Enhancement of human neutrophil adherence by synthetic leukotriene constituents of the slow-reacting substance of anaphylaxis

The functionally predominant constituents of the slow-reacting substance of anaphylaxis (SRS-A), designated leukotrienes C4 and D4 (LTC4 and LTD4), as well as the leucocyte chemotactic factor leukotriene B4 (LTB4) enhance the adherence of human neutrophils to Sephadex G-25. Enhancement of neutrophil adherence was significant at leukotriene concentrations of 3 X 10(-9) M -3 X 10(-7) M, and reached a maximum level for each of the leukotrienes that was similar in magnitude to that evoked by the neutrophil chemotactic peptide N-formyl-methionyl-leucylphenylalanine (FMLP). The leukotrienes and FMLP elicited optimum increases in neutrophil adherence within 1-2 min at 37 degrees. Indomethacin inhibited the increase in neutrophil adherence evoked by LTC4 and LTD4 and the concurrent elevation in the concentration of endogenous thromboxane B2. The smooth muscle contractile and vasoactive factors LTC4 and LTD4, which lack chemotactic activity for leucocytes, are as active as LTB4 in stimulating human neutrophil adherence, and the effect may be mediated in part by neutrophil-derived thromboxane A2.     

A comparative study on the release of leukotrienes and histamine by guinea pig lung and trachea after challenge with antigen or stimulation with ionophore A23187 or melittin

The release of leukotrienes and histamine from guinea pig lung and trachea after immunological and nonimmunological stimulation were compared. Antigen, ionophore A23187 and melittin caused the release of leukotriene (LT)B4, LTC4, LTD4 and LTE4 from lung and trachea as determined by reverse-phase high performance liquid chromatography (RP-HPLC) and bioassay. The release of LTB4 by lung and trachea was maximum after 5 min of ionophore stimulation (128 +/- 40 and 142 +/- 29 pmol/g tissue, respectively). Lung, but not trachea, also released the 20-OH-LTB4 and 20-COOH-LTB4. The release of LTC4 by lung tissues was maximum after 5 min, whereas maximal tracheal responses occurred at 10 min (27 +/- 11 and 9 +/- 3.5 pmol/g tissue, respectively). Maximal release of LTD4 by lung and trachea respectively occurred after 10 and 15 min (103 +/- 21 and 20 +/- 6 pmol/g tissue, respectively). The release of LTD4 in response to ionophore by both tissues decreased after 15 min, whereas the release of LTE4 continued to increase. Release of leukotrienes from melittin stimulated lung was 2-3-fold less than in ionophore stimulation. In contrast, tracheal responses to melittin and ionophore for the release of LTB4 were equivalent, whereas release of peptidoleukotrienes in response to melittin was approximately 50% that resulting from ionophore. Antigen challenge was the least potent stimulus for LTB4 release in both tissues, whereas it was at least as potent as melittin for the release of peptidoleukotrienes. The release of histamine by lung tissue was approximately 2-3-fold greater than by trachea (7 +/- 1 and 2 +/- 0.5 nmol/g tissue, respectively) after 5 min of stimulation with either ionophore, melittin or antigen. These data demonstrate that lung tissues and trachea respond to immunologic stimulations by releasing the mediators of inflammation and immediate hypersensitivity. The lung releases peptidoleukotrienes and histamine 2-5-fold greater than the trachea, whereas the release of LTB4 in both tissues are approximately equal.     

(9-[4-acetyl-3-hydroxy-2-n-propylphenoxy) methyl]-3-(1H-tetrazol-5-yl)-4H-pyrido [1,2-a] pyrimidin-4-one), AS-35, inhibits leukotriene synthesis

AS-35, (9-[4-acetyl-3-hydroxy-2-n-propylphenoxy) methyl]-3-(1H-tetrazol-5-yl)-4H-pyrido[1, 2-a] pyrimidin-4-one), was developed as a leukotriene (LT) receptor antagonist, which also inhibited IgE-mediated release of leukotrienes (LTs). We have investigated the action of AS-35 on the enzyme activities which are involved in the synthesis of LTC(4) and LTB(4) (LT-synthesizing enzymes); cytosolic phospholipase A(2) (cPLA(2)), 5-lipoxygenase (5-LO), leukotriene (LT)C(4) synthase and LTA(4) hydrolase. AS-35 dose-dependently inhibited IgE- and A23187-stimulated production of LTC(4) by up to 71.5-84.8% and that of LTB(4) by 48.3-49.2% at 2. 5x10(-5) M. The assays for cPLA(2)(-), 5-LO-, LTC(4) synthase- and LTA(4) hydrolase-activities revealed that the inhibition is attributable to suppression of cPLA(2), 5-LO and LTC(4) synthase but not LTA(4) hydrolase. We have also studied the action of AS-35 on the release of beta-hexosaminidase (beta-HEX) as a marker of preformed mediators. AS-35 had only weak inhibitory action on the release of beta-HEX. The results indicate that anti-allergic action of AS-35 is predominantly attributable to its inhibition of LT synthesis by suppressing three consecutive enzymes for LTC(4) synthesis.     

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

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

Preventive effect of a novel leukotrienes antagonist ONO-1078 on leukotriene C4- and D4-induced human bronchial smooth muscle contraction

To study whether a novel leukotrienes antagonist ONO-1078 (4-oxo-8-[4-phenylbutyloxy) benzoylamino]-2-(tetrazol-5-yl)-4H-1-benzopyran hemihydrate) prevents leukotrienes C4- and D4-(LTC4, LTD4) induced human bronchial smooth muscle contraction, we examined the bronchial contractile response to LTC4 and LTD4 in the presence or in the absence of ONO-1078 in human bronchial strips. We prepared 4 strips from each of 5 patients undergoing lobectomy of the lung because of lung cancer. We mounted the strips in organ baths and measured the contractile response to LTC4 and LTD4 from 10(-11) M to 10(-7) M after incubation with ONO-1078 (10(-8) M, 10(-7) M and 10(-6) M) and without ONO-1078. ONO-1078 shifted the dose response curve to LTC4 to higher concentration in dose dependent fashion, and it significantly inhibited the contractile response to LTC4 and LTD4. In the presence of L-serine borate complex (45 mM), an inhibitor of gamma-glutamyl transpeptidase, ONO-1078 significantly inhibited the LTC4-induced contraction. On the other hand, ONO-1078 had no effect on the contractile response to acetylcholine. These results suggest that ONO-1078 is a specific antagonist of LTC4 and LTD4 receptors in human bronchial smooth muscle. Because the effect of LTC4 and LTD4 is thought to be an important part of the pathogenesis of bronchial asthma, our results also suggest that ONO-1078 may be a useful prophylactic drug for bronchial asthma.     

Inhibition of calcium ionophore-stimulated leukotriene generation from intact human neutrophils by captopril

Effects of captopril on the formation of leukotrienes (LTs) from stimulated intact human neutrophils were investigated. Neutrophils were stimulated with 1 microM calcium ionophore A23187 for the generation of LTs. A reverse phase high performance liquid chromatography technique and UV spectroscopy were used to detect and quantitate the released LTs namely, LTB4. LTC4. delta6-trans-LTB4 and delta6-trans-12-epi-LTB4. Preincubation of neutrophils with captopril significantly reduced LTB4 formation in a concentration-dependent manner, as compared to diluent-treated control cells. Since LTA4 is the substrate for both LTB4 and LTC4, thus in presence of captopril, shunting of LTA4 from synthesis of LTB4 was not directed to LTC4 formation. This finding was evidenced by the significant decrease of LTC4 production under the influence of high concentration of the drug. Formation of LTB4 stereoisomers, delta6-trans-LTB4 and delta6-trans-12-epi-LTB4 was not markedly altered by captopril. In subsequent experiments, when neutrophils were stimulated with A23187 after preincubation with exogenous arachidonic acid (75 microM), and treatment with captopril, similar findings were obtained for LTB4 and LTC4. Meanwhile, formation of the nonenzymatic hydrolysis products of LTA4 tended to rise reaching significant level in case of delta6-trans-LTB4, at the high concentration of captopril. These results demonstrate that captopril is an inhibitor of enzymatically generated LTs produced by intact human neutrophils, being more potent against LTB4. These effects of captopril on LTs are not mediated via an inhibition of arachidonic acid formation from membrane phospholipids. It could be suggested that captopril, at doses used clinically. could inhibit the generation of LTB4 without affecting LTC4. Consequently, these findings might account for possible antiinflammatory activity for captopril, and further suggest that some of the observed side effects of captopril might not be related to an overproduction of LTC4.     

Immunological and non-immunological release of leukotrienes and histamine by guinea-pig heart

Fragments of sensitized guinea-pig heart (1 g wet weight) were incubated with 5 micrograms/ml of antigen for up to 30 min, and the incubation media were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) for the presence of leukotrienes LTB4, LTC4, LTD4 and LTE4. Maximum release of LTB4, LTC4 and LTD4 was observed after 15 min (32.8 +/- 4, 8 +/- 2 and 9.5 +/- 2.5 pmol/g tissue wet weight, respectively, mean +/- SEM). At the same time, histamine was also released, reaching a maximum at 5 min (290 +/- 63 pmol/g tissue) as determined by radioenzymatic assay. Similarly, the non-sensitized guinea-pig heart stimulated with the ionophore A23187 (4 microM) released LTB4, LTC4, LTD4 and LTE4, as well as the 5-hydroxy-eicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE as determined by RP-HPLC. The release of LTB4, LTC4 and LTD4 was at a maximum after 10-15 min of stimulation (63 +/- 8.4, 10.7 +/- 2 and 17.5 +/- 4 pmol/g tissue). The ionophore also stimulated the release of histamine in heart tissue, with a peak maximum after 5 min (325 +/- 77 pmol/g tissue). These data demonstrate that heart as well as pulmonary tissues release significant amounts of leukotrienes and histamine during immunological or non-immunological challenges.     

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

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

Modulation of leukotriene formation by cellular composition and exogenous leukotriene A4.

We investigated the interactions of exogenous leukotriene A4 (LTA4) with isolated cells in the presence or absence of cellular stimuli. The majority of isolated cells are able to transform exogenous LTA4 into LTB4 as well as LTC4. In eosinophils, LTA4 induced 15-hydroxy-eicosatetranoic acid formation and was converted into LTB4. The Ca-ionophore-induced generation of LTB4 from polymorphonuclear leukocytes or from the cell fraction containing lymphocytes, monocytes and basophils was significantly suppressed with LTA4 while the formation of LTC4 was increased. Conversely, the Na-fluoride- and fMLP-induced generation of LTB4 was significantly increased. Our results suggest that the stimulus and the cellular composition determine the pattern of the generated inflammatory mediators.     

Effects of the new 5-lipoxygenase inhibitor E6080 on leukotriene release in vitro.

Fundamental studies were conducted to examine the release of histamine and leukotriene (LT) C4 from lung fragments of guinea pigs and the effects of E6080 on the release of LTB4 and LTC4 from lung fragments or inflammatory cells. The release of histamine and LTs showed large interindividual variations and a marked dependence on experimental conditions. Addition of 10 mM L-cysteine significantly increased LTC4 release compared with that in its absence (about 1.7 times, in terms of mean value). E6080 inhibited antigen-stimulated LTB4 and LTC4 release from passively sensitized human (IC50: LTB4 0.08 microM, LTC4 0.2 microM) and guinea-pig lung fragments (IC50: LTC4 1.1 microM). The LTB4 and LTC4 releases from healthy human polymorphonuclear leukocytes (calcium ionophore A23187) and from allergic patients' leukocytes (basophils, antigen) were inhibited by E6080 with IC50 values of below 1.0 microM. Furthermore, the LTC4 release from rat alveolar macrophages (silica particles) was inhibited by E6080 with an IC50 of 0.2 microM. The potent inhibition by E6080 might be a result of the inhibition of 5-lipoxygenase, since 5-lipoxygenase in rat basophilic leukemia cell was inhibited by E6080 with an IC50 of 0.2 microM. The results confirm the potent inhibitory effects of E6080 on the release of LTs.     

Effect of cations on leukotriene release: requirements for the metabolism of peptido-leukotrienes (leukotrienes C4, D4) by human polymorphonuclear granulocytes

Stimulation of human polymorphonuclear granulocytes with opsonized zymosan leads to a time-dependent release of the leukotrienes LTB4, 6-trans-LTB4, 12-epi-6-trans-LTB4 and LTC4 measured by HPLC analysis and LTC4 radioimmunoassay. The amounts of leukotrienes released on stimulation with opsonized zymosan are lower than those obtained with the calcium ionophore A23187. Opsonized zymosan as stimulus required higher calcium concentrations to obtain optimal leukotriene release as compared with the calcium ionophore. Magnesium in the presence of calcium increased the leukotriene formation with opsonized zymosan. Addition of the peptido-leukotrienes LTC4, LTD4 to the unseparated, opsonized zymosan-prestimulated cell suspension leads to the generation of 6-trans-LTB4, 12-epi-6-trans-LTB4 and a metabolite which is more polar than LTC4. The rate of LTC4 metabolism is strongly dependent on the time of prestimulation as well as on the stimuli used for cell triggering, e.g. opsonized zymosan, phorbol-12-myristate-13-acetate, calcium ionophore A23187 or formyl-methionyl-leucyl-phenylalanine. Inhibitors of the oxidative burst decreased LTC4 metabolism. Thus, the peptido-leukotriene LTC4 is metabolized by two pathways: the enzymes gamma-glutamyl-transpeptidase and dipeptidase transform LTC4 into LTD4 and LTE4; these enzymes are present within the supernatants of stimulated cells; transformation of LTC4 into LTB4-isomers occurs in the presence of stimulated cells.     

Enhanced granulocyte cytotoxicity by mediators derived from anti-IgE-stimulated human leucocytes.

Basophil-containing leucocyte fractions stimulated with an anti-human IgE, F(ab')2, generated histamine and the leukotrienes LTB4 and LTC4 with significant correlations between LTB4 and histamine (P less than 0.01; n = 26) and LTC4 and histamine release (P less than 0.001; n = 29) in the cell-free supernatants (SN). SN from these anti-IgE-treated cells enhanced the cytotoxicity of eosinophils and neutrophils (against complement-coated schistosomula of Schistosoma mansoni) in vitro. When SN were fractionated by reverse phase-high performance liquid chromatography (RP-HPLC), the enhancing activity for neutrophils was almost totally confined to fractions having LTB4 immunoreactivity (co-eluting as a single peak with the synthetic LTB4 marker). In contrast, the LTB4-containing fraction had minimal effects on eosinophil cytotoxicity, whereas synthetic histamine gave comparable enhancement to the unfractionated SN. The generation of LTs (but not histamine), as well as enhanced neutrophil cytotoxicity from basophil-containing leucocytes by anti-IgE treatment, was maximally inhibited by the 5-lipoxygenase inhibitors U-60, 257 and BW755C. Conversely, the cyclooxygenase inhibitor indomethacin did not significantly affect LT release, nor did it affect the subsequent cytotoxicity enhancing activity of SN from such cells. These results indicate that LTB4 and LTC4 are released from basophils, together with histamine, by IgE-dependent mechanisms, LTB4 enhances the cytotoxicity of bystander neutrophils, and histamine (and to a lesser extent, LTB4) augments eosinophil cytotoxicity.     

Generation and Metabolism of Leukotrienes and Release of Histamine from Human Dispersed Tonsillar Cells

We studied the generation and metabolism of leukotrienes (LT) and the release of histamine by human tonsillar cell suspensions. Human tonsils were dissected and mechanically dispersed. This procedure yielded a single cell suspension with 1.6 +/- 0.5 X 10(8) cells/g tissue consisting of 97.3 +/- 0.4% lymphocytes, 1.4 +/- 0.3% granulocytes, 1.3 +/- 0.3% macrophages/monocytes, and 0.03 +/- 0.02% mast cells/basophils. The cells were stimulated either with Ca-ionophore A 23187, melittin, or anti-human IgE. Determination of the 5-lipoxygenase products LTB4 and LTC4 was performed with specific radioimmunoassays (RIA), and histamine release was measured by the fluorophotometric technique. A time- and dose-dependent release of the mediators was monitored. LTB4 exceeded the amount of LTC4 in the supernatants. The concentration of leukotrienes ranged between 0.8 and 5.4 ng LTB4/1 X 10(8) cells or 0.5 and 1.5 ng LTC4/1 X 10(8) cells, depending on the stimulus. Histamine release after stimulation ranged between 25 and 35% of the total histamine content, whereas buffer controls amounted to 17%. The incubation of the cells (1 X 10(8) with exogenously added LTB4 resulted in the formation of omega-oxidated products (20-OH and 20-COOH-LTB4) and a novel unpolar metabolite, as identified by thin layer chromatography. This metabolite was not immunoreactive in the LTB4-RIA used. LTC4 and LTD4 were converted into LTE4 when added either to sonicated cells or to the cell-free supernatants of prestimulated tonsillar cells, indicating the release of gamma-glutamyltranspeptidase and dipeptidase, respectively. Our data clearly demonstrate the generation and metabolism of the 5-lipoxygenase products LTB4 and LTC4 as well as the release of histamine from human dispersed tonsillar cells, suggesting that they have a modulatory function with respect to the inflammatory potential at local sites.     

Modulation of leukotriene release from human polymorphonuclear leucocytes by PMA and arachidonic acid

Stimulation of human neutrophils (PMN) with Ca ionophore A23187, opsonized zymosan and formyl-L-methionyl-L-leucyl-phenylalanine (FMLP) led to a time- and dose-dependent release of LTB4, 20-OH-LTB4, 20-COOH-LTB4, 6-trans-LTB4, 12-epi-6-trans LTB4 and LTC4, as detected by reverse-phase HPLC. Preincubation of the PMN suspension in the presence of Ca2+ and Mg2+ with phorbol-12-myristate-13-acetate (PMA) did not release leukotrienes by itself, but modulated the subsequent Ca ionophore-induced leukotriene release. The release of LTC4, 20-OH-LTB4 and 20-COOH-LTB4 was significantly decreased. Lesser effects were observed for the release of LTB4 and the non-enzymatic LTB4 isomers. In contrast, opsonized zymosan and FMLP enhanced the release of LTB4 and LTB4-omega-oxidation products from cells pretreated with PMA. With arachidonic acid as prestimulus, the amounts of the LTB4 isomers (6-trans-LTB4 and 12-epi-6-trans-LTB4) were enhanced significantly on subsequent stimulation with Ca ionophore. Prestimulation of lymphocytes, monocytes and basophilic granulocytes (LMB) with PMA had no significant effects on the ionophore-induced release of LTC4 and LTB4. PMN, but not LMB, suspensions prestimulated with PMA convert exogenously added LTC4 to LTB4 isomers and LTC4 sulphoxide. Our data suggest that preincubation of human granulocytes with PMA modified leukotriene release by activation or inhibition of different metabolic pathways for LTC4 and LTB4.     

Inhibitory effect of amlexanox (AA-673) on the immunological and non-immunological release of histamine or leukotrienes

The effect of amlexanox on the non-immunological or immunological release of histamine or leukotrienes (LTs) from passively sensitized human lung fragments and atopic human leukocytes was investigated and compared with those of AA-861, tranilast, azelastine and disodium cromoglycate. 1) Amlexanox at concentrations of 10(-7)-10(-4) M showed an inhibition of histamine, LTB4, LTC4, LTD4 and LTE4 release from passively sensitized human lung fragments in a concentration-dependent fashion. A selective and competitive inhibitor of the 5-lipoxygenase activity, AA-861 modestly affected the histamine release and potently suppressed the any LT release at 10(-7) and 10(-6) M. Antiallergic drugs, tranilast and disodium cromoglycate also suppressed these chemical mediator release, but the inhibition potency was somewhat weaker than that of amlexanox. 2) Ca ionophore A23187-induced release of LTB4 and LTC4 from atopic human leukocytes was slightly enhanced up to 10(-6) M of amlexanox. However, 10(-4) M of the drug strongly diminished both of LT release. From these results, it is suggested that amlexanox is a clinically effective drug for atopic diseases, especially allergic asthma and rhinitis.     

Preferential generation of leukotriene C4 by human eosinophils.

The leukotriene generating capacities of ionophore stimulated human eosinophils and neutrophils were compared using specific radioimmunoassays for LTB4 and LTC4. Mixed granulocyte preparations (neutrophils and eosinophils) produced both LTB4 and LTC4 in a time-dependent fashion which was maximal at 10 and 15 min, respectively. Following the separation of eosinophils (greater than 75%) and neutrophils (greater than 90%) by metrizamide gradients, LTC4 production was predominantly from eosinophils, whereas neutrophils were the principal source of LTB4. The concentrations of leukotrienes produced by the eosinophil and neutrophil rich cell preparations were directly proportional to the concentration of ionophore. Following purification of eosinophil derived products by RP-HPLC the LTC4 immunoreactivity corresponded to the elution profile of a synthetic LTC4 marker. Furthermore, in 32 atopic subjects (21 bronchial asthmatics and 11 non-asthmatics) the amounts of LTC4 produced by unseparated leucocytes were directly proportional to the percentage of eosinophils in the total cell suspension. Preferential generation of LTB4 by neutrophils was also demonstrated by immunoreactivity of ionophore stimulated supernatants subjected to RP-HPLC, as well as by its characteristic u.v. absorbance and GC-MS profile and the ability to promote directional neutrophil locomotion (chemotaxis). These experiments support the concept that eosinophils accumulate in tissues partly as a result of the response to neutrophil derived LTB4, and that these cells contribute to the production of sulphidopeptide leukotrienes with subsequent amplification of the acute allergic response.     

Release of leukotrienes and histamine by the isolated anaphylactic heart

The purpose of this study was to demonstrate the ability of heart tissue to release the mediators of anaphylaxis after antigenic challenges. Guinea pigs were sensitized with ovalbumin. Hearts were excised, perfused in a langendorff apparatus, and challenged with a bolus injection of ovalbumin. Analysis of the perfusates demonstrated the presence of histamine as determined by radioenzymatic assay. Histamine release was observed to be maximum after 2 min (8 +/- 1 nmol) of perfusion, then decreased to baseline level. The heart also released LTB4, LTC4, LTD4, and LTE4 as determined by high performance liquid chromatography and bioassays. The release of LTC4 occurred rapidly, reaching maximum after 2 min (4.2 +/- 1 pmol) and then returned to baseline level. Although the release of LTD4 paralleled the release of LTC4, it reached a maximum after 5 min (7.7 +/- 2 pmol). LTE4 was detected after 10 min and was undetectable after 15 min. Maximum release of LTB4 was observed after 5-10 min (15 +/- 3 pmol) and was no longer detectable after 15 min. These results indicate that the isolated sensitized heart undergoing antigenic challenge releases leukotrienes and histamine suggesting the cardiac anaphylaxis might occur by the locally released mediators.     

Arachidonic acid metabolism in guinea pig eosinophils: synthesis of thromboxane B2 and leukotriene B4 in response to soluble or particulate activators

The arachidonic acid metabolism of guinea pig eosinophils isolated from either peritoneal cavity or bronchoalveolar lavages was studied by reverse-phase high-performance liquid chromatography. The purified eosinophils (95-100%) from either source released thromboxane B2 (TxB2), luekotriene B4 (LTB4) and 5-hydroxy eicosatetraenoic acid (5-HETE) following calcium ionophore A23187 stimulation. Quantification by radioimmunoassay indicated that maximal mediator output from the stimulated peritoneal cells was reached at 3 min after stimulation. The increase in production of TxB2 and LTB4 was correlated to increasing calcium ionophore A23187 concentration up to 1.0 micrograms/ml. In addition to calcium ionophore, the guinea pig peritoneal cells were also activated by f-met-leu-phe, phorbol 12-myristate, 13-acetate (PMA), and to lesser extent platelet-activating factor (PAF) to produce TxB2. LTB4 synthesis was stimulated by calcium ionophore, by f-met-leu-phe, as well as by unopsonized glucan, a particulate phagocytotic stimulus. The guinea pig eosinophils do not synthesize sulfidopeptide leukotrienes because of the absence of the specific LTA4 glutathione S-transferase. These results suggest that the guinea pig eosinophils differ from the human circulating eosinophils in the synthetic capacity of lipid mediators derived from arachidonic acid metabolism. This difference may be important in the understanding of the role of the eosinophils in inflammatory reactions such as that which occurs in the bronchial tissues of asthmatics.     

Endogenous arachidonic acid metabolism by calcium ionophore A23187-stimulated lamb lungs: effect of hypoxia

We have determined eicosanoid production from endogenous arachidonic acid by neonatal lamb lungs stimulated with calcium ionophore A23187 during normoxia and hypoxia. Lungs of lambs 19 to 25 d of age were isolated and perfused with cell-free Krebs' bicarbonate buffer at a flow rate of 15 to 20 ml/kg/min. After 30 min of equilibration in a recirculating system, A23187 was added to the perfusate in a 5-microM concentration and perfusion continued for 15 min more. Eicosanoids were measured in perfusate and lung homogenate supernatant. Cyclooxygenase metabolites prostaglandin (PG) E2, thromboxane A2, and PGI2, were measured by radioimmunoassay, and 5-lipoxygenase metabolites leukotrienes (LT) B4, C4, D4, and E4 by high performance liquid chromatography. During normoxia, all three cyclooxygenase metabolites were present in perfusate, but only PGI2 and thromboxane A2 were present in lung homogenate supernatant. Prostacyclin constituted 50% of all the cyclooxygenase products measured. LTC4 and LTD4 were detected in both perfusate and lung homogenate supernatant with little production of LTE4 and LTB4. During hypoxia, the profile of cyclooxygenase products was unchanged and prostacyclin production was not increased. However, the profile of leukotriene metabolites was altered. LTC4 synthesis was markedly reduced. The synthesis of LTE4 and LTB4 was increased 10-fold, with most of the leukotrienes being retained in lung tissue. We conclude that hypoxia significantly alters leukotriene metabolism of endogenous arachidonic acid by calcium ionophore-stimulated lungs. The increased production by stimulated lungs during hypoxia of LTE4, a substance that may increase lung capillary permeability, and that of LTB4, a powerful chemoattractant, may be important contributing factors to lung injury.