Azurophilic Enzyme Research Articles

Elevated Leukocyte Azurophilic Enzymes in Human Diabetic Ketoacidosis Plasma Degrade Cerebrovascular Endothelial Junctional Proteins.

Diabetic ketoacidosis in children is associated with vasogenic cerebral edema, possibly due to the release of destructive polymorphonuclear neutrophil azurophilic enzymes. Our objectives were to measure plasma azurophilic enzyme levels in children with diabetic ketoacidosis, to correlate plasma azurophilic enzyme levels with diabetic ketoacidosis severity, and to determine whether azurophilic enzymes disrupt the blood-brain barrier in vitro. Prospective clinical and laboratory study. The Children's Hospital, London Health Sciences Centre. Pediatric type 1 diabetes patients; acute diabetic ketoacidosis or age-/sex-matched insulin-controlled. Acute diabetic ketoacidosis in children was associated with elevated polymorphonuclear neutrophils. Plasma azurophilic enzymes were elevated in diabetic ketoacidosis patients, including human leukocyte elastase (p < 0.001), proteinase-3 (p < 0.01), and myeloperoxidase (p < 0.001). A leukocyte origin of human leukocyte elastase and proteinase-3 in diabetic ketoacidosis was confirmed with buffy coat quantitative real-time polymerase chain reaction (p < 0.01). Of the three azurophilic enzymes elevated, only proteinase-3 levels correlated with diabetic ketoacidosis severity (p = 0.002). Recombinant proteinase-3 applied to human brain microvascular endothelial cells degraded both the tight junction protein occludin (p < 0.05) and the adherens junction protein VE-cadherin (p < 0.05). Permeability of human brain microvascular endothelial cell monolayers was increased by recombinant proteinase-3 application (p = 0.010). Our results indicate that diabetic ketoacidosis is associated with systemic polymorphonuclear neutrophil activation and degranulation. Of all the polymorphonuclear neutrophil azurophilic enzymes examined, only proteinase-3 correlated with diabetic ketoacidosis severity and potently degraded the blood-brain barrier in vitro. Proteinase-3 might mediate vasogenic edema during diabetic ketoacidosis, and selective proteinase-3 antagonists may offer future vascular- and neuroprotection.

Sepsis‐Associated Proteinase 3 Induces Endothelial Permeability

Sepsis is a systemic inflammatory response to known or suspected infection, and associated with microvascular dysfunction and capillary leak. The cause of capillary leak may be due to substances released by activated polymorphonuclear neutrophils (PMNs) that adhere to microvascular endothelial cells. Following binding, PMN often degranulate releasing azurophilic enzymes (e.g. elastase and proteinase 3 (PR3)) at the endothelial surface and into plasma. We hypothesized that the actions of these enzymes, particularly PR3 contribute to the loss of endothelial integrity.Patients with severe sepsis (sepsis with evidence of at least one organ dysfunctioning), were recruited on admission to the intensive care unit. Patient samples were paired with age and sex‐matched controls. The mean age was 50 ± 14 years, 3/10 males for both sepsis and controls. Septic patient pathogens were determined to be 4 gram positive, 4 gram negative and 2 culture negative; survival outcomes were 9/10 patients surviving. We employed ELISAs to determine plasma PR3 concentrations and in vitro permeability assays using Texas Red‐Dextran flow to determine the effect of PR3 on human umbilical vein endothelial cell (HUVEC) monolayer integrity.The sepsis plasma PR3 concentration was significantly higher (p &lt; 0.001) compared to controls (55.4 ±8.8 vs 7.7 ±2 ng/mL). For in vitro permeability assays, we employed concentrations of PR3 higher than the plasma measurements in order to more closely mimic conditions at the site of degranulation. 1 hour of PR3 treatment (5 μg/mL) significantly increased HUVEC permeability to Texas Red‐Dextran compared to controls.The loss of endothelial integrity secondary to PR3 release by adhered PMNs could result in microvascular dysfunction and capillary leak. Endothelial injury could also exacerbate further PMN recruitment. Since other enzymes are contained in the same granules as PR3, they may also have significant actions on endothelial cells, these will be studied in the future.We conclude that neutrophil degranulation, as evidenced by increased PR3 plasma concentrations, can lead to increased endothelial permeability through a mechanism which in part involves the actions of PR3.Support or Funding InformationFunded by the Lawson Health Research Institute Internal Research Fund.

Activation, Apoptosis, and Clearance of Neutrophils in Wegener's Granulomatosis

Wegener's granulomatosis (WG) is strongly associated with the presence of antineutrophil cytoplasmic autoantibodies (ANCAs). Within WG these ANCAs are usually (80-90%) directed against the azurophilic enzyme proteinase 3, the so called PR3-ANCA. A pathophysiological role for these autoantibodies, supported by numerous in vitro and in vivo studies, is specifically based on their capacity to bind and activate neutrophils and potentially may damage vessels. In this review, the pathogenic potential of different developmental stages of the neutrophil in the pathogenesis of WG is discussed. After release from the bone marrow into the circulation, neutrophils can be primed by TNFalpha and become attached to locally activated endothelium. Once attached to the endothelium, ANCAs can fully activate these primed neutrophils. In this activation process, the degree of activation after stimulation with PR3-ANCAs associates with the level of PR3 expression on the membrane of the neutrophil. Following activation, infiltrated neutrophils become apoptotic with further membrane expression of PR3. In WG patients, clearance of apoptotic neutrophils can be disturbed due to the opsonization of PR3-expressing apoptotic neutrophils with PR3-ANCAs, thereby perpetuating inflammation by the release of proinflammatory cytokines during clearance; or it may favor autoimmunity by PR3 presentation in an inflammatory environment. Furthermore, the presence of ANCAs and the release of the vessel-related pentraxin PTX3 may lead to the persistence of late apoptotic neutrophils in tissues, thereby inducing leukocytoclastic lesions that are characteristic in patients with WG. All together, alive neutrophils as well as apoptotic neutrophils play a key role in different inflammatory phenomena seen in patients suffering from WG.

Antineutrophil cytoplasmic antibodies: Major autoantigens, pathophysiology, and disease associations

Antineutrophil cytoplasmic antibodies (ANCA) are important serological markersfor the primary systemic vasculitides, including microscopic polyarteritis and necrotizing crescentic glomerulonephritis. Numerous reports have established the clinical utility of ANCA titer in monitoring disease activity, relapses, and response to treatment. ANCA, detected by indirect immunofluorescence (IIF) assays using patient's serum and ethanol-fixed human neutrophils, produce two common fluorescent staining patterns: cytoplasmic (C-ANCA), involving a 29-kD neutral serine protease termed proteinase 3 (PR3), and perinuclear (P-ANCA), the result mainly of myeloperoxidase (MPO), but occasionally by other components of the azurophilic granules including lysozyme, elastase, cathepsins, and lactoferrin. Some sera contain granulocyte-specific antinuclear antibodies (GS-ANA), which require formaldehyde fixation of neutrophils to cross link cytoplasmic antigens for distinguishing between ANCA and the GS-ANA by IIF. Positive IIF is confirmed by Western blot analysis or specific enzyme-linked immunosorbent assay for PR3, MPO, and other neutrophil granule antigens. The C-ANCA pattern is highly specific for Wegener's granulomatosis, a disease characterized by granulomatous inflammation, necrotizing and crescentic glomerulonephritis, and vasculitis; P-ANCA is found in sera of individuals with vasculitis, glomerulonephritis, and several other diseases. ANCA are predominantly immunoglobulin (Ig)G isotype, but may be IgM and IgA. Various pathophysiologic mechanisms have been proposed involving ANCA-mediated neutrophil activation in a hypothetical model of vasculitic diseases: positive signals via the FcγRII (CD32) receptor after IgG-ANCA binding to membrane-associated PR3, relevant cytokines, production of adhesion molecules on both activated neutrophils and endothelial cells, and the release of neutrophil reactive oxygen species and degranulation causing endothelial cell damage. Interference of C-ANCA with PR3 proteolysis and PR3 inhibition physiologically by the α 1-proteinase inhibitor may have a pathogenic role. No convincing data have been reported for the existence of autoreactive T lymphocytes reactive to any degree with the neutrophil azurophilic enzymes. Studies of various drug- and infectious agent-related diseases and ANCA may contribute to understanding the mechanism(s) involved in some vasculitides.

Functional activation of human neutrophils by recombinant monocyte-derived neutrophil chemotactic factor/IL-8.

Monocyte-derived neutrophil chemotactic factor (MDNCF)/IL-8, a novel cytokine, distinct from IL-1 and TNF was recently purified and cloned. This study was performed to investigate the biologic effect of recombinant MDNCF/IL-8 on human polymorphonuclear neutrophils (PMN) by assessment of their growth inhibitory activity against Candida albicans. The chemoattractant, FMLP was used as a positive control. We demonstrated that MDNCF/IL-8, similar to FMLP, effectively enhanced PMN-mediated anti-Candida activity. MDNCF/IL-8, from 1.0 to 1000 ng/mol, enhanced PMN-mediated anti-Candida activity, whereas FMLP was effective from 10(-10) to 10(-7) M. The optimal dose of MDNCF/IL-8 for PMN stimulation was 10 ng/ml which equalled the optimal chemoattractant dose. MDNCF/IL-8 itself, like FMLP, had no direct effect on Candida growth at any concentration and it stimulated antifungal activity only in PMN but not in monocytes. Interestingly, MDNCF/IL-8 failed to stimulate directly the production of superoxide from PMN or prime the respiratory burst of PMN exposed to FMLP. However, MDNCF/IL-8 was capable of releasing azurophilic enzymes from cytochalasin B-treated PMN into the extracellular space. Enhancement of PMN anti-Candida activity and release of azurophilic enzymes from PMN by MDNCF/IL-8 were inhibited in the presence of colchicine, which is a known inhibitor of degranulation. These results suggest that MDNCF/IL-8 induced antifungal action of PMN via oxygen-independent pathways. Furthermore, MDNCF/IL-8 induction of anti-Candida action by PMN was inhibited by pretreatment with Bordetella pertussis toxin, suggesting that enhancement of PMN antifungal activity by MDNCF/IL-8, as well as by FMLP, may be mediated by a GTP-binding protein.