Structural Requirements for Activation of the 5-Oxo-6E,8Z,11Z,14Z-eicosatetraenoic Acid (5-Oxo-ETE) Receptor:Identification of a Mead Acid Metabolite with PotentAgonist Activity
Pranav Patel, Chantal Cossette, Jaganmohan R. Anumolu, Sylvie Gravel, Alain Lesimple,Orval A. Mamer, Joshua Rokach, and William S. PowellClaude Pepper Institute and Department of Chemistry, Florida Institute of Technology, Melbourne, Florida (P.P., J.R.A., J.R.);Meakins-Christie Laboratories, Department of Medicine, McGill University, Montreal, Quebec, Canada (C.C., S.G., W.S.P.);and Mass Spectrometry Unit, McGill University, Montreal, Quebec, Canada (A.L., O.A.M.)
Received December 5, 2007; accepted February 20, 2008
ABSTRACTThe 5-lipoxygenase product 5-oxo-6E,8Z,11Z,14Z-eicosatet-raenoic acid (5-oxo-ETE) is a potent chemoattractant for neu-trophils and eosinophils, and its actions are mediated by theoxoeicosanoid (OXE) receptor, a member of the G protein-coupled receptor family. To define the requirements for activa-tion of the OXE receptor, we have synthesized a series of5-oxo-6E,8Z-dienoic acids with chain lengths between 12 and20 carbons, as well as a series of 20-carbon 5-oxo fatty acids,either fully saturated or containing between one and five doublebonds. The effects of these compounds on neutrophils (calciummobilization, CD11b expression, and cell migration) and eosin-ophils (actin polymerization) were compared with those of5-oxo-ETE. The C12 and C14 analogs were without appreciableactivity, whereas the C16 5-oxo-dienoic acid was a weak partialagonist. In contrast, the corresponding C18 analog (5-oxo-18:2)
was nearly as potent as 5-oxo-ETE. Among the C20 analogs,the fully saturated compound had virtually no activity, whereas5-oxo-6E-eicosenoic acid had only weak agonist activity. Incontrast, 5-oxo-6E,8Z,11Z-eicosatrienoic acid (5-oxo-20:3) andits 8-trans isomer were approximately equipotent with 5-oxo-ETEin activating granulocytes. Because of the potent effects of 5-oxo-20:3, we investigated its formation from Mead acid (5Z,8Z,11Z-eicosatrienoic acid), which accumulates in dietary essential fattyacid deficiency, by neutrophils. The main Mead acid metaboliteidentified was 5-hydroxy-6,8,11-eicosatrienoic acid, followed by5-oxo-20:3 and two 6-trans isomers of leukotriene B3. We con-clude that optimal activation of the OXE receptor is achieved with5-oxo-ETE, 5-oxo-18:2, and 5-oxo-20:3, and that the latter com-pound could potentially be formed under conditions of essentialfatty acid deficiency.
Metabolism of arachidonic acid by the 5-lipoxygenase (5-LO)pathway leads to the formation of leukotriene (LT) B4, LTC4,LTD4, and 5-HETE (Funk, 2001). LTB4, acting through theBLT1 receptor, is a potent activator of neutrophils and lympho-cytes. LTD4 interacts with the cysteinyl-LT1 and cysteinyl-LT2
receptors to stimulate smooth muscle contraction, cytokine re-lease from leukocytes, and various other responses. Although
This work was supported by the Canadian Institutes of Health Research(Grant MOP-6254; to W.S.P.), the Quebec Heart and Stroke Foundation, theJT Costello Memorial Research Fund, and the National Institutes of Health(Grant HL81873; to J.R.). J.R. received support from the National ScienceFoundation for the AMX-360 (Grant CHE-90-13145) and Bruker 400 MHz(Grant CHE-03-42251) nuclear magnetic resonance instruments.
Article, publication date, and citation information can be found athttp://jpet.aspetjournals.org.
5-HETE itself has only relatively weak biological activities, it isoxidized to the potent granulocyte chemoattractant 5-oxo-ETEby the action of 5-hydroxyeicosanoid dehydrogenase (5-HEDH)(Powell and Rokach, 2005). 5-HEDH is a microsomal enzymethat is dependent on NADP� as an electron acceptor. Thislimits its activity because NADP�, in contrast to its reducedform NADPH, is only present at low concentrations within cells.Thus, 5-oxo-ETE synthesis from arachidonic acid requires bothactivation of 5-LO in conjunction with increased NADP� levels,which can be induced by oxidative stress and, in phagocyticcells, by activation of the respiratory burst. 5-HEDH is presentin most types of leukocytes (Powell and Rokach, 2005) as well asendothelial (Erlemann et al., 2006) and epithelial cells (Erle-mann et al., 2007).
5-Oxo-ETE induces a variety of rapid responses in neutro-phils and eosinophils, including calcium mobilization, actinpolymerization, CD11b expression, and L-selectin shedding(Powell and Rokach, 2005). It also stimulates superoxideproduction and degranulation in neutrophils primed withtumor necrosis factor-� (O’Flaherty et al., 1994) or granulo-cyte macrophage colony-stimulating factor (O’Flaherty et al.,1996). It is a potent chemoattractant for both neutrophils(Powell et al., 1993) and eosinophils (Powell et al., 1995a) andinduces transendothelial migration of eosinophils (Dallaireet al., 2003). It also has modest chemoattractant effects onmonocytes and stimulates actin polymerization (but not cal-cium mobilization) (Sozzani et al., 1996) and granulocytemacrophage colony-stimulating factor release (Stamatiou etal., 2004) in these cells. When administered in vivo to BrownNorway rats by intratracheal instillation, 5-oxo-ETE elicitspulmonary eosinophilia (Stamatiou et al., 1998). In humans,it induces the infiltration of both eosinophils and neutrophilsinto the skin (Almishri et al., 2005). In addition to its effectson inflammatory cells, 5-oxo-ETE has been reported to block
the induction of apoptosis in prostate tumor cells by MK886(Ghosh and Myers, 1998) and selenium (Ghosh, 2004).
The biological effects of 5-oxo-ETE are mediated by the Gi
protein-coupled oxoeicosanoid (OXE) receptor (Hosoi et al.,2005). This receptor is expressed on eosinophils, neutrophils,and monocytes (Hosoi et al., 2002; Jones et al., 2003) and hasalso been reported to be present on prostate tumor cells butnot on normal prostate epithelial cells (Sundaram and Ghosh,2006). Metabolism of 5-oxo-ETE by a variety of pathways,including reduction back to 5-HETE by 5-HEDH, reduction ofthe 6,7-double bond by a �6-reductase, conversion to 5-oxo-12-HETE by 12-lipoxygenase, and �-oxidation by LTB4 20-hydroxylase, results in dramatic reductions in OXE-receptoragonist activity (Powell and Rokach, 2005). However, therelationship of biological activity to carbon chain length andthe presence or absence of the �8, �11, and �14 double bondsis unknown. Because such knowledge is critical for the designof potent OXE-receptor agonists and antagonists, we pre-pared a series of 5-oxo-ETE analogs with different chainlengths and numbers of double bonds and investigated theirpotencies in eliciting biological responses in neutrophils andeosinophils.
Materials and MethodsOxygenated Fatty Acids. 5-Oxo-ETE was prepared by chemical
synthesis as described previously (Khanapure et al., 1998). 5-Oxo-20:5 was prepared by incubation of the corresponding 5-hydroxycompound (5-HEPE; obtained from Cayman Chemical, Ann Arbor,MI) with a microsomal fraction from human neutrophils in thepresence of NADP� as described previously (Powell et al., 1995b). Allof the other 5-oxo fatty acids shown in Fig. 1 as well as 5-hydroxy-20:3 were prepared by total chemical synthesis using methods thatwill be published separately. The structures were confirmed by nu-clear magnetic resonance and mass spectrometry. All compounds
Fig. 1. Structures of 5-oxo-ETE analogs tested in thepresent study. The abbreviations refer to those used inFigs. 2 to 5 and 7.
were purified by reversed-phase high-performance liquid chromatog-raphy (RP-HPLC) within 1 month of use. 13-HODE was prepared byoxidation of linoleic acid with soybean lipoxygenase type 1B (Sigma-Aldrich, St. Louis, MO) (Hamberg and Samuelsson, 1967).
Other Reagents. Phorbol 12-myristate 13-acetate (PMA) waspurchased from Sigma-Aldrich, whereas NADP� and A23187 wereobtained from Roche Diagnostics (Laval, QC, Canada) and Calbio-chem (La Jolla, CA), respectively. Dimethyl sulfoxide was purchasedfrom Fisher Scientific (Nepean, ON, Canada).
Preparation of Neutrophils. Neutrophils were purified fromwhole blood as described previously using dextran 500 (from Leu-conostoc; Sigma-Aldrich) to remove red blood cells followed by cen-trifugation over Ficoll-Paque (GE Healthcare, Baie d’Urfe, QC Can-ada) to remove mononuclear cells and hypotonic lysis of anyremaining red blood cells (Powell et al., 1992). The neutrophils weresuspended in phosphate-buffered saline (PBS).
Measurement of Calcium Mobilization in Neutrophils. In-tracellular calcium was measured in neutrophils loaded with indo-1acetoxymethyl ester (Invitrogen, Carlsbad, CA) as described previ-ously (Powell et al., 1996). The cells were washed and resuspended inPBS. Five minutes before commencing data acquisition, Ca2� andMg2� were added to give final concentrations of 1.8 and 1 mM,respectively. After stabilization of the baseline, fluorescence wasmeasured using a Deltascan 4000 spectrofluorometer (Photon Tech-nology International, Birmingham, NJ) with a temperature-con-trolled cuvette holder equipped with a magnetic stirrer.
Evaluation of CD11b Expression in Neutrophils. CD11b ex-pression was evaluated by flow cytometry (Powell et al., 1997) usinga FACSCalibur instrument (BD Biosciences, San Jose, CA). Unfrac-tionated leukocytes, prepared by treatment of whole blood with dex-tran followed by hypotonic lysis as described above, were incubatedfor 10 min with various concentrations of 5-oxo fatty acids. The cellswere then stained with fluorescein isothiocyanate-labeled anti-CD11b (Beckman Coulter, Fullerton, CA), and neutrophils wereidentified on the basis of their forward- and side-scatter properties.
Measurement of Actin Polymerization in Eosinophils. F-actin was measured using unfractionated leukocytes prepared asdescribed above. The leukocytes were first treated with PC5-labeledanti-CD16 (Beckman Coulter) to label neutrophils and then incu-bated with 5-oxo fatty acids for 20 s. The cells were then fixed withformaldehyde and stained by treatment with a mixture of lysophos-phatidylcholine and NBD-phallacidin (Invitrogen) as described pre-viously (Monneret et al., 2002). F-actin was then measured by flowcytometry in eosinophils, which were identified as a population ofcells with high side scatter and low expression of CD16.
Neutrophil Migration. Neutrophil migration was measured us-ing 48-well microchemotaxis chambers (Neuro Probe Inc., CabinJohn, MD) and Sartorius cellulose nitrate filters (8-�m pore size,140-�m thickness; Neuro Probe Inc.) as described previously (Powellet al., 1996). 5-Oxo fatty acids were added to the bottom wells, andneutrophils were added to the top wells. After 2 h, the filters wereremoved and stained, and the numbers of cells on the bottom sur-faces were counted in five different fields at a magnification of 400�for each incubation, each of which was performed in triplicate.
Analysis of Metabolites of Mead Acid. A suspension of neutro-phils (2 � 106 cells/ml) in PBS containing 1.8 mM Ca2� and 1 mMMg2� was incubated with Mead acid (50 �M; Cayman Chemical) inthe presence of A23187 (5 �M) and PMA (50 nM). After varioustimes, the incubations were terminated by addition of methanol (0.65ml) containing 0.15% trifluoroacetic acid and cooling to 0°C. Afteraddition of 13-HODE (100 ng) as an internal standard, the productswere analyzed by automated precolumn extraction coupled to RP-HPLC (Powell, 1987) using a Waters Alliance system (Waters, Mil-ford, MA). Products were quantitated by comparing the areas of theirpeaks of UV absorbance at their �max with that of 13-HODE. Theconditions for preparative experiments were similar, with the excep-tion that 108 neutrophils in 20 ml were used, and the products wereextracted manually on a C18 Sep-Pak (Waters) (Powell, 1980). A
Novapak C18 column (3.9 � 150 mm; Waters) was used as thestationary phase, whereas the mobile phase was a linear gradientover 40 min between water/acetonitrile/methanol/acetic acid (60:30:10:0.02) and water/acetonitrile/methanol/acetic acid (10:38:52:0.02)with a flow rate of 1 ml/min.
Mass Spectrometry. Mead acid metabolites were identified byFourier transform mass spectrometry (FTMS) as described previ-ously (Erlemann et al., 2007) using an IonSpec 7.0 Tesla instrument.MS/MS experiments were performed after isolating the monoisotopicparent ion using 500 ms of sustained off-resonance (1 kHz) irradia-tion collision-induced dissociation and a 90-ms nitrogen gas pulse.
Data Analysis. With the exception of EC20 and IC50 values, thevalues are expressed as the means � S.E. of data from n independentexperiments, as indicated in the figure legends. The concentration-response data are normalized and are expressed as percentages ofthe response to 1 �M 5-oxo-ETE for each individual experiment.“EC20” values are the concentrations of 5-oxo fatty acids that induceda response equal to 20% of the response to 1 �M 5-oxo-ETE (whichwas the maximal response to 5-oxo-ETE in the case of actin poly-merization). IC50 values are the concentrations of 5-oxo fatty acidsthat inhibited the response to 5-oxo-ETE (10 nM) by 50%. Both EC20
and IC50 values are presented as geometric means with the 95%confidence intervals shown in brackets. The statistical significance ofdifferences in EC20 and IC50 values and maximal responses wasdetermined by one-way repeated-measured analysis of variance us-ing the Bonferroni test as a multiple comparison method. Although itwas not possible to test all of the compounds in the same experiment,a complete concentration-response curve to 5-oxo-ETE was done ineach case. For the purposes of statistical analysis, the results foreach of the 5-oxo fatty acids were compared with those for 5-oxo-ETEonly from the same experiments.
ResultsEffects of 5-Oxo-ETE Analogs on Calcium Mobiliza-
tion in Neutrophils. We investigated the effects of the5-oxo-ETE analogs illustrated in Fig. 1 on cytosolic calciumlevels in indo-1-loaded human neutrophils. Various concen-trations of 5-oxo-ETE analogs were first added, followed 90 slater by 5-oxo-ETE (10 nM) to determine whether the ini-tially added compound induced homologous desensitizationor antagonized the response to 5-oxo-ETE. Digitonin wasthen added to determine the maximal fluorescence responsein the presence of a saturating concentration of calcium (Fig.2). When 5-oxo-ETE was added 90 s after addition of vehicle,a strong calcium response was observed (Fig. 2A). This wasdiminished only slightly by prior addition of 5-oxo-14:2 (10�M), which did not itself induce any detectable calcium tran-sient (Fig. 2B). In contrast, 5-oxo-16:2 (10 �M) elicited amodest calcium transient and completely abolished the re-sponse to subsequent addition of 5-oxo-ETE (Fig. 2C). Onethousand-fold lower concentrations of 5-oxo-20:3 (Fig. 2D)and 5-oxo-20:4 (i.e., 5-oxo-ETE; Fig. 2E) induced nearly iden-tical strong calcium responses and dramatically reduced theresponse to 5-oxo-ETE when it was added 90 s later.
The concentration-response curves for the effects of 5-oxo-ETE and other 5-oxo fatty acids on calcium mobilization areshown in Fig. 3. All of the compounds tested had 5-oxosubstituents, but, for simplicity, only the carbon chainlengths and numbers of double bonds are indicated by thelabels. The response to 5-oxo-ETE itself seems to be biphasic,tending toward a plateau at 1 �M but then increasing sub-stantially at the highest concentration tested (10 �M) (Fig.3A). A number of the other analogs tested also exhibited thistype of concentration-response relationship, in which a pla-
teau did not seem to have been reached by a concentration of10 �M. For this reason, it was not possible to calculateprecise EC50 values. Instead, EC20 values were calculated,but there was still considerable variability for some com-pounds that had low maximal responses (Table 1).
Figure 3A shows the effects on calcium mobilization of aseries of 5-oxo-6-trans-8-cis dienoic acids between 12 and 20carbons compared with that of 5-oxo-ETE. Among these di-enoic acids, 5-oxo-18:2 gave the strongest response and wasapproximately 20 times more potent than 5-oxo-20:2. 5-Oxo-16:2 was much less active and seemed to have a considerablyreduced maximal response compared with the longer-chaincompounds. Neither 5-oxo-14:2 nor 5-oxo-12:2 induced a de-tectable calcium response. In Fig. 3B, the effects of 20-carbon5-oxo fatty acids containing different numbers of doublebonds are shown. 5-Oxo-20:3 is nearly as active as 5-oxo-ETE, but the response drops off considerably as the numberof double bonds is reduced, with 5-oxo-20:1 having only weakagonist activity, and 5-oxo-20:0 displaying no detectable ac-tivity at the highest concentration tested (10 �M). The effectsof three different 5-oxo-20:3 isomers and two 5-oxo-monoenoicfatty acids are compared in Fig. 3C. The responses to 5-oxo-20:3(�6E,8Z,11Z) and 8-trans-5-oxo-20:3 (�6E,8E,11Z) are quite sim-ilar, with the exception that the 8-cis isomer tends to elicit astronger response at lower concentrations, whereas the8-trans isomer seems to induce a slightly higher maximalresponse. As observed for the dienoic acid series, 5-oxo-18:1induces a stronger response than its C20 homolog.
Desensitization of 5-Oxo-ETE-Induced Calcium Mo-bilization by 5-Oxo Fatty Acids. The ability of each of the5-oxo fatty acids to desensitize neutrophils to subsequentaddition of 5-oxo-ETE (10 nM) was investigated. The rank
order of potency for desensitization was similar to that foragonist activity. However, in this case, all of the analogsinduced complete desensitization to 5-oxo-ETE, with the ex-ception of 5-oxo-12:2, 5-oxo-14:2, and 5-oxo-20:0. Parallel con-centration-response curves were obtained with well defined,maximal responses, permitting calculation of IC50 values(see Fig. 6). 5-Oxo-ETE, 5-oxo-20:3, 8-trans-5-oxo-20:3, and5-oxo-18:2 all had very similar concentration-response curves(Fig. 3, D–F) with IC50 values of approximately 3 nM.�6E,8Z,14Z-5-Oxo-20:3 and 5-oxo-20:2 were approximately fiveto eight times less potent than 5-oxo-ETE in inducing desen-sitization to 5-oxo-ETE, whereas 5-oxo-16:2, 5-oxo-18:1, and5-oxo-20:1 were approximately 100 to 150 times less potent.Only very modest responses were observed for 5-oxo-14:2 and5-oxo-20:0 at the highest concentration tested (10 �M),whereas 5-oxo-12:2 was without any detectable activity (Fig.3, D and E).
Up-Regulation of CD11b by 5-Oxo-ETE Analogs. Theeffects of 5-oxo fatty acids on the surface expression of CD11bby neutrophils were also examined. In general, the resultswere similar to those obtained for calcium mobilization, with5-oxo-ETE, 5-oxo-20:3, 8-trans-5-oxo-20:3, and 5-oxo-18:2 allbeing potent inducers of CD11b expression (Fig. 4, A–C) withEC20 values between approximately 2 and 5 nM (Table 1).The response to 5-oxo-ETE tended toward a plateau at 1 �Mbut then increased sharply at 10 �M (Fig. 4A). This patternwas observed to an even greater extent for 8-trans-5-oxo-20:3(Fig. 4C) but not for 5-oxo-20:2 (Fig. 4A). 5-Oxo-20:2 was abetter inducer of CD11b expression (EC20, 10 nM) than cal-cium mobilization (EC20, 38 nM) (Fig. 4A). However, it in-duced a maximal response that was clearly lower than that to5-oxo-ETE. We also investigated the effect of 5-oxo-20:5 onCD11b expression and found it to be slightly less potent than5-oxo-ETE (Fig. 4B). Unlike all of the other 5-oxo fatty acids,we prepared 5-oxo-20:5 biosynthetically by incubating5-HEPE with neutrophil microsomes, and we did not havesufficient quantities to test concentrations higher than 1 �M.5-Oxo-18:1 was nearly 10 times more potent than 5-oxo-20:1in inducing CD11b expression (Fig. 4C).
Effects of 5-Oxo Fatty Acids on Actin Polymerizationin Eosinophils. The potencies of 5-oxo fatty acids in induc-ing actin polymerization in eosinophils were determined us-ing flow cytometry. These cells were identified in unfraction-ated leukocyte preparations on the basis of minimal labelingwith anti-CD16 and high side scatter. The use of unfraction-ated leukocytes has the advantage of reducing the number ofmanipulations to which the cells are subjected, thus lessen-ing the chances of activation during cell preparation. Al-though it is theoretically possible that the responses of eo-sinophils could have been mediated by the release ofstimulating factors from neutrophils, this would seem un-likely because of the very rapid actin polymerization re-sponse, which was determined after incubation with agonistsfor only 20 s.
In contrast to their effects on calcium mobilization andCD11b expression, the responses to most of the compoundstested reached plateaus. However, the maximal responses tosome of the analogs were considerably lower than that to5-oxo-ETE, which increased the levels of F-actin (i.e., poly-merized actin) to 75 � 5% above the basal level. In contrast,5-oxo-16:2 only increased F-actin levels to 23 � 3% abovebaseline (p � 0.001; Fig. 5A), whereas the maximal responses
Fig. 2. Effects of 5-oxo fatty acids on calcium mobilization in neutrophils.Indo-1-loaded neutrophils were first treated with either vehicle (A),5-oxo-14:2 (B), 5-oxo-16:2 (C), 5-oxo-20:3 (D), or 5-oxo-ETE (E), and fluo-rescence was measured using excitation and emission wavelengths of 331and 410 nm, respectively. After 1.5 min, 5-oxo-ETE (20:4) was added,followed 1.5 min later by digitonin (0.1%). Abbreviations are as defined inFig. 1. The numbers in parentheses are the concentrations in nanomolar.
observed for 5-oxo-20:1 and 5-oxo-18:1 were 33 � 3% (p �0.001) and 48 � 2% (p � 0.001) above baseline, respectively(Fig. 5C). The maximal responses to �6E,8Z,14Z-5-oxo-20:3(43 � 18% above control; p � 0.001) and 5-oxo-20:2 (37 � 5%
above control; p � 0.001) were also lower than that to 5-oxo-ETE by approximately 40% (Fig. 5, C and A, respectively).
Because of the variability in the maximal responses amongthe different analogs, EC20 rather than EC50 values were
Fig. 3. Concentration-response curves for the effects of 5-oxo-ETE analogs on calcium mobilization in neutrophils. A, effect of chain length on calciummobilization: 5-oxo-12:2 (f; n � 4), 5-oxo-14:2 (�; n � 4), 5-oxo-16:2 (Œ; n � 4), 5-oxo-18:2 (ƒ; n � 6), 5-oxo-20:2 (E; n � 6), and 5-oxo-ETE (●; n � 14).B, effect of the number of double bonds of 5-oxo C20 fatty acids: 5-oxo-20:0 (ƒ; n � 3), 5-oxo-20:1 (�; n � 6), 5-oxo-20:2 (E; n � 6), 5-oxo-20:3 (�; n �8), and 5-oxo-ETE (●; n � 14). C, effects of other 5-oxo-ETE analogs on calcium mobilization: 5-oxo-18:1 (�; n � 4), 5-oxo-20:1 (�; n � 6),5-oxo-�6,8,14-20:3 (�; n � 4), 5-oxo-20:3 (�; n � 8), and 8-trans-5-oxo-20:3 (Œ; n � 5). The results are expressed as percentages � S.E. of responses to1 �M 5-oxo-ETE for each individual experiment. D to F, desensitization of neutrophils to 5-oxo-ETE (10 nM)-induced calcium mobilization. The resultsare expressed as the percentage of inhibition � S.E. of the response to 5-oxo-ETE (10 nM), compared with the response after prior addition of vehicle,as shown in Fig. 2. The symbols and values for n are the same as in A to C.
TABLE 1Potencies of 5-oxo-fatty acids in stimulating neutrophil and eosinophil responsesCD11b expression, Ca2� mobilization, and desensitization to 5-oxo-ETE (10 nM)-induced Ca2� mobilization were measured in neutrophils, whereas actin polymerization wasmeasured in eosinophils. The values are geometric means (in boldface) with confidence intervals (in parenthesis) and were calculated from the individual experimentsrepresented in Figs. 3 to 5.
determined (Table 1). The effects of carbon chain length onpotency are shown in Fig. 6A. The corresponding IC50 valuesfor inhibition of 5-oxo-ETE-induced calcium mobilization (cf.Fig. 3D) are shown for comparison. Among the 5-oxo-dienoicacids tested, 5-oxo-18:2 was the most potent, having an EC20
(1.9 nM) significantly lower than that of its C20 homolog5-oxo-20:2 (EC20, 10 nM; p � 0.02) and slightly lower thanthat of 5-oxo-ETE (EC20, 3.1 nM; N.S.) (Fig. 6A). Similarresults were obtained for the desensitization by 5-oxo fattyacids of 5-oxo-ETE-induced calcium mobilization in neutro-phils, with 5-oxo-ETE and 5-oxo-18:2 being more potent than5-oxo-20:2. Of the two monoenoic acids tested, the C18 fattyacid (5-oxo-18:1; EC20, 132 nM) was also more potent instimulating actin polymerization than the corresponding C20
compound (5-oxo-20:1; EC20, 289 nM; p � 0.05) (Fig. 6A).The relationship between the number of olefinic double
bonds and agonist potency is shown in Fig. 6B. At least onedouble bond is required for appreciable agonist activity, be-
cause 5-oxo-20:0 has only a very modest effect at the highestconcentration tested (10 �M) (Fig. 5B). In this series, themost potent substance was 5-oxo-20:3, which had an EC20
value (1.8 nM) significantly lower than that of 5-oxo-ETE(EC20, 3.1 nM) (p � 0.05). In general, the IC50 values forcalcium desensitization paralleled the EC20 values for actinpolymerization, with the exception that 5-oxo-20:3 and 5-oxo-ETE had similar potencies (Fig. 6B).
Effects of 5-Oxo-20:3 and Related Compounds onNeutrophil Migration. Because of the high potency of5-oxo-20:3 in eliciting various responses in neutrophils andeosinophils, we investigated its chemotactic effects on neu-trophils. Both 5-oxo-20:3 and 8-trans-5-oxo-20:3 stronglystimulated neutrophil chemotaxis, with concentration-re-sponse curves that were virtually indistinguishable from thatof 5-oxo-ETE (Fig. 7) and EC50 values between 30 and 50 nM.The maximal response for 5-oxo-20:2 was approximately 25%lower than that for 5-oxo-ETE (p � 0.05).
Fig. 4. Effects of 5-oxo-ETE analogs on the surface expression of CD11b by neutrophils. A, effect of modification of chain length: 5-oxo-12:2 (f; n �3), 5-oxo-14:2 (�; n � 3), 5-oxo-16:2 (Œ; n � 4), 5-oxo-18:2 (ƒ; n � 5), 5-oxo-20:2 (E; n � 8), and 5-oxo-ETE (●; n � 13). B, effect of the number of doublebonds of 5-oxo C20 fatty acids: 5-oxo-20:0 (ƒ; n � 4), 5-oxo-20:1 (�; n � 5), 5-oxo-20:2 (E; n � 8), 5-oxo-20:3 (�; n � 4), 5-oxo-ETE (●; n � 13), and5-oxo-20:5 (�; n � 4). C, effects of other 5-oxo-ETE analogs on CD11b expression: 5-oxo-18:1 (�; n � 5), 5-oxo-20:1 (�; n � 5), 5-oxo-�6,8,14-20:3 (�;n � 5), 5-oxo-20:3 (�; n � 4), and 8-trans-5-oxo-20:3 (Œ; n � 7). The results are expressed as percentages � S.E. of responses to 1 �M 5-oxo-ETE foreach individual experiment.
Fig. 5. Effects of 5-oxo fatty acids on actin polymerization in eosinophils. A, effect of modification of chain length: 5-oxo-12:2 (f; n � 4), 5-oxo-14:2 (�;n � 4), 5-oxo-16:2 (Œ; n � 4), 5-oxo-18:2 (ƒ; n � 5), 5-oxo-20:2 (E; n � 4), and 5-oxo-ETE (●; n � 13). B, effect of the number of double bonds of 5-oxoC20 fatty acids: 5-oxo-20:0 (ƒ; n � 4), 5-oxo-20:1 (�; n � 4), 5-oxo-20:2 (E; n � 4), 5-oxo-20:3 (�; n � 5), 5-oxo-ETE (●; n � 13), and 5-oxo-20:5 (�; n �4). C, effects of other 5-oxo-ETE analogs on actin polymerization: 5-oxo-18:1 (�; n � 4), 5-oxo-20:1 (�; n � 4), 5-oxo-�6,8,14-20:3 (�; n � 4), 5-oxo-20:3(�; n � 5), and 8-trans-5-oxo-20:3 (Œ; n � 5). The results are expressed as percentages � S.E. of responses to 1 �M 5-oxo-ETE for each individualexperiment.
Formation of 5-Oxo-20:3 from Mead Acid by Neutro-phils. Because of the potent effects of 5-oxo-20:3 on neutro-phils and eosinophils, we investigated the possibility that itcould be formed from Mead acid (i.e., 5Z,8Z,11Z-eicosatrie-noic acid) by neutrophils. Mead acid was incubated withneutrophils in the presence of the calcium ionophore A23187and PMA, and the products were analyzed by RP-HPLC (Fig.8A). The major product had a retention time (tR) of 32.9 minand a �max at 235 nm (Fig. 8B), and cochromatographed withauthentic chemically synthesized 5-hydroxy-6,8,11-eicosa-trienoic acid (5-hydroxy-20:3). Two less polar products ab-sorbing at 237 and 235 nm (tR 26.2 and 29.8 min, respec-tively), probably other monohydroxy metabolites, were alsoformed, but their identities were not determined. Three ma-jor products absorbing at 280 nm were also detected (Fig.
8A). The least polar of these (tR, 35.3 min) had a �max at 280nm (Fig. 8B) and cochromatographed with authentic 5-oxo-20:3. Two more polar products with tRs of 13.7 and 14.8 minhad identical UV spectra typical of conjugated trienes with�max values at 258, 269, and 279 nm (Fig. 8B). These productsare presumably identical to 6-trans-LTB3 and 12-epi-6-trans-LTB3, which have previously been reported to be formed fromMead acid by neutrophils (Stenson et al., 1984).
The time course for the formation of the four productsdiscussed above is shown in Fig. 8C. All of these productswere formed very rapidly and reached maximal levels byapproximately 2 min. 5-Hydroxy-20:3 was the major productat all time points, followed by 5-oxo-20:3 and the two 6-transisomers of LTB3.
To provide more conclusive evidence for the identities of thetwo major products as 5-hydroxy-20:3 and 5-oxo-20:3, thesesubstances were purified by RP-HPLC after incubation of Meadacid with neutrophils and analyzed by mass spectrometry.Analysis of the material in the peak attributed to 5-hydroxy-20:3 by high-resolution electrospray ionization-FTMS in massspectrometry mode gave an intense M-1 ion at m/z 321.2435(C20H33O3
�1requires 321.2435). Collision-induced dissociation of
this ion gave a series of product ions (Fig. 9A) with m/z 303.2328(M-1-H2O; C20H31O2
requires 115.0401). The ionsat m/z 115 and 205 are formed by cleavage of the bond betweencarbons 5 and 6 and are indicative of the presence of a hydroxylgroup at C5. This product ion spectrum is virtually identical tothat obtained for authentic chemically synthesized 5-hydroxy-20:3 (data not shown).
FTMS analysis of the material in the HPLC peak attrib-uted to 5-oxo-20:3 gave an intense M-1 ion at m/z 319.2279(C20H31O3
�1requires 319.2279), which, when subjected to col-
lision-induced dissociation (Fig. 9B), gave a series of productions at m/z 301.2174 (M-1-H2O; C20H29O2
requires 111.0452). The ions at m/z 113 and 205formed by cleavage between carbons 5 and 6 are consistent withthe presence of an oxo group at carbon 5. This mass spectrum isnearly identical to that obtained for authentic 5-oxo-20:3 (datanot shown).
DiscussionThe results of the present study clearly show that the OXE
receptor is selective for 5-oxo fatty acids containing 18 or 20carbons and double bonds in the 6 and 8 positions. Compar-ison of a series of 5-oxo-6E,8Z-dienoic acids with carbon chainlengths between 12 and 20 revealed, somewhat surprisingly,that the C18 compound 5-oxo-18:2 was the most potentamong this group (Fig. 6A). Of the two 5-oxo-monoenoic acidsinvestigated, the C18 fatty acid 5-oxo-18:1 was also morepotent than its C20 counterpart 5-oxo-20:1. Of the C20 com-
Fig. 6. EC20 values for the effects of 5-oxo fatty acids on actin polymer-ization and 5-oxo-ETE-induced calcium mobilization. A, effects of modi-fication of chain length in series of 5-oxo-6E,8Z-dienoic acids (●, E) and5-oxo-6E-monoenoic acids (Œ, �) on actin polymerization in eosinophils(●, Œ, �) and desensitization to 5-oxo-ETE-induced calcium mobilizationin neutrophils (E, �, ƒ). The values obtained for 5-oxo-ETE (�, ƒ) areshown for comparison. B, EC50 values for the effects of C18 (Œ, �) and C20(●, E) 5-oxo fatty acids on actin polymerization (Œ, ●) and inhibition of5-oxo-ETE-induced calcium mobilization (E, �). The data for the C20 fattyacid with three double bonds is for 5-oxo-20:3. The values shown in A andB are geometric means, which were calculated from the data from eachindividual experiment represented in Figs. 3 (D–F) and 5 as describedunder Materials and Methods.
Fig. 7. Neutrophil migration in response to 5-oxo-ETE and other 5-oxoC20 fatty acids. Neutrophil migration was measured using 48-well micro-chemotaxis chambers as described under Materials and Methods. Neu-trophils were placed in the top chambers, whereas 5-oxo-ETE (●; n � 6),5-oxo-20:3 (�; n � 6), 8-trans-5-oxo-20:3 (ƒ; n � 5), and 5-oxo-20:2 (�; n �4) were placed in the bottom wells. The data are presented as percentagesof the maximal response to 5-oxo-ETE and are means � S.E.
pounds tested, 5-oxo-20:3 and its 8-trans isomer were themost potent in stimulating actin polymerization in eosino-phils (Fig. 6B). These compounds were equipotent with 5-oxo-ETE in stimulating chemotaxis and desensitization of theOXE receptor in neutrophils, and they were slightly lesspotent in inducing surface expression of CD11b and calciummobilization in these cells. The optimum number of doublebonds is thus 3 or 4, with greater (e.g., 5-oxo-20:5) or fewer(e.g., 5-oxo-20:2) double bonds resulting in reduced potency.Furthermore, the �11 double bond is clearly much more im-portant than the �14 double bond, because 5-oxo-20:3(�6E,8Z,11Z) is considerably more potent than �6E,8Z,14Z-5-oxo-20:3 in inducing all of the responses investigated. Loss of the�14 double bond likewise has little impact on the effects ofleukotrienes on their receptors. LTD3 was reported to beequipotent with LTD4 in stimulating contraction of guineapig ileum (Hammarstrom, 1981), whereas LTB3 was nearlyas potent as LTB4 in activating neutrophils (Evans et al.,1985a).
The dramatically reduced potency caused by shorteningthe carbon chain to 16 carbons (5-oxo-16:2) suggests that thehydrophobic �-end of the molecule is important for recogni-tion by the OXE receptor. This is in agreement with ourprevious finding that introduction of a polar hydroxyl groupat C20 (5-oxo-20-HETE) results in a reduction in potency ofnearly 100-fold (Powell et al., 1996). It is interesting to note
that 5-oxo-16:2 seems to be a partial agonist, because itinduced maximal responses that were only 34 � 3% (actinpolymerization) and 20 � 8% (calcium mobilization) of thoseto 5-oxo-ETE. In contrast, 5-oxo-16:2 completely blocked5-oxo-ETE-induced calcium mobilization. It is possible thatfurther modification of the structure of this compound couldresult in a compound with antagonist properties.
The lack of activity of short-chain analogs of 5-oxo-ETEsuggests that short-chain lipid peroxidation products such as5-oxovaleric acid, which can be released from peroxidizedphospholipids by platelet-activating factor (PAF) acetyl hy-drolase (Stremler et al., 1989), would not activate the OXEreceptor. In contrast, peroxidized phospholipids containingshort-chain aldehydes have been shown to activate the PAFreceptor (Smiley et al., 1991).
The presence of double bonds in the 6 and 8 positions of5-oxo fatty acids is clearly important for biological activity. Inthe absence of any carbon-carbon double bonds (5-oxo-20:0),there is only a small degree of activity at the highest concen-tration tested (10 �M). The importance of the �6 double bondis consistent with the lack of activity of 6,7-dihydro-5-oxo-ETE (i.e., 5-oxo-8Z,11Z,14Z-eicosatrienoic acid), which wepreviously reported to be a metabolite of 5-oxo-ETE formedby a calcium/calmodulin-dependent olefin reductase in neu-trophils (Berhane et al., 1998). Although the presence of asingle double bond in the 6 position (i.e., 5-oxo-20:1) clearlyincreased activity, this compound was still approximately100 times less potent than 5-oxo-ETE. The further addition ofa �8 double bond resulted in markedly increased biologicalactivity. However, the actual configuration of this doublebond seems to be of much lesser importance, because therewas little difference between the potencies of 5-oxo-20:3(�6E,8Z,11Z) and 8-trans-5-oxo-20:3 (�6E,8E,11Z). This is a littlesurprising, because alteration of the configuration of thisdouble bond would be expected to affect the orientation of thedistal, hydrophobic portion of the molecule, which, as notedabove, is important for a strong interaction with the OXEreceptor. This suggests that there is sufficient flexibility inthe ligand to permit the terminal part of the molecule (�C18–C20) to interact appropriately with the binding site on thereceptor. We previously found that alteration of the configu-ration of the �8 double bond of 5-oxo-ETE (i.e., 8-trans-5-oxo-ETE) reduced potency by approximately 6-fold comparedwith 5-oxo-ETE. The greater impact of this modification inthe case of 5-oxo-ETE may be due to the presence of the �14
double bond, which, although not required for agonist activ-
Fig. 8. Metabolism of Mead acid by neutrophils. A,chromatogram of metabolites formed after incuba-tion of neutrophils (2 � 106 cells/ml) with Mead acid(50 �M) in the presence of A23187 (5 �M) and PMA(50 nM) for 10 min. The products were analyzed byRP-HPLC as described under Materials and Meth-ods. 5h-20:3, 5-hydroxy-20:3; 5o-20:3, 5-oxo-20:3; 6t-B3, 6-trans-LTB3, and 12e-6t-B3, 12-epi-6-trans-LTB3. B, UV spectra of the major metabolites ofMead acid. The UV spectrum shown for 6-trans-LTB3 is identical to that for 12-epi-6-trans-LTB3. C,time courses for the formation of Mead acid metab-olites. The incubation conditions are as describedabove. Values are means � S.E. (n � 4).
Fig. 9. Mass spectra of the product ions obtained from the major metab-olites formed after incubation of Mead acid with neutrophils as describedin the legend to Fig. 8. A, mass spectrum of the product with a tR of 32.9min and maximal absorbance at 235 nM (i.e., 5-hydroxy-20:3) shown inFig. 8A. B, mass spectrum of the product with a tR of 35.3 min andmaximal absorbance at 280 nM (i.e., 5-oxo-20:3) shown in Fig. 8A.
ity, might restrict the flexibility of this part of the molecule,reducing its ability to compensate for the lack of a �8 cisdouble bond.
The concentration-response curves for the effects of 5-oxo-ETE on calcium mobilization and CD11b expression seem tobe biphasic, tending toward maxima at �1 �M and thenincreasing markedly at 10 �M. We conducted a limited num-ber of experiments at higher concentrations of 5-oxo-ETE (upto 100 �M), which resulted in substantially greater, but morevariable, responses, but we have not pursued this further. Asimilar pattern was observed for 5-oxo-18:2 and 8-trans-5-oxo-20:3. On the other hand, the responses to some of theother analogs, such as �6E,8Z,14Z-5-oxo-20:3 and 5-oxo-20:2,seemed to reach maxima, at least in the case of CD11bexpression. In contrast, the concentration-response curvesfor the effects of most of the 5-oxo fatty acids on actin poly-merization in eosinophils reached maxima within the concen-tration range tested. Similar results were obtained with neu-trophils (data not shown). The most likely explanation for thebiphasic response is that 5-oxo-ETE is a weak activator ofanother unrelated receptor at very high, presumably non-physiologic, concentrations. Activation of this second recep-tor would then result in increased calcium mobilization andCD11b expression, without having an appreciable effect onactin polymerization. We previously found that the ability ofagonists to stimulate calcium mobilization and CD11b ex-pression in eosinophils did not necessarily parallel their abil-ity to promote actin polymerization, with 5-oxo-ETE elicitingrelatively stronger actin responses compared with other ago-nists such as PAF (Powell et al., 1999) and eotaxin (Powell etal., 2001).
Although it would seem conceivable that the responses to5-oxo fatty acids that we observed are mediated principallyby the OXE receptor, it is possible that interaction with otherreceptors could have contributed to some extent, as discussedabove. Moreover, the degree of stimulation of OXE and non-OXE receptor-mediated responses may not have been thesame for all of the compounds tested. The contribution ofresponses mediated by other receptors should be reduced byfocusing more on the responses to lower concentrations ofagonists by using EC20 rather than EC50 values. Further-more, the results obtained for desensitization of 5-oxo-ETE-induced calcium mobilization used a low concentration of5-oxo-ETE (10 nM), which would minimize activation of re-ceptors other than the OXE receptor. Thus, inhibition of thisresponse by 5-oxo fatty acids is probably due to their effectson the OXE receptor. The actin polymerization response tothese compounds in eosinophils is also probably a good indi-cator of OXE receptor activation, because responses to all ofthe more potent agonists reached plateaus by approximately1 �M. In general, there is very good agreement in the relativepotencies of the analogs tested with respect to the differentresponses (Table 1).
Because of the potency of 5-oxo-20:3 in activating neutro-phils and eosinophils, we were interested in determiningwhether it could be synthesized from a polyunsaturated fattyacid (PUFA) precursor, namely the �9 PUFA 5,8,11-eicosa-trienoic acid, otherwise known as Mead acid. In contrast to�6 and �3 PUFA, which are essential fatty acids that mustbe supplied by the diet, �9 fatty acids are formed de novo bymammalian cells. In conditions of essential fatty acid defi-ciency, Mead acid accumulates in tissue lipids (Mead, 1956).
Although it cannot be converted to prostanoids by cyclooxy-genase because it lacks the 14,15-double bond, it can beconverted to mono- and dihydroxyeicosatrienoic acids by thisenzyme (Elliott et al., 1986). In contrast, Mead acid can beconverted to leukotrienes (Hammarstrom, 1981) and 5-hy-droxy-20:3 (Wei et al., 1985) by 5-LO. Although both arachi-donic acid and Mead acid are transformed to LTA4 and LTA3,respectively, by 5-LO, these two substances are metabolizeddifferently from one another. Both LTA4 and LTA3 are goodsubstrates for LTC synthase (Hammarstrom, 1981), whereasonly LTA4 is extensively metabolized by LTA hydrolase, andLTA3 inhibits this enzyme (Evans et al., 1985b). Thus, in-stead of being metabolized to LTB3 by neutrophils, Meadacid-derived LTA3 is nonenzymatically converted principallyto 6-trans-LTB3 and 12-epi-6-trans-LTB3 by these cells.
The major product formed from Mead acid by neutrophilsin the present study was 5-hydroxy-20:3, the identity ofwhich was confirmed by mass spectrometry. Substantialamounts of the two 6-trans isomers of LTB3 were also de-tected. In addition, 5-oxo-20:3 was identified by comparisonof its properties and mass spectrum with those of the authen-tic chemically synthesized compound. This is the first reportof this compound from either a biological or chemical source,and the first documentation of its potent biological effects.Because neutrophils are unable to synthesize significantamounts of LTB3 or LTC3 from Mead acid, it would seem thatthe major biologically active 5-LO product formed from thisPUFA by these cells is 5-oxo-20:3.
In conclusion, C18 and C20 5-oxo-�6,8 fatty acids are potentactivators of the OXE receptor-mediated activation of humanneutrophils and eosinophils. The most potent compoundstested were 5-oxo-ETE, 5-oxo-20:3, 8-trans-5-oxo-20:3, and5-oxo-18:2. Neutrophils convert the �9 PUFA Mead acid toone of these products, 5-oxo-20:3, which could potentially beformed and act as a proinflammatory mediator in situationsof dietary essential fatty acid deficiency.
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