Development of Oxidase activity by human bone marrow granulocytes

Abstract

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.

Evaluation of the expression of NADPH oxidase components during maturation of HL-60 clone 15 cells to eosinophilic lineage

“…In contrast with our observations, it was previously shown, using the NBT assay, that the activity of NADPH oxidase was not expressed until the metamyelocyte stage using bone-marrow cells from individuals with disorders, such as miliary tuberculosis, idiopathic thrombocytopenic purpura, chronic renal failure, and multiple myeloma [13]. The discrepancy between the previous results and our results may be because of the difference in the cells used (from individuals with disorders vs. healthy volunteers) and/or assay conditions for NBT assay (0.2% NBT and 100 ng/ml PMA vs. 0.04% NBT and 500 ng/ml PMA).…”

“…During maturation, acquisition of O 2 Ϫ -generating activity was determined by nitro blue tetrazolium (NBT) assay, which detects reduction of NBT to formazan by superoxide oxide. The assay was performed based on the method of Zakhireh and Root [13] with a slight modification by incubating cells (1ϫ10 6 cells/ml, 200 l) with 0.04% NBT in PBS containing 1 mM CaCl 2 and 1 mM MgCl 2 in the presence or absence of 500 ng/ml PMA at 37°C for 30 min in a Lab-Tak chamber slide (Nalge-Nunc International, Naperville, IL), and then formazan-containing cells were monitored. Furthermore, NADPH oxidase activity was assayed by cytochrome c reduction [20]; the assay mixtures consisted of 1 ϫ 10 6 cells/ml, 60 M cytochrome c, 1 mM CaCl 2 , 1 mM MgCl 2 , and 1 g/ml PMA with or without 20 g/ml superoxide dismutase in a total volume of 400 l PBS.…”

“…The importance of each of these oxidase components is demonstrated in chronic granulomatous disease (CGD), which is an inherited disease characterized by mutations that result in the loss or inactivation of one of the core subunits of NADPH oxidase, with the failure of O 2 Ϫ production and a marked increase in the susceptibility of affected patients to bacterial and fungal infections [9,12]. Previous studies suggested that activity of the oxidase was not expressed in immature neutrophil precursors using bone-marrow cells from patients with no clinical evidence of neutrophil-functional disorders [13]. Furthermore, it is known that following maturation of HL-60 cells to neutrophils, superoxide-generating activity is obtained [14], and NADPH oxidase components are expressed [15].…”

Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation

“…indicating that bone marrow myeloid precursors can reduce NBT (27) and ingest paraffin droplets (l) and that other morphologically immature leukemia cell lines generate Oz (12) . We used glucose oxidation as a measure of HMPS activity to examine changes in the respiratory burst.…”

Metabolic basis of benzene toxicity

“…MPO requires hydrogen peroxide as a substrate for effective turnover; the concentration of hydrogen peroxide available to support MPOmediated metabolism in bone marrow is unknown. Granulocytic respiratory burst activity is known to generate superoxide and hydrogen peroxide (44) and approximately 8% of bone marrow neutrophils demonstrated respiratory burst activity in the absence of exogenous stimulation (45). Long-term bone marrow cultures have also been shown to produce both superoxide and hydrogen peroxide (46), suggesting that in situ production of hydrogen peroxide may occur in bone marrow.…”