Role Of Neutrophil Elastase Research Articles

Abstract 264: Neutrophil elastase (NE) inhibition enhances metastasis free survival by altering regulation of tumor intrinsic cytoskeletal and cellular adhesion pathways in murine breast cancer models

Abstract Background: Neutrophil elastase (NE), a serine protease exclusively secreted in neutrophils, is a crucial mediator of chronic inflammation and tumor progression. Studies from our group reveal that NE may be a prognostic marker of metastasis. In a cohort of 192 breast cancer patients (58% ER/PR+ve, 20%-HER-2 +ve and 22% TNBC), our results show that higher infiltration of NE-positive Tumor Associated Neutrophils (TANs), is associated with a decrease in recurrence free survival (hazard ratio=3.4, 95% CI, 1.1-5.5), regardless of breast cancer subtype Further, we observe that the genetic deletion of Elane (encoding NE) inhibits lung metastasis in in vivo models of breast cancer. Yet the precise mechanism(s) by which NE promotes tumorigenesis and metastasis of breast cancer remains to be elucidated. To address this gap in knowledge, we have identified the tumor-intrinsic and -extrinsic mechanisms by which NE promotes metastasis. Methods: The role of NE in breast cancer metastasis was assessed using Elane+/+ and Elane-/- mice in FVB/NJ genetic background, bearing PyMT tumors (orthotopic and spontaneous) respectively. These mice develop lung metastasis in 80-90% of the tumor-bearing mice within 1-3 months of primary tumor initiation. To assess metastasis-free survival (MFS), mice received orthotopic engraftment of PyMT tumors, followed by resection and subsequent monitoring for metastasis. The efficacy of the NE inhibitor AZD9668 was assessed in Elane+/+ mouse models by treating the mice with 100mg/kg B.I.D treatments. Preliminary results: Genetic ablation of NE (Elane-/-) in the PyMT models, reduced lung metastasis by ~90% (Lung metastatic index =21.8 vs. 2.7, respectively; p=0.0044). Survival studies in FVB Elane-/- showed a MFS benefit of 345 days compared to controls, which succumbed to metastasis-related death in 46 days post primary tumor resection. RNA-sequencing analysis of PyMT- Elane+/+ and Elane-/- tumors showed differential regulation of tumor intrinsic actin cytoskeleton and integrin signaling pathways between the two genetic backgrounds. These NE-regulated pathways are critical for cell-to-cell contact and tissue integrity, explaining the delay in metastasis in the Elane-/- mice. 100mg/kg daily treatment of AZD9668 reduced lung metastasis in PyMT mice by 94% compared to vehicle-treated mice (0.49+/- 0.21% vs. 0.03+/-0.01%; p=0.05) and significantly reduced instances of primary tumor recurrence. Conclusions: Collectively, our studies suggest that genetic and pharmacological ablation of NE reduces metastasis and extends MFS in in vivo models of breast cancer. Our preclinical studies presented here are likely to provide the much-needed rationale for the use of this class of NE inhibitors as a viable treatment strategy for the metastatic breast cancer. Citation Format: Amriti R. Lulla, Said Akli, Lucas D. Warma, Natalie W. Fowlkes, Kelly K. Hunt, Xiayu Rao, Jing Wang, Stephanie S. Watowich, Khandan Keyomarsi. Neutrophil elastase (NE) inhibition enhances metastasis free survival by altering regulation of tumor intrinsic cytoskeletal and cellular adhesion pathways in murine breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 264.

The role of neutrophil elastase in aortic valve calcification

BackgroundCalcific aortic valve disease (CAVD) is the most commonly valvular disease in the western countries initiated by inflammation and abnormal calcium deposition. Currently, there is no clinical drug for CAVD. Neutrophil elastase (NE) plays a causal role in inflammation and participates actively in cardiovascular diseases. However, the effect of NE on valve calcification remains unclear. So we next explore whether it is involved in valve calcification and the molecular mechanisms involved.MethodsNE expression and activity in calcific aortic valve stenosis (CAVD) patients (n = 58) and healthy patients (n = 30) were measured by enzyme-linked immunosorbent assay (ELISA), western blot and immunohistochemistry (IHC). Porcine aortic valve interstitial cells (pVICs) were isolated and used in vitro expriments. The effects of NE on pVICs inflammation, apoptosis and calcification were detected by TUNEL assay, MTT assay, reverse transcription polymerase chain reaction (RT-PCR) and western blot. The effects of NE knockdown and NE activity inhibitor Alvelestat on pVICs inflammation, apoptosis and calcification under osteogenic medium induction were also detected by RT-PCR, western blot, alkaline phosphatase staining and alizarin red staining. Changes of Intracellular signaling pathways after NE treatment were measured by western blot. Apolipoprotein E−/− (APOE−/−) mice were employed in this study to establish the important role of Alvelestat in valve calcification. HE was used to detected the thickness of valve. IHC was used to detected the NE and α-SMA expression in APOE−/− mice. Echocardiography was employed to assess the heat function of APOE−/− mice.ResultsThe level and activity of NE were evaluated in patients with CAVD and calcified valve tissues. NE promoted inflammation, apoptosis and phenotype transition in pVICs in the presence or absence of osteogenic medium. Under osteogenic medium induction, NE silencing or NE inhibitor Alvelestat both suppressed the osteogenic differentiation of pVICs. Mechanically, NE played its role in promoting osteogenic differentiation of pVICs by activating the NF-κB and AKT signaling pathway. Alvelestat alleviated valve thickening and decreased the expression of NE and α-SMA in western diet-induced APOE−/− mice. Alvelestat also reduced NE activity and partially improved the heart function of APOE−/−mice.ConclusionsCollectively, NE is highly involved in the pathogenesis of valve calcification. Targeting NE such as Alvelestat may be a potential treatment for CAVD.

Tissue neutrophil elastase contributes to extracorporeal shock wave lithotripsy-induced kidney damage and the neutrophil elastase inhibitor, sivelestat, attenuates kidney damage with gratifying immunohistopathological and biochemical findings: an experimental study

Although the efficacy of extracorporeal shock wave lithotripsy (ESWL) has been well established within the literature, debate continues on the safety of the procedure while focusing on cellular injury and its long-term consequences. Here, we describe the role of neutrophil elastase (NE) in ESWL-related rat kidney damage and investigate the protective effects of sivelestat, an inhibitor of NE, during the early and late phases. Four groups including control, ESWL alone, ESWL with sivelestat 50mg/kg and ESWL with treatment of 100mg/kg, each consisting of ten rats were created. Biochemical parameters of kidney function and damage and immunohistopathological findings were compared in the early (72h after ESWL) and late (1week after ESWL) periods between the groups. During the early period, serum and urine creatinine levels and urine kidney injury molecule-1 (KIM-1) levels and the KIM-1/creatinine ratio increased in rats treated with ESWL compared to the control group. Furthermore, increased tissue inflammation, ductal dilatation and hemorrhage, and glomerular, tubular, and interstitial damage with increased NE staining were also detected in the ESWL treatment group. During the late phase, although urine KIM-1 levels remained stable at high levels, other parameters showed significant improvements. On the other hand, the administration of sivelestat 50mg/kg decreased serum creatinine and urine KIM-1 and KIM-1/creatinine levels significantly in rats treated with ESWL, during the early and late periods. Significant decreases in tissue inflammation, tubular, and interstitial tissue damage were also observed during the early period. In conclusion, ESWL-related kidney tissue damage occurs primarily during the early period, and NE is involved in this process. On the other hand, the NE inhibitor sivelestat attenuated this ESWL-induced kidney damage.

Abstract P3-10-02: Neutrophil elastase as a therapeutic target to inhibit metastasis in breast cancer

Abstract Background: Chronic inflammation, a hallmark of cancer, is associated with poor prognosis in human various malignancies. Predominantly, tumor associated neutrophils (TANs) and myeloid-derived suppressor cells (MDSCs) are immune cells well characterized to promote inflammation, support tumor growth and metastasis, by secreting chemokines, serine proteases and reactive oxygen species. Increased TANs and MDSCs are predictive markers of both tumor progression and metastasis, suggesting that their inhibition may provide viable anti-tumor strategies. Neutrophil elastase (NE), a serine protease exclusively secreted by TANs and MDSCs, is a crucial mediator of inflammation and tumor progression. Recent preliminary studies from our group suggests that genetic deletion of Elane (encoding NE) inhibits lung metastasis in in vivo models of breast cancer. Yet the precise mechanism(s) by which NE promotes tumorigenesis and metastasis of breast cancer remains to be elucidated. To address this gap in knowledge, the current focus of our work is to identify the tumor-intrinsic and -extrinsic mechanisms by which NE promotes metastasis. Methods: To determine the prognostic significance of NE, we analyzed 192 breast cancer patients (58% ER/PR+ve, 20%-HER-2 +ve and 22% TNBC) for infiltration of TANs. To examine the role of NE in breast cancer metastasis, we have generated Elane+/+ and Elane-/- mice in BALB/c and FVB/N genetic backgrounds, bearing 4T1 and PyMT tumors (orthotopic and spontaneous) respectively. Each of these mouse models develop lung metastasis in 80-90% in tumor-bearing mice within 1-3 months of primary tumor initiation. Results: Immunohistochemical staining of NE, a marker of TANs shows that higher infiltration of NE-positive TANs is associated with recurrence free survival with a hazard ratio of 3.4 (95% CI, 1.1-5.5). Genetic ablation of NE (Elane −/−) in the PyMT and 4T1 models strongly inhibits lung metastasis. Specifically, the number of lung foci was reduced by ~90% in PyMT Elane−/− mice compared to controls (21.8 vs. 2.7, respectively; p=0.0044). Similarly, ~50% decrease in the number of lung foci was observed in the 4T1 Elane−/− mice compared to control (7.8 vs. 3.4, respectively; p=0.0281). Comparison of inflammatory factors in the tumor microenvironment revealed that Csf3 (G-CSF), Cxcr2 (CXCR2) and Cxcl1 (CXCL1/KC) were significantly elevated in tumors from PyMT/4T1 Elane+/+ mice vs. PyMT/4T1 Elane-/- animals. Mechanistically, our result implies NE-dependent recruitment of immunosuppressive subsets (CXCL1 and related chemokines), thus sustaining inflammation at the primary tumor site. NE- dependent secretion of CXCL1 can further enrich for cancer stem-like cells (CSCs) via CXCR2 signaling, thus aiding metastasis. Lastly, we interrogated the efficacy reversible NE inhibitor AZD9668 in Elane+/+ mouse models. 100mg/kg daily treatment of AZD9668 reduced lung metastasis in PyMT mice by 94% compared to vehicle-treated mice (0.49+/- 0.21% vs. 0.03+/-0.01%; p=0.05). Conclusions: Collectively, our studies suggest that genetic and pharmacological ablation of NE reduces metastasis in in vivo models of breast cancer. NE inhibitors thus far have only been clinically tested in COPD patients and their efficacy in breast cancer remains to be evaluated. Our preclinical studies presented here are likely to provide the much needed rationale for the use of this class of NE inhibitors as a viable treatment strategy for the metastatic breast cancer. Citation Format: Amriti Rajender Lulla, Said Akli, Taylor T Chrisikos, Marc A. Pina, Yifan Zhou, Haiyan Li, Stephanie S Watowich, Khandan Keyomarsi. Neutrophil elastase as a therapeutic target to inhibit metastasis in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-02.

Knockout of neutrophil elastase protects against western diet induced nonalcoholic steatohepatitis in mice by regulating hepatic ceramides metabolism

Previous studies reported increased expression and activity of neutrophil elastase (NE) in NASH. However, the role of NE in nonalcoholic steatohepatitis (NASH) remained unclear. Wild type (WT) and NE knockout (NE KO) mice were fed with western diet (WD) for 24 weeks to establish NASH model. The severity of liver injury in NASH mice was assessed by biochemical analysis, liver triglyceride (TG) quantitation and histological scoring. The gene and protein expression was detected by quantitative real-time PCR, immunohistochemical and immunofluorescence staining (IHC/IF) and western blot analysis. After 24 weeks, WD induced WT (WD-WT) mice had significantly up-regulated NE protein in the liver. Moreover, body weight, liver/body weight, serum and hepatic TG, liver histological score, and hepatic inflammatory factors expression were significantly higher in WD-WT mice than those in low fat diet (LFD) induced WT mice, and these effects were markedly improved by NE KO. In addition, we found incereased expression of ceramides and serine palmitoyltransferase subunit 2 (SPT2) in WD-WT mice could be reversed by NE KO. Up-regulating expression of ceramides and SPT2 by active NE treatment was also found in mice primary hepatocytes. Collectively, these findings indicated that NE KO ameliorated WD induced NASH, and this beneficial effect was due, at least in part, to the potential of NE in regulating hepatic ceramides metabolism.

Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin-dependent neutrophil lysis.

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA-treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA-treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA-induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.

Overexpression of MTA1 inhibits the metastatic ability of ZR-75-30 cells in vitro by promoting MTA2 degradation

BackgroundAs the first member of the metastasis-associated protein (MTA) family, MTA1 and another MTA family member, MTA2, have both been reported to promote breast cancer progression and metastasis. However, the difference and relationship between MTA1 and MTA2 have not been fully elucidated.MethodsTranswell assays were used to assess the roles of MTA1 and MTA2 in the metastasis of ZR-75-30 luminal B breast cancer cells in vitro. Immunoblotting and qRT-PCR were used to evaluate the effect of MTA1 overexpression on MTA2. Proteases that cleave MTA2 were predicted using an online web server. The role of neutrophil elastase (NE) in MTA1 overexpression-induced MTA2 downregulation was confirmed by specific inhibitor treatment, knockdown, overexpression and immunocytochemistry, and NE cleavage sites in MTA2 were confirmed by MTA2 truncation and mutation. The effect of MTA1 overexpression on the intrinsic inhibitor of NE, elafin, was detected by qRT-PCR, immunoblotting and treatment with inhibitors.ResultsMTA1 overexpression inhibited, while MTA2 promoted the metastasis of ZR-75-30 cells in vitro. MTA1 overexpression downregulated MTA2 expression at the protein level rather than the mRNA level. NE was predicted to cleave MTA2 and was responsible for MTA1 overexpression-induced MTA2 degradation. NE was found to cleave MTA2 in the C-terminus at the 486, 497, 542, 583 and 621 sites. MTA1 overexpression activated NE by downregulating elafin in a histone deacetylase- and DNA methyltransferase-dependent manner.ConclusionsMTA1 and MTA2 play opposing roles in the metastasis of ZR-75-30 luminal B breast cancer cells in vitro. MTA1 downregulates MTA2 at the protein level by epigenetically repressing the expression of elafin and releasing the inhibition of neutrophil elastase, which cleaves MTA2 in the C-terminus at multiple specific sites.

Neutrophil Elastase Contributes to the Early Molecular Abnormalities of the Retina in Diabetes

Introduction: Previous studies by our lab indicate that neutrophils are involved with capillary degeneration in diabetic retinopathy (DR). The elastase is a serine protease in neutrophils that plays an important role in a variety of inflammatory diseases, such as chronic obstructive pulmonary disease. We are investigating the potential role of NE in the development of DR. Method: The activity of neutrophil elastase (NE) was selectively inhibited (by daily sivelestat) or deleted (Elane-/-) from diabetic mice, and the effects of NE loss were compared to the effects of inhibition of general protease activity (over-expression of protease inhibitor, alpha-1 antitrypsin). Molecular abnormalities associated with the development of DR, including retinal superoxide production, expression of proteins associated with inflammation, and leukocyte-mediated cytotoxicity of retinal endothelial cells were used to determine the effects of NE in diabetic mice. Result: At two months of diabetes, selective inhibition of NE or deletion of the elastase both inhibited diabetes-induced superoxide production and inflammation in the retina. Both also inhibited leukostasis and/or leukocyte mediated damage to endothelial cells in cell cytotoxicity assays. In contrast, over-expression of alpha-1 antitrypsin resulted in increased retinal superoxide production and leukocyte activity in cytotoxicity assays in both diabetic and nondiabetic animals. Conclusion: NE may play an important role in the development of DR. Disclosure H. Liu: None. T. Kern: None.

Biomarkers in Alpha-1 Antitrypsin Deficiency Chronic Obstructive Pulmonary Disease.

Biomarkers of pathogenesis in chronic obstructive pulmonary disease (COPD) can significantly accelerate drug development. In COPD related to alpha-1 antitrypsin deficiency, the role of neutrophil elastase and its inhibition by alpha-1 antitrypsin protein focused interest on elastin degradation and the development of pulmonary emphysema. Amino acids desmosine and isodesmosine are unique cross-links in mature elastin fibers and can serve as biomarkers of elastin degradation when measured in body fluids. This review gives a perspective on what has been learned by the earliest measurements of desmosine and isodesmosine followed by later studies using methods of increased sensitivity and specificity and the meaning for developing new therapies. Also included are brief statements on the biomarkers fibrinogen, CC-16, and Aa-Val-360 in COPD.

Neutrophil Elastase Differentially Regulates Interleukin 8 (IL-8) and Vascular Endothelial Growth Factor (VEGF) Production by Cigarette Smoke Extract

Inflammation by IL-8-induced neutrophil recruitment and apoptosis of epithelial cells by decreased expression of VEGF have been suggested as one of the complicated pathogenic mechanisms of chronic obstructive pulmonary disease (COPD). The role of neutrophil elastase (NE) in the development of COPD is also well known. However, little is known about how they interact. The objective of this study was to elucidate the effect of NE on cigarette smoke extract (CSE)-induced IL-8 and VEGF production and its molecular mechanism in bronchial epithelial cells. CSE increased both IL-8 and VEGF production in human bronchial epithelial cells (BEAS-2B). Although NE significantly enhanced CSE-induced IL-8 production, it suppressed VEGF production. This differential regulation was not CSE-specific. The effect of NE on IL-8 production, but not VEGF, was ERK-dependent. Interestingly, in contrast to decreased VEGF protein expression, NE accelerated VEGF transcription by CSE, suggesting post-translational modification. When cells were incubated with purified NE, it was detected in the cytoplasm, suggesting the intracellular translocation of NE. Furthermore, NE fragmented recombinant human VEGF in vitro but not recombinant human IL-8. These results indicate that VEGF down-regulation is due to direct degradation by NE, which is translocated into cells. Similar to in vitro cell experiments, elastase treatment increased CSE-induced IL-8; however, it suppressed VEGF production in bronchoalveolar lavage fluid of CSE-treated mice. Moreover, elastase treatment enhanced CSE-induced emphysema in mice. Considering the actions of IL-8 and VEGF, our results suggest that NE contributes to the pathogenesis of COPD by enhancing inflammation and apoptosis.

Abstract 1422: Neutrophil Elastase plays a key role in epithelial-mesenchymal transition and metastasis in triple-negative breast cancers

Abstract Background: Triple negative breast cancers (TNBCs) account for about 15% of breast cancer diagnosis in the USA, and occur at a higher percentage among African-American women (about 34%). Although they constitute a small percentage, the lack of targeted therapy of these hormone receptor-negative cancers has led to poor prognosis and overall survival. The main cause of TNBC-associated death is tumor metastasis from the primary site to distant sites. Characterization of drivers of EMT, one of the major drivers of metastasis could potentially identify therapeutic targets to prevent and potentially block metastasis. Interactions between tumor cells and inflammatory cells, including tumor-infiltrating neutrophils (TILs) play an important role in initiating and driving the process of metastasis. Our group previously reported that Neutrophil Elastase (NE), a secreted elastin from neutrophils as a mitogen that promotes tumor growth and activation of EGFR and TLR4 signaling pathways. We have also shown that molecular and pharmacological inhibition of NE leads to 90% reduction in metastatic foci in the lung. In this study, we interrogated the role of NE in EMT and its ability to affect key cellular functions involved in tumor metastasis, using TNBC cell lines as our model system. Methods: Using a genetic knockdown model of NE in murine and several human breast cancer cell lines we performed cellular proliferation, migration and invasion assays to evaluate its role in essential EMT-associated functions. We also interrogated the specific roles of the epithelial junction protein E-cadherin and mesenchymal transcription factor Snail in the ability of NE to drive a mesenchymal phenotype in the breast cancer cells. Results: Silencing of NE in all cells examined to date led to a significant increase in E-cadherin, a junction protein associated with an epithelial phenotype. We also observed a significant decrease at the mRNA level of Snail, a major transcription factor associated with epithelial-mesenchymal transition and Axl, a receptor tyrosine kinase that is highly associated with mesenchymal cells. Performing functional assays, we showed that NE silencing had a significant effect on cellular invasion through Matrigel. Studies are on going to validate the effects on cellular invasion and EMT mediated by exogenous NE uptake by the breast cancer cells. Conclusions: In summary, we identified NE as a potential driver of EMT and a mesenchymal phenotype in breast cancer cell lines. NE promotes cellular invasion and causes upregulation of factors associated with EMT. Studies are on going to characterize the major pathways downstream of NE that mediate the effects and to determine the function of exogenous NE that tumor cells are exposed to in their microenvironment. This study will lead to identification of mechanisms of NE-mediated effects on EMT and potential therapeutic targets of tumor metastasis. Citation Format: Kavitha Balaji, Chun-Hui Su, Said Akli, Kelly K. Hunt, Khandan Keyomarsi. Neutrophil Elastase plays a key role in epithelial-mesenchymal transition and metastasis in triple-negative breast cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1422. doi:10.1158/1538-7445.AM2015-1422

The role of neutrophil elastase and its inhibitors in acute respiratory distress syndrome: an update

The role of neutrophil elastase and its inhibitors in acute respiratory distress syndrome: an update

Neutrophils Increase or Reduce Parasite Burden in Trypanosoma cruzi-Infected Macrophages, Depending on Host Strain: Role of Neutrophil Elastase

Neutrophils are involved in the initial steps of most responses to pathogens and are essential components of the innate immune response. Due to the ability to produce and release various soluble mediators, neutrophils may participate in the regulation of the inflammatory response. Little is known about the role of neutrophils during protozoan infections including infection by Trypanosoma cruzi. In the present study we investigated the importance of inflammatory neutrophils on macrophage activation and T. cruzi replication in vitro, in cells obtained from BALB/c mice and C57Bl/6 mice. Co-cultures of BALB/c apoptotic or live neutrophils with infected peritoneal macrophages resulted in increased replication of the parasites and in the production of TGF-β and PGE2. The treatment with anti-TGF-β neutralizing antibody and COX inhibitor blocked the parasite replication in vitro. On the other hand, co-cultures of T. cruzi infected macrophages with live neutrophils isolated from C57BL/6 mice resulted in decreased number of trypomastigotes in culture and increased production of TNF-α and NO. The addition of anti-TNF-α neutralizing antibody or elastase inhibitor resulted in the abolishment of macrophage microbicidal effect and increased parasite replication. Addition of elastase to infected macrophages reduced the replication of the parasites, and on the other hand, addition of a selective inhibitor of iNOS increased parasite growth, suggesting the role of NO in this system. Our findings reveal that neutrophils may regulate T. cruzi experimental infection and determine susceptibility and resistance to infection.

Role of Neutrophil Elastase in the Development of Pulmonary Vascular Injury and Septic Shock in Rats

Role of Neutrophil Elastase in the Development of Pulmonary Vascular Injury and Septic Shock in Rats

Role of neutrophil elastase in lung injury induced by burn–blast combined injury in rats

ObjectiveNeutrophil elastase (NE) takes part in the pathogenesis of acute lung injury. However, its role in lung injury of burn–blast combined injury is unclear. Our objective was to assess the role of NE, and effect of sivelestat, a specific NE inhibitor, in lung injury induced by burn–blast combined injury in rats. MethodsOne hundred and sixty male Sprague-Dawley rats were randomly subjected to burn–blast combined injury (BB) group, burn–blast combined injury plus sivelestat treatment (S) group or control (C) group. Blood gas, protein concentration and NE activity in bronchoalveolar lavage fluid (BALF), pulmonary myeloperoxidase (MPO) activity, serum concentrations of TNF-α and IL-8, etc. were investigated from 0h to 7d post-injury. ResultsIn BB group, PaO2 decreased, while NE activity in BALF, total protein concentration in BALF, pulmonary MPO activity and W/D ratio, serum concentrations of TNF-α and IL-8 increased with neutrophil infiltration, progressive bleeding and pulmonary oedema. Compared with BB group, sivelestat treatment decreased the NE activity and ameliorated the above indexes. ConclusionSivelestat, exerts a protective effect in lung injury after burn–blast combined injury through inhibiting NE activity to decrease pulmonary vascular permeability, neutrophil sequestration, and production of TNF-α and IL-8.

Proteinase 3 activity in sputum from subjects with alpha-1-antitrypsin deficiency and COPD

Chronic obstructive pulmonary disease (COPD) is associated with tissue damage believed to result from an imbalance between serine proteinases and their inhibitors. Although the role of neutrophil elastase (NE) has been studied, it is likely that other proteinases play a role. The importance of proteinase 3 (PR3) has not been established, as specific substrates have only recently been available. We studied clinically stable subjects with either alpha-1-antitrypsin (A1AT) deficiency or usual COPD with chronic bronchitis. Sol phase sputum was analysed for PR3 activity and concentration, NE activity and concentration, concentrations of airway inhibitors (A1AT, secretory leukoproteinase inhibitor and elafin) and markers of neutrophilic inflammation. 12 patients were also studied during exacerbations. PR3 activity was present in most sputum samples and greater than NE activity (which was largely undetectable) in both subject groups (A1AT deficiency median PR3 128 nM, interquartile range (IQR) 33-558 nM; NE 0 nM, IQR 0-0 nM; p=0.0043; COPD PR3 22 nM, IQR 0-103 nM; NE 0 nM, IQR 0-0 nM; p=0.015). PR3 activity was greater during exacerbations than in the stable state (p=0.037) and correlated with markers of neutrophilic inflammation. The regular identification of PR3 activity in sputum from stable subjects with A1AT deficiency or usual COPD suggests it may play a greater role in the pathophysiology than previously thought.

Urinary Trypsin Inhibitor Attenuates Seawater-Induced Acute Lung Injury by Influencing the Activities of Nuclear Factor-ĸB and Its Related Inflammatory Mediators

Background: Few data are available on the role of neutrophil elastase (NE) and nuclear factor-ĸB (NF-ĸB) in the course of seawater drowning-induced acute lung injury (SWD-ALI), and there is no evidence on the value of giving urinary trypsin inhibitor (UTI) in the case of SWD-ALI. Objective: To investigate the role of NF-ĸB and NE in the pathogenesis of SWD-ALI and whether UTI treatment can attenuate SWD-ALI in rabbits. Methods: Rabbits were randomly assigned to control, seawater drowning, and UTI treatment groups. The rabbits in the control group only suffered from intubation, whereas the rabbits in the seawater drowning group and the UTI treatment group received arterial injection of normal saline without/with 50,000 U/kg body weight of UTI after instillation of seawater into an endotracheal catheter. The activities or contents of NF-ĸB, MPO, NE, TNF-α, and IL-10 in lung tissue were measured by nonradioactive EMSA, biochemical methods, and ELISA, respectively. Results: After the seawater challenge, all of the rabbits demonstrated immediate drops in arterial PaO₂/FiO₂ and pronounced pulmonary edema and inflammatory cell infiltration with evidence of an increase in the ratio of wet weight to dry weight, lung permeability index, lung injury scores, and the activities or contents of NF-ĸB, NE, MPO, TNF-α, and IL-10. UTI treatment markedly attenuated lung histopathological changes with evidence of a decrease in all of the parameters, except for upregulation of IL-10. Arterial PaO₂/FiO₂ was significantly improved after 6 h of UTI treatment. Conclusion: These results suggest that NF-ĸB and NE play an important role in SWD-ALI. UTI protects against SWD-ALI, at least partly, through inhibition of the enhanced local activity of NF-ĸB, contents of TNF-α and NE, and infiltration of neutrophils and promotion of the level of IL-10.

Administration of a specific inhibitor of neutrophil elastase attenuates pulmonary fibrosis after acute lung injury in mice

ABSTRACTExcess production of neutrophil elastase contributes to the pathogenesis of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, the role of neutrophil elastase in the repair process following ALI/ARDS is not well understood. The objective of this study was to evaluate the effect of neutrophil elastase on the process of tissue repair after acute lung injury in mice. C57BL/6 mice were exposed to sublethal irradiation followed by intranasal instillation of lipopolysaccharide (LPS) to generate a model of impaired lung repair. The authors assessed the histopathology, lung mechanics, and total lung collagen content 7 days after irradiation and/or LPS-induced injury with daily administration of a neutrophil elastase inhibitor. The number of inflammatory cells in the bronchoalveolar lavage fluid (BALF) was also evaluated. In addition, the concentration of activated transforming growth factor (TGF)-β1 in the BALF and the expression of phospho-SMAD2/3 were investigated. Irradiated and LPS-treated mice developed pulmonary fibrosis after injury. The neutrophil elastase inhibitor significantly decreased the collagen deposition in lung parenchyma and improved the static lung compliance of injured lungs. Administration of the neutrophil elastase inhibitor also decreased the accumulation of neutrophils in the BALF, TGF-β1 activation, and expression of phospho-SMAD2/3. The authors conclude that inhibiting neutrophil elastase protects against the development of lung fibrosis after acute injury. In addition, these data suggest that this neutrophil elastase inhibitor has therapeutic potential for the fibroproliferative phase of ALI/ARDS.

Neutrophil extracellular traps

Neutrophil extracellular traps

No role for neutrophil elastase in influenza-induced cellular recruitment, cytokine production or airway hyperresponsiveness in mice

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 50μL 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.