Azurophilic Granules Of Neutrophils Research Articles

Neutrophil myeloperoxidase harbors distinct site-specific peculiarities in its glycosylation

Anti-neutrophil cytoplasmic autoantibodies (ANCAs) are directed against lysosomal components of neutrophils. ANCAs directed to proteinase 3 and myeloperoxidase (MPO) in particular are associated with distinct forms of small vessel vasculitides. MPO is an abundant neutrophil-derived heme protein that is part of the antimicrobial defense system. The protein is typically present in the azurophilic granules of neutrophils, but a large portion may also enter the extracellular space. It remains unclear why MPO is frequently the target of antibody-mediated autoimmune responses. MPO is a homodimeric glycoprotein, posttranslationally modified with complex sugars at specific sites. Glycosylation can strongly influence protein function, affecting its folding, receptor interaction, and backbone accessibility. MPO potentially can be heavily modified as it harbors 5 putative N-glycosylation sites (10 in the mature dimer). Although considered important for MPO structure and function, the full scope and relative abundance of the glycans attached to MPO is unknown. Here, combining bottom-up glycoproteomics and native MS approaches, we structurally characterized MPO from neutrophils of healthy human donors. We quantified the relative occupancy levels of the glycans at each of the five sites and observed complex heterogeneity and site-specific glycosylation. In particular, we detected glycosylation phenotypes uncommon for glycoproteins in the extracellular space, such as a high abundance of phosphorylated high-mannose species and severely truncated small glycans having the size of paucimannose or smaller. We hypothesize that the atypical glycosylation pattern found on MPO might contribute to its specific processing and presentation as a self-antigen by antigen-presenting cells.

The effect of myeloperoxidase isoforms on biophysical properties of red blood cells.

Myeloperoxidase (MPO), an oxidant-producing enzyme, stored in azurophilic granules of neutrophils has been recently shown to influence red blood cell (RBC) deformability leading to abnormalities in blood microcirculation. Native MPO is a homodimer, consisting of two identical protomers (monomeric MPO) connected by a single disulfide bond but in inflammatory foci as a result of disulfide cleavage monomeric MPO (hemi-MPO) can also be produced. This study investigated if two MPO isoforms have distinct effects on biophysical properties of RBCs. We have found that hemi-MPO, as well as the dimeric form, bind to the glycophorins A/B and band 3protein on RBC's plasma membrane, that lead to reduced cell resistance to osmotic and acidic hemolysis, reduction in cell elasticity, significant changes in cell volume, morphology, and the conductance of RBC plasma membrane ion channels. Furthermore, we have shown for the first time that both dimeric and hemi-MPO lead to phosphatidylserine (PS) exposure on the outer leaflet of RBC membrane. However, the effects of hemi-MPO on the structural and functional properties of RBCs were lower compared to those of dimeric MPO. These findings suggest that the ability of MPO protein to influence RBC's biophysical properties depends on its conformation (dimeric or monomeric isoform). It is intriguing to speculate that hemi-MPO appearance in blood during inflammation can serve as a regulatory mechanism addressed to reduce abnormalities on RBC response, induced by dimeric MPO.

MPO as a Novel Susceptibility Gene in Myeloid Malignancies

Myeloperoxidase (MPO) on chromosome 17q22 codes for a heme-containing protein produced following commitment and during myeloid differentiation. Ultimately, mature neutrophil azurophilic granules have MPO as their major component. Consistently, high MPO expression, suggestive of a more differentiated type, has been associated with favorable outcomes in acute myeloid leukemia (AML). MPO deficiency is an extremely rare autosomal recessive disorder caused by any of a number of known germline variants, though the propensity to develop myeloid malignancy has not been reported for affected individuals. Somatic MPO mutations have not be identified in myeloid neoplasia (MN). In an effort to identify novel susceptibility genes and variants, we analyzed whole-exome sequence data from the germlines of 690 MN patients. Among all genes examined, MPO carried the highest burden of variants deemed pathogenic or likely pathogenic under the American College of Medical Genetics (ACMG) criteria; 19/690 MN patients (2.7%) carried a pathogenic/likely pathogenic MPO variant. Carriers of pathogenic MPO variants had higher rates of cytogenetic lesions, particularly -5 and complex karyotypes (Fig. A). The pathogenic variants found in our cohort are dominated by two substitutions: a splice site variant (c.2031-2A>C; eleven patients), and a missense variant (c.C1705T, p.R569W; seven patients) (Fig. B). Multi-species alignment shows that both of the corresponding wild-type alleles are highly conserved. Furthermore, both variants have significantly higher frequencies in our cohort (0.8% and 0.51%, respectively), as well as in the recently-published Beat AML patient cohort (0.8% and 0.6%), as compared to control populations (0.38% and 0.13% in gnomAD; OR 1.9, 95% CI 1.1-3.5, P<0.05 for c.2031-2A>C and OR 3.6, 95% CI 1.9-8.0, P<.0005 for p.R569W) (Fig. C). Of note is that the c.2031-2A>C variant has previously been reported in a family with hereditary MPO deficiency, with a splice variant-induced retention of intron 11 in MPO messenger RNA, inducing a premature stop codon. Another study showed that the R569W variant results in maturation arrest in the processing of MPO precursors. Analysis of the 3D structure of MPO shows that the R569W variant will abrogate heme binding (Fig. D). We next sought to validate our findings in an independent cohort. To this end, we queried next-generation DNA and RNA sequence from 1,119 myeloid malignancy patients from the Munich Leukemia Laboratory (MLL). Similar to our discovery cohort and the Beat AML group, the c.2031-2A>C variant had significantly higher allele frequency in confirmatory cohort (0.8%) than in population controls (0.38%), though the R569W did not (Fig. C). We found that MPO expression is significantly lower in carriers of the c.2031-2A>C allele (P = 0.024), as expected since the variant affects mRNA processing. There have been no prior reports of MPO as a MN susceptibility gene, but it is an appealing candidate. Taken together with evidence from earlier studies characterizing the impact of the variants highlighted here, our results support the hypothesis that inherited disruption of MPO confers myeloid malignancy susceptibility. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

The N125S polymorphism in the cathepsin G gene (rs45567233) is associated with susceptibility to osteomyelitis in a Spanish population.

Osteomyelitis is a bone infection, most often caused by Staphylococcus aureus, in which neutrophils play a key role. Cathepsin G (CTSG) is a bactericidal serine protease stored in the neutrophil azurophilic granules. CTSG regulates inflammation, activating matrix metalloproteinases (MMPs), and coagulation. Lactoferrin (LF), a neutrophil glycoprotein, increases CTSG catalytic activity and induces inflammation. The aim of this study was to analyze a potential association between a CTSG gene polymorphism (Asn125Ser or N125S, rs45567233), that modifies CTSG activity, and could affect susceptibility to, or outcome of, bacterial osteomyelitis. CTSG N125S polymorphism was genotyped in 329 osteomyelitis patients and 415 controls), Blood coagulation parameters, serum CTSG activity, LF, MMP-1, MMP-13, and soluble receptor activator for nuclear factor κ B ligand (sRANKL) levels were assessed in carriers of the different CTSG genotypes. CTSG N125S (AG) genotype was significantly more frequent among osteomyelitis patients than controls (15.5% vs. 9.4%, p = 0.014). CTSG N125S variant G allele (AG +GG) was also more frequent among osteomyelitis patients (8.1% vs. 4.7%, p = 0.01). Serum CTSG activity and LF levels were significantly higher in osteomyelitis patients carrying the G allele compared to those with the AA genotype, (p<0.04). Serum MMP-1 was lower in the G allele carriers (p = 0.01). There was no association between these genotypes and clinical characteristics of osteomyelitis, or coagulation parameters, MMP-13, and sRANKL serum levels. Differences in the CTSG gene might enhance osteomyelitis susceptibility by increasing CTSG activity and LF levels.

Association of monocyte myeloperoxidase with incident cardiovascular disease: The Atherosclerosis Risk in Communities Study.

Myeloperoxidase (MPO) is a heme-containing peroxidase found in azurophilic granules of neutrophils and monocytes. Epidemiological studies have reported greater plasma MPO concentration to be associated with increased incidence of several cardiovascular diseases (CVD), but the association of intracellular monocyte MPO (mMPO) with CVD is unclear. The prospective population-based Atherosclerosis Risk in Communities (ARIC) cohort study measured mMPO using flow cytometry in 1,465 participants. The association of mMPO with incident cardiovascular disease (CVD, comprising incident coronary heart disease (CHD), heart failure, stroke, peripheral artery disease, and cardiovascular mortality) was examined over a median 9.6 years of follow-up (n = 290 CVD events). There was no statistically significant association between mMPO and all incident CVD events in either age, sex, and race-adjusted proportional hazards models (HR (95% CI) across tertiles of mMPO: 1, 1.09 (0.76, 1.57), and 0.78 (0.52, 1.15), P-trend = 0.21) or adjusted for other major CVD risk factors (HR (95% CI): 1, 1.17 (0.81, 1.69), and 0.87 (0.58, 1.29), P-trend = 0.50). There also was no association between mMPO tertiles and incident CHD, heart failure, or all-cause mortality, examined separately. In conclusion, intracellular monocyte myeloperoxidase was not associated with incident cardiovascular disease in this prospective population-based study.

Evaluation of the Functional Condition of Human Blood Neutrophills Based on the Registrationof Active Halogen Forms Production

The development of adequate methods for monitoring the functional state of neutrophils is an urgent task due to their important role in the development of the inflammatory response. In this study, the functional activity of neutrophils has been assessed using a complex approach, including the registration of generation of ROS by neutrophils as a result of the functioning of the NADPH oxidase complex; degranulation of azurophilic granules; as well as production of AFG, formed in reactions catalyzed by the enzyme azurophilic granules of neutrophils – MPO in the presence of substrates (Н2О2 and halide ions).

New Insights into the Role of Zinc Acquisition and Zinc Tolerance in Group A Streptococcal Infection.

Zinc plays an important role in host innate immune function. However, the innate immune system also utilizes zinc starvation ("nutritional immunity") to combat infections. Here, we investigate the role of zinc import and export in the protection of Streptococcus pyogenes (group A Streptococcus; GAS), a Gram-positive bacterial pathogen responsible for a wide spectrum of human diseases, against challenge from host innate immune defense. In order to determine the role of GAS zinc import and export during infection, we utilized zinc import (ΔadcA ΔadcAII) and export (ΔczcD) deletion mutants in competition with the wild type in both in vitro and in vivo virulence models. We demonstrate that nutritional immunity is deployed extracellularly, while zinc toxicity is utilized upon phagocytosis of GAS by neutrophils. We also show that lysosomes and azurophilic granules in neutrophils contain zinc stores for use against intracellular pathogens.

Myeloperoxidase Exocytosis from Activated Neutrophils in the Presence of Heparin

Exocytosis of myeloperoxidase (MPO) from activated neutrophils has been investigated in the presence of the anionic polysaccharide heparin. The optimal concentration of heparin (0.1 U/mL), which did not cause additional activation of cells (lack of augmentation of lysozyme exocytosis from specific and azurophilic granules), was determined. After preincubation of cells with heparin (0.1 U/mL) MPO exocytosis from neutrophils was stimulated by various activators (fMLP, PMA, plant lectins CABA and PHA-L) and was higher as compared to the effects of the activators alone. Experiments performed using MPO isolated from leukocytes have shown that heparin in the range of concentrations 0.1–50 U/mL had no effect on MPO peroxidase activity. Thus, the use of heparin at a concentration of 0.1 U/mL avoids the artifact caused by the “loss” of MPO due to its binding to neutrophils and increases the accuracy of the method of registration of degranulation of neutrophil azurophilic granules based on determination of the MPO concentration or its peroxidase activity in cell supernatants.

Native and myeloperoxidase-oxidized low-density lipoproteins act in synergy to induce release of resolvin-D1 from endothelial cells

Background and aimsOxidation of native low-density lipoproteins (LDLs-nat) plays an important role in the development of atherosclerosis. A major player in LDL-nat oxidation is myeloperoxidase (MPO), a heme enzyme present in azurophil granules of neutrophils and monocytes. MPO produces oxidized LDLs called Mox-LDLs, which cause a pro-inflammatory response in human microvascular endothelial cells (HMEC), monocyte/macrophage activation and formation of foam cells. Resolvin D1 (RvD1) is a compound derived from the metabolism of the polyunsaturated fatty acid DHA, which promotes resolution of inflammation at the ng/ml level. MethodsIn the present study, we used liquid chromatography–mass spectrometry (LC-MS/MS) to investigate the synthesis of RvD1 and its precursors - 17(S)-hydroxy docosahexaenoic acid (17S-HDHA) and docosahexaenoic acid (DHA) - by HMEC, in the presence of several concentrations of Mox-LDLs, copper-oxidized-LDLs (Ox-LDLs), and native LDLs or in mouse plasma. The LC-MS/MS method has been validated and applied to cell supernatants and plasma to measure production of RvD1 and its precursors in several conditions. ResultsMox-LDLs played a significant role in the synthesis of RvD1 and 17S-HDHA from DHA compared to Ox-LDLs. Moreover, Mox-LDLs and LDLs-nat acted in synergy to produce RvD1. In addition, different correlations were found between RvD1 and M1 macrophages, age of mice or Cl-Tyr/Tyr ratio. ConclusionsThese results suggest that although Mox-LDLs are known to be pro-inflammatory and deleterious in the context of atherosclerosis, they are also able to induce a pro-resolution effect by induction of RvD1 from HMEC. Finally, our data also suggest that HMEC can produce RvD1 on their own.

Exocytosis of myeloperoxidase from activated neutrophils in the presence of heparin

Exocytosis of myeloperoxidase (MPO) from activated neutrophils in the presence of the anionic polysaccharide heparin was studied. It was determined that the optimal concentration of heparin (0.1 u/ml), at which there is no additional activation of cells (absence of amplification of exocytosis of lysozyme contained in specific and azurophilic granules). It was found that after preincubation of cells with heparin (0.1 u/ml) the exocytosis of MPO from neutrophils activated by various stimulants (fMLP, PMA, plant lectins CABA and PHA-L) increased compared to that under the action of activators alone. In addition, it was shown that heparin in the range of concentrations 0.1-50 u/ml did not affect on the peroxidase activity of the MPO isolated from leukocytes. Thus, the use of heparin at a concentration of 0.1 u/ml avoids the artifact caused by the "loss" of MPO in a result of its binding to neutrophils, and increases the accuracy of the method of registration the degranulation of azurophilic granules of neutrophils based on determination of the concentration or peroxidase activity of MPO in cell supernatants.

Myeloperoxidase can differentiate between sepsis and non-infectious SIRS and predicts mortality in intensive care patients with SIRS

BackgroundSystemic inflammatory response syndrome (SIRS) is a clinical syndrome following inflammation. Clinically, it is difficult to distinguish SIRS following an infection, i.e., sepsis, from non-infectious SIRS. Myeloperoxidase is a hemeprotein stored in the neutrophil azurophilic granules and is one of the main pillars of neutrophil attack. Therefore, we hypothesized that myeloperoxidase can differentiate between sepsis and non-infectious SIRS in patients with systemic inflammatory response syndrome in the intensive care unit (ICU).MethodsAn observational single-center cohort study was conducted measuring myeloperoxidase in patients with SIRS in the first 48 h after admission. The outcomes were established using predefined definitions. Thirty-day mortality was retrospectively assessed.ResultsWe found significantly higher levels of myeloperoxidase in patients with sepsis and septic shock compared to patients without sepsis (60 ng/ml versus 43 ng/ml, P = 0.002). Myeloperoxidase levels were related to 30-day mortality (P = 0.032), and high MPO levels on top of a high APACHE IV score further increased mortality risk.ConclusionsWe show that myeloperoxidase is a potentially novel biomarker for sepsis in the ICU. Myeloperoxidase could eventually help in diagnosing sepsis and predicting mortality. However, more research is necessary to confirm our results.

Profile of the ProAxsis active neutrophil elastase immunoassay for precision medicine in chronic respiratory disease

ABSTRACTIntroduction: Neutrophil elastase (NE) is a 29kDa serine protease released from the azurophilic granules of neutrophils. It may be directly involved in the pathogenesis and disease progression in cystic fibrosis, bronchiectasis and COPD through the degradation of airway elastin and by impairing host defence.Areas covered: Measurement of NE activity has emerged as a promising biomarker strategy in inflammatory lung disease. The authors review studies where NE activity has been linked with clinical outcomes such as lung function decline, exacerbation frequency or other cross-sectional and longitudinal markers of disease severity. In this article the evidence for NE measurement, and the strengths and weaknesses of a commercially available immunoassay which can specifically detect NE activity in human biological samples such as sputum and bronchoalveolar lavage are reviewed.Expert commentary: NE is a promising biomarker for stratifying severity disease. NE also appears to be responsive to antibiotic and other treatments, potentially therefore allowing it to be used as an indicator of treatment response in clinical trials.

Toolbox of Fluorescent Probes for Parallel Imaging Reveals Uneven Location of Serine Proteases in Neutrophils.

Neutrophils, the front line defenders against infection, express four serine proteases (NSPs) that play roles in the control of cell-signaling pathways and defense against pathogens and whose imbalance leads to pathological conditions. Dissecting the roles of individual NSPs in humans is problematic because neutrophils are end-stage cells with a short half-life and minimal ongoing protein synthesis. To gain insight into the regulation of NSP activity we have generated a small-molecule chemical toolbox consisting of activity-based probes with different fluorophore-detecting groups with minimal wavelength overlap and highly selective natural and unnatural amino acid recognition sequences. The key feature of these activity-based probes is the ability to use them for simultaneous observation and detection of all four individual NSPs by fluorescence microscopy, a feature never achieved in previous studies. Using these probes we demonstrate uneven distribution of NSPs in neutrophil azurophil granules, such that they seem to be mutually excluded from each other, suggesting the existence of unknown granule-targeting mechanisms.

Neutrophil azurophilic granule exocytosis is primed by TNF-α and partially regulated by NADPH oxidase.

Neutrophil (polymorphonuclear leukocyte) activation with release of granule contents plays an important role in the pathogenesis of acute lung injury, prompting clinical trials of inhibitors of neutrophil elastase. Despite mounting evidence for neutrophil-mediated host tissue damage in a variety of disease processes, mechanisms regulating azurophilic granule exocytosis at the plasma membrane, and thus release of elastase and other proteases, are poorly characterized. We hypothesized that azurophilic granule exocytosis would be enhanced under priming conditions similar to those seen during acute inflammatory events and during chronic inflammatory disease, and selected the cytokine TNF-α to model this invitro. Neutrophils stimulated with TNF-α alone elicited intracellular reactive oxygen species (ROS) generation and mobilization of secretory vesicles, specific, and gelatinase granules. p38 and ERK1/2 MAPK were involved in these components of priming. TNF-α priming alone did not mobilize azurophilic granules to the cell surface, but did markedly increase elastase release into the extracellular space in response to secondary stimulation with N-formyl-Met-Leu-Phe (fMLF). Priming of fMLF-stimulated elastase release was further augmented in the absence of NADPH oxidase-derived ROS. Our findings provide a mechanism for host tissue damage during neutrophil-mediated inflammation and suggest a novel anti-inflammatory role for the NADPH oxidase.

Secretion from Myeloid Cells: Secretory Lysosomes.

Many cells of the myeloid lineage use an unusual secretory organelle to deliver their effector mechanisms. In these cells, the lysosomal compartment is often modified not only to fulfill the degradative functions of a lysosome but also as a mechanism for secreting additional proteins that are found in the lysosomes of each specialized cell type. These extra proteins vary from one cell type to another according to the specialized function of the cell. For example, mast cells package histamine; cytotoxic T cells express perforin; azurophilic granules in neutrophils express antimicrobial peptides, and platelets von Willebrand factor. Upon release, these very different proteins can trigger inflammation, cell lysis, microbial death, and clotting, respectively, and hence deliver the very different effector mechanisms of these different myeloid cells.

Myeloperoxidase Stimulates Neutrophil Degranulation.

Myeloperoxidase, heme enzyme of azurophilic granules in neutrophils, is released into the extracellular space in the inflammation foci. In neutrophils, it stimulates a dose-dependent release of lactoferrin (a protein of specific granules), lysozyme (a protein of specific and azurophilic granules), and elastase (a protein of azurophilic granules). 4-Aminobenzoic acid hydrazide, a potent inhibitor of peroxidase activity of myeloperoxidase, produced no effect on neutrophil degranulation. Using signal transduction inhibitors (genistein, methoxyverapamil, wortmannin, and NiCl2), we demonstrated that myeloperoxidase-induced degranulation of neutrophils resulted from enzyme interaction with the plasma membrane and depends on activation of tyrosine kinases, phosphatidylinositol 3-kinases (PI3K), and calcium signaling. Myeloperoxidase modified by oxidative/halogenation stress (chlorinated and monomeric forms of the enzyme) lost the potency to activate neutrophil degranulation.

Relationship between glycemic control and histochemical myeloperoxidase activity in neutrophils in patients with type 2 diabetes.

BackgroundMyeloperoxidase (MPO) is a lysosomal hemoprotein found in the azurophilic granules in neutrophils. Myeloperoxidase plays an important role in oxygen-dependent killing of bacteria, fungi, virus and malignant cells. Diabetes mellitus (DM) is listed among conditions that may lead to secondary MPO deficiency in neutrophils but inconsistent results concerning MPO activity in diabetic patients have been reported in the literature. In this study, we aimed to evaluate the relationship between glycemic control in patients with type 2 DM and MPO activity in neutrophils from a histochemical perspective.MethodsThe study included 40 patients with type 2 DM with poor glycemic control, 30 patients with type 2 DM with good glycemic control and 31 healthy controls. Peripheral blood smears were analyzed for each patient included in the study. Myeloperoxidase dye was used for staining. Myeloperoxidase ratios in neutrophil were evaluated for proportions of staining with MPO in 100 neutrophils in each smear. SPSS 16.0 version was used for statistical analyses.ResultsMyeloperoxidase ratios in neutrophils were 70 (58.5–80) in type 2 DM patients with poor glycemic control compared to 80 (73.75–90) in those with good glycemic control and 88 (78–92) in healthy controls. The DM group with poor glycemic control was statistically significantly different from the other groups (p < 0.001).ConclusionsPoor glycemic control in diabetic patients results in decreased MPO activity in neutrophils histochemically.

The voltage-gated proton channel Hv1/VSOP inhibits neutrophil granule release.

Neutrophil granule exocytosis is crucial for host defense and inflammation. Neutrophils contain 4 types of granules, the exocytotic release of which is differentially regulated. This exocytosis is known to be driven by diverse mediators, including calcium and nucleotides, but the precise molecular mechanism remains largely unknown. We show in the present study that voltage-gated proton (Hv) channels are necessary for the proper release of azurophilic granules in neutrophils. On activation of NADPH oxidase by PMA and IgG, neutrophils derived from Hvcn1 gene knockout mouse exhibited greater secretion of MPO and elastase than WT cells. In contrast, release of LTF enriched in specific granules was not enhanced in these cells. The excess release of azurophilic granules in Hv1/VSOP-deficient neutrophils was suppressed by inhibiting NADPH oxidase activity and, in part, by valinomycin, a potassium ionophore. In addition, Hv1/VSOP-deficient mice exhibited more severe lung inflammation after intranasal Candida albicans infection than WT mice. These findings suggest that the Hv channel acts to specifically dampen the release of azurophilic granules through, in part, the suppression of increased positive charges at the plasma membrane accompanied by the activation of NADPH oxidase in neutrophils.

Synthesis and pharmacological characterization of 2-aminobenzaldehyde oxime analogs as dual inhibitors of neutrophil elastase and proteinase 3

Proteinase 3 (Pr3), and human neutrophil elastase (HNE) are two major neutrophilic serine proteases (NsPs) expressed in neutrophil azurophil granules. Emerging data suggest that excessive release of proteases mediates tissue damage, and therefore prolonged neutrophil accumulation has an important role in the pathogenesis of many diseases. Thus, HNE and Pr3 inhibitors may prove to be targets for the generation of agents in the treatment of neutrophilic inflammatory disease. Sivelestat is the only commercially available selective HNE inhibitor. Therefore, sivelestat was chosen as the model structure in an attempt to obtain more potent anti-NsPs agents. In the present study, a series 2-aminobenzaldehyde oxime and 2-aminobenzoate analogs were synthesized and their inhibitory effects on NsPs (CatG, Pr3, and HNE) were determined, respectively. The results of structure–activity relationships studies concluded that a hydroxyl oxime moiety plays an important role in ligand–enzyme affinity through hydrogen bonding. As compound 6 had more potency and showed dual inhibitory effects on NE and Pr3, both in vitro and in vivo experiments were carried out to evaluate its selectivity, effects in cell-based assays, and efficacy in models of inflammation and damage. Compound 6 had the potential to reduce paw edema induced by LPS and HNE, as well as acute lung injury, and may be approved as a candidate for the development of new agents in the treatment of neutrophilic inflammatory diseases.

The neutrophil serine protease PR3 induces shape change of platelets via the Rho/Rho kinase and Ca2 + signaling pathways

IntroductionProteinase 3 (PR3) is released from neutrophil azurophilic granules and exerts complex effects on the inflammatory process. PR3 catalyzes the degradation of a number of macromolecules, but the consequences on blood cells are less well defined. In the present study, the effect of PR3 on human platelets was thoroughly investigated. MethodsThe experiments were performed on washed platelets freshly isolated from blood donated by healthy human volunteers. Platelets shape change and aggregation was measured on a Chrono-Log aggregometer. The phosphorylated form of MYPT1 was visualized by immunostaining. Platelet activation was further evaluated by flow cytometry. ResultsPR3 induced platelet shape change but not aggregation. Flow cytometry analysis showed that PR3 induced no P-selectin expression or binding of fibrinogen to the platelets, and it did not change the activation in response to PAR1- or PAR4-activating peptides or to thrombin. Furthermore, Fura-2 measurement and immuno-blotting analysis, respectively, revealed that PR3 stimulated small intracellular Ca2+ mobilization and Thr696-specific phosphorylation of the myosin phosphatase target subunit 1 (MYPT1). Separate treatment of platelets with the Rho/Rho kinase inhibitor Y-27632 and the intracellular Ca2+ chelator BAPTA/AM reduced the shape change induced by PR3 whereas concurrent treatment completely inhibited it. ConclusionThe data shows that the neutrophil protease PR3 is a direct modulator of human platelets and causes shape change through activation of the Rho/Rho kinase and Ca2+ signaling pathways. This finding highlights an additional mechanism in the complex interplay between neutrophils and platelets.