Neutrophil Elastase In Tissue Research Articles

The Potential of Molecular Remission: Tissue Neutrophil Elastase Is Better Than Histological Activity for Predicting Long-Term Relapse in Patients With Ulcerative Colitis in Endoscopic Remission.

Growing interest exists in deep remission, beyond clinical and endoscopic remission, to enhance long-term prognosis in patients with ulcerative colitis (UC). Our study aimed to evaluate the risk of relapse according to tissue expression levels of calprotectin and neutrophil elastase (NE) in patients with quiescent UC. Rectal biopsies were performed on 218 patients with UC in clinical and endoscopic remission. Histological activity was prospectively scored using the Robarts Histological Index. Tissue calprotectin and NE levels were evaluated using immunohistochemistry. Optimal tissue calprotectin and NE cutoffs for relapse were determined using log-rank analysis. Cox proportional hazard analyses evaluated relapse risk factors. Tissue calprotectin and NE levels were significantly higher in patients with histological activity than in those in histological remission (P < .001). The optimal cutoffs of tissue calprotectin and NE for relapse were 10.61 and 22.08permm2, respectively. The 3-year clinical relapse risk was significantly lower in the low-tissue NE group than in the high-tissue NE group (P = .009); however, it did not differ between the low- and high-tissue calprotectin group (P = .094). In multivariate analyses, a low level of tissue NE expression was independently associated with a lower risk of 3-year clinical relapse (adjusted hazard ratio = 0.453, 95% confidence interval = 0.225-0.911, P = .026), unlike histological index and tissue calprotectin. In patients with UC who have achieved clinical and endoscopic remission, tissue expression of NE is a better predictor of long-term relapse than histological activity.

Preclinical evaluation of [11C]GW457427 as a tracer for neutrophil elastase

IntroductionNeutrophils are part of the innate immune system and function as a first line of defense against invading microorganisms. Overactivity of the immune system may result in a devastating immuno-inflammation with extensive damage to tissue leading to organ damage and/or failure. The literature suggests several human diseases in which neutrophil elastase (NE) is postulated to be important in the pathophysiology including inflammatory bowel disease (IBD), chronic obstructive pulmonary disorder (COPD), abdominal aortic aneurysms (AAA), breast and lung cancer, and recently also in Sars-cov-2 virus infection (Covid-19). In particular, the lungs are affected by the destructive power of the protease neutrophil elastase (NE). In this paper, we report the pre-clinical development of a selective and specific positron emission tomography (PET) tracer, [11C]GW457427, as an in vivo biomarker for the study of NE, now available for human studies. Methods[11C]GW457427 was produced by methylation of GW447631 using [11C]methyl triflate and GMP validated production and quality control methods were developed. Chemical purity was high with no traces of the precursor GW611437 or other uv-absorbing compounds. A method for the determination of intact [11C]GW457427 in plasma was developed and the binding characteristics were evaluated in vitro and in vivo. An animal model for lung inflammation was used to investigate the specificity and sensitivity of the [11C]GW457427 tracer for neutrophil elastase (NE) in pulmonary inflammation, verified by blockade using two structurally different elastase inhibitors. Results[11C]GW457427 was obtained in approximately 45% radiochemical yield and with a radiochemical purity higher than 98%. Molar activity was in the range 130–360 GBq/μmol. Binding to NE was shown to be highly specific both in vitro and in vivo and a significantly higher uptake of tracer was found in a lipopolysaccharide mouse model of pulmonary inflammation compared with control animals. The uptake in lung tissue measured as standardized uptake value (SUV) strongly correlated with tissue NE content as measured by ELISA. In vitro studies also showed specific tracer binding in aortic tissue of patients with abdominal aorta aneurysm (AAA). The rate of metabolism in rats was appropriate considering the critical balance between available tracer for binding and requirement for blood clearance with about 40% and 20% intact [11C]GW457427 in plasma at 5 and 40 min, respectively. Radioactivity was cleared from blood and organs in control animals with mainly hepatobiliary excretion with distribution in the intestines and the urinary bladder; but without retention of the tracer in healthy organs of interests such as the lung, liver, kidneys or in the cardiovascular system. A dosimetry study in rat indicated that the whole-body effective dose was 2.2 μSv/MBq with bone marrow as the limiting organ. It is estimated that up to five PET-CT investigations could be performed in humans without exceeding a total dose of 10 mSv. Conclusion[11C]GW457427 is a promising in vivo PET-biomarker for NE with high specific binding demonstrated both in vitro and in vivo. A GMP validated production method including quality control has been developed and a microdosing toxicity study performed with no adverse signs. [11C]GW457427 is currently being evaluated in a First-In-Man PET study.

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.

1700-P: Exogenous a1-Antitrypsin A Enhances Insulin Sensitivity in Mice Fed a High-Fat Diet by Attenuation of Neutrophil Infiltration in Epididymal Fat

Neutrophil elastase (NE) is a serine protease expressed by neutrophils which is inhibited endogenously by α1-Antitrypsin A (AAT). Obesity is associated with increased plasma NE:AAT ratios and adipose tissue NE accumulation, which may drive obesity-associated metabolic dysfunction. Exogenous AAT is now an FDA approved treatment for AAT deficiency, therefore we investigated whether AAT treatment may also have potential therapeutic benefits for diet induced metabolic dysfunction. Standard Chow and high-fat diet (HFD) fed male C57Bl6 mice were randomized to receive 3x weekly IP injections of either AAT (2mg) or vehicle (PBS) for 10 weeks. Chow fed mice treated with recombinant AAT showed no differences in plasma NE, body weight or metabolic phenotype compared to vehicle treated control mice. However, during HFD feeding AAT treatment attenuated increases in plasma NE and white adipose tissue (WAT) NE and neutrophil accumulation without affecting circulatory neutrophil levels or body weight. Consistent with NE knockout mice being partially protected from HFD glucose intolerance, treatment of HFD fed mice with AAT enhanced whole body insulin sensitivity which was attributed to higher insulin-dependent p-AktSer473 and reduced inflammation markers in WAT but no other peripheral tissues. Treatment of 3T3L1 adipocytes with recombinant NE impaired insulin-stimulated glucose uptake andp-AktSer473, suggesting that inhibition of NE in WAT is the primary mechanism through which AAT treatment enhances insulin sensitivity. Collectively, our data suggests AAT may play a potential role in mitigating diet-induced neutrophil infiltration in WAT insulin resistance. Disclosure R.F. D’Souza: None. J.S.T. Woodhead: None. S.W. Masson: None. S.L. James: None. C. Hedges: None. T.L. Merry: None. Funding Health Research Council of New Zealand

Celastrol alleviates arthritis by modulating the inflammatory activities of neutrophils

ObjectiveNeutrophils play an important role in the pathogenesis of rheumatoid arthritis (RA). In this study, we used the adjuvant-induced arthritis murine model to evaluate the efficacy of celastrol on neutrophil-mediated inflammation in RA. MethodsFreund's complete adjuvant-induced arthritis was used as the murine model of RA. Celastrol was intraperitoneally administrated daily after onset of the disease. The joint diameter and inflammatory score were evaluated daily during the treatment period. Myeloperoxidase (MPO) and neutrophil elastase (NE) activities were evaluated by immunohistochemical analyses. Quantitative PCR and enzyme-linked immunoabsorbent assay were used to quantify the expression of cytokines. The expression of apoptosis-related proteins Bcl-2, Bax and caspase-3 were evaluated by western blot. ResultsCelastrol suppressed inflammation in joints of arthritic mice and diminished the expression of MPO and NE in the joint tissue. Celastrol significantly inhibited the expression of TNFα and IL-6 induced by LPS in neutrophils in a dose-dependent manner in vitro. Moreover, celastrol induced apoptosis of LPS-stimulated neutrophils by increasing the expression of Bax and cleaved caspase-3 while decreasing Bcl-2 expression. ConclusionOur findings show that celastrol significantly alleviates murine arthritis by modulating the inflammatory activities of neutrophils. These results indicate that celastrol could serve as an alternative or adjunct modality for the treatment of RA.

Ovine forestomach matrix biomaterial is a broad spectrum inhibitor of matrix metalloproteinases and neutrophil elastase

Proteases play a critical role in the ordered remodelling of extracellular matrix (ECM) components during wound healing and tissue regeneration. However, the usually ordered proteolysis is compromised in chronic wounds due to over-expression and high concentrations of matrix metalloproteinase's (MMPs) and neutrophil elastase (NE). Ovine forestomach matrix (OFM) is a decellularised extracellular matrix-based biomaterial developed for tissue regeneration applications, including the treatment of chronic wounds, and is a heterogeneous mixture of ECM proteins and proteoglycans that retains the native structural and functional characteristics of tissue ECM. Given the diverse molecular species present in OFM, we hypothesised that OFM may contain components or fragments that inhibit MMP and NE activity. An extract of OFM was shown to be a potent inhibitor of a range of tissue MMPs (IC50 s = 23 ± 5 to 115 ± 14 µg/ml) and NE (IC50 = 157 ± 37 µg/ml), and was more potent than extracts prepared from a known protease modulating wound dressing. The broad spectrum activity of OFM against different classes of MMPs (i.e. collagenases, gelatinases and stromelysins) may provide a clinical advantage by more effectively addressing the protease imbalance seen in chronic wounds.

Cellular Uptake of Soluble Neutrophil Elastase Increases Cyclin E (CCNE) Isoform Expression and Significantly Augments Susceptibility of Breast Cancer Cells to Cytolysis by CCNE-Specific Cytotoxic T Lymphocytes

AbstractAbstract 2090We have shown that cytotoxic T lymphocytes (CTL) with specificity for the cyclin E (CCNE) derived HLA-A2-restricted peptide CCNE144-152 (ILLDWLMEV) specifically lyse myeloid and lymphoid leukemia in proportion to CCNE overexpression. Full length (FL) CCNE is also overexpressed in most solid tumors, including breast cancer where it is a poor prognostic factor. In addition, leukemia and many breast cancers express tumor-specific low molecular weight (LMW) isoforms of CCNE that result from post-translational processing of the FL protein. Neutrophil elastase (NE), derived from the primary granules of neutrophils, cleaves FL CCNE into LMW forms and NE has been identified in breast cancer tissue. Therefore, we hypothesized that CCNE may also be a breast cancer tumor antigen because the CCNE144-152 peptide is contained within the overexpressed FL and LMW forms, and that effective T cell immunity could be amplified by increased availability of LMW forms within tumor cells exposed to NE. The link between innate immunity, inflammation, and tumor immunity is poorly understood, and this mechanism could explain a role for tumor-infiltrating inflammatory cells in breast cancer. To test this, we elicited CCNE-CTL from peripheral blood lymphocytes of HLA-A2+ healthy donors by weekly stimulation with CCNE-pulsed T2 cells and low-dose IL-2. After 21 days, cytotoxicity of target cells by the lymphocytes was tested with a standard 4-hour calcein AM-based assay. The CCNE-CTL specifically lysed T2 cells pulsed with CCNE (30%) but not non-pulsed T2 cells (0%) at an effector:target (E:T) ratio of 20:1 (p < 0.01). Next, we tested whether CCNE-CTL killed HLA-A2+ MDA-MB-231 (231) breast cancer cells. First, we confirmed that FL CCNE and LMW forms were expressed in 231 cells, while low expression of FL CCNE and no expression of the LMW forms was observed in benign epithelial cells by Western blot. Next, we showed that CCNE-CTL mediated 49% lysis of 231 breast cancer cells at an E:T ratio of 20:1. To look for in vivo evidence of CCNE recognition, we studied peripheral blood lymphocytes from breast cancer patients by flow cytometry with CCNE/HLA-A2 tetramers and anti-CD8 antibodies. In 3 of 4 breast cancer patients we identified CCNE-CTL, with no detectable CCNE-CTL in healthy controls. Together, these results confirm that CCNE is also a tumor antigen in breast cancer. NE, which cleaves CCNE and is expressed in breast cancer tissue, may be produced endogenously by breast cancer cells, or exogenously by inflammatory cells in the tumor microenvironment. Therefore, we studied 231 cells and three other breast cancer cell lines (MDA-MB-453, MCF-7, and HER18) for NE expression. RT-PCR performed with NE-specific primers showed that none of the cells expressed NE mRNA and Western blot showed no NE protein expression, suggesting that NE in tumor tissue derives from neutrophils or other inflammatory cells. To determine whether NE is taken up by breast cancer cells, we used flow cytometry to show that 231 cells pulsed with soluble NE took up an increasing amount of NE and was maximal by 24 hours when intracellular NE expression in 231 cells was comparable to that of HL60 leukemia cells that express high levels of NE. In addition, LMW isoforms of CCNE were increased in NE-pulsed 231 cells, by Western blot. Importantly, CCNE-CTL specific lysis of NE-pulsed 231 cells was 2-fold higher compared to nonpulsed 231 (46% versus 22% at E:T 10:1, p = 0.01). Taken together, these data show that overexpressed CCNE144-152 is a novel breast cancer peptide antigen. Furthermore, exogenous NE is taken up by breast cancer cells, increasing LMW forms of CCNE and enhancing the susceptibility of breast cancer cells to CCNE-CTL-mediated cytolysis. This study links a specific enzyme secreted by neutrophils in the innate immune response to tumors to a specific adaptive immune response against breast cancer, and it suggests that immunotherapy targeting CCNE is warranted. Disclosures:No relevant conflicts of interest to declare.

Prevention of Ischemia/Reperfusion-Induced Accumulation of Matrix Metalloproteinases in Rat Lung by Preconditioning With Nitric Oxide

Pulmonary ischemia/reperfusion (I/R) injury is associated with degradation of structural proteins. Preconditioning by short-term inhalation of nitric oxide (NO) ameliorates some of the severe consequences of an I/R cycle. The aim of this study was to evaluate the effects of NO preconditioning on I/R-induced changes of matrix metalloproteinase (MMP) activity. Left lung in situ ischemia in rats was maintained for 1 h, followed by reperfusion for 30 min or 4 h. In the NO group, animals inhaled NO (15 ppm) for 10 min directly before ischemia. Changes of expression or activity of MMPs (MMP-2, MMP-7, MMP-9, MMP-14) and of neutrophil elastase (NE) in bronchoalveolar lavage fluid (BALF), lung tissue, and arterial plasma were analyzed by zymography and Western blotting. Western blotting was also used to detect tissue inhibitors of matrix proteases, the extracellular metalloproteinase inducer (EMMPRIN or CD147), and endostatin, a proteolytic collagen fragment. Ischemia resulted in an increase of lavagable MMP activity (12.3-fold MMP-2, 8.1-fold MMP-7) at 30 min reperfusion. The activity of MMP-9 and NE in lung tissue progressively increased with time, whereas MMP-14 and MMP-2 were constant. Inhalation of NO prevented the early increase of MMP-2 and MMP-7 in BALF, but the level of MMP-9 and NE in tissue was not affected. The expression of tissue inhibitors of matrix proteases and EMMPRIN did not respond to any treatment. The release of endostatin proceeded in parallel to the level of MMPs in BALF. Significant correlations between MMP-9 and myeloperoxidase in lung tissue and between MMP-2/MMP-7 and plasma protein extravasation were found. The early rise of MMP-2 and MMP-7 in BALF resulted from plasma protein extravasation, whereas MMP-9 and NE were imported into lung tissue via leukocyte invasion. The effect of NO inhalation on lavagable MMPs was secondary to the sealing of the permeability barrier.

Neutrophil elastase and cancer

This mini-review summarizes our recent experimental and clinical studies on neutrophil elastase (NE) and cancer based on our original view point. Neoplasms metastasize as a result of a complex series of events. This process requires various degradative enzymes including proteases. NE has broad substrate specificity under physiological conditions, and excessive NE results in digestion of not only elastin, but also other extracellular matrix proteins. Several cell lines from human breast cancer and human lung cancer produce immunoreactive NE. The amount of immunoreactive NE in tumor tissue is an independent prognostic indicator of patients with breast cancer and lung cancer. Furthermore, a specific NE inhibitor completely suppressed growth of cancer cells transplanted into severe combined immunodeficiency mice. The use of NE inhibitor would seem to be a promising way to prevent the invasion and metastasis of cancer.