Cathepsin-G and leukocyte elastase inactivate human tumor necrosis factor and lymphotoxin

doi:10.1016/0008-8749(91)90275-G

Cellular Immunology

Volume 135, Issue 2, July 1991, Pages 299-313

https://doi.org/10.1016/0008-8749(91)90275-G Get rights and content

Abstract

The addition of either cathepsin-G or leukocyte elastase to endotoxin-stimulated human peripheral blood monocytes decreased the immunoreactive tumor necrosis factor (TNF) detected in culture supernatants in a concentration-dependent manner. Both enzymes also induced a loss of supernatant cytolytic activity as determined on the WEHI-164 target cell line. Incubation of recombinant human TNF and lymphotoxin (LT) with either cathepsin-G or leukocyte elastase resulted in a loss of cytokine bioactivity. Examination of enzyme-treated recombinant cytokines by gel electrophoresis revealed that cathepsin-G cleaved LT into a 12.6-kDa fragment and leukocyte elastase fragmented LT into a 14.1-kDa product. On Western blots cathepsin-G and leukocyte elastase degraded TNF into 11- and 7.6-kDa fragments, respectively. Incubating leukocyte elastase with plasma elastase inhibitor α-1-antitrypsin prevented the loss of recombinant TNF bioactivity and blocked the degradation of this cytokine. This study suggests that two of the most abundant neutrophil proteases, cathepsin-G and leukocyte elastase, may be important regulators of TNF and LT bioactivity.

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Neutrophil elastase modulates cytokine expression: Contribution to host defense against pseudomonas aeruginosa-induced pneumonia
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Citation Excerpt :
At any rate, detection of cleaved TLR-4 and active NE (Fig. 1A) suggest that both proteins co-localize in infected lungs and support the hypothesis that NE could target TLR-4 in vivo. A number of in vitro studies have suggested that proteolytic modifications of cytokines by neutrophil serine proteases including NE change their biologic activities (15, 25). In the present work, we show that in the setting of P. aeruginosa lung infection, NE deficiency resulted in changes of protein levels of a variety of cytokines coinciding with increased mortality of mutant mice to infection.
There is accumulating evidence that following bacterial infection, the massive recruitment and activation of the phagocytes, neutrophils, is accompanied with the extracellular release of active neutrophil elastase (NE), a potent serine protease. Using NE-deficient mice in a clinically relevant model of Pseudomonas aeruginosa-induced pneumonia, we provide compelling in vivo evidence that the absence of NE was associated with decreased protein and transcript levels of the proinflammatory cytokines TNF-α, MIP-2, and IL-6 in the lungs, coinciding with increased mortality of mutant mice to infection. The implication of NE in the induction of cytokine expression involved at least in part Toll-like receptor 4 (TLR-4). These findings were further confirmed following exposure of cultured macrophages to purified NE. Together, our data suggest strongly for the first time that NE not only plays a direct antibacterial role as it has been previously reported, but released active enzyme can also modulate cytokine expression, which contributes to host protection against P. aeruginosa. In light of our findings, the long held view that considers NE as a prime suspect in P. aeruginosa-associated diseases will need to be carefully reassessed. Also, therapeutic strategies aiming at NE inhibition should take into account the physiologic roles of the enzyme.
No role for neutrophil elastase in influenza-induced cellular recruitment, cytokine production or airway hyperresponsiveness in mice
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Citation Excerpt :
Whilst all three NSPs are known for their degradative properties and their ability to cause tissue damage, each has the ability to modulate the actions of cytokines in a different manner (Korkmaz et al., 2008). On one hand PR3 is required for the activation of TNF-α (Robache-Gallea et al., 1995) while NE and CG both degrade mature TNF-α (Scuderi et al., 1991). TNF-α is an important inflammatory cytokine and it has been shown that inhibition of TNF-α results in reduced pulmonary inflammation and less severe immunopathology in influenza-infected mice (Hussell et al., 2001; Peper and Van Campen, 1995).
Previous studies have suggested that in vitro modulation of neutrophil chemokines and inflammatory cytokines by neutrophil elastase (NE) does not translate to the in vivo setting. We aimed to test the role of NE in the recruitment of neutrophils, cytokine production and lung function responses to respiratory viral infection. To address this, we inoculated neutrophil elastase (NE^−/−) deficient and wild-type (WT) 129Sv mice with 50 μL of 10^4.5 pfu Influenza A/Mem71 (H3N1) or a control preparation. Mice were subjected to methacholine (MCh) challenge at 3–4 days post-infection during the peak of cellular inflammation. Inflammation, protein content and cytokines (TNF-α and MIP-2) were assessed in bronchoalveolar lavage fluid. Influenza-infected mice had a heightened responsiveness to MCh, increased cellular inflammation, increased protein leak and altered cytokine production, none of which were influenced by the absence of NE. These data demonstrate that NE does not modulate neutrophil recruitment, cytokine production, epithelial permeability or responsiveness to bronchoconstricting agents during acute influenza infection in mice.
Cathepsin G: Roles in antigen presentation and beyond
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Contributions from multiple cathepsins within endosomal antigen processing compartments are necessary to process antigenic proteins into antigenic peptides. Cysteine and aspartyl cathepsins have been known to digest antigenic proteins. A role for the serine protease, cathepsin G (CatG), in this process has been described only recently, although CatG has long been known to be a granule-associated proteolytic enzyme of neutrophils. In line with a role for this enzyme in antigen presentation, CatG is found in endocytic compartments of a variety of antigen presenting cells. CatG is found in primary human monocytes, B cells, myeloid dendritic cells 1 (mDC1), mDC2, plasmacytoid DC (pDC), and murine microglia, but is not expressed in B cell lines or monocyte-derived DC. Purified CatG can be internalized into endocytic compartments in CatG non-expressing cells, widening the range of cells where this enzyme may play a role in antigen processing. Functional assays have implicated CatG as a critical enzyme in processing of several antigens and autoantigens. In this review, historical and recent data on CatG expression, distribution, function and involvement in disease will be summarized and discussed, with a focus on its role in antigen presentation and immune-related events.
Neutrophil serine proteases fine-tune the inflammatory response
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Neutrophil serine proteases are granule-associated enzymes known mainly for their function in the intracellular killing of pathogens. Their extracellular release upon neutrophil activation is traditionally regarded as the primary reason for tissue damage at the sites of inflammation. However, studies over the past several years indicate that neutrophil serine proteases may also be key regulators of the inflammatory response. Neutrophil serine proteases specifically process and release chemokines, cytokines, and growth factors, thus modulating their biological activity. In addition, neutrophil serine proteases activate and shed specific cell surface receptors, which can ultimately prolong or terminate cytokine-induced responses. Moreover, it has been proposed that these proteases can impact cell viability through their caspase-like activity and initiate the adaptive immune response by directly activating lymphocytes. In summary, these studies point to neutrophil serine proteases as versatile mediators that fine-tune the local immune response and identify them as potential targets for therapeutic interventions.
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Neutrophil elastase (NE), a serine protease released from the azurophil granules of activated neutrophil, proteolytically cleaves multiple cytokines, and cell surface proteins. In the present study, we examined whether NE affects the biological abilities of angiogenic growth factors such as basic-fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). NE degraded bFGF and VEGF in a time- and concentration-dependent manner, and these degradations were suppressed by sivelestat, a synthetic inhibitor of NE. The bFGF- or VEGF-mediated proliferative activity of human umbilical vein endothelial cells was inhibited by NE, and the activity was recovered by sivelestat. Furthermore, NE reduced the bFGF- or VEGF-induced tubulogenic response of the mice aortas, ex vivo angiogenesis assay, and these effects were also recovered by sivelestat. Neutrophil-derived NE degraded potent angiogenic factors, resulting in loss of their angiogenic activity. These findings provide additional insight into the role played by neutrophils in the angiogenesis process at sites of inflammation.
Role of protein-tyrosine phosphatase SHP<inf>2</inf> in focal adhesion kinase down-regulation during neutrophil cathepsin G-induced cardiomyocytes anoikis
2006, Journal of Biological Chemistry
Inflammatory cells and their proteases contribute to tissue reparation at site of inflammation. Although beneficial at early stages, excessive inflammatory reaction leads to cell death and tissue damage. Cathepsin G (Cat.G), a neutrophil-derived serine protease, has been shown to induce neonatal rat cardiomyocyte detachment and apoptosis by anoikis through caspase-3 dependent pathway. However the early mechanisms that trigger Cat.G-induced caspase-3 activation are not known. This study identifies focal adhesion kinase (FAK) tyrosine dephosphorylation as an early mechanism that regulates Cat.G-induced anoikis in cardiomyocytes. Both FAK tyrosine phosphorylation at Tyr-397 and kinase activity decrease rapidly upon Cat.G treatment and was associated with a decrease of FAK association with adapter and cytoskeletal proteins, p130^Cas and paxillin, respectively. FAK-decreased tyrosine phosphorylation is required for Cat.G-induced myocyte anoikis as concurrent expression of phosphorylation-deficient FAK mutated at Tyr-397 or pretreatment with a protein-tyrosine phosphatase (PTP) inhibitor, pervanadate, blocks Cat.G-induced FAK tyrosine dephosphorylation, caspase-3 activation and DNA fragmentation. Analysis of PTPs activation shows that Cat.G treatment induces an increase of SHP₂ and PTEN phosphorylation; however, only SHP₂ forms a complex with FAK in response to Cat.G. Expression of dominant negative SHP₂ mutant markedly attenuates FAK tyrosine dephosphorylation induced by Cat.G and protects myocytes to undergo apoptosis. In contrast, increased SHP₂ expression exacerbates Cat.G-induced FAK tyrosine dephosphorylation and myocyte apoptosis. Taken together, these results show that Cat.G induces SHP₂ activation that leads to FAK tyrosine dephosphorylation and promotes cardiomyocyte anoikis.

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^☆: Supported by a grant from the Green Valley Associates in Cancer Research.

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Cathepsin-G and leukocyte elastase inactivate human tumor necrosis factor and lymphotoxin☆

Abstract

J. Biol. Chem

Lancet

Anal. Biochem

Anal. Biochem

J. Biol. Chem

J. Biol. Chem

Biochem. Biophys. Res. Commun

Cell

Cancer Immunol. Immunother

Science

Nature

Nature

Surg. Gynecol. Obstet

J. Exp. Med

Science

J. Exp. Med

J. Leukocyte Biol

Arch. Surg

Arch. Surg

J. Clin. Invest