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• W2052257525 abstract "Elafin is a serine protease inhibitor produced by epithelial and immune cells with anti-inflammatory properties. Research has shown that dysregulated protease activity may elicit proteolytic cleavage of elafin, thereby impairing the innate immune function of the protein. The aim of this study was to generate variants of elafin (GG- and QQ-elafin) that exhibit increased protease resistance while retaining the biological properties of wild-type (WT) elafin. Similar to WT-elafin, GG- and QQ-elafin variants retained antiprotease activity and susceptibility to transglutaminase-mediated fibronectin cross-linking. However, in contrast to WT-elafin, GG- and QQ-elafin displayed significantly enhanced resistance to degradation when incubated with bronchoalveolar lavage fluid from patients with cystic fibrosis. Intriguingly, both variants, particularly GG-elafin, demonstrated improved lipopolysaccharide (LPS) neutralization properties in vitro. In addition, GG-elafin showed improved anti-inflammatory activity in a mouse model of LPS-induced acute lung inflammation. Inflammatory cell infiltration into the lung was reduced in lungs of mice treated with GG-elafin, predominantly neutrophilic infiltration. A reduction in MCP-1 levels in GG-elafin treated mice compared to the LPS alone treatment group was also demonstrated. GG-elafin showed increased functionality when compared to WT-elafin and may be of future therapeutic relevance in the treatment of lung diseases characterized by a protease burden. Elafin is a serine protease inhibitor produced by epithelial and immune cells with anti-inflammatory properties. Research has shown that dysregulated protease activity may elicit proteolytic cleavage of elafin, thereby impairing the innate immune function of the protein. The aim of this study was to generate variants of elafin (GG- and QQ-elafin) that exhibit increased protease resistance while retaining the biological properties of wild-type (WT) elafin. Similar to WT-elafin, GG- and QQ-elafin variants retained antiprotease activity and susceptibility to transglutaminase-mediated fibronectin cross-linking. However, in contrast to WT-elafin, GG- and QQ-elafin displayed significantly enhanced resistance to degradation when incubated with bronchoalveolar lavage fluid from patients with cystic fibrosis. Intriguingly, both variants, particularly GG-elafin, demonstrated improved lipopolysaccharide (LPS) neutralization properties in vitro. In addition, GG-elafin showed improved anti-inflammatory activity in a mouse model of LPS-induced acute lung inflammation. Inflammatory cell infiltration into the lung was reduced in lungs of mice treated with GG-elafin, predominantly neutrophilic infiltration. A reduction in MCP-1 levels in GG-elafin treated mice compared to the LPS alone treatment group was also demonstrated. GG-elafin showed increased functionality when compared to WT-elafin and may be of future therapeutic relevance in the treatment of lung diseases characterized by a protease burden. Mature elafin is a 6 kDa serine protease inhibitor largely generated via proteolytic processing of trappin-2 (pre-elafin), primarily by the mast cell-derived protease tryptase.1Guyot N Zani ML Berger P Dallet-Choisy S Moreau T Proteolytic susceptibility of the serine protease inhibitor trappin-2 (pre-elafin): evidence for tryptase-mediated generation of elafin.Biol Chem. 2005; 386: 391-399Crossref PubMed Scopus (40) Google Scholar,2Schalkwijk J Wiedow O Hirose S The trappin gene family: proteins defined by an N-terminal transglutaminase substrate domain and a C-terminal four-disulphide core.Biochem J. 1999; 340: 569-577Crossref PubMed Scopus (171) Google Scholar Elafin was originally isolated from human bronchial secretions in 1985 by Kramps and Klasen3Kramps JA Klasen EC Characterization of a low molecular weight anti-elastase isolated from human bronchial secretion.Exp Lung Res. 1985; 9: 151-165Crossref PubMed Scopus (24) Google Scholar under the name of LMI-5000 and subsequently by Schalkwijk et al.4Schalkwijk J Chang A Janssen P De Jongh GJ Mier PD Skin-derived antileucoproteases (SKALPs): characterization of two new elastase inhibitors from psoriatic epidermis.Br J Dermatol. 1990; 122: 631-641Crossref PubMed Scopus (97) Google Scholar and Wiedow et al.5Wiedow O Schröder JM Gregory H Young JA Christophers E Elafin: an elastase-specific inhibitor of human skin. Purification, characterization, and complete amino acid sequence.J Biol Chem. 1990; 265: 14791-14795Abstract Full Text PDF PubMed Google Scholar in 1990 from human psoriatic skin under the name SKALP and elafin, respectively. In addition, elafin has also been reported to be expressed by macrophages and neutrophils.6Mihaila A Tremblay GM Human alveolar macrophages express elafin and secretory leukocyte protease inhibitor.Z Naturforsch C. 2001; 56: 291-297Crossref PubMed Scopus (41) Google Scholar Work to date has shown that elafin acts as a multifunctional host defence protein with antimicrobial, antiprotease, and immunomodulatory properties.7Simpson AJ Maxwell AI Govan JR Haslett C Sallenave JM Elafin (elastase-specific inhibitor) has anti-microbial activity against gram-positive and gram-negative respiratory pathogens.FEBS Lett. 1999; 452: 309-313Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar,8Zani ML Baranger K Guyot N Dallet-Choisy S Moreau T Protease inhibitors derived from elafin and SLPI and engineered to have enhanced specificity towards neutrophil serine proteases.Protein Sci. 2009; 18: 579-594PubMed Google Scholar,9Butler MW Robertson I Greene CM O’Neill SJ Taggart CC McElvaney NG Elafin prevents lipopolysaccharide-induced AP-1 and NF-kappaB activation via an effect on the ubiquitin-proteasome pathway.J Biol Chem. 2006; 281: 34730-34735Crossref PubMed Scopus (64) Google Scholar,10Baranger K Zani ML Chandenier J Dallet-Choisy S Moreau T The antibacterial and antifungal properties of trappin-2 (pre-elafin) do not depend on its protease inhibitory function.FEBS J. 2008; 275: 2008-2020Crossref PubMed Scopus (86) Google Scholar,11Williams SE Brown TI Roghanian A Sallenave JM SLPI and elafin: one glove, many fingers.Clin Sci (Lond). 2006; 110: 21-35Crossref PubMed Scopus (224) Google Scholar,12Scott A Weldon S Taggart CC SLPI and elafin: multifunctional antiproteases of the WFDC family.Biochem Soc Trans. 2011; 39: 1437-1440Crossref PubMed Scopus (76) Google Scholar Elafin expression is upregulated at various sites of inflammation by a range of proinflammatory mediators6Mihaila A Tremblay GM Human alveolar macrophages express elafin and secretory leukocyte protease inhibitor.Z Naturforsch C. 2001; 56: 291-297Crossref PubMed Scopus (41) Google Scholar,13Pfundt R Wingens M Bergers M Zweers M Frenken M Schalkwijk J TNF-alpha and serum induce SKALP/elafin gene expression in human keratinocytes by a p38 MAP kinase-dependent pathway.Arch Dermatol Res. 2000; 292: 180-187Crossref PubMed Scopus (80) Google Scholar,14Sallenave JM Shulmann J Crossley J Jordana M Gauldie J Regulation of secretory leukocyte proteinase inhibitor (SLPI) and elastase-specific inhibitor (ESI/elafin) in human airway epithelial cells by cytokines and neutrophilic enzymes.Am J Respir Cell Mol Biol. 1994; 11: 733-741Crossref PubMed Scopus (274) Google Scholar,15Bingle L Tetley TD Bingle CD Cytokine-mediated induction of the human elafin gene in pulmonary epithelial cells is regulated by nuclear factor-kappaB.Am J Respir Cell Mol Biol. 2001; 25: 84-91Crossref PubMed Scopus (57) Google Scholar and may provide protection against neutrophilic proteases.16Pfundt R van Ruissen F van Vlijmen-Willems IM Alkemade HA Zeeuwen PL Jap PH et al.Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.J Clin Invest. 1996; 98: 1389-1399Crossref PubMed Scopus (137) Google Scholar Elafin is recognized as a potent inhibitor of a restricted set of serine proteases, namely neutrophil elastase (NE) and proteinase-3 (PR3).4Schalkwijk J Chang A Janssen P De Jongh GJ Mier PD Skin-derived antileucoproteases (SKALPs): characterization of two new elastase inhibitors from psoriatic epidermis.Br J Dermatol. 1990; 122: 631-641Crossref PubMed Scopus (97) Google Scholar,5Wiedow O Schröder JM Gregory H Young JA Christophers E Elafin: an elastase-specific inhibitor of human skin. Purification, characterization, and complete amino acid sequence.J Biol Chem. 1990; 265: 14791-14795Abstract Full Text PDF PubMed Google Scholar,17Schalkwijk J de Roo C de Jongh GJ Skin-derived antileukoproteinase (SKALP), an elastase inhibitor from human keratinocytes. Purification and biochemical properties.Biochim Biophys Acta. 1991; 1096: 148-154Crossref PubMed Scopus (67) Google Scholar,18Wiedow O Lüademann J Utecht B Elafin is a potent inhibitor of proteinase 3.Biochem Biophys Res Commun. 1991; 174: 6-10Crossref PubMed Scopus (113) Google Scholar Trappin-2 and elafin have a number of transglutaminase (TG) reactive residues and therefore can be linked covalently to various extracellular matrix proteins such as fibronectin by tissue transglutaminases and retain potent antiprotease activity.19Guyot N Zani ML Maurel MC Dallet-Choisy S Moreau T Elafin and its precursor trappin-2 still inhibit neutrophil serine proteinases when they are covalently bound to extracellular matrix proteins by tissue transglutaminase.Biochemistry. 2005; 44: 15610-15618Crossref PubMed Scopus (54) Google Scholar,20Nara K Ito S Ito T Suzuki Y Ghoneim MA Tachibana S et al.Elastase inhibitor elafin is a new type of proteinase inhibitor which has a transglutaminase-mediated anchoring sequence termed “cementoin”.J Biochem. 1994; 115: 441-448PubMed Google Scholar,21Molhuizen HO Alkemade HA Zeeuwen PL de Jongh GJ Wieringa B Schalkwijk J SKALP/elafin: an elastase inhibitor from cultured human keratinocytes. Purification, cDNA sequence, and evidence for transglutaminase cross-linking.J Biol Chem. 1993; 268: 12028-12032Abstract Full Text PDF PubMed Google Scholar,22Baranger K Zani ML Labas V Dallet-Choisy S Moreau T Secretory leukocyte protease inhibitor (SLPI) is, like its homologue trappin-2 (pre-elafin), a transglutaminase substrate.PLoS One. 2011; 6: e20976Crossref PubMed Scopus (19) Google Scholar Due to its cationic nature, it is postulated that trappin-2 displays antibacterial properties via disruption of bacterial cell membranes.7Simpson AJ Maxwell AI Govan JR Haslett C Sallenave JM Elafin (elastase-specific inhibitor) has anti-microbial activity against gram-positive and gram-negative respiratory pathogens.FEBS Lett. 1999; 452: 309-313Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar,10Baranger K Zani ML Chandenier J Dallet-Choisy S Moreau T The antibacterial and antifungal properties of trappin-2 (pre-elafin) do not depend on its protease inhibitory function.FEBS J. 2008; 275: 2008-2020Crossref PubMed Scopus (86) Google Scholar In addition, it has been demonstrated that trappin-2 and mature elafin can bind and neutralize lipopolysaccharide (LPS)23McMichael JW Roghanian A Jiang L Ramage R Sallenave JM The antimicrobial antiproteinase elafin binds to lipopolysaccharide and modulates macrophage responses.Am J Respir Cell Mol Biol. 2005; 32: 443-452Crossref PubMed Scopus (54) Google Scholar,24Guyot N Butler MW McNally P Weldon S Greene CM Levine RL et al.Elafin, an elastase-specific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis.J Biol Chem. 2008; 283: 32377-32385Crossref PubMed Scopus (71) Google Scholar thereby suppressing macrophage TNF-α production.23McMichael JW Roghanian A Jiang L Ramage R Sallenave JM The antimicrobial antiproteinase elafin binds to lipopolysaccharide and modulates macrophage responses.Am J Respir Cell Mol Biol. 2005; 32: 443-452Crossref PubMed Scopus (54) Google Scholar In human monocytic cells, mature elafin inhibited LPS-induced production of monocyte chemotactic protein-1 (MCP-1) and activation of both activator protein-1 (AP-1) and nuclear factor κB (NF-κB) via disruptions to the ubiquitin proteasome pathway.9Butler MW Robertson I Greene CM O’Neill SJ Taggart CC McElvaney NG Elafin prevents lipopolysaccharide-induced AP-1 and NF-kappaB activation via an effect on the ubiquitin-proteasome pathway.J Biol Chem. 2006; 281: 34730-34735Crossref PubMed Scopus (64) Google Scholar In the healthy lung, antiproteases such as trappin-2/elafin are present providing the lung with a powerful anti-inflammatory screen. However, in diseases such as adult respiratory distress syndrome (ARDS), chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF), this protease–antiprotease balance is tipped in favor of proteases leading to dysregulated extracellular protease activity resulting in lung damage. A number of pathogen and host proteases have the ability to cleave elafin.25Guyot N Bergsson G Butler MW Greene CM Weldon S Kessler E et al.Functional study of elafin cleaved by Pseudomonas aeruginosa metalloproteinases.Biol Chem. 2010; 391: 705-716Crossref PubMed Google Scholar,26Kerrin A Weldon S Chung AH Craig T Simpson AJ O’Kane CM et al.Proteolytic cleavage of elafin by 20S proteasome may contribute to inflammation in acute lung injury.Thorax. 2013; 68: 315-321Crossref PubMed Scopus (13) Google Scholar,27Kantyka T Latendorf T Wiedow O Bartels J Gläser R Dubin G et al.Elafin is specifically inactivated by RgpB from Porphyromonas gingivalis by distinct proteolytic cleavage.Biol Chem. 2009; 390: 1313-1320Crossref PubMed Scopus (24) Google Scholar,28Brown A Farmer K MacDonald L Kalsheker N Pritchard D Haslett C et al.House dust mite Der p 1 downregulates defenses of the lung by inactivating elastase inhibitors.Am J Respir Cell Mol Biol. 2003; 29: 381-389Crossref PubMed Scopus (82) Google Scholar Increased NE activity is present in various respiratory diseases including ARDS, COPD, and CF and may overwhelm the protective antiprotease levels within the lung. Indeed, we have found evidence of proteolytic cleavage of elafin by NE in patients with CF with established Pseudomonas aeruginosa infection.24Guyot N Butler MW McNally P Weldon S Greene CM Levine RL et al.Elafin, an elastase-specific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis.J Biol Chem. 2008; 283: 32377-32385Crossref PubMed Scopus (71) Google Scholar Elevated NE levels during Pseudomonas infection in the CF lung also leads to cleavage and inactivation of the related antiprotease secretory leucoprotease inhibitor.29Weldon S McNally P McElvaney NG Elborn JS McAuley DF Wartelle J et al.Decreased levels of secretory leucoprotease inhibitor in the Pseudomonas-infected cystic fibrosis lung are due to neutrophil elastase degradation.J Immunol. 2009; 183: 8148-8156Crossref PubMed Scopus (90) Google Scholar An imbalance in NE and trappin-2/elafin levels has also been reported in ARDS and COPD patients with secondary bacterial infections.26Kerrin A Weldon S Chung AH Craig T Simpson AJ O’Kane CM et al.Proteolytic cleavage of elafin by 20S proteasome may contribute to inflammation in acute lung injury.Thorax. 2013; 68: 315-321Crossref PubMed Scopus (13) Google Scholar,30Wang Z Chen F Zhai R Zhang L Su L Lin X et al.Plasma neutrophil elastase and elafin imbalance is associated with acute respiratory distress syndrome (ARDS) development.PLoS One. 2009; 4: e4380Crossref PubMed Scopus (54) Google Scholar,31Mallia P Footitt J Sotero R Jepson A Contoli M Trujillo-Torralbo MB et al.Rhinovirus infection induces degradation of antimicrobial peptides and secondary bacterial infection in chronic obstructive pulmonary disease.Am J Respir Crit Care Med. 2012; 186: 1117-1124Crossref PubMed Scopus (190) Google Scholar In early 2013, the US Food and Drug Administration (FDA) granted elafin orphan drug designation for the prevention of inflammatory complications associated with transthoracic esophagectomy.32Shaw L Wiedow O Therapeutic potential of human elafin.Biochem Soc Trans. 2011; 39: 1450-1454Crossref PubMed Scopus (50) Google Scholar However, proteolytic cleavage of elafin could attentuate its anti-inflammatory and antiprotease functions and limit the efficacy of elafin in clinical trials in conditions such as ARDS, CF, and COPD. To address this, we have synthesized novel variants of elafin, and we hypothesize that these variants will be more resistant to NE cleavage and therefore of significant utility in the treatment of pulmonary inflammation in diseases characterized by a NE burden. As shown in Figure 1, mutations to the coding sequence for mature elafin, as denoted by the arrows positioned on the WT-elafin sequence, were made at the two previously described NE cleavage sites in order to generate the GG- and QQ-elafin variants.24Guyot N Butler MW McNally P Weldon S Greene CM Levine RL et al.Elafin, an elastase-specific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis.J Biol Chem. 2008; 283: 32377-32385Crossref PubMed Scopus (71) Google Scholar For GG-elafin, the codons coding for valines at position 5 and 9 of the elafin amino acid sequence were mutated to generate glycine residues. For QQ-elafin, the codons coding for valines at position 5 and 9 of the elafin amino acid sequence were mutated to generate glutamine residues. These residues were selected as elastase cleavage after glycine and glutamine is rare (MEROPS database). Neither amino acid substitution had any effect on the isolelectric point and net charge of the protein. As shown in Table 1, the Ki of both mutated elafin variants (GG and QQ) against human NE and PR3 were very closely matched to that of WT-elafin. Similarly, addition of equimolar amounts of each elafin molecule (WT, GG, and QQ) to NE resulted in a significant reduction (P < 0.01) in the turnover of NE substrate compared to NE alone (data not shown) indicating effective 1:1 stoichiometric inhibition of NE by all three elafin proteins.Table 1Comparison of the antiprotease activity of elafin variantsNeutrophil elastase K1 (mol/l)Proteinase 3 Ki. (mol/l)WT-elafin8 × 10-111.2 × 10-10GG-elafin2 × 10-115.25 × 10-10QQ-elafin3 × 10-115.1 × 10-10WT, wild type. Open table in a new tab WT, wild type. To compare the proteolytic susceptibility of GG- and QQ-elafin to WT-elafin, we incubated the three elafin proteins with pooled Pseudomonas-positive CF bronchoalveolar lavage fluid (BALF) over a time period of 0, 2, and 8 hours, and the cleavage products were assessed by western blot analysis (Figure 2a). WT-elafin was rapidly cleaved by CF BALF as denoted by the presence of a double band at 2 hours which was almost completely degraded by 8 hours. In contrast, GG-elafin was completely resistant to cleavage by Pseudomonas-positive CF BALF even after 8 hours of incubation. There was some evidence of cleavage of QQ-elafin by CF BALF, although there was still a significant portion of intact QQ-elafin present after 8 hours. The susceptibility of the elafin variants to proteolysis by NE was also compared (Figure 2b). Similar to the BALF results in Figure 2a, WT-elafin was rapidly cleaved by NE, whereas GG- and QQ-elafin remain relatively intact over the course of the experiment. However, following the 60 minutes of incubation, GG-elafin exhibited enhanced resistance to cleavage when compared to QQ- and WT-elafin. Overall, these data indicate that the GG-elafin variant is more resistant to proteolytic cleavage when compared to QQ- and WT-elafin. Elafin can interact with the LPS of Gram-negative bacteria and modulate cellular responses after LPS stimulation.9Butler MW Robertson I Greene CM O’Neill SJ Taggart CC McElvaney NG Elafin prevents lipopolysaccharide-induced AP-1 and NF-kappaB activation via an effect on the ubiquitin-proteasome pathway.J Biol Chem. 2006; 281: 34730-34735Crossref PubMed Scopus (64) Google Scholar,23McMichael JW Roghanian A Jiang L Ramage R Sallenave JM The antimicrobial antiproteinase elafin binds to lipopolysaccharide and modulates macrophage responses.Am J Respir Cell Mol Biol. 2005; 32: 443-452Crossref PubMed Scopus (54) Google Scholar,24Guyot N Butler MW McNally P Weldon S Greene CM Levine RL et al.Elafin, an elastase-specific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis.J Biol Chem. 2008; 283: 32377-32385Crossref PubMed Scopus (71) Google Scholar Therefore, the ability of the elafin variants to bind and neutralize LPS were investigated. As illustrated in Figure 3a, QQ-elafin variant retained the highest LPS-binding ability when compared to the WT- and GG-elafin. GG-elafin also bound to LPS substantially more than the WT-elafin, although not to the same extent as the QQ-elafin variant. These findings suggest that the introduction of mutations in the GG- and QQ-elafin variants enhances the LPS binding activity of both molecules compared to WT-elafin. As mentioned previously, elafin can be cross-linked to extracellular matrix proteins such as fibronectin via the action of TG.19Guyot N Zani ML Maurel MC Dallet-Choisy S Moreau T Elafin and its precursor trappin-2 still inhibit neutrophil serine proteinases when they are covalently bound to extracellular matrix proteins by tissue transglutaminase.Biochemistry. 2005; 44: 15610-15618Crossref PubMed Scopus (54) Google Scholar,20Nara K Ito S Ito T Suzuki Y Ghoneim MA Tachibana S et al.Elastase inhibitor elafin is a new type of proteinase inhibitor which has a transglutaminase-mediated anchoring sequence termed “cementoin”.J Biochem. 1994; 115: 441-448PubMed Google Scholar,21Molhuizen HO Alkemade HA Zeeuwen PL de Jongh GJ Wieringa B Schalkwijk J SKALP/elafin: an elastase inhibitor from cultured human keratinocytes. Purification, cDNA sequence, and evidence for transglutaminase cross-linking.J Biol Chem. 1993; 268: 12028-12032Abstract Full Text PDF PubMed Google Scholar,22Baranger K Zani ML Labas V Dallet-Choisy S Moreau T Secretory leukocyte protease inhibitor (SLPI) is, like its homologue trappin-2 (pre-elafin), a transglutaminase substrate.PLoS One. 2011; 6: e20976Crossref PubMed Scopus (19) Google Scholar GG-elafin exhibited comparable binding to fibronectin when compared to WT-elafin in the presence of the TG (Figure 3b). Furthermore, the QQ-elafin variant demonstrated a significant increase in binding to fibronectin when compared to WT-elafin (P < 0.05). A similar trend was also observed when compared to GG-elafin; however, this was found to be nonsignificant. Peripheral blood monocytes (PBMs) and U937 monocytic cells were pretreated with WT-elafin and each elafin variant (10 μg/ml) prior to LPS stimulation. Secreted IL-8 levels in cell-free supernatants were quantified by enzyme-linked immunosorbent assay (ELISA). PBMs (Figure 4a) and U937s (Figure 4b) pretreated with GG-elafin secreted significantly lower levels of IL-8 compared to LPS alone stimulated controls. Furthermore, although WT-elafin and QQ-elafin decreased LPS-induced IL-8 release from PBMs and U937s, this was not significant suggesting that the GG-elafin variant has augmented anti-inflammatory properties over the parental molecule. Given the preservation of binding capabilities to extracellular matrix proteins and LPS, and also the increased resistance to proteolytic cleavage, GG-elafin was selected for further validation experiments in vivo. Leading on from the in vitro studies which demonstrated significant anti-inflammatory properties of GG-elafin compared to WT-elafin, the effects of WT- and GG-elafin in an in vivo model of LPS-induced acute lung inflammation were investigated (Figure 5). Treatment of mice with WT-elafin resulted in a nonsignificant decrease in inflammatory cell infiltration in response to LPS (Figure 5a,b). However, treatment of mice with GG-elafin resulted in a significant reduction in LPS-induced neutrophil infiltration into the lung when compared to mice treated LPS alone (Figure 5a; P < 0.01). In order to assess alveolar-capillary barrier permeability induced by LPS, total protein concentrations in BALF were quantified. BALF protein levels were decreased in the WT-elafin (not significant) and GG-elafin (Figure 5c; P < 0.01) treated mice when compared to that of the LPS alone treated mice. In agreement with in vitro observations (Figure 4), these findings suggest that GG-elafin has augmented anti-inflammatory activity over the parental WT-elafin molecule. Given the observed effects of elafin on neutrophilic infiltration into the lung, chemokine levels in BALF were investigated (Figure 6). There was no difference in the levels of KC and MIP-2 in either the GG-elafin or the WT-elafin treated mice when compared to the LPS alone treated mice (Figure 6a,b). However, there was a significant difference in BALF MCP-1 levels from mice treated with the GG-elafin compared to those which received the LPS treatment alone as shown in Figure 6c (P < 0.01). In contrast, mice treated with WT-elafin did not exhibit a significant reduction in MCP-1 compared to mice treated with LPS alone (Figure 6c). To investigate the effects of elafin on LPS-induced protease burden, we measured NE activity in BALF. Overall, NE activity was undetectable in the majority of samples. Low turnover of substrate was detected only in a number of the LPS samples (112.5 pmol 7-amino-4-methylcoumarin (AMC)/μg protein ± 42.7 pmol AMC/μg protein), and no activity was detected in the LPS+WT or LPS+GG BAL samples, which suggests that both forms of elafin are able to inhibit elastase activity in vivo. As a surrogate marker of protease activity, we measured levels of endostatin in the BALF from our study by ELISA. As illustrated in Figure 6d, LPS challenge upregulated the levels of endostatin in BALF compared to the saline groups. Although both WT- and GG-elafin inhibited the generation of endostatin in comparison to the LPS alone group, only the GG-elafin was significantly lower versus LPS alone, which correlates well with our neutrophil counts in Figure 6a. Elafin has been previously shown to possess potent inhibitory properties toward the neutrophil serine proteases, NE and PR3. Given its low molecular weight and potent antiprotease activity, it has been postulated that elafin could have potential therapeutic relevance in a number of diseases characterized by a protease burden.16Pfundt R van Ruissen F van Vlijmen-Willems IM Alkemade HA Zeeuwen PL Jap PH et al.Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.J Clin Invest. 1996; 98: 1389-1399Crossref PubMed Scopus (137) Google Scholar,32Shaw L Wiedow O Therapeutic potential of human elafin.Biochem Soc Trans. 2011; 39: 1450-1454Crossref PubMed Scopus (50) Google Scholar However, it has previously been demonstrated by our group that elafin is susceptible to proteolysis by NE in the lungs of CF patients chronically infected with P. aeruginosa.24Guyot N Butler MW McNally P Weldon S Greene CM Levine RL et al.Elafin, an elastase-specific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis.J Biol Chem. 2008; 283: 32377-32385Crossref PubMed Scopus (71) Google Scholar Therefore, the development of a more cleavage-resistant elafin molecule using directed mutagenesis may offer an attractive strategy for the development of elafin as a therapy. The generation of the QQ- and GG-elafin variants in this study confirmed enhanced cleavage resistance over the native WT-elafin molecule when exposed to Pseudomonas-infected CF BALF. Furthermore, the QQ-elafin variant demonstrated a significantly increased affinity for fibronectin in the presence of transglutaminase when compared to WT-elafin. The increased binding affinity of the QQ-elafin is likely due to the introduction of glutamine residues,22Baranger K Zani ML Labas V Dallet-Choisy S Moreau T Secretory leukocyte protease inhibitor (SLPI) is, like its homologue trappin-2 (pre-elafin), a transglutaminase substrate.PLoS One. 2011; 6: e20976Crossref PubMed Scopus (19) Google Scholar but this requires further validation. In addition, we have demonstrated increased LPS binding and a reduction in LPS-induced IL-8 production in monocytic cells preincubated with GG-elafin compared to WT-elafin. These data led us to consider if GG-elafin may have additional anti-inflammatory effects in vivo compared to WT-elafin. With the use of the LPS-induced mouse model of acute lung inflammation, we have demonstrated that the GG-elafin variant possessed enhanced anti-inflammatory characteristics when compared to the parent WT-elafin molecule as demonstrated by the noted reduction in inflammatory cells within the lungs, in particular, neutrophils. It is acknowledged that neutrophilic influx characterizes the early stages of acute pulmonary inflammation and is associated with disruption of the alveolar-capillary barrier and lung tissue damage.33Maus U von Grote K Kuziel WA Mack M Miller EJ Cihak J et al.The role of CC chemokine receptor 2 in alveolar monocyte and neutrophil immigration in intact mice.Am J Respir Crit Care Med. 2002; 166: 268-273Crossref PubMed Scopus (164) Google Scholar,34Maus U Huwe J Maus R Seeger W Lohmeyer J Alveolar JE/MCP-1 and endotoxin synergize to provoke lung cytokine upregulation, sequential neutrophil and monocyte influx, and vascular leakage in mice.Am J Respir Crit Care Med. 2001; 164: 406-411Crossref PubMed Scopus (71) Google Scholar Although a near onefold reduction in neutrophilia was observed in the mice receiving LPS+GG elafin compared to those receiving LPS alone, this may not be sufficient to reduce lung damage in the host. Further studies are required to more carefully evaluate the effect of a onefold reduction perhaps using more advanced in vivo models of lung damage. NE activity as measured using the substrate AAPV-AMC was only detected in a nu" @default.
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• W2052257525 date "2015-01-01" @default.
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• W2052257525 title "A Functional Variant of Elafin With Improved Anti-inflammatory Activity for Pulmonary Inflammation" @default.
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