Enhanced generation of leukotriene B4 and superoxide radical from calcium ionophore (A23187) stimulated human neutrophils after priming with interferon-alpha

The present study was designed to investigate the effect of interferon-alpha ( INF-alpha) on the production of leukotrienes (LTs) and superoxide radicals when intact human neutrophils were stimulated with calcium ionophore (A23187). A reverse phase high performance liquid chromatography and UV spectroscopy were employed to detect and quantitate the released LTs; namely LTC4, LTB4 and its trans isomers, 6-trans LTB4 and 12-epi-6-trans LTB4. Preincubation of intact human neutrophils at 37 degrees C for 30 min with INF-alpha and stimulation with calcium ionophore A23187 for 1 min enhanced significantly the formation of LTB4. Preincubation of intact human neutrophils with INF-alpha and subsequent stimulation with calcium ionophore A23187 also enhanced significantly superoxide radical generation that reduced nitroblue tetrazolium into blue formazan. The in vivo effect of INF-alpha in rats demonstrated that the higher dose of INF-alpha that induced superoxide radical and LTB4 by A23187 stimulated intact human neutrophil in vitro, also induced a significant decrease in white blood cells and RBCs started at 4 h after i.p administration. The differential white blood cell count revealed that, the prime target for INF-alpha is the white blood cells of myeloid origin. These results might demonstrate the modulatory effects of INF-alpha on granulocyte functions.     

Pharmacological effect of KC-404 on leukotriene release from human leukocytes induced by ionophore A23187

Release of leukotriene (LT) B4, LTC4 and histamine from human peripheral mixed leukocytes stimulated with calcium ionophore A23187 was evaluated. The amounts of LTB4 and LTC4 in supernatants were analyzed by high performance liquid chromatography and histamine was measured by an automated fluorometric technique. A time-course study demonstrated the characteristic patterns of the release of these mediators. LTB4 and LTC4 were more slowly released than histamine and the release of these LTs was inhibited by KC-404, a novel anti-asthmatic drug which has no effect on histamine release.     

Horse eosinophil degranulation induced by the ionophore A23187. Ultrastructure and role of phospholipase A2

Horse eosinophils stimulated with the calcium ionophore A23187 were examined by transmission and scanning electron microscopy. Secretion was characterized by granule movement to the cell periphery and fusion of adjacent granules. The granules became swollen and less electron-dense as their contents were released into large intracellular vacuoles, which opened to the outside of the cell through surface pores. A23187-induced eosinophil peroxidase (EPO) release, as measured by guaiacol oxidation, was blocked by eicosa-5,8,11,14-tetraynoic acid (ETYA) (which inhibits both the cyclooxygenase and lipoxygenase pathways of arachidonic acid metabolism) but not by indomethacin (which inhibits only the cyclooxygenase pathway). Highly purified porcine phospholipase A2 induced noncytotoxic eosinophil degranulation (as measured by the release of EPO without the concomitant release of the cytoplasmic marker lactate dehydrogenase), which was blocked by pretreatment of the enzyme with the phospholipase A2 inhibitor 4-bromophenacyl bromide. These results suggest that calcium-dependent activation of phospholipase A2 and generation of lipoxygenase products of arachidonic acid metabolism are important in the initiation of eosinophil degranulation.     

Release of leukotriene B4 from human neutrophils after interaction with nontypeable Haemophilus influenzae.

Opsonization of nontypeable Haemophilus influenzae with antibody is critical for the interaction between the organism and human polymorphonuclear leukocytes (PMNs). Nontypeable H. influenzae opsonized in fresh antibody-positive serum induced the release of 42.5 +/- 17.9 ng of leukotriene B4 per ml from PMNs after 20 min of incubation at 37 degrees C. On the other hand, opsonization of the organisms in fresh antibody-negative serum stimulated the release of significantly smaller amounts of leukotriene B4 by the PMNs. Simultaneous determinations of phagocytosis demonstrated similar patterns of response. A small amount (26.7 +/- 7.6%) of unopsonized nontypeable H. influenzae was phagocytosed by PMNs during 20 min of incubation at 37 degrees C. In contrast, 89.3 +/- 2.0% of nontypeable H. influenzae opsonized in fresh antibody-positive serum was phagocytosed during the same incubation period (P less than 0.001). Removal of complement through heat inactivation at 56 degrees C for 30 min did not significantly affect phagocytosis. These data suggest that the humoral immune response to nontypeable H. influenzae plays an important role in the inflammatory process and may contribute to the production of middle ear effusions in otitis media.     

Leukotriene A4 modulates generation of leukotriene B4 and sulphidopeptide leukotrienes by human neutrophils.

We investigated the influence of exogenous leukotriene A4 (LTA4) on the reactivity of polymorphonuclear leucocytes (PMN). PMN were either prestimulated with LTA4 or incubated simultaneously with LTA4 and the Ca ionophore A23187 or sodium fluoride (NaF). The Ca ionophore A23187 and NaF induced generation of LTB4 from PMN was significantly diminished in the presence of LTA4 while the formation of LTC4 was enhanced. In contrast, preincubation of cells with LTA4 followed by subsequent stimulation with NaF synergistically increased the LTB4 generation from PMN. LTA4, either alone or in combination with the calcium ionophore A23187 or NaF, decreases GTPase activity in human PMN. This decrease was abolished when LTA4 pretreated cells were subsequently stimulated with NaF, but not with calcium ionophore A23187, suggesting a regulatory role of LTA4 on G-proteins. The results demonstrate dual functions of LTA4: it serves as a substrate for the generation of leukotrienes and also regulates the susceptibility of human PMN for subsequent response.     

Studies on the uptake, binding and metabolism of leukotriene B4 by human neutrophils.

Human polymorphonuclear granulocytes (PMN) generate the inflammatory mediator leukotriene B4 (LTB4) as a response to cell activation. In addition, PMN inactivate LTB4 by omega-oxidation resulting in the formation of 20-OH- and 20-COOH-LTB4. The transport of exogenous LTB4 to the metabolizing enzymes is mediated via high- and low-affinity receptor subsets. Uptake of [3H]LTB4 by the cells was carried out in a time-dependent fashion, reaching maximal values after 5 min of incubation. No additional uptake of [3H]LTB4 then occurred. Prestimulation of PMN with phorbol myristate acetate or sodium fluoride resulted in the loss of high- and low-affinity receptors. Deactivating concentrations of LTB4 specifically reduced the high-affinity receptor subset. Prestimulation of PMN with cytochalasin B or with the membrane fluidizer butanol shifted the low-affinity receptors to the high-affinity state. The polyene antibiotic amphotericin B shifted high-affinity receptors to the low-affinity subset. The changes in the receptor expression pattern correlated with the respective conversion rate of exogenously added LTB4. Our results suggest that the distribution of high- and low-affinity receptors is regulated by GTP-binding proteins, the activation of protein kinase C and the organization of the membrane bilayer. In this way, human neutrophils control the respective level of the lipid mediator LTB4.     

Inhibition of leukotriene B4 in neutrophils by lipoxins A4 and B4

Lipoxins are derived from the oxygenation products of arachidonic acid in human leukocytes. They have exhibited selective biological effects different from those of other eicosanoids. We have examined the effect of lipoxin A₄ and B₄ (LXA₄, LXB₄) on the production of leukotriene B₄ (LTB₄) in human neutrophils. Cultured human polymorphonuclear leukocytes were preincubated with LXA and B and their ability to inhibit LTB₄ generation was assessed after incubation with calcium ionophore A23187. We found that the pretreatment of neutrophils with lipoxins inhibit the release of LTB₄ by A23187 stimulated PMNs. Our data suggests that LXA₄ and B₄ can contribute to immunosuppression in an inflammatory state via the inhibition of LTB₄ synthesis.     

A linear correlation between histamine release and degranulation of human basophils by specific antigen or the ionophore A23187

Simultaneous kinetic measurements have been made of basophil degranulation and of histamine release. Both antigen and ionophore A23187 were studied. Cells from the same donor under the same conditions undergo both reactions at a similar rate. Regression analysis indicated a statistically highly significant correlation between these two parameters for activation by either antigen or ionophore. In addition, the mean slope of the regression lines approached unity indicating a close direct correspondence between the number of cells degranulated and the quantity of histamine released. At any time during histamine release almost all of the cells identifiable as basophils were confluently stained and only rarely were partly degranulated cells observed. These results suggest that: (1) histamine release is not synchronous for populations of basophils; (2) individual cells are fully activated to begin histamine release at different times during the release period and then release all or almost all of their histamine in a short time relative to the total release period; (3) some cells release essentially none of their histamine and the number of these cells varies among donors and experimental conditions accounting in large measure for the incompleteness of histamine release.     

Ultrastructure of isolated human mast cells during histamine release induced by ionophore A 23187.

In the presence of calcium, the ionophore A 23187 (10(-7) TO 10(-6) M) causes a dose-dependent histamine release from isolated human mast cells. The accompanying degranulation process is characterized by a formation of channels of fused mast cell granules and by an exocytotic extrusion of altered granule material. Simultaneously, large numbers of newly formed 70 A filaments occur. These filaments probably have a key function in secreting human mast cells.     

Phosphatidylserine externalization in human sperm induced by calcium ionophore A23187: relationship with apoptosis, membrane scrambling and the acrosome reaction

BACKGROUND: Translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is a modification of the lipid architecture occurring in sperm. This is one of the earliest signs of apoptosis that can be monitored by the calcium-dependent binding of annexin V. METHODS AND RESULTS: Flow cytometric analysis of annexin V binding was performed. Calcium ionophore A23187 led to a significant increase in the proportion of living sperm with PS exposure: 7.3 3.2% of cells in the untreated ejaculate versus 47.5 5.6% of cells after 1 h of incubation with A23187. Conversely, diminution of mitochondrial membrane potential [DiOC6(3)/propidium iodide (PI) assay], caspase activation [fluorescein isothiocyanate (FITC)-Val-Ala-Asp-fluoromethylketone (VAD-FMK)/PI assay], increased plasma membrane permeability (Yo-Pro-1/PI assay) and increased DNA fragmentation [TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay], which are among the main signs of apoptosis, were not observed in sperm, even after 4 h of incubation with A23187. However, A23187 significantly increased the proportion of sperm with plasma membrane scrambling and with a reacted acrosome, as detected with the merocyanine 540 probe (M540) and the monoclonal anti-human CD46-PE antibody respectively. CONCLUSIONS: Our results suggest that PS exposure in human sperm, as induced by A23187, is mainly related to the acrosome reaction rather than to apoptosis.     

Release of leukotriene C4 from human eosinophils and its relation to the cell density

Human eosinophils and neutrophils were isolated from 20 ml of peripheral blood by dextran sedimentation, centrifugation with lymphoprep, and density gradient centrifugation with Percoll. The incubation of neutrophils of purity higher than 99% (density: 1.080-1.085 g/ml) with calcium ionophore A23187 for 20 min resulted in the release of a little amount of leukotriene C4 (2.3 +/- 0.6 ng/10(6) cells) as measured with a commercial radioimmunoassay kit. On the other hand, the release from eosinophils was over 10 times that from neutrophils. When the releasability of leukotriene C4 from eosinophils was examined in relation to the cell density, the eosinophils with lower density were observed to produce greater amounts of leukotriene C4.     

A T-lymphocyte-derived factor that enhances IgG-dependent release of leukotriene B4 (LTB4) from human neutrophils.

We describe a human blood mononuclear cell (MNC)-derived leukotriene release enhancing factor (LREF) which significantly increased IgG-dependent leukotriene B4 (LTB4) generation by neutrophils. MNCs incubated with phytohaemagglutinin (PHA) or anti-CD3 monoclonal antibody and PHA-stimulated ER+ lymphocytes produced a 150-350% increase in LTB4 generation from IgG-stimulated neutrophils. With PHA, maximal activity was observed 48 hr after culture in serum-free medium. LREF was relatively stable when exposed to low pH (pH 2) or heat (56 degrees, 60 min). Following progressive purification by gel filtration (FPLC, Superose 12) and chromatofocusing (FPLC, Mono P) LREF was associated with proteins of molecular weight of 35-40 kD and a pI of 5.1-5.5. Partially purified LREF did not contain detectable amounts of interleukin 2 (IL-2) or interferon-gamma (IFN-gamma). Although high concentrations of recombinant granulocyte-macrophage colony stimulating factor (rGM-CSF) (greater than 2 ng/ml) and recombinant tumour necrosis factor (rTNF) (greater than 100 U/ml) gave a slight (60%) enhancement of LTB4 generation, this was considerably less than that of partially purified LREF (350%). Our results suggest that human lymphocyte-derived LREF may play a role in the amplification of inflammatory reactions involving sensitized lymphocytes, neutrophils and lipid mediators.     

Recombinant human tumour necrosis factor (rTNF)2 enhances leukotriene biosynthesis in neutrophils and eosinophils stimulated with the Ca2+ ionophore A23187

Recombinant human tumour necrosis factor alpha (rTNF) did not cause the release of leukotrienes (LT) by human neutrophils and eosinophils. It did, however, prepare neutrophils and eosinophils for enhanced production of LT after activation with the calcium ionophore A23187. The effect was observed with 10(2) to 10(4) units/ml of rTNF which enhanced LTB4 production by ionophore-stimulated neutrophils, by 40 to 400%, while 10(3) to 10(4) units/ml of rTNF increased LTC4 production by ionophore-stimulated eosinophils by 30 to 120%. The enhancement was observed when granulocytes were incubated for 5 to 10 min with rTNF before stimulation with the ionophore. Increased leukotriene production was attributed to the induction of fatty acid hydrolase and/or 5-lipoxygenase activities on the basis of the increment in the total counts released after 3H-arachidonic acid labelling of neutrophils pretreated with rTNF as compared to control medium.     

Activation of gastro-intestinal smooth muscle induced by the calcium ionophore A23187

Summary Tension development was recorded in isolated smooth muscle preparations from the guineapig, namely circular strips from the fundus and antrum region of the stomach, and taenia coli. The calcium ionophore A23187 (2·10^–6–2·10^–5 mol/l) induced maximum activity in fundus and taenia coli, and in antrum an activity slightly smaller than that obtained with acetylcholine (ACh) (5·10^–6 mol/l). The ionophore-induced activity could be suppressed by so-called calcium antagonists: D600 (3·10^–6 mol/l) suppressed the ionophore-induced activity of taenia coli completely; phasic and tonic components in the stomach preparations were selectively suppressed by D600 and sodium nitroprusside (10^–6 mol/l), respectively.     

Conductance rise induced by the calcium ionophore A23187 in unfertilized eggs of tilapia.

Current-clamp measurements on unfertilized tilapia eggs gave linear current-voltage relations in the range 0 to -120 mV. Eggs had low resting potentials (-22 mV) and high specific membrane resistances (500 k omega cm2) and specific membrane capacitances (0.9 microF cm-2) indicating no marked folding of their plasma membrane. Application of A23187 induced a small depolarization and a conductance rise, the response having a reversal potential of about -16 mV. The results suggest that tilapia eggs have a calcium-gated conductance probably activated at fertilization.     

Zymosan-induced leukotriene B4 generation by human neutrophils is augmented by rhTNF-alpha but not chemotactic peptide.

Tumour necrosis factor (TNF) is a mediator of inflammation that has been shown to enhance neutrophil responses to soluble and particulate stimuli. The release of leukotriene B4 (LTB4) by human neutrophils stimulated by unopsonized zymosan was measured in the presence or absence of recombinant human TNF-alpha (rhTNF-alpha) preincubation. There was a threefold increase in the LTB4 response at an optimal TNF concentration of 10(-9) M and an optimal preincubation time of 10-20 min. A similar time and dose dependency was observed for CR3 receptor expression and for the release of the secondary lysosomal granule marker, vitamin B12-binding protein. In contrast, the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP), although stimulating an increase in both CR3 receptor number and in particle phagocytosis, failed to induce an increase in LTB4 release in response to zymosan. In addition, the present study demonstrated that, unlike FMLP, the exocytotic mechanism for secondary granule release by rhTNF-alpha functioned in the absence of a rise in cytosolic free calcium. Furthermore, it was independent of changes in cyclic nucleotide concentrations and did not require an intact cytoskeleton. Thus the capacity of rhTNF-alpha to amplify the neutrophil response to zymosan through the CR3 receptor appears to be related to the amplification of post-membrane events as well as to an increase in the number of functionally active receptors.     

Interferon-alpha enhances the production of leukotriene B4 in murine peritoneal macrophages stimulated by opsonized zymosan.

Murine peritoneal macrophages pretreated with interferon (IFN)-alpha and then stimulated by opsonized zymosan produced two to three times more LTB4 than untreated macrophages. However, PGE2 production was not changed by IFN-alpha. Meanwhile, IFN-gamma did not affect the production of LTB4 and PGE2. From the results it is considered that IFN-alpha can modulate inflammation or host defence through the production of LTB4.     

Conversion of leukotriene A4 by neutrophils and platelets from patients with atopic dermatitis.

The generation of arachidonic acid-derived inflammatory mediators from unstimulated and stimulated neutrophils (PMN) and platelets in the presence of exogenous LTA4 has been studied in patients with atopic dermatitis (AD) as well as in healthy volunteers. PMN were stimulated with the interleukins IL-3, IL-8, C5a, and the Ca-ionophore A23187. In addition, NaF and thrombin were used to stimulate platelets. The release of leukotriene (LT)B4, 20-COOH- and 20-OH-LTB4, cysteinyl-leukotrienes and 12-HETE was measured. The proinflammatory mediator release from PMN and platelets of patients with AD was significantly higher as compared to the control group. The spontaneous conversion of LTA4 by PMN and platelets was markedly enhanced in patients with AD. Different results with receptor-specific and non-specific stimuli (Ca-ionophore A23187) in the presence of exogenous LTA4 were obtained. The results indicate a higher state of activation for enzymes involved in leukotriene formation. Furthermore, the production of 12-HETE by platelets from patients with AD was enhanced in unstimulated and stimulated cells. Our data emphasize that neutrophils and platelets may play an important role in the pathogenesis of AD by an increased responsiveness to receptor-specific stimuli and cell-cell interaction via LTA4.     

Production of leukotriene B4 and 5-hydroxyeicosatetraenoic acid by human neutrophils is inhibited by Pseudomonas aeruginosa phenazine derivatives.

Pyocyanin, a phenazine pigment produced by Pseudomonas aeruginosa, and its metabolite 1-hydroxyphenazine inhibited leukotriene B4 and 5-hydroxyeicosatetraenoic acid production by up to 70% in human neutrophils stimulated with the calcium ionophore A23187 (5 microM). This potential anti-inflammatory effect was dose dependent and occurred at low concentrations (10 to 50 microM) that did not inhibit neutrophil viability.