Biomedical subjects

S W Edwards

Publications and source records attributed to S W Edwards.

At least 73 records · Page 4Linked to original sources

PubMed1994

Role of Fc gamma receptors in the activation of neutrophils by soluble and insoluble immunoglobulin aggregates isolated from the synovial fluid of patients with rheumatoid arthritis.

OBJECTIVES: Synovial fluid from patients with rheumatoid arthritis contains both soluble and insoluble immunoglobulin aggregates which activate reactive oxidant production in human neutrophils. The objectives were to determine the roles played by Fc gamma receptors in activation of neutrophils by these complexes. METHODS: Pronase treatment was used to remove Fc gamma RIII from the neutrophil surface and blocking monoclonal antibodies were used to prevent the binding of complexes to Fc gamma RII and Fc gamma RIII. RESULTS: When Fc gamma RIII was removed from the cell surface by pronase treatment, activation by the soluble aggregates did not occur [mean (SD) inhibition 89 (16)%, n = 6] whereas activation via the insoluble aggregates was less affected [34 (16)%, n = 6]. Blocking the binding to Fc gamma RIII with antibodies decreased activation in response to the soluble aggregates [mean (SD) inhibition 71 (22)%, n = 8] but again had a lower effect on activation by the insoluble aggregates [40 (17)%, n = 9]. When binding to Fc gamma RII was blocked, activation via the soluble aggregates was substantially inhibited [mean (SD) 93 (13)%, n = 8] whereas that via the insoluble aggregates was inhibited to a much lesser extent [28 (38)%, n = 9]. When Fc gamma RII and III were simultaneously blocked, activation by the insoluble aggregates was only inhibited by 45% [(19), n = 5]. CONCLUSION: These data thus indicate that activation of human neutrophils by soluble immunoglobulin aggregates from rheumatoid synovial fluid occurs via cooperative occupancy of both Fc gamma RII and III: perturbation of binding to either of these receptor classes will abrogate activation.

Antibodies, Monoclonal↗

PubMed1994

Interferon-gamma enhances monocyte cytotoxicity via enhanced reactive oxygen intermediate production. Absence of an effect on macrophage cytotoxicity is due to failure to enhance reactive nitrogen intermediate production.

Interferon-gamma (IFN-gamma) enhanced the cytotoxic capability of freshly isolated human blood monocytes but failed to enhance the tumoricidal competence of monocyte-derived macrophages. Treatment of monocytes with IFN-gamma (100 U/ml) caused a significant increase (P < 0.001) in lucigenin-dependent chemiluminescence and O2- production stimulated by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) during the first few days in culture but IFN-gamma was unable to prevent the decline to negligible levels of chemiluminescence and O2- production which occurred during the later days in vitro. Culture of monocytes in the presence of IFN-gamma had no effect on phorbol 12-myristate 13-acetate (PMA)-stimulated O2- production. However, IFN-gamma decreased PMA-stimulated lucigenin-dependent chemiluminescence during the first 24 hr in vitro but then significantly enhanced (P < 0.001) chemiluminescence after 2-4 days in culture. IFN-gamma was unable to prevent the eventual decline to undetectable levels in PMA-stimulated chemiluminescence during the later days in vitro. Nitrite production by macrophages was unaffected by IFN-gamma treatment. It is concluded therefore, that IFN-gamma enhanced the cytotoxicity of freshly isolated human blood monocytes by increasing reactive oxygen intermediate generation but was unable to enhance the tumoricidal competence of macrophages as reactive nitrogen intermediate production was unaffected.

Acridines↗

PubMed1993

Changes in mechanisms of monocyte/macrophage-mediated cytotoxicity during culture. Reactive oxygen intermediates are involved in monocyte-mediated cytotoxicity, whereas reactive nitrogen intermediates are employed by macrophages in tumor cell killing.

Freshly isolated human blood monocytes were spontaneously cytotoxic toward K562 tumor cells. During culture of the monocytes in vitro cytotoxicity decreased during the first 48 h but tumoricidal competence was restored after 3 to 4 days in vitro. These changes were accompanied by changes in both reactive oxygen intermediate generating capacity and reactive nitrogen intermediate production. Lucigenin-dependent chemiluminescence stimulated with either FMLP or PMA declined during the first 2 days in culture and was negligible during the later days in culture. Superoxide radical production in response to either FMLP or PMA remained fairly constant for the first few days in vitro and then declined. NO2- concentration in monocyte-conditioned medium was fairly constant during the first few days in vitro but increased after 6 days. The return to tumoricidal competence after 3 to 4 days in culture was decreased by the addition of NG-monomethyl-L-arginine. These results indicate that reactive oxygen intermediates are employed by monocytes in the killing of tumor cells. However, after maturation of monocytes to macrophages, this mechanism becomes less important and reactive nitrogen intermediates are employed in mediating macrophage cytotoxicity.

Arginine↗

PubMed1993

The effects of GM-CSF on myeloperoxidase release in normal and myelodysplastic neutrophils.

When purified control neutrophils were primed with GM-CSF, a significant increase in FMLP-induced MPO release was observed (mean +/- S.E.M., 3.4 +/- 0.8 mU/10(7) unprimed cells compared to 6.5 +/- 1.1 mU/10(7) primed cells, p < 0.001). This MPO release was greatly augmented by Cytochalasin B (Cy B), but after the addition of Cy B the priming effects of GM-CSF became less obvious. Exposure to GM-CSF without FMLP did not enhance MPO release. Within whole blood, FMLP produced negligible MPO release, but priming with GM-CSF prior to FMLP always resulted in a significant increase in MPO release. Myelodysplastic neutrophils released similar amounts of MPO in response to FMLP, compared with control cells (3.4 +/- 0.8 mU/10(7) control cells compared to 2.7 +/- 0.3 mU/10(7) MDS cells, p > 0.05). Priming with GM-CSF produced an increase in FMLP-stimulated MPO release comparable with control cells. In terms of total MPO content, although some MDS patients exhibited low levels, as a group there was no significant difference from controls (169 +/- 21 mU/10(7) control cells compared with 157 +/- 19 mU/10(7) MDS cells). These findings suggest that MPO activity is not a universal defect in MDS and cannot account for the defects in respiratory burst activity in these neutrophils.

Anemia↗

PubMed1993

Effect of cytotoxic drugs on mature neutrophil function in the presence and absence of granulocyte-macrophage colony-stimulating factor.

The effects of the cytotoxic drugs, adriamycin, cyclophosphamide, daunomycin (daunorubicin), prednisolone, actinomycin D, azacytidine and vincristine at concentrations of 1 microM on mature neutrophil function were examined. Up to 5 h incubation with adriamycin, azacytidine, cyclophosphamide, daunomycin and prednisolone had no effect on either luminol chemiluminescence or superoxide secretion. However, after 15 min or 1 h (but not 5 h) incubation vincristine enhanced fMet-Leu-Phe stimulated chemiluminescence, whilst after 5 h incubation with actinomycin D the ability of neutrophils to generate reactive oxidants in response to all stimuli tested was impaired: after 5 h incubation with adriamycin reactive oxidant production was also impaired, but only when fMet-Leu-Phe was used as stimulant. All of the drugs tested except azacytidine inhibited neutrophil oxidant production after 5 h incubation in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Actinomycin D and cyclophosphamide also inhibited GM-CSF stimulated protein biosynthesis. These data indicate that cytotoxic drugs may compromise the potentially beneficial effects of CSFs on mature neutrophil function during therapy.

Antineoplastic Agents↗

PubMed1993

Activation of neutrophils by soluble and insoluble immunoglobulin aggregates from synovial fluid of patients with rheumatoid arthritis.

OBJECTIVES: Previous work has shown that synovial fluid isolated from patients with active rheumatoid arthritis contains soluble (not sedimented by centrifugation at 11,600 g for two minutes) and insoluble (sedimented by centrifugation at 11,600 g for two minutes) immunoglobulin aggregates that are capable of activating reactive oxidant production by bloodstream neutrophils. The purpose of this study was to determine which of these types of immunoglobulin aggregates activated the secretion of reactive oxygen metabolites and granule enzymes from neutrophils. METHODS: Cell free synovial fluid (from patients with rheumatoid arthritis) was added to neutrophils isolated from blood of healthy controls that had been incubated in the presence and absence of granulocyte-macrophage colony stimulating factor (GM-CSF). Reactive oxidant production was measured by luminol chemiluminescence (which detects both intracellular and extracellular oxidant production) and by cytochrome c reduction (which measures superoxide secretion). RESULTS: The soluble aggregates only activated neutrophils that were previously primed, and activated a rapid and transient burst of reactive oxidant secretion. On the other hand, the insoluble aggregates activated primed and unprimed neutrophils with similar efficacy and most of the oxidants generated (especially in unprimed cells) were intracellular. The soluble aggregates, but not the insoluble aggregates, also activated the secretion of myeloperoxidase from neutrophils that had either been pretreated with cytochalasin B or primed with GM-CSF. CONCLUSION: It is thus proposed that these soluble immunoglobulin aggregates are responsible for activation of the release of tissue damaging granule enzymes and reactive oxidants from primed neutrophils within the rheumatoid joint.

Adult↗

PubMed1993

Receptor expression in synovial fluid neutrophils from patients with rheumatoid arthritis.

OBJECTIVES: The aim of this study was to determine if neutrophils isolated from the blood and synovial fluid of patients with rheumatoid arthritis had patterns of receptor expression resembling those of blood neutrophils from controls which had been activated and primed in vitro. METHODS: Fluorescence activated cell sorting was used to measure receptor expression in paired blood and synovial fluid neutrophils from patients and in control neutrophils exposed to phorbol myristate acetate and granulocyte-macrophage colony stimulating factor. RESULTS: There was no significant difference in the patterns of receptor expression in blood neutrophils from patients and healthy controls, but neutrophils in the synovial fluid had been primed and activated within the joint. About 50% of rheumatoid synovial fluid neutrophil samples expressed Fc gamma RI, a high affinity receptor for monomeric IgG, which is only expressed in neutrophils exposed to cytokines. CONCLUSIONS: Synovial fluid neutrophils are activated and primed within the inflamed joint and hence their ability to respond to activating factors such as immune complexes will be modulated. As the expression of Fc gamma RI requires active biosynthesis, this work indicates that selective gene activation occurs when neutrophils are recruited into rheumatoid joints.

Antigens, CD↗

PubMed1993

Comparison of measured and computed epicardial potentials from a patient-specific inverse model.

This study reports the first direct comparison of measured and computed epicardial potentials in which the specific anatomy of a test subject has been used to calculate the inverse electrocardiographic model. It is now feasible to obtain low-noise body surface potential maps and to incorporate accurate anatomic data into inverse procedures for the purpose of computing epicardial potential distributions. The direct verification of computed human epicardial distributions remains an important goal. The experiment reported here obtained direct measurements from six transcutaneous pacing wires that were attached to points on the epicardial surface of the human heart in an intact subject. From the same subject, a magnetic resonance scan was used to produce a specific thoracic model consisting of 5-mm cubes. The forward model uses the finite difference method to compute a forward transfer matrix that relates each of 26 epicardial regions to body surface measurements. The inverse computation was performed by zero-order Tikhonov regularization. Body surface potentials were used in the inverse procedure to compute epicardial potentials, which were then compared with direct epicardial measurements. The computed epicardial potentials were compared to the measured ones by correlation, which gave an amplitude-independent measure of similarity. Amplitude differences and time delays in computed potentials were observed, but the morphologic trend was generally well recovered. The results obtained indicate the sensitivity of the inverse model to a number of factors. The robustness of computed epicardial distributions to errors in assumed lung conductivity is shown. Results from a nonpatient-specific, but realistic, torso model are presented.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiac Pacing, Artificial↗

PubMed1992

Activation of neutrophil reactive-oxidant production by synovial fluid from patients with inflammatory joint disease. Soluble and insoluble immunoglobulin aggregates activate different pathways in primed and unprimed cells.

Cell-free synovial fluid from patients with rheumatoid arthritis stimulated the NADPH oxidase activity in human neutrophils, which reached a peak 15-20 min after addition. Insoluble immunoglobulin aggregates isolated from these fluids activated a similar pattern of oxidase activity. However, when synovial fluid was added to neutrophil suspensions which had been previously exposed to granulocyte-macrophage colony-stimulating factor, the stimulated oxidase activity was biphasic, in that an additional transient activity was observed which reached a peak within 5 min of addition. The additional neutrophil-stimulating activity could not be sedimented by centrifugation at 330,000 g-min, and only activated oxidase activity in neutrophils which had previously been primed. The neutrophil-stimulating activity in this soluble fraction was removed by Protein A affinity chromatography, and activity was recovered in eluates from this column. Thus activity in this soluble fraction from synovial fluid is attributed to the presence of soluble immunoglobulin aggregates. Whereas oxidase activity stimulated by the isoluble immunoglobulin aggregates was inhibited by staurosporine (and hence largely dependent on the activity of protein kinase C), the activity stimulated by the soluble immunoglobulin aggregates was staurosporine-insensitive. The soluble immunoglobulin aggregates were present at significantly higher levels in synovial fluids from patients with rheumatoid arthritis compared with those from other joint arthropathies. Thus rheumatoid synovial fluids possess heterogeneous immunoglobulin aggregates which activate neutrophils via distinct molecular pathways. As neutrophils within rheumatoid joints are primed, the soluble immunoglobulin aggregates are likely to be of importance in disease pathology.

Alkaloids↗

PubMed1992

Chemiluminescence of human bloodstream monocytes and neutrophils: an unusual oxidant(s) generated by monocytes during the respiratory burst.

Maximal rates of O2- and H2O2 production by human bloodstream monocytes activated during the respiratory burst by phorbol ester were only about 10% of those of neutrophils. Furthermore, monocytes possess only about 5% of the myeloperoxidase activity of neutrophils and so can only produce low levels of HOCl and related compounds. These combined reductions in O2- generating ability and lower myeloperoxidase levels result in low levels of luminol chemiluminescence stimulated during the respiratory burst of monocytes. However, although monocytes generate much lower levels of O2- and H2O2 than neutrophils, these cells produce comparable rates of PMA-stimulated lucigenin chemiluminescence. Hence, this assay does not accurately reflect the production of either of these two oxidants by activated phagocytes, and further lucigenin must react with some other oxidant(s) via a process which leads to photon emission. This oxidant(s) is not O2-, H2O2, .OH, 1O2 or NO, but is derived from O2- generated during the respiratory burst and is generated in greater quantities by activated monocytes compared with neutrophils. Thus, lucigenin chemiluminescence is an indirect measure of superoxide release.

Acridines↗

PubMed1992

Neutrophil function in whole blood and after purification: changes in receptor expression, oxidase activity and responsiveness to cytokines.

Neutrophil function and plasma membrane receptor expression was measured in cell suspensions isolated by two separate procedures and in unfractionated whole blood. When cells were prepared by a combined dextran/ficoll procedure, their ability to generate reactive oxidants in response to fMet-Leu-Phe was greater than in corresponding cells isolated by a one-step procedure on Mono-Poly Resolving Medium (M-PRM). Cells prepared by both methods could be primed in vitro by rGM-CSF, but the priming ratio was greater in cells prepared by the latter method. The ability of neutrophils in whole blood to generate reactive oxidants in response to fMet-Leu-Phe was extremely low, but this was increased by more than 10 fold if the blood was pre-incubated with rGM-CSF. Similarly, expression of CD 11b and CD 16 was very low (or undetectable) in neutrophils in whole blood, but this was rapidly increased upon priming. Activation by PMA resulted in a down regulation of CD 16 expression as the receptor was shed from the cell surface. Neutrophils isolated by either the dextran/ficoll or the M-PRM method showed increased expression of receptors compared with those in whole blood, although this expression was lower in cells isolated by the latter method. These data indicate that the isolation procedures used to obtain purified neutrophils prime both receptor expression and oxidase function, although these effects are minimalised in isolation procedures using M-PRM. Furthermore, as CD 16 expression on neutrophils in whole blood is rapidly up-regulated during priming, it seems likely that, as for complement receptors, rapidly-mobilisable intracellular stores of this receptor exist.

Antibodies, Monoclonal↗

PubMed1992

Interactions between human monocytes and tumour cells. Monocytes can either enhance or inhibit the growth and survival of K562 cells.

Human bloodstream monocytes can kill cultured tumour cells (K562), as assessed by specific release of 51Cr from the targets and by inhibition of 3H-thymidine incorporation. Confluent monolayers of monocytes were required for maximal cytotoxicity, and the density of the K562 cells was also an important factor. For example, when K562 cells were seeded at high cell densities, they were killed during incubation with monocytes, but when seeded at low cell densities their growth and survival was enhanced during culture with monocytes. The factor(s) which promoted the survival and division of low density K562 cultures was endogenously secreted from monocytes as it was present in monocyte-conditioned medium, whereas the cytotoxic factor(s) were only expressed during co-culture of monocytes with K562 cells. Conditioned medium from HL 60, U-937, HeLa and K562 could also enhance the growth and survival of low density K562 cultures, and a similar effect was also observed upon the addition of catalase and superoxide dismutase to such cultures. Thus, the monocyte:target ratio is important in determining whether monocytes exhibit cytotoxic or growth-promoting effects and hence tumour-derived or monocyte-derived reactive oxidant species may play a role in tumour cell cycle regulation.

Catalase↗

PubMed1992

Stimulation of neutrophils by insoluble immunoglobulin aggregates from synovial fluid of patients with rheumatoid arthritis.

Insoluble immunoglobulin aggregates present in the synovial fluid of patients with rheumatoid arthritis have been examined for their ability to activate reactive oxidant and granule enzyme secretion from bloodstream neutrophils. These insoluble complexes activated luminol chemiluminescence, but did not activate O2-, H2O2 or granule enzyme secretion and did not activate lucigenin chemiluminescence, which also measures reactive oxidant secretion. Hence, the luminol chemiluminescence detected after activation by insoluble immunoglobulin aggregates must be due to intracellularly generated reactive oxidants, i.e. produced within phagolysosomes. Because reactive oxidant and granule enzyme secretion has occurred within rheumatoid joints, other mechanisms of neutrophil activation must exist.

Arthritis, Rheumatoid↗

PubMed1992

Sequential phospholipase activation in the stimulation of the neutrophil NADPH oxidase.

Stimulation of human neutrophils with the chemotactic peptide fMet-Leu-Phe results in activation of a rapid, transient burst of oxidant secretion, which reaches a maximal rate by about 1 min after stimulation. This phase of oxidant secretion is then followed by intracellular oxidant production, which is detected by luminol chemiluminescence but not by assays such as cytochrome c reduction or scopoletin oxidation. The rapid phase of oxidant secretion requires increases in intracellular free Ca2+ and phospholipase A2 activity, but not the activities of phospholipase D or protein kinase C. In contrast, intracellular oxidant production requires the activities of phospholipase D and protein kinase C. A model is thus proposed suggesting the sequential activation of different phospholipases which activate oxidase molecules on the plasma membrane or else from the membranes of specific granules.

Calcium↗

PubMed1992

Inhibition of neutrophil oxidant secretion by D-penicillamine: scavenging of H2O2 and HOCl.

D-Penicillamine inhibited oxidant secretion from human neutrophils after activation by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe), as assessed by luminol dependent chemiluminescence. In contrast, this drug had little effect on either intracellular oxidant production or lucigenin dependent chemiluminescence activated by the same agonist. The drug was shown to scavenge both H2O2 and HOCl in a cell free luminol chemiluminescence system, though its ability to scavenge HOCl was greater than that for H2O2. Both these oxidants could oxidise the drug, but again HOCl was more potent than H2O2. When D-penicillamine was oxidised by exposure to H2O2 it could no longer serve as a scavenger of secreted oxidants from neutrophils. These data suggest that in vivo the preferential scavenging of HOCl may be important under pathological conditions where secreted myeloperoxidase may be functional.

Humans↗

PubMed1991

Protein kinase C-dependent and -independent activation of the NADPH oxidase of human neutrophils.

The protein kinase C inhibitor, staurosporine, inhibited NADPH oxidase activity of human neutrophils activated by phorbol myristate acetate. However, this inhibitor had no effect on either the initiation or the maximal rate of O2- secretion activated by the chemotactic peptide, fMet-Leu-Phe, but resulted in a more rapid termination of oxidant production. Similarly, staurosporine had no effect on the rapid (1 min) increase in luminol-dependent chemiluminescence activated by fMet-Leu-Phe, but the second (intracellular) phase of oxidant production was inhibited. The initial burst of oxidant production during phagocytosis was similarly protein kinase C-independent, but again the later phases of oxidase activity were staurosporine-sensitive. Neutrophils loaded with Quin-2 at concentrations sufficient to act as a Ca2+ buffer could not secrete O2- in response to fMet-Leu-Phe; although the initial (protein kinase C-independent) burst of luminol chemiluminescence was not observed in fMet-Leu-Phe-stimulated Ca2(+)-buffered cells, the second phase of (protein kinase C-dependent) oxidant production was largely unaffected. Hence, the initial burst of oxidant production activated by fMet-Leu-Phe, opsonized zymosan, and latex beads is independent of the activity of protein kinase C-dependent intracellular activation processes, but the activity of this kinase is required to extend or sustain the duration of oxidant production.

Alkaloids↗

PubMed1991

Synthesis of redox derivatives of lysine and related peptides containing phenothiazine or tris(2,2'-bipyridine)ruthenium(II).

Boc-L-Lysine derivatives and lysine-containing peptides bearing the electron donor 10H-phenothiazine (PTZ) or the redox chromophore tris(2,2'-bipyridine)ruthenium(II) dication ([Rub3]2+, where b is 2,2'-bipyridine) have been synthesized and characterized. SeO2 oxidation (53% yield) of 4,4'-dimethyl-2,2'-bipyridine, Ag2O oxidation (85% yield) of the monoaldehyde, complexation (96% yield) of 4'-methyl-2,2'-bipyridine-4-carboxylic acid (m-OH) with Rub2Cl2, activation (81% yield) of the acid [Rub2m-OH]2+ (PF6-)2, and condensation (83% yield) of the succinimido ester [Rub2m-OSu]2+ (PF6-)2 with Boc-Lys furnished the protected redox-chromophore module [Boc-Lys(Rub2m)-OH]2+ (PF6-)2 in 29% overall yield over five steps. The first two steps constitute the first practical synthesis of the monocarboxylic acid m-OH (45% overall yield). Also prepared were m-OSu, Boc-Lys(m)-OH, Boc-Lys(m)-OCH3, and [Rub2m-NHCH3]2+ (PF6-)2. Activation (91% yield) of 3-(10H-phenothiazine-10)propanoic acid (PTZpn-OH) and condensation (92% yield) of the succinimido ester PTZpn-OSu with Boc-Lys furnished the protected electron-donor module Boc-Lys(PTZpn)-OH (84% overall yield). The latter was used in solid-phase syntheses of two redox tripeptides, CH3CO-Ala-Lys(PTZpn)-Ala-OH and [Rub2m-Ala-Lys(PTZpn)-Ala-OH]2+ (PF6-)2. The electrochemical properties of these redox amino acids and peptides were similar to those of PTZpn-OH, [Rub2m-OH]2+ (PF6-)2, or [Rub2m-NHCH3]2+ (PF6-)2. Lys(PTZpn), [Lys(Rub2m)]2+ (PF6-)2, and other redox modules may be useful for engineering light-harvesting proteins, photovoltaic cells, and other molecular electronic devices.

2,2'-Dipyridyl↗

PubMed1991

Neutrophils isolated from the synovial fluid of patients with rheumatoid arthritis: priming and activation in vivo.

The oxidative metabolism of neutrophils isolated from the bloodstream and synovial fluid of 16 patients with rheumatoid arthritis was compared by measuring the ability of neutrophils to generate luminol dependent chemiluminescence and to secrete O2-. Measurements of receptor mediated--that is, N-formyl-methionyl-leucyl-phenylalanine stimulated--activation or receptor and second message independent--that is phorbol myristate acetate stimulated--activation showed that synovial fluid neutrophils had biochemical characteristics to suggest that they had been either up-regulated (primed) or down-regulated (activated) in vivo. These conclusions were confirmed by comparison of these responses with the changes in oxidative metabolism observed during in vitro priming and activation of control neutrophils: synovial fluid neutrophils possessed lower levels of myeloperoxidase than paired bloodstream cells, and unlike bloodstream cells could not be primed in vitro. These data thus suggest that synovial fluid neutrophils have been exposed to both priming and activating agents within rheumatoid joints.

Adult↗