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Biomedical subjects

D A Bass

Publications and source records attributed to D A Bass.

At least 55 records · Page 3Linked to original sources
PubMed1985

Independent stimulation of membrane potential changes and the oxidative metabolic burst in polymorphonuclear leukocytes.

Early events of stimulus-response coupling in polymorphonuclear leukocytes (PMNL) reportedly include membrane depolarization as a necessary antecedent to oxidative responses. However, depolarization by nonspecific means (ionophores) is insufficient to elicit an oxidative burst. This apparent conflict might be related to whether depolarization is due to membrane receptor-mediated stimulation of PMNL. We used two fluorescent probes and dual laser flow cytometry to monitor both membrane potential and oxidative product formation in individual PMNL, following stimulation by phorbol myristate acetate (PMA) or formylmethionyl-leucyl-phenylalanine (fMLP). Dipentylindocarbocyanine [di-I-C5(3)] is one of a family of dyes that partition between cells and aqueous media as a function of transmembrane potential. The dye appeared stable in the presence of PMNL oxidative products. Oxidation of intracellularly trapped, nonfluorescent dichlorofluorescin (DCFH) to fluorescent dichlorofluorescein (DCF) provided a quantitative assessment of oxidative metabolism (H2O2 production) of stimulated PMNL. Incubating PMNL with both fluorochromes resulted in stable red [di-l-C5(3)] and green (DCF) fluorescence when examined on a Cytofluorograph. Upon stimulation by 0.03 to 0.1 nmol/L PMA, PMNL showed a unimodal apparent depolarization (decrease in di-l-C5(3) fluorescence). Oxidative activity (increased DCF fluorescence) was first seen at a concentration of PMA of 0.17 nmol/L, higher than that required to elicit depolarization. This oxidative burst appeared as a dose-dependent, graded response. Thus, receptor-mediated membrane depolarization, although antecedent to the onset of the oxidative burst, was not in itself sufficient to trigger the oxidative metabolic response. When PMNL were isolated by centrifugation through Ficoll-metrizoate, fMLP caused an apparent depolarization of a variable subpopulation of the cells. However, such purified PMNL appeared relatively unstable and often depolarized spontaneously. PMNL studied without centrifugation through Ficoll-metrizoate were stable. Moreover, fMLP stimulation of such cells did not cause membrane depolarization but did stimulate a two- to six-fold increase in DCFH oxidation. Apparently, membrane depolarization may antecede oxidative responses in PMNL, but appears to depend upon the method of cell preparation and the nature of the stimulus.

Carbocyanines↗
PubMed1984

Co-localization of superoxide generation and NADP formation in plasma membrane fractions from human neutrophils.

In order to resolve discrepancies in the literature concerning the subcellular localization of NADPH oxidase, we disrupted human neutrophils by nitrogen cavitation and fractionated the subcellular organelles on a discontinuous sucrose density gradient. The lightest fraction was 20- to 40-fold enriched for plasma membranes as determined by the marker enzymes alkaline phosphatase and phosphodiesterase I as well as by the ratio of lipid phosphorus to protein. There was a significant decrease in the specific activities of the granule markers myeloperoxidase, lysozyme, and beta-glucuronidase. An intermediate fraction was enriched in membrane markers but not to the extent the lightest fraction was enriched. This fraction contained more granular contamination, as shown by the marker enzymes. In contrast, the densest bands of the gradient were enriched for granule markers with little contamination by plasma membrane. Superoxide generation and NADP formation were primarily associated with the two membrane-enriched fractions from polymorphonuclear leukocytes stimulated with phorbol myristate acetate. The NADP formation associated with a dense granule fraction observed previously in our laboratory was probably due to a cyanide-stimulated oxidation of NADPH by myeloperoxidase.

Alkaline Phosphatase↗
PubMed1984

Flow cytometric quantitation of oxidative product formation by polymorphonuclear leukocytes during phagocytosis.

Stimulation of the oxidative metabolic burst of human polymorphonuclear leukocytes (PMNL) may occur by an all-or-none trigger mechanism or by a graded response to increasing stimulation of an individual cell. If the proposed all-or-none mechanism occurred during phagocytosis, a PMNL would expend all of its metabolic potential at once, yet PMNL can proceed to ingest multiple organisms. This study employed dual laser flow cytometry to correlate the number of cell-associated organisms with oxidative product formation in individual PMNL. Intracellular oxidation of nonfluorescent 2',7'-dichlorofluorescein (DCFH) to highly fluorescent 2',7'-dichlorofluorescein (DCF) provided a quantitative assay of H2O2-dependent oxidative product formation generated by the cell's oxidative metabolic burst. Staphylococcus aureus were fixed and stained with Texas red to allow simultaneous monitoring of bacteria (red fluorescence, greater than 580 nm) and DCF (green fluorescence, 510 to 550 nm) content of each cell. Computer correlation of bacterial and DCF fluorescence allowed determination of the DCF formation by PMNL containing specific numbers (0 to 15) of bacteria. Oxidative product formation was directly related to the number of bacteria ingested in a time-dependent manner (mean per cell of 6.4, 12.8, 19.1, and 24.4 attomoles (amol) DCF formed per cell per bacterium after 15, 30, 45, and 60 min, respectively. Opsonization of bacteria with fresh normal serum (primarily C3b opsonization) or with specific IgG demonstrated qualitatively similar responses, except that the response per IgG-opsonized organism was, on the average, more than twice the response to bacteria opsonized with serum. Thus, sequential phagocytosis of multiple bacteria elicits an incremental oxidative response of human PMNL.

Blood Physiological Phenomena↗
PubMed1984

Characterization of the effect of influenza virus on polymorphonuclear leukocyte membrane responses.

Depressed chemotactic activity of polymorphonuclear leukocytes (PMNL) infected with influenza virus could be due to changes occurring at the plasma membrane. The present study examined the effect of unopsonized influenza virus on chemotaxis, adherence, receptor binding, shape change, membrane fluidity, and release of specific granules from PMNL. Chemotactic activity of PMNL under-agarose to the chemoattractants, zymosan-activated serum ( ZAS ) and N-formyl-methionyl-leucyl-phenylalanine (fMLP), and adherence of PMNL to a plastic surface were markedly decreased in virus-treated cells as compared to control cells. The binding of fMLP to the PMNL was increased in virus-treated cells compared with control cells. Exposure of cells to virus, ZAS , or fMLP caused 35%-50% of the cells to become bipolar in shape, whereas less than 5% of the cells exposed to buffer became bipolar. Influenza virus did not alter membrane fluidity as measured by electron spin resonance spectroscopy with the probe 5-doxyl stearate. Virus-treated PMNL stimulated with FMLP or Staphylococcus aureus exhibited a marked decrease in the amount of lactoferrin released into phagosomes, onto the cells' outer membrane, and into the extracellular medium as compared to control cells. The possible relationship between inhibition of lysosomal enzyme degranulation and decreased chemotactic activity and adherence of PMNL is discussed.

Cell Adhesion↗
PubMed1983

Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation.

We have developed a quantitative assay to monitor the oxidative burst (H2O2 production) of polymorphonuclear leukocytes (PMNL) using single cell analysis by flow cytometry, and have examined whether PMNL respond to membrane stimulation with an all-or-none oxidative burst. During incubation with normal neutrophils, dichlorofluorescin diacetate diffused into the cells, was hydrolyzed to 2',7'-dichlorofluorescin (DCFH) and was thereby trapped within the cells. The intracellular DCFH, a nonfluorescent fluorescein analogue, was oxidized to highly fluorescent 2',7'-dichlorofluorescein (DCF) by PMNL stimulated by phorbol myristate acetate (PMA). That the oxidative product was DCF was shown by excitation/emission spectra and by mass spectrometry of the product from PMA-stimulated PMNL. Normal resting and PMA-stimulated PMNL oxidized 6.9 +/- 0.7 and 160 +/- 13 attomoles DCF per cell, respectively, in 15 min. Absence of calcium and magnesium ions and/or addition of 2 mM EDTA did not inhibit DCF formation by PMNL stimulated by 100 ng/ml PMA. Since EDTA prevented aggregation of PMNL (even when stimulated by 100 ng/ml PMA), which would prevent accurate flow cytometric analysis, further experiments were performed with EDTA in the medium. A close correlation between average DCFH oxidation and hexose monophosphate shunt stimulation was demonstrated using cells from patients whose PMNL had oxidative metabolic defects of varying severity. Intracellular DCFH was also oxidized by reagent H2O2 or oxygen derivatives generated by glucose oxidase + glucose or by xanthine oxidase + acetaldehyde; DCFH oxidation by these systems was inhibited by catalase but unchanged by superoxide dismutase. The data indicate that the DCFH oxidation assay is quantitatively related to the oxidative metabolic burst of PMNL, and they strongly suggest that the reaction is mediated by H2O2 generated by the PMNL. Incubation of PMNL with varying concentrations of PMA caused graded responses by all PMNL present; i.e., 1 ng/ml PMA caused a mean response of 34% maximal with a single population of responding PMNL (rather than 66% resting and 34% fully stimulated as predicted by the all-or-none hypothesis). Thus, with these assay conditions, oxidative product formation by PMNL occurs as a graded response to membrane stimulation by PMA.

Flow Cytometry↗
PubMed1982

Influenza A virus-induced polymorphonuclear leukocyte dysfunction.

Previous studies have shown that influenza A virus can activate the polymorphonuclear leukocyte (PMN) respiratory burst and that upon subsequent stimulation of the cell there is depressed metabolic function. We examined the mechanism by which influenza virus causes PMN dysfunction by measuring the effect upon the chemiluminescent activity of cells of varying the type of influenza virus used, the period of time that cells were exposed to virus, and the secondary stimulus that was used. The various types of intact influenza virus elicited different amounts of chemiluminescent activity, but when cells were subsequently stimulated with phorbol myristate acetate, each virus caused equivalent depression of the PMN response. Purified glycoproteins incorporated into a liposome structure similarly stimulated the PMN chemiluminescence, yet did not induce PMN dysfunction. Depressed PMN function was noted after as little as 5 min of incubation of cells with virus and occurred to both receptor-dependent (zymosan, N-formylmethionyl-leucyl-phenylalanine, and phorbol myristate acetate) and -independent (calcium ionophore A23187) stimuli.

Humans↗
PubMed1982

Inhibition of neutrophil lysosome-phagosome fusion associated with influenza virus infection in vitro. Role in depressed bactericidal activity.

The present study examined the effect of various unopsonized strains of influenza A virus on release of myeloperoxidase (MPO) and acid phosphatase in polymorphonuclear leukocytes (PMNL). These results were correlated with the effect that these same viruses had on bactericidal activity in PMNL. Several strains of virus inhibited the fusion of azurophil granules with phagosomes containing Staphylococcus aureus. These same strains inhibited the extracellular release of MPO from PMNL (39-59%) and caused depressed killing (42-77%). In contrast, one of the influenza viruses (X-47a) did not inhibit PMNL MPO release or killing. The data indicate a close relationship between the ability of influenza virus to ablate normal intracellular lysosome-phagosome fusion with subsequent depression of bactericidal functions of PMNL.

Blood Bactericidal Activity↗
PubMed1982

Antigen-mediated release of eosinophil growth stimulating factor from Trichinella spiralis sensitized spleen cells: a comparison of T. spiralis stage-specific antigen preparations.

When non-adherent, Trichinella spiralis-sensitized mouse spleen cells were challenged in vitro with T. spiralis antigens, an eosinophil growth factor (Eo-GSF) was released into the culture medium. This factor was assayed by its ability to initiate eosinophil production in liquid cultures of syngeneic, non-adherent marrow cells obtained from unsensitized mice. Extracts of each parasite stage as well as excretory-secretory (ES) products of adult and muscle larva stages were compared for their ability to stimulate spleen cells to release Eo-GSF. All stages and ES products had this ability but most of the preparations had unique dose-optima and there was a very wide range with regard to the optimum dose (in microgram protein/ml): (i) preadult stage, 1 x 10(-5); (ii) muscle stage ES products, 1 x 10(-3); (iii) muscle stage, 1 x 10(-2); (iv) adult stage, 1 x 10(-2); (v) adult ES products, 1 x 10(-1); and (vi) newborn stage, 1.0. When the Eo-GSF-containing conditioned media derived from spleen cell cultures exposed to the optimum dosages were tested on the same population of marrow cells, three potency groups were identified. The rank order of potency was: muscle stage ES products greater than preadult, newborn and adult stages greater than muscles stage and adult ES products. Preliminary experiments revealed that this ranking was not maintained with regard to the release of neutrophil and macrophage growth factors by these preparations.

Aging↗
PubMed1981

Role of Ca2+ and Mg2+ in neutrophil hexose transport.

The influence of extracellular Ca2+ and Mg2+ on the transport of 2-deoxy-[3H]glucose into human polymorphonuclear neutrophils was studied. Omission of these cations from the cell suspensions had little effect on resting hexose uptake. Furthermore, the addition of the bivalent cation chelator, EDTA, depressed uptake only slightly. Similarly, neither cation was essential for the enhanced 2-deoxy-D-[3H]glucose uptake stimulated by two chemotactic factors (C5a and N-formylmethionylleucylphenylalanine) and arachidonic acid: enhanced uptake was only partially depressed by the omission of Ca2+ and Mg2+ from the suspensions and was still prominent in the presence of EDTA. Two other neutrophil stimulants, the ionophores, A23187 and ionomycin, also enhanced hexose uptake but their actions were heavily dependent upon extracellular bivalent cations and were totally abrogated by EDTA. In all instances, extracellular Ca2+, but not Mg2+, supported optimal enhanced hexose transport induced by stimuli. Activation of 2-deoxy-D-[3H]glucose uptake by each of the five stimuli was totally blocked by cytochalasin B (a blocker of carrier-mediated hexose transport) and D-glucose but not by L-glucose. The data indicate, therefore, that a variety of neutrophil stimulants activate carrier-mediated hexose transport. Although this transport can be triggered by the movement of extracellular Ca2+ into the cell (as exemplified by the action of the two ionophores), such Ca2+ movement is not required for the actions of chemotactic factors or arachidonic acid. Other mechanisms, such as a rearrangement of intracellular Ca2+, may be involved in mediating the activation of hexose transport induced by the latter stimuli.

Biological Transport, Active↗
PubMed1981

Neutrophil responses to platelet-activating factor.

1-O-Alkyl-2-O-acetyl-sn-glycerol-3-phosphorylcholine (i.e., platelet-activating factor) was prepared and confirmed to possess potent platelet aggregating activity. It was also potent in aggregating and degranulating rabbit and human neutrophils. When injected into rabbits, the lipid induced profound neutropenia, thrombocytopenia, and anaphylactic symptoms. The lyso derivative of this lipid, 1-O-alkyl-sn-glycerol-3-phosphorylcholine, was inactive or several orders of magnitude weaker in inducing these responses. The acetylated lipid appears to be a potent stimulator of both platelets and neutrophils. Its anaphylactic-like toxicity may be related, at least in part, to its ability to aggregate or otherwise stimulate these cells.

Animals↗
PubMed1981

Stimulation of hexose uptake by human eosinophils with chemotactic factors.

The ability of several stimuli, all of which have been reported to stimulate eosinophil motility, to augment uptake or extracellular deoxyglucose (DOG), a glucose analogue, was studied. DOG uptake was stimulated in a concentration-dependent manner by the more potent chemotaxins--zymosan-activated serum, partially purified C5a. N-formyl-methionyl-leucyl-phenylalanine, and 5- and 11-hydroxyeicosatetraenoic acids. Less potent stimuli--the eosinophil chemotactic factor of anaphylaxis, histamine, and prostaglandin E2--did not stimulate DOG uptake. However, the correlation between the abilities of the agents to stimulate motility and DOG uptake was not completely uniform. Prostaglandin F2 alpha was a potent stimulant of DOG uptake yet is known to enhance only weakly chemokinesis and to be not chemotactic for eosinophils. The combined data from this and other studies indicate that these stimuli elicit specific and different sets of functional responses from eosinophils.

Arachidonic Acids↗
PubMed1981

Activation of lysosomal acid phosphatase of eosinophil leukocytes.

Acid phosphatase (AP) within the specific granules of eosinophils is held in a latent, inactive form prior to degranulation. Eosinophils responded to membrane stimuli with activation of AP throughout the granules of the cell, prior to degranulation. Activation occurred within the time required for fixation of the cells by glutaraldehyde. Whereas resting normal eosinophils demonstrated AP activity in 7.8 +/- 1.2 per cent of the granules, cells stimulated with opsonized staphylococci demonstrated activity in 78.9 +/- 5.0 per cent of the granules, and the amount of lead phosphate reactant deposited per active granule was considerably more intense in the stimulated than in the unstimulated cells. A similar stimulation of AP activity in eosinophils occurred with exposure to the surface-active agent phorbol myristate acetate or the calcium ionophore A23187. Activation was not altered by 5 microgram. per ml. of cytochalasin B or 4 x 10(-6) M colchicine. These aspects of the response closely parallel the stimulation of oxidative metabolism of eosinophils; however, the two were separable. Eosinophils obtained from a patient with chronic granulomatous disease, although unable to activate the normal oxidative metabolic burst, demonstrated a normal activation of AP.

Acid Phosphatase↗
PubMed1981

Effects of quercetin on magnesium-dependent adenosine triphosphatase and the metabolism of human polymorphonuclear leukocytes.

The bioflavinoid quercetin was found to exert at least three separate effects on human polymorphonuclear leukocytes. (1) Concentrations of approximately 100 microM inhibited the membrane-associated magnesium adenosine triphosphatase by 60%-80% in either broken cell preparations or intact cells. Lineweaver-Burk plots showed the inhibition to be uncompetitive in nature. (2) Similar concentrations of quercetin inhibited respiratory burst activity of the cells as measured by oxygen consumption, glucose oxidation, or iodination of protein. All inhibitions were dose-dependent and were observed with either opsonized zymosan or phorbol myristate acetate as stimulus. (3) Quercetin likewise inhibited the transport of the nonmetabolizable hexose, 3H-2-deoxyglucose. These observations are most consistent with the hypothesis that quercetin exerts a generalized effect at the level of the cell membrane of the neutrophil.

Adenosine Triphosphatases↗