Myeloperoxidase Pathway Research Articles

Dynamics of redox signaling in aging via autophagy, inflammation, and senescence.

Review paper attempts to explain the dynamic aspects of redox signaling in aging through autophagy, inflammation, and senescence. It begins with ROS source in the cell, then states redox signaling in autophagy, and regulation of autophagy in aging. Next, we discuss inflammation and redox signaling with various pathways involved: NOX pathway, ROS production via TNF-α, IL-1β, xanthine oxidase pathway, COX pathway, and myeloperoxidase pathway. Also, we emphasize oxidative damage as an aging marker and the contribution of pathophysiological factors to aging. In senescence-associated secretory phenotypes, we link ROS with senescence, aging disorders. Relevant crosstalk between autophagy, inflammation, and senescence using a balanced ROS level might reduce age-related disorders. Transducing the context-dependent signal communication among these three processes at high spatiotemporal resolution demands other tools like multi-omics aging biomarkers, artificial intelligence, machine learning, and deep learning. The bewildering advancement of technology in the above areas might progress age-related disorders diagnostics with precision and accuracy.

Genetic variants influencing elevated myeloperoxidase levels increase risk of stroke.

Primary intracerebral haemorrhage and lacunar ischaemic stroke are acute manifestations of progressive cerebral microvascular disease. Current paradigms suggest atherosclerosis is a chronic, dynamic, inflammatory condition precipitated in response to endothelial injury from various environmental challenges. Myeloperoxidase plays a central role in initiation and progression of vascular inflammation, but prior studies linking myeloperoxidase with stroke risk have been inconclusive. We hypothesized that genetic determinants of myeloperoxidase levels influence the development of vascular instability, leading to increased primary intracerebral haemorrhage and lacunar stroke risk. We used a discovery cohort of 1409 primary intracerebral haemorrhage cases and 1624 controls from three studies, an extension cohort of 12 577 ischaemic stroke cases and 25 643 controls from NINDS-SiGN, and a validation cohort of 10 307 ischaemic stroke cases and 29 326 controls from METASTROKE Consortium with genome-wide genotyping to test this hypothesis. A genetic risk score reflecting elevated myeloperoxidase levels was constructed from 15 common single nucleotide polymorphisms identified from prior genome-wide studies of circulating myeloperoxidase levels (P < 5 × 10-6). This genetic risk score was used as the independent variable in multivariable regression models for association with primary intracerebral haemorrhage and ischaemic stroke subtypes. We used fixed effects meta-analyses to pool estimates across studies. We also used Cox regression models in a prospective cohort of 174 primary intracerebral haemorrhage survivors for association with intracerebral haemorrhage recurrence. We present effects of myeloperoxidase elevating single nucleotide polymorphisms on stroke risk per risk allele, corresponding to a one allele increase in the myeloperoxidase increasing genetic risk score. Genetic determinants of elevated circulating myeloperoxidase levels were associated with both primary intracerebral haemorrhage risk (odds ratio, 1.07, P = 0.04) and recurrent intracerebral haemorrhage risk (hazards ratio, 1.45, P = 0.006). In analysis of ischaemic stroke subtypes, the myeloperoxidase increasing genetic risk score was strongly associated with lacunar subtype only (odds ratio, 1.05, P = 0.0012). These results, demonstrating that common genetic variants that increase myeloperoxidase levels increase risk of primary intracerebral haemorrhage and lacunar stroke, directly implicate the myeloperoxidase pathway in the pathogenesis of cerebral small vessel disease. Because genetic variants are not influenced by environmental exposures, these results provide new support for a causal rather than bystander role for myeloperoxidase in the progression of cerebrovascular disease. Furthermore, these results support a rationale for chronic inflammation as a potential modifiable stroke risk mechanism, and suggest that immune-targeted therapies could be useful for treatment and prevention of cerebrovascular disease.

FEATURES OF LUMINOL- AND LUCIGENIN-INDUCED CHEMILUMINESCENCE OF NEUTROPHILIC GRANULOCYTES IN PATIENTS WITH CHRONIC RHINOSINUSITIS

Studies of functional activity of neutrophilic granulocytes from patients with chronic rhinosinusitis was performed using luminol- or lucigenin-dependent chemiluminescence. We have revealed increased production of reactive oxygen species (ROS) both under basal conditions, and in zymozan-induced chemiluminescent reaction, employing luminol as an activator, and determining total functional activity of neutrophils. Meanwhile, in lucigenin-induced chemiluminescence associated with production of superoxide anion radicals, a delayed ROS production was revealed, both in spontaneous and zymozan-induced response. One may suggest that, when studying these pathological events in neutrophils, ROS production in the cells proceeds mostly by myeloperoxidase pathway.

Myeloperoxidase levels predict accelerated progression of coronary atherosclerosis in diabetic patients: Insights from intravascular ultrasound

ObjectiveWhile inflammation has been proposed to contribute to the adverse cardiovascular outcome in diabetic patients, the specific pathways involved have not been elucidated. The leukocyte derived product, myeloperoxidase (MPO), has been implicated in all stages of atherosclerosis. The relationship between MPO and accelerated disease progression observed in diabetic patients has not been studied. MethodsWe investigated the relationship between MPO and disease progression in diabetic patients. 881 patients with angiographic coronary artery disease underwent serial evaluation of atherosclerotic burden with intravascular ultrasound. Disease progression in diabetic (n = 199) and non-diabetic (n = 682) patients, stratified by baseline MPO levels was investigated. ResultsMPO levels were similar in patients with and without diabetes (1362 vs. 1255 pmol/L, p = 0.43). No relationship was observed between increasing quartiles of MPO and either baseline (p = 0.81) or serial changes (p = 0.43) in levels of percent atheroma volume (PAV) in non-diabetic patients. In contrast, increasing MPO quartiles were associated with accelerated PAV progression in diabetic patients (p = 0.03). While optimal control of lipid and the use of high-dose statin were associated with less disease progression, a greater benefit was observed in diabetic patients with lower compared with higher MPO levels at baseline. ConclusionsIncreasing MPO levels are associated with greater progression of atherosclerosis in diabetic patients. This finding indicates the potential importance of MPO pathways in diabetic cardiovascular disease.

Clinical significance of a myeloperoxidase gene polymorphism and inducible nitric oxide synthase expression in cirrhotic patients with hepatopulmonary syndrome

The clinical significance of a myeloperoxidase (MPO) gene polymorphism and inducible nitric oxide synthase (iNOS) expression in cirrhotic patients with hepatopulmonary syndrome (HPS) was explored. Enrolled subjects were divided into three groups according to their disease/health conditions: the HPS group (cirrhotic patients with HPS; n=63), the non-HPS group (cirrhotic patients without HPS; n=182), and the control group (healthy subjects without liver disease; n=35). The distribution of the MPO -463 G/A genotype and its relationship with iNOS expression in a typical cell block from ascitic fluid were detected by immunohistochemistry and polymerase chain reaction-restricted fragment length polymorphism analysis (PCR-RFLP). In the HPS group, the partial pressure of oxygen in blood and ascitic fluid was significantly decreased (8.95+/-1.58 kPa and 6.81+/-0.95 kPa, respectively; both P<0.01), while the partial pressure of carbon dioxide significantly increased (4.62+/-0.20 kPa and 5.92+/-0.45 kPa, respectively; P<0.01). MPO and iNOS levels were significantly increased in the HPS group as compared with the non-HPS group. These increases were even more remarkable in ascitic fluid (41.36+/-11.62 and 13.23+/-4.81 microg/L; 10.27+/- 3.20 and 4.95+/-1.12 microg/L) than in blood (16.66+/-5.24 and 4.87+/-1.73 microg/L; 5.79+/-2.31 and 2.35+/-0.84 microg/L). The distribution of the MPO genotypes GG, GA, and AA were 76.2%, 22.2% and 1.6% in the HPS group, and 57.7%, 37.9% and 4.4% in the non-HPS group (P<0.05). The expression of iNOS was significantly higher in patients with the G alleles (G/G and G/A) (61.54%, 48/78) than in patients with A alleles (G/A and A/A) (38.46%, 30/78) (P<0.01). It was suggested that the expression levels of iNOS and MPO were correlated with HPS-induced hypoxemia. The MPO-463 G/A mutation might be a protective factor that prevents the development of HPS. The MPO might be involved in the regulation of iNOS expression. In humans, MPO pathways, the iNOS/NO system, and their interaction might have an impact on the occurrence and development of HPS.

Oxidation of apolipoprotein A-I by myeloperoxidase impairs the initial interactions with ABCA1 required for signaling and cholesterol export

A key cardioprotective effect of high-density lipoprotein involves the interaction of its major protein, apolipoprotein A-I (apoA-I) with ATP-binding cassette transporter A1 (ABCA1), a macrophage cholesterol exporter. ApoA-I is thought to remove cholesterol from macrophages by a cascade of events. First it binds directly to ABCA1, activating signaling pathways, and then it binds to and solubilizes lipid domains generated by ABCA1. HDL isolated from human atherosclerotic lesions and blood of subjects with established coronary artery disease contains elevated levels of 3-chlorotyrosine and 3-nitrotyrosine, two characteristic products of myeloperoxidase (MPO), a heme protein secreted by macrophages. Here we show that chlorination (but not nitration) of apoA-I by the MPO pathway impairs its ability to interact directly with ABCA1, to activate the Janus kinase 2 signaling pathway, and to promote efflux of cellular cholesterol. In contrast, oxidation of apoA-I has little effect on its ability to stabilize ABCA1 protein or to solubilize phospholipids. Our results indicate that chlorination of apoA-I by the MPO pathway selectively inhibits two critical early events in cholesterol efflux: (1) the binding of apoA-I to ABCA1 and (2) the activation of a key signaling pathway. Therefore, oxidation of apoA-I in the artery wall by MPO-generated chlorinating intermediates may contribute to atherogenesis by impairing cholesterol efflux from macrophages.

The benzene metabolite para-benzoquinone is genotoxic in human, phorbol-12-acetate-13-myristate induced, peripheral blood mononuclear cells at low concentrations

Benzene is one of the most prominent occupational and environmental pollutants. The substance is a proven human carcinogen that induces hematologic malignancies in humans, probably at even low doses. Yet knowledge of the mechanisms leading to benzene-induced carcinogenesis is still incomplete. Benzene itself is not genotoxic. The generation of carcinogenic metabolites involves the production of oxidized intermediates such as catechol, hydroquinone and para-benzoquinone (p-BQ) in the liver. Further activation to the ultimate carcinogenic intermediates is most probably catalyzed by myeloperoxidase (MPO). Yet the products of the MPO pathway have not been identified. If an oxidized benzene metabolite such as p-BQ was actually the precursor for the ultimate carcinogenic benzene metabolite and further activation proceeds via MPO mediated reactions, it should be possible to activate p-BQ to a genotoxic compound in vitro. We tested this hypothesis with phorbol-12-acetate-13-myristate (PMA) activated peripheral blood cells exposed to p-BQ, using the cytokinesis-block micronucleus test. Addition of 20–28 ng/ml PMA caused a significant increase of micronuclei at low and non-cytotoxic p-BQ concentrations between 0.04 and 0.2 μg/ml (0.37–1.85 μM). Thus with PMA or p-BQ alone no reproducible elevation of micronuclei was seen up to toxic concentrations. PMA and p-BQ induce micronuclei when administered jointly. Our results add further support to the hypothesis that MPO is a key enzyme in the activation of benzene.

The Effects of Iron Deficiency on Neutrophil/Monocyte Oxidative Burst Response in Children.

Background: Microbial killing killing of by phagocytic cells by iron containing myeloperoxidase pathway is part of the immune system. We aimed to investigate the effects of iron deficiency anemia (IDA) on oxidative burst response of phagocytic cells and understand if the effect is reversible after iron supplementation.Material and methods: There were 57 IDA patients and 31 healthy children as the control group that were aged between 6 months-12 years with similar demographic status. IDA patients were classified according to the severity of their anemia as follows; group 1 (Hb<8 gr/dl), group 2 (Hb≥8 gr/dl and Hb≤10 gr/dl), and group 3 (Hb>10 gr/dl). Neutrophil and monocyte oxidative burst response of each three groups of IDA and control group were compared by flow cytometry. IDA group were given oral iron supplementation (3 mg/kg-Ferrosanol) therapy. On day 15 of iron supplementation therapy neutrophil and monocyte oxidative burst response of each three groups of IDA were rechecked and compared to the control group. In order to minimize the discrepancies observed in functional studies, the oxidative burst index was calculated as the proportion of oxidative burst response in IDA group to the simultaneous oxidative burst response in the control group.Results: The number of infection episodes observed in the last three months was significantly higher in the IDA group (0–13 episodes) than the control group (1–2 episodes) (p=0.05). Neutrophil (p=0.1) and monocyte (p=0.02) oxidative burst responses in IDA group were found to be less than the control group. On day 15 of iron supplementation therapy, these values were observed to increase to the control group's values. Evaluation of neutophil and monocyte burst indexes at the diagnosis of IDA revealed that the neutrophil burst indexes in the Hb ≤10 gr/dl group (p=0.006), and monocyte burst indexes in the Hb <8 gr/dl group (p=0.01) were significantly low. The initially observed significant differences in oxidative burst responses disappeared at day 15 of iron supplementation therapy in all hemoglobin groups.Conclusion: Correction of the differences in oxidative burst response after iron supplementation therapy implies the fact that IDA might be the reason for changes in neutophil and monocyte burst indexes.

Thematic review series: The Pathogenesis of Atherosclerosis The oxidation hypothesis of atherogenesis: the role of oxidized phospholipids and HDL

For more than two decades, there has been continuing evidence of lipid oxidation playing a central role in atherogenesis. The oxidation hypothesis of atherogenesis has evolved to focus on specific proinflammatory oxidized phospholipids that result from the oxidation of LDL phospholipids containing arachidonic acid and that are recognized by the innate immune system in animals and humans. These oxidized phospholipids are largely generated by potent oxidants produced by the lipoxygenase and myeloperoxidase pathways. The failure of antioxidant vitamins to influence clinical outcomes may have many explanations, including the inability of vitamin E to prevent the formation of these oxidized phospholipids and other lipid oxidation products of the myeloperoxidase pathway. Preliminary data suggest that the oxidation hypothesis of atherogenesis and the reverse cholesterol transport hypothesis of atherogenesis may have a common biological basis. The levels of specific oxidized lipids in plasma and lipoproteins, the levels of antibodies to these lipids, and the inflammatory/anti-inflammatory properties of HDL may be useful markers of susceptibility to atherogenesis. Apolipoprotein A-I (apoA-I) and apoA-I mimetic peptides may both promote a reduction in oxidized lipids and enhance reverse cholesterol transport and therefore may have therapeutic potential.

Lysine Residues Direct the Chlorination of Tyrosines in YXXK Motifs of Apolipoprotein A-I When Hypochlorous Acid Oxidizes High Density Lipoprotein

Oxidized lipoproteins may play an important role in the pathogenesis of atherosclerosis. Elevated levels of 3-chlorotyrosine, a specific end product of the reaction between hypochlorous acid (HOCl) and tyrosine residues of proteins, have been detected in atherosclerotic tissue. Thus, HOCl generated by the phagocyte enzyme myeloperoxidase represents one pathway for protein oxidation in humans. One important target of the myeloperoxidase pathway may be high density lipoprotein (HDL), which mobilizes cholesterol from artery wall cells. To determine whether activated phagocytes preferentially chlorinate specific sites in HDL, we used tandem mass spectrometry (MS/MS) to analyze apolipoprotein A-I that had been oxidized by HOCl. The major site of chlorination was a single tyrosine residue located in one of the protein's YXXK motifs (where X represents a nonreactive amino acid). To investigate the mechanism of chlorination, we exposed synthetic peptides to HOCl. The peptides encompassed the amino acid sequences YKXXY, YXXKY, or YXXXY. MS/MS analysis demonstrated that chlorination of tyrosine in the peptides that contained lysine was regioselective and occurred in high yield if the substrate was KXXY or YXXK. NMR and MS analyses revealed that the N(epsilon) amino group of lysine was initially chlorinated, which suggests that chloramine formation is the first step in tyrosine chlorination. Molecular modeling of the YXXK motif in apolipoprotein A-I demonstrated that these tyrosine and lysine residues are adjacent on the same face of an amphipathic alpha-helix. Our observations suggest that HOCl selectively targets tyrosine residues that are suitably juxtaposed to primary amino groups in proteins. This mechanism might enable phagocytes to efficiently damage proteins when they destroy microbial proteins during infection or damage host tissue during inflammation.

Taurine: new implications for an old amino acid

Taurine is a semi-essential amino acid and is not incorporated into proteins. In mammalian tissues, taurine is ubiquitous and is the most abundant free amino acid in the heart, retina, skeletal muscle, brain, and leukocytes. In fact, taurine reaches up to 50 mM concentration in leukocytes. Taurine has been shown to be tissue-protective in many models of oxidant-induced injury. One possibility is that taurine reacts with hypochlorous acid, produced by the myeloperoxidase pathway, to produce the more stable but less toxic taurine chloramine (Tau-Cl). However, data from several laboratories demonstrate that Tau-Cl is a powerful regulator of inflammation. Specifically, Tau-Cl has been shown to down-regulate the production of pro-inflammatory mediators in both rodent and human leukocytes. Taurolidine, a derivative of taurine, is commonly used in Europe as an adjunctive therapy for various infections as well as for tumor therapy. Recent molecular studies on the function of taurine provide evidence that taurine is a constituent of biologic macromolecules. Specifically, two novel taurine-containing modified uridines have been found in both human and bovine mitochondria. Studies investigating the mechanism of action of Tau-Cl have shown that it inhibits the activation of NF-κB, a potent signal transducer for inflammatory cytokines, by oxidation of IκB-α at Met 45. Key enzymes for taurine biosynthesis have recently been cloned. Cysteine sulfinic acid decarboxylase, a rate-limiting enzyme for taurine biosynthesis, has been cloned and sequenced in the mouse, rat and human. Another key enzyme for cysteine metabolism, cysteine dioxygenase (CDO), has also been cloned from rat liver. CDO has a critical role in determining the flux of cysteine between cysteine catabolism/taurine synthesis and glutathione synthesis. Taurine transporter knockout mice show reduced taurine, reduced fertility, and loss of vision due to severe apoptotic retinal degeneration. Apoptosis induced by amino chloramines is a current and important finding since oxidants derived from leukocytes play a key role in killing pathogens. The fundamental importance of taurine in adaptive and acquired immunity will be unveiled using genetic manipulation.

Myeloperoxidase-associated tyrosine nitration after intratracheal administration of lipopolysaccharide in rats.

Previous studies have shown that lipopolysaccharide-induced inflammation in the lung results in tyrosine nitration. The objective of this study was to evaluate the contribution of myeloperoxidase and peroxynitrite pathway to the tyrosine nitration in lipopolysaccharide-administered lungs of rats that were otherwise untreated or leukocyte-depleted by cyclophosphamide or received inhaled nitric oxide (NO). The authors analyzed the immunoreactivity of inducible nitric oxide synthase (iNOS), nitrotyrosine (a product of the myeloperoxidase or peroxynitrite pathway), and chlorotyrosine (a byproduct of the myeloperoxidase pathway) by use of specific antibodies. The number of neutrophils in bronchoalveolar lavage fluid (BALF) and levels of myeloperoxidase activity in lung homogenates were also measured. Lipopolysaccharide enhanced the immunoreactivity of iNOS, nitrotyrosine, and chlorotyrosine in alveolar wall cells, alveolar macrophages, and neutrophils. Leukocyte depletion by cyclophosphamide and inhibition of leukocyte accumulation in the lungs by NO inhalation did not eliminate the increase in iNOS immunoreactivity in alveolar macrophages after lipopolysaccharide treatment, but nitrotyrosine and chlorotyrosine were not produced in these cells. Tyrosine nitration in response to lipopolysaccharide was associated with increases in neutrophil count in BALF and in myeloperoxidase activity in lung homogenates, whereas NO inhalation suppressed the neutrophil count in BALF and reduced tyrosine nitration and chlorination. These findings suggest that myeloperoxidase pathway has a role in tyrosine nitration in the lungs of lipopolysaccharide-treated rats, and that NO inhalation during early phase of inflammation does not increase but rather decreases tyrosine nitration and chlorination, possibly by reducing neutrophil sequestration.

Oxidized amino acids: culprits in human atherosclerosis and indicators of oxidative stress1,2

Oxidized low-density lipoprotein (LDL) is implicated in atherogenesis, but the mechanisms that oxidize LDL in the human artery wall have proven difficult to identify. A powerful investigative approach is mass spectrometric quantification of the oxidized amino acids that are left in proteins by specific oxidation reactions. Comparison of these molecular fingerprints in biological samples with those produced in proteins by various in vitro oxidation systems can indicate which biochemical pathway has created damage in vivo. For example, the pattern of oxidized amino acids in proteins isolated from atherosclerotic lesions implicates reactive intermediates generated by myeloperoxidase, a major phagocyte enzyme. These intermediates include hypochlorous acid, tyrosyl radical, and reactive nitrogen species, each of which generates a different pattern of stable end products. Despite this strong evidence that myeloperoxidase promotes LDL oxidation in vivo, the antioxidant that has been tested most extensively in clinical trials, vitamin E, fails to inhibit myeloperoxidase pathways in vitro. Because the utility of an antioxidant depends critically on the nature of the pathway that inflicts tissue damage, interventions that specifically inhibit myeloperoxidase or other physiologically relevant pathways would be more logical candidates for the prevention of cardiovascular disease. Moreover, levels of oxidized amino acids in urine and plasma might reflect those in tissues and therefore identify individuals with high levels of oxidative stress. Trials with such subjects would seem more likely to uncover effective antioxidant therapies than trials involving the general population.

Advanced glycation end products activate endothelium through signal-transduction receptor RAGE: a mechanism for amplification of inflammatory responses.

The products of nonenzymatic glycation and oxidation of proteins, the advanced glycation end products (AGEs), form under diverse circumstances such as aging, diabetes, and kidney failure. Recent studies suggested that AGEs may form in inflamed foci, driven by oxidation or the myeloperoxidase pathway. A principal means by which AGEs alter cellular properties is through interaction with their signal-transduction receptor RAGE. We tested the hypothesis that interaction of AGEs with RAGE on endothelial cells enhances vascular activation. AGEs, RAGE, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin are expressed in an overlapping manner in human inflamed rheumatoid synovia, especially within the endothelium. In primary cultures of human saphenous vein endothelial cells, engagement of RAGE by heterogeneous AGEs or Nepsilon(carboxymethyl)lysine-modified adducts enhanced levels of mRNA and antigen for vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and E-selectin. AGEs increased adhesion of polymorphonuclear leukocytes to stimulated endothelial cells in a manner reduced on blockade of RAGE. AGEs, through RAGE, may prime proinflammatory mechanisms in endothelial cells, thereby amplifying proinflammatory mechanisms in atherogenesis and chronic inflammatory disorders.

Proteolytic inactivation of alpha-1-antitrypsin by human neutrophils: involvement of multiple and interlinked cell responses to phagocytosable targets.

Neutrophil polymorphonuclear leukocytes (PMN) can inactivate the PMN-elastase inhibitor alpha-1-antitrypsin (A1AT) proteolytically, by using metalloproteinases normally stored as zymogens in myeloperoxidase (MPO)-negative granules. Supernatants from opsonized zymosan (OPZ)-triggered human PMN cleaved and inactivated human A1AT through a process inhibitable by metal-chelators, suggesting that the interaction of PMN with OPZ leads to the extracellular availability of active metalloenzymes. During OPZ-triggering, PMN used approximately 80% of the generated hydrogen peroxide (H2O2) to produce HOCl by means of the MPO pathway, while the remainder was catabolized by PMN themselves. No H2O2 was available as free compound in the extracellular environment and hydroxyl (.OH) or .OH-like radicals were not generated. The selective deletion of single components of the HOCl-generating MPO pathway resulted in the generation of PMN supernatants free of active metalloenzymes but rich of the corresponding zymogens. Similar results were obtained by replacing normal PMN with cells from a patient with hereditary MPO deficiency. No evidence was obtained for the intervention or contribution of .OH-like radicals, serine-proteinases and oxidized glutathione in the transformation of the zymogens into enzymes able to inactivate A1AT. On concluding, PMN undergoing phagocytosis release MPO in amount sufficient to handle the extracellular pool of the generated H2O2 entirely, leading to the generation of equimolar amounts of HOCl. In turn, HOCl or a similar compound derived from it interacts with concomitantly released metallozymogens, switching on their A1AT inactivating potential without the apparent contribution of other PMN-derived molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Nimesulide as a downregulator of the activity of the neutrophil myeloperoxidase pathway. Focus on the histoprotective potential of the drug during inflammatory processes.

Neutrophils, recruited to tissue sites of inflammation, release a variety of oxidants and enzymes, which are responsible for tissue damage. Among the oxidants released are potent chlorinated compounds, such as hypochlorous acid and chloramines, which induce tissue cell damage and inactivate protease inhibitors, particularly alpha 1-antitrypsin, the specific inhibitor of neutrophil elastase. In studying a rational approach to the pharmacological control of neutrophil-mediated tissue injury, we investigated the activity of the anti-inflammatory drug nimesulide. This agent reduced the function of the myeloperoxidase pathway (which generates hypochlorous acid), by exerting a cell-directed inhibitory activity, as shown by measurement of superoxide anion and hydrogen peroxide production. Nimesulide also inactivated hypochlorous acid directly and protected alpha 1-antitrypsin from the neutrophil-mediated oxidation. Thus, neutrophil elastolytic activity may be attenuated by nimesulide-spared alpha 1-antitrypsin. The prevention of oxidative inactivation of alpha 1-antitrypsin by nimesulide strictly correlates with the drug's ability to suppress the extracellular availability of hypochlorous acid. Taken together, these data suggest that nimesulide may prevent tissue injury at sites of inflammation by maintaining natural host protective systems.

Biotransformation of para-aminobenzoic acid and salicylic acid by PMN

Para-aminobenzoic acid (PABA) is an essential cofactor for the production of folic acid in bacteria and has mild anti-inflammatory activity. We have recently reported that salicylic acid and benzoic acid are oxidized by stimulated granulocytes Polymorphonuclear Neutrophils (PMN). The oxidation of salicylate appears mediated by a potent oxygen metabolite generated during the respiratory burst which is dependent primarily on superoxide (O 2 −) for its production. These background studies with the salicylate group of drugs suggested that PABA might be similarly metabolized by PMN. In these studies, we demonstrate that PABA is metabolized by stimulated PMN. However, in contrast to the biochemical mechanism involved in the metabolism of salicylate, our scavenger studies indicate that PABA is metabolized primarily by the myeloperoxidase pathway. Our results may explain the mild anti-inflammatory actions of the drug and suggest that the degradation of PABA by PMN at an inflammatory site may limit the availability of PABA for bacterial growth.

The anti-inflammatory drug nimesulide rescues alpha-1-proteinase inhibitor from oxidative inactivation by phagocytosing neutrophils.

When neutrophils are recruited to tissue sites and exposed to phagocytosable targets, they release oxidants which may be responsible for the local inactivation of alpha-1-proteinase inhibitor (A1PI). Consequently, A1PI becomes incapable of inhibiting the proteolytic activity of elastase, released at the same time by neutrophils as a result of leakage from phagocytic vacuoles. In the present paper we show that phagocytosing neutrophils inactivate A1PI via a process inhibitable by chemical agents known to interfere with the hypochlorous acid (HOCl)-generating myeloperoxidase pathway. The anti-inflammatory drug nimesulide (NMS), which is able to efficiently limit the extracellular availability of HOCl in the neutrophil surroundings, was found to prevent the inactivation of A1PI by neutrophils. The results provide evidence for a possible way to control neutrophil elastase activity by rescuing its natural inhibitor (A1PI) at inflamed tissue sites during infectious and noninfectious processes.

Cytoprotection against neutrophil-delivered oxidant attack by antibiotics

In the present study we have investigated the effect of six antibiotics (penicillin G, ceftazidime, cephotaxime, cephoperazon, ampicillin and piperacillin) on the neutrophil cytolytic activity by using a system constituted of phorbol-12-myristate-13-acetate-triggered neutrophils and 51Cr-labelled lymphoblastoid Daudi target cells. The results demonstrate that five of these drugs (ceftazidime, cephotaxime, cephoperazon, amplcillin and piperacillin) are capable of inhibiting the neutrophil cytolytic activity by inactivating the hypochlorous acid (HOC1) generated extracellularly by the myeloperoxidase pathway and crucial to the target cell lysis. Penicillin G had no effect on neutrophil-mediated cytolysis. Thus, these data demonstrate that ceftazidime, cephotaxime, cephoperazon, amplcillin and piperacillin lower the neutrophil-mediated target cell damage by a HOCl-scavenging mechanism, suggesting a possible cytoprotective role for these drugs during infections.

Tumor necrosis factor primes neutrophils for hypochlorous acid production

Tumor necrosis factor (TNF) has a weak direct effect on neutrophil oxidative metabolism and primes neutrophils for oxidant release in response to other stimuli. We examined the effect of recombinant human TNF alpha (rTNF alpha) on production of hypochlorous acid (HOCl) by human neutrophils. TNF alone, even at concentrations of 1,000 U/ml, did not stimulate HOCl production. In contrast, rTNF alpha, in a dose-dependent manner, primed neutrophils for HOCl production in response to the weak agent unopsonized zymosan. rTNF alpha concentrations as low as 10 U/ml resulted in a fivefold increase in HOCl in this system. rTNF alpha-primed cells also exhibited increased phagocytosis. Priming in this model system occurred regardless of whether cells were preincubated with rTNF alpha before addition of zymosan or coincubated with both rTNF alpha and zymosan. rTNF alpha priming for HOCl production could not be washed away and required a lag period of approximately 10 min. rTNF alpha priming was not dependent on extracellular Ca2+ and Mg2+. Preincubation experiments demonstrated that rTNF alpha priming was not inhibited by the microfilament blocker cytochalasin B. Although the mechanism remains unclear, these findings demonstrate that rTNF alpha has an important priming effect on the neutrophil myeloperoxidase pathway.