15-Hydroxyeicosatetraenoic Acid Inhibits Superoxide Anion Generation by Human Neutrophils: Relationship to Lipoxin Production

Human neutrophils can aggregate, degranulate, and release mediators of inflammation including oxygen radicals and lipoxygenase (LO)-derived products of arachidonic acid. The regulation of 5– and 15-lipoxy-genases appears to be important since their products (e.g. leukotrienes and lipoxins) display unique spectra of bioactions. Addition of 15-HETE. a product of the 15-LO, to neutrophils in suspension dramatically shifted the LO products generated and led to a dose-dependent increase in lipoxins, while the production of leukotriene B₄ and its μ-oxidation products (i.e. 20-COOH-LTB₄ and 20-OH-LTB₄) was inhibited. Exogenous 15-HETE also dose-dependently inhibited the generation of superoxide anions induced by either the chemotactic peptide f-met-leu-phe or the divalent cation ionophore A23187. Neither lipoxin A, nor lipoxin B₄ (10^?8?10^?6M) inhibited O₂^?_? generation induced by either f-met-leu-phe or A23187. These results indicate that in addition to serving as a substrate for lipoxin generation, 15-HETE also inhibits superoxide anion generation by human neutrophils. Together they provide further evidence to suggest that products of the 15-lipoxygenase may serve a regulatory role at inflammatory loci.     

Leukotriene B4 biosynthesis by alveolar macrophages

Resting alveolar macrophages in culture synthesized small amount of leukotriene B₄. This synthesis was increased 2.5 fold following phagocytic stimulation by zymosan, and was increased 12.6 fold after stimulation with calcium and calcium ionophore A23187. The leukotriene B₄ synthesis could be completely inhibited by nordihydroguaiaretic acid (10^?5M). Phorbol myristate acetate, a membrane perturbant, has no effect on leukotriene B₄ production by macrophages.     

6,9-deepoxy-6,9,-(phenylimino)-delta 6,8-prostaglandin I1, (U-60,257), a new inhibitor of leukotriene C and D synthesis: in vitro studies

Addition of the calcium inophore, A 23187, and cysteine to isolated mononuclear cells from rat peritoneal washings causes a marked increase in the formation of thromboxane B2 (TxB2) along with the formation of leukotrienes C and D (LT's). The formation of LT's in this system was inhibited by 6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin I1, U-60,257, or its methyl ester, U-56,467, (ID50 4.6 and 0.31 microM, respectively). There was no inhibition of TxB2 formation. By contrast, two structurally-related compounds, PGI2 and its stable analog, 6-beta-PGI1, did not affect the formation of either LT's or TxB2. The inhibition of LT formation by U-60,257 was rapidly reversed after removal of this compound from the cells. U-60,257 did not inhibit the cyclooxygenase of human polymorphonuclear leukocytes. Nor did it inhibit formation of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) in human platelets. On the other hand, U-60,257 inhibited glutathione S-transferase activity of rat basophil leukemia cells (ID50, 37 microM), suggesting that this compound may inhibit the last step in LTC biosynthesis. In addition to inhibiting LT synthesis, U-60,257 also appears to be a competitive inhibitor of the action of LT on the guinea pig ileum, although this inhibition requires a higher drug concentration than those ordinarily encountered during assay for LT's in U-60,257-treated incubations.     

A possible requirement for arachidonic acid lipoxygenation in the mechanism of phagocytic degranulation by human neutrophils stimulated with aggregated immunoglobulin G

Aggregated immunoglobulin G (AggIgG) caused a concentration-dependent extracellular release of granule-associated lysozyme and myeloperoxidase (MPO) from human neutrophils. Generation of the 5-lipoxygenase product of arachidonic acid (AA) metabolism, 5(S),12(R)-dihydroxy-6,14-cis,8,10-trans-eicosatetraenoic acid [leukotriene B4 (LTB4)], by neutrophils is exposed to AggIgG occurred in the presence but not absence of exogenous AA. U-60,257B (piriprost potassium), an inhibitor of leukotriene synthesis, caused a dose-related suppression of LTB4 production and granule exocytosis by AggIgG-treated cells. These data suggest that a lipoxygenase product of AA metabolism may mediate AggIgG-induced phagocytic release of granule constituents from neutrophils.     

Studies on the mechanism of formation of the 5S,12S-dihydroxy-6,8,10,14(E,Z,E,Z)-icosatetraenoic acid in leukocytes

Incubation of peripheral blood leukocytes with arachidonic acid (and ionophore A23187) led to the formation of leukotriene B₄, Δ⁶-trans-leukotriene B₄, Δ⁶-trans-12-epi-leukotriene B₄, 5-hydroxy-icosatetraenoic acid, 12-hydroxy-icosatetraenoic acid and of 5S,12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-icosatetraenoic acid (5S,12S-DiHETE). Incubation of leukocytes with leukotriene A₄ resulted in the formation of leukotriene B₄ and of its two Δ⁶-trans-isomers but not of the 5S,12S-DiHETE. ¹⁸O₂ labeling experiments have shown that the hydroxyl groups at C5 and C12 in the 5S,12S-DiHETE are derived from molecular oxygen. The tetraacetylenic analog of arachidonic acid was found to be a potent inhibitor of the formation of the 5S,12S-DiHETE whereas it potentiated the synthesis of the 5-hydroxy acid and of leukotriene B₄. Addition of the 12-hydroxy-icosatetraenoic acid to leukocytes, or of the 5-hydroxy-icosatetraenoic acid to a suspension of platelets caused the formation of the 5S,12S-DiHETE. It is concluded that the 5S,12S-DiHETE is not derived from leukotriene A₄ but is a product of the successive reactions of arachidonic acid with two lipoxygenases of different positional specificities.     

Evaluation of inhibitors of eicosanoid synthesis in leukocytes: Possible pitfall of using the calcium ionophore A23187 to stimulate 5′ lipoxygenase

The effect on arachidonate metabolism of two compounds (BW755C and benoxaprofen) which have been reported to inhibit 5′ lipoxygenase in leukocytes has been evaluated in human polymorphonuclear leukocytes (PMN) stimulated with the calcium ionophore A23187 and serum-treated zymosan (STZ). The syntheses of leukotriene B₄ (LTB₄) and thromboxane B₂ (TXB₂) from endogenous substrate were determined by specific radioimmunoassays as indicators of 5′ lipoxygenase and cyclo-oxygenase activity in the PMN respectively. Benoxaprofen inhibited the synthesis of leukotriene B₄ by human PMN stimulated with the calcium ionophore A23187, but it was approximately 5 times less potent than BW755C. However, benoxaprofen (IC₅₀ 1.6 × 10⁻⁴M) was approximately 100 times less potent than BW755C (IC₅₀ 1.7 × 10⁻⁶M) at inhibiting leukotriene B₄ synthesis induced by serum-treated zymosan. Both drugs inhibited thromboxane synthesis by leukocytes stimulated with A23187 or serum-treated zymosan at similar concentrations (approximately 5 × 10⁻⁶M). The data obtained using STZ as stimulus are consistent with previous studies and indicate that benoxaprofen is a relatively selective inhibitor of cylco-oxygenase. However, this selectivity was far less apparent when A23187 was used as a stimulus to release the eicosanoids which suggests that this inhibition could be via an indirect mechanism and therefore A23187 should be used with caution as a stimulus of 5′ lipoxygenase for evaluating inhibitors of eicosanoid synthesis.     

The synthesis of 6,9-deepoxy-6,9-N-phenylimino-Δ-prostaglandin I1 a novel inhibitor of leukotriene C and D synthesis

The pyrrole analog of prostacyclin, 6,9-deepoxy-6,9-N-phenylimino-Δ^6,8-prostaglandin I₁ was synthetized from PGF₂α methyl ester. This pyrroloprostacyclin (U-60, 257) and its methyl ester (U-56, 467) have been shown to inhibit leukotriene C/D biosynthesis and antagonized leukotriene C/D contractions in vitro. Antigen induced bronchopulmonary changes in monkeys and guinea pigs are inhibited by U-60, 257 in vivo.     

Superoxide anion and hydrogen peroxide production by chemically elicited peritoneal macrophages--induction by multiple nonphagocytic stimuli

The ability of a number of stimulants to activate an oxidative burst (OB) in oil-elicited guinea pig peritoneal exudate macrophages (MPs) was examined. The parameters of the OB were the generation and extracellular release of Superoxide anions (O₂^?) and hydrogen peroxide (H₂O₂). We found that: (1) The cocarcinogen and skin irritant phorbol myristate acetate (PMA) was the most potent OB activator—The weak cocarcinogen 4-O-methyl PMA was a proportionally less effective OB activator; (2) The lectins concanavalin A (Con A) and wheat germ agglutinin (WGA), but not soybean, Lotus, and pokeweed lectins, were also quite effective OB activators—The ability of Con A to stimulate O₂^? production was abolished by succinylation and could be prevented by the presence of α-methyl-D-mannoside; (3) Other stimulators of an OB in MPs were: N-formyl-methionyl peptides, opsonized zymosan, the Ca²⁺ ionophore A23187, phospholipase C, NaF, antimacrophage antibody, microtubule-disrupting drugs, and sodium nitroprusside—O₂^? generation induced by A23187 (but not that stimulated by PMA) was dependent on extracellular Ca²⁺; (4) The amount of O₂^? produced per cell was higher at low cell densities; (5) The addition of Superoxide dismutase (SOD) to the medium totally prevented the detection of O₂^? and augmented twice the amount of H₂O₂ recovered; (6) Pretreatment of MPs with the SOD inhibitor sodium diethyldithiocarbamate had no effect on the release of O₂^? but blocked H₂O₂ release in a dose-dependent manner. These data were interpreted as indicating that the bulk of H₂O₂ was derived by enzymatic dismutation of O₂^?; (7) The common mechanism by which such a variety of stimuli provoke an OB in MPs was not elucidated. No evidence was found to suggest a role for a cyclic nucleotide messenger.     

Human Red Cells Enhance the Formation of 5-Lipoxygenase-Derived Products by Neutrophils

Upon activation, human neutrophils generate 5-lipoxygenase products which are involved in inflammation as well as other physiological and pathophysiological processes. We have examined the influence of red cells on the generation of lipoxygenase-derived products by neutrophils utilizing high pressure liquid chromato-graphy system which permitted quantitation of SHETE, leukotriene B₄ (and its isomers) and the omega oxidation products of leukotriene B₄ (20-hydroxyleukotriene B₄, 20-carboxyleukotriene B₄) within the same sample. Co-incubation of red cells with neutrophils (50:1, red cells:neutrophils) resulted in a 722 percent increase in 5-hydroxyeicosatetraenoic acid production and a slight increase in leukotriene B₄ and its omega oxidation products which were not accompanied by increases in 15-hydroxyeicosatetraenoic acid production. The role of the sulfhydryl status of the red cell and its ability to scavenge hydrogen peroxide were assessed in relationship to the interaction of red cells on the neutrophil-derived lipoxygenase products. Together, these findings indicate that red cells can regulate the levels of lipid-derived mediators produced by neutrophils. Moreover, they suggest that red cell-neutrophil interactions may be of importance in inflammatory reactions.     

Activation of the human neutrophil respiratory burst with zymosan-activated serum

Zymosan-activated serum (ZAS) stimulated a time- and concentration-dependent generation of superoxide anion (O₂^?) by human neutrophils. O₂^? production was rapid with maximum generation occurring 2 minutes after cell exposure to ZAS. O₂^? generation is markedly reduced if cells are not preincubated with cytochalasin B prior to contact with ZAS. The amount of O₂^? produced by ZAS stimulated neutrophils was enhanced in the presence of extracellular calcium. However, the intracellular calcium antagonist, 8-(N,N-diethylamino)-octyl-(3,4,5-trimethoxy) benzoate hydrochloride (TMB-8), caused a dose-related inhibition of ZAS-elicited O₂^? production. Neutrophils pretreated with ZAS were desensitized to the subsequent exposure to this stimulus. The fact that pretreatment of neutrophils with ZAS did not diminish the capacity of these cells to generate O₂^? in response to 1-0-hexadecyl/octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), N-formyl-methionyl-leucyl-phenylalanine (FMLP) or 5(5),12(R)-dihydroxy-6,14-cis-8,10-transeicosatetraenoic acid (LTB₄), demonstrates the stimulus specific nature of ZAS-induced desensitization. Thus, ZAS, which contains the complement-derived neutrophil activator, C5a, a naturally occurring phlogistic mediator, represénts a relevent probe for investigating neutrophil function.     

Effect of leukotrienes, bradykinin and calcium ionophore (A 23187) on bovine endothelial cells: Release of prostacyclin

Incubation of the bovine endothelial cell line, CPAE, with the calcium ionophore (A23187), bradykinin (BK), leukotriene D₄ (LTD₄) or leukotriene C₄ (LTC₄) resulted in concentration dependent increases in prostacyclin release measured as 6-ketoprostaglandin F_1α. The kinetics of induction of prostacyclin synthesis differed among the agents studied. Statistically significant increases in prostacyclin were observed one minute after treatment with A23187, at slightly longer times with bradykinin and after approximately three minutes with the leukotrienes. Two other leukotrienes were tested. Both leukotriene B₄ and leukotriene E₄ (LTE₄) were inactive at con- centrations up to 10 μM. The induction of prostacyclin synthesis by LTC₄ and LTD₄ was inhibited by cycloheximide and actinomycin-D. The effect of BK was inhibited by cycloheximide but not by actinomycin-D. Induction by A23107 was not inhibited by either actinomycin-D or cycloheximide. The results suggest that these agents induced the increases in prostacyclin synthesis by different mechanisms.     

Formation of leukotrienes and other hydroxy acids during platelet-neutrophil interactions in vitro

Interactions of human platelets with neutrophils were studied in suspensions of [³H]arachidonate-labeled platelets and unlabeled neutrophils stimulated with ionophore A23187. Several radioactive arachidonate metabolites, not produced by platelets alone, were detected, including [³H]-labeled leukotriene B₄ (LTB₄), dihydroxyeicosatetraenoic acid (DHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). When [³H]12-HETE, a platelet product, was added to stimulated neutrophils, DHETE was formed. Similarly, when [³H]5-HETE, a neutrophil product, was added to stimulated platelets, DHETE was the major product. These results suggest that upon stimulation: 1) platelet-derived arachidonate may serve as precursor for the neutrophil-derived eicosanoids LTB₄ and 5-HETE, and 2) that platelet-derived 12-HETE can be converted to DHETE by human neutrophils. The present investigation documents cell-cell interactions via the lipoxygenase pathway, which may be important in hemostasis, thrombosis and inflammation.     

Products of 5-lipoxygenase and myeloperoxidase activities are increased in young male cigarette smokers

Abstract

The significance of 5-lipoxygenase and myeloperoxidase activities has not been extensively studied among young male smokers. Leukotriene B₄, 20-hydroxy-leukotriene B₄, 20-carboxy-leukotriene B₄ and 3-chlorotyrosine were measured in plasma and urinary samples of young male smokers at 8 hours following cigarette abstinence and an hour after cigarette smoking. Leukotriene B₄ and 3-chlorotyrosine were determined in neutrophils isolated from these individuals. The levels of these markers were compared with those of age-matched controls. In vitro studies were performed to evaluate the production of leukotriene B₄ and 3-chlorotyrosine from human neutrophils following exposure to nicotine and cotinine. Thirty male smokers (mean age, 27.4 years) and 28 male non-smokers (mean age, 28.7 years) were studied. Plasma levels of leukotriene B₄, 20-carboxy-leukotriene B₄ and 3-chlorotyrosine were higher in smokers than in non-smokers; leukotriene B₄ and 20-carboxy-leukotriene B₄ levels increased further an hour after cigarette smoking. Peripheral neutrophils isolated from smokers showed greater expressions of myeloperoxidase and 5-lipoxygenase activities compared with non-smokers, while plasma leukotriene B₄ and 3-chlorotyrosine were correlated significantly with high-sensitivity C-reactive protein and plasma nicotine concentrations. Exposure of human neutrophils to nicotine and cotinine resulted in a higher production of leukotriene B₄ and 3-chlorotyrosine. To conclude, leukotriene B₄ and 3-chlorotyrosine levels are increased in young male cigarette smokers. These results suggest that cigarette smoking aggravates neutrophil-mediated inflammation by modulating the activities of myeloperoxidase and 5-lipoxygenase pathways.     

Leukotriene B4 is a complete secretagogue in human neutrophils: a kinetic analysis

The interactions have been studied of leukotriene B₄ (LTB₄) and 20-COOH-LTB₄ with human neutrophils (PMN). Kinetic studies, utilizing continuous recording techniques, showed that LTB₄ activates PMN with respect to aggregation, mobilization of membrane-associated Ca²⁺, $\underset{\dot{}}{?}$ generation, and degranulation within seconds of exposure. Dose-response studies indicate 1) that LTB₄ is much more potent than its dicar?ylic acid derivative (20-COOH-LTB₄) or its all trans-isomer, and 2) that PMN responses to these agents are largely dependent upon pretreatment of the cells with cytochalasin B. These properties were similar to those of the microbial ionophores, ionomycin and A23187. Results demonstrate that LTB₄ rapidly activates PMN and indicate that LTB₄ serves as a complete secretagogue. Moreover, they provide additional evidence that oxidized fatty acids activate human PMN.     

Generation of leukotriene B4 and leukotriene E4 from porcine pulmonary artery

When chopped porcine pulmonary arteries were incubated with calcium ionophore A23187 (1) in the presence of indomethacin there was a time dependent generation of a substance which produced contractions of superfused strips of guinea-pig ileum smooth muscle (GPISM) which were indistinguishable from those induced by LTD₄. This material however had a different retention time from LTD₄ when subjected to HPLC and co-chromatographed with synthetic LTE₄. In addition to LTE₄ a substance which had properties indistinguisable from those of LTB₄ when assayed on a combination of guinea-pig lung parenchymal strips (GPP) and GPISM (2) was generated from the pulmonary artery. This substance co-chromatographed with synthetic LTB₄. The adventitia and intima were the richest source of LTE₄, the adventitia releasing slightly more than the intima. The output of LTB₄ and LTE₄ was inhibited by 6,9-deepoxy-6,9-(phenylimino)-Δ^6,8 prostaglandin I₁ (U-60,257). Nordihydroguaiaretic acid (NDGA) inhibited the generation of LTE₄.     

C2-ceramide primes specifically for the superoxide anion production induced by N-formylmethionylleucyl phenylalanine (fMLP) in human neutrophils

Activated sphingomyelinases release ceramide molecules believed to be involved in intracellular signalling. The present study investigated whether soluble C₂-ceramide modulates some of the effects of N-formylmethionylleucyl phenylalanine (fMLP) and other agonists on human neutrophils (or polymorphonuclear leukocytes-PMN); principally superoxide anion (O₂⁻) production. The preincubation of PMN for 15 min with C₂-ceramide increased by up to almost 3-fold the amounts of O₂⁻ generated in response to 0.1 and 1 μM fMLP. Priming was detected at C₂-ceramide concentrations of 2 μM to 4 μM per million PMN. Though less potent than C₂-ceramide, C₆-ceramide (N-hexanoylsphingosine) could prime for O₂⁻ generated in response to 0.1 μM fMLP, with maximal effects obtained at 10–20 μM. In contrast, micromolar concentrations of sphingosine, dihydroceramide, and ceramide-phosphate, failed to exert any potentiating effect on fMLP-induced O₂⁻ generation. As expected, TNF-α (1000 U/ml), also primed for fMLP-induced O₂⁻ production; however, the combination of TNF-α and C₂-ceramide showed no additive effect. Moreover, S. aureus sphingomyelinase (0.1 U/ml), was unable to reproduce the priming effects of C₂-ceramide and TNF-α. C₂-ceramide at 2 μM did not enhance the production of O₂⁻ induced by 100 nM recombinant human interleukin-8 (IL-8), leukotriene B₄ (LTB₄), platelet-activating factor (PAF) or 20 mM sodium fluoride (NaF). Furthermore, C₂-ceramide (2 μM) did not enhance the mobilization of calcium, the release of arachidonic acid or the accumulation of phosphatidylethanol, induced by 100 nM fMLP. This suggests that probably neither phospholipases C, A₂ or D (PLC, PLA₂, PLD) were involved in the priming effect by C₂-ceramide. However, C₂-ceramide inhibited in a dose-related manner the production of O₂⁻ induced by phorbol 12-myristate 13-acetate (PMA) and mezerein. Furthermore, PMA-stimulated PLD activity was also significantly reduced by a preincubation of PMN with C₂-ceramide. The priming O₂⁻ production by C₂-ceramide could involve yet unidentified mechanisms specific for fMLP, or it might imply that cytokines such as TNF-α have different mechanisms than C₂-ceramide.     

Retinoid-induced inhibition of bosinophil LTC4 production

Naturally occuring and synthetic retinoids demonstrate a marked antiinflammatory effect when employed in such disorders as acne and psoriastis. This effect may result in part from their inhibition of release of potent mediators (e.g. eicosanoids) by inflammatory cells. In this study, we examined the effect of eight retinoids (tretinoin, isotretinoin, retinol, retinal, acitretin, retinyl palmitate, etretinate, Ro 15–0778) on the release of leukotriene (LT)C₄, an important lipid mediator generated by eosinophils. Tretinoin, isotretinoin, retinol, retinal, and acitretin at 10⁻⁵ M or 10⁻⁴ M concentrations inhibited LTC₄ release by A23187-stimulated horse eonsinophils in vitro; 10⁻⁴ M retinyl palmitate was also inhibitory. However, 10⁻⁵ M etretinate augmented A23187-induced LTC₄ release, and the arotinoid Ro 15–0778 had no effect on LTC₄ production. These data suggest that selected retinoids may have potential use in the reduction of LTC₄ generation by eosinophils. This inhibition could be beneficial in the theraphy of such diseases as bronchial asthma in which release of LTC₄ may be involved in the inflammtory process.     

Purification and characterisation of leukotriene A4 hydrolase from rat neutrophils

Leukotriene A₄ hydrolase was rapidly and extensively purified from rat neutrophils using anion exchange and gel filtration high-pressure liquid chromatography. The enzyme which converts the allylic epoxide leukotriene A₄ to the 5,12-dihydroxyeicosatetraenoic acid leukotriene B₄ was localized in the cytosolic fraction and exhibited an optimum activity at pH 7.8 and apparent K_m for leukotriene A₄ between 2 · 10^?5 and 3 · 10^?5 M. The purified leukotriene A₄ hydrolase was shown to have a molecular weight of 68 000 on sodium dodecylsulfate polyacrylamide gel electrophoresis and of 50 000 by gel filtration. The molecular weight and monomeric native form of this enzyme are unique characteristics which distinguish leukotriene A₄ hydrolase from previously purified epoxide hydrolases.     

Inhibition of the neutrophil oxidative response to a chemotactic peptide by inhibitors of arachidonic acid oxygenation

N-formylmethionylphenylalanine stimulates a short burst of antimycin A-insensitive O₂ uptake, O₂^? production and hexosemonophosphate shunt oxidation of glucose by guinea pig peritoneal neutrophils. The stimulated oxidative metabolism, as well as release of lysosomal enzymes ± cytochalasin B, are inhibited by 5,8,11,14-eicosatetraynoic acid (ID₅₀ 1.5 × 10^?5 M). High concentrations of indomethacin inhibit the peptide-stimulated oxidations (ID₅₀ 1.6 × 10^?4 M) while acetylsalicylic acid (2.5 × 10^?3 M) does not. Digitonin-stimulated oxidative metabolism and enzyme release are not inhibited by 5,8,11,14-eicosatetraynoic acid or indomethacin at concentrations that depress effects of the N-formylated peptide.     

Theophylline infusion modulates prostaglandin and leukotriene production in man

Although theophylline has been used in the treatment of asthma for decades, it is not a first line choice any more. It is a well-known bronchodilator, but was recently discovered also to be an anti-inflammatory, immunomodulatory and bronchoprotective agent. Therefore we wanted to establish the role of theophylline on prostaglandin and leukotriene production, which plays a part in the pathogenesis of asthma. Theophylline was infused (bolus 5 mg/kg in 15 min and infusion 0.4 mg/kg/h for 1 h 45 min) into healthy volunteers. Thromboxane B₂, prostaglandin E₂ and leukotriene E₄ were measured from the A23187-stimulated whole blood samples and stable metabolites of thromboxane A₂; prostacyclin and leukotriene E₄ were measured from urine. Theophylline increased prostaglandin E₂ production and decreased leukotriene E₄ production ex vivo in whole blood, thus increasing the prostanoid/leukotriene ratio. It did not change thromboxane B₂ production stimulated by either spontaneous clotting or A23187 in the whole blood. Theophylline had hardly any effect on in vivo thromboxane, prostacyclin and leukotriene E₄ production measured as urinary metabolites, 11-dehydro-thromboxane B₂, 2,3-dinor-6-keto-prostaglandin F_1α and leukotriene E₄, respectively. Serum theophylline concentrations were at the lower level of normal therapeutic range during the infusion. The increase in PGE₂ and the decrease in LTE₄ synthesis ex vivo may offer a new explanation for the mode of antiasthmatic action of theophylline. It is notable that this phenomenon occurs at low serum theophylline concentrations. These results confirm the idea that theophylline has an anti-inflammatory and bronchoprotective action and support the use of theophylline as a therapeutic agent in asthmatic patients.