Leukotriene B4 levels from stimulated neutrophils from healthy and allergic subjects: effect of platelets and exogenous arachidonic acid

Leukotriene B4 (LTB4) levels were measured in peripheral blood neutrophils from allergic and healthy donors after stimulation by calcium ionophore A 23187. This level was higher in neutrophils from allergic subjects than in neutrophils from healthy subjects in the presence as well as in the absence of exogenous arachidonic acid. Platelets from allergics increased LTB4 levels from neutrophils from allergics but not levels in those from healthy donors. Moreover, platelets from healthy subjects reduced LTB4 in neutrophils from both groups. These results suggest that biochemical differences exist in neutrophils and platelets from allergics which contribute to changes in arachidonic acid metabolism via the 5-lipoxygenase pathway. In addition, they support the concept that platelets may play an important role in the regulation of neutrophil LTB4 levels, possibly by affecting the 5-lipoxygenase activity during the course of allergic inflammatory reactions.     

Leukotriene biosynthesis by polymorphonuclear leukocytes from two patients with chronic granulomatous disease.

Polymorphonuclear leukocytes (PMNL) isolated from two patients with chronic granulomatous disease (CGD) were tested for their ability to metabolize arachidonic acid to lipoxygenase products including 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4). Analyses of incubations of these PMNL with arachidonic acid and the calcium ionophore A23187 did not differ from simultaneous controls in the production of LTB4, other 5,12-dihydroxy-eicosatetraenoic acids, or monohydroxy-eicosatetraenoic acids. The clinical diagnosis of CGD was confirmed in both cases by determination of PMNL chemiluminescence. Leukocytes from both patients failed to generate active oxygen species in response to either LTB4 or formyl-methionyl-leucyl-phenylalanine. The observation of arachidonic acid oxidation in the absence of superoxide anion precludes a role for the active oxygen species in this metabolic process. These studies clearly dissociate the ionophore-induced leukocyte respiratory burst from the oxidation of arachidonate to the leukotrienes. In addition, the defect of CGD appears to be unrelated to the ability of PMNL to carry out arachidonate oxygenation.     

Bacterial lipopolysaccharides prime human neutrophils for enhanced production of leukotriene B4

Neutrophils can be "primed" for an enhanced respiratory burst by lipopolysaccharide (LPS) in concentrations measurable in patients with septic shock. Leukotriene B4 (LTB4) is the primary eicosanoid product of neutrophils and is felt to be a mediator of host defense and inflammation. We investigated the in vitro effects of LPS on neutrophil production of LTB4 and the omega-oxidation metabolites of LTB4. Incubation of neutrophils with LPS in concentrations ranging from 0.01 to 100 ng/ml did not result in production of LTB4 or metabolites in the absence of a second stimulus. Priming neutrophils with LPS and then stimulating with opsonized zymosan, phorbol-myristate-acetate or a low concentration of the calcium ionophore A23187 resulted in enhanced production of LTB4. LPS priming of neutrophils occurred in a concentration dependent manner. LPS did not result in LTB4 production in response to the chemoattractant peptide FMLP. LPS priming of neutrophils had no effect on cytosolic calcium concentrations of resting or zymosan-stimulated cells. These results suggest that LPS might effect host defense and tissue injury by potentiating the effect of other stimulants on neutrophil production of LTB4. This LPS induced enhancement may represent an important pathogenetic pathway in patients with gram negative sepsis.     

Effects of Phorbol Myristate Acetate on the Metabolism and Ultrastructure of Neutrophils in Chronic Granulomatous Disease

Previous investigations have demonstrated that phorbol myristate acetate (PMA), the active principle of croton oil, stimulates alterations in normal polymorphonuclear leukocytes (PMN) that resemble closely the changes that develop in the cells after phagocytosis of bacteria. The present study has compared the effects of PMA and heat-killed bacteria on the oxygen uptake, glucose oxidation, nitroblue tetrazolium (NBT) reduction, and ultrastructure of normal neutrophils and PMN from six patients with chronic granulomatous disease (CGD). PMA stimulated oxygen consumption, hexose monophosphate shunt activity, and NBT reduction in normal cells but failed to produce similar effects in CGD neutrophils. However, PMA did induce formation of cytoplasmic vacuoles in the CGD cells similar to those observed in normal neutrophils. The results indicate that PMA is a useful nonparticulate agent for distinguishing between normal and CGD neutrophils and for studying basic mechanisms of phagocytosis in normal and abnormal PMN.     

The influence of phorbol myristate acetate on the metabolism of neutrophils from carriers of sex-linked chronic granulomatous disease.

The present investigation has compared the influences of phorbol myristate acetate (PMA) and heat-killed bacteria (HKB) on oxygen consumption and glucose oxidation by polymorphonuclear leukocytes (PMN) from carriers of sex-linked chronic granulomatous disease (CGD). PMA or HKB caused neutrophils from CGD carriers, considered as a group, to consume oxygen and oxidize glucose-1-14C at rates that were statistically distinguishable from rates of normal controls and affected CGD hemizygotes. PMA at a final concentration of 1.0 micrograms per milliliter wass more effective and reproducible than a ratio of 50 HKB: 1 PMN in discriminating the partial abnormality of carrier PMN from normal PMN. Moreover, a deficiency in glucose oxidation by the PMN of one individual carrier was detectable using PMA stimulation when no defect was apparent with HKB. Results of the present investigation confirm and extend previous observations which have demonstrated the similarity in responses of PMA-treated normal and CGD PMN to the reactions produced by particulates under similar conditions.     

Basophil Histamine Release and Leukotriene (LTB4 - LTC4) Production in Cluster Headache

Histamine release and leukotrienes (LTB4 and LTC4) production from circulating basophil have been studied in 13 patients with episodic cluster headache (CH) during the remission phase of symptoms, and in 9 normal subjects. Cell suspensions of basophils were stimulated with scalar dilutions of anti-IgE, f-met-peptide and Ca2+ ionophore A23187. Histamine was measured by an automated fluorimetric method; LTB4 and LTC4 with RIA in individual cases, and with Reverse-Phase HPLC in the two pools obtained from the supernatants of CH patients and controls. Mean values of histamine release in patients with CH were significantly lower when compared to those obtained in control subjects after stimulation with anti-IgE at the three dilutions used. LTC4 mean levels measured in CH patients were significantly lower than those of supernatants from controls after stimulation with 0.05 gamma/ml of A23187. A reduction of LTC4 and LTB4 levels in CH patients was also observed in R-P HPLC, which showed different elution patterns in the two groups. The histamine release in individual cases was related to leukotriene production: LTC4 levels were significantly (p less than .05) higher in "high histamine releasers" than in "low histamine releasers". Our results indicate that CH patients have complex abnormalities of histamine release and of leukotriene production during the painless phase of the disease.     

Impaired respiratory burst contributes to infections in PKCδ-deficient patients

Patients with autosomal recessive protein kinase C δ (PKCδ) deficiency suffer from childhood-onset autoimmunity, including systemic lupus erythematosus. They also suffer from recurrent infections that overlap with those seen in patients with chronic granulomatous disease (CGD), a disease caused by defects of the phagocyte NADPH oxidase and a lack of reactive oxygen species (ROS) production. We studied an international cohort of 17 PKCδ-deficient patients and found that their EBV-B cells and monocyte-derived phagocytes produced only small amounts of ROS and did not phosphorylate p40^phox normally after PMA or opsonized Staphylococcus aureus stimulation. Moreover, the patients’ circulating phagocytes displayed abnormally low levels of ROS production and markedly reduced neutrophil extracellular trap formation, altogether suggesting a role for PKCδ in activation of the NADPH oxidase complex. Our findings thus show that patients with PKCδ deficiency have impaired NADPH oxidase activity in various myeloid subsets, which may contribute to their CGD-like infectious phenotype.     

Leukotriene B4 level in stimulated blood neutrophils and alveolar macrophages from healthy and asthmatic subjects. Effect of beta-2 agonist therapy

Leukotriene B4 levels were measured after stimulation by calcium ionophore A23187: (i) in peripheral, neutrophils (PMN) from allergic asthmatics, rhinitis and healthy subjects; (ii) in macrophages collected by bronchoalveolar lavage. LTB4 levels in PMNs were significantly higher in non-treated allergic asthmatics and non-treated subjects with rhinitis compared to controls. Beta-2 agonist-treated asthmatics showed a significantly decreased LTB4 production which was not different from those of controls. In vitro, LTB4 production decreased significantly after PMN incubation with Salbutamol (10(-6) mol l-1). LTB4 produced by AM collected by BAL was measured in non-treated (n = 5) and treated (n = 11) asthmatics with inhaled beta-2 agonist. AM collected from all controls and non-treated asthmatics produced LTB4. By contrast, no production of LTB4 was observed in the treated group. LTB4 production decreased when normal AM were incubated in vitro with Salbutamol (10(-8) mol l-1). These results suggest that biochemical differences occur in PMN and macrophages from subjects treated with beta-2 agonist, presumably in changing the 5-lipoxygenase pathway.     

Use of lipophilic probes of membrane potential to assess human neutrophil activation. Abnormality in chronic granulomatous disease.

Previous studies using membrane potential sensitive probes have provided evidence that chemotactic factors elicit membrane potential changes in normal human neutrophils (PMN). In addition to stimulation of PMN motility, chemotactic factors also stimulate degranulation and superoxide ion (O-2) generation and it has been suggested that alteration of membrane potential activates these events (Korchak, H. M., and G. Weissmann. 1978. Proc, Natl, Acad, Sci. U. S. A. 75: 3818--3822). To further define the inter-relationship of these functions, studies were done with two indirect probes of membrane potential, 3-3''-dipentyloxacarbocyanine and triphenylmethylphosphonium ion (TPMP+) using PMN from normal subjects, from patients with abnormal O-2 production (chronic granulomatous disease [CGD]), and from patients with defective degranulation and/or chemotaxis (Cheddiak-Higashi syndrome and patients with elevated immunoglobulin (Ig)E and recurrent staphylococcal infections). The stimuli used were the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) and the secretagogues ionophore A23187 and phorbol myristate acetate (PMA). The results obtained with 3-3''-dipentyloxacarbocyanine and TPMP+ were comparable. The apparent membrane potential changes elicited by f-Met-Leu-Phe and PMA in normal PMN were reduced or entirely absent in PMN obtained from patients with CGD but normal in PMN from other patients. PMN from patients with CGD had normal calculated resting membrane potentials and normal responses elicited by the potassium ionophore valinomycin. The responses to calcium ionophore A23187 were only slightly impaired. The abnormality of the elicited response of CGD cells of f-Met-Leu-Phe and PMA could not be attributed to the absence of O-2, hydroxyl radical, singlet oxygen, or hydrogen peroxide acting on the probes. Instead this abnormality appears to be associated with a dysfunction in the normal molecular mechanism(s) stimulated upon neutrophil activation. The data suggest chemoattractant alteration of membrane potential in normal PMN is related to activation of oxidative metabolism but the relationship to chemotaxis and degranulation remains to be established.     

佛波酯对LPS／IFN-γ诱导的巨噬细胞活性氧产生的影响

目的研究佛波酯（PMA）对脂多糖／γ-干扰素（LPS／IFN-γ）诱导的巨噬细胞RAW264．7活性氧产生的影响。方法于实验前0、3、6、9、12、18h,用1μg／mL的LPS和100U／mL IFN-γ诱导处于对数生长期的RAW264．7细胞,采用蛋白质印迹技术检测细胞中一氧化氮合酶（iNOS）的表达;对经LPs／IFN-γ诱导12h的RAW264．7细胞加入终浓度为200ng／mL的PMA,应用激光共聚焦显微技术观察RAW264．7细胞中活性氧的产生。结果LPS／IFN-γ可使RAW264．7细胞的诱导型iNOS表达增加,同时PMA可刺激经LPS／IFN-γ诱导后的RAW264．7细胞产生呼吸爆发,细胞的荧光强度较未刺激组增强（P〈0．01）。结论PMA可促使经LPS／IFN-γ诱导的RAW264．7细胞产生大量活性氧。     

Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Partial purification and identification as a cytochrome P-450.

Human polymorphonuclear leukocytes (PMN) not only synthesize and respond to leukotriene B4 (LTB4), but also catabolize this mediator of inflammation rapidly and specifically by omega-oxidation. To characterize the enzyme(s) responsible for omega-oxidation of LTB4, human PMN were disrupted by sonication and subjected to differential centrifugation to yield membrane, granule, and cytosol fractions (identified by biochemical markers). LTB4 omega-hydroxylase activity was concentrated (together with NADPH cytochrome c reductase activity) only in the membrane fraction (specific activity increased 10-fold as compared to whole sonicates, 41% recovery). Negligible activity was detected in granule or cytosol fractions. LTB4 omega-hydroxylase activity in isolated PMN membranes was linear with respect to duration of incubation and protein concentration, was maximal at pH 7.4, had a Km for LTB4 of 0.6 microM, and was dependent on oxygen and on reduced pyridine nucleotides (apparent Km for NADPH = 0.5 microM; apparent Km for NADH = 223 microM). The LTB4 omega-hydroxylase was inhibited significantly by carbon monoxide, ferricytochrome c, SKF-525A, and Triton X-100, but was not affected by alpha-naphthoflavone, azide, cyanide, catalase, and superoxide dismutase. Finally, isolated PMN membranes exhibited a carbon monoxide difference spectrum with a peak at 452 nm. Thus, we have partially purified the LTB4 omega-hydroxylase in human PMN and identified the enzyme as a membrane-associated, NADPH-dependent cytochrome P-450.     

Activation of guinea pig eosinophil respiratory burst by leukotriene B4: role of protein kinase C

Leukotriene B4 (LTB4) and the protein kinase C activator, 4-beta-phorbol dibutyrate (PDBu), both induced a pronounced and concentration-dependent stimulation of hydrogen peroxide (H2O2) generation by purified guinea pig peritoneal eosinophils in the concentration range 1 nM-1 microM. The LTB4 response was inhibited competitively by the specific LTB4 receptor antagonist, U-75302, with a KB of 25 nM, while the concentration-response curves for both stimuli were shifted rightwards (3.8-fold and 2.8-fold for LTB4 and PDBu, respectively) by the competitive protein kinase C inhibitor, 1-O-hexadecyl-2-O-methylglycerol at a concentration of 300 microM. LTB4 appears, therefore, to induce respiratory burst in eosinophils via a receptor-mediated mechanism involving protein kinase C.     

Oxidative degradation of leukotriene C4 by human monocytes and monocyte-derived macrophages

Freshly isolated 2-h adherent normal human monocytes, when stimulated, degrade added leukotriene C4 (LTC4) by a myeloperoxidase (MPO) and H2O2-dependent mechanism. Among the stimuli effective in this regard are phorbol myristate acetate (PMA), the calcium ionophore A23187, opsonized zymosan, and N-formyl-methionine-leucine-phenylalanine (FMLP) when combined with cytochalasin B. The predominant products formed are the all-trans isomers of LTB4, 5-(S), 12-(R)-6-trans-LTB4 and 5-(S),12-(S)-6-trans-LTB4. Degradation is inhibited by azide and catalase, but not by superoxide dismutase. LTC4 degradation does not occur when MPO-deficient monocytes are used, unless MPO is added. Stimulated monocytes from patients with chronic granulomatous disease also are unable to degrade LTC4 under these conditions. Normal monocytes maintained in culture lose their ability to degrade LTC4. The addition of MPO to monocyte-derived macrophages increases degradation, particularly when the monolayers are pretreated with gamma-interferon. The oxidative degradation of LTC4 is a capacity shared by neutrophils, eosinophils, and mononuclear phagocytes, and may be an important mechanism for the modulation of leukotriene activity in inflammatory lesions.     

The role of reactive oxygen species in thromboxane b2 generation by polymorphonuclear leukocytes

These studies evaluated further the relationship between the metabolism of reactive oxygen species (ROS) and prostaglandins in human granulocytes. Our experiments examined (1) the effects of several scavengers of ROS on thromboxane B2 (TXB2) production by zymosan-stimulated PMNs, (2) the capacity of the granulocytes of patients with chronic granulomatous disease (CGD) to produce TXB2, and finally (3) the generation of oxygen radicals in PMNs stimulated to produce TXB2 by the enzyme phospholipase A2. Our results confirm that both zymosan- and PMA-stimulated PMNs release increased amounts of TXB2. This enhanced production of TXB2 by normal PMNs could not be impaired and, in fact, appeared to be enhanced by scavengers of ROS. The PMNs of one patient with CGD produced TXB2 in an amount similar to those of healthy persons, whereas the TXB2 produced by the PMNs of a second patient was markedly increased. Finally, the enzyme phospholipase A2 stimulated TXB2 production in PMNs without stimulating the production of ROS. These data indicate that the activation of prostaglandin metabolism in PMNs is not dependent on the simultaneous production of ROS by these cells. However, the simultaneous production of ROS may be associated with an alteration of prostaglandin metabolism.     

Leukotriene B4 Generation by Polymorphonuclear Leukocytes: Possible Involvement in the Pathogenesis of Headache

SYNOPSIS
Leukotriene B₄ (LTB₄) levels in plasma and its generation in vitro from isolated polymorphonuclearleukocytes (PMN) were measured in migraine and cluster headache patients during and between painattacks. The LTB₄plasma levels in cluster headache patients during an attack were significantly higherthan in patients between attacks. There was a positive correlation between the time of sampling from thebeginning of attack and the LTB₄level. The LTB₄ plasma levels in migraine patients during and betweenattacks did not differ significantly from control levels. The results suggest that LTB₄appears rapidly in thecirculation at the beginning of a cluster attack and thereafter declines in amount. LTB₄release from PMNwas induced by stimulation with the calcium ionophore A23187. The release of LTB₄was significantlyhigher in migraine patients during attacks on stimulation with a submaximal dose of A23187 0.5 μM. LTB₄ release induced by A23187 in migraine patients during symptom-free intervals and cluster headachepatients did not differ from healthy controls. LTB₄ release was not affected by 5HT and NA inconcentrations up to 10^-4M. The results suggest that LTB₄is more easily generated in migraine patientsduring attacks. These studies on isolated cells provide a suitable model for the investigation of themetabolism of lipoxygenase derivates in headache patients.     

Increased leukotriene B4 synthesis in immune injured rat glomeruli

We examined glomerular synthesis of the 5-lipoxygenase metabolite, LTB4, in normal and immune-injured rat glomeruli. Glomeruli isolated from normal rats and from rats with nephrotoxic serum nephritis (NSN), passive Heymann nephritis (PHN) and cationic bovine gamma globulin (CBGG)-induced glomerulonephritis were incubated with the calcium ionophore A23187 (3 microM). Lipids in the glomeruli and media were extracted with ethyl acetate, and were purified and fractionated by HPLC. Immunoreactive-LTB4 (i-LTB4) was determined by radioimmunoassay on HPLC fractions with a detection limit of 50 pg of i-LTB4. A large peak of i-LTB4 that comigrated with authentic LTB4 was found exclusively in glomeruli isolated from the CBGG-injected rats. Addition of the lipoxygenase inhibitor BW755C (50 micrograms/ml) to glomerular incubation resulted in greater than 90% inhibition of i-LTB4. Synthesis of i-LTB4 by glomeruli from normal, NSN and PHN rats was undetectable. Glomerular LTB4 synthesis by CBGG-injected rats was confirmed by radiometric HPLC and by gas chromatography mass-spectroscopy (GC-MS) analysis. In order to rule out synthesis of LTB4 by neutrophils entrapped in the glomeruli, a group of rats received 1,000 rad total body x irradiation, with shielding of the kidneys before induction of CBGG glomerulonephritis. Despite greater than 95% reduction in total leukocyte count, glomerular synthesis of LTB4 remained enhanced. Augmented glomerular synthesis of the proinflammatory lipid, LTB4, in the CBGG model of glomerular disease could have an important role in the development of glomerular injury and proteinuria.     

Increased urinary excretion of LTB4 and omega-carboxy-LTB4 in patients with Zellweger syndrome.

The metabolic inactivation of leukotrienes proceeds by beta-oxidation from the omega-end. We investigated the importance of peroxisomes and mitochondria in LTB4 oxidation in vivo. LTB4 and its oxidation products were analysed after high-performance liquid chromatography separation by immunoassays and gas chromatography-mass spectrometry in the urine of patients with Zellweger syndrome, patients with long-chain acyl CoA dehydrogenase deficiency, and healthy controls. LTB4 (median 97; range 35-238 nmol/mol creatinine) and its omega-oxidation product omega-carboxy-LTB4 (median 898; range 267-4583 nmol/mol creatinine) were present and significantly increased in the urine of all patients with Zellweger syndrome compared to the controls (P <0.01). In contrast, LTB4 and omega-carboxy-LTB4 were below the detection limit (< 5 nmol/ mol creatinine) in patients with long-chain acyl CoA dehydrogenase deficiency and healthy controls. The beta-oxidation product omega-carboxy-tetranor-LTB3 was neither detectable in the urine of patients with Zellweger syndrome, patients with long-chain acyl CoA dehydrogenase deficiency nor in the controls (< 5 nmol/mol creatinine). Analysis of urinary leukotrienes represents an additional diagnostic tool in peroxisome deficiency disorders. Furthermore, these results clearly underline the essential role of peroxisomes in the oxidation of LTB4 in humans.     

Neutrophils from p40phox-/- mice exhibit severe defects in NADPH oxidase regulation and oxidant-dependent bacterial killing

The generation of reactive oxygen species (ROS) by the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex plays a critical role in the antimicrobial functions of the phagocytic cells of the immune system. The catalytic core of this oxidase consists of a complex between gp91(phox), p22(phox), p47(phox), p67(phox), p40(phox), and rac-2. Mutations in each of the phox components, except p40(phox), have been described in cases of chronic granulomatous disease (CGD), defining their essential role in oxidase function. We sought to establish the role of p40(phox) by investigating the NADPH oxidase responses of neutrophils isolated from p40(phox-/-) mice. In the absence of p40(phox), the expression of p67(phox) is reduced by approximately 55% and oxidase responses to tumor necrosis factor alpha/fibrinogen, immunoglobulin G latex beads, Staphylococcus aureus, formyl-methionyl-leucyl-phenylalanine, and zymosan were reduced by approximately 97, 85, 84, 75, and 30%, respectively. The defect in ROS production by p40(phox-/-) neutrophils in response to S. aureus translated into a severe, CGD-like defect in the killing of this organism both in vitro and in vivo, defining p40(phox) as an essential component in bacterial killing.     

Reactive oxygen species generation by Kupffer cells and blood monocytes of mice infected with Plasmodium berghei and the chloroquine treatment

Generation of reactive oxygen species (ROS) by blood monocytes and Kupffer cells of normal and Plasmodium berghei infected mice treated at different levels of parasitaemia with chloroquine, were studied. Cells isolated at lower level of parasitaemia (less than 2%) produced ROS within the range of normal animals, whereas ROS production by the cells isolated from the animals at higher level of parasitaemia (greater than 20%), was significantly higher even without stimulation with latex particles. The ROS generation capacity of both normal and infected animals was less after the chloroquine treatment. This inhibitory effect of chloroquine may be beneficial in protecting the host from the adverse effect of reactive oxygen species by controlling their overproduction.     

Phorbol myristate acetate-induced lung injury: Involvement of reactive oxygen species

Using lucigenin-enhanced chemiluminescence, isolated rat lungs perfused with physiological salt-Ficoll solution were studied to test whether phorbol myristate acetate (PMA)-induced lung injury was mediated by reactive oxygen species (ROS). PMA (0.03 pg ml-I) caused small but significant increases in lung ROS levels and pulmonary arterial perfusion pressure (Ppa) but did not induce lung oedema. PMA (0.15 pg ml-I) induced lung oedema with large increases in ROS production and Ppa. Superoxide dismutase (SOD) inhibited the increases in ROS, Ppa, and lung oedema. Catalase and dimethylthiourea inhibited lung oedema but did not attenuate the increases in ROS and Ppa entirely. Indomethacin attenuated lung oedema partially but did not inhibit the increases in ROS and Ppa. These data indicate that PMA-induced lung injury is dependent on PMA concentration and ROS are responsible for such lung injury. Thromboxane plays a minor role for PMA-induced lung injury. The different effects of oxygen radical scavengers suggest that different radical species contribute to the increased pulmonary vascular response and lung injury.