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.
To examine the relationship between binding of surface-active stimuli to human neutrophils and activation of the superoxide (02.) and peroxide (H202) forming oxidase(s) of these cells. oxygen (02) consumption and 02. and H202 release were measured following stimulation by plant lectins. Both concanavalin A (Con-A) and phytohemagglutinin (PHA) were more active than other lectins in stimulating the respiratory activity of neutrophils. When the cells were pretreated with cytochalasin B (Cyto-B). recovery of extracellular 02 and H202 was increased by 3-10-fold. as found previously with particulate stimuli. Whereas cytochalasin B reduced 02 consumption by cells triggered with particulate stimuli. cytochalasin B treatment of cells produced more than a threefold increase in 02 consumption by cells triggered with plant lectins. The stimulatory effect of cytochalasin B on the respiration of Con-A-or PHA-treated cells was rapid. with an enhancement observed within 60 sec. With Con-A-stimulated cells. it could be inhibited by alpha-methyl-D-mannoside (AMM). indicating that it was specifically due to an activity mediated by the lectin as opposed to some other action of cytochalasin B. To correlate these events with binding of the lectin to the cells. 3H-Con-A was employed. Pretreatment of cells with Cyto-B increased 3H-Con-A binding in direct parallel to the stimulation of respiration. Similar results were found when the Cyto-B was added in the middle of an incubation period. Both the increased binding and respiratory activity of the cytochalasin-B-treated cells could be inhibited by the simultaneous addition of AMM. These findings support the concept that the degree of activation of the neutrophil oxidase(s) is a direct function of the extent of binding of the initial stimulus. They also suggest that ongoing stimulus binding is a prerequisite for continued respiratory activity. The precise nature of the lectin binding sites ' 'uncovered" in Cyto-B-treated cells and their potential relationship to sites of oxidase activity or regulation remains to be determined.
To study the stage of development of oxidase activity responsible for the respiratory burst in human granulocytes. we investigated the effect of phorbol myristate acetate (PMA) and fluoride (F) on nitroblue tetrazolium (NBT) reduction by bone marrow and circulating granulocytic cells in 1 3 subjects without leukocyte functional disorders and a patient with chronic granulomatous disease (CGD). Myeloblasts. promyelocytes, and myelocytes did not reduce NBT. Metamyelocytes showed minimal activity that improved with further maturation of the cell. Morphologically mature bone marrow polymorphonuclear leukocytes (PMNs) had significantly less NBT-reducing capacity than peripheral blood cells. Inhibition of phagocytosis by cytochalasin B completely abolished dye reduction under resting conditions. PMAstimulated NBT reduction, however, was not affected. Thus, ingestion of NBT was not a prerequisite for its reduction. None of the PMNs from the CGD patient reduced NBT. These results indicate that either oxidase activity or the ability of cells to respond to activating stimuli does not develop until the metamyelocyte stage. and furthermore, a disparity between the morphological and functional maturation exists in bone marrow granulocytes.
Fresh blood, stored blood and granulocyte concentrates were passed through 170-µm and microaggregate blood filters to determine the degree of complement activation that occurs during transfusion of citrated blood products. Complement activation was assessed by measurement of C3 conversion using crossed immunoelectrophoresis and by assessment of C5a using a leukocyte aggregation functional assay. Prefiltration, fresh or stored blood products showed 0-1% C3 activation. Postfiltration, the degree of C3 conversion did not change for fresh blood or granulocyte concentrates. For stored whole blood, the degree of C3 conversion increased slightly to 2-3%. Prefiltration results for all samples showed a low level of C5a which did not change after passage through any filter. Serum incubated with filter material at 37°C showed 2-10% C3 conversion. In contrast, results with citrated plasma showed less than 3% conversion of C3. We conclude that although some filter materials may activate complement in serum, filtration of citrated blood products through microaggregate blood filters induces little complement activation.
Three monoclonal antibodies (MAb)--OKMI, 7C3, and 60.3-- immunoprecipitated a common 170-kd neutrophil membrane antigen closely associated with, or identical to, the C3bi receptor (CR3). Despite binding to a common receptor, these antibodies displayed marked differences in their effects on C3bi-mediated neutrophil function as assessed by the binding and ingestion of opsonized zymosan and the subsequent triggering of the respiratory burst. Antibody 7C3 caused a time-dependent, irreversible inhibition of the neutrophil oxidative response to opsonized zymosan that correlated with capping of the bound antibody. In contrast, antibody 60.3 caused an immediate inhibition of the neutrophil oxidative response to opsonized zymosan that required the continuous presence of exogenous antibody to achieve the maximal inhibitory effect. Antibody OKMI demonstrated minimal inhibition of O2- release. Despite their functional differences, binding of either 7C3 or 60.3 led to up-regulation of new antigen, presumably from intracellular sites as previously described using OKMI. Crossed immunoprecipitations of radiolabeled neutrophil lysates indicated that each MAb bound to different antigens near or within the CR3 complex. Thus three MAb binding to the neutrophil CR3 receptor each caused receptor up- regulation but had markedly different functional effects on the cell.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.