lipoxin-b4 and 15-hydroxy-5-8-11-13-eicosatetraenoic-acid

The unstable epoxide leukotriene (LT) A(4) is a key intermediate in leukotriene biosynthesis, but may also be transformed to lipoxins via a second lipoxygenation at C-15. The capacity of various 12- and 15-lipoxygenases, including porcine leukocyte 12-lipoxygenase, a human recombinant platelet 12-lipoxygenase preparation, human platelet cytosolic fraction, rabbit reticulocyte 15-lipoxygenase, soybean 15-lipoxygenase and human eosinophil cytosolic fraction, to catalyze conversion of LTA(4) to lipoxins was investigated and standardized against the ability of the enzymes to transform arachidonic acid to 12- or 15-hydroxyeicosatetraenoic acids (HETE), respectively. The highest ratio between the capacity to produce lipoxins and HETE (LX/HETE ratio) was obtained for porcine leukocyte 12-lipoxygenase with an LX/HETE ratio of 0.3. In addition, the human platelet 100000xg supernatant 12-lipoxygenase preparation and the human platelet recombinant 12-lipoxygenase and human eosinophil 100000xg supernatant 15-lipoxygenase preparation possessed considerable capacity to produce lipoxins (ratio 0.07, 0.01 and 0.02 respectively). In contrast, lipoxin formation by the rabbit reticulocyte and soybean 15-lipoxygenases was much less pronounced (LX/HETE ratios <0.002). Kinetic studies of the human lipoxygenases revealed lower apparent K(m) for LTA(4) (9-27 microM), as compared to the other lipoxygenases tested (58-83 microM). The recombinant human 12-lipoxygenase demonstrated the lowest K(m) value for LTA(4) (9 microM) whereas the porcine leukocyte 12-lipoxygenase had the highest V(max). The profile of products was identical, irrespective of the lipoxygenase used. Thus, LXA(4) and 6S-LXA(4) together with the all-trans LXA(4) and LXB(4) isomers were isolated. Production of LXB(4) was not observed with any of the lipoxygenases. The lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate was considerably more efficient to inhibit conversion of LTA(4) to lipoxins, as compared to the inhibitory effect on 12-HETE formation from arachidonic acid (IC(50) 1 and 50 microM, respectively) in the human platelet cytosolic fraction.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arachidonic Acid; Blood Platelets; Enzyme Inhibitors; Humans; Hydroxyeicosatetraenoic Acids; Leukocytes; Leukotriene A4; Lipoxins; Rabbits; Reticulocytes; Swine

This study investigates the production of interleukin (IL-)1beta by cultured human bone marrow stromal cells. RT-PCR experiments indicate that two-thirds of cultures constitutively express IL-1beta mRNA transcripts. Their cell-associated IL-1beta levels are elevated after stimulation with tumour necrosis factor (TNF-)alpha but not with cytokines such as IL-1alpha, IL-3, IL-4, IL-6, IL-7, IL-10, SCF, G-CSF, M-CSF and TGF-beta or lipid mediators such as PGE2, LTB4, LXA4, LXB4, 12-HETE, 15-HETE and PAF. Addition of IL-4, but not IL-10 or TGF-beta, reduces the TNF-alpha-induced cell-associated IL-1beta. IL-1beta is never detected in bone marrow stromal cell supernatants whatever the stimulant added. In conclusion the pro-inflammatory molecule TNF-alpha stimulates bone marrow stromal cell-associated IL-1beta levels while the anti-inflammatory cytokine IL-4 reduces the TNF-alpha-induced effect. These results strengthen the key regulatory role of IL-4 on the production of haematopoietic cytokines by human bone marrow stromal cells.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Bone Marrow Cells; Cells, Cultured; Cytokines; Dinoprostone; Humans; Hydroxyeicosatetraenoic Acids; Interleukin-1; Leukotriene B4; Lipid Metabolism; Lipoxins; Platelet Activating Factor; Stromal Cells

Asthma is characterized by chronic airway inflammation resulting from overproduction of pro-inflammatory mediators, such as leukotrienes (LT). The authors questioned the biosynthetic capacity of asthmatic patients for lipoxins (LX) and 15-epimer lipoxins (15-epi-LX), endogenous regulators of inflammatory responses that inhibit pro-inflammatory events. Levels of LXA4, 15-epi-LXA4 and LTC4 were determined in 14 clinically characterized aspirin-intolerant asthmatics (AIA), 11 aspirin-tolerant asthmatics (ATA) and eight healthy volunteers using a stimulated whole blood protocol. Both LXA4 and 15-epi-LXA4 were generated in whole blood activated by the divalent cation ionophore, A23187. Higher levels of LXA4 were produced in ATA than either AIA or healthy volunteers. Exposure of AIA whole blood to interleukin-3 prior to A23187 did not elevate their reduced capacity to generate LXA4. Generation of a bronchoconstrictor, LTC4, was similar in both AIA and ATA. Consequently, the ratio of LXA4:LTC4 quantitatively favoured the bronchoconstrictor for AIA and differed from both ATA and healthy subjects. In addition, the capacity for 15-epi-LXA4 generation was also diminished in AIA, since whole blood stimulated in the presence of aspirin gave increased levels only in samples from ATA. The present results indicate that asthmatics possess the capacity to generate both lipoxins and 15-epimer-lipoxins, but aspirin-intolerant asthmatics display a lower biosynthetic capacity than aspirin-tolerant asthmatics for these potentially protective lipid mediators. This previously unappreciated, diminished capacity for lipoxin formation by aspirin-intolerant asthmatic patients may contribute to their more severe clinical phenotype, and represents a novel paradigm for the development of chronic inflammatory disorders.

Topics: Adult; Aspirin; Asthma; Chromatography, High Pressure Liquid; Enzyme-Linked Immunosorbent Assay; Female; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene C4; Lipoxins; Male; Middle Aged; Stereoisomerism

The influence of lipoxygenase metabolites of arachidonic acid on proliferation and differentiation of CD34(+) cells was studied. Their effects on the CFU-GM and BFU-E progenitors were investigated by culture of CD34(+) cells in liquid or semisolid medium. Only 12-HETE (1 microM) stimulated the [(3)H]thymidine as well as BrdU incorporation and increased the number of cell divisions (PKH2 tracking). Addition of 12-HETE and 15-HETE but not of LXA(4), LXB(4), LTB(4), and LTC(4) to liquid cultures of CD34(+) cells for 3 and 8 days reduced in a time-dependent manner the number of CFU-GM and BFU-E. Both HETEs also increased the percentage of glycophorin A(+) cells while they reduced the percentage of CD34(-)/CD33(+) cells after 3 and 5 days of liquid cultures. These results show that HETE treatment stimulates proliferation and accelerates the differentiation of CD34(+) cells, mostly toward the erythroid lineage.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Antigens, CD34; Antigens, Differentiation; Arachidonic Acids; Cell Count; Cell Division; Colony-Forming Units Assay; Culture Media; Dose-Response Relationship, Drug; Erythroid Precursor Cells; Erythropoietin; Hematopoietic Stem Cells; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxins; Lipoxygenase; Methylcellulose; Solutions; Thymidine; Time Factors

The objectives of this work are to investigate the incorporation of arachidonic acid (AA) in the human myeloma cell lines OPM2, U266 and IM9, and to assess the effect of AA and lipoxygenase products of AA on their growth. The kinetics of acylation of [3H]AA indicates that myeloma cells incorporate AA into their membrane phospholipids and triglycerides. PLA2-treatment and base hydrolysis experiments confirm that [3H]AA is incorporated unmodified in U266, IM9 and OPM2 phospholipids, and is linked by an ester bond. Prelabeling-chase experiments indicate no trafficking of labeled AA among the various phospholipid species. Addition of AA and lipoxygenase products of AA (leukotriene B4 and C4, lipoxin A4 and B4, 12- and 15-hydroxyeicosatetraenoic acid) have no effect on U266, IM9 and OPM2 proliferation assessed by [3H]thymidine incorporation into DNA. In conclusion, while human myeloma cells readily incorporate AA in their membrane phospholipids and triglycerides, AA and lipoxygenase products are not important modulators of their proliferation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Acylation; Arachidonic Acid; Cell Division; Cell Membrane; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Leukotriene C4; Lipoxins; Lipoxygenase; Membrane Lipids; Multiple Myeloma; Phospholipids; Triglycerides; Tumor Cells, Cultured

Arachidonic acid (AA) metabolites derived from both cyclooxygenase (COX) and lipoxygenase (LOX) pathways transduce a variety of signals related to cell growth. Here, we report that the AA LOX pathway also functions as a critical regulator of cell survival and apoptosis. Rat Walker 256 (W256) carcinosarcoma cells express 12-LOX and synthesize 12(S)- and 15(S)-hydroxyeicosatetraenoic acids as their major LOX metabolites. W256 cells transfected with 12-LOX-specific antisense oligonucleotide or antisense oligonucleotides directed to conserved regions of LOXs underwent time- and dose-dependent apoptosis. Likewise, treatment of W256 cells with various LOX but not COX inhibitors induced apoptotic cell death, which could be partially inhibited by exogenous 12(S)- or 15(S)-hydroxyeicosatetraenoic acids. The W256 cell apoptosis induced by antisense oligos and LOX inhibitors was followed by a rapid downregulation of bcl-2 protein, a dramatic decrease in the bcl-2/bax ratio, and could be suppressed by bcl-2 overexpression. In contrast, p53, which is wild type in W256 cells, did not undergo alterations during apoptosis induction. The results suggest that the LOX pathway plays an important physiological role in regulating apoptosis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Apoptosis; Arachidonate 12-Lipoxygenase; Base Sequence; Cell Division; Cell Line; Cell Survival; Female; Homeostasis; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Leukotriene C4; Leukotrienes; Lipid Peroxides; Lipoxins; Masoprocol; Molecular Sequence Data; Oligonucleotides, Antisense; Plasmids; Rats; Transfection

Eicosanoids derived from lipoxygenase (LO)-catalyzed reactions play important roles in pulmonary inflammation. Here, we examined formation of LO-derived products by human alveolar macrophages (HAM). HAM converted [1-14C]-arachidonic acid to a product carrying 14C-radiolabel that was identified as 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) by physical methods. 15-LO mRNA was demonstrated in HAM by reverse transcription-polymerase chain reaction. Incubation of HAM for 3 d with interleukin 4(IL-4) before exposure to [1-14C]arachidonic acid led to both increased mRNA for 15-LO and a 4-fold increase in 15-HETE formation. In contrast, 5(S)-hydroxy-6-trans-8,11,14-cis-eicosatetraenoic acid generation was not significantly altered by prior exposure to IL-4. Additionally, lipoxins (LXA4 and LXB4) were detected from endogenous substrate, albeit in lower levels than leukotriene B4 (LTB4), in electrochemical detection/high performance liquid chromatography profiles from HAM incubated in the presence and absence of the chemotactic peptide (FMLP) or the calcium ionophore (A23187). Exposure of HAM to leukotriene A4 (LTA4) resulted in a 2-fold increase in LXA4 and 10-fold increase in LXB4. These results demonstrate the presence of 15-LO mRNA and enzyme activity in HAM and the production of LXA4 and LXB4 by these cells. Along with 5-LO-derived products, the biosynthesis of 15-LO-derived eicosanoids by HAM may also be relevant in modulating inflammatory responses in the lung.

Topics: Arachidonate 15-Lipoxygenase; Base Sequence; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Interleukin-4; Leukotrienes; Lipoxins; Macrophages, Alveolar; Molecular Sequence Data; Oligodeoxyribonucleotides; RNA, Messenger

Human alveolar macrophages (AM) from bronchoalveolar lavage of asthmatic patients (AP) and healthy volunteers (HS) were compared for their respective capacities to produce lipoxins and leukotrienes when stimulated by calcium ionophore A23187 with or without 15(S)-HETE. The metabolites were analyzed using an isocratic RP-HPLC system and their formation profiles evaluated on the basis of chromatographic behaviour, UV spectral characteristics and co-elution with synthetic standards. Without 15-HETE, AM from AP produced more LTB4 and 5-HETE than those from HS. In the presence of 15-HETE, human AM were able to produce 5,15-diHETE and lipoxins. Moreover, the total amount of lipoxins synthesized by AM from AP was 2 fold higher than that synthesized by AM from HS, thus showing an enhanced cell activation via the 5-lipoxygenase (5-LO) pathway. These results presented AM as in vitro 15-HETE metabolizing cells and suggested some hypothesis about human AM 5-LO regulation mechanism. The enhanced 5-LO activity in AM from AP suggested that in vivo they could participate in cell to cell interaction mechanisms involved in inflammatory lung diseases and might also take up and transform 15-HETE predominantly released by airway epithelial cells.

Topics: Adult; Asthma; Bronchoalveolar Lavage Fluid; Calcimycin; Cells, Cultured; Chromatography, High Pressure Liquid; Epithelial Cells; Epithelium; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxins; Lung; Macrophage Activation; Macrophages, Alveolar; Middle Aged

1. The effects of some prostanoids, leukotrienes, lipoxins and lipoxin precursors (15-HETE, 15-HPETE) were examined in guinea-pig isolated basilar arteries. 2. The potency order among the prostanoids to elicit contraction was U44069 greater than prostaglandin B2 greater than prostaglandin F2 alpha greater than prostaglandin E2. Leukotriene C4 and D4 were approximately equipotent with prostaglandin B2. 3. Lipoxin A4 and B4 elicited small contractions (4% of the contractile response to 124 mM K+ at 3 x 10(-6) M), which were significantly (P less than 0.02) enhanced by indomethacin. The contractile responses to 15-HETE and 15-HPETE varied considerably (2-102% and 2-56% at 3 x 10(-6) M, respectively) between different vascular segments. 4. Among the leukotrienes, lipoxins and lipoxin precursors, only lipoxin A4 elicited a relaxation, albeit small and transient. 5. In summary, all examined eicosanoids contracted the guinea-pig basilar artery, although the responses to the lipoxins were small but significantly enhanced by cyclooxygenase inhibition.

Topics: Animals; Basilar Artery; Dose-Response Relationship, Drug; Female; Guinea Pigs; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Lipoxins; Lipoxygenase; Male; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Vasoconstriction

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 15-Lipoxygenase; Bronchi; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotrienes; Lipoxins; Nasal Polyps; Neutrophils

Alveolar macrophages (AM) obtained both from specific viral antibody-positive (VA+) rats and from specific viral antibody-negative (VA-) rats were compared for their respective capacity to produce lipoxins (LX) in a given experimental condition. Both cell preparations were incubated with (15S), 15-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) in the presence of calcium ionophore A23187. The reaction mixtures were extracted and chromatographed on reverse-phase high performance liquid chromatography (RP-HPLC). Profiles of LX formation in each group were evaluated on the basis of chromatographic behavior, UV spectral characteristics (lambda max at 301 nm with shoulders at 289 nm and 317 nm), and co-elution with synthetic lipoxin A4 (LXA4) and lipoxin B4 (LXB4) standards as well as with authentic materials. The AM harvested from VA+ rats (AM-VA+) produced consistently detectable levels of LX (i.e., LXA4 and LXB4), whereas the AM from VA- rats (AM-VA-) produced no detectable LX under the same experimental condition. These results offer a possibility that elevated capacity of the AM-VA+ to produce LX may be associated with previous exposures to selected specific viral pathogens and/or with viral infections. Considering the distinct biologic properties of LX, the apparent production of the enhanced levels of LX as shown in this model system may play an important role in both pathophysiology and immunoregulatory network in virus-induced pulmonary disorder.

Topics: Animals; Antibodies, Viral; Calcimycin; Chromatography, High Pressure Liquid; Hydroxyeicosatetraenoic Acids; Lipoxins; Macrophages; Male; Pulmonary Alveoli; Rats; Rats, Inbred Strains; Spectrophotometry, Ultraviolet

Human neutrophils from peripheral blood may physically interact with platelets in several settings including hemostasis, inflammation, and a variety of vascular disorders. A role for lipoxygenase (LO)-derived products has been implicated in each of these events; therefore, we investigated the formation of lipoxins during coincubation of human neutrophils and platelets. Simultaneous addition of FMLP and thrombin to coincubations of these cells led to formation of both lipoxin A4 and lipoxin B4, which were monitored by reversed-phase high pressure liquid chromatography. Neither stimulus nor cell type alone induced the formation of these products. When leukotriene A4 (LTA4), a candidate for the transmitting signal, was added to platelets, lipoxins were formed. In cell-free 100,000 g supernatants of platelet lysates, which displayed 12-LO activity, LTA4 was also transformed to lipoxins. Platelet formation of lipoxins was inhibited by the LO inhibitor esculetin and partially sensitive to chelation of Ca2+, while neither acetylsalicylic acid nor indomethacin significantly inhibited their generation. In contrast, neutrophils did not transform LTA4 to lipoxins. Cell-free 100,000 g supernatants of neutrophil lysates converted LTA4 to LTB4. These results indicate that neutrophil-platelet interactions can lead to the formation of lipoxins from endogenous sources and provide a role for platelet 12-LO in the formation of lipoxins from LTA4.

Topics: Arachidonate 12-Lipoxygenase; Arachidonate Lipoxygenases; Blood Platelets; Calcium; Cell Communication; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene A4; Leukotrienes; Lipoxins; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Stereoisomerism; Thrombin

Chopped human nasal polyps and bronchial tissue produced lipoxin A4 and isomers of lipoxins A4 and B4, but not lipoxin B4, after incubation with exogenous leukotriene A4. In addition, these tissues transformed arachidonic acid to 15-hydroxyeicosatetraenoic acid. The capacity per gram of tissue to produce lipoxins and 15-hydroxyeicosatetraenoic acid was 3-5-times higher in the nasal polyps. Neither tissue produced detectable levels of lipoxins or leukotrienes after incubation with ionophore A23187 and arachidonic acid. Co-incubation of nasal polyps and polymorphonuclear granulocytes with ionophore A23187 led to the formation of lipoxins, including lipoxins A4 and B4. The results indicate the involvement of an epithelial 15-lipoxygenase in lipoxin formation in human airways.

Topics: Arachidonate 15-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Bronchi; Calcimycin; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotrienes; Lipoxins; Nasal Polyps; Neutrophils

Topics: Animals; Drug Synergism; Granuloma; Hydroxyeicosatetraenoic Acids; Lipoxins; Male; Models, Biological; Potassium Permanganate; Rats; Rats, Inbred Strains

Granulocytes from human eosinophilic donors were incubated with arachidonic acid or 15-hydroxyeicosatetraenoic acid (15-HETE) and stimulated with the ionophore A23187. The eicosanoids were extracted with reversed-phase cartridges and subjected to RP-HPLC analysis. When extracts from eosinophil-enriched populations were analysed and compared with extracts from human neutrophils, three additional peaks were detected which coeluted with 15-hydroxy-delta 13-trans-15H derivatives of leukotriene C4, D4 and E4 in different HPLC systems. The recorded absorbance spectra of the eluted compounds and the standards were identical and showed a maximum at 307 nm which is characteristic for a conjugated tetraene system with a bathochromic shift by the sulfur moiety in alpha-position to the tetraene system. The compound which coeluted with the 15-hydroxy-LTC4 standard was treated with gamma-glutamyltransferase and converted to the corresponding leukotriene D4 derivative. The results indicate that interaction between the 5- and 15-lipoxygenase pathways leads to the formation of a new series of arachidonic acid metabolites in human eosinophils. Since the biosynthetic route is similar to that of lipoxin A4 and lipoxin B4, we suggest the trivial names lipoxin C4, D4 and E4.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Eosinophils; Granulocytes; Humans; Hydroxyeicosatetraenoic Acids; Lipoxins; Spectrophotometry, Ultraviolet

Lipoxygenase (LO) products generated by human PMN were examined utilizing a gradient-HPLC and rapid spectral detector which permitted continuous UV-spectral monitoring of leukotrienes, lipoxins and related oxygenated products of arachidonic acid. When exposed to the ionophore A23187, PMN generated LTB4 and its omega-oxidation products as well as LXA4, LXB4, and 7-cis-11-trans-LXA4 from endogenous sources. Addition of 15-HETE changed the profile of products generated by activated PMN and led to a time- and dose-dependent increase in lipoxins and related compounds while the production of LTB4 and its omega-oxidation products was inhibited. Results of time-course and radiolabel studies revealed that 15-HETE is rapidly transformed within 15 s to 5,15-DHETE and conjugated tetraene-containing products, and that the inhibition of leukotriene formation followed a similar time-course. In contrast, PMN did not generate either lipoxins or related products from 5-[3H]HETE, nor did 5-HETE block leukotriene formation. Stimulated PMN generated 5,15-DHETE from exogenous 5-HETE, while in the absence of ionophore, 5-HETE was transformed to 5,20-HETE. These results indicate that PMN can generate lipoxins and related products from endogenous sources and that 15-HETE and 5-HETE are transformed by different routes.

Topics: Calcimycin; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Leukotrienes; Lipoxins; Neutrophils; Spectrophotometry, Ultraviolet

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-lipoxygenases 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 omega-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 A4 nor lipoxin B4 (10(-8)-10(-6) M) 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.

Topics: Calcimycin; Free Radicals; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Lipoxins; Neutrophils; Superoxides

Addition of (15S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) and the ionophore A23187 (2.5 microM) to human neutrophils led to the formation of both lipoxin A4 and lipoxin B4 as well as a novel 5,6,15-trihydroxyeicosatetraenoic acid. The new compound was identified using an improved isolation and detection system and its basic structure was determined by physical methods. On the basis of biosynthetic considerations, geometric isomers of lipoxin A4 and lipoxin B4 were prepared by total synthesis. Comparison of these synthetic materials with the neutrophil-derived product showed that the new compound is (5S,6R,15S)-trihydroxy-9,11,13-trans-7-cis-eicosatetraenoic acid or the 7-cis-11-trans-isomer of LXA4 (7-cis-11-trans-LXA4). LXA4, 11-trans-LXA4, 7-cis-LXA4 and 7-cis-11-trans-LXA4 all evoked dose-dependent (0.1-10 microM) contractions of the guinea pig lung strip, whereas 6-cis-LXB4 and 6-cis-8-trans-LXB4 relaxed this preparation. LXA4 and 7-cis-LXA4 were approx. 10-times more potent than the compounds with 11-trans geometry. However, all four double-bond isomers of LXA4 caused contractions which, based upon pharmacological evidence, appeared to involve specific activation of the same site as cysteinyl-containing leukotrienes. In conclusion, 7-cis-11-trans-LXA4 was isolated and identified as a novel biologically active eicosanoid formed by human neutrophils.

Topics: Airway Resistance; Animals; Chemical Phenomena; Chemistry; Gas Chromatography-Mass Spectrometry; Guinea Pigs; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Lipoxins; Neutrophils; Stereoisomerism

The pure lipoxygenases from rabbit reticulocytes and soybeans convert a variety of substrates (arachidonic acid, 15-HPETE, 15-HETE, 5-HETE, various DiHETE isomers) to trihydroxy eicosanoids containing a conjugated tetraene system (lipoxins). In general, the methyl esters are better substrates for lipoxin formation than are the free acids. Lipoxygenase inhibitors (5,8,11,14-eicosatetraynoic acid, nordihydroguaiaretic acid) strongly inhibit the lipoxin formation. The complete stereochemistry of the lipoxin B formed from 15S-HETE methyl ester has been established by co-chromatography with authentic standards on various types of HPLC columns, by GC/MS analysis, by gas liquid chromatography of the ozonolysis fragments of the menthoxy carbonyl derivatives and 1H-NMR studies. The molar absorption coefficient of the conjugated tetraenes was measured as epsilon 301 = 53,000. The lipoxins formed from 15-HETE and various DiHETE isomers are formed exclusively via the oxygenation pathway as shown by experiments under an 17O2 atmosphere and/or by anaerobic incubations. Our results indicate that lipoxins can be synthesized via lipoxygenase-catalyzed sequential oxygenation of polyenoic fatty acids and their hydro(pero)xy derivatives.

Topics: Animals; Esters; Glycine max; Hydroxy Acids; Hydroxyeicosatetraenoic Acids; Lipoxins; Lipoxygenase; Oxidation-Reduction; Rabbits; Reticulocytes; Spectrophotometry, Ultraviolet; Substrate Specificity

Porcine leukocytes incubated with an isoenzyme of phospholipase A2 (PLA2) (isolated from snake venom) produced several trihydroxytetraene- containing compounds which were derived from endogenous sources of arachidonic acid. The formation of these compounds was dose-dependent with an EC50 of approximately 1.25 X 10(-8) M. At this concentration of the isoenzyme and time of exposure the cells remained viable as determined by the exclusion of trypan blue. The compounds were purified by HPLC and their identities were determined by physical criteria which included U.V. spectrometry, GC/MS and by comparison with both synthetic and authentic materials. The biologically derived compounds proved to be lipoxin B (5S, 14R, 15S-trihydroxy-6, 10, 12-trans-8-cis-eicosatetraenoic acid) and its two structural isomers (8-trans-LXB and 14S-8-trans-LXB). Of interest, only small amounts of lipoxin A and its isomers were found in these incubations. Results of the present study indicate that porcine leukocytes can generate lipoxin B and its isomers from endogenous sources of arachidonic acid. Moreover, they suggest that certain PLA2 isoenzymes may initiate the formation of lipoxins and related compounds.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Isoenzymes; Leukocytes; Lipoxins; Phospholipases; Phospholipases A; Phospholipases A2; Swine

The pure reticulocyte lipoxygenase converts 15LS-hydroxy-5,8,11,13(Z,Z,Z,E)-icosatetraenoic acid (15LS-HETE) methyl ester to a complex mixture of products containing 5DS,14LR,15LS-trihydro(pero)xy-6E,++ +8Z,10E,12E-icosatetraenoate methyl ester (lipoxin B methyl ester), 5DS,15LS-DiH(P)ETE methyl ester and four 8,15LS-DiH(P)ETE methyl ester isomers [DiH(P)ETE = dihydro(pero)xy-icosatetraenoic acid]. After a short incubation period (15 min) 5DS,15LS-DiH(P)ETE methyl ester was found to be the main product, whereas after a 3-h incubation lipoxin B methyl ester was the predominant product. The reaction shows a remarkable stereoselectivity since only small amounts of other trihydroxy tetraenes are formed. Anaerobiosis, heat inactivation of the enzyme, or incubation in the presence of lipoxygenase inhibitors (icosatetraynoic acid, nordihydroguaiaretic acid) completely abolished the reaction. The complete steric structure of the major tetraene product (lipoxin B methyl ester) was established by ultraviolet spectroscopy, HPLC on four different types of columns, gas chromatography/mass spectrometry, gas/liquid chromatography of the ozonolysis fragments of the menthoxycarbonyl derivatives, and by 400-MHz 1H-NMR. Atmospheric oxygen was incorporated at carbon-5 and carbon-14 into the major product. 5DS,15LS-DiH(P)ETE methyl ester was shown to be an intermediate in the synthesis. Lipoxin B was also formed during the oxygenation of arachidonic acid, 15LS-HETE and 5DS,15LS-DiHETE. The results presented here indicate that lipoxin B can be formed by pure lipoxygenases via a sequential oxygenation of arachidonic acid or its hydro(pero)xy derivatives.

Topics: Animals; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Lipoxins; Lipoxygenase; Magnetic Resonance Spectroscopy; Oxidation-Reduction; Rabbits; Reticulocytes; Stereoisomerism

Lipoxin B (LXB) was prepared by incubation of (15S)-15-hydroperoxy-5,8,11-cis-13-trans-icosatetraenoic acid (15-HPETE) with human leukocytes. Comparison with a number of trihydroxyicosatetraenes prepared by total synthesis showed that biologically derived LXB is (5S,14R,15S)-5,14,15-trihydroxy-6,10,12-trans-8-cis-icosatetraenoi c acid. Two isomers of LXB were identified by using an improved isolation procedure. These compounds were shown to be (5S,14R,15S)-5,14,15-trihydroxy-6,8,10,12-trans-icosatetraenoic acid (8-trans-LXB) and (5S,14S,15S)-5,14,15-trihydroxy-6,8,10,12-trans-icosatetraenoic acid [(14S)-8-trans-LXB]. Experiments with 18O2 showed that formation of LXB and its two isomers occurred with incorporation of molecular oxygen at C-5 but not at C-14. These results together with the finding that (15S)-hydroxy-5,8,11-cis-13-trans-icosatetraenoic acid (15-HETE) is a precursor of LXB compounds in activated leukocytes suggest that 15-hydroxy-5,6-epoxy-7,9,13-trans-11-cis-icosatetraenoic acid or its equivalent is a common intermediate in the biosynthesis of LXB and its two isomers.

Topics: Arachidonic Acids; Blood Platelets; Calcimycin; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Isomerism; Leukocytes; Leukotrienes; Lipid Peroxides; Lipoxins; Spectrophotometry, Ultraviolet; Stereoisomerism

Lipoxin A (5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic acid) and lipoxin B (5D,14,15-trihydroxy-6,8,10,12-eicosatetraenoic acid), two newly isolated compounds derived from the oxygenation of arachidonic acid in human leukocytes, inhibit the cytotoxic activity of human natural killer (NK) cells. Dose-response studies showed that both lipoxin A and lipoxin B inhibit, at submicromolar concentrations (ID50 10(-7) M), NK cell activity assayed against K562 target cells. Prostaglandin E2 (PGE2) also inhibited cytotoxicity, whereas both 15-HETE (5(S)-hydroxy-5,8,11,13-eicosatetraenoic acid) and leukotriene B4 (synthetic and biologically derived) were ineffective. PGE2 stimulated a time- and dose-dependent increase in intracellular cAMP, which was accompanied by a decrease in NK target cell binding. Lipoxin A and lipoxin B did not elevate intracellular cAMP, nor did they inhibit target cell binding. Together these findings suggest that lipoxin A and lipoxin B abrogate NK cell cytotoxicity at a step distal to target effector cell recognition. In contrast, PGE2 appears to exert its effect, at least in part, on cytotoxicity indirectly by decreasing the binding between target and effector cells (in vitro). Moreover, they suggest that novel oxygenated derivatives of arachidonic acid (i.e., lipoxin A, lipoxin B) may regulate the activities of NK cells.

Topics: Binding Sites; Binding, Competitive; Cell Line; Cyclic AMP; Cytotoxicity, Immunologic; Dinoprostone; Humans; Hydroxyeicosatetraenoic Acids; Intracellular Fluid; Killer Cells, Natural; Leukemia, Erythroblastic, Acute; Lipoxins; Prostaglandins E