Intracellular Elastase Research Articles

Rationally Designed Two-Photon Ratiometric Elastase Probe for Investigating Inflammatory Bowel Disease.

Elastase, a serine protease, plays important roles in our body in food digestion and defence against pathogens. Particularly, the elastase present in neutrophils is directly associated with inflammatory bowel disease (IBD). Through a rational approach, we have developed a fluorescent elastase probe that has multiple advantages for biological applications including two-photon ratiometric imaging capability. Using the probe, which is capable of detecting intracellular elastase activity associated with inflammation, we have investigated elastase level changes in various mouse organs under an IBD condition for the first time. The results reveal notably higher elastase levels in the liver and duodenum of the healthy mice than in the other investigated organs. Under the IBD condition, we observed significant elastase level changes in the liver, duodenum, colon, and lung. The downregulation of elastase in the liver under the IBD condition suggests migration of neutrophils into the upregulated organs. The notable upregulation of elastase in the duodenum is explained by self-production of elastase, in addition to the neutrophil migration from the liver. We have observed little elastase level changes in selected organs of immune-deficient mice raised under the normal and IBD conditions, which supports the neutrophil migration as the reason for perturbed elastase activity in the healthy mice. The results also suggest an important role of the liver in maintaining the immune response associated with the inflammation-induced elastase level changes. The probe offers an ideal tool for mapping neutrophil migration in body. Further understanding of the elastase-associated biology and diagnosis of IBD by monitoring affected organs are anticipated using the probe.

Therapeutic Effects of Dipterocarpus tuberculatus with High Antioxidative Activity Against UV-Induced Photoaging of NHDF Cells and Nude Mice.

To investigate the therapeutic effects of methanol extracts of Dipterocarpus tuberculatus Roxb. (MED) against UV-induced photoaging, we assessed for alterations in the antioxidant activity, anti-apoptotic effects, ECM modulation, skin appearances, and anti-inflammatory response in normal human dermal fibroblast (NHDF) cells and nude mice orally treated with MED. High levels of tannin content and high free radical scavenging activity to DPPH were determined in MED, while seven active components, namely, gallic acid, bergenin, ellagic acid, ε-viniferin, asiatic acid, oleanolic acid, and 2α-hydroxyursolic acid, were identified using LC–MS analyses. UV-induced alterations in the NO concentration, SOD activity, and Nrf2 expression were remarkably recovered in MED-treated NHDF cells. Moreover, the decreased number of apoptotic cells and G2/M phase arrest were observed in the UV + MED-treated groups. Similar recoveries were detected for β-galactosidase, MMP-2/9 expression, and intracellular elastase activity. Furthermore, MED treatment induced suppression of the COX-2-induced iNOS mediated pathway, expression of inflammatory cytokines, and inflammasome activation in UV-radiated NHDF cells. The anti-photoaging effects observed in NHDF cells were subsequently evaluated and validated in UV + MED-treated nude mice through skin phenotypes and histopathological structure analyses. Taken together, these results indicate that MED exerts therapeutic effects against UV-induced photoaging and has the potential for future development as a treatment for photoaging.

Dual Transport of Active Substances with a Layer-by-Layer-Based Drug Delivery System to Terminate Inflammatory Processes.

Conventional therapies for chronic inflammation with high dose application of active agents are often accompanied with severe side effects so that other therapeutical strategies shall be developed to be less physically demanding but still highly efficient. Locally applied Layer-by-Layer (LbL) microcarriers transporting a low, but efficient dosage of active agents directly into the inflamed tissue offer a gentle therapy option. Here, the inhibition of highly degradative enzyme human neutrophile elastase (HNE) is adressed, which is produced and secreted by neutrophile granulocytes (PMNs) in the progress of inflammation. The protected transport and release of its natural inhibitor α1-antitrypsin (AT) as a constituent of the microcarrier's biopolymer multilayer allows for an efficient inhibition of extra- and intracellular elastase. The HOCl scavenger molecule cefoperazone, which preserves AT activity, as an additional multilayer constituent induces a much higher efficacy of the inhibitor. The successful assembly of both agents in different layers of the multilayer and the subsequent HNE inhibition in PMNs is investigated. The parallel application of cefoperazone leads to an enhanced inhibitory effect even with reduced AT amount and reduced carrier:cell ratio. It is demonstrated that the modular assembly strategy of LbL carriers allows for efficient synergistic effect of active agents in inflammatory process.

Protective effect of green tea catechin against urban fine dust particle-induced skin aging by regulation of NF-κB, AP-1, and MAPKs signaling pathways

The increase in ambient fine dust particles (FDP) due to urbanization and industrialization has been identified as a major contributor to air pollution. It has become a serious issue that threatens human health because it causes respiratory diseases and skin aging. In the present study, the protective effect of the green tea catechin, (−)-epigallocatechin gallate (EGCG), against FDP (ERM-CZ100)-stimulated skin aging in human dermal fibroblasts (HDFs) was investigated. The results demonstrate that EGCG significantly and dose-dependently scavenged intracellular reactive oxygen species (ROS) in and increased the viability of FDP-stimulated HDFs. In addition, EGCG dose-dependently recovered collagen synthesis and inhibited intracellular elastase and collagenase activities. Moreover, EGCG decreased the expression of human matrix metalloproteinases (MMPs) via regulation of nuclear factor kappa B (NF-κB), activator protein 1 (AP-1), and mitogen-activated protein kinases (MAPKs) signaling pathways in FDP-stimulated HDFs. This study suggests that EGCG is a potential anti-aging candidate that can be used for FDP-induced skin aging as a therapeutic agent itself or as an ingredient in pharmaceutical and cosmeceutical products.

Free circulating active elastase contributes to chronic inflammation in patients on hemodialysis.

Atherosclerosis and cardiovascular complications are prevalent among patients undergoing chronic hemodialysis (HD). In this population, peripheral polymorphonuclear leukocytes (PMNLs) are primed, releasing proinflammatory mediators such as elastase. Elastase is normally inhibited by a specific inhibitor, avoiding undesirable degradation of cellular and extracellular components. This study tested the hypothesis that in states of noninfectious inflammation, elastase is released by PMNLs and acts in an uncontrolled manner to inflict vascular damage. Blood was collected from patients undergoing HD and healthy controls (HC). PMNL intracellular and surface expressions of elastase were determined by quantitative real-time PCR, Western blotting, and flow cytometry. The elastase activity was evaluated using a fluorescent substrate. The levels of serum α1-antitrypsin (α1-AT), the natural elastase inhibitor, were determined by Western blot. Free active elastase was elevated in HD sera, whereas the levels of α1-AT were decreased compared with HC. The levels of the intracellular elastase enzyme and its activity were lower in HD PMNLs despite similar expression levels of elastase mRNA. Elastase binding to PMNL cell surface was higher in HD compared with HC. The increased circulating levels of free active elastase released from primed HD PMNLs together with the higher cell surface-bound enzymes and the lower levels of α1-AT result in the higher elastase activity in HD sera. This exacerbated elastase activity could lead to the endothelial dysfunction, as hypothesized. In addition, it suggests that free circulating elastase can serve as a new biomarker and therapeutic target to reduce inflammation and vascular complications in patients on hemodialysis.

Ubiquinol Effects on Antiphospholipid Syndrome Prothrombotic Profile: A Randomized, Placebo-Controlled Trial.

Antiphospholipid syndrome (APS) leukocytes exhibit an oxidative perturbation, directly linked to alterations in mitochondrial dynamics and metabolism. This disturbance is related to the patients' prothrombotic status and can be prevented by in vitro treatment with coenzyme Q10. Our aim was to investigate short-term effects of in vivo ubiquinol (reduced coenzyme Q10 [Qred]) supplementation on markers related to inflammation and thrombosis in APS through a prospective, randomized, crossover, placebo-controlled trial. Thirty-six patients with APS were randomized to receive Qred (200 mg/d) or placebo for 1 month. Thirty-three patients with APS completed the intervention, which increased plasma coenzyme Q10. Qred improved endothelial function and decreased monocyte expression of prothrombotic and proinflammatory mediators, inhibited phosphorylation of thrombosis-related protein kinases, and decreased peroxides and percentage of monocytes with depolarized mitochondria; mitochondrial size was increased, and mitochondrial biogenesis-related genes were upregulated. Qred ameliorated extruded neutrophil extracellular traps in neutrophils and downregulated peroxides, intracellular elastase, and myeloperoxidase. Nanostring microRNA profiling revealed 20 microRNAs reduced in APS monocytes, and 16 of them, with a preponderance of cardiovascular disease-related target mRNAs, were upregulated. Monocytes gene profiling showed differential expression of 29 atherosclerosis-related genes, 23 of them changed by Qred. Interaction networks of genes and microRNAs were identified. Correlation studies demonstrated co-ordinated effects of Qred on thrombosis and endothelial function-associated molecules. Our results highlight the potential of Qred to modulate the overexpression of inflammatory and thrombotic risk markers in APS. Because of the absence of clinically significant side effects and its potential therapeutic benefits, Qred might act as safe adjunct to standard therapies in APS. URL: http://www.clinicaltrials.gov. Unique identifier: NCT02218476.

Differential effector responses by circulating/blood and tissue/peritoneal neutrophils following burn combined with Enterococcus faecalis infection

Recently we found that superimposition of Enterococcus faecalis infection on burn injury caused an eruption of host mortality not seen with either individual challenge. We hypothesized that the Enterococcus bacteria, and/or factors related to these organisms, aggravate burn-induced modulations in host defense by neutrophils. Our study focuses on alterations in neutrophils' oxidative, proteolytic, and adhesive functions and transendothelial migration of neutrophils in burn rats inoculated with E. faecalis. Rats were subjected to burn (30% total body surface area) and then intra-abdominally inoculated with E. faecalis (10(4)CFU kg(-1) b.w). Polymorphonuclear neutrophils (PMNs) were harvested from circulating/blood and tissue/peritoneal cavity at day-2 post injury. Extracellular release of O(-)(2) anion production was determined by luminometry, and intracellular production of reactive oxygen species was measured by digital imaging technique. Fluoroscan analysis and confocal microscopy determined intracellular elastase production. The expression of adhesion molecule CD11b/CD18 was performed by flow cytometry. Calcein AM-labeled PMNs were co-cultured with TNF-α-stimulated rat lung microvascular endothelial cells, and their ability to adhere was assessed by fluorometry and digital imaging and finally, chemotaxis was measured by neutrophil transmigration assays. The results showed differential effector responses by circulatory and/or tissue PMNs. Tissue/peritoneal PMNs produced more O(-)(2), less intracellular elastase, and increased expression of CD11b/CD18 accompanied with increased adhesivity of MIP-2-stimulated PMNs to endothelial cells as compared to circulatory/blood PMNs. This differential effect was more pronounced following burn plus E. faecalis infection, indicating that the combined injury changed neutrophil functions.

Trophoblast- and Vascular Smooth Muscle Cell-Derived MMP-12 Mediates Elastolysis during Uterine Spiral Artery Remodeling

During the first trimester of pregnancy, the uterine spiral arteries are remodeled, creating heavily dilated conduits that lack maternal vasomotor control but allow the placenta to meet an increasing requirement for nutrients and oxygen. To effect permanent vasodilatation, the internal elastic lamina and medial elastin fibers must be degraded. In this study, we sought to identify the elastolytic proteases involved in this process. Primary first-trimester cytotrophoblasts (CTBs) derived from the placenta exhibited intracellular and membrane-associated elastase activity; membrane-associated activity was primarily attributable to matrix metalloproteinases (MMP). Indeed, Affymetrix microarray analysis and immunocytochemistry implicated MMP-12 (macrophage metalloelastase) as a key mediator of elastolysis. Cultured human aortic smooth muscle cells (HASMCs) exhibited constitutive membrane-associated elastase activity and inducible intracellular elastase activity; these cells also expressed MMP-12 protein. Moreover, a specific inhibitor of MMP-12 significantly reduced CTB- and HASMC-mediated elastolysis in vitro, to 31.7 ± 10.9% and 23.3 ± 8.7% of control levels, respectively. MMP-12 is expressed by both interstitial and endovascular trophoblasts in the first-trimester placental bed and by vascular SMCs (VSMCs) in remodeling spiral arteries. Perfusion of isolated spiral artery segments with CTB-conditioned medium stimulated MMP-12 expression in medial VSMCs. Our data support a model in which trophoblasts and VSMCs use MMP-12 cooperatively to degrade elastin during vascular remodeling in pregnancy, with the localized release of elastin peptides and CTB-derived factors amplifying elastin catabolism.

JOHANSON-BLIZZARD-SYNDROM - UBR-1 TARGET IDENTIFICATION BY PROTEOME ANALYSIS AND PATHOPHYSIOLOGICAL RELEVANCE

Introduction: Johanson-Blizzard syndrome (JBS; OMIM 243800), an autosomal recessive multisystem disorder, is characterized by congenital abnormalies including prenatal pancreatic inflammation and later exocrine pancreatic insufficiency. JBS is associated with mutations in the UBR1 gene on chromosome 15q14-21.1, which encodes one of the E3 ubiquitin ligases of the N-end rule pathway. UBR1 is part of the ubiquitin-dependent proteolytic pathway whose substrates include proteins with destabilizing N-terminal residues. The UBR1-dependent mechanism that causes pancreatic damage in JBS is unknown and was studied here. Methods: Ubr-1-deficient mice generated by targeted disruption of the Ubr-1 gene (Mol Cell Biol. 2001; 21:8007-21) were obtained from Y.T. Kwon, Univ. of Pittburgh, USA. Acute pancreatitis was induced by caerulein (50 μg/h, i.p.). Proteome analysis of pancreatic tissue from caerulein treated WT and UBR-1 knock-out animals was performed to identify disease associated targets and those candidates were further characterised in functional assays. Results: Proteome analysis in UBR-1-ko and WT animals after caerulein treatment with a focus on destabilizing N-terminal residues revealed a significant accumulation of pancreatic proteases such as chymotrypsin B, anionic trypsin and pancreatic elastase. Furthermore, we found an up-regulation of ER-stress proteins and inflammation related proteins. Phenotypic characterisation of pancreatitis after 8h revealed significantly increased lipase levels in UBR-1-ko animals, a significantly increased histology score (WT 2.04+/−0,78; UBR-1 ko 2.79 +/−0.72, p = 0.023) and significantly increased elastase activity 8h after the onset of pancreatitis. In isolated pancreatic acini we found a significant increase in intracellular elastase activation upon supramaximal CCK stimulation in UBR-1 ko animals associated with a significant rise in the rate of necrosis. Conclusion: Experimental pancreatic injury in UBR-1 ko-animals was followed by significantly greater local and systemic inflammation suggesting a vital function of UBR1 in the defense against pathologic damage - and JBS as an inflammatory disorder due to an inadequate UBR1 defense. Proteom analysis and functional data from isolated pancreatic acini showed an accumulation of activated proteases and hereby support a crucial role of proteases as underlying pathomechanism for the pancreatic phenotype in JBS.

Innate immunity defects in Hermansky-Pudlak type 2 syndrome

AbstractAdaptor protein-3 (AP-3) is an ubiquitous cytoplasmic complex that shuttles cargo proteins from the trans-Golgi and a tubular-endosomal compartment to endosome-lysosome–related organelles. Lack of the β3A subunit of this complex causes Hermansky-Pudlak syndrome type 2, an autosomal recessive disease characterized by partial albinism, prolonged bleeding tendency, and immunodeficiency. To investigate the pathogenesis of immunodeficiency, we studied natural killer (NK) cells and neutrophil functions in 2 previously unreported siblings affected by Hermansky-Pudlak type 2 syndrome. In both patients we observed a dramatic reduction of cytolytic activity of freshly isolated and of IL-2–activated NK cells. Levels of perforin were reduced in unstimulated NK cells, thereby accounting for the impairment of NK cytolitic activity. In addition, analysis of neutrophils in these patients demonstrated that intracellular elastase content was largely reduced while CD63 expression on plasma membrane was substantially increased. Taken together, these observations suggest that type 2 Hermansky-Pudlak syndrome is characterized by defects of innate immunity.

Polymorphonuclear neutrophils and Escherichia coli proteases involved in proteolysis of casein during experimental E. coli mastitis

Abstract An Escherichia coli mastitis model was used to characterize enzymes involved in bovine mammary tissue damage and proteolysis in milk. One-quarter each of four cows were inoculated with a suspension (104 cfu mL−1) of E. coli P4:O32. Blood and milk were collected before inoculation and for 216 h afterwards. Intracellular elastase, collagenase and cathepsin activities were measured by flow cytometry of peripheral blood leukocytes and milk polymorphonuclear neutrophils (PMNs). Leukopenia occurred in peripheral blood 9 h after infection, concomitant with an increase in somatic cell count in milk. Milk PMNs had lower activity of cathepsins and collagenase than peripheral blood PMNs. In parallel, milk samples were studied by zymography, and several proteases were detected in mastitic milk. These activities increased after infection, to reach a peak in 6 h. However, total protease profiles and plasmin activities differed. It was concluded that proteases released by PMNs and E. coli contribute to proteolysis of casein during mastitis, as well as plasmin.

Granulocyte functions and changes in ability with age in newborns; Report no. 2: Activation of granulocyte functions by cytokines

The aim of the present study was to examine the function of granulocytes in newborns from the perspective of granulocyte activation by cytokines. Granulocytes were stimulated with tumor necrosis factor-alpha, granulocyte-macrophage colony stimulating factor (GM-CSF) or granulocyte colony stimulating factor, and the reactivity of granulocytes to these substances was then compared between newborns (umbilical cord blood, peripheral blood obtained at age 5 days and at age 1 month) and peripheral blood obtained from adults. In addition, the expression of cytokine receptors on the surface of granulocytes was measured for each age group. The amplification of CD11b expression on the surface of granulocytes and suppression of l-selectin expression were weaker for cord blood regardless of which cytokine was added. In addition, the increases in the activity of intracellular elastase when stimulated with tumor necrosis factor-alpha or GM-CSF were significantly lower for cord blood. Moreover, the expression of GM-CSF receptors and granulocyte colony stimulating factor receptors on the surface of granulocytes was lower for cord blood, and this expression approached the level found in adults as age increased. Granulocytes during early infancy exhibit low reactivity to inflammatory cytokines, and this was considered to be one of the factors contributing to the higher incidence of serious bacterial infections in infants.

Endostatin expression in a pancreatic cell line is modulated by a TNFα-dependent elastase

Endostatin, an inhibitor of angiogenesis, is a 20 kDa fragment of the basement membrane protein, collagen XVIII. The formation of endostatin relies upon the action of proteases on collagen XVIII. TNFα, produced by activated macrophages, is a multifunctional proinflammatory cytokine with known effects on endothelial function. We postulated that TNFα may modulate the activities of proteases and thus regulate endostatin formation in pancreatic cells. Collagen XVIII/endostatin mRNA was expressed in one pancreatic cell line, SUIT-2, but not in BxPc-3. The 20 kDa endostatin was found in the cell-conditioned medium of SUIT-2 cells. Precursor forms only were found in the cells. Exogenous endostatin was degraded by cellular lysates of SUIT-2 cells. Elastase activity was found in cell extracts but not the cell-conditioned media of SUIT-2 cells. Incubation of SUIT-2 cells with TNFα increased intracellular elastase activity and also increased secretion of endostatin into the medium. We conclude that endostatin is released by SUIT-2 cells and that increases in intracellular elastase, induced by TNFα, are correlated with increased secretion. Endostatin is however susceptible to degradation by intracellular proteases and if tissue injury accompanies inflammation, endostatin may be degraded, allowing angiogenesis to occur.

Dynamic aspects of granulocyte activation in acute pancreatitis

We demonstrated the dynamic aspects of granulocyte activation in rat severe acute pancreatitis which was induced by cerulein and aggravated following lipopolysaccharide (LPS) injection. Pancreatitis induced by cerulein increased intracellular elastase activity of granulocytes in the blood. However, significant systemic cytokinemia was not provoked under such conditions. After induction of severe pancreatitis by LPS, intracellular elastase activity of circulating granulocytes decreased markedly and immediately. The timing of this decrease was concomitant with induction of systemic hypercytokinemia and granulocytes migration into the lung. Overall results provide the following implications: (1) circulating granulocytes are activated by the induction of mild pancreatitis; (2) the activation of granulocytes is mediated by other factors than systemic cytokinemia, such as locally produced cytokine; (3) those priming granulocytes immediately and significantly migrate from the circulation into extravascular space by the induction of endotoxemia; (4) the migration of granulocytes, in turn, may be mediated by systemic cytokinemia.

Assessment by flow cytometry of peripheral blood leukocyte enzymatic activities in HIV patients

Leukocyte enzymatic activities are important in non-specific protection against bacterial infections, but traditional methods for the detection of intracellular enzymatic activities rely on cumbersome and complex assays. The development of specific substrates, which become fluorescent upon degradation of the biomolecule after its passive entry into intact cells, permits a simplified evaluation of leukocyte enzymatic activities. We have used this method to assess intracellular elastase, collagenase and cathepsin D activities of peripheral blood leukocytes using flow cytometry in a series of HIV patients and healthy controls. Monocytes displayed the highest enzymatic activities for the three proteases tested. In HIV-infected patients, the collagenase and cathepsin D activities of monocytes were significantly lower, whereas the elastase and cathepsin D activities of polymorphonuclear cells were elevated. Slightly higher elastase activity was detected in the lymphocytes of patients. This study demonstrates the feasibility of this new method for the study of intracytoplasmic enzymatic activities. Significant variations were observed in the peripheral blood of HIV-infected patients and different patterns were especially evident in monocytes and polymorphonuclear cells.

Neutrophil-related host response in hidradenitis suppurativa: a pilot study in patients with inactive disease.

Host-defence defects in hidradenitis suppurativa patients have been suspected, but not proven. Activated neutrophils can destroy the surrounding tissues by a release of reactive oxygen species and active proteases. Peripheral neutrophils from 15 female patients (mean age 46, range 27-57 years) in an inactive state of their hidradenitis suppurativa, were studied and compared with 15 age-matched healthy female controls. There were no significant differences between patients and controls in the assessments of intracellular elastase activity, total content of antigenic elastase or release of elastase. Furthermore, no differences were found in total content and membrane expression of the receptors measured. The generation of free oxygen radicals, after stimulation with the protein kinase C activator phorbol myristate acetate, was significantly higher in the patients than in the controls, while there was no difference after Fc-receptor-mediated stimulation. Dysfunctional neutrophils might be involved in the pathogenesis of hidradenitis suppurativa, but the findings should be interpreted with caution because of the small number of observed cases.

Inhibitory effect of serine protease inhibitors on neutrophil-mediated endothelial cell injury

To investigate the inhibitory effect of serine protease inhibitors (SPI) on neutrophil-mediated endothelial cell (EC) injury, we analyzed the in vitro cytotoxicity of radiolabeled human umbilical vein EC (HUVEC) mediated by neutrophils in the presence of SPI. The EC injury was inhibited dose-dependently by urinary trypsin inhibitor (ulinastatin, UTI) and ONO-5046, which have the ability to inactivate neutrophil elastase, but not by gabexate mesilate, nafamostat mesilate, aprotinin, and argatroban, which have no ability to inactivate neutrophil elastase. In addition, when UTI and ONO-5046 were added to the tumor necrosis factor alpha-primed neutrophils alone, they showed a dose-dependent inhibition of the intracellular elastase activity, but the other SPI did not, for either flow cytometry or confocal microscopy. Therefore, UTI and ONO-5046 may protect EC against the neutrophil-mediated injury not only by inactivating the extracellular elastase secreted by neutrophils, but also by acting directly on neutrophils and suppressing the production and secretion of activated elastase from them.

Elastase activity enhances the adhesion of neutrophil and cancer cells to vascular endothelial cells.

Elastase activity in cancer cells has been reported to promote their metastasis. Hence, we analyzed the influence of elastase activity of cancer cells on their responsive adhesion to vascular endothelial cells. Human pancreatic (S2-007, S2-013, S2-020, S2-028) and colonic (COLO205) cancer cell lines were used. S2-007, S2-013, and S2-020 possess high elastase activity, whereas S2-028 and COLO205 have low elastase activity. Adhesive reactions of these cancer cells and neutrophils to TNFalpha-activated HUVEC were analyzed. Bound cells onto HUVEC were counted after incubation for 10 min. The effects of suppression of elastase activity by ZD8321, a potent elastase inhibitor, and supplementation of human neutrophil elastase (NE) on the adhesive reactions were also analyzed. In addition, E-selectin expression on HUVEC and concentrations of soluble E-selectin in the medium were measured. Adhesion of cells with high intracellular elastase activity to TNFalpha-activated HUVEC was suppressed by ZD8321. On the other hand, adhesion of cells with low elastase activity was enhanced by exogenous NE. Expression of E-selectin, a key molecule in leukocyte-endothelial cell interaction, on HUVEC was increased by NE. Soluble E-selectin concentration in the medium increased after the adhesive reaction between neutrophils and HUVEC. This increase was thought to be due to the shedding of cell surface E-selectin. Such responses were inhibited by ZD8321. Elastase activity has a biological function of stimulating both the E-selectin expression on HUVEC and the resultant adhesive reaction of cancer cells with them. Inhibition of elastase activity is a potent strategy for controlling cancer metastasis.

Expression of intracellular elastase activity in peripheral neutrophils from patients with adult periodontitis.

This study aimed to investigate whether circulating neutrophils from patients with periodontitis contain more catalytically active elastase than neutrophils from healthy controls. The amount of IL-1beta in these cells was also analyzed and the correlation with elastase activity was tested. The periodontitis group consisted of 15 subjects with marked periodontal destruction. The healthy control group consisted of 15 subjects with no clinical signs of periodontal destruction. The elastase activity and the IL-1beta content in the cells were measured with flow cytometry using a specific substrate and antibodies, respectively. The plasma concentration of IL-1beta and the total content of antigenic elastase in the crushed cells were measured with an ELISA. The elastase activity per neutrophil was significantly higher in the patients than in the controls, while the total antigenic elastase content did not differ. The % of cells positively stained for IL- 1beta was somewhat higher in the patient group. Significantly higher amounts of IL-1beta per sample, estimated by multiplying the % of stained cell with the amount of staining per cell, were found in the patient group. A significant correlation between IL-Ibeta and elastase activity was noted in the patient group (R=0.6, p=0.001), but not in the control group. In conclusion, peripheral neutrophils from patients with adult periodontitis express more active elastase and total amounts of IL-1. The similar amounts of antigenic elastase suggests that this higher activity is possibly due to some kind of priming/activation already in the circulation.

A Leukocyte Elastase Inhibitor Reduces Thrombin-Induced Pulmonary Oedema in the Rat: Mechanisms of Action

The effect of a selective leukocyte elastase inhibitor, ICI 200,355, on thrombin-induced pulmonary oedema was studied in rats. Thrombin administration produced an increase in lung weight (P<0.05), wet weight/dry weight ratio (P<0.05), and relative lung water content (P<0.05). The lung weight increase was reduced by the elastase inhibitor in doses of 2000, 200 and 20 μg/kg per h (P<0.05), but not by 2 μg/kg per h. A dose of 20 μg/kg per h seems to be optimal, since 10-fold and 100-fold increases in dose did not further improve the effect. Free elastase activity in lung tissue was higher after thrombin infusion than in controls, but was not depleted by the elastase inhibitor in vivo (P<0.05). This elastase activity in the lung was, however, inhibited by the elastase inhibitor in vitro, indicating that the inhibitor can block extracellular, but not intracellular elastase activity. Thrombin infusion resulted in a significant decrease in plasma elastase inhibitory capacity (P<0.05), which was depleted by the elastase inhibitor (20 μg/kg per h) (P<0.05). Myeloperoxidase activity was significantly increased in lung tissue after thrombin infusion (P<0.05). Lung myeloperoxidase activity 5 min after thrombin infusion was not affected by the elastase inhibitor, but the inhibitor induced a further increase in myeloperoxidase as seen 90 min after thrombin infusion, indicating that the effect of this inhibitor on pulmonary oedema is not due to reduction of leukocyte infiltration in the lungs, but may partly be exerted by prevention of neutrophil destruction.