A myeloperoxidase-specific assay based upon bromide-dependent chemiluminescence of luminol.

Measurement of myeloperoxidase (MPO; EC 1.11.1.7) activity is often used as a marker of neutrophil infiltration into tissues. However, most enzymatic assays for MPO are susceptible to interference from other peroxidases (including eosinophil peroxidase, EPX) and hemoproteins (such as hemoglobin and myoglobin) present in the tissues. In this report, we describe a bromide-dependent chemiluminescence (Br-CL) assay that uses luminol as a chemiluminescence probe. The assay can distinguish between MPO and nonspecific peroxidase reactions. The MPO-specific reaction is believed to proceed in two steps: (i) the enzymatic generation of hypobromous acid (HOBr) from KBr and H(2)O(2) at pH 5 and (ii) the spontaneous reaction of HOBr and H(2)O(2) with luminol to give a Br-CL signal. The assay is sufficiently sensitive to allow detection of MPO in <100 human neutrophils. Other peroxidases and hemoproteins do not interfere with the Br-CL signal. Although EPX can also oxidize bromide to generate HOBr, activities of MPO and EPX can be distinguished at different pHs. As a demonstration of the utility of the Br-CL assay, MPO activity was measured in murine tumors known to be infiltrated with neutrophils. A statistically significant correlation was seen between MPO activity and histological neutrophil counts in the tumors (r = 0.69, P < 0.01, n = 14). The assay should have wide application for measuring the neutrophil content of tissues.     

Production of brominating intermediates by myeloperoxidase. A transhalogenation pathway for generating mutagenic nucleobases during inflammation

The existence of interhalogen compounds was proposed more than a century ago, but no biological roles have been attributed to these highly oxidizing intermediates. In this study, we determined whether the peroxidases of white blood cells can generate the interhalogen gas bromine chloride (BrCl). Myeloperoxidase, the heme enzyme secreted by activated neutrophils and monocytes, uses H2O2 and Cl(-) to produce HOCl, a chlorinating intermediate. In contrast, eosinophil peroxidase preferentially converts Br(-) to HOBr. Remarkably, both myeloperoxidase and eosinophil peroxidase were able to brominate deoxycytidine, a nucleoside, and uracil, a nucleobase, at plasma concentrations of Br(-) (100 microM) and Cl(-) (100 mM). The two enzymes used different reaction pathways, however. When HOCl brominated deoxycytidine, the reaction required Br(-) and was inhibited by taurine. In contrast, bromination by HOBr was independent of Br(-) and unaffected by taurine. Moreover, taurine inhibited 5-bromodeoxycytidine production by the myeloperoxidase-H2O2-Cl(-)- Br(-) system but not by the eosinophil peroxidase-H2O2-Cl(-)-Br(-) system, indicating that bromination by myeloperoxidase involves the initial production of HOCl. Both HOCl-Br(-) and the myeloperoxidase-H2O2-Cl(-)-Br(-) system generated a gas that converted cyclohexene into 1-bromo-2-chlorocyclohexane, implicating BrCl in the reaction. Moreover, human neutrophils used myeloperoxidase, H2O2, and Br(-) to brominate deoxycytidine by a taurine-sensitive pathway, suggesting that transhalogenation reactions may be physiologically relevant. 5-Bromouracil incorporated into nuclear DNA is a well known mutagen. Our observations therefore raise the possibility that transhalogenation reactions initiated by phagocytes provide one pathway for mutagenesis and cytotoxicity at sites of inflammation.     

Myeloperoxidase-Halide-Hydrogen Peroxide Antibacterial System

An antibacterial effect of myeloperoxidase, a halide, such as iodide, bromide, or chloride ion, and H(2)O(2) on Escherichia coli or Lactobacillus acidophilus is described. When L. acidophilus was employed, the addition of H(2)O(2) was not required; however, the protective effect of catalase suggested that, in this instance, H(2)O(2) was generated by the organisms. The antibacterial effect was largely prevented by preheating the myeloperoxidase at 80 C or greater for 10 min or by the addition of a number of inhibitors; it was most active at the most acid pH employed (5.0). Lactoperoxidase was considerably less effective than was myeloperoxidase when chloride was the halide employed. Myeloperoxidase, at high concentrations, exerted an antibacterial effect on L. acidophilus in the absence of added halide, which also was temperature- and catalase-sensitive. Peroxidase was extracted from intact guinea pig leukocytes by weak acid, and the extract with peroxidase activity had antibacterial properties which were similar, in many respects, to those of the purified preparation of myeloperoxidase. Under appropriate conditions, the antibacterial effect was increased by halides and by H(2)O(2) and was decreased by catalase, as well as by cyanide, azide, Tapazole, and thiosulfate. This suggests that, under the conditions employed, the antibacterial properties of a weak acid extract of guinea pig leukocytes is due, in part, to its peroxidase content, particularly if a halide is present in the reaction mixture. A heat-stable antibacterial agent or agents also appear to be present in the extract.     

Morpho‐functional modifications of human neutrophils induced by aqueous cigarette smoke extract: comparison with chemiluminescence activity

Cigarette smoking plays an important role as a cause of morbidity and mortality in humans, involving respiratory, cardiovascular, digestive and reproductive systems. Tobacco smoke contains a large number of molecules, some of which are proven carcinogens. Although not fully understood, polymorphonuclear leukocytes seem to play a crucial role in the mechanisms by which tobacco smoke compounds are implicated in smoke‐related diseases. In this paper the effects of an aqueous cigarette smoke extract on the expression of adhesion molecules of polymorphonuclear leukocytes together with the changes in the cell morphology have been related to the chemiluminescence activity. The results obtained show that polymorphonuclear leukocytes treated with aqueous cigarette smoke extract are significantly impaired, as suggested by the changes of chemiluminescence activity, of membrane receptors (CD18, CD62), myeloperoxidase expression and of cell morphology. Altogether the present data indicate that treated polymorphonuclear leukocytes are ineffectively activated and therefore unable to phagocytize zymosan particles. Copyright © 2010 John Wiley & Sons, Ltd.     

The effect of chloride on the redox and EPR properties of myeloperoxidase

Myeloperoxidase was purified from human polymorphonuclear leukocytes and the effect of chloride upon the EPR and potentiometric properties was studied. The redox titration between the ferrous and ferric states of the enzyme yielded n = 1 Nernst plots between pH 9 and 4, with clear isosbestic points in the optical spectra during the redox change. The midpoint potential (Em) between the ferric and ferrous forms of the enzyme exhibited a pH-dependent change between pH 4 and 9, and the effect of added chloride ion indicated that Cl- competed with OH- for a binding site on the enzyme. Interestingly, the pH dependence of the Em indicated that the overall redox reactions of the enzyme was: ferric myeloperoxidase + 2e- + 1H+ = ferrous myeloperoxidase. Myeloperoxidase exhibited a rhombic high spin EPR signal which exhibited reduced rhombicity upon the binding of chloride. Our results strongly suggest that chloride binds to the sixth coordination position of the chlorin iron in myeloperoxidase by replacing the water which is the sixth ligand in the resting state. It is also concluded that the two iron centers are identical and that there is no interaction between them.     

Phagocytes produce 5-chlorouracil and 5-bromouracil,two mutagenic products of myeloperoxidase,in human inflammatory tissue

Oxidative damage to DNA has been implicated in carcinogenesis during chronic inflammation. Epidemiological and biochemical studies suggest that one potential mechanism involves myeloperoxidase, a hemeprotein secreted by human phagocytes. In this study, we demonstrate that human neutrophils use myeloperoxidase to oxidize uracil to 5-chlorouracil in vitro. Uracil chlorination by myeloperoxidase or reagent HOCl exhibited an unusual pH dependence, being minimal at pH approximately 5, but increasing markedly under either acidic or mildly basic conditions. This bimodal curve suggests that myeloperoxidase initially produces HOCl, which subsequently chlorinates uracil by acid- or base-catalyzed reactions. Human neutrophils use myeloperoxidase and H2O2 to chlorinate uracil, suggesting that nucleobase halogenation reactions may be physiologically relevant. Using a sensitive and specific mass spectrometric method, we detected two products of myeloperoxidase, 5-chlorouracil and 5-bromouracil, in neutrophil-rich human inflammatory tissue. Myeloperoxidase is the most likely source of 5-chlorouracil in vivo because halogenated uracil is a specific product of the myeloperoxidase system in vitro. In contrast, previous studies have demonstrated that 5-bromouracil could be generated by either eosinophil peroxidase or myeloperoxidase, which preferentially brominates uracil at plasma concentrations of halide and under moderately acidic conditions. These observations indicate that the myeloperoxidase system promotes nucleobase halogenation in vivo. Because 5-chlorouracil and 5-bromouracil can be incorporated into nuclear DNA, and these thymine analogs are well known mutagens, our observations raise the possibility that halogenation reactions initiated by phagocytes provide one pathway for mutagenesis and cytotoxicity at sites of inflammation.     

Myeloperoxidase inactivation in the course of catalysis of chlorination of taurine.

Myeloperoxidase (donor: hydrogen-peroxide oxidoreductase, EC 1.11.1.7) was isolated from leukocytes of patients with chronic granulocyte leukemia. In the presence of H2O2 and Cl- at pH 4.0-6.6 the myeloperoxidase catalyses chlorination of taurine to monochloramine taurine and simultaneously undergoes inactivation. The myeloperoxidase inactivation rate depends on the concentration of H2O2 and Cl-: both the initial rate of chlorination and myeloperoxidase inactivation rate increase with increasing concentration of H2O2. However, an increase in concentration of Cl- results in a decrease in enzyme inactivation. At a given H2O2 concentration, myeloperoxidase inactivation is a first order reaction, which implied that the enzyme may react with a substrate a limited number of times.     

Metabolic activity of phagocytes in experimental sporotrichosis

Phagocytosis plays an important role as a protective mechanism against infections, since polymorphonuclear leukocytes (PMN) and macrophages are the first cellular lines opposed to agressive microorganisms. In patients with sporotrichosis a diminished capability of killing engulfed yeast by their PMN has been described, but the origin of this deficiency remains unknown.In this work, partial aspects of the oxidative metabolism of PMN leukocytes and peritoneal macrophages of mongolian gerbils experimentally infected with sporotrichosis were studied. For this purpose the nitroblue tetrazolium (NBT) test as described by Baehner and Nathan (1) and myeloperoxidase activity measured according to Kaplow's method were utilized.The PMN and macrophages of mongolian gerbils infected with sporotrichosis showed increased reduction of NBT when compared with the phagocytic cells of normal ones, as is usually observed in most infections. Myeloperoxidase activity was diminished in both PMN and macrophages, but this diminution was statistically significant only in PMN leukocytes. These results show that part of the oxidative mechanisms of phagocytic cells can be impaired in experimental sporotrichosis, and could be correlated with the diminished fungicidal activity of PMN leukocytes obtained from patients infected with sporotrichosis.     

Hypochlorite- and hypobromite-mediated radical formation and its role in cell lysis.

Activated leukocytes generate the potent oxidants HOCl and HOBr via the formation of H(2)O(2) and the release of peroxidase enzymes (myeloperoxidase, eosinophil peroxidase). HOCl and HOBr are potent microbiocidal agents, but excessive or misplaced production can cause tissue damage and cell lysis. In this study it is shown that HOBr induces red blood cell lysis at approximately 10-fold lower concentrations than HOCl, whereas with monocyte (THP1) and macrophage (J774) cells HOCl and HOBr induce lysis at similar concentrations. The role of radical formation during lysis has been investigated by EPR spin trapping, and it is shown that reaction of both oxidants with each cell type generates cell-derived radicals. Red blood cells exposed to nonlytic doses of HOCl generate novel nitrogen-centered radicals whose formation is GSH dependent. In contrast, HOBr gives rise to nitrogen-centered, membrane-derived protein radicals. With lytic doses of either oxidant, protein (probably hemoglobin)-derived, nitrogen-centered radicals are observed. Unlike the red blood cells, treatment of monocytes and macrophages with HOCl gives significant radical formation only under conditions where cell lysis occurs concurrently. These radicals are nitrogen-centered, cell-protein-derived species and have parameters identical to those detected with red blood cells and HOBr. Exposure of these cells to HOBr did not give detectable radicals. Overall these experiments demonstrate that HOCl and HOBr react with different selectivity with cellular targets, and that this can result in radical formation. This radical generation can precede, and may play a role in, cell lysis.     

Myeloperoxidase binds to non-vital spermatozoa on phosphatidylserine epitopes

The heme protein myeloperoxidase is released from stimulated polymorphonuclear leukocytes, a cell species found in increasing amounts in the male and female genital tract of patients with genital tract inflammations. Myeloperoxidase binds only to a fraction of freshly prepared human spermatozoa. The number of spermatozoa able to bind myeloperoxidase raised considerably in samples containing pre-damaged cells or in acrosome-reacted samples. In addition, myeloperoxidase released from zymosan-stimulated polymorphonuclear leukocytes was also able to bind to pre-damaged spermatozoa. The ability of spermatozoa to bind myeloperoxidase coincided with the binding of annexin V to externalized phosphatidylserine epitopes indicating the loss of plasma membrane integrity and with the incorporation of ethidium homodimer I. Myeloperoxidase did not interact with intact spermatozoa. Annexin V and myeloperoxidase bind to the same binding sites as verified by double fluorescence techniques, flowcytometry analyses as well as competition experiments. We demonstrated also that myeloperoxidase is eluted together with pure phosphatidylserine liposomes or liposomes composed of phosphatidylserine and phosphatidylcholine in gel filtration, but not with pure phosphatidylcholine liposomes. In conclusion, myeloperoxidase interacts with apoptotic spermatozoa via binding to externalized phosphatidylserine indicating a yet unknown role of this protein in recognition and removal of apoptotic cells during inflammation.     

Peroxidase-mediated irreversible binding of arylamine carcinogens to DNA in intact polymorphonuclear leukocytes activated by a tumor promoter

Addition of the tumor promoter phorbol myristate acetate to polymorphonuclear leukocytes results in the oxidation of the arylamine carcinogens; [14C]benzidine, N-[14C]methylaminoazobenzene and [14C]aminofluorene to reactive intermediate(s) that bind irreversibly to the leukocyte DNA. The binding was dependent on oxygen and was decreased by sulfhydryl inhibitors and phenolic antioxidants that inhibit the respiratory burst triggered by the phorbol myristate. Both the binding and the respiratory burst were increased by azide, presumably as a result of intracellular catalase inhibition. However higher concentrations of azide and cyanide prevented binding without affecting the respiratory burst indicating that myeloperoxidase is a catalyst for the binding. Granules isolated from the activated leukocytes and H2O2 catalyzed a cyanide sensitive benzidine binding to calf thymus DNA. Myeloperoxidase and H2O2 also catalysed extensive binding of these arylamines to calf thymus DNA. The leukocytes appear to be a useful model cell for studying one electron oxidation-catalyzed carcinogen activation.     

Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid.

Myeloperoxidase catalyses the conversion of H2O2 and Cl- to hypochlorous acid (HOCl). It also reacts with O2- to form the oxy adduct (compound III). To determine how O2- affects the formation of HOCl, chlorination of monochlorodimedon by myeloperoxidase was investigated using xanthine oxidase and hypoxanthine as a source of O2- and H2O2. Myeloperoxidase was mostly converted to compound III, and H2O2 was essential for chlorination. At pH 5.4, superoxide dismutase (SOD) enhanced chlorination and prevented formation of compound III. However, at pH 7.8, SOD inhibited chlorination and promoted formation of the ferrous peroxide adduct (compound II) instead of compound III. We present spectral evidence for a direct reaction between compound III and H2O2 to form compound II, and for the reduction of compound II by O2- to regenerate native myeloperoxidase. These reactions enable compound III and compound II to participate in the chlorination reaction. Myeloperoxidase catalytically inhibited O2- -dependent reduction of Nitro Blue Tetrazolium. This inhibition is explained by myeloperoxidase undergoing a cycle of reactions with O2-, H2O2 and O2-, with compounds III and II as intermediates, i.e., by myeloperoxidase acting as a combined SOD/catalase enzyme. By preventing the accumulation of inactive compound II, O2- enhances the activity of myeloperoxidase. We propose that, under physiological conditions, this optimizes the production of HOCl and may potentiate oxidant damage by stimulated neutrophils.     

Salivary myeloperoxidase of young adult humans

Leukocytes, principally polymorphonuclear leukocytes (PMNs), enter the oral cavity where they release a portion of their constituents, including myeloperoxidase, into oral fluids. A greater number of PMNs in the oral cavity are associated with oral inflammation. However, the quantitative contribution of the PMN to oral fluids, including saliva, during various conditions is poorly understood. An assay method based on the adsorbance loss at 278 nm from the reaction of the myeloperoxidase product hypochlorous acid with monochlorodimedon to yield dichlorodimedon was developed for the quantitation of salivary myeloperoxidase. Myeloperoxidase was determined in supernatants of whole saliva obtained at low and moderate flow rates and in parotid saliva collected during moderate and pronounced stimulation from young adults with minimal oral inflammation. The greatest myeloperoxidase activity was in whole saliva supernatants collected at low flow rates where PMN products have an opportunity to accumulate. Lesser quantities of myeloperoxidase were found in both the whole saliva supernatants and parotid saliva obtained at the faster flow rates. Low flow rate whole saliva supernatants contained about 25% of the myeloperoxidase in the PMNs which enter the oral cavity. Myeloperoxidase is responsible for a significant portion (15-20%) of the total peroxidase activity in supernatants of whole saliva obtained at low flow rates. Preliminary results indicate that young adults with phenytoin-associated gingival overgrowth or who smoke have more myeloperoxidase activity in low flow rate whole saliva.     

Inhibition of myeloperoxidase by salicylhydroxamic acid.

Salicylhydroxamic acid inhibited the luminol-dependent chemiluminescence of human neutrophils stimulated by phorbol 12-myristate 13-acetate or the chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe). This compound had no inhibitory effect on the kinetics of O2.- generation or O2 uptake during the respiratory burst, but inhibited both the peroxidative activity of purified myeloperoxidase and the chemiluminescence generated by a cell-free myeloperoxidase/H2O2 system. The concentration of salicylhydroxamic acid necessary for complete inhibition of myeloperoxidase activity was 30-50 microM (I50 values of 3-5 microM) compared with the non-specific inhibitor NaN3, which exhibited maximal inhibition at 100-200 microM (I50 values of 30-50 microM). Whereas taurine inhibited the luminol chemiluminescence of an H2O2/HOC1 system by HOC1 scavenging, this compound had little effect on myeloperoxidase/H2O2-dependent luminol chemiluminescence; in contrast, 10 microM-salicylhydroxamic acid did not quench HOC1 significantly but greatly diminished myeloperoxidase/H2O2-dependent luminol chemiluminescence, indicating that its effects on myeloperoxidase chemiluminescence were largely due to peroxidase inhibition rather than non-specific HOC1 scavenging. Salicylhydroxamic acid prevented the formation of myeloperoxidase Compound II, but only at low H2O2 concentrations, suggesting that it may compete for the H2O2-binding site on the enzyme. These data suggest that salicylhydroxamic acid may be used as a potent inhibitor to delineate the function of myeloperoxidase in neutrophil-mediated inflammatory events.     

Reaction of 3′,5′-di-O-acetyl-2′-deoxyguansoine with hypobromous acid

Hypobromous acid (HOBr) is formed by eosinophil peroxidase and myeloperoxidase in the presence of H₂O₂, Cl^?, and Br^? in the host defense system of humans, protecting against invading bacteria. However, the formed HOBr may cause damage to DNA and its components in the host. When a guanine nucleoside (3′,5′-di-O-acetyl-2′-deoxyguansoine) was treated with HOBr at pH 7.4, spiroiminodihydantoin, guanidinohydantoin/iminoallantoin, dehydro-iminoallantoin, diimino-imidazole, amino-imidazolone, and diamino-oxazolone nucleosides were generated in addition to an 8-bromoguanine nucleoside. The major products were spiroiminodihydantoin under neutral conditions and guanidinohydantoin/iminoallantoin under mildly acidic conditions. All the products were formed in the reaction with HOCl in the presence of Br^?. These products were also produced by eosinophil peroxidase or myeloperoxidase in the presence of H₂O₂, Cl^?, and Br^?. The results suggest that the products other than 8-bromoguanine may also have importance for mutagenesis by the reaction of HOBr with guanine residues in nucleotides and DNA.     

Spontaneous lung chemiluminescence upon paraquat administration

In vivo rat lung chemiluminescence was measured at different times after a single injection of either 30 or 60 mg paraquat/kg b.w. The lungs were isolated to determine myeloperoxidase (index of polymorphonuclear leukocytes), lung wet weight (lung edema) and malondialdehyde (lipid peroxidation). The highest chemiluminescence was reached 30 hours after injection of 30 mg/kg or 6 hours after a 60 mg/kg dose. The peak chemiluminescence was coincident with the maximum concentration of myeloperoxidase and lung wet weight suggesting that most chemiluminescence was the consequence of polymorphonuclear activation after migration to the injured areas.     

Mechanism of inhibitory action of ketone bodies on the production of reactive oxygen intermediates (ROIS) by polymorphonuclear leukocytes.

We determined an effect of acetoacetic acid (AcAc) and 3-hydroxybutyrate (3-OHB) on the production of reactive oxygen intermediates (ROIs) in polymorphonuclear leukocytes from healthy volunteers. Both AcAc and 3-OHB inhibited the luminol-dependent chemiluminescence (LDCL) activities assessed with initial slope and the inhibition rates were about 42%, 44% respectively by AcAc and 3-OHB when the leukocytes were preincubated with 10 mM AcAc or 3-OHB for 60 minutes. The LDCL activity was reduced by 16% and 42% following the addition of 1mM and 10 mM AcAc. The similar reduction of the LDCL activity was observed in the addition of 3-OHB. Either 3-OHB or AcAc failed to show a significant reduction of myeloperoxidase (MPO) activity. However, both 3-OHB and AcAc dose-dependently inhibited superoxide anion (O2-) production, measured by using cytochrome c. These data provided evidence that both 3-OHB and AcAc suppress neutrophil oxidative metabolism with respect with O2- production.     

Processing of a newly identified intermediate of human myeloperoxidase in isolated granules occurs at neutral pH

Myeloperoxidase is a major component of specialized lysosomes known as azurophil granules in polymorphonuclear leukocytes or neutrophils. The processing of myeloperoxidase in human HL-60 promyelocytic leukemia cells was studied by pulse-labeling cells in culture with [35S]methionine followed by immunoprecipitation and identification of myeloperoxidase polypeptides from cell fractions after various chase intervals. These studies revealed the presence of a previously unidentified intermediate with Mr 74,000 which kinetically followed the appearance of a larger Mr 81,000 intermediate. Using an in vitro lysosomal preparation the newly identified Mr 74,000 intermediate was directly converted within protected granules to mature forms of myeloperoxidase (Mr 63,000 and 60,000). This conversion occurred optimally at pH 7.5 and was not inhibited by lysosomotropic agents (chloroquine, NH4Cl) or protonophores (monensin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone). Furthermore, the uptake of radiolabeled amines indicated a neutral intragranular environment (pH 7.35-7.67) which remained unchanged in the presence and absence of 1 mM ATP or 2.5 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone. We conclude that, in contrast to other lysosomal pathways, the final proteolytic cleavage of myeloperoxidase does not require an acidic environment.     

Inactivation of alpha(2)-Macroglobulin by Activated Human Polymorphonuclear Leukocytes

The proteolytic activity of trypsin releases the dye Remazol Brilliant Blue from its high molecular weight substrate, the skin powder (Hide Powder Azure, Sigma), with an increase in absorbance at 595 nm. Active alpha(2)- macroglobulin (80 mug/ml) totally inhibits the proteolytic activity of trypsin (14 mug/ml) by trapping this protease. But after a 20 min incubation of alpha(2)-macroglobulin at 37 degrees C with 2 x 10(6) human polymorphonuclear leukocytes activated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (10(-7) M) and cytochalasin B (10(-8) M), 100% of trypsin activity was recovered, indicating a total inactivation of alpha(2)-macroglobuHn. Incubation with granulocyte myeloperoxidase also inactivates alpha(2)-macroglobulin. Hypochlorous acid, a by-product of myeloperoxidase activity, at a concentration of 10(-7) M also inactivates alpha(2)-macroglobulin, which indicates that an important cause of alpha(2)-macroglobulin inactivation by activated polymorphonuclear leukocytes could be the activity of myeloperoxidase.     

The Fluorescein-derived Dye Aminophenyl Fluorescein Is a Suitable Tool to Detect Hypobromous Acid (HOBr)-producing Activity in Eosinophils

The specific detection of peroxidase activity in human granulocytes is essential to elucidate their role in innate immune responses, immune regulation, and inflammatory diseases. The halogenating activity of myeloperoxidase in neutrophils can be determined by the novel fluorescent probe aminophenyl fluorescein (APF). Thereby non-fluorescent APF is oxidized by HOCl to form fluorescein. We successfully verified that APF equally detects the hypobromous acid (HOBr)-producing activity of eosinophil granulocytes. This was revealed by three different approaches. First, we investigated the conversion of non-fluorescent APF into fluorescein by HOCl and HOBr by means of fluorescence and mass spectrometry approaches. Thereby comparable chemical mechanisms were observed for both acids. Furthermore in vitro kinetic studies were used to detect the halogenating activity of myeloperoxidase and eosinophil peroxidase by using APF. Here the dye well reflected the different substrate specificities of myeloperoxidase and eosinophil peroxidase regarding chloride and bromide. Finally, peroxidase activities were successfully detected in phorbol ester-stimulated neutrophils and eosinophils using flow cytometry. Thereby inhibitory studies confirmed the peroxidase-dependent oxidation of APF. To sum up, APF is a promising tool for further evaluation of the halogenating activity of peroxidases in both neutrophils and eosinophils.