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The antimycobacterial role of eosinophil peroxidase (EPO), one of the most abundant granule proteins in human eosinophils, was investigated. Our data indicate that purified EPO shows significant inhibitory activity towards Mycobacterium tuberculosis H37Rv. On a molar basis, this activity was similar to that exhibited by neutrophil myeloperoxidase (MPO) and was both dose and time dependent. In contrast to the activity of MPO, which requires H(2)O(2), EPO also exhibited anti-M. tuberculosis activity in the absence of exogenously added peroxide. Morphological evidence confirmed that the mechanism of action of EPO against mycobacteria differs from that of MPO. While MPO kills M. tuberculosis H37Rv exclusively in the presence of hydrogen peroxide, it does not induce morphological changes in the pathogen. In contrast, EPO-treated bacteria frequently had cell wall lesions and eventually underwent lysis, either in the presence or in the absence of H(2)O(2).

Inhibition of growth or eradication of experimentally induced tumors has been shown to be accompanied by infiltration of eosinophils and macrophages into the tumor mass. Since macrophages are important mediators of host antitumor activity, the possibility arises that a collaboration may exist between these two cell types in the control of tumor growth. In this study, we report the effect of eosinophil peroxidase (EPO), a basic protein contained in eosinophils that binds to several cell types including macrophages, on tumor necrosis factor (TNF) production and hydrogen peroxide release by human monocyte-derived macrophages. After incubation with EPO, the macrophages produced large amounts of TNF and displayed an enhanced phorbol 12-myristate 13-acetate-triggered hydrogen peroxide release. These effects were accompanied by an increased cell protein content and by morphologic changes leading the large, round macrophages of the control cultures to become elongated, pear-like or spindle shaped cells after treatment with EPO. The stimulatory effect of EPO on hydrogen peroxide release was insensitive to addition of exogenous catalase, a H2O2-degrading enzyme, suggesting that an extracellular catalytic activity of EPO was not involved. In addition, myeloperoxidase, the homologous peroxidase of neutrophils with a catalytic activity similar to that of EPO, was ineffective. The EPO-induced effects differed in several aspects from the effects of lipopolysaccaride and interferon-gamma, two well-known macrophage activators. These findings provide supportive evidence for a functional interrelationship between eosinophils and macrophages that may be physiologically relevant in the tumoricidal activity of macrophages.

Five eosinophil peroxidase (EPO)-deficient subjects were identified from 131,000 peripheral blood samples examined for routine automated analysis. The EPO-deficient eosinophils of these subjects met the main criteria established for EPO deficiency: absent or strongly decreased reaction for peroxidase, absent or strongly decreased staining with Sudan Black, and an increased ratio of the granule core volume to the total granule volume. In this report we show that this granule alteration is caused mainly by a decrease of its volume, particularly of the matrix, and that two other matrix proteins, eosinophil cationic protein and eosinophil derived neurotoxin, appear to be present in normal amounts in the EPO-deficient granules.

The degranulation of neutrophils and eosinophils is frequently monitored by assaying myeloperoxidase (MPO) and eosinophil peroxidase (EPO) activity in the cell-free supernatant of degranulating cells, after removal of the cells by centrifugation. This procedure leads to underestimation of the extent of degranulation, since both peroxidases tend to stick to cell surfaces, to test tube walls, and to particulate stimuli used to elicit degranulation, because of their highly cationic nature. In this paper we describe a method for assaying MPO and EPO secretion in whole cell suspensions that avoids separation of the cells from the incubation medium. The least toxic and thus safest among the sensitive peroxidase substrates, 3,3',5,5'-tetramethylbenzidine (TMB), was employed for peroxidase assay. The method we describe here is applied to the detection of peroxidase release by neutrophil and eosinophil cell suspensions incubated in either polypropylene test tubes or flat-bottomed microtiter plate wells. Because of the omission of the centrifugation step, the TMB method offers two major advantages over the currently used techniques: (1) higher estimates of degranulation, which permits the use of a smaller number of cells (in the microassay version, 150,000 neutrophils and 50,000 eosinophils) and smaller amounts of the secretagogues, and (2) rapidity, since the degranulation assay can be performed immediately on completion of the cell incubation with the secretagogue.

The eosinophils from bone granuloma, bone marrow, and peripheral blood of a patient with Hand-Schüller-Christian disease (HSCD) were studied by electron microscopy and cytochemistry. Impressive eosinophil degranulation was observed. Extracellular release of eosinophil peroxidase (EPO) and EPO binding to surrounding cells were seen in the granuloma and bone marrow. Cells with peroxidase-positive plasma membrane were also observed in peripheral blood. The pattern of eosinophil degranulation showed quite different features from those described so far. In the granuloma, the process begins with intracytoplasmic release of the granule matrix content, as revealed by both extensive extragranular accumulation of EPO and progressive decrease of the matrix electron density. Core dissolution follows thereafter, leading to complete disappearance of the granules. At the end of the process, the cells show rupture of the plasma membrane and release of their content into the surrounding environment. This pattern of secretion was also observed in blood and marrow eosinophils of the patient. In view of the previously reported findings that EPO binding to inflammatory cells influences their functions, EPO release and binding to surrounding cells in HSCD may play a role in the evolution of the inflammatory lesion in the disease.

Biochemical assays for peroxidase activity do not usually distinguish between different peroxidases. The guaiacol assay, for example, which is one of the most commonly used assays for peroxidase activity, is sensitive to both eosinophil peroxidase (EPO) and the peroxidase of neutrophils, i.e., myeloperoxidase (MPO), thus preventing distinction of the two peroxidases in mixed neutrophil-eosinophil populations. In this paper we describe a simple and sensitive method for selective assays of EPO in EPO-MPO mixtures or mixed populations of neutrophils and eosinophils. The method is based on the peroxidase-mediated oxidation of homovanillic acid (HVA) under appropriate assay conditions in which EPO is still very active in catalyzing the reaction whilst MPO-mediated HVA oxidation is almost undetectable. Optimal assay conditions were as follows: pH 10.5, 10 microM hydrogen peroxide, 0.8 mM HVA and an incubation time of 120 min at 37 degrees C. Under these conditions the assay permits EPO activities as low as 0.025 guaiacol U/ml to be measured even in the presence of 0.175 guaiacol U/ml of MPO. In mixed neutrophil-eosinophil cell suspensions the test permits the detection of as few as 5 X 10(3) eosinophils even in the presence of about 700 X 10(3) neutrophils (eosinophils: neutrophils ratio 1:140) with no appreciable interference by the latter cells. The method described here has been applied to studies of human EPO deficiency and proved to be successful in the identification of individuals with partial EPO deficiency, which is not feasible with non quantitative methods (for example, cytochemistry) or unselective biochemical assay of peroxidase activity.

The effects of the interactions between eosinophil peroxidase (EPO) and neutrophils were studied. It is shown that binding of EPO to neutrophils results on the one hand in reversible inhibition of EPO peroxidase activity and, on the other, in an increased neutrophil aggregation and neutrophil adhesion to endothelial cell monolayers. After prolonged periods of exposure to EPO, neutrophils also show a decreased ability to exclude the vital dye erythrosin B. The reversible inhibition of EPO activity may represent a means of keeping under control the oxidative potential of EPO, while the increased neutrophil aggregation and adherence to endothelial cells suggest that EPO may influence at least two key events of inflammation, that is, the margination of neutrophils and/or their accumulation in the inflammatory site.

We have recently shown that human neutrophils bind and internalize human eosinophil peroxidase (EPO) but not myeloperoxidase (MPO). In the present work, we studied the interactions of human EPO and MPO with other cells that may be involved in the inflammatory process, i.e., lymphocytes, monocytes, platelets, fibroblasts, and endothelial cells. The results indicate that EPO is bound by all the cell types considered, but is efficiently internalized only by lymphocytes, monocytes, and endothelial cells. Conversely, MPO binds appreciably only to fibroblasts and endothelial cells, although with a lower affinity than EPO, but its internalization by any of the cell types studied is hardly detectable. Furthermore, both peroxidases bind strongly to collagen fibers, whereas only EPO binds to elastin. The results suggest that EPO, owing to its high cytophilia, exerts its biological activity close to the site at which it is released from the eosinophil.

Quantitation of myeloperoxidase (MPO) activity by guaiacol peroxidation (GP) assay is profoundly affected by the peroxidase present in eosinophils (EPO) that contaminate the granulocyte suspensions. Inclusion of 3-amino-1,2,4-triazole (AMT) in the GP assay permits quantitation of MPO activity in mixed neutrophil-eosinophil suspension because of the differential inhibition of EPO and MPO by AMT. Results show that: (1) the peroxidase activity of eosinophil-free granulocyte suspensions is not appreciably affected by AMT; (2) in the presence of AMT the peroxidase activities of granulocyte preparations containing different numbers of eosinophils are similar on a neutrophil basis, regardless of the number of eosinophils and correspond with the activity of eosinophil-free granulocyte suspensions; (3) AMT almost completely inhibits the activity of partially purified EPO, only slightly affecting the catalytic activity of partially purified MPO; (4) AMT completely inhibits the residual peroxidase activity of granulocyte suspensions from MPO-deficient subjects contributed by contaminating eosinophils. The GP assay in the presence of AMT was used to study the pattern of hereditary transmission of MPO deficiency. The genealogy derived on the basis of this assay was compatible with an autosomal recessive inheritance, in agreement with previously reported results, while no definite pattern of inheritance could be established by use of the GP assay without AMT. We suggest that the GP assay supplemented with AMT is the method of choice for detection of MPO deficiency, particularly partial deficiency.

A simple procedure to purify human eosinophil peroxidase (EPO) is described. The method uses pure anucleated granule-rich eosinophil fragments (cytosomes) as a suitable starting material from which EPO can be quickly isolated. The enzyme obtained by this procedure has both the biochemical and the spectral properties of EPO and shows a reasonable degree of purity, as judged by its rz value. This procedure, besides its simplicity and reproducibility, offers at least two other advantages over the methods currently used for EPO purification, the possibility of isolating EPO from small amounts of normal human blood and a very high recovery of the enzyme activity.

A cytochemical analysis was carried out for study of the interaction between human eosinophil peroxidase (EPO) and human neutrophils. To this end, neutrophils with a genetic deficiency of myeloperoxidase (MPO) were used to avoid the otherwise inevitable interference of the high endogenous MPO activity of normal neutrophils. The data show that human neutrophils incubated with EPO (1 GU/ml) rapidly bind the enzyme all over the cell surface and internalize it in small vesicles. Part of bound EPO concentrates in a limited area on the cell surface and is then internalized by means of coarse tubular channels. Fusion of the small vesicles to each other or possibly with the tubular channels gives rise ultimately to EPO-containing multivesicular bodies, which, after 30 minutes of incubation, are the only peroxidase-positive structures in the cytoplasm. Under identical experimental conditions, no binding of human MPO to the neutrophils was detected. At concentrations 10 times as high as those used for EPO, a minority of neutrophils bound MPO, but the binding pattern remained diffuse on the plasma membrane and the internalization was negligible. It seems, therefore, that the EPO trapping system of human neutrophils exhibits specificity at least among leukocyte peroxidases. Furthermore, it operates at much lower concentrations of EPO than those reported for EPO uptake by mast cells and basophils. The uptake of EPO by neutrophils may serve to sequester a potentially toxic agent, thus limiting damage to the tissue in eosinophil-rich inflammatory lesions.