Subcellular localization and dynamics of components of the respiratory burst oxidase.

Membrane and cytosolic factors cooperate to generate NADPH-oxidase. The study of the syndrome of NADPH-oxidase deficiencies, chronic granulomatous disease, has enabled the identification of two membrane factors: a flavin adenine dinucleotide flavoprotein and a b cytochrome. The nature of the cytosolic components is still unknown, but a 47-kD protein, whose phosphorylation occurs in parallel with the generation of a respiratory burst in intact cells, seems to be one of the cytosolic factors.

Lactoferrin-mediated transfer of iron to intracellular ferritin in human monocytes.

Interactions of 125I-59Fe-lactoferrin with human monocytes were studied. After 4 hours of incubation, the uptake of 59Fe exceeded that of 125I. In dissociation studies the cellular 59Fe-activity was only partly dissociable during 16 h, whereas the 125I-activity could be released nearly completely.

Reduced function of neutrophils during routine postoperative glucose infusion.

Postoperative infusion of carbohydrate solution leads to moderate fall in the serum concentration of inorganic phosphate. The possible significance of this fall for cellular function as expressed by the neutrophils was investigated in 16 patients undergoing elective cholecystectomy. On postoperative day 2 all received 1 l 10% glucose intravenously to which in eight (randomly chosen) cases 10 mmol phosphate buffer/l had been added.

Polymorphonuclear leucocyte dysfunction during short term metabolic changes from normo- to hyperglycemia in type 1 (insulin dependent) diabetic patients.

Polymorphonuclear leucocyte (PMN) ingestion of particles coated with lipopolysaccharide (LPS) from Escherichia coli was compared to other PMN functions in seven patients with insulin dependent diabetes mellitus (IDDM) during short-term controlled metabolic changes from normo- to hyperglycemia without ketoacidosis. Factors known to interfere with PMN functions were excluded. PMN ingestion of particles coated with both LPS and bovine serum albumin became reduced from normo- to hyperglycemia.

Prevention of tissue damage: inhibition of myeloperoxidase mediated inactivation of alpha 1-proteinase inhibitor by N-acetyl cysteine, glutathione, and methionine.

The ability of the sulphur compounds, N-acetyl cysteine, Methionine, and Glutathione to prevent inactivation of alpha 1-proteinase inhibitor by Myeloperoxidase-H2O2-Cl--system was investigated in vitro with purified components. The Myeloperoxidase system, or its main product HOCl by itself, readily abrogated the ability of alpha 1-proteinase inhibitor to inhibit elastase. This inactivation of alpha 1-proteinase inhibitor was effectively prevented by micromolar concentrations of N-acetyl cysteine, Methionine and reduced Glutathione, whereas oxidized Glutathione was much less effective.

Chemoattractant-regulated mobilization of a novel intracellular compartment in human neutrophils.

A novel mobilizable intracellular compartment was identified in human neutrophils by latent alkaline phosphatase activity. This compartment is mobilized to the plasma membrane much more readily than any identified granule subset and has kinetics of up-regulation in the membrane similar to those reported for a variety of receptor proteins. Triton X-100 permeabilization of both intact human neutrophils and subcellular fractions obtained by density-gradient centrifugation revealed that 70 percent of the alkaline phosphatase is located in an intracellular compartment distinct from primary, secondary, and gelatinase granules and from the plasma membrane.

Development of the phagocytic and cidal capacity during maturation of myeloid cells: studies on cells from patients with chronic myelogenous leukaemia.

Myeloid cells from peripheral blood of patients with chronic myelogenous leukaemia were isolated and fractionated by density gradient centrifugation using Lymphoprep gradient followed by discontinuous Percoll gradients. Six fractions were obtained, each enriched in one of the morphologically identifiable types of myeloid cells from myeloblasts to polymorphonuclear neutrophils. Each of these cell types were functionally and biochemically characterized.

Stimulated mobilization of monocyte Mac-1 and p150,95 adhesion proteins from an intracellular vesicular compartment to the cell surface.

Monocytes were stimulated to increase their cell surface quantity of leukocyte adhesion proteins p150,95 and Mac-1 by the chemoattractant formyl-methionyl-leucyl-phenylalanine, or other mediators such as platelet-derived growth factor, tumor necrosis factor, C5a, and leukotriene B4. Dose-response curves indicated variations in the sensitivity of monocytes and granulocytes to these mediators. These increases were independent of protein synthesis and half-maximal at 2 min.

Beta 2-microglobulin in neutrophils: an intragranular protein.

The subcellular localization of beta2-microglobulin (beta 2m) in human neutrophils was determined by an enzyme-linked immunosorbent assay on subcellular fractions obtained by Percoll density gradient centrifugation of neutrophils disrupted by nitrogen cavitation. The neutrophils were found to contain 160 ng beta 2m/mg protein. Approximately two-thirds co-located with the markers for specific granules and was released from intact cells during degranulation, whereas one-third of the beta 2m was located together with markers of the plasma membrane.

Lactic acid concentration and acid-base balance in cerebrospinal fluid: observations of diagnostic importance in non-Hodgkin lymphoma.

Measurements of lactic acid concentration and gas analysis were performed in lumbar cerebrospinal fluid from 36 patients without malignant central nervous system involvement and four patients with meningeal dissemination of non-Hodgkin lymphoma. The upper lactic acid concentration in controls of 2.48 mmol/l was exceeded in all four patients, also in cases with low blast counts and normal protein and glucose content.

Anomalous neutrophil granule distribution in a patient with lactoferrin deficiency: pertinence to the respiratory burst.

Neutrophils from a patient with lactoferrin deficiency were examined and the quantity and subcellular localization of protein markers were determined on Percoll density gradients. Distribution of azurophilic and specific granule markers was abnormal in that azurophilic granules were lighter than normal and appeared in the fraction of the gradient where normally the specific granules sediment. The specific granule membrane markers, cytochrome b-235 and its associated flavoprotein, were abnormally distributed in the gamma fraction, the site of the plasma membrane marker alkaline phosphatase.

Neutrophils autoinactivate secretory products by myeloperoxidase-catalyzed oxidation.

The neutrophil response to inflammatory stimuli involves the formation of reactive oxygen species and secretion of granule enzymes. In studying secretion of vitamin B12 binding protein by human neutrophils, we noted a major decrease in total recoverable activity from the extracellular fluid plus the stimulated cells (54% of resting cells). Recovery of B12 binding protein from neutrophils exposed to phorbol myristate acetate or opsonized zymosan was significantly enhanced on addition of heme enzyme inhibitors (azide, cyanide) or catalase or when halide-free medium was used.

The respiratory burst of phagocytosis: biochemistry and subcellular localization.

Using nitrogen cavitation and Percoll density gradient centrifugation for subcellular centrifugation of human neutrophils, approximately 90% of the low potential b-cytochrome, unique for phagocytes, as well as 50% of the flavoproteins in normal neutrophils were found in a granule fraction which co-sedimented with the specific granules. Upon stimulation of the intact cells with phorbol myristate acetate, both the b-cytochrome and the flavoprotein translocated from this granule fraction to the fractions which contained the plasma membranes and the NADPH oxidase activity. In neutrophils from two patients with chronic granulomatous disease, both the b-cytochrome and the flavoprotein of the granules were absent, but flavoprotein was present in normal amounts in the membrane and cytosol fractions.

Proton secretion by stimulated neutrophils. Significance of hexose monophosphate shunt activity as source of electrons and protons for the respiratory burst.

Phagocytosis by neutrophils is accompanied by a burst in O2 consumption and activation of the hexose monophosphate shunt (HMPS). Proton secretion equal to the amount of O2 consumed is an additional feature of the respiratory burst, but its source has not been identified, nor has the source of all electrons donated to O2 in the respiratory burst. We chemically quantitated total CO2 generation in human neutrophils and found that proton secretion elicited by phagocytosis was accompanied by a stoichiometric increase in CO2 generation.

Development of the superoxide-generating system during differentiation of the HL-60 human promyelocytic leukemia cell line.

Utilizing the induced differentiation of HL-60 promyelocytic leukemia cells as a model of myeloid maturation, we examined the development of the superoxide-generating system, focusing on NADPH oxidase activity, membrane depolarization, and cytochrome b content. NADPH oxidase activity, measured as NADPH-dependent superoxide production, increased with both spontaneous and N,N-dimethylformamide-induced differentiation. Activity in particulate fractions from induced HL-60 cells and human peripheral blood polymorphonuclear leukocytes was proportional to their relative rates of superoxide production, but activity from uninduced cells was surprisingly high: one-third that from induced cells, despite only 7% their rate of superoxide generation.

Bactericidal mechanisms of the human neutrophil. An integrated biochemical and morphological model.

The bactericidal activity of the human neutrophil is dependent on a coordinated series of events by which the bacteria become confined to a vacuole. Fusion of the azurophil and specific granules with the phagocytic vacuole results in secretion of BPI, the primary oxygen independent bactericidal protein, and of myeloperoxidase into the phagolysosome. Simultaneously, an electron transport chain, the NADPH oxidase, is activated in the membrane of the phagolysosome, resulting in generation of H2O2, which together with myeloperoxidase and Cl- forms a highly bactericidal agent.

Subcellular localization of the human neutrophil NADPH oxidase. b-Cytochrome and associated flavoprotein.

Human neutrophils were fractionated by nitrogen cavitation and Percoll density centrifugation, and the subcellular localization of FAD-flavoprotein, b-cytochrome, NADH-cytochrome b5 reductase, and NADPH-dependent cytochrome c reductase were determined in normal cells, cells from two patients with chronic granulomatous disease (CGD), and normal cells that had been stimulated with phorbol myristate acetate. In normal cells, a FAD-flavoprotein is found in a 1:2 molar ratio, with cytochrome b in the fractions containing the specific granules. Triton X-114 phase distribution indicates that the b-cytochrome but not the b-cytochrome-associated flavoprotein is an integral membrane protein.