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.     

15-Lipoxygenation of leukotriene A(4). Studies Of 12- and 15-lipoxygenase efficiency to catalyze lipoxin formation

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.     

Synthesis of lipoxins and other lipoxygenase products by macrophages from the rainbow trout, Oncorhynchus mykiss

Rainbow trout macrophages maintained in short term culture when incubated with either calcium ionophore, A23187, or opsonized zymosan synthesize a range of lipoxygenase products including lipoxins and leukotrienes. These cells are unusual in that they generate more lipoxin than leukotriene following such challenge. The main lipoxin synthesized was lipoxin (LX) A4. This compound was identified by cochromatography with authentic standard during reversephase high performance liquid chromatography, by ultra violet spectral analysis, radiolabeling following incorporation of [14C]arachidonic acid substrate into macrophage phospholipids, and gas chromatography electron impact mass spectrometry of the methyl ester, trimethylsilyl ether derivative. Other 4-series lipoxins synthesized by trout macrophages were identified as 11-trans-LXA4, 7-cis-11-trans-LXA4, and 6(S)-LXA4. These cells also produced 5-series lipoxins tentatively identified as LXA5, 11-trans-LXA5 and possibly 6(S)-LXA5. No LXB4 or LXB5 was, however, detected. The dynamics of leukotriene and lipoxin release were also determined. Lipoxin generation was slower than leukotriene generation the latter reaching a maximum after 30 min of exposure to ionophore (5 microM, 18 degrees C) compared with 45 min for the former.     

The effects of lipoxin A and lipoxin B on functional responses of human granulocytes

Lipoxin A and lipoxin B (LXA and LXB) are formed from arachidonic acid by leukocyte 5- and 15-lipoxygenases. We have assessed the effects of synthetic lipoxins on functional responses of human granulocytes. LXA stimulated migration at 1 nM. The effect was highly stereospecific, since e.g. 6S-LXA and LXB were less active than LXA. Neither synthetic LXA nor several of its stereoisomers provoked degranulation or aggregation. LXB and its isomers did not induce any of these functional responses. These results indicate that migratory granulocyte responses to LXA are highly stereospecific.     

Nomenclature of lipoxins and related compounds derived from arachidonic acid and eicosapentaenoic acid

Oxygenated derivates of arachidonic acid and eicosapentaenoic acid which contain conjugated tetraene structures and are non-cyclized C20 carboxylic acids were first isolated and characterized from human and porcine leukocytes (Serhan, C.N. et al, 1984, Biochem. Biophys. Res. Commun. 118, 943-949; Wong, P.Y.-K., et al, 1985, Biochem. Biophys. Res. Commun. 126, 765-775). The trivial names lipoxins and lipoxenes have been introduced for compounds belonging to each of these series. Here, we propose that tetraene-containing compounds derived from arachidonic acid be denoted as lipoxins (LX) of the four series (i.e. lipoxin A4 or LXA4 and lipoxin B4 or LXB4) and those derived from eicosapentaenoic be termed lipoxins of the five series (i.e. lipoxin A5 or LXA5 and lipoxin B5 or LXB5).     

Lipoxin formation in fish leucocytes

Adherent leucocytes, consisting of mainly macrophages, isolated from the haemopoietic head kidney of five species of fish were challenged with calcium ionophore and the resulting lipoxygenase products were separated and identified by reverse-phase high performance liquid chromatography. Of the fish examined, only adherent leucocytes from the Atlantic salmon and mirror carp generated lipoxins. Atlantic salmon leucocytes synthesized mainly lipoxin (LX) A4/LXA5 and 11-trans-LXA4/11-trans-LXA5, while mirror carp produced both LXA4 and LXB4 and their isomers but no 5-series lipoxins. This variation in lipoxin generation suggests that there are differences in the mode(s) of biosynthesis of these compounds between the two species of fish.     

Transformation of leukotriene A4 to lipoxins by rat kidney mesangial cell

Incubation of rat mesangial cells with leukotriene A4 in the presence of calcium ionophore A23187 led to a substrate dependent formation of lipoxin and its isomers. The major metabolite coeluted with authentic lipoxin A4 (LXA4) and lipoxin B4 (LXB4) in RP-HPLC system, and possessed a characteristic U.V. spectrum and C-value which were identical to authentic standards. GC/MS analysis on LXA4 further demonstrates that the mesangial cell derived LXA4 was identical to that reported by Serhan et al. (1) as LXA4 [5(S), 6,(R), 15(S)-trihydroxy7,9,13-trans-11-cis-eicosatetraenoic acid]. The formation of LXA4 was linear with substrate (LTA4) concentration. No similar products occurred in boiled controls. Incubation of mesangial cell with 15-HPETE failed to produce any lipoxin-like material. The absence of LX-like substance following incubation of 15-HPETE with mesangial cells suggested that 5-lipoxygenase activity is not expressed in mesangial cells under these conditions. The generation of LXA4 from LTA4 in mesangial cells suggested that there is an active 15- or 12- lipoxygenase activity in the kidney. The production of LX may play an important role in the regulation of renal function and the response to inflammatory stimuli.     

Lipoxin A4 and lipoxin B4 stimulate the release but not the oxygenation of arachidonic acid in human neutrophils: dissociation between lipid remodeling and adhesion

The profiles of actions of lipoxin A4 (LXA4) and lipoxin B4 (LXB4), two lipoxygenase-derived eicosanoids, were examined with human neutrophils. At nanomolar concentrations, LXA4 and LXB4 each stimulated the release of [1-14C]arachidonic acid from esterified sources in neutrophils. Lipoxin-induced release of [1-14C]arachidonic acid was both dose- and time-dependent and was comparable to that induced by the chemotactic peptide f-met-leu-phe. Time-course studies revealed that lipoxin A4 and lipoxin B4 each induced a biphasic release of [1-14C]arachidonic acid, which was evident within seconds (5-15 sec) in its initial phase and minutes (greater than 30 sec) in the second phase. In contrast, the all-trans isomers of LXA4 and LXB4 did not provoke [1-14C]AA release. Lipoxin-induced release of arachidonic acid was inhibited by prior treatment of the cells with pertussis toxin but not by its beta-oligomers, suggesting the involvement of guaninine nucleotide-binding regulatory proteins in this event. Dual radiolabeling of neutrophil phospholipid classes with [1-14C]arachidonic acid and [3H]palmitic acid showed that phosphatidylcholine was a major source of lipoxin-induced release of [1-14C]arachidonic acid. They also demonstrated that lipoxins rapidly stimulate both formation of phosphatidic acid as well as phospholipid remodeling. Although both LXA4 and LXB4 (10(-8)-10(-6) M) stimulated the release of [1-14C]arachidonic acid, neither compound evoked its oxygenation by either the 5- or 15-lipoxygenase pathways (including the formation of LTB4, 20-COOH-LTB4, 5-HETE, or 15-HETE). LXA4 and LXB4 (10(-7) M) each stimulated the elevation of cytosolic Ca2+ as monitored with Fura 2-loaded cells, albeit to a lesser extent than equimolar concentrations of FMLP. Neither lipoxin altered the binding of [3H]LTB4 to its receptor on neutrophils. In addition, they did not stimulate aggregation or induce adhesion of neutrophils to human endothelial cells. Results indicate that both LXA4 and LXB4 stimulate the rapid remodeling of neutrophil phospholipids to release arachidonic acid without provoking either aggregation or the formation of lipoxygenase-derived products within a similar temporal and dose range. Together they indicate that LXA4 and LXB4 display selective actions with human neutrophils and suggest that these eicosanoids possess unique profiles of action which may regulate neutrophil function during inflammation.     

Mesenteric vascular responses to i.v. administration of lipoxin A4 and lipoxin B4 in the conscious rat

Lipoxin A4 and lipoxin B4 are newly discovered lipoxygenase-interacting products of leukocytes which might have a role in cardiovascular events associated with anaphylaxis. We have tested this possibility by systemic administration of both LXA4 and LXB4 to the conscious rat while monitoring systemic and regional hemodynamic changes. LXA4 and LXB4 (1-100 micrograms/kg) produced dose-dependent constriction of the mesenteric vessels, up to +123 +/- 23% and +50 +/- 9% for LXA4/B4, respectively. Dose-related changes were not observed in arterial blood pressure, heart rate, renal (LXB4) and hindquarter blood flow. We suggest that LXA4 and LXB4 might affect selective vascular beds, such as the mesenteric vessels, and contribute to variations in blood flow in specific pathophysiological states.     

Lipoxins and aspirin-triggered 15-epi-lipoxins are the first lipid mediators of endogenous anti-inflammation and resolution

Lipoxins (LXs) or the lipoxygenase interaction products are generated from arachidonic acid via sequential actions of lipoxygenases and subsequent reactions to give specific trihydroxytetraene-containing eicosanoids. These unique structures are formed during cell-cell interactions and appear to act at both temporal and spatially distinct sites from other eicosanoids produced during the course of inflammatory responses and to stimulate natural resolution. Lipoxin A4 (LXA4) and lipoxin B4 (LXB4) are positional isomers that each possesses potent cellular and in vivo actions. These LX structures are conserved across species. The results of numerous studies reviewed in this work now confirm that they are the first recognized eicosanoid chemical mediators that display both potent anti-inflammatory and pro-resolving actions in vivo in disease models that include rabbit, rat, and mouse systems. LXs act at specific GPCRs as agonists to regulate cellular responses of interest in inflammation and resolution. Aspirin has a direct impact in the LX circuit by triggering the biosynthesis of endogenous epimers of LX, termed the aspirin-triggered 15-epi-LX, that share the potent anti-inflammatory actions of LX. Stable analogs of LXA4, LXB4, and aspirin-triggered lipoxin were prepared, and several of these display potent actions in vitro and in vivo. The results reviewed herein implicate a role of LX and their analogs in many common human diseases including airway inflammation, asthma, arthritis, cardiovascular disorders, gastrointestinal disease, periodontal disease, kidney diseases and graft-vs.-host disease, as well as others where uncontrolled inflammation plays a key role in disease pathogenesis. Hence, the LX pathways and mechanisms reviewed to date in this work provide a basis for new approaches to treatment of many common human diseases that involve inflammation.     

Lipoxin generation by human megakaryocyte-induced 12-lipoxygenase.

Eicosanoid biosynthesis was examined with a human megakaryocytic cell line (Dami). Megakaryocytes incubated with [1-14C]arachidonic acid and either ionophore A23187 or thrombin generated both thromboxane and 12-hydroxyheptadecatrienoic acid (HHTrE). Exposure to phorbol myristate acetate (PMA) for 1 through 9 days induced differentiation and revealed an increase in the conversion of [1-14C]arachidonate to cyclooxygenase- and lipoxygenase (LO)-derived products. The LO-derived product was identified as 12S-HETE by its physical characteristics including GC/MS and chiral column SP-HPLC. PMA-treated Dami cells did not generate 5-HETE, leukotrienes or lipoxins from exogenous arachidonic acid while they did convert leukotriene A4 (LTA4) to lipoxin A4, lipoxin B4 and their respective all-trans isomers. In addition, COS-M6 cells transfected with a human 12-lipoxygenase cDNA and incubated with either arachidonic acid or LTA4 generated 12-HETE and lipoxins, respectively. The lipoxin profile generated by transfected COS-M6 cells incubated with LTA4 was similar to that generated by the PMA-treated Dami cells. Results indicate that human megakaryocytes can transform arachidonate and LTA4 to bioactive eicosanoids and that the 12-lipoxygenase appears upon further differentiation of these cells. In addition, they indicate that the 12-LO of human megakaryocytes and the 12-LO expressed by transfected COS cells can generate both lipoxins A4 and B4. Together they suggest that the human 12-LO can serve as a model of LX-synthetase activity with LTA4.     

Characterization of lipoxins by combined gas chromatography and electron-capture negative ion chemical ionization mass spectrometry: formation of lipoxin A4 by stimulated human whole blood

The lipoxins are a recent addition to the family of bioactive products derived from arachidonic acid. Here, we have prepared pentafluorobenzyl ester, trimethylsilyl ether derivatives of lipoxin A4, lipoxin B4 and pentadeuterolipoxin A4 and have characterized these products by electron-capture negative ion chemical ionization gas chromatography/mass spectrometry (NICI GC/MS). Lipoxin A4 (5S,6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosa-tetraenoic acid; LXA4) was quantified following extraction from whole blood by stable isotopic dilution utilizing deuterium-labeled LXA4 as internal standard and selected ion monitoring of the [M--pentafluorobenzyl] anions. Studies with a second tritiated internal standard (e.g. [11,12-3H]LXA4) also showed that the recovery of LXA4 was greater than 80% following solid-phase extraction from whole blood, and greater than 90% from isolated cells. In addition, neither isolated neutrophils nor platelets oxidatively metabolized [11,12-3H]LXA4 when incubated in the presence or absence of stimuli. Whole blood incubated with either the ionophore of divalent cations (A23187), thrombin, or thrombin plus the chemotactic peptide formylmethionyl-leucine-phenylalanine generated both LXA4 and thromboxane, which were quantified by stable isotope dilution. The ratio of thromboxane to LXA4 formed by stimulated whole blood ranged from approximately 2:1 to 20:1. These results indicate that the lipoxins display suitable characteristics as their respective pentafluorobenzyl ester, trimethylsilyl ether derivatives for quantification by electron-capture NICI GC/MS. Moreover, they provide evidence that LXA4 can be generated from endogenous sources in whole blood following exposure to physiologically relevant stimuli.     

Lipoxin recognition sites. Specific binding of labeled lipoxin A4 with human neutrophils.

Lipoxin A4 stimulates rapid lipid remodeling and a pertussis toxin-sensitive release of arachidonic acid in polymorphonuclear leukocytes (PMN) (Nigam, S., Fiore, S., Luscinskas, F.W., and Serhan, C.N. (1990) J. Cell. Physiol. 143, 512-523) and has been shown to inhibit leukocyte responses in several systems. To examine the basis underlying these actions, we have prepared [11,12-3H]lipoxin A4 (LXA4) and characterized its interactions with human PMN. Time course studies (0-90 min) with intact PMN demonstrated cell association of 3H label which was specific and reversible. PMN bound [3H]LXA4 with a Kd of 0.5 +/- 0.3 nM, representing approximately 1,830 sites/PMN, and the Hill plot value of 1.9 suggests cooperative binding. [3H]LXA4 binding was stereoselective since neither leukotriene B4 (LTB4), lipoxin B4 (LXB4), (6S)-LXA4, 11-trans-LXA4, nor SKF 104353 competed for [3H]LXA4-specific binding while LTD4 and LTC4 partially competed. Subcellular fractionation revealed that specific binding with [3H]LXA4 was associated with membrane (42.1%)-, granule (34.5%)-, and nuclear (23.3%)-enriched fractions, a distribution distinct from that of [14,15-3H] LTB4 binding. [11,12-3H]LXA4-specific binding was modulated by guanosine analogs, suggesting the involvement of G proteins. A fluorescent LXA4 derivative (methyl-7-methoxycoumarin-LXA4) competed with [3H]LXA4 binding to intact PMN and showed specific and reversible binding as monitored by flow cytometric analysis. These results indicate that PMN possess specific recognition sites for LXA4 which may mediate its actions.     

Agonist-dependent generation of lipoxins from rat basophilic leukemia cell (RBL-1)

Incubation of RBL-1 cells in the presence of 15-HPETE and various agonists generated lipoxins and several isomers. Addition of either A23187, fMLP or PMA modulated the number of isomers and amount of lipoxins produced. Administration of A23187 yielded the largest amount of product (5.3 +/- 1.6 micrograms per 10(8) cells) and generated a total of six and three isomers of LXA4 and B4, respectively. This was 2-fold greater than fMLP, which produced a total of two isomers of LXA4 and LXB4. Addition of PMA generated only LXA4 (0.68 +/- 0.26 micrograms). This is similar to the control receiving only 15-HPETE. Biologically derived LXA4 (3 nM) isolated from RBL-1 incubations contracted a rat tail artery preparation to 12% of the maximum induced by phenylephrine (0.125 microM), whereas LXA4 standard (3 nM) elicited 17.6% of the maximum contraction. These results indicate that RBL-1 cells can utilize exogenous 15-HPETE to generate biologically active lipoxins. Further, the yield and isomers of lipoxins can be modified by different agonists.     

Lipoxins: Eicosanoids carrying intra-and intercellular messages

The lipoxins are a recent addition to the family of biologically active products derived from arachidonic acid. Compounds of this series contain a conjugated tetraene structure and can be generated by the actions of the major lipoxygenases of human tissues (5-, 12-, and 15-LO's). Biosynthesis of the lipoxins from cellular sources of unesterified arachidonic acid is triggered by the initial actions of either the 15-LO or 5-LO followed by additional reactions. Recent results indicate that lipoxins are also generated by receptor-mediated events during cell-cell interactions with the transcellular metabolism of key intermediates. Lipoxin A₄ and lipoxin B₄ each possess a unique spectrum of biological activities unlike those of other eicosanoids in bothin vivo andin vitro systems. Lipoxin A₄ stimulates changes in the microvasculature and can block some of the proinflammatory effects of leukotrienes (in vivo). Lipoxin A₄ and lipoxin B₄ both inhibit natural killer cells (in vitro), and lipoxin B₄ displays selective actions on hematopoietic cells. The finding that lipoxin A₄ activates isolated protein kinase C suggests that it may also serve an intracellular role in its cell of origin before it is released to the extracellular milieu. Thus, cell-cell interactions, along with multiple oxygenations by lipoxygenases, generate compounds that can regulate cellular responses by serving as both intra- and intercellular messages.     

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 × 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.     

Anti-inflammatory actions of lipoxin A4 and aspirin-triggered lipoxin are SOCS-2 dependent

Control of inflammation is crucial to prevent damage to the host during infection. Lipoxins and aspirin-triggered lipoxins are crucial modulators of proinflammatory responses; however, their intracellular mechanisms have not been completely elucidated. We previously showed that lipoxin A4 (LXA4) controls migration of dendritic cells (DCs) and production of interleukin (IL)-12 in vivo. In the absence of LXA4 biosynthetic pathways, the resulting uncontrolled inflammation during infection is lethal, despite pathogen clearance. Here we show that lipoxins activate two receptors in DCs, AhR and LXAR, and that this activation triggers expression of suppressor of cytokine signaling (SOCS)-2. SOCS-2-deficient DCs are hyper-responsive to microbial stimuli, as well as refractory to the inhibitory actions of LXA4, but not to IL-10. Upon infection with an intracellular pathogen, SOCS-2-deficient mice had uncontrolled production of proinflammatory cytokines, decreased microbial proliferation, aberrant leukocyte infiltration and elevated mortality. We also show that SOCS-2 is a crucial intracellular mediator of the anti-inflammatory actions of aspirin-induced lipoxins in vivo.     

Lipoxin A4 inhibits acute edema in mice: implications for the anti-edematogenic mechanism induced by aspirin

Lipoxin A4 (LXA4) is a lipid mediator that plays an important role in the resolution of inflammation. However, the role of LXA4 and aspirin (ASA)-triggered lipoxins (ATLs) in inflammatory edema formation remains unclear. Here, we investigated the inhibitory role played by LXA4 in the carrageenan-induced and other inflammatory mediator-induced edematogenic response in mice, and also assessed the role of ATLs in the anti-edematogenic action of aspirin. Our results showed that LXA4 (1-20 ng/paw or 5 microg/kg i.p.) was effective in inhibiting carrageenan-induced paw edema from 30 min to 2 h. LXA4 (10 ng/paw) was also able to acutely inhibit PAF-, histamine-, PGE2- or bradykinin-induced paw edema, as well as the PAF-induced myeloperoxidase activity increase in the paws. Likewise, LXA4 (10 ng/cavity) also inhibited the pleural edema triggered by histamine (1h), and this response was not followed by leukocyte accumulation. Of note, the lipoxin receptor (ALX-r) antagonist Boc2 (butoxycarbonyl-Phe-Leu-Phe-Leu-Phe, 200 ng/paw) significantly reverted the anti-edematogenic effect of ASA (300 mg/kg p.o.) against carrageenan, PAF, PGE2 and BK, without affecting the anti-edematogenic action caused by indomethacin (3 mg/kg i.p.) in the carrageenan-induced paw edema. Collectively, our results demonstrate for the first time that LXA4 displays an acute and rapid onset anti-edematogenic activity that does not discriminate among different pro-inflammatory stimuli, an effect that is most likely independent of its action on the leukocyte influx. Finally, the present study demonstrates that ATLs exert a very important role in the acute anti-edematogenic action of ASA.     

On the mechanism of transcellular lipoxin formation in human platelets and granulocytes

Endogenous arachidonic acid was converted to lipoxins A4, B4 and (6S)-lipoxin A4, in ionophore-A23187-stimulated mixtures of human platelets and granulocytes, while no lipoxins were formed when these cells were incubated separately. However, pure platelet suspensions transformed exogenous leukotriene A4 to lipoxins, including lipoxin A4 and (6S)-lipoxin A4, but not lipoxin B4. This compound was produced exclusively in the presence of granulocytes. A common unstable tetraene intermediate in lipoxin formation, 15-hydroxy-leukotriene A4 [5(6)-epoxy-15-hydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid], was indicated by trapping experiments with methanol. Thus, identical profiles of less polar tetraene-containing derivatives were formed from leukotriene A4 in platelet suspensions, from exogenous 15-hydroxyeicosatetraenoic acid in granulocyte suspensions and from endogenous substrate in mixed platelet/granulocyte suspensions. Evidence for the involvement of 12-lipoxygenase in platelet-dependent lipoxin formation was obtained. Thus, lipoxin synthesis from leukotriene A4 and 12-hydroxyeicosatetraenoic acid production from arachidonic acid by human platelets was equally inhibited by 15-hydroxyeicosatetraenoic acid with 50% inhibition obtained at 7.0 microM and 8.2 microM, respectively. In experiments with subcellular preparations from platelets, lipoxin synthesis was observed in both the particulate and soluble fraction and was paralleled by the 12-lipoxygenase activity. Furthermore, lipoxin formation from leukotriene A4 in platelet sonicates was dose-dependently inhibited by exogenous arachidonic acid. Finally, 12-lipoxygenase-deficient platelets from a patient with chronic myelogenous leukemia were totally unable to produce lipoxins from exogenous or granulocyte-derived leukotriene A4. It is concluded that the transcellular lipoxin synthesis is dependent on the platelet 12-lipoxygenase and proceeds via the unstable intermediate, 15-hydroxy-leukotriene A4. This tetraene epoxide is transformed to lipoxin B4 by a granulocyte epoxide hydrolase activity or to lipoxin A4 and lipoxins A4/B4 isomers by enzymatic or nonenzymatic hydrolysis.     

Aspirin-triggered lipoxin A4 and B4 analogs block extracellular signal-regulated kinase-dependent TNF-alpha secretion from human T cells

Lipoxins (LX) and their aspirin-triggered 15-epimer endogenous isoforms are endogenous anti-inflammatory and pro-resolution eicosanoids. In this study, we examined the impact of LX and aspirin-triggered LXA(4)-stable analogs (ATLa) on human T cell functions. 15-epi-16-(p-fluoro)phenoxy-LXA(4) (ATLa(1)) blocked the secretion of TNF-alpha from human PBMC after stimulation by anti-CD3 Abs, with the IC(50) value of approximately 0.05 nM. A similar action was also exerted by the native aspirin-triggered 15-epi-LXA(4), a new 15-epi-16-(p-trifluoro)phenoxy-LXA(4) analog (ATLa(2)), as well as LXB(4), and its analog 5-(R/S)-methyl-LXB(4). The LXA(4) receptor (ALX) is expressed in peripheral blood T cells and mediates the inhibition of TNF-alpha secretion from activated T cells by ATLa(1). This action was accomplished by inhibition of the anti-CD3-induced activation of extracellular signal-regulated kinase, which is essential for TNF-alpha secretion from anti-CD3-activated T cells. These results demonstrate novel roles for LX and aspirin-triggered LX in the regulation of T cell-mediated responses relevant in inflammation and its resolution. Moreover, they provide potential counterregulatory signals in communication(s) between the innate and acquired immune systems.