Oxidation and uptake of LDL by monocyte-derived macrophages from blood of patients with IHD

The main goal of this study was to test our hypothesis that monocyte-derived macrophages of patients with ischemic heart disease (IHD, MP_IHD) are in vivo prestimulated (primed) or stimulated cells. Their capacity for LDL oxidation and uptake exceeds that of macrophages from healthy donors (MP_N). Monocytes were isolated from blood of 18 healthy donors and 25 IHD patients and LDL preparations were obtained from plasma of 16 healthy donors (LDL_N) and 15 patients with familial hypercholesterolemia (LDL_H). Aerobic incubation of LDL_N or LDL_H with MP_IHD resulted in earlier accumulation (by 1 h) of TBARS in LDL, earlier aggregation of LDL (1 h vs 3 h in the case of MP_N), more pronounced LDL apoB fragmentation as well as increased LDL uptake with the increase in accumulation of total cholesterol (TCh; by 1.8–2.1-fold, p < 0.05–0.01) and the decrease in cell viability compared with MP_N (p < 0.01). MP_IHD and MP_N exhibited more effective oxidation and uptake of LDL_H than LDL_N and in most tests this capacity (to oxidize and uptake LDL) increased under hypoxic conditions. These results demonstrate that macrophages of IHD patients are in vivo stimulated cells and that this stimulation, especially in combination with hypercholesterolemic LDL and local or generalized hypoxia, represent serious predisposition for onset or progression of atherosclerosis in IHD patients. The express test model based on MP_IHD may be used for estimation of monocyte/macrophage stimulation in IHD patients as well as for optimization of drug therapy and screening new antiatherosclerotic and antiischemic drugs.     

α-tocopherol modulates tyrosine phosphorylation in human neutrophils by inhibition of protein kinase C activity and activation of tyrosine phosphatases

α-Tocopherol augmentation in human neutrophils was investigated for effects on neutrophil activation and tyrosine phosphorylation of proteins, through its modulation of protein kinase C (PKC) and tyrosine phosphatase activities. Incubation of neutrophils with α-tocopherol succinate (TS) resulted in a dose-dependent incorporation into cell membranes, up to 2.5 nmol/2 × 10⁶ cells. A saturating dose of TS (40 μmol/l) inhibited oxidant production by neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan (OZ) by 86 and 57%, as measured by luminol-amplified chemiluminescence (CL). With PMA, TS inhibited CL generation to a similar extent to staurosporine (10 nmol/l) or genistein (100 μmol/l), and much more than Trolox (40 μmol/l). With OZ, TS inhibited CL to a similar extent to Trolox. Neutrophil PKC activity was inhibited 50% or more by TS or staurosporine. The enzyme activity was unaffected by genistein or Trolox, indicating a specific interaction of α-tocopherol. TS or Trolox increased protein tyrosine phosphorylation in resting neutrophils, and as with staurosporine further increased tyrosine phosphorylation in PMA-stimulated neutrophils, while the tyrosine kinase (TK) inhibitor genistein diminished phosphorylation. These effects in resting or PMA-stimulated neutrophils were unrelated to protein tyrosine phosphatase (PTP) activities, which were maintained or increased by TS or Trolox. In OZ-stimulated neutrophils, on the other hand, all four compounds inhibited the increase in tyrosine-phosphorylated proteins. In this case, the effects of pre-incubation with TS or Trolox corresponded with partial inhibition of the marked (85%) decrease in PTP activity induced by OZ. These results indicate that α-tocopherol inhibits PMA-activation of human neutrophils by inhibition of PKC activity, and inhibits tyrosine phosphorylation and activation of OZ-stimulated neutrophils also through inhibition of phosphatase inactivation.     

α-tocopherol modulates tyrosine phosphorylation in human neutrophils by inhibition of protein kinase C activity and activation of tyrosine phosphatases

-Tocopherol augmentation in human neutrophils was investigated for effects on neutrophil activation and tyrosine phosphorylation of proteins, through its modulation of protein kinase C (PKC) and tyrosine phosphatase activities. Incubation of neutrophils with -tocopherol succinate (TS) resulted in a dose-dependent incorporation into cell membranes, up to 2.5 nmol/2 × 10⁶ cells. A saturating dose of TS (40 μmol/l) inhibited oxidant production by neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan (OZ) by 86 and 57%, as measured by luminol-amplified chemiluminescence (CL). With PMA, TS inhibited CL generation to a similar extent to staurosporine (10 nmol/l) or genistein (100 μmol/l), and much more than Trolox (40 μmol/l). With OZ, TS inhibited CL to a similar extent to Trolox. Neutrophil PKC activity was inhibited 50% or more by TS or staurosporine. The enzyme activity was unaffected by genistein or Trolox, indicating a specific interaction of -tocopherol. TS or Trolox increased protein tyrosine phosphorylation in resting neutrophils, and as with staurosporine further increased tyrosine phosphorylation in PMA-stimulated neutrophils, while the tyrosine kinase (TK) inhibitor genistein diminished phosphorylation. These effects in resting or PMA-stimulated neutrophils were unrelated to protein tyrosine phosphatase (PTP) activities, which were maintained or increased by TS or Trolox. In OZ-stimulated neutrophils, on the other hand, all four compounds inhibited the increase in tyrosine-phosphorylated proteins. In this case, the effects of pre-incubation with TS or Trolox corresponded with partial inhibition of the marked (85%) decrease in PTP activity induced by OZ. These results indicate that -tocopherol inhibits PMA-activation of human neutrophils by inhibition of PKC activity, and inhibits tyrosine phosphorylation and activation of OZ-stimulated neutrophils also through inhibition of phosphatase inactivation.     

Luminol‐, isoluminol‐ and lucigenin‐enhanced chemiluminescence of rat blood phagocytes stimulated with different activators

Luminol‐, isoluminol‐ or lucigenin‐enhanced chemiluminescence (CL) was used to measure the production of reactive oxygen species by rat blood leukocytes. Opsonized zymosan (OZ), phorbol‐12‐myristate‐13‐acetate (PMA), calcium ionophore A23187 (Ca‐I) or N‐formyl‐Met‐Leu‐Phe (fMLP) were used as activators. The CL signal of isolated blood leukocytes decreased in rank order of luminol > isoluminol > lucigenin. The kinetic pro?les of luminol‐ and isoluminol‐enhanced CL were similar upon stimulation by each activator tested. The remarkably higher luminol and isoluminol CL responses were obtained after OZ stimulation when compared with other activators. However, when lucigenin was used, the PMA‐ and OZ‐stimulated CL were comparable. The presence of plasma increased OZ‐activated CL because of the enhanced phagocytosis of OZ. This was demonstrated by determining the phagocytosis of the ?uorescent OZ using a ?ow cytometer. In contrast, the presence of plasma decreased PMA‐activated CL, due to the antioxidant properties of plasma as determined by the CL method. As far as whole blood is concerned, only OZ activated luminol‐enhanced CL was reliable. Blood volumes over 5 µL decreased CL activity due to the scavenging ability of erythrocytes. The results suggest that 0.5 µL whole blood is suf?cient for routine luminol‐enhanced CL analysis of whole blood oxidative burst in rats. Copyright © 2004 John Wiley & Sons, Ltd.     

Measurement of chemiluminescence from neutrophils in a 96-well microplate using Lumi Box U-800 II

We have developed a microplate photon counting system based on a cooled charge-coupled device (Lumi Box U-800 II) jointly with Maikurotekku Nition Company (Chiba, Japan). The system makes it possible to quantify chemiluminescence (CL) in a 96-well microplate automatically and simultaneously in a single experiment. We studied the measurement conditions for a luminol-dependent CL assay from neutrophils stimulated with opsonized zymosan (OZ) using this system. Parameters examined included the effect of OZ dose per well, mixing speed, mixing time and detection time on CL responses. The results indicated that this system allows the measurement of CL from phagocytes on a large number of samples using small amounts of sample and regents. © 1997 John Wiley & Sons, Ltd.     

Identification of distinct activation pathways of the human neutrophil NADPH-oxidase

The stimulation of the human neutrophil NADPH-oxidase is initiated by a variety of agonists, which appear to utilize more than one activation pathway. We have discerned that opsonized zymosan (OZ) stimulates O2- release by a mechanism distinct from that of phorbol myristate acetate (PMA). PMA differs from OZ stimulation in its susceptibility to H-7 (a protein kinase inhibitor) inhibition of O2- release and the lack of PMA-initiated release of radiolabeled arachidonic acid ([3H]AA) from prelabeled cells. That AA release was linked to O2- generation in OZ-stimulated cells was suggested by the finding that mepacrine, a phospholipase inhibitor, exhibits parallel dose response inhibition for both O2- generation and [3H]AA release, whereas mepacrine did not significantly inhibit the O2- generation induced by PMA. The specific involvement of phospholipase A2 (PLA2) in the release of AA was indicated by the lack of release of [3H]oleate, which is not released by PLA2 in intact cells; [3H]AA released from phosphatidylinositol and phosphatidylcholine and not accompanied by the formation of [3H]-arachidonyl phosphatidic acid, thus eliminating the involvement of phospholipase C; and the inhibition of [3H]AA release by p-bromophenacyl bromide, a specific PLA2 inhibitor. The reduction of O2- formation by inhibitors of AA metabolism (BW755C, acetylsalicylic acid, and indomethacin) further supports a linkage between AA release and O2- generation. That [3H]AA release, like O2- generation, in OZ-stimulated cells was calcium dependent further differentiates OZ from calcium-independent PMA activation. These studies in toto suggest that OZ stimulation of the NADPH-oxidase differs from PMA, in that the particulate stimulus is PLA2 mediated and independent of protein kinase C.     

Effect of sulphite on the oxidative metabolism of human neutrophils: Studies with lucigenin- and luminol-dependent chemiluminescence

To assess the effect of sulphite on the oxidative metabolism of human neutrophils, chemiluminescence (CL) measurements were performed using lucigenin and luminol as chemiluminigenic probes. Lucigenin-dependent CL was used for measuring superoxide anion (O) production, and luminol-dependent CL was used for determination of myeloperoxidase (MPO)-connected processes. With sulphite concentrations of 0.01 to 1 mmol/L, resting neutrophils showed an up to sixfold increase of lucigenin-dependent CL, but only a 1.9-fold increase of luminol-dependent CL. Subsequent stimulation of sulphite-treated neutrophils with phorbol myristate acetate (PMA) (soluble stimulant) or zymosan (particulate stimulant) resulted in an additional significant increase of lucigenin-dependent CL compared to stimulated control cells, whereas luminol-dependent CL increased slightly by 0.01 mmol/L sulphite and decreased then continuously. Sulphite concentrations above 1 mmol/L decreased both lucigenin- and luminol-dependent CL of resting and PMA- or zymosan-stimulated neutrophils. Lucigenin-dependent CL of sulphite-treated and subsequently stimulated neutrophils was strongly inhibited by extracellularly added superoxide dismutase, whereas luminol-dependent CL was markedly reduced by the MPO inhibitor azide. The intracellular activity of MPO in neutrophils stimulated with PMA in the presence of sulphite (2 mmol/L) was reduced by 55%. Sulphite (0.1 mmol/L) also inhibited strongly the activity of MPO in a cell-free system. These results indicate that micromolar concentrations of sulphite exert a stimulating effect on the O production of neutrophils extracellularly, but have an inhibitory effect on MPO-catalysed reactions intracellularly.     

Human serum albumin modified under oxidative/halogenative stress enhances luminol-dependent chemiluminescence of human neutrophils

It is shown that human serum albumin, previously treated with HOCl (HSA-Cl), enhances luminol-dependent chemiluminescence of neutrophils activated by phorbol-12-myristate-13-acetate (PMA). The enzyme-linked immunosorbent assay revealed that addition of HSA-Cl to neutrophils promotes exocytosis of myeloperoxidase. Inhibitor of myeloperoxidase — 4-aminobenzoic acid hydrazide, without any effect on lucigenin-dependent chemiluminescence of neutrophils stimulated with PMA, effectively suppressed luminol-dependent chemiluminescence (IC₅₀ = 20 μM) under the same conditions. The transfer of the cells from medium with HSA-Cl and myeloperoxidase to fresh medium abolished an increase in PMA-induced luminol-dependent chemiluminescence, but not the ability of neutrophils to respond to re-addition of HSA-Cl. A direct and significant (r = 0.75, p < 0.01) correlation was observed between the intensity of PMA stimulated neutrophil chemiluminescence response and myeloperoxidase activity in the cell-free media after chemiluminescence measurements. These results suggest the involvement of myeloperoxidase in the increase of neutrophil PMA-stimulated chemiluminescence response in the presence of HSA-Cl. A significant positive correlation was found between myeloperoxidase activity in blood plasma of children with severe burns and the enhancing effects of albumin fraction of the same plasma on luminol-dependent chemiluminescence of PMA-stimulated donor neutrophils. These results support a hypothesis that proteins modified in reactions involving myeloperoxidase under oxidative/halogenative stress, stimulate neutrophils, leading to exocytosis of myeloperoxidase, a key element of halogenative stress, and to closing a “vicious circle” of neutrophil activation at the inflammatory site.     

Luminol-dependent chemiluminescence in bovine eosinophils and neutrophils: Differential increase of intracellular and extracellular chemiluminescence induced by soluble stimulants

Luminol chemiluminescence was used to detect activation of the respiratory burst oxidase in bovine eosinophils and neutrophils. Extracellular and intracellular chemiluminescence were measured by supplementing the medium with horseradish peroxidase and catalase, respectively. Pure bovine eosinophils (> 90%), maximally stimulated with 1 nmol/l phorbol 12-myristate-13-acetate (PMA) showed ten times more extracellular luminol-dependent chemiluminescence (CL) than maximally stimulated pure bovine neutrophils (> 96%). Extracellular CL from eosinophils was preferably induced over intracellular CL by both PMA (27-fold difference) and platelet-activating factor (PAF) at 2 μmol/l (9-fold difference), but not by calcium ionophore A23187 (15 μmol/l). Time course information was used in the following experiments to distinguish between the mode of action of various stimulants. A progressively longer lag period was observed in eosinophil suspensions treated with decreasing doses of PMA, whereas platelet-activating factor induced a dose-dependent increase in the maximum response with no change in time to peak CL. The time course of extracellular CL was almost identical to intracellular CL for all stimulants tested, providing no evidence to suggest that extracellular CL stems from a different enzyme system than intracellular CL. Eosinophils generated most extracellular CL when stimulated with PMA, whereas neutrophils were most efficiently stimulated with A23187, which induced intracellular CL in eosinophils as well as in neutrophils. This accords with the greater tendency of neutrophils to ingest and kill microorganisms, whereas eosinophils are armed to destroy large extracellular targets.     

Nitric oxide synthase inhibitors decrease human polymorphonuclear leukocyte luminol-dependent chemiluminescence

Nitric oxide synthase (NOS) inhibitors have been reported to modulate luminol-dependent chemiluminescence (CL) in rat macrophages, whereas the potent oxidant peroxynitrite (ONOO^-) was shown to react with luminol to yield CL in a cellfree system. We evaluated the role of the -arginine/NOS pathway in luminol CL by phorbol ester-activated human polymorpho-nuclear (PMN) leukocytes using the NOS inhibitors N^G-monomethyl- -arginine ( -NMMA) and N-iminoethyl- -omithine ( -NIO). Nitric oxide (·NO) release was determined by oxidation of oxymyoglobin. In addition, the effect of NOS inhibitors on superoxide anion O₂^-) production was measured. Luminol CL was notably diminished by -NMMA in a dose-dependent manner. Superoxide dismutase (SOD) also decreased luminol CL and -NMMA potentiated light emission decrease produced by SOD. Nitric oxide and O₂^·- production was significantly decreased by -NMMA; moreover, luminol-dependent CL but not O₂^·- production was attenuated by -NIO. These data suggest that products of catalytic activity of both ·NO synthase and NADPH oxidase are required to elicit maximal luminol CL in this system. These studies demonstrate that the NOS synthase pathway is involved in luminol CL by human PMN, and they suggest that ONOO would be an unrecognized mediator in this phenomenon.     

Sesquiterpene lactone from Wunderlichia crulsiana inhibits the respiratory burst of leukocytes triggered by distinct biochemical pathways

The sesquiterpene lactone tubiferin was chemically purified from the brazilian native plant Wunderlichia crulsiana and identified by NMR and GC/MS data. Its ability to inhibit the respiratory burst of peritoneal inflammatory polymorphonuclear leukocytes (PMN) stimulated upon addition of phorbol miristate acetate (PMA), opsonized zymosan (OZ), and N-formyl-methionyl-leucyl-phenylalanine (fMLP) was evaluated. The tubiferin inhibition was more pronounced when PMN were stimulated through the protein kinase C pathway (PMA) compared to the alternative complement pathway (OZ). The inhibition when PMN were triggered by a chemoattractant stimulus (fMLP) was similar to that achieved with OZ-stimulated phagocytes. Tubiferin showed dose-dependent effects on the PMN respiratory burst triggered by the three different substances, and also decreased substantially the carrageenan-induced mice paw edema.     

STUDIES ON NITRIC OXIDE FREE RADICALS GENERATED FROM POLYMORPHONUCLEAR LEUKOCYTES (PMN) STIMULATED BY PHORBOL MYRISTATE ACETATE (PMA)

The interaction of NO and O^?₂free radicals generated from PMA (phorbol myristate acetate)-stimulated PMN (polymorphonuclear leukocytes) was studied by a nitroxide spin trap, DMPO (5,5-dimethyl-1-pyrroline-1-oxide). It was found that addition of L-arginine to the system would significantly decrease the trapped O^?₂by DMPO and addition of N^G-monomethyl-arginine (NGMA) would significantly increase the trapped O^?₂by DMPO. It was proved that the formation of ONOO^?by the reaction of NO and O^?₂was the main reason for the decrease of trapped O^?₂in the experiment with xanthine/xanthine oxidase and irradiation of riboflavin systems. The yield of NO during this process was calculated. The generation dynamic of NO was studied by a luminol-dependent chemiluminescence technique and it was found that after stimulation of PMN by PMA, there would be an immediate, significant chemi-luminescence, which came mainly from the active oxygen free radicals generated by PMN. If L-arginine was added to this system, the chemiluminescence would increase about 100-fold, but NGMA inhibited the increase of the chemiluminescence. Ten minutes after addition of L-arginine, this increase did not change, the chemiluminescence peak decreased gradually, but the half life increased. The ESR and chemiluminescence properties of NO and ONOO^?synthesized were also studied in model systems.     

Luminol and diazoluminomelanin as indicators of HL-60 cell differentiation

Summary This paper describes use of a novel substituted melanin which is useful in detection of differentiating leukemia cells and their membranes. Comparisons of luminol-(5-amino-2,3-dihydro-1,4-phthalazinedione) and diazoluminomelanin (DALM)-mediated chemiluminescence (CL) were made with various types of differentiated and undifferentiated HL-60 whole cells, cell lysates, and membrane fractions. Luminol had a greater CL response than DALM with HL-60 promyelocytic stem cells and differentiated macrophage-like or neutrophil-like whole cell and cell lysate preparations. However, DALM showed markedly greater CL than luminol for membrane fractions derived from each cell type. The greatest luminol-dependent CL was observed for cell types high in myeloperoxidase (MPO). The greatest DALM-mediated CL was seen with cell types that are high in MPO or strong producers of superoxide (O_2-) anions. In some cases, significant differences in CL could also be distinguished on the basis of inducing agent used [i.e. dimethylsulfoxide, all-trans retinoic acid or 12-o-tetradecanoylphorbol-13-acetate]. Both luminol- and DALM-dependent CL were strongly inhibited by preincubation of cellular preparations with 3-amino-l-tyrosine (a component of DALM). Taken together, these data suggest that the reaction mechanism of luminol favors interaction with cytoplasmic MPO whereas that of DALM favors membrane interactions. Thus, both reagents may be of use in assays to detect differentiating leukocytes or their cellular components.     

Myeloperoxidase-Based Chemiluminescence of Polymorphonuclear Leukocytes and Monocytes

Luminol and lucigenin chemiluminescence (CL) responses produced by separated human blood polymorphonuclear leukocytes (pmn) and monocytes (mono) have been studied following stimulation with the surface-receptor agonist fMLP (a synthetic chemotactic peptide) and the protein kinase C activator phorbol myristate acetate (PMA). Pmn produced two- to threefold the luminol CL and superoxide anion (O₂⁻) levels of mono; lucigenin CL was similar for both cell-types. The myeloperoxidase (MPO) inhibitor salicylhydroxamic acid (SHA) abrogated luminol but not lucigenin CL in both cell types, but did not further inhibit the already grossly subnormal luminol CL responses seen with MPO-deficient cells which produced normal lucigenin CL. SHA also profoundly inhibited the luminol CL response in a cell-free MPO–H₂O₂ system. Mono lucigenin CL does not appear to specifically measure O₂⁻ production. These data show that luminol CL provides a useful measure of pmn and also mono MPO activity. However, analysis of the effects of various reactive oxygen species (ROS) scavengers, assessed on phagocyte and cell-free CL systems (both MPO–H₂O₂ and superoxide generating) suggest that the luminol CL signal is not entirely dependent on MPO activity.     

Temperature influence on stimulated PMN respiratory burst.

Body temperature can modulate the pathogenesis of infectious, metabolic and autoimmune diseases. This effect has been attributed to several hypothesized mechanisms. Body temperature could play an important role in influencing some cellular functions of human white blood cells. In this work we examined the temperature effect on the respiratory burst in human neutrophils. Human polymorphonuclear leucocytes (PMN) were obtained from heparinized venous blood by dextran sedimentation and erythrocyte lysis with NH4Cl (0.87%). Granulocytes were stimulated with opsonized zymosan (OZ), formyl-methionyl-leucyl-phenylalanine (FMLP), phorbol myristate acetate (PMA), and monosodium urate (MSU) crystals at different temperatures (26, 37, 39, 40, 42 degrees C). The technique of luminol dependent chemiluminescence (CL) was used as indicator of oxygen free radicals (OFR) release by stimulated cells. OFR production from PMN stimulated with OZ, PMA, FMLP was higher at 37 degrees C than at 26, 39, 40, 42 degrees C (p < 0.001 OZ stimulated PMN at 40-42 degrees C; p < 0.05 PMA stimulated PMN at 42 degrees C. Significantly different from 37 degrees C value). OFR release from PMN stimulated with MSU crystals was significantly increased at 39 degrees C compared to 37 degrees C value (p < 0.001). This effect could not only be attributed to temperature influence on neutrophil activity. The specific polymorphonuclear leukocyte response to the microcrystals and the temperature influence on chemical and physical characteristics of the crystals may play an important role. We are now studying the temperature effect on activity of PMN exposed to others crystals.     

Reactive Oxygen Metabolite Production is Inhibited by Histamine and H 1 -antagonist Dithiaden in Human PMN Leukocytes

The study evaluated the distinction between extracellular and intracellular production of reactive oxygen metabolites (ROM) in isolated polymorphonuclear leukocytes (PMNL) stimulated with opsonised zymosan (OZ) and investigated its modulation by the endogenous mediator histamine (0.1-100 &#119 mol/l) and by the H 1 -antagonist dithiaden (1-100 &#119 mol/l). For this observation, a modified luminol and an isoluminol amplified chemiluminescence (CL) technique were used. Our results showed that PMNL activated with OZ responded with a respiratory burst accompanied by both extra- and intracellular generation of ROM. Histamine and dithiaden significantly decreased both the extra- and intracellular component of chemilumiescence stimulated with OZ. While dithiaden decreased both the extra- and intracellular part of CL with the same potency, histamine decreased preferentially the extracellular part of CL. The fact that histamine as well as the H 1 -antagonist dithiaden decreased the respiratory burst indicates that not only histamine receptors but also non-receptor mechanisms could be involved in the reduction of CL. Interaction with enzymes (NADPH-oxidase, myeloperoxidase, phospholipase A 2 ) or interference with PMNL membrane structure may well result in reduction of the chemiluminescence signal.     

Change in the chemiluminescence reactivity pattern during in vitro differentiation of human monocytes to macrophages

We determined the luminol-enhanced chemiluminescence (CL) of fresh human monocytes and monocytes cultured for 1-14 days in vitro, within hydrophobic membranes, using a variety of stimuli known to trigger the respiratory burst of phagocytes. It was assured that CL emerged from an adherent subpopulation of mononuclear cells; polymorphonuclear leukocytes (PMN) contaminating mononuclear leukocytes (MNL) contributed little, if anything, to the CL response of MNL. Typical response patterns were established for fresh monocytes triggered by phorbol 12-myristate 13-acetate (PMA), zymosan, the Ca2+ ionophore A 23187, antibody-coated erythrocytes and Sendai virus. Differentiation in vitro into macrophages was associated with a general decrease in magnitude of the CL peak, in an overproportional decrease of the A23187 triggered response and in a complete loss of the response to Sendai virus--a loss which could not be prevented by addition of myeloperoxidase (MPO). In contrast to monocyte CL, macrophage CL was resistant to sodium azide, indicating its MPO-independent origin. Macrophage-type reactivity was obtained at day 4 of culture. Activation of macrophages with recombinant interferon-gamma for the last 2 days of culture was associated with a quantitative (approx. threefold) increase of the CL signal, although qualitatively the same reactivity pattern was obtained as with control macrophages. In contrast to luminol-dependent CL, the lucigenin-dependent CL response of macrophages was greater than that of monocytes, an increase which was particularly prominent for PMA stimulation.     

Reactive oxygen metabolite production is inhibited by histamine and H1-antagonist dithiaden in human PMN leukocytes

The study evaluated the distinction between extracellular and intracellular production of reactive oxygen metabolites (ROM) in isolated polymorphonuclear leukocytes (PMNL) stimulated with opsonised zymosan (OZ) and investigated its modulation by the endogenous mediator histamine (0.1-100 μmol/l) and by the H 1 -antagonist dithiaden (1-100 μmol/l). For this observation, a modified luminol and an isoluminol amplified chemiluminescence (CL) technique were used. Our results showed that PMNL activated with OZ responded with a respiratory burst accompanied by both extra- and intracellular generation of ROM. Histamine and dithiaden significantly decreased both the extra- and intracellular component of chemilumiescence stimulated with OZ. While dithiaden decreased both the extra- and intracellular part of CL with the same potency, histamine decreased preferentially the extracellular part of CL. The fact that histamine as well as the H 1 -antagonist dithiaden decreased the respiratory burst indicates that not only histamine receptors but also non-receptor mechanisms could be involved in the reduction of CL. Interaction with enzymes (NADPH-oxidase, myeloperoxidase, phospholipase A 2 ) or interference with PMNL membrane structure may well result in reduction of the chemiluminescence signal.     

Priming Effect of Pseudomonal Leukocidin on Chemiluminescence Response of Rabbit Polymorphonuclear Leukocytes

To clarify effects of pseudomonal leukocidin (42.5 kd) on chemiluminescence (CL) production of polymorphonuclear leukocytes (PMNs), rabbit PMNs were stimulated by zymosan or phorbol myristate acetate (PMA) after pretreatment with the leukocidin, which by itself stimulated little chemiluminescence response. The extent of CL responses stimulated by zymosan or PMA was respectively 5.3- or 3.5-fold greater in leukocidin (1.5 μg/ml)-pretreated PMNs than in non-pretreated ones. The priming effect of the leukocidin was greater than that of G-CSF and related to some steps before NADPH oxidase activation. The increased CL productions might be related to tissue damages caused by pseudomonal infections in vivo.     

Luminol-dependent chemiluminescence in antibody-sensitized neutrophils stimulated with protein A-bearing staphylococci

When mouse polymorphonuclear leukocytes (PMNs) sensitized with rabbit antibody to mouse Ehrlich ascites tumor cells were stimulated by Staphylococcus aureus Cowan I cells, a conspicuous luminol-dependent chemiluminescence was observed in the absence of opsonin. The profile of the chemiluminescence (CL) response evoked by staphylococcal cells from antibody-sensitized PMNs had two peaks. An initial peak, observed within 1 min after stimulation, was sharp and high and a second peak, observed about 5 min after stimulation, was low and extended. The CL response of antibody-sensitized PMNs stimulated by S. aureus Cowan I cells was dose-dependently blocked by preincubation with soluble SpA. Cells of a mutant derived from S. aureus Cowan I strain with trace amounts of cell-bound SpA failed to stimulate the antibody-sensitized PMNs to generate the CL response. The antibody-sensitized PMNs were found to phagocytize SpA-bearing S. aureus cells even in the absence of opsonic serum. These results suggest that the observation presented here might provide a useful tool for the investigation of CL response of PMNs.