Active MPO Research Articles

Recurrent ocular toxoplasmosis is associated with interferon-gamma deficiency possibly due to genetic origin

ObjectiveOcular toxoplasmosis (OT) can cause posterior uveitis; causes of recurrent OT are not well understood. We explored clinical, immunological and genetic properties associated with recurrent OT.Methods and analysisA recurrent OT...

Myeloperoxidase enzyme and Ferric-reducing antioxidant power concentrations in lean and obese dogs.

The study aimed to assess plasma Myeloperoxidase enzyme (MPO) and Ferric-reducing antioxidant power (FRAP) in obese dogs and compare them with ideal body weight dogs. Thirty-two dogs were distributed into two groups according to a 9-point body condition score (BCS), as follows: Control group (n = 16), dogs with a BCS of 4 or 5; Obese group (n = 16), dogs with a BCS of 8 or 9. Plasma MPO and FRAP assays, neutrophil count, lipid profile (cholesterol and triglycerides), and systolic blood pressure (SBP) were evaluated in both groups. The duration of obesity was defined based on history. The obese group showed higher values for body weight, BCS, SBP, neutrophil count, triglycerides, and MPO than the Control group. A positive correlation was observed between MPO concentrations and BCS and body weight. FRAP concentrations showed a positive correlation with the duration of obesity. The results suggested that an inflammatory state caused by obesity may promote increased neutrophil count and MPO concentrations, besides the positive correlation between MPO with BCS and body weight. The obesity in dogs promoted slight active MPO elevation, influenced by body weight, BCS, and neutrophil count. The FRAP assay did not show the expected reduction and, therefore, needs further investigation.

A novel glucocorticoid-free maintenance regimen for anti-neutrophil cytoplasm antibody-associated vasculitis.

Glucocorticoids (GCs) are a mainstay of treatment for patients with ANCA-associated vasculitis (AAV) but are associated with significant adverse effects. Effective remission induction in severe AAV using extremely limited GC exposure has not been attempted. We tested an early rapid GC withdrawal induction regimen for patients with severe AAV. Patients with active MPO- or PR3-ANCA vasculitis or ANCA-negative pauci-immune glomerulonephritis were included. Induction treatment consisted of two doses of rituximab, 3 months of low-dose CYC and a short course of oral GC (for between 1 and 2 weeks). Clinical, biochemical and immunological outcomes as well as adverse events were recorded. A total of 49 patients were included, with at least 12 months of follow-up in 46. All patients achieved remission, with decreases observed in creatinine, proteinuria, CRP, ANCA level and BVAS. Three patients requiring dialysis at presentation became dialysis independent. Two patients required the introduction of maintenance GC for treatment of vasculitis. Overall outcomes were comparable to those of two matched cohorts (n = 172) from previous European Vasculitis Society (EUVAS) trials, but with lower total exposure to CYC and GCs (P < 0.001) and reduced rates of severe infections (P = 0.02) compared with the RITUXVAS (rituximab versus cyclophosphamide in AAV) trial. We found no new cases of diabetes in the first year compared with historic rates of 8.2% from the EUVAS trials (P = 0.04). Early GC withdrawal in severe AAV is as effective for remission induction as the standard of care and is associated with reduced GC-related adverse events.

S115 The effects of hypoxia on neutrophil-mediated tissue damage in the lung

Sites of infection and inflammation are profoundly hypoxic, requiring neutrophils to function under low oxygen tensions. Although neutrophils are well adapted and can rely on glycolytic metabolism, hypoxia still impairs...

Myeloperoxidase associated with neutrophil extracellular traps is active and mediates bacterial killing in the presence of hydrogen peroxide

A variety of inflammatory stimuli induces NETs. These structures consist of a network of chromatin strands associated with predominately granule proteins, including MPO. NETs exhibit antimicrobial activity, which is proposed to augment the more-established mechanism of phagosomal killing. They may also be detrimental to the host in situations such as chronic inflammation or severe sepsis. The objective of this study was to establish whether MPO associated with NETs is active and able to kill bacteria. Neutrophils were stimulated with PMA to release NETs. Peroxidase activity measurements were performed and showed that enzymatically active MPO was released from the neutrophils, 2-4 h after stimulation, concomitant with NET formation. Approximately 30% of the total cellular MPO was released, with the majority bound to the NETs. The bound enzyme retained its activity. Staphylococcus aureus were not killed when added to preformed NETs under our assay conditions. However, addition of H(2)O(2) to the bacteria in the presence of NETs resulted in MPO-dependent killing, which was observed with NETs in situ and with NETs when they were removed from the neutrophils by limited DNase digestion. Our results show that the enzymatic activity of MPO on NETs could contribute to antimicrobial activity or tissue injury when NETs are released from neutrophils at sites of infection or inflammation.

Differentiation Dependent Dynamics of Histone Modifications during Myelopoiesis.

Covalent modifications on histones are epigenetic changes that play critical roles in control of gene expression. Most studies correlating specific histone modifications with transcriptional activity have been performed in yeast, and little is known about their dynamics during hematopoietic differentiation. We examined the dynamics of histone modifications and chromatin accessibility during all-trans retinoic acid (ATRA) induced differentiation of promyelocytes down the neutrophil lineage. As a model system, we used the human promyelocytic NB4 cell line, which undergoes neutrophil differentiation in response to ATRA. Using chromatin immunoprecipitation (ChIP) and quantitative PCR, we measured changes in dimethyl K4 (2MeH3K4), trimethyl K4 (3MeH3K4) and acetyl lysine 9 (Ac9H3K9) of histone H3 in the promoters of 3 genes that undergo transcriptional activation (Defensin-a, C/EBP-b and RAR-b), 1 gene that undergoes transcriptional downregulation (Myeloperoxidase), one gene that is constitutively active (GAPDH) and 1 gene that is silent (Albumin) during ATRA-induced differentiation. We correlated the changes in histone modifications with the gene expression pattern of these genes. AcH3K9 levels correlated with active gene transcription. At time 0, levels of AcH3K9 were enriched 50-fold and 100-fold over input in the MPO and GAPDH promoters, respectively, but only 5-fold on the C/EBP-b, Def-a and RAR-b promoters. Consistent with this finding, levels of AcH3K9 increased to 40-fold over input within 24h of differentiation in the Def-a and C/EBP-b promoters. On the silent albumin promoter, AcH3K9 levels never increased over input after ATRA. For the methylation patterns on H3K4, however, the findings were quite revealing. As expected, on the active MPO promoter, 2MeH3K4 was enriched 60-fold. However, 2MeH3K4was also present at high levels (15-30-fold) on the promoters of unexpressed Def-a, C/EBP-b and RAR-b suggesting that silent genes that are “primed” for activation are enriched for 2MeH3K4, consistent with previous data in yeast. After differentiation with ATRA, 2MeH3K4 went up only 3-fold for Def-a and C/EBP-b. The most surprising changes were found in 3MeH3K4 levels and in chromatin modification at the RAR-b promoter. Consistent with previous data showing that 3MeH3K4 is associated with gene activation, time 0 levels of 3MeH3K4 were enriched 80-fold and 150-fold over input in the MPO and GAPDH promoters, respectively, and were at background levels in the C/EBP-b, Def-a, and RAR-b promoters. After differentiation, however, although there was a significant increase in 3MeH3K4 levels within 12 hours in the Def-a promoter, 3MeH3K4 never was present on the C/EBP-b promoter, despite a huge increase in gene transcription, as well as significant and rapid increases in AcH3K9 and 2MeH3K4. Perhaps the most significant findings, however, were at the promoter of the RAR-b gene. RAR-b is unique amongst the genes studies in that it is directly bound by the PML-RAR-a fusion protein. This promoter was not detectable by PCR in ChIP assays after ATRA addition. To test the hypothesis that histones were lost on the RAR-b promoter during ATRA-induced differentiation, we used MNase digestion of chromatin. Nucleosome loss was confirmed by a decrease in precipitated RAR-b promoter DNA within 24 hours of ATRA addition. In contrast, exon1 of RAR-b was modified in a manner similar to Def-a and C/EBP-b. These findings indicate that despite the seemingly coordinately regulated increase in transcription of multiple genes upon myeloid differentiation, the chromatin modifications on the promoters of these genes are regulated quite differently.

Neutrophil myeloperoxidase revisited: it's role in health and disease

Human myeloperoxidase (MPO; EC 1.11.1.7) is a specific heme (Fe3+) peroxidase, present in high concentrations in the azurophilic granules of neutrophils. Its amino acid and genomic sequences have been elucidated, and recombinant MPO is produced from genetically engineered mammalian cells. This peroxidase has the unique activity of chlorination, generating hypochlorous acid (HOCl) from hydrogen peroxide and chloride anion, but also chlorine and monoatomic chlorine. By interacting with other enzymes of neutrophils and reacting with the products of neutrophil activation, MPO also produces other reactive oxygen species (singlet oxygen, hydroxyl radical, nitrosyl and nitryl chloride, etc.). In phagolysosomes, MPO acts together with NADPH oxidase and proteases for the destruction of the ingested organisms, by binding to the microorganism walls and producing locally HOCl, which is particularly active against the polysaccharidic capsules. MPO activity influences the transduction of the cellular signal (activation of NF-κB, chlorination of tyrosyl residues on essential enzymes, etc.) and modulates the functions of cells: it decreases the killer activity of NT lymphocytes and, after internalization, it enhances the microbial activity of macrophages. MPO is taken up by endothelial cells. MPO deficiency is the most common neutrophilic lysosomal enzyme deficiency, but usually without apparent increased susceptibility to infection or altered inflammatory response. MPO has been recognized to be responsible for the oxidation and chlorination of low density lipoproteins, contributing to the early stage of atherosclerosis. In disease with excessive and uncontrolled inflammatory reaction, MPO can be released in the extracellular milieu where it becomes cytotoxic for neighboring cells (oxidant stress) and oxidizes tissues and proteins (thiol oxidation, oxidation and chlorination of lipids and amino acids, etc.). Out of the neutrophil, the activity of MPO would be quickly inhibited by proteins; however, active MPO has been measured in broncho-alveolar lavage fluids from patients with acute lung injury. This specific enzyme, thus, presents a double role of essential host protection when acting into the phagocytes and of host damage when released in the extracellular milieu.

Cytotoxicity towards human endothelial cells, induced by neutrophil myeloperoxidase: protection by ceftazidime

We investigated the effects of the antibiotic ceftazidime (CAZ) on the cytolytic action of the neutrophil myeloperoxidase–hydrogen peroxide–chloride anion system (MPO/H2O2/Cl−). In this system, myeloperoxidase catalyses the conversion of H2O2 and CI− to the cytotoxic agent HOCl. Stimulated neutrophils can release MPO into the extracellular environment and then may cause tissue injury through direct endothelial cells lysis. We showed that human umbilical vein endothelial cells (HUVEC) were capable of taking up active MPO. In presence of H2O2 (10−4 M), this uptake was accompanied by cell lysis. The cytolysis was estimated by the release of 51Cr from HUVEC and expressed as an index of cytotoxicity (IC). Dose dependent protection was obtained for CAZ concentrations ranging from 10−5 to 10−3 M;this can be attributed to inactivation of HOCl by the drug. This protection is comparable to that obtained with methionine and histidine, both of which are known to neutralize HOCl. This protection by CAZ could also be attributed to inactivation of H2O2, but when cytolysis was achieved with H2O2 or O2- generating enzymatic systems, no protection by CAZ was observed. Moreover, the peroxidation activity of MPO (action on H2O2) was not affected by CAZ, while CAZ prevented the chlorination activity of MPO (chlorination of monochlorodimedon). So, we concluded that CAZ acts via HOCl inactivation. These antioxidant properties of CAZ may be clinically useful in pathological situations where excessive activation of neutrophils occurs, such as in sepsis.

Cellular and extracellular myeloperoxidase in pyogenic inflammation

We explored the effect of in vitro phagocytosis and in vivo inflammation on the MPO content of functioning neutrophils and on the ability of these cells to export active MPO into the extracellular environment. After ingestion of staphylococci, neutrophils retained 52% of their MPO and released 8% into the medium in active form; the remaining 40% of their MPO could no longer be detected. During bacterial infection induced by intradermally injecting staphylococci, neutrophils harvested from minced infected lesions contained 52% of the MPO of circulating neutrophils that had not reached the lesions. Extracellular fluid from the lesions contained active MPO secreted by the neutrophils, and concentrations of 10–45 U/ml were detected. These data demonstrate that functioning neutrophils can lose approximately half of their MPO. In vitro, 4%-8% of neutrophilic MPO appears in the extracellular space and 40% is inactivated. In vivo, the MPO content of inflammatory neutrophils also decreases, and MPO appears in the extracellular fluid in active form where it is available to participate in a variety of physiologic processes.

Cellular and Extracellular Myeloperoxidase in Pyogenic Inflammation

We explored the effect of in vitro phagocytosis and in vivo inflammation on the MPO content of functioning neutrophils and on the ability of these cells to export active MPO into the extracellular environment. After ingestion of staphylococci, neutrophils retained 52% of their MPO and released 8% into the medium in active form; the remaining 40% of their MPO could no longer be detected. During bacterial infection induced by intradermally injecting staphylococci, neutrophils harvested from minced infected lesions contained 52% of the MPO of circulating neutrophils that had not reached the lesions. Extracellular fluid from the lesions contained active MPO secreted by the neutrophils, and concentrations of 10-45 U/ml were detected. These data demonstrate that functioning neutrophils can lose approximately half of their MPO. In vitro, 4%-8% of neutrophilic MPO appears in the extracellular space and 40% is inactivated. In vivo, the MPO content of inflammatory neutrophils also decreases, and MPO appears in the extracellular fluid in active form where it is available to participate in a variety of physiologic processes.