Cysteinyl-leukotrienes and their receptors in asthma and other inflammatory diseases: critical update and emerging trends.

Cysteinyl-leukotrienes (cysteinyl-LTs), that is, LTC4, LTD4, and LTE4, trigger contractile and inflammatory responses through the specific interaction with G protein-coupled receptors (GPCRs) belonging to the purine receptor cluster of the rhodopsin family, and identified as CysLT receptors (CysLTRs). Cysteinyl-LTs have a clear role in pathophysiological conditions such as asthma and allergic rhinitis (AR), and have been implicated in other inflammatory conditions including cardiovascular diseases, cancer, atopic dermatitis, and urticaria. Molecular cloning of human CysLT1R and CysLT2R subtypes has confirmed most of the previous pharmacological characterization and identified distinct expression patterns only partially overlapping. Interestingly, recent data provide evidence for the immunomodulation of CysLTR expression, the existence of additional receptor subtypes, and of an intracellular pool of CysLTRs that may have roles different from those of plasma membrane receptors. Furthermore, genetic variants have been identified for the CysLTRs that may interact to confer risk for atopy. Finally, a crosstalk between the cysteinyl-LT and the purine systems is being delineated. This review will summarize and attempt to integrate recent data derived from studies on the molecular pharmacology and pharmacogenetics of CysLTRs, and will consider the therapeutic opportunities arising from the new roles suggested for cysteinyl-LTs and their receptors.

A patient with neurological symptoms and abnormal leukotriene metabolism: a new defect in leukotriene biosynthesis.

A 15-year-old male patient presented with mental retardation, mild motor impairment, and partial deafness. Biochemical investigations showed an abnormal urinary profile of leukotrienes. Concentration of leukotriene D(4) (LTD(4)), which is usually not detectable, was highly increased, whereas LTE(4), the major urinary metabolite in humans, was completely absent. These data suggest membrane-bound dipeptidase deficiency, a new defect in leukotriene biosynthesis on the step of LTE(4) synthesis, as underlying defect.

Roflumilast inhibition of pulmonary leukotriene production and bronchoconstriction in ovalbumin-sensitized and -challenged Guinea pigs.

This study investigated the effects of roflumilast, a PDE4 inhibitor, on slow-reacting substance of anaphylaxis (SRS-A)-mediated bronchoconstriction and pulmonary leukotriene (LT) release in ovalbumin (OVA)-sensitized and -challenged guinea pigs. Animals were treated with roflumilast orally (0.04, 0.12, 0.4, or 4 mg/kg) or placebo 1 hour before OVA challenge. Bronchoconstriction was quantified by measuring airway conductance (Gaw) and dynamic lung compliance (Cdyn). Roflumilast significantly attenuated the decrease in Gaw (50% inhibitory dose [ID50] = 0.33 mg/kg) and Cdyn (ID50 = 0.25 mg/kg) in a dose-dependent manner and significantly inhibited Cys-LT (ID50 = 0.06 mg/kg) and LTB4 (ID50 = 0.05 mg/kg) release versus placebo-treated animals. Roflumilast did not affect LTD4-induced bronchoconstriction. These findings support the role of roflumilast as an anti-inflammatory treatment for asthma.

Autocrine regulation of cord blood-derived human mast cell activation by IL-10.

BACKGROUND: Ligation of the high-affinity receptor for IgE on human mast cells (MCs) induces the release of proinflammatory mediators, including vasoactive amines and cytokines (TNF-alpha, IL-5, and IL-8). Moreover, we have recently shown that IL-10 inhibits the release of proinflammatory mediators by activated MCs. OBJECTIVE: We investigated whether human cord blood-derived MCs (CBMCs) could produce IL-10 and whether this production could inhibit their activation in an autocrine fashion. METHODS: IL-10 synthesis by resting or activated human MCs derived from cord blood progenitors was investigated in cell supernatants or by using immunostaining and RT-PCR methods. In addition, the effect of IL-4 on such synthesis was also studied. Anti-IL-10-neutralizing antibodies were used to investigate the validity of the hypothesis of an autocrine regulation of MCs by IL-10. Finally, the presence of specific receptors for IL-10 was searched on human CBMCs by using flow cytometric analysis. RESULTS: Human CBMCs spontaneously synthesize and release IL-10, and this synthesis is increased after IgE/anti-IgE stimulation. In addition, the presence of IL-10 in resting or in activated MCs was proved by immunostaining. Interestingly, the release of IL-10 was also increased after incubation of the cells with IL-4. Besides, the use of neutralizing antibodies against IL-10 confirmed that IL-10 released inhibited MC activation in an autocrine fashion. Finally, the presence of specific receptors for this cytokine was observed on the membranes of our population of human CBMCs. CONCLUSION: Taken together, our data are in favor of an autocrine regulation pathway through synthesis and release of IL-10 by human MCs. Such an autoregulatory mechanism is, to our knowledge, the first described for these elements.

Cysteinyl leukotrienes are involved in angiotensin II-induced contraction of aorta from spontaneously hypertensive rats.

OBJECTIVE: Non specific lipoxygenase inhibitors have been reported to reduce the in vitro constrictor response and the in vivo pressor effect of angiotensin II in rats. The aim of this study was to assess the role of cysteinyl leukotrienes, in the vascular response to angiotensin II in spontaneously hypertensive rats (SHR). METHODS: Rings of thoracic aorta from SHR and normotensive Wistar-Kyoto rats (WKY) were compared in terms of contractile responses and release of cysteinyl leukotrienes in response to angiotensin II. RESULTS: Pretreatment with the specific 5-lipoxygenase inhibitor AA861 10 microM reduced the efficacy of angiotensin II in intact and endothelium-denuded aorta from SHR (% inhibition vs. control: 65+/-12.6% with endothelium (n=6), P<0.05; 43+/-7.2% without endothelium (n=6), P<0.05) but not in aorta from WKY. In addition, in aorta from SHR, the CysLT(1) receptor antagonist MK571 1 microM reduced by 55+/-6.1% (n=6, P<0.05) the contractile effects of angiotensin II in rings with endothelium but not in endothelium-denuded rings. Angiotensin II induced a 8.6+/-2.1-fold increase in cysteinyl leukotriene production in aorta rings from SHR with endothelium which was prevented by the AT(1) receptor antagonist losartan 1 microM but not by the AT(2) receptor antagonist PD123319 0.1 microM. In aorta rings from WKY, cysteinyl leukotriene production remained unchanged after exposition to angiotensin II. The cysteinyl leukotrienes (up to 0.1 microM) induced contractions in aorta rings from SHR but not from WKY. CONCLUSIONS: These data suggest that cysteinyl leukotrienes, acting at least in part on endothelial CysLT(1) receptors, are involved in the contractile response to angiotensin II in isolated aorta from SHR but not from WKY.

Leukotriene C4 synthase: a critical enzyme for the biosynthesis of SRS-A.

Leukotriene (LT) C4 synthase catalyzes the conjugation of LTA4 with reduced glutathione (GSH) to form LTC4, the parent compound of cysteinyl leukotrienes. It is a 18 kDa protein that functions as homodimer. Cloning of LTC4 synthase cDNA reveals amino acid homology with 5-lipoxygenase activating protein (FLAP) and newly identified microsomal glutathione S-transferase II (mGST-II) but not with cytosolic GSTs or mGST-I. LTC4 synthase gene contains 5 exons and four introns. This gene has been localized to the long arm of human chromosome 5 at the region of 5q35 which is in close proximity to the cluster of genes that are involved in inflammation and asthma. Mutagenic studies reveals that amino acid residues Arg-51 and Tyr-93 are critical for catalytic function. Arg-51 was proposed to open the epoxide ring of LTA4 and Tyr-93 to provide the thiolate anion of GSH.

Alteration of n-3 fatty acid composition in lung tissue after short-term infusion of fish oil emulsion attenuates inflammatory vascular reaction.

OBJECTIVES: To investigate whether modulation of the fatty acid profile can be achieved by the short-term infusion of a fish oil emulsion which may attenuate the pulmonary response to inflammatory stimulation. Changes of fatty acid pattern in-lung tissue and perfusate were analyzed and correlated with physiologic data after a 3-hr infusion of fish oil in comparison with a soybean oil preparation. DESIGN: Prospective, randomized, controlled trial. SETTING: Experimental laboratory in a university teaching hospital. SUBJECTS: Forty standard breed rabbits of either gender. INTERVENTIONS: Isolated lungs from anesthetized rabbits were ventilated and recirculation-perfused (200 mL/min) with 200 mL of cell-free buffer solution to which either 2 mL of saline (control, n = 6), 2 mL of a 10% soybean oil preparation (n = 6), or 2 mL of a 10% fish oil emulsion (n = 6) were added. Samples of perfusate and lung tissue were collected for analysis of fatty acid composition. Tissue and perfusate fatty acid composition were analyzed by capillary gas chromatography. To study metabolic alterations in states of inflammatory stimulation, lungs of each group were stimulated with small doses of the calcium ionophore, A23187 (10(-8) M), during the 180-min lipid perfusion period and again after washing out the lipids by exchanging the perfusion fluid. Pulmonary arterial pressure and lung weight gain were monitored, and eicosanoids were analyzed in the perfusate. MEASUREMENTS AND MAIN RESULTS: Free eicosapentaenoic acids increased several-fold in lung tissue and perfusate during a 3-hr infusion with fish oil. The intravenously administered n-3 fatty acids were rapidly hydrolyzed, as indicated by the appearance of substantial quantities of eicosapentaenoic acid in the perfusate free fatty acid fraction. This increase of perfusion levels of eicosapentaenoic acid was paralleled by an attenuated pressure increase and edema formation due to calcium ionophore challenge and an altered eicosanoid spectrum determined in the perfusate compared with soybean oil-treated lungs. CONCLUSION: Short-term n-3 lipid application (fish oil emulsion) exerts anti-inflammatory effects on lung vasculature, which may be due to the metabolism of eicosapentaenoic acid resulting in the generation of less potent inflammatory eicosanoids.

[Effect of tremulacin on actions of SRS-A and histamine].

The effect of tremulacin (TRC) extracted from Mao Bai Yang (Folia Populus tomentosa Carr) on actions of SRS-A and histamine were investigated by using isolated guinea pig ileum and spectrofluorometric assay. TRC was found to inhibit the contraction of isolated guinea pig ileum induced by histamine and SRS-A, in a dose-dependent manner with IC50 of 1.78 x 10(-4) mol.L-1 and 2.51 x 10(-4) mol.L-1, respectively. TRC at the dose of 10(-4) mol.L-1 inhibited SRS-A release from sensitized isolated guinea pig lung. While at the dose of 10(-5) mol.L-1 inhibited histamine release from the peritoneal mast cells in sensitized rats. These results indicate that inhibition of the release of histamine and SRS-A may play an important role in the mechanism of antiinflammatory actions of TRC.

Phorbol ester-induced leukotriene biosynthesis and tumor promotion in mouse epidermis.

In mouse skin in vivo the irritant and hyperplasiogenic tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) strongly increased the epidermal content of the cysteinyl leukotrienes LTC4, LTD4 and LTE4, but not of leukotriene LTB4. This effect was completely suppressed by the selective leukotriene biosynthesis inhibitor MK-886. Intragastric administration of MK-886 prevented phorbol ester-induced ear edema, but not epidermal hyperproliferation and tumor promotion. These data indicate that leukotrienes are involved in the pro-inflammatory effects of the phorbol ester, whereas its hyperproliferative and tumor-promoting activities do not depend on 5-lipoxygenase-catalyzed leukotriene formation. This action differs from several non-selective inhibitors of lipoxygenases that were found to inhibit tumor promotion in initiated mouse skin.

Inhibition of human IgE synthesis by anti-IgE antibodies requires divalent recognition.

We used a selection of well-characterized murine monoclonal anti-IgE antibodies to investigate their effect on human in vitro IgE synthesis. We found anti-IgE antibodies that either inhibited or enhanced interleukin-4 plus anti-CD40-induced in vitro IgE synthesis in peripheral blood mononuclear cells (PBMC). This differential activity was isotype specific as neither IgM nor IgG synthesis were affected. Interestingly, only coding IgE mRNA was down-regulated, whereas germ-line epsilon RNA expression was not influenced by anti-IgE monoclonal antibody (mAb). On purified B cells all anti-IgE mAb inhibited interleukin-4 plus anti-CD40-induced IgE synthesis, implying a role of non-B cells for the enhancing activity observed in PBMC. Using Fab and F(ab')2 of an inhibitory anti-IgE mAb we could show that divalent recognition was required for inhibition of IgE synthesis.

Simplified sulfidoleukotriene ELISA using LTD4-conjugated phosphatase for the study of allergen-induced leukotriene generation by isolated mononuclear cells and diluted whole blood.

By using leukotriene D4 (LTD4)-labeled alkaline phosphatase (LTD4-AP) and a mouse monoclonal anti-sulfidoleukotriene (sLT) antibody (1A-LDR1), we developed a solid-phase competition ELISA for sLTs. The detection limit of this assay was 6.3 +/- 1.2 pg sLT/100 microliters (mean +/- SEM; n = 10). Intraassay variations at 15, 50 and 150 pg sLT/well (9.8, 13.5 and 12.3%, respectively), the corresponding interassay variations (32.1, 11.9 and 18.8%, respectively) and the good correlation with a commercial radioimmunoassay (r = 0.97; n = 43) confirmed the validity of this ELISA. Furthermore, the assay had a low background and gave a 70-90% recovery of LTD4 added to medium containing up to 10% serum. In contrast to another sLT ELISA using the same antibody but based on competition with solid-phase LTE4-BSA, our assay is about 7-fold more sensitive. It also requires about 40 times less leukotriene for conjugate production than the previous method for the same number of determinations. The assay allows measurement of sLTs generated by small numbers of basophils present in isolated mononuclear cells or diluted whole blood in response to allergen, and allows simple and rapid determination of basophil reactivity to suspected allergens.

Regulation of human lung mast cell function by phosphatase inhibitors.

Okadaic acid, a cell-permeant inhibitor of serine/threonine phosphatases (PP) was found to attenuate the IgE-mediated release of both performed (histamine) and newly formed (leukotriene C4) mediators from human lung mast cells (HLMC). Optimal inhibition (82 +/- 6%) of the IgE-triggered release of histamine was observed after a 2-h incubation of HLMC with okadaic acid (1 microM). Shorter (< 2 h) incubation times and lower (< 1 microM) concentrations of okadaic acid were less effective at inhibiting histamine release from HLMC. The extent to which okadaic acid prevented histamine release was highly dependent upon the strength of the stimulus with lower levels of stimulation more readily modulated. The efficacy of okadaic acid as an inhibitor was not dependent upon the nature of the stimulus because matched levels of histamine release (approximately 30%) induced by either IgE-mediated processes or the calcium ionophore A23187 were attenuated to comparable extents. A series of analogues of okadaic acid and a structurally-distinct PP inhibitor, calyculin, were also found to inhibit the IgE-induced release of mediators from HLMC with the following rank order of potency; calyculin (approximate IC50; 0.015 microM) > okadaic acid (0.2 microM) > okadaol (3.3 microM) > nor-okadaone (> 10 microM). These same PP inhibitors displayed a similar rank order of activity for the inhibition of mediator release induced by the ionophore A23187. Extracts of purified HLMC were found to liberate 32P from radiolabeled glycogen phosphorylase and this PP activity was inhibited by both low (2 nM) and high (5 microM) concentrations of okadaic acid suggesting that HLMC contain both PP type 2A (PP2A) and PP type 1 (PP1). The okadaic acid, analogues of okadaic acid, and calyculin attenuated mediator release at concentrations and with a rank order of activity that closely parallels their activities as inhibitors of PP suggests that PP are important in the regulation of HLMC function. This contention is further supported by the finding that PP activities are constitutively associated with HLMC and that these activities could be inhibited by okadaic acid.

Rat hepatocytes generate peptide leukotrienes from leukotriene A4.

The biosynthetic mechanism of peptide leukotrienes was studied in rat liver. A catalytic activity to synthesize leukotriene (LT) C4 from LTA4 (LTC4 synthesis activity) was shown to be associated mainly with the microsomal fraction of hepatocytes, but also to a small extent with nonparenchymal cells including Kupffer cells, suggesting that the hepatocytes have an ability to generate peptide LTs. Stimulation of the isolated hepatocytes with calcium ionophore (A23187) did not cause any significant production of peptide LTs, whereas addition of LTA4 induced a remarkable generation of peptide LTs in a dose-dependent manner. Addition of LTA4 also augmented the peptide LT production by Kupffer cells, but its amount was much smaller than that by the hepatocytes under the same culture conditions. Coincubation of the hepatocytes and Kupffer cells, following A23187 stimulation, elicited a markedly enhanced production of peptide LTs even without any addition of LTA4, whereas the LT production in the coincubation system was suppressed almost completely by treating the Kupffer cells with a specific inhibitor of 5-lipoxygenase, AA861 (10 microM). An enzymatic cooperation between the hepatocytes and Kupffer cells may play an important role in generating peptide LTs in the liver.

Alteration of eicosanoid production in the sensitized guinea pig lung by CS-518.

The effects of CS-518 (sodium 2-(1-imidazolylmethyl)-4,5-dihydrobenzo [b]thiophene-6-carboxylate), a thromboxane A2 synthase inhibitor, on changes in arachidonic acid metabolism were investigated in the lung of actively sensitized guinea pigs. Antigen challenge enhanced the production of thromboxane A2 as well as histamine and peptide leukotrienes in lung fragments. Exogenous leukotriene D4 also stimulated significant thromboxane A2 production in the non-sensitized lung in vitro. CS-518 was effective in preventing the thromboxane A2 production induced by either antigen or leukotriene D4, and the IC50 values were 90 and 7.5 ng/ml (320 and 27 nM), respectively. CS-518 markedly potentiated the production of prostaglandin E2 and I2 with slight inhibition of leukotriene formation, but indomethacin significantly stimulated leukotriene production. When CS-518 was administered orally, it induced long-lasting inhibition of thromboxane A2 production and potentiation of prostaglandin I2 production in guinea pig lung. Thus, CS-518 not only inhibited thromboxane production but also improved the change in arachidonic acid metabolism in the guinea pig bronchoalveolar tissue during allergic reaction in vivo as well as in vitro, which suggests amelioration of the asthmatic condition.

Enhanced gastric mucosal leukotriene B4 synthesis in patients taking non-steroidal anti-inflammatory drugs.

The effects of longstanding non-steroidal anti-inflammatory drug (NSAID) treatment on gastric mucosal synthesis of leukotriene B4 (LTB4), leukotriene C4 (LTC4), and prostaglandin E2 (PGE2) was studied. Gastric antral biopsies in 65 patients with arthritis taking NSAIDs and 23 control patients were taken and eicosanoid concentrations, stimulated by vortex mixing or calcium ionophore, were measured by radioimmunoassay. Median gastric mucosal synthesis of LTB4 was increased in patients taking NSAIDs compared with non-users: (0.9(0.2-2.5) pg/mg v 0 (0-0.6) pg/mg (p < 0.001)). These differences persisted when subgroups of patients were analysed according to Helicobacter pylori colonisation or degree of mucosal injury. Synthesis of LTB4 was strongly associated with the presence of type C (chemical) gastritis. Increased synthesis of LTC4 was associated with Helicobacter pylori colonisation but not NSAID use. Synthesis of PGE2 was decreased in patients taking NSAIDs compared with control patients (p < 0.001). Enhanced gastric mucosal synthesis of LTB4 in patients taking NSAIDs may represent a primary effect of these drugs and could be implicated in the pathogenesis of gastritis and ulceration associated with NSAIDs.

Cellular and chemical mediators of type 1 hypersensitivity in calves infected with Ostertagia ostertagi: histamine, prostaglandin D2, prostaglandin E2 and leukotriene C4.

Plasma histamine, prostaglandin E2 (PG) D2, PGE2, and leukotriene (LT) C4 levels were determined in 26 Holstein steers before and after natural or experimental infection with Ostertagia ostertagi. Post-infection abomasal lymph was also assayed for PGD2, PGE2, and LTC4. Histamine determinations were performed on abomasal tissue from three locations. Results showed that: (1) tissue histamine levels increased in steers with type 2 ostertagiosis, (2) lymphatic PGD2 and PGE2 levels were increased, probably as a result of macrophage activity, (3) lymphatic LTC4 levels increased in steers with type 1 ostertagiosis, and were correlated with tissue eosinophilia, and (4) plasma levels of PGD2, PGE2, LTC4 and histamine were not useful for predicting worm burdens. These findings suggest a functional role for eosinophils and mast cells in the pathophysiology of ostertagiosis, through mediation of a type 1 hypersensitivity reaction.

A prodrug of a 2,6-disubstituted 4-(2-arylethenyl)phenol is a selective and orally active 5-lipoxygenase inhibitor.

BI-L-226, a 2,6-disubstituted 4-(2-arylethenyl)phenol, is a potent and selective 5-lipoxygenase inhibitor which shows excellent inhibition of antigen-induced leukotriene generation in the lung of cynomolgus monkeys by aerosol administration, although little activity has been observed by the p.o. route. The facile synthesis of the succinate ester BI-L-357, however, results in a prodrug which has p.o. activity between 10 to 30 mg/kg in an ex vivo whole blood model of leukotriene B4 generation in both squirrel and cynomolgus monkeys. In addition, the prodrug is effective in inhibiting pulmonary leukotriene C4 production in antigen-challenged cynomolgus monkeys in the same dose range. Plasma levels of the parent compound in the monkey after p.o. administration of 30 mg/kg are 25-fold higher than the IC50 needed for in vitro inhibition of leukotriene B4 in whole blood. Absolute bioavailability of the parent compound was 50%. The prodrug concept therefore extends the potential of this class of compounds to inflammation sites mediated by 5-lipoxygenase not readily treated by topical administration.

Inter-relationships between inflammatory mediators released from colonic mucosa in ulcerative colitis and their effects on colonic secretion.

Metabolites of arachidonic acid have been implicated in the pathophysiology of ulcerative colitis-they can stimulate intestinal secretion, increase mucosal blood flow, and influence smooth muscle activity. The influence on the mucosal transport function of culture medium in which colonic mucosal biopsy specimens had been incubated was investigated using rat stripped distal colonic mucosa in vitro as the assay system. Colonic tissue from patients with colitis and from control subjects was cultured. Medium from inflamed tissue contained more prostaglandin E2 (PGE2) and leukotriene D4 (LTD4) and evoked a greater electrical (secretory) response in rat colonic mucosa than control tissue medium. In inflamed tissue, cyclo-oxygenase inhibition (indomethacin) attenuated PGE2 but increased LTD4 production; conversely lipoxygenase inhibition (ICI 207968) inhibited LTD4 production but enhanced PGE2 output. Each inhibitor alone enhanced the electrical response in the rat colon. Inhibition of both enzymes (indomethacin plus ICI 207968) caused a fall in both PGE2 (82%) and LTD4 (89%) production and in the electrical response (57%). Inflamed tissue treated with a phospholipase A2 inhibitor (mepacrine) produced less PGE2, LTD4, and electrical responses when compared with inflamed tissue, either untreated (91%, 92%, and 79% respectively) or treated with cyclo-oxygenase and lipoxygenase inhibition. Incubation with bradykinin stimulated eicosanoid release and electrical response, while a bradykinin antagonist caused a modest inhibition. Analysis of these observations suggests that a combination of arachidonic acid derivatives accounts for about half the secretory response. Other products of phospholipase A2 activity are probably responsible for much of the remainder, leaving up to 20% the result of types of mediator not determined in this study.

Effects of exogenous eicosapentaenoic acid on generation of leukotriene C4 and leukotriene C5 by calcium ionophore-activated human eosinophils in vitro.

Exogenous eicosapentaenoic acid (EPA) has been compared with exogenous arachidonic acid (AA) for its ability to modulate the oxidative metabolism of membrane-derived arachidonic acid by the 5-lipoxygenase pathway in ionophore-activated human eosinophils, and for its suitability as a parallel substrate in this pathway. Products were quantitated by specific RIA and tetraene and pentaene leukotrienes (LT) were separated by reverse-phase HPLC. Eosinophils were preincubated with control buffer, exogenous EPA or AA and stimulated optimally with 10 microM calcium ionophore (A23187) for 15 min. Mean generation of LTC4 in the absence of fatty acid was 6.0 = 1.1 ng/10(6) eosinophils (mean = SEM, n = 5). In the presence of EPA, the amount of LTC4 generated rose to peak at 16.5 +/- 1.9 ng/10(6) eosinophils at 10 micrograms/ml EPA and then fell to 8.3 +/- 3.1 ng/10(6) cells at 40 micrograms/ml EPA. The EPA derivative, LTC5 was first detectable at 5 micrograms/ml EPA with 4.8 +/- 1.2 ng/10(6) cells and gradually rose with increasing dose of EPA to be maximal at 40 micrograms/ml with 12.7 +/- 2.2 ng/10(6) cells. Identity of the LTC5 was confirmed by an identical retention time to synthetic LTC5 standard, immunoreactivity to a specific antibody against LTC4 and LTC5 and a typical UV absorbance spectrum. When eosinophils were preincubated with AA and similarly stimulated, LTC4 generation gradually increased from a baseline of 6.7 +/- 0.7 ng/10(6) cells in the absence of fatty acid to reach a maximum of 12.9 +/- 0.8 ng/10(6) cells at 40 micrograms/ml of AA. Total LTC generation was nearly twofold more with cells incubated with EPA than with cells incubated with AA (p < 0.05). Thus, EPA does not suppress LTC generation from eosinophils but stimulates it at lower doses and is a substrate for LTC5 generation.

Lack of increased numbers of low-density eosinophils in the circulation of asthmatic individuals.

The density distribution pattern of eosinophils over discontinuous isotonic Percoll gradients from the blood of normal, asymptomatic allergic and non-allergic asthmatic individuals was investigated. There was a completely identical distribution pattern between the investigated groups. Analysis of the expression of surface markers for complement receptors CR1 and CR3 and immunoglobulin G receptor on eosinophils derived from the density bands 1.080, 1.085 and 1.090 g/ml supported this finding since they did not reveal differences in expression between the bands within one group but also not between the three groups. Eosinophils of the various density bands were further purified and stimulated in vitro to produce leukotriene C4 (LTC4) by the calcium ionophore A23187 or serum treated zymosan. Equal amounts of LTC4 were synthesized by the eosinophils of the various density bands within one group. However, it appeared that the eosinophils of all density bands of allergic and non-allergic asthmatics synthesized significantly more LTC4 than the eosinophils from normal individuals (five- to tenfold). Probably this indicates in vivo priming of the eosinophils in asthmatic individuals which is not reflected by a change in density. Control experiments, dealing with possible artifacts due to the isolation procedure or the patient selection, to find differences in distribution patterns over discontinuous Percoll density gradients of the eosinophils of asthmatic compared to normal individuals failed to show such a difference. Therefore, the density distribution pattern of eosinophils over these gradients does not reflect cell activation, whereas LTC4 formation clearly does. This could mean that LTC4 formation is a more sensitive parameter for cell activation than density distribution or cell surface marker expression.