Leukotriene B4 metabolism in neutrophils of patients with chronic granulomatous disease: phorbol myristate acetate decreases endogenous leukotriene B4via NADPH oxidase-dependent mechanism

We studied the effect of phorbol myristate acetate (PMA) on endogenous leukotriene B4 (LTB4) metabolism of calcium ionophore A23187-stimulated human neutrophils. Preincubation of normal neutrophils with PMA significantly suppressed the recovery of endogenous LTB4 induced by A23187. PMA did not suppress the recovery of LTB4 produced by neutrophils from patients with chronic granulomatous disease (CGD), which is known to be defective in NADPH oxidase activation to produce reactive oxygen species (ROS). PMA inhibited the formation of omega-oxidation products of LTB4, but enhanced arachidonic acid release in normal and CGD neutrophils. Furthermore, 5-lipoxygenase activity of 10,000 x g supernatants from normal neutrophils pretreated with PMA was equivalent to that of the controls. Decrease in LTB4 recovery was not attributed to the suppression of the intracellular Ca2+ increase. Thus, it is suggested that reactive oxygen species (ROS) produced by PMA may directly affect endogenous LTB4 and convert it into metabolite(s) distinct from omega-oxidation products.     

Accumulation of leukotriene C4 and histamine in human allergic skin reactions.

To determine whether lipoxygenase products of arachidonic acid metabolism are released in vivo during human allergic cutaneous reactions, we serially assayed chamber fluid placed over denuded skin sites for the presence of both C-6 peptide leukotrienes (e.g., LTC4, LTD4, and LTE4) and leukotriene B4 (LTB4), using radioimmune assay and HPLC separation, and compared it to histamine (assayed radioenzymatically) in 13 atopic and two nonatopic volunteers. Skin chamber sites challenged with ragweed or grass pollen antigen (250-750 protein nitrogen units/ml) for the first hour and phosphate-buffered saline (PBS) for the next 3 h were assayed hourly and compared to sites challenged with PBS alone. As assessed by HPLC, LTC4 composed greater than 85% of the C-6 peptide leukotriene released at any skin site, whereas little LTD4 or LTE4 was detected. LTC4 was present in significantly greater concentrations at antigen sites as compared to PBS-challenged sites throughout the 4-h period. Minimal concentrations of LTB4 were found throughout this time period and were not different at antigen or PBS sites. Histamine was present in significantly greater concentrations at antigen rather than PBS sites, but the pattern of release was different from that of LTC4. Peak histamine release invariably occurred during the first hour and decreased progressively thereafter, whereas the greatest amounts of LTC4 were detected during the 2nd to 4th hours. The amount of LTC4 accumulating at the site was dependent upon the dosage of antigen used in the epicutaneous challenge. We have demonstrated in this study that of the leukotrienes assessed LTC4 is released in the greatest quantity in situ during in vivo allergic cutaneous reactions and that it is present at such sites for at least 4 h after antigen challenge. Since intradermal injection of LTC4 in humans induces wheal and flare responses that persist for hours, our findings support the hypothesis that LTC4 is an important mediator of human allergic skin reactions.     

Studies on the release of leukotrienes and histamine by human lung parenchymal and bronchial fragments upon immunologic and nonimmunologic stimulation. Effects of nordihydroguaiaretic acid, aspirin, and sodium cromoglycate

Fragments of human lung parenchyma or bronchi were studied by high performance liquid chromatography, gas chromatography-mass spectrometry, and bioassay for the biosynthesis of 5-lipoxygenase metabolites of arachidonic acid, and by radioenzymatic assay for the release of histamine, upon immunologic and nonimmunologic stimulation. Human lung parenchyma were passively sensitized with serum from timothy-positive allergic patients (radioallergosorbent test, 30-40%) and challenged with 0.5 microgram/ml of timothy allergen. Analysis of the incubation media showed the presence of LTB4, LTC4, LTD4, LTE4, and histamine. Maximum release of LTB4 and LTD4 was observed after 15 min of challenge (92.8 +/- 21, and 67.8 +/- 14 pmol/g tissue wet weight, respectively; mean +/- SEM) whereas maximum release of LTC4 was observed after 5 min of challenge (25 +/- 7.1 pmol). In parallel to leukotriene formation, histamine was released rapidly and reached a maximum after approximately 15 min of challenge (2.85 +/- 0.76 nmol/g tissue). When fragments of human lung parenchyma were stimulated with ionophore A23187 (4 microM), we observed a profile of leukotriene and histamine release similar to that seen in response to the allergen. Ionophore A23187 stimulated the release of two- to fivefold greater amounts of leukotrienes and histamine than did the allergen. Release of LTC4 and histamine was maximal after 5 min of stimulation (83 +/- 22.2 and 5.2 +/- 0.95 nmol/g tissue, respectively), whereas LTB4 and LTD4 release reached a maximum after 15 min (438 +/- 66.6 and 205 +/- 68 nmol/g tissue, respectively). In addition, human lung parenchyma metabolized LTB4 into omega-OH-LTB4 and omega-COOH-LTB4. This tissue also released 5-hydroxy-eicosatetraenoic acid (5-Hete), 12-Hete, and 15-Hete. Fragments of human lung bronchi also released a similar profile of leukotrienes (except LTC4) and histamine when challenged with the allergen or ionophore A23187. Maximum release of LTB4 and LTD4 by allergen or ionophore stimulation was observed after approximately 15 min (40 +/- 7.5 and 21 +/- 8 pmol/g tissue, respectively, upon allergen challenge; 100 +/- 13 and 47 +/- 10.6 pmol/g tissue, respectively, upon ionophore stimulation). The maximum release of histamine by bronchi was observed after approximately 15 min of allergen challenge and 5 min of ionophore stimulation (2.25 +/- 0.65 and 3.15 +/- 0.9 nmol/g tissue, respectively). The release of leukotrienes but not of histamine by human lung parenchyma upon both allergen and ionophore challenge was inhibited by nordihydroguaiaretic acid (NDGA) (ID50, 2 X 10(-6)M).(ABSTRACT TRUNCATED AT 400 WORDS)     

Leukotrienes LTB4 and LTC4 in thermally injured patients' plasma and burn blister fluid

Radioimmunoassays for 5(S), 12(R)-dihydroxyeicosa-6, 14-cis, 8,10-trans-tetraenoic acid (leukotriene B4, LTB4) and 5(S)-hydroxy-6(R)-L-gamma-glutamyl-L-cysteinyl-glycinyleicosa-7,9- trans, 11,14-cis-tetraenoic acid (leukotriene C4, LTC4) have been used to quantify the concentrations of these arachidonic acid metabolites in plasma (12 patients) and blister fluid (6 patients) of burned patients. The results of this study demonstrate that, in general, burn plasma LTB4, and LTC4 were not elevated; however, in individual cases, transient high levels of leukotrienes were observed. Correlation between leukotriene "peaks" and the clinical course could not be established. High levels of LTB4 and LTC4 in burn blister fluid suggest their participation in local inflammatory reactions.     

Leukotriene synthesis by gastrointestinal tissue and its pharmacologic modification

Tissues of the gastrointestinal tract synthesize leukotriene (LT) B4 and the sulfidopeptide-leukotrienes LTC4, LTD4 and LTE4 from endogenous substrate. Formation of leukotrienes was demonstrated using radioimmunoassay, high pressure liquid chromatography (HPLC) and bioassay. Under basal conditions the gastrointestinal tissues released minor amounts of leukotrienes only. Formation of lipoxygenase-derived products of arachidonic acid metabolism was, however, significantly increased in the presence of various stimuli. Thus, significant amounts of LTB4 and of sulfidopeptide-leukotrienes were released from colonic and gastric mucosa of guinea-pigs sensitized against ovalbumin when incubations were carried out in the presence of antigen. Antigen-induced leukotriene formation was not found in the muscularis propria and subserosal of ovalbumin-sensitized guinea-pigs. Release of cyclooxygenase-derived metabolites of arachidonic acid, on the other hand, was most abundant in the subserosal layer of the guinea-pig colon and was not influenced by the immunological reaction. Inhibitors of cyclooxygenase, such as indomethacin, reduced gastrointestinal formation of prostaglandins, but not of leukotrienes. Inhibitors of 5-lipoxygenase, however, significantly decreased leukotriene formation. Synthesis of LTB4 and of sulfidopeptide-leukotrienes was also found in human colonic mucosal tissue, using the divalent cation-ionophore A23187 as stimulating agent. HPLC analysis demonstrated that the sulfidopeptide-leukotrienes released were composed of a mixture of LTC4, LTD4 and LTE4. In addition, human colonic mucosal tissue contained high activities of enzymes that rapidly convert LTC4 to LTE4. As in most biological systems LTE4 is less active than LTC4 and LTD4 degrading enzymes might represent a local inactivating mechanism. Mucosal tissue of patients with Crohn's disease synthesized considerably more LTB4 and sulfidopeptide-leukotrienes than non-inflamed mucosa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Macrophages cultured in vitro release leukotriene B4 and neutrophil attractant/activation protein (interleukin 8) sequentially in response to stimulation with lipopolysaccharide and zymosan

The capacity of lipopolysaccharide (LPS), zymosan, and calcium ionophore A23187 to induce neutrophil chemotactic activity (NCA), leukotriene B4 (LTB4), and neutrophil attractant/activation protein (NAP-1) release from human alveolar macrophages (AM) retrieved from normal nonsmokers was evaluated. LPS induced a dose-dependent release of LTB4 that began by 1 h, 4.0 +/- 3.2 ng/10(6) viable AM; peaked at 3 h, 24.7 +/- 13.5 ng/10(6) viable AM; and decreased by 24 h, 1.2 +/- 1.0 ng/10(6) viable AM (n = 8). Quantities of LTB4 in cell-free supernatants of AM stimulated with LPS were determined by reverse-phase high-performance liquid chromatography and corresponded well with results obtained by radioimmunoassay. By contrast, NAP-1 release began approximately 3-5 h after stimulation of AM with LPS, 197 +/- 192 ng/ml, and peaked at 24 h, 790 +/- 124 ng/ml. Release of NAP-1 was stimulus specific because A23187 evoked the release of LTB4 but not NAP-1, whereas LPS and zymosan induced the release of both LTB4 and NAP-1. The appearance of neutrophil chemotactic activity in supernatants of AM challenged with LPS for 3 h could be explained completely by the quantities of LTB4 present. After stimulation with LPS or zymosan for 24 h, AM had metabolized almost all generated LTB4. Preincubation of AM with nordihydroguiaretic acid (10(-4) M) completely abolished the appearance of NCA, LTB4, and NAP-1 in supernatants of AM challenged with LPS. Therefore, LPS and zymosan particles were potent stimuli of the sequential release of LTB4 and NAP-1 from AM.     

Release of leukotrienes, induced by the Ca++ ionophore A23187, from human lung parenchyma in vitro

When chopped human lung is stimulated with the Ca++ ionophore A23187 (0.25-10 microM) leukotriene (LT) B4, LTD4 and LTE4 are found in the incubation medium according to different patterns. LTD4 is released promptly and its levels increase up to 45 min after the stimulus (A23187, 10 microM) and decline later (120 min); LTE4 formation follows a sigmoidal shape and continues to accumulate even 2 hr after the challenge; LTB4 levels reach a plateau at 45 min. LTC4 was undetectable in most experiments but it was found to accumulate when reduced glutathione (10 mM) was added. Addition of exogenous LTC4 to unstimulated fragments of human lung shows that an effective interconversion to LTD4 and LTE4 takes place: A23187 stimulates formation of LT and cyclo-oxygenase products dose dependently; a statistically significant formation of LT occurs at A23187 concentration of 1 microM whereas thromboxane B2 (TXB2) and 6-K-prostaglandin F1 alpha levels increased significantly only at 2.5 microM A23187. Pretreatment with U-60257 (100 microM) prevented formation of LT without a concomitant increase of TXB2 levels. Indomethacin (1.5 microM) blocked the release of 6-K-prostaglandin F1 alpha and TXB2 without a shift of arachidonic acid towards LT-like activity. Addition of exogenous LTC4 did not trigger synthesis of TXB2 or 6-K-prostaglandin F1 alpha. Our results indicate that reduced glutathione levels and the activity of the enzymes involved in LT biosynthesis and/or metabolism play an important role in controlling the pattern of LT release from human lung.     

Isoproterenol-induced inhibition of immunoglobulin E-mediated release of histamine and arachidonic acid metabolites from the human lung mast cell

The inhibitory effect of isoproterenol was examined on the release of histamine, leukotriene C4 (LTC4), and prostaglandin D2 (PGD2) from human lung mast cells. Isoproterenol was more potent in inhibiting anti-immunoglobulin (Ig) E-induced LTC4 and PGD2 release than histamine release from the dispersed lung cell preparations (2-5% mast cells). The negative log molar EC50 values of isoproterenol for inhibiting histamine, LTC4 and PGD2 release were 8.2 +/- 0.2, 8.9 +/- 0.2 and 8.7 +/- 0.3, respectively (mean +/- S.E.M., n = 8). Isoproterenol seldom inhibited histamine release by more than 60%, but usually abolished the release of LTC4 and PGD2. The potency of propranolol (KB = 6 X 10(-10) M) was the same for competitively antagonizing isoproterenol mediated inhibition of histamine, LTC4 or PGD2 release. Using the irreversible beta adrenergic receptor antagonist, bromoacetylalprenololmenthane, the estimated dissociation constant of isoproterenol was 2 X 10(-7) M irrespective of whether inhibition of anti-IgE-induced histamine or LTC4 release was examined. When the divalent cation ionophore A23187 was used to induce histamine release, isoproterenol had either no effect or potentiated the release. In contrast, isoproterenol was capable of virtually abolishing A23187-induced LTC4 release. Qualitatively similar effects of isoproterenol were observed on purified mast cell preparations (greater than 80% mast cells); however, the potency of isoproterenol in inhibiting mediator release from these preparations was reduced. The results demonstrate that there is a substantial receptor reserve for isoproterenol-mediated inhibition of histamine, LTC4 and PGD2 release from the human lung mast cell. The intrinsic efficacy of isoproterenol in inhibiting anti-IgE-induced release of arachidonic acid metabolites is approximately 3 times greater than that for inhibiting histamine release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proinflammatory lipoxygenase products from peripheral mononuclear cells in patients with rheumatoid arthritis

The formation of 5-lipoxygenase products (5-hydroxyeicosatetraenoic acid [5-HETE], leukotriene B4 [LTB4], and leukotriene C4 [LTC4]) by polymorphonuclear and mononuclear leukocytes isolated from peripheral blood of patients with rheumatoid arthritis was evaluated and compared with the data obtained from a group of control subjects. Although the levels of arachidonic acid metabolites via 5-lipoxygenase pathway by stimulated polymorphonuclear cells were comparable between patients and controls, mononuclear leukocytes from patients synthesized, when stimulated, significantly greater amounts of 5-HETE, LTB4, and LTC4 than did cells isolated from normal subjects. In addition, the release of superoxide anion, stimulated by either a particulate or a soluble stimulus, was increased in mononuclear cells from patients. The enhanced capacity of peripheral mononuclear leukocytes isolated from patients with rheumatoid arthritis to generate proinflammatory metabolites of arachidonic acid and oxygenated species with bactericidal and tissue-damaging properties may contribute to the pathogenesis of this complex disease.     

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.     

Relative contribution of leukotriene B4 to the neutrophil chemotactic activity produced by the resident human alveolar macrophage

Human alveolar macrophages release chemotactic activity for neutrophils, providing a role for alveolar macrophages in regulating inflammation in the lung. As alveolar macrophages produce large amounts of leukotriene B4 (LTB4), a chemotactically active lipoxygenase product of arachidonic acid, we investigated the contribution of LTB4 to the total neutrophil chemotactic activity produced by these cells. Normal human alveolar macrophages were recovered by bronchoalveolar lavage from healthy volunteers and incubated either with the calcium ionophore A23187 for 1 h, or with opsonized zymosan particles or latex beads for 3 h. Nordihydroguaretic acid (NDGA), a relatively specific lipoxygenase inhibitor, blocked the release of neutrophil chemotactic activity after all three stimuli in a dose-dependent manner. This correlated with blockade of LTB4 production as measured by high performance liquid chromatography using freshly isolated alveolar macrophages, as well as blockade of [3H]LTB4 production by macrophages prelabeled with [3H]arachidonate. Molecular sieve chromatography using Sephadex G-50 confirmed that essentially all of the chemotactic activity in the stimulated macrophage supernatants co-eluted with authentic [3H]LTB4, and that NDGA completely blocked the chemotactic activity in the eluting fractions. Readdition of authentic LTB4 (1 X 10(-7) M) to the NDGA-blocked macrophage supernatants restored the chemotactic activity in the supernatants. The macrophage supernatants did not contain platelet-activating factor-like activity, as measured by the stimulation of [3H]serotonin release from rabbit platelets, and by high performance liquid chromatography. NDGA did not change the protein-secretion profiles of fresh alveolar macrophages, or of macrophages prelabeled with [35S]methionine. The complement (C) components C5adesarg were not detected in any of the supernatants by radioimmunoassay. Concentration of the supernatants by positive pressure filtration (5,000-D membrane) did not augment chemotactic activity in the stimulated supernatants or uncover chemotactic activity in the NDGA-blocked supernatants. As with the 3-h studies, when alveolar macrophages were incubated overnight with opsonized zymosan, all of the increase in chemotactic activity could also be blocked by NDGA. These data indicate that LTB4 is the predominant neutrophil chemotactic factor secreted by the normal resident human alveolar macrophage in response to two major types of stimuli, calcium fluxes across the cell membrane and the phagocytosis of opsonized particulates.     

Differential regulation of human basophil and lung mast cell function by adenosine.

Adenosine was found to modulate the activity of the human basophil and lung mast cell (HLMC) differently. In the basophil, adenosine inhibited the anti-IgE stimulated release of histamine and leukotriene C4 (LTC4) and increased total cell cyclic AMP (cAMP) levels. Substituted adenosine analogs had a rank order potency of: N-ethylcarboxamideadenosine (NECA) greater than 2-chloroadenosine greater than R-phenylisopropyladenosine for the inhibition of immunoglobulin E-triggered mediator release from the basophil and increases in cAMP levels. The adenosine receptor antagonist, 8-phenyltheophylline, antagonized both the NECA-induced inhibition of mediator release and elevations in cyclic nucleotide. The purinergic transport inhibitor, dipyridamole, reversed the inhibition by adenosine of histamine release but not LTC4 generation, suggesting that these two actions are mechanistically separable. Dipyridamole failed to modify the adenosine-induced elevation in cAMP. In contrast to the findings in the basophil, the response to adenosine in the HLMC was biphasic in nature. Thus, at low concentrations of the nucleoside, adenosine potentiated the release of histamine and LTC4 from immunologically activated HLMC, whereas at higher concentrations a counteractive inhibitory process was observed. Analogs of adenosine had the same effects on HLMC; NECA was more potent than R-phenylisopropyladenosine for both the potentiating and inhibitory components of the biphasic response. Low concentrations of adenosine analogs, which potentiated secretion, initiated modest elevations in cAMP levels, whereas higher concentrations, which inhibited secretion, significantly augmented cAMP levels. Although R-phenylisopropyladenosine was almost as potent as NECA at elevating cAMP in HLMC, it was not as efficacious. The NECA-induced modulation of HLMC mediator release and elevations in cAMP were antagonized by 8-phenyltheophylline.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histamine secretion from human skin slices induced by anti-IgE and artificial secretagogues and the effects of sodium cromoglycate and salbutamol

Human cutaneous mast cells show functional differences from their counterparts in other tissues. Following passive sensitization with 1% atopic serum for 30 min at 37 degrees C human skin slices released histamine after challenge with anti-human IgE in a concentration dependent manner. Maximum release of 14 +/- 2% was achieved with a 1/10 dilution of anti-IgE. Passive sensitization with 10% atopic serum increased the secretory response to anti-IgE but histamine release was only concentration related over the entire 1/1000 to 1/10 dilution range in half of the specimens studied, the remainder showing high dose tolerance to anti-IgE. Negligible histamine release occurred with anti-IgE challenge of slices which had not been passively sensitized. The histamine releasing ability of A23187 in human skin slices was similar to that observed in lung and adenoidal mast cells being concentration dependent over the range 0.1-3 microM with a maximum release of 25 +/- 3%. In contrast to human lung and adenoidal mast cells, poly-L-lysine and compound 48/80 induced histamine release from skin slices. Poly-L-lysine induced a concentration-dependent release of histamine over the range 0.01-10 microM with a maximum of 27 +/- 3%. The response to compound 48/80 was variable, releasing in some but not all specimens. Histamine release caused by anti-IgE, A23187 and poly-L-lysine was shown to be dependent upon extracellular calcium while release stimulated by compound 48/80 was calcium independent. The chemotactic peptide, formyl-methionyl-leucyl-phenylalanine, over the range 0.01-10 microM failed to release histamine from skin slices.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

ATP-dependent transport of leukotrienes B4 and C4 by the multidrug resistance protein ABCC4 (MRP4)

The proinflammatory mediators leukotriene (LT) B(4) and LTC(4) must be transported out of cells before they can interact with LT receptors. Previously, we identified the multidrug resistance protein ABCC1 (MRP1) as an efflux pump for LTC(4). However, the molecular basis for the efflux of LTB(4) was unknown. Here, we demonstrate that human ABCC4 mediates the ATP-dependent efflux of LTB(4) in the presence of reduced glutathione (GSH), whereby the latter can be replaced by S-methyl GSH. Transport studies were performed with inside-out membrane vesicles from V79 fibroblasts and Sf9 insect cells that contained recombinant ABCC4, with vesicles from human platelets and myelomonocytic U937 cells, which were rich in endogenous ABCC4, but ABCC1 was below detectability. Moreover, human polymorphonuclear leukocytes contained ABCC4. K(m) values for LTB(4) were 5.2 muM with vesicles from fibroblasts and 5.6 muM with vesicles from platelets. ABCC4, with its broad substrate specificity, also functioned as an ATP-dependent efflux pump for LTC(4) with a K(m) of 0.13 muM in vesicles from fibroblasts and 0.32 muM in vesicles from platelets. However, GSH was not required for the transport of this glutathionylated leukotriene. The transport of LTC(4) by ABCC4 explains its release from platelets during transcellular synthesis. ATP-dependent transport of LTB(4) and LTC(4) by ABCC4 was inhibited by several organic anions, including S-decyl GSH, sulindac sulfide, and by the LTD(4) receptor antagonists montelukast and 3-(((3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)-((3-dimethyl-amino-3-oxopropyl)-thio)-methyl)thio)propanoic acid (MK571). Thus, as an efflux pump for the proinflammatory mediators LTB(4) and LTC(4), ABCC4 may represent a novel target for anti-inflammatory therapies.     

Defective histamine release in chronic urticaria.

Histamine release from peripheral blood leukocytes challenged with anti-human IgE was studied in patients with chronic urticaria and nonatopic controls. 19 of 23 controls, but only 6 of 20 patients, released over 20% of the total available leukocyte histamine. The response to anti-IgE concentrations of 1.66, 0.33, 0.066, and 0.013 mug antibody N/ml was significantly lower in patients than in controls. Serum IgE levels were significantly higher in the patients but total histamine content of about 10(7) leukocytes was not. Deuterium oxide (D2O) greatly increased histamine release (in both groups), indicating that the anti-IgE interacted with the basophils of urticaria patients. Passive sensitization of leukocytes with biologically active IgE was achieved in both patients and control subjects whose cells responded to anti-IgE, but was not achieved in either patients or control subjects whose cells were nonresponsive to anti-IgE challenge. 125I-anti-IgE autoradiographic studies revealed no obvious quantitative abnormality in the amount of basophil-bound IgE in chronic urticaria patients. Ionophore stimulation of aliquots of the same leukocytes used for anti-IgE challenge demonstrated that the urticaria patients'' basophils were capable of releasing normal amounts of histamine. Leukocyte cyclic AMP levels in the two groups were not significantly different either in base-line levels or in responsiveness to stimulation with isoproterenol. These data indicate that chronic urticaria patients have a (acquired?) defect in leukocyte histamine release that occurs after the anti-IgE-IgE interaction, but before the actual (second-stage) release process, and that is comparable to the phenomenon of desensitization.     

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.     

Multiple actions of the leukotriene B4 receptor antagonist SC-41930.

7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8- propyl-2H-1-benzopyran-2-carboxylic acid (SC-41930), a leukotriene B4 (LTB4) receptor antagonist with anti-inflammatory activity in animal models of colitis, was evaluated for effects on superoxide, LTB4 and prostaglandin E2 production. SC-41930 inhibited human neutrophil (PMN) superoxide generation maximally stimulated by f-Met-Leu-Phe (IC50 4 microM) and C5a (IC50 approximately 12 microM). Moreover, postreceptor stimulation of superoxide production by NaF (a G protein activator), but not by phorbol myristate acetate, was significantly inhibited by SC-41930, indicating that SC-41930 may act via attenuation of a G protein-mediated signal transduction. SC-41930 also inhibited A23187-stimulated LTB4 production (IC50 5.3 microM) in human PMN as well as LTB4 (IC50 2.1 microM) and prostaglandin E2 (IC50 2.9 microM) production in HL-60 cells. When coinjected intradermally (400 micrograms/site), SC-41930 inhibited A23187-stimulated increases in LTB4 levels in guinea pig skin. SC-41930 inhibited human synovial phospholipase A2 (IC50 72 microM), A23187-stimulated 5-hydroxy-eicosatetranoic acid production in human PMN (IC50 8.5 microM), and rat peritoneal leukotriene A4 hydrolase (IC50 20 microM), but not ram seminal vesical cyclooxygenase. The results suggest that the anti-inflammatory activity of SC-41930 could be attributed to postreceptor inhibition of inflammatory mediator production by PMN and other cells in addition to antagonism of PMN LTB4 receptors.     

Effect of filtration and storage of platelet concentrates on the production of the chemotaxins C5a, interleukin 8, tumor necrosis factor alpha, and leukotriene B4

BACKGROUND: The production in platelet concentrates (PCs) of C3 activation products (C3bc), terminal complement complex (TCC), and chemotaxins C5a, interleukin (IL)-8, tumor necrosis factor alpha (TNFalpha), and leukotriene B4 (LTB4) and the proposed reduction in concentration of the chemotaxins by white cell reduction were examined. STUDY DESIGN AND METHODS: Samples were collected from supernatants of PCs produced by apheresis (apheresis PCs) or from buffy coats (BC PCs) immediately after the production, after white cell-reduction filtration on Day 1, and after 5-day storage, and examined by enzyme immunoassays. RESULTS: Complement was activated in all PCs during storage, and the concentration of activation products was not influenced by prestorage filtration. In prestorage white cell-reduced BC PCs, only C3bc levels increased. Levels of IL-8, TNFalpha, and LTB4 increased during storage of apheresis PCs, but not in filtered units, except for LTB4. In contrast, levels of IL-8 decreased after storage of filtered BC PCs. C5a correlated significantly with IL-8, which also correlated with TNFalpha and LTB4. CONCLUSION: Both C5a and TNFalpha generation in apheresis PCs seem to induce white cell IL-8 production, which mediates cellular LTB4 release. Prestorage white cell reduction is recommended for reducing chemotactic cytokine and leukotriene levels in all PCs. Production of BC PCs is recommended to achieve less complement activation, which is not affected by filtration.