Release of immunoreactive human neutrophil proteinase 4, normally and in peritonitis.

A specific enzyme-linked immunoassay (ELISA) has been developed for the determination of neutrophil proteinase 4 (NP4) in human plasma/serum and tissue fluids. Comparison of the sequence for the first 20 N-terminal amino acids of NP4, neutrophil elastase and cathepsin G shows that NP4 is distinct from the other two proteases. However, all three show considerable homology. Neither elastase nor cathepsin G show any immunoreactivity when tested in the present ELISA. Normal human plasma contains about 38 micrograms/l of NP4, identified as alpha 1-proteinase inhibitor complexes. This represents about 50% of the total amount of NP4 released in plasma. The remaining 50% is bound by alpha 2-macroglobulin. Blood coagulation leads to a rapid release of NP4 from the leukocytes. Peritonitis is accompanied by a pronounced release of NP4, as shown by a three-to 10-fold increase of NP4 plasma levels and by the NP4 level in peritoneal exudates, which reaches about 40 mg/l in severe cases.     

Release of immunoreactive canine leukocyte elastase normally and in endotoxin and pancreatitic shock.

A specific ELISA has been developed for the determination of alpha 1-proteinase inhibitor-bound leukocyte elastase in canine plasma and tissue fluids. Comparison of the sequence of the first 16 N-terminal amino acids of the isolated canine leukocyte elastase to other elastases indicated moderate homology with porcine pancreatic elastase and pronounced identity with human leukocyte elastase. Normal canine plasma contains about 66 micrograms/l leukocyte elastase measured as elastase alpha 1-proteinases inhibitor complexes. This represents about 70% of the total amount of leukocyte elastase released in plasma. The remaining 30% is bound by alpha 1 alpha 2-macroglobulin. Blood coagulation leads to a rapid release of elastase from the leukocytes. Slow intravenous infusion of a lethal dose of endotoxin into dogs is followed by a marked drop in leukocyte count and a simultaneous rapid increase in plasma leukocyte elastase concentration reaching a plateau level of 2-3 mg/l plasma. Bile-induced pancreatitis in dogs is accompanied by successive increases in leukocyte elastase levels in plasma as well as in peritoneal exudates, reaching a level of about 15 mg/l in the exudates during the late stages of disease.     

Concentration of leukocyte elastase in plasma and polymorphonuclear neutrophil extracts in type 2 diabetes.

The concentration of leukocyte elastase/alpha1-proteinase inhibitor complexes in plasma and polymorphonuclear neutrophil extracts, and plasma trypsin inhibitory capacity were determined in 88 patients with type 2 diabetes and 47 control subjects. Higher values of these variables were found in patients as compared to controls (p < 0.001). The concentration of elastase was higher in obese patients than in lean ones (p < 0.05 for plasma, p < 0.01 for polymorphonuclear leukocytes). Only leukocyte elastase levels were significantly higher in the group with both micro- and macroangiopathy in comparison to the group with microangiopathy (p < 0.01) or macroangiopathy (p < 0.05) alone. Poor short-term glycaemic control was associated with higher elastase concentration in plasma and neutrophils (p < 0.05). The present study demonstrates that measurements of plasma polymorphonuclear neutrophil elastase level can be considered as a marker of development of diabetic angiopathy.     

Semen polymorphonuclear neutrophil leukocyte elastase as a diagnostic and prognostic marker of genital tract inflammation--a review.

Elastase is a protease released by polymorphonuclear neutrophils (PMN) during the inflammatory process. Since 1987, seminal elastase-inhibitor complex (Ela/alpha1-PI) has been proposed as a marker of male silent genital tract inflammation. Measured by immunoassay in seminal plasma, Ela/alpha1-PI at a cut-off level of > or = 230 microg/l, is useful in the detection of genital tract inflammation. The prevalence of increased seminal Ela/alpha1-PI in infertile men is significantly higher than that observed in fertile men. The Ela/alpha1-PI level is positively correlated with other seminal fluid markers of male genital tract inflammation: reduced semen volume, citric acid, fructose, and increased albumin, complement component C3, caeruloplasmin, immunoglobulins IgG and IgA, and cytokines interleukins-8 and -6. A higher seminal Ela/alpha1-PI level is significantly associated with tubal damage in female partners. After antibiotic therapy, a decrease of Ela/alpha1-PI level is observed. The presence of tubal damage in the partner may negatively affect the response to antibiotic treatment. A higher seminal Ela/alpha1-PI is associated with lower percentage of sperm with single-stranded deoxyribonucleic acid (DNA) and better fertilization rate in in vitro fertilization. Besides infertility, the determination of Ela/alpha1-PI is useful to confirm the presence of prostate and other male accessory gland bacterial inflammation. Screening for PMN Ela/alpha1-PI is easy to perform and reproducible and is a reliable quantitative test for diagnosis and prognosis of silent genital tract inflammation of couples. Moreover, sequential determinations allow the follow-up of inflammation during and after therapy.     

Release of granulocyte elastase and complement changes during hysterectomy

Complement activation and release of granulocyte elastase has been previously reported during major vascular surgery and has been ascribed to plasma/cell interactions with foreign surfaces or to administration of blood or plasma. The effect of uncomplicated general anaesthesia and surgery, not requiring blood or plasma, on complement activation and signs of proteinase release was assessed by measuring C3d and elastase-alpha-1 PI, respectively, in nine patients undergoing elective hysterectomy. There was a minor decrease in plasma C3d during anaesthesia, surgery and in the post-operative period caused by the diluting effect of intravenous fluids. Elastase alpha-1 PI remained largely unchanged until the first post-operative day, when a significant increase was seen (p less than 0.05); on the second and third day significant elevated values were still observed. Release of granulocyte elastase seems to be a generalized response to surgical trauma and may be a part of the endocrine-metabolic stress response.     

Beneficial effect of an inhibitor of leukocyte elastase (EPI-hNE-4) in presence of repeated lung injuries

Persistence of alveolar neutrophil influx and activation may enhance the fibrotic process after acute lung injury. On the other hand, elastase has an antimicrobial activity and could participate in neutrophil migration, both events being critically important in host defense, explaining the controversial issue of therapeutic elastase inhibition in the setting of acute lung injury. We assessed the effect of a neutrophil elastase inhibitor, EPI-hNE-4, in single (bleomycin, 1.2 mg/rat intratracheally) and repeated (bleomycin, 1.2 mg/rat plus endotoxin and 1 mg/kg intratracheally 24 h later) lung injuries to assess the role of neutrophil in fibrosis. Subsequently, the effect of EPI-hNE-4 on bacterial clearance was evaluated during Pseudomonas aeruginosa-induced pneumonia. In the single injury model, despite a dramatic reduction of alveolar neutrophil influx with EPI-hNE-4 treatment, no significant inhibition of the decrease in respiratory system compliance, an index of lung fibrosis, was demonstrated at day 14. In the repeated injury model, EPI-hNE-4 treatment afforded a significant protective effect on compliance and alveolar inflammation at day 14. During bacterial pneumonia, EPI-hNE4 did not modify alveolar neutrophil recruitment or bacterial clearance from bronchoalveolar lavage fluid and lung homogenate. In conclusion, EPI-hNE-4, a specific inhibitor of leukocyte elastase, afforded a partial protective effect on the respiratory system compliance during repeated lung injuries, and had no detrimental effect during a gram-negative bacterial pneumonia.     

The inhibitory complex of human alpha 1-proteinase inhibitor and human leukocyte elastase is a neutrophil chemoattractant

An inhibitor-proteinase complex consisting of human alpha 1-PI and human leukocyte elastase is chemotactic for human neutrophils. The chemotactic activity is optimal at 1 nM and is associated only with the alpha 1-PI portion of the complex. Neither HLE in the complex, free HLE, nor native alpha 1-PI possesses chemotactic activity for human neutrophils. alpha 1-PI in complex is hydrolyzed at the Met-358-Ser-359 bond. The chemotactic activity is associated with the Mr 4,200 fragment of alpha 1-PI that has Ser-359 as its NH2 terminus. The region of the HLE-alpha 1-PI complex that stimulates chemotaxis appears to be the same as that of the Mr 4,200 fragment generated by hydrolysis of the Pro-357-Met-358 bond during proteolytic inactivation of alpha 1-PI. The data suggest the presence of a neutrophil surface receptor bound by alpha 1-PI after the formation of a complex with HLE or after proteolytic degradation. This receptor may play a role in clearance of these modified alpha 1-PI molecules.     

Pathobiochemical significance of granulocyte elastase complexed with proteinase inhibitors: effect on glycosaminoglycan metabolism in cultured synovial cells

Interactions between elastase inhibitor complexes and synovial cells are of special interest, since, in chronic joint diseases, granulocytes release large amounts of elastase into the synovial fluid and connective tissue, where the proteinase is bound to alpha 1-proteinase inhibitor and alpha 2-macroglobulin. To study the effect of elastase-alpha 2-macroglobulin and elastase-alpha 1-proteinase inhibitor complexes on the glycosaminoglycan metabolism of cultured synovial cells, we determined the distribution of [3H]glucosamine-labelled hyaluronate, which represents the main synthesized glycosaminoglycan, and of 35SO4(2-)-labelled chondroitin sulphate into the intracellular, pericellular and extracellular compartments of the cell culture. Exposure of the synovial cells to elastase-alpha 2-macroglobulin complexes leads to an enhanced synthesis and secretion of hyaluronate, and chondroitin sulphate, and also induces a rise of the fibronectin concentration in the medium. Analogous but less pronounced effects are observed in the presence of elastase-alpha 1-proteinase inhibitor complexes. Native uncomplexed elastase, however, causes no significant changes in hyaluronate metabolism. An increase of prostaglandin E2 in the culture medium during incubation with elastase inhibitor complexes occurs in parallel to the stimulatory effect on glycosaminoglycan metabolism. Our results demonstrate that elastase, whose enzymic activity is inactivated by the formation of complexes with alpha 1-proteinase inhibitor or alpha 2-macroglobulin, nevertheless acts as an inflammatory mediator, which in vitro induces metabolic changes closely resembling the in vivo findings in inflammatory joint diseases.     

Ergotism and factitious hypotension associated with interaction of ergotamine with CYP3A4 inhibitors

Background. Although uncommon, severe ergotism continues to occur. The purpose of this study is to describe causes and clinical effects of ergotism in recent years. Methods. This is an observational case series with data obtained retrospectively from all patients with ergotism referred to Ramathibodi Poison Center in Bangkok, Thailand from January 2006 to August 2013. Result. Twelve cases of ergotism were identified. All cases involved ergotamine 1 mg/caffeine 100 mg combination tablets. Nine cases (75%) were precipitated by drug–drug interactions with CYP3A4 inhibitors. The other cases involved suicidal attempt (2 cases) and pediatric unsupervised ingestion (1 case). Ten patients (83%) had signs of peripheral vascular insufficiency. Five of these patients initially had factitiously low or unmeasurable blood pressure using non-invasive technique and had paradoxical increase following intravenous vasodilator administration. Two patients required partial foot amputations due to gangrene. Two patients, including a 15-month-old boy with an unsupervised ingestion, died. Discussion. In this series, most cases of severe ergotism were associated with interaction with CYP3A4 inhibitors, which increase ergotamine bioavailability. Factitious low blood pressure in these cases was likely caused by severe vasospasm. Conclusion. Critical ergotism continues to occur in Thailand, most commonly associated with the drug–drug interactions.     

Levels of calprotectin (leukocyte L1 protein) during apheresis.

The plasma concentration of calprotectin was measured before, during and after apheresis in patients with Guillain-Barré Syndrome (GBS), Waldenstr?m's syndrome or hypercholesterolaemia and in healthy donors of platelets. Increased calprotectin levels were found after plasma exchange in the Waldenstr?m's syndrome patients, probably caused by release of the protein from activated leukocytes. The decreased calprotectin values observed in the other patients, may be due to plasma dilution. Unexpectedly, the GBS patients were found to have high initial calprotectin levels in plasma but not in cerebrospinal fluid. In donors, normal and unchanged calprotectin concentrations were found throughout.     

The design and synthesis of potent inhibitors of hepatitis C virus NS3-4A proteinase

Hepatitis C virus (HCV) is the cause of the majority of transfusion-associated hepatitis and a significant proportion of community-acquired hepatitis worldwide. Infection by HCV frequently leads to persistent infections that result in a range of clinical conditions including an asymptomatic carrier state, severe chronic active hepatitis, cirrhosis and, in some cases, hepatocellular carcinoma. The HCV genome consists of a single-stranded, positive sense RNA containing an open reading frame of approximately 9060 nucleotides. This is translated into a single polyprotein of approximately 3020 amino acids (C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B), which in turn is processed by a series of host and viral proteinases into at least 10 cleavage products. The N-terminal portion of the NS3 protein encodes a serine proteinase that is responsible for the cleavage at the NS3-4A, NS4A-4B, NS4B-5A and NS5A-5B junctions. The 54 amino acid NS4A protein is a cofactor that binds to the NS3 protein and enhances its proteolytic activity. This report describes the expression of a recombinant NS3-4A proteinase fusion protein in Escherichia coli and the in vitro characterization of the enzyme activity using synthetic peptide substrates. It then demonstrates how these results were employed to guide the design of potent inhibitors of this enzyme.     

Remodeling of neutrophil phospholipids with 15(S)-hydroxyeicosatetraenoic acid inhibits leukotriene B4-induced neutrophil migration across endothelium.

5-Lipoxygenase products, such as leukotrienes, are important stimuli for leukocyte-mediated tissue injury in acute inflammation. 15-Hydroxyeicosatetraenoic acid (15-HETE) is an eicosanoid generated by a variety of cell types via the actions of 15-lipoxygenases and, in addition, cyclooxygenases and epoxygenases. 15-HETE levels are frequently elevated at sites of inflammation, and extracellular 15(S)-HETE is esterified rapidly into neutrophil (PMN) phospholipids in vitro to levels that are comparable with arachidonic acid. We present evidence that remodeling of PMN phospholipids with 15(S)-HETE stereoselectively inhibits PMN migration across endothelium in response to leukotriene B4 (LTB4) and other chemoattractants. Esterified 15(S)-HETE causes a striking reduction in the affinity of LTB4 cell-surface receptors for their ligand and inhibition of LTB4-triggered stimulus-response coupling. As a result of these actions, esterified 15(S)-HETE attenuates the cytoskeletal rearrangements and CD11/CD18-mediated adhesive events that subserve directed locomotion of PMN across endothelium. These observations indicate that products of the 5-lipoxygenase and 15-lipoxygenase pathways can exert counterbalancing influences on PMN trafficking across endothelium. They suggest that 15(S)-HETE may be a potent endogenous inhibitor of PMN-endothelial interactions in vivo and serve to limit or reverse acute inflammation.     

Augmented release of human leucocyte lactoferrin (and elastase) during coagulation

Lactoferrin, an iron binding protein, present in specific granules of neutrophils has been shown to be released concomitantly with elastase, when neutrophils are activated in vitro. Using enzyme-linked immunosorbent assay, lactoferrin release has been studied during blood coagulation, a more physiological system for studying the in vivo neutrophil activation in vitro. The assay has a lower detection limit of 1.0 ng/ml with intra and interassay coefficients of variation of 8 and 14% respectively. Median plasma and serum lactoferrin levels were 144 and 705 ng/ml respectively. The difference was statistically significant at p less than 0.0001. The median elastase proteinase inhibitor complex (EPIC) levels in plasma and serum were 85 and 176.5 ng/ml respectively. This difference was also highly significant at p less than 0.0001. There was no correlation between these parameters in the plasma samples. However, there was a statistically significant correlation between these measurements in sera at p less than 0.001 (Kendall's rank correlation). Release of lactoferrin and elastase-A1PIC during coagulation reflects the activation of neutrophils in vivo, especially during inflammation.     

Release of PMN elastase, TGF-beta1 and neopterin during blood storage; unfiltered versus filtered blood.

Release of inflammatory mediators from blood cells during prestorage leukocyte filtration may result in recipient immune suppression. To investigate the effects of prestorage leukocyte filtration on the quality of blood components, twenty-four blood units were collected from healthy donors and randomised into 3 groups. Eight units were stored as whole blood, eight units were separated into plasma, red blood cells (RBC) and buffy coat and eight units were collected and filtered through the ASAHI RZ 2000 leukocyte filter and separated into plasma and RBC. The units were stored for 35 days. Samples were collected weekly for analyses of polymorphonuclear elastase (PMN elastase), transforming growth factor-beta1 (TGF-beta1) and neopterin. PMN elastase and neopterin increased during storage of whole blood and RBC. From the beginning and throughout storage, PMN elastase was increased in filtered plasma as compared with unfiltered plasma. Filtration per se did not influence the neopterin concentration in plasma or RBC. TGF-beta1 increased in plasma and RBC during storage. In filtered plasma, an elevation of the TGF-beta1 concentration was observed from the start of storage. The TGF-beta1 levels were higher in filtered plasma compared with unfiltered plasma. Prestorage leukocyte filtration increased the release of PMN elastase and TGF-beta1 in plasma and RBC.     

Receptor-mediated binding and internalization of leukocyte elastase by alveolar macrophages in vitro.

Radioiodinated leukocyte elastase was found to bind rapidly and specifically to alveolar macrophages in vitro. In contrast to the binding of pancreatic and bacterial proteases, leukocyte elastase binding did not require the presence of alpha 2 macroglobulin. The binding was inhibited by an excess of unlabeled enzyme and was saturable by increasing elastase concentrations. Leukocyte elastase binding thus met criteria for receptor-mediated binding, with and estimated association constant of 4.97 x 10(5) M-1 and an estimated total of 640 x 10(6) binding sites/cell. It differed from the previously described binding of lysosomal glycosidases to macrophages in that it was insensitive to trypsin pretreatment, did not require calcium ions, and was not inhibited by yeast mannan. High-resolution autoradiography indicated that the cell-associated radiolabeled leukocyte elastase was rapidly incorporated into phagolysosomes. Macrophage binding may have a role in clearance of leukocyte elastase from tissue sites where alpha 2 macroglobulin is absent or present in low concentration. Thus, enzyme uptake by alveolar macrophages may be an important factor in the amelioration of lung tissue injury by extracellular leukocyte elastase.     

Development of an assay for in vivo human neutrophil elastase activity. Increased elastase activity in patients with alpha 1-proteinase inhibitor deficiency.

Leukocyte extracts contain enzymes that digest fibrinogen and release a fibrinopeptide A-containing fragment. This study was undertaken to identify the responsible proteinase and to characterize the fibrinopeptide A-containing fragment so that it could be used as an index of enzyme activity. Both the fibrinogenolytic activity and the release of the fibrinopeptide A-containing fragment mediated by the leukocyte extracts were shown to be due to human neutrophil elastase (HNE) by the following criteria: activity was completely blocked by a specific HNE inhibitor or by adsorbing HNE from the extracts with a monospecific antibody and reconstitution with purified HNE restored the ability to release the fibrinopeptide A-containing fragment. This fragment was not released by a variety of other proteinases or by HNE-inhibitor complexes indicating that, at least with respect to the enzymes tested, it is a specific product of HNE and its release requires the free enzyme. By separating the products of HNE digestion of fibrinogen using high performance liquid chromatography, identifying the immunoreactive fractions and subjecting them to amino acid analysis, the fragment was identified as A alpha 1-21, indicating an HNE cleavage site at the Val(A alpha 21)-Glu(A alpha 22) bond. The mean plasma A alpha 1-21 level was markedly higher in patients with alpha 1-proteinase inhibitor deficiency as compared to healthy controls (0.2 nM vs. 7.9 nM; P less than 0.0001), consistent with increased in vivo HNE activity in these individuals.