Role Of Myeloperoxidase Research Articles

The role of myeloperoxidase and superoxide anion in the luminol- and lucigenin-dependent chemiluminescence of human neutrophils

The amount of chemiluminescence (CL) or light that is emitted from human polymorphonuclear leukocytes (PMN) during phagocytosis or activation by soluble stimuli is dependent on the emission of photons from the oxidation of particulate or bystander molecules. Because the compounds luminol and lucigenin yield photons with high quantum efficiency these agents have been introduced to sensitively assess PMN-CL. Since there is limited information about the pathways involved in the chemiluminescence of these compounds, we investigated the role of both myeloperoxidase (MPO) and superoxide anion (· O 2 −) in luminol-and lucigenin-PMN-CL. We compared the CL between normal and MPO- deficient PMN using zymosan for phagocytosis and N-formylmethionyleucylphenylalanine (FMLP) as a soluble stimulus. Our data demonstrated that luminol-CL was dependent on the presence of MPO and independent of · O 2 − generation during phagocytosis but independent of MPO during FMLP activation. In contrast lucigenin-CL was independent of MPO during both phagocytosis and FMLP activation and appeared to reflect · O 2 − production. Consequently, dependent on the type of activation, it appears that luminol- and lucigenin-CL are generated via different oxidative pathways and may serve as potentially useful tools to differentiate the redox activity of phagocytic cells.

Kinetics and mechanism of the bactericidal action of human neutrophils against Escherichia coli

A mutant strain of Escherichia coli (E. coli ML-35) was used to follow the kinetics of phagocytosis, perforation of the bacterial cell envelope, and inactivation of bacterial proteins by human neutrophils. This particular E. coli mutant strain has no lactose permease, but constitutively forms the cytoplasmic enzyme beta-galactosidase. This implies that the artificial substrate ortho-nitrophenyl-beta-D-galactopyranoside cannot reach the beta-galactosidase unless the bacterial cell envelope has been perforated. Thus, the integrity of the E. coli envelope can be measured simply by the activity of beta-galactosidase with this substrate. Indeed, ingestion of E. coli ML-35 by human neutrophils was followed by perforation of the bacteria (increase in beta-galactosidase activity). Subsequently, the beta-galactosidase activity decreased due to inactivation of the enzyme. With a simple mathematical model and a curve-fitting computer program, we have determined the first-order rate constants for phagocytosis, perforation, and beta-galactosidase inactivation. With 32 normal donors, we found an interdonor variation in these rate constants of 20% to 30% (SD) and an assay variance of 5%. The perforation process closely correlated with the loss of colony-forming capacity of the bacteria. This new assay measures phagocytosis and killing in a fast, simple, and accurate way; it is not hindered by extracellular bacteria. Moreover, this method also measures the postkilling event of inactivation of a bacterial protein, which permits a better detection of neutrophils deficient in this function. The assay can also be used for screening neutrophil functions without the use of a computer program. A simple calculation suffices to detect neutrophil abnormalities. Neutrophils from patients with chronic granulomatous disease (CGD) showed an impaired rate of perforation and thus also of inactivation. Neutrophils from myeloperoxidase-deficient patients or from a patient with the Chediak-Higashi syndrome only showed a retarded inactivation of beta-galactosidase, but normal ingestion and perforation. The role of myeloperoxidase in the killing process is discussed. Although myeloperoxidase does not seem to be a prerequisite for perforation, it probably plays a role in bacterial destruction by normal cells, because the inactivation of bacterial proteins seems strictly myeloperoxidase dependent.

Kinetics and Mechanism of the Bactericidal Action of Human Neutrophils Against Escherichia coli

Abstract A mutant strain of Escherichia coli (E. coli ML-35) was used to follow the kinetics of phagocytosis, perforation of the bacterial cell envelope, and inactivation of bacterial proteins by human neutrophils. This particular E. coli mutant strain has no lactose permease, but constitutively forms the cytoplasmic enzyme beta-galactosidase. This implies that the artificial substrate ortho-nitrophenyl-beta-D- galactopyranoside cannot reach the beta-galactosidase unless the bacterial cell envelope has been perforated. Thus, the integrity of the E. coli envelope can be measured simply by the activity of beta- galactosidase with this substrate. Indeed, ingestion of E. coli ML-35 by human neutrophils was followed by perforation of the bacteria (increase in beta-galactosidase activity). Subsequently, the beta- galactosidase activity decreased due to inactivation of the enzyme. With a simple mathematical model and a curve-fitting computer program, we have determined the first-order rate constants for phagocytosis, perforation, and beta-galactosidase inactivation. With 32 normal donors, we found an interdonor variation in these rate constants of 20% to 30% (SD) and an assay variance of 5%. The perforation process closely correlated with the loss of colony-forming capacity of the bacteria. This new assay measures phagocytosis and killing in a fast, simple, and accurate way; it is not hindered by extracellular bacteria. Moreover, this method also measures the postkilling event of inactivation of a bacterial protein, which permits a better detection of neutrophils deficient in this function. The assay can also be used for screening neutrophil functions without the use of a computer program. A simple calculation suffices to detect neutrophil abnormalities. Neutrophils from patients with chronic granulomatous disease (CGD) showed an impaired rate of perforation and thus also of inactivation. Neutrophils from myeloperoxidase-deficient patients or from a patient with the Chediak-Higashi syndrome only showed a retarded inactivation of beta-galactosidase, but normal ingestion and perforation. The role of myeloperoxidase in the killing process is discussed. Although myeloperoxidase does not seem to be a prerequisite for perforation, it probably plays a role in bacterial destruction by normal cells, because the inactivation of bacterial proteins seems strictly myeloperoxidase dependent.

Role of myeloperoxidase in the respiratory burst of human neutrophils

Myeloperoxidase (MPO), a heme enzyme present in the primary granules of polymorphonuclear leukocytes (PMNs), has been demonstrated to participate in the oxygen-dependent microbicidal activity of these cells. Evidence for the importance of MPO in this role comes in part from studies of normal PMNs treated with the heme enzyme inhibitor, sodium azide. MPO has also been suggested to regulate the respiratory activity of PMNs during phagocytosis. The role of MPO in PMN oxygen metabolism was examined by studying parameters of the respiratory burst of PMNs from a number of unrelated MPO-deficient subjects; in addition, the ability of heme enzyme inhibitors to duplicate the MPO-deficient state was studied by treating normal and MPO-deficient cells with these compounds. MPO-deficient PMNs were found to have a time-dependent hypermetabolic response as assessed by measurement of oxygen consumption, superoxide generation, hydrogen peroxide release, and hexose monophosphate shunt activity. Catabolic pathways for hydrogen peroxide were normal, suggesting the increased recovery of oxygen metabolites reflects increased production rather than decreased catabolism of H2O2. These observations support the concept that MPO may play an important role in terminating the respiratory burst of normal PMNs. The three heme enzyme inhibitors studied--sodium azide, potassium cyanide, and 3-aminotriazole--differed greatly in the degree to which they inhibited various enzymatic systems in the PMN. Nonetheless, as a group, they exerted qualitatively similar effects on oxygen metabolism of normal and of MPO-deficient PMNs. This indicates that many of the mechanisms by which heme enzyme inhibitors influence PMN metabolism are independent of the inhibition of MPO. Conclusions from studies using such treatment of PMNs should be interpreted with caution.

Role of Myeloperoxidase in the Respiratory Burst of Human Neutrophils

Myeloperoxidase (MPO), a heme enzyme present in the primary granules of polymorphonuclear leukocytes (PMNs), has been demonstrated to participate in the oxygen-dependent microbicidal activity of these cells. Evidence for the importance of MPO in this role comes in part from studies of normal PMNs treated with the heme enzyme inhibitor, sodium azide. MPO has also been suggested to regulate the respiratory activity of PMNs during phagocytosis. The role of MPO in PMN oxygen metabolism was examined by studying parameters of the respiratory burst of PMNs from a number of unrelated MPO-deficient subjects; in addition, the ability of heme enzyme inhibitors to duplicate the MPO-deficient state was studied by treating normal and MPO-deficient cells with these compounds. MPO-deficient PMNs were found to have a time-dependent hypermetabolic response as assessed by measurement of oxygen consumption, superoxide generation, hydrogen peroxide release, and hexose monophosphate shunt activity. Catabolic pathways for hydrogen peroxide were normal, suggesting the increased recovery of oxygen metabolites reflects increased production rather than decreased catabolism of H2O2. These observations support the concept that MPO may play an important role in terminating the respiratory burst of normal PMNs. The three heme enzyme inhibitors studied--sodium azide, potassium cyanide, and 3-aminotriazole--differed greatly in the degree to which they inhibited various enzymatic systems in the PMN. Nonetheless, as a group, they exerted qualitatively similar effects on oxygen metabolism of normal and of MPO-deficient PMNs. This indicates that many of the mechanisms by which heme enzyme inhibitors influence PMN metabolism are independent of the inhibition of MPO. Conclusions from studies using such treatment of PMNs should be interpreted with caution.

Further evaluation of luminol-enhanced luminescence in the diagnosis of disorders of leukocyte oxidative metabolism: role of myeloperoxidase.

Chemiluminescence can be used to identify defects in the oxidative metabolism of granulocytes. This procedure has recently been adopted for use with microliter quantities of whole blood, appropriate for prenatal or neonatal study. Although the contribution of myeloperoxidase to the chemiluminescence assay has been noted, the possible diagnostic confusion between chronic granulomatous disease of childhood (which is rare and severe) and myeloperoxidase deficiency (which is common and of little clinical consequence) has not been stressed. We report a father and his infant daughter whose cells emitted no light in the luminol-enhanced luminescence assay; both patients are totally peroxidase deficient. These results emphasize the hereditary nature of myeloperoxidase deficiency, and the possibility for erroneous diagnosis of chronic granulomatous disease of childhood based on the luminol-enhanced luminescence test.

Role of myeloperoxidase in luminol-dependent chemiluminescence of polymorphonuclear leukocytes.

When polymorphonuclear leukocytes (PMNL) and soluble or particulate matter interact, the cells produce chemiluminescence, linked to activation of the oxidative metabolism of the cells. PMNL isolated from a patient with a myeloperoxidase deficiency were found to produce almost no luminol-dependent chemiluminescence, despite a pronounced production of superoxide anions (O2-). The chemotactic peptide formylmethionyl-leucyl-phenylalanine induced a two-peak chemiluminescence response in control PMNL. The response was modified, both in magnitude and in time-course, when the cells were incubated at 22 degrees C for 120 min. Addition of purified myeloperoxidase to the PMNL lacking this enzyme, before stimulus addition, resulted in a chemiluminescence response. In the response to formylmethionyl-leucyl-phenylalanine, only one peak, corresponding to the initial peak of control PMNL, was found. This indicated that luminol-dependent chemiluminescence is dependent on and directly related to the presence of myeloperoxidase in PMNL and that both intra- and extracellularly located myeloperoxidase has to be taken into account when interpreting the cellular response assayed as chemiluminescence.

Role of myeloperoxidase and bacterial metabolism in chemiluminescence of granulocytes from patients with chronic granulomatous disease.

Phagocytosis of catalase-positive microbes by normal polymorphonuclear neutrophils results in increased metabolism as required for microbicidal action. Chemiluminescence is a product of the associated oxygenation reactions. Neutrophils from patients with chronic granulomatous disease are capable of phagocytizing catalase-positive microbes, but there is no associated respiratory burst, microbicidal action is greatly decreased, and chemiluminescence is not detected. However, these defective neutrophils can kill catalase-negative, H2O2-generating bacteria. In the present study, chemiluminescence by neutrophils from patients with chronic granulomatous disease after phagocytosis of H2O2-generating streptococci was detected. Acid extracts of myeloperoxidase from either control or patient neutrophils also yielded chemiluminescence in the presence of streptococci, but not in the presence of catalase-positive microbes.

Comparison of Myeloperoxidase Activity in Leukocytes from Normal Subjects and Patients with Chronic Granulomatous Disease

A study was undertaken to compare myeloperoxidase (MPO) activity in leukocytes from normal subjects and those with chronic granulomatous disease (CGD) and to eliminate further consideration of MPO as the oxidase responsible for the post-phagocytic respiratory burst and subsequent oxidase microbicidal sequence lacking in leukocytes from patients with CGD. With use of granule fractions isolated from a number of samples, MPO activity in several MPO-mediated biochemical systems (peroxidase assays, protein iodination, and amino acid decarboxylation) was measured. No difference was demonstrated between granule fraction preparations from normal subjects and those with CGD. These data show that MPO activity is normal in CGD leukocytes and are inconsistent with a role for MPO in initiating the post-phagocytic respiratory burst.

Ethylene formation by polymorphonuclear leukocytes. Role of myeloperoxidase.

Ethylene formation from the thioethers, beta-methylthiopropionaldehyde (methional) and 2-keto-4-thiomethylbutyric acid by phagocytosing polymorphonuclear leukocytes (PMNs) was found to be largely dependent on myeloperoxidase (MPO). Conversion was less than 10% of normal when MPO-deficient PMNs were employed; formation by normal PMNs was inhibited by the peroxidase inhibitors, azide, and cyanide, and a model system consisting of MPO, H2O2, chloride (or bromide) and EDTA was found which shared many of the properties of the predominant PMN system. MPO-independent mechanisms of ethylene formation were also identified. Ethylene formation from methional by phagocytosing eosinophils and by H2O2 in the presence or absence of catalase was stimulated by azide. The presence of MPO-independent, azide-stimulable systems in the PMN preparations was suggested by the azide stimulation of ethylene formation from methional when MPO-deficient leukocytes were employed. Ethylene formation by dye-sensitized photooxidation was also demonstrated and evidence obtained for the involvement of singlet oxygen (1O2). These findings are discussed in relation to the participation of H2O2, hydroxyl radicals, the superoxide anion and 1O2 in the formation of ethylene by PMNs and by the MPO model system.

Decay of oxyperoxidase and oxygen radicals; a possible role for myeloperoxidase

The formation of superoxide anion during the decay of oxyperoxidase to ferric peroxidase was detected by using a spectrophotometric assay based on the use of adrenaline. The finding that peroxidase is a potential source of superoxide suggests a possible role for myeloperoxidase in leucocytes.

Myeloperoxidase of human neutrophilic granulocytes as chlorinating enzyme.

Two pH‐dependent spectral forms of myeloperoxidase have been described. Their absorption maxima in the Soret region were at 432 nm and 426 nm for acid and neutral pH, respectively. Chloride exerts an influence on the spectrum of peroxidase, mainly in acidic medium. Dissociation constants for the myeloperoxidase chloride complex and Km values for chloride in the chlorination reaction catalyzed by myeloperoxidase were determined. It was found that the affinity of chloride ions to the enzyme decreases with an increase of pH.Catalytic activity of myeloperoxidase in the reaction of incorporation of 36Cl into aliphatic amino compounds such as taurine, β‐alanine, ethanolamine and diethanolamine was investigated. Primary amino groups were chlorinated to N‐mono‐ or dichloramines, depending on the pH of the medium. The method of determination of 36Cl‐labelled chlorocompounds in the presence of excess Na36Cl was described.The role of myeloperoxidase as chlorinating enzyme in the mechanism of the bactericidal effect on bacteria phagocytized by neutrophilic granulocytes is discussed.

Leukocyte myeloperoxidase deficiency and disseminated candidiasis: the role of myeloperoxidase in resistance to Candida infection.

The neutrophils and monocytes of a patient with disseminated candidiasis were found to lack detectable levels of the lysosomal enzyme myeloperoxidase (MPO), although they had normal levels of other granule-associated enzymes. Leukocytes from one of the patient's sisters also lacked detectable MPO; leukocytes from his four sons contained approximately one-third of mean normal peroxidase levels. Neither the patient nor his affected relatives had experienced frequent or unusual bacterial infections. The phagocytic activity of the patient's MPO-deficient neutrophils was intact, and the cells displayed normal morphologic and metabolic responses to phagocytosis. In contrast to normal leukocytes which killed 30.5+/-7.3% of ingested Candida albicans in 1 hr, however, the patient's neutrophils killed virtually none. His leukocytes also failed to kill the strain of C. albicans recovered from his lesions, as well as other Candida species. These MPO-deficient neutrophils killed Serratia marcescens and Staphylococens aureus 502A at an abnormally slow rate, requiring 3-4 hr to achieve the bactericidal effect attained by normal leukocytes after 45 min. No other abnormalities in his cellular or humoral immune responses were demonstrated. These findings suggest that hereditary MPO deficiency is transmitted as an autosomal recessive characteristic, that the homozygous state conveys enhanced susceptibility to disseminated candidiasis, and that MPO is necessary for candidacidal activity in human neutrophils. Although lending support to the suggested bactericidal role of MPO in leukocytes, the data indicate that alternative bactericidal mechanisms, effective in the absence of MPO, are functionally dominant in the human neutrophil.