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2.
Sci Rep ; 10(1): 9766, 2020 06 17.
Article in English | MEDLINE | ID: mdl-32555384

ABSTRACT

The blood-brain barrier (BBB) is a dynamic component of the brain-vascular interface that maintains brain homeostasis and regulates solute permeability into brain tissue. The expression of tight junction proteins between adjacent endothelial cells and the presence of efflux proteins prevents entry of foreign substances into the brain parenchyma. BBB dysfunction, however, is evident in many neurological disorders including ischemic stroke, trauma, and chronic neurodegenerative diseases. Currently, major contributors to BBB dysfunction are not well understood. Here, we employed a multicellular 3D neurovascular unit organoid containing human brain microvascular endothelial cells, pericytes, astrocytes, microglia, oligodendrocytes and neurons to model the effects of hypoxia and neuroinflammation on BBB function. Organoids were cultured in hypoxic chamber with 0.1% O2 for 24 hours. Organoids cultured under this hypoxic condition showed increased permeability, pro-inflammatory cytokine production, and increased oxidative stress. The anti-inflammatory agents, secoisolariciresinol diglucoside and 2-arachidonoyl glycerol, demonstrated protection by reducing inflammatory cytokine levels in the organoids under hypoxic conditions. Through the assessment of a free radical scavenger and an anti-inflammatory endocannabinoid, we hereby report the utility of the model in drug development for drug candidates that may reduce the effects of ROS and inflammation under disease conditions. This 3D organoid model recapitulates characteristics of BBB dysfunction under hypoxic physiological conditions and when exposed to exogenous neuroinflammatory mediators and hence may have potential in disease modeling and therapeutic development.


Subject(s)
Blood-Brain Barrier/pathology , Endothelium, Vascular/pathology , Hypoxia/physiopathology , Inflammation/physiopathology , Models, Biological , Neurons/pathology , Organoids/pathology , Anti-Inflammatory Agents/pharmacology , Astrocytes/drug effects , Astrocytes/metabolism , Astrocytes/pathology , Biological Transport , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Cell Membrane Permeability , Cytokines/metabolism , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Humans , Neurons/drug effects , Neurons/metabolism , Organoids/drug effects , Organoids/metabolism , Oxidative Stress
3.
Am J Respir Cell Mol Biol ; 23(1): 37-44, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10873151

ABSTRACT

Secretory phospholipase A(2) (sPLA(2)) enzymes contribute to inflammatory injury in human lungs by several mechanisms, including eicosanoid production and hydrolytic damage to surfactant phospholipids. Several distinct sPLA(2) genes have been described in human tissue but little is known regarding their presence, localization, or function(s) within lungs. We hypothesized that sPLA(2)s would have cell-specific distributions within lung. We used reverse transcriptase/polymerase chain reaction to identify sPLA(2) messenger RNAs (mRNAs) in adult human lung tissue. Resulting complementary DNA (cDNA) sequences indicated that total lung extracts contained mRNA for Groups IB, IIA, V, and X sPLA(2). An epithelial cell line, BEAS cells, expressed only Groups IIA, V, and X. We used these cDNAs to clone these enzymes, especially the recently described Group X and Group V enzymes. Digoxigenin-labeled complementary RNA probes were used to determine localization of each sPLA(2) by in situ hybridization of human lung. Hybridization was strongly positive for Group X and Group V in airway epithelial cells, which failed to hybridize Group IB or IIA probes. Although four known mammalian sPLA(2) isotypes were expressed in lung, only Group X and Group V sPLA(2) mRNAs appear uniquely expressed in airway epithelium, suggesting they could provide a mechanism of pulmonary surfactant hydrolysis during lung injury.


Subject(s)
Epithelial Cells/enzymology , Lung/enzymology , Phospholipases A/metabolism , Animals , Bronchi/cytology , Bronchi/enzymology , COS Cells , Cell Line , Cloning, Molecular , Group II Phospholipases A2 , Histocytochemistry , Humans , Hydrolysis , In Situ Hybridization , Isoenzymes/genetics , Isoenzymes/metabolism , Lung/cytology , Organ Specificity , Phospholipases A/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Substrate Specificity , Transfection
4.
Clin Rev Allergy Immunol ; 17(1-2): 5-26, 1999.
Article in English | MEDLINE | ID: mdl-10436856

ABSTRACT

The metabolism of AA reflects a carefully balanced series of biochemical pathways. The level of free arachidonate in a cells is controlled by de novo synthesis, dietary uptake, and transcellular metabolism. Lysophospholipids are key controlling substrates for a variety of acyl transferase and transacylase reactions, whose combined effect is to remodel cellular membranes placing AA in up to 20 different molecular species of phospholipids. PLA2 enzymes, both cytosolic and secretory, can release AA for subsequent metabolism via lipoxygenase, COX, and cytochrome P450 enzymes into a variety of eicosanoid products. Reactions are often tissue- and cell-specific, and provide a spectrum of inflammatory mediator release in which many of the molecular details remain to be elucidated.


Subject(s)
Arachidonic Acid/metabolism , Inflammation Mediators/metabolism , Animals , Humans , Lung/metabolism , Lung/pathology , Lung Injury , Models, Immunological
5.
Biochim Biophys Acta ; 1394(2-3): 224-34, 1998 Nov 02.
Article in English | MEDLINE | ID: mdl-9795228

ABSTRACT

We have shown previously that both 1,2-diacylglycerol (AAG) and 1-O-alkyl-2-acylglycerol (EAG) prime neutrophil release of arachidonic acid via uncharacterized phospholipases A2. Therefore, we investigated the actions of EAG and AAG specifically on neutrophil cytosolic (cPLA2) and secretory (sPLA2) phospholipase A2s. We hypothesized that AAG as a protein kinase activator would activate cPLA2 via phosphorylation events. EAG is antagonistic to the AAG activation of PKC, thus it was not expected to act via phosphorylation of cPLA2. Neutrophils were primed with either AAG or EAG and then stimulated with fMLP. When neutrophils were primed with 5-20 microM 1,2-diacylglycerol, a shift was observed in cPLA2 migration on SDS-PAGE gels, consistent with phosphorylation of the protein. This gel shift was not seen after exposure to EAG. AAG also caused a parallel increase in enzymatic activity of cPLA2 that was not seen with EAG. We also investigated whether either diglyceride would cause similar priming or direct secretion of sPLA2. Both AAG and EAG directly caused significant secretion of neutrophil sPLA2. EAG also increased the release of sPLA2 in cells subsequently stimulated with fMLP. Thus, AAG activated cPLA2 and stimulated secretion of sPLA2. In contrast, EAG did not activate cPLA2, but directly activated secretion of sPLA2. We also demonstrated that human synovial fluid sPLA2 increased AA release from resting and fMLP-stimulated neutrophils. Given that diglycerides prime for release of AA, PAF, and LTB4, these current data support the hypothesis that such priming may be mediated by phosphorylation dependent (cPLA2) or phosphorylation independent (e.g. secretion of sPLA2) events.


Subject(s)
Diglycerides/pharmacology , Neutrophils/enzymology , Phospholipases A/metabolism , Arachidonic Acid/metabolism , Blotting, Western , Cytosol/enzymology , Enzyme Activation/drug effects , Gas Chromatography-Mass Spectrometry , Humans , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Phospholipases A2 , Phosphorylation , Synovial Fluid/enzymology
6.
Am J Physiol ; 275(4): L740-7, 1998 10.
Article in English | MEDLINE | ID: mdl-9755106

ABSTRACT

Hydrolysis of surfactant-associated phospholipids by secretory phospholipases A2 is an important potential mechanism for surfactant dysfunction in inflammatory lung diseases. In these conditions, airway secretory phospholipase A2 (sPLA2) activity is increased, but the type of sPLA2 and its impact on surfactant function are not well understood. We examined in vitro the effect of multiple secretory phospholipases A2 on surfactant, including their ability to 1) release free fatty acids, 2) release lysophospholipids, and 3) increase the minimum surface tension (gammamin) on a pulsating bubble surfactometer. Natural porcine surfactant and Survanta were exposed to mammalian group I (recombinant porcine pancreatic) and group II (recombinant human) secretory phospholipases A2. Our results demonstrate that mammalian group I sPLA2 hydrolyzes phosphatidylcholine (PC), producing free fatty acids and lysophosphatidylcholine, and increases gammamin. In contrast, mammalian group II sPLA2 demonstrates limited hydrolysis of PC and does not increase gammamin. Group I and group II secretory phospholipases A2 from snake venom hydrolyze PC and inhibit surfactant function. In summary, mammalian secretory phospholipases A2 from groups I and II differ significantly from each other and from snake venom in their ability to hydrolyze surfactant-associated PC.


Subject(s)
Biological Products , Phosphatidylcholines/metabolism , Phospholipases A/metabolism , Pulmonary Surfactants/chemistry , Pulmonary Surfactants/metabolism , Animals , COS Cells , Fatty Acids, Nonesterified/metabolism , Group II Phospholipases A2 , Humans , Hydrolysis , Lung/enzymology , Lysophospholipids/metabolism , Mammals , Pancreas/enzymology , Phospholipases A2 , Recombinant Proteins/metabolism , Snake Venoms , Substrate Specificity , Surface Tension , Swine , Transfection
7.
Biochim Biophys Acta ; 1389(3): 273-84, 1998 Jan 23.
Article in English | MEDLINE | ID: mdl-9512652

ABSTRACT

Cytokines alter neutrophil (PMN) function during inflammation, and Tumor Necrosis Factor (TNF) in vitro primes PMN such that receptor-mediated stimulation causes markedly enhanced release of arachidonic acid. We hypothesized that two Ca(2+)-dependent PLA2's in PMN might be activated during priming of the cell, thus affecting arachidonate release. A low molecular weight, secretory PLA2 was identified by enzymatic activity in the cell free supernates of primed or stimulated PMN, and in PMN disrupted by nitrogen cavitation. The enzymatic activity was calcium-dependent, acid stable, destroyed by dithiothreitol, and blocked by anti-sPLA2 antibodies. TNF caused secretion of sPLA2 and also caused an increase in cell-associated sPLA2 enzymatic activity. Activation and release were maximal with fMLP stimulation of TNF-primed PMN. Neutrophils also contained a cytosolic PLA2 (cPLA2) characterized by enzymatic activity which was calcium dependent, enhanced by dithiothreitol, and blocked by anti-cPLA2 antibody. TNF caused a doubling of cPLA2 enzymatic activity which was associated with phosphorylation of the enzyme as judged by a migration shift on Western blots. Thus, TNF priming of human PMN caused marked increase in fMLP stimulated AA release in parallel to enhanced activity of two different PLA2's.


Subject(s)
N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/physiology , Phospholipases A/blood , Tumor Necrosis Factor-alpha/pharmacology , Arachidonic Acid/blood , Cell Separation , Cytosol/enzymology , Enzyme Activation , Humans , In Vitro Techniques , Kinetics , Neutrophils/drug effects , Neutrophils/enzymology , Phospholipases A/metabolism , Phospholipases A2
8.
Biochim Biophys Acta ; 1347(2-3): 219-30, 1997 Aug 16.
Article in English | MEDLINE | ID: mdl-9295167

ABSTRACT

In human neutrophils, the choline-containing phosphoglycerides contain almost equal amounts of alkylacyl- and diacyl-linked subclasses. In contrast to phosphatidylinositol hydrolysis which yields diacylglycerol, hydrolysis of choline-containing phosphoglycerides by phospholipase D coupled with phosphohydrolase yields both alkylacyl- and diacylglycerol. While diacylglycerol activates protein kinase C, alkylacylglycerol does not, and its role is unclear. Yet previous studies have shown that exogenous alkylacyl- and diacylglycerols can prime for the release of radiolabeled arachidonic acid (AA) in intact neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine. We have now examined the effects of both diacylglycerol (1-oleoyl-2-acetylglycerol; OAG) and alkylacylglycerol (1-O-hexadecyl-2-acetylglycerol; EAG) on the activation of mitogen-activated protein (MAP) kinase and the 85-kDa cytosolic phospholipase A2 (cPLA2) in human neutrophils. We observed that while OAG could effectively activate p42 and p44 MAP kinases along with cPLA2 in a time- and concentration-dependent manner, EAG could not. A novel p40 MAP kinase isoform is also present and activated in response to OAG treatment; the behavior of this MAP kinase isoform is discussed. The activation of cPLA2 and MAP kinase by 20 microM OAG could be inhibited by pretreatment with 1 microM GF-109203X, a selective inhibitor of protein kinase C. Although only OAG activated cPLA2, both OAG and EAG primed for the release of AA mass as determined by gas chromatography/mass spectrometry. The priming of AA release by OAG may be explained by the phosphorylation of cPLA2 through the activation of protein kinase C linked to MAP kinase. However, priming by EAG appears to involve a separate mechanism that is dependent on a different PLA2. Our results support a role for phospholipase D-derived products modulating the activation of cPLA2, further supporting the idea of cross-talk among various phospholipases.


Subject(s)
Diglycerides/pharmacology , Neutrophils/drug effects , Phospholipases A/metabolism , Protein Kinases/metabolism , Arachidonic Acid/analysis , Cytosol/enzymology , Enzyme Activation/drug effects , Humans , Mitogen-Activated Protein Kinase Kinases , Neutrophils/enzymology , Phospholipases A2 , Phosphorylation
9.
Am J Respir Crit Care Med ; 155(2): 421-5, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9032172

ABSTRACT

Phospholipases A2 (PLA2) hydrolyze phospholipids resulting in the release of fatty acids including arachidonic acid (AA) and lysophospholipids. AA, in turn, serves as a substrate for the synthesis of leukotrienes which can cause bronchoconstriction and airways edema and appear to be important mediators of clinical asthma. Further, lysophospholipids may be cytotoxic and/or impair the function of surfactant. We examined the release of secretory PLA2 (sPLA2) and AA into the airways after antigen challenge in 16 subjects with allergic asthma. Asthmatic subjects underwent bronchoscopy with bronchoalveolar lavage (BAL) before and after inhaled antigen challenge; in addition, a single BAL, without inhaled antigen, was performed in 10 control subjects. BAL was obtained at 4 h (n = 7), the time of the late asthmatic response (LAR) (n = 5), or 24 h (n = 4) after challenge. There was no difference between normal and asthmatic subjects in either BAL fluid (BALF) sPLA2 activity or AA concentration at baseline. Both sPLA2 and AA increased after antigen challenge (p < 0.01 and 0.05, respectively). These changes were most marked 4 h after challenge (p < 0.03 for both). sPLA2 may play an important role in the generation of AA in patients with asthma.


Subject(s)
Arachidonic Acid/metabolism , Asthma/metabolism , Bronchoalveolar Lavage Fluid/chemistry , Phospholipases A/metabolism , Asthma/diagnosis , Bronchial Provocation Tests , Gas Chromatography-Mass Spectrometry , Humans , Methacholine Chloride , Phospholipases A2 , Respiratory Function Tests
10.
J Exp Med ; 182(5): 1181-90, 1995 Nov 01.
Article in English | MEDLINE | ID: mdl-7595189

ABSTRACT

Increasing evidence suggests that the metabolism of arachidonic acid (AA) may be different in inflammatory cells isolated from blood or migrating into tissues. To explore the possibility that changes in AA metabolism between blood and tissue inflammatory cells could be due in part to a different content or distribution of AA in glycerolipid classes, we studied these parameters in six human inflammatory cells isolated from blood (eosinophils, monocytes, neutrophils, and platelets) or from the lung tissue (mast cells and macrophages). Lung cells generally had a higher total cellular content of AA than that found in the blood cells. In addition, both mast cells and macrophages had a large endogenous pool of AA associated with triglycerides (TG), containing 45 and 22% of their total cellular AA, respectively. To address the hypothesis that cells migrating into the lung had a higher cellular level of AA and a larger AA pool in TG, we studied neutrophils isolated from the bronchoalveolar lavage (BAL) of patients with adult respiratory distress syndrome. BAL neutrophils had a fourfold increase in cellular AA as compared with blood neutrophils and contained 25% of their AA in TG versus 3% in blood neutrophils. BAL neutrophils also had a higher number of cytoplasmic lipid bodies (8 +/- 3/cell) relative to blood neutrophils (2 +/- 1/cell). High concentrations of free AA were also found in the cell-free BAL fluid of adult respiratory distress syndrome patients. To explore whether changes in BAL neutrophils may be due to the exposure of the cells to high concentrations of exogenous AA found in BAL, we incubated blood neutrophils in culture with AA (10-100 microM) for 24 h. Neutrophils supplemented with AA had a 10-fold increase in the amount of AA associated with TG and a sixfold increase in the number of lipid bodies. In addition, supplementation with AA induced a dose-dependent formation of hypodense cells. Taken together, these data indicate that human inflammatory cells undergo a fundamental and consistent remodeling of AA pools as they mature or enter the lung from the blood. These biochemical and morphological changes can be mimicked in vitro by exposing the cells to high levels of AA. This mechanism may be responsible for the changes in AA mobilization and eicosanoid metabolism observed in tissue inflammatory cells.


Subject(s)
Arachidonic Acid/metabolism , Blood Cells/physiology , Lung/pathology , Mast Cells/physiology , Phagocytes/physiology , Triglycerides/metabolism , Adult , Arachidonic Acid/pharmacology , Blood Platelets/physiology , Bronchoalveolar Lavage Fluid/cytology , Cell Movement , Chemotaxis, Leukocyte , Eosinophils/physiology , Humans , Inflammation , Lipid Metabolism , Lung/metabolism , Macrophages/physiology , Monocytes/physiology , Neutrophils/physiology , Pulmonary Edema/pathology
11.
Biochim Biophys Acta ; 1258(2): 135-44, 1995 Sep 14.
Article in English | MEDLINE | ID: mdl-7548176

ABSTRACT

This investigation examined the concept that arachidonic acid (AA) serves as a second messenger in stimulation of the respiratory burst and degranulation of polymorphonuclear neutrophils (PMN). The main support for this idea is from observations that reagent AA, added to cell suspensions, stimulates the respiratory burst and degranulation and these events are blocked by PLA2 inhibitors. We verified that exogenously-added AA stimulated release of O2-, myeloperoxidase (MPO), and lysozyme (LZ), but this required amounts of AA which approximated the critical micellar concentration. This suggested that such administration of AA might act as an extracellular agonist, similar to particulate stimuli, rather than acting as a second messenger as might occur following mobilization of AA from cellular membranes. To investigate the role of fatty acids released by hydrolysis of cellular phospholipids, exogenously-added group I, II or III PLA2's were used to mobilize fatty acids from cellular membranes. Mole quantities of cell-associated free fatty acids were measured by negative ion chemical ionization gas chromatography/mass spectrometry. AA mobilization in response to exogenous PLA2 was dose- (0.1 to 10 U/ml PLA2) and time-dependent (peak at 1 to 2 min with a reduction by 4 min). Resting neutrophils contained < 10 pmol free AA/10(7) PMN; the receptor-mediated agonist N-formyl-methionyl-leucyl-phenylalanine (fMLP) alone did not increase these values. Exogenously-added PLA2 generated large quantities of free AA in control and fMLP-treated cells (462 +/- 122 and 2097 +/- 176 pmol/10(7) PMN, respectively); however, this did not induce O2-, nor did it augment the level of O2- stimulated by fMLP. Also, PLA2 caused no degranulation and did not alter degranulation induced by fMLP. PLA2 also did not alter O2- or degranulation responses in primed PMN. The data indicate that mobilization of AA from cellular phospholipids neither stimulates nor modulates the respiratory burst or degranulation of PMN.


Subject(s)
Arachidonic Acid/metabolism , Neutrophils/metabolism , Respiratory Burst , Superoxides/metabolism , Cell Degranulation , Chemotactic Factors/pharmacology , Fatty Acids/metabolism , Humans , In Vitro Techniques , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Peroxidase/metabolism , Phospholipases A/pharmacology , Phospholipases A2 , Respiratory Burst/drug effects
13.
J Periodontal Res ; 24(4): 242-6, 1989 Jul.
Article in English | MEDLINE | ID: mdl-2528623

ABSTRACT

Recombinant human interleukin-2 (IL-2) was studied to determine effects on acid production by individual osteoclasts in situ on mouse calvarial bones. This analysis was performed using a microspectrofluorimetric technique to quantify acid production in individual cells. Radioactive calcium release was determined using calvarial bones in a standard tissue culture system. This allowed us to correlate changes in acid production with a measure of bone resorption. IL-2 stimulated acid production and bone resorbing activity. Both effects were inhibited by calcitonin. No stimulation of bone resorption occurred when IL-2-containing test media was incubated with a specific anti-IL-2 antibody and ultrafiltered. Our data demonstrated a correlation between acid production and bone resorbing activity in mouse calvaria exposed to parathyroid hormone (PTH). The data obtained from cultured mouse calvaria exposed to IL-2 demonstrated similar stimulatory effects to those seen during PTH exposure. These data suggest that calvaria exposed to IL-2 in vitro have increased osteoclastic acid production corresponding with increased bone resorption.


Subject(s)
Bone Resorption/metabolism , Bone and Bones/metabolism , Calcium/pharmacokinetics , Interleukin-2/pharmacology , Osteoclasts/drug effects , Acids/metabolism , Acridine Orange , Animals , Bone Resorption/physiopathology , Calcitonin/pharmacology , Calcium Radioisotopes , Culture Techniques , Mice , Mice, Inbred C57BL , Osteoclasts/metabolism , Parathyroid Hormone/antagonists & inhibitors , Parathyroid Hormone/pharmacology , Recombinant Proteins
14.
J Immunol ; 136(3): 860-6, 1986 Feb 01.
Article in English | MEDLINE | ID: mdl-3001188

ABSTRACT

Stimulated human polymorphonuclear leukocytes (PMNL) have a marked increase in oxidative metabolism, producing reduced oxygen species (e.g., H2O2) that mediate bacterial killing. Previously, quantitation of metabolic responses of PMNL from patients with acute infections employed assays that measure mean activity of the entire PMNL population; such studies reported a modest and highly variable increase in oxidative metabolic responses of such "toxic" PMNL compared with normal cells. To assess metabolic capability of PMNL from 51 patients with acute bacterial infection, we employed a quantitative flow cytometric assay of H2O2-dependent oxidative product formation, the intracellular oxidation of 2',7'-dichlorofluorescin (DCFH). After stimulation by phorbol myristate acetate, the PMNL of patients demonstrated an increase in mean DCFH oxidation (315 +/- 14 and 180 +/- 4.5 amol/cell, patients and controls). Hexose monophosphate shunt activation was similarly increased in stimulated PMNL from bacteremic patients. These data are comparable with previous studies of mean metabolic activities of toxic PMNL. However, these mean values underestimate the quantitative responses of the hyperresponsive ("primed") PMNL within a mixture of normal and primed PMNL in the patients' blood. The flow cytometric assay demonstrated that the PMNL of the patients were composed of two populations. One population of PMNL had normal oxidative responses; the other "primed" population had up to 4.6 times the oxidative product formation of normal cells. Similar priming of circulating PMNL was caused by infection with gram-positive or gram-negative staining bacteria or by Candida species. The proportion and oxidative ability of the primed PMNL occurred independently of the number of juvenile neutrophil forms and independently of "toxic" morphologic changes of Wright's-stained PMNL. On the average, 40% of the PMNL of patients were primed, but the size of the primed PMNL population varied widely between patients (range 0 to 80%). This variable subpopulation may explain the variability of mean responsiveness of the PMNL of patients reported previously. Moreover, the marked increase in oxidative metabolic capability of the primed PMNL may be a significant component of the host response to acute infection. It could also contribute to the damage to host tissues such as pulmonary vascular endothelium during bacteremia.


Subject(s)
Fluoresceins/blood , Neutrophils/metabolism , Oxygen/blood , Sepsis/blood , Acute Disease , Binding, Competitive , Cytoplasmic Granules/metabolism , Cytosol/metabolism , Extracellular Space/metabolism , Flow Cytometry , Fluoresceins/biosynthesis , Free Radicals , Humans , In Vitro Techniques , Neutrophils/classification , Oxygen/toxicity , Permeability , Peroxidase/blood
15.
Blood ; 65(1): 233-40, 1985 Jan.
Article in English | MEDLINE | ID: mdl-3965049

ABSTRACT

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.


Subject(s)
Neutrophils/metabolism , Oxygen/blood , Carbocyanines/pharmacology , Flow Cytometry , Humans , Hydrogen Peroxide/blood , In Vitro Techniques , Membrane Potentials/drug effects , Microscopy, Fluorescence , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/drug effects , Tetradecanoylphorbol Acetate/pharmacology
16.
J Immunol ; 130(4): 1910-7, 1983 Apr.
Article in English | MEDLINE | ID: mdl-6833755

ABSTRACT

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.


Subject(s)
Flow Cytometry , Hydrogen Peroxide/blood , Neutrophils/metabolism , Fluoresceins/pharmacology , Granulomatous Disease, Chronic/blood , Humans , Membrane Potentials/drug effects , Neutrophils/drug effects
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