Charlotte Edenius

Transcellular conversion of endogenous arachidonic acid to lipoxins in mixed human platelet-granulocyte suspensions

Incubation of mixed human platelet/granulocyte suspensions with ionophore A23187 led to a platelet dependent formation of several lipoxin isomers from endogenous substrate. The major metabolite coeluted with authentic lipoxin A4 (5(S), 6(R), 15(S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid) in several HPLC-systems and showed an identical UV-spectrum. Furthermore, a similar profile of lipoxins was formed in pure platelet suspensions incubated with exogenous leukotriene A4 (5(S) -5, 6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid). The conversion of exogenous leukotriene A4 to lipoxin A4 was markedly increased in the presence of ionophore A23187.

Lipoxin formation in human nasal polyps and bronchial tissue

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.

Formation and proliferative effects of lipoxins in human bone marrow.

Lipoxins A4 and B4 together with the all-trans lipoxin (LX) isomers were produced by normal human bone marrow cell suspensions after incubation with ionophore A23187. Both LXA4 and LXB4 enhanced the growth of myeloid progenitor cells in semisolid agar in the presence of suboptimal concentrations of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). Lipoxin A4 at 10(-10) M stimulated the colony formation in 13 out of 15 tested human bone marrows with a mean (+/- SEM) increase of 47 +/- 11% (p = 0.001). A similar stimulatory effect was observed after addition of LXB4 (10(-10) M). The monohydroxyeicosatetraenoic acids 5-, 12- and 15-HETE did not affect colony growth. In addition, LXA4 (10(-8) M) efficiently counteracted the increased colony formation induced by leukotriene C4 (10(-10) M), suggesting an antagonistic relationship between these lipoxygenase products. The results support a role for lipoxins in the regulation of human myelopoiesis.

Phorbol ester‐induced suppression of leukotriene C4 synthase activity in human granulocytes

The effect of the protein kinase C activator, phorbol 12‐myristate 13‐acetate (PMA), on the metabolism of exogenous leukotriene (LT)A4 in human granulocytes was investigated. After incubation with LTA4 decreased levels of LTC4 but not LTB4 were observed in granulocyte suspensions pretreated with PMA. This finding could in part be ascribed to oxidative metabolism of LTC4, since PMA induced a rapid degradation of exogenously added LTC4. After blocking of LTC4 metabolism with the H2O2 scavenger catalase, a PMA‐provoked suppression of the conversion of LTA4 to LTC4 was observed, indicating PKC‐dependent regulation of LTC4 synthase activity. This effect, as well as PMA‐induced degradation of LTC4 was presented by specific protein kinase C inhibitors.

Uncoupled regulation of leukotriene C4 synthase in platelets from aspirin-intolerant asthmatics and healthy volunteers after aspirin treatment.

Background We have reported that thromboxane A2 induces suppression of leukotriene (LT) C4 synthase activity in human platelets.

Conversion of 5,6-dihydroxyeicosatetraenoic acids A novel pathway for lipoxin formation by human platelets

Leukotriene A4 may be metabolized to 5(S),6(R)‐ and 5(S),6(S)‐dihydroxy‐7,9‐trans‐11,14‐cis‐eicosatetraenoic acids by enzymatic or non‐enzymatic hydrolysis. Incubation of human platelet suspensions with these dihydroxy acids led to the formation of lipoxin A4 and 6(S)‐lipoxin A4 via lipoxygenation at C‐15. Furthermore, human platelets converted the two 5(R),6(S)‐ and 5(R),6(R)‐dihydroxy‐7,9‐trans‐11,14‐cis‐eicosatetraenoic acids to tetraene‐containing trihydroxyeicosatetraenoic acids. In contrast, leukotrienes C4, D4 and E4 were not transformed to cysteinyl‐lipoxins, Time‐course studies of leukotriene A4 metabolism in human platelet suspensions indicated lipoxin formation via two pathways: (i) direct conversion of leukotriene A4, leading to formation of the lipoxin intermediate 15‐hydroxy‐leukotriene A4; and (ii) 15‐lipoxygenation of the 5(S),6(R)‐ and 5(S),6(S)‐dihydroxyeicosatetraenoic acids. The results demonstrate that lipoxygenation at C‐15 of 5,6‐dihydroxy‐7,9,11,14‐eicosatetraenoic acids may be an alternative novel pathway for platelet‐dependent lipoxin formation.

The influence of aspirin on release of eoxin C4, leukotriene C4 and 15-HETE, in eosinophilic granulocytes isolated from patients with asthma.

Background: The effect of aspirin on the release of key arachidonic acid metabolites in activated eosinophils from subjects with aspirin-intolerant asthma (AIA) has not been investigated previously, despite the characteristic eosinophilia in AIA. Methods: Peripheral blood eosinophils were isolated from four groups of subjects: healthy volunteers (HV; n = 8), mild asthma (MA; n = 8), severe asthma (SA; n = 9) and AIA (n = 7). In the absence or presence of lysine-aspirin, eosinophils were stimulated with arachidonic acid or calcium ionophore to trigger the 15-lipoxygenase-1 (15-LO) and 5-lipoxygenase (5-LO) pathways, respectively. 15(S)-hydroxy-eicosatetraenoic acid (15-HETE) and eoxin C4 (EXC4) were measured as 15-LO products and leukotriene (LT)C4 as a product of the 5-LO pathway. Results: Activated eosinophils from patients with SA and AIA produced approximately five times more 15-HETE than eosinophils from HV or MA patients. In the presence of lysine-aspirin, eosinophils from AIA, MA and SA patients generated higher levels of 15-HETE than in the absence of lysine-aspirin. Furthermore, in the presence of lysine-aspirin, formation of EXC4 was also significantly increased in eosinophils from AIA patients, and LTC4 synthesis was increased both in AIA and SA patients. Conclusions: Taken together, this study shows an increased release of the recently discovered lipid mediator EXC4, as well as the main indicator of 15-LO activity, 15-HETE, in activated eosinophils from severe and aspirin-intolerant asthmatics, and also elevated EXC4 and LTC4 formation in eosinophils from AIA patients after cellular activation in the presence of lysine-aspirin. The findings support a pathophysiological role of the 15-LO pathway in SA and AIA.

Evidence for a novel pathway of leukotriene formation in human platelets.

The metabolism of arachidonic acid via lipoxygenase-catalyzed reactions in washed human platelets was investigated. In addition to the previously discovered lipoxygenase metabolites, 12-hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, 8,15-dihydroxyeicosatetraenoic acid and 14,15-dihydroxyeicosatetraenoic acid, several other products were formed. The compounds were all dihydroxylated metabolites of arachidonic acid, containing a conjugated triene structure, and identified as 11,12-dihydroxyeicosatetraenoic acid (two isomers) and 5,12-dihydroxyeicosatetraenoic acid (four isomers). The identification was based on ultraviolet spectroscopy and gas chromatography-mass spectrometry of native and hydrogenated compounds. Stereochemical analysis of the hydroxyl groups of the 5,12-dihydroxyeicosatetraenoic acids and experiments with 18O2 indicated that the compounds were formed by the 12-lipoxygenase pathway, probably via an unstable epoxide.

Stimulation of lipoxin synthesis from leukotriene A4 by endogenously formed 12-hydroperoxyeicosatetraenoic acid in activated human platelets

Human platelets are devoid of 5-lipoxygenase activity but convert exogenous leukotriene A4 (LTA4) either by a specific LTC4 synthase to leukotriene C4 or via a 12-lipoxygenase mediated reaction to lipoxins. Unstimulated platelets mainly produced LTC4, whereas only minor amounts of lipoxins were formed. Platelet activation with thrombin, collagen or ionophore A23187 increased the conversion of LTA4 to lipoxins and decreased the leukotriene production. Maximal effects were observed after incubation with ionophore A23187, which induced synthesis of comparable amounts of lipoxins and cysteinyl leukotrienes (LTC4, LTD4 and LTE4). Chelation of intra- and extracellular calcium with quin-2 and EDTA reversed the ionophore A23187-induced stimulation of lipoxin synthesis from LTA4 and inhibited the formation of 12-hydroxyeicosatetraenoic acid (12-HETE) from endogenous substrate. However, calcium did not affect the 12-lipoxygenase activity in the 100,000 x g supernatant of sonicated platelet suspensions. Furthermore, the stimulatory effect on lipoxin formation induced by platelet agonists could be mimicked in intact platelets by the addition of low concentrations of arachidonic acid, 12-hydroperoxyeicosatetraenoic acid (12-HPETE) or 13-hydroperoxyoctadecadienoic acid (13-HPODE). The results indicate that the elevated lipoxin synthesis during platelet activation is due to stimulated 12-lipoxygenase activity induced by endogenously formed 12-HPETE.

Metabolism of Granulocyte-Derived Leukotriene A4 in Human Platelets and Respiratory Tissue: Transcellular Formation of Lipoxins and Leukotrienes

Leukotriene (LT)A4, the unstable intracellular intermediate in leukotriene biosynthesis, may be released to the extracellular space by activated leukocytes (1). As a consequence, the metabolism of LTA4 is not restricted to cells with 5-lipoxygenase activity, but can also be exerted by other cell types equipped with LTA4-metabolizing enzymes. Thus, LTA4, released by 5-lipoxygenase expressing cells, may be converted to LTB4 by surrounding erythrocytes (2), endothelial cells (3) or lymphocytes (4), all possessing LTA4 hydrolase activity. Similarly, mast cells (1), endothelial cells (3, 5) and smooth muscle cells (6) have been demonstrated to convert LTA4 to cysteinyl-containing leukotrienes. The present chapter describes some of our recent data regarding the metabolism of synthetic or granulocyte-derived LTA4 in human platelets and respiratory tissue leading to formation of cysteinyl-containing leukotrienes and lipoxins (LX).