Mast cell mediators prostaglandin-D2 and histamine activate human eosinophils.

Airway damage secondary to eosinophil activation is thought to contribute to the development of asthma. Using the fluorescent dye FURA-2 to measure the concentration of cytosolic calcium, we found that supernatants from anti-IgE-stimulated human lung mast cells increased cytosolic calcium in human eosinophils. We then examined the major mast cell mediators (histamine, PGD2, platelet-activating factor (PAF), eosinophil chemotactic factor of anaphylaxis (ECF-A), leukotriene (LT)C4 and LTB4) for their ability to increase cytosolic calcium in eosinophils. We found that both PAF (5 x 10(-9) to 5 x 10(-6) M) and PGD2 (two of five donors responsive at 1 x 10(-9) M) were potent stimuli for calcium mobilization. LTB4 (10(-8), 10(-7) M) and histamine were also active, although higher concentrations of histamine were required to see a response (3 x 10(-7) to 10(-5) M). LTC4, val-ECF-A, and ala-ECF-A were inactive. The effects of PGD2 and histamine were specific for eosinophils, although LTB4 and PAF increased calcium in both neutrophils and eosinophils. The histamine-induced increase in intracellular calcium was not blocked by the H1 or H2 antagonists pyrilamine or cimetidine (10(-4) M), respectively; however, the response to 10(-6) M histamine was completely blocked by the specific H3 antagonist thioperamide (10(-6) M). To evaluate the relative contribution of these stimulatory mast cell mediators on the calcium mobilizing activity in supernatants from anti-IgE-stimulated human lung mast cell (HLMC), we examined the effect of supernatants from HLMC pretreated with indomethacin and/or the 5-lipoxygenase pathway inhibitor MK886. These supernatants were added to FURA-2-loaded eosinophils that had been preincubated with thioperamide and/or the PAF antagonist WEB-2086. We found that the increase in eosinophil calcium in response to supernatants from anti-IgE-stimulated-HLMC was totally inhibited only when the mast cells were challenged in the presence of indomethacin and MK886, and the eosinophils were preincubated with thioperamide. WEB-2086 had little effect. When we examined the effect of these mediators on eosinophil secretory function, we found that PGD2 (not histamine) primed eosinophils for enhanced release of LTC4 in response to the calcium ionophore A23187. We conclude that the activation of eosinophils by PGD2 and other mast cell products may contribute to airways inflammation that is characteristic of asthma.     

Leukotriene C4 production from human eosinophils in vitro. Role of eosinophil chemotactic factors on eosinophil activation

We studied the role of naturally occurring eosinophil chemotactic factors on leukotriene (LT)C4 production from highly purified (87.1 +/- 2.4%) normodense eosinophils. Platelet activating factor (PAF) directly induced LTC4 production from eosinophils in a dose (10(-9) to 10(-5) M) and a time-dependent manner. PAF (10(-5) M) induced 0.74 +/- 0.08 ng of LTC4 production/10(6) eosinophils. However, lyso-PAF, eosinophil chemotactic factor of anaphylaxis, and LTB4 failed to induce LTC4 production within the tested range. Furthermore, the pre-incubation of eosinophils with 5 micrograms/ml of cytochalasin B did not alter the chemotactic factor-induced LTC4 production. When eosinophils were stimulated by the submaximal concentration (1 microgram/ml) of calcium ionophore A23187, the pre-incubation of eosinophils with 10(-6) M or 10(-5) M of PAF, or 10(-5) M of eosinophil chemotactic factor of anaphylaxis significantly enhanced LTC4 production up to 163.9 +/- 17.5% (p less than 0.05), 279.2 +/- 32.9% (p less than 0.01) and 165.2 +/- 21.2% (p less than 0.05) of the control, respectively. However, the pre-incubation with lyso-PAF or LTB4 failed to enhance A23187-induced LTC4 production. The pre-incubation of eosinophils with phosphatidyl serine also failed to enhance A23187-induced LTC4 production. However, the direct stimulation of protein kinase C by PMA enhanced the submaximal concentration of A23187-induced LTC4 production from eosinophils up to 179.5 +/- 20.9% (p less than 0.05) of the control. Our findings indicate that PAF and ECF-A work not only as chemotactic factors but also induce a functionally active state of eosinophils probably through their post-receptor mechanisms, and contribute to the inflammatory processes.     

In vitro and in vivo chemotaxis of guinea pig leukocytes toward leukotriene B4 and its w-oxidation products

Leukotriene B4 (LTB4), 20-OH-LTB4, and 20-COOH-LTB4 were studied for their relative activities towards guinea pig peritoneal eosinophils and neutrophils during in vitro chemotaxis in modified Boyden chambers. The leukotrienes were also injected into guinea pig skin, and the cellular infiltrate in 4 hour biopsies was evaluated histologically. Eosinophils migrated more actively than neutrophils towards LTB4 in vitro, while in vivo, more neutrophils were observed. 20-OH-LTB4 was markedly less active than LTB4 in vivo and in vitro, and 20-COOH-LTB was barely active at all. Crude ionophore-stimulated neutrophil supernatants (ECF) were more active towards eosinophils than towards neutrophils, both in vivo and in vitro, compared to the pure leukotrienes. The data confirm the potent chemotactic properties of LTB4 for eosinophils and neutrophils, with less activity of its w-metabolites.     

A novel dioxygenation product of arachidonic acid possesses potent chemotactic activity for human polymorphonuclear leukocytes

We have found that a novel dioxygenation product of arachidonic acid, 8(S),15(S)-dihydroxy-5,11-cis-9,13-trans-eicosatetraenoic acid (8,15-diHETE), possesses chemotactic activity for human polymorphonuclear leukocytes comparable to that of leukotriene B4. Authentic 8,15-diHETE, identified by gas chromatography-mass spectrometry, was prepared by treating arachidonic acid with soybean lipoxygenase and was purified by reverse-phase high performance liquid chromatography. Using a "leading front" assay, 8,15-diHETE exhibited significant chemotactic activity at a concentration of 5.0 ng/ml. Maximum chemotactic activity was observed at a concentration of 30 ng/ml. The 8,15-diHETE generated by mixed human leukocytes after stimulation with arachidonic acid and the calcium ionophore, A23187, exhibited quantitatively similar chemotactic activity. Two synthetic all-trans conjugated isomers of 8,15-diHETE, however, were not chemotactic at concentrations up to 500 ng/ml. In contrast to its potent chemotactic activity, 8,15-diHETE (at concentrations up to 10 micrograms/ml) was relatively inactive with respect to its ability to provoke either degranulation or generation of superoxide anion radicals by cytochalasin B-treated leukocytes. Both leukotriene B4 and 8,15-diHETE may be important mediators of inflammation.     

Identification of 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid as a novel and potent human eosinophil chemotactic eicosanoid.

Incubation of human eosinophils with arachidonic acid led to the formation of a novel and potent eosinophil chemotactic lipid (ECL) (Morita, E., Schr?der, J.-M., and Christophers, E. (1990) J. Immunol. 144, 1893-1900). To test the working hypothesis of whether ECL could have been formed via eosinophil-arachidonic acid 15-lipoxygenase we investigated whether other arachidonic acid 15-lipoxygenases such as soybean lipoxygenase I catalyze formation of a similar ECL. In the presence of hemoproteins and soybean lipoxygenase I arachidonic acid is converted to an ECL, which has physicochemical properties similar to those found for the eosinophil-derived ECL. Purification of this ECL by high performance liquid chromatography revealed that ECL is structurally different from well known eosinophil chemotactic eicosanoids such as leukotriene B4, 5,15-(6E,8Z,11Z,13E)-dihydroxyeicosatetraenoic acid (5,15-diHETE), and (8S,15S)-(5Z,9E,11Z,13E)-dihydroxyeicosatetra eno ic acid ((8S,15S)-diHETE). UV spectra of this ECL with absorbance maxima at 230 and 278 nm revealed the presence of two independent chromophores such as a conjugated oxodiene and a conjugated diene. Catalytic hydrogenation of ECL methyl ester led to the formation of 5,15-dihydroxyarachidic acid methyl ester. Reduction of ECL with sodium borohydride produced a product which is identical with authentic (5S,15S)-(6E,8Z,11Z,13E)-diHETE. Formation of an ECL monomethoxime derivative supports the conclusion that this highly potent eosinophil chemotactic eicosanoid is structurally identical with 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid.     

Further studies on the mechanism of action of leukotrienes and histamine on guinea pig lung parenchyma. Role of calcium, phospholipase and methyltransferase

The contractile activity of leukotriene B4 (LTB4), leukotriene D4 (LTD4) and histamine on strips of guinea pig lung parenchyma was shown to be dependent on the calcium concentrations of the Krebs solution. The calcium channel blocker verapamil (2.0 to 15 microM) had an additive effect on the inhibitory activity of low calcium (0.1 mM) on contractions of guinea pig parenchyma to leukotrienes and histamine. Cobalt chloride, a divalent cation, also produced dose-dependent reductions of the myotropic activities of LTB4, LTD4 and histamine. An antagonist of calmodulin, trifluoperazine (1-200 microM), dose-dependently inhibited the contractile activity of the three agonists on the parenchyma strip. The IC50 of this compound for inhibition of histamine was much lower (2-3 microM) than the IC50 for inhibition of leukotrienes (75 microM). Valinomycin, a potassium ionophore, also interfere with the contractile activities of leukotrienes and histamine whereas a blocker of sodium channel, tetrodotoxin, had no effect on the activity of these agonists. Furthermore, an inhibitor of methyltransferase, 3-deazaadenosine, significantly diminished the responses of the parenchyma to leukotrienes and histamine. These results confirmed the important role of extracellular and intracellular calcium in the myotropic activity of leukotrienes and histamine in guinea pig lungs and showed that compounds which interfere either directly or indirectly with calcium mobilization into the lung smooth muscles, decreased the tissue responsiveness.     

Stereospecificity of leukotriene B4 and structure-function relationships for chemotaxis of human neutrophils

The chemotactic activity of leukotriene B4 (5S, 12R Dihydroxy 6, 14 cis, 8, 10 trans eicosatetraenoic acid) (LTB4) was examined by using a sensitive Boyden-chamber assay. The activity of LTB4 was compared to other biosynthetic stereoisomers: 5S, 12R Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (6-trans LTB4); 5S, 12S Dihydroxy 6, 8, 10 trans 14 cis eicosatetraenoic acid (12-epi-6-trans LTB4), 5S, 12S DiHETE; the metabolic product 20-Hydroxy LTB4 (20-OH LTB4); methylated LTB4 (Methyl-LTB4), and the related monoHETE 5-HETE and 12-HETE. The compounds were purified by several steps of reverse phase and straight phase HPLC. The LTB4 exhibits measurable chemotactic activity at 10(-9) M with maximal activity at 10(-7) M and an ED50 of 10(-8) M. The LTB4 isomers and monoHETE were less chemotactic than previously reported. The monoHETE (5-HETE and 12-HETE), the isomer 12-epi-6-trans LTB4, and 5S, 12S DiHETE fail to attract neutrophils at levels between 10(-6) and 10(-5) M. If these compounds are chemotactic, then activity is at least four orders of magnitude less than that of LTB4. The isomer 6-trans LTB4 at 10(-6) to 10(-5) M induced chemotaxis with an extrapolated ED50 value of 10(-5) M, indicating that a trans for cis change in configuration at position 6 reduces the chemotactic activity of LTB4 by 1000-fold. Conversely, the metabolic product 20-OH LTB4 is at least as active as the native compound LTB4. Methylation of the carboxyl group of LTB4 reduces its chemotactic activity by two orders of magnitude. These results indicate a high degree of stereospecificity for the LTB4 receptor with strict dependence on hydroxyl group, and triene configuration and considerable dependence on the carboxyl group. Modification at the aliphatic omega end of the LTB4 molecule has a minimal effect on function, suggesting that the hydrophobicity of this portion of the molecule is not important for optimal activity. Furthermore, we propose that metabolic products of LTB4 may be of greater importance than LTB4 as physiologic inflammatory mediators in vivo.     

Characterization and biologic properties of 5,12-dihydroxy derivatives of eicosapentaenoic acid, including leukotriene B5 and the double lipoxygenase product

Leukotriene B5 (LTB5) and three stereoisomers were prepared biosynthetically from eicosapentaenoic acid and compared with the analogous derivatives of arachidonic acid for their chemotactic and aggregating effects on human neutrophilic polymorphonuclear leukocytes. Leukotriene B4 (LTB4), LTB5, and the 6-trans-diastereoisomers of each were generated by activating polymorphonuclear leukocytes with the calcium ionophore A23187 in the presence of 14C-labeled and unlabeled arachidonic acid or 14C-labeled and unlabeled eicosapentaenoic acid, respectively. The double lipoxygenase products, (5S,12S)-6-trans-8-cis-LTB4 and (5S,12S)-6-trans-8-cis-LTB5, were generated from 5S-hydroxyeicosatetraenoic acid and racemic 5-hydroxyeicosapentaenoic acid intermediates by incubation with platelet sonicates. The products of each reaction were isolated by reverse-phase-high performance liquid chromatography and identified by their retention times relative to the appropriate totally synthetic standards, ultraviolet absorption spectra, immunoreactivity in a radioimmunoassay for LTB4, and, for all but the double lipoxygenase products, by incorporation of radiolabel from the specific polyunsaturated fatty acid source. When the concentration of LTB5 eliciting maximum chemotactic response of human polymorphonuclear leukocytes, 50 ng/ml (1.5 X 10(-7) M), and that eliciting a maximum aggregation response, 20 ng/ml (5.9 X 10(-8) M), were compared with the interpolated values of LTB4 eliciting comparable effects, the potency of LTB5 relative to LTB4 was approximately 1:8 as a chemotactic agent and about 1:20 as an aggregating agent. The double lipoxygenase products and the resolved 6-trans-diastereoisomers of the pentaene and tetraene series were about 2 logs less active as chemotactic factors than LTB4 and only (5S,12S)-6-trans-8-cis-LTB4 had even minimal aggregating activity.     

Biological activity of leukotriene B4 analogs: inhibition of guinea pig eosinophil migration in vitro by the 2,6-disubstituted pyridine analogs U-75,302 and U-75,485.

A "late phase" antigen-induced bronchoalveolar eosinophilia has been demonstrated in ovalbumin sensitized guinea pigs (1,2). This in vivo response to antigen inhalation can be inhibited by a 2,6-disubstituted pyridine analog of LTB4, U-75,302(2) (3). In the present study, the mechanism of the drug action was studied by assessing the activity of U-75,302 and a second analog, U-75,485 to displace [3H]-leukotriene B4 binding at the guinea pig eosinophil membrane, as well as their action as chemoattractants or inhibitors of the directional migration of guinea pig eosinophils in vitro. Radioligand competition experiments demonstrated that both analogs interacted strongly with the high affinity LTB4 binding sites on guinea pig eosinophil membrane. Both analogs are powerful chemoattractants for guinea pig eosinophils since they induced directional migration of guinea pig eosinophils when administered alone. In addition, when the cells were treated with either analog and their chemotaxis response was measured in response to a natural chemoattractant, both U-75,302 and U-75,485 at concentrations of 0.1 to 100 microM dose dependently inhibited the LTB4 induced chemotaxis response. The EC50s obtained for U-75,302 and U-75,485 as inhibitors of LTB4 induced guinea pig eosinophil chemotaxis were estimated to be 11.5 +/- 5.5 microM and 5.4 +/- 2.5 microM respectively. Under the same conditions, they had no significant effect upon eosinophil migration induced by zymosan activated plasma at concentrations below 100 microM. We suggest that the inhibition of antigen-induced eosinophil infiltration in guinea pig airway in vivo by U-75,302 or U-75,485 may be a result of partial antagonism or desensitization at the LTB4 receptor level of guinea pig eosinophils.     

Low molecular weight eosinophil chemotactic factor in granulomatous liver of murine schistosomiasis.

An acidic peptide, preferentially chemotactic for eosinophils, was extracted from livers of mice infected with Schistosoma mansoni. Sephadex G-25 column chromatography showed that the majority of the eosinophil chemotactic activity was detected in the fractions just after elution of the molecular marker vitamin B12 (m.w. 1355.4). This activity began to appear in the livers of some mice 5 weeks after infection. Peak activity was detected at 8 to 12 weeks after infection and persisted at least until 16 weeks. It was sensitive to carboxypeptidase-A. By Dowex-1 anion exchange chromatography, the activity eluted as a narrow peak at pH 3.1 TO 2.6 as shown for eosinophil chemotactic factor of anaphylaxis (ECF-A). The activity was also detected in a broad peak at pH 6.3 to 3.7. Unlike ECF-A, the activity was stable to boiling in both acid and alkali. These findings suggest that granulomatous liver of murine schistosomiasis-derived eosinophil chemotactic factor (ECF-G) may play a specific role in eosinophil accumulation in this chronic inflammation.     

An in vivo chemotaxis assay in the dog trachea: evidence for chemotactic activity of 8,15-diHETE

We describe a new in vivo chemotaxis assay in the dog trachea using a double-balloon endotracheal catheter. When inflated, the two balloons isolate a segment of trachea, which is perfused through Silastic tubes using a peristaltic pump. After instilling a chemotactic agent, the perfusate is sampled periodically to permit characterization of the chemotactic response. We anesthetized four mongrel dogs and ventilated them mechanically through the double-balloon catheter. Two mediators, leukotriene B4 (LTB4) and 8S,15S-dihydroxyeicosatetraenoic acid (8,15-diHETE) were tested in each dog by perfusing the trachea with each mediator in Hanks' balanced salt solution (HBSS) containing ethanol and antibiotics. Aliquots were removed for differential cell counts at fixed time intervals over a 4-h period. Control experiments performed in each dog with the identical concentrations of ethanol and antibiotics in HBSS showed no cellular response before 180 min. At 240 min, the cell counts were 86 +/- 28 (SE) granulocytes/microliter (n = 4). In contrast, both LTB4 and 8,15-diHETE gave a significant cellular response at 120 min (309 +/- 125 and 141 +/- 41 granulocytes/microliter, respectively; P less than 0.05) but did not differ significantly from each other. These results suggest that both LTB4 and 8,15-diHETE can incite inflammatory responses in the dog trachea in vivo. Furthermore, the double-balloon catheter technique promises to be a useful in vivo chemotaxis assay.     

Activation of guinea pig eosinophils by human recombinant IL-5. Selective priming to platelet-activating factor-acether and interference of its antagonists.

The potential role of platelet-activating factor (PAF)-acether and of IL-5 as an eosinophil-proliferating, activating, and/or recruiting mediator in asthma led us to study the effects of human (h) rIL-5 (hrIL-5) and PAF-acether, alone or combined, on isolated guinea pig eosinophils. Two populations of eosinophils were separated from peritoneal lavages of polymyxin B-treated guinea pigs upon a discontinuous metrizamide gradient: one of low density (between 20 and 22% of metrizamide, purity: 63 +/- 3%, n = 27) and another of normal density (between 22 and 24% of metrizamide, purity: 87 +/- 2%, n = 16). Chemotactic activity was evaluated on a micro-Boyden chamber, results being expressed as the number of migrating eosinophils (mean +/- SEM) at 40 microns through a cellulose nitrate filter (3 microns pore size) in the presence of the agonist or of the solvent alone. hrIL-5 dose-dependently stimulated normodense eosinophil chemotaxis, reaching a peak at 500 ng/ml (98 +/- 21 migrating eosinophils, n = 5, p less than 0.05). These eosinophils also responded to PAF-acether and to LTB4 and not to FMLP, hrTNF alpha, and LPS. Eosinophil preincubation with hrIL-5 increased significantly the migration by PAF-acether (173 +/- 23 migrating eosinophils with PAF-acether 10 nM after preincubation with hrIL-5 500 ng/ml vs 69 +/- 10 after preincubation with buffer alone, p less than 0.01) and failed to enhance migration by LTB4 or to uncover an activity for FMLP. Migration by PAF-acether was antagonized when the cells were preincubated with the antagonists BN 52021 and WEB 2086, which also inhibited migration by hrIL-5. Eosinophils were auto-desensitized by and to PAF-acether or LTB4, but were not cross-desensitized to each other. Eosinophils desensitized to PAF-acether failed to migrate with hrIL-5, but those desensitized to LTB4 responded to hrIL-5 as controls. hrIL-5 failed to induce the elevation of intracellular free calcium concentration and superoxide anion generation from basal values, whereas preincubation of eosinophils with hrIL-5 induced a significant increase in the rise in intracellular free calcium concentration and in superoxide anion generation by 10 nM PAF-acether but not by LTB4. In conclusion, the in vivo eosinophil migration in allergy may involve hrIL-5, particularly associated to PAF-acether.     

Functional properties of guinea pig eosinophil leukotriene B4 receptor.

It is currently thought that pulmonary eosinophils play a proinflammatory role in bronchial asthma. Leukotriene B4 (LTB4) is being considered as an important mediator in regulating eosinophil function because of its potent activities in inducing leukocyte chemotaxis, chemokinesis, degranulation, and aggregation. Because the LTB4 receptor has not been characterized in eosinophils, we report in this study the presence of a functional high affinity receptor for LTB4 on guinea pig (GP) eosinophils. Scatchard analysis of saturation binding studies yielded a Kd of 1.4 +/- 0.2 nM (mean +/- SEM, n = 3) and a Bmax of 1.6 +/- 0.4 pmol/mg of protein for LTB4 in GP eosinophil membranes. A linear Scatchard plot was obtained, suggesting that GP eosinophil membranes expressed only a single high affinity LTB4 receptor population. Saturation binding studies in whole cells also yielded a linear Scatchard plot, with a Kd of 2.8 +/- 0.96 nM (mean +/- SEM, n = 4) and a Bmax of 4 x 10(4) +/- 6 x 10(3) receptors/cell. Competitive binding studies using several compounds with structures similar to that of LTB4 showed that these agents bound to the receptor in the following descending order of affinity (Ki, nM): LTB4 (0.96) less than TB3 (1.0) greater than 20-hydroxy-LTB4 (3.5) greater than 12(R)-hydroxy-5,8,14-cis,10-trans-eicosatetraenoic acid (20) greater than 12(S)-hydroxy-5,8,14-cis,10-trans-eicosatetraenoic acid (231) greater than 20-carboxy-LTB4 (350) greater than 5(S),12(S)-dihydroxy-6,10-trans,8,14-cis-eicosatetraenoic acid (541). This rank order of potency in binding affinity correlates closely with the ability of these compounds to induce both chemotaxis and superoxide anion generation. Analysis of the structure-activity relationship suggests that the 12R-hydroxyl group and a cis double bond at the C-6 position are important for optimal agonist binding to the LTB4 receptor present in GP eosinophil membranes. The results suggest that LTB4 may be an important chemoattractant for eosinophils in GP and may induce the release of reactive oxygen species from this cell.     

Metabolism of 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid and other 5(S)-hydroxyeicosanoids by a specific dehydrogenase in human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNL) convert 6-trans isomers of leukotriene B4 (LTB4) to dihydro metabolites (Powell, W.S., and Gravelle, F. (1988) J. Biol. Chem. 263, 2170-2177). In the present study we investigated the mechanism for the initial step in the formation of these products. We found that the 1,500 x g supernatant fraction from human PMNL converts 12-epi-6-trans-LTB4 to its 5-oxo metabolite which was identified by mass spectrometry and UV spectrophotometry. The latter compound was subsequently converted to the corresponding dihydro-oxo product, which was further metabolized to 6,11-dihydro-12-epi-6-trans-LTB4, which was the major product after longer incubation times. The 5-hydroxyeicosanoid dehydrogenase activity is localized in the microsomal fraction and requires NADP+ as a cofactor. These experiments therefore suggest that the initial step in the formation of dihydro metabolites of 6-trans isomers of LTB4 is oxidation of the 5-hydroxyl group by a microsomal dehydrogenase. Studies with a variety of substrates revealed that the microsomal dehydrogenase in human PMNL oxidizes the hydroxyl groups of a number of other eicosanoids which contain a 5(S)-hydroxyl group followed by a 6-trans double bond. There is little or no oxidation of hydroxyl groups in the 8-, 9-, 11-, 12-, or 15-positions of eicosanoids, or of the 5-hydroxyl group of LTB4, which has a 6-cis rather than a 6-trans double bond. The preferred substrate for this enzyme is 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5(S)-HETE) (Km, 0.2 microM), which is converted to 5-oxo-6,8,11,14-eicosatetraenoic acid. Unlike 5(S)-HETE, 5(R)-HETE is a poor substrate for the 5(S)-hydroxyeicosanoid dehydrogenase, indicating that in addition to exhibiting a high degree of positional specificity, this enzyme is also highly stereospecific. In addition to 5(S)-HETE and 6-trans isomers of LTB4, 5,15-diHETE is also a good substrate for this enzyme, being converted to 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid (5-oxo-15-hydroxy-ETE). The oxidation of 5(S)-HETE to 5-oxo-ETE is reversible since human PMNL microsomes stereospecifically reduce 5-oxo-ETE to the 5(S)-hydroxy compound in the presence of NADPH. 5-Oxo-ETE is formed rapidly from 5(S)-HETE by intact human PMNL, but because of the reversibility of the reaction, its concentration only reaches about 25% that of 5(S)-HETE.     

Chemotaxis of human neutrophils and eosinophils towards leukotriene B4 and its 20-w-oxidation products in vitro

Peripheral blood neutrophils and eosinophils from 70 patients and controls were studied for their in vitro chemotactic and chemokinetic responses towards synthetic leukotriene B4 (LTB4), 20-OH-LTB4 and 20-COOH-LTB4. All three factors induced chemotaxis and chemokinesis of cells. 20-OH-LTB4 was always less and 20-COOH-LTB4 even less active than the parent compound. Cells from patients with atopic eczema and T cell lymphoma moved less than cells from normal controls or from patients with psoriasis. In the presence of LTB4, 20-OH-LTB4 and buffer alone, more eosinophils than neutrophils moved to the lower side of the filter, while this did not occur with platelet activating factor as chemoattractant. Studies of neutrophil and eosinophil chemotaxis in the presence of LTB4 should therefore always take into account a high variability of the quantitative response which is donor and disease dependent.     

11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid: an endothelium-derived 15-lipoxygenase metabolite that relaxes rabbit aorta

Previous studies indicate that 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA), an endothelium-derived hyperpolarizing factor in the rabbit aorta, mediates a portion of the relaxation response to acetylcholine by sequential metabolism of arachidonic acid by 15-lipoxygenase, hydroperoxide isomerase, and epoxide hydrolase. To determine the stereochemical configuration of the endothelial 11,12,15-THETA, its activity and chromatographic migration were compared with activity and migration of eight chemically synthesized stereoisomers of 11,12,15(S)-THETA. Of the eight isomers, only 11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid comigrated with the biological 11,12,15-THETA on reverse- and normal-phase HPLC and gas chromatography. The same THETA isomer (10(-7)-10(-4) M) relaxed the rabbit aorta in a concentration-related manner (maximum relaxation = 69 +/- 5%). These relaxations were blocked by apamin (10(-7) M), an inhibitor of small-conductance Ca2+-activated K+ channels. In comparison, 11(S),12(R),15(S),5(Z),8(Z),13(E)-THETA (10(-4) M) relaxed the aorta by 22%. The other six stereoisomers were inactive in this assay. With use of the whole cell patch-clamp technique, it was shown that 10(-4) M 11(R),12(S),15(S),5(Z),8(Z),13(E)-THETA increased outward K+ current in isolated aortic smooth muscle cells by 119 +/- 36% at +60 mV, whereas 10(-4) M 11(R),12(R),15(S),5(Z),8(Z),13(E)-THETA increased outward K+ current by only 20 +/- 2%. The 11(R),12(S),15(S),5(Z),8(Z),13(E)-THETA-stimulated increase in K+ current was blocked by pretreatment with apamin. These studies suggest that 11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid is the active stereoisomer produced by the rabbit aorta. It relaxes smooth muscle by activating K+ channels. The specific structural and stereochemical requirements for K+ channel activation suggest that a specific binding site or receptor of 11,12,15-THETA is involved in these actions.     

Increased biosynthesis of platelet-activating factor in activated human eosinophils

1-Alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase catalyzes the conversion of biologically inactive lysophospholipid to bioactive platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) by an acetylation reaction. The activity of this enzyme in eosinophils isolated from patients with eosinophilia is stimulated (up to 4-fold) in a dose-, time-, and Ca2+/Mg2+-dependent manner after exposure to the eosinophil chemotactic factor of anaphylaxis (ECF-A), C5a, formyl-methionylleucylphenylalanine (fMLP), or ionophore A23187. The three naturally occurring chemotactic factors (ECF-A, C5a, and fMLP) cause a rapid and transient increase of enzyme activity, with a maximum at 1 or 3 min, whereas ionophore A23187 maintains an elevated level for up to 15 min. The activity of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase, an enzyme that catalyzes the breakdown of PAF to lyso-PAF, is not affected by C5a, fMLP, or ionophore A23187. The presence of PAF in eosinophils was established by demonstrating the lipid nature of the compound, the RF value being identical with that of synthetic 1-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine on thin layer chromatograms, and by its ability to induce serotonin release from rabbit platelets. Furthermore, ECF-A, C5a, fMLP, and ionophore A23187 all induce the secretion of PAF from eosinophils. These findings suggest that the generation and release of PAF could be a consequence of eosinophil chemotactic activation and may thus function in inflammatory and allergic reactions in which eosinophils participate.     

Structural requirements for chemotactic activity of leukotriene B4 (LTB4)

LTB4 (5s, 12R dihdroxy-6, 14-CIS-8, 10-trans-eicosatetraenoic acid) formed in activated neutrophils by lipoxygenation of arachidonic acid is an extremely potent chemotaxin. We examined structural requirements for chemotactic and aggregatory activity of the ligand using synthetic LTB4 and several of its isomers. Additionally we examined the potency of two analogs, nor- and homo-LTB4. Dose response curves for neutrophil chemotaxis to these compounds were obtained using a modified Boyden chamber. The mean distance cells moved into the filter was determined after 30 minutes. Peak chemotactic activity of LTB4 was at 10(-7)M. At higher concentrations, chemotactic activity was decreased. The shape of the dose response curve was similar to that of FMLP except that maximum chemotaxis to LTB4 was consistently greater than chemotaxis to FMLP. A mixture of the two epimers at c-5 and c-12 shifted the response curve to the right but did not lower maximum activity. Increasing or decreasing the chain by one carbon between the first hydroxyl group and the carboxyl group also shifted the response curve to the right without lowering maximal activity. Changing the 6 double bond from cis to trans has a greater effect. Activity was only detectable at high concentrations and maximum activity achieved was less than 50% that of LTB4. Thus the chain length between the carboxyl and C-5 hydroxyl groups, the c-5 and c-12 absolute stereochemistry and the stereochemistry of the delta6 double bond are all important structural features for chemotactic activity with delta6 stereochemistry apparently having the greatest contribution. The relative potencies of these compounds in inducing aggregation were comparable to their chemotactic potencies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leukotriene B4 is an indirectly acting vasoconstrictor in guinea pig aorta via an inducible type of BLT receptor

Leukotriene B(4) (LTB(4)) is a potent leukocyte chemoattractant recently implicated in the pathogenesis of atherosclerosis. The aim of this study was to assess the effects of LTB(4) on isolated aortic preparations. Rings of guinea pig aorta were challenged with LTB(4) for recording mechanical responses and measurements of mediator release, and LTB(4) receptor (BLT(1)) expression was assessed by RT-PCR. Single concentrations of LTB(4) induced concentration-dependent contractions that were inhibited by treatment with antihistamines, indomethacin, or the thromboxane receptor antagonist BAYu3405 as well as by denudation of endothelium. In addition, LTB(4) increased the release of histamine and thromboxane in the bath. The contractions induced by LTB(4) were inhibited by either the unselective BLT receptor antagonist ONO-4057 or the selective BLT(1) receptor antagonist U-75302. Pretreatment with all-trans-retinoic acid enhanced the contractions and the release of histamine induced by LTB(4), without affecting either the contractions induced by histamine or the histamine release evoked by calcium ionophore A23187. Analysis by RT-PCR indicated the expression of a BLT(1) receptor in the guinea pig aorta and that BLT(1) receptor mRNA was upregulated after treatment with retinoic acid. These results suggest that LTB(4) contracts the guinea pig aorta via an indirect mechanism involving the release of histamine and thromboxane and that this BLT(1) receptor-mediated response can be upregulated by all-trans-retinoic acid.     

Leukotrienes cause eosinophil emigration into conjunctival tissue

The ability of LTB4, LTC4, the 5S,6R and 5R,6S LTD4 stereoisomers, and LTE4 to evoke leukocyte infiltration into the conjunctiva was demonstrated in the guinea pig by histological and light microscopy techniques. LTD4 and LTE4 demonstrated a dose-dependent and predominantly eosinophilic infiltrate over the selected dose range (10 ng to 1000 ng), while there was only a minimal response to LTC4. LTB4 produced marked eosinophil infiltrates only at the highest dose; scattered neutrophil infiltrates were also noted at the high dose of LTB4. The 5R,6S LTD4 stereoisomer did not evoke any leukocyte infiltration. The SRS-A antagonist, FPL 55712, abolished peptidoleukotriene-induced eosinophil emigration, and indomethacin pre-treatment had no inhibitory effect, indicating direct mediation of this response by LTs. Histamine caused a comparable eosinophilia over a dose range of 10 micrograms to 1000 micrograms. LT-induced eosinophil emigration was directed to the conjunctival epithelium; the cells appeared intact and no tissue damage was observed. These results may have relevance in the areas of allergic conjunctivitis and asthma research.