disorders such as erysipelas and sepsis are specifically desensitized to complement factor C5a stimulation but not to stimulation with other stimuli like N-formyl-methionyl-leucyl-phenylalanine (FMLP), interleukin-8 (IL-8), leukotriene B4 (LTB4), or platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). In this study, we raised the question whether factors released from polymorphonuclear leukocytes (PMNs) can specifically down-regulate C5a-dependent neutrophil functions. When neutrophils were preincubated with either neutrophil lysates or neutrophil degranulation supernatants, a complete inhibition of C5a-stimulated beta-glucuronidase release and chemotaxis could be observed, whereas FMLP-, IL-8-, LTB4- or PAF-dependent functions were not affected. Serine protease inhibitors like phenylmethylsulfonyl fluoride, antileukoprotease, or elafin abolished this effect. High-performance liquid chromatography of neutrophil degranulation supernatants revealed pronounced inhibition of C5a-dependent neutrophil functions in fractions exerting elastase or cathepsin G activity, but not in fractions exerting proteinase 3 activity. Using purified human leukocyte elastase (HLE), C5a responses like intracellular calcium influx, beta-glucuronidase release, and chemotaxis were also specifically inhibited. Our experiments show that the release of HLE or cathepsin G from neutrophils specifically down-regulates the responsiveness of neutrophils to C5a. Elastase and cathepsin G may therefore play an important role in the down-regulation of acute inflammation.
Circulating human neutrophils from patients with severe inflammatory disorders such as erysipelas and sepsis are specifically desensitized to complement factor C5a stimulation but not to stimulation with other stimuli like N-formyl-methionyl-leucyl-phenylalanine (FMLP), interleukin-8 (IL-8), leukotriene B4 (LTB4), or platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). In this study, we raised the question whether factors released from polymorphonuclear leukocytes (PMNs) can specifically down-regulate C5a-dependent neutrophil functions. When neutrophils were preincubated with either neutrophil lysates or neutrophil degranulation supernatants, a complete inhibition of C5a-stimulated beta-glucuronidase release and chemotaxis could be observed, whereas FMLP-, IL-8-, LTB4- or PAF-dependent functions were not affected. Serine protease inhibitors like phenylmethylsulfonyl fluoride, antileukoprotease, or elafin abolished this effect. High-performance liquid chromatography of neutrophil degranulation supernatants revealed pronounced inhibition of C5a-dependent neutrophil functions in fractions exerting elastase or cathepsin G activity, but not in fractions exerting proteinase 3 activity. Using purified human leukocyte elastase (HLE), C5a responses like intracellular calcium influx, beta-glucuronidase release, and chemotaxis were also specifically inhibited. Our experiments show that the release of HLE or cathepsin G from neutrophils specifically down-regulates the responsiveness of neutrophils to C5a. Elastase and cathepsin G may therefore play an important role in the down-regulation of acute inflammation.
Any process that stops, prevents, or reduces the frequency, rate or extent of the chemical reactions and pathways resulting in the formation of interleukin-8.
J. Leukoc. Biol. 72, 538-545 (2002)[PubMed:12223522]
CD40 is a crucial element in the process of fibroblast activation. We demonstrated that treatment of human gingival fibroblast (HGF) with human leukocyte elastase (HLE), a neutrophil serine protease, down-regulated the expression of CD40 and binding to the CD40 ligand (CD40L) using flow cytometry. The other neutrophil serine proteases, cathepsin G and proteinase 3, exhibited markedly less activity for CD40 reduction. The CD40 reduction by HLE was also observed in skin and lung fibroblasts, but not in monocytes, macrophages, and dendritic cells. The reduction resulted from direct proteolysis by HLE on the cell surface, because HLE reduced CD40 on fixed HGF and also on cell lysates and membranes. HLE treatment of HGF decreases interleukin (IL)-8 and macrophage chemoattractant protein-1 production by HGF when stimulated by CD40L, but not by IL-1alpha, suggesting that HLE inhibited a CD40-dependent cell activation. These results suggest that HLE possesses an anti-inflammatory effect for the HGF-mediated inflammatory process.
Any process that activates or increases the frequency, rate or extent of the chemical reactions and pathways resulting in the formation of interleukin-8.
The sequestration of neutrophils in the lung and the release of proinflammatory mediators, including neutrophil elastase, are responsible for sepsis-induced microvascular permeability and alveolar epithelial cell damage. To assess the underlying mechanism, human neutrophil elastase (0.01-0.5 microg/ml) was added to cultured A549 epithelial cells in the presence or absence of inhibitors. IL-8 was analyzed by ELISA or by RT-PCR to measure the IL-8 synthesis capacity. Mitogen-activated protein kinase (MAPK) activity was detected by Western blot analysis. Neutrophil elastase dose-dependently increased IL-8 release from cultured A549 epithelial cells. Pretreatment with a specific elastase inhibitor, elastase inhibitor II (at 0.5, 5, and 50 microg/ml), dose-dependently inhibited neutrophil elastase-induced IL-8 release. The activities of MAPK, p38, and extracellular signal-regulated kinase (ERK) were upregulated by neutrophil elastase. Nuclear transcriptional factor-kappa B (NF-kappaB) and activator protein 1 (AP-1) were also activated. These responses were significantly inhibited by elastase inhibitor II. A specific inhibitor of p38 MAPK (SB203580) and an NF-kappaB inhibitor (pyrrolidine dithiocarbamate), but not an ERK inhibitor (PD 98059), significantly inhibited neutrophil elastase-induced IL-8 release and mRNA expression. The specific tyrosine kinase inhibitor, genistein, and the protein kinase C (PKC) inhibitor, Ro 31-8220, also inhibited IL-8 release and mRNA expression as well as p38 and NF-kappaB activation. There was no significant effect by the protein kinase A inhibitor, H-89, on neutrophil elastase-induced IL-8 synthesis or p38 MAPK activation. Our results indicate that neutrophil elastase activates p38 MAPK which upregulates NF-kappaB and AP-1 activities, thus inducing IL-8 mRNA expression and protein synthesis. Tyrosine kinase and PKC are implicated in neutrophil elastase activation of the MAPK pathway.
The sequestration of neutrophils in the lung and the release of proinflammatory mediators, including neutrophil elastase, are responsible for sepsis-induced microvascular permeability and alveolar epithelial cell damage. To assess the underlying mechanism, human neutrophil elastase (0.01-0.5 microg/ml) was added to cultured A549 epithelial cells in the presence or absence of inhibitors. IL-8 was analyzed by ELISA or by RT-PCR to measure the IL-8 synthesis capacity. Mitogen-activated protein kinase (MAPK) activity was detected by Western blot analysis. Neutrophil elastase dose-dependently increased IL-8 release from cultured A549 epithelial cells. Pretreatment with a specific elastase inhibitor, elastase inhibitor II (at 0.5, 5, and 50 microg/ml), dose-dependently inhibited neutrophil elastase-induced IL-8 release. The activities of MAPK, p38, and extracellular signal-regulated kinase (ERK) were upregulated by neutrophil elastase. Nuclear transcriptional factor-kappa B (NF-kappaB) and activator protein 1 (AP-1) were also activated. These responses were significantly inhibited by elastase inhibitor II. A specific inhibitor of p38 MAPK (SB203580) and an NF-kappaB inhibitor (pyrrolidine dithiocarbamate), but not an ERK inhibitor (PD 98059), significantly inhibited neutrophil elastase-induced IL-8 release and mRNA expression. The specific tyrosine kinase inhibitor, genistein, and the protein kinase C (PKC) inhibitor, Ro 31-8220, also inhibited IL-8 release and mRNA expression as well as p38 and NF-kappaB activation. There was no significant effect by the protein kinase A inhibitor, H-89, on neutrophil elastase-induced IL-8 synthesis or p38 MAPK activation. Our results indicate that neutrophil elastase activates p38 MAPK which upregulates NF-kappaB and AP-1 activities, thus inducing IL-8 mRNA expression and protein synthesis. Tyrosine kinase and PKC are implicated in neutrophil elastase activation of the MAPK pathway.
Neutrophils and their derived elastase are abundant in chronic inflammatory responses of asthma. This study aimed to investigate the mitogenic effect of elastase on airway smooth muscle (ASM) cells and the implicated signal transduction pathway. Near confluent cultured human ASM cells were treated with human neutrophil elastase (HNE, 0.01 to 0.5 microg/ml) or vehicle for 24 hours with or without extracellular signal-regulated kinase (ERK) inhibitor (PD98059, 30 microM), p38 kinase inhibitor (SB203580, 10 microM) or elastase inhibitor II (100 microg/ml). The ASM cell numbers were counted by a hemocytometer and DNA synthesis was assessed by flowcytometry. Western blots analysis for the expression of ERK, p38 and cyclin D1 was determined. HNE dose-dependently increased ASM cell numbers and the percentage of cells entering S-phase of cell cycle. This response was abolished by neutrophil elastase inhibitors and attenuated by PD98059, but not SB203580. HNE increased ERK phosphorylation and cyclin D1 expression. Pretreatment with PD98059 significantly inhibited elastase-induced cyclin D1 activity. The increased ASM cellular gap and cell shape change by proteolytic activity of HNE may be contributory to ERK activation and therefore cell proliferation. Our results demonstrate that HNE is mitogenic for ASM cells by increasing cyclin D1 activity through ERK signaling pathway.
The chemical reactions and pathways resulting in the breakdown of a protein by the destruction of the native, active configuration, with or without the hydrolysis of peptide bonds.
Components of kinin-forming systems operating at inflammatory sites are likely to interact with elastase that is released by recruited neutrophils and may, at least temporarily, constitute the major proteolytic activity present at these sites. The aim of this work was to determine the effect of kininogen degradation by human neutrophil elastase (HNE) on kinin generation by tissue and plasma kallikreins. We show that the digestion of both low molecular mass (LK) and high molecular mass (HK) forms of human kininogen by HNE renders them essentially unsusceptible to processing by human urinary kallikrein (tissue-type) and also significantly quenches the kinin release from HK by plasma kallikrein. Studies with synthetic model heptadecapeptide substrates, ISLMKRPPGFSPFRSSR and SLMKRPPGFSPFRSSRI, confirmed the inability of tissue kallikrein to process peptides at either termini of the internal kinin sequence, while plasma kallikrein was shown to release the kinin C-terminus relatively easily. The HNE-generated fragments of kininogens were separated by HPLC and the fractions containing internal kinin sequences were identified by a kinin-specific immunoenzymatic test after trypsin digestion. These fractions were analyzed by electrospray-ionization mass spectrometry. In this way, multiple peptides containing the kinin sequence flanked by only a few amino acid residues at each terminus were identified in elastase digests of both LK and HK. These results suggest that elastase may be involved in quenching the kinin-release cascade at the late stages of the inflammatory reaction.
According to the hitherto accepted view, neutrophils kill ingested microorganisms by subjecting them to high concentrations of highly toxic reactive oxygen species (ROS) and bringing about myeloperoxidase-catalysed halogenation. We show here that this simple scheme, which for many years has served as a satisfactory working hypothesis, is inadequate. We find that mice made deficient in neutrophil-granule proteases but normal in respect of superoxide production and iodinating capacity, are unable to resist staphylococcal and candidal infections. We also show that activation provokes the influx of an enormous concentration of ROS into the endocytic vacuole. The resulting accumulation of anionic charge is compensated for by a surge of K+ ions that cross the membrane in a pH-dependent manner. The consequent rise in ionic strength engenders the release of cationic granule proteins, including elastase and cathepsin G, from the anionic sulphated proteoglycan matrix. We show that it is the proteases, thus activated, that are primarily responsible for the destruction of the bacteria.
Any process that results in a change in state or activity of a cell or an organism (in terms of movement, secretion, enzyme production, gene expression, etc.) as a result of an ultraviolet radiation (UV light) stimulus. Ultraviolet radiation is electromagnetic radiation with a wavelength in the range of 10 to 380 nanometers.
Solar elastosis is characterized by accumulation of large amounts of material staining similarly to elastin in the dermis. The nature of this material and the process responsible for its accumulation are still unknown. Elastolytic proteases have important functions in the catabolism of the interstitial matrix and can also generate, by the digestion of the interstitial proteins, soluble peptides which can induce collagen and elastin synthesis and deposition. We investigated whether (i) elastolytic enzymes can be detected in samples from sun-exposed and non-exposed skin, and (ii) ultraviolet (UV) rays influence the production of elastolytic activities in cultured dermal fibroblasts. Immunoelectron microscopy showed a positive reaction for neutrophil elastase and cathepsin G in fibroblast-like cells from specimens of sun-exposed areas. Little or no reaction was found in biopsies of sun-protected skin. Fibroblast cultures from sun-exposed skin expressed higher levels of hydrolytic activity against synthetic substrates of elastases and cathepsin G than those obtained from sun-protected areas. Irradiation with UVA strongly stimulated the production of these activities in fibroblasts from sun-protected sites. No significant change was detected in parallel sets of cultures after UVB irradiation. Inhibition experiments indicated that the elastase-like activity expressed by fibroblasts can be attributed to at least two enzymes.
Enzyme which catalyzes hydrolysis reaction, i.e. the addition of the hydrogen and hydroxyl ions of water to a molecule with its consequent splitting into two or more simpler molecules.
Proteolytic enzyme with a serine residue (Ser) in its active site. The reactivity of the serine residue is ensured by the vicinity of a histidine and an aspartate residue (catalytic triad), all three residues are required for the charge relay system to take place.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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